Kinetic enzymatic method for automated determination of HDL cholesterol in plasma

A sensitive fixed-time kinetic enzymatic method for the measurement of high density lipoprotein cholesterol is described, as adapted for the Cobas Bio centrifugal analyser (Hoffmann LaRoche). The Boehringer Mannheim cholesterol esterase/cholesterol oxidase/peroxidase/3,4-dichlorophenol kinetic reagent was modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid which reacts with hydrogen peroxide and 4-aminophenazone to produce a quinone-imine dye with a greater molar absorptivity than that produced with phenol. The method has been developed for the determination of HDL fractions isolated with polyethylene glycol 6000, for which a reagent of high sensitivity is required. The method is linear to at least 3.88 mmol/l of HDL cholesterol and the coefficients of variation for within-run and day-to-day precision were less than 3.0%. Correlation of the kinetic HDL cholesterol method with an equilibrium method was good (R = 0.9980). The assay is rapid, inexpensive and large numbers of specimens can be processed conveniently.