NMR-based metabolomics on human plasma and saliva: application to cardiac ischemia and sport exercise

Metabolomics, one of the ‘omic’ sciences in systems biology, is the global assessment and validation of endogenous small-molecule metabolites within a biologic system. Analysis of these key metabolites in body fluids has become an important role to monitor the state of biological organisms and is a widely used diagnostic tool for disease. In this thesis, we present two applications of this approach using nuclear magnetic resonance (NMR) as analytical technique: metabolomics of coronary artery disease and sport medicine. With the growing appreciation of the metabolic basis of cardiovascular disease (CVD), metabolomics has become a powerful investigative tool. Among CVD, cardiac ischemia is a severe pathological condition characterized by a reduced oxygen flow to the heart that can lead to myocardial infarction. Classical diagnostic methods are affected by low sensitivity or by high dose of radiation. Here we present a non-invasive metabolomics profiling of cardiac ischemia fingerprint in human plasma. Plasma samples of ischemic and not ischemic patients were collected before and after a cardiac stress test. Multivariate data analysis was used to evaluate metabolic fingerprint discrimination among patients. Unexpectedly, we found a significant difference already in the basal samples between not ischemic and ischemic patients undergoing the cardiac stress test. In addition, several biochemical patterns noted in our metabolomic data were consistent with results of prior experimental and physiologic studies on cardiovascular metabolism. We mainly observed an alteration of the gut microbiome- derived metabolites, such as TMAO and acetate, the amino acids profile and the creatine-creatinine energy system. These results suggest that metabolism is already anticipating the outcome of the stress test and can help in determining a risk factor useful for physicians to diagnose cardiac conditions. Assessing health, sport, performance and recovery of professional athletes, which represent a broad physiological function, requires monitoring simultaneously a large number of biomarkers, making the use of metabolomics particularly interesting in this context. Considerable attention has also been placed on the use of saliva as a potentially useful biofluid to follow metabolic changes during exercise, mainly due to its non-invasive collection. However, understanding changes in metabolite concentrations in saliva occurring during a soccer match meets significant challenges, such as the nature of the exercise itself, which is far from controlled and homogeneous for all players. Saliva samples of seventeen soccer players were analyzed before and after an official match. Two different ways of normalizing data are discussed, using total proteins and total metabolite concentrations. Changes in markers related to energy, hydration status, amino acids and other compounds were found. The limits and advantages of using saliva to define the systemic responses to exercise are examined, both in terms of data normalization and interpretation, and the time that the effect lasts in this biofluid. Our work focuses mainly on three different aspects: the effect that time sampling has on the observed effect, the type of normalization that is necessary to perform in order to cope with changes in water content, and the metabolic response that can be observed using saliva.