Cholesterol Synthesis, Transport, and Excretion

Biomedical Importance of Cholesterol

• Occurrence and Distribution: Cholesterol is found in virtually all tissues and in plasma. It exists in two primary forms: free cholesterol (unesterified) and cholesteryl ester (the storage form, combined with a long-chain fatty acid).

• Structural Role: As an amphipathic lipid, cholesterol is an essential structural component of cell membranes. It is vital for maintaining proper membrane permeability and fluidity. It is also an essential component of the outer layer of plasma lipoproteins.

• Metabolic Precursor: Cholesterol serves as the precursor for all other steroids in the body, which include:

  • Corticosteroids.

  • Sex hormones.

  • Bile acids.

  • Vitamin D.

• Source and Transport:

  • Dietary cholesterol is found in animal-derived foods such as egg yolk, meat, liver, and brain.

  • Plasma low-density lipoprotein (LDL) acts as the vehicle supplying cholesterol and cholesteryl ester to various tissues.

  • Free cholesterol is removed from tissues by high-density lipoprotein (HDL) and transported to the liver for elimination or conversion into bile acids, a process known as reverse cholesterol transport.

• Pathologic Significance: Cholesterol is a major constituent of gallstones. Its most significant pathological role is in the development of atherosclerosis in vital arteries, leading to coronary, cerebrovascular, and peripheral vascular disease.

Biosynthesis of Cholesterol from Acetyl-CoA

• General Characteristics:

  • Slightly more than half of the body's cholesterol (approximately $700\,mg/d$) is synthesized de novo; the remainder comes from the diet.

  • The liver and intestines each account for approximately $10\%$ of total synthesis in humans.

  • Synthesis occurs in the endoplasmic reticulum and cytosolic compartments of virtually all nucleated cells.

• The Five Basic Stages: All 27 carbon atoms of cholesterol are derived from acetyl-CoA through the following stages:

  1. Synthesis of mevalonate from acetyl-CoA.

  2. Formation of isoprenoid units from mevalonate via the loss of $CO_2$.

  3. Condensation of six isoprenoid units to form squalene.

  4. Cyclization of squalene into lanosterol, the parent steroid.

  5. Formation of cholesterol from lanosterol.

Detailed Biochemical Steps of Synthesis

• Step 1: Biosynthesis of Mevalonate:

  • This process is extramitochondrial.

  • Two molecules of acetyl-CoA condense to form acetoacetyl-CoA, catalyzed by cytosolic thiolase.

  • Acetoacetyl-CoA condenses with a third acetyl-CoA molecule to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), catalyzed by HMG-CoA synthase.

  • HMG-CoA is reduced to mevalonate by NADPH, catalyzed by HMG-CoA reductase. This is the principal regulatory step and the site of action for statin drugs.

• Step 2: Formation of Isoprenoid Units:

  • Mevalonate is sequentially phosphorylated by three kinases using ATP.

  • After decarboxylation, the active 5-carbon isoprenoid unit, isopentenyl diphosphate, is formed.

• Step 3: Synthesis of Squalene:

  • Isopentenyl diphosphate isomerizes to dimethylallyl diphosphate.

  • Condensation occurs: One isopentenyl diphosphate and one dimethylallyl diphosphate form geranyl diphosphate ($10\text{ carbons}$).

  • A second isopentenyl diphosphate condenses to form farnesyl diphosphate ($15\text{ carbons}$).

  • Two farnesyl diphosphate molecules condense at the diphosphate end to form squalene ($30\text{ carbons}$). This reaction, catalyzed by squalene synthetase, eliminates inorganic pyrophosphate ($PP_i$) and requires NADPH reduction.

• Derivative Pathways from Farnesyl Diphosphate:

  • Farnesyl diphosphate is a precursor to dolichol (requires up to 16 additional units) and ubiquinone (requires 3 to 7 additional units).

  • Prenylation of GTP-binding proteins with farnesyl or geranylgeranyl ($20\text{ carbons}$) residues facilitates membrane anchoring and protein-protein interactions.

• Step 4: Formation of Lanosterol:

  • Squalene is converted to squalene 2,3-epoxide by squalene epoxidase (a mixed-function oxidase in the ER).

  • Cyclization into lanosterol is catalyzed by oxidosqualene-lanosterol cyclase. This involves methyl group transfers (C14 to C13 and C8 to C14).

• Step 5: Final Production of Cholesterol:

  • Occurs in the membranes of the ER.

  • Methyl groups on C14 and C4 are removed to form 14-desmethyl lanosterol and then zymosterol.

  • The double bond at C8-C9 moves to C5-C6 in two steps, forming desmosterol (24-dehydrocholesterol).

  • The side chain double bond is reduced to produce the final 27-carbon cholesterol molecule.

Regulation of Cholesterol Synthesis

• HMG-CoA Reductase Control:

  • The enzyme is inhibited by mevalonate (immediate product) and cholesterol (end product).

  • Dietary cholesterol intake reduces hepatic de novo synthesis.

• Transcriptional Regulation:

  • Cholesterol and its metabolites repress HMG-CoA reductase mRNA transcription via inhibition of Sterol Regulatory Element-Binding Protein (SREBP) transcription factors.

  • Insulin-induced gene (Insig) proteins, induced by insulin, inhibit SREBP activation and promote HMG-CoA reductase degradation in the ER.

• Posttranslational and Hormonal Modification:

  • Insulin and thyroid hormone increase HMG-CoA reductase activity.

  • Glucagon and glucocorticoids decrease its activity.

  • The enzyme is inactivated by phosphorylation via AMP-activated protein kinase (AMPK). AMPK itself is activated by AMPK kinase (AMPKK) and allosterically by AMP.

Cellular Cholesterol Balance

• Factors Increasing Intracellular Cholesterol:

  • Uptake of lipoproteins through LDL receptors v v,1 or scavenger receptors (e.g., CD36).

  • Direct uptake of free cholesterol from cholesterol-rich lipoproteins into the cell membrane.

  • De novo synthesis.

  • Hydrolysis of stored cholesteryl esters by cholesteryl ester hydrolase.

• Factors Decreasing Intracellular Cholesterol:

  • Efflux to HDL via transporters ABCA1, ABCG1, or SR-B1.

  • Esterification for storage by ACAT (acyl-CoA:cholesterol acyltransferase).

  • Utilization for synthesis of steroid hormones or bile acids.

• Role of the LDL Receptor:

  • LDL receptors are located in clathrin-coated pits. LDL is internalized via endocytosis and hydrolyzed in lysosomes.

  • Influx of cholesterol inhibits transcription of genes for HMG-CoA synthase, HMG-CoA reductase, and the LDL receptor itself through the SREBP pathway.

  • PCSK9 (Proprotein convertase subtilisin/kexin type 9) regulates receptor levels by targeting them for degradation, preventing recycling to the cell surface.

Plasma Transport of Cholesterol

• Lipoprotein Profiles: Humans carry most plasma cholesterol in LDL particles.

• Absorption: Dietary cholesteryl ester is hydrolyzed to cholesterol in the gut. $80\%$ to $90\%$ of absorbed cholesterol is re-esterified in the intestinal mucosa and incorporated into chylomicrons.

• Remnant Delivery: $95\%$ of chylomicron cholesterol is delivered to the liver in remnants.

• LCAT and Reverse Transport: Lecithin:cholesterol acyltransferase (LCAT), associated with apo A-I in HDL, esterifies cholesterol. This creates a gradient that draws cholesterol from tissues into HDL.

• CETP Mechanism: Cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester from HDL to VLDL, IDL, and LDL in exchange for triacylglycerol. This allows LCAT activity to continue without product inhibition.

Synthesis and Excretion of Bile Acids

• Fecal Elimination: Cholesterol is excreted in the bile as free cholesterol or bile acids. Coprostanol, formed by intestinal bacteria, is the primary fecal sterol.

• Primary Bile Acids: These are cholic acid and chenodeoxycholic acid, synthesized in the liver.

  • CYP7A1 (Cholesterol $7\alpha$-hydroxylase): The microsomal cytochrome P450 enzyme that catalyzes the first and rate-limiting step of bile acid synthesis. It requires oxygen, NADPH, and cytochrome P450.

  • Alternative Pathway: CYP27A1 (sterol 27-hydroxylase) in mitochondria also contributes to primary bile acid synthesis.

• Conjugation: Primary bile acids are conjugated with glycine or taurine in liver peroxisomes (typical ratio is $3:1$). These ions are assumption-based "bile salts" in alkaline bile ($pH\,7.6-8.4$).

• Secondary Bile Acids: Intestinal bacteria perform deconjugation and $7\alpha$-dehydroxylation to produce deoxycholic acid and lithocholic acid.

• Enterohepatic Circulation:

  • While fat absorption occurs in the first $100\,cm$ of the small intestine, bile acids are absorbed in the ileum.

  • Approximately $98\%$ to $99\%$ of bile acids return to the liver via the portal circulation.

  • The bile acid pool ($3-5\,g$) cycles $6$ to $10$ times per day.

  • Feedback Regulation: Farnesoid X receptor (FXR) is activated by bile acids (especially chenodeoxycholic acid) to suppress transcription of the CYP7A1 gene.

Clinical Correlation: Atherosclerosis and Cholesterol

• Risk Factors: Plasma cholesterol levels exceeding $5.2\,mmol/L$ and elevated blood triacylglycerols promote atherosclerosis.

• Pathological States: Hyperlipidemia associated with diabetes mellitus, hypothyroidism, and lipid nephrosis increases the risk of severe atherosclerosis.

• Predictive Indicators: The ratio of LDL:HDL is a critical parameter; low HDL2 levels are inversely correlated with coronary heart disease.

• Dietary Factors:

  • Substituting saturated fats with monounsaturated or polyunsaturated ($\omega6$ or $\omega3$) fatty acids lowers serum cholesterol.

  • Saturated fats (e.g., butter, beef fat, palm oil) contribute to smaller, cholesterol-rich VLDL particles.

  • Sucrose and fructose elevate triacylglycerols more than other carbohydrates.

• Lifestyle Factors:

  • Protective factors: Regular exercise (raises HDL, lowers LDL), moderate alcohol consumption (increases apo A-I and HDL), and estrogen in premenopausal women.

  • Negative factors: Smoking, hypertension, obesity, emotional stress, and coffee drinking.

• Pharmacotherapy:

  • Statins: Atorvastatin, simvastatin, fluvastatin, and pravastatin; inhibit HMG-CoA reductase and upregulate LDL receptors.

  • Ezetimibe: Blocks intestinal cholesterol absorption via Niemann-Pick C1-like 1 protein.

  • PCSK9 Inhibitors: Alirocumab and evolocumab; prevent LDL receptor degradation.

  • Fibrates: Clofibrate, gemfibrozil, and nicotinic acid; lower VLDL secretion and triacylglycerols.

Inherited Disorders of Lipoprotein Metabolism (Dyslipoproteinemias)

• Abetalipoproteinemia: Defect in loading apo B with lipid; results in no chylomicrons, VLDL, or LDL. Associated with intestinal malabsorption and low blood acylglycerols.

• Tangier Disease / Fish-eye Disease: HDL deficiency. Tangier presents with near absence of HDL and low LDL.

• Familial Lipoprotein Lipase Deficiency (Type I): Deficiency of LPL or apo C-II; results in slow clearance of chylomicrons and VLDL; low LDL and HDL.

• Familial Hypercholesterolemia (Type IIa): Defective LDL receptors or mutated apo B-100 ligand; results in severe hypercholesterolemia and premature atherosclerosis.

• Familial Type III Hyperlipoproteinemia: Abnormality in apo E (presence of E2 isoform only); leads to chylomicron and VLDL remnant accumulation.

• Familial Hypertriacylglycerolemia (Type IV): VLDL overproduction; often associated with glucose intolerance, obesity, and alcoholism.

• Familial LCAT Deficiency: Block in reverse cholesterol transport; results in abnormal LDL (Lipoprotein X) and nascent disk-shaped HDL.

• Familial Lipoprotein(a) Excess: High levels of Lp(a) (bound to LDL); associated with atherosclerosis and thrombosis due to plasminogen homology inhibiting fibrinolysis.

• Risk Factors: Plasma cholesterol levels exceeding $5.2 \text{ mmol/L}$ and elevated blood triacylglycerols promote atherosclerosis.

• Pathological States: Hyperlipidemia associated with diabetes mellitus, hypothyroidism, and lipid nephrosis increases the risk of severe atherosclerosis.

• Predictive Indicators: The ratio of LDL:HDL is a critical parameter; low HDL2 levels are inversely correlated with coronary heart disease.

• Dietary Factors: Substituting saturated fats with monounsaturated or polyunsaturated fatty acids lowers serum cholesterol. Saturated fats (e.g., butter, beef fat, palm oil) contribute to smaller, cholesterol-rich VLDL particles. Sucrose and fructose elevate triacylglycerols more than other carbohydrates.

• Lifestyle Factors: - Protective factors: Regular exercise (raises HDL, lowers LDL), moderate alcohol consumption (increases apo A-I and HDL), and estrogen in premenopausal women. - Negative factors: Smoking, hypertension, obesity, emotional stress, and coffee drinking.

• Pharmacotherapy: Statins (e.g., atorvastatin, simvastatin) inhibit HMG-CoA reductase and upregulate LDL receptors; Ezetimibe blocks intestinal cholesterol absorption; PCSK9 inhibitors (e.g., alirocumab, evolocumab) prevent LDL receptor degradation; fibrates (e.g., clofibrate, gemfibrozil) lower VLDL secretion and triacylglycerols.