A synthesis and quantification method for endogenous metabolites dimethylguanidino valeric acid

Dimethylguanidino valeric acid (DMGV) is a group of endogenous metabolites derived from arginine-containing proteins and is associated with several metabolic disorders. Latest studies have identified the stereoisomers of DMGV: asymmetric dimethylguanidino valeric acid (ADGV) and symmetric dimethylguanidino valeric acid (SDGV), which are derived from asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), respectively. However, the lack of commercial standards has hampered research into the molecular mechanisms of DMGV and its potential clinical application as biomarkers. Reported chemical synthesis methods have low yields and require specialized chemical synthesis skills and apparatus. Therefore, we aimed to develop a practical and efficient method to synthesize DMGV stereoisomers and determine their precise concentration profile in healthy subjects. A novel biocatalytic method for synthesizing DMGV using alanine-glyoxylate aminotransferase 2 (AGXT2) was developed. A metabolite panel including eight DMGV-related metabolites was also established for human plasma samples using HPLC-MS/MS, and its performance was comprehensively evaluated, especially in terms of quantitative sensitivity, precision and accuracy. Compared with the reported chemical synthesis methods, the biocatalytic approach demonstrates superior yield, conversion rate, and product purity, while being easy to implement in biological laboratories. The established DMGV quantification method has been well validated and successfully applied to measure the contents of these metabolites, especially the concentration profile of ADGV in the plasma of healthy individuals. To sum up, this study provides an efficient and practical biocatalytic approach for DMGV synthesis and determines the levels of DMGV in healthy subjects. These findings will undoubtedly promote future mechanistic studies of DMGV and its future clinical applications.