5 methylquinoxaline

Methylglyoxal (40 % in water), bovine serum albumin (BSA, fraction V), aminoguanidine, 2-methylquinoxaline, 5-methylquinoxaline, o-phenylenediamine, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu’s phenol reagent, and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade.

Methylglyoxal (40 % in water), isoferulic acid (IFA, 3-hydroxy-4-methoxycinnamic acid), bovine serum albumin (BSA, fraction V), aminoguanidine, 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB), 2-deoxy-D-ribose, 2-methylquinoxaline, 5-methylquinoxaline, o-phenylenediamine, thiobarbituric acid and cupric sulfate (CuSO₄) were purchased from Sigma-Aldrich (St.Louis, MO, USA). L-lysine, 2,4-dinitrophenyl hydrazine (DNPH) and guanidine hydrochloride were obtained from Himedia (Mumbai, India), Ajax Finechem (Taren Point, Australia) and Fluka (Steinheim, Germany), respectively. OxiSelect™ N^ε-(carboxymethyl) lysine (CML) ELISA kit was acquired from Cell Biolabs (San Diego, CA, USA). QIAprep Spin Miniprep kit was obtained from Qiagen (Venlo, Netherlands) and cytochrome c was purchased from Affymetrix (Santa Clara, CA, USA). All other chemicals used were of analytical grade.

MG and pyruvate levels were measured by converting these metabolites into quinoxaline derivatives using 1,2-diaminobenzene (Merck) and methods already detailed [9 (link), 26 (link)]. For utilization in enzymatic studies and intracellular quantification, MG from a commercial 40% solution was repurified by vacuum distillation (Sigma, USA) to eliminate polymerized MG and impurities [27 (link)]. The distilled MG concentration was calculated to be 0.5565 M, which was stoichiometrically confirmed by 2-methylquinoxaline (Sigma) using a high-performance liquid chromatography (HPLC) system. Analytical grade pyruvate (Sigma) was purchased and used without further purification. Quinoxaline derivatives of MG and pyruvate (i.e., 2-methylquinoxaline and 2-hydroxy-3-methylquinoxaline) were obtained using a solid-phase extraction (SPE) cartridge (Waters) containing a C18 resin. The resulting derivatives were separated using an Agilent 1200 series HPLC system with a Zorbax Eclipse XDB-C18 analytic column (Agilent, 150 × 4.6 mm) at a wavelength of 336 nm with an internal standard 5-methylquinoxaline (Sigma).