N-acetyl-beta-D-glucosaminidase assay in urine: urea inhibition

Abstract

Urea inhibition of urinary N-acetyl-beta-D-glucosaminidase (NAG) was examined with human isoenzymes and total enzyme in urine. The fluorometric substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide (MUNAG) and the colorimetric substrate m-cresolsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide (MCPNAG) were used to determine substrate kinetics and apparent urea inhibition kinetics. Apparent KmS of 0.47 (MUNAG) and 0.35 mmol/L (MCPNAG) for human NAG isoenzyme A and 0.46 (MUNAG) and 0.30 mmol/L (MCPNAG) for human NAG isoenzyme B were determined. Apparent Kis of approximately 100 mmol/L for urea inhibition of both isoenzymes in either substrate and apparent Kis of 79 (MUNAG) and 91 mmol/L (MCPNAG) for NAG in dilute urine samples were found. The potential for urea inhibition at physiological concentrations of urea and at normal assay dilutions therefore exists. Inhibition at these dilutions was minimal when substrate concentrations of 2 x Km or greater were used, but the substrate MUNAG at 1/2 x Km gave the highest sensitivity and lower blanks and allowed better quantitation of low activity samples. Within-run CVs of 3.2% with MUNAG and 10% with MCPNAG were found for low activity samples.