Advanced analytical method developments and their applications to food and biological samples for the study of the role of diet in health

Resumen

Nutrition directly influences the human physiology and the state of health of the individual, being able to avoid the appearance of diseases or predisposing to suffer certain pathologies. The composition of food is of vital importance; however, it is complicated to establish links between the bioavailability of the intaken foodstuff and their effects on health. Amino acids (AAs) and hydroxy acids are chiral compounds. In humans, proteins are made of L-AAs. It was supposed that the human being did not produce endogenously D-AAs. Nowadays, their presence is attributed mainly to diet, metabolism of the intestinal microbiota and to de novo synthesis. Abnormally high levels of D-AAs have been linked to various diseases. This thesis sought the development of new methodologies as well as the improvement and adaptation of previously selected methods, based on liquid chromatography coupled to mass spectrometry (LC-MS) and supercritical fluid chromatography coupled to mass spectrometry (SFC-MS), for the analysis of samples derived from human nutritional studies, mainly food and biological samples, to expand the scarce current knowledge around the chiral profile of the diet and their role in health. An indirect approach for quantitative chiral analysis using LC-MS/MS was validated for cooked food samples and applied to real independent meal samples (edible part of whole breakfast, lunch and dinner). This method was adapted to study the presence of D- and L-AAs in serum and urine of healthy volunteers who participated in a randomised, controlled, crossover clinical trial. The proportion of chiral AA was investigated in meals, serum and urine and changes in their amount were related to the degree of healthiness of two different diets. Additionally, the presence of chiral AAs was investigated in faecal matter in two very different populations, following two types of diets, vegan and omnivore, which are in fact opposite diets. Moreover, to improve the understanding of the components of the food reaching the microflora, believed to be responsible for producing most of the D-AAs detected in biological samples, gastric and duodenal samples were collected from healthy people one hour after a specific diet was consumed. AAs were quantified in the three samples, food, stomach and duodenum, to track the changes in the chirality of the foodstuffs along the gastrointestinal tract. This dissertation provides data and tools to progress in the study of the role of certain chiral compounds in health, starting with their abundance in the diet and following their evolution through the digestion process. Absolute quantification, as well as the ratio of each enantiomer of chiral metabolites, has been determined to fill the necessity of analytical methodologies with extreme sensitivity in targeted metabolomic approaches for chiral compounds in food and in bioanalysis. Chirality of AAs changes within meals (breakfast, lunch and dinner) and between diets with different degree of healthiness. Besides, different concentrations of D- and L-AAs are found in human biological samples, some of them directly related to the diet and some not. Direct and indirect approaches were assayed to develop a method for chiral analysis of hydroxy acids by SFC-MS. Advantages and disadvantages of both strategies are described. A chiral derivatizing agent was used capable of reacting not only with chiral molecules but also non-chiral hydroxy acids and AAs. Derivatives were elucidated based on “in-source fragmentation” and accurate mass tools. Finally, a method was developed and validated for the analysis of compounds with medium and low polarity in cooked meals, with different degree of healthiness, by LC-MS with accurate mass to provide new insights about whole meal patterns. Characterisation of the profile of compounds detected with this methodology is presented for four diets together with the identifications of many of those compounds which appeared statistically significant between the most extreme diets. Nutritional studies involving metabolic profiling strategies can benefit from this method.