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Transcript
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 Cholesterol
2
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 Catabolism
4
Contents of The Lecture
•
•
•
•
•
5
Cholesterol Recycling And Excretion
Principle of the Test
Materials
Method
Results
• 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
What is Cholesterol?.....cont.
-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.
7
What is Cholesterol?.....cont.
• 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.
8
What is Cholesterol?......cont.
-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.
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.
Cholesterol
Structure
13
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 13.
5. A
14
5
double bond.
Structure of Cholesterol
C
A
15
B
D
Structure of Cholesteryl Ester
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.
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 (5.136.13 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
Sources of Cholesterol
- Cholesterol in the intestine comes
from diet, bile, intestinal secretions.
Therefore cholesterol has 2 major
sources:
-Exogenous
-Endogenous
19
Major sources of liver cholesterol
1. Dietary cholesterol
3. Cholesterol synthesized in extrahepatic tissues
2. De novo synthesis in liver
Chylomicron remnants
HDL
Liver Cholesterol
Pool
Secretion of HDL and
VLDL
20
Conversion to bile
acids/salts
Free cholesterol secreted in
bile
Major routes by which cholesterol leaves liver
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
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.
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
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.
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
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.
.31
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
De Novo Synthesis Of Cholesterol
• Mevalonate Synthesis….cont.
2- The next step requires the enzyme βhydroxy-β - methylgularyl-Co.A (HMG-CoA)
synthase. This enzyme catalyzes the
condensation of a third acetyl-CoA with βketobutyryl-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.
34
De Novo Synthesis of
Cholesterol
HMG.Co.A
Sythase
35
HMG.Co.A
Reductase
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.
37
2. Condensation of six isoprenoid units
form squalene (30C).
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
HMG.Co.A
Sythase
40
HMG.Co.A
Reductase
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
HMG.Co.A
Reductase
43
The RateLimitting
Step in
De Novo
Synthesis
of
Cholesterol
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.
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 HMGCoA reductase is regulated by some dietary
factors.
45
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
46
acids.
Regulation
of
Cholesterol
Synthesis
By
Feed-Back
Inhibition
of these
Substances
( Intermediates)
47
Regulation of Cholesterol
Synthesis …..cont.
3. Hormonal Regulation: Both
Glucagon and glucocorticoids
decrease HMG-CoA reductase
activity.
48
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.
49
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.
50
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.
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
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
53 chylomicrons .
Structure
of Lipoproteins
54
Structure of Chylomicrons
55
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
56the liver.
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
57 to supply the body’s needs for triglycerides and
cholesterol.
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 synthesis
Cholesterol synthesized
in extrahepatic tissues
Liver cholesterol
pool
Secretion of HDL
and VLDL
60
Free cholesterol
In bile
Conversion to bile salts/acids
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
• 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).
64
The Fate
of
Unused
LDL
65
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”
“Good”
(non-HDL(
68
Good Vs. Bad Lipoprotein
•
69
plasma lipoproteins , there are good and
bad forms of lipoproteins in terms of their
effect in the formation of atherosclerotic
plaque.
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
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.
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
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+
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
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.
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
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.
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. Samples
5. Test Tubes
6. Pipettes
7. Cuvettes
8. Spectrophotometer
9. Water bath
92
Method
1- Label 7 test tubes for test 1(A,B)test 2 (C,D) standard
(E,F) and blank (O) pipette the following
Sample 1
(A,B)
0.1 ml
Sample 2
(C,D)
93
(B)
0.1 ml
Standard
Cholesterol
Distilled
Water
Cholesterol 4 ml
reagent
(E,F)
0.1 ml
0.1 ml
4 ml
4 ml
4ml
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
Results
Sample 1
A=
B=
mg/dl
mg/dl
Sample 2
C=
D=
mg/dl
mg/dl
Standard cholesterol
E=
F=
96
mg / dl
mg/dl