Plasma nitrate and nitrite are increased by a high nitrate supplement, but not by high nitrate foods in older adults

Introduction

According to the most recent projections from the United States Census Bureau the number of individuals over the age of 65 years will double from ~35 million in 2000 to 71 million in 2030[1]. Maintaining function in this population is a public health priority. In older adults, the degree of vascular disease correlates with good health and function, and improvements in vascular function are linked to improved quality of life[2]. A key aspect of vascular endothelial dysfunction is a reduced ability to make nitric oxide (NO) via L-arginine dependent pathways by endothelial nitric oxide synthase. It is well documented that NO enhances blood flow, decreases platelet aggregation, improves mitochondrial function, and diminishes cellular vascular adhesion[3,4]. Nitric oxide is also produced independently of the L-arginine-NOS (nitric oxide synthetase) pathway through the nitrate/nitrite/NO cycle. Until recently nitrite was thought to be biologically inert[5]. However, it has been shown that supraphysiological amounts of plasma nitrite increase forearm blood flow and this activity is enhanced under hypoxic conditions[6].

Substantial elevations in plasma nitrite can occur through increasing dietary nitrate intake[7]. Absorbed nitrate is concentrated in the salivary gland and subsequently reduced to nitrite by symbiotic, oral bacteria; a portion of the nitrite is then absorbed and found in plasma[7,8]. In the United States, dietary intake of nitrate is around 40-100 mg/day, with 85% coming from vegetables[9]. Dietary nitrate can be greatly enhanced, reaching an intake of over 1200 mg (nearly 20 mmoles) by making appropriate selections of high nitrate containing foods as part of the DASH diet (Dietary Approaches to Stop Hypertension). This is more than 5 fold above the World Health Organizations Acceptable Daily Intake of 3.7 mg nitrate/kg body weight /day and more than 2 fold above the United States Environmental Protection Agency of 7.0 mg nitrate/kg body weight/day for a 60 kg individual[10].

Little is known about the effect of dietary nitrate intake on the nitrate/nitrite/NO cycle in older adults. This is an important gap in the literature since the cycle is known to be affected by a number of factors that may be altered in older adults, including achlorhydria, medications that alter gastric pH, dry mouth, oral bacteria populations, and intestinal health. In addition, the potential health implications of increasing dietary nitrate to raise plasma nitrate and nitrite are critically important for health and function in older adults. For example, dietary supplementation of nitrate has been shown to reduce blood pressure[11,12], increase exercise performance[13], and alter cerebral perfusion[14].

Currently, the effects of single meals and repetitive days of diets with different nitrate levels on the kinetics of plasma nitrate and nitrite concentrations have not been described in detail in older adults. Pragmatically it would be useful to know if, and for how long, plasma nitrate/nitrite was elevated throughout the day following a high nitrate meal and/or supplement because it may have implications for the timing of physical activity for older adults with poor vascular function. Therefore, the primary objective of this study was to examine, in 8 sedentary older adults, the effect of a 3-day control vs. high nitrate diet, with and without a beverage supplement (beetroot juice) high in nitrate, on the kinetics of plasma nitrate and nitrite concentrations using a randomized four period cross-over controlled design. All participants received all four treatments. We also examined the effect of the four treatments on blood pressure. We hypothesized that the high nitrate diet alone would raise plasma nitrate/nitrite and lower blood pressure compared to the control diet. Furthermore, it was hypothesized that plasma nitrate and nitrite would be elevated and blood pressure lowered from the high nitrate supplement vs. no supplement, and that the high nitrate diet would potentiate this effect as compared to the control diet.

Methods and materials

Outcome measures

Statistical analyses

Demographic and baseline characteristics were summarized using means and standard deviations for continuous measures and frequencies and relative frequencies for categorical measures. The fixed diet and supplement effects on plasma nitrate, plasma nitrite, systolic blood pressure, and diastolic blood pressure were analyzed using mixed models. The day and hour effects were treated as fixed to allow for testing of daily and hourly time trends, while within-individual variability was assumed a random source of variation. Nitrate and nitrite means are summed over all days, time points, and individuals, while blood pressure measures are summarized for only the 2-hour post- meal time points. The models assume a compound symmetric covariance structure to accommodate the small sample size. All figures present the model-adjusted least square mean of nitrate and nitrite values over time. All statistical analyses and graphs were executed using SAS v9.2. All statistical comparisons were performed assuming a Type I error rate of 0.05, and pairwise comparisons maintained this error rate using Tukey's method for multiple comparisons.

Results

Ten individuals were recruited for the study and signed a consent to participate. Eight participants (5 women, 3 men) completed all testing visits and all analyses are based on these data. There were two dropouts due to recurring scheduling conflicts and time burden. The mean age of the participants was 72.5±4.7 years. The MMSE ranged from 27 to 30 (28.5±0.9). Mean body mass was 81.4±15.1 kg and mean body mass index was 28.5±3.9 kg/m². The SPPB ranged from 6 to 11 (9.6±1.6) indicating that all participants were experiencing some lower extremity mobility dysfunction. Study participants reported no hypertension, diabetes, or chronic kidney disease and were not on any medications known to influence the study outcomes.

In general, the beetroot juice supplement was well accepted. The mean score for enjoying the taste of the beverage was 2.5 with only one participant rating it below neutral. Side effects included gastrointestinal upset (n=1) and headache (n=1). Beeturia was reported by all but one individual.

Discussion

This is the first trial to examine the kinetics of plasma nitrate and nitrite across multiple days following dietary manipulation of nitrate using foods and a beverage supplement in older adults. A surprising result of this study was that there was no difference in plasma nitrate or nitrite between the high nitrate diet compared to the control diet, contrary to our original hypothesis. Also, the high nitrate diet alone did not appear to potentiate the effect of the supplement on plasma nitrate or nitrite levels. Therefore our data did not support our research hypothesis. However, we demonstrated that a high nitrate supplement consumed at breakfast substantially elevated both plasma nitrate and nitrite levels over the course of the day regardless of the diet. We did not observe any difference in systolic or diastolic blood pressure between the four treatments. Therefore our hypothesis that the high nitrate diet alone or when combined with the supplement would lower blood pressure compared to the control condition was not supported. We suggest the reader uses caution in the interpretation of these blood pressure data given the small sample size.

The level of nitrate contained in the beetroot juice supplement was 6.5 mg/kg body weight vs. 1.9 mg/kg body weight in the high nitrate diet and 0.5 mg/kg body weight in the control diet. In comparison, the World Health Organizations Acceptable Daily Intake is 3.7 mg nitrate/kg body weight /day and the United States Environmental Protection Agency is 7.0 mg nitrate/kg body weight/day. The addition of the beetroot juice supplement to the high nitrate diet increased total nitrate intake to 8.4 mg/kg body weight, about 20% above the United States Environmental Protection Agency acceptable intake, and to 7.0 mg nitrate/kg body weight for the control+supplement treatment.

From a practical standpoint, our data suggest that in the diets provided to our participants, the level of nitrate in the diet has minimal impact on plasma nitrate and nitrite, but that a high nitrate source (beetroot juice) consumed with the breakfast meal can significantly increase plasma nitrate and nitrite throughout the day. Our findings have real-world significance for older adults who seek to use beetroot juice with conditions such as peripheral arterial disease, or more generally, in those with impaired vascular function or vascular disease who wishes to engage in physical activity. Previous work by Kenjale and colleagues showed that individuals with peripheral arterial disease who consumed beetroot juice three hours prior to an exercise session had improved physical function and increased time to onset of claudication pain during an exercise test as compared to consuming a low nitrate containing beverage[20]. Our data show that plasma nitrate and nitrite are significantly increased throughout the day with a single dose of beetroot juice. Thus older adults may not need to concern themselves with timing the intake of a high nitrate supplement prior to an exercise session or a bout of moderate-to-high intensity physical activity. It is likely that this would lead to better adherence to a treatment regimen.

We showed that plasma nitrate peaked 1-2 hours after the morning meal following the consumption of the high nitrate supplement. These peak values and changes following the supplement approximated the levels reported earlier by us[14] and Webb and colleagues[17]. Interestingly, the level of nitrate in the beetroot juice product used by Webb et al., was about 2.5 times higher than the beetroot juice in our two studies. Furthermore, there was about a 2 fold higher plasma peak change in nitrite for our current work vs. that of Webb et al. The change in plasma nitrite concentrations reported by Webb et al., were about 0.2 μM occurring 3-5 hours following the beetroot juice supplement as compared to the nearly 1.0 μM change in our work for the control diet+supplement treatment. The difference between studies in plasma nitrite may be related to the conditions under which the supplement was consumed; we had participants consume it with a meal in both of our studies, whereas it is not stated this was the case in the work by Webb and colleagues. Also, the amount of nitrite in the supplements (and diet) differed, which may have contributed to the difference; Webb et al., reported < 0.05 μM nitrite in their beetroot juice, whereas our product had 0.3 μM of nitrite and our diets contributed an additional 0.06 and 3.4 μM for the breakfast meals of the control and high nitrate diets, respectively. Finally, differences in the results may also be due to the differences in demographics and potential health conditions between the two studies. Webb et al. recruited a younger and leaner cohort (mean age = 25 years; BMI = 22 kg/m²) compared to our sample.

There was a progressive decline in plasma nitrate throughout the day, although it remained substantially elevated above baseline levels. In the supplement conditions, the rise in plasma nitrite tracked the rise in plasma nitrate during the initial part of the day with the exception that in the high nitrate diet+supplement treatment there was a substantial rise in plasma nitrite concentration during the first several hours before they matched the control diet+supplement levels. Furthermore, in the supplement conditions, plasma nitrite showed a secondary rise following the evening meal beginning at the 7.5 hour time point. We believe this may be due to an increase in saliva production from the evening meal, subsequent conversion of nitrate to nitrite, and then the swallowing of the saliva while eating. The two supplemented treatments had higher plasma nitrate at these times which may have been due to a higher concentration of nitrate in the salivary glands. If so, when saliva production increased at a meal, this would have led to conversion of nitrate to nitrite by oral bacteria and a subsequent rise in plasma nitrite. Changes in plasma nitrite from baseline were higher for high nitrate diet+supplement compared to the other conditions. The observation of a larger change in plasma nitrite for high nitrate diet+supplement vs. control diet+supplement is surprising considering there was no difference in the levels of substrate for plasma nitrate between the two treatments. This might indicate a more efficient conversion of nitrate to nitrite in the high nitrate diet+supplement condition; however pursuing this possibility is beyond the scope of the study.

Previous work with dietary nitrates has shown that consumption of an acute dose of nitrate isolated in a supplement or as a whole food product, such as beetroot juice, increases plasma nitrate, and subsequently plasma nitrite[11-13,17,21-25]. These previous studies were conducted primarily to investigate the acute effect of ingestion of nitrate on blood pressure and exercise performance following a single acute dose of nitrate supplement, or up to 15-days of a supplement. We manipulated nitrate intake through changes across the whole diet for three consecutive days. Sobko and colleagues also manipulated nitrate via the diet, but their study was conducted in healthy participants who consumed a traditional Japanese diet with foods high in nitrates[26] (see reference for list of their specific foods). They observed elevated plasma and saliva levels of nitrate and nitrite on day 10 following a 10-hour overnight fast; however, they did not perform serial measures of plasma nitrate and nitrite to study the intra- and inter-day kinetics of the dietary manipulations. Our study is the first to look at the changes in plasma nitrate and nitrite at multiple time points across several consecutive days of treatment. Our study adds unique information to the literature in that we examined older adults and manipulated the nitrate intake through both the diet and supplement over several days.

Surprisingly, the high nitrate diet alone did not elevate either plasma nitrate or nitrite and high nitrate diet+supplement did not elevate plasma nitrate/nitrite above that of control diet+supplement. We hypothesized that the high nitrate diet alone would result in a significant rise in plasma nitrate compared to the control diet alone. This was based on our calculations of the nearly 4 fold higher nitrate load in the high nitrate diet compared to the control diet. In comparison to other work[16], the 2.5 mmoles in the high nitrate diet is lower than the single dose administration of 4 mmoles of nitrate that has been found to cause physiological effects and increases in plasma nitrate and nitrite. Apparently our diet dose was not sufficient to significantly raise plasma nitrate and nitrite above the control diet. Furthermore, the amount was spread across the three meals and snacks throughout the day and may have not provided a large enough bolus dose of nitrate at any meal or snack to raise plasma levels of either nitrate or nitrite. The meal with the largest nitrate dose from foods alone (not including the beetroot juice supplement) was at dinner with about 1.25 mmoles of nitrate or one-half of the daily nitrate dose. This was not sufficient to significantly increase plasma nitrate/nitrite compared to the control diet. A second possibility is that consumption of nitrate in whole foods (both cooked and raw) may provide a lower bioavailability of nitrate vs. from a beverage. However, we do not believe this is a satisfactory explanation because van Velzen and colleagues demonstrated an absolute bioavailability of nitrate of 100% from several high nitrate containing vegetables (cooked spinach, raw lettuce, and cooked beetroot) when participants consumed a single dose of the product[27].

Based on earlier work, we hypothesized that the high nitrate diet and supplement, both together and separately, would lower resting blood pressure compared to the control diet and no supplement interventions[11,16,17]. Previously, Kapil and colleagues showed that a nitrate dose of 5.5 mmoles delivered in beetroot juice lowered diastolic blood pressure by over 4 mm Hg 2.5-3 hours after drinking the beverage as compared to water[16]. Systolic and diastolic blood pressures were also reduced by up to 10 mm Hg at nitrate daily doses ranging from 5.2 to 22.5 mmoles[13,16,17,20,21,28-30]. These were primarily in young, healthy adults. We did not observe a nitrate treatment effect on blood pressure which may have been related to variability in blood pressure measures and the small sample size of our study. We used a single measure of the blood pressure at each of the time points whereas others have obtained 3 or more measurements and used the mean of these as the data point. Additionally, our sample of older adults was quite healthy and we excluded participants with hypertension. This may have blunted the effect of the nitrate treatments (both diet and supplement).

The hypotensive effect found in DASH studies may be as much attributable to inorganic nitrate found in certain vegetables and fruits as to the high potassium, polyphenols, and calcium content of the DASH diet. Based on analysis by Hord et al., dietary nitrate levels could easily reach over 16 mmoles (1000 mg; 12 mg/kg body weight)[10] in a DASH dietary pattern by astutely choosing high nitrate containing vegetables and fruit. In comparison, our high nitrate diet (without the supplement) was only 2.5 mmoles. With the documented health benefits from the DASH diet and diets abundant in vegetables and fruits, it has been suggested that the current World Health Organization and United States Environmental Protection Agency Acceptable Daily Intake for nitrate needs to be reevaluated[10,31].

There are limitations to this study. The small sample of older adults in this study was ostensibly healthy and individuals were screened for disorders or drugs that may have interfered with the metabolism of nitrate and nitrite. This information was obtained from self-report but it is possible that participants had some underlying disease and may have misreported medications. The low inter-subject variability suggests that this was not a major concern. Participants were also asked to refrain from using antibacterial mouthwash for the duration of the study but may not have done so. It seems highly unlikely that participants, who were blind to the study purpose, could have manipulated the data via medications/mouthwash, to suppress the effect of the high nitrate diet. The level of nitrate in the high nitrate diet treatment may have led to the lack of response from this treatment alone. Developing a higher nitrate diet menu may provide differences in dietary treatments. Finally, since we excluded individuals with conditions common in the older adult population, caution should be exercised when generalizing these data.

This study confirms that the nitrate-nitrite cycle is intact in ostensibly healthy older adults when a high nitrate supplement is consumed in a single dose over a 30-minute period. In summary, in healthy older adults, a high nitrate supplement consumed at breakfast increased plasma nitrate and nitrite levels throughout the day. This observation may have practical utility for the timing of physical activity and a high nitrate supplement for older adults with vascular dysfunction.