Clinical Chemistry · 1982 · 2.9K citations · 3 references
Life SciencesBiochemistryAnalytical ChemistryLipid BiochemistryMetabolismBioanalytical Method ValidationLipid MetabolismLipid OxidationLipid PeroxidationLipoprotein AnalysisTriglyceride MetabolismBlank MeasurementChromogenic DetectionGlycerol MetabolismHydrogen Peroxide GenerationStandard Curve ConstructionTriglyceride Analysis In Body FluidsTriglyceride BreakdownTriglyceride Hydrolysis
The assay hydrolyzes serum triglycerides with lipase, then measures the released glycerol via glycerol kinase and L‑α‑glycerol‑phosphate oxidase to generate hydrogen peroxide, which is detected by horseradish peroxidase in a 3,5‑dichloro‑2‑hydroxybenzenesulfonic acid/4‑aminophenazone chromogenic system, completing in 15 min at room temperature with a single stable reagent. The method yields a high‑absorbance chromogen at 510 nm, enabling linear calibration up to 13.6 mmol L⁻¹, 100.1 % recovery, within‑run CV ≤1.6 % and between‑run CV ≤3.0 %, and operates with a 1:150 sample/reagent ratio without a blank, making it suitable for automation.
In this direct colorimetric procedure, serum triglycerides are hydrolyzed by lipase, and the released glycerol is assayed in a reaction catalyzed by glycerol kinase and L-alpha-glycerol-phosphate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is monitored in the presence of horseradish peroxidase with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogenic system. The high absorbance of this chromogen system at 510 nm affords useful results with a sample/reagent volume ratio as low as 1:150, and a blank sample measurement is not needed. A single, stable working reagent is used; the reaction is complete in 15 min at room temperature. The standard curve is linear for triglyceride concentrations as great as 13.6 mmol/L. Average analytical recovery of triglycerides in human sera is 100.1%, and within-run and between-run precision studies showed CVs of less than or equal to 1.6 and less than or equal to 3.0%, respectively. The method is suitable for automation.
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