PROTEIN MODIFICATION BY THE DEGRADATION PRODUCTS OF ASCORBATE - FORMATION OF A NOVEL PYRROLE FROM THE MAILLARD REACTION OF L-THREOSE WITH PROTEINS

Ascorbate (vitamin C) degradation products can undergo non-enzymatic g lycation (Maillard reaction) with proteins to form highly crosslinked structures with brown pigmentation and characteristic fluorescence. Pr oteins in the body, especially the long-lived proteins develop similar changes during aging and diabetes. Several studies have shown excessi ve degradation of ascorbate in plasma in diabetes, and in ocular lens during aging and cataract formation. Recent studies have suggested tha t ascorbate degradation products-mediated glycation plays a role in le ns pigmentation and cataract formation. However, the precise chemical nature of ascorbate-specific advanced glycation end-products are not k nown. Here, we report the purification and characterization of a glyca tion end-product derived from one of the major degradation products of ascorbate, L-threose. This compound was characterized to be lhyl)-2-f ormyl-4-hydroxymethyl-1-pyrrolyl)hexanoic acid (formyl threosyl pyrrol e or FTP) formed by the condensation of E-amino group of lysine with t wo molecules of threose. Formation of FTP occurred rapidly in the incu bation of threose and lysine and reached plateau level within a day. W e have developed a sensitive assay for its quantification in proteins based on enzyme digestion followed by HPLC. Ribonuclease A and human l ens crystallins incubated with L-threose showed time- and sugar concen tration-dependent increases in FTP, reaching 8.2 and 2.48 nmol per mg protein, respectively after one week of incubation. Human plasma prote ins showed a peak with identical retention time as that of purified FT P under two different HPLC conditions. FTP may be used as a sensitive marker to assess ascorbate-mediated protein glycation and modification s in aging and diabetes.