Cholesterol

1

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 Cholesterol2

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

3

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 Catabolism4

Contents of The Lecture

• Cholesterol Recycling And Excretion

• Principle of the Test

• Materials

• Method

• Results

5

• What is Cholesterol?

-Cholesterol is the most prominent member of steroid family of lipids

-Cholesterol is present in tissues and in plasma either as free cholesterol or as a storage form, combined with a long chain fatty acid as cholesteryl ester.

6

-The term lipid applies to a class of compounds that are soluble in organic solvents and nearly insoluble in water.

-Cholesterol is an amphipathic lipid, and , therefore, it becomes an essential structural component of membranes and of the outer layer of plasma membranes.

What is Cholesterol?.....cont.

7

• Only a limited number of numerous different lipids are of clinical and analytical importance and cholesterol is one of these lipids that measuring its concentration is of a clinical and analytical importance since cholesterol is associated with cardiac vascular diseases.

What is Cholesterol?.....cont.

8

-Cholesterol is found principally in animals and humans, where it is also the main sterol.

-Small quantities are sythesized in other eukaryotes, such as plants and fungi.

-Virtually all human cells and body fluids contain some cholesterol.

What is Cholesterol?......cont.

9

•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.

-Cholesterol, like other steroids, is

a derivative of the cyclic hydrocarbon

Perhydrocyclopentanophenanthrene.10

Perhydrocyclopentanophenanthrene

11

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 .

12

Figure.1.

CholesterolStructure

13

- 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 13.

5. A 5 double bond.

Structure of Cholesterol….cont.

14

Structure of Cholesterol

A

DC

B

15

Cholestryl esters: Much of the plasma cholesterol is in an esterified form (with a fatty acid attached at carbon 3) which makes the structure even more hydrophobic.

Structure of Cholesteryl Ester

16

Cholestryl Ester Structure

17

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 200-239 mg/dL (5.13-6.13 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.

18

- Cholesterol in the intestine comes from diet, bile, intestinal secretions. Therefore cholesterol has 2 major sources:

-Exogenous

-Endogenous

Sources of Cholesterol

19

Liver CholesterolPool

2 .De novo synthesis in liver

Chylomicron remnants

Secretion of HDL and VLDL

Free cholesterol secreted in bile

Conversion to bile acids/salts

HDL

1 .Dietary cholesterol 3 .Cholesterol synthesized in extrahepatic tissues

Major sources of liver cholesterol

Major routes by which cholesterol leaves liver20

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.

21

• 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.

What Are The Exogenous Sources Of

Cholesterol?........cont.

22

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 esterases which exisit in pancreatic and small intestinal secretions.

23

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.

24

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.

25

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

26

-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

27

-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.

28

-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.

Endogenous Sources Of Cholesterol ……cont.

29

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.

30

• 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.   

.

31

The Main Role of the Liver in the Production of the Majority of the Body

Cholesterol Pool ……cont.

Cholesterol Synthesis

• Similar to ketogenic pathway

• Occurs in cytosol

• Requires NADPH and ATP

• Highly regulated

• 80 % in liver, ~10% intestine, ~5% skin

32

•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.

33

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.

De Novo Synthesis Of Cholesterol

• Mevalonate Synthesis….cont.

34

De Novo Synthesis of Cholesterol

35

HMG.Co.AReductase

HMG.Co.ASythase

Biosynthesis of Cholesterol …..cont.

• After the Formation of Mevalonate.

The biosynthesis of cholesterol (from mevalonate) may be divided into 4 setps.

36

Biosynthesis of Cholesterol……cont.

• After the Formation of Mevalonate ……cont.

1.Formation of isoprenoid (isoprene units)

,isopentenyl pyrophosphate (5C) from mevalonate by loss of CO2.

2. Condensation of six isoprenoid units form squalene (30C).

37

Cholesterol Synthesis

38

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.

39

De Novo Synthesis of Cholesterol

40

HMG.Co.AReductase

HMG.Co.ASythase

Cholesterol Synthesis

41

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.

42

The Rate-Limitting

Step inDe Novo

Synthesis of

Cholesterol

43

HMG.Co.AReductase

•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.

Biosynthesis of Cholesterol…..cont.

The Rate-Limitting Step ……cont.

44

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.

45

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 acids.

Regulation of Cholesterol Synthesis…..cont..

46

47

Regulation of

Cholesterol Synthesis

By Feed-Back Inhibition of these

Substances )Intermediates(

3. Hormonal Regulation: Both Glucagon and glucocorticoids decrease HMG-CoA reductase activity.

Regulation of Cholesterol Synthesis …..cont.

48

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.

Regulation of Cholesterol Synthesis …..cont.

49

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.

Regulation of Cholesterol Synthesis……cont.

50

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.

Regulation of Cholesterol Synthesis….cont.

51

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. 

52

• 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 chylomicrons.

Cholesterol is Transprtedfrom Intestine to Liver inChylomicrons

53

Structure of Lipo-proteins

54

Structure of Chylomicrons

55

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 the liver. 

Cholesterol is Transprted from Intestine to Liverin Chylomicrons……cont.

56

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 to supply the body’s needs for triglycerides and cholesterol.

57

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

58

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.

59

Sources of Cholesterol

Diet De novo synthesisCholesterol synthesized in extrahepatic tissues

Liver cholesterolpool

Free cholesterolIn bile

Conversion to bile salts/acidsSecretion of HDLand VLDL60

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.

61

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.  

62

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)

63

The Fate of Unused LDL

64

• 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). 

65

The Fate of

Unused LDL

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.

66

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

67

Classification of Lipoproteins

“Bad ”))non-HDL

“Good”

68

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. 

69

Hypercholesterolemia Causes Atherosclerosis

70

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. 

71

Cholesterol is

Transported by Different Lipoproteins

72

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.

73

HOW TO TREAT HYPERCHOLESTEROLEMIA

74

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.

75

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.

76

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.

HOW TO TREAT HYPERCHOLESTEROLEMIA

….Cont.

77

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.

78

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.

79

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+

Biological Functions of

Cholesterol …..cont.

80

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.81

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.

82

Sex Hormones Derived from Cholesterol

83

Adrenal Gland Hormones

84

Cholesterol Catabolism

- The ring structure of cholesterol cannot be metabolized to CO2 and H2O in humans.

85

• 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.

Cholesterol Catabolism

86

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 200-400mg /d.

• These in turn are 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.[

87

• 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.

Cholesterol Recycling And Excretion

88

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.

89

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)

90

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.

91

Materials

1.Cholesterol reagent (Acetic anhydride + acetic acid)

2.Sulphuric acid 95-97%3.Standard cholesterol (300 mg/dl)4.Samples5.Test Tubes6.Pipettes7.Cuvettes8.Spectrophotometer9.Water bath

92

Method1- 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)(B)

Sample 10.1 ml

Sample 20.1 ml

Standard Cholesterol

0.1 ml

Distilled Water

0.1 ml

Cholesterol reagent

4 ml4 ml4 ml4ml

93

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.

94

Results

Absorbance of samples X 300 = ----------- mg/dl

Absorbance of standard

95

ResultsSample 1

A = mg/dl

B = mg/dl

Sample 2

C = mg/dl

D = mg/dl

Standard cholesterol

E = mg / dl

F = mg/dl

96