Interference by ascorbic acid in test systems involving peroxidase. I. Reversible indicators and the effects of copper, iron, and mercury.

I describe the mechanism whereby ascorbic acid can hamper test systems involving a peroxide-generating system, peroxidase, and a benzidine-type indicator. In test media such as urines, abnormally high concentrations of ascorbic acid may give rise to false negatives in the determination of analytes such as glucose. Absorbance measurements in solution or reflectance measurements on commercially available paper reagent strips demonstrate either inhibition of visible activity by ascorbic acid or a lag time in the development of oxidized indicator color. The duration of the lag time is proportional to the ascorbic acid concentration, is inversely proportional to the enzyme concentration, and is also affected by concentrations of hydrogen peroxide and o-tolidine indicator. The same results were seen in both citrate buffer pH 5 and phosphate buffer pH 7. Because the complete system (o-tolidine indicator, hydrogen peroxide, and peroxidase) must be present if the ascorbate is to be oxidized rapidly, this indicates that ascorbic acid inhibits color development by re-reducing oxidized indicator as fast as it is formed; the o-tolidine then acts catalytically in oxidizing ascorbic acid. Added Cu2+ and Fe3+, both known to react with ascorbic acid, had measurable but small effects on the system. In contrast, Hg2+ abolished the ascorbic acid-elicited lag time, even when present in near-stoichiometric concentration. Hg2+ showed little inhibitory effect on the activity of either glucose oxidase or peroxidase. Presumably it rapidly oxidizes ascorbic acid to dehydroascorbate. The reaction of mercuric ion with ascorbate was measured by reflectance measurements of paper reagent strips in addition to absorbance measurements of solution assays; equivalent results were obtained. Incorporation of Hg2+ into reagent strips can thus render both strips and solution diagnostic tests insensitive to interfering substances such as ascorbic acid.