Identification of novel biomarkers of altered homeostasis

Resumen

Poor eating habits exert a direct impact on the incidence of chronic diseases of high global prevalence, such as cardiovascular and fatty liver diseases. Nevertheless, the beneficial effects of a correct nutrition are principally effective in the opening phase of the metabolic disorder, being able to prevent and even reverse its development. By contrast, current biomarkers have a delayed response, contributing to the late diagnosis of the disease and encouraging the use of pharmacological therapies. These treatments can also be expensive and lead to serious side effects for the patients. Therefore, it is essential to find new risk markers that assist on early diagnosis. Only with these novel biomarkers able to determine the primarily metabolic disturbances that lead into a plethora of diseases, nutrition can be efficient in disease therapy. The recent emergence of the omics sciences along with the improvement of the equipment and tools for the processing of large volumes of data, provide the adequate support for biomarker search. In this context, the present doctoral thesis was aimed to identify novel early biomarkers useful in the diagnosis of metabolic disorders derived for altered nutrition and lifestyle. For this purpose, non-targeted metabolomics was applied to the analysis of plasma samples from rodents fed different diets and subject to behavioral intervention (e.g. hyperphagia, physical activity). Thus, glycerol-based lysophospholipids (Lyso-PLs), namely lysophosphatidylcholines (Lyso-PCs) and lysophosphatidylethanolamines (Lyso-PEs), were identified as putative circulating biomarkers for the progression of lipid disorders. These compounds, besides being structural components of the biological membranes, can act as bioactive molecules in a broad range of physiological and pathological events (e.g. reproduction, inflammation or apoptosis). Furthermore, through the periodic administration of a chemical treatment (Poloxamer 407, a hyperlipidemic agent), it was excluded the possibility that Lyso-PLs were actually diet-consumption biomarkers. The expression analyses carried out in rodents revealed that the pharmacological intervention is able to induce dyslipidemia acting through liver mechanisms other than the high-fat diet. Nonetheless, both types of intervention affected genes that are involved in the regulation of extracellular Lyso-PL levels. Therefore, the metabolic determination of circulating Lyso-PLs can also be used as an indicator of etiology. Once Lyso-PLs were selected as biomarkers, their distribution and metabolism were studied exhaustively through the use of targeted metabolomics techniques. For this task, it was necessary to develop a specific method of quantification for Lyso-PLs in biological samples. Because of this, we were able to identify thirty-one metabolites belonging to the categories of interest. Importantly, multivariate statistical analysis showed that the indicative trace left by Lyso-PL molecules in plasma is representative of the overall metabolome of animals, which is composed by thousands of circulating metabolites. The multicompartmental analysis of the lysophospholipidome confirmed the liver as an essential organ in the metabolism of Lyso-PLs. Thus, the excess of fat of the diet caused a generalized lowering of the plasma levels along with an intrahepatic modification of lyso forms through processes of desaturation and elongation of acyl chains. These findings were associated with incipient processes of steatosis in the liver of animals, while circulating transaminases were unchanged. As a result, the comprehensive assessment of lysophospholipidome in plasma appears to be of great relevance for the subclinical non-invasive diagnosis of fatty liver. Finally, the adequacy of Lyso-PLs as biomarkers of early pathology was determined in a small cohort of humans, which were grouped according to their transaminase levels, but within the range considered as healthy. The metabolomics analysis of plasma samples confirmed the findings in rodents, indicating that the mechanisms involved in the early progression of steatosis might be similar. Although further research must be conducted on humans, the results of the present doctoral thesis are of considerable interest in the early diagnosis of metabolic disorders. As a conclusion, we propose Lyso-PLs, including Lyso-PCs and Lyso-PEs, as suitable candidates for biomarkers of risk of fatty liver in humans.