Cholest s er e o r l o ١

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1 Cholesterol ١

2 Contents of The Lecture What is Cholesterol? Structure of Cholesterol Structure of Cholesteryl Ester Normal Cholestrol Level Sources of Cholesterol What Are The Exogenous Sources Of Cholesterol? Absorption Of Exogenous Cholesterol ٢

3 Contents of The Lecture Endogenous Sources Of Cholesterol The Main Role of the Liver in the Production of the Majority of the Body Cholesterol Pool Cholesterol Synthesis Regulation of Cholesterol Synthesis. Cholesterol is Transprted from Intestine to Liver in Lipoproteins ٣

4 Contents of The Lecture How to Utilize Cholesterol? Lipoprotein lipase The Fate of Unused LDL HDL (Good Cholesterol) What Happens if Cholesterol Gets High? How to Treat Hypercholestrolemia? Biological Functions of Cholesterol Cholesterol Catabolism ٤

5 Contents of The Lecture Cholesterol Recycling And Excretion Objective of the Exp. Principle of the Test Materials Method Results ٥

6 What is Cholesterol? Cholesterol is the most prominent member of steroid family of lipids. Cholesterol is found principally in animals and humans, where it is also the main sterol. ٦ Small quantities are sythesized in other eukaryotes, such as plants and fungi..

7 What is Cholesterol?...cont. ٧ Virtually all human cells and body fluids (including plasma) contain some cholesterol either as free cholesterol or as a storage form: cholesteryl ester in which cholesterol is combined with a long chain fatty acid ( at C-3), which makes the structure of cholesterol even more hydrophobic.

8 What is Cholesterol?...cont. ٨ Cholesterol is an amphipathic lipid, and, therefore, it becomes an essential structural component: 1. Of membranes of cellular organelles. 2. Of the outer layer of plasma membranes.

9 What is Cholesterol?...cont. Measuring cholesterol concentration is of clinical and analytical importance since: High levels of plasma cholesterol is associated with Cardiac Vascular Diseases. ٩

10 Structure of Cholesterol - Cholesterol is a sterol because it s a steroid and alcohol at the same time. Cholesterol is a solid alcohol of high molecular weight. ١٠ Cholesterol, like other steroids, is a derivative of the cyclic hydrocarbon Perhydrocyclopentanophenanthrene.

11 Perhydrocyclopentanophenanthrene ١١

12 Structure of Cholesterol.cont. Cholesterol consists of four rings. These rings are identified by the first four letters of the alphabet. - The carbons of cholesterol which are 27 are numbered in the sequence shown in figure 1. ١٢

13 Figure.1. Cholesterol Structure ١٣

14 Structure of Cholesterol.cont. - In addition to the : 1. Basic ring structure 2. Cholesterol contains a hydroxyl group at C-3 3. An aliphatic chain of 8 carbons at C-17 of ring D. 4. Methyl groups at C-10 and C A 5 double bond. ١٤

15 Structure of Cholesterol C D A B ١٥

16 Structure of Cholesteryl Ester Cholesteryl Esters Much of the plasma cholesterol is in an esterified form. ١٦

17 ١٧ Cholesteryl Ester Structure

18 Normal Cholestrol Level Desirable total blood cholesterol level less than 200 mg/dl (5.13 mmol/l). About 50% of all adults fall into this cholesterol level. Borderline high risk total blood cholesterol level mg/dl ( mmol/l). About 33% of adults fall into this cholesterol level. High risk total blood cholesterol level 240 mg/dl (6.14 mmol/l) and over. You have twice the risk of coronary heart disease. About 17% of adults are in this cholesterol level. ١٨

19 Sources of Body Cholesterol Intestine Cholesterol Cholesterol in the intestine comes from diet, bile, and intestinal secretions. Therefore intestine cholesterol comes from 2 major sources: (1) Exogenous: ( diet ) ١٩ (2) Endogenous: ( bile and intestinal secretions)

20 Major Sources of Liver Cholesterol 1. Dietary cholesterol Chylomicron remnants 3. Cholesterol synthesized in extrahepatic tissues 2. De novo synthesis in liver HDL Liver Cholesterol Pool Secretion of HDL and VLDL ٢٠ Free cholesterol secreted in bile Conversion to bile acids/salts Major routes by which cholesterol leaves liver

21 Sources of Body Cholesterol Liver Cholesterol ٢١ Cholesterol in the liver comes from 2 major sources: (1) Exogenous: ( diet ) (2) Endogenous: (de novo synthesis in liver and cholesterol synthesized in extrahepatic tissues

22 What Are The Exogenous Sources of Cholesterol? As a typical product of animal metabolism, cholesterol occurs in foods of animal origin such as egg yolk, meat, liver, seafood, whole fat dairy products and brain. These animal products provide the bulk of dietary cholesterol. ٢٢

23 What Are The Exogenous Sources of Cholesterol?...cont. However the fat found in food is composed mainly of triglycerides of about 98 99% and only the remaining 1% to 2% of the lipids include cholesterol and other lipids. ٢٣

24 Absorption Of Exogenous Cholesterol Practically cholesterol in the intestine is present in the unesterified (free) form, since esterified cholesterol is rapidly hydrolyzed in the intestine by cholesterol esterase which exists in ٢٤ pancreatic + small intestinal secretions.

25 Intestinal Absorption of Exogenous Cholesterol cont In order to be absorbed, cholesterol is soluibilized by formation of mixed micelles containing : (1) Unesterified cholesterol (2) Fatty acids (3) Monoglycerides (monoacylglycerols) (4) Phospholipids (lysolecithin) (5) Conjugated bile acids. These micelles also facilitate cholesterol transport across the luminal cell surfaces. ٢٥

26 Absorption Of Exogenous Cholesterol.cont. The liver and gut are connected by a cystic duct which empties bile from the gallbladder into the gut. The bile is formed in the liver, and stored in the gall bladder until a meal with fat enters the upper small intestine. Bile solubilize cholesterol (and other lipids) make them readily absorbed by intestinal cells. ٢٦

27 Absorption Of Exogenous Cholesterol cont In order to be absorbed, cholesterol is soluibilized by formation of mixed micelles containing : (1) unesterified cholesterol, (2) fatty acids, (3) monoglycerides (monoacylglycerols), (4) phospholipids (lysolecithin) and (5) conjugated bile acids. These micelles also facilitate cholesterol transport across the luminal cell surfaces. ٢٧

28 Absorption Of Exogenous Cholesterol.cont. In the absence of bile acids, digestion and absorption of both cholesterol and triglycerides are severely impaired. On the average 30-60% of dietary and intestinal cholesterol is absorbed daily, to a maximum of ~1 g/d when the oral intake reaches 3 g/d ٢٨

29 -Endogenous Sources Of Cholesterol Cholesterol is synthesized by virtually all tissues in humans. Liver is the major site of cholesterol synthesis. Intestine is also a main site of cholesterol synthesis after the liver ٢٩

30 -Endogenous Sources Of Cholesterol..cont. Adrenal cortex and reproductive tissues including ovaries, testes and placenta make the largest contributions to the body s cholesterol pool after the liver and intestine. ٣٠

31 Endogenous Sources Of Cholesterol cont. -In adults the liver and intestinal wall (gut) propably supply over 90% of the plasma cholesterol of endogenous origin. -Almost all animal tissues synthesize cholesterol from acetyl Co.A. ٣١

32 The Main Role of the Liver in the Production of the Majority of the Body Cholesterol Pool Of endogenous cholesterol, up to 75% of cholesterol in the body is produced by the liver. ٣٢

33 The Main Role of the Liver in the Production of the Majority of the Body Cholesterol Pool cont. In other words, the liver synthesizes cholesterol even in the total absence of cholesterol in the diet. With an increased intake of cholesterol in the diet, there is a somewhat reduced synthesis of cholesterol in the liver, keeping the total cholesterol pool roughly constant.. ٣٣

34 Cholesterol Synthesis Similar to ketogenic pathway Occurs in cytosol Requires NADPH and ATP Highly regulated 80 % in liver, ~10% intestine, ~5% skin ٣٤

35 De Novo Synthesis Of Cholesterol -Mevalonate Synthesis: 1- The sequence of cholesterol biosynthesis begins with a condensation in the cytosol of two molecules of acetyl Co.A. A reaction catalyzed by thiolase. ٣٥

36 De Novo Synthesis Of Cholesterol Mevalonate Synthesis.cont. 2- The next step requires the enzyme β- hydroxy-β - methylgularyl-co.a (HMG-CoA) synthase. This enzyme catalyzes the condensation of a third acetyl-coa with β- ketobutyryl-coa to yield HMG CoA. 3- HMG-CoA is then reduced to mevalonate by HMG-CoA reductase. The activity of this reductase is primarily responsible for control of the rate of cholesterol biosynthesis. ٣٦

37 De Novo Synthesis of Cholesterol HMG.Co.A Sythase HMG.Co.A Reductase ٣٧

38 Biosynthesis of Cholesterol..cont. After the Formation of Mevalonate. The biosynthesis of cholesterol (from mevalonate) may be divided into 4 setps. ٣٨

39 Biosynthesis of Cholesterol cont. After the Formation of Mevalonate cont. 1. Formation of isoprenoid (isoprene units),isopentenyl pyrophosphate (5C) from mevalonate by loss of CO 2. ٣٩ 2. Condensation of six isoprenoid units form squalene (30C).

40 ٤٠ Cholesterol Synthesis

41 Biosynthesis of Cholesterol cont. After the Formation of Mevalonate.cont. 3. Cyclization of squalene give rise to the parents steroid, lanosterol by cyclase. 4. Formation of cholesterol from lanosterol (30C) by removal of 3 methyl groups. ٤١

42 De Novo Synthesis of Cholesterol HMG.Co.A Sythase HMG.Co.A Reductase ٤٢

43 ٤٣ Cholesterol Synthesis

44 Biosynthesis of Cholesterol..cont. The Rate-Limitting Step Cholesterol synthesis is controlled by regulation of the rate of mevalonate. Synthesis, i.e., the synthesis is controlled by regulation of HMG-CoA reductase. ٤٤

45 The Rate- HMG.Co.A Reductase Limitting Step in De Novo Synthesis of Cholesterol ٤٥

46 Biosynthesis of Cholesterol..cont. The Rate-Limitting Step cont. Therefore HMG-CoA is an important intermediate for the biosynthesis of cholesterol. -The biosynthesis of cholesterol is catalyzed by enzymes in the cytosol and enzymes including HMG Co.A reductase, bound to the endoplasmic reticulum. ٤٦

47 Regulation of Cholesterol Synthesis. 1. Dietary Factors: Reduced synthesis of cholesterol during starvation is accompained by a decrease in the activity of the enzyme. Therefore, we can say that hepatic HMG- CoA reductase is regulated by some dietary factors. ٤٧

48 Regulation of Cholesterol Synthesis..cont.. 2. Feedback Inhibition: HMG-CoA reductase in liver is inhibited by mevalonate, the immediate product of the pathway, and by cholesterol, the main product. However, it is only hepatic synthesis that is inhibited by dietary cholesterol. Feedback control of hepatic cholesterogenesis is also mediated, directly or indirectly by bile ٤٨ acids.

49 Regulation of Cholesterol Synthesis By Feed-Back Inhibition of these Substances ( Intermediates) ٤٩

50 Regulation of Cholesterol Synthesis..cont. 3. Hormonal Regulation: Both Glucagon and glucocorticoids decrease HMG-CoA reductase activity. ٥٠

51 Regulation of Cholesterol Synthesis..cont. 4. Sterol- Mediated regulation of transcription: The synthesis of cholesterol is also regulated by the amount of cholesterol taken up by the cells during lipoprotein metabolism. ٥١

52 Regulation of Cholesterol Synthesis cont. 4. Sterol- Mediated regulation of transcription cont. 1) Chylomicron remnants internalized by liver cells, and 2) low density lipoproteins (LDL) internalized by cells of the liver and peripheral tissues, to provide cholesterol, which causes a decrease in transcription of the HMG-CoA reductase gene, leading to a decrease in the de novo synthesis of cholesterol. ٥٢

53 Regulation of Cholesterol Synthesis.cont. 5. Inhibition by Drugs (statins): Lovastatin and mevastatin are reversible, competitive inhibitors of HMG-CoA reductase, They are used to decrease plasma cholesterol levels in patients with hypercholestrolemia. ٥٣

54 Cholesterol is Transprted from Intestine to Liver in Lipoproteins The liver and the gut (intestine) are the two major organs of lipoprotein synthesis and transport. ٥٤

55 Cholesterol is Transprted from Intestine to Liver in Chylomicrons Bile salts in the bile help in emulsifying the fats so they can be more easily digested and absorbed in the gut. Within the epithelial cells of the gut wall, a combination of triglycerides, and cholesterol, and fatty acids are coated with protein to form large lipoproteins known as ٥٥ chylomicrons.

56 Structure of Lipoproteins ٥٦

57 ٥٧ Structure of Chylomicrons

58 Cholesterol is Transprted from Intestine to Liver in Chylomicrons cont. Chylomicrons Chylomicrons are absorbed into the lymphatic system, which eventually drains into the circulation. Chylomicrons then goes to the liver where they are broken down to contribute to the triglyceride and the cholesterol pool within ٥٨the liver.

59 Cholesterol is Transprted from Liver to Other Body Tissues by VLDL Lipoproteins The use of cholesterol by the liver is to supply cholesterol for the rest of the body s needs. In order to do this, cholesterol from the liver s pool is combined with triglycerides and coated with a special protein that makes it soluble in our blood. These rather large molecular weight structures are called very low density lipoproteins (VLDL=Cholestrol+ Cholestryl ester + TG + additional lipids), VLDL is released from the liver into the circulation ٥٩ to supply the body s needs for triglycerides and cholesterol.

60 Structure of Lipoproteins: VLDL,IDL,LDL,and HDL ٦٠

61 How to Utilize Cholesterol? The liver s cholesterol pool, regardless of the source, is used in two major ways. 1. It s used to form bile salts. ٦١

62 Sources of Cholesterol Diet De novo synthesis Cholesterol synthesized in extrahepatic tissues Liver cholesterol pool Secretion of HDL and VLDL ٦٢ Free cholesterol In bile Conversion to bile salts/acids

63 How to Utilize Cholesterol? The liver s cholesterol pool, regardless of the source, is used in two major ways cont. 2. Exporting cholesterol to extra-hepatic tissues in VLDL lipoproteins. ٦٣

64 Lipoprotein Lipase Lipoprotein lipase is an enzyme found throughout the body, with high amounts in the arterial walls. Lipoprotein lipase helps to strip off triglycerides from the VLDL. Those triglycerides can be used in various tissues throughout the body. ٦٤

Lipoprotein lipase cont. As triglycerides are removed from the VLDL, the VLDL gets smaller and becomes enriched with a higher percentage of its composition as cholesterol.

65 Lipoprotein lipase cont. As triglycerides are removed from the VLDL, the VLDL gets smaller and becomes enriched with a higher percentage of its composition as cholesterol. In this stage the lipoproteins are referred to as intermediate density lipoproteins,(idl = VLDL- some of TG). Further removal of triglycerides from IDL enriches the percentage of cholesterol even more, and results in an even more compact molecule called low density lipoprotein (LDL=IDL all of the TG) ٦٥

66 The Fate of Unused LDL The regulated receptors on the liver will help return some of the cholesterol to the liver from unused LDL.. LDL is the so-called bad cholesterol and genetic differences in these regulated receptors can result in the accumulation or large amounts of LDL in the circulation (familial hyper-cholesterolemia). ٦٦

67 The Fate of Unused LDL ٦٧

68 HDL Good Cholesterol Another lipoprotein formed by the liver is called high density lipoprotein (HDL), sometimes referred to as the good cholesterol. High density lipoprotein is formed with very small amounts of cholesterol, small amounts of triglyceride and a special protein coat within the liver that makes it distinct from the VLDL, also formed in the liver. ٦٨

69 HDL Good Cholesterol..cont. The HDL in the circulation acts like a sponge in picking up excess cholesterol from tissues that normally metabolize cholesterol but are receiving more than they can possibly use. An enzyme important in this process of reverse cholesterol transport is called lecithin-cholesterol acyl transferase. LCAT stimulates reverse cholesterol transport from tissues that have excessive amounts of cholesterol into the HDL molecules ٦٩

70 Classification of Lipoproteins Bad (non-hdl) Good ٧٠

71 Good Vs. Bad Lipoprotein plasma lipoproteins, there are good and bad forms of lipoproteins in terms of their effect in the formation of atherosclerotic plaque. ٧١

72 Hypercholesterolemia Causes Atherosclerosis ٧٢

73 Maturing HDL As HDL absorbs cholesterol from many tissues it becomes mature and it returns to the liver. The lipoprotein coat of HDL helps the liver to recognize it, and direct the cholesterol to the liver pool. ٧٣

74 Cholesterol is Transported by Different Lipoproteins ٧٤

75 What Happens if Cholesterol Gets High? Increases the risk of vascular disease..mainly IHD. What are the risk factors of IHD? -Hyperlipidemia ( hypercholesterolemia) -Hypertension. -Diabetes mellitis. -Smoking. - Obesity - +ve family history. ٧٥

76 HOW TO TREAT HYPERCHOLESTEROLEMIA ٧٦

77 HOW TO TREAT HYPERCHOLESTEROLEMIA 1) INCREASE YOUR "GOOD" CHOLESTEROL LEVELS: High-density lipoproteins (HDL) or "good" cholesterol, carries bad cholesterol away from the arteries and heart and back to the liver where it can be used by the body. Replacing saturated fats with mono- or poly- unsaturated fats found in nuts and vegetable oils will help raise HDL levels. ٧٧

78 HOW TO TREAT HYPERCHLESTLEMIA.Cont. 2) MAINTAIN A HEALTHY BODY WEIGHT: Individuals who are overweight should aim to decrease their weight with regular physical activity and a balanced, healthy diet. Weight management and cholesterol control go hand in hand. ٧٨

79 HOW TO TREAT HYPERCHOLESTEROLEMIA.Cont. 3) EAT A DIET HIGH IN FIBER: Fiber binds to excess cholesterol in the intestine and removes it from the body. Soluble fiber found in oatmeal and whole grains can help to control cholesterol levels. ٧٩

80 Cholesterol Metabolism - Biological functions: Because of the well established positive association between plasma cholesterol concentration and coronary heat disease (CHD), it may be thought that of cholesterol as a harmful substance. ٨٠

81 Biological Functions of Cholesterol..cont. - Cholesterol is essential for normal functioning of the organism because it is: 1. An essential structural component of the membranes of all animal cells and sub -cellular particles. ٨١

82 Biological Functions of Cholesterol..cont. I- Cholesterol increases the fluidity of plasma membranes. II. It is required to establish proper membrane permeability, it reduces the permeability of plasma membrane to H + and Na + ٨٢

83 Biological Functions of Cholesterol...cont. 2. An obligatory precursor of bile acids. In the liver, cholesterol is converted to bile. Bile contain bile salts which solubilize fats in the digestive tract and aid in the intestinal absorption of fat molecules as well as fat soluble vitamins: Vitamin A, Vitamin D, Vitamin E and Vitamin K. ٨٣

84 Biological Functions of Cholesterol...cont. 3. A precursor of all steroid hormones : I) Sex hormones: progesterone, estrogens, and testosterone and their derivatives. II) Adrenal gland hormones : cortisol and aldesteron. ٨٤

85 Sex Hormones Derived from Cholesterol ٨٥

86 ٨٦ Adrenal Gland Hormones

87 Cholesterol Catabolism - The ring structure of cholesterol cannot be metabolized to CO 2 and H 2 O in humans. ٨٧

88 Cholesterol Catabolism After cholesterol enters the cell, the esters are hydrolyzed by the action of specific lysosomal esterases. The lack or malfunction of these lysosomal enzymes results in intracellular accumulation of cholesteryl esters and produces a clinical disorder known as cholesteryl ester storage disease. ٨٨

89 Cholesterol Recycling And Excretion Cholesterol is oxidized by the liver into a variety of bile acids (about 30 / d). The bile acid synthesis rate average mg /d. Bile salts are bile acids conjugated with glycine, taurine, glucuronic acid, or sulfate. A mixture of conjugated and non-conjugated bile acids along with cholesterol itself is excreted from the liver into the bile. Approximately 95% of the bile acids are reabsorbed from the intestines and the remainder lost in the feces. [ ٨٩

90 Cholesterol Recycling And Excretion The excretion and reabsorption of bile acids forms the basis of the enterohepatic circulation which is essential for the digestion and absorption of dietary fats. Under certain circumstances, when more cholesterol is concentrated, as in the gallbladder, cholesterol crystallises and is the major constitute of most gallstones. ٩٠

91 Principle of the Test. -In this experiment cholesterol is estimated by a chemical method rather than an enzymatic method. -This method is qualitative and quantitative one. ٩١

92 Principle of the Test..cont. - In this method: Cholesterol reacts as a typical alcohol with strong, concentrated acid. The products are green-colored substances, mainly cholestapolyenes and cholestapolyene carbonium ions (Liebermann Burchard reaction) ٩٢

93 Principle of the Test.cont. -In this procedure cholesterol reacts with : 1. Acetic acid and acetic anhydride which act as solvents and dehydrating agents. 2. Sulfuric acid that used as a dehydrating and oxidizing reagent. - The absorbance of the green compound is then measured at 610nm. ٩٣

94 Materials 1.Cholesterol reagent (Acetic anhydride + acetic acid) 2.Sulphuric acid 95-97% 3.Standard cholesterol (300 mg/dl) 4. Samples 5. Test Tubes 6. Pipettes 7. Cuvettes 8. Spectrophotometer 9.Water bath ٩٤

95 Method 1- Label 7 test tubes for test 1(A,B) test 2 (C,D) standard (E,F) and blank (O) pipette the following (A,B) (C,D) (E,F) (Blank) Sample ml Sample ml Standard Cholesterol 0.1 ml Distilled Water 0.1 ml Cholesterol reagent 4 ml 4 ml 4 ml 4ml ٩٥

96 Method cont. 2- Stand for 20 minutes 3- Pipette 1.0 ml of sulphuric acid into each tube running the acid cautiously down the side of the tube. 4- Place the tube in a water bath at room temperature. 5- After around 5 minutes remove from the water bath and shake vigorously to remove any adhered proteins to the walls of the tubes, 6- Stand for 10 minutes then measure the absorbance of the samples against the blank at 610nm. ٩٦

97 Results Sample 1 Absorbance A = B = Sample 2 Absorbance C = D = Standard Cholesterol Absorbance E = F = ٩٧

98 Results Absorbance of samples X 300 = mg/dl Absorbance of standard The average of normal levels of cholesterol is mg/dl ٩٨

99 ٩٩

Cholesterol metabolism. Function Biosynthesis Transport in the organism Hypercholesterolemia

Cholesterol metabolism. Function Biosynthesis Transport in the organism Hypercholesterolemia Cholesterol metabolism Function Biosynthesis Transport in the organism Hypercholesterolemia - component of all cell membranes - precursor of bile acids steroid hormones vitamin D Cholesterol Sources: dietary