Discovery of free glycated amines and glycated urea as potential markers of diabetic nephropathy

The role of protein glycation in the pathogenesis of diabetes is well established. Akin to proteins, free amino acids and other small-molecule amines are also susceptible to glycation in hyperglycemic conditions and may have a role in the pathogenesis of the disease. However, the information on glycation of free amino acids and other small- molecule amines is relatively obscure. In the quest to discover small molecule glycated amines in the plasma, we have synthesized glycated amino acids, glycated creatine, and glycated urea, and by using a high-resolution accurate mass spectrometer, a mass spectral library was developed comprising the precursor and predominant fragment masses of glycated amines. Using this information, we report the discovery of glycation of free lysine, arginine, and leucine/isoleucine from the plasma of diabetic patients. This has great physiological significance as glycation of these amino acids may create their deficiency and affect vital physiological processes such as protein synthesis, cell signaling and insulin secretion. Also, these glycated amino acids could serve as potential markers of diabetes and its complications. On the other hand, accumulation of creatinine and urea in the plasma act as biomarkers of diabetic nephropathy diagnosis. For the first time, we report the detection of glycated urea in diabetes, which is confirmed by matching the precursor and fragment masses with the in vitro synthesized glycated urea by using 12C6 and 13C6-glucose. Further, we quantified glycated urea detected in two forms, monoglycated urea (MGU) and diglycated urea (DGU), by a targeted mass spectrometric approach in the plasma of healthy, diabetes, and diabetic nephropathy subjects. Both MGU and DGU showed a positive correlation with clinical parameters such as blood glucose and HbA1c. Given that urea gets converted to glycated urea in hyperglycemic conditions, it is crucial to quantify MGU and DGU along with the urea for the diagnosis of diabetic nephropathy.