23.2 Specimen Requirements
Serum (fasting) or plasma (EDTA or heparin may be used but not oxalate,fluoride,
or citrate). Serum is stable for 5–7 days at 4C. Results with EDTA plasma are 3%
lower than serum results.
23.3 Methodology....................................
Cholesterol is estimated by Zak ferric chloride- sulfuric acid method.
23.4 Principle........................................
Ferric chloride in acetic acid precipitates the serum proteins, and the cholesterol
liberated reacts with conc. H 2 SO 4 to make a chromophore (colored complex). The
reaction seems to involve oxidation, dehydration, and sulfonation. The amount of
color produced is directly proportional to the cholesterol present in the sample which
is measured at 550 nm. The reaction involves“3-hydroxy-5ene”part of the choles-
terol molecule, which isfirst dehydrated to form cholesta 3,5-diene and then
oxidized by the H 2 SO 4 to link two molecules together as bis-cholesta-3,5-diene.
This compound can be sulfonated by the H 2 SO 4 to produce mono- and disulfonic
acids which are highly colored. In presence of more H 2 SO 4 and ferric ions, the
disulfonic acids are preferentially formed and are red in color. The color thus
produced is measured at 550 nm.
23.5 Reagents........................................
1.Precipitating reagent: 0.05% FeCl 3 in glacial acetic acid
2.Conc. H 2 SO 4
3.Cholesterol standard: 100 mg % in glacial acetic acid
25.5 Procedure
- Take a set of seven test tubes and label them as blank, standard (S 1 -S 5 ), and test
(T). - Dispense 20, 40, 60, 80, and 100μl of 100 mg % cholesterol standard into the
tubes labelled as (S 1 – S 5 ), respectively, and make the volume 3 ml with
precipitating reagent. - In the blank tube, add 3 ml precipitating reagent.
94 23 To Estimate the Amount of Total Cholesterol in Serum