Direct determination of total serum cholesterol by use of double-wavelength spectrophotometry.

We describe a simple, accurate method for direct determination of total cholesterol in serum. Systematic investigation of a previously described modified Liebermann-Burchard reagent has indicated the necessity of accounting for both bilirubin interference and decreased specificity owing to exothermia. Double-wavelength spectrophotometry was used to optically null out bilirubin as an interfering factor, whereas adding serum to the cold reagent increases its specificity for the cholesterol color reaction. Comparison of 106 cholesterol values with those obtained by the procedure of Abell et al. [J. Biol. Chem. 195, 357 (1952)] yielded a correlation coefficient greater than 0.99; our inter-run coefficient of variation of polled laboratory serum was 1.7%.