Development of a direct LC-ESI-MS method for the measurement of human serum carnosinase activity

Carnosine (β-alanyl-L-histidine) is a natural peptide that have been described as a potential pharmacological agent owing to some positive outcomes from several pharmacological tests in animal models of human diseases. However, carnosine has limited activity in humans since the peptide upon absorption is rapidly hydrolyzed in the serum by the enzyme carnosinase (i.e. CN1; E.C. 3.4.13.20). Over the years the main approaches aimed at limiting carnosine hydrolysis have been focused on obtaining CN1-stable derivatives with an increased bioavailability and unmodified or enhanced activity. Only recently the hypothesis of co-administration of carnosine and selective inhibitors of CN1 have been proposed. Such an approach requires reliable methods for screening the effect on carnosine hydrolysis rate operated by CN1 in a throughput scale allowing to test from few compounds up to whole compound libraries. The only assay with such features available in literature relies on ortho-phtalaldehyde (OPA) derivatization of the hydrolysis product (i.e. histidine), followed by a fluorimetric read. Herein, we propose an alternative method based on a direct measurement of the residual substrate by liquid chromatography-mass spectrometry (LC-MS). The assay demonstrated to be reliable since gave results comparable to literature data concerning the hydrolysis rate of carnosine as determined into human serum. Moreover, the method was quite flexible and easily adaptable to a substrate change, as demonstrated by the measurement of the hydrolysis rate of all the natural analogs of carnosine. In this context the data collected for anserine suggest that our method looked more reliable and substrate change can undergo an underestimation of hydrolytic activity in OPA -based assays.