Ingestion of vegetables rich in inorganic nitrate (NO3-) content has emerged as an effective method, via the formation of a nitrite (NO2-) intermediate, for acutely elevating vascular nitric oxide (NO) levels. As such a number of beneficial effects of NO3- ingestion have been demonstrated including the suggestion that platelet reactivity is reduced. I initially investigated whether inorganic NO3- supplementation might also reduce platelet reactivity in healthy volunteers and have determined the mechanisms involved in the effects seen. I conducted a randomised crossover study in 24 (12 of each sex) healthy subjects assessing the acute effects of potassium nitrate capsules (KNO3, 8 mmol) vs placebo (KCl) control capsule ingestion on platelet reactivity. Inorganic NO3- ingested via supplementation raised circulating NO3- and NO2- levels in both sexes and attenuated ex vivo platelet aggregation responses to adenosine diphosphate (ADP) and, albeit to a lesser extent, collagen but not epinephrine in male but not female volunteers. These inhibitory effects were associated with a reduced platelet P-selectin expression and elevated platelet cyclic guanosine monophosphate (cGMP) levels. In addition, I have shown that NO2- reduction to NO occurs at the level of the erythrocyte and not the platelet. These results demonstrate that inorganic NO3- ingestion, whether via the diet or through supplementation, results in a modest decrease in platelet reactivity in healthy males. I then sought to examine the effects of 6 weeks daily intake of NO3--rich beetroot juice versus a placebo NO3--deplete juice on endothelial and platelet function in a cohort of otherwise healthy non-diabetic untreated hypercholesterolaemics. In this randomised double blind placebo controlled parallel study 69 subjects were recruited. The primary end point was change in endothelial function determined using ultrasound flow-mediated dilatation (FMD). Secondary endpoints included change in pulse wave analysis (PWA), aortic pulse wave velocity (aPWV), platelet P-selectin and platelet monocyte aggregate (PMA) expression and plasma, urine and salivary NO3- and NO2- levels. Baseline characteristics, including lipid levels, were similar between the groups. Dietary NO3- caused an improvement in FMD of ~24% from 4.6%±2.2% to 5.7%±2.6% in the treatment group (p<0.001) not seen in the placebo group (4.5%±1.9% versus 4.3%±1.8% p=0.07). This improvement in FMD was also noted following acute administration of dietary NO3-. Small but significant improvements also occurred in aPWV and PWA augmentation index (p=0.04). The % of platelet monocyte aggregates was significantly reduced in the NO3- limb by 7.6% versus an increase of 10.1% in the placebo group (p=0.004). No adverse effects of dietary NO3- were detected. In this study population, chronic dietary NO3- ingestion improves endothelial function, vascular stiffness and platelet markers of atherogenesis in a cohort of hypercholesterolaemics who are otherwise at increased risk of cardiovascular disease (CVD). This thesis provides strong support for assessment of the potential of dietary NO3- as a primary prevention strategy to prevent atherothrombotic and atherogenic complications in larger cohorts.

Tesi qualificada amb un EXCEL.LENT "Cum Laude", per a l'obtenció del títol de doctor per la Universitat de Barcelona amb menció europea / Nitric oxide or nitrogen monoxide (NO) is a tiny free radical gas. The discovery of this intriguing molecule has revolutionized physiology and pharmacology research during the last 20 years. Currently, it is known that NO is endogenously synthesized by several molecules and tissues via two pathways: the synthase-dependent pathway and the synthase-independent pathway. In the first, the amino acid L-arginine is the main donnor of NO synthesis. In the second, inorganic nitrate is the main substrate for the synthesis of this molecule. Interestingly, both pathways are directly connected. While the synthase-dependent pathway is oxygen dependent, the synthase-independent metabolic route is greatly facilitated under hypoxia conditions. Thus, these mechanisms of NO production regulate levels of NO in the tissues. An adequate production of NO is important because it plays an essential role in mechanisms related with vasodilatation and blood flow distribution. Additionally, NO modulates other important functions in the human body such as mitochondrial respiration and immune mechanisms. For all these reasons, the interest for dietary NO donors have increased during the last years. It has been suggested that the consumption of food rich in L-arginine or in inorganic nitrate may enhance NO availability in the human body. This hypothesis has not been unnoticed in exercise physiology. In fact, it has been suggested that supplementation with NO donors may improve the cardio-respiratory response, as well as the tolerance to endurance exercise in humans. However, there is a lack of studies analyzing this issue. Therefore, the aim of this doctoral thesis was to assess the effect of L-arginine and inorganic nitrate in the cardio-respiratory and metabolic response of healthy humans. To develop this aim, three studies and one review were carried out. In the first, it was found that L-arginine supplementation during three days at several doses, between 5.5 and 20.5 g/day was not effective to increase plasma markers of NO, as well as the cardio-respiratory and metabolic response during endurance test. In the second study we found that acute dose of inorganic nitrate supplementation (10 mg/kg of body mass) raised significantly plasma levels of nitrate and nitrite. However, this effect did not report an improvement in the cardio-respiratory response at low-to-moderate intensities of exercise. However, at maximal work loads of exercise dietary nitrate induced significantly reduction of oxygen consumption (VO2peak) compared with placebo. Other cardio-respiratory parameters, as well as blood lactate concentration did not differ between nitrate and placebo. In addition, exercise performance measured as time to exhaustion during an incremental test did not increase compared with placebo. All these findings together suggested that at higher intensities of exercise energy production became more efficient after inorganic nitrate ingestion. Accordingly, in the third study it was analyzed the effect of dietary inorganic nitrate ingestion for three days during endurance exercise in a cycle ergometer at high intensity (time-trial of 40-min). Results of this study showed that nitrate supplementation did not increase significantly plasma levels of nitrite, as well as enhance performance in healthy subjects. Interestingly, a significant, negative correlation was found between change in nitrite and endurance capacity measured as VO2peak during the exercise test. These results indicated that the effect of dietary nitrate ingestion was lower in subjects with high cardiovascular capacity compared with subjects with poor tolerance capacity to endurance exercise. This fact is very important, since it is known that endurance training increase values of VO2peak in sedentary population and this fact is correlated with lower incidence of cardiovascular diseases. These and other important conclusions of these studies are included in the last work of this thesis which was a review article. / L’òxid nítric (NO) es un radical lliure alliberat per diverses molècules i teixits en l’organisme humà. El descobriment d’aquesta intrigant molècula ha revolucionat la recerca en el camp de la fisiologia i la farmacologia durant els últims 20 anys. Actualment, es coneix que la alliberació de NO per part de les cèl•lules endotelials estimula el procés de vasodilatació. A més, també es coneix que aquesta molècula es un important regulador de la respiració mitocondrial i del sistema immunològic. Totes aquestes funcions han generat un gran interès per els precursors nutricionals de NO. En l’àmbit de la fisiologia de l’exercici físic s’ha suggerit que la suplementació amb alguna d’aquestes substancies (L-arginina o nitrat inorgànic) pot millorar la tolerància a l’exercici físic de resistència. No obstant, hi ha molta controvèrsia en els resultats dels estudis que han analitzat aquesta hipòtesi. Per tant, l’objectiu principal d’aquesta tesi doctoral va ser analitzar els efectes dels principals precursors de NO, L-arginina i nitrat inorgànic, en la resposta cardiorrespiratòria i metabòlica durant l’exercici físic de caràcter aeròbic en humans. Per dur a terme aquest objectiu es van realitzar 3 estudis i una revisió bibliogràfica. Els principals resultats d’aquests estudis van mostrar que la suplementació de L-arginina en diferents dosis no va ser efectiva per augmentar el marcadors plasmàtics de NO, així com, la resposta cardiorrespiratòria i metabòlica durant un exercici físic aeròbic en intensitats moderades. En referència als nitrat inorgànic, es va observar que la suplementació augmenta els nivells d’aquests compostos en plasma. No obstant, aquest fet no es va correlacionar amb una millora de la tolerància a l’exercici físic de resistència. A més, es va observar una correlación negativa i significativa entre l’augment dels nitrits plasmàtics i la potència aeròbica màxima (VO2max). Tots aquests resultats van ser àmpliament tractats en l’últim treball (revisió bibliogràfica) d’aquesta tesi. En resum, l’ingesta nutricional de L-arginina i/o nitrat inorgànic no resulta efectiva per millorar la resposta cardiorrespiratòria i la tolerància a l’exercici físic de resistència en humans sans i entrenats físicament.