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Nitrate-Rich Vegetables Increase Plasma Nitrate and Nitrite Concentrations and Lower Blood Pressure in Healthy Adults

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  • FrieslandCampina Innovation Centre Wageningen

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Background: Dietary nitrate is receiving increased attention due to its reported ergogenic and cardioprotective properties. The extent to which ingestion of various nitrate-rich vegetables increases postprandial plasma nitrate and nitrite concentrations and lowers blood pressure is currently unknown. Objective: We aimed to assess the impact of ingesting different nitrate-rich vegetables on subsequent plasma nitrate and nitrite concentrations and resting blood pressure in healthy normotensive individuals. Methods: With the use of a semirandomized crossover design, 11 men and 7 women [means ± SEMs: age: 28 ± 1 y, body mass index (BMI, in kg/m(2)): 23 ± 1; exercise: 1-10 h/wk] ingested 4 different beverages, each containing 800 mg (∼12.9 mmol) nitrate: sodium nitrate (NaNO3), concentrated beetroot juice, a rocket salad beverage, and a spinach beverage. Plasma nitrate and nitrite concentrations and blood pressure were determined before and up to 300 min after beverage ingestion. Data were analyzed using repeated-measures ANOVA. Results: Plasma nitrate and nitrite concentrations increased after ingestion of all 4 beverages (P< 0.001). Peak plasma nitrate concentrations were similar for all treatments (all values presented as means ± SEMs: NaNO3: 583 ± 29 μmol/L, beetroot juice: 597 ± 23 μmol/L, rocket salad beverage: 584 ± 24 μmol/L, spinach beverage: 584 ± 23 μmol/L). Peak plasma nitrite concentrations were different between treatments (NaNO3: 580 ± 58 nmol/L, beetroot juice: 557 ± 57 nmol/L, rocket salad beverage: 643 ± 63 nmol/L, spinach beverage: 980 ± 160 nmol/L;P= 0.016). When compared with baseline, systolic blood pressure declined 150 min after ingestion of beetroot juice (from 118 ± 2 to 113 ± 2 mm Hg;P< 0.001) and rocket salad beverage (from 122 ± 3 to 116 ± 2 mm Hg;P= 0.007) and 300 min after ingestion of spinach beverage (from 118 ± 2 to 111 ± 3 mm Hg;P< 0.001), but did not change with NaNO3 Diastolic blood pressure declined 150 min after ingestion of all beverages (P< 0.05) and remained lower at 300 min after ingestion of rocket salad (P= 0.045) and spinach (P= 0.001) beverages. Conclusions: Ingestion of nitrate-rich beetroot juice, rocket salad beverage, and spinach beverage effectively increases plasma nitrate and nitrite concentrations and lowers blood pressure to a greater extent than sodium nitrate. These findings show that nitrate-rich vegetables can be used as dietary nitrate supplements. This trial was registered atclinicaltrials.govasNCT02271633.
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The Journal of Nutrition
Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Nitrate-Rich Vegetables Increase Plasma Nitrate
and Nitrite Concentrations and Lower Blood
Pressure in Healthy Adults
1–3
Kristin L Jonvik,
4–6
Jean Nyakayiru,
4,6
Philippe JM Pinckaers,
4
Joan MG Senden,
4
Luc JC van Loon,
4,5
and Lex B Verdijk
4
*
4
Nutrition and Toxicology Research Institute Maastricht, School of Nutrition and Translational Research in Metabolism, Maastr icht
University Medical Centre, Maastricht, Netherlands; and
5
Institute of Sport and Exercise Studies, Hogeschool van Arnhem en Nijmegen,
University of Applied Sciences, Nijmegen, Netherlands
Abstract
Background: Dietary nitrate is receiving increased attention due to its reported ergogenic and cardioprotective
properties. The extent to which ingestion of various nitrate-rich vegetables increases postprandial plasma nitrate and
nitrite concentrations and lowers blood pressure is currently unknown.
Objective: We aimed to assess the impact of ingesting different nitrate-rich vegetables on subsequent plasma nitrate and
nitrite concentrations and resting blood pressure in healthy normotensive individuals.
Methods: With the use of a semirandomized crossover design, 11 men and 7 women [means 6SEMs: age: 28 61 y,
body mass index (BMI, in kg/m
2
): 23 61; exercise: 1–10 h/wk] ingested 4 different beverages, each containing 800 mg
(;12.9 mmol) nitrate: sodium nitrate (NaNO
3
), concentrated beetroot juice, a rocket salad beverage, and a spinach
beverage. Plasma nitrate and nitrite concentrations and blood pressure were determined before and up to 300 min after
beverage ingestion. Data were analyzed using repeated-measures ANOVA.
Results: Plasma nitrate and nitrite concentrations increased after ingestion of all 4 beverages (P< 0.001). Peak plasma nitrate
concentrations were similar for all treatments (all values presented as means 6SEMs: NaNO
3
:583629 mmol/L, beetroot
juice: 597 623 mmol/L, rocket salad beverage: 584 624 mmol/L, spinach beverage: 584 623 mmol/L). Peak plasma nitrite
concentrations were different between treatments (NaNO
3
:580658 nmol/L, beetroot juice: 557 657 nmol/L, rocket salad
beverage: 643 663 nmol/L, spinach beverage: 980 6160 nmol/L; P= 0.016). When compared with baseline, systolic blood
pressure declined 150 min after ingestion of beetroot juice (from 118 62 to 113 62mmHg;P<0.001)androcketsalad
beverage (from 122 63to11662mmHg;P=0.007)and300minafteringestionofspinachbeverage(from11862to11163
mm Hg; P<0.001),butdidnotchangewithNaNO
3
.Diastolicbloodpressuredeclined150minafteringestionofallbeverages(P<
0.05) and remained lower at 300 min after ingestion of rocket salad (P=0.045)andspinach(P=0.001)beverages.
Conclusions: Ingestion of nitrate-rich beetroot juice, rocket salad beverage, and spinach beverage effectively increases
plasma nitrate and nitrite concentrations and lowers blood pressure to a greater extent than sodium nitrate. These findings
show that nitrate-rich vegetables can be used as dietary nitrate supplements. This trial was registered at clinicaltrials.gov
as NCT02271633. J Nutr doi: 10.3945/jn.116.229807.
Keywords: nitrate, nitrite, blood pressure, bioavailability, beetroot, rocket salad, spinach
Introduction
Dietary nitrate, often consumed with beetroot juice as its carrier,
has become a popular supplement due to its reported ergogenic
(1–5) and cardioprotective (6–8) properties. These beneficial
effects of dietary nitrate have been attributed to its capacity to
increase the bioavailability of NO. NO represents an important
signaling molecule in the human body and plays a key role in
several physiological processes by regulating blood flow, muscle
contractility, glucose and calcium homeostasis, and mitochon-
drial respiration and biogenesis (9). Nitrate and nitrite have
traditionally been viewed as inactive by-products of NO me-
tabolism through the NO synthase–dependent pathway. How-
ever, research from the 1990s showed that a reverse pathway
exists whereby nitrate and nitrite can be reduced back into
1
Supported by Dutch Technology Foundation STW grant 12877.
2
Author disclosures: KL Jonvik, J Nyakayiru, PJM Pinckaers, JMG Senden, LJC
van Loon, and LB Verdijk, no conflicts of interest.
3
Supplemental Table 1 and Supplemental Figures 1 and 2 are available from the
‘‘Online Supporting Material’’ link in the online posting of the article and from the
same link in the online table of contents at http://nutrition.org.
6
These authors contributed equally to this work.
*To whom correspondence should be addressed. E-mail: Lex.verdijk@
maastrichtuniversity.nl.
ã2016 American Society for Nutrition.
Manuscript received January 4, 2016. Initial review completed January 21, 2016. Revision accepted March 7, 2016. 1 of 8
doi: 10.3945/jn.116.229807.
The Journal of Nutrition. First published ahead of print April 13, 2016 as doi: 10.3945/jn.116.229807.
Copyright (C) 2016 by the American Society for Nutrition
at UNIVERSITEITS MAASTRICHT on April 15, 2016jn.nutrition.orgDownloaded from
NO (10, 11). There is now a general consensus that dietary
nitrate ingestion can strongly increase plasma nitrate con-
centrations. Circulating nitrate is subsequently actively taken
up by the salivary glands and concentrated in the saliva,
where it can be reduced to nitrite by facultative anaerobic
bacteria in the oral cavity. After swallowing, nitrite enters the
circulation and can be further reduced to NO via various
pathways (12–14).
Multiple studies have reported increased plasma nitrate and
nitrite concentrations after ingestion of nitrate in the form of
sodium nitrate (4, 5, 15–17). Similar effects have been reported
using (concentrated) beetroot juice (1, 2, 18–23). While there are
several other nitrate-rich food sources, including green leafy and
root vegetables (24), research on the pharmacokinetic and
physiological effects of nitrate supplementation has mainly ap-
plied either sodium nitrate or beetroot juice as a nitrate donor.
One of the consequences of the postprandial increase in plasma
nitrate and nitrite availability is a decrease in resting blood
pressure, which has been reported after ingestion of both sodium
nitrate (7) and beetroot juice (6, 8). As of yet there is little
research on the optimal way of supplementing dietary nitrate.
Literature has reported a dose-response relation between the
amount of dietary nitrate ingested and the rise in plasma nitrate
and nitrite concentrations, as well as the reduction in oxygen
cost of submaximal exercise (18) and the reduction in blood
pressure (25). Apart from the dose of nitrate ingested, it is
unknown which other factors influence the bioavailability of
nitrate and nitrite, and subsequent performance and clinical
outcomes (26). Although various nitrate-rich sources are avail-
able, no studies, to our knowledge, have directly compared the
extent to which the actual source of dietary nitrate may affect
the pharmacokinetic and physiological effects upon ingestion.
In the present study we assessed the acute pharmacokinetic
and blood pressure–lowering effects of ingesting various nitrate-
rich sources. Therefore, recreationally active participants ingested
800 mg nitrate provided as sodium nitrate, concentrated beetroot
juice, rocket salad, and spinach, after which plasma nitrate and
nitrite concentrations and resting blood pressure were determined
for up to 300 min after ingestion.
Methods
Participants and ethical approval. Twenty-two healthy, adult, and
nonhypertensive participants were recruited to take part in this study,
which was conducted between September and December 2014. Exclu-
sion criteria were as follows: current or recent smoking (<6 mo), current
or recent beetroot juice (or other nitrate) supplementation (<1 mo),
resting blood pressure >140/90 mm Hg, BMI <18 or >30 kg/m
2
, age <18
or >45 y, and consumption of chronic medications. To exclude the
possible confounding effect of either extremely high or low training
status on NO metabolism, recreationally active participants (exercise:
1–10 h/wk) were recruited. This study was approved by the medical
ethical committee of the Maastricht University Medical Centre,
Maastricht, Netherlands; followed the principles of the Declaration of
Helsinki; and was registered at clinicaltrials.gov as NCT02271633.
After being informed of the purpose and potential risks of the study, all
participants provided written informed consent.
Study design. Using a semirandomized crossover design, we investi-
gated the impact of ingestion of 800 mg dietary nitrate provided in 4
different sources on subsequent plasma nitrate and nitrite concentrations
and resting blood pressure. Over a 5-wk period, participants were
required to report to the laboratory on 5 occasions, consisting of a
screening session (visit 1) and 4 experimental test days (visits 2–5). The 4
sources all contained 800 mg (;12.9 mmol) nitrate and were provided as
sodium nitrate (NaNO
3
)
7
, concentrated beetroot juice, a rocket salad
(arugula) beverage, and a spinach beverage. In order to expose all partic-
ipants to vegetable beverages from the same batch, a single test day was
designated for the rocket salad treatment (all participants on 1 d) and also
a single test day for the spinach treatment (all participants on 1 d). As
such, we could only randomize the order of the other 2 treatments, and
defined this procedure as semirandomized. Thus, the NaNO
3
and beetroot
juice treatments were randomly administered before and/or after the
rocket salad and spinach beverage treatments (random number genera-
tor). The test days were interspaced by a 7-d washout period.
Experimental protocol. During a screening session, eligibility for
participation in the study was assessed. Standard medical questionnaires
were administered, and blood pressure was determined to rule out
hypertension. After a 10-min rest period, blood pressure was measured
4 times by using an automated cuff (Omron Healthcare Inc), with the last
3 measurements averaged to obtain mean blood pressure. Body mass
(digital balance scale; accuracy: 0.1 kg) and height (wall-mounted
stadiometer; accuracy: 0.1 cm) were measured with participants stand-
ing barefoot and dressed lightly.
On the 4 experimental test days, the participants arrived in the
morning after an overnight fast. After a 10-min rest period, blood
pressure was measured as described above. Subsequently, a catheter was
inserted into an antecubital vein for repeated venous blood draws. After
a baseline blood sample was obtained, participants consumed a stan-
dardized breakfast, immediately followed by the treatment beverage.
Repeated blood draws were performed at 30, 60, 120, 150, 180, 240,
and 300 min after the ingestion of the beverages. The time 300 min was
chosen to capture the peak and the subsequent begin of decline in plasma
nitrate and nitrite concentrations (26), without the need for subsequent
additional food intake (i.e., lunchtime). Resting blood pressure was also
measured at 150 and 300 min after beverage ingestion. In addition, a
gastrointestinal tolerance questionnaire was administered at baseline
and at 150 min after beverage ingestion. To limit any effect of circadian
blood pressure fluctuation throughout the day (27), all test days were
performed at the exact same time of the day for each participant (all
participants starting between 0800 and 0900).
Study treatments. With previous work suggesting a minimally required
dose of ;500 mg nitrate to induce acute blood pressure–lowering effects
(6, 7, 15, 25, 28, 29), and dose-response relations observed for the
improvement in blood pressure (25) and other physiological parameters
(18), we applied a treatment dose of 800 mg nitrate to optimize the
chance of detecting effects on blood pressure. Although above the
current acceptable daily intake (ADI) level of 3.7 mg !kg
21
!d
21
(30),
this dose falls well within the range that was used previously in acute and
multiday nitrate supplementation trials (including the above-mentioned
studies), providing 496–1488 mg.
The 4 beverages were sodium nitrate (NaNO
3
dissolved in water),
concentrated beetroot juice (Beet It; James White Drinks), a fresh rocket
salad (arugula) beverage, and a fresh spinach beverage. The beetroot
juice, rocket salad, and spinach beverages were analyzed for nitrate
content using chemiluminesence as described below for plasma analysis
to ascertain that an exact dose of 800 mg nitrate was provided for each
treatment. Beetroot juice was diluted 2000 times in distilled water
(18 MU) before being analyzed for nitrate content. Both rocket salad and
spinach beverages were blended into a smoothie-like beverage upon
arrival from the store. Adapted from a previously described method (31),
the vegetable beverages were extracted for nitrate and nitrite analysis.
The beverage (500 mg) was weighed accurately in a screw cap glass tube.
Then 5 mL distilled water and 5 mL methanol were added, and the tube
was capped and shaken vigorously for 15 min, before being centrifuged
(4!C) for 5 min at 1000 3g. The supernatant was filtered through a
polyvinylidene difluoride filter (0.22 mm), and the filtrate was diluted in
distilled water until the concentrations fell within the measurement
range, before being analyzed for nitrate and nitrite content. Beetroot
juice, rocket salad, and spinach beverages were analyzed for nitrate
7
Abbreviations used: cGMP, cyclic guanosine-5’-monophosphate; iAUC, incre-
mental AUC; NaNO
3
, sodium nitrate.
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content in duplicate within 3% variance, and calculations were per-
formed to determine the exact amount of beverage providing 800 mg of
nitrate. This resulted in an amount of 1.1 g NaNO
3
(140 mL), 116 g
beetroot juice (106 mL), 196 g rocket salad beverage (225 mL), and 365 g
spinach beverage (400 mL). The beverages were stored at 4!Cand
provided to the participants within 24 h after analysis for nitrate content.
Nutritional composition of the treatments is provided in Supplemental
Table 1.
Physical activity and dietary standardization. In the 48 h leading up
to the first experimental test day (visit 2), participants recorded their
dietary intake and physical activity and refrained from strenuous
exercise or labor. Participants replicated their diet and physical activities
in the 48 h before the following 3 test days (visits 3–5). Participants
avoided caffeine and alcohol for 12 and 24 h before each test day,
respectively. To prevent any attenuation in the reduction of nitrate to
nitrite in the oral cavity by commensal bacteria, participants refrained
from using any antibacterial mouthwash/toothpaste and chewing gum,
and avoided tongue-scraping during the intervention period (14). No
restrictions were set for the intake of nitrate-rich foods (21, 23) during
the intervention period. On the evening before each test day, all partic-
ipants consumed a standardized dinner that was adapted to their body
weight (53 kJ/kg, providing 57% of energy from carbohydrate, 27%
from fat, and 16% from protein, composed of a mixed meal of potato,
chicken and vegetables, orange juice, crackers, and yogurt). Furthermore, all
participants received the same standardized breakfast (39 kJ/kg, providing
68% of energy from carbohydrate, 18% from fat, and 14% from protein,
composed of bread, butter, cheese, jam, crackers, and orange juice) on the
morning of each test day before ingestion of the beverage. The ad libitum
consumption of water was registered during the first test day (visit 2) and
replicated during the subsequent test days (visits 3–5).
Plasma analysis. Blood samples were collected in lithium-heparin
containing tubes for plasma nitrate and nitrite analysis, and centrifuged
immediately at 1000 3gfor 5 min, at 4!C. Aliquots of plasma were
frozen and stored at 280!C for subsequent analysis. Determination of
plasma nitrate and nitrite concentrations was performed using the
chemiluminescence technique, which has been described previously (32).
In short, plasma nitrate and nitrite concentrations are determined by
their reduction to NO. The spectral emission of electronically excited
nitrogen dioxide, from the NO reaction with ozone, is detected by a
thermoelectrically cooled, red-sensitive photomultiplier tube, housed in
a Sievers gas-phase chemiluminescence NO analyzer (Sievers NOA 280i;
Analytix). Inter- and intra-assay CVs were 4.2% and 1.1% for plasma
nitrate and 4.9% and 4.7% for plasma nitrite.
Statistical methods. For the power calculation we used a difference in
plasma nitrite concentrations of 50 nmol/L as primary outcome. With a
crossover design, sample size was calculated with a power of 95%, a
significance level of 0.008 (to adjust for Bonferroni’s corrected post hoc
testing), and a drop-out rate of 10%. The final number of participants to
be included after screening was calculated to be 22. Plasma nitrate and
nitrite concentrations and resting blood pressure were analyzed using a
2-factor (time 3treatment) repeated-measures ANOVA. Additionally,
1-factor repeated-measures ANOVA was used to determine differences in
peak concentrations, time to peak values, and incremental AUC (iAUC)
for both plasma nitrate and nitrite concentrations, between the different
treatments. For each treatment, peak concentrations were defined as the
highest measured values at any time point for each individual. Time to
peak was defined as the time point at which the individual reached the
highest plasma nitrate and nitrite concentrations. iAUC was calculated by
multiplying each time period (i.e., 30 or 60 min) by the mean increase
above baseline for that period, summing all time periods. This was done
for each individual and each treatment separately. Although we did not
specifically aim to study any sex effects on the response to dietary nitrate
ingestion, the individual plasma nitrate and nitrite graphs appeared
substantially different between men and women upon visual inspection.
Therefore, as a secondary analysis, we included sex as a between-subjects
factor in the ANOVA analyses described above. As another secondary
analysis, the change in blood pressure from baseline was compared
between groups using 1-factor repeated-measures ANOVA. For all analyses,
statistical significance was set at P<0.05,andanyinteractionormaineffect
was subsequently analyzed using a Bonferroni’s corrected post hoc
test. All data were analyzed using SPSS 22.0 (SPSS Inc.) and are presented as
means 6SEMs.
Results
Four of the 22 participants dropped out during the study due to
failure to comply with the protocol (incomplete ingestion of 1 of
the beverages, within the given time frame of 15 min). General
characteristics of the remaining 18 participants are reported in
Table 1.
Plasma nitrate. Plasma nitrate concentrations at different time
points are presented in Figure 1A. Baseline plasma nitrate con-
centrations were similar for all 4 treatments (NaNO
3
:666
6mmol/L, beetroot juice: 61 65mmol/L, rocket salad beverage:
63 66mmol/L, and spinach beverage: 69 66mmol/L; P= 0.56).
A significant time 3treatment interaction (P< 0.001) was
observed; although plasma nitrate increased to similar peak con-
centrations after ingestion of all 4 beverages (NaNO
3
:5836
29 mmol/L, beetroot juice: 597 623 mmol/L, rocket salad
beverage: 584 624 mmol/L, and spinach beverage: 584 6
23 mmol/L, P= 0.65), differences in nitrate concentrations
were observed at specific time points (t=30,60,120,240,and
300 min; Figure 1A). Furthermore, there was a significant treat-
ment effect for time to peak (P< 0.001; Figure 1B), and post
hoc analysis showed that after ingestion of rocket salad and
spinach beverages, time to peak was later than both NaNO
3
and
beetroot juice (all P< 0.001). As a result, the iAUC was signif-
icantly different between the treatments (P< 0.001; Figure 1C).
Post hoc analysis showed that the iAUC for plasma nitrate con-
centrations after ingestion of NaNO
3
was smaller than beetroot
juice (P= 0.038) and greater than rocket salad beverage (P=
0.08), but not different from spinach beverage (P= 0.08).
Moreover, iAUC for plasma nitrate after ingestion of beetroot
juice was higher than both rocket salad and spinach beverages
(both P< 0.001).
Plasma nitrite. Plasma nitrite concentrations at different time
points are presented in Figure 2A. Baseline plasma nitrite concen-
trations were similar for all 4 treatments (NaNO
3
:131624 nmol/L,
beetroot juice: 135 620 nmol/L, rocket salad beverage: 115 614
nmol/L, and spinach beverage: 155 625 nmol/L; P=0.37).A
significant time 3treatment interaction (P=0.001)showedthat
although plasma nitrite increased after ingestion of all 4 beverages
(all P<0.001),differencesinnitriteconcentrationswereobservedat
specific time points (t=30,60,120,150,and180min;Figure2A).
There were no significant differences in time to peak between the
treatments (P=0.08;Figure2B).However,therewasasignicant
TABLE 1 Participants!characteristics of healthy adults who
ingested 4 different nitrate sources
1
All Men Women
n18 11 7
Age, y 28 6129622662
Height, cm 181 62 185 62 174 63
Body mass, kg 76 6382636664
BMI, kg/m
2
23 61 24 61 22 61
1
Values are means 6SEMs.
Dietary nitrate source 3 of 8
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treatment effect for peak nitrite concentrations (NaNO
3
:5806
58 nmol/L, beetroot juice: 557 657 nmol/L, rocket salad beverage:
643 663 nmol/L, and spinach beverage: 980 6160 nmol/L; P=
0.016). Although peak nitrite concentration was numerically greater
for the spinach beverage, differences were not significant in the post
hoc test (P=0.08,P=0.12,andP=0.20,forspinachbeverage
compared with beetroot juice, NaNO
3
,androcketsaladbeverage,
respectively). The iAUC for plasma nitrite concentrations was
significantly different between treatments (P=0.003;Figure2C)
and was higher after ingestion of spinach beverage compared with
NaNO
3
(P=0.025),beetrootjuice(P=0.035),androcketsalad
beverage (P=0.046),withnodifferencesbetweenNaNO
3
,
beetroot juice, and rocket salad beverage.
Blood pressure. A significant time 3treatment interaction was
observed for both systolic (P= 0.017; Figure 3A) and diastolic
(P= 0.010; Figure 3B) blood pressure. Separate analyses showed
a decrease in systolic blood pressure from baseline to 150 min
after ingestion of both beetroot juice (P< 0.001) and rocket
salad beverage (P= 0.007; Figure 3C). After ingestion of spinach
beverage, systolic blood pressure was significantly lower at
300 min than at baseline values (P< 0.001; Figure 3C). In
contrast, no changes in systolic blood pressure were observed
after ingestion of NaNO
3
(P= 0.11; Figure 3C). The change in
systolic blood pressure from baseline to 150 min (Figure 3C) was
significantly different between treatments (P= 0.022), with post
hoc analysis showing a significant difference for NaNO
3
com-
pared with beetroot juice (P= 0.022) and NaNO
3
compared
with rocket salad beverage (P= 0.001). No differences were
FIGURE 1 Plasma nitrate concentrations (A), time to peak plasma
nitrate concentrations (B), and iAUC for plasma nitrate concentrations
(C) in 18 healthy adults ingesting 4 different nitrate sources. Values
are means 6SEMs, n= 18. (A) *Significant difference between
treatments, P,0.05. (B, C) Labeled means without a common letter
differ, P,0.05. iAUC, incremental AUC; NaNO
3
, sodium nitrate.
FIGURE 2 Plasma nitrite concentrations (A), time to peak plasma
nitrite concentrations (B), and iAUC for plasma nitrite concentrations
(C) in 18 healthy adults ingesting 4 different nitrate sources. Values
are means 6SEMs, n= 18. (A) *Significant difference between
treatments, P,0.05. (B, C) Labeled means without a common letter
differ, P,0.05. iAUC, incremental AUC; NaNO
3
, sodium nitrate.
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observed in the change in systolic blood pressure from baseline
to 300 min between treatments (P= 0.21), despite the strong
reduction for the spinach beverage treatment only (Figure 3C).
For diastolic blood pressure, separate analyses showed a
decrease from baseline to 150 min after ingestion of all
beverages (NaNO
3
,P= 0.022; beetroot juice and rocket salad
beverage, P< 0.001; and spinach beverage, P= 0.002; Figure
3D). However, at 300 min after ingestion, diastolic blood
pressure remained only significantly lower than baseline values
in the rocket salad beverage (P= 0.045) and spinach beverage
(P= 0.001; Figure 3D) treatments. Despite these observations,
no differences were observed in the actual change in diastolic
blood pressure from baseline to 150 and 300 min between
treatments (P=0.12andP= 0.23, respectively).
Sex. Plasma nitrate and nitrite concentrations and iAUC for
men and women separately are presented in Supplemental
Figures 1 and 2. Subanalysis including sex as a between-subjects
factor showed that the baseline (P= 0.020), peak value (P<
0.001), and iAUC (P= 0.005) for plasma nitrate concentrations
were higher in the women than in the men for all 4 treatments.
For plasma nitrite concentrations, no sex differences were ob-
served at baseline. Although peak values and iAUC for plasma
nitrite concentrations appeared higher in the women than the
men, this difference reached statistical significance for only the
spinach beverage treatment (P= 0.010 and P= 0.016, respec-
tively). Despite these sex differences in the plasma nitrate and
nitrite responses, no differences in blood pressure responses to
nitrate ingestion were observed between men and women.
Side effects. No serious adverse events were reported. Inges-
tion of the 4 beverages was well tolerated by most of the
participants. Four participants (1 man and 3 women) did not
manage to ingest the total volume of the rocket salad or spinach
beverages within the given time frame of 15 min and were
therefore excluded from the study. Gastrointestinal complaints
were reported by 2 participants after ingestion of the rocket
salad beverage and by 1 participant after ingestion of the spinach
beverage. No gastrointestinal complaints were reported for the
NaNO
3
and beetroot juice beverages, respectively. One partic-
ipant reported headache at baseline and throughout the test day
after ingestion of the spinach beverage.
Discussion
The present study demonstrated that acute ingestion of 800 mg
nitrate from beetroot juice, rocket salad beverage, and spinach
beverage elevated plasma nitrate concentrations to the same
extent (;9-fold) as the ingestion of an identical dose from
sodium nitrate. Although ingestion of all 4 nitrate sources led
to substantial increases in plasma nitrite concentrations, the
increase was greater (;6-fold compared with ;4-fold) after
ingestion of spinach than after ingestion of the other nitrate
sources. In contrast to the ingestion of sodium nitrate, inges-
tion of beetroot juice, rocket salad beverage, and spinach bev-
erage resulted in a significant lowering of both systolic and
diastolic blood pressure in this group of healthy, normotensive
individuals.
Over the past decade, beetroot juice has become an increas-
ingly popular dietary supplement due to its high nitrate content
and the associated ergogenic and cardioprotective properties.
Research in this area has mainly used the ingestion of beetroot
juice and sodium nitrate as dietary intervention. Both sources
have been shown to substantially increase plasma nitrate and
nitrite concentrations, improve exercise performance (1–5), and
lower blood pressure (6–8) in healthy individuals. However, it
remains unclear whether differences exist in the pharmacoki-
netic and physiological responses to the ingestion of different
dietary nitrate sources. We assessed the acute effects of ingestion
of various nitrate-rich sources on plasma nitrate and nitrite con-
centrations and resting blood pressure. In our study, ingestion of
FIGURE 3 BP reported as absolute
systolic (A), absolute diastolic (B),
change in systolic from baseline (C),
and change in diastolic from baseline
(D) in 18 healthy adults ingesting 4
different nitrate sources. Values are
means 6SEMs, n= 18. For reasons
of clarity, treatment differences are
displayed only in panels C and D.
*Significant change from baseline
within treatment, P,0.05.
#
Change
from baseline significantly different
compared with NaNO
3
,P,0.05.
BP, blood pressure; NaNO
3
, sodium
nitrate.
Dietary nitrate source 5 of 8
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exactly 800 mg of nitrate from all 4 sources led to similar
increases (;9-fold) in plasma nitrate concentrations (Figure 1A).
Our data lend further support to the suggestion by Van Velzen
et al. (33) that dietary nitrate from different vegetable sources can
be effectively absorbed, reaching ;100% bioavailability in the
circulation. The time to peak plasma nitrate concentrations after
sodium nitrate and beetroot juice ingestion (Figure 1B) were
similar to that shown in earlier studies using these sources (18,
32). The longer time needed to reach peak plasma nitrate con-
centrations after ingestion of rocket salad and spinach bever-
ages may be related to their higher fiber content and the larger
volume ingested (Supplemental Table 1), likely resulting in a
slower gastric emptying (34). Hence, the optimal timing of sup-
plementation may slightly differ between various nitrate sources.
More important, ingestion of dietary nitrate from all 4 sources
seemed to be equally effective in increasing plasma nitrate
concentrations.
In line with the data on plasma nitrate concentrations, sub-
stantial increases in plasma nitrite concentrations were observed
after ingestion of all 4 beverages (Figure 2A). Both the increases
in plasma nitrite concentrations (;4-fold) and time to reach
peak values (;180–240 min) are in agreement with previous
work supplementing ;500–1000 mg nitrate in the form of
sodium nitrate (15) or beetroot juice (2, 20–22). In contrast with
the plasma nitrate concentrations, time to reach peak plasma
nitrite concentrations did not differ between treatments (Figure
2B). This could imply that the speed of bioconversion of nitrate
to nitrite is not merely driven by the rate at which nitrate appears
in the circulation. Furthermore, the total release of nitrite into
the blood may be affected by the composition of the specific
nitrate source. In this context, we observed a remarkable initial
increase in plasma nitrite concentrations after ingestion of
spinach (Figure 2A), which was much stronger than the other
beverages. This substantial increase in plasma nitrite (;4-fold
within 30 min) is unlikely to be completely explained by the
endogenous conversion of dietary nitrate (32). Alternatively, the
initial increase in plasma nitrite after spinach ingestion may be
attributed to the higher nitrite content found in spinach than in
the other vegetables (35). In agreement, analysis of the 4
provided beverages for nitrite content revealed substantially
greater nitrite content in the spinach beverage (;100 mg) than in
the rocket salad beverage (;20 mg), beetroot juice (<1 mg), and
sodium nitrate (<1 mg). Although this may partly explain the
greater initial increase in plasma nitrite concentrations after
ingestion of the spinach, plasma nitrite concentrations continued
to increase throughout the test day. In fact, the increase in
plasma nitrite concentrations from 30 min after ingestion to
peak values was similar for all 4 beverages (Figure 2A). As such,
to our knowledge, our findings are the first to show that the
effective uptake of dietary nitrate and the total bioconversion of
the ingested nitrate into nitrite do not seem to depend on the
source of dietary nitrate.
Plasma nitrite represents a precursor for the NO synthase–
independent formation of NO, which can then act as a
vasodilatory agent thereby lowering blood pressure (6). Previous
work in healthy populations has shown reductions in blood
pressure after acute beetroot juice (6, 8, 20, 22, 29), several-day
beetroot juice (1, 2, 23), or acute spinach (36, 37) ingestion. The
blood pressure–lowering effects of sodium nitrate have been
studied less extensively. We are the first to compare the blood
pressure–lowering effects after ingestion of various nitrate-rich
vegetable sources and sodium nitrate, using identical nitrate
doses. We observed a more pronounced decrease in blood
pressure after ingestion of the vegetable beverages (systolic:
;5–7 mm Hg, diastolic: ;4–8 mm Hg; Figure 3C,D) than
sodium nitrate (only diastolic: ;2–4 mm Hg). In line with our
findings, previous work reported that despite substantial in-
creases in plasma nitrate and nitrite concentrations, sodium
nitrate only decreased diastolic blood pressure, whereas systolic
blood pressure remained unaffected (7, 15). It has been specu-
lated that other compounds in vegetables (such as vitamin C,
potassium, and polyphenols) may contribute and/or act syner-
gistically with nitrate, enhancing its blood pressure–lowering
effects. For example, it has been shown that vitamin C increases
the reduction of nitrite to NO (38, 39), which may potentiate the
blood pressure–lowering effects at a given nitrate/nitrite concen-
tration. Additionally, 2 studies using potassium nitrate observed
positive effects on both systolic and diastolic blood pressures
(25, 28), suggesting that the combination of potassium and
nitrate may be more effective than sodium and nitrate. Obvi-
ously, all vegetable beverages in the present study contained
substantial amounts of macro- and micronutrients that were not
present in the sodium nitrate beverage (Supplemental Table 1). As
such, the principle of a synergistic effect between nitrate and
other nutritional compounds may explain why the vegetable bev-
erages are more effective than sodium nitrate in lowering blood
pressure, despite similar increases in plasma nitrate and nitrite
concentrations. Yet, it remains to be established what specific
mechanisms may underlie a potentially more effective conver-
sion of nitrite into bioactive NO, thereby explaining the more
pronounced blood pressure–lowering effects after ingestion of
different nitrate-rich vegetables.
A discordance between changes in plasma nitrite concentra-
tions and subsequent physiological effects was recently also
observed by Larsen et al. (17). Despite a substantial increase in
plasma nitrite concentrations, intravenous nitrite infusion did
not lead to a decreased resting metabolic rate as observed after
ingestion of dietary nitrate (17). In accordance, the initial high
plasma nitrite concentrations after spinach ingestion did not
translate into any additional reduction in blood pressure in the
present study (Figure 3C). Overall, it appears that plasma nitrite
per se is not the key driver, nor the key indicator of the
physiological effects induced through the nitrate-nitrite-NO
pathway. Although nitrite represents an important intermediate,
Larsen et al. (17) suggest that nitrate-derived bioactive nitrogen
oxides other than nitrite could be the final mediators of the
observed effects. Ultimately, itis the actual bioavailability of NO
that is likely driving any physiological effects (32). In this
respect, cyclic guanosine-5’-monophosphate (cGMP) has been
suggested to be the most sensitive indicator of NO bioactivity
(25). As such, cGMP should be included in future studies
investigating the effects of dietary nitrate, including potential
differences between various dietary nitrate sources.
The major strength of the present study lies in the within-
subject comparison of the 4 treatments, providing exactly 800
mg of nitrate with each treatment, under fully standardized
conditions. By testing each participant at the same time of day
for all 4 treatments, we bypassed the effects of circadian fluctu-
ation of blood pressure, ensuring a valid comparison between
treatments. However, because blood pressure is known to rise
throughout the morning (27), we cannot rule out the possibility
that we may have overlooked (in the case of sodium nitrate) or
simply underestimated the blood pressure–lowering effect of
each treatment. We chose not to burden the participants with
a control trial because our primary aim was to study the
pharmacokinetics of plasma nitrate and nitrite. It is clearly estab-
lished that plasma nitrate and nitrite do not change upon placebo
ingestion (20, 22, 40, 41). As a further limitation, we did not
6 of 8 Jonvik et al.
at UNIVERSITEITS MAASTRICHT on April 15, 2016jn.nutrition.orgDownloaded from
statistically power to investigate any potential sex differences.
However, subanalysis of the data indicated higher plasma nitrate
and nitrite concentrations in women than in men (Supplemental
Figures 1, 2). Of note, these sex differences did not translate
to any differences in the blood pressure–lowering effects after
nitrate ingestion. The sex differences in plasma nitrate and
nitrite concentrations could partly be explained by differences in
body mass and/or distribution volume of plasma. Previous studies
have also reported higher plasma nitrate and/or nitrite responses
in women than in men, whereas a subsequent reduction in platelet
reactivity and increase in platelet cGMP (42) as well as a reduction
in blood pressure (25) was more pronounced in men than in
women. As a potential explanation, it has been suggested that
there may be sex differences in the bacterial colonization of the
tongue, likely affecting nitrate reductase activity (25). Again, it
appears that not nitrate/nitrite per se but rather the effective
bioconversion into NO is the final mediator of any physiological
effects. As such, sex differences in the response to dietary nitrate
ingestion, including cGMP, may represent a key target for future
research.
The present work clearly shows that various natural food
sources, here provided as vegetable beverages, can be used
effectively as dietary nitrate donors, with no differences in
postprandial plasma nitrate and nitrite concentrations. Further-
more, all vegetable beverages induced a substantial reduction in
blood pressure, despite the recruitment of healthy, normotensive
individuals. Our findings are relevant for the development of
nutritional strategies to increase dietary nitrate intake either to
enhance sports performance or facilitate clinical and/or health
benefits (43). Obviously, the minimal nitrate dose needed to
achieve beneficial effects should be investigated. We provided a
dose exceeding the recommended acceptable daily intake of
3.7 mg !kg
21
!d
21
(30), but well within the range used in studies
showing blood pressure–lowering effects (6, 7, 15, 25, 28, 29).
There is an ongoing debate on potential health risks of long-term
exposure to high nitrite and nitrate, especially with regard to the
formation of low-molecular-weight N-nitrosamines, and asso-
ciated cancer risk. Given the acute setting in the present study
(i.e., determining plasma and blood pressure responses up to 300
min after a single nitrate dose), we chose not to burden our
participants with multiple 24-h urine collections to measure
nitroso-compound formation. Furthermore, previous work sug-
gests that there is no relevance of measuring nitroso formation in
plasma after acute nitrate supplementation (32). Although the
risk of long-term exposure to nitrite and nitrate and developing
cancers is weak at best, and the cardiovascular benefits have
been suggested to outweigh the risks (43), any potential risk still
needs to be carefully considered. Therefore, we propose that
future work investigating (long-term) exposure to high nitrate
intakes should include measurements of nitroso-compound
formation. In the present study, we did not observe any acute
serious adverse effects. A few participants reported minor
gastrointestinal complaints after ingestion of the rocket salad
or spinach beverages, likely related to the total volume to be
ingested rather than the nitrate dose. For practical use of various
nitrate sources, future studies should focus on optimizing the
volume, consistency, and palatability of various nitrate sources.
In conclusion, ingestion of 800 mg nitrate provided as
sodium nitrate, beetroot juice, rocket salad beverage, and
spinach beverage substantially increases plasmanitrateand
nitrite concentrations. Ingestion of nitrate from beetroot
juice, rocket salad beverage, and spinach beverage lowers
blood pressure to a greater extent than ingestion of the same
amount of nitrate provided as sodium nitrate. These findings
imply that nitrate-rich vegetables can be used as dietary
nitrate supplements.
Acknowledgments
KLJ, JN, LJCvL, and LBV designed the research study; KLJ, JN,
PJMP, and JMGS conducted the research trials; KLJ, JN, PJMP,
and LBV analyzed the data; and KLJ had primary responsibility
for the final content. All authors wrote the paper, and read and
approved the final manuscript.
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... Although there are several other nitrate-rich food sources, including green leafy and root vegetables (25) , research on the pharmacokinetic and physiological effects of nitrate supplementation has mainly used either sodium nitrate or BRJ as a nitrate donor. Very few studies have investigated the effect of more than one high nitrate source (10,26) . A recent crossover study found that (beetroot, rocket salad or spinach) juice increased plasma nitrate and lowered BP to a greater extent than sodium nitrate (26) after a single exposure. ...
... Very few studies have investigated the effect of more than one high nitrate source (10,26) . A recent crossover study found that (beetroot, rocket salad or spinach) juice increased plasma nitrate and lowered BP to a greater extent than sodium nitrate (26) after a single exposure. A further crossover trial investigated the effect of a high nitrate diet and control diet on BP for 1 week (3) . ...
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The suitability of groundwater and agricultural products for human consumption requires determining levels and assessing the health risks associated with potential pollutants. Here, particularly pollution with nitrate still remains a challenge, especially for those urban areas suffering from insufficient sewage collection systems, resulting in contaminating soil, endangering food safety, and deteriorating drinking water quality. In the present study, nitrate concentrations in the commonly consumed fruit and vegetable species were determined, and the results, together with the groundwater nitrate levels, were used to assess the associated health risks for Mashhad city residents. For this assessment, 261 water samples and 16 produce types were used to compute the daily intake of nitrate. Nitrate in groundwater was analyzed using a spectrophotometer, and produce species were examined using High-Performance Liquid Chromatography. Ward’s hierarchical cluster analysis was applied for categorizing produce samples with regard to their nitrate content. Additionally, to account for the sanitation hazards associated with groundwater quality for drinking purposes, total coliform and turbidity were also assessed using the membrane filter (MF) technique and a nephelometer, respectively. Nitrate concentrations exceeded the prescribed permissible limits in 42% of the groundwater wells. The outcomes also exhibit significantly higher nitrate accumulation levels in root-tuber vegetables and leafy vegetables compared to fruit vegetables and fruits. Using cluster analysis, the accumulation of nitrate in vegetables and fruits was categorized into four clusters, specifying that radish contributes to 65.8% of the total content of nitrate in all samples. The Estimated Daily Intake (EDI) of nitrate and Health Risk Index (HRI) associated with consumption of groundwater exceeded the prescribed limit for the children’s target group in Mashhad’s south and central parts. Likewise, EDI and HRI values for produce consumption, in most samples, were found to be in the tolerable range, except for radish, lettuce, and cabbage, potentially posing risks for both children and adult consumers. The total coliforms in groundwater were found to violate the prescribed limit at 78.93% of the sampling locations and were generally much higher over the city’s central and southern areas. A relatively strong correlation ( R ² = 0.6307) between total coliform and nitrate concentrations suggests the release of anthropogenic pollution (i.e., sewage and manure) in the central and southern Mashhad.
... In healthy adults, nitrate-rich vegetables increase plasma nitrate and nitrite levels [23]. Shepherd [19,20]. ...
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Nitrate determination from fresh plant material is common in routine analysis, but comparisons between extraction solutions, detection techniques or combinations of both show that there is a wide range in performance in methods determining plant nitrate. In this paper, the performance of methanol:water (1:1, V:V) as an extraction solution was compared against water and KCl as extractants. Measurement results of methanol:water extracts were consistently higher than the water or KCl results. Even compared to a recommended nitrate determination method, using dried material, significantly higher results were obtained with methanol:water as an extraction solution. This has consequences on the interpretation of internal quality of produce, like some vegetables, where maximum allowable nitrate concentrations are put forward in various countries. Furthermore, the results of the methanol:water extracts of two common determination techniques, i.e., segmented flow analysis and HPLC detection technique, were highly comparable.
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Recent studies surprisingly show that dietary inorganic nitrate, abundant in vegetables, can be metabolized in vivo to form nitrite and then bioactive nitric oxide. A reduction in blood pressure was recently noted in healthy volunteers after dietary supplementation with nitrate; an effect consistent with formation of vasodilatory nitric oxide. Oral bacteria have been suggested to play a role in bioactivation of nitrate by first reducing it to the more reactive anion nitrite. In a cross-over designed study in seven healthy volunteers we examined the effects of a commercially available chlorhexidine-containing antibacterial mouthwash on salivary and plasma levels of nitrite measured after an oral intake of sodium nitrate (10mg/kg dissolved in water). In the control situation the salivary and plasma levels of nitrate and nitrite increased greatly after the nitrate load. Rinsing the mouth with the antibacterial mouthwash prior to the nitrate load had no effect on nitrate accumulation in saliva or plasma but abolished its conversion to nitrite in saliva and markedly attenuated the rise in plasma nitrite. We conclude that the acute increase in plasma nitrite seen after a nitrate load is critically dependent on nitrate reduction in the oral cavity by commensal bacteria. The removal of these bacteria with an antibacterial mouthwash will very likely attenuate the NO-dependent biological effects of dietary nitrate.
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Purpose: The power asymptote (critical power [CP]) and curvature constant (W') of the power-duration relationship dictate the tolerance to severe-intensity exercise. We tested the hypothesis that dietary nitrate supplementation would increase the CP and/or the W' during cycling exercise. Methods: In a double-blind, randomized, crossover study, nine recreationally active male subjects supplemented their diet with either nitrate-rich concentrated beetroot juice (BR; 2 × 250 mL·d, ∼8.2 mmol·d nitrate) or a nitrate-depleted BR placebo (PL; 2 × 250 mL·d, ∼0.006 mmol·d nitrate). In each condition, the subjects completed four separate severe-intensity exercise bouts to exhaustion at 60% of the difference between the gas exchange threshold and the peak power attained during incremental exercise (60% Δ), 70% Δ, 80% Δ, and 100% peak power, and the results were used to establish CP and W'. Results: Nitrate supplementation improved exercise tolerance during exercise at 60% Δ (BR, 696 ± 120 vs PL, 593 ± 68 s; P < 0.05), 70% Δ (BR, 452 ± 106 vs PL, 390 ± 86 s; P < 0.05), and 80% Δ (BR, 294 ± 50 vs PL, 263 ± 50 s; P < 0.05) but not 100% peak power (BR, 182 ± 37 vs PL, 166 ± 26 s; P = 0.10). Neither CP (BR, 221 ± 27 vs PL, 218 ± 26 W) nor W' (BR, 19.3 ± 4.6 vs PL, 17.8 ± 3 kJ) were significantly altered by BR. Conclusion: Dietary nitrate supplementation improved endurance during severe-intensity exercise in recreationally active subjects without significantly increasing either the CP or the W'.