Identificação de AGEs na saliva como método de diagnóstico do diabetes mellitus

Abstract

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by high blood glucose levels that result from absolute or relative insulin deficiency. One of the main mechanisms that leads to complications involved in DM is the formation of advanced glycation end products (AGEs). The glycation process refers to a sequence of non-enzymatic reactions that begins when reducing sugars react with proteins, lipids or nucleic acids, generating AGEs, which can alter tissue integrity. The objective of the study was to quantify the AGEs in saliva by the in vitro glycation assay, thus enabling the development of a new method for diagnosing DM. Bovine serum albumin (BSA), lysozyme, saliva and salivary α-amylase were glycated with fructose and methylglyoxal (MGO). Fluorescence analyzes (350nmex and 420nmem), salivary α-amylase activity, oxidative damage, SDS-PAGE, Western blotting to detect salivary α-amylase, and characterization by ATR-FTIR spectroscopy were performed. The fluorescence analysis registered increases in intensity in all glycated samples, except in saliva with fructose. The enzymatic activity of salivary α-amylase was reduced after glycation with fructose and MGO. The oxidative damages generated by fructose and MGO were determined by the significant increase in the content of carbonylated proteins in all glycated samples, except in the glycated saliva with fructose. The sulfhydryl content showed a significant reduction in the samples of lysozyme with fructose and MGO and saliva with MGO. In the electrophoretic profile it was possible to observe the appearance of a new band close to 95kDa in BSA with MGO. The glycated lysozyme revealed bands with molecular weights of 28, 36 and 55kDa after glycation. In the saliva a decrease in the intensity of the bands with molecular weights 80, 78, 56, 30 e 14kDa was observed in the glycated samples, this decrease being more accentuated in the saliva with MGO. Western blotting performed with the saliva samples showed a reduction in the concentration of salivary α-amylase, given in % of pixel density, after glycation with MGO. The use of ATR-FTIR allowed to evaluate and distinguish peaks in the spectra in BSA and lysozyme glycated with fructose and MGO, and in glycated saliva with fructose. From the results it is possible to conclude that saliva is a fluid that can be used as an indicator of the presence of AGEs. Thus, the identification of AGEs in saliva represents an alternative to evaluate the evolution of diabetes and the occurrence of chronic complications of this disease.