Dl glyceraldehyde

AGEs were prepared as previously described by Okazaki et al. [30 (link)]. Briefly, 50 mg/ml bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) was incubated with 0.1 M DL-glyceraldehyde (Sigma-Aldrich) in 0.2 M phosphate buffer (pH 7.4) at a 37°C sterilized incubator for 1 week, then exhaustively dialyzed against phosphate-buffered saline (pH 7.4) for three days. Nonglycated BSA was prepared at the same time, except that no DL-glyceraldehyde was added. Fluorescence strength of AGE solution was detected at an excitation/emission wavelength of 370/440 nm, which is fortyfold higher than the BSA control.

Human recombinant aldose reductase from Escherichia coli, d,l-glyceraldehyde, NADPH, NaH₂PO₄, Na₂HPO₄ and Tolrestat were purchased from Sigma Aldrich.

Streptozotocin (STZ), DL-glyceraldehyde, and analytical-grade solvents were purchased from Sigma–Aldrich (St. Louis, MO, USA). NADPH tetrasodium salt and 2-mercaptoethanol were purchased from Carl Roth GmbH (Karlsruhe, Germany).

DL-Glyceraldehyde, reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), bovine serum albumin, methylglyoxal, 2,2-diphenyl-1-picrylhydrazyl (DPPH), aminoguanidine, L-ascorbic acid, and quercetin used in this study were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.

AR was purchased from Prospec-TanyTechnogene Ltd. (Israel). AR activity was evaluated as previously described [48 (link), 49 (link)]. Briefly, the reaction mixture was composed of enzyme solution (20 mmol·L^-1, 20 μL), NADPH (0.104 mmol·L^-1, 50 μL; Italian Roth, Italy), DL-glyceraldehyde (10 mmol·L^-1, 50 μL; Sigma, U.S.), buffer phosphate (0.1 mol·L^-1), and selected concentrations of test compounds. The reaction was initiated by the addition of DL-glyceraldehyde as the substrate, and PBS was used as the blank control. The use of NAPDH, in parallel to AR activity, was monitored at room temperature for 10 min at 40 s intervals at 340 nm in an EnSpire^™ Multimode Plate Reader. AR inhibition was expressed as inhibition rate (%) = [1−(A₂ − A₀)/(A₁ − A₀)] ×100% [50 (link)], where A₀ represents the decrease in NADPH absorbance without AR, substrate, or compounds; A₁ represents the decrease in NADPH absorbance prior to the test compounds addition; A₂ represents decrease in NADPH absorbance following the test compounds addition. All assays were performed in triplicate.

dl-Glyceraldehyde, the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), bovine serum albumin (BSA), sodium phosphate, methyl glyoxal, quercetin, DPPH, l-ascorbic acid, aminoguanidine, and methanol (MeOH) used in this study were purchased from Sigma (St. Louis, MO, USA). human recombinant aldose reductase (HRAR) was purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals and reagents used were of analytical grade.