A sensitive reaction for dilute cholesterol determinations

A sensitive reaction for the peroxidase-coupled sequence of the determination of a dilute total cholesterol mixture of free and esterified forms is described. Substitution of a chlorinated auxochrome of phenol made water soluble by sulfonation through a synthetic procedure previously described created a severalfold enhancement factor which magnified considerably the sensitivity of one equilibrium reaction over the other. This enabled the determination of dilute cholesterol solutions to be carried out with high absorbance signals across the range of 0.0–10.0 mg/liter (0–0.0259 mmol/liter) of cholesterol, a range which includes and exceeds the normal values one would encounter in dilute solutions such as cerebrospinal fluid. Important potential interference factors including increased protein concentrations and calcium and magnesium were considered and studied. The formidable interacting compound, bilirubin, which is competitive with the 4-aminoantipyrene and sodium 2-hydroxy-3, 5-dichlorobenzenesulfonate mixture in the peroxidase sequence was also studied, its interference characteristics were determined and an alternative methodology suggested for obviating this perturbing effect. It is believed that the simple substitution of the auxochrome derivative is a useful contribution not only here but in studies presently ongoing involving needed sensitivity for cholesterol fractions which are considerably lower than the serum total cholesterol concentrations most commonly determined.