Objective: To measure serum zinc concentration by means of carbonic anhydrase reactivation using an automated analyzer.
Methods: The zinc content of carbonic anhydrase (CA), whose cofactor is zinc, was removed by dialysis against pyridine 2 to 6 dicarboxylic acid and a pure apoenzyme was obtained. Serum proteins were precipitated with trichloroacetic acid (TCA) solution and the supernatant fraction of the sample was used to determine the zinc concentration. The negative effects of the precipitant on CA activity in the assay were completely removed, reaction conditions for maximal CA activity were provided and the color of the product was enhanced and stabilized. P-nitrophenyl acetate was used as the substrate and the change of absorbance of p-nitrophenol which was produced was followed at 400 nm. The initial rate of the esterase activity of CA was measured by using an automated analyzer. Analytical performance characteristics of the assay were determined. The zinc concentrations in serum samples of healthy subjects and patients were measured.
Results: The enzymatic assay is accurate, sensitive, specific and is not affected by other metals. There was excellent agreement with the results obtained using an atomic absorption spectrophotometer (AAS) (y = 0.98X + 0.18, r = 0.99). Serum zinc concentrations were found to be higher in patients with vivax malaria, and lower in patients with cutaneous leishmaniasis than in healthy subjects.
Conclusion: The enzymatic method is suitable for semiautomated measurement of serum zinc concentration.