CHROMATOGRAPHIC EVIDENCE FOR PYRRALINE FORMATION DURING PROTEIN GLYCATION IN-VITRO AND IN-VIVO

Pyrraline (formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine) is an adv anced Maillard reaction product formed from 3-deoxyglucosone in the no n-enzymatic reaction between glucose and the epsilon-amino group of ly sine residues on proteins. Although its presence in vivo as well as in in vitro incubations of proteins with sugars has been documented by i mmunochemical methods using polyclonal and monoclonal antibodies, its formation in proteins has recently been questioned by similar methodol ogy. To clarify this issue, we investigated pyrraline formation in pro teins following alcaline hydrolysis and quantitation by high-performan ce liquid chromatography on a C-18 reverse-phase column. Time- and sug ar concentration-dependent increase in pyrraline formation was noted i n serum albumin incubated with either 100 mM glucose or 50 mM 3-deoxyg lucosone. Formation of pyrraline from 3-deoxyglucosone was rapid at sl ightly acidic pH, confirming its synthetic pathway through this Mailla rd reaction intermediate. Low levels of pyrraline(< 10 pmol/mg protein ) were also detected in a pool of human skin collagen by this method, but no age effect was apparent. Using a slightly different approach, p yrraline-like material was detected in human plasma proteins following enzyme digestion and analysis by high performance liquid chromatograp hy. Plasma from diabetic patients showed a significant increase in pyr raline-like material compared to controls. The levels in diabetic and normal individuals were 21.6 +/- 9.56 and 12.8 +/- 5.6 pmol per mg pro tein, respectively (P = 0.005), reflecting thereby the elevated levels of the immediate precursor of pyrraline, 3-deoxyglucosone, in diabeti c plasma.