Synthesis of Cholesterol and Bile Salts/Acids

Question 1

Influx and Efflux of Liver Cholesterol

Answer 1

• the liver plays an essential role in maintaing human cholesterol homeostasis
• the sources of the liver’s cholesterol pool include dietary cholesterol and that synthesized de novo by extrahepatic tissues and the liver itself
• cholesterol leaves the liver as unmodified free cholesterol in the bile, or by conversion to bile acids/salts secreted into the intestinal lumen, or as VLDL secreted into the circulation
• the balance of influx and efflux is not perfect and can lead to a deposition of cholesterol in the endothelial lining of blood vessels

Question 2

Cholesterol Sturcture

Answer 2

• four planar hydrocarbon rings called the steroid nucleus
• it has an eight carbon hydrocarbon attached to carbon 17, of the D ring; a hydroxyl group attached to carbon 3 of the A ring; and a double bond between carbon 5 and 6 of the B ring
• most cholesterol in the plasma is esterified to a fatty acid at carbon 3. The cholesteryl ester is even more hydrophobic than cholesterol itself

Question 3

Cholesterol and Cell membranes

Answer 3

• sterols appear to have evolved to fill the flickering spaces between the fatty acyl chains in membrane bilayers
• the hydroxyl group of cholesterol gives the otherwise hydrophobic molecule its amphiphilic character
• the steroid rings and hydrocarbon tail intercalate between the fatty acid chain of the phospholipids, within the membrane bilayer, while the polar OH group is in line with the polar head groups and in contact with the aqueous environment
• at physiologic conditions cholesterol increases the packing within the hydrophobic core of the bilayer, thereby increasing the mechanical strength while decreasing the permeability and fluidity of the membrane

Question 4

Sterols

Answer 4

• cholesterol is the major form in animal tissues of a family of molecules called sterols
• contain four fused hydrocarbon rings, 8 to 10 carbon atoms in the hydrocarbon tail attached to carbon 17 and a hydroxyl group at carbon 3
• while ~40% of dietary cholesterol is absorbed by humans only 5% of plant sterols are absorbed
• Beta-sitosterol, a plant sterol, and excess cholesterol upon entering the enterocytes is actively transported back into the intestinal lumen by two members of the ATP Binding Cassette family of transporters
• when either transporter is defective that causes the rare autosomal recessive condition of sitosterolemia
• the accumulated Beta-sitosterol and cholesterol in the enterocytes eventually enter the blood stream Sitosterolemia
• this explains the increased cardiovascular morbidity in people with this disorder
• the drug ezetimbe blocks cholesterol intestinal absorption through the enterocyte brush border

Question 5

Cholesterol Basics

Answer 5

• cholesterol is synthesized by virtually all cells, except RBCs, in the human
• the majority is synthesized by the liver, intestines, adrenal cortex and reproductive tissues
• similar to fatty acid synthesis all carbons in cholesterol are provided by acetyl CoA and NADPH provides the reducing equivalents
• the pathway requires energy that is supplied by hydrolysis of the thioester bond of acetyl CoA and the terminal phosphate of ATP
• the reactions occur on the cytoplasmic surface of the smooth ER and require ER membrane and cytosolic enzymes

Question 6

Acetyl CoA to HMG CoA

Answer 6

• first two molecules of acetyl coA condense, with loss of one CoA, to form Acetoacetyl CoA
• a third molecule of acetyl coA is added by HMG-CoA synthase forming 3-Hydroxy-3-methylglutaryl (HMG) CoA
• the cytosolic form of HMG-CoA synthase, not the mitochondrial isoenzyme, catalyzes this reaction

Question 7

HMG CoA to Mevalone

Answer 7

• key regulatory step
• the next rate limiting and key regulatory step, in the synthesis of cholesterol, is the conversion of HMG CoA to mevalonate
• the reaction is catalyzed by HMG CoA reductase
• the reaction requires 2 molecules of the NADPH as the reducing agent
• CoA is released making the reaction irreversible
• the enzyme is an integral membrane protein of the smooth ER with a catalytic domain facing the cytoplasm
• there is inhibition of enzyme expression by excess cholesterol

Question 8

Mevalonate to Cholesterol

Answer 8

• mevalonate is converted to 5-pyrophosphomevalonate in two steps each of which transfers a phosphate group from ATP
• a five carbon isoprene unit, isopentenyl pyrophosphate (IPP), is formed by the decarboxylation of 5-pyrophosphomevalonate. The reaction requires ATP. IPP is the precursor of a family of molecules, the isoprenoids. Cholesterol is a sterol isoprenoid. Nonsterol isoprenoids include dolichol, coenzyme Q and vitamin K
• IPP is isomerixed to 3,3-dimethylallyl phosphosphate (DPP)
• IPP and DPP condense to form ten-carbon geranyl pyrophosphate (GPP)
• a second molecule of IPP then condenses with GPP to form 15- carbon farnesyl pyrophosphhate (FPP). Covalent attachment of farnesyl to proteins, a process known as prenylation is one mechanism for anchoring proteins to plasma membranes
• two molecules of FPP combine, releasing pyrophosphate, and are reduced forming the 30-carbon compound squalene. Squalene is formed from six isoprenoid units (takes 18 ATP)
• squalene is converted to the sterol lanosterol by a sequence of reactions catalyxed by ER associated enzymes that use molecular oxygen and NADPH. The hydroxylation of squalene triggers the cyclization of the structure to lanosterol
• conversion of lanosterol to cholesterol is a multistep, ER-associated process involving shortening of the side chain, oxidative removal of methyl groups, reduction of double bonds and migration of double bond

Question 9

Smith-Lemli-Opitz Syndrome

Answer 9

• an autosomal recessive disorder of choleterol biosynthesis, iscaused by a partial deficiency of 7-dehydrocholesterol-7-reductase, the enzyme that reduces the double bond in 7-dehydrocholesterol (7-DHC) thereby converting it to cholesterol
• SLOS is one of several multisystem, embryonic malformation syndrome associated with impaired cholesterol synthesis

Question 10

Regulation of HMG CoA Reductase

Answer 10

• expression of the HMG CoA Reductase gene is under the control of a transcription factor SREBP-2 which binds the cis acting sterol regulatory element. SREBP-2, in its inactive form, is an integral ER membrane protein
• it associates with another ER protein SCAP
• when cholesterol levels are low the SREBP-2-SCAP complex moves to the Golgi where it stimulates specific cleavage of SREBP resulting in a soluble fragment that is activated SREBP transcription factor
• the SREBP transcription factor enters the nucleus, binds SRE and stimulates the expression of HMG CoA reductase mRNA transcripts, increasing expression of the enzyme and cholesterol synthesis
• when cholesterol is at high concentration it binds to the sterol sensing domain of SCAP, which binds to additional ER proteins (insigs, insulin induced gene products) which anchor the SREBp-2-SCAP complex to the ER membrane. As a result cholesterol synthesis decreases

Question 11

Enzyme Degradation of HMG CoA reductase

Answer 11

• when cholesterol levels are high they also bind to the sterol-sensing domain of the reductase itself
• this causes binding of the reductase to insigs in the ER membrane which triggers ubiquitination and proteasomal degradation of the enzyme, leading to reduced cholesteol biosynthesis

Question 12

Phosphorlyation/Dephosphorylation of HMG CoA reductase

Answer 12

• the reductase is controlled by AMP-activated protein kinase and a phosphoprotein phosphatase
• the phosphorylated form of enzyme is inactive and the dephosphorylated form is active
• when ATP is low, and AMP high the enzyme is inactive and cholesterol synthesis is reduced

Question 13

Hormones HMG CoA reductase

Answer 13

-insulin and thyroxine upregulate expression of the HMG CoA reductase gene while glucagon and glucocorticoids down regulate expression

Question 14

Statin Drugs

Answer 14

-portions of statins clearly resemble HMG CoA. However the bulky hydrophobic groups of the inhibitors differ from the CoA moiety of the substrate

• they can serve as competitive inhibitors of HMG CoA Reductase
• they lower plasma levels of cholesterol

Question 15

Degradation of Cholesterol

Answer 15

• the ring structure of cholesterol is not metabolized to CO2 and water in humans
• the Sterol Nucleus is eliminated from the body by conversion to bile acids and bile salts
• a small percentage of cholesterol is eliminated in the feces or by secretion into the bile which carries it to the intestine for elimination
• some of the cholesterol in the intestine is modified by bacteria before excretion
• the primary products made are isomers of coprostanol and cholestanol, reduced forms of cholesterol
• the two compounds above and cholesterol make up a majority of neutral fecal sterols

Question 16

Bile Acid Structure

Answer 16

• bile consists of a mixture of bile salts, phosphatidyl choline and other organic and inorganic molecules
• bile can pass from the liver where it is produced, to the duodenum through the bile duct or can be stored in the gall bladder when not immediately needed for digestion
• bile acids contain the sterioid nucleus ring structure with two or three hydroxyl groups and a hydrocarbon side chain with a terminal carboxyl group
• the carboxyl group has a pKa of 6 which is approximate pH of the duodenum lumen. So in the lumen 50% of the molecules are protenated (bile acids) and other 50% is deprotenated (bile salts)
• bile salts and acids have their OH groups below the plain of the sterol ring structure and their methyl groups. The result is that the bile acids and salts have a polar face and a nonpolar face
• as a result they can act as emulfsifying agents in the intestines preparing complex lipids for diestion by pancreatic digetive enzymes
• two most common primary bile acids are shown

Question 17

Bile Acid Synthesis

Answer 17

• bile acid synthesis is a multi-step process involving multiple organelles in hepatic cells
• in these steps OH groups are added to the sterol ring structure, the double bond in the B ring is reduced, the hydrocarbon chain is shortened by 3 carbons introducing a carboxyl group to the end of the chain
• the rate limiting step is the addition of the hydroxyl group at carbon 7 of cholesterol converting it to 7-alpha hydroxylcholesterol
• the reaction is catalyzed by cholesterol-7-alpha hydroxylase and requires O2 and NADPH
• expression of this enzme is down regulated by bile acids

Question 18

Conjugated Bile Salts

Answer 18

• before bile acids leave the liver they are conjugated to either to serine or taurine (a product of systeine catabolism)
• an amide bond forms between the carboxyl group of the bile acid and the amino group of serine or taurine
• the structures formed are glycocholic and glycochenocholic acid and taurocholic and taurochenocholic acids
• the ratio of glycine to taurine form is 3:1
• the addition of carboxyl group (glycine) or a sulfate group (taurine) lower the pKa and are therefore the bile salts are fully ionized at the alkaline pH of bile
• the conjugated ionized bile salts are better detergents than the bile acids because of their increased amphipathic nature
• only the conjugated forms are found in bile

Question 19

Intestinal Flora

Answer 19

• bacteria in the intestine can remove glycine and taurine from conjugated bile salts
• they can also remove the hydroxyl group from carbon 7 producing secondary bile acids

Question 20

Enterohepatic Circulation of Bile Salts

Answer 20

• the 0.5 grams/day of primary and secondary bile salts (<3%) lost in the feces is compensated for by the 0.5 grams/day synthesized from cholesterol in the liver
• the liver secretes bile salts into the bile
• they are reabsorbed in the terminal ileum of the intestine by a Na+-bile salt cotransporter and returned to the blood by a different transporter
• albumin binds and transports the bile salts in the blood
• hepatocytes take up the bile salts from the blood utilizing an isoform of the Na+-bile cotransporter
• the continuous process of release of bile salts synthesized in the liver into bile; their passage through the bile duct to the duodenum where some is deconjuated and dehydroxylated to secondary bile salts; their movement to the ileum where 95% is returned to the liver via the portal vein and 5% becomes part of fecal excretion is termed enterohepatic circulation

Question 21

Cholesterol Gallstone Disease: Cholelithiasis

Answer 21

• the bile salts are produced in the liver and secreted into the bile. They are stored in the gallbladder until released into the intestine during a meal. Thereupon they act as detergents preparing complex lipids for digestion
• the movement of cholesterol into the bile must be accompanied by bile salt and phospholipid secretion
• if the dual secretion process is disrupted by a decrease of bile salt production or increased cholesterol secretion this causes an imbalance where cholesterol cannot be sufficiently
• the result is the precipitation of cholesterol and formation of gall stones, a disorder called cholelelithiasis
• the treatment of choice is laproscopic cholecystectomy