Cardiovascular disease (CVD) is one of the leading causes of mortality worldwide (Joseph et al., 2017). In 2013 there were > 54 million deaths (95% uncertainty interval, 53.6-56.3 million) globally, and 32% were occasioned to cardiovascular disease (GBD, 2015). Hypertension and obesity are a major risk factor for cardiovascular disease (Roth el al., 2017). In 2015, 603.7 million adults were obese around the world, and the overall prevalence of obesity was 12% (GBD, 2017). Interestingly, the hallmark and shared feature of CVD and metabolic syndrome is the reduced bioavailability of nitric oxide (NO) signaling (Newsholme et al., 2009).
Non-pharmacological intervention (lifestyle modification) offers an attractive alternative for preventing and treating hypertension, and nutritional strategies and exercise are widely recommended (Samadian et al., 2016). Exercise has been considered an important approach for reducing cardiovascular risk, promoting clinically acute and chronic effects on blood pressure (BP). According to the American College of Cardiology, regular physical exercise reduces systolic BP (SBP) and diastolic BP (DBP) by 4-5 mmHg and 2-4 mmHg, respectively (Whelton et al., 2018). In fact, even a single bout of exercise can induce short-term reductions on BP, a phenomenon called post-exercise hypotension (Brito et al., 2015).
In addition to exercise, numerous clinical trials have shown beneficial cardiovascular effects of several different nutrients found in vegetables (Houston, 2010; Ravera et al., 2016; Lennon et al., 2017). More recently, attention has been directed to other possible elements in vegetables that may have a role, including inorganic nitrate (Bondonno et al., 2018), that is present in some vegetables such as spinach, beetroot and celery and have important bioactive phytochemical with cardioprotective properties (Hobbs et al., 2013; Gee and Ahluwalia, 2016). Because the nitrate concentration in vegetables is dependent of different factors such as the nitrate content of the soil where the vegetables are grown or the presence or absence of nitrogen-based fertilizers (Gee and Ahluwalia, 2016), the majority of studies have used either nitrate-rich beetroot juice or sodium nitrate to verify the effects on performance or on health. Oral supplementation with inorganic nitrate or nitrate-containing foods exert pleiotropic, beneficial vascular effects, through mechanisms that involves NO bioavailability restoration in a process known as the nitrate-nitrite-NO pathway (Koch et al., 2017).
A classic systematic review with meta analysis showed reductions in SBP and DBP of -4.4 mmHg and -1.1 mmHg, respectively, after short-term inorganic nitrate and beetroot juice supplementation (Siervo et al., 2013). Interestingly, one study showed that a single dose of nitrate administered in beetroot juice acutely reduces BP in hypertensive patients, with average reduction of 11.2 mmHg and 9.6 mmHg in systolic and diastolic ambulatory BP, respectively (Ghosh et al., 2013). Despite these reports, no study to date has evaluated whether dietary nitrate in the form of beetroot juice can enhance the reduction of BP that occurs following a single session of exercise. This may be especially relevant in individuals who have an increased cardiovascular risk profile, but have not yet developed hypertension, such as obese individuals.
Thus, the aim of this study was to examine the effects of dietary nitrate supplementation (nitrate-rich beetroot juice) on the post-exercise ambulatory BP in obese males. It was hypothesized that the supplementation with nitrate-rich beetroot juice, prior to an exercise bout, would enhance the post-exercise hypotension effect compared to a low-nitrate drink.
Design and participants
This study was a prospective single-center, randomized, crossover-controlled trial carried out from February to June 2017 at the nutritional assessment laboratory of the University. The following inclusion criteria were considered in the study: males, aged between 20 and 30 years old, obese (body mass index--BMI--between 30 and 40 kg/[m.sup.2] with increased body adiposity), free of cardiovascular disease including hypertension, able to perform exercise according to the Physical Activity Readiness Questionnaire (PARQ) (Shephard, 1988), but physically inactive. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The study protocol was approved by the Human Research Ethics Committee at Federal University of Rio Grande do Norte (Protocol No. 1.808.205, CAAE 59716216.8.0000.5292) and was prospectively registered on a publicly accessible database (http://www.ensaiosclinicos.gov.br/rg/RBR-5gkhhj/). The study design followed CONSORT guidelines (Schulz et al., 2010).
Subjects attended the research laboratory on four different occasions before noon and after a 12-hour fasting period. The aim of the first day was to obtain data for sample characterization, to perform the maximum incremental test (to determine the intensity of the exercise on the days of the experimental sessions) and perform a familiarization session. Anthropometric evaluation and body composition assessment were performed after blood collection for biochemical characterization of the sample and fasting nitrite and nitrate concentrations (NOx). After that, subjects received a standardized meal determined by an experienced nutritionist (to avoid perform the exercise test in fast) and performed the maximal exercise test. Finally, group allocation was randomly carried out by means of a lottery in order to determine the order of the experimental sessions for each subject without his knowledge, and with an interval of five days between sessions (washout period). Subjects then underwent controlled experimental trials in the following three visits: 1) juice rich in nitrate (beetroot juice) with exercise (BJE), 2) fruit soda low in nitrate with exercise (FSE), and 3) session with mineral water and without exercise--control (CON).
Subjects were instructed to maintain their habitual diet and activity during the study. At 48-hour pre-assessment day the participants were asked to abstain from any kind of exercise, consuming alcohol, caffeine and nitrate-rich food (Curtis et al., 2015). Subjects were additionally asked to avoid using mouthwash and chewing gum in the 48h period prior to testing, because it can reduce oral bacterial nitrate reductase activity (Govoni et al., 2008).
Anthropometric measures and body composition assessment
Body weight was measured using a digital scale (BKH200FN, Balmak[R], Santa Barbara D'oeste, Brazil). Height was measured by a portable stadiometer (Personal Caprice Portatil, Sanny[R], Sao Paulo, Brazil) with a precision of 1 mm. Nutritional status was classified according to World Health Organization (WHO, 2014). The waist circumference measurement was performed using inelastic tape (Cescorf[R], Porto Alegre, Brazil) at the midpoint between the lower costal margin and the iliac crest (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001). Body fat percentage was assessed by dual-energy X-ray absorptiometry (DEXA) (GE, Medical Systems, Madison, WI, USA).
Maximal graded exercise test
Subjects performed a maximal graded exercise test on a motorized treadmill (RT350, Movement[R], Pompeia, Brazil) to determine their maximal heart rate. The maximal graded exercise test started at 4 km/h followed by fixed increments of 1 km/h every minute. Heart rate (HR) was monitored throughout the test using a HR monitor (RS800CX, Polar[R], Finland) and recorded at the end of each minute. The highest HR value observed during the test was considered as the HRmax. Rating of perceived exertion (RPE) was also monitored and recorded at the end of each minute according to the Borg scale 6-20 (Borg, 1982). The end of the test was determined by the presence of at least one of the following criteria: i) HR > 100% estimated for age; ii) RPE > 18; or iii) when participants voluntarily stopped (Howley et al., 1995). On that day, mean resting heart rate was also measured during a five-minute period in a seated position in order to determine the heart rate reserve (heart rate max--resting heart rate), which was the method used to prescribe the intensity of the aerobic exercise sessions. BP was measured in triplicate by digital sphygmomanometer (Visomat[R], comfort 20/40), with intervals of 1 minute between them after 5 minutes of rest in the sitting position with their legs uncrossed, back supported and arm placed on a table with the palm facing up (American College of Sports Medicine, 2009).
The experimental pre-session protocol was identical for all three visits, only differing in the drink offered. The volunteers were instructed not to change their diet and avoid foods high in nitrates the day before the experimental sessions. Participants attended the laboratory after a 12-hour overnight fasting. Resting blood pressure (BP) was collected before the standard meal according to the 7th Brazilian Arterial Hypertension Guideline (Malachias, 2016) using the oscillometry method (HEM-7200, OMRON, USA). A standardized meal (cheese sandwich and fruit, 84% carbohydrates, 14% protein and 2% lipids) was offered 70 minutes prior exercise, with an energy value of 15% of the individual daily energy, according to the energy recommendations for physically inactive people (Brazil, 2006). Additionally, 200ml of the drink corresponding to the experimental session was offered 10 minutes after the end of the meal (i.e., 60 minutes prior to exercise session or control session). This amount of food and timing was in accordance with current guidelines (Thomas et al., 2016).
The drinks were: 1) beetroot juice rich in nitrate (two shots of Beet It Sport[R], James White Drinks Ltd., Ipswich, UK; 70mL each shot with a total 400mg nitrate) for the BJE...