4. Discussion
gastrointestinal tract, T3.DA3S
The identification of new biomarkers of food and nutrient bioavailability has developed fast over the past two decades and could potentially provide new tools for dietary intake assessment. In this context, metabolomics has opened new opportunities, since new putative biomarkers are frequently identified by the metabolic profiling of body fluids following the ingestion of several foods [To the best of our knowledge, this study is the first NMR-based study that identified and quantified the urinary biomarkers of red beetroot juice intake and comprehensively investigated the combined effect of red beetroot juice and physical activity on healthy individuals.This is a pilot study, and the number of subjects was sufficient for a crossover experimental design. Moreover, we examined the same group in several different conditions and time points, for a total of fifteen urine samples for each volunteer.In agreement with a previous mass-spectrometry study, DA3S and 4MP2CA were the identified urinary biomarkers related to RBJ intake [Sulfate conjugation is an important pathway for the detoxication of xenobiotics, and previous studies have shown that in plasma more than 98% of dopamine is sulfated [The sulfonation of phenolic hydroxyl groups is catalyzed by the SULT1 family, which is a group of cytosolic sulfotransferases present in the intestine epithelium, liver, and kidneys [The present results showed a significant increment of urinary DA3S two times higher at T2 comparing exercise and rest, with the RBJ intake being equal. Furthermore, the level of DA3S increased, peaking at T3.DA3S is an endogenous metabolite, but a 20-fold increment was observed comparing the RBJ intake with placebo intake, with the physical activity intensity being equal. These results were in agreement with the increase in dopamine sulfate observed in cyclists exercising after banana intake, contributing to observed increment of antioxidant capacity [Interestingly, at T3 the urine of all subjects was colored, indicating the presence of a coloring molecule, probably betanin. This datum resulted more intense after physical activity than at rest and was not recognized during the placebo experiment (4MP2CA, the other suggested biomarker of RBJ intake, is a decarboxylation product of 4-methylpyridine-2,6-dicarboxylic acid, a catabolite of betalamic acid produced in the gastrointestinal tract (The degradation of betanin by the decarboxylation route was found to be dependent on heating, pOThe transport through the intestinal mucosa of 4-methylpyridine-2,6-dicarboxylic acid or 4MP2CA is not known, but a transcellular transport could be hypothesized, analogous to that reported for pyridine compounds mediated by organic anion transporters or monocarboxylate transporters [The urinary metabolic profile of RBJ with respect to placebo intake was also characterized by higher levels of 3-HIB and Gln, with the workload exercise being equal.The biomarker 3-HIB is a biomarker for valine catabolism in skeletal muscle [Regarding Gln, physical exercise can differentially affect Gln production in muscle and its availability in plasma, decreasing Gln availability following strenuous exercise [The metabolic profiles of volunteers after physical activity and RBJ intake showed significant variations in four metabolites compared to RBJ intake at rest. The variations were represented by an increase in Hyp and 4-HBz levels and a decrease in 1-MNA and 2PY levels.Hyp is a nucleotide degradation product involved in purine metabolism. Purine metabolites are more sensitive markers of training status and better performance predictors than typical biochemical and physiological indicators [In our study, the levels of Hyp significantly changed depending on exercise, but not on RBJ intake. In order to evaluate the possible effect of RBJ intake on muscular metabolism, further studies should be performed using a larger amount of juice intake or a higher intensity workload, as reported in other experimental protocols [The metabolite 4-HBz is found to be a product of microbial phenol metabolism [After RBJ intake, it was found that 4-HBz was significantly higher after exercise compared to rest. After exercise, this metabolite remained unchanged both in RBJ and placebo intake. For all the above, it is possible to hypothesize that physical activity plays a role in the metabolism of aromatic compounds.Urinary levels of 1-MNA and 2PY were significantly lower in subjects after physical activity compared to the same subject at rest, with the RBJ intake being equal.At rest and at fast, a borderline negative correlation between 1-MNA and nicotinamide riboside (precursor of NAD+) was found (