Single-cuvet sequential determination of triglyceride and cholesterol.

I describe a sequential method for kinetic assay of serum triglyceride and cholesterol in a single cuvet. Triglyceride is assayed first, by an ultraviolet enzymic method with ATP and NADH as the driving reagents (Clin Chem 21:1627-1629, 1975); cholesterol is then assayed with use of cholesterol oxidase, peroxidase, and the Trinder reaction. Excess NADH left by the triglyceride assay is removed by adding either pyruvate or glycerol to the cholesterol reagent; the addition prevents interference of NADH with nascent H2O2, and does not affect the apparent Km of cholesterol oxidase or the pseudo-first-order reaction of the cholesterol assay. The method, tested in both a random-access analyzer (Hitachi 705) and a batch analyzer (Olli C/D), at three concentrations, gave the following within-run CVs:3.0, 1.6, 1.5% for cholesterol; 4.0, 4.2, 5.4% for triglyceride. The corresponding between-run CVs were 12.5, 9.0, 8.1% and 12.8, 11.1, 8.9%. The calibration was linear to at least 8.0 g/L for both cholesterol and triglyceride. Interference from bilirubin, hemoglobin, and certain drugs is almost negligible. Correlation studies with reference and routine methods showed r values ranging from 0.985 to 0.995 for cholesterol, 0.984 to 0.991 for triglyceride.