AN IMPROVED KINETIC METHOD FOR MEASUREMENT OF URINARY ALANINE AMINOPEPTIDASE (AAP) ACTIVITY AS A NEPHROTOXIC MARKER

This study examines the effect of various factors that influence alanine aminopeptidase (EC 3.4.11.2; AAP) activity in untreated urine samples, using L-alanine- p -nitroanilide as substrate. An improved kinetic method for assay of AAP activity without pretreatment of urine samples was obtained. The results show that the final concentration of 6 mmol/ L L-alanine- p -nitroanilide and 325 mmol/ L tris(hydroxymethyl)aminomethane (pH 7.90 at 30 C) and the sample/ final reaction volume ratio of 1/ 25 to be optimum. The within-run coefficient of variation (CV) was 1.40%; the between-run CV averaged 3.11%. Reference values were established for AAP activity of untimed urine samples obtained from 810 healthy persons. The normal reference interval (mean 1 SD) was 0.81-38.64 (12.76 6.88) U/ g of creatinine. Freezing the untreated urine sample resulted in a considerable loss of AAP activity. However, when urinary AAP activity is measured by the present method, there is no need for urine and reagent blanks; assay of the enzyme in urine is possible using one reagent solution. Therefore, the present method is suitable for use with various automated analyzers. Finally, the present method requires only 30muL of untreated urine samples, so it is suitable for evaluating of nephrotoxicity in human (especially neonates and infants) and small animal models such as rats.