In the last 20 years or so there has been renewed interest in the potential of inorganic nitrate (NO₃ ^-) to control blood pressure in humans [1]. Dietary inorganic nitrate is absorbed rapidly and completely in the proximal small intestine with 100% bioavailability [2]. Approximately 25% of the nitrate circulating in the plasma is then concentrated in the salivary glands and secreted into the mouth where around 20% (or ≈ 5 – 8% of intake) is converted to nitrite (NO₂ ^-) by commensal bacteria on the tongue and subsequently swallowed [3]. Upon reaching the stomach the NO₂ ^- is either absorbed directly or reduced to nitric oxide (NO) as a result of the acidic environment of the stomach [3–5]. Endogenously produced NO and NO₂ ^- are vasoprotective agents with the ability to increase vasodilation, decrease blood pressure (BP) and improve cardiovascular function [6]. Reduced endogenous NO production is associated with hypertension [7] and there is evidence to support the hypothesis that the NO and NO₂ ^- produced as a result of dietary NO₃ ^- could induce health benefits [8].

The individual daily intake of dietary nitrate has been estimated to be ≈81–106 mg/d (not including loses from washing, peeling and cooking) in the typical Western diet, with vegetables contributing approximately 80% of this value [9, 10]. The vegetables with the highest nitrate contents (>250 mg/ 100 g fresh weight) are celery, cress, chervil, lettuce, red beetroot, spinach and rocket [11]. Green leafy vegetables have recently been shown to be among the foods most beneficial in the prevention of coronary heart disease and ischemic stroke [12, 13]. This effect has been postulated to be due to the high inorganic NO₃ ^- content of these vegetables [14–16].

Beetroot is a rich source of dietary NO₃ ^-[11] and a number of studies have investigated its potential for reducing blood pressure in humans [8, 17–20], which appears to be more potent in men.

Although beetroot appears to be a promising means of lowering blood pressure, previous studies in free-living subjects have been small in size and routinely used a controlled diet in combination with administering the beetroot juice. It is not clear whether a blood pressure-lowering effect may be seen when beetroot juice is consumed on top of a normal diet. The present study examines the use of beetroot juice as a means of regulating blood pressure in free-living individuals whilst not imposing any dietary restrictions.

Individual BP changes from baseline after each treatment showed a drop of 4.6 mmHg with BJ and 3.4 mmHg with PL at 3-h, 6.2 mmHg and 2.2 mmHg respectively at 6-h and 4.5 mmHg and 2.3 mmHg respectively, at 24-h. Statistically the 6-h difference was a trend overall (P=0.064), with men showing a difference of −4.7 mmHg, (P=0.1) and women a difference of −2.5 mmHg (P=0.5). In the planned ANCOVA in men only BJ treatment was significantly different from placebo (P=0.007) and a treatment covariate interaction was observed (P=0.024). In those subjects with little variation in baseline BP measures there was a clear effect of BJ. In those subjects with a large variation in baseline BP measures, SBP did not change with BJ and increased with PL, although the difference between the drinks was smaller.

In a post hoc analysis of outliers, those men with large drops in BP (≥ 20 mmHg SBP) at 6-h following drink consumption on either treatment (n =2) were removed. In the remaining 13 men the 6-h difference between PL and BJ of 4.9 mmHg was statistically significant (P=0.042).

It has been postulated that the inclusion of dietary nitrates in the form of beetroot-derived foods may be useful in the regulation of normal BP due their high inorganic NO₃ ^- content. The present contribution is the first study, to the authors’ knowledge, to examine the effect of beetroot juice on BP in free-living individuals in the absence of dietary restrictions, such as a low nitrate diet. Dietary restrictions in studies with beetroot juice and BP remove confounding dietary factors that may have an effect (negative or positive) on BP, thus making interpretation of study results more straightforward. The drawback, however, is that beetroot juice as a nutritional intervention to regulate BP would likely be consumed as part of a normal diet, not as part of a low nitrate diet or in the absence of other dietary components (e.g. coffee, alcohol, black tea) that may affect BP [21–23]. It was therefore uncertain whether there is any clinically relevant benefit from beetroot juice supplementation on BP in the unregulated home environment.

Unsurprisingly, there was a large degree of variation in the BP readings for a given individual both across the two measurement days and throughout each day. This may have been a result of the free-living nature of the study. Whilst there was no significant difference in baseline SBP or DBP between men and women, the trend of BJ lowering BP was stronger in men than in women and a planned separate analysis in men showed that BJ lowered SBP by 4–5 mmHg at 6-h after ingestion. It is uncertain whether these differences between the sexes was a result of gender per se, or whether the older age of the women (48.9 ± 3.1 y) compared to the men (36.2 ± 2.9 y) may have influenced the variation seen. It is also notable that approximately half of the women (n=8) who participated in the study took prescription medication, whilst none of the men did. The crossover design of the study should have eliminated any individual variation in blood pressure due to any medication taken daily on the two 24 h periods that BP was measured. However, the possibility of the effect of medication or diet (e.g. sodium intake) on the outcomes of the trial cannot be discounted and ideally the experiment should be repeated in non-medicated individuals and the dietary intake recorded. Two of the studies in the literature investigating beetroot juice and BP that used both men and women did not report results by gender [8, 20]. The third such study, by Kapil and colleagues tested 24 mmol of KNO₃ (or KCl) in capsules in a double-blind crossover study in 8 males (baseline BP 126/73 mmHg) and 12 females (baseline BP 102/67 mmHg) and found that the KNO₃ lowered systolic BP by 9.4 mmHg at 6-h and diastolic by 6 mmHg at 2.5-h (systolic lowering was 6 mmHg at this time point) [19]. Females had no significant fall in blood pressure (3-4/5 mmHg reduction only) despite absolute plasma nitrite rises twice those seen in men. Some of these differences may have been due to the large difference in baseline BP (difference in SBP = 24 mmHg, P<0.001) between men and women in that particular study as it was noted that decreases in BP were correlated with baseline BP (r= 0.66 to 0.7). In the present study, baseline BP readings were remarkably similar between men (132/80 ± 2.2/1.8 mmHg) and women (133/83 ± 2.3/1.7 mmHg). It is difficult to ascertain if there is in fact a gender-specific response to dietary nitrates.

The drop of 4–5 mmHg observed in the study reported here at 6-h after consumption in men is smaller and with a later peak drop in BP than in other controlled studies with beetroot juice. In an open-label and unblinded study, Webb et al. demonstrated that 500 ml of beetroot juice containing 45 mmol/L (or 2.79 g/L) of NO₃ ^- lowered BP by a maximum of 10/4.8 mmHg at approximately 3-h (p<0.001; n=9 m, 5 f; mean baseline BP: 109/71 mmHg) and the effect persisted in SBP for 24-h [8]. Although it was stated that the effect on BP was related to the increase in plasma nitrite this only accounted for about 7% of the variance. In that study, volunteers were measured seated in clinic and asked to refrain from caffeine-containing drinks or foods with a high nitrate content (green leafy vegetables, beetroot) for 12-h prior to the study and were fasting on the morning of the study.

More recently, Hobbs and workers [18] conducted a single-blind, randomized, controlled, crossover study and observed an almost dose-dependent drop of 20.5/14.6 mmHg at 2-3 h postprandial using beetroot juice containing 5.7 mmol NO₃ ^- relative to the control (water), and 22.2/18.3 mmHg with twice that dose of NO₃ ^-. Once again, subjects (n=18 M, mean baseline BP: 131/82 mmHg) were fasted for 12-h and the diet was restricted (no alcohol or and caffeine) in the 24-h prior to the test and were provided with a standard evening meal for the night before each visit and for the lunch and dinner of the study day. Subjects were also instructed not to take any dietary supplements, vitamins or minerals for 1-wk prior to the study or during the intervention period. Subjects were also required to meet a large number of inclusion criteria including not smoking, not exercising more than 3 times / wk (<20 min/session) and not consuming > 150 ml alcohol / wk. The highly controlled nature of this study may have been responsible for the particularly large decreases in BP observed.

Two other studies have also reported a reduction in BP from beetroot juice, but the number of study participants was small. In an open-label study in 9 volunteers (gender not stated) Kapil et al. tested 250 ml beetroot juice (5.5 mmol NO₃ ^-) relative to a water control [19]. A peak reduction in SBP of 5.4 ± 1.5 mmHg was found, whilst diastolic blood pressure changes were not significant. Both plasma NO₃ ^- and NO₂ ^- were elevated for 3-h following beetroot juice ingestion. In another study, Vanhatalo et al. tested 8 volunteers (n=5 M, 3 F; baseline SBP and DBP of 127 ± 6 and 72 ± 5 mmHg, respectively) with 0.5 l beetroot juice daily, in two equal doses in the morning and evening, for 5 days against a placebo (low-calorie blackcurrant juice cordial with negligible NO₃ ^-content) in a randomized, crossover study with a 10-d washout period [20]. On day 5 of the treatment, SBP and DBP were reduced on the beetroot juice by approximately 4% relative to the placebo. In the present study, a single dose of BJ was administered and the effect measured over the next 24-h. It is possible that a cumulative beneficial effect may occur if the BJ was consumed on a daily basis. Further studies are needed to confirm if this is the case and to determine what the optimal daily dose should be in free-living adults. The BJ and PL used here contained 28% apple juice. It is plausible that the apple juice may have had some contrary effect on BP, thus partially negating or cancelling the effect of the beetroot on lowering BP. While the literature actually supports a BP lowering effect of quercetin (found in large amounts in apples) [24], the experiment could be repeated using pure beetroot juice to eliminate any possible effect of other components present in the BJ.

As a higher baseline BP has been correlated with a larger reduction in BP following BJ ingestion [19] it is possible that hypertensive patients could benefit more from beetroot juice consumption than the group of subjects studied here. The volunteers used here were healthy, but unlike a number of other studies, they included both genders, a broad age range (23 – 68 y) and exhibited higher baseline BP values (mean 132/81 ± 1.6/1.2 mmHg). The results presented here are therefore more generalizable to the population as a whole in Western countries, especially given the free-living nature of the study. Using an even larger group of volunteers, with different characteristics (e.g. on average, lower / higher BMI or age) would be further beneficial, as the group studied here were on average middle-aged and slightly overweight. Although it was not undertaken in the present study, it would also be preferable to measure plasma NO₃ levels before and after consumption of the BJ and PL in order to establish a causative relationship between the NO₃ present in beetroot and the effect on plasma NO₃ levels leading to a reduction in BP. This would assist in eliminating other possible causes of the effect seen, such as the diuretic property of beetroot.

In conclusion, it was demonstrated here that in free-living people consuming an unrestricted diet and a single dose of 500 g of beetroot and apple juice, a trend to lower blood pressure by 4–5 mmHg at 6-h was observed (significant only in men after adjustment for baseline variation). A reduction in SBP in the magnitude of 5 mmHg has been correlated to a cardiovascular mortality reduction of approximately 10% at the population level [25]. Additional studies with beetroot and apple juice in larger groups of free-living men and women are needed to fully assess the efficacy such a dietary intervention at a public health level in the treatment of cardiovascular disease and to determine the exact mechanism of action.