A colorimetric method is described for the determination of biological fluid triglycerides content.
In this single reagent enzymatic procedure for specific determination of triglycerides in serum, hydrolysis of triglycerides is catalysed by lipase to produce glycerol.
Then the released glycerol is assayed in a reaction catalysed by glycerol kinase and glycerol phosphate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is monitored in the presence of horseradish peroxidase with 4 – amino phenazone as the chromogenic system. The sample – reagent volume ratio is 1:150 and absorbance of this chromogen system at 510nm affords useful results. A single stable working reagent is used. The reaction is completed in 15 minutes at room temperature. The standard curve is linear for triglyceride concentrations as great as 13.5 mili mol per litre. within – run and between – run precision studies showed coefficient variations of 1.6 and 3% respectively.
This method is suitable for manual and automation.
Background: Determination of triglycerides in serum is a routine test which is done in diagnostic laboratories. There are several chemical methods for doing this test. Our enzymatic method is direct and more accurate.
Materials and methods: Main chemicals which have been used for this reseacrch were:
glycerokinase, glycerolphosphate oxidase, lipase, peroxidase, aminophenazone, adenosine – 5– triphosphate, potassiumferrocyanide, these chemicals were purchased from sigma co. potassium dihydrogen and monohydrogen phosphate , magnesium chloride and triton from merck. Other chemicals were anlalytical grade.
The working reagent is a solution in phosphate buffer and enzymes. Then 10 µL of sample (serum or plasma) is added to 1.5 mL of working reagent. After 15 min the absorbance is measured at 510 nm vs the reagent blank.
Results: we assessed the linearity and sensitivity of reagent responses for a suitable diluted triglyceride concentration solution, 0 to 13.5 mmol/L. Coefficient correlation was 0.999. The analytical recovery of triglyceride assay in concentrations ranging from 1 to 13.5 mmol/L was 100.7, 100.6 and 99% respectively.
Color stability was tested with use of three different pools of human sera and with the working glycerol standard, no important changes in absorbance were detected during 60 min observation after 15 min incubation time at 22 ْC and 37 ْC.
Conclusion : Determination of triglycerides in biological fluids by the use of enzymes is direct and accurate. This proposed method also offers rapid execution and good reliability and recommended for large scale preparations.