3.Working triglyceride reagent
Add 250μl of enzyme reagent to 30 ml of buffer solution. Mix gently, and keep the
reagent for at least 10 min at room temperature before use. The working reagent is
stable for 6 weeks at 2– 8 C or for 3 days at 15– 25 C when protected from light.
4.Triglyceride standard: 200 mg/dl
35.3.3 Procedure
Label three tubes as test (T), standard (S), and blank (B). Then add 10μl of sample to
the tube labelled as“T.”Add 10μl of 200 mg/dl triglyceride standard to the tube
labelled as“S.”Add 1 ml of working triglyceride reagent to all the tubes, mix the
contents, and incubate for 5 min at 37C. Then measure the absorbance of the
samples against the reagent blank within 60 min.
35.3.4 Precautions
- Lipemic specimen causes turbidity of the sample mixture leading to false eleva-
tion of results. - The test is linear up to a triglyceride concentration of 100 mg/dl. Samples with a
higher concentration have to be dilutedfive times with saline and retested.
Multiply the result by 5.
35.4 HDL Estimation
HDL is a small particle consisting of 50% protein (mostly contain apo A but also
some amount of apo C and apo E), 20% cholesterol (mostly esterified), 30%
phospholipids, and only traces of triglycerides. HDL is often subdivided into
HDL 2 and HDL3,varying in density, particle size, composition, and possibly also
physiologic role.
35.4.1 Principle
Chylomicrons, LDL, and VLDL are precipitated from the sample by adding
phosphotungstic acid and magnesium ions to the sample (this precipitating reagent
precipitates all the lipoprotein which has apo B). Centrifugation leaves only the HDL
in the supernatant. The cholesterol content is determined by enzymatic method
(cholesterol oxidase/peroxidase method).
142 35 To Measure Lipid Profile in Serum Sample