THE DETERMINATION of TOTAL

CHOLESTEROL in SERUM
 ZAK METHOD
Principle: Based on the red violet colour which is proportional to the amount of
cholesterol and formed after the reaction of cholesterol melted in acetic acid,FeCl3
and sulfuric acid
Material: Serum

Experimental procedure:
 1. In a centrifuge tube, add 1 ml of serum, 4 ml of iron chloride chloride and stir.
 2. Add 2 ml of acetic acid, 2 ml of sulfuric acid, and mix to another test tube by
taking 2 ml of supernatant from this tube.
 3. At the same time, in another test tube, add 2 ml of iron chloride chloride, 2 ml
of acetic acid, 2 ml of sulfuric acid as a blind test and stir.
 4. This solution is read at 560 nm in the spectrophotometer. Using the standard
curve graph, the amount of cholesterol in the serum is evaluated as % mg.
 Preparation of Standard Curve Graph
Blank TUBE 1 TUBE 2 TUBE 3 TUBE 4 Sample

Cholesterol - 0.5mL 1mL 1.5mL 2mL -

standart solution
(%10mg)
FeCl3 indicator 2mL 2mL 2mL 2mL 2mL -

Serum filtrate - - - - - 2mL

Sulfuric acid 2mL 2mL 2mL 2mL 2mL 2mL

Glacial acetic 2mL 1.5mL 1mL 0.5mL - 2mL

acid
% mg of 0 102.5 205 307.5 410 -
cholesterol in
serum
Standart graphic

Normal Total Cholesterol Values for Zak method= %110-250 mg

 TESTS for KETONE BODIES in URINE
LANGE TEST (for acetone):
 Principle: Sodium nitroprussiate is formed a colored complex with acetone.

 Material: Urine

Experimental procedure
 1. 5 ml of urine is collected in tube and then 0.5 ml of glacial acetic acid and 0.5 ml of
10% sodium nitroprusside solution is added.
 2. The tube is kept in an inclined state and 2 ml of ammonium hydroxide is added to
bring a layer to the plate
 3. Purple ring formation between the two liquids indicates the presence of acetone.
TESTS for KETONE BODIES in URINE
GERHARD TEST (for acetoacetic acid ):

Principle: Acetoacetic acid is formed red color with iron chloride 3 chloride
Material: Urine

Experimental procedure:
 1. 5 mL of urine is collected into a test tube. A few drops of FeCl3 are added.
 The formation of cherry or reddish red color indicates the presence of
acetoacetic acid in urine.
 References

 Biochemistry pratice book (2004)