Carnosine, a histidine containing dipeptide, exerts beneficial effects by scavenging reactive carbonyl compounds (RCCs) that are implicated in pathogenesis of diabetes. However, the reduced carnosine levels may aggravate the severity of diabetes. The precise quantification of carnosine levels may serve as an indicator of pathophysiological state of diabetes. Therefore, we have developed a highly sensitive targeted multiple reaction monitoring (MRM) method for quantification of carnosine in human plasma samples. Various mass spectrometry parameters such as ionization of precursor, fragment abundance and stability, collision energy, tube lens offset voltage were optimized to develop a sensitive and robust assay. Using the optimized MRM assay, the lower limit of detection (LOD) and limit of quantification (LOQ) for carnosine were found to be 0.4 nM and 1.0 nM respectively. Standard curves were constructed ranging from 1.0 nM to 15.0 mM and the levels of carnosine in mice and human plasma were determined.Further, the MRM assay was extended to study carnosine hydrolyzing activity of human carnosinases, the serum carnosinase (CN1) and the cytosolic carnosinase (CN2). CN1 showed three folds higher activity than CN2. The MRM assay developed in this study is highly sensitive and can be used for basal plasma carnosine quantification, which can be developed as a novel marker for scavenging of RCCs in diabetes. Fig. 1 Scheme of plasma carnosine extractions and quantification using MRM method.This journal is Fig. 4 (A) CN1 and CN2 activity. CN1 and CN2 activity was studied using 2.0 mM carnosine and 2.0 mg of either CN1 or CN2 enzyme. The per cent residual carnosine is plotted. Effect of EDTA on CN1/CN2 was compared by the same method. The control assay was performed without any enzyme. (B and C) Activity curves of CN1 and CN2. Five different concentrations of either CN1 or CN2 were incubated with 2.0 mM of carnosine and plotted against the amount of percent carnosine hydrolysis at each concentration.768 | RSC Adv., 2020, 10, 763-769This journal is
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.