Quantificação da Nε-carboximetilisina em formulas enterais e parenterais através de Elisa e LC-MS/MS

Abstract

The advanced glycation endproducts (AGEs) constitute a large variety of compounds formed from nonenzymatic amino-carbonyl interactions between reducing sugars or oxidized lipids and proteins, nucleic acids or aminophospholipids. The AGEs formation in foods and biological systems is a topic of increasing interest for the science, since they are involved in proinflammatory and pro-oxidative effects related to the pathogenesis of chronic degenerative diseases such as diabetes, Alzheimer's disease and renal failure. The Nε-carboxymethyllisine (CML) was the first AGE identified in foods and, since then, has been the compound of choice in studies on which a single product is used as an AGE marker. Immunochemical or instrumental methods are available for the AGEs determination, but they present limitations and there is not an ideal method yet. Thus, in order to compare and optimize different analytical methods, this study aimed to determine the CML content on enteral and parenteral nutrition formulas by ELISA and LC-MS/MS. So, in order to compare and optimize different analytical methods, this study aimed to determine the content of CML in nutritional enteral and parenteral formulas by ELISA and by liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS). Thus, 5 parenteral and 17 enteral commercially available formulations were investigated. All samples studied showed detectable levels of CML, regardless of the method of analysis used. The parenteral formulations presented CML contents measured by ELISA ranging from 529.9 ± 33.47 to 1948.88 ± 3.68 ng CML/mL of sample and showed positive linear correlations to their content in lipids (0.9259) and carbohydrates (0.9426), but they were not submitted to the LC-MS/MS analysis due to the impossibility of applying the established purification protocol for this group of samples. Enteral formulations analyzed by ELISA presented CML contents ranging from 1076.91 ± 76.87 to 55950.71 ± 1891.29 ng CML/mL of sample and showed positive correlations to its contents in carbohydrate (0.6057), lipid (0.5264) and protein (0.6157). They showed a variation from 0.09 to 0.503 μg CML/mg of protein of the diets when analyzed by LC-MS/MS and there was no correlation between CML and the variables "lipid", "carbohydrate" or "protein" calculated for this group. The investigation conducted during the samples preparation prior to injection into the LC-MS/MS showed a significant loss of CML during the different stages of the protocol, compromising the reliability of the results obtained through this method of analysis, while the comparison between the results obtained through different methods applied to similar samples showed the reliability of the anti-CML ELISA test used in this experiments. The results of this study indicate the need for improvement of AGEs analysis protocols in food and should guide future research on this area. The determination of reliable methods of detection, which enable the measurement of AGEs structures in body fluids, tissues and also in food, in a sensitive, specific, fast and not too expensive way is a challenge that remains present on this field.