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