THE DETERMINATION Of TOTAL CHOLESTEROL İn SERUM and TEST FOR KETONE BODIES in URINE

THE DETERMINATION Of TOTAL CHOLESTEROL İn SERUM and TEST FOR

KETONE BODIES in URINE

Research Assıstant NURI OZMEN

 75% of the cholesterol in circulation is esterified with fatty acids, the rest as free cholesterol.

 In organism, cholesterol is synthesized in many tissues

except erythrocyte, but the tissue from which it is

synthesized is liver (about 80%).

 Normal Total Cholesterol Values: 110-250 % mg

 Hypercholesterolemia occurs when cholesterol increases in blood:

 infectious hepatitis and portal cirrhosis,

 Nephritis,

 Pancreatic diseases,

 Hypothyroid and myxedema,

 Xanthomatosis

 Hypocholesterolemia occurs as a result of the decrease of cholesterol in the blood:

 Hyperthyroidism

 Increased lipolysis with the effect of estrogen hormone

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,FeCl

and sulfuric acid.

Cholesterol in glacial acetic acid gives a red colour with ferric chloride and apolar

sulphuric acid. This reaction has been employed by ZAK’S to estimate the cholesterol in an unknown serum sample.

Material: Serum

Experimental procedure:

 1. For the preparing serum filtrate, ın a centrifuge tube, add 0.4 ml of serum, 4 ml of iron chloride chloride and stir.

 2. 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 standart

solution (%10mg) - 0.25 mL 0.5mL 1mL 1.5mL -

FeCl₃ indicator 2mL 2mL 2mL 2mL 2mL -

Serum filtrate - - - - - 2mL

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

Glacial acetic acid 2mL 1.75 ml 1.5mL 1mL 0.5mL 2mL

% mg of cholesterol

in serum 0 51.25 102.5 205 307.5 -

Standart Graphic

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

KETON BODIES

 The main sources of energy are broken down to acetyl CoA as a result of metabolism.

 Acetyl CoA enters the crepe cycle to observe energy.

 Cholesterol synthesis plays a role in the synthesis of fatty acids,

 A number of acetyl CoA is converted to ketone bodies through a series of reactions (ketogenesis).

 Ketone bodies:

 Acetoacetic acid

 B-hydroxy butyric acid

 Acetone

 In the organism, ketone bodies accumulate, this is called ketosis. Ketosis increases the ketone bodies in the blood.

Increasing the concentration of ketone bodies in the blood is called ketoneemia or hyperketonemia. Ketone bodies that increase in blood flow into the urine. This is called ketonuria.

 Accumulation of ketone bodies in the blood causes a

condition called acidosis.

 Ketosis is seen in pathological and physiological conditions:

Diabetes,

Diet (high fat , low carbohydrate dietary)

Pregnancy

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. 2 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. 2 mL of urine is collected into a test tube. A few drops of 10% FeCl

are added.

 The formation of cherry or reddish red color indicates the presence of acetoacetic acid

in urine.

References

 Biochemistry pratice book (2004)