Background: Urinary enzymes, including the brush border enzyme alanine aminopeptidase (AAP), are reliable biomarkers of nephrotoxicity and represent early indicators of tubular damage. The aim of the present study was to develop and validate an automated urinary AAP activity assay.
Methods and results: For this purpose, we adapted a photometric test using the substrate L-alanine-4-nitroanilide to a dedicated clinical chemistry analyser. Due to the unavailability of calibrators with established AAP activity, we assessed the AAP activity of a commercially available control material using the photometric test. This material was subsequently employed as a calibrator for the automated assay. Quality assurance was accomplished with independent control materials. A fast gel filtration step of urine samples using Sephadex G50 was demonstrated to be necessary before AAP analysis to avoid underestimation of enzyme activity due to ammonia and amino acids. We compared the automated assay with a manual photometric assay and obtained excellent correlation between both methods (r2=0.946, n=50).
Conclusions: We conclude that the newly developed automated assay represents a user-friendly and analytical tool to determine urinary AAP activity in a medium- to high-throughput laboratory. Potential applications include clinical diagnostic work and toxicological studies.