Experiment 6 Determination Of Cholesterol In HDL

1.Aim And request Comprehend the method of determination of cholesterol in HDL Learn about with the importance of HDL in vivo and its clinical significance.

2.Principle lipids are normally transported together with protein components. lipoproteins are composed of the lipid components and a number of apolipoproteins; in these amphiphilic proteins, the hydrophobic residues face the lipid core of the lipoprotein, while the hydrophilic side is facing the aqueous medium.

Phosphotungstic acid and magnesium chloride can precipitate CM、VLDL and LDL of the serum, then remove them with centrifugation. Extract cholesterol with anhydrous alcohol and acetic ether to precipitate protein, and then centrifuge to remove precipitation. Add development solvent (concentrated sulfuric acid- iron trichloride), which can react with cholesterol and produce stable purple compound. The shade of purple is in direct proportion to the amount of cholesterol. Using the standard cholesterol solution known concentration as control can determine the amount of cholesterol in HDL.

HDL centrifugation centrifugation HDL protein Phosphotungstic acid, magnesium chloride HDL CM VLDL LDL HDL CM VLDL LDL centrifugation anhydrous alcohol, acetic ether cholesterol centrifugation HDL protein

Stable purple compound. cholesterol concentrated sulfuric acid- iron trichloride Stable purple compound. cholesterol

3.PROCEDURE ⑴ Prepare for the HDL extract tube（H extract tube）: Put exact 0.5 ml serum and sodium phosphotungstic acid 0.25ml into a centrifuge tube, mix, then put magnesium chloride 0.25ml, mix thoroughly, centrifugate 4000r/min for 10min to get the upper liquid 0.2ml for use, as the HDL extract tube. ⑵ Prepare for the total cholesterol extract tube (T extract tube): Put serum and distilled water 0.1ml each.

⑶ Extract cholesterol：Put extract solution 1ml into the T and H centrifuge tube respectively，shake it thoroughly，then centrifugate 4000r/min for 3min, get the supernatant to determine the cholesterol. ⑷ Development: Mark 4 dry tubes with number, perform according to the following table as below.

HDL extract tube（H extract tube） Get the supernatant 0.2ml sodium phosphotungstic acid 0.25ml; magnesium chloride 0.25ml centrifugate 4000r/min for 10min Serum 0.5 ml H extract tube total cholesterol extract tube (T extract tube): serum and distilled water 0.1ml each T extract tube

Extract cholesterol： Get the supernatant extract solution 1ml centrifugate 4000r/min for 3min H extract tube H tube extract solution 1ml Get the supernatant 0.6ml centrifugate 4000r/min for 3min T extract tube T tube

reagent amount: ml blank tube(B) standard tube(S) H tube T tube HDL upper liquid(extraction) 0.6 total cholesterol supernatant 0.6 extract solvent 0.5 standard cholesterol solution 0.5 distilled water 0.1 0.1 development solvent 1.5 1.5 1.5 1.5 shake thoroughly concentrated sulfuric acid 1.5 1.5 1.5 1.5 Shake the tubes immediately, and then place them in room temperature for 10 minutes. Adjust the spectrophotometer to zero with blank tube. Determine the absorbance of each tube under 550 nm wavelength.

4.Result and Calcuation ODH ODT ODS cholesterol of HDL(mg/dl) = × 200 ; total cholesterol (mg/dl) = × 200 (standard cholesterol solution 200 mg/dl) REFERENCE VALUE: total cholesterol in serum :2.59-6.47mmol/L cholesterol in HDL: male 41.1-62.3mg/dl; female 41.7-67.3mg/ml ODH ODS ODT ODS