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1 number 29 Done by Ali Yaghi Corrected by Shahd Alqudah Doctor Faisal Al-Khatibe In this lecture we will continue the steps of synthesizing cholesterol. in the previous sheet we reached the step of forming squaline. P age1

squaline is a compound composed of 30 carbons, it is a hydrocarbon molecule(formed from carbon+ hydrogen only),therefore it is hydrophobic and nonpolar.

2 squaline is a compound composed of 30 carbons, it is a hydrocarbon molecule(formed from carbon+ hydrogen only),therefore it is hydrophobic and nonpolar. Note: In the cell, squaline it is maintained in the soluble form by binding to a protein. Steps of synthesizing cholesterol from squaline: 1- Adding oxygen to squaline to form squaline 2,3-epoxide 2- Cyclation of squaline2,3-epoxide to form Lanosterol 3- Reduction of 7-dehydrocholesterol to form cholesterol squaline The first step of synthesizing cholesterol from squaline is adding oxygen by an oxygenase enzyme to form squaline 2,3 epoxide (oxygen is connected to carbon 2 and 3, which makes it unstable as the angle is 60 degree). The next step is the cyclation step by cyclase enzyme to form lanosterol in several steps reaction involved in the closure of the rings. Note: The triangle(epioxide) makes it very unstable that s what drives the cyclation reaction. P age2

3 Note: Lanosterol is composed of 30 carbons: 4 rings (3 of them are 6membered rings connected to each other, and the last one is a five membered ring) with a hydrophobic tale. Note: Lanosterol is the first steroid intermediate. Note: The conversion of lanosterol to cholesterol is a multistep process involving shortening of the side chain, oxidative removal of methyl groups, reduction of double bonds, and migration of a double bond. we have to understand the last step of forming cholesterol from lanosterol only. The other steps aren't required from us. The last step of synthesizing cholesterol is the reduction of 7- dehydrocholestrol (the last intermediate) by NADPH into cholesterol Note: 7-dehydrocholestrol is a precursor in synthesis of vitamin d (synthesized in the body upon exposure to sunlight ). Revision for synthesizing cholesterol: P age3

4 1-3 Acetyl co-a condensed to give HMG CoA 2- HMG CoA is reduced by HMG CoA reductase to Mevalonate. 3- Mevalonate can be phosphorylated by the addition of 2 phosphates then decarboxylation to isopentenyl Pyrophosphate that has 5 carbons. 4- Isopentenyl Pyrophosphate can be condensed with its isomer to give Geranyl Pyrophosphate 5- Geranyl Pyrophosphate is condensed with another isopentenyl Pyrophosphate to give FarnesylPyrophosphate (C15 ). 6- Condensing 2 FarnesylPyrophosphate together forms Squaline 7- Squaline is modified further to give squaline 2,3 epoxide. 8- Cyclase enzyme converts squaline 2,3 epoxide to lanosterol. 9- Several reactions happen to form 7-dehydrocholestrol (the last intermediate) dehydrocholestrol is reduced by NADPH-dependent reductase. Note: Condensation step happens by removing pyrophosphate (which drives this reaction) and joining 2 compounds together. Synthesis of bile acids Note: Bile acids can be synthesized from cholesterol in similar steps. As example we are going to talk about cholic acid Notes: 1- Cholic acid differs from cholesterol by having 2 extra hydroxyl groups ( on c7 & c12). 2- The side chain is reduced from 8 carbons in cholesterol to 5 carbons in cholic acid. 3- And the side chain is oxidized to carboxyl group (carboxylic acid). 4- The dashed line means that the hydroxyl group is below the compound(organic chemistry). This means that cholic acid has 2 sides, one is hydrophilic composed of OH and the other is hydrophobic composed of CH3. Therefore cholic acid is an amphipathic molecule that helps in emulsification. 5- pka for cholic acid is close to 6. This explains how that molecule is found in the protonated and deprotonated (cholate) form. P age4

5 The synthesis of cholic acid is catalyzed by cholesterol 7 alpha hydroxylase enzyme which adds hydroxyl group on carbon 7. Note: 1- This is the rate limiting step 2- This step is inhibited by cholic acid (end product) and activated by cholesterol. Synthesis of Bile salts: ** Glycine can be added to cholic acid and will be named glycocholic acid ( a bile salt), and the COO- of glycocholic acid is much stronger (pka is 2) than cholic acid because of the prescence of the amide group. This means that glycocholic acid will be found mainly in the ionized form (a bile salt). **Taurine (sulfuric acid has SO4) can be added to chenodeoxycholic acid (bile acid) to form taurochenodeoxycholic acid (bile salt) Note: Taurine can be formed from cystine metabolism Note: Chenodeoxycholic acid has 2 hydroxyl groups only (OH on carbon 12 may or may not be there). Note: at least we have 2 bile acids (cholic acid, chenodeoxycholic acid) these are called primary bile acids. When they are combined with Glycine or Taurine they become primary bile salts. The Enterohepatic circulation. P age5

1- Bile salts are secreted through the bile either in the gall bladder or small intestine( In the small intestine, the primary bile salts can act as emulsifier).

6 1- Bile salts are secreted through the bile either in the gall bladder or small intestine( In the small intestine, the primary bile salts can act as emulsifier). 2- Primary bile salts become separated from glycine and taurine and get converted to primary bile acids. 3- Primary bile acids are converted by some bacteria to secondary bile acids by removing a hydroxyl group from carbon 7. Note: both primary and secondary bile acids can work as emulsifiers. 4- In the Ilium, the primary and secondary bile acids can be reabsorbed through the portal vein and go back to the liver 5- In the liver, primary and secondary bile acids become conjugated again to Glycine and Taurine to resynthesize the primary and secondary bile salts. Note: Every day it is estimated that g of bile salt are rotating many times in the enterohepatic circulation. Each time, some of the primary and secondary bile salts (about.5 g) escape the enterohepatic circulation and they are excecreated in the feaces. Note: The excreted.5 g is compensated by the synthesis of new primary and secondary bile salts from cholesterol. [.5 g are synthesized from cholesterol and excreted in feaces each time] Lowering cholesterol levels: Why do we need that? It was found that high level of cholesterol is associated with atheloscelerosis(it is a risk factor). But don't forget that cholesterol is essential for our cells and the absence of it causes Distortions. Note: in normal adults the total cholesterol is 200 mg/100 ml P age6

Lowering Cholesterol Level in the Plasma Dietary 1- decrease Cholesterol intake 2- increase PUSFA / SFA (poly unsaturated fatty acids/ saturated fatty acids) 3- increase Fiber intake 4-daily

7 Lowering Cholesterol Level in the Plasma Dietary 1- decrease Cholesterol intake 2- increase PUSFA / SFA (poly unsaturated fatty acids/ saturated fatty acids) 3- increase Fiber intake 4-daily Ingestion of Plant Steroid Esters Inhibition of Synthesis Decrease Enterohepatic Circulation of Bile Acids Notes about dietary ways of decreasing cholesterol intake: 1- If we decrease cholesterol intake, synthesis will increase. 2- Fibers are indigestible such as cellulose 3- Plant Steroid Esters lowers cholesterol absorption 4- These are not effective ways for lowering cholesterol levels. 5- Inhibition of synthesis is the most effective way How can we inhibit cholesterol synthesis? HMG CoA reductase enzyme inhibition will inhibit cholesterol synthesis Simvastatin drug has a similar shape for HMG so it competes with HMG for the enzyme in a competitive inhibition. There are many drugs that can be used to inhibit the HMG reductase enzyme, all of them end with statin ( such as atrovastatin,..) Bile sequestering agents We know that liver converts cholesterol to bile acids, and this conversion is inhibited by bile acids, so if we ingest a substance that binds to bile acids such as (cholestyramine) it will prevent their reabsorbing. As a result, the amount of bile acids secreted in the feces will increase and the reabsorbed will decrease. The decreasing of reabsorbing of bile acids will increase the conversion of cholesterol to bile acids and as a result decreases the amount of cholesterol. P age7

Esterification of Cholesterol in the Cells Note: Cholesterol ester is a storage form of cholesterol, while cholesterol is found in the plasma memberane. How it is done?

8 Esterification of Cholesterol in the Cells Note: Cholesterol ester is a storage form of cholesterol, while cholesterol is found in the plasma memberane. How it is done? 1- The form of fatty acid that is used in esterfication is the active form (fatty acyl co-a) 2- The fatty acyl co-a is added to OH of the cholesterol by the enzyme acyl co-a cholesterol:acyl transferase(because it is transferring acyl group from acyl coa to cholesterol). 3- The esterified cholesterol is never used in the plasma membrane because it lost its amphipathicity (it is esterified). The esterification of cholesterol in the plasma The esterification of cholesterol happens also in the plasma, but in the plasma there is no Acyl CoA transferase (it is found only in the cells), so the esterification happens in the HDL. The source of cholesterol that will be esterified in the plasma is HDL, and the source of fatty acids (to make acyl co-a ) is the phospholipids (lecithin). P age8

9 Fatty acid at number 2 is transferred from lecithin to cholesterol by the enzyme Lecithin:Cholesterol Acyl Transferase (LCAT) which is found in the HDL. Note: What is remained from lecithin is called lysolecithin (lysophosphatidic acid)[ the fatty acid is taken from c2] Regulation of Cholesterol Synthesis (regulation of the enzyme HMG co-a) Feedback inhibition: If the cholesterol amount is high, it will inhibit HMG CoA reductase enzyme (the third enzyme in synthesizing cholesterol) Regulation of Gene Expression Covalent Modification Hormonal Regulation Proteolytic Regulation 1- Gene expression: The gene of any enzyme is found on the DNA in the nucleus of all cells, but not all cells will express and transcripe this gene. The transcription of the gene and producing mrna is activated by transcriptional factors (proteins bind to DNA that allow the gene to be transcribed). There is something called sterol regulatory element(sre) that is a sequence of DNA found just before the gene.the gene can't be transcribed unless it has this regulatory element. The transcriptional factor of the gene that produces HMG CoA reductase is called SREBP (sterol regulatory element binding protein). The SREBP binds to SRE and initiate the transcription of the gene. When cholesterol level is decreased, the SREBP will dissociate from the ER and bind to the SRE on the DNA, this leads to transcription of the gene and synthesis of mrna which will be translated to HMG co-a reductase enzyme. 2- Covalent modification: HMG CoA reductase enzyme is present in 2 states: Active( dephosphorylated ) and inactive(phosphorylated). The addition of phosphate group to HMG CoA is catalyzed by enzyme kinase, and the removal of is is catalyzed by phosphatase. P age9

Note: Protein kinase is activated by AMP, when AMP level increases, it binds to protein kinase leading to phosphorylation of the HMG co-a reductase.

10 Note: Protein kinase is activated by AMP, when AMP level increases, it binds to protein kinase leading to phosphorylation of the HMG co-a reductase. Note: Adding phosphate group means low glucose level (energy level is low so it is not the time to make cholesterol which is important for cell division, growth, etc.. which happen when the body is in high energy state ) Note: AMP dependent protein kinase is regulated by insulin/glucagon and epinephrine ratio. If the insulin level is high, it will activate protein phosphatase(remove phosphate group from HMG co-a reductase) and inhibit protein kinase. 3- Hormonal regulation: High Glucagon: Phosphorylated Form ( kinase) High Insulin: Dephosphorylated Form ( Phosphatase) 4- Proteolitic regulation: high cholesterol level increases the rate of degredation of the HMG CoA reductase enzyme(proteolysis) Transport of Cholesterol in the Blood It is mentioned in previous sheets *** Extra hepatic tissue receives LDL from liver by endocytosis - Dietary cholesterol is transported from small intestine to the liver by chylomicron remnants. P age10

-VLDL is produced in liver and it carries cholesterol and then it is converted to IDL by removing TAGs. - IDL is taken into the liver. - LDL is taken by extrahepatic tissues and liver.

11 -VLDL is produced in liver and it carries cholesterol and then it is converted to IDL by removing TAGs. - IDL is taken into the liver. - LDL is taken by extrahepatic tissues and liver. *** When we measure the cholesterol, we measure the total level, but we can't know the risk factor in this way. The most important thing is to know the level of LDL and HDL LDL endocytosis 1- The LDL receptor is clustered on a region of the plasma membrane called the pit, and these pits are coated (from the inside) with protein called Clathrin. This allows the LDL to enter the cell with the help of Apolipoprotein B- 100 receptor. 2- The next step is endocytosis and a coated vesicle is formed. P age11

12 3- The Clathrin is recycled, and the vesicles can be joined together and form an endosome. 4- The ph in the vesicle is decreased by continuous pumping of protons into the vesicles. The increased acidity leads to separation of apolipoprotein from the receptor. 5- The receptors are recycled to the plasma membrane and the endosome is combined with the lysosome (which has hydrolytic enzymes) that degrades the lipids and the apolipoproteins. The digestion of endosome produces: 1- Aminoacids result from apolipoprotein degradation. 2- Fatty acids are from cholesterol ester degradation. 3- And free cholesterol: used to make cell membranes or to make bile salts in the liver orto make steroid hormones. Notes: 1- The oversupply of the cholesterol stimulates the enzyme ACAT which stimulates the storage of cholesterol and inhibition of HMG CoA reductase (to inhibit the synthesis of cholesterol). 2- It also inhibits the synthesis of LDL receptors. 3- If the LDL remains out of the cell for a long time, it might get damaged by oxidation. If it is damaged by oxidation, it won't be taken by LDL receptors which leads to its accumulation. 4- In order not to accumulate, there is another receptor that is found on the macrophages is called macrophage scavenger receptor. This receptor can bind to damaged LDL or LDL can't be taken by LDL receptors. 5- This receptor isn't regulated by the amount of cholesterol, so macrophages can take LDL until it is converted to foam cells. 6- Accumulation of foam cells in the subendothelial space is an Early evidence of atherosclerosis. Familial Hypercholesterolemia It is an increment of cholesterol because of genetic disease in some families. It can be inherited from one parent of from both of them. Homozygotes 680 mg/dl (from both parients) [no LDL receptors] P age12

13 Heterozygotes 300 mg/dl (from one parient) [half amount of normal LDL receptors] The cause of this disease is the absence of LDL receptor or abnormal receptors. Which can't carry the endocytosis. Accumulation of IDL leads to increase of LDL, which causes rapid forming of foam cells. The patient dies from myocardial infarction before the age of 20 in homozygotes. (الدكتور حكا ادرسوهم من الكتاب) HDL Origin: Liver and Intestine It has Nascent Discoid Shapeand it has a lot of phospholipids. The green is phospholipids The red dots are apoliporpotein A1 After the synthesis of HDL, it stores more cholesterol inside it by cholesterol esterification (which esterifies cholesterol on the membrane and stores it inside the cell ),and the HDL becomes spherical shape. HDL ends in the liver, so we can say that HDL transports cholesterol different tissues to the liver, so it is extremely important and it is a sign of good health. unlike the LDL that its increment is a sigh of bad health (because ir transports cholesterol from the liver to different tissues and blood vessels). P age13

Cholesterol Metabolism

Cholesterol Metabolism Lippincott s Illustrated Review Chapter 18 Steroid Nucleus 1 2 Cholesterol was isolated from gall bladder stones in 1774 3 Sources and Elimination of Cholesterol Synthesis: 1000