Aging Aging

210 Requena et al.Fig. 1. Structure of pentosidine.that pentosidine would have a significant effect on collagen structure.Pentosidine is, however, an excellent biomarker for nonenzymatic modificationof long-lived proteins by the Maillard reaction, providing insight into overallrole of the Maillard reaction in aging and disease. Concentrations ofpentosidine in plasma protein and collagen are elevated in several pathologicalconditions, especially in end-stage renal disease (2,8,10), and, to a lesser extent,in diabetes (2–4,6,8,11,16,17), rheumatoid arthritis (12,13,18), atherosclerosis,and neurodegenerative diseases (19). The elevation in pentosidine concentrationin these diseases is attributed to increased precursor concentrations,resulting from either increased oxidative stress and/or decreased detoxificationof reactive carbonyl intermediates in the Maillard reaction.This chapter describes a procedure for the preparation, purification, andcalibration of a pentosidine standard, followed by a general method for measuringpentosidine by reverse-phase high-performance liquid chromatography(RP-HPLC) with fluorescence detection. This method is suitable for collagensamples, which produce clean chromatograms. In these cases, pentosidine isnormalized to the hydroxyproline (20) or lysine content of the sample, or to theamount of protein. More complex samples, such as tissue extracts, plasma, andserum, yield noisy chromatograms in which pentosidine cannot always beresolved from other components. In these cases, the basic procedure ismodified to include a simple clean-up procedure using sulfopropyl–Sephadex(SP-Sephadex) solid-phase extraction columns, based on a modification ofthe method of Takahashi et al. (10). In these cases, pentosidine concentrationis expressed per mole of lysine, per milligram of protein, or per milliliter ofserum or plasma. In general, to facilitate comparisons between proteins, itis more convenient to express the data as moles of pentosidine per mole oflysine.