ARTICLE

Acute dietary nitrate supplementation does not augmentsubmaximal forearm exercise hyperemia in healthy young menJin-Kwang Kim, David J. Moore, David G. Maurer, Daniel B. Kim-Shapiro, Swati Basu,Michael P. Flanagan, Ann C. Skulas-Ray, Penny Kris-Etherton, and David N. Proctor

Abstract: Despite the popularity of dietary nitrate supplementation and the growing evidence base of its potential ergogenicand vascular health benefits, there is no direct information about its effects on exercising limb blood flow in humans. Wehypothesized that acute dietary nitrate supplementation from beetroot juice would augment the increases in forearm bloodflow, as well as the progressive dilation of the brachial artery, during graded handgrip exercise in healthy young men. In arandomized, double-blind, placebo-controlled crossover study, 12 young (22 ± 2 years) healthy men consumed a beetroot juice(140 mL Beet-It Sport, James White Juice Company) that provided 12.9 mmol (0.8 g) of nitrate or placebo (nitrate-depleted Beet-ItSport) on 2 study visits. At 3 h postconsumption, brachial artery diameter, flow, and blood velocity were measured (Dopplerultrasound) at rest and during 6 exercise intensities. Nitrate supplementation raised plasma nitrate (19.5-fold) and nitrite(1.6-fold) concentrations, and lowered resting arterial pulse wave velocity (PWV) versus placebo (all p < 0.05), indicating absorp-tion, conversion, and a biological effect of this supplement. The supplement-associated lowering of PWV was also negativelycorrelated with plasma nitrite (r = –0.72, p = 0.0127). Despite these systemic effects, nitrate supplementation had no effect onbrachial artery diameter, flow, or shear rates at rest (all p ≥ 0.28) or during any exercise workload (all p ≥ 0.18). These findingssuggest that acute dietary nitrate supplementation favorably modifies arterial PWV, but does not augment blood flow or brachialartery vasodilation during nonfatiguing forearm exercise in healthy young men.

Key words: inorganic nitrate, vascular function, pulse wave velocity.

Résumé : En dépit de la popularité des suppléments de nitrate alimentaire et du nombre croissant d’études probantes au sujetde son potentiel ergogène et de ses bienfaits en santé vasculaire, il n’y a pas d’études directes chez des humains au sujet de seseffets sur le débit sanguin des membres a l’effort. Nous posons l’hypothèse selon laquelle un supplément de nitrate alimentaireprovenant du jus de betterave accroît le débit sanguin dans l’avant-bras et suscite une dilatation graduelle de l’artère brachialeau cours d’un exercice de préhension manuelle d’intensité croissante chez de jeunes hommes en bonne santé. Selon un devisaléatoire a double insu dans une étude croisée avec groupe placebo de contrôle, douze jeunes hommes (22 ± 2 ans) en bonne santéconsomment du jus de betterave (140 mL Beet-It Sport, James White Juice Company) procurant 12,9 mmol (0,8 g) de nitrate ou unplacebo (boisson Beet-It Sport vidée de son nitrate) en deux séances distinctes. Trois heures après la consommation, on évalue parultrasonographie Doppler le diamètre, le flot et le débit sanguin de l’artère brachiale au repos et au cours de six intensitésd’exercice. La supplémentation en nitrate suscite une augmentation de la concentration plasmatique de nitrate (19,5 fois) et denitrite (1,6 fois) et une diminution de la vélocité de l’onde pulsatile (« PWV ») dans l’artère au repos comparativement a lacondition de placebo (p < 0,05 pour toutes les différences), ce qui indique l’absorption, la conversion et l’effet biologique de cesupplément. La diminution de la PWV associée a la supplémentation est inversement reliée a la concentration plasmatique denitrite (r = –0,72, p = 0,0127). Même en présence de ces effets systémiques, la supplémentation n’a pas d’effet sur le diamètre, leflot et le taux de cisaillement de l’artère brachiale au repos (p ≥ 0,28 pour toutes les différences) et a toutes les intensitésd’exercice (p ≥ 0,18 pour toutes les différences). D’après ces observations, la simple supplémentation en nitrate alimentairemodifie favorablement la PWV artérielle, mais n’améliore pas le débit sanguin ou la vasodilatation de l’artère brachiale au coursd’un exercice non épuisant de l’avant-bras chez de jeunes hommes en bonne santé. [Traduit par la Rédaction]

Mots-clés : nitrate inorganique, fonction vasculaire, vélocité de l’onde pulsatile.

IntroductionNitric oxide (NO) plays a key contributing role in the modula-

tion of blood vessel tone at rest and under conditions of increasedmetabolic demand, such as exercise (Heinonen et al. 2011; Joynerand Casey 2009; Joyner and Tschakovsky 2003). As a result, therehas been a growing interest in the use of dietary interventions

that increase bioavailable NO as both cardiovascular health-promoting (Kapil et al. 2010; Lundberg et al. 2011; Machha andSchechter 2011) and ergogenic aids (Bailey et al. 2009; Bescos et al.2012; Jones et al. 2011; Lansley et al. 2011; Vanhatalo et al. 2011). Inparticular, dietary supplementation with inorganic salts or high-nitrate containing foods (e.g., beetroot juice) has recently grown

Received 21 June 2014. Accepted 2 October 2014.

J.-K. Kim and D.J. Moore. Intercollege Graduate Degree Program in Physiology, The Pennsylvania State University, University Park, PA 16802, USA.D.G. Maurer. Department of Kinesiology, 105 Noll Laboratory, The Pennsylvania State University, University Park, PA 16802, USA.D.B. Kim-Shapiro and S. Basu. Department of Physics and the Translational Science Center, Wake Forest University, Winston-Salem, NC 27109, USA.M.P. Flanagan. Penn State Hershey College of Medicine, Hershey, PA 10733, USA.A.C. Skulas-Ray and P. Kris-Etherton. Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA 16802, USA.D.N. Proctor. Intercollege Graduate Degree Program in Physiology, The Pennsylvania State University, University Park, PA 16802, USA; Department ofKinesiology, 105 Noll Laboratory, The Pennsylvania State University, University Park, PA 16802, USA.Corresponding author: David N. Proctor (e-mail: dnp3@psu.edu).

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Appl. Physiol. Nutr. Metab. 40: 1–7 (2015) dx.doi.org/10.1139/apnm-2014-0228 Published at www.nrcresearchpress.com/apnm on 15 October 2014.

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in popularity for its potential blood pressure-lowering and aero-bic exercise performance-enhancing effects. With regard to thelatter function, many of the studies performed in humans havelargely focused on the influence of dietary nitrate on exercising mus-cle metabolism, such as a putative ability to reduce the oxygen costof muscle contraction (Bailey et al. 2009, 2010; Bentley et al. 2014;Bescos et al. 2012; Jones et al. 2011; Larsen et al. 2011). However, evi-dence from a rodent model suggests that dietary nitrate supplemen-tation may also improve the perfusion of active skeletal musclesduring exercise (Ferguson et al. 2013b). Ferguson et al. (2013b) haverecently demonstrated that consumption of nitrate-rich beetrootjuice augments hindlimb skeletal muscle blood flow and vascularconductance during treadmill running in rats. In humans, 2 studieshave investigated the effects of intra-arterially infused doses of so-dium nitrite on forearm exercise hyperemia, but these studies in-volve supra-physiological doses of nitrite. Therefore, it is unclearwhether increasing plasma nitrite within the physiological range bydietary nitrate supplementation can impact vascular function dur-ing exercise. To our knowledge, no studies have investigated theinfluence of dietary nitrate supplementation on exercise hyperemiaor local vascular responses to exercise in humans.

The purpose of the present study was to determine whether anacute dose of nitrate-rich beetroot juice augments forearm exer-cise hyperemia, as well as brachial artery dilation during gradedhandgrip exercise in healthy young adults. We hypothesized thatthis nitrate-rich supplement would augment the increases in fore-arm blood flow, as well as the progressive dilation of the brachialartery, during graded handgrip exercise based on the potent va-sodilator effects of intra-arterially infused nitrite in the humanforearm (Cosby et al. 2003; Dejam et al. 2007) and the effects ofbeetroot juice consumption on blood flow to the exercising ro-dent hindlimb (Ferguson et al. 2013b).

Materials and methods

ParticipantsYoung (age, 22 ± 2 years; range, 19–24 years) healthy men were

recruited from the local university community. Interested volun-teers provided written informed consent prior to enrollment. Onthe same day, medical screening was performed to determineeligibility, followed by familiarization with the study procedures.Participants were selected if they were low- to moderately active(i.e., <3 days/week of exercise), free of overt chronic disease asassessed by clinician-reviewed medical history questionnaire andvenous blood chemistry (hematologic, liver, and kidney function),and met the following criteria: (i) resting blood pressure between110/60 and 140/90 mm Hg, (ii) body mass index between 18.5 and30 kg/m2, (iii) fasting plasma glucose <100 mg/dL, (iv) fasting plasmalow-density lipoprotein <130, and/or high-density lipoprotein>40 mg/dL, (v) nonsmoker, (vi) not taking any cardiovascular medica-tions, and (vii) had not donated blood or blood products in the past3 months. Characteristics of the 12 participants that completed thisstudy are shown in Table 1. All procedures were approved by theOffice of Research Protections at Pennsylvania State University inagreement with the guidelines set forth by the Declaration ofHelsinki.

Study designAfter the baseline medical screening visit, participants at-

tended 2 study visits that were at least 5 days apart, during whichthey consumed 140 mL (two 70-mL bottles) of either a concentratedbeetroot juice supplement (BRnitrate; Beet-It Sport, James WhiteDrinks Ltd., Ipswich, UK) or the equivalent nitrate-depleted placebo(BRplacebo). The order of these 2 study visits (nitrate visit vs. placebovisit) was randomized by clinical support staff, with primary studyinvestigators blinded to the treatment order during testing and dataanalysis. Each 70-mL Beet-It Sport beverage has an average nitratecontent of 6.45 mmol (0.4 g), equivalent to approximately 400 mL of

beetroot juice. Therefore, the expected dose of inorganic nitrate ad-ministered to each subject during their BRnitrate visit was 12.9 mmol(0.8 g).

Participants followed their usual diet (i.e., they did not follow anitrate-restricted diet), but were instructed to refrain from alco-hol and dietary supplements (48 h) and to arrive at each study visitafter an overnight fast (water only). They were asked to abstainfrom caffeine consumption (12 h) and from aerobic or resistancetraining (24 h) prior to each study visit. Importantly, participantswere instructed not to brush their teeth or to use mouthwash themorning of each study visit to prevent killing anaerobic nitrate-reducing bacteria on the dorsal surface of the tongue, which areessential for the conversion of nitrate-to-nitrite following beet-root juice ingestion (Gladwin et al. 2005; Webb et al. 2008). Bothstudy visits took place at approximately the same time of daystarting between 0700 and 1000 hours.

Upon arrival at the Clinical Research Center a venous bloodsample was collected for determination of baseline plasma nitrateand nitrite concentration. Participants then consumed two 70-mLbottles of BRnitrate or BRplacebo. Two hours and 30 min after consum-ing the nitrate supplement or placebo, a second venous blood sam-ple was taken to determine the postconsumption change in plasmanitrate and nitrite because peak plasma nitrite concentration inhealthy younger men is reported to occur 2 h postconsumption andremain elevated above baseline until 8 h postconsumption (Wylieet al. 2013). Following the second venous blood sample, participantswere escorted to the Vascular Aging and Exercise laboratory forvascular testing.

Pulse wave velocity (PWV)Brachial-to-ankle PWV (PWVBA) was determined by a vascular

profiling machine (VP2000, Colin Corp., Komaki, Japan) after15 min of supine rest, which was approximately 3 h post-BRnitrateor BRplacebo consumption. Briefly, PWVBA was calculated by divid-ing the estimated distance between the brachial and posteriortibial artery (determined from the participants’ height by the vas-cular profiling machine) by the pulse transit time between the2 sites. Reported PWV values are the mean of the left and rightmeasurements.

Graded handgrip exerciseHandgrip exercise was performed with each subject’s left hand

using a custom-built pulley-based device. Subjects lied supinewith their elbow extended to facilitate Doppler Ultrasound imag-ing of their brachial artery. Following 1 min of resting brachialartery hemodynamic measurements, subjects performed rhyth-mic contractions of their forearm muscles at a controlled rate

Table 1. Subject characteristics.

Young men

Subjects, n 12Height, cm 179±5Weight, kg 79±8Age, years 22±2BMI, kg/m2 25±2Glucose, mg/dL 91±5Cholesterol, mg/dL 145±20HDL, mg/dL 52±7LDL, mg/dL 78±16Triglycerides, mg/dL 77±27Systolic blood pressure, mm Hg 120±7Diastolic blood pressure, mm Hg 64±7HR, bpm 58±11Handgrip MVC, kg 50±7

Note: Values are means ± SE. BMI, body mass index;HDL, high-density lipoprotein; HR, heart rate; LDL, low-density lipoprotein; MVC, maximal voluntary contraction.

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(30 contractions per minute, 1-s contraction/1-s relaxation) andrange of motion (10-cm excursion of the pulley wire with eachcontraction). Each participant completed six 3-min bouts of exer-cise with progressively increasing weight (200, 400, 600, 800, 1000,and 1200 g), with 1 min of quiet rest between bouts. The gradedhandgrip exercise protocol began approximately 3 h after con-sumption of the BRnitrate or BRplacebo.

Vascular responses to exerciseDiameter and blood flow velocity of the brachial artery were mea-

sured using Doppler ultrasound (HDI 5000, Philips; Bothell, Wash.,USA). For blood flow velocity, the artery was insonated at a constantangle of 60° with the sample volume adjusted to cover the width ofthe artery. Velocity measurements were sampled in real time(400 Hz) using a data acquisition system (Powerlab, AD Instruments;Castle Hill, Australia). Mean blood velocity was calculated from thefinal 30 s of the third minute during each bout. High-resolutiondiameter measurements (6 MHz probe) were taken from 15-s record-ings performed during baseline rest and immediately following themeasurement of flow velocity during each exercise bout. Imageswere recorded directly to computer using a video capture device andsoftware (Dazzle Video Creator, Pinnacle; Mountain View, Calif.,USA). Brachial artery diameter was determined as the average acrossthe cardiac cycle using edge-detection software (Brachial AnalyzerSoftware, Medical Imaging Applications; Coralville, Iowa). Forearmblood flow at each work rate was calculated by multiplying the cross-sectional area of the brachial artery with mean blood velocity usingthe equation � (diameter/2)2 × velocity × 60. The dilatory response ofthe brachial artery (% increase above resting baseline) to gradedhandgrip exercise was examined as a function of workload and esti-mated shear rate. All vascular analyses were conducted by a singleinvestigator who was blinded to the participant and supplementorder.

Plasma [nitrate/nitrite] analysisVenous blood samples were drawn into EDTA tubes (10 mL K2

EDTA tubes, BD Vacutainer, Franklin Lakes, N.J., USA) and immedi-ately centrifuged at 3200 r/min (1590g) and 4 °C for 10 min. Plasmawas then extracted and stored in –80 °C freezer for later analysis ofnitrate and nitrite concentrations. The ENO-20 analyzer (EICOM, SanDiego, Calif., USA) with a sensitivity of 0.1 pmol for nitrate and nitritewas used to measure nitrate and nitrite concentrations in the plasmasamples. Briefly, plasma was mixed with an equal volume of 100%methanol and centrifuged at 13 000 r/pm (12 000g) for 10 min. Sam-ples were then loaded into a 96-well plate. Nitrate and nitrite werethen separated via column chromatography and individually reactedwith a Griess reagent, synthesizing a diazo compound. The absor-bance of this red diazo compound was then read at a wavelength of540 nm using a visible light detector.

Statistical analysisData were analyzed using IBM SPSS Statistics for Windows (ver-

sion 20.0.; IBM Corp., Armonk, N.Y., USA). Comparisons of restingsupine blood pressures and arterial PWV between the BRnitrate andBRplacebo visits were performed using paired t tests. Paired t testwas also performed to compare plasma nitrate and nitrite concen-trations between the BRnitrate and BRplacebo for both baseline and2.5 h postconsumption. Two-factor ANOVA with repeated mea-sures was used to assess the effects of nitrate supplementation onvascular responses during graded handgrip exercise. The associationbetween PWV and plasma nitrite concentration was determined bysimple linear regression. All data are presented as means ± SE. Avalue of p < 0.05 was considered statistically significant.

ResultsSubject characteristics are provided in Table 1. Ingestion of the

beetroot juice supplement and its nitrate-depleted placebo waswell tolerated by all participants with no side effects reported.

Concentrated beetroot juice ingestion raised plasma nitrateand nitrite (Fig. 1)

Prior to beverage ingestion, resting baseline plasma nitrate(p = 0.79) and nitrite (p = 0.92) concentrations were not differentbetween visits. Two hours and 30 min following ingestion of theBRplacebo, plasma nitrate (p = 0.09) and nitrite (p = 0.40) concentra-tions were not elevated above baseline. However, plasma nitrateand nitrite concentrations were significantly elevated followingingestion of the BRnitrate (nitrate, 19.5-fold; nitrite, 1.6-fold; bothp < 0.03).

Acute nitrate supplementation and resting arterialhemodynamics

Resting supine systolic (120 mm Hg vs. 119 mm Hg, p = 0.24) anddiastolic (63 mm Hg vs. 63 mm Hg, p = 0.30) blood pressures,which were measured approximately 3 h after beverage consump-tion, were not different between BRnitrate or BRplacebo visits. How-ever, simultaneously measured PWV was significantly lower 3 hafter consuming the BRnitrate beverage (1065 ± 32 cm/s) comparedwith the BRplacebo (1106 ± 28 cm/s; p = 0.03, Fig. 2a). Moreover, asignificant negative correlation (r = –0.72, p = 0.01) was observedbetween the difference in postconsumption plasma nitrite con-centrations and the corresponding difference in PWV measuredbetween the 2 days (Fig. 2b).

Acute nitrate supplementation does not alter forearmexercise hyperemia (Fig. 3)

Resting brachial artery blood flow measured immediately priorto graded handgrip exercise was similar between BRplacebo andBRnitrate visits (p = 0.55). As expected, there was an observed in-crease in blood flow across exercise intensities (p = 0.001). However,exercising forearm blood flow between BRplacebo and BRnitrate visitswas similar at each exercise intensity (all p > 0.05).

Fig. 1. Plasma nitrate (a) and nitrite (b) concentrations at baseline(white bars) and 2.5 h after (hashed bars) ingestion of nitrate-rich(BRnitrate) or nitrate-depleted (BRplacebo) beetroot juice. �M, �mol/L.Data are presented as means ± SE. *, Significant difference frombaseline of same treatment, p < 0.05.

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Nitrate supplementation does not alter the brachial arterydilatory response to exercise (Fig. 4)

Resting brachial artery diameter measured immediately priorto graded handgrip exercise was similar between BRplacebo andBRnitrate visits (p = 0.67). Brachial artery diameter and calculatedshear rate increased in an intensity-dependent manner followingtreatment with both the BRplacebo and BRnitrate (p < 0.0001), withno significant difference in these responses between treatments(p ≥ 0.70).

DiscussionThe primary objective of the current study was to determine

whether acute dietary nitrate supplementation augments fore-arm blood flow and conduit vasodilator responses during graded

Fig. 2. Brachial-to-ankle pulse wave velocity (PWVBA) measured at 3 hafter ingestion of nitrate-rich (BRnitrate; solid bar) or nitrate-depleted(BRplacebo; white bar) beetroot juice (a), and correlation of PWVBA

difference and plasma nitrite concentration difference betweenpostconsumption of BRnitrate and BRplacebo supplementation (b). �M,�mol/L. Data are presented as means ± SE. *, Significant difference frombaseline of same treatment, p < 0.05.

Fig. 3. Brachial artery blood flow during graded forearm exerciseperformed 3.5 h after ingestion of nitrate-rich (BRnitrate; filledcircles) or nitrate-depleted (BRplacebo; unfilled circles) beetroot juice.Data are presented as means ± SE for each workload.

Fig. 4. Brachial artery diameter (a), percent change in diameter (b),and shear rate (c) during graded forearm exercise performed 3.5 h afteringestion of nitrate-rich (BRnitrate; filled circles) or nitrate-depleted(BRplacebo; unfilled circles) beetroot juice. Data are presented as means ±SE for each workload.

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handgrip exercise. Dietary nitrate supplementation in the form ofa single dose of highly concentrated beetroot juice raised plasmanitrate (19.5-fold) and nitrite (1.6-fold) concentrations, and low-ered resting PWV versus placebo (all p < 0.05). These differencesindicate that there was absorption, conversion, and a systemicbiological effect of the ingested nitrate, respectively, in theseyoung healthy subjects. However, in contrast with our hypothesis,this supplement had no obvious effects on forearm blood flow orbrachial artery dilatory responses of healthy young men to theexercise protocol we used.

Nitrate supplementation and forearm exercise hyperemiaThe lack of any observable effects of acute nitrate supplemen-

tation on handgrip exercise blood flow in the present study wereunexpected, especially considering the relatively high dose of ni-trate we administered (BRnitrate visit) and the significant role ofnitric oxide in forearm exercise hyperemia in healthy youngeradults as previously demonstrated using nitric oxide synthase (NOS)inhibition with L-NG-monomethyl arginine (L-NMMA) (Schrage et al.2004; Wray et al. 2011). The dose of ingested nitrate (800 mg) and theincrease in plasma nitrate (19.5-fold) were well within the range ofprior studies, demonstrating improved exercise performance andbeneficial muscle metabolic effects in healthy adults (Bescos et al.2012; Vanhatalo et al. 2011; Wylie et al. 2013). However, the conver-sion of nitrate to nitrite in the present study (1.6-fold increase innitrite) appears low in relation to most of these prior reports (2.5- to4-fold increase). The absolute increase in venous nitrite concentra-tion after our subjects consumed the BRnitrate supplement averaged210 nmol/L (360 nmol/L at baseline, 570 nmol/L at 2 h postconsump-tion). This increase in venous nitrite concentration is just below thevenous nitrite concentration difference (220 nmol/L) associated withthe threshold for increased forearm blood flow induced by intra-arterial infusion of (sodium) nitrite in healthy adults (Dejam et al.2007). Collectively, these findings raise the possibility that the singledose of dietary nitrate we administered, and any subsequent intra-vascular conversion to nitric oxide, may have been insufficient toimpact exercise-induced hyperemia in the forearm of these subjects.

Exercise intensity is another important factor to consider wheninterpreting the vascular effects of nitrate supplementation. Re-cent evidence in humans (Casey and Joyner 2011) and rodent mod-els (Copp et al. 2013) implicate an intensity-dependent role for NOon muscle blood flow responses to exercise (i.e, greater impact ofNO synthase blockade at higher intensities), including the rhyth-mic handgrip exercise model we employed (Wray et al. 2011). Oursubjects completed 6 steady-state work rates that resulted in amodest �4-fold increase in forearm blood flow above baseline(Fig. 3), but we did not have them exercise at high (i.e., fatiguing)intensities, conditions which would likely have promoted in-creased activation of the nitrate–nitrite–NO signaling pathway.Thus, it is possible that contraction intensities during our hand-grip exercise protocol were not high enough to tax local (micro-vascular) oxygen supply in relation to metabolic demand, at leastin subjects with no vascular or metabolic limitations and averagegrip strength (Table 1). No direct information regarding the influ-ence of dietary nitrate supplementation on exercising limb bloodflow in humans is available for comparison; however, the effects ofdietary nitrate supplementation on active limb muscle oxygenation(near-infrared spectroscopy-based measurements) and metabolicperturbation (i.e., fall in pH, rates of phosphocreatine degradationand inorganic phosphate accumulation, accumulation of lactate) aremore evident during high versus low-intensity exercise, particularlyunder conditions of hypoxia (Bailey et al. 2009; Masschelein et al.2012; Vanhatalo et al. 2011). Recent observations of enhanced localoxygenation following dietary nitrate supplementation in exercisingcalf muscles of peripheral arterial disease patients (Kenjale et al. 2011)and in low (but not high) oxidative hindlimb muscles of exercisingrodents (Ferguson et al. 2013b), is further evidence that the vasculareffects of dietary nitrates are more evident when there is a greater

mismatch between local muscle oxygen supply versus demand. Itremains to be determined whether nitrate supplementation aug-ments conduit or microcirculatory responses in exercising limbmuscles during higher intensity, intermittent, and/or blood-flowrestricted (isometric) contractions, i.e., nonsteady-state condi-tions that more closely mimic activities of daily life.

Nitrate supplementation and brachial artery vasodilationWray et al. recently reported that nitric oxide plays a highly

significant role in the brachial artery dilator response to gradedhandgrip exercise in young healthy adults (Wray et al. 2011), whichthey observed as a 70% reduction in this response during local endo-thelial NOS inhibition with L-NMMA. The use of a similar handgripexercise protocol (6 × 3-min stages with 1-min recoveries) resulted ina shear stress response (Fig. 4C) comparable with that of Wray et al.(2009) in young men, and afforded us the opportunity to deter-mine if acute nitrate supplementation impacts this graded, nitricoxide-dependent dilatory response. Our results suggest that a sin-gle, moderately high dose (800 mg) of inorganic nitrate does notaffect exercise-induced, endothelium-mediated increases in bra-chial artery diameter in healthy young men.

All previous studies examining the effects of acute dietary ni-trate supplementation on endothelial vasodilator function in hu-mans have utilized the brachial artery dilator response followinga 5-min period of forearm cuff ischemia (the flow-mediated dila-tion (FMD) test), the noninvasive standard measure of endothelialfunction (Thijssen et al. 2011). Upon release of the occlusion cuff,there is a brief, uncontrolled increase in blood flow through thebrachial artery. While the size of the FMD response (peak % dila-tion of the brachial artery above baseline) is prognostic for theoccurrence of cardiovascular events, this response depends on themagnitude and duration of the vascular shear stimulus, a variablethat to our knowledge has not been reported in any dietary nitratesupplementation studies to date. Failure to quantify the actual shearstimulus for vessel dilation makes it difficult to compare brachialFMD responses between groups and interpret the response to inter-ventions. This omission could contribute to the variable findings inthe literature with some studies reporting substantial increases inbrachial FMD following nitrate supplementation (Heiss et al. 2012)and others very small (Bondonno et al. 2012) or no changes (Bahraet al. 2012; Kapil et al. 2010; Kenjale et al. 2011; Webb et al. 2008). Inthe present study, we chose to utilize the graded handgrip exercisemodel to evaluate this question as it provides sustained, wide-ranging endothelial shear rates through the brachial artery and pro-duces intensity-dependent vasodilation, which was recently shownto be nitric oxide-mediated in younger healthy adults (Wray et al.2011). The lack of any observable BRnitrate versus BRplacebo differencesin the estimated shear rate within the brachial artery, or in theconventional assessment of percentage of brachial artery dilation,suggests that an acute dose (800 mg) of dietary nitrate does not alterbrachial artery dilator function in healthy young men. It is impor-tant to note, as indicated above, that dietary interventions to aug-ment the conversion of nitrite to NO might not result in improvedhyperemic or vasodilatory responses during exercise at lower (mildto moderate) intensities, particularly in subjects with no apparentendothelial dysfunction.

The lack of any nitrate supplementation-associated alterationsin resting brachial artery blood flow or vessel diameter, or bra-chial artery dilation in response to exercise, are surprising giventhe reduction we observed in PWV. The reasons for this are un-clear as alterations in resting PWV are often observed when sys-temic (nitric oxide-mediated) vasomotor tone is acutely altered(Fok et al. 2012). However, Bahra et al. (2012) also observed a re-duction of PWV in the absence of any changes in brachial FMDafter nitrate supplementation (potassium nitrate salt ingestion) ina group of healthy men and women aged 18–45 years. Interest-ingly, the reduction of PWV that we observed in the present studywas correlated with changes in plasma nitrite concentration (Fig. 2B),

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suggesting that there was a hemodynamic effect associated with theconsumption of this concentrated beetroot juice supplement.

Experimental considerationsStrengths of the current investigation include the use of a double-

blind, randomized cross-over study design in a carefully screenedsample of healthy younger men. This was also 1 of only 2 studiesexamining vascular outcomes of dietary nitrate supplementationthat used a “true placebo” (i.e., nitrate-depleted beetroot juice; e.g.,Gilchrist et al. 2013) and one of very few acute supplementationstudies that did not restrict subjects’ diets of nitrate containing foodsprior to supplementation (i.e., nitrate-free run in diet; e.g., Coles andClifton 2012). The use of a true placebo that differs only in nitratecontent, along with standardized pre-visit instructions to avoid caf-feine, exercise, and oral anti-bacterial hygiene (teeth brushing,mouthwash) is arguably the most rigorous means of testing the ef-fects of dietary nitrate supplementation per se on vascular function.

There are several limitations of the present study that should beconsidered alongside these strengths. First, the present results arespecific to graded submaximal forearm exercise and cannot beextrapolated to large muscle (leg or whole body) exercise. Second,we measured plasma nitrite concentration at baseline and 2.5 hpostconsumption of beetroot juice, but not immediately before orduring the graded handgrip exercise protocol. Therefore, it is un-clear whether the current exercise protocol was sufficient to re-duce nitrite to nitric oxide within the exercising limb. Finally, weassessed maximal isometric grip strength (MVC, Jamar device),but not the peak dynamic power output of our subjects’ forearmmuscles using our testing device (pulley-based handgrip device).Thus we were unable to quantify either the contraction intensityor the “relative” exercise intensity associated with each work levelof our graded forearm exercise protocol. This is an importantconsideration given the intensity-dependent role of nitric oxidefor muscle blood flow responses to exercise (Ferguson et al. 2013a;Wray et al. 2011) and the ergogenic/metabolic effects of dietarynitrate supplementation (Bailey et al. 2009; Bescos et al. 2012;Jones et al. 2011; Lansley et al. 2011; Vanhatalo et al. 2011). Toaddress this issue, we closely compared brachial artery blood flow,shear rate, and dilator responses between our subjects and thehandgrip exercise study of Wray et al (2011), which examined thenitric oxide dependency of these responses in healthy youngadults using a protocol with a similar number of exercise stages(n = 6) and work/rest durations (3 min/1 min). Comparisons be-tween these 2 studies revealed a similar range of hyperemic (up to�4-fold above rest), shear rate (300–400 s−1), and conduit dilator(6%–10%) responses that include workloads extending to a rangethat Wray et al. demonstrated (via L-NMMA infusion) to be nitricoxide-dependent (Wray et al 2011). Collectively, this suggests thatthe range of workloads we examined were sufficiently intense todetect the local vascular effects of dietary nitrate supplementa-tion in the population we studied.

As indicated above, the conversion of nitrate to nitrite on theBRnitrate visit was less than we expected and is a potential expla-nation for the null effects on forearm exercise blood flow. How-ever, the 1.6-fold increase in plasma nitrite concentration weobserved is comparable in magnitude and time course with pre-vious dietary nitrate supplementation studies reporting signifi-cant effects on resting hemodynamic outcomes (Bahra et al. 2012;Kapil et al. 2010; Webb et al. 2008). Given this, and our use of aningested nitrate dose (800 mg) recently shown to elicit maximalblood pressure-lowering effects in younger men (Wylie et al. 2013),it was surprising to find no evidence of a nitrate treatment effecton resting blood pressure in the present study. However, estab-lishing the effects of dietary nitrate supplementation on restingblood pressure was not the focus of our study. Consequently, weonly performed these measures once (3 h postconsumption) ateach study visit, as opposed to performing serial measurementsbefore and after consumption on both days. It is possible that day

to day variability in resting blood pressure may have limited ourability to detect differences in resting blood pressure on the pla-cebo versus nitrate supplement visit. Reported reductions in rest-ing systolic and/or diastolic blood pressure following acuteingestion of nitrate supplements such as beetroot juice are rela-tively small in absolute terms (–4.4 mm Hg and –1.1 mm Hg, re-spectively; Siervo et al. 2013), particularly in healthy subjects withlow resting blood pressures. It is also important to note that thereare several published studies, including those in healthy youngeradults, that have not observed a nitrate supplementation treat-ment effect on resting blood pressure (Cermak et al. 2012; Heisset al. 2012; Larsen et al. 2006, 2010; Miller et al. 2012).

ConclusionsTo the best of our knowledge, the present study is the first to

show a correlation between the changes in plasma nitrite concen-tration and PWV with dietary nitrate supplement and to evaluatedirectly the effects of dietary nitrate supplementation on humanexercise hyperemia. The results indicate that a single 800-mg doseof inorganic nitrate, delivered in the form of concentrated beetrootjuice, lowers PWV, but does not augment dilation of the brachialartery or blood flow to the forearm muscles during graded, nonfa-tiguing handgrip exercise in healthy young men. Further investiga-tion into the effects of dietary nitrate supplementation on restingand exercise induced vascular responses, particularly in populationswith reduced nitric oxide bioavailability and under conditions ofgreater muscle metabolic demand, is warranted. While these find-ings are limited to healthy young men, and cannot be extrapolated tolarge muscle, dynamic exercise, this study provides important,methodological information for future investigations of the effectsof dietary nitrate-supplementation on exercise-induced vascular re-sponses.

AcknowledgementsFunding was provided from Social Sciences Research Institute

at Penn State University (to D.N.P.) and HL058091 (to D.K.S.). Inaddition, the project described was supported by the NationalCenter for Research Resources and the National Center for AdvancingTranslational Sciences, National Institutes of Health (NIH), throughgrant UL1 TR000127. The content is solely the responsibility of theauthors and does not necessarily represent the official views of theNIH.

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