Measurement of Human Serum Ceruloplasmin by Its p-Phenylenediamine Oxidase Activity

Abstract

Optimum reaction conditions were evaluated for assay of serum ceruloplasmin by measurement of its p-phenylenediamine oxidase activity. The pH optima of p-phenylenediamine oxidase activities of human and rat ceruloplasmins were 5.45 and 5.2, respectively. The p-phenylenediamine oxidase activity of rat ceruloplasmin was markedly inhibited by phosphate (0.1 mol/liter), that of human ceruloplasmin only slightly. These reaction conditions were judged to be optimum for the ceruloplasmin assay: (a) substrate: p-phenylenediamine dihydrochloride, 8.9 mmol/liter; (b) buffer: acetate, 0.1 mol/liter; (c) chloride concentration: 21 mmol/liter; (d) serum dilution: 31-fold; (e) incubation: 37°C for 30 min; (f) spectrophotometry: 530 nm (vs. a "serum blank" containing NaN₃). Using these reaction conditions, we devised an improved technique for measuring serum ceruloplasmin. The coefficients of variation of replicate analyses by this technique were 1.25% (for "within-run" precision) and 2.8% (for "day-to-day" precision). The mean concentration of ceruloplasmin in sera from 29 healthy men was 0.315 g/liter (SD = ± 0.049, range = 0.233 to 0.402).