Methylglyoxal (MG) is a highly reactive dicarbonyl compound involved in the formation of advanced glycation endproducts (AGE). Levels of MG are elevated in patients with type-2 diabetes mellitus (T2DM), and AGE have been implicated in the progression of diabetic complications. The antihyperglycemic drug metformin (MF) has been suggested to be a scavenger of MG. The present work examined and characterized unequivocally the resulting scavenged product from the metformin–MG reaction. The primary product was characterized by 1H, 13C, 2D-HSQC, and HMBC NMR and tandem mass spectrometry. X-ray diffraction analysis determined the structure of the metformin and MG-derived imidazolinone compound as (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yI)guanidine (IMZ). A LC-MS/MS multiple reaction monitoring method was developed to detect and quantify the presence of IMZ in metformin-treated T2DM patients. Urine from >90 MF-treated T2DM patients was analyzed, with increased levels of MF directly correlating with elevations in IMZ. Urinary MF was detected in the range of 0.17 μM to 23.0 mM, and simultaneous measurement of IMZ concentrations were in the range of 18.8 nM to 4.3 μM. Since plasma concentrations of MG range from 40 nM to 4.5 μM, the level of IMZ production may be of therapeutic significance. Thus, in addition to lowering hepatic gluconeogenesis, metformin also scavenges the highly reactive MG in vivo, thereby reducing potentially detrimental MG protein adducts, with subsequent reductions in diabetic complications.

Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC-MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan-HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients.

Advanced glycation end products (AGE) are a possible link between hyperglycemia and the development of diabetic complications. Careful control of AGE could be vital to improve diabetic complications. Biomarkers to evaluate control of AGE are needed to guide development of emerging therapies. Using an unbiased proteomics approach, dicarbonyl (methylglyoxal, glucosone, and 3‐deoxyglucosone) on arginine (R) and oxidative markers on methionine residues on serum albumin were studied in 120 subjects with varying degrees of type‐2 diabetes mellitus (T2DM) with or without metformin treatment. Trypsin digested delipidated serum samples were analyzed by multiple reaction monitoring on a QTRAP mass spectrometer, based on transitions that were validated in vitro. Strong signals for dicarbonyl modification were observed at R186, R257, and R428. Statistical analyses of the dicarbonyl‐R in relation to known T2DM clinical markers including HbA1c, fasting glucose, and waist circumference were performed between groups. Dicarbonyl markers were significantly higher in T2DM (n=47) compared to non‐T2DM (n=46), and certain markers were significantly decreased in T2DM/metformin compared to T2DM (n=26; p<0.001 [post hoc]). The study reveals that dicarbonyl adducts may be a useful biomarker for monitoring the progression of T2DM and metformin treatment efficacy. (P30ES006694, R24DK083948, T32ES016652, T32ES007091).

Reactive dicarbonyls such as methylglyoxal (MG) are elevated in type‐2 diabetes mellitus (T2DM) patients and their ability to covalently modify proteins contributes to diabetic complications. The T2DM first‐line drug metformin (MF) significantly reduces diabetes‐related endpoints and mortality more effectively than other glucose‐lowering medications. We have examined whether, in addition to its ability to reduce hepatic gluconeogenesis, MF directly scavenges dicarbonyls as an additional mechanism to reduce T2DM complications. We synthesized a MF/MG cyclized product (183 mw) according to a published method, and characterized the product by ESI‐MS/MS mass spectrometry (MH+, 184 m/z; Agilent 6490) and 13C and 1H NMR (Bruker AVIII‐400). Using an LC‐MS‐based multiple reaction monitoring analysis we measured MF and cyclized metabolite (CM) in human urine with nM sensitivity of detection. The CM was detected in all 30+ MF‐treated T2DM subjects analyzed to date. Quantitation of CM in MF subjects is ongoing, using creatinine or specific gravity normalization. Correlations between CM levels and diabetic complications will be determined. The data reveal that urine from every T2DM patient treated with MF contains this MG scavenging product. The role of the CM in the reduction of diabetic complications warrants further study. Grant Funding Source: Supported by DK090958, ABRC, ES016652, T32ES007091, P30ES006694.