Triglycerides (GPO/PAP)

INTENDED USE
NS Biotec triglycerides reagent is intended for the in vitro quantitative determination of triglycerides in serum and plasma on both automated and manual systems.

ASSAY PRINCIPLE
Triglycerides(C6H8O6) are generally determined by a combination of hydrolysis to glycerol and free fatty acids and measurement of the amount of glycerol released. The most commonly used methods involve alkaline hydrolysis and either chemical or enzymatic measurement of glycerol. Chemical means of analysis generally rely on measurement of the product of periodate oxidation of glycerol. Eggstein and Kreutz developed an enzymatic method for measuring glycerol released from triglycerides by alkaline hydrolysis. This method was based on the coupled reaction sequence catalyzed by glycerol kinase, pyruvate kinase, and lactate dehydrogenase. A method for complete enzymatic hydrolysis to triglycerides avoiding the need for serum pretreatment was described by Bucolo and David, using a combination of lipase and at least one proteolytic enzyme. Wahlefeld reported that certain esterases could be combined with a lipase to achieve complete triglycerides hydrolysis. Both methods employed a coupled enzymatic reaction sequence to measure glycerol. NS Biotec triglycerides reagent combines the use of lipoproteinlipase, glycerol kinase, and glycerol phosphate oxidase with the peroxidase/4- chlorophenol/4-aminoantipyrine system of Trinder for the measurement of triglycerides in human serum. The series of reactions involved in the assay system are as follows:

1. C6H8O6 are hydrolyzed by lipoprotein lipase (LPL) to glycerol and fatty acids.

2. Glycerol is then phosphorylated to glycerol-3-phosphate by ATP in a reaction catalyzed by glycerol kinase (GK).

3. The oxidation of glycerol-3-phosphate is catalyzed by glycerol phosphate oxidase (GPO) to form dihydroxyacetone phosphate and hydrogen peroxide (H2O2).

4. In presence of peroxidase (POD), the hydrogen peroxide (H2O2) formed effects the oxidative coupling of 4 – chlorophenol and 4- aminoantipyrine (4-AAP) to form a red-colored quinoneimine dye.