Biochemistry

Marrow Edition 8 - MBBS First Year

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Instructions

• Notes are to be used in conjunction with Marrow videos.

Please note:
• The information in this book has been printed based on the transcript of the Marrow videos. This
book has to be used in conjunction with the Marrow videos and not as a standalone material.

• The information contained in this book is for educational purposes only. The content provided is

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not intended to substitute for professional medical advice, diagnosis or treatment.

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• This book cannot be sold separately. It has been made available to only select eligible users who

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have an active subscription to Marrow videos.

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• The text, images, slides, and other materials used in this book have been contributed by the
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faculty, who are subject matter experts. We have merely reproduced them as video transcripts in
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this book.
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• The notes have been consciously designed in a way that is concise and revisable. To ensure this,
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we have intentionally added only the most relevant modules and images that are needed for you.
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• The notes contain blank spaces primarily for labelling diagrams, completing cycles and more to
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promote active engagement and reinforce learning.

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• Reasonable care has been taken to ensure the accuracy of the information provided in this book.
Neither the faculty nor Marrow takes any responsibility for any liability or damages resulting from
applying the information provided in this book.

All Rights Reserved

No part of this publication shall be reproduced, copied, transmitted, adapted, modified or stored in any form or
by any means, electronic, photocopying, recording or otherwise.

©Marrow
Contents

Basic Biochemistry
Cell and Subcellular Organelles 1

Enzymes
Introduction to Enzymes 8

Classification of Enzymes 12

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Mechanisms of Action of Enzymes  19

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Enzyme Kinetics 22

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Enzyme Inhibition 26
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Concept of Enzyme Regulation 29
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Enzyme Regulation 31
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Clinical Enzymology 36
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Chemistry and Metabolism of Carbohydrates

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Chemistry of Carbohydrates 42
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Glycosaminoglycans 50
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Digestion of Carbohydrates 56
Glucose Transporters 58

Glycolysis : Part 1 61

Glycolysis : Part 2 66

Pyruvate Dehydrogenase 69

Glycogen Metabolism 72

Glycogen Storage Disorders 77

Gluconeogenesis 82

Minor Metabolic Pathways 87

Regulation of Blood Glucose 94

Diabetes Mellitus : Part 1 99

Diabetes Mellitus : Part 2 104

Diabetes Mellitus : Part 3 108

Chemistry and Metabolism of Lipids

Chemistry of Lipids 112

Phospholipids 117

Digestion and Absorption of Lipids 123

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Lipid Metabolism in Fasting State 126

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Ketone Body Synthesis 135

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Lipid Metabolism in Fed State 139
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Lipoproteins and its Metabolism 147
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Dyslipidemia 155
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Chemistry and Metabolism of Proteins

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Chemistry of Amino Acids 162

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Digestion and Absorption of Proteins 169

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Plasma Proteins and Immunoglobulins 172

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Formation, Transport and Detoxification of Ammonia  181

Urea Cycle and its Disorders 185

Aromatic Amino Acids 189

Sulphur Containing Amino Acids 196

Tryptophan 201

Miscellaneous Amino Acids 204

Heme Metabolism 214

Hemoglobin Part : 1 221

Hemoglobin : Part 2 227

Extracellular Matrix
Fibrous Proteins 232

Integration of Metabolism and Biological Oxidation

Integration of Metabolism 235

Krebs Cycle 243

Electron Transport Chain 248

Vitamins and Nutrition

Fat Soluble Vitamins 252

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Hematopoietic Vitamins 267

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Energy Releasing Vitamins 272

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Vitamin B6 and C 19 277
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Protein-Energy Malnutrition and Obesity 281

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Minerals and Acid Base Balance

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Minerals : Part 1 285

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Minerals : Part 2 294

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Minerals : Part 3 302

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Acid Base Balance 308

Acid Base Disorders 316

Molecular Biology
Chemistry of Nucleotides 326

Metabolism of Nucleotides 329

Structure and Organisation of DNA 337

DNA Replication and Repair 341

Transcription 349

Translation 355
Regulation of Gene Expression 360

Hybridisation Techniques 367

Recombinant DNA Technology 375

Amplification and Sequencing Techniques 384

Mutation 395

Organ Function Tests

Liver Function Tests 400

Renal Function Tests 407

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Thyroid Function Tests 418

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Xenobiotics and Miscellaneous

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Xenobiotics 19 426
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Oncogenesis, Alcohol and Free Radicals 429

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 Cell And Subcellular Organelles 1

CELL AND SUBCELLULAR ORGANELLES ----- Active space -----

Properties & Types of Cells  00:00:20

Properties :
• Basic unit of any organism (Unicellular or multicellular).
• Dynamic in nature : Grow, divide, specialize to other forms.

Prokaryote vs. Eukaryote :

Prokaryote Eukaryote

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Size Small Large

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Cell membrane Rigid cell wall Fluid plasma membrane

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Sub cellular organelles - Distinct organelles +

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Nucleus 19
Ill-defined nucleoid Well-defined nucleus
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Mitochondrial energy metabolism - +

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Cell division Fission (M/c) Mitosis

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Cytoplasmic organelles/cytoskeleton - +
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Subcellular Organelles 00:02:20

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NUCLEUS
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Structure :
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Outer : Continuous with endoplasmic reticulum

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2 membranes
Inner : Numerous pores +

DNA coiled with chromatin

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 2 Basic Biochemistry

----- Active space ----- Function : Note :

• Site of DNA & RNA synthesis. • Absent nucleus : RBC
• Nucleolus : rRNA processing. • Prominent nucleus : Lymphocyte & sperms
ENDOPLASMIC RETICULUM (ER)
Identified by George Palade.

Structure :
Cisternae : Flattened
interconnecting
channels extending
from outer nuclear
membrane to
plasma membrane

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Types & Functions :

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Rough ER : Studded with ribosomes Smooth ER : AKA microsomes

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• Protein synthesis & sorting • Steroid hormone synthesis

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• Drug metabolism
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Marker enzyme : Glucose-6-phosphatase.

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GOLGI APPARATUS
Structure : 1
Network of flattened smooth
2 membranes & vesicles

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Medial cisterns

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Route of proteins from ER to target site :
Cis cisterns Medial cisterns Trans cisterns Secretory vesicles.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Cell And Subcellular Organelles 3

Functions : ----- Active space -----

• N & O Glycosylation of proteins. 
(Enzymatic addition of carbohydrate residues to proteins).
• Protein sorting.

Marker enzyme : Galactosyl transferase.

LYSOSOMES
Structure :
• Contains hydrolytic enzymes : Digest proteins, nucleic acids, polysaccharides, lipids.
• Acidic pH.

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Function :
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Lysosome + phagosome Phagolysosome : H ydrolyze/digest waste products & foreign particles.

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Marker enzyme : Cathepsin & acid phosphatase.

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Clinical significance :
1. Gout : Accumulation of monosodium urate crystals Damage the lysosome
Released enzymes
initiate inflammation
& pain.
2. Cathepsins : Lysosomal proteases that enhance tumor metastases.
3. Silicosis : Rupture of lysosomes Release of hydrolytic enzymes
Fibroblast activation.
4. Inclusion cell (I-cell) disease : Protein targeting defect in which enzymes
lack mannose-6-phosphate.
5. Lysosomal storage disorder : Sphingolipidosis, mucopolysaccharidosis, etc.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 4 Basic Biochemistry

----- Active space ----- MITOCHONDRIA

Structure :
• Spherical/oval/rod-like structure.
• 2 membranes Outer.
Inner : Forms folds/Cisternae.

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Functions :
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1. IMM (Inner Mitochondrial Membrane) contains ETC (Electron Transport Chain)

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Generate ATP.

2. Matrix contains enzymes for :

• TCA cycle.
• Heme synthesis.
• Urea cycle.

Marker enzyme : ATP synthase.

Clinical Significance :
Oxphos : Defect in mitochondrial oxidative phosphorylation
1. Leigh syndrome.
2. MERRF : Myoclonic Epilepsy & Ragged Red Fibers.
3. MELAS : Mitochondrial Encephalopathy Lactic Acidosis Stroke-like syndrome.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Cell And Subcellular Organelles 5

PEROXISOME ----- Active space -----

Structure : Granular matrix lined by single membrane.
Function :
1. Free radical scavenging : Mediated by
peroxidase & catalase.
2. Oxidation of VLCFA (Very Long Chain Fatty Acids).

Marker enzyme : Catalase. Structure of Peroxisome

Clinical Significance :
Peroxisomal targeting & biogenetic disorders :
1. Zellweger syndrome.
2. Adrenoleukodystrophy. Defect in VLCFA oxidation

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3. Primary hyperoxaluria.
Accumulation of VLCFA

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4. Refsum’s disease : Defect in a-oxidation.

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MARKER ENZYMES
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Organelle Marker Enzyme
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Mitochondria (In IMM)

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Lysosomes
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Golgi apparatus
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Microsomes
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Cytoplasm
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Plasma Cell Membrane 00:20:15

Fluid Mosaic Model :

• Proposed by Singer & Nicolson (1972).
• Bilayered arrangement of phospholipids.
• Amphipathic phospholipids Polar head : Towards the extracellular & cytoplasmic side.
Hydrophobic tail : Forms the core.
• Diameter :
Head : 10 Å
Each leaflet : ~25 Å
Tail : 15 Å
Total thickness : ~50-80 Å

Arrangement of Phospholipids

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 6 Basic Biochemistry

----- Active space ----- Fluid nature :

• Permits : • Restricts: Flip flop movement
- Free lateral movement. (E.g : Apoptosis)
- Endocytosis & exocytosis.

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Fluid & mosaic model

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Mosaic pattern : Formed by

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• Phospholipids & sphingolipids. • Carbohydrates.
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• Proteins. • Cholesterol.
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Membrane Phospholipids :
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Partial lipid asymmetry

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Outer monolayer Inner monolayer :

• Phosphatidylcholine (Glycerophospholipid) • Phosphatidylethanolamine
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• Sphingomyelin (Sphingophospholipid) • Phosphatidylserine

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• Phosphatidylinositol
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Membrane Cholesterol :
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• Amphipathic in nature <Tm (Melting temperature) :↑Fluidity

• Alters fluidity of the membrane & permeability.
>Tm : ↓Fluidity.

Membrane Proteins :

Peripheral proteins Integral proteins

Integral proteins :
• Deeply embedded in bilayer.
• Bound by hydrophobic interactions/
van der Waals forces.
• Transmembrane proteins :
Span the whole bilayer.
• Eg : Glucose transporter,
GPCR, Insulin receptor. Integral proteins
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Cell And Subcellular Organelles 7

Peripheral Proteins : ----- Active space -----

• Attached to the membrane surface via electrostatic bonds to lipids/integral
proteins.
• Bound weakly.
• Released from the membrane by ionic solvent.
• Present on extracellular or cytosolic side.
• Eg : Spectrin, Ankyrin, Band 4.1 of RBC membrane.

Melting Temperature :
• Temperature at which membrane shifts from a solid gel to fluid state.
• Determined by :
1. Length of aliphatic hydrocarbon chain
2. Degree of unsaturation : Directly proportional to fluidity.

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3. Cis/trans forms of unsaturated fatty acids : Trans forms a/w ↓fluidity.

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Functions of Plasma Membrane :

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1. Protects the cell from the external environment. 19
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2. Anchors the cytoskeleton to provide shape to the cell.
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3. Selective permeability to substances.

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4. Maintains cell potential.

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5. Cell recognition : Mediated via certain markers.

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6. Tight junctions : Binds cells together.

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7. Signal transmission via receptors.

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Marker Enzyme :
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1. Na+-K+ ATPase.
2. 5’ Nucleotidase.
3. Adenylyl cyclase.

Clinical Significance :
• Insulin resistance : Occurs d/t impaired ligand-receptor interaction.
• Excess cholesterol.
• Trans fatty acid. ↓Membrane fluidity Impairs ligand-receptor interaction.
• ↑Saturated fatty acids.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 8 Enzymes

----- Active space ----- INTRODUCTION TO ENZYMES

Enzymes
Specialised proteins that can act as biological catalysts.

Exception :
Ribozyme : RNA acts as enzymes.
Ribozymes Function
Ribosome
• 28S rRNA Peptide bond synthesis.

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• Peptidyl transferase

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Sn RNA

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Splicing of exons : post-transcriptional

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Group II introns modification of mRNA.

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Ribonuclease P 19
Post-transcriptional modifications of tRNA.
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Enzymes, Co-enzymes and Co-factors

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Types of enzymes :
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Simple Complex
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Only protein component Protein component + Non-protein component

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Apoenzyme Apoenzyme Co-enzyme Co-factor

Properties of enzymes :
Enzymes are proteins.
• Nitrogen : 16% by weight.
• Heat labile.
• Precipitated by protein precipitating agents.

Co-enzyme :
• Second substrate or co-substrate.
• Mostly B-complex vitamins.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Introduction to Enzymes 9

Properties of co-enzyme : ----- Active space -----

• Heat stable.
• Low molecular weight organic molecule.

Examples :
Active form Reactions involved.
• Oxidative decarboxylation.
Thiamine (B1) Thiamine pyrophosphate (TPP)
• Transketolase.
• Oxidative decarboxylation.
• Redox reaction :
Flavine adenine dinucleotide (FAD)
Riboflavin (B2) - Complex I of electron
FMN
transport chain (ETC).

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- Predominantly FMN.

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• Oxidative decarboxylation :

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Nicotinamide adenine dinucleotide
Predominantly NAD+
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Niacin (B3) (NAD+), Nicotinamide adenine
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dinucleotide phosphate (NADP+)
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(dehydrogenase)
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Pantothenic acid (B5) Co-enzyme A Transfer of acyl group

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• Transamination.
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Pyridoxine (B6) Pyridoxal phosphate (PLP)

• Trans-sulfuration.
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Folic acid (B9) Tetrahydrofolate (THFA) One carbon transfer.

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Methyl B12 Homocysteine methyl transferase

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cobalamin (B12)
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Adenosyl B12 Methyl-malonyl CoA mutase

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Lipomide
Lipoate - Oxidised form oxidative decarboxylation
- Reduced form
Ascorbic acid (C) Ascorbate Hydroxylation reaction

Co-factor :
• Inorganic molecules.
• Predominantly minerals.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 10 Enzymes

----- Active space ----- Types

Metalloenzyme : Metal activated enzyme :

• Metal & apoenzyme tightly integrated. • Metal not tightly integrated with enzyme.
• Eg : • Presence of metal is required for enzyme action.
- Cu in tyrosinase. -Eg: Ca2+ required for action of lipase.
- Zn2+ in Carbonic anhydrase.
Carboxy peptidase.
Prosthetic group :
Co-enzyme/ Metalloenzyme (Co-factor) tightly integrated to enzyme.

Holoenzyme  00:17:29

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• Completed form of complex enzyme.

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• Apoenzyme + co-enzyme/ co-factor.

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Examples : 19
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Metals Enzyme Function

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Carbonic anhydrase. Transport of CO2.

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Carboxypeptidase A & B . Digestion of proteins.

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Alcohol dehydrogenase. Retinol Retinal (Vision).

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Removal of phosphate in alkaline

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Alkaline phosphatase.
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medium .
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Zinc
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ALA dehydratase. Synthesis of heme.

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Adenosine deaminase. Purine catabolism.

Free radical scavenging : Anti-
Cytosolic superoxide dismutase (SOD).
oxidant.
Lactate dehydrogenase. Anaerobic glycolysis.
• Kinase.
• Phosphatase.
Magnesium Transfer of phosphate.
• Mutase.
• Enolase.
Heme iron : Complex III & IV of ETC
-
(present in cytochrome).
Iron
• Nitric oxide synthase. • Synthesis of nitric oxide.
• Peroxidase, catalase. • Free radical scavengers.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Introduction to Enzymes 11

----- Active space -----

Metals Enzyme Function

Tryptophan dioxygenase -
Iron Non-heme iron : Complex I & II of ETC
-
(found as Fe-S complex)
• Kinase, phosphatase
• Arginase
Manganese -
• Ribonucleotide reductase
• Mitochondrial SOD
Purine catabolism :
• End product : Uric acid

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Molybdenum Xanthine oxidase
• Deficiency of molybdenum:

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Hypouricemia.

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• Pyruvate kinase
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Potassium 19 -
• Na+-K+ ATPase
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Melanin synthesis :
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Tyrosinase • Deficiency of copper :

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Hypopigmentation.
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Copper Complex IV ETC Energy productions

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Collagen synthesis :
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Lysyl oxidase • Deficiency of copper: Bleeding

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manifestations.
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Nickel Urease Not seen in humans

• Lecithinase
Calcium -
• Lipase
Glutathione peroxidase Free radical scavenger (Anti-oxidant)
Thioredoxin reductase -
Selenium
Deiodinase Thyroid hormone synthesis
Selenoprotein P -

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 12 Enzymes

----- Active space ----- CLASSIFICATION OF ENZYMES

OVERVIEW

Trivial name of enzyme :

• Named after reaction mechanism (M/c) /substrate.
• Can be common for 2 enzymes.

IUBMB Classification of Enzyme :

(International Union of Biochemistry and Molecular Biology)

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Enzyme commission/class/code number : 4 digits.

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Eg : 1 1 1 1 (Alcohol dehydrogenase).

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1 1 1 19 1
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1st digit : 2nd digit : 3rd digit : 4th digit :

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Class Subclass Subsubclass Unique number for every enzyme

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Classes of enzymes :
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7 Classes mnemonic : Operation Theatre Has Low Intensity LighT

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• Oxidoreductase. • Isomerase.
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• Transferase. • Ligase.
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• Hydrolase. • Translocase (added on August 2018).

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• Lyase.

Class I: Oxidoreductases  00:08:07

Enzyme that catalyze oxidative reduction reactions.

SUBCLASS 1 : DEHYDROGENASES (DH)

• Catalyze transfer of hydrogen elements (H+, H-, H2) & electrons to an acceptor
in a coupled oxidation-reduction reaction.

Acceptors (Co-enzymes) :

Flavoproteins
• FAD FADH2
• Catalyzing enzymes

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Classification of Enzymes 13

- Acyl CoA DH (in β-oxidation). ----- Active space -----

- Succinate DH (in Tricarboxylic acid cycle).
- Glycerol-3-P DH (Mitochondrial).

Nicotinamide

NADP+ NADPH NAD+ NADH

Catalyzing Enzymes Catalyzing enzymes

• enzymes in HMP shunt • Most other enzymes.
pathway
- Glucose-6-phosphate DH.
- 6-Phosphogluconate DH.

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- Cytoplasmic Isocitrate DH.

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- Malic enzyme.

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SUBCLASS - 2 : OXIDASES
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Substrate Product + H2O / H2O2
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(catalyze H2 transfer)
(H2 acceptor is O2)
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Eg :
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• Cytochrome C oxidase • Mono amino oxidase.

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- Complex IV of electron • L-Amino acid oxidase. Produce

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transport chain. • Xanthine oxidase.

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H2O2
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- O2 gets reduced to H2O.

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SUBCLASS 3 : OXYGENASE
Add O2 directly to the substrate.

Types :
Monooxygenase/ mixed function oxidase Dioxygenase
Features • Enzymes adding 1 atom of O2 • Enzymes adding both atoms of O2
• Most are hydroxylases • Homogentisate dioxygenase
• Eg : • Tryptophan dioxygenase
- Phenylalanine hydroxylase aromatic
Examples - Tyrosine hydroxylase amino acid
- Tryptophan hydroxylase hydroxylases
- 7α hydroxylase
- Cytochromes

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 14 Enzymes

----- Active space ----- SUBCLASS 4 : HYDROPEROXIDASES

Types :
Peroxidase Catalase
Substrate H2O2 > organic peroxide
Eg : Glutathione (GSH) H2O2
2H2O2
H2O2 GSH (reduced)
Catalase
Glutathione peroxidase
Electron H2O GSSG (oxidised) 2H2O
acceptor +
O2

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• Ascorbate

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• Quinones

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• Cytochrome C

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Class II : Transferases and Class III : Hydrolases

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CLASS II : TRANSFERASES
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Transfer of functional groups (except hydrogen) to acceptors.

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Examples
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• Transaminase
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• Transketolase
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(enzymes with trans prefix)

kinases Phosphorylases
Catalyse transfer of PO43- Glycogen phophorylase
Glycogen Glycogen
from ATP (organic molecule) (n) Glu-1 PO43- (n-1)
Pi
Glucokinase/ (inorganic
Hexokinase PO43-)
a) Glucose/Galactose Galactose/Glucose
6-phosphate n=number of glucose molecules
ATP ADP
Phosphofructokinase
b) Fructose 6-phosphate (PFK)
Fructose 1,6
ATP ADP bisphosphate

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Classification of Enzymes 15

CLASS III : HYDROLASES ----- Active space -----

Catalyze hydrolytic cleavage of covalent bonds (C-C, C-N, C-O)
Eg :
1) Digestive enzymes

Biomolecule Covalent bond Digestive enzymes

Carbohydrates Glycosidic linkage Amylase, maltase, lactase,
sucrase
Protein Peptide bond • Protease : Trypsin,
chymotrypsin, elastase,
pepsin
• Peptidase

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Nucleic acid 3’-5’ Phosphodiester bond Exonuclease

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Lipids Ester bond Lipase, esterase

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2) Phosphatase 3) Arginase (in urea cycle)

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G-6 Pase 19
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G 6 PO4 Glucose
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H20 Pi
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F-1,6-B Pase
F-1,6 BF Fructose 6P
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H20 Pi
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Class IV Lyases
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Examples :
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1) Cleave covalent bond without adding water

Eg : Aldolase

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 16 Enzymes

----- Active space ----- 2) Form double bonds by atom elimination,+

Eg : Enolase

3) Add groups across double bonds/form single bond

Eg : Fumarase

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4) All enzymes with lyase suffixes

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Eg : HMG CoA lyase, argininosuccinate lyase

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5) Decarboxylases :
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Type of decarboxylation
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Simple decarboxylation Oxidative Decarboxylation

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Class: Oxidoreductases
Class: Lyases
Enzyme Subclass : Dehydrogenases
Subclass : decarboxylase
• All are multienzyme complexes
• Histidine PLP Histamine • Pyruvate (3C) Acetyl CoA (2C)
CO2 NAD+ CO2 NADH

• Tryptophan PLP Tryptamine

• α ketoglutarate (5C) Succinyl CoA (4C)
Examples • Tyrosine PLP Tyramine CO
NAD+ NADH 2
• Glutamate PLP GABA
CO2
• Dihydroxy phenylalanine
• Branched chain ketoacid (nC) Corresponding
(DOPA) PLP Dopamine Acyl CoA ((n-1)C)
NAD NADH
+
Epinephrine + Norepinephrine (n=number of Carbon atoms)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Classification of Enzymes 17

----- Active space -----

Simple decarboxylation Oxidative Decarboxylation

• 5 coenzymes
- Thiamine pyrophosphate (TPP)
- Coenzyme A
Coenzyme Vitamin B6 (PLP) - Lipoate/lipomide
- FAD
- NAD+

Pyridoxine (B6) dependent

seizures in neonates
Pathogenesis : B6 deficiency

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aspect Lack of

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neurotransmitters

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Class V: Isomerases, Class VI: Ligases, Class VII: Translocases

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CLASS V : ISOMERASES
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• Catalyze structural or geometric isomer formation.

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Subclasses
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Subclass Examples
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Phosphohexose isomerase
• G 6 PO4 (6C) F6PO4 (6C)
1) Isomerases
• Dihydroxyacetone P04 (3C) Phosphotriose isomerase Glyceraldehyde 3 P04 (3C)

2) Mutase
• G 6 PO4Phosphoglucomutase G 1 PO4
Intra molecular transfer of
functional groups. • 3PG
Phosphoglycerate mutase
2PG (phosphoglycerate)
3) Racemase • D alanine L-alanine
Create D and L isomers • D glucose L-glucose
from each other.
Note : Kinase: Transfers PO43- from 1 substrate to another.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 18 Enzymes

----- Active space -----

CLASS VI : LIGASES
Coupling of 2 molecules with ATP hydrolysis.

Subclass 1
Biotin dependent carboxylase

• Pyruvate (3C) Pyruvate carboxylase Oxaloacetate (4C)

ATP ADP
• ATP hydrolysis.
• Biotin (B7) dependent.
• Acetyl CoA (2C) Acetyl CoA carboxylase Malonyl CoA (3C)
ATP ADP
• CO2fixation.

• Propionyl CoA (3C) Propionyl CoA carboxylase Methyl Malonyl CoA (4C)

om
ATP ADP

l.c
ai
Biotin independent carboxylation :

gm
• Carbamoyl phosphate Synthetase (CPS) I - urea cycle.
@
• CPS II-Pyrimidine synthesis. 19
55
na

• Gamma carboxylation (Vit K required).

ee

• Malic enzyme.
m
ar

• AIR carboxylase (in de novo purine synthesis).

um
itk

Subclass II :
am

• All enzymes with synthetase suffix.

|
w
ro

• Hydrolysis of ATP involved.

ar
M

• Eg :
©

- Arginosuccinate synthetase.
- Carbamoyl phosphate synthetase.

CLASS VII : TRANSLOCASES

• Transfer of molecules & ions across membrane.
• Eg :
- Ca2+ channel.
- H+ pump.
- K+ channel.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Mechanisms of Action of Enzymes 19

MECHANISMS OF ACTION OF ENZYMES ----- Active space -----

Active site

Reaction Thermodynamics 00:03:05

om
l.c
Types :

ai
Reactions

gm
@
19
55
Uncatalysed Catalysed (On adding enzymes)
na

Transition state
ee

• Transition state lowered

m
ar

Eact reduced.
um

• ∆G0 Unchanged
itk

∆G+
am

Gs Eact
Gibbs (free) energy

Transition
w

Reactants
ro

∆rG 0 state
ar
M
©

Gp
Free Energy

Products

∆G0
Reaction coordinate
Gs : Free energy of substrate
Gp : Free energy of product

∆G+/ Eact : Free energy, substrate has to Progress of reaction

acquire to enable reaction (formation of
products)

∆G0 : Free energy change = Gp - Gs

If negative Thermodynamically
favourable reaction

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 20 Enzymes

----- Active space ----- Activation energy barriers :

Energy barrier Enzymatic effect

• Entropy (Randomness) of system ↓entropy
• Shell of hydration

Substrate
Desolvation of substrate
Enzyme from shell of hydration
Shell of hydration
Active site

• Improper alignment of enzyme & substrate Proper alignment

om
l.c
Enzyme & Substrate Interactions 00:19:47

ai
gm
Theories explaining alignment of enzyme and substrate

@
19
55
Features
na

Only substrate complementary to active site can bind.

ee

1. Emil Fischer Template Theory/

m

Drawback : Can’t explain dynamic changes of a

ar

Lock & key Mechanism

um

reaction
itk

If active site complementary to transition state

am

2. William Jencks & Linus Pauling

|

Theory
w
ro

more products are formed

ar
M
©

3. Induced Fit theory by Daniel

Koshland

Advantage : Explain dynamic changes accompanying a

reaction

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Mechanisms of Action of Enzymes 21

Mechanisms of lowering activation energy ----- Active space -----

Features Example
Reactants come in bond forming
Catalysis by proximity -
distance
Catalytic residue
Aspartate protease
Acid base catalysis Acid Base enzyme :
• Pepsin
Proton donors Proton acceptors
Serine proteases :
Transient covalent bond b/w enzyme • Trypsin.
Covalent catalysis
and substrate • Chymotrypsin

om
l.c
• Elastase

ai
gm
Metal enables alignment of enzyme
Metal ion catalysis Zn in carboxypeptidase
@
towards substrate
19
55
Enzymes in lytic reactions
na

Enzyme Strains substrate

ee

Catalysis by strain
m

Lysing of substrate
ar

Lyases Hydrolases
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 22 Enzymes

----- Active space ----- ENZYME KINETICS

Enzyme kinetics
Depends on

Rate/Velocity of Factors affecting enzyme

reaction activity
Rate of reaction  00:02:22

Reversible reaction : r1 = Rate of forward reaction.

A+B r1 P+Q r2 = Rate of backward reaction.

om
(Substrates) r2 (Products) K = constant.

l.c
ai
At Equilibrium :

gm
@
• r1 = r2
• k1 [A][B] = K2 [P][Q] 19
55

• K1 = Keq (Equilibrium constant) = [P][Q] = [Products]

na
ee

K2 [A][B] [Substrate]
m

when [Product] = [Substrate] keq = 1

ar
um
itk

Factors affecting enzyme activity  00:07:10

am
|

1. SUBSTRATE CONCENTRATION
w
ro

Vi/V0 = Initial velocity of reaction

ar

No further
M

V0 [S] = Substrate concentration

change in velocity (2)
©

(1) : First order kinetics : V0 ∝ [S]

Vmax (2) : Zero order kinetics : V0 not
All active sites proportional to [S]
saturated
1/2 Vmax
(1)

[S] at 1/2Vmax [S]

Michaelis Constant (Km) :

• Constant for an enzyme substrate pair (Signature of enzyme substrate pair).
• Km ∝ 1
Affinity of enzyme towards substrate

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Enzyme Kinetics 23

Applications : ----- Active space -----

a)
Hexokinase Glucokinase
Km 0.2 mM 10 mM
Affinity for substrate
High Low
(Glucose)
High (Well fed
[S] required Low
state)
b)
GLUT-2 GLUT-3
Site Liver, Pancreas Brain
Km High Low

om
affinity to glucose Low High

l.c
ai
gm
Active state Well fed state Fasting state

@
Michaelis-Menten Equation : 19
55

V × [S]
na

V1 = max
ee

Km + [S]
m
ar
um

Km + [S]
Reciprocal : 1 =
itk

V1 Vmax × [S]
am
|

Km [S]
w

1 =V +
ro

V1 [S] Vmax[S]
ar

max
M
©

1 Km 1 + 1
V1 = × [S]
Vmax Vmax

Y axis Constant (a) X axis Constant (b)

Lineweaver Burk Plot / Double Reciprocal plot :

• At y intercept : x=0
Hence, y = 1
1 Vmax
Vi
• At x intercept : y = 0
Hence x = -1
Y intercept Km

X intercept 1
[S]

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 24 Enzymes

----- Active space ----- ENZYME CONCENTRATION

Reaction velocity ∝ [E]
V0
V0 ∝ [E]
velocity of
reaction

Enzyme [E]
concentration

TEMPERATURE

om
l.c
ai
V0 Bell shaped curve

gm
Optimum Temperature (OT)

@
Vmax • Temperature at which Vmax is attained.
19
• In humans : 35-40°C
55
na

• ↑kinetic energy of reactants

ee
m

• ↑velocity till Vmax Temperature > 45-55°C

ar

Denaturation of enzymes
um
itk
am

OT Temperature
|
w
ro
ar

Temperature Coefficient :
M

For every 10°C rise in temp Velocity doubles (Till Vmax).

©

Q10 = 2.
PH OF MEDIUM
Optimum PH :
Vmax • PH at maximum velocity.
V0 • In humans : 5-9
- Physiological pH : 7.4.
Bell shaped

Optimum [S]
PH

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Enzyme Kinetics 25

Catalytic Constant  00:37:12 ----- Active space -----

Catalytic Constant (Kcat) :

E+S ES P
AKA Turnaround number. ES E Free enzymes (E)
Vmax ES Enzyme substrate
Kcat = Et = E + Es complex (Es)
Et Total enzyme concentration
Application :
• Catalytic efficiency of enzyme : Kcat .
Km
• ↑ Kcat ↑ Efficiency of enzyme.

Other Catalytic Constants :

k1 ES k2
E+S Products

om
k-1 Complex

l.c
ai
kd : Dissociation constant. k1 : Rate constant of association of E and S.

gm
k-1 : Rate constant of dissociation of ES complex.

@
K-1 Tendency to dissociate
kd = 19
k2 : Rate of product formation Irreversible.
55
K1 Tendency to associate
na

ka : Association constant.
ee
m

K
ar

ka = 1
um

K-1
itk
am

Summary :
|

K1
1. Keq = [Product]
w

4. Ka =
ro

[Substrate] K-1
ar
M

2. Km = [Substrate] at 1/2 Vmax. K

©

V 5. Kd = -1
K1
3. Kcat = max
Et

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 26 Enzymes

----- Active space ----- ENZYME INHIBITION

Types of enzyme inhibition 00:01:15

1. Inhibition by external agents 2. Responsible for physiological regulation

• Competitive inhibition. • Allosteric inhibition.
• Non-competitive inhibition. • Feedback inhibition.
• Uncompetitive inhibition.
• Suicide inhibition.

COMPETITIVE, NON-COMPETITIVE AND UNCOMPETITIVE INHIBITION

• : Uninhibited

om
• : Inhibited

l.c
ai
gm
Competitive inhibition Non-competitive inhibition Uncompetitive inhibition

@
Inhibitor • Competes for the • Binds to a distinct site. • Cannot bind to free
same active site where 19
• Not a structural analogue of enzyme.
55
na

substrate binds. substrate. • Binds to ES complex.

ee

• Structural analogue of E+S ES complex →P E+S ES complex →P

m

substrate. + + X
ar

↑ +
um

I I I I
itk
am

EI complex ESI complex ESI complex

|
w

No products Less products

ro

Less products
ar
M
©

Reversibility Reversible : Mostly irreversible. -

If ↑[S] Substrate binds
to active site Reaction
happens.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Enzyme Inhibition 27

----- Active space -----

Competitive inhibition Non-competitive inhibition Uncompetitive inhibition

Kinetic V 0 V 0 V 0
properties Vmax Vmax
Vmax=
Vmax’ Vmax’ Vmax’
Vmax Vmax
Vmax 2 2
2 Vmax’
Vmax’
2
2

Km Km’ Km=Km’ [S]

Km’ Km [S]
[S]

om
Km’ > Km → Km’ = αKm Vmax’ < Vmax Vmax’ < Vmax

l.c
(α : Constant) Km unchanged Km’< Km

ai
gm
Vmax unchanged

@
19
55
Lineweaver 1
na

Burk Plot V0
ee
m
ar

X-shaped graph
um

Y-intercept= 1
itk

Vmax V-shaped graph Parallel graph

am

X-intercept = -1 1
|
w

Km [S]
ro
ar
M

X & Y intercept X-intercept moves closer to X-intercept unchanged. Both Y and X intercept move
©

zero. Y-intercept moves away from away from zero.

Y-intercept remains the same zero.
Examples 1. Most pharmacological Most poisons : Phenylalanine X Placental ALP
drugs : • Complex IV of Electron
• Methotrexate X DHF transport chain :
reductase - Cyanide X Cyt C oxidase
• Statins X HMG CoA - CO X Cyt C oxidase
reductase. • Fluoride X Enolase.
• Dicoumarol X Vit. K • Iodoacetate X
epoxide. glyceraldehyde-3-
• Ethanol X Alcohol phosphate dehydrogenase.
dehydrogenase (used • Fluoroacetate X Aconitase
in methanol poisoning :
prevents formation of
formaldehyde.)
2. Poisons : Malonate X
Succinate degydrogenase.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 28 Enzymes

----- Active space ----- SUICIDE INHIBITION

• AKA Mechanism based inactivation.
• Irreversible inhibition.
Unreactive inhibitor
Uses mechanism of
Enzyme
enzyme activity
Binds to active site

Reactive inhibitor

om
Irreversibly inhibits enzyme

l.c
Suicide inhibition

ai
Examples

gm
• Allopurinol →
X Xanthine oxidase (xo)

@
- Use : Treatment of 19
55
na

hyperuricemia/gout
ee

- MOA : Hypoxanthine
m
ar
um

XO
itk
am

Xanthine
|

XO Allopurinol
w
ro

Alloxanthine Irreversibly inhibits XO

ar
M

Uric acid
©

Uric acid not produced

• Aspirin →
X Cyclooxygenase.
• Difluoromethylornithine (DFMO) →
X Ornithine decarboxylase

FEEDBACK INHIBITION

A B C D (end product).
E1 E2 E3

Feedback Inhibition
• Prevents unwanted
accumulation of product.
• Optimal concentrations
maintained.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Concept of Enzyme Regulation 29

CONCEPT OF ENZYME REGULATION ----- Active space -----

Covalent Modification 00:01:00

Sites of phosphorylation
Hydroxyl group containing amino-acids:
- Serine (M/C)
- Threonine
- Tyrosine

om
Mechanism

l.c
ai
gm
@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Examples
Enzyme Insulin : Glucagon ratio High activity in
Phosphofructokinase (in glycolysis) High DP state
Fructose-1,6-bisphosphate (in
Low P state
gluconeogenesis)
Glycogen synthase High DP state
Glycogen phosphorylase (in
Low P state
glycogenolysis)
Pyruvate dehydrogenase (link
High DP state
between glycolysis & TCA cycle)
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 30 Enzymes

----- Active space -----

Enzyme Insulin : Glucagon ratio High activity in

Acetyl CoA carboxylase (Fatty acid
High DP state
synthesis)
HMG CoA reductase (Cholesterol
High DP state
synthesis)
Hormone sensitive lipase (hydrolysis
Low P state
of stored TAG in adipose tissues)

Allosteric Regulation 00:16:31

om
l.c
Feed Forward reaction Feedback inhibition

ai
gm
+ -

@
↑[Substrate] Forward reaction ↑[Product] Forward reaction
19
55
na

Enzyme Allosteric activator Allosteric inhibitor

ee
m

Phosphofructokinase
ar

(In glycolysis) Products of glycolysis:

um

Substrates in glycolysis
itk

• ATP
• 5’ AMP
am

• Low pH (d/t lactic acid)

• Fructose-6-
|
w

Fructose-6- phosphate Fructose-1,6- • Citrate (formed from

ro

phosphate
ar

bisphosphate acetyl CoA)

M

(Product)
©

Acetyl CoA carboxylase

• Malonyl CoA (product)
Citrate (substrate) • Acyl CoA (fatty acid
product)
Acetyl CoA Malonyl CoA
ALA synthase - Heme (product)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Enzyme Regulation 31

ENZYME REGULATION ----- Active space -----

Control of enzyme
Enzyme quantity synthesis

Enzyme Regulation (Long term : Days) Control of enzyme

degradation
Allosteric regulation
Enzyme quality/Intrinsic
Catalytic activity (Short
term : min to hrs) Covalent modification

om
Regulation of Enzyme Quantity  00:01:05

l.c
ai
gm
1. CONTROL OF ENZYME SYNTHESIS

@
a) Heme synthetic pathway 19 b) Cholesterol synthetic pathway
55
na
ee
m
ar
um

Nucleus
itk

↑ heme
am

Cytosol
(-)
|

Production of
w

ALA synthase
ro
ar

protein
M
©

↓ heme
synthesis

Trigger ↑ heme level ↑ cholesterol level (↑ Dietary cholesterol)

Response Inhibition of enzyme synthesis at the gene level
2. CONTROL OF ENZYME DEGRADATION
For Short lived proteins (Eg : Cyclins).

Ubiquitin Proteasomal pathway :

Function : Degradation of aberrant/Defective proteins.
Dysfunction : Protein degradation/Folding disorders Accumulation of misfolded
proteins.
Eg :
• Neurodegenerative disorders : Alzheimer’s, Parkinson’s disease.
• β Thalassemia.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 32 Enzymes

----- Active space -----

Regulation of Enzyme Quality  00:07:10

1. COVALENT MODIFICATION/HORMONAL REGULATION

Enzymes regulated by : Addition/Removal of functional groups Formation/
Breaking of covalent bonds.

Types :
Covalent modification
Irreversible Zymogen activation
Reversible (Formation and breakdown of Covalent bonds)
(Proteolytic cleavage)
Phosphorylation - ADP
Gastrointestinal enzymes. Clotting factors. Acetylation Methylation
Dephosphorylation. ribosylation.
Eg :
Eg :

om
• Chymotrypsinogen
• Plasminogen Most versatile M/c covalant

l.c
(inactive) - -
(inactive) mechanism. modification.

ai
Chymotrypsin (active).

gm
Plasmin (active).
• Trypsinogen Trypsin.

@
19
55
Phosphorylation (P) - Dephosphorylation (DP) :
na

Insulin : Glucagon ratio

ee
m
ar
um

High in well-fed state Low in fasting/stressed state

itk
am
|

Dephosphorylates (DP) the phosphorylates (P) the rate

w
ro

rate limiting enzyme (active limiting enzyme (active

ar
M

state). state).
©

Fasting state
Glucagon Epinephrine (Ligand)
(Ligand) Adenylyl
t e in r cyclase
ro
G-p recept
o Fed state
Plasma pled (+)
cou rotein
Insulin
membrane G-p ATP CAMP 5’AMP (+)
(3P) (1P)
Phosphodiesterase
CAMP dep
Proteinkinase A
P (+)

Enzyme Phosphorylated
enzyme
Protein phosphatase

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Enzyme Regulation 33

Examples : ----- Active space -----

Enzyme Insulin : Glucagon ratio High activity in

Phosphofructokinase (In glycolysis) high DP state
Fructose-1, 6-Bisphosphatase (In gluconeogenesis) low P state
Glycogen Synthase high DP state
Glycogen Phosphorylase (In glycogenolysis) low P state
Pyruvate Dehydrogenase (link b/w glycolysis and TCA cycle) high DP state
Acetyl CoA Carboxylase (Fatty acid synthesis) high DP state
HMG CoA Reductase (Cholesterol synthesis) high DP state
Hormone Sensitive Lipase (Hydrolysis of stored TAG in
low P state
adipose tissue)

om
Note : Insulin (-) Hormone Sensitive Lipase (HSL).

l.c
ai
Hence in Diabetes, ↑ HSL.

gm
@
2. ALLOSTERIC REGULATION 19
55
na

substrate
ee

Catalytic/
m

Active site
ar
um

Enzyme
itk

Modifier
am

Allosteric site
|
w
ro
ar

Allosteric modifiers
M
©

Positive Negative
Induces Induces
Favourable conformational Unfavourable conformational
changes to catalytic site changes to catalytic site

Substrate binds. Substrate cannot bind.

Features :
• Modifier need not be a structural analogue of substrate (Similar to non-
competitive inhibition).
• Most Allosteric enzymes : Multisubunit enzymes
Eg. : Quaternary structure of Hemoglobin (Hb).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 34 Enzymes

----- Active space ----- • Cooperative Binding :

Enzyme : 4
active sites
Substrate
• Does not follow Michaelis
Favours Leads Menten kinetics.
Binding of substrate (Oxygen) • Velocity against [s]
to 1 active site Binding of other Oxygen atoms to
other active site in same enzyme (Hb) Sigmoidal curve kinetics.
• Allosteric enzymes key regulatory/Rate limiting enzyme in metabolic
pathways (Ex. ALA Synthase).

saturation of active sites no further

om
velocity
binding of substrate

l.c
(Vo)

ai
gm
Coopertive binding

@
19
55
Increase in Vo at
na

high rate
ee
m
ar
um

Substrate concentration [s]

itk
am

Effect of Allosteric activators & Inhibitors

|
w

On addition of allosteric activator allosteric Inhibitor

ro
ar

Curve shift to left Shift to right

M
©

K0.5 ↓ ↑
Affinity towards substrate ↑ ↓
State of enzyme Relaxed Taut
Example (Binding of O2 to Hb) (Unloading of O2 from Hb)
V0
: On adding allosteric
Vmax
activators
: On adding allosteric
inhibitor

1/2 Vmax

K0.5 K0.5/S0.5 K0.5 [S]

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Enzyme Regulation 35

K0.5/S0.5/Binding constant : ----- Active space -----

• [S] at 1/2 Vmax.
• 50% of active sites saturated with substrate.

Classification of Allosteric enzymes :

K series V series
Vmax constant ↓
K0.5 ↑ constant
Kinetic Similar to Non-Competitive
Similar to competitive inhibition
property inhibition

Examples of Allosteric enzymes :

om
Allosteric Inhibitor Allosteric Activator

l.c
Enzyme

ai
(mostly products) (mostly substrate)

gm
@
ALA Synthase Heme -
19
55
Aspartate Transcarbamoylase
na

CTP (Pyrimidine) ATP (Purine)

(In pyrimidine synthesis)
ee
m

HMGCoA Reductase Cholesterol -

ar
um

Phospho FructoKinase Citrate, ATP AMP, F2, 6-BP

itk

Acetyl CoA Carboxylase Acyl CoA Citrate

am
|

Citrate Synthase ATP -

w
ro

Carbamoyl Phosphate
ar

- NAG
M

Synthetase-I
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 36 Enzymes

----- Active space ----- CLINICAL ENZYMOLOGY

Isoenzymes  00:02:15

Physically distinct forms of the same enzyme (Catalyze the same reaction).

Properties of isoenzymes :

Properties Examples

Products of different genes Salivary amylase and pancreatic amylase

om
• Lactate dehydrogenase (LDH) 1-5

l.c
Subunits may be different
• Creatine kinase (CK) 1-3

ai
gm
Different electrophoretic • LDH - 1 fastest, LDH - 5 slowest

@
mobility • CK - 1 19
fastest, CK - 3 slowest
55
na

Isoenzyme of α2 ALP (alkaline phosphatase) :

ee

Differ in heat stability • Heat stable

m
ar

• Heat labile
um

Gluco kinase (specific to glucose) and Hexokinase (acts

itk

Differ in substrate specificity

am

on all hexoses)
|

Isocitrate dehydrogenase (ICH) :

w
ro

Differ in cofactor
• Cytoplasmic : Requires NADP+
ar

requirement
M

• Mitochondrial : requires NAD+

©

Note :
• Functional enzymes : Perform a function in the blood.
Eg :
- Lipoprotein Lipase : Hydrolyses triacylglycerol from lipoproteins.
- Coagulation factors.
• Non functional enzymes :
- No function in blood.
- Usually intracellular Normal cell turnover low levels in serum
Tissue damage/necrosis high level in serum

diagnostic markers
- Eg. LDH, CK.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Clinical Enzymology 37

Isoenzymes as Diagnostic Markers : ----- Active space -----

Identification of

Location of Injury Severity of Injury

Liver Heart Bone Irreversible injury

Reversible injury
Eg : Eg : Eg : ALP (Alkaline ↑ Plasma membrane ↑ Mitochondrial
• ALT (Alanine • AST (Aspartate phosphatase) permeability permeability
amino amino
transaminase) transaminase) ↑ Levels of cytoplasmic ↑ Levels of mitochondrial
• CK-MB (Creatine enzyme in blood enzyme in blood
Kinase muscle,

om
Bone)

l.c
ai
EXAMPLES OF ISOENZYMES

gm
@
1. Lactate Dehydrogenase (LDH) : 55
19
Reaction : Pyruvate LDH Lactate.
na

H M
ee

Site: Cytoplasm.
m
ar
um

Structure : M H
itk

• Tetramer.
am

H LDH-3 : H2M2 Subunits

• 2 types of subunits
|

M
w
ro

Isoenzymes of LDH :
ar
M

Mobility in
©

Tissue
Isoenzyme Subunits electrophoretogram % in Serum
Localization
(towards anode)
LDH - 1 H4 Fastest Heart 30%
LDH - 2 H3M1 Faster RBC 35%
LDH - 3 H2M2 Intermediate/Fast Brain 20%
LDH - 4 H1M3 Slower Liver and 10%
LDH - 5 M4 Slowest Skeletal muscle 5%
2. Creatine Kinase (CK) :
Reaction : Creatine CK Creatine Phosphate. Structure : Dimer.
Site : Cytoplasm except CK -Mt : Located in Mitochondria
M B
Enters bloodstream if
CK - 2 : MB Subunits
irreversible injury
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 38 Enzymes

----- Active space ----- Isoenzymes of CK :

Mobility in Tissue % in
Isoenzyme Subunits
electrophoretogram Localization Serum
CK - 1 BB Fastest Brain 1%
CK - 2 MB Intermediate Heart 5%
CK - 3 MM Slowest Skeletal Muscle 80%

3. Alkaline phosphatase (ALP) :

Reaction : Substrate with phosphate ALP Hydrolyzed product + P O43 (Inorganic).
(Alkaline
medium)
Isoenzymes of ALP :

om
Isoenzymes Location Clinical Significance

l.c
ai
Membrane of epithelial Elevated in Extrahepatic obstruction to bile flow

gm
α-1-ALP
cells of biliary canaliculi (Cholestasis); e.g., Stone, Carcinoma

@
Heat labile Hepatic Sinusoidal cells 19Marker of hepatic injury
55
na

α-2 • Most stable ALP

ee

Heat stable Placenta

• Inhibited by Phenylalanine
m
ar

• Marker of bone formation

um
itk

• Elevated in :
am

Pre-beta ALP Osteoblasts of bone - Paget’s disease

|
w

- Vit D deficiency
ro
ar

- Hyperparathyroidism (1° and 2°)

M

Elevated in
©

• Ulcerative colitis
Gamma ALP Intestinal cells
• Hepatitis (d/t impaired clearing from plasma by
sinusoidal cells of liver)
Leukocyte ALP Leukocytes Associated with leukemia

Note : Isoenzymes similar to Placental ALP

• Regan Isoenzyme/(Carcino Placental Enzyme)
- Origin from germ cells Elevated in malignant
• Nagao Isoenzyme tumors
• Kasahara Isoenzyme

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Clinical Enzymology 39

Clinical Applications  00:24:42 ----- Active space -----

1. CARDIAC BIOMARKERS
Any chemical compound (enzyme or other biomolecule) elevated in cardiac injury.

Examples :
• Cardiac Troponin : Stays in the blood for • CK-MB : First enzyme to rise.
longest time • AST.
- Trop T. • LDH.
- Trop I.

• Brain Natriuretic peptide (BNP) : Marker of volume overload d/t circulatory/

cardiac failure.

om
• Myoglobin :

l.c
ai
- First biomarker to rise (starts to rise at 2hrs).

gm
- Non specific.

@
• Hs CTn (High Sensitivity Cardiac Troponin) : 19
55

- Detection by Monoclonal Antibody assay : Very sensitive Detects upto

na
ee

<1 ng/l.
m
ar
um

Blood levels :
itk
am

Marker Start to rise Peak Return to baseline

|

CK-MB 4-8 hours 24 hours 48 - 72 hours

w
ro

Trop T 4-6 hours 24 hours 7 - 10 days

ar
M

Trop I 4-6 hours 24 hours 7 - 10 days

©

LDH 24 hours 3-6 days -

AST 12 hours 48 hours 4-5 days

Blood level X : CK-MB

X : Troponin T

1 2 3 4 5 6 7 8 9 10
Days

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 40 Enzymes

----- Active space -----

2. ENZYME PROFILE IN LIVER DISEASE
Markers of
Markers ofhepatic Injury :
liver disease Markers of cholestasis (obstruction to bile flow)
ALT ↑ specificity for GGT (gamma 5’
AST ALP
liver injury glutamyl transferase) nucleotidase
• Cell membrane Cell
Cell membrane
• Smooth membrane
Cytoplasmic and of biliary
Location Cytoplasmic endoplasmic of biliary
mitochondrial canalicular
reticulum canalicular
epithelial cells
(microsomes) cells
Fatty liver :
• ↑ ALT
• other enzymes
normal
Specificity for liver injury : ALT > AST

om
AST : ALT Ratio (AAR)

l.c
• Less specific for

ai
gm
cholestasis
Features

@
<1 (ALT>AST) >1 (AST>ALT) Specific marker • Marker of More specific
and
of extra hepatic 19
alcoholic hepatitis/ than ALP and
causes of • Chronic viral hepatitis • Alcoholic hepatitis : D/t
55
cholestasis heavy drinkers/ GGT
na

elevation • Non alcoholic fatty mitochondrial injury

ee

liver disease phenobarbital (d/t

m

• Toxic hepatitis ↑ AST > ↑ ALT microsomal injury)

ar
um

(marked enzyme • Hepatic cirrhosis : D/t

itk

elevation) impaired clearance of

am

• Acetaminophen AST from circulation

|

toxicity by sinusoidal cells

w

• Liver neoplasia
ro
ar
M
©

Aminotransferase level
Ischemic or
toxic liver
Acute viral injury
Alcoholic hepatitis
Aminotransferase level, IU/L

liver Autoimmune
disease hepatitis

Chronic
Liver hepatitis
cirrhosis
Reference
range

Elevation of ASt & ALT : Higher values in acute

conditions of liver

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Clinical Enzymology 41

3. OTHER SYSTEMIC APPLICATIONS ----- Active space -----

a) Enzyme profile in prostate disorders :
Acid phosphatase : Prostate specific antigen (PSA)
• Tartrate labile. AKA kallikrein related peptidase-3.
• Less specific. • More specific.
• normal cutoff level : <4ng/ml.

Note : Leukocytes & spleen Tartrate resistant acid phosphatase.

b) Enzyme profile in pancreatic disorders :

Serum amylase : Non specific (Also ↑ in salivary gland disorders).
Serum lipase : More specific.

c) Markers of bone disease :

om
l.c
ai
Bone formation markers Bone resorption markers

gm
Origin Osteoblast osteoclast
@
• pre beta ALP 19
• N-Telopeptide of Type 1 Collagen
55
na

Examples • Osteocalcin • C-Telopeptide of Type 1 Collagen

ee

• Propeptide of type 1 collagen • Urine Free Deoxypyridinoline

m
ar
um

d) Bio Markers of Acute Kidney Injury :

itk

• Kidney injury molecule-1 (KIM-1). • Microalbuminuria.

am

• Neutrophil gelatin associated lipocalin • Osteopontin.

|
w
ro

(NGAL). • Liver fatty acid binding protein.

ar

• Interleukin 8. • Sodium - hydrogen exchanger

M
©

• ALT. isoforms.
• Glutathione S-transferase (GST). • Exosomal fetuin.
• GGT.

4. ENZYME MARKERS OF CELL ORGANELLES

Organelle Marker enzyme

• 5’ Nucleotidase
Plasma membrane • Adenylyl cyclase
• Na+ K+ ATPase
Endoplasmic reticulum Glucose-6 phosphatase
Golgi apparatus Galactosyl transferase
Mitochondria ATP synthase
Lysosomes Cathepsin

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 42 Chemistry and Metabolism of Carbohydrates

----- Active space ----- CHEMISTRY OF CARBOHYDRATES

General formula : Cn(H2O)n (Hydrates of carbon).

Definition : Aldehyde or ketone derivative of a polyhydroxy-alcohol.
Example :
CH2OH CH2OH CHO
| | |
C=O CH OH CH OH

|
|
| | |
CH2OH CH2OH CH2OH
Ketone derivative Polyhydroxy Aldehyde derivative

om
(Ketose) alcohol (Aldose)

l.c
ai
Types :

gm
@
1. Monosaccharides.
2. Disaccharides. 19
55
na

3. Oligosaccharides.
ee
m

4. Polysaccharides.
ar
um
itk

Types of Carbohydrates  00:05:19

am
|

MONOSACCHARIDES
w
ro

One sugar unit.

ar
M
©

Examples :

No. of Carbon Atoms Aldoses Ketoses

1. Triose (3C) Glyceraldehyde Dihydroxy acetone
2. Tetrose (4C) Erythrose Erythrulose
Ribulose, Xylulose (Epimers of
3. Pentose (5C) Ribose, Xylose (Epimers of ribose), Arabinose
ribulose)
4. Hexose (6C) Glucose, Galactose, Mannose Fructose

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Carbohydrates 43

DISACCHARIDES ----- Active space -----

2 monosaccharides with a glycosidic linkage (Covalent bond).

Examples :
Reducing Disaccharides (Free functional group)
Name Monomer units Glycosidic linkage
Maltose Glucose + Glucose α 1,4
Isomaltose Glucose + Glucose α 1,6
Lactose Galactose + Glucose β 1,4
Lactulose (Synthetic) Galactose + Fructose α1, β4
Non reducing Disaccharides (No free functional group)
Trehalose Glucose + Glucose α 1,1

om
Sucrose Glucose + Fructose α1, β2

l.c
ai
gm
OLIGOSACCHARIDES

@
3-10 monosaccharides.
19
55
na

POLYSACCHARIDES
ee

>10 monosaccharides.
m
ar
um

Types :
itk

Polysaccharides
am
|
w
ro
ar

Homopolysaccharides : Heteropolysaccharides :
M
©

• One type of monomer • More than one type of monomer

• Examples : • Examples :
- Starch - Chitin - Glycosaminoglycans (GAG)
- Glycogen - Inulin - Pectin
- Cellulose - Dextran - Agarose
Glycogen :
Storage carbohydrate in animals (AKA animal starch).

Structure : Linkage :
• Branched polymer of α D glucose. α(1→6)
Glycogen in mammal :
• Center : Single reducing end (Glycogenin)
• Terminal portions : Multiple non reducing ends. α(1→4)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 44 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Starch :

Storage carbohydrate in plants.

Structure :
Starch

Amylose : Amylopectin :
• soluble • insoluble
• linear, no branches • multiple branches
Linkage :
• α(1→4) : Linear. • α(1→6) : Branched.

Other Polysaccharides :

om
l.c
ai
Monosaccharide unit Significance

gm
1. Inulin Fructose (Fructosan) Inulin clearance test : Assessment of GFR
@
2. Chitin N-Acetyl Glucosamine 19 Exoskeleton of crustaceans
55
na

3. Pectin Galacturonic acid Dietary fibre

ee
m
ar

4. Dextrin Product of hydrolysis of starch :

um

Glycogenolysis (Limit dextrins)

itk
am

• Plasma volume expander

|

5. Dextran α D Glucose • Size exclusion chromatography

w
ro

• Dental plaque
ar
M
©

Dietary fibres :
AKA non starch polysaccharides.

Constituents :
• Remnants of edible parts of plants.
• Major fibre : Cellulose (Made of β D Glucose)
Human intestine lacks cellulase Metabolism

Cannot hydrolyse β1→4 linkage

No digestion/absorption of fibre

Partial/complete fermentation in large intestine

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Carbohydrates 45

Types : ----- Active space -----

Soluble Insoluble (Crude fibres)

Eg : Eg :
Gums (Funugreek) Cellulose
Mucilage Hemicellulose
Pectin Lignin (Not fermented)
RDA : 40 g/2000 Kcal.
Energy : 2 Kcal/g.

Uses :
• Soften and ↑ fecal bulk.
• Regular bowel movements.

om
• Sequestration of bile salts.

l.c
ai
gm
↓ cholesterol
@
• Improve satiety. 19
55
na

• Prebiotic : Substrate for colonisation of probiotic bacterial strains.

ee

• ↑ glucose tolerance.
m
ar

• Gums and pectin ↓ Postprandial blood glucose levels.

um
itk
am

Structures of Carbohydrates  00:33:00

|
w
ro

RING STRUCTURE
ar
M

a) Glucose :
©

H O| 6 CH2OH
1C
=

| 5 O
H 2C OH
Linkage of C1 and C5
|
|

|
HO 3C H (In solution form) 4 1
|
|

|
H 4C OH OH OH OH
|
|

| 3 2
H 5C OH
|
|

|
6
CH2OH OH
Pyranose ring :
Straight chain : • 6 membered ring
AKA Fischer projection. • AKA glucopyranose

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 46 Chemistry and Metabolism of Carbohydrates

----- Active space -----

b) Fructose :
C
1
| 0
C=O
2
|
C Linkage of C2 and C5
3
| 5 2
C
4
|
C OH 4 3
|
5
| Furanose ring :
6
C
Straight chain 5 membered (4 C + 1 O2)

ISOMERISM
Chirality :

om
Asymmetrical carbon atoms :

l.c
ai
H O

gm
|
1C
H 1C OH
=

|
|
| C,C,C,C |
H 2C OH 2 3 4 5 19 H 2C OH O C1, C2, C3, C4, C5
|
|

|
|
| |
55
na

HO 3C H HO 3C H
|
|

|
|
| |
ee

H 4C OH H 4C OH
m
|
|

|
| | |
ar

H 5C OH H 5C
um
|
|

| |
CH OH CH OH
itk

6 2 6 2
am

Straight chain Ring structure

|
w

Le Bel-Van’t Hoff formula :

ro
ar

• Used to calculate total number of isomerisms possible.

M

• Formula = 2n.
©

n : Number of asymmetric carbon atoms.

Classification :
Isomerism

Structural Stereo-
isomerism isomerism

D&L d&l
isomerism Anomerism Epimerism isomerism
Stereoisomerism : Aka optical isomerism

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Carbohydrates 47

D and L isomerism : ----- Active space -----

• Isomerism at the penultimate/reference carbon atom.
• Examples : D Glucose and L Glucose.
CHO CHO
1
| 1
|
C C

|
|
2
| 2
|
C C

|
|

3
| 3
|
C Reference/ C

|
|

4
| 4
| Note :
H 5C OH Penultimate carbon OH 5C H

|
|
|
|

| | • Most carbohydrates : D isomers

6
CH2OH 6
CH2OH
• Most amino acids : L isomers
D Glucose L Glucose

Anomerism :

om
• Isomerism at the functional carbon atom.

l.c
ai
• Types : α and β anomers.

gm
@
• Examples :
a. C1 of Glucose. 19
55
na

b. C2 of Fructose.
ee
m

OH OH OH
ar
um

4 6 4 6 4 6 Above
O OH O
itk

OH S 5 OH R 5 OH S 5
am

R 2 H R 2 R 2 OH
3 3 1 H 3
|

OH R OH R OH R
1S 1 R
w

S S S
ro

OH OH O OH
OH H
ar
M

α glucose Anomerism in glucose β glucose

©

Below
Epimerism :
• Isomerism at any carbon atom apart from the functional and penultimate
carbon atoms.
• Examples :
1
CHO 1
CHO 1
CHO Allose : E pimer of
| | |
OH 2C H H 2C OH H 2C OH glucose at
|
|

|
|

|
|

| | |
HO 3C H HO 3C H HO 3C H C-3.
|
|

|
|

|
|

| | |
H 4C OH H 4C OH OH 4C H
|
|

|
|

|
|

| | |
H 5C OH H 5C OH H 5C OH
|
|

|
|

|
|

| | |
6
CH2OH 6
CH2OH 6
CH2OH
D-Mannose D-Glucose D-Galactose
(Epimer at C2) (Epimer at C4)
Epimerism
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 48 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Optical isomerism :

Dextrorotation :
• Clockwise/rightward
deflection.
• ‘d’ or (+).
Light Polariser Glucose (AKA
Dextrose)
Levorotation :
• Anticlockwise/leftward
deflection.
Light Polariser Fructose (AKA • ‘l’ or (-).
Levulose)

om
Oxidation and Reduction Reactions  00:51:49

l.c
ai
Oxidation of carbohydrates :

gm
Sugars Oxidation Sugar acid.
@
19
55
Examples :
na

1. Aldehyde group Oxidation Aldonic acid (Eg : Gluconic acid).

ee
m

2. 6th carbon atom Oxidation Uronic acid (Eg : Glucuronic acid).

ar
um

3. 1st and 6th carbon atom Strong Oxidation Saccharic acid

itk
am

(Eg : Glucosaccharic acid).

|
w

Note :
ro

Glucose oxidase method : Glucose Oxidation Gluconic acid

ar
M
©

• Used in the estimation of glucose.

Reduction of carbohydrates :
Sugar Reduction Sugar alcohol.

Examples :
1. Glucose Reduction Sorbitol Diabetic cataract.
Aldose reductase
(In lens, seminal fluid)
Reduction
Sorbitol
2. Fructose
Mannitol

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Carbohydrates 49

3. Mannose Reduction Mannitol (Given IV to ↓ intracranial pressure) ----- Active space -----

4. Galactose Reduction Dulcitol/Galactitol Causes Oil drop cataract (In galactosemia).

Note :
1. Monosaccharide with single asymmetric carbon atom : Glyceraldehyde (C2).
2. Ketoses : 1 asymmetric carbon atom less than corresponding aldoses.
3. Monosaccharide with no asymmetric carbon : Dihydroxyacetone.
4. Amino acid with no asymmetric carbon : Glycine.
5. Predominant form of glucose in free form : β-D-Glucopyranose.

om
l.c
ai
gm
@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 50 Chemistry and Metabolism of Carbohydrates

----- Active space ----- GLYCOSAMINOGLYCANS

Glycosaminoglycans  00:00:50

Long, unbranched heteropolysaccharide made up of repeating disaccharide unit.

Disaccharide

Amino sugar Acidic sugar (COO-)

om
Glucosamine Galactosamine Glucuronic acid Iduronic acid

l.c
ai
gm
Epimers
@
19
55
Properties & functions :
na

• Negatively charged d/t :

ee
m

- Acetyl group CH3COO-

ar

Attach to amino group

um

- Sulfate group SO42-

itk

- Acidic group contains COO-.

am

• Like charges repel each other :

|
w

- Slippery nature of mucus secretion.

ro
ar

- Mobility of joint (Cartilage, bones, synovial fluid are rich in GAGs).

M
©

• Compressibility & lubrication :

- D/t ability to attract water.
- Helps in joint resilience.

Shrinking on releasing H2O Swelling on absorption of H2O

Action of GAG with water

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycosaminoglycans 51

TYPES ----- Active space -----

Disaccharide units GAG Location Significance

N-acetyl glucosamine
• Not covalently attached to
proteins
• No sulphate group
Skin, synovial fluid, loose
• Facilitates cell migration :
+ Glucuronic acid Hyaluronic acid connective tissue, bone,
- Wound repair
cartilage
- Tumor mets
- Embryogenesis
• Compressibility of cartilage
• Transparency of cornea (KS1)

om
+ Galactose
Keratan Cornea (KS1), loose

l.c
(Non-uronic acid or • No uronic acid

ai
sulphate (KS) connective tissue (KS II)

gm
acidic sugar) • Most heterogeneous GAG

@
N-acetyl galactosamine 55
19
Chondroitin • Most abundant GAG
na

+ Glucuronic acid Bone, cartilage, CNS

ee

sulphate • Compressibility of cartilage

m

• Most widely distributed

ar
um

• Structure of sclera
itk

Dermatan Skin, eye (structural • Known as atherogenic GAG

am

+ Iduronic acid
sulfate component of sclera) - Synthesized from smooth
|
w
ro

muscle attract LDL

ar
M

atherosclerosis
©

Glucosamine
• Receptor in plasma membrane
• Present in synaptic vesicles
Skin,
• Anchor LPL (lipoprotein lipase) in
Heparan glomerular
+ Glucuronic acid the endothelial surface
sulphate basement
• Charge selectiveness of GBM
membrane (GBM)
- Repels the entry of albumin
into the filtrate
• Anticoagulant
- Binds to anti-thrombin III
+ Iduronic acid Heparin Mast cells, lungs, skin. • Only intracellular GAG
• Dislodge LPL from its anchoring
site

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 52 Chemistry and Metabolism of Carbohydrates

----- Active space ----- STRUCTURE

GAG (95%) + protein (5%)

Proteoglycan
Glycosaminoglycans are usually attached to
proteins to form proteoglycans

GAG
Small Stalk :
Attach GAG to
core protein

om
made of

l.c
ai
gal-gal-xyl

gm
@
19
55
na
ee
m

Bottle brush shape of Proteoglycan monomer

ar
um
itk
am
|
w
ro
ar
M
©

Proteoglycan aggregate
Present in extracellular matrix

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycosaminoglycans 53

Mucopolysaccharidosis (MPS)  00:21:37 ----- Active space -----

GAGs are AKA mucopolysaccharides.

Process Location
Synthesis Rough endoplasmic reticulum (RER), Golgi apparatus
GAGs
Degradation Hydrolase Lysosomes
X Accumulation of GAG : Mucopolysaccharidosis Lysosomal
Defect in lysosome (MPS) storage disorder
Autosomal recessive inheritance except hunter disease (X-linked recessive).

GENERAL CLINICAL MANIFESTATIONS

Face :

om
• Gargoylic facies : Vertical facies

l.c
- Frontal bossing.

ai
gm
- Depressed nasal bridging.

@
- Gingival hypertrophy Upper respiratory
55
19
- Large tongue tract infection
na
ee
m

copious nasal discharge

ar

Gargoylic facies corneal clouding

• Corneal clouding (not present in all MPS).
um
itk

• Intellectual disability (not present in all MPS).

am

GIT :
|

• Visceromegaly.
w
ro

• Umbilical hernia.
ar
M
©

Hand :

Claw hand

Autosomal recessive Umbilical

inheritence hernia

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 54 Chemistry and Metabolism of Carbohydrates

----- Active space ----- OTHER COMMON FEATURES

Radiological features :

Dysostosis multiplex Beaking of Bullet shaped middle phalanx

vertebra
Degeneration in vertebra & metacarpals

om
l.c
Histological feature :

ai
gm
Inclusion body in leukocytes : Reilly body inclusions.

@
TYPES OF MPS 19
55
na

MPS Type Gene Enzyme defect

ee
m

Hurler’s disease MPS IH IDA α L iduronidase

ar
um

Hunter’s disease MPS II IDS α L iduronate sulfatase

itk

Sanfilippo disease (M/c) MPS III - Enzyme that degrades heparan sulfate
am
|

SPECIFIC FEATURES
w
ro
ar

Mental Corneal Leukocyte Present in all MPS :

M

MPS Visceromegaly • Coarse facial features.

©

retardation clouding inclusion

IH + + + + • Short stature.
II + - + + • Dysostosis multiplex.
III + - + +
Natowicz syndrome (Mps 1x) : Defect in hyaluronidase.

TREATMENT

Rx MPS
Stem cell therapy IH
I : Aldurazyme
Enzyme replacement therapy
II : Elaprase
Substrate reduction therapy III : Flavinoids

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycosaminoglycans 55

Inclusion cell (I-cell) disease  00:38:39 ----- Active space -----

Lysosomal protein targeting disorder.

RER Synthesis of protein : Enzymes of lysosome

+
N-acetyl glucosamine transfer of X Synthesis of mannose 6 phosphate
phosphotransferase phosphate group X
to mannose
Transport of lysosomal enzymes Plasma
Deficiency
into lysosomes
Accumulation of

om
Formation of mucopolysaccharides

l.c
inclusion bodies in lysosomes

ai
gm
@
Features of MPS
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 56 Chemistry and Metabolism of Carbohydrates

----- Active space ----- DIGESTION OF CARBOHYDRATES

Sites & Enzymes of Carbohydrate Digestion  00:01:20

Site Enzyme Bond/substrate hydrolyzed Digestion products

Salivary
Mouth • Hydrolyzes α-1-4 linkages 1. Oligosaccharides :
α-amylase
• Cannot digest : 2. Maltose :
- Terminal α-1-4 linkages 3. Maltotriose :
Pancreatic

om
Intestine - α-1-6 linkages 4. α-limit dextrins :
α-amylase

l.c
ai
gm
Disaccharidases :

@
1. Sucrase Sucrose Glucose + fructose
19
55
na

Intestine : maltase
Maltose Glucose + glucose
ee

Microvilli of subunit
m
ar

brush border 2. Isomaltase

um

Isomaltose : α 1-6 linkage Glucose + glucose

itk

cells
am

3. Lactase Lactose : β 1-4 linkage Galactose + glucose

|
w

4. Trehalase Trehalose Glucose + glucose

ro
ar
M
©

Note :
• Digestion in mouth : Short & incomplete (Completed in jejunum).
• Salivary α-amylase : Inactivated by acidic gastric juices.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Digestion of Carbohydrates 57

Applied Biochemistry 00:09:00 ----- Active space -----

Lactose Intolerance :
Types :
• Congenital : Lactase deficiency from birth.
• Acquired : D/t ↓ production of lactase (>6-7 y/o) D/t ↓ milk consumption.

Pathophysiology : Lactase
Lactose Glucose + galactose

Accumulation of lactose

Bacterial fermentation

om
l.c
Short chain fatty acids + gases.

ai
gm
@
C/f :
• Bloating. 19
55
na

• Abdominal pain.
ee

• Osmotic diarrhoea.
m
ar

• Flatulence.
um
itk

Rx :
am

• Curd : Lactobacilli + .
|

Rich in lactase.
w

• Yeast.
ro
ar
M

Sucrose Intolerance (Sucrase-Isomaltase Deficiency) :

©

Types :
• Primary : Congenital.
• Secondary : Acquired.

C/f : Manifests early in life.

• Bloating.
• Abdominal pain.
• Flatulence.
• Vomiting.
• Diarrhoea.

Rx :
• Sucrose free diet.
• Enzyme replacement therapy : Yeast-derived sacrosidase.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 58 Chemistry and Metabolism of Carbohydrates

----- Active space ----- GLUCOSE TRANSPORTERS

Glucose is hydrophilic.
G
Plasma membrane

Carrier protein
Cell
Types  00:01:29

om
Glucose transporters

l.c
ai
gm
@
Sodium dependent (SGLT) Sodium independent (GLUT)
19
55
SGLT :
na
ee

• Sodium - glucose symport.

m

• Unidirectional.
ar
um

• Secondary active transport :

itk
am

- Sodium : Along concentration gradient.

|

- Glucose : Against concentration gradient.

w
ro

Types :
ar
M
©

Types Location Function

Luminal side of intestine Absorption of glucose
SGLT-1
Proximal renal tubules
Re-absorption of glucose
SGLT-2 Proximal renal tubules

Clinical applications :
ORS : SGLT-1 in intestine is a sodium - glucose symport.
Renal glycosuria :
• Mutation in SLC5A2 gene Affects • Renal threshold : ↓.
SGLT-2 Defective reabsorption of • Oral glucose tolerance test (OGTT) :
glucose in renal tubules Excretion. Test to rule out renal glycosuria.
• Blood glucose : (N). • Renal threshold for excretion : 180 mg/dL.
• Benedict’s test : +ve. Note :
(Urine glucose : +ve). In DM, blood & urine glucose : ↑.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Glucose Transporters 59

Glifozines (Oral hypoglycemic agents : OHA) : ----- Active space -----

• SGLT-2 inhibitors.
• Prevents reabsorption of glucose Glucosuria ↓ Blood glucose.
• S/E : Urinary tract infection. Extracellular compartment
GLUT : ‘Pong’
• Bidirectional. Plasma (1) (2) Transports
glucose from high
membrane GLUT
• Along the concentration gradient. conc. to low conc.
‘Ping’
• Ping-pong mechanism. Intracellular compartment
Ping-pong mechanism
• Facilitated carrier mediated
transport.
• Passive process. simple diffusion

om
Facilitated carrier

l.c
Rate of

ai
mediated transport

gm
transport hyperbolic

@
plateau after
19
55
an initial phase
na

Conc. of solute
ee

Simple vs Facilitated diffusion

m

Types :
ar
um

Types Location Features

itk
am

Brain, placenta, kidney, • Widely distributed

GLUT-1
• Absorption of glucose
|

retina, colon, RBC

w
ro

• β-cells of pancreas β-cells Secrete insulin

ar

• Sinusoidal cells of liver High Km

M

GLUT-2 • Serosal surface of Low affinity

©

(In fed state) Glycolysis Pyruvate

intestine Liver
• Proximal renal tubules Stored as glycogen
• Neurons (Inside BBB) Low Km, High affinity :
GLUT-3
• Placenta, Kidney Obligatory requirement of glucose
Heart, skeletal muscles,
GLUT-4 Insulin dependent glucose transporter
adipose tissue
• Intestine : Luminal
GLUT-5 surface Fructose transporter
• Testis & sperm
Pseudogene (Gene not expressed),
GLUT-6 Spleen, leukocyte
No transporter function
Liver smooth endoplasmic
GLUT-7 Transport glucose in SER
reticulum (SER)
GLUT-8 Blastocyst -
Urate transporter
GLUT-9 Intestine & kidney
Defect in GLUT-9 1° gout
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 60 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Note :

Heart, skeletal muscle : Also gains energy from fatty acids.
Adipose tissues : Also gains energy from triacylglycerol & fatty acids.

Overview of Glucose Transporter  00:25:25

Intestinal cell 3Na+

SGLT-1 Na+-K+-ATPase pump
Na+
Glucose 2K+ Glut-2
(2° active transport)
Fructose
Fructose
GLUT G
5 Blood vessel

om
l.c
ai
gm
@
19
55
GLUT-2 GLUT-2
na
ee
m

stored as Production of ATP

ar

↑ Insulin
glycogen
um

↑ insulin glucagon ratio

Liver
itk

Pancreas
am

GLUT-4 GLUT-4
|

GLUT-4
w
ro
ar
M

Adipose
©

tissue

Skeletal
muscle Heart

↓ Blood glucose

Glycemic index :
Glucose > Fructose
Transported by SGLT-1 GLUT-5
Conc. gradient Against Along
Absorption Complete Incomplete

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycolysis : Part 1 61

GLYCOLYSIS : PART 1 ----- Active space -----

Glucose Metabolism 00:00:54

In fed state :
↑ Blood glucose
Entry via GLUT-2
Liver

Undergoes glycolysis
Forms
Pyruvate

om
In the presence Pyruvate
Link reaction

l.c
of O2 dehydrogenase

ai
gm
Acetyl CoA

@
19
55
Enters TCA cycle FADH2 and Enters electron transport ATP synthesis
na

NADH synthesis
ee

chain (ETC)
m

2) If excess glucose remains Glycogen synthesis.

ar
um

Glucose Glucose 6 phosphate Glycogen.

itk

3) If excess glucose still remains :

am
|

Entry via ↑ Glucose

w

GLUT-2
ro
ar
M

Pancreas Glucose 6 phosphate Forms excess Acetyl CoA

©

Insulin secretion Enters HMP shunt Undergoes fatty acid (FA) synthesis
pathway
↑ level of GLUT4 in : FA
• Heart Forms NADPH Glycerol
• Adipose tissue Triacyl glycerol (TAG)
• Skeletal muscle FA synthesis
Transported into blood via VLDL
Storage of excess glucose
Stored in adipose tissue
Glycolysis Pathway 00:07:30

AKA Embden Meyerhoff Parnas pathway.

Site :
Organ : All organs.
Organelle : Cytoplasm.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 62 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Significance :

• The only pathway that is both aerobic and anaerobic.
• RBC :
- Glucose is the only metabolic fuel in both fed & fasting state.
- Lacks mitochondria Anaerobic glycolysis.
- Glycolytic enzyme defect Hemolysis.
• Heart (Low glycolytic capacity) : • Skeletal muscle (High glycolytic capacity) :
Ischemia Exercise/Exertion
Hypoxia ↓ O2
Necrosis Anaerobic glycolysis
MI

om
AEROBIC GLYCOLYSIS

l.c
ai
gm
Overview :

@
Preparatory phase : 55
19
• ATP utilized. Glucose (6C)
na

• Stages :
ee

Splits into 2 Triose phosphates (3C)

m

- Stage of splitting.
ar
um

- Stage of phosphorylation. Dihydroxy acetone Glyceraldehyde 3

itk

phosphate (DHAP) phosphate

am

Pay off phase :

|

ATP
w
ro

• ATP is generated.
ar

Glyceraldehyde 3 Pyruvate
• Stages :
M

phosphate
©

- Oxidative phosphorylation. NADH

- Substrate level phosphorylation. Enters ETC
ATP synthesis
Preparatory phase :
Glucose
ATP Hexokinase/Glucokinase
ADP
Glucose 6 Phosphate
Phosphohexose isomerase
Fructose 6 Phosphate
ATP Phosphofructokinase (PFK )
ADP 1

Fructose 1,6 bisphosphate

Aldolase
DHAP Phosphotriose Glyceraldehyde 3 phosphate
isomerase

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycolysis : Part 1 63

Hexokinase vs. Phosphofructokinase : ----- Active space -----

Characteristics Hexokinase Phosphofructokinase

Regulatory/flux Rate limiting/committed/
1) Mediates
generating step bottleneck step
Commits glucose to Commits Fructose-1,6
2) Action
cellular metabolism - bisphosphate to glycolysis

Hexokinase vs Glucokinase :

Characteristics Hexokinase Glucokinase

1) Isoforms I, II, III, IV Hexokinase IV
2) Km value Low High

om
3) Affinity High Low

l.c
ai
Inducible by insulin

gm
4) Inducible/constitutive Constitutive
(Postprandial)

@
5) Inhibited by G6P Yes 19 No
55
na
ee

Pay off phase :

m

Glyceraldehyde 3 phosphate
ar
um

Pi NAD+ Glyceraldehyde 3 phosphate dehydrogenase

1
itk

NADH ETC 2.5 ATP

am

1,3 bisphosphoglycerate (1,3 BPG)

|
w

ADP
ro

2 ATP 1,3 BPG kinase

ar
M

3 phosphoglycerate (3 PG)
©

PG mutase
2 phosphoglycerate
Mg2+
H20 Enolase (Lyase)
Mn2+

Phosphoenol pyruvate (PEP)

ADP Pyruvate kinase
2 ATP
Pyruvate

1,3 BPG kinase: The only reversible kinase

1 Oxidative phosphorylation (The only step where inorganic phosphate (Pi) is added).
2 Substrate level phosphorylation.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 64 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Energetics :

• Glyceraldehyde 3 phosphate dehydrogenase : 5 ATP (from 2 NADH)
• 1,3 Bisphosphoglycerate kinase : 2 ATP
• Pyruvate kinase : 2 ATP
• Hexokinase : -1 ATP
• Phosphofructokinase : -1 ATP
Net ATP : 7 ATP

Note :
I) In RBC, NADH accumulation (D/t lack of mitochondria) is prevented by LDH :
LDH
NADH NAD+

om
2) Energetics of 1 Glucose undergoing aerobic oxidation :

l.c
ai
a) Aerobic Glycolysis : 7 ATP.

gm
b) 2x (Pyruvate PDH Acetyl CoA) : 2x2.5 ATP = 5 ATP.
@
c) 2x (TCA cycle) : 2 x 10 ATP = 20 ATP. 19
55
na

Net : 32 ATP.
ee
m
ar

ANEROBIC GLYCOLYSIS
um

Initial steps similar to aerobic glycolysis.

itk
am

Pyruvate
|

NADH Lactate dehydrogenase (LDH)

w

NAD+
ro
ar

Lactate
M
©

Energetics :
• 1,3 BPG kinase : 2 ATP
• Pyruvate kinase : 2 ATP
• Hexokinase : -1 ATP
• Phosphofructokinase : -1 ATP
Net ATP : 2 ATP

INHIBITORS OF GLYCOLYSIS

1) Iodoacetate : 2) Arsenate :
a) Glyceraldehyde 3 a) Glyceraldehyde 3
phosphate dehydrogenase. phosphate dehydrogenase.
b) ↓ availability of Pi.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycolysis : Part 1 65

3) Fluoride : ----- Active space -----

a) Enolase.
b) ↓ availability of Mg 2+, Mn 2+.

Note :
Vacutainer for blood glucose estimation (Grey colored) : Contains sodium fluoride
oxalate mixture.

CLINICAL APPLICATIONS

1) Pyruvate kinase deficiency :

• 2nd m/c enzyme deficiency.
• Causes hemolysis.

om
l.c
2) Aldolase deficiency :

ai
gm
↓ glycolysis Hemolysis.

@
Note : 19
55

M/c enzyme deficiency : G6PD deficiency.

na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 66 Chemistry and Metabolism of Carbohydrates

----- Active space ----- GLYCOLYSIS : PART 2

Rapaport Luebering Cycle  00:00:24

Bypass of glycolysis pathway.

AKA RL shunt/2,3-BPG shunt.
Takes place in RBC.
In RBC :
90% : Anaerobic glycolysis of Glucose Pyruvate Lactate
10% : RL shunt Glucose

om
Hexokinase

l.c
ai
Glucose-6-phosphate

gm
2 ATP used

@
19
Fructose-6-phosphate
55
na

Phosphofructokinase
ee

Fructose-1,6-bisphosphate
m
ar
um

Glyceraldehyde-3-phosphate
itk
am
|

1,3-BPG BPG mutase 2,3-BPG

w
ro

se
ADP 1,3- BPG ata
ar

p h
hos
M

2x kinase X
Gp
©

BP
ATP 2,3- H2O
Pi
3-PG

PEP
2 ATP Pyruvate kinase
Pyruvate

Lactase
Net ATP : Zero.
2,3- BPG :
• Shifts O2 dissociation curve to right ↓ Affinity of O2 to Hb Unloading of O2
in tissues.
• Leads to taut state/ low affinity state of Hb in tissues.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Glycolysis : Part 2 67

Regulation of glycolysis 00:07:37 ----- Active space -----

Hormonal regulation :

Fed state Fasting state

High insulin : glucagon ratio Low insulin : Glucagon ratio
Dephosphorylated enzymes Phosphyralated enzyme
Inactive PFK-2
Fructose-6-phosphate
PFK- II Fructose-2,6-bisphosphatase

Favours glycolysis Fructose-2,6-bisphosphate

Inhibit glycolysis
Activates

om
PFK-1 (Allosteric enzyme)

l.c
ai
gm
PFK II (Phosphofructokinase II) : Regulatory enzyme in glycolytic pathway.

@
19
55
Allosteric regulation :
na
ee

Regulatory enzymes of
m

Allosteric activator Allosteric inhibitor

ar

glycolysis
um
itk

Hexokinase - ATP (Product)

am

• Fructose-6-phosphate ATP
|
w

Phosphofructokinase - I (substrate) Citrate (Product)

ro

• 5’ AMP (substrate)
ar

Fr-6-P Fr-1,6-BP Low pH

M

• Fructose-2,6-bisphosphate
©

(D/t ↑ lactate)
Pyruvate kinase -
ATP (Product)

Applications in cancer 00:15:21

Warburg hypothesis :
By Otto Warburg in 1924.
Cancer cells +O High uptake of glucose.
2
↑Glucose → Lactate : Aerobic fermentation/Glycolysis
Net ATP production : 2
Mitochondrial intermediates :
• Acetyl CoA Biosynthetic pathway (Consumes ATP) Cachexia.
• Citrates

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 68 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Normal cell : Glucose Cancer cells :

Phosphoenol pyruvate
Isomeric forms of
pyruvate kinase PKM1 PKM2
PKM1 : 7 ATP PKM2 :
• Tetramer. • Dimer.
• High catalytic activity. • Low catalytic activity.
Pyruvate
Acetyl CoA Lactate

om
2 x 10 ATP 5 ATP 2 ATP

l.c
ai
gm
TCA cycle

@
Total : 32 ATP 19
Total : 2 ATP
55
na

To compensate :
ee

↑Glucose enters cancer cells Aerobic glycolysis

m
ar
um

PET screening of cancer cells :

itk

PET : Positron Emission Tomography.

am

Fluorodeoxy glucose administered Uptake by cancer cells ↑Concentration

|
w

in cells Inference : Active lesion.

ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Pyruvate Dehydrogenase 69

PYRUVATE DEHYDROGENASE ----- Active space -----

Fates of pyruvate : 1. Pyruvate dehydrogenase (PDH)

Lactate 2. Pyruvate carboxylase
NAD+ 3. Alanine amino transferase
(4) 4. Lactate dehydrogenase (LDH)
NADH
Alanine Transamination Pyruvate (1) Acetyl CoA
PLP (3)
NAD+ NADH
(2) ATP
Biotin, Co2 ADP

om
Oxaloacetate (4C)

l.c
ai
gm
Pyruvate dehydrogenase  00:04:17

@
Site: Mitochondria. 19
55
na

Transport : Pyruvate symporter (Cytoplasm Mitochondria).

ee
m

Reaction : Oxidative decarboxylation.

ar

CO2
um

Pyruvate Acetyl CoA

itk

(3C) (2C)
am

NAD+ NADH
|
w
ro

ETC
ar
M
©

ATP
PDH complex :
Multienzyme complex :
Pyruvate CO2
1. PDH.
2. Dihydrolipoamide transacetylase.
(1) 3. Dihydrolipoamide dehydrogenase.
Thiamine Hydroxyethyl TPP
pyrophosphate (TPP)

(2)
Oxidized lipoamide Acetyl lipoamide

FADH2 CoA
NAD
+ (3) (2)
Reduced Acetyl CoA
FAD
lipoamide
NADH
+H
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 70 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Enzyme Coenzyme

1. PDH TPP (Vit. B1)
2. Dihydrolipoamide transacetylase Lipoamide, CoA (Vit. B5)
3. Dihydrolipoamide dehydrogenase FAD, NAD+

Note :
Oxidative decarboxylation reactions :
1. PDH.
2. α Ketoglutarate dehydrogenase (TCA cycle).
3. Branched chain ketoacid dehydrogenase.

Regulation of PDH :
1. Hormonal regulation : 2. Allosteric regulation :

om
l.c
↑ Insulin: Glucagon ratio ↑ Acetyl CoA: CoA ratio OR

ai
gm
↑ ATP: ADP ratio OR

@
Covalent modification of PDH ↑ NADH: NAD+ ratio
19
55
na

Dephosphorylation Accumulation of products

ee
m
ar

Activation of PDH Inhibition of PDH

um
itk
am
|

Significance of PDH :
w

PDH
ro

1. Pyruvate Acetyl CoA (one way valve).

ar

(irreversible)
M
©

2. Cannot be circumvented by any other enzyme.

3. Acetyl CoA can never be converted to pyruvate Can never be a substrate
for gluconeogenesis.
4. Fat cannot be converted to glucose
• Exceptions : glycerol, propionyl CoA.
5. ↑Carbohydrates Glucose Pyruvate PDH Acetyl CoA Fatty acids (FA)
Glycerol

Triacyl glycerol (TAG)

(stored in adipose tissue)
6. Irreversible link between glycolysis and TCA cycle :

Glucose via glycolysis Pyruvate PDH Acetyl CoA

Link reaction

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Pyruvate Dehydrogenase 71

7. Chronic alcoholism : ----- Active space -----

Thiamine deficiency

↓ PDH activity ↓ α-ketoglutarate dehydrogenase activity

↓ NADH ↓ TCA cycle

↓ ATP
(energy depletion)

Clinical Application

om
PDH deficiency (M/c deficiency in the PDH complex)

l.c
a. Pyruvate PDH Acetyl CoA

ai
gm
@
Lactate 55
19
na
ee

Lactic acidosis
m

b. Glycolysis
ar

↓ oxidative pathways
um
itk

PDH in the brain

am
|

TCA cycle Neurological deficits

w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 72 Chemistry and Metabolism of Carbohydrates

----- Active space ----- GLYCOGEN METABOLISM

Branched polymer of α-D-glucose :

Linear : α 1-4 linkage

Branches : α 1-6 linkage
Functions :
Storage form of glucose d/t :

om
• Compact structure.

l.c
ai
• Multiple non-reducing ends Fast release of glucose.

gm
• Less osmolar :
@
- Glucose is hydrophilic 19
Attracts water into cell Cell lysis.
55
na

- Glycogen prevents cell lysis.

ee
m

Liver vs muscle glycogen :

ar
um
itk

Liver Muscle
am

Source of Blood glucose ATP during exertion

|
w

Total content Less Highest

ro
ar

% by tissue weight 10% 2%

M
©

Glycogen Metabolism 00:04:34

GLYCOGEN SYNTHESIS
Occurrence :
Well fed state Insulin Stored as glycogen.
Excess carbohydrates
Sites :
Liver & skeletal muscle.
Organelle: Cytoplasm.
Steps :
I. Synthesis of UDP glucose (Active glucose donor).
II. Synthesis of linear polymer.
III. Branching of linear polymer.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Glycogen Metabolism 73

Glycogenin : ----- Active space -----

• Primer for glycogen synthesis.
• Polypeptide : 37 kDa.
• Multiple tyrosine residues Undergoes Glycosylation.
I. Synthesis of UDP glucose :
Glucose
Hexokinase
Glucose 6-phosphate
Phosphoglucomutase (Isomerase)
Glucose 1-phosphate
Uridine triphosphate (UTP)
Pyrophosphate (PPi)

om
UDP glucose

l.c
ai
gm
@
II. Synthesis of linear polymer :
19
55
na

Glycogenin + (UDP glucose)n 7-8 glucose residues added

ee
m
ar
um

(UDP glu)n
itk

Glycogen synthase
am

UDP
|

11 glucose residues
w
ro
ar
M

III. Branching of the linear polymer : Stops synthesis

©

Branching
Branching enzyme :
α-1→4, 1→6
glucan transferase
Branching at multiple points
α 1-4 linkage
α 1-6 linkage Complex structure of glycogen

GLYCOGENOLYSIS
• Early fasting Low insulin : Glucagon Glycogenolysis of hepatic glycogen.
(4- 16 hrs without food)
• Glycogen stores depleted by 16-18 hrs.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 74 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Sites :

Liver, muscle.
Organelle:
• Cytoplasm.
• Lysosomes (1-2%) Enzymes : Acid α-glucosidase/acid maltase.
• Smooth endoplasmic reticulum.
Steps :
Glycogen phosphorylase
Pyridoxal phosphate
Debranching enzyme :
(Pi)n (Glu 1 PO4)n
Bifunctional enzyme
A. α 1 41 4 glucan transferase

om
cuts trisaccharide residue

l.c
ai
B. α 1 6 glucosidase

gm
@
19
55
B A
Linear polymer
na
ee
m

Free glucose
ar
um

Conversion of glucose-1-phosphate to free glucose :

itk

Glucose 1 phosphate
am

Phosphoglucomutase • Glycogen synthesis

|
w

G-6-phosphate • Glycogenolysis
ro
ar
M
©

In smooth endoplasmic In muscle G-6-Pase is absent

reticulum of liver (& in adipose tissues)

• Gluconeogenesis G-6-p enters

G6P • Glycogenolysis Glycolysis
Glucose 6 O2
phosphatase
T1 Glucose T2 + -
• Transporters: T1, T2 Anaerobic glycolysis (Exercise)
Aerobic glycolysis
• G-6-P not immediately converted to free glucose

ATP produced : Utilisation :

1. 1,3 BPG = 2 ATP 1. PFK = 1 ATP
PK: pyruvate kinase 2. Pk = 2 ATP (HK is bypassed)
PFK: phosphofructokinase Total: 3 ATP produced from
HK: hexokinase muscle glycogen
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Glycogen Metabolism 75

Regulation of Glycogen Metabolism 00:31:59 ----- Active space -----

HORMONAL REGULATION
Fasting state :
Liver
Glucagon Epinephrine (Flight, fight response)
Muscle
Attach GPCR
Muscle
Release of G-protein
+ (+) Insulin
Adenylyl cyclase PDE
ATP cAMP 5’ cAMP
Block cAMP dependent
cAMP dependent pathway

om
protein kinase A

l.c
ai
Glycogen Glycogenolysis

gm
synthesis

@
P P
19
Glycogen Glycogen
55
Glycogen Glycogen synthase
na

synthase phosphorylated phosphorylase phosphorylase

ee

(Active) (P attached) (Inactive) (Active)

m
ar
um
itk

+
am

Insulin Phosphatase
|
w
ro

Activates phosphodiesterase
ar
M

Action of insulin :
©

• Dephosphorylation of glycogen synthase & phosphorylase.

• Activation of phosphodiesterase.

Regulation at various conditions :

Stress/exercise : Extreme anoxia :
Myosin ATPase
Ca2+ from sarcoplasmic reticulum Release of 5’ AMP

Ca2+ - Calmodulin dependent kinase Binds with glycogen phosphorylase

Glycogen phosphorylase Phosphorylated glycogen phosphorylase
+P Active without phosphorylation
Starts
Active glycogenolysis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 76 Chemistry and Metabolism of Carbohydrates

----- Active space -----

GLYCOGEN ALLOSTERIC REGULATION
A) Liver :
Glycogen

Glucose 6-P
Glucose 6-P
ATP
Glycogen Glycogen Glycogen
Glycogenolysis
phosphorylase synthase synthesis

Glucose

om
l.c
ai
Glucose-1-phosphate

gm
@
B) Muscle : 19
55
na
ee
m

Glycogen
ar
um

Glucose 6-P
itk

Glucose 6-P
am

ATP
|
w

Glycogen
ro

Glycogenolysis Glycogen Glycogen

ar

synthesis
M

phosphorylase synthase
©

AMP

Ca2+

Glucose-1-phosphate

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycogen Storage Disorders 77

GLYCOGEN STORAGE DISORDERS ----- Active space -----

Liver glycogen Muscle glycogen

Function Source of blood glucose Source of ATP for muscles
Symptoms d/t defect Fasting hypoglycemia Exercise intolerance

Liver GSD  00:03:44

Type Name/Features Enzyme Defect

Ia Von Gierke’s Glucose 6 phosphatase : M/c GSD
Features : Transporter of glucose 6

om
Ib Type Ia + Neutropenia + Recurrent phosphate in smooth endoplasmic

l.c
ai
bacterial infection reticulum

gm
@
Cori’s/Forbes disease/Limit
III
dextrinosis 19
Debranching Enzyme
55
na

IV Anderson disease/Amylopectinosis Branching enzyme

ee
m

VI Her’s disease Hepatic glycogen phosphorylase

ar
um
itk

Muscle GSD  00:06:35

am
|
w

Hypertrophic cardiomyopathy (HCM)

ro
ar

Present Absent
M
©

Type V/Mc Ardle’s

Type II/Pompe’s disease
disease Type VII/
GSD (Also, Lysosomal storage
• M/c Muscle GSD. Tarui’s disease
disorder)
• M/c GSD in adolescent.
• Acid maltase
Enzyme AKA Muscle glycogen Erythrocyte/
Defect - Acid α glucosidase phosphorylase muscle PFK-1
- Acid α 1-4 glucosidase

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 78 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Features of Various GSD  00:09:26

Von - Gierke’s disease/TI GSD :

Glycogen
P Glycogen
G1PO4 Phosphorylase
Other glucose source : OAA G6PO4 HMP shunt pathway
Glucose 6 Phosphatase
Ribose

X
Glucose
↓ Glycogenolysis
Purine
Catabolism
Fasting hypoglycemia
Uric acid
OAA : Oxaloacetic acid.

om
l.c
Hyperuricemia

ai
gm
Triacyl glycerol

@
19
55
Glucose 6 phosphate
na

Fatty acid Glycerol

ee

Excess acetyl CoA (-)

m

Oxidation Pyruvate
ar

↑ Acetyl Co-A (2C)

um

Pyruvate dehydrogenase
itk
am

↓ OAA Acetyl Co-A ↑ Lactate

+
|

↓ OAA d/t Fatty acid,

w

(4C)
ro

depletion (in cholesterol synthesis Lactic

ar

↓ TCA cycle ↑ Ketone body

M

gluconeogenesis) acidosis
©

synthesis Hyperlipidemia

Ketosis : Rothera’s
test +ve
Clinical features :
• Chubby cheeks. • Renomegaly (d/t fat deposition
• Thin extremities. around kidney).
• Massive hepatomegaly. • No splenomegaly.

Investigations :
• S. Glucose ↓↓. • Liver enzymes : AST, ALT = Normal.
• Rothera’s test +ve. • Liver biopsy : Normal glycogen ↑.
• S. uric acid ++. • IV glucagon challenge : No rise in
• S. lactate ++. blood glucose.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Glycogen Storage Disorders 79

Cori’s disease/T3GSD : ----- Active space -----

Debranching enzyme
↑ Limit dextrin X Linear polymers.

Clinical features :
• Hypoglycemia (Moderate).
• Accumulation of abnormal glycogen.
• Non-progressive cirrhosis.

Investigation :
• S. Glucose ↓. • Liver enzyme : AST, ALT = ↑↑.
• Rothera’s test -ve. • Liver biopsy : Accumulation of
• S. uric acid abnormal glycogen (limit dextrin).
Normal

om
• S. lactate

l.c
ai
IV glucagon challenge :

gm
IV glucagon Promotes glycogenolysis.
@
19
55
Rise of blood glucose after bolus of IV glucagon :
na
ee
m

Von Gierke’s Cori’s

ar
um

Fed Rise in blood glucose

No rise in blood glucose
itk

Fasting No rise in blood glucose

am
|

Anderson’s disease/Type IV GSD/Amylopectinosis :

w
ro

Accumulation of linear polymers (Amylopectin like substance).

ar
M
©

Clinical features :
• Hypoglycemia.
• Hepatomegaly d/t accumulation of amylopectin like material (abnormal
glycogen).
• Progressive cirrhosis Liver failure :
- Portal hypertension.
- Oesophageal varices.
- Death around 5yrs (d/t liver failure).

Investigation :
• S. glucose ↓. • S. lactate : Normal.
• Uric acid • Liver enzymes : ALT, AST = ↑↑.
• Ketone bodies Normal • Liver biopsy : Amylopectin-like material.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 80 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Pompe’s Disease/Type II GSD :

• Also a lysosomal storage disorder.
• Defect in lysosomal degradation of glycogen.

Clinical features :
• Feeding difficulties. • Hypertrophic cardiomyopathy.
• Failure to thrive. • Death around 2 yrs, (D/t cardiac failure).
• Hypotonia.

Investigation :
• S. glucose : ↓. • S. lactate : Normal.
• Uric acid • Liver enzymes : Normal.
Normal
• ketone bodies • S. creatine kinase, LDH : ↑

om
l.c
McArdle’s disease :

ai
gm
Clinical features :

@
• Exercise intolerance. 19
55

• Second wind phenomenon : Ability to exercise after a period of rest.

na
ee

• Rhabdomyolysis.
m
ar

• Myoglobinuria.
um

Muscle
itk

Glycogen X
am

phosphorylase
|

Glycogen Limit dextrin

w
ro
ar

Pi G1PO4
M
©

G6PO4 Pyruvate Lactate

Investigation :
• S. glucose during exercise : ↓.
• S. lactate : ↓.
• S. creatine kinase, LDH : ↑↑.
• Liver enzymes : ALT, AST : Normal.

Tarui’s disease/Type VII GSD :

Glycogen PFK-1 X Glycolysis Pyruvate Lactate.

Clinical features :
• Hemolysis (+). • Rhabdomyolysis.
• No second wind phenomenon. • Myoglobinuria.
• Exercise intolerance.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Glycogen Storage Disorders 81

Type 0 GSD : ----- Active space -----

• Defect in glycogen synthase.
• No accumulation of glycogen.
• No hepatomegaly.
• Death in intrauterine period.

Fanconi Bickel syndrome :

Well fed s tate : X
GLUT-2
Glucose
Glycogen

Defect in GLUT-2 Defect in glycogen synthesis

om
Other features of GSD :

l.c
• Liver GSD + Neurological manifestation : Type IV.

ai
gm
- Anterior horn cells, brain cells are affected.

@
• Liver GSD + Abnormal glycogen : Type III, IV. 19
55
na

Summary :
ee
m
ar

Rothera’s S.uric Creatine

um

S.Glucose S.lactate ALT/AST

test acid kinase
itk
am

Von Gierkes ↓ + ↑ ↑ N
N
|
w

Pompe’s ↓/N ↑
ro
ar

Cori’s N
M

↓ ↑ N
©

Anderson
- N
McArdle N/↓ ↓ ↑
VI ↓ N N N
VII N ↓ ↑

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 82 Chemistry and Metabolism of Carbohydrates

----- Active space ----- GLUCONEOGENESIS

Introduction :
• Source of blood glucose during 16-48 hrs of fasting.
• Low Insulin : Glucagon ratio.

Definition :
The process of synthesis of glucose from non-carbohydrate substrates.

Site :
Organ : Liver, kidney.

om
Organelle : Cytoplasm, Mitochondria, Smooth endoplasmic reticulum(SER).

l.c
ai
gm
Substrates  00:03:44

@
19
55
1) Glucogenic amino acids : Mainly alanine.
na
ee

Glucose Glucose
m
ar
um

Pyruvate
itk
am

Pyruvate
|
w

Transamination
ro

Alanine
ar

Liver
M

Alanine
©

Skeletal muscle
Glucose Alanine/ Cahill cycle
2) Lactate :
Source : Skeletal muscle and RBC.
Glucose
Glucose

Pyruvate
Pyruvate

Lactate Liver
Lactate
Skeletal muscle
Glucose lactate/ Cori’s cycle

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Gluconeogenesis 83

3) Glycerol : ----- Active space -----

Triacyl glycerol (TAG) Fatty acid (FA)
Glycerol
Glycerol kinase
Glycerol-3-phosphate

Glycerol-3-phosphate dehydrogenase
Dihydroxyacetone phosphate

Glucose Glyceraldehyde-3-phosphate

om
4) Propionyl CoA :

l.c
ai
Source : Odd chain FA.

gm
@
Propionyl CoA carboxylase 19
Propionyl CoA (3C) D-Methyl malonyl CoA (4C)
55
ATP, Biotin, CO2
na
ee

Racemase
m

Methyl malonyl CoA mutase

ar

Succinyl CoA L- Methyl malonyl CoA

um

Vit. B12
itk
am
|

Oxaloacetate Glucose
w
ro
ar

Note :
M
©

1. Acetyl CoA is not a substrate of gluconeogenesis.

2. Vitamin B12 deficiency ↑Serum methyl malonyl CoA Differentiate Vit.
B12 and Folic acid deficiency.

Enzymes  00:11:43

• Reversible enzymes of glycolysis : Common to gluconeogenesis.

• Irreversible enzymes of glycolysis: Reversed by key enzymes of
gluconeogenesis.
Irreversible enzymes of glycolysis Key enzymes of gluconeogenesis
Glucose Cytoplasm
Hexokinase/ Glucose-6-phosphate Glucose-6-phosphatase Glucose
Glucokinase
Glucose-6-phosphate SER H2O Pi T2
T1
G-6-P Glucose
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 84 Chemistry and Metabolism of Carbohydrates

----- Active space -----

Irreversible enzymes of glycolysis Key enzymes of gluconeogenesis

Fructose-6-phosphate (F-6-P) Fructose- 1,6- bisphosphate (F-1,6-BP)
H20
Phosphofructokinase (PFK1 ) F-1,6-Bisphosphatase
Pi
Fructose-1,6-bisphosphate (F-1,6-BP) Fructose-6-Phosphate (F-6-P)
Phosphoenol pyruvate (PEP)
Mitochondria
Pyruvate kinase
Pyruvate Pyruvate carboXylase Oxaloacetate (OA)
Pyruvate ATP (4C)
(3C) Biotin ADP
Aspartate Malate
CO2

om
Cytoplasm

l.c
Malate-aspartate shuttle Aspartate Malate

ai
gm
@
OA (4C)
19
55
GTP PEP carboxykinase
na

1. Decarboxylation
ee

GDP 2. Phosphorylation
m
ar
um

PEP (3C)
itk
am

Regulation  00:22:38
|
w
ro

1. Hormonal Regulation :
ar
M

Fasting state:
©

• ↓Insulin : Glucagon ratio.

• Enzymes are in phosphorylated state : Active.

2. Allosteric regulation :
a. Acetyl CoA : Allosteric activator of pyruvate carboxylase (Rate limiting
enzyme of gluconeogenesis).
b. Fructose-2,6-bisphosphate :
- Allosteric activator of PFK1 (Enzyme of glycolysis).
- Allosteric inhibitor of Fructose-1,6-bisphosphatase (Rate limiting enzyme
of gluconeogenesis).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Gluconeogenesis 85

3. Reciprocal regulation of glycolysis and gluconeogenesis : ----- Active space -----

a. Well-fed state :
Fructose-6-phosphate (F-6-P) F-1,6-BP
PFK2

F-2,6-BP (-) F-1,6-bisphosphatase

Enters glycolysis F-6-P No gluconeogenesis

b. Fasting state :
PFK2 is inactive No inhibition of F-1,6-BPase
F-1,6-BP

om
l.c
ai
F-6-P

gm
@
Enters gluconeogenesis 19
55
na

Clinical applications :
ee
m
ar

1. Raw egg :
um
itk

Raw egg Contains avidin Inhibits biotin ↓Activity of pyruvate carboxylase

am

(Rate limiting enzyme of gluconeogenesis)

|
w
ro
ar

↓Gluconeogenesis
M
©

2. Biguanides :
Biguanides Inhibits Pyruvate carboxylase (PC)

Pyruvate Oxaloacetate

Lactate

Lactic acidosis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 86 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Pathway  00:31:00

2 x Lactate Lactate Pyruvate Pyruvate carboxylase OA PEP carboxykinase PEP

dehydrogenase
ATP ADP GTP GDP

3-PG 2-phosphoglycerate (2-PG)

ATP
1,3-BPG kinase

ADP
1,3- bisphosphoglycerate (1,3-BPG)

om
l.c
ai
Glyceraldehyde-3-phosphate DHAP

gm
@
19
55
na

F-1,6-BP F-1,6-BPase F-6-P G-6-P G-6-Pase Glucose

ee
m

P� H2O P� H2O
ar
um
itk
am

Note :
|
w

NADH utilization /= ATP utilization.

ro
ar

NADH production = ATP production.

M
©

Energetics :
• Pyruvate carboxylase : 1 ATP.
• PEP carboxykinase : 1 ATP (GTP)
• 1,3- Bisphosphoglycerate kinase : 1 ATP.
• Total : 3 ATP (Per molecule of lactate).
• Net consumption : 2 x 3ATP = 6 ATP (2 molecules of lactate).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minor Metabolic Pathways 87

MINOR METABOLIC PATHWAYS ----- Active space -----

Galactose  00:00:10

Sources : Functions :
Lactose (Milk sugar). 1. Conversion to glucose and glycogen.
2. Synthesis of lactose, GAG, proteoglycans.

Metabolism :
Site : Liver, fibroblasts, RBC.

om
Pathway : Galactose

l.c
(1)

ai
gm
Galactose 1-phosphate UDP Glucose 1. Galactokinase
(2) (3) (3)
@
Glucose 1-phosphate UDP Galactose 2. Galactose 1-phosphate uridyl
19
55
transferase (GALT)
na

Glucose 6 phosphate : GAG, Lactose

ee

3. UDP Hexose epimerase

m

Used for :
ar

• Glycolysis
um
itk

• Glycogen synthesis
am
|
w

Classic galactosemia :
ro
ar

Deficiency of galactose 1-phosphate uridyl transferase.

M
©

Biochemical defect :
↓ Galactose 1-phosphate uridyl transferase
after it is Galactose Aldose Dulcitol/Galactitol
Accumulation of Galactose 1-phosphate
depleted ↑ Galactose deposition in reductase (Osmotically
the eyes active)
↓ Inorganic P04 (Pi)
Oil drop cataract

↓ ATP ↓ Phosphorylation of
Glycogen phosphorylase Rate-limiting enzyme (RLE)
Effects on the brain in glycogenolysis
↓ Glycogenolysis
Intellectual disability

Hepatomegaly
Fasting hypoglycemia Accumulation of glycogen Liver failure
in the liver Oil drop cataract
Jaundice

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 88 Chemistry and Metabolism of Carbohydrates

----- Active space -----

Clinical features :
• Age of onset : First 2 weeks of life (Precipitated by breastfeeding).
• Feeding difficulties, failure to thrive.
• Liver failure, vomiting, jaundice, hepatomegaly.
• Intellectual disability.
• Neonatal sepsis (M/c organism : E.coli).
Investigations :
Test Result Inference
1. Urine Benedict’s test Positive Reducing sugar (+)
2. Glucose oxidase test Negative Not glucose
3. Mucic acid test (Specific test) Positive Galactose (+)
Newer tests : Enzyme studies, genetic mutation test.

om
l.c
Treatment :

ai
gm
1. Stop breastfeeding.

@
2. Lactose free diet upto 4-5 years. 19
55
After 4-5 yrs : Activation of
na

Galactose 1 phosphate
ee

Galactose 1 phosphate pyrophosphorylase

m

Galactose
ar

Non Classic galactosemia :

um
itk
am

Enzyme defect Clinical features

|

Galactokinase Oil drop cataract

w
ro

UDP hexose epimerase Benign, variable presentation

ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minor Metabolic Pathways 89

Fructose  00:19:30 ----- Active space -----

Sources :
1. Sucrose (Cane sugar). 3. Fruit juices.
2. Honey. 4. Glucose (Through polyol pathway).

Metabolism :
Site : Liver.
Pathway: Fructose (1) Fructose 1-phosphate

(2) 1. Fructokinase
Glyceraldehyde Dihydroxy acetone Deficiency Essential fructosuria
phosphate (DHAP) 2. Aldolase B

om
Deficiency H ereditary fructose

l.c
intolerance

ai
gm
Glyceraldehyde-3-phosphate

@
19
55
Pyruvate
na
ee

No regulatory step in fructose metabolism : Fructose Pyruvate Acetyl CoA Fatty acid (FA)
m
ar
um

Dyslipidemia ↑ TAG
Note :
itk
am

1. Aldolase A : Found in muscle; part of glycolysis.

|

2. PFK-1 : Regulatory step in glucose metabolism.

w
ro
ar

Hereditary fructose intolerance :

M
©

Deficiency of Aldolase B.

Biochemical defect :
↓ Aldolase B ↑ Fructose excreted d/t No cataract
r
afte ion low renal
let
↑ Fructose 1 phosphate dep threshold
↓ availability of Pi

same clinical effects as

galactosemia

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 90 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Investigations :

Test Result Inference
1. Urine Benedict’s test Positive Reducing sugar
2. Glucose oxidase test Negative Not glucose
3. Seliwanoff’s test/Rapid furfural test Positive Fructose(+)
(Specific tests)
Newer tests : Enzyme studies, genetic mutation test.

Clinical features :
• Age of onset : 6 months (Precipitated by weaning diet).
• Similar to galactosemia, but no cataract.

Treatment :

om
l.c
Sucrose free diet.

ai
gm
Note :
@
Galactosemia vs Hereditary fructose intolerance : 19
55
na

Galactosemia HFI
ee
m

Age of onset 2 weeks 6 months

ar
um

Cataract Present Absent

itk
am

Enzyme defect GALT Aldolase B

|

Accumulating substance Galactose 1 Po4 Fructose 1 Po4

w
ro

Treatment Lactose free diet Sucrose free diet

ar
M

Duration of Rx up to 4-5 yrs age lifelong

©

Essential fructosuria :
Defect in fructokinase.

Biochemical defect :
Defect in fructokinase ↑ Fructose Excreted d/t Fructosuria
low renal
threshold
Hexose Monophosphate Pathway  00:34:18

• AKA HMP shunt/6 Phosphogluconate/Pentose phosphate/Dickens-Horecker

pathway.
• No ATP generation.
Site : Cytoplasm.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minor Metabolic Pathways 91

PATHWAY ----- Active space -----

Oxidative Phase Non oxidative
• Irreversible Phase
• Generates • Reversible
NADPH (M/c • Generates
source) Pentoses

Oxidative Phase :
Glucose 6 phosphate (G6P) H2O2 GSH NADP+
NADP+ (1) (2)
(1) H2O GS SG NADPH
NADPH
6 Phosphogluconate 1. Glutathione peroxidase (Needs Se).
NADP+ 2. Glutathione reductase (Needs NADPH) :
(2)

om
NADPH CO2 GSSG : oxidised glutathione.

l.c
Ribulose 5 phosphate GSH : Reduced glutathione.

ai
gm
@
1. G6P dehydrogenase (G6PD) : RLE 2. Maintains the reduced state of iron in
2. 6 Phosphogluconate dehydrogenase hemoglobin : 19
55
na

Iron Oxidised state Methemoglobin Cyanosis

ee
m
ar

3. Reductive biosynthesis of FA and steroids :

um

Site : liver, adrenal cortex, gonads,

itk
am

adipose tissue.
|
w
ro

Functions of NADPH :
ar
M

1. Free radical scavenging :

©

• Prevents hemolysis (Maintains RBC membrane integrity).

• Prevents lens cataract.
Non - oxidative phase :
Function : Note :
Synthesis of pentoses (Used in DNA Erythrocyte transketolase activity :
synthesis) : indicator of thiamine levels.
• Major source.
• Sites : Organs with rapid cell
turnover :
1. Mucosal cells 2. Skin.
of intestine. 3. Bone marrow.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 92 Chemistry and Metabolism of Carbohydrates

----- Active space -----G6PD DEFICIENCY

Biochemical defect :
↓ G6PD Inability of haem
to combine with globin
↓ NADPH
Cross linking of globin (D/t
reactive sulfhydryl groups)
↓ Free radical Oxidation of iron in
scavenging hemoglobin Precipitation of globin
Hemolysis ↑ Methemoglobin
Heinz bodies Splenic circulation Bite cells
Cyanosis • Blue colour on supravital staining (Crystal violet)
Anemia Jaundice

om
Clinical Features :

l.c
ai
• X linked recessive disorder.

gm
@
• M/c enzyme deficiency in humans.
• Common in the Middle East and Mediterranean 19
55
na

regions and areas with ↑ prevalence of

ee
m

P. falciparum.
ar
um

Precipitating factors : Primaquine, chloroquine, Sulfa

itk

drugs, Fava beans (Favism).

am

Presentation : Hemolytic anemia, jaundice,

|
w

methemoglobinemia.
ro
ar

Bite cells
M
©

Uronic Acid Pathway  01:02:08

Introduction :
Oxidative pathway for glucose.

Site :
• Organ : Liver. • Organelle : Cytoplasm.

Functions :
1. Uronic acid synthesis : 2. Synthesis of pentoses : 3. Synthesis of ascorbic
Glucuronic acid : Minor source. acid :
a. Conjugation of Not seen in humans and
bilirubin. higher primates (Absence
b. Synthesis of GAGs. of L-gulonolactone
oxidase).
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Minor Metabolic Pathways 93

Essential pentosuria : ----- Active space -----

Benign.
Deficiency : Xylitol dehydrogenase AKA Xylulose reductase.

Biochemical defect : Investigations :

↓ Xylitol dehydrogenase 1. Benedict’s test : Positive.
2. Bial’s test : Positive (Specific to pentoses).
↑ L Xylulose

Urinary excretion

Polyol Pathway  01:05:26

om
Sorbitol

l.c
dehydrogenase
Glucose Aldose reductase

ai
Sorbitol Fructose

gm
@
NADPH NADP+ NAD+ NADH 19
55
na
ee

Significance :
m

1. Diabetes Deposition of sorbitol :

ar
um

• Lens Diabetic cataract.

itk

• Nerves Diabetic neuropathy.

am

• kidneys
|

Diabetic nephropathy.
w
ro

2. Seminal vesicle : Fructose is important for sperm motility.

ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 94 Chemistry and Metabolism of Carbohydrates

----- Active space ----- REGULATION OF BLOOD GLUCOSE

Significance of blood glucose :

• Metabolic fuel for vital organs like the brain.
• Regulated by finely tuned homeostatic mechanisms.

Normal levels :
• Fasting blood sugar (FBS) : 70-100 mg/dL.
• 2 hour post prandial blood sugar (PPBS) : ≤140 mg/dL.

Sources of Blood Glucose

om
00:03:10

l.c
ai
gm
Stage Time period Source

@
Well fed state Within 1-4 hours of food intake Dietary glucose
19
55
Hepatic glycogenolysis : Glycogen
na

Early fasting 4-16 hrs without food intake

stores depleted within 16-18 hours
ee
m

Fasting 16-48 hrs without food intake Gluconeogenesis

ar
um

Fatty acid oxidation

itk

Prolonged fasting/
am

2-5 days without food intake

starvation Ketone body synthesis
|
w
ro
ar

Prolonged starvation >5 days without food intake Muscle proteolysis

M
©

Hormonal Regulation 00:08:00

Mechanism of normalizing
State Blood glucose levels Hormone
blood glucose levels
β cells of pancreas • ↑Glucose uptake by ↑GLUT 4
Well fed state ↑
• ↑Glucose utilization
Insulin
α cells of pancreas • ↓Glucose uptake by ↓GLUT 4
Fasting state ↓ • ↑Glucose release
Glucagon • ↑Glucose synthesis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Regulation of Blood Glucose 95

Classification : ----- Active space -----

Hyperglycemic hormones : Hypoglycemic hormone :

• Glucagon. Insulin.
• Epinephrine.
• Glucocorticoids.
• Growth hormone.
• ACTH.
• Thyroxine.

Well-Fed State - ↑ Insulin : Glucagon Ratio 00:11:25

Blood Glucose Regulation by Insulin :

om
↑Blood glucose levels

l.c
ai
gm
β cells of pancreas
@
19
55
Insulin : Activation of various pathways by
na
ee

dephosphorylation of enzymes (High insulin-glucagon ratio)

m
ar
um

+ -
itk

↑GLUT4
am
|

↑Glucose uptake by : Pathways Pathways

w
ro

• Heart. utilizing glucose generating glucose

ar
M

• Skeletal muscle.
©

↑Glucose utilization
• Adipose tissue.

Covalent modification by insulin : Goal ↓Blood glucose.

Metabolic pathway Effect of insulin d/t dephosphorylation
Glycolysis ↑
Glycogen synthesis ↑
Glycogenolysis ↓
Gluconeogenesis ↓
↑
+ Glycerol
↑PDH
Lipogenesis Excess glucose Pyruvate Acetyl CoA Fatty acid

Stored as TAG

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 96 Chemistry and Metabolism of Carbohydrates

----- Active space ----- ORGAN MAPPING

Effects on organs upon release of insulin from the β cells of pancreas due to
↑blood glucose.

Glucose
en Glycolysis
Glycog
sis
synthe G6PO4

Glycogen
Pyruvate

PDH

om
l.c
Fatty acid Acetyl CoA TCA

ai
gm
Lipogenesis + Glycerol
@
19 Cells of :
55
TAG
na

: Adipose.
ee
m

: Liver & skeletal muscle.

ar
um
itk

: Common to all 3.
am
|
w
ro

Organ Glucose uptake via Effect on metabolic pathways

ar
M

• Glycogen synthesis
©

• Glycolysis
Liver GLUT 2
• PDH (Pyruvate dehydrogenase)
• TCA cycle
• Glucose uptake by ↑GLUT 4
• Glycolysis
Adipose tissue GLUT 4
• PDH
• Fatty acid synthesis & ↑lipogenesis
• Glucose uptake by ↑GLUT 4
• Glycolysis
Skeletal muscle GLUT 4 • PDH
• TCA cycle
• Glycogen synthesis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Regulation of Blood Glucose 97

Fasting (Post-Absorptive) State : ↓Insulin : Glucagon Ratio 00:23:39 ----- Active space -----

Blood Glucose Regulation by Glucagon :

No dietary glucose

α cells of pancreas

Glucagon : Activation of various pathways by phosphorylation of enzymes

(Low insulin-glucagon ratio)

↓Activity of GLUT4 • ↓Glucose utilization.

• ↑Glucose generation.

om
↓Glucose uptake.

l.c
ai
gm
@
Covalent modification by glucagon : Goal 19
↑Blood glucose.
55
na
ee

Metabolic pathway Effect of glucagon d/t phosphorylation

m

Glycolysis
ar

↓
um

Glycogenolysis Early fasting (4-16 hrs) : ↑

itk
am

Gluconeogenesis
|

(Non-carbohydrate Glucose) Fasting (16-18 hrs) : ↑

w
ro

substrates
ar
M

Prolonged fasting/Starvation (>2 days) : ↑

©

Stored TAG
Glycerol
↑Hormone sensitive lipase
Fatty acid
Lipolysis
β oxidation ↑Lipolysis
Acetyl CoA

Ketone body synthesis : Metabolic fuel

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 98 Chemistry and Metabolism of Carbohydrates

ORGAN MAPPING
----- Active space -----
Effects on organs upon release of glucagon from α cells of pancreas d/t ↓ blood
glucose.
Adipose : Skeletal muscle : Liver :

Lipolysis : Glycogen
Stored TAG ↑Glycogenolysis
Glycogen
↑ HSL Glycerol G6PO4
Glycogenolysis
Fatty acid Glycolysis
Glucose Released into blood
β oxidation Pyruvate
Gluconeogenesis
Acetyl CoA
Alanine Non-carbohydrate

om
substrates

l.c
Ketone body synthesis (Gluconeogenic

ai
gm
substrate)

@
19
55
na

Note : End product of glycogenolysis in skeletal muscle is G-6-PO4 & not glucose
ee
m

due to absence of glucose-6-phosphatase.

ar
um

Organ Effect on metabolic pathways

itk
am

• Glycogenolysis
Liver
|

• Gluconeogenesis
w
ro
ar

• GLUT4
M

• Glucose uptake
©

Adipose tissue
• HSL (Hormone sensitive lipase)
• Lipolysis
• GLUT4
Skeletal muscle • Glucose uptake
• Glycogenolysis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Diabetes Mellitus : Part 1 99

DIABETES MELLITUS : PART 1 ----- Active space -----

Diabetes mellitus :
• ‘Diabetes’ (Aretaeus of Cappadocia) : “To siphon”.
• ‘Mellitus’ (Thomas Willis) : “Sweet urine”.
• Metabolic disorder with hyperglycemia d/t absolute/relative insulin deficiency.
• Complex interaction b/w genetic & environmental factors.

Classification of Diabetes Mellitus (DM) 00:03:36

CLASSIFICATION

om
l.c
ai
gm
Type I DM Type II DM Diabetes prone state Secondary to other causes :

@
19 • Hyperthyroidism.
55
• Pancreatic disease.
na
ee

• Drug induced.
m
ar

• Endocrinopathies :
um

Cushing’s ds, acromegaly etc.

itk
am

Gestational DM : Impaired glucose Impaired fasting

|
w

Hyperglycemia diagnosed tolerance. glycemia.

ro
ar

during pregnancy.
M
©

Type 1 v/s Type 2 DM :

Type 1 DM Type 2 DM
↓ Secretion of insulin by ↓ Biological response to insulin :
Etiology
β cells of pancreas Insulin resistance
Onset < 30 yrs > 40 yrs
Insulin dependence + -
(Obsolete criteria) Insulin dependent DM (IDDM) Non-insulin dependent DM (NIDDM)
Ketosis Prone Less prone
Genetic predisposition Usually - Family h/o +
Circulating antibodies Autoantibodies + -
Plasma insulin levels ↓ ↑

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 100 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Metabolic Derangements in Diabetes Mellitus 00:09:50

Carbohydrate Metabolism :
• ↓ Glucose uptake
+ Hyperglycemia
• ↓ Glucose utilization

Absolute/relative insulin deficiency

Persistent low insulin/glucagon ratio

(Simulated fasting state inspite of hyperglycemia)

↓ GLUT 4 ↓ Glucose utilization :

om
(Insulin dependent glucose transporter) • Glycolysis : ↓.

l.c
ai
• Glycogen synthesis : ↓.

gm
@
↓ Glucose uptake by : • ↓Activity of pyruvate dehydrogenase.
19• Glycogenolysis : ↑
55
na

• Gluconeogenesis : ↑
ee
m

Heart. Skeletal Adipose (Synthesis of glucose from

ar

muscle. tissue.
um

non-carbohydrate substrates).
itk
am

Hyperglycemia.
|
w
ro
ar
M

Lipid Metabolism :
©

Absolute/relative insulin deficiency Note :

-
Insulin HSL
Low insulin/glucagon ratio
(Simulated fasting state in spite of hyperglycemia)
+
Hormone sensitive lipase (HSL)
TCA
TAG
Depletion of OAA d/t
Gluconeogenesis Hydrolysis ↑ Lipolysis
Ketone body Acetyl CoA Fatty acid Glycerol
b oxidation
synthesis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Diabetes Mellitus : Part 1 101

Outcome : ----- Active space -----

• ↑ Activity of HSL. • ↑ Fatty acid oxidation.
• ↑ Lipolysis. • ↑ Ketone body synthesis.

Protein Metabolism :
• ↑ Transamination.
• ↑ Protein catabolism (Degradation).
• ↓ Protein synthesis.

Symptoms of Diabetes Mellitus 00:25:13

Cardinal Symptoms :

Polyuria Polydipsia Polyphagia

om
l.c
Symptom ↑ Frequency of micturition ↑ Thirst ↑ Appetite

ai
gm
Glycosuria ↑Protein, carbohydrate &
ECF contraction
@
(Glucose in urine) lipid catabolism
19 +
55
Pathophysiology Thirst centers
na

Osmotic diuresis ↑Demand for nutrients

ee
m

↑Thirst
Polyuria ↑Appetite
ar
um

Other Symptoms :
itk
am

Prone to infections : D/t immunosuppression.

|

• Skin infections (Eg : Boils). • UTI (Urinary tract infection).

w
ro
ar
M
©

Boils
Acanthosis nigricans : Black pigmentation at the nape of neck, axilla etc.

Black pigmentation

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 102 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Laboratory Diagnosis 00:31:00

Diagnosis of DM Follow up/Monitoring/Laboratory diagnosis of

complications.

DIAGNOSIS OF DIABETES MELLITUS

Done to confirm DM in suspected cases with symptoms.

Investigations :
1. Blood glucose estimation.
a. Fasting blood sugar (FBS) : 70-100 mg/dL.
b. Post prandial blood sugar (PPBS) : ≤ 140 mg/dL.
2. Glycated hemoglobin (HbA1c).
3. Oral glucose tolerance test (OGTT).

om
l.c
ai
American Diabetes Association 2017 Criteria :

gm
@
Impaired glucose tolerance
19 Diabetes
Normal
55
(Pre-diabetes) mellitus
na
ee

FBS 70-100 mg/dL 100-125 mg/dL > 126 mg/dL

m
ar

1 hour PPBS (Peak value) < 160 mg/dL - -

um

2 hour PPBS < 140 mg/dL 140-199 mg/dL ≥ 200 mg/dL

itk
am

HbA1c < 5.6% 5.6 - 6.4% ≥ 6.5%

|
w

Guidelines for investigations :

ro
ar

• FBS ≥ 126 mg/dL on > 1 occasions.

M
©

• 2-hour PPBS > 200 mg/dL on a single occasion.

• ↑ FBS or ↑ PPBS at the same occasion.
• RBS (Random blood sugar) > 200 on > 1 occasions.
• HbA1c > 6.5% at any occasion.
Glycated Hemoglobin :
• Best index to monitor long term control of glucose.
• Glycation : Non-enzymatic addition of glucose to proteins (Hb).

Note :
Glycosylation :
• Enzymatic addition of glucose to proteins.
• Sites : Endoplasmic reticulum, golgi apparatus.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Diabetes Mellitus : Part 1 103

Maillard reaction : Formation of HbA1c. ----- Active space -----

(Aldimine linkage)

Added to NH2 Hb N Glucose

Excess glucose N-terminal of Hb
b globin
Amadori
chain
rearrangement (Irreversible)
Hemoglobin Ketoamine linkage
(Seen in HbA1c)

HbA1c :
• Glucose attached to N-terminal valine of Hb.
• M/c assessed fraction of glycated hemoglobin.
• Normal value : < 6.5%.

om
• Significance :

l.c
ai
Determines glycemic control of the past 6-8 wks > 10-12 wks. (~ Lifespan

gm
@
of RBCs).
Estimated average glucose (EAG) : (HbA1c x 28.7) - 46.7. 19
55
na
ee

FOLLOW-UP/DIAGNOSIS OF COMPLICATIONS
m
ar

Clinical Assessments :
um

• BP assessment : Quarterly.
itk
am

• Ophthalmological examination : Biannually/annually.

|

• Neurological evaluation : Annually.

w
ro

• Foot examination for diabetic foot.

ar
M
©

Lab Assessments :
• Fasting lipid profile : Biannually.
• Renal function tests (RFT) : Annually.
• Microalbumin in urine : Annually.
• Glycemic control : FBS, PPBS, HbA1c (Every visit).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 104 Chemistry and Metabolism of Carbohydrates

----- Active space ----- DIABETES MELLITUS : PART 2

Glucose tolerance :
• Ability of the body to metabolize exogenous/dietary glucose &
bring it back to baseline level.
• Assessed in a clinical setting by OGTT.

Oral Glucose Tolerance Test (OGTT) 00:02:33

OGTT : Measuring glucose levels at frequent intervals for 2-2.5 hours after
administering an oral glucose load.

om
l.c
ai
Indications of OGTT :

gm
1. Suspected case of diabetes mellitus with inconclusive FBS values.

@
2. During pregnancy : 19
55

• H/o miscarriages.
na
ee

• Excessive weight gain.

m
ar

• H/o baby weight >4 kgs.

um

3. Renal glycosuria
itk
am

Contraindications of OGTT :
|
w

1. Confirmed case of diabetes mellitus.

ro
ar

2. Monitoring of diabetes mellitus.

M
©

3. Acute illness.

Preparation of the patient :

• Good carbohydrate diet : 3 days prior to the test.
• Exercise.
To be avoided
• Drugs affecting blood glucose.
• Previous day dinner at 8 pm.
12 hours fasting
• Report for testing at 8 am.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Diabetes Mellitus : Part 2 105

Procedure of classical OGTT : ----- Active space -----

Zero hour (1st sample) blood & urine sample taken

Glucose load given :

• Adults : 75 g of anhydrous glucose in 250 ml water. Small, frequent sips to
• Children : 1.7 g/kg body weight. avoid nausea/vomiting

Blood & urine samples collected half hourly for 2½ hours

Samples evaluated for blood glucose levels & presence of

glucose in urine.

om
Glucose Tolerance Test (GTT) Curves 00:09:01

l.c
ai
gm
Blood glucose (mg/dL)
@
19
55
350
na
ee

300
m
ar

250
um

200 Renal
itk

threshold
am

150
|
w

100
ro
ar

50
M
©

0 hr 1 hr 2 hr 3 hr

0 h (FBS) 1 h (Peak) 2h
:N  ormal GTT curve 70-100 <160 <140
: Impaired glucose tolerance 100-126 - 140-200
Peak
: D iabetes mellitus >126 >200
(>>200)

Renal threshold of glucose :

• Refers to the minimum level of blood glucose that results in glycosuria
(Excretion of glucose in urine).
• Normal value : 170-180 mg/dL.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 106 Chemistry and Metabolism of Carbohydrates

----- Active space ----- ABNORMAL GTT

Impaired Glucose Tolerance :
FBS & 2hr PPBS values above the normal range
but below the diabetic levels.
Pre-diabetic
Impaired Fasting Glycemia :
FBS values above the normal range (>100 mg/dL)
but below the diabetic levels (<126 mg/dL).

Alimentary Glycosuria:
Seen in patients with hyperthyroidism/post-gastrectomy.

Findings :

om
Blood glucose Urine glucose

l.c
Fasting Normal -

ai
gm
1 hr >160 mg/dL +

@
2 hr 19
Normal -
55
na
ee

Renal Glycosuria :
m
ar

• Glycosuria + despite normal blood glucose values.

um

• Absolute indication for OGTT.

itk
am

Cause : Mutation in the gene for SGLT-2 (Renal tubules)

|
w
ro
ar

Lowering of renal threshold.

M
©

GTT Variants 00:23:36

Mini GTT :
• WHO recommendation.
• Only 2 samples taken : Zero hour sample & 2 hour PPBS.

Glucose Challenge Test :

Indication : Screening test during pregnancy.

Procedure :
• Done b/w 22-24 weeks of pregnancy.
• Oral glucose load : 50g of anhydrous glucose.
• Fasting : Not required.
• Sample : 2 hours post glucose load.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Diabetes Mellitus : Part 2 107

Interpretation : ----- Active space -----

• <140 mg/dL : Normal value.
• >140 mg/dL GTT.

IV GTT :
Indication : Malabsorption.

Procedure :
• 12 hour fasting.
• 25g glucose in 100 mL sterile distilled water administered.
• Blood sample collected at 10 min intervals for 1 hour.

Interpretation :
Normal : Blood glucose value <100 mg/dL by 45-60 mins.

om
l.c
ai
Corticosteroid Stressed GTT :

gm
Indication : Pre-diabetes & diabetes prone states.

@
19
55
Procedure :
na

• 100g steroid : Given in 2 divided doses orally, 2 hours prior to the test
ee
m

Followed by
ar
um

Glucose load.
itk

• Samples : 1 hour & 2 hour post glucose load.

am
|

Interpretation : Normal values.

w
ro

• 1 hour : <180 mg/dL.

ar
M

• 2 hour : <160 mg/dL.

©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 108 Chemistry and Metabolism of Carbohydrates

----- Active space ----- DIABETES MELLITUS : PART 3

Complications of Diabetes Mellitus  00:00:30

Acute :
1. Diabetic ketoacidosis (DKA).
2. Hyperosmolar non-ketotic coma (HONK)/
hyperglycemic hyperosmolar state (HHS).
3. Lactic acidosis : Occurs d/t hypoxia

om
↑Anaerobic glycolysis.

l.c
ai
gm
4. Hypoglycemia : Occurs d/t insulin overdose.

@
Chronic : 19
55
na

Microvascular :
ee

• Diabetic nephropathy.
m
ar

• Diabetic retinopathy.
um

• Diabetic neuropathy.
itk
am

• Diabetic gastropathy.
|
w

Macrovascular :
ro
ar

• Coronary artery disease (CAD).

M
©

• Cerebrovascular accidents (CVA).

• Peripheral vascular disease (PVD).

Others :
• Diabetic cataract.
• Autonomic dysfunction.

Diabetic Ketoacidosis (DKA) 00:05:11

• Elevated ketone bodies In blood : Ketonemia.

Excreted in urine : Ketonuria.
• Normal level of ketone bodies in blood : < 1 mg/dL.
• Ketosis is seen in uncontrolled diabetes mellitus.
• Ketosis : Ketonemia + Ketonuria.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Diabetes Mellitus : Part 3 109

Biochemical Reasoning of DKA : ----- Active space -----

↑↑Blood glucose Absolute/relative deficiency of insulin

Low insulin-glucagon ratio

↓ Glucose uptake via ↑ Activity of hSL Liver :

GLUT4 by : (Hormone sensitive lipase) • ↑Glycogenolysis.
• Heart. • ↑Gluconeogenesis
TAG
• Muscle.
• Adipose tissue. Glycerol Fatty acid Depletion of OAA
(Oxaloacetate).
β oxidation

om
l.c
Acetyl CoA

ai
gm
Ketone body (KB) +

@
synthesis 19 TCA
55
na

Net effect :
ee
m

1. Hyperglycemia.
ar
um

2. ↓ Glucose uptake.
itk

3. ↓ Glucose utilization.
am

4. ↑ Activity of hSL.
|
w

5. ↑ Ketone body synthesis.

ro
ar
M

Clinical Manifestations :
©

Metabolic acidosis :
↑ Ketone bodies High anion gap metabolic acidosis (↑H+)

↑H+(Acidosis) + HCO3-

H2CO3
Carbonic anhydrase
Expelled in air CO2 + H2O
Respiratory compensation by hyperventilation.

Kussmaul’s respiration : Deep sighing respiration + ↑Rate of respiration.

Fruity smell in breath : D/t acetone Volatile KB, excreted through lungs.
Osmotic diuresis : D/t glycosuria.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 110 Chemistry and Metabolism of Carbohydrates

----- Active space ----- Dehydration & coma :

Hyperglycemia Glycosuria Osmotic diuresis

ECF contraction

Dehydration
if untreated
Coma.
Electrolyte imbalance :
1. Hyperkalemia :
Causes

om
Insulin deficiency Acidosis : ↑H+ Renal injury

l.c
ai
H+

gm
Expulsion of K +
↓ K+ excretion.

@
K+
out of the cell. 19
Cell K+ efflux
55
na
ee
m

2. Hyponatremia :
ar

Hyperglycemia
um
itk

Shift of water from intracellular to

am

extracellular space (Osmotic effect)

|
w
ro

Dilutional hyponatremia.
ar
M
©

Laboratory Diagnosis :
• Blood glucose : 250 – 600 mg/dL.
• Rothera’s test : Positive.
• ABG analysis : High anion gap metabolic acidosis (HAGMA).
• Serum electrolytes : Hyperkalemia, hyponatremia.

Treatment :
1. Fluid replacement : 0.9% saline.
2. Administer short acting insulin (To ↓K+).
3. Correction of electrolyte imbalance.
4. Treat the underlying cause.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Diabetes Mellitus : Part 3 111

Hyperglycemic Hyperosmolar State (HHS) 00:24:00 ----- Active space -----

Biochemical Reasoning of HHS :

Relative insulin deficiency + ↓Fluid intake

Severe hyperglycemia

Osmotic diuresis

Intravascular volume depletion.

Characteristic features :
• No ketosis.

om
• Blood osmolality > 350 mOsmol/L.

l.c
ai
gm
DKA vs HHS 00:26:23

@
19
55

DKA HHS
na
ee

Blood glucose 600 – 1200 mg/dL

m
ar

Serum sodium N - Slightly ↓

um
itk

Serum K+ N
am

Osmolality Normal : 300 - 320 mOsm/mL : 330 - 380 mOsm/mL

|
w
ro

Blood and Urine KB

ar
M

N - Slightly ↑
©

Anion gap
(HCO3 : N )

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 112 Chemistry and Metabolism of Lipids

----- Active space ----- CHEMISTRY OF LIPIDS

Definition and Classification  00:00:35

Definition of lipids :
• Heterogeneous group of compounds which are soluble in non polar solvents
(Eg : chloroform, ether) and insoluble in polar solvents.
• Related physically rather than chemically.

Bloor’s Classification :
1. Simple lipids : 3. Derived lipids :

om
Esters of acids (fatty acids) and alcohol Derived from simple/compound lipids.

l.c
ai
(glycerol). Glycerol

gm
Eg : TAG

@
Eg : Triacylglycerol (TAG). Fatty acid (FA)
2. Compound lipids : 19
55
4. Miscellaneous lipids :
na

Esters of acids, alcohol and another

ee

Steroid hormones, fat soluble vitamins.

m

group.
ar

Eg : Phospholipid, lipoproteins, glycolipids.

um

o
itk

CH2 O C R1 Acyl group

=

CH2OH RICOOH
am

|
|
|

| | o
CH OH + R2COOH CH O C R2
=
|

o
|

|
w

|
|
|

|
ro

CH2 OH R3COOH 3H20 CH2 O C R3

=
ar
|

|
|
|

Non polar
M

Glycerol 3 FA TAG
©

Hydrophobic
Triacyl glycerol AKA fat
Fatty Acids  00:06:30

CLASSIFICATION
1. Based on number of carbon atoms : R COOH
|

a. Short chain FA (2-6).

b. Medium chain FA (8-14).
Hydrocarbon chain Hydroxyl group
c. Long chain FA (>16).
• Non polar • Some polar nature
d. Very long chain FA (>20-22).
General formula

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Lipids 113

2. Based on the presence of double bond : ----- Active space -----

FA

Saturated FA (SFA) : Unsaturated FA :

No double bond Double bond present

Monounsaturated FA (MUFA) Polyunsaturated FA (PUFA)

Common saturated FA :
saturated FA Source
Short chain FA
Acetic acid (2c) Vinegar

om
1. Propionic acid (3C)

l.c
2. Butyric acid (4C)

ai
gm
Butter
3. Valeric acid (5C)

@
4. Caproic acid (6C) 19
55

Medium chain FA
na
ee

1. Lauric acid (12c)

m

Coconut oil (richest source)

ar

2. Myristic acid (14c)

um
itk

Long chain FA
am

1. Palmitic acid (16c)

Animal fat
|
w

2. Stearic acid (18c)

ro
ar

Common unsaturated FA :
M
©

Unsaturated FA Source
Mono unsaturated FA
1. Palmitoleic acid (16c :1 double bond)
Vegetable oils
2. Oleic acid
(18c :I double bond) (richest sources : Mustard oil, rapeseed oil)
3. Elaidic acid
Polyunsaturated FA
1. Linoleic acid (18C : 2 double bonds) Safflower oil, sunflower oil
α α : Flaxseed oil (richest source)
2. Linolenic acid γ (18C : 3 double bonds) γ : Oil of evening primrose
3. Arachidonic acid (20c : 4 double bonds) Body fat
4. Timnodonic acid (EPA) (20C : 5 double bonds) Fish oil, algal oils
5. Cervonic acid (DHA) (22c : 6 double bonds) Fish oil, algal oils, breast milk
EPA : Eicosa pentaenoic acid
DHA : Docosa hexaenoic acid
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 114 Chemistry and Metabolism of Lipids

----- Active space ----- • Highest PUFA : Safflower oil. • Highest α linolenic acid : Flaxseed oil.
• 2nd highest PUFA : Sunflower oil. • Highest MUFA : Mustard/rapeseed oil.
• Least PUFA : Coconut oil. • Highest MCFA : Coconut oil.

3. Based on source of FA :
a. Essential FA : b. Semi essential FA :
• Only source : diet. • Synthesized from linoleic acid.
• Examples : • Examples :
a. Linoleic acid (most essential FA). a. Arachidonic acid.
b. α Linolenic acid. b. γ linolenic acid.
• Deficiency :
- Follicular hyperkeratosis Toad skin.

om
NUMBERING OF FA

l.c
Omega numbering : > Delta numbering :

ai
gm
Starts from the tail (methyl end). Starts from the head (Carboxyl) end.

@
ω3 (1st double bond at C3) 19 Δ7 (1st double bond at C7)
55
na

1 2 3 4 5 6 7 8 9 10
CH3 - CH2 - CH = CH - CH2 - CH2 - CH2 - CH2 - CH2 - COOH
ee

10 9 8 7 6 5 4 3 2 1
m
ar

ω3 FA : ω6 FA :
um

• Examples : Mnemonic ATC • Examples : Mnemonic GLA

itk
am

- Alpha linolenic acid. - Gamma linolenic acid.

|

- Timnodonic acid. - Linoleic acid.

w
ro

- Cervonic acid. - Arachidonic acid.

ar
M

• Significance : • Significance :
©

- ↓ Cardiovascular risk. ↑ Linoleic acid

- ↓ Platelet aggregation.
- ↓ Inflammation. Arachidonic acid (source of
- Infant brain development (DHA). prostaglandins, leukotrienes)
- ↓ Risk of mental illness (ADHD,
depression). Inflammation
- ↓ Risk of degenerative diseases
Note :
(Rheumatoid arthritis, Alzheimer’s).
ω6 : ω3 ratio :
- Protective in Type 2 diabetes,
• Ideal (1 : 1 to 2 : 1).
cancer and non alcoholic fatty liver
• Recommended (3 : 1 to 4 : 1).
disease.
• Western diet (50 : 1).
• Indian diet (16 : 1).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Lipids 115

DOCOSA HEXAENOIC ACID ----- Active space -----

AKA Cervonic acid.
Sources : Significance :
Breast milk, fish oils, algal oils. a. Infant brain development.
b. Retina development.
• Low DHA Retinitis pigmentosa.
• DHA crosses placental barrier

Effective if prescribed during pregnancy

Trans Fatty Acid  00:33:07

om
l.c
ai
Unsaturated FA

gm
@
19
55

Cis form : Trans form :

na
ee

Similar groups on the same Similar groups on opposite

m
ar

side of the double bond sides of the double bond

um
itk
am

Sources :
|

1. Partial hydrogenation of vegetable oils

w
ro
ar
M

Solidified vegetable oils

©

• cake butter
• margarine

Advantages Disadvantages
• ↑ Shelf life • ↑ Trans FA (TFA)
• ↓ risk of rancidity

2. Deep frying.
3. Reheating of oil.
4. Heating oil at very high temperatures.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 116 Chemistry and Metabolism of Lipids

----- Active space ----- Disadvantages :

1. ↓ Fluidity of membrane 2. Dyslipidemia (↑TAG, ↓HDL, ↑LDL).
3. ↑ Cardiovascular risk.
↑ Resistance of plasma 4. Causes essential FA deficiency.
membrane receptors 5. ↑ Inflammation (Risk of arthritis).

Insulin resistance

Note :
• Cis FA ↑ fluidity of membrane.
• Daily allowance of TFA : 2-7g/day.

Medium chain FA vs. Unsaturated FA :

om
l.c
MCFA UFA

ai
gm
Richest source coconut oil Sunflower oil

@
1. No double bond
19
55
na

Advantages No conversion of cis Trans Essential FA

ee
m

2. Absorption into portal veins does

ar
um

not need lipoproteins

itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Phospholipids 117

PHOSPHOLIPIDS ----- Active space -----

Definition : Compound lipids with a phosphoric acid.

Glycerophospholipid
Types
Sphingophospholipid (Sphingomyelin)

Glycerophospholipids  00:01:32

Components : Glycerol
1. Glycerol o
CH2- O -C - R

=
Diacylglycerol (DAG)

om
2. 2 fatty acids (FA) o FA

l.c
CH - O -C -R

=
3. Phosphoric acid (PA).

ai
gm
4. Nitrogenous/Non-nitrogenous base. CH2- O -P -Base

@
Structure
19
Classification :
55
na

Based on
ee
m
ar
um

Nitrogenous base : Non-nitrogenous base :

itk
am

• Lecithin. • Phosphatidyl inositol.

|

• Cephalin. • Cardiolipin.
w
ro

• Phosphatidyl serine. • Phosphatidyl glycerol.

ar
M
©

Glycerophospholipid Constituent Features

• Simplest glycerophospholipid.
Phosphatidic Acid (PA) DAG + Phosphate • Found in cell membrane.
• No nitrogenous/Non-nitrogenous base.
• Storehouse of choline.
Cell membrane.
Lecithin/Phosphatidyl • Most abundant phospholipid in
PA + choline Lung surfactant.
choline Eg : Dipalmitoyl phosphatidyl choline (DPPC) :
Fatty acid Palmitic acid.
Cephalin/Phosphatidyl PA + • Component of cell membrane.
ethanolamine ethanolamine • Involved in blood coagulation.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 118 Chemistry and Metabolism of Lipids

----- Active space -----

Glycerophospholipid Constituent Features

• First isolated from cardiac muscle.
• Found in inner mitochondrial membrane.
• Only antigenic phospholipid.

Cross-reacts with antibodies of Treponema pallidum

Cardiolipin/
PA + glycerol + False positive result in tests for Syphilis
Diphosphatidyl
PA • Associations :
glycerol
a. Cardioskeletal myopathy/Barth syndrome :

om
- Mitochondrial disorder.

l.c
- Cardiomyopathy + skeletal myopathy.

ai
gm
b. Aging.

@
c. Hypothyroidism. 19
55
d. Cardiac failure.
na
ee

Phosphatidyl serine PA + serine Function : Programmed cell death (Apoptosis.)

m
ar

: Mediator of second messengers in hormonal pathways

um

Eg : PIP2 IP3 + DAG

itk
am

Phosphatidyl inositol PA + inositol

|

Secondary messenger for signal

w
ro

Cascade pathways of hormones

ar
M
©

Sphingophospholipid (Sphingomyelin)  00:14:25

• Amino alcohol derived from serine + palmitic acid.

• Components : Sphingosine + FA + Phosphate + Nitrogenous base C1
|
ceramide o C 2 NH2
|

|
R C C3 OH
=

• Function : Component of
|
|
|

Palmitic acid Serine

Outer layer of Myelin sheath of White matter Structure
Specialized structures
plasma membrane nerve fibers of brain (Lipid rafts)
GLYCOLIPID/GLYCOSPHINGOLIPID
• Has an additional
Sphingolipid Glycosphingolipid
carbohydrate in place of
phosphate group. Sphingosine + +
• Not a phospholipid. phosphate group + -

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Phospholipids 119

Components : ----- Active space -----

Sphingosine + FA + carbohydrate.
ceramide
Classification :
Glycosphingolipid Constituents Examples
• Galactocerebroside (In neural tissues)
Cerebroside Ceramide + Monosaccharide
• Glucocerebroside (In extraneural tissues)
Ceramide + Disaccharide/
Globoside Lactosyl ceramide
Oligosaccharide
Ceramide + Oligosaccharide • GM3 : Simplest ganglioside
Ganglioside
+ • GM1 : Receptor for cholera toxin in
(GM1, GM2, GM3)

om
N-Acetyl neuraminic acid (NANA) intestine

l.c
ai
G : Ganglioside.

gm
M : Monosialo (NANA) containing.
@
NANA : Type of sialic acid (Derivative of carbohydrate). 19
55
na
ee

Sphingolipidoses  00:27:37
m
ar
um

Lysosomal-storage disorder.
itk

Accumulating
am

Disorder Defective enzyme Clinical Features

substance
|
w

Frontal bossing
ro

β-galactosidase
Depressed nasal bridge
ar

(GM1 GM2) Typical facies :

M

Low-set ears
©

1. GM1 Long philtrum

GM1 • Blindness
gangliosidosis • Angiokeratoma
• Hepatosplenomegaly
• Intellectual disability
Angiokeratoma • Cherry red spot
2. GM2 gangliosidosis :
β-hexosaminidase A
(GM2 GM3) • Neurological deficits
• Cherry-red spot in macula & retina
a. Tay-Sach’s
• Hyperacusis
disease
GM2 • No visceromegaly
• Muscle weakness

c. Sandhoff’s
β-hexosaminidase A & B Hepatosplenomegaly ±
disease

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 120 Chemistry and Metabolism of Lipids

----- Active space ----- Note : β-hexosaminidase : Isoforms A : 1α & 1β subunit

B : 2β subunits
Mutation of subunit α Affects only isoform A
β Affects both isoforms (A & B)

Disorder Accumulating substance Defective enzyme Clinical Features

• Opisthotonus
• Severe neurological deficits
• Cherry red spot in macula
• No visceromegaly
β-galactocerebrosidase/ • Globoid cell inclusions (Engorged
Krabbe’s macrophages in brain)
β-galactosidase
disease (Globoid Galactocerebroside in neural
(Galactocerebroside
cell inclusion tissues
disease)

om
Ceramide)

l.c
ai
gm
@
19
55
β-glucocerebrosidase/ • Abdominal distension/
na

β-glucosidase visceromegaly
ee
m

(Glucocerebroside • Accumulation in bone marrow

ar

- Pain
um

ceramide) - Pathological
in long
itk

fractures bones
am

- Pancytopenia
|

4. Gaucher’s
Glucocerebroside in - Thrombocytopenia
w

disease (M/c)
ro

extraneural tissues Bleeding manifestations

ar

• X-ray : Erlenmeyer flask

M
©

deformity
• Bone marrow : Crumpled/
Bone marrow : Crumpled wrinkled tissue paper
tissue paper appearance appearance
• No intellectual deficits
• No cherry red spot

Treatment of Gaucher’s disease :

1. Enzyme replacement therapy with :
• Imiglucerase recombinant acid β-glucosidase.
• Velaglucerase-α.
• Taliglucerase-α.
2. Oral substrate reduction therapy with Miglustat (↓es glucosylceramide synthase).
3. Bone marrow transplantation :

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Phospholipids 121

----- Active space -----

Disorder Accumulating substance Defective enzyme Clinical Features

sphingomyelinase • Neurological deficits
• Difficulty in walking, clumsiness
• Dystonias
5. Niemann Pick sphingomyelin in monocyte • Cherry red spot in macula
disease macrophage system • Zebra body inclusions in brain

Painful joint
swelling with
6. Farber’s nodules (Resembles
ceramide ceramidase
disease

om
rheumatoid arthritis)

l.c
ai
gm
7. Fabry’s • X-linked recessive disorder

@
Globotriaosylceramide α-galactosidase
disease 19 Only males affected
55
• Angiokeratoma
na

• Corneal & lenticular opacities (Whorled

ee

appearance in lens)
m
ar

• Fabry’s crisis: Agonising pain & swelling of

um

proximal joints.
itk

• Hypohydrosis : ↓ sweating ( Heat

am

intolerance)
|

• Maltese-cross appearance of urinary

w

Corneal opacity
ro

sediments
ar
M

Treatment of Fabry’s disease : Recombinant α-galactosidase.

©

Enzyme replacement therapy with Agalsidase-β (Fabrazyme).

Agalsidase-α (Replagal).
Fabry’s disease.
Note : D/d for maltese cross appearance of urinary sediments
Nephrotic syndrome.
Wolman’s disease :
Lysosomal storage disorder/Cholesterol ester storage disorder.
Defective Enzyme : Acid lipase.
Clinical Features :
• Relentless vomiting.
• Watery-green diarrhoea.
• Hepatosplenomegaly.
• Peripheral neuropathy.
• Calcification of Adrenals (Pathognomonic). Calcification of adrenals
Not a Sphingolipidosis.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 122 Chemistry and Metabolism of Lipids

----- Active space ----- High yield points :

• All are autosomal recessive except Fabry’s disease (XLR).
• Cherry red spot in all except Fabry’s disease.
Gaucher’s disease.
• Mental retardation absent in Fabry’s disease.
Gaucher’s Type 1 disease.
• Crumpled tissue paper appearance : Gaucher cell. Note :
Fabry’s disease. Type 1 is the m/c
• Angiokeratoma
GM1 Gangliosidosis. variant of Gaucher’s
• Resembles Rheumatoid arthritis : Farber’s disease. disease.
• Calcification of adrenals : Wolman’s disease (Not a sphingolipidosis).
• Maltese-cross appearance of urinary sediments : Fabry’s disease.

om
• Inclusions Zebra body : Niemann Pick.

l.c
Globoid : Krabbe’s disease.

ai
gm
Summary :

@
19
55
Disease Enzyme Defect
na
ee

GM1 gangliosidosis β-galactosidase

m

Tay Sachs’s disease β-hexosaminidase A

ar
um

Sandhoff’s disease β-hexosaminidase A & B

itk
am

Krabbe’s disease β-galactocerebrosidase/β-galactosidase

|

Niemann Pick type 1 sphingomyelinase

w
ro

Gaucher’s disease β-glucocerebrosidase/β-glucosidase

ar
M

Metachromatic leukodystrophy Aryl sulfatase A

©

Farber’s disease Ceramidase

Wolman’s disease Acid lipase
Fabry’s disease α-galactosidase

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Digestion and Absorption of Lipids 123

DIGESTION AND ABSORPTION OF LIPIDS ----- Active space -----

Digestion 00:00:30

STOMACH
Lingual lipase Gastric lipase
From mouth • Produced by : Chief cells
Source
Mostly seen in infants • Stimulated by : Gastrin
Active at pH 2 - 2.5 (Stomach) 5 - 5.5
Digestion/ 30% triglycerides 30% of TAG

om
l.c
Hydrolysis of (Short chain fatty acids) (Short & long chain FA)

ai
gm
@
INTESTINE
19
55
Enzymes :
na

Pancreatic lipase with colipase : Cholesterol esterase :

ee
m

• Colipase necessary for action. Cholesterol ester

ar
um

• Cannot act on FA on 2nd position.

itk

Cholesterol + FA
am

TAG
|

FA Lipase Phospholipase A2 :
w
ro

Phospholipids
ar

2,3-DAG (Diacylglycerol)
M
©

FA Lipase
Lysophospholipids + FA
2-MAG (Monoacylglycerol) (78%)
Isomerase (Shifts FA from 2nd 1st position)
1-MAG (Monoacylglycerol)
Lipase
Glycerol + Fatty Acid (FA)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 124 Chemistry and Metabolism of Lipids

----- Active space ----- Role of Bile Salt :

Emulsification :
• Detergent action :
Large droplet of fat Small droplets.
• Mechanism : ↓Surface tension.
• Advantage :
↑Surface area to facilitate
digestion.

Products of Digestion :
• 2-MAG. • Fatty acid.
• 1-MAG. • Cholesterol.

om
• Glycerol. • Lysophospholipids.

l.c
ai
gm
Absorption
@
00:09:40
19
55
na

Step 1 : Formation of Micelle

ee
m

• Products of lipid digestion Bile salt Mixed micelle.

ar
um

• Ready to be absorbed through intestinal mucosa.

itk

Cholesterol
am

Phospholipid
|
w

Mixed micelle
ro
ar
M

Step 2 : Re-esterification of FA to TAG

©

• Mixed micelle enter intestinal cell. 2

• Location : Endoplasmic reticulum.
• Re-esterification of products with
3
glycerol and FA to form TAG again.
- 2 MAG TAG 1
- 3 FA + Glycerol TAG

Step 3 : Formation of chylomicrons

TAG + Apo B48 Lipoproteins (Chylomicrons).
Chylomicrons are then secreted.
They also carry fat soluble vitamins (A, D, E, K).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Digestion and Absorption of Lipids 125

Summary 00:15:00 ----- Active space -----

Mixed
micelle

om
l.c
ai
gm
@
19
55
na
ee

Micelle enters mucosal

m
ar

cell and resterified in

um

chylomicrons
ER to form TAG
itk
am
|
w
ro
ar
M
©

Note : Short chain FA and medium chain FA can be directly absorbed to portal vein.
They do not need micelle formation or re-esterification.

Disorders of Digestion and Absorption of Lipids 00:17:20

Defective digestion : Defective absorption : Defective digestion

• Steatorrhea : • Coeliac disease, & absorption :
Undigested fat is Crohn’s disease. • Block in common
excreted. • Digestion complex. bile duct.
• Unsplit fat in faeces. • Split fat in faeces. • ↓Bile salt.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 126 Chemistry and Metabolism of Lipids

----- Active space ----- LIPID METABOLISM IN FASTING STATE

Lipolysis  00:01:12

Hydrolytic cleavage of TAGs stored in adipose tissue (in fasting state).

Steps : Regulation :
Triacyl glycerol (TAG)
Activators of hsl Inhibitors of hsl
Acyl Hormone sensitive lipase • Glucagon • Insulin
2,3 Diacyl glycerol (DAG) • Epinephrine • Niacin

om
Acyl Hormone sensitive lipase (hsl) • Glucocorticoids • Prostaglandin

l.c
• ACTH E1 (PGE1)

ai
2 Monoacyl glycerol (MAG)

gm
• TSH

@
Acyl 2 MAG esterase • 19
Melanocyte stimulating
55
Glycerol hormone
na
ee

• Thyroxine
m
ar
um

Fatty Acid Oxidation  00:05:56

itk
am

Types :
|
w

β oxidation : α oxidation :
ro
ar

(Major). (Minor).
M

ω oxidation :
©

Eg : Saturated FA. Eg : Branched chain FA.

(if β oxidation is defective).
Unsaturated FA.
Very long chain FA (VLCFA).
Odd chain FA.
β OXIDATION
• The process by which FA is cleaved successively to form a 2 carbon unit called
acetyl CoA with release of energy.
• M/c FA oxidation. • m/c FA to undergo β oxidation :
Palmitic acid (16C).
Overview :
a. Cutting/Cleaving Acetyl CoA TCA cycle.
b. Oxidation of β carbon atom.
c. Generation of reducing equivalents Electron transport chain Energy.
(ETC)
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Lipid Metabolism in Fasting State 127

----- Active space -----

α
• Successive cleaving between α and
CH3 β COOH β carbon atoms.
• Successive oxidation of β carbon
Acetyl CoA atom [CH COOH].
2
(2C)

Site :
• Organ : liver, adipose tissue, skeletal muscle.
• Organelle : Mitochondria.

Steps :
1. Preparatory : 2. β oxidation.
a. Activation of FA.

om
b. Transportation of activated FA into mitochondria.

l.c
ai
Role : Provides ATP for gluconeogenesis.

gm
@
Stage of fasting 19
1° metabolic fuel
55

1. Early fasting (4-16hrs) Glycogenolysis

na
ee

2. Fasting (16-48hrs) Gluconeogenesis

m
ar

3. Prolonged fasting/
um

starvation TAGs FA oxidation Acetyl CoA Ketone bodies

itk
am

Activation of FA :
|
w
ro

Site : Cytoplasm
ar

Acyl coA synthetase/

M
©

Reaction : FA thiokinase Acyl

CoA (contains Vit. B5)
ATP AMP
Acyl CoA

The only step requiring energy (2 Pi) in FA oxidation.

Transportation of activated FA :
Carnitine :
• FA transporter.
• Synthesized from lysine and methionine.
• FA with <14 carbon atoms : Do not need carnitine.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 128 Chemistry and Metabolism of Lipids

----- Active space ----- FA Acyl CoA Malonyl Cytoplasm

CoA
CAT I / (RLE) Outer mitochondrial
Thiokinase
CPT I membrane

Acyl CoA + Carnitine Acylcarnitine Intermembrane space

CAT II / Inner mitochondrial

CACT
CPT II membrane

Acylcarnitine Carnitine + AcylCoA Acylcarnitine Matrix

om
β oxidation

l.c
ai
gm
CAT : Carnitine acyl transferase.

@
CPT : Carnitine palmitoyl transferase : 19
55
- Gateway of beta oxidation.
na

CPT 1
ee

- Rate limiting enzyme (RLE).

m

CACT : Carnitine acylcarnitine translocase.

ar
um
itk

β oxidation Pathway :
am

Acyl CoA
|
w

FAD
ro

Acyl CoA dehydrogenase

ar

FADH2
M

α β unsaturated FA (Enoyl CoA)

©

H2O
Hydratase
β hydroxy acyl CoA
NAD+ β OH acyl CoA
NADH dehydrogenase
β ketoacyl CoA
Thiolase
Acetyl CoA (2C)
Acyl CoA (-2C)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipid Metabolism in Fasting State 129

Energetics : ----- Active space -----

Eg : Palmitic acid (16C) : No. of acetyl CoA = No. of carbon atom
• No. of acetyl CoA = 16/2 = 8 2
No. of β oxidation = No. of carbon atom -1
80 ATP 2
(1 acetyl CoA TCA cycle 10 ATP)
• No. of β oxidation = 8-1 = 7

28 ATP
1 NADH 2.5 ATP
1 β oxidation
1 FADH2 1.5 ATP
4 ATP
• Total ATP = 80 + 28

om
= 108 ATP

l.c
• ATP utilised = 2 ATP (Activation of FA).

ai
gm
• Net ATP = 106 ATP.

@
Regulation : 19
55
na

Gateway/RLE : CPT I Malonyl CoA (Product of acetyl CoA carboxylase (RLE of

ee
m

FA synthesis)).
ar
um

Fed state Starving

itk
am

↑ Insulin : Glucagon ratio ↓ Insulin : Glucagon ratio

|
w
ro
ar

FA synthesis No FA synthesis
M
©

(+) Acetyl CoA carboxylase (-) Acetyl CoA carboxylase

↑ Malonyl CoA ↓ Malonyl CoA

↓ β oxidation ↑ β oxidation

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 130 Chemistry and Metabolism of Lipids

Oxidation of VLCFA :
----- Active space -----
VLCFA (>20C) :
VLCFA

VLCFA

Modified
H2O2 β oxidation Octanoyl CoA
Acetyl CoA
Octanoyl CoA β oxidation
Octanoyl CoA (8C)

Peroxisome Mitochondria

om
Oxidation of unsaturated FA :

l.c
ai
In mitochondria Energetics :

gm
α

@
1 FADH2 less per double bond
19
55
na

CH3 β COOH 1.5 ATP less per double bond

ee

Acetyl CoA β oxidation

m
ar

α
um

α β unsaturated FA
itk
am

CH3 β COOH
|

Acyl CoA dehydrogenase step

w

bypassed for every double bond

ro
ar

in even position
M
©

β oxidation

Oxidation of odd chain FA :

In mitochondria
CH3 - CH2 - CH2 - CH2 - CH2 - CH2 - COOH
β oxidation
CH3 - CH2 - CH2 - CH2 - CH2
β oxidation
CH3 - CH2 - CH2 -
Propionyl CoA (3C)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipid Metabolism in Fasting State 131

Propionyl CoA (Glucogenic) Note : ----- Active space -----

Biotin ATP Vit. B12 deficiency
CO2 Propionyl CoA carboxylase
D Methyl malonyl CoA ↑ methyl malonic acid (Marker
Racemase of Vit. B12 deficiency)
L Methyl malonyl CoA
L methyl malonyl CoA mutase (+) B12
Succinyl CoA

Oxaloacetate

Pyruvate

om
l.c
ai
Glucose

gm
@
α OXIDATION
Oxidation of α carbon atom. 19
55
na

Site :
ee
m

• Peroxisomes.
ar

• Smooth endoplasmic reticulum (SER).

um
itk
am

Features :
|

• No acetyl CoA synthesis.

w
ro

• No ATP generation.
ar
M

• FA with branch at β carbon atom.

©

Eg : Phytanic acid (Green leafy vegetables and dairy products).

ω OXIDATION
Oxidation of ω carbon atom. CH3 COOH
ω oxidation
Site : SER (Microsomes).
Features : No ATP generation.
COOH COOH
Dicarboxylic acid

Disorders Of FA Oxidation  00:53:17

MCAD DEFECT
Deficiency of medium chain acyl CoA dehydrogenase (MCAD).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 132 Chemistry and Metabolism of Lipids

----- Active space ----- Biochemical defect :

• Palmitic acid (16C)
β oxidation
14C FA
β oxidation MCAD deficiency
12C FA
• In MCAD deficiency, • MCAD deficiency
14C FA
ω oxidation ↓ β oxidation
Dicarboxylic
acid synthesis
(No ATP generation) ↓ Acetyl CoA ↓ ATP

om
l.c
↓ Pyruvate ↓ Gluconeogenesis

ai
gm
carboxylase activity

@
19
(acetyl CoA is Fasting
55
allosteric activator) hypoglycemia
na
ee

• In prolonged starvation Metabolic fuel : Ketone bodies

m
ar

MCAD deficiency ↓ Acetyl CoA ↓ Ketone body synthesis Fatal.

um
itk

Clinical features :
am

May present as sudden infant death syndrome (SIDS).

|
w
ro

Treatment :
ar
M

Frequent meals (To avoid fasting) : Low fat high carbohydrate diet.
©

JAMAICAN VOMITING SICKNESS

• Precipitated by consumption of ackee fruit.
• Common in West Africa.
Biochemical defect :
Acyl CoA Unripe ackee fruit
Acyl CoA dehydrogenase
β oxidation Hypoglycin
↓ β oxidation

↓ ATP ↓ Acetyl CoA

↓ Gluconeogenesis ↓ Ketone body synthesis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Lipid Metabolism in Fasting State 133

Clinical features : ----- Active space -----

1. Sudden onset vomiting.
2. Features of hypoglycemia
- Fatal if not treated immediately.

REFSUM’S DISEASE
Defect in phytanoyl CoA oxidase/hydroxylase.

Biochemical defect :
↓ Phytanoyl CoA oxidase activity

↓ α oxidation

om
↑ Phytanic acid

l.c
ai
Clinical features :

gm
@
• Mostly asymptomatic.
19
• If symptomatic :
55
na

a. Retinitis pigmentosa.
ee
m

b. Ichthyosis.
ar
um

c. Peripheral neuropathy.
itk

d. Cardiac arrhythmia.
am
|
w
ro
ar
M
©

Ichthyosis

Retinitis pigmentosa : fundus autofluorescence

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 134 Chemistry and Metabolism of Lipids

----- Active space -----

Investigations :
↑ Serum phytanic acid.

Treatment :
Restrict phytanic acid (Green leafy vegetables, dairy products).

Zellweger Syndrome  01:05:30

AKA cerebrohepatorenal syndrome.

Biochemical defect :
Mutation in PEX gene RER proteins :
• Synthesised in rough endoplasmic
reticulum.

om
No production of peroxisomal
• E.g :

l.c
targeting sequences (PTS)

ai
- Peroxisomal enzymes.

gm
@
RER proteins do not attach to PTS - Mitochondrial proteins.
19 - Nuclear proteins.
55
na

- Plasma membrane proteins.

ee

RER proteins do not ↑ RER proteins in

m
ar

reach peroxisome the serum

um
itk
am

↓ VLCFA ↓ α oxidation ↓ peroxisome

|
w

oxidation biogenesis
ro
ar
M

Clinical features :
©

Similar to Down’s syndrome :

• Mongoloid facies. • High forehead.
• Hypertelorism. • Brushfield spots.
• Frontal bossing. • Intellectual disability.
• Unslanting palpebral fissure.
Investigations :
1. Peroxisomal ghost : Empty peroxisome.
2. ↑ VLCFA.
3. ↑ Phytanic acid.
4. ↓ Peroxisome synthesis.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Ketone Body Synthesis 135

KETONE BODY SYNTHESIS ----- Active space -----

Metabolism of Ketone Bodies 00:00:25

SYNTHESIS
Trigger :
Converted to
Prolonged fasting/ starvation Fatty acid Acetyl CoA Ketone bodies
(Source of blood
Site : glucose)
Exclusively in liver mitochondria.

om
Steps :

l.c
ai
gm
Fatty acid

@
β oxidation
19
55
Acetyl CoA (2C) Acetyl CoA (2C)
na
ee

Acetoacetyl CoA (4C) : Starting substrate for KB synthesis

m
ar

Combines HMG CoA synthase : Rate limiting enzyme

um
itk
am

HMG CoA (6C)

|
w

Lysis HMG CoA lyase

ro
ar
M

Acetoacetate : Primary KB
©

Forms 20 ketone bodies

Spontaneous reaction NADH
CO2 β OH butyrate DH
NAD+
Acetone : β hydroxy(OH) butyrate
• Volatile.
• Excreted through lungs.

If Ketosis : Fruity smell in breath

• Neutral KB (No energy produced by lysis).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 136 Chemistry and Metabolism of Lipids

----- Active space ----- UTILISATION

Prerequisite :
Mitochondria.
Site :
All organs except :
• Liver : D/t absence of thiophorase enzyme.
• RBC : D/t absence of mitochondria.

Steps :
β hydroxy(OH) butyrate
NAD+
Via electron transport chain
NADH 2.5 ATPs produced

om
TCA cycle
Acetoacetate

l.c
Succinyl coA Total energy produced :

ai
CoA transferase/

gm
Succinyl CoA CoA
Substrate level thiophorase
a. From β hydroxy (OH) butyrate :

@
thiokinase
Succinate • Expected : 2.5 + 10 + 10 =
phosphorylation 19
55
(SLP) Succinate Acetoacetyl CoA (4C) 22.5 ATPs.
na

• D/t lack of SLP :

ee
m

Split 22.5-1 = 21.5 ATPs.

ar

1 ATP produced
um

Does not occur 1 Acetyl CoA 1 Acetyl CoA b. From acetoacetate :

itk

10 + 10 - 1 = 19 ATPs.
am

Enters TCA Enters TCA

cycle cycle
|
w
ro

10 ATPs 10 ATPs
ar
M
©

β hydroxy (OH) butyrate : Acetoacetate ratio

• Normal = 1 : 1.
• Ketosis (D/t starvation or DKA) = 6:1 (β OH butyrate is predominant).

Clinical Correlation of KBs 00:15:00

STARVATION KETOSIS
Mechanism :
• ↓blood glucose ↑glucagon activity.
↓insulin activity.
• Starvation : ↓Glut 4 No gluçose transport into adipose cell

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Ketone Body Synthesis 137

----- Active space -----

Glucose
Triacyl Step 1 via β Produce Used in
Glut 4 glycerol stores oxidation ATP gluconeogenesis

Fatty
Fatty acid TCA cycle (Does not occur because
Glycerol acid ds
e starvation ↓OAA)
Step 2 Ne AA
O

Adipose cell Ketone body synthesis

Supplied
(Predominates in starvation)

om
to vital

l.c
ai
↑ KBs organs

gm
eg : Brain
@
Liver cell
DIABETIC KETOACIDOSIS (DKA) 19
55
na

Mechanism :
ee

↓insulin (Absolute/relative deficiency)

m
ar
um

↓ GLUT-4
itk
am

Glucose not absorbed into adipose tissue

|
w

↓insulin
Further ↑blood glucose
ro
ar
M

↑ glucagon action
©

↑↑blood glucose
GLUT 2
↓insulin Blood glucose (Bidirectional)
Glucose
↑ activity of hormone ↑ glucose ↑ gluconeogenesis
Glut 4 sensitive lipase (HSL)
TCA cycle
A
Triacyl glycerol OA (Does not occur d/t ↓OAA)
Acetyl CoA
Fatty Ketone body synthesis
Glycerol oxidation
acid Fatty acid (Predominates
(+)
in DKA)
Adipose cell
Liver cell

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 138 Chemistry and Metabolism of Lipids

----- Active space ----- Lab diagnosis :

1. Rothera’s test : 2. Ketosticks :
Colour code : Indicates
amount of KBs in urine

Purple ring at the

junction of 2 liquids :
Positive reaction

3. Gerhardt’s test :
Postive for Ketone bodies : Only positive for acetoacetate.
• Aceto acetate. 4. β OH butyrate DH based enzymatic test :
• Acetone. Detects β OH butyrate.

om
l.c
ai
gm
Not excreted in urine in

@
ketoacidosis 19
55
(Added to urine in lab)
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipid Metabolism in Fed State 139

LIPID METABOLISM IN FED STATE ----- Active space -----

Fatty Acid Synthesis  00:00:48

Overview :
Well fed state

↑ Insulin : Glucagon ratio

Carbohydrate Pyruvate Acetyl CoA

om
De novo synthesis :
Fatty acid (FA)

l.c
synthesis • Elucidated by Feodor Lynen

ai
gm
• Aka Lynen’s spiral

@
19
Site :
55
na
ee

Organs : Organelle :
m
ar

• Liver. • Cytoplasm (Extramitochondria).

um

• Adipose tissue.
itk
am

• Kidney.
|

• Brain.
w
ro

• Lactating mammary glands.

ar
M
©

Substrate :
Acetyl CoA.

Source of Acetyl CoA :

Pyruvate dehydrogenase
Pyruvate Acetyl CoA (Mitochondrial).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 140 Chemistry and Metabolism of Lipids

----- Active space ----- Steps :

1. Transportation of acetyl CoA : Via citrate

Mitochondria

Pyruvate PDH Acetyl CoA(2C)

Citrate Oxaloacetate (4C)
synthase
Citrate (6c)

TCA transporter

Cytoplasm

om
Citrate (6c)

l.c
ai
gm
Oxaloacetate ATP citrate lyase

@
19
Acetyl CoA (2c)
55
na

2. Acetyl CoA carboxylase :

ee
m

Acetyl CoA (2c)

ar

Biotin ATP
um

CO2 Acetyl CoA carboxylase Rate limiting step

ADP
itk
am

Malonyl CoA (3c)

|
w
ro

3. Fatty acid synthase (FAS) complex :

ar
M

• Multifunctional enzyme complex.

©

• X shaped.
• Homodimer : Each monomer contains 6 enzyme activities.

Reduction unit : ER
- Ketoacyl reductase (KR) ER KR KR
- Dehydratase (DH) ACP
ACP
- Enoyl reductase (ER)
DH

DH

TE
TE
Releasing unit:
KA
- Thioesterase (TE) KAS S
MAT MAT

Condensing unit:
- Malonyl acetyl
Fatty acid synthase complex transacylase (MAT)
- Keto acyl synthase
(KAS)
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Lipid Metabolism in Fed State 141

Reactions : ----- Active space -----

1. Condensation :
+ Acetyl (2C) Acetyl (2C)

MAT KAS

KAS ketoacyl (4C)

+ Malonyl (3C) Malonyl (3C) Acetyl (2C)
FAS complex CO2

2. Reduction :
(1) KR (NADPH)
(2) DH

om
(3) ER (NADPH)

l.c
ai
gm
@
Ketoacyl (4C) 19
Acyl (4C)
55
na

3. Repeat condensation and reduction :

ee
m
ar

+ Acyl (4C) Acyl (4C) Acyl (4C)

um
itk

MAT KAS
am
|

CO2
w
ro

+ Malonyl (3C) Malonyl (3C) Acetyl (2C)

ar
M

KAS
©

(1) KR (NADPH)
(2) DH
(3) ER (NADPH)

Palmitoyl CoA Acyl (6C) Ketoacyl (6C)

Palmitic adic

4. Releasing :

TE + Palmitoyl CoA

Palmitoyl CoA
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 142 Chemistry and Metabolism of Lipids

----- Active space ----- Cofactors :

FAS Complex : Acetyl CoA carboxylase :
1. NADPH. 1. Biotin.
2. Manganese. 2. Bicarbonate (Source of CO2)

Regulation :
Regulatory enzyme : Acetyl CoA carboxylase.
Allosteric regulation :
Allosteric activator : Citrate (Substrate) .
Allosteric inhibitor : Long chain acyl CoA (Product).
Hormonal regulation :
A) Well fed state B) Fasting state

om
l.c
↑ Insulin : Glucagon ratio ↓ Insulin : Glucagon ratio

ai
gm
@
Dephosphorylation of Phosphorylation of
acetyl CoA carboxylase 19 Acetyl CoA carboxylase
55
na
ee

Activation of acetyl CoA Inactivation of Acetyl

m
ar

carboxylase CoA carboxylase

um
itk
am

↑ FA synthesis ↓ FA synthesis
|
w
ro

Cholesterol Synthesis  00:24:48

ar
M

Cholesterol :
©

• Not a metabolic fuel.

• Exclusive animal sterol.
Functions of cholesterol :
Anabolic role :
• Steroid hormone synthesis.
• Vitamin D synthesis.

Sites :
Organs : Organelles :
• Liver. • Cytoplasm.
• Adrenal cortex. • Endoplasmic reticulum (ER).
• Gonads.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipid Metabolism in Fed State 143

Sources : ----- Active space -----

Dietary cholesterol.
Acetyl CoA (Carbohydrates Pyruvate Acetyl CoA).

Steps :

Acetyl CoA (2C) + Acetyl CoA (2C)

Thiolase
Acetoacetyl CoA (4C) + Acetyl CoA (2C)
HMG CoA synthase (Cytoplasmic)
HMG CoA (6C)
NADPH HMG CoA reductase (RLE)

Mevalonate

om
l.c
Isoprenoid unit (5C) + Isoprenoid unit (5C)

ai
gm
@
Geranyl pyrophosphate (10C) + Isoprenoid unit (5C)
55
19
na

Farnesyl pyrophosphate (15C) + Farnesyl pyrophosphate(15C)

ee
m
ar

Squalene (30C)
um
itk
am

Lanosterol
|
w
ro

Zymosterol
ar
M
©

Desmosterol

Cholesterol (27C)

Steroid hormones Bile acids Vitamin D

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 144 Chemistry and Metabolism of Lipids

----- Active space ----- Regulation :

Rate limiting enzyme (RLE) : Hormonal regulation :
HMG CoA reductase (Inhibited by statins). Well fed state

Feedback regulation : ↑ Insulin : Glucagon ratio

Dietary cholesterol
Dephosphorylation of HMG CoA
Suppresses enzymes of cholesterol Reductase
synthesis
Activation of HMG CoA Reductase

↑ Cholesterol synthesis

om
l.c
ai
STEROID HORMONE SYNTHESIS

gm
@
Pathway : 19
55
Cholesterol side chain cleavage :
na
ee
m

ACTH
ar

(cAMP)
um

P450scc
itk
am

Cholesterol (27C) Cytochrome P450 (21C)Pregnenolone + isocaproaldehyde (6C)

|
w

side chain cleavage

ro

enzyme Mother of steroid hormones

ar
M

Basic steroid hormone structures :

©

17β-D-Estradiol Testosterone Cortisol Progesterone

Estrane group (C18) Androstane group (C19) Pregnane group (C21)
Pathway of steroid hormone synthesis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipid Metabolism in Fed State 145

Biosynthesis : ----- Active space -----

Pregnenolone Progesterone

ee 3β-Hydroxysteroid dehydrogenase and δ5,4 isomerase

17α-Hydroxylase 17α-Hydroxylase

17α-Hydroxypregnenolone 17α-Hydroxyprogesterone
Delta 4 pathway (Major)

Delta 5 pathway (minor)

17,20-Lyase 17,20-Lyase

Dehydroepiandrosterone

om
Androstenedione

l.c
ai
gm
17β-Hydroxysteroid 17β-Hydroxysteroid

@
dehydrogenase 19 dehydrogenase
55
na

Δ5-Androstenediol Testosterone
m

Aromatase
ar
um

17β estradiol
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 146 Chemistry and Metabolism of Lipids

----- Active space ----- Bile Acid Synthesis  00:47:42

Bile acid : Degradation product of cholesterol.

Site : liver, intestine.

Steps :
7α hydroxylase (RLE)
Cholesterol 7 OH cholesterol C holic acid >
(CYP7A1) 1° bile
NADPH Chenodeoxycholic
acids
Vit. C acid
Glycine
Conjugation Taurine

2° bile acids

om
l.c
Common bile duct

ai
gm
@
55
19 Intestine :
Cholic acid Chenodeoxycholic
na
ee

acid
m

Deconjugation
ar
um

+
itk

Dehydroxylation
am

Deoxycholic acid > Lithocholic acid

|
w

(98-99%) minimal
ro
ar
M

Enterohepatic
©

circulation

Liver

Bile acid and bile salt : Bile acid sequestrant :

Bile acid • Used in management of hyper-cholesterolemia.
In alkaline • Bile acid + Sequestrant
medium
Ionisation Excretion of bile acid
Na+/K+
↑ Cholesterol metabolism and excretion
Na+/K+ salt of bile acid

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipoproteins and its Metabolism 147

LIPOPROTEINS AND ITS METABOLISM ----- Active space -----

Lipoproteins 00:01:03

Plasma lipids + Protein Lipoproteins

(Insoluble in blood) • Compound lipid
• Examples : • Can be transported in blood
- Triacyl glycerol (TAG).
- Cholesterol.
- Cholesterol ester.

om
- Phospholipids.

l.c
- Fatty acids (FA).

ai
gm
@
19
STRUCTURE
55
na
ee
m
ar

Core :
um

• Made of hydrophobic lipids.

itk
am

• No polar component.
|

Layer of amphipathic lipids • TAG, Cholesterol ester.

w
ro

• Both hydrophobic and hydrophilic.

ar
M

• Slightly polar.
©

• Cholesterol, Phospholipids. Layer of complex proteins

• Apoproteins/Apolipoproteins

Lipoprotein structure
Peripheral Integral

TYPES

Chylomicron : Highest lipid content, Lowest density.

Very low density lipoprotein (VLDL).
Size Low density lipoprotein (LDL).
Intermediate density lipoprotein (IDL).
High density lipoprotein (HDL) : Highest protein content, Highest density.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 148 Chemistry and Metabolism of Lipids

----- Active space ----- Chylomicron 00:08:57

Synthesis : Intestine. Density : Lowest (Maximum lipid content) ; Most buoyant.

Function : Transports exogenous/ Lipid content : Mostly exogenous TAG.
dietary lipids (TAGs) to Apoprotein : ApoB48 (Unique to chylomicron),Apo C2 , Apo A1 ,
peripheral organs. Apo E, Apo A2 , Apo A4 , Apo C1, Apo C3.
Size : Largest.

Metabolism :
Formation of nascent chylomicron :
Intestinal cells Microsomal TG transfer
Intestinal protein (MTTP/ MTP)
lumen

om
l.c
ai
TAG

gm
Fatty Acids FA + Glycerol TAG
(FA) Binds to

@
SER 19 Apo B48
55
na

Nascent chylomicron
ee

Smooth endoplasmic
m

reticulum
ar
um

Pathway :
itk

Mature chylomicron :
am

Nascent chylomicron Transported in blood vessels

|

Intestine Apo E
w

Generates Apo B48 Apo B48

ro

Apo C2
ar

activates
M

TAG Apo C2 TAG Peripheral

©

E C2 organ

Lipoprotein lipase Adipose

tissue

Remnant chylomicron :
Low TAG content Causes hydrolysis of Taken
Chylomicron TAG in chylomicron up into
E Apo E acts Apo B48 loses Apo C2 adipose
Liver as ligand in order to cell
TAG Glycerol Fatty acids
preserve
E
Receptor for Apo E remaining TAG
Remnant chylomicron content
taken up into liver :
Receptor Mediated
Endocytosis
TAG : Tri acyl glycerol

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipoproteins and its Metabolism 149

Lipoprotein lipase : ----- Active space -----

Site : Vascular endothelium of vessels surrounding peripheral organs.
E.g. : Adipose tissue.
VLDL : LDL :
Synthesis : Liver. Synthesis : VLDL.
Function : Function : Transport cholesterol.
• Transports endogenous TAG to ( 30% to peripheral organs Harmful)
peripheral organs. Lipid content : Maximum cholesterol
• VLDL IDL LDL (Lipoprotein and cholesterol ester content.
cascade pathway) Apoprotein : Apo B100
Lipid content : Endogenous TAG.
Apoprotein : Apo B100 , Apo C2, Apo E.

om
l.c
Liver cell

ai
Metabolism of VLDL, IDL and LDL

gm
@
Formation of VLDL : FA + Glycerol
SER 19
MTTP
55
TAG TAG
na
ee

Binds to
m

Apo B 100
ar
um

Nascent VLDL
itk
am

Lipoprotein Cascade Pathway : VLDL IDL LDL

|
w

Nascent VLDL
ro
ar

B - 100 Apo E
M

B - 100
Generates Apo C2
©

Nascent VLDL TAG / Apo C2 TAG / activates

C C
Liver
E C2 Peripheral
organ
TAG : Triacylglycerol
C : Cholesterol Adipose
Lipoprotein lipase tissue
IDL : Intermediate density lipoprotein

Remnant VLDL/ IDL

Low TAG content
Causes hydrolysis of Taken
B - 100 VLDL loses TAG in VLDL up into
Apo C2 peripheral
TAG /
in order to organ
C Glycerol Fatty acids
preserve
E
remaining TAG
content
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 150 Chemistry and Metabolism of Lipids

----- Active space -----

Fates of IDL

Uptake into liver via OR • Apo E removed.

Receptor mediated • TAG hydrolyzed by hepatic
endocytosis and endothelial lipase
B - 100
Forms LDL
TAG / C
B - 100
E
C
Acts as ligand

E
Receptor

om
l.c
for Apo E

ai
gm
@
Liver
19
55
Fate of LDL
na
ee
m
ar
um

70 % taken 30 % taken up by
itk

up by liver Extrahepatic tissues

am

Apo B100 acts

|
w

as ligand
ro
ar
M

Receptor
©

C C
mediated
B - 100 endocytosis B - 100
Receptor for B100
Receptor for B100
Extrahepatic

No mechanism to
tissues

Cholesterol Liver convert cholesterol

to bile acids
Bile acids
Cholesterol deposits in coronary,
carotid vessels, etc
Excreted
Risk for thrombosis Harmful
Not harmful

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipoproteins and its Metabolism 151

HDL : ----- Active space -----

Synthesis : Liver, Intestine.
Function : Reverse cholesterol transport.
Lipid content : Highest phospholipid content.
Size : Smallest.
Density : Highest.
Apoprotein : Apo A1, Apo A2 , Apo A4 , Apo C, Apo D, Apo E.
Transporters of cholesterol :
Transport cholesterol into HDL.
1. ABC (ATP binding cassette) A1.
2. ABC G1.
3. SR (Scavenger receptor) B1.

om
l.c
Lecithin cholesterol acyl transferase (LCAT) :

ai
gm
• Transports FA from lecithin to cholesterol.

@
19
55
• Lecithin + Cholesterol (Amphipathic)
na

LCAT
ee
m

Lysolecithin + Cholesterol ester (Hydrophobic).

ar
um

Metabolism :
itk
am
|

Cholesterol
w
ro
ar
M

Excreted Bile acids Discoidal HDL Intestine

©

Liver
SRB 1 Apo A1 LCAT
ABC A1

Spherical Spherical ABC G 1

Cholesterol
HDL 2 HDL 3
SRB 1
Peripheral
organs

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 152 Chemistry and Metabolism of Lipids

----- Active space ----- Pathway :

Synthesize
Discoidal HDL/HDL2
• Polar end present on surface.
Liver
Bile acids • Non-polar end present inferiorly.
Excreted

CE
A1
↑ PL
SRB 1
↑C Apo A1
Transporter of
Amphipathic
Synthesize Intestine
CE into liver activates
(Polar + Non
polar end)

om
l.c
Excess CE

ai
gm
unloaded

@
from HDL to Cholesterol LCAT Cholesterol ester
19
55
liver
na
ee
m
ar
um

• Hepatic lipase
itk

• Endothelial lipase Internalising

am

of hydrophobic
|

+
w

cholesterol ester Cholesterol deposited

ro
ar

by LDL
M
©

Spherical HDL 2 Spherical HDL 3

A1 A1 ABC A1
↑↑↑ CE HDL3 - HDL2 C Transport C Peripheral
Cycle CE ABC G 1
C tissue
PL PL SRB 1
LCAT action :
C CE

PL : Phospholipid.
C : Cholesterol.
CE : Cholesterol ester.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Lipoproteins and its Metabolism 153

ELECTROPHORETIC PATTERN : ----- Active space -----

Origin Chylomicron

LDL (β-fraction)
Mobility VLDL (Pre β-fraction)

IDL (Broad β-fraction)

HDL (α-fraction) Maximum mobility
Anode (↑ protein content)
(+)
OTHER LIPOPROTEINS

om
a. Lipoprotein a :

l.c
• Similar to LDL.

ai
gm
• AKA Little rascal.

@
Apoprotein : 55
19
ApoB 100
na

Disulphide
ee
m

bond
ar

Apo A
um

Apo a : Plasminogen analogue

itk
am

Inhibits fibrinolysis
|
w
ro

↑ Risk of coronary artery disease

ar
M
©

Fibrin Plasmin Fibrin degradation

Fibrinolysis
products (FDP)

b. Lipoprotein X :
Liver

Cholesterol Bile acids Excreted

in bile
Cholestasis ↓ excretion of cholesterol in bile

↑ cholesterol
Phospholipid

Lipoprotein X (Indicator of cholestasis)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 154 Chemistry and Metabolism of Lipids

----- Active space ----- Functions of Apolipoproteins 00:45:28

Apo C I : Inhibits cholesterol ester transfer protein.

Apo C 2 : Activates lipoprotein lipase (LPL).
Apo C3 : Inhibits lipoprotein lipase.
Apo AI : Activates LCAT.
Apo A2 : Inhibits lipoprotein lipase.
Apo A5 : Facilitates binding of chylomicron and VLDL to LPL.
Apo D5 : Associated with neurodegenerative diseases like Parkinson’s disease.
Apo E 4: • Arginine rich.
• Associated with Late onset Alzheimer’s disease.

om
l.c
ai
gm
@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Dyslipidemia 155

DYSLIPIDEMIA ----- Active space -----

Dyslipidemia

Hyperlipoproteinemia : Hypolipoproteinemia :
↑ Lipoprotein levels ↓ Lipoprotein levels
Hyperlipoproteinemia 00:00:25

Classification :
Fredrickson classification :

om
Type I

l.c
Type IV

ai
gm
With hypertriglyceridemia Type V

@
Hyperlipoproteinemia With hypercholesterolemia 19 Type II
55
na
ee

With hypertriglyceridemia Type III

m
ar

and hypercholesterolemia
um
itk

Type I Hyperlipoproteinemia :
am

• AKA familial chylomicronemia syndrome.

|
w

• Autosomal recessive.
ro
ar

• Presents in childhood.
M
©

Biochemical defects :
• LPL deficiency.
• Apo C2 defect
Mature VLDL Mature chylomicron
LPL LPL
↓LPL activity Remnant VLDL Remnant chylomicron

↑ Mature chylomicron > ↑ Mature VLDL

• ↑ serum TAGs.
Plasma lipid
• Normal serum cholesterol

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 156 Chemistry and Metabolism of Lipids

----- Active space ----- Clinical features :

1. Pancreatitis : Recurrent pain abdomen.

2. Lactescent (Milky white) plasma : 3. Eruptive xanthomas : 4. Lipemia retinalis :

D/t ↑ chylomicron. Yellowish raised papules • Deposition of lipid in
retinal vessels.
• Seen on fundoscopy.

om
Discrete Clusters

l.c
ai
gm
Type II Hyperlipoproteinemia :

@
• AKA familial hypercholesterolemia.
19
55
• M/c hyperlipoproteinemia.
na

• Mostly autosomal dominant inheritance.

ee
m

• Presents in adolescence (Especially heterozygous type).

ar
um

• Prevalence : 1 in 311.
itk
am

Biochemical defects :
|
w

Defect in LDL receptor/ Apo B100

ro
ar
M
©

↓ LDL clearance

↑ LDL Tendon
xanthoma
• ↑ Serum cholesterol
Plasma lipid
• Normal serum TAGs

Clinical features :
1. Coronary artery disease (CAD).
2. Cerebrovascular accident (CVA).
3. Clear plasma.
4. Solitary tendon xanthoma : Corneal
M/c site : Achilles tendon. arcus
5. Corneal arcus : white ring around the cornea
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Dyslipidemia 157

Types : ----- Active space -----

Familial Hypercholesterolemia

Autosomal dominant ADH Type II ADH Type III Autosomal recessive Sitosterolemia
Hypercholesterolemia (ADH) (Rare)
Type 1 (M/c type).

LDL Clearance pathway :

PCSK9 (Doesn’t allow separation of

LDL-ApoB100 from LDL receptors)

om
LDL

l.c
ai
Clathrin coated

gm
ApoB100 pit
@
LDL receptor 19
55
na

LDL receptor
ee
m

adaptor protein
ar

Endosomes
um
itk
am
|

Lysosomes
w
ro
ar
M
©

Recycled
Degraded Degraded

Site of mutation Disorder

1 LDL receptor ADH Type I
2 Apo B100 ADH Type II
3 LDL receptor adaptor protein Autosomal recessive hypercholesterolemia
4 PCSK9 (Gain of function mutation) ADH Type III
5 ABC G5, G8 Sitosterolemia

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 158 Chemistry and Metabolism of Lipids

----- Active space ----- Plant sterol clearance :

Intestinal Intestinal cell

lumen ABCG 5 • Plant sterol (Sitosterol):
- Incompatible with human body.
Plant sterol - May be ingested through diet.
(Dietary) • ATP Binding Cassette (ABC) G 5 and G 8 :
ABCG 8 - Present in liver and intestinal cells.
- Transport plant sterols back into
intestinal lumen for excretion.
ABCG 8

Bile duct Plant

ABCG 5 sterol

om
l.c
Liver

ai
gm
Mutation of ABCG8/ABCG5 ↓ Excretion of plant sterols ↑ Plant sterols
@
19
55
na
ee

Feedback inhibition of LDL

m
ar
um
itk

Sitosterolemia ↑ Cholesterol ↓ Transcription of LDL receptor

am
|
w
ro
ar

Type III Hyperlipoproteinemia :

M
©

AKA familial dysbetalipoproteinemia, remnant removal disease, broad β disease.

Biochemical defect : Clinical features :

ApoE mutation
1. Tuberoeruptive xanthoma :
↓ Uptake of remnant chylomicron Yellowish raised lesions with
and VLDL bunch of grape appearance.
2. Palmar xanthoma.
↑ Remnant chylomicron and VLDL
3. Slight ↑ risk of CAD.
↑ serum TAG Plasma
↑ serum cholesterol lipid

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Dyslipidemia 159

Hypolipoproteinemia 00:30:36 ----- Active space -----

AKA Tangier disease (Common in Tangier region).

Biochemical defect :
Mutation in ABCA1

↓ HDL synthesis
Clinical features :
1. Enlarged greyish/orange tonsil 2. Hepatosplenomegaly

om
l.c
ai
gm
@
19
55
na
ee

3. Peripheral neuropathy
m
ar
um
itk

Abetalipoproteinemia 00:32:40
am
|
w

Biochemical defect
ro
ar

Defect in MTTP
M
©

Inhibits synthesis of VLDL

and chylomicron from TAG

↓VLDL ↓ Chylomicron
(Pre β)
↓ Fat soluble vitamins
↓ IDL (↓ Vitamin K)
(Broad β)
↓ γ carboxylation of
↓ LDL (β) clotting factors

Bleeding
manifestations
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 160 Chemistry and Metabolism of Lipids

----- Active space ----- Clinical features :

1. Diarrhea : D/t malabsorption.
2. Failure to thrive.
3. Neurological manifestations.
4. Progressive pigmentary retinopathy.

Progressive pigmentary retinopathy

on fundus autofluorescence

om
l.c
5. Acanthocytes on peripheral smear

ai
gm
@
19
55
na
ee
m
ar
um
itk
am

6. Bleeding manifestations
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Dyslipidemia 161

LCAT Deficiency 00:36:33 ----- Active space -----

LCAT deficiency

Complete Partial

Norum disease : Fish eye disease :

• May progress to end Benign
stage renal disease.
• Complete deficiency
of LCAT

om
l.c
ai
gm
Lecithin Cholesterol ester
LCAT

@
+ +
19
55
Cholesterol Lysolecithin
na
ee
m
ar
um
itk

↑ Lecithin ↓ Cholesterol ester ↓ HDL

am

↑ Cholesterol ↓ Lysolecithin
|
w
ro
ar
M

Treatment of Hyperlipoproteinemia : Newer Modalities 00:39:00

©

1. Type I hyperlipoproteinemia :
Lipogene tiparvovec :
• Gene therapy treatment.
• Gain-of-function variant of LPL.
• Intramuscular injection using a vector (adeno associated viral vector).

2. Type II hyperlipoproteinemia :
a. Lomitapide :
Inhibits MTTP ↓ VLDL ↓ IDL ↓ LDL.
b. Mipomersen :
• Antisense oligonucleotide therapy against apoB.
• ↓ ApoB activity ↓ VLDL ↓ IDL ↓ LDL.
c. VERVE 101 :
Genome editing based on CRISPR cas9.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 162 Chemistry and Metabolism of Proteins

----- Active space ----- CHEMISTRY OF AMINO ACIDS

Amino acid :
• Most amino acids are α amino acids.
• Non-α-amino acids : β-alanine, β-aminoisobutyrate, γ-aminobutyrate.

H
|
Amino NH2 α C COOH Acid

|
|

|
R
Variable side chain

om
l.c
Classification of Amino Acids  00:02:18

ai
gm
@
BASED ON SIDE CHAIN
I. Aliphatic : 19II. Hydroxyl (OH) group :
55
na

• Serine.
ee

• Threonine.
m
ar

Simple : Branched chain : • Tyrosine.

um

• Glycine • Leucine
itk
am

• Alanine • Isoleucine
|

• Valine
w
ro
ar

III. Sulphur (S) containing : Iv. Acidic (-COOH) :

M

• Cysteine. • Glutamic acid (Glutamate).

©

• Methionine. • Aspartic acid (Aspartate).

v. Amides (CONH2) : vI. Basic (-NH2) :
• Glutamine. • Histidine.
• Asparagine. • Arginine.
• Lysine.

vII. Aromatic : VIII. Imino acid : (NH is a part of the ring)

• Benzene ring : Phenylalanine. • Pyrrolidine ring : Proline.
• Phenol ring : Tyrosine.
• Indole ring : Tryptophan (Benzene + Pyrrole).
Imidazole ring (2N) : Histidine.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Amino Acids 163

BASED ON SIDE CHAIN CHARACTERISTICS ----- Active space -----

Polar (Water soluble) Non-polar (Water insoluble)

Branched Chain :
Charged Uncharged • Leucine
Acidic : Amides : • Isoleucine
• Aspartate • Glutamine • Valine
• Glutamate • Asparagine Aromatic :
Basic : Hydroxyl group : • Phenylalanine
• Histidine • Serine • Tyrosine
• Arginine • Threonine • Tryptophan

om
• Lysine Simple : Glycine Simple : Alanine

l.c
Sulphur containing

ai
Sulphur containing : Cysteine

gm
: Methionine

@
19 Imino acid : Proline
55
na

BASED ON METABOLIC FATE

ee
m
ar
um

Purely ketogenic : Both ketogenic and glucogenic : Purely glucogenic :

itk

Lysine, Leucine Phenylalanine, Isoleucine, All other amino acids

am

Tyrosine, Tryptophan
|
w
ro
ar
M

BASED ON NUTRITIONAL REQUIREMENT

©

Essential : Semi-essential : Non-essential :

Methionine, Threonine, Tryptophan, Arginine All other amino acids
Valine, Isoleucine, Leucine,
Phenylalanine, Histidine, Lysine
Mnemonic : METT VIL PHLy

Standard & Derived Amino Acid  00:16:37

Standard vs derived aminoacid :

Standard amino acid Derived amino acid
Codon + -
Modification Cotranslational Post translational
Eg Lysine Hydroxylysine
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 164 Chemistry and Metabolism of Proteins

----- Active space ----- STANDARD AMINO ACIDS :

Selenocysteine Pyrolysine
Protein forming amino
21st 22nd
acid
Codon UGA (Stop codon) Re-coding Coding codon UAG
Precursor amino acid Serine Cysteine Se Seleno cysteine Lysine
Enzymes containing Thioredoxin reductase, deiodinase, glutathione
-
the amino acids peroxidase, selenoprotein P & glycine reductase
Mnemonic : Serena’s sister selena coming from Uganda.

DERIVED AMINO ACIDS :

Seen in proteins Not seen in proteins

om
l.c
Amino acid Characteristics Amino acid Characteristics

ai
gm
Protein : Collagen
- Ornithine
@
Hydroxylysine Formed by : Post-translational 19 Intermediate in
- Argininosuccinate
55
Hydroxyproline modification, hydroxylation (Needs urea cycle
na

- Citrulline
Vit-C)
ee
m

Derived from : Glutamate

ar
um

Function : Gamma carboxylation of

itk

clotting factors II, VII, IX, x, protein C,

am

Gamma carboxy
protein S (Needs Vit K) Intermediate of
|

glutamate
w

Homoserine,
• Matrix glutamic acid residue
ro

sulfur containing
ar

Homocysteine
(Matrix GLA), nephrocalcin, amino acid
M
©

osteocalcin
Desmosine Derived from lysine, found in elastin
Methyl lysine Derived from lysine, found in myosin

Properties of Amino Acid  00:26:12

1. Isomerism :
• Central carbon atom of amino acid : Chiral/asymmetric carbon atom
- Isomers D & L.
- Most aminoacids : L-form.
- Amino acid with no optical activity/asymmetric H
|
carbon atom : Glycine. NH2 C COOH
|

|
|

H
Note :
Most carbohydrates : D-form.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Chemistry of Amino Acids 165

2. Absorbs UV light 250-290 nm : ----- Active space -----

• Max. absorption : 280 nm
- Tryptophan > phenylalanine > tyrosine : D/t conjugated ring.
• Colourless : Do not absorb visible spectrum.

Note :
Substances that absorb UV light :
• DNA : 260 nm.
• NAD+ : 340 nm.
• Porphyrin : 400 nm, presence of Soret band.

3. Exist in different charged state :

Isoelectric point (pI) : pK1 + pK2 ; pK1 : Ionisation constant of COOH (COO-)

om
2 pK2 : Ionisation constant of NH2 (NH3+).

l.c
ai
gm
a. At pH = pI : Zwitter ionic state b. pH < pI

@
• Positive charge = Negative charge Protonated/positive charge
19
55
• No net charge No shell of
na

hydration Max. precipitability c. pH > pI

ee
m

min. solubility Deprotonated/negative charge

ar
um

• No mobility in electric field

itk
am

4. Buffering capacity :
|

Buffer : Solution that resist change in pH on addition of acid or alkali.

w
ro

Henderson Hasselbach equation :

ar
M

pH = pKa + log base (Or log ionised )

©

acid unionised

Titration curve :
a. Compound with single ionizable group, Eg : Acetic acid.
A : [Unionised] form of compound.
C B : [Ionised form] = [Unionised form].
Hence, pH = pKa Max. buffering
capacity :
PH
B
- Imidazole group of histidine : Has max.
buffering capacity (As pH of blood = pH
Partially ionized state of histidine).
C : Fully ionized form of compound.
A
Amount of alkali (OH-) added

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 166 Chemistry and Metabolism of Proteins

----- Active space -----

b. Compound with two ionisable groups :

II
pI = pK1 + pK2
PK2
PI 2
PH I pH = pI : Least buffering
PK1
pH = pKa : Max buffering

Addition of OH-

Protein  00:53:04

om
H H H H
| | | |

l.c
NH+
C COOH + NH3 C COOH
+
NH +
C CO NH C COOH

ai
|

gm |
|
|

|
| |
|

|
3
|

|
3
R1 R2 R1 R2

@
19 Peptide bond
55
Uncharged
na
ee

Partial double bond

m

Transisomeric form
ar
um
itk

STRUCTURE OF PROTEINS
am
|
w

Primary Secondary Tertiary Quaternary

ro
ar
M

• Linear sequence • 3-30 consecutive amino • 3 dimensional structure • Aggregation of

©

of amino acid acid for a polypeptide >1 polypeptide

• Peptide bond • Geometrical arrangement Eg : Eg : 2α-2β of Hb
• Covalent bond - Domain
- Rossmann fold in
oxidoreductase
- Immunoglobulin fold

α-helix : β-sheets : Joins two 2° structures

• M/c 2° structure • Extended zigzag
• Most stable • 2nd m/c 2° structure
• Right handed helical • Interchain hydrogen Loops Turns
structure bond
• Intrachain hydrogen bond
Long segment Short segment

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Amino Acids 167

• 3° & 4° structures are bound by non-covalent bonds, Vanderwaals’ force, ----- Active space -----
hydrophobic interaction.
• Subunit interaction : Specific to quaternary.
• Amino acid that disrupts α-helix : Proline
- Present only in 1st turn of α-helix.
• Amino acid that induces bends in α-helix : Glycine.

PROTEIN FOLDING
1° 2° 3° 4°

Molecular chaperones :
Auxiliary Proteins that help in protein folding.
Eg: - Heat shock proteins

om
- BiP : Immunoglobulin heavy chain binding protein.

l.c
- GRP-94 : Glucose regulated protein.

ai
gm
- Calreticulin

@
Calcium binding protein
- Calnexin 19
55
Note : Calpain Not a chaperone.
na
ee
m

Enzymes assisting folding :

ar
um

• Protein disulfide isomerase.

itk

• Peptidyl prolyl isomerase.

am
|

Protein misfolding diseases :

w
ro

1. Prion disease.
ar
M

2. Prion related protein disease :

©

- Alzheimer’s disease. - Huntington’s disease.

- Parkinson’s disease. - Fronto temporal dementia (FTD).
- Beta thalassemia. - Amyotrophic lateral sclerosis (ALS).
- Cystic fibrosis. - Dementia with Lewy bodies (DLB).
3. Amyloidosis.

PROTEIN DEGRADATION

Lysosomal degradation :
• ATP independent mechanism.
• Seen in :
- Long lived proteins.
- Extracellular proteins.
- Membrane protein.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 168 Chemistry and Metabolism of Proteins

----- Active space ----- Proteasomal degradation :

• Requires ATP.
• Seen in :
- Short lived proteins.
- Misfolded proteins.
• Presence of PEST sequence :
- Proline
Proteasomes
- E-glutamate binds with
+ degrades protein
- Serine 4 ubiquitin
into smaller peptides
- Threonine

PROTEIN DENATURATION
• Preservation of 1° structure : Covalent bonds are intact.

om
• Loss of 2°, 3°, 4° structure : Loss of biological activity.

l.c
ai
gm
@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Digestion and Absorption of Proteins 169

DIGESTION AND ABSORPTION OF PROTEINS ----- Active space -----

Digestion of Proteins 00:00:36

PROTEOLYTIC ENZYMES
• Break peptide bonds.
• Includes Proteases/Peptidases Class : Hydrolases.

Peptidase :

om
Endopeptidase : Exopeptidase :

l.c
ai
• Acts on peptide bond Acts on peptide bonds at the

gm
within polypeptide chain. ends of the polypeptide chain.
@
• Eg : Most proteases. 19
55
na

Carboxypeptidase : Aminopeptidase :
ee
m

Acts at the carboxyl end. Acts at the N-terminal.

ar
um

SITES OF PROTEIN DIGESTION

itk
am

Stomach :
|

1. Rennin/Chymosin :
w
ro

• Seen in infants (Absent in adults).

ar
M

• Casein (Milk protein) Rennin

©

Para casein.
Curdling of milk

2. Pepsin :
Chief cells Pepsinogen HCl Pepsin
(Stomach) (Zymogen : Inactive) (Active)

Proteins Pepsin Peptones + proteoses.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 170 Chemistry and Metabolism of Proteins

----- Active space ----- Pancreas :

Pancreatic juice : Contains zymogens.
• Trypsinogen : Acts at peptide bond formed by basic aa. (Arginine & lysine).
• Chymotrypsinogen : Acts at peptide bond formed by aromatic aa.
(Eg : Phenylalanine, tryptophan)
• Proelastase : Acts at peptide bond formed by small neutral aa (Alanine).
• Carboxypeptidase (Exopeptidase) : Produces small peptides.

Activation of zymogens :
Microvilli of intestine Enterokinase/enteropeptidase

Trypsinogen Trypsin

om
Trypsin

l.c
• Chymotrypsinogen Chymotrypsin.

ai
gm
Trypsin

@
• Proelastase Elastase.
19
55
na

Small Intestine :
ee
m

Succus entericus (Intestinal juice) :

ar

• Tripeptidase.
um

Completes protein digestion :

itk

• Dipeptidase.
am

• Aminopeptidase. Small peptides Amino acids.

|
w
ro
ar
M

Absorption of Proteins 00:08:54

©

Sodium dependent ATPase (Symports) :

Various sets for absorption of :
• Neutral amino acids.
• Basic amino acids (Arginine, lysine).
• Imino acids (Proline).
• Acidic amino acids (Aspartic acid, glutamic acid).
• Beta alanine.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Digestion and Absorption of Proteins 171

GAMMA GLUTAMYL CYCLE/ MEISTER’S CYCLE ----- Active space -----

Glutathione mediated process. aa : Amino acid

Amino acid

Cell membrane
Gamma Glutamyl
Transpeptidase/Transferase (GGT) : Transfers gamma glutamyl
of glutathione to aa

Glutathione Cysteinyl glycine Gamma glutamyl - aa

om
(Gamma-glutamyl-

l.c
cysteinyl-glycine) Glycine + Cysteine

ai
gm
aa

@
19 Glutamate
55
na
ee

Gamma-glutamyl-
m
ar

cysteine
um
itk
am

Note :
|
w
ro

Glutathione contains :
ar
M

1. γ Glutamic acid.
©

2. Cysteine.
3. Glycine.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 172 Chemistry and Metabolism of Proteins

----- Active space ----- PLASMA PROTEINS AND IMMUNOGLOBULINS

Plasma and Serum  00:01:00

Anticoagulant
• Albumin
Centrifugation Supernatant : Plasma • Globulin
• Fibrinogen

om
l.c
ai
Blood sample

gm
Clot retractor

@
(No anticoagulant) Supernatant : Serum • Albumin
19 • Globulin
55
na

Plasma - Fibrinogen = Serum

ee
m

Salting Out :
ar
um

Fractioning of plasma proteins:

itk

• Albumin.
am

• Globulin.
|
w

• Fibrinogen.
ro
ar
M

Electrophoresis :
©

Fractioning of serum proteins based on electric charge.

Method : Creating an electric field in a support medium (Agarose gel).

- Agarose Gel +
Cathode Anode

Point of application P rotein with maximum negative charge (Albumin)

migrates the fastest & furthest.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Plasma Proteins and Immunoglobulins 173

----- Active space -----

• α1-antitrypsin
55% • Cortisol binding protein
2.4% • Retinol binding protein

6-12% • α2-macroglobulin
• Haptoglobin
8-12% • Ceruloplasmin

12-22% • Transferrin
• Hemopexin
• Prothrombin

om
l.c
ai
gm
@
19
55
na
ee
m
ar

Abnormal Electrophoretic Patterns :

um
itk

Nephrotic Multiple Hepatic Chronic

am

syndrome myeloma cirrhosis infection

|
w
ro

• ↓ Albumin, β, γ • ↓ Albumin
ar

• ↑ α2 band • ↑ γ globulin
M
©

• All protein lost through urine M band (Monoclonal (Compensatory) ↑ γ globulin :

except α2-macroglobulin. band) at γ1 region. • β bridging (β fuses with γ1) wide & smooth
(Sharp spike) rise.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 174 Chemistry and Metabolism of Proteins

----- Active space ----- Functions of Plasma Proteins 00:14:32

Colloid Osmotic Pressure :

Albumin provides effective oncotic pressure.

Starling’s hypothesis :
Vessel

H2O

Hydrostatic force Colloid osmotic/Oncotic pressure

Intra Extravascular H2O Extra Intravascular

35 mmHg
15 mmHg
Capillary

om
Hydrostatic pressure

l.c
25 mmHg

ai
25 mmHg

gm
Colloid osmotic pressure

@
Arterial end : 19 Venous end :
55
na

• Hydrostatic P > Oncotic P. • Oncotic P > Hydrostatic P.

ee

• Water moves to • Water moves back to

m
ar

extravascular space. intravascular space.

um
itk

↓ Albumin :
am

• ↓Colloid oncotic pressure.

|
w

• Water expelled to extravascular space “Edema”.

ro
ar
M

Transport Proteins :
©

Plasma protein Compound being transported

• Bilirubin • Free fatty acid
Albumin
• Ca 2+
• Steroid hormones
• Thyroxine
Transthyretin
• Retinol
• Thyroxine
Thyroid binding globulin
• T3, T4
Cortisol binding globulin Steroids
Haptoglobin Extracellular hemoglobin
Hemoperin Heme
Transferrin Fe3+
Ceruloplasmin Cu2+

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Plasma Proteins and Immunoglobulins 175

Buffering Action : ----- Active space -----

Albumin :
• Major contributor in blood.
• Mediated by imidazole group in histidine.

Nutritional Purpose :
Albumin : Taken up by some cells for nutrition.

Other Functions :
• Hormones : Erythropoietin.
• Clotting factors.
• Defense mechanism : Immunoglobulins.

Clinical Applications of Plasma Proteins

om
00:23:54

l.c
ai
Kernicterus :

gm
• Albumin bound bilirubin Cannot cross Blood-Brain Barrier (BBB).
@
• ↑↑↑Unconjugated bilirubin (↓Binding sites) : Lipophilic 19
Can enter BBB
55
na

Kernicterus.
ee
m

Protein Bound Calcium :

ar
um

Binding site : Binding site :

itk

For Ca2+ For H+

am
|
w
ro
ar

Protein
M
©

Alkalosis : Acidosis :
• ↓H +
• ↑H +

• ↑binding site for Ca2+ • ↓binding site for Ca2+

↓Free/ionized Ca2+ ↑Free/Ionized Ca2+

Hypocalcemia Hypercalcemia
Edema :
↓Colloid osmotic pressure : H2O shifts to extravascular space.

NORMAL VALUES
• Total protein : 6-8 g/dl.
• Serum albumin : 3.5-5 g/dl.
• Serum globulin : 2.5-3.5 g/dl.
• Albumin globulin ration (A : G) : 1.5-2.5.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 176 Chemistry and Metabolism of Proteins

----- Active space ----- HYPOALBUMINEMIA

Causes :

↓Synthesis : ↑Loss : ↓Dietary intake :

• Hepatitis. • Intestine : P rotein losing Nutritional.
• Cirrhosis. enteropathy.
• Hepatocellular • Kidney : ↑
 glomerular permeability
carcinoma. (Proteinuria).

↓Albumin
↑Compensatory production of γ globulin.
• Reason : γ globulin synthesized from plasma cells (All other plasma proteins : Liver).

om
• Result : Reversal of A : G ratio.

l.c
ai
gm
@
HYPERGAMMAGLOBULINEMIA
Causes : 19
55
na

• Plasma cell tumors : Multiple myeloma.

ee
m

• Compensatory to ↑ d/t hypoalbuminemia.

ar

• Infection.
um
itk
am

MULTIPLE MYELOMA
|

Pathophysiology :
w
ro

Monoclonal neoplasm arising from plasma cell.

ar
M
©

↑γ globulin

↑Synthesis of abnormal immunoglobulin

(Impaired fusion of heavy chain and light chain)

Light chain excreted in urine :

Bence Jones Protein (BJP)
Note : Plasma cell Progenitor cell of B-cell lineage.
Forms
B lymphocyte.
Diagnosis :
Serum electrophoresis : M band in γ region.
Immunofixation electrophoresis : Type of immunoglobulin (M/c : IgG).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Plasma Proteins and Immunoglobulins 177

Urine BJP : ----- Active space -----

• Bradshaw’s test : Urine + HCl White ring at the junction.
• Heat test :
- Principle : BJP precipitates b/w 45-60°C.
- Procedure : Urine in test tube is heated.
Forms precipitate (45-60°C).

Further heating dissolves precipitate (>60°C).

On cooling, precipitate appears (45-60°C).

Later disappears (<45°C).

Serum tests :

om
• ↑Serum alkaline phosphatase : ↑D/t bone marrow involvement Lysis of

l.c
ai
bone.

gm
@
• ↑Serum calcium : D/t lysis of bone.
• 19
55
↑ESR.
na

• ↑Serum free light chain assay (Quantitative).

ee
m

• Serum β2 microglobulin : Determines prognosis.

ar
um

Acute Phase Reactants 00:39:50

itk
am

Plasma proteins that can increase/decrease in response to infection/inflammatory

|
w
ro

condition.
ar
M
©

Positive acute phase reactants Negative acute phase reactants

• CRP : C - reactive protein • Albumin
• Ceruloplasmin • Transthyretin (Prealbumin)
• Alpha 1 antitrypsin • Transferrin
• Alpha 2 macroglobulin

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 178 Chemistry and Metabolism of Proteins

----- Active space ----- Immunoglobulins 00:41:30

Plasma proteins synthesized by plasma cells in response to antigen.

STRUCTURE
Contain 2 heavy chains & 2 light chains
Variable region : Towards N terminal.
N terminal Constant region : Towards carboxyl end.
Hinge region : Connects CH1 & CH2.
VH VH
VL VL Disulphide bonds : Connect hinge region.
CH1 CH1 Fab region :
CL
CL Hinge region • Towards N terminal.
• Seen in variable region of light & heavy chain.

om
CH2 CH2
• Function : Ag binding.

l.c
ai
Fc region (Fraction crystallizable) :

gm
CH3 CH3
Carboxyl end
• Function : Complement binding region.
@
19
55
Key :
na

V : Variable L : Light chain

ee
m

C : Constant H : Heavy chain

ar
um
itk

Types of Heavy Chain & Ig Classification : Types of Light Chains :

am

• g : IgG k (Kappa)
|

Either 2k or 2l
w

• a : IgA
ro

l (Lambda)
ar

• μ : IgM
M
©

• d : IgD
• e : IgE
PEPSIN & PAPAIN DIGESTION
Action of Papain on Ig : Action of Pepsin on Ig :
F(ab’)2

Fab
Papain
site Pepsin Fragments
Fc site

• Cleaves below disulfide bond. • Cleaves after disulfide bond.

• 2 Fab fragments : 2 Fab. • Single Fab fragment (Fab)2
• Single Fc fragment. • Fc fragment cut at multiple sites.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Plasma Proteins and Immunoglobulins 179

TYPES OF IMMUNOGLOBULIN ----- Active space -----

Ig Structure Features
• Classical Y shaped structure
• Monomer
• Versatile : Can perform all functions of immunoglobulins
• Most abundant (75-80%) in serum
IgG
• Main antibody in secondary immune response
• Only class of IgG that crosses placenta
• Effective activator of classical complement pathway
• Involved in opsonization (Fc)

• Types :
- Serum IgA : monomer

om
- Secretory IgA : Dimer joined by J chain

l.c
IgA • Main effector of mucosal immune system

ai
gm
• Most abundant Ig in body secretions : Saliva, tears,

@
colostrum and gastrointestinal secretions
19
• Complement fixation -
55
na
ee
m
ar

• Heavy chain : μ
um

• Largest Ig : Pentamer joined by J chain

itk

• First Ig synthesized by fetus

am

IgM
• Ig involved in primary immune response
|
w

• Most effective activator of classical complement

ro

pathway
ar
M
©

• Monomer
• Heavy chain : δ type
• Low levels in serum
IgD
• Function uncertain
• Primarily found on B cell surface : Receptor for Ag
• Complement fixation -
• Monomer
• Heavy chain : ε
• Cytophilic AB
• Least common Ig in serum
IgE
• Involved in allergic reactions : Binds to basophils and
mast cells Release histamine.
• Role in parasitic helminthic diseases
• Complement fixation -

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 180 Chemistry and Metabolism of Proteins

----- Active space ----- Summary :

• M/c in serum :
• L/c in serum :
• Largest :
• Pentamer :
• Dimer :
• Fixes complement :
• Present in secretion :
• Shape of monomer :
• Crosses placenta :
• IgG not crossing placenta :
• Primary immune response :

om
• Secondary immune response :

l.c
• With J chain :

ai
gm
• With secretory piece :

@
- Synthesized in mucosal cells. 19
55
- Protects against proteolytic enzymes.
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Formation, Transport and Detoxification of Ammonia 181

FORMATION, TRANSPORT AND DETOXIFICATION ----- Active space -----

OF AMMONIA

NH2 H Co2
α | α
O=C-H NH2- C - COOH NH2-CH2
| |
(R) Aminoacid (R) Carboxylic acid (R)
NH2 Co2
α ketoacid Amino acid Biologically Important amines
PLP
Pyruvate Alanine Tyrosine Tyramine.
Oxaloacetate Aspartate Tryptophan Tryptamine.

om
α-ketoglutarate (α KG) Glutamate Glutamate Gamma amino butyric

l.c
ai
acid.

gm
Histidine Histamine.
@
Co2 19
55
na

DOPA Dopamine Epinephrine, norepinephrine.

ee

PLP
m

Deficiency of PLP :
ar
um

• ↓ GABA (neurotransmitter)
itk

Pyridoxine dependent seizures

• ↓ Epinephrine, Norepinephrine
am
|
w
ro

Handling of amino group  00:06:24

ar
M

Major source of amino group :

©

α-amino acid Release NH3 (toxic)

NH3 NH3
α KG Glutamate Glutamine
(Intermediate in
Co2
TCA cycle)
GABA
(Inhibitory neurotransmitter)
• α-ketoglutarate (KG) : Replenishes glutamate.
↓ α KG ↓ TCA cycle.
• ↑ NH3 ↓Glutamate
↑ Glutamine Attracts water into brain Edema + Encephalopathy.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 182 Chemistry and Metabolism of Proteins

----- Active space ----- Transamination  00:10:33

Transfer of amino group from aminoacid to ketoacid, to generate a new aminoacid

& a ketoacid.
α-Amino acid α-ketoacid
(Substrate 1) (Substrate 2)
α-keto-acid α-amino acid
(Product 1) (Product 2)

Site : All Organs. Characteristics :

- Organelle : Cytoplasm. • Reversible.
Co-enzyme : PLP (B6). • NH3 is not freely released.
• Enzyme : Transaminase/

om
Aminotransferase.

l.c
ai
gm
Eg :

@
a. Alanine α-ketoglutarate
19
55
PLP Alanine aminotransferase (ALT)/SGPT
na

Pyruvate Glutamate
ee
m
ar
um

b. Aspartate α-KG
itk

PLP Aspartate aminotransferase (AST)/SGOT

am

Oxaloacetate Glutamate
|
w

(OA)
ro
ar

Specific Non-specific
M
©

Properties :
1. Specific for one pair of substrate (Alanine to pyruvate, aspartate to
oxaloacetate), non-specific for the other pair (α KG to glutamate).
2. Concentrate toxic amino group as non-toxic glutamate.
3. Bi-bi reaction (2 substrate, 2 product) : Ping pong mechanism.
• Transaminase.
• Oxidation-reduction reaction.
4. Biosynthesis of nutritionally non-essential aminoacid.
• α KG Glutamate.
• Pyruvate Alanine.
• OA Aspartate.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Formation, Transport and Detoxification of Ammonia 183

Transamination of non-alpha amino acids : ----- Active space -----

δ Ornithine aminotransferase.

Deficiency : Gyrate atrophy of retina & choroid.

Rx :
• Substrate reduction : Limit ornithine and its source (Arginine).
• Supplement PLP (vit B6).

Aminoacids not undergoing transamination :

• Proline.
• Hydroxyproline.
• Lysine.
• Threonine.

om
l.c
TRANSPORT OF AMMONIA

ai
gm
In brain & other organs :

@
Glutamine
Source: synthetase 19
55
α-amino acid detoxified Glutamate Glutamine (transport form of
na

Other sources :
ee

ammonia)
m

Amino sugar, Purines, ATP ADP

ar

NH3 Glutamine synthetase :

um

pyrimidine, Porphyrin
• Ligase.
itk
am

• First line trapping of ammonia.

|

• Takes place in mitochondria.

w
ro

In skeletal muscle :
ar
M

α-amino acid α-KG Pyruvate

©

α-ketoacid Glutamate Alanine (Transport form of ammonia)

Fate of glutamine and alanine :
Glutamine & alanine Alanine Liver Glutamate
Glutaminase
NH3 Urea Cycle
Pyruvate α-KG
Glutamine

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 184 Chemistry and Metabolism of Proteins

----- Active space ----- Oxidative Deamination  00:32:32

Site : Liver & Kidney.

- Organelle : Mitochondria Note : Only glutamate can undergo
Glutamate significant oxidative deamination.
dehydrogenase(GDH)
Glutamate α KG
NH3 urea cycle
NAD(P+) NAD(P)H
(freely released)
• Reversible reaction :
Allosteric

Inhibitor : Activator :

om
NADH, ATP, GTP ADP

l.c
ai
gm
Transdeamination : Transamination Coupled with oxidative deamination.

@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Urea Cycle and its Disorders 185

UREA CYCLE AND ITS DISORDERS ----- Active space -----

Aka Kreb’s Henseleit cycle, Ornithine cycle, urea bi-cycle, Kreb’s bi-cycle.
Fumarate

Urea cycle Kreb’s/TCA cycle

Aspartate
Urea : NH2 - CO - NH2
Source : NH3 Respiratory CO2 Aspartate from TCA cycle

om
Site : Liver.

l.c
Organelle : Cytoplasm & mitochondria.

ai
gm
Note : Reactions taking place in both cytoplasm & mitochondria.

@
Mnemonic : PUBG 19
55

• Pyrimidine synthesis. • Blood : Heme synthesis.

na
ee

• Urea cycle. • Gluconeogenesis.

m
ar
um

Reactions of Urea Cycle 00:05:32

itk
am
|

CO2 + NH3
w
ro
ar

I. Carbamoyl phosphate synthetase I (CPSI) : Rate limiting

M

Mitochondria 2 ATP
enzyme
©

2 ADP
NAG
Carbamoyl phosphate TCA cycle
II. Ornithine
Aspartate
Ornithine transcarbamoylase (OTC) Citrulline
Ornithine (VII) Citrin transporter(VI)
transporter
Citrulline + Aspartate
Ornithine
Urea Arginase (V) 1 ATP
1 AMP AS synthetase (III)
H2O Argininosuccinate (AS)
Cytoplasm Arginine
AS lyase (1v)

Fumarate (C4)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 186 Chemistry and Metabolism of Proteins

----- Active space -----

Energy expenditure :

Enzyme ATP High energy phosphates

CPS I 2 2
AS Synthetase 1 2
Total Direct : 3 ATP 4 high energy PO4= 4 ATP equivalence

Regulation :
• No hormonal regulation.
• ↑ protein in diet Induces enzymes of urea cycle.
• Allosteric regulation :
- NAG : N-acetyl glutamate.

om
- Obligate allosteric activator.

l.c
• Compartmentation : Partly cytoplasm & mitochondria.

ai
gm
@
Urea cycle disorders 19 00:16:17
55
na

Depiction Defect Disorder

ee
m

I CPS I Hyperammonemia Type I

ar
um

II OTC Hyperammonemia Type II

itk

III AS synthetase Citrullinemia Type 1

am

IV AS Lyase Argininosuccinic aciduria

|
w
ro

V Arginase Argininemia
ar
M

VI Citrin transporter Citrullinemia Type 2

©

VII Ornithine transporter HHH syndrome

Hyperammonemia type II :
• M/c urea cycle disorder (40%).
• X-linked recessive partially dominant (only males affected).

shunted for
Carbamoyl phosphate Pyrimidine synthesis

Excretion of pyrimidines Accumulation of orotic acid

in urine
Orotic aciduria

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Urea Cycle and its Disorders 187

Hyperammonemia and plasma glutamine : ----- Active space -----

• Glutamine is the transport form of ammonia.
• Hyperammonemia Hyperglutaminemia.
↑ NH3 + Glutamate ↑ glutamine
HHH syndrome :
• Accumulation of ornithine in cytoplasm : Hyperornithinemia
• Accumulation of NH3 : Hyperammonemia
• Carbamoyl phosphate + Lysine Homocitrulline (Accumulation) Homocitrullinemia

Arginemia :
• Least hyperammonemia.

om
• Spastic diplegia/quadriplegia.

l.c
• Scissoring gait : Characteristic.

ai
gm
@
Argininosuccinic aciduria :
19 Scissoring of lower limb
55
• Trichorrhexis nodosa : Brittle hair.
na

- Alopecia, soft, pliable, easily pluckable hair, tufted hair.

ee
m
ar

CLINICAL FEATURES OF UREA CYCLE DISORDER

um

Encephalopathy :
itk
am

• Depletion of α-ketoglutarate.
|

• ↑ GABA.
w
ro

• Accumulation of water in brain Edema.

ar

Trichorrhexis nodosa
M

Respiratory alkalosis :
©

Tachypnea CO2 washout ↓ H2CO3 Respiratory alkalosis.

Hyperammonemia
In neonates :
Feeding difficulties, failure to thrive, lethargy, convulsions, coma.

MANAGEMENT
Investigation :
↑/N : urea cycle disorder
1. pH of blood
↓: Organic aciduria
2. Serum ammonia levels : ↑
3. Tandem mass spectrometry : Gold standard of all metabolic disorders.
(or)
High performance/pressure liquid chromatography (HPLC) : High sensitivity.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 188 Chemistry and Metabolism of Proteins

----- Active space -----

• Interpretation :

Specific AA↑↑↑ No ↑ in specific AA

- ↑Citrulline : Citrullinemia. Do step 4

- ↑ Ornithine : HHH syndrome.
- ↑ Arginine : Argininemia.
- ↑ Arginosuccinate : Arginosuccinic. aciduria

↑↑ : Hyperammonemia type II
4. Plasma orotic acid
N : Hyperammonemia type I
Treatment:

om
Arginine supplementation : C/I in arginase defect.

l.c
• First line d/t

ai
gm
@
Arginase
- Arginine 19
Ornithine (catalytic role)
55
na

Urea
ee

- Activator of NAG synthase : Allosteric activator of urea cycle.

m
ar

Acylation therapy
um

• Sodium benzoate + glycine Benzoyl glycine Excretion

itk
am

Glycine synthase
|
w

NH3 +CO2 + 1C group

ro
ar
M

• Phenyl butyrate Phenylacetate + glutamine Phenylacetyl glutamine

©

(Prodrug) (transport of NH3 / ammonia (water soluble)

scavenger)
Excreted in urine

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Aromatic Amino Acids 189

AROMATIC AMINO ACIDS

----- Active space -----

Salient features :
Phenylalanine : Tyrosine :
• Essential amino acid. • Non essential amino acid (synthesized from phenylalanine).
• Both ketogenic & glucogenic. • Both ketogenic and glucogenic.
• Non polar. • Least non polar among aromatic amino acids.

Metabolism :
Phenyl alanine Hydroxylase (PAH)
Phenyl alanine Tyrosine

Catabolic pathway Anabolic pathway

om
• Ketogenic. • Melanin.

l.c
ai
• Glucogenic. • Catecholamines.

gm
• Thyroid hormones.
@
Conversion of phenylalanine to Tyrosine : 19
55
na
ee

PAH
m

Phenylalanine Tyrosine
ar

• Irreversible reaction.
um
itk

• PAH is a monooxygenase.
am

• Metabolic functions of phenylalanine : Carried out by tyrosine.

|
w
ro

Catabolic Pathway
ar

00:05:25
M
©

02 H2O
PAH
Phenyl alanine Tyrosine Tyrosine transaminase Para-hydroxyphenyl pyruvate (PHPP)
PLP
PHPP hydroxylase
Tetrahydrobiopterin (BH4) (BH2) Dihydrobiopterin (4-hydroxyphenyl pyruvate
Dihydrobiopterin reductase dioxygenase)
NADP + NADPH Homogentisic acid
Homogentisate
oxidase/ dioxygenase
(Dihydroxyphenyl acetate
dioxygenase)
FAA Fumaryl MAA Maleylacetoacetate (MAA)
Fumarate + Acetoacetate
hydrolase isomerase
(Glucogenic) (Ketogenic) acetoacetate
(FAA)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 190 Chemistry and Metabolism of Proteins

----- Active space ----- Metabolic disorders :

1. Classic phenylketonuria (Type I) : Defect in phenylalanine hydroxylase.
2. Non classic phenylketonuria (Type II and III) : Defect in dihydrobiopterin. reductase.
3. Non classic phenylketonuria (Type IV and V) : Defect in GTP
Tetrahydrobiopterin.
4. Alkaptonuria : Defect in homogentisate oxidase/ dioxygenase (AKA
Dihydroxyphenyl acetate dioxygenase).
5. Tyrosinemia :
Type I : Defect in FAA hydrolase.
Type II : Defect in tyrosine transaminase.
Type III : Defect in PHPP hydroxylase (AKA 4-hydroxy phenyl pyruvate dioxygenase).
6. Hawkinsinuria :

om
• Partial defect in PHPP hydroxylase.

l.c
• Swimming pool odour +.

ai
gm
CLASSIC PHENYLKETONURIA
@
Biochemical defect : 19
55
na

↓catecholamines
Neurological
ee
m

Defect in Phenylalanine hydroxylase No tyrosine ↓thyroid hormones deficits

ar
um

synthesis
↓ melanin Hypopigmentation
itk
am

Phenylalanine Transamination Phenylpyruvate (Ketoacid)

|
w
ro
ar
M

Phenyl lactate Phenyl acetate

©

Mousy/ barny/ musty/

wolf like body odour

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Aromatic Amino Acids 191

Alternative mechanism of neurological deficits : ----- Active space -----

Phenylalanine > Tyrosine, Tryptophan

↑entry of phenylalanine into the brain

through common transporters

↓tyrosine and tryptophan in the brain

↓neurotransmitters

Neurological deficits
Clinical features :

om
• M/c amino acid metabolic disorder.

l.c
ai
• Autosomal recessive.

gm
Presentation :
@
• Intellectual disability. 19
• Severe vomiting in infants (D/d : Pyloric stenosis).
55
na

• Hypopigmentation. • Mousy / barny / wolf like / musty body odour.

ee

• Agitation. • Hyperactivity.
m
ar
um

Investigations :
itk

1. Guthrie’s test :
am

AKA Bacterial inhibition test

|
w
ro

Bacillus subtilis (obligatory need for phenylalanine)

ar
M
©

Growth in blood culture

Indicates phenylalanine +
2. Blood phenylalanine estimation :
• 2-6 mg/dL : Normal.
• >20 mg/dL : Poor prognosis.
3. Enzyme studies.
4. Ferric chloride test :
Presence of phenyl pyruvic acid in urine

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 192 Chemistry and Metabolism of Proteins

----- Active space ----- 5. Tandem mass spectrometry : Gold standard.

6. High performance liquid chromatography.
Treatment :
1. Phenylalanine restricted diet (Cassava based diet).
2. Tyrosine supplementation.
3. Synthetic tetrahydrobiopterin (Sapropterin dihydrochloride / KUVAN).
4. Concentrate of large neutral amino acids (↑tyrosine and tryptophan)
↑entry of tyrosine and tryptophan into brain ↑neurotransmitters
ALkAPTONURIA
Garrod’s tetrad :
• Cystinuria. • Alkaptonuria. • Albinism. • Pentosuria.

om
Biochemical defect :

l.c
ai
Defect in homogentisate oxidase/ dioxygenase (AKA dihydroxyphenyl acetate

gm
dioxygenase)
@
Homogentisate Diverted to 19
Benzoquinone acetate
55
form
na
ee
m

Maleyl acetic acid Alkaptone bodies

ar
um
itk

Urinary excretion
am

Accumulation in Accumulation in cartilage

|

intervertebral disc & elastic tissue

w
ro

Black discoloration of
ar
M

urine on standing/ Ochronosis Black pigmentation of

©

alkalanisation
Ochronotic arthritis Skin Sclera Pinna

Backache
Clinical features :
Age of onset : Middle age
Presentation :
• Low backache.
• Black pigmentation of skin, sclera, pinna.
• Darkening urine (on standing/alkalanisation).
• No intellectual disability.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Aromatic Amino Acids 193

Investigations : ----- Active space -----

1. Ferric chloride test :
Positive test : Blue/black colour.
2. Silver nitrate test : Black colour precipitate
3. Alkalinisation of urine.
4. Benedict’s test : Positive.
5. X-ray :
• Parrot beak appearance. • Bamboo like spine.
Treatment :
Nitisinone (NTBC) :
Inhibits PHPP hydroxylase and ↓ Homogentisate.

om
TYROSINEMIA

l.c
Type 1 :

ai
gm
AKA Hereditary/hepatorenal tyrosinemia.

@
• M/C type of tyrosinemia. 55
19
• Resembles porphyria.
na
ee

Biochemical defect :
m
ar

Defect in FAA hydrolase ↑ FAA Succinyl acetone Hepatic and renal toxicity
um
itk

Presentation : Hepatic & renal failure.

am

Treatment : Nitisinone.
|
w
ro
ar

Type 11 :
M

AKA oculocutaneous tyrosinemia/Richner Hanhart syndrome.

©

Biochemical defect :
Defect in tyrosine transaminase.
Presentation :
• Ocular : Corneal ulcer (Poor staining with fluroscein) Corneal opacity
• Cutaneous : Non pruritic hyperkeratotic plaques on soles and palms.
Type 111 :
AKA neonatal tyrosinemia
• L/C type of tyrosinemia.
• Biochemical defect : PHPP hydroxylase.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 194 Chemistry and Metabolism of Proteins

----- Active space ----- Anabolic Pathway 00:52:57

CATECHOLAMINES
Synthesis :
Steps :
Tyrosine Tyrosine Dihydroxy DOPA decarboxylase Dopamine
hydroxylase PLP Dopamine β
phenylalanine
hydroxylase
(DOPA) Norepinephrine
N methyl SAM
transferase SAH
Site :
Epinephrine
• Epinephrine : Adrenal medulla (80%)
• Norepinephrine : Extra adrenal sites

om
Nerve endings.

l.c
ai
Sympathetic ganglia.

gm
@
Degradation : 19
55
Homovanillic acid (HVA) Dopamine
na
ee

COMT
m

) Norepinephrine
(MAO Normetanephrine
ar

Vanillyl mandelic
um

Mono
am
itk

acid (VMA) oxida ine Catecholamine O

methyl transferase Epinephrine
am

se Metanephrine
(MAO
)
|

(COMT)
w
ro

Pheochromocytoma :
ar

• Neuroendocrine tumour.
M
©

• AKA the great masquerader (Variable clinical presentation).

Biochemical defect :
Tumour in adrenal medulla ↑ catecholamines ↑ degradation products

• ↑24 hr urine VMA

• ↑ metanephrine
Clinical features :
Classic triad :
Palpitations

Hypertension
Headache Profuse sweating
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Aromatic Amino Acids 195

Investigations : ----- Active space -----

1. 24 hour urinary tests :
a. VMA (Most specific).
b. Catecholamines.
c. Fractionated metanephrines (Most sensitive).
d. Total metanephrines.
2. Blood tests :
a. Catecholamines.
b. Free metanephrines (most sensitive).
MELANIN
Synthesis :
Steps :

om
Tyrosine

l.c
ai
Cu Tyrosinase

gm
DOPA
@
Cu Tyrosinase 19
55
na

Dopaquinone
ee
m
ar

Melanin
um
itk

Site : Melanosomes in melanocytes in stratum basale layer of dermis

am

Albinism :
|
w
ro

Autosomal recessive.
ar
M

Biochemical defect :
©

Deficiency of tyrosinase Absence of melanin Depigmentation

Note : In PKU Hypopigmentation is d/t ↓tyrosine
(But dietary tyrosine Melanin)

Clinical features :
• Milky white skin.
• Milky white hair.
• Photophobia.
• Lacrimation.
Function :
Pigmentation of skin and hair.
• Tyrosinase : Contains copper.
• Copper deficieny Hypopigmentation.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 196 Chemistry and Metabolism of Proteins

----- Active space ----- SULPHUR CONTAINING AMINO ACIDS

Cysteine & Methionine  00:00:30

Cysteine Methionine
H H
| |
NH2- C - COOH NH2-C - COOH
| |
SH CH2
Structure |
Sulfhydryl/Thiol/ CH2
|
S
Thioalcohol group | Thioether linkage

om
CH3

l.c
ai
• Glucogenic • Glucogenic

gm
Type of • Polar aminoacid • Non-polar (Terminal group
@
amino acid • Non-essential (Can be 19 is CH3)
55
na

synthesized from methionine) • Essential

ee
m

HOMOCYSTEINE
ar
um

S-adenosyl methionine (SAM) :

itk

Homocysteine + Methyl group = Methionine

am
|
w
ro

Adenosine ThioEther linkage : Covalent bond

ar
M

S-adenosyl methionine
©

Metabolic pathway : Methionine adenosyl transferase

Methionine S-adenosyl methionine

(Principle methyl donor)
THFA Methyl
B12
N5 CH3
Methyl B12
THFA Homocysteine
methyl Transferase/ Homocysteine S-adenosyl homocysteine
Methionine Synthase +
serineS-adenosyl
Cystathionine + B homocysteinase
6
β synthase
Cystathionine
Cystathionase + B
6
Homoserine + Cysteine
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Sulphur Containing Amino Acids 197

Disorders : ----- Active space -----

Defect in Disorder
Transport of methionine Oast house syndrome/Smith strang disease
Primary hypermethioninemia :
MAT
Boiled cabbage smell
Cystathionine β synthase Classic homocysteinuria
Cystathionase Cystathioninuria
• Methylene THFA reductase
Non-classic homocysteinuria
• Methyl B12
Reabsorption of cysteine in
Cystinuria
kidney
Deficiency of B12/Folic acid/B6 ↑ Homocysteine CAD/CVA.

om
l.c
Folate trap :

ai
Methionine

gm
THFA B12

@
Trapped N5 Methyl THFA Me B12 19
B12 deficiency
55

Homocysteine
na
ee

THFA starvation/
m
ar

Functional deficiency
um

of THFA
itk
am

Folic acid deficiency

|
w
ro
ar

Classical homocysteinuria :
M

Biochemical defect :
©

Arachnodactyly : Elongated fingers

• ↑ Homocysteine in blood.
• ↑ Homocysteine in urine.
• ↓↓ Cysteine synthesis.
Clinical features :
• Asymptomatic (Initially). • Mental Retardation.
• Skeletal deformities : • Developmental delay.

Tall stature High arched palate Pectus Carinatum Pectus excavatum

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 198 Chemistry and Metabolism of Proteins

Genu varum.
----- Active space -----
- knee
Genu valgum.
- Pes cavus/planus.
- Scoliosis.

Pes cavus
• Dislocation of lens • Thromboembolism :

om
l.c
Risk factor for atherosclerosis

ai
gm
@
19
55
na
ee
m
ar
um
itk

Ectopia lentis :
am

Downward & medial

|
w

Note :
ro
ar

D/d : Marfan syndrome

M

• No mental retardation.
©

• Superotemporal dislocation of lens.

Lab diagnosis :
• Cyanide nitroprusside test : Magenta color Positive test.
• Enzyme studies.
• Genetic mutation studies.
Treatment :
• Cysteine supplementation.
• Methionine (Substrate) restriction.
• High dose of Vit B6 (Co-enzyme of cystathionine β synthase).
• Vit B9 , B12 supplementation (Augments Homocysteine Methionine).
• Betaine supplementation
Trimethyl glycine methyl donor Homocysteine Methionine.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Sulphur Containing Amino Acids 199

Classic vs Non-classic homocysteinuria : ----- Active space -----

Classic Non-classic
Defect CBS B9 , B12 MTHFR
Cysteine ↓ Normal

Cystinuria :
Defect : Dibasic amino acid transporter in kidney.
• Reabsorb dibasic aminoacid : COLA
- Cystine
- Ornithine
Excreted
- Lysine
- Arginine

om
• Part of Garrod’s tetrad.

l.c
ai
gm
Cystinosis :

@
Lysosomal storage disorder.
19
55
Defect :
na

• H+ driven lysosomal cystine transporter.

ee
m

• CTNS gene Cystinosin.

ar
um

Clinical manifestations :
itk

• Liver failure. • Corneal opacity.

am

• Kidney failure. • Bone marrow suppression.

|
w
ro

Specialised products from cysteine :

ar
M

• Betamercaptoethanolamine (Component of Co-A).

©

• Co-A.
• Pantothenic acid.
• Taurine : Conjugation of bile acids.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 200 Chemistry and Metabolism of Proteins

----- Active space ----- Glutathione  00:39:56

Tripeptide, Pseudopeptide/Atypical peptide.

3 amino acids : Glutamic acid + Cysteine + Glycine.
AKA Gamma glutamyl cysteinyl glycine (GSH).

(G) (SH)
Functions of glutathione :
1. Amino acid transport :
• Meister’s cycle/Gamma glutamyl cycle.
• Used in intestine, brain, kidney.
• Utilizes ATP.

om
2. Free radical scavenging :

l.c
Reduced glutathione NADP+

ai
H2O2 1. Glutathione peroxidase

gm
1. 2.
H2O Oxidized glutathione NADPH + H+ 2. Glutathione reductase
@
19 HMP shunt
55
na
ee

3. Conjugation / Phase II reactions :

m

Used in xenobiotics (Preservatives, drugs).

ar
um
itk

4. Coenzyme role :
am

Maleyl acetoacetate Cis-trans isomerase Fumaryl acetoacetate.

|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Tryptophan 201

TRYPTOPHAN ----- Active space -----

Chemistry : Molecular structure :

• Aromatic amino acid. H
• Non polar amino acid. |
NH2-C -COOH
• Essential amino acid : Cannot be |
CH2
synthesised in vivo. |
C =CH
| Pyrrole ring
C = NH Benzopyrrole/
Benzene ring Indole ring

om
Metabolism of Tryptophan 00:02:10

l.c
ai
gm
FATE OF TRP Tryptophan (Trp)Trp pyrrolase

@
19
55
na

Anabolic fate : Catabolic fate (Both ketogenic & glucogenic)

ee

Tryptophan
m

Quinolinate phophoribosyl
ar

Trp hydroxylase Gluconeogenesis Ketone body synthesis

um

transferase (QPRTase)
itk
am

Niacin (Vitamin B3) : 5' OH Trp

• Trp : Only amino acid PLP Decarboxylase
|
w
ro

that can be converted Serotonin

ar

to a vitamin
M

(Neurotransmitter)
©

SAM
N-methyl transferase
Melatonin
(Neurotransmitter responsible
for biological rhythm)

Catabolic fate-Trp Trp pyrrolase is a heme containing enzyme

Donor of formyl group (Formyl tetrahydrofolic acid) 1 carbon atom donor

Vit. B6 deficiency can lead to (Branch of Niacin (Vit. B3) deficiency

catabolic pathway diverting to anabolism) : : Pellagra
Required for kynureninase enzyme Xanthurenic aciduria :
Niacin is an endogenous vitamin Trp enters alternative
metabolic pathway
Conversion factor : 60 mg of Trp to 1 mg of Niacin
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 202 Chemistry and Metabolism of Proteins

----- Active space ----- Neurotransmitters from Trp :

Serotonin Melatonin
Site of Argentaffin cells : Intestine,
Pineal gland
synthesis platelets, mast cells, brain.
• Vasoconstriction. • Biological rhythm :
• Neurotransmitter. ↓ melatonin Insomnia
Function • Temperature regulation. • Neurotransmitter.
• Mood elevation. • Antioxidant (Free radical
• Gastrointestinal tract motility. scavenger).

Note :
• Candle light

om
• Incandescent light ↑ production of melatonin

l.c
ai
gm
@
Biochemical Defects 19 00:21:27
55
na

1. Carcinoid syndrome/tumour :
ee
m

Neuroendocrine tumour of Argentaffin cells.

ar
um

Pathogenesis and features :

itk
am

Enters
|

Blood Trp Argentaffin cells ↑ production of serotonin

w
ro
ar
M

• Intermittent flushing D/t ↑ tachykinins vasodilation.

©

• Diarrhea.
• Sweating.
• Lacrimation.
• Feeling of warmth.
• Facial edema.

↓ production of Niacin

Pellagra like symptoms : Cutaneous photosensitivity Deep red/violaceous

• Rashes pruritis erythematous rash in sun exposed areas

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Tryptophan 203

Diagnosis : ----- Active space -----

• ↑ Serum serotonin.
• ↑ 24-hr 5-hydroxy indole acetic acid (5HIAA) : Degradation product of serotonin.
• Neuroendocrine markers :
- Serum chromogranin A.
- Neuron specific enolase.
- Serum synaptophysin.

2. Hartnup disease :

Etiopathogenesis and features :

Mutation of SLC6A19 gene

om
BOAT-1 protein (Trp transporter)

l.c
defect in intestine & kidney

ai
gm
↓ serotonin synthesis Neurological manifestations :
Excess Trp Not absorbed ↓ blood Trp

@
• Intermittent ataxia
19 ↓ niacin synthesis
55
• Wide based gait
na

• Dermatitis Photosensitive dermatitis

ee

Bacterial • Diarrhoea
m

decomposition • Dementia
ar
um

Cutaneous photosensitivity (M/c symptom) :

itk

• Rashes in sun exposed areas

am

• Casal's necklace
|
w

Indoxyl compounds (Blue coloured) : Absorbed

Renal Excreted in urine Bluish discolouration
ro

System
• Indigoblue
ar

circulation circulation of diaper

M

• Indican
©

Intestinal lumen Bloodstream

Note : Most cases are asymptomatic.

Diagnosis :
• ↓ S. serotonin.
• ↓ Niacin levels.
• Obermeyer test for indican.

Treatment : Lipid soluble esters of Trp Easily absorbed into bloodstream.

3. Drummond syndrome/Blue diaper syndrome :

BOAT-1 transporter defective only in intestine.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 204 Chemistry and Metabolism of Proteins

----- Active space ----- MISCELLANEOUS AMINO ACIDS

Simple Amino Acids 00:00:20

GLYCINE
Properties :
• Simplest amino acid.
• Polar amino acid.
• Non-essential.
• Purely glucogenic.
• Optically inactive.

om
l.c
ai
Synthesis :

gm
Glycoxylate Threonine

@
(α-keto acid of glycine) 19

e
55

las
ldo
na

ea
ee

nin
m

reo
ar

1C
Th
um

Serine Glycine CO + NH3+ 1 C group

itk

Serine hydroxy Glycine (M/C : N2 N methylene THFA)

(3C) (2C)
am

5 10
methyl transferase synthase
|

• Needs PLP and Folate

w
ro
ar

Metabolic Functions :
M

a) Synthesis of :
©

Entirely
1. C4, C5 & N7 of Purine (Glycine converted Purine)
2. Heme
3. Glutathione : Glutamic acid + Cysteine + Glycine
4. Collagen :
- Every 3rd amino acid : Glycine.
- Most abundant amino acid in collagen (33%).
5. Creatinine :
- Synthesized from 3 amino acids
(Glycine + Arginine + Methionine).
- Application : Serum creatinine levels in RFT.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Miscellaneous Amino Acids 205

----- Active space -----

Glycine + Arginine

Guanidinoacetate Site : Kidney

Methyl S-adenosyl Methionine Methionine
transferase (SAM)
SA Homocysteine
Creatine Site : Liver
Creatinine 1 ATP
Kinase
1 ADP
Creatine Phosphate/Phosphagen :
High energy compound
1 ADP

om
• Spontaneous (no enzyme needed)
• Substrate level phosphorylation.

l.c
1 ATP

ai
• Lohmann’s reaction.

gm
Creatinine

@
Creatinine synthesis
19
55
b) Conjugation reactions :
na
ee

• Phase II Conjugation :
m

Benzoic Acid Benzoyl CoA+Glycine Hippuric Acid (Excreted in urine)

ar
um

• Conjugating agent for bile acids (eg : Sodium glycocholate)

itk
am

Note : ATP donors for sprint

|
w

• 1st 3-4 seconds : Creatinine

ro
ar

• Later : Glycogen
M
©

Hyperoxaluria

metabolic defect :
Alanine Pyruvate

Glycine Glycoxylate
PLP
Glycoxylate alanine
Enzyme defect
aminotransferase
↑Accumulation of Glycoxylate

↑Oxalate

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 206 Chemistry and Metabolism of Proteins

Causes :
----- Active space ----- Hyperoxaluria

Primary : Secondary :
Inherited causes • B6deficiency
(Inborn errors of metabolism) (also causes xanthurenic aciduria) ↑Oxalate
• Type 1 Hyperoxaluria : • Vit C excess
• Ethylene glycol poisoning Oxaluria
Glycoxylate alanine amino
transferase defect. • Methoxy flourine Oxalate
• Enteric hyperoxaluria renal
stones
Serine
SERINE
H

om
Properties :

l.c
NH2 C CooH

ai
• Non-essential

gm
CH2
• Polar (d/t OH group)
@
• Glucogenic 19 OH
55
na
ee

Hydroxyl group containing

Note : Phosphorylation sites :
m
ar

Hydroxyl group of Serine > Threonine > Tyrosine amino acid (m/c site of
um

Phosphorylation)
itk

Metabolic Functions :
am

• 1° donor of 1C group
|
w

• Synthesis of Cysteine
ro
ar

• Synthesis of Phosphatidyl serine (phospholipid)

M
©

• On decarboxylation Ethanolamine
• Choline and Betaine synthesis
• Precursor of Selenocysteine.
Serine Glycine
(loses 1 carbon) 1C
metabolism
THFA N5 N10 Methylene THFA

1st entry point in 1 carbon metabolism

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Miscellaneous Amino Acids 207

Basic Amino Acids 00:22:18 ----- Active space -----

Common properties
• All are polar.
• Metabolic fate : Glucogenic.

HISTIDINE

Properties :
• Only polar aromatic amino acid.
• Imidazole ring : Maximum buffering action in blood.

Derivatives of Histidine :
• Carnosine (β-alanine + Histidine) Present in • Ergothionine

om
• Anserine (methylated carnosine) skeletal muscle. • Homocarnosine (GABA + Histidine)

l.c
• Histamine (via decarboxylation) : Allergic

ai
• FIGLU

gm
reactions

@
19
55
Synthesis of FIGLU : FIGLU : Biochemical test for Megaloblastic
na
ee

Histidine anemia due to B9 deficiency.

m
ar
um

Urocanate
itk
am

Imidazole propionate
|

1 C group
w
ro

Formiminoglutamic acid
ar

(FIGLU) THFA (B9)

M

If deficiency of B9
©

Formimino THFA FIGLU excretion in urine

Glutamic acid
Histidine load test :
Differentiates between B9 and B12
deficient megaloblastic anemia.

ARGININE

Properties :
• Most basic amino acid (most -NH2 groups).
• Polar amino acid.
• Semi essential amino acid.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 208 Chemistry and Metabolism of Proteins

----- Active space ----- Derivatives of Arginine :

Arginase • Urea
via Urea Cycle • Ornithine
• Reactive free radical.
• Gaseous molecule.
• 2nd messenger in hormonal
NO synthase (NOS) Nitric Oxide
Arginine pathways cGMP.
Requires: (NO)
• Endothelium derived relaxing
• Heme factor Vasodilator :
• NADPH - Rx of Pulmonary HTN.
- Rx of Impotence.
Along with Glycine Creatinine • Released by Glyceryl nitrate
and Methionine (used in angina).

om
LYSINE

l.c
ai
gm
Properties : Note : Amino acids that do

@
• Contains Epsilon amino group 19 not undergo transamination :
55
• Does not undergo transamination. • Lysine
na
ee

• Proline
m

• Hydroxyproline
ar

Derivatives :
um

• Threonine
itk
am

Histones : • Abundant in basic amino acids (Lysine, Arginine).

|

Pyrrolysine
w

• Positively charged.
ro

Coded by UAG
ar

(Stop codon)
M
©

Lysine Desmosine : found in crosslinks of

Along with
methionine elastin. (Derived amino acid)
Hydroxylation
Carnitine (Vit C required) Deficiency of Vit C

↓Hydroxylysine, hydroxyproline
Hydroxylysine
• Found in collagen (absent in elastin) Collagen defect

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Miscellaneous Amino Acids 209

Acidic Amino Acids 00:35:41 ----- Active space -----

ASPARTIC ACID

Synthesis : Synthesis
From Oxaloacetate and Asparagine : Oxaloacetate Aspartate Asparagine
Transamination

Functions :
• Pyramidine ring synthesis :
• Urea cycle : synthesis of urea C
TCA Cycle 4
C
NH3 CO2 Aspartic Acid 3 5

om
NH2 CO2 NH2
2

l.c
6
C

ai
gm
• Purine ring synthesis : 1

@
N
19 Given by Aspartate
55
Aspartate
na

N1
ee
m
ar
um
itk
am
|
w

Canavan Disease :
ro
ar

Biochemical defect : Deficiency of Asparto-acylase

M
©

Deficiency N-acetyl aspartate

accumulates in CSF, blood,
N-acetyl aspartate Asparto-acyclase Aspartate
and urine.

Features :
• Gross developmental delay • Distorted mitochondria
• Macrocephaly • Severe leukodystrophy
• Persistent head lag

GLUTAMIC ACID
Synthesis : From α-ketoglutarate (α-KG)

α-ketoglutarate Forms Glutamic Acid Forms Glutamine

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 210 Chemistry and Metabolism of Proteins

----- Active space ----- Functions :

• N-acetyl glutamate (NAG) : Allosteric activator of Carbamoyl Phosphate
Synthetase I (pacemaker enzyme of Urea cycle).
• Glutathione synthesis : Glutamic acid + Cysteine + Glycine required.
• GABA formation : Glutamate Gamma amino butyric acid (GABA)

CO2
Branched Chain Amino Acids (BCAAs) 00:43:11

Examples : Metabolic Fate :

• Leucine • Valine Glucogenic
• Isoleucine • Leucine Ketogenic
• Valine • Isoleucine Both glucogenic

om
and ketogenic

l.c
Properties :

ai
gm
• All are essential

@
• All are non-polar
19
55
na
ee

REACTIONS :
m
ar

BCKD : Multi enzyme (E) complex.

um

BCAA
• E1 : Branched chain ketoacid (BCK) decarboxylase
itk

I. Transamination • E2 : Dihydrolipomide (DHL) transacylase

am

Coenzyme : PLP • E3 : Dihydrolipomide (DHL) dehydrogenase

|
w
ro

Note :
ar

Branched chain
M

• α -ketoglutarate dehydrogenase Multienzyme

©

keto acid • Pyruvate dehydrogenase complexes

NAD+ • BCKD
Branched
chain keto acid II. Oxidative decarboxylation E2 +E3 are
dehydrogenase common.
(BCKD) CO2 E1 variable.
NADH • Class I oxidoreductase enzymes
Acyl group • Catalysis of oxidative decarboxylation
FAD reactions.
• 5 co-enzymes :
III. FAD dependent dehydrogenation - Thiamine pyrophosphate (Vit B1)
FADH2 - Lipomide
- CoA (Vit B5)
Product - FAD (VIT B2)
- NAD+ (Vit B3)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Miscellaneous Amino Acids 211

MAPLE SYRUP URINE DISEASE (MSUD) ----- Active space -----

Biochemical defect : Defect in BCKD

Gene Component coded MSUD Type

E1 α Type 1A MSUD
E1 - BCK decarboxylase
E1β Type 1B MSUD
E2 DHL Transacylase Type 2 MSUD
E3 DHL Dehydrogenase Type 3 MSUD
Clinical Features :
• Difficulty feeding
• Convulsions

om
• Lethargy

l.c
ai
• Coma

gm
• Periods of hypertonicity alternating with hypotonicity (repetitive, like boxing/ bicycling)
@
19
55
Urine : sweet smell of burnt sugar/caramel/maple syrup
na
ee

Lab diagnosis :
m
ar

• Blood and urine : ↑BCAA, ↑BCKA (d/t defective BCKD)

um
itk

• Dinitrophenyl Hydrazine Test (DNPH)

am

• Rothera’s test
|
w
ro
ar

Treatment :
M

• Restrict BCAAs
©

• Supplement B1 (co-enzyme) DNPH Test

Positive : Yellow precipitate
• Liver transplant (extreme cases)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 212 Chemistry and Metabolism of Proteins

----- Active space ----- Miscellaneous Points 00:55:50

Disorders Enzyme Defect

Albinism Tyrosinase
MSUD BCKD
Isovaleryl CoA DH
Isovaleric Aciduria
(in Leucine metabolism)
Cystathionine β-synthase
Homocystinuria
(Vit B6/PLP needed)

Amino Aciduria Defective Enzyme

om
Phenylketonuria Phenylalanine Hydroxylase

l.c
ai
Homogentisate oxidase/

gm
Alkaptonuria dioxygenase/Dihydroxy

@
phenylacetate dioxygenase
19
55
Fumaryl Aceto acetate
na

Tyrosinemia type I
hydrolase
ee
m

Tyrosinemia type II Tyrosine transaminase

ar
um

Parahydroxyphenyl Pyruvate
Tyrosinemia type III
itk

Hydroxylase (Dioxygenase)
am
|

Laboratory tests in Amino Aciduria :

w
ro
ar

Test Aminoaciduria/Tumor
M
©

Ferric Chloride Test PKU/Alkaptonuria

DNPH Test MSUD
Guthrie Test PKU
Obermeyer Test Hartnup’s Disease
Cyanide Nitroprusside Test Homocystinuria
LaBrosse VMA Spot Test Pheochromocytoma
5-HIAA Carcinoid Syndrome

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Miscellaneous Amino Acids 213

Peculiar odours in Inborn Errors of Metabolism : ----- Active space -----

Disease Urine Odour

Glutaric Acidemia (Type II) Sweaty feet
Hawkinsuria Swimming pool
Isovaleric Aciduria Sweaty feet
MSUD Maple syrup
Hypermethioninemia Boiled cabbage
Multiple Carboxylase Deficiency Tomcat urine
Oasthouse Urine Disease Hops-like urine
Phenylketonuria Mousy/Musty
Trimethylaminuria/Fish Odour
Rotting fish

om
Disease

l.c
Tyrosinemia Boiled cabbage/Rancid butter

ai
gm
@
Trimethylaminuria/Fish Odour Disease : 19
55
na
ee

Biochemical defect : Defect in Trimethylamine monooxygenase (flavoprotein)

m
ar
um
itk

FAD requiring enzyme

am
|
w

Trimethylamine containing compounds not

ro
ar

catabolised
M

(Eg: Choline)
©

↑Trimethylamines

Rotting fish smell in urine

Aggravating factor : Consumption of foods rich in choline.
Eg : Nuts, Egg yolk.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 214 Chemistry and Metabolism of Proteins

----- Active space ----- HEME METABOLISM

Heme 00:00:40

Metalloporphyrin. 3 4
Porphyrin : 2 5
1
• Formed by 4 pyrrole rings joined by methenyl bridges. NH
Pyrrole
• Precursors : Porphyrinogens (Colorless). (5 membered ring)
• Forms Soret band : Sharp band at 400nm due to absorption Diagnostic for detecting porphyrin
• Emits red fluorescence on Wood’s lamp UV illumination accumulation in body fluids.
Use : Cancer phototherapy

om
• Porphyrin injected into tumor cells to destroy tumor cells.

l.c
ai
• Mechanism : Porphyrin Sunlight Free radicals released

gm
Destroys

@
lysosome Cutaneous photosensitivity
19
55
Types of Porphyrin :
na
ee

U : Uroporphyrin
m

C : Coproporphyrin
ar
um

P : Protoporphyrin
itk
am

Structure of heme :
|
w

Metal (Fe2+) + porphyrin (protoporphyrin)

ro
ar
M
©

Ferroprotoporphyrin (Iron in centre) = heme

Heme containing proteins :
• Hemoglobin • Catalase
• Myoglobin • Nitric oxide synthase
• Cytochrome C, p450 • Tryptophan pyrrolase

Heme Biosynthesis 00:09:04

Synthesis All tissues in body Porphobilinogen

Predominantly : Liver & erythrocyte (Monopyrrole)
precursors of bone marrow Iron
Organelle :
Partly cytoplasmic & partly mitochondrial Heme (Tetrapyrrole)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Heme Metabolism 215

Steps : ----- Active space -----

Succinyl CoA + glycine (Starting substrates)
ALA synthase (Requires active Vit B6-PLP)
Amino-levulinic acid (ALA)
ALA dehydratase
(2x ALA) Porphobilinogen (PBG) : First monopyrrole synthesised
HMB synthase/PBG deaminase/
Uroporphyrinogen I synthase
(4 x PBG) Hydroxymethylbilane (Linear tetrapyrrole)
Uroporphyrinogen III synthase

om
Uroporphyrinogen III : First porphyrin synthesized (Cyclic tetrapyrrole)

l.c
ai
Uroporphyrinogen decarboxylase

gm
Coproporphyrinogen III

@
Coproporphyrinogen oxidase 19
55
na

Protoporphyrinogen III
ee
m

Protoporphyrin oxidase
ar
um

Protoporphyrin III
itk
am

Fe2+ Ferrochelatase
|

Heme
w
ro
ar

ALA Synthase :
M

• Rate limiting enzyme of heme synthesis.

©

• Inducible enzyme : ↓ heme/hematin (Oxidized product of heme)

Induces gene for ALA synthase

↑ ALA synthase

Factors affecting heme synthesis :

Drugs metabolised by cytochromes :

• ↓ Cyt ↓ heme Induce gene for ALA synthase

↑Intermediates

Aggravate porphyria

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 216 Chemistry and Metabolism of Proteins

----- Active space ----- Lead :

• Inhibit ALA dehydratase, ferrochelatase.
• Lead poisoning : ↑ ALA Abdominal pain.

INH :
• Antitubercular drug.
• ↓ availability of PLP Rx : Supplementation of PLP.

Porphyria 00:22:01

• D/t acquired/hereditary enzyme deficiencies in heme biosynthesis.

• ↑ Production & excretion of porphyrins.
MODE OF INHERITANCE

om
Predominantly AD, except :

l.c
• Congenital erythropoietic porphyria

ai
gm
• ALA dehydratase deficient porphyria AR

@
• Erythropoietic protoporphyria (EPP) 19
55

• X-linked protoporphyria : X-linked

na
ee
m

CLINICAL FEATURES :
ar
um
itk

Substances Manifestations
am

Neurovisceral manifestations :
|
w

• Convulsions
ro

↑ ALA & PBG

ar

• Psychiatric manifestations
M
©

• Abdominal pain
↑ Porphyrins Cutaneous photosensitivity

Glucose & Porphyria :

Administration of glucose to relieve attacks of porphyrias :

Metabolite from glucose prevents induction of ALA synthase gene.

In starvation :
• Lack of glucose Induces ALA synthase gene.
• Precipitate attack of porphyria by ↑ accumulation of intermediates.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Heme Metabolism 217

----- Active space -----

Enzyme
Porphyria Features
Deficiency
• AKA plumboporphyria : D/t resemblance of
ALA dehydratase deficient
ALA dehydratase lead poisoning
porphyria (ADP)
• AKA Doss porphyria
Hydroxymethyl • ↑ ALA & ↑ PBG
bilane synthase/ • M/c acute porphyria
Acute intermittent porphyria
PBG deaminase/ • Neurovisceral manifestation
UPG I synthase • No photosensitivity
Congenital erythropoietic • ↑ hydroxymethylbilane

om
porphyria (CEP) Spontaneous

l.c
ai
AKA Günther’s disease Uroporphyrinogen I Accumulated

gm
@
UPG III synthase 19 Coproporphyrinogen I
55
• Non-immune hydrops
na
ee

• Brownish discoloration of teeth

m

• Erythrodontia : Red fluorescence on

ar
um

Erythrodontia illuminating teeth with UV light

itk
am

Porphyria cutanea tarda • M/c porphyria

|

(PCT) • Most readily treatable porphyria

w

Uroporphyrinogen
ro

• Cutaneous porphyria (Blisters in sun-

ar

(UPG)
M

exposed areas)
©

decarboxylase
• Hemochromatosis causes PCT (Iron inhibits
Cutaneous porphyria uroporphyrinogen decarboxylase)
• Autosomal recessive
Erythropoietic • M/c porphyria in children
Ferrochelatase
Protoporphyria • Non-blistering photosensitivity
• Skin swelling and eczematous rash
Hereditary coproporphyria CPG oxidase -
Varigate porphyria PPG oxidase -
Gain of function
X-linked protoporphyria mutation of ALA -
synthase
ALA synthase • No porphyria
Sideroblastic anemia
B6 deficiency • X-linked disorder
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 218 Chemistry and Metabolism of Proteins

----- Active space ----- INVESTIGATIONS

• Ehrlich’s test : Test for porphobilinogen & urobilinogen (In hemolytic anemia).
• Hoesch test.
• Watson Schwartz test (For PBG).
• Soret band at 400 nm light.
• Red fluorescence in UV light.

Heme Catabolism 00:43:45

PATHWAY
Stages :
I : In Reticuloendothelial system : Heme Bilirubin
II : In liver: conjugation of bilirubin

om
l.c
III : In intestine: conversion to urobilinogen

ai
In RE system

gm
Microsomal hemoxygenase

@
system. Heme 19
55
NADPH, O2 (Only process in human body with
CO
na

Hemeoxygenase endogenous CO production)

ee

Biliverdin
m
ar

NADPH
um

Biliverdin reductase
itk

Bilirubin (Yellow colored)

am
|
w

Binds to albumin & transported in blood

ro
ar
M

Taken up by liver cells

©

Liver
Albumin
Bilirubin enters cell

Intracellular binding by proteins (to prevent

loss of bilirubin back into blood)
UDP glucuronyl transferase
Conjugation of bilirubin
Secretion into bile duct :
• Active process: using transporters MRP-2 & MOAT
(MRP-2 : Multidrug
• Rate limiting step
resistance protein-2,
Intestine Deconjugation of bilirubin by β-glucuronidase MOAT : Multispecific
(Produced by intestinal bacteria) organic anion
transporter)
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Heme Metabolism 219

----- Active space -----

Intestine Deconjugation of bilirubin by β-glucuronidase

(Produced by intestinal bacteria)

Bilirubin
Enterohepatic 20% Urobilinogen (UBG)
circulation 80%
Portal vein kidney Stercobilinogen Excreted through feces
Liver Excreted in urine

Lab Diagnosis of Jaundice 00:52:16

om
Van den Bergh test :

l.c
ai
To detect Bilirubin :

gm
@
• Direct : Bilirubin + Ehrlich’s Diazo reagents Reddish purple azo compound.
• Indirect : Unconjugated bilirubin 19
Extraction with alcohol Gives color.
55
na

(Water insoluble)
ee
m
ar

Delta bilirubin/Biliprotein :
um

• Conjugated bilirubin covalently linked to albumin.

itk
am

• Half-life : 12-14 days (T½ of unbound conjugated bilirubin : 4 hrs).

|

• Delayed clearance from plasma.

w
ro
ar

Urine tests for jaundice :

M
©

Testing Component Disease

Conjugated bilirubin (Bile pigment) in
Fouchet’s test
urine
Hay test :
2 test tubes with urine & Obstructive
distilled water (control) + jaundice
Bile salt
sulfur powder Sulfur
sinks d/t ↓ surface
tension
Urine sample + Ehrlich Hemolytic
Urobilinogen (Pink color)
reagent jaundice

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 220 Chemistry and Metabolism of Proteins

----- Active space ----- Differentiating types of jaundice :

Prehepatic (Hemolytic) Hepatic Obstructive

Direct / Conjugated bilirubin N ↑
Indirect / Unconjugated N/↑
↑ N
bilirubin
Urine bile salt
- +
Urine bile pigment +/-
Urine UBG + -
ALT: Alanine
aminotransferase
↑↑ N
AST: Aspartate N

om
aminotransferase

l.c
ALP: Alkaline phosphatase ↑ ↑↑↑

ai
gm
@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Hemoglobin Part : 1 221

HEMOGLOBIN PART : 1 ----- Active space -----

Structure & Types of Hemoglobin  00:00:30

Structure of Hemoglobin :
• Quaternary structure.
• 4 globin chains : 2α, 2β (Adult Hemoglobin : HbA1).
- Alpha subunit : 141 amino acids, chromosome 16.
- Beta subunit
- Gamma subunit 146 amino acids, chromosome 11.

om
- Delta subunit

l.c
• Each globin chain carries 1 heme (Prosthetic group). Hemoglobin

ai
gm
• Heme (Ferroprotoporphyrin) : Iron (Fe2+) + Protoporphyrin.

@
• Heme : Binds to oxygen 1 Hb : 4 Heme 19
Carries 4 oxygen.
55
na

Normal levels : Note : g/dL used for :

ee
m

• Males : 14-16 g/dL. • Hemoglobin.

ar
um

• Females : 13-15 g/dL. • Protein in blood.

itk
am

Types of Hemoglobin :
|
w

Types of hemoglobin Constituent Normal adult blood

ro
ar

Adult Hb (HbA1) 2α + 2β 97%

M
©

HbA2 2α + 2δ 2%
HbF 2α + 2γ 1%
Structure of Iron :
Ferrous state (Fe2+) :
Can form 6 coordinate bonds :
• 4 bonds :
Nitrogen of each pyrrole ring.
• 5th bond :
Imidazole group of histidine (Proximal histidine).
• 6th bond :
Oxygen molecule (Attached to distal histidine).

6 coordinate bonds of Fe2+

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 222 Chemistry and Metabolism of Proteins

----- Active space ----- Ferric form (Fe3+) :

• Meth hemoglobin : Oxidized state of iron.
• 6th coordinate lost No oxygen carrying capacity.

Embryonic Hemoglobin :
• Hemoglobin Gower 1 (ζ2, ε2).
• Hemoglobin Gower 2 (α2,ε2).
• Hemoglobin Portland (ζ2,γ2).

Synthesis :
• Site before 8 weeks : Yolk sac.
• At 4-5 weeks :
- ζ & ε chain ↓.

om
- α & γ chain ↑ : HbF (Compensatory).

l.c
• After birth :

ai
gm
- HbF ↓.

@
- HbA ↑. 19
55
na
ee

Haemoglobinopathies 00:14:04
m
ar
um

• Inherited disorders in globin chain due to genetic mutation, resulting in

itk

alteration in amino acid sequence.

am

• Phenotypical manifestation :
|
w

Changes in :
ro
ar

a. Physical properties.
M
©

b. Chemical properties.
c. Function.

CLASSIFICATION

Structural haemoglobinopathies : ↓ Synthesis :

• Qualitative. • Quantitative.
• Eg : • Eg : Thalassemia syndromes.
- Sickle syndromes.
- Unstable hemoglobins.
- Hb with abnormal oxygen affinity.
- Structural variants that lead to
thalassemia.
- Asymptomatic.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Hemoglobin Part : 1 223

Sickle Cell Disease 00:17:30 ----- Active space -----

Sickle cell hemoglobin (HbS) : M/c Hb variant.

Molecular Defect :
Occurs at 6th codon of β globin gene.
Normal Sickle cell disease
DNA coding strand : GAG GTG

Template strand : CTC CAC

mRNA : GAG GUG

Replaced by
Amino acid : Glutamic acid Valine
(Hydrophobic aa)

om
(Hydrophilic aa)

l.c
Mutations :

ai
gm
• Point mutation (Missense).

@
• Base substitution. 19
55
• Non-conservative mutation.
na
ee

• Partially acceptable missense mutation.

m
ar
um

HbS during Deoxygenation :

itk

Sticky patch of HbS

am

Deoxygenated state
|
w
ro

Hb polymerization
ar
M

Ca2+ influx H2O efflux

©

K+ efflux
RBC sickling
(Shrunken d/t loss of water)

Hemolysis (Membrane damage) : Dense and rigid RBC :

Hemolytic anemia & jaundice Microvascular occlusion :

Vasoocclusive crisis

HbS polymerization Sickling of RBC

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 224 Chemistry and Metabolism of Proteins

----- Active space ----- PHENOTYPIC CLASSIFICATION

Based on mutation of one or both alleles of globin chain.
Sickle Cell Trait (βAβS) Sickle Cell Disease (βSβS)
Zygosity Heterozygous Homozygous
Allele Only one allele mutated Both alleles mutated
• 50% RBC normal
RBC All RBCs completely sickled
• Hypoxia induces sickling
Symptoms Mild symptoms Severe symptoms
CLINICAL FEATURES
• Hemolytic anemia.
• Hemolytic jaundice.

om
• Hepatosplenomegaly (d/t ↑ RBC destruction).

l.c
ai
gm
Vasoocclusive crisis/Sickle cell crisis :

@
• Bone : M/c (Hand/foot pain) 19 • Brain
55
• Lung • Spleen
na
ee

• Liver
m
ar
um

DIAGNOSIS
itk

Hemoglobin Electrophoresis :
am
|

Condition Finding
w
ro

Normal
ar
M

• Fastest moving & longest : HbA

©

• Near origin : HbA2

• In between : HbF

Sickle cell trait • Normal Hb (HbA) + HbS present

• HbA leading
- Glutamic acid : More negative  oves faster
M
towards anode.
• HbS lagging
- Valine : Neutral aa Slower movement
(Less negative charge)
Sickle cell disease • No HbA
• Strong band of HbS +
• Compensatory ↑ in HbF (α2, γ2)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Hemoglobin Part : 1 225

Sickling Test : ----- Active space -----

Done for sickle cell trait

Procedure : Peripheral smear + Sodium dithionite Sickling of RBCs

(Induces hypoxia)

om
l.c
PEDIGREE ANALYSIS

ai
gm
Normal (AA) & Sickle Cell Trait (AS) : Sickle Cell Trait & Sickle Cell Disease (AS x SS) :

@
19
55
AA AS AS SS
na
ee
m
ar

AS AS SS
um

AA AS AS AA SS
itk

• 50% SC trait (Carriers).

am

• 50% SC trait.
• 50% SC disease.
|

• 50% normal.
w
ro
ar

TREATMENT
M

• Repeated blood transfusion.

©

• Anti-sickling agents : Hydroxyurea.

• HbF inducers : Sodium butyrate.
• Splenectomy (To ↓ hemolysis).

OTHER HEMOGLOBIN VARIANTS

Hb Molecular change Features
• 2nd most prevalent variant
• More prevalent in West Bengal
Hemoglobin E β26 : Glutamic acid Lysine
• Heterozygous : Asymptomatic
• Homozygous : ↑ RBC fragility
Hemoglobin C β6 : Glutamic acid Lysine -
Hemoglobin D
β121 : Glutamic acid Glutamine -
Punjab
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 226 Chemistry and Metabolism of Proteins

----- Active space ----- Other Structural Hemoglobinopathies 00:39:20

Unstable Hb :
Hb Zurich :↑ tendency to denature and form molecular aggregates

↑ hemolysis.

Hb Variants with Increased O2 Affinity :

Hb Chesapeake :
Hb has difficulty unloading O2

Oxygen dissociation • Tissue hypoxia.

curve shifted to left. • Compensatory polycythemia.

om
l.c
ai
gm
Hb Variants with Decreased O2 Affinity :

@
Hemoglobin M : 19
55
• Substitution of proximal (5th coordinate)/distal (6th coordinate) histidine of
na
ee

alpha or beta chains.

m

• Types :
ar
um

- Hb M Boston (Alpha 58 histidine Tyrosine).

itk
am

- Hb M Hyde Park (Beta 92 histidine Tyrosine).

• ↓ Oxygen binding
|

Cyanosis.
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Hemoglobin : Part 2 227

HEMOGLOBIN : PART 2 ----- Active space -----

Thalassemia  00:00:18

• Group of disorders characterized by inadequate synthesis of α or β globin

chains which are otherwise structurally normal.
• Quantitative defect.

Genesis of Alpha & Beta Chain :

α chain β chain

om
Genes 4 genes : 2 from each parent 2 genes : 1 from each parent

l.c
ai
gm
Synthesis 8-10 weeks of gestation ~ 38 weeks (Near birth)

@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Hemoglobin chains

Classification :
Based on type of chain affected

Alpha thalassemia : Beta thalassemia :

• Deletion of α genes • Deletion of β genes
• ↓ α chains • ↓ β chains

Based on zygosity

Heterozygous : Homozygous : Variable :

β thalassemia minor. β thalassemia major. β thalassemia intermedia.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 228 Chemistry and Metabolism of Proteins

----- Active space ----- Alpha Thalassemia 00:08:50

Affects all types of Hb Severe form.

Molecular defect : Large gene deletions ↓ synthesis of α globin chains.

Variants :
Defect Clinical features
Silent carrier Only 1 α gene deleted Asymptomatic
α thalassemia trait 2 α genes deleted Asymptomatic - mild symptoms
• 3 α genes deleted
Hb H disease (β4) Moderate - severe symptoms
• β chains form tetramers
α Thalassemia major/ • All 4 α genes deleted Hypoxia : D/t ↑ O2 affinity of g chains

om
• g chains form tetramer (No a)

l.c
Hydrops fetalis

ai
(Hb Bartz γ4) (No normal Hb) Death in utero

gm
@
19
55
β Thalassemia 00:16:04
na
ee

• More common (As α thalassemia : Highly fatal).

m
ar

• ↓/Absence of β chains Compensatory ↑HbA2 & ↑HbF.

um
itk

Molecular Defects :
am
|

β˚ : No β chain synthesis.
w
ro

β+ : ↓ β chain synthesis.
ar
M
©

Genetic Mutation :

Splicing mutation : Splicing of exons in mRNA affected.

Point mutation/ Promoter region mutation : Point mutation at promoter site.

Frame shift mutation
Chain termination mutation : Frameshift mutation causing premature
termination of protein synthesis.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Hemoglobin : Part 2 229

Effects d/t ↓b Globin Chain : ----- Active space -----

↓ β globin chain synthesis

↓ HbA (α2, β2) ↑ α chain synthesis

RBC with ↓ Hb α chain aggregate

Microcytic hypochromic anemia. Damage RBC membrane : Hemolytic anemia

Ineffective erythropoiesis Extramedullary erythropoiesis

(Bone marrow) (Liver + spleen)

om
Bone marrow expansion • Hepatomegaly.

l.c
• Splenomegaly.

ai
gm
Skeletal deformities

@
19
55

X-ray skull : Crewcut appearance

na

Hemolytic facies/
ee

Chipmunk facies. (Spinous projection from outer table of skull :

m
ar

New bone formation).

um
itk
am

β THALASSEMIA MAJOR/COOLEY’S ANEMIA

|
w

Most severe form.

ro
ar
M

Clinical Features :
©

• Age of onset : 1st year of life.

• Anemia : ↓ Hb causing tissue hypoxia.
- Fatigue.
- Dyspnea.
• Cyanosis (Tissue hypoxia).
• Compensatory bone marrow expansion :
- Stunted growth. Chipmunk facies
- facies.
• Extramedullary erythropoiesis : ,
.

Diagnosis :
Peripheral smear : anemia.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 230 Chemistry and Metabolism of Proteins

----- Active space ----- Haemoglobin electrophoresis :

Condition Electrophoresis strip Finding

Normal • Strong HbA band

• ↓ HbA band
β thalassemia trait • Marked ↑HbA2 band
• ↑ HbF

• No HbA band
β thalassemia major
• Markedly ↑HbA2 and HbF

om
l.c
ai
Treatment :

gm
@
• Blood transfusion (Repeated transfusion Iron toxicity).
19
• Desferrioxamine (Iron chelating) : Subcutaneous infusion.
55
na

• Bone marrow transplant.

ee
m

• Splenectomy.
ar
um

BETA THALASSEMIA MINOR/TRAIT

itk
am

• Heterozygous : One gene normal. • Microcytic hypochromic anemia.

|

• No clinical symptoms. • Compensatory ↑ in HBA2 or HBF.

w
ro
ar
M

Structural Hemoglobinopathies vs. Thalassemia 00:35:36

©

Structural hemoglobinopathies Thalassemia

Type defect defect
Normal globin chains in
Defect
in normal amount
Variant Alpha chain < Beta chain variants Alpha or beta thalassemia
Hb (g4) (b4)

Hemoglobin Derivatives 00:40:00

Formation :
• Combination of various ligands with heme OR
• Change in oxidation state of iron.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Hemoglobin : Part 2 231

TYPES ----- Active space -----

Normal Hb derivatives : Abnormal Hb derivatives :

• Oxy Hb (oxygenated) : Dark red • Met Hb : Dark brown
• Deoxy Hb (deoxygenated) : Purple • CO Hb : Cherry red
• Sulf Hb : Green
Oxy Hemoglobin : Deoxy Hb :
• 95% of total Hb. • <5% of total Hb.
• Transport of oxygen. • >5% causes cyanosis.

Carboxy Hb :
• Hb combined with carbon monoxide.

om
• CO : High affinity to Hb

l.c
( x 200 times that of O2).

ai
gm
• Unsuitable for oxygen transport.

@
• Fire accidents in closed spaces 19
55
na

↑CO present
ee

Carboxy Hb
m

CO poisoning (Lethal)
ar
um

Met Hb :
itk
am

Iron in Hb is Fe2+ Fe3+

|
w
ro

Met Hb reductase enzyme system : Reduce met Hb in RBCs back to ferrous state.
ar

• NADH dependent (Cytochrome B5 reductase) : 75%.

M
©

• NADPH dependent system : 20%.

• Glutathione dependent Met Hb reductase : 5%.
Methemoglobinemia :
• Occurs when Met Hb blood levels exceed 1%.
• Markedly ↓ oxygen binding and transport capacity.
- Fe2+ : 6th coordinate bond attaches to oxygen.
- Fe3+ : Loss of 6th coordinate bond.
• Manifestation : Cyanosis.
Sulfhemoglobin : Cyanosis
• Action of hydrogen sulfide on oxy Hb.
• Cause : Drugs.
- Sulfonamide. - Dapsone.
- Phenacetin.
• Irreversible : Cannot be converted back to oxy Hb.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 232 Extracellular Matrix

----- Active space ----- FIBROUS PROTEINS

Proteins

Globular Fibrous
(Functional) (Structural)

Collagen Elastin Keratin Fibrillin

Collagen  00:01:31

om
l.c
Most abundant protein in humans & in Extracellular matrix.

ai
gm
STRUCTURE
@
19
55
Polyproline α chain : Triple helix (2° structure) :
na

• Glycine X - Y repeats. • 3 α chains. Quarter staggered

ee
m

- X, Y : Hydroxyproline • Each twists in left handed arrangement (3° structure) :

ar
um

& hydroxylysine direction. Lateral arrangement of

itk

• Turns in left handed • 3 together turn in right handed triple helix.

am

direction. direction to form triple helix.

|
w

• Each chain ~ 1000 amino acids.

ro

1/4th distance away from

ar

first layer
M
©

Triple helix : Right handed twisted chain Quarter staggered arrangement

SYNTHESIS

I. Intracellular Synthesis :
Site : Fibroblast, Rough endoplasmic reticulum.
In ribosome : Procollagen formed.
1. Formation of pre-pro alpha chains with signal sequence.
2. Hydroxylation of Proline, Lysine in presence of Vitamin C.
3. Glycosylation : Addition of carbohydrate residue to hydroxylysine residues.
4. Formation of disulphide bond.
5. Formation of triple helix.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Fibrous Proteins 233

Procollagen packed into the secretory vesicles of golgi apparatus. ----- Active space -----

Triple helix exported to extracellular matrix.

II. Extracellular Synthesis :
• Cleavage of N and C terminal polypeptide.
• Collagen fibrils assembled to quarter staggered arrangement.
• Formation of covalent cross links (D/t aldol condensation) :
Cu in Lysyl oxidase : Required for covalent bond formation.
Applied Aspect :
Deficiency of Cu Affects covalent cross links Menke’s disease.

Role of Copper on Vit C :

om
Vit C Copper

l.c
Enzyme formation Prolyl & Lysyl hydroxylase Lysyl oxidase

ai
gm
Oxidative deamination of lysyl

@
Reaction Hydroxylation
19
residues Aldol condensation
55

Function Triple helix Covalent cross links

na
ee
m

Types :
ar
um

Types Tissue Remarks

itk
am

• Most abundant. Note :

|

I Connective tissue, Bones • Present in hard & soft

w

Recurring &
ro

tissue.
ar

most abundant
M

II Cartilage, Vitreous humor - aminoacid present

©

IV Basement membranes - in collagen : Glycine.

• Present in microfibrils.
VI Connective tissue • Defective in Bethlem
myopathy.
Defective in Epidermolysis
VII Anchoring fibrils
Bullosa.

Disorders :
Type of collagen Disease
• Osteogenesis imperfecta
Type I • Osteoporosis
• Ehlers-Danlos syndrome type VII
• Severe chondrodysplasia
Type II
• Osteoarthritis
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 234 Extracellular Matrix

----- Active space ----- Type of collagen Disease

Type III Ehlers-Danlos syndrome type IV
Type IV Alport syndrome
Type VII Epidermolysis bullosa (Dystrophic type)
Type X Schmid metaphyseal dysplasia
Lysyl oxidase Menke’s disease : ATP 7A gene

Other Fibrous Proteins  00:23:33

ELASTIN
Lysine Desmosine Crosslink formation.

Differentiating features from collagen :

om
l.c
Absence of :

ai
gm
• Triple helix formation.

@
• Glycine x-y repeats.
19
55
• Hydroxylation & Glycosylation.
na
ee

Associated disorders :
m
ar

• Williams-Beuren syndrome.
um

• Cutis laxa (Laxity of skin).

itk
am

KERATIN :
|
w

Site : Outer layer of skin, nails & hair.

ro
ar

Formation : Alpha helix crosslinked by disulphide bond.

M
©

Rich in : Cysteine (↑ Cysteine Harder keratin).

Associated disorders :
Epidermolysis bullosa :
• Classical type.
• Defect in keratin 5.

FIBRILLIN :
• Glycoprotein.
• Component of myofibrils.
• Function : Scaffolding of elastin.

Type Defect Disease

Fibrillin 1 Marfan’s syndrome
Fibrillin 2 Congenital Contractural Arachnodactyly

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Integration of Metabolism 235

INTEGRATION OF METABOLISM ----- Active space -----

Well Fed State/Postprandial/Absorptive Phase 00:00:10

• 2-4 hrs after meal : Digestion and absorption of food.

• ↑ in plasma levels of glucose, amino acids, fatty acids.
• Metabolic pathways directed towards storage.
• Hormone : Insulin.

Insulin Secretion :
Begins to rise : At blood glucose >70 mg/dL or >3.9 mmol/L.

om
l.c
ai
GLUT-2 :

gm
• High Km : Low affinity for glucose.

@
• Only transport glucose when in high levels. 19
55
na

Insulin :
ee
m

• Synthesized in rough endoplasmic reticulum.

ar
um

• Packaged in golgi apparatus into vesicles.

itk

• Assessment of level of C-peptide = Level of insulin.

am

Glucose
|
w

GLUT-2 β cell of pancreas

ro
ar

Glucokinase
M

Glucose Glucose-6-phosphate
©

ATP
Pyruvate
ATP/ADP ratio↑

Closure of ATP sensitive K+ channel

Membrane depolarization

Opening of voltage gated Ca2+ channel

Ca influx

Insulin + C-peptide Insulin + C-peptide

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 236 Integration of Metabolism and Biological Oxidation

----- Active space ----- Functions of Insulin :

↑Protein translocation :
• ↑GLUT-4 level.
• ↑Insulin receptor level.

↑Gene transcription : Of Glucokinase.

↑Enzyme activity :
• Phosphodiesterase.
Dephosphorylates regulatory enzymes.
• Phosphatase.

Liver in Fed State :

Liver : Glucose consumer in fed state.

om
l.c
ai
↑Glucose

gm
@
GLUT-2 55
19
Glucokinase/Hexokinase
na

Glucose G-6-P
ee

Glycogen synthesis HMP shunt

m
ar

Glycolysis
um
itk

Glycogen Pyruvate NADPH

am

PDH
|
w
ro

Acetyl CoA
ar

ATP
M

FA synthesis
©

Lipogenesis TAG carried

TCA FA TAG
Glycerol by VLDL

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Integration of Metabolism 237

Adipose Tissue : ----- Active space -----

GLUT-4 : Insulin dependent transporter.
Glucose

GLUT-4
Glucose G-6-P

Pyruvate
PDH
Acetyl CoA FA Glycerol
ATP

om
TAG Stored in adipose tissue

l.c
ai
d/t inhibition of hormone-

gm
@
sensitive lipase by insulin
19
55
Skeletal Muscle :
na

Glucose
ee
m
ar

GLUT-4
um
itk

Glucokinase/Hexokinase
am

Glucose G-6-P
|
w

Glycogen synthesis
ro
ar

Glycolysis
M
©

Pyruvate
PDH
Amino acid Protein synthesis↑
Transamination ATP FA synthesis
Oxidative deamination
Lipogenesis

Note :
Transamination & oxidative deamination :
Removal of amino group Carbon skeleton Anabolic functions.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 238 Integration of Metabolism and Biological Oxidation

----- Active space ----- Brain :

• Obligatory requirement of glucose.
• Energy production via oxidative pathways only.
GLUT-3 :
• Present in neurons.
• Highest affinity, Lowest Km.

GLUT-1 GLUT-3

Aerobic glycolysis

om
l.c
ai
gm
PDH

@
19
55
na

FA synthesis
ee
m
ar
um

(Structural component)
itk
am
|
w

METABOLIC FUEL IN FED STATE

ro
ar
M

Organ Fuel
©

• Brain
Glucose
• RBC
• Liver
• Adipose tissue Glucose >> FFA (Free fatty acid)
• Skeletal muscle
• Heart FFA > Glucose (D/t low glycolytic capacity)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Integration of Metabolism 239

Fasting State/Post Absorptive Phase 00:27:30 ----- Active space -----

Energy source : Stored glycogen, TAG.

Stage Duration within food intake
Early fasting 4-16 hrs
Fasting 16-48 hrs
Prolonged fasting or starvation 2-5 days
Prolonged starvation >~5 days

Early Fasting :
• without food.
• Glycogen stores : Depleted in 16-18 hrs.

om
l.c
Source of glucose : Hepatic glycogenolysis.

ai
gm
Hepatic glycogenolysis
Glycogen G6P G6 Phosphatase Glucose
@
19
55
Note :
na
ee

Muscle lack G6 Phosphatase Cannot release free glucose directly.

m
ar

Fasting :
um
itk

• without food.
am

• ↓Glycogen stores : ↓Glycogenolysis.

|
w
ro

Source of glucose : Gluconeogenesis

ar
M

• Production of glucose from non-carb substrates Glycerol.

©

Alanine.
• ATP dependent pathway. Lactate.
Adipose tissue Muscle :
• Alanine (Major gluconeogenic
TAG
amino acid).
Lipolysis HSL( + glucagon) • Lactate.
FA + Glycerol
β oxidation
Acetyl CoA Gluconeogenesis

ATP
TCA cycle

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 240 Integration of Metabolism and Biological Oxidation

----- Active space ----- Starvation :

• without food intake.
• Gluconeogenesis : D/t depletion of non-carb substrates.
Metabolic fuel : Ketone bodies.
TAG

Note :
β oxidation Oxaloacetic acid (OAA) depleted d/t
↑gluconeogenesis in fasting stage.

↓OAA

om
↓TCA cycle Ketone body

l.c
ai
synthesis↑

gm
(Liver)
@
19
55
na
ee

Prolonged Starvation :
m
ar

Muscle protein (Structural)

um

Catabolism
itk
am

Amino acids
|
w
ro
ar

Carbon skeleton
M
©

Catabolic role (Causes cachexia)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Integration of Metabolism 241

FASTING STAGE IN ORGANS ----- Active space -----

Glucose
Liver :
Ketone bodies for other organs

Glycogen

Glucose G6P

ATP TCA Ketone bodies

Muscle Alanine Pyruvate Acetyl CoA

om
Lactate

l.c
Glycerol

ai
gm
@
+ 19
55
na

( + Glucagon)
ee
m
ar

Adipose tissue TAG

um
itk
am

Adipose Tissue :
|

↓Insulin : ↓GLUT-4 activity.

w
ro

Glucose
ar
M
©

TAG
Lipolysis HSL ( + Glucagon) Liver
Gluconeogenesis
β-oxidation Excess FA Ketone body synthesis

TCA cycle
Energy for
adipose tissue

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 242 Integration of Metabolism and Biological Oxidation

----- Active space ----- Skeletal Muscle :

↓Insulin : ↓GLUT 4 activity.
Glucose

Glycogenolysis
Glycogen G-6-P Pyruvate Alanine
(Glucogenic amino acid) Liver
Proteolysis
Protein Amino acid

Sources of energy in muscle :

1. FA Oxidation Acetyl CoA

om
TCA ATP.

l.c
2. Ketone bodies from liver.

ai
gm
@
Brain :
Available glucose 19
55

KB
na
ee
m

Ketone bodies
ar
um

Glucose Lysis
itk
am

Acetyl CoA TCA ATP

|
w
ro
ar
M

METABOLIC FUEL IN FASTING

©

Organ Early fasting/fasting Starvation

Brain Glucose/Ketone bodies (20%)
Glucose Available glucose;
RBC
No glucose RBC lysis
Amino acid, FFA
Liver FFA > Glucose
(Never ketone bodies)
Adipose
Heart FFA > Glucose FFA > ketone bodies
Skeletal muscle

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Krebs Cycle 243

KREBS CYCLE ----- Active space -----

• Aka TCA (Tricarboxylic acid) Cycle/Citric Acid Cycle.

• Final common oxidative metabolic cycle for carbohydrates, lipids & proteins.
Carbohydrates Proteins Lipids

Glycolysis Ketogenic amino acids β-oxidation

Pyruvate

Acetyl CoA (2C) + Oxaloacetate (4C)

om
l.c
ai
gm
TCA cycle

@
19
55
Electron transport chain
na
ee
m

ATP
ar
um

TCA cycle
itk

00:03:35
am
|

Site : All organs with mitochondria.

w
ro
ar

Organelle : Mitochondria.
M
©

Location of enzymes: Matrix of mitochondria.

Exception: Succinate dehydrogenase in inner mitochondrial membrane (Complex II

of ETC)

Oxaloacetate:
• Important intermediate of TCA cycle.
• Has catalytic function.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 244 Integration of Metabolism and Biological Oxidation

----- Active space ----- Steps

Regeneration of oxaloacetate Oxidation of acetyl CoA

NADH Oxaloacetate+ Acetyl CoA
NAD+
(4C) (2C)

Malate Citrate synthase

dehydrogenase Regulatory enzyme
Malate Citrate (6C)

• Lyase Aconitase
• 2-step reaction : +Fe2+

om
- Dehydration

l.c
ai
Fumarase
- Rehydration

gm
Lyase

@
19
55
na
ee

Fumarate Isocitrate (6C)

m

• Regulatory enzyme
ar

Isocitrate
um

• 1st oxidative dehydrogenase

itk

decarboxylation
am
|

Succinate dehydrogenase
w
ro

• 2nd oxidative
ar
M

decarboxylation.
©

NAD+
• Regulatory enzyme.
• Multi-enzyme complex (like NADH
FADH2
FAD pyruvate dehydrogenase) CO2
Succinate α-ketoglutarate (KG)
Substrate level phosphorylation α-KG (5C)
Succinate thiokinase dehydrogenase
NAD+
ATP/GTP Succinyl CoA
ADP/GDP NADH
CO2

Note :
FAD → FADH2 takes place in:
• Succinate dehydrogenase
• Acyl CoA of β-oxidation.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Krebs Cycle 245

Enzyme inhibitors ----- Active space -----

Enzyme Inhibitors Type of inhibition

Fluoroacetate
Aconitase Non-competitive
(Flourine + acetyl CoA)
α-KG DH Arsenite (Inhibit SH- group enzyme) -
Succinate dehydrogenase Malonate Competitive

Energetics
(2.5 x 3 NADH) + (1.5 X 1 FADH2) + 1 ATP = 10 ATP

Significance
• Metabolic traffic circle.

om
• Final common oxidative cycle.

l.c
ai
• Amphibolic cycle Catabolic : Acetyl CoA 2CO2

gm
Anabolic Citrate (6C) Fatty acid
@
α-KG (5C) 19
Glutamate GABA
55
na

Succinyl CoA (4C) Heme synthesis

ee

• Anaplerotic role :
m
ar

- Filling up reaction
um

- Replenish depleted intermediates (6C, 5C, 4C)

itk
am

Eg:
|
w

1. Pyruvate (extra-hepatic) Pyruvate carboxylase Oxaloacetate

ro

ATP CO2 +Biotin

ar
M
©

2. Glutamine Glutamate
Arginine α-KG
Proline
3. Methionine
Threonine
Succinyl Co-A
Isoleucine
Valine
4. Phenylalanine
Fumarate
Tyrosine
5. Alanine Pyruvate Acetyl CoA

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 246 Integration of Metabolism and Biological Oxidation

----- Active space ----- Recent Advances 00:29:40

Mutation of isocitrate dehydrogenase (mutant ICDH)

Isocitrate Mutant ICDH 2-hydroxyglutarate Inhibit TET gene

↑DNA methylation ↑Histone modifcation

Alter epigenomes

Cancer :

om
• Cholangiocarcinoma

l.c
• AML

ai
gm
• Sarcoma

@
19
55
na

Drugs inhibiting mutant ICDH: Sidenibs

ee
m
ar

Mutant Succinate Dehydrogenase

um

Causes:
itk
am

• Familial glioblastoma.
|

• Familial pheochromocytoma.
w
ro
ar
M

Vitamins Used & Regulation of TCA Cycle 00:32:08

©

VITAMINS USED IN TCA CYCLE

Vitamins Function
B5 Part of CoA
B2 As FADH2 in succinate dehydrogenase
B3 NADH formation
B1 α-KG dehydrogenase

REGULATION
• No hormonal regulation (both in fed & fasting stages)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Krebs Cycle 247

ATP/ADP ratio ----- Active space -----

NADH/NAD+ ratio

Decreased Increased

↑TCA cycle ↓TCA cycle

Regulatory enzymes
• Citrate synthase
• Isocitrate dehydrogenase
• α-KG dehydrogenase
• Pyruvate dehydrogenase (Glycolysis links to TCA cycle)

om
l.c
ai
gm
@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 248 Integration of Metabolism and Biological Oxidation

----- Active space ----- ELECTRON TRANSPORT CHAIN

1. Oxidation : Loss of electrons.

2. Reduction : Gain of electrons.
3. Redox couple : Compounds that can exist in both oxidised and reduced state.
Ex : NAD+/NADH, FAD/FADH2, Fe+3/Fe+2 in heme in cytochrome, FMN/FMNH2.
4. Redox potential : The ability of a redox couple to transfer electrons.
Low redox potential transfers electrons High redox potential
Exergonic process

om
Free energy

l.c
ai
Series of redox couples arranged in ascending order of redox potential.

gm
Site : Inner mitochondrial membrane (IMM).
@
19
55
na

Complexes  00:05:01
ee
m
ar

Succinate
complex
um

II Fumarate Intermembrane
NADH 4H NAD
+ +
2H+
itk

e 4H+ space(IMS)
am

e- complex (Final e- Fo
e
|

-
complex I e -
complex III O2 acceptor) complex
w

e - IV 1mm
ro

CoQ Cyt c e- V
H2O
ar

F1
M

4H+ 4H+ 2H+

©

Matrix
Complex I : CoQ : Complex II :
• Portal of entry of • Mobile electron carrier. • portal of entry of
electrons from a • AKA ubiquinone. electrons from a
reducing equivalent. reducing equivalent.
• AKA NADH • AKA succinate Q
dehydrogenase, NADH oxidoreductase.
CoQ oxidoreductase. • Pumps no H+ ions.
• Pumps 4 H+ ions into
Components :
Intermembrane space.
1. FAD/FADH2.
Components : 2. Fe-S complex.
1. FMN/FMNH2.
2. Fe-S complex.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Electron Transport Chain 249

Complex III : Cytochrome C : Complex IV : ----- Active space -----

• AKA CoQ cytochrome C • Mobile electron carrier. • Irreversible.
oxidoreductase. • AKA cytochrome c
• Pumps 4 H ions into
+
oxidase.
IMS. • Pumps 2H+ ions into
Components : IMS.
1. Cytochrome b. Components :
2. Cytochrome c1. 1. Cytochrome a/a3.
3. Rieske Fe-S complex. 2. Copper A Copper B
(CuA CuB) centre.

Complex V/ATP Synthase complex :

om
l.c
ai
gm
@
19
55
Proton channel
na

Contains 10 c disc proteins

ee
m

• Rotatory
ar

(only
um

mobile
itk

subunit) (9 subunits)
am

• Connects
|

F0 to F1
w

site of ATP synthesis

ro
ar

Pathway 
M

00:21:46
©

Complex I III IV
Complex II III IV
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 250 Integration of Metabolism and Biological Oxidation

----- Active space ----- Oxidative Phosphorylation :

• Chemiosmotic theory by Peter Mitchell. • Electron transfer
• Each complex : Proton pump.
• Oxidation Phosphorylation H+ pumped into
Complex Complex V intermembrane space
Coupling through
I-IV (ADP ATP)
H+ gradient Potential difference
created

H+ pumped back into matrix

Oxidative phosphorylation
Drives ATP synthase complex

om
Inhibitors of Respiratory Chain  00:30:49

l.c
ai
gm
Inhibitors of respiratory chain

@
19
55
Inhibitors of electron chain Inhibitors of oxidative Uncouplers
na
ee

phosphorylation (Complex V)
m

No movement of electrons Redirect H+ from ATP

ar
um

• No ATP synthesis synthase complex to another

itk

• Slight ↑O2 consumption channel

am

No O2 No H+ gradient
(initial)
|

consumption
w

No O2 consumption (late) No H+ gradient

ro

No ATP synthesis
ar
M

• No ATP synthesis
©

• ↑ O2 consumption (initial)
No O2 consumption (later)
Inhibitors of electron chain :
At Complex I : At Complex II : At Complex III : At Complex IV :
(B/w NADH and CoQ) : 1. Malonate. (B/w Cyt b and (Cyt c oxidase) :
1. Rotenone. 2. Thenoyl trifluoro Cyt c) : 1. Carbon monoxide.
2. Amobarbital. acetone. 1. Antimycin A. 2. Cyanide.
3. Piericidin A. 3. Carboxin. 2. British Anti Lewisite 3. H2S.
(Dimercaprol). 4. Sodium azide.

Inhibitors of oxidative phosphorylation :

At ADP/ATP transporter : At Fo subunit : At F1 subunit :
Atractyloside. 1. Oligomycin. Aurovertin.
2. Venturicidin.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Electron Transport Chain 251

Uncouplers : ----- Active space -----

Physiological : Chemical :
1. Thermogenin (uncoupling protein 1). 1. 2, 4 dinitrophenol.
2. Thyroxine. 2. Dinitrocresol.
3. Long chain fatty acid. 3. Fluoro carbonyl cyanide phenyl
hydrazine (FCCP).
4. Aspirin (High dose).

Note :
• Thermogenin is found in brown fat of :
- Hibernating animals.
- Neonates.
• Protective against hypothermia :

om
Transfer of electrons in ETC Release of energy

l.c
ai
Thermogenin Inhibits Blocks ATP Energy released

gm
@
phosphorylation production as heat
19
55
na

Non-shivering
ee
m

thermogenesis
ar
um

Effect of compounds on ATP synthesis and O2 consumption :

itk
am

In an isolated mitochondria, various compounds were added and effects were :

|

1. ADP + Pi.
w

3
ro

2. Succinate.
ar

3. Cyanide
M

O2 ATP synthesis (inhibits Complex IV).

©

consumption
2
1

1. Succinate.
4 2. ADP + Pi.
3
3. Oligomycin/Venturicidin
O2 ATP synthesis (inhibits Fo).
4. Dinitrophenol (uncoupler).
consumption
2
1

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 252 Vitamins and Nutrition

----- Active space ----- FAT SOLUBLE VITAMINS

Vitamins : Overview 00:00:10

Definition : Organic compounds present in small amounts in various food substances

needed for growth and maintenance of the body.

Sources :
Supplemented in diet.
Exceptions :

om
Endogenously synthesised vitamins

l.c
ai
gm
@
By body In body by intestinal flora
19
55
• Niacin : From Tryptophan. • Vitamin K.
na

• Vitamin D : From 7-dehydrocholesterol. • Pantothenic acid.

ee
m

• Biotin.
ar
um

Classification :
itk
am

Fat Soluble Water Soluble

|
w

Along with chylomicron Chylomicron not required.

ro

1. Absorption
ar

(Lipoprotein) (Absorbed directly to plasma)

M
©

2. Excretion Not excreted in urine Excreted in urine.

3. Storage Stored in Liver Not stored.
Properties
Rare except : • Niacin ( B3 )
4. Toxicity Present
• Vitamin B6
• Multiple functions.
• Vit K : Coenzyme
5. Functions Act as coenzymes
for gamma
carboxylase.
Hematopoietic :
Energy releasing : Others :
Vit B9 (Folate)
Examples Vitamin A, D, E, K Vitamin B1 , B2 , B3 , Vit B6 ,
and Vit B12
B5 , Biotin. Vit C
(Cobalamin)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Fat Soluble Vitamins 253

Vitamin A 00:06:46 ----- Active space -----

FORMS
Pro vitamin A Preformed Vit A
Dietary
Plant Sources Animal Sources
Source :
Retinoids
(All compounds related to retinol)
Retinal Retinoic acid Retinol
• All trans retinal. • All trans retinoic
β − carotene • 11 - Cis retinal : acid.
Examples
(Carotenoids) Present in • 13-cis retinoic

om
l.c
Rhodopsin acid.

ai
• 9-cis retinoic

gm
@
11 - Cis retinal + acid.
19
55
Opsin
na
ee

Structure 2 Bionone rings Single Bionone ring ( )

m
ar
um

CHO COOH
itk
am
|

β − carotene Aldehyde Acid Alcohol

w
ro
ar

• Regulation of
M
©

gene expression
(Act at nuclear
receptors)
Later converted to Control
Function Vision Reproduction
active Vit A. Growth,
Morphogenesis & Cell
differentiation
• Steroid hormone
like function.
Note : Non Pro Vitamin A Carotenoids

Lutein and Zeaxanthin Lycopene

• Prevents macular degeneration. • Prevents prostate cancer.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 254 Vitamins and Nutrition

----- Active space ----- METABOLISM

1. Absorption, Storage and Transport to target site :

β carotene Retinol : Absorbed

forms
Converted to • Stored as Retinyl
Retinol carried Ester (Palmitate).
in Chylomicron • Site : Perisinusoidal/
Retinol Ito cells.
Blood vessels Liver
Binds with Insoluble
fatty acids in blood

Retinyl ester

om
Retinal carried as

l.c
Tri molecular Complex
Intestinal cell

ai
gm
Target

@
Transthyretin Retinol Retinol binding
site 19
(Also transports protein (RBP)
55
na

thyroxine)
ee

Transported in blood
m
ar
um

2. Wald’s Visual Cycle :

itk
am

Regeneration of 11 - cis retinal.

|
w
ro
ar
M

Rhodopsin Rhodopsin
©

Multistep Multiple isomers

Opsin process formed Bathorhodopsin

11 cis retinal all trans retinal Lumirhodopsin

RETINAL
EPITHELIUM Metarhodopsin I
11 cis retinal all trans retinal
Metarhodopsin II
BLOOD (Generates nerve impulses)
11 cis retinal all trans retinal

NADH LIVER NAD+

Oxidation
NAD+ NADH
11 cis retinol all trans retinol
Isomerisation
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Fat Soluble Vitamins 255

3. Generation of Nerve Impulses : ----- Active space -----

Opsin :
• G-protein Coupled Receptor (GPCR).
• 7 Transmembrane protein

11 - cis Retinal 11 - trans Retinal

Binds to Light Stimulus

Opsin Opsin
Photoisomerisation

Inactive GPCR Active GPCR

om
Liberates

l.c
ai
gm
@
19
55
Active G protein :
na

Transducin
ee
m
ar
um
itk

Combines with
am
|
w

Active PDE Inactive

ro

Converted to
ar

Phosphodiesterase
M

(PDE)
©

c GMP 51 GMP Closes Hyperpolarisation

(2nd messenger) Na+ channel
Generates nerve impulses.

FUNCTIONS
1. Vision.
2. Regulation of gene expression : By retinoic acid.
3. Normal reproduction : By Retinol.
4. Maintenance of Normal skin & mucosa.
5. Antioxidant properties.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 256 Vitamins and Nutrition

----- Active space ----- VITAMIN A DEFICIENCY

• M/C vitamin deficiency.
• Preventable cause of blindness.

Eye Manifestations :

Symptoms :
• Nyctalopia (Night blindness) : First symptom.
• Loss of vision to green light.

Signs :
1. Conjunctival Xerosis 2. Bitot’s spot :
Progresses to
• Triangular raised lesions in bulbar conjunctiva.
• Characteristic feature of Vit A deficiency.

om
Corneal Xerosis

l.c
ai
gm
@
19
55
Bitot’s
na

spot
ee
m
ar
um
itk
am
|
w
ro
ar

3. Corneal ulcer &

M
©

Keratomalacia

Skin and Mucous secreting epithelium manifestations :

1. Epithelial metaplasia &
hyperkeratinization.

Obstruction of adnexal glands

Follicular hyperkeratosis/
Toad skin/Phrynoderma.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Fat Soluble Vitamins 257

----- Active space -----

2. Squamous metaplasia of
• Upper respiratory tract.
• Urinary tract.

3. Loss of taste sensation.

om
WHO Classification :

l.c
ai
gm
Stage Clinical Manifestations

@
XN 19
Night blindness
55
na

X1A Conjunctival Xerosis

ee
m
ar

X1B Bitot’s spots

um
itk

X2 Corneal Xerosis
am

X3A Corneal ulcer / Keratomalacia < 1/3rd surface

|
w
ro

X3B Corneal ulcer / Keratomalacia ≥ 1/3rd surface

ar
M
©

XS Corneal scarring
XF Fundal xerophthalmicus

THERAPEUTIC USES :
Treatment of Form used
β-carotene Prevents cutaneous
Cutaneous Porphyria
(Good antioxidant) photosensitivity
Promyelocytic
All trans-retinoic acid Differentiation therapy
leukemia
• Cystic acne
13 - cis retinoic acid S/E : Teratogenicity in
• Childhood
(Isotretinoin) reproductive age group.
Neuroblastoma

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 258 Vitamins and Nutrition

----- Active space ----- TOXICITY :

Acute Toxicity : Chronic Toxicity : Pregnancy :

• Seen in Arctic explorers. • Long term intake of • Teratogenic.
(D/t consumption of polar bear liver ↑ Vit A) > 50,000 IU/day.
• Organelle affected : Lysosomes. • Effects on
• Effects on : Bones : Bony exostosis.
Brain : Pseudotumour cerebri Liver : Hepatomegaly
Progresses to
• ↑ Intracranial tension findings. Cirrhosis

om
l.c
(Resembles tumor) Non-specific :

ai
gm
- Dizziness - Weight loss.

@
- Vomiting - Anorexia.
19
55
- Headache
na

Skin : Exfoliative dermatitis.

ee
m

Liver : Hepatomegaly.
ar
um

Lipid profile : Hyperlipidemia.

itk
am

RDA AND SOURCES

|
w

Recommended Daily Allowance (RDA) :

ro

Sources
ar
M

Demographic RDA ( µg of retinol )

©

Children Animal sources Plant sources

400 • Fish oil : Halibut liver oil • Carrot : Richest source.
(Age 1-6 yrs)
• Meat • Green leafy vegetable
Women 600 • Egg
Men 600 • Cheese
Pregnancy 800 • Milk
Lactation 950

Note :
• 1 IU of Vit A : 0.3 µg of retinol.
• Assay of Vit A :
a. Dark adaptation time ↑ (Assessment of Nyctalopia)
b. Carr-Price reaction.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Fat Soluble Vitamins 259

Vitamin D 00:35:20 ----- Active space -----

• Group of sterols with hormone like function Act on Nuclear receptors.

SOURCES
• Food Limited source unless fortified.
- Exception : Fish.
• Sunshine : Richest source (aka Sunshine vitamin).
• Endogenously synthesised vitamin.
Forms

Ergocalciferol ( Vit D2 ) Cholecalciferol ( Vit D3 )

om
a. Plant sources. Animal sources

l.c
ai
b. Fungal ( Ergot ) For commercial purposes.

gm
@
METABOLISM 19
55
Site of Synthesis : Stratum corneum of skin
na
ee

Sunlight : UVB : 290-315nm

m
ar
um

Stratum Corneum
itk
am

7 - dehydrocholesterol
|
w

Other dietary Cholecalciferol ( Vit D3 ) Transported in blood

ro

sources • Major source :

ar

by Vit D binding protein

M

• Fat soluble.
©

• Insoluble in blood.

Cholecalciferol
Hydroxylation
25 - OH - CC 25 - OH - Cholecalciferol
Hydroxylation (25 - OH - CC)
1,25 - OH - CC
Biologically most potent Liver
and active form
If sufficient 24
stores of hydroxylase
Vit D
Calcitroic acid Excreted in urine
(Inactive)
Kidney

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 260 Vitamins and Nutrition

----- Active space ----- Hydroxylations :

25 α Hydroxylase 1 α Hydroxylase
Enzyme 24 α Hydroxylase activity
activity activity

Site Liver Kidney Kidney

25-(OH) CC : used 1,25 (OH)2 CC / 24, 25 DH CC /
Product
for assay of Vit D Calcitriol Calcitroic acid
• Calcitroic acid : Inactive.
• Rate limiting step.
Features - • Found when ↑ blood Ca2+
• Favoured by PTH.
& excreted in urine.

om
FUNCTION

l.c
ai
gm
Calcium and Phosphorus Regulation :

@
Performed by Vit D and parathyroid. 19
55
na

• VitD ↑ Blood Ca2+

ee

↑ Blood P
m
ar
um

• PTH ↑ Blood Ca2+

itk

↓ Blood P
am
|
w

Effect on Intestine :
ro
ar
M

PTH
©

+
25 (OH) D 1,25 (OH)2 D
Hydroxylation
in Kidney

Acts on Intestine

↑ Level of Calcium Binding ↑ Level of Calcium Transport

Protein (Calbindin 9k) Channel ( TRPV 5 )
TRPV : Transient receptor
potential vanilloid family
↑ Absorption of Ca2+ from intestine

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Fat Soluble Vitamins 261

Effect on Kidney : ----- Active space -----

+
a. 1,25 (OH)2 D ↑ PO43- Reabsorption ↑ Serum PO43-
from DCT

↑ Level of Calcium Transport ↑ Level of Calcium Binding

Channel ( TRPV 6 ) protein (Calbindin 28k)

↑ Ca2+ Reabsorption of DCT

↑ Serum Ca2+

om
l.c
+
b. PTH Ca2+ Reabsorption

ai
gm
@
-
PO43- Reabsorption ↑ Excretion of PO43-
19
55
(Phosphaturic)
na
ee

Effect on Bones :
m
ar

PTH & 1, 25 (OH)2 D

um

+
RANK
itk

RANK ligand
am

receptor
Pro Mature
↑ Bone resorption
|

Osteoblast Osteoclast Osteoclast

Matures
w

Binds to
ro
ar
M

Demineralization
©

(Ca shifts out of bone)

2+

Raised Serum Ca2+

Summary of Actions on Ca2+ and PO43- :
Vit D PTH
↑ Reabsorption of Ca2+ and P
• ↑ Reabsorption of Ca2+ ↑ S. Ca2+.
Kidney
• Phosphaturic ↓ S. PO43-
S. Phosphate, S. Calcium
Bone Release of Ca2+. from bone ↑ S. Ca2+.
• No direct action.
↑ Absorption of Ca2+.
• ↑ 1 α hydroxylation ↑ 1, 25 (OH)2 CC
Intestine
↑ S. Ca2+.
↑ Absorption of Ca2+. from intestine

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 262 Vitamins and Nutrition

----- Active space ----- Vitamin D Deficiency 00:51:30

Effect on Bone :
Normal : Bone components

Protein matrix : Deposited Mineral : Ca2+ and PO43-

Osteoid on (Hydroxyapatite)
+

Vitamin D

Vitamin D deficiency : Inadequate mineralization of matrix

om
l.c
Time of occurence

ai
gm
@
Before closure After closure
of epiphyses 19 of epiphyses
55
na
ee
m

Rickets Osteomalacia
ar
um
itk
am

BIOCHEMICAL DEFECTS & CHANGES OF RICKETS

|
w

1. Nutritional rickets/ Osteomalacia :

ro
ar
M

↓ 1,25 (OH)2 vit D and 25 (OH) vit D

©

↓ Serum Ca2+

2° Hyperparathyroidism
PTH is
+
↓ S. phosphate
phosphaturic
1 - α hydroxylation

Normalises / Increases 1, 25(OH)2 vit D

Normalises Serum Ca2+

Hence, check for S.PTH if S.Ca2+ normal

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Fat Soluble Vitamins 263

2. Other types : ----- Active space -----

Rickets type Biochemical defect

Mutation of gene encoding for 1 α Hydroxylase
Vitamin D Dependent Rickets Type 1 /
Pseudo-Vitamin D Resistant Rickets
↓ Biological active form of Vit D
Mutation in Vit D receptor
Vitamin D dependent Rickets Type 2
/ True Vitamin D Resistant Rickets
Vit D cannot act on target organs
Mutation in PHEX gene

↑ FGF-23 activity
X linked Hypophosphatemic Rickets

om
l.c
Phosphaturia

ai
gm
@
Hypophosphatemia (↓ S. phosphate)
19
55
Mutation in proteases which degrade FGF 23
na
ee

Autosomal Dominant
m

↑ FGF-23
ar

Hypophosphatemic Rickets
um
itk

↓ S. phosphate
am

Mutation in Dentin matrix protein |

|
w
ro

Autosomal Recessive
ar

↑ FGF-23
M

Hypophosphatemic Rickets
©

↓ S. phosphate

Note :
FGF-23 (Fibroblast growth factor-23) : Phosphatonin

↓1-α-Hydroxylase ↓ Apical expression of ↓P reabsorption

Na+/P cotransporter in PCT
↓-1,25-(OH)2D.

↑ P excretion.
• PHEX gene -
FGF-23.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 264 Vitamins and Nutrition

----- Active space ----- SKELETAL MANIFESTATIONS OF RICKETS

Wind swept deformity : Harrison’s sulcus : Rachitic rosary : Genu Genu

• Varum deformity in Pulled up sternum (Bead like swelling of valgum varum
knee. with diaphragm. costochondral junction).
• Valgum deformity in

om
other knee.

l.c
ai
Normal Genu valgum Genu varum

gm
• Cupping

@
Concavity at metaphysis end
19
55
• Splaying
na

Widening of metaphyseal plate

ee

Knee • Fraying
m
ar

>7o Medial Irregularities at borders

um

5-7 deviation of of metaphyses

itk

o
of tibia • White line of Frankel
am

valgus
|

Deformities at Knee Joint X-ray features of Rickets

w
ro
ar
M

RDA, Toxicity, Assay of Vit D 01:04:39

©

Recommended Daily Allowance (RDA) :

Demographic RDA
Children 10 µg/day = 400 IU

Adults 5 µg/day = 200 IU

In pregnancy 10 µg/day = 400 IU

Note : Richest dietary source of vitamin D Halibut liver oil.

Toxicity : Calcinosis ↑ Ca2+ deposition in blood vessels.
Assay : Measurement of :
• 25 (OH) Cholecalciferol.
• Serum Osteocalcin
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Fat Soluble Vitamins 265

Vitamin E 01:07:24 ----- Active space -----

Properties and function :

• Most potent naturally occurring antioxidant : α Tocopherol.
• Lipophilic chain breaking antioxidant

Protects LDL from oxidation Protects PUFA in membranes

Feature of deficiency :
• Axonal degeneration.
• Peripheral neuropathy.
• Spinocerebellar ataxia.
• Hemolytic anemia : Hemolysis of RBC membrane PUFA

om
l.c
ai
gm
Cell integrity not maintained.

@
• Ophthalmic features :
19
55
- Pigmentary retinopathy.
na

- Ophthalmoplegia.
ee
m

- Nystagmus.
ar
um

Uses :
itk

Treatment of :
am

• Retrolental fibroplasia.
|
w

• Intermittent claudication.
ro
ar

• Bronchopulmonary dysplasia.
M
©

• Intraventricular haemorrhage.
• Slowing of aging.

RDA :
Male : 10 mg/day.
Female : 8 mg/day.
Pregnancy : 10 mg/day.
Lactation : 12 mg/day.

Toxicity :
Interferes with Platelet aggregation
Vitamin K

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 266 Vitamins and Nutrition

----- Active space ----- Vitamin K (Coagulation vitamin) 01:07:30

Naphthoquinol derivative.
Forms

Vitamin K1 : Phylloquinone Vitamin K2 : Menaquinone Vitamin K3 : Menadione

Dietary source. • Synthesized by • Synthetic.
Bacterial flora. • Water soluble.

Function :
Post Translational Gamma Carboxylation (Maturation) of :
a. Clotting factors : c. Protein S.
• Factor II (Prothrombin) d. Osteocalcin (Protein in bone).

om
• Factor VII e. Nephrocalcin (Protein in kidney).

l.c
• Factor IX

ai
f. Product of gene gas-6.

gm
• Factor X g. Matrix Glutamic acid (Gla) protein.
@
b. Protein C 19
55
na

Vitamin K Cycle
ee
m
ar
um
itk
am
|

Reduced vitamin K Epoxide of Vitamin K

w
ro

(Active) (Inactive)
ar
M
©

g
clin

Vitamin K Warfarin,
cy

epoxidase Dicoumarol
Re

Oxidised
Note :
vitamin K
Orlistat : Affects function of Vitamin K.
Deficiency :
• Clotting affected Bleeding :↑ Prothrombin time (PT)
↑ Clotting time (CT)
• Common in premature babies d/t
- Low fat reserves
- Liver immature Supplementation with
- Gut sterile Vitamin K needed
- Breast milk : Poor source of Vit. K

Toxicity : Hemolysis Hyperbilirubinemia Kernicterus.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Hematopoietic Vitamins 267

HEMATOPOIETIC VITAMINS ----- Active space -----

Folic Acid (vitamin B9)  00:00:50

Active form : Sources :

Tetrahydrofolic acid (THFA). Vegetarian diet : Green leafy vegetables :
Raw>cooked.

If heated > 10 mins Loss of 95% active folate

FUNCTION

om
Carries 1C groups : Eg :

l.c
ai
• Formyl (-CHO). • Methenyl (-CH=).

gm
• Methyl (-CH3). • Formimino (-CH=NH).
@
• Methylene (-CH2-). 19
55
na
ee

Carbon Metabolism and applied aspects :

m
ar
um

Donors of 1C Carriers of 1C Function/End points

itk
am

Glycine cleavage system Methyl

a. Glycine Methylene B12
|

Methionine
w

THFA THFA
ro

Serine hydroxy
ar

Methyl
M

b. Serine (3c) methyl transferase 1C Free THFA Homocysteine

con
©

ve B12
to THFA Methenyl rts
Glycine (2C)
THFA
c. Histidine (3c) Via FIGLU Forminino dUMP TMP DNA synthesis
THFA
d. tryptophan Formyl THFA Purine synthesis

dUMP : Deoxy Uridine Mono Phosphate

TMP : Thiamine Mono Phosphate

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 268 Vitamins and Nutrition

----- Active space ----- DEFICIENCY

Features :
Megaloblastic anemia
Pathogensis :
B12 deficiency THFA deficiency Folate Trap ↓TMP synthesis

Impaired DNA synthesis

Nucleo cytoplasmic
asynchrony of erythroid • ↓ Nuclear maturation
Ineffective erythropoiesis
precursors (Cells with • Normal cytoplasmic
high proliferation) maturation

om
Megaloblasts in Hypersegmented

l.c
different stages of neutrophil

ai
gm
maturation (Erythroid Macro-ovalocytes

@
precursor)
Bone Marrow 19 Peripheral smear
55
na

• Neural tube defects : Spina Bifida, Anencephaly d/t folate

ee
m

deficiency in pregnancy folate prophylaxis.

ar
um

• Homocysteinemia
itk

- Normal pathway :
am

N5 Methyl THFA B12 methionine S-Adenosyl

|

Principle CH3
w

methionine
ro

donor
ar

Methyl (SAM)
M

THFA Homocysteine
B12
©

- If B9 deficiency Methyl group not • Methionine not formed

given to homocysteine • ↑Homocysteine
• ↓SAM

Transmethylation reactions
Investigations : affected
• S. Folate (or red cell Folate) : ↓
• Histidine load Test : Histidine

FIGLU if deficiency of B9 FIGLU excreted

THFA in urine
Formimino THFA
Glutamic acid
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Hematopoietic Vitamins 269

3. Serum Homocysteine. ----- Active space -----

4. ↑
 Serum Aminoimidazole Carboxamide Ribose 5 Phosphate (AICAR) : Intermediate
in purine synthesis.
↓ B9 Impaired ↓ Purine synthesis Accumulation of
donation of intermediate
1C group AICAR
Note :
Antifolate : 1. Folinic acid : 5 formyl THFA
to prevent deficiency
• ↓Availability of THFA 2. Leucovorin : Racemic mixture of
Supplement with
• Eg : Drugs like folinic acid
methotrexate Leucovorin rescue

om
Vitamin B12 (Cobalamin) 

l.c
00:23:40

ai
gm
Source : Animal Sources. Pyrrole

@
19 ring
55
ABSORPTION AND TRANSPORT
N N
na
ee

4.35%
m

Protein Cobalt
ar

Diet : Protein bound B12 present

B12
um

Cobalophilins/R- Secrete N N
itk

Binders/Haptocorrins Salivary glands

Mouth :
am
|
w

Gastric parietal cells

ro

Stomach : Pepsin
ar

Digests Secrets pepsin and IF

M
©

Cobalophilin
bound Vit B12
Intrinsic
Factor (IF)
Duodenum : Digests
Pancreatic enzymes

Intrinsic factor
bound cobalamin
Jejunum :
Intrinsic factor
Receptor Cubulin
Transporter : Transcobalamin
Ileum : II (TC-II) > I
TC-II Reaches target organ
Site of Absorption Portal vein Transcobalamin receptor :
Megalin

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 270 Vitamins and Nutrition

----- Active space ----- Active Forms of B12 :

Coenzymes Using B12 B12 deficiency
L-methyl malonyl CoA
Adenosyl B12 ↑ Serum methyl malonyl
Methylmalonyl CoA mutase
(Ado B12) CoA : Only in B12 deficiency
Succinyl CoA
Methyl Homocysteine
B12
THFA Homocysteine methyl
↑ Serum Homocysteine in
Methyl B12 Methyl transferase/methionine
THFA B12 and B9 deficiency
B12 synthase
Methionine
B12 DEFICIENCY

om
l.c
Causes :

ai
gm
1. Nutritional : Strict Vegans Can use Curd Lactobacillus synthesizes B12.
Supplementations
@
2. Gastric Causes : 19
55

• Autoimmune gastritis (Pernicious anemia) ↓ Intrisic factor (IF).

na
ee

• Gastrectomy No parietal cells No IF.

m
ar
um

3. Intestinal Causes : Any ileal pathology.

itk

• Crohn’s Disease.
am

• Stagnant Loop Syndrome.

|
w

• Fish tape worm (Diphyllobothrium latum)

ro
ar
M
©

Consumes B12

Biochemical Defects and Features :

1. Methyl THFA THFA
B12 ↓THFA
deficiency
Impaired DNA synthesis

megaloblastic Anemia
• Proximal cause of megaloblastic anemia : Folic acid decifiency (Even if B12
deficiency present).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Hematopoietic Vitamins 271

2. Methyl Malonic Acid SuccinylCoA ----- Active space -----

B12 ↑MMA
deficiency
↑Propionic acid

Abnormal Neuronal lipids

Myelin breakdown

• Neurological manisfestations : Only in B12

deficiency
• Subacute combined degeneration

Investigations :

om
• Serum Cobalamin.

l.c
ai
• Serum Homocysteine (also for B9 deficiency).

gm
@
• Urine Homocystine.
• Serum Methyl Malonate : Exclusive B12 deficiency. 19
55
na

• Schilling test.
ee

• Peripheral smear similar to B9 deficiency.

m
ar
um

Mx of Megaloblastic anemia :
itk
am

• 1st rule out Vitamin B12 deficiency before supplementing Folic acid.
|
w

- If folic acid given 1st in B12 deficiency :

ro

Folic acid (+) Methyl THFA Converted to THFA

ar

Further depletes B12 stores

M
©

Exacerbation of neurological symptoms

Hence always Supplement B12 then B9.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 272 Vitamins and Nutrition

----- Active space ----- ENERGY RELEASING VITAMINS

Thiamine (Vitamin B1) 00:00:14

Source :
Aleurone layer of cereal : Required for carbohydrate metabolism.
Lost by Preserved in
Polishing rice • Parboiled rice.
• Brown rice.

om
Coenzyme role :

l.c
Multienzyme complex for oxidative decarboxylation

ai
• Pyruvate DH

gm
Active form : Thiamine Carbohydrate

@
Co enzyme • α ketoglutarate DH metabolism
pyrophosphate
for 19
55
• Branched chain keto DH
na

Transketolase reaction : In non-oxidative phase of

ee
m

Note : HMP Shunt pathway (In carbohydrate metabolism)

ar
um

B6/ PLP : Cofactor in amino acid metabolism.

itk
am

DEFICIENCY
|
w

Risk factors :
ro

• Alcohol intake -
ar

Absorption of thiamine.
M
©

• Staple diet of polished rice.

Manifestations :
Dry Beri Beri : Wet Beri Beri :
Peripheral nervous system effects : Cardiovascular effects :
a. Symmetric motor and sensory a. Peripheral edema.
neuropathy. b. High output cardiac failure.
b. Loss of reflex. c. Dyspnoea.
c. Muscle cramp. d. Cardiomegaly.
d. Muscle atrophy (severe cases). e. Pulmonary edema.
Wernicke’s Encephalopathy (WE) : Wernicke’s Korsakoff’s Syndrome :
a. Confusion. a. Symptoms of WE +
b. Horizontal nystagmus. b. Dementia +
c. Ophthalmoplegia Ptosis. c. Confabulatory psychosis
d. Truncal ataxia Unsteady gait.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Energy Releasing Vitamins 273

Investigations : ----- Active space -----

• Erythrocyte transketolase level.
• Urinary B1 excretion.

Riboflavin (Vitamin B2) 00:09:25

OVERVIEW
Introduction :
AKA Warburg Yellow Enzyme.
• ↑ intake via supplements Can cause yellow urine.
• Heat stable.
• Redox vitamin Function Oxidative decarboxylation.

om
Active forms and functions :

l.c
Acyl coA DH (In β oxidation)

ai
gm
Succinate DH (In TCA cycle)
a. FAD (Flavoproteins) Co enzyme
@
for
19
Glycerol 3-PO4 DH (Mitochondrial)
55
3 multienzyme complexes :
na
ee

• Pyruvate DH.
m

• α-ketoglutarate DH.
ar
um

• Branched Chain keto acid DH.

itk
am

b. FMN Redox couple in Complex I of Electron Transport Chain.

|
w
ro

DEFICIENCY
ar

Features :
M
©

Corneal
Vascularisation

Fissures in Angular Beefy tongue/ magenta colored

tongue stomatitis tongue d/t loss of surface papilla
• Cheilosis.
• Lacrimation.
Biochemical assessment :
• Erythrocyte Glutathione reductase activity (Flavoprotein) : After giving FAD in vitro.
• Urinary riboflavin excretion.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 274 Vitamins and Nutrition

----- Active space ----- Niacin (Vitamin B3) 00:16:03

OVERVIEW
Source :
Endogenously synthesized by tryptophan :
• 60 mg Tryptophan 1 mg Niacin.
Active forms :
1. NAD+ cofactor for All dehydrogenases except :
• Acyl coA DH.
• Succinate DH.
cofactor for • HMP Shunt Pathway oxidative phase
2. NADP+
(NADPH generating

om
- G6PDH
reactions)

l.c
- 6 PGDH

ai
NADP+ NADPH • Malic enzyme

gm
@
• Cytoplasmic isocitrate dehydrogenase
55
19
na

cofactor for Reductases

ee

3. NADPH
m

(NADPH utilising
Eg :
ar
um

reactions) • Enoyl reductase.

itk

• HMG CoA reductase.

am

• Dihydrofolate reductase.
|
w

DEFICIENCY OF NIACIN : PELLAGRA

ro
ar

Features :
M
©

4D’s
• Dermatitis Photosensitive dermatitis Symmetrical erythematous
exposed areas. rash in sun
• Diarrhea.
• Dementia.
• Death (rare).
• Depressive psychosis (rare).
Conditions causing pellagra like symptoms :
Hartnup’s disease : ↓absorption of Tryptophan (Trp) ↓ blood Trp ↓ B3.
Carcinoid Syndrome : Tryptophan Enters argentaffin cells Converted to
serotonin Less Trp available for B3 Synthesis.
Staple diet Maize/ Corn : Niacin in bound form.
Jowar/Sorghum : ↑ leucine content (-) QPRTase ↓ Synthesis
of B3.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Energy Releasing Vitamins 275

B6 deficiency : ----- Active space -----

B6 required for Kynureninase enzyme Used in Synthesis of B3.
↓ B6 ↓ Kynureninase ↓ B3 synthesis.
TOXICITY
Develops in those taking niacin Rx for other conditions.
Features :
• Flushing (Prostaglandin mediated) Rx • Premedication with Aspirin
• PGD2 antagonist : Laropiprant
• Glucose intolerance. • Gastric irritation.
• Hyperuricemia Gout. • Fulminant hepatitis.
• Macular edema.

om
Note : B vitamins causing toxicity B6 and B3.

l.c
ai
THERAPEUTIC USE

gm
Lipid modifying drugs :
@
• ↓ Triacylglycerol. • ↓ LDL. 19
55
na

• ↑ HDL.
ee
m
ar

Panthothenic Acid (Vitamin B5) & Biotin (Vitamin B7)

um

00:26:45
itk

VITAMIN B5 /PANTOTHENATE
am
|

Etymology : Pantos Everywhere .

w
ro

Sources :
ar
M

• Coenzyme A :
©

- Acetyl CoA.
- Acyl CoA.
- HMG CoA.
• Acyl carrier protein in fatty acid synthase complex.
Deficiency : Nutritional Melalgia / Gopalan’s burning foot syndrome
VITAMIN B7/ VITAMIN H/ BIOTIN
Active form :
Carboxybiocytin

Co enzyme role :
Carboxylation
• Pyruvate carboxylase ABC enzymes
• Propionyl coA carboxylase ATP, B7,
• Acetyl coA carboxylase CO2
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 276 Vitamins and Nutrition

----- Active space -----

Note :
Biotin independent carboxylation :
• Gamma carboxylation (Vitamin K dependent) : Clotting factors 11, V11, IX, X.
• Carbamoyl phosphate synthetase.
• Malic enzyme.
• AIR carboxylase (In purine synthesis pathway).
Deficiency :
Risk factor : Raw egg consumption Avidin (In egg white) - Biotin (In egg yolk)
Features :
• Depression/Hallucination.
• Seborrheic dermatitis : Nose, eye, mouth Scaling, erythematous rash.
• Fatigue.

om
Mechanism of fatigue :

l.c
ai
a. Early morning exercise :

gm
@
↓ Biotin ↓ Pyruvate carboxylase activity ↓ Synthesis of
19
55
oxaloacetate (Enzyme in gluconeogenesis) Impaired gluconeogenesis
na

Exercise in early morning Hypoglycemia & tiredness

ee
m

b. Exercise in fed state :

ar
um

• Pyruvate carboxylase needed Oxaloacetate + Acetyl CoA Enters TCA.

itk

• If ↓ B7 ↓ Oxaloacetate ↓ energy production Fatigue.

am
|

Investigation :
w
ro

• Urinary biotin concentration.

ar
M

• Serum & urine propionic acid level

©

Propionic acid B7 Methyl malonic acid

If deficient ↑accumulation of propionic acid.
• ↓ activity of B7 dependent enzymes in lymphocytes (For carboxylation reaction).
Note :
Leiner’s disease : Defect in biotinidase enzyme ↓ active form of biotin.
Therapeutic uses of biotin :
Added to
1. Labelling of DNA : B7 DNA Probe.
2. Elisa test : B7 labelled reagents used.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Vitamin B6 and C 277

VITAMIN B6 AND C ----- Active space -----

Vitamin B6 (Pyridoxine)  00:00:20

Source :
Pyridoxine
Pyridine ring Pyridoxal Pyridine derivatives
Pyridoxamine

Active form :

om
Pyridoxal phosphate (PLP).

l.c
ai
gm
Functions :

@
Coenzyme role : 19
55
1. Transamination : 3. Transulfuration :
na
ee

Eg : Eg :
m

• Alanine aminotransferase (ALT). • Cystathione β synthase.

ar
um

• Aspartate aminotransferase (AST). • Cystathionase.

itk

2. Decarboxylation :
am

4. Tryptophan metabolism : Kynureninase.

Eg :
|

CO2 5. Heme synthesis : ALA synthase.

w
ro

• Histidine Histamine.
ar

CO2 6. Glycogenolysis : Glycogen phosphorylase.

M
©

• DOPA Dopamine. Note : 80% of PLP in the body- Muscle.

CO2
• 5-OH Tryptophan 5-OH Tryptamine (Serotonin)

Deficiency manifestations :

1. Pyridoxine dependent seizures : 2. Sideroblastic anemia :

• PLP : Coenzyme for ALA synthase (rate
limiting enzyme in heme synthesis).

• Iron laden mitochondria in perinuclear

region of erythroblast.
Sideroblasts on peripheral
smear

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 278 Vitamins and Nutrition

----- Active space ----- 3. Pellagra like symptoms :

• Tryptophan Kynureninase
PLP Niacin.
• ↓ PLP ↓ Niacin Pellagra like symptoms.

4. Hormone dependent cancers :

• Physiology : • Vit.B6 deficiency :
Hormone + Receptor ↓ Vitamin B6

Hormone receptor complex ↑ Hormone activity

Vitamin B6
Binds to DNA Cancers
Eg :
Regulated hormone • Ca Prostate.

om
activity • Ca uterus.

l.c
ai
• Ca breast.

gm
@
5. Personality changes : 19
55
na

• Depression.
ee

• Confusion.
m
ar
um

Urinary metabolites in Vitamin B6 deficiency :

itk
am

Xanthurenic acid
|
w
ro

Kynureninase
ar

(Tryptophan metabolism)
M
©

B6 deficiency
Glyoxalate alanine Cystathianione
aminotransferase β synthase

Oxaluria Homocystinuria

Biochemical assay :
Enzyme activity : Erythrocyte transaminase.
Load test : Tryptophan load test (Excretion of xanthurenic acid).
Direct measurement : Estimation of Vitamin B6 levels in blood.

Toxicity :
Sensory neuropathy.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Vitamin B6 and C 279

Note : ----- Active space -----

• Vitamin B6 Affects most enzyme activity in RDA of Vit. B6 depends
amino acid metabolism on protein intake
• ↑ Protein intake Requires ↑ Vit.B6 intake

Vitamin C  00:12:50

AKA Ascorbic acid, antiscorbutic factor.

Source :
Uronic acid pathway
Glucose Vitamin C

Not seen in humans and higher primates

om
(D/t lack of L-Gulonolactone oxidase)

l.c
ai
gm
Functions :

@
1. Collagen synthesis : Triple helix 19
3. Tyrosine metabolism : PHPPhydroxylase.
55

formation through prolyl and lysyl 4. Tryptophan metabolism :Hydroxylation reaction.

na
ee

hydroxylases. 5. Bile acid synthesis : 7 α hydroxylase.

m
ar

2. Iron absorption : 6. Steroid hormone synthesis.

um

Ferrireductase (Vitamin C) 7. Cholesterol synthesis.

itk
am

Cofactor function
Fe+3 Fe+2
|
w
ro
ar

Intestinal absorption
M
©

Vit. C deficiency Anemia.

Deficiency manifestations :
A. Scurvy :

1. Splinter hemorrhages 2. Hemarthrosis 3. Petechiae

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 280 Vitamins and Nutrition

----- Active space -----

4. Bleeding gums 5. Anemia on peripheral smear 6. Scorbutic rosary

• Beaded appearance at the
B. Barlow’s disease : costochondral junction
• Angulated Differentiates from
AKA Infantile scurvy. • Tender rachitic rosary
At 6-12 months (weaning period)

om
Require vitamin C supplementation

l.c
ai
gm
Miscellaneous  00:19:05

@
1. Urinary metabolites in vitamin deficiencies :19
55
na
ee

Vitamin deficiency Urinary metabolite

m
ar

a. Methyl malonic acid

um

1. Vitamin B12
b. Homocystine
itk
am

a. Homocystine
|

2. Folic acid b. FIGLU (Metabolite of histidine)

w
ro

c. AICAR (Purine metabolism)

ar
M

2. B complex vitamins with toxicity : 5. Vitamins phosphorylated in active

©

a. Niacin. form :
b. Pyridoxine. a. Pyridoxine.
3. Vitamins with antioxidant property : b. Thiamine.
a. Vitamin C. 6. Vitamin in coenzyme A :
b. Vitamin E. Pantothenic acid.
c. β carotene. 7. Vitamin with β alanine :
(Also have pro-oxidant property) Pantothenic acid.
4. Sulphur containing vitamins :
a. Biotin.
b. Thiamine.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Protein-Energy Malnutrition and Obesity 281

PROTEIN-ENERGY MALNUTRITION AND OBESITY ----- Active space -----

Protein Energy Malnutrition (PEM)  00:01:00

Syndromes associated with moderate to severe undernutrition.

TYPES OF PEM
• Marasmus
• Kwashiorkor
Marasmus

om
• Severe deficiency of both dietary energy and protein.

l.c
ai
• Primary calorie deficiency Secondary protein deficiency.

gm
@
Clinical features :
19
55
• Age : <1 year.
na

• Severe wasting of fat and muscle.

ee

Marasmus
m

• Loss of subcutaneous fat : Baggy pant appearance

ar
um

(Loose skin over buttocks).

itk

• General attitude : Alert.

am
|

Biochemical features :
w
ro

• Hypoglycemia.
ar
M

• No hypoalbuminemia : No edema.
©

(Hepatic synthesis of plasma proteins : Normal) Baggy pant appearance

Kwashiorkor
Deficiency of protein with adequate calorie intake (M/c : At the onset of weaning).
• ↓Protein intake.
↓Protein synthesis
• Essential amino acid deficiency

Clinical features :
• Age : 1-5 years of age.
• ‘Fat sugar baby appearance’ : Pot belly (Protruding abdomen)
- Gross edema.
- Muscle wasting + : Unnoticed due to edema.
• Crazy pavement dermatitis
(Peeling, cracking, denudation of skin). Pot belly appearance

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 282 Vitamins and Nutrition

----- Active space ----- Biochemical features :

• Hypoalbuminemia : s. Albumin <2 g/dL.
• Immunoglobulin level ↑ : D/t ↑ susceptibility to infection.
• Fatty liver : ↓Lipoprotein synthesis.
• Blood glucose : N .

Marasmus v/s Kwashiorkor :

Marasmus Kwashiorkor
Deficiency
s. Albumin ↓
Blood glucose
Fatty liver

om
Age of onset <1 year

l.c
ai
gm
Edema -

@
Skin 19
Dry
55
appearance
na

General attitude Apathy +

ee
m
ar
um

Obesity
itk

00:10:40
am

• State of overnutrition : ↑Calorie intake + ↓Energy expenditure.

|
w

• Abnormal growth of adipose tissue d/t :

ro
ar
M
©

↑Fat cell size ↑Fat cell number

(OR)
(Hypertrophy) (Hyperplasia)

OBESITY INDICATORS
Based on Body Weight :
Body Mass Index (BMI)/Quetelet index :
• Normal : 18.5-24.99
BMI = Weight (kg)2 • Overweight : ≥25
[Height (m)]
• Obesity : ≥30
Brocas index : Height (in cm) - 100.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Protein-Energy Malnutrition and Obesity 283

Waist Circumference/Waist Hip Ratio : ----- Active space -----

Midpoint between lower border of ribcage and iliac crest.
• Males : <90 cm.
• Females : <80 cm.

OBESITY RELATED PATHOLOGIES

Endocrine system :
Cardiovascular system : • Metabolic syndrome/Syndrome X/
• ↑Risk of coronary artery disease Insulin resistance syndrome
• ↑Risk of peripheral vascular disease • Polycystic ovarian syndrome
• Dyslipidemia • Type II diabetes mellitus
• Varicose vein • ↑Risk of hypertension

om
l.c
ai
Musculoskeletal system : Psychologic :

gm
• ↑Risk of hyperuricemia & gout • Depression
@
Obesity
• Low backache 19 • Social stigma
55
na

• Osteoarthritis • Low self-esteem

ee
m
ar
um

Gastrointestinal : Neurologic :
itk

• Gastro esophageal reflux disease • ↑Risk of cerebrovascular accidents

am

• ↑Risk of fatty liver disease • ↑Risk of dementia

|
w
ro

(MASLD/NAFLD)
ar

• ↑Risk of cholelithiasis
M
©

Note :
• MASLD : Metabolic dysfunction Associated Steatotic Liver Disease.
• NAFLD : Non Alcoholic Fatty Liver Disease.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 284 Vitamins and Nutrition

----- Active space ----- Metabolic Syndrome :

• AKA Syndrome /Insulin resistance syndrome.
• Group of metabolic abnormalities a/w resistance.

NCEP and ATP III Criteria :

National Cholesterol Education Program and Adult Treatment Plan III criteria for
metabolic syndrome :
1. Central obesity : Waist circumference Males >90 cm
Females >80 cm
2. Hypertriglyceridemia : Serum TAG >150 mg/dL.
3. Low HDL Males : <40 mg/dL Any 2 or 3 +
Females : <50 mg/dL
4. Hypertension Systolic BP >130 mmHg (OR) Metabolic

om
Diastolic BP >85 mmHg syndrome

l.c
ai
5. Fasting blood glucose : >100 mg/dL.

gm
@
TREATMENT OF OBESITY 19
55
• Lifestyle modifications :
na
ee

- Diet.
m
ar

- Exercise.
um

- Yoga.
itk
am

• Drugs.
|

• Bariatric surgery : ↓Stomach volume.

w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 1 285

MINERALS : PART 1 ----- Active space -----

Classification :
Macrominerals Microminerals
Requirement > 100 mg/day < 100 mg/day
• Iron (Fe)
• Calcium (Ca)
• Iodine (I)
• Sodium (Na)
• Copper (Cu)
• Magnesium (Mg)
• Manganese (Mn)
Examples • Potassium (K)
• Zinc (Zn)

om
• Phosphorus (P)

l.c
• Molybdenum (Mo)
•

ai
Chlorine (Cl)

gm
• Selenium (Se)
• Sulphur (S)

@
• Fluorine (F)
19
55
na
ee

Calcium : Introduction 00:02:20

m
ar
um

Body composition :
itk
am

Total Ca : 1-1.5 kg 99% : Bones & teeth.

|

1% : Body fluids.
w
ro
ar

Distribution :
M
©

• Total plasma Ca : 8.5-10.5 mg/dL.

• Free/Ionized Ca :
- 5 mg/dL.
- Metabolically active form.

Sources :
• Milk (Richest source) > Egg, meat, fish, vegetables.
• Cereals : Poor source.

Required Daily Allowance (RDA) :

Adult : 1000 mg/day.
Pregnancy & lactation : 1500 mg/day.
Children : 750 mg/day.

Sources of calcium

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 286 Minerals and Acid Base Balance

----- Active space ----- METABOLISM

Absorption :
Site : Duodenum 1st & 2nd part.
Mode :
• Active transport.
• Ca-ATPase transporter.

Affecting factors :

↑Absorption : ↓Absorption :
• ↑Vitamin D : ↑Calbindin (Ca transporter). • Phytates & oxalates
• Acidity. (Green leafy vegetables).

om
• Parathyroid hormone (PTH): ↑1α-hydroxylase activity. • ↑Phosphorus.

l.c
+ • Malabsorption.

ai
gm
25-hydroxy cholecalciferol 1,25 dihydroxy cholecalciferol/

@
calcitriol (Active form). 55
19
FUNCTIONS
na
ee

Activation of Enzymes :
m
ar

Mediated through calmodulin :

um

Direct activation
Calcium calmodulin dependent kinase
itk
am

• Pancreatic lipase
• Glycogen phosphorylase
|

• Rennin : Acts on casein (Milk

w
ro

• Adenyl cyclase
ar

protein)
• Pyruvate kinase
M

• Factors of coagulation cascade

©

Actions in Muscles & Nerves :

Muscle fibers : Nerves :

Excitation & contraction. Nerve impulse transmission :
Presynaptic to postsynaptic region.

Hormonal Secretion :
Hormones Role of calcium
Insulin Opening of voltage-gated Ca2+ channels
PTH ↓Serum Ca ↑PTH
Calcitonin -

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 1 287

Action as Second Messenger : ----- Active space -----

Mediated pathways IP3 (Inositol triphosphate)
GPCR (G-protein-coupled receptors)

Coagulation Pathway :
Roles of Ca :
• Factor IV (Ca itself).
IX IXa
• Conversions X Xa
III IIIa
• Stable fibrin clot formation.

Actions on Myocardium :

om
Prolongation of systole :

l.c
Ca interacts with troponin C Contraction.

ai
gm
@
Note : Hypercalcemia Heart arrests in systole. IP3-DAG pathway
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Bone & Teeth :

Major inorganic component of bone/teeth : Hydroxyapatite (Complex calcium salt).

Note : Cells in bone mineralization ↑ by osteoblasts

↓ by osteoclasts

Calpains :
Calcium-dependent proteolytic enzyme (Cysteine proteases).

Function :
Cellular functions :
• Cell cycle progression. • Cell fusion.

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 288 Minerals and Acid Base Balance

----- Active space ----- Regulation of Blood Calcium 00:21:11

Factors :

Major regulators : Others :

• Vitamin D • pH
Hormonal/endocrine
• PTH • Phosphate
regulation
• Calcitonin • Age

VITAMIN D
Active form : 1, 25 dihydroxycholecalciferol/Calcitriol.
Effects : ↑Serum Ca & P.

om
l.c
ai
Actions :

gm
Intestine : ↑Transcription of calcium transporters.
@
• ↑Calbindin 19
55
↑Ca absorption.
na

• ↑Ca ATPase channel

ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Calcium absorption in intestines

Kidneys :
↑Transcription of calcium transporters ↑Reabsorption of Ca & P.

Bones :
↑Osteoclast activity Bone demineralization ↑Serum Ca.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 1 289

Vitamin D Metabolism & Action : ----- Active space -----

7-Dehydrocholesterol Skin Vitamin D3 (Cholecalciferol)

Liver 25-Hydroxylase
25-Hydroxycholecalciferol
Kidney 1α Hydroxylase
1,25-Dihydroxycholecalciferol (Calcitriol)

Intestine : Kidney : Bones :

↑Ca & P absorption ↑Ca & P reabsorption ↑Mobilization of Ca

Maintain blood Ca & P.

om
l.c
PARATHYROID HORMONE (PTH)

ai
gm
Source : Chief cells of parathyroid gland.

@
Effects : 19
55
• ↑Serum Ca.
na
ee

• ↓Serum P.
m
ar

Parathyroid glands (Posterior view)

um
itk
am

Mechanism of Action :
|
w

↓Blood Ca
ro
ar

Sensed by
M
©

Calcium sensitive receptor (Ca SR)

PTH released

Bone : Kidney : Intestine (Indirect) :

↑Bone demineralization • ↑Ca reabsorption • ↑1α hydroxylase activity :
• ↑P excretion (Phosphaturic) 25-hydroxycholecalciferol
↑Blood Ca
1, 25 dihydroxycholecalciferol

• ↑Ca absorption
• ↑P absorption

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 290 Minerals and Acid Base Balance

----- Active space ----- CALCITONIN

Source : Parafollicular cells in thyroid gland.
Effect : ↑Bone mineralization ↓Blood Ca.

Mechanism of Action :
Thyroid gland
Secretes
Calcitonin
Acts on

Intestine : Kidney : Bones :

↓Ca absorption ↓Ca reabsorption • ↓Osteoclast activity
• ↑Bone mineralization

om
(Tonic to bone)

l.c
ai
gm
CALCIUM HOMEOSTASIS

@
Calcitonin 19 Acts on
55
Releases
na
ee
m

Bone : Kidney :
ar

Thyroid gland
• Ca reabsorption
um

Mineralization
itk

• Ca excretion
am

Stimulates
|

High
w
ro

ases
Decre
ar
M

ases Blood Ca
Incre
©

Sensed by
Low
Bones : Kidneys : Intestine :
Stimulates • Ca Ca absorption (In parathyroid glands)
reabsorption
• Vitamin D
activation
Acts on Releases
PTH

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 1 291

OTHER FACTORS ----- Active space -----

Serum Albumin :
• ↓Serum albumin ↓Protein bound Ca
↓Total Ca.
Ionized Ca : Normal
• For every 1 g/dL ↓ in serum albumin Total Ca ↓ by 0.8 mg/dL.

Blood pH :
Serum albumin binds H+ & Ca2+.
Acidosis : ↑H+ binding ↓Ca binding sites ↑Free/ionized Ca Hypercalcemia.
Alkalosis : ↓H+
↑Ca binding to albumin ↓Free/ionized Ca Hypocalcemia.

Blood Phosphate :
Inverse relationship b/w Ca & P.

om
l.c
Ionic product of serum Ca & P : Constant.

ai
gm
Normal adult : Children :

@
• Ca : 10 mg/dL, P : 4 mg/dL. 19
• Ca levels are high.
55
• Ionic product : (Ca) x (P) = 40. • Ionic product : 50.
na
ee
m

Clinical Significance
ar

00:45:54
um
itk
am

HYPERCALCEMIA
|

Causes :
w
ro

Main cause : Primary hyperparathyroidism.

ar
M

Secondary causes :
©

• Ectopic PTH secreting tumor.

• Multiple myeloma.
D/t ↑bone lytic activity.
• Metastatic carcinoma of bones.
• Prolonged immobilization.

Clinical Features :
Non-specific : Fatigue, confusion, depression.
Heart : Cardiac arrest at systole.
Bones Osteoporosis.
Pathological fracture.
Pancreas : Ectopic calcification.
Kidneys Polyuria, polydipsia.
Renal calculi.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 292 Minerals and Acid Base Balance

----- Active space ----- Investigations :

ECG findings :
• Short QT intervals.
• Arrhythmias.

Blood results :
• Ca & alkaline phosphatase (ALP) : ↑ Hypercalcemia :
Short QT interval
• Phosphate level : ↓

Treatment :
• Hydration.
• IV furosemide : ↑Ca excretion.
• Rx of underlying cause.

om
l.c
ai
gm
HYPOCALCEMIA

@
Serum Ca <8.5 mg/dL : Mild tremors.
19
55
<7.5 mg/dL : Tetany.
na
ee

Causes :
m
ar

• Vitamin D deficiency.
um

• Accidental removal of parathyroid gland during thyroidectomy.

itk
am

• Calcitonin secreting tumor.

|

• Malabsorption.
w
ro

• Chronic renal failure.

ar
M

• Alkalosis.
©

Clinical Features :
• Peripheral neuropathy.
• Tremors & tetany.
D/t neuromuscular excitability.
• Muscular spasm.
• Cardiac arrest.

Signs of tetany :
• Carpopedal spasm : D/t involuntary muscle contractions of hands & feet.
• Trousseau’s sign : BP cuff tied around forearm Inflation Spasm of hands.
• Chvostek’s sign : Tapping of area over facial nerve Causes Facial muscle
twitching.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 1 293

----- Active space -----

Carpopedal spasm Trousseau’s sign Chvostek’s sign

Investigations :
ECG findings : Long QT interval.

om
Blood results :

l.c
• ↓Serum Ca.

ai
gm
• ↑Serum P.

@
19
Hypocalcemia : Long QT interval
55
Treatment :
na

• Adequate dietary intake & supplementation : Ca & vitamin D.

ee
m

• Adequate sunlight exposure.

ar
um

• Avoid hypomagnesemia.
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 294 Minerals and Acid Base Balance

----- Active space ----- MINERALS : PART 2

Introduction to Iron  00:01:20

Total body iron : 3-5 gm.

Distribution 75% : Blood.

25% : Bone, muscle & liver.

Required daily allowance (RDA) :

• Adults : 20 mg/day.

om
• Children : 20-30 mg/day.

l.c
ai
• Pregnant/lactating : 40 mg/day.

gm
@
Iron containing :
19
55
na
ee

Proteins : Enzymes :
m
ar

• Hemoglobin. • All complexes in

um

• Myoglobin. electron transport chain (ETC).

itk
am

• Cytochromes. • Cytochromes.
|

• Catalase :
w
ro

Free radical scavenging enzyme.

ar
M

• Nitric oxide synthase :

©

For nitric oxide synthesis (Vasodilation).

• Tryptophan pyrrolase.

Sources :
• Green leafy vegetables & animal products :
Meat (Mainly liver) > cereals & pulses.
• Cooking in iron utensils (Jaggery).

Note :
Very poor source of iron Milk.
• Unweaned infants : May develop iron deficiency anemia.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 2 295

IRON METABOLISM : OVERVIEW ----- Active space -----

Absorption :
Site : Duodenum & proximal jejunum.
Dietary iron : Fe3+
• ↓pH
• SH containing enzymes
• Ferric reductase + Vit C
Iron state absorbed : Fe2+

Absorbed via : DMT-1

(Divalent metal transporter)

Affecting factors :

om
l.c
ai
↑Absorption : ↓Absorption :

gm
@
• Ascorbic acid (↑Ferrireductase, vit C). • Phytates & oxalates.
19
• Acidity. • Excess Ca, Cu, Zn, Pb
55
na

• SH containing enzymes. ↑Occupation of DMT-1

ee
m

↓ Fe2+ absorption.
ar
um
itk

Maintenance of Iron Homeostasis :

am

• Maintained at level of absorption.

|
w

• Iron : One way element (Not excreted).

ro
ar
M

Mucosal Block Theory :

©

Depleted iron stores Adequate iron stores

↑Iron absorption. ↓Iron absorption.

Regulation of Iron Metabolism 00:14:18

IRON ABSORPTION
Site : Enterocytes of duodenum & proximal jejunum.
Phases :
• Luminal.
• Mucosal (Cellular phase).
• Basolateral phase.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 296 Minerals and Acid Base Balance

----- Active space -----

Dietary iron
Intestinal lumen

Heme iron Non-heme

(Animal meat) iron (Fe3+)
Ferric reductase
Heme (Vitamin C,
transporter duodenal Converts to
cytochromes)

Fe2+
Transported to blood
DMT-1 via ferroportin

om
Enters 2+ Hephaestin
Fe Fe
3+

l.c
ai
Enterocyte +

gm
Heme & Fe2+ Apotransferrin
@
converts to 19
55
na

Fe3+ Transferrin
ee

(Transport form)
m

+ Apoferritin
ar

Combines with
um

Stored in intestinal cell as transferrin receptor

itk

Ferritin (Storage form)

am

Target site
|
w
ro
ar
M
©

Iron absorption : Mucosal phase

Storage

Utilisation
by tissues

Iron absorption : Luminal phase

Note :
Hephaestin : Copper containing enxyme.
Iron absorption : Basolateral phase
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Minerals : Part 2 297

REGULATION OF IRON METABOLISM ----- Active space -----

Sites of regulation :
Iron absorption at enterocyte

Consumption by erythroid precursor cells Storage at hepatocyte

Hepcidin :
• Small peptide (25 amino acids).
• Acute-phase reactant.

Synthesized by : Liver cells.

Function : Involved in killing bacteria.
Role in iron metabolism : Downregulates DMT-1 & ferroportin ↓Iron absorption.

om
l.c
ai
Hepcidin levels

gm
@
Increases : 19 Decreases :
55
na

• Iron overload. • Anemia.

ee

• Acute inflammation. • Iron deficiency.

m
ar
um

Transferrin Receptor & Ferritin :

itk
am

Reciprocal relationship.
|
w
ro
ar
M
©

(Storage)
Excess
iron

Iron Excess Low iron levels

Ferritin ↑ ↓
Transferrin receptor levels ↓ ↑

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 298 Minerals and Acid Base Balance

----- Active space ----- Proteins Associated with Iron Metabolism 00:31:30

Transferrin :
Structure :
• Glycoprotein.
• Beta 1 globulin.

Synthesized : In liver.

Role in iron metabolism :

• Transport form of serum iron : Fe3+ + apotransferrin.
• AKA diferric transferrin : 1 transferrin molecule carries 2 Fe3+.

Half life : 7–10 days.

om
l.c
Target sites :

ai
gm
• Reticuloendothelial system.

@
• Erythroid precursors in bone marrow. 55
19
na

Normal transferrin level : 250 mg/dL.

ee
m

TIBC (Total iron binding capacity) : Normally 400 µg/dL.

ar
um
itk

Transferrin saturation :
am

Serum iron
• in %.
|
w

TIBC
ro

• Normal : 30–33% or 1/3rd of transferrin.

ar
M
©

Iron deficiency anemia :

• ↑TIBC.
• ↓Transferrin saturation.

Ferritin :
Role in iron metabolism :
• Storage form of iron : Apoferritin + Fe3+.
• One ferritin contains ≥4000 Fe3+.
Present in :
Ferritin
Liver, bone marrow, spleen, mucosal cells & macrophages.

Significance :
• Index of iron stores.
• Early iron deficiency anemia : ↓Serum ferritin.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 2 299

Normal level : 25–325 ng/mL. ----- Active space -----

Clinical significance :
• Acute phase reactant.
• Ferritin levels :
- Increased : Iron overload (Hemochromatosis).
- Decreased : Iron deficiency anemia.

Iron Excretion & Conservation 00:38:45

IRON EXCRETION
• Iron : One way element.
• Never excreted in urine.

om
Modes of excretion :

l.c
ai
• Through faeces : Unabsorbed iron.

gm
• Denudation of mucosal cells.
@
• Blood loss. 19
55
na

• Desquamation.
ee
m

IRON CONSERVATION
ar
um

RBC lysis
itk
am

Hb Hb-Haptoglobin complex : Iron reutilised

|
w

Taken up by liver Kupffer cells

ro
ar

Globin removed
M
©

Heme Heme-hemopexin complex : Iron reutilised

Taken up by hepatocytes
Porphyrin removed

Iron Iron-transferrin complex Storage as ferritin

Disorders of Iron Metabolism 00:44:52

IRON DEFICIENCY ANEMIA

30% of world population are anaemic.

Effects :
Children : Learning ability impairment (Irreversible).
Adults : Work capacity impairment.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 300 Minerals and Acid Base Balance

----- Active space ----- Etiology :

Nutritional deficiency :
• Low socioeconomic status.
• Excessive junk food intake.

Repeated pregnancy.
Gastric mucosa atrophy : ↓HCl ↓Iron absorption.
Blood loss : Menorrhagia.
Hookworm infestation.

Clinical Manifestations :
• Gastric mucosa atrophy.
• Plummer-Vinson syndrome (Precancerous condition) :

om
Atrophy of oral cavity + esophagus Dysphagia.

l.c
• Improper functioning of iron-requiring enzymes (Electron transport chain).

ai
gm
• Apathy, irritability.

@
• Poor scholastic performance. 19
55
na

Signs :
ee
m

• Pallor : Lower conjunctiva, tongue & palms.

ar
um

• Koilonychia : Spooning of nails.

itk

• Brittle nails.
am

Brittle nails
|
w
ro
ar

Koilonychia : Spooning of nails

M
©

Pallor : Lower conjunctiva Pallor : Tongue

Laboratory Findings :
Peripheral smear : Microcytic hypochromic anemia.
↓ ↑
• Hb
• TIBC
• Serum ferritin
• Soluble transferrin
• Serum iron
receptor levels
• Transferrin saturation

Peripheral smear :
Microcytic hypochromic anemia

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 2 301

Treatment : ----- Active space -----

Oral iron supplementation.
National Nutritional Anemia Prophylaxis program :
• Pregnant : 100 mg Fe + 500 μg folic acid.
• Children : 20 mg Fe + 100 μg folic acid.

IRON OVERLOAD
Hemochromatosis :
Etiology :
• Hereditary : HFE gene mutation.
• Thalassemia : Ineffective erythropoiesis.

om
• Repeated blood transfusions
↑↑Absorption.

l.c
• Parenteral iron therapy

ai
gm
• Dietary : Bantu siderosis

@
- Consumption of alcohol made in iron utensils. 55
19
- Bantu tribe in Africa.
na
ee

Hemosiderosis :
m
ar

Hemosiderin :
um

• Formed : Partial deproteinization by lysosomes.

itk
am

• Aggregates in liver, spleen & bone marrow.

|

• Seen in iron overload.

w
ro

• Features :
ar
M

- More insoluble
©

Compared to ferritin.
- Slow release of iron

Etiology : Repeated blood transfusions.

• Hemophilia.
• Thalassemia.

Clinical Triad :
Cirrhosis

Bronze
diabetes Diabetes mellitus
Skin pigmentation
Management :
• Phlebotomy.
• Desferrioxamine : Iron chelation.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 302 Minerals and Acid Base Balance

----- Active space ----- MINERALS : PART 3

Copper  00:01:00

Body copper : 100 mg.

FUNCTIONS
Cytochrome C oxidase : ETC complex IV.

Lysyl oxidase : Formation of Covalent crosslinks in collagen.

Enzyme
Desmosine crosslinks in elastin.
cofactor

om
Tyrosinase : Synthesis of melanin.

l.c
ai
gm
Superoxide dismutase (Cytoplasmic) : Antioxidant.

@
Constituent of proteins 19
Cerruloplasmin Transport of copper.
55

Hephaestin : Ferroxidase activity : Fe2+ Fe3+.

na
ee
m

RDA & SOURCES

ar
um

RDA : 1.5 - 3 mg/day.

itk
am

Sources :
|

• Meat.
w
ro

• Vegetables.
ar
M

• Nuts.
©

• Cereals.

ABSORPTION
Normal blood level : 75-150 μg/dL.

Transporters :
Site : Intestine Mucosal cell Blood
7A Cu2+
Ctr
Cu2+ Cu2+ 7B Albumin

Copper transporters (Ctr) ATP 7A & 7B : Copper

binding ATPase
Zinc :
• ↓ Cu absorption.
• Rx : Copper toxicity & Wilson’s disease.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 3 303

CLINICAL SIGNIFICANCE ----- Active space -----

Wilson’s Disease :
Defect : ATP 7B gene Code for Cu binding ATPase ATP 7B.

Pathophysiology :
↑ Cu2+ ATP 7B
↑Cu in free form
+
Liver Cu2+ + Apo ceruloplasmin
Deposits in other sites
Ceruloplasmin.

Brain Liver Eye

Neurological : Liver toxicity : Kayser Fleishner ring :

om
• Tremor. Signs of failure + . • Rusty brown ring (Copper deposit)

l.c
• C/f similar to around cornea.

ai
gm
Parkinson’s disease. • Pathognomonic : Seen in 99%.

@
19
55
Diagnosis : Treatment :
na
ee

• ↑S. Cu .2+
• Zinc.
m
ar

• ↓S. Ceruloplasmin. • Penicillamine (Chelation).

um

• ↑Urinary Cu .2+
itk
am

• Liver Cu2+ estimation : Gold standard.

|
w

Menke’s Disease/Menke’s kinky/Steely Hair Syndrome :

ro
ar

Detect :
M

• ATP 7A gene
©

Code for Cu binding ATPase ATP 7A.

• X-linked.
Pathophysiology :
↓ Intestinal Cu2+ absorption Defect in Lysyl oxidase Collagen disorder.
Diagnosis : ↓ S. Cu2+.

EFFECTS OF COPPER DEFICIENCY

Copper Deficiency Anemia :
Pathophysiology :
Cu2+ deficiency
↓ALA synthase activity Hephastin : ↓Ferroxidase activity

↓Heme synthesis. ↓Iron metabolism.

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 304 Minerals and Acid Base Balance

----- Active space ----- Peripheral smear : Microcytic normochromic/hypochromic anemia.

Elastin and Collagen Defect :

↓ Lysyl oxidase activity ↓Covalent crosslinks in collagen.
↓Desmosine crosslinks in elastin.
Hypopigmentation :
• ↓Tyrosinase activity : ↓Melanin Hypopigmentation of skin & hair.
• Flag type of hair :
Cu2+ deficiency : Hypopigmented hair strands
N Cu2+ levels : Normal hair colour

Zinc

om
00:19:56

l.c
ai
RDA : 10 - 15 mg/day.

gm
Sources :
@
• Shellfish. 19
55
na

• Meat.
ee

• Nuts.
m
ar

• Cereals.
um
itk

FUNCTIONS
am

Zn containing enzymes :
|
w

• Carboxy peptidase.
ro
ar

• Carbonic anhydrase.
M
©

• ALA dehydralase.
• ALP.
• Alcohol dehydrogenase.

Insulin : Stabilizes insulin Used in insulin preparations.

Gusten : Zn containing protein involved in taste sensation.

Sperm : Motility.

Antioxidant : Cofactor for superoxide dismutase.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 3 305

CLINICAL SIGNIFICANCE ----- Active space -----

Acrodermatitis Enteropathica :
• Autosomal recessive.
• ↓Zn absorption.

Clinical features :
• Rashes around mouth &
perineal region.
• Diarrhea.
• Alopecia. Rashes around mouth Rashes around perineal region

Leukonychia : Alzheimer’s Disease :

Cu2+ deficiency ↑Neurodegeneration.

om
l.c
Hypogeusia :

ai
gm
↓Gusten : ↓Taste sensation.

@
19
55
Zinc Toxicity :
na

• Occupational hazard : Welder’s

ee
m

White spots on nails exposed to Zn fumes.

ar
um

• Poisoning : Rat poison.

itk
am

Flourine 00:25:12
|
w
ro

Safe limit : < 1ppm. Note : 1 ppm

ar

• 1 part per million.

M
©

• l g fluorine in 1 million g of water.

Function :
Flouroapatite layer : Prevent tooth decay.

Flourosis (Toxicity) :
Flourine levels > 2 ppm.
Teeth mottling
Signs & symptoms :
Fluorine levels Systemic effects
>2 ppm Intestinal discomfort
>5 ppm Teeth mottling
>20ppm Osteoporosis, Brittle bone, genu valgum

Genu valgum
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 306 Minerals and Acid Base Balance

----- Active space ----- Selenium  00:28:18

RDA : 50-100 μg/day.

Selenium Containing Enzymes :

• Glutathione peroxidase :
Antioxidant/free radical scavenging Relation with Vit E :
• Thioredoxine reductase. Selenium : Antioxidant property +
• Deiodinase (T4 T3 conversion).
• Selenoprotein P. ↓Requirement of Vit E.

DEFICIENCY DISORDERS
Disorder Features

om
• Prevalent in China (Soil deficient in selenium)

l.c
Keshan disease
• Endemic cardiomyopathy : Cardiomegaly, arrhythmia

ai
gm
• D/t selenium/iron deficiency
@
Kashinbeck disease 19
• Chronic degenerative disorder of joints
55
na

Hypothyroidism • ↓Action of deiodinase : ↓T4 T3 conversion

ee
m
ar
um

Magnesium 00:32:28
itk
am

RDA : 400 mg/day.

|
w

Normal serum level : 1 - 2.2 mg/dL

ro
ar

(4th abundant cation : M/c in intracellular fluid).

M
©

Enzymes requiring Mg2+ :

• Kinases : ATP complexed with Mg2+.
• Alkaline phosphatase.

CLINICAL SIGNIFICANCE
Hypomagnesemia Hypermagnesemia
• ↑Aldosterone
• ↑Loss through intestine : • ↑Intake of antacid
Cause Malabsorption/diarrhea • Rectal enema
• Nasogastric suction/vomiting • Renal failure : ↓Excretion
(Hospitalized patients)
Clinical • Neuromuscular irritability • Neuromuscular depression
features • Cardiac arrhythmias • Respiratory depression

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Minerals : Part 3 307

Mg and Ca : ----- Active space -----

• Mg : Required for section of PTH.
• ↓Mg ↓PTH ↓Blood calcium.
• Rx of hypocalcemia : Includes correction of hypomagnesemia.

Other Minerals 00:37:20

Mineral Function Deficiency disorders

Cofactor for : • Impaired :
• Kinase - Growth
• Glucosyl transferase - Skeletal development
Manganese • Mitochondrial SOD - Reproduction
• Arginase, carboxylase,enolase - Lipid & carbohydrate

om
• Phosphoglucomutase metabolism

l.c
ai
• Ribonucleotide reductase • Upper body rash

gm
@
Nickel
(Absorption : Lungs)
Cofactor for urease 19 -
55
na
ee

Core component of
Cobalt Macrocytic anemia
m

adenosyl cobalamin
ar
um

Potentiates the action of insulin :

itk

Chromium Impaired glucose tolerance

am

↑ Glucose tolerance
|

Iodine Thyroid hormone synthesis Thyroid enlargement, cretinism

w
ro
ar

Cofactor for :
M

• Severe neurologic
• Xanthine oxidase :
©

abnormalities
Molybdenum Xanthine Uric acid
• Xanthinuria :
• Sulfite oxidase
No conversion to uric acid
• Aldehyde oxidase

SUMMARY
Mineral Feature Deficiency
• Constituent of
Zinc Impaired
• insulin
Chromium insulin action Impaired
Selenosis  breath • (Chronic joint disease)
Selenium
(D/t dimethyl selenide) • (Endemic cardiomyopathy)

Note : Highest concentration of Zn Hippocampus and prostatic secretion.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 308 Minerals and Acid Base Balance

----- Active space ----- ACID BASE BALANCE

Biochemical reactions : Sensitive to pH changes Blood pH maintained at

7.38 to 7.42.

Basics of Acid Base Balance  00:02:00

Base : Proton acceptor.

Bronsted (1923).
Acid : Proton donor.
Strong acid : Ionize almost completely in aqueous solution (Eg : HCl).
Weak acid : Ionize partially in aqueous solution (Eg : Acetic acid).

om
Constants :

l.c
ai
Dissociation/Equilibrium constant (Ka) :

gm
@
Vf
HA Vr H+ + A- 19 Vf : Velocity of forward reaction
55
na

• Vf ∝ [HA] Vf = Kf [HA] Vr : Velocity of reverse reaction

ee

• Vr ∝ [H+][A-] [HA] : Substrate concentration

m

Vr = Kr [H+][A-]
ar

• At equilibrium, Vf = Vr K : Rate constant

um
itk
am

Kf [HA] = Kr [H+][A-]
|
w
ro

Kf = [H+][A-] = Ka
ar
M

Kr [HA]
©

lonisation constant (pKa) :

pKa = -Log Ka.

Henderson-Hasselbalch Equation :
Relation b/w pH, pKa of weak acid & concentrations of acid & base of a buffer
solution is given by :
pH = pKa + log [Salt] .
[Acid]
Applications :
• Calculation of one parameter when the other two are known.
• Assess acid-base status of a patient.
• Assess limits of compensation in a patient with acid-base imbalance.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Acid Base Balance 309

pH (Puissance/Potenz/Power of Hydrogen) : ----- Active space -----

Introduced by SPL Sorensen (Danish chemist) in 1909.
pH = -log [H+].

pH scale : Neutral
0 7 14
Acidic Alkaline
pH measurement :
1. pH paper/disc : Color of paper dipped in solution measured against the color
gradient.

om
l.c
ai
gm
@
19
55
na

pH paper pH disc
ee

2. Indicators : Solutions that change color in response to pH.

m
ar
um
itk
am
|
w
ro
ar
M
©

Chlorophenol red Thymol blue

3. pH meter (Latest) : Digital reading of pH using potential difference b/w 2

electrodes d/t H+ ions.

pH meter

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 310 Minerals and Acid Base Balance

----- Active space ----- Buffer Solutions :

Definition : Solutions that resist changes in pH when an acid/alkali is added.
Types :
1. Solution of weak acid & its salt with a strong base.
2. Solution of weak base & its salt with a strong acid.

Examples :
• Bicarbonate buffer : H2CO3 (Carbonic acid) + NaHCO3.
• Acetate buffer : CH3COOH (Acetic acid) + CH3COONa.
• Phosphate buffer : NaH2PO4 (Acidic phosphate) + Na2HPO4 (Basic phosphate).

Buffering range :
B

om
A : [Unionized form] > [Ionised form]

l.c
B : [Ionised form] > [Unionised form]

ai
gm
[OH-] added

Buffering pKa (lonisation constant) : pH at which [Unionized form] = [Ionised form]

@
range pKa (Partially ionised solution)
19
55
na
ee
m
ar

A
um

pH
itk

Titration curve of CH3COOH (Weak

am

acid with single ionisable group)

|
w
ro

• Buffering capacity : The capacity of a solution to maintain pH even when an

ar
M

acid/alkali is added.
©

• Buffering range :
- Range of pH at which there is maximum buffering capacity.
- Formula : pKa ± 1.

Regulation of body pH :
Body buffers Respiratory regulation Renal regulation
1st line 2nd line 3rd line

Body Buffers 00:22:33

Action : Quick, but temporary.

Acts on : ↑Acid load d/t various metabolic process.

Prerequisites :
• Acid load not too high. • Alkali reserve is maintained.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Acid Base Balance 311

Examples : ----- Active space -----

Extracellular fluid (ECF) Intracellular fluid (ICF) Erythrocyte

1St buffer Bicarbonate buffer Phosphate buffer Hemoglobin buffer
2nd buffer Phosphate buffer Protein buffer Bicarbonate buffer
3rd buffer Protein buffer Bicarbonate buffer Phosphate buffer

Bicarbonate Buffer (NaHCO3/H2CO3) :

Most important body buffer.

Application of Henderson Hasselbatch equation :

pH = pKa + log [Salt] . pKa of H2CO3 = 6.1
[Acid]
[Salt] = [HCO-3] = 24 mmol/L
pH = 6.1+ log [24]

om
[1.2] [Acid] = [H2CO3] = 1.2 mmol/L

l.c
pH = 6.1+1.3 Log 20 = 1.3

ai
gm
pH = 7.4

@
19
55
• pH of blood is maintained when [HCO-3] = 20 [H2CO3].
na

• [HCO-3] : Maintained by renal system .

ee
m

(AKA Metabolic component) Determines alkali reserve.

ar
um

• [H2CO3] : Regulated by respiration (AKA Respiratory component).

itk
am

Action during acid-base imbalance :

|

1. Acidosis (↑H+) :
w
ro

Carbonic
ar

H2CO3 Anhydrase (CA)

M

H+ + HCO-3 H2O + CO2 Expelled through lungs via

©

hyperventilation

Any excess H+ handled by bicarbonate buffer CA washout CO2 washout

2. Alkalosis (↓H+) :
H2CO3 + H+ + HCO3 Excreted through kidneys (Renal regulation).

Maintains acid levels.

Alkali reserve : Ability of bicarbonate to meet acid load.

Determined by HCO-3 H2CO3 : HCO-3 ratio = 1:20 Maintains Normal physiological
conditions.

Phosphate Buffer (Na2HPO4/NaH2PO4) :

Effective buffer (pKa = 6.8 is close to physiological pH).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 312 Minerals and Acid Base Balance

----- Active space ----- Application of Henderson-Hasselbach equation :

[Na2HPO4]
• pH = pKa + log
[NaH2PO4]
[Na2HPO4] [Na2HPO4]
• 7.4 = 6.8 + log Needs to be 0.6 = 4.
[NaH2PO4] [NaH2PO4]
• Hence, pH of blood is maintained when [Na2HPO4] = 4 [NaH2Po4].

Protein Buffer :
Protein Main site of action
Albumin ECF
Hemoglobin (Hb) Erythrocyte

om
Buffering ability : D/t ionisable side chain (Imidazole of histidine pKa = 6.1).

l.c
ai
Hb buffer :

gm
• 2/3rd of total protein buffering capacity.

@
19
• Needs buffering action to transport CO2 as HCO-3 (Isohydric transport of CO2).
55
na

Relative Significance of Intracellular & Entracellular Buffers :

ee
m
ar

Tissue cells RBC ECE

um

Hb H2CO3 PO4 Proteins

itk

Buffers PO4 buffer > Proteins buffer > H2C03 buffer

am

buffer buffer buffer buffer

|

52% 6% 40% 1% 1%
w

Significance
ro

Intracellular : 58% Extracellular : 42%

ar
M
©

Respiratory Regulation of pH 00:46:23

Acts by adjusting concentration of carbonic acid :

[H2CO3] ∝ pCO2 ∝ Respiratory rate (RR) Change in RR Leads to Change in [H2CO3].

Mechanism :
1. Acidosis (↓pH ; ↑[H+]) :
↓pH Respiratory centre Hyperventilation CO2 (from H2CO3) ↑pH
stimulated d/t pH (↑RR) expelled
sensitive chemoreceptors
2. Alkalosis (↑pH ; ↓[H+]) :
↑pH Hypoventilation (↓RR) CO2 retention ↑ H2CO3

H+ + HCO-3
↑pH Excreted via kidneys
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Acid Base Balance 313

Summary : ----- Active space -----

Acidosis Alkalosis
Effect on H+ ↓ ↑
CO2 Expelled through lungs Retained
RR ↑ ↓

Renal Regulation of pH 00:54:00

Action : Slow, but complete. Acts on : Mainly ECF pH.

Significance :
1. Excretion of acid.
2. Maintains alkali reserve by effective reabsorption of HCO-3.

om
l.c
Mechanism :

ai
gm
1. Excretion of H+
Bicarbonate buffer.
@
2. Reabsorption of filtered bicarbonate 55
19
3. Excretion of titratable acid Phosphate buffer (Urinary buffer).
na
ee

4. Secretion of NH3 Ammonia mechanism.

m
ar

Excretion of H+ :
um
itk

Trigger :
am

1. Metabolic acidosis (A/w ↓ HCO-3). 2. Respiratory acidosis (↑pCO2).

|
w
ro

Significance : Excretion of H+ HCO-3 reabsorption Alkali reserve maintained.

ar
M

Site : Proximal Convoluted Tubule (PCT).

©

Steps :
Plasma PCT cell Tubular lumen
Na+ Na+ reabsorbed Na+
3. 2. Na H exchanger
+ +
HCO-3 reabsorbed
HCO-3 H+ H+ Excreted
Alkali reserve
maintained. H2CO3
Carbonic
Anhydrase (CA)
1. C02 + H20

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 314 Minerals and Acid Base Balance

----- Active space ----- Reabsorption of Filtered HCO-3 :

Significance : No net H+ excretion.
Site : PCT.

Steps :
Plasma PCT cell Tubular lumen
NaHCO3 (Filtered HCO-3)
Na+ Na+ Na+ HCO-3
HCO-3 reabsorbed HCO-3 H+ H+
H2CO3
Alkali reserve H2CO3
CA
maintained.

om
CA C02 + H20
1. C02 + H20

l.c
ai
gm
@
Excretion of Titratable Acid :
Basic phosphate Converted to Acid phosphate. 19
55
na
ee

Significance :
m
ar

H+ excretion + HCO-3 reabsorption Acidification of urine Requires PO4 buffer.

um
itk

Site : Distal Convoluted Tubule (DCT) & Collecting Duct (CD)

am
|

Steps:
w
ro

Plasma PCT cell Tubular lumen

ar
M

Na2HPO4 (Basic phosphate, pH = 7.4)

©

Na+ Na+ Na+ NaHPO-4

HCO-3 reabsorbed HCO-3 H+ H+
NaH2PO4 (Acidic phosphate, pH = 5.4)
Alkali reserve H2CO3
maintained CA Maintains urinary pH at 5.4-7.4
C02 + H20
(AKA Urinary buffer)

Titrable acid :
• Amount of strong alkali (Eg : N/10 NaOH) required to titrate 1 litre of urine to pH
7.4.
• Inhibited by : Acetazolamide (Carbonic anhydrase inhibitor).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Acid Base Balance 315

Excretion of Ammonium Ions : ----- Active space -----

Site : Throughout renal tubules, most prominent in PCT during acidosis.

Steps :
Plasma PCT cell Tubular lumen
Glutamine Glutaminase Glutamate
NH3 NH3
Na+ Na+ Na +

HCO-3 reabsorbed H+
HCO-3 H+
NH+4
Alkali reserve
H2CO3
maintained. Excreted along with H+

om
CA

l.c
C02 + H20

ai
gm
@
Miscellaneous
19 01:13:00
55
na

1. Acidosis a/w hyperkalemia :

ee

H+ excretion Accompanied by K+ reabsorption

m

Acidosis a/w hyperkalemia (↑K+).

ar
um

2. pH & Ca2+
itk

• In alkalosis
am

↑Protein bound Ca+2 ↓Ionized (Free/active) Ca2+

|
w

Hypocalcemia.
ro
ar

• Acidosis Release of protein bound Ca2+ ↑Free Ca+2 Hypercalcemia.

M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 316 Minerals and Acid Base Balance

----- Active space ----- ACID BASE DISORDERS

NORMAL PARAMETERS

Blood pH : Respiratory parameters : Metabolic (Renal) parameters :

7.38-7.42. • pCO2 : 35-45 mmHg. [HCO3-] : 22-26 meq/L (mmol/L).
• [H2CO3] : 1.2 mmol/L.

Euphemia :
• State in which normal pH is maintained.

om
• Maintained by metabolic and respiratory components.

l.c
ai
gm
Henderson Hasselbalch equation :

@
19
55
[HCO3-]
pH = pKa + log
na

[H2CO3] Respiratory Metabolic

ee
m

[HCO3 ]
-
ar

= 20 pH = 7.4
um

[H2CO3]
itk

7.6 7.4 7.2

am

pH
|
w
ro

Classification and Compensation 00:05:30

ar
M
©

CLASSIFICATION
Acid base disorders

Acidosis : Alkalosis :
pH <7.38 pH >7.42

Metabolic Respiratory Metabolic Respiratory

acidosis acidosis alkalosis alkalosis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Acid Base Disorders 317

Algorithm to Identify Primary Acid Base Disturbance : ----- Active space -----

1. pH of the blood : 2. Respiratory parameter : 3.  Metabolic parameter :

pH : 7.38-7.42 pCO2 : 35-45 mmHg S.HCO3- : 22-28 meq/L

↓ ↑ ↑ ↓ ↓ ↑
<7.38 : >7.42 : >45 : <35 : <22 : >28 :
Acidosis. Alkalosis. Respiratory Respiratory Metabolic Metabolic
acidosis. alkalosis. acidosis. alkalosis.

COMPENSATORY MECHANISM
Done to normalize HCO3-/H2CO3 ratio.

om
Respiratory compensation Metabolic compensation

l.c
ai
Cause Metabolic/renal disorder Respiratory disorder

gm
@
Response Immediate + partial Slow + complete
19
55
Result Partial correction Almost complete
na
ee
m

Note :
ar
um

Permanent compensation : Only by correction of cause.

itk
am

Metabolic Acidosis 00:12:40

|
w
ro

Due to primary deficiency of bicarbonate.

ar
M
©

CLASSIFICATION
Based on anion gap

High anion gap Normal anion gap

metabolic acidosis. metabolic acidosis. 7.6 7.4 7.2
pH
M. acidosis
Anion Gap :
• Normally in ECF : Sum of cations = Sum of anions (Maintain electrical neutrality)

Measurable : Measurable :
Na and K
+ +
Cl- and HCO3-
(95% of cations). (86% of anions only).
• Unmeasured anions constitute the anion gap.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 318 Minerals and Acid Base Balance

----- Active space ----- Calculation : Difference b/w measured cations and anions.
A.G = [Na+ + K+] - [HCO3- + Cl-]
K+ Anion gap Misc Organic acids
Normal value : 12 ± 2 mmol (10-14 mmol/L).
Contribution
Unmeasurable anions : HCO3- from the
anionic charge
• Organic acids : Major constituent. Na+ of albumin
- Ketone bodies.
Cl-
- Lactic acid.
- Propionic acid.
• Albumin. Cations Anions
Calculation example :

om
(Na+ + K+) - (HCO3- + Cl-)

l.c
• Na+ : 136

ai
• K+ : 4

gm
(136 + 4) - (24 + 104)

@
• HCO3- : 24
19
Anion gap : 12 mmol/L
55
• Cl- : 104
na
ee
m

Causes of High & Normal Anion Gap Acidosis :

ar

High anion gap metabolic acidosis Normal anion gap metabolic acidosis
um

• ↑Organic acid production : • Diarrhea :

itk
am

- Diabetic ketoacidosis - Loss of HCO3- Maintains

|

- Starvation ketosis - Loss of Na and K

+ + the gap
w
ro

- Lactic acidosis • Hyperchloremic acidosis

ar
M

• Renal failure : - Renal tubular acidosis (RTA)

©

↓H+ excretion OR ↓HCO3- reabsorption - Carbonic anhydrase inhibitor

• Organic aciduria therapy (Acetazolamide)
• Methanol poisoning

↑Anion gap d/t ↓HCO3- Normal anion gap d/t ↑Cl- reabsorption

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Acid Base Disorders 319

RTA : ----- Active space -----

Failure of tubules to :
• Excrete H+ (↑H+)
• Reabsorb HCO3- (↓HCO3-)

Cl- is reabsorbed (↑Cl-)

Hyperchloremic acidosis.

COMPENSATION

Respiratory Compensation :
[HCO3-] (↓)
Acidosis (↓HCO3-) log

om
[H2CO3]

l.c
ai
Stimulation of respiratory centre

gm
@
• ↑Respiratory rate : Hyperventilation 19 Kussmaul’s
55
na

• ↑Depth of respiration. respiration

ee
m
ar

CO2 washout
um
itk

[HCO3-] (↓)
am

↓H2CO3 log Balanced

|

[H2CO3] (↓)
w
ro
ar
M

Renal Compensation :
©

Ammonia mechanism :
• ↑H+ excretion.
NH3 + H+ NH4
• ↑HCO3- generation
Phosphate buffer mechanism :
NaHPO4 + H+ NaH2PO4

LAB FINDINGS
• pH :↓
• S. HCO3- : ↓
• pCO2 : N Compensation
↓
• S. H2CO3 : N

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 320 Minerals and Acid Base Balance

----- Active space ----- CLINICAL FEATURES

• H/o uncontrolled DM/starvation/diarrhea/renal failure.
• Kussmaul’s breathing : Hyperventilation.
• Neuromuscular weakness :
Hypercalcemia (↑ Ionised Ca2+ : ↓Neuromuscular junction depolarization).
• Cardiac arrhythmias & cardiac arrest risk : D/t hyperkalemia.

Hypercalcemia in acidosis :
• Albumin has binding sites for H+ and Ca2+.
• H+ ↑ Ca binding ↓ Free Ca2+ ↑

Hyperkalemia in acidosis :
H+ enters cell K+ exits outside.

om
l.c
TREATMENT

ai
gm
• Symptomatic Rx.

@
• IV bicarbonate. 55
19
• Correct electrolyte imbalance.
na

• Treatment of underlying cause.

ee
m
ar
um

Respiratory Acidosis 00:41:16

itk
am

Primary excess of carbonic acid d/t CO2 retention as a result of hypoventilation.

|
w
ro

Causes :
ar

• Impaired functioning of respiratory centre.

M
©

• Lung diseases :
- Bronchopneumonia.
- COPD (Chronic obstructive pulmonary disease).
- Bronchial asthma.
• Chest injury. 7.6 7.4 7.2
• Drug affecting respiratory centre : Narcotic pH
drugs (Morphine). R. acidosis
• Respiratory muscle paralysis :
- Polio.
- GBS (Guillain-Barre syndrome).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Acid Base Disorders 321

COMPENSATION ----- Active space -----

Respiratory compensation not possible.
Renal Compensation :
[HCO3-]
Acidosis (↑H2CO3) log
[H2CO3] (↑)
• ↑Excretion of H+
• ↑Reabsorption of HCO3-

[HCO3-] (↑)
↓H+, ↑HCO3- log Balanced
[H2CO3] (↑)
CLINICAL FEATURES
• H/o COPD/bronchial asthma/bronchopneumonia/morphine overdose/chest injury.

om
• ↓Respiratory rate.

l.c
ai
• Hypotension, coma.

gm
• Hypercapnia (Retention of CO2) : Peripheral vasodilatation, tachycardia,
@
tremors. 19
55
na
ee

LAB FINDINGS
m

• pH :↓
ar
um

• S. HCO3 : N Compensation ↑
-
itk

• pCO2 :↑
am

• S. H2CO3 : ↑
|
w
ro
ar

TREATMENT
M

• Treat underlying cause.

©

• Provide mechanical ventilation.

Metabolic Alkalosis 00:48:50

Primary excess of HCO3- : D/t loss of acid or gain of base.

Types :

Chloride responsive : Chloride resistant :

D/t loss of acidic contents : • Excess aldosterone :
• Vomiting. Conn’s syndrome.
• Pyloric stenosis. • Excess glucocorticoids :
• Nasogastric suction. Cushing’s syndrome. 7.6 7.4 7.2
• Drugs : pH
Loop diuretics (Furosemide). M. Alkalosis
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 322 Minerals and Acid Base Balance

----- Active space ----- Mechanism :

Chloride responsive : Chloride resistant :

Loss of gastric contents : ↑Aldosterone or ↑glucocorticoids
• ↑Fluid loss (Primary)
• ↑Na+ & Cl- loss

Contraction of ECF

↑Secretion of renin & aldosterone

(Secondary)

om
↑Na+ reabsorption

l.c
ai
+

gm
@
↑ H excretion, K+ excretion
+

19
55
na

↑HCO3- reabsorption.
ee
m
ar

COMPENSATION
um
itk

Respiratory Compensation :
am

[HCO3-] (↑)
Alkalosis (↑HCO3-) log
|

[H2CO3]
w
ro
ar

Hypoventilation : ↓ Respiratory rate (RR)

M
©

CO2 retention
[HCO3-] (↑)
↑H2CO3 log Balanced
[H2CO3] (↑)

Renal Compensation :
↑Excretion of excess HCO3-.

CLINICAL FEATURES
• H/o vomiting/pyloric stenosis/Conns syndrome/Cushings syndrome.
• Hypoventilation.
• Hypocalcemia :
- ↓H+ ↑Binding sites available for Ca2+ in albumin.
- Hyperexcitability (Tetany : Carpopedal spasm).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Acid Base Disorders 323

LAB FINDINGS ----- Active space -----

• pH :↑
• S. HCO3 : ↑
-

• pCO2 : N Compensation
↑
• S. H2CO3 : N

TREATMENT
• Correct electrolyte imbalance.
- IV NaCl (Chloride responsive).
- IV KCl : To correct hypokalemia.
• Treat underlying cause.

Respiratory Alkalosis

om
01:01:30

l.c
Primary deficit of carbonic acid d/t ↓pCO2 as a result of hyperventilation.

ai
gm
@
Causes :
19
55
• Psychogenic : M/c cause.
↑RR, CO2 washout
na

• Anxiety.
ee
m

• Hypoxia : High altitude.

ar
um

• Salicylate poisoning.
itk
am

COMPENSATION
7.6 7.4 7.2
|

Respiratory compensation not possible.

w

pH
ro

R. alkalosis
ar

Renal Compensation :
M

[HCO3-]
©

Acidosis (↓H2CO3) log

[H2CO3] (↓)
• ↓Reabsorption of HCO3-
• ↓Excretion of H+

[HCO3-] (↓)
↑H , ↓HCO
+ -
log Balanced
3
[H2CO3] (↓)

CLINICAL FEATURES
• ↑Respiratory rate : Hyperventilation.
• Hypocalcemia : Tetany.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 324 Minerals and Acid Base Balance

----- Active space ----- LAB FINDINGS

• pH :↑
• S. HCO3 : N Compensation ↓
-

• pCO2 :↓
• S. H2CO3 : ↓

TREATMENT
• Symptomatic treatment.
• Correct electrolyte imbalance.
• Treat underlying cause.

Summary 01:08:04

om
l.c
NORMAL VALUES

ai
gm
Parameter Value
@
pH 19
55
na

Bicarbonate
ee
m

Chloride 104 (96-106) mmol/L

ar
um

Potassium 3.5-5 mmol/L

itk

Sodium 136-145 mmol/L

am

pO2 95 (85-100) mmHg

|
w
ro

pCO2 40 mmHg
ar
M

Anion gap
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Acid Base Disorders 325

----- Active space -----

Causes : Causes :
HAGMA NAGMA •
• • •
• • Hyperchloremic • Drug
• Organic aciduria acidosis • Respiratory muscle paralysis
• Methanol poisoning

Compensation : Compensation :
Respiratory : Respiratory Renal :
acidosis • reabsorption
•

om
(Kussmaul’s breathing) excretion

l.c
HCO3-↓ pCO2↑

ai
gm
C/f : C/f :

@
• 19 ↓ •
55

• Hypercalcemia • Hypercapnia
na
ee

pH
m
ar
um

Diagnosis Diagnosis
itk

& Rx ↑ & Rx
am
|
w

HCO3-↑ pCO2↓
ro

C/f : C/f :
ar

• Hypoventilation • Hyperventilation
M
©

• Metabolic •
alkalosis

Compensation : Compensation :
• HCO3- reabsorption
• H+ excretion

Cause : Cause :
• Chloride resistant : Hyperventilation :
•
• Chloride responsive : •
• Hypoxia : High altitude
• Salicylate poisoning

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 326 Molecular Biology

----- Active space ----- CHEMISTRY OF NUCLEOTIDES

Building Blocks of Nucleic Acids 00:00:26

Nucleosides :
Nucleotides : Bond : β N-glycosidic bond.
Monomers of nucleic acids (DNA and RNA). Structure : Nitrogenous base +
Bond : 3’-5’ phosphodiester bond. Pentose sugar.
Structure : Nitrogenous base Purines
Pyrimidines
+
Ribose
Pentose sugar
Deoxyribose

om
+

l.c
Phosphate group

ai
gm
@
Nitrogenous base : 55
19
na

Purines :
ee
m

Adenine and Guanine (Mnemonic : Guardian Angels are Pure)

ar
um

NH2 O
N N N
itk

6 7 6 7
am

N1 6 7 N1 N1
5 5 5
|

8 8 8
w

2 4 2 4 2 4
ro

9 3 9 NH2 3 9
ar

3
N N N N N
M

N
Purine (Two heterocyclic rings) Adenine (6-aminopurine) : Guanine (2-Amino-6-oxo purine)
©

Nitrogenous base with the

least oxygen
Pyrimidines :
Cytosine, Uracil and Thymine (Mnemonic: CUT)
NH2 O O
N3 4 5 N3 4 5 N 4 CH3
Deamination Methylation 3 5
2 6 2 6 2 6
O 1 O 1 O 1
N N N
Cytosine : Uracil : Thymine :
2-oxo,4-amino pyrimidine • 2,4-dioxo pyrimidine 2,4-dioxo,5 methyl pyrimidine
• Only in RNA, not DNA
Vit B1
Note : Thiamine
Pyrimidine ring +

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Chemistry of Nucleotides 327

Formation of Nucleic Acids 00:09:17 ----- Active space -----

Formation of nucleosides : Formation of nucleotides :

NH2 NH2 N
N
N1 6 7 N1 6 7
5 5
8 Purine 8 Purine
2 4 2 4
3 9 3 9
N N N o N
o Ester 5’ β N-glycosidic bond
β N-glycosidic bond PO4 PO4 PO4 HC
HOH2C5’ bond 2 4’ C1’
4’
C 1
’
Pentose sugar
Acid anhydride bond 3’ 2’
3’ 2’ Pentose sugar
• N9 of purine + C1 of pentose sugar.

om
• N1 of pyrimidine + C1 of pentose sugar.

l.c
ai
gm
Acid anhydride bond :

@
Energy rich Releases energy when broken 19
55
na
ee

E.g.: Adenine + Ribose Adenosine AMP ADP ATP

m

PO4 PO4 PO4

ar
um

Formation of nucleic acids :

itk

NH2
am

N
N1
|

N
w
ro
ar
M
©

N N NH2
o N
N2
P-O-CH2 5
’
N
(5’ end of N1)
3’ N N NH2 N
OH o N3
P-O-CH2 5’ N

3’ N N
OH o
P-O-CH2 5’
3’ → 5’ phosphodiester
bond formed by DNA 3’
polymerase (3’ end of N3) OH

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 328 Molecular Biology

----- Active space -----

Nucleic acid sequencing : From 5’ position to 3’ position.

Polarity in nucleic acids :

Polarity is due to the ionisable groups (P, OH) at either ends

Base Base Base

Ionisable P Sugar P Sugar P Sugar OH Ionisable

Free phosphate group 3’→5’ phosphodiester Free hydroxyl group

om
at 5’ end bond at 3’ end

l.c
ai
Base sequence : 5’

gm
3’

@
Examples : 19
55
na

Deoxyribose
ee

Nitrogenous base Nucleoside Ribose monophosphate

m

monophosphate
ar
um

Adenosine monophosphate
Adenine Adenosine d. AMP
itk

(AMP)
am

Guanine Guanosine GMP d. GMP

|
w
ro

Uracil Uridine UMP —

ar
M

Hypoxanthine Inosine IMP d. IMP

©

Xanthine Xanthosine XMP —

Cytosine Cytidine CMP d. CMP

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Metabolism Of Nucleotides 329

METABOLISM OF NUCLEOTIDES ----- Active space -----

Purine Metabolism  00:00:30

DE NOVO SYNTHESIS • 2C, 1 N atom.

Site : • Entirely used.
All organs except :
• Brain
• RBC’s Depend solely on
• Leucocytes (WBCs) salvage pathway

om
l.c
ai
Steps of synthesis : Purine ring

gm
@
Phosphoribosyl
Ribose 5 phosphate PRPP synthetase 19
55
pyrophosphate (PRPP)
na

ATP AMP
ee

Glutamine H2O
m
ar

Rate limiting enzyme : PRPP glutamyl amidotransferase

um

Glutamate PP1 (2 phosphate groups)

itk
am

Phosphoribosyl amine
|
w

Regulatory PRPP glutamyl amidotransferase

ro

steps PRPP synthetase

ar
M

Inosine monophosphate (IMP)

©

• First purine nucleotide formed.

• Purine base : Hypoxanthine.
Amino group
from aspartate
IMP dehydrogenase

Adenosine monophosphate (AMP) Xanthosine monophosphate (XMP)

Amino group
from glutamine

Guanosine monophosphate (GMP)

Addition of atoms to Ribose 5 P Product : Purine nucleotide (Purine + Sugar +PO4)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 330 Molecular Biology

----- Active space ----- Fate of PRPP

• Purine synthesis.
• Pyrimidine synthesis.
• NAD+ synthesis (Involves QPRTase)

SALVAGE PATHWAY
• Purine nucleosides. Recycled to form
Purine nucleotides
• Purine bases.
+ PO4 Donates
Phosphoribosylation + Ribose PRPP

Recycled

Cell lysis Release DNA On lysis Purine Purine Purine

om
releases
base

l.c
nucleotide nucleoside

ai
gm
Recycled

@
Uric acid
19Phosphorylation (PO4)
55
na

Phosphoribosylation of purines :
ee
m

1. Adenine APRTase AMP

ar

APRTase :
um

PRPP PPi (Adenine Phosphoribosyl transferase)

itk
am

2. Hypoxanthine HGPRTase IMP HGPRTase :

|
w

PRPP PPi (Hypoxanthine Guanine Phosphoribosyl transferase)

ro
ar

3. Guanine GMP
M

HGPRTase
©

4. Adenosine Adenosine Kinase AMP

ATP ADP
5. Guanosine GMP
Guanosine Kinase

Use :
• Effective recycling of nucleotides.
• Conservation of energy.
• ln organs without de novo purine synthesis.
PURINE CATABOLISM
Site :
• Liver : Blood purines catabolised.
• Intestine : Dietary purines catabolised.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Metabolism Of Nucleotides 331

Organelle : Cytoplasm. ----- Active space -----

End product : Uric acid.
Steps of Catabolism
Adenosine (Purine nucleoside)
Adenosine Guanosine
deminase (1) (Purine nucleoside)
Inosine Ribose 1 PO4
Purine nucleoside
ribose transferase (2)
Pi Pi
Hypoxanthine Guanine
Xanthine
Guanine
oxidase (XO)(3)
Allopurinol deminase

om
Xanthine NH3

l.c
Xanthine

ai
gm
oxidase (3)

@
Uric acid 55
19
Defects :
na
ee

1. Adenosine deaminase defect :

m
ar
um

Severe Combined Immuno Deficiency

itk

• Both B cells and T cells affected.

am

2. Purine nucleoside ribose transferase defect

|
w
ro
ar
M

Immunodeficiency
©

• Only T cells affected.

• B cells normal.
3. Xanthine Oxidase defect

• ↑ accumulation of xanthine Xanthinuria and hypouricemia

• Hyperuricemia : Rx : Allopurinol (Inhibits Xanthine oxidase).
Lesch Nyhan Syndrome
Biochemical defect : Defect in salvage pathway
• Normal : Hypoxanthine/Guanine HGPRTase Inosine monophosphate /Guanosine monophosphate
(purines) PRPP PPi
• LN syndrome : HGPRTase defect ↑purines catabolyses Uric acid

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 332 Molecular Biology

----- Active space ----- Features :

• Hyperuricemia.
• Neurological manifestations (brain dependent on salvage pathway).
• Compulsive self mutilation.
Rx :
• Allopurinol (Prevents
hyperuricemia by inhibiting XO).
• High fluids : Prevents uric acid
stones.
• Alkalinisation of urine : ↑ excretion
of uric acid. Self mutilating behaviour
• Symptomatic Mx.

om
l.c
ai
gm
Kelley Seegmiller Syndrome :
@
Biochemical defect : Partial HGRTase deficiency. 19
55
na
ee

Gout
m

00:25:10
ar
um

Hyperuricemia
itk
am

Group of disorders presenting with Uric acid nephrolithiasis

|

Acute inflammatory arthritis

w
ro

ETIOLOGY
ar
M

Primary Secondary
©

• Enzyme defects ↑ Production of uric

↓Excretion of uric
- ↑PRPP synthetase activity. acid (D/t ↑ cell turn
acid
- ↑PRPP glutamyl amidotransferase over)
activity

↑purine catabolism ↑uric acid

• Other causes :
Cause • Renal failure.
- Lesch Nyhan syndrome.
• Malignancy. • Lactic acidosis.
- Von Gierke’s disease (glycogen storage
• Anticancer drugs. • Diuretics (Eg
disorder) :
Thiazides).
↑ Glucose -6-P04
via HMP shunt

↑ purine catabolism ↑ uric acid

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Metabolism Of Nucleotides 333

----- Active space -----

Aggravating factors :
• Alcohol intake :
Alcohol Alcohol dehydrogenase Aldehyde
NAD + NADH
Carbohydrate

Glucose
With alcohol
↑ alcohol ↑ NADH accumulation (-) Pyruvate Lactate Acidify urine
intake ( ↑ NADH : NAD+ ratio) NAD +
Pyruvate dehydrogenase

om
NADH ↑ uric acid

l.c
crstallisation

ai
Acetyl CoA

gm
@
19 Gout
55
na

• ↑ dietary intake of purine (eg : Meat)

ee
m

CLINICAL FEATURES
ar
um

1. Acute gout :
itk

• Acute inflammatory arthritis : Pain

am

Proximal joints affected : 1st Metatarsophalangeal joint Swelling

|
w
ro

(First to be affected) Redness

ar

• Uric acid nephrolithiasis

M
©

Abdominal pain

Uric acid stones

2. Chronic gout :
• Tophi : Monosodium urate crystals accumulated

Soft tissue swellings

Tophi

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 334 Molecular Biology

----- Active space ----- MANAGEMENT

Definitive diagnosis : Synovial joint aspirate Polarised light microscopy :
• Monosodium urate
crystals.
• Needle shaped.
Treatment : • Negatively birefringent.
• ↑ fluid intake.
• Alkalinise urine.
• Allopurinol.
• Anti inflammatory drugs : eg. Colchicine.
• Uricosuric drugs : eg. Probenecid.
• Avoid precipitating factors : Alcohol, high fructose diet, meat.

om
l.c
Pyrimidine Metabolism 

ai
00:35:45

gm
@
BIOSYNTHESIS
Site : Liver. 19
55
na

Organelle : Cytoplasm &

ee
m

mitochondria.
ar
um
itk

C From aspartic
From
am

4
N3 C acid
|

glutamine 5
w
ro

Respiratory CO2 C2 6C
ar

1
M

N
©

Sources of Pyrimidine Ring

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Metabolism Of Nucleotides 335

Steps : ----- Active space -----

CO2 + glutamine Note : CPS 1 In urea cycle

Carbamoyl phosphate synthetase 11
Carbamoyl phosphate
In Aspartic acid Aspartate transcarbamoylase Forms multifunctional enzyme
cytoplasm (parts of a single polypeptide)
Carbamoyl aspartic acid
Dihydro orotase

Dihydro orotic acid

NAD+
In Dihydro orotate dehydrogenase
NADH

om
mitochondria

l.c
Orotic acid

ai
Ribose PRPP

gm
Phopshate Orotate phosphoribosyl transferase

@
PPi 19
55
na

OMP
ee
m

OMP decarboxyase
ar

CO2
um

UMP
itk
am

Ribonucleotide reductase
dUDP
|

UDP
w
ro
ar

dUMP
M

UTP
©

Thymidylate N5N10 methyleneTFHA

CTP synthetase
synthase (methyl donor)
CTP dTMP
Applied aspects :
5 Fluorouracil : Inhibits duMP dTMP
Anticaner drugs
Methotrexate inhibits DHFR (dihydrofolate reductase)

Prevents regeneration of N5 N10 methylene THFA

Note :
• Purine Nucleotide synthesised first.
• Pyrimidine Ring synthesised first Ribose + PO4 added

Nucleotides formed.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 336 Molecular Biology

----- Active space -----

PYRIMIDINE CATABOLISM
End products :
• Cytosine Forms Water soluble Excreted in urine
β alanine
• Uracil
• Does not accumulate.
• Thiamine Forms β amino iso • No symptoms in case of excess
butyrate pyrimidine synthesis.
• ↑ pyrimidine synthesis ↑ accumulation of orotic Orotic aciduria
acid (Intermediate)
Hereditary Orotic Aciduria :
Defect in Denovo synthetic pathway of pyrimidines ↓ pyrimidine synthesis

om
↓ DNA synthesis

l.c
ai
Types

gm
@
Type 1 19 Type 11
55
na

• Defect in UMP synthase : Defect in OMP decarboxylase

ee

Bifunctional enzyme
m
ar
um
itk

Orotate phosphoribosyl Orotidine monophosphate

am

transferase decarboxylase
|
w
ro
ar
M

Features :
©

• ↓ DNA synthesis Macrocytic hypochromic anemia : Does not respond to

B12 /iron/ folate therapy.
• Bone marrow Megaloblasts.
• Failure to thrive.
Hyperammonemia Type 11 :
(Urea cycle disorder)
Defect in ornithine transcarbamylase (OTC)

Carbamoyl phosphate enters cytoplasm

↑ pyrimidine synthesis

↑ orotic acid

Orotic aciduria
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Structure & Organisation of DNA 337

STRUCTURE AND ORGANISATION OF DNA ----- Active space -----

Structure of DNA 00:03:10

Salient features :
• Two polydeoxyribonucleotide strands (formed by base pairs).
- Antiparallel arrangement (one strand 3’ 5’, another 5’ 3’).
• Right handed spiral. 5’
3’
• Primary structure : 3’ 5’ phosphodiesterase bond.
Watson Crick base pairing rule :

om
Thickness :
• Base pairs linked by hydrogen bonds. /3.4 Å

l.c
ai
• Adenine always pairs with thymine : 2 hydrogen

gm
: Deep
bonds (A=T)

@
Pitch :
• Cytosine always pairs with guanine : 3 hydrogen 1934 Å / 3.4nm
55

bonds (G--C)
na
ee

: Shallow
m

Chargaff’s rule : 3’
ar
um

Number of Purines (Adenine, Guanine) = Number of 5’

itk

Pyrimidines (Thymine, Cytosine)

am

Base stacking :
|
w

• Van der waal’s force, hydrophobic interaction. (D/t non polar, aromatic nature
ro
ar

of bases)
M

• Number of bases per turn : 10.5 bases per turn.

©

Major and minor grooves :

Sites of protein-DNA interaction.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 338 Molecular Biology

----- Active space ----- Types and Properties of DNA 00:12:37

6 types : A, B, C, D, E, Z
Right handed
A form B form (most abundant) Z form
Handedness Right Right Left
Base pair 11 bp / turn 10.5 bp /turn 12 bp / turn
• Broad and short. • Longer and thinner
• Elongated & thin
• Form of DNA preferred in • Most stable
Characteristic • Backbone of DNA is
solution devoid of water. • High degree of hydration &
zigzag in Z form.
• High salt concentration. low salt concentration
Z DNA : Predominantly contains pyrimidines alternating with purines, E.g. : CGCG

om
l.c
DENATURATION & MELTING OF DNA

ai
gm
Two strands separating into component strands.

@
19
55
Features :
na

• Breaking of hydrogen bonds.

ee
m

• Disruption of base stacking.

ar
um

• Phosphodiester bond, covalent bonds intact.

itk

• No alteration of 1˚ structure.
am

• Modification of 2˚ and 3˚ structures.

|
w

• Decreased viscosity.
ro
ar

• Heterochromicity : ↑in absorbance of UV light (260 nm).

M

• ↑Optical absorbance of DNA at 260 nm (UVC band).

©

- >40 % hyperchromicity : Denatured DNA.

- UVB band : Required for synthesis of DNA.
Note : Other important wavelengths
• 400 nm : Absorbed by porphyrin.
• 280 nm : Aromatic amino acids. E.g. : Tryptophan, Phenylalanine & Tyrosine.
Properties of denaturation :
Melting temperature (Tm) : Mid-point of range of temperature separating two
strands of DNA
Factors affecting Tm :
• ↑G-C bp : ↑temperature (d/t hydrogen bonds) = ↑Tm
• Formamide : Destabilise hydrogen bond = ↓Tm

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Structure & Organisation of DNA 339

Salt concentration & DNA denaturation : ----- Active space -----

10 fold increase in monovalent metal ion ↑Tm by 16.6˚ C
Note :
1. Mitochondrial DNA
• dS circular DNA
• Features :
- 16,569 bp 22 tRNA
- 37 structural genes 2 rRNA
13 proteins code for ETC protein
- High mutation rate : d/t
a. Close proximity to ETC (↑ free radicals).
b. No histones.

om
c. No repair enzymes (mutations hit exons).

l.c
d No introns.

ai
gm
- Non mendellian cytoplasmic/Maternal/Matrilinear

@
inheritance :
- Unique genetic code. 19
55
na
ee
m
ar
um

Codons Nuclear code Mitochondrial code

itk
am

AUA Isoleucine Methionine (Start codon)

|

UGA Stop codon Tryptophan

w
ro

AGA, AGG Arginine Stop codon

ar
M

2. Nuclear DNA : 1 haploid set contains 3 x 109 bp 3 billion bps.

©

Organization of DNA 00:28:15

1st level : DNA double helix 2 nm diameter

↔

Histone octamer
2nd level : 10 nm chromatin made up of nucleosomes +
DNA double helix
3rd level : 30 nm chromatin condensed & non condensed

metaphase chromosomes.
SECOND LEVEL ORGANISATION
Histones :
• Most abundant chromatin.
• DNA + histones (proteins) organised chromatin.
• Basic (+ve charge) proteins.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 340 Molecular Biology

----- Active space ----- • conserved among species (Amino acid sequence in all species).
• 5 classes :
Type Classes Location
Core histones H2A, H2B, H3, H4 Histone octamer
Linker histones H1 Linker DNA

H2A H2B
• Abundant in arginine and lysine. H3 H4
• Histone protein
dimerises to form
H2A H2B
Ionic Histone octamer (+ve charge)
+ H3 H4
bond DNA double helix wind (-ve charge)

om
Histone octamer
• Nucleosome :

l.c
ai
- 10 nm chromatin fibrin 3o nm chromatin further metaphase chromosome

gm
coiling
- Beads on a string appearance.
@
19
55
Histone octamer :
na
ee

• Left handed.
m

• ~ 2/1.75 turns of double helical chains.

ar
um

• ~ 146 bp.
itk
am

EUCHROMATIN VS HETEROCHROMATIN
|
w
ro

Euchromatin Heterochromatin
ar
M

Activity Transcriptionally active Less transcriptionally active

©

Organised Less organised Highly organised

Chromatin stain Less dense Dense
Chromatin Permissive Repressive

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 DNA Replication and Repair 341

DNA REPLICATION AND REPAIR ----- Active space -----

Salient features :
• S phase of cell cycle.
• Both strands of parent strands act as template.
• DNA synthesis : 5I 3I.
• DNA replication is bidirectional.
• DNA polymerase requires primer for DNA synthesis in replication.
• Semidiscontinuous :
- Leading strand : Continuous.

om
- Lagging strand : Discontinuous.

l.c
ai
5I 3I

gm
Discontinuous

@
3I 5I
19
Continuous
55
5I 3I
na

3I
ee

5I
m
ar

• Semiconservative :
um

- Half of the parent strand is conserved.

itk
am
|

Daughter strand :
w
ro

Complementary to parent strand.

ar
M
©

Parent strand

Steps 00:08:10

I. Identification of origin of replication (ori).

II. Unwinding of DNA.
III. Formation of replication fork.
IV. DNA synthesis (Leading & lagging strand).

I. Identification of origin of replication (ori) :

Ori is the fixed point for initiation of replication.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 342 Molecular Biology

----- Active space ----- Types :

• E. coli : Ori c.
• λ phage : Ori λ.
• Yeast : Autonomous replication sequence (ARS).
• Humans : Similar to ARS, multiple ori.

Process :
Ori
Ori binding Unwinding of
AT rich
protein AT rich region
region/DUE

Binding of SSB to unbound region.

om
(Prevents reannealing of DNA)

l.c
ai
gm
DUE : DNA unwinding element.

@
SSB : Single stranded binding protein.
19
55
na
ee
m

II. Unwinding of DNA :

ar
um

Helicase : Unwinding of DNA.

itk

Topoisomerase :
am

• Nicking / Resealing enzyme.

|
w

• Prevents topological constraints during unwinding.

ro
ar

Types :
M
©

Type I Type II
- Nick in one strand of DNA. - Nick in both strands.
- No ATP required. - ATP required.
- Eg : DNA gyrase (In bacteria).

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 DNA Replication and Repair 343

III. Formation of replication fork : ----- Active space -----

Replication bubble

5I 3I

3I 5I

Branching point forms replication fork

om
IV. DNA synthesis

l.c
ai
gm
5I 3I

@
19
55
Leading Lagging
na

strand strand
ee
m

Replication fork 5I
ar

3I
um
itk

Leading strand synthesis :

am

• Primase : Adds RNA primer on the DNA. 5I 3I

|
w
ro

• DNA polymerase : Continuous formation of DNA Polymerase

ar

H
M

DNA strand.
©

Leading strand
- In prokaryotes : DNA polymerase III. Primase
- Same direction as helicase H . 3 RNA primer
I
5I
Lagging strand synthesis :
• Discontinuous.
• Direction opposite to helicase.
1. Synthesis of RNA primer by primase.
2. Synthesis of Okazaki fragments by DNA polymerase 111.
3. Removal of RNA primer & gap filling by DNAP I (In prokaryotes).
- Okazaki fragments act as primer to fill.
4. DNA ligase seals the nicks.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 344 Molecular Biology

----- Active space ----- 5I 3I

H
RNA primer
Lagging strand

Okazaki fragment
5I
3I DNA Polymerase

5I 3I
H Lagging strand
Nick

Okazaki 5I
3I

om
fragment

l.c
ai
gm
Enzymes 00:32:20

@
Helicase : Unwinding DNA, requires ATP. 19
55
na

Topoisomerase : Relieves the strain in DNA. Note :

ee

Primase : Synthesis of primer.

m

SSB : Protein acts prior

ar

DNAP III : Synthesis of DNA in leading strand.

um

to helicase enzyme.
DNAP I : • Removal of primer in lagging strand.
itk
am

• Filling of gaps.
|

DNA Ligase : Sealing of nick, requires ATP.

w
ro
ar

PROKARYOTIC DNAP
M
©

Enzyme Function
• Removal of primer I & gap filling.
DNAP I
• Major DNA repair.
DNAP II • DNA repair.
• Synthesis of leading strand.
DNAP III
• Synthesis of Okazaki fragments in lagging strand.

• DNAP I, II, III : Proofreading.

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 DNA Replication and Repair 345

EUKARYOTIC DNAP ----- Active space -----

Types : α, β, γ, δ, ε. Enzyme Function
DNAP α Primase activity
DNAP β Major DNA repair
DNAP γ Mitochondrial DNA synthesis
DNAP δ Lagging strand synthesis
DNAP ε Leading strand synthesis
DNAP γ, δ, ε : Proof reading function.

Additional points :
• Most processive : DNA polymerase III.

om
- Synthesis of maximum number of nucleotides.

l.c
ai
• Kornberg’s enzyme : DNA polymerase I.

gm
@
- Discovered in E. coli by Arthur Kornberg.
• Klenow fragment : 19
55
na

- DNA polymerase I with no 51 31 exonuclease activity.

ee
m

- Using in recombinant DNA technology & Sanger’s sequencing.

ar
um
itk

DNA repair mechanism 00:44:15

am
|
w

Defects in DNA Repair mechanism Disorders associated

ro
ar
M
©

Double strand break

repair mechanism • Severe combined
1. Non homologous immunodeficiency (SCID).
end joining • Radiosensitive SCID.
• Double strand breaks (NHEJ)
(DSB).
• Single strand breaks. • Ataxia telangiectasia like
• Intrastrand cross links. disorder.
• Nijmegen break syndrome.
2. Homologous
• Bloom’s syndrome.
recombination
• Werner syndrome.
(HR)
• Rothmund-Thomson syndrome.
• Breast cancer susceptibility
(BRCA 1, 2).

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 346 Molecular Biology

----- Active space -----

Defects in DNA Repair mechanism Disorders associated

Large defects :
• Bulky adducts.
• Xeroderma pigmentosum (XP).
• Pyrimidine dimers Nucleotide excision
• Cockayne syndrome.
- UV rays 260nm. repair (NER)
• Trichothiodystrophy.
( Major cause for M/c
Thymidine defect )
Small defect :
Abasic sites Base excision repair
MUTYH-associated polyposis
(Absence of a base in the (BER)
DNA)

om
Mismatch repair Hereditary non-polyposis colorectal

l.c
Base mismatch

ai
(MMR) cancer (HNPCC) / Lynch Syndrome

gm
@
Note : 19
55
na

• Adenosine deaminase defect also causes SCID.

ee
m

• Xeroderma pigmentosa presentation :

ar
um

- Blisters in sun exposed areas, high susceptibility to skin cancers.

itk
am

Enzymes involved in prokaryotic DNA repair:

|
w
ro

Repair mechanism Enzyme

ar
M

Uvr ABC excinuclease.

Nucleotide excision repair (NER)
©

(Uv specific endonuclease)

N glycosylase.
Base excision repair
Apurinic (Abasic) endonuclease.
• Endonuclease
GATC methylation : Differentiation
Mismatch repair + Parent strand.
− Daughter strand.
• Mut H, Mut L, Mut S proteins
Nucleotide excision repair
(In humans) Helicase activity of transcription factor ( Tf II H ) :
Bulky lesions like pyrimidine Product of two XP − B, XP − D
dimers

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 DNA Replication and Repair 347

Note : ----- Active space -----

• Endonuclease : Cuts phosphodiesterase bonds from within.
• XP : Mutation in any gene from XPA to XPG.

Double strand break repair mechanisms :

Homologous recombination Non-homologous end joining repair

Occurs in Yeast Mammals
Homologous
Required Not required
chromosomes
Phase of cell
S & G2 /M phase G0/G 1 phase
cycle

om
l.c
Enzymes involved in DSB repair :

ai
gm
Non-homologous end joining : ku protein

@
19
55
na
ee
m
ar

• Attaches to the break in DNA.

um

• ↑ DNAP kinase (DNAPK) Joins the ends.

itk
am

TELOMERE & TELOMERASE

|
w
ro

Telomere :
ar
M

• Ends of the chromosome.

©

Hexanucleotide
• Tandem repeat TTAGGG
At the 31 end.
End replication error :
One primer Gap at 51 end of No replication at 31 Shortening of DNA.
removed. daughter strand. end of parent strand. (If uncorrected)

• Hayflick limit ≥ 50 cell divisions No further division.

- Cause for aging.
Telomerase :
• Ribonucleoprotein.
• Components :
1. Intrinsic RNA template :
- Telomerase binds to 31 end of DNA.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 348 Molecular Biology

----- Active space -----

2. Reverse transcriptase activity : RNA DNA.
- Extension of 3 end of parent strand.
1

• Site : Stem cells, germ line cells, hematopoietic cells, lymphocytes, cancer cells.
- Has immense replicating potential.
- No aging / No Hayflick limit.
• Absent in somatic cells.
- Shortening of DNA d/t absent replicating potential.
↑ Cancer
• Telomerase
↓ Progeria : Premature aging.
• Chemotherapeutic agents : Drugs inhibiting telomerase.

om
l.c
ai
gm
@
19
55
na
ee
m
ar
um
itk
am
|
w
ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Transcription 349

TRANSCRIPTION ----- Active space -----

DNA Transcription RNA : 1st step of gene expression.

5’ 3’ Coding/plus/sense strand 5’ T A C G T A 3’ In coding stands
3’ 5’ Template/minus/ 3’ A T G C A T 5’ In template strand
antisense strand 5’ U A C G U A 3’ In RNA
5’ RNA 3’

Enzymes of Transcription  00:07:25

om
RNA POLYMERASE

l.c
ai
gm
In Prokaryotes :
Sigma subunit
@
• Single type of RNA polymerase (RNAP).
19 + Holoenzyme
55
• Does not require primer.
na

• Holoenzyme (Multisubunit enzyme). Core enzyme

ee
m
ar

β subunit :
um

• Magnesium binds to β subunit.

itk
am

• Catalytic subunit : Adds successive ribonucleotides.

|
w

σ subunit :
ro
ar

• Binds to promoter (TATA box).

M
©

In Eukaryotes :
Types : RNAP I, RNAP II, RNAP III.

RNAP I RNAP II RNAP III

Sensitivity to alpha amanitin
Least Maximum Intermediate
(Mushroom toxin)
mRNA, miRNA, snRNA, tRNA, 5SrRNA snRNA
Major products rRNA : most abundant
lncRNA, circRNA (few)
rRNA : Ribosomal
snRNA : Small nuclear
lncRNA : Long non-coding
circRNA : Circular

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 350 Molecular Biology

----- Active space ----- Promoters & Enhancers of Transcription  00:15:49

PROMOTERS :
• Short conserved sequence in the coding strand.
• Specifies the start site of transcription.
Promoter
5’ 3’ Coding strand
Upstream
Upstream
elements +1 Downstream elements
-3 -2 -1 +2 +3 +4 +5
3’ 5’ Template strand
Start site
Prokaryotic promoters :
-35 Sequence Pribnow box
5’ 3’ -10bp refers to

om
TGTTGACA TATAAT

l.c
-35 bp -10 bp 10 bp away from

ai
3’ 5’

gm
start site

@
Eukaryotic promoters : 55
19
Tata box/Goldberg
na

CAAT box hogness box

ee

5’ 3’
m

GC box CAAT TATAAA Identifies + 1 site

ar

-70/-80 bp -25 bp
um

+1 site
itk

3’ 5’
am
|

ENHANCERS :
w
ro

• DNA elements.
ar
M

• Enhance transcription.
©

• Orientation : Either direction (5’ 3’/3’ 5’).

• May be embedded within target promoter.

Transcription Cycle  00:33:39

1. Template binding & closed promoter complex :

Attachment of σ subunit of RNA polymerase with unbound form of DNA.

2. Open promoter complex :

Unwinding of the DNA.

3. Chain initiation :
• RNA polymerase remains fixed to promoter.
• β subunit reaches +1 site Synthesis of RNA in 5’ 3’.
• Addition of ribonucleotides until 11 to 20 nm.
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 Transcription 351

4. Promoter clearance : ----- Active space -----

• Removal of σ subunit from RNA polymerase.
• RNA polymerase detaches from promoter.
• β subunit reaches further deoxyribonucleotides.

5. Chain elongation :
Addition of ribonucleotides RNA elongation.
ρ dependent
6. Chain termination
ρ independent
ρ dependent termination : 5’ 3’
ρ factor : 3’ 5’
5’ ρ 3’
• ATPase activity.
• Unwinding enzyme.

om
Rut Site :
• Binds with newly synthesized RNA at “rut site”.

l.c
ρ utilisation site

ai
• Function : Destabilize the newly synthesized RNA from

gm
ρ-Dependent termination

@
DNA.
19
55

ρ independent termination :
na

5’ 3’
ee

• No ρ factor. (GC)n (GC)n AAAAA

m

3’ 5’
ar

• Termination signal facilitates Detaches

um

UUUU from DNA

termination :
itk

Intrastrand
am

- GC rich region : Codes for GC C G basepairing

|

sequence.
w

G C
ro

- Intra strand base pairing

ar

C G
M

Forms hairpin G C
©

structure Destabilizes RNA. Hairpin structure

ρ-Independent termination

Post Transcriptional Modification  00:44:32

Occurs in all eukaryotic RNA & Prokaryotic RNA (Except mRNA).

Site : Nucleus (Nucleolus) > Cytoplasm.
Methods :
• 5’ capping.
• 3’ poly A tailing.
• Splicing of exons & removal of introns.
• Methylation.
• Alternate/differential RNA processing.
• RNA editing.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 352 Molecular Biology

----- Active space ----- 5’ CAPPING

Newly synthesized RNA : Primary transcript aka Heteronuclear RNA (hnRNA) :

Guanosine (Guanine + ribose) Methylation

7th Nitrogen S-adenosyl methionine 7-Methyl
Attaches to
guanosine
5’ terminal of RNA guanylyl transferase
Nucleus Cytoplasm

3’ POLY A TAILING
5’ Cap
5’ 3’
AAUAAA-20 +
1. Cut by Endonuclease

om
2. Addition of 40 -200 adenosine

l.c
ai
gm
Functions of 5’ capping & 3’ poly A tailing :

@
• Prevent the attack by 5’ to 3’ exonucleases.
19
55
• Stabilise mRNA.
na

• Helps initiation of translation.

ee
m
ar

SPLICING OF EXONS & REMOVAL OF INTRONS

um

5’ splice sites 3’ splice site

itk
am
|

Exon-1 Intron (A) Exon-2

w
ro
ar

Exon-intron Adenosine :
M

junction Branch site

©

Spliceosome :
• Helps in RNA splicing.
• Multicomponent structure.
snRNA (Ribozyme) :
• Transcribed by RNAP II.
• Rich in uracil (U1, U2, U4, U5, U6).

Small nuclear ribonucleoprotein (snRNP)/snurps :

• snRNA + protein = snRNP.
• snRNP combines with primary transcript (5’ splice site) = Spliceosome.

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 Transcription 353

5’ splice sites 3’ splice sites ----- Active space -----

Exon-1 Intron (A) Exon-2

Sn RNP cuts 5' Splice site
3’ splice sites

Exon-1 Intron (A) Exon-2

2’ OH
3’ OH 5’ Po4
2' OH of adenosine forms 2' → 5' phosphodiester bond
3’ splice sites

Exon-1 Exon-2
'2'-5' phosphodiester bond

om
3’ OH

l.c
Nucleophilic attack on 3'splice site by 3’ OH

ai
gm
Intron : Lariat
@
Exon-1 Exon-2
19 Structure
55
na

3'-5' phosphodiester bond

ee
m
ar

ALTERNATE/DIFFERENTIAL RNA PROCESSING

um

Total ~ 20,000 genes Codes 1 lakh proteins.

itk
am
|

Selective splicing :
w
ro

Diverse set of proteins from same gene. A B C D

ar
M

Selective splicing
©

Alternate 5’ splice site :

5’ splice site
A B C D
Absence of this region

Alternate 3’ splice site :

3’ splice site
A B C D
Absence of this region

Alternate polyadenylation site :

Varied site of polyadenylation (AAuAAA).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 354 Molecular Biology

----- Active space ----- RNA EDITING

• Chemical modification in mRNA.
• Exception to central dogma
(Central dogma : DNA mRNA Protein).
• Example :
Apo B gene Apo B gene
in liver in intestine

Apo B mRNA CAA Apo B mRNA (CAA present)

0 CAA
100% translated 0
apo B-100 (Protein) cytidine
uracil Cytidine deaminase
NH3

om
UAA = Stop codon
0

l.c
48% translated

ai
gm
Truncated (Protein)

@
19
55
na
ee

Types of RNA  01:08:37

m
ar
um
itk
am

Protein coding RNA : Non-protein coding RNA :

|

Poly A tail (+)

w

mRNA
ro

Poly A tail (-) :

ar

Large non-coding : Small non-coding :

M

• Histones
©

• rRNA : Translation • tRNA : Translation

• Long non-coding RNA • Small nuclear RNA (snRNA)
: spliceosome

Additional Points :
Self splicing introns : hn RNA with no intron : Disorder associated with
• Group II introns. Histone gene. snurps :
• Ribozyme Systemic lupus
• Undergoes RNA erythematosus
Splicing. (Autoimmune disorder).

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 Translation 355

TRANSLATION ----- Active space -----

• Process of synthesis of protein from RNA.

• Site :
a. Rough endoplasmic reticulum. b. Free ribosome.

Genetic Code  00:02:28

The relationship between a sequence of DNA and a sequence of amino acid in the
corresponding polypeptide.
Representation of amino acids :

om
Sequence of 3 nucleotides (Triplet)

l.c
ai
gm
(4)3 64 codons 20 amino acids
@
19
55
Codon :
na
ee

A triplet sequence in mRNA representing specific amino acids.

m
ar

Stop/Terminator codons :
um
itk

Out of 64 codons, 3 are stop codons :

am

a. UAA (Ochre).
|
w

b. UGA (Opal) : Can be recoded to selenocysteine.

ro
ar

c. UAG (Amber) : Can be recoded to pyrrolysine.

M
©

Note :
1. Amino acids represented by a single codon :
• Methionine (AUG). • Tryptophan (UGG).
2. Amino acids represented by maximum number of codons : Serine, Leucine,
Arginine (6 codons).

Salient features of genetic code :

1. Degenerate/Redundant :
• Amino acids represented by >1 codon.
• Degeneracy lies on the 3rd base :
Eg : UUU UUC

Phenylalanine
2. Unambiguous : No two amino acids are represented by the same codon.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 356 Molecular Biology

----- Active space ----- 3. Non-overlapping :

Eg : AUG GUG AAA GUU

4. Non-punctuated : Translation only stops at stop codons.

5. Universal :
• An amino acid is represented by the same codon across species.
• Exception : Mitochondrial DNA.

6. Initiator codon :
AUG
Methionine (Eukaryotes).
N-formyl Methionine (Prokaryotes).

om
l.c
Ribosome 

ai
00:15:37

gm
• Cellular machinery for protein synthesis.
@
• rRNA + Specific proteins. 19
55
na
ee

Structure : Note :
m
ar

80S Ribosome (Eukaryotes) 28s rRNA :

um

• Ribozyme.
itk

• Peptidyl transferase activity.

am
|

60s Subunit 40s Subunit

w
ro
ar
M

(28s, 5.8s, 5s) rRNA (18s rRNA + 30 proteins)

©

+
50 proteins

Transfer RNA  00:17:51

Features :
• tRNA AKA soluble RNA (sRNA).
• 1 tRNA contains 74-95 nucleotides.
• RNA with largest number of unusual bases.
• Only RNA containing thymine (Ribothymidine : Pseudouridine arm).

Shape :
2° structure : Clover leaf shaped.
3° structure : Inverted L shaped.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Translation 357

Structure : ----- Active space -----

Acceptor arm :
• 3 unpaired nucleotides CCA at
3’ end.
• Accepts amino acids.

DHU arm : Pseudouridine (tΨc) arm :

• Contains dihydrouracil. • Contains thymine.
• Recognizes specific • Binds to ribosome.
aminoacyl tRNA
synthetase enzyme.

om
Acceptor arm Aminoacyl

l.c
+ tRNA

ai
gm
Amino acid

@
Anticodon arm :
19
• Nucleotide sequence complimentary to codon.
55
• Binds to the codon.
na
ee

Note :
m
ar

For every enzyme : Unique aminoacyl tRNA synthetase enzyme.

um

Wobbling
itk
am

Phe tRNA
Wobbling : Phe
|
w

Can bind
ro

Codon-Anticodon interaction at 3rd base is not strict.

ar

to UUU or
M

UUC
©

Degeneracy.

31 tRNAs can combine with 61 codons.

Hence reduction of number of tRNA is possible
d/t wobbling. AAA AAA
MRNA UUU UUC

Steps of Translation  00:30:58

1. Charging of tRNA.
2. Initiation : Needs initiation factors (IF in prokaryotes, eIF in eukaryotes).
3. Elongation : Needs elongation factors (EF).
4. Termination : Needs releasing factors (RF).

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 358 Molecular Biology

----- Active space ----- Charging of tRNA :

DHU arm recognizes specific aminoacyl tRNA synthetase.
ATP
(2 Pi utilised)
AMP
Amino acid attaches to acceptor arm.
Initiation :
Marker sequence : Steps :
• Helps in identifying initiator codon Step 1 : Disassembly of ribosomal units.
(AUG). Step 2 : Formation of 43S preinitiation
• Prokaryotes : Shine Dalgarno Step 3 : Formation of 48S initiation
sequence complex.
• Eukaryotes : Kozak consensus Step 4 : Formation of 80S initiation

om
sequence. complex.

l.c
• First AUG after marker sequence is

ai
gm
start codon.

@
GTP Binds with 19
55
eIF-2
na
ee

Binary complex
m
ar

eIF-2
um

GTP
Initiator methionine
itk
am

tRNA mRNA
Ternary complex
|

Free site
w

Bound to initiator tRNA

ro

40S subunit Vacant

ar
M
©

43S Pre-initiation complex

Binds with mRNA

48S Initiation complex

60S Subunit

80S Initiation Complex GTP leaves Used for energy

Elongation :
Catalyzed by elongation factors (EF).

Step 1 : Binding of amino acyl TRNA

tRNA to A site Anticodon arm

Elongation requires : Binds to

• Elongation factor 1. A site
80s initiation
• 1 GTP (Hydrolysed for energy). complex
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Translation 359

Step 2 : Formation of peptide bond ----- Active space -----

No energy needed.

Binds with
(+) Peptidyl transferase : Present in 28s rRNA Present in 60s subunit
On Asite
On P site

complex.

Step 3 : Translocation of ribosome on mRNA

om
Requires :

l.c
ai
• Elongation factor 2.

gm
@
• Hydrolysis of I GTP.
• Ribosome moves by 1 codon length on mRNA. 19
55
na

• A site becomes free again Can accept new aminoacid & tRNA.
ee
m

• E site receives exiting tRNA polypeptide from P site.

ar
um
itk

New tRNA with

am

amino acid binds

|
w
ro

to A site
ar
M
©

Step 4 : Termination
Complexed with :
a. RF-3.
b. GTP (For hydrolysis Energy release).
c. Peptidyl transferase.

Energetics :
Charging of tRNA (tRNA tRNAaa) : 2Pi +
EF1 (Binding of tRNAaa to A site) : 1 GTP +
EF2 (Translocation) : 1 GTP.
1 Peptide bond synthesis = 4 high energy PO4.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 360 Molecular Biology

----- Active space ----- REGULATION OF GENE EXPRESSION

I. Level of transcription : II. Level of DNA (Gene) :

• By induction & repression. • Gene switching.
• Operon concept : Lac operon. • Gene rearrangement.
• Gene silencing.
• Gene amplification.

Constitutive/House keeping gene :

• Genes expressed at a constant rate in almost all cells.
• Required for basal cellular function.

om
Eg : Enzymes for glycolysis.

l.c
ai
Inducible gene :

gm
• Genes expressed under special circumstances.
@
19
Increases d/t an activator/inducer.
55
• Response
na

Decrease d/t a repressor.

ee
m
ar
um

Lac Operon  00:06:25

itk
am

Components :
|
w
ro

Regulator/Inhibitor gene Promoter gene Operator gene Structural gene

ar
M
©

Function :
Metabolism of lactose in E.coli.
Pathway : Structural gene
Regulator/Inhibitor gene Promoter gene Operator gene Lac Z Lac y Lac A

Produces Permease : Thiogalactose

β galactosidase : Permits entry of transacetylase
Catabolism of lactose into cell
Lactose
Catabolite repression :
Occurs d/t the presence of CRP/CAP.
CRP : Catabolite repressor protein/CAMP receptor protein.
CAP : Catabolite activator protein.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Regulation of Gene Expression 361

Action of CAP/CRP : ----- Active space -----

Activator protein Initiates transcription of lac operon.
Mechanisms :
• Binds to promoter of structural gene of lac operon.
• Interact with C terminal domain of α subunit of RNAP.
CAP is a positive regulator of lac operon.

Role of CAP :
↑ Glucose availability (-) Lac operon
Catabolic repressor
(Irrespective of presence of lactose)
Fasting stage : Glucagon ↑ cAMP Binds with CRP/CAP activated
Binds with

om
Lac operon active ↑ Expression of structural gene Promoter site

l.c
ai
gm
Fed state : ↓ cAMP CAP inactive Lac operon inactive.
@
Glucose 19
55
na
ee
m

Absent
ar

Present
um
itk

Inhibit adenylyl cyclase Adenylyl cyclase active

am
|
w

↓ cAMP ↑ cAMP
ro
ar
M

Lac operon inactive Lac operon active

©

Some scenarios of lac operon :

Inactive lac operon : Active lac operon : Inactive lac operon :
Glucose ++ Glucose -- Glucose ++
Lactose - Lactose ++ Lactose ++

Lac I gene Lac I gene cAMP ↓

Produces repressor protein Produces repressor protein CAP inactive

Binds with operator site Binds with allolactose

Inhibits RNA polymerase Inactive repressor

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 362 Molecular Biology

----- Active space ----- GENE REARRANGEMENT :

Rearrangement of various gene segments to form unique immunoglobulins.
Eg : Immunoglobulin
VDJ gene segments of B
lymphocyte during development

Unique variable region

109-1011 different immunoglobulins

from single gene

GENE AMPLIFICATION :
Process of increasing number of genes available for transcription.

om
l.c
Eg :

ai
gm
• Dihydrofolate Tetrahydrofolate.

@
Dihydrofolate reductase
19
55
na

Methotrexate
ee

• Leads to ↑ in gene producing dihydrofolate reductase R esistant to

m
ar

methotrexate.
um
itk

GENE SWITCHING :
am

One gene is switched off and a closely related gene takes up its function.
|
w
ro

Eg :
ar
M

• Intrauterine : Genes for alpha & gamma chains of Hb.

©

• Infancy : Genes for alpha & beta chains of Hb.

• Immune response 1° : IgM active.
2° : IgG active.
Transposons :
• Aka jumping genes.
• Discrete mobile DNA sequence that transports itself to other locations within the
genome.
• Discovered by Barbara McClintock.
• Regulate gene expression by activating or inactivating genes.
• Causes insertion/deletion mutations.
• Constitute >50% DNA elements Transposons/Retroposons.

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 Regulation of Gene Expression 363

Retrotransposons : ----- Active space -----

DNA sequence moves from one segment to another through an RNA intermediate.

GENE SILENCING :
The process of switching off genes.

Mechanisms :
• Epigenetic mechanisms.
• RNA interference by miRNA and siRNA.

miRNA & siRNA :

miRNA siRNA
• Silencing RNA
Name Micro RNA

om
• Small interfering RNA

l.c
• Small non-coding single stranded RNA.

ai
gm
Features
• 21 to 25 nucleotide length

@
Endogenously from pri-micro 19
55
Source Exogenous(Or endogenous)
na

RNA gene
ee

Post-transcriptional regulation ds RNA ssRNA siRNA

m
ar

Function of gene expression by affecting Post transcriptional regulation/

um

mRNA stability RNA Interference

itk
am

Epigenetics :
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• Reversible heritable chemical modification of DNA or chromatin.

ro
ar

• Does not alter DNA sequence.

M
©

Types :
1. DNA methylation : Modification in DNA.
2. Histone modification in histone.
Mechanism :
S-adenosyl methionine (Methyl donor)

Methyl transferase

Methylation of cytosine residues at CpG islands.

• CpG : C and G attached by phosphodiesterase bond
• Found in promoter regions.
- Initiation of transcription by binding of RNA polymerase

Genes silenced
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 364 Molecular Biology

----- Active space -----

Histone modification :
Histone acetylation :
• Classical epigenetic modification.
• 8 histones + DNA bound around it : Nucleosome.
• Acetylation of lysine group of histones Activation of genes E uchromatin
formation.
• Acetyl group : -ve charge.
• Histones : +ve charge.
Acetylation Addition of
↑ -ve charge ↓ Condensation
by histone acetyl transcription mRNA Protein
↓ +ve charge of chromatin
transferase factors
Histone deacetylation :

om
Deacetylation Inactivates genes Heterochromatin formation.

l.c
ai
Prevents binding

gm
Removal of acetyl group ↑ +ve charge ↑ Chromatin Condensation :

@
by histone deacetylase of transcription
of histone 19
Heterochromatin
factors
55
na
ee
m

Generation of siRNA & miRNA 

ar

00:40:55
um

Generation of miRNA :
itk
am

Pri miRNA gene

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ro
ar

Pri miRNA (Inactive)

M

Endonuclease
©

Cuts the ends

DROSHA, DGCR8
Pre-miRNA

Nuclear pore : Exportin

Transports outside the nucleus

TRBP- dicer Endonucleases
miRNA duplex
ds, 21-22 nucleotide
Argonaute Protein 2
Cassette like Protein
RISC : RNA induced silencing complex
ss, 21-22 nucleotide

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 Regulation of Gene Expression 365

Generation of siRNA : ----- Active space -----

ds RNA
TRBP- dicer
Cuts ends of RNA

ds, siRNA duplex

Argonaute
Protein 2
ss, siRNA
21-22 nucleotide

Post Transcriptional Gene Regulation  00:45:30

om
RNA interference or RNAi :

l.c
ai
gm
miRNA : Binds to

@
55
19 3’ seeding sequence in
3’ UTR of mRNA
na
ee

mRNA Poly A tail

m
ar

5’ 3’ untranslated
um

7 methyl 5’ untranslated
region
itk

region (UTR)
am

guanosine cap
|
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miRNA - mRNA base pairing

ro
ar
M
©

Perfect Imperfect

mRNA degradation Translation arrest

mRNA Protein

RNAi

Gene silencing/Gene knockdown

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 366 Molecular Biology

----- Active space -----

Pathological effects of miRNA :
• RISC : Graveyard of mRNA.
• mRNA of Oncogene in 3’ untranslated region of mRNA :
Failure of oncogene expression

Degradation Arrest of translation

Protective against cancer

• Oncosuppressors : miRNA that degrades oncogenic mRNA.
• Oncogenic miRNA/Oncomirs : miRNA that degrades mRNA of tumour suppressor

om
genes.

l.c
mRNA of tumor suppressor genes

ai
gm
Degraded

@
Suppression of tumor suppressor gene 55
19
na

Causes cancer
ee
m

Oncogenic miRNA causes ↓ oncosuppressor miRNA.

ar
um
itk

Functions of epigenetic modification :

am

• Regulation of specific gene expression.

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• X chromosome inactivation (In females) : Facultative heterochromatin.

ro
ar

• Genomic imprinting.
M

• Aging process.
©

Diseases :
• Cancer.
• Fragile X syndrome.
• Genomic imprinting disorder.

Investigation :
• Methylation specific PCR.
• DNA chromatin immunoprecipitation (ChIP).
• Bisulphite Sequencing in DNA sequencing.
• Methylation sensitive restrictive endonuclease digestion.

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 Hybridisation Techniques 367

HYBRIDISATION TECHNIQUES ----- Active space -----

Blotting Techniques 00:03:00

SOUTHERN BLOT

Principle : DNA - DNA hybridisation based on Watson-Crick base pairing rule.

Technique :
DNA Isolation from Fragmented DNA Separated DNA
blood sample clumped together strands

om
Treated with DNA electrophoresis :

l.c
restriction enzyme -Agarose Gel, or

ai
gm
-Polyacrylamide gel
(PAGE)

@
Probe finds 19
55
complementary DNA
na
ee

strand & detects

m

fluorescence/ Denatured
ar
um

radioactivity
itk

Single stranded DNA

am

Treated with radio/

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fluorescent labelled probe Blot to nitrocellulose/

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ar

nylon membrane
M

*
©

Probe : Known oligonucleotide sequence

Uses :
• Detect specific viral/bacterial DNA.
• Screening of inborn errors of metabolism.
- Base substitutions.
- Indels (Insertions, deletions).
- Trinucleotide expansion.
• As a part of DNA fingerprinting /RFLP to detect DNA after PCR.

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 368 Molecular Biology

----- Active space ----- NORTHERN BLOT

Detects specific RNA.

Technique :

RNA Isolation
from sample Separated by Blot to nitrocellulose
electrophoresis membrane
(Agarose/PAGE)

om
l.c
Probe binds if

ai
*

gm
complementary • Adding of labelled cDNA probe (radio/
RNA present

@
fluorescence labelled)
19
* • cDNA : complementary DNA to the RNA
55
na

sequence.
ee
m
ar

Uses :
um

• Detects specific RNA (eg : detection of HIV viral RNA).

itk
am

• Study of gene expression.

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ro
ar

WESTERN BLOT / IMMUNOBLOT

M
©

Detects specific protein/antigen.

Technique : Protein on membrane

Protein/ antigen isolated
from sample and separated Blot to nitrocellulose/
using agarose gel/PAGE nylon membrane
electrophoresis.

Detects antigen if present

on the sample via antigen
antibody reaction.
* Antigen detection by
Eg : Testing for HbSAg using * adding radio/fluorescent
complementary antibody probe. labelled antibody probe.
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 Hybridisation Techniques 369

SOUTH-WESTERN BLOT/OVERLAY BLOT ----- Active space -----

Detects DNA-protein interaction.

Technique :

Protein on membrane
Protein isolated from sample
and separated using agarose Blot to nitrocellulose/
gel/ PAGE electrophoresis. nylon membrane.

Detects specific

om
DNA-protein

l.c
interaction.

ai
gm
Treated with
*
@
DNA probe.
* 19
55
na
ee
m
ar

SLOT-BLOT/DOT-BLOT
um

Sample applied directly on nylon/nitrocellulose membrane using blotting apparatus.

itk
am
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Microarray Techniques
ro

00:15:38
ar
M

Prerequisites : Thousands of immobilised known oligonucleotides in different wells

©

in an area ≤ microscopic slide.

Based on contents of the slide :

• DNA microarray
• cDNA microarray
• Protein microarray
• SNP (single nucleotide polymorphism) microarray.

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 370 Molecular Biology

----- Active space ----- DNA MICROARRAY/DNA CHIP

Technique :
Binds to complementary
oligonucleotide if found
*
Adding fluorescently
labelled unknown DNA Hybridisation occurs
*
known oligonucletides in
different wells

Fluorescence
Reports about Loaded to
detected in a
the unknown DNA computer
particular well

om
l.c
Advantage :

ai
gm
Multiple unknown DNAs can be added to the same chip

@
19
MUltiple samples studied together
55
na

Uses :
ee

• Detection of mutations.
m
ar

• Genome sequencing.
um
itk

• Unknown DNA detection.

am
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ro

cDNA MICROARRAY
ar
M

Arrangement of complementary DNAs (cDNA)

©

Technique
Binds to complementary
oligonucleotide if found
*
Adding fluorescently
labelled unknown RNA Hybridisation occurs
*
Known cDNA in different wells

Fluorescence
Reports about Loaded to
detected in a
the unknown RNA computer
particular well
Use :
Studying of gene expression in cancer patients (via harvesting tissue RNA).

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 Hybridisation Techniques 371

PROTEIN MICROARRAY ----- Active space -----

Technique :
Antigen-antibody
reaction occurs
*
Adding fluorescently
labelled unkown antigen
*
Known antibody in different wells

Fluorescence
Reports about Loaded to

om
detected in a
unknown antigen computer

l.c
particular well

ai
gm
Uses :

@
• Detection of unknown antigens and antibodies. 19
55
• Study of proteomics (entire set of proteins expressed in an organism).
na
ee

• Development of monoclonal antibodies.

m

• Study of antigen expression in various disorders.

ar
um
itk
am
|

ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (CGH)

w
ro
ar

Prerequisites :
M
©

Test DNA : Red Normal DNA : Green

fluorescence fluorescence
Genome chip : contains
fragments of entire genome
of an organism

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 372 Molecular Biology

----- Active space ----- Technique :

Considering a genome fragment with 100 nucleotides (nt) :

a) Normal : Test and normal

genome bind equally

100nt 100nt 50nt

100nt Both bind with 50nt

100nt on well
A A

Test and normal genome Creates yellow

added to well fluorescence in well

om
l.c
ai
100nt

gm
@
b) Gene deletion : 19
55
na
ee

100nt 100nt 75nt

m
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50nt 25nt
itk
am

B B
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Test and normal genome • Creates green

ro

added to well fluorescence in well

ar
M

• Indicates gene deletion

©

100nt
c) Gene amplification :

100nt 100nt 25nt

200nt 75nt

C C

Test and normal genome • Creates red

added to well fluorescence in well
• Indicates gene amplification

100nt

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 Hybridisation Techniques 373

Use of Array CGH : ----- Active space -----

• Assessment of disorders with unknown etiology : • Detect gene deletion
• Eg : • Detect gene amplification.
- Dysmorphic features. • Compare 2 genomes
- Cancer. (Eg : Cancer vs Normal
- Autism (mental retardation). genome)
• Detect aneuploidy : FISH > Array CGH.

Drawback : Cannot detect structural abnormalities without loss of genetic elements.

Eg : Balanced translocation FISH is the preferred technique.

Karyotyping 00:34:18

om
Karyotyping

l.c
ai
gm
@
Conventional 19
Molecular FISH
55
na

FISH (FLUORESCENT IN-SITU HYBRIDISATION)

ee

AKA Spectral Karyotying Produces a spectrum of colors.

m
ar

(conventional karyotyping Produces only black & white)

um
itk
am

Definition :
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ro

Simple detection of specific genetic material in a morphologically intact tissue (in-

ar

situ within nucleus of cell) using fluorescent probes.

M
©

Technique :
• 23 distinct mixtures of 5 flourophores.
• Each chromosome labelled with unique colours.
• Dividing cell needed Performed at metaphase Spreading out
of chromosomes on slide (Metaphase arrest).

Findings :
a) Normal sample :
• Each chromosome painted with a unique colour.

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 374 Molecular Biology

----- Active space ----- b) Test sample :

Chr. 5 gene deletion
Chr. 10 gene amplification
Exchange of genes between
Chr. 9 and Chr. 22 (balanced
translocation)

Cannot be detected in array

CGH
Uses :
• Detect aneuploidy.
• Detect structural abnormalities.
• Detect chromosomal deletion and duplication.

om
• Locate newly detected gene in its correct chromosomal loci.

l.c
ai
gm
@
Types :
19
55
na

Metaphase FISH Interphase FISH/ Nuclear

ee
m

• During metaphase. • Between 2 cell divisions.

ar

Timing
um

• Dividing cell. • Non-dividing cell.

itk
am

Sister chromatids
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ro

View
ar

Centromere
M

Normal : 2x Chr. Trisomy : 3x Chr.

©

21 21
• Chromatids seen seperately. • No seperate chromatids seen.

• Rapid results.
Time consuming • Use :
Time for results
(Culture & wait for metaphase) - Cancer detection.
- Prenatal screening.

Sensitivity Lower Higher

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 Recombinant Dna Technology 375

RECOMBINANT DNA TECHNOLOGY ----- Active space -----

In vivo amplification technique inside host cell.

Target DNA Combines with Vector DNA.

A.k.a DNA cloning.

Restriction enzyme (RE) 00:01:30

RESTRICTION ENDONUCLEASE (MOLECULAR SCISSORS)

om
Belong to class 3 enzymes : Hydrolases.

l.c
ai
gm
Types :

@
Type 1 19 Type 2
55
na

Cuts at random sites Cuts at specific palindromic sites

ee

Not commonly used for Employed in recombinant DNA

m
ar

recombinant DNA technology technology as molecular scissors

um
itk
am

Type 2 :
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Forming sticky ends Forming blunt ends

ar
M

Sticky/staggered/cohesive end
©

5’ G A A T T C 3’ Overhang present
5’ G A A T T C 3’ Internal
cleavage
3’ C T T A A G 5’ 3’ C T T A A G 5’

Nomenclature :
Eco : Name of bacterial source
E.g. : EcoR 1 R : Strain of bacteria
1 : Unique number

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 376 Molecular Biology

----- Active space ----- Examples :

Palindromic Site Bacteria
GAATT
EcoR I Sticky ends E. coli
TTAAA
AGATCT
BamH 1 Sticky ends Bacillus amyloliquifaciens H
TCTAGA
AAGCTT
Hind III Sticky ends Haemophilus influenza
TTCGAA
GTTAAC
Hpa I Blunt ends Haemophilus parainfluenza
CAATTG
CTGCAG
Pst I Sticky ends Providencia stuartii
GACGTC

om
l.c
ai
RECOMBINASES

gm
@
Alternative/adjunct to restriction enzyme
19
55
Examples :
na
ee
m

Example Host Specific site

ar
um

Cre recombinase Bacteria Lox P site on bacterial genome

itk

INT protein Lambda phage λatt site on phage genome

am

Flp recombinase Yeast FRT site on yeast genome

|
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ro

Technique :
ar
M
©

Target DNA

Bacterial
host cell Recombinase identifies particular site : Recombinase enables
Site specific recombination incorporation of target DNA
into host genome
Bacterial genome
Host cell

Recombinant DNA

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 Recombinant Dna Technology 377

Application of restriction endonucleases 00:11:10 ----- Active space -----

RESTRICTION MAP
Unique DNA band pattern obtained by treating an individual’s DNA with a specific
restriction enzyme.

Technique :

Restriction
enzymes

2 restriction sites 3 restriction sites

om
l.c
ai
On electrophoresis

gm
@
Unique band 19
55
pattern for
each individual
na
ee
m
ar
um

Normal variation in restriction fragment length between individuals : Polymorphism.

itk
am

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

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Inherited difference in pattern of restriction map on digestion by specific RE.

ro
ar
M

Technique : Treatment of individual’s DNA with specific restriction enzyme.

©

Restriction fragments created.

Compared to other individual’s map.

Uses :

Detect mutations (Abnormal DNA variations) :

β globin gene : Band 2 = 0.2
Band 1 = 1.15 kbp kbp Restriction
1
sites
5’ 3’ 2
Normal On cleavage by Mst II ↑ 1 ↑ 2 3 ↑ (kbp = kilo
3
allele (Restriction enzyme) Mst II base pair)
Mst II Mst II

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 378 Molecular Biology

----- Active space ----- • sickle cell allele 1 restriction site abolished by mutation On cleavage by
Mst II

Single band = 1.35 kbp

5’↑ ↑ 3’
R1, R2 : Restriction sites R1 R2

Detect zygosity via pedigree analysis :

P1 : Sickle cell trait P2 : Sickle cell trait

(Heterozygous)

om
P1, P2 : Parents

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O1, O2, O3 : Offsprings

ai
gm
@
19
55
O1 : Heterozygous O2 : Normal O3 : Homozygous
na
ee

On cleavage by Mst II :
m
ar
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P1 O1 O2 O3 P2
itk

From sickle cell allele 1.35 kb - - - -

am

1.15 kb - - - -
|

From normal allele

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0.2 kb - - - -
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ar
M
©

DNA FINGERPRINTING

Procedure :
Visible fragments

Restriction + Radio labelled

enzyme probes

Sample DNA fragmented Southern blotting : DNA fingerprint

DNA isolated
(Hair, skin) DNA bands not visible.

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 Recombinant Dna Technology 379

Application : ----- Active space -----

• Crime scenes :
DNA fragments from :

Crime scene DNA Suspects’ DNA

Compared with

Suspect 1 Suspect 2 : Suspect 3

Matches
crime scene
• Paternity testing

om
Criminal

l.c
ai
DNA FOOTPRINTING

gm
to Protein

@
d
Use : Detects DNA-protein binding. 19 de
ad
55
Radiolabelling
na

Procedure :
ee
m
ar
um

Fragmented and
itk
am

divided into 2
halves
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ro
ar

Genomic DNA -A -B
M
©

DNase DNase
Cleaving of DNA Cleaving of DNA except for
protein bounded fragments
(undergo lysis)

DNA electrophoresis
-A -B

Protein bounded segments

missing : Footprints of the
protein.

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 380 Molecular Biology

----- Active space ----- Vectors 00:30:16

TYPES

Sources :
Origin Source Example
• Plasmid
Bacterial
Natural plasmids • Cosmid
Phage Phage DNA
Bacterial based genome BAC (Bacterial artificial chromosome)
Artificial plasmids Based on E. coli bacteriophage PAC (P1 phage artificial chromosome)
Based on yeast YAC (Yeast artificial chromosome)

om
l.c
Plasmid :

ai
gm
• Small double stranded circular DNA outside the genome.

@
• A.k.a. episome, epigenome. 55
19
na

Use : Confer antibiotic resistance.

ee
m
ar

DNA insert size : 0.1 - 10 kbp incorporated into vector.

um
itk

Phages (Bacterial virus) :

am

• Viruses that infect bacteria

|
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• Linear DNA
ro
ar

• Chimeric/Recombinant DNA : Vector DNA + target DNA

M
©

Enter host cell

Lytic phase :
• Multiplication of recombinant
DNA insert size : 10 - 20 kbp.

Cosmids :
Plasmids containing cohesive end site/cos site.
Cos site Packs phage DNA into Enables phage cycle
phage particles
Cosmid

DNA insert size : 35 -50 kbp

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 Recombinant Dna Technology 381

Artificial chromosome : ----- Active space -----

Can incorporate large DNA inserts.

DNA insert size :

• BAC, PAC : 50 - 250 kbp
• YAC : 500 - 3000 kbp

Steps of recombinant DNA technology 00:36:51

Isolate specific DNA to clone Identify suitable vector

Sticky ends Complementary to each other.

Both acted on by same restriction enzyme

om
(cuts at palindromic site)

l.c
ai
gm
Synthesis of chimeric DNA (recombinant DNA)

@
19
55
Introduction into host cell
na

cDNA divides inside host cell

ee
m

Bacterial DNA
ar

Forms recombinant clones

um
itk

Plasmid
am
|
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ro

Screening for recombinant vectors : Multiple methods Antibiotic sensitivity (m/c)

ar
M
©

Selection/isolation of specific clones of target DNA from vector.

Antibiotic sensitivity screening :

Target gene

Plasmid Acted upon by Plasmid + Target gene Plasmid

Amp Tet PST I Amp Amp vector
vector vector

Amipicillin Ampicillin : Resistant Can be used

Resistant
Tetracyclin Tetracycline : sensitive for isolation of
colonies with
target gene.

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 382 Molecular Biology

----- Active space ----- Gene library 00:42:19

Collection of recombinant clones from a specific source.

Types :
Genomic DNA library cDNA library
Genomic DNA
Origin mRNA of a gene

Genomic DNA Harvesting mRNA from various cells

Restriction enzymes (Eg. : Beta Islet cells for insulin gene).

Gene A Gene B Gene C Gene D

Reverse transcriptase

om
Complimentary DNA

l.c
ai
gm
Cloned

@
Formation 19
55
na
ee

Cloned
m
ar
um
itk
am
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c DNA c DNA c DNA c DNA

ro
ar

• Used only for small genomes (e.g. : Bacterial). • No introns more meaningful.
M

Features
• Human genome too large. • Insert size : low.
©

CRISPR CAS 9
Clustered regularly interspersed short palindromic sequence associated gene 9
Origin :
Bacterial defence system : Acquired/adaptive immunity. encodes for
Cas endonuclease (Cas 9)
Use :
Novel genome editing technology
• Therapeutic : First FDA approved CRISPR Cas 9 Casgevy (By Vertex
Pharmaceuticals) for treatment of sickle cell anemia.
• Targeted mutagenesis (Create a mutation).
• Gene knock out (Removing a specific gene from genome).
• Modulating gene expression :
- via CRISPR Cas 9.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Recombinant Dna Technology 383

Mechanism : ----- Active space -----

Cas9-gRNA complex : Mutated DNA :

Cas 9 endonuclease

Mutation

gRNA binding site

gRNA (Guide RNA) :
Complementary to mutated region
Step I : gRNA binds to mutated region Unwinding

om
l.c
ai
gm
@
19
55
na
ee

Step II : Cas 9 endonuclease cuts mutated DNA out of genome.

m
ar
um
itk
am
|

Defective DNA
w
ro
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M

Step III : DNA repaired by endogenous mechanism (Non-homologous end joining >
©

homologous recombination) Most crucial step.

Normal DNA without mutation

Note : CRISPR Cas 9 is better than gene therapy as gene rejection absent.

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 384 Molecular Biology

----- Active space ----- AMPLIFICATION AND SEQUENCING TECHNIQUES

Polymerase Chain Reaction  00:00:51

In vitro test tube based technique.

Exponential amplification : Number of amplified products = 2n ; n : Number of PCR
cycles.
Instrument : Thermocycler.
Amplified product : Amplicon.
Flanking sequence Flanking sequence
STEPS
Step 1 : Denaturation

om
2 Primers added

l.c
ai
gm
>90°C temperature

@
Forward Reverse
primer primer 19
55
na
ee

Step 2 : Annealing
m
ar

• 54°C temperature.
um

Bind to 3' end in flanking sequence

itk
am

Extension of primer using

|
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Taq polymerase
ro

Step 3 : Extension
ar
M

• 72°C Temperatur
©

(68-75°C).
• Taq polymerase
required. I cycle of PCR : produces 2 target DNAs.
• Mgcl2, KCl added.

After 1st Cycle PCR

After 2nd Cycle PCR

Exponential amplification
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Amplification & Sequencing Techniques 385

TYPES ----- Active space -----

1. Reverse transcriptase PCR (RTPCR) :

Amplifies any kind of RNA.

Enzyme : Tth polymerase

• Thermostable.
• Derived from Thermus thermophilus (Bacteria dwelling in hot springs).
• Activity :
- DNA polymerase.
- Reverse transcriptase (RNA DNA).

Technique :

om
RNA Index :

l.c
Reverse transcriptase cDNA-complimentary DNA.

ai
gm
RNA

@
cDNA
RNAse H (Removes RNA) 19
55
na

CDNA
ee
m
ar

ds DNA Undergoes PCR

um
itk

2. Real time PCR :

am

• Quantitative PCR (qPCR).

|
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ro

• Simultaneous amplification and detection of amplifical products in exponential

ar
M

phase (Unlike conventional PCR).

©

Methods of quantification :
1) Intercalating dyes : Only bind to double stranded DNA.
Eg :
• Ethidium bromide (Mutagenic).
• SYBR green (More popular).
Less hazardous
2) Sequence specific probes.
Eg :
• Taqman probe.
• Molecular beacon.
• Fluorescence resonance evergy transfer (FRET) probe.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 386 Molecular Biology

----- Active space ----- Mechanism :

Intercalating dye (SYBR Green) : Taqman probe :
dsDNA dsDNA
Denaturation Denaturation
unbound SYBR green SSDNA
SS DNA
SSDNA
SS DNA Taqman Primer
probe
Primer binding Primer and probe
Fluorophore Quencher binding
Fluorophore and

om
Extension
quencher aligned

l.c
ai
Extension (No fluorescence)

gm
dsDNA with bound SYBR green :

@
• Emits fluorescence 55
19
exponentially
na
ee

Fluorophore and
m

Easily detected
ar

quencher not aligned

um

(Emits fluorescence)
itk
am

Ct value :
|
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ro
ar
M

Fluorescence curve
©

RFU
(Relative
fluorescence
unit)
Ct
Threshold value
2 4 6 8 10 12 14 16 18 20 22
PCR cycle number

• Threshold value : • Ct (Cycle threshold) :

RFU value at which PCR cycle number at
fluorescence is which the threshold
detected. value is crossed.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Amplification & Sequencing Techniques 387

Using Ct values to determine relative initial sample DNA : ----- Active space -----
• Sample 1 : Ct1 = 8 • Initial DNA in Sample 2 = N1 = 256 = 32
No. of amplified products Initial DNA in Sample 1 N2 8
(N1) = 2Ct1 = 28 • Interpretation : Sample 2 has 32 times
N1 = 256 mole initial DNA than Sample 1 Early
• Sample 2 : Ct2 = 3 detection of flourescence in Sample 2.
No. of amplified produces
(N2) = 2Ct2 = 23
N2 = 8
1
• Initial DNA ∝
Ct
Applications of PCR :

om
1. Detect and quantify infectious agent : Even if it is latent.

l.c
2. Accurate diagnosis of mutation : Produces DNA fragment for subsequent

ai
gm
analysis by other molecular technique.

@
3. Detect allelic polymorphism :
19
55
• Single base pair changes.
na

• Indels to gene amplification.

ee
m

4. Quantitative RNA analysis :

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• Using RTPCR/rRTPCR (Real-time).

itk

• Used in COVID virus detection.

am
|

3. Droplet digital PCR (dd PCR) :

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3rd generation quantitative PCR.

ar
M
©

Mechanism :
Sample

Turned into droplets

(Each droplet : Micro reactor)

Detection and quantification

of genetic material in each
droplet

Advantage: 
Accurate detection and quantification of low abundant targets.
(↑ amplification & ↑ sensitivity).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 388 Molecular Biology

----- Active space ----- Techniques :

Multiple droplets created
from 1 sample drop

Positive droplets.
(With DNA)

Negative droplets
(Without DNA)
Single DNA
drop from Sample partitioned Multiple PCR reactions
sample into many reactants

om
l.c
Multiple read outs

ai
gm
@
Absolute 19 Negative
55
quantification read outs
na
ee

possible
m
ar

Positive read
um

outs
itk
am
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ro

4. Multiplex PCR :
ar
M
©

Multiple random primers

Denatured DNA

Simultaneous
amplification of
multiple targets using
random primers
Advantage : Time saving.
Disadvantage : Not Specific.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Amplification & Sequencing Techniques 389

5. Nested PCR : ----- Active space -----

• More specific amplification technique .
• Two sets of primers

Outer primer Inner primer

Normal PCR
Amplicon
Technique : 1st set of PCR product
Flanking Target
Target sequence
DNA DNA
Denatured 2nd set of PCR
Outer Inner

9@2nd set of PCR

Denature Primer Primer

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added added

l.c
ai
gm
Undergoes
1st set of PCR 1
55
2nd set of
amplification
na

Amplification
ee

1st set of
m

product
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itk
am
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Mechanism of specificity :
ro
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2nd set of amplification occurs only if outer primer binds to the

M
©

correct target DNA

Multiple specific copies of target DNA

Other Amplification Techniques  00:36:29

1) Based on temperature :

Thermal cycling Isothermal cycling

(Varying temperature) (Constant temperature)

PCR Ligase chain Nucleic acid sequence Branched DNA technique

reaction (LCR) based analysis (NASBA) (bDNA technique)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 390 Molecular Biology

----- Active space -----

2) Based on the component amplified :

Target amplification Probe/primer amplification Signal amplification (Signal

(Target DNA amplified) (Probe/primer attached to attached to target
target DNA amplified) amplified)

PCR NASBA
bDNA technique
LCR Qβ replicase
Multiplex ligation probe amplification (MLPA) :
Annealing of 2 adjacent oligonucleotide probes (MLPA probes) to a segment of
genomic DNA followed by quantitative PCR
Reverse

om
primer

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Forward Probe sequence Sizing

ai
gm
primer (Complimentary to target DNA) sequence
Fluorophore

@
19
55
na

5'ML probe 3'ML probe

ee
m

Can be ligated
ar
um

Uses :
itk

1. Detect small and Large deletions and duplications.

am

2. Detect copy number variation (CNV).

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ro

3. Detect mutations in disorders and prenatal screening.

ar

Eg : α globin gene locus deletion in α Thalassemia.

M
©

Technique :
• MLPA involves complete genome (Multiple segment bind to probe simultaneously)
:
Genomic DNA

Step I : Denaturation

Step II : Hybridisation
(Binding) of both probes

5’ MLPA 3’ MLPA probe

probe Step III : Ligation
of probes

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Amplification & Sequencing Techniques 391

----- Active space -----

Step III : Ligation
of probes

Step IV Amplification & detection.

Second strand for amplification :

Ligated probe

Sequencing Techniques Types  00:46:09

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Types :

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1) Maxam Gilbert sequencing : 3) Pyrosequencing :

gm
@
• Chemical cleavage. • Chain addition techinque.
• Only for small DNA fragments. 19
• More sensitive than Sanger’s
55
na

sequencing.
ee
m

2) Sanger’s sequencing : 4) Next generation sequencing :

ar

• Controlled chain termination. • Most recent.

um
itk

• Can be automated Faster • Multiple samples in a short span of

am

results. time.
|
w
ro
ar

SANGER’S SEQUENCING
M

Invented by Frederick Sanger.

©

Prerequisites :
• Deoxynucleotides. • klenow polymerase.
• Dideoxynucleotides. • Primers.
Principle :

No OH group at 3’ :
Cannot form 3’-5’ phosphodiester bond

Inhibits pairing of further deoxynucleotide

Termination of chain growth

OH group at 3’ position :
Forms 3’-5’ phosphodiester band with nucleotide

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 392 Molecular Biology

----- Active space ----- Procedure : Result :

Contents of test tube : ddNTP Chain termination at
• DNA to be sequenced. dd ATP Any T in sequence
• dNT (ATP/GTP/CTP/TTP). dd GTP Any c in sequence
• dd NT (ddATP/ddUTP/ddCTP/ddTTP). dd CTP Any G in sequence
• Klenow polymerase
dd TTP Any A in sequence
dNTP and ddNTP binds to sequence

Fragments formed at termination

site (No. of base pairs depends on
site of ddNTP attachment)

om
Interpretation of results :

l.c
ai
gm
Position can be determined
1
@
by corresponding dideoxy
2
3 19
55
nucleotides and their
4
na

5 fluorescences.
ee

6
m

Eg :
ar

7
um

8 Position of G : 1, 6, 10 (Yellow
9
itk

10 fluorescence of ddCTP).
am

11
|

12
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Simultaneous sequencing
M
©

Position T A C G
Lane ddA ddT ddG ddc

DNA Ladder :
Contains all the 12 fragments

Capillary electrophoresis :
• Automated sequencing.
• Yields quick results.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Amplification & Sequencing Techniques 393

Pyrosequencing 01:01:45 ----- Active space -----

Chain addition technique.

Principle : Uses :
Sequential addition of nucleotides Detection of :
1. Single nucleotide polymorphism.
Release of pyrophosphate 2. Indels.
3. CNV.
Used to generate light (Via enzymes) for 4. DNA methylations (If bisulphite
detection step is added).
Disadvantage : Only short stretch of DNA can be analysed.
Procedure :
Sample contains one of dNTP :

om
l.c
A G G

ai
C T C T

gm
T C G A A

@
19PPi
DNA primer
55
Does not (Pyrophosphate)
na

bind Used for

ee

+ ATP
dATP dATP
m

Adenosine-5- synthesis of
ar

dATP Luciferase
um

dATP dATP phosphosulphate

dATP
itk

Light
am

Sample pool containing

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dATP Detected by
ro
ar

NEXT GENERATION SEQUENCING

M

Pyrogram
©

• Revolutionised genomic research.

• Massive parallel sequencing in short period of time.
• Entire human genome can be sequenced within a Single day.

Applications :
1) Clinical genetics :
a. Identifies more mutations than simple Sanger’s sequencing.
Eg :
• Substitution • Translocation
• Inversion • Indels
b. Used in population based studies.
c. Study of heterogenous DNA.
2) Microbiology :
Identification of pathogens by genomic definition rather than conventional
characterisation.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 394 Molecular Biology

----- Active space -----

3) Oncology : Large scale cancer genome profiling

Patient tailored therapeutics.

Hybridoma Technique 01:15:35

• Developed by Georges Kohler and Caeser Milstein in 1975.

• Technique of synthesizing monoclonal antibody.
Clinical application :
Diagnostic : Therapeutic :
Eg : ELISA. Eg : Rituximab, Trastuzumab.
Technique :
Antigen Multiple myeloma cells :

om
Formation of antibodies • Immense replicating potential.

l.c
ai
specific to antigen • HGPRTase negative (Depends on

gm
@
Isolation of B-cells other cells for salvage pathway).
19
55
na
ee
m
ar
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itk
am
|
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ro
ar

Fusion of B-lymphocytes & multiple myeloma cells

M

by polyethylene glycol (PEG)

©

Transferred to HAT medium

Only fused cells survive HAT medium :
Contains :
Unfused MM cells Unfused B-cells • Hypoxanthine
die d/t lack of die d/t limited Test for specific • Aminopterine (Folate
salvage pathway replication power antibody antagonist) :
- Blocks de novo purine
synthesis
Expansion of positive clones
2 methods • Thymidine

In vitro In vivo : Injected into mice

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Mutation 395

MUTATION ----- Active space -----

Mutation : Permanent change in primary nucleotide sequence regardless of its

functional consequence.
• Incidence : <1% of population.
Polymorphism : • Incidence >1% of population.
• Normal DNA variations.
Epigenetics : • Chemical modifications of DNA/chromosomes.
• Reversible.
• Heritable.
• No change in nucleotide sequence.

om
l.c
Types of mutations

ai
00:02:28

gm
@
Mutations :
19
55
Class Group Type
na
ee

Synonymous
m

• No change in encoded amino Silent

ar
um

acid/ polypeptide sequence.

itk

Missense
am

Base
• Different amino acid coded.
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substitution
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• E.g. Sickle cell anemia (Glutamic

• M/C mutation Non-synonymous
ar

acid Valine).
M

• Change in encoded amino acid.

©

Nonsense
• Stop codon coded.
• E.g. UGA, UAG, UAA.
TYPES OF MISSENSE MUTATIONS
A. Based on functionality of protein coded :
1. Acceptable missense mutation :
• No clinical symptoms
β chainExample :
Codon Amino acid Protein
Normal AAA or AAG 61 position : Lysine
st
Normal Hb
Hb Hikari
Altered AAU or AAC Aspartic acid
• No clinical symptoms.
• Altered electrophoretic mobility.
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 396 Molecular Biology

----- Active space ----- 2. Partially acceptable missense mutation :

• Clinical symptoms present.
• No change in function of encoded protein.
Example :
β chain Codon
Amino acid Protein
6th position :
Normal GAA or GAG Normal Hb
Glutamic acid
HbS Carries O2
Altered GUA or GUG Valine
Sickling of RBC Hemolysis
(Symptomatic)

om
3. Unacceptable missense mutations :

l.c
Loss of function of protein coded

ai
gm
Example :

@
α chain Codon Amino acid 19 Protein
55
na

E7 Histidine
ee

Normal CAU or CAC • Give 6 valences to Fe2+. Normal Hb

m
ar

• 6th valency Holds O2.

um
itk

E7 Tyrosine Meth Hb (Hb M)

am

Altered UAU or UAC • Loss of 6 valency in Fe ex : HbM Boston

th 2+.
|

• Cannot bind to O2 • Cannot carry O2

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ro
ar
M

Note :
Base substitutions
©

Transition Transversion
• 1 purine another purine • Purine Pyrimidine
• 1 pyramidine another pyramidine e.g. GAG GUG

Adenine Uracil
(Purine) (Pyrimidine)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Mutation 397

B. Based on newly coded amino acid : ----- Active space -----

Missense mutations

Non conservative/ Non homologous Conservative/ Homologous

1 amino acid 1 amino acid

Replaced by Replaced by

Amino acid with different Similiar amino acid

characteristics Ex : • Aspartic acid Glutamic acid
Acidic (Both acidic and polar)
Ex : Glutamic acid
Polar • Valine Isoleucine

om
(Both branched chain & non polar)

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ai
Branched chain amino acid

gm
Valine
Non polar
@
19
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na

Insertion, Deletion and Nonsense Mutations

ee

00:16:40
m
ar

Insertion : Addition of nucleotide/s to sequence.

um

Deletion : Removal of nucleotide/s from sequence.

itk
am

Frameshift mutation : Alteration of reading frame.

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Insertion Deletion
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ar

Non Non
M

Frameshift Frameshift
©

frameshift frameshift
Number Multiples Multiples
of bases of 3 (Entire of 3 (Entire
Non multiples of 3 Non multiples of 3
added/ codon codon
removed added) removed)
Normal mRNA : Normal mRNA :
AUG CAA UGG UUA GCA UUU AUG CAA UGG UAA CCA UUU
Normal protein : Normal protein :
Met-Gln-Trp-Leu-Ala-Phe Met-Gln-Trp-Leu-Ala-Phe
Examples
Mutant mRNA : Mutant mRNA :
AUG -CAA-UGG-UCU-AGC-AUU AUG-CAA-UGG-UAG-CAU
Mutant protein : Mutated protein :
Met-Gln-Trp-Serine-Serine-Ile Met-Gln-Trp-Stop

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 398 Molecular Biology

----- Active space ----- Non sense mutation :

Base substitution Causes Premature termination of amino acid sequence
Example
β chain Codon Amino acid Protein
Normal UAC Tyrosine Normal Hb
Altered UAG Stop codon Thalassemia
Note : If insertion/ deletion causes formation of stop codon NOT a
nonsense mutation

Mutation Detection Techniques 00:24:17

Methods :

om
l.c
Detects
Numerical (aneuploidy) or

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1. Cytogenetic analysis

gm
structural abnormality in

@
19 chromosome
55
Karyotyping Fluorescent in situ
na

hydridisation (FISH)
ee
m
ar

2. Methods detecting point mutations/ Small deletions/Insertions :

um
itk

Methods Remarks
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a. DNA Sequencing : Gold standard

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Expensive
ro
ar

Sanger’s sequencing (M/C)

M
©

b. Restriction Fragment Length If mutation creates/abolishes a

Polymorphism (Low sensitivity) restriction site
Detects alterations in
c. DNA Electrophoresis techniques electrophoretic mobility d/t
mutation
d. Oligonucleotide Specific Hybridization
-
(OSH)
e. RNA ase cleavage -
Genotyping of SNP
f. Micro Array (DNA chip)
(Single Nucleotide Polymorphism)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Mutation 399

Procedures : ----- Active space -----

Procedures to detect

Alteration in DNA sequence Alterations in length of DNA

Ex : Small insertions/ deletions/ point mutations Ex : • Copy number variations.
(like HbS) • Triplet nucleotide repeats.
Initial procedure : PCR
Causes
Amplicon length Real time PCR Multiplex ligation
Amplification of DNA analysis (quantitative) probe analysis
Next (MLPA)

DNA sequencing techniques Restriction

om
l.c
Fragment

ai
gm
Length
Pyrosequencing Sanger’s

@
polymorphism
sequencing 19
55
(RFLP)
na
ee
m
ar
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itk
am
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ro
ar
M
©

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 400 Organ Function Tests

----- Active space ----- LIVER FUNCTION TESTS

Functions of Liver and Classification of LFT  00:02:02

FUNCTIONS OF LIVER

Synthesis : Metabolic pathways :

• Plasma proteins. • Glycolysis.
• Glucose. • FA oxidation.
• Cholesterol. • TCA cycle.

om
• TAG. • Protein catabolism.

l.c
ai
• Lipoprotein (VLDL, HDL).

gm
@
Liver
55
19
na

Detoxification and conjugation

ee
m

Storage : (Excretion) :
ar
um

• Fat soluble vitamins. • Bilirubin.

itk

• Vitamin B12 • Cholesterol Bile acids.

am

(Water soluble vitamin). • Drugs (Xenobiotic reactions).

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ar
M

CLASSIFICATION OF LFT
©

Blood investigations Liver enzyme profile Urine investigations Special investigations

Detoxification : Hepatocellular injury : • u. Bile salt • s. Ceruloplasmin
• s. Bilirubin • s. ALT • u. Bile pigment • Alpha fetoprotein
Transaminases
• Blood ammonia • s. AST • u. Urobilinogen • Alpha-1-antitrypsin
Synthetic: Cholestasis: • Ferritin
• s. Albumin • s. ALP • Iron
• Prothrombin time • s. Gamma glutamyl • Transferrin
transferase
• s. 5’ Nucleotidase

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Liver Function Tests 401

Blood Investigations 00:08:33 ----- Active space -----

SERUM BILIRUBIN
Normal levels :
• Total : 0.2-0.8 mg/dL.
• Conjugated : 0-0.2 mg/dL.
• Unconjugated : 0.2-0.6 mg/dL.

Van den Bergh Reaction :

Method :
Diazotized sulfanilic acid + Bilirubin Azobilirubin (Purple colored complex).
(Sulfanilic acid in HCl
and sodium nitrate)

om
l.c
ai
Reactions :

gm
@
19 Diazotized sulfanilic acid
55
na

+ Sample
ee
m
ar
um
itk
am

Colour
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change
ro
ar

Immediate color change Color on adding alcohol Color deepens on adding alcohol
M
©

Reaction Direct reaction Indirect reaction Biphasic reaction

Indicated Indirect/unconjugated Both conjugated &
Direct/conjugated bilirubin
bilirubin bilirubin unconjugated bilirubin

Interpretation :
Bilirubin
Jaundice Cause/Reaction
Total Direct Indirect
↑Hemolysis
Pre-hepatic • Sickle cell anemia ↑ N ↑
• Hereditary spherocytosis
Hepatic Biphasic reaction ↑ ↑ ↑
Post hepatic/ Obstruction : Conjugated
↑ ↑ N
obstructive bilirubin accumulation and spill

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 402 Organ Function Tests

----- Active space ----- Hepatic jaundice :

• Early stage : Predominant ↑indirect bilirubin.
• Later stages :
Biliary canaliculi obstruction (D/t inflammation) ↑Direct bilirubin.

SERUM ALBUMIN
Significance :
• Assess synthetic function :
- All plasma proteins are synthesized by liver.
- Exception : Immunoglobulin (Synthesized by B lymphocytes).
• Most abundant plasma protein.
• Long half life : 20 days.
- Chronic liver disease ↓Albumin level.

om
- Differentiate b/w acute & chronic condition.

l.c
ai
gm
A/G Ratio :

@
• Normal albumin : 3.5-5 g/dL 19
Normal Albumin Globulin ratio : 1.5-2.5.
55
• Normal globulin : 2-3.5 g/dL
na
ee
m

Reversal of A/G ratio :

ar
um

• Liver disease : ↓Albumin, N globulin.

itk

• Multiple myeloma (Plasma cell neoplasia) : ↑γ globulin.

am
|

PROTHROMBIN TIME (PT)

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• Clotting factors : Synthesized in liver.

ar
M

• Normal : 9-11 sec.

©

• Liver disease ↓Clotting factor ↑PT.

Liver Enzyme Profile 00:26:50

TEST FOR HEPATOCELLULAR INJURY

Enzyme & reaction Normal value Significance Raised in
s. ALT (SGPT) • Very high levels :
Alanine transaminase Acute hepatitis (Infection/toxins)
More • Mild-moderate ↑ :
Alanine ALT Pyruvate 10-35 IU/L
specific - Chronic hepatitis
α ketoglutarate Glutarate - Cirrhosis
- Hepatocellular carcinoma (HCC)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Liver Function Tests 403

----- Active space -----

Enzyme & reaction Normal value Significance Raised in

s. AST (SGOT) • Alcoholic liver disease :
Aspartate transaminase AST > ALT ↑
Aspartate AST Oxaloacetic acid 8-25 IU/L Less specific • Hepatitis
• Cirrhosis
α KG Glutamate • HCC
AST/ALT ratio 0.8 - >2 : Alcoholic liver disease

TESTS FOR CHOLESTASIS

TESTS FOR CHOLESTASIS
Normal

om
Enzyme & function Significance Raised in
levels

l.c
ai
• α 1 ALP : Specific marker of

gm
@
obstruction • Very high level : Cholestasis
19
• α2 heat labile ALP : Specific
55
s. ALP - Intrahepatic
na

Alkaline phosphatase : 40-125 marker of liver injury - Extrahepatic

ee
m

Removal of PO4 group IU/L • Non-specific : Secreted by • Mild-moderate ↑ :

ar
um

in alkaline medium many other sites - Hepatitis

itk

- β ALP : Osteoblast - HCC

am

- γ ALP : Intestine
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Alcohol intake
ro
ar
M
©

s. GGT Microsomal (SER) injury : • Isolated ↑ :

10-30
Gamma glutamyl GGT migrates to plasma Alcohol consumption
IU/L
transferase membrane • ↑GGT + ↑ALP : Cholestasis

Released to blood
• Enzyme in plasma • Highly ↑ : Obstructive liver
2-10
s. 5’ Nucleotidase membrane disease
IU/L
• More specific than ALP • Moderately ↑ : Hepatitis

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 404 Organ Function Tests

----- Active space -----

ALGORITHM OF ENZYMES IN LIVER DISEASE

Liver enzymes

• ALT ↑↑ • ALT N
• AST ↑ • AST N
• ALP N • ALP ↑↑

AST/ALT Obstructive liver disease

>2 <2

om
Alcoholic liver disease Hepatocellular injury

l.c
ai
gm
s. Albumin
@
19
55
na

N ↓
ee
m

Acute condition Chronic condition

ar
um
itk

Urine Tests 00:46:48

am
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Test Procedure Interpretation

ro
ar
M

Sprinkle sulfur powder

©

Bile salt :
Obstructive jaundice
Hay’s test
Sample Powder sinks d/t detergent
action of bile salts
Filter paper with barium sulfate precipitate Conjugated bilirubin
Bile pigment : + Fouchet’s reagent in urine : Choluria
Fouchet’s test
Bluish green color (Positive). Obstructive jaundice

Urine + Ehrlich reagent

Hemolytic/
urobilinogen :
prehepatic jaundice
Ehrlich test Sample Pink color
(Urobilinogen)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Liver Function Tests 405

Obstructive Liver Disease : Hemolytic Jaundice : ----- Active space -----

RBC lysis
Obstruction Hemolysis :
↑↑Bilirubin
Portosystemic
Bile salt pigments spill shunt :
to systemic circulation ↑Conjugated ↑↑Urobilinogen
bilirubin in liver in circulation
Enterohepatic
circulation Excreted
by
kidney
↑↑Urobilinogen UB
in intestine

om
l.c
↑Urobilinogen

ai
Excreted in urine in urine

gm
@
19
55
na

Note :
ee
m

• Unconjugated bilirubin ↑ : Water insoluble Not excreted in urine.

ar

• Ehrlich’s test : Negative in obstructive jaundice

um
itk
am

Conjugated bilirubin Intestine.

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M

SUMMARY
©

Jaundice Hay’s test Ehrlich’s test Fouchet’s test

Prehepatic +++
Hepatic ± ± ±
Posthepatic +++ +++

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 406 Organ Function Tests

----- Active space ----- Summary 00:58:20

Jaundice :

Prehepatic : Hepatic : Post hepatic :

Hemolysis : • :
• • Cirrhosis • CBD/Gallbladder
• • Cancer of head of pancreas
• G6PD deficiency

INTERPRETATION OF RESULTS

om
Jaundice

l.c
Test Parameter

ai
Prehepatic Hepatic Posthepatic

gm
Total bilirubin ↑ ↑ ↑
@
Direct bilirubin 19 Biphasic ↑↑
55
na

Blood tests Indirect bilirubin ↑↑ reaction

ee
m

s. Albumin
ar
um

Prothrombin time
itk

Liver s. ALT N N
am

enzyme s. AST N N
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panel
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s. ALP N ↑↑↑
ar
M

u. Bile salt (Hay’s) - +/-

©

Urine tests u. Urobilinogen (Ehrlich’s) +/- -

u. Bile pigment (Fouchet’s) - +/-

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Renal Function Tests 407

RENAL FUNCTION TESTS ----- Active space -----

Renal functions & RFT Classification  00:01:30

Functions of Kidney :
Excretion : Hormone :
• Water • Erythropoctin
• Salt • Activation of Vit D :
• Metabolic waste 25-HydroxyCholecalciferol
1α hydroxylase

om
Homeostasis : 1,25-Dihydroxy cholecalciferol

l.c
ai
• Acid base balance Metabolic :

gm
@
• Electrolyte balance • Gluconeogenesis
19
55
na

Classification of Renal Function Tests (RFT) :

ee
m
ar
um

To screen for renal disease : To assess renal function :

itk

• Complete urine analysis. • Glomerular function tests :

am

• Serum electrolytes. - GFR. - Permeability.

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• Tubular function tests.

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ar
M

Urine Analysis 00:05:22

©

Physical Characteristics :
Character Normal Abnormal
• Polyuria: >3000 mL/d.
Volume 1.5 L/d • Oliguria: <400 mL/d.
• Anuria : <100 mL/d.
• Yellow : Jaundice.
• Red : Blood in urine.
Color Amber
• Reddishbrown : Hemoglobin in urine.
• Black : Alkaptonuria.
Odor Aromatic -
Specific gravity 1.015 - 1.025 (Measure of solutes) -
PH 5.5 - 7.5 -

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 408 Organ Function Tests

----- Active space ----- Chemical Characteristics :

Abnormal constituents : Normal constituent :

• Sugar. Urobilinogen.
• Ketone bodies.
• Bile salt.
• Bile pigment.
• Blood.

S. Creatinine, S. Urea, BUN 00:09:30

SERUM CREATININE
Creatinine Phosphate Creatinine

om
(Muscle) (Excreted in urine).

l.c
ai
gm
Normal level : 0.7 - 1.4 mg/dL

@
Method of Estimation : 19
55
na
ee

Jaffe’s reaction (Chemical method) : Enzymatic method :

m

• M/c
ar

Creatininase enzyme used.

um

• Procedure : Alkaline medium

itk

(Sample + Picric acid)

am
|
w
ro

Creatine picrate : Tautomer

ar
M
©

Color : Orange Red

Color measured by colorimeter.

Increased Creatinine :
• Glomerulonephritis.
• Pyelonephritis.
• Renal failure.
• Obstruction to urinary tract.

BLOOD UREA
• End product of protein catabolism : Disposes toxic NH3.
• Synthesized in liver Excreted in urine.
Normal level : 20 - 40 mg/dL.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Renal Function Tests 409

Pre-renal factors affecting estimation : ----- Active space -----

• ↑Protein intake.
• ↑Protein breakdown (Eg : Fever).
• Dehydration.

Method of estimation :
• Chemical method : M/c (Diacetyl Monoxime method).
• Enzymatic method.

Abnormal Urea :

↑ Urea : ↓ Urea :
• ↓Protein intake.

om
Pre-renal : Renal : Post-renal : • Hepatic failure

l.c
• Acute glomerulo (↓Synthesis of urea).

ai
• Dehydration Obstruction d/t

gm
• ↑Protein intake nephritis. • Stones. • Overhydration.

@
• ↑Protein breakdown • Pyelonephritis. 19
• Strictures.
55

• Renal failure. • Prostate :

na
ee

- Enlargement.
m
ar

- Tumor.
um
itk

BLOOD UREA NITROGEN (BUN)

am

Urea expressed in terms of nitrogen.

|
w
ro

Calculation :
ar
M

• Molecular weight Urea : 60 mg.

©

Nitrogen : 28 mg s. Urea
BUN X 2.14 = s. Urea BUN =
Urea (wt) 60 2.14
• = = 2.14
Nitrogen (Wt) 28
Azotemia :
• ↑s. Urea + ↑s. Creatinine.
• Seen in renal failure.

NPN (Non Protein Nitrogen substances) :

• Urea.
• Uric acid.
• Creatinine.
• Urobilinogen.
• Indican.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 410 Organ Function Tests

----- Active space ----- Clearance Test 00:24:20

Markers of

Glomerular filtration : Glomerular permeability :

Clearance test. Proteinuria.
FILTRATION
• Functional unit : Nephron.
• Blood flow : Afferent arteriole Glomerular capillaries Efferent arteriole.
Renal plasma flow :625 mL/min
1. Ultra filtration of renal plasma :
Glomerular Bowmans

om
l.c
capillaries capsule

ai
gm
GFR : 125 mL/min = 170-180 L/day

@
2 Reabsorption : Tubules Blood 19
55
na

3. Secretion : Blood Tubules

ee
m

Urine : 1.5 L/day

ar
um

4. Excretion : Filtration - Reabsorption + Secretion.

itk
am
|
w
ro
ar
M
©

Process of filtration

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Renal Function Tests 411

CLEARANCE ----- Active space -----

• Volume of plasma cleared of a substance per unit time.
• Expressed as mL/min.

Calculation :
Cs : Clearance of substance ‘s’
UXV U : Concentration of substance in urine
Cs =
P P : Concentration of substance in blood
V : Urine flow rate (Volume in mL/min)

Ideal Substance for Clearance Test :

• Completely filtered Endogenous :
• Neither reabsorbed nor secreted • Creatinine (M/c) : Near ideal.

om
• Excreted only through urine • Urea.

l.c
• Constant blood levels

ai
Exogenous :

gm
• Inulin (Gold standard) : Ideal.

@
19
55
na

Creatinine Clearance :
ee
m

Method :
ar
um

• Give 100 mL water.

itk

• After 30 min, emply bladder (Discard).

am

• After 60 min, void and collect all urine.

|
w

• Note urine volume.

ro
ar

• Collect a blood sample.

M

• U, V, P measured.
©

Calculation :

Uncorrected : Corrected :
UXV U X V X 1.73
Cc = Cc =
P PXA
• Corrected to body surface area.
• Used for children, tall/short people.
• A : Body surface area.
• 1.73 : Average body surface area of an adult.

Normal value :
• Males : 95 - 115 mL/min
10% secreted (Slightly lower than GFR).
• Females : 85 - 110 mL/min

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 412 Organ Function Tests

----- Active space ----- Advantages & disadvantages :

Advantages Disadvantages
• Endogenous substance
• Near ideal :
• 10% secreted tubular component.
- completely filtered
• Creatinine blind area :
- Not reabsorbed
- Cannot measure slight ↓GFR.
- Small amount secreted
- 40 - 70 mL/min/1.73
• Formed spontaneously (No enzyme required)
• Constant Blood values : Proportional to muscle mass
Creatinine coefficient :
• Urinary creatinine expressed in mg/kg body weight.

om
• Males : 20 - 28 mg/kg.

l.c
• Females : 15 - 21 mg/dL.

ai
gm
@
Estimated GFR/eGFR : 19
55
na

Indication : Chronic kidney disease patients.

ee

Advantage : Timed urine collection not required.

m
ar
um

Cockcroft Gault equation :

itk
am

eGFR (mL/min) = 140 - Age (yrs) X weight (kg) X 0.85 (If female)
|

72 x s. Creatinine (mg/dL)
w
ro
ar

MDRD :
M

• Modification in Diet in Renal Disease.

©

• Better measure.
• More complicated.

Cystatin C :
• Cysteine protease inhibitor.
• Newer marker for GFR.

Advantage : Advantage over creatinine:

• Completely filtered. • Does not depend on age, sex or
• Neither reabsorbed nor secreted. muscle mass.
• Detect ↓GFR in creatinine blind area.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Renal Function Tests 413

Urea Clearance Test : ----- Active space -----

Volume of plasma cleared of urea/mL per unit time.

Less preferred :
• Clearance affected by pre-renal factors.
• Urea is reabsorbed (Curea< GFR).

Calculation :
• Maximum urea clearance :

Curea =

- Normal 75 mL/min
• Standard urea clearance :

om
l.c
- When urine flow rate <2 mL/min.

ai
gm
U X √V

@
Curea =
P 19
55
na

- Normal : 54 mL/min.
ee

• Inulin :
m
ar

- Gold standard.
um

- Polymer of fructose.
itk
am

Advantage : Disadvantage :
|
w

• Completely filtered. Extraneous/Exogenous substance.

ro
ar

• Neither reabsorbed nor secreted.

M
©

Normal value : 125 mL/min (Same as GFR)

Glomerular Permeability Test 00:52:08

Urinary protein concentration :

Normal value : <150 mg/day (insignificant) :
Albumin : <30 mg/day.
α1 microglobulin.
Tam Horsfall protein/Uromodulin :
• Most abundant protein in human urine.
• Produced from thick ascending loop of henle.

↑Protein : ↑Glomerular permeability (As normally proteins are not filtered).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 414 Organ Function Tests

----- Active space ----- PROTEINURIA

Types of Proteinuria :
Proteinuria Pathology Condition
• Nephrotic syndrome :
Anasarca (Generalised edema)

• ↑Vascular permeability
Glomerular Hypoalbuminemia Proteinuria >3g/day
• Damaged glomerulus
• Acute glomerular nephritis
• Nephropathy :
- Diabetes mellitus
- Hypertension

om
↓Tubular reabsorption

l.c
ai
• Fanconi syndrome

gm
Tubular
Low molecular weight protein • Nephrotoxic drugs
@
present in urine. 19
55
na

• Hemoglobinuria : ↑Hb
ee

Excess low molecular weight

• Myoglobinuria : ↑Myoglobin
m

Overflow
proteinuria
ar

• Multiple myeloma : BJP (Bence Jones Proteins)

um
itk

Urogenic Inflammation in lower urinary tract Urinary tract infections

am

Microalbuminuria :
|
w

• Minimal albumiuria/Paucialbumiuria : 30 - 300 mg/day (Normal : <30 mg/d)

ro
ar

• Not detected by usual tests.

M
©

Inference : Early damage to glomerular permeability.

• Early Diabetic nephropathy.
• Early hypertensive nephropathy

Significance : Reversible at this stage on Rx.

Significant albuminuria : >300 mg/day

ALBUMIN-CREATININE RATIO :
Concentration of albumin (mg/dL)
A : C (mg/g) = x 1000
Concentration of creatinine (g/dL)
Normal value :
• Males : <23 mg/g of creatinine.
• Females : <32 mg/g of creatinine.
Advantage : Spot urine collection (24 hr urine collection not required).
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Renal Function Tests 415

BENCE JONES PROTEINS ----- Active space -----

• Low molecular weight proteins :
- Light chain of immunoglobulin.
- kappa or lambda.
• Overflow proteinuria : Seen in plasma cell neoplasia (Multiple myeloma).

Tests for BJP :

• Heating test : Urine in test tube is heated

Forms precipitate (45-60°C)

Further heating dissolves precipitate

om
On cooling precipitate appears (Around 60°C).

l.c
ai
• Dipstick test.

gm
@
Tubular Function Tests 19 01:06:00
55
na
ee
m

Measurement of Concentration Water dilution Urinary acidification

ar
um

specific gravity tests

itk
am

SPECIFIC GRAVITY MEASUREMENT

|

• Measurement of concentration of solute in solution (Urine).

w
ro

• Reflects the Ability of tubules to concentrate urine.

ar
M
©

Instrument : Urinometer.
Normal value : 1.015 - 1.025.

Note : Osmolarity Concentration of osmotically active substance in a solution

Interpretation :
Increased Decreased
• Overhydration
• Diabetes insipidus
• Severe dehydration
• Chronic renal conditions :
• Diabetes Mellitus
- Chronic glomerulonephritis
- Chronic renal failure

Isosthenuria :
Plasma osmolarity = Urine osmolarity (Fixed urine specific gravity : 1.008 - 1.014)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 416 Organ Function Tests

----- Active space ----- Cause :

• >75% tubular function loss.
• Chronic renal failure.
• Overuse of diuretics.

CONCENTRATION & DILUTION TEST

Principle Procedure
Fluid intake restricted for 15 hrs
↓Water intake
Concentration Collect early morning sample
↑Tubular reabsorption of water
test
Measure specific gravity : >0.125.
Concentrated urine.

om
(Concentrated urine)

l.c
↑Water intake After emptying bladder, 1200 mL water is given

ai
gm
@
Dilution test ↓Tubular reabsorption of water 55 Collect 4 hourly urine separately
19
na

Dilute urine. Measure specific gravity : <1.003 (Diluted urine).

ee
m
ar

URINE ACIDIFICATION TESTS

um

Acid Loading Test :

itk
am

Principle :
|

• ↑Excretion of H+.
w
ro

• ↑Reabsorption of HCO3..
ar
M
©

Procedure :
• Give NH4C1 capsule orally.
• Collect urine hourly for 8 hours.
• Detect pH.
NH4CL NH4+ + Cl-
Detoxified by liver H+ HCl
Urea (↓Urinary pH : <5.5)
C/I : Hepatic failure.

Note : Acid Base balance maintained by

• Blood buffer.
• Renal system.
• Respiratory system

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Renal Function Tests 417

Summary 01:20:20 ----- Active space -----

DERANGED RFT CASES

Nephrotic syndrome :
• M/c affects children.
• Nephrotic range proteinuria Hypoalbuminema Edema.
• Dyslipidemia : ↑ total cholesterol, ↑TAG.

Acute glomerulonephritis/Nephritic syndrome :

• Hematuria.
• Proteinuria.
• Hypertension.
• Predisposing factor

om
(Eg : Streptococcal infection : skin/respiratory).

l.c
ai
gm
Acute renal failure :

@
• Edema (Puffiness of face).
19
55
• Oliguria or anuria.
na

• Sudden onset :
ee
m

- Conditions causing severe dehydration.

ar
um

- Drugs (Eg : NSAIDs).

itk

- Severe bacterial infection.

am

• Nausea/Vomiting.
|
w
ro

Chronic Renal failure :

ar
M

• Edema (Puffiness of face).

©

• Oliguria or anuria.
• Slow Onset :
- Diabetes mellitus.
- Hypertension.
RFT Summary :

Screening for renal disease : Glomerular function tests : Tubular function tests :
• Complete : Clearance test : •
- Physical characteristics. • clearance. • Urine
- Chemical characteristics. • clearance. • Urine
• s. • Urine
• s.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 418 Organ Function Tests

----- Active space ----- THYROID FUNCTION TESTS

Thyroid Hormones  00:00:34

Thyroid Gland :
Butterfly-shaped gland located in the front of the neck.

Thyroid Hormone Synthesis :

om
Thyroid gland

l.c
Thyroglobulin : Present in thyroid follicle.

ai
gm
Contains tyrosine residues Iodination Thyroid hormone.
@
19
55
Blood Epithelial cells of thyroid follicle Thyroid follicle
na
ee
m
ar

Na+ I- transporter
um

Na+ Na+ 2 Pendrin -

itk

1 I TPO I
am

I-
I- 3
I-
|
w

I-
ro

I-
ar

Thyroglobulin (Tg) Exocytosis

M

TSH TSHR Nucleus Tg

©

T3, T4 Excreted
T3, T4

Site of Iodination :3rd & 5th position of phenol

Anterior Pituitary ring in tyorsine residues

TPO
4 Organification : I + Tyrosine TPO MIT
2I + Tyrosine 3,5 DIT/DIT

MIT + DIT TPO Triiodothyronine (T3)

TSH : Thyroid stimulating 5 Coupling : TPO
hormone DIT + DIT Tetraiodothyronine (T4)
TSHR : Thyroid stimulating
hormone receptor
MIT : Mono iodo-tyrosine
DIT : Di-iodo tyrosine
TPO : Thyroid peroxidase
Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024
 Thyroid Function Tests 419

Regulation of Thyroid Hormone Synthesis : ----- Active space -----

Occurs via hypothalamus-pituitary-thyroid axis.
Hypothalamus
-
TRH
+
Negative feedback Pituitary
-
TSH
+
Thyroid gland
T3, T4

om
Formation of T3 & T4 :

l.c
Organification :

ai
gm
Addition of I- to the phenol ring of tyrosine in thyroglobulin.

@
19
55
OH OH OH
na

I I I
ee
m
ar
um
itk
am

Thyroglobulin (Tg) Monoiodotyrosine Di-iodotyrosine

|
w
ro
ar

Coupling :
M
©

OH OH
I I
I I

O
I I
DIT
+
OH
I I
3,5,3’,5’ Tetraiodothyronine (T4) :
• Prohormone.
• 80% of thyroid hormone produced.

DIT

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 420 Organ Function Tests

----- Active space ----- OH

I I
OH
I H

MIT
O
I I
+
OH
I H
3,5,3’ Tri-iodo thyronine (T3) :
• Active hormone.
• 20% of thyroid hormone produced.

om
DIT

l.c
ai
gm
@
Peripheral deiodination :
Deiodinase 19
55
T4 T3
+ Selenium
na
ee
m

Deiodinase : Selenocysteine containing enzyme

ar
um
itk

Thyroid Function Test 00:13:47

am
|
w

Anterior pituitary hormone : S. TSH

ro
ar
M

Thyroid hormone :
©

• S. total T3, T4.

• S. free T3, T4(FT3, FT4).

Antithyroid antibodies :
• Anti TPO (Thyroid peroxidase).
• Anti thyroglobulin.
• TRAb (TSH receptor stimulating antibody).

Others :
• Radioactive iodine uptake.
• TRH response.
• S. thyroglobulin.
• S. thyroid binding globulin.
• S. cholesterol (Non-specific).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Thyroid Function Tests 421

TSH ----- Active space -----

• Trophic hormone.
• Glycoprotein with 2 subunits : α, β.
- α : Non-specific (Common to LH, FSH, hCG).
- β : Specific.
• Reliable marker of TFT.
- Marked variation with minimal change in thyroid hormone.
- N TSH : Rules out thyroid dysfunction.
• Diurnal variation :
- Peak : Early morning.
- ↓ at noon.
Normal range : 0.35 - 4.50 mIU/mL.

om
l.c
THYROID HORMONES

ai
gm
@
Characteristics Normal range
19
55
Total T4 • Total molar concentration of T4 = 100 x T3 4.6 - 10.5 µg/dL
na
ee

• 0.02% of total T4 , 3 x FT3

m

Free T4 • Correlates with the clinical status of the 0.8 - 2.7 ng/dL
ar
um

patient
itk
am

20% : Direct secretion

|

Total T3 80% : From peripheral conversion 70 - 204 ng/dL

w
ro

• More accurate, less expensive than FT3

ar
M

• 0.3% of total T3
©

Free T3 210 - 440 pg/dL

• Unbound & bioactive form

rT3 (Reverse T3) :

• Deiodination by D3 (Isoform of deiodinase) at 5th position.
• Biologically inactive.

ANTI-THYROID ANTIBODIES

Anti TPO
Anti Tg Autoimmune thyroiditis (Hashimoto’s).

TRAb : Autoimmune thyroiditis (Grave’s disease).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 422 Organ Function Tests

----- Active space ----- OTHER TESTS

TRH Test :
Distinguishes b/w pituitary or hypothalamic causes.

Procedure :
TRH administration

TSH N No response

Hypothalamic cause Pituitary cause

Radioactive Iodine Uptake (RAIU) :

RAIU = Total counts administered

om
Counts measured by scanning

l.c
ai
Iodine uptake by thyroid gland

gm
@
19
55
↑ ↓ Elsewhere
na
ee

Anatomical defect :
m

Hyperactive thyroid Hypoactive thyroid

ar

Ectopic or hemithyroid
um
itk

Radioisotopes : Tc-99, I- 123, I- 131.

am
|
w
ro
ar
M
©

A. Normal B. Graves disease

C. Toxic multinodular goitre D. Toxic adenoma

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Thyroid Function Tests 423

Thyroglobulin : ----- Active space -----

Follicular cells of thyroid Direct exocytosis Non-iodinated thyroglobulin.

Normal level : 3 - 40 ng/mL.

Cholesterol :
• Hypothyroidism :
↑Cholesterol levels (D/t ↓degradation of cholesterol carrying lipoprotein).
• Not diagnostic.

Disorders of Thyroid Gland 00:36:05

om
Hyperthyroidism Hypothyroidism

l.c
ai
gm
@
Primary Central Subclinical
19
55
(Secondary/tertiary)
na
ee

HYPOTHYROIDISM
m
ar
um

Central
Primary Subclinical
itk

Secondary Tertiary
am

Disorders affecting
|
w

thyroid gland :
ro
ar

• Autoimmune thyroiditis
M

Autoimmune
©

(Hashimoto’s) Hypothalamic cause

Causes Pituitary cause thyroiditis :
• Thyroidectomy (Rare)
Minimal symptoms
• Radiation exposure
• Iodine deficiency
• Drugs
T4, fT4 ↓ ↓ ↓
Low normal
T3 ↓ - -

↓ (D/t interrupted ↓ (D/t interrupted

↑↑ (D/t ↓ Negative
TSH feedback loop at feedback loop at ↑↑
feedback)
pituitary) hypothalamus)

Antibodies : TRH response : TSH response :

Others -
Anti TPO, Anti Tg Low (No response) + (↑ TSH)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 424 Organ Function Tests

----- Active space ----- Clinical Features :

• In children : Cretinism.
• In adults : Myxedema.

Symptoms & Signs :

• Weight gain. • Delayed ankle jerk.
• Dry coarse skin. • Enlarged thyroid gland : Goitre.
• Cold intolerance • Bradycardia.
• Slow response, sluggishness. • Constipation.
• Heavy menstrual bleeding.

Features of Myxedema :

om
Dry, sparse hair

l.c
ai
gm
Periorbital edema

@
19
55
Puffy face due to edema
na
ee
m
ar
um

Myxoedema
itk

HYPERTHYROIDISM
am

Central
|

Primary Subclinical
w
ro

Secondary Tertiary
ar

•
M

Autoimmune : Grave’s disease

• Autoimmune
©

• Toxic multinodular goitre (MNG)

Hypothalamic (Grave’s disease)
Causes • Toxic thyroid adenoma Pituitary tumour
cause (Rare) • Minimal
• Metastatic carcinoma of thyroid
symptoms
• Iodine excess
T4, fT4 ↑ ↑ ↑
High normal
T3 ↑ ↑ ↑
N /↑
(D/t interrupted
TSH ↓(D/t Negative feedback) ↑ ↓
negative
feedback)
TRH response : TSH response :
Others TRab : + (Grave’s) -
-
+ (↑ TSH)

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Thyroid Function Tests 425

T3 Toxicosis : ----- Active space -----

• fT4 : N
• T3 : ↑↑ ↓ TSH.

Clinical Features :
• Weight loss. • Tremor.
• Sweating. • Variable gland enlargement.
• Heat intolerance. • Tachycardia.
• Restlessness • Diarrhea.
• Menstrual irregularities :
Oligomenorrhea.

om
Eye Features :

l.c
• Proptosis : Bulging of eyeball.

ai
gm
• Red eye.

@
• Infection of cornea/sclera 19 Proptosis
55
na
ee

Algorithm for Evaluation 01:12:35

m
ar
um

TSH
itk
am

↓ N
|

↑
w
ro
ar

T3, T4 No further T3, T4

M
©

Primary evaluation Primary (Overt)

↑ ↓
hyperthyroidism hypothyroidism
Normal thyroid gland
Subclinical Subclinical
High N Low N
hyperthyroidism hypothyroidism
Central Central (2˚)
↓ ↑
hypothyroidism hyperthyroidism
Antibodies

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 426 Xenobiotics and Miscellaneous

----- Active space ----- XENOBIOTICS

Biotransformation  00:00:55

Parent substance Chemical reaction New substance.

(Site : Body)

Types of Biotransformation :

Bioactivation/Toxication : Detoxification :
Parent compound Parent compound

om
l.c
Toxic compound. Less toxic compound.

ai
gm
Xenobiotic Reactions :

@
• Biotransformation of xenobiotics. 19
55

• M/c : Detoxification.
na
ee
m

Xenobiotics :
ar
um

• Compounds that are foreign to the body.

itk

• Eg :
am

- Accidental ingestion of substances (Poisons).

|
w

- Drugs.
ro
ar

- Products of bacterial metabolism in the body.

M
©

Phases of Detoxification 00:05:06

PHASE I REACTION
Compound rendered more reactive by introducing groups that can be conjugated
with a conjugating agent (Eg : Glucuronic acid, glutathione).

Eg :
Mnemonic : RHODE
• Reduction.
• Hydroxylation/Hydrolysis.
• Oxidation.
• Desulfuration.
• Epoxidation.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Xenobiotics 427

Oxidation : ----- Active space -----

Enzyme Characteristics
i) Hydroxylase • Requires Cytochrome P450
(Monooxygenase/ • Eg : Toluene Benzyl alcohol
Mixed function oxidase) NADPH NADP+
Oxidase • Eg :
Aldehyde DH
- Alcohol Alcohol DH Acetaldehyde Acetate
ii) Dehydrogenase (DH)
- Methanol Formaldehyde
Toxication
- Ethylene glycol Oxalic acid
• Requires Cytochrome P450

om
• Eg :

l.c
Reduction Reductase - Nitrobenzene Aniline

ai
gm
- Picric acid Picramic acid

@
19
- Paranitrophenol Para aminophenol
55
• Hydrolytic cleavage by covalent bond breakage
na
ee

(By addition of water)

m
ar

Hydrolysis Hydrolase • Eg :
um

- Aspirin Salicylic acid

itk
am

- Acetanilide Aniline
|
w
ro
ar

PHASE II REACTION
M

Produces water soluble, polar compounds Excreted through bile/urine.

©

Conjugation :
M/c phase II reaction.
Conjugating agent Compounds
Glucuronic acid Bilirubin, benzoic acid, barbiturates
Sulphate Steroid, indole compounds
Cysteine + Glutathione Halides & epoxides
Acetic acid Isoniazid, sulfanilamide
Pyridine Methylpyridine
Methylation
Mercaptophenol
Phenylacetic acid :
Glutamine
• Use : Rx of Hyperammonemia

Biochemistry` • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 428 Xenobiotics and Miscellaneous

----- Active space ----- PHASE III REACTION

• Rare.
• Conjugation of products of phase II.
Eg :
Conjugation with glutathione : M/c type of phase III.

om
l.c
ai
gm
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Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Oncogenesis, Alcohol and Free Radicals 429

ONCOGENESIS, ALCOHOL AND FREE RADICALS ----- Active space -----

Oncogenesis  00:00:50

CANCER
Definition (UICC : International Union Against Cancer) :
Disturbance in growth characterised by excessive proliferation of cells without
apparent relation to physiological demands of the organs involved.

Causes :
Multifactorial disorder :

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• Genetic.

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• Hormonal.

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• Environmental.
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• Metabolic.
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• Physical.
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• Chemical.
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Mutagens :
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• Substances that ↑ rate of mutation.

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• All carcinogens are mutagens.

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Eg :
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Compounds Examples
Benzopyrene (Cigarette smoke),
Chemical Aromatic hydrocarbons
chloranthrenes
mutagens
Nitroso compounds Dimethyl nitrosamine
Natural compound - Aflatoxin

Antimutagens :
• AKA antioxidants.
• ↓ progression of carcinogenesis.

Eg :
• Vit A. • Curcumin.
• Vit E. • Flavonoids.
• Vit C. • Phenolic compounds (Fruits).

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 430 Xenobiotics and Miscellaneous

----- Active space ----- Protooncogene :

Normal cellular gene Promotes cellular proliferation.

Eg :
• Growth factor : FGF (Fibroblast growth factor), EGF ( Epidermal growth factor).
• Growth factor receptors.
• Signal transduction.

Oncogene :
Protooncogene
Mutation or overexpression
Oncogene

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Uncontrolled cell proliferation.

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Eg. :

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Oncogene Virus 55
19 Product
abl Abelson leukemia virus Tyrosine kinase
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erb-B Erythroblastosis virus EGF

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Myc Myelocytoma virus DNA binding protein

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Sis Simian sarcoma virus PDGF (Platelet derived growth factor)

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Ras Rat sarcoma GTPase

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Oncosuppressor Gene :
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Protective factor against cancer.

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Short form Expansion

p53 Gene for protein 53
BRCA Familial breast cancer
DCC Deleted in colon cancer
RB Retinoblastoma
WT Wilms tumour

Tumour Markers :
• Biomarkers. released from the tumour.
• Use : Dx, prognosis, monitoring of cancer.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Oncogenesis, Alcohol and Free Radicals 431

Examples of tumor markers : ----- Active space -----

Markers Cancer
Alpha feto protein (AFP) Hepatocellular carcinoma, germ cell tumours
Carcinoembryonic antigen GIT tumours
Beta hCG Choriocarcinoma
CA-125 Ovarian cancer of epithelial origin
CA 19.9 Ca pancreas
PSA (Prostate specific antigen) Ca prostate
Estrogen receptor Breast cancer
Progesterone receptor Uterine cancer

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Enzymes as tumour markers :

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Enzymes Cancers

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ALP Bone tumours
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PSA
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Prostate cancer
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Prostate specific acid phosphatase

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NSE (Neuron Specific Enolase) Neuroendocrine tumours

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Alcohol 00:11:50
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METABOLISM
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Sites :
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Cytoplasm : Predominant site.

Alcohol Acetaldehyde
dehydrogenase dehydrogenase
Alcohol (Ethanol) Acetaldehyde Acetate.
NAD+ NADH NAD+ NADH
Microsome :
Alcohol (Ethanol) CYP2E1 (MEOS) Acetaldehyde.
NAD+ NADPH
MEOS (Microsomal Ethanol Oxidation System) :
Protective factor against alcohol toxicity in certain populations.
Peroxisome :
Alcohol (Ethanol) Acetaldehyde.
H20 H202 Free radical handled by Catalase

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 432 Xenobiotics and Miscellaneous

----- Active space ----- ACUTE EFFECTS OF ALCOHOL

D/t ↑ NADH/NAD+ Ratio :
1. ↑ Anaerobic glycolysis :
PDH
Glucose Pyruvate Acetyl CoA TCA cycle.
Anaerobic NADH NAD+ NADH
LDH
glycolysis NAD+
↑↑ NADH
Lactate
PDH : Pyruvate dehydrogenase
Lactic acidosis LDH : Lactate dehydrogenase

Precipitate gout d/t

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crystallization of uric acid.

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2. ↑Gluconeogenesis.

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3. ↓ TCA cycle :
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- Isocitrate dehydrogenase (ICDH) NAD+
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α-ketoglutarate dehydrogenase (α KGDH) - -

↑ NADH
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- Malate dehydrogenase (MDH) NADH

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4. Ketogenesis & fatty liver :

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↑ Acetate
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↑ Acetyl CoA
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↑ Fatty acid

Fatty liver. Ketone body synthesis.

Free Radicals & Antioxidants 00:21:48

FREE RADICALS
• Molecule or molecular fragment that contains one 0r more unpaired electron in
its outer orbit.
• Represented as R°.

Oxygen Free Radicals/Reactive Oxygen Species (ROS) :

Complete reduction :
02 + 4e- 4H 2H20.
+

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Oncogenesis, Alcohol and Free Radicals 433

Incomplete reduction : Generates ROS. ----- Active space -----

02 + 1e-

O2- : Superoxide radical

1e-
2H+
H202 : Hydrogen peroxide (Not a free radical)
1e-
1H+
OH° : Hydroxy radical (Most potent free radical).

Sources :
• Electron leakage from electron transport chain (ETC).

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• Oxidation reactions :

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- Xanthine oxidase

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- α-oxidation in peroxisome Produces H202.
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- L aminoacid oxidase 19
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• Respiratory burst :
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- Site : Neutrophils, macrophage.

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- Enzymes : NADPH oxidase, Myeloperoxidase.

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NADPH oxidase
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202 NADPH 202- H2O2 Myeloperoxidase HOCl (Hypochlorous acid)

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NADP
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OH° Kills bacteria.

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• Transition metals : Cu+, Co2+, Ni2+, Fe2+.

• Nitric oxide (Free radical) Generates hydroxyl radical.

ANTIOXIDANTS
Antioxidants/Free radical scavenging

Preventive Chain breaking

Reduce rate of chain initiation : Interferes with chain propogation :

• Glutathione peroxidase & • Superoxide dismutase.
reductase. • Vit E.
• Catalase. • Uric acid.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 434 Xenobiotics and Miscellaneous

----- Active space ----- Glutathione Peroxidase & Reductase :

H202 GSH (reduced) NADP+

↓OH° Glutathione Glutathione
production peroxidase reductase

H20 GS - SG (oxidised) NADPH HMP shunt

pathway

Catalase :
In peroxisome : 2H2O2 Catalase 2H2O + 02.

Superoxide Dismutase (SOD) :

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Cytosol Mitochondria

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Metabolism : 02- + 02- + 2H+ SOD H2O2 + 2H+

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Other Chain Breaking Antioxidants :

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Lipophilic Hydrophilic
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Hydrophobic core : Inside biomembrane Cytoplasm : Water soluble

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• Vit E/α-tocopherol :
- Most potent, natural antioxidant
• Ascorbate/Vit C
Eg - Potentiator : Selenium
• Uric acid
• Beta carotene
• Ubiquinone

EFFECTS OF FREE RADICALS

Pathogenesis :
• DNA mutation : Aging, cell death, cancer.
• Membrane permeability : ↑ free radicals RBC membrane damage

Hemolysis.
• Lipid peroxidation (In biomembrane).
• Loss of protein function.

Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024

 Oncogenesis, Alcohol and Free Radicals 435

Diseases produced : ----- Active space -----

Site Disease
Eye Retinopathy Macular degeneration, cataract
RS Bronchial asthma, respiratory disorders
Liver Fatty liver
GIT Gastric ulcer, colitis
Brain Dementia, degenerative diseases (Alzheimer’s ds, Parkinson’s ds)
Heart Cardiac failure, arrhythmia
Kidney Kidney failure
Skin Accelerated aging, Wrinkle, pigmentation, sagging

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Biochemistry • v1.0 • Marrow 8.0 MBBS - First Year • 2024