Enzymic Determination of the Free Cholesterol Fraction of High-Density Lipoprotein in Plasma with Use of 2,4,6-Tribromo-3-hydroxybenzoic Acid

A highly sensitive enzymic colorimetric reagent is described for determination of the free cholesterol fraction of high-density lipoprotein (HDL), which represents about 20 % of the total cholesterol content of this lipoprotein. For greater sensi-tivity with respect to cholesterol, I used 2,4,6-tribromo-3-hydroxybenzoic acid instead of phenol in the cholesterol oxidase/peroxidase/4-aminoantipyrine reagent system. This allows determination of the free cholesterol fraction of HDL isolates prepared with polyethylene glycol 6000, a method for precipitating /3-lipoprotein that involves a twofold dilution of plasma. The reagent, adapted for use with a Cobas-Bio centrifugal analyzer, results in between-run and within-run CVs of <3 % and a linearity to at least 400 mg of HDL free cholesterol per liter. Comparison with results by Tririder’s cholesterol method, which measures cholest-4-en-3-one a