Diagnostic Test Kit

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In the reaction, Triglyceride is hydrolyzed to glycerol and free fatty acids by lipase. In the presence of adenosine triphosphate (ATP) and glycerol kinase (GK), the glycerol is phosphorylated to glycerol-3-phosphate. Glycerol-3- phosphate is then oxidized by glycerol phosphate oxidase (GPO) to yield hydrogen peroxide (H2O2). The hydrogen peroxide causes oxidative coupling of 4-chlorophenol and 4- aminoantipyrine, producing a red colored quinoneimine dye complex. The increase in absorbance at 500 nm due to the formation of the quinoneimine dye is directly proportional to the concentration of Triglycerides in the sample.

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Cholesterol esterases (CHE) hydrolyze the cholesterol esters into free cholesterol. Cholesterol oxidase (CHOD) oxidizes the cholesterol into cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide reacts with a mix. of 4- aminoantipyrene and phenol in the presence of peroxidase enzyme (POD) & converts the reactants into a red quinone dye.

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Chylomicrons, LDL, HDL and VLDL are precipitated from serum by pohosphotungastate in the presence of divalent ctions, such as magnesium. The HDL cholesterol remains unaffected in the supernatentant and is estimated using total cholesterol reagent.

Sserum/Plasma/Total cholesterol  Phosphotungstate  HDL(LDL+VDL)

Cholesterol esterases (CHE) hydrolyze the cholesterol esters into free cholesterol. Cholesterol oxidase (CHOD) oxidizes the cholesterol into cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide reacts with a mix. of 4- aminoantipyrene and phenol in the presence of peroxidase enzyme (POD) & converts the reactants into a red quinone dye.

The cholesterol present in the sample originates a colored complex according to the following reaction:           

Cholesterol Ester+H₂O           CHE                    Cholesterol + Fatty Acids

Cholesterol +O₂                 CHOD                

  4-Cholesterol-3-One +H₂O₂

 2H₂O₂+4-Aminoantipyrine + Phenol         POD            Quinoneimine-4H₂O

 The intensity of the color is proportional to the cholesterol concentration in the sample.

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Sugars with reducing property arising out of the presence of a potential aldehyde or keto group are called reducing sugars. Ex. Glucose, galactose, lactose, maltose etc. 3,5 dinitrosalicylic acid (DNSA) is reduced to 3-amino-5-nitrosalicylic   acid   in   the   presence   of   reducing   sugars   in   an   alkaline solution. The reducing sugar is oxidized to sugar acid and Rochelle’s salt reduces the tendency to dissolve oxygen by increasing the ion concentration in the solution. Phenol increases the amount of color produced during the colr-developing reaction. Sodium bisulphate stabilizes the color obtained and reacts with any oxygen present in the medium. Alkaline medium is required for the re-ox reaction between DNSA and glucose, of other reducing sugars.

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