Chemical and Immunological Characterization of Oxidative Nonenzymatic Protein Modifications in Dialysis Fluids

Abstract

Glucose degradation products (GDP) in dialysis fluids may induce nonenzymatic protein modifications, the chemical nature and biological properties of which should be better defined. To characterize nonenzymatic protein modifications present in glucose-based peritoneal dialysis fluids (PDF) and to evaluate the relationship between concentrations of GDP and the derived nonenzymatic modifications, and the potential of PDF for generating these modifications in vitro. The presence, distribution, and content of several nonenzymatic protein modifications in PDF were evaluated by immunological methods, by HPLC, and by gas chromatography-mass spectrometry (GC/MS). Peritoneal dialysis fluid-induced oxidative stress in cells was evaluated by flow cytometry. The potential of PDF for generating oxidative and glycoxidative modifications was examined by immunological and cross-linking analyses. The albumin present in PDF is modified by carboxymethyllysine (CML). GC/MS analyses of PDF proteins confirmed the presence of CML and demonstrated the occurrence of carboxyethyllysine, malondialdehyde lysine, and oxidation-derived semialdehydes. Furthermore, their concentrations in PDF proteins were significantly higher than those in plasma proteins (in all cases, p < 0.02). The concentration of pyrraline, a non-oxidative advanced glycation end-product, increased with dwell time up to 6 hours (p < 0.03). The PDF induced cellular free-radical production, which was partially inhibited by the Maillard reaction inhibitor aminoguanidine (p < 0.001). The potential to generate oxidative and glycoxidative modifications demonstrated an inverse relationship with dwell time (p < 0.05). The PDF was able to induce collagen cross-linking in a close relationship with GDP concentration. (1) PDF contains non-oxidative and several oxidative nonenzymatic protein modifications in higher concentrations than plasma. (2) Peritoneal dialysis fluid induces oxidative stress in vitro, which can be partially inhibited by aminoguanidine. (3) These properties are directly related to GDP concentration. (4) Peritoneal dialysis fluid is able to generate glycoxidative and oxidative damage to proteins in vitro in a dwell-time dependent fashion.