Download 1. Sources of cholesterol • diet ~ 30 % • biosynthesis ~ 70% 2. Types

1. Sources of cholesterol
• diet ~ 30 %
• biosynthesis ~ 70%
2. Types of cholesterol found in the body.
• Free cholesterol functions as essential structural
component of the bi-layer membranes and of outer
monolayer of plasma protein. It is transported in the
circulation by lipoprotein.
• Esterified cholesterol is stored in most tissue. It is
transported in the body in the hydrophobic core of the
lipoprotein.
3. Types of lipoprotein
• LDL mediates cholesterol ester and free cholesterol into
many tissues.
• HDL mediates removal of free cholesterol from the tissues
and transports it to the liver for conversion to bile acids.
4. Synthesis of Cholesterol
In the cytoplasm
Citrate-------ÆAcetyl CoA-------Æ HMg CoA
Lyase
thiolase
In the smooth Endoplasmic Reticulum
HMG CoA -------ÆMelavonateÆÆÆCholesterol
HMG CoA reductase
a. Acetyl CoA precursor for cholesterol is produced either
from dietary fat or glucose.
b. The reaction from Acetyl CoA to cholesterol occur in the
cytoplasm and on the smooth endoplasmic reticulum of
the cell.
c. The rate determining step is the conversion of HMG CoA
to mevalovate. The step is catalyzed by HMG CoA
reductase.
1
d. The four stages of cholesterol synthesis is summarized
below.
Figure 1. Stages of cholesterol synthesis.
5. Regulation of cholesterol synthesis – checks and balances
a. HMG CoA reductase –increase or decrease of activity.
• In a fasting state, the activity of the enzyme can be
decreased through phosphorylation of the enzyme. The
trigger is the elevated concentration of glucagon in the
circulation.
• After eating, insulin activates the enzyme by indirectly
causing dephosphorylation of the enzyme by a protein
phophatase.
b. HMG CoA reductase - reducing amount.
• Normal/high cholesterol represses the formation of HMG
CoA reductase, lowering the amount of enzyme.
2
Mechanism:
• pre SREBP-1 (Sterol Regulatory Element Binding
Protein) is trapped in the endoplasmic reticulum when
cell free cholesterol is high because the protease needed
to cleave the pre-portion is inhibited.
• When the free cholesterol falls, the protease is active and
the pre- SREBP-1 is cleaved to liberate a fragment
SREBP-1 that enters the nucleus. This factor then binds
as a dimmer to SRE-1(sterol responsive element) of the
HMG CoA reductase and LDL receptor genes to activate
transcription of the enzyme.
Figure 3 Regulation of HMG CoA reductase activity.
• The LDL receptor binds the LDL apoprotein B100, and
the LDL with its receptor is taken up by intact by
endocytosis. The receptor returns to the membrane
while LDL is broken down in the lysosome. The
3
protein is degraded to amino acids and the cholesterol is
hydrolyzed.
• If the LDL receptor is exceeded due to elevated
circulating LDL, then cholesterol esters will be
deposited in the artery walls instead of being taken up
normally by peripheral cells.
6. Metabolism of cholesterol
• Esterification of cholesterol is catalyzed by ACAT
(AcylCoA:Cholesterol Acyltransferase) for intracellular
storage and LCAT (Lecithin:Cholesterol Acyltransferase)
for transport by HDL
• Cholesterol ultimately enters the liver and is excreted in
the bile as cholesterol or bile acids. The enterohepathic
circulation allows bile acids used in lipid adsorption to be
conserved. The 5 % bile acids not absorbed is excreted in
the feces.
• Synthesis of bile acid, cholic acid and chenodeoxycholic
acid, are synthesized in the liver from a common
precursor, 7ά-hydroxycholesterol – the rate limiting step.
Its production is catalyzed 7ά-hydroxylase.
4
Figure 4 Fates of cholesterol and the role of ACAT and
LCAT.
• High levels of cholesterol activate ACAT to promote
esterification of cholesterol for cell storage.
• Bile acids repress 7ά-hydroxylase and this induced by
cholesterol.
7. Serum cholesterol reduction through drug and supplement
therapy.
a. Statins inhibits HMG CoA reductase.
b. Bile salt sequestering resins (cholestyramine) by blocking
the readsorption of bile acid causing a chain of events.
• More cholesterol is converted to bile acids.
• Lower liver cholesterol increases the available LDL
receptor.
• With more LDL receptors available, there is increase
hepatic uptake of LDL, decreasing plasma
cholesterol.
c. Policosanol is a mixture of fatty alcohols derived from the
wax of sugarcane.
• The components of policosanol include 1octacosanol, 1-dotriacontanol, 1-triacontanol, 1tetracosanol, 1-tetratriacontanol, 1-hexacosanol, 1heptacosanol and 1-nonacosanol.
• Inhibiting cholesterol synthesis at a point between
the formation of acetate and mevalonate.
• Exerting no direct inhibition on HMG-CoA
reductase .
• Significantly increasing LDL receptor dependent
processing as demonstrated by increasing the
incorporation of LDL into the hepatocyte and
stimulating its catabolism.
5
Figure 3 to 4 from http://www.biochem.arizona.edu/classes/bioc801/notes/lec29.pdf
Procedure:
Safety caution:
Ethanol is flammable. Absolutely on flames.
Part A. Extraction of cholesterol from food.
• Do all of Part A in the hood.
• Split the combined supernatant into two centrifuge tubes.
• #7 add 2.5 mL of 15% Na2SO4 into each of the centrifuge
and spin.
• You will need 4 centrifuge tubes total.
• Water bath is setup in the hood.
• You will need 9 cuvets for Parts A and B
• #9 20 uL will be measure with a micropipette, use the same
1 mL pipet to measure 0.1 and .6 of the extracts.
Part B. Preparation of Standard Curve
Your results will be as good as your standard curve. Measure
accurately.
• Cholesterol reacts with FeCl3 to form a colored complex.
The higher concentration of cholesterol, the deeper the color
of the complex.
• The standard and sample solutions are placed in the Spec 20.
The amount of light transmitted through the solutions is
measured.
• % Transmission is converted to Absorbance, A.
A = -log (%T/100)
• The concentrations vs. Absorbance is plotted in Excel.
6
Standard curve
y = 0.3165x
0.7
absorbance
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.5
1
1.5
2
2.5
cholesterol in ug/mL
• The slope equation is determined by Excel.
• The cholesterol in ug/mL is determined by plugging in the
absorbance of the samples and solving for x. “y” is
absorbance, and x is the cholesterol concentration.
7
8