Interference of bromide in the Zak ferric chloride-sulfuric acid cholesterol method, and means of eliminating this interference.

HE AUTHORS’ LABORATORY has employed the modified Zlatkis method (1) of Zak and associates (2) for the routine determination of serum cholesterol fractions for over a year with generally satisfactory results. However, ester fractions greater than 80 per cent of the total cholesterol values were occasionally obtained. Recently, an analysis of the serum “total cholesterol” from a patient with acute brominism (serum bromide, 50 mEq./L.) resulted in the formation of a reddish-orange instead of the typical purple color. The “free cholesterol” fraction of this serum had the usual appearance. Subsequent investigation showed that the ferric chloride-sulfuric acid color reaction for cholesterol gives erroneously high results in the presence of even traces of bromide. This interference is a likely explanation for the high ester fractions previously mentioned. Table 1 shows the effect of various quantities of NaBr added to a cholesterol standard. The interference is measurable with NaBr concentrations as low as 5 pig. or less per tube-levels at which the color appears a normal purple to the eye. In Table 2 are results of cholesterol determinations with varying amounts of NaBr added to human sera. The values for total cholesterol are incorrect and the ester fraction (calculated by subtracting the free cholesterol from the total) is in similar positive error. The free cholesterol levels are essentially unaffected because the cholesterol digitonide precipitate is freed of bromide contamination by decanting and washing with acetone. Iodide also gives a color comparable to that of bromide. Several experiments showed that neither bromide nor iodide affects the