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 sensitivity 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 beta-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 less than 3% and a linearity to at least 400 mg of HDL free cholesterol per liter. Comparison with results by Trinder's cholesterol method, which measures cholest-4-en-3-one at 232 nm, showed good correlation (r = 0.9829, slope 1.0001, and y-intercept +2.4797 mg/L). With the manual procedure for HDL free cholesterol, between-batch and within-batch CVs were less than 5%, and results correlated well with those by the automated method (r = 0.9975, slope 0.9839, and y-intercept +2.4327 mg/L). The mean (and SD) HDL free cholesterol for 123 men was 96.8 (30.6) mg/L and for 122 women 136.4 (36.8) mg/L, indicating a distinct sex-related difference, similar to that found for HDL total cholesterol. HDL free cholesterol in plasma may therefore be a potential new predictor of coronary heart disease.
Moshides
J S
JS
Department of Clinical Chemistry, Prince of Wales Hospital, Randwick, N.S.W., Australia.
D000328:Adult; D000368:Aged; D000675:Ampyrone; D001322:Autoanalysis; D001970:Bromobenzoates; D002784:Cholesterol / Q000097:blood*; D002789:Cholesterol Oxidase; D008076:Cholesterol, HDL / Q000097:blood*; D003327:Coronary Disease / Q000097:blood*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates; D008297:Male; D008875:Middle Aged; D009195:Peroxidase; D011092:Polyethylene Glycols; D011786:Quality Control; D012016:Reference Values; D012044:Regression Analysis; D013053:Spectrophotometry
D001970:Bromobenzoates; D008076:Cholesterol, HDL; D062385:Hydroxybenzoates; D000675:Ampyrone; C043713:2,4,6-tribromo-3-hydroxybenzoic acid; D011092:Polyethylene Glycols; D002784:Cholesterol; D002789:Cholesterol Oxidase; D009195:Peroxidase