BIOCHEMISTRY

for

B.Sc Nursing Students

(Revised, Updated and Enlarged)

Other Books by the Same Author

• Textbook of Biochemistry, 2nd ed.

• A Textbook of Biochemistry for Dental Students, 4th ed.

• Food & Nutrition (Medical, Nursing and Allied Sciences), 2nd ed.

• MCQs in Biochemistry

• Essentials of Pharmaceutical Biochemistry

BIOCHEMISTRY

for

B.Sc Nursing Students

(Revised, Updated and Enlarged)

FOURTH EDITION

HARBANS LAL
Ph.D., FIAO, FACBI, FSOBSI
Senior Professor & Head, Department of Biochemistry,
Maharaja Agrasen Medical College, Agroha (Hisar)
Former Sr. Professor, PGIMS, Rohtak
Ex WHO Fellow

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Preface to the
Fourth Edition

It gives me pleasure to present the fourth edition of the book “Biochemistry for B.Sc. Nursing Students”. A major goal continues to provide the book to the students of nursing that describes and illustrates the basics of biochemistry in a concise interesting manner. All the chapters have been thoroughly revised and updated. A large number of new figures and tables exhibiting the significance of biochemistry in nursing have been added in each chapter. The relevant information about clinical significance has been given in boxes.

I am highly thankful to the faculty and the students of nursing for their feedback, suggestions and help. I am also thankful to Dr. Anisha and Mrs. Preeti for their ever available help in the completion of this book. Mr. S.K. Jain and his team from CBS Publishers & Distributors Pvt. Ltd., New Delhi, deserve special mention for their helpful suggestions and bringing out this edition of the book in a very short time.

January 2016

HARBANS LAL
hl.biopgimsr@gmail.com

About the Book

Biochemistry for B.Sc Nursing Students, (Revised, Updated and Enlarged) endeavors to convey knowledge of the time-old principles of biochemistry supplemented with the modern developments in a simple, precise and lucid manner.

• The contents have been thoroughly revised and updated so as to give a recent concept of the subject to the nursing students, including the significance of biochemistry in this field.

• The whole subject, written as per the syllabus of the Nursing Council of India, has been divided into 19 chapters, including a chapter on sample collection and reference values.

• All the chapters have been added with new figures and tables. Keeping in mind the fact that the entire syllabus of biochemistry has to be covered in just 30 lectures, only the most essential chemical structures have been elucidated.

• Important terms have been highlighted in all the chapters.

About the Author

Harbans Lal is Senior Professor and Head, Department of Biochemistry, Maharaja Agrasen Medical College, Agroha, Hisar, and ex-Senior Professor, Department of Biochemistry, Postgraduate Institute of Medical Sciences, Rohtak, Haryana. He has nearly 42 years of experience of teaching biochemistry to various classes including MD, MSc, MBBS, BDS, BSc MLT and BSc Nursing. Besides teaching, he has supervised nearly 75 PhD and MD theses, written seven books, and published about 150 research papers in various international and national journals of repute.

Dr Harbans Lai is also Associate Editor, Journal of Biochemistry Letters, and reviewer of several international and national journals of biochemistry. He was a visiting faculty at Louisiana State University Medical Centre, New Orleans, USA, and Department of Biosciences, MD University, Rohtak. He is recipient of several international and national awards; fellowships of World Health Organization, Association of Clinical Biochemists of India and Society of Biological Scientists of India; Seth GS Medical College and KEM Hospital Oration Award; and Best Teacher Award for excellence in teaching at PGIMS, Rohtak.

CHAPTER
1

Introduction to
Biochemistry and its Significance

Biochemistry, sometimes also called biological chemistry, is the study of chemical processes within and relating to the living organisms.

HISTORY OF BIOCHEMISTRY

• The dawn of biochemistry may have been the discovery of the first enzyme diastase (these days also called as amylase) in 1833 by Payen.

• Thereafter, Buchner contributed the first demonstration of a complex biochemical process outside a cell in 1896, i.e. alcoholic fermentation in cell extracts of yeast.

• Although the term “biochemistry” seems to have been first used in 1882, it is generally accepted that the formal coinage of biochemistry occurred in 1903 by Neuberg, a German chemist.

• Since then biochemistry has advanced, especially after the mid-20th century, with the development of the new techniques such as chromatography, X-ray diffraction, NMR spectroscopy, radio-isotopic labelling and electron microscopy, etc. These techniques allowed for the discovery and detailed analysis of many molecules and metabolic pathways of the cell such as glycolysis and the Krebs cycle (citric acid cycle), etc.

BIOCHEMISTRY AND MOLECULAR BIOLOGY

Another significant historic event in biochemistry is the discovery of the gene and its role in the transfer of information in the cell. This part of biochemistry is often called molecular biology.

• In the 1950s, Watson, Crick, Franklin and Wilkins were instrumental in solving the structure of DNA and suggesting its relationship with the genetic transfer of information.

• In 1958, Beadle and Tatum showed that one gene produces one enzyme.

• In 1988, Pitchform was the first person convicted of murder with DNA evidence, which led to the growth of the use of molecular biology in forensic science.

• More recently, Fire and Mello discovered the role of RNA interference (RNAi) in the silencing of gene expression. Thus, depending upon the exact definition of the terms used, molecular biology can be thought of a branch of biochemistry or biochemistry as a tool to investigate and study molecular biology.

APPLICATIONS OF BIOCHEMISTRY

These days biochemistry has become so successful at explaining the living processes in almost all the areas of life sciences, viz. agriculture, nutrition and medicine.

• In agriculture, biochemists investigate and try to discover the ways to improve crop cultivation, yield and nutritive values of the food grains and vegetables and fruits.

• In nutrition, they study how to maintain health and the effects of nutritional deficiencies.

• In medicine, biochemists investigate the causes and cure of diseases.

BIOCHEMISTRY AS THE SCIENTIFIC BASIS OF MEDICINE

Biochemistry is beyond all other branches of science and forms a scientific basis of medicine.

• Human biochemistry focuses on the biochemistry of human beings and their medical illnesses. For example, there are more than twenty different chemical reactions known to be involved, in sequence, in the conversion of glucose to carbon dioxide and water but we are now able to say that a particular one is affected by the available supply of insulin.

• Clinicians think of biochemistry, chiefly as a collection of analytical procedures whose results they find useful in diagnosis, e.g. the diagnosis of hyperglycemia or renal glycosuria is dependent on blood glucose determination; gout on the estimation of uric acid; hyperparathyroidism on serum calcium concentration while nephrosis with respect to the estimation of serum proteins, etc.

SIGNIFICANCE OF BIOCHEMISTRY IN NURSING

A review of the application of biochemistry in nursing needs to mention of many therapeutic agents whose existence was first demonstrated in laboratory experiments. Moreover, these preparations, in usable forms, were first achieved by biochemical techniques, e.g. the hormone insulin, all the vitamins and many other products of therapeutic significance. If an infant is always vomiting after giving milk, she or he probably, has galactose intolerance. Similarly, if an infant is born with jaundice, her or his liver is not fully functional. When color of the infant’s urine turns dark, probably he/she suffers from an amino acid metabolic disorder. Anemic children, older, elder or geriatric persons can be explained with biochemistry, e.g. intake of Fe, hemolytic disorder (G6PD deficiency or pyruvate kinase deficiency) or a chronic renal disease. Many such conditions thus, can be explained on the basis of biochemistry.

CHAPTER
2

The Cell

The cell is a structural and functional unit of life. All animals and plants are made up of a large number of such units, in a manner to the utilization of bricks in the construction of a building.

Living cells are divided into two groups, i.e. the prokaryotic cells and the eukaryotic cells. As their name suggests (pro = prior to; karyot = nucleus; eu = true), the fundamental difference between them is the absence or presence of a true nucleus.

PROKARYOTIC CELLS

The simplest form of the cell is a prokaryotic cell. Prokaryotes, e.g. bacteria are unicellular and have one of the three basic shapes, viz. spheroidal (Cocci), rodlike (Bacilli) and helically-coiled (Spirella).

• A prokaryotic cell is small in size (1 to 10 nm), relatively simple in structure and has only a single membrane, called cell membrane, which is usually surrounded by a rigid cell wall of characteristic structure.

• There may or may not be a surrounding capsule.

• Besides, there is a single chromosome comprised of a molecule of double helical DNA which is densely coiled to form a nuclear zone.

• Reproduction is by asexual division. The best characterized prokaryotic cell is Escherichia coli.

• Some prokaryotes possess pili and flagella for adhesion and movement, respectively (Fig. 2.1).

Fig. 2.1. A prokaryotic cell.

EUKARYOTIC CELLS

Animals, plant, fungi and protozoa are called eukaryotes which may be unicellular or multi-cellular.

• Eukaryotic cells are one to ten thousand times large in size and are more complex in structure than the prokaryotic cells.

• They may vary from one tissue to another with respect to their functions, e.g. the liver parenchymal cell, adipose cell, nerve cell, renal tubular cell, white blood cell, etc.

• Generally, a eukaryotic cell has a well-defined membrane-bound nucleus containing several chromosomes.

• Their chromosomes undergo replication of DNA during mitosis and get separated into daughter chromosomes, i.e. these cells reproduce by cell division.

• A typical eukaryotic cell contains various organelles such as a nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, etc. (Fig. 2.2).

Fig. 2.2. A eukaryotic cell.

Differences between Prokaryotic and Eukaryotic Cell Structure

Major differences between prokaryotic and eukaryotic cell structures are given in Table 2.1.

Table 2.1. Major differences between prokaryotic and eukaryotic cell structures

Parameters | Prokaryotic cells | Eukaryotic cells
Cell size | Small | Large
Overall organization | Simple | Complex
Boundary | Cell membrane and cell wall, sometimes surrounded by a capsule | Cell membrane
Subcellular entities | Few | Many
Nucleus | They have a single nucleus which contains double helix DNA that is present in a poorly defined region called nucleoid | Well defined nucleus with a membrane and multiple DNA double helices organized into chromatin
Reproduction | Asexual | Sexual
Examples | Bacteria, blue-green algae | Plant and animal cells

STRUCTURE AND FUNCTIONS OF INTRACELLULAR COMPONENTS

THE NUCLEUS

The nucleus is the largest component of the cell, containing DNA organized into separate chromosomes and is surrounded by a membrane called nuclear membrane.

• The nuclear membrane consists of two layers which are separated by an intermembrane space termed as perinuclear space (cisterns). The outer membrane though is continuous with the endoplasmic reticulum but the two layers of the nuclear membrane are fused together at several places producing nuclear pores for the exchange of materials between the nucleus and the cytoplasm.

• The nucleus is filled with the nucleoplasm which has a discrete body called nucleolus and a thread-like structure called chromatin (Fig. 2.3).

Fig. 2.3. The nucleus.

• Nucleolus: The number of nucleoli may vary from one cell type to another. The genes for three of the four ribosomal RNA molecules are located in the nucleolus. Nucleoli are rich in RNA and disappear during cell division.

• Chromatin: It contains most of the cellular DNA in association with basic proteins, termed histones. At the time of cell division, the chromatin is organized into small thread-like structures called chromosomes. Human somatic cell contains 23 pairs of chromosomes.

Important Functions of the Nucleus

Important functions of the nucleus include:

• Control of cell division (DNA replication).

• Protein synthesis (by controlling the synthesis of RNA).

The regulation of DNA synthesis and other functions of the nucleus are severely disturbed in some pathological conditions such as cancer.

DNA and DNA polymerase are the markers of the nucleus.

THE ENDOPLASMIC RETICULUM

Endoplasmic reticulum is a system of membranes (lipid bilayer structures) with a network of vesicular spaces. This network is present throughout the cytoplasmic matrix and grows by its own synthesis. These membranes run parallel to each other creating channels which are called cisternae. The interior of the endoplasmic reticulum thus is well connected with perinuclear spaces and through pores on the cell surface, with the extracellular space. Cisternae have a role in the exchange of materials between the cell and the extracellular fluid. The surface of the endoplasmic reticulum may or may not bear ribosomes. Accordingly, endoplasmic reticulum is of two types:

• Rough endoplasmic reticulum (RER): It is also called as the granular type of endoplasmic reticulum since it has small granules attached to it. These granules are termed as ribosomes.

• Smooth endoplasmic reticulum (SER): It is also called as the agranular type of endoplasmic reticulum since it consists of the membranous structure only and does not contain ribosomes on its outer surface. The SER has enzymes for the biosynthesis of lipids and glycoproteins. Further, SER are very important in hepatocytes where these are primarily concerned with oxidative metabolism and for the detoxification of many drugs and other toxic organic molecules.

Glucose-6-phosphatase is a marker enzyme for the endoplasmic reticulum.

THE RIBOSOMES

Ribosomes consist of ribonucleoprotein particles of two sizes, i.e. 50S and 30S in prokaryotes or 60S and 40S in eukaryotes. Because of their high RNA content, ribosomes are the site of protein synthesis.

• Ribosomes on the RER are associated with the synthesis of proteins for export from the cell.

• Free ribosomes, on the other hand, are present in the cytoplasm and synthesize proteins for use within the cell.

RNA is used as a marker for the ribosomes.

THE GOLGI APPARATUS

The Golgi apparatus is a smooth membrane system with vacuoles. It is rich in lipids and is considered to be the site where secretions from other organelles are brought and assembled.

• The newly synthesized proteins are also transferred from RER and stored in the Golgi apparatus, temporarily.

• Some of the synthesized proteins also undergo post-translational modifications within the Golgi apparatus and thereafter, are transported to different destinations. The Golgi apparatus is thus especially active in cells which produce proteins for export. They form secretory granules for the proteins after their synthesis on the ribosomes.

Galactosyl transferase is a marker enzyme for the Golgi apparatus.

THE MITOCHONDRIA

The mitochondria are the major organelle of a eukaryotic cell lacking any direct structural relationship with other organelles and contain its own DNA (Fig. 2.4).

• A mitochondrion produces energy in the form of ATP for the cellular functions and is thus called as a power house of the cell. Thus, depending upon energy requirement of the cell, mitochondria may vary in size, shape and number, from cell to cell.

• Besides producing energy, mitochondria also help to control the level of calcium in the cytoplasm.

• Most of the cells contain several hundred mitochondria.

Fig. 2.4: A mitochondrion.

Mitochondrial membrane

It is a double-layered structure where the two layers are separated from each other by 50–100 Å intermembrane space. Several enzymes especially those involved in the nucleotide metabolism are located here.

• The outer membrane of the mitochondria has a smooth structure. It is composed of both lipids and proteins and is freely permeable to most of the small molecules. Several enzymes involved in lipid metabolism, such as the enzymes for fatty acid elongation, glycerol phosphate acyltransferase and phospholipase A, are associated with the outer membrane of the mitochondria.

• The inner membrane of the mitochondria has a denser structure. It has more proteins than lipids. The inner membrane has extensive irregular foldings, called cristae. Cytochromes, the enzymes of electron transport chain and flavoproteins are localized within the inner membrane of the mitochondria.

Matrix

The intra-mitochondrial space (enclosed within the inner membrane) is called mitochondrial matrix. This chamber contains enzymes for β-oxidation of fatty acids, citric acid cycle and glutamate dehydrogenase. In addition, mitochondria also have DNA, referred to as mtDNA.

Mitochondrial DNA: Mitochondria are the only cellular organelles that contain their own chromosomal DNA (mtDNA), which is maternally inherited. Human mtDNA is a small double stranded circular molecule (about 16,000 base pairs), encoding 13 polypeptides that are integrated into the inner mitochondrial membrane along with other polypeptides encoded by nuclear genes. In addition, it encodes 2 rRNAs and 22 tRNAs that are used in protein synthesis within the organelle. mtDNA differs from nuclear DNA in several aspects.

Comparison between nuclear DNA and mtDNA

Comparison between nuclear DNA and mtDNA is given in Table 2.2.

Table 2.2. Comparison between nuclear DNA and mitochondrial DNA (mtDNA)

Parameters | Nuclear DNA | mtDNA
Location | Nucleoplasm | Mitochondrial matrix
Inheritance | Both paternal and maternal | Maternal
Shape | Linear | Circular
Base pairs | 3 × 109 | 16 × 10³
Introns | Present | Absent
Histones | Present | Absent
Encoded proteins | Used within the cell as well as exported | Used within mitochondria
Repair mechanisms | Well developed | Poorly developed
Mutation rate | Low | High

In contrast to nuclear DNA, the mtDNA is exposed to high levels of mutagenic free radicals and is not protected by the usual DNA repair mechanisms. This results in mutations in mtDNA that affect mitochondrial structure and function, leading to various muscular and neurological disorders, such as Leber’s hereditary optic neuropathy, mitochondrial encephalopathy, chronic progressive external opthalmoplegia, mitochondrial encephalopathy lactic acidosis and stroke like episode (MELAS), etc.

Succinate dehydrogenase and glutamate dehydrogenase are marker enzymes for mitochondria.

THE LYSOSOMES

Lysosomes (derived from the Greek words lysis, meaning to loosen and soma, body) are the membrane-bound cell organelle found in animal cells. They are absent in red blood cells. Lysosomes are structurally and chemically spherical vesicles containing hydrolytic enzymes which are capable of breaking down all kinds of biomolecules including proteins, lipids, carbohydrates, nucleic acids and cellular debris.

• Lysosomes are known to contain more than fifty different enzymes which are all active at an acidic environment of about pH 5. Thus, lysosomes act as waste disposal system of the cell by digesting unwanted materials in the cytoplasm both from outside of the cell and obsolete components inside the cell.

• Further, lysosomes are responsible for cellular homeostasis for their involvements in secretion, plasma membrane repair, cell signaling and energy metabolism, which are related to health and diseases.

• Depending on their functional activity their sizes can vary.

Acid phosphatase is a marker enzyme of the lysosomes.

Suicide Bags

Lysosome is a bag (oval, rounded) that is loaded with digestive enzymes which, when activated, act upon cellular structures and demolish them leading to cell death. They are referred to as suicide-bags due to their role in autolysis, a process by which a biological cell self-destructs, i.e. during the period of starvation it engulfs or eats the whole cell.

Residual Bodies

Cytoplasmic vacuole containing the leftover products of digestion (e.g. membrane fragments or ferritin-like particles) after fusion with the contents of a lysosome is referred to as a residual body. Residual bodies are either secreted by the cell via exocytosis (generally in macrophages) or become lipofuscin granules that remain in the cytosol indefinitely. Longer-living cells like neurons and muscle cells usually have a higher concentration of lipofuscin than other more rapidly proliferating cells.

LYSOSOMAL STORAGE DISEASES

Lysosomes are responsible for a group of genetically inherited disorders called lysosomal storage diseases (LSD). They are a type of inborn errors of metabolism caused by malfunction of one of the enzymes. The rate of incidence is estimated to be 1 in 5,000 live births.

The primary cause is deficiency of an acidic hydrolase while there are conditions of defects in lysosomal membrane proteins that fail to transport the enzyme or nonenzymatic soluble lysosomal proteins. The initial effect is accumulation of specific macromolecules or monomeric compounds inside the endosomal–autophagic–lysosomal system. This results in abnormal signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking, ultimately leading to pathogenic disorders. The organs most affected are brain, viscera, bone and cartilage. There is no direct medical treatment to cure LSDs.

Gaucher’s disease: Gaucher’s disease is due to deficiency of the enzyme glucocerebrosidase. Consequently the enzyme substrate, the fatty acid glucosylceramide accumulates, particularly in white blood cells. This in turn affects spleen, liver, kidneys, lungs, brain and bone marrow. The disease is characterized by bruises, fatigue, anemia, low blood platelets, osteoporosis and enlargement of the liver and spleen.

THE PEROXISOMES

Peroxisomes, also called microbodies, contain certain oxidative enzymes, e.g. uric acid oxidase, D-amino acid oxidase and catalase, etc.

• The major function of the peroxisomes is in hepatocytes where they are involved in the oxidation of fatty acids by a modified α-oxidation pathway.

ZELLWEGER SYNDROME

Absence of the functional peroxisomes results in a rare autosomal recessive disease referred to as Zellweger syndrome. It is characterized by abnormalities of the several organs due to the decreased levels of plasmalogens.

• Besides, peroxisomes also represent a very primitive cellular organelle which provides protection to the cell from the toxicity of hydrogen peroxide (H₂O₂).

Catalase and uric acid oxidase are marker enzymes of the peroxisomes.

THE CYTOSOL

The cytosol or cell-sap is a structureless material filling the cell (aqueous matrix) in which all the cellular organelles float.

• It is a colloidal solution of proteins containing nearly 70% water.

• Besides proteins, the cytosolic fraction also contains various enzymes for glycolysis, gluconeogenesis and HMP shunt, and a variety of organic as well as inorganic substances such as glucose, potassium and magnesium.

• The cytosol is in contact with all the cellular organelles and an important vehicle for the transport of metabolites from one organelle to the other.

• The cytosol of all eukaryotic cells also contains a network of fibres, collectively called cytoskeleton, which includes microtubules, intermediate filaments and microfilaments.

Lactate dehydrogenase is a marker enzyme of the cytosol fraction of the cell.

THE PLASMA MEMBRANE

Plasma membrane or cell membrane is the outer membrane of the cell. It is in contact with the extracellular matrix. Structure and functions of the cell membrane are described separately (Chapter 3).

MICROSCOPY

Because of their small size, various types of the cells cannot be seen by a naked eye. These can only be observed with the aid of an instrument called microscope, which provides a magnified image of a tiny object. Microscopes give a wide range of magnifications.

They are of two types, called simple microscopes and compound microscopes.

Simple Microscope

A simple microscope works like a biconvex lens while a compound microscope employs two separate lens systems for greater magnification. There are two basic types of compound microscopes referred to as the optical or light microscope and the electron microscope.

Optical Microscope

An optical microscope uses optical lens and light wave for magnification. Widely used microscopes have two lenses. The lens system nearest the specimen is called objective. It forms primary image of the specimen and magnifies a definite number of times. The second lens system is called the eye piece or ocular lens, and is mounted in a holder known as body tube. Eye-piece further magnifies the image formed by the objective. Image seen by eye thus has a magnification of the two lens systems. Other components of the microscope include stage, revolving nose piece, coarse adjustment knob, mirror and base. This type of microscope is called bright filed microscope and magnifies about 1000 to 1500 times (Fig. 2.5).

Fig. 2.5. A microscope.

Electron Microscope

Besides, there is also a more sophisticated version called electron microscope. In electron microscope a magnification of 10,000 to 50,000 can be obtained and objects as small as 1–2 micron can be photographed. In principle, it works by using light of short wavelength to increase resolving power. A powerful electron beam is passed in vacuum through magnetic field, which replaces usual optical system of microscope and image of the material under examination is photographed. Its lenses are circular electromagnets whose focus varies as strength of the applied magnetic field.

Microscopy

Microscopy refers to the process of the use of a microscope. To use a microscope, place a slide over the hole in the center of the stage. Adjust the mirror until it reflects maximum amount of light through the specimen with the low objective in position. Look through the eye piece and slowly adjust the objective with coarse adjustment until the specimen is in approximate focus. Bring the specimen to sharp focus with the fine adjustment. Focus under low power and then under high power or oil immersion. Diaphragm and condenser should be manipulated each time for critical illumination of the object to be examined.

SOME IMPORTANT QUESTIONS

1. Describe structure, functions and biochemical markers of various subcellular entities of a eukaryotic cell.

2. Explain:

(i) Mitochondria as a power house of the cell.

(ii) Functions of the rough endoplasmic reticulum.

(iii) Differences between nuclear and mitochondrial DNA.

3. Write notes on:

(i) The nucleus

(ii) Ribosomes

(iii) The Golgi apparatus

(iv) Matrix

(v) Suicide bags

(vi) Age-pigments

(vii) Lysosomal storage diseases

(viii) Mitochondria and chloroplasts

(ix) Lysosomes and peroxisomes

(x) Microscopy

CHAPTER
3

Structure and
Functions of
Cell Membrane

All living cells are surrounded by a highly viscous yet flexible structure called cell membrane which, in animal cells, is also called as the plasma membrane. In addition to the cell membrane, eukaryotic cells also contain internal membrane systems (nuclear membrane, mitochondrial membrane, lysosomal membrane, etc.) which form specialized compartments within the cell. Cell membrane and other membranes are collectively referred to as biological membranes that determine which substances are to enter or exit from the enclosed region.

COMPOSITION OF A BIOLOGICAL MEMBRANE

Biological membranes are composed of lipids, proteins and carbohydrates. Different membranes within the cell and between cells, however, have different composition (Fig. 3.1).

Fig. 3.1. Composition of biological membranes.

LIPIDS

Lipids form more than 50% of the total membrane constituents. Membrane lipids comprise of both, hydrophobic as well as hydrophilic regions and thus are termed as amphipathic molecules. Various types of lipids are present in biological membranes such as fatty acids, phospholipids and cholesterol.

• Fatty acids have a polar head group and a nonpolar tail that may be saturated or unsaturated (Fig. 3.2).

Fig. 3.2. A fatty acid.

• Phospholipids form the major proportion of the lipid component of cell membranes (Fig. 3.3).

Fig. 3.3. A phospholipid.

• Besides, free/unesterified cholesterol is also present.

There are also qualitative differences between different classes of lipids and the individual lipids in different membranes within the cells as well as between the cells, e.g. plasma membrane has higher concentration of cholesterol and sphingolipids while intracellular membranes primarily contain glycerophospholipids with little sphingolipids or cholesterol.

In an aqueous solution, amphiphilic molecules form structurally ordered aggregates such as micelles and bilayers, which form structural basis of a biological membrane.

Micelles: A micelle is a spheroidal aggregate where a large number of amphiphilic molecules, e.g. soaps and detergents are arranged in such a way that their hydrophilic groups (at the globular surface) interact with the aqueous solvent while the hydrophobic groups are associated at the center, i.e. away from the solvent (Fig. 3.4).

Fig. 3.4. A micelle.

Micelles are formed when the cross-section of the hydrophilic head group exceeds that of the hydrophobic tails. This molecular arrangement eliminates unfavorable contact between water and hydrophobic tails of amphiphiles.

Lipid bilayers

Bilayers are formed when the cross-section of the hydrophilic head group of amphiphilic lipids are equal to that of the hydrophobic tails. Lipid bilayers are the key structures in biological membranes. A lipid bilayer exists as a sheet, i.e. an expanded planer-aggregate, in which hydrophobic regions of phospholipids are protected from the aqueous environment while the hydrophilic regions are immersed in water (Fig. 3.5).

Fig. 3.5. A lipid bilayer.

These are extremely stable structures which are held together by noncovalent interactions of the hydrocarbon chains and ionic interactions of the charged head groups with water. Lipophilic molecules such as cholesterol intercalate between fatty acyl residues of phospholipids and have minimal contact with the aqueous phase.

Liposomes

A lipid bilayer when closes on itself and forms a spherical vesicle, which separates the external environment from the internal compartment is called a liposome. These are a special type of vesicles surrounded by lipid bilayer and are of clinical significance.

• As the outer structures of the liposomes are similar to a biological membrane, liposomes serve as models of biological membranes.

• When administered intravenously, liposomes are absorbed by many cells through fusion with the plasma membrane. This has a profound clinical application because the technique can be used to deliver those compounds to a patient which cannot be absorbed when administered orally. Thus, they are used as vehicles for drug delivery, e.g. liposomal amphotericin B is commercially available for the treatment of systemic fungal infections in immunocompromised patients. Liposomes have also been prepared with enzymes and DNA.

PROTEINS

Proteins form another major portion of a biological membrane and play several roles such as the structural components, transport systems, enzymes and recognition sites for hormones. Proteins concentration may vary from about 20% in the myelin sheath to about 80% in the inner membrane of the mitochondria. Membrane proteins may be classified, by their mode of interaction with the membrane, as integral membrane proteins, peripheral membrane proteins or lipid-linked proteins.

Integral membrane proteins

These proteins have the following characteristics:

• They span the cell membrane from one side to the other and are also called as transmembrane proteins or intrinsic proteins, e.g. glycophorin A which spans the erythrocyte membrane.

• These are the amphiphilic molecules tightly bound to the membrane by hydrophobic interactions.

• Some proteins contain α-helical structure consisting primarily of hydrophobic amino acids. This, in turn, forms a transmembrane sequence which has three domains, i.e. a sequence exterior to the cell containing the – NH₂ terminal end, a transmembrane sequence and a sequence extending into the cell with the −COOH terminal end.

• They can be separated from the membrane only by drastic treatment with certain agents that disrupt membranes such as organic solvents or detergents.

Peripheral membrane proteins

These proteins have the following characteristics:

• They are embedded on any one side of the membrane and are also called as extrinsic proteins, e.g. cytochrome c which is present in the outer surface of the inner mitochondrial membrane.

• They are immersed in the membrane only partially and are weakly bound to the hydrophilic region of the specific integral proteins.

• They may have various modes of attachment, e.g. some bind to integral proteins such as an antigen. On the other hand, some peripheral proteins such as cytochrome b5 have sequences of hydrophobic amino acids at one end of the peptide chain which serve as an anchor in the membrane lipid.

• They can be released by relatively mild procedures that leave the membrane intact, such as by treatment with a salt solution of high ionic strength or by extremes of pH.

CARBOHYDRATES

Some of the membrane proteins and lipids bear short chains of carbohydrates (oligosaccharides), covalently attached either to a protein as glycoprotein or to a lipid as glycolipid.

Carbohydrate content of biological membranes may vary between 3–10%. Oligosaccharide chains are normally located on the outer surface of the membrane or on the terminal side of the endoplasmic reticulum.

STRUCTURE OF A BIOLOGICAL MEMBRANE

As described above, some of the proteins span the lipid bilayer whereas others are immersed only partially. This is called as the fluid mosaic model because the membrane is fluid in consistency, consisting of a mosaic of proteins and lipids which are free to drift about in the plane of the membrane (Fig. 3.6).

Fig. 3.6. Fluid mosaic model of a biological membrane.

It has following characteristics:

• It has a sheet-like structure forming a closed boundary and has a thickness of about 7–10 nm.

• It forms non-covalent, asymmetric assemblies of amphiphilic lipids and proteins.

• It is electrically polarized (inside negative with respect to outside).

Membrane fluidity

Membrane fluidity refers to the flexible nature of the membrane, i.e. it can shrink or expand. Membrane fluidity is regulated by several factors:

1. Presence of unsaturated fatty acids – Since cis-double bonds cause fatty acyl chains to bend (i.e. form kink), the membrane is less tightly packed and therefore more fluid in nature. Further, higher the unsaturation more is the fluidity.

2. Increased Ca²⁺ decreases membrane fluidity.

3. Cholesterol content of the biomembrane affects its fluidity.

FUNCTIONS OF A BIOLOGICAL MEMBRANE

Biological membranes perform several functions:

1. Compartmentalization: It separates two different microenvironments, e.g. the cell membrane separates the intracellular compartment from the extracellular matrix or extracellular fluid.

2. Cell shape: Plasma membrane proteins have a structural role, i.e. maintain shape of the cell and define its boundaries.

3. Cell movement: Specific arrangement of plasma membrane proteins is critical in controlling the movements of some cells, e.g. movement of neutrophils from the intravascular to the extravascular compartment requires a change in cell shape as well as movement, known as diapedesis.

4. Enzymes: Many of the membrane proteins are enzymes and are located either within or on the cell membrane. The inner mitochondrial membrane is essential for localization and correct orientation of the respiratory enzymes within it for their maximum efficiency. The membrane of the endoplasmic reticulum plays an important role in localizing lipophilic substrates for their oxidation by the enzymes present therein.

5. Receptor molecules: Membrane proteins act as recognition sites such as hormone receptors for insulin or glucagon, or immunoreceptors present on B lymphocytes for the recognition and response to foreign antigens, etc.

6. Transport of substances: Membrane proteins regulate transport of molecules such as amino acids, glucose and various ions.

7. Signal transduction: Various membrane proteins help in the transmission of signals such as transmission of nerve impulses.

TIGHT JUNCTION

Tight junctions, also known as occluding junctions, are the closely associated areas of the two cells whose membranes join together forming a virtually impermeable barrier to the fluid. It is a type of junctional complex present only in vertebrates. They are located at the very apical end of the junction complex between the adjacent epithelial cells (Fig. 3.7).

Fig. 3.7. Tight junction.

• Three main transmembrane protein families are found in tight junctions. These include occludins, claudins and junctional adhesion molecules (JAMs). Integral proteins of the tight junction form continuous fibrils that completely encircle the cell, like a gasket and makes contact with the neighboring cells on all sides. As a result of it, tight junction serves as a barrier to the free diffusion of water and solutes from the extracellular compartment on one side of an epithelial sheet to the other side.

• Tight junctions are also present between the endothelial cells that line walls of the capillaries. They are particularly evident in the brain where they form blood brain barrier which prevents substances from passing from bloodstream into the brain. Although small ions and water may not penetrate blood-brain barrier, cells of the immune system pass across the endothelium through these junctions.

• Tight junctions are specialized sites of contact that block solutes from diffusing between the cells in an epithelium. A simple epithelium like the lining of the intestine or the lungs is composed of a layer of cells that adhere tightly to one another to form a thin cellular sheet. Solutes cannot diffuse freely through this layer because of the specialized contacts called tight junctions.

Functions of Tight Junctions

1. Tight junctions hold the cells together.

2. They act as barrier and fence, and thus prevent passage of the molecules and ions through the space between plasma membranes of adjacent cells so that the materials must actually enter the cells by diffusion or active transport only, in order to pass through the tissue.

3. Tight junctions also help to distinguish the apical and basolateral domains of the cell by acting as intracellular barriers to macromolecules and proteins between these two regions.

4. They also maintain apicobasal polarity via protein complexes.

SOME IMPORTANT QUESTIONS

1. Discuss composition of a biological membrane.

2. Describe:

(i) Fluid mosaic model of cell membrane

(ii) Important functions of biological membranes

(iii) Micelles

(iv) Lipid bilayer

(v) Liposomes

(vi) Integral membrane proteins

(vii) Peripheral proteins

(viii) Tight junctions

CHAPTER
4

Transport of
Substances Across
the Cell Membrane

Transport of substances across the cell membrane can be described, in a functional sense, according to the number of molecules transported as well as the direction of their movement (Fig. 4.1).

Fig. 4.1. Transport of substances with respect to the number of molecules and the direction of movement.

Uniport: The process which allows the movement of one type of molecules in only one direction, e.g. glucose uptake in erythrocytes.

Co-transport: The process where transfer of one solute depends upon the simultaneous or sequential transfer of the other. It includes:

Symport: When two types of the molecules move in the same direction, it is called symport, e.g. the Na⁺-glucose transporter-1 (SGLT1) or the Na⁺-amino acid transporters in the cells lining the small intestine and proximal renal tubules.

Antiport: When two types of the molecules move in opposite directions, it is called antiport, e.g. the Na⁺-K⁺ antiport, the Na⁺-Ca²⁺ transporter and the Cl⁻-HCO₃⁻ antiport.

TRANSPORT SYSTEMS

There are three types of transport systems (Fig. 4.2).

Fig. 4.2: Transport systems.

DIFFUSION

Some solutes like O₂, N₂, CO₂, NO, urea, etc., move across the cell membrane by diffusing down an electrochemical gradient and do not require metabolic energy. This is called diffusion, simple diffusion or passive diffusion.

• It is limited by the thermal agitation of the specific molecule and by the concentration gradient across the membrane.

• The direction of flow is always from a higher to a lower concentration and the net movement of a molecule from one side to the other continues until concentration on each side is at chemical equilibrium.

• Diffusion of a substance may occur through channels or pores.

Channels

Channels are pore-forming membrane proteins whose functions include establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane, controlling the flow of ions across secretory and epithelial cells, and regulating cell volume (Fig. 4.3).

Fig. 4.3. Channel.

• Ion channels are located within the plasma membrane of nearly all cells and many intracellular organelles.

• These are often described as narrow tunnels that allow only ions of a certain size and/or charge to pass through. This characteristic is called selective permeability. For example, sodium or potassium ion channels. However, some channels may be permeable to the passage of more than one type of ion, typically sharing a common charge, may be positive (cations) or negative (anions).

• Ion channels are integral membrane proteins, typically formed as assemblies of several individual proteins.

• Ion channels are key components in a wide variety of biological processes that involve rapid changes in cells such as cardiac, skeletal and smooth muscle contraction, epithelial transport of nutrients and ions, T-cell activation and pancreatic β-cell insulin release.

CHANNELOPATHIES

Channelopathies include a number of genetic disorders which disrupt normal functioning of the ion channels and have disastrous consequences for the organism, e.g. cystic fibrosis. It is caused by mutations in the CFTR gene, which is a chloride channel.

Osmosis: Osmosis refers to the spontaneous net movement of solvent molecules through a partially permeable membrane into a region of higher solute concentration, in the direction that tends to equalize the solute concentrations on the two sides (Fig. 4.4).

Fig. 4.4. Osmosis.

It may be described as a physical process in which any solvent moves across a semipermeable membrane, i.e. the membrane which is permeable to the solvent but not the solute, separating two solutions of different concentrations. Osmosis is a vital process in biological systems as biological membranes are semipermeable. When a cell is submerged in water, the water molecules pass through the cell membrane from an area of low solute concentration to high solute concentration.

Suppose an animal cell is placed in a solution of sugar or salt in water.

• If the medium is hypotonic relative to the cell cytoplasm, the cell will gain water through osmosis. This means that the cell will swell and may even burst.

• If the medium is isotonic there will be no net movement of water across the cell membrane.

• If the medium is hypertonic relative to the cell cytoplasm the cell will lose water by osmosis. Essentially, this means that it will shrivel.

Aquaporins: Aquaporins selectively conduct water molecules in and out of the cell while preventing the passage of ions and other solutes. Also known as water channels, aquaporins are integral membrane pore proteins. There are several types of aquaporins in mammals and six of these are located in the kidneys. Mutations in the aquaporin-2 gene cause hereditary nephrogenic diabetes insipidus in humans.

Ionophores

An ionophore is a lipid-soluble molecule usually synthesized by microorganisms to transport ions across the lipid bilayer of the cell membrane (Fig. 4.5). The two broad classifications of ionophores are:

Fig. 4.5. Ionophore.

1. Chemical compounds (mobile ion carriers) that bind to a particular ion, shielding its charge from the surrounding environment and thus facilitating its crossing to the hydrophobic interior of the lipid membrane.

2. Channel formers that introduce a hydrophilic pore into the membrane, allowing ions to pass through while avoiding contact with the membrane’s hydrophobic interior.

Ionophores disrupt the transmembrane ion concentration gradients required for the proper functioning and survival of microorganisms and have antibiotic properties. They are produced naturally by a variety of microbes and act as a defence against competing microbes. Many antibiotics, particularly the macrolide antibiotics, are ionophores that exhibit high affinities for Na⁺ or K⁺, e.g., valinomycin is selective for potassium ions (K⁺), ionomycin for calcium ions (Ca²⁺), etc.

Facilitated Diffusion

Facilitated diffusion refers to the transport of solutes through membrane transport proteins without the expenditure of metabolic energy. As they cannot freely diffuse through the membrane and are transported with the help of some specific carrier molecules called transport proteins. Transport proteins may act as carriers, permeases, porters, translocases or transporters. Examples of the transport proteins which mediate facilitated diffusion include:

1. Glucose transporters (GLUT): It is a group of transport proteins present in the plasma membrane of mammalian cells for the transport of D-glucose, e.g. GLUT1, GLUT2 etc. These are referred to as glucose permeases. They translocate several D-sugars by a uniport mechanism.

2. Cl⁻/HCO₃⁻ exchanger: It is an anion exchanger protein for the antiport movement of Cl⁻ and HCO₃ in RBC membrane.

3. ATP-ADP translocase: An antiport for the exchange of anions (ADP³⁻ and ATP⁴⁻) between the cytosol and the mitochondrial matrix, located in the inner mitochondrial membrane.

The process can operate either uni-directionally or bidirectionally and the net flux across the membrane occur down a concentration gradient, i.e. the molecules flow from a higher to the lower concentration. Transport of a solute molecule occurs by the ping pong mechanism (Fig. 4.6).

Fig. 4.6. Facilitated diffusion – A ping pong mechanism.

Active Transport

In this process, the transport protein moves a specific molecule against the concentration gradient, i.e. from a lower concentration to the higher concentration, with the expenditure of metabolic energy. Thus, it is a process that requires energy which, in most cases, is coupled to the hydrolysis of ATP.

Active transport can be classified into two groups as:

• Primary active transport, and

• Secondary active transport.

Primary Active Transport

It is a process that requires energy which is provided by the direct hydrolysis of ATP. This type of transport system has the same characteristics as the passive transport system but it is an endergonic process. Examples of such transporters include membrane-bound ATPases that translocate cations. They are further classified as:

• P Type transporters: The transporter protein is phosphorylated and dephosphorylated during the transport activity.

• V Type transporters: These are present in the membrane of the lysosomes, the Golgi vesicles and the secretory vesicles, and are responsible for acidification of the interior of these vesicles.

• F Type transporters: These are present in mitochondria and are involved in ATP synthesis.

Primary active transporters are important in the maintenance of electrochemical gradient in biological systems. They consume nearly one third of the total energy expenditure of a cell. Examples of primary active transport systems include Na⁺/K⁺-ATPase, Ca²⁺-ATPase, H⁺/K⁺-ATPase, etc.

Na⁺/K⁺-ATPase: Na⁺/K⁺-ATPase, also referred to as Na⁺-K⁺ pump, is an antiport that moves three Na⁺ ions out of the cell in exchange of two K⁺ ions into the cell, with the concomitant hydrolysis of intracellular ATP. The pump is an integral part of the membrane and has binding sites for, both ATP and Na⁺ on the cytoplasmic side and K⁺ on the extracellular side of the membrane (Fig. 4.7).

Fig. 4.7. Na⁺-K⁺ pump.

Drugs such as cardiac glycosides, e.g. digoxin and ouabain, inhibit Na⁺-K⁺ pump and are of clinical importance in the management of cardiac failure.

• Normally, Na⁺-K⁺-ATPase in the cardiomyocyte membrane moves Na⁺ out of the cell in exchange for K⁺. Low cytosolic Na⁺ in turn maintains Na⁺-gradient for the functioning of the Na⁺-Ca²⁺ exchanger so that low cytosolic Ca²⁺ levels are maintained.

• In a failing heart, the aim of the treatment is to make the myocardium contract with greater force. This is achieved with digitalis, which, by inhibiting Na⁺-K⁺-ATPase, abolishes the Na⁺-gradient. Consequently, Ca²⁺ cannot exit the cell and attains sufficiently high intracellular level to mediate excitation-contraction coupling and generate greater contractile force. As more blood is pumped out, symptoms of heart failure are alleviated (Fig. 4.8).

Fig. 4.8. Inhibition of Na⁺-K⁺ pump with digitalis.

Ca²⁺-ATPase: Ca²⁺-ATPase, also called Ca²⁺ pump, actively pumps two Ca²⁺ ions out of the cytosol at the expense of ATP hydrolysis. The mechanism of Ca²⁺-ATPase resembles that of the Na⁺/K⁺-ATPase.

Ca²⁺ ions act as an important intracellular messenger, also referred to as second messenger. It regulates various cellular processes such as muscle contraction, release of neurotransmitters and glycogen breakdown. It is also an important activator of the oxidative metabolism. In order to maintain low cytosolic Ca²⁺ concentration, it is actively transported out of the cell across the plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum. In eukaryotes, Ca²⁺ transporter is regulated by the cytosolic Ca²⁺ level through a calcium binding protein called calmodulin.

H⁺/K⁺-ATPase: H⁺/K⁺-ATPase, also called proton pump, is an antiporter. It is important in the secretion of H⁺ ions in the luminal membrane. As H⁺ ions are pumped out, K⁺ ions, which enter the cells, are subsequently externalized. Since chloride channels secrete Cl⁻ ions into the lumen, the overall product, i.e. HCl is secreted by the parietal cells in the gastric mucosa.

Excess production of HC1 can damage the gastric mucosa and may lead to peptic ulcer. Drugs such as omeprazole act as proton pump inhibitors and are used clinically.

Secondary Active Transport

In secondary active transport, also called ion gradient driven active transport, free energy of the electrochemical gradient, which is generated by an ion pumping ATPase, drives the transport of a substance such as sugar or amino acid against its concentration gradient. Na⁺-glucose transport system, amino acid transport systems, etc., are the examples of the secondary active transport systems.

Na⁺-glucose transport system: In this process, Na⁺/K⁺-ATPase (as mentioned above) located in the basolateral membrane, pumps out the Na⁺ ions which in turn enter the cell (proximal renal tubules or the small intestine) with glucose, i.e. the uptake of glucose and sodium is secondarily dependent on the Na⁺ gradient that is maintained primarily by Na⁺/K⁺-ATPase. Accordingly, glucose enters intestinal epithelial cells by active transport using the electrogenic Na⁺-glucose transporter-1 (SGLT1) in the apical membrane (Fig. 4.9).

Fig. 4.9. Secondary active transport of glucose in the apical membrane.

This also forms the principle of oral rehydration therapy. As we know, main ingredients of the oral rehydration solution (ORS) include glucose, NaCl or NaHCO₃, KCl and water. On administration of ORS, glucose and Na⁺ are reabsorbed by the sodium-glucose transporter-1 (SGLT1) in the apical membrane of enterocytes. This in turn increases the osmolarity thereby favoring the absorption of water, osmotically. In this manner, absorption of glucose accompanied by the obligatory increase in the absorption of NaCl and water helps to compensate for the diarrhoeal losses of water and solutes and in turn treat dehydration.

Amino acid transport systems: Amino acids are transported by luminal epithelial cells of the intestine by the Na⁺-dependent pathway similar to the Na⁺-dependent glucose transport system. Several brush-border specific transport systems have been identified for the uptake of different groups of amino acids in the luminal membrane.

Defects in the amino acid transport systems can lead to several pathological conditions such as Hartnup’s disease. Hartnup’s disease is due to a decrease in the transport of neutral amino acids including tryptophan in the epithelial cells of the intestine and the renal tubules. It is characterized by pellagra-like symptoms.

TRANSPORT OF LARGE MOLECULES

Cells also transport certain macromolecules across the plasma membrane by mechanisms which involve vesicle formation with or from the plasma membrane and are referred to as endocytosis and exocytosis. All these processes require energy (ATP), Ca²⁺ and the cytoskeletal system for proper functioning.

Endocytosis

Endocytosis is a mechanism for the uptake of large molecules such as polysaccharides, proteins or polynucleotides into the cytoplasm. In this process:

• Firstly, a region of the plasma membrane invaginates, enclosing a small volume of the extracellular fluid and its contents within a bud, and generates endocytotic vesicle.

• The vesicle then pinches-off, as fusion of the plasma membrane seals the neck of the vesicle at the original site of invagination. The resulting small vesicle is called an endosome.

• It moves into the interior of the cell and delivers its contents to some other organelle, bound by a single membrane, e.g. a lysosome, by fusion of the two membranes.

• This hybrid vesicle is called a secondary lysosome. Due to the presence of hydrolytic enzymes, the macromolecular contents are digested to their monomers such as amino acids, simple sugars or nucleotides, which then diffuse out of the vesicle in the cytoplasm.

There are two general types of processes referred to as endocytosis, viz. phagocytosis and pinocytosis.

• Phagocytosis (or cell eating): It occurs only in specialized cells like macrophages and granulocytes for the ingestion of large particles such as bacteria, viruses, etc.

• Pinocytosis: Pinocytosis may be simple or receptor mediated.

Simple pinocytosis (or cell drinking) leads to cellular uptake of fluid and its contents as a result of invagination of the plasma membrane (Fig. 4.10).

Fig. 4.10. Pinocytosis.

Receptor mediated pinocytosis, also called absorptive pinocytosis, is a very selective type of pinocytosis that occurs in coated pits, lined with the protein clathrin, resulting in the formation of the clathrin-coated vesicles.

• The high affinity receptors permit selective concentration of the ligand from the medium, e.g. low density lipoproteins (LDL), transferrin, etc., and the ligand bound receptors are subsequently internalized by means of the coated-pits containing clathrin.

• The coated vesicle may fuse with lysosomes, the contents are digested and clathrin is recycled back to the membrane (Fig. 4.11).

Fig. 4.11. Receptor-mediated pinocytosis.

In case of some hormones, clathrin is not required for receptor-mediated pinocytosis. The internalized vesicle fuses with another organelle such as Golgi complex, i.e. no secondary lysosomes are formed and the process is known as adsorptive pinocytosis.

Exocytosis

Exocytosis is the reverse of endocytosis. It involves contact of two inside surface mono-layers from the cytosolic side and the release of macromolecules to the exterior of a cell. A secretory vesicle in the cytoplasm, originating in the Golgi complex or the endoplasmic reticulum, moves to the inner surface of the plasma membrane and fuses with it, releasing the vesicular contents outside the membrane (Fig. 4.12).

Fig. 4.12. Exocytosis.

The secreted/exocytosed molecules may have either of three possible fates:

1. They become a part of the cell membrane surface, e.g. antigens.

2. They become a part of the extracellular matrix, e.g. collagens.

3. They enter the blood and carried to distant sites, e.g. hormones like insulin.

GIBBS DONNAN MEMBRANE EQUILIBRIUM

Gibbs Donnan membrane equilibrium, also called Gibbs Donnan effect or simply Donnan membrane equilibrium, refers to the behavior of charged particles in a semipermeable membrane that sometimes fail to distribute evenly across two sides of the membrane.

It works on the principle that when two solutions of differing concentrations are separated by a semipermeable membrane their concentrations equalizes as a result of diffusion. Diffusion occurs when substances move from areas of high concentration to low concentration down a concentration gradient. However, if there is an impermeable solute in one of the solutions such as A⁻, concentration of the solution does not equalize. Concentration of the solution with impermeable solute remains high even at equilibrium (Fig. 4.13).

Fig. 4.13. Donnan membrane equilibrium.

Clinical usefulness of the Donnan Membrane Equilibrium

The Donnan effect can be applied to living cells since their cell membranes are selectively permeable, which means that they allow some molecules to pass through while keeping others out. Living cells contain impermeable anionic colloids which are mostly made up of proteins and organic phosphates that cannot cross the cell membrane. As a result of it, there is a high concentration of non-diffusible anions across the cell membrane thus creating the Donnan Equilibrium. This means that there are more ions inside the cell than outside. When a semipermeable membrane separates a nondiffusible substance such as protein, from diffusible substances, the diffusible anions and cations are distributed on the two sides of the membrane so that the products of their concentrations are equal and that the sum of the diffusible and non-diffusible anions on either side of the membrane is equal to the sum of the concentrations of diffusible and nondiffusible cations; the unequal distribution of diffusible ions thus produced, creates a potential difference across the membrane (membrane potential).

DIALYSIS

Dialysis is the process of separating molecules in solution by the differences in their rates of diffusion through a semipermeable membrane such as dialysis tubing. Typically a solution of several types of molecules is placed into a semipermeable dialysis bag such as a cellulose membrane with pores and the bag is sealed. The sealed dialysis bag is placed in a container of a different solution or pure water. Molecules small enough to pass through the tubing (often water, salts and other small molecules) tend to move into or out of the dialysis bag, in the direction of decreasing concentration. Larger molecules (often proteins, DNA or polysaccharides) that have dimensions significantly greater than the pore diameter are retained inside the dialysis bag (Fig. 4.14).

In medicine, dialysis is a process for removing waste and excess water from the blood and is used primarily as an artificial replacement for the lost kidney function in people with renal failure. Dialysis is done by using a special fluid called dialysate.

Dialysate, a mixture of pure water and chemicals, is carefully controlled to pull wastes out of the blood without removing substances, the body needs. A semipermeable membrane keeps the blood apart from the dialysate. This membrane lets the wastes and fluid in the blood flow through into the dialysate. There are two main types of kidney dialysis: hemodialysis and peritoneal dialysis.

Fig. 4.14. Dialysis.

HEMODIALYSIS

In hemodialysis, blood from a peripheral artery of the patient is allowed to flow on one side of a semipermeable membrane while an artificially prepared fluid of known solute concentration, called dialysate, flows by the other side but in opposite direction . Proteins present in the blood are the large non-diffusible ions. The solute concentration in the dialysate is so designed as to maximize diffusion of water and solutes across the membrane. The net effect is the reduction of toxic molecules to low levels in the blood while improving the levels of the essential ones. The purified blood is returned through a peripheral vein (Fig. 4.15).

Peritoneal dialysis: In peritoneal dialysis the basic procedure is the same. In this case the dialysateisrunthroughatubeintotheperitoneal cavityandtheperitonealmembraneitselfserves as the semipermeable membrane.The dialysate isretained forsome time and eventually drained out through the tube, removing the excess of water and the waste products.

Fig. 4.15. Hemodialysis.

SOME IMPORTANT QUESTIONS

1. Describe

(i) Ion channels

(ii) Transport disorders

(iii) Channelopathies

(iv) Ionophores

(v) Facilitated diffusion

(vi) Osmosis

(vii) Aquaporins

(viii) Na⁺, K⁺-pump

(ix) Primary active transport

(x) Secondary active transport

(xi) Gibbs Donnan membrane equilibrium

(xii) Dialysis

(xiii) Hemodialysis

(xiv) Exocytosis

(xv) Endocytosis

CHAPTER
5

Cytoskeleton

Cytoskeleton is a system of protein fibers that pervades cytoplasm.

Functions

It performs several functions:

1. Cytoskeleton provides structural support that determines shape of the cell and resists forces that tend to deform it.

2. It functions as an internal framework that is responsible for positioning various organelles within interior of the cell.

3. It provides a network that directs the movement of materials and organelles within the cells, e.g. delivery of mRNA to specific parts of the cell, movement of membranous carriers from the endoplasmic reticulum to the Golgi complex, etc.

4. It functions as the force-generating apparatus that moves cells from one place to another, e.g. locomotion of sperm, white blood cells, fibroblasts, etc.

5. Cytoskeletal elements make up the apparatus that is responsible for separating chromosomes during mitosis and meiosis, and for splitting the parent cell into two daughter cells during cytokinesis.

Composition

Cytoskeleton is composed of three distinct types of fibrous structures called microtubules, intermediate filaments and microfilaments. Each of it is a polymer of protein subunits which are held together by weak, non-covalent bonds. Each cytoskeletal element of different non-muscle cells such as an epithelial cell, a nerve cell and a dividing cell, has distinct properties and perform variety of functions (Table 5.1).

Table 5.1. Properties of cytoskeletal elements

MICROTUBULES

Microtubules are stiff, hollow, tubular structures that are 25 nm in diameter. They are assembled from protein tubulins that are arranged in longitudinal rows, called protofilaments. Tubule dimers that are polymerized into a microtubule contain a GTP molecule that is hydrolyzed soon after its incorporation into the polymer. The microtubules of the epithelial and nerve cells function primarily in support and organelle transport whereas the microtubules of the dividing cell form the mitotic spindle are required for chromosome segregation.

Microtubules perform various functions:

1. Microtubules provide structural support that determine cell shape and resist forces that tend to deform it.

2. They also provide the internal framework responsible for positioning various organelles.

3. Microtubules also provide a network of tracks that direct movement of materials and organelles within cells, e.g. delivery of mRNA from the nucleus to the ribosomes, movement of molecules destined for exocytosis, etc.

4. They also form a part of the locomotor machinery for the movement of cell as a whole, e.g. spermatozoa, WBC, fibroblasts, etc.

5. Microtubules also help in spindle formation during chromosome segregation and cell division.

Accessory proteins

Certain accessory proteins are required for the proper functioning of microtubules. These include:

• Microtubule associated proteins (MAP), e.g. tau protein. Tau proteins influence many properties of microtubules including their flexibility and stability. An abnormally high level of phosphorylation of tau protein is implicated in several neurodegenerative disorders, e.g. Alzheimer’s disease.

• Motor proteins such as kinesins and dyneins that generate the forces required for intracellular movements as well as the movements of the entire cell. To achieve this, the motor proteins convert the chemical energy of ATP into mechanical energy.

Microtubule Assembly

The assembly of microtubules from ab-tubulin dimers occurs in two distinct phases. These include a slow phase of nucleation in which a small portion of the microtubule is initially formed, followed by a rapid phase of elongation.

The nucleation is controlled by specialized structures called microtubules-organizing centres that govern the number of microtubules, their polarity and number of protofilaments that make up their walls as well as the time and location of their assembly. Some external factors interfere with the functions of microtubules by promoting disassembly, e.g. cold temperature, elevated Ca²⁺ concentration, and drugs like colchicine and vincristine.

• Colchicine is very beneficial in acute attack of gout involving the joints because it binds to the protein tubulin and rapidly leads to disassembly of the microtubules.

• Vincristine is an alkaloid that binds to tubulin dimers thus, inhibiting the assembly of microtubules. This arrests mitosis in metaphase and therefore, vincristine is a useful anticancer drug.

INTERMEDIATE FILAMENTS

Intermediate filaments are strong rope-like fibers that are approximately 10 nm in diameter. Depending on the cell type, they are composed of a variety of different globular protein subunits, capable of assembling into similar types of filaments.

Intermediate filaments are often connected by long wispy cross-bridges consisting of fibrous protein called plectin. The basic unit of intermediate filament is a tetramer formed by two dimers which may be homodimers or heterodimers. They provide mechanical stability to cells that may be required for specialized tissue-specific functions.

Intermediate filaments include keratin filaments and neurofilaments:

• Keratin filaments constitute the primary structural proteins of epithelial cells, including epidermal cells, liver hepatocytes and pancreatic acinar cells.

• Neurofilaments: Cytoplasm of neurons contains loosely packed bundles of intermediate filaments called neurofilaments whose long axis is oriented parallel to that of the nerve cell axon.

Intermediate filaments have tissue specific functions which are more important in some cells than in others.

MICROFILAMENTS

Microfilaments are 8 nm in diameter and are composed of a double helical polymer of the protein actin. Actin is one of the major contractile proteins of the muscle cells. In the presence of ATP, actin monomers polymerize to form a flexible, helical filament called actin filament or F-actin. They play a key role in virtually all types of contractility and motility within cells.

Depending on the type of cell and the activity, actin filaments can be organized into highly ordered arrays, loose ill-defined networks or tightly held bundles. Before being incorporated into a filament, an actin monomer binds a molecule of ATP that is subsequently hydrolyzed to ADP as the actin filament grows. This is due to the ATPase activity of actin. The bulk of an actin filament thus, consists of ADP-actin subunits. The molecular motors of actin filaments are the myosin family of proteins.

Myosins are generally divided into two groups:

1. Type I (unconventional myosins): Myosin I molecules have a single globular head and a variable tail length, and are implicated in cell motility and organelle transport.

2. Type II (conventional myosins): These were first identified in muscles and contain a long, rod-shaped tail attached at one end to two globular heads. The head binds the actin filament, hydrolyzes ATP and undergoes conformational changes required for generating force during muscle contraction as well some non-muscle activities like cytokinesis.

SOME IMPORTANT QUESTIONS

1. What is cytoskeleton? Name various cytoskeletal elements.

2. Briefly discuss the differences between various cytoskeletal elements.

3. Write note on:

(i) Microtubule assembly

(ii) Myosins

CHAPTER
6

Acid Base Balance

ACID

Acid is a substance whose dissociation in water releases hydrogen ions (H⁺). Since the H⁺ ion does not contain neutron and is essentially equivalent to a proton, an acid therefore is often referred to as a proton donor. Addition of an acid to a solution thus, increases the concentration of free H⁺ ions in the solution. This, in turn, produces a more acidic solution and results in a decrease in pH.

An acid, e.g. HA, in aqueous solution reversibly dissociates into H⁺ ion and a conjugate base (A⁻). This dissociation of HA thus can be represented symbolically by the following equation:

HA → H⁺ + A⁻

where A⁻ represents an anion (a conjugate base) that remains when H⁺ (a cation) is released. This tendency of an acid to give up H⁺ ions depends upon its strength.

A strong acid such as HCl completely dissociates in an aqueous solution as follows:

HCl → H⁺ + Cl⁻

On the other hand, a weak acid gives up H⁺ ions less rapidly and does not dissociate completely. A solution of a weak acid thus, contains both dissociated ions as well as undissociated molecule. Most of the acids produced in the living systems are weak acids.

BASE

A base releases hydroxyl ions (OH⁻) in an aqueous solution and decreases its H⁺ ions concentration by accepting or binding with the free H⁺ ions. This in turn results in increase in pH of the solution.

A strong base such as sodium hydroxide (NaOH) dissociates completely as follows:

NaOH → Na⁺ + OH⁻

The OH⁻ ions, so produced, accepts H⁺ ions and result in the formation of water. This in turn, lowers the concentration of the H⁺ ions in the solution.

OH⁻ + H⁺ ⇌ H₂O

A weak base such as bicarbonate (HCO₃⁻) can also utilize H⁺ ions and thus lowers H⁺ ions concentration of the solution.

HCO₃⁻ + H⁺ ⇌ H₂CO₃

Amount of an acid or a base is expressed in terms of equivalents. One mole of HCl contains one equivalent of acid and neutralizes one equivalent of base. On the other hand, one mole of sulfuric acid (H₂SO₄) contains two equivalents of acid since sulfuric acid can donate two H⁺ ions and requires two moles of NaOH to neutralize it.

AMPHOTERIC SUBSTANCES

Some substances such as amino acids and proteins can act as acid as well as base. These substances are referred to as amphoteric substances, e.g. glycine.

H₂N – CH₂ – COOH ⇌ H₃N⁺ – CH₂ – COO⁻

It acts as an acid when its ammonium group donates the H⁺ ion:

On the other hand, glycine acts as a base when its carboxylate group accepts H⁺ ion:

pH

pH is defined as the negative logarithm to the base 10 of the molar concentration of the hydrogen ions (H⁺) in a solution:

pH = – log [H⁺]

or = log (1/[H⁺])

The pH of the solution is inversely related to free H⁺ ions concentration, i.e. [H⁺]. Low pH indicates high H⁺ concentration while high pH indicates low H⁺ concentration.

If we add a strong base such as NaOH to water, the OH⁻ ions from NaOH will combine

with the H⁺ ions supplied by water. This in turn will shift the equilibrium towards the undissociated water and lower its free H⁺ ions concentration thereby resulting in increase in pH.

The scale of pH measurement varies from 0–14.

If pH of a solution is below 7, the solution is said to be acidic; if equal to 7 it is referred to as neutral, and if its pH is greater than 7 the solution is said to be alkaline or basic in nature.

BUFFERS

A buffer is a solution, which resists change in pH; i.e. it minimizes the change in the H⁺ ions concentration following the addition of an acid or a base.

A buffer can be prepared by mixing a weak acid, e.g. HA, with its conjugate base (A⁻). When a strong acid such as HCl is added to it, the conjugate base A⁻ reacts with the H⁺ ion, neutralizes it and forms HA. On the other hand, if a base is added, HA gets dissociated to give H⁺ ions and the conjugate base A⁻, as follows:

Dissociation constant (Ka) of the weak acid can be expressed as follows:

Larger the value of Ka, stronger the acid is, because most of the acid gets dissociated and converted to H⁺ and A⁻. On the other hand, if the Ka value is small, less acid is dissociated, being a weak acid.

A buffer has maximum buffering capacity at its pKa (–log Ka), i.e. when its acidic and basic forms are present in equal concentrations. Its acid (protonated) form reacts with the added base while the basic (unprotonated) form neutralizes the added acid.

Amino Acids as Buffers

Since an amino acid, e.g. alanine, contains two ionizable groups, one of which is the α-carboxyl group (α-COO⁻) while the other is the α-amino group (α-NH₃⁺). pKa of the α-COO⁻ group (called pKa₁) is 2.4 while that of the α-NH₃⁺ group (called pKa₂) is 9.8.

On titration with NaOH from pH 0 to 12, both of its ionizable groups are sequentially titrated. In the first stage of titration, i.e. at very low pH, the predominant ionic species is the protonated form. At pH 2.4 (pKa₁), since equimolar concentrations of the proton donor and the proton acceptor species are present, it proves to be a good buffer. The second stage of titration corresponds to the removal of a proton from the −NH₃⁺ group and accordingly, at pH 9.0 (pKa₂) it again has a good buffering capacity.

Thus, when dissolved in water, alanine exists as a dipolar ion or zwitterion in which the carboxyl group is unprotonated (−COO⁻) while the amino group is protonated (−NH₃⁺). The pH at this stage is 6.2, i.e. halfway between pKa₁ (pKa of the carboxyl group, 2.4) and pKa₂ (pKa of the amino group, 9.8).

The quantitative relationship between the concentration of a weak acid, e.g. HA, and its conjugate base, i.e. A⁻, is described by an equation, referred to as Henderson-Hasselbalch equation.

HENDERSON-HASSELBALCH EQUATION

The general dissociation of a weak acid such as a carboxylic acid is given by the equation 1:

HA ⇌ H⁺ + A– (1)

where HA is the protonated form or the conjugate acid (associated form) and A⁻ is the unprotonated form or the conjugate base (the dissociated form).

The dissociation constant (Ka) for such a weak acid is defined as the equilibrium constant for the dissociation reaction of the acid, by equation 2:

The hydrogen ion concentration [H⁺] of a solution of a weak acid then can be calculated by rearranging this equation, to equation 3:

A negative logarithm of the above can be expressed, as equation 4:

Since pH is the negative logarithm of [H⁺], multiplying the equation 4 by −1, arrives at the equation 5 which is referred to as the Henderson-Hasselbalch equation:

where pH is the negative logarithm of [H⁺], i.e. – log [H⁺], pKa is equal to the negative logarithm of dissociation constant (Ka), i.e. as – log Ka.

Henderson-Hasselbalch equation can be used for the analysis of acid base equilibrium systems. The extent of protonation of acidic and basic functional groups and therefore the net charge will vary with the pKa of the functional group and the pH of the solution. For example, for alanine, which has two functional groups with pKa₁ of 2.4 and pKa₂ of 9.8, the net charge varies with pH from −1 to +1. At a point intermediate between the two pKa values (6.2), it has a net zero charge. This pH is called its isoelectric point (pI).

The effectiveness of a buffer, in minimizing changes in pH, depends upon two factors:

1. The pK’a of the buffer in relation to the desired pH, e.g. pK’a of the imidazole group of histidine in hemoglobin is close to 7.4 and thus, it is an ideal buffer in the blood.

2. The concentration of the buffer: Greater the concentration of the buffer more is its ability to bind or release H⁺.

BODY BUFFERS

Metabolism of food consumed in our diet results in the production of CO₂ as well as acids, in the body. For example, most carbohydrates and fats are normally, completely oxidized to CO₂ and water. Metabolism of dietary protein results in the production of strong acids, etc. In the pulmonary capillary blood, CO₂ reacts with water and forms carbonic acid (H₂CO₃). A zinc-containing enzyme carbonic anhydrase, which also catalyzes the dissociation of carbonic acid to H⁺ and HCO₃⁻, catalyzes this reaction. Dissociation of H₂CO₃ or association of H⁺ and HCO₃⁻ to form H₂CO₃ occurs, instantaneously.

H₂O + CO₂ ⇌ H₂CO₃ H⁺ + HCO₃⁻

Despite metabolic production of acids in the body, blood and intestinal fluid pH is maintained with in the normal limits in a healthy person. Here, first line of defense of pH consists of chemical buffers that are present in the extracellular and the intracellular fluids, and the bone while the second line of defense is the lungs that maintain blood pH by disposing off CO₂.

Blood plasma is a mixed buffer system and has three major buffers, i.e. HCO₃⁻ / CO₂, HPO₄2–/H₂PO₄– and protein–/H.protein.

1. Phosphate buffer: Phosphate buffer has a pK of 6.8. It is a good buffer for stabilizing the pH of the blood. It is also an important buffer in the urine.

2. Protein buffer: A protein contains many different ionizable groups, each one of which can donate or accept H⁺. Protein comprises of the largest amount of buffer in the body. For example, albumin acts as a buffer in the blood plasma. An important example of the intracellular protein buffer is hemoglobin (Hb).

3. Bicarbonate/CO₂ buffer: Bicarbonate/CO₂ buffer has special importance in buffering pH in the extracellular fluid, due to the reason that:

(i) The components of this buffer system, i.e. HCO₃⁻ and CO₂, are present in large quantities. In the course of metabolism, a normal adult produces about 300 L of CO₂ every day. Further, there is also an appreciable concentration of HCO₃⁻ in the extracellular fluid.

(ii) This buffer pair is quite effective since it operates as an open system, i.e. either of its components can be removed or added. Though CO₂, truly is not an acid but is an acid anhydride, since it readily reacts with water and forms H₂CO₃, which ionizes to H⁺ and HCO₃⁻.

The sum of the two equations is:

CO₂ + H₂O ⇌ H⁺ + HCO₃⁻

As concentration of water, generally, does not alter much, equilibrium expression for the above reaction can be represented as:

K’ eq = ([H⁺] [HCO₃⁻]/[CO₂])

where [CO₂] represents concentration of dissolved CO₂. Since concentration of a gas, in a solution, is directly proportional to its partial pressure, therefore, concentration of the dissolved CO₂ in the blood is proportional to pCO₂.

Two components of the buffer can be easily regulated. Whereas lungs control arterial blood pH by regulating pCO₂, the kidney controls plasma HCO₃–.

MAINTENANCE OF ACID BASE BALANCE

Normally, lungs and kidney stabilize extracellular fluid pH.

Regulation by the Lungs

The respiratory system controls partial pressure of CO₂ and thus minimizes pH changes in the arterial blood.

• Normally, CO₂ is expired at the same rate at which it is produced and thus pCO₂ is maintained.

• If blood becomes acidic by the addition of the fixed acids, pulmonary ventilation is increased. In the state of hyperventilation, CO₂ is flushed-out at a rate greater than the rate at which it is produced and consequently pCO₂ falls. This reduction in arterial blood pCO₂, in turn, results in fall in H⁺ concentration of the blood. The reactions are pulled to the left, which in turn lowers HCO₃⁻ concentration and reduces acidic shift in the blood.

• On the other hand, if blood becomes more alkaline, changes occur in the opposite direction. Accordingly, hypoventilation takes place, blood pCO₂ rises, and these reactions are pushed to the right, which in turn results in a high carbonic acid concentration in the blood and a less alkaline shift in blood pH. Hypoventilation, however, is limited because it causes retention of CO₂, which further stimulates ventilation.

Buffering of acid-base disturbances by the respiratory response although is rapid but is relatively coarse. It brings blood pH close to normal but can not eliminate a fixed acid or base from the body. So, it can not restore the pH to normal.

Regulation by the Kidney

Renal response to an acid-base disturbance though is slow but more accurate. The kidney regulates acid-base balance by controlling bicarbonate reabsorption and secreting acid. Both of these processes depend upon the formation of H⁺ and HCO₃⁻ from CO₂ and H₂O with in the tubule cells.

Secretion of H⁺ ions: Normally, H⁺ formed in the reaction is actively secreted into the tubular fluid, in exchange for Na⁺. Within the tubule cell, Na⁺ as well as HCO₃⁻ is pumped out of the cell into the interstitial fluid. Sodium uptake by the tubular cell is partly passive, flowing down the electrochemical gradient and partly active via Na⁺–H⁺ antiport system (Fig. 6.1).

Fig. 6.1. Secretion of H⁺.

Mechanisms of the removal of the secreted H⁺ ions

H⁺ ions that are secreted into the tubular fluid can be removed by any of the three mechanisms, suggesting that three processes are involved in the acidification of urine.

1. By reabsorption of the filtered bicarbonate,

2. By excretion of titratable acids, and

3. Excretion of ammonium ions.

All the three mechanisms involve H⁺ secretion and are associated with the addition of bicarbonate to the peritubular capillary blood by the kidney tubules.

Reabsorption of the Filtered Bicarbonate

H⁺ ions that are secreted into the tubules (in exchange for Na⁺) combine with bicarbonate that has been filtered by the glomeruli. This, in turn, leads to the formation of carbonic acid in the tubular fluid. Dissociation of this acid to CO₂ and water is catalyzed in the lumen by carbonic anhydrase. The CO₂ rapidly diffuses through the cell membrane. The secretion of H⁺, thus, reabsorbs the filtered HCO₃⁻, indirectly (Fig. 6.2).

Fig. 6.2. By the reabsorption of the filtered bicarbonate.

Excretion of Titratable Acid

As reabsorption of sodium bicarbonate proceeds, the tubular fluid becomes depleted of HCO₃⁻ and the pH drops. Following this, H⁺ ions are taken up by the phosphate buffer. At this stage, hydrogen ions that are secreted into the tubular fluid (in exchange for Na⁺) react with the phosphate buffer:

HPO₄²⁻ + H⁺ → H₂PO₄⁻

As a result of it, basic form of phosphate (HCO₄2–) is converted to acidic form (H₂PO₄–). Besides phosphate, which is the most important buffer in the urine, other ions are also important in the excretion of H⁺. For example, in diabetic ketoacidosis when pH of the urine reaches at its minimum (about 4.4), acetoacetate and β-hydroxybutyrate pass through the glomerular filtrate and appear in the tubular fluid. Under these conditions these ions also serve as buffer.

Excretion of titratable acids thus, not only eliminates H⁺ from the body but also restores depleted plasma bicarbonate reserve since for each meq of H⁺ excreted as titratable acid, a new HCO₃⁻ ion is added to the blood (Fig. 6.3).

Fig. 6.3. By the excretion of the titrable acids.

The amount of the acid excreted (as the acid component of a urinary buffer) can be measured by the titration of the urine back to the normal pH of the plasma (to pH 7.4). The quantity of the base so required is equivalent to the quantity of the acid excreted and is referred to as titratable acidity of urine.

Excretion of Ammonia

The third fate of H⁺ in the tubular fluid is neutralization by NH₃. Ammonia is mainly synthesized in the proximal tubule from amino acids, predominantly glutamine. It readily gets diffused through the cell membrane and combines with the secreted H⁺ and forms NH₄⁺. NH₄⁺ is usually accompanied by an anion, e.g. chloride, in the urine. For each H⁺ excreted in the urine as NH₄⁺, an equivalent quantity of HCO₃⁻ is added to the blood (Fig. 6.4).

Fig. 6.4. Excretion as ammonium ion.

Elimination of NH₄⁺ in the urine contributes to net acid excretion, as NH₄⁺ is normally a major urinary acid. Nearly, one-half to two-thirds of our daily acid load is excreted as NH₄⁺.

Ability of the kidney to synthesize NH₃, from glutamine, is increased under conditions of excess acid in the body.

The sum of the titrable acidity and NH₄⁺ comprises total acidity of urine (total acid excretion).

ACID BASE IMBALANCE

Blood pH, compatible with life, varies from 6.8 to 7.8 while pH of the arterial blood normally varies from 7.35 to 7.45.

Acid Base Disorders: If arterial blood pH is acidic (pH below 7.35), it results in acidosis and leads to acidemia. Acidosis may be a result of the excess of acid or deficiency of alkali in the body.

If blood pH is more alkaline than 7.45, it results in alkalosis (alkalemia). Alkalosis is due to excess of alkali or deficiency of acid in the body. Accordingly, there are four types of acid base disturbances (Table 6.1):

• Respiratory acidosis,

• Respiratory alkalosis,

• Metabolic acidosis, and

• Metabolic alkalosis.

The CO₂ is volatile and normally, excreted by the lungs. Inability of the lungs to excrete CO₂ adequately leads to respiratory acidosis or alkalosis. On the other hand, if the problem is an excess or deficit of HCO₃–, due to a fixed acid or base, metabolic (non-respiratory) disturbances of acid base balance are present.

ACIDOSIS

Acidosis refers to a decrease in pH of the blood. It includes respiratory acidosis and metabolic acidosis.

Respiratory acidosis

Respiratory acidosis is due to inadequate ventilation (hypoventilation) by the alveoli. This in turn results in the retention of CO₂ and fall in blood pH.

Alveolar hypoventilation occurs when depth or rate of respiration is diminished such as in airways obstruction or a pulmonary disease. It results in a rise in pCO₂. As pCO₂ rises, plasma pH drops and HCO₃⁻ concentration rises.

Kidney compensates for the respiratory acidosis by increasing the output of H⁺ in the urine. The filtered HCO₃– in turn is reabsorbed and the increased amounts of H⁺ combine with the urinary buffers and are excreted in the urine.

Metabolic acidosis

Metabolic acidosis is characterized by the accumulation of nonvolatile acids (gain of fixed acids other than H₂CO₃) and loss of HCO₃–

The gain of metabolically produced acid may be due to several reasons such as:

(i) Renal failure, due to reduced excretion of metabolically produced acids.

(ii) Uncontrolled diabetes mellitus, due to the production of large quantities of ketone bodies.

(iii) Inadequate circulation, due to cardiogenic or hemorrhagic shock, which in turn promotes anaerobic glycolysis and increases production of lactic acid.

(iv) Heavy exercise, resulting in the production of lactic acid, or

(v) Ingestion of certain acidifying agents such as ammonium chloride.

Metabolic acidosis may also be due to the loss of HCO₃⁻ from the body such as due to diarrhea, or abnormal excretion of HCO₃ in the urine.

Two compensatory mechanisms are usually available to deal with:

1. Due to the acidic pH, respiratory system is stimulated. This in turn lowers pCO₂ and hence carbonic acid concentration in the blood. Respiratory compensation though is prompt but usually does not restore the blood pH to normal.

2. The other mechanism, which begins almost instantly, is the renal compensation. The kidney increases H⁺ excretion. This, however, takes time and alone is not adequate to return HCO₃⁻ concentration (and the pH) to normal.

Table 6.1. Changes in arterial blood values and compensatory responses in different types of acid base disturbances

ALKALOSIS

Alkalosis refers to an increase in pH of the blood. It includes respiratory alkalosis and metabolic alkalosis.

Respiratory alkalosis

Respiratory alkalosis is a result of hyperventilation which may be caused by anxiety, central nervous system injury involving the respiratory centre, salicylate poisoning or fever. This in turn causes a fall in arterial blood pCO₂. At high altitude also, due to decrease in total atmospheric pressure, alveolar pCO₂ falls and there is chronic respiratory alkalosis.

The kidney compensates for respiratory alkalosis, by excreting HCO₃– in the urine. Low pCO₂ leads to decreased H⁺ secretion by the tubular epithelium. This in turn results in reduced reabsorption of the filtered bicarbonate and increases its excretion in the urine. This loss of HCO₃– normal. in the urine results in further decrease in plasma HCO₃– and brings plasma pH towards

Metabolic alkalosis

Metabolic alkalosis is characterized by the intake of excess alkali, i.e. by the gain of strong base or HCO₃–, or abnormal loss of acid other than H CO . For example, ingestion of excess sodium bicarbonate used in treating gastric ulcer or oxidation of organic anion such as citrate, lactate and acetate.

A common cause of abnormal loss of acid causing metabolic alkalosis is prolonged vomiting or gastric lavage since loss of gastric contents results in net addition of HCO₃⁻ to the blood. It may also be a result of rapid loss of body water, e.g. in diuresis which in turn may result in temporary rise in HCO₃⁻ in the plasma and the extracellular fluid.

Characteristic arterial blood changes include a primary increase in plasma HCO₃⁻ concentration, a rise in pH and a compensatory rise in pCO₂. Metabolic alkalosis is seldom long lasting.

In metabolic alkalosis, as a primary defect is increase in plasma HCO₃– concentration, immediate physiological compensation is hypoventilation, followed by increased renal excretion of HCO₃⁻.

BIOCHEMICAL ASSESSMENT OF ACID BASE BALANCE

Disturbances of acid base balance are accompanied by the characteristic changes in blood pH, pCO₂ and HCO₃–. Besides the above parameters, base excess and anion gap are also calculated.

Base Excess

Base excess is defined as the amount of acid which is required to titrate the blood to the pH value of 7.4 at pCO₂ of 40 mmHg (at 37°C). If pH of the blood is low, base excess will be negative.

Anion Gap

Anion gap is a difference between the sum of Na⁺ and K⁺ (cations) from the sum of Cl⁻ and HCO₃– (anions).

Anion gap thus, represents plasma anions other than Cl⁻ and HCO₃⁻ which are not routinely measured.

Anion gap = (Na⁺ + K⁺) – (Cl⁻ + HCO₃⁻)

Normal value for the anion gap is in the range of 12–16 meq/L. This is a most commonly used parameter to establish differential diagnosis for metabolic acidosis.

Profile of the Blood Gases

Results of acid base measurements are considered in the light of the clinical findings as well as other types of investigations such as plasma creatinine, urea, Na⁺ and K⁺. Normal values for various parameters related to acid base status are shown in Table 6.2.

Table 6.2. Normal blood values for some parameters related to acid base status

Parameter | Normal value
pH | 7.35 to 7.45
pCO₂ | 31–45 mmHg
HCO₃⁻ | 22–26 meq/L
Base excess | −2.0 to + 3.0 mmol/L
Anion gap | 10 to 20 mmol/L
Serum creatinine | 0.8 to 1.3 mg/dl
Blood urea | 15 to 40 mg/dl
Serum electrolytes |
Na⁺ | 136 to 145 meq/L
K⁺ | 3.5 to 5.0 meq/L
Cl⁻ | 98 to 107 meq/L

SOME IMPORTANT QUESTIONS

1. Define buffer. What are the important buffers present in the body? Explain the role of various body buffers in the regulation of blood pH.

2. Define pH. How pH of blood is regulated?

3. Explain:

(i) Role of kidney in the regulation of blood pH.

(ii) Various mechanisms of H⁺ secretion.

(iii) Disturbances of the acid base balance.

(iv) Biochemical assessment of acid base balance.

(v) Role of lungs in the regulation of blood pH.

4. Write notes on:

(i) pH

(ii) Henderson-Hasselbalch equation

(iii) Chemical buffers found in the body

(iv) Titratable acidity

(v) Respiratory acidosis

(vi) Respiratory alkalosis

(vii) Metabolic acidosis

(viii) Metabolic alkalosis

(ix) Base excess

(x) Anion gap

CHAPTER
7

Enzymes and Conenzymes

Enzymes are organic molecules that are produced in the living organisms and increase the rate of a biochemical reaction without being utilized in the overall process. They are non-dialysable, colloidal particles which are thermolabile proteins with highly specific catalytic activity. An enzyme increases rate of a chemical reaction by lowering its free energy barrier that separates a substrate (reactant) from the product.

Most of the enzymes are produced with in the cell of a particular tissue and function there only. Such enzymes are called intracellular enzymes, e.g. enzymes of the glycolysis, citric acid cycle, fatty acid synthesis, etc. On the other hand, there are certain enzymes, which are produced by the cells of a particular tissue from where they are liberated but function in some other tissues. Such enzymes are called extracellular enzymes, e.g. proteolytic enzymes trypsin, chymotrypsin, etc. They are secreted in the pancreatic juice for their action in the small intestine.

PROENZYMES

Most of the intracellular enzymes are secreted in their active form called zymase. Some of the enzymes, however, are secreted in their inactive form called proenzyme or zymogen. After it comes in contact with certain activating agent, the proenzyme undergoes some modifications and is converted to active enzyme (zymase).

The activating agent can be H⁺, some other enzyme or the active form of the zymogen itself. For example, gastric juice contains pepsin which is secreted as pepsinogen (a zymogen). Pepsinogen is changed to its active form pepsin by the H⁺ of the gastric juice. Once pepsin has been formed, it acts on pepsinogen and catalyses its conversion to pepsin. This process is referred to as autocatalysis (Fig. 7.1).

Fig. 7.1. Conversion of a proenzyme to active enzyme.

Table 7.1 lists some of the proenzymes, their activators and the active enzymes.

Table 7.1. Some of the proenzymes, their activators and active enzymes

Proenzyme | Activator | Active enzyme
Pepsinogen | H⁺, pepsin | Pepsin
Trypsinogen | Trypsin, enterokinase | Trypsin
Chymotrypsinogen | Trypsin | Chymotrypsin
Procarboxypeptidase | Trypsin | Carboxypeptidase
Proelastase | Trypsin | Elastase

CHEMICAL NATURE OF ENZYMES

All enzymes are protein in nature and have large molecular weights. Some RNA molecules called ribozymes, also have catalytic activity.

Ribozymes: A ribozyme is an RNA molecule, which exhibits highly substrate-specific catalytic activity. They catalyze hydrolysis of phosphodiester bonds with in the RNA molecules and play a key role in intron splicing, in the conversion of pre-mRNA to mature mRNA, e.g. RNase P from E. coli.

Although, most of the enzymes are simple proteins and have a single polypeptide chain, there are various enzyme which have more than one catalytic activity and are called multimeric enzymes. They may be grouped as oligomeric enzymes or multi-enzyme complexes.

Oligomeric Enzymes

These are the multimeric proteins, i.e. have more than one protein subunits, which have a catalytic activity only when associated with each other. These multi-subunit enzymes are called oligomeric enzymes. For example, phosphorylase, hexokinase, and lactate dehydrogenase, each one is a tetramer, i.e. each one of these enzymes has four subunits. Phosphofructokinase, fructose-1,6-bisphosphatase, and creatine phosphokinase are dimers, i.e. each one of these enzymes has two protein subunits.

Multi-enzyme Complexes

These are the single polypeptide chains with several catalytic sites. Due to multi-enzyme activities associated with a single protein, these enzymes are called multi-enzyme complexes. They act on a substrate and catalyze series of reactions, e.g. pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, etc. Each of these enzymes is a complex having three different enzyme activities. Fatty acid synthetase is a multi-enzyme complex with seven enzyme activities.

PROPERTIES OF ENZYMES

ENZYME SPECIFICITY

Enzymes are highly specific, interacting with only one or a few substrates and catalyzing one type of a chemical reaction only. They exhibit several types of catalytic specificities such as stereo-specificity, reaction specificity, etc.

Stereo-specificity

As we know that there can be many optical isomers of a compound but it is only one of the isomers which can act as a substrate for a particular enzyme. This is called stereo-specificity. For example, L-amino acid oxidase will oxidize only L-amino acids. Similarly, succinate dehydrogenase will act only on succinate and not on malate (Fig. 7.2).

Fig. 7.2. Stereo-specificity.

Reaction specificity

Though a substrate can be used in several reactions but a particular enzyme will catalyze only one of these reactions. This is referred to as reaction specificity. For example, oxaloacetate can be converted to aspartate as well as to malate but the two reactions are catalyzed by two separate enzymes and not by the same enzyme (Fig. 7.3).

Fig. 7.3. Reaction specificity.

The extent of substrate specificity may vary from enzyme to enzyme. An enzyme may have either an absolute specificity or a relative specificity for its substrate:

Absolute Specificity: An enzyme may have an absolute specificity for its substrate and not bind with any other substrate, e.g. urease which is specific for urea only.

Relative Specificity: An enzyme may be specific either to a particular group or type of bond:

Group specificity: It refers to the action of enzymes which hydrolyze some specific groups only. For example, pepsin hydrolyses a protein chain only at aromatic amino acid residues while trypsin at basic amino acids.

Bond specificity: It refers to the action of those enzymes which specifically hydrolyze a particular type of bond only. For example, proteases hydrolyze peptide bonds in proteins; glycosidases hydrolyze glycosidic bonds in polysaccharides while esterases hydrolyze only the ester bonds in lipids.

COENZYMES AND COFACTORS

Several enzymes require the presence of some non-protein organic substances or metal ions for their activity. These enzymes are referred to as holo-enzymes (conjugated proteins). The protein part of such an enzyme is called apoenzyme (apo-protein) while the non-protein component is called prosthetic group.

Apo-enzyme (protein) + prosthetic group (non-protein component) = Holo-enzyme

If the prosthetic group is removed, enzyme loses its biological activity and becomes inactive.

If the prosthetic group is an organic compound, it is called coenzyme while inorganic ions are called cofactors.

COENZYMES

A coenzyme is a dialyzable, thermostable, low-molecular weight, organic substance, which may be referred to as co-substrate or second substrate.

Generally, coenzymes are the derivatives of the B complex group of vitamins, e.g. TPP (coenzyme form of vitamin B₁), FMN and FAD (derivatives of B₂), pyridoxal-5-phosphate (coenzyme form of vitamin B₆), etc. (Table 7.2).

Table 7.2. B Complex vitamins, their coenzymes and some of the reactions catalyzed by them

Vitamin | Coenzyme form | Reaction catalyzed
Thiamin | TPP | Pyruvate → Acetyl CoA
|  | α-Ketoglutarate → Succinyl CoA
Riboflavin | FMN, FAD | α-Amino acids → α-Keto acids
|  | Succinate → Fumarate
Niacin | NAD⁺, NADP⁺ | Lactate → Pyruvate
|  | Glucose-6-P → 6-P-Gluconolactone
Pyridoxine | Pyridoxal-5-P | Alanine + α-Ketoglutarate → Pyruvate + Glutamate
|  | Tyrosine → Tyramine
Pantothenic acid | CoA | Pyruvate → Acetyl CoA
|  | Fatty acid →Fatty acyl CoA
Biotin | Active biotin | Acetyl CoA → Malonyl CoA
|  | Pyruvate → Oxaloacetate
Folate | Tetrahydrofolic acid | Glycine → Serine
|  | Homocysteine → Methionine
B₁₂ | Deoxyadenosylcobalamin/Methylcobalamin | Methylmalonyl CoA → Succinyl CoA
|  | Homocysteine → Methionine
Lipoic acid | Lipoic acid | Pyruvate → Acetyl CoA
|  | α-Ketoglutarate → Succinyl CoA

COFACTORS

Several enzymes require certain metal ions called cofactors for their activity, e.g. Mg²⁺, Zn²⁺ or Cl⁻, etc.

A metal ion may be either tightly bound to the enzyme (metalloenzymes) or loosely associated with it (metal-associated enzymes).

Metalloenzymes: When a metal ion forms an integral part of the enzyme, such enzymes are referred to as metalloenzymes. For example, zinc is an essential component of superoxide dismutase. Some of the metalloenzymes and their cofactors are shown in Table 7.3.

Table 7.3. Some metalloenzymes

Metalloenzyme | Cofactor
Cytochromes, peroxidases, catalase | Fe
Ascorbic acid oxidase, cytochrome oxidase, tyrosinase | Cu
Aldehyde oxidase, xanthine oxidase, nitrate reductase | Mo
Carbonic anhydrase, lactate dehydrogenase, carboxypeptidase | Zn
Glutathione peroxidase | Se

Metal-associated enzymes: In certain enzymes a metal ion may be loosely associated with it, forming a metal-enzyme complex. Such enzymes are called metal-associated enzymes. They require the presence of the particular ions for their activity, e.g. Cl⁻ (as NaCl) acts as an activator for salivary amylase. Table 7.4 lists metal-associated enzymes and the associated metal.

Table 7.4. Some metal-associated enzymes

Metal Ion | Enzyme
Ca | Lipase, ATPase, succinate dehydrogenase
Co | Lecithinase, peptidases
Mg | Phosphorylase, enolase, DNA polymerase
Mn | Arginase, phosphoglucomutase, cholinesterase
Cl | Salivary amylase
K | Pyruvate kinase

NOMENCLATURE OF ENZYMES

Enzymes are usually named by the suffix “-ase” with the name of the substrate or the catalytic action of the enzyme, e.g. lipase catalyzes the hydrolysis of lipids, fatty acid synthase catalyzes the synthesis of fatty acids, etc. These names are called as the trivial names. While assigning a trivial name to an enzyme, no systematic rules are followed.

E.C. Number

International Union of Biochemistry and Molecular Biology (IUBMB) adopted a scheme for the rational naming of each enzyme and suggested a functional classification. According to IUBMB, each enzyme is assigned a four-digit systematic code, commonly known as Enzyme Commission number (E.C. number).

The first digit of the code denotes main class of the enzyme, which is according to general type of the chemical reaction an enzyme catalyzes.

Its second digit characterizes the sub-class, which may be based on the nature of the chemical group removed or transferred, or any bond spliced or formed.

Its third digit indicates the sub-sub-class and describes detailed subdivision of each sub-class.

The last digit of the E.C. number indicates serial number of the particular enzyme in the sub-sub-class. For example, 1.1.1.1 is the enzyme code for alcohol dehydrogenase, which is the first enzyme of the sub-sub-class 1, of the sub-class 1, of the class 1.

CLASSIFICATION OF ENZYMES

According to the enzyme commission, enzymes are classified into six classes, as follow:

1. Oxidoreductase

Oxidoreductases are involved in oxidation and/ or reduction of the substrate, i.e. they catalyze addition of oxygen, or removal of hydrogen or electrons. Their sub-classes include dehydrogenase, oxidase, oxygenase, etc.

Examples of the class oxidoreductase are alcohol dehydrogenase, xanthine oxidase, glutathione reductase, etc.

2. Transferase

Transferases catalyze transfer of a particular group from one substrate to another. On the basis of the group transferred, transferases are sub-classified as aminotransferases, phospho-transferases, etc.

Glutamate oxaloacetate transaminase, hexokinase, glucose-1-phosphate uridyltrans-ferase, etc., are some of the examples of the class transferase.

3. Hydrolase

Hydrolases bring about hydrolysis of a substrate with the addition of a molecule of water. According to the nature of the group or bond hydrolyzed, hydrolases are sub-classified as peptidases, glycosidases, etc.

Examples of the class hydrolase include glucose-6-phosphatase, amylase, pepsin, etc.

4. Lyase

Lyases catalyze splitting of a substrate into two products without the addition of water. They are sub-classified on the basis of the linkage splitted. Its various sub-classes include lyases catalyzing the breakdown of the C–C, C–N or C–S bond, etc.

Examples of the class lyase include fumarase, pyruvate decarboxylase, histidase, etc.

5. Isomerase

Isomerases are involved in the conversion of one isomeric form of the substrate into another, i.e. in isomerisation of the substrate. Racemases, epimerases and mutases, etc., are the sub-classes of the class isomerase.

Examples of the class isomerase include phosphohexose isomerase, phosphoglucomutase, alanine racemase, etc.

6. Ligase

Ligases are involved in the synthesis of a product by linking two substrates together. This joining of the two substrates requires energy which is coupled with the hydrolysis of some high-energy bond. Its various sub-classes include ligases that can form a C–C, C–N or C–S bond.

Examples of the class ligase include glutamine synthetase, acetyl CoA carboxylase, glutathione synthetase, etc. (Table 7.5).

Table 7.5. Classification of enzymes with examples

Class of enzyme | Examples
Oxidoreductase | Alcohol dehydrogenase, xanthine oxidase, catalase
Transferase | Glutamate pyruvate transaminase, serine hydroxymethyl transferase, hexokinase
Hydrolase | Glucose-6-phosphatase, carboxypeptidase, pepsin
Lyase | Pyruvate decarboxylase, aldolase, histidase
Isomerase | Phosphotriose isomerase, phosphoglucomutase, alanine racemase
Ligase | Glutamine synthetase, acetyl CoA carboxylase, glutathione synthetase

MECHANISM OF ENZYME ACTION

Basic Enzyme Reaction

Enzymes are catalysts and increase the speed of a chemical reaction without undergoing any permanent chemical change. They are neither used in the reaction nor appear as reaction products.

The basic enzymatic reaction can be represented as follows:

S + E → P + E

where E represents the enzyme catalyzing the reaction, S the substrate (the substance being changed) and P the product of the reaction.

Energy Levels

As we know that for most chemical reactions to proceed, some form of energy is needed. This quantity of energy is referred to as the energy of activation. It is the magnitude of the activation energy which determines how fast the reaction will proceed. It is believed that enzymes lower the activation energy for the reaction they catalyze.

The enzyme reduces the path of the reaction. This shortened path requires less energy for each molecule of substrate converted to product. Given a total amount of available energy, more molecules of substrate would be converted when the enzyme is present than when it is absent. Hence, the reaction is said to go faster in a given period of time.

The Enzyme Substrate Complex

The substrate and the enzyme form some intermediate substance which is known as the enzyme substrate complex (ES). The reaction can be represented as:

S + E → ES → P + E

This indicates that the enzyme first combines in some way with the substrate and then returns to its original form after the reaction is completed.

Chemical Equilibrium

Most of the enzyme catalyzed reactions do not go to completion. This is due to reversibility of most of the reactions. In general:

where, k⁺¹ is the forward reaction rate constant and k–1 is the rate constant for the reverse reaction.

Combining the two reactions and applying this relationship to enzymatic reactions, allows the equation:

Various models have been proposed to explain this binding of the substrate with the enzyme. These include lock and key model and induced-fit model.

LOCK AND KEY MODEL

Complementarity between the enzyme and the substrate is the basis of the lock and key model of enzyme-substrate binding. In this model, during the process of formation of the enzyme-substrate complex, substrate gets attached to the active site on the enzyme through some specific groups as a key fits into its lock. This mechanism of enzyme activation was proposed by Emil Fischer and is called lock and key model.

Active site of the enzyme may be a free hydroxyl group of serine or phenol group of tyrosine, SH-group of cysteine or an imidazole group of histidine. Formation of the specific ionic pair contributes towards the recognition of the substrate by the enzyme.

INDUCED-FIT MODEL

Induced-fit model explains that though the substrate-binding sites on the enzyme are preformed but these sites undergo conformational changes after binding of the enzyme with the substrate.

During its binding with the enzyme, the substrate induces some changes at the active site of the enzyme, by changing the configuration of the protein, so as to attain a shape where the substrate can fit conveniently. Enzyme-substrate complex is formed by multiple bondings of the substrate with the enzyme such as by covalent bonds, hydrogen bonds and electrostatic bonds. This mechanism of enzyme activation was proposed by Koshland and is called induced-fit model (Fig. 7.4).

Enzyme, which requires a coenzyme, does possess a site for the attachment of the coenzyme also.

Fig. 7.4. Induced-fit model of the formation of the enzyme-substrate complex.

ENZYME KINETICS

An enzyme exhibits maximum activity only under optimal conditions. Enzyme activity is markedly affected by several factors such as temperature, pH, concentration of the enzyme or the substrate, or the presence of activator/ inhibitor.

Effect of Temperature

Rate of an enzyme catalyzed reaction increases with the increase in temperature of the reaction mixture. It is due to the reason that an increase in temperature increases the energy of activation of the chemical system, which in turn increases reaction velocity. It is, however, possible up to certain temperature only, called optimum temperature. Optimum temperature of an enzyme is defined as the temperature at which its activity is maximum.

For most of the enzymes optimum temperature is near the body temperature, i.e. 37°C. With the rise in temperature of the reaction mixture beyond the optimum temperature of the enzyme, enzyme, being protein, gets denatured and looses its biological activity.

Relationship of the enzyme activity to the change in temperature is represented by a bell-shaped curve, peak of which represents optimum temperature (Fig. 7.5).

Fig. 7.5. Effect of temperature on enzyme activity.

Effects of pH

The pH of the reaction medium also affects reaction velocity. The pH at which enzyme activity is maximum, is defined as its optimum pH. Relationship of the enzyme activity to the pH is represented by a bell-shaped curve, which has its peak at optimum pH (Fig. 7.6).

Fig. 7.6. Effect of pH on enzyme activity.

Optimum pH of an enzyme depends upon the ionization state of the enzyme as well as the substrate. Change in pH affects ionization state of the enzyme thereby decreasing the number of its active sites. Hence, at extremely low or high pH, enzyme gets denatured. Optimum pH for most of the enzymes is in the neutral range, i.e. around 7.0. Optimum pH for pepsin is about 2.0 while optimum pH for the enzymes of the pancreatic juice is near 8.0.

Effect of Substrate Concentration

With a fixed quantity of the enzyme, reaction rate is proportional to the concentration of the substrate. This is, however, true only up to a certain point, the point at which its velocity is maximum (Vmax) and a further increase in the substrate concentration will not increase the rate of the reaction (Fig. 7.7).

Fig. 7.7. Effect of substrate concentration on enzyme activity.

Michaelis and Menten derived a relationship between substrate concentration and reaction velocity, and described an equation called Michaelis-Menten equation, which is as follows:

where V₀ is the initial reaction velocity, Vmax maximum velocity of the reaction, [S] concentration of the substrate, and Km a constant, which is referred to as Michaelis constant.

Km is defined as the substrate concentration at which the reaction velocity is half of the maximum velocity, i.e. Km = Vmax/2.

Effect of Enzyme Concentration

Reaction velocity is also affected by the concentration of the enzyme. Reaction velocity is proportional to the concentration of the enzyme present in the reaction mixture. With a two-fold increase in the concentration of the enzyme, the enzyme will combine with double the amount of the substrate, and the amount of the product so formed will also be two-folds. This relationship, however, holds true up to certain limit, i.e. only for the lower concentration of the enzyme but with the adequate concentration of the substrate (Fig. 7.8).

Fig. 7.8. Effect of enzyme concentration on enzyme activity,

ENZYME INHIBITION

Phenomenon of decrease in the rate of the enzymatic reaction brought about by the addition of some substances is called enzyme inhibition. An inhibitor may affect enzyme activity either competitively or noncompetitively.

COMPETITIVE INHIBITION

A competitive inhibitor competes with the substrate for binding at the active sites of the enzyme. Chemical structure of such an inhibitor closely resembles the substrate, e.g. malonate, which inhibits the action of succinate dehydrogenase, has structure similar to succinate.

The inhibitor forms the complex with the enzyme, which is called as the enzyme-inhibitor complex (EI) and inhibits the action of the enzyme.

Binding of enzyme with the competitive inhibitor and the substrate depends upon the relative concentrations of the two and is usually a reversible process. By increasing the concentration of the substrate, degree of inhibition can be reduced. Presence of the competitive inhibitor increases the Km without any change in Vmax of the enzymatic reaction (Fig. 7.9).

Several drugs are used as competitive inhibitors.

Fig. 7.9. Effect of a competitive inhibitor on enzyme activity.

DRUGS AS COMPETITIVE INHIBITORS

Due to their structural similarity with the substrate, several drugs are used as inhibitors of the enzyme in a particular metabolic pathway. For example, sulfanilamide (a sulfa drug) is an antibacterial agent and resembles p-aminobenzoic acid, structurally. It is used as a competitive inhibitor of the enzyme dihydropteroic acid synthase in bacteria. Similarly, methotrexate, a structural analog of folate, competitively inhibits dihydrofolate reductase and is used in the treatment of childhood leukemias (Fig. 7.10).

Table 7.6 lists some of the competitive inhibitors and the enzymes inhibited by them.

Fig. 7.10: Some drugs (structural analogs) used as competitive inhibitors.

Table 7.6. Some enzymes, their substrates and competitive inhibitors

Enzyme | Substrate | Competitive inhibitor
Succinate dehydrogenase | Succinate | Malonate
Carbonic anhydrase | Carbonate | Acetazolamide
Dihydrofolate reductase | Dihydrofolate | Amethopterin (methotrexate)
L-Histidase | L-Histidine | D-Histidine
Xanthine oxidase | Hypoxanthine | Allopurinol
HMG CoA reductase | HMG CoA | HMG

NONCOMPETITIVE INHIBITION

Several substances react covalently with the functional groups that are present on the active site of the enzyme and thus inactivate the enzyme, non-competitively. For example, enzymes which have free SH-groups in their molecules are inhibited by substances like iodoacetate or p-chloromercuro-benzoate that bind SH-groups and inactivate the enzyme. Heavy metals like Hg2+ and Ag+ also inactivate some of the enzymes. This type of inhibition is usually irreversible and makes active sites of the enzyme ineffective.

A noncompetitive inhibitor may also bind at a site other than the substrate-binding site. In such cases either the enzyme or the enzyme-substrate complex binds the inhibitor. As a result, both binary (EI) as well as ternary (ESI) complexes are formed. ESI may break down to form a product but at a slower rate, a reversible noncompetitive inhibitor thus decreases Vmax but does not change the Km (Fig. 7.11).

Some antimetabolites act as noncompetitive inhibitors.

Fig. 7.11. Effect of a noncompetitive inhibitor on enzyme activity.

REGULATION OF ENZYME ACTIVITY

Regulation of the enzyme activity is essential for the coordination of numerous metabolic processes. Besides changes in the substrate concentration, enzyme activity is also regulated by modifying the activity of the existing enzyme molecules by a number of processes which include allosteric regulation, covalent modifications and induction/repression.

ANTIMETABOLITES

Certain derivatives of purines and pyrimidines, called antimetabolites, act as noncompetitive inhibitors and are used as chemo-therapeutic agents, e.g. 5-fluorouracil. It is an analog of thymine but inhibits thymidylate synthetase, noncompetitively (Fig. 7.12).

Fig. 7.12: Thymine and its analog 5-fluorouracil (an antimetabolite, which is used as a noncompetitive inhibitor).

ALLOSTERIC REGULATION

Oligomeric enzymes, having two or more subunits, have two different sites, which are called the catalytic site or regulatory site (also known as the allosteric site) and the substrate-binding site (active site). These two sites are located apart from each other on two different sub-units of the oligomeric enzymes. Such enzymes are called allosteric enzymes or regulatory enzymes. Allosteric enzymes frequently catalyze the committed step that is generally present in the beginning of the pathway. For example, HMG CoA reductase (a regulatory enzyme of cholesterol biosynthesis), aspartate transcarbamoylase (of pyrimidine synthesis), etc.

Certain substances, called allosteric modulators or effectors, bind reversibly to such an enzyme at the allosteric site and regulate its activity. An interaction with the effector molecule brings about conformational changes at the catalytic site of the enzyme. An effector molecule may either activate an enzymatic reaction (called positive effector or allosteric activator) or inhibit it (called allosteric inhibitor), and is referred to as allosteric regulator.

Due to interaction between the substrate and the activator/inhibitor, a characteristic sigmoid curve (S-shaped curve) is obtained. Presence of an allosteric activator (+) shifts the curve to the right whereas an allosteric inhibitor (−) shifts the curve to the left (Fig. 7.13).

Fig. 7.13. Effect of an allosteric activator/inhibitor on enzyme activity.

Table 7.7 lists some of the enzymes that are allosterically regulated along with their allosteric activators/inhibitors.

Table 7.7. Some allosteric enzymes with their allosteric activators/inhibitors

Enzyme | Allosteric activator | Allosteric inhibitor
Hexokinase | ADP | Glucose-6-P, ATP
Isocitrate dehydrogenase | ADP | Glucose-6-P, ATP
Glutamate dehydrogenase | ADP | ATP, NADH
Pyruvate carboxylase | Acetyl CoA | ADP

COVALENT MODIFICATION

Several enzymes are regulated by covalent modification, most frequently by the addition or removal of a phosphate group from serine, threonine or tyrosine residues of the enzyme. Protein kinases catalyze phosphorylation of enzymes while phosphoprotein phosphatases remove phosphate groups from the phosphorylated enzymes. Depending upon the specific enzyme, either the phosphorylated (such as for glycogen phosphorylase) or the dephosphorylated (e.g. glycogen synthase) enzyme is active.

INDUCTION/REPRESSION

Cells can also regulate the amount of an enzyme, usually by altering its rate of synthesis. Increased (induction) or decreased (repression) synthesis of the enzyme in turn regulates its activity. Enzymes that are required only at one stage of development or under selected physiological conditions are regulated in this way. For example, elevated levels of insulin, as a result of high blood glucose level, cause increased synthesis of the key enzymes that are involved in glucose utilization.

ISOENZYMES

Isoenzymes (isozymes) are the physically distinct forms of an enzyme, which have same specificity but differ from each other with respect to their structure, electrophoretic mobility, immunological properties or source, e.g. lactate dehydrogenase, creatine phosphokinase, alkaline phosphatase, etc.

LACTATE DEHYDROGENASE

Lactate dehydrogenase (LDH) is a tetramer, i.e. it has four polypeptide subunits. Its each subunit may be one of the two types, known as the H (heart) type and the M (muscle) type. LDH thus exists in serum in five distinct forms (isoenzymes) with different proportions of the H and the M subunits. LDH₁ has four polypeptide chains of the H type (H₄) while LDH₅ has four subunits of the M type (M₄). Other forms include LDH₂ (H₃M), LDH₃ (H₂M₂) and LDH₄ (HM₃).

LDH₁ and LDH₂ are predominant in the myocardium and erythrocytes while LDH₄ and LDH₅ are predominant in liver and skeletal muscle. LDH1 increases between l2 to 24 hours following myocardial infarction. LDH₅ is diagnostic of secondary congestive liver involvement.

CREATINE PHOSPHOKINASE

Creatine phosphokinase (CPK), also referred to as creatine kinase (CK), has three isoenzymes. Each of its isoenzyme is a dimer, i.e. composed of two subunits which may either be of M (muscle) type or B (brain) type. Hence it has three isoenzymes which are called CPK₁ (BB) that is mainly found in the brain; CPK₂ (MB) found in the myocardium; and CPK₃ (MM) found in skeletal muscle.

CPK₂ is present in very small amount in serum. Its level rises with in 4 hours, reaching a maximum value with in 1 day, of myocardial infarction.

ALKALINE PHOSPHATASE

Alkaline phosphatase exists in three forms. Different tissues contain a distinct form of the enzyme. Major portion of the enzyme in serum is from liver whose level rises in post-hepatic jaundice.

In growing children the serum enzyme is mainly from bone and is related to increased osteoblastic activity.

During the last trimester of pregnancy there is an increase in serum alkaline phosphatase which is primarily of the placental origin. This form of the enzyme is heat-stable; hence placental enzyme is also called heat-stable alkaline phosphatase.

DIAGNOSTIC SIGNIFICANCE OF ENZYMES

Measurement of several enzymes helps in the diagnosis of various diseases:

TRANSAMINASES

Serum levels of aspartate aminotransferase (AST, also called glutamate oxaloacetate transaminase or GOT) and alanine aminotransferase (ALT, also called glutamate pyruvate transaminase or GPT) have clinical significance. In routine, these enzymes are also called SGOT and SGPT, respectively.

In viral hepatitis and other forms of the liver disease serum GOT and GPT levels are raised even before the clinical signs and symptoms of the disease appear. Levels of both the enzymes may reach values up to 100 folds the upper normal limits. Their peak levels are seen between seven and twelve days of a liver disease. Thereafter, their activities gradually decrease and reach towards normal between three to five weeks. Although, serum levels of both the enzymes (GPT and GOT) are elevated, GPT is more liver specific.

Five to ten-folds rise also occurs in both the enzymes in patients with primary or metastatic carcinoma of the liver.

After myocardial infarction (MI) increased GOT activity appears in serum as its concentration is highest in the cardiac muscle. Since GPT concentration is less than GOT in cardiac muscle, there is comparatively lesser rise in GPT. Peak level of GOT is reached after 18 to 24 hours and returns towards normal by fourth or fifth day.

GOT and occasionally GPT levels are elevated in progressive muscular dystrophy.

CREATINE PHOSPHOKINASE

CPK or CK (creatine kinase) activity is elevated in all types of muscular dystrophies. Usually, it is only CK₃ that is present in serum in dystrophies and myopathies. However, in a neurogenic muscle disease such as myasthenia gravis, its activity remains with in normal limits.

Measurement of CK₂ in serum is used in the diagnosis of acute MI where initial rise occurs with in 4 to 6 hours. Peak levels are observed after approximately 24 hours and returns towards normal with in 48 to 72 hours.

Serum CK activity also increases in patients with acute cerebrovascular diseases. This is entirely due to CK₃.

LACTATE DEHYDROGENASE

Rise in serum LDH starts 12–18 hours after the onset of acute MI with a peak after 48 to 72 hours. Levels return towards normal after 6 to 10 days. Rise in total LDH in patients with MI parallels a rise in LDH₁.

Marked elevation in total LDH activity, up to 50 times the upper normal value, may be observed in megaloblastic anemia. Elevation in LDH activity is also observed in liver diseases, renal diseases, and malignancy.

ALKALINE PHOSPHATASE

Estimation of serum alkaline phosphatase (ALP) is of significance in the diagnosis of hepatobiliary diseases and bone diseases that are associated with increased osteoblastic activity. Amongst various bone diseases, highest levels are seen in Paget’s disease (nearly 10–25 times of the upper normal limit). A moderate rise is also observed in osteomalacia.

In intrahepatic obstruction of the bile flow, increase in serum ALP is nearly 2–3 times of the upper normal limit only.

DIAGNOSTIC SIGNIFICANCE OF ENZYMES

Measurement of certain enzymes in serum is helpful in the diagnosis of various diseases. The level of a particular enzyme increases in serum when it is released in large amounts due to the damage to the tissue. Some of these enzymes also have a prognostic significance.

Some of the enzymes, which have a diagnostic significance, include transaminases, alkaline phosphatase, etc.

Some of the enzymes, which have diagnostic significance along with their principal tissue of origin and clinical application, are listed in Table 7.8.

Table 7.8. Some enzymes of diagnostic significance with their principal source and clinical uses

Enzyme | Principal source | Clinical situation in which altered
Acid phosphatase | Prostate and RBC | Increases in carcinoma of prostate
Alkaline phosphatase | Liver, bone, placenta and kidney | Increases in obstructive jaundice, rickets, Paget’s disease, and hyperparathyroidism
Aldolase | Skeletal muscle and heart | Increases in muscular dystrophies and acute liver diseases
Amylase | Salivary glands, pancreas and ovaries | Increases in acute pancreatitis, intestinal obstruction and acute parotitis. Decreases in acute liver diseases
Ceruloplasmin | Liver | Increases in cirrhosis. Decreases in Wilson’s disease
Choline esterase | Liver | Increases in nephrotic syndrome and organo-phosphorus poisoning. Decreases in acute liver diseases
Creatine phosphokinase | Skeletal muscle, brain and heart | Increases in myocardial infarction and muscular dystrophies
Glutamate oxaloacetate transaminase (GPT) | Liver, skeletal muscle, heart, kidney and RBCs | Increases in myocardial infarction, toxic liver cell necrosis, acute liver diseases, and muscle diseases
γ-Glutamyl transferase | Liver and kidney | Increases in alcoholism and hepatobiliary diseases
Lactate dehydrogenase | Heart, liver, skeletal muscle, RBCs and platelets | Increases in acute myocardial infarction, acute hepatitis, and muscle disorders
Lipase | Pancreas | Increases in acute pancreatitis and pancreatic carcinoma. Decreases in liver diseases and diabetes mellitus
5’-Nucleotidase | Hepatobiliary tract | Increases in liver diseases, obstructive jaundice, and hepatic carcinoma
Trypsin | Pancreas | Increases in acute pancreatic diseases

γ-GLUTAMYL TRANSPEPTIDASE

Serum γ-glutamyl transpeptidase (γ-glutamyl transferase or γ-GT) primarily originates from the hepatobiliary system. Its activity is increased in all liver diseases.

Elevated level of γ-GT is of clinical significance in detecting an alcohol-induced liver disease. Its levels are increased not only in patients with alcoholic cirrhosis but also in majority of people who are heavy drinkers.

AMYLASE

Estimation of amylase in serum and urine is of significance in diagnosis of the diseases of the pancreas.

In acute pancreatitis, a transient rise (4–6 folds increase) in serum amylase occurs with in 2–12 hours of onset of the disease and return towards normal by third or fourth day.

SOME IMPORTANT QUESTIONS

1. What are enzymes? Classify them. Discuss factors affecting enzyme activity.

2. Define enzyme, coenzyme and cofactor. Discuss role of B complex vitamins as coenzymes.

3. Define an enzyme. What are isoenzymes? Discuss importance of enzymes in clinical diagnosis.

4. What are allosteric enzymes? How their activity is modulated? Give examples.

5. Give brief account of factors affecting enzyme activity.

6. Discuss briefly :

(i) Various types of enzyme inhibition, giving examples.

(ii) Apoenzyme, holoenzyme and coenzyme with examples.

(iii) Classify enzymes giving examples.

7. Write notes on:

(i) Diagnostic significance of enzymes.

(ii) Noncompetitive inhibition of enzyme activity

(iii) Isoenzymes and their importance

(iv) Proenzymes

(v) Competitive inhibitors

(vi) Enzyme specificity

(vii) Coenzymes

(viii) Classification of enzymes

(ix) Ribozymes

(x) Allosteric regulation

(xi) Covalent modification of enzyme activity

(xii) Induction and repression of enzyme activity

CHAPTER
8

Chemistry and
Metabolism of
Carbohydrates

Carbohydrates, also called saccharides (Greek: Sakcharon, sugar), are the most abundant biological molecules containing C, H and O, which are combined in the ratio of 2 : 1, i.e. as (CH₂O)n where n is 3 or more. They may be defined as the aldehyde or ketone derivatives of higher polyhydroxyalcohols or their anhydrides.

CLASSIFICATION OF CARBOHYDRATES

Depending upon the number of the monomer units present in a molecule, carbohydrates are classified as monosaccharides, disaccharides, oligosaccharides and polysaccharides (Fig. 8.1).

Fig. 8.1. Classification of carbohydrates.

MONOSACCHARIDES

Monosaccharides are simple sugars, which can join in several ways to form di, oligo and polysaccharides. These are the aldehyde or ketone derivatives of the straight chain polyhydroxyalcohols which contain 3 or more carbons.

Classification of the Monosaccharides

Monosaccharides are divided into different groups, according to:

• Chemical nature of their carbonyl group or

• Number of carbon atoms in the chain (Table 8.1).

Table 8.1. Classification of monosaccharides

According to chemical nature of the carbonyl group: According to chemical nature of the carbonyl group monosaccharides are divided into two groups as aldoses and ketoses:

Aldoses: The sugar is an aldose when it has an aldehyde (–CHO) group on carbon-l, e.g. glucose.

Ketoses: The sugar is a ketose when it has a ketone (–CO) group at carbon-2, e.g. fructose.

Some of the ketoses are named by inserting ‘-ul’ before the suffix ‘-ose’ in the name of the corresponding aldose, e.g. erythrulose. It is a ketose corresponding to erythrose, which is an aldose.

According to number of carbon atoms: According to number of carbon atoms monosaccharides are designated as trioses, tetroses, pentoses, hexoses, etc.:

Trioses: Trioses are the smallest monosaccharides with three carbon atoms, e.g. glyceraldehyde (an aldotriose) and dihydroxyacetone (a ketotriose). Both, are of physiological significance, as their phosphate esters, i.e. glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, occur as intermediates of glycolysis.

Tetroses: Tetroses have four carbon atoms, e.g. erythrose (an aldotetrose) and erythrulose (a ketotetrose). Erythrose, as erythrose-4-phosphate, occurs as an intermediate in hexose monophosphate (HMP) shunt.

Pentoses: Pentoses contain five carbon atoms, e.g. ribose (an aldopentose) and xylulose (a ketopentose). Both of these occur as intermediates of the HMP shunt. Ribose is also a constituent of nucleotides that are present in RNAs.

Hexoses: Hexoses contain six carbon atoms, e.g. glucose (an aldohexose) and fructose (a ketohexose). Glucose, commonly known as dextrose, is used as a source of energy in the body. Galactose is a constituent of milk sugar lactose. Fructose is a constituent of the common sugar sucrose.

Monosaccharides of biological importance are exhibited in Fig. 8.2.

Fig. 8.2. Some monosaccharides of biological importance.

STRUCTURAL PROPERTIES

Isomerism

Monosaccharides are optically active compounds as they can rotate plane of polarized light. This is characteristic of substances having a tetrahedral carbon (asymmetric carbon), i.e. a carbon that has four different atoms or groups attached to it.

Stereoisomerism

A compound having one asymmetric carbon occurs in two isomeric forms both, of which are non-superimposable mirror images of each other. These two molecules are referred to as the spatial isomers or stereoisomers (enantiomers). Both the enantiomers rotate the plane of the polarized light to the same degree but in opposite direction.

D and L Forms

According to Fischer, two isomers of the parent compound of the carbohydrate family, i.e. glyceraldehyde, are designated as D-glyceraldehyde and L-glyceraldehyde. D or L form of a monosaccharide is determined with respect to its spatial relationship with glyceraldehyde.

In the Fischer projection, glyceraldehyde with an –OH group around the asymmetric center (i.e. on the α carbon atom or the carbon atom adjacent to the carbon atom with the terminal primary alcoholic group) on the right hand side, when the −CHO group is at the top, is designated as D-glyceraldehyde. Its non-superimposable mirror image, which has –OH group around the asymmetric center on the left hand side, is designated as L-glyceraldehyde (Fig. 8.3).

Fig. 8.3. D-and L-isomers of glyceraldehyde.

Addition of a successive carbon atom in the form of −CHOH (the secondary alcoholic group) to the parent compound (D-glyceraldehyde) gives rise to the family of D sugars. Thus, D sugars have same absolute configuration at the asymmetric center (farthest from their carbonyl group) as does D-glyceraldehyde (e.g. the −OH at carbon 5 of D-glucose on the right hand, which is similar to the −OH group at carbon 2 of D-glyceraldehyde). Since each new carbon atom (as a secondary alcoholic group) is added as an asymmetric carbon, number of the isomers increase with the increasing number of the carbon atoms as asymmetric carbons. Number of isomers so formed thus depends upon the number of asymmetric carbons in the molecule. It is represented by the formula 2n since number of asymmetric carbon atoms (n) in a glucose molecule is four therefore, glucose has 2⁴ (16) stereoisomers.

Every sugar, thus, exists in D- or L-form, each being mirror image of the other.

D form of a monosaccharide though is present in abundance in mammals, L isomers of some monosaccharides, e.g. L-xylulose, are also found in nature.

Dihydroxyacetone (a ketotriose) is a monosaccharide that does not occur as D or L isomer.

Optical Activity

An isomer, which rotates plane of polarized light to the right is designated as dextrorotatory (d or +) while its isomer that rotates plane of polarized light to the left is designated as levorotatory (l or –).

A chiral molecule with D configuration thus can be either dextrorotatory [D (+)] or levorotatory [D (–)] indicating its structural relationship to D or L glyceraldehyde but exhibiting different optical rotations.

A mixture containing equal amounts of the two isomers is called racemic mixture or dl-mixture.

Ring Structures of Monosaccharides

Monosaccharides normally occur in ring conformation. When D-glucose forms a six-membered ring, a hemiacetal bond is formed between the aldehyde group of carbon-2 and the hydroxyl group of carbon-5. Thus, cyclization of glucose results in the formation of the ring that is analogous to the structure of pyran, which is a six-membered ring containing five carbons and oxygen. Sugars with the pyran ring are designated as pyranoses and that cyclization of D-glucose yields a cyclic hemiacetal, referred to as glucopyranose.

A five-membered ring with four carbons and oxygen is called furan. Smallest monosaccharide having a furan ring is ribose. Linear form of D-fructose cyclizes between the carbonyl group of carbon 2 and the alcoholic group of carbon 5 and forms a hemiketal, referred to as fructofuranose (Fig. 8.4).

Fig. 8.4. Pyran and furan rings.

Various ring structures of sugars were proposed by Haworth and are called Haworth structures.

Anomers

When a monosaccharide cyclizes, its carbonyl carbon atom, called the anomeric carbon also becomes asymmetric and exhibits two configurations, which are referred to as the α-form and the β-form. Such stereoisomers that differ in configuration at the anomeric carbon, i.e. at carbon-1 in an aldose or carbon-2 in a ketose are called anomers. Both the anomers have different physical and chemical properties, including optical rotation (Fig. 8.5).

Fig. 8.5. α and β anomers of fructose.

Two anomers of glucose are freely inter-convertible in an aqueous solution. At equilibrium, D glucose is a mixture of β-anomer and α-anomer, which are found in a ratio of approximately 2:1.

Mutarotation

Interconversion of two anomers also results in a change in optical rotation of glucose. During equilibrium optical rotation of D (+) glucose slowly changes and reaches a value of 52.5°. This phenomenon of change in optical rotation of D (+) glucose in an aqueous solution and reaching a constant value, due to interconversion of the two anomers, is called mutarotation (Fig. 8.6).

Fig. 8.6. Mutarotation of glucose.

Epimers

Sugars that differ in configuration of its –H and −OH groups around only one of the optically active carbons are called epimers. For example, D-mannose differs in configuration around carbon 2 with respect to D glucose. Similarly, D galactose has different configuration than D glucose around carbon 4. D Mannose and D galactose therefore, are the two epimers of D glucose (Fig. 8.7).

Fig. 8.7. Epimers of glucose.

DERIVED SUGARS

Several derivatives of monosaccharides such as acids, alcohols, amino acid derivatives and methylglycosides also occur in the body.

ACID SUGARS

Oxidation of carbon-l of an aldose converts its aldehyde group to the carboxyl group and forms an acid which is called aldonic acid, e.g. D-gluconic acid.

On the other hand, oxidation of the primary alcoholic group at carbon 6 of an aldose produces uronic acid, e.g. D-glucuronic acid (Fig. 8.8).

Fig. 8.8. D-glucuronic acid.

SUGAR ALCOHOLS

Sugars reduce under mild conditions and produce cyclic polyhydroxyalcohols called alditols, e.g. ribitol (a component of FMN and FAD) and xylitol (a sweetener used in the formation of gums and candies).

AMINO SUGARS

Amino sugars such as glucosamine and galactosamine are formed when the −OH group at carbon-2 is substituted by the −NH₂ group (Fig. 8.9).

Fig. 8.9. Glucosamine.

Some of the amino sugars also occur as their acetylated derivatives. Several derivatives of the amino sugars are found in glycosaminoglycans such as in hyaluronic acid, chondroitin sulfates, erythromycin, etc. N-acetylneuraminic acid and its derivatives are called sialic acids. They are important constituents of glycoproteins and glycolipids.

GLYCOSIDES

When carbon-1 of sugar reacts with an alcohol such as methanol, it yields a compound called methylglycoside. Methylglycosides are found in glycosaminoglycans.

DISACCHARIDES

Disaccharides consist of two similar or dissimilar monosaccharides which are linked together by a glycosidic bond. Maltose, lactose and sucrose are the disaccharides which have biological importance.

Maltose

Maltose [α-D glucopyranosyl-(1,4) α-D glucopyranose] consists of two α-D glucopyran units which are linked together by α-1, 4-glycosidic linkage. Accordingly, in maltose, the −OH group at carbon 4 of a glucose molecule is linked to the −OH group at the carbon l (the anomeric carbon) of the other glucose molecule. This in turn leaves a free −OH at the anomeric carbon, i.e. carbon-l, of the first glucose molecule suggesting that maltose is a reducing disaccharide (Fig. 8.10).

Fig. 8.10. Maltose.

Maltose is usually obtained as one of the hydrolyzed products of some polysaccharides such as starch and glycogen. Maltose can be hydrolyzed to its monosaccharides, i.e. two glucose units, by the enzyme maltase in the intestinal lumen.

Isomaltose: Partial hydrolysis of glycogen and starch produce isomaltose. This is also a reducing sugar and contains two glucose units which are linked by the α-1, 6 glycosidic linkage.

Lactose

Lactose [β-D-galactopyranosyl-(1,4) β-D glucopyranose)] consists of a molecule each of β-D galactopyranose and β-D-glucopyranose which are linked together by β-1, 4-glycosidic linkage. The glycoside bond links carbon-1 of galactose to carbon-4 of glucose. This in turn, leaves a free anomeric carbon on the glucose residue. Thus, lactose is a reducing disaccharide (Fig. 8.11).

Fig. 8.11. Lactose.

Lactose is found in milk, hence it is also called milk sugar. It is synthesized in the mammary glands during lactation from where it is secreted into the milk.

Lactose is hydrolyzed by the intestinal enzyme lactase (β-D-galactosidase) to its monosaccharides, i.e. glucose and galactose.

Sucrose

Sucrose [α-D-glucopyranosyl-(1,2)-β-D fructofuranose] consists of one molecule each of α-D glucopyranose and β-D fructofuranose which are linked by α-1, 2-glycosidic linkage (Fig. 8.12).

Fig. 8.12: Sucrose.

Since the carbonyl groups of both the monosaccharides, i.e. carbon-l of glucose and carbon-2 of fructose participate in the formation of the glycosidic bond and that as such there is no free carbonyl carbon on any of its monosaccharides, hence sucrose is a non-reducing sugar.

Sucrose, also known as invert sugar, is a most abundant disaccharide, which is found in plants. It is also referred to as table sugar or cane sugar. It is hydrolyzed to its monosaccharides, i.e. glucose and fructose by the intestinal enzyme sucrase, which is also referred to as invertase.

OLIGOSACCHARIDES

Oligosaccharides are the carbohydrates built by the linking of more than two (generally three to ten) monosaccharide units by O-glycosidic bonds, e.g. maltotriose, raffinose, etc. Most of these are not digested in human beings. High extent of O-linked oligosaccharides occurs in mucin.

Oligosaccharides are usually attached to proteins at the sequences that form surface loops or turns. Some oligosaccharides also play structural roles. A single protein may contain several N- and O-linked oligosaccharide chains. Glycosylation may affect structure, stability or activity of a protein.

POLYSACCHARIDES

Polysaccharides, also called glycans, are polymers of large number (usually more than ten) of monosaccharides, which are linked together by glycosidic bonds. They are sparingly soluble in cold water but form a colloidal solution in hot water. Polysaccharides are neither sweet in taste nor have reducing properties.

Polysaccharides are classified as homopolysaccharides and heteropolysaccharides.

Homopolysaccharides

Homopolysaccharides or homoglycans are the polymers of the identical monosaccharide units. Common homopolysaccharides of biological significance are starch, glycogen and cellulose (polymers of glucose) and inulin (a polymer of fructose).

Starch: Starch is stored as reservoir of food in cereals (wheat, maize, etc.) and tubers (potato, etc.), in plants. Starch consists of two polysaccharides units called α-amylose and amylopectin. Starch has both α-(1 → 4) and α-1 → 6 linkages (Fig. 8.13).

Fig. 8.13. Starch.

α-Amylose is a linear polymer of glucose. In amylose, glucose residues are linked together by α-(1 → 4) glycosidic bonds.

Amylopectin is a branched polymer of glucose. It has both, α-1 → 4 linkage (in the straight chain) as well as α-1 → 6 linkage (at the branching point). Branching occurs after every 24 to 30 glucose residues.

Starch is a main source of carbohydrate in human diet. Amylase, present in the saliva and the pancreatic juice, randomly hydrolyses α-1 → 4 glycosidic bonds of starch and degrades it to a mixture of maltose, maltotriose and dextrins.

Glycogen: Glycogen is a polysaccharide that is found in animals. It is also called animal starch. Though, glycogen is found in all the cells but is most prevalent in muscle and liver.

Glycogen has both α-1 → 4 as well as α-1 → 6 glycosidic linkages. It is a highly branched structure and branching occurs more frequently, i.e. after every 10–12 glucose residues (Fig. 8.14).

Fig. 8.14. Glycogen.

In the liver, glycogen is used as a readily available source of glucose during starvation.

Table 8.2 lists various differences between starch and glycogen.

Table 8.2. Differences between starch and glycogen

Starch | Glycogen
It is a plant reserve food that is mostly stored in seeds. | It is an animal reserve food that is mainly stored in the liver and muscle.
It is less branched. Branching occurs after nearly 20–30 glucose units. | It is highly branched. Branching occurs after nearly 10–12 glucose units.
It consists of amylose and amylopectin. | No such structures are present.
It is sparingly soluble in cold water and forms a paste in hot water. | It forms an opalescent solution in water.
It gives violet color with iodine. | It gives brown to red color with iodine.

Cellulose: Cellulose is a component of plant cell wall. It is a linear polymer having β-D glucosyl-(1 → 4) α-D glucose repeating units. Cellulose thus has β-1(1 → 4) glycosidic linkages.

Cellulose cannot be digested by human beings because they lack cellulases, i.e. the enzymes which are capable of hydrolyzing β-(1 → 4) linkages. Cellulose, though can not be utilized by human beings, it forms a considerable part of the vegetarian food and adds to the bulk of feces.

Inulin: Inulin is a linear homopolymer of β-D fructose. It is found in the bulbs of dahlia, onion and garlic. Inulin is not utilized by human beings but is of biological significance since it is used in the determination of glomerular filtration rate (GFR).

Dextrans: Dextrans are highly branched homopolymers of glucose having 1→ 6, 1→4 and 1→3 glycoside linkages. Various micro-organisms grown in a sucrose media produce dextrans. They have molecular weight between 1–4 millions. They are used for intravenous infusion, as volume expanders in the treatment of hypovolumic shock.

Chitin: Chitin is present in the exoskeleton of insects. It is composed of N-acetyl-glucosamine residues which are linked by β-1 → 4 glycosidic linkages.

Heteropolysaccharides

Heteropolysaccharides, also called hetero-glycans, contain repeating disaccharide units, which bear negatively charged groups. They are present in the connective tissue, e.g. cartilage, tendon, skin and blood vessel walls. They are linked to proteins and are also called glycosaminoglycans. Glycosaminoglycan-protein complexes are also referred to as proteoglycans.

Glycosaminoglycans

Most important glycosaminoglycans are hyaluronic acid, chondroitin sulfates, heparin, etc.

Hyaluronic acid: Hyaluronic acid is composed of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine. This polysaccharide chain is the longest of the glycosaminoglycans (GAG) and is different from others as it is the only GAG which is non-sulfated. It is not covalently attached to protein and is not limited to animal tissues but is also found in bacteria. It is an important GAG component of connective tissue, synovial fluid and vitreous humor of the eye. Viscoelastic behaviour of hyaluronate solution makes them excellent biological shock absorbers and lubricants.

Chondroitin sulfates: Chondroitin sulfates contain glucuronic acid and N-acetylgalacto-samine with sulfate on the hydroxyl group of either carbon-4 (chondroitin-4-sulfate) or carbon-6 (chondroitin-6-sulfate). They are major components of the cartilage where they bind collagen and hold fibers in a tight, strong network. They are also found in aorta, tendons & ligaments.

Dermatan sulfate: Dermatan sulfate is derived from chondroitin by enzymatic epimerization of carbon-5 of glucuronate residues to iduronate residues. Thus, dermatan sulfate contains L-iduronic acid (with variable amounts of glucuronic acid) and N-acetyl-galactosamine. It is found in skin, blood vessels, tendon and heart valves.

Heparan sulfate: Heparan sulfates are not constructed of identical disaccharides but exhibit structural diversity. They consist of glucuronic or iduronic acid and glucosamine. Some glucosamines are acetylated and may have N- and O-sulfate groups. It is a ubiquitous cell-surface component as well as extracellular substance in blood vessel walls and brain. Heparan sulfates, which function directly at the cell surface, interact with a variety of proteins, including important growth factors and their receptors (such as fibroblast growth factors and their receptors). The formation of ternary complexes of heparan sulfate with fibroblast growth factor and fibroblast growth factor receptor initiates signaling processes.

Heparin: Heparin consists of glucuronic or iduronic acid and glucosamine. Most of its glucosamine units are N-sulfated. Sulfate is also bound to the hydroxyl group on carbon-2 of uronic acid residues and carbon-3 or carbon-6 of glucosamine. It is a highly charged polymer found in the intracellular granules of mast cells that occur in arterial walls, especially in liver, lungs and skin. Heparin is widely used, clinically, to inhibit blood clotting, e.g. in post-surgical patients.

Keratan sulfates: Keratan sulfate is the most heterogeneous group of glycosaminoglycans as its sulfate content is variable. It contains smaller amounts of fucose, mannose, N-acetylglucosamine and sialic acid. The repeating disaccharide, generally, is N-acetylglucosamine and galactose. Sulfate content is variable and may be present on hydroxyl group at carbon-6. Keratan sulfates are linked to protein either by an N-linked (keratan sulfate I) or O-linked (keratan sulfate II) oligosaccharides. Keratan sulfate I is found in cornea while keratan sulfate II occurs in loose connective tissue proteoglycan aggregates with chondroitin sulfate.

USES OF CARBOHYDRATES

1. Carbohydrates provide significant fraction of energy in the diet of most organisms.

2. They also act as storage form of energy in the body.

3. Carbohydrates also serve as the components of a cell membrane that mediates intracellular communication.

4. They also serve as structural components of many organisms, including cell wall of bacteria, exoskeleton of insects and fibrous cellulose portion of plants.

METABOLISM OF CARBOHYDRATES

Principle sources of carbohydrates include starch from cereals, pulses, potato, rice, etc.; lactose from milk and sucrose from table sugar. Glucose may not form an essential part of diet since all the dietary carbohydrates can be readily converted to glucose in the body.

DIGESTION AND ABSORPTION OF CARBOHYDRATES

Digestion of Carbohydrates

Digestion of carbohydrate begins in the mouth where salivary amylase hydrolyses starch into smaller polysaccharides and maltose. Chewing stimulates the production of saliva and mixes salivary amylase with the food. Only about 5% of the starch is broken down by the time the food is swallowed. When carbohydrate enters the stomach, acidity of the stomach stops the action of salivary amylase by denaturing the enzyme.

As stomach contents enter small intestine, the pancreas secretes pancreatic amylase through the pancreatic duct into the small intestine. Pancreatic amylase continues the digestion of starch, breaking it into maltose. Meanwhile, brush border disaccharidases; break various disaccharides into monosaccharides (Table 8.3).

Dietary fiber moves to the large intestine where bacteria partially ferment these undigested carbohydrates and produce gas and a few short-chain fatty acids. Soluble fiber softens the stool and makes it easier to pass. Insoluble fiber resists bacterial activity and passes unchanged to add bulk to stool.

Table 8.3. Digestion of carbohydrates

Enzyme
Site of action
Catalytic action

Salivary amylase
Mouth
Starch/Glycogen → Partially hydrolyzed dextrins, oligosaccharides, isomaltose and maltose

Pancreatic amylase
Small intestine
Partially hydrolyzed dextrins/oligosaccharides → Dextrins, maltose, isomaltose, maltotriose

α-Dextrinase
Small intestine
α-Limit dextrins → Maltotriose, glucose

Disaccharidases
Small intestine

Sucrase

Sucrose → Glucose and fructose

Lactase

Lactose → Glucose and galactose

Maltase

Maltose/Maltotriose → Glucose

Isomaltase

Isomaltose → Glucose

Absorption of Carbohydrates

Different monosaccharides are absorbed into the mucosal cell lining the small intestine by two mechanisms. Whereas fructose is absorbed by facilitated diffusion, the process of active transport absorbs glucose and galactose. Sodium linked glucose transporter called SLGT-1 helps in this process.

In the villi, absorbed monosaccharides pass through the intestinal mucosal cells and enter the liver via the portal vein. Within the liver, galactose and fructose are also converted to glucose.

METABOLISM OF GLUCOSE

Sources and Fate in the Body

Glucose is the major monosaccharide, which enters the liver. Thereafter, it is converted to glycogen and stored in the liver and muscle. It is also oxidized in the liver to produce energy (Fig. 8.15).

Fig. 8.15. Metabolic fates of glucose.

Various metabolic pathways, which utilize glucose, include glycolysis, glycogenesis and pentose phosphate pathway.

On the other hand, gluconeogenesis and glycogenolysis are the pathways which generate glucose.

GLYCOLYSIS

Glycolysis is also called Embden-Meyerhof Parnas pathway or the Embden-Meyerhof Pathway. It is a process of catabolism of glucose either in the presence of oxygen, such as in the liver (aerobic glycolysis), or lack of oxygen, such as in skeletal muscle (anaerobic glycolysis).

Glycolytic enzymes are present in the extra-mitochondrial compartment of the cell. Although aerobic glycolysis occurs in most of the tissues like liver and kidney, anaerobic glycolysis occurs only in muscle (Fig. 8.16).

Various reactions of the glycolytic pathway are shown in Fig. 8.17.

Fig. 8.17. Glycolysis.

1. Phosphorylation of Glucose: Glucose is activated, irreversibly, to glucose-6-phosphate by glucokinase or hexokinase, in the presence of ATP and Mg²⁺ (Fig. 8.18). Hexokinase is allosterically inhibited by glucose-6-phosphate and has high affinity (low Km) for glucose. This enzyme is present in all the extrahepatic tissues.

Fig. 8.18. Phosphorylation of glucose.

In the liver, in the fed state, this reaction is catalyzed by glucokinase which is an inducible enzyme. Its activity is induced by glucose since it has low affinity (high Km) for glucose and its function is to remove glucose from blood in the fed state.

Fig. 8.16. Aerobic and anaerobic glycolysis.

2. Glucose-6-phosphate is isomerized, in a freely reversible reaction, to fructose-6-phosphate by the enzyme phosphohexose isomerase.

3. Fructose-6-phosphate is then phosphorylated, by an allosteric enzyme phosphofructokinase, to form fructose-1, 6-bisphosphate.

This is an irreversible reaction. It requires ATP, which acts as a co-substrate as well as an allosteric inhibitor of the enzyme.

4. Fructose-1, 6-bisphosphate (a hexose) is cleaved by aldolase to 3-phosphoglyceraldehyde (glyceraldehyde-3-phosphate) and dihydroxyacetonephosphate, the two trioses.

5. Subsequently, only 3-phosphoglyceraldehyde enters the pathway. Hence, dihydroxyacetone phosphate is also converted to 3-phosphoglyceraldehyde by the enzyme phosphotriose isomerase.

Glycerol can also enter the glycolytic pathway through this reaction.

6. In the presence of NAD⁺ and inorganic phosphate (Pi), glyceraldehyde-3-phosphate dehydrogenase oxidizes 3-phosphoglyceraldehyde to 1, 3-bisphosphoglycerate.

7. In the next step, 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate. This reaction is catalyzed by the enzyme phosphoglycerate kinase.

In this reaction, there is production of ATP. It is called substrate level production of ATP (i.e. substrate level phosphorylation). Arsenic acid inhibits phosphorylation at this step and thus, ATP production.

8. Thereafter, 3-phosphoglycerate is converted to 2-phosphoglycerate by the enzyme phosphoglycerate mutase.

2,3-Bisphosphoglycerate (2,3-BPG) acts as coenzyme for this reaction.

9. Enolase converts 2-phosphoglycerate to a high-energy compound called phosphoenolpyruvate.

Enolase requires Mg²⁺ or Mn²⁺ for its activity but is inhibited by fluoride. Since fluoride inhibits this reaction, therefore, fluoride is added to the blood, as a preservative, which is collected for glucose estimation.

10. Subsequently, phosphoenolpyruvate is converted to pyruvate, irreversibly. This reaction is catalyzed by the enzyme pyruvate kinase. It also phosphorylates ADP to ATP.

Deficiency of pyruvate kinase leads to hemolytic anemia.

Regulation of Glycolysis

There are three irreversible steps which are catalyzed by hexokinase, phosphofructokinase and pyruvate kinase. These are the sites of the regulation of glycolysis.

Insulin stimulates these enzymes and increases utilization of glucose by glycolysis. On the other hand, glucagon inhibits this process.

FATE OF PYRUVIC ACID

1. Oxidative Decarboxylation to Acetyl CoA

Under aerobic conditions pyruvate is transported into mitochondria where it is oxidatively decarboxylated to acetyl CoA by the enzyme pyruvate dehydrogenase complex (Fig. 8.19).

Fig. 8.19. Conversion of pyruvate to acetyl CoA.

Pyruvate dehydrogenase complex requires five coenzymes, i.e. TPP, CoA, NAD⁺, FAD and lipoic acid. Insulin stimulates its activity whereas it is inhibited by acetyl CoA, NADH and increased ATP/ADP ratio.

This enzyme is also inhibited by arsenate and mercuric ions as well as by dietary deficiency of thiamin. Inhibition of the enzyme results in accumulation of pyruvate and lactate, resulting in lactic acidosis. This in turn may manifest several neurological disturbances.

2. Reduction to Lactate

Under anaerobic conditions pyruvate is reduced to lactate. This reaction is catalyzed by the enzyme lactate dehydrogenase which requires NADH + H⁺.

Tissues such as skeletal muscle, when function under hypoxic conditions, produce large amount of lactic acid.

ATP PRODUCTION BY GLYCOLYSIS

Number of ATP produced during glycolysis varies, depending upon the condition.

Under Aerobic State

Under aerobic condition, NADH produced during the conversion of 3-phosphoglycerate to 1,3-bisphosphoglycerate enters electron transport chain and releases 3 ATP. Thus, additionally, 6 ATP are produced per molecule of glucose. Total yield under aerobic conditions, therefore, is 10 ATP while the net yield is 8 ATP.

Under Anaerobic State

Since each of the energy-yielding steps, i.e. conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate and phosphoenolpyruvate to pyruvate produce one ATP, total 2 ATP are produced per molecule of triose or 4 molecules of ATP per molecule of hexose (Fig. 8.20).

Fig. 8.20. Energy-yielding steps in glycolysis.

As 2 ATP are used in the initial two reactions of the process, i.e. conversion of glucose to glucose-6-phosphate and fructose-6-phosphate to fructose-1, 6-bisphosphate, net energy yield per molecule of glucose is 2 ATP.

GLUCONEOGENESIS

Gluconeogenesis (neoglucogenesis) is the process of the formation of glucose from various non-carbohydrate sources such as the glucogenic amino acids, lactate, glycerol or propionate. It is not a reversal of glycolysis. Gluconeogenesis takes place in the fasting state or on a low carbohydrate diet, particularly in the liver and some other tissues, which are solely dependent on glucose for their energy demand. It does not take place in muscle.

Gluconeogenesis thus, enables maintenance of blood glucose when all the dietary glucose has been absorbed and oxidized. This process is essential since blood glucose level has to be maintained to support metabolism of the tissues that use glucose as the primary substrate such as the brain, red blood cells and lens.

Gluconeogenesis, however, is not truly a reversal of glycolysis since there are three irreversible reactions in the glycolytic pathway (Fig. 8.21).

These reactions are bypassed by an alternate set of reactions, which include:

Fig. 8.21. Irreversible reactions in the glycolytic pathway.

Conversion of Pyruvate to Phosphoenolpyruvate

For the reversal of the pyruvate to phosphoenol pyruvate, pyruvate is first converted to oxaloacetate by the enzyme pyruvate carboxylase, in mitochondria.

Thereafter, malate dehydrogenase converts oxaloacetate to malate, which is freely transported across the mitochondrial membrane. Malate thus, comes out from the mitochondria to the cytosol where it is converted back to oxaloacetate by the same enzyme. This also reduces NAD⁺ to NADH + H⁺. This enzyme thus, not only transports oxaloacetate (via malate) but also reducing equivalents from mitochondria into cytosol. Reactions of gluconeo-genesis thus occur in the cytosol as well as mitochondria.

In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by the enzyme phosphoenolpyruvate carboxykinase (PEP-carboxykinase).

Conversion of Fructose-1,6-Bisphosphate to Fructose-6-Phosphate

Fructose-1,6-bisphosphate is converted to fructose-6-phosphate by the enzyme fructose-1, 6-bisphosphatase.

Conversion of Glucose-6-Phosphate to Glucose

Glucose-6-phosphate is converted to glucose by the enzyme glucose-6-phosphatase. This enzyme is not found in the muscle and adipose tissue.

SUBSTRATES FOR GLUCONEOGENESIS

Lactate

Lactate is produced during the process of anaerobic glycolysis. For its conversion to glucose, firstly, lactate is oxidized to pyruvate. This reaction is catalyzed by the enzyme lactate dehydrogenase, which requires NAD⁺.

The direction of LDH reaction depends on the relative intracellular concentrations of pyruvate and lactate, and on the ratio of NADH/ NAD⁺, in the cell. The formation of lactate is the major fate of pyruvate in several tissues such as red blood cells, lens and cornea of the eye, etc.

Subsequently, pyruvate is converted to glucose by the reversal of the glycolytic reactions.

Glycogenic Amino Acid

In the liver, various glucogenic amino acids transfer their α-amino group by transamination and release carbon skeletons, which form intermediates of the citric acid cycle (Table 8.4).

Table 8.4. Conversion of glucogenic amino acids to the citric acid cycle intermediates

Amino Acid | Citric acid cycle intermediate formed
Gly, Ala, Ser, Thr, Cys, Trp and HO-Pro | Pyruvate
Arg, His, Glu, Gln and Pro | α-Ketoglutarate
Phe and Tyr | Fumarate
Val, Ile and Met | Succinyl CoA

These citric acid cycle intermediates in turn can form glucose by reversal of the glycolytic reactions via pyruvate.

Glycerol

Glycerol is produced as a result of lipolysis in the adipose tissue. Glycerol kinase converts it to α-glycerolphosphate in the liver, which is later reduced by a dehydrogenase to dihydroxyacetone phosphate and can enter in glycolysis at this point (Fig. 8.22).

Fig. 8.22. Formation of glucose from glycerol.

Propionate

Oxidation of fatty acids containing odd number of carbon atoms, in addition to acetyl CoA also produces a molecule of propionyl CoA, particularly in ruminants. In human beings, propionyl CoA is obtained from Ile, Val and Thr. It is converted to D-methylmalonyl CoA by propionyl CoA carboxylase. D-Methylmalonyl CoA is then converted to L-methylmalonyl CoA and finally, by an isomerase to succinyl CoA, which is an intermediate of the citric acid cycle. Succinyl CoA in turn can be converted to glucose via pyruvate (Fig. 8.23).

Fig. 8.23. Formation of glucose from propionate.

TRICARBOXYLIC ACID CYCLE

Tricarboxylic acid cycle is also called citric acid cycle or the Krebs cycle. It is a process of oxidation of acetyl CoA (active acetate) to CO₂ and H₂O.

During the course of the oxidation of acetyl CoA, reducing equivalents are produced, which enter respiratory chain and generate large amount of ATP. Reactions of the Krebs cycle are shown in Fig. 8.24.

Fig. 8.24. Tricarboxylic acid cycle.

1. In the first step, acetyl CoA (formed from pyruvate under aerobic condition) combines with oxaloacetate and forms citric acid (a tricarboxylic acid).

This reaction is catalyzed by the enzyme citrate synthase, also called as the condensing enzyme.

2. Citrate is then rearranged to cis-aconitate, which is subsequently changed to isocitrate. Both of these steps are catalyzed by aconitase.

Conversion of citrate to isocitrate is inhibited by fluoroacetate.

3. In the presence of isocitrate dehydrogenase, isocitrate is converted to oxalosuccinate, which is subsequently decarboxylated to α-ketoglutarate.

4. a-Ketoglutarate undergoes oxidative decarboxylation and gets converted to succinyl CoA.

This reaction is similar to the conversion of pyruvate to acetyl CoA. α-Ketoglutarate dehydrogenase complex, which also requires five coenzymes, i.e. TPP, NAD⁺, FAD, coenzyme A and lipoic acid, catalyzes this reaction.

5. Succinyl CoA is converted to succinate, by the enzyme succinate thiokinase.

During this reaction, a molecule of GTP is formed. This is known as substrate level phosphorylation, as a high-energy molecule is formed at the substrate level.

6. Succinate is converted to fumarate, by the enzyme succinate dehydrogenase.

Due to structural similarities between malonate and succinate, malonate inhibits succinate dehydrogenase, competitively.

7. With the addition of a molecule of water, by the enzyme fumarase (fumarate hydratase), fumarate is converted to L-malate.

8. Finally, malate dehydrogenase, in the presence of NAD⁺, converts malate to oxaloacetate.

Energy yield from Tricarboxylic Acid Cycle

As a result of oxidation of one molecule of acetyl CoA in the Krebs cycle, three molecules of NAD⁺ and one molecule of FAD are reduced.

Reducing equivalents (from NADH + H⁺) enter respiratory chain and result in the production of three molecules of ATP. Similarly, FADH₂ yields 2 ATP. Besides, there is also substrate level production of GTP.

Thus, total ATP yield, per molecule of acetyl CoA, is 12 ATP (Table 8.5).

Table 8.5. Energy production in citric acid cycle

Reaction | Reducing equivalents produced as | Number of ATP produced
Isocitrate → α-Ketoglutarate | NADH + H⁺ | 3
α-Ketoglutarate → Succinyl CoA | NADH + H⁺ | 3
Succinyl CoA → Succinate | – | 1
Succinate → Fumarate | FADH₂ | 2
Malate → Oxaloacetate | NADH + H⁺ | 3
| Total | 12

As mentioned above, conversion of pyruvate to acetyl CoA also generates NADH + H⁺ and gives 3 ATP. Thus, total number of ATP produced from oxidation of pyruvate is 15.

Since 2 molecules of pyruvate are formed from one molecule of glucose, therefore, in addition to energy yield during aerobic glycolysis (8 ATP) a molecule of glucose also produces 30 ATP via the Krebs cycle. Thus, a total of 38 ATP are obtained when a molecule of glucose is completely oxidized to CO₂ and H₂O under aerobic conditions, i.e. via glycolysis and Krebs cycle.

Biological Significance of Tricarboxylic Acid Cycle

Citric acid cycle has a dual role, i.e. it is important in oxidation as well as synthetic processes. It is thus amphibolic in nature.

It is catabolic for the oxidation of carbohydrates, lipids and proteins, as they are completely oxidized to CO₂ and H₂O, and release energy.

It is also important in the anabolic reactions, as various intermediates of the cycle can be used for the biosynthesis of the nonessential amino acids. Various intermediates of the cycle are also potentially glucogenic and thus, can give rise to glucose in liver and kidney (Fig. 8.25).

Fig. 8.25. Biological significance of tricarboxylic acid cycle.

GLYCOGEN METABOLISM

GLYCOGEN SYNTHESIS

Glycogen synthesis (Glycogenesis) is the process of the conversion of glucose to glycogen. Although, it is operative in several tissues, liver and muscle are the main organs for the synthesis of glycogen. Glycogenesis is stimulated by insulin.

Various reactions of glycogenesis are shown in Fig. 8.26.

Fig. 8.26. Glycogenesis.

1. Glucose is first activated (phosphorylated) to glucose-6-phosphate.

In the liver, glucokinase converts most of the glucose into glucose-6-phosphate, in the fed state. This is an inducible enzyme and has greater specificity for its substrate.

In the muscle and other tissues, this reaction is catalyzed by hexokinase.

2. Glucose-6-phosphate is epimerised to form glucose-1-phosphate, by the enzyme phosphoglucomutase.

3. Glucose-1-phosphate reacts with UTP and gets converted to uridinediphosphate glucose (UDP-Glu). The reaction is catalyzed by UDPG pyrophosphorylase (glucose-1-phosphate uridyltransferase).

Pyrophosphate, so released during this process, is immediately hydrolyzed to two molecules of inorganic phosphate, by pyrophosphatase.

4. From UDP-Glu, glucose is transferred to the glycogen primer (i.e. the preformed oligosaccharide). This reaction is catalyzed by the enzyme glycogen synthase.

Glycogen primer is formed on a protein known as glycogenin.

The incoming glucose is linked to the primer at the non-reducing end, by 1,4-a-glycosidic linkage and results in elongation of the pre-existing branch. This results in the formation of an unbranched glycogen.

Both, glycogen primer and UDP-Glu are the substrates for glycogen synthase, which is a key enzyme of glycogenesis.

5. Once the straight chain containing nearly11 glucose residues is formed, branching enzyme removes a block of 7–8 glycosyl residues from the growing chain and transfers it to the neighboring chain (Fig. 8.27).

This branch point again grows with the addition of the glucose molecules at the 1→4 linkage. This in turn results in the formation of a highly branched glycogen structure.

Fig. 8.27: Formation of branching in glycogen.

DEGRADATION OF GLYCOGEN

Glycogen breakdown (glycogenolysis) is the process of the conversion of glycogen to either glucose-6-phosphate in muscle or free glucose in liver and kidney (Fig. 8.28).

1. In the first step, glucose molecules are sequentially removed from glycogen as glucose-1-phosphate. This reaction is catalyzed by the enzyme phosphorylase. It is the rate limiting step of this pathway.

Phosphorylase hydrolyzes the α-1,4-glycosidic bonds and removes glucose units, as glucose-1-phosphate, until nearly four glucose residues are left. After the action of phosphorylase, glycogen is partially hydrolyzed, leaving limit dextrin.

Fig. 8.28: Glycogenolysis.

2. Limit dextrin is further hydrolyzed by the debranching enzyme to glucose-1-phosphate and free glucose.

Debranching enzyme is a bifunctional enzyme. Its first unit has glycosyltransferase, i.e. 1→4 α-D-glucontransferase [α-(1→4) transglycosylase], activity in which a strand of three glucosyl residues is removed from the four glucosyl residue branch of the molecule and is attached to the nonreducing end of another branch. This reaction forms a new α(1→4) linkage and thus, three more glucose units become available for the phosphorylase reaction.

The α(1→6) bond, which is linking the remaining glucosyl residue on the branch point to the main chain, is hydrolyzed by the second unit of the debranching enzyme that is referred to as amylo-α-1,6-glucosidase. This glucosyl residue is released as free glucose, rather than as glucose-1-phosphate, leaving the unbranched glycogen molecule (Fig. 8.29).

Cooperative and repetitive actions of phosphorylase and debranching enzyme result in complete hydrolysis of glycogen.

During glycogenolysis, glucose-1-phosphate and glucose are liberated in the ratio of approximately 10:1.

Fig. 8.29. Debranching.

3. Glucose-1-phosphate, produced as a result of the action of phosphorylase, is converted to glucose-6-phosphate by the enzyme phosphoglucomutase.

In the muscle, glucose-6-phosphate is the end product of glycogen breakdown and is used in the glycolytic pathway to liberate energy that is required during exercise. Thus, glycogenolysis is followed by glycolysis, due to the absence of the enzyme glucose-6-phosphatase in the muscle.

4. In liver and kidney, glucose-6-phosphate is further hydrolyzed to glucose. This reaction is catalyzed by glucose-6-phosphatase.

Glycogen, which is stored in the liver, is thus converted to free glucose and is used to maintain blood glucose level in a hypoglycemic state.

In the muscle, end product of glycogen breakdown, during glycolysis, is glucose-6-phosphate and not glucose. This is due to absence of the enzyme glucose-6-phosphatase. This, glucose-6-phosphate, is used for energy production during exercise via anaerobic glycolysis and results in the production of lactate. Lactic acid is transported to the liver and reforms glucose by the process of gluconeogenesis and becomes available for the muscle and other tissues. This is called Cori cycle.

Cori cycle: Process of the transport of lactate from the muscle to the liver, for its conversion into glucose, which again goes to the muscle and reforms glycogen, for its re-utilization during exercise is called lactic acid cycle or Cori cycle. It results in the net exchange of glycogen via glucose-6-phosphate and lactate in the two tissues, i.e. the muscle and the liver (Fig. 8.30).

Fig. 8.30. Cori cycle.

PENTOSE PHOSPHATE PATHWAY

Pentose phosphate pathway, also called hexose monophosphate shunt (HMP shunt), is the second major pathway for the metabolism of glucose. Enzymes for this pathway are localized in the cytosol, while reducing equivalents are accepted by NADP⁺, instead of NAD⁺. This pathway is operative in many tissues such as liver, erythrocytes, lactating mammary glands, testes and adipose tissue. Rate of the HMP shunt reactions is increased by insulin.

GLYCOGEN STORAGE DISEASES

This is a group of inherited disorders that are associated with glycogen metabolism where an abnormal type or quantity of the glycogen is deposited in different tissues.

According to the deficiency of the enzyme involved, there are several types of glycogen storage diseases (Table 8.6).

Table 8.6. Glycogen storage diseases

In the overall process, six molecules of glucose are utilized to give six molecules of CO₂ and six molecules of pentoses. The pentoses are subsequently, rearranged to give four molecules of fructose-6-phosphate and two molecules of glyceraldehyde-3-phosphate. Two trioses also form a hexose by the reversal of glycolysis thus, regenerating five molecules of hexoses (Fig. 8.31).

Fig. 8.31. HMP shunt – an overview.

1. Firstly, glucose is activated to glucose-6-phosphate, by the enzyme hexokinase.

2. Thereafter, glucose-6-phosphate is changed to 6-phosphogluconolactone and subsequently, to 6-phosphogluconate, by the enzyme glucose-6-phosphate dehydrogenase. This enzyme is induced by insulin. During this process NADP⁺ is reduced to NADPH + H⁺ (Fig. 8.32).

Fig. 8.32. Conversion of glucose-6-phosphate to 6-phosphogluconate.

3. In the next reaction, a hexose is converted to a pentose. In this process, 6-phosphogluconate is oxidized by the enzyme 6-phosphogluconate dehydrogenase, to form an intermediate, called 3-keto-6-phosphogluconate. This reaction also generates NADPH + H⁺.

Subsequently, by the enzyme 6-phosphogluconate decarboxylase, a carbon is removed from 3-keto-6-phosphogluconate, as CO₂, leaving a pentose called ribulose-5-phosphate (Fig. 8.33).

Fig. 8.33. Conversion of 6-phosphogluconate to ribulose-5-phosphate.

These two enzymes, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, are the regulatory enzymes of this shunt.

4. In the second stage of the shunt, ribulose-5-phosphate changes to other pentoses.

Ribulose-5-phosphate is changed to ribose-5-phosphate, by the enzyme ribose-5-phosphate ketoisomerase, and to xylulose-5-phosphate by the enzyme ribulose-5-phosphate epimerase (Fig. 8.34).

Fig. 8.34. Conversion of ribulose-5-phosphate to other pentoses.

5. In the next reaction, carbon skeletons of the two pentoses are rearranged. In this process two pentoses are converted to a hexose and a tetrose (Fig. 8.35).

Fig. 8.35. Conversion of pentoses to a hexose and a tetrose.

Firstly, a transketolase transfers a ketol group (the first 2 carbon atoms) from xylulose-5-phosphate to ribose-5-phosphate, forming sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate. Transketolase requires TPP as a coenzyme.

Subsequently, an aldol group (3 carbons moiety) is transferred by the enzyme trans-aldolase, from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate, forming fructose-6-phosphate and erythrose-4-phosphate.

6. In the next reaction, transketolase transfers a ketol group (2 carbons moiety) from xylulose-5-phosphate to erythrose-4-phosphate, forming fructose-6-phosphate and glyceraldehyde-3-phosphate. It converts a pentose and a tetrose to a hexose and a triose (Fig. 8.36).

Fig. 8.36: Conversion of a pentose and a tetrose to a hexose and a triose.

BIOLOGICAL IMPORTANCE OF PENTOSE PHOSPHATE PATHWAY

In the Liver

1. Generation of NADPH: Two molecules of NADP are reduced to NADPH, per molecule of glucose-6-phosphate, in the pentose phosphate pathway. NADPH, so produced, is used for the various anabolic reactions, such as synthesis of fatty acids and cholesterol.

2. Source of pentoses: Pentose phosphate pathway is also important source of pentoses, which are required for the biosynthesis of nucleic acids (Fig. 8.37).

Fig. 8.37. Biological significance of HMP shunt.

In erythrocytes

NADPH is utilized by glutathione reductase in the erythrocytes, to maintain the reduced state of glutathione and prevent their hemolysis. This reaction is important for the detoxification of H₂O₂ which is produced in the erythrocytes. If accumulates, H₂O₂ decreases their life span and causes hemolysis (Fig. 8.38).

Fig. 8.38. Significance of NADPH production in RBCs.

METABOLISM OF OTHER HEXOSES

Although, glucose is the most widely used monosaccharide, fructose and galactose are also important fuels and can be funneled into the glycolytic pathway.

METABOLISM OF FRUCTOSE

Fructose can enter the glycolytic pathway by any of the two routes:

1. Most of the ingested fructose is metabolized by the liver, using the fructose-1-phosphate pathway.

The first step is the phosphorylation of fructose to fructose-1-phosphate by fructokinase.

Fructose-1-phosphate is then splitted into glyceraldehyde and dihydroxyacetone phosphate, an intermediate in glycolysis. This aldol cleavage is catalyzed by a specific fructose-1-phosphate aldolase.

Glyceraldehyde is then phosphorylated by triose kinase to glyceraldehyde-3-phosphate, a glycolytic intermediate.

2. In other tissues, fructose can be phosphorylated to fructose-6-phosphate by hexokinase.

In hereditary fructose intolerance, fructose can not be converted to glucose because of a defect in the gene encoding fructose-1,6-bisphosphate aldolase. Nutrition therapy involves the elimination of fructose and sucrose from the diet.

METABOLISM OF GALACTOSE

Galactose is converted into glucose-6-phosphate in four steps:

1. The first reaction is the phosphorylation of galactose to galactose-1-phosphate by galactokinase.

GALACTOSEMIA

The disruption of galactose metabolism is referred to as galactosemia. The most common form of galactosemia is an inherited deficiency of galactose-1-phosphate uridyl transferase activity. Afflicted infants fail to thrive and vomit or have diarrhoea after consuming milk. In addition, enlargement of the liver and jaundice are common, sometimes progressing to cirrhosis. The blood galactose level is markedly elevated and galactose is also found in the urine. The absence of the enzyme transferase in red blood cells is a definite diagnostic criterion. Cataract is formed since the transferase is not active in the lens of the eye and in the presence of aldol reductase the accumulating galactose is reduced to galactitol. Since galactitol is osmotically active, water diffuses into the lens, instigating the formation of cataract.

The most common treatment is to remove galactose (and lactose) from the diet. Although elimination of galactose from the diet prevents liver disease and cataract development, the majority of patients still suffer from central nervous system malfunction. Female patients also display ovarian failure.

2. Galactose-1-phosphate than acquires uridyl group from uridine diphosphate glucose (UDP-glucose), an intermediate in the synthesis of glycosidic linkages, in glycogenesis. This reaction is catalyzed by galactose-1-phosphate uridyl transferase, and produces UDP-galactose and glucose-1-phosphate.

3. The galactose moiety of UDP-galactose is then epimerized to UDP-glucose by UDP-galactose-4-epimerase.

This is a reversible reaction and is important in the reverse direction also, for the conversion of UDP-glucose into UDP-galactose. UDP-galactose is essential for the synthesis of galactosyl residues in complex polysaccharides and glycoproteins, if the amount of galactose in the diet is inadequate to meet these needs.

4. Finally, glucose-1-phosphate (formed from galactose) is isomerised to glucose-6-phosphate, by phosphoglucomutase.

LACTOSE INTOLERANCE

Many adults are unable to metabolize the milk sugar lactose and experience gastrointestinal disturbances, if they drink milk. This condition is referred to as lactose intolerance or hypolactasia. It is most commonly caused by a deficiency of the enzyme lactase, which cleaves lactose into glucose and galactose. The lactose is a good energy source for micro-organisms in the colon and they ferment it to lactic acid while also generating methane and hydrogen gas. The gas produced creates uncomfortable feeling of gut distension and flatulence. The lactate produced by the microorganisms is osmotically active and draws water into the intestine, resulting in diarrhoea. If severe enough, the gas and diarrhoea hinder the absorption of other nutrients such as fat and protein. The simplest treatment is to avoid the consumption of milk and milk products (containing lactose).

REGULATION OF BLOOD GLUCOSE

Blood glucose level is maintained with in the normal physiological range of 60–90 mg per 100 ml in the fasting (post-absorptive) state by various hormones and by the kidney.

BY HORMONES

Several hormones regulate blood glucose concentration. While insulin lowers blood glucose, various other hormones increase blood glucose level (Fig. 8.39).

Fig. 8.39: Regulation of blood glucose by hormones.

Hormone which has Hypoglycemic Action

Insulin is the only hormone which has hypoglycemic action. It is released into the blood stream under the direct influence of hyperglycemia. It is a polypeptide containing 2 chains and is produced by β-cells of the islets of Langerhans of the pancreas.

Insulin lowers blood glucose concentration by increasing uptake of glucose by the extrahepatic tissues. Insulin also promotes glycolysis and glycogenesis, both in the liver and the muscle. At the same time, insulin also suppresses glycogenolysis in the liver and the kidney. Besides, insulin also suppresses gluconeogenesis.

Insulin also stimulates synthesis of various enzymes such as glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ATP-citrate lyase, etc. Insulin also represses synthesis of pyruvate carboxylase; PEP-carboxykinase and fructose-1,6-bisphosphatase.

Insulin also promotes transport of amino acids across the cell membrane and thus stimulates protein synthesis.

Insulin also inhibits ketogenesis and promotes lipogenesis.

Hormones which have Hyperglycemic Action

Several hormones have hyperglycemic action. These are called diabetogenic hormones and include glucagon, epinephrine, glucocorticoids, growth hormone and thyroxine.

Glucagon: Glucagon is produced by a-cells of the islets of Langerhans of pancreas. Its synthesis is stimulated under the influence of hypoglycemia. It acts only in the liver and promotes glycogenolysis. It also promotes gluconeogenesis from amino acids and lactate.

Epinephrine: Epinephrine, also called adrenaline, is secreted by adrenal medulla. It is a hormone in the first line of defense against hypoglycemia. It is a derivative of tyrosine.

Epinephrine promotes glycogenolysis, both in the liver and muscle. Epinephrine also promotes gluconeogenesis in the liver. It also inhibits release of insulin from the pancreas and decreases transport and utilization of glucose in different tissues. At the same time, epinephrine also stimulates secretion of glucagon.

Glucocorticoids: Glucocorticoids are steroids secreted by adrenal cortex. They are antagonists to insulin.

Cortisol is a major glucocorticoid present in blood. It stimulates protein catabolism in the muscle and promotes gluconeogenesis in the liver. It also stimulates lipolysis and inhibits glucose oxidation, by the liver. Cortisol also facilitates actions of other hyperglycemic hormones (permissive effects) such as glucagon, epinephrine and growth hormone, which further inhibit uptake and utilization of glucose by the peripheral tissues.

Growth Hormone: Anterior pituitary secretes growth hormone. Its secretion is stimulated under the influence of hypoglycemia. Prolonged administration of the growth hormone stimulates secretion of insulin. As a result of increased insulin secretion, β-cells are exhausted producing diabetes like situation. Growth hormone also decreases glucose uptake and utilization by muscle. It also promotes lipolysis in the adipose tissue.

Thyroxine: Thyroxine is secreted by the thyroid gland. It stimulates intestinal absorption of glucose and promotes glycogenolysis as well as gluconeogenesis in the liver. Thyroxine also causes destruction of insulin. Hyperthyroidism is generally associated with hyperglycemia and mild diabetes.

BY KIDNEY

Kidney also regulates blood glucose concentration, which is continuously filtered by the glomeruli and is completely reabsorbed by the renal tubules. However, when blood glucose concentration exceeds 180 mg/dl (renal threshold value); tubules fail to reabsorb all the filtered glucose, some of which starts appearing in the urine.

INVESTIGATIONS AND THEIR INTERPRETATIONS

Blood glucose concentration in a normal adult remains in the range of 60–100 mg/100 ml. When the value exceeds, it is called hyperglycemia. Persistent hyperglycemia may be a result of diabetes mellitus, which is detected by estimating blood glucose concentration during fasting. To confirm the diagnosis, fasting and postprandial (after meals) samples are analyzed. Sometimes, it may also be essential to perform glucose tolerance test (GTT).

DIABETES MELLITUS

Diabetes mellitus is characterized by persistent hyperglycemia with or without glycosuria (glucosuria). This is the commonest endocrine disease caused by the deficiency in secretion or action of insulin. Besides insulin, several diabetogenic hormones influence metabolism of glucose.

Diabetes mellitus is defined as a state of chronic hyperglycemia which may be a result of genetic and/or environmental factors. It is a multifactorial disease. There are two major clinical classes of diabetes mellitus which are referred to as:

• Insulin Dependent Diabetes Mellitus and

• Noninsulin Dependent Diabetes Mellitus

Insulin dependent diabetes mellitus

Insulin dependent diabetes mellitus (IDDM) or type I diabetes is also called juvenile-onset diabetes because it usually appears in the childhood or in young age group (commonly less than 40 years of age). There is absolute deficiency of insulin, due to a gradual depletion of β-cells of the pancreas, which may get destroyed by an autoimmune process.

IDDM is characterized by hyperglycemia, hyperlipoproteinemia (raised chylomicron and VLDL) and severe ketoacidosis. This in turn suggests that besides defects in carbohydrate metabolism, there are abnormalities in fat and protein metabolism also, in such patients.

Hyperglycemia in IDDM is a result of inability of the insulin-dependent tissues, to take up glucose as well as due to accelerated hepatic gluconeogenesis from amino acids (derived from muscle protein).

Increased lipolysis (in the adipose tissue) and accelerated fatty acid oxidation (in the liver) result in ketoacidosis.

Insulin deficiency also reduces lipoprotein lipase activity, thereby resulting in hyperchylo-micronemia.

Patients with IDDM can usually be recognized by abrupt appearance of polyuria (frequent urination), polydypsia (excessive thirst) and polyphagia (excessive hunger), often triggered by the stress or some other illness. These symptoms are usually accompanied with fatigue, weight loss and weakness.

Its diagnosis is confirmed by high fasting blood glucose, commonly accompanied with ketoacidosis. Insulin injection though does not cure the disease but promotes glucose uptake by the tissues and inhibits gluconeogenesis, lipolysis and proteolysis.

Life span of the patient is reduced as a result of the degenerative complications such as kidney malfunction, nerve impairment and cardiovascular disease.

Hyperglycemia also leads to blindness through retinal degeneration and glycosylation of the lens proteins which in turn cause cataract.

Non-insulin dependent diabetes mellitus

Majority of the diabetic patients (over 80%) suffer from non-insulin dependent diabetes mellitus (NIDDM) or type II diabetes. It is also called maturity-onset-diabetes since it usually occurs in the middle age group (usually more than 40 years of age), who are generally obese. Occurrence of the disease is almost completely determined by the genetic factors. NIDDM develops gradually, without obvious symptoms. Metabolic alterations are milder than IDDM.

NIDDM is characterized by hyperglycemia, often with hypertriglyceridemia. Inspite of high level of insulin, glucose levels are poorly controlled because of the lack of normal response to insulin. Insulin resistance in these patients may be due to increased expression of the tumor necrosis factor-α, in the adipocytes of obese individuals. Hyperglycemia is mainly a result of the poor peripheral utilization of glucose, especially in muscle.

Ketoacidosis does not develop because adipocytes remain sensitive to the effect of insulin. Rapid de novo synthesis of fatty acids and VLDL lead to hypertriglyceridemia without hyper-chylomicronemia.

Blood glucose concentration is much higher than normal, particularly after a meal.

Weight reduction and dietary modifications often correct hyperglycemia of type II diabetes. Hypoglycemic agents such as sulphonylureas, may be required to achieve a satisfactory fall in blood glucose level.

Salient features of two types of diabetes mellitus are shown in Table 8.7.

For any form of diabetes, metabolic derangement is due to elevated glucagon/insulin ratio. In addition, there may be weight loss in IDDM.

Metabolic complications of the uncontrolled diabetes include ketoacidosis and coma in IDDM, hyperosmolar non-ketotic coma in NIDDM, or hypoglycemia due to insulin overdose.

Long-term complications of the disease include acute myocardial infarction (diabetic patients often present with painless acute myocardial infarction, i.e. silent MI), due to accelerated atherosclerosis and coronary heart disease, nephropathy, retinopathy, neuropathy, infections and delayed wound healing.

Diabetic ketoacidosis: Diabetic ketoacidosis is a common feature observed in patients with insulin dependent diabetes mellitus. This is due to the severe deficiency of insulin with excess of glucagon and other hyperglycemic hormones such as epinephrine, cortisol and growth hormone.

Plasma free fatty acid concentration is increased. This in turn leads to increased production of ketone bodies by the liver.

There is also marked hyperglycemia, ketonemia, ketonuria and water and electrolyte imbalance.

Various features of the disease can be corrected by insulin administration.

Table 8.7. Salient features of two types of diabetes mellitus

| IDDM | NIDDM
Type of Diabetes | Juvenile onset (Type I) | Maturity onset (Type II)
Prevalence | About 20% | About 80%
Age of onset | <40 years | >40 years
Body habitués | Normal to wasted (weight loss) | Obese
Plasma insulin | Low (due to β-cell destruction) | Normal to high (insulin resistance)
Plasma glucagon | High (suppressible) | High (resistant)
Acute complications | Ketoacidosis | Hyperosmolar coma (ketoacidosis is rare)
Insulin therapy | Responsive | Resistant
Oral hypoglycemic agents | Unresponsive | Responsive

Estimation of Fasting and Postprandial Blood Glucose Levels

International Committee of Diabetologists has recommended the estimation of blood glucose in the overnight fasting and postprandial samples (2 hours after 75 g of a glucose load) to confirm the diagnosis of diabetes mellitus. Accordingly, an individual is said to be normal if fasting blood glucose value is less than 100 mg/100 ml and the 2-hours’ value is below 140 mg/100 ml. A fasting value of 126 mg/100 ml or higher and the 2 hours’ value of more than 200 mg/100 ml is diagnostic of diabetes mellitus. Fasting value between 100–125 mg/100 ml and/or the 2 hours’ value between 140–200 mg/100 ml is referred to as impaired glucose tolerance (Table 8.8).

Table 8.8. Criteria for the evaluation of the glucose tolerance

GTT | Fasting plasma glucose (mg/dl) | Two hours value after 75 g of glucose (mg/dl)
Normal | < 100 | < 140
Impaired GT | 100–125 | ≥ 140 – ≤ 199
Diabetes mellitus | > 126 | ≥ 200

Glucose Tolerance Test

Glucose tolerance test (GTT) is performed by giving a load of glucose, orally. It is helpful in confirming the diagnosis of diabetes in those patients whose fasting blood glucose concentration is in the upper normal limit, with transient or sustained glycosuria without the symptoms of diabetes or with family history of the disease.

Procedure: For GTT, fasting venous blood sample (after 12–16 hours of fast) is drawn, in a fluoride-oxalate tube. Thereafter, 75 g of glucose (about 1 g/kg body weight) is given in nearly 200–300 ml of water. Blood samples are again drawn after every 30 minutes, till 2 to 2½ hours. Urine specimen may also be collected simultaneously. Each blood sample is analyzed and glucose tolerance curve is plotted (Fig. 8.40).

Fig. 8.40. Glucose tolerance curves.

Interpretation: Fasting blood glucose concentration, maximum blood glucose value after a glucose load, and the rate at which it returns to the fasting, gives an idea about the disease. For a normal individual, fasting blood glucose value should be within the normal limit. It is increased by about 50% within 60 minutes and thereafter returns towards fasting after two hours. Glycosuria is not observed in any of the urine sample.

Any deviation from the above is referred to as an abnormal glucose tolerance curve. In a patient with diabetes mellitus, fasting blood glucose level is above normal. In addition, the rise in blood glucose with time may be higher and the level may not return towards fasting. In addition, glucose may be present in urine. If glucose tolerance curve is normal but there is glycosuria, it is referred to as renal glycosuria.

SOME IMPORTANT QUESTIONS

1. Define carbohydrate. Give their classification. Explain stereoisomerism of carbohydrates.

2. What are polysaccharides? Classify them.

3. Differentiate between starch and glycogen.

4. Write notes on :

(i) Cellulose

(ii) Anomers

(iii) Epimers

(iv) Inulin

(v) Homopolysaccharides

(vi) Heteropolysaccharides

5. Outline reactions of the citric acid cycle.

Give energetic of the cycle and explain the amphibolic role of this cycle.

6. Outline pentose phosphate pathway reactions. Discuss significance of the pathway in different tissues.

7. Give outline of glycolysis. What are its regulatory steps? What is the energy yield of this pathway?

8. Define glycogenolysis. Outline reactions of this pathway.

9. What is glycogenesis? Give reactions of glycogenesis.

10. Explain :

(i) Why aerobic glycolysis releases more energy than anaerobic glycolysis?

(ii) How glycolysis is reversed?

(iii) Citric acid cycle and its energetics.

11. Discuss briefly :

(i) Regulation of blood glucose

(ii) Glycogenolysis

(iii) Metabolism of fructose

(iv) Metabolism of galactose

12. Write notes on :

(i) Diabetes mellitus

(ii) Glycogenesis

(iii) Gluconeogenesis

(iv) Glycogen storage diseases

(v) Energetics of the citric acid cycle

(vi) Oxidation of pyruvate to acetyl CoA

(vii) von Gierke disease

(viii) Cori cycle

(ix) Digestion and absorption of carbohydrates

(x) Differentiate between type I and type II diabetes mellitus

(xi) Galactosemia

(xii) Lactose intolerance

(xiii) Fructose intolerance

(xiv) Diabetic ketoacidosis

(xv) Glucose tolerance test

CHAPTER
9

Chemistry and
Metabolism of
Lipids

Lipid (Greek: lipos, fat) is a heterogeneous group of hydrophobic organic molecules found in cells. They are sparingly soluble in water but are soluble in organic solvents such as acetone, ether, alcohol and chloroform. Lipids, therefore, can be easily separated from other biological materials after their extraction with some organic solvent.

CLASSIFICATION OF LIPIDS

Lipids exhibit greater structural variability and constitute a category of substances that are similar only in that they are largely hydrophobic and soluble in non-polar solvents. They are classified into three groups as simple lipids, compound lipids and derived lipids (Fig. 9.1).

Simple Lipids

Simple lipids are esters of fatty acids with alcohol. These include neutral fats, oils and waxes.

Neutral fats and oils are the mixtures of triacylglycerols (triglycerides) whose fatty acid composition may vary with source. They differ only in that fats are solid while oils are liquid at room temperature.

Fig. 9.1. Classification of lipids.

Neutral fats are obtained from animal sources. They have, comparatively, more number of saturated fatty acids. They also have higher melting points.

On the other hand, oils are obtained from plant seeds. They are usually rich in unsaturated fatty acids and have low melting points.

Esters of long chain fatty acids with high molecular weight monohydroxy aliphatic alcohols are called waxes. They are used in the manufacturing of wax-polish.

Compound Lipids

In addition to fatty acids and alcohol, compound lipids also contain an amphipathic group. For example, lipids conjugated with phosphoric acid are called phospholipids.

Derived Lipids

They are obtained after hydrolysis of simple and compound lipids. They include fatty acids, glycerol, etc.

FATTY ACIDS

Fatty acids are the carboxylic acids with a long hydrocarbon side chain which may be saturated or unsaturated with one or more double bonds.

Major source of the circulating free fatty acids is adipose tissue. Free fatty acids enter plasma after intraplasmic hydrolysis of triacylglycerols in chylomicrons and very low density lipoproteins. Although plasma concentration of free fatty acids is low, they have rapid turnover and are important source of energy for many tissues. Since they have limited solubility in water, they are transported with plasma albumin which has specific binding sites for free fatty acids.

PROPERTIES OF FATTY ACIDS

1. Generally, short chain fatty acids are liquid while long chain fatty acids are solid at room temperature.

2. Fatty acids present in various oils react with alkali and form salts. Salts of various fatty acids are used as soaps and emulsifying agents.

3. Unsaturated fatty acids undergo reduction and are converted to saturated fat. For example, hydrogenation of vegetable oil results in the formation of ghee.

4. Unsaturated fatty acids exhibit cis-trans isomerism due to presence of double bonds.

5. Various fatty acids can be separated from a mixture by gas liquid chromatography.

NOMENCLATURE OF FATTY ACIDS

Nomenclature of a fatty acid may be either according to delta (δ) system or omega (ω) system.

Delta System of Nomenclature

Carbon atoms in a fatty acid are numbered beginning with the carboxyl carbon (−COOH) as carbon number 1. The next carbon, to which the carboxyl group is attached, is designated as carbon number 2. This is also called as α-carbon atom. Accordingly, carbon 3 is referred as the β-carbon, and so on. This system of numbering the carbons is called delta (δ) system of nomenclature.

Omega System of Nomenclature

Carbon atoms are numbered beginning the methyl terminal (CH₃₋) end where a double bond first appears. This carbon atom, i.e. the carbon atom of the terminal CH₃ group is called omega (ω) carbon and this system of nomenclature is called omega (ω) system. Recently, replacement of the symbol “ω” by the letter “n” has also been recommended (Fig. 9.2).

Fig. 9.2. Nomenclature of a fatty acid.

Most of the naturally occurring fatty acids have even number of carbon atoms because they are synthesized from acetyl CoA, i. e. by the combination of two carbon units. They are divided into three groups as short chain fatty acids (containing 4–8 carbons), medium chain fatty acids (with 10–14 carbons) and long chain fatty acids (having 16 or more carbons). They have different physicochemical properties and are metabolized in the body in different ways.

In plants and higher animals, predominant fatty acids are those containing 16 or 18 carbon atoms such as palmitic acid, oleic acid, linoleic acid and stearic acid.

Saturated Fatty Acids

Saturated fatty acids end in word “-anoic”, e.g. octadecanoic acid (stearic acid), a fatty acid with 18 carbon atoms. Saturated fatty acids are highly flexible molecules and can assume a wide range of conformation because there is relatively free rotation around each of their C–C bonds. Saturated fatty acids are commonly found in animal fat, e.g. butter.

Saturated fatty acids of common biological significance are listed in Table 9.1.

Table 9.1. Saturated fatty acids

Unsaturated Fatty Acids

Unsaturated fatty acids end with word “-enoic”, e.g. octadecenoic acid (oleic acid), a fatty acid having 18 carbon atoms and one double bond.

Widely used convention to name an unsaturated fatty acid is to indicate the number of carbon atoms, followed by the number of double bonds with their position in the chain, such as octadecenoic acid (18:1; 9), a fatty acid with 18 carbons and one double bond at carbon-9, i.e. between carbon-9 and 10 (Fig. 9.3).

Fig. 9.3. Oleic acid – an unsaturated fatty acid.

In δ system, number followed by the symbol “Δ” indicates the number of double bonds. This is followed by a superscript, which indicates the number of carbon atom having a double bond. For example, linoleic acid that has 18 carbons and two double bonds at carbons-9 and 12 is written as 18:2 Δ9,12 (Fig. 9.4).

According to ω-system of nomenclature, linoleic acid is designated as ω-6 fatty acid because the double bond that is close to the ω-end is present on the sixth carbon.

Double bonds may be present in cis- or trans-orientation. Naturally occurring fatty acids, predominantly, have cis-double bonds. Unsaturated fatty acids are chemically more reactive than the saturated fatty acids. Fluidity of the lipid increases with the degree of unsaturation.

Polyunsaturated Fatty Acids

Fatty acids obtained from plant seeds usually contain two or more double bonds. These fatty acids are called polyunsaturated fatty acids (PUFAs). In these fatty acids, double bonds generally occur after every three carbon atoms. They are mainly obtained from plants oils, e.g. soybean oil, sunflower oil, groundnut oil, etc.

Fig. 9.4. Linoleic acid.

Physiological Role of PUFAs

1. Three PUFAs, i.e. linoleic acid, linolenic acid and arachidonic acid form essential component of the diet since they are necessary for growth as well as normal health. Hence, they are called essential fatty acids.

2. They are also important constituent of a biological membrane.

3. They are also used for esterification of cholesterol and thus help in its transport and metabolism. Particularly, consumption of the fat containing ω-6 fatty acids helps in lowering plasma cholesterol.

4. Diet rich in ω-3 fatty acids promotes reduction in plasma triacylglycerols.

5. Arachidonic acid also acts as a precursor of eicosanoids such as prostaglandins, thromboxanes and leukotrienes.

TRIACYLGLYCEROLS

Triacylglycerols (also called triglycerides) or neutral fats are fatty acid triesters of glycerol (glycerin) which is a trihydric alcohol.

Glycerol with one molecule of fatty acid is called monoacylglycerol, with two molecules of fatty acids a diacylglycerol and with three molecules of fatty acids a triacylglycerol (Fig. 9.5).

Fig. 9.5. Glycerol and a triacylglycerol.

A molecule of triacylglycerol may contain three similar or dissimilar fatty acids which may be saturated and/or unsaturated. All natural fats and oils, invariably, are mixed triacylglycerols. Plant oils are usually rich in unsaturated fatty acids.

Triglycerides are named according to the presence of the fatty acid residues attached to glycerol, e.g. 1-palmitoyl-2-arachidonoyl-3-stearoylglycerol is a triacylglycerol which has palmitic acid at carbon-1, arachidonic acid at carbon-2 and stearic acid at carbon-3 of glycerol. Generally, with the primary alcoholic group of a triacylglycerol, i.e. at α-positions (at carbon-1 and carbon-3 of glycerol), there are saturated fatty acids while at carbon-2 there is an unsaturated fatty acid.

Triacylglycerols are transported in plasma in chylomicrons and very low density lipoproteins.

PROPERTIES OF TRIACYLGLYCEROLS

1. Triacylglycerol is the storage form of energy in the body. In animals, fat cells (adipocytes) are important for the synthesis and storage of triacylglycerols. Average fat content of a normal human being is about 21% in men and 26% in women. This in turn may allow them to survive for 2–3 months.

2. They are hydrolyzed by lipases to free fatty acids and glycerol (Fig. 9.6).

3. Naturally occurring fat, particularly from animal sources, develop unpleasant odor and taste, if stored for a long period under moist conditions. This is called rancidity. Antioxidants, e.g. vitamin E, can prevent development of rancidity.

Fig. 9.6. Hydrolysis of a triacylglycerol.

4. Unsaturated fatty acids found in triacylglycerol accept halogens such as iodine, at the double bond. This process is termed as halogenation. It is a measure of the degree of unsaturation.

PROSTAGLANDINS

Prostaglandins and the related compounds thromboxanes and leukotrienes are collectively referred to as eicosanoids. They are synthesized from polyunsaturated fatty acids with 20 carbons. Arachidonic acid is the immediate precursor of the predominant class of prostaglandins in man. Prostaglandins exist in virtually every mammalian tissue and act as local hormones.

There are three classes of prostaglandins referred to as A, E and F. They are distinguished by the functional group on the cyclopentane ring. A series has a, β-unsaturated ketone, E series contain β-hydroxy ketone while F series has 1, 3-diol. Subscript indicates number of double bonds in the side chain.

Prostaglandins of series 2 are derived from arachidonic acid and are the principal prostaglandins in man. They are of great significance, biologically. They are natural mediators of inflammation. Synthetic prostaglandins are very effective in inhibiting gastric acid secretion in patients with peptic ulcer. PGE2 and PGF2 have been used to induce parturition. PGI2 inhibits platelet aggregation.

PHOSPHOLIPIDS

Phospholipids are a heterogeneous group of compounds, which contain one or more phosphoric acid residues and a polar group that may be a nitrogenous base, an amino acid or a polyhydroxy-alcohol. In addition, they also contain one or two long chain fatty acids.

They are amphipathic in nature, i.e. they contain both, a hydrophilic head and a long hydrophobic tail.

Hydrophilic domain interacts with the aqueous environment and contains a phosphate group plus an alcohol, e.g. serine, choline, etc.

Hydrophobic domain forms the nonaqueous environment and contains a long chain fatty acid or fatty acid derived hydrocarbon.

PHYSIOLOGICAL ROLE OF PHOSPHOLIPIDS

1. Phospholipids are predominant lipids of a cell membrane. Membrane phospholipids also function as a reservoir for intracellular messengers. They also serve as anchor for some proteins in a cell membrane.

2. Phospholipids are also component of lung surfactants.

3. They are also essential component of the bile and help in regulation of serum cholesterol.

CLASSIFICATION OF PHOSPHOLIPIDS

Phospholipids are classified into several groups (Table 9.2):

Table 9.2. Classification of phospholipids

Glycerophospholipids

• Lecithin (Phosphatidylcholine)

• Cephalins (Phosphatidylethanolamine and phosphatidylserine)

• Phosphatidylinositols

• Cardiolipins and plasmalogens

Sphingolipids

• Sphingomyelins

• Cerebrosides

• Gangliosides

Glycerophospholipids

Phospholipids which are the derivatives of glycerol are called glycerophospholipids or phosphoglycerides. They are major components of a biological membrane. They are the derivatives of phosphatidic acid.

Phosphatidic acid consists of a molecule of glycerol, two fatty acids and a molecule of phosphoric acid (Fig. 9.7).

Fig. 9.7. Phosphatidic acid.

When phosphoric acid is attached at carbon 3 of glycerol, it is referred to as glycerol-3-phosphate. Its carbon-1 and 2 are esterified with two fatty acids. Usually, a saturated fatty acid is attached at carbon-1 (α-carbon) while commonly a polyunsaturated fatty acid is attached at carbon-2 (β-carbon) of glycerol.

Phosphoryl group of phosphatidic acid may be linked either to a nitrogenous compound such as ethanolamine, choline, serine or to some other group like inositol. Various glycerophospholipids include phosphatidylethanolamine (cephalin), phosphatidylcholine (lecithin), phosphatidylinositol, etc.

Lecithins

Lecithins contain choline (a nitrogenous base) at carbon-3. They are the surface active agents and help in emulsification of fat. They are widely distributed in the brain, nerve cells, sperm and egg yolk. Various hydrolytic enzymes, known as phospholipases, act at different sites of glycerophospholipids and hydrolyze them. For example, phospholipase A₂ removes a fatty acid from carbon-2 and forms lysophospholipid, called lysolecithin (Fig. 9.8).

Fig. 9.8. Lecithin and lysolecithin.

Cephalins

Cephalins are glycerophospholipids where (instead of choline present in the lecithin) either ethanolamine or serine is present as a nitrogenous base. Cephalins are present in erythrocyte membrane, brain and many other tissues.

Phosphatidylinositols

Phosphatidylinositols are the glycerophospholipids containing inositol instead of a nitrogenous base. They are found mainly in the brain.

Plasmalogens

Plasmalogens are the glycerophospholipids in which carbon-1 of glycerol is linked to an unsaturated fatty acid by an ether linkage (in the cis-configuration) rather than a saturated fatty acid linked by the ester linkage. Plasmalogens are present in cardiac and skeletal muscles and semen.

Sphingolipids

Sphingolipids do not contain glycerol but are the derivatives of an unsaturated amino alcohol which is referred to as sphingosine. Sphingolipids have 18 carbon atoms and one double bond.

N-Acyl derivatives of sphingosine are called ceramides. They are the parent compounds of most of the sphingolipids, e.g. sphingomyelins, cerebrosides and gangliosides.

Sphingomyelin: Sphingomyelins are ceramides which contain either phosphocholine or phosphoethanolamine, and are called sphingo-phospholipids. Myelin sheath, that surrounds and electrically insulates many nerve cell axons, is rich in sphingomyelin.

Cerebrosides

Cerebrosides are glycosphingolipids. They contain ceramide which is attached to a sugar residue, such as a galactose or glucose. Hence, cerebrosides are ceramide monosaccharides. Most prevalent cerebrosides are galacto-cerebrosides and glucocerebrosides.

A number of the cerebrosides are found in white matter of the brain and in the myelin sheath of nerves. They are named with respect to the presence of the fatty acid, e.g. a galactosyl-ceramide called cerebron contains cerebronic acid. Similarly, nervon has an unsaturated derivative of lignoceric acid which is referred to as nervonic acid.

Gangliosides

Gangliosides are the most complex glycolipids. They are ceramides that contain oligosaccharides including hexosamines (glucosamine or galactosamine) and at least one molecule of N-acetylneuraminic acid (NANA or sialic acid).

Gangliosides are present in large amount in ganglion cells of the central nervous system, particularly, in the nerve endings. More than 60 gangliosides are known to occur. Each one of it has different oligosaccharide head groups. Gangliosides are designated as GM₁, GM₂, GM₃, etc.

STEROIDS

The most common steroid is cholesterol. It is a derivative of cyclopentanoperhydrophen-anthrene ring also called steroid nucleus, which consists of four fused, non-polar rings that are labelled as A, B, C, and D.

Cholesterol is a most abundant steroid found in animals. It has 27 carbons, a hydroxyl group at carbon 3, a double bond between carbons 5 and 6, two methyl groups at carbons 18 and 19, and a side chain at carbon 17 (Fig. 9.9).

Fig. 9.9. Structure of the steroid ring and cholesterol.

Because of the presence of the OH group, cholesterol is called a steroid alcohol (sterol). Due to the presence of this group cholesterol can form an ester with a long chain fatty acid and form cholesterol esters.

USES OF LIPIDS

1. Lipids are important dietary constituents of food due to their high calorific value.

2. Dietary lipid is also a source of fat soluble

vitamins and essential fatty acids.

3. Lipids also serve as energy stores, e.g. fat stored in adipose tissue.

4. Fat present in the subcutaneous tissue acts as an insulating material.

5. Lipids, in the form of a bilayer, together with proteins are important components of biological membranes.

6. Lipids are also involved in signaling events.

METABOLISM OF LIPIDS

DIGESTION AND ABSORPTION OF LIPIDS

Digestion and Absorption of Triacylglycerols and Phospholipids

Digestion of Triacylglycerols and Phospholipids

Digestion of triglycerides is initiated in the mouth with chewing and by the action of the lingual lipase. Thereafter, with the action of gastric lipase, and churning and contraction by the stomach, nearly 30% of the dietary triglycerides get degraded to di-glycerides and free fatty acids, with in 2 to 3 hours. Contents from the stomach pass into the small intestine. Presence of the fat in the small intestine stimulates the release of two hormones, i.e. cholecystokinin (CCK) and secretin from the duodenal cells. CCK signals the gallbladder to contract and release bile, down the bile duct and into the duodenum. Bile contains large quantity of bile salts and lecithin. Secretin signals the pancreas to release pancreatic juice which is rich in pancreatic lipase.

Bile emulsifies fat and breaks fat globules (clumps of triglycerides) into small pieces and keep them suspended in solution. This in turn increases total surface area for the action of pancreatic lipase. Pancreatic lipase hydrolyzes all the accessible triglycerides, within minutes, into monoglycerides and free fatty acids. Bile salts surround the hydrolyzed products of fat digestion and form water soluble globules with a fatty core, called micelles. Micelles transport monoglycerides and free fatty acids through the watery intestinal environment to the brush border of the intestinal mucosal cells for absorption.

Similarly, phospholipases hydrolyze phospholipids and release monoglycerides, free fatty acids, phosphate and a nitrogenous substance (Table 9.3).

Table 9.3. Various enzymes catalyzing the digestion of lipids

Enzyme | Site of action | Action of the enzyme
Lingual lipase (important in neonates) | Mouth | Triglycerides → Fatty acids, monoglycerides, glycerol
Lipase/Colipase | Pancreas | Triglycerides → Fatty acids, monoglycerides, glycerol
Phospholipase A/B | Pancreas | Lecithin → Fatty acid, lysolecithin
Cholesterol esterase | Pancreas | Cholesterol esters → Cholesterol, fatty acids
Retinyl ester hydrolase | Pancreas | Retinyl esters → Retinol, fatty acids
Monoglyceride lipase | Small intestine | Monoglycerides → Glycerol, fatty acids
Lecithinase | Small intestine | Lecithin → Fatty acids, glycerol, phosphoric acid, choline

Absorption of Triacylglycerols and Phospholipids

Most fat absorption takes place in the duodenum or jejunum of the small intestine.

Micelles carry monoglycerides and long chain fatty acids to the surface of the microvilli in the brush border and diffuse into the intestinal cells. The unabsorbed bile salts return to the interior of the small intestine for the transport of more monoglycerides and fatty acids. Bile salts are absorbed in the ileum and return, via the portal vein, to the liver where they are again secreted into the bile. This recycling pathway for bile, i.e. from the liver to the intestine and from the intestine to the liver is called enterohepatic circulation.

As monoglycerides and fatty acids pass into the intestinal cells, they reform triglycerides. Most of the triglycerides as well as cholesterol and phospholipids combine with apoproteins and form chylomicrons. Chylomicrons make their way to the central lacteal of the villi where they enter the lymph system, to be propelled through the thoracic duct, and emptied into vein in the neck (Fig. 9.10).

Fig. 9.10. Absorption of lipids in the intestine.

Glycerol and medium and short chain fatty acids are absorbed directly into the blood stream. Dietary fat begins to appear in the blood stream after 1 to 2 hrs of meal, with a peak after 3 to 5 hrs. It is generally cleared by 10 hrs.

Absorption of fat is drastically reduced in the absence of bile salts. As a result of it, bulk of the unabsorbed fat is excreted in the stool. The production of stool with an abnormally high amount of fat is referred to as steatorrhea. Steatorrhea may follow diseases of malabsorption such as cystic fibrosis, radiation therapy or digestive tract surgery. In such conditions triglycerides with medium chain fatty acids are often given in the diet. This is due to the reason that medium chain fatty acids are comparatively more water soluble than long chain fatty acids and thus can be emulsified more readily. Breast milk is a rich source of medium and short chain fatty acids.

Digestion and Absorption of Sterols

Sterols remain, mostly, unchanged by digestion. Their absorption is also relatively poor. Cholesterol gets esterified prior to its absorption. Dietary fat increases absorption of cholesterol where as plant sterols and dietary fiber decrease cholesterol absorption.

METABOLISM OF FATTY ACIDS

Breakdown of fatty acids

Major pathway for the catabolism of fatty acids occurs in the mitochondria. This pathway is called β-oxidation and was proposed by Knoop.

β-Oxidation

In this process, oxidation of fatty acids occurs at the β-carbon atom (carbon-3) and two carbon fragments are successively removed from the carboxyl end of fatty acyl CoA. This in turn results in the elimination of two terminal carbon atoms as acetyl CoA, thereby leaving fattyacyl CoA that has two carbons less than the original fatty acid (Fig. 9.11).

Activation of Fatty Acid

A long chain fatty acid (fatty acid containing >12 carbons) enters a cell where it is converted to its CoA derivative, by the enzyme long-chain fatty acyl CoA synthetase (thiokinase) in the cytosol.

Transport of Active Fatty Acid into Mitochondria

The process of β-oxidation occurs in the mitochondrial matrix. Thus, fatty acid must be transported across the mitochondrial membrane. Short chain fatty acids can cross the inner mitochondrial membrane and are activated to their CoA derivatives within the mitochondria. However, it is impermeable to a long chain fatty acid. A long chain fatty acid is transported, from cytosol to the mitochondrial matrix, with the help of a specialized carrier called carnitine. This process is called carnitine shuttle (Fig. 9.12).

Fig. 9.12. Role of carnitine in the transport of a fatty acid.

In this process, an acyl group is first transferred from the cytosolic CoA to carnitine, by the enzyme carnitine palmitoyl transferase-I (CPT-I). This enzyme is associated with the outer mitochondrial membrane and is also referred to as carnitine acyl transferase-I (CAT-I). This reaction forms acylcarnitine and releases CoA. Thereafter, acyl carnitine is transported into the mitochondrial matrix, by a protein called carnitine acyltranslocase. Finally, CPT-II (CAT-II), which is present in the inner mitochondrial membrane, catalyzes transfer of the acyl group, from acylcarnitine to CoA, into the mitochondrial matrix and releases carnitine.

Fig. 9.11. The process of β-oxidation of a fatty acid.

Carnitine is obtained from meat. A small amount of it can also be synthesized in the liver and kidney, from lysine and methionine. Carnitine deficiency can be observed in patients with a liver disease, malnutrition or in strict vegetarians. Congenital deficiency of CPT-I or CPT-II may also be observed. Such a deficiency can result in the decreased ability to use long chain fatty acids by the tissues.

The Process of β-oxidation

The process of β-oxidation consists of four reactions, which result in shortening of the fatty acid chain by two carbons. It includes:

1. Oxidation: After activation of the fatty acid such as palmitic acid, and its entry into the mitochondrial matrix, flavoprotein containing acyl CoA dehydrogenase removes two hydrogens from the α- and β-positions of the fatty acyl CoA and produces FADH₂. The fatty acyl CoA is changed to α, β-unsaturated fatty acyl CoA (enoyl CoA).

2. Hydration: In the next step, enoyl CoA hydratase adds a molecule of water at the double bond and converts the enoyl CoA to β-hydroxyacyl CoA.

3. Oxidation: Thereafter, β-hydroxyacyl CoA dehydrogenase oxidizes β-hydroxyacyl CoA to β-ketoacyl CoA.

4. Cleavage: In the last step, acyl CoA: acyltransferase, also called thiolase, in the presence of CoA results in thiolytic cleavage of the β-ketoacyl CoA into acetyl CoA and fattyacyl CoA which has two carbons less than the original fattyacyl CoA.

These four steps are repeated, i.e. the fattyacyl CoA again undergoes above four sets of reactions, starting with acyl CoA dehydrogenase. Each cycle produces acetyl CoA, FADH₂ and NADH. Thus, the fatty acid is sequentially degraded to acetyl CoA.

SUDDEN INFANT DEATH SYNDROME (SIDS)

Medium-chain fatty acyl CoA dehydrogenase deficiency leads to an autosomal recessive disorder, which is referred to as sudden infant death syndrome (SIDS) or Reye’s syndrome. This in turn reduces fatty acid oxidation. Features of the disease include vomiting, lethargy and coma, accompanied with the hypoketotic hypoglycemia and dicarboxylic aciduria. Symptoms appear after more than 12 hours of fast and are more complicated on prolonged starvation, usually with in two years of life. Treatment includes a carbohydrate rich diet.

Energy Production from β-Oxidation

When a molecule of palmitic acid (C₁₅H₃₁COOH) undergoes β-oxidation, it releases 8 molecules of acetyl CoA in seven rounds. In every round, one molecule, each of FADH₂ and NADH + H⁺ are produced, which in turn generate 2 and 3 ATP, respectively.

Thus, a total of 35 ATP are produced during this process. In addition, each molecule of acetyl CoA, when oxidized via the citric acid cycle, produces 12 ATP. Therefore, additionally, 96 ATP are produced from 8 molecules of acetyl CoA. Hence, a total of 131 ATP are produced from one molecule of palmitic acid.

As two high energy phosphate bonds are hydrolyzed from one molecule of ATP, which is changed to AMP, thus a total of 2 ATP are used during activation of the fatty acid.

Therefore, there is a net yield of 129 ATP when one molecule of palmitic acid (fatty acid with 16 carbons) is completely oxidized.

Oxidation of Fatty Acids with Odd Number of Carbon Atoms

β-Oxidation of a saturated fatty acid with odd number of carbon atoms proceeds in the same way as a saturated fatty acid with even number of carbons, until final three carbons are reached. Thus, in addition to acetyl CoA, it also produces a molecule of propionyl CoA (containing 3 carbons), which is used as follows:

1. Firstly, propionyl CoA is carboxylated by the enzyme propionyl CoA carboxylase, to D-methylmalonyl CoA. This enzyme requires biotin (Fig. 9.13).

Fig. 9.13: Conversion of propionyl CoA to succinyl CoA.

2. D-Methylmalonyl CoA is then converted to L-methylmalonyl CoA, by the enzyme methylmalonyl CoA racemase.

3. Finally, L-methylmalonyl CoA is converted to succinyl CoA by the enzyme methylmalonyl CoA mutase. This enzyme requires deoxyadenosylcobalamin (coenzyme form of vitamin B₁₂).

Oxidation of Unsaturated Fatty Acids

Unsaturated fatty acids are activated, transported across the inner mitochondrial membrane and undergo β-oxidation, by the process similar to that of a saturated fatty acid. However, since the double bonds of the naturally occurring unsaturated fatty acids are found in cis-configuration (compared to those produced during β-oxidation, which have trans-configuration) hence, three additional enzymes are required for their oxidation. These include:

1. Enoyl CoA isomerase,

2. Dienoyl CoA reductase, and

3. 3,2-Enoyl CoA isomerase

Thus, in addition to the other enzymes of β-oxidation, these three enzymes are also required for the oxidation of the unsaturated fatty acids (Fig. 9.14).

Fig. 9.14. Oxidation of an unsaturated fatty acid.

ALTERNATE OXIDATIVE PATHWAYS

α-Oxidation of Fatty Acids

Fatty acids are also oxidized by another process, called α-oxidation, in microsomes, in the liver and brain.

During α-oxidation, a fatty acid is metabolized by α-hydroxylation, followed by dehydrogenation and decarboxylation (Fig. 9.15).

Fig. 9.15. The process of α-oxidation of a fatty acid

In this process, there is sequential removal of a carbon atom as CO₂, from the carboxyl end of the fatty acid. This process neither produces CoA intermediates nor generates ATP.

ω-Oxidation of Fatty Acids

In this process, oxidation of the medium and long chain fatty acids occurs from both the ends.

Firstly, the end methyl group (–CH₃) of the acyl chain, called w-carbon, is oxidized and forms a dicarboxylic acid. This reaction is catalyzed by the enzyme w-hydroxylase, which requires cytochrome P₄₅₀.

REFSUM DISEASE

Genetic deficiency of α-hydroxylase enzyme (a monooxygenase) results in an increase in phytanic acid in the serum and its accumulation in several tissues. This in turn leads to a neurological disorder called Refsum disease.

This dicarboxylic acid thereafter, under-goes β-oxidation from both the ends, simultaneously (Fig. 9.16).

Fig. 9.16. The process of ω-oxidation of a fatty acid.

METABOLIC FATES OF ACETYL COA

Acetyl CoA is mainly used in the Krebs cycle.

Besides Krebs cycle, acetyl CoA can also be used for the synthesis of fatty acids and cholesterol. A small quantity of it is also converted to ketone bodies in the liver (Fig. 9.17).

Fig. 9.17. Metabolic fates of acetyl CoA.

SYNTHESIS OF FATTY ACIDS

Mammals can synthesize a major portion of the saturated as well as monounsaturated fatty acids. First, a saturated, straight chain, 16 carbon containing fatty acid, i.e. palmitic acid is synthesized, which is subsequently changed to other fatty acids.

The process of fatty acid synthesis is called de novo synthesis. In human beings, fatty acid synthesis occurs, primarily in the cytosol, in the liver, lactating mammary glands and to a lesser extent in the adipose tissue.

DE NOVO SYNTHESIS

Acetyl CoA, which is produced in the mitochondria, first comes out of the mitochondria to the cytosol, where fatty acid synthesis occurs. Mitochondrial membrane, however, is impermeable to CoA. Thus, first step in fatty acid synthesis is the transport of the acetate units, from mitochondria to the cytosol. For this purpose, acetyl CoA condenses with oxaloacetate and forms citrate. Citrate is then translocated from mitochondria to the cytosol, where it is cleaved by the enzyme ATP-citrate lyase and releases acetyl CoA and oxaloacetate, in the cytosol (Fig. 9.18).

Fig. 9.18. Transport of acetyl CoA from mitochondria to cytosol.

Conversion of Acetyl CoA to Malonyl CoA

Acetyl CoA is used in fatty acid synthesis after its carboxylation and conversion to malonyl CoA. In the cytosol, acetyl CoAis carboxylated tomalonyl CoA by a biotin-containing enzyme, referred to as acetyl CoA carboxylase. This coenzyme is covalentlylinkedtothelysylresidueoftheenzyme, which requires ATP and CO₂ (as HCO₃⁻).

Prolonged consumption of high calorie diet increases synthesis of the enzyme acetyl CoA carboxylase while fasting or low calorie diet decreases its synthesis.

Synthesis of Palmitic Acid

An enzyme referred to as fatty acid synthase catalyzes the remaining reactions of fatty acid synthesis, in eukaryotes. It is a multifunctional, dimeric enzyme, composed of the two identical subunits. Its each monomer is a multicatalytic polypeptide with seven different enzyme activities plus a domain that covalently binds a molecule of 4’-phosphopantotheine. During fatty acid synthesis, 4’-phosphopantotheine carries acetyl and acyl units on its terminal thiol (–SH) group.

In prokaryotes, fatty acid synthase is a multienzyme complex and the 4’-phosphopantotheine domain is present on a separate protein, referred to as acyl carrier protein (ACP).

Individually, different enzyme activities of the fatty acid synthase complex are designated as acetyl CoA-ACP acetyl transacylase, malonyl CoA-ACP transacylase, β-ketoacyl-ACP synthase (condensing enzyme), β-ketoacyl ACP reductase, β-hydroxyacyl ACP dehydra-tase and enoyl-ACP reductase. These enzyme activities are separate catalytic domains that are present on each subunit of the multicatalytic fatty acid synthase complex.

Fatty acid synthase contains two-SH groups, called pantotheinyl-SH group, which is referred to as the ACP-SH, and the thiol-SH group that is designated as the CYS-SH.

1. Fatty acid synthesis starts with the transfer of the molecule of acetate, from acetyl CoA to the ACP-SH. This reaction is catalyzed by the acetyl CoA-ACP acetyl transacylase domain of the fatty acid synthase complex.

This two-carbon fragment is transferred to the CYS-SH, which is a temporary holding site.

2. At the same time, malonyl CoA-ACP transacylase domain of the fatty acid synthase transfers three carbon malonate unit from malonyl CoA to the vacant -SH group of ACP.

3. In the next step, malonyl group loses CO₂ and transfers the acetyl group, by b-ketoacyl ACP synthase domain of the fatty acid synthase, which in turn causes condensation of the two acetyl group on the CYS–SH and forms a four carbon acetoacetyl unit that is attached to the ACP domain.

4. β-Ketoacyl ACP reductase domain of the fatty acid synthase reduces keto group of the acetoacetyl-S-ACP to β-hydroxybutyryl-S-ACP (β-hydroxyacyl-S-ACP).

5. β-Hydroxyacyl-ACP dehydratase domain of the fatty acid synthase removes a molecule of H₂O from β-hydroxyacyl-S-ACP and introduces a double bond in it, to form crotonyl-S-ACP (α,β-unsaturated acyl-S-ACP).

6. Crotonyl-S-ACP is reduced by the enoyl-ACP reductase domain of the fatty acid synthase and is converted to butyryl-S-ACP (acyl-S-ACP).

As a result of these reactions, a four carbon compound is formed whose three terminal carbons are fully saturated and remain attached to ACP.

These steps are again repeated, beginning with the transfer of the butyryl chain to the CYS-SH. This cycle of the reactions is repeated five more times, each time incorporating two carbon units, that are derived from malonyl CoA and are attached to the growing fatty acid chain at the carboxyl end. When the chain length reaches 16 carbons, the process is terminated with the formation of palmitoyl-S-ACP. Thereafter, palmitoyl thioesterase cleaves the thioester bond with the release of palmitate (Fig. 9.19).

Reducing equivalents (NADPH + H⁺), which are used in the biosynthesis of fatty acids, are derived from the pentose phosphate pathway.

Regulation of Fatty Acid Synthesis

The rate-limiting step in the synthesis of fatty acids is the conversion of acetyl CoA to malonyl CoA. This reaction is catalyzed by the enzyme acetyl CoA carboxylase which is stimulated by citrate.

Citrate, allosterically, activates acetyl CoA carboxylase, while a long chain fatty acyl CoA acts as an allosteric inhibitor.

Insulin also stimulates activity of this enzyme. A high carbohydrate diet increases production of acetyl CoA, which in turn increases the citrate pool. Thus, a high carbohydrate diet leads to increased fatty acid synthesis. Insulin also reduces cAMP level, inhibits activation of the hormone sensitive lipase and favors lipogenesis.

On the other hand, glucagon increases cAMP level, activates lipase activity and inhibits fatty acid synthesis. Glucagon thus, promotes release of fatty acids from the adipose tissue (lipolysis) and opposes lipogenesis.

Fatty acid synthase is the other key enzyme of the palmitate biosynthesis. A high carbohydrate diet or a fat free diet stimulates fatty acids synthase activity. On the other hand, high fat diet, fasting and glucagon decrease enzyme synthesis.

Phosphorylated sugars, e.g. glucose-6-phosphate, also activate this enzyme.

Fig. 9.19: The process of De novo synthesis of palmitic acid.

SYNTHESIS OF OTHER FATTY ACIDS

Palmitate, produced by the fatty acid synthase catalyzed reactions can be modified by:

• Chain elongation, and/or

• Desaturation.

Chain Elongation

Chain elongation of a fatty acid occurs in the mammalian system by the addition of two carbon units in the mitochondria and the endoplasmic reticulum.

In the Mitochondria

Chain elongation in the mitochondria takes place by the successive addition of acetyl CoA, as two carbon units, where both NADH as well as NADPH can be used as a source of the reducing equivalents.

This is the process of the reversal of β-oxidation with the exception that NADPH-linked enoyl CoA reductase replaces FAD-linked acyl CoA dehydrogenase.

Firstly, acyl CoA condenses with acetyl CoA and forms β-ketoacyl CoA, which is subsequently reduced to β-hydroxyacyl CoA. Removal of a molecule of water converts β-hydroxyacyl CoA to α,β-unsaturated fattyacyl CoA (enoyl CoA), which is finally reduced to the higher member of the fatty acid (Fig. 9.20)

Fig. 9.20. Chain elongation of a fatty acid in mitochondria.

In the Endoplasmic Reticulum

The process of chain elongation in the endoplasmic reticulum is similar to the process of de novo synthesis of fatty acid, as occur in the cytosol. It utilizes malonyl CoA as a source of two carbon unit but elongation occurs as a CoA derivative of the fatty acid rather than the ACP-derivative.

In most of the tissues, chain elongation in the endoplasmic reticulum converts palmitate to stearate.

Brain, however, has additional elongation capabilities, allowing it to produce very long chain fatty acids (up to 24-carbon atoms). These fatty acids are required for the synthesis of complex lipids, with in the brain.

Desaturation

Desaturation of a fatty acid (addition of the cis double bond) occurs in higher animals, in the endoplasmic reticulum, by the enzyme desaturases, which are the mixed function oxidases. Desaturation reaction requires NADPH and O₂ (Fig. 9.21).

Fig. 9.21. Desaturation of a fatty acid.

Several polyunsaturated fatty acids can be synthesized through additional desaturation and elongation. Human beings, however, lack the ability to introduce a double bond prior to carbon-9, i.e. between carbon-9 and the ω-carbon of the chain.

SOURCES OF FAT DEPOTS

The hydrolyzed products of lipids absorbed by enterocytes migrate to the endoplasmic reticulum where resynthesis of complex lipids takes place. The fatty acids are stored in the liver and the adipose tissue (fat depot) as triacylglycerols.

METABOLISM OF TRIACYLGLYCEROLS

Synthesis of Triacylglycerols

Fatty acids are stored in the liver and adipose tissue in collaboration with glycerol, as triacylglycerols. The process of biosynthesis of triacylglycerol is called lipogenesis.

Lipogenesis occurs mainly in the liver and the adipose tissue. It requires activation of free fatty acid and α-glycerolphosphate and the re-esterification of glycerolphosphate (Fig. 9.22).

Fig. 9.22. The process of synthesis of triacylglycerols.

1. Activation of Fatty acid: Free fatty acid, in the presence of ATP and CoA, is activated to fatty acyl CoA (acyl CoA) by the family of fatty acyl CoA synthetases (thiokinases).

2. Synthesis of glycerol phosphate: There are two pathways for the production of glycerol phosphate:

(i) In the liver and the adipose tissue, L-α-glycerol-3-phosphate is formed by the reduction of dihydroxyacetone phosphate, an intermediate of the glycolytic pathway. This reaction is catalyzed by glycerol-3-phosphate dehydrogenase.

(ii) Alternatively, in the tissues such as liver, lactating mammary glands, kidney and intestinal mucosa, glycerol is converted directly, to L-α-glycerol-3-phosphate, by the enzyme glycerol kinase.

3. Synthesis of triacylglycerol: After activation of the fatty acid and the synthesis of L-α-glycerolphosphate, triacylglycerol is synthesized by the re-esterification of L-α-glycerol phosphate:

(i) Firstly, a molecule of acyl CoA combines with α-glycerolphosphate and forms 1-acylglycerol-3-phosphate (lysopho-sphatidic acid). This reaction is catalyzed by the enzyme glycerol-3-phosphate acyltransferase.

(ii) Thereafter, 1-acylglycerol-3-phosphate is converted to 1,2-diacylglycerolphosphate (phosphatidic acid) by the enzyme 1-acylglycerol-3-phosphate acyltransferase, also called lysophosphatidyl-acyltransferase.

(iii) Phosphatidic acid is then hydrolyzed to 1, 2-diacylglycerol by the enzyme phosphatidic acid phosphohydrolase, also called phosphatidic acid phosphatase.

In many tissues such as intestine, liver, kidney, aorta and lactating mammary glands, especially in the intestinal mucosa, 1, 2–diacylglycerol is also formed from 2-monoacylglycerol, since monoacylglycerol is directly absorbed in the intestinal mucosa. This reaction is catalyzed by the enzyme monoacylglycerol acyltransferase.

(iv) Finally, diacylglycerol is esterified to form triacylglycerol. This reaction is catalyzed by the enzyme diacylglycerol acyltransferase.

Generally, palmitic acid is present at position 1 while oleic acid may be found at positions 2 and 3 of the triacylglycerol, in adipose tissue, in human beings.

Breakdown of Triacylglycerols

Only a little amount of triacylglycerol is stored in the liver. Most of it is exported, packed with cholesteryl esters, cholesterol, phospholipids and apolipoproteins, as very low density lipoprotein.

Triacylglycerol is stored in adipose tissue, in the cytosol of the cell. It serves as depot fat, which is ready for mobilization, when body requires it as fuel.

Fatty acids that are stored in the adipose tissue in the form of triacylglycerol are mainly oxidized for energy. Mobilization of the stored fat requires hydrolytic release of the fatty acids and glycerol, from triacylglycerols. The enzyme called hormone-sensitive lipase, which removes fatty acids from carbon-1 and/or 3 of a triacylglycerol, initiates this process. Additional lipases, which are specific for diacylglycerols and monoacylglycerols, remove the remaining fatty acids (Fig. 9.23).

Fig. 9.23. Breakdown of triacylglycerols.

Utilization of these fatty acids, however, varies from tissue to tissue, depending upon the metabolic status of the tissue. Impairment in fatty acid oxidation may lead to hypoglycemia.

ROLE OF LIVER IN LIPID METABOLISM

1. Liver is the primary tissue for de novo synthesis of fatty acids. This pathway occurs in the absorptive period because dietary calorie intake exceeds energy expenditure and fatty acid synthesis is favoured by the availability of substrate.

2. Triacylglycerol synthesis is favoured in the liver because fatty acyl CoA is available, both from de novo synthesis from acetyl CoA and from hydrolysis of the triacylglycerols component of chylomicron remnants, which are removed from the blood by hepatocytes.

3. The liver packages triacylglycerols into VLDL particles that are secreted into blood for use by extrahepatic tissues, particularly adipose tissue and muscle. VLDLs are produced in the liver. They are composed pre-dominantly of triacylglycerols (approximately 60%) and their function is to carry this lipid from the liver to the peripheral tissues. VLDLs are secreted directly into the blood by the liver as nascent VLDL particles containing apo B-100.

4. Chylomicron remnants are rapidly removed from the circulation by the liver whose cell membrane contains lipoprotein receptors that recognize apo E. Chylomicron remnants bind to these receptors and are taken into the hepatocytes by receptor-mediated endocytosis. Their cholesteryl esters and triacylglycerols are hydrolysed and metabolised in the liver.

5. Liver, in addition to intestine, adrenal cortex, and reproductive tissues, makes the largest contribution to the body’s cholesterol pool. Liver also plays a central role in the regulation of the body’s cholesterol homeostasis. For example, cholesterol enters the liver pool and is eliminated from the liver as free cholesterol in the bile or converted to bile salts that are secreted into the intestinal lumen. It also serves as a component of plasma lipoproteins, which are transported to the peripheral tissues.

6. Bile acids are synthesized in the liver. The most common resulting compounds are cholic acid and chenodeoxycholic acid, called primary bile acids. Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine. These new structures are called bile salts. Bile salts are more effective detergents than bile acids because of their enhanced amphipathic nature. Bile salts provide the only significant mechanism for cholesterol excretion, both as a metabolic product of cholesterol and as a solubilizer of cholesterol in bile.

7. The oxidation of fatty acids derived from adipose tissue is the major source of energy in hepatic tissue, during prolonged fasting.

8. The liver is also unique to synthesize and release ketone bodies (primarily 3-hydroxy-butyrate) to the blood, for use as fuel by peripheral tissues.

FATTY LIVER

Fatty liver, also known as fatty liver disease (FLD) or steatosis hepatitis, is a reversible condition where large vacuoles of triacylglycerols accumulate in liver cells via the process of steatosis. Despite having multiple causes, fatty liver can be considered a single disease that occurs worldwide in those with excessive alcohol intake and who are obese (with or without effects of insulin resistance). The condition is also associated with other diseases that influence fat metabolism. Eating fatty food does not, by itself, produce fatty liver.

Though fatty liver is commonly associated with alcohol intake or metabolic syndrome (diabetes, hypertension and dyslipidemia) but can also be due to any one of these causes which include metabolic (abetalipoproteinemia, glycogen storage diseases, acute fatty liver of pregnancy, lipodystrophy); nutritional (malnutrition, severe weight loss, gastric bypass); drugs and toxins (methotrexate, glucocorticoids, environmental hepatotoxins, e.g. phosphorus, toxic mushroom); or other causes (inflammatory bowl disease, HIV).

The diagnosis of steatosis is made when fat in the liver exceeds 5–10% by weight. The patient may have an enlarged liver or minor elevation of liver enzyme tests.

The treatment of fatty liver is related to the cause. It is important to remember that simple fatty liver may not require treatment. The benefit of weight loss, dietary fat restriction and exercise in obese patients is inconsistent. Reducing or eliminating alcohol intake can improve fatty liver due to alcohol toxicity. Controlling blood sugar may reduce the severity of fatty liver in patients with diabetes.

Lipotropic factors

A lipotropic factor is defined as a substance which prevents or removes an accumulation of excess of fat in the liver. These include choline, betaine and other related compounds, choline precursors, such as methionine and inositol. In addition to playing important role in the mobilization and utilization of dietary fat, lipotropic factors act as the body’s natural emulsifiers, holding blood lipids in solution and resisting lipid deposition within the cardiovascular system. Sufficient lipotropic factors can also keep homocysteine levels in check to support cardiovascular health, including the health of the arterial walls and blood lipids.

The body is able to synthesize lipotropic factors when given all the ingredients, i.e. choline, inositol, betaine, folic acid and B vitamins.

METABOLISM OF KETONE BODIES

Acetoacetate, β-hydroxybutyrate and acetone are collectively referred to as ketone bodies (acetone bodies).

FORMATION OF KETONE BODIES

The process of the formation of ketone bodies is called ketogenesis.

In a normal man, concentration of the ketone bodies is usually less than 3 mg/100 ml of blood. In certain conditions such as during prolonged starvation or impaired glucose oxidation, like diabetes mellitus, when fat becomes the source of energy and lipolysis is accelerated, there is increased production of acetyl CoA. This, in turn, can not be fully utilized through Krebs cycle (due to lack of oxaloacetate) and is converted to ketone bodies.

Clinical condition resulting from the increased synthesis of ketone bodies is called ketosis. They are diffused into the blood. Increased concentration of ketone bodies in the blood is called ketonemia. This in turn also results in their increased excretion in the urine, which is referred to as ketonuria.

Formation of ketone bodies occurs in the mitochondria, in the liver since liver has a highly active system for the production of acetoacetate, which is the principal ketone body.

Firstly, 2 molecules of acetyl CoA condense and form acetoacetyl CoA, which further reacts with one more molecule of acetyl CoA and forms HMG CoA. Thereafter, HMG CoA lyase splits HMG CoA to acetyl CoA and acetoacetate (Fig. 9.24).

Fig. 9.24. Formation of acetoacetate.

Acetoacetate is the parent compound that is synthesized in the body. In the mitochondria, a part of it is reduced by NADH-dependent β-hydroxybutyrate dehydrogenase, to β-hydroxybutyrate. Some of the acetoacetate is also spontaneously decarboxylated, by a non-enzymatic reaction, to acetone (Fig. 9.25).

Fig. 9.25. Conversion of acetoacetate to other ketone bodies.

UTILIZATION OF KETONE BODIES

Acetoacetate and β-hydroxybutyrate, which are produced by the liver, can be used as a source of energy in the peripheral tissues such as kidney and muscle. Although glucose is the major fuel for brain in the well nourished state but during starvation even brain starts utilizing ketone bodies.

The process of the oxidation of ketone bodies is called ketolysis. It includes:

KETOSIS

Both, acetoacetate and b-hydroxybutyrate are the strong acids. Slowly they deplete the alkali reserve of the body and cause metabolic acidosis. Hence, this condition is also called ketoacidosis.

Ketosis occurs in severe diabetes, starvation or on high fat, low carbohydrate diet.

Under normal conditions, acetone formation is negligible but when acetoacetate accumulates, such as in severe diabetic ketoacidosis, the amount of acetone in the blood increases to the extent that it can be detected in the breath of the patient.

In the patients with insulin dependent diabetes mellitus, major metabolic derangements include marked hyperglycemia, ketonemia and ketonuria. It may also be associated with water and electrolyte imbalance.

Oxidation of β-Hydroxybutyrate

A mitochondrial enzyme, β-hydroxybutyrate dehydrogenase, oxidizes β-hydroxybutyrate to acetoacetate.

Oxidation of Acetoacetate

Acetoacetate is oxidized after its activation to acetoacetyl CoA. Since liver does not contain this enzyme, ketone bodies can not be oxidized in the liver.

Acetoacetate reacts with succinyl CoA, in the presence of the enzyme transacylase (aceto-acetate-succinyl CoA transferase, also called thiophorase), and is converted to acetoacetyl CoA (Fig. 9.26).

Fig. 9.26. Conversion of acetoacetate to acetoacetyl CoA.

Thereafter, acetoacetyl CoA is hydrolyzed to two molecules of acetyl CoA, by the enzyme thiolase (β-ketothiolase).

Acetone is oxidized very slowly, mainly through lungs.

METABOLISM OF CHOLESTEROL

Cholesterol is derived from diet. It is also synthesized in various tissues in the body. About 50% of the normal intake of dietary cholesterol is absorbed by the small intestine while the rest of it is excreted in the feces.

Nearly 0.3 g of cholesterol is absorbed from the diet, daily. Ingested cholesterol is absorbed with other lipids and is incorporated into chylomicrons and VLDL. More than 80% of it is esterified in the intestinal mucosa and is transported with lipoproteins.

Cholesterol is excreted from the body either as fecal neutral sterol or as bile acid. About 600–750 mg of cholesterol is lost each day as bile acids, 500–600 mg in fecal neutral sterols, 75–100 mg is shed by the skin and nearly 35–50 mg is converted into steroid hormones.

An adult man contains about 1 g of cholesterol/Kg body weight. Nearly 25% of cholesterol is present in the membrane of the nervous system, as a major component of myelin sheath. It is also a major component of plasma membrane. Cholesterol is also a metabolic precursor of various substances such as steroid hormones (glucocorticoids and mineralocorticoids), vitamin D and bile acids and salts.

Egg, meat and milk are the important dietary sources of cholesterol. It is also synthesized in the body by nearly all cells. The amount of cholesterol synthesized each day, in man, is at least twice to that of ingested in the diet.

BIOSYNTHESIS OF CHOLESTEROL

A large quantity of cholesterol (about 1 g/day) is synthesized in the extramitochondrial compartment of the cell. Important sites for cholesterol biosynthesis include liver, skin, intestine, adrenal cortex and reproductive tissues, including ovaries, testes and placenta.

All the carbon atoms of cholesterol are derived from acetate (acetyl CoA), which is obtained from several sources such as oxidation of long chain fatty acids, ketogenic amino acids and glucose (via pyruvate).

De novo synthesis of cholesterol takes place in the body, as shown in Fig. 9.27.

Fig. 9.27. De novo synthesis of cholesterol.

1. First, two molecules of acetyl CoA condense to form acetoacetyl CoA.

This reaction is catalyzed by the enzyme acetoacetyl CoA thiolase.

2. In the presence of the enzyme β-hydroxy-β-methylglutaryl CoA synthase (HMG CoA synthase), acetoacetyl CoA further condenses with another molecule of acetyl CoA and forms β-hydroxy-β-methylglutaryl CoA (HMG CoA).

3. An NADPH dependent enzyme, HMG CoA reductase, converts HMG CoA to mevalonic acid.

This is the regulatory step in cholesterol biosynthesis.

4. Thereafter, there is stepwise transfer of two g-phosphate groups, from two molecules of ATP. These reactions are catalyzed by two kinases, called mevalonate kinase (enzyme I) and phosphomevalonate kinase (enzyme II).

Subsequently, decarboxylation takes place by the enzyme decarboxylase (pyrophosphomevalonate decarboxylase). Thus, phosphorylation and decarboxylation of mevalonic acid forms isopentenyl pyrophosphate (5C), which is also called active isoprenoid unit.

5. In the next step, the isoprenoid unit (isopentenyl pyrophosphate) is isomerised to another isoprenoid unit designated as 3,3,-dimethylallylpyrophosphate, by the enzyme isopentenyl pyrophosphate isomerase.

6. Stepwise condensation of the three isoprenoid units (5C units) leads to the formation of a 15-carbon unit, called farnesyl pyrophosphate (15C).

First, a molecule each of isopentenyl pyrophosphate and 3,3-dimethylallylpyro-phosphate condense together to from a 10-carbon unit called geranylpyrophosphate (10C), which further condenses with another molecule of isopentenyl pyrophosphate and forms farnesylpyrophosphate (15C). This reaction is catalyzed by the enzyme geranyl-transferase.

7. Fusion of the two molecules of farnesylpyro-phosphate forms a 30-carbon compound called squalence (30C).

8. By ring closure and removal of the three methyl groups, squalene is converted to cholesterol, which has 27 carbons.

Regulation of Cholesterol Biosynthesis

Dietary cholesterol, feedback inhibition and its disposal from the liver, regulate cholesterol synthesis.

Dietary cholesterol: Rate of de novo synthesis of cholesterol is inversely related to the amount of dietary cholesterol. When dietary cholesterol intake is reduced, cholesterol synthesis is increased in the liver and the intestine, to meet needs of the other tissues.

On the other hand, when dietary cholesterol intake is increased, its synthesis is reduced.

Feed back inhibition: Cholesterol inhibits its own synthesis by feed back inhibition. The enzyme HMG CoA reductase regulates cholesterol synthesis. Cholesterol inhibits the activity of HMG CoA reductase, by suppressing its synthesis and promoting inactivation.

Disposal by the liver: Liver removes cholesterol by different processes:

1. Esterification of cholesterol: Both, HDL and lecithin:cholesterol acyltransferase (LCAT) are important for the removal of cholesterol from the body.

LCAT is a plasma enzyme which is produced mainly by the liver. It transfers one fatty acid from carbon 2 of lecithin to the 3-hydroxy group of cholesterol. This reaction is freely reversible and utilizes cholesterol, which is present in HDL.

Cholesterol esters, so formed, diffuse into the core of the HDL particle where they are transported from the tissues and plasma, to the liver, which is the only organ that is capable of metabolizing and excreting cholesterol.

This is referred to as the reverse transport of cholesterol since HDL provides the vehicle for the transport of cholesterol, from the peripheral tissues to the liver.

In a normal healthy adult, on a low cholesterol diet, about 1300 mg of cholesterol is returned to the liver, each day.

2. Excretion of cholesterol: Free cholesterol as well as bile acids/salts are excreted in the bile.

3. Incorporation of cholesterol into VLDL: In the liver, cholesterol is incorporated into VLDL and is secreted into circulation.

UTILIZATION OF CHOLESTEROL

Free cholesterol is present in abundance, in bile. It is also present in large amounts, in the myelin structure of the brain. Cholesterol also occurs in the esterified form in the cell membrane.

Cholesterol is also a precursor of the bile acids, steroid hormones and vitamin D (Fig. 9.28).

Fig. 9.28. Utilization of cholesterol.

BILE ACIDS

Bile salts are glycine and taurine conjugates of bile acids. Bile acids contain 24 carbons with two or three hydroxyl groups and a side chain that has a carboxyl group, which is not fully ionized at physiological pH. They are amphipathic in nature.

Primary Bile Acids

Bile acids are synthesized in the liver, from cholesterol. Most common bile acids, that are synthesized in the liver, are called cholic acid and chenodeoxycholic acid. They are also referred to as primary bile acids. Before they leave liver, they get conjugated with a molecule of either glycine or taurine (derived from cystine) by an amide bond, which is formed between the carboxyl group of the bile acid and the amino group of glycine/taurine. These conjugates, i.e. glycocholate and glycochenodeoxycholate as well as taurocholate and taurochenodeoxy-cholate are referred to as bile salts. Normally, the ratio of glycine to taurine conjugates is approximately 3:1. Because of their enhanced amphipathic nature, bile salts are more effective detergents than bile acids.

Secondary Bile Acids

Bacteria, in the intestine, remove glycine and taurine from bile salts and regenerate bile acids. These bacteria also remove a hydroxyl group from primary bile acids, producing deoxycholic acid (from cholic acid) and lithocholic acid (from chenodeoxycholic acid). These compounds are called secondary bile acids.

Mixture of primary and secondary bile acids and salts is observed, primarily, in the ileum. They are actively transported from the intestinal mucosal cells into the portal blood and are removed by liver parenchymal cells. Liver again converts both, primary and secondary bile acids into bile salt, by conjugation with glycine and taurine, and secrets them into bile.

This process of secretion of bile salts into bile, their passage through the duodenum, where some of them are re-converted to bile acids, and their subsequent return to the liver is termed as enterohepatic circulation.

Bile salts have detergent properties and are essential for digestion and absorption of lipids.

STEROID HORMONES

Steroid hormones are also derived from cholesterol. Cholesterol is first converted to pregnenolone, which is subsequently used for the synthesis of steroid hormones, in specific tissues. Steroid hormones are divided into two groups: (i) sex and progestational hormones and (ii) hormones of the adrenal cortex.

Hormones secreted by the adrenal cortex are grouped as glucocorticoids and mineralo-corticoids that exhibit various metabolic effects. Principal glucocorticoid is cortisol (hydro-cortisone), which act predominantly in inter-mediary metabolism of carbohydrates, lipids and proteins. Principal mineralocorticoid is aldosterone that predominantly regulates the metabolism of sodium and potassium.

VITAMIN D

Vitamin D, also called calciferol, is a steroid alcohol (sterol). It is synthesized in the skin from 7-dehydrocholesterol, which is also called provitamin D (Chapter 12).

TRANSPORT OF LIPIDS

Lipids as are not soluble in the aqueous plasma, hence they are transported in the body in association with proteins. These protein lipid complexes are called lipoproteins or proteo-lipids. They are globular micelle like particles consisting of nonpolar core of triacylglycerols and cholesterol esters, which are surrounded by the amphiphilic coating that consists of proteins, cholesterol and phospholipids.

LIPOPROTEINS

Classification of Lipoproteins

Lipoproteins are classified into different groups as follows:

Chylomicrons: They are very large particles that are secreted into lymphatics by mucosal cells of small intestine. About 90% of their mass is triglycerides, which together with cholesterol esters and fat soluble vitamins form a core of the particle. Its surface layer is made of free cholesterol, phospholipids and apoproteins (apo B48 apo AI and apo AIV).

Very Low Density Lipoproteins: Very low density lipoproteins (VLDL) are large particles, which are secreted into the blood stream, by the hepatocytes. Fifty to sixty percent of their mass is triacylglycerols. They contain relatively more cholesterol esters than chylomicrons. Their surface coat contains free cholesterol and phospholipids, a single molecule of apo B-100 and other apoproteins. VLDL is transformed into intermediate density lipoproteins (IDL).

They are referred to as IDL (also called LDL₁) since their density as well as lipid composition are in between the VLDL and the LDL. Major apoproteins of IDL are apo C and apo B100. IDL is either removed by liver or converted to LDL₂, also called low density lipoproteins (LDL).

Low Density Lipoproteins: Low density lipoproteins (LDL) has a density range of 1.019–1.063. It contains a single molecule of apo B-100 and carries about 75% of total cholesterol, in human plasma. Its major function is to transport cholesterol from liver to the extrahepatic tissues.

High Density Lipoproteins: High density lipoprotein (HDL) fraction is very heterogeneous. It is commonly separated into two major groups called HDL₂ (d 1.063–1.125) and HDL₃ (d 1.125–1.21). Further, there occur at least two types of HDL₂ and three types of HDL₃. Major apoproteins found in HDL include apo AI, apo AII, apo AIV, apo C, apo D and apo E. Various lipoproteins can be separated according to their density by ultracentrifugation. They can also be separated according to their protein content, by electrophoresis (Table 9.4).

Table 9.4. Physical properties and chemical composition of human serum lipoproteins

APOLIPOPROTEINS

Protein components of lipoproteins are called apolipoproteins or apoproteins. They serve as the structural components of lipoproteins, provide recognition sites for cell surface receptors and serve as activators for enzymes involved in lipoprotein metabolism.

Various apolipoproteins are divided into different classes from A to E. At least nine of them are known to occur in human serum lipoproteins. They are referred to as AI, AII, B-48, B-100, CI, CII, CIII, D and E. All of them except B-100 are water soluble.

Apolipoprotein B-100 (apo B-100) is one of the largest monomeric protein, which is synthesized by the liver and is present in the LDL.

METABOLISM OF LIPOPROTEINS

Metabolism of Chylomicrons

Chylomicrons are triglyceride rich particles that are assembled in the intestinal mucosal cells. They carry dietary triacyglycerols, cholesterol, and cholesteryl esters to peripheral (extrahepatic) tissues.

Intestinal mucosal cells release nascent chylomicrons, which contain apo B-48 that is synthesized in the intestine. These nascent chylomicrons receive apo E and apo C from the circulating HDL. Apo CII in turn activates lipoprotein lipase and hydrolyzes triacylglycerols to monoacylglycerols, free fatty acids and glycerol. As a result of hydrolysis of triacylglycerol, which is the major component of chylomicrons, they decrease in size and increase in density. Thereafter, apo C is returned to HDL and the leftover particles are called chylomicron remnants. Chylomicron remnants bind to apo E receptors on hepatocytes. They are then internalized and their lipids, including cholesteryl esters, are either utilized or stored, intracellularly.

A small amount of the released free fatty acids enters the circulation and contributes to plasma free fatty acids pool.

Metabolism of Very Low Density Lipoproteins

Very low density lipoproteins (VLDL), also called pre β-lipoproteins, are composed of triacylglycerols like chylomicrons but their function is to transport lipids from liver to the peripheral tissues.

VLDL is released from the liver as nascent VLDL, similar to the release of nascent chylomicrons from the intestinal mucosa. Nascent VLDL particles contain apo B-100 and apo A-1. Subsequently, they acquire apo C-II and apo E from the circulating HDL.

During circulation, lipoprotein lipase hydrolyses triacylglycerols and releases free fatty acids, which are taken up by the peripheral tissues. Apo C and apo E are transferred to HDL. In addition, cholesteryl esters, which are synthesized in the plasma, as a result of the action of lecithin cholesterol acyltransferase (LCAT), are transferred from HDL to VLDL and IDL via cholesteryl ester transfer protein (CETP). As a result of it, their particle size is reduced and density is increased. VLDL particles are, hence converted into VLDL remnants, which are also designated as intermediate density lipoproteins (IDL).

IDL particles are removed by the liver or converted to the LDL.

Metabolism of Low Density Lipoproteins

Low density lipoproteins (LDL) contain high concentration of cholesterol. They retain apo B-100 and lose other apoproteins to HDL. Primary function of LDL is to transport cholesterol from liver to the extrahepatic tissues.

LDL binds to its receptor, which are present in the cell membrane. The LDL receptors are negatively charged glycoproteins that are clustered in the pits on the cell membrane and recognize apo B-100, which in turn acts as a ligand between the IDL and its receptor. The intracellular side of the pit is coated with a protein called clathrin, which stabilizes shape of the pit.

Ability of the tissues to take up LDL depends upon the rate of transcapillary transport as well as the number of receptors. The number of these receptors is regulated by cholesterol content of the cell.

After binding, LDL particles are internalized by endocytosis, in the form of vesicles. These vesicles rapidly loose clathrin coat, fuse with the other vesicles and form endosomes (large vesicles). This in turn results in separation of LDL particles from their receptors.

Receptors are recycled whereas endosome contents are degraded; resulting in release of cholesterol, free fatty acids, phospholipids and amino acids.

Free cholesterol is used for membrane biosynthesis or converted to steroid hormones. Excess of the cholesterol, however, is re-esterified by acylcholesterol acyltransferase (ACAT) and is stored.

In man, 70–80% of LDL is removed by LDL receptors while rest of it is removed by the non-specific endocytosis via the scavenger pathway. The latter is also a receptor mediated process involving phagocytic cells, in the reticuloendothelial system.

Metabolism of High Density Lipoproteins

High density lipoprotein (HDL) particles are synthesized in the liver and are released into the blood stream, by exocytosis (Fig. 9.29).

Secretary HDL particles are called nascent HDL and are discoidal in shape. Nascent HDL is composed of apo A, apo E, phospholipids and free cholesterol.

There also occur transfer of apo C from the chylomicrons and VLDL to nascent HDL, which is converted to mature HDL, called HDL₃.

From HDL₃, cholesteryl esters (produced by the action of LCAT) are shifted to VLDL and IDL. At the same time, HDL₃ acquires triacylglycerols and is converted to HDL₂.

This is an important mechanism for the removal of excess cholesterol from the peripheral tissues and is referred to as the reverse transport of cholesterol.

Fig. 9.29. Metabolism of lipoproteins.

HDL performs several functions:

1. HDL serves as a circulating reservoir of apo C-II, which in turn is transferred to the chylomicrons and VLDL as an activator of lipoprotein lipase.

2. It removes free cholesterol from the extrahepatic tissues and esterifies it with the help of lecithin cholesterol acyltransferase (LCAT), which is present in plasma. HDL thus, transports cholesterol from the extrahepatic tissues to liver.

3. HDL transfers cholesteryl esters to VLDL and LDL, in exchange of triacyglycerols and phospholipids from there.

4. HDL also carries cholesteryl esters to the liver.

In the liver, HDL is degraded and releases cholesterol, which is either repacked in the lipoproteins or is converted to bile acids and secreted into bile, for its removal from the body. Elevated levels of HDL in plasma thus, protect the body against atherosclerosis.

INVESTIGATIONS AND THEIR INTERPRETATIONS

Serum Cholesterol

Cholesterol content in normal human blood varies from 130 to 220 mg/100 ml, being equally distributed between plasma and erythrocytes. Only about 30% of the circulating cholesterol occurs free, while the remaining of it exists in the form of cholesterol esters. Usually, a long chain fatty acid is attached to the −OH group (by an ester linkage), on carbon 3 of the ring A of cholesterol.

ATHEROSCLEROSIS

Atherosclerosis (arteriosclerosis) is a disease of arterial wall. It is characterized by the accumulation of cholesterol and its esters in cells, which are derived from monocyte-macrophage line smooth muscle cells. This in turn results in the formation of atherosclerotic plaque, which narrows the blood vessels and serves as the site of thrombus formation (Fig. 9.30).

High calorie diet or diet rich in saturated fat and cholesterol, hypercholesterolemia, sedentary life style, stress and strain, obesity, hypertension and smoking are important contributory factors favourable in the development of atherosclerosis.

Risk of developing the disease is directly related to the concentration of the LDL-cholesterol, in plasma. It is also inversely related to the plasma concentration of HDL-cholesterol. Therefore, LDL-cholesterol is called bad cholesterol while HDL-cholesterol is referred to as good cholesterol.

Drugs like cholestyramine bind bile salts and promote their excretion in stool. This in turn increases the rate of bile salt synthesis and LDL uptake by the liver. Lovastatin, the drug that is used in the treatment of hyperlipidemia, is an inhibitor of the enzyme HMG CoA reductase. Hence, it decreases endogenous synthesis of cholesterol and stimulates LDL uptake (via LDL receptors) by the tissues.

Dietary recommendations

1. Reduce the intake of fat. Not more than 30% of total energy intake should come from fat.

2. Reduce the use of animal fat. Not more than one third of the total fat (less than 10% calories/ day) should come from saturated animal fat while the remainder should be unsaturated (plant) fat.

3. Reduce the intake of cholesterol. Dietary cholesterol should be less than 300 mg/day.

4. Trans fatty acid intake should limit to less than 1% calories/day.

5. Increase omega-3 fatty acid intake from dietary or supplementary fish oils.

6. Eat five or more servings of fruits and vegetables/day.

7. Reduce salt intake.

Other Life Style Interventions

In addition to dietary advice, other life style interventions include:

– Promotion of physical activity, e.g. adults should perform at least 30 minutes of moderate activity (brisk walking), at least 5 days per week.

– Limit sedentary activities and give up smoking.

Fig. 9.30. Atherosclerosis.

Lipid Profile

Serum lipids and lipoproteins should be measured after a 10 hours fast because chylomicrons normally may be present in plasma up to 10 hours after a meal.

Serum begins to appear hazy when the level of triglycerides reaches above 200 mg/dl. Chylomicrons can be readily detected because they form a white supernatant layer, after refrigeration. For differentiation of disorders of lipoproteins, triglycerides and total cholesterol are routinely measured. The measurement of plasma total cholesterol is a first line screening test. Most of the cholesterol present in plasma is found in LDL. Similarly, plasma triglycerides concentrations are a matter of the sum of total of VLDL and IDL. To obtain a more accurate picture of lipid metabolism, we measure or calculate cholesterol present in the LDL or HDL fractions.

DISORDERS OF PLASMA LIPOPROTEINS

Hyperlipoproteinemias

Increased rate of synthesis or reduced clearance of lipoproteins from blood may result in a group of disorders, referred to as hyperlipidemias or hyperlipoproteinemias.

Usually, they are detected by measuring plasma triacylglycerols and cholesterol (Table 9.5).

Table 9.5. Primary hyperlipoproteinemias

Type |  | Elevated lipoprotein fraction
I | Exogenous hyperlipidemia (Exogenous hypertriglyceridemia) | Chylomicron
IIa | Hypercholesterolemia | LDL
IIb | Combined hyperlipidemia | LDL + VLDL
III | Remnant hyperlipidemia | Remnant VLDL
IV | Endogenous hyperlipidemia | VLDL
V | Mixed hyperlipidemia | VLDL + chylomicrons

Hypolipoproteinemias

Abetalipoproteinemia: This is due to the absence of apo B. As a result of it, all the plasma lipids, particularly, triacylglycerols are reduced. Fat malabsorption is present from birth. There is also poor appetite, vomiting, diarrhea and failure to thrive in neonates.

Familial Hypobetalipoproteinemia: Low level of apo B results in moderate reduction of plasma lipids. VLDL and LDL are low while HDL concentrations are variable.

Tangier’s Disease: It is the HDL deficiency state where plasma triacylglycerols and VLDL levels are normal or moderately increased. LDL is rich in triacylglycerols. Plasma total cholesterol as well as LDL-cholesterol levels are reduced by about 2/3rd while HDL-cholesterol is extremely low. Apo AI is around 1% of the concentration normally found in plasma and apo AII about 5–8% of the normal. Besides apo AI and apo AII, there is also a moderate reduction in other apoproteins.

The fasting lipid profile, i.e. the concentration of total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides, provides information about the main lipid markers of cardiovascular risk. LDL-cholesterol to HDL-cholesterol ratio indicates the balance between cholesterol transport to and from peripheral tissues. A ratio above 5 indicates an increased cardiovascular risk.

Dividing the value of triglycerides by 5, gives the concentration of VLDL-cholesterol in plasma.

VLDL–Cholesterol (mg/dl) = Triglycerides / 5

LDL–Cholesterol concentration in plasma can also be calculated from the value of total cholesterol, triglycerides and HDL-cholesterol, by the equation, called Friedwald equation, as follows. :

LDL-Cholesterol (mg/dl) = Total cholesterol – [HDL cholesterol + Triglycerides / 5]

A large number of specialized investigations, including ultracentrifugation, apolipoprotein estimation and enzymes, and molecular genetic studies may occasionally be helpful.

Other biochemical cardiovascular risk factors or markers include troponin, myoglobin, homocysteine and C-reactive protein.

In about 95% of the patients with primary hypercholesterolemia, plasma cholesterol concentration is usually raised, as the abnormality is due to a combination of dietary factors and a number of genetic abnormalities in handling cholesterol.

In patients with familial hypertriglyceridemia, plasma triglycerides (VLDL) concentration is increased since this condition is associated with defects either in the production or in the catabolism of VLDL.

In familial combined hyperlipidemia, there may be increase in plasma LDL only, plasma VLDL only, or both.

SOME IMPORTANT QUESTIONS

1. What are lipids? Classify them. Give biological significance of lipids.

2. What are fatty acids? Classify them. Write biological significance of polyunsaturated fatty acids.

3. Outline β-oxidation of fatty acids. How much energy is produced when a molecule of palmitic acid is oxidized to acetyl CoA?

4. Outline de novo synthesis of fatty acids. What is the source of reducing equivalents? How de novo fatty acid synthesis differs from fatty acid elongation in the microsomes and the mitochondria?

5. Outline cholesterol synthesis. Add a note on its relationship with atherosclerosis.

6. Describe briefly:

(i) Classification of lipids with suitable examples

(ii) Plasmalogens

(iii) Sphingolipids

(iv) Chemistry and functions of cholesterol

(v) Cerebrosides

(vi) Role of carnitine in fatty acid oxidation

(vii) Degradation of cholesterol

(viii) Process of β-oxidation of fatty acids

(ix) Synthesis of cholesterol and its regulation

(x) Oxidation of fatty acids containing odd number of carbon atoms

7. Write notes on:

(i) Ketone bodies

(ii) ω-oxidation

(iii) Atherosclerosis

(iv) De novo synthesis of fatty acids

(v) Sudden infant death syndrome

(vi) α-oxidation of fatty acids

(vii) Refsum disease

(viii) Lipofuscin

(ix) Metabolic fates of acetyl CoA

(x) Metabolic fates of cholesterol

(xi) LCAT

(xii) Diabetic ketoacidosis

(xiii) PUFA

(xiv) Triacylglycerols

(xv) Lecithins

(xvi) Lipotropic factors

(xvii) Phospholipids

(xviii) Bile salts

(xix) Lipoproteins

(xx) Apoproteins

CHAPTER
10

Chemistry and
Metabolism of
Amino Acids and Proteins

Protein (Greek: proteios means primary) is the class of natural products, which contain nitrogen. It is now well known that proteins are at the center of action in biological processes and are essential structural components of a cell. They are most abundant and functionally diverse molecules present in all living systems.

All proteins, from whatever source it may be, are polymers of small units called amino acids. Though, a large number of amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins. Since amino acids are hydrolyzed products of proteins, they are also called building blocks of proteins.

AMINO ACIDS

Commonly occurring amino acids are designated as α-amino acids because each amino acid has a carboxylic group (−COOH), a primary amino group (−NH₂) and a distinctive side chain (R-group), which is present on the α-carbon atom.

General structure of an amino acid is shown in Fig. 10.1.

Fig. 10.1. General structure of an amino acid.

BIOLOGICAL SIGNIFICANCE OF AMINO ACIDS

1. Amino acids are important dietary source of nitrogen.

2. They are used in protein biosynthesis.

3. Glucogenic amino acids, e.g. alanine, serve as a source of energy.

4. Some of the amino acids are also used in the biosynthesis of various specialized products. For example:

(i) Glycine is a precursor of creatine, glutathione, heme, bile acids and purines.

(ii) Sulfur-containing amino acids are the source of sulfur for the body.

Cysteine is also used in the biosynthesis of glutathione while methionine is important in one-carbon metabolism.

(iii) Aromatic amino acids are the precursors of thyroid hormones, catecholamines (hormones of the adrenal medulla, i.e. dopamine, norepinephrine and epinephrine), and melanin (a brown-black pigment in skin).

(iv) Aspartate and glutamate participate in transamination reactions.

(v) Aspartate and glutamine are used in the biosynthesis of purines and pyrimidines.

CLASSIFICATION OF AMINO ACIDS

Amino acids may be classified according to the properties of their side chains. They are also classified according to their nutritional requirements.

BASED ON STRUCTURE OF THEIR SIDE CHAIN

According to Charge

According to the charge on their side chain (R-group attached to the α-carbon atom) amino acids may be electrically neutral, or may have a positive or negative charge. Thus, based on the ionic charges, amino acids are classified as:

Neutral Amino Acids

These amino acids are neutral in reaction and have one amino group (−NH₂) and one carboxyl group (−COOH). These amino acids are also referred to as monoamino mono-carboxylic acids.

According to the presence of the R group, these amino acids are further subdivided into aliphatic and aromatic amino acids:

Aliphatic Amino Acids

Aliphatic amino acids are further grouped as:

1. Simple Amino Acids: These amino acids have either hydrogen or a methyl group in the aliphatic side chain.

Glycine has hydrogen (H) whereas alanine has a methyl group (–CH₃) as a side chain (Fig. 10.2).

Fig. 10.2. Glycine and alanine.

2. Branched Chain Amino Acids: These amino acids have an aliphatic side chain, which is branched. These are valine, leucine and isoleucine (Fig. 10.3).

Fig. 10.3. Branched chain amino acids.

3. Hydroxy Amino Acids: These amino acids have a hydroxy (–OH) group in its aliphatic side chain. These are serine and threonine (Fig. 10.4).

Fig. 10.4: Hydroxy group containing amino acids.

4. Sulfur-containing Amino Acids: In addition to C, H, O and N, sulfur-containing amino acids also contain sulfur (S) in their side chain. These include cysteine and methionine.

Cysteine has a thiol group (–SH). Through the −SH group, two molecules of cysteine can link with each other and form dicysteine, which has a disulfide bond (–S–S–). Dicysteine is also referred to as cystine.

Methionine has a methyl group attached to sulfur (CH₃–S–), in its side chain (Fig. 10.5).

Fig. 10.5. Sulfur-containing amino acids.

Aromatic Amino Acids

These amino acids have an aromatic ring in their side chains. Aromatic amino acids include phenylalanine (alanine attached to a phenyl group), tyrosine (4-hydroxyphenyl-alanine) and tryptophan (alanine attached to an indole ring – Fig. 10.6).

Fig. 10.6. Aromatic amino acids.

Acidic Amino Acids

An acidic amino acid has one amino group but more than one, generally two, carboxyl groups. Hence these amino acids are also referred to as monoamino dicarboxylic acids. These are aspartic acid and glutamic acid (Fig. 10.7).

Besides, there are two amino acids, which occur as the amides of the above two amino acids.

Amide of aspartic acid is called asparagine while that of glutamic acid is called glutamine (Fig. 10.7).

Fig. 10.7. Acidic amino acids and their amides.

Basic Amino Acids

Basic amino acids have one carboxyl group with either more than one amino group in its aliphatic side chain or a heterocyclic ring, which is basic in reaction. These are lysine, arginine and histidine.

Lysine has two −NH₂ groups while arginine has a guanidino group in the aliphatic side chain.

Histidine has a heterocyclic ring called imidazole ring (Fig. 10.8).

Fig. 10.8. Basic amino acids.

Imino Acid

In addition to the above amino acids that are found in most of the proteins, some proteins, e.g. collagen, are rich in proline.

Proline does not contain a primary α-amino group (−NH₂) in its side chain but has a secondary imino group (–NH–), which forms a part of the heterocyclic ring. Hence, proline is not an amino acid but imino acid (Fig. 10.9).

Fig. 10.9. Proline—an imino acid.

However, for uniformity, proline is included in the list of α-amino acids. Proline can be converted to its 4-hydroxy derivative, called hydroxyproline, in the body.

Various naturally occurring amino acids with their commonly used three letter abbreviations are shown in Table 10.1.

Table 10.1. Classification of amino acids

Amino acid | Abbreviation*
NEUTRAL AMINO ACIDS |
Simple Amino Acids |
Glycine | Gly
Alanine | Ala
Branched Chain Amino Acids |
Valine | Val
Leucine | Leu
Isoleucine | Ile
Hydroxy Amino Acids |
Serine | Ser
Threonine | Thr
Aromatic Amino Acids |
Phenylalanine | Phe
Tyrosine | Tyr
Tryptophan | Trp
Sulfur-Containing Amino Acids |
Cysteine | Cys
Methionine | Met
ACIDIC AMINO ACIDS |
Aspartic acid | Asp
Glutamic acid | Glu
Amides of the Acidic Amino Acids |
Asparagine | Asn
Glutamine | Gln
BASIC AMINO ACIDS |
Lysine | Lys
Arginine | Arg
Histidine | His
IMINO ACID |
Proline | Pro

* Three letters abbreviation is widely used. This, generally, includes first three letters of an amino acid.

Based on Polarity

According to polarity, amino acids are classified into two groups:

Amino Acids with Nonpolar Side Chain

Amino acids with a nonpolar side chain neither release protons nor participate in hydrogen/ionic bond formation. These amino acids are glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline.

Amino Acids with Polar Side Chain

Some of the amino acids have uncharged polar side chain with a zero net charge at neutral pH, e.g. serine, threonine and tyrosine. They have a hydroxy group, which can participate in hydrogen bond formation. The hydroxy group also serves as a site for the attachment of phosphate or an oligosaccharide.

Asparagine and glutamine have a charged polar side chain as they contain an additional amide group, which also participates in a reaction.

Based on Dietary Requirement

According to nutritional requirement, amino acids are divided into two groups, referred to as essential amino acids and nonessential amino acids (Table 10.2).

Table 10.2. Classification of amino acids according to nutritional requirement

Essential | Semi-essential | Non-essential
Methionine | Arginine | Glycine
Threonine | Histidine | Alanine
Tryptophan |  | Serine
Valine |  | Cysteine
Isoleucine |  | Tyrosine
Leucine |  | Aspartate
Phenylalanine |  | Glutamate
Lysine |  | Asparagine
|  | Glutamine
|  | Proline

Essential Amino Acids

As all amino acids cannot be synthesized in the body and are to be essentially included in the diet, hence those amino acids which are to be essentially included in the diet are called indispensable or essential amino acids.

There are eight amino acids which are essential for adult human beings. These are methionine, threonine, tryptophan, valine, isoleucine, leucine, phenylalanine and lysine.

Semi-Essential Amino Acids

Arginine and histidine are not essential for a normal adult human being. However, under certain conditions, e.g. growth, pregnancy and lactation (when their requirement is increased) or under conditions such as a high grade fever and chronic infections (when their catabolism is increased), arginine and histidine also become dietary essential. Hence, these two amino acids are called semi-essential amino acids.

Non-Essential Amino Acids

Remaining all the amino acids, since can be synthesized by a normal human being and are not dietary essential, are called non-essential amino acids.

PROPERTIES OF AMINO ACIDS

Isomerism

All the naturally occurring amino acids except glycine, have at least one asymmetric carbon atom and are optically active. They have L-configuration.

Acid Base Properties

Amino acids exhibit acid base properties. An amino acid may have either a positive charge or a negative charge. At low pH, an amino acid exists in the cationic form as its amino group is protonated and is present as −NH₃⁺. As the pH rises, carboxyl group loses its proton and the ampholyte form appears. With further increase in pH, the amino group is deprotonated and the anionic form is formed. At physiological pH (pH 7.4), carboxyl group of the amino acids are dissociated (R–COO⁻) while their amino groups are protonated (R–NH₃⁺).

Such ionized molecules, which have both negative as well as positive charges, are called ampholytes. Molecules with equal number of ionizable groups, i.e. when the number of cations is equal to the number of anions, are called zwitterions.

The pH at which a molecule exists as a zwitterion is called isoelectric pH or isoelectric point (pI).

At pH more than its pI, an amino acid is negatively charged while at pH less than its pI, it is positively charged (Fig. 10.10).

Fig. 10.10. Various ionic forms of an amino acid.

Buffer Action

Amino acids in an aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, acidic and basic amino acids also contain an ionizable group in its side chain. Hence, amino acids also act as buffers.

PROTEINS

Most important property of amino acids is the formation of a peptide bond (–CO–NH–). During this process, the carboxyl group of an amino acid reacts with the amino group of the other amino acid. This is a condensation reaction and results in the formation of an amide linkage, called a peptide bond (Fig. 10.11).

Fig. 10.11. Formation of peptide bond.

When several amino acids are linked together, they form polypeptides and proteins. Each polypeptide has a free α-amino group on the left hand side of the peptide chain. This end of the protein chain is referred to as N-terminal (amino terminal) end. The protein chain also has a free α-carboxylic group on the right hand side of the peptide chain. This end of the protein chain is referred to as C-terminal (carboxyl terminal) end (Fig. 10.12).

Fig. 10.12. A polypeptide.

BIOLOGICAL SIGNIFICANCE OF PROTEINS

With respect to their biological importance, proteins are divided into several groups, as shown in Table 10.3.

1. Structural Proteins: Some of the proteins, e.g. collagens (found in bone and cartilage), keratins (found in hair and nail), etc., form an essential part of particular structures with in the body

2. Enzymes: Some of the proteins such as pepsin, catalyze biological reactions and act as enzymes.

3. Hormones: Some of the proteins act as hormones, e.g. insulin. They regulate metabolic processes with in the body.

4. Transporters: Some of the proteins, e.g. hemoglobin, ceruloplasmin, etc., serve as carriers for the transport of certain substances with in the body and are called transport proteins.

5. Receptors: Proteins such as hormone receptors, act as receptors for certain hormones and play an important role in signal transduction.

6. Storage Proteins: Some of the proteins bind to a substance for its storage in different tissues in the body, e.g. ferritin for the storage of Fe³⁺.

7. Antibodies: Proteins such as γ-globulin, act as antibodies and provide immunity.

Table 10.3. Biological importance of proteins

Biological role of protein | Examples
As structural proteins | Collagen, keratins, etc.
As enzymes | Pepsin, amylase, etc.
As hormones | Insulin
As transporters | Hemoglobin
As receptors | Hormone receptors
As storage molecules | Ferritin
In immune response | γ-Globulin

CLASSIFICATION OF PROTEINS

Depending upon their solubility and physical properties, proteins are classified into three classes (Table 10.4).

Table 10.4. Classification of proteins

Simple proteins | Conjugated proteins | Derived proteins
Albumins | Nucleoproteins | Primary derived proteins
Globulins | Glycoproteins |
Prolamines | Phosphoproteins | Secondary derived proteins
Glutelins | Metalloproteins |
Scleroproteins | Lipoproteins |
Histones |  |

Simple Proteins

Based on their solubility, simple proteins are divided into several groups, as follows:

1. Albumins: Albumins are found in serum, milk, egg, etc. They are soluble in water as well as salt solutions.

2. Globulins: Globulins are found in serum, milk and egg along with albumin. They are sparingly soluble in water but soluble in a neutral salt solution.

3. Prolamines: Prolamines, e.g. gliadin of wheat or zein of maize, are insoluble in water but soluble in 60–80% of alcohol.

4. Glutelins: Glutelins, e.g. gluten of wheat or oryzenin of rice, are soluble in dilute acids and alkalies.

5. Scleroproteins: They are highly insoluble.

Scleroproteins are present in supporting tissues such as bones, cartilages, etc. They are also found in protective tissues like skin, hair, nail, etc.

6. Histones: Histones, e.g. nucleohistones, are rich in basic amino acids. They are soluble in water.

Conjugated Proteins

Proteins are conjugated with a nonprotein substance, called prosthetic group. Such proteins are referred to as conjugated proteins. For example, nucleoproteins (histones conjugated with nucleic acids), glycoproteins (proteins conjugated with carbohydrates), phosphoproteins (phosphoric acid attached through serine or threonine in the protein, such as casein in milk), metalloproteins (metal-containing proteins such as ceruloplasmin, a copper containing protein) and lipoproteins (lipid-associated proteins).

Derived Proteins

These are the derivatives of simple proteins such as denatured and coagulated proteins (primary derived proteins) or those produced by cleavage of the peptide bonds (secondary derived proteins).

PROTEIN STRUCTURE

Variations in the number and order of amino acids, i.e. the manner in which various amino acids are arranged in a polypeptide chain, reflect structure of a protein. There are four levels of protein structure, referred to as primary, secondary, tertiary and quaternary levels of protein structure.

Primary Structure

Primary structure of a protein refers to the order and sequence of amino acids in a protein chain (Fig.10.13).

Fig. 10.13. Primary structure of a protein,

Some proteins such as insulin, are synthesized as a single polypeptide and are later cleaved into two or more chains. These proteins have intra-chain as well as inter-chain disulfide linkages.

Secondary Structure

Secondary structure of a protein refers to folding and twisting pattern of a polypeptide chain, i.e. the spatial arrangement of a polypeptide. Secondary structures include α-helix and β-sheets.

α-Helix

Twisting the backbone of a polypeptide chain forms coil or helix. As a result of its folding, hydrogen bonds may also be formed between the −CO and the −NH groups, with in the polypeptide chain. These types of linkages are called intra-chain peptide linkages.

The helix may be α-helix or β-helix.

α-Helix refers to right handed folding of the protein chain. It is the most stable conformation of the polypeptide chain, e.g. α-keratin. There are 3.6 amino acid residues per turn of the helix (Fig. 10.14).

Left-handed helix generally does not occur in proteins.

Fig. 10.14: A right handed α-helix structure.

β-Pleated Sheet

The second regular conformation found in proteins is referred to as β-sheet structure. β-Denotes the second regular structure that was described after α-helix. The conformation, in which β-structure is optimally hydrogen bonded, has rippled or pleated-edge on appearance. Therefore, this conformation is also called pleated sheet-like structure.

Such proteins, e.g. wool, may contain 2 to 12 polypeptides in β conformation. Each strand may contain up to 15 residues. β-Sheet orientation is formed due to stretching of the polypeptide chains, as a result of the formation of hydrogen bonds between −CO and −NH groups of the neighboring polypeptides. These types of linkages are called intra chain peptide linkages (Fig. 10.15).

Fig. 10.15. β-Pleated sheet structure.

In these types of proteins, polypeptide chains may run either in antiparallel or parallel directions.

Antiparallel β-Sheet: It is formed when two polypeptide chains run in the opposite directions.

Parallel β-sheet: It is formed when two chains run in the same direction.

Various forces which stabilize secondary structure of a protein, include hydrogen bonds, hydrophobic interactions, electrostatic interactions and van der Waals forces.

Tertiary Structure

Tertiary structure involves intramolecular folding of the polypeptide chain thereby forming a compact three dimensional structure which has a specific shape.

Electrovalent linkages, hydrogen bonds, disulfide bridges, van der Waals forces and hydrophobic interactions maintain tertiary structure of proteins (Fig. 10.16).

Fig. 10.16. Tertiary level of protein structure.

Depending on their overall morphology, proteins have been classified as either globular or fibrous.

Globular Proteins

Globular proteins have a spheroidal shape and vary in size. They have relatively high water solubility and function as catalysts, transporters and regulators of metabolic pathways and gene expression. Their shapes are the result of complex interactions between secondary, tertiary and some times quaternary structural elements. Major types of the secondary structural elements, i.e. the α-helices and the β-sheets, occur in globular proteins in varying proportions and combinations. Certain groupings of the secondary structural elements, called super-secondary structures or motifs, also occur in many unrelated globular proteins. Some proteins such as cytochromes, consist of only α-helices spanned by short connecting links. Others, such as immunoglobulin folds, have a large proportion of β-sheets and are devoid of the α-helices. Most of the proteins, such as lactate dehydrogenase, however, have significant amounts of both types of the secondary structures. Most common globular proteins are hemoproteins, such as hemoglobin, myoglobin, cytochromes and catalase.

Fibrous Proteins

Fibrous proteins exhibit special mechanical properties. They characteristically, contain large amounts of the regular secondary structures and have long cylindrical (rod-like) shapes. They have low solubility in water. Fibrous proteins have a protective, connective or supportive role in living organisms, e.g. collagen, keratin and tropomyosin, etc. These are found as components of the skin, connective tissue, blood vessel walls and sclera and cornea of the eye.

Quaternary Structure

Multi subunit proteins contain several identical and/or different chains, where each one of the peptide chains is referred as a subunit. These polypeptide subunits are associated with a specific geometry. Spatial arrangement of these subunits is referred to as the quaternary structure of proteins (Fig. 10.17).

Fig. 10.17. Quaternary level of protein structure.

Proteins with more than one subunit are called oligomeric proteins. For example hemoglobin, is an oligomeric protein with four subunits called α₂β₂.

Disintegration of the monomeric subunits of a multimeric protein results in the loss of its biological activity.

HEMES AND CHLOROPHYLL

Heme

Heme consists of one atom of ferrous iron (Fe²⁺) and a tetrapyrrole ring called protoporphyrin IX. Heme is a prosthetic group found in hemoglobin, a globular protein, which is present in high concentration in red blood cells. Hemoglobin binds oxygen in lungs and transports it to different parts in the body. Besides hemoglobin, heme is also found in myoglobin and various cytochromes (Chapter 14).

Chlorophyll

Like heme group of globins and cytochromes, protoporphyrin IX is also a precursor of chlorophyll, which is a principal photoreceptor in photosynthesis. It, however, differs from heme, as in chlorophyll the central metal ion is Mg²⁺ rather than Fe2. Further, in chlorophyll, a cyclopentane ring (as ring V) is fused to the pyrrole ring III.

PROTEIN DENATURATION

Usually, proteins are soluble in water due to their organized structures. On heating or adding a strong acid, protein looses its native structure and gets precipitated. This is referred to as denaturation. Usually, on denaturation, protein looses its biological activity since denaturation results in unfolding and disorganization of its secondary and tertiary structures.

Denaturing agents include heat, mechanical force, detergents, strong acids or bases and the ions of heavy metals such as lead and mercury. Under ideal conditions, denaturation may be reversible, however, most proteins, once denatured remain permanently disordered. Denatured proteins are often insoluble and therefore precipitate from the solution.

PRECIPITATION OF PROTEINS

Proteins are charged, high molecular weight, colloidal particles and can be precipitated from the solution by a number of compounds.

Salting-out: Proteins can be precipitated by the addition of a salt solution. This process is known as salting out. Highly soluble salts like NaCl and (NH₄)₂SO₄ are generally, used for this purpose. Albumin is more soluble in water than globulins hence globulins get precipitated at half the saturation by ammonium sulphate while albumin precipitates at full saturation.

Precipitation of protein by a salt solution is, apparently, due to neutralization of the charges as well as dehydration of the molecule. Protein, which has been precipitated by a salt solution, remains native and usually redissolves, when put back in the solvent.

Precipitation by other reagents: Since proteins are not soluble in organic solvents, they can also be precipitated with the addition of some nonaqueous solvents like alcohol, acetone, etc. Proteins get precipitated due to dehydration of the molecule.

By heavy metals: Most commonly used metals as protein precipitants include metal salts such as lead acetate, mercuric chloride, barium chloride, zinc sulfate, etc. Proteins get precipitated due to adsorption of the metallic cations by the negatively charged colloidal particles of the protein.

By alkaloid reagents: Proteins can also be precipitated by the addition of some alkaloid reagent. Precipitation by an alkaloid reagent is due to the formation of an insoluble salt between the anions of the reagent and the positively charged protein particles.

By heating: By heating protein gets coagulated. This is referred to as heat coagulation. Coagulation is the maximum at isoelectric pH.

PROPERTIES OF PROTEINS

Based on Size and Charge

Size is measured in terms of molecular weight (MW), in Daltons. One Dalton is atomic mass of proton or neutron. There is wide range of weights varying up to thousands of kD.

Each protein has numerous charges, such as amino terminus (may be positively charged), carboxy terminus (may be negatively charged) and the amino acid side chains (R groups) may carry any charge. The net charge on a protein is the sum of all the individual charges. The net charge on the protein varies with pH. At the pI, the protein has no net charge, i.e. the number of positive charges is equal to the number of negative charges. At low pH, the protein becomes more positively charged while at high pH the protein becomes more negatively charged.

SEPARATION TECHNIQUES

Chromatography

Chromatography is a technique commonly used for the separation of closely related compounds from a mixture. In this technique, separation is effected by differences in the equilibrium distribution of components between the two immiscible phases, called stationary phase and the mobile phase. Differences in equilibrium distribution are the result of nature and degree of interaction of components between the two phases. The stationary phase is a porous medium through which sample percolates under the influence of a moving solvent called mobile phase. There are number of interactions between the sample and the stationary phase. Chromatography techniques are classified based on the stationary phase used, such as paper chromatography, thin layer chromatography, column chromatography, gas liquid chromatography and high pressure liquid chromatography.

Electrophoresis

Electrophoresis refers to the process of migration of charged particles under the influence of electric current. According to the separation medium used, it may be grouped as paper electrophoresis, starch gel electrophoresis, etc.

Amino Acid Sequencing

Amino acid sequencing can be done by Edman degradation where phenylisothiocyanate reacts with the terminal amino group and sequentially removes one amino acid from the amino end of a peptide. In this process, phenylisothiocyanate reacts with the terminal amino group of the peptide and forms phenylisothiocarbamoyl derivative, which cyclizes under mildly acidic conditions to give phenylthiohydantoinamino acid, leaving the peptide which is shorter by one amino acid. These phenylthiohydantoinamino acids can be subsequently identified by chromatography.

PROTEINS AS COLLOIDS

Certain substances in solution such as proteins, are not true molecules but aggregates of small peptides that are held together by noncovalent interactions. They, infact, are large, discrete molecules that do not diffuse through parchment or animal membrane although they form homogenous or heterogeneous solution. These substances are called colloids. They are electrically charged, which is of importance in stabilizing a colloidal solution.

A colloid is a type of chemical mixture where one substance is dispersed evenly throughout another. The particles of the dispersed substance are only suspended in the mixture, unlike a solution, where they are completely dissolved within. This occurs because the particles in a colloid are larger than in a solution, small enough to be dispersed evenly and maintain a homogenous appearance but large enough to scatter light and do not dissolve. Because of this dispersal, some colloids have the appearance of solution. A colloidal system consists of two separate phases: a dispersed phase (or internal phase) and a continuous phase (or dispersion medium). A colloidal system may be solid, liquid or gaseous.

PLASMA PROTEINS

Plasma contains over 300 proteins. Albumin, globulins and fibrinogen are the major plasma proteins, all of which are synthesized in the liver (except γ-globulins). Others include apolipoproteins (present in lipoproteins), protein hormones (such as insulin, prolactin, etc.), enzymes and coagulation proteins. Total plasma protein concentration varies from 6.3–8.0 g/dl, out of which albumin constitutes 3.7–5.3 g/dl while globulins constitute 1.8–3.7 g/dl. The range for the various globulin fractions are – α₁ plus β₂ = 0.4–1.4 g/dl, β = 0.5–1.3 g/dl and γ = 0.6-1.5 g/dl.

A/G Ratio

A/G ratio refers to plasma albumin: globulin ratio. The normal plasma A/G ratio is 1.2–2.0:1, which may be altered in a number of conditions. For example, an increase in total plasma proteins occurs in dehydration, due to prolonged diarrhea, vomiting and diabetes insipidus. Because of hemoconcentration, both albumin and globulins are increased proportionately so that A/G ratio remains unaltered.

A decrease in total plasma proteins is generally due to hypoalbuminemia as seen in liver cirrhosis, nephrotic syndrome, kwashiorkor, etc. It may be accompanied by either no increase in globulins or an increase in globulins that is proportionately less than the fall in albumin, resulting in a reversal of A/G ratio.

Functions of Plasma Albumin

Albumin performs several functions:

• Maintenance of plasma colloidal osmotic pressure and hence the regulation of blood volume.

• Transport of various molecules such as long chain fatty acids, unconjugated bilirubin, steroids, Cu2+ and drugs like salicylates, barbi-turates, penicillin, sulfonamides and warfarin.

• Plasma antioxidant because of the presence of sulfhydryl (-SH) groups.

• Protein reserve in case of nutritional depletion.

Functions of Plasma Globulins

α and β-Globulins transport various molecules such as hormones, copper, iron, etc. (Table 10.5).

γ-Globulins constitute the defense proteins that combat antigenic challenge.

Table 10.5. Transport proteins

Proteins | Molecule transported
Albumin | Cu²⁺, Fe³⁺, bilirubin, free fatty acids, steroids, heme
Ceruloplasmin | Copper
Transferrin, ferritin, hemosiderin | Iron
Haptoglobin, hemopexin | Free hemoglobin
Thyroid binding globulin | Thyroid hormones
Cortisol binding globulin | Cortisol
Sex hormone binding globulin | Androgens, estrogens

Separation of Plasma Proteins

In routine, plasma proteins are separated by paper electrophoresis.

Paper Electrophoresis: In paper electrophoresis, a strip of filter paper is taken and about 10 μl of serum is applied along a thin line, near the cathode. The strip is dipped in a buffered solution (barbitone buffer, pH 8.6) and is laid on a platform. The apparatus is covered with a sheet, to minimize evaporation. Two electrodes are also dipped in the two compartments containing buffer. Current is supplied through a power supply, which either supplies constant current and/or voltage. The current is carried by the charged particles and depending upon the charge, proteins migrate towards either anode or cathode. Since at pH 8.6, all the proteins carry a net negative charge and tend to migrate towards anode. Thus, on passing a constant current (about 1–2 mA per strip), different proteins migrate at different rates. At the end, their positions are revealed by some dye such as bromophenol blue, which stains proteins. Different proteins form separate bands on the paper strip.

PLASMA PROTEIN DISORDERS

Hypoalbuminemia

It refers to low plasma albumin and may arise due to several reasons such as protein malnutrition (e.g. kwashiorkor and marasmus), malabsorption (e.g. inflammatory bowel disease), impaired synthesis in chronic liver diseases (e.g. cirrhosis), excessive losses in urine (e.g. nephrotic syndrome) or increased catabolism (e.g. major surgery, trauma, sepsis and malignancy).

Hyperglobulinemia

Increase in globulins occurs most commonly in advanced liver disease (e.g. cirrhosis), chronic infectious diseases (e.g. tuberculosis or viral hepatitis) or multiple myeloma.

In the normal serum five bands are observed. Since albumin carries largest number of the negative charges, it moves farthest while γ-globulin has less negative charges, it moves least. Starting from anode, these bands are designated as albumin, α₁-globulin, α₂-globulin, β-globulin and γ-globulin. Electrophoresis of plasma proteins helps in the diagnosis of several diseases (Fig. 10.18).

Fig. 10.18. Plasma proteins pattern on electrophoresis in some of the diseases.

METABOLISM OF AMINO ACIDS AND PROTEINS

Proteins are degraded to amino acids, which contribute to the amino acid pool. They are used for protein biosynthesis. Excess of them are also used for energy. They are also the important source of nitrogen for the body.

DIGESTION AND ABSORPTION OF PROTEINS

Digestion of Proteins

Digestion of proteins begins in the gastrointestinal tract and is carried out by proteases secreted by the stomach, the pancreas and the small intestine:

• Digestion of proteins starts in the stomach by the action of proteolytic enzyme pepsin, at pH of the gastric juice (1.5–2.5). Pepsin is secreted by the cells of the gastric mucosa in the form of a zymogen (proenzyme), called pepsinogen, which is activated by hydrogen ions of the gastric juice (HCl) and later autocatalytically by pepsin itself.

• Proteolytic products of pepsin pass on to the small intestine where they are attacked by proteases that are secreted by the pancreas, i.e. trypsin, chymotrypsin and carboxypeptidases.

• Trypsin is secreted in the zymogen form as trypsinogen, which is converted to trypsin by another enzyme called enterokinase (enteropeptidase) that acts as an activator of trypsinogen and is secreted by the intestinal mucosa. Once trypsin is formed, it acts on trypsinogen and converts it to trypsin, autocatalytically.

• Chymotrypsin is also synthesized and secreted as a zymogen, designated as chymotrypsinogen. It is activated by trypsin.

• Pancreatic juice also contains collagenase and elastase, which specifically act on collagen and elastin, respectively.

• In addition to the above endopeptidases, exo-peptidases are also secreted by the pancreatic juice, in the zymogen forms. These include procarboxypeptidase A and procarboxypeptidase B. They are also activated by trypsin. These two enzymes attack peptide linkages, specifically those having a free carboxyl group. Carboxypeptidase A hydrolyzes the C-terminal amino acid in peptides, particularly of those where the terminal amino acid is an aromatic or aliphatic amino acid, while carboxypeptidase B hydrolyzes the terminal peptide bond of the basic amino acids (arginine and lysine).

• The digestion of proteins thereafter, is completed by the action of aminopeptidases and dipeptidases which are secreted by mucosa of the small intestine.

Any part of the protein that is not hydrolyzed in the small intestine enters the large intestine and excreted in the feces.

Absorption of Amino Acids

The end products of digestion of proteins, i.e. amino acids, are absorbed from the small intestine into the portal blood. Absorption of L-amino acids is a Na⁺-dependent active transport and requires ATP and specific transport proteins. Pyridoxal phosphate (vitamin B₆) is also required for the absorption of amino acids.

There are several transport systems for the uptake of different amino acids. These include:

1. a transport system for neutral amino acids with short or polar side chains such as Ala, Ser and Thr;

2. for neutral amino acids with an aromatic or hydrophobic side chain such as Phe, Tyr, Met, Val, Leu, Ile;

3. for imino acids, i.e. Pro and hydroxyproline (HO-Pro);

4. for Lys, Arg and Cystine (Cys-Cys);

5. for acidic amino acids, i.e. Asp and Glu;

6. for β-amino acids, e.g. β-Ala and taurine; and

7. for dipeptides, e.g. peptide-1 (pep-1), glycylsarcosine, etc. These dipeptides are absorbed through the intestinal cells where they are hydrolyzed by dipeptidases to the constituent amino acids.

Besides, the small intestine of the fetus and the neonate can absorb intact proteins by endocytosis (pinocytosis).

Absorption of amino acids may be affected in specific transport disorders or other diseases involving the small intestine or the immune system, such as Hartnup’s disease and cystinuria.

AMINO ACIDS TRANSPORT DISORDERS

Hartnup’s disease: It is a genetic lesion in epithelial amino acid transport, named after the family in which the disease was first recognized. Transport and absorption of neutral amino acids including that of tryptophan, is decreased in the epithelial cells of the small intestine and proximal tubules of the kidney. The intestinal defect results in malabsorption of free amino acids from the diet. The inability to reabsorb amino acids by the kidney manifests in the excretion of neutral amino acids including tryptophan and other indole derivatives in the urine, resulting in neutral amino aciduria. Symptoms of the disease are mainly those due to tryptophan amino acid and nicotinamide deficiencies. Due to tryptophan deficiency there are pellagra like features, such as dermatitis, diarrhoea and dementia. Women are more susceptible to pellagra since kynurenine hydroxylase (an enzyme of the tryptophan niacin pathway) is inhibited by estrogen.

Cystinuria: It involves the carrier for sulfur-containing amino acid, cystine and the basic amino acids, i.e. lysine and arginine.

NITROGEN BALANCE

Comparison of nitrogen intake (dietary protein) to nitrogen excretion (sum of all sources of nitrogen excretion, such as in urine and feces, from the skin and hair, and in body fluids) gives a measure of nitrogen balance and indicates protein status of the body.

When nitrogen intake exceeds nitrogen excretion, body is said to be in a positive nitrogen balance. Positive nitrogen balance means body is adding protein, as in the case for growing children, pregnant women and people recovering from protein deficiency or illness.

When nitrogen excretion exceeds nitrogen intake, body is said to be in negative nitrogen balance. This means that body is losing protein, such as during starvation, on weight loss diets, during fever, severe illness or infections.

When nitrogen intake is equal to nitrogen excretion, the nitrogen balance is zero and body is in nitrogen equilibrium, such as in healthy individuals. There is no net gain or loss of the body protein.

Nitrogen Fixation

It is a process that converts atmospheric N₂ to a biologically useful form. This is done by few strains of bacteria (called diazotrophs), such as cyanobacteria. They produce enzyme nitrogenase, which catalyzes the reduction of N₂ to NH₃. This enzyme is not found in human beings.

Nitrogen Assimilation

In bacteria and plants (but not in animals), nitrogen (as ammonia) is assimilated (incorporated) into α-ketoglutarate, to produce glutamate. Once it is assimilated as glutamate, it can be used in the biosynthesis of other amino acids by transamination, in human beings.

CATABOLISM OF AMINO ACIDS

There are two processes by which the α-amino group can be removed from amino acids. These are referred to as transamination and deamination.

TRANSAMINATION

Transamination is a process of transfer of the α-amino group from an amino acid to a keto acid. As a result of it, the amino acid is converted to a keto acid while the keto acid is converted to an amino acid (Fig. 10.19).

Fig. 10.19.: Transamination reaction.

Enzymes, which catalyze transamination, are called transaminases (aminotransferases). Although, liver contains transaminases for all the amino acids (except for threonine and lysine), most important of these transaminases are glutamate pyruvate transaminase (GPT, also called alanine aminotransferase or ALT) and glutamate oxaloacetate transaminase (GOT, also called aspartate aminotransferase or AST). These two transaminases catalyze the following reactions:

An obligate amino acid or the α-keto acid, which participates in these reactions, is glutamate or α-ketoglutarate.

During the process of transamination, the amino acid first reacts with the coenzyme pyridoxal-5-phosphate (PLP) and forms Schiff’s base. Coenzyme in turn becomes protonated and is subsequently hydrolyzed to release the α-keto acid of the corresponding amino acid while retaining the α-amino group (−NH₂). Pyridoxal-5-phosphate thus gets aminated and is converted to pyridoxamine-5-phosphate. Thereafter, α-keto acid combines with the coenzyme bound amino group (pyridoxamine-5-phosphate) which in turn releases the keto acid as a new amino acid and regenerates the coenzyme (Fig. 10.20).

Fig. 10.20. Role of Vitamin B₆ in transamination.

DEAMINATION

Process of removal of the α-amino group from amino acids is referred to as deamination. Liver and kidney are the main organs, which are responsible for the deamination of amino acids. Deamination may take place either by oxidative or nonoxidative process.

Oxidative Deamination

Liver and kidney contain several enzymes, which catalyze deamination of amino acids, such as amino acid oxidases, glutamate dehydrogenase, etc.

Amino Acid Oxidases: L-amino acid oxidase is linked to FMN (the prosthetic group) and catalyzes the oxidation of an L-amino acid producing α-keto acid, ammonia and water, as the final products (Fig. 10.21).

Fig. 10.21. Oxidative deamination.

Some of the tissues also contain FAD-linked D-amino acid oxidases which catalyze oxidation of glycine and the D-amino acids.

Glutamate Dehydrogenase: Glutamate is produced from α-ketoglutarate by the enzyme glutamate dehydrogenase (GDH). This enzyme is also important for the release of ammonia from glutamate, when glutamate is degraded and used for gluconeogenesis.

Glutamate dehydrogenase thus, catalyzes reversible set of reactions. NADPH is used as coenzyme in the synthetic reaction while NAD⁺ is used in the degradative reaction (Fig. 10.22).

Fig. 10.22: Reactions catalyzed by glutamate dehydrogenase.

DIAGNOSTIC SIGNIFICANCE OF TRANSAMINASES

Serum GOT and GPT have diagnostic significance. They are present in large amounts in cardiac muscle and liver, respectively. Although serum levels of both the enzymes are raised in heart as well as liver diseases, SGOT is markedly raised in myocardial infarction while SGPT is more increased in liver disorders such as viral hepatitis or toxic liver necrosis (Table 10.6).

Table 10.6. Diagnostic significance of transaminases

Enzyme | Comparatively more raised in
SGOT | Cardiac disorders, e.g. myocardial infarction
SGPT | Liver disorders, e.g. viral hepatitis

Non-oxidative Deamination

Dehydratase, sulfhydrase and histidase catalyze deamination of serine, cysteine and histidine, respectively. They catalyze dehydration followed by nonoxidative deamination of the amino acid, spontaneously, (Fig. 10.23).

Fig. 10.23. Non-oxidative deamination.

METABOLIC FATES OF AMMONIA

Ammonia is removed as glutamine from the brain. It is also removed as urea, by urea cycle, from the liver.

TRANSPORT OF AMMONIA AS GLUTAMINE

From the brain, ammonia is removed as glutamine, which is synthesized by the enzyme glutamine synthetase. Glutamine is transported to the kidney, where glutaminase removes the amide group from glutamine as ammonia, which is excreted in the urine (Fig. 10.24).

Fig. 10.24. Synthesis and degradation of glutamine.

Urea Cycle

Urea cycle is the process by which ammonia, that is a highly toxic substance, is converted to urea, which is a less toxic, water soluble, excretory waste product, in the liver (Fig. 10.25).

1. In the first step, ammonia reacts with carbon dioxide and forms carbamoylphosphate, in the mitochondria.

This reaction is catalyzed by the enzyme carbamoylphosphate synthetase (CPS), which requires N-acetylglutamate as coenzyme.

2. Thereafter, ornithine transcarbamoylase transfers carbamoyl group from carbamoylphosphate to ornithine and forms citrulline.

3. In the presence of the enzyme argininosuccinate synthetase, citrulline combines with L-aspartate and forms argininosuccinate.

4. Subsequently, argininosuccinase hydrolyzes argininosuccinate to arginine and fumarate.

Fumarate, which is released in this reaction, enters mitochondria where it is converted to oxaloacetate via citric acid cycle.

Hence, urea cycle is coupled with the Krebs cycle, both of which were discovered by Sir Hans Krebs and Coworkers.

5. In the last reaction, arginine is hydrolyzed, by the enzyme arginase, to ornithine and urea.

Ornithine is transported to mitochondria and reenters the cycle.

This is the mechanism for the excretion of nitrogen in humans and other primates.

Two atoms of nitrogen, in urea, are derived from two different sources, i.e. from NH₃ and the amino group of aspartate.

Fig. 10.25. Urea cycle.

Regulation of urea cycle

N-acetylglutamate acts as an allosteric activator for the enzyme carbamoylphosphate synthetase.

Induction of the urea cycle enzymes occurs when either the delivery of ammonia or amino acids, to the liver, is increased. Thus, a high protein diet and starvation result in induction of enzymes of the urea cycle.

DEFECTS IN THE UREA CYCLE ENZYMES

Since ammonia is very toxic, a metabolic disorder of urea cycle may result in coma and may be potentially fatal.

Defects in various enzymes of the urea cycle have been reported (Table 10.7).

Table 10.7. Disorders of urea cycle

Enzyme | Disorder
Carbamoylphosphate synthetase | Hyperammonemia type I
Ornithine transcarbamoylase | Hyperammonemia type II
Argininosuccinate synthetase | Citrullinemia
Argininosuccinase | Argininosuccinic aciduria
Arginase | Hyperargininemia

CATABOLISM OF CARBON SKELETON OF AMINO ACIDS

A large proportion of the amino acids, within the cells, are incorporated into proteins, which are constantly being synthesized and degraded. Excess of these dietary amino acids are not simply excreted but are degraded to common metabolites and are used as metabolic fuel.

Carbon skeletons of amino acids, after the removal of α-amino group as ammonia, form amphibolic intermediates, which can be converted to either glucose or ketone bodies. For example, transamination of alanine, glutamate and valine forms pyruvate, α-ketoglutarate and succinyl CoA, respectively. As these are the intermediates of the citric acid cycle and can be converted to glucose by gluconeogenesis, these amino acids are called glucogenic amino acids.

Catabolism of some other amino acids such as phenylalanine, in addition to an intermediate of the citric acid cycle, also forms acetyl CoA or acetoacetyl CoA, which are the precursors of the ketone bodies. Therefore, these amino acids are referred as both, glucogenic and ketogenic.

Leucine is purely a ketogenic amino acid as the end products of its catabolism are acetyl CoA and acetoacetyl CoA. Lysine is also a ketogenic amino acid as the end product of its catabolism is acetoacetyl CoA.

End products of the catabolism of different amino acids and their metabolic fates are shown in Table 10.8.

Table 10.8. Metabolic fate and end products of catabolism of amino acids

Metabolic fate of amino acids | End products of their catabolism
Glucogenic |
Gly, Ala, Ser, Cys, HO-Pro | Pyruvate
Glu, Gln, Pro, Arg, His | α-Ketoglutarate
Val, Met | Succinyl CoA
Asp, Asn | Oxaloacetate
Glucogenic and Ketogenic
Ile | Succinyl CoA and Acetyl CoA
Phe and Tyr | Fumarate and Acetoacetyl CoA
Trp | Pyruvate and Acetoacetyl CoA
Thr | Pyruvate and Acetyl CoA
Ketogenic |
Leu | Acetyl CoA and Acetoacetyl CoA
Lys | Acetoacetyl CoA

CATABOLISM OF AROMATIC AMINO ACIDS

CONVERSION OF PHENYLALANINE TO TYROSINE

As men and most animals cannot synthesize aromatic ring hence, phenylalanine is dietary essential. Under normal conditions, dietary phenylalanine that is not incorporated into proteins is converted to tyrosine.

Conversion of phenylalanine to tyrosine is catalyzed by phenylalanine hydroxylase (Fig. 10.26).

Fig. 10.26. Conversion of phenylalanine to tyrosine.

CATABOLISM OF TYROSINE

As tyrosine can be synthesized from phenylalanine, tyrosine is dietary nonessential.

In the liver, tyrosine is transaminated to p-hydroxyphenylpyruvate, by tyrosine amino-transferase (tyrosine transaminase). It is an inducible enzyme whose synthesis is increased by glucocorticoids as well as tyrosine.

In the next step, a copper containing enzyme p-hydroxyphenylpyruvate oxidase, converts p-hydroxyphenylpyruvate to homogentisic acid.

p-Hydroxyphenylpyruvate oxidase is a complex which requires ascorbic acid and catalyses decarboxylation, oxidation, migration of the side chain as well as its hydroxylation.

Subsequently, an iron-containing enzyme, homogentisic acid oxidase, oxidizes homogentisic acid.

Subsequent reactions catalyzed by maleyl-acetoacetate isomerase and fumaryl-acetoacetate hydrolase result in the formation of the end product of tyrosine catabolism, i.e. fumarate and acetoacetate (Fig. 10.27).

Fig. 10.27. Degradation of tyrosine in the liver.

Metabolic fates of Tyrosine

Phenylalanine and tyrosine also serve as precursors for a number of biologically active compounds, in different tissues (Fig. 10.28).

Fig. 10.28. Metabolic fates of tyrosine.

Synthesis of Amino Acid Hormones

Synthesis of Catecholamines

In the adrenal medulla, tyrosine is converted to norepinephrine (noradrenaline) and epinephrine (adrenaline), which are the two hyperglycemic hormones, secreted by the gland.

Tyrosine is first converted to dihydroxy-phenylalanine (DOPA), by tyrosine hydroxy-lase. Thereafter, DOPA-decarboxylase converts DOPA to dopamine, which enters storage granules as the active neurotransmitter. Dopamine is then converted to norepinephrine, by dopamine-β-hydroxylase.

Norepinephrine is a hormone, present in sympathetic nerve endings. Its synthesis is regulated by plasma tyrosine in the brain.

Methylation of norepinephrine, by phenyl-ethanolamine-N-methyltransferase (PNMT), forms epinephrine. In this reaction active methionine (S-adenosylmethionine) is used as a methyl donor (Fig. 10.29).

Fig. 10.29. Synthesis of catecholamines.

These aromatic amines, with a catechol ring (3,4-dihydroxyphenyl ring), i.e. dopamine, norepinephrine and epinephrine, are collectively, called catecholamines.

Catecholamines have general modulatory effects on overall brain functions, such as mood and arousal. These include norepinephrine (noradrenaline), its methylated derivative called epinephrine (adrenaline) and dopamine. Norepinephrine is a major transmitter in the sympathetic nervous system. Stimulation of these nerves is responsible for various features of the fight or flight response such as stimulation of the heart rate, sweating and vasoconstriction in the skin and bronchodilation.

Epinephrine is produced by the adrenal medulla, under the influence of acetylcholine containing nerves, analogous to the sympathetic preganglionic nerves. It has stimulatory effects on glycogen metabolism in the liver.

Norepinephrine and epinephrine are released from storage vesicles in the adrenal medulla, in response to stress, exercise, cold and low levels of blood glucose. They increase degradation of glycogen and triacylglycerols as well as increase blood pressure and the output of the heart.

Dopamine is an intermediate in the synthesis of norepinephrine. It is a neurotransmitter in nerves that interconnect the nuclei of the basal ganglia in the brain and controls voluntary movement. Damage to these nerves causes Parkinson’s disease, which is characterized by tremor and difficulties in initiating and controlling movement.

Synthesis of Thyroid Hormones

In the thyroid gland, iodination of tyrosyl residues of the thyroglobulin, results in the synthesis of thyroid hormones.

Synthesis of Melanin

In the melanocytes, tyrosine is converted to melanin.

Tyrosine is first converted to DOPA and then to dopaquinone, by the enzyme tyrosinase (a copper containing protein). Subsequently, dopaquinone forms melanin, a brown-black pigment, which is found in the skin, hair and retina (Fig. 10.30).

Fig. 10.30. Synthesis of melanin.

CATABOLISM OF TRYPTOPHAN

Tryptophan is an essential amino acid containing an indole ring. The major pathway for the degradation of tryptophan in the liver is called kynurenine anthranilate pathway.

DISORDERS OF PHENYLALANINE AND TYROSINE METABOLISM

Phenylketonuria

Autosomal recessive deficiency of hepatic phenylalanine hydroxylase results in a condition called phenylketonuria.

The alternative pathway catabolizes an excess of phenylalanine, which cannot be converted to tyrosine. It is transaminated to phenylpyruvic acid, which is subsequently reduced to phenyllactic acid as well as decarboxylated to phenylacetic acid (Fig. 10.31).

Due to increased urinary excretion of phenylpyruvic acid (a phenylketone), this disease is called phenylketonuria. Increased levels of phenylketoacids in the blood cause mental retardation and convulsions.

Such infants are given low phenylalanine diet and under these conditions tyrosine becomes dietary essential.

Tyrosinemias

A complete or partial deficiency of tyrosine aminotransferase may result in tyrosinemia, which in turn leads to accumulation and excretion of tyrosine and its metabolites. Tyrosinemias are of two types:

Tyrosinemia type I: It is called hepatorenal tyrosinemia. It is caused by the deficiency of fumarylacetoacetate hydrolase and is characterized by liver failure, renal tubular dysfunction, rickets and polyneuropathy.

Tyrosinemia type II: It is also called oculocutaneous tyrosinemia. It is due to the deficiency of tyrosine transaminase. It results in eye and skin lesions and mental retardation.

Alkaptonuria

Hereditary deficiency of homogentisic acid oxidase causes alkaptonuria. Such individuals excrete homogentisic acid in their urine. Homogentisic acid, in turn, gets auto-oxidized and is subsequently polymerized to form intensely dark substance, referred to as alkapton.

Dark urine is the only consequence of this condition, in early life. Homogentisate is slowly oxidized and deposited in the bone, connective tissue and other organs. This condition is called ochronosis because of ochre color of the pigment.

Albinism

Lack of tyrosinase, in the melanocytes, affects the synthesis of melanin and leads to the formation of white patches on the skin and iris in the eyes. This condition is referred to as albinism.

Lack of the pigment in the skin makes these albinos more sensitive to sunlight and increases the risk of carcinoma of skin. Lack of the pigment in the eyes contributes to photophobia.

Various disorders of phenylalanine and tyrosine metabolism are shown in Table 10.9.

Table 10.9. Disorders of phenylalanine and tyrosine metabolism

Metabolic disorder | Deficient enzyme
Phenylketonuria | Phenylalanine hydroxylase
Tyrosinemia type I | Fumarylacetoacetate hydrolase
Tyrosinemia type II | Tyrosine transaminase
Alkaptonuria | Homogentisic acid oxidase
Albinism | Tyrosinase

Fig. 10.31. Metabolism of phenylalanine in phenylketonuria.

Kynurenine Anthranilate Pathway

• In this pathway, degradation of tryptophan begins with the opening of the indole ring, which in turn results in the formation of N-Formylkynurenine. This reaction is catalyzed by a heme containing enzyme tryptophan pyrrolase, also called as tryptophan oxygenase, which is a dioxygenase. This enzyme is found in the liver and is induced by glucocorticoids and glucagon.

• With the removal of the formate, formylkynurenine is converted to kynurenine, by the enzyme formylase.

• Subsequently, kynurenine is hydroxylated to 3-hydroxykynurenine, by the enzyme kynurenine hydroxylase.

• With the cleavage of alanine from side chain, by the enzyme kynureninase, 3-hydroxy-kynurenine is converted to 3-hydroxy-anthranilate.

• Subsequent oxidation of 3-hydroxy-anthranilate, by the enzyme 3-hydroxy-anthranilate oxidase, forms an unstable intermediate, which is decarboxylated to 2-amino-3-carboxymuconate-6-semialdehyde.

• Through a number of steps, it is then converted to acetoacetyl CoA (Fig. 10.32).

Fig. 10.32. Kynurenine-anthranilate pathway.

Other Metabolic Fates of Tryptophan

Metabolic fates of tryptophan are shown in Fig. 10.33.

Fig. 10.33. Metabolic fates of tryptophan.

Synthesis of NAD

Kynurenine anthranilate pathway is also important for the synthesis of NAD⁺. In the liver and some other tissues, tryptophan is catabolized to an unstable intermediate referred to as 2-acroleyl-3-aminofumarate. Nonenzymatic cyclization of its forms quinolinic acid. There-after, quinolinate phosphoribosyltransferase (QPRT) transfers a ribonucleotide moiety from phosphoribosylpyrophosphate (PRPP) and leads to the synthesis of nicotinate mononucleotide, which is finally converted to NAD⁺ (Fig. 10.34).

Fig. 10.34. Synthesis of NAD.

Synthesis of Serotonin

In platelets, brain and intestinal epithelium, tryptophan is hydroxylated by tryptophan hydroxylase to 5-hydroxytryptophan, which is subsequently decarboxylated to 5-hydroxy-tryptamine (serotonin), by a decarboxylase (Fig. 10.35).

Fig. 10.35. Synthesis of serotonin.

Serotonin is a neurotransmitter and causes contraction of smooth muscle of arterioles and bronchioles.

Synthesis of Melatonin

Acetyltransferase, present in the pineal gland and the retina, synthesizes melatonin (N-acetyl-5-methoxytryptamine) from tryptophan (Fig. 10.36).

Fig. 10.36. Synthesis of melatonin.

Melatonin is a sleep inducing substance. It is synthesized mostly at night and is involved in the regulation of circadian rhythm. Melatonin, in turn, may inhibit the synthesis and secretion of other neurotransmitters such as dopamine and GABA.

INVESTIGATIONS AND THEIR INTERPRETATIONS

Serum Proteins

Serum proteins form a complex mixture consisting of mainly albumin and globulins, each with varied functions. The concentration of serum total proteins is 6–8 g/100 ml. Its concentration in serum is influenced by the nutritional state, hepatic function, renal function, disease states like multiple myeloma and metabolic errors.

Causes of low total protein concentration (hypoproteinemia) are overhydration, excessive loss of protein mainly albumin through the kidney in the nephrotic syndrome and from the skin after burns, loss through the intestine in protein losing enteropathy, decreased synthesis of proteins due to severe dietary protein deficiencies like kwashiorkor, severe liver disease or severe malabsorption.

Causes of raised total protein concentration (hyperproteinemia) are dehydration, hemoconcentration like in diarrhea, chronic inflammatory conditions, cirrhosis of liver, autoimmune diseases like rheumatoid arthritis.

Blood Urea

Urea is the end product of protein metabolism. Its normal concentration in blood is 20 to 40 mg/ 100 ml. The protein content of the diet influences blood urea concentration. Blood urea concentration may be low in late pregnancy, in starvation and in a diet grossly deficient in proteins.

Increase in blood urea may occur in various conditions, which may be classified as pre-renal, renal and post-renal.

Pre-renal causes: Dehydration (such as in severe vomiting, intestinal obstruction or severe diarrhea) may lead to high blood urea. Moderate increase is seen in diabetic coma, severe burns, post-operative stages, fever and high protein diet.

Renal causes: Blood urea concentration is increased in all forms of kidney disease such as acute glomerulonephritis, nephrosis, malignant hypertension and chronic pyelonephritis.

Post-renal causes: Conditions such as stones in the urinary tract, stricture of the urethra, enlarged prostate and tumors of the bladder, when urine flow is obstructed, cause reduction in glomerular filtration rate and a corresponding increase in blood urea.

SOME IMPORTANT QUESTIONS

1. What are proteins? Classify proteins, based on their solubility.

2. What are amino acids? Classify them.

3. Outline urea cycle. What is the site of urea synthesis? What is the need for urea synthesis in the body?

4. Describe various mechanisms of the removal of ammonia from amino acids.

5. How is ammonia removed from the body?

6. Explain how tyrosine is metabolized. Describe biologically important compounds derived from it?

7. Describe various fates of phenylalanine and tyrosine in the body.

8. Name enzymes and coenzymes of the following reactions :

(i) Synthesis of carbamoylphosphate

(ii) Conversion of phenylalanine to tyrosine

9. Discuss briefly:

(i) α-Helix

(ii) β-Sheets

(iii) Quaternary structure of proteins

(iv) Protein conformation

(v) Biological significance of amino acids

(vi) Aromatic amino acids

(vii) Sulfur-containing amino acids

(viii) Essential amino acids

(ix) Branched chain amino acid

(x) Basic amino acids

(xi) Classification of amino acids according to nutritional requirement

(xii) Reactions of urea cycle

(xiii) Synthesis of urea and its abnormalities

(xiv) Transamination and its importance

(xv) Metabolism of phenylalanine

(xvi) Processes of the removal of NH₃ from amino acids

10. Write notes on:

(i) Fibrous proteins

(ii) Globular proteins

(iii) Protein denaturation

(iv) Zwitterion

(v) Alkaptonuria

(vi) Catabolism of tyrosine

(vii) Phenylketonuria

(viii) Disposal of ammonia from the body

(ix) Deamination

CHAPTER
11

Nucleic Acids and
Protein Synthesis

Nucleic acids are the macromolecules, which occur in cells in association with basic proteins, such as histones and protamines. They are strongly acidic in nature and are negatively charged at physiological pH.

Repeated monomer units, called nucleotides, are the building blocks of nucleic acids, as amino acids to proteins or monosaccharides to polysaccharides.

NUCLEOTIDES

A nucleotide has 3 constituents and consists of phosphoric acid, pentose sugar and nitrogenous base.

SUGARS

Presence of the pentose sugar defines the type of the nucleic acid. There are two classes of nucleic acids called deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). As the name suggests, deoxyribonucleic acid contains deoxyribose whereas ribonucleic acid contains ribose.

Ribose

Ribose has three −OH groups, one each at carbon atoms 1, 2 and 3 of the pentose sugar.

Deoxyribose

Deoxyribose does not contain oxygen at the second carbon and thus has only two −OH groups, one each at carbon numbers 1 and 3. Deoxyribose thus, is also called 2-deoxyribose (Fig. 11.1).

Fig. 11.1. Ribose and deoxyribose.

Sugar molecule is linked to the nitrogenous base on one side through carbon number one (C-1) and to phosphoric acid on the other side through carbon number five (C-5).

NITROGENOUS BASES

Nitrogenous bases found in nucleotides are the derivatives of either a purine or a pyrimidine.

Purine is a nine-member ring containing 5 carbon and 4 nitrogen atoms. On the other hand, pyrimidine is a six-member ring with 4 carbon and 2 nitrogen atoms (Fig. 11.2).

Fig. 11.2. Purine and pyrimidine rings.

Various nitrogenous bases have different substitutions on the parent heterocyclic rings.

Purines

There are two types of purine bases found in nucleic acids. These are referred to as adenine (6-aminopurine) and guanine (2-amino-6-oxypurine).

Pyrimidines

Pyrimidine bases found in nucleic acids are cytosine (2-oxy-4-amino pyrimidine), uracil (2,4-dioxypyrimidine) and thymine (2,4-dioxy-5-methylpyrimidine). Thymine can also be referred to as 5-methyluracil (Fig. 11.3).

Fig. 11.3. Purine and pyrimidine bases found in nucleic acids.

Out of the three pyrimidine bases, only two are found in a nucleic acid. Usually, cytosine and uracil are found in RNA while cytosine and thymine are found in DNA.

Unusual nitrogenous bases

Besides the above nitrogenous bases (2 purines and 2 pyrimidines) that are commonly found in nucleic acids, there are few others, which may also be found in some of the nucleic acids. For example, 5-methylcytosine, 5-hydroxymethyl-cytosine, pseudouracil (where structural positions of N-1 and C-5 of the uracil are inter-changed and that instead of N-1, C-5 is linked to ribose), N⁶-methyladenine, N⁷-methylguanine, etc., are found in some RNAs. These nitrogenous bases are referred to as unusual nitrogenous bases, as they are not usually found in nucleic acids (Fig. 11.4).

Fig. 11.4. Some unusual bases found in nucleic acids.

Some Purine Derivatives of Metabolic Significance

Certain purine derivatives of metabolic significance are also found either as intermediates during purine biosynthesis such as hypoxanthine and xanthine, or as end product of purine catabolism, such as uric acid (Fig. 11.5).

Fig. 11.5: Some purine derivatives of metabolic significance.

NUCLEOSIDES

A nucleoside has two components, i.e. it consists of a nitrogenous base and a pentose sugar. The molecule of sugar (ribose or deoxyribose) is linked to the nitrogenous base with the N-glycosidic linkage.

To differentiate the carbon of the sugar from the carbon of the nitrogenous base, position of the carbon of the sugar is represented with a dash mark, called prime (′). These carbons are represented as C-1′, C-5′, etc. On the other hand, carbon and nitrogen of the nitrogenous base are representedwithouttheprimesign,as N-1,C-2,etc.

First carbon of the sugar (C-1′) is mostly linked to N-1 of the pyrimidine or N-9 of the purine:

Ribonucleosides

Nucleosides which contain ribose are called ribonucleosides. Accordingly, ribonucleosides of adenine, guanine, cytosine and uracil are designated as adenosine, guanosine, cytidine and uridine, respectively (Fig. 11.6).

Fig. 11.6. Ribonucleosides.

Deoxyribonucleosides

Nucleosides containing deoxyribose are designated as deoxyribonucleosides and are written with the prefix deoxy or d. Thus, deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine are the nucleosides containing deoxyribose with adenine, guanine, cytosine and thymine, respectively. Since thymine is usually found only in DNA and is linked to deoxyribose, its nucleoside is simply referred to as thymidine instead of deoxythymidine (Fig 11.7).

Fig. 11.7. Deoxyribonucleosides.

NUCLEOTIDES

A nucleotide has 3 constituents and consists of a nitrogenous base, a pentose sugar and phosphoric acid.

Sugar molecule (ribose or deoxyribose) is linked to the nitrogenous base on one side and to phosphoric acid on the other.

C-1′ of the sugar is generally linked to the nitrogenous base through N-1 of the pyrimidine or N-9 of the purine. Although any hydroxy group (–OH) of the sugar may be esterified with phosphoric acid but commonly C-5′ of sugar is linked to phosphate.

Ribonucleotides

Nucleotides of ribose are called ribonucleotides. Four ribonucleotides are generally obtained on hydrolysis of RNA. These are designated as adenosine monophosphate or adenylate (AMP), guanylate (GMP), cytidylate (CMP) and uridylate (UMP). Some of these are shown in Fig. 11.8.

Fig. 11.8. Ribonucleotides.

Deoxyribonucleotides

Deoxyribonucleotides are obtained on the hydrolysis of DNA and are designated as dAMP, dGMP, dCMP and dTMP (Fig. 11.9).

Fig. 11.9. Deoxyribonucleotides.

Nitrogenous bases, nucleosides and nucleotides, which are commonly obtained on the hydrolysis of nucleic acids, are given in Table 11.1.

Table 11.1. Nitrogenous bases, nucleosides and nucleotides, commonly obtained on the hydrolysis of nucleic acids

Nitrogenous bases | Nucleosides (Base + Sugar) | Nucleotides (Base + Sugar + P)
RNA |  |
Adenine | Adenosine | AMP
Guanine | Guanosine | GMP
Cytosine | Cytidine | CMP
Uracil | Uridine | UMP
DNA |  |
Adenine | Deoxyadenosine | dAMP
Guanine | Deoxyguanosine | dGMP
Cytosine | Deoxycytidine | dCMP
Thymine | Thymidine | TMP

NUCLEOTIDE DERIVATIVES HAVE BIOLOGICAL SIGNIFICANCE

Besides the nucleotides of adenine, guanine, cytosine, uracil and thymine, which are generally obtained as hydrolyzed products of nucleic acids, their di and triphosphates such as ADP, ATP, GDP, GTP, CTP, UTP, etc., also occur in the body and have biological significance. For example, they are used in the biosynthesis of nucleic acids. In addition, ATP is also used as a high energy phosphate compound in various energy requiring processes. CTP is used in the biosynthesis of phosphoglycerides.

Besides, various sugar derivatives of UDP, such as UDP-glucose and UDP-galactose, are important in the interconversion of glucose and galactose. UDP-glucuronic acid is used in conjugation reactions.

Cyclic nucleoside monophosphates of adenine and guanine, i.e. cyclic-3′,5′-AMP (cAMP) and cyclic-3′,5′-GMP (cGMP), mediate series of reactions and act as intracellular signals. S-adenosylmethionine (active methionine) serves as a methyl donor). NAD⁺, NADP⁺, FAD, etc., are used as coenzymes (Fig. 11.10).

Fig. 11.10. Some nucleotide derivatives of biological significance.

POLYNUCLEOTIDES

Polymerization of the monomeric units, i.e. the nucleotides, leads to the formation of a polynucleotide (nucleic acid).

Nucleotides are linked together by 3′,5′-phosphodiester linkage where hydroxy group of the sugar molecule of one nucleotide is linked to the hydroxy group of the sugar moiety of the other nucleotide.

On one side of the polynucleotide there is a free 5′-hydroxy or phosphate while on the other end there is a free 3′-phosphate or hydroxy group. These are referred to as 5′-end and 3′-end, respectively.

Conventionally, first letter of the base, i.e. A, G, C or T, is written to represent a nucleotide. Since each nucleotide is linked through the phosphate moiety (p), thus pApGpT represents that there is AMP with phosphate at the 5′ terminus and TMP with a free hydroxy group at the 3′ terminus (Fig. 11.11).

Fig. 11.11. A polynucleotide.

Two types of the polymers of nucleotides found in nature are called deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They are essential for the storage and transfer of genetic information as well as protein synthesis.

DEOXYRIBONUCLEIC ACID

Deoxyribonucleic acid (DNA) stores genetic information and is called chemical basis of heredity. DNA is a large molecule that occurs in various sizes with few hundred to several million nucleotides. It is a double stranded polymer of deoxyribonucleotides, which are linked together by phosphodiester bonds.

STRUCTURE OF DNA

Watson-Crick Model

Watson and Crick described various features of the structure of DNA. It is called Watson-Crick Model of DNA. They described that:

1. DNA is a polynucleotide of deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxy-cytidylate (dCMP), and thymidylate (TMP).

2. Information content of DNA resides in the sequence in which various deoxyribonucleotides are arranged.

3. The polymer possesses a polarity. Its left-hand terminal has a free hydroxyl group or phosphate. This terminal is called 5′-terminus. The right-hand terminal also has a free phosphate or a hydroxyl group that is referred to as the 3′-terminus.

4. DNA is a double stranded molecule, i.e. it has two strands (polynucleotide sequences or chains), which run antiparallel to each other. Here one strand runs in 5′→3′ direction while the other strand runs in 3′→5′ direction (Fig. 11.12).

Fig. 11.12: Double stranded DNA.

5. Both the polynucleotide chains are wound around a common axis forming a right handed helix, which is nearly 20Å in diameter (Fig. 11.13).

Fig. 11.13. DNA double helix.

6. The helix has a pitch of 34 Å and has10 base pairs (bp) per turn.

7. Both the strands of the helix are held together by the hydrogen bonds, which are formed between a purine and a pyrimidine. This base pairing is very specific as A is always linked to T with two hydrogen bonds while G to C with three hydrogen bonds.

8. DNA molecule thus, has equal number of adenine and thymine residues as well as guanine and cytosine residues. This relationship is known as Chargaff’s rule.

9. Bases occupy core of the helix while sugar-phosphate chain runs along the periphery. This in turn forms two grooves of unequal width. These are called the major groove and the minor groove.

Types of DNA

There are 3 different structural forms of DNA, referred to as B-DNA, A-DNA and Z-DNA.

B-D NA: This is the most common biologically active form of DNA, which has structural features as defined by Watson and Crick, i.e. it has two antiparallel polynucleotide strands that are wound right handed, around a common axis.

Bases occupy core of the helix. Each of its base pair has nearly same width that accounts for the symmetry of the molecule. Helix has 10 base pairs (bp) per turn and a pitch of 34 Å.

A-DNA: Under dehydrating conditions B-DNA can undergo reversible conformational change and produces a structure, which is referred to as A-DNA. It becomes wider and flattens into a right handed helix. A-DNA has 11 bp per turn and a pitch of 28 Å with an axial hole. Further, plane of its base pair is tilted 20° with respect to its axis. A-DNA has deep major groove and a very shallow minor groove.

Z-DNA: Z-DNA has 12 bp per turn, a pitch of 45 Å and a deep major groove but no identifiable minor groove. It has complementary polynucleotides with alternating purines and pyrimidines, such as poly d (GC).d (AT) or poly d (AC).d (GT), assuming a Z (zig zag) conformation.

RIBONUCLEIC ACIDS

Three main classes of RNA are known to occur in cells. They are known as messenger RNA, ribosomal RNA and transfer RNA. Though all the three types of RNA are involved in protein biosynthesis, they differ from each other with respect to size, composition and location within the cell, as well as functions.

Types of RNA

Messenger RNA

Messenger RNA (mRNA) is a group of RNAs, which is most heterogeneous. They are highly elongated and short lived molecules that are synthesized within the nucleus, as a result of transcription.

The mRNA transcript has 5′→3′ polarity, which is complementary to the coding strand of the gene (DNA molecule) with the difference that mRNA contains uracil, in place of thymine found in DNA (Fig. 11.14).

Fig. 11.14. mRNA.

An mRNA conveys message (information) from gene to the protein synthesizing machinery. This information is present in mRNA, in the form of sequences of nucleotides, as triplets, called codons. Each mRNA serves as a template on which a specific sequence of amino acids is polymerized to form a specific protein, which is the ultimate product of gene. They are generally stable in eukaryotes but quite unstable in prokaryotes.

In mammalian cells, transcription product of DNA is a large molecule, called heterogeneous nuclear RNA (hnRNA), which undergoes post-transcriptional changes and gets converted to mRNA that is released into the cytosol, for protein biosynthesis.

Transfer RNAs

Transfer RNA (tRNA) molecules are much smaller in size and consist of nearly 75 to 90 ribonucleotides. Total tRNAs comprises of about 15 per cent of the total cellular RNA. It is also known as soluble RNA (sRNA) because it is found in the soluble fraction of the cell.

There are about 50 different types of tRNA, each one of which fulfils its role as an adapter molecule and has a sequence of three nucleotides, called anticodon. Each tRNA binds an amino acid and locates itself at the correct codon for the translation of information that is present in the sequence of nucleotides on the mRNA and is translated into the specific amino acids, forming a protein chain.

Various tRNAs though differ from each other with respect to their nucleotide sequences, as a class they have many features in common:

1. The nucleotide sequence (primary structure) allows extensive folding and intramolecular base pairing, to form a secondary structure, giving it a cloverleaf like structure.

2. All tRNA molecules, in addition to four usual bases (A, G, C and U), have several unusual (modified) bases, e.g. 7-methylguanine, thymine, dihydrouracil, pseudouracil, hypo-xanthine, etc.

3. All tRNA molecules have 4 arms, which are referred to as the amino acid acceptor arm, anticodon arm, DHU arm and TφC arm.

• Amino acid acceptor arm: Amino acid acceptor arm has 7 base paired stem, which terminates in CCA (5′→3′ sequence) whose A (the adenosyl moiety) accepts an amino acid. The carboxy group of the amino acid forms an ester linkage with the 3′-hydroxy group of the adenosyl moiety.

• Anticodon arm: The anticodon arm has 5 base pairs and a loop, which has a triplet, i.e. a sequence of three nucleotides (called anticodon) that is complementary to the codon. This anticodon is responsible for the specificity of the tRNA molecule.

• DHU arm: DHU arm has an unusual base, called dihydrouracil (DHU or UH₂), in its loop along with a 3 or 4 base paired stem.

• TφCarm:TφCarmhasasequenceofthymine (T), pseudouracil (φ) and cytosine (C), in its loop along with a stem having 5 base pairs.

• Extra arm: In addition to above four arms, tRNAs also have an extra arm, which has a small loop of 3–5 base pairs (Fig.11.15).

Fig. 11.15: tRNA.

Ribosomal RNAs

Ribosomal RNA (rRNA) molecules are present in ribosomes, in association with several polypeptides. Ribosomes are found in the cytosol of the cell and contain nearly 80% of the total RNA that is found in a cell. Both, mRNA and tRNA interact on ribosomes to translate the information, which is transcribed from the gene, into a specific protein.

Mammalian ribosomes are very complex in nature with a sedimentation velocity of 80S. Each ribosome has 2 subunits that are referred to as the large subunit (60S) and the small subunit (40S) (Fig. 11.16).

Fig. 11.16. Ribosomes.

The large subunit contains 3 different types of rRNA, which are designated as 5S, 5.8S and 28S rRNA. It also has more than 50 polypeptides. The small subunit has an RNA, which is referred to as 18S rRNA and nearly 30 polypeptides.

DIFFERENCES BETWEEN DNA AND RNA

DNA differs from RNA in several aspects (Table 11.2).

Table 11.2. Differences between DNA and RNA

| DNA | RNA
Sugar | Deoxyribose | Ribose
Bases | A, G, C and T | A, G, C and U
Strands | Double stranded | Usually single stranded
Purine/pyrimidine ratio | A = T and | No ratio
| G = C, also |
| A + G = C + T |

BIOLOGICAL SIGNIFICANCE OF NUCLEIC ACIDS

Nucleic acids perform various functions, such as that:

• DNA makes copies of it and thus transfers genetic information from one generation to the next. This process is known as replication.

• DNA also determines properties of a living cell and regulates biological information, by controlling protein synthesis through RNA.

The information first passes from DNA to RNA. This process is referred to as transcription. Thereafter, the sequence of bases from RNA is translated into the sequence of amino acids in a protein. This process is referred to as translation.

Thus, DNA serves as a template both, for the replication of information into the daughter DNA as well as for the transcription of this information into RNA and its subsequent translation into protein (Fig. 11.17).

Fig. 11.17. Functions of DNA.

GENETIC CODE

Genetic code is a dictionary that gives a correspondence between the sequences of four nucleotide bases that are found in mRNA to the sequence of twenty amino acids, which occur in peptides.

Each word of the genetic code is composed of three letters (nucleotide bases) and is referred to as codon. This is due to the reason that if one nucleotide codes for an amino acid, four nucleotides will code for (4)¹, i.e. four amino acids, only. If there are two nucleotides in a code, it will translate (4)², i.e. 16 amino acids, only. Hence, a 3:1 correspondence of nucleotides to amino acids with (4)³, i.e. 64 combinations, is there to code 20 amino acids.

Codons are present on the mRNA language as the nucleotides (U, C, A and G) and are written in a sequence from the 5′-end to the 3′-end. Codons thus, are the permutations of four bases, which are taken in the sets of three. These 64 combinations, of three bases each, define genetic code (Table 11.3).

Table 11.3. Genetic code

Salient features of the genetic code are described in Table 11.4.

Table 11.4. Salient features of genetic code

Degeneracy | Multiple codons code for same amino acid
Unambiguous | A specific codon codes for only a single amino acid
Non-overlapping | Reading of the genetic code occurs without overlapping
Punctuation | Message is read continuously without any break between the sequence of codons until a nonsense codon is reached
Universality | The same codon is used in all the living organisms, with only few exceptions in the mitochondria

Wobble Hypothesis

Each tRNA carries a specific amino acid only, which has three bases anticodon sequence. However, there are 61 codons, which designate an amino acid while most of the cells have nearly 50 tRNA species. This in turn suggests that certain variations from the standard base pairing in codon-anticodon interactions occur and that all the codons may not be used equally. Some codons may be read more efficiently by an anticodon, while some of the codons may be used less efficiently. This suggests that many amino acids can be carried by more than one tRNA species and it is also possible that the degenerate codon can be read by more than one tRNA.

The possibility by which one anticodon recognizes more than one codon for the specific amino acid, is described by the hypothesis that was given by Crick and is referred to as Wobble hypothesis. This explains that a base at the 5′- end of the anticodon (the first base of the anticodon or the third base of the codon) is not as well defined, with respect to its spatial configuration, as the other two bases. Thus, first two positions of the codon predominate in tRNA selection and that the third position, called degenerate position, is less important in this regard.

PROTEIN SYNTHESIS

The process of protein biosynthesis is called translation. It is the biochemical translation of the genetic information, from the four letter language of nucleotides to the twenty letter language of amino acids.

Protein synthesis (translation) requires mRNA, several tRNAs, ribosomes and various protein factors. Besides the activation of amino acids, the process of protein synthesis occurs in three stages, i.e. initiation, elongation and termination, along with the release of the polypeptide.

Activation of Amino Acids

For their incorporation into the polypeptide chain, amino acids are activated and attached to the appropriate tRNA carrier molecules. Linkage of the amino acid to its tRNA requires ATP and is catalyzed by the enzyme aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetase is a group of enzymes, each one of which is specific for an amino acid and its appropriate tRNA.

During the process of amino acid activation, a high energy complex (amino acid-AMP-enzyme complex) is formed as the intermediate. Each synthetase then recognizes the tRNA and transfers amino acid from the amino acid-AMP-enzyme complex to the corresponding tRNA (Fig. 11.18).

Fig. 11.18. Activation of amino acid.

Aminoacyl tRNA synthetase not only carries a correct amino acid but also edits protein synthesis, since wrong acylated-tRNA is recognized by the enzyme and removed.

During translation mRNA sequence is read in 5′→3′ direction whereas, due to codon-anticodon interaction, amino acids are incorporated from the amino terminal end to the carboxy terminal end.

Initiation of Protein Synthesis

Initiation of protein synthesis, in eukaryotes, involves assembly of various components of the translation system, i.e. two ribosomal subunits, mRNA, aminoacyl-tRNA specified by the codon, GTP and several initiation factors that facilitates assembly of the initiation complex.

First, the initiation codon (AUG) is recognized by the initiation tRNA (that carries methionine). This is facilitated by eIF-2a (eukaryotic initiation factor-2a), which binds to GTP and methioninyl-tRNA (Met-tRNA Met) and forms a ternary complex. Subsequently, it requires 40S ribosomal subunit that is associated with eIF-3.

Mammalian eIF-3 is a complex protein that has 8 different polypeptides. It is also called anti-association factor, since it blocks the association of the 40S ribosomal subunit with the 60S subunit.

A complex, comprising of the eIF-2a.Met-tRNAiMet. GTP, eIF-3.40S rRNA, eIF-4c and some more protein factors, is formed. This complex in turn binds to mRNA and forms preinitiation complex. In addition, eIF-4a and eIF-4b are required to unwind the secondary structure, in mRNA. It also requires eIF-4f (also called the cap-binding protein) that helps to place the message on the 40S subunit. During this process, a molecule of ATP is hydrolyzed and helps to locate and place the initiation sequence in mRNA.

The final pre-initiation complex thus, includes 40S ribosomal subunit, eIF-2a.Met-tRNAiMet. GTP ternary complex, correctly oriented mRNA and several protein factors.

Formation of this initiation complex proceeds with the involvement of the 60S subunit and eIF-5.

eIF-5, first interacts with the pre-initiation complex, GTP is hydrolyzed and eIF2a.GDP, eIF-3 and other factors are released. The 40S.Met-tRNAiMet.mRNA complex interacts with the 60S subunit and initiation factor eIF-4d, to generate 80S ribosome with the mRNA and initiator tRNA, which is correctly positioned with in the ribosome.

The eIF-2a.GDP complex so released interacts with the guanine nucleotide, eIF-2b and GTP, to regenerate eIF-2a.GTP, for another round (Fig. 11.19).

Fig. 11.19. Initiation of protein synthesis.

In E. coli, binding and positioning of the mRNA on the 30S ribosomal subunit, is facilitated by complementary base pairing between the pyrimidine rich sequence of 8 nucleotides, in the 16S RNA and a purine rich sequence of 10 nucleotides, which is designated as Shine-Dalgarno Sequence, present on mRNA.

Chain Elongation

Chain elongation ensures selection of the proper aminoacyl tRNA and movement of mRNA along with the associated tRNA through the decoding region of the ribosome.

The initiating methioninyl-tRNA is placed at such a position that it occupies the donor site, also called the peptidyl site or P-site, on the ribosome so that its methioninyl residue may be donated (transferred) to the free α-amino group of the incoming aminoacyl-tRNA. Aminoacyl-tRNA, specified by the next codon on the mRNA, is bound at the acceptor site, also called the aminoacyl site or A-site, on the ribosome.

Selection of the correct aminoacyl-tRNA is enhanced by elongation factor-1 (EF-1).

EF-1α component of the EF-1 first, forms a ternary complex with the aminoacyl-tRNA and GTP. This EF1α.aminoacyl-tRNA.GTP complex then binds on the ribosome and if codon anticodon interaction is correct, the aminoacyl-tRNA is placed at the A-site. During this process, GTP is hydrolyzed and EF-1α.GDP complex is dissociated. The initiating methioninyl-tRNA and the incoming aminoacyl-tRNA are now properly positioned on the ribosome on the P-site and the A-site, respectively. Their anticodons are paired with the successive codons on the mRNA (in the decoding region of the small subunits) while amino acids are present on the peptidyltransferase site of the large subunit.

For the formation of the peptide bond, peptidyltransferase catalyzes attachment of the α-amino group of the aminoacyl-tRNA to the carboxyl carbon of the methioninyl-tRNA. This in turn results in the transfer of methionine to the amino group of the aminoacyl-tRNA, which occupies a hybrid position, i.e. its anticodon remains in the 40S subunit on the A-site, while the acceptor-end along with the attached polypeptide is present in the 60S sub-unit, on the P-site. The anticodon of the deacylated tRNA remains in the 40S on the P-site while the acceptor is located in the 60S, towards the upper end, which may also be called the exit site (E-site).

mRNA and the dipeptidyl-tRNA at the 40S are now repositioned by the elongation factor-2 (EF-2), also called translocase, for another elongation cycle to proceed. EF-2 moves the mRNA and the dipeptidyl-tRNA in the codon anticodon register, from the A-site to the P-site. During this process, a molecule of GTP is hydrolyzed and the A-site becomes vacant. Simultaneously, the deacylated methioninyl-tRNA also moves to the E-site, which only exists in the 60S subunit.

Ribosomes now enter the new cycle. At each step, peptidyltransferase transfers the growing polypeptide from the carrier tRNA to the α-amino group of the amino acid residue of the aminoacyl tRNA, specified by the next codon and with the hydrolysis of GTP, stepwise elongation of the chain results in the completion of the polypeptide (Fig. 11.20).

Fig. 11.20. Chain elongation.

Termination

The presence of the chain terminating codon (UAG, UAA, or UGA) on the A-site does not promote binding of the tRNA. Instead, a non-ribosomal protein, called releasing factor (eRF), binds the ribosome as eRF.GTP complex.

The peptide-tRNA ester linkage is cleaved by the enzyme peptidyltransferase and poly-peptide is released from the carrier tRNA. Dissociation of the eRF from ribosome requires the hydrolysis of GTP and releases the ribosome to dissociate into two subunits. eRF is reutilized while the ribosomes re-enter the cycle, at the initiation stage (Fig. 11.21).

Fig. 11.21. Termination of protein synthesis.

Differences in the process of protein synthesis between prokaryotes and eukaryotes are shown in Table 11.5.

Table 11.5. Differences in the process of protein biosynthesis between prokaryotes and eukaryotes

Prokaryotes | Eukaryotes
The mRNA is usually polycistronic. | The mRNA is monocistonic. It also contains a poly-A tail.
Ribosomes are present in the free form. They are 70S in size, comprising of 50S and 30S subunits. | Ribosomes are present in the free form as well as bound to endoplasmic reticulum. They are 80S in size, consisting of 60S and 40S subunits.
Three initiation factors are required in the process. | One initiation factor is known to participate in the process.
N-Formylmethioninyl-tRNA binds to the start codon. | Met-tRNA is not formylated.
Three elongation factors are required. They are referred to as EF-Tu, EF-Ts and EF-G. | Two elongation factors are required. They are referred to as eEF-1 and eEF-2.
Termination of the process requires three releasing factors, which are referred to as RF-1, RF-2 and RF-3. | Termination of the process requires one releasing factor, which is designated as eRF.

INHIBITORS OF PROTEIN SYNTHESIS

Many antibiotics and toxins are known to inhibit the process of protein biosynthesis. They are useful in clinical practice, as therapeutic agents (Table 11.6).

Table 11.6. Inhibitors of protein biosynthesis

Streptomycin: It binds to the small subunit of the prokaryotic ribosome and interferes with initiation of protein synthesis. It also causes mis-reading of the mRNA.

Neomycin and Gentamycin: They interact with the small ribosomal subunit and cause mis-translation.

Tetracyclin: It binds directly to the ribosome and interferes in aminoacyl-tRNA binding.

Puromycin: It structurally, resembles an aminoacyl-tRNA. It thus, binds at the ribosomal A-site and acts as acceptor in peptidyltransferase reaction. Puromycin thus, prematurely terminates translation, leading to release of peptidylpuromycin complex.

Chloramphenicol: It directly inhibits peptidyltransferase, by binding the transferase.

Erythromycin: It interferes with the translocation process, on prokaryotic ribosomes.

Fusidic acid: It interferes in the process of translocation, by inhibiting EF-G (GTP-dependent translocase), in prokaryotes.

Cycloheximide: It inhibits elongation process, in eukaryotes.

Diphtheria toxin: It is a protein, which is produced by Corynebacterium diphtheriae that inhibits the process of translocation, in eukaryotes.

Toxins that attack RNAs and inhibit protein synthesis

Ricin: It is obtained from the castor bean. It cleaves adenine from rRNA, in the large subunit and thus inactivates the ribosome in eukaryotes.

α-Sarcin: It is a fungal toxin. It cleaves rRNA at the single site, with in the large subunit of the ribosome and inactivates the ribosome in eukaryotes.

Colicin E3: It is a toxin, which is obtained from E. coli. It has ribonuclease activity and cleaves 16S rRNA of the 30S ribosomal subunit, near the mRNA-binding sequence and the decoding region. It thus, inactivates small subunit of the ribosome and stops initiation of protein synthesis.

POST-TRANSLATION MODIFICATIONS

After synthesis of the polypeptide chain, several proteins undergo various changes. Since these modifications occur after translation of the process, these changes are referred to as post-translation modifications.

Post-translation modifications include partial proteolysis of the protein chain or covalent modifications with the addition of one or more groups on amino acids, in the protein chain.

Partial Proteolysis of the Protein Chain

Some proteins particularly, those destined for secretion from the cell, are synthesized as the large precursors that are not functionally active, e.g. insulin, several zymogens, etc. A small portion of the protein is removed by specialized endoproteases, which in turn results in the release of a biologically active protein.

Some of them are cleaved in endoplasmic reticulum or Golgi apparatus while others in developing secretory vesicles or after secretion. For example:

Conversion of preproinsulin to insulin: Insulin is synthesized as preproinsulin, which enters endoplasmic reticulum where signal peptidase cleaves the signal peptide and converts it to proinsulin. Proinsulin is then transported to the Golgi apparatus where it is packed into the secretory granules. The C-peptide (connecting-peptide) is removed by proteolysis and mature insulin is secreted (Fig. 11.22).

Fig. 11.22: Conversion of preproinsulin to proinsulin.

Abnormally processed insulin (proinsulin) is released into the circulation in an autosomal dominant condition, referred to as familial hyperproinsulinemia.

Activation of the zymogen: Proteolysis also leads to activation of the zymogen, such as proteases. Inactive zymogen precursors are packed in the storage granules and activated by proteolysis, e.g. trypsinogen is cleaved to trypsin or chymotrypsinogen is converted to chymotrypsin.

Covalent Modifications

As we know only 20 amino acids are encoded genetically. Post-translational modifications however, result in the attachment of some groups on these amino acids. Modification of the amino acid may occur in several ways, such as:

Phosphorylation: Phosphorylation may occur on the hydroxyl group of serine or threonine, in a protein, e.g. reversible phosphorylation of glycogen phosphorylase and glycogen synthase by protein kinase and protein phosphatase, respectively.

Glycosylation: Plasma membrane proteins or some other proteins, which are to be secreted from the cell, have carbohydrate chains attached to the hydroxyl group of serine or threonine by O-linkage or to the NH₂ group of asparagine by N-linkage. Stepwise addition of sugar molecules (glucose, galactose, mannose, fucose, etc.) occurs in the endoplasmic reticulum and Golgi apparatus. Glycosylation of the protein thus, forms a glycoprotein such as membrane bound glycoproteins.

Methylation: Methylation of the α-amino group of lysine occurs in histones. This in turn modulates their interaction with the DNA.

Hydroxylation: Proline and lysine residues in collagen are extensively hydroxylated in the endoplasmic reticulum.

GLYCOSYLATION OF PROTEINS

Glycosylation refers to an enzymatic reaction in which a carbohydrate (a glycosyl donor) is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor). The majority of proteins synthesized in rough endoplasmic reticulum undergo glycosylation. Commonly produced glycans include:

1. N-linked glycans: The glycosyl moiety is attached to the nitrogen of asparagine or arginine side chains.

2. O-linked glycans: The glycosyl moiety is attached to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine or hydroxyproline side chains.

3. Phosphoglycans: The glycosyl moiety is linked through the phosphate of a phosphoserine.

Many of the proteins secreted from cells, including those present in the serum, are glycoproteins. Carbohydrate attachment is important for processing, stability and targeting of these proteins. Protein glycosylation takes place inside the lumen of the endoplasmic reticulum and in the Golgi complex.

All N-linked oligosaccharides have, in common, a pentasaccharide core, consisting of three mannose and two N-acetylglycosamine residues. The N-linked glycosylation begins in the endoplasmic reticulum and continues in the Golgi complex whereas the O-linked glyco sylation takes place exclusively in the Golgi complex. The Golgi complex is the major sorting center of the cell from where proteins proceed to lysosomes, secretory granules or the plasma membrane, according to signals encoded within their amino acid sequences (Fig. 11.23).

Fig. 11.23. Protein glycosylation.

Carbohydrate groups have key roles in the function of many glycoproteins, particularly, as binding sites in their interactions with other macromolecules. They also add in the proper folding of the protein, to which they are attached. The addition of sugar to an oligosaccharide chain is catalyzed by a family of membrane bound enzymes, called glycosyltransferases. These enzymes transfer a specific monosaccharide from a nucleotide sugar, such as GDP-mannose or UDP-N-acetylglucosamine, to the growing end of the carbohydrate chain.

Errors in glycosylation can result in severe inherited human diseases called congenital diseases of glycosylation (CDGs). They are usually identified through blood tests that detect abnormal glycosylation of serum proteins. In I-cell disease, a lysosomal storage disease, there is deficiency of phosphotransferase. This in turn, results in large inclusion of undigested glycosaminoglycans and glycolipids in lysosomes. Patients suffer sever psychomotor retardation and skeletal deformities.

PROTEIN TARGETING

Many of the proteins, whose synthesis occurs in the cytoplasm of eukaryotic cells, are destined to perform their functions within specific cellular organelles. Such proteins usually contain amino acid sequences that direct these proteins to their final location. Proteins destined for secretion from the cell are targeted to RER-associated ribosomes by the presence of an N-terminal hydrophobic signal sequence. Nuclear proteins contain a nuclear localization signal whereas mitochondrial proteins have a mitochondrial entry sequence (Fig. 11.24).

Fig. 11.24. Protein targeting.

Free ribosomes synthesize proteins that remain within the cell, either within the cytoplasm or directed to organelles such as the nucleus, mitochondria and chloroplasts. Ribosomes bound to the endoplasmic reticulum usually, synthesize proteins destined to leave the cell or are in corporated into the cell membrane. These proteins fall into three major classes, i.e. secretary proteins (proteins exported by the cell), lysosomal proteins and proteins spanning the plasma membrane.

As the proteins are synthesized, they fold to form their three dimensional structures in the lumen of the endoplasmic reticulum. Finally, they are sorted and transported to their final destination. Transport is mediated by transport vesicles that bud off the endoplasmic reticulum. Transport proteins carry the cargo proteins to the Golgi complex where the vesicles fuse and deposit the cargo inside the complex. There, the cargo proteins are modified, e.g. the attachment of carbohydrates. From the Golgi complex, transport vesicles carry the cargo proteins to their final destination, e.g. plasma membrane, lysosomes or cell exterior.

SOME IMPORTANT QUESTIONS

1. Describe various features of the Watson-Crick Model of DNA.

2. Describe various types of RNAs with their functions.

3. What is a nucleotide? Name different nucleotides found in DNA. Describe various nucleotides of biological significance found in the body.

4. Describe the process of protein synthesis with diagrams.

5. Explain significance of post-translational modifications, with examples.

6. Write notes on :

(i) Structure and functions of tRNA

(ii) Differences between DNA and RNA

(iii) Various nitrogenous bases found in nucleic acids

(iv) Unusual nitrogenous bases

(v) Genetic code

(vi) B-DNA

(vii) A-DNA

(viii) Z-DNA

(ix) Nucleosides

(x) Nucleotides

(xi) hnRNA

(xii) Different types of DNA

(xiii) Structural features of tRNA

(xiv) Messenger RNA

(xv) Salient features of genetic code

(xvi) Protein synthesis and its inhibitors

(xvii) Wobble hypothesis

(xviii) Inhibitors of protein synthesis

(xix) Post-translational modifications

CHAPTER
12

Vitamins

Vitamins are the organic constituents of food, which are called as the protective accessory food factors since these are essential for life and well-being. Vitamins, in general, can not be synthesized by the body and have to be supplied in diet though in very small quantities.

Vitamins are classified into two groups, as fat-soluble and water soluble.

WATER SOLUBLE VITAMINS

Water soluble vitamins include vitamin B complex and vitamin C.

Typical deficiency symptoms for most of the water soluble vitamins, particularly those of vitamin B complex, include dermatitis, glossitis, cheilosis and diarrhea, besides some of the neurological symptoms such as peripheral neuropathy and malaise.

VITAMIN B COMPLEX

Vitamin B complex includes a group of compounds most of which are synthesized in the gastrointestinal tract by the microbial flora.

Derivatives of these vitamins act as coenzymes. These vitamins are not stored in the body and thus their toxicity is generally not observed.

Most of these vitamins are heat stable except vitamin B₁, which is heat labile.

Two members of vitamin B complex, i.e. folic acid and vitamin B₁₂, are important hemopoietic factors as they help in the prevention of anemia.

Various members of vitamin B complex include thiamin (B₁), riboflavin (B₂), niacin, pyridoxine (B₆), pantothenic acid, biotin, choline, lipoic acid, folic acid and cyanocobalamin (B₁₂).

THIAMIN

Thiamin is a first member of vitamin B complex, therefore it is also known as vitamin B₁. Due to its role in the prevention of beriberi, thiamin is also called antiberi-beri factor. Since thiamin has an important role in the nervous system, it is also called aneurine.

Structurally, thiamin consists of the pyrimidine ring that is linked to a thiazole ring.

Thiamin occurs in nature as thiamin hydro-chloride. Though thiamin is heat labile, it is quite stable in the acidic medium (Fig. 12.1).

Fig. 12.1. Thiamin.

Absorption, Transport, Storage and Metabolism

Thiamin is absorbed from the proximal small intestine by active transport (in low doses) and passive diffusion (in high doses).

It is phosphorylated in many tissues by specific kinases into the diphosphate (TPP) and triphosphate esters. The activated TPP is carried to the liver by the portal circulation. Approximately 90% of circulating thiamin is carried as TPP by erythrocytes. Tissues retain thiamine as phosphate esters, most of which are bound to proteins (chiefly albumin). Tissue levels of thiamin vary, with no appreciable storage of the vitamin.

TPP is catabolised by a phosphorylase to yield TMP. Along with it small amount of several other excretory metabolites are also produced and excreted in the urine.

Biochemical Functions

Thiamin plays an important role as a coenzyme in several biochemical reactions. Thiamin pyrophosphate (TPP), i.e. diphosphorylated form of the thiamin, is referred to as the coenzyme of vitamin B₁.

TPP plays an important role in carbohydrate metabolism. It is used as a coenzyme in oxidative decarboxylation of α-keto acids, e.g.

TPP also acts as a coenzyme with transketolase, an enzyme of the hexose monophosphate shunt.

Deficiency Symptoms

Thiamin deficiency causes beri-beri.

Requirements

Average daily requirement for thiamin is 1.2–1.8 mg/day. Its daily requirement, however, depends upon carbohydrate intake and has been defined as 0.4–0.5 mg/1000 Kcal.

Requirement is increased during fever and in hyperthyroidism, alcoholics and pregnant women.

Sources

Rich sources of thiamin include cereal grains, yeast, liver, kidney and heart.

RIBOFLAVIN

Riboflavin (B₂) is also called lactoflavin, due to its high content in milk. It is sensitive to light and ultraviolet irradiation. Riboflavin is heat stable in acidic as well as neutral medium.

Riboflavin has an isoalloxazine ring, which is attached to ribitol side chain (Fig. 12.3).

Fig. 12.3. Riboflavin.

Absorption, Transport, Storage and Metabolism

Riboflavin is absorbed in the proximal small intestine in the free form, by a carrier mediated process that requires ATP. Because most foods contain the vitamin in its coenzyme forms, FMN and FAD, absorption occurs only after the hydrolytic cleavage of free riboflavin from its various flavoprotein complexes, by phosphatases.

Riboflavin is transported in the plasma as free riboflavin and FMN, both of which are mainly bound to plasma proteins, primarily albumin. A specific riboflavin binding protein also functions in the transplacental movement of the vitamin. The vitamin is transported in its free form into cells by a carrier-mediated process. It is not stored in any useful amount in the body.

Riboflavin is converted to its coenzyme form FMN by ATP-dependent phosphorylation, by flavokinase. Most of FMN is them converted to FAD by FAD-pyrophosphorylase. Both steps are regulated by thyroid hormones, ACTH and aldosterone.

Most excess riboflavin is excreted as such in the urine.

BERI-BERI

Beri-beri refers to a cluster of symptoms caused primarily by a nutritional deficiency of thiamin (vitamin B₁). Historically, beri-beri has been endemic in regions dependent on polished (white or dehusked) rice. Symptoms of beri-beri include weight loss, emotional disturbances, impaired sensory perception, weakness and pain in the limbs, and periods of irregular heart rate. Edema (swelling of bodily tissues) is common. In advanced cases, the disease may cause high output cardiac failure and death.

Beri-beri is divided into three historical classifications:

Dry beri-beri: It specially affects peripheral nervous system. There is wasting and partial paralysis resulting from damaged peripheral nerves. It is also referred to as endemic neuritis (Fig 12.2).

Fig. 12.2. Beri-beri.

Dry beri-beri is characterized by difficulty in walking, loss of sensation (numbness) in hands and feet, loss of tendon reflexes, loss of muscle function or paralysis of the lower legs, mental confusion/speech difficulties, pain, involuntary eye movements and vomiting.

Wet beri-beri: It specially affects the cardiovascular and other bodily systems. Wet beri-beri affects the heart and circulatory system. It is sometimes fatal, as it causes a combination of heart failure and weakening of the capillary walls, which causes the peripheral tissues to become edematous.

Wet beri-beri is characterized by increased heart rate, vasodilation leading to decreased systemic vascular resistance and high output cardiac failure besides elevated juglar venous pressure, dyspnea (shortness of breath) on exertion and peripheral oedema (swelling of lower legs).

Infantile beri-beri: Infantile beri-beri usually occurs between two and six months of age, in children whose mothers have inadequate thiamine intake. It also affects the children of mal-nourished mothers. In the acute form, the baby develops dyspnea and cyanosis, and soon dies of heart failure.

Infantile beri-beri is characterized by hoarseness (where the child makes moves to moan but emits no sound or just faint moans caused by nerve paralysis), weight loss (becoming thinner and then marasmic as the disease progresses), vomiting, diarrhea, occasionally convulsions in the terminal stages, pale skin, edema, ill temper and alterations of the cardiovascular system, especially tachycardia (rapid heart rate).

Wernicke’s encephalopathy: It refers to beri-beri which may be caused by shortcomings other than inadequate intake, e.g. diseases or operations on the digestive tract, alcoholism, dialysis, genetic deficiencies, etc. Wernicke’s disease is one of the most prevalent neurological or neuro-psychiatric diseases.

Biochemical Functions

Riboflavin is used as a coenzyme in various biochemical reactions. Coenzyme forms of riboflavin include FMN and FAD.

FMN (flavin mononucleotide) has a molecule of riboflavin and phosphoric acid. FAD (flavin adenine dinucleotide) is a dinucleotide of FMN and AMP (Fig.12.4).

Fig. 12.4. FMN and FAD.

The coenzyme forms an integral part of the flavoprotein containing enzymes and participates in several oxidation reduction reactions.

FMN is used as a coenzyme with cytochrome C reductase and L-amino acid oxidases.

FAD is used as a coenzyme with succinate dehydrogenase, glycerol-3-phosphate dehydrogenase, sphingosine reductase, glycine oxidase, xanthine oxidase, D-amino acid oxidases and acyl CoA dehydrogenase. FAD is important in the metabolism of carbohydrates, lipids as well as proteins.

Deficiency Symptoms

In humans, signs and symptoms of riboflavin deficiency include cracked and red lips, inflammation of the lining of mouth and tongue, mouth ulcers, cracks at the corners of the mouth (angular cheilosis) and a sore throat (Fig. 12.5).

Fig. 12.5. Angular cheilosis.

Its deficiency may also cause dry and scaling skin, fluid in the mucous membranes and iron deficiency anemia. The eyes may also become bloodshot, itchy, watery and sensitive to bright light.

Requirements

Average daily requirement of riboflavin is 1–2 mg/day (0.5–0.6 mg/1000 Kcal).

Sources

Milk, liver and kidney are rich sources of vitamin B₂.

NIACIN

Niacin is also known as pellagra preventive factor (anti pellagra factor) because of its role in the prevention of pellagra.

Vitamin niacin refers to two compounds, both of which are the derivatives of pyridine ring. They are referred to as nicotinic acid and nicotinamide (Fig. 12.6).

Fig. 12.6. Niacin vitamins.

Absorption, Transport, Storage and Metabolism

Niacin in many animal foods consists mostly of coenzyme forms NAD and NADP, which are digested to release nicotinamide and nicotinic acid, the forms in which it is absorbed. In plant foods, it is found in covalently bound complexes with small peptides and carbohydrates. These forms are collectively referred to as niacytin and become bioavailable through alkaline hydrolysis.

Nicotimamide and nicotinic acid are absorbed in the stomach and small intestine by carrier-mediated facilitated diffusion. These are transported in the plasma in free solution and taken up by most tissues through passive diffusion. Niacin is retained in tissues by being converted, primarily to NAD but also to NADP.

The de novo synthesis of NAD and NADP also occur through quinolinic acid, from tryptophan. The conversion of tryptophan to niacin depends on the amount of tryptophan and niacin ingested, and pyridoxine status.

NAD and NADP are catabolized by hydrolysis to yield nicotinamide, which can be deaminated into nicotinic acid or methylated to yield 1-methyl-nicotinamide.

Biochemical Functions

Niacin acts as a coenzyme for several biochemical reactions. Two coenzyme forms of niacin include NAD (nicotinamide adenine dinucleotide) and its phosphorylated from, called NADP (nicotinamide adenine dinucleotide phosphate).

NAD⁺ is used as a coenzyme with malate dehydrogenase, which converts malate to oxaloacetate and lactate dehydrogenase (LDH), which converts lactate to pyruvate.

NADP⁺ is used as a coenzyme with isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase.

Both NAD⁺ and NADP⁺ can be used with glutamate dehydrogenase.

Deficiency Symptoms

Niacin deficiency is common in persons whose staple food is maize and jowar. Such individuals may develop pellagra-like symptoms since these grains are rich in isoleucine but deficient in tryptophan. This amino acid imbalance reduces biosynthesis of niacin in the body.

Deficiency of niacin can lead to a disease called pellagra.

PELLAGRA

Pellagra is a vitamin deficiency disease most frequently caused by a chronic lack of niacin in the diet. It can also be caused by decreased intake of tryptophan and possibly, by excessive intake of leucine. It may also result from alterations in protein metabolism, in disorders such as carcinoid syndrome.

Pellagra is classically described by the four Ds, i.e. diarrhea, dermatitis, dementia and death. A more comprehensive list of symptoms includes high sensitivity to sunlight (Fig. 12.7); dermatitis, alopecia (hair loss), edema (swelling); insomnia, weakness, mental confusion; ataxia (lack of coordination), paralysis of extremities, peripheral neuritis (nerve damage); diarrhea and eventually dementia. Prolonged deficiency of niacin may result in death.

Fig. 12.7. Pellagra.

Requirements

Average daily requirement of niacin is 12–20 mg (5.5–6.5 mg/1000 Kcal).

Sources

Rice polish, yeast, liver, poultry and green vegetables are good sources of niacin.

Milk and milk products though are not important sources of niacin but have high tryptophan contents and thus are important in the prevention of pellagra.

Biosynthesis from Tryptophan

This vitamin can be synthesized in the liver from tryptophan. Sixty mg of tryptophan is converted to one mg equivalent of niacin. This (60:1) is also called tryptophan niacin conversion ratio.

PYRIDOXINE

Pyridoxine, pyridoxal and pyridoxamine, all have vitamin B₆ activity. They contain a pyridine ring and are called vitamers of vitamin B₆ (Fig. 12.8).

Fig. 12.8. Compounds having vitamin B₆ activity.

Absorption, Transport, Storage and Metabolism

Vitamin B₆ is absorbed by passive diffusion, primarily in the jejunum and ileum. Absorption is driven by phosphorylation to form PLP and pyridoxamine phosphate, and then by protein binding of these metabolites in the intestinal mucosa and blood.

The predominant form of the vitamin in the blood is PLP, most of which is driven from the liver after metabolism by hepatic flavoenzymes. Muscle is the largest depot containing 80 to 90% of the total body vitamin stores, in the form of PLP bound to glycogen phosphorylase.

The vitamers B₆ are readily, metabolically, interconverted by phosphorylation dephosphorylation, oxidation reduction and amination deamination reactions. In the liver, PLP is dephosphorylated and oxidized by FAD and NAD-dependent enzymes to yield 4-pyridoxic acid and other inactive metabolites that are excreted in the urine.

Biochemical Functions

Vitamin B₆ acts as a coenzyme in several biochemical reactions. Coenzyme form of vitamin B₆ is referred to as pyridoxal-5-phosphate or PLP (Fig. 12.9).

Fig. 12.9. Pyridoxal-5-phosphate.

Pyridoxal-5-phosphate performs several biochemical functions:

1. Pyridoxal-5-phosphate is a coenzyme for glutamate pyruvate transaminase (GPT, also called alanine aminotransferase or ALT) and glutamate oxaloacetate transaminase (GOT, also called aspartate aminotransferase or AST). In these reactions, PLP acts as an acceptor of the amino group from an amino acid, thereby forming the corresponding α-keto acid and pyridoxamine phosphate. Followed by reversal of the process, pyridoxamine releases the amino group to another α-keto acid and forms the corresponding amino acid (Fig. 12.10).

Fig. 12.10. Role of vitamin B₆ as a coenzyme.

2. Pyridoxal-5-phosphate is also used with dehydratases and desulfhydrases for the metabolism of hydroxy and sulphur-containing amino acids.

3. Pyridoxal-5-phosphate is used as a coenzyme for decarboxylases, e.g. glutamate decarboxylase, which forms γ-aminobutyric acid from glutamic acid; dopa decarboxylase that forms dopamine from dopa; and 5-hydroxytryptophan decarboxylase which forms serotonin from 5-hydroxytryptophan. Thus, vitamin B₆ regulates neuronal activity.

4. Pyridoxal-5-phosphate is also required by the enzyme kynureninase. Thus, it is important in the conversion of tryptophan to niacin.

5. Pyridoxal-5-phosphate is also required in the synthesis of heme, in the formation of δ-aminolevulinic acid by the enzyme δ-aminolevulinic acid synthase.

6. Pyridoxal-5-phosphate is also essential in glycogen metabolism in the muscle and liver, as it is required as a coenzyme by phosphorylase.

7. Pyridoxal-5-phosphate is also essential for chain elongation of unsaturated fatty acids, e.g. in the conversion of linoleic acid to arachidonic acid.

8. Vitamin B₆ is also required for the absorption of amino acids.

Deficiency Symptoms

Deficiency symptoms include cheilosis, glossitis and hypochromic anemia.

In infants, deficiency of B₆ affects central nervous system and results in convulsions, demyelination of the peripheral nerves and degeneration of axons.

Pyridoxine has also been shown to be helpful in the treatment of nausea and vomiting, during pregnancy, radiation therapy and muscular dystrophy.

Deficiency of vitamin B₆ has been shown to occur in patients with tuberculosis who are taking antitubercular drug INH (isonicotin hydrazide) since this drug acts as an antagonist to vitamin B₆ (Fig. 12.11).

Fig. 12.11. Vitamin B₆ and INH.

Requirements

Daily requirement of vitamin B₆ is approximately 1–2 mg.

Sources

Rich sources of vitamin B₆ are egg, fish, green leafy vegetables and cereals.

PANTOTHENIC ACID

Pantothenic acid is heat stable in the acidic medium. It has a molecule of pantoic acid, which is linked to β-alanine (Fig. 12.12)

Fig. 12.12: Pantothenic acid.

Absorption, Transport, Storage and Metabolism

Panthothenic acid exists in foods mostly as CoA and ACP, which are hydrolysed to phosphopantotheine and then converted to pantothenic acid.

Panthothenic acid is absorbed by passive diffusion and active transport in the jejunum.

It is then transported in free form in the plasma and taken up by diffusion into erythrocytes, which carry most of the vitamin in the blood.

Panthothenic acid is taken up by cells of peripheral tissues by a sodium-dependent active transport process in some issues and by facilitated diffusion in others. Within the cells, the vitamin is converted to CoA, which is the predominant form in most tissues.

CoA and ACP are degraded to yield free pantothenic acid and other metabolites. The vitamin is excreted mainly in the urine, as free pantothenic acid. About 15% of daily intake is oxidized completely and excreted through the lungs, as carbon dioxide.

Biochemical Functions

Pantothenic acid is an important constituent of coenzyme A (Fig. 12.13).

Fig. 12.13. Coenzyme A.

Coenzyme A also contains adenine and a sulfhydryl group (–SH), which is contributed by β-mercaptoethanolamine.

Due to the presence of the −SH group, coenzyme A is also designated as HS-CoA (CoA).

Pantothenic acid as well as coenzyme A performs several functions:

1. Pantothenic acid, as 4-phosphopantotheine, forms prosthetic group of the acyl carrier protein, which participates in fatty acid synthesis.

2. CoA acts as a carrier for the acetyl group as acetyl CoA, also called active acetate and is used in the biosynthesis of acetylcholine (a transmitter at the nerve synapse), cholesterol, etc.

3. CoA is also used for oxidative decarboxylation of pyruvate, which is converted to acetyl CoA and enters the Krebs cycle.

4. CoA is also essential in the metabolism of branched chain amino acids.

5. CoA is also required for the activation of fatty acids and their oxidation.

6. CoA is also required for the activation of succinate and thus in the biosynthesis of heme.

Deficiency Symptoms

Pantothenic acid deficiency, in human beings, affects nervous system as well as digestive system. Its deficiency has also been shown to result in adrenal insufficiency.

Deficiency symptoms include headache, fatigue, impaired motor activity, muscle cramps and gastrointestinal disturbances.

Pantothenic acid is also used in the burning-feet syndrome, and healing of ulcers and bedsores.

Requirements

Average daily requirement of pantothenic acid is 5–10 mg.

Sources

Rich sources of pantothenic acid are yeast, liver, egg, potato, whole cereals and legumes.

BIOTIN

Biotin is also called as anti-egg white injury factor since it protects animals against toxicity of raw egg white.

Structurally, biotin is a cyclic derivative of urea (ureido ring), which is fused to the thiophene ring (Fig. 12.14).

Fig. 12.14. Biotin.

Absorption, Transport, Storage and Metabolism

Biotin, in foods, is largely protein bound. It is released by proteolytic digestion to yield free biotin, biocytin or biotin peptide. Intestinal biotinidase releases free biotin from biocytin and biotin peptide. Free biotin is absorbed in the proximal small intestine, primarily by carrier mediated diffusion.

Biotin is transported in the plasma, primarily as free biotin. It is taken into cells by a specific carrier mediated process.

Vitamin is rapidly excreted in the urine as free biotin as well as its oxidized products, as biotin sulfoxides.

Biochemical Functions

Biotin binds by the amide linkage to e-amino group of the lysyl residue of the apoenzyme and forms a holoenzyme, which is referred to as biocytin (biotin-containing enzyme).

Biocytin accepts CO₂, as HCO₃⁻ and forms a complex called active biotin (carboxybiocytin-enzyme complex).

Biotin is required as a coenzyme in several reactions, involving CO₂ fixation, which are catalyzed by various carboxylases, such as:

1. Carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase.

2. Conversion of propionyl CoA to succinyl CoA (via methylmalonyl CoA), by propionyl CoA carboxylase.

3. Conversion of acetyl CoA to malonyl CoA, which is used in the formation of long chain fatty acids, by acetyl CoA carboxylase.

Deficiency Symptoms

Deficiency of biotin may cause mild dermatitis, nausea and loss of appetite, muscular pain and anemia.

Biotin deficiency is not generally seen, as intestinal flora can synthesize it.

Biotin deficiency is induced by the ingestion of large amount of raw egg white, which contains a heat labile biotin binding protein, called avidin. Avidin binds to biotin and prevents its absorption. Heating egg white prevents this effect since heating denatures avidin.

Requirement

Daily requirement of biotin is 15–30 μg.

Sources

Rice polish, whole cereals, milk, yeast and organ meat such as liver and kidney, are the good sources of biotin.

FOLIC ACID

Folic Acid and the related compounds are referred to as folacin since it is derived from the word folium (means leaf).

Folic acid is also called pteroylglutamic acid. It has a pteridine nucleus, a molecule of p-aminobenzoic acid and glutamate in its structure. Number of glutamate molecules may vary from one to seven (Fig. 12.15).

Fig. 12.15. Folic acid.

Absorption, Transport, Storage and Metabolism

Folate in foods is present in polyglutamate forms, which are hydrolysed to monoglutamate, by deconjugases in the brush border and intracellular mucosa. Monoglutamate forms of folic acid, such as 5-methyltetrahydrofolic acid and 5-formyltetrahydrofolic acid, are absorbed by active transport, mainly in the jejunum. When ingested in large amounts, folate can also be absorbed by passive diffusion.

Folate, taken up by the intestinal mucosal cell, is reduced to tetrahydrofolate (FH₄), which can either be transferred to the portal circulation or converted to 5-methyl.FH₄, before entering the circulation. Monoglutamate derivatives from plasma are taken up by the cells using an energy-dependent process with a specific folate binding protein or a carrier-mediated anion exchange process. Within cells, FH₄ is methylated to 5-methyl.FH₄, which is rertained, intracellularly. The liver is the most important depot for folate, containing about half of the total body’s store as polyglutamates of 5-methyl.FH₄ and 10-formyl.FH₄. Tissues with higher rates of cell division, e.g. intestinal mucosa, have high concentrations of 10-formyl.FH₄.

Folates are metabolized in three ways:

1. Reduction to one of the several derivatives with single-carbon units, substituted at the N-5 or N-10, or both positions of the pterin ring. The main source of the single carbon fragment is via serine hydroxymethyltrans-ferase to produce 5, 10-methylene.FH₄.

2. Tissue folates turnover, by cleavage of their pteridine and para-aminobenzoyl polyglutamate moieties.

3. The later are further degraded to a variety of water soluble side chain metabolites that are excreted in the urine and bile.

Biochemical Functions

Folic acid is used as a coenzyme, called tetrahydrofolate (FH₄). During its synthesis, folate is first reduced by folate reductase to dihydrofolate (FH₂) and subsequently by dihydrofolate reductase to FH₄ (Fig. 12.16).

Fig. 12.16. Conversion of folic acid to FH₄.

Tetrahydrofolate (FH₄) acts as an acceptor as well as donor of the one-carbon unit (1-C) in several reactions, in the metabolism of amino acids and nucleotides.

Sources of the one-carbon moiety include histidine (formimino group), serine (hydroxymethyl group) and choline and methionine (methyl group).

One-carbon moiety carried by FH₄ may be accepted as carbon 2 and 8 in the biosynthesis of purine ring; or in the conversion of glycine to serine, uracil to thymine, homocysteine to methionine, and in the biosynthesis of choline.

Some of these reactions also require vitamin B₁₂.

Deficiency Symptoms

Folic acid deficiency may also result in macrocytic (megaloblastic) anemia, which is accompanied by leucopenia.

Other deficiency symptoms, such as psychosis with mental retardation, are due to combined deficiency of folic acid and vitamin B₁₂.

Recently, folic acid deficiency (in early pregnancy) has also been implicated in neural tube defects.

Folic acid deficiency may occur in stressful conditions, during pregnancy, intestinal malabsorption, such as steatorrhea and during antibiotic therapy.

Requirement

Average daily requirement of folic acid for an adult is 150–300 μg.

Sources

Green leafy vegetables such as spinach and cabbage, yeast, liver and kidney are good sources of folic acid.

VITAMIN B₁₂

Vitamin B₁₂ is also called the extrinsic factor of Castle. Due to the presence of the cyano group and the cobalt atom, vitamin B₁₂ is also referred to as cyanocobalamin.

The central ring structure of the vitamin has a corrin nucleus. Cobalt is linked to the cyano group as well as to the nitrogen of the imidazole ring, which is esterified through aminopropanol and propionic acid to ring IV of the corrin nucleus (Fig. 12.17).

Fig. 12.17: Vitamin B₁₂.

Absorption, Transport, Storage and Metabolism

Vitamin B₁₂ is bound to protein in food and is released from it by pepsin digestion in the stomach. The vitamin then combines with R proteins (cobalphilins) in the stomach and moves into the small intestine. In the small intestine, the R proteins are hydrolysed and intrinsic factor (IF), a specific binding protein for B₁₂ produced in the stomach, binds the cobalamin. From the IF-vitamin B₁₂ complex, the vitamin is taken into the erythrocyte by a process involving binding to a specific membrane receptor on the ileal brush border.

After absorption, cobalamin binds to the plasma R proteins, known as transcobalamins (TCs, i.e. TCI, TCII and TCIII). TCII is the main transporter protein for newly absorbed cobalamins. Cellular uptake of vitamin B₁₂ is mediated by a specific TC receptor that internalizes the TC-vitamin complex. TC is degraded in the lysosomes and the free vitamin is released for binding the vitamin B₁₂-dependent enzymes.

In adequately nourished individuals, vitamin B₁₂ is stored in appreciable amounts, mainly in the liver, most of which is in the form of adenosylcobalamin.

Vitamin B₁₂ is metabolically active only as deoxyadenosylcobalamin or methylcobalamin.

Free cobalamins are excreted intact by renal and biliary routes.

Biochemical Functions

Vitamin B₁₂ is used as a coenzyme in a number of reactions. There are two coenzyme forms of the vitamin. These are called methylcobalamin and deoxyadenosylcobalamin, which are referred to as cobamides.

Methylcobalamin is used as a coenzyme in the formation of methionine from homocysteine. Vitamin B₁₂ thus, maintains methionine stores of the body and spares FH₄ for the biosynthesis of purines and pyrimidines.

Deoxyadenosylcobalamin is important in the metabolism of branched chain fatty acids as well as fatty acids containing odd number of carbon atoms, by converting methylmalonyl CoA to succinyl CoA, by methylmalonyl CoA isomerase.

Deficiency Symptoms

Vitamin B₁₂ deficiency can result in methylmalonic aciduria. Measurement of the urinary excretion of methylmalonic acid is used to confirm deficiency of this vitamin.

Vitamin B₁₂ deficiency is not generally observed due to its wide spread nature in animal tissues as well as its synthesis by microbial flora. Deficiency, however, can occur in geriatric persons particularly, in those who are strict vegetarians.

Its deficiency may also occur on its prolonged reduced intake, intestinal mal-absorption, atrophy of the gastric mucosa or due to the deficiency of the intrinsic factor, e.g. in pernicious anemia.

Requirements

Daily requirement of vitamin B₁₂ is 1–3 μg.

Sources

Liver, kidney, egg, fish and milk are the good sources of vitamin B₁₂.

VITAMIN C

Vitamin C is also called ascorbic acid, anti-scorbutic factor or antiscurvy factor.

Majority of the animals can synthesize vitamin C from glucuronate but men, primates and guinea pigs can not synthesize it.

Vitamin C is readily absorbed from the intestinal tract. Its absorption is reduced during gastrointestinal infections and in achlorhydria.

Though it is stored in the liver and the adrenals, highest concentration is found in the adrenal cortex.

Vitamin C is destroyed by heat.

Absorption, Transport, Storage and Metabolism

Dietary vitamin C is absorbed to the extent of 80–90%, by active transport and passive diffusion.

Ascorbic acid is transported in the plasma, in the reduced form, in free solution. It is taken up by cells through a glucose transporter and a specific active transport system. Dehydroascorbic acid is readily reduced to ascorbate in the cells. The glucose transporter based system of uptake is stimulated by insulin and inhibited by glucose.

The vitamin is concentrated, primarily as dehydroascorbic acid, in many vital organs, particularly the adrenals, brain and eye.

Ascorbic acid is oxidized to dehydroascorbic acid. Subsequently, it undergoes irreversible hydrolysis to CO₂ and several five-carbon fragments (such as xylose) or is oxidized to oxalic acid.

Biochemical Functions

Vitamin C has an en-diol group at the second and the third carbons, due to which it is sensitive to oxidizing agents. Oxidation of ascorbic acid converts it to dehydroascorbic acid (Fig. 12.18).

Fig. 12.18. Vitamin C.

Due to its oxidation reduction properties, ascorbic acid performs several biological functions in the body:

1. Ascorbic acid is required in the hydroxylation of amino acids particularly, the aromatic amino acids.

2. Due to its role in the hydroxylation of proline and lysine (in collagen), ascorbic acid is required for the repair of the ground substance.

3. Vitamin C is essential for the absorption of iron from the gastrointestinal tract as well as its release from ferritin.

4. Vitamin C has stimulating effect on phagocytic activity of leucocytes. It thus, provides resistance to the body.

5. Ascorbic acid is also required for the synthesis of the steroid hormones in the adrenal cortex. It thus, helps in lowering blood cholesterol level.

Deficiency Symptoms

Prolonged deficiency of vitamin C causes scurvy.

Because of its role in absorption, transport and storage of iron, vitamin C deficiency may also cause anemia.

Requirement

Daily requirement of vitamin C, for adults, is 40–60 mg.

Sources

Amla is a richest source of vitamin C. Citrus fruits, green chilies, guava and tomato are also good sources of vitamin C.

Hypervitaminosis

Vitamin C may be toxic, if taken in large doses. More than 2 g, in a single dose, may result in abdominal pain, diarrhea and nausea. More than 3 g/day may lead to urolithiasis due to the formation of oxalate stones.

Various water soluble vitamins, their coenzyme forms, major biochemical functions, principal deficiency diseases and daily requirements are shown in Table 12.1.

Table 12.1. Water soluble vitamins, coenzyme forms, biochemical role, deficiency diseases and daily requirement for adults

FAT SOLUBLE VITAMINS

Fat soluble vitamins are vitamin A, D, E and K. They are absorbed in the lumen, in the presence of fat and are emulsified with bile.

Their precursors are called provitamins, which mostly occur in plants.

Due to fat soluble nature of these vitamins, they are stored in the liver and several other tissues. Large doses of these vitamins particularly, for a fairly longer duration, may result in hypervitaminosis leading to various toxic symptoms.

SCURVY

Scurvy is characterized by multiple hemorrhages. Early symptoms of the disease include swollen, spongy and bleeding gums; loosening of teeth and pain in joints (Fig. 12.19).

Fig. 12.19. Scorbutic gums.

In severe cases, there may also be bleeding from nose, gastrointestinal tract and genitourinary tract.

Infants may lose appetite and body weight with painful tenderness of the extremities, bleeding from gums and mucous membrane, and cessation of osteogenesis of long bones.

VITAMIN A

Vitamin A is also called anti-night blindness factor. It has a characteristic 6-carbon ring, known as β-ionine ring, which is attached to a long hydrocarbon side chain (Fig. 12.20).

Fig. 12.20. Vitamin A.

Vitamin A (retinoids) actually refers to three preformed compounds that exhibit metabolic activity, i.e. the alcohol (retinol), the aldehyde (retinaldehyde or retinal) and the acid (retinoic acid). Stored retinol is often esterified to a fatty acid, usually palmitate, and is called retinylpalmitate. These retinyl esters are also usually found complexed with proteins, in foods. These active forms of vitamin A exist in only animal products.

In addition to these preformed forms found in animal products, plants contain a group of compounds collectively known as carotenoids, which can yield retinoids when metabolized in the body. Although a large number of carotenoids exist in food, naturally as antioxidants, only a few have significant vitamin A activity. The most important of these is β-carotene. The amount of vitamin A available from dietary carotenoids depends on the efficiency of absorption and its conversion to retinol. Absorption of carotenoids varies greatly from 5–50% and is affected by other dietary factors such as the digestibility of the proteins complexed with the carotenoids, and the level and type of dietary fat.

Vitamin A alcohol is also referred to as retinol. It may be converted to aldehyde (retinal, also called retinene) as well as to acid (retinoic acid). All the forms of the vitamin are found in different tissues, in animals.

Provitamin A: Carotenoids are called provitamin A. They are found in plants and have an additional ionine ring. Depending upon the presence of the second ionine ring, provitamins are designated as α, β and γ-carotenes.

β-Carotene has highest vitamin A activity, as it gives 2 molecules of vitamin A (retinal). On the other hand, α- and γ-carotenes give only one molecule. Provitamins (carotenoids) are absorbed in the lumen and converted to retinal in the intestinal epithelium. Carotenes are splitted at the center of the hydrocarbon chain and give rise to active vitamin A with in the intestinal wall.

Conversion of β-carotene to retinal requires an enzyme referred to as dioxygenase (Fig. 12.21)

Fig. 12.21. Conversion of β-carotene to retinal.

Absorption, Transport, Storage and Metabolism

Before the absorption of vitamin A or its carotenoid provitamins, proteases present in the stomach hydrolyze the proteins that are complexed with these compounds. In addition, lipases hydrolyze retinyl esters to retinol and free fatty acids, in the small intestine.

Retinoids and carotenoids are incorporated into the micelles, along with other lipids, for passive absorption into the mucosal cells of the small intestine. Once in the intestinal mucosal cells, retinol is bound to a cellular retinol-binding protein and re-esterified into retinyl esters. Carotenoids and retinyl esters are incorporated into chylomicrons, for transport into the lymph and eventually the bloodstream, or may be cleaved into retinal which is then reduced to retinol and re-esterified into retinylesters. Chylomicron remnants deliver retinylesters to the liver. These esters are immediately hydrolyzed into retinol and free fatty acids. Retinol in the liver has three metabolic fates: First, retinol may bind to cellular retinol-binding protein, which controls free retinol concentration.

Second, retinol may be re-esterified to form retinyl esters, mostly retinylpalmitate, for storage.

Finally, retinol may be bound to retinol-binding protein, leaves the liver and enters the blood where another protein, transthyretin, attaches, forming a retinol-RBP-transthyretin complex that transports retinol in the blood to the peripheral tissues. The retinol-RBP-transthyretin complex delivers retinol to other tissues via cell surface receptors.

In addition to being esterified for storage, the transport form of retinol can also be oxidized into retinal and then into retinoic acid, or conjugated into retinyl glucuronide or retinyl phosphate. After retinoic acid is formed, its chain is shortened and it is converted to the forms, that are readily excreted in the urine. Intact forms of the vitamin are excreted only in the bile and feces.

Biological Functions

1. ROLE IN VISUAL PROCESS

Vitamin A aldehyde (retinal) is essential for vision, both in rods as well as cones:

• In rods: Rod cells contain a conjugated transmembrane protein, called rhodopsin, which consists of an apoprotein opsin and a nonprotein prosthetic group, called 11-cis-retinal (11-cis-retinene). It is essential for vision in dim light.

When light falls on retina, rhodopsin is bleached and generates all-trans-retinal, which can not form a complex with opsin. All-trans-retinal is reduced to all-trans-retinol by the enzyme all-trans-retinol dehydrogenase (retinene reductase). It is then transported to liver and stored there. In the liver, it is isomerized to 11-cis-retinol by an isomerase. After its delivery to the retina, 11-cis-retinol is changed to 11-cis-retinal, which again conjugates with opsin and reforms rhodopsin, in dark. This is called visual cycle (Fig. 12.22).

Fig. 12.22. Visual cycle.

1. In cones: Similar to rhodopsin, which is present in rods for the black and white vision, cones contain 3 different pigments that are sensitive to three primary colors, i.e. porphyropsin (for red), iodopsin (for green) and cyanopsin (for blue). Prosthetic group in these pigments is the same, i.e. 11-cis-retinal.

2. Vitamin A deficiency leads to reduction in gluconeogenesis. This is due to decreased synthesis of steroid hormones particularly, synthesis of corticosterone and cortisol.

3. Vitamin A also plays a role in cholesterol biosynthesis.

4. Vitamin A deficiency causes keratinization of the mucosal membrane and decreases synthesis of glycosaminoglycans.

5. Vitamin A deficiency impairs spermato-genesis and affects cell growth and differentiation.

6. Deficiency of vitamin A also reduces synthesis of membrane glycoproteins.

7. Vitamin A is also required in the maintenance of integrity of the tissues lining eyes, gastrointestinal tract, genitourinary tract and respiratory system and ensures optimal functioning of the immune system.

Deficiency Symptoms

Vitamin A deficiency is observed, primarily due to its low intake. Its deficiency is aggravated by infections, which reduces its absorption and utilization. Vitamin A deficiency affects many tissues:

1. It is most detrimental for eyes. Deficiency of the vitamin may result in xerophthalmia, which is characterized by drying of conjunctiva and cornea, followed by destruction of cornea, leading to blindness.

2. Vitamin A deficiency also affects osteoblastic activity and results in defective resorption of bone, affecting growth.

3. Deficiency of vitamin A also results in xerosis and keratinization of the mucous membrane, resulting in dry and rough skin. Keratinization of the mucous membrane increases its susceptibility to infections. Keratinization of the urinary tract may lead to urolithiasis.

4. Vitamin A deficiency also results in certain abnormalities of the reproductive system such as degeneration of testes, abortions and production of the malformed-offspring. Keratinization of epithelium of the semini-ferous tubules may also lead to infertility.

XEROPHTHALMIA

Xerophthalmia occurs when body store of vitamin A is exhausted or supply fails to meet its requirement. Gradual depletion of vitamin A store results in a condition referred to as xerophthalmia. It may manifest as night blindness, conjunctival xerosis, Bitot’s spot, corneal xerosis and ulceration, keratomalacia, and corneal scar.

1. Night blindness: First symptom of vitamin A deficiency is night blindness, i.e. an individual cannot see in dark. Since retinal is essential for the synthesis of rhodopsin (by rods) and vitamin A deficiency interferes with the production of rhodopsin, thus deficiency of vitamin A impairs rod functions and results in night blindness. Night blindness responds to vitamin A therapy, rapidly.

2. Bitot’s spot: Bitot’s spot means accumulation of foamy-cheesy material on the conjunctiva, often in association with other signs of xerophthalmia, such as conjunctival xerosis. Although spots may differ in size, location or shape, they have similar appearance.

3. Corneal xerosis and ulceration: When cornea becomes dry, this condition is referred to as xerosis. If the disease is not treated at this stage, it can progress with in hours to ulcer of the cornea.

4. Keratomalacia: If the disease is not treated in its early stage, a corneal ulcer can lead to melting or wasting of cornea. This is referred to as keratomalacia. It indicates destruction of a part or complete corneal stroma, which in turn results in permanent structural alterations.

5. Corneal scar: Keratomalacia can lead to perforation of the cornea. At this stage a corneal scar is formed, which however, remains forever (Fig. 12.23).

Fig. 12.23. Xerophthalmia.

Requirements

Average daily requirement of vitamin A for an adult is 2000–3500 IU (about 750–1000 μg).

Daily requirement of β-carotene is 4000–6000 IU (about 2000 μg).

Sources

Liver oil, fish-liver oil, egg, milk and milk products are the good sources of vitamin A.

On the other hand, papaya, carrots, sweet potatoes and green leafy vegetables are the good sources of β-carotene.

Hypervitaminosis

If taken in large quantities for a longer period, vitamin A is toxic since it is a fat soluble vitamin and stored in the liver.

Toxicity symptoms include skin abnormalities such as dry and rough skin, scaly dermatitis, headache, nausea, vomiting and loss of appetite, hepatomegaly, painful joints and several bone abnormalities.

VITAMIN D

Vitamin D (calciferol) is a sterol. It is also called anti-rachitic factor and considered to be a pro-hormone. It is present in nature in several forms, two of which are most common. These are referred to as vitamin D₂ and D₃. Vitamin D₂ (ergocalciferol) is of plant origin and can be obtained from ergot plant. It can also be synthesized in the laboratory by irradiating ergosterol. Vitamin D₃ (cholecalciferol) is of animal origin (Fig. 12.24).

Fig. 12.24. Vitamin D₃.

Absorption, Transport, Storage and Metabolism

Dietary vitamin D is incorporated with other lipids into micelles and absorbed with lipids into the intestine, by passive diffusion. Inside the absorptive cells, the vitamin is incorporated into chylomicroms, enters the lymphatic system and subsequently enters the plasma.

It is then delivered to the liver, by chylomicron remnants or to the specific carrier vitamin D-binding protein, or transcalciferin. The efficiency of this absorption process is about 50%.

Vitamin D, synthesized in the skin from cholesterol, enters the capillary system and is transported to the peripheral tissues. Little vitamin D is stored in the liver.

Provitamin D

A derivative of the cholesterol, called 7-dehydrocholesterol, is a precursor of vitamin D₃ in animals. It is also called provitamin D. It is synthesized in the human beings and is converted to vitamin D3 by UV light, in the skin. This process is referred to as photobiogenesis.

Active Vitamin D

Vitamin D is activated by two sequential hydroxylations. The first step takes place in the liver where it is hydroxylated, by cytochrome P450-dependent 25-hydroxylase, to 25-hydroxycholecalciferol [25(OH)₂.D₃].

From liver, 25-hydroxycholecalciferol is transported to kidney where it is further hydroxylated at position 1, by the mitochondrial enzyme 1-α-hydroxylase, forming 1,25-dihydroxycholecalciferol [1, 25(OH)₂.D₃]. This is the physiologically active form of the vitamin, which functions as a hormone and is essential for calcium homeostasis (Fig. 12.25).

Fig. 12.25. Activation of vitamin D.

Due to presence of the 3-OH groups, 1,25(OH)₂. D₃ is also called calcitriol.

1-α-Hydroxylase activity is present in the renal tubules, bone and placenta. Parathyroid hormone (PTH), serum phosphorus and calcitriol regulate its activity. Production of 1,25(OH)₂.D₃ is also regulated by serum calcium.

Biological Functions

Most important function of active vitamin D is to regulate serum calcium level.

Role of Vitamin D in Calcium Metabolism

Calcitriol induces synthesis of calcium-binding protein, Ca²⁺-dependent ATPase and alkaline phosphatase, which are essential for the regulation of serum calcium and phosphorus.

Net result is achieved through its action in the intestine bone and kidney:

• In the intestine, due to stimulation of the synthesis of the calcium-binding protein, 1, 25(OH)₂.D₃ increases intestinal absorption of calcium as well as phosphorus.

• In the bone, calcitriol stimulates synthesis of osteocalcin, which in turn mobilizes calcium from bone, i. e. it causes demineralization of bone, and

• In the kidney, active vitamin D increases tubular reabsorption of calcium and excretion of phosphorus.

Overall effect of the three processes, thus is to increase serum calcium level towards normal.

Deficiency Symptoms

Deficiency of vitamin D, generally, does not occur, as it can be synthesized in the body. Deficiency however, may occur due to lack of sunshine. Vitamin D deficiency leads to rickets in children and osteomalacia in adults.

Requirements

Average daily requirement is 200 IU (5–10 μg) of cholecalciferol.

Sources

As vitamin D can be synthesized in the body by the action of the ultraviolet light on the skin, hence sunshine is the best source of vitamin D. Besides sunshine, rich sources of vitamin D are milk, butter, egg yolk, fish oil and liver oil.

RICKETS AND OSTEOMALACIA

Rickets

Rickets is a disease, which occurs during infancy and childhood. It is a nutritional disorder due to the deficiency of vitamin D. Besides vitamin D, exposure of the body to sunlight has both, preventive as well as curative effects.

Symptoms such as enlarged wrists, ankles and poor structural development, affecting long bones, are frequently seen.

Ends of the long bones become soft and bulky, particularly characteristic of the bowed-legs and knock-knees. There may also be extreme stunting of growth (Fig. 12.26).

Deformities may also appear in bones of the skull, chest and spine. Teeth may erupt late and are deformed.

Biochemically, a patient with rickets shows decrease in serum calcium and phosphorus along with increase in serum alkaline phosphatase and increased excretion of inorganic phosphorus in urine.

Product of serum calcium and phosphorus, which is normally 40 (10 mg calcium × 4 mg phosphorus), is decreased to below 30, in such children.

Osteomalacia

Deficiency of vitamin D in adults result in a condition called osteomalacia, also termed as adult rickets. It is commonly observed in females during pregnancy and lactation, or in those who are not exposed to sunlight, may be because of their customs wearing Purdah. Bone, particularly of the pelvic region, shows characteristic deformities, which in turn may complicate parturition.

Fig. 12.26. Rickets.

Hypervitaminosis

Extremely large doses of vitamin D, over a long period, may lead to hypercalcemia, hyperphosphatemia, anorexia, nausea, vomiting and diarrhea besides bone pain and metastatic calcification of the soft tissues, including kidney, myocardium, pancreas and uterus.

VITAMIN E

Several compounds belonging to the group of tocopherols, such as α, β, γ and δ tocopherols, exhibit vitamin E activity. Amongst them, α-tocopherol has highest biological activity as well as widest distribution. It is an alcohol with a phytol and trimethylhydroquinone. Commer-cially, vitamin E is available both as a natural substance as well as a synthetic compound. Natural vitamin E is d-α-tocopherol and is a single stereoisomer. On the other hand, synthetic vitamin E is dl-α-tocopherol, which is produced commercially (Fig. 12.27).

Fig. 12.27. Vitamin E (α-Tocopherol).

Absorption, Transport, Storage and Metabolism

Vitamin E is absorbed in the upper small intestine by micelle-dependent diffusion only after hydrolysis of the esterified forms by esterases, at the duodenal mucosa. Absorption efficiencies range from 20–70%.

Absorbed vitamin E is incorporated into chylomicrons and is transported into the general circulation via lymph. Vitamin E, delivered to the liver, is incorporated into VLDL using a transport protein specific for vitamin E. In the plasma, tocopherol is also partitioned into LDL and HDL where it may protect the lipoproteins from oxidation. The cellular uptake of vitamin E can occur either as a receptor-mediated process or as a process mediated by lipoprotein lipase. Within the cell, intracellular transport of the tocopherol requires an intracellular tocopherol-binding protein.

Vitamin E is primarily oxidized into the biologically inactive tocopheryl quinone, which can be reduced to tocopheryl hydroquinone. Glucuronic acid conjugates of the hydro-quinones are secreted in the bile and excreted in the feces. This is major route of elimination of the vitamin.

Biological Functions

Most important function of vitamin E is as an antioxidant since it has the ability to quench free radicals, which are generated by metabolic processes and environmental pollutants.

Deficiency Symptoms

Vitamin E deficiency results in a range of degenerative diseases.

Its deficiency is seen in patients with abetalipoproteinemia who can not form chylomicrons and VLDL. As a result of abetalipo-proteinemia, vitamin E can neither be absorbed nor distributed.

Deficiency of vitamin E is also seen in children with fat malabsorption, where it results in severe progressive neuropathy and retinopathy.

Hepatic necrosis, muscular dystrophy, defects in vascular system and certain reproductive defects are also known to occur due to deficiency of vitamin E, in various animal species.

Requirements

Daily requirement of vitamin E for an adult man is 5–10 mg (about 15 IU).

Requirement of vitamin E varies depending upon the level and nature of physical activity, diet, psychological stress, exposure to pollutants and intake of polyunsaturated fatty acids (PUFA). Normally, 0.4 mg of vitamin E is required per g of PUFA.

Sources

Tocopherols are present in small quantities in green leafy vegetables such as spinach and lettuce, milk and milk products and egg yolk. Oils, such as corn oil, cottonseed oil, and sunflower oil, are the rich sources of vitamin E.

VITAMIN K

The name vitamin K has been derived from the Danish word ‘Koagulation’ since it has an important role in blood clotting.

Various compounds related to 2-methyl-1, 4-napthoquinone have been shown to possess vitamin K activity.

• The most important form of vitamin K is K₁, also called phylloquinone. It occurs naturally and is found from plant sources. Vitamin K₁ has phytyl side chain. (Fig. 12.28).

Fig. 12.28. Vitamin K₁ (Phylloquinone).

• Vitamin K₂, also called menaquinone or farnequinone), is synthesized by the intestinal bacteria. It has farnesyl side chain.

• Vitamin K₃, also called menadione, is a synthetic compound. It is water soluble and gets easily absorbed on parenteral administration. Vitamin K which is commercially available for therapeutic use, is menadione.

Absorption, Transport, Storage and Metabolism

Phylloquinone (K₁) is absorbed by an energy-dependent process, in the small intestine. However, the menaquinone (K₂) and menadione (K₃) are absorbed in the small intestine and colon by passive diffusion.

The absorbed vitamin is incorporated into chylomicrons in the lymph and taken to the liver where they are incorporated into VLDL and subsequently delivered to the peripheral tissues by LDL.

Vitamin K is found in low concentration in many tissues where it is localized in cellular membrane. Most tissues contain phylloquinone and menaquinone.

Phylloquinone can be converted to menaquinone by successive bacterial dealkylation and realkylation before absorption. Side chain shortening and oxidation produce metabolites that are excreted in the faeces via the bile, frequently as glucuronic acid conjugates and catabolise phylloquinone and menaquinone. Menadione is metabolized more rapidly. It is excreted, primarily in urine as a phosphate, sulphate or glucuronide derivative.

Biological Functions

Vitamin K is essential for maintenance of the normal levels of various blood clotting factors such as prothrombin, factor VII, factor IX and factor X. These proteins are synthesized in the liver in their inactive forms and get converted to their biologically active forms by γ-carboxylation, with the help of vitamin K.

Role of Vitamin K in Gamma-Carboxylation

Formation of clotting factors requires vitamin K-dependent carboxylation of glutamic acid residues of proteins (Fig.12.29).

Fig. 12.29: Role of vitamin K in γ-carboxylation.

This forms a mature clotting factor that contains γ-carboxyglutamate (Gla) and is capable of subsequent activation.

Deficiency Symptoms

Deficiency of vitamin K is seen in newborns, due to their sterile intestine, particularly in premature infants who show reduced levels of vitamin K, prothrombin and Christmas factor.

Common deficiency symptoms include cutaneous and intramuscular hemorrhages with bluish-red coloration in different parts of the body.

Requirements

Its requirement is not well established in adults but it is suggested that 20–100 μg/day is sufficient to meet its daily need. It is advised to administer a single prophylactic dose of 1 mg of vitamin K₁ to a newborn, on the first day of life.

Sources

Green leafy vegetables such as spinach, lettuce and cabbage, are the rich sources of vitamin K. Fish, liver and skeletal muscle are also good sources of this vitamin.

Vitamin K Antagonists

Anticoagulants such as dicumarol and warfarin, due to their structural similarities with vitamin K₁, act as antagonists (anti-vitamins) of vitamin K. They act as competitive inhibitors for the enzymes, which are concerned with the activation of prothrombin and other coagulation factors, and require vitamin K for their activation.

Table 12.2 lists various fat soluble vitamins, their biological functions, deficiency diseases, source and daily requirements for adults.

Table 12.2.

SOME IMPORTANT QUESTIONS

1. Name fat soluble vitamins? Mention their dietary sources, biological functions and deficiency disorders.

2. Describe chemistry, biological functions and deficiency manifestations of vitamin A.

3. How is vitamin D synthesized in the body? Describe its biochemical role.

4. Describe dietary sources, biological functions and deficiency manifestations of folic acid and vitamin B₁₂.

5. Given an account of chemistry, dietary sources, biological functions and deficiency manifestations of vitamin C.

6. Describe coenzyme forms and biochemical roles of:

(i) Riboflavin

(ii) Pantothenic acid

(iii) Biotin

(iv) Vitamin B₆

(v) Thiamin

(vi) Niacin

7. Discuss briefly:

(i) Role of pyridoxal phosphate in amino acid metabolism

(ii) Structure and functions of vitamin D

(iii) Role of vitamin A in vision

8. Write notes on:

(i) Keratomalacia

(ii) Pellagra

(iii) Beri-beri

(iv) Scurvy

(v) Cobamide coenzymes

(vi) Calcitriol

(vii) Role of vitamin D in calcium metabolism

(viii) Role of vitamin K in gamma-carboxylation

(ix) Pyridoxine

(x) Wernicke’s encephalopathy

(xi) Provitamin A

(xii) Xerophthalmia

(xiii) Provitamin D

(xiv) Active vitamin D

(xv) Rickets

(xvi) Osteomalacia

(xvii) Vitamin K antagonists

(xviii) Hypervitaminosis

CHAPTER
13

Minerals

SODIUM

Adult human body contains about 100 g of sodium, as sodium ion (Na⁺). Nearly half of it is found in the extracellular fluid while other half is intracellular. A small amount is also found in bones.

Na⁺ is the main extracellular cation, which exists in the body in association with several anions, such as chloride, bicarbonate, phosphates, lactate and proteins. It is the Na⁺, K⁺-ATPase, which maintains high extracellular concentration of Na⁺.

Sodium is freely filtered by the glomeruli. About 70% of the filtered sodium is reabsorbed in the proximal tubule, 15% in the loop of Henle, 5% in the distal convoluted tubule while the remaining 10% by the collecting duct.

Active reabsorption of Na⁺ in the proximal tubule also results in the passive reabsorption of Cl⁻ and HCO₃⁻ along with water. Reabsorption of Cl⁻ and HCO₃⁻ with Na⁺ helps to maintain electrical neutrality of the extracellular fluid while reabsorption of water ensures normal osmotic pressure.

Biological Functions

1. Regulation of Osmotic Pressure: Sodium plays a dominant role in the maintenance of osmotic pressure of different body fluids and protects body against excessive fluid loss.

2. Regulation of Acid Base Balance: Sodium salts, with the corresponding weak acids, form chief buffer system of the extracellular fluid and thus play an important role in the regulation of pH of the body fluids.

3. Absorption of Monosaccharides and Amino Acids: Na⁺ also plays an important role in the absorption of monosaccharides as well as amino acids from the small intestine.

4. Nerve Transmission: Sodium is also important in the maintenance of neuromuscular irritability and excitability.

5. Maintenance of Blood Viscosity: Sodium is also important in the maintenance of blood viscosity.

6. Electrolyte and Water Balance: Sodium is also important in the maintenance of water and electrolyte balance.

7. Na⁺ also acts as a cofactor for some enzymes, such as Na⁺, K⁺-ATPase.

Requirements

Daily requirement of sodium (as sodium chloride), for a normal adult, varies from 5 to 15 g.

Sources

Most important source of sodium is the common salt. Meat, sea foods, milk, cheese and bread are good sources of sodium. Plant foods are low in sodium.

POTASSIUM

Potassium (K⁺) is the chief cation of the intracellular fluid. Maintenance of its normal level is essential for life. Potassium is freely filtered at the glomeruli.

Biological Functions

1. Potassium helps to maintain normal osmotic pressure of different body fluids and thus maintains water and electrolytes balance.

2. Potassium salts also form buffer system of the intracellular fluid and thus play an important role in the regulation of pH of the body fluids.

3. It is also important in the maintenance of normal neuromuscular irritability and excitability.

4. Bases in the alkaline digestive juices, such as pancreatic juice and bile, contain potassium salts.

5. Storage of cell protein and glycogen requires potassium.

6. Potassium also acts as a cofactor for several enzymes, such as pyruvate kinase, Na⁺, K⁺-ATPase, etc.

Requirements

Normal daily intake of potassium, for a normal adult, has been recommended as 4 g.

Sources

Potassium is widely distributed in plant and animal sources as chloride, phosphate and carbonate salts of inorganic and organic acids, and proteins.

Coffee, cocoa, dried beans, wheat bran, most of the vegetables such as potato and tomato, dairy products, fish, eggs, meat, and fruits like banana, peach and orange, are good sources of potassium.

CALCIUM

Amongst various minerals, calcium (Ca) is found in highest concentration in the body. Nearly all cells need it. An adult man contains nearly 1.0–1.2 Kg of calcium. About 99% of it is present in the skeletal tissue, i.e. in bones and teeth, while remaining of it is found in soft tissues, such as muscles and nervous tissue.

Normal serum calcium level is 9–11 mg/ 100 ml. It is present in three forms:

1. Ionized calcium: About 45–50% of the total serum calcium is present in the ionized form as Ca²⁺ ions. Ionized calcium is freely exchangeable between soft tissues, extracellular fluid and blood.

2. Protein-bound calcium: Nearly 45–50% of the total serum calcium is bound to protein, and

3. Calcium salts: About 5–10% of calcium is present in the form of calcium salts, i.e. calcium bound with citrate and/or phosphate.

Absorption of Calcium

Calcium is absorbed by all parts of the small intestine but most rapid absorption occurs in the acidic medium, in the duodenum. Absorption is slow in the remainder of the small bowl because of the alkaline pH.

Only about 30% of the ingested calcium is absorbed by adults. Calcium is absorbed by two mechanisms, i.e. by:

1. Active transport which operates predominantly at low luminal concentration and is controlled by the action of calcitriol, and

2. Passive transport that occurs along the entire length of the small intestine.

Absorption of calcium, within the gut, is influenced by several factors.

Factors Affecting Calcium Absorption

1. Calcium absorption is impaired in vitamin D deficiency.

2. Excess of phosphate reduces calcium absorption.

3. Phytic acid and related substances, which are found in unrefined cereals, interfere with calcium absorption.

4. Excessive amounts of fat, fiber and oxalate reduce calcium absorption, due to the formation of the insoluble salts.

5. Higher amounts of protein and lactose in the diet and acidic pH promote calcium absorption.

Excretion

About 50% of the ingested calcium is excreted in urine. Almost equal amount is secreted into the intestine and lost in the feces. A small amount (about 15 mg/day) is also lost through skin.

Regulation of Serum Calcium

Maintenance of serum calcium, within the normal limits, is called calcium homeostasis.

Ionized calcium (Ca²⁺) is freely exchangeable between soft tissues, extracellular fluid and blood. It is called diffusible calcium and performs most of the functions of calcium. It is regulated by parathyroid hormone (PTH), vitamin D and calcitonin (Fig. 13.1).

Fig. 13.1. Calcium homeostasis.

Parathyroid Hormone: PTH plays a key role in the regulation of Ca²⁺. If Ca²⁺ concentration falls, secretion of PTH is increased. PTH acts directly on the osteoclasts and increases the dissolution of bone minerals. This process is called bone resorption.

PTH also increases reabsorption of Ca²⁺ by the kidneys.

Overall effect, of the above two processes, is to elevate serum Ca²⁺.

PTH also stimulates 1-α-hydroxylase activity in the kidneys, which in turn activates vitamin D and converts it to 1,25-dihydroxy-cholecalciferol (calcitriol).

Active Vitamin D: It increases serum Ca²⁺ concentration by promoting intestinal absorption of Ca²⁺, by increasing the synthesis of calcium-binding protein.

Calcitonin: Its action is to lower serum Ca²⁺ level by promoting deposition of calcium by the osteoblasts. It is secreted when serum Ca²⁺ level is increased.

PTH and calcitonin, thus act in opposite directions.

Biological Functions

Calcium is one of the important elements, which is present in the body. It performs several functions:

1. Calcification of the growing bones and teeth, and maintenance of mature bones.

2. Calcium acts as an activator for several enzymes, e.g. adenylate cyclase, ATPase, protein kinase, etc.

3. Calcium is essential for clotting of blood.

4. It is also required for contraction of muscle.

5. Calcium is also required to regulate permeability of the capillary walls and excitability of the nerve fibers.

6. It is also required for the secretion of various hormones. It also acts as a second messenger.

7. Calcium decreases membrane fluidity.

8. Ca²⁺ also regulates citric acid cycle. It activates pyruvate dehydrogenase phosphatase, which in turn activates pyruvate dehydrogenase complex and produces acetyl CoA. It also activates isocitrate dehydrogenase and a-ketoglutarate dehydrogenase activities.

9. Ca²⁺ also activates phosphorylase kinase through its binding to calmodulin and increases rate of glycogen breakdown.

Deficiency Disorders

Hypocalcemia may be caused by the deficiency of vitamin D as well as in hypo-parathyroidism. Prolonged hypocalcemia may lead to faulty calcification of bones, such as rickets (in children) and osteomalacia (in adults).

Decrease in blood calcium may also result in a condition called tetany, which is characterized by abnormal muscle spasm, and osteoporosis.

TETANY

Tetany or tetany seizure is a medical sign, consisting of the involuntary contraction of muscles which may be caused by disease or other conditions that increase the action potential frequency of muscle cells or the nerves that innervate them (Fig. 13.2).

Hypocalcemia is the primary cause of tetany. Low calcium levels, in the extracellular fluid, increase the permeability of neuronal membranes to sodium ions, causing a progressive depolarization which increases the possibility of action potentials. This occurs because calcium ions interact with the exterior surface of sodium channels in the plasma membrane of nerve cells.

Fig. 13.2. Tetany.

Vitamin D is essential for absorption and utilization of calcium. Vitamin C is needed for the formation of bone matrix. Magnesium and phosphorus are essential since they also form an essential part of the bone structure. Vitamin K, copper, zinc, manganese and boron are also important in bone formation.

Requirements

Nearly 800 mg of calcium is required by an adult, daily. During pregnancy and lactation, its requirement is increased to about 1.2 g/day.

OSTEOPOROSIS

Osteoporosis is a condition characterized by a decrease in the density of bone, decreasing its strength and resulting in fragile bones. Osteoporosis, literally, means abnormally porous bone that is compressible like a sponge. This disorder of the skeleton weakens the bone and results in frequent fractures (breaks) of the bone (Fig. 13.3).

Fig. 13.3. Osteoporosis.

Normal bone is composed of protein, collagen and calcium, all of which give bone its strength. Bones that are affected by osteoporosis can break (fracture) with relatively minor injury that normally would not cause a bone to fracture. Decreased calcium intake, particularly during menopause, leads to increased fragility and fractures of bones, mainly of the extremities and the pelvis. It can be retarded by increased supply of calcium along with the estrogen therapy. Besides calcium, an adequate diet sufficient in all the nutrients along with regular exercise is important for preventing osteoporosis.

Sources

Milk and milk products are rich sources of calcium. Egg yolk, dry fruits, legumes, cabbage, cauliflower and leafy vegetables are also good source of calcium.

PHOSPHORUS

Phosphorus (P) is widely distributed in most of the tissues. A normal adult body contains nearly 600–800 g of phosphorus. About 75% of it is present, in combination with calcium, in bones, teeth and skeletal tissues. Blood, muscle and nerve tissue also contain fairly good amount of phosphorus.

It is present in the body in two forms, as inorganic phosphorus and organic phosphorus.

Inorganic phosphorus is essential for the formation and development of bone and teeth. It helps in ossification and gets deposited as calcium phosphate. It is also present in soft tissues and various body fluids, as sodium and potassium phosphates.

Phosphorus containing organic compounds includes phosphate esters of monosaccharides, such as glucose-6-phosphate, dihydroxyacetone phosphate, phospholipids, nucleic acids, high-energy compounds and various coenzymes like TPP, NADP⁺, etc.

Absorption of Dietary Phosphorus

Most of the phosphates are absorbed in the inorganic state. Organically bound phosphate is hydrolyzed in the lumen of the intestine and is released as inorganic phosphate, primarily through the action of pancreatic and intestinal phosphatases.

Bioavailability of phosphate depends on the form of the dietary phosphate and pH. In general, efficiency of phosphate absorption is 60%-70% in adults, which is almost twice of calcium.

Biological Functions

Phosphorus performs various functions in the body:

1. Phosphorus, along with calcium, is essential for mineralization of bone and teeth. It forms a part of the hydroxyapatite, which is laid down in bone.

2. Phosphorus also plays an important role in the maintenance of acid base balance thus; it helps in the regulation of pH of body fluids.

3. Phosphorus is also found in phospholipids, which are essential for the transport and metabolism of lipids. Phospholipids also form an integral part of the cell membrane.

4. Phosphorus also forms an important constituent of nucleic acids, in the cell nucleus and thus, is essential for cell multiplication and protein biosynthesis.

5. Inorganic phosphorus (Pi) as well as organic esters of phosphoric acid, such as ADP, glucose-6-phosphate, etc., are necessary for metabolism of carbohydrates and thus in the production of energy.

6. As a component of several coenzymes, phosphorus also participates in various enzyme-catalyzed reactions.

Requirements

Daily requirement of phosphorus, for normal adult, is nearly 50% higher than calcium. It varies from 1.0 to 1.5 g/day.

Sources

Phosphorus is found in foods containing phosphoproteins, nucleoproteins, phospholipids and glycerophosphates as well as in the form of inorganic phosphates, chiefly of Ca and Na.

Calcium rich diets, such as milk, cheese and beans, are also the rich sources of phosphorus. Eggs, cereals, fish and meat are also good sources of phosphorus.

IRON

Total quantity of iron (Fe) in a normal adult human body is about 4 g.

Iron may be incorporated in the protoporphyrin ring, both in Fe²⁺ or Fe³⁺ form. The Fe²⁺-protoporphyrin complex is called heme while the Fe³⁺-protoporphyrin complex is designated as hematin.

Iron-containing Proteins found in the body

Ferritin: Ferritin is an iron containing protein, which is involved in the storage of iron.

Protein part of the ferritin is called apoferritin. Its synthesis is regulated by the concentration of free intracellular iron.

Apoferritin controls absorption of iron as it has rapid turnover. It is converted to ferritin, high concentration of which blocks absorption of iron in the mucosal cells. On the other hand, when more iron is to be absorbed, apoferritin is rapidly synthesized and becomes readily available. Thus, ferritin acts both as sink for iron during its excess intake as well as source of iron during its deprivation.

Transferrin: Transferrin is a nonheme iron protein involved in the transport of iron. It is a serum glycoprotein (β₁-globulin) and is synthesized in the liver. Protein part of the transferrin is designated as apotransferrin. It has affinity for Fe³⁺.

Hemosiderin: Excess of iron stimulates storage capacity of the newly synthesized apoferritin, which may be partially degraded by the proteolytic enzymes. This in turn results in the release of iron oxide micelles forming insoluble aggregates, which is designated as hemosiderin. These insoluble aggregates are trapped in the secondary lysosomes.

In a normal healthy subject, very little of hemosiderin is detectable. Quantity of hemosiderin, as brownish granules in cells, increases during iron overload.

Absorption, Transport and Storage

Dietary iron exists in two chemical forms:

• Heme Iron: It is found in hemoglobin, myoglobin and some enzymes. Heme iron is absorbed across the brush border (mucosa) of the intestinal absorbing cells (enterocytes) after it is obtained from the animal sources. After the heme enters cytosol, its ferrous iron is enzymatically removed from the ferro-porphyrin complex and combines with apoferritin, to form ferritin, similar to free nonheme iron. Ferritin carries it from brush border to the basolateral membrane of the absorbing cells. Brush border iron transporter, called divalent metal transporter 1 (DMT 1), transports ferrous iron. Ferric iron is reduced by the brush border enzyme ferric reductase, for its absorption (Fig. 13.4).

Efficiency of iron absorption (from gut lumen to blood) is nearly 5% to 15% of the iron (both heme and non-heme iron).

Fig. 13.4. Mechanism of iron absorption.

• Nonheme Iron: Nonheme iron is found predominantly in plant foods. It is also found in animals as nonheme enzymes and ferritin. Nonheme proteins include ferritin, transferrin, cytochromes and iron-sulphur proteins. It is transported by transferrin, from intestinal absorbing cells, to various tissues.

Factors affecting Iron Absorption

1. Iron absorption increases with its intake.

2. Iron is more effectively absorbed in the ferrous form than the ferric form.

3. Amino acids (such as cysteine) and reducing substances (e.g. vitamin C) stimulate iron absorption.

4. Due to the presence of certain complex forming agents, such as oxalates, phosphates, carbonates, phytates and fiber, iron from vegetables and cereals is poorly absorbed.

5. Iron status of the body also affects its absorption. Absorption of dietary iron varies inversely with iron stores of the body.

6. Hydrochloric acid secreted by the gastric mucosa enhances iron absorption by stimulating digestion of protein, which in turn releases protein-bound iron.

Storage: Between 1200–1500 mg of iron is stored in the body as ferritin and hemosiderin. Thirty percent of body’s iron is stored in the liver, 30% in the bone marrow and rest in the spleen and muscles.

Excretion: Iron is only lost through bleeding and in very small amounts through defecation, sweat and normal exfoliation of hair and skin. Unabsorbed iron is lost in the feces. Iron is not excreted in urine. Daily iron loss is approximately 1 mg.

Biological Functions

Iron plays number of roles in the body:

1. As a component of hemoglobin and myoglobin, iron is required for transport of O₂ and CO₂.

2. Several enzymes contain heme iron as a part of their prosthetic group, e.g. catalase, peroxidase, tryptophan pyrrolase, etc.

3. As a component of cytochromes, iron is required for electron transport chain and oxidative phosphorylation.

4. As a component of the lysosomal enzyme myeloperoxidase, iron is required in phagocytosis.

5. Iron is also an essential component of several non-heme proteins, such as transferrin, ferritin and iron-sulphur proteins (ferridoxins).

Deficiency Symptoms

Most common iron deficiency disorder is anemia, which is a common feature in children, adolescent girls and females of the child bearing age, particularly in developing countries.

ANEMIA

Anemia is characterized by reduced number of circulating erythrocytes and/or their hemoglobin contents. Anemia may be of three types:

Microcytic anemia: In this condition, erythrocytes become small in size and have reduced content of hemoglobin. Their number may also be reduced but to a smaller extent.

It is caused by the deficiency of iron. Conditions like pregnancy, hookworm infections and complete or partial gastrectomy may lead to iron deficiency anemia.

Macrocytic anemia: In macrocytic anemia, size of the erythrocytes is increased while their number is reduced, thereby presenting a characteristic picture of pernicious anemia. It is usually caused by the deficiency of vitamin B₁₂ and/or folic acid.

Normocytic anemia: In this condition, size as well as hemoglobin content of the erythrocytes remains normal but their number is reduced. It is usually due to hemorrhages.

Children become dull and inactive, have loss of appetite and poor growth. Deficiency of iron in females also reduces their work capacity.

Iron deficiency is also associated with reduced immunocompetence.

Requirements

Recommended dietary allowance for iron is about 10 mg for normal adult males. For females the requirement is 18–20 mg/day, particularly during the reproductive stage. This is on account of increased losses during menstruation and on account of increasing demand of the body during pregnancy and lactation. Daily requirement of iron is increased to nearly 30 mg/day during pregnancy.

Sources

Food cooked in iron cookwares is the best source of iron.

Animal foods, such as meat, fish and egg, are good sources of heme iron.

Green leafy vegetables, cereals and legumes (whole grains) are good sources of non-heme iron. Milk is a poor source of iron.

Iron Toxicity

Iron overload may lead to a condition called hemochromatosis (hemosiderosis). It may result in deposition of iron in many tissues, particularly in storage organs, such as liver and spleen, leading to hepatomegaly, pancreatic and cardiac dysfunctions, and skin pigmentation.

Hemochromatosis may be a result of failure of the regulation of iron absorption, excess dietary intake of iron, alcoholic cirrhosis, chronic pancreatitis or excessive blood transfusion.

Biological functions, deficiency disorders, requirements and dietary sources of other minerals are shown in Table 13.1.

Table 13.1. Minerals, their important biological functions, sources, daily requirements, deficiency disorders and toxicity symptoms

SOME IMPORTANT QUESTIONS

1. Describe dietary sources, daily requirement and biological functions of calcium.

2. Describe role of parathyroid hormone, vitamin D and calcitonin in calcium metabolism.

3. Name iron containing proteins that are found in the body and explain their functions. Also describe the disorders related to iron metabolism.

4. Describe:

(i) Calcium homeostasis

(ii) Factors affecting absorption of iron

(iii) Biological functions of phosphorus

(iv) Role of zinc in the body

5. Write notes on:

(i) Biological functions of calcium

(ii) Tetany

(iii) Ferritin

(iv) Transferrin

(v) Hemosiderin

(vi) Anemia

(vii) Hemosiderosis

(viii) Factors affecting absorption of calcium.

CHAPTER
14

Metabolism of Heme

Hemoglobin is a globular protein, which is present in high concentration in red blood cells. It binds oxygen in the lungs and transports it to different cells in the body. A molecule of hemoglobin consists of four heme groups along with the four polypeptide chains, which are synthesized separately and subsequently, bind to four heme groups.

SYNTHESIS OF HEME

Heme consists of one atom of ferrous iron (Fe²⁺) and a tetrapyrrole ring, called protoporphyrin IX. It is synthesized from eight residues of glycine and succinyl CoA (Fig. 14.1).

Fig. 14.1. Synthesis of heme.

In the biosynthesis of heme, some of the reactions occur in mitochondria while others in the cytosol.

1. In the first step, succinyl CoA condenses with glycine and forms δ-aminolevulinic acid (ALA), in the mitochondria.

This reaction is catalyzed by the enzyme δ-aminolevulinic acid synthase (ALA synthase), which requires pyridoxal phosphate (PLP) as a coenzyme. This is the rate limiting step of heme synthesis.

Increased levels of ALA synthase, along with the reduced levels of some enzymes of the heme biosynthetic pathway, result in a group of disorders referred to as porphyrias.

2. In the next step, two molecules of δ-aminolevulinic acid condenses, asymmetrically and form porphobilinogen.

This reaction is catalyzed by the enzyme aminolevulinic acid dehydratase (ALA dehydratase).

ALA dehydratase is a zinc-containing enzyme, which occurs in the cytosol. It consists of eight subunits, four of which interact with the substrate.

ALA dehydratase is sensitive to heavy metals particularly, lead. ALA levels increase in lead poisoning.

3. Porphobilinogen undergoes deamination, by the enzyme porphobilinogen deaminase (hydroxymethylbilane synthase or uroporphyrinogen I synthase) and is converted to a linear tetrapyrrole, called hydroxymethylbilane.

Decreased activity of porphobilinogen deaminase along with the increased activity of ALA synthase results in a condition called acute intermittent porphyria.

4. Spontaneously, with ring closure, hydroxymethylbilane is converted to uroporphyrinogen I.

Alternatively, uroporphyrinogen III synthase converts hydroxymethylbilane to uroporphyrinogen III (Fig. 14.2).

Fig. 14.2. Conversion of porphobilinogen to uroporphyrinogens.

Only, type I and type III isomers are found in nature. Porphyrins having symmetric arrangement of the substituent groups on four pyrrol rings are designated as type I while those having asymmetric substitutions are referred to as type III porphrinogens (Fig. 14.3).

Fig. 14.3. Type I and type III uroporphyrinogens.

Under normal conditions, uroporphyrinogen formed is, almost exclusively, type III. Heme is also a type III porphyrin (Fig. 14.4).

Fig. 14.4. Heme.

Abnormality of the reticulocyte uroporphyrinogen III synthase causes an inherited disease called erythropoietic porphyria. It is associated with marked cutaneous light sensitization and increased synthesis of uroporphyrinogen I and coproporphyrinogen I in the bone marrow.

5. Uroporphyrinogen I and III are converted to the respective coproporphyrinogens by the enzyme uroporphyrinogen decarboxylase.

Iron salts inhibit uroporphyrinogen decarboxylase.

Genetic deficiency of uroporphyrinogen decarboxylase leads to a disease, known as porphyria cutanea tarda, which shows cutaneous manifestations, primarily, sensitivity to light. The manifestation is expressed in patients who take drugs that cause an increase in porphyrin synthesis or in individuals who consume large amount of alcohol. This, in turn, leads to accumulation of iron, which further inhibits uroporphyrinogen decarboxylase.

6. In the next reaction, coproporphyrinogen III enters the mitochondria and is converted to protoporphyrinogen IX. This reaction is catalyzed by the enzyme coproporphyrinogen oxidase, which requires molecular oxygen. This enzyme does not act on coproporphyrinogen I.

(These porphyrins are identified as IX since they were designated ninth in a series of the postulated isomers.)

Inherited deficiency of the enzyme coproporphyrinogen oxidase leads to a form of the hepatic porphyria, known as hereditary coproporphyria.

7. Protoporphyrinogen oxidase acts on protoporphyrinogen IX and converts it to protoporphyrin IX, in the mitochondria.

Protoporphyrin IX is water insoluble. Its excessive amounts are excreted by the biliary system into the intestinal tract. Deficiency of the enzyme, protoporphyrinogen oxidase, leads to a condition called variegate porphyria.

8. In the final step of heme synthesis, ferrous iron (Fe²⁺) is incorporated into protoporphyrin IX. This reaction is catalyzed by the enzyme ferrochelatase, which requires some reducing substances.

This enzyme is sensitive to heavy metals, especially lead. It is also sensitive to iron deprivation.

Inherited deficiency of the enzyme ferrochelatase results in a disease, referred to as hereditary protoporphyria.

PORPHYRIAS

Porphyria is due to abnormality in the pathway of heme biosynthesis. It may be genetic or acquired. Six major types of porphyrias have been described, five of which are inherited as autosomal dominant (Table 14.1).

Table 14.1. Derangement in porphyrin metabolism in different types of porphyrias

Regulation of Heme Biosynthesis

ALA synthase occurs in two forms. These are called hepatic (ALA-S1) and erythrocytic (ALA-S2) enzymes.

ALA-S1 controls the rate-limiting step of heme synthesis. Further, heme acts both, as a repressor of synthesis of the enzyme as well as its inhibitor.

HEME BREAKDOWN

Red blood cells have limited life span of approximately 100–120 days. These senescent cells are recognized by their membrane changes, removed and engulfed by the reticuloendothelial system, at the extravascular site. Degradation of red blood cell occurs in spleen, bone marrow, liver and lymph glands.

HEMOGLOBINOPATHIES

Patients with unstable hemoglobin develop hemoglobinopathies with characteristic features, such as anemia, reticulocytosis, splenomegaly and urobilinuria. Hemoglobinopathies are a heterogeneous group of single gene disorders.

A mutant gene may result in the substitution of one amino acid, in one type of the polypeptide chain, by another amino acid, or loss of one or more amino acid residues of a polypeptide chain. This, in turn, results in the formation of structurally abnormal hemoglobin. In some cases, the change may be clinically insignificant while in others it may cause a serious disease. The most common hemoglobinopathy is sickle cell disease.

Hemoglobin C: Hemoglobin C is another variant of HbA₁ where glutamic acid, normally present at the sixth position of the β-chain of HbA₁, gets replaced by lysine. This, in turn, results, in the formation of aggregates and reduces survival time for red blood cells. This form of hemoglobinopathy exhibits more limited pathological effects and is commonly found among certain black African population.

Sickle Cell Anemia

Sickle cell anemia or sickle cell disease is a hereditary blood disorder, characterized by red blood cells that assume an abnormal, rigid, sickle shape (Fig. 14.5).

Fig. 14.5. Red blood cells in sickle cell anemia.

This is a consequence of point mutation. Red blood cells of such individuals have hemoglobin S (HbS), which differs from that of the normal adult hemoglobin (HbA₁) with respect to only one amino acid in their β-chains, i.e. glutamic acid normally present at the sixth position of the β chain of HbA₁ gets replaced with valine in HbS. This is due to a change in a single nucleotide within the codon (from GAA or GAG, which code for glutamic acid, to GUA or GUG, which code for valine). This missense mutation results in a mutant gene, which is present in homozygous state and results in sickle cell disease. Such a mutation is mostly seen in some Blacks, (in some parts of the world), particularly where malaria is widespread. Hemoglobin S though can bind and release oxygen but abnormally. Heterozygous individuals do not exhibit symptoms of sickle cell anemia except under extreme conditions of hypoxia, due to the reason that such individuals have 50% of HbA₁ and 50% of HbS in their red blood cells.

Degradation of heme occurs mainly, in the liver. If degradation of red blood cells occurs in the tissues other than the liver, hemoglobin is transported to the liver by means of hapto-globulin.

After the aged red blood cells are recognized by macrophages, they are rapidly engulfed by the phagocytes and form phagosomes. They fuse with the primary lysosomes and form secondary lysosomes. Lysosomal cathepsin results in complete degradation of the cellular proteins, including globin of hemoglobin, to the constituent amino acids, which are utilized for general metabolic needs.

Heme is degraded in the reticuloendothelial cells, to a linear tetrapyrrole (biliverdin IXa), by the microsomal enzyme system, which is designated as heme oxygenase. This enzyme requires molecular oxygen and NADPH, and is induced by heme. Heme oxygenase catalyzes the cleavage of α-methenyl bridge which is quantitatively converted to carbon monoxide (CO) that is trapped by the hemoglobin and eventually exhaled.

Biliverdin is reduced to bilirubin by the enzyme biliverdin reductase (Fig 14.6).

One gram of hemoglobin yields about 35 mg of bilirubin.

Fig. 14.6. Breakdown of hemoglobin.

BILIRUBIN

Bilirubin is an orange-yellow pigment, derived from the breakdown of red blood cells in the liver, spleen and bone marrow. Its daily production, in men, averages from 250–300 mg. Approximately, 85% of this is derived from the heme moiety of hemoglobin, which is released from the erythrocytes that are destroyed in the reticuloendothelial cells while rest of it is formed from catabolism of other heme containing proteins, such as myoglobin, cytochromes and other heme containing enzymes.

Bilirubin, normally present in the blood, is bound to albumin and is transported to the liver.

Hepatocytes trap bilirubin by means of a specific binding protein, called ligandin. In the hepatocytes, bilirubin gets conjugated with UDP-glucuronate, which is derived from the oxidation of UDP-glucose. This reaction is catalyzed by UDP-glucuronyltransferase (Fig. 14.7).

Fig. 14.7: Conjugation of bilirubin.

THALASSEMIAS

Thalassemia is a family of related genetic disorders that arise due to the deletion of one or more globin-like genes in either the globin gene cluster or a defect in transcription, and/or processing of mRNA of the globin gene. If there is reduced synthesis or total lack of synthesis of α-globin mRNA, the disease is classified as α-thalassemia. On the other hand, if β-globin mRNA level is affected, it is called β-thalas-semia.

One to four α-globin genes may be missing in the patients with α-Thalassemia. If one α-globin gene is missing, the condition is called α-Thalassemia 1 (α-thal 1). When two α-globin genes are missing, it is called α-thal 2. Both the conditions are associated with mild to moderate anemia. On the other hand, if three α-globin genes are missing it results in the synthesis of more β-globin molecules, forming a tetramer containing four β-globin sub-units. This condition is called HbH disease. When all the four α-globin genes are absent, it results in a fatal condition called hydrops fetalis.

β-Thalassemias also exhibits different degree of severity and can be caused by a variety of defects or deletions.

In the normal bile, bilirubin diglucuronide is the major form of excreted bilirubin with only a small amount of the bilirubin monoglucuronide. As bilirubin diglucuronide is much more water soluble than free bilirubin, transferase thus facilitates the excretion of bilirubin, via bile duct, into the intestine.

As bilirubin diglucuronide is poorly absorbed by the intestinal mucosa, glucuronide residues are released in the terminal ileum and large intestine, by intestinal β-glucuronidases and by the enzymes produced by anaerobic bacteria. It is reduced to colorless linear tetra-pyrroles, called stercobilinogen, mesobilinogen and urobilinogen. These compounds are collectively referred to as urobilinogens.

A large portion of urobilinogens are excreted in the feces.

Majority of these re-circulating pigments are taken up by the liver and re-excreted in the bile. Some of the urobilinogens (only up to 2%) are also reabsorbed passively from the colon and return to the liver via the portal venous blood. This is referred to as enterohepatic circulation (Fig. 14.8).

Fig. 14.8. Metabolism of bilirubin.

Most of the reabsorbed urobilinogen is taken up by the liver and is re-excreted in the bile. A small portion of urobilinogen (2–5%) however, escapes hepatic extraction, reaches the peripheral circulation and is excreted in the urine.

Serum thus contains two different forms of bilirubin, which are referred to as:

1. Unconjugated (lipid soluble) form that is bound to albumin and is transported from the reticuloendothelial system to the liver, and

2. Conjugated (water soluble) form which is regurgitated from the liver into the plasma.

Normal value, for the sum of the unconjugated and conjugated forms, is 0.1–1.0 mg/100 ml of serum or plasma.

Normally, almost all the bilirubin in plasma is unconjugated.

Disorders of bilirubin metabolism lead to hyperbilirubinemia where serial measurement of bilirubin is helpful in knowing the severity of a liver disease.

Bilirubin fractionation is also helpful in differential diagnosis of jaundice.

JAUNDICE

Jaundice is a physical sign characterized by yellow appearance of the patient and is a most characteristic clinical manifestation of hyperbilirubinemia. It results from the deposition of bilirubin (bile pigment) in the skin, mucous membrane and sclera of the patient (Fig. 14.9).

Fig. 14.9. Jaundice.

Jaundice is apparent clinically, when serum bilirubin level is more than 2 mg/dL. If serum bilirubin is below 2 mg/dL, it is called latent jaundice (subclinical jaundice) since it is not detectable at this stage, clinically.

There are three types of jaundice, referred to as pre-hepatic, hepatic and post-hepatic jaundice. Biochemical differentiation of the three types of jaundice is given in Table 14.2

Table 14.2. Differential diagnosis of different types of jaundice based on various biochemical tests

Prehepatic Jaundice

Pre-hepatic jaundice is also called hemolytic jaundice. It is characterized by the excessive presence of unconjugated bilirubin. It may be a result of increased production of unconjugated bilirubin such as in hemolysis, decreased uptake of unconjugated bilirubin across the hepatocyte membrane as in Gilbert’s syndrome, or decreased biotransformation such as in neonatal jaundice, Criggler-Najjar syndrome, etc.

Hemolysis: In disorders associated with hemolysis (hemolytic anemia), rate of bilirubin production is increased, which may exceed the amount that can not be removed by the liver.

Gilbert’s syndrome: This is a heterogeneous group of disorders inherited as autosomal recessive trait. Several defects include the deficiency of bilirubin glucuronyltransferase, a defect in hepatic uptake of bilirubin, or a decrease in red cell survival.

Neonatal jaundice: Neonatal jaundice is also called physiological jaundice of the newborn. Every infant exhibits some transient unconjugated hyperbilirubinemia (about 5 mg/dL) between the second and the fifth day of life, as at this stage hepatic glucuronyltransferase activity is not fully developed. Activity of the enzyme increases within 2 weeks after birth when serum bilirubin returns to normal.

Neonatal jaundice is more pronounced in a premature infant.

Unconjugated bilirubin levels above 18 mg/dL lead to deposition of bilirubin in the lipid-rich basal ganglia. This is referred to as kernicterus.

Criggler-Najjar syndrome: This disorder is known to exist in two forms:

Type I: It is clinically more severe form of the disease. It is due to complete absence of glucuronyl transferase.

Type II: It has moderate clinical findings and is due to partial deficiency of the enzyme. In both the conditions unconjugated bilirubin is significantly raised.

Hepatic Jaundice

Hepatic jaundice is due to a primary disease. Plasma, usually, shows elevated levels of both conjugated as well as unconjugated bilirubin, e.g. in viral hepatitis, liver cirrhosis, etc. There is also an increase in SGOT and SGPT levels.

Elevated levels of conjugated as well as unconjugated bilirubin along with the dark-brown pigmentation, in the liver cells, is also observed in a benign, autosomal recessive condition known as Dubin-Johnson syndrome.

Inhalation, ingestion or parenteral administrations of several pharmacological and chemical agents, such as carbon tetrachloride, acetaminophen, isoniazid or chlorpromazine, which result in liver injury, also lead to hepatic jaundice.

Posthepatic Jaundice

Post-hepatic jaundice is also referred to as obstructive jaundice or post-hepatic cholestasis. It is caused by obstruction of the bile duct, may be due to gallstones, carcinoma of the head of pancreas or carcinoma of the bile duct. Serum of such a patient shows excessive amount of conjugated bilirubin. It is also characterized by the presence of the excessive amount of bilirubin (bile pigment) in the urine.

SOME IMPORTANT QUESTIONS

1. Describe the process of heme synthesis. How it is regulated?

2. What is porphyria? Describe various types of porphyrias.

3. Describe the process of heme degradation.

4. Write notes on:

(i) Hemoglobin S

(ii) Sickle cell anemia

(iii) Hemoglobinopathies

(iv) Thalassemias

(v) Jaundice

CHAPTER
15

Water and Electrolyte
Balance

Adequate hydration (water) and normal levels of electrolytes are important for proper functioning of the body.

WATER

Water is a very special kind of nutrient as it is consumed at more frequent intervals and in greater amounts as compared to other nutrients. Besides:

• It is the most abundant substance present in the body.

• It is more important than food, as if one is given water but no food, the individual may survive several weeks inspite of the loss of most of the body fat and 50% of the tissue proteins. On the other hand, a loss of about 10% of the body water may cause illness whereas a further loss of 10% of body water may even cause death.

• Increased fluid intake is very beneficial for individuals with renal calculi, urinary tract infections and chronic obstructive respiratory disease.

Human body consists of about 50–70% of its weight as water. At birth, about 75% of total body mass is water which is reduced to nearly 60% for an average adult male. The value falls to about 50% thereafter. Due to relatively high fat content, females have nearly 5% less water.

Most tissues contain more than 70% water though bone and adipose tissue contain relatively lesser amounts. This is due to the reason that the proportion of water in a tissue or in the whole body depends upon the proportion of fat in the tissue (Table 15.1).

Table 15.1. Water contents of different tissues

Tissue | Water content (% of total weight)
Whole body | 60–70
Muscles | 75–80
Nervous tissue | 70–85
RBC | 60
Bones | 25
Adipose tissue | 20

Physiological Functions of Water

Water is needed for various physicochemical processes in the body:

1. It is a common solvent in which most of the chemical reactions of the body occur.

2. It participates in various metabolic processes within the cell.

3. It transports vital substances via blood, lymph and other body fluids.

4. It is also essential for the elimination of waste products from the body.

5. Water intake helps to maintain water content of the faeces and thus promotes regular bowel movement.

6. Increased water intake prevents urinary tract infections and kidney stones, by increasing the flow of urine.

7. It helps to provide lubrication in various strategic locations of the body, e.g. synovial fluid between the joints, mucus in the respiratory passage, etc.

8. As the latent heat of evaporation of water is high, it assists in the regulation of body temperature.

Sources and Requirements

Most of the foods, beverages, and many fruits and vegetables are good sources of water. Water is also available to the body as a result of metabolic breakdown of food, for energy. The amount of water produced by the oxidation of nutrients however, depends upon the type of the nutrient used in the body, e.g. 100 g of fat produces 107 ml, 100 g of carbohydrates produce 56 ml while 100 g of proteins produce 41 ml of water. On an average, the amount of endogenous water, produced by an individual, is nearly 300 ml per day.

Daily need for water varies widely, with variations in several characteristics of the person, e.g. body surface area, amount of body fat, metabolic rate, physical activity and environmental factors such as temperature, humidity, altitude, clothing, etc. The minimum requirement for a resting healthy 70 Kg adult man, at moderate environmental temperature and humidity, is about 1700–1800 ml/day. Normal activity increases this requirement by 700–800 ml/day. Thus, a total of about 2500 ml of water is required daily. This in turn suggests that under normal circumstances about 1 ml/Kcal is a reasonable allowance for an adult. A higher requirement, about 1.5 ml/Kcal, is recommended for healthy infants.

Regulation of Body Water

In a normal adult, the quantity of water intake is balanced by that which is eliminated from the body every day, thus maintaining a state of equilibrium, called water homeostasis (Fig. 15.1).

Fig. 15.1. Regulation of body water.

Water balance is thus regulated by two mechanisms, i.e. by its intake as well as output.

Water intake

Volume of the liquid ingested is regulated by thirst, which is under the control of thirst centre. Minimum requirement for water, however, is highly variable and depends upon several other factors, such as:

• Obligatory losses: There is an obligatory but variable loss of water in insensible perspiration, breath and faeces. Sweating can greatly increase fluid loss and thus its demand.

• Disease conditions: Fluid intake is also associated with physical condition and injury, e.g. fluid loss increases due to watery diarrhoea or blood loss, etc. Human body has receptors in the right atrium, called baroreceptors, which are sensitive to changes in osmotic pressure and blood volume. During certain pathological conditions, e.g. congestive heart failure, hepatic cirrhosis, nephrotic syndrome, etc., baroreceptors detect a decrease in blood volume despite an overall expansion of the ECF. This excess fluid in the ECF compartment gets collected in the tissues and cause edema.

• Dietary factors: Sodium tends to hold water so that extra sodium (salt) intake may increase water content of the body. Carbohydrate content of the diet affects both sodium and water balance. Increased amount of carbohydrate in the diet increases water retention while low carbohydrate intake results in a loss of body fluid. Serum proteins influence water balance by their osmotic effect since proteins draw water from the interstitial fluid into the blood. If a low protein diet is consumed for a longer duration, serum albumin levels fall to such an extent that fluid accumulates between the cells, and thus total body water is increased.

Water output

Water is removed from the body in the form of urine, faeces, perspiration and expiration:

• Urine: It is an important medium for the elimination of water, present in the body, in excess of its requirement. Normally, about 1.2 to 1.5 litre of water is excreted in the form of urine, daily.

• Feces: Normally, about 80–150 ml of water is excreted in the feces of an adult, daily. The value may increase, considerably, on a high vegetable diet, particularly in the presence of diarrhoea.

• Perspiration: The amount of water lost in the perspiration varies enormously. When the environmental temperature or humidity increases, sweat glands become active and thus in summer, excessive water is lost via perspiration. Sweat removes a hypotonic solution, which in addition to water also contains Na⁺ and Cl⁻. Insensible perspiration is the process whereby the body loses water continuously from the skin surface and lungs. Contrary to the visible perspiration which removes hypotonic solution, insensible perspiration consists solely of water. Normally, from an adult human being about 300–700 ml of water is eliminated by this route, daily.

• Expiration: Nearly 500 ml of water is lost daily, in the exhaled air.

Failure to regulate water balance results in dehydration.

DEHYDRATION

Water deficit or volume depletion is referred to as dehydration. However, pure water deficit is rare and hence in clinical practice, the term ‘dehydration’ indicates water plus electrolyte deficit. Several conditions, such as diarrhea, uncontrolled diabetes mellitus, diabetes insipidus, overdose of diuretics, heat stroke etc., may lead to dehydration. Fluid and electrolyte status of these patients should be corrected at the earliest, by employing suitable oral (oral rehydration solution) or intravenous (isotonic saline, dextrose saline, Ringer lactate) fluids.

Distribution of Water in Different Compartments

Total body water can be divided into two major compartments, called extracellular fluid (ECF) and intracellular fluid (ICF). In a young adult man, two third of the body water is in the ICF and one third in the ECF (Fig. 15.2).

Fig. 15.2. Distribution of body water in different compartments.

The two fluids also differ in terms of their electrolyte composition. Their total solute concentrations (osmolality), however, are equal because of the high water permeability of most cell membranes, so that an osmotic difference between cells and ECF rapidly disappears. Electrolyte concentrations of the body fluid in different compartments are shown in Table 15.2.

Table 15.2. Balance of cations and anions concentrations in ECF and ICF (meq/l)

Electrolytes | ECF (meq/L) | ICF (meq/L)
Cations |  |
Na⁺ | 140 | 35
K⁺ | 5 | 123
Ca²⁺ | 5 | 15
Mg²⁺ | 5 | 2
Total | 155 | 175
Anions |  |
Cl⁻ | 104 | 5
PO₄³⁻ | 2 | 80
SO₄²⁻ | 1 | 10
Protein | 16 | 70
HCO₃⁻ | 27 | 10
Organic acids | 5 | –
Total | 155 | 175

Extracellular Fluid

Extracellular fluid (ECF) is composed of fluid outside the cells. It is further subdivided into two compartments, which are separated from each other by the endothelium of blood vessels.

Plasma: The plasma is the ECF found within the vascular system. It is the fluid portion of the blood in which blood cells are suspended. Plasma constitutes approximately 5% of the body volume (about 3.5L) for an average 70 Kg man.

Most important ions in plasma are Na⁺, K⁺, Ca²⁺, Cl⁻, HCO₃⁻ and phosphates. In addition, glucose and urea contribute to plasma osmolality. The total plasma concentration of cations is about 150 meq/L (or mmol/L), out of which Na⁺ is, approximately 140 meq/L while potassium is about 5 meq/L. Most abundant plasma anions are Cl⁻ and HCO₃⁻ with average concentrations of nearly 104 and 27 meq/L, respectively.

Because concentrations of Na⁺, K⁺, Cl⁻ and HCO₃⁻ provide most relevant information about the osmotic status, hydration and pH of the body, they are commonly analyzed in the plasma or serum in an electrolyte profile.

An increase in the concentration of one anion is accompanied either by a corresponding decrease in other anions or increase in one or more cations or both so total the electrical neutrality is invariably maintained.

Interstitial fluid and lymph: The interstitial fluid and lymph are considered together because they cannot be separated easily. Both are ultrafiltrates of plasma. The interstitial fluid directly bathes most body cells while the lymph is the fluid within lymphatic vessels. The water of the interstitial fluid and lymph comprises 10.5 L of the ECF.

Plasma is separated from the interstitial fluid by endothelial lining of the capillaries, which act as a semipermeable membrane and allows the passage of water and diffusible solutes but not compounds of the large molecular mass, such as proteins. Oncotic pressure, exerted by the plasma proteins, determines the distribution of water between plasma and the interstitial fluid.

The plasma, interstitial fluid and lymph are nearly identical in composition except for the higher protein concentration in the plasma.

Transcellular fluid: An additional ECF compartment, the transcellular fluid, is small but physiologically important, amounting to 1–3% of body weight. It includes the cerebrospinal fluid, aqueous humor, secretions of the gastrointestinal tract and associated organs (saliva, gastric juice, bile, pancreatic juice, intestinal juice, etc.), renal tubular fluid and bladder urine, synovial fluid and sweat. In these cases, the fluid is separated from the plasma by an epithelial cell layer, in addition to a capillary endothelium. The epithelial layer modifies the electrolyte composition of the fluid so that transcellular fluids are not plasma ultrafiltrates (as are interstitial fluid and lymph). There is a constant turnover of transcellular fluids, i.e. they are continuously formed and absorbed or removed. Impaired formation, abnormal loss from the body or blockage of fluid removal can have serious consequences.

Intracellular fluid

Intracellular fluid (ICF) is composed of the fluid within the trillions of cells in our body.

The volume of the ICF constitutes nearly 66% of total body volume. Composition of ICF markedly differs from that of ECF. These two compartments are separated from each other by a cell membrane, which in turn allows the composition of these fluids to differ. The composition differences are a consequence of the Gibbs-Donnan equilibrium and active and passive transports of ions. The transport of small molecules across the impermeable membrane is possible due to the existence of specific transport proteins, including ion pumps. Amongst several ionic pumps, Na⁺/K⁺-ATPase shows greatest transport activity.

Potassium is the main intracellular cation (about 110 meq/L). There is also a substantial amount of magnesium within the cells. The main anions in the ICF are phosphates and proteins.

METABOLISM OF ELECTROLYTES

Major electrolytes are the ionic forms of essential minerals, specifically sodium (Na⁺), potassium (K⁺) and chloride (Cl-). Metabolism of electrolytes in described in detail in Chapter 13.

Their functions are described in Table 15.3.

Table 15.3. Functions of major electrolytes

Sodium (Na⁺)

• Movement of fluid from one compartment to another

• Hydrogen ion balance of ECF

• Maintenance of osmotic pressure of ECF

• GI and renal absorption of glucose and amino acids

• Normal muscle irritability and contractility

• Normal cell permeability

Potassium (K⁺)

• Normal neuronal excitability

• Contractility of muscle

• Intracellular osmotic pressure

• Intracellular hydrogen ion balance

• GI secretions

• Cofactor for enzymes

• Storage of proteins and glycogen

Chloride (Cl⁻)

• Maintenance of osmotic pressure of ECF

• Hydrogen ion balance of ECF

• Component of HCl of gastric juice

• Chloride shift

SODIUM

The adult human body contains nearly100 g of sodium, as sodium ions (Na⁺). About half of this is found in the extracellular fluid while the remaining in the cells and bone. Sodium is the major cation in the ECF. It exists in the body in association with several anions, such as chloride, bicarbonate, phosphate, lactate and protein. High concentration of Na⁺ alongwith low K⁺ in the extracellular fluid is maintained by Na⁺/K⁺-ATPase (Na⁺/K⁺-pump).

Alterations in Sodium Homeostasis

Hyponatremia

It is defined as plasma Na⁺ concentration <135 mEq/L. It is the most common disorder of body fluid and electrolyte balance. Most often, it reflects a problem of too much water, not too little Na⁺, in the plasma. Drinking large quantities of water rarely causes hyponatremia because of the large capacity of the kidney to excrete dilute urine. If, however, plasma anti-diuretic hormone (ADH) is not decreased when plasma osmolality is decreased or if the ability of the kidney to dilute the urine is impaired, hyponatremia may develop even with a normal water intake.

Because Na⁺ is the major solute in the plasma, hyponatremia is usually associated with hypo-osmolality. However, hyponatremia may also occur with a normal or even elevated plasma osmolality.

General symptoms include muscle cramps, lethargy, fatigue, disorientation, headache, anorexia, nausea, agitation, hypothermia, seizures and coma. These symptoms, mainly neurological, are a consequence of the swelling of brain cells, as plasma osmolality falls. Excessive brain swelling may be fatal or may cause permanent damage.

Hypernatremia

Hypernatremia is defined as plasma Na⁺ concentration >150 meq/L. It is always hyperosmolar and is characterized by edema, azotemia and anemia.

Accumulation of Na⁺ in the body has been shown to result in a concomitant rise in blood pressure, referred to as essential hypertension. Symptoms are primarily neurological, which include tremors, irritability, confusion and coma.

POTASSIUM

Potassium is the chief cation in the ICF. Potassium is completely absorbed from the gastrointestinal tract. Its functions are outlined in Table 15.3.

Alterations in Potassium Homeostasis

Disorders of acid base balance, water balance and administration of some drugs are often accompanied by alterations in potassium balance, either potassium deficit (hypokalemia) or excess (hyperkalemia).

Hypokalemia

Hypokalemia is defined as serum potassium concentration <3.5 meq/L. It is seen in individuals taking potassium losing diuretics (carbonic anhydrase inhibitors, loop diuretics and thiazides), those with severe liver disease, gastrointestinal losses and metabolic alkalosis. Hyperaldosteronism can cause excess excretion of potassium, accompanied by increased retention of Na⁺, which leads to depletion of plasma K⁺. Prolonged hypokalemia, in turn, further causes damage to the kidney.

Both cardiac and skeletal muscles are affected by hypokalemia. Deficiency symptoms include muscular weakness, mental apathy, cardiac arrhythmias (irregular heart beat), tachycardia (heart beat more rapid than usual), hypotension, paralysis, bone fragility, etc.

Hyperkalemia

Hyperkalemia is defined as serum potassium concentration >5.5 meq/L. Hyperkalemia arises when K⁺ excretion by the kidney is depressed, such as in acute renal failure. Acidosis causes leakage of K⁺ from the cell, which may increase plasma K⁺ while leaving the cells depleted of K⁺. Potassium-sparing diuretics may lead to hyperkalemia. Early signs of cardiac damage include arrhythmia followed by a slowing of the heart rate, ventricular fibrillation and arrest in diastole.

Hyperkalemia may lead to CNS depression, mental confusion, numbness or tingling of extremities and weakness of respiratory muscles, resulting in difficulty in breathing and speech. High concentration of potassium is also toxic to the muscles and may cause paralysis.

CHLORIDE

Chloride (Cl⁻) is the most abundant anion in the ECF. Normal plasma chloride concentration varies between 95–107 meq/L. The highest concentration of chloride is found in the CSF. Its functions are outlined in Table 15.3.

Alterations in plasma Cl⁻ concentration generally parallel with those in plasma Na⁺ except in the presence of some acid base disturbances. The determination of plasma Cl⁻ concentration, thus is useful in the differential diagnosis of acid base disturbances, for calculating the anion gap.

Alterations in Chloride Balance

Hypochloremia

Decrease in plasma Cl⁻ concentration, called hypochloremia, occurs in:

• Persistent diarrhea and prolonged vomiting. The chloride content of the gastric juice is normally about 150 mmol/L. Patients who lose large volume of gastric secretion, e.g. due to pyloric stenosis, often show a marked fall in plasma Cl⁻. Loss of HCl, in turn, results in compensatory increase in plasma HCO₃⁻. Hence, these patients develop metabolic alkalosis and are often dehydrated.

• Metabolic acidosis, such as diabetic ketoacidosis and renal failure.

• In respiratory alkalosis, due to hyperventilation which leads to elimination of CO₂ with consequent increase in blood HCO₃– and a compensatory decrease in Cl⁻.

• Extreme sweating.

• Salt-losing nephritis, e.g. chronic pyelonephritis, which is probably due to defective occur in patients who develop respiratory acidosis. tubular reabsorption.

• In patients with Addison’s disease, due to reduced renal tubular reabsorption of chloride.

Hyperchloremia

Increased plasma Cl⁻ concentration, called hyperchloremia, occurs with dehydration, in patients with acute renal failure, renal tubular acidosis, metabolic acidosis associated with prolonged diarrhoea and loss of sodium bicarbonate, diabetes insipidus and adrenocortical hyperfunction (Cushing’s syndrome).

EDEMA

Due to the retention of water along with sodium, both sodium and water move from blood to the interstitial fluid, thereby allowing the maintenance of a relatively normal blood volume. When fluid retention is observable, the condition is called edema.

Edema often takes the form of puffiness in fingers and ankles or sometimes on the entire body. Retention of sodium and water, as in patients with congestive heart failure, increases workload of the heart and precipitates acute heart failure. A serious consequence of acute heart failure may result in congestion in the lungs (pulmonary edema) and thus may lead to respiratory distress.

Dietary modifications particularly, reduced sodium intake, often in conjunction with diuretic therapy, is helpful in the treatment of such conditions, which are associated with the excessive retention of sodium as well as water. However, when diuretic therapy is used along with sodium restriction, special attention should be paid to the need of potassium and other minerals whose excretion is also affected by the diuretic. Use of loop diuretics, such as furosemide and thiazides, is known to cause depletion of potassium, magnesium and zinc.

Some increase in plasma chloride may also occur in patients who develop respiratory acidosis.

In severe diarrhoea, there is a loss of HCO₃⁻while in respiratory acidosis there is retention CO₂ due to shallow breathing. Thus, reduced levels of HCO₃⁻ result in compensatory hyperchloremia.

Chloride Shift

At the normal pH (7.4) of blood, carbonic acid (a constituent of the bicarbonate buffer) dissociates into H⁺ and HCO₃⁻ (bicarbonate ions). Some of the HCO₃⁻ combine with K⁺ present in the red blood cells and form potassium bicarbonate while the remainder is diffused from the cell to the plasma. The electroneutrality of plasma, caused by the shifting of HCO₃⁻ from the red blood cells, is balanced by the shift of Cl⁻ from plasma into red blood cells. This process is known as chloride shift.

LABORATORY EVALUATION OF WATER AND ELECTROLYTE DISORDERS

The following measurements are performed for the assessment of water and electrolyte balance:

1. Serum electrolyte concentration – it includes sodium, potassium, chloride and bicarbonate.

2. Serum creatinine and urea concentration

3. Urine volume, osmolality and sodium concentration

4. Serum osmolality

SOME IMPORTANT QUESTIONS

1. What are the important physiological functions of water in the body?

2. Discuss various factors responsible for the regulation of body water.

3. Describe physiological functions of sodium.

4. Discuss important physiological functions of potassium.

5. Describe physiological functions of chloride.

6. Write note on:

(i) Intracellular fluid

(ii) Extracellular fluid

(iii) Hyponatremia

(iv) Hypernatremia

(v) Hypokalemia

(vi) Hyperkalemia

(vii) Anion gap

(viii) Chloride shift

(ix) Hypochloremia

(x) Hyperchloremia

(xi) Dehydration

(xii) Edema

CHAPTER
16

Immunochemistry
Balance

The immune system is a complex system; consisting of the tissues, cell and molecules and genes involved in the recognition, reaction with and elimination of foreign and non-self substances that may disturb the homeostasis of the body. Such substances are called antigens.

The ability of the body to protect itself from infection by microbes, parasites and other foreign substances, that can be referred to as non-self, is called immunity.

NONSPECIFIC AND SPECIFIC IMMUNE RESPONSE

Immune response can be categorized as being either nonspecific or innate, and specific or adaptive.

Nonspecific Immune Response: Non-specific immune response is the body’s first line of defense. It uses physicochemical barriers, such as the skin and mucosal epithelia, and their associated secreted products, e.g. sweat, mucus and acid. It also involves inflammatory response.

Specific Immune Response: The immune system is the specific response to antigen and includes cell-mediated immunity and humoral immunity.

Immunity is divided into natural immunity and acquired immunity.

Natural Immunity

Natural, native or innate immunity is the nonspecific immunity, which is inborn and unchanging, resulting from the genetic nature of the host. For example, humans are naturally immune to many of the infectious diseases that affect animals.

Acquired Immunity

Acquired immunity is further divided into active and passive immunity.

Active Immunity: Active or adaptive immunity results from development of the immune response when an individual is stimulated by an antigen, such as those associated with invading microorganisms. This response is specific to a particular antigen.

Passive Immunity: This immunity is received by a non-immune individual from an immune individual, e.g. injection of a human with tetanus antitoxin, which has been produced in horse, to protect against tetanus.

Cell-mediated immunity (cellular immunity) is a form of acquired immunity that is dependent on the action of various types of T lymphocytes. It is particularly important in defending against those diseases caused by intracellular pathogens (e.g. viruses), facultative intracellular parasites (e.g. Mycobacterium tuberculosis), some bacteria, fungi, protozoan and helminths. This is specific and mediated by T lymphocytes (except NK cells) that recognize MHC-bound antigens.

ANTIGEN

Substances that interact with the immune system and elicit an immune response are referred to as immunogens or antigens. Any substance that is capable of stimulating an immune response is referred to as immunogen. In contrast, those substances that are capable of stimulating specific immune responses by binding with specific lymphocytes or antibodies are defined as antigens. The features of an antigen that make it immunogenic include foreignness, high molecular weight and chemical complexity. A wide variety of macromolecules, such as proteins, lipoproteins, peptides, glycoproteins, many polysaccharides and some nucleic acids that are foreign to the individual, can act as antigens.

Hapten: Low molecular weight substances, such as amino acids, sugars and small polymers that by themselves are not able to initiate antibody formation but can combine with specific carrier molecules (e.g. a protein) and induce a specific antibody response, are called haptens.

THE IMMUNE SYSTEM

The immune system consists of a complex network of different cells and their products that interact in the immune response. The components, comprising the immune system, arise in the fetal bone marrow between the third and sixth month of fetal life. Cells called stem cells arise and multiply in the bone marrow to yield two major cells lines, i.e. the hemopoietic cells and the lymphopoietic cells.

The hemopoietic System

Stem cells give rise to hemopoietic stem cells, which give rise to the following cell types:

Megakaryocytes: These cells give rise to platelets (thrombocytes), which are involved in blood clotting and inflammation.

Erythroid stem cells: These differentiate into red blood cells, which have no role in the immune system.

Granulocyte-monocyte precursor cells: These differentiate into various cells of the myeloid series (granular leucocytes and mononuclear phagocytes), depending on various growth or colony-stimulating factors.

Granulocytes include four cell types, i.e. basophils, mast cells, neutrophils (poly-morphs) and eosinophils. The granulocytemonocyte common precursors also give rise to monocytes and macrophages.

The Lymphopoietic System

Lymphoid Organs: The primary lymphoid organs serve as the site of maturation for lymphocytes, and include the bone marrow and the thymus.

The secondary lymphoid organs, the spleen and lymph nodes, are responsible for lymphocyte interaction with antigen, other immune cells and accessory cells. The lymph nodes are located throughout the body. In addition, tonsils, adenoids, the vermiform appendix and Peyer’s patches of the ileum region of the small intestine, all consist of lymphoid tissue. Clusters of lymphoid tissue, like the Peyer’s patches, are also found in mucous and other membranes and throughout various tissues (Fig. 16.1).

Fig. 16.1. Lymphoid organs.

Stem cells give rise to lymphopoietic stem cells, which differentiate into T and B lymphocytes after processing in the thymus and bursa equivalent (bursa, bone marrow or fetal liver), respectively.

T Lymphocytes: T Lymphocytes (T for thymus) or T cells comprise about 75% of lymphocytes in blood. They are the main components of cell-mediated immunity and (like B cells) they are derived from precursor lymphoid stem cells in the bone marrow. The T cells have surface antigens that make possible their identification. The T cells further differentiate in the thymus, as follows:

Cytoxic T Cells: Cytoxic T cells (Tc cells) are involved in killing foreign cells. They recognize foreign antigens, imbeded in MHC class I molecules. They possess the surface antigen CD8 and produce the cytokine γ-interferon.

Helper T Cells: These cells have a surface antigen called CD4. Helper T cells (Th cells) are required for the production of normal levels of antibody by B cells and aid in development of cell-mediated immunity. The various functions of the Th cells are due to cytokines, interleukin-2 (IL-2), IL-4, IL-6, IL-10 and γ-interferon. There are two subsets of Th cells, referred to as Th1 (for macrophage activation) and Th2 (for antibody production).

Delayed Hypersensitivity Cells: Delayed hypersensitivity cells (Td cells) have the surface antigen CD4. They produce several cytokines whose functions include attracting macrophages and other defensive cells. Td cells are also involved in the rejection of transplant tissue, some allergic reaction and immunity to neo-plasms.

Suppressor T Cells: Suppressor T cells (Ts cells) produce cytokines and other effects that suppress the immune response.

Besides these T cells, natural killer cells and macrophages also play an important role in cell mediated immunity.

Natural Killer Cells: Natural killer cells (Nk cells) are the cytotoxic lymphocytes, which are neither T cells nor B cells but are of lymphoid lineage. They are able to kill tumor and virus infected cells without specific antigen stimulation. They attack those cells that have reduced or altered major histocompatability complex expression and their mechanism of killing is similar to that of cytotoxic T Cells.

Macrophages: Macrophage is the main phagocyte of tissues, organs and such serous membranes as the pleura and peritoneum. Macrophages have a role in all phases of the immune response. They are involved in the initial defense of the host by engulfing microbes and in response to the later, secreting cytokines that attract more phagocytes to the area.

Macrophages function as antigen presenting cells (APC), which determine the epitopes available to T cells that ultimately decide the immune response toward a particular antigen. Macrophages are effector cells in cell-mediated immunity and once activated by cytokines present in the area, they serve to produce additional cytokines, in addition to antitumor and antimicrobial activities. Macrophages play crucial role in inflammation, tissue reorganization and tissue repair.

B Lymphocytes: Most B lymphocytes (B for bursa) or B cells migrate from the bone marrow and liver to secondary lymphoid organs and tissues throughout the body. Roughly 20% remain in the circulation. B lymphocytes can transform into plasma cells, which produce antibodies and thus are responsible for humoral immunity. Plasma cells are found mainly in the spleen and lymphoid tissue, and only rarely in the blood, however, antibodies readily enter the circulation.

The Complement System

The complement system consists of at least twenty proteins that interact in a complex reaction sequence, referred to as the complement cascade. The proteins of the system are referred to collectively as complement (C) because their action complements certain antibody-mediated reactions. Proteins of the complement system make up about 5 per cent of the serum proteins and are designated by numbers following C, ranging from C1 through C9.

The various proteins act in a sequence or cascade in numerical order where each protein activates the next one in the series, often by cleaving it. The resulting components have new functions. The complement system employs two pathways to achieve its ends, called the classical pathway and the alternative pathway (Fig. 16.2).

Fig. 16.2. The complement system.

The classical pathway is initiated by the binding of antibodies to antigen, while the alternative pathway is initiated by cell-wall polysaccharides of the kind found in bacteria and fungi. Both the classical and alternative pathways lead to the cleavage of C3 into fragments C3a and C3b. These initiate cytolysis, inflammation and opsonization.

Cytolysis: The most important function of the complement cascade is to destroy foreign cells. This is accomplished by damaging the cell membrane to the point that the cellular contents leak out. This process is called cytolysis.

Inflammation: Inflammation is the localized tissue reaction to injury, caused by physical and chemical agents, and by introduction of foreign substances to tissues, including microorganisms. Components C3a to C5a contribute to acute inflammation. Their binding to mast cells, basophils and blood platelets results in the release of histamine, which increases the permeability of blood vessels.

Inflammation is characterized by redness, pain, heat and swelling. The functions of inflammation are:

1. To destroy and remove a deleterious agent, if possible;

2. If it cannot be removed, to sequester the injury to a localized area within the body; and

3. To repair any damage that may have resulted from the injury.

Inflammation is the body’s response to injury or tissue damage. It involves the interaction of the microvasculature, circulating blood cells, cells in the tissues and their secreted products. Cells involved in inflammation include those that usually circulate in the blood and are involved in phagocytic and secretory responses, such as polymorphonuclear leucocytes (neutrophil, eosinophil and basophil), mononuclear phagocytes (monocytes), lymphocytes, platelets and endothelial cells as well as those that are tissue-based, which include mast cells and macro-phages.

All the cells involved in inflammatory response synthesize and secrete a wide variety of soluble chemical substances termed inflammatory mediators, such as acute-phase reactants like C-reactive protein (CRP) and components of the complement system, etc.

Opsonization: It is the promotion of phagocytosis by specific antibody in conjunction with complement. Component C3b coats foreign cells and interact with special receptors on phagocytes, to promote phagocytosis.

MOLECULES OF IMMUNE RESPONSE

Cytokines

Cytokines are soluble molecules that mediate interactions between cells. These are a diverse group of small, soluble proteins produced by leucocytes that mediate a variety of immune functions. Those cytokines produced by lymphocytes are sometimes called lymphokines.

Generally, they are secreted by one cell and bind to specific receptors on the corresponding target cells. Cytokines may also bind to the cells that produce them and have self-stimulation (autocrine) functions that result in such processes as cell division and protein synthesis, and ultimately differentiation and clonal proliferation.

Those cytokines that mediate interactions between leucocytes are called interleukins (ILs). Cytokines other than interleukins include interferon (IFN), granulocyte-monocyte colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF).

Cytokines are antigen nonspecific glycoproteins that are rapidly secreted by the cells that produce them, rather than stored. Thus, they are very short-lived and are typically in minute quantities. These compounds are responsible for delayed type hypersensitivity responses and the recruitment of large number of monocytes to a site of infection. Each cell is capable of producing multiple cytokines while each cytokine also has a unique function. Each cytokine can affect many different types of cells, depending upon the cytokine receptors, present on a particular cell.

Over-expression of cytokines can have a devastating effect on the immune response. For example, overproduction of TNF by macrophages can lead to septic shock.

Cytokines are soluble mediators of the inflammatory and immune response. They are produced by a variety of cells and tissues, are peptides or glycoprotein in nature and are active at concentration between 10⁻⁹-10⁻¹⁵ molar. They act by interacting with receptors on the surface of their target cells.

Cytokines may be grouped as interferons, interleukins and chemokines.

Besides the prostaglandins and leukotrienes, cytokines form another group of inflammatory mediators.

Major Histocompatibility Complex

The collection of genes coding for the self-marking proteins or major histocompatibility antigens is referred to as major histocompatibility complex (MHC). These antigens occur on the surface of all the body’s cells and identify them as belonging to the body and not foreign. Some MHC antigens appear on the surface of cells of the immune system.

The human MHC region is known as the HLA (human leucocyte antigen) region and the corresponding genes are located on chromosome 6. Although HLA complex pertains to the humans, sometimes MHC and HLA antigens are the terms that are used interchangeably.

The MHC is subdivided into three regions, known as Class I, Class II and Class III. MHC Class I and Class II are associated with the stimulation of T cells. Class III MHC contains an assemblage of twenty different genes, some cytokines, heat-shock proteins and complement factors.

MHC Class I Antigens

In humans, the Class I genes contain three loci, i.e. A, B and C. These genes encode a transmembrane protein that possesses three extracellular domains, designated as α₁, α₂ and α₃. Chemically, class I antigens are glycoproteins composed of a heavy chain and a light chain, known as β₂-microglobulin.

MHC class I molecules are transplantation antigens and are expressed on most of the nucleated cells in mammals. These include HLA-A, HLA-B and HLA-C antigens. The β-micro-globulin is essential for the expression of the MHC Class I molecules on the surface of the cell. Expression of the MHC Class I molecule on the cell surface can be either up-regulated (increased expression) or down-regulated (decreased expression) by a variety of stimuli.

When interacting with the T cell receptors, the CD8 molecule interacts with the invariant region of the MHC class I molecule. These transplantation antigens are the main determinants, which would decide whether a graft is to be accepted or rejected. If the transplantation antigens of a donor match the antigens found on the recipient, the transplant is successful.

MHC Class II Molecules

These are also known as immune responsiveness antigens. In humans, the MHC class II region includes three genes that are referred to as DP, DQ and DR. Each of these encodes an β chain and a β chain. MHC class II molecules are expressed constitutively on the surface of macrophages, monocytes, B cells, dendritic cells and thymic epithelial cells. The expression of MHC class II can be induced on other cells by IFN?.

The CD4 molecule of the T cell receptors interacts with the invariant region of the MHC class II molecule.

HLA TYPING

HLA compatibility has to be determined prior to transplantation, paternity testing and any HLA associated diseases. This is referred to as HLA typing and is done with the help of serological tests, such as lymphocyte cytotoxicity tests and mixed lymphocyte culture.

Lymphocyte Cytotoxicity Tests

These tests are done for matching class I antigen in which the antisera are obtained from multi-parous women (that have given birth to many children) who are immunized against particular set of HLA antigens. Such women have a high titer of antibodies against HLA antigens.

In principle, the test involves using lymphocytes, acting as antigens. When the antiserum containing an antibody to antigen present on the lymphocyte is reacted in the presence of complement, an antigen antibody reaction takes place with the binding of complement. The complement induces cell lysis and the number of cells killed is counted by a staining reaction with eosin. Only the cells that are killed, take up the dye while the viable cells remain unstained. The extent of lymphocytotoxicity is estimated and the lymphocyte antigens are identified, by using different groups of antisera.

The latest technique utilizes monoclonal antibodies in HLA typing and polymerase chain reaction (PCR) is used to study variation of DNA sequence in an allele of HLA locus.

Mixed Lymphocyte Culture

This technique has been devised to identify class II antigens by using pure B lymphocyte population that is homozygous for the particular HLA-D allele.

The principle of the assay involves culturing of lymphocytes in the presence of antigens, different from those present on the lymphocytes. The cells are stimulated to divide, resulting in blast formation. This happens when lymphocyte populations are heterozygous. Blast cells are metabolically very active, in which DNA synthesis is enhanced. The rate of DNA synthesis can be estimated by tritiated thymidine (³H-thymidine). The donor lymphocytes are treated with mitomycin C to prevent their division, in response to recipient’s lymphocytes.

HLA tests show that there will be no proliferation in case of identical siblings whereas in case of non-identical siblings proliferation of lymphocytes would occur.

IMMUNOGLOBULINS

Immunoglobulins form a related but enormously diverse group of proteins called globulins, which have antibody activity. They are synthesized and secreted by plasma cells, in response to invasion by an antigen. They are glycoprotein in nature and are present in the serum and some tissue fluids of all the mammals. Immunoglobulins constitute approximately 20% of the total plasma proteins.

Some of them are carried to surface of the B cells where they act as receptors for the specific antigens while others are found in free form in blood or lymph. Contact between B cells and the antigen is needed to convert B cells into the antibody forming cells, also called plasma cells, which secrete large amount of antibodies.

Antibodies are highly specific for the three-dimensional conformation of the epitope.

Structure

• A molecule of immunoglobulin has Y-shaped structure with two arms and a stem.

• It has four polypeptide chains, which are bound by −S–S-linkages. They comprise of two small subunits called L chains and two large subunits called H chains.

• Each subunit has a variable region, which is located towards the N-terminal end and constant region(s) that are located towards the C-terminal end.

• Its two arms are called the Fab fragments whereas the stem is called Fc fragment.

• Both the arms are linked to the stem by a flexible region, called hinge region.

An Immunoglobulin has Four Polypeptide Chains

An immunoglobulin is a basic unit comprising of four polypeptide chains. Out of these, there are two identical small polypeptides, called L-chains (light chains) and two identical large polypeptides, which are designated as H-chains (heavy chains). They occur in light-heavy-heavy-light arrangement. Its four chains are joined with each other by disulfide bonds (–S– S–) and form, roughly, a Y-shaped molecule, which has two arms and a stem (Fig. 16.3).

Fig. 16.3. Structure of an immunoglobulin.

Light chains

The smaller polypeptides (about 25 kD each) are designated as the light chains (L-chains). Each L chain has approximately 220 amino acids.

Two types of the L chains may occur in immunoglobulins. They are designated as kappa (κ-chain) and lambda (λ-chain).

Though any type of the L-chain may be found in an immunoglobulin molecule but, generally, an immunoglobulin has two identical chains, i.e. it has either two κ-chains (κ₂) or two λ-chains (λ₂) but never one λ-chain and one λ-chain (κλ).

These are referred to as the isotypes and are found in all the individuals. In human immunoglobulins, κ-chains are more prevalent than the λ-chains.

Heavy chains

The larger subunits that are found in the immunoglobulins are called heavy chains (H-chains).

Five different types of the H chains, each containing 440–450 amino acids, are found in immunoglobulins, in human beings. Molecular mass of an H-chain may vary from 50 to 75 kD.

Various types of the H chains are designated as gamma (γ), alpha (α), mue (μ), delta (δ) and epsilon (ε). Each one of the H-chain is specific to a class of the immunoglobulin. Thus, depending upon the type of the H-chain, they are called immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE). The H-chain determines the class and subclass of the immunoglobulin molecule.

Both types of the chains, i.e. the L-chain and the H-chain, are synthesized separately and are subsequently assembled to form a mature immunoglobulin tetramer molecule (L₂H₂), within the plasma cells. During assembly, each one of the two-H chains is associated with the two-L chains as well as with each other by −S– S–linkages.

An L-chain is joined to the H-chain in such a way that they run parallel to each other. Since L-chain is only about half the size of the H-chain (as an L-chain contains nearly 220 amino acids as compared to 440–450 present in the H-chain), L chain is therefore, associated with only NH₂-terminal half of the H-chain.

Various chains in an immunoglobulin molecule are folded into discrete regions, called domains. There are two domains in the light chain while depending on their class, four or five domains are found in a heavy chain.

Each Polypeptide has Two Regions

Each chain has two regions called constant region and variable region.

Variable Regions: Amino acid sequences that are found in the NH₂-terminal half of the L chains and one-fourth of the H chains are highly variable between the different immunoglobulin molecules. Therefore, these NH₂-terminal regions are called variable regions (V regions). They are highly heterogeneous and vary to the extent that no two V regions, from different persons, have identical amino acid sequences.

The V regions of the L-chains and the H-chains are designated as the VL region and the VH region, respectively.

Constant Regions: Half of the L-chain towards the COOH-terminal end and three-fourths of the H-chain towards the COOH-terminal end has homologous amino acid sequences, i.e. these segments are homologous with the same segments of the other L- or H-chains found in the same class of immunoglobulin. These regions, with a homologous primary structure, are called constant regions (C region). The C regions of the L-chains and the H-chains are designated as the CL region and the CH region, respectively.

CH Region is further subdivided into three parts, each comprising of about 110 amino acids. These are designated as CH¹, CH² and CH³, respectively. Each such segment of the CH region is homologous to each other as well as to the CL region. Each of these segments (sequence repeats) contains an intra chain disulphide bond.

The μ type and κ type of the H chains have four CH regions instead of three found in others. V regions of the immunoglobulin is involved in recognition and binding of the antigen with the antibody. This ability of the antibody resides in several short sequences, which are located in small loops within the V region. These short loops are termed as hypervariable regions (hypervariable sequences).

Only 5–10 amino acids in each of the hypervariable sequence contribute to the antigen-binding site (ab site). These hypervariable sequences (the antigen-binding sites) are complementary to the topology of the antigen and are designated as complementary-determining regions. Three hypervariable sequences are commonly found in the VL region. A VH region may have three or four hypervariable sequences, depending upon the type of the antibody.

Amino acid segments separating different hypervariable sequences from each other are referred to as the framework regions.

Characteristics of the CH Region

1. CH Region determines the antibody class of an immunoglobulin.

2. This region of the immunoglobulin molecule provides a site for the binding of the complement protein.

3. These are also the sites for the antibodies to cross the placental membrane.

4. Depending upon the class of an immunoglobulin, variable amounts of carbohydrates (2–15%) are attached to the CH² region.

5. Amino acid sequences in CH region, in some cases, also promote polymerization of the basic molecular structure of the immunoglobulin molecule.

In a polymeric structure, basic molecular structures are covalently linked to each other around a peptide, called joining subunit or J chain.

Immunoglobulins of the IgA class generally occur as dimmers while IgM occur as pentamers (Fig. 16.4).

Fig. 16.4. IgA and IgM molecules.

PROTEOLYTIC DEGRADATION OF AN IMMUNOGLOBULIN MOLECULE

An immunoglobulin molecule, such as IgG, can be cleaved with the enzyme papain, which hydrolyzes it into three fragments of approximately 50 kD each. Two of these fragments are identical and represents two arms. These are the fragments where an antigen binds and are called Fab fragments. The third fragment represents the stem. Since this fragment can be easily crystallized, it is called Fc fragment (Fig. 16.5).

Fab Fragment

An Fab fragment has entire L-chain and half of the H chain (comprising of the VH and the CH¹ regions). Each of the Fab fragments can bind to an antigen with the affinity similar to that of the intact antibody. Because there are two Fab domains, a molecule of an immunoglobulin can bind two molecules of the antigen, simultaneously. Thus, an antibody is said to be divalent.

Fc Fragment

The Fc fragment consists of the C-terminal halves of the two-H chains, i.e. the CH² and CH³ regions, which are joined together by disulfide bridges.

All antibodies thus are bifunctional, i.e. in addition to the antigen binding, they also exhibit one or more effector functions, such as complement activation and cell binding (in the Fc region).

Hinge Region

Two Fab fragments of the immunoglobulin are connected to the Fc fragment by a flexible region, referred to as the hinge region. It is present between the CH¹ and CH² regions and confers flexibility to the molecule. This in turn allows two Fab fragments to move independently and help them to bind two antigens, simultaneously, that may be present at different distances, apart from each other. The hinge region has one or more cysteine residues, which provide interchain disulfide bridges. It also controls interactions between different parts of an immunoglobulin.

Proteolytic cleavage by pepsin: Action of pepsin on an immunoglobulin molecule results in a fragment that has 2 Fab regions only, i.e. F(ab)₂ while the Fc fraction is hydrolyzed to oligopeptides and amino acids (Fig. 16.5).

Fig. 16.5. Degradation of an immunoglobulin molecule.

CLASSIFICATION OF IMMUNOGLOBULINS

There are five classes of immunoglobulins called IgG, IgA, IgM, IgD and IgE. Each one it differs from other with respect to the type of its H chain.

Various immunoglobulin classes have different physiological functions (Table 16.1).

Table 16.1. Immunoglobulin (Ig) classes

Immunoglobulin G

Immunoglobulin G (IgG) is a major immunoglobulin produced by the plasma cells. It accounts for 70–75% of the total immunoglobulins found in serum.

IgG is the most important class of immunoglobulins that occurs in secondary immune response and is distributed evenly between the intravascular and the extra vascular pools.

Antibodies of the IgG class are produced in response to most bacteria and viruses. These antibodies bind to macrophages and neutrophils, and promote phagocytosis.

IgG has four subclasses, which are referred to as IgG₁, IgG₂, IgG₃ and IgG₄. Structurally, these subclasses differ, primarily, in the hinge region. It is the IgG1 and IgG₃ both of which bind firmly to Fc receptors of phagocytic cells and activate K cells (killer monocytes).

In human beings, IgG molecules of all the subclasses cross placenta and confer high degree of passive immunity to the newborn.

The major effector mechanism of the human IgG₁ and IgG₃ subclasses is the activation of the classical pathway of a complement.

Immunoglobulin A

Immunoglobulin A (IgA) accounts for about 15–20% of the total serum immunoglobulins.

In human beings, more than 80% of the IgA occurs as a monomer but in most other mammals, serum IgA is mainly dimeric.

IgA has two subclasses, designated as IgA₁ and IgA₂. In human serum, IgA1 is the predominant subclass. It accounts for 75–90% of total IgA in many secretions, such as nasal secretion, tear, saliva and milk. In the colon IgA₂ predominates.

Secretory IgA: Secretory IgA (sIgA) is the most important form of IgA. It is called secretory IgA since it is the predominant immunogloulin in seromucus secretions, such as saliva, colo-strums, milk and tracheobronchial and genitourinary secretions. It exists, mainly in the dimeric form, in association with another protein that is known as a secretory component. sIgA may be of either of the two subclasses, i.e. it may be IgA₁ or IgA₂.

sIgA is assembled during an active transport process, as the locally produced dimeric IgA passes across the mucosal epithelium. It is the sIgA, which is found in the bronchial, nasal and intestinal mucous secretions, tear, milk and colostrum.

sIgA provides initial defense against invading viral and bacterial pathogens, prior to their entry into the internal spaces. Its deficiency is the commonest disorder, which results in recurrent infections of the sinus and respiratory tract.

Immunoglobulin M

Immunoglobulin M (IgM) accounts for nearly 5–10% of the total circulating immunoglobulins. Most of the IgM in serum is a pentamer consisting of five Y-shaped monomeric molecules, which are arranged around the J chain.

Two other features characterize an IgM molecule, i.e. an abundance of oligosaccharide units, which are associated with the μ chain and an additional peptide chain called joining chain (J-chain) that assist the process of polymerization, prior to its secretion.

IgM is the first immunoglobulin which is synthesized by the cell, on introduction of an antigen into the host. IgM thus, is called the first line of defense. It is the only immunoglobulin that a neonate normally synthesizes.

IgM is the predominant antibody in primary immune response. It is largely confined to the intravascular pool and is frequently associated with the immune response to antigenically complex blood-borne infectious organisms.

Once bound to its target, IgM is a potent activator of the classical pathway of a complement.

Immunoglobulin D

Immunoglobulin D (IgD) accounts for less than 1% of the total serum immunoglobulins. It is the major component of the surface membrane of many B cells and may play a role in antigen-triggered lymphocyte differentiation though its primary function is unknown.

Immunoglobulin E

Immunoglobulin E (IgE) is normally present in blood in minute concentrations. It is found on the surface membrane of basophils and mast cells, in all individuals.

It also sensitizes cells on the mucosal surfaces, such as conjunctival, nasal and bronchial mucosa. It protects the host against parasites and has been implicated in allergic reactions. When antigen cross-links some of the attached IgE molecules, mast cells are stimulated to release histamine and other vasoactive amines, which, in turn, are responsible for vascular permeability and smooth muscle contraction.

IgE is commonly associated with the allergic diseases, such as hay fever and asthma.

ANTIBODY PRODUCTION

Both, L chains and H chains are synthesized independently and form an immunoglobulin molecule.

The immune system has a potential to produce large number of different antibodies, which can react with almost every antigen that an organism encounters. A human, however, does not contain as many numbers of genes so that each one of it can code for an individual immunoglobulin. This is achieved by generating great diversity in the antibodies that are produced by the B cells.

Generation of such immense antibody diversity is achieved by recombination of a limited number of gene segments, to produce vast number of variable domains.

QUANTITATIVE DETERMINATION OF IMMUNOGLOBULINS

Agglutination Tests

This is the binding of particulate antigen by antibody, resulting in an observable clumping or aggregation. Antigens, both soluble and particulate, can be adsorbed to carrier red cells. When these coated red cells are mixed with specific antibody, hemagglutination takes place. This test is used for the diagnosis of a number of diseases.

Immunodiffusion

It is based on the principal that when an antigen is mixed with a specific antiserum, they form an antigen-antibody complex which precipitates out. The amount of the precipitate depends on the relative concentration of the antigen and the antibody.

This is a precipitin test in which soluble antigen is placed in a well, in an agar gel containing antibody. The antigen and antibody diffuse towards one another and a specific reaction results in a visible precipitate within 48 hours. A precipitin ring is formed, the diameter of which is proportional to the concentration of the antigen.

Immunoelectrophoresis

In this technique, a sample containing mixture of proteins (antibodies) is subjected to electrophoresis in agar gel, which contains an antigen. Proteins migrate to various positions in the gel by an electric current. Thereafter, antiserum to a specific immunoglobulin is added into a horizontal trough, in agar gel. Development of the precipitin arc identifies the presence of the protein of interest.

Rocket immunoelectrophoresis is used to quantify antigen. In this technique, an agarose gel is prepared containing a predetermined concentration of antibody. Wells are cut into the agarose, once it has solidified, and are filled with antigen. An electric current is then passed through the gel. Antigen-antibody complexes are formed in the shape of rockets. The height of the rocket is proportional to the amount of antigen in the well.

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

In enzyme-linked immunosorbent assay (ELISA), an enzyme (usually peroxidase or phosphatase) is linked chemically, to the antibody. Antigenic component, to be assayed, is attached to the solid surface on the ELISA plate. Thereafter, enzyme linked antibodies are allowed to bind to the antigen, followed by washing of the excessive antibodies. After the addition of a suitable substrate, the substrate is converted to a coloured reaction product, which can be read in an ELISA reader.

In sandwich ELISA, antigen specific antibodies are bound to the plate. These are able to bind antigen with high affinity. A separate enzyme linked antibody that recognizes a different epitope of the immobilized (first) antibody, is then used to detect the bound antigen.

SOME IMPORTANT QUESTIONS

1. What are immunoglobulins? Describe the structure of an immunoglobulin.

2. Classify immunoglobulins. Describe the function of each class of immunoglobulin.

3. Write notes on:

(i) Hinge region

(ii) Secretory IgA

(iii) HLA typing

(iv) ELISA

(v) Major histocompatibility complex

(vi) Complement system

(vii) Lymphoid organs

CHAPTER
17

Free Radicals and
Antioxidants

The elemental oxygen (O₂), as we all know, is essential for life. About 90% of it is used via oxidative phosphorylation while the other 10% in enzymatic hydroxylation and oxygenation reactions. A small fraction of the oxygen is converted to inherently toxic forms, such as superoxide and hydrogen peroxide, etc. These are the highly reactive forms of oxygen and are referred to as free radicals or reactive oxygen species (ROS). They are a source of chronic damage to biomolecules, within the tissues.

FREE RADICALS

In biological redox reactions involving O₂, oxygen is activated by redox active metal ions, such as iron and copper, which are present in metalloenzymes and other proteins, e.g. hemoglobin, myoglobin, etc. These metal ions provide one electron at a time to oxygen and activate it to strong oxidizing forms.

As shown in Fig. 17.1, a number of reactive oxygen species, also called free radicals, are formed from partial reduction of molecular oxygen. The first product is the anion radical, called superoxide (O₂·). Reduction of superoxide yields hydroperoxide in the form of H₂O₂. Reduction of H₂O₂ causes a cleavage reaction that releases hydroxyl radical (OH·). The end product of complete reduction of oxygen is water.

Fig. 17.1. Formation of reactive oxygen species from molecular oxygen.

ROS are formed by three mechanisms in vivo, i.e. by reaction of oxygen with decompart-mentalised metal ions, as a side reaction of electron transport, or by normal enzymatic reactions of electron transport, e.g. by mono-amine oxidase (in dopamine metabolism), fatty acid oxidases (in the peroxisomes), etc.

Various reactive oxygen species are formed continuously as by-products of aerobic metabolism through reactions with drugs and environmental toxins or when the level of antioxidants is diminished. This in turn creates a condition called oxidative stress. Reactive oxygen species can cause serious chemical damage to DNA, proteins and unsaturated lipids and can lead to cell death. ROS have also been implicated in a number of pathological processes, including reperfusion injury, cancer, inflammatory disease and aging.

Damage Produced by Free Radicals

Among the family of reactive oxygen species, hydroxyl radical (OH.–) is the most reactive and damaging. Its half life is measured in nano-seconds. It reacts with biomolecules primarily by hydrogen abstraction and addition reactions. One of the most sensitive sites of free radical damage is the cell membrane, which is rich in readily oxidized polyunsaturated fatty acids.

The hydroxyl radical abstracts a hydrogen atom from a polyunsaturated fatty acid, setting off a chain of lipid peroxidation reactions. Lipid peroxides, formed in this reaction, degrade to form characteristic products, such as malondial-dehyde (MDA). These compounds react with proteins to form adducts and cross links, known as advanced lipoxidation end-products (ALE).

ROS also react with carbohydrates to form dicarbonyl compounds that react with protein to form cross links and adducts, known as glycoxidation products or advanced glycation end-products (AGE). ROS also cause some residual damage to the genome, in the form of mutations in DNA. As a result of oxidative stress, MDA adducts, i.e. ALE and AGE, increase in several chronic conditions, such as atherosclerosis, Alzeheimer’s disease and diabetes.

ANTIOXIDANT DEFENSE SYSTEMS

There are several levels of protection against oxidative damage by antioxidant enzymes and vitamins.

Enzymatic Defenses

There is a group of enzymes that act to detoxify the precursors of hydroxyl radicals. These include catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx).

Catalase detoxifies H₂O₂ and is found in large amount in peroxisomes:

SOD detoxifies superoxide to the less toxic H₂O₂:

There are two classes of SOD. One is Mn.SOD isoenzyme, which is found in the mitochondria while the other is Cu–Zn.SOD, which is widely distributed in all the compartments of the cell.

Glutathione peroxidase (GPx) reduces both, H₂O₂ and lipid hydroperoxides to water and a lipid alcohol, respectively. GPx requires reduced glutathione (GSH) as a co-substrate.

GSH is tripeptidethiol (γ-glutamylcysteinyl-glycine) containing glutamate, cysteine and glycine. It is found in all the cells and can chemically detoxify H₂O₂. This reaction is catalyzed by GPx, which is a selenium containing enzyme. In this reaction, GSH is oxidized to GSSG (oxidized glutathione), which is not protective. The cell regenerates GSH from GSSG by an NADPH-dependent enzyme, called glutathione reductase. NADPH is produced by the pentose phosphate pathway.

Antioxidant Defenses in Erythrocytes

ROS are formed continuously in the erythrocytes and initiate lipid peroxidation reaction, which can lead to loss of membrane integrity, causing cell lysis. They are well protected against oxidative stress, as the cell has high concentration of GSH. Moreover, in erythrocytes, glucose is mainly oxidized by the pentose phosphate pathway, which supplies NADPH.

ANTIOXIDANT VITAMINS

Three vitamins, i.e. vitamin A, C and E, provide the other line of defense against oxidative damage and are called antioxidant vitamins.

Vitamin C (ascorbate) in the aqueous phase while vitamin E (α- and γ-tocopherols) in the lipid phase, act as chain-breaking antioxidants. Vitamin C reduces superoxide and lipid peroxyl radicals. It also has a role in reduction and recycling of vitamin E. In response to severe oxidative stress, vitamin E is maintained at constant concentration in the lipid phase until all the vitamin C is consumed. Thus, they do not propagate radical damage and are recycled.

Vitamin A is a lipophilic antioxidant. It is a potent singlet oxygen scavenger and protects against damage from sunlight in the retina and skin. Consumption of food rich in ascorbate, vitamin E and β-carotene has been correlated with a reduced risk for certain types of cancers and decreased frequency of certain other chronic health problems.

SOME IMPORTANT QUESTIONS

1. Write notes on:

(i) Reactive oxygen species

(ii) Oxidative stress

(iii) Free radicals

(iv) Antioxidant defenses

(v) Antioxidant vitamins

CHAPTER
18

Specialized Proteins

COLLAGEN

Collagens comprise a family of fibrous glycoproteins that are present only in extracellular matrices. Collagen is the most abundant protein in the human body. It has a long rigid structure in which three polypeptides, called α-chains, are wound around one another in a rope-like triple helix. Collagens are found throughout the body. Their types and organization are determined by the structural role, collagen plays in a particular organ.

In some tissues, collagen may be dispersed as a gel that gives support to the structure, as in the extracellular matrix or the vitreous humor of the eye. In other tissues, collagen may be bundled in tight, parallel fibers that provide great strength, as in tendons.

In the cornea of the eye, collagen is stacked so as to transmit light with a minimum of scattering. Collagen of bone occurs as fibers arranged at an angle to each other so as to resist mechanical sheer from any direction.

Types of Collagen

It is a superfamily of proteins, which include 27 distinct types. Although there are many differences among the members of the collagen family, all share at least two important structural features:

1. All collagen molecules are trimers consisting of three polypeptide chains called α-chains.

2. All three polypeptide chains of a collagen molecule are wound around each other to form a unique, rod-like triple helix, e.g. collagen type I is αI₂α₂, i.e. it has two αI and one α₂ chains. Type II collagen is αI₃, i.e. it contains 3α1 chains, etc.

Based on their location and functions in the body, collagens are organized into 3 groups:

1. Fibril-forming Collagens: These are linear polymers of fibrils with characteristic bonding patterns and have rope-like triple-stranded helix, e.g. type I, type II and type III. Type I collagen fibers are found in supporting elements of high tensile strength, such as in skin, bone, tendon, blood vessels and cornea. Type II fibers are found in cartilaginous structures, such as cartilage, intervertebral disk and vitreous body. Type III collagens occur in more distensible tissues, such as blood vessels and fetal skin.

2. Network forming Collagens: They form a three-dimensional mesh, e.g. type IV and VII collagens. Type IV collagens are thin, sheet or mesh-like structures that function as semipermeable basement membranes found in the kidney and the lung. Type VII collagens are found beneath stratified squamous epithelia.

3. Fibril-associated Collagens: These collagens link collagen fibrils to one another as well as to other components in the extracellular matrix, e.g. type IX and type XII collagens. Type IX collagens are found in cartilage whereas type XII collagen occurs in tendon and ligaments.

Structure and Composition

Collagen is a fibrous protein, which has an elongated triple-helical structure. It is rich in proline and glycine, both of which are important in the formation of the triple-stranded helix. Proline facilitates the formation of the helical conformation of each α-chain because of its ring structure that causes kinks in the peptide chain. Glycine is found in every third position of the polypeptide chain, forming part of a repeating sequence Gly-X-Y-. It may be due to the reason that glycine is the smallest amino acid and fits into the restricted space where three chains of the helix come together. X is frequently proline while Y may be proline or lysine, which gets hydroxylated and occurs as hydroxyproline or hydroxylysine.

Biosynthesis of Collagen

1. The polypeptide precursors of the collagen molecules are formed in fibroblasts (or osteoblasts of bone and chondroblasts of cartilage) and secreted into the extracellular matrix. The newly synthesized polypeptide precursor of a-chains contains a special amino acid sequence at the N-terminal ends. This signal sequence facilitates the binding of ribosomes to the rough endoplasmic reticulum and directs the passage of the polypeptide chain into the cisternae of the RER. The signal sequence is rapidly cleaved in the endoplasmic reticulum to yield a precursor called pro-α-chain.

2. The pro-α-chain is processed by a number of enzymes within the lumen of the RER while the polypeptides are still being synthesized. Proline and lysine residues found in the Y-position are hydroxylated by prolylhydroxy-lase and lysylhydroxylase, respectively. This is a post-transciptional modification, and the hydroxylation reactions require molecular oxygen and vitamin C. Hydroxyproline is important in stabilizing the triple-helical structure of collagen because it maximizes interchain hydrogen bond formation. In case of vitamin C deficiency, collagen fibers can-not be cross-linked, greatly decreasing the tensile strength of the assembled fiber, such as in scurvy.

Scurvy is a disease that results from a deficiency of vitamin C and is characterized by inflamed gums and tooth loss, poor wound healing, brittle bones and the weakening of the lining of blood vessels, causing internal bleeding.

3. Some hydroxylysine residues are enzymatically glycosylated with glucose or glycosyl-galactose.

4. After hydroxylation and glycosylation, pro-α-chains forms procollagen. Procollagen molecules are translocated to the Golgi apparatus where they are packaged in secretory vesicles. The vesicle fuses with the cell membrane, causing the release of procollagen molecule into the extracellular space.

5. After their release, the procollagen molecules are cleaved by N- and C-procollagen peptidases, which remove the terminal propeptides, releasing triple-helical molecules.

6. Individual collagen molecules, spontaneously, associate to form fibrils.

7. Lysyl oxidase, oxidatively deaminates some of the lysyl and hydroxylysyl residues, resulting in the formation of allysine and hydroxyallysine. These reactive aldehydes condense with lysyl or hydroxylysyl residues in neighbouring collagen molecules to form covalent cross-links.

Degradation of Collagen

Normal collagens are highly stable molecules. However, connective tissue is constantly being remodeled, often in response to growth or injury of the tissue. Collagen fibrils are broken down with the help of specific metaloproteinases, called collagenases. These enzymes cleave the molecule into smaller fragments, which are further degraded to the constituent amino acids by other proteinases.

ELASTIN

Elastin is a connective tissue protein with rubber-like properties. It is the predominant protein of the elastic fibers in the extracellular matrices of blood vessels, lungs, ligaments and spine.

Structure and Synthesis of Elastin

Elastin is a polymer synthesized from a precursor called tropoelastin. Tropoelastin is a linear polypeptide synthesized on the rough endoplasmic reticulum and secreted by the cell into the extracellular space. Tropoelastin does not undergo post-translational modification. During the assembly process, some of the lysyl side chains of the tropoelastin polypeptides are oxidatively deaminated by lysyl oxidase, generating allysine in specific sequences. The reactive aldehyde of allysine condenses with other allysine or with unmodified lysine, from the same or on different tropoelastin polypeptide chains, to form desmosine cross-link heterocyclic structures. As a result, elastin can stretch in any direction giving elasticity to the connective tissue.

Elastin is rich in glycine, proline, lysine and valine. One in every seventh of its amino acids is valine. Its primary structure consists of alternating hydrophilic and hydrophobic, lysine and valine rich domains. Lysines are involved in intermolecular cross linking while the weak interactions between valine residues in the hydrophobic domains impart elasticity to the molecule.

Elastin is an insoluble, polymeric, amorphous core covered with a sheath of microfibrils, the predominant constituent of which is the glycoprotein fibriltin.

ABNORMALITIES IN COLLAGEN SYNTHESIS

A large number of mutations have been identified in genes coding for collagen protein or processing enzymes. The following diseases can occur as a result of defective collage synthesis:

1. Mutations in the gene for either the pro 1- or pro 2-α chains of type I collagen can result in structurally abnormal pro α-chains that can prevent folding of the protein into a triple-helical conformation. This is called osteogenesis imperfecta (OI), also known as brittle bone syndrome. Extremely fragile bones, thin skin and weak tendons characterize it. It is a heterogeneous group of inherited disorders distinguished by bones that easily bend and fracture. Type I osteogenesis imperfecta, called osteogenesis imperfecta tarda, is present in early infancy with fractures secondary to minor trauma. Type II osteogenesis imperfecta, called osteogenesis imperfecta congenita, is more severe and patients die in utero or in the neonatal period of pulmonary hypoplasia.

2. A deficiency of any collagen processing enzyme, e.g. lysyl hydroxylase or procollagen peptidase, results in Ehlers-Danlos syndrome. This disorder is a heterogeneous group of generalized connective tissue disorders that result from inheritable defects in the metabolism of fibrillar collagen molecules. These patients show defects in collagen type I and type III fibrils. They have hyperflexible joints and highly extensible skin.

3. Mutation in type IV collagen gene occurs in patients with Alport syndrome, an inherited kidney disease in which the glomerulus basement membrane is disrupted.

DISORDERS OF ELASTIN METABOLISM

Marfan syndrome

Mutations in the fibriltin gene results in a genetic disease of connective tissues, called Marfan syndrome. People with the disease have typically tall stature, long arms and legs, and long spidery fingers. The disease in a mild form causes loose joints, deformed spine, floppy mitral valves and eye problems, such as lens dislocation. In severely affected individuals, the aorta wall is prone to rupture because of defects in elastic fiber formation.

Emphysema

Elastin is degraded by the enzyme elastase. This enzyme is a powerful protease that is released into the extracellular space and degrades elastin of alveolar walls and other structural proteins, in a variety of tissues. α1-Antitrypsin (α₁-AT), produced primarily by liver and other tissues, such as monocytes and alveolar macrophages, inhibits a number of proteolytic enzymes including elastase. This in turn prevents elastin degradation in the alveolar walls. A deficiency of α₁-AT can cause emphysema, which results from the destruction of the connective tissue of alveolar walls.

KERATIN

Keratin is a protein used by numerous groups of animals as a structural element and is a classic example of a fibrous protein. It is formed by keratinocytes, living cells which make up a large part of skin, hair, nails and other keratin-containing parts of the body. The cells slowly push their way upwards, eventually dying and forming a protective layer of cells. Thousands of these cells are shed every day and the process can be accelerated by various medical conditions, such as psoriasis. Damage to the external layer of keratin can cause skin, hair and nails to look unhealthy or flaky. The condition known as dandruff results when the scalp sheds such cells. Hair and nails on humans, especially, tend to become dry and brittle because the dead keratin is being pushed to great lengths. By eating foods like gelatin and keeping hair and nails moist, they can be grown out while still remaining healthy. In general, the thicker the layer of keratin, the healthier the hair or nail is, because the dead cells outside protect the living cells at the core. Keeping the external layer of keratin moisturized will also keep it healthy and prevent cracking and splitting.

There are two main forms of keratin, α-keratin and β-keratin. The α-keratin is seen in humans and other mammals while β-keratin is present in birds and reptiles. However, β-keratin is harder than α-keratin. Structurally α-keratin has α-helical coiled structure while β-keratin has twisted β-sheet structure.

In humans, the keratin family of proteins is divided into the soft epithelial keratins or cytokeratins and the hard hair keratins.

Keratins are intermediate filaments. Like all intermediate filaments, keratin proteins form filamentous polymers in a series of assembly steps beginning with dimerization; dimers assemble into tetramers and octamers, and eventually, the current hypothesis holds, into unit-length-filaments (ULF) capable of annealing end-to-end into long filaments. During the process of epithelial differentiation, cells become cornified as keratin protein is incorpo-rated into longer keratin intermediate filaments. Eventually, the nucleus and cytoplasmic organelles disappear, metabolism ceases and cells undergo a programmed death as they become fully keratinized. In many other cell types, such as dermis, keratin functions in the cytoplasm to mechanically stabilize the cell against physical stress. It does this through connections to desmosomes, cell-cell junctional plaques and hemidesmosomes, cell-basement membrane adhesive structures.

Cells in the epidermis contain a structural matrix of keratin, which makes this outermost layer of the skin almost waterproof, and along with collagen and elastin, gives skin its strength. Rubbing and pressure cause keratin to proliferate with the formation of protective calluses, useful for athletes and on the fingertips of musicians who play stringed instruments. Keratinized epidermal cells are constantly shed and replaced.

Keratins contain a high proportion of the smallest of the 20 amino acids, i.e. glycine and alanine. In addition to intra and intermolecular hydrogen bonds, keratins have large amounts of the sulphur-containing amino acid cysteine, required for the disulphide bridges that confer additional strength and rigidity by permanent, thermally-stable crosslinking. Human hair is approximately 14% cysteine. The pungent smell of burning hair and rubber is due to the sulfur compounds. Extensive disulfide bonding contributes to the insolubility of keratins.

The more flexible and elastic keratins of hair have fewer interchain disulfide bridges than the keratins in mammalian fingernails, hooves and claws, which are harder and more like their analogs in other vertebrate classes. Hair and other α-keratins consist of α-helically-coiled single protein strands (with regular intra-chain hydrogen bonding), which are then further twisted into superhelical ropes that may be further coiled. The β-keratins of reptiles and birds have β-pleated sheets twisted together, then stabilized and hardened by disulfide bridges.

Some infectious fungi, such as those which cause athlete’s foot and ringworm, feed on keratin.

MYOSINS

Myosins are a large family of motor proteins found in eukaryotic tissues. They are responsible for actin-based motility while hydrolyzing ATP. Molecular motors of actin filaments comprise of members of the myosin superfamily.

Thick filaments are composed, almost entirely of myosin, which are placed ‘end-to-end’ with the light chains, striking out in the form of double heads.

Myosin consists of six polypeptide chains which include two identical heavy chains and two pairs of two different types of the light chains.

Two types of the light chains are described as regulatory light chains and essential light chains.

Each myosin head associates with an essential light chain and a regulatory light chain.

Regulatory light chains play a role in the control of muscle contraction by Ca²⁺ ions whereas the essential light chains are required for ATPase activity of the myosin head. Thus, both types of light chains are involved in the regulation of muscle contraction.

The N-terminal half of each of the heavy chain forms an elongated globular head to which one subunit, each of the two light chains binds. The C-terminal half of the heavy chain forms a long fibrous α-helical tail, two of which associate to form a left-handed coil. Taken together, a molecule of myosin, thus, consists of a long rod like segment with two globular heads (Fig. 18.1).

Fig. 18.1. Myosin.

Types of Myosins

A large number of divergent myosin genes have been discovered throughout eukaryotes. Thus, although myosin was originally thought to be restricted to muscle cells (hence, “myo”), there is no single “myosin” but rather a huge superfamily of genes whose protein products share the basic properties of actin binding, ATP hydrolysis (ATPase enzyme activity) and force transduction. Virtually all eukaryotic cells contain myosin isoforms. Some isoforms have specialized functions in certain cell types (such as muscle) while other isoforms are ubiquitous.

About 20 classes of myosin have been distinguished on the basis of the sequence of amino acids in their ATP-hydrolyzing motor domains. The different classes of myosin also differ in structure of their tail domains. Tail domains have various functions in different myosin classes, including dimerization and other protein-protein interactions.

Myosin I: Myosin I functions as monomer in vesicle transport. It has only one heavy chain with a single globular motor domain. Its relatively short tail lacks the heptad repeats that would be involved in dimerization via formation of a coiled coil.

Myosin II: Myosin II was first studied for its role in muscle contraction but it functions also in non-muscle cells. It includes two heavy chains and two light chains.

The globular motor domain of each heavy chain catalyzes ATP hydrolysis and interacts with actin. Each heavy chain continues into a tail domain in which heptad repeat sequences promote dimerization by interacting to form a rod-like β-helical coiled-coil.

It also contains 4 light chains (2 per head), which bind the heavy chains in the neck region between the head and tail. These light chains are often referred to as the essential light chain and the regulatory light chain. The binding site for each light chain is an isoleucine, glutamine sequence motif. Its light chains are similar in structure to calmodulin but in many organisms have lost the ability to bind Ca²⁺. However, the calmodulin-like light chains of some myosins do bind Ca²⁺.

In muscle cells, it is myosin II that is responsible for producing the contractile force. Here, the long coiled-coil tails of the individual myosin molecules join together, forming the thick filaments of the sarcomere. The force producing head domains stick out from the side of the thick filament, ready to walk along the adjacent actin-based thin filaments in response to the proper chemical signals.

In non-muscle cells, myosin II (the type in muscle sarcomeres) is often found to be associated with actin filament bundles.

Myosin II of smooth muscle as well as non-muscle cells may be regulated by phosphorylation of its regulatory light chains. Dephosphorylation stabilizes an inhibited bent conformation in which the motor domains contact distal tail domains preventing formation of bipolar complexes.

Regulation by Ca²⁺ varies, depending on the type of myosin, the tissue and the organism. For example, some myosins are regulated by binding of Ca²⁺ to calmodulin-like light chains, in the neck region. A complex of tropomyosin and troponin (which includes a calmodulin-like protein) regulates actin-myosin interaction in skeletal muscle sarcomeres. Caldesmon, a protein regulated by phosphorylation and by Ca²⁺, controls actin-myosin interaction in smooth muscle.

Myosin V: Myosin V is an unconventional myosin motor, which is functional as a dimer. It translocates along actin filaments traveling towards the barbed end of the filaments. Myosin V is thought to be critical in vesicle movement from the center of the cell to the periphery.

It has two heavy chains like myosin II, but myosin V has a longer neck region that has 6 binding sites for calmodulin light chains. Its shorter coil region is followed by a globular domain, at the end of each heavy chain tail.

Myosin VI: Myosin VI is an unconventional myosin motor, which is functional as a dimer. It translocates along actin filaments, traveling towards the pointed end of the filaments. It is the only myosin motor class known to translocate towards the pointed end. Myosin VI is thought to transport endocytic vesicles into the cell. Myosin VI is found to be either monomeric or dimeric, under different conditions.

Myosin VII: Myosin VII is an unconventional myosin with two ferm domains in the tail region. It has a very short coil region. Myosin VII is required for phagocytosis, spermato-genesis and stereocilia formation in mice and zebrafish.

Myosin X: Myosin X is an unconventional myosin motor, which is functional as a dimer. In mammalian cells, the motor is found to localize to filopodia. Myosin X walks towards the barbed ends of filaments, preferentially on bundles of actin rather than single filament. It is the first myosin motor to exhibit this behavior.

Myosin XV: Myosin XV is a motor that is involved in the structuring and motion of stereocilia in the inner ear. It is thought to be functional as a monomer.

All of these genes are not active in humans.

LENS PROTEINS

The lens is also known as the aquula (Latin, a little stream, dim. of aqua, water) or crystalline lens. In human, the refrective power of the lens, in its natural environment, is approximately one-third of the eye’s total power.

Crystalline lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. The lens, by changing shape, functions to change the focal distance of the eye so that it can focus on objects at various distances, thus allowing a sharp real image of the object of interest to be formed on the retina. This adjustment of the lens is known as accommodation. Accommodation is similar to the focusing of a photographic camera via movement of its lenses. The lens is more flat on its anterior side than on its posterior side.

The lens is bathed on one side by aqueous humor and supported on the other side by vitreous humor. It has no blood supply but is metabolically active. It obtains nutrients from and eliminates waste into the aqueous humor. The lens is mostly water and proteins.

The human eye lens consists of a highly concentrated mix of several proteins:

• The majority of vertebrate lens proteins are – α, β and γ crystallins. They have defined functions and are generally expressed in other tissues.

The α and β crystallins are small heat shock proteins (sHSP) or chaperons that function to help maintain lens proteins in their native, unaggregated states. The α-crystallin is a major lens protein, comprising up to 40% of total lens proteins where its structural function is to assist in maintaining the proper refractive index in the lens. In addition to its structural role, chaperons prevent the clumping of proteins under stress conditions. The chaperon-like function of α-crystallin helps in preventing the formation of large light-scattering aggregates and possibly cataract. Protective proteins prevent these proteins from aggregating and clumping. If this protective function fails, lens blurs and the patient develops cataract.

Their highest expression is in eye lens but they also occur in other tissues such as brain, skeletal and cardiac muscle. Malfunctions of the αβ-crystallin in tissue cells can give rise to cancer and neurological defects, including Alzheimer’s disease.

• There are also albuminoids, enzymes and membrane proteins that are synthesized in an epithelial layer around the edge of the lens. These proteins maintain a clear crystalline state and are sensitive to changes in oxidation-reduction, omolarity, excessively increased concentration of metabolites and UV irradiation.

SOME IMPORTANT QUESTIONS

1. What is collagen? How they are synthesized? Describe some abnormalities of collagen biosynthesis.

2. Write notes on:

(i) Collagen

(ii) Osteogenesis imperfecta

(iii) Ehlers-Danlos syndrome

(iv) Elastin

(v) Marfan syndrome

(vi) Keratin

(vii) Myosins

(viii) Lens proteins

CHAPTER
19

Sample Collection &
Reference Values

SAMPLE COLLECTION

COLLECTION OF BLOOD

Biochemical tests are performed on any kind of body fluid but mostly on blood, plasma or serum. The type of test required dictates what type of sample is to be used? Proper sample collection and handling is an integral part of obtaining a valid and timely laboratory test result. Specimen must be obtained using proper phlebotomy technique, collected in the proper container, correctly labeled (in the presence of the patient) and promptly transported to the laboratory.

Timing of Collection

Blood composition is significantly altered after consuming food, which consequently alters many clinical chemistry tests.

• In routine, the basal state, i.e. the early morning (approximately 12 hours after the last ingestion of food) is recommended for determining concentration of the various body constituents, such as glucose, lipoproteins, cholesterol, triglycerides, electrolytes and proteins.

• However, for glucose alone, fasting of four hours is sufficient.

• When the primary lipids of interest are serum total cholesterol and high density lipoprotein-cholesterol, blood can be taken at any time of the day, i.e. with the subject even nonfasting.

• It is necessary to provide the patient, in advance, with appropriate collection instructions and information on fasting, diet and medication restrictions, when necessary.

Position of the Subject

Position of the subject can influence results of some of the analytes thus, standardization of the position is necessary. It is, generally, recommended that all blood samples should be drawn in a sitting position and that the participant remain in sitting position for 15 minutes prior to blood collection. This waiting period will allow equilibration of the concentration of blood components.

Venipuncture

Venipuncture, venopuncture or venepuncture, in medicine, is the process of obtaining intravenous access for blood sampling of venous blood. This procedure is performed by medical laboratory technologists, medical practitioners, phlebo-tomists and other nursing staff. It is one of the most routinely performed invasive procedure carried out to obtain blood for diagnostic purposes as well as to monitor level of blood components.

There are many ways in which blood can be drawn from a vein. The method varies with the age of the patient, equipment available and tests required:

• Venipuncture, using a needle and tube, is the most common method for the collection of blood.

• Blood collection can also be done with an evacuated tube system called vacutainer, which consists of a plastic hub, a hypodermic needle and a vacuum tube.

• On some occasions, blood may be collected by puncturing the skin with a retractable lancet device.

Use of Tourniquet: Prolonged venous occlusion can cause changes in the concentration of blood constituents. Therefore, use of a tourniquet should be minimized. If a tourniquet is used to search for a vein, it should be released before withdrawal of blood begins. In any case, the use of a tourniquet should be limited to less than one minute.

Site of Venipuncture: Preferably blood should not be collected from the arm that is used for blood pressure measurement, i.e. blood should usually be drawn from the left arm.

• Blood samples should be taken from the vein in the antecubital fossa.

• Before blood collection, the subject should remove tight clothes that might constrict the upper arm.

• During blood collection, the arm should rest on some supportive prop.

• Though larger median cubital and cephalic veins are the usual choice but the basilic vein on the dorsum of the arm or dorsal hand veins are also acceptable.

• Foot veins are a last resort because of the higher probability of complications.

Venipuncture Procedure: The venipuncture procedure is complex, requiring both knowledge and skill to perform. Several essential steps are required for every successful collection procedure:

1. The first step to the collection is to positively identify the patient by two forms of identification, i.e. ask the patient to state and spell – his/her name and give you his/her age. Check these against the requisition form.

2. Check the requisition form for requested tests, patient information and any special requirements.

3. Gather the tubes and supplies that one will need for the withdrawal of blood.

4. Position the patient in a chair, or sitting or lying on a bed.

5. Wash your hands.

6. Select a suitable site for venipuncture by placing the tourniquet 3 to 4 inches above the selected puncture site on the patient. Do not put the tourniquet on too tightly or leave it on the patient for longer than 1 minute.

7. Put on non-latex gloves and palpate for a vein.

8. When a vein is selected, clean the area in a circular motion, beginning at the site and working outward. Allow the area to air dry. After the area is cleaned, it should not be touched or palpated again. If you find it necessary to re-evaluate the site by palpation, the area needs to be re-cleaned before the venipuncture is performed.

9. Grasp the patient’s arm firmly using your thumb to draw the skin taut and anchor the vein.

10. Swiftly insert the needle through the skin into lumen of the vein. The needle should form 15–30 degree angle with the arm surface (Fig. 19.1).

Fig. 19.1. Collection of blood sample.

11. Avoid excess probing.

12. When the last tube is filling, remove the tourniquet.

13. Remove the needle from the patient’s arm, using a swift backward motion.

14. Place gauze immediately on the puncture site. Apply and hold adequate pressure to avoid formation of a hematoma. After holding pressure for 1–2 minutes, tape a fresh piece of gauze to the puncture site.

15. Dispose of the contaminated materials/ supplies in designated containers.

Disposal of the Syringe, Needle and other Material

Needle disposal boxes should be available for all personnel, drawing blood samples.

• Needles should be released from adapters directly to the needle disposal box.

• Needles should never be re-sheathed after use.

• The disposal boxes should not be allowed to become overfull to avoid potential hazard.

Whole Blood, Serum or Plasma

It is imperative that the in vivo state of a constituent remains unchanged after withdrawal from the body fluid of a patient to obtain a valid medical laboratory result. Historically, serum is the preferred assay material for determining extracellular concentrations of the constituents in blood. These days even plasma is preferred for many, but not all, laboratory investigations.

Whole blood: Blood is a bodily fluid that delivers necessary substances, such as nutrients and oxygen to the cells and transports metabolic waste products away from the cells.

• Whole blood is composed of blood cells suspended in plasma. The blood cells are mainly red blood cells (also called RBCs or erythrocytes) and white blood cells, including leukocytes and platelets.

• In a venous, arterial or capillary blood sample, concentrations and properties of cellular and extracellular constituents remain relatively unaltered when compared with their in vivo state.

• Anticoagulation in vitro stabilizes the constituents in a whole blood sample for a certain period of time.

Serum: Serum is the undiluted, yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. This is most easily done by centrifugation, which packs the denser blood cells and platelets to the bottom of the centrifuge tube, leaving the liquid serum fraction resting above the packed cells. Serum is used for many tests. For this purpose:

• Blood is collected into a tube without any anticoagulant and then allowed to clot.

• Leave the container at room temperature for at least 30 minutes to separate serum from blood cells. This period is short when coagulation has been activated.

Plasma: Plasma is the cell-free supernatant of blood containing anticoagulant, obtained after centrifugation. It constitutes about 55% of the blood fluid. It is mostly water (92% by volume) and contains proteins, glucose, mineral ions, hormones and carbon dioxide. It is obtained by centrifugation before clotting of blood occurs.

• Plasma samples can be centrifuged directly after sample collection, unlike serum in which coagulation is completed after 30 minutes.

• Nearly 15 to 20 % more volume can be obtained than that of serum, from the same volume of blood.

• Blood collected in additive tubes (anticoagulant tubes) must be mixed, by gently inverting the tube several times, to ensure even distribution of the additive in the specimen. For this purpose, tilt the tube repeatedly (do not shake and avoid foaming) immediately after filling to mix the sample thoroughly with the anticoagulant.

• Only the recommended quantity of the anticoagulant should be added to avoid error in result.

Use of anticoagulants

Anticoagulants are the additives that inhibit blood and/or plasma from clotting, ensuring that the constituent to be measured is not significantly changed. Anticoagulation occurs by binding calcium ions such as by EDTA or citrate, or by inhibiting thrombin activity, e.g. by heparin. Commonly used anticoagulants that are mixed with blood immediately after sample collection, include:

EDTA: Dipotassium (K₂), tripotassium (K₃) or disodium (Na₂) salt of EDTA (ethylene diamine tetraacetic acid) is used in a concentration of 1.2 to 2.0 mg/mL of blood, in a powdered form. It strongly and irreversibly chelates calcium ion to prevent blood from clotting.

Some of tests are interfered with the addition of EDTA, especially potassium while using K₂ EDTA. In such a case heparin should be used, as an alternative. Tubes with liquid EDTA reduce the risk of hemolysis that sometimes may occur with the tube using EDTA in the powder form.

Citrate: Trisodium citrate or acid-citrate-dextrose with 0.100 to 0.136 mol/L (3–4%) citric acid (as buffered-citrate, pH 5.5 to 5.6) is used in the liquid form, in the tube for coagulation tests as well as in blood transfusion bags. A mixture of one part of citrate with nine parts of blood is recommended for coagulation tests while one part of citrate is mixed with four parts of blood to determine the erythrocyte sedimentation rate (ESR).

Citrate binds calcium ions but not as strongly as EDTA. Correct proportion of the anticoagulant solution is important to avoid excessive dilution of the blood.

Oxalate: Oxalate acts by the mechanism similar to that of citrate. It is an anticoagulant used in fluoride oxalate tubes for glucose and lactate determinations.

Heparin: About 12 to 30 IU/mL of unfractionated sodium, lithium or ammonium salt of heparin is recommended, to obtain heparinized plasma. Calcium-titrated heparin, at a concentration of 8 to 12 IU/mL of blood, is recommended for the determination of ionized calcium.

Fluoride: Fluoride tubes are used for blood glucose determination.

Hemolytic, icteric or lipemic sample

Laboratory tests are affected by several endogenous and exogenous factors in the sample matrix. The relevant observation should be mentioned for each sample, e.g. hemolytic, icteric, opalescent and turbid or lipemic, if a relevant color or turbidity is seen.

Hemolytic Sample: Hemolysis is defined as release of the intracellular components of erythrocytes and other blood cells into the extracellular fluid of blood. After separation of the blood cells, hemolysis may be visible by the red color of serum or plasma. Cell constituents, particularly those with an intracellular concentration of 10 times higher than the extracellular concentration, will increase in plasma/ serum during hemolysis, e.g. potassium, lactate dehydrogenase, aspartate aminotransferase, etc. Sample associated with greater degree of hemolysis may also interfere with other laboratory tests.

Icteric Sample: Bilirubin occurs in plasma as a free molecule as well as covalently bound to albumin. In addition, water soluble bilirubin conjugates also exist as mono and diglucuro-nides. Conjugated bilirubin also appears in urine when present at increased concentration in blood. In patients with proteinuria, bilirubin bound to albumin can also appear in urine.

Bilirubin interferes in oxidase/peroxidase based test systems. Proportionally to its concentration, bilirubin reacts with H₂O₂ which is formed in the test system. This in turn causes systematically lower results in enzymatic procedures that are used for the measurement of glucose, cholesterol, triglycerides, uric acid and creatinine.

Bilirubin also competitively interferes with dyes binding to albumin.

Lipemic Sample: Lipemia is a turbidity of serum or plasma, which is caused by the elevated lipoproteins concentration and is visible by the eye. A sufficiently transparent sample container must be used to detect lipemia. Post-centrifugal coagulation of serum samples of heparinized patients can also be the cause of turbidity.

Most often, lipemia results from increased triglyceride concentration in plasma/serum. This can be due to food intake, altered lipid metabolism or infusion of lipids. After intestinal absorption, triglycerides are present in plasma, as chylomicrons, for 6 to 12 hours. Lipemia in plasma or serum is visually observed at triglycerides concentration above 300 mg/dL. In whole blood triglycerides concentration above 1000 mg/dL cause turbidity that is detected by visual inspection. Centrifugation for 10 minutes at 1000 g is effective when chylomicrons cause turbidity.

Sequence of Filling the Tubes for various biochemical tests

The order, in which various tubes are filled, is determined by the risk of contamination and coagulation. The following sequence for filling tubes with blood from a patient is recommended to avoid contamination, i.e. serum, citrate, heparin and EDTA-containing tubes. So, EDTA samples should be drawn last and plain tubes first.

The recommended order is:

1. Blood for blood culture

2. Tubes for serum (avoid serum as first tube when electrolytes shall be measured)

3. Citrate-filled tubes

4. Heparin-filled tubes

5. EDTA-containing tubes, and

6. Tubes-containing additional stabilizers (e.g. fluoride as glycolytic inhibitor).

Another consideration that might affect the order of tube filling is the priority of the assay for which the tubes are needed.

If there are any problems with blood flow during its withdrawl, e.g. collapsing vein, the procedure should be discontinued and an attempt should be made on the other arm.

Depending on the scheduled analyses, tubes should be filled in the following order (Table 19.1).

Table 19.1. Sequence of filling the tubes for various biochemical tests

Type of analysis | Specimen type | Type of tube
Lipids | Serum | 10 ml, plain tube
Glucose | Plasma | 4 ml, tube filled with glycolytic inhibitors, i.e. potassium oxalate and sodium fluoride
Glycatedhemoglobin | Whole blood | 3 ml, tube with anticoagulant K₂ EDTA

Before the subject leaves the examination site and the rack is moved, all the tubes should be labeled with the subject identification code.

Storage and Transport of the Sample

Non-centrifuged samples should not be stored at room temperature for the longer time. Do not freeze whole blood samples before or after centrifugation. Samples may be refrigerated to 4°C.

For transport, samples should be properly marked and transfer lists should be kept in order to check for possible loss of the sample.

Collection of Urine

For the quantitation of creatinine, protein, 17-ketosteroids or VMA in urine, 24 hours sample is collected, in 2 litres bottle, with few crystals of thymol.

For calcium 10 ml of 6 N HCl is added in the bottle.

First morning and midstream urine sample (about 25–30 ml) is collected for Bence Jones protein as well as urobilinogen.

REFERENCE VALUES

The range of reference values of some important constituents of blood, urine, C.S.F and gastric juice are shown for male adults, in the fasting state, in conventional units, in Tables 19.2 – 19.5.

Table 19.2. Reference values for some of the constituents in urine

Constituents | Normal excretion
D-xylose | 1–2 g/5 hrs
Total nitrogen | 10–20 g/24 hrs
Creatinine | 1.0–2.0 g/24 hrs
Calcium | 0.1–0.7 g/24 hrs
Chloride | 110–250 meq/24 hrs
Sodium | 40–220 meq/24 hrs
Potassium | 25–125 meq/24 hrs
Phosphorus | 0.6–1.5 g/24 hrs
Urea | 20–35 g/24 hrs
Uric Acid | 0.5–1.0 g/24 hrs

Table 19.3. Reference values for some of the constituents in C.S.F

Constituents | Normal concentration
Total Protein | 15–40 mg/dL
Glucose | 50–75 mg/dL
Chloride (as NaCl) (118–132 mmol/L) | 700–750 mg/dL

Table 19.4. Reference values for some of the parameters in gastric juice

Parameter
Normal range

Volume
2–3 L/24 hrs

pH
1.6–1.8

Titrable acidity (Fasting Sample)
15–35 meq/L

Basal Acid Output

Males

1.0–5.0 meq/hrs

Females

0.2–3.8 meq/hrs

Maximal Acid Output (After Histamine/Pentagastrin)

Males

18–28 meq/hrs

Females

11–21 meq/hrs

Table 19.5. Reference values of some of the constituents of blood

Constituents | Reference range
Common constituents |
Bilirubin (total) | 0.2–0.8 mg/dL
Calcium (total) | 9–11 mg/dL
Cholesterol (total) | 140–220 mg/dL
Creatinine | 0.8–1.3 mg/dL
Glucose (fasting) | 60–100 mg/dL
Phosphorus (inorganic) | 3.0–4.5 mg/dL
Urea | 15–40 mg/dL
Uric acid | 3–7 mg/dL
Proteins |
Total serum proteins | 6.0–8.3 g/dL
Serum albumin | 3.5–5.2 g/dL
Serum globulins | 2.5–3.5 g/dL
A/G Ratio | 1.1–1.7
Lipid profile |
Serum triglycerides | 50–160 mg/dL
Total cholesterol | 140–220 mg/dL
HDL-Cholesterol | 30–60 mg/dL
LDL-Cholesterol | 60–160 mg/dL
VLDL-Cholesterol | upto 32 mg/dL
Electrolytes |
Sodium | 136–145 meq/L
Potassium | 3.5–5.0 meq/L
Chloride | 98–107 meq/L
Enzymes |
Acid phosphatase | 1–3.5 KA Units/L
Alkaline phosphatase | 3–13 KA Units/L
Amylase | 60–180 Somogyi U/L
LDH | 100–190 U/L
SGOT | upto 40 U/L
SGPT | upto 40 U/L
CPK (Total) | 20–90 U/L
Hormones |
Cortisol | 5–25 mg/dL (8 AM)
T₃ | 70–200 ng/dL
T₄ | 5–12 ng/dL
TSH | 0–5 mU/ml
Acid Base Analysis and Blood Gases |
pH | 7.35–7.45
Total CO₂ | 22–28 mmol/L
Bicarbonate (actual) | 22–26 mmol/L
pCO₂ | 31–45 mm of Hg
pO₂ | 85–95 mm of Hg
Oxygen saturation | 96–100%
Anion gap | 10–20 mmol/L
[(Na⁺ + K⁺) – (Cl⁻ + HCO₃⁻)] Base excess | −2 to +3 mmol/L