Advanced glycation end products (AGEs) as pathology biomarkers: microwave assisted synthesis and biological quantitative analysis by LC-FTMS

Introduction The Maillard reaction is a complex set of reactions, initially between carbonyl groups of reducing sugars and amino groups. Reactions of sugars with aminoacids or proteins are of great interest in food chemistry and in pathology. In fact AGEs accumulate in long lived tissue proteins in correlation with the development of complications in aging, diabetes, atherosclerosis and multiple sclerosis. Our research strategy has therefore focused on the quantification of specific AGEs biomarkers (pentosidine, glyceraldehyde derived pyridinium: GLAP) which could be associated with early pathology syndromes on rats fed high fat and fructose. In this work we synthesized pentosidine and GLAP by microwave assisted reactions in order to obtain standard compounds at improved yields and optimize the LC-FTMS quantitative analysis method. Methods In this study we initially developed a synthetic procedure to obtain standard pentosidine and GLAP by MW assisted synthesis. Male Wistar rats were given high cholesterol-fructose (HCF) diet for 15 weeks; diabetic and untreated rats were used as control. This diet is relevant to human nutrition because it mimics a common Western diet with high consumption of soft drinks. Control animals were fed with a standard diet. Plasma samples of both groups were analyzed after protein precipitation and hydrolysis. LC-HRMS analysis were accomplished on a HPLC-LTQ-Orbitrap instrument, with an electrospray interface. Analytical performances (sensitivity, recovery, repeatability) were compared with that of other MS analyzers (ion trap, triple quadrupole) and of a fluorescence detector. Preliminary data Diabetes was accompanied by a significant increase in different classes of AGEs. Among the different types of AGEs found, we concentrated on pentosidine and GLAP. In the first phase of this work we developed a method to measure pentosidine in plasma samples via gradient LC-HRMS after protein precipitation with trichloroacetic acid and protein hydrolysis with hydrochloric acid or protease. A full validation protocol was prepared verifying the effect of different precipitation and hydrolysis methods and evaluating stability of pentosidine using different sample preparation approaches. Analytical standard compounds were then synthesized via microwave assisted reactions in polar solvents (dimethylformamide and buffered water). The reaction yields were significantly larger than in the case of the analogous reactions in buffered water in physiological conditions. GLAP (at the moment the compound is not commercially available) was isolated and its structure was eventually elucidated using NMR spectroscopy and high resolving power MSn. In the second phase of the work, quantitative analysis of the obtained biomarkers was carried out on plasma samples of treated animals, showing significant differences between controls, early pathological and diabetic individuals. Novel aspects Identification, synthesis and measurement of biomarkers for early pathology.