Chromatographic Quantitation of Plasma and Erythrocyte Pentosidine in Diabetic and Uremic Subjects

Abstract

Pentosidine is a fluorescent advanced Maillard/glycosylation product and protein cross-link present in elevated amounts in skin from diabetic and uremic subjects. A high-performance liquid chromatographic (HPLC) assay was developed to quantitate pentosidine in plasma and erythrocytes and other tissue proteins with low levels of pentosidine. High protein content and presence of basic amino acids and O2 during acid hydrolysis led to the formation of fluorescent artifacts that could be separated from true pentosidine through combined reverse-phase ion-exchange HPLC. No true pentosidine was formed during acid hydrolysis of ribated protein, suggesting that Amadori products do not generate artifactual pentosidine during hydrolysis. With the combined reverse-phase ion-exchange chromatographic assay, we found a 2.5-fold (P < 0.001) and a 23-fold (P < 0.001) elevation of mean ± SD plasma protein pentosidine in diabetic (2.4 ± 1.2 pmol/mg) and uremic (21.5 ± 10.8 pmol/mg) subjects compared with healthy (0.95 ± 0.33 pmol/mg) subjects. Pentosidine in hemolysate was normal in diabetes but dramatically elevated in uremia (0.6 ± 0.4 pmol/mg hemoglobin, P < 0.001). Although the precise nature of the pentosidine precursor sugar is unknown, plasma pentosidine may be a useful marker for monitoring the biochemical efficacy of trials with aminoguanidine or other treatment modalities. Furthermore, pentosidine in plasma proteins may act as a signal for advanced glycosylation end product-mediated receptor uptake by macrophages and other cells and contribute to accelerated atherosclerosis in diabetes and uremia.