Influence of nonspecific reaction on determination of H2O2 using Trinder reagents

We examined two enzymatic reagents for cholesterol measurement that contained cholesterol oxidase. One of the reagents gave higher values by up to 11 mg/dl in samples of 109 out of 3119 patients examined. We found that the positive errors were caused by a nonspecific reaction that has not previously been recognized in the determination of H(2)O(2) using Trinder reagents, and we further discovered its mechanism.

We compared the total cholesterol concentration in 3119 patients between the 2 reagents. By examining the characteristics of the interferrent-susceptible reagent and affected patients' samples, we identified the cause and mechanism of the influence of the nonspecific reaction.

The existence of potassium ferrocyanide in the reagent gave rise to positive errors when the concentration of unsaturated iron binding capacity (UIBC) in the patients' sample was <70 microg/dl. Moreover, ceruloplasmin in the patients' sample was found to cause a nonspecific reaction and its influence was proportional to the serum copper concentration. The two types of reagents compared in the present study included buffers with different metal chelating capacities; the one with the lower capacity gave an erroneous measurement. The buffer in the reagent without influence had been acting as a chelating agent. Therefore, the influence of the interferrent-susceptible reagent could be prevented by addition of a chelating agent (e.g. EDTA).

Trinder reagents containing potassium ferrocyanide have been widely used for clinical diagnostic tests such as creatinine, uric acid and so on. These reagents would have similar problems. Some test items might have a greater influence than cholesterol reagent. Physicians should be aware of the risks of diagnostic mistakes due to these errors of measurement.