35.4.2 Reagents
1.Precipitating reagent: This contains 0.55 mM phosphotungstic acid and 25 mM
magnesium chloride.
2.Cholesterol reagent: Same as that used in cholesterol estimation.
- Working precipitating reagent prepared by mixing precipitating reagent four parts
with one part distilled water.
35.4.3 Procedure
Mix 500μl of working precipitating reagent with 200μl serum, and allow it to stand
at room temperature for 10 min. Centrifuge this reagent at 4000 rpm for 10 min or at
12000 rpm for 3 min. After centrifugation, separate the clear supernatant within 2 h,
and determine the cholesterol content. The supernatant may be stored up to 5 days at
2 – 25 C.
Label two tubes as test and blank. Pipette 100μl of supernatant into the tube
labelled as“test.”Add 100μl of distilled water into blank tube. Then add 1 ml
cholesterol reagent to both tubes and mix. Now, incubate the tubes at 37C for 5 min
or room temperature for 10 min. Measure absorbance at 500 nm against blank in a
spectrophotometer, and calculate the HDL cholesterol.
35.4.4 Precautions
- The sample should be collected from fasting patient and free from hemolysis.
- After precipitation the supernatant should be clear and estimate the HDL choles-
terol within 2 h after precipitation with enzymatic method only. - Serum from the blood clot should be separated as rapidly as possible. HDL
cholesterol remains stable in serum for 6 days at 2– 25 C and 4 months at 20 C.
35.5 LDL Estimation..................................
LDL is also called bad cholesterol since it transports cholesterol from the liver to
peripheral tissues. It constitutes about 50% of the total lipoprotein mass in human
plasma. The particle size is much smaller than that of the triglyceride-rich lipopro-
tein. Cholesterol accounts for about half of LDL mass; most of it is esterified. About
25% of LDL mass is protein, mostly apoB-100 with traces of apo C.
35.5.1 Principle
Firstly, polyvinyl sulfate is added to sample to precipitate the LDL, and the choles-
terol content of HDL, VLDL, and chylomicron is measured. LDL cholesterol is
35.5 LDL Estimation 143