Unit 4: Biochemistry of Lipids

Unit 4: Biochemistry of Lipids

Introduction to Lipids

Lipids are a heterogeneous group of organic compounds that are insoluble in water but soluble in nonpolar organic solvents (ether, chloroform, benzene). They are essential components of living cells with diverse functions including energy storage, structural components of membranes, signaling molecules, and thermal insulation. In pharmaceutical sciences, lipids are crucial for drug formulation, delivery systems, and as targets for therapeutic intervention.

Major Functions of Lipids:

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1. Definition and Classification of Lipids

1.1 Definition

Lipids are defined as hydrophobic or amphipathic small molecules that originate entirely or partially from carbanion-based condensations of thioesters (fatty acids, polyketides) and/or carbocation-based condensations of isoprene units (prenols, sterols).

1.2 Classification Based on Reactivity and Complexity

A. Simple Lipids (Esters of fatty acids with alcohols)

B. Complex Lipids (Contain additional groups like phosphate, nitrogenous bases, carbohydrates)

C. Derived Lipids (Obtained from hydrolysis of simple/complex lipids)

D. Miscellaneous Lipids

1.3 Classification Based on Saponification

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2. Essential Fatty Acids (EFA)

2.1 Definition

Essential fatty acids are fatty acids that cannot be synthesized by the human body and must be obtained from the diet. The human body lacks the enzymes (desaturases) to introduce double bonds beyond the Δ9 position.

2.2 Types of Essential Fatty Acids

2.3 Omega-3 Fatty Acids

Major Types:

• α-Linolenic acid (ALA) : Plant-derived, short-chain
• Eicosapentaenoic acid (EPA) : Marine-derived, 20 carbons
• Docosahexaenoic acid (DHA) : Marine-derived, 22 carbons

Physiological Functions:

• Cardiovascular: Reduce triglycerides, antiarrhythmic, antithrombotic, lower blood pressure
• Anti-inflammatory: Reduce production of inflammatory cytokines and eicosanoids
• Neurological: Essential for brain development and function; DHA is major structural lipid in gray matter and retina
• Immune modulation: Affect immune cell function

Pharmaceutical Applications:

• Prescription omega-3 products (Lovaza, Vascepa) for hypertriglyceridemia
• Dietary supplements for cardiovascular health
• Potential benefits in inflammatory conditions (rheumatoid arthritis, inflammatory bowel disease)

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3. Eicosanoids

3.1 Definition

Eicosanoids are signaling molecules derived from 20-carbon polyunsaturated fatty acids (primarily arachidonic acid). They act locally (autocrine or paracrine) and are involved in inflammation, fever, pain, blood pressure regulation, and reproduction.

3.2 Classes of Eicosanoids

3.3 Synthesis of Eicosanoids

A. Release of Arachidonic Acid: Arachidonic acid is esterified in membrane phospholipids (primarily phosphatidylcholine and phosphatidylinositol). Its release is the rate-limiting step, catalyzed by:

• Phospholipase A₂ (PLA₂) : Releases arachidonic acid directly from phospholipids
• Phospholipase C (PLC) : Produces diacylglycerol, which is cleaved by diacylglycerol lipase

B. Pathways:

                    Membrane Phospholipids
                             |
                             | Phospholipase A₂
                             ↓
                    Arachidonic Acid (20:4)
                    /                   \
                   |                     |
          COX Pathway                 LOX Pathway
        (Cyclooxygenase)             (Lipoxygenase)
                   |                     |
        ┌──────────┴──────────┐    ┌─────┴─────┐
        ↓                     ↓    ↓           ↓
   Prostaglandins        Thromboxanes    Leukotrienes  Lipoxins
   (PGD₂, PGE₂, PGF₂α)     (TXA₂)        (LTA₄, LTB₄, LTC₄, LTD₄, LTE₄)
        ↓
   Prostacyclin (PGI₂)

C. COX Isoforms:

3.4 Pharmacological Relevance of Eicosanoids

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4. Reactions of Fatty Acids and Lipids

4.1 Chemical Reactions of Fatty Acids

4.2 Reactions of Lipids in Biological Systems

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5. Pharmaceutical Importance of Lipids

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6. Digestion, Absorption, Metabolism, and Excretion of Lipids

6.1 Digestion of Lipids

A. Oral Phase:

• Lingual lipase (from lingual glands) – active in stomach (acid-stable)
• Minor digestion in mouth and stomach (especially for short- and medium-chain triglycerides in milk)

B. Gastric Phase:

• Gastric lipase (from gastric mucosa) – optimal pH 4-6
• Digests 10-30% of dietary triglycerides, especially those with short/medium chains

C. Intestinal Phase (Major Site) :

Emulsification: Bile salts (cholic acid, chenodeoxycholic acid conjugated with glycine or taurine) reduce interfacial tension, breaking large fat globules into smaller emulsion droplets (micelles of 4-6 nm).

6.2 Absorption of Lipids

A. Mixed Micelle Formation:

• Bile salts surround lipid digestion products (2-monoglycerides, free fatty acids, cholesterol, lysophospholipids, fat-soluble vitamins)
• Mixed micelles diffuse through unstirred water layer to brush border

B. Cellular Uptake:

• Lipid components diffuse across microvillus membrane (passive diffusion)
• Short/medium-chain fatty acids (C<12) enter portal blood directly (bound to albumin)
• Long-chain fatty acids, 2-monoglycerides, cholesterol, and lysophospholipids enter enterocytes

C. Intracellular Reesterification: In smooth endoplasmic reticulum of enterocytes:

• Fatty acids activated to fatty acyl-CoA (acyl-CoA synthetase)
• 2-Monoglyceride pathway: resynthesis of triglycerides via monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT)
• Cholesterol esterified by acyl-CoA:cholesterol acyltransferase (ACAT)

D. Chylomicron Formation and Secretion:

• Triglycerides, cholesterol esters, phospholipids, and fat-soluble vitamins packaged with apolipoprotein B-48 (apoB-48) to form chylomicrons
• Chylomicrons are large lipoprotein particles (80-500 nm)
• Exported via exocytosis into lacteals (lymphatic vessels) → thoracic duct → bloodstream

6.3 Transport of Lipids in Blood (Lipoproteins)

Lipoprotein Structure: Core of hydrophobic lipids (triglycerides, cholesterol esters) surrounded by amphipathic monolayer of phospholipids, free cholesterol, and apolipoproteins.

Classes of Lipoproteins:

Apolipoprotein Functions:

• Structural: Maintain lipoprotein integrity (apoB, apoA)
• Ligand for receptors: ApoB-100 (LDL receptor), ApoE (LDL receptor, LRP)
• Cofactors for enzymes: ApoC-II activates lipoprotein lipase; ApoA-I activates LCAT

Key Enzymes in Lipoprotein Metabolism:

6.4 Metabolism of Lipids in Tissues

A. Adipose Tissue:

• Uptake: Fatty acids from chylomicrons/VLDL via LPL
• Storage: Re-esterified to triglycerides
• Mobilization: Hormone-sensitive lipase (HSL) activated by glucagon, epinephrine (cAMP); inhibited by insulin
• Release: Free fatty acids bound to albumin; glycerol (cannot be re-esterified due to lack of glycerol kinase)

B. Muscle (including heart) :

• Uptake: Fatty acids from lipoproteins or albumin
• Oxidation: Fatty acids → acetyl-CoA via β-oxidation → TCA cycle → ATP
• Regulation: Fatty acid oxidation inhibited by glucose (glucose-fatty acid cycle, Randle cycle)

C. Liver:

• Uptake: Remnants of chylomicrons (via apoE receptor), free fatty acids from blood, synthesis from carbohydrates
• Metabolism: Oxidation (to acetyl-CoA), ketogenesis, synthesis of triglycerides and phospholipids
• Export: VLDL (triglycerides), bile (cholesterol, bile acids)

6.5 Excretion of Lipids

• Bile: Cholesterol excreted in bile (as free cholesterol or converted to bile acids)
• Feces: Unabsorbed dietary lipids, cholesterol, bile acids, and bacterial lipids
• Skin: Sebum (waxes, triglycerides, cholesterol)
• Urine: Minimal (lipids not normally filtered; lipiduria indicates pathology)

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7. Oxidation of Fatty Acids

7.1 Activation of Fatty Acids (Cytosol)

Fatty acid + CoA + ATP → Fatty acyl-CoA + AMP + PPᵢ

Enzyme: Acyl-CoA synthetase (thiokinase) Location: Outer mitochondrial membrane, peroxisomes, ER Energy cost: 2 ATP equivalents (ATP → AMP = 2 ATP)

7.2 Transport into Mitochondria (Carnitine Shuttle)

Regulation: CPT I is rate-limiting; inhibited by malonyl-CoA (product of fatty acid synthesis), preventing simultaneous synthesis and oxidation.

7.3 β-Oxidation (Mitochondrial Matrix)

β-Oxidation is a spiral pathway that cleaves two-carbon units (acetyl-CoA) from the carboxyl end of fatty acyl-CoA.

Four Reactions per Cycle:

Products per Cycle:

• 1 Acetyl-CoA
• 1 FADH₂
• 1 NADH

Complete Oxidation of Palmitate (C16:0) :

• 7 cycles → 8 acetyl-CoA
• 7 FADH₂ → 7 × 1.5 = 10.5 ATP
• 7 NADH → 7 × 2.5 = 17.5 ATP
• 8 acetyl-CoA → 8 × 10 = 80 ATP (via TCA)
• Total: 108 ATP
• Minus activation cost: -2 ATP
• Net: 106 ATP

7.4 Oxidation of Unsaturated Fatty Acids

Unsaturated fatty acids require additional enzymes:

7.5 Oxidation of Odd-Chain Fatty Acids

Odd-chain fatty acids (e.g., C15, C17) yield propionyl-CoA in the final cycle.

Propionyl-CoA Metabolism:

Propionyl-CoA
       │ (propionyl-CoA carboxylase - requires biotin)
       ↓
D-Methylmalonyl-CoA
       │ (methylmalonyl-CoA epimerase)
       ↓
L-Methylmalonyl-CoA
       │ (methylmalonyl-CoA mutase - requires vitamin B₁₂ as coenzyme B₁₂)
       ↓
Succinyl-CoA (enters TCA cycle)

Clinical Relevance: Vitamin B₁₂ deficiency leads to accumulation of methylmalonic acid (methylmalonic aciduria) and neurological symptoms.

7.6 Peroxisomal β-Oxidation

• Location: Peroxisomes (for very-long-chain fatty acids >C22, branched-chain fatty acids, bile acid intermediates)
• Enzymes differ: First step uses acyl-CoA oxidase (produces H₂O₂, which is degraded by catalase)
• Products: Acetyl-CoA and chain-shortened fatty acids that are transferred to mitochondria for further oxidation
• Clinical relevance: Zellweger syndrome (peroxisome biogenesis disorders) – accumulation of very-long-chain fatty acids

7.7 α-Oxidation

• Location: Peroxisomes
• Function: Oxidizes fatty acids with methyl branch at β-carbon (e.g., phytanic acid from chlorophyll)
• Enzyme: Phytanoyl-CoA hydroxylase (requires iron and 2-oxoglutarate)
• Clinical relevance: Refsum disease (defect in α-oxidation) – phytanic acid accumulates, causing neurological symptoms

7.8 ω-Oxidation

• Location: Endoplasmic reticulum (minor pathway)
• Function: Hydroxylation of terminal (ω) carbon by cytochrome P450 (CYP4A family)
• Products: Dicarboxylic acids, which can be excreted or undergo β-oxidation from both ends
• Significance: Becomes important in conditions of impaired β-oxidation

7.9 Dysregulation of Fatty Acid Oxidation

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8. Biosynthesis of Fatty Acids (Lipogenesis)

8.1 Overview

Fatty acid synthesis occurs primarily in liver, adipose tissue, and lactating mammary gland. It is the reverse of β-oxidation but occurs in cytoplasm and uses different enzymes and cofactors.

8.2 Substrates and Requirements

• Carbon source: Acetyl-CoA (from glucose via pyruvate, or from amino acids)
• Reducing power: NADPH (from pentose phosphate pathway, malic enzyme)
• Bicarbonate: Required for carboxylation (as CO₂)
• Energy: ATP

8.3 Transport of Acetyl-CoA to Cytosol (Citrate Shuttle)

Acetyl-CoA from mitochondria cannot cross the inner membrane; it is transported via:

1. Citrate synthase (mitochondria): Oxaloacetate + acetyl-CoA → citrate
2. Citrate transported to cytosol via tricarboxylate transporter
3. ATP-citrate lyase (cytosol): Citrate + CoA + ATP → acetyl-CoA + oxaloacetate + ADP + Pi
4. Oxaloacetate → malate (malate dehydrogenase) → pyruvate (malic enzyme, generates NADPH) → back to mitochondria

8.4 Formation of Malonyl-CoA (Committed Step)

Acetyl-CoA + CO₂ + ATP → Malonyl-CoA + ADP + Pi

Enzyme: Acetyl-CoA carboxylase (ACC) Cofactor: Biotin Regulation:

• Activated by citrate (allosteric)
• Inactivated by phosphorylation (AMPK, PKA)
• Insulin promotes dephosphorylation (activation); glucagon promotes phosphorylation (inactivation)

8.5 Fatty Acid Synthase Complex (FAS)

Mammalian FAS is a multifunctional enzyme dimer (homodimer) containing all seven catalytic activities on a single polypeptide. The growing fatty acid chain is attached to acyl carrier protein (ACP) domain via phosphopantetheine group.

Steps of Fatty Acid Synthesis:

Product: Palmitate (C16:0) is the primary product; released by thioesterase.

Stoichiometry for Palmitate: 8 Acetyl-CoA + 7 ATP + 14 NADPH → Palmitate + 8 CoA + 7 ADP + 7 Pi + 14 NADP⁺

8.6 Elongation and Desaturation

Elongation:

• Microsomal (ER) : Extends palmitate to longer chains (up to C24) using malonyl-CoA and NADPH
• Mitochondrial: Minor pathway, uses acetyl-CoA

Desaturation:

• Stearoyl-CoA desaturase (Δ9 desaturase) : Introduces cis double bond at Δ9 position (palmitoleic acid, C16:1; oleic acid, C18:1)
• Humans lack Δ12 and Δ15 desaturases → cannot synthesize linoleic and α-linolenic acids (essential fatty acids)

8.7 Regulation of Fatty Acid Synthesis

8.8 Dysregulation of Fatty Acid Synthesis

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9. Cholesterol Synthesis and Its Dysregulation

9.1 Overview

Cholesterol is a 27-carbon steroid essential for membrane structure, precursor for bile acids, steroid hormones, and vitamin D. All carbons come from acetyl-CoA.

9.2 Sites of Synthesis

• Liver: 50% of total body synthesis
• Intestine: 15%
• Skin, adrenal cortex, reproductive tissues: Variable

9.3 Pathway of Cholesterol Synthesis (Cytosol and ER)

Stage 1: Synthesis of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA)

2 Acetyl-CoA → Acetoacetyl-CoA (thiolase) Acetoacetyl-CoA + Acetyl-CoA → HMG-CoA (HMG-CoA synthase)

Stage 2: Formation of Mevalonate (Committed Step)

HMG-CoA + 2 NADPH → Mevalonate + CoA + NADP⁺

Enzyme: HMG-CoA reductase Location: Smooth ER Rate-limiting step; major regulatory point Inhibitors: Statins (e.g., atorvastatin, simvastatin) – competitive inhibitors

Stage 3: Isoprenoid Synthesis

Mevalonate → Isopentenyl pyrophosphate (IPP) → Dimethylallyl pyrophosphate (DMAPP) (Requires phosphorylation by kinases and decarboxylation)

Stage 4: Condensation to Squalene

IPP + DMAPP → Geranyl pyrophosphate (GPP, C10) GPP + IPP → Farnesyl pyrophosphate (FPP, C15) 2 FPP → Squalene (C30) (squalene synthase)

Stage 5: Cyclization to Cholesterol

Squalene → Squalene 2,3-epoxide → Lanosterol (first sterol) → (19 steps) → Cholesterol

9.4 Regulation of Cholesterol Synthesis

9.5 Dysregulation of Cholesterol Metabolism

9.6 Pharmacological Targeting of Cholesterol Synthesis

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10. Biosynthesis of Neutral Lipids

10.1 Triacylglycerol (Triglyceride) Synthesis

A. Liver and Adipose Tissue:

1. Glycerol-3-phosphate formation:

   • From glucose: Dihydroxyacetone phosphate (from glycolysis) → Glycerol-3-phosphate (glycerol-3-phosphate dehydrogenase)
   • From glycerol: Glycerol + ATP → Glycerol-3-phosphate (glycerol kinase – present in liver, absent in adipose tissue)

2. Acylation steps:

   • Glycerol-3-phosphate + fatty acyl-CoA → Lysophosphatidic acid (glycerol-3-phosphate acyltransferase, GPAT)
   • Lysophosphatidic acid + fatty acyl-CoA → Phosphatidic acid (acylglycerol-3-phosphate acyltransferase, AGPAT)

3. Dephosphorylation:

   • Phosphatidic acid → 1,2-Diacylglycerol (phosphatidate phosphatase, PAP)

4. Final acylation:

   • 1,2-Diacylglycerol + fatty acyl-CoA → Triacylglycerol (diacylglycerol acyltransferase, DGAT)

B. Intestine (2-Monoglyceride Pathway) :

• Dietary 2-monoglyceride + fatty acyl-CoA → 1,2-Diacylglycerol (monoacylglycerol acyltransferase, MGAT)
• Then DGAT as above

10.2 Phospholipid Synthesis

A. Phosphatidic Acid Pathway:

• Phosphatidic acid + CTP → CDP-diacylglycerol (CDP-diacylglycerol synthase)
• CDP-diacylglycerol + serine → Phosphatidylserine (then decarboxylated to phosphatidylethanolamine)
• CDP-diacylglycerol + inositol → Phosphatidylinositol

B. Kennedy Pathway (for PC and PE) :

• Choline → Phosphocholine → CDP-choline → Phosphatidylcholine (from diacylglycerol)
• Ethanolamine follows similar pathway

10.3 Sphingolipid Synthesis

• Serine + Palmitoyl-CoA → 3-Ketosphinganine → Sphinganine → Ceramide (addition of fatty acid)
• Ceramide + Phosphocholine (from PC) → Sphingomyelin
• Ceramide + UDP-glucose → Glucocerebroside (glycolipids)

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11. Ketone Bodies

11.1 Definition

Ketone bodies are water-soluble molecules produced by the liver from acetyl-CoA during periods of low carbohydrate availability (starvation, fasting, prolonged exercise, uncontrolled diabetes). They serve as an alternative energy source for extrahepatic tissues (brain, muscle, heart).

11.2 Types of Ketone Bodies

11.3 Ketogenesis (Synthesis)

Location: Liver mitochondria

Pathway:

1. 2 Acetyl-CoA → Acetoacetyl-CoA (thiolase)
2. Acetoacetyl-CoA + Acetyl-CoA → HMG-CoA (HMG-CoA synthase – different isoform from cholesterol synthesis)
3. HMG-CoA → Acetoacetate + Acetyl-CoA (HMG-CoA lyase)

Acetoacetate can be:

• Reduced to β-hydroxybutyrate (β-hydroxybutyrate dehydrogenase, using NADH)
• Spontaneously decarboxylated to acetone

11.4 Ketolysis (Utilization)

Location: Mitochondria of extrahepatic tissues (not liver)

Pathway:

1. β-Hydroxybutyrate → Acetoacetate (β-hydroxybutyrate dehydrogenase)
2. Acetoacetate + Succinyl-CoA → Acetoacetyl-CoA + Succinate (succinyl-CoA:acetoacetate CoA transferase, SCOT – absent in liver)
3. Acetoacetyl-CoA → 2 Acetyl-CoA (thiolase) → TCA cycle

11.5 Regulation of Ketogenesis

11.6 Dysregulation: Ketoacidosis

Diabetic Ketoacidosis (DKA) :

• Occurs in Type 1 diabetes (or severe Type 2) with insulin deficiency
• Unrestrained lipolysis → massive fatty acid release to liver
• Glucagon high → stimulates ketogenesis
• Overproduction of ketone bodies exceeds tissue utilization
• Acetoacetate and β-hydroxybutyrate are acids → metabolic acidosis
• Acetone causes fruity breath odor

Clinical features: Nausea, vomiting, abdominal pain, Kussmaul respirations (deep, rapid breathing), confusion, coma

Laboratory findings: Hyperglycemia, metabolic acidosis (low pH, low bicarbonate), elevated anion gap, positive serum ketones, elevated β-hydroxybutyrate

Alcoholic Ketoacidosis:

• Chronic alcohol use, poor nutrition, vomiting
• Ethanol metabolism depletes NAD⁺, impairs gluconeogenesis → hypoglycemia
• Fatty acids mobilized and converted to ketones

Starvation Ketoacidosis:

• Mild, self-limited ketosis after prolonged fasting (usually not severe acidosis)

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12. Energetics of Various Metabolic Processes of Lipids

Energy Density Comparison:

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13. Brief Overview of Lipid Metabolic Disorders

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Summary Tables

Table 1: Classification of Lipids

Table 2: Major Lipoproteins

Table 3: Enzymes of Fatty Acid Oxidation and Synthesis

Table 4: Disorders of Lipid Metabolism

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References

1. Lippincott Williams & Wilkins. (2020). Lippincott's illustrated reviews: Biochemistry. (Chapters on Lipid Metabolism)

2. Berg, J. M., Tymoczko, J. L., & Gatto, G. J. (2019). Stryer's biochemistry (8th ed.). W. H. Freeman and Company. (Chapters on Lipids and Lipid Metabolism)

3. Nelson, D. L., & Cox, M. M. (2017). Lehninger principles of biochemistry (7th ed.). W. H. Freeman and Company. (Chapters on Fatty Acid Catabolism, Lipid Biosynthesis, and Cholesterol)

4. Rodwell, V. W., Bender, D. A., Botham, K. M., Kennelly, P. J., & Weil, P. A. (2017). Harper's illustrated biochemistry (31st ed.). McGraw-Hill Education. (Chapters on Lipid Metabolism and Disorders)

5. Devlin, T. M. (2016). Textbook of biochemistry with clinical correlations (8th ed.). Wiley-Liss. (Chapters on Lipid Metabolism and Clinical Correlations)

6. Scriver, C. R., et al. (2001). The metabolic and molecular bases of inherited disease (8th ed.). McGraw-Hill. (For lipid storage diseases and other inborn errors)

7. Kumar, V., Abbas, A. K., & Aster, J. C. (2020). Robbins & Cotran pathologic basis of disease (10th ed.). Elsevier. (Clinical correlations for lipid disorders)

8. Brown, M. S., & Goldstein, J. L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science, 232(4746), 34-47. (Nobel Prize work on LDL receptor)

9. Endo, A. (1992). The discovery and development of HMG-CoA reductase inhibitors. Journal of Lipid Research, 33(11), 1569-1582. (Discovery of statins)

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Recommended Textbooks for Further Reading:

• Lippincott Williams & Wilkins. (2020). Lippincott's illustrated reviews: Biochemistry. (Excellent for visual summaries and clinical notes on lipid metabolism)
• Nelson, D. L., & Cox, M. M. (2017). Lehninger principles of biochemistry (7th ed.). (Detailed mechanistic explanations)
• Rodwell, V. W., et al. (2017). Harper's illustrated biochemistry (31st ed.). (Strong clinical emphasis)