BIOCHEM 3.1 - CHOLESTEROL & BILE SALTS

• These notes cover cholesterol and bile salts, including their synthesis, uses, metabolism, role in health/disease, and related enzymes.

Cholesterol

• Found in all biological membranes
• Contributes to the fluidity of membranes at different temperatures.
• Low temperatures lead to increased fluidity.
• High temperatures (body temperature) can decrease fluidity.
• Involved in the creation of cholesterol-rich patches in the membrane, reducing permeability
• Serves as a precursor to steroid hormones, vitamin D, and bile salts.

Cholesterol Synthesis

• Primarily synthesized from Acetyl-CoA in the liver, with some synthesis occurring in the intestines, adrenal cortex, and gonads.
• ATP and Acetyl-CoA are needed as starting point.
• NADPH from the pentose phosphate pathway is vital for redox reactions during the synthesis.
• Formation involves multiple intermediate steps and ring formation.
• The first committed step is the formation of mevalonic acid, which occurs in the cytosol.
• Begins with a condensation process similar to ketone body formation, using HMG-CoA.
• The rate-limiting step involves HMG-CoA Reductase (HMGR).
• The reaction requires 2 NADPH.
• HMGR is embedded in the ER.

HMGR Regulation

• Regulation occurs through feedback inhibition, phosphorylation, degradation rate, and hormones.
• Phosphorylation:
• When phosphorylated by AMP-dependent kinase (AMPK), HMGR is inactive.
• HMGR is active when not phosphorylated.
• Degradation Rate:
• HMGR contains a sterol-sensing domain.
• High cholesterol levels cause binding and ubiquitylation of HMGR, leading to its degradation.
• Hormone Regulation:
• Insulin and triiodothyronine promote HMGR activity.
• Glucagon and cortisol inhibit HMGR activity.
• Statins inhibit HMGR by binding to the HMG-CoA binding site.

Ring Formation

• Squalene, synthesized by squalene synthase, forms a ring structure.
• Oxygen is inserted into the structure via squalene monooxygenase.
• Ring closure is catalyzed by oxidosqualene cyclase.
• This forms lanosterol.

Squalene Monooxygenase

• Activity changes based on cholesterol availability.
• MARCH 6 ubiquitylates the enzyme to target it for degradation.
• When cholesterol synthesis is needed:
• Low cholesterol and high squalene, degradation is reduced.
• Lanosterol synthesis can progress.
• High squalene levels drive lanosterol synthesis even in the presence of high cholesterol levels.

Final Steps of Cholesterol Synthesis

• Multiple enzymatic reactions are needed to produce cholesterol from lanosterol.
• This includes:
• Decarboxylation, which requires O2 and produces CO2.
• Isomerization via isomerase.
• Reduction, requiring NADPH at each step from lanosterol to cholesterol.

Need for Carrier Proteins

• Several steps of the process result in hydrophobic molecules.
• Carrier proteins are required, beginning with the production of squalene, to manage hydrophobic molecules.
• Squalene Binding Protein and Sterol Binding Protein are needed.

Cholesterol Transport and Esterification

• Cholesterol is excreted by the liver.
• Free cholesterol makes up ~30% of circulating cholesterol and has low solubility in water.
• Cholesterol esters are cholesterol plus long-chain fatty acids like linoleic acid, and they possess even lower solubility in water.
• Cholesterol Esterification:
• In cells, esterification occurs via acyl-CoA:cholesterol acyltransferase (ACAT) in the mitochondrial membrane.
• Cholesterol esters are stored in the ER as lipid droplets.
• In plasma, esterification occurs via cholesterol-lecithin acyltransferase.
• Cholesterol esters are found in chylomicrons and distributed to tissues in VLDL and LDL.

ACAT Inhibitors

• acyl-CoA:cholesterol acyltransferase (ACAT) overexpression is linked to increased cholesterol ester formation in cancer.
• Inhibiting ACAT can be a useful target against cancer and may be useful in treatment of Alzheimer's Disease.

Cholesterol Homeostasis

• Summary of synthesis and homeostasis:
• Making 1 mole of cholesterol requires 18 moles of acetyl-CoA, 36 moles of ATP, and 16 moles of NADPH.
• Balancing Free Cholesterol:
• Increase Concentration: synthesis, breakdown of intracellular cholesterol esters by hydrolase activity, diet (uptake of LDL via receptors/increase in LDL receptor concentration on cell surface).
• Decrease Concentration: inhibition of synthesis, decreased concentration of LDL receptors, esterification via ACAT, release to HDL, conversion to bile acids or steroid hormones, decreased HMG-CoA availability and/or activity, high membrane cholesterol concentration.

Bile Acids and Bile Salts

• Bile acids are the most abundant metabolic product of cholesterol.
• Hepatic parenchymal cells synthesize cholic and chenodeoxycholic acids.
• Conjugation of cholic and chenodeoxycholic acids via amide linkages with glycine or taurine produces the 4 primary bile acids.
• Secondary bile acids are formed by bacteria in the intestine.
• Sodium or potassium ion addition yields bile salts at physiological pH.
• The terms Bile Salts and Bile Acids are usually interchangeable.
• Bile synthesis occurs in the liver, with direct secretion to the duodenum or storage in the gall bladder.

Bile and Digestion

• Bile includes bile salts with water, phospholipids, cholesterol, and excretory products like bilirubin.
• Gastrointestinal hormones modulate bile secretion:
• Hepatocrinin causes secretion of bile.
• Cholecystokinin causes emptying of the gallbladder.
• Hormones, released because of partially digested foods in the duodenum, lead to bile secretion.
• Bile acts as detergents to assist in lipid emulsification.
• Most bile acids are recycled to the liver (deconjugated and reabsorbed) via enterohepatic recirculation.

Case Study 1

• A 50-year-old male experiences pain in the upper right quadrant of the abdomen with vomiting after eating fatty foods.
• The biochemical abnormality is raised alkaline phosphatase, nearly double the upper limit of 260 U/L.
• Elevated alkaline phosphatase indicates cholestasis.
• This can be an indicator transport function where alkaline phosphatase expression from bile duct epithelial cells is increased due to bile duct obstruction.
• Gallstones are caused by excess cholesterol.
• A cholesterol to phospholipid ratio greater than 1:1 will mean decreased cholesterol solubilization.
• Excess cholesterol leads to crystallization around insoluble nuclei, causing high bile and the crystallization of gallstones.

Case Study 2

• A 21-year-old reports heaviness in chest after walking, along with shortness of breath. EKG, chest X-ray, and cardiac enzymes do not reveal pathology.
• BP is elevated (151/99mmHg), with a pulse of 97 bpm and respirations of 20/min.
• History of smoking (3 packs per day), obesity (BMI 39.7), and family history of coronary heart disease. Diagnosis:
• Stable angina (chest pain or discomfort with activity or emotional distress).
• Follow-up testing revealed insulin resistance and mild dyslipidemia.
• An exercise stress test revealed:
• High BP (196/80mmHg vs 136/84 mmHg at rest).
• Increased pulse to 176 bpm.
• Nonsustained ventricular tachycardia.
• Echocardiography and coronary tomography angiography (CTA) revealed atherosclerosis.
• Updated diagnosis: Coronary Heart Disease (CHD).

Atherosclerosis

• Atherosclerosis involves enough information to generate an entire book.
• Formation involves many components, including:
• Inflammation, cholesterol, cholesterol-trafficking, oxidation of VLDL and LDL.
• Oxidative stress, formation of foam cells.
• 1° & Messenger Inflamm Cyto/Chemokines.
• Cellular Adhesion Molecules.
• Plaque Destabilization.
• Plaque Rupture.
• Acute Phase Reactants.