The 4-hydroxybenzoate/4-aminophenazone chromogenic system used in the enzymic determination of serum cholesterol

Abstract

A single reagent, containing cholesterol oxidase, cholesterol esterase, peroxidase, 4-hydroxybenzoate, and 4-aminophenazone, is used in determining serum cholesterol. Analysis time is 15 min, and the standard curve is linear to 6.0 g/liter. Analytical recovery of cholesterol was 100.1 ± 0.4%. Within-run precision (CV) was ≤ 1.4%, between-run ≤ 4.8%. Comparison with results by a Liebermann-Burchard method gave a linear regression of y = 1.08x - 0.05, with a correlation coefficient (r) of 0.985. Comparison with the Roeschlau enzymic method gave y = 1.02x + 0.01 (r = 0.958). Comparison with the enzymic method of Allain et al. gave y = 1.01x - 0.00 (r = 0.995). The following substances do not interfere up to the indicated concentrations (mg/liter): hemoglobin (5000), bilirubin (100), reduced glutathione (150), L-cysteine (400), urea (3000), creatinine (200), uric acid (200), D-glucose (10,000), L-ascorbic acid (50), acetylsalicylic acid (500), L-dopa (10), erogthioneine (1000), 2,5-dihydroxybenzoic acid (20), and 3,4-dihydroxybenzoic acid (10). Stored in an amber-colored bottle, the working reagent is stable for three months at 2-8°C and for three weeks at 25°C.