PATHWAYS OF FORMATION OF GLYCOXIDATION PRODUCTS DURING GLYCATION OF COLLAGEN

Glycoxidation products (GOPs), such as N-epsilon-(carboxymethyl)lysine (CML) and pentosidine, are formed during reaction of glucose with pro tein under oxidative conditions in vitro. It is uncertain whether thes e GOPs are derived from oxidation of Amadori adducts on protein or fro m oxidation of glucose or intermediates formed prior to the Amadori re arrangement. To address this question, we reacted collagen with 250 mM glucose in 200 mM phosphate buffer, pH 7.4, under antioxidative condi tions, yielding a protein rich in Amadori adducts, but with only trace s of GOPs. This ''preglycated'' collagen was then exposed to [C-13(6)] glucose under oxidative conditions, producing both natural and [C-13(2 )]-CML. At 200 mM phosphate buffer, [C-13(2)]-CML was the major produc t, even at low (5 mM) [C-13(6)]glucose concentration, a limited role f or Amadori compounds in formation of CML in high phosphate. The relati ve yields of natural and [C-13(2)]-CML varied with phosphate concentra tion, becoming similar at more physiological (10 mM) phosphate. We con clude that during glycation of proteins at high phosphate concentratio ns in vitro, GOPs are formed primarily by oxidation of free glucose or rapidly-formed intermediates preceding the Amadori rearrangement, suc h as carbinolamine or Schiff base adducts. In contrast, at lower phosp hate and glucose concentrations in vivo, the Amadori adduct may be the more significant precursor of GOPs. The fact that glycoxidation react ions proceed by multiple routes must be considered in the development of therapeutic approaches for inhibiting the Maillard reaction in diab etes.