Construction and validation of a method for detecting DPP-IV activity and tissue distribution in C57 mice based on a fluorescent probe.

Dipeptidyl peptidase-IV (DPP-IV) is critical for drug metabolism and physiological regulation. In terms of differences in DPP-IV among species, mouse and human DPP-IV share a high degree of similarity, and mice are typically used as the optimal animal model for conducting the relevant pharmacological evaluations and biological function studies. Herein, a detection method is developed using the fluorescent probe GP-BAN and mouse tissue S9 fraction (enzyme source) to rapidly quantify DPP-IV activity in mouse tissues. The method was validated for specificity, linearity and precision, and the metabolite BAN showed good linearity in the 0-20 µM range using a weighted (1/x) least squares linear regression model (r2 = 0.9996), with an LOD of 5.5 nM and LOQ of 16.7 nM. In C57 mice, the DPP-IV activity in 14 tissues/organs (including the liver, kidney, thymus, and small intestine) differed significantly: the thymus had the highest activity (2.64 nM µg-1 protein per min), followed by the liver, kidney and small intestine. Enzyme kinetics showed that the Km of GP-BAN for mouse DPP-IV was 34.05 µM, which is close to that of human liver microsomes (HLM, 41.46 µM), indicating cross-species substrate binding consistency. ELISA confirmed that DPP-IV protein expression correlated positively with activity in the liver, kidney and thymus (r > 0.92, p < 0.001). This sensitive, species-translatable assay and its organ-specific activity/kinetic data support mouse models in preclinical DPP-IV studies, improving cross-species extrapolation predictability in physiological research.