Lipid Metabolism
Lipids are the most energy-dense macronutrients, storing more than twice the energy per gram compared to carbohydrates or proteins. This section explores the oxidation and synthesis of fatty acids, the formation of ketone bodies, the assembly of complex lipids, and the metabolism of cholesterol—a molecule central to membrane biology, steroid hormone production, and cardiovascular disease.
Energy Storage
Fatty acids yield ~9 kcal/g vs ~4 kcal/g for carbohydrates, making adipose tissue the body's primary energy reserve.
Signaling Molecules
Eicosanoids, sphingolipids, and phospholipids serve as critical signaling molecules regulating inflammation and cell fate.
Clinical Relevance
Cholesterol metabolism underpins atherosclerosis, and statins are among the most widely prescribed drugs worldwide.
Learning Objectives
After completing Part IV, you should be able to:
- •Describe the activation, transport, and stepwise β-oxidation of fatty acids, and calculate the ATP yield from palmitate
- •Explain the role of acetyl-CoA carboxylase as the committed step of fatty acid synthesis and its regulation
- •Compare and contrast the enzymology of β-oxidation and fatty acid synthesis
- •Outline the pathways of ketogenesis and ketolysis and explain the metabolic logic of ketone body production during fasting
- •Describe the synthesis of triacylglycerols, phospholipids, and sphingolipids
- •Explain eicosanoid biosynthesis and the mechanism of action of NSAIDs
- •Trace the mevalonate pathway for cholesterol biosynthesis and explain regulation by SREBPs
- •Distinguish the major lipoprotein classes and describe the LDL receptor pathway
- •Discuss the pathogenesis of atherosclerosis and the pharmacological basis of lipid-lowering therapies
Chapter 18
Fatty Acid Oxidation (β-Oxidation)
Fatty acid oxidation is a major source of energy for many tissues, particularly cardiac muscle, skeletal muscle during prolonged exercise, and the liver. The process involves the sequential removal of two-carbon units from the carboxyl end of fatty acyl chains, generating acetyl-CoA, FADH₂, and NADH that feed into the citric acid cycle and oxidative phosphorylation.
18.1 Fatty Acid Activation
Before a fatty acid can be oxidized, it must be activated by attachment to coenzyme A. This reaction is catalyzed by acyl-CoA synthetase (also called fatty acid thiokinase), located on the outer mitochondrial membrane. The reaction proceeds in two steps:
Step 1 — Formation of acyl-adenylate:
Step 2 — Transfer to CoA:
Net reaction:
The pyrophosphate (PPi) produced is immediately hydrolyzed by pyrophosphatase, driving the reaction to completion. Because ATP is converted to AMP (not ADP), the energetic cost of activation is equivalent to 2 ATP. This cost must be subtracted from the total ATP yield of fatty acid oxidation.
Key Concept — Why 2 ATP Equivalents?
ATP → AMP breaks two high-energy phosphoanhydride bonds. Regenerating ATP from AMP requires two phosphorylation steps: AMP → ADP (via adenylate kinase) and ADP → ATP (via oxidative phosphorylation). Thus the net cost is 2 ATP equivalents, not just one. This is a common point of confusion in examinations.
18.2 The Carnitine Shuttle
Long-chain acyl-CoA molecules cannot cross the inner mitochondrial membrane directly. Instead, the acyl group is transferred to carnitine for transport via a three-step shuttle system:
Step 1: CPT-I (Carnitine Palmitoyltransferase I)
Located on the outer face of the inner mitochondrial membrane, CPT-I transfers the acyl group from CoA to carnitine, forming acylcarnitine and releasing free CoA. CPT-I is the rate-limiting step of fatty acid oxidation and is inhibited by malonyl-CoA, the first committed intermediate of fatty acid synthesis.
Step 2: Carnitine-Acylcarnitine Translocase
This antiporter in the inner mitochondrial membrane shuttles acylcarnitine into the matrix while simultaneously exporting free carnitine to the intermembrane space.
Step 3: CPT-II (Carnitine Palmitoyltransferase II)
Located on the matrix side of the inner membrane, CPT-II regenerates acyl-CoA from acylcarnitine and intramitochondrial CoA. The released carnitine returns to the cytosol via the translocase.
Clinical Correlation — CPT Deficiencies
CPT-II deficiency is the most common inherited disorder of fatty acid oxidation. The adult myopathic form presents with recurrent episodes of rhabdomyolysis triggered by prolonged exercise, fasting, or cold exposure. The severe neonatal form can cause hypoketotic hypoglycemia, cardiomyopathy, and liver failure. Medium-chain fatty acids (C6–C12) bypass the carnitine shuttle because they can cross the inner membrane independently, explaining why medium-chain triglyceride (MCT) supplementation is therapeutic.
18.3 The β-Oxidation Spiral
Once inside the mitochondrial matrix, acyl-CoA undergoes a repetitive four-step cycle that removes two carbons per round as acetyl-CoA. Each cycle consists of:
Oxidation (Acyl-CoA Dehydrogenase)
A trans-α,β double bond is introduced between C-2 and C-3, producing trans-Δ²-enoyl-CoA. The electrons are transferred to FAD → FADH₂, which passes them to the electron-transferring flavoprotein (ETF) and ultimately to ubiquinone in the respiratory chain.
Hydration (Enoyl-CoA Hydratase)
Water is added across the double bond in a stereospecific manner to form L-3-hydroxyacyl-CoA (also called L-β-hydroxyacyl-CoA).
Oxidation (3-Hydroxyacyl-CoA Dehydrogenase)
The hydroxyl group at C-3 is oxidized to a ketone, producing 3-ketoacyl-CoA (β-ketoacyl-CoA). NAD⁺ serves as the electron acceptor, generating NADH.
Thiolysis (β-Ketothiolase / Thiolase)
A molecule of CoA-SH attacks C-3, cleaving the bond between C-2 and C-3. This releases acetyl-CoA and an acyl-CoA that is two carbons shorter than the original substrate.
18.4 Complete Oxidation of Palmitate
Palmitate (C16:0) is the prototypical fatty acid used to calculate ATP yield. With 16 carbons, it requires 7 rounds of β-oxidation to generate 8 acetyl-CoA molecules.
Mathematical Deep Dive — ATP Yield from Palmitate
Overall equation for β-oxidation of palmitoyl-CoA:
From β-oxidation directly:
- 7 FADH₂ × 1.5 ATP/FADH₂ = 10.5 ATP
- 7 NADH × 2.5 ATP/NADH = 17.5 ATP
From 8 acetyl-CoA via TCA cycle:
- 8 × 3 NADH × 2.5 = 60 ATP
- 8 × 1 FADH₂ × 1.5 = 12 ATP
- 8 × 1 GTP = 8 ATP
Total: 10.5 + 17.5 + 60 + 12 + 8 = 108 ATP
Minus activation cost: 108 − 2 = 106 ATP net per palmitate
18.5 Odd-Chain and Unsaturated Fatty Acid Oxidation
Odd-chain fatty acids (common in ruminant fats and some marine lipids) undergo normal β-oxidation until the final round, which produces one molecule of propionyl-CoA (3 carbons) instead of acetyl-CoA. Propionyl-CoA is converted to succinyl-CoA via three enzymatic steps:
- Propionyl-CoA carboxylase (biotin-dependent): propionyl-CoA → D-methylmalonyl-CoA
- Methylmalonyl-CoA racemase: D-methylmalonyl-CoA → L-methylmalonyl-CoA
- Methylmalonyl-CoA mutase (vitamin B₁₂-dependent): L-methylmalonyl-CoA → succinyl-CoA
Unsaturated fatty acids require additional enzymes:
- Enoyl-CoA isomerase — converts cis-Δ³ bonds to trans-Δ² bonds (for monounsaturated FAs like oleate)
- 2,4-Dienoyl-CoA reductase — reduces 2,4-dienoyl intermediates that arise from polyunsaturated FAs (requires NADPH)
For Graduate Students — Peroxisomal β-Oxidation
Very-long-chain fatty acids (≥C22) are first shortened in peroxisomes before mitochondrial β-oxidation can proceed. Peroxisomal β-oxidation uses acyl-CoA oxidase (which transfers electrons directly to O₂, producing H₂O₂ rather than feeding the ETC), making it less energy-efficient. The H₂O₂ is detoxified by catalase. Defects in peroxisomal biogenesis cause Zellweger syndrome, characterized by accumulation of very-long-chain fatty acids, severe neurological dysfunction, and hepatomegaly.
Key Concept — Comparison of β-Oxidation and Fatty Acid Synthesis
| Feature | β-Oxidation | Fatty Acid Synthesis |
|---|---|---|
| Location | Mitochondrial matrix | Cytoplasm |
| Acyl carrier | Coenzyme A | ACP (acyl carrier protein) |
| Electron carriers | FAD, NAD⁺ | NADPH |
| C2 donor/product | Acetyl-CoA (product) | Malonyl-CoA (donor) |
| Hydroxyl intermediate | L-isomer | D-isomer |
| Hormonal activation | Glucagon, epinephrine | Insulin |
Clinical Correlation — Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency
MCAD deficiency is the most common inborn error of fatty acid oxidation, with an incidence of approximately 1 in 15,000. Patients cannot oxidize medium-chain fatty acids (C6–C12) and present with hypoketotic hypoglycemia during fasting or illness. The condition is detected by newborn screening via elevated octanoylcarnitine (C8) on tandem mass spectrometry. Treatment involves avoidance of fasting and provision of adequate carbohydrate intake during illness.
Chapter 19
Fatty Acid Synthesis
While β-oxidation breaks down fatty acids in the mitochondria, fatty acid synthesis (lipogenesis) occurs in the cytoplasm, primarily in the liver and adipose tissue. The pathway uses a different set of enzymes, different electron carriers (NADPH rather than NADH/FADH₂), and builds the carbon chain two carbons at a time from malonyl-CoA—not from acetyl-CoA directly.
19.1 Acetyl-CoA Carboxylase (ACC)
The committed and rate-limiting step of fatty acid synthesis is the carboxylation of acetyl-CoA to malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACC). This enzyme uses biotin as a prosthetic group and requires ATP and bicarbonate:
ACC exists in two forms in mammals: ACC1 (cytoplasmic, provides malonyl-CoA for fatty acid synthesis) and ACC2(associated with the outer mitochondrial membrane, produces malonyl-CoA that inhibits CPT-I).
Key Concept — Regulation of ACC
Activators (Stimulate Synthesis)
- Citrate (allosteric activator — promotes polymerization)
- Insulin (dephosphorylation via protein phosphatase)
- High carbohydrate diet (increased citrate, insulin)
Inhibitors (Block Synthesis)
- Palmitoyl-CoA (allosteric inhibitor — feedback)
- Glucagon / Epinephrine (phosphorylation via AMPK)
- AMP-activated protein kinase (AMPK) phosphorylation
19.2 Fatty Acid Synthase (FAS)
In mammals, fatty acid synthase is a large homodimeric multifunctional enzyme (a single polypeptide chain containing all seven catalytic activities plus an acyl carrier protein domain). Each monomer contains a 4'-phosphopantetheine prosthetic group that serves as a flexible arm to shuttle intermediates between active sites.
The seven sequential reactions in each elongation cycle are:
Acetyl transacylase (AT)
Loads acetyl group onto the Cys-SH of β-ketoacyl synthase (KS)
Malonyl transacylase (MT)
Loads malonyl group onto the phosphopantetheine-SH of ACP
β-Ketoacyl synthase (KS)
Condensation of acetyl (or growing acyl chain) with malonyl group; CO₂ released; drives reaction forward
β-Ketoacyl reductase (KR)
Reduction of β-keto group to β-hydroxy using NADPH
β-Hydroxyacyl dehydratase (DH)
Dehydration to form trans-α,β-enoyl intermediate
Enoyl reductase (ER)
Reduction of double bond using NADPH to form saturated acyl chain
Thioesterase (TE)
After 7 cycles, releases the completed palmitate (C16) from FAS
Notice that the reactions of FAS are essentially the reverse of β-oxidation, but with important differences: NADPH replaces NADH/FADH₂ as the electron donor, and the intermediates are bound to ACP rather than CoA.
19.3 Overall Stoichiometry
Mathematical Deep Dive — Palmitate Synthesis
The overall equation for palmitate synthesis from acetyl-CoA:
Accounting:
- 8 acetyl-CoA provide 16 carbons for the palmitate chain
- 7 ATP are consumed by acetyl-CoA carboxylase (7 malonyl-CoA from 7 of the 8 acetyl-CoA; the first acetyl-CoA is the primer)
- 14 NADPH are used (2 per elongation cycle × 7 cycles): one for the β-ketoacyl reductase, one for the enoyl reductase
19.4 The Citrate Shuttle
Acetyl-CoA is produced in the mitochondrial matrix (from pyruvate dehydrogenase or β-oxidation), but fatty acid synthesis occurs in the cytoplasm. Acetyl-CoA cannot cross the inner mitochondrial membrane directly. Instead, it is exported as citrate:
- In the matrix: Acetyl-CoA + oxaloacetate → citrate (citrate synthase)
- Citrate exits the mitochondria via the citrate transporter
- In the cytoplasm: ATP-citrate lyase cleaves citrate → acetyl-CoA + oxaloacetate
- Oxaloacetate → malate (malate dehydrogenase, using NADH)
- Malate → pyruvate + CO₂ (malic enzyme, generating NADPH)
- Pyruvate re-enters the mitochondria via the pyruvate carrier
The malic enzyme reaction provides a portion of the NADPH required for fatty acid synthesis. The remainder comes from the pentose phosphate pathway(oxidative phase).
19.5 Elongation and Desaturation
Palmitate (C16:0) is the primary product of FAS, but cells require fatty acids of varying chain lengths and degrees of unsaturation:
- Elongation occurs on the smooth ER membrane using malonyl-CoA and NADPH, adding 2 carbons per cycle (e.g., C16 → C18 → C20)
- Desaturation is performed by fatty acyl-CoA desaturases (Δ9, Δ6, Δ5) which introduce cis double bonds. Mammals cannot introduce double bonds beyond C-9 (counting from the carboxyl end)
Clinical Correlation — Essential Fatty Acids
Because mammals lack Δ12 and Δ15 desaturases, linoleic acid(18:2, ω-6) and α-linolenic acid (18:3, ω-3) must be obtained from the diet—they are essential fatty acids. Linoleic acid is the precursor of arachidonic acid (20:4, ω-6), which is the substrate for prostaglandin and leukotriene synthesis. Deficiency causes dermatitis, poor wound healing, and growth retardation.
For Graduate Students — De Novo Lipogenesis in Disease
Increased de novo lipogenesis (DNL) is a hallmark of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), and many cancers. The transcription factors SREBP-1c (induced by insulin) and ChREBP (activated by glucose metabolites) coordinately upregulate ACC, FAS, and other lipogenic enzymes. Pharmacological inhibitors of ACC (e.g., firsocostat) and FAS (e.g., TVB-2640) are in clinical trials for NASH and cancer treatment.
Chapter 20
Ketone Bodies
Ketone bodies—acetoacetate, D-β-hydroxybutyrate, and acetone—are water-soluble fuels synthesized in the liver from acetyl-CoA during periods when fatty acid oxidation is high and oxaloacetate is diverted to gluconeogenesis (fasting, starvation, uncontrolled diabetes, prolonged exercise).
20.1 Ketogenesis
Ketogenesis occurs exclusively in the hepatic mitochondrial matrix. The pathway begins with the condensation of two molecules of acetyl-CoA and proceeds through the HMG-CoA intermediate:
Step 1: Thiolase (Acetoacetyl-CoA Thiolase)
Two molecules of acetyl-CoA condense to form acetoacetyl-CoA.
Step 2: HMG-CoA Synthase (Rate-Limiting)
A third acetyl-CoA is added to acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This is the rate-limiting step of ketogenesis. Note: This is the mitochondrial HMG-CoA synthase, distinct from the cytoplasmic isoform used in cholesterol synthesis.
Step 3: HMG-CoA Lyase
HMG-CoA is cleaved to produce acetoacetate and acetyl-CoA.
Step 4: β-Hydroxybutyrate Dehydrogenase
Acetoacetate is reduced to D-β-hydroxybutyrate using NADH. This is the predominant ketone body in the blood (ratio of β-hydroxybutyrate:acetoacetate reflects the mitochondrial NADH/NAD⁺ ratio).
Acetone is formed by the spontaneous, non-enzymatic decarboxylation of acetoacetate. It is volatile and exhaled via the lungs, giving the characteristic fruity breath odor in diabetic ketoacidosis.
Mathematical Deep Dive — Net Ketogenesis
Net reaction for β-hydroxybutyrate formation:
Note that HMG-CoA synthase consumes one acetyl-CoA, but HMG-CoA lyase regenerates one, so the net consumption is 2 acetyl-CoA per ketone body.
20.2 Ketolysis (Ketone Body Utilization)
Extrahepatic tissues (brain, heart, skeletal muscle, kidney cortex) can oxidize ketone bodies for energy. The liver cannot utilize ketone bodies because it lacks succinyl-CoA:3-oxoacid CoA transferase(thiophorase/SCOT), the key enzyme for ketolysis.
- D-β-Hydroxybutyrate → Acetoacetate (β-hydroxybutyrate dehydrogenase, NAD⁺ → NADH)
- Acetoacetate + Succinyl-CoA → Acetoacetyl-CoA + Succinate (thiophorase/SCOT)
- Acetoacetyl-CoA + CoA-SH → 2 Acetyl-CoA (thiolase)
The 2 acetyl-CoA molecules then enter the TCA cycle for complete oxidation.
Key Concept — Why Can't the Liver Use Ketone Bodies?
The liver lacks thiophorase (SCOT), the enzyme needed to activate acetoacetate back to acetoacetyl-CoA. This makes metabolic sense: the liver is the producer of ketone bodies, and consuming its own product would create a futile cycle. This metabolic compartmentalization ensures ketone bodies are exported as fuel for other tissues during fasting.
20.3 Regulation of Ketogenesis
Ketogenesis is regulated at multiple levels:
- Substrate supply: Increased fatty acid mobilization from adipose tissue (driven by low insulin, high glucagon) provides more acetyl-CoA
- Malonyl-CoA levels: Low malonyl-CoA (due to decreased ACC activity during fasting) relieves CPT-I inhibition, increasing β-oxidation and acetyl-CoA production
- Oxaloacetate depletion: Gluconeogenesis draws oxaloacetate away from the TCA cycle, diverting acetyl-CoA toward ketogenesis
- HMG-CoA synthase: Transcriptionally induced during fasting by PPARα; also regulated by succinylation (inhibitory)
Clinical Correlation — Diabetic Ketoacidosis (DKA)
In uncontrolled type 1 diabetes, the absence of insulin leads to:
- Unrestrained lipolysis → massive fatty acid release from adipose tissue
- Increased β-oxidation → excess acetyl-CoA production
- Accelerated gluconeogenesis → OAA depletion
- Overwhelming ketogenesis → blood ketone levels rise 30–50 fold
Acetoacetate and β-hydroxybutyrate are organic acids. Their accumulation causes a metabolic acidosis (blood pH can drop below 7.0), leading to Kussmaul breathing (deep, rapid respirations to blow off CO₂), dehydration from osmotic diuresis, electrolyte imbalances (especially potassium), and potentially coma and death without insulin treatment.
Mathematical Deep Dive — ATP Yield from β-Hydroxybutyrate
When β-hydroxybutyrate is oxidized in extrahepatic tissues:
- β-Hydroxybutyrate → Acetoacetate: generates 1 NADH (= 2.5 ATP)
- Acetoacetate → 2 Acetyl-CoA: costs 1 succinyl-CoA (= −1 GTP = −1 ATP)
- 2 Acetyl-CoA via TCA: 2 × 10 = 20 ATP
Total: 2.5 − 1 + 20 = 21.5 ATP per β-hydroxybutyrate
Key Concept — Ketone Bodies as Brain Fuel
During prolonged fasting (beyond 2–3 days), ketone bodies progressively replace glucose as the brain's primary fuel. At peak adaptation, ketone bodies can supply up to 60–70% of the brain's energy needs. This adaptation is critical for survival because it dramatically reduces the need for gluconeogenesis from amino acids, thereby preserving lean muscle mass. Without ketone body utilization, the body would deplete its protein reserves within 2–3 weeks of starvation. With ketoadaptation, survival can extend to 60–70 days (assuming adequate fat stores and hydration).
For Graduate Students — Therapeutic Ketosis
The ketogenic diet (very low carbohydrate, high fat) induces a controlled state of ketosis in which blood β-hydroxybutyrate levels reach 1–5 mM (compared to <0.3 mM in the fed state and >25 mM in DKA). Ketone bodies serve as alternative fuels for the brain, reducing glucose dependence. This has proven therapeutic value in drug-resistant epilepsy, particularly in children. Emerging evidence suggests benefits in neurodegenerative diseases (Alzheimer's, Parkinson's) and certain cancers, though mechanisms remain under investigation. β-Hydroxybutyrate also functions as a signaling molecule, inhibiting class I histone deacetylases (HDACs) and activating the GPR109A receptor on immune cells.
Chapter 21
Complex Lipids
Beyond simple fatty acids and their energy metabolism, cells require a diverse array of complex lipids for membrane architecture, signal transduction, and intercellular communication. This chapter covers the synthesis and function of triacylglycerols, phospholipids, sphingolipids, and eicosanoids—bioactive lipid mediators with profound effects on inflammation, hemostasis, and immunity.
21.1 Triacylglycerol Synthesis
Triacylglycerols (TAGs) are the principal storage form of metabolic energy. Their synthesis occurs primarily in the liver, adipose tissue, and intestinal mucosa via the glycerol-3-phosphate pathway (Kennedy pathway for TAG synthesis):
- Glycerol-3-phosphate acyltransferase (GPAT): Acylation at sn-1 position → lysophosphatidic acid
- 1-Acylglycerol-3-phosphate acyltransferase (AGPAT): Acylation at sn-2 → phosphatidic acid
- Phosphatidic acid phosphatase (lipin): Removal of phosphate → 1,2-diacylglycerol (DAG)
- Diacylglycerol acyltransferase (DGAT): Acylation at sn-3 → triacylglycerol
The glycerol-3-phosphate backbone comes from two sources: (1) reduction of dihydroxyacetone phosphate (DHAP) from glycolysis, or (2) phosphorylation of glycerol by glycerol kinase (primarily in the liver—adipose tissue has minimal glycerol kinase activity, which is why adipocytes require glucose for TAG synthesis).
Key Concept — Phosphatidic Acid as a Branch Point
Phosphatidic acid (PA) is a critical metabolic branch point. Dephosphorylation by lipin yields DAG, which leads to TAG synthesis (energy storage). Alternatively, PA can be converted to CDP-diacylglycerol, the precursor for phosphatidylinositol, phosphatidylglycerol, and cardiolipin. The balance between these fates is regulated by nutritional and hormonal signals: insulin promotes TAG synthesis, while CDP-DAG pathway activity ensures membrane lipid homeostasis.
21.2 Phospholipid Synthesis (Kennedy Pathway)
Phospholipids are the primary structural components of biological membranes. The major glycerophospholipids— phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)—are synthesized via the Kennedy pathway (discovered by Eugene Kennedy in the 1950s):
Phosphatidylcholine (PC) Synthesis
- Choline kinase: Choline + ATP → Phosphocholine + ADP
- CTP:phosphocholine cytidylyltransferase (rate-limiting): Phosphocholine + CTP → CDP-choline + PPi
- CDP-choline:DAG cholinephosphotransferase: CDP-choline + DAG → PC + CMP
Phosphatidylethanolamine (PE) Synthesis
Follows an analogous pathway using ethanolamine instead of choline. PE can also be converted to PC by three sequential methylations using S-adenosylmethionine (SAM), catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT), which is quantitatively significant in the liver.
Phosphatidylserine (PS) Synthesis
In mammalian cells, PS is synthesized primarily by base-exchange reactions: PS synthase 1 exchanges the choline head group of PC for serine, and PS synthase 2 exchanges the ethanolamine of PE for serine. PS is concentrated on the inner leaflet of the plasma membrane; its appearance on the outer leaflet is an "eat-me" signal for phagocytosis of apoptotic cells.
21.3 Sphingolipid Metabolism
Sphingolipids are built on a sphingoid base backbone (sphingosine) rather than glycerol. They are critical components of membrane lipid rafts and serve as potent signaling molecules.
De novo sphingolipid synthesis:
- Serine palmitoyltransferase (rate-limiting): Palmitoyl-CoA + Serine → 3-Ketosphingosine + CoA-SH + CO₂
- 3-Ketosphingosine → Sphinganine (reduction by NADPH)
- Sphinganine → Dihydroceramide (N-acylation by ceramide synthase)
- Dihydroceramide → Ceramide (desaturation by dihydroceramide desaturase)
Ceramide is the central hub of sphingolipid metabolism. It can be converted to:
- Sphingomyelin: Ceramide + phosphatidylcholine → sphingomyelin + DAG (sphingomyelin synthase)
- Glucosylceramide: Ceramide + UDP-glucose → glucosylceramide (glucosylceramide synthase) — precursor to gangliosides
- Sphingosine-1-phosphate (S1P): Ceramide → sphingosine (ceramidase) → S1P (sphingosine kinase)
Clinical Correlation — Sphingolipid Storage Diseases
Deficiencies in lysosomal enzymes that degrade sphingolipids cause accumulation of specific intermediates, resulting in sphingolipidoses(lysosomal storage diseases):
| Disease | Deficient Enzyme | Accumulated Lipid |
|---|---|---|
| Gaucher disease | β-Glucocerebrosidase | Glucocerebroside |
| Niemann-Pick (A/B) | Sphingomyelinase | Sphingomyelin |
| Tay-Sachs | Hexosaminidase A | GM2 ganglioside |
| Fabry disease | α-Galactosidase A | Globotriaosylceramide |
| Krabbe disease | Galactocerebrosidase | Galactocerebroside |
| Metachromatic leukodystrophy | Arylsulfatase A | Sulfatide |
Gaucher disease is the most common sphingolipidosis and was the first disease treated with enzyme replacement therapy (ERT). Substrate reduction therapy (e.g., miglustat, eliglustat) is an alternative approach.
21.4 Eicosanoids
Eicosanoids are 20-carbon bioactive lipid mediators derived from arachidonic acid (20:4, ω-6), which is released from membrane phospholipids by phospholipase A₂ (PLA₂). The three major classes of eicosanoids are produced by different enzymatic pathways:
Cyclooxygenase (COX) Pathway → Prostaglandins & Thromboxanes
COX enzymes catalyze the first committed step: arachidonic acid → PGH₂ (prostaglandin H₂), which is then converted to specific prostanoids by downstream synthases:
- PGE₂: Pain, fever, inflammation, vasodilation
- PGI₂ (prostacyclin): Vasodilation, inhibits platelet aggregation
- PGF₂α: Uterine contraction, bronchoconstriction
- TXA₂ (thromboxane A₂): Vasoconstriction, platelet aggregation
Lipoxygenase (LOX) Pathway → Leukotrienes
5-Lipoxygenase (5-LOX) converts arachidonic acid to 5-HPETE and then to leukotriene A₄ (LTA₄):
- LTB₄: Neutrophil chemotaxis and activation
- LTC₄, LTD₄, LTE₄ (cysteinyl leukotrienes): Bronchoconstriction, vascular permeability — key mediators of asthma
Cytochrome P450 Pathway
CYP epoxygenases produce epoxyeicosatrienoic acids (EETs), which are vasodilatory and anti-inflammatory. CYP ω-hydroxylases produce 20-HETE, a vasoconstrictor.
Clinical Correlation — COX Enzymes and NSAIDs
COX-1 is constitutively expressed in most tissues and produces prostaglandins involved in homeostatic functions: gastric mucosal protection (PGE₂), renal blood flow, and platelet aggregation (TXA₂). COX-2is inducible and upregulated at sites of inflammation by cytokines and growth factors.
- Aspirin: Irreversibly acetylates Ser-530 in the COX active site. At low doses (81 mg/day), it preferentially inhibits platelet COX-1 (platelets cannot synthesize new protein), providing antiplatelet effects. At higher doses, it inhibits both COX-1 and COX-2.
- Ibuprofen/Naproxen: Reversible, non-selective COX inhibitors. Anti-inflammatory, analgesic, antipyretic. GI side effects from COX-1 inhibition.
- Celecoxib: Selective COX-2 inhibitor. Reduced GI toxicity but increased cardiovascular risk (decreased PGI₂ without decreased TXA₂ tips balance toward thrombosis).
- Corticosteroids: Inhibit phospholipase A₂ (via lipocortin/annexin A1), blocking release of arachidonic acid and thus reducing synthesis of all eicosanoids.
For Graduate Students — Specialized Pro-Resolving Mediators (SPMs)
Resolution of inflammation is not merely a passive decay of pro-inflammatory signals but an active process driven by specialized pro-resolving mediators (SPMs). These include resolvins (from EPA and DHA via COX-2 and LOX), protectins (neuroprotectin D1 from DHA), and maresins (from DHA via 12-LOX in macrophages). SPMs promote clearance of debris, counter-regulate pro-inflammatory mediator production, and enhance tissue repair. Aspirin-triggered resolvins are produced when aspirin acetylates COX-2, redirecting its catalytic activity toward R-epimer products with enhanced pro-resolving activity. This may partly explain aspirin's anti-inflammatory benefits beyond simple COX inhibition.
Chapter 22
Cholesterol Metabolism
Cholesterol is an essential molecule—a component of cell membranes, the precursor to steroid hormones, bile acids, and vitamin D—yet its excess contributes to atherosclerosis and cardiovascular disease. Understanding cholesterol biosynthesis, regulation, and transport is fundamental to both biochemistry and clinical medicine.
22.1 The Mevalonate Pathway
Cholesterol is synthesized from acetyl-CoA in a complex pathway of more than 30 enzymatic reactions. The pathway can be conceptually divided into five stages:
Stage 1: Mevalonate Synthesis (C2 → C6)
- 2 Acetyl-CoA → Acetoacetyl-CoA (thiolase)
- Acetoacetyl-CoA + Acetyl-CoA → HMG-CoA (HMG-CoA synthase, cytoplasmic isoform)
- HMG-CoA → Mevalonate (HMG-CoA reductase — rate-limiting, NADPH-dependent)
Stage 2: Activated Isoprene Units (C6 → C5)
Mevalonate is phosphorylated three times and decarboxylated to form isopentenyl pyrophosphate (IPP, the "active isoprene unit") and its isomer dimethylallyl pyrophosphate (DMAPP). Three ATP molecules are consumed.
Stage 3: Squalene Synthesis (C5 → C30)
Six isoprene units are assembled: IPP + DMAPP → geranyl-PP (C10) → farnesyl-PP (C15). Two farnesyl-PP molecules condense head-to-head to form squalene (C30), catalyzed by squalene synthase (requires NADPH).
Stage 4: Lanosterol Formation (C30)
Squalene is epoxidized by squalene monooxygenase (requires O₂, NADPH) and then cyclized by oxidosqualene cyclase (lanosterol synthase) to form lanosterol—the first sterol intermediate.
Stage 5: Cholesterol (C30 → C27)
Lanosterol undergoes ~19 additional reactions (removal of 3 methyl groups, reduction of the double bond in the side chain, and isomerization of the remaining double bond) to yield cholesterol. These reactions occur on the ER membrane.
Mathematical Deep Dive — Energetic Cost of Cholesterol
The overall biosynthesis of one cholesterol molecule requires:
This enormous energetic investment explains why cholesterol biosynthesis is so tightly regulated. The body synthesizes approximately 800–1000 mg of cholesterol per day, primarily in the liver, while dietary intake contributes 200–300 mg/day.
22.2 Regulation of Cholesterol Synthesis
Cholesterol biosynthesis is regulated primarily at the level of HMG-CoA reductase through multiple mechanisms:
SREBP-SCAP-Insig Pathway
Sterol regulatory element-binding proteins (SREBPs)are transcription factors that activate genes for cholesterol and fatty acid synthesis. When cholesterol is abundant, SREBP is retained in the ER membrane by SCAP-Insig interaction. When cholesterol is low, SCAP escorts SREBP to the Golgi, where it is cleaved by Site-1 and Site-2 proteases, releasing the active N-terminal domain that translocates to the nucleus and activates transcription of HMG-CoA reductase, the LDL receptor, and other targets.
Enzyme Degradation (ERAD)
When sterol levels are high, Insig proteins bind to HMG-CoA reductase directly, promoting its ubiquitination and proteasomal degradation via ER-associated degradation (ERAD). This can reduce enzyme levels by >90% within hours.
Phosphorylation by AMPK
AMP-activated protein kinase (AMPK) phosphorylates and inactivates HMG-CoA reductase when cellular energy is low, linking cholesterol synthesis to energy status. Dephosphorylation by protein phosphatase 2A (PP2A) reactivates the enzyme.
Clinical Correlation — Statins
Statins (atorvastatin, rosuvastatin, simvastatin, etc.) are competitive inhibitors of HMG-CoA reductase and are among the most widely prescribed drugs worldwide. By inhibiting cholesterol synthesis, statins trigger compensatory upregulation of LDL receptors (via SREBP activation), which increases LDL clearance from the blood.
Clinical trials have demonstrated that statins reduce LDL cholesterol by 30–50%, decrease cardiovascular events by 25–35%, and reduce cardiovascular mortality. The "statin paradox" is that blocking HMG-CoA reductase actually causes compensatory upregulation of the enzyme (via SREBP activation), but the net effect is still reduced cholesterol synthesis because the drug concentration exceeds the increased enzyme levels.
Side effects include myopathy (1–5%), rhabdomyolysis (rare), elevated liver transaminases, and a modest increase in new-onset diabetes. Statin intolerance affects approximately 10–15% of patients, often due to muscle symptoms.
22.3 Bile Acid Synthesis
Bile acid synthesis is the major route of cholesterol elimination. In the liver, cholesterol is converted to the primary bile acids cholic acid and chenodeoxycholic acid by a series of modifications including 7α-hydroxylation (rate-limiting, catalyzed by cholesterol 7α-hydroxylase / CYP7A1), ring modifications, and side-chain oxidation.
Bile acids are conjugated with glycine or taurine to form bile salts, secreted into bile, stored in the gallbladder, and released into the duodenum to emulsify dietary lipids. In the ileum, ~95% of bile salts are reabsorbed (enterohepatic circulation) via the apical sodium-dependent bile acid transporter (ASBT). Only ~5% (400–600 mg/day) are excreted in feces—this represents the only significant route for cholesterol elimination from the body.
Bile acid sequestrants (cholestyramine, colesevelam) bind bile acids in the intestinal lumen, preventing their reabsorption. This forces increased bile acid synthesis from cholesterol, upregulates hepatic LDL receptors, and lowers plasma LDL cholesterol.
22.4 Lipoproteins: Transport of Lipids in Blood
Because lipids are hydrophobic, they are transported in blood as lipoproteins—spherical particles with a hydrophobic core (triacylglycerols, cholesterol esters) surrounded by an amphipathic shell (phospholipids, free cholesterol, apolipoproteins).
| Lipoprotein | Origin | Primary Lipid | Key Apolipoproteins | Function |
|---|---|---|---|---|
| Chylomicrons | Intestine | Dietary TAG | ApoB-48, ApoC-II, ApoE | Deliver dietary lipids |
| VLDL | Liver | Endogenous TAG | ApoB-100, ApoC-II, ApoE | Export hepatic TAG |
| IDL | VLDL remnant | TAG + Cholesterol | ApoB-100, ApoE | Intermediate → LDL |
| LDL | IDL remnant | Cholesterol esters | ApoB-100 | Deliver cholesterol to tissues |
| HDL | Liver, Intestine | Cholesterol esters | ApoA-I, ApoA-II | Reverse cholesterol transport |
Key enzymes in lipoprotein metabolism:
- Lipoprotein lipase (LPL): On capillary endothelium; activated by ApoC-II; hydrolyzes TAG in chylomicrons and VLDL, releasing fatty acids for tissue uptake
- Hepatic lipase: On hepatic sinusoidal endothelium; converts IDL to LDL; remodels HDL
- LCAT (Lecithin-Cholesterol Acyltransferase): On HDL; esterifies free cholesterol using a fatty acid from phosphatidylcholine, allowing HDL to accumulate cholesterol esters in its core
- CETP (Cholesteryl Ester Transfer Protein): Transfers cholesterol esters from HDL to VLDL/LDL in exchange for TAG
22.5 The LDL Receptor Pathway
The LDL receptor pathway, elucidated by Michael Brown and Joseph Goldstein (Nobel Prize, 1985), is the principal mechanism for cellular uptake of cholesterol:
- LDL receptors on the cell surface bind ApoB-100 on LDL particles
- Receptor-LDL complexes cluster in clathrin-coated pits
- Internalization by receptor-mediated endocytosis → coated vesicles → endosomes
- In the acidic endosome (pH ~5), LDL dissociates from the receptor
- Receptors recycle back to the cell surface (~150 times during their lifespan)
- LDL is delivered to lysosomes; cholesterol esters are hydrolyzed by acid lipase
- Free cholesterol enters the cell and triggers regulatory responses
Intracellular cholesterol exerts three regulatory effects:
- Suppresses SREBP processing → decreased HMG-CoA reductase and LDL receptor transcription
- Activates ACAT (acyl-CoA:cholesterol acyltransferase) → esterification for storage
- Suppresses LDL receptor gene transcription directly
Clinical Correlation — Familial Hypercholesterolemia
Familial hypercholesterolemia (FH) is caused by mutations in the LDL receptor gene (or less commonly in ApoB-100 or PCSK9). Heterozygous FH (1 in 250–500) causes LDL cholesterol of 300–500 mg/dL and premature coronary artery disease by age 40–50. Homozygous FH (1 in 1,000,000) causes LDL >600 mg/dL and myocardial infarction in childhood or adolescence.
Brown and Goldstein classified five classes of LDL receptor mutations: Class 1 (no synthesis), Class 2 (defective transport from ER to Golgi), Class 3 (defective LDL binding), Class 4 (defective internalization/clustering in coated pits), and Class 5 (defective receptor recycling). Their work established the paradigm of receptor-mediated endocytosis.
Clinical Correlation — PCSK9 Inhibitors
PCSK9 (proprotein convertase subtilisin/kexin type 9) is a secreted protein that binds to the LDL receptor on the cell surface and promotes its lysosomal degradation (preventing recycling). Gain-of-function PCSK9 mutations cause hypercholesterolemia; loss-of-function mutations lower LDL cholesterol and protect against coronary disease. Monoclonal antibodies against PCSK9 (evolocumab, alirocumab) reduce LDL cholesterol by an additional 50–60% on top of statin therapy and have been shown to reduce cardiovascular events. Inclisiran, a small interfering RNA (siRNA) targeting PCSK9 mRNA, was approved in 2021 and requires only twice-yearly dosing.
22.6 Atherosclerosis
Atherosclerosis is a chronic inflammatory disease of arterial walls driven by the accumulation of modified lipoproteins. The "response-to-retention" hypothesis outlines the pathogenesis:
LDL Retention
LDL particles enter the subendothelial space and are retained by binding to extracellular matrix proteoglycans. Small, dense LDL particles are particularly atherogenic.
LDL Modification
Retained LDL undergoes oxidative modification by reactive oxygen species (ROS) and enzymatic modification by lipoxygenases, myeloperoxidase, and secretory phospholipase A₂.
Endothelial Activation
Oxidized LDL (oxLDL) activates endothelial cells, which express adhesion molecules (VCAM-1, ICAM-1) and secrete chemokines (MCP-1), recruiting monocytes.
Foam Cell Formation
Monocytes differentiate into macrophages, which take up oxLDL via scavenger receptors (SR-A, CD36)—receptors not downregulated by cholesterol. Lipid-laden macrophages become foam cells.
Plaque Progression
Smooth muscle cells migrate from the media, proliferate, and secrete extracellular matrix, forming a fibrous cap. The core contains lipid, necrotic foam cells, cholesterol crystals, and calcification.
Plaque Rupture
Thin-cap fibroatheromas with active inflammation are vulnerable to rupture. Matrix metalloproteinases (MMPs) secreted by macrophages degrade the fibrous cap. Rupture exposes thrombogenic material, triggering acute thrombosis—the proximate cause of myocardial infarction and stroke.
Key Concept — HDL and Reverse Cholesterol Transport
HDL mediates reverse cholesterol transport—the only pathway for returning cholesterol from peripheral tissues to the liver for excretion as bile acids. Nascent HDL (discoidal, lipid-poor ApoA-I) acquires free cholesterol from cells via the ABCA1 transporter. LCAT esterifies this cholesterol, converting discoidal HDL to mature spherical HDL. Cholesterol esters are delivered to the liver either directly (via the SR-BI receptor) or indirectly (via CETP transfer to LDL/VLDL, followed by hepatic LDL receptor uptake). Higher HDL cholesterol levels are epidemiologically associated with lower cardiovascular risk, though pharmacological HDL-raising strategies (e.g., CETP inhibitors) have yielded mixed results in clinical trials.
For Graduate Students — Beyond LDL: Lp(a) and Residual Risk
Lipoprotein(a) [Lp(a)] is an LDL-like particle with an additional apolipoprotein(a) covalently linked to ApoB-100 by a disulfide bond. Lp(a) levels are largely genetically determined (by the LPA gene locus) and are an independent, causal risk factor for atherosclerotic cardiovascular disease. Lp(a) is resistant to statin therapy. Novel therapies targeting Lp(a) include antisense oligonucleotides (pelacarsen) and siRNAs (olpasiran, lepodisiran), which reduce Lp(a) levels by 80–98% and are in phase III cardiovascular outcomes trials. Triglyceride-rich lipoprotein remnants and apolipoprotein CIII are also emerging as important targets for addressing residual cardiovascular risk after LDL lowering.
Part IV Summary — Key Takeaways
β-Oxidation degrades fatty acids in the mitochondrial matrix, producing acetyl-CoA, FADH₂, and NADH. Palmitate yields a net 106 ATP—far more than glucose (30–32 ATP).
Fatty acid synthesis occurs in the cytoplasm using NADPH and is regulated at the ACC step. Malonyl-CoA links synthesis and oxidation by inhibiting CPT-I.
Ketone bodies are water-soluble fuels produced by the liver during fasting. They spare glucose for the brain but can cause life-threatening acidosis in uncontrolled diabetes.
Complex lipids include phospholipids, sphingolipids, and eicosanoids. Defects in sphingolipid degradation cause lysosomal storage diseases. Eicosanoids mediate inflammation and are targets of NSAIDs.
Cholesterol metabolism is tightly regulated by SREBP signaling. Lipoproteins transport lipids in blood; LDL delivers cholesterol to tissues while HDL mediates reverse transport. Statins and PCSK9 inhibitors are the cornerstones of lipid-lowering therapy.
Quick Reference — Key Enzymes of Part IV
CPT-I
Rate-limiting for β-oxidation; inhibited by malonyl-CoA
Acetyl-CoA Carboxylase (ACC)
Rate-limiting for FA synthesis; biotin-dependent
HMG-CoA Synthase (mitochondrial)
Rate-limiting for ketogenesis
HMG-CoA Reductase
Rate-limiting for cholesterol synthesis; statin target
Cholesterol 7α-Hydroxylase (CYP7A1)
Rate-limiting for bile acid synthesis
Lipoprotein Lipase (LPL)
Hydrolyzes TAG in chylomicrons/VLDL; activated by ApoC-II