Experiment 6 Determination Of Cholesterol I

n 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.

CM VLDL

LDL HDL

Phosphotungstic acid,

magnesium chloride

centrifugation

CM VLDL LDL

HDL

HDL

anhydrous alcohol, acetic ether

centrifugation

protein

cholesterol

cholesterol

concentrated sulfuric acid- iron trichloride Stable

purple compound.

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）

Serum 0.5 ml

sodium phosphotungstic acid 0.25ml; magnesium chloride 0.25ml

centrifugate 4000r/min for 10min

Get the supernatant 0.2ml

H extract tube

total cholesterol extract tube (T extract tube):

serum and distilled water 0.1ml each

T extract tube

Extract cholesterol：

H extract tube

T extract tube

extract solution 1ml

centrifugate 4000r/min for 3min

extract solution 1ml

centrifugate 4000r/min for 3min

Get the supernatant

Get the supernatant

H tube

T tube

0.6ml

0.6ml

reagent amount: ml blank tube(B) standard tube(S) H tube T tubeHDL upper liquid(extraction) 0.6 total cholesterol supernatant 0.6extract 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 thoroughlyconcentrated 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

ODS

ODH

ODT

ODS