Polarographic Method for Rapid Microdetermination of

Cholesterol concentrations in serum are enzymatically determined rapidly by use of a polarographic oxygen analyzer with a circuit modified to record simultaneous-ly the amount and rate of oxygen consumption. The final assay system, assessed from the oxygen con-sumption value that we found to be optimum, consists of 1 ml of sodium phosphate buffer (0.6 mol/liter, pH 7.0) containing NaN3 (10 mg/liter), Triton X-100 surfac-tant (10 mI/liter), 0.4 U of cholesterol ester hydrolase, and 0.6 U of cholesterol oxidase. Oxygen consumption and cholesterol concentration are linearly related to 8.0 g/liter, and only 10 tl of serum is required. Replicate analyses of pooled serum by the present method dem-onstrated the following inter-run precision: mean = 1731 mg/liter, SD 22.3 mg/liter, CV 1.3%. Biliru-bin and ascorbic acid were without effect on the present method, unlike the enzymatic colorimetric methods. AddItional Keyphrases: pediatric chemistry #{149}intermethod comparison Because of clinical importance and analytical dif-ficulties, determination of serum cholesterol has been the subject of numerous investigations. Colorimetric reactions for cholesterol determination have been plagued with a long list of difficulties: lack of speci-ficity, difficulty in standardization, variable reactivi-ty of esters, unstable and corrosive reagents, and many interfering substances. Recently, the specificity provided by enzyme reagents has been introduced into cholesterol determinations (1-4). In the nzy-matic cholesterol-measuring systems adopted in the