Abstract

GLYCATION BY GLUCOSE AND KETONE BODIES OF AMINOPHOSPHOLIPIDS FROM HUMAN TISSUES IS INHIBITED BY L-ARGININE AND CREATINE. I. ERYTHROCYTE MEMBRANE

José D. Méndez*

Abstract

The aim of this study was to evaluate the in vitro effect of L-arginine and creatine on glycation by glucose and ketone bodies of aminophospholipids from human erythrocyte membranes. Aminophospholipids were isolated by selective extraction from human erythrocyte membranes. Purity of aminophospholipids was confirmed by thin layer chromatography. When the effect of L-arginine or creatine was tested, 10 mM concentration of these substances was used. Emulsions were prepared in 0.1 M phosphate buffer, pH 7.4 containing 11 mM glucose, 3 mM β-hydroxybutyrate or 3 mM acetoacetate, respectively and incubated at 37 °C in dark during 61days. Samplings were made at days 0, 12, 19, 26, 48 and 61. Samples were analyzed by thin layer chromatography, fluorescence techniques and ultraviolet spectroscopy. Three intense spots with Rfs of 0.097, 0.50, 0.902, respectively were revealed, in addition to other spots with low intensity. Glycation was higher for glucose followed by β-hydroxybutyrate and acetoacetate. Fluorescence analysis registered into two ranks of excitation and emission; 320/460, and 355/460 nm, respectively showed that the fluorescence increased as a function of incubation time. L-arginine and creatine interfere with the glycation process depending on the glycating agent. Relevant spectral changes from 290 to 200 nm were revealed by ultraviolet spectroscopy when aminophospholipids and ketone bodies were combined. Glycation process was affected by L-arginine and creatine. L-arginine effect was better on samples containing β-hydroxybutyrate. The effect of creatine was similar when it was added to aminophospholipids combined with glucose, β-hydroxybutyrate and acetoacetate, respectively. This study provides evidence that ketone bodies react with aminophospholipids of erythrocyte membranes forming fluorescent advanced glycation end products as do it glucose. Glycation of aminophospholipids was prevented by L-arginine and creatine.

Keywords: Acetoacetate, Aminophospholipids, L-Arginine, Creatine, Glycation, ?-hydroxybutyrate, Hyperglycemia.