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Biochemistry ; Questions and Answers Book · January 2013 DOI: 10.13140/RG.2.1.3676.0168



1 author: Nalluri Mallikarjuna Rao Vishnu Dental College 30 PUBLICATIONS 62 CITATIONS SEE PROFILE

Available from: Nalluri Mallikarjuna Rao Retrieved on: 16 June 2016

Questions and Answers



Questions and Answers

Questions and Answers


Questions and Answers


Questions and Answers

Questions and Answers

Dr. N. MALLIKARJUNA RAO Professor & HOD Department Of Biochemistry Vishnu Dental College, Bhimavaram - 534202.


Questions and Answers

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First Edition : 2013

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Printed & Designed by : M.M. PRINTERS, Anjananagara, Bangalore-560091.


Questions and Answers

Preface This book is written to help student in their preparation for examinations. It meets needs of first year M.B.B.S., B.D.S., B.Sc.(N), B.P.T., M.Sc (Medical) and second year B.Pharm students. Topics prescribed by Various Health Science, Universities in India Vijayawada are included in the book. In this book questions and answers are given for 21 topics. Complex pathways are presented in a easy to remember way. This book is written in such way that learning of questions and answers given in each chapter makes student to acquire concept or theme of that topic simultaneously. The book contains 495 questions. Of this answers are provided to 249 questions remaining are model questions. Answers to 54 essay questions, 110 short questions and 85 very short or brief questions are given in this book. Answers are given in simple language with necessary diagrams or illustrations. Model questions given enhances students ability to answer questions with alteration. I am grateful to Sri K. Prasanna Kumar of Seekay Publications for publishing the book.




Questions and Answers


Questions and Answers

Contents 1. Cell, Membrane and Transport


2. Carbohydrates


3. Proteins, Plasma Proteins, Aminoacids and Peptides


4. Lipids


5. Enzymes


6. Nucleotides and Nucleic acids


7. Biological oxidation


8. Carbohydrate Metabolism


9. Lipid Metabolism


10. Protein and Amino acid Metabolism


11. Porphyrin and hemoglobin Metabolism


12. Nucleotide Metabolism


13. Replication, Transcription and Translation


14. Vitamins


15. Minerals


16. Water, electrolytes and acid –base balance


17. Nutrition and Energy Metabolism


18. Hormones


19. Organ function Tests


20. Xenobiotics


21. Cancer



Questions and Answers

CHAPTER - 1 | Cell Membrane & Transport



Cell Membrane & Transport 1. Describe common structural and functional features of eukaryotic cell. A. 1. Though mammals contain many types of cells which differ in function, shape, size etc., they have common features. 2. All types of cells contain nucleus, membrane and sub cellular components etc. 3. Each cell component has uniqe structure and function. Nucleus 1. It is located in the centre of most of the cells. It is surrounded by double layered membrane in which pores are present. 2. Pores present in the membrane permits exchange of material between nucleus and other structures of cell. 3. The outer membrane of nucleus is continuous with other membrane. 4. Chromosomes are present in the nucleus of human and other mammalian cells. 5. Chromatin is the substance present in chromosomes. 6. Chromatin is nucleoprotein which consist of DNA and proteins. 7. Nucleus also contain some amount of RNA. 8. DNA and RNA present in nucleus are carriers of genetic information. NUCLEUS

Outer Nuclear Membrane Chromosome

Inner Nuclear Membrane

Nuclear Pore Mitochondria: 1. Like nucleus it is also surrounded by double layered membrane. 2. The inner membrane forms folds which are named as cristae. 3. Knob like structures are present in cristae. 4. Matrix is the name given to space within inner membrane.



Questions and Answers

5. Number of mitochondria varies from one organ to other. 6. Mitochondria is the power house of the cell. 7. Size and shape of mitochondria depends on the function of organ in which they are present. 8. Electron transport chain, citric acid cycle, β-oxidation, ketone body formation, pyruvate oxidation, few of heme biosynthesis and urea cycle enzymes are present in mitochondria. MITOCHONDRIA Outer Membrane

Chistae Knob Inner Membrane Matrix

Endoplasmic reticulum: This membranous net work is divided into smooth endoplasmic reticulum and rough endoplasmic reticulum. Smooth endoplasmic reticulum: It is also known as microsomal fraction of cell. It appears smooth due to the absence of ribosomes. It is site of hydroxylation reactions of drugs and steroids etc.

Rough Endoplasmic Reticulum


Smooth Endoplasmic Reticulum

Rough endoplasmic reticulum: It is continuation of outer nuclear membrane. It appears rough due to presence of ribosomes. It

Nucleus Ribosome

is the site of protein synthesis.

Golgi complex: It is another membranous net work present in cell. It is involved in secretion of proteins, formation of other cellular components and in glycosylation of proteins.


Golgi Complex

CHAPTER - 1 | Cell Membrane & Transport

Lysosomes: They are vesicle like membrane surrounded structure present in cytoplasm. They are involved in hydrolysis of internalized foreign molecules as well as endogenous substances. Since lysosomes are involved in the removal of endogenous substances they are called as suicide bags of cell. Peroxisomes: Are membranous vesicles found in cytosol. They are involved in hydrogen peroxide metabolism. Cytosol: Soluble portion of the cell is called as cytosol. It contains enzymes of glycolysis, HMP shunt, aminoacid and fatty acid activation, fattyacid synthesis, and few enzymes of porphyrins and urea synthesis.

2. Write note on chemical constituents of cell. A. Chemical constituents of life forms (Cells):Cells contain various organic as well as inorganic molecules and water. a. Organic substances : They form major part of cell. There are two type of organic molecules. Macro molecules are nucleic acids, proteins, lipids and carbohydrates. Amino acids, fatty acids, peptides, vitamins, monosaccharides, nucleotides, hormones and coenzymes are small organic molecules. b. Inorganic molecules : They are present as anions and cations. They are sodium, potassium, calcium, magnesium, bicarbonate, chloride, phosphate etc. c. Water: It is the most predominant molecule of cell.

3. Write about structure of cell membrane. A. 1. Membranes are non covalent assemblies of lipids and proteins with carbohydrates attached. 2. They are gel or semi fluid or semi solid structures. 3. Membrane lipids are organized in a bilayer form in which proteins are embeded. 4. The two sides of membrane are different i. e. molecular composition of cytosolic side of membrane differs from extra cellular side. Membrane lipids : 1. Lipids present in membrane are


Protein Lipid

phospholipids, cholesterol and glycolipids. 2. Phosphalipids and glycolipids form

Membrane Bilayer


membrane bilayer. 3. The proportion of phospholipid and glycolipid in membrane is different in membranes.

4. Membrane lipids are in constant motion.



Questions and Answers

Membrane proteins: 1. There are two types of membrane proteins. 2. They are peripheral membrane proteins and integral membrane proteins. 3. The protein content is different in membranes. 4. The peripheral membrane proteins are present on membrane surface. 5. The integral membrane proteins occupy membrane bilayer. Fluid mosaic model: 1. It is model proposed for membrane structure. 2. Membrane is of fluid in nature. 3. Lipids forms bilayer. 4. The membrane proteins float in the lipid bilayer. 5. Membrane proteins interact extensively with lipids present in bilayer. 6. Surface of the membrane appears as that of mosaic surface. Mosaic Surface Lipid Bilayer


Protein Fluid Mosaic Model

4. Describe transport of molecules across cell membrane with examples. A. For the trans port of molecules across membrane several mechanisms exist. Membrane transport: Two or more types of transport mechanism are involved in movement of molecules across membrane. They are A. Simple or passive diffusion B. Mediated transport A. Simple diffusion: It is transport of molecules down the concentration gradiant. It does not require either energy or carrier. Examples : Absorption of xylose and mannose. B. 1. Facilitated or mediated transport: This type of transport requires carrier molecule. The carrier molecule is responsible for moving molecules from out side of cell to in side or vice versa. It does not require energy. Mechanism of transport of molecules by carrier involves conformational change in carrier molecule. The carrier molecule exist in two states and has binding site for solute molecule. In the native state the binding site of carrier molecule is exposed to high concentration of solute.


CHAPTER - 1 | Cell Membrane & Transport

The solute molecule binds to carrier molecule at its binding site. This is followed by conformational change in the carrier molecule which exposes solute to low concentration. Solute molecule is released and carrier molecule comes back to native state. Membrane Conformational

Solute Outside





Binding Site

1. Glucose uptake by adipocytes, erythrocytes 2. Fatty acid uptake by enterocytes 3. Transport of glucose from enterocyte into blood

2. Active transport: It transport solute molecules against concentration gradient i. e. from low concentration to high concentration. It is accompanied by hydrolysis of ATP. Examples:

1. Na+/K+ – ATPase 2. Ca2+-ATPase of muscle. 3. H+/K+-ATPase of stomach.

3. Secondary active transport: In this type of transport energy required for movement of solute molecule is derived from movement of another solute molecule down concentration gradient. Hence it is called as cotransport. Carrier is symporter. Examples:

1. Glucose uptake by enterocyte 2. Aminoacid uptake by enterocyte

5. Define ionophores and ion channels. Give examples. A. Ionophores: Ionophores form pores in membrane which allows movement of ions across membranes. Examples: 1. Gramicidin. 2. Valinomycin. 3. Diphtheria toxin. Ion channels: Ion channels are pores (channels) present in membrane that allow movement of ions across membrane. Examples : 1. Sodium (Na+) channel. 2. Pottasium (K+) channel. 3. Calcium (Ca2+) channel. 4. Cholirde (Cl-)channel.

6. Write differences between facilitated transport and active transport. A. Differences between facilitated transport and active transport:



Questions and Answers

Facilitated transport

Active transport

1. Transport molecules down the concentration

1. Transport molecules against gradient.

concentration gradient. 2. Requires no energy.

2. Requires energy.

3. Carrier is saturated

3. No carrier saturation.

4. Influenced by hormones

4. Not under hormonal influence.

7. What are the functions of cell membrane? A. 1. Membranes separates cell from its surroundings. 2. Shape of cell depends on membrane. 3. Cell interacts with environment through the membrane. 4. Membranes act as permeability barriers. 5. Membranes are involved in energy production. 6. Flow of molecules form cell into surroundings and vice versa is regulated by membranes. 7. Formation of various cell organelles requires membrane. Other model questions are

8. Write note on mitochondria structure and functions. 9. Write briefly on nucleus/ nucleolus. 10. Write about cytomembranes of a eukaryotic cell. 11. Define facilitated transport and active transport. Give examples for each. 12. Write about membrane lipids and membrane proteins. 13. Explain features of fluid mosaic membrane model with help of a diagram 14. Facilitated transport.


CHAPTER - 2 | Carbohydrates



Carbohydrates 1. Classify carbohydrates. Give examples for each class. Add note on the function of each example. A. Carbohydrates classification: Carbohydrates are classified into a. Monosaccharides. b. Oligo saccharides, c. Polysaccharides based on their carbon chain length. Monosaccharides: 1. Monosaccharides are carbohydrates which


can not be hydrolyzed to small molecules.


H – C – OH



2. Monosaccharide containing three to seven CH2OH

carbons with functional aldehyde or keto group are present in nature.


CH2OH Dihydroxy Acetone

3. They are aldotriose, keto triose, aldo tetrose, keto tetrose, aldopentose, ketopentose, aldohexose, ketohexose and aldoheptose, ketoheptose. 4. Glyceraldehyde and dihydroxy acetone are aldotriose and ketotriose respectively. The phosphorylated forms are metabolic intermediates. 5. Erythrose is an example for aldotetrose and erythrulose is an example for ketotetrose. Erythrose phosphate is metabolic intermediate. 6. Aldopentose and ketopentose are ribose and ribulose respectively. Ribose is constituent of nucleic acids. Ribulosephosphate is metabolic

CHO H – C – OH HO – C – H

CH2OH C–O HO – C – H

intermediate. 7. Aldohexoses are glucose, galactose and mannose. Fructose and sedoheptulose are ketohexose and ketoheptose respectively.

H – C – OH

H – C – OH

H – C – OH

H – C – OH



8. Glucose is present in our blood and gives rise to energy on oxidation.


Fructose 007


Questions and Answers

9. Galactose is a constituent of lactose and has function like glucose. 10. Phosphorylated sedoheptulose is metabolic intermediate. Oligosaccharides :

They consist of few monosaccharides. They are disaccharides, trisaccharide etc.


Monosaccharide Glycosidic Bond Disaccharide




Tri Saccharide Glycosidic Bond Disaccharides: 1. Disaccharide consist of two monosaccharide units. 2. Glycosidic bond joins individual monosaccharides. Maltose, lactose and sucrose are examples. Name





Glucose+ Glucose

α (1→4)

Malt, barley



β (1→ 4)




α, β (1→ 2)

Sugarcane, honey, fruit juices.

Functions: All disaccharides yields energy after their hydrolysis to constituent monosaccharides. Polysaccharides: 1. Polysaccharides are made up of more than ten monosaccharide units. 2. They are polymers of monosaccharides. 3. They are divided into a. Homopolysaccharides b. Heteropolysaccharides.

Homopolysaccharides: 1. They are made up of only one type of monosaccharide. 2. So building block of homopolysaccharide is only one type. 3. They are glycogen, starch, cellulose, inulin, dextrin etc. Starch: 1. It consist of two components. A major amylose and minor amylopectin components.


CHAPTER - 2 | Carbohydrates

2. Amylose is a linear polymer of glucose in which monomeric glucose units are joined by α (1, 4) linkages. It has helical secondary structure. 3. Amylopectin has branched structure. 4. In the linear part glucose units are joined by α (1, 4) linkage. At the branch point glucose units are held by α (1→6) linkage. 5. For every 20-30 glucose units a branch point is present in amylopectin. 6. The secondary structure of amylopectin is random coil due to branches. 7. Starch is common polysaccharide in our diet. It is a storage polysaccharide present in our food stuffs like rice, wheat, pulses, tubers, grains etc. AMYLOPECTIN Glu







a (1 Z 4) Glycosidic Bond











a (1 Z 6) Glu Glycosidic Bond Glu

a (1 Z 4) Glycosidic Bond Glu

Glu-Glucose Glycogen: 1. The structure of glycogen is like that of amylopectin part of starch. 2. Glucose units are held by α (1→4) likages in straight chain part and at branch point α (1→6) glycosidic bond is present between glucose units. 3. Though the glycogen structure is similar to amylopectin the number of branch points are more. 4. Branching occurs for every 6 glucose units.

a (1Z6) linkage

a (1Z4) linkage




Questions and Answers

5. It is present in humans and other mammals. 6. It is also known as animal starch because in animals it serve as reserve food or stored material 7. It is present in liver and skeletal muscle in more amounts. Heteropolysaccharides: 1. They are made up of more than one type of monosaccharide. 2. Usually a disaccharide which is made up of more than one type of monosaccharide serve as building block or repeating unit. 3. Hyaluronicacid, heparin, chondroitin sulfate, keratan sulfate etc. are examples for heteropolysaccharides. 4. Their composition and functions are given below. Name



1. Hyaluronicacid

- ( — β-glucuronicacid-

Lubricant in synovial fluid

N-acetylglucosamine-)n –

and in eye.

2. Chondroitinsulfate – (–β- glucuronicacid-N- acetyl

3. Heparin

Structural component of

Glucuronicacid sulfate-)n —

bone, tendon and cartilage

– (–Iduronicacid- glucosamine sulfate –

Anti coagulant

Glucuronicacid – glucoosamine sulfate-)n – 4. Dermatan sulfete

– (– Iduronicacid- N-acetyl

Component of bone & skin

Galactosamine sulfate–)n – 5. Keratan sulfate

– (– galactose-N-acetyl

Components of cartilage &

Galactosamine sulfate-)n –

loose connective tissue

2. Define carbohydrates. Write their biological functions. A. Carbohydrates are defined as poly hydroxy alcohols with functional aldehyde or keto group. Functions: 1. They are major energy source for man. 2. They function as reserve food material in man and plants. 3. They are components of connective tissues, bone, cartilage, skin, membrane and nerve tissue. 4. They are components of blood group substances, nucleic acids etc. 5. Carbohydrate derivatives are vitamins, antibiotics and drugs

3. Define enantiomers or optical isomers. Give examples. A. Enantiomers (optical isomers): Optical isomers of a compound are called as enantiomers. D and L glucose are examples for optical isomers. 010

CHAPTER - 2 | Carbohydrates

4. Define epimers and anomers. Give examples. A. Epimers: 1. They differ in the configuration of –OH and H groups on 2nd, 3rd and 4th carbon atoms of monosaccharide. 2. Glucose and galactose are epimers. 3. They differ in the configuration of –OH and H groups on fourth carbon atom. 4. Like wise glucose and mannose. Anomers: 1. Anomers differ in configuration of –OH and-H groups on first or anomeric carbon of sugar. 2. α –glucose and β – glucose are two anomers of glucose. 3. In α- glucose –OH is present on right side whereas in β –glucose it is present on left side.

5. Write note on mutarotation. A. Mutarotation: 1. Due to the presence of asymmetric carbon glucose exhibits optical activity and rotates plane polarized light. 2. Optical rotation of a solution containing α –D-glucose is +112◦. 3. But on standing the rotation decreases and reaches +52. 5◦ and no more change occurs due to equilibrium. 4. β –D-glucose also exhibits this change in optical rotation when allowed to stand in a solution. 5. This compound initially show +19◦ of rotation and it gradually increases to +52. 5◦. 6. This phenomenon is called as mutarotation. 7. It is due to change of glucose form pyranose ring form to open chain form. α-D-Glucose +120

D –Glucose ↔ +52. 5

β-D- Glucose

Pyranose ring form Open chain form

+19◦ Pyranose ring form.

6. Write briefly about paper chromatography. A. 1. It is most widely used separation technique. 2. It is used for the separation of closely related compounds from mixture. 3. It is based on partition principle of the compounds to be separated between two phases. 4. The mixture to be separated is applied on whatman No:1 filter paper over a short distance from one end. 5. The paper serve as support for stationary phase of solvent system.



Questions and Answers

6. The solvent system consist of n-butanol, aceticacid and water in the ratio of 4:1:5. 7. The paper is dipped in the solvent system and solvent is allowed to flow over applied sample. 8. The water is absorbed by filter paper and serve as stationary phase. 9. The organic solvent that moves over the paper is known as mobile phase. 10. Compounds which are more soluble in organic solvent move faster. 11. The relative mobility of the compounds during chromatography depends on the partition coefficients of the compounds in two solvent phases. 12. So similar compounds which have different partion coefficients move to different extents. 13. The ratio of the distance moved by compound to the distance moved by solvent is known as Rf values. 14. Rf values are different for different solvent systems. 15. Compounds are identified by staining.

Sample Application

16. Aniline or silver nitrate are used to stain


carbohydrates after separation. 17. Among carbohydrates glucose moves faster followed by galactose.


18. Paper chromatography is also used for separation of


Solvent Front

amino acids. Other model questions are

7. Write a note on monosaccharides. 8. Define disaccharides. Give examples. 9. How sucrose and maltose differ with respect to a. Structure b. Source c. Function 10. Write briefly about structure and functions of starch and glycogen. 11. What are polysaccharides? Classify. Give examples. 12. Write briefly about mucopolysaccharides 13. Write four functions of carbohydrates. 14. Write composition and functions of a. Hyaluronicacid b. Heparin. 15. Write composition and function of lactose.


CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids



Proteins, Plasma Proteins, Peptides & Aminoacids PROTEINS

1. Classify proteins based on composition giving examples for each class. A. Proteins classification, based on composition. According to this proteins are classification into a. Simple proteins. b. Conjugated proteins. c. Derived proteins. a. Simple proteins:Are those proteins which yields only aminoacids on hydrolysis. Ex:Trypsin, plasma albumin, pepsin etc. b. Conjugated proteins: 1. Are those proteins that yields aminoacids and other organic or inorganic molecules or non protein molecules on hydrolysis. 2. The non protein molecule is called as prosthetic group. 3. Usually conjugated proteins are named according to the name of non protein. 4. Some examples are tabulated below. Conjugated protein

Non protein part


1. Heme proteins



2. Glycoproteins



3. Flavoproteins


Succinate dehydrogenase

4. Nucleoproteins



5. Phosphoproteins



6. Lipoporteins


Various classes of lipoproteins like VLDL, HDL

7. Metalloproteins



c. Derived Proteins: Are those proteins that are derived from partial hydrolysis of simple or conjugated proteins. Gelatin, Peptone and proteose are examples.



Questions and Answers

2. Classify proteins on the basis of solubility giving examples for each class. A. Classification of proteins based on their solubility. According to this proteins are classified into a. Albumins. b. Globulins. c. Glutelins. d. Protamins. e. Histones. f. Prolamines. g. Sclero proteins. a. Albumins:Are those proteins that are soluble in water as well as salt solutions. Egg albumin, plasma albumin and lactalbumin are examples. b. Globulins: Are proteins weakly soluble in water but soluble in salt solutions. Ovoglobulins, plasma globulin and lactoglobulins are examples. c. Glutelins: Are proteins soluble in mild acids and alkalis. zein, glutenin and oryzenin are examples. d. Protamines: Are proteins soluble in water and ammonia. Fish proteins like salmine and sturine are examples. e. Histones: Are those proteins which are soluble in water and dilute acids. Histones present in chromosomes are examples. f. Prolamines:Are proteins insoluble in water and alcohol but soluble in dilute alcohol. Plant proteins zein and gliadin are examples. g. Sclero Proteins: Are proteins insoluble in water, acids and alkalis. Animal proteins keratin, elastin and collagen are examples.

3. Describe structural organization of proteins. OR What is primary, secondary, tertiary and qua ternary structure of proteins? Explain each one giving examples. . A. Primary structure of proteins : 1. Aminoacid sequence of a protein is known as primary structure of protein. 2. Peptide bonds and disulfide bonds are involved in primary structure. H2N




Ala - Alanine Gly - Glycine Phe - Phenyl Alanine Lys - Lysine





Peptide Bond






Tyr - Tyrosine His - Histidine Val - Valine Leu - Leucine

CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids

Primary structure of insulin: 1. It consist of two polypeptide chains. 2. They are A chain and B chain. 3. Inter chain di sulfide bonds links two chains. 4. Further an intra chain disulfide bond is present in A chain. 5. Glycine is the N-terminal aminoacid and aspargine is the C-terminal amino acid in A chain. 6. In the B chain alanine is C-terminal amino acid and phenyl alanine is the N-terminal aminoacid. Intrachain Disultide Bond S S | | CYS CYS CYS | S Inter | Chain Disulfide Bond S | CYS

CYS - Cysteine CYS | S | S | CYS

A Chain

B Chain

INSULIN Secondary structure of protein: 1. Two dimentional folding of polypeptide chain is known as secondary structure of protein. 2. The folding of protein chain can be ordered or disordered. 3. The ordered secondary structures are α-helix and β-pleated sheet. 4. The disordered secondary structures are random coil and reverse turn or β-turn. H2N

Alpha (α)Helix It is the secondary structure found in α-Keratin of hair, nails and epidermis of the skin.

Structural features of α-helix: a. Coiling of polypeptide or protein chain along long axis produce α-


helix. b. α-helix is stabilized by intra chain hydrogen bonding. c. Peptide bonds are involved in hydrogen bonding. d. C=O and –N-H groups of peptide bond participate in hydrogen bonding.



Questions and Answers

e. There are 3. 6 aminoacids in one turn of α-helix. f. Peptide bonds that are four aminoacid residues away participate in hydrogen bonding i. e. -N-H of second aminoacid residue and –C=O of sixth aminoacids are involved in hydrogen bonding. g. α-helix of fibrous proteins is right handed. h. α-helix is destabilized by hydrophobic aminoacids. I. In contrast aromatic aminoacids stabilizes α-helix. j. α-helical regions are found in several other proteins. Beta (β)Pleated Sheet When two or more polypeptide chains line side by side along long axis beta pleated sheet is formed. Adjacent segments of a protein or polypeptide chain may also form secondary structure.

Polypeptide Chain 1 Polypeptide Chain 2 Beta Pleated Sheet (Parallel) Structural features of β-Pleated Sheet a. Polypeptide chains are fully extended along long axis in beta pleated sheet. b. Inter chain hydrogen bonds stabilizes beta pleated sheet. c. Based on direction β-pleated sheet is divided into i. Antiparallel β-pleated sheet and ii. Parallel β-pleated sheet. d. In antiparallel β-pleated sheet polypeptide chains run in opposite direction. e. In parallel β-pleated sheet polypeptide chains run in same direction. f. Pleated sheet is found in many proteins. Albumin and hemoglobin of blood contains βpleated sheet. g. Antiparallel β pleated sheet is found in β-Keratin of silk fibroin, spider web and amyloid protein found in the brain of Alzheimer's disease patients. h. β-pleated sheet content varies among proteins. Tertiary structure of protein: 1. It is formed due to three dimentional folding of polypeptide chain of protein in space. 2. Tertiary structure of protein contains ordered and disordered secondry structures i. e. α-Helix, β-pleated sheet, random coil conformation etc.


CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids

Forces involved in maintenance: 1. Several non covalent bonds stabilizes tertiary structure. 2. Usually it refers to native conformation of a protein. 3. Internal hydrogen bonds, electrostatic, hydrophobic and van der Waals interactions are bonds that keep tertiary structure intact. 4. In the case of proteins that are made up of only one polypeptide chain tertiary structure is the final level of protein structure. Quaternary structure of protein: 1. Proteins which are made up of more than one polypeptide chain contains quaternary structure. 2. Such proteins are known as oligomeric proteins and constituent polypeptide chains are referred as sub units or protomers.

COOH Sub Unit

NH2 Quaternary Structure

Tertiary Structure

Hemoglobin, creatine phosphokinase. Lactate dehydrogenase etc are examples for proteins with quaternary structure. Hemoglobin and lactate dehydrogenase are made up of four subunits whereas creatine phosphokinase contains two sub units.

4. Define proteins. Write their functions. A. Proteins are polymers of aminoacids. The aminoacids are joined by peptide bonds. Hence they are also called as polypeptides. Amino Acid1

Amino Acid2

Peptide Bond

Amino Acid3

Amino Acid4

Amino Acidn

Protein (Polypeptide)

Functions of Proteins: 1. Proteins are present in body. They are structural components of tissues, cells etc. 2. Proteins function as hormones 3. Proteins functions as enzymes 4. Proteins regulate gene expression. 5. Proteins are involved in muscle contraction



Questions and Answers

6. Proteins perform transport functions. 8. Proteins are used as nutrients. 7. Proteins act as buffers. 9. Proteins act as reservoir of minerals 10. Proteins act as infective agents.

5. Classify proteins based on their shape. A. Protein classification on the basis of shape. According to this proteins are divided into a. Fibrous proteins b. Globular proteins. a. Fibrous Proteins: Are proteins in which polypeptide chains are elongated. Keratin, collagen and elastin are examples. b. Globular Proteins: Are proteins in which polypeptide chains are folded into globular or spherical shape. Hemoglobin, albumin and trypsin are examples.

6. Write briefly about protein denaturation. A. 1. Denaturation of protein is loss of native conformation.

Heat Protein

Denatured Protein

2. Denatured proteins exhibit properties which are not shown by native protein. 3. They are A. Loss of biological function. B. Solubility changes. C. Susceptible to enzyme action. D. Increased chemical reactivity. E. Dissociation of subunits in case of oligomeric protein. Methods of protein denaturation By several ways proteins are denatured. They are 1. By exposing protein to extreme acidic or alkaline PH. 2. High temperature. 3. Use of detergents like sodium dodecyl sulfate (SDS). 4. By treating with strong acids like trichloroacetic acid (TCA), Tungstic acid and picric acid. 5. Exposing to ultraviolet light. 6. Using salts like urea and guanidinium chloride at high concentration. 7. Vigorous shaking. 8. Ultrasonication. 9. Heavy metal exposure like lead, arsenic, mercury etc. 10. By organic solvents like acetone, alcohol etc.


CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids

Clinical Importance: 1. Protein denaturation is part of estimation of blood constituents. 2. Plasme protein separation involves protein denaturation. 3. Isolation of protein or enzyme from mixture of proteins involves denaturation. Examples for protein denaturetion: 1. Exposure of egg albumin to high temperature leads to formation of coagulum. 2. Sweet tasting protein monellin loses its property on denaturation.

7. Write methods for determination of protein primary structure. A. Primary structure of protein is determined by a. Sanger's method. b. Edman's method. Sanger's method: 1. In this 1-fluoro-2, 4-dinitrobenzene (FDNB) is used to determine primary structure of protein. 2. FDNB reacts with free aminogroup of protein to produce yellow 2, 4 – dinitrophenyl residue of aminoacids which are identified after chromatographic separation. 3. Since FDNB reacts with other amino acids only one aminoacid is determined at a time with this method FDNB + Protein

Identification of amino Acid

Dinitrophenyl Derivative


Edman's meathod: 1. In this method also primary structure is elucidated from N-terminus. 2. However complete sequence of protein is obtained by repeating several times with Edman's reagent. 3. Unlike Sanger's method Edman's reagent reacts with only one aminoacid and rest of the aminoacids remain intact. 4. Edman's reagent (Phenylisothiocyanate)reacts with free aminogroup in presene of acid to produce phenylthiohydantoins which are estimated by using chromatography. PLASMA PROTEINS

8. Describe composition and functions of various plasma proteins. A. Several structurally and functionally different proteins are present in plasma. They are albumin and various components (fractions) of globulins.



Questions and Answers

Albumin: It contributes osmotic pressure in blood. It is involved in maintenance of blood volume. One gm of albumin can hold 18ml of fluid in blood. It is involved in transport of several substances. Further it binds to various substances and drugs. Fatty acids and bilirubin are transported by albumin. Several hormones also transported by albumin Sex hormones and glucocorticoids are transported by albumin. Albumin function as buffer. Peripheral tissues use albumin as nutrient. Alpha, (α1) alpha2 (α2), beta (β)and gamma (g) globulins are components of globulin fraction of plasma. Further each of subglobulin fraction consist of several proteins. α1-globulins α1-antitrypsin and α1-acid glycoprotein are principle components of this fraction. Other components are α-lipoprotein, prothrombin, α1-fetoprotein, thyroxine binding and retinol binding proteins. α1-antitrypsin: It accounts for more than 90% of α-globulin fraction. It is an inhibitor of trypsin, chymotrypsin, elastase etc. It prevents action of proteases on pulmonary tissue and other tissues. Lack of α1-antitrypsin results in emphysema. α-Lipoprotein: It is involved in transport of lipid (cholesterol)from peripheral tissues to liver for removal. Prothrombin: It is one of the blood clotting factors and involved in blood coagulation. α1-fetoprotein:As the name implies it is the protein present in foetal blood and its presence in adult blood indicates liver cancer. It is considered as tumor marker for liver cancer. Thyroxine and retinol binding proteins are involved in the transport of thyroxine and vit. A respectively. α2-globulins: α2-macroglobulin, haptoglobulin, erythropoietin, ceruloplasmin and pseudo choline esterase are present in this fraction. α2-Macroglobulin: It is an inhibitor of proteases. It combines with proteases to form complex which is then easily removed from circulation. Haptoglobulin: It is involved in the transport of hemoglobin. It combines with hemoglobin to from complex. Erythropoietin: It is required for formation of reticulocytes. Ceruloplasmin: It is also known as ferrooxidase. It is a copper containing protein. Pseudo choline estrase: It is an enzyme present in blood. β-globulins β-lipoprotein, transferrin and complement-3 are components of this fraction. β-Lipoprotein : It is involved in the transport of lipids from liver to peripheral tissues.


CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids

Transferrin: It is involved in the transport of iron. Complement-3:It is one of the component of complement system. Îł-globulins Immunoglobulins are major component of this fraction. C-reactive protein is another component of this plasma protein fraction. C-Reactive protein: It is produced in inflammatory condition. Immunoglobulins They are involved in defence function. They are antibodies present in serum. They are produced when foreign molecules or antigens enters inside body. Structure: 1. Generally an Immunoglobulin is made up of 4 polypeptide chains. The molecular weight of this is about 150000 daltons. 2. Two types of polypeptide chains are present. Two heavy or H chains and two light or L chains. 3. Each H chain molecular weight is 50, 000 and contains 450 aminoacids. 4. Molecular weight of L chain is about 25, 000 and contains 220 aminoacids. 5. The H chains contains variable region at N terminus [VH] and three constant regions at C terminus [CH1, CH2 and CH3]. 6. In the L chain one variable region (VL) at N terminus and constant region (CL) at C terminus exist. 7. The aminoacid sequence varies in variable regions of H and L chains and largely depends on class or type of immunoglobulin. 8. However constant regions of H and L chain aminoacid sequence is constant or same in various types of immunoglobulins 9. The variable regions recognizes antigens. Shape 1. Overall shape of immunoglobulin is that of Y. 2. Two H chains intertwines to form base of Y. 3. Arm of the Y is formed by joining L chains to H chains. 4. Most of the immunoglobulins contains carbohydrate in CH2 region. 5. Several intra and inter chain disulfide bonds maintain Y shape.

–S-S– Light Chain

immunoglobulins are classified into three major classes and two

Heavy Chain

minor classes. Ig G, Ig A and IgM are major classes. Ig and IgE are minor


Classification: Based on composition of H and L chains

classes. Not only composition, size, shape, distribution and function



Questions and Answers

also varies among various classes of immunoglobulins. Each class of immunoglobulin contains unique H chain based on which they are named. The different H chains are g (Gamma), α (Alpha), μ (mu), δ (Delta) and ε (Epsilon). However in all five classes of immunoglobulins only two types of L chains are found. They are κ (kappa) and λ (lambda). 1. Ig G Class 1

Structure : It consist of two g type H chains and two L chains of K or lambda type. So it is designated as g2 L2 or g2 K2 or g2λ2. Function: It is major immunoglobulin of serum. It is


the major antibody of new born. Ig G binds to foreign cells or antigens which increases their susceptibility for elimination.

Ig G dimer

2. IgA Class Structure: It consist of two alpha type H chains and two κ or λ type L chains. Hence it is designated as α2 L2. It may exist as multimer of the basic unit. Polypeptide chains like SC and J are also found. They are involved in joining of monomers. Function: It accounts about 10-20% of immunoglobulins. It is chief of antibody of mucosal cells, secretions of lungs and gut where it combines with antigen thus protecting them from harmful antigens. 3. Ig M Class


Structure: It consist of two μ type H chains and two L chains. Hence it is designated as μ2L2. This basic unit exist as multimer like Ig A class. Most common



occurence is in the pentameric form (μ2 L2) 5. SC and J components also may occur. Function: Ig M on B- Lymphocytes act as receptor for antigens. Complement fixation requires Ig M. About 5



– 10% total immunoglobulins is Ig M type. 4. Ig D Class

Ig M Pentamer

Structure: It is made up of two δ type H chains and two C chains. It is designated as δ2 L2. Function: It is involved in alternate pathway of complement fixation. It accounts only 0. 5% of total immunoglobulins. 5. Ig E Class Structure: It is made up of two ε type H chains and two L chains. It is designated as ε2 L2.


CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids

Function:It is involved in anaphylactic response. Among all classes of immounoglobulins it is least concentrated. However in allergic reactions its concentration may increase. They may be found in mucous secretions of lung and gut.

9. Write briefly about Bence-Jones protein. A. Bence –jones proteins are found in urine of multiple myeloma patients. They are derived from immunoglobulin light chain. They are detected in urine based on their behaviour on heating. These proteins precipitate at 40- 600C and dissolves at boiling point. Further cooling re precipitates and boiling re dissolves.

10. Write a note on acute phase proteins. A. 1. α1-antitrypsin, haptoglobulin, ceruloplasmin, complement -3, fibrinogen and Creactive proteins are known as acute phase proteins. 2. In acute inflammation their concentration in plasma increases. 3. Interleukin released by macrophases at site of injury induces synthesis of these proteins by liver. 4. In plasma levels of these proteins during inflammation raises at different rates. Creactive protein raises initially. 5. This is followed by raise in α1-anti trypsin. At the end complement-3 level raises. PEPTIDES

11. Define peptide. How they are formed ? Give examples. A. 1. Peptides are compounds containing peptide bonds. 2. Peptides are formed due to inter action between carboxyl group of one aminoacid with amino group of other aminoacids. 3. Peptide bond formation involves loss of one water molecule. 4. Glutathione, thyrotrophin releasing hormone, enkaphalins, oxytocin, vasopressin are examples for peptides.

H2N – CH – COOH – H2N – CH – COOH | | R2 R1

H2O H2N – CH – CONH – CH – COOH | | R1 R2 Peptide Group

12. Define dipeptide, tripeptide and penta peptide. Give examples. A Dipeptide: A dipeptide is made up of two aminoacids which are joined by single peptide bond. carnosine and anserine are examples.



Questions and Answers




Peptide Bond

Tripeptide : It is composed of three aminoacids. Two peptides bonds connect these aminoacids. Glutathione and thyrotrophin releasing hormone are examples. AminoAcid1




Peptide Bond

Pentapeptide : Five aminoacids are linked by four peptide bonds. Enkaphains are examples.

13. Write the glutathione composition, short form and functions. A. Glutathione : It consist of glutamate, cysteine and glycine. It is written as glutamatecysteine- glycine. G-SH is short form. It is a reducing agent. It undergo dimerization on loss of hydrogen. G –S-S-G is oxidized form. It is involved in the maintenance of -SH groups on proteins on reduced form. In red blood cells (R. B. C.) it is involved in the elimination of hydrogen peroxide. It participates in detoxification. It is involved in hormone secretion and apoptosis.

14. Define cyclic peptide and toxic peptide. Give examples. A. Cyclic peptide (s):It is formed when amino and carboxyl terminals of the peptide are joined by peptide bond. Antibiotic gramicidin –S and tyrocidin are examples. Toxic peptides: Are peptides acting as toxins. α-Amanitin is toxic peptide present in mush rooms which is responsible for mush room poisoning. AMINOACIDS

15. Classify aminoacids based on side chain and ring structure giving examples. A. Aminoacids are classified into seven major classes based on side chains. a) Aliphatic aminoacid s: Are those which contain aliphatic side chains. Glycine, alanine, valine, leucine and isoleucine are examples for aliphatic aminoacids. The latter three aminoacids are also known as branched chain aminoacids. H | H2N – C – COOH | H Glycine

H | H2N – C – COOH | CH3 Alanine

H | H2N – C – COOH | CH CH3 CH3 Valine

H | H2N – C – COOH | CH2 | CH CH3 CH3 Leucine


CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids

b) Hydroxy aminoacids :Are those aminoacids that contain sulfhydryl groups in side chain. Serine and threonine are examples for hydroxyl aminoacids. H | H2N – C – COOH | CH – CH3 | CH2 | CH3

H | H2N – C – COOH | CH2 – OH

H | H2N – C – COOH | CH – OH | CH3

H | H2N – C – COOH | CH2 – SH

H | H2N – C – COOH | CH2 | CH2 – S | CH3






c) Sulfur containing aminoacids : These aminoacids contain sulfhydryl groups in side chain. They are cysteine, methionine and cystine. H | H2N – C – COOH | CH2 – COOH

H | H2N – C – COOH | CH2 – CONH2

H | H2N – C – COOH | CH2 | CH2 – COOH

H | H2N – C – COOH | CH2 | CH2 – CONH2





d) Acidic aminoacids : Side chains of these aminoacids contain acidic groups or their amides. They are glutamate, glutamine, aspartate and aspargine. H | H2N – C – COOH | CH2 | NH CH2 | CH2 – NH – C – NH2


H | H2N – C – COOH | CH2 | NH N

H | H2N – C – COOH | CH2 | CH2 | CH2 | CH2 – NH2



e) Basic aminoacids: Basic groups are present in side chains of these aminoacids. They are arginine, lysine, hydroxyl lysine and histidine. H | H2N – C – COOH | CH2

H | H2N – C – COOH | CH2


Phenyl Alanine


H | H2N – C – COOH | CH2 | N H Tryptophan



Questions and Answers

f) Aromatic aminoacids : Aromatic rings are present in the side chains of these aminoacids. They are phenylalanine, tyrosine and tryptophan. OH


COOH Proline

N COOH H Hydroxy Proline

g) Iminoacids: Are those aminoacids in which amino group is replaced by imino group. They are proline and hydroxy praline.

16. Define an aminoacid. Write their functions. A. Aminoacids are acids containing aminogroups. They are building blocks of proteins and peptides present in humans and other living organisms.

Amino Group

H | H2N – C – COOH | R

Acid Group

R-Side Chain

Amino Acid

Functions: Free aminoacids are found in blood and cells of humans. Hormones, purines, pyrimidines, heme, some vitamins, creatine etc found in body are derived from aminoacids.

17. Classify aminoacids based on reaction in solution.


CHAPTER - 3 | Proteins, Plasma Proteins, Peptides & Aminoacids

A. Semi essential aminoacids: Semi essential aminoacids are synthesized in the body to some extent. They are histidine and arginine. Unusual or rare aminoacids or Non protein aminoacids: These aminoacids are not found in proteins. But they have other functions. Examples are i). Intermediates of urea cycle i. e. ornithine. citrulline and argininosuccinate. ii). taurine. iii). Gamma aminobutyric acid (GABA). iv). Beta (β)-alanine. v). Pantothenic acid.

20. Explain charge or acid base properties of aminoacids. A. 1. Depending on pHof surroundings an aminoacid can exist as cation or positively charged molecule, anion or negatively charged molecule and zwitter ions. 2. Zwitter ion carries no net charge It contains equal number of positive charges and negative charges. 3. Further aminoacids act as acids or bases. When alkali is added aminoacid act as acid by donating proton. Aminoacid act as base by accepting a proton from acid. 4. At nutral pH aminoacid functional groups amino and carboxyl groups exist in ionized form. 5. The amino group exist in protonated -NH3+form and carboxyl group in the dissociated –COO-form this is known as zwitter ionic form. 6. In strong acidic conditions –COOH remains undissociated i. e. aminoacid exist as cation. 7. In strong alkaline condition proton from –NH3+is lost i. e. aminoacid exist as anion. Anion ← H AlkalineP

Zwitterion Neutral PH

cation acidicPH

8. The PHdependence of charge of aminoacid is used for separation of aminoacids.

21. Define isoelectric point of aminoacid. How it is determined? Write its importance. A. 1. At iso electric point aminoacid exist as zwitter ion.

H | H2N – C – COO– | R

+ H | H3N – C – COO– | R

+ H | H3N – C – COOH | R

2. The isoelectric point of an aminoacid having one carboxyl group and one amino group is obtained by dividing Pkvalues of these groups with 2. 3. At isoelectric point aminoacids or proteins have minimum solubility. 4. This is exploited for separation of proteins or aminoacids from mixture.

22. Define Pk values of amino acid.. Write the significance of it. A. 1. It is PH at which un dissociated and dissociated forms of a group are present in equal



Questions and Answers

amounts. 2. For example Pk of amino group of aminoacid is designated as Pkam. It is PHat which dissociated (-NH2) and undissociated (-NH3+) are found in equal amounts. 3. Like wise Pk of acid groups of aminoacid is designated as Pka. 4. The Pk values indicates strength of groups. 5. Low Pkvalues indicates more ionizing power. High Pk values indicates less ionizing power.

Other model questions are 23. Write a note on immunoglobulins. 24. Write briefly about conjugated proteins. 25. What are derived proteins ? Give examples. 26. What is meant by secondary structure of proteins? Give examples. 27. Write about quaternary structure of proteins giving examples. 28. Write about structural feature of α –helix. 29. Write a note on ß -pleated sheet. 30. Draw general structure of immunoglobulins. Label its parts. 31. Write briefly about Ig E and Ig M. 32. Write briefly about tertiary structure of protein. 33. Define primary structure of protein. Write a method for its determination. 34. Write a note on albumin. 35. Write very briefly about aromatic aminoacids. 36. Write note on α 1- globulins. 37. Write clinical importance of alpha fetoprotein and macroglobulin. 38. Name sulphur containing aminoacids and basic aminoacids. 39. Classify immunoglobulins. Give structural and functional aspect of each class. 40. Name three charged forms of aminoacid. When the aminoacid assumes these states? 41. Write a note on α 2- globulins. 42. Write a note on ß-globulins. 43. Write a note on the general structure of immunoglobulins. 44. Non protein aminoacids 45. Protein structure


CHAPTER - 4 | Lipids



Lipids 1. Classify lipids. Give examples for each class along with functions. A. Classification: Based on composition lipids are classified into I. Simple lipids II. Compound lipids and III. Derived lipids. I. Simple lipids : Esters of fatty acids with alcohol are known as simple lipids. Fats and waxes are simple lipids. a. Fats: 1. Are esters of fatty acids with glycerol. 2. Triglycerides, diglycerides and mono glycerides are fats. 3. Triglyceride is also called as tri acyl glyccrol. 4. In triglycerides three fatty acids are esterified to three hydroxyl groups of glycerol. 5. In diglycerides two of the hydroxyl groups of glycerol are esterified with glycerol. 6. Only one fatty acid is esterified to any one of hydroxyl group of glycerol in monoglycerides. Esterbond CH2 – OH | CH – OH | CH2 – OH

CH2 – O – COR1 | CH – O – COR2 | CH2 – O – COR3

CH2 – O – COR1 | CH – O – COR2 | CH2 – OH

CH2 – O – COR1 | CH – OH | CH2 – OH





COR1, COR2, COR3 - Acyl Groups Functions: 1. They are mainly involved in storage function. 2. Adipose tissue present under skin contains triglycerides. In the abdomen, thighs and in mammary gland, adipose tissue containing triglycerides is present.



Questions and Answers

3. Obese people contain more triglycerides. 4. Women contain more triglycerides than men. 5. In hibernating animals, seals and penguins triglycerides are more. 6. Fat under the skin has dual roles. It function as energy store as well as insulator against cold. b. Waxes: Are esters of fatty acids with long chain alcohols. Wool and bees wax are waxes known well. Wool is ester of fatty acid with long chain alcohol lanosterol and agnosterol. Bees wax is an ester of fatty acid with myricyl alcohol. Functions: 1. Waxes form protective layer over the skin, fur, feathers of animals. Shiny appearance of fruits, leaves of plants are due to waxes. 2. Waxes are hard at low temperature and soft at high temperature. 3. Wool a wax of animal origin is used as protection against low temperature or cold. Woolen clothing protect us from cold for this reason. 4. Waxes act as water barrier for animal, plants, birds etc. II. Compound lipids: Are esters of fatty acids with alcohol containing additional groups and nitrogenous bases. They are further subdivided based on alcohol present. They are glycerophospho lipids and sphingolipids. In glycero phospholipids glycerol is alcohol and sphingosine is alcohol in sphingolipids. A. Glycerophospholipids: 1. In which two fatty acids are esterified to two hydroxyl groups and nitrogenous base bearing phosphate is esteri fied to third hydroxyl group of glycerol. 2. Glycerophospholipid lacking nitrogenous base is known as phosphatidicacid. 3. Some glycerophospholipids are considered as derivatives of phosphatidic acid and they are named accordingly. 4. Phosphatidyl choline, phosphatidyl serine, phosphoatidyl ethanolamine and phosphatidyl inositol are examples for glycerophospholipids. 5. Due to the presence of phosphate they are often referred as phospholipids. i) Phosphatidyl choline: It consist of glycerol, two fatty acids esterified to first and second hydroxyl groups. Phosphate is esterified to third hydroxyl group. Nitrogenous base choline is esterified to phosphate. Lecithin is the alternate name for this glycerophospholipid. ii) Phosphatidyl serine : It is an aminophospholipid. Serine an aminoacid is attached to phosphate which is esterified to third hydroxyl of glycerol. First and second hydroxyl groups of glycerol are esterified with two fatty acids. Cephalin is alternate name for this phospholipid. 030

CHAPTER - 4 | Lipids

iii) Phosphatidyl inositol : Sugar alcohol inositol is esterified to phosphate of phosphatidic acid. CH2 – O – COR1 | CH – O – COR2 | CH2 – O – Phosphocholine

CH2 – O – COR1 | CH – O – COR2 | CH2 – O – Phosphoserine

CH2 – O – COR1 | CH – O – COR2 | CH2 – O – Phospho Ethanolamine

Phosphatidyl Choline

Phosphatidyl Serine

Phosphatidyl Ethanolamine

Functions: 1. Phosphatidyl choline is major lipid present in cell membrane. It is also present in egg yolk and plasma lipoproteins. 2. Cephalin is also component of cell membrane, lipoproteins and nervous tissue. 3. Cell membrane contains phosphatidyl inositol. 4. Inositol triphosphate (IP3) which is involved in signal transducution is derivative of phosphatidyl inositol. B. Sphingolipids: They consist of an aminoalcohol sphingosine, fatty acid, nitrogenous base and additional groups. They are subdivided into a. Sphingomyelins. b. Glycolipids a. Sphingomyelins: 1. They are made up of fatty acid linked to sphingosine by amide bond and phosphoryl choline which is esterified to sphingosine. 2. Due to presence of phosphate sphingomyelins are also considered as phospholipids. Sphingomyelin

Fatty acid




Functions: 1. Sphingomyelins occur in myelin sheath of nervous tissue. 2. They are most abundant sphingolipids. 3. They are also present in grey matter. 4. Cell membrane also contain sphingomyelin. b. Glycolipids: They are subdivided into two groups. 1. Cerebrosides. 2. Gangliosides.



Questions and Answers

Cerebrosides: a. They consist of sphingosine, fatty acid and carbohydrate or sugar. b. Usually they are named according to sugar present. c. For example if glucose is the sugar present in a cerebroside then it is called as glucocerebroside. d. Similarly galacto cerebroside contain galactose sugar. e. In some cerebrosides sulfate is esterified to sugar moiety. f. They are known as sulfatides or sulfolipids. Cerebroside

Fatty acid




Fatty acid




Gangliosides: a. They are most complex of all compound lipids. b. They are made up of sphingosine, fattyacid, oligosaccharide and sialic acid. c. The oligo saccharides contain aminosugar and acetylated aminosugars. Ganglioside





Functions: 1. White matter of the brain and myelin sheath of nerves contain cerebrosides. 2. Grey matter contain gangliosides. 3. Gangliosides serve as receptors for toxins, hormones etc. 4. Cerebrosides and gangliosides are also present in non neural tissues. 5. Gangliosides are also involved in cell cell recognition, growth and differentiation and carcinogenesis. III Derived lipids : Hydrolysis of simple and compound lipids produce derived lipids. Fatty acids, steroids, fat soluble vitamins and glycerol are examples for derived lipids. Fatty acids : Hydrolysis of triglycerides yield fatty acids. They are acids containing long hydrocarbon chain. Many fatty acids are identified in nature. They are subdivided into a. Saturated fatty acids. b. Unsaturated fatty acids based on nature of hydrocarbon chain. a. Saturated fatty acids: 1. The hydrocarbon chain of these fatty acids is saturated. 2. No double bonds occur. 3. Saturated fatty acids containing up to 20 carbons are identified. 4. More important are palmitic acid, stearic acid and arachidonic acids.


CHAPTER - 4 | Lipids

CH3 – CH2 – CH2 – CH2 – – – – – – CH2 – COOH Hydro Carbon Chain

Acid Group

Saturated Fatty Acid b. Unsaturated fatty acids: 1. They contain double bonds in hydrocarbon chain. 2. Unsaturated fatty acids containing up to 30 carbons are identified. 3. They are subdivided into mono unsaturated fatty acids and polyunsaturated fatty acids (PUFA) based on number of double bonds. 4. Mono unsaturated fatty acids are palmitoleic acid and oleic acid. They contain one double bond. 5. Poly unsaturated fatty acids are linoleic, linolinic and arachidonic acids. They contain many double bonds. CH3 – CH2 – – – – CH = CH – CH2 – – – – CH = CH – – – – CH2 – COOH Double Bond Unsaturated Fatty Acid Functions: 1. Fatty acids are source of energy for humans like glucose. 2. Fatty acids are components of nervous tissue, lipoproteins etc. 3. Poly unsaturated fatty acids are essential fatty acids. 4. They are required for the synthesis of eicosanoids. 5. They are also components of cell membrane. Steroids : They contain complex fused ring system which is also known as steroid nucleus. Fused ring system contains four rings collectively known as cyclopentanoperhydrophenan threne ring. Cholesterol is an example for steroid which is steroid alcohol. Functions : 1. It is most abundant steroid in animals. 2. About 200g of cholesterol is present in human adult. 3. Nervous tissue is rich in cholesterol. 4. Egg yolk is also rich in cholesterol. 5. Cholesterol is used for the formation of vitamins and steroid hormones. 6. Vit. D is derivative of cholesterol.





Questions and Answers

7. Glucocorticids, mineralo corticoids, male sex hormones, female sex hormones are derivatives of cholesterol.

2. Define lipids. Write briefly about their functions. A. Lipids are organic substances soluble only in organic solvents like chloroform, ether and benzene but insoluble in water. Functions: 1. Lipids are structural components of cell membrane and nervous tissue. 2. Lipids present in myelinated nerves act as insulators for propagation of depolarization wave. 3. Lipids present under skin act as thermal insulator against cold. 4. Lipids are energy source for man like carbohydrates. 5. Lipids like steroids function as hormones. 6. Lipids present around kidney act as padding and protect kidney from mechanical injuries. 7. Lipids serve as vitamins. 8. Lipids are part of lipoproteins present in blood plasma. 9. Absorption of fat soluble vitamins requires lipids. 10. Essential fatty acids a kind of lipids are essential for life. 11. Lipids act as microbicides and fungicides. 12. Some lipids function as surfactants. 13. Lipids are involved in immune response. 14. Lipids act as mitogens. 15. Some lipids serve as precursors for the formation of complex lipids. 16. Due to its high energy and water output on oxidation mammals including humans prefer to store energy in the form of lipid only

3. Write a note on structure and function of lyso phospholipids. A. Partial hydrolysis of glycerophospholipids yield lysophospholipids. Hence they contain only one acyl group instead of two acyl groups and phosphorylated nitrogenous base. Functions : They are produced as intermediates during phospholipid biosynthesis. Lyso lecithin a derivative of lecithin is present in cobra venom. It is a strong hemolysing agent.

4. Write a note on plasmalogens. A. They are also glycerophospholipids. They contain unsaturated fatty alcohol in the place of first fatty acid at first hydroxyl group. Because of this an ether linkage is found on first carbon instead of usual ester linkage. Phosphorylated nitrogenous bases are usually choline, serine, ethanolamine etc.


CHAPTER - 4 | Lipids

Functions: 1. They are structural components of tissues like brain, muscle and heart. 2. Platelet activating factor is a plasmalogen. 3. In cancer cells plasmalogen content is more.

5. Write a note on dipalmitoyl lecithin. A. 1. It consist of two palmitic acid residues esterified to first and second carbon atoms of glycerol and phosphocholine on third carbon. 2. In the lung it serve as surfactant. 3. It is involved in the maintenance of shape of alveoli of lungs. 4. It is synthesized only after 30 weeks of gestation. 5. Hence its deficiency occurs in premature infants and causes respiratory distress syndrome (RDS).

6. Write the composition and clinical importance of Cardiolipin. A. 1. Cardiolipin is a double phosphoglycerolipid. 2. Two phosphatidic acids are esterified to first and third carbons of glycerol. 3. It is structural component of inner mitochondrial membrane. 4. It shows immunological properties. 5. It is found in cardiac muscle hence the name. 6. It is useful in the diagnosis of syphilis.

7. What are lipoproteins ? Write their composition and general structure. A. 1. Lipoproteins are lipid and protein complexes present in plasma. 2. The protein part of lipoprotein is called as apolipoprotein or apoprotein. 3. Non covalent bonds keep lipid and apoprotein together. Composotion: 1. Triglycerides, free and esterified cholesterol and phospholipids are major lipids present in lipoproteins. 2. However proportions of these lipids in various classes of lipoproteins differs. 3. Composition of apoprotein differs among lipoproteins. 4. Also proportions of proteins in various classes of lipoproteins differs. 5. Five types of apoproteins are known so far. 6. They are apoprotein A, or apo A, apoB, apoC, apoD,

Apoprotein CORE

and apo E. ApoF, apoG and apo H are also found. 7. Some of them has subtypes also.

Lipids Lipoprotein

8. ApoB is largest of all.



Questions and Answers

Structure: 1. Lipoproteins have spherical to oval shaped structure. 2. Non polar hydrophobic lipids lies in the central core. 3. They are surrounded by more polar hydrophilic apolipo proteins and lipids. 4. The outer polar coat solubilizes inner non polar lipids in aqueous environment of plasma. 8. Write separation, classification and functions of lipoproteins. A. Separation and classification: 1. Lipoproteins are separated by ultracentrifugation and electrophoresis. 2. Ultra centrifugation separates lipoproteins based on their density. 3. Density of a lipoprotein is inversely related to lipid content. 4. So higher the lipid content of lipoprotein then lower its density. 5. Ultracentrifugation separates lipoproteins into 4 classes. 6. They are1. Chylomicrons. 2. Very low density lipoproteins (VLDL). 3. Low density lipoproteins (LDL) and 4. High density lipoproteins (HDL). 7. Electrophoretic separation of lipoproteins is based on differences in mobilities. 8. The plasma lipoprotein electrophoresis gives four bands which corresponds to chylomicrons, α-lipoprotein, preβ-lipoprotein and β-lipoprotein. LDL



Lipoprotein Electrophore sis

Chylomicrons b-Lipo Protein



Lipo Protein


Functions: 1. Chylomicrons are involved in the transport of dietary triglycerides from intestine to liver. 2. Very low density lipoproteins (VLDL)are involved in the transport of endogenous triglycerides from liver to peripheral tissues. 3. Low density lipoproteins (LDL) are involved in the transport of cholesterol from liver to peripheral tissues. 4. High density lipoproteins (HDL) are involved in the transport of cholesterol form peripheral tissues to liver.


CHAPTER - 4 | Lipids

5. Some apoproteins have functions other than structure. They act as activators or inhibitors of enzymes of lipid metabolism.

9. What are prostaglandins? Name them. Write their functions. A. Prostaglandins (PG) are derived from prostanoic acid. It is a cyclic compound with two side chains. The cyclic ring is cyclopentane ring. Many types of prostaglandins are found. They differ in substituent groups on cyclo pentane ring. Some known prostaglandins are PGA, PGB, PGC, PGD, PGE, PGF, PGG and PGH. COOH

Cyclo Pentane Ring

Sidechain 1 Sidechain 2

CH3 Prostanoic Acid

Functions: 1. Prostaglandins have several effects on cardiovascular system. a. They act on heart and increases cardiac output and myocardial contraction. b. They are involved in maintenance of arterial pressure and vascular tone. c. Some prostaglandins act as antihypertensive agents. They lowers blood pressure. 2. Prostaglandins act on central nervous system. They are involved in sedation and tranquilizing effect in cerebral cortex. 3. Prostaglandins influences excretory functions of kidneys. They facilitates elimination of sodium, potassium and chloride ions. They also influences urine volume. 4. Prostaglandins act on respiratory system. a. They dilates bronchi. b. They act as anti asthmatics. c. They relieve nasal congestion. 5. Prostaglandins act on digestive system. a. They decrease acid secretion in stomach. b. They are useful in peptic ulcer treatment. 6. Prostaglandins have actions on reproductive system. a. They cause contraction of uterine muscle. b. They are useful in inducing abortions. c. They have role in fertility. 7. Prostaglandins play role in metabolism. Through cAMP they mediate their action. cAMP level alteration affects lipid as well as carbohydrate metabolism. 8. Some prostaglandins are involved in inflammation. 9. Haematopoietic system also influenced by prostaglandins. a. They inhibit platelet aggregation.



Questions and Answers

b. Some promote clot formation. c. Some cause platelet aggregation. 10. Prostaglandins promotes tooth movement by increasing resorption.

10. Define micelles, mixed micelles and Liposome and lipid bilayer. Write the importance of each one. A. These liquid structures are generated by amphipathic molecules which contain both hydrophobic as well as hydrophilic parts. Micelles : Are formed when amphipathic molecules are present beyond critical concentration in aqueous medium. They are sphere shaped aggregates of amphipathic molecules. Bile salts form micelles which are required for lipid digestion. Mixed micelles: Are formed when micelles of one type of lipids combines with other lipids. In the intestine bile salt micelles combines with products of lipid digestion to form mixed micelles. Mixed micelle formation is essential for digestion and absorption of lipids. Liposome: Is formed when a lipid bilayer cyclizes i. e. two ends of lipid bilayer joins. They are used as carriers of drugs or genes in case of gene therapy. Lipid bilayer:Is formed when phospholipids are present in water and oil mixture. Cell membrane is a lipid bilayer. Other model questions are

11. Write briefly about triglycerides. 12. Write the importance of cholesterol. 13. What are glycerophospholipids? Give examples. Mention their functions. 14. Write a note on phospholipids. 15. Write the composition and function of sphingomyelin. 16. What are glycolipids? Give examples. 17. Write composition and function of gangliosides. 18. What are derived lipids? Give examples. 19. Classify fatty acids. Give examples for each class. 20. Write the functions of prostaglandins. 21. Essential fatty acids. 22. Eicosanoids


CHAPTER - 5 | Enzymes



Enzymes 1. Classify enzymes. Give examples for each class along with reaction and cofactors involved. A. Classification: Based on the type of reaction they catalyzes enzymes are classified into six major classes. All classes of enzymes with examples are given below. 1. Oxidoreductases: They oxidizes or reduces substrates using an hydrogen acceptor or donor. Glutamate dehydrogenase is an example which catalyzes below given reaction. Glutamate+ NAD+H2O → α-ketoglutarate +NADH+H+ + NH4. Succinate dehydrogenase that catalyzes below given reaction is another example. Succinate +FAD →Fumarate + FADH2. 2. Transferases: They transfer group between substrates. Transaminase catalyze transfer of amino group from one aminoacid to ketoacid as shown below. Alanine+ α-Ketoglutarate→ Pyruvate + Glutamate Glucokinase catalyses transfer of phosphate from ATP to glucose as shown Glucose +ATP→ Glucose-6-phosphate + ADP. 3. Hydrolases: These enzymes hydrolyzes glycosidic bond or ester bonds etc. Amylase catalyzes hydrolysis of glycosidic bonds of starch. Amylase Starch +H2O

Hydrolytic products.

Pepsin catalyzes hydrolysis of peptide bonds of proteins Pepsin Protein+H2O

Hydrolytic products.

4. Lyases:They catalyzes splitting of substrates by using mechanism other then hydrolysis and generates double bonds in products HMG- CoA lyase is an example.



Questions and Answers

HMG-CoA lyase HMG-CoA


Citrate lyase is another example. Citrate+ ATP+CoA→Oxaloacetate +Acetyl-CoA+ADP+Pi 5. Isomerases: They catalyzes formation of functional, optical and geometrical isomers. Phosphohexose isomerase inter converts functional isomers. Glucose-6- phosphate→Fructose -6-phosphate. Maleyl acetoacetate cis-trans isomerase catalyzes inter conversion of geometric isomers. Maleyl acetoacetate→ Fumaryl acetoacetate. 6. Ligases:These enzymes catalyzes formation of new compounds by linking two compounds using energy. Arginino succinate synthase is an example. Citrulline+ Aspartate+ ATP→ Argininosuccinate+ AMP+PPi Propionyl –CoA carboxylase is another example. Propionyl-CoA+CO2+ ATP →D-Methyl malonyl- CoA+ADP+Pi

2. Define enzymes. Write an enzymatic reaction and properties of enzymes. A. Enzymes are biological catalysts. They fasten the chemical reactions in living organisms. An enzyme catalyzed reaction consist of substrate, enzyme and product. Substrate is substance on which enzyme act. Substrate

Enzyme Product.

Enzyme properties: Enzymes are proteins and they are not consumed in the reaction. Enzymes are usually high molecular weight substance. Molecular weight of enzymes ranges form thousands to millions. Enzymes are able to cut big molecules to small molecules. Conversely enzymes form big molecules by joining small molecules. Enzymes are more efficient than man made catalysts and they have enoromous power of catalysis.

3. Explain how enzymes accelerate reactions in living organisms? A. 1. Enzymes accelerate reactions like that of catalyst because enzymes are catalysts. 2. The acceleration of reaction by catalyst is explained with transition state theory. 3. When enough energy is supplied reactant of a reaction is converted to product.


CHAPTER - 5 | Enzymes

4. It involves formation of transition state of reactant.

Transition State

5. Usually transition state is unstable so reactant get converted to stable product. 6. In presence of catalyst reactant


attains transition state much easily and requires less energy. 7. In

Non Enzyme catalyzed

Enzyme Catalyzed

Ground State

presence of enzymes transition state is attained very rapidly and requires

Uncatalyzed A C T I V A T I O N


Progress of Reaction

very less energy. 8. The amount of energy required by reactant to attain transition state is known as activation energy. 9. Thus enzymes accelerate reactions by lowering activation energy.

4. Write about nomenclature and EC number of enzymes. A. Enzyme name consist of two parts. The first part indicates substrate name the second part ends with “ase� and indicates type of reaction enzyme catalyzes. EC Numbre : It is a enzyme code number given to an enzyme. It has four digits. The first digit indicates major class, second digit indicates sub class, third digit refers to sub subclass and final digit indicates specific enzyme.

5. Define active site of enzyme. Write its characteristics. A. Active site: It is part of the enzyme that is needed for enzyme action or catalysis. Characteristics of active site: It has two parts. a. Catalytic site : Part of active site that brings about catalysis. b. Binding site : Part of active site that binds to substrate. Aminoacids that makes active site are far


away in the absence of substrate. In the presence of


substrate active site aminoacid that are apart comes


closely and orient in specific manner to form precise


active site. Active site is three dimentional and are


clefts within enzyme molecule. Serine, histidine,


Binding Site

Catalytic Site

aspartate, cysteine, glutamate etc usually make up active site. 6. Write about active site models of an enzyme. A. Two models are proposed for active site of enzyme.



Questions and Answers

1. Lock and key model: As the name implies shape of the active site and substrate are complementary like that of lock and key in this model. Complementary nature of active site and substrate shape allows formation of tight enzyme substrate complex to yield product and free enzyme. However this model fails to explain reversible enzyme catalyzed reactions due to rigid shape of active site.

Active site


Enzyme (E)


Substrate (S)

(ES) Complex



2. Induced fit model: In this model rigid nature of active site is avoided. Enzyme active site is flexible in this model. Further in the absence of substrate active site is not in proper form. Binding of substrate to enzyme induces conformational change in enzyme molecule. As a result precise active site forms to favour tight binding between enzyme and substrate and catalysis. Since enzyme is unstable in induced conformation it returns to native state in the absence of substrate. This model allows formation of enzyme product complex to favour the formation of substrate in the case of reversible enzyme catalyzed reactions.

Active site


Enzyme (E)


Substrate (S)

(ES) Complex



7. Explain influence of various factors on enzyme catalyzed reaction with suitable diagrams and examples. A. Enzyme catalyzed reactions are affected by many factors. They are 1. Substrate concentration. 2. Temparature 3. Hydrogen ion concentration. 4. Enzyme concentration. 5. Cofactors and inhibitors.


CHAPTER - 5 | Enzymes

1. Substrate concentration: Initial velocity (VO) of enzyme reaction increases proportionately in the beginning with increasing substrate concentration (S). Further increase in substrate concentration leads to slight increase in initial velocity and reaches maximum (Vmax). Beyond that increase in substrate concentration has no effect on velocity of enzyme reaction. The plot of (S) versus VO is a rectangular hyperbola. It is known as Michaleis plot.


Vmax 2 Vo O



Michaleis-Menton Equation:It is mathematical expression for Michalies plot relating substrate concentration, initial velocity and maximum velocity. Vmax (S) VO =

Where Km= Michaleis constant. Km+ (S)

From this equation Michaleis constant is obtained. From Michaleis plot substrate concentration that produces maximum velocity is difficult to obtain. But at least substrate concentration that produces half maximal velocity is possible to know. So by substituting this in Michaleis – Menton equation we get. Vmax

Vmax (S) =


Km+ (S)

On cross multiplication Km+2 (S)=S

i. e. Km= (S).

Michaleis constant: It is substrate concentration that produces half maximal velocity. Km significance: a. Measurment of enzyme activity requires knowledge of Km. It provides substrate concentration range for proper measurement of enzyme activity. b. Km indicates affinity of enzyme towards substrate. Km and affinity are inversely related. High Km indicates low affinity and low Km indicates high affinity. c. Km values of enzyme are needed for use as drugs and reagents.



Questions and Answers

2. Temperature: Enzymes work optimally at a particular temperature. Above or below that temperature enzyme exhibits low activity. Optimum Temperature: It is temperature at which enzymes are optimally active. For most of the enzymes. Optimum temperature is temperature of cell where it exist. Hence optimum temperature for most of the mammalian enzymes is 37â—Ś C. Enzyme activity increases as temperature is increased until optimum temperature is reached. Beyond that enzyme activity decreases with increasing temperature. Plot of enzyme activity versus temperature is bell shaped curve. Some of plant derived enzymes and enzymes of thermophilic bacteria have optimum temperature close to boiling point.














37 Temperature (°C)



7 PH


3. Hydrogen ion concentration: Like optimum temperature enzymes requires a particular PH for optimum activity. This is known as optimum PH. For most of the enzymes optimum PH ranges from 5-8 or PHof body or cell in which it occurs. However enzymes with alkaline optimumPH or acidic optimum PHare known. When PH and enzyme activity are plotted a bell shaped curve is obtained. 4. Enzyme concentration : The rate of product formation in an enzyme catalyzed reaction is proportional to concentration of enzyme. The plot of enzyme concentration and rate of product formation is straight line passing through origin. 5. i. Inhibitors: These substances if present in enzyme catalyzed reaction they inactivate enzyme. As a result rate of product formation may decrease or not occur. ii. Cofactors: Several enzymes can work only in presence of some non protein molecules. In the absence of these molecules enzyme catalysis may be slowed down or not take place. COMPETITIVE INHIBITION 1. It is a kind of reversible enzyme inhibition. 2. It occurs in presence of competitive inhibitor. 3. The competitive inhibitor is structurally similar to the substrate. Hence it competes with substrate to bind at active site.


CHAPTER - 5 | Enzymes

4. Binding of inhibitor at active site blocks formation of product. 5. By increasing substrate concentration this type of enzyme inhibition is masked. 6. In presence of competitive inhibitor Km of an enzyme increases i. e, affinity decreases. However Vmax is not altered. 7. The interaction of enzyme inhibitor and substrate is shown as equation below. E+S





E +P

I = Inhibitor


P = Product

Example: Classical example for competitive inhibition is inhibition of succinate dehydrogenase by Malonate which is structurally related to substrate succinate. Vmax

Substrate Only Inpresence of competitive inhibitor

Vmax 2 Vo O


Competitive Inhibitor Competitive Inhibition

Km Km (S)

COOH | Succinate CH2 Dehydrogenase Succinate | + FAD Fumarate + FADH2 COOH CH2 (-) | (-) Inhibition | CH2 COOH Competitive Malonate | Inhibtor COOH Applications Competitive inhibitors are used in medicine as 1) Antibiotics, 2). Anti cancer agents 3). Drugs for treating metabolic diseases. Antibiotics : Competitive inhibitors used as antibiotics to treat bacterial infections are mainly sulfonamides or sulfa drugs. Most of these drugs contains sulfanilamide an analogue of p- amino benzoic acid. For growth bacteria need vitamin folic acid. p- amino benzoic acid is required for formation of folic acid. Sulfonilamide competitively inhibit enzyme involved in synthesis of folic acid using p- amino benzoic acid. This results in block in folic acid formation. Lack of folic acid leads to arrest of bacterial growth.



Questions and Answers

Ăž-Amino benzoic Acid + Precursor

Block in folic Acid Formation

(-) Sulfonila Mide

Arrest of Bacterial Growth

Anti cancer agents: Several competitive inhibitors are used as anti cancer agents. Folic acid analogs are most notable among them. Rapidly growing cancer cells requires folicacid for nucleic acid formation. Dihydrofolate reductase is competitively inhibited by folic acid analogs like aminopterin and amethopterin. They are used in the treatment of blood cancer. Inhibition of dihydrofolate reductase results in block in folic acid formation. This in turn affect nucleic acid synthesis. Lack of nucleic acids leads to arrest of cancer growth.

Reductase Folic Acid (F)


Block in Nucleic Acid Formation

(-) Aminopterin

Arrest of Cancer Growth

Competitive inhibitors in treatment of metabolic diseases: Competitive inhibitors are used in the treatment of gout, atherosclerosis, hypertension etc. i) Gout is disease due to excessive production of uric acid. It is treated using allopurinol, a competitive inhibitor of enzyme xanthine oxidase involved in uric acid production. Inhibition of xanthine oxidase leads to decreased uric acid production.


xanthine oxidase (-) Allopurinol


x anthine oxidase (-) Allopurinol

Reduced Uric Acid Formation

Gout Cured

ii) Lovastation is competitive inhibitor of enzyme HMG - CoA reductase involved in cholesterol production. In atherosclerosis cholesterol is present in excess. When used lovastatin blocks cholesterol production. This leads to arrest of advancement of atherosclerosis.


CHAPTER - 5 | Enzymes

HMG-CoA Reductase HMG-CoA

(-) Lova Statin

Block in Mevalonate Formation

Reduced Cholesterol Formation

Arrest of Atherosclerosis Progress

iii) Captopril, lisinopril and enalapril are competitive inhibitors of angiotensin converting enzyme involved in blood pressure regulation. They are used in the treatment of hypertension.

High Blood Pressure or Hypertension

Angio tensin converting Enzyme

Normal Blood Pressure

(-) captopril, Lisinopril NON COMPETITIVE INHIBITION 1. It is another type of enzyme inhibition. 2. Most of the cases are irreversible enzyme inhibition. 3. Non competitive inhibitors are not structural analogs of substrates. 4. They bind enzyme at site other than active site. Hence no competition occurs between substrate and inhibitor to bind at active site. 5. Substrate can bind to enzyme inhibitor complex. Rate of formation of product from these complexes is affected. 6. So in non competitive inhibition Km remains same but Vmax is altered. 7. The interaction of enzyme, substrate, inhibitor is written as E+S









E+P (SLOW); I=Inhibitor, P=Product. E+P (SLOW).


Substrate Only

Vmax In presence of Non Competitive Inhibitor

Vmax 2 Vmax 2 Vo O



Non Competitive Inhibitor Km




Questions and Answers

Examples Several non competitive inhibitors irreversibly inactivate enzymes. So they are often known as


CHAPTER - 5 | Enzymes

transfer of one carbon units between substrates. Formimino group of formiminoglutamate is transferred to FH4as shown below. Formiminoglutamate+FH4

Glutamate + Formimino FH4.

Formimino group of formiminoFH4 is later transfered to other substrates. d. Coenzymes involved in transfer of groups: Methyl cobamide coenzyme of vit. B12 is involved in methyl group transfer. CoenzymeA coenzyme of pantothenic acid is involved in CoA transfer reactions. Methionine synthase transfers methyl group of methyl cobamide as shown below. Homocysteine +methylcobamide

Methionine + cobamide

Acyl-CoA synthatase catalyzes transfer of CoA to fatty acid as shown below. Fatty acid +CoA+ATP


e. Nucleotide coenzymes : Many nucleotides function as coenzymes. They are ATP, GTP, CTP, ADP, GDP, CDP, PAPS and SAM. Metals Enzymes in which metal is part of enzyme molecule are known as metalloenzymes and metal is attached through coordinate bond. More over metal takes part in catalysis. Removal of metal leads to loss of catalytic activity of enzymes. Cytochrome oxidase, catalase, succinate dehydrogenase are examples for iron metallo enzymes. Metal dependent enzymes Are those enzymes in which metal is not part of enzyme molecule but it is required for catalysis. It act as bridge between enzyme and substrate. In the absence of metal enzyme is unable to form enzyme substrate complex. Hexokinase, galactokinase and pyruvate kinase are dependent on magnesium for activity. Metal activated enzymes In presence of metals activity of these enzymes is increased to many folds. In the absence of metal they catalyze reaction but at low rate. Chloride is an activator of amylase and angiotensin converting enzyme. Calcium is an activator of trypsin.

8. Define isoenzymes. Give examples. How they are separated? A. Isoenzymes are multiple forms of an enzyme. They catalyze same reaction but differ in physicochemical properties. They may occur among organs, species. They are present in blood and other fluids. Isoenzymes of several dehydrogenases, transaminases and phosphatase are identified. Lactate dehydrogenase Isoenzymes Lactate dehydrogenase is a oligomeric enzyme. It is a tetramer. Made up of two types of sub



Questions and Answers

units. Isoenzymes of lactate dehydrogenase differ in quaternary structure or sub unit composition. Sub units present in lactate dehydrogenase are H and M type. Different isoenzymes of lactate dehydrogenase and their composition is given below. Name of isoenzyme

Sub unit composition


H4 o H H H H


H3M or H H H


H2 M2 or H H M M


HM3 or H MMM


M4 or MMMM

Separation of isoenzymes: Electrophoresis is used for separation of lactate dehydrogenase isoenzymes. When serum is subjected to electrophoresis at PH8. 6 the five isoenzymes of lactate dehydrogenase separates into 5 bands. The five isoenzymes bands corresponds to LDH1, LDH2 LDH3, LDH4, and LDH5. Alkaline phosphatase Isoenzymes: Iso enzymes of alkaline phosphatase are tissue or organ specific. Four organ specific isoenzymes are known. They can be separated on electrophoresis. The four organ specific isoenzymes are derived from bone, intestine, liver and placenta. These isoenzymes of alkaline phosphatase differ in composition. They are glycoproteins. The carbohydrate content of isoenzymes is different. Creatine phosphokinase Isoenzymes: Three isoenzymes exist for creatine phosphokinase. Creatine phosphpokinase is a oligomeric protein contain two sub units. Isoenzymes of creatine phosphokinase differ in quaternary structure. Sub unit composition varies among isoenzymes of creatine phosphokinase. Two types of sub units are found in isoenzymes. They an M and B. The subunit composition of three isoenzymes is given below.


Subunit Composition







9. Write about regulation enzyme activity by covalent modification. A. 1. By covalently attaching group to enzyme molecule its activity is regulated. 2. Phosphate and nucleotide are groups used to regulate enzyme by covalent attachment. 3. Serine residue of enzyme molecule is site of phosphorylation. Tyrosine residue of enzyme molecule is site of nucleotide attachment. 4. Phosphorylation is catalyzed by protein kinase and adenyl transferase catalyzes nucleotide attachment.


CHAPTER - 5 | Enzymes

5. Glycogen synthase and glutamine synthatase are two enzymes whose activity is regulated by attachment of phosphate and nucleotide respectively. Glycogen synthase (High active)+ATP Glutamine synthetase (More active)+ATP

Phosphorylated glycogen synthase (Less active)+ADP Glutamine synthatase-AMP (Less active)+PPi

10. Write an essay on clinically important enzymes. A. Estimation of enzymes in blood and other body fluids in normal and disease conditions is important for diagnosis and prognosis. Under normal conditions blood contains some enzymes. These enzyme are divided into a. Functional enzymes. b. Non functional enzymes. Functional enzymes: These enzymes are present in significant amounts in blood because they have physiological function. Non functional enzymes: These enzymes are present in blood only in small amounts under normal conditions. But concentration of these enzymes increases when organs are damaged due to disease or injury. The amount of enzyme present is proportional to extent of disease. Hence estimation of enzyme in blood is used to confirm diagnosis that is made by physical examination. Further estimation of enzymes in blood is also used to know effectiveness of treatment. There fore measurement of enzyme levels is both diagnostic as well as prognostic importance. In the case of secretory enzymes block in secretory route causes increase in levels of these enzymes. Apart from serum, cerebrospinal fluid (CSF), Synovial fluid, peritonial fluid and amniotic fluid are used for measurement of enzyme levels. Some routinely measured clinically important enzymes levels in normal condition and associated pathological states are given below. 1. Aminotransferases or Transaminases: Two important aminotransferases or transaminases are aspartate transaminase (AST) and alanine transaminase (ALT). The normal AST level in blood is about 3-20 units /litre (U/L). ALT normal level is 4-20U/L. These enzymes are also known as SGOT (serum glutamate oxaloacetate transaminase)and SGPT (Serum glutamate pyruvate transaminase). These two enzymes differ in distribution among tissues. Heart is rich in AST. However liver contains both of them in equal amounts. There fore in acute infective hepatitis both enzymes are elevated. The levels of these enzymes reaches peak value following infection and return to normal level in a week. AST level is increased in myocardial Infarction or heart attack and hence it is estimated in diseases specific to heart. ALT level rises in diseases of liver because ALT is more in liver only. Some of the liver diseases associated with raise in ALT level are alcoholic cirrhosis, biliary obstruction, cancer and toxic



Questions and Answers

hepatitis. In lung disease both transaminases in serum are elevated. Skeletal muscle is another organ that contain significant amount of ALT. Hence ALT level increases in diseases affecting skeletal muscle like muscular dystrophy and muscle injury. 2. Alkaline Phosphatase: Normal level of this enzyme in blood is 20 -90 U/L. Rickets, obstructive jaundice, hyper parathyroidism, bone cancer and cancer are some diseases associated with increased level of this enzyme in blood. Liver secretes this enzyme into bile. So block to flow of bile causes increase in blood level of this enzyme. Hence in obstructive jaundice level of enzyme increases by ten fold. In intestinal disorders, lung and kidney damage. Leukaemia, congestive heart failure and Hodgkins disease also the enzyme level is more. 3. Lactate Dehydrogenase (LDH) : Normal level of this enzyme is 70 to 90 U/L. Serum LDH level increases mainly in heart attack or myocardial infarction. The level of this enzyme in serum increases within 24 hrs of heart attack and reaches maximum level in 2 to 3 days and returns to normal in 7 days. Acute hepatitis, pernicious anaemia, megaloblastic anaemia, muscular dystrophy and blood cancer levels of this enzyme is more. 4. Creatine Phosphokinase (CPK) : Normal level of this enzyme is 12 to 60 U/L. Skeletal muscle contains more of this enzyme. Hence it is elevated in skeletal muscle diseases like muscular dystrophy, muscle injury and Polio myositis. Severe muscular exercise may raise plasma CPK level. It is also elevated in other than diseases of skeletal muscle like hypothyroidism, tetanus, etc. 5. Acid Phosphotase : This enzyme is concentrated in prostate gland. Normal level of this enzyme is 2. 5-12U/L. Its level is mainly elevated in prostate cancer. In bone diseases and breast cancer also level of this enzyme is increased. 6. Gamma glutamyltranspeptidase (GGT): Normal level of this enzyme in plasma is upto 30U/L. Like alkaline phosphatase this enzyme is secreted into bile. Hence in liver disease like obstructive jaundice, alcoholic cirrhosis level of this enzyme is increased. In brain lesions level of this enzyme is elevated. 7. Isocitrate dehydrogenase (ICDH): Apart from plasma this enzyme is found in cerebrospinal fluid (CSF) also. Hence measurement of this enzyme is useful in diseases affecting brain. In the plasma normal level of this enzyme is upto 5U/L. In inflammatory conditions level of this enzyme is elevated. In acute infective hepatitis and toxic hepatitis level of this enzyme is increased. However in obstructive jaundice level of this enzyme is more elevated than in brain tumors. 8. Amylase: This is a secretory enzyme. It is secreted by pancreas and parotid gland. Normal level of this enzyme in plasma is 800- 1800U/L. It is mainly elevated when there is block in its secretory route. So in acute pancreatitis and parotitis level of this


CHAPTER - 5 | Enzymes

enzyme is more. Further in intestinal obstruction and mumps also the enzyme level is increased in plasma. 9. Lipase:It is secreted by pancreas along with amylase. The level of this enzyme in plasma is about 150U/L. It is mainly increased in diseases affecting pancreas like acute pancreatitis and cancer of the pancreas. In other abdominal diseases also level of this enzyme is elevated. They are abdominal lesions, peritonitis, intestinal obstruction, perforated peptic ulcer etc. ,

11. Write a note on clinically important isoenzymes. A. High plasma enzyme level may not indicate severity of disease and organ involved because plasma enzyme is derived from several tissues. But iso enzyme level indicates organ involved in disease because isoenzymes are organ specific. Further like enzymes distribution of isoenzymes among organs varies. If an organ is diseased more isoenzyme of that organ enters plasma. Estimation of that isoenzyme level in plasma is used to confirm organ affected. Thus isoenzyme estimation is useful in differential diagnosis. 1. Isoenzymes of Lactate Dehydrohydrogenase or LDH isoenzymes; The five isoenzymes of LDH differ in their distribution. Each isoenzyme has unique source. The proportion of isoenzymes in serum is also different. Heart is rich in LDH1 so LDH1 in serum is mostly derived from heart. Likewise LDH5 in serum is derived from skeletal muscle because it is rich in LDH5. Liver contains LDH2 to LDH5in different proportions. LDH1 level in serum increases when heart muscle is damaged as occurs in myocardial infarction. Hence measurement of LDH1 isoenzyme in serum is more sensitive index of myocardial damage than total LDH activity. Like wise LDH5 is more sensitive index of skeletal muscle damage. 2. Isoenzymes of creatine phosphokinase or CPK isoenzymes: Plasma CPK activity is contribution of three isoenzymes i. e. CPK1, CPK2, CPK3. The CPK2 isoenzyme accounts for about 2% of total CPK in normal people. But it increase by ten times (20%) with in few hours of myocardial infarction. There fore CPK 2 estimation serve as better index of heart attack. 3. Isoenzymes of alkaline phosphatase: Plasma alkaline phosphatase activity is due to four of its isoenzymes. The four isoenzymes of alkaline phosphatase are organ specific. They are derived from bone, liver, placenta and intestine. Alkaline phosphatase isoenzymes measurement is useful in differential diagnosis. In metastatic carcinoma liver lesions are differentiated from bone lesions by measuring alkaline phosphatase isoenzymes.

12. Write a note on allosteric enzymes.



Questions and Answers

A. 1. Allosteric enzymes consist of many sub units. 2. Their activity increases in presence of activator and decreases in presence of inhibitors. 3. Activators and inhibitors binds at allosteric other than substrate binding site. 4. Further allosteric inhibitors are not structurally related to substrates of allosteric enzymes. Kinetics 1. Allosteric enzymes exhibit kinetics different from classical Michaelis- Menton kinetics. 2. A sigmoidal shape curve is obtained when substrate concentration and initial velocity are plotted instead of rectangular hyperbola. 3. Further the curve shifts to right in presence of allosteric inhibitor and to left in presence of allosteric activator. 4. The sigmoidal curve also indicates a rapid change in initial velocity in presence of substrate alone, allosteric inhibitor and allosteric activator. Example

Allosteric Activator Substrate only Allosteric Inhibitor V



1. Aspartate carbamoyltransferase (ACT) is classical example for allosteric enzymes. 2. It catalyzes formation of carbamoyl aspartate from carbamoyl phosphate and aspartate as shown below. Carbamoyl phosphate + aspartate

carbamoyl aspartate +phosphate

3. CTP is allosteric inhibitor and ATP is allosteric activator. 4. ATP converts less active ACTase to high active form. 5. In contrast CTP converts high active form to less active form. ATP and CTP bind at separate sites other than substrate binding sites.

13. Write a note on pro enzymes.


CHAPTER - 5 | Enzymes

A. 1. Pro enzymes are inactive precursor forms of enzymes. 2. They are also known as zymogens. 3. Limited proteolysis removes few amino acid residues or a portion of pro enzyme molecule which results in conversion of pro enzyme to active enzyme. 4. Enzymes of protein digestion and blood clotting factors are synthesized in inactive proenzyme forms. 5. They are converted to active form when physiological need arises. 6. At acidic PH pepsinogen is converted to pepsin in stomach. It involves cleavage of peptide bonds. Pepsinogen → Pepsin 7. Pencreatic proteases are produced in proenzymes form. 8. Enterokinase initiates conversion of these proenzymes to enzyme. It converts trypsinogen to trypsin initially. 9. The remaining pancreatic proenzymes are converted to enzymes by trypsin. It converts chymotrypsinogen, proelastase, pro phospholipase, and procarboxypeptidase to chymotrypsin, elastase, phospholipase and carboxypeptidase respectively. 10. In presence of factor X and V prothrombin is converted to thrombin which in turn converts fibrinogen to fibrin during blood clotting. Enterokinase


Trypsinogen → Trypsin Chymotrypsinogen → Chymotrypsin Proelastase → Elastase

Prothrombin → Thrombin

Pepsingen → Pepsin pH 1.5

14. Explain enzyme induction and repression with examples. A. Induction:In presence of an inducer synthesis of inducible enzymes is more. It is known as induction. Example: Usually in E. coli lactase is produced in small amounts. If E. coli is grown in lactose containing medium synthesis of lactase increases. So lactose acting as inducer increases synthesis of lactase which is an inducible enzyme. Repression: In presence of repressor synthesis of enzymes required for repressor formation is blocked. This is known as repression. Example: When histidine is present in S-typhi medium synthesis of enzymes involved in formation histidine is blocked. So histidine acting as repressor blocks its own synthesis.

15. Explain co operativity phenomenon.




Questions and Answers

1. Cooperativity phenomenon is proposed to explain this rapid change in velocity of allosteric enzyme. 2. According to this the allosteric enzyme exist in two forms a 'T' tensed less active state and 'R' relaxed high active state. 3. Binding of substrate to 'T' form is slow and causes conformational change to 'R' form. 4. Further binding of substrate to 'R' form is rapid. 5. Allosteric inhibitor stabilizes enzyme in T form where as allosteric activator stabilizes enzyme in R form.

Other model questions are

16. Define cofactors. Classify. Give examples. Write reaction for each example. 17. Explain competitive inhibition with examples. 18. Describe enzyme inhibition. 19. How competitive inhibitors are useful in clinical medicine? 20. Write a note on influence of substrate concentration on enzymatic reaction. 21. Define Km. Write its importance. 22. Explain effect of the following on enzymatic reaction A. Hydrogen ion concentration. B. Temperature. 23. Write about competitive inhibitors used as chemotherapeutic agents. 24. In what diseases following enzymes level in blood is more A. SGOT B. Alkaline phosphatase 25. Write about enzyme poisons. 26. Write an essay on clinical enzymology. 27. Write a note on non competitive inhibition.


CHAPTER - 5 | Enzymes

28. Define proenzyme, isoenzyme and coenzyme. Give examples for each. 29. Write the effect of substrate concentration on enzyme reaction. 30. What is meant by feedback inhibition. Give examples. 31. Write about cardiac enzymes and isoenzymes. 32. Define metalloenzymes, metal dependent enzymes and metal activated enzymes. Give examples for each. 33. Define functional enzymes and non functional enzymes. Give examples. 34. Write briefly about LDH isoenzymes. 35. Write the diagnostic importance of serum transaminases.


CHAPTER - 6 | Nucleotides and Nucleic Acids



Nucleotides and Nucleic Acids NUCLEOTIDES

1. Define nucleoside. Give examples. A. Nucleoside: It consist of nitrogenous base and sugar. Nucleosides are classified based on nitrogenous base present. They are purine nucleosides and pyrimidine nucleosides. Purine nucleoside: They contain purine bases adenine and guanine. Adenosine is nucleoside of adenine and guanosine is the nucleoside of guanine. Adenosine






Pyrimidine nucleosides : These nucleosides are composed of pyrimidine bases. Cytosine, Uracil and thymine are pyrimidine nitrogenous bases. Cytidine is nucleoside of cytosine. Uridine and thymidine are nucleosides of uracil and thymine respectively. Cytidine Thymidine

Cytosine Thymine






2. Define nucleotide. Give examples. A. Nucleotides: They are phosphorylated nucleosides. A nucleotide consist of nitrogenous base, sugar and phosphate. Nucleotide

Purine or Pyrimidine base



Purine nucleotides:In these nucleotides nitrogenous base is purine. They are adenosine monophosphate (AMP), adenosine diphosphate (ADP)and ATP. Like wise guanosine monophosphate (GMP), guanosine di phosphate (GDP) and GTP. Adenine




Pyrimidine nucleotides: These nucleotides contain pyrimidine nitrogenous bases like thymine, cytosine and uracil. They are cytidine monophosphate (CMP), cytidine di phosphate (CDP) and CTP. Likewise TMP, TDP, TTP;UMP, UDP, UTP. Cytosine





CHAPTER - 6 | Nucleotides and Nucleic Acids

3. Write functions of nucleosides and nucleotides. A. Nucleotides and nucleosides have several important biological functions. 1. Nucleotides are involved in signal traduction. 2. Nucleotides are required for the formation of nucleic acids. 3. Nucleotides are high energy compounds. 4. Nucleotides are components of some water soluble vitamin coenzymes. 5. Nucleotides serve as second messengers. Many hormones mediate their action through second messengers. 6. Some nucleotides function as donors of sugars, nitrogenous compounds and phosphates. 7. Nucleosides function as carriers or donors of groups. 8. Nucleoside analogs are used as anti cancer agents. 9. Some nucleotides function as alarmones. They alaram cell when something goes wrong in the cell.

4. What are unusal nucleosides? Give examples. A. Unusual nucleosides: Are those nucleosides in which nitrogenous base and sugar are unusal. Ribothymidine and pseudouridine are examples of unusual nucleosides. Ribothymidine consist of thymine and ribose. It is present in ribonucleic acids (RNA) which is not usually found. Pseudouridine is a unusual nucleoside of uracil. In this nucleoside carbon –carbon bonding occurs between uracil and ribose instead of usually carbon –nitrogen bond.

5. Write about Synthetic analogs of purines, pyrimidines and nucleosides A. Some synthetic purine and pyrimidine analogs are used as anticancer agents and antiviral agents. Purine analogs are mercaptopurine, thioguanine, aminopurine etc. Pyrimidine analog is 5- fluro uracil. Nucleoside analogs are used as anticancer agents, antiviral agents and mutagens. Deazauridine, 6-aza uridine, ara-A, ara-C and fluro deoxyuridine are nucleoside analogs used as anticancer agents. Azidothymidine (AZT), dideoxy cytidine and iododeoxyuridine are used as anti viral agents. Bromodeoxy uridine is used as mutagen.

6. W rite about Pharmacologically important purines A. Caffeine of coffee, theophylline of tea and theobromine of tea are some purines of pharmacological importance. Caffeine and theophylline act as CNS stimulants. Inhalers used by asthma patients contains theophylline. It releives nasal and bronchial congestion of these patients.



Questions and Answers

7. Describe nucleotides of biochemical (Physiological) importance A. Cells present in various organs of human body and other mammals contain several free nucleotides. These free nucleotides are involved in many biochemical or biological process that are given below. i. Adenine nucleotides and their physiological importance 1. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) are most important adenine nucleotides. 2. ATP, ADP, and AMP are high energy compounds. 3. ATP is popularly called as 'energy currency' of cell. Energy exchange in biochemical reactions occurs through ATP. 4. ADP is required for the formation of ATP in electron transport chain and in energy yielding reactions. 5. cAMP, a cyclic nucleotide of adenine is known as second messenger. Many hormones action occurs through cAMP. 6. Many coenzymes of water soluble vitamins contain adenine nucleotides. For example NAD+, FAD, NADP, coenzyme A and cobamide coenzymes. 7. PAPS (Phospho adenosine phosphosulfate) serve as donor of sulfate in biosynthetic reactions. 8. ATP is required for replication and protein biosynthesis. 9. Some adenosine nucleotides are involved in blood pressure and platelet function. 10. Diadenosine nucleotides are neurotransmitters. 11. Oligoadenylate mediates action of interferon. 12. Poly adenylate serve as tail of mRNA ii. Guanine nucleotides and their physiological importance 1. Like ATP, ADP; Guanosine triphosphate (GTP) and guanosine diphosphate (GDP) also exist in cells. 2. GTP and GDP are high energy compounds. 3. Cyclic GMP or cGMP mediates actions of several hormones. 4. GTP and GDP are components of G-proteins which are involved in signal transduction of several physiological processes like taste, odor, vision, metabolic regulation etc. 5. GTP is required for replication and protein biosynthesis. 6. Guanine nucleotides are required for catalytic function of ribonucleic acids or ribozymes. 7. Mucopolysacharide formation requires guanine nucleotides.


CHAPTER - 6 | Nucleotides and Nucleic Acids

iii. Cytosine nucleotides of physiological importance 1. CTP (Cytidine triphosphate), CDP (Cytidine diphosphate) and CMP (Cytidine monophsphate), are high energy compounds. 2. Cyclic CMP or cCMP also occurs in cells. 3. CDP, CMP serve as donor of nitrogenous compounds during biosynthesis. 4. CMP-NANA serve as donor of NANA in the biosynthesis of gangliosides. 5. CDP- choline serve as donor of choline in phospholipids biosynthesis. iv. Uracil nucleotides of physiological importance 1. UTP (Uridine triphosphate), UDP (Uridine diphosphate) and UMP (Uridine monophosphate) are high energy compounds. 2. UDP- Glucuronic acid serve as donor of glucuronic acid in the synthesis of mucopolysacharides, bilirubin diglucuronide and detoxification reactions. 3. UDP is carner of sugar and aminosugars needed for synthesis of glycogen, gangliosides, glycoproteins etc. v. Thymine nucleotides of physiological importance 1. TTP (thymidine triphosphate), TDP (thymidine diphosphate) and TMP (thymidine monophosphate) are high energy compounds. 2. TTP and d TTP are used for the synthesis of nucleic acids. vi. Hypoxanthine and xanthine nucleotides of physiological importance Hypoxanthine and xanthine are two purine bases not found in nucleic acids. But their nucleotides have important role in metabolism. 1. I DP (inosine diphosphate), IMP (inosine monophosphate) are nucleotides of hypoxanthine. They are high energy compounds. 2. IMP is intermediate in purine nucleotide biosynthesis. 3. XMP (xanthosine monophosphate) is an intermediate in purine nucleotide biosynthesis.

8. What are nucleic acids? Classify. Write their composition. A. Nucleic acids are acidic substances present in nucleus. Two types of nucleic acids are found in cells. They are deoxy ribonucleic acid (DNA) and ribonucleic acid (RNA). Pentose sugar in DNA is deoxy ribose where as in RNA it is ribose. Both DNA and RNA are polymers of nucleotides and often referred as polynucleotides. Due to deoxy ribose nucleotides present in DNA are known as deoxy ribonucleotides. They are designated as dADP, dATP; dGDP;dGTP, dTTP, dTDP, dCTP, dCDP etc. RNA contains ribonucleotides they are designated as ATP, ADP;GTP, GDP;CTP, CDP;UTP, UDP etc.



Questions and Answers


9. Describe structure and function of DNA. A. DNA structure 1. It consist of two poly nucleotide chains. 2. These polynucleotide chains coil along long axis in the form double helix. 3. Each polynucleotide is made up of four types of nucleotides. 4. Individual nucleotides are joined by phosphodiester bonds. 5. Four types of nucleotides are present in two chains. They are adenylicacid, guanylic acid, cytidylic acid and thymidylic acid. 6. Each poly nucleotide chain or strand has direction or polarity and 5'and3'ends. These ends may be in either free form or phosphorylated form. 7. Sugar and phosphate forms back bone of two strands. 8. The two strands are complementary to each other. 9. Base composition of a strand is complementary to opposite strand. If thymine is found in one strand adenine appears in opposite strand and vice versa. Like wise if guanine appears in one strand cytosine is found in opposite strand and vice versa. 10. Further bases of opposite strands are involved in pairing. It is popularly known as base pairing rule. Adenine of one strand pairs with thymine of opposite strand through two hydrogen bonds. Guanine of a strand pairs with cytosine of opposite strand through three hydrogen bonds. 11. Due to the presence of three hydrogen bonds GC pair is stronger than AT pair. 12. This base pairing makes copying mechanism simple and easier. 13. Complementary nature of two strands and base pairing rule are most outstanding features of Watson-Crick model. 51


Hydrogen Bonds A : Adenine


G : Guanine

C : Cytosine T : Thymine

C====G 31


14. The base pairs are stacked. The pitch of the helix is 34 Aâ—Śand contain ten base pairs. The width of the helix is 20 Aâ—Ś.


CHAPTER - 6 | Nucleotides and Nucleic Acids

15. Due to the presence of hydrogen bonds through out molecule DNA is highly stable. 16. Major and minor grooves are present on the double helix. 17. Watson- Crick model DNA is known as B-DNA. Functions 1. DNA is genetic material of living organisms. It contains all the information needed for the development of entire organism or individual. 2. DNA is transferred from parent to offspring or generation to generation. 3. DNA contains information required for formation of individuals proteins. 4. Information is present in DNA in the form of genes. 5. Amount of DNA present in the cell of an organism depends on complexity of organisms. 6. Human cells contain more DNA than bacterial cells or viruses. 7. DNA amount in given cell is independent of nutritional or metabolic state of the organism. 8. DNA flows from generation to generation in any given species. 9. DNA determines physical fitness of an organism or susceptibility to disease.

10. How many types of RNA are there? Name them. Write structure and function of each one. A. There are three types of RNAs in cells. They are present in prokaryotes as well as eukaryotes. They are 1) Messenger RNA or mRNA 2) Transfer RNA or tRNA 3) Ribosomal RNA or rRNA. Messenger RNA 1. Majority of mRNA molecules are linear polymers. 2. They contain about 1000-10, 000 nucleotides. 3. They have 3' or 5' free or phosphorylated ends 4. Life span of m RNA molecules varies from few Hair pin Loop Poly A Tail

minutes to days. 5. Some RNAs have secondary


structure. 6. Intra strand base pairing





among complementary bases leads to folding of linear molecules into hair pin like secondary structure. 063


Questions and Answers

7. In some m RNA at 5'and 3' ends special nucleotides or sequences occurs. 8. Poly A tail is present in some mRNAs at 3' end. 9. At 5' end some mRNA are capped. Methylated GTP is cap. 10. At 5' and an AG rich shine – Dalgarno sequence is present in some mRANs. Functions: 1. mRNA carries genetic information from nucleus to cytoplasm. 2. Generally one mRNA contains information for formation one protein. 3. The sequence of mRNA is complementary to strand from which it is copied. 4. In mRNA genetic information is present in the form of genetic code. 5. Occassionaly one mRNA contains information for the formation of more than one protein. Transfer RNA (tRNA) It is smallest of all RNAs and contains up to 1000 nucleotides. It contains several unusual bases like pseudouridine, dihydro uracil, mythylated adenine and guanine and isopentenyl adenine etc. Due to intra strand base pairing between complementary bases tRNA molecules exist in characteristic secondary structure shape. Secondary structure of tRNAs is in the form of clover leaf. Structural features of clover leaf 1. It contains an aminoacid arm at 3' end CCA is characteristic sequence of aminoacid arm. 2. An arm containing unusual pseudouridine and ribothymidine. Hence it is known as TφC arm in which pseudouridine is indicated with psi (φ) symbol. 3. An anticodon arm containing IGC sequence. Generally this arm recognizes codon on mRNA. 4. Dihydrouridine (UH2) arm or DHU arm that contains dihydrouracil. 5. A guanine containing 5'end. 6. An extra arm in some tRNAs exist between TφC arm and anti codon arm.

A31 C 51





φ C

Extra Arm

Anticodon Arm



φ C Arm

CHAPTER - 6 | Nucleotides and Nucleic Acids

Functions 1. It serve as adaptor molecule in protein biosynthesis. It carries aminoacids to site of protein synthesis. 2. For every aminoacid one specific tRNA molecule exist. 3. Stability of eukaryotic and prokaryotic tRNA varies. Ribosomal RNA (rRNA) It is found in combination with proteins in ribosomes. It contains about 100-600 nucleotides. Prokaryotic and eukaryotic ribosomes contain several RNA that differ in sedimentation coefficient. Due to intra strand base pairing between complementary bases secondary structures are found in rRNA molecules. They are known as domains. 16S rRNA with 1500 nucleotides has four major domains. Functions 1. It is involved in initiation of protein synthesis. 2. It is required for the formation of ribosomes.

11. Write very briefly about organization of eukaryotic DNA. A. In the nucleus of eukaryotes DNA is present as nucleo protein chromatin which is combination of DNA and basic proteins histones. In eukaryotes chromatin is present as chromosomes. Each eukaryotic cell contains 23 pairs of chromosomes and one DNA molecule is present in each chromosome. Chromatin has beaded structure. Beads

Linker DNA



Bead is a nucleosome in which DNA is coiled around basic protein histone octamer. The nucleosomes are joined by linker DNA as well as histones.

12. Explain the following A. DNA denaturation B. Mitochondrial DNA A. DNA denaturation Exposure to heat leads to separation of strands. On cooling strands coils. Formation of DNA molecules due to base pairing between two strands is known as annealing. It is very useful in genetic engineering particularly in techniques based on hybridization.



Questions and Answers


Cool Strands




B. Mitochondrial DNA It is DNA present in mitochondria of eukaryotic cell. It is different from DNA present in nucleus. Mitochondrial DNA base composition differs from nuclear DNA. Usually mitochondrial DNA is circular double stranded molecule.

13. Write differences between DNA and RNA A. Differences between DNA and RNA DNA


1. Double stranded molecule.

1. Single stranded molecule

2. Found in combination with proteins.

2. Except rRNA other RNAs exist as free molecules

3. Pentose sugar is deoxyribose.

3. Pentose sugar is ribose.

4. Sum of the purine bases is equal to

4. Some of purine bases is not equal to sum of

sum of pyrimidine bases.

pyrimidine bases.


A+G ≠C+T

5. Pyrimidine base uracil is absent.

5. Thymine a pyrimidine base is not usually found.

6. Only one form of DNA predominantly

6. More than three types of RNAs occurs.

occurs. 7. Resistant to alkaline hydrolysis.

7. Easily hydrolyzed by alkali.

8. Modified bases are usually absent.

8. Unusual and modified bases are found.

9. Lacks catalytic activity.

9. Some RNAs act as enzymes or posses catalytic activity.

14. Define ribosome. Classify them. Write their composition. Draw diagrams. A. They are complexes of proteins and nucleic acids. They are large molecules compared to proteins and nucleic acids. They are classified based on sedimentation coefficients (S). Even ribonucleic acid components of ribosomes are identified based on sedimentation coefficients. . There are two types of ribosomes. They are70S ribosome of prokaryote and 80S ribosome of eukaryotes. 066

CHAPTER - 6 | Nucleotides and Nucleic Acids


34 Proteins + 5S and 23S RNA


21 Proteins + 16 S RNA


The 70S ribosome contains a large 50S sub unit and small 30S subunit. The 50S sub unit consist of 34 proteins and two RNAs or 23S and 5S RNA. The 30S subunit contains 21 proteins and one 16S RNA. The 80S ribosome contains a large 60S subunit and small 40S subunit.

15. Define a plasmid. Give example. How they are useful? A. Plasmids are circular DNA molecules present in antibiotic resistance bacteria. pBR322 of an intestinal bacteria E. Coli is an example.


Plasmids contain genes for inactivation of antibiotics. They are used as vectors in genetic engineering. Other model questions are

16. Name nucleoside and nucleotides of adenine and cytosine. Write their importance. 17. Define nucleoside. List purine and pyrimidine nucleosides. 18. Give examples for cyclic nucleotides. Write their importance. 19. Write a note on tRNA. 20. List purine nucleotides and their functions. 21. Give examples for pyrimidine nucleotides. Mention their functions. 22. Write a note on mRNA. 23. Write about synthetic purine and pyrimidine analogs of clinical importance. 24. Define chromatin and nucleosome. Explain how they are related? 25. Write the functions of nucleic acids. 26. Purine and pyrimidine bases. 067

CHAPTER - 7 | Biological Oxidation



Biological Oxidation 1. Define biological oxidation. Write its importance. Explain the role of oxygen in this process. A. Biological oxidation is related to utilization of respiratory oxygen (O2) in living organisms. Biological oxidation is a major way of regenerating coenzymes which are reduced in metabolic pathways. It is final aspect of all energy yielding compounds in the body Oxygen act as final electron acceptor in the respiratory chain and get reduced to water. Oxygen is directly associated with biological oxidation reactions in the body. Formation of several new compounds and removal of toxins are dependent oxygen. . Oxygen is a source for the formation of reactive oxygen species (ROS) and super oxide etc. However oxygen is extremely toxic to cells at high concentration. This property of oxygen is exploited in cancer therapy by combining with radiation.

2. What are high energy compounds? Give examples. Mention their other roles. A. High energy compounds are those compounds that yield large amount of free energy on hydrolysis. The energy yield is expressed as standard free energy change. ∆G01is the symbol used for standard free energy change. Usually compounds that yield more than 7. 3 kcal / mol of energy are considered as high energy compounds. Nucleoside mono, di and triphosphates, phosphocreatine, thiol esters, enol phosphates and acylphosphates are few such high energy compounds. 1. Nucleoside phosphates :ATP, ADP and AMP are adenine based nucleoside phosphates. ATP is involved in the energy transfer in living systems. It is known as energy currency of cell. Energy released in exergonic reactions is used to form ATP and in endergonic reactions energy released on ATP hydrolysis is consumed. Exergonic ADP + Pi

ATP Reaction

Endergonic ATP ADP+Pi reaction 068

CHAPTER - 7 | Biological Oxidation

So ATP is link between energy yielding and energy consuming reactions. In addition energy released on ATP hydrolysis is used for muscle contraction, cell motility, transport of ions across membrane etc. GTP, GDP, CTP, CDP, TTP, TDP ; UTP and UDP are nucleoside di and triphosphates that serve as high energy compounds in living systems. 2. Acyl phosphates : They are formed from two types of acids. For example 1, 3–bis phosphoglycerate is the combination of glyceric acid and phosphoric acid. So it is mixed anhydride. On hydrolysis it yields about 12 kcal /mol energy and 3- phosphoglycerate is product. OH O |


P -O-CH2 –CH—C - O - (P)

P - Phosphate

1, 3-Bis phosphoglycerate 3. Enol phosphates: They are esters of enols with phosphoric acid. Phospho enol pyruvate (PEP) is an example for enol phosphate. On hydrolysis it yields about 15 kcal / mol energy and pyruvate is product. O– P | CH2= C – COOH PEP 4. Thio esters: They are esters of thiol with acid. Acetyl –CoA is an example for thiol ester. On hydrolysis 7. 5 kcal /mol energy is released and acetic acid is product. O || CH3 – C ~ S-CoA 5. Phosphocreatine: It is a guanidinium group containing high energy compound present in skeletal muscle. On hydrolysis it yields 10 kcal / mol energy and creatinine is product. NH H


P – N – C – N – CH2 – COOH | CH3 Creatine phosphate



Questions and Answers

3. Write a note on cytochromes of electron transport chain. A. They are heme proteins persent in electron transport chain. However cytochromes also exist outside of electron transport chain. Cytochromes of electron transport chain are cyt b, cyt c1, and cyt c. Cytochromes b and c, are integral membrane proteins where as cyt c is peripheral membrane protein. Cyt b and cyt c1 are components cytochrome reductase of electron transport chain. During electron transfer in respiratory chain iron of cytochromes undergo oxidation reduction s. The iron oscillates between Fe2+and Fe3+ states Cytochrome oxidase It is final component of electron transport chain. It consist of two cytochromes. They are cytochrome a or Cyt a and Cyto chrome a3 or Cyt a3. Each cytochrome contain two metal ions. They are iron and copper. These metal ions participates in oxidation and reduction reactions. Iron oscillates between Fe2+ and Fe3+ and copper oscillates between Cu+and Cu2+. Cu+ is is reduced form and Cu2+ is oxidized form. In the respiratory chain cytochrome oxidase catalyzes transfer of electrons from Cyt c to molecular oxygen.

4. Write very briefly about cytochrome P450 and ubiquinone A. Cytochrome P450 is cytochrome outside electron transport chain. Cyt P450 is also a heme protein. It is component of mixed function oxidases or mono oxygenases or hydroxylases. Cyt P450 directly interacts with oxygen. Iron of Cyt P450 participates in oxidation and reduction reactions. Two types of Cyt P450 dependent hydroxylases are identified. They are 1. Microsomal Cyt P450 hydroxylase. 2. Mitochondrial Cyt P450 hydroxylase. Ubiquinone It is also known as coenzyme Q or CoQ. It is non protein component of electron transport chain. It is mobile carrier of electron transport chain. It collects hydrogens or electrons from NADH and FADH 2 and transfers to cytochromes. It participates in oxidation and reductions reactions via semiquinone

5. Write about Iron sulfur proteins. A. They are proteins containing iron sulfur clusters. They are present in respiratory chain. Some occur outside respiratory chain also. In these proteins iron is complexed with organic as well as inorganic sulfur. Cysteine residues of proteins contributes organic sulfur. The iron of these proteins is referred as non heme iron (NHI). Iron and sulfur are involved in oxidation reduction reactions. Iron oscillates between Fe2+ (ferrous) and Fe3+ (ferric) state. Ferrous is reduced state and ferric is oxidized state of iron. In electron transport chain or respiratory chain iron sulfur (Fe:S) proteins transfer electrons from NADH –CoQ reductase and succinate- CoQ reductase to ubiquinone.


CHAPTER - 7 | Biological Oxidation

6. Define redox reactions and redox potantial. Write the symbol for redox potential. What is its importance in respiratory chain? A. Redox reactions: Oxidation and reduction reactions are known as redox reactions. The oxidant and reductant of redox reactions are known as redox pair. Redox potential:Electromotive force (e. m. f) of a redox pair is known as redox potential. It is indicated with symbol Eo1. Redox potential of a redox pair indicates ability of redox pair to either gain or loose electrons. Further redox potential has critical role in arrangement of components of respiratory chain. Location of specific component of respiratory chain depends on its redox potential. Starting components of respiratory chain have negative redox potential where as terminal components of respiratory chain have positive redox potential. Further ATP synthesis in respiratory chain also depends on redox potential difference between redox pair of respiratory chain. Approximately 0. 15volts potential difference is required for one ATP formation.

7. Describe Respiratory chain (RC) or Electron transport Chain (ETC) A. Electron transport chain consist of several components. These components are involved in electron transfer and they are arranged in sequence. They carry electrons from NADH to final electron acceptor oxygen. Most of the components of electron transport chain are proteins. However some non protein components also act as carriers of electrons. In the respiratory chain electrons flow from NADH to Co Q and then to cytochromes. From there they move to molecular oxygen. From FAD electrons flow to CoQ. The position of particular component in the respiratory chain is determined by redox potential of that component. Usually initial components of have negative redox potential and terminal components have positive redox potential. Hence due to redox potential difference electrons flows from negative to positive components in the respiratory chain. Electrons from substrate like malate and glutamate flow to NAD. This electron transfer is catalyzed by NAD linked dehydrogenases. However electrons from substrates like pyruvate and α-ketoglutarate flow to NAD via FAD. Coenzyme Q collects electrons from FAD linked dehydrogenases like acyl –CoA dehydrogenases, succinate dehydrogenase etc. NAD



Cyt b


Cyt c

Cyt a

Cyt a3


Recent research indicated presence of complexes in electron transport chain rather than individual components. Respiratory chain consist of four complexes and three mobile carriers. They are complexes I to V and mobile carriers are NAD, CoQ, cyt c and molecular oxygen. Complex I. is NADH- dehydrogenase or NADH –CoQ reductase. This complex also contains iron sulfur centers and FMN. So electrons collected by NAD from substrates is transferred to CoQ by this complex I through FMN and iron sulfur centre. This results in 071


Questions and Answers

oxidation of NAD. Complex II is succinate –CoQ reductase. It transfers electrons from substrates like succinate through FAD and iron sulfur centres to Co Q. . Now from CoQ electrons are transferred to cyt c via cyt b, cyt c1 and iron sulfur centre by complex III which is also known as cytochrome reductase. This lead s to oxidation of CoQ. Complex IV is cytochrome oxidase. . It transfers electrons from cyt c to final electron acceptor molecular oxygen. As a result cyt c is oxidized and oxygen is reduced to water. Succinate Complex ІІ NAD+

Complex І


Complex ІІІ

Cyt C

Complex ІV


Oxidative phosphorylation Synthesis of ATP using energy released when electrons flow in the respiratory chain from NAD+ to oxygen is oxidative phosphorylation. It consist of two processes an oxidation and phosphorylation. These two processes are coupled. It is different from substrate level phosphorylation with respect to location, mechanism, susceptibility to inhibitors etc. Substrate level phosphorylation is not combination of two processes and hence it does not involves coupling. Further it is insensitive to inhibitors. It occurs in metabolic pathways and located outside of mitochondria and does not involve electron transfer. In constrast ATP synthesis in respiratory chain is associated with three complexes of respiratory chain which is present in mitochondria. Complex І or NADH – CoQ reductase, complex ІІІ or CoQ –cyt c reductase and com plex ІV or cytochrome oxidase of respiratory chain are involved in ATP synthesis. Flow of electrons through these complexes causes synthesis of ATP. ATP Synthase When electrons flow in respiratory chain from NAD to oxygen ATP is generated. ATP synthase or F0F1 ATPase




present in inner mitochondrial membrane catalyzes formation of ATP from ADP and Pi. It is an integral membrane protein. This enzyme consist of two subunits. F0 subunit and F1. It has knob like structure. Head of the knob


ATP Synthase


is F1subunit. F0 is the base of the knob which is embedded in membrane. F1subunit has catalytic activity. F0 subunit is proton channel. When electrons move from outside to inside of the membrane through F0 subunit F1subunit catalyzes ATP formation from ADP and Pi using energy released. Anti biotic oligomycin blocks ATP synthase catalyzed ATP synthesis.


CHAPTER - 7 | Biological Oxidation

Mechanism of oxidative phosphorylation Chemiosmotic model: This model is proposed to explain mechanism of oxidative phosphorylation in respiratory chain by P. Mitchell. Novel element in this model is proton translocation from matrix to out side of mitochondrial membrane as electrons flow in respiratory chain. Due to this a proton gradient is generated across inner mitochondrial membrane. This in turn leads to development of potential difference also across mitochondrial membrane. Thus electron flow in respiratory chain leads to development of electrochemical gradient across inner mitochondrial membrane. Return of protons into matrix through proton channel of ATP synthase leads to ATP synthesis. Protons are driven into matrix from outside by electro chemical gradient. (H+) Protons out side Membrane

Electron Transport Chain

ATP Synthase

in side (H+) Protons ADP+Pi


Energy that is released when electrons pass through F0 subunit of ATP synthase is used by F1subunit for the synthesis of ATP. This proton extrusion occurs at the three complexes of respiratory chain. About 3 to 4 protons are extruded at each complex. This gives rise a PHdifference of about 0. 05 units across inner mitochondrial membrane. This is equal to 0. 15 volts potential difference at each complex which is sufficient for ATP formation. Since there are three complexes in respiratory chain electrons flow from NAD to oxygen generates 3ATP molecules. Latest research indicates synthesis of 2. 5 ATP only P:O Ratio: It is ratio of number of ATP formed in the electron transport chain per atom of oxygen consumed. When electrons flow from NAD to O2 3 ATP are formed per atom of oxygen consumed hence P, O ratio is 3. Like wise P, O ratio is 2 when electrons flow from FAD to O2 (oxygen). According to new research P, O ratio is 2. 5 and 1. 5 respectively Inhibitors of Respiratory chain Respiratory chain is inhibited by two types of inhibitors. They are a) Inhibitors of oxidative phosphorylation. b) Uncouplers. a) Inhibitors of oxidative phosphorylation: Inhibitors specific to each complex (site) of oxidative phosphorylation are found. Amytal, a sedative; rotenone a fish poison and piericidin an antibiotic inhibits oxidative phosphorylation at complex І or site 1. Antimysin A an antibiotic and BAL an antidote for arsenic poisoning inhibit oxidative phosphorylation at complex ІІІ or site 2. Cyanide, carbon monoxide, hydrogen sulfide and azide inhibit oxidative phosphorylation at complex ІV or site 3. 073


Questions and Answers

b) Uncouplers: These compounds uncouples or dissociates oxidation from phosphorylation in the respiratory chain. Due to this ATP synthesis is not possible even through oxidation is possible. Some uncouplers are 2, 4 dinitro phenol, dinitro cresol, salicylanilides, pentachrophenol and CCCP.

8. Write a note on substrate level phosphorylation 1. It is synthesis of ATP without involvement of respiratory chain 2. A high energy compound is generated in a pathway from which ATP is synthesized. 3. For example in glycolysis 1, 3 - bis phosphoglycerate is generated initially. Then phosphoglycerate kinase catalyzes ATP formation by converting 1, 3 – phosphoglycerate to 3-phosphoglycerate. 4. Likewise formation of ATP by pyruvate kinase of same pathway.

9. What are oxygenases ? Classify. Give examples for each. A. Oxygenases are those enzymes which catalyzes incorporation of oxygen directly into substrate molecules. Two types of oxygenases are known. They are a) Dioxygenases b) Mono oxygenases a) Dioxygenases : Are those enzymes that incorporate two atoms of oxygen molecules into substrate. Tryptophan dioxygenase and homogentisate dioxygenase are examples. b) Mono oxygenases: Are those enzymes that incorporate only one atom of oxygen molecule into substrate. Another atom of oxygen is reduced to water. Phenylalanine hydroxylase, trytophan hydroxylase and cytochrome P450 hydroxylases are examples for monooxygenases.

10. Write briefly about A) Hydrogen peroxide B) Superoxide A. Hydrogenperoxide Hydrogen peroxide is formed from reactions of riboflavin dependent aerobic dehydrogenases and oxidases. It may be also formed from reduction of oxygen to water and from superoxide dismutase. Hydrogen peroxide is toxic to cells so it must be eliminated. Hydroperoxidases are type of enzymes involved in removal of hydrogen peroxide. They are catalase and peroxidase. In erythrocytes glutathione peroxidase eliminates hydrogen peroxide.


CHAPTER - 7 | Biological Oxidation


Catalase 2H2O

2H2O + O2

H2O2 + H2


In macrophages hydrogen peroxide is produced as part of their normal function. Much of the generated hydrogen peroxide is used to produce free radicals like hypochlorite which kills bacteria that were engulfed. B. Superoxide Superoxide formation from oxygen occurs on addition of one electron. Superoxide is toxic to cells. It generates free radicals which are extremely toxic to cells. So it is eliminated by superoxide dismutase. Superoxide dismutase -

O2 + e

– 2

Superoxide O


2O2 + 2H


O2 + H2 O2

However in macrophages superoxide is produced from oxygen by adding electrons. NADPH oxidase adds electrons. The superoxide formed in turn generates free radicals hypochlorite and hydroxyl radicals which kills bacteria. Hence superoxide has role in phagocytosis of macrophages.

Other model questions are

11. Define oxidative phosphorylation and substrate level phosphorylation. Write their differences. 12. Write the principle of chemiosmotic hypothesis. 13. What are uncouplers? Give examples. 14. Write the components of electron transport chain in the order. Show sites of ATP synthesis. 15. Write about inhibitors of oxidative phosphorylation. 16. Write about a) ATP synthase b) Free radicals. 17. Subsrate level phosphorylation 18. Write about P:O ratio.



Questions and Answers



Carbohydrate Metabolism 1. Name food carbohydrates. How they are digested and absorbed ? Add a note on diseases associated with these processes. A. Food carbohydrates : Carbohydrates present in food are polysaccharides, disaccharides and very small amounts of monosaccharides. Polysaccharides are present in plant and animal diets. Cereals like rice, wheat, vegetables and roots like potato, tapioca contain starch, dextrin and inulin. Glycogen is present mainly in animal meat. Milk, cane sugar and malt contain disaccharides like lactose, sucrose and maltose. Bakery products, honey, sweets and fruits may contain monosaccharides. Carbohydrate digestion: Is a process that hydrolyzes food polysaccharides to their constituent monosaccharides. In the mouth : Carbohydrate digestion is initiated in mouth by salivary amylase present in saliva. Though its action is limited it converts polysaccharides like starch, glycogen and dextrin to maltose and oligosaccharides by hydrolyzing α 1, 4 glycosidic bonds. It has optimum PH of 7. 0 and requires chloride for optimum activity. Amylase Starch or glycogen or dextrin

Maltose + Oligo saccharides.

In the stomach:Due to absence of carbohydrate breaking enzymes in gastric juice no digestion of carbohydrate occurs in the stomach. In the duodenum: Pancreatic amylase is major carbohydrate digesting enzyme in duodenum. It hydrolysis alpha (α), 1, 4 glycosidic bonds of polysaccharides and converts them to maltose, maltotriose, limit dextrin and oligosaccharides. α- dextrin is another name for limit dextrin, It contains α 1, 6 glycosidic bonds. Amylose part of starch is converted to maltose, maltotriose and oligosaccharides. Amylase Amylose

Maltose + Maltotriose + Oligosaccharides.

Amylopectin is converted to limit dextrin, oligosaccharides, maltose and maltotriose. 076

CHAPTER - 8 | Carbohydrate Metabolism

Amylase Amylopectin

Limit dextrin + Maltose + Maltotriose.

Pancreatic amylase has optimum PH 7-8 and chloride is its activator. In the small intestine: Succus entericus which is secretion of small intestinal cells contains enzymes like disaccharidases, α-dextrinase or isomaltase that hydrolyzes disaccharides and limit dextrin to constituent monosaccharides. Isomaltase catalyzes hydrolysis of α 1, 6 glycosidic bonds of limit dextrin and converts to oligosaccharide and glucose. α-dextrinase Limit dextrin Oligosaccharide + Glucose Maltase catalyzes hydrolysais α (1, 4) glycosidic bonds from one end of oligosaccharide and releases glucose. Maltase Oligosaccharide

Glucose + Oligosaccharide shorter by one glucose unit.

The action of maltase on oligosaccharide continues until a disaccharide is formed (maltose). Oligosaccharide shorter by glucose Disaccharide Finally disaccharide containing two glucose units is also hydrolyzed to glucose. Maltase Maltose Glucose+ Glucose. Sucrase catalyzes hydrolysis of sucrose to glucose and fructose. Sucrase Sucrose

Glucose + Fructose.

Lactase hydrolyzes lactose of diet to glucose and galactose. Lactase Lactose


Thus dietary (food) polysaccharides are converted to their constituent monosaccharides. Carbohydrates absorption: The products of carbohydrate digestion are absorbed by a. Passive diffusion and b. Facilitated transport or secondary active transport. Passive diffusion :Mannose and xylose are absorbed by simple diffusion. Jejunum is site of absorption. Absorbed monsaccharides reaches liver through portal circulation. Facilitated transport or Secondary active transportt: Glucose, galactose and fructose are absorbed in jejunum by facilitated transport. These absorbed monosaccharides reaches liver through portal venous system. A carrier protein is involved in the absorption. It is called as translocase and present in enterocyte membrane. It transports glucose along with sodium. Hence it is known as symporter. Energy needed is supplied by movement of sodium.



Questions and Answers




Enterocyte Cytosol


In the initial stage it is present on external surface or luminal side of enterocyte. There are two binding sites on this carrier protein one glucose binding site and another for sodium binding. When these sites are occupied by glucose and sodium it moves to cytosolic side and releases sodium and glucose into cytosol. Glucose and galactose diffuses into blood from cytosol. However sodium is extruded by pump mechanism which is dependent on ATP. The carrier molecule returns to its original place to transport another glucose molecule. Disorders of carbohydrate digestion and absorption They are due to either defective enzymes or defective transporters. Some of them are given below. Lactose intolerance:It is due to deficiency of lactase. Hence patients of this disease fail to utilize lactose present in diet. Acumulation of lactose in the intestine leads to diarrhoea and abdominal pain, flatulence etc. due to fermentation of lactose by intestinal bacteria. Isomaltase and sucrase deficiency: It occurs in childhood. Isomaltase and sucrase are deficient. Disacchariduria:It is characterized by excretion of disaccharides in urine. It is due to deficiency of disaccharidases. Malabsorption syndromes of monosaccharides: They are due to defective transporter. Due to defective transporter absorption of monosaccharides is impaired.

2. Define glycolysis. Describe reactions of this process. Add a note on its energetics. A. Glycolysis is the degradation of glucose to pyruvate or lactate by a sequence of enzyme catalyzed reactions. Glycolysis takes place in the cytosol of most of cell types. In the skeletal muscle end product of glycolysis is lactate. It is known as anaerobic glycolysis. Reactions of glycolysis: 1. Glucose enters glycolysis by phosphorylation catalyzed by hexokinase. It is ATP dependent irreversible reaction. Glucose is phosphorylated on 6thcarbon. Magnesium ion (Mg2+) is required for this reaction and glucose -6-phosphate is product. In liver


CHAPTER - 8 | Carbohydrate Metabolism

glucokinase is present. However it phosphorylates glucose only after a meal when blood glucose level is more. 2. Isomerization of glucose -6-phosphate of first reaction to fructose -6-phosphate is second reaction and this reaction is freely reversible. Phosphoglucoisomerase catalyzes this reaction. Glucose


Hexokinase Glucose-6-phosphate + ADP Mg2+ (1) Phosphoglucose


Fructose-6-phosphate Isomerase (2)

3. Another ATP dependent phosphorylation of fructose -6- phosphate occurs in the third reaction which is catalyzed by phosphofructo kinase. Fructose -1, 6-bis phosphate is product and like first reaction this is also an irreversible reaction requiring ATP and magnesium ion. Up to this stage of glycolysis two high energy bonds are utilized. 4. Aldolase A splits fructose-1, 6-bis phosphate to two triose phosphates namely glyceraldalhyde-3- phosphate and dihydroxy acetone phosphate. Phosphofructokinase

Aldolase A

Fructose-6-phosphate +ATP

Fructose1, 6 bisphosphate 2+




ADP Glyceraldehyde-3-phosphate + Dihydroxy acetone phosphate 5. A reversible isomerization converts dihydroxy acetone phosphate to glyceraldehyde 3- phosphate which is catalyzed by triose phosphate isomerase. Triose phosphate isomerase Glyceraldehyde -3-phosphate dihydroxy acetone phosphate (5) Thus one glucose molecule is converted to two three carbon glyceraldehyde-3phosphate molecules. 6. An NAD+ dependent glyceraldehyde -3- phosphate dehydrogenase catalyzes oxidation and phosphorylation of glyceraldehyde-3-phosphate to an high energy 1, 3-bis phosphoglycerate. It is a reversible reaction and inorganic phosphate is required. Glyceraldehyde3-phosphate dehydrogenase Glyceraldehyde-3-phosphate+ NAD+

1, 3Pi



bis phosphoglycerate + NADH+H .



Questions and Answers

7. The high energy 1, 3-bis phosphoglycerate serve as source of energy for formation of ATP from ADP in this irreversible reaction catalyzed by magnesium dependent phosphoglycerate kinase. 3-phosphoglycerate is product of this reaction. Substrate level phosphorylation occurres. 8. A mutase shifts phosphate of 3- phosphoglycerate to the 2nd positition in this reversible reaction. Phosphoglyceratekinase 1, 3-bis phosphoglycerate+ADP

3-Phosphoglycerate Mg2+ (7) ATP

Phosphoglycerate mutase 2-phosphoglycerate. (8) 9. A high energy compound is generated in this reaction from 2- phosphoglycerate by enolase. Phospho enol pyruvate is product of this reversible reaction. Manganese or magnesium ions are required. Another substrate level phosphorylation occurres here 10.

Synthesis of ATP from ADP once again occurs in this reaction of glycolysis. The reaction is irreversible and requires magnesium. Phosphoglyceratekinase catalyzes this reaction and pyruvate is product of this reaction. If glycolysis ends with pyruvate then it is called as aerobic glycolysis. 2-phosphoglycerate


Enolase Pyruvatekinase phosphoenol pyruvate Pyruvate +ATP 2+ 2+ Mg (9) (10) Mg ADP

In the skeletal muscle lactate is formed from pyruvate on reduction catalyzed by lactate dehydrogenase. NADH produced in reaction (6) serve as source of hydrogen. In the erythrocytes also lactate is formed from pyruvate. It is a reversible reaction. Lactate Pyruvate+NADH+H+

Lactate + NAD+ Dehydrogenase (11)

Energetics: ATP formation in glycolysis of skeletal muscle is given below. Anaerobic glycolysis 1. Number of ATP formed by phosphoglycerate kinase


2. Number of ATP formed by Pyuvate kinase.


3. ATP consumed by hexokinase and phosphofructokinase

-2 Net 2


CHAPTER - 8 | Carbohydrate Metabolism

So net formation of glycolysis of skeletal muscle = 2. Generally glycolysis that yields lactate is considered as anaerobic glycolysis. There fore anaerobic glycolysis that occurs in skeletal muscle and erythrocytes generates 2ATP molecules per molecule of glucose. Aerobic glycolysis ATP formation in glycolysis of hepatocyte is given below. It is considered as aerobic glycolysis. NADH generated in 6threaction of glycolysis is oxidized in respiratory chain because it is not used for formation of lactate from pyruvate. 1. Number of ATP s generated by phosphoglyceratekinase


2. Number of ATPs generated by pyruvate kinase.


3. Number of ATP s generated by respiratory chain oxidation


4. ATP s consumed by hexokinase and phosphofructokinase

-2 Net 8

Thus the aerobic glycolysis generates 8 ATP molecules. According to new research only 7 ATPs are generated.

3. Write the significance of glycolysis. A. Glycolysis meets energy requirements of all kinds of cells. Anaerobic glycolysis mainly supplies energy to rapidly contracting skeletal muscle. Dietary fructose and a lactose are also metabolized by this path way. Glycolysis also supplies precursors for other pathways. For example pyruvate is used for alanine formation and dihydroxyacetone is used for triglyceride formation. In erythrocytes deficiency of pyruvate kinase causes hemolytic anaemia.

4. Write the fate of pyruvate OR Write about pyruvate dehydrogenase complex. A. Under aerobic conditions pyruvate is transported into mitochondria by a transporter present in mitochondrial membrane. In the mitochondria it is oxidatively decarboxylaed to acetyl –CoA by a multi enzyme complex pyruvate dehydrogenase (PDG) complex. Three enzymes are present in this complex. First enzyme is pyruvate dehydrogenase and contains TPP as prosthetic group. It is designated as E1 –TPP. Second enzyme dihydrolipoyl transacylase and containstwo sulfhydryl groups SH contributed by lipoicacid. It is written as E2- lipoamide

S and E2-Lipoamide



The first one is reduced form and latter one is oxidized form. The third enzyme is dihydrolipoyl dehydrogenase and contains FAD as cofactor. It is written as E3 –FAD. The first enzyme decarboxylates pyruvate and remaining hydroxyethylidine moiety is bound to TPP.



Questions and Answers

(1) Pyruvate+E1-TPP

E1-TPP- hydroxyethylidine+CO2.

The second enzyme transfers hydroxyethylidine to one of sulfur of oxidized lipoamide and releasing E1-TPP. It also shifts acetyl group to coenzyme A to form acetyl –CoA and reduced lipoamide. E1-TPP S



E2-lipoamide S



S-Acetyl E2-lipoamide



Acetyl-CoA+E2 –lipoamide (2)


The third enzyme regenerates oxidized lipoamide by transferring hydrogen to NAD via FAD. SH


E3 –FAD +E2 –Lipoamide

E2-lipoamide SH

E3 – FADH2 + NAD+


+E3 –FADH2 S



Fate of NADH: It is oxidized in respiratory chain. Three ATP (2. 5)s are generated.

5. Describe Citric acid cycle. A. It is a cyclic arrangement of reactions which convert acetyl-CoA to carbon dioxide. Oxidation of acetyl-CoA is accompanied by energy output. It is also known as Krebs cycle or tricarboxylic acid (TCA) cycle. Enzymes of this cycle are present in mitochondrial matrix. Reactions of TCA cycle: 1. Citricacid cycle reactions begins with formation of tricarboxylic acid citrate from acetyl –CoA and oxaloacetate catalyzed by citrate synthase a condensing enzyme. It is an irreversible reaction. Citrate Synthase Oxaloacetate + Acetyl -CoA

Citrate + CoASH. (1)

2. In the second reaction citrate is isomerized to isocitrate via cis aconitate and involves loss and addition of water. This reaction is catalyzed by aconitase an iron containing protein. It is a reversible reaction. Aconitase Citrate

Aconitase Cis-aconitate



2 H2O



CHAPTER - 8 | Carbohydrate Metabolism

3. Dehydrogenation and decarboxylation of isocitrate occurs in the third reaction of the cycle. It is catalyzed by NAD+ dependent isocitrate dehydrogenase. Isocitrtate is converted to α-ketoglutarate via oxalosuccinate. It is a reversible reaction. Isocitrate Dehydrogenase Isocitrate +NAD+ oxalosuccinate (3) (3) + NADH+H


4. Like pyruvate in this reaction α-ketoglutarate undergoes oxidative decarboxylation catalyzed by α-ketoglutarate dehydrogenase complex. It requires lipoic acid, TPP, CoA, FAD and NAD. It is an irreversible reaction. Succinyl-CoA an high energy compound and NADH are products of this reaction. α-ketoglutarate Dehydrogenase α-ketoglutarate +NAD+

Succinyl-CoA + NADH +H+ Co2 (4)

5. High energy compound GTP is formed in this reaction. It is catalyzed by succinate thiokinase. It also requires magnesium ions and it is an irreversible reaction. Succinate is product. It is another example for substrate level phosphorylation. 6. Dehydrogenation of succinate to fumarate occurs in this reaction. It is catalyzed by FAD dependent succinate dehydrogenase. It is reversible. Succinate Succinate Thiokinase dehydrogenase Succinyl-CoA +GDP Succinate Fumarate +FADH2 (5) (6) GTP FAD 7. Addion of water to fumarate by fumarase occurs in this reaction. Malate is the product and it is a reversible reaction. 8. Finally oxaloacetate is regenerated from malate by NAD dependent malate dehydrogenase. It is a reversible reaction. Fumarase Malate Dehydrogenase Fumarate +H2O Malate oxaloacetate +NADH+H+ (7) (8) + NAD The reactions of cycle starts again using oxaloacetate and it continues as long as acetyl-CoA molecules are available. Energetics: Amount of ATP generated by citric acid cycle per molecule of acetylCoA oxidation is given below.



Questions and Answers

1. Respiratory chain oxidation of NADH generated isocitrate dehydrogenase


2. Respiratory chain oxidation of FADH generated by succinate dehydrogenase


3. Respiratory chain oxidation of NADH generated by α-ketoglutarate dehydrogenase


4. Respiratory chain oxidation of NADH generated by Malate dehydrogenase.


5. GTP generated by succinate thiokinase is equal to one ATP.

1 Total 12

There fore 12 ATPs are produced in citric acid cycle when one acetyl- CoA is oxidized. Only 10 ATPs are produced as per latest research. Regulation: Citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are regulatory enzymes. They are subjected to allosteric regulation. ATP and NADH are allosteric (regulators) or inhibitors and ADP is allosteric activator. So Citric acid cycle rate depends on cellular levels of ATP and NADH. Significance: It is final common metabolic pathway for oxidation of carbohydrates, lipids and proteins. Intermediates of TCA cycle are used for anabolic reactions. Fatty acids, cholesterol, aminoacids and porphyrins are compounds formed from citric acid cycle intermediates.

7. Describe glycogen metabolism. A. It consists of A. Glycogenesis B. Glycogenolysis Glycogensis: It is the formation of glycogen from glucose. It occurs in almost all cells but liver and skeletal muscle are major organs. Reactions : 1. Glucose -6- phosphate of glycolysis conversion to glucose -1-phosphate is the first reaction of glycogenesis. It is a reversible reaction and catalyzed by phosphoglucomutase. 2. Formation of active sugar UDP- glucose occurs in second reaction. It is an irreversible reaction It is catalyzed by UDPG – pyrophosphorylase and uses UTP as energy source. The inorganic pyrophosphate (PPi) released is converted to two molecules of inorganic phosphate (2Pi) by pyrophosphatase.


CHAPTER - 8 | Carbohydrate Metabolism

phospho gluco UDPG- Pyro mutase Phosphorylase Glucosse-6-phosphate Glucose-1-phosphate UDP- glucose (UDPG) (1) (2) UTP ppi 3. UDP-glucose or active glucose serve as donor of glucose units. A glycogen primer accepts glucose from UDP- glucose which is catalyzed by glycogen synthase and involves glycosidic linkage of type 1, 4. The action of glycogen synthase continues until glycogen primer is elongated by 6-11glucose residues. Glycogen Synthase Primer glycogen +UDP-glucose

UDPG UDP primer glycogen (n+1)residues



UDP Elongated primer glycogen. 4. Now new branch is created in primer glycogen by transferring oligosaccharide containing six glucose residues from newly formed fragment to adjacent chain of primer glycogen. This reaction is catalyzed by branching enzyme. It involves hydrolysis and formation of glycosidic linkages of Îą 1, 4 type and Îą 1, 6 type respectively. Branching Enzyme Elongated primer glycogen primer glycogen with new branch. (4) 5. Further elongation of new branch by glycogen synthase and further branching by branching enzyme leads to formation of glycogen molecule. Glycogen Synthase Primer glycogen with new branch Elongation of new branch of glycogen. (5) Further Glycogen with elongated new branch

Glycogen. Branching


Significance: Glycogen formation occurs immediately after meal. Glycogen formed in liver and skeletal muscle function as stored form of energy. Glycogenolysis: It is degradation of glycogen to glucose or lactate. It occurs in liver and skeletal muscle. Reactions: 1. Degradation of glycogen is initiated by enzyme phosphorylase. It hydrolyzes 1, 4 glycosidic bonds from an end of a branch of glycogen and releases glucose as glucose -1- phosphate. Its action continues until four glucose residues remain on either side of branch point. Action of phosphorylase converts glycogen to limit dextrin.



Questions and Answers

Phosphorylase Glycogen Glucose -1- phosphate +glycogen shorter by one glucose residue (1) Pi Glycogen with one glucose less

Glucose -1-phosphate+ glycogen

(1) With 4 glucose residues on either side of branch point. 2. In this reaction a trisaccharide chain containing 3glucose units of partly hydrolyzed branch is transferred to adjacent branch to expose 1, 6 glycosidic bond at branch point. It is catalyzed by glucan transferase. It involves breaking as well as formation of glycosidic bonds. 3. A debranching enzyme hydrolyzes 1, 6 glycosidic bond. This results information of glycogen with one branch less. 4. Now further action of phosphorylase continues on another branch and this is followed by glucan transferase and debranching enzyme. Thus the combined action of these enzymes results in degradation of glycogen to glucose 1-phosphate. Glucan transferase Glycogen with glucose 4 units on either side of branch point Debranching Enzyme Glycogen with exposed glycogen with one branch short Branch 3 Glucose- 1- phosphate



5. Glucose-1- phosphate is converted to glucose -6-phosphate by phosphoglucomutase in this reaction. 6. Free glucose is released from glucose -6-phosphate in liver by the action of glucose -6phosphatase. However in muscle glucose -6- phosphate enters glycolysis and get converted to lactate. Phosphogluco glucose-6 Mutase phosphatase Glucose-1- phosphate glucose -6- phosphate glucose+Pi (6) (6) liver glycolysis Glucose-6-phosphate

lactate. Muscle


Significance : Glycogenolysis in liver meets body glucose requirements in between meals and starvation. In the skeletal muscle glycogenolysis meets energy requirement in between meals. 086

CHAPTER - 8 | Carbohydrate Metabolism

Glycogen storage diseases Glycogen metabolism is defective in several diseases due to deficiency of enzymes of either glycogen formation or break down. Some are given below. a. von Gierk's disease or type І glycogen storage disease : It is due to deficiency of glucose-6phosphatase. This results in accumulation of glycogen in liver, kidney etc. Hence enlargement of liver occurs. Symptoms are hypoglycemia, hyper uricemia, hyper lipidemia and ketosis. b. Pompe's disease or TypeІІ glycogen storage disease:It is due to defective glycogen break down in lysosomes. Lysosomes contain α-glucosidase which usually hydrolyzes glycogen in normal people. Lack of this enzyme leads to accumulation of glycogen in lysosomes of all types of cells occurs. It is a fatal condition. In heart accumulation leads to cardiomegaly. Children born with this defect may die in second year of life due to cardiorespiratory failure. c. Coris disease or TypeІІІ glycogen storage disease :In this disease glycogenolys is blocked due to deficiency of debranching enzyme and limit dextrin accumulates in liver. Hence this condition is also known as limit dextrinosis. d. Anderson's disease or Type ІV glycogen storage disease:This condition is characterized by accumulation of amylopectin an intermediate of glycogenesis in liver. It is due to deficiency of branching enzyme. In other organs like heart and spleen also accumulation of amylopectin occurs. It is a serious disease. Since amylopectin accumulation occurs in organs this condition is also called amylopectionosis. e. McArdle's syndrome or TypeV glycogen storage disease: In this disorder glycogen accumulates in skeletal muscle of affected persons due to lack of phosphorylase. Lactic acid production is not increased in muscle after exercise indicating block in glycogenolysis. Muscle cramps and diminished tolerance to exercise are symptoms. f. Her's disease or TypeVІ glycogen storage disease: This condition is characterized by accumulation of glycogen in liver due to deficiency of phosphorylase of glycogenolysis.

8. Describe hexose monophosphate (HMP) shunt pathway. A. Enzymes of HMP shunt pathway are present in cytosol of liver, adipose tissue, blood cells mainly red blood cells and neutrophils of white blood cells, adrenal cortex, testis, ovaries, lactating mammary gland, thyroid etc. In the skelatal muscle this pathway is less active. It is also known as direct oxidative pathway and pentose phosphate pathway. Reactions: 1. Glucose-6-phosphate of glycolysis is starting compound of this pathway. Dehydrogenation of this molecule by NADP dependent glucose-6-phosphate dehydrogenase is initial reaction of this pathway. 6-phosphogluconolactone and NADPH are products. Magnesium or calcium ions are also needed for this reaction.



Questions and Answers

2. Lactonase hydrolyzes lactone to 6-phosphogluconate in presence of magnesium, manganase or calcium ions. Glucose-6-phosphate Dehydrogenase lactonase Glucose-6- phosphate 6-phosphoglucorolactone (1) (2) + NADP+ NADPH+H Phosphogluconate.


3. Another NADP dependent dehydrogenation converts 6 phosphogluconate to pentose phosphate. It occurs in two steps. In the first step phosphogluconate is dehydrogenated at 3carbon to 3-keto-6- phosphogluconate and NADPH is formed. In the second step spontaneous decarboxylation of 3-keto -6-phosphogluconate yields ribulose phosphate. phospho gluconate Dehydrogenase + 6-phosphogluconate+NADP 3-keto-6-phosphogluconate (3) NADPH+H+ Ribulose- 5- phosphate.



4. Isomerization of ribulose-5-phosphate to ribose-5-phosphate occurs in this reaction. 5. Another molecule of ribulose-5-phosphate is epimerized to xylulose 5-phosphate in this reacton. pentose phosphate pentosephosphate Isomerase Epimerase Ribose-5- phosphatre Ribulose-5- phosphate xylulose-5-phosphate. (4) (5) Rearrangements between two pentose phosphates in subsequent reactions generates intermediates of glycolysis. 6. A two carbon fragment glycaldehyde of xylulose-5-phosphate transfer to ribose-5phosphate yields 7 carbon sedoheptulose-7-phosphate and glyceraldehydes -3- phosphate. This reaction is catalyzed by TPP and magnesium dependent transketolase enzyme. Trans ketolase Xylulose-5-phosophate+ Ribose-5- phosphate Sedoheptulose-7- phosphate TPP (6) +Glyceraldehyde -3-phosphate. 7. In this reaction sedoheptulose -7-phosphate is converted to 4 carbon erythrose-4phosphate and fructose -6- phosphate. It involves transfer of 3 carbon dihydroxy acetone phosphate moiety of seven carbon sugar to another 3 carbon sugar glyceraldehydes -3phosphate. The reaction is catalyzed by transaldolase.


CHAPTER - 8 | Carbohydrate Metabolism

Trans aldolase Sedoheptulose-7-phosphate+glyceraldehyde -3- phosphate Erythrose -4phosphate+ Fructose -6-phpsphate. (7) 8. This is another trans ketolase catalyzed reaction. Erythrose -4- phosphate is converted to fructose -6-phosphate by transferring 2 carbons fragment from xylulose -5-phosphate by transketolase. The remaining three carbon fragment of xylulose -5-phosphate is released as glyceraldehyde -3- phosphate. TPP and magnesium ions are required. Transketolase Erythrose -4-phosphate +xylulose -5 phosphate Glyceraldehyde-3-phos TPP Mg2+ (8) Phate + Fructose -6-phosphate. Thus pentose phosphates generated are converted to fructose -6-phosphate and glyceraldehyde-3-phosphate which enters glycolysis for further utilization. Significancte: 1. NADPH produced is used for the biosynthesis of fatty acids, cholesterol. deoxyribonucleotides, bile acids, glutamate, hormones and detoxification by cytochrome P450 hydroxylase. 2. In erythrocytes NADPH is used for removal of hydrogen peroxide by glutathione and conversion of methemoglobin to normal hemoglobin. 3. In neutrophils NADPH is used for superoxide biosynthesis. 4. Pentose phosphates are used for nucleic acid and nucleotide biosynthesis. 5. This pathway convertes glucose to directly Co2 and hence it is called as direct oxidative pathway of glucose. 6. Pentose of nucleic acid breakdown are used for energy production after they are converted to intermediates of glycolysis by this pathway. 7. Xylulose of uronicacid pathway is either converted to glucose or intermediates of this pathway. 8. Glucose -6-phosphate dehydrogenase deficiency:It is sex linked inherited disease of HMP shunt pathway. In these individuals a tenfold less active glucose -6-phosphate dehydrogenase is produced in erythrocytes. They appear normal until they are exposed to certain drugs. In presence of antimalarial drug primaquine, sulfonamide antibiotics and painkiller aspirin the less active enzyme becomes inactive. As a result NADPH production is blocked and susceptibility of erythrocytes to hemolysis increases. Therefore affected individuals develop hemolytic anaemia on exposure to those drugs. Fava beans also cause this disease. However favism is the name given to this disease.



Questions and Answers

9. Define gluconeogenesis. Write reactions involved in the formation of glucose from lactate. A. Gluconeogenesis is the synthesis of glucose from non carbohydrates like pyruvate of amino acids, lactate, glycerol etc. Formation of glucose from lactate It involves participation enzymes of glycolysis, citric acid cycle, key enzymes of gluconeogenesis and cytosolic malate dehydrogenase. The enzymes are present in mitochondria and cytosol. Key enzymes of gluconeogenesis:These enzymes makes reversal of glycolysis. They by pass irreversible reactions of glycolysis which prevent reversal of glycolysis. They are 1. Pyruvate carboxylase. 2. Phosphophenol pyruvate carboxykinase. 3. Fructose -1, 6-bis phosphatase 4 Glucose -6-phosphatase. Site:Gluconeogenesis mainly occurs in liver. Reactions of gluconeogenesis: 1. Lactate dehydrogenase converts lactate to pyruvate using NAD as hydrogen acceptor pyruvate so formed enters mitochondria through specific transporter present in inner mitochondrial membrane. 2. In the mitochondria pyruvate carboxylase converts pyruvate to oxaloacetate. It is a ATP and biotin dependent carboxylation. Lactate Pyruvate Dehydrogenase Carboxylase Lactate +NAD+ pyruvate Oxaloacetate+ADP+Pi (1) (2) NADH+H+ ATP Co2 Biotin 3. Oxaloacetate formed in mitochondria must enter cytosol where enzymes of glycolysis are present. But oxaloacetate is impermeable to mitochondrial membrane. Malate dehydrogenase of TCA cycle converts oxaloacetate to malate which is permeable to mitochondrial membrane. 4. In the cytosol oxaloacetate is regenerated from malate by cytosolic malate dehydrogenase. Malate Malate Dehydrogenase Dehydrogenase Oxaloacetate+NADH+H+ NAD++ Malate (3) (4) Oxaloacetate+NADH+H+. 5. Phosphoenol pyruvate an intermediate of glycolysis is formed from oxaloacetate in this reaction catalyzed by phosphoenol pyruvate carboxykinase (PEPCK). It requires GTP as energy source. 090

CHAPTER - 8 | Carbohydrate Metabolism

6. Enolase of glycolysis converts phosphoenol pyruvate to 2-phosphoglycerate. PEPCK Oxaloacetate+GTP

Enolase Phosphoenol pyruvate


2-phosphoglycerate. (6)

GDP 7. Another enzyme of glycolysis phosphoglycerate mutase converts 2-phosphoglycerate to 3phosphoglycerate. 8. Phosphoglyceratekinase of glycolysis converts 3- phosphoglycerate to 1, 3bisphosphoglycerate. Phosphoglycerate phosphoglycerate Mutase kinase 2-phosphoglycerate 3-phosphoglycerate 1, 3-bis Phosphoglycerate. (7) (8) ATP ADP 9. Glyceraldehyde -3-phosphate dehydrogenase of glycolysis forms glyceraldehyde-3phosphate from 1, 3-bisphosphoglycerate. 10. Triose phosphate isomerase of glycolysis converts a molecule of glyceraldehyde-3phosphate to dihydroxy aectone phosphate. Glyceraldehyde-3-phosphate Dehydrogenase (10) 1, 3-bisphosphoglycerate+NADH+H+ Glyceraldehyde-3-phosphate 9 Pi Isome + Dihydrooxyacetone phosphate. NAD rase 11. Reversible action of aldolase of glycolysis generates fructose-1, 6-bisphosphate from glyceraldehydes -3-phosphate and dihydroxy acetone phosphate. Aldolase Glyceraldehyde -3-phosphate +Dihydroxyacetone phosphate Bis phosphate.

Fructose -1, 6(11)

12. Fructose-1, 6-bisphosphatase of gluconeogenesis generates fructose-6-phosphate from fructose-1, 6-bis phosphate. 13. Glucose-6-phosphate is formed from fructose-6-phosphate by action of phosphohexose isomerase of glycolysis. Fructose-1, 6-bis phosphohexose Phosphatase Isomerase Fructose-1, 6-bisphosphate Fructose-6-phosphate Glucose (12) (13) -6-phosphate. Pi




Questions and Answers

Glucose-6- phosphatase last key enzyme of gluconeogenesis generates glucose from. Glucose-6- phosphate by hydrolyzing phosphate. Glucose-6-phosphatase Glucose -6-phosphate

Glucose + Pi (14) H2o

Significance:During fasting and starvation gluconeogenesis meets body glucose requirement. Gluconeogenesis is the only source of glucose to organs like brain, skeletal Muscle, , erythrocytes etc. If gluconeogenesis is blocked brain dysfunction occurs. Gluconeogenesis clears metabolic waste product like lactate. Excess aminoacids of dietary origin are converted to glucose by gluconeogenesis.

10. Write reactions of Uronic acid pathway. A. Reactions: 1. UDP-glucose of glycogenesis serve as starting compound of this pathway. To give UDPglucuronic acid UDP-glucose undergoes 4 electron transfer reaction. It is catalyzed by UDP- glucose dehydrogenase and 2NADH are generated. 2. Glucuronic acid is formed from UDP glucuronic acid on hydrolysis catalyzed by hydrolase. UDP-glucuronic acid serve as active or donor of glucuronic acid. UDPG Dehydrogenase UDP-glucose+2NAD+ UDP- glucuronicacid (1) 2NADH+2H+

Hydrolase Glucuro (2)UDP nic acid

3. An NADPH dependent reduction of glucuronic acid to gulonic acid by gulonate dehydrogenase occurs in this reaction. 4. Oxidation of L-gulonate to 3-keto –L-gluconate by NAD+ dependent dehydrogenase is four th reaction. Dehydro Genase Glucuronicacid + NADPH+H+

NAD+ L-gulonate



NADP+ Gluconate +NADH+H+. 5. A decarboxylase converts 3-keto –L-gulonate to L-xylulose by removing a carbon of 3- Keto –L- gulonate as carbon dioxide. 6. An NADPH dependent reduction of L-xylulose to xylitol by dehydrogenase is sixth reaction.


CHAPTER - 8 | Carbohydrate Metabolism

3-Keto- L- gulonate

Decarboxylase Dehydrogenase L-xylulose Xylitol+NADP+ (5) (6) CO2 NADPH +H+

7. A ketopentose D-Xylulose is formed from xylitol in this reaction catalyzed by dehydrogenase. It involves removal of hydrogen by NADP+ from Xylitol. 8. Finally xylulose -5- phosphate an intermediate of HMP shunt is formed by phosphorylation of xylulose. Dehydrogenase Xylitol +NADP+

Xylulose kinase D-Xylulose Xylulose-5-phosphate (7) (8) + ADP + NADPH+H ATP

11. Write the significance of uronic acid pathway. A. Glucuronic acid is used for synthesis of mucopolysacharides, detoxification, conjugation with bilirubin, steroid hormone etc. In plants and mammals other than man Vit. C is synthesized from gulonate by this pathway. This pathway utilizes glucuronic acid of endogenous origin for energy production. Dietary xylitol is utilized by this pathway.

12. Write about essential pentosuria A. This inherited disease is characterized by excretion L-xylulose in urine. It is due to deficiency of enzyme xylitol dehydrogenase. Due to lack of this enzyme L-xylulose can not be converted to xylitol and it accumulates in blood and get excreted in urine. Drugs like barbiturate and paracetamol increases utilization of glucose by this pathway.

13. Trace route of fructose conversion to glucose and pyruvate. A. In liver fructose is converted to either glucose or intermediates of glycolysis. However in skeletal muscle and adipose tissue fructose is converted to intermediates of glycolysis. Reactions: 1. Fructose metabolism begins with phosphorylation. In liver fructokinase phosphorylates fructose to fructose -1-phosphate. In skeletal muscle and adipose tissue hexo kinase phosphorylates fructose to fructose -6-phosphate. This enters glycolysis. Magnesium ions are required. Hexokinase Glycolysis


Fructokinase Fructose+ATP


Fructose -1- phosp (1) Phosphate+ADP.

ADP 093


Questions and Answers

2. Aldolase B present in liver splits fructose -1-phosphate to glyceraldehyde and dihydroxy acetone phosphate. 3. Aldehyde kinase in liver converts glyceraldehyde to glyceraldehyde -3-phosphate. ATP is phosphate donor. Aldolase Fructose-1-phosphate

Dihydroxyacetone phosphate+glyceraldehyde. (2)

Glyceraldehyde+ATP Aldehydekinase Glyceraldehyde -3-phosphate. (3) 4. Now glucose is formed from glyceraldehyde -3-phosphate and dihydroxy acetone phosphate by reversal of glycolysis and gluconeogenesis. Alternatively they can be used for energy production by remaining reactions of glycolysis. Pyruvate

Glyceraldehyde-3-phosphate +dihydroxy acetone phosphate. (4)

Gluconeogenesis Glucose

14. Write about inherited disease of fructose metabolism. A. Inherited diseases of fructose metabolism are 1. Essential fructosuria : It is due to deficiency of fructo kinase. Hence fructose utilization is blocked. Fructosuria and fructosemia develops on consumption of fructose containing diets. 2. Hereditary fructose intolerance:It is due to deficiency of aldolase B. People affected with this condition appear normal until they are exposed to fructose containing diets. Consumption of fructose causes vomiting and diarrhoea in these individuals. Hence they dislike sweets. Hypoglycemia, fructosemia, fructosuria develops on consumption of fructose. Other symptions are jaundice, liver enlargement, kidney disease and growth retardation.

15. Describe Galactose metabolism. A. Galactose is converted to glucose in liver. Further galactose is required for synthesis of lactose, galactolipids and mucopolysacharides. Reactions: 1. Initial reaction of galactose utilization is phosphorylation catalyzed by galactokinase. ATP and magnesium ions are required and galactose-1-phosphate is product. 2. Transfer of galactose to UDP-glucose replacing glucose occurs in this reaction. Glucose is released as glucose-1-phosphate. Reaction is catalyzed by galactose-1-phosphate uridyl transferase. 094

CHAPTER - 8 | Carbohydrate Metabolism

Galactose-1-phosphate Uridyl transferase Galactose+ATP Galactose-1-phosphate UDP (1) (2) ADP UDP-Glucose -Galactose +Glucose-1-Phosphate. Galactokinase

3. UDP-Galactose serve as donor galactose for synthesis of lactose, galactose containing lipids etc. Alternatively it is converted to UDP –glucose by UDP galactose epimerase. It occurs in two steps and involves participation of NAD+. First UDP-galactose undergoes dehydrogenation to 4-keto –UDP galactose and NADH is produced. In the second step reduction of 4-keto –UDP-galactose by NADH results in formation of UDP-glucose and NAD+. Epimerase UDP –galactose +NAD


Epimerase 4-keto-UDP-galactose+NADH+H




UDP-Glucose+NAD+. 4. From UDP-glucose, glucose is liberated as glucose -1-phosphate after in corporation into glycogen followed by phosphorylase action. (4) UDP-glucose

(4) glycogen

Glucose -1- phosphate


Galactosemia :This inherited disease is due to deficiency of galactose-1-phosphate uridyl transferase. So galactose utilization in affected persons is blocked. Accumulation of galactose leads to galactosemia and galactosuria. Vomiting and diarrhoea occurs on consumption of milk. Cataract of eye due to accumulation of galactitol a reduced product of galactose, mental retardation, jaundice and liver failure are other symptoms. Continued intake of galactose may lead to death. By withdrawing galactose containing products in diet death can be prevented. Adult galactosemics tolerate milk due to development of other routes of galactose utilization.

16. What is normal blood glucose level? How it is regulated? A. Normal blood glucose level: The normal blood glucose level is 60-90 mg% in post absorptive conditions. After a meal blood glucose level raises to 110-130 mg%. It is known as post prandial blood glucose level. In fasting blood glucose level falls to 50-60mg%. In normal people this level is brought back to normal level. How ever the blood glucose level in normals is determined by a. Rate of entry of glucose into blood from various routes. b. Rate of removal of glucose from blood by various pathways. Blood glucose sources: Dietary carbohydrates are digested and products glucose, fructose, galactose reach liver. In the liver galactose and fructose are also converted to glucose.



Questions and Answers

Dietary carbohydrates keep blood glucose level with in limits up to 3hours after food in take. Liver glycogenolysis meets blood glucose requirements up to 10 hour after food in take. Liver gluconeogenesis meets blood glucose requirements up to 36 hours after food in take and beyond that period if food is not taken. Blood glucose removal: Pathway of carbohydrate metabolism uses glucose in various ways. a. Glycolysis use glucose for energy b. Glycogenesis use glucose for glycogen formation. c. HMP shunt use glucose for NADPH and pentose production. d. Uronic acid pathway use glucose for uronic acid production. e. Glucose is used for fat formation. A finely regulated homeostatic mechanism maintains stable blood glucose level in which liver, extra hepatic tissues and various hormones are involved. They maintain stable glucose level either by affecting glucose sources or glucose removal. Role of liver:Liver plays crucial role in maintenance of stable blood glucose level. Live rcells (hepatocytes)are freely permeable to glucose. Movement of glucose across hepatocyte membrane is not influenced by insulin. When blood glucose level rises liver brings down to normal level by converting excess glucose into glycogen, fat and pentoses. Similarly when blood glucose level is below normal liver rises blood glucose level to normal by forming glucose from glycogen (glycogenolysis) and non carbohydrate sources (Gluconeogenesis). Extra hepatic tissues involved in blood glucose regulation or homeostasis are skeletal muscle, kidney and erythrocytes. These extra hepatic tissues are not freely permeable to glucose. Skeletal muscle:When the blood glucose level rises skeletal muscle lowers by converting glucose to glycogen. If the blood glucose level falls below normal it indirectly contributes to blood glucose by supplying lactate. During starvation muscle aminoacids particularly alanine is used for glucose formation. Kidney:When blood glucose level falls below normal kidney contributes to blood glucose through gluconeogenesis. If the blood glucose level is above normal kidney brings down to normal by eliminating glucose in urine. Erythrocytes:When the blood glucose level is high they remove glucose through HMP pathway, 2-3-bis phosphoglycerate cycle and glycolysis. If glucose level falls it contributes to blood glucose by supplying lactate. Hormones:Many hormones are involved in maintenance of stable blood glucose level. Based on their action on blood glucose level they are divided in to two types. They are 1. Hypoglycemic hormones and 2. Hyper glycemic hormones.


CHAPTER - 8 | Carbohydrate Metabolism

Hypoglycemic hormone As the name implies this hormone lower blood glucose level. Insulin is the only known hormone of this category. Insulin :Insulin is secreted by beta cells of islets of Langerhans in response to increased blood glucose level or hyperglycemia. Insulin plays crucial role in the regulation of blood glucose level. It lowers blood glucose level by a. Increasing up take of glucose by peripheral tissues like skeletal muscle and adipose tissue. In the muscle excess glucose is converted to glycogen and in adipose tissue fat is synthesized. b. Increasing utilization of glucose by various pathways. Insulin increases rate of glycolysis, HMP shunt, fatty acid synthesis, pyruvate dehydrogenase complex and glycogenesis. At same time it decreases rate of glycogenolysis and gluconeogenesis. Activitives of enzymes of glycolysis, HMP shunt, glycogenesis, fatty acid synthesis is increased by insulin. Activities of enzymes of gluconeogenesis and glycogeneolysis are decreased by insulin. Hyper glycemic hormones As the name implies these hormones raises blood glucose level. Glucagon, epinephrine (norepinephrine), glucocorticoids, anterior pituitary hormones and thyroid hormones are hyperglycemic hormones. Glucagon:It is another hormone produced by pancreas. Alpha cells of islets of Langerhans secretes this hormone in response to hypoglycemia. It is an antagonist of insulin. It increases blood glucose level by a. Promoting gluconeogenesis in liver. b. Inhibiting glycogenesis. Epinephrinc (Nor epinephrine) : Adrenal medulla secretes these hormones in response to hypoglycemia. It increases blood glucose level by a. Increasing gluconeogenesis in liver. b. Inhibiting glycogenesis. c. Stimulating glycogenolysis. Glucocorticoids: Adrenal cortex secretes glucocorticoids into blood stream. They increase blood glucose level by a. Reducing glucose utilization by peripheral tissues. b. Enhancing gluconeogenesis by inducing formation of enzymes of gluconeogenesis. Anterior pituitary hormones:Anterior pituitary gland increases blood glucose level by secreting two hormones. They are growth hormone and adereno corticotrophic hormone (ACTH). Growth hormone:Secretion of growth hormone occurs as response to hypoglycemia. It increases blood glucose level by i. Inhibiting uptake of glucose by peripheral tissues. ii. Promoting fat mobilization. iii. Liver gluconeogenesis.



Questions and Answers

ACTH: It increases blood glucose level by producing glucocorticoids and acting on glycogen metabolism. Thyroid hormone:Thyroxine increases blood glucose level by i. Affecting glucose utilization by peripheral tissues. ii. Affecting glucose absorption in intestine.

17. Write about 2. 3-bis phosphoglycerate cycle. or Rapoport-Leubering cycle A. This cycle is active in erythrocytes. It deals with formation and degradation of 2, 3-bis phosphoglycerate. Formation of 2, 3 –bis phosphoglycerate (2, 3 –BPG): Phosphoglycerate mutase catalyzes formation of 2, 3 –BPG from 1, 3-bis phosphoglycerate of glycolysis. Glycolysis

1, 3 –bis phosphoglycerate

2, 3-bis phosphoglycerate.

Degradation of 2, 3 –BPG: 2, 3-BPG is degraded to 3-phosphoglycerate by a phosphatase. Further fate of 3-phosphoglycerate occurs in glycolysis. 2, 3-bis phosphoglycerate



Significance:In erythrocytes 2, 3-BPG helps in unloading of oxygen by hemoglobin. 18. Explain glucose- lactate cycle OR Cori cycle A. In this cycle lactate that is produced in rapidly contracting skeletal muscle enters blood stream because it is a dead end of glycolysis. Through blood stream it reaches liver where it is converted to glucose through gluconeogenesis. Glucose so formed enters blood stream and reaches skeletal muscle for utilization. Thus the liver supplies glucose to skeletal muscle which in turn supplies lactate to liver. These reactions constitutes coricycle or glucose –lactate cycle. Skeletal muscle Glucose

Blood Lactate

Liver Lactate Gluconeogenesis




19. Define Diabetes mellitus. Classify. Write about each class. Mention clinical and biochemical symptoms. A. Diabetes mellitus is disease due to lack of action of insulin and characterized by elevated blood glucose level and glucose in urine. There are two types of diabetes mellitus. I. Type І Diabetes mellitus or insulin dependent diabetes mellitus (IDDM) or Juvenile onset diabetes mellitus :As the name implies it appears in young people. The age of affected people is always below 30 years. It accounts about 20% of diabetic cases. Usually individuals of this disease are thin or lean and appears as under nourished. It is due to absence of insulin. Hence patients of this disease are treated with insulin injections.


CHAPTER - 8 | Carbohydrate Metabolism

ii. Type ІІ diabetes mellitus or non insulin dependent diabetes mellitus (NIDDM)or Adults on set diabetes mellitus :As the name implies it appears in adults. The age of affected people is always above 30years. It accounts about 80% of diabetes cases. Usually individuals of this disease are obese. It is due to lack of insulin action i. e. insulin is present but due to lack of insulin receptors its action is lost. Hence patients of this disease cannot be treated with insulin injections. Biochemical and clinical symptoms: In acute diabetic patients following biochemical and clinical symptoms are seen. a. Hypoglycemia b. Glycosuria c. Polyuria d. Increased hunger (Polydipsia) e. Increased thrist (polyphasia) f. Ketosis in type І diabetes g. Weight loss h. Delayed wound healing. i. Keto acidosis. j. Coma and death.

20. Describe Glucose Tolerance Test (GTT) A. The ability of body to oxidize a load of glucose given is referred as glucose tolerance. This test is used to distinguish normal people from people with increased or decreased tolerance that occurs in diseases like diabetes mellitus, hormonal disorders etc. Procedure: After over night or 12 hour of fasting GTT is done. Fasting blood and urine samples are collected. The subject is asked to drink 200 ml water which contain test dose of glucose. A standard dose of 50gm of glucose or 0. 75 -1. 5 gm per Kg body weight is usually dissolved in 200ml water. The time is noted and for every 30 minutes blood and urine samples are collected for two and half hours. Glucose in the blood samples and urine samples is determined. Usually blood glucose level is measured quantitatively and qualitative Bendicts test is used for urine sugar analysis. The blood glucose values are plotted against time. 300

Severe Diabetes

200 Blood Glucose mg%


Mild Diabetes

100 Normal 50 0


60 90 Time



Normal glucose tolerance (response):The fasting blood glucose level is with in range of 6090mg%. The blood glucose level reaches a peak with in 30 to 60 minutes after consuming glucose test dose. The peak value is 110-130mg%. The initial rise is due to absorption of glucose. However increased blood glucose level returns to normal at the end of 2 hours due to increased glucose utilization. None of the urine samples contain glucose because the blood glucose level is below renal threshold for glucose which is 175mg%. 099


Questions and Answers

Decreased glucose tolerance :Diabetes mellitus is mainly responsible for the diminished glucose tolerance. Fasting blood glucose values are above 120mg % and depends on severity of disease. After test dose of glucose, blood glucose level rises sharply and extent of increase is more than that seen in normal people. The most striking is high blood glucose level even after 2 hours. In mild diabetes minimum one urine sample contains glucose. All urine samples contain glucose in the case of severe diabetes. Decreased glucose tolerance also occurs in Cushing's syndrome, thyrotoxicosis, hyper activity of pituitary gland and in liver disease. Increased glucose tolerance: Increased tolerance is seen in addison's disease, myxedema, cretinism, hypopituitarism etc. In cases with impaired glucose absorption also increased tolerance occurs. Sprue, celiac disease, and idiopathic steatorrhea are some intestinal disorders associated with increased tolerance. Usually a flat glucose tolerance curve is obtained.

21. Write about glycosurias. A. Glycosurias are conditions associated with excretion of glucose and or sugars in urine. Most common disease is diabetes mellitus. Renal diabetes is another disease of glycosuria where excretion of sugar or glucose in urine is due to defective reabsorption of glucose in renal tubules. Other glycosurias are 1. Lactosuria : It is characterized by excretion of lactose in urine. It occurs in pregnancy. 2. Galactosuria: It is associated with excretion of Galactose in urine. It occurs in galactosemia. 3. Fructosuria: It occurs in hereditary fructose intolerance and associated with excretion of fructose in urine. 4. Pentosuria: It occurs in essential pentosuria and characterized by excretion of pentose xylulose in urine.

22. Test for reducing substances in urine. A. Benedict's test is used for detection of reducing substances in urine. It involves boiling of urine with Benedict's qualitative reagent. Depending on amount of reducing sugar in urine variety of colors are produced. Reducing monosaccharides and disaccharides are detected in urine by performing this test. Under alkaline conditions reducing sugar decomposes to enediols which reduces cupric to red cuprous oxide.

Other model questions are

23. Write about enzymes involved in carbohydrate digestion. 24. Write about diseases of carbohydrate digestion and absorption. 100

CHAPTER - 8 | Carbohydrate Metabolism

25. Write a note on a) Lactose intolerance b) Disaccharidases

26. Write irreversible reactions of glycolysis. 27. Write the significance of citric acid cycle. 28. Define glycogenesis. Write the reactions of this process. 29. Explain how glycogen is degraded? 30. Write a note on glycogen storage diseases. 31. Write the significance of HMP shunt pathway. 32. Write about glucose -6-phosphate dehydrogenase deficiency. 33. Define key enzymes of gluconeogenesis. Write reactions they catalyze. 34. Write significance of gluconeogenesis. 35. Write biochemical symptoms and enzyme defect in the following a) Galactosemia b) Hereditary fructose intolerance c) Essential pentosuria

36. Write sources and routes of of blood glucose. 37. Write the role of liver in blood glucose level maintenance. 38. What is the action of insulin on blood glucose level? 39. Write the extra hepatic tissues role in blood glucose regulation. 40. Explain how hyperglycemic hormones raises blood glucose level. 41. Define glucose tolerance. What conditions it is altered? 42. Expand IDDM and NIDDM. Write briefly about any one of them. 43. Write normal response of glucose tolerance test. 44. McArdle syndrome 45. von Gierk's disease 46. Direct oxidative pathway of glucose.


CHAPTER - 9 | Lipid Metabolism



Lipid Metabolism 1. Name lipids present in diet. Explain how they are solubilized, digested and absorbed? A. Lipids present in the diet are triglycerides, phospholipids, Cholesterol and its esters, fatty acids, glycolipids, carotenes and sterols other than cholesterol. They are present in plant and animal food stuffs. They are vegetable oils, or cooking oils of plant origin and eggs, meat, cheese, milk, butter and fat of animal origin. Solubilization of lipids: Since lipids are insoluble in aqueous environment of lumen digestion of lipids requires their initial solubilization. Bile salts present in bile are responsible for solubilization of dietary lipids. Bile salts form emulsion with dietary lipids. They increases surface of lipid at water inter phase for the action of enzymes. Digestion of lipids Lipid digestion : Hydrolysis of triglycerides, phospholipids and cholesterol esters to glycerol, free fatty acids, mono acylglycerol and cholesterol is known as digestion of lipids. In mouth :Due to lack of favourble conditions no digestion of lipid occurs in the mouth. In the stomach :Mechanical emulsification of food lipids allows action of gastric lipase to some extent. However acidic environment limit action of enzymes on lipids. In the small intestine :Major part of lipid digestion occurs in small intestine by pancreatic enzymes. Pancreatic juice contains lipase, cholesterol esterase and phospholipase. Lipase as such not able to interact with emulsion particle containing dietary lipids. Colipase which is also present in pancreatic juice and bile salts aids lipid digestion by lipase. Pancreatic lipase hydrolyzes ester bonds of 1, 3 carbons of triglycerides. 2monoacylglycerol and free fatty acids are formed. lipase Triglyceride

2-monoacylglycerol + Free fattyacids. colipase

Majority of 2-monoacylglycerol about 72% comes out of emulsion particle and forms mixed micelles. The remaining about 28% is converted to 1-mono acylglycerol by an isomerase. 102

CHAPTER - 9 | Lipid Metabolism


mixed micelles 72%

Isomerase 2-monoacylglycerol 1-monoacylglycerol. 28% Lipase act on 1-monoacylglycerol and hydrolyzes about 22% of monoacylglycerol to glycerol and free fatty acids. The remaining 6%monoacylglycerol is absorbed as such. Lipase Absorbed

1-monoacylglycerol 6%

glycerol + Free fatty acids 22%

Cholesterol esters are hydrolyzed by cholesterol esterase to cholesterol and free fatty acids. Phospholipase A2 hydrolyzes phospholipids ester bond on 2 carbon to form lysophospholipid and free fatty acids. cholesterol Cholesterol ester

cholesterol + Free fatty acid esterase

Phospholipase A2 Phospholipids

lyso phospholipids + Free fatty acid

Lysophospholipase act on lysophospholipid and forms glycero phocholine and fatty acid. Lysophospholipase Lyso phospholipids

Glycerophosphocholine +Free fatty acid

Lipid absorption In the proximal part of jejunum 2-monoacyglycerol, free cholesterol, fatty acids, lysophospholipids interact with bile salt micelles and forms mixed micelles. To the brush border membrane mixed micelles carry these products of digestion where they are absorbed through specific transporter present in enterocyte membrane. Bile Lysophospholipids + monoacylglycerol + cholesterol+ Free fatty acids Salts Micelles Specific Mixed micelles Brush border Cytosol of enterocyte Membrane Transporter

2. Write the fate of absorbed lipids in intestine. Explain how they enter circulation? A. In the enterocyte of intestine mono acylglycerol, free fatty acids are converted to triglycerides. Lysophospholipids are converted to phospholipids by esterification. Cholesterol esters are formed from cholesterol and free fatty acids.



Questions and Answers

Monoacylglycerol + Fatty acids


Lysophospholipid +Fatty acid


Cholesterol + Fatty acids

Cholesterol ester

In the enterocyte triglycerides, phospholipids and cholesterol ester formed combines with proteins to form lipoproteins chylomicrons. These chylomicrons are released into lymph of intestinal lymphatics. Due to absorption of dietary lipids intestinal lymph appears milky and is called as chyle. Finally chylomicrons enters systematic circulation through thoracic duct. apo Triglycerides, phospholipids, cholesterol ester

Chylomicrons Lipoproteins

Intestinal lymphatics

Thoracic duct


For the triglycerides and cholesterol ester synthesis only long chain fatty acid are used. So absorbed short and medium chain fatty acids with glycerol directly enters portal venous blood.

3. Write about diseases associated with lipid absorption or digestion. A. Chyluria :People affected with this disease excretes milky urine due to abnormal connection between intestinal lymphatics and urinary tract. Since only long chain fatty acids are used for resynthesis of lipids it is corrected by replacing diet with short and medium chain containing fatty acids. Chylous fistule is another name for this disease. Chylothorax : It is characterized by accumulation of milky pleural fluid in the pleural space of lungs due to abnormal connection between lungs pleural space and intestinal lymphatics. The condition is corrected by supplying diet containing short and medium chain fatty acids. Pancreatitis : In this condition bile flow is obstructed. As a result digestion of lipid is affected. Cholestasis : In this condition bile flow is blocked. Since bile is required for fat digestion, in cholestasis lipid digestion is affected.

4. Describe fatty acid oxidation. A. Fatty acids are oxidized in the mitochondria of several types of cells. Liver cells, adipocytes, cardiac myocytes, renal cells, Pulmonary cells, muscle cells, and to some extent in neuronal cells of brain. Fatty acid oxidation involves. i. Initial activation in cytosol or outer mitochondrial membrane. ii. Translocation of activated fatty acids into mitochondria iii. Beta (Ă&#x;) –oxidation in mitochondria. 104

CHAPTER - 9 | Lipid Metabolism

i. Fatty acid activation :It involves conversion of fatty acid into corresponding CoA form. Acyl-CoA synthetases converts fatty acids to acyl-CoAs using ATP, CoASH and magnesium ions. They are also called as thiokinases. They are present in outer mitochondrial membrane. ATP is converted to AMP and pyrophosphate (PPi). Pyrophosphatase converts pyrophosphate to phosphate. Acyl-CoA




Fatty acid +ATP

Acyl-CoA + PPi



Mg + AMP ii. Transport of acyl-CoAs into mitochondria : Acyl - CoAs are impermeable to inner mitochondrial membrane. Carnitine translocates activated fatty acids from out side to matrix of mitochondria. It begins with transfer of acyl-CoA to carnitine catalyzed by carnitine acyl transferase-І (CAT-І) present in outer mitochondrial membrane. Acyl carnitine is product of this reaction. Carnitine- acylcarnitine transloc present in inner mitochondrial membrane translocates acyl-carnitine into matrix of mitochondria. In the matrix of mitochondria carnitine-acyl transferase –ІІ (CAT-ІІ) transfers acyl residue to CoA from acyl carnitine and free carnitine is released. To complete the translocation process translocase pumps back carnitine to out side of mitochondria. Carnitine – acyl trans ferase-І (CAT-І) Acyl-CoA+carnitine Acyl-carnitine + CoA Outer mitochondrial Membrane Trans locase Acyl-carnitine

Acyl - carnitine in matrix Inner mitochondrial membrane

carnitine acyltransferase-ІІ (CAT-ІІ) Acyl-carnitine+CoA Acyl-CoA+ carnitine. Matrix of mitochondria Translocase Carnitine

carnitine out side of mitochondria. Carnitine- acylcarnitine

iii. Beta oxidation of fatty acids : As the name implies fatty acid oxidation involves sequential removal of two carbon fragments from carboxy terminus by cleaving fatty acid at beta carbon. An acyl-CoA shorter by two carbon atoms and an acetyl-CoA are



Questions and Answers

products. Reactions of beta oxidation continues until acyl-CoA is completely converted to acetyl-CoA. Acyl-CoA

Beta oxidation Acetyl –CoA + Acyl-CoA shorter by two carbons Beta Oxidation

Acyl-CoA shorter by four carbons


Reactions : 1. First reaction of beta oxidation is dehydrogenation of acyl-CoA by FAD dependent acyl CoA dehydrogenase. Acyl –CoA is converted to enoyl-CoA and FAD H2 isformed. 2. Hydratase catalyzes addition of water across double bond in the second reaction. ß –hydroxyacyl-CoA is product. Acyl-CoA Dehydrogenase Acyl-CoA+FAD

Hydratase Enoyl – CoA

ß-Hydroxy acyl-CoA.


(2) FADH2


3. An NAD+ dependent dehydrogenation occurs in this reaction. Beta hydroxy acyl-CoA dehydrogenase catalyzes this reaction and ß-ketoacyl-CoA is product. 4. Clevage of ß – ketoacyl-CoA at beta carbon by ß – ketothiolase (thiolase) in this reaction yields acyl-CoA that is shorter by two carbons and acetyl-CoA. ß-hydroxyacyl-CoA Dehydrogenase ß-hydroxyacyl-CoA+NAD+

ß-ketoacyl-CoA+ NADH+H+ (3)

ß-ketothiolase ß-ketoacyl-CoA+CoA

Acetyl-CoA+Acyl-CoA shorter by two carbons. (4)

Acyl-CoA shorter by two carbons enters beta oxidation reactions and thus the cycle continues until acetyl-CoA is produced from the acyl- CoA. Energetics of beta oxidation: Energy production by beta oxidation process taking palmitic acid as an example. Since palmitic acid is 16 carbon saturated fatty acid it under goes beta oxidation process seven times and produces 8 Acetyl-CoA s, 7FADH2 and 7 NADH. Acetyl –CoA is completely oxidized in citric acid cycle. FADH2 and NADH are oxidized by respiratory chain. Amount of ATP generated when palmitic acid is oxidized by beta oxidation.


CHAPTER - 9 | Lipid Metabolism

1. ATP generated by oxidation of 8 Acetyl-CoA s in citric acid cycle.

08x12 = 96

2. ATP generated by oxidation of 7 FADH2 in respiratory chain

07x02 = 14

3. ATP generated by oxidation of 7NADH in respiratory chain

07x03 = 21 131

ATP consumed for activation of fatty acid

-2 Net =


Therefore complete oxidation of palmitic acid produces 129 ATP molecules. As per latest concepts only 106 ATPs are generated.

5. Define Alpha oxidation. Write reactions of this process. Name disease associated. A. As the name implies Alpha oxidation involves oxidation of fatty acid by sequential removal of one carbon units from carboxy terminus after cleaving fatty acid at alpha carbon atom. It occurs in peroxisomes. It does not generate energy and requires no CoA intermediates. Reactions: 1. A monoxygenase brings about hydroxylation of alpha carbon of fatty acid in first reaction. Hydroxy fatty acid is produced. 2. Dehydrogenation and oxidative decarboxylation converts hydroxy fatty acids shorter by one carbon atom. Refsum's disease : It is due to block in α- oxidation of phytanic acid. So phytanic acid accumulates in blood and liver. Main symptoms are peripheral neuropathy, abonormalities in skin and bone. Symptoms disappear on consuming phytanic acid free diet.

6. Write about omega oxidation. A. As the name implies Omega (ω) oxidation involves oxidation of fatty acid by oxidizing omega (ω) carbon to carboxylic group. It occurs in smooth endoplasmic reticulum. Reactions : 1. A cyt P450 dependent mixed function oxidase first catalyzes hydroxylation of carbon of fatty acid. ω– hydroxy fatty acid is product. 2. Further oxidation at ω-carbon generates di carboxylic acid which under goes beta oxidation.

7. Name ketone bodies. Describe metabolism of ketone bodies. A. Ketone bodies are acetone, acetoacetic acid and beta hydroxybutyric acid. Ketone body metabolism consist of a. Ketogenesis and b. Ketolysis. Ketogenesis : Synthesis of ketone bodies is known as ketogenesis. It occurs in liver. Acetyl CoA is precursor.



Questions and Answers

Reactions : 1. Ketogenesis begins with condensation of two acetyl –Co A molecules catalyzed by thiolase. Acetoacetyl-CoA is product. Thiolase 2 Acetyl –CoA

Acetoacetyl –CoA+ CoA. (1)

2. Acetoacetate forms from acetoacetyl- CoA by two ways. a. In this route acetoacetyl –CoA condenses with another molecule of acetyl –CoA to form beta hydroxy beta methyl glutaryl-CoA (HMG-CoA) catalyzed by HMG –CoA synthase. A lyase catalyzes splitting of HMG –Co A to acetoacetate and acetyl CoA. HMG - CoA Synthase Acetoacetyl-CoA+Acetyl- CoA beta hydroxybeta methyl glutaryl -CoA+ 2a CoA. HMG CoA lyase HMG-CoA

acetoacetate+ Acetyl-CoA. 2a

b. In another route decarboxylation of acetoacetyl- CoA by deacylase yields aceto acetate Deacylase Acetoacetyl-CoA Acetoacetate+ CoA. 2b 3. ß- hydroxybutyrate is formed from acetoacetate. NADH dependent dehydrogenase catalyzes this reaction. Dehydrogenase Aceto acetate +NADH +H+

beta hydroxybutyrate +NAD+ (3)

4. Spontaneous decarboxylation of acetoacetate yields acetone. Spontaneous Acetoacetate

Acetone+CO2. (4)

Significance : Under certain conditions citric acid cycle is unable to produce energy from entire acetyl-CoA generated either from beta oxidation or pyruvate. This excess acetyl –CoA are converted to ketone bodies in liver. . Liver distributes ketone bodies thus generated among various organs. So ketogenesis allow distribution of


CHAPTER - 9 | Lipid Metabolism

excess fuel (Acetyl –CoA) among organs. These ketone bodies produced in liver reaches various organs through systemic circulation. They are taken by peripheral tissues for utilization. Ketolysis : Ketolysis is the degradation of ketone bodies. It occurs in cardiac muscle, brain, kidney and to some extent by skeletal muscle. Utilization of acetoacetate Reactions : 1. Activation of aceto acetate is first reaction of its utilization. Acetoacetyl-CoA synthase an ATP and magnesium dependent enzyme converts aceto acetate to corresponding CoA. AMP and PPi are products. PPi is further hydrolyzed by pyrophospahtase. 2. Thiophorase or acetoacetate- succinyl-CoA transferase transfer CoA from succinyl –CoA to acetoacetate. This is another mode of activation. Acetoacetyl-CoA Synthase Acetoacetate+ ATP +CoA Acetoacetyl –CoA + AMP+PPi Pyro phosphatase (1) PPi 2Pi. (1) Thiophorase Acetoacetate+succinyl- CoA

Acetoacetyl-CoA+ succinate. (2)

A thiolase cleaves acetoacetyl –CoA to two molecules of acetyl-CoA. These acetylCoA s are utilized by citric acid cycle. Thiolase AcetoacetylCoA+ 2 acetyl –CoA CoA (3)

TCA cycle


Utilization of beta hydroxy butyrate Beta hydroxy butyrate is utilized by two ways. Reactions: 1. A dehydrogenase converts beta hydroxy butyrate to aceto acetate which is used for energy production as detailed above. 2. In a minor route a synthetase activates beta hydroxybutyrate to beta hydroxyl butyryl –CoA. Dehyrogenation of beta hydroxybutyryl-CoA yields acetoacetyl-CoA. dehydrogenase ß- hydroxybutyrate +NAD


NADH+H+Aceto acetate (1)

TCA Cycle.



Questions and Answers

Synthetase Dehydrogenase ß- hydroxybutyryl -CoA Acetoacetyl CoA. (2) (2) CoA The acetoacetyl-CoA is converted acetyl-CoA as described above. ß-hydroxybutyrate

Utilization of Acetone Utilization of acetone by peripheral tissues is slow. Usually it is removed in urine or as Co2 through lungs. Significance : Some tissues like cardiac tissue and kidney prefers ketone bodies for energy production than glucose. Ketone body utilization for energy production is more significant in prolonged starvation. Regulation of ketone body metabolism : 1. Ketogenesis largely depends on mobilization of free fatty acids. 2. CAT-І is mainly involved in controlling ketone body formation. In fed conditions CAT –І activity is more so more acetyl –CoA is formed from beta oxidation. Hence ketogenesis is more in starvation. Medical importance : 1. Normal blood ketone body level is 1mg%. Under normal conditions ketone body formation is balanced by their utilization. So ketone body level in blood remains constant. 2. Ketosis: If ketogenesis is more than ketolysis accumulation of ketone bodies in blood occurs. It is known as ketonemia. Excess ketone bodies are excreted in urine. It is known as ketonuria. Ketonemia and ketonuria gives rise to ketosis. Symptoms are headache, vomiting and coma. Kotosis occurs in i. Prolonged starvation. ii. Diabetes mellitus iii. von Geirke's disease iv. Fever. v. Severe muscular exexcise. 3. Ketoacidosis: It occurs in uncontrolled diabetes mellitus and in prolonged starvation due to depletion of blood bicarbonate. To maintain normal blood pH ketone bodies are usually neutralized by bicarbonate buffer. But ketone bodies are produced in excess in uncontrolled diabetes mellitus. So more bicarbonate is needed for neutralization and blood bicarbonate depletion occurs. This leads to decreased blood PH i. e. Acidosis. The condition is called as keto acidsis because acidosis is due to more ketone bodies.

8. Describe fatty acid biosynthesis De novo. A. Site :Cytosol of liver, adipose tissue, lung, mammary gland, brain and kidney contain enzyme system for fatty acid biosynthesis. Precursor : Acetyl-CoA of pyruvate oxidation, NADPH of HMP shunt and cytosolic malic enzyme are precursors of fatty acid synthesis.


CHAPTER - 9 | Lipid Metabolism

Transport of acetyl-CoA : Acetyl-CoA which is precursor of fatty acid biosynthesis is formed in mitochondria. But fatty acid synthesis occurs in cytosol and acetyl-CoA is impermeable to mitochondrial membrane. Since mitochondrial membrane is permeable to citrate, acetyl –CoA enters cytosol in the form of citrate. 1. As a part of citric acid cycle, citrate is formed form acetyl-CoA. A tricarboxylate transporter present in mitochondrial membrane transports citrate out of mitochondria. 2. In the cytosol acetyl- CoA is regenerated by ATP : Citrate lyase from citrate. Citrate




Acetyl—CoA+ oxaloacetate


Acetyl-CoA + ATP


Oxaloacetate + ADP + Pi 3. Oxaloacetate is converted to malate by cytosolic malate dehydrogenase. 4. A cytosolic malic enzyme converts malate to pyruvate in presence of NADP+. Malate Dehydro





Pyruvate + CO2 +NADPH + H+.

Malate (3) +

(4) +




The transport of acetyl-CoA is accompanied by formation of NADPH in cytosol. This NADPH and acetyl –CoA are used for fatty acid synthesis.

Fatty acid synthase complex In the cytosol fatty acid synthase complex synthesizes fatty acids by using acetyl –CoA and NADPH. This multi enzyme complex is dimer consisting two monomers or subunits. Each monomer has two sulfhydryl (SH)groups, activities of seven enzymes and an acyl carrier protein (ACP). Phosphopantothein of ACP contributes one-SH at one end and another –SH is contributed by cysteine residue of one of seven enzymes. The two monomers are arranged in head to fail manner. Cysteine –SH of one monomer is in close proximity with phosphopantothein –SH of another monomer. Individual monomers are inactive only dimer is active. Functional unit consist of one half one monomer and complementary half of another monomer. Hence two fatty acids are produced at a time.



Questions and Answers















Fatty acid synthase complex. Reactions : Multi enzyme complex uses only one acetyl-coA as such rest of acetyl – CoA s are used in the form of malonyl-CoA. Formation of malonyl-CoA : Acetyl- CoA is converted to malonyl-CoA by carboxylation which depends on biotin and energy. Acetyl-CoA carboxylase is the enzyme that catalyzes this reaction. Acetyl-CoA Carboxylase Acetyl-CoA + ATP+ CO2

Malonyl-CoA+ ADP+ Pi Biotin

Fatty acid synthase reactions :The availability of acetyl –CoA, NADPH initiates fatty acid synthase dependent reactions of de novo fatty acid synthesis. 1. First reaction of fatty acid synthase complex is transfer of acetyl –CoA to Cysteine-SH of fatty acid synthase complex which is catalyzed acetyl trans acylase. Acetyl trans Acylase Acetyl –CoA + Fatty acid synthase complex Acetyl enzyme complex + CoA (1) 2. A molecule of malonyl –CoA is transferred to pan-SH of other monomer of fatty acid synthase complex. Malonyl trans acylase catalyzes this reaction. Malonyl trans Acylase Acetyl enzyme complex + malonyl –CoA Acetyl –malonyl enzyme +CoA. (2) 3. A condensing enzyme ß- ketoacyl synthase catalyzes condensation of acetyl and malonyl groups. A keto acyl enzyme complex is formed. Cysteine –SH group of the monomer becomes free ketoacyl Synthase Acetyl –molonyl enzyme complex

ß-ketoacyl enzyme complex. (3)

4. An NADPH dependent ketoacyl reductase reduces ketoacyl group to hydroxyacyl group. ß-hydroxyacyl enzyme is formed. 112

CHAPTER - 9 | Lipid Metabolism

ketoacyl reductase ß-ketoacyl enzyme complex +NADPH +H+ ß- hydroxyl acyl enzyme +NADP. (4) 5. In this reaction a water molecule is removed from ß-hydroxyacyl enzyme by hydratase. Enoyl enzyme is formed. 6. Another NADPH dependent reaction of enoyl reductase generates butyryl enzyme. The four carbon butyryl moiety is on the phosphopantotheine-SH of enzyme complex. Hydratase Enoyl Reductase ß- hydroxyacyl enzyme Enoyl enzyme Butyryl enzyme +NADP+. (5) (6) H2O NADPH+H+ 7. All of the above multi enzyme reactions are repeated six times incorporating malonyl –CoA each time to generate palmitoyl moiety. 8. Finally palmitoyl moiety is released from multi enzyme complex by last enzyme of complex thioesterase. Thioesterase Butyryl enzyme

palmitoyl enzyme

(7) palmitic acid + fatty acid synthase complex.


Regulation of fatty acid synthesis : De novo biosynthesis of fatty acids is subjected to both allosteric and hormonal regulation. Acetyl CoA carboxylase is regulatory enzyme. Allosteric control : Acetyl –CoA carboxylase exist in two forms an active form and inactive form. Polymer of acetyl –CoA carboxylase is a active form. Monomer is inactive form. Citrate is a allosteric activator and long chain acyl-CoA is allosteric inhibitor. Further activity of acetyl-CoA carboxylase is inversely related to plasma free fatty acid level. Hence in starvation and diabetes due to increased fatty acid level synthesis of fatty acids is inhibited. Hormonal regulation :Glucagon inhibits fatty acid synthesis where as insulin promotes fatty acid synthesis. These hormones act by cAMP mediated phosphorylation of acetylCoA carboxylase.

9. Explain how triglyceride are synthesized? A. Synthesis of triglycerides : Triglycerides are synthesized in liver, adipose tissue, and intestine. In liver and adipose tissue dihydroxyacetone phosphate of glycolysis is used for triglyceride biosynthesis. However liver is able to synthesize triglycerides from glycerol also. In intestine triglycerides are formed from monoacylglycerol pathway. Synthesis of triglycerides from glycerol and dihydroxy acetone phosphate : 1. In liver glycerol and dihydroxy acetone phosphate are converted to glycerol 3- phosphate.



Questions and Answers

The former reaction is catalyzed by kinase and latter is catalyzed by NADH dependent dehydrogenase. Glycerokinase Glycerol

Dehydrogenase Glycerol-3-phosphate Dihydroxy acetone phosphate

(1) ATP

(1) NAD+



2. Now incorporation of fatty acids into glycerol-3- phosphate occurs. Activated long chain fatty acids of both saturated and unsaturated are used. Lysophosphatidate is product. Acyl transferase catalyzes this reaction. 3. Another fatty acid incorporation leads to phosphotidate formation. Acyl Transferase Glycerol-3- phosphate

Acyl Transferase Lysophosphatidate

(2) Acyl-CoA CoA


(3) Acyl-CoA CoA

4. Removal of phosphate from phosphatidate by phosphatase yields 1, 2-diglyceride. 5. In the intestine monoacyl glycerol is converted to 1, 2 –diglyceride by incorporation of fatty acid. Phosphatase Acyl Transferase Phosphatidate 1, 2 – diglyceride Monoacyl glycerol. (4) (5) pi CoA Acyl-CoA 6. Transfer of another acyl-CoA to 1, 2 –diglyceride by transferase produces triglyceride. Acyltrans ferase 1, 2- diglyceride

Triglyceride+CoA. (6) Acyl-CoA

Significance:Triglyceride biosynthesis is linked to fatty acid biosynthesis. In well fed state triglyceride biosynthesis is more. In starvation and diabetes triglyceride biosynthesis is less

10. Explain triglyceride degradation or lipolysis. Add a note on hormonal action on lipolysis. A. Hormone sensitive lipase present in adipose tissue hydrolyzes triglycerides to free fatty acids and di or monoglycerides. Di or monoglyceride lipase hydrolyzes monoglycerides or diglycerides to glycerol and fatty acids. Hormone sensitive Triglycerides free fatty acids +mono or diglycerides Lipase 114

CHAPTER - 9 | Lipid Metabolism

Mono or diglyceride Mono or diglycerides Glycerol+ free fatty acids. Lipase. Triglyceride breakdown is more in energy deficient and stress conditions. In starvation and diabetes also triglyceride breakdown is more. Action of hormones on lipolysis Hormones like insulin, glucagon, epinephrine, nor epinephrine, glucocorticoids, growth hormone etc. affects triglyceride breakdown. Glucagon, epinephrine and glucocorticoids stimulates lipolysis. Insulin antagonizes lipolysis. As the name implies these hormones affect activity of hormone sensitive lipase. It exist in two forms an active form and an inactive form. Lipolytic hormones keeps this enzyme in active form by promoting cAMP dependent phosphorylation. In contrast, insulin suppresses lipolysis by inhibiting cAMP dependent phosphorylation. Phosphorylation Hormone sensitive lipase (inactive)

hormone sensitive lipase (active)

(+) Epinephrine, glucagon etc



of hormone sensitive


lipase (+) activation (-) inhibition

11. Define fatty liver. What are the causes ? A. Abnormal accumulation of lipid or fat in the liver is known as fatty liver. Usually lipid content of liver does not exceed 5% but in fatty liver the lipid content raises to 25-30 %. Several factors cause abnormal accumulation of lipid. They are 1. Increased free fatty level in plasma :Mobilization of fat causes increased plasma free fatty acid level. These excess fatty acids are taken up by liver and converted to triglycerides. However proteins required for the formation of lipoprotein VLDL occurs at normal rate. This leads to accumulation of lipid in liver. Raised plasma free fatty acid level occurs in i. Diabetes ii. High fat diet. iii. Starvation. iv. Malnutrition. Hence fatty liver occurs in all these conditions 2. Due to block in lipoprotein production :If lipoprotein production particulerly VLDL is blocked due to lack of substances required for its formation fatty liver occurs even at normal rate of triglyceride synthesis. Because for triglyceride movement from liver to peripheral tissues VLDL is needed. However supply of deficient substance prevents fat accumulation.



Questions and Answers

12. Define lipotrophic factors. Give examples. A. Lipotrophicfactors : Are those substances or compounds that prevent accumulation of fat in liver. They are choline, methionine, betaine, vit. E, pyridoxine, poly unsaturated fatty acids (PUFA) and pantothenicacid. They cure fatty livers.

13. Describe cholesterol biosynthesis. A. Biosynthesis of cholesterol : Cholesterol biosynthesis occurs in cytosol and microsomes of most of the cells of the body. Some of the organs of cholesterol biosynthesis are liver, adrenal cortex, testis, ovaries, brain, placenta, skin and blood vessels. Precursors :Acetyl-CoA of pyruvate, aminoacids and fatty acids and NADPH of HMP shunt are precursors for cholesterol formation. Reactions : 1. Condensation of two acetyl –CoA molecules catalyzed by ß- keto thiolase is the first reaction of cholesterol biosynthesis. 2. Aceto Acetyl-CoA formed in the initial reaction condenses with another molecule of acetyl-CoA catalyzed by HMG-CoA synthase. In this reaction HMG- CoA serve as source of isoprenoid units of cholesterol biosynthesis. ß-keto HMG-CoA Thiolase synthase 2Acetyl –CoA Acetoacetyl – CoA HMG-CoA + CoA (1) (2) CoA Acetyl-CoA 3. Reduction of HMG-CoA by an NADPH dependent HMG-CoA reductase is the third reaction. Mevalonate is product. 4. An ATP dependent phosphorylation of mevalonate by mevalonate phosphotransferase occurs in this reaction. Mevalonate-5- phosphate is product. HMG-CoA HMG-CoA



Mevalonate phosphotransferase

(3) 2NADPH+2H






5. Another phosphorylation catalyzed by a kinase is fifth reaction. Mevalonate-5-pyrophosphate is product. kinase Mevalonate-5- phosphate + ATP

Mevalonate –5-pyrophosphate+ ADP (5)

6. An ATP dependent decarboxylation catalyzed by decarboxylase converts mevalonate5- pyrophosphate to isopentenyl pyrophosphate (IPP). 116

CHAPTER - 9 | Lipid Metabolism

Decarboxylase Mevalonate-5- pyrophosphate +ATP Isopentenyl pyrophosphate +ADP + (6) Pi + Co2 7. Isomerization of isopentenyl pyrophosphate to dimethyl allyl pyrophosphate (DMAP) by an isomerase occurs in this reaction. Isomerase Isopentenyl pyrophosphate

Dimethyl allyl pyrophosphate. (7)

8. The remaining reactions of cholesterol biosynthesis is carried out by two isoprenoid isomers. A condensation reaction between two isomers catalyzed by prenyl transferase generates geranyl pyrophosphate. Prenyl Transferase Dimethyl allyl pyrophosphate + isopentenyl pyrophosphate (8) Geranyl pyrophosphate + PPi. 9. Gerenyl pyrophosphate condenses with one molecule of isopentenyl pyrophosphate catalyzed by farnesyl pyrophosphate synthase. Farnesyl pyrophosphate is product. Farnesyl pyrophosphate synthase Gerenyl pyrophosphate + isopentenyl pyrophosphate Farnesyl (9) pyrophosphate + PPi. 10. Two molecules of farnesyl pyrophosphate undergo condensation in this reaction. Squalene synthase catalyzes this reaction. Squalene is product. 11. NADPH dependent squalence monoxygenase catalyzes oxidation of squalene to squalene -2-, 3-epoxide. Squalene Synthase Farnesyl pyrophosphate

Squalene mono Oxygenase Squalene Squalene-2, 3(10) (11) PPi NADPH+H+O2

epoxide+NADP++H2O. 12. Formation of lanosterol by cyclization of squalene-2, 3 –epoxide occurs in this reaction. It is catalyzed by squalene oxidocyclase. Squalene oxido Squalene-2, 3- epoxide






Questions and Answers

13. Removal of three methyl groups and double bond shifting leads desmosterol formation from lanosterol. 14. Finally cholesterol is formed from desmosterol Lanosterol

Desmosterol (13)

cholesterol. (14)

15. In the skin cholesterol is formed from lanosterol via 7-dehydro cholesterol. Lanosterol

7- dehydrocholesterol (15)

cholesterol. (15)

14. Write about cholesterol transport in the body. A. It consist of a. Transport of dietary and hepatic cholesterol b. Extra hepatic tissue cholesterol transport. a. Transport of dietary and hepatic cholesterol :Dietary cholesterol is transported to liver after incorporation into chylomicrons. Cholesterol formed in the intestine is transported to liver in the same way. In the liver cholesterol is released from chylomicrons. In the liver dietary cholesterol and cholesterol synthesized are incorporated into VLDL and LDL and they are secreted into plasma. However LDL contains highest proportion of cholesterol. Through receptor mediated endocytosis LDL are taken up by extra hepatic tissues where cholesterol is released. The released cholesterol may be stored or used for cell membrane. LDL cholesterol is known as bad cholesterol because its accumulation leads to atherosclerosis. b. Extra hepatic tissue cholesterol transporter or reverse cholesterol transport : Extra hepatic tissue free cholesterol is esterified to fatty acid of HDL lecithin by cholesterol lecithin acyl transferase. As a result lecithin is converted to lysolecithin. Cholesterol+ fatty acid of HDL lecithin.

Cholesterol ester +lysolecithin.

The cholesterol ester formed migrates into core of HDL and transported to liver. In the liver cholesterol is eliminated as bile acids. This cholesterol transport is known as reverse cholesterol transport. HDL cholesterol is known as good cholesterol because transport of peripheral tissue cholesterol by HDL to liver lowers plasma cholesterol level.

15. Write normal plasma cholesterol level. In what conditions it is elevated? How plasma cholesterol level is lowered? A. Normal plasma cholesterol level is about 150-200mg %. Plasma cholesterol level is elevated in atherosclerosis, coronary artery disease, diabetes, nephrotic syndrome, hypothyroidism, obstructive jaundice and xanthomatosis. Some drugs are used to lower cholesterol level in blood. They are known as cholesterol lowering drugs or


CHAPTER - 9 | Lipid Metabolism

hypocholesterolemic drugs. Lovastatin is competitive inhibitor of HMG-CoA reductase. Compactin or mevastatin is another inhibitor used to lower cholesterol level in plasma. Nicotinicacid, D-thyroxine, questran, clofibrate etc. are also used to lower plasma cholesterol level. Other model questions are

16. Name the enzymes of lipid digestion. 17. Explain the role of bile salts in lipid digestion and absorption. 18. How products of lipid digestion are absorbed and transported? 19. Define beta oxidation. Write the reactions of this process. 20. Explain the role of carnitine in fatty acid oxidation. 21. Write the energetics of palmitic acid oxidation. 22. How HMG-CoA is formed? Write its fate. 23. What is normal ketone body level in plasma? In what diseases it is elevated? 24. Write a note on ketosis and ketoacidosis. 25. Explain how ketone bodies are formed? 26. Write about utilization of of ketone bodies. 27. Write about the structural features of fatty acid synthase complex. 28. Write the role of citrate in fatty acid synthesis. 29. Write about fatty livers. 30. Briefly write on lipotrophic factors. 31. Explain roles of HDL and LDL in cholesterol transport. 32. Write the reaction catalyzed and importance of LCAT. 33. How triglycerides are formed in adipose tissue? 34. Write the role of NADPH in lipid metabolism. 35. Write about utilization of acetyl –CoA in lipid s formation. 36. Blood cholesterol level lowering drugs.


CHAPTER - 10 | Protein and Aminoacid Metabolism



Protein and Aminoacid Metabolism 1. Name protein sources of food. Explain digestion of dietary proteins, A. Food Proteins : Animal foods like meat, eggs, milk, fish and plant foods like cereals, legumes, nuts, pulses, vegetables and fruits are protein sources in diet. Digestion and absorption of proteins Protein digestion : Hydrolysis of dietary proteins into amino acids is known as protein digestion. In the mouth : No protein digestion takes place in mouth due to lack of protein digesting enzymes. In the stomach : In acid environment of stomach dietary protein undergo de naturation. This acid induced protein denaturation aids protein digestion. Pepsin is protein splitting enzyme present in gastric juice. It is active in the acid environment of stomach i. e pH 1. 52. 5. It hydrolyzes peptide bonds of proteins and specific for peptide bonds in which amino group is contributed by acidic or aromatic amino acids. Pepsin converts proteins to peptones and proteoses. Pepsin Protein

Proteoses + peptones.

pH 1. 5-2. 5 In the infant stomach rennin is present. It causes coagulation of milk. In the small intestine : Small intestine is the major site of protein digestion. Succus entericus of small intestine and pancreatic juice contains several proteases and peptidases. These enzymes converts peptones and proteoses to amino acids. Proteases present in pancreatic juice are trypsin, chymotrypsin, elastase, collagenase and carboxy peptidase. Except carboxy peptidase all other proteases are endopeptidase. They act on peptones and proteoses and convert them to oligopeptides. Trypsin Proteoses

chymotrypsin Oligopeptides, peptones


Carboxy peptidase is an exopeptidase. It hydrolyzes peptide bonds of proteins from


CHAPTER - 10 | Protein and Aminoacid Metabolism

carboxy terminus and release one amino acid and polypeptide shorter by one amino acid. The action of carboxy peptidase continues until a di peptide is formed. Carboxy Protein

Amino acid + protein shorter by one amino acid peptidase


Protein shorter by one amino acid

Di peptide

+Amino acids Aminopeptidase is an exopeptidase and hydrolyzes peptide bond of oligo peptide from amino terminus and releases an amino acid and oligopeptide shorter by one amino acid. The action of amino peptidase continues on oligo peptide until it is converted to dipeptide. Aminopep Oligopeptide

Oligopeptide shorter by amino acid + aminoacid tidase Amino peptidase

Oligo peptide shorter by one amino acid

Amino acids + dipeptide.

Di peptidase hydrolyzes dipeptide into amino acids. Dipeptidase Dipeptides

Amino acids.

2. How products of protein digestion are absorbed? Mention about disorders associated. A. Protein digestion products absorption : Amino acids produced from dietary proteins in the lumen are absorbed into portal blood. Mediated transport or secondary active transport is major mechanism of amino acid absorption. Various classes of amino acids are absorbed by different carriers present in enterocyte membrane. There are five different carriers for five different classes of amino acids. For neutral amino acids one carrier, methionine and phenyl alanine another carrier, acidic amino acids third carrier, basic amino acids fourth carrier and fifth carrier for iminoacids. All these carriers are symporters like glucose transporter. They allow sodium transport along with aminoacids. In some diseases aminoacid absorption and protein digestion are affected. They are a. Celiac disease : It is due to absorption of oligo peptides of wheat protein gluten. These peptides are produced from gluten by action of protein digesting enzymes. Further they act as antigens and produce immune response in children. Symptoms are inflammation and atrophy of intestinal mucosa. This results in impaired absorption in the small intestine. b. Non tropical sprue :It is due to absorption of gluten of oat oligo peptides and symptoms are similar to those of celiac disease. Gluten free diet consumption relieves symptoms.



Questions and Answers

c. Hartnup disease : In this condition aromatic amino acid carrier in the intestine is defective. So their absorption is blocked. d. Pancreatitis : In this condition protein digestion is affected due to block in flow of pancreatic juice which contains enzymes of protein digestion.

3. Explain with examples various types of aminoacid deamination. A. Amino acid deamination is removal of amino group of amino acids. It is the first step of amino acid degradation. It occurs by several ways. They are a. Transamination followed by oxidative deamination. b. Oxidative deamination c. Non oxidative deamination A. Transamination followed by oxidative deamination :It involves initial transfer of amino group of aminoacid to α-ketoglutarate followed by oxidative deamination of glutamate that is formed by trans amination. Transaminases catalyzes transfer of amino group of amino acids to α-ketoglutarate. They are present in most of the tissues. They require pyridoxal phosphate as coenzyme. Among many transaminases alanine transaminase and aspartate transaminase are most important. Both of them transfer amino groups of alanine and aspartate to α-ketoglutarate. Alanine Transaminase Alanine+α-ketoglutarate

Pyruvate + glutamate P. Po4 Aspartate

Aspartate +α-ketoglutarate

Oxaloacetate + glutamate. Transaminase

The amino group that is collected is removed from glutamate as ammonia by oxidative deamination catalyzed by glutamate dehydrogenase. Glutamate Dehydrogenase Glutamate +H2O + NADP+ α-ketoglutarate + Ammonia + NADPH +H+. B. Oxidative deamination :This type of deamination of amino acids is catalyzed by amino acid oxidases. They are of two types. a. D-amino acid oxidase b. L- amino acid oxidase. L- amino acid oxidase catalyzes oxidative deamination of all amino acids except glycine and it is FMN dependent enzyme. D- amino acid oxidase acts on glycine and it is an FAD dependent enzyme. These enzymes first oxidizes amino acid to an imino acid which is followed by hydrolytic loss of ammonia. Further they produce hydrogen peroxide.


CHAPTER - 10 | Protein and Aminoacid Metabolism

L-Amino acid oxidase L- Amino acid

Imino acid FMN FMNH2

α-keto acid + Ammonia H2O

D-Amino acid oxidase D- Amino acid

Imino acid

α-keto acid + Ammonia.



FMNH2 + O2

FMN + H 2 O2

FADH2 + O2

FAD +H2 O2.

Non oxidative deamination : Specific enzymes catalyzes non oxidative deamination of aminoacids. Serine dehydratase catalyzes non oxidative deamination of serine to pyruvate. Cysteine desulfhydrase catalyzes conversion of cyteine to pyruvate. Threonine dehydratase catalyzes conversion of threonine to α- ketobutyrate. Serine dehy Serine

pyruvate + Ammonia dratase Cysteine


Pyruvate+ Ammonia+ H2S

desulfhydrase Threonine Threonine

α- ketobutyrate +Ammonia.


4. Write normal plasma ammonia level. How ammonia is transported from brain and skeletal muscle to liver ? A. Normal plasma ammonia level is 10-20 µg %. Ammonia produced in peripheral tissue and brain is transported to liver in the form of amino acids alanine and glutamine. More over ammonia in free form is toxic to central nervous system. In the skeletal muscle ammonia is used for the formation of glutamate from α-ketoglutarate by the reversal of glutamate dehydrogenase. Trans amination transfers aminogroup to pyruvate to form alanine. Glutamate α- ketoglutarate + ammonia +NADPH+ H


Glutamate +H2O + NADP+




Questions and Answers

Alanine Glutamate+ pyruvate

α-keto glutarate +Alanine

Transaminase From the brain and other peripheral tissues ammonia produced is transported to liver in the form of glutamine. Glutamine synthetase catalyzes this reaction. Glutamine Glutamate + ammonia +ATP

Glutamine +ADP+Pi. Synthetase

5. How ammonia is removed from alanine and glutamine in liver? A. In the liver, kidney and intestine ammonia is removed from glutamine by glutaminase. Glutaminase Glutamine


In the liver ammonia is removed from alanine by transamination followed by glutamate dehydrogenase action. Alanine + α-ketoglutarate

Pyruvate +glutamate

α-ketoglutarate +NH3.

6. Describe Urea cycle. Add a note on disorders of urea cycle. A. Urea cycle is present in liver. Enzymes of this cycle are located in mitochondria and cytosol of hepatocytes. It converts toxic ammonia to non toxic urea. First two reactions occurs in mitochondria and remaining three reactions takes place in cytosol. All the intermediates of urea cycle are amino acids with out codons. Formation of urea from ammonia requires energy in the form of ATP. For the formation of urea molecule only one ammonia molecule is used as such another ammonia molecule is contributed by amino group of aspartate. Carbon dioxide or bicarbonate serve as source of carbon for urea formation. Since reactions of urea cycle are proposed by Krebs and Henseleit it is known as Krebs- Heneseleit cycle. Reactions : 1. Condensation of ammonia and bicarbonate at the expense of two high energy bonds to form carbamoyl phosphate is the first reaction of urea cycle. Mitochondrial carbamoyl phosphate synthetase–І (CAPS-І) catalyzes this reaction. N-acetyl glutamate and magnesium are cofactors required. Carbamoyl phosphate Synthetase-І (1) Ammonia +Bicarbonate+2ATP N-acetylglutamate


Carbamoyl phosphate + Pi+2ADP.

CHAPTER - 10 | Protein and Aminoacid Metabolism

2. Carbamoyl phosphate condenses with ornithine in the second reaction. The reaction is catalyzed by ornithine trans carbamoylase and citrulline is the product. Ornithine trans Carbamoylase Carbamoyl phosphate +ornithine

Citrulline +Pi. (2)

Since cytosol is the site for remaining reactions of urea cycle citrulline comes out of the mitochondria through a transporter present in mitochondrial membrane. 3. In the cytosol condensation of citrulline and aspartate yields arginino succinate. It is catalyzed by arginino succinate synthetase and two high energy bonds are used. ATP is hydrolyzed to AMP and PPi. Arginino Succinate Synthetase Citrulline +aspartate + ATP

Arginino Succinate +AMP+ PPi. (3)

PPi is further hydrolyzed by pyrophosphatase. Pyrophosphatase PPi

2Pi. (3)

4. Cleavage of arginino succinate by arginino succinase occurs in this reaction. Arginino Succinase Arginino Succinate

Arginine +Fumarate. (4)

5. Finally ornithine is regenerated from arginine by arginase releasing urea. Arginase Arginine

Urea +ornithine. (5)

Ornithine enters mitochondria for continuation of urea cycle through a transporter present in mitochondrial membrane. Disorders of urea cycle Diseases due to deficiency of enzyme of urea cycle are known as urea cycle disorders. They are inherited diseases. Ammonia toxicity occurs in this diseases because conversion of ammonia to urea is blocked. Some clinical symptoms commonly seen in these cases are vomiting, irritability, lethargy, mental retardation, seizures, coma and death. Some of them are given below.



Questions and Answers

1. Hyper ammonemia Type –І: Carbamoyl phosphate synthetase is deficient in this condition. So ammonia accumulates because its conversion to carbamoyl phosphate is blocked. Mental retardation is major symptom of this disorder. 2. Hyper ammonemia Type –ІІ : Ornithine trans carbamoylase is deficient in this condition. Among urea cycle disorders it is most common. So carbamoyl phosphate accumulates and diverted to pyrimidine nucleotide formation. As a result in urine intermediates of pyrimidine nucleotide formation like orotic acid and uracil are excreted. 3. Citrullinemia :Arginino succinate synthetase is absent in this condition. So citrulline accumulates in blood due to block in its utilization and leads to citrullinemia. Excess citrulline is excreted in urine. 4. Arginino Succinic aciduria :This condition is due to absence of arginino succinase. Hence arginino succinic acid accumulates in blood and get excreted in urine. 5. Hyper argininemia :It is due to deficient arginase. So arginine conversion to urea and ornithine is blocked and accumulation in blood leads to excretion in urine. 6. HHH Syndrome: It is due to defective ornithine transporter. So ornithine accumulates and carbamoylation of lysine occurs leading to formation of homocitrulline. Hyper ornithinemia, hyper ammonemia and homocitrullineia are seen in affected persons and hence name HHH syndrome.

7. Define carbon skeletons of aminoacids. Classify aminoacids giving examples based on fate of their carbon skeletons. A. After removal of amino group of amino acid the remaining structure of a amino acid is the carbon skeleton. Twenty different amino acids gives rise to twenty different carbon skeletons. Based on fate of carbon skeleton amino acids are classified into 1. Glucogenic amino acids. 2. Ketogenic aminoacids. 3. Glucogenic and ketogenic amino aids. 1. Glucogenic aminoacids: Are those aminoacids whose carbon skeletons are converted to either glucose or intermediates of TCA cycle. Final products of these aminoacids are pyruvate, oxaloacetate, α-ketoglutarate, fumarate and succinate. Examples :Glycine, alanine, valine, serine, threonine, aspartate, glutamate, aspargine, glutamine, cysteine, methionine, histidine, arginine and proline. 2. Ketogenic aminoacids :Are those amino acids whose carbon skeletons are converted to either fat like substance or intermediates of fatty acid oxidation. Final products of these aminoacids are acetyl –CoA or acetoacetyl –CoA.


CHAPTER - 10 | Protein and Aminoacid Metabolism

Examples : Leucine is the only ketogenic amino acid. Isoleucine, phenylalanine, tyrosine, tryptophan and lysine are also ketogenic amino acids. 3. Glucogenic and ketogenic amino acids :Are those amino acids whose carbon skeletons are converted to glucose or intermediates of TCA cycle or fat like substances. Final products of these amino acids are pyruvate, oxaloacetate, succinate, fumarate, αketoglutarate, acetyl- CoA and acetoacetyl –CoA. Examples : Lysine, Phenyl alanine, tyrosine, tryptophain and isoleucine.

8. Describe glycine Metabolism. A, Glycine metabolism consist of a. Glycine synthesis b. Glycine degradation. Glycine synthesis 1. Glycine synthesis from serine :Serine trans hydroxy methylase converts serine to glycine. Tetrahydrofolate is coenzyme. Serine trans Hydroxymethylase Serine

Glycine + methylene FH4 FH4

2. Transamination of glyoxalate yields glycine. Pyridoxal phosphate is coenzyme. Transaminase Glyoxalate + Glutamate

Glycine + α-ketoglutarate. P. Po4

3. Glycine –choline cycle generates glycine from choline. 4. Glycine is synthesized from threonine by serine trans hydroxyl methylase. Glycine degradation 1. Glycine conversion to ammonia and carbon dioxide by glycine synthase is major route of glycine degradation in mammals and birds. Liver mitochondria contains this enzyme. Tetrahydrofolate, NAD+ and lipoicacid are cofactors. It is also known as Glycine cleavage system. It is made up of three enzymes and H- protein. Lipoic acid is attached to H-protein by amide linkage like pyruvate dehydrogenase complex. The three enzymes are a. Glycine dehydrogenase b. Aminomethyl transferase c. Lipoamide dehydrogenase. Glycine Synthase CO2 + NH4+NADH+ H++ methenyl-FH4.

Glycine + FH4 +NAD Lipoicacid



Questions and Answers

2. Glycine is degraded to pyruvate after its conversion to serine by reversal of serine trans hydroxymethylase. Serine dehydratase catalyzes formation of pyruvate from serine. Serine Trans Glycine

Serine dehydratase Serine


hydroxymethylase 3. Oxidative deamination of glycine by D- aminoacid oxidase is third route of glycine degradation. Oxidative D-amino acid Glycine

decarboxylation Glyoxalate


one carbon pool.

Oxidase Co2 Biologically important Compounds formed form glycine Glycine is required for the formation of many important compounds. They are 1. Heme formation 2. Purine ring formation 3. Creatine formation. 4. Glutathione formation 5. Hippuric acid formation. 6. Bile acid formation. 7. Collagen synthesis 8. Serine formation. 9. Glucose formation. 10. One carbon pool. Diseases of glycine metabolism Some inherited diseases are due to defective glycine metabolism. Deficiencies of enzymes of Glycine metabolism affects glycine metabolism. Defective enzymes are produced by defective genes. Some inherited diseases of glycine metabolism are 1. Glycinuria :In this rare genetic disorder glycine is excreted in urine even though plasma glycine level is normal. Re absorption of glycine in renal tubules is defective due to defective trans porter. Hence glycine excreted in urine. 2. Primary hyper oxaluria :In this condition excess amount of oxalate about 15-60mg/ day is excreted in urine even though dietary oxalate is as usual. Glyoxalate utilization by transaminase as well as its conversion to formate is blocked. Hence glyoxalate accumulates and get oxidized to oxalate which is excreted in urine. Calcium present in urine combines with oxalate to form calcium oxalate crystal. These crystals deposit in the kidney and urinary tract. Therefore symptoms are bilateral urolithiasis due to stones in both ureters, nephro calcinosis due to stones in kidney and recurrent urinary tract infections. Affected individual die at child hood or early adult life due to renal failure or hypertension. 3. Non ketotic hyper glycinemia :This condition is characterized by excess glycine in blood and urine. Glycine synthase is defective in this condition. Symptoms are severe mental retardation and death of affected occurs in infancy. 128

CHAPTER - 10 | Protein and Aminoacid Metabolism

9. Write about biological roles of glutamine and glutamate A. a. Glutamate biological roles : 1. In amino acid metabolism glutamate has key role. It act as amino group source for formation of nonessential amino acids. In amino acid catabolism it act as collecting point of amino groups. 2. Glutamate is required for synthesis of glutathione, glutamine, N- acetyl glutamate and glucose. 3. Gamma amino butyric acid (GABA):Glutamate gives rise to this compound on de carboxylation catalyzed by pyridoxal phosphate dependent glutamate decarboxylase. It is an inhibitory neurotransmitter present in synaptic vesicles. 4. Gamma carboxylation :Glutamate of many proteins are carboxylated at gamma carbon. This carboxylation of glutamate at gamma carbon has role in blood clotting and bone formation. 5. Folic acid contains several glutamate residues. 6. Glutamate is neuro trans mitter. b. Glutamine biological roles 1. Several compounds amino group is derived from amide group of glutamine. They are purine nucleotides, pyrimidine nucleotides, amino sugars and co enzyme NAD. 2. Glutamate is synthesized from glutamine. 3. Detoxification reactions use glutamine particularly conjugation reactions. 4. Histidine and tryptophan synthesis needs glutamine. 5. In blood glutamine is present in high concentration about 10mg. 6. Glutamine has role in acid base balance. In kidney glutamine contributes ammonia.


Trace reactions of histidine catabolism.

A. Reactions of histidine break down : 1. Non oxidative de amination of histidine by histidase or histidine ammonia lyase occurs in the first reaction of its breakdown. Urocanic acid is the product of this reaction. 2. A water molecule addition by hydratase or urocanase is the second reaction and 4imidazolone-5-propionate is the product of this reaction. Histidase Histidine

Urocanase Urocanic acid

4-Imidazolone-5- propionate.


(2) NH4


3. In this reaction imidazolone propionate is cleaved to formimino glutamate (FIGLU) by an hydrolase.



Questions and Answers

4. Transfer of formimino group to one carbon carrier tetrahydrofolate (FH4)by formimino transferase occurs in this reaction. Glutamate is product. 4-imidazolone-5-propionate

Hydrolase (3)

Formimino glutamate Transferase (FIGLU)


(4) Formimino FH4

5. Finally α- keto glutarate is generated from glutamate by transaminase Transaminase Glutamate + pyruvate α-keto glutarate +alanine. (5)

11. Name biologically important compounds formed from histidine A. 1. Decarboxylation of histidine yields histamine. 2. Histidine is required for the synthesis of ergothionine, carnosine, anserine etc. 3. Glucose is formed from α-keto glutarate of histidine.

12. Write about FIGLU excretion test. A. FIGLU excretion Test : It is a folic acid deficiency test. Since conversion of FIGLU to glutamate is dependent on folicacid, in folic acid deficiency this reaction is blocked. This leads to accumulation and excreation of FIGLU in urine. The test involves administration of test dose of histidine to patient under investigation. Excretion of more of FIGLU in urine by patient indicates folic acid deficiency.

13. Describe catabolism of cysteine. Add a note on its sulfur fate. A. Cysteine degradation is brought about by two pathways. I. Dioxygenase pathway. II. Transaminase pathway. I. Reactions of dioxygenase pathway : In mammals it is the major route of cysteine degradation. 1. Incorporation of two atoms of oxygen in presence of NAD (P) H and iron by dioxygenase yields cysteine sulphinate. 2. Cysteine sulphinate has two metabolic fates. i. Direct desulphination of cysteine sulfinate yields sulfite and pyruvate. ii. Transamination followed by desulfination catalyzed by trans aminase and desulfinase respectively produce pyruvate. Dioxygenase Cysteine +o2 + NAD (P)H cysteine sulfinate +NAD(P)+ (1) Desulfinase Cysteinyl sulfinate

Pyruvate + sulfite. 2i


CHAPTER - 10 | Protein and Aminoacid Metabolism

Transaminase Cysteine sulfinate + α-ketoglutarate

Sulfinyl pyruvate +glutamate. (2 ii)

Desulfinase Sulfinyl pyruvate

pyruvate + sulfite. (2 ii)

II. Reactions of transaminase pathway : 1. A transaminase present in liver and kidney of mammals transfers amino group of cysteine. This results in formation of mercapto pyruvate. Transaminase Cysteine +α- keto acid

α-Amino acid + mercapto pyruvate (1)

2. The mercapto pyruvate has at least two metabolic fates. i. A dehydrogenase converts mercapto pyruvate to mercapto lactate which is excreted in urine Dehydrogenase +

Mercapto lactate + NAD +.

Mercapto pyruvate + NADH +H

(2 i) ii. In another route sulfur of mercapto pyruvate is released as hydrogen sulfide. (2 ii) Mercapto pyruvate

Pyruvate + Hydrogen sulfide.

Fate of sulfur of cysteine : In liver and kidney sulfide is converted to sulfite. Sulfite oxidase present in liver mitochondria oxidizes sulfite to sulfate. This enzyme is coupled to cytochrome c of electron transport chain through cytochrome b5. The sulfate is excreted in urine Sulfite oxidase Sulphide





(2) O2

Some part of sulphate is converted to active sulphate PAPS. PAPS is donor of sulphate for formation of sulfo lipids and glycosamino glycans. PAPS is also donor of sulphate involving conjugation reactions of steroids, drugs etc. After conjugation with sulfate they are excreted in urine and contributes to organic or ethereal sulfate of urine. Sulfate +ATP Urine



Sulfolipids PAPS



Glycosamino glycans Urine.



Questions and Answers

14. Write about biologically important compounds formed from cysteine. A. 1. Cysteine is required for synthesis of several biologically important compounds. They are a. Glutathione b. Coenzyme A c. Taurine d. Fatty acid synthase complex e. Cystine f. Glucose. 2. Cysteine contributes to urinary inorganic and organic sulfate. 3. Cysteine is involved in detoxification reactions.

15. Write reactions of methionine catabolism A. Methionne is degraded to cysteine and propionyl –CoA. Reactions of methionine breakdown : 1. In the first reaction of methionine degradation active methionine or S- adenosyl methionine (SAM) is formed. The reaction is catalyzed by S-adenosyl methionine synthase ATP serve as donor of adenosine moiety and energy source. SAM is a high energy compound and serve as donor of methyl groups. PPi is hydrolyzed by pyrophosphatase. SAM synthase Methionine +ATP

S- adenosyl methionine (SAM)+PPi, PPi


(1) 2. Methyl transferase transfers methyl groups of SAM to an acceptor to form S-adenosyl homo cysteine (SAH). Methyl Transferase S-adenosyl methionine +Acceptor

S-adenosyl homo cysteine + (2)


Methylated acceptor. 3. In this reaction an hydrolase converts S- adenosyl homo cyteine to homo cysteine and adenosine. Hydrolase S-adenosyl homocysteine (SAH) +H2 O

Adenosine+ homo cysteine. (3)

4. Condensation of homocysteine with serine catalyzed by pyridoxal phosphate dependent cystathionine synthase is the fourth reaction. Cystathionine is product.


CHAPTER - 10 | Protein and Aminoacid Metabolism

Cystathionine Synthase Homocysteine+ Serine

Cystathionine. P. Po4 (4)

5. Liberation of cysteine occurs in this reaction. Another pyridoxal phosphate dependent cystathioninase splits cystathionine to cysteine and homoserine. Cystathioninase Cystathionine

Cysteine +Homoserine. P. Po4 (5)

6. Removal of amino group homoserine by deamination forms α-ketobutyrate in this reaction. Deaminase Homo serine

α – ketobutyrate + ammonia. (6)

7. α-ketobutyrate dehydrogenase multi enzyme complex brings about oxidative decarboxylation of α- ketobutyrate. Propionyl-CoA is product. This reaction is similar to oxidative decarboxylation of pyruvate and α-ketoglutarate. α-ketobutyrate Dehydrogenase Α-ketobutyrate + CoA

Propionyl- CoA+CO2. (7)

8. Propionyl –CoA is converted to succinyl –CoA which enters TCA cycle. (8) Propionyl - CoA



TCA cycle.

Write about important compounds formed from methionine :

A. 1. Methyl groups of active methionine is used in the synthesis of several compounds and detoxification reactions. Transfer of methyl group of active methionine by methyl transferase to an acceptor is called as trans methylation. Examples of compounds synthesized by trans methylation are given below. 1. Guanido acetate


2. Nor epinephrine


3. Acetyl serotonin


4. Ethanolamine



Methylated DNA, RNA.

2. Methionine gives rise to cysteine. 3. Glucose is synthesized from methionine. 133


Questions and Answers

4. N-formyl methionine serve as initiating amino acid of protein synthesis. 5. Polyamine formation also requires methionine.

17. Write a note on diseases of methionine break down. A. Diseases of methionine breakdown :These diseases are due to production of defective enzymes of methionine metabolism. They are inherited diseases and genes are defective. 1. Homocystinuria and homo cysteinemia :This disease is characterized by high levels of homocystine an oxidized product of homo cysteine in urine and blood. It is due to deficiency of cystathionine synthase. So homo cysteine is not converted to cystathionine and hence it accumulates. Symptoms are thrombosis, mental retardation and eye lesions. 2. Cystathionineuria :It is characterized by elevated levels of cystathionine in blood and excretion in urine. It is due to block of cystathioninase catalyzed reaction. 3. Hyper methioninemia : It is due to deficiency of S- adenosyl methionine synthase. So methionine is not converted to S- adenosyl methionine and accumulates in blood.

18. Decribe catabolism of Phenyl alanine and Tyrosine. A. Phenyl alanine and tyrosine are degraded to fumarate and acetoacetate. Catabolism of phenyl alanine involves its conversion to tyrosine. Hence tyrosine break down pathway is same as that of phenyl alanine. In other words single pathway is responsible for the break down of both phenyl alanine and tyrosine. Reactions : 1. First reaction of phenyl alanine breakdown is its conversion to tyrosine by phenyl alanine hydroxylase. This reaction uses hydrogen from tetrahydro biopterin (THB) instead of known hydrogen donors. Transfer of hydrogen leads to formation of dihydrobiopterin (DHB). An NADPH dependent reduction converts DHB to THB. Phenyl alanine Hydroxylase Phenyl alanine +o2 +THB Tyrosine +DHB+H2O. (1) DHB+NADPH+H+


2. Tyrosine undergoes trans amination in this reaction. Para hydroxy phenyl pyruvate is product. Transaminase Tyrosine+ Îą-ketoglutarate phydroxy phenyl pyruvate +glutamate. (2)


CHAPTER - 10 | Protein and Aminoacid Metabolism

3. A vit. C dependent dioxygenase known as para hydroxy phenyl pyruvate hydroxylase converts para hydroxy phenyl pyruvate to homogentisic acid which involves decarboxylation and hydroxylation. Para hydroxy Phenyl pyruvate Hydroxylase Para hydroxy phenyl pyruvate + O2

Homogentisic acid. +C02 (3)

4. Another dioxygenase called as homogentisic acid oxidase cleaves benzene ring of homo gentisic acid to form maleyl aceto acetate. Homogentisic Acid oxidase Homogentisic acid + O2 Maleyl acetoacetate. (4) 5. Isomerization by an isomerase converts maleyl aceto acetate to fumaryl acetoacetate. Glutathione also required. Isomerase Maleyl aceto acetate

Fumaryl aceto acetate (5)

6. An hydrolase spilts fumaryl aceto acetate to fumarate and aceto acetate. Hydrolase Fumaryl aceto acetate +H2 O

Fumarate +aceto acetate. (6)

19. Write about com pounds synthesized from tyrosine. A. 1. Tyrosine is required for formation of catacholamines i. e. epinephrine and nor epinephrine. 2. Thyroid hormones are synthesized from tyrosine. 3. Tyrosine is required for synthesis of melanin. 4. Glucose and fat or ketone bodies synthesis occurs from tyrosine. 5. Protein synthesis requires phenyl alanine and tyrosine.

20. Write a note on various metabolic diseases of phenyl alanine and tyrosine A. Several metabolic diseases of phenyl alanine and tyrosine are known. They are due to defectives enzymes produced by defective genes and hence they are inherited diseases. 1. Phenylketonuria :This condition is characterized by excretion of phenyl ketone i. e. phenyl pyruvate in urine. It is due to deficiency of phenyl alanine hydroxylase. So



Questions and Answers

phenyl alanine is not converted to tyrosine and it accumulates in tissues. By other catabolic routes it is converted to phenyl pyruvate, phenyl lactate and phenyl acetate. All are excreted in urine. Symptoms are mental retardation and convulsions. 2. Tyrosinemia :In this condition tyrosine accumulates in blood due to lack of transaminase. It undergoes other routes of catabolism and converted to p-hydroxy phenyl acetate and N-acetyl tyrosine. They are excreted in urine along with tyrosine. Symptoms are skin and eye lesions, mental retardation etc. 3. Neonatal tyrosinemia : In this condition tyrosine accumulates in blood and excreted in urine. It is due to defective p-hydroxy phenyl pyruvate hydroxylase. So para hydroxy phenyl pyruvate conversion to homo gentisate is blocked. Accumulation and excretion para hydroxy phenyl pyruvate occurs. 4. Alkaptonuria : It is characterized by excretion of homo gentisic acid in urine. Further urine tuns dark on standing due to polymerization on of oxidative products of homo gentisic acid. On exposure to O2 homogentisic acid is oxidized to quinones. Due to deficiency of enzyme homogentisic acid oxidase homogentisic acid is not converted to maleyl aceto acetate. So accumulation of homogentisic acid in blood and excretion in urine occurs. Symtoms are connective tissue pigmentation and arthritis. 5. Tyrosinosis : In this condition plasma tyrosine level is elevated. It may be due to deficiency of isomerase and hydrolase. Tyrosine undergoes other routes of break down and products are excreted in urine. Symptoms are cabbage like odor, vomiting and diarrhoea.

21. Trace reactions of tryptophan catabolism. A. Tryptophen is degraded to alanine and aceto acetyl –CoA. Reactions: 1. Tryptophan dioxygenase opens indole ring of tryptophan in the initial reaction of tryptophan catabolism. N-formyl kynurenine is product. Tryptophan Dioxygenase Tryptophan +O2

N- Formyl kyn urenine (1)

2. Hydrolysis of N- formyl kynurenine by formylase removes formyl group as formate and kynurenine is produced. Formylase N-Formyl kynurenine

kynurenine +Formate. H2 O (2)


CHAPTER - 10 | Protein and Aminoacid Metabolism

3. An NADPH dependent kynurenine monooxygenase catalyzes hydroxylation of kynurenine. 3- hydroxy kynurenine is product. Kynurenine Mono oxygenase Kynurenine +NADPH+ H+ + O2 3- hydroxy kynurenine + NADP++H2 O (3) 4. An hydrolase kynureninase hydrolyzes hydroxy kynurenine to alanine and 3- hydroxy anthranilic acid. Pyridoxal phosphate is required for this reaction. 3- Hydroxy kynurenine +H2O

kynureninase Alanine +3- hydroxy anthranilic acid. (4)

5. A dioxygenase opens phenyl ring of hydroxy anthranilate and inserts two oxygen atoms to yield 2- amino -3- carboxymuconic acid semialdehyde. Dioxygenase 3-hydroxy anthrarilic acid +o2 2-Amino-3-carboxy muconic acid semi aldehyde. (5) 6. Decarboxylation of product of above reaction by decarboxylase yield 2-Aminomuconic acid semialdehyde. Co2 is released. Decarboxylase 2-Amino-3-carboxy muconic acid semialdehyde 2- Amino muconic acid semi (6) aldehyde +Co2. 7. Semi aldehyde is converted to 2- amino muconic acid by NAD+ dependent dehydrogenase. dehydrogenase 2- amino muconic acid semi aldehyde +NAD+ 2- Amino muconic acid (7) +NADH+H+ 8. NADPH dependent reduction of 2- Amino muconic acid by a reductase yield α-keto adipic acid. Reductase 2- Amino muconic acid +2NADPH+ 2H α-ketoadipic acid +2 NADP+. (8) +

9. Oxidative decarboxylation of α-keto adipate by α- keto acid dehydrogenase generates glutaryl-CoA. The enzyme is similar to pyruvate and α-keto glutarate dehydrogenases.



Questions and Answers

α-ketoacid Dehydrogenase α-ketoadipate + CoA + NAD+

Glutaryl- CoA+ NADH+H++ Co2. (9)


An FAD dependent dehydrogenation of glutaryl –CoA by dehydrogenase yields gluta conyl –CoA. Dehydrogenase Glutaryl- CoA +FAD

Glutaconyl –CoA + FADH2. (10)


Glutaconyl-CoA undergoes decarboxylation to crotonyl- CoA.


An hydratase adds water to crotonyl – CoA to yield beta hydroxy butyryl CoA. This reaction is analogous to ß- oxidation reaction. Decarboxylase Glutaconyl –CoA


Crotonyl –CoA




Co2 13.


An NAD dependent dehydrogenase converts beta hydroxy butyryl- CoA to aceto acetyl –CoA. Dehydrogenase Beta hydroxy butyryl- CoA +NAD+

Acetoacetyl –CoA + NAD +H+. (13)


Finally acetyl-CoA is generated from acetoacetyl –CoA by thiolase. TCA cycle oxidizes acetyl-CoA generated. Thiolase Acetoacetyl –CoA

2Acetyl-CoA CoA

TCA cycle.


22. Write organs and reactions involved in creatine formation. A. Synthesis of Creatine :It is mainly synthesized in liver, kidney and pancreas. Three amino acids are involved in creatine synthesis. They are arginine, glycine and methionine. Reactions : 1. Transfer of guanidine group of arginine to glycine by transamidinase is the first reaction. It occurs in kidney. Guanido acetate is product. 2. Trans methylation involving S- adenosyl methionine (SAM)as methyl donor yields creatine. In the liver this reaction takes place.


CHAPTER - 10 | Protein and Aminoacid Metabolism

Transmethylase (SAH) Transamidinase Arginine

ornithine + Guanido acetate






3. In the skeletal muscle creatine is converted to creatine phosphate by creatine phosphokinase (CPK). CPK Creatine +ATP

Creatine phosphate +ADP. (3)

23. How creatinine is formed? Write its fate. A. Creatinine formation :In the skeletal muscle phospho creatine serve as reservoir of energy. It is nonenzymatically converted to creatinine. Fate of creatinine Creatinine diffuses from muscle and excreted in urine as waste product. About 1-1. 5gm of creatinine excreted per day. Non enzymatic Creatine phosphate





24. Name compounds synthesized from Tryptophan. A. Several biologically important compounds are formed from Tryptophan. They are niacin, melatonin, serotonin, skatole and indole. Niacin is one of the water soluble vitamin. About 1 mg of niacin is synthesized from 60 mg of Tryptophan. Melatonin is hormone secreted by pineal gland. Serotonin is neurotransmitter. In the intestine skatole and indole are formed from Tryptophan by bacterial action. They are responsible for characteristic odor of feces.

25. Write about a)Maple syrup urine disease b) Xanthurenic aciduria A. a) Maple syrup urine disease : It is an inherited disease of branched chain aminoacid metabolism. It is due to lack of Îą-ketoacid dehydrogenase which converts alpha keto acids of three branched chain aminoacids to corresponding Co As. This leads to accumulation of Valine, Leucine, Isoleucine and their ketoacids in blood and these ketoacids are reduced to hydroxy acids which are excreted in urine. So urine gives burnt sugar smell due to hydroxy acids and hence name of the disease as maple syrup urine disease. Mental problems and vomiting are other symptoms. b) Xanthurenicaciduria: It occurs in vit. B6 or pyridoxine deficiency. So kynureninase is less active and conversion of 3-hydroxy kynurenine to alanine and 3-hydroxy



Questions and Answers

anthranilic acid is blocked. Through alternative pathways 3-hydroxy kynurenine is converted to xanthurenic acid and get excreted in urine. Other model questions are

26. Write about enzymes of protein digesting enzymes. 27. Write difference between oxidative and non oxidative deamination. 28. Write sources and utilization of ammonia. 29. Name enzymatic defects in a. Argininosuccinicaciduria b. Citrullinemia

30. Define glucogenic aminoacids. Give examples. 31. What are ketogenic aminoacids? Give examples. 32. Write differencec between glucogenic and ketogenic aminoacids. 33. Name biologically important compounds formed from cysteine. 34. Write the biochemical changes in a. Glycinuria b. Primary hyperoxaluria.

35. How SAM is formed ? Write its importance. 36. What is transmethylation? Give examples. 37. Write enzymatic defects in a. Phenylketonuria b. Alkaptonuria


CHAPTER - 11 | Porphyrin and Hemoglobin Metabolism



Porphyrin and Hemoglobin Metabolism 1. What are porphyrins ? Write their functions and properties. A. Porphyrins Porphyrins are derivatives of porphin. Porphin consist of four pyrrole rings in cyclic form. Hence it is known as tetra pyrrole. The four pyrrole rings are joined by methenyl bridges. Four pyrrole rings have eight substituent positions. Naturally occurring porphyrins differ in substituent positions or groups. Functions 1. Porphyrins are components of heme proteins. Heme is a metallo porphyrin. Iron is the metal present in heme. 2. Heme proteins are hemoglobin, myoglobin, cytochromes, cytochrome oxidase, cyto chrome P450 hydroxylases and enzymes of tryptophan and oxygen metabolisms. They are tryptophan dioxygenase, cyclo oxygenase, catalase, peroxidase etc. Properties 1. Isomers of porphyrins : Porphyrins exist in isomeric forms due to various side chains present in different substituent groups and arrangement of side chains. For example uroporphyrin with two types of side chains acetate (A) and propionate (P) exist in four isomers. They are type І, type ІІ, type ІІІ and type IV. In uroporphyrin type І all side chains are arranged symmetrically and in other types A, P are arranged asymmetrically. In nature most common are type І and type ІІІ. In type ІІІ the side chains on third pyrrole ring are arranged in different manner. 2. Color :All porphyrins are colored molecules. 3. Porphyrinogens : They are reduced forms of porphyrins. For example uroporphyrinogen І is

reduced form of uroporphyrin

І. All porphyrinogens are

colorless molecules. 4. Light absorption : All porphyrins absorb light at 400nm as well as in visible region. Light absorption property is used in identification of porphyrins.



Questions and Answers

2. How heme is synthesized ? Add a note on regulation of heme biosynthesis. A. Site:Except mature erythrocytes most of cells are capable of synthesizing heme. However liver and bone narrow are considered as major organs involved in heme production. About 20% is generated by liver and 80% by bone narrow. Reactions of heme synthesis. :Enzymes of heme biosynthesis are present in mitochondria and cytosol. First and last three reactions occurs in mitochondria and rest of the reactions takes place in cytosol. Glycine and succinyl –CoA are precursors for heme production. 1. Aminolevulinic acid or ALA synthase initiates heme biosynthesis and condenses succinyl –CoA and glycine to aminolevulinic acid. It occurs in two steps. a. First step involves formation of α-amino-ß- keto adipic acid. Pyridoxal phosphate is required. b. Decarboxylation of α- amino-ß- keto adipic acid yields δ-aminolevulinic acid in the second step. ALA synthase Succinyl-CoA +glycine

α-Amino ß- keto adipic acid (1a) PLP

δ(l b)

CoA Aminolevulinic acid + co2. 2. Formation of first pyrrole porphobilinogen (PBG) is the second reaction. It is catalyzed by ALA dehydratase. It forms pyrrole ring by eliminating a water molecule from two molecules of ALA. ALA Dehydratase δ-Aminolevulinic acid +δ –Aminolevulinic acid Porphobilinogen (2) (PBG) H2o 3. Uroporphyrinogen synthase –І or porphobilinogen deaminase (PBG Dase) condenses four PBG molecules in a step wise manner to form hydroxymethyl bilane. PBGD ase 4porphobilinogen

Hydroxymethyl bilane +4 NH3 (3)

4. In presence of uroporphyrinogen ІІІ cosynthase hydroxymethyl bilane is converted to uroporphyrinogen ІІІ. Uroporphyrinogen ІІІ cosynthase Hydroxymethyl bilane

Uroporphyrinogen ІІІ (4)


CHAPTER - 11 | Porphyrin and Hemoglobin Metabolism

Alternatively hydroxy methylbilane undergoes spontaneous (non – enzymatic) cyclization to form uroporphyrinogen І. Non enzymatic Hydroxymethylbilane

Uroporphyrinogen І.

5. Decarboxylation of acetate side chains of uroporphyrinogen ІІІ by uroporphyrinogen ІІІ decarboxylase yields coprophyrinogen ІІІ. Decarboxylase Uroporphyrinogen ІІІ

Coproporphyrinogen ІІІ +4co2. (5)

6. In the mitochondria protoporphyrinogen IX is formed from coproporphyrinogen ІІІ. The reaction is catalyzed by coproporphyrinogen ІІІ oxidase. In this reaction decarboxylation and oxidation of propionic acid side chains on І and ІІ pyrroles occurs. Coproporphyrinogen ІІІ oxidase Coproporphyrinogen ІІІ

Protoporphyrinogen IX +2CO2. (6)

7. Protoporphyrinogen oxidase catalyzes oxidation of protoporphyrinogen IX to protoporphyrin IX in this reaction in presence of molecular oxygen. Protoporphyri nogen oxidase Protoporphyrinogen IX +O2

Protoporphyrin IX. (7)

8. Heme formation occurs in this reaction. Heme synthase catalyzes formation of heme from protoporphyrin IX by inserting Fe2+ (Iron) in to protoporphyrin IX. Heme synthase 2+

Protoporphyrin IX + Fe

Heme. (8)

Regulation of heme synthesis :Heme synthesis is subjected to regulation by 1. Feed back inhibition 2. Repression and de repression. ALA synthase is regulatory enzyme. In the feed back inhibition heme end product of the pathway inhibits ALA synthase. In repression heme act as corepressor and combines with aporepressor to from repressor which inhibits expression of ALA synthase. In de repression, heme is diverted to detoxification enzymes synthesis like cytochrome P450 system in presence of drugs like barbiturates. So repression is relieved.



Questions and Answers

3. Define porphyries. Classify. Give examples for each class. Write clinical and biochemical abnormalities of each. A. Porphyrias They are inherited diseases of heme biosynthesis. They are characterized by accumulation and excretion of porphyrins or their precursors in plasma and urine respectively. Classification of porphyrias: Based on cells or organs affected porphyrias are classified into 1. Erythropoietic porphyrias and. 2. Hepatic porphyrias. Erythropoietic porphyrias A. Congenital or hereditary erythropoietic porphyria : This condition is characterized by accumulation and excretion of uro and coproporphyrinogens of Type І in erythrocytes and urine respectively. Further urine of the diseased persones turns to red due to conversion of uro and coproporphyrinogens І to corresponding porphyrins. Uroporphyrinogen ІІІ cosynthase is defective in this condition. Clinical symptoms are photo sensitivity, pink bones and teeth, hemolytic anaemia etc. b. Erythropoietic protoporphyria :It is due to deficiency of heme synthase. So protoporphyrin IX is excess in erythrocytes and protoporphyrin excretion in feces is increased. Photo sensitivity is main symptom. Liver problems and anaemia may develop later. Hepatic porphyrias a. Acute intermittant porphyria (AIP) : Affected individuals excrete large amounts of PBG and its precursors in urine. It is due to deficiency of uroporphyrinogen І synthase. As a result PBG and its precursors accumulates in plasma. Urine of the patients turn to dark due to polymerization of PBG in urine. Clinical symptoms are abdominal pain, neuropsychiatric symptoms but no photosensitivity. b. Variegata porphyria (VP) : Patients of this disease excrete PBG, ALA, Uro and coproporphyrins in urine. The urine may be colored due to presence of uro and coproporphyrins. Protoporphyrinogen oxidase is deficient and ALA synthase may be more active in this disease. Photosensitivity is main symptom. Other symptoms vary. c. Hereditary coproporphyria (HCP): This condition is characterized by excretion of large amounts of coproporphyrinogen ІІІ in urine and feces due to deficiency of coproporphyrinogen ІІІ oxidase. Photo sensitivity is main symptom. Urine may be colored. d. Porphyria cutanea tarda (PCT) :This is due to block in conversion of uroporphyrinogen ІІІ to coproporphyrinogen ІІІ by uroporphyrinogen decarboxylase. Hence uroporphyrinogen ІІІ accumulates in blood and excreted in urine. Due to presence of uroporphyrinogen ІІІ urine appears pink. Photosensitivity is major symptom.


CHAPTER - 11 | Porphyrin and Hemoglobin Metabolism

4. Write normal plasma bilirubin level. How bilirubin is formed? Explain its fate in liver and intestine. A. Normal plasma bilirubin level is 1mg%. Formation of bilirubin It is formed from heme of heme proteins. In liver microsomes a complex enzyme system heme oxygenase converts heme to biliverdin in presence of NADPH and o2. Heme oxygenase Heme+ o2 + NADPH + H+ Biliverdin + NADP + +CO+Fe2+ +H2O The biliverdin is reduced to bilirubin by NADPH dependent biliverdin reductase Biliverdin+NADPH+H+

Biliverdin Reductase

Bilirubin+ NADP+

Bilirubin formed in reticulo endothetial cells (REC) is released into circulation. Since free bilirubin is insoluble in plasma albumin combines and forms complex. Further metabolism occurs in liver. At sinusoidal surface of hepatocyte bilirubin dissociates from albumin and enters hepato cyte which is mediated by a transporter present in membrane of hepatocyte. Thus bilirubin enters cytosol of hepatocyte. Albumin Bilirubin A

Sinusoidal Surface Bilirubin –Albumin complex Bilirubin ↓ albumin.

Transporter Bilirubin

Bilirubin in hepatocyte cytosol

Now in the cytosol bilirubin is bound to ligandin and z or y protein which carries bilirubin to microsomes where it is detoxified by conjugation with glucuronic acid. Ligandin Bilirubin in cytosol

Bilirubin in microsomes. Z or y protein

First one molecule of glucuronic acid is transferred from UDP –glucuronic acid catalyzed by UDP –glucuronyl transferase. Bilirubin monoglucuronide is product. Transferase Bilirubin +UDP – glucuronic acid

Bilirubin monoglucuronide (BMG) UDP

BMG is converted to bilirubin diglucuromide (BDG) by adding one more glucuronic acid. Transferase BMG + UDP-Glucuronic acid Intestinal metabolism of bilirubin

Bilirubin diglucuronide (BDG) UDP



Questions and Answers

Most of conjugated bilirubin is secreted in bile. In the large intestine glucuronide is removed by hydrolases and bilirubin is released. Intestinal bacteria converts bilirubin to urobilinogen which is reabsorbed and re excreted into bile. However a small part enters circulation and excreted in urine by kidney. Majority of urobilinogen is eliminated in feces and it is popularly known as stercobilinogen. Bacterial Bilirubin glucuronide

Bilirubin + glucuronide. Hydrolases

Bacterial Enzymes Bilirubin

Circulation Urobilinogen



Feces Urobilinogen


5. Define hyper bilirubinemias. Classify. Give examples for each class. A. Hyper bilirubinemias Are conditions associted with increased plasma bilirubin level. They are of two types A. Conjugated hyper bilirubinemias B. Un conjugated hyper bilirubinemias. A. Conjugated hyper bilirubinemias : They are characterized by more of conjugated bilirubin in blood. Examples are Dubin- Johnson syndrome or chronic idiopathic jaundice and Rotor syndrome. B. Un conjugated hyper bilirubinemias : In these diseases unconjugated bilirubin level in plasma is elevated. Examples are neonatal physiological jaundice, Crigler-Najjar syndrome and Gilbert's disease.

6. What is jaundice? How it is classified? Write about each class. A. Jaundice is yellowish discoloration of sclera and skin due to excess bilirubin level. Classification Jaundice is classified into a. Prehepatic jaundice. b. Hepatic jaundice. c. Post hepatic jaundice based on causes. a. Pre hepatic jaundice :Hemolytic jaundice is the other name given to this condition. It is mainly due to increased hemolysis and hence name. Excess hemolysis leads to formation of excess bilirubin. But liver is unable to conjugate excess bilirubin. So accumulation of unconjugated free bilirubin occurs and plasma free bilirubin level is elevated. Increased hemolysis is seen in hemoglobinopathies, incompatable blood transfusion, hereditary spherocytosis and in malaria. In glucose -6- phosphate dehydrogenase deficiency cases administration of drugs like primaquine, aspirin and sulfonamide cause excess hemolysis.


CHAPTER - 11 | Porphyrin and Hemoglobin Metabolism

b. Hepatic jaundice: It is also known as hepato cellular jaundice. Because liver cell damage is the main cause for this type of jaundice. Viral infections, toxins, chemicals damage liver cells. Hepatitis virus, mushroom poisons, chloroform, carbon tetra chloride and phosphorus damage hepatocytes. Antibiotics use and in cirrhosis also hepatocytes are damaged. Functions of damaged hepatocytes are impaired. So hepatocytes may not be able to conjugate or secrete bilirubin though the bilirubin production is normal. If conjugation is impaired plasma level of unconjugated bilirubin is elevated. If secretion of conjugated bilirubin is affected its level in plasma is elevated. Hence in hepatic jaundice both conjugated and free bilirubin levels are increased. Post hepatic jaundice : It is also known as obstructive jaundice. Bile duct obstruction causes this disease. Stones in gall bladder and cancer of head of pancreas cause bile duct obstruction. Due to block in bile flow conjugated bilirubin secreted into bile returns to blood. Hence conjugated bilirubin level is elevated in obstructive jaundice.

7. Write principle and types of van den Bergh reaction. How it is useful in jaundice diagnosis. A. van den Bergh Reaction It is based on Ehrlich's reaction. It is used for measurement of plasma bilirubin. In this reaction a purple red color is produced. It is due to coupling of bilirubin with diazo reagent or diazotized sulphanilic acid. Two types of van den Bergh reactions are known. a. Direct van den Bergh reaction :It measures conjugated bilirubin only. In this reaction purple color is produced when conjugated bilirubin reacts directly with diazo reagent. b. Indirect van der Bergh Reaction :It measures un conjugated bilirubin only. In this reaction purple color is produced when un conjugated bilirubin reacts with diazo reagent in presence of methanol. van den Bergh Reaction is used in differential diagnosis of jaundice. Since un conjugated bilrubin is more in hemolytic jaundice indirect van den Bergh reaction is obtained with this serum. In contrast direct van den Bergh Reaction is obtained with obstructive jaundice serum. Therefore a positive indirect van den Bergh Reaction is used to confirm hemolytic jaundice and positive direct van den Bergh reaction is used to confirm obstructive jaundice.

8. Write normal hemoglobin level in blood. Explain its structure and functions. A. Hemoglobin concentration in blood is 12-15 gm%. Structure :Hemoglobin present in adults is known as hemoglobin Aor Hb A. It is a conjugated protein. It consist of globin as protein and heme as non protein part. It is a



Questions and Answers

tetramer. It consist of 4 polypeptide chains of two types. They are α chains two and ß – chains two. Each polypeptide chain is attached to one heme group. α –chain consist of 141 amino acids and ß-chain consist of 146 amino acids. Total 574 amino acids are present in hemoglobin molecule. It has molecular weight of about 64, 400 daltons. In each sub unit hydrophobic amino acids are present in interior. Hydrophilic amino acids are present on outer surface which makes it soluble in water. Heme is in hydrophobic interior. The sub units inter act in a unique way unlike sub units like α1, ß1 and α2, ß2 inter act extensively and like sub units i. e α1, α2 ; ß1, ß2 interacts weekly. Over all shape is spherical. Function : It transports oxygen from lungs to tissues. One molecule of hemoglobin carries 4 molecules of oxygen molecules. Hemoglobin also transports co2 from tissues to lungs. Hemoglobin serve as major blood buffer.

9. Write very briefly about glycosylated hemoglobin. A. Glycosylated hemoglobin HbA1C This type of hemoglobin contain glucose units. They are attached to ß-chain at Nterminus. It is a non enzymatic attachment. Usually the rate of glycosylated hemoglobin formation depends on blood glucose level. In normal people it accounts about 4-7% of total hemoglobin. It increases up to 20% in diabetic cases.

10. Write a note on sickle cell hemoglobin. A. Sickle cell hemoglobin HbS 1. It is most commonly occurring and severe abnormal hemoglobin. 2. It differs from normal hemoglobin in one amino acid residue of ß –chain. 3. In this hemoglobin valine replaces glutamate at 6position of ß chain. 4. This makes sickle cell hemoglobin more positive. 5. Further this change in one amino acid affects erythrocyte shape. 6. Erythrocytes containing sickle cell hemoglobin remain normal due to oxygenation 7. Deoxygenation leads to aggregation of sub units due to hydrophobic valine in beta chain. 8. It induces characteristic sickle shape. 9. Erythrocyte with altered shape have decreased life span and hence they undergo hemolysis. 10. Therefore sickle cell hemoglobin causes sickle cell anaemia.

11. Write briefly on a) Hemoglobinopathies b) Thalassemias A. a) Hemoglobinopathies are group of inherited diseases associated with production of


CHAPTER - 11 | Porphyrin and Hemoglobin Metabolism

hemoglobin with altered composition. Only composition of α and β chains of globin part is altered and prosthetic group remains unchanged. They are due to mutations in genes coding Alpha and beta chains. Hemoglobin with altered composition is called as abnormal hemoglobin or hemoglobin variant. In homozygous individuals only abnormal hemoglobin is produced where as in heterozygous individuals both normal and abnormal hemoglobins are produced. Production of abnormal hemoglobin triggers hemolysis. Hence moderate to severe hemolytic anemia occurs in susceptible people like infants, children, young girls and in pregnant women. Hb S, Hb E and Hb D are some abnormal hemoglobins seen in hemoglobinopatheis. b) Thalassemias are group of inherited diseases in which total synthesis of one of the globin chain is blocked. Hence they are named according to synthesis of chain blocked. They are of two types 1) α- Thalassemia 2) β- Thalassemia 1) α- Thalassemia : In this condition α- chain synthesis is blocked. So Hb A is not formed. The β-chain form abnormal HbH (β4). So it is also known as HbH disease. Further formation of abnormal HbH triggers hemolysis and anemia develops in affected individuals. 2) β- Thalassemia : In this condition β-chain synthesis is blocked. Hence synthesis of HbA is affected. The α- chain is unable to form tetramer but forms large inclusion (Heinz) bodies which triggers hemolysis leading to anemia in affected individuals. The condition is called as Cooley's anemia or β-thalassemia major in homozygotes and β-thalassemia minor in heterozygotes Other model questions are

12. Write formation of bilirubuin. 13. Write a note on Hemoglobin. 14. Write about acute intermittent porphyria. 15. HbS. 16. Hb A1C 17. Crigler-Najjer syndrome 18. Cooley's anemia


CHAPTER - 12 | Nucleotide Metabolism



Nucleotide Metabolism 1. Describe denovo synthesis of purine nucleotides. A. Denovo purine nucleotide biosynthesis Site:Liver cytosol contains all the enzymes required for purine nucleotide biosynthesis. Purine nucleotide formation involves construction of purine ring on ribose phosphate. Sources of purine ring : Purine ring is generated by incorporating carbon and nitrogen atoms from various sources. Carbons 4, 5 and 7 are derived from glycine ; carbon 2 and 8 are derived from N-10 formyl FH4 and N-5, 10 methenyl FH4 respectively ; carbon dioxide contributes to carbon 6, nitrogen 1 is from aspartate ; nitrogen 3 and 9 are from amide of glutamine.


7 Glycine N

6 C

Co2 1N




C8 FH4



N9 Glutamine

Reactions : 1. Phosphoribosyl pyrophosphate (PRPP) formation from ribose -5- phosphate is the first reaction. PRPP synthetase catalyzes this reaction. ATP and Mg2+ are required. AMP is formed from ATP. PRPP Synthetase Ribose -5-phosphate +ATP

5- phosphoribosyl pyrophosphate + AMP. 2+ (

Mg 1) 2. Phosphoribosyl amido transferase catalyzes formation of phosphoribosyl amine by transferring amide of glutamine.


CHAPTER - 12 | Nucleotide Metabolism

Amido Transferase 5-phosphoribosyl pyrophosphate +glutamine

Phosphoribosyl amine + (2)

Glutamate +PPi.

3. 5-Phosphoribosyl glycinamide is formed in this reaction from glycine and phosphoribosyl amine. An ATP dependent glycinamide kinosynthetase catalyzes this reaction. Synthetase Phosphoribosyl amine + ATP + Glycine + ADP +Pi.

phosphoribosyl glycin amide (3)

4. Formylation by trans formylase generates phosphoribosyl –N- formyl- glycinamide from phosphoribosyl glycinamide. Transformylase 5-Phosphoribosyl glycinamide Amide+FH4.

Phosphoribosyl -N-formyl glycin(4)

5. Transfer of amide group of glutamine again to carbonyl oxygen of glycinamide is the fifth reaction. This ATP dependent reaction is catalyzed by 5- phosphoribosyl –Nformyl glycinamidine synthetase. 5-phosphoribosyl –N- formyl glycinamidine is product of this reaction. glycinamidine synthetase 5- phosphoribosyl –N- formyl glycinamide + ATP + glutamine (5) 5- phosphoribosyl –N- formyl glycinamidine + ADP + Pi + glutamate. 6. An ATP dependent imidazole ring of purine formation occurs in this reaction. 5phosphoribosyl amino imidazole synthetase brings this reaction. Imidazole Synthetase 51 –phosphoribosyl -5-.

5- phosphoribosyl –N- formyl glycinamidine +ATP (6) amino imidazole + ADP +Pi + H2O.

7. Carboxylation of amino imidazole ring by carboxylase is seventh reaction. Carboxylase 1

51- phosphoribosyl –5-

5 – phosphoribosyl -5- amino imidozole + CO2 (7) Amino imidazole-4-carboxylate.



Questions and Answers

8. An ATP dependent transfer of amino group of aspartate to carboxyl introduced in above reaction takes place in presence of synthetase Synthetase 1

5 – phosphoribosyl – 5-amino imidazole -4-carboxylate + ATP +Aspartate (8) 1

5 -phosphoribosyl -4- (N- succino carboxamide)- 5- Amino imidazole. 9. A lyase eliminates fumarate from product of above reaction to form 51- phosphoribosyl 4- carboxamide -5-amino imidazole. Lyase 51- phosphoribosyl -4- (N-succino carboxamide)- 5- Amino imidazole


phosphoribosyl -4- carboxamide -5 amino imidazole + Fumarate. 10. Formylation once again by another transformylase incorporates formyl group to form 51-phosphoribosyl -4-carboxamide-5- formamido imidazole. Trans formylase 5 - phosphoribosyl -4- carboxamide -5- amino imidazole 51- phosphoribosyl (10) +N-10 formyl FH4. 4-carboxamide-5-Formamido imidazole + FH4. 1

11. A cyclohydrolase catalyzes ring closure by dehydration to yield first purine nucleotide inosine mono phosphate (1MP). Cyclohydrolase 1

5 - phosphoribosyl -4- carboxamide -5-form amido imidazole

inosine (11)

monophosphate (IMP) + H2O. Synthesis of AMP and GMP from IMP :Synthesis of AMP and GMP from IMP occurs by two separate pathways. Formation of AMP from IMP occurs as shown below Adenylo succinate Synthetase IMP +aspartate+GTP

Adenylo succinate + GDP + Pi. (1)

Adenylo succinase Adenylo succinate

AMP + Fumarate. (2)

Formation of GMP from IMP occurs as below shown.


CHAPTER - 12 | Nucleotide Metabolism

dehydrogenase IMP +NAD+ + H2O

xanthosine mono phosphate (XMP) + NADH + H+. (1)

GMP synthetase XMP+ ATP +glutamine GMP + glutamate +AMP +PPi. (2) Formation of ADP, GDP and ATP, GTP. AMP +ATP









d GTP.

2. Write about Pyrimidine nucleotide synthesis denovo. A. De novo pyrimidine nucleotide biosynthesis Site :Cytosol of liver cells. In pyrimidine nucleotide biosynthesis pyrimidine ring is formed first then pentose phosphate is added later. Pyrimidine ring is generated from aspartate and carbamoyl phosphate. Reactions : 1. Carbamoyl phosphate synthetase –ІІ (CAPS –ІІ) catalyzes formation of carbamoyl phosphate from glutamine amide nitrogen and carbon dioxide. ATP is required for this reaction. CAPS-ІІ Co2 + glutamine + 2ATP

Carbomoyl phosphate + 2ADP + Pi. (1)

2. Reaction of aspartate and carbamoyl phosphate in presence of aspartate trans carbamoylase (ATC ase) yields carbomyl aspartate + Pi. ATC ase Carbomyl phosphate +Aspartate

Carbamoyl aspartate + Pi. (2)

3. An intra molecular water elimination leads to pyrimidine ring formation from carbamoyl aspartate. Dihydro oratase catalyzes this reaction. Dihydroorotase Carbamoyl aspartate

Dihydroorotate + H2o. (3)

4. An NAD+ dependent dehydrogenase converts dihydro orotate to orotic acid. Dehydrogenase +

Orotic acid + NADH +H+.

Dehydro orotate +NAD




Questions and Answers

5. Orotidine monophosphate (OMP) is formed from orotic acid in this reaction. Orotate phosphoribosyl transferase catalyzes transfer of ribose -5- phosphate to orotate from PRPP. Phosphoribosyl Transferase Orotate +PRPP

Orotidine mono phosphate (OMP) +PPi. (5)

Pyro phosphatase PPi


6. First pyrimidine nucleotide uridine mono phosphate (UMP) is generated from OMP by decarboxylase. Decarboxylase Orotidine mono phosphate

Uridine mono phosphate (UMP) + CO2. (6)

Synthesis of CTP and dTTP from UMP UMP +ATP


UTP + Glutamine +ATP UDP



CTP +glutamate + ADP + Pi.






3. Write about salvage pathways of purine nucleotides. A. Salvage pathways are active in tissues which lack de novo pathways. Blood cells and brain are dependent on these pathways. These pathways use pre formed purine bases of either exogenous and endogenous origin. They uses even nucleosides for nucleotide formation. Purine salvage pathways :They are involved in the conversion of purine bases, purine nucleosides into nucleotides. 1. Hypoxanthine guanine phosphoribosyl transferase (HGPRT ase) converts hypoxanthine and guanine to IMP and GMP respectively. PRPP act as donor of ribose phosphate. HGPRT ase Hypoxanthine + PRPP

IMP +PPi ; Guanine + PRPP


2. Adenine is converted to AMP by adenine phosphoribosyl transferase Adenine + PRPP

AMP + PPi.

3. Adenosine and guanosine are converted to AMP and GMP by kinases. Adenosine Adenosine +ATP

AMP +ADP. kinase


CHAPTER - 12 | Nucleotide Metabolism

Guanosine Guanosine + ATP

GMP +ADP. kinase

4. Write a note on Pyrimidine salvage pathways. A. Pyrimidine salvage pathways are: 1. Pyrimidine phosphoribosyl transferase (PPRT ase) salvages free pyrimidine bases using PRPP as donor of ribose phosphate. PPRT ase Thymine + PRPP PPi


Uracil +PRPP



2. Pyrimidine nucleosides are salvaged by kinases. Uridine kinase Uridine +ATP


Deoxythymidine +ATP

deoxycytidine +ATP



5. Write about inhibitors of nucleotide biosynthesis. A. Inhibitors of nucleotide biosynthesis are useful as anti bacterial, antiviral and anticancer agents. Antibacterial agents : Sulfa drugs like sulfanilamide and tri methoprim are used to treat bacterial infections. They are folic acid analogs and block folic acid dependent reactions of nucleotide biosynthesis in bacteria. Bacterial growth is impaired due to lack of folic acid. Thus bacterial infection is controlled. Anticancer agents : Several inhibitors of nucleotide biosynthesis are used as anti cancer agents. For example folic acid analogs aminopterin and amethopterin work as anti cancer agents by blocking folic acid dependent reactions. Azaserine and acivicin block glutamine involving reactions. Mercapto purine and fluorouracil work by blocking purine and pyrimidine nucleotide formation. Anti viral agents : Acyclovir and AZT (azothymidine) are antiviral agents.

6. Wrie about a) Orotic acid uria b) Lesch-Nyhan syndrome. A. They are diseases of nucleotide formation. Defective nucleotide formation causes diseases. a) Orotic acid uria :It is an inherited disease of pyrimidine nucleotide formation. It is due to lack of orotate phosphoribosyl transferase and decarboxylase. It is characterized by more orotic acid in blood and its excretion in urine. Clinical symptoms are growth retardation and anaemia.



Questions and Answers

b) Lesch – Nyhan syndrome :HGPRT ase is deficient in this condition. More of purine nucleotides are produced. Symptoms are self mutilation, mental retardation, anemia and hyper uricemia.

7. How purine nucleotides are degraded? A. Site : Liver is the major site of purine nucleotide degradation. Reactions: 1. Nucleases generate purine nucleotides from nucleic acids. Nuclease Nucleic acids


Nucleic acids


(1) 2. AMP is converted to inosine by two routes. a. Deamination of AMP by adenylate deaminase followed by dephosphorylation. Adenylate






Inosine + Pi.


2a NH3

b. Dephosphorylation followed by deamination. Nucleotidase catalyzes phosphate removal from AMP and adenosine deaminase (ADA) catalyzes removal of amino group of adenosine. Adenosine Nucleotidase AMP

deaminase Adenosine


Inosine +NH3 2b

pi 3. A nucleotidase converts GMP to guanosine. Nucleotidase GMP

guanosine +Pi. 3

4. Now inosine and guanosine are converted to xanthine by two separate routes. a. Transfer of ribose by nucleoside phosphorylase followed by oxidation. Xanthine oxidase (XO) catalyzes latter reaction. Nucleoside Phosphorylase Inosine +Pi

Ribose -1- phosphate + Hypoxanthine. 4a


CHAPTER - 12 | Nucleotide Metabolism

Xanthine Oxidase Hypoxanthine + o2 + H2 O

Xanthine + H2 O2. (4b)

b. Transfer of ribose by nucleoside phosphorylase followed by deamination. Guanase catalyzes latter reaction. Nucleoside Phosphorylase Guanosine + Pi

Ribose -1- phosphate + guanine. (4b)

Guanase Guanine

Xanthine + NH3. (4b)

5. Finally uric acid which is end product of purine degradation is formed from xanthine by action of xanthine oxidase. Xanthine oxidase Xanthine + H2 O + O 2

Uric acid + H2 O2. (5)

8. Write normal plasma uric acid. Write about conditions with raised uric acid level. A. Normal plasma uric acid level is below 6mg%. Gout : It is common disease of purine nucleotide degradation. Plasma uric acid level is elevated which is characteristic sign of gout. Symptoms : Deposition of uric acid crystals occurs in soft tissues because uric acid is less soluble in aqueous environment of body fluids. Tophi is the name given to urate crystals that are found in joints, cartilage of fingers and toes. Arthritic type gouty attacks occurs in affected individuals. Causes : 1. Over production of uric acid causes gout. Increased purine nucleotide production leads to excessive uric acid production. It occurs in HGPRT ase deficiency, Hyper active PRPP synthetase, leukaemia, von Gierke's disease and polycythemia. 2. Impaired removal of uric acid by kidneys causes gout. It is called as renal gout. It occurs due to defective

uric acid transport in renal tubular cells and




Questions and Answers

9. Write very briefly about SCIDD. A: It is usually known as

severe combined immunodeficiency disease (SCIDD). It is due to

adenosine deaminase deficiency. Due to lack of this enzyme DNA synthesis is decreased and lymphocytes do not mature. Affected persons are susceptible to infections.

Other model questions are

10. How inosine monophosphate is synthesized? 11. Write the formation of ATP and GTP from IMP. 12. Write purine ring. Label sources of its various elements. 13. Write the formation of UMP. 14. Draw structure of Pyrimidine ring. Label sources of its various elements.


CHAPTER - 13 | Replication, Transcription & Translation



Replication, Transcription & Translation REPLICATION

1. Define replication. Explain process of replication. A Replication :Synthesis of new DNA is known as replication. Process of replication :Replication begins at specific locations of DNA. It is known as ori C. It involves DNA un winding, initiation, elongation and several protein factors. Ori C is a sequence of 245 basepairs (bp) in E. coli Chromosome. It has binding sites for DNA binding proteins. 1. Un winding of DNA occurs due to binding of dna A a DNA binding protein to ori C. 2. Further binding of dna B and dna C to DNA facilitates unwinding. dna B is helicase. It causes un winding of DNA. dna A DNA

dna B DNA –dna A

Unwinding of DNA. dna C

3. Single strand binding proteins (SSBP) bind to unwound DNA and stabilizes single strand by preventing rewinding. SSBP DNA with a unwinding strand

DNA with stabilized unwound strand.

4. Un winding of DNA creates super coils and prevent further unwinding. DNA gyrase removes super coils by creating negative super coils which facilitates replication. DNA gyrase DNA un winding

super coils in DNA

Negative super coils in DNA

Replication Favoured. 5. Another strand of DNA is also stabilized by SSBP. A replication fork is created. Binding of SSBP DNA with one

to another strand

Replication fork.

Unwound strand 159


Questions and Answers

6. At replication fork a RNA primer is formed by action of RNA – polymerase or primase. When the RNA primers are elongated to about 10- 200 bases DNA polymerase ІІІ form new DNA in 51

31direction by polymerizing deoxy nucleotide triphophates (dNTPs).

RNA poly Replication Fork of DNA

DNA polynease ІІІ RNA primer at


replication fork

New DNA strand dNTPS

Newly formed DNA strand is known as leading strand. 7. DNA polymerase ІІІ is able to synthesize both DNA stands but in different ways because it cannot polymerize nucleotides in 31

51 direction.

8. It is able to synthesize leading strand continuously. It synthesizes other strand in a discontinuous manner. It is known as lagging strand. Fragments of this strand are known as okazaki fragments. 9. DNA polymerase - І fills up gaps between okazaki fragment to form lagging strand. DNA polymerase ІІІ Replication fork with leading strand

DNA polymeraseI

okazaki fragment dNTPS

okazaki fragment with gaps filled. 10. DNA ligase joins okazaki fragments. Lagging strand is formed. DNA ligase okazaki fragments with

Lagging strand gaps filled.

Gaps filled 11. RNA primers are removed by exonuclease activity of DNA polymerase –І.




Leading Strand




DNA Polymerase III 1






3 5 Okazaki Fragments

RNA Primer





CHAPTER - 13 | Replication, Transcription & Translation

2. Write about inhibitors of replication and their medical importance. A. Inhibitors of replication :Several compounds are capable of blocking replication. They are known as inhibitors of replication. Some of them are a. Cytosine arabinoside b. Actinomycin D c. Acyclovir. Cytosine arabinoside : It is a nucleoside analog of cytosine and contain modified pentose. Arabinose is present instead of usual ribose. Introduction of this compound blocks elongation of replication. Actinomycin D : It inferfers with interaction of DNA bases because it contains an hydrophobic ring. This leads to destabilization of DNA and replication is blocked. Acyclovir : It is another nucleoside analog. It is an analog of guanosine with three carbon sugar instead of usual ribose. It inhibits polymerase action. Medical Importance : Inhibitors of replication are used as anti cancer, antibacterial and antiviral agents. Cytosine arabinoside and actinomycin D are anticancer agents. Acyclovir is antiviral agent. Bleomycin is an antibiotic. It inhibits replication by breaking bonds of DNA. TRANSCRIPTION

3. Define transcription. Explain process of transcription. A. Transfer of genetic information from DNA to RNA or synthesis of RNA from DNA is known as transcription. Process of Transcription: Transcription involves enzymes, protein factors, initiation, elongation and termination. Initiation. 1. Certain regions of DNA serve as signals for the synthesis of RNA. They are known as promoter sites. 2. RNA polymerase is enzyme involved in RNA synthesis. It contains five subunits. Two alpha, two beta and one sigma subunits. RNA polymerase recognizes promoter site with the help of sigma factor (subunit). It binds to template strand of DNA at promoter site. This binding leads to unwinding of DNA and initiation of RNA formation. Sigma subunit DNA promoter site

RNA polymerase binds promoter


of Template strand. 3. RNA polymerase has two binding site. One for purine nucleotides and another for any nucleotide. Assuming that ATP binds to purine nucleotide site and pyrimidine nucleotide UTP binds to another site first phosphodiester bond formation occurs between ATP and UTP. Thus chain growth is initiated. After this the sigma factor is released. 161


Questions and Answers

ATP, UTP Unwound Template strand subunit released.

Chain growth initiated


Elongation : 1. RNA polymerase continues polymerization of ribo nucleotide triphosphates (rNTPs)as directed by template strand. DNA unwinds ahead of new RNA chain and newly formed RNA or nascent RNA is base paired to templates strand. RNA polymerase New chain of RNA growth Formation of new RNA or nascent RNA. rNTPS 2. Rewinding of DNA leads to dissociation of nascent RNA from template strand during elongation. Rewinding of DNA Nascent RNA Dissociation of nascent RNA from template strand. Termination : 1. Certain regions of DNA serve as signals for termination of RNA synthesis. They are known as termination signals. 2. Formation of hair pin loop in nascent RNA occurs when termination signal is transcribed by RNA polymerase. Termination Nascent RNA Hairpin loop formation in nascent RNA. Signal Specific terminator protein Rho (e) recognizes hair pin loop in nascent RNA. This terminator protein has helicase activity also. So it separates base paired nascent RNA from template strand of RNA. This type of termination of RNA synthesis is known as Rho (e) dependent termination. On terminatipon of RNA synthesis RNA polymerase leaves DNA molecule. Rho protein Nascent RNA with Separation of nascent RNA Hair pin loop transcription

Termination of

RNA polymerase leaves DNA.


5 31




y Fo



l New

NA ed R

51 31

RNA Polymerase

CHAPTER - 13 | Replication, Transcription & Translation

3. Write about Inhibitors of Transcription A. Several compounds block RNA formation. They are known as inhibitors of transcription. Some of them are antibiotics. Toxins also act by blocking transcription. Toxins are mushroom toxin Îą- amanitin that causes mushroom poisoning and aflatoxin produced by fungus of ground nut that causes liver cancer. Rifamycin and rifampicin are antibiotics used in the treatment of tuberculosis work by inhibiting transcription. Actinomycin D is anti cancer agent used in treatment of Wilm's tumor work by inhibiting transcription.

4. Write a note on post transcriptional modifications A. They convert precursor RNA to functional RNAs. All three types of RNAs are produced as precursors. Messenger RNA or mRNA: It is synthesized as precursor RNA known as heterogenous RNA or hn RNA. Post transcriptional modifications it undergo are a. Capping at 51 end b. Poly adenylational at 31 end. c. Splicing: Eukaryotic hn RNA contains non functional sequences known as introns. Functional sequences of hn RNA are known as exons. Splicing removes introns of hn RNA and joins exons to generate functional m RNA. Splicing of hn RNA requires small nuclear RNA or sn RNA and splicing proteins. tRNA or rRNA are also generated from precursors by post transcriptional modifications. TRANSLATION

5. Define genes and write on genetic code A. Genes of DNA contain code words for amino acids. Genes are segments of DNA that contain information for protein formation or poly peptide synthesis. The code words of amino acids are known as genetic code. These code words are transferred to m RNA and t RNA by transcription. So m RNA and tRNA contain amino acid code words. Characteristics of genetic code : 1. It is triplet code. Two types of code words exist. They are codon and anti codon. Both codon and anti codon consist of sequence of three nucleotides. Codons are present on m RNA and anti codon is present on t RNA. Codon and anticodon are complementary in nature. 2. Each aminoacid is coded by codon. For example UUU code for phenyl alanine, GGG for glycine and CCC for proline. 3. Some codons serve as initiating codons for protein synthesis. AUG is an example for initiating codon of protein synthesis.



Questions and Answers

4. Some codons do not code for any amino acid and they cause termination of protein synthesis. They are known as termination codons or nonsense codons. They are UAA, UAG or UGA. 5. The codon is universal. Codon of an amino acid is identical in all species. 6. A codon codes only one amino acid. However an amino acid may have more than one codon. 7. Genetic code is comma less. 8. Base pairing occurs between bases of codon and anticodon.

6. Define translation. Describe translation A. Synthesis of proteins using information present in RNAs is known as translation. Mechanism of translation involves initiation, elongation, termination, ribosomes, initiation factors (IFs), elongation factors (EFs) and releasing factors (RFs). Activation of aminoacids :Prior to utilization for protein formation aminoacid must be activated. Activation of aminoacid provides energy needed for peptide bond formation. Activation involves attachment of amino acid to t RNA. Amino acyl –t RNA synthetase is enzyme involved in activation of amino acid. ATP is required. It is converted to AMP and PPi. Pyrophosphatase converts PPi to in organic phosphate. Aminoacyl tRNA Amino acid + t RNA +ATP Aminoacyl- tRNA (AA-tRNA)+AMP+PPi. Synthetase Pyrophosphatase PPi


Mechanism of Translation Initiation: 1. It begins with binding of initiation factors (IFs) to 30S ribosomal subunit. Three initiation factors are required. They are IF-1, IF-2 and IF -3. First IF-3 binds with 30S subunit. IF-2 combines with GTP to form IF-2-GTP complex. IF-1 and IF-2-GTP complex binds to IF-3 containing 30S ribosomal subunit. 30S ribosomal subunit +IF-3

IF-3-30S subunit IF-2 +GTP

IF-3-30S +IF-1 + IF-2-GTP

30S-IF-3 -IF-1-IF-2-GTP.


2. Now mRNA and aminoacyl-tRNA (AA-tRNA)can bind to 30S ribosomal subunit containing GTP and initiation factors. The mRNA

combines 30Sribosomal subunit. The initiating

tRNA containing first amino acid AA1 -tRNA joins mRNA -30S subunit complex through codon anti codon base pairing to form initiation complex. Release of IF-3 accompanies this complex formation.


CHAPTER - 13 | Replication, Transcription & Translation

AA1- tRNA 30S-IF-3 -IF-1-IF-2-GTP+mRNA

m RNA-30S-IF-3-IF-1-IF-2-GTP

AA1-tRNA-mRNA-30S-IF-1-IF-2-GTP + IF-3 Initiation complex. The initiation complex has high affinity to wards 50S ribosomal subunit and binds to one of 50S subunit from pool. Joining of 50S subunit with initiation complex causes hydrolysis of GTP to GDP and Pi and release of IF-1 and IF-2 and leading to formation of 70S initiation complex. In the 70S initiating complex peptidyl site or P site of ribosome is occupied by initiating amino acyl-tRNA (AA1-tRNA)and aminoacyl site or A site of ribosome is free. AA1-tRNA-mRNA-30S-IF-1-IF-2-GTP+50S AA1-tRNA (P site)-A site (Free)-70S –mRNA + GDP + Pi + IF-1 + IF-2 Initiation complex. Elongation : By sequential addition of aminoacids new polypeptide chain is elongated. t RNA s carry aminoacids. Elongation factors EF-Tu, EF-Ts, EF-G and GTP are required. 1. tRNA carrying second aminoacid (AA2-tRNA) to be incorporated cannot directly combine with 70S initiation complex. It requires elongation factor EF-Tu and GTP. AA2 – tRNA combines with EF-Tu-GTP complex and interacts with 70S initiation complex. A site of 70S ribosome is occupied by AA2 –tRNA with concomitant hydrolysis of GDP and Pi. EFTu-GDP complex dissociates from 705 ribosome. AA2 -tRNA AA1-tRNA (Psite)-A site -70S –mRNA + EF-Tu-GTP AA1 – tRNA (Psite)-AA2 –tRNA (Asite)-70S-m RNA + EF-Tu-GDP + Pi. 2. Presence of two aminoacyl – tRNA s on P and A sites of 70S ribosome respectively leads to first peptide bond formation. Peptidyl transferase activity of 50S ribosome subunit catalyzes peptide bond formation between AA1 and AA2 Aminoacids. It is accompanied by shifting of dipeptide (AA1-AA2) to A site of ribosome. The empty tRNA remains in P site. Peptidyl AA1 –tRNA (Psite)-AA2-tRNA (Asite)-70S-mRNA Transferase tRNA (Psite)-AA1- AA2- tRNA (Asite)-70S-mRNA. 3. The incoming tRNA carrying third amino acid (AA3 –tRNA) cannot bind to Psite. It can bind only to Asite of 70S ribosome. So tRNA carrying dipeptide (dipeptidyl-tRNA or AA1AA2 –tRNA)is translocated to free Psite in presence of elongation factor EF-G and GTP. First EF-G combines with GTP to form EF-G-GTP complex. This complex inter acts with 70S ribosome. This leads to release of free tRNA from Psite and shifting of dipeptidyl tRNA from Asite to Psite. During this shifting mRNA also moves by three nucleotides and third 165


Questions and Answers

codon appears in Asite. Hydrolysis of GTP to GDP and Pi provides energy for this process and EF-G is released. EF-G+GTP

EF-G-GTP complex. EF-G-GTP

tRNA (Psite)-AA1 - AA2 – tRNA (Asite)-70S-m RNA AA1-AA2 –tRNA- (Psite)-A site -70S-m RNA + EF – G + GDP + Pi + t RNA Regeneration of EF-Tu-GTP: EF-Tu- GTP regeneration is essential for continuation of elongation or protein synthesis. Second elongation factor EF-Ts is involved in this process. EF-Ts reacts with EF-Tu – GDP complex replaces GDP and forms EF-Tu- EF-Ts complex. EF-Ts+EF –Tu- GDP


Now GTP reacts with EF-Ts –EF –Tu complex displaces EF-Ts to form. EF-Tu-GTP complex. EF-Ts is released. GTP EF-Ts – EF-Tu


EF-Tu-GTP complex availability

leads to continuation of

elongation process. The

elongation process is repeated many times adding one aminoacid each time until termination codon is encountered in Asite. Elongation repeated n times AA1 – AA2 - tRNA (Psite)-Asite -70S-mRNA AA1 – AA2 ------------- AAn tRNA (Psite)-A site -70S-mRNA Termination : 1. Exposure of termination codon on Asite signals termination of polypeptide formation. Releasing factors (RF)and GTP are required. There are three releasing factors RF-1, RF-2 and RF-3. They recognizes termination codon on A site and bind to site where tRNA binds. 2. Polypeptide chain is separated from tRNA by hydrolysis of ester bond. Ribosomal peptidyl transferase activity of ribosomes causes this hydrolysis. Further GTP is hydrolyzed to GDP and Pi. This results in release of mRNA and tRNA from ribosomes. Dissociation of ribosomes to 30S and 50S subunits takes place immediately. AA1- AA2 .............. tRNA (Psite)-Asite – 70S –mRNA + RF + GTP AA1 – AA2 .............. AAn-tRNA (Psite)–RF- GTP (Asite)-70S-mRNA. AA1- AA2 .............. + tRNA + 50S + 30S + mRNA + RF + GDP + Pi. Polypeptide (Protein)


CHAPTER - 13 | Replication, Transcription & Translation









31 5






n Comp

itiatio 70S In


New Polypaptide AA1







+ Termination

+ tRNA

31 51


30 S

50 S



7. Write about Inhibitors of protein synthesis A. Many antibiotics are inhibitors of translation. They work by inhibiting translation at differents stages. Even some toxins work by blocking protein synthesis. Antibiotics are puromycin, tetracyclins, chloramphenicol, erythromycin, streptomycin, tunicamycin and cycloheximide. Diphtheria toxin that causes diphtheria in children inhibit translation.

8. Write a note on post translational modifications A. Nascent proteins that comes out of protein synthesizing machinery may not be functionally active. They are converted to active or functional proteins by post translational modifications. Some of them are phosphorylation, glycosylation, hydroxylation, methylation, proteolytic modifications, carboxylation and iodination.

9. Explain DNA damage and DNA repair mechanisms. A. Since DNA is genetic material for survival of species through generations integrity of DNA must be maintained. However DNA damage can result from action of environmental, physical, chemical, and biological agents. Therefore species evolved mechanisms for the removal of damaged DNA. Excision repair : An exinuclase cuts damaged DNA and removes it. DNA polymerase І synthesizes new DNA in that gap. Then DNA ligase integrates new DNA into native DNA. Enzymatic photo reactivation :It is involved in the removal of thymine dimer formed by exposure of DNA to ultra violet light. A photolyase get activated on exposure to normal light which cleaves linkage between two thymine bases.

10. Write very briefly about diseases of DNA repair. A. Defective DNA repair mechanisms leads to diseases.



Questions and Answers

Xeroderma pigmentosum: It is due to defective enzyme of excision repair. Characteristic sign is development of skin cancer in affected people on exposure to ultra violet light of sunlight.

11. Explain salient features of Lac operon model A. Lac operon deals with regulation of gene expression at transcriptional and translational level. It is a model proposed to explain enzyme induction and repression related to lactose metabolism. Hence the name lac operon. An operon consist of four types of genes on DNA segment. The four types of genes are structural genes, promoter gene (p), regulatory gene (i) and operator gene (o). They are located adjacent to each other. The structural genes are composed of three genes. They are z, y and a and present side by side. These structural genes codes for enzymes required for lactose metabolism. They code for galactosidase, permease and trans acetylase. Regulatory gene regulates operon. It codes a regulatory protein known as repressor. i





Ry gene

ter gene gene




operator Structural genes DNA segment

When repressor molecule is produced it binds to operator gene and prevents transcription of structural genes by RNA polymerase. So enzymes of lactose metabolism are not produced or repressed. This occurs in the absence of lactose. Therefore absence of lactose causes repression of enzymes that utilize it. When the lactose is present it combines with repressor molecule and form complex. This prevents binding of repressor to operator gene. So operator gene is free. RNA polymerase binds to operator gene and transcribes genes of lactose metabolism. As a result enzymes of lactose metabolism are produced or induced and cells use lactose. Therefore in presence of lactose (inducer)enzyme induction occurs.

12. Define recombinant DNA. How it is obtained? Write applications of this technology. A. Recombinant DNA is combination of DNA from two different species or organisms. Preparation of recombinant DNA : Restriction enzymes are used for recombinant DNA preparation. DNAs of two species or organisms are cut with same restriction enzyme to generate sticky ends. DNA having sticky ends combines with another DNA containing same sticky ends. Then a DNA ligase links these two DNA of different origin leading to formation of recombinant DNA.


CHAPTER - 13 | Replication, Transcription & Translation

Restriction Enzyme Human DNA

Human DNA with Stick ends

DNA Ligase

Rest of DNA

Restriction Enzyme Recombinant DNA Bacterial DNA

Bacterial DNA with Sticky ends

Applications: 1. Recombinant DNA technology is popularly known as genetic engineering. It is used to produce pharmaceuticals or drugs. 2. Hormone insulin, interferons, growth hormones, blood clotting factors etc. are produced using recombinant DNA technology. 3. It is used for development of DNA vaccines. 4. Gene therapy is another area where principles of recombinant DNA is utilized.

13. Write about PCR. A. Polymerase chain reaction is known as PCR. In this technique DNA replication takes place in cyclic manner in laboratory. Bacterial DNA polymerase which is stable to heat is used for DNA amplification. Each cycle doubles the DNA amount. The product of the first cycle becomes the template of the next cycle. . However replication of DNA by thermophilic bacterial Taq DNA polymerase requires primers. Two oligonucleotide primers are synthesized by using appropriate methods. These primers bind to DNA that is to be amplified (replicated) at specific sequences on opposite strands. In the first step of a cycle DNA to be amplified is exposed to heat to separate strands. In the second step of the cycle primers are ligated to separated DNA strands at appropriate place. In the third step of cycle Taq DNA polymerase is added to double initial two strands. Thus at completion of one cycle initial amount of DNA is doubled. Likewise the initial DNA amount is amplified to several times. Twenty cycles of PCR can amplify particular DNA segment to 105 or more times Applications 1. PCR amplifies DNA rapidly and selectively 2. Very small amount (50-100 bp) of DNA from single cell or sperm cell or hair follicle can be amplified to large quantities by PCR. 3. PCR is used to detect infectious agents in the body because infections are due to presence of (viral or bacterial) foreign DNA.



Questions and Answers

4. PCR is used for prenatal diagnosis of genetic diseases a which are due to alterations in DNA like sickle cell anaemia, hemophilia etc. 5. PCR is used to detect certain cancers like leukemia, thyroid cancer etc. 6. PCR is used for tissue typing which is essential for organ transplantation. 7. In the forensic work amplification of little DNA recovered from the suspect or from the crime site by PCR allows generation of sufficient DNA for finger printing. 8. DNA recovered from archaelogical materials (sites) is amplified by PCR and used to study evolution or civilization. 9. PCR can be used to create extinct animals like dinosaurs by amplifing DNA recovered from fossil materials. DNA to be Amplified

Strand Separation Primers

Taq DNA Polymerase

2nd Cycle

3rd Cycle

4 Strands 16 Strands

8 Strands

14. Write a note on restriction endonucleases. A. 1. These enzymes cut both DNA strands at specific sites. 2. They are present in prokaryotes and absent in eukaryotes. 3. They can not cut DNA of cell of their origin. 4. They hydrolyze only foreign DNA molecules. Since they restrict the entry of foreign DNA into host DNA by cutting foreign DNA they are referred as restriction enzymes. 5. Several restriction enzymes are isolated from bacteria and viruses. 6. They differ in structural requirement, cleavage site and produces DNA fragments with characteristic ends. 7. They are named from their origin. For example restriction endonuclease of E. coli is known as EcoRI. 8. Restriction endonuclease of Para influenza which is known as Hpa I


CHAPTER - 13 | Replication, Transcription & Translation

Applications :They are useful for a) Analysis of chromosome structure. b) Isolation of genes. c) DNA sequencing. d) Recombinant DNA technology. e) RFLP (Restricted fragment length polymorphism).

15. Write about Blotting or Hybridization techniques. A. Southern Blot, Northern Blot and Western Blot are known as blotting or hybridization techniques. Southern Blot 1. This technique is used for identification of DNA fragment of interest in sample of DNA. 2. First DNA sample is cleaved by restriction endonuclease into small fragments. 3. By agarose gel electrophoresis DNA fragment of interest is separated from rest of fragments. 4. Then DNA of interest is transferred on to nitrocellulose membrane by blotting. 5. A radiolabeled cDNA probe which recognizes DNA of interest is added. 6. Finally hybrid of cDNA and DNA of interest is visualized as band on x-ray film. Northern Blot 1. This technique is used for identification of RNA from sample 2. RNA ase is used to cut given RNA sample into small fragments 3. RNA of interest is separated from rest by performing agarose gel electrophoresis 4. Separated RNA is transferred to nitro cellulose membrane by blotting. 5. Radiolabelled cDNA probe is added which recognizes RNA of interest and form hybrid. 6. The hybrid is visualized as band on x-ray film. Western Blot 1. It is used to identify protein of interest from mixture of proteins. 2. By agarose gel electrophoresis desired protein is separated from remaining proteins. 3. To nitrocellulose membrane separated protein is transferred by blotting. 4. Radiolabelled monoclonal antibody probe is added which recognizes and form hybrid with desired protein. 5. The hybrid is visualized as band on x-ray film.



Questions and Answers

Other model questions are

16. Write about okazaki fragments. 17. How DNA polymerases are used in replication ? 18 Write about transcription factors. 19. Write a note on genetic engineering. 20. Write about factors of translation. 21. Write about formation and functions of RNA primers. 22 Write about genes of lac operon model. 23. Write a note on replication fork. 24. RNA polymerases


CHAPTER - 14 | Vitamins



Vitamins 1. Define vitamins. Classify. Give examples for each class. A. Vitamins are small organic molecules which are not synthesized in the body and hence must be present in diet. They are classified into a. Fat soluble vitamins and b. Water soluble vitamins. Fat soluble vitamins are Vit. A, Vit. D, Vit. E and Vit. K. Water soluble vitamins are Vit. C and members of Vit. B complex. B complex vitamins are thiamine or Vit. B1, riboflavin, niacin, pyridoxine, folic acid, cyanocobalamin or Vit. B12, Biotin and pantothenic acid.

2. Write differences between water soluble and fat soluble vitamins. A. The water soluble and fat soluble vitamins differ in several properties. They are a. Solubility : Fat soluble vitamins are soluble in fats or organic solvents only. Water soluble vitamins are soluble in water. b. Bile salts : Fat soluble vitamins dependent on bile salts for their absorption. But water soluble vitamins does not require bile salts for absorption. c. Storage : Liver stores all fat soluble vitamins. Except vit. B12. other water soluble vitamins are not stored. d. Cooking conditions : Fat soluble vitamins are stable to normal cooking conditions. But water soluble vitamins are unstable to normal cooking conditions. e. Excretion route : Fat soluble vitamins are excreted in feces but water soluble vitamins are excreted in urine.

3. Write chemistry, functions, deficiency symptoms, sources and daily requirements of Vitamin A A. Chemistry : Three compounds retinol (an alcohol), retinal (an aldehyde) and retinoic acid (an acid) exhibit vit. A activity. They are also known as retinoids. These three form of vit A are derived from a 20 carbon compound which contains Ă&#x;- ionine ring and isoprenoid side chain. Double bonds are present in side chain. 173


Questions and Answers

Isomerism : Due to presence of double bonds in side chain Vit. A exhibits isomerism. The two isomeric forms are all trans retinol and 11-cis –retinol.



ß-ionine ring –isoprenoid chain with double bonds. Vit. A Functions : 1. Retinal is required for normal vision as well as color vision. Visual (Walds) cycle : This cycle begins with dissociation of rhodopsin ends with regeneration of rhodopsin. Rods present in the eye contains vit. A containing visual pigment rhodopsin. It is a conjugated protein and composed of 11-cis retinal and protein opsin. When light or photon strikes 11-cis- retinal is converted to all trans retinal. The apo protein opsin dissociates. The conversion of rhodopsin to all trans retinal and opsin involves several intermediates. Meta rhodopsin is one such intermediate. It generates nerve impulse by interacting with G- proteins present in rods plasma membrane. Light Rhodopsin

All trans retinal + opsin

Nerve impulse.

Photons The all trans retinal is reduced to all trans retinol by NAD dependent reductase. A small amount of all trans retinal may be isomerized to 11-cis –retinal by an isomerase. Isomerase 11-cis-retinal

Reductase NADH

All trans retinal

All trans retinol. NAD

The 11-cis retinal combines with opsin to regenerate rhodopsin. 11-cis –retinal + opsin


However for normal vision this small amount of rhodopsin is inadequate so constant supply of Vit. A1 is required for total regeneration of rhodopsin. From circulation rod cells take up all trans retinol and isomerize to 11-cis –retinol by isomerase rod cells Diet



All trans trans retinol


CHAPTER - 14 | Vitamins

In retina an NAD+ dependent alcohol dehydrogenase converts 11-cis- retinol to 11-cis –retinal. Dehydro Genase Retina +

11-cis-retinal + NADH+ + H +

11-cis-retinol +NAD

With availability of 11-cis- retinal in sufficient quantities more of rhodopsin is formed in retina. Retina 11-cis-retinal+opsin


2. vit. A is required for growth and reproduction. 3. vit. A function as steroid hormone. 4. vit. A is required for glycoprotein synthesis. 5. vit. A promotes differentiation. 6. vit. A is required for integrity of epithelial alls of mucous layers of gastrointestinal, urinary, respiratory, skin and salivary glands etc. 7. vit. A is required for nervous tissue growth and function. 8. vit. A is required for bone and tooth formation. Vit. A deficiency symptoms 1. Nyctalopia or night blindness : It is a major Vit. A deficiency symptom. Affected persons are unable to see in dim light or night light. So they are equal to blind during night. Hence the name night blindness. It is common in people taking low Vit. A. If night blindness is not treated it progresses to xerophthalmia. Bitot's spots in conjuctive appear in children. If xerophthalmia is not controlled it leads to keratomalacia in which corneal epithelium is degenerated. Finally ulceration of cornea may lead to total blind ness. 2. Reproductive disorders like degeneration of testis, malformation and resorption of foetus occurs. 3. Mucous linings of respiratory, reproductive, salivary and lacrimal glands are keratinized. The condition is known as hyper kerotosis. 4. Keratinization of skin also occurs (Xeroderma). 5. Nervous tissue growth is affected. 6. Bone and tooth formation becomes defective. 7. Kidneys are degenerated. Sources : Vit. A occurs in plant and animals. Plants : In plant foods Vit. A is present as carotenes. Red palm oil is excellent source. Leafy vegetables, yellow colored vegetables and fruits also contain vit. A. Amarnath leaves, curry leaves, coriander leaves, spinach, cabbage and drumsticks are good sources. Yellow pigmented



Questions and Answers

vegetables like carrot, pumpkin, sweet potato, tomato and bottle gourd are good sources. Yellow fruits like mangoes, papaya, pineapple, jackfruit, bananas, oranges also contain vit. A. Animals : In animal foods vit. A is present as retinol esters. Fish liver oils like halibut liver oil, shark liver oil and cod liver oil are excellent sources. Liver of poultry and farm animals contain vit. A. Dairy products buttermilk and eggs also contain vit. A. Daily allowance : Adults : 2500 International units, one international unit=0. 3µg; 750ug of retinol or 3mg carotene.

4. Write chemistry, functions, deficiency symptoms, sources and daily requirements of Vitamin D A. Chemistry : Two cholesterol derivatives ergo calciferol also known as vit. D2 and cholecalciferol also known as vit. D3 exhibits vit. D activity.





Chole Calciferol Vit. D3

Ergocalciferol Vit. D2

Functions : 1. 1, 25-dihydroxy cholecalciferol is physiologically active form of vit. D. It is also known as calcitriol. Synthesis of calcitriol : In liver cytochrome P450 dependent hydroxylase forms 25-hydroxy cholecalciferol from cholesterol. Liver Cholecalciferol + NADPH+O2

25- hydroxy cholecalciferol + NADP+ + H2O. Cyt P450

Through the circulation it reaches kidney where it is converted calcitriol by α –hydroxylase which is also dependent on cyt P450 and NADPH 25-hydroxy cholecalciferol +NADPH +O2

1, 25-dihydroxy

Cyt P450 Kidney Cholecalciferol + NADP+ + H2 O.


CHAPTER - 14 | Vitamins

2. Calcitriol increases calcium and phosphate absorption in the intestine. In the intestine calcium absorption by calcitriol is promoted by two mechanisms. a. In the intestine calcitriol increases synthesis of calcium binding proteins (CBP)which promotes calcium absorption. From the plasma calcitriol enters intestinal cells. In the cytosol of enterocyte it combines with receptor to form complex. Receptor Plasma


calcitriol –receptor complex

calcitriol This complex migrates into nucleus and interacts with DNA. As a result expression of gene of calcium binding protein occurs. Nucleus Calcitriol- receptor


DNA- calcitriol- receptor complex


complex. Translation mRNA of CBP


calcium binding protein

Increased calcium absorption.

b. Calcitriol promotes phosphate absorption in the intestine by different mechanism which requires glucose and sodium. 3. Calcitriol promotes synthesis of osteocalcin, a calcium binding protein of bone. Osteocalcin is required for bone formation and mineralization of bone. 4. Handling of calcium and phosphorus by kidney is influenced by calcitriol. It decreases excretion of calcium and phosphorus by kidney. 5. Normal muscle tone is maintained by calcitriol. Vit. D deficiency symptoms 1. Rickets : Vit. D deficiency causes rickets in children. In rickets bone and teeth formation are affected. Skull, chest, spine, leg and pelvic deformities are commonly seen. Parietal and frontal bossing and craniotabes are skull deformities. Pigeon breast is deformity of chest. Bow legs or knock kees are deformities seen in legs. Usually in low income group children vit. Ddeficiency occurs. 2. Osteomalacia : In adults vit. D deficiency causes osteomalacia. Pregment women and women in pardha are prone to this disease. Skeletal pain and deformities of spine, legs, pelvis, ribs etc. are seen. 3. Osteoporosis : In old people vit. D deficiency causes osteoporosis. Porous bones and bone pains are common symptoms. Sources : Marine fish liver oils are excellent sources. Halibut liver oil, shark liver oil, cod liveroil are good sources. Other fish like sardines, egg and butter contain small amounts.



Questions and Answers

Daily allowances : Adult : 5ug of vit. D or 200 international units. Pregnant and lactating women : 10ug or 400 international units.

5. Write chemistry, functions, deficiency symptoms, sources and daily requirements of Vitamin E. A. Chemistry : Tocopherols are compounds that exhibit Vit. E activity. There are four types of to copherols. They are α-tocopherol, ß-tocopherol, g-tocopherol and δ-tocopherol. Among all α-tocopherol is most potent. These tocopherols are derivatives of tocol which is composed of chromane ring and phytyl side chain.


a - Tocopherol

Functions : 1. Vit. E functions as biological antioxidant. It is present in high concentrations in tissues that are exposed to high oxygen tension like lungs, eyes and erythrocytes. It act as free radial scavenger. It is present in membranes of cells, cell organelles and in cytosol. Protects membrane lipids particularly poly unsaturated fatty acids (PUFA) from peroxidation by peroxy radical. Peroxyradical is formed from PUFA in the membrane. PUFA peroxy radical initiates free redial chain reaction. Tocopherol eliminates peroxy radical and thus protects membrane lipids. Peroxy radical + Tocopherol

Hydroperoxide + Tocopherol free redial

Tocopherol free redial + peroxy redical

Hydroperoxide + oxidized products

of tocopherol. Hydroperoxide and oxidized product of tocopherol are eliminated. 2. Vit. E is known as fertility factor for lower animals. It is required for sperm formation in male rats and foetal development in female rats. 3. Vit. E is involved in control of muscle tone. 4. Vit. E promotes heme proteins synthesis. 5. Dietary carotenes and Vit. A are protected from oxidation by vit. E. Vit. E deficiency symptoms 1. Hemolytic anelmia in children is main symptoms of vit. E deficiency. Since vit. E protects erythrocytes from oxidative damage deficiency of Vit. E increases susceptibility of erythrocytes to hemolysis. 2. Since vit. E is required for fertility, sterility is symptom of vit. E deficiency in male rats and foetal resorption in female rats. 178

CHAPTER - 14 | Vitamins

3. Vit. E Deficiency causes muscular dystrophy in animals like rat, rabbit, lamb etc. Sources : Vegetable oils like ground nut oil, rice bran oil, sunflower oil, cotton seed oil, mustard oil, palm oil and cereal germ oils like wheat germ oil, corn germ oil are good sources. Daily requirement : Adults : About 10mg of vit. E per day. In pregnancy and lactation about 1213 mg of vit E is required pe day.

6. Define Pro vitamins. Give examples. How they are converted to vitamins. A. They are precursor forms of vitamins. For example carotene, 7-dehydrocholesterol and ergosterol are provitamin forms of vit. A and vit. D respectively. A dioxygenase converts Ă&#x;carotene of diatary origin to 2 molecules of Vit. A retinal. Dioxygenase Ă&#x;-carotene

2 retinal. O2

In presence of ultra violet (UV) light provitamins of vit. D are converted to corresponding vitamins. UV light 7-dehydrocholesterol


7. Write chemistry, functions, deficiency symptoms, sources and daily requirements of Vitamin K. A. Chemistry : Phylloquinone is the major from of vit. K present in plants. It is also known as vit. K1 Menaquinone is the vit. K present in animals. It is termed as vit. K2 Both are derivatives of naphthoquinone containing different side chains. Vit. K3 is menadione. It is syntheticanalog of vit. K.

3 Vit. K1 Functions : 1. Vit. K is required for post translational modifications of blood clotting factors like prothrombin, proconvertin, stuart factor and christamas factors. 2. It is required for carboxylation of these blood clotting factors. Gamma carbon of glutamate is site of carboxylation. Hence it is also known as g- (Gamma) carboxylation. A carboxylase adds 179


Questions and Answers

carbon dioxide to gamma carbon of glutamate (glu) residue of clotting factors in presence of reduced vit. K1 to form gamma carboxylated glutamate (gla). Vit. K1 is converted to epoxide. Carboxylase Glutamate (glu) + Reduced vit. K1 + co2 + o2

Blood clotting factor.

Of blood clotting factor Gamma carboxylated (Gla) + Reduced vit. K1 epoxide + H2 o. Reduced vit. K1 is regenerated from vit. K1 epoxide by epoxide reductase in presence of NADPH +H+. Reductase +

Vit. K1 epoxide +NADPH +H

Reduced vit. K1+ NADP+.

This post translational modification is essential for blood clotting. It promotes binding of calcium to gamma carboxyl groups of prothrombin. This leads to conversion of prothrombin to thrombin. This vit. K dependent Carboxylation is known as vit. K cycle. 3. Another calcium binding protein osteocalcin in bone also under goes vit. K dependent gamma carboxylation of glutamate residues. A vit. K dependent carboxylase catalyzes this reaction. Vit. K deficiency symptoms 1. Rare in adults. However in pre Mature babies vit. K deficiency occurs. Main symptom is haemorrhage due to increased prothrombin time. 2. Prolonged use of antibiotics may produce vit. K deficiency in adults. Sources : Green leafy vegetables like cabbage, spinach, turnipgreens, cauliflower, green peas and soybean peas are good sources. Poultry products like eggs, liver dairy products like cheese and butter are also good sources. Daily requirement : Adults : 70-140mg/day, pregnancy and lactation 150-200 mg/day.

8. Write chemistry, functions, deficiency symptoms, sources and daily requirements of Vitamin-C A. Chemistry : Ascorbic acid, a sugar acid is known as vit. C. It undergoes oxidation easily in at mospheric oxygen to dehydroascorbic acid. O2 Ascorbic acid

Dehydroascorbic acid

High temperature, acid and alkali promotes oxidation. Oxidized vit. C is functionally less active.


CHAPTER - 14 | Vitamins

Functions :


1. Vit. C is biological antioxidant. It eliminates free radicals. It is free radical scavenger. Dietary vit. C þrevents oxidation of carotenes, vit. E and B complex vitamins present in diet. 2. Vit. C is required for bone and teeth formation. 3. It is involved in post translational modification of collagen. It is required for hydroxylation of proline and lysine residues of collagen. 4. It is required for the absorption of iron. It maintains iron in ferrous form. 5. It also participates in metabolic reactions. 6. Hydroxylation of steroid hormone requires vit. C 7. Bile acid formation requires vit. C

C | HO – C O HO – C | C | HO – C – H | CH2OH Ascorbic Acid or Vit. C

8. Catecholamines formation requires vit. C 9. Tyrosine metabolism requires vit. C. 10. Carnitine biosynthesis requires vit. C. 11. In high doses it reduces severity of cold and other infections. Vit. C deficiency symptoms 1. Scurvy is main vit. C deficiency disease. Usually it occurs in mariners who stays on sea for prolonged time. 2. Scurvy symptoms are internal hemorrhages and prone to bone fractures and infections. 3. Since vit. C is required for collagen synthesis capillaries are prone to ruptures. 4. Delayed wound healing, swollen gums and joints, loose teeth and anemia are also seen in scurvy affected people. 5. In infants vit. C deficiency causes infantile scurvy. Sources : Citrus fruits like lemon, orange, pineapple and grapes are good sources. Amla also known as Indian goose berry is excellent source. Other fruits like guava, papaya, mango, apples and bananas also contain this vitamin in good amounts. Among vegetables tomato, cabbage, cauliflower, potato and leafy vegetables coriander leaves, amaranth leaves and spinach leases also contain vit. C Daily requirement : Adult : 60-80 mg/per day.

9. Write functions and deficiency symptoms of thiamin A. Functions : 1. Thiamin pyrophosphate (TPP)or thiamin diphosphate (TDP) is active form of thiamin. It serve as coenzyme of oxidative decarboxylation reactions and transketolase reactions.



Questions and Answers

CH2 – CH2 – O – (P) – O – (P) CH2 – N


S CH3 N Thiamin Diphosphate (TDP)

a. Pyruvate dehydrogenase and α-ketoglutarate and α-keto adipate dehydrogenase reactions TDP function as coenzyme. Pyruvate

Acetyl- CoA, α-ketoglutarate TPP

a- ketoadipate

Succinyl –CoA TPP

Glutaryl-CoA. TPP Transketolase

b. Xylulose -5-phosphate +ribose -5- phosphate

sedoheptulose-7- phosphate TPP

+glyceraldehyde -3- phosphate. Deficiency symptoms : 1. Thiamin deficiency occurs in polished rice consuming areas. Beri beri is major deficiency symptom. Two types of beri beri are identified. a. Wet beri beri : In this type of beri beri cardiovascular system is affected. Edema is seen in lower limbs, trunk, face etc. b. Dry beri beri : Only central nervous system is affected. No edema. Affected people are unable to walk and usually bed ridden. 2. Thiamin deficiency in infants causes infantile beri beri. 3. In birds thiamin deficiency causes polyneuritis.

10. Write functions and deficiency symptoms of riboflavin. A. Functions : 1. FMN and FAD are active forms of riboflavin. FMN is flavin mononucleotide and FAD is flavin adenine dinucleotide. FMN and FAD function as carriers of hydrogen atoms in oxidation and reduction reactions. Flavin – Ribitol – O – P – O – P – Ribose – Adenine Flavin Adenine Dinucleotide (FAD)


CHAPTER - 14 | Vitamins

A. Aminoacid oxidase, NADH –CoQ reductase are enzymes requiring FMN. B. Succinate dehydrogenase and acyl-CoA dehydrogenase requires FAD as coenzyme. Deficiency Symptoms : 1. Riboflavin deficiency causes angular stomatitis, cheliosis and glossitis in humans. Dermatitis of certain parts like nasolabial regions, urogenital regions also seen. 2. In experimental animals riboflavin deficiency causes eye lesions and growth impairment.

11. Write functions and deficiency symptoms of Niacin A. Functions 1. Niacin is converted to NAD (Nicotinamide adenine dinucleotide) and NADP (Nicotinamide adenine dinucelotide phosphate) in the body. NAD and NADP act as carriers of hydrogen atoms in oxidation and reduction reactions.

CONH2 N Niacin Adenine – Ribose – O – P – O – P – O – Ribose - Nicotinamide (Niacin) Nicotinamide Ademine Dinucleotide (NAD) a. Some NAD dependent enzymes are malate dehydrogenase, isocitrate dehydrogenase, glyceraldehyde-3- phosphate dehydrogenase etc. b. NADP dependent enzymes are glucose -6- phosphate dehydrogenase, glutamine reductase etc. Deficiency symptoms : 1. Pellagra is deficiency symptom of niacin in man. It is common in maize eating countries. Dermatitis, diarrhea and dementia are characteristic symptoms of pellagra. 2. Niacin deficiency in experimental animals causes black tongue. 3. In some cases stomatitis and glossitis i. e. Inflammtion of oral cavity and tongue.

12. Write functions and deficiency symptoms of pyridoxine. A. Functions : Pyridoxal phosphate (PLP) is active form of pyridoxine. It serve as coenzyme of enzymes involved in transamination, decarboxylation, nonoxidative deamination, desulfration, transsulfuration etc. reactions.



Questions and Answers









Pyridoxal Phosphate

1. Pyridoxal phosphate is coenzyme of several transaminases like alanine transaminase, aspartate transaminase etc. 2. Pyridoxal phosphate is coenzyme of glutamate decarboxylase, dopamine decarboxylase etc. 3. Pyridoxal phosphate is coenzyme of trans sulfuration enzymes like cystathionine synthase, cystathionine lyase etc. 4. Pryidoxal phosphate is coenzyme of desulfuration enzyme cysteine desulfhydrase. 5. Pyridoxal phosphate is coenzyme of non oxidative deamination enzymes like serine dehydratase. 6. Pyridoxal phosphate is part of phosphorylase of glycogenolysis. 7. Pyridoxal phosphate is coenzyme of ALA synthase, kynureninase of tryptophan metabolism. Deficiency symptoms : 1. Microcytic hypochromic anaemia is common symptom due to decreased heme synthesis. 2. Epileptic from convulsions occurs in children of pyridoxine deficiency due to decreased production of g-aminobutyric acid (GABA). 3. Growth impairment, decreased mental ability, convulsions, skin lesions etc. are seen in experimental pyridoxine deficiency humans and other animals.

13. Write functions and deficiency symptoms of Biotin A. Functions : Biotin is the only water soluble vitamin that function as coenzyme as such. It is coenzyme for carboxylases that catalyze carboxylation reactions. Acetyl-CoA carboxylase of fatty acid biosynthesis, propionyl –CoA carboxylase and pyruvate carboxylase of gluconeogenesis are some enzymes requiring biotin as coenzyme. Deficiency symptoms : 1. Since biotin is well distributed in foods its deficiency rarely occurs in humans. However in breast fed infants dermatitis occurs due to low biotin content in breast milk.


CHAPTER - 14 | Vitamins

2. In experimentally induced biotin deficiency neurological problems like depression, anemia, muscular pain, loss of hair and dermatitis are observed.

14. Write functions and deficiency symptoms of Folic acid A. Functions : Coenzyme form of folic acid is tetrahydrofolic acid FH4.






CH2 – N – H


– C – N – Glutamate H

OH Folic Acid Synthesis of FH4 : An NADPH dependent dihydrofolate reductase reduces folic acid to tetrahydrofolic acid in two steps. Dihydrofolic acid is intermediate. Reductase +

Folic acid +NADP +H


Dihydrofolic acid

Tetrahydrofolic Acid.




FH4 + NADP+.

1. FH4 actas carrier of one carbon units. It carries one carbon moieties like methyl, formyl, formimino and formate groups. In degradative pathways FH4 with one carbon unit is generated. In anabolic pathways one carbon is transferred to an acceptor. Transferase a. Formimino glutamate+FH4 b. Methyl FH4 +cobalamin c. Formyl FH4

Formimino FH4 + Glutamate Methyl cobalamin +FH4

C -2 of Purine ring

d. Methenyl FH4

carbon 8 of purine ring.

Deficiency symptoms : 1. Megaloblastic anaemia is major folic acid deficiency in man. Folic acid deficiency affects rapidly growing cells like bone marrow cells, intestinal cells because nucleotide formation requires folic acid. 2. Thrombocytopenia 3. Macrocytic hyperchromic anemia and leucopenia 4. Diarrhoea and weakness 5. Growth impairment, intestinal ulceration and anemia are deficiency symptoms in experimental animals.



Questions and Answers

15. Write chemistry, absorption, transport, functions, deficiency symptoms, sources and daily allowance of Vitamin B12 A. Chemistry : It consist of corrin ring CH3

with central cobalt atom. Corrin ring is tetrapyrrole. To the central cobalt



various groups are attached. So Vit. B12 with cyanide is known as cyano


cobalamin. Vit. B12 with hydroxyl N

group attached to central cobalt is


called as hydroxy cobalamin. If


methyl group is attached then it is

Methyl Cobalamin

called as methyl cobalamin. Deoxy adenosine containing Vit. B12 is called

as deoxy adenosyl cobalamin. Most common is cyano cobalamin. Absorption and Transport : Absorption of Vit. B12 requires intrinsic factor. It is secreted by parietal cells of stomach and it is a glycoprotein. Dietary Vit. B12 combines with intrinsic factor (IF) to form complex. Dietary Vit. B12 + Intrinsic factor

Vit. B12 - IF complex.

Through a receptor mediated mechanism this complex is absorbed in the ileum. The complex dissociates in ileal cells to IF and Vit. B12. Receptor Vit. B12 –IF

Vit. B12 –IF Ilealcells

Vit. B12 + IF

A Vit. B12 Transport protein known as transcobalamin in ileal cells transport Vit. B12 to various tissues of the body. Transcobalamin Vit. B12 in ileal cell Functions :

Vit. B12 – Transcobalamin complex


Vit. B12 is required as coenzyme by many enzymes. They are known as

cobamide coenzymes. Two cobamide common enzymes are methyl cobamide or methyl cobalamin and deoxy adenosyl cobamide or deoxy adenosyl cobalamin. 1. Methyl cobalamin is coenzyme of methionine synthesis. Methionine Synthase Homocysteine +Methyl-cobalamin Methionine +cobalamin. 2. Methyl malanonyl –CoA mutase requires deoxy adenosyl cobalamin as coenzyme. Methylmalonyl –CoA 186

Succinyl –CoA.

CHAPTER - 14 | Vitamins

Deficiency Symptoms : 1. Megaloblastic anemia with neurological disturbances is main symptoms of Vit. B12 deficiency. In Vit. B12 deficiency methionine sythesis is blocked. So neurotransmitter formation is affected. This results in neurogical problems like numbness in feet and hands and spinal cord degeneration. 2. Since Intrinsic factor is required for Vit. B12 deficiency lack of this factor also causes vit. B12 deficiency. However it is called as pernecious anemia. Here also three systems like erythropoietic, gastro intestinal and neurological system are affected. Sources : Vit. B12 is present mainly in animal sources. Organ meat like liver, kidney, brain, dairy products eggs and fish are good sources. Milk and milk products also contain this vitamin. Daily requirement : 3mg per day.

16. Write functions and deficiency of Pantothenic acid A. Functions : 1. Pantothenic acid is required for the synthesis of coenzyme A (CoA). CoA is involved in metabolism of carbohydrates, lipids and proteins. 2. Fatty acid synthase complex contains phosph pantotheine that serve as acyl carrier. Formation of coenzyme (CoA) and phosphopantotheine 1. Synthesis of Co A from pantothenic acid involves phosphorylation, cysteinylation, decarboxylation, adenosylation and phosphorylation. Phosphorylation Pantothenic acid

Cysteinylation phosphopantothenate

De carboxylation Phosphopanto theinyl cysteine



DephosphocoenzymeA phosphopantotheine

fatty acid synthase Phosphorylation

Dephosphocoenzyme A

coenzymeA (CoA).

Deficiency : Burning feet, fatigue, restlessness and abdominal cramps are noticed in experimentally deficiency induced human subjects. 2. In other animals experimental pantothenic acid deficiency causes dermatitis, growth impairment and neurological symptoms.



Questions and Answers

17. Write a note on Anti vitamins A. They are antagonists of vitamins. They cause vitamin deficiency. Some anti vitamins are used as drugs. They are present in foods. Some drugs are anti vitamins. 1. Avidin present in raw egg white binds biotin and prevent its absorption. This leads to biotin deficiency. 2. Vit. K antagonists are used as anti coagulants. Dicoumarol and warfarin are Vit. k antagonists used as anticoagulants. 3. Folic acid antagonists are used as anti cancer drugs. They are aminopterin and amethopterin. 4. Isoniazid is used in treatment of tuberculosis. It prevents formation of pyridoxal phosphate. So pyridoxine deficiency is likely to occur in isoniazid treatment. 5. Thiaminase destroys thiamin of foods. Hence thiamin deficiency occurs. Other model questions are

18. Visual cycle 19. Nyctalopia 20. Calcitriol 21. Rickets 22. Vit. K cycle. 23. Tocopherol 24. Biological antioxidants 25. Beri Beri 26. Coenzyme functions of thiamin 27. Write reactions involving coenzymes of riboflavin. 28. Write coenzyme form of niacin. Write reactions involving the coenzyme. 29. Scurvy 30. Avidin 31. Pellagra 32. Pyridoxine 33. Folicacid 34. PLP 35. Absorption of vit. B12 36. RDA of vit. C & A 188

CHAPTER - 15 | Minerals



Minerals 1. Define minerals. Classify. Give examples for each class. A. Minerals are small inorganic molecules. They are not produced in the body. So diet is main source. Based on requirement they are classified into. a. Bulk minerals : Are those minerals that are required in more than 100 mg per day. They are calcium, phosphorus, sodium, potassium, chloride and magnesium. b. Trace minerals : Are those minerals that are required in less than 100 mg per day. They are iron, iodine, fluorine, manganese, copper, zinc, selenium, molybdenum, cobalt and chromium.

2. Write functions of minerals. A. Functions of minerals : 1. Minerals are component of soft tissues like liver, kidney, lung etc. 2. Minerals are components of bone structure. 3. Minerals are required for several essential functions like blood coagulation, muscle contraction, neuro transmission, membrane potential and blood pressure. 4. Minerals are involved in acid base balance. 5. Minerals are required for actions of enzymes. 6. Minerals are structural components of hemoglobin, cytochromes, vitamins, nucleic acids etc. 7. Minerals are involved in hormone action. 8. Minerals are involved in transport of gases.

3. Write absorption, functions, deficiency symptoms, sources and daily requirements of Calcium A. Absorption : Duodenum and first part of jejunum are major sites of calcium absorption. Calcitriol promotes calcium absorption against concentration gradient by active transport mechanism. Factors affecting calcium absorption : Absorption of calcium in the intestine is influenced by several factors. 189


Questions and Answers

1. Calcium binding protein (CBP) : In the intestinal cells calcium binding protein is synthesized. Calcitriol promotes synthesis of this protein. This protein is known as calbindin and increases calcium absorption in the intestine. 2. Dietary calcium and phosphorus ratio affects calcium absorption. High phosphate decreases calcium absorption. 3. High fibre in diet interferes with calcium absorption. 4. PH or hydrogen ion concentration : Calcium absorption requires acid or neutral PH. Calcium absorption is less under alkaline or high PHconditions. 5. Calcium absorption depends on fat digestion and absorption. If fat digestion and absorption are impaired then calcium absorption is decreased. 6. Calcium absorption is affected by protein content in diet. Low protein in diet decreases calcium absorption. High protein in diet increases calcium absorption. 7. Calcium absorption is decreased in presence of salts of oxalates. Because calcium combines with oxalates and forms insoluble calcium oxalate. 8. Calcium absorption is decreased if phytic acid is present. Cereals of diet contains phytic acid. Functions : Calcium is involved in several important physiological processes. 1. Calcium is required for muscle contraction. 2. Neurotransmission is dependent on calcium. 3. Bone and teeth formation requires calcium. Calcium is major constituent of bone and teeth. 4. Blood clotting is another process depends on calcium. During blood clotting calcium is required for conversion of prothrombin to thrombin. 5. Actions of several hormones are mediated through calcium. Hence calcium is known as secondary messenger of hormone action. 6. Calcium is required for cell motility involving cellular activities like mitosis, migration etc. 7. It is required for action of cytosolic calcium dependent proteases calpains. 8. Many enzymes requires calcium for their action. For example enzymes of HMP shunt like glucose -6- phosphate dehydrogenase, lactonase, phosphogluconate dehydrogenase etc. 9. Enzyme activation also dependent on calcium. For example trypsin activation occurs in presence of calcium. 10. In some marine organisms calcium triggers bioluminescence. Jelly fish exhibits this property. 11. Calcium is involved in membrane structure and function. 12. Membrane transport requires calcium. Membrane integraty is maintained in presence of calcium.


CHAPTER - 15 | Minerals

Deficiency : Deficiency of calcium causes growth impairment, osteoporosis, tetany, hyperplasia of para thyroid glands. Sources : Milk is rich calcium source. Green leafy vegetables, eggs, fish, vegetables and fruits are fair sources. Dairy requirement : Adults : 800mg/day, children : 1200 mg / day.

4. What is normal plasma calcium level? Write about regulation of plasma calcium A. Normal plasma calcium level is 9-11mg %. Calcium exist in three forms in plasma. Physiologically active form or ionized form : It accounts for 50-60%of total calcium. It is only responsible for most of physiological actions of calcium. Protein bound : It is bound to protein like albumin. It is about 35-40% of total calcium. Non protein molecules like citrate, phosphate and bicarbonate also found in bound form with calcium. It accounts for about 5% of total calcium. Stable calcium is required for majority of its actions. So calcium level must be finely regulated. Many hormones and substances are responsible for maintenance of constant calcium level. They control calcium level within normal limits by acting at different sites. They are intestine, bone and kidney. Para thyroid hormone (PTH), calcitonin and calcitriol are substances and hormones involved in regulation of plasma calcium level. Parathyroid hormone secretion : Secretion of parathyroid hormone by parathyroid gland depends on calcium level in plasma. Decreased plasma serum calcium level is sensed by calcium receptors present in parathyroid cells. They inturn react with G- proteins present in membrane of parathyroid cells. The G- proteins activate phospholipase –C to produce inositol triphosphate (IP3)which raises intracellular calcium by acting on calcium stores and opening calcium channels. The raised intra cellular calcium leads to binding of calcium to intracellular calcium binding protein calmodulin to form calcium calmodulin complex This complex increases

cAMP by inhibiting phosphodiesterase. Increased

intracellular cAMP cause

release of PTH. Low plasma calcium level Calcium receptors G- Protein G-proteins Calcium stores Phospholipase –C Inositol triphosphate (IP3) Increased Calcium channels Calmodulin Intracellular calcium

calcium –calmodulin

More cAMP

PTH release.

complex. Para thyroid actions : A receptor present in membrane surface transport PTH into target cells. PTH target tissues are bone, kidney and intestine. It increases plasma calcium level by acting on these organs In bone it increases movement of calcium from bone by promoting



Questions and Answers

dissolution of bone matrix. This action of PTH is mediated by cAMP. By acting on adenylate cylase PTH increases cAMP. In the kidney PTH increases calcium reabsorption. In the intestine PTH

increases calcium absorption by promoting formation of calcitriol. The

calcitriol also promotes calcium absorption in the kidney. Thus the combined actions of PTH and calcitriol raises plasma calcium level to normal. With restoration of plasma calcium level to normal actions of PTH and calcitriol are inhibited. PTH






Release of calcium

Raised plasma calcium level

increased calcium absorption More reabsorption of calcium

Raised calcium level. Raised calcium level.

Calcitonin : It is another hormone involved in calcium regulation. It is produced by thyroid gland. It is an antagonist of para thyroid hormone. Secretion of calcitonin by c cells of thyroid gland is stimulated by raised plasma calcium level. On release calcitonin decreases plasma calcium by acting on bone. A receptor mediated process translocates calcitonin into osteoclasts of bone. In the osteoclasts calcitonin decreases release of calcium from bone. It prevents bone resorption by osteoclasts. cAMP mediates action of calcitonin. Its level increases in presence of calcitonin.

5. Write about diseases associated with calcium level in plasma. A. Diseases of plasma calcium leval a. Hypocalcemia : Decrease in plasma calcium level is known as hypocalcemia. It occurs in vit. D deficiency, hypopara thyroidism, renal insufficiency, rickets and osteomalacia. Decrease in plasma calcium level causes tetany. Clinical symptoms of tetany are hyper excitability of nerves of face and extremities and muscular spasms. b. Hypercalcemia : Increased plasma calcium level is known as hypercalcemia. It occurs in hyper parathyroidism, vit. D toxicity etc. In infants idiopathic hypercalcemia occurs. Clinical symptoms are cardiac abnormalities, neurological disturbances.

6. Write the functions of phosphate A. 1. Phosphate is present in humans to an extant of about 500-700gm. More of it is present in bone and teeth. 2. In the body phosphate is present in two forms. An inorganic form found in bone and teeth which is complexed with calcium and magnesium. Another is organic form. It is present as organic compounds in the cells and cell membranes. 3. In the bone and teeth it is present as hydroxyl apatite. 4. It is a component of nucleotides and nucleic acids. 5. High energy compounds like ATP, GTP, CTP etc contain phosphate. 6. Phospholipids formation requires phosphate. 7. Phosphate is component of blood buffers.


CHAPTER - 15 | Minerals

8. Formation of some water soluble vitamins coenzymes involves phosphate like TPP, pyridoxal phosphate etc. 9. Several cellular functions as well as enzyme regulation involves addition or removal of phosphate. 10. Degradation of several compounds involves phosphate containing intermediates. 11. Second messenger molecules cAMP, cGMP are phosphate containing substances. 12. Milk protein casein is phosphoprotein i. e. phosphate containing conjugated protein.

7. Write normal phosphate level in blood and disorders of it. A. Blood phosphate : Normal plasma phosphate level is 2. 5- 4. 5mg%. Blood phosphate level is higher in children. It is about 4-6mg%. Two types of blood phosphate level alterations occurs. Hypophosphatemia : Blood phosphate level is below normal. It occurs in hyper parathyroidism, vit. D deficiency, Fanconi syndrome etc. Hyperphosphatemia : Blood phosphate level is elevated. It occurs in hypo para thyroidism and vit. D toxicity. Since kidney filters phosphate from plasma in renal failure and nephritis hyper phosphatemia occurs.

8. Write a note on Sodium functions. A. Functionsof sodium 1. Sodium is present in high concentration in extra cellular fluids than intracellular fluids. 2. Sodium is constituent of buffers of blood. So it is required for proper hydrogen or pH homeostasis. 3. It is involved in nerve impulse transmission. 4. It is required for muscle contraction. 5. It is essential for the absorption of glucose in intestine. 6. Intestinal absorption of amino acids involves sodium ion co transport. 7. It is constituent of bile salts. 8. It is required for the action ATPases or pumps. 9. It is essential for growth of tissues.

9. Write functions of Potassium A. 1. Potassium is present in high concentration in intracellular fluid than extra cellular fluid. Hence it is major cation inside cell. 2. It is involved in membrane potential maintenance. 3. Potassium is involved in muscle function. 4. It has role in nerve impulse transmission. 193


Questions and Answers

5. It is essential for bile salt formation 6. It is required for the action of ATPase. 7. Tissue growth requires potassium 8. Storage of glycogen in liver requires potassium 9. Muscle glycogen storage involves use of potassium

10. Name functions of Chloride A. Functions of chloride : 1. It is constituent of hydrochloric acid (HCl) produced by parietal cells of stomach. 2. It is more in extra cellular fluid than intracellular fluid. 3. It is required for proper erythrocyte function i. e chloride shift. 4. It is involved in maintenance of plasma volume. 5. It is activator of some enzymes like amylase, angiotensim converting enzyme etc. 6. It is involved in neurotransmission.

11. Mention Magnesium functions A. Magnesium Functions : 1. Like potassium magnesium is more in intracellular fluid than extra cellular fluid. 2. It is required for bone and teeth formation. 3. It is essential for kinases catalyzed reactions in carbohydrate metabolism, protein metabolism, nucleic acid metabolism. 4. It is also required for other enzymes like RNA polymerase, glucose-6-phosphate dehydrogenase, enolase, transketolase etc. 5. It also act as activator of some enzymes.

12. Write absorption, transport, storage, functions, deficiency symptoms, sources and daily requirement of Iron A. Absorption : Iron occurs in the bound state in plant foods in the ferric (Fe3+) form. However in stomach acid environment iron dissociates and get reduced to ferrous form (Fe2+). Vit. C and cysteine are compounds that promote iron reduction. In the stomach an iron combining protein gastroferrin is also involved in iron absorption. An unknown mechanism is responsible for the absorption of gastroferrin and ferrous iron in duodenum and jejunum. In animal foods iron occurs in heme. Heme is absorbed as such in the mucosal cells of small intestine. In the enterocyte iron is released from heme. In the mucosal cells a copper containing enzyme ceruloplasmin forms ferric iron by oxidation from ferrous iron. This ferric iron undergoes to storage and transport.


CHAPTER - 15 | Minerals

Acidic pH Plant foods iron

vit. C

ferric iron

Ferrous iron

Intestinal mucosal cells.

Enterocyte Anlmal food iron

protoporphyrin +ferrous iron.

ceruloplasmin Ferrous iron

Ferric iron

storage and transport

Regulation of iron absorption : Hepcidin a 25 aminoacid peptide synthesized by liver regulates iron absorption. If sufficient iron is absorbed further absorption is blocked by hepcidin. Hemojuvelin another peptide also influences iron absorption by regulating expression of hepcidin. Iron transport : Iron enters plasma from mucosal cells of intestine aided by ferroportin. In the plasma an iron transport protein apotransferrin is present. It combines with iron to form transferrin. Iron is transported to various sites by this transferrin. plasma Iron of intestine


Various site of body.

Apo transferrin Storage of iron : Liver. spleen, bone marrow and intestine are the organs where iron is stored. These tissue contain iron storage protein apo ferritin. It combines with ferric iron to form ferritin and stored. Fe3+ Apoferritin



Functions : 1. Iron is required for the formation of hemoglobin, myoglobin and cytochromes. 2. Hemoglobin and myoglobin transport oxygen. Oxygen is attached to iron of these molecules. 3. Cytochromes iron is involved in electron transfer or oxidation reduction reactions. 4. Iron is required for the formation of iron sulfur proteins. 5. Iron of these proteins participate in oxidation reduction reactions. 6. Iron is constituent of several enzymes. 7. Tryptophan dioxygenase, homogentisic acid oxidase, xanthine oxidase, catalase, cytochrome p 450 – hydroxylase enzymes etc are examples for iron containing enzymes. 8. Lactoferrin present in milk is iron containing protein. Deficiency : Since iron is essential for production of blood anemia is main symptom of iron deficiency. Popularly it is called as iron deficiency anemia. Skin of affected people acquires pale yellow color. Microcytic hypochromic erythrocytes are seen in blood. Usually children,



Questions and Answers

pregnant women, adolescent girls are susceptible to this disease. Other clinical symptoms are fatigue, breathlessness and giddiness. It is major nutritional problem in developing countries. Sources : Green leafy vegetables like spinach, coriander leaves, palak etc and cereals, legumes and jaggary are good sources. Organ meat like liver, kidney, spleen etc and farm products like eggs and fish are fair sources. Daily requirement : 10mg/day, females : 18mg/day. In pregnancy and lactation it is about 30mg/day.

13. Write functions and deficiency symptoms of iodine. A. Iodine Functions : 1. It is constituent of thyroid hormones. 2. Thyroxine and triiodotyronine (T3) synthesis involves iodine utilization. 3. Thyroid hormones are essential for mental development as well as physical development. Deficiency symptoms : 1. Goitre, Swollen thyroid gland is major symptom of iodine deficiency. It is seen more in sub Himalayan regions of India. It occurs in other developing countries like China. 2. In children iodine deficiency causes decreased mental abilities. 3. In adults iodine deficiency causes apathy. 4. Radiation effects susceptibility is more in iodine deficiency. 5.

Mental disturbances, hypothyroidism and iodine induced hyper thyroidism are other symptoms of iodine deficiency.

14. Write functions and deficiency symptoms of Zinc A. Functions : 1. Zinc is an integral part of more than 2oo enzymes. Zinc metalloenzymes are involved in carbohydrate metabolism, aminoacid metabolism, bone metabolism, nucleic acid metabolism, electrolyte and blood pressure maintenance, gas transport and super oxide metabolism. Some examples are lactate and malate dehydrogenase of carbohydrate metabolism, carboxy pepetidase of protein digestion, DNA and RNA polymerase of nucleic acid biosynthesis, alkaline phosphatase of bone metabolism, angiotensinconverting enzyme of electrolyte and blood pressure regulation, carbonic anhydrase of gas transport and super oxide dismutase of superoxide metabolism. 2. Zinc is constituent of zinc finger proteins which are involved in regulation of gene expression. 3. Gustin is a zinc containing protein involved in taste bud development.


CHAPTER - 15 | Minerals

4. Zinc is required for stability of hormone insulin. 5. Zinc is involved in maintenance of structure of ribosomes and chromatin. 6. It is involved in immune system function. 7. It is essential for tissue growth, development and regeneration. 8. For normal reproduction zinc is needed. 9. Bone and muscle formation involves participation of zinc. Deficiency symptoms : 1. Zinc deficiency occurs in a rare genetic disease acrodermatitis enteropathy. In this disease zinc deficiency is due to block in absorption of zinc. Clinical symptoms are acral dermatitis and diarrhoea. 2. Hypogonadism and growth retardation are symptoms of zinc deficiency. 3. Loss of taste occurs in zinc deficiency. 4. Dermatatis and immune dysfunction are observed in experimentally induced zinc deficiency.

15. Mention functions and deficiency symptoms of Fluorine A. Functions : 1. Fluorine is required for bone and teeth formation. In the teeth it is present in dentin in fluoroapatite form. 2. Fluorine protects teeth enamel from action of bacterial acids. In the oral cavity fluorine act as inhibitor of bacterial enzymes. Bacteria use glycolysis for energy production. Since fluorine inhibits glycolytic enzymes bacterial growth is prevented. Therefore in oral cavity fluorine prevent solubilization of enamel of teeth by acids produced by bacteria. 3. In adults fluorine prevents osteoporosis. 4. In elder people progressive loss of hearing occurs as age advances. Fluorine may slow down this process. Deficiency symptoms : 1. Dental caries is the major symptom of fluorine deficiency. 2. Dental caries is characterized by cavities on tooth surface due to loss of enamel.

16. Write a note on Fluorosis and defluoridation. A. Consumption of drinking water containing excess fluorine leads to fluorosis. Fluorosis is seen in several states of this country. It affects people of all ages. It is major health problem in several districts of Andhra Pradesh. There are two types of fluorosis. They are 1. Dental fluorosis and 2. Skeletal fluorosis. Dental fluorsis : Molted teeth is major symptom. Due to loss of enamel teeth appears dull, patches and cavities are produced on surface of teeth. 197


Questions and Answers

Skeletal fluorosis : Knock knees or genu valgum is main skeletal deformity seen. Major joints becomes stiff and painful. Neurological disturbances due to spine deformities also occurs. Defluoridation : It is process used to remove excess fluorine present in drinking water. Several defluoridation equipments and portable units are developed by various agencies. Most of them use absorbents and activated charcoal to eliminate excess fluorine from water.

Other model questions are

17. Write about factors affecting calcium absorption. 18. Iodine deficiency disorders. 19. Iron absorption 20. Transport and storage of iron. 21. Calcium functions 22. Functions of iron 23. Iron deficiency 24. Fluorosis 25. Functions of fluorine 26. Serum calcium homeostasis 27. Trace elements 28. Zinc 29. Iron containing proteins 30. Zinc containing proteins.


CHAPTER - 16 | Water, Electrolytes & Acid-Base Balance



Water, Electrolytes & Acid-Base Balance 1. Write functions of Water A. WaterFunctions : 1. Human body is nothing but a water reservoir. About 80% of body mass is occupied by water. 2. For all forms of life water is essential constituent. 3. Semi solid nature of human body is due to enormous amount of water present. 4. Every cell of human body and all other life forms contain water. 5. Water is medium in which all cellular events occurs. 6. Water is required for enzyme action. 7. Transport of several molecules requires water. 8. Body temperature is regulated by water. 9. Water aids folding of proteins, nucleic acids, enzyme etc. 10. Ions required for biochemical reactions of cells is provided by water.

2. Write about Maintenance of water balance and associated disorders. A. In a normal healthy individual water intake equals water out put. Water intake per day is about 2500ml. Drinking water contributes to about 1200ml, food water is about 1000ml and water of metabolisms is about 300ml. Water out put per day is about 2500 ml. Urine is major route of water elimination. About 1200ml of water is removed in urine. Other routes of water removal are through lungs, skin and feces. The body water homeostasis is achieved by combined action of hormonal and other factors. ADH is hormone involved in water homeostasis. Water intake and water out put are two factors involved in water balance maintenance. Decrease in water content in the body leads to stimulation of thirst centre and thirst in caused. At the same time another centre in the hypothalamus is stimulated anti diuretic hormone is released. Water is consumed due to thirst. Through circulation ADH reaches kidney and act on renal tubular cells. As a result more absorption of water takes place in kidney. So by the combined action of ADH and thirst centre water balance is attained. Exactly reverse occurs when there is excess water in body. Due to excess water thirst centre and other centre in hypothalamus are inhibited. Water in take is stopped. ADH secretion is inhibited. Absence of ADH leads to 199


Questions and Answers

elimination of water through kidney. These mechanisms bring back water content to normal state. Thus the body water content of healthy adult is maintained. Diseases of water balance : 1. Edema : Excess water in body leads to edema. It is also known as over hydration. It occurs in excess secretion of ADH, water intoxication, cancer, drugs and overdose of intravenous fluids. 2. Dehydration : In this condition water content of body is decreased. It occurs in vomiting, diarrhoea, hypothalamus lesions, diabetes insipidus etc.

3. Define Electrolytes. Classify. Give examples for each class. A. Charged solute molecules are known as electrolytes. There are two types of electrolytes. Inorganic and organic solutes. Usually inorganic solutes are considered as electrolytes. Inorganic solutes are further divided into cations and anions. Cations : They are positively charged inorganic solutes. Sodium (Na+), Potassium (K+), Calcium (Ca2+), and magnesium (Mg2+), are examples. Anions : They are negatively charged inorganic solutes. Sulphate (So42-), Phosphate (Po43-), Chloride (Cl-) and bicarbonate (HCO3-) are examples. Organic electrolytes are mainly anions. They are contributed by protein, organic acid and organic phosphate.

4. Name plasma electrolytes. Write their normal level in plasma and functions A. Plasma electrolytes includes both anions and cations. Anions of blood plasma : Bicarbonate, chloride, phosphate, sulphate, iodine and fluoride are anions present in plasma. Normal plasma bicarbonate level is 24-30meq/ L. Normal chloride level in plasma is 100-110 meq/L. Cations of blood plasma : Sodium, potassium, calcium, magnesium, iron and copper are cations present in plasma. Normal plasma sodium level is 135-145 meq/L. Normal potassium level in plasma is 3-5 meq/L. Functions of electrolytes : 1. Electrolytes are essential for several physiological process. 2. Electrolytes are involved in membrane potential maintenance. 3. Neuro muscular excitability and nerve impulse trans mission involves electrolytes. 4. Electrolytes are required for bone formation. 5. Enzyme catalysis is dependent on electrolytes. 6. Blood clotting is another physiological process dependent on electrolytes.

5. Write about electrolyte balance maintenance and disorders associated. A. In the body electrolyte balance is maintained by many mechanisms. 1. Sodium pump is one mechanism of maintaining sodium balance. It keeps high extra cellular sodium level against low intracellular sodium. 200

CHAPTER - 16 | Water, Electrolytes & Acid-Base Balance

2. Aldosterone is hormone involved in handling of sodium and potassium by kidney. It favours sodium absorption and potassium excretion by kidney. 3. Bicarbonate concentration is maintained by kidney. Kidney also maintains electrolyte balance by eliminating salts. 4. Electrolyte concentration in body is also affected by diet, water intake and salt in take. Diaseases of electrolyte balance : Electrolyte balance is disturbed due to loss of body fluids. It occurs in diarrhoea, vomiting, burns, hemorrhages and sun stroke.

6. Describe Acid base balance or Hydrogen ion (PH) Homeostasis A. Normal persons blood pH ranges from 7-35-7. 45. This range is maintained by specific systems. These systems keeps blood pH within normal range by neutralizing acid or bases produced in the body. If they are not neutralized then hydrogen homeostasis or acid –base balance is disturbed which is not good for the well being of an individual. Hydrogen ion homeostasis : Since proper pH is required body processes maintenance of pH with in normal range is essential. Several systems participates in body hydrogen homeostasis. They are 1. Buffer systems present in body. 2. Lungs 3. Kidneys. Buffer systems They are present in blood, plasma, extracellular fluids, intracellular fluid and tissues of the body. Buffer is composed of weak acid and its salt or conjugate base. Buffer resist change in PH of medium in which they are present when acid or base is added. Buffers of blood plasma 1. Bicarbonate and carbonic acid buffer : It has major role in the maintenance of blood PH. It is present in high concentration in blood. It function as an effective buffer in controlling pH by maintaining conjugate (bicarbonate)and weak acid (carbonic acid) ratio 20 : 1. The blood pH is with in normal limits as long as this ratio is maintained. Mechanism of action of bicarbonate buffer : In the blood bicarbonate exist as sodium bicarbonate (NaHCO3). When acid is added NaH CO3 reacts with acid and reduces its strength i. e convert to weak acid. For example aceto acetic acid when added it is converted to sodium acetate. NaHCO3 +Acetoacetate

Sodium aceto acetate + carbonic acid.

The carbonic acid formed is weak acid compared to acetoacetate. So blood pH changes very little and ratio of bicarbonate and carbonic acid alters. This is restored to normal level or ratio by eliminating carbonic acid from lungs. Thus the ratio of bicarbonate and carbonic acid returns to normal and blood pH remains 7. 4. 2. Phosphate buffer and protein buffer : These buffers are present in blood at low concentrations. Hence their role in blood pH regulation is limited. However at normal blood pH phosphate buffer is found as more effective than bicarbonate buffer.



Questions and Answers

Erythrocytes Buffers : In erythrocytes hemoglobin is present at high concentration. Hemoglobin (Hb) and its protonated form (HHb) constitutes buffer system in erythrocytes. It is known as hemoglobin (Hb/HHb) buffer system. High hemoglobin concentration make hemoglobin buffer system as major buffer of blood. Further constituent aminoacids particularly histidine make hemoglobin an effective buffer at blood pH. Respiratory system or lungs Lungs have an important role in blood pH homeostasis. They maintain blood pH by affecting carbonic acid part of bicarbonate buffer. They control carbonic acid concentration in blood by carbon dioxide partial pressure in plasma. Further formation of carbonic acid from carbon dioxide is catalyzed by carbonic anhydrase present in lungs. Decrease in blood pH occurs due to neutralization of acids by bicarbonate. As a result ratio of bicarbonate and carbonic acid is altered. Now respiratory centre is stimulated more of carbon dioxide is eliminated and carbonic acid formation is decreased. This leads to restoration of bicarbonate and carbonic acid ratio to normal and blood pH returns to normal. Thus hyper ventilation compensates blood pH fall. When the blood pH increases exactly reverse process occurs. Due to raise in blood pH carbonic acid concentration in blood decreases. This leads to change in bicarbonate and carbonic acid ratio. To bring blood pH to normal respiratory rate decreases and more carbonic acid is formed due to increased partial pressure of carbon dioxide. At the same time ratio of bicarbonate and carbonic acid also returns to normal. So hypoventilation compensates for raise in blood pH. Kidneys Kidneys play major role in maintenance of constant level of blood pH. It regulates blood pH by several mechanisms. 1. Bicarbonate absorption : In proximal tubule of kidney bicarbonate of plasma filtrate is absorbed. Usually luminal membrane is not permeable to bicarbonate. It enters tubular cells in the form of carbon dioxide which is freely permeable. In the luman bicarbonate combine with proton to form carbonic acid which is decomposed to water and carbon dioxide. H++HCO3 -



H2O + Co2

Enters luminal cell.

In tubular cells carbonic anhydrase rehydrates carbon dioxide to carbonic acid which dissociates to bicarbonate and proton. The bicarbonate diffuses into blood. Carbonic Co2 + H2 o


An Hydrase


Blood plasma


2. Excretion of hydrogen ions : Kidney regulates blood pH by eliminating hydrogen in distal tubules. In tubular cells hydrogen ion is formed from dissociation of carbonic acid. As mentioned above bicarbonate diffuses into plasma and hydrogen is eliminated into luman. In the lumen hydrogen combines with (NaHPO4) monosodium hydrogen phosphate to form


CHAPTER - 16 | Water, Electrolytes & Acid-Base Balance

mono sodium dihydrogen phosphste (NaH2 PO4) which is excreted in urine. Due to this the urine pH becomes acidic in distal tubules lumen. HCO3NaHPO4 Distal tubular cells




NaH2 PO4


luman Ammonia excretion : By eliminating ammonia in urine kidney plays important role in acid base balance. In renal cells ammonia is released from glutamine by the action of glutaminase enzyme. Glutamine enters renal cells from blood plasma. Plasma


Glutaminase renal cells Ammonia Glutamate

Ammonia formed in renal cells diffuses into lumen where it combines with proton to form ammonium ion. The presence of ammonium ion makes urine acidic. Proton Ammonia

Ammonium ion


Acidic Urine.

Lumen Thus kidney regulates blood pH by absorbing bicarbonate, eliminating hydrogen ion and excreting ammonium ion. Further kidney also recover sodium along with bicarbonate. Therefore kidneys are not only involved in acid base balance but also in electrolyte balance.

7. Explain functions of body normal pH A. 1. For the action of enzymes appropriate pH is essential. If pH is altered then enzyme action is affected. This disturbs body homeostasis. 2. For transport of solute molecules in the body proper pH is required. 3. For the maintenance of structure of folded state of proteins, enzymes and nucleic acids proper pH is required. Alteration in pH causes structural changes in these biomolecules which in turn affects functions of these macro molecules. 4. Proper pHis required for maintenance of structure of coenzymes and metabolites.

8. Write about Acid base imbalance or Acid base disturbances A. Two types of acid base disturbances are known. They are acidosis and alkalosis. a. Acidosis : It occurs due to low blood pH. It is due to accumulation of acids. It is further sub divided into 1. Metabolicacidosis and 2. Respiratory acidosisbased on underlying cause.



Questions and Answers

1. Metabolic acidosis : Most common acid base disturbance is metabolic acidosis. More acids are produced by metabolism. This leads to decreased bicarbonate concentration. It occurs in uncontrolled diabetes mellitus and starvation. Loss of bicarbonate due to diarrhoea and vomiting also cause metabolic acidosis. Increased elimination of bicarbonate by kidneys also leads to metabolic acidosis. Ingestion of acids and decreased elimination of proton by kidneys also leads to metabolic acidosis. 2. Respiratory acidosis : Plasma partial carbon dioxide pressure is more due to abnormal lung function. Decreased respiration or hypoventilation occurs due to depression of respiratory centre. Sedatives like morphine and barbiturates depress respiratory centre. Hypoventilation also occurs due to obstruction to air passage. In pnumonia, emphysema, asthma air passage is narrowed. Therefore respiratory acidosis occurs in diseases of lung and respiratory centre depression. b. Alkalosis : It occurs due to high blood pH. It is due to accumulation of alkali. It is further subdivided into 1. Metabolic alkalosis 2. Respiratory alkalosis. 1. Metabolic alkalosis : Bicarbonate concentration is more in blood. It occurs due to loss of HCl. More HCl is lost in prolonged vomiting. Excessive excretion of ammonia by kidney also leads to metabolic alkalosis. Ingestion of alkali cause metabolic alkalosis. 2. Respiratory Alkalosis : Partial pressure of carbon dioxide is less. It occurs due to hyperventilation. When respiratory centre is stimulated hyperventilation occurs. Hyper ventilation occurs at high altitudes, head injury, drug poisoning, fever and anxiety. Other model questions are

9. Blood buffers 10. Role of kidneys in acid base balance 11. Acidosis 12. ADH 13. Water intoxication. 14. Alkalosis 15. Serum electrolytes 16. Normal levels of electrolytes in blood 17. Write normal blood PH range. Explain mechanisms of its regulation. 18. Metabolic acidosis 19. Respiratory acidosis


CHAPTER - 17 | Energy Metabolism and Nutrition



Energy Metabolism and Nutrition 1. Define calorie. How energy values of foods are measured? Write calorific values of common foods. A. Calorie (C) or Kilocalorie (Kcal) or nutritional calorie (C) is energy unit currently in use. It is defined as amount of heat energy required to raise temperature of 1Kg of water by 1o c. Direct colorimetric methods are used to determine energy values of foods. Energy out put of food stuff is estimated by measuring heat production. The human subject is asked to stay in a steel insulated chamber. When food is oxidized in his body heat is produced which is transferred to water through chamber. Energy out put of food stuff is calculated from temperature differences between in coming and out going water. The calorific values of some common foods are carbohydrates (1g) -4C ;Lipids (1g) -9C ; proteins (1g) -4C;bread (1kg) -2630C; sugar (1kg) -4100C and cake (1kg) -4000C.

2. Write a note on Respiratory quotient A. It is defined as ratio of amount of carbon dioxide produced to amount of oxygen consumed when food is oxidized in side the body. Amount of carbon dioxide produced Respiratory quotient (R. Q) = –––––––––––––––––––––––––––––––––– Amount of oxygen consumed Significance : 1. Carbohydrate, fat and protein (R. Q) values are 1, 0. 7 and 0. 8 respectively. 2. R. Q of mixed diet is about 0. 85. 3. Type of food being oxidized for energy production in the body can be known from R. Q values. 4. In some diseases like diabetes mellitus and starvation R. Q values decreases due to change of food used for energy production in the body.



Questions and Answers

3. Write about BMR. A. Basal metabolic rate (BMR) : It is defined as heat out put of an individual having normal body temperature who is on fasting for the last 12 hours and lying in complete mental and physical and emotional rest. BMR of an individual is obtained using formula 24C/day /kg. Normal BMR Values for male and female are 37. 5C/sqm/hr and 35C/sqm/hr respectively. Factors affecting BMR : 1. Age, surface area, sex and environment influences BMR. 2. In children BMR is high but it is low in old people. 3. BMR increases with increasing surface area. 4. Males has high BMR than females. 5. In cold weather BMR is high but in hot climate BMR is low. 6. Physiological conditions and exercise also affects BMR. In pregnancy and lactation BMR is high but it is low in sleep. BMR increases with exercise. 7. In several pathological conditions BMR is either increased or decreased. 8. In fever, hyperthyroidism and cushing's syndrome BMR is increased. 9. In starvation, hypothyroidism and addison's disease BMR is decreased.

4. Specific dynamic action of food (SDA) A. Specific dynamic action (SDA) of food : It is defined as extra amount of energy produced over normal energy out put of food stuff when it is oxidized in the body. For example when carbohydrate of 100C is oxidized in the body 106C of energy is produced. The extra 6C of carbohydrate oxidation is SDA of carbohydrate. Traditionally SDA is expressed as per centage. Hence SDA of carbohydrate is 6% like wise SDA of other foods like protein and fat is 30% and 4% respectively. For mixed diet SDA is 6-19%. This extra energy comes from tissue metabolism. Since mixed diet is most commonly consumed to compensate for this loss of energy by tissues about 10% of total calories are added to daily energy requirement of an individual.

5. Calculate your energy requirement per day. A. Energy requirement of college student per day Daily energy need of 18 years old college student with weight of 70Kg has been determined. A college student is a sedentary worker. His daily life consist of three phases. Each phase has duration of 8 hours. They are a. Sleep. b. Personal activities which consist of 4 hours of light work and 4 hours of moderate work. c. Sedentary work (college work). Energy requirement for sleep is calculated as 600calaries (BMR X body surface area X 8 = 44C X 1. 7 X 8). Energy requirement for personal activities is calculated as 1120calaries (1. 5C X 206

CHAPTER - 17 | Energy Metabolism and Nutrition

70 X 4 + 2. 5 X 70 X 4). For 8 hours of college work energy requirement is calculated as 840 calories (1. 5 X 70 X 8). Total energy requirement is obtained by taking sum of three energy requirements. It is about 2560C(600+840+1120). Thus a college student requires about 2500 C per day. In the case of adult sedentary worker the daily energy requirement is 2500C and 2000C for men and women respectively. For moderate worker daily energy requirement is 3000C for men and 2500C for women. An adult male heavy worker needs about 4500C per day where as female heavy worker needs about 3000 calories per day. The energy requirement of pregnant and lactating women is more than sedentary worker. About 2300 calories and 2700 calories are required by pregnant and lactating women respectively.

6. Define nutrients. Give examples. A. Nutrients : These are essential substances present in food which are not produced in the body. They are required for growth and maintenance of the body. Six major types of nutrients are identified. They are 1. Carbohydrates

2. Lipids. 3.

Proteins. 4. Vitamins. 5. Minerals and 6. Water.

7. Write about proximate principles of food A. Carbohydrates, lipids and proteins are known as proximate principles of food and they yield energy. 1. Carbohydrates : Food carbohydrate contributes to about 60% of body energy requirement in many countries. Carbohydrates present in diet are majority of them are polysaccharides and to some extant disaccharides. Cereals, legumes, vegetables like potatoes, sweet potatoes etc and other fruits like apples, bananas etc and meat, garlic etc. are source for polysaccharides. Starch, glycogen and inulin are polysaccharides present in diet. Among all the polysaccharides starch is present in high concentration. Further amylopectin content varies among starches of different origin. Glycogen is absent in plant foods. In animal foods glycogen content is less. Milk is major source of disaccharide lactose. In the infants lactose contributes to 50% of energy requirement. Table sugar is major source of disaccharide sucrose. Other source of sucrose are sweets, bakery products like jams, jellies, syrups, ice creams etc. Fruits juices, honey and beverages contain sucrose. Some biscuits contain glucose. Fructose is present some fruit juices. Daily requirement : For proper health about 100gm of carbohydrate must be present in diet. However for short periods carbohydrate is not essential because it is synthesized from protein and lipids. 2. Lipids : Fat is common name for dietary lipids. Food fat contributes to 30% of energy requirement of people in majority of countries. However in industrialized nations fat 207


Questions and Answers

may contribute to more i. e about 40-50% of energy requirement. Fat of plants as well as animal origin is present in diet. Animal fat tissue (Adipose tissue) is major source for fat in diet. Ground nut oil, rice bran oil, sunflower oil, coconut oil, soybean oil, corn oil, palm oil etc are other oils that contributes to fat in diet. Further nuts, cereals and pulses of food also contain lipid. Triglycerides, fatty acids, phospholipids and cholesterol are lipids present in dietary fats. Animal foods are rich in cholesterol. Other functions of dietary fat is to supply essential fatty acids and fat soluble vitamins. Fat reduces size of diet and improves palatability of food. Dietary fat is immediate source of energy also apart from glucose. Further dietary fat is responsible for fullness feeling. Daily requirement : Diet must contain about 30-50gm of fat per day. And 5gm of essential fatty acids. 3. Proteins : Protein in diet supplies about 10-15% of energy demand of body. However in rich industrialized countries proteins contributes more to energy requirement. In the case of low income people of developing countries protein contributes to less of energy requirement. Animal meat, fish, eggs, milk, cereals and pulses are sources for protein in diet. Vegetables and fruits contain less of protein. Main function of dietary protein is to replace protein lost due to turn over. By supplying essential amino acids dietary proteins replaces nitrogen lost from the body. Another function of dietary protein is maintenance of nitrogen balance. Daily requirement : About 70gm of protein is required per day.

8. Define balanced diet. Write balanced diet for a sedentary worker. A. It is a diet containing all six types of nutrients in adequate amounts to meet energy requirement as well as nutritional requirement of an individual. Further recommended proportion of carbohydrates, fats and proteins in balanced diet are 70%, 20% and 10% respectively. Balanced diet of a sedentary worker whose energy demand is 2500 C per day must contain 440gm of carbohydrate, 50gm of fat, 70gm of protein and vitamins, minerals and water in adequate amounts. However a balance diet of this nature is difficult to prepare because foods we consume consist of cereals, legumes, vegetables and fruits etc in which carbohydrates, fats and proteins are together. Hence composition of balanced diet for vegetarians consisting of cereals, legumes, vegetables etc. is cereals, 450gm;pulses, 70gm; green vegetables, 100gm; other vegetables, 175gm;milk, 250ml; fruit, 30gm; fat, 45gm and sugar 40gm.

9. Write about Malnutrition A. Consumption of food or diet low in calories or proteins or both is called as malnutrition. People of developing countries are mostly exposed to malnutrition. Civil war, famines or


CHAPTER - 17 | Energy Metabolism and Nutrition

drought, earth quakes, sunamies etc may cause malnutrition in general population. Children and pregnant women are most susceptible. Two types of malnutrition are well known. They are a. Marasmus b. Kwashiorkor. Marasmus : It occurs due to consumption of food low in protein and calories. Children below 2years of age are most affected. Weight of children affected with disease is below 60% of normal. It is due to feeding infants diet low in calories and proteins after withdrawn from breast feeding. Main clinical symptoms are severe loss of body fat and muscle protein, head is big, dry skin, growth and mental retardation are common. Kwashiorkor : It occurs due to consumption of insufficient or inadequate protein only. Children below 2years of age are most affected. Weight of children affected with this disease in usually below 80% of normal weight. Usually it occurs in children when they are given traditional foods after with drawn from breast feeding. These traditional foods are of mostly plant based foods rich in starch but lacks adequate protein. So these foods meet only energy requirement of body but protein requirement is not met. Low economic group children are most affected. Clinical symptoms are edema, pot belly or distended abdomen, dermatitis, anaemia, diarrhoea and susceptible to infections.

10. Write briefly about dietary fibre A. Dietary fibre is mainly of plant origin. Indigestible plant polysaccharides like cellulose, hemicellulose, pectin, gums and lignin constitutes dietary fibre. Importance of dietary fibre in human

health is recently recognized. Dietary fibre has several protective,

preventive and curative effects. It is required for good health. The incidence of colonic diseases like ulcerative colitis, piles, constipation and irritable bowel syndrome decreases with use of fibre in the diet. Metabolic diseases like diabetes mellitus, obesity, coronary artery disease, hypertension etc incidence decreases with use of fibre diet. Dietary fibre lowers blood glucose, cholesterol and triglyceride levels. However absorption of glucose, cholesterol and some minerals is slow in presence of dietary fibre.

11. Write a note on biological value A. Biological value (BV) is defined as percentage of absorbed nitrogen that is retained by the body. N retained Biological value (BV) = ––––––––––– N absorbed

x 100

Significance The biological value of a protein measures the quantity of dietary protein used by animal for growth and maintenance of body function. Biological value of protein may also indicates essential amino acid content, digestibility of protein and availability of digested products for



Questions and Answers

absorption. Biological values for some proteins are 96% for egg, 80% for milk and meat proteins and 60% for rice and wheat proteins. Based on biological values proteins are classified as 1. Good quality proteins 2. Poor quality proteins Good quality proteins have high biological values. Examples are animal derived proteins Poor quality proteins have low biological values. Most of plant derived proteins come under this category. Other model questions are

12. PEM 13 Kwashiorkor 14. Dietary fibre 15. PCM


CHAPTER - 18 | Hormones



Hormones 1. Define hormone. Write chemical nature of hormones. A. Hormones are substances produced by various cells or glands of the body. They act on cells which are

far away

from site or gland of their origin. Usually blood carries these

hormones from site of production to site of action. Organs on which hormones act are known as target tissues. Endocrine glands produce hormones. Chemical nature :Hormones are proteins, steroids, organic substances, peptides and amino acid derivatives.

2. Describe general mechanisms of Hormone action A. 1. Generally hormones are chemical substances with messages. 2. In target tissues these messages are translated into biological effects. 3. Several steps may be involved in translation of these messages and number of steps depends on type of hormones. 4. Further second messenger molecules present in target tissues mediate hormone action in some cases. 5. Translation of message begins with binding of hormones to receptor present on membrane to form hormone receptor complex. 6. The remaining steps involved in signal transduction process varies from one hormone to another a. For example adrenal medullary hormones like catecholamines the hormone receptor complex inter act with class of membrane proteins G-proteins to produce second messenger molecules. G- proteins are heterotrimers containing three different subunits Gα, Gß and Gγ and binding sites for GTP and GDP. Signal transduction of hormones involves association and dissociation of subunits. In resting state GDP is bound to Gα subunits of G- protein. It is designated as GDP –G protein. The hormone –receptor complex causes conformational change in GDP- G protein. This lead to dissociation of Gß and Gγ subunits and exchange of GTP for GDP. Now G-Protein designated as Gα-GTP.



Questions and Answers

Hormone GDP-G protein Membrane receptor Hormone receptor complex Gα- GTP + Gß and Gγ. Now Gα-GTP activates enzymes like adenylate cyclase, phospholipase C to produce second messenger molecules like cAMP and inositol triphosphate (IP3). Gα – GTP Inactive adenylate cyclase Active adenylate cyclase Active adenylate cyclase ATP


In active phospholipase C

Active phospholipase C

Active phospholipaseC PI P2

Inositol tri phosphate (IP3).

These second messenger molecules produce final biological responses by acting on enzymes of biological process. Enzymes cAMP, IP3 Biological response. Proteins b. In the case of steroid hormones and thyroid hormones hormone – receptor complex is translocated into nucleus where it binds selective genes and alters gene expression. The increased expression of genes lead to biological effect. Translocation Hormone –receptor Nucleus complex Gene product c. In the case of

Gene expression

Biological response. hormones like insulin binding of hormone to receptor leads to

activation of tyrosine kinase activity of receptor. Tyrosine kinase phosphorylates tyrosine residues of receptor to form phosphorylated receptor. This inturn phosphorylates insulin receptor substrate (IRS). Binding of phosphorylated IRS to several proteins, enzymes leads to final biochemical effect. Hormone

Receptor Hormone receptor

Activated tyrosine kinase

Activation of receptor tyrosine kinase

Phosphorylated receptor

phosphorylation of IRS

Binding Phosphorylated IRS

Enzymes, proteins To


Biochemical effect.

CHAPTER - 18 | Hormones

3. Name hormones of pancreas. Write their actions. A. Insulin, glucagon and somatostatin are hormones of pancreas. They are proteins. Actions of Insulin Insulin affects carbohydrate metabolism, lipid metabolism and protein metabolism. Carbohydrate Metabolism :It increases glucose entry into peripheral tissues like muscle and adipose tissue by promoting translocation of glucose transporter. It stimulates glycogensis by activating glycogen synthase of glycogenesis. It suppresses gluconeogenesis. It increases rate of glycolysis by activating regulatory enzymes of glycolysis. All these actions of insulin lowers blood glucose level. Insulin Adipose tissue, muscle

Translocation of glucose transporter

more glucose entry.

Insulin Glycogen synthase

Activation of glycogen synthase

Increased glycogenesis

Insulin Key enzymes of gluconeogenesis

Suppression of key enzyme


gluconeogenesis. Insulin Regulatory enzymes of glycolysis Enzymes

Activation of regulatory

Increased glycolysis rate.

Lipid Metabolism :Insulin promotes lipogenesis in adipose tissue. It increases fatty acid biosynthesis by activating acetyl –CoA carboxylase. Insulin Adipose tissue

Increased lipogenesis Insulin

Acetyl –CoA carboxylase (native)

Active acetyl –CoA carboxylase

Increased fatty acid biosynthesis. By increasing uptake of glucose by adipose tissue insulin promotes triglyceride biosynthesis. Insulin Adipose tissue

Activation of HMG –CoA reductase

cholesterol synthesis

increased. Protein Metabolism :Insulin is an anabolic hormone. It promotes protein synthesis and retards protein breakdown. Decreased protein breakdown


Increased protein synthesis.



Questions and Answers

Actions of glucagon Glucagon is an antagonist of insulin. It raises blood glucose level by promoting glycogenolysis and gluconeogenesis. It inhibits fatty acid synthesis by inhibiting acetyl –CoA carboxylase. By activating hormone sensitive lipase glucagon promotes lipolysis. Cholesterol synthesis is inhibited by glucagon. Actions of Somatostatin It prolongs gastric emptying. It decreases acid secretion by stomach. It decreases secretion of pancreatic enzymes. Absorption of glucose is decreased by somatostatin.

4. Name hormones of Adrenal cortex. How they are synthesized ? Mention their functions. A. Aderenal cortex produces glucocorticoids and mineralo corticoids. All of them are steroids and produced from cholesterol. Gluco corticoids :Cortisol is the major glucocorticoid. They affects carbohydrate, lipid and protein metabolism. Biosynthesis of cortisol : It beings with cholesterol. Progesterone is an important intermediate. Cholesterol







Carbohydrate Metabolism :Glucocorticoids promote gluconeogenesis by increasing availability of glucogenic amino acids

and by increasing activity of key enzymes of

gluconeogenesis. They promote glycogenesis by activating glycogen synthase. Glucocortucoids raises blood glucose levels by these actions. Lipid Metabolism :Glucocorticoids promotes lipolysis by activating hormone sensitive lipase. At high concentration they promote lipogenesis. Protein Metabolism :Under normal conditions glucocorticoids stimulates protein metabolism. At excess concentration they promote protein breakdown. Other functions of glucocoticoids are immuno suppressive action, anti inflammatory action and maintenance of blood pressure. Mineralo corticoids : 1. Aldosterone promotes sodium retention in distal convoluted tubules. 2. It promotes sodium retention by increasing formation of proteins involved in sodium absorption. Synthesis of aldosterone :Cholesterol is converted to aldosterone via progesterone as out lined below


CHAPTER - 18 | Hormones



Deoxy corticosterone





5. Name hormones of adrenal Medulla. Write their synthesis and functions. A. Hormones of adrenal medulla are adrenaline or epinephrine and nor adrenaline or nor epinephrine. They are also called as catecholamines. They are amino acid derivatives. Biosynthesis :Catecholamines are synthesized from amino acid tyrosine. Reactions : 1. Hydroxylation of tyrosine by tyrosinase initiates catecholamine formation. Dihydroxy phenyl alanine (DOPA) is product. 2. In the second reaction a pyridoxal phosphate dependent decarboxylase converts DOPA to dopamine. Tyrosinase Tyrosine


dihydroxy phenyl alanine (Dopa) O2 (1)

Dopamine. (2) Co2

3. Another vit. C dependent hydroxylation of dopamine by hydroxylase generates nor adrenaline. 4. A transmethylation reaction using SAM as methyl donor converts nor adrenaline to adrenaline Hydroxylase Dopamine


Nor adrenaline

Adrenaline +SAH.

Vit. c (3)

(4) SAM

Actions of catecholamines :The catecholamines act through two classes of receptors. They are α –adrenergic and ß –adrenergic receptors. These two classes consist of two sub classes. They are α1, α2 and ß1, ß2. Epinephrine can bind to both types of receptor but nor epinephrine binds to only α-receptors. 1. In liver and muscle catecholamines increases glycogenolysis. 2. They cause contraction of smooth muscle. 3. In liver they inhibit glycogenesis 4. By activating hormone sensitive lipase they promote lipolysis. 5. They cause myocardial contraction. 6. They increase gluconeogenesis in liver. 7. They raise blood pressure by acting on smooth muscle of blood vessels and heart.



Questions and Answers

6. Name thyroid Hormones. How they are formed? Mention their functions. A. Thyroid gland produces thyroxine (T4) and triiodotyronine (T3). They are amino acid tyrosine derivatives. Biosynthesis :Tyrosine serve as precursor of thyroid hormones. Reactions : 1. Iodination of tyrosine of thyroglobulin (TG) initiates thyroid hormone synthesis. Mono iodotyrosine (MIT) and diiodotyrosine (DIT) bound to TG are products. І Tyrosine –TG

Monoiodotyrosine (MIT) –TG+ Diiodotyrosine (DIT)-TG. (1)

2. In the second reaction MIT and DIT condense to form tri iodotyromine (T3)-TG and tetraiodo tyrosine (T4) –TG. Condensation MIT + DIT

Triiodotyronine (T3) – TG +Tetraiodotyronine (T4) –TG. (2)

3. Proteolysis generates T3 and T4 from thyroglobulin. Proteolysis T3 –TG



T4 – TG


T4 (Thyroxine) (3)



Functions : 1. Thyroid hormones are involved in maintenance of Basal Metabolic Rate (BMR) 2. They cause positive nitrogen balance by increasing protein synthesis. 3. They are required for over all development of humans. 4. They influences blood glucose through an unknown mechanism. 7. Name hormones of Gonads. Write their chemical nature, biosynthesis and actions. A. Gonads produce sex hormones. Ovaries produce estrogen and progesterone. Testes produce testosterone. Chemical nature:They are all steroids. They are synthesized from cholesterol. Biosynthesis of testosterone: Cholesterol is converted to testosterone. Cholesterol Andostenediol


Pregnenolone Testosterone.

Hydroxy pregnenolone

CHAPTER - 18 | Hormones

Actions of Testosterone 1. It is an anabolic steroid hormone. It regulates gene expression. 2. It is required for spermatogenesis and for development of secondary sex characteristics. 3. It is involved in sexual differentiation. 4. It determines male pattern behaviour by influencing brain regions sensitive to this hormone. Synthesis of progesterone and oestrogen :Cholesterol is converted to oestrogen via progesterone and testosterone. Cholesterol



Progesterone Testosterone

Hydroxy progesterone Estrogen.

Actions of oestrogen 1. It is an anabolic steroid hormone. It regulates gene expression and growth. 2. It is required for maturation of ovarian follicle and for development of tissues involved in reproduction. 3. It promotes mammary gland maturation. 4. It is required for menstrual cycle. Actions of Progesterone 1. It prepares uterine epithelium for implantation of fertilized ovum. 2. Progesterone decreases peripheral blood flow and reduces body temperature

8. Write about factors regulating hormone action. A. Hormone action is regulated by two ways a) Regulation of hormone action by releasing and trophic hormones Higher centres of brain regulate hormone action by release or secretion or inhibition of hormones by endocrine glands. Depending on needs of organism or individual a particular hormone is produced or inhibited and this signal is delivered to target gland through chemical substances known as releasing factors or release inhibiting factors and trophic hormones. Releasing factors or release inhibiting factors are produced by hypothalamus where as trophic hormones are produced by anterior pituitary gland Usually releasing factors or release inhibiting factors of hypothalamus reach anterior pituitary gland through direct circulatory connection where as trophic hormones of pituitary reaches target gland through blood circulation. The releasing or release inhibiting factors produced by hypothalamus are thyrotrophin releasing factor (TRF), growth hormone release inhibiting factor (GRF) or somatostatin, corticotrophin releasing factor (CRF) and gonadotrophin releasing factor (GRF).



Questions and Answers

When hypothalamus is activated by higher brain centres releasing or release inhibiting factors are produced. In response to hypothalamic signal through releasing or release inhibiting factor anterior pituitary either generates trophic hormone or stops its release. The trophic hormone if released acts on target endocrine glands to produce hormone. For example thyrotrophin releasing factor (TRF) when released by hypothalamus in response to higher centre stimulation acts on anterior pituitary to release thyrotropic or thyroid stimulating hormone (TSH). This in turn acts on thyroid gland to release thyroxine. Hypothalamus




Anterior Pituitary TSH

b) Regulation of hormone action by feed back inhibition This is another way of controlling hormone action. Usually hormone action is self limiting. It controls its own production. For example hormones of adrenal cortex inhibit release of CRF and ACTH Release of CRF

Release of ACTH Inhibition

Adrenal Cortex Inhibition Hormones

Other model questions are

9. Mechanism of steroid hormone action 10. Adrenal cortical hormones 11. G-Proteins 12. cAMP 13. Mechanism of insulin action. 14. Catacholamines 15. Glucagon. 16. Thyroid hormones 17. Insulin 18. Glucocorticoids 19. Regulation of hormone action


CHAPTER - 19 | Organ Function Tests



Organ Function Tests 1. Write about Kidney Function Tests (KFT) A. Since urine is product of kidney function, urine analysis is most common kidney function test. 1. Urine Volume : In kidney diseases urine volume decreases. Urine out put during day time and night time is not same. Usually more urine is passed in the day time and less in night time. Higher urine output in night time is suggestive of kidney dysfunction. 2. Urine specific gravity : If urine contains glucose or protein specific gravity increases. If water re absorption in renal tubules is defective due to absence of ADH specific gravity of urine decreases. 3. Urine proteins : Albumin is absent in normal persons urine. If glomerular permeability increases albumin appears in urine. It is indicative of kidney disease. Since kidney eliminates several substances from blood in kidney diseases levels of these substances is affected. 1. Blood urea, uric acid and creatinine : The levels of these substances increases in blood with progressing kidney disease. 2. Blood Phosphorus : Kidney is involved in phosphorus excretion. Phosphorus excretion by kidney decreases in renal disease. Hyper phosphatemia (more phosphorus in blood ) occurs in chronic nephritis which may ultimately lead to renal failure. 3. Serum protein electrophoresis : It is useful in diagnosis of nephrotic syndrome. In electro phoretogram albumin and gamma globulin bands size decreases and Îą- globulin band size increases. 4. Blood cholesterol : In nephrotic syndrome blood cholesterol level is more. 5. Serum Cystatin C : It is a Cysteine protease inhibitor. It is cleared by kidneys. It is elevated in kidney dysfunction. Apart from blood and urine analysis kidney function is assessed by specific tests. These tests may assess function of kidney as whole or its parts like glomerulus and renal tubules.



Questions and Answers

1. Concentration Test : It is test for renal tubular function. This test indicates concentrating ability of kidney. Specific gravity of urine is measured after administration of anti diuretic hormone (ADH). If specific gravity is normal then kidney function is normal. If specific gravity is decreased it is suggestive of impaired concentrating ability of kidney. 2. Dilution test : It is another test that assesses renal tubular function. It this test a fixed dose of water is given. Then urine sample is collected. Volume and specific gravity of urine sample are determined. If volume of urine is equal to volume of water given it indicates normal kidney function. In normals the specific gravity of urine collected is below normal. Clearance Tests Since glomerulus is major functional unit of kidney and involved in filtration clearance tests are used to assess glomerular filtration rate (GFR). The word clearance is defined as ml of plasma from which a substance is cleared (eliminated) by kidneys in a minute. The cleared substance is found in urine. Then clearance is calculated by estimating that substance in blood and urine. If kidney function is not normal clearance values decreases. Some common clearance tests are detailed below. Urea clearance test (UCT) Procedure : After a normal break fast about 200ml of water is given to patient under investigation. Immediately urine is collected and discarded. After an hour his urine and blood samples are collected. Urea level is estimated in the samples. Calculation : U V Urea clearance (ml/min) = –––––– P U = Urine urea concentration P = Blood urea concentration V = Urine out put per minute. Significance : Normal urea clearance value is 75ml/ min. Low clearance value suggest loss of kidney function that occurs due to diseases. Creatinine Clearance Test (CCT) Procedure : Initially the patient under investigation is given 500ml of water to hydrate his body completely. After one hour urine sample is collected and discarded. Again urine is collected after 4 hours and urine volume is noted. Blood sample is also collected. Then blood and urine samples creatinine is determined.


CHAPTER - 19 | Organ Function Tests

Calculation : UV Creatinine clearance ml/ min = ––––––– P U = Creatinine concentration in urine V = Urine volume per minute P = Creatinine concentration in blood. Significance : Creatinine clearance normal value is 90-110 ml/min/ 1. 73m2 body surface area. Low values are obtained when GFR is decreased due to diseases of kidney and pre renal diseases.

2. Describe Liver Function Tests (LFT) A. Liver is involved in most of the metabolisms of body. It has role in hemoglobin, protein, lipid, carbohydrates and xenobiotics metabolisms. So they are affected in liver diseases. By measuring various products of these metabolisms in blood liver function is assessed. Hemoglobin (Bile pigment ) Metabolism 1. Serum bilirubin : Secretory function of liver is assessed by measuring serum bilirubin. Van den Bergh Test : It is used to measure conjugated and unconjugated bilirubin level in blood. a. Conjugated bilirubin : It is more in obstructive jaundice. b. Un conjugated bilirubin : In pre hepatic jaundice it is more. c. Conjugated and unconjugated bilirubin : In hepatic jaundice both are elevated. 2. Urine bilirubin : In normal people urine bilirubin is absent. It is found in urine in post hepatic or obstructive type jaundice. Fouchet's test is used for urine bilirubin. 3. Urine urobilinogen : Urobilinogen in urine increases in pre hepatic jaundice. Decrease in urine urobilinogen occurs in post hepatic or obstructive jaundice. Ehrlich's test detects urobilinogen in urine. Protein Metabolism These functional tests mostly assesses synthetic function of liver because many proteins, urea and ammonia are handled by liver. 1. Serum albumin : Since liver synthesizes albumin low albumin level indicates liver disease like cirrhosis. 2. Serum globulins : Increased globulin level occurs in blood in liver disease like cirrhosis, auto immune hepatitis, biliary cirrhosis etc. Method : Biuret method is used to estimate proteins in serum.



Questions and Answers

3. Serum protein electrophoresis : Changes that takes place in various plasma proteins is assessed by electrophoresis. In cirrhosis size of albumin and gamma globulin bands decreases. Band sizes of other proteins remains same. 4. Prothrombin time (PT) : Prothrombin a blood clotting factor is synthesized by liver. Decreased prothrombin synthesis increases prothrombin time. Hence increased prothrombin time suggests liver dysfunction. In obstructive jaundice also prothrombin time increases. 5. Alanine transaminase (ALT) : It is an enzyme involved in amino acid break down in liver and other tissues. Since liver is rich in ALT this test is specific for liver dysfunction. ALT is more in hepatitis. In alcoholic cirrhosis also ALT is more. 6. Gamma glutamyl trans peptidase (GGT) : It is an enzyme involved in peptide metabolism. In all liver diseases the level of this enzyme is more in blood. Elevation is more marked in biliary tree obstruction. It is also more in alcoholic cirrhosis, fatty liver and infective hepatitis. 7. Blood Urea : Liver is the only organ involved in synthesis of urea. So low urea level in blood is suggestive of liver diseases. This test also assesses synthetic capacity of liver. 8. Blood Ammonia : Liver maintains normal ammonia level by converting it to urea. It is eliminated by kidney. Ammonia is toxic but urea is nontoxic. So liver detoxifies ammonia to urea. Hence high ammonia level in blood suggests impaired liver function. Lipid Metabolism 1. Serum bile acids : Liver converts cholesterol to bile acids and secretes into bile. So this test assesses secretory and excretory function of liver. Serum bile acid level increases in hepatitis and cirrhosis. In cholestasis or obstruction to bile flow or biliary tract bile acid level is elevated to a greater extent. 2. Blood Cholesterol : Increased level of cholesterol is suggestive of liver disease because liver catabolizes cholesterol to various products. In obstructive jaundice cholesterol level in blood is increased. 3. Urine bile salts : Liver produce bile acids from cholesterol and secretes into bile. In the bile, bile acids generates bile salts which are eliminated through bile. In normal people bile salts are absent in urine. Presence of bile salts is suggestive of obstructive jaundice. Hays test is used to detect bile salts in urine. Carbohydrate Metabolism Functional tests based on carbohydrate metabolism assesses synthetic function of liver. Liver is responsible for the conversion of galactose to glucose. So in liver disease this capacity of liver is disturbed.


CHAPTER - 19 | Organ Function Tests

Galactose Tolerance Test (GATT) : It measures rate of conversion of galactose to glucose by liver. Procedure : A dose of

galactose is injected into blood. Then blood samples are collected for

every ten minutes for about 30 minutes. Then blood samples are analyzed for galactose. Significance : Normally within 10 to 15 minutes galactose is cleared by liver. Decreased clearance indicates liver dysfunction due to diseases like cirrhosis or hepatitis. Xenobiotics Metabolism Many xenobiotics are cleared by liver. So extent of removal of given xenobiotic from blood is directly related to liver dys function. Some xenobiotics used to assess liver function are amino pyrine, indocyanin, bromosulfophthalein, rose Bengal and sodium benzoate. Retention of the administered xenobiotic in blood suggests liver dysfunction. Nucleotide Metabolism 51 – Nucleotidase : It is an enzyme of nucleotide breakdown. It is present in bile canaliculus and bound to membrane. It is increased in blood when there is obstruction to bile flow. Hence elevated level of this enzyme suggests cholestasis. Mineral Metabolism Alkaline phosphatase : It is an enzyme involved in hydrolysis of organic phosphates at alkaline pH. Portal vascular system and sinusoids are rich in this enzyme. However bile canaliculi contain less. In portal vascular endothelium it exist as membrane bound enzyme. It generally passes into bile. So in extra hepatic cholestasis it is elevated significantly and in intra hepatic cholestasis moderately. However in infective hepatitis it may be normal. In metastasis also it is elevated. Serum hepcidin and prohepcidin : Hepcidin and prohepcidin are proteins related to iron metabolism. In cirrohosis serum prohepcidin level is lowered. Serum hepcidin level indicates degree of liver dysfunction.

3. Write a note on Thyroid function test A. Thyroid function is assessed by determining thyroid hormones level in blood. 1. Thyroid hormone profile : It is most common thyroid function test in our country. Thyroid hormones measured in blood are thyroid stimulating hormone (TSH), thyroxine (T4 ) and tri iodotyronine (T3 ). Many methods are available to determine these hormones in blood. Enzyme linked immuno absorbent assay (ELISA ) and radio immuno assay (RIA) methods are currently in use. In this test directly blood is collected from subject under investigation and result are compared with hormonal status of normal person. In normal people T4 ranges from 4. 0 -6.



Questions and Answers

0 mg/ 100 ml. T3 level is100-250ng /100ml. Normal TSH ranges from100-300 m U/100 ml. Hyper thyroidism : T3, T4 level is more in hyper thyroidism. But TSH level decreases in hyper thyroidism. Hypothyroidism : T3, T4 levels diminishes in hypothyroidism. However TSH level is raised in this condition. All three hormone levels diminishes in hypothyroidism that occurs due to diseases of hypothalamus and pituitary gland. 2. Thyrotrophin releasing hormone (TRH) stimulation test : In this test TRH is administered intravenously in a fixed dose. Then blood is collected and TRH is measured. Normal levels of TRH indicates hyperthyroidism. Higher TRH level indicates hypothyroidism. 3. Blood cholesterol : Normal blood cholesterol level is 150-200mg%. Its level is more in hypothyroidism, In contrast level of cholesterol is less in hyper thyroidism. Other model questions are

4. Enumerate liver function tests and kidney function tests. Give detailed procedure f or any one. 5. Clearance tests 6. Liver function tests in jaundice 7. Creatinine clearance tests 8. Differntial diagnosis of jaundice 9. Enzyme tests in liver dysfunction 10. Urine urobilinogen in jaundice 11. Glomerular filtration tests 12. Urea clearance test 13. Urine protein 14. Bile salts in urine 15. Hemolytic jaundice 16. LFT 17. KFT 18. Van den Bergh test 19. PT 20. TRH Stimulation test


CHAPTER - 20 | Xenobiotics



Xenobiotics 1. What are xenobiotics? A. Many substances that are used in agriculture, food processing, drugs, environmental pollutants, toxins etc may enter into human body through food and air. They may be pesticides used in agriculture, food additives, food colors of food processing, drugs taken as part of treatment of illness, insecticides used to eliminate domestic pests, adulteration of food with cheaply available varieties and microbial contamination gives rise to microbial toxins. Generally all these are referred as xenobiotics

2. What is detoxification? Write its significance. A. Detoxification is word used for the chemical transformation of xenobiotics or toxins or poisons or drugs or chemicals to less toxic form. . Since they cause damage to body they are eliminated from body. The elimination involves chemical modifications which reduces toxicity and increases solubility of xenobiotics. Hence detoxification may be considered as protective mechanism. It is one way of protecting body from potential carcinogens

3. Explain biotransformation and lethal synthesis. A. Biotrans formation is another similar word for detoxification. Sometimes a less toxic substance may be converted to highly toxic substance by biotransformation. Then it is known as lethal synthesis

4. Write about different types of detoxification reactions with examples. A. Liver is the major organ involved in detoxification. Due to detoxification of drugs and their elimination from blood to maintain a level of drug in the body the drug must be taken in fixed timings and in fixed dose. Other wise it may not be effective in controlling diseases. Liver detoxifies xenobiotics by four types of reactions. They are 1. Hydrolysis, 2. Oxidation, 3. Hydroxylation, 4. Conjugation. A xenobiotic is detoxified by any one of these reaction types or combination. Generally detoxification of highly hydrophobic substances, potentially carcinogenic substances and formation of procarcinogens or carcinogens involves more than one type of these reactions.



Questions and Answers

1. Hydrolysis : a. Drug like aspirin which is used as pain killer or analgesic is detoxified by hydrolysis. An esterase hydrolyses aspirin. Esterase Aspirin

Acetic acid + salicylic acid H2 O

b. Drugs that work as sedatives or anesthetic like atropine is hydrolyzed. Atropine +H2 o

Tropine +tropicacid.

c. Digitalis a cardiac glycoside is hydrolyzed. Digitals

Aglycone + carbohydrate

d. Pethidine is hydrolyzed to ethanol and meperidinic acid. e. Phenacetin a pain killer and anti inflammatory drug is hydrolyzed to acetic acid and phenetidine. f. Procaine an anesthetic is hydrolyzed to dimethyl amino ethanol and para amino benzoic acid. g. Acetanilide is hydrolyzed to acetic acid and aniline. 2. Oxidation : a. Ethyl alcohol an essential part of beverages or hot drinks is detoxified by oxidation. An alcohol dehydrogenase converts it to an aldehyde which inturn oxidized to acetic acid by aldehyde oxidase. Ethyl alcohol


Acetic acid.

b. Methanol an adulterant of ethyl alcoholic drinks is also detoxified by oxidation, initially it is converted to aldehyde which is oxidized. Methanol


Formic acid.

c. In the intestine tryptophan is converted to skatole and indole by enzymes of intestinal flora. They are responsible for characteristic odor or smell of feces. They are detoxified by oxidation. Skatole is oxidized to skatoxyl and indole is oxidized to indoxyl. d. Benzaldehyde and toluene are oxidized to benzoic acid. e. Codeine a common ingradiant of cough syrups is detoxified by oxidation to morphine and formaldehyde. 3. Hydroxylation :Detoxification by hydroxylation is brought about by microsomal cyt P450 hydroxylase system. Pentobarbitol an anticonvulsant is hydroxylated to hydroxy pentobarbital.


CHAPTER - 20 | Xenobiotics

Cy t P450 Pentobarbitol

Hydroxy pentobarbital.


H2 O

Butazone is detoxified by hydroxylation to hydroxybutazone. Steroid hormone containing drugs are eliminated by hydroxylation. Many anti inflammatory drugs contain steroid hormones. Steroids

Hydroxy steroids.

Quinones are detoxified by hydroxylation Quinone


Reduction : Rarely toxins are eliminated by reduction. a. Desulfiram an inhibitor of aldehyde dehydrogenase is detoxified to diethyl dithio carbomic acid. b. Chloroamphenicol or chloromycetin an antibiotic is detoxified by reduction. c. Picric acid is reduced to picramic acid. 4. Conjugation :Many drugs are detoxified by conjugation. Variety of compounds are used to combine with drug. Some of them are glucuronic acid, sulfate, acetyl-CoA, methyl group, glutathione, aminoacids like glycine, glutamine etc. a. An antibiotic chloroamphenicol is conjugated with glucuronic acid. b. Morphine a sedative or pain killer is detoxified with glucuronic acid. Morphine + UDP – Glucuronic acid

Conjugated product +UDP.

c. Chloral hydrate a sedative is detoxified by conjugation with glucuronic acid. UDP- Glucuronic acid+ chloral hydrate

UDP + Conjugated product.

d. Phenol is detoxified by conjugation with sulphate Phenol +PAPS

Phenyl sulphate +PAP.

e. Skatoxyl and indoxyl are detoxied by conjugation with sulphate. f. Antibiotic sulfanilamide is detoxified by conjugation with acetate. Sulfanilamide + Acetyl –CoA

Acetyl sulfanilamide+ CoA.

g. Glutathione is used to conjugate several hydrocarbons with substituted halogens. Difluoro benzene is conjugated with glutathione. Diflorobenzene + glutathione

Mercapturic acid.

h. Salicylic acid is conjugated with glycine. Salicylic acid +glycine

Salicyluric acid.



Questions and Answers

I. Indole acetic acid is conjugated with glutamine. Indole acetic acid +glutamine

indoleacetyl glutamine.

j. Nicotinic acid is detoxified by methylation. Nicotinic acid + SAM

Methyl nicotinic acid +SAH.

k. Arsenic is detoxified by methylation. Arsenic +SAM

Methyl arsenic acid.

5. Name phase I and phaseII detoxification reactions. A. The three types of detoxification reactions hydrolysis, oxidation and hydroxylation are also known as phase I detoxification reactions. Conjugation detoxification reactions are known as phase II detoxification reactions. Other model questions are

6. Detoxification 7. Xenobiotics 8. Detoxification by conjugation 9. How aspirin and phenol are detoxified? 10. How skatole and indole are detoxified?


CHAPTER - 21 | Cancer


21 Cancer

1. Define cancer, metastasis and invasion. A. Cancer is malignant growth or uncontrolled growth of cells. Malignant growth of cell is also called as tumor. Cancer of a particular organ or tissue develops when the cells of that organ have lost growth control. Metastasis and invasion is spreading of cancer from organ of its origin to other organs. Cancer cells of an organ enters blood stream then enters other organs and turns other normal organs into malignant tumors.

2. Write about cancer genes A. Cancer genes are related to development of cancer as well as prevention of cancer. They are oncogenes, proto oncogenes and tumour suppressor genes. Oncogenes : Are genes responsible for development of cancer. Proto oncogenes : They are precursors of oncogenes. They are converted to oncogenes by activation. Both cellular and viral oncogenes are found. Examples for oncogenes and proto oncogenes are given below. 1. Cellular oncogene that causes rat sarcoma is designated as c-ras oncogene. Like wise cras proto oncogene. 2. Viral oncogene that causes rat sarcoma is designated as v-ras oncogene. Like wise vras proto oncogene. Tumour suppressor genes (TSG)

: They are present in normal healthy people.

Products of them prevent cancer development. They encode proteins involved in several cellular processes. They are 1. Enzymes that participate in DNA repair 2. Proteins that promote apoptosis 3. Receptors or signal transducers for hormones or developmental signal that inhibit cell proliferation



Questions and Answers

4. Intra cellular proteins involved in cell cycle progression. 5. Check point control proteins that arrest cell cycle if DNA is damaged. Mutations in these proteins leads to loss of function. In many cancers tumor suppressor genes have deletions or point mutations that prevent protein synthesis or lead to synthesis of non functional protein. Examples are ATM, ATR, Ch K 1, Ch K2, Rb, P16 and P53

3. What is carcinogenesis? Write about different types of Carcinogenesis A. Cancer forming process is known as carcinogenesis or tumorigenesis. Cancer is primarily due to DNA damage or damage of genes. DNA damage may result from the action of biological, chemical, physical and environmental agents on DNA. Incidence of cancer also depends on the genetic make up of an individual. By several ways carcinogenesis occurs in humans and other animals. Usually they are named according causative agent or factor. Different types of carcinogenesis are given below 1. Biological agents that cause cancer or biological (viral) carcinogenesis : Some DNA and RNA viruses are carcinogenic and hence they are called as oncogenic viruses. When normal cells are cultured with oncogenic viruses, the normal cells are transformed into cancer (tumour) cells. Oncogenes of the viruses are responsible for the development of cancer. Examples:

1. Hepatitis B virus cause liver cancer in humans. 2. Retro viruses also cause cancer in humans.

2. Chemical carcinogens or mutagens or chemical carcinogenesis: Many chemical substances cause mutations in DNA. They are called mutagens. Some times this mutation in DNA may convert normal cell to cancer cell. Then they are called as carcinogens. Examples: 1. Cigarette smoke causes lung cancer in humans. 2. Aflatoxins are carcinogens. 3. Nitrosamine, Benzapyrins and asbestos also cause cancer. 3. Physical agents that cause cancer or physical carcinogenesis: Exposure to radiation may damage DNA. UV light exposure causes mutation in DNA of skin cells. Mutant DNA mediates carcinogenesis by activation of oncogenes which leads to development of cancer of skin or multiple tumours of skin.

4. Write a note on tumour markers. A. Cancer ells produce abnormal substances. Usually these substances are not produced by normal cells. The abnormal substances produced by the cancer cells are enzymes, hormones and proteins. These substances are released into blood by cancer cells. As a result their level in blood rises. Measurement of these substances in blood or serum provides useful information about cancer. Hence, they are called as tumour markers. 230

CHAPTER - 21 | Cancer

Some important tumour markers are 1. Feto protein (AFP): It is a plasma protein and usually absent in normal people plasma. It is tumour marker for liver cancer and germ cell cancer. 2. Calcitonin: It is a hormone. It is tumour marker for thyroid cancer. 3. Carcino embryonic antigen (CEA): It is a protein and it is tumour marker for lung cancer, breast cancer, colon cancer and pancreas cancer. 4. Human chorionic gonodotropin (HCG): It is tropic hormone. It is tumour marker for germ cell cancer and trophoblast cancer. 5. Acid phosphatase: It is tumour marker for prostate cancer. 6. High mobility group chromosomal proteins (HMGCP): They are family of nonhistone chromosomal proteins that serve as architectural elements in chromatin. In normal tissues these proteins are expressed at very low levels. Their level is elevated in many human cancers. This small molecular weight protein's expression is increased in neoplastic transformation of cells and metastatic of tumour progression. They can serve as novel diagnostic tumour markers. 7. Prostate specific antigen(PSA): It is tumour marker for prostate cancer 8. CA125: It is cancer marker.

5. Write a note on anti cancer agents. A. Several compounds are able to prevent growth of cancer cells by blocking nucleotide or nucleic acid formation which are necessary for cell multiplication. They are used as anticancer agents. Mercaptopurine, fluorouracil, methotrexate, acivicin, azaserine etc. are examples of anticancer agents. Other model questions are

6. Mutagens 7. Viral carcinogenesis 8. Alfa feto protein 9. Tumour suppressor gene 10. PSA




Questions and Answers

Biochemistry Questions and Answers Ø This book contains questions, answers and model questions.

Ø The book is written in such way that learning of question and answers given in each chapter makes student to a enquire concept of that topic simultaneously.

Ø Answers are given in simple language with illustrations.

Ø Complex path ways are presented in a easy to remember way.

Ø This book helps students in their preparation for examination.

Ø The book contains 495 questions and answers to 249 questions. Answers to essay questions, short and very smart questions are given in this book.

Dr. N. Mallikarjuna Rao

ISBN 978-81-924169-3-9

# 225/B, 9th ‘A’ Main, Vijayanagar, Bengaluru - 560 040. Ph.: +91 80 2340 7999 Fax : +91 80 2330 2032

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