ESTIMATION OF CHOLESTREOL IN BLOOD SCHOENHEIMER AND SPERRY METHOD:

ESTIMATION OF CHOLESTEROL IN BLOOD

SCHOENHEIMER AND SPERRY METHOD:

PRINCIPLE:

                 blood or serum is extracted with an alcohol-acetone mixture which removes cholesterol and other lipids and precipitates protein. The solvent is removed by evaporation in a boiling water bath. The residue is dissolved in chloroform and the color is developed by the Liebermann-Burchard reaction and compared with the standard

REAGENTS REQUIRED:

1. alcohol: an acetone mixture (1:1)
2. chloroform (AR)
3. acetic anhydride
4. concentrated H2SO4
5. standard cholesterol solution (0.1mg/ml) in chloroform

PROCEDURE:

place 10 ml of alcohol: an acetone solvent in a centrifuge tube and add 0.2ml of serum or blood. cork the tube and immerse in a boiling water bath with shaking until the solvent begins to boil. Remove the tube and continue the shaking for a further 5 minutes. Coll to room temp, and centrifuge it at 1500rpm for 5 minutes take the supernatant fluid in a small beaker and evaporate to dryness in a boiling water bath. Cool and dissolve the residue in 6ml of chloroform and transfer it to a dry “tt” (T). at the same time label to other “tt” standard(S) and blank (B). add 6ml of cholesterol standard into the standard tube and 6ml of chloroform into the blank tube. Add 2ml of anhydride and 0.1ml of conc H2SO4 to all the tubes and thoroughly mix. Leave the tubes in the dark for 15min and read the absorbance at 680nm with a spectrophotometer against the reagent blank

serum cholesterol (mg/100ml)=absorbance of test/absence of std X 300.

CLINICAL SIGNIFICANCE:

 cholesterol is a steroid that is influenced by heredity, nutrition, endocrine function, and integrity of vital organs such as the liver and kidney.

NORMAL VALUES:

total cholesterol- 130 to 250 mg%
HDL cholesterol:
male: 30 – 60 mg %
female 35 – 75 mg %