Determination of serum catalase activity on a centrifugal analyzer by an NADP/NADPH coupled enzyme reaction system

Abstract

We developed a simple, kinetic method for the determination of catalase activity in which i) the enzyme catalyzes the peroxidation of ethanol by hydrogen peroxide to acetaldehyde and water, and ii) the acetaldehyde so formed is rapidly oxidized to acetic acid and NADPH by the addition of an excess of NADP+ and aldehyde dehydrogenase. The rate of NADPH production was monitored at 340 nm in a COBAS centrifugal analyzer. The reaction was linear to 800 U/L or a delta A of 0.020/min. Using human serum pools containing 80 and 460 U/L of peroxidase activity, the within-run coefficients of variation (CV) were 1.9 and 1.3%, respectively. Between-run CV values were 5% for both pools. The reference range for sera from 72 males and 52 females was 23 to 158 U/L (mean + 2 SD) by log normal transformation. The activity in red cells was 600 U/g hemoglobin but did not change the reference range appreciably provided that serum without visible hemolysis was used. Preliminary observations on sera from nine patients with various pancreatic disorders showed a poor correlation between the activities of catalase (peroxidase) and amylase in serum. The reasons for this discrepancy are under investigation.