An improved method for evaluation of nephrotoxicity by assay of urinary N-acetyl-beta-D-glucosaminidase (NAG) activity

Reaction conditions of N-acetyl-beta-D-glucosaminictase (EC 3.2.1.30; NAG) determination in untreated urine samples were studied, using 4-nitrophenyl-N-acetyl-beta-D-glucosaminide (PNP-NAG) as substrate. Final concentrations of 8 mmol of PNP-NAG, 25 mmol of acetate, or 100 mmol of citrate (pH 4.80 at 37 degrees C) per liter and a sample/reagent volume ratio of 1/18 were found to be optimum. The within-run and between-run precision was excellent, with CVs averaging 2.67 and 4.55%, respectively. Reference values for urinary NAG activity assayed by this method were established for untimed human urine specimens from 206 healthy subjects. The normal reference interval (mean +/- 1SD) was 0.15-15.05 (4.51 +/- 3.02) U per gram creatinine. By this method, urinary NAG activity can be measured over a wide range (up to 200 U/L) in untreated urine samples with high sensitivity. Urine samples need no pretreatment, and two reagent solutions are enough for measurement of NAG activity The method is thus suitable for use with various automated analyzers. Finally, this method requires only to 25 mu L Of untreated urine. Therefore, it is appropriate for evaluating nephrotoxicity in human (especially neonates and infants) and small animal models by assay of urinary NAG activity.