Acute Effects of Dietary Nitrate on Exercise Tolerance, Muscle Oxygenation, and Cardiovascular Function in Patients With Peripheral Arterial Disease

in International Journal of Sport Nutrition and Exercise Metabolism
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  • 1 Maastricht University Medical Centre
  • | 2 HAN University of Applied Sciences
  • | 3 Radboud University Medical Center
  • | 4 Radboudumc
  • | 5 Liverpool John Moores University

Previous studies have used supplements to increase dietary nitrate intake in clinical populations. Little is known about whether effects can also be induced through vegetable consumption. Therefore, the aim of this study was to assess the impact of dietary nitrate, through nitrate-rich vegetables (NRV) and beetroot juice (BRJ) supplementation, on plasma nitrate and nitrite concentrations, exercise tolerance, muscle oxygenation, and cardiovascular function in patients with peripheral arterial disease. In a randomized crossover design, 18 patients with peripheral arterial disease (age: 73 ± 8 years) followed a nitrate intake protocol (∼6.5 mmol) through the consumption of NRV, BRJ, and nitrate-depleted BRJ (placebo). Blood samples were taken, blood pressure and arterial stiffness were measured in fasted state and 150 min after intervention. Each intervention was followed by a maximal walking exercise test to determine claudication onset time and peak walking time. Gastrocnemius oxygenation was measured by near-infrared spectroscopy. Blood samples were taken and blood pressure was measured 10 min after exercise. Mean plasma nitrate and nitrite concentrations increased (nitrate; Time × Intervention interaction; p < .001), with the highest concentrations after BRJ (494 ± 110 μmol/L) compared with NRV (202 ± 89 μmol/L) and placebo (80 ± 19 μmol/L; p < .001). Mean claudication onset time and peak walking time did not differ between NRV (413 ± 187 s and 745 ± 220 s, respectively), BRJ (392 ± 154 s and 746 ± 176 s), and placebo (403 ± 176 s and 696 ± 222 s) (p = .762 and p = .165, respectively). Gastrocnemius oxygenation, blood pressure, and arterial stiffness were not affected by the intervention. NRV and BRJ intake markedly increase plasma nitrate and nitrite, but this does not translate to improved exercise tolerance, muscle oxygenation, and/or cardiovascular function.

Peripheral arterial disease (PAD) is caused by atherosclerosis, which leads to arterial obstruction and impaired blood flow to the lower extremities, claudication pain, and severe exercise intolerance (Hamburg & Creager, 2017). Pathophysiological factors contributing to PAD include endothelial cell dysfunction and decreased nitric oxide (NO) bioavailability, which contributes to intermittent or chronic tissue ischemia, especially under conditions of increased demand of tissue for blood flow and oxygen delivery (Hamburg & Creager, 2017). Nitric oxide is an important physiological signaling molecule that can modulate many processes crucial to health and exercise tolerance, such as regulation of blood flow and muscle contractility (Cooper & Giulivi, 2007; Stamler & Meissner, 2001). As a consequence, there has been increased interest in the role of dietary nitrate (NO3), as a biologically active NO donor. Increased consumption of dietary nitrate has been reported previously to enhance muscle efficiency, fatigue resistance, and exercise performance (Regimbal et al., 2020) in healthy individuals, with some indication of similar benefits in diseased populations (Jones et al., 2018). Furthermore, dietary nitrate has been shown to result in both acute and chronic reductions in resting blood pressure and other markers of vascular health, such as arterial stiffness and ischemia reperfusion injury (Bondonno et al., 2016; Lauer et al., 2008). Vascular pathologies, including PAD, peripheral tissue maladaptations to chronic under perfusion, and disuse (coupled with endothelial dysfunction and a decreased bioavailability of NO; Lauer et al., 2008), may provide an ideal target for dietary nitrate interventions. However, only three studies explored the use of dietary nitrate as an NO donor in PAD (Bock et al., 2018; Kenjale et al., 2011; Woessner et al., 2018). One study found that acute beetroot juice (BRJ) supplementation delays the onset of claudication pain during walking, which in turn resulted in an improved time to exhaustion (Kenjale et al., 2011). Another study demonstrated that 8 weeks of daily inorganic nitrate supplementation increased 6-min walking distance as well as calf blood flow and vasodilation in response to reactive hyperemia (Bock et al., 2018). Woessner et al. (2018) showed that inorganic nitrate supplementation prior to exercise rehabilitation (i.e., three times per week for 12 weeks) produced increases in pain-free exercise tolerance.

Increasing dietary nitrate can also be achieved through enhancing habitual vegetable intake (e.g., arugula, bok choy, spinach, rhubarb). Interestingly, some studies in healthy populations suggest that habitual “whole” vegetable intake may provide similar or greater health benefits than supplementation (via concentrated BRJ), potentially due to the presence of various polyphenols and antioxidants in vegetables (Jonvik et al., 2016a; van der Avoort et al., 2020). As such, the principle of eating a variety of vegetables may be superior to a “single product” supplementation strategy in lowering blood pressure, a suggestion reflected in a recent systematic review (Natarajan et al., 2019). However, research into the potential clinical effects of increasing nitrate intake through whole vegetables in patient groups is currently lacking. Therefore, the current study has been conducted to investigate the effects of a dietary approach for the treatment of PAD. If successful, the results could influence current medical practice for these patients, while also providing mechanistic insights into potential physiologic targets for other lifestyle interventions.

The primary aim of this study was to compare the impact of the consumption of a single meal containing ∼400 mg (∼6.5 mmol) of dietary nitrate versus nitrate supplementation (via concentrated BRJ) on exercise tolerance (claudication onset time [COT] and maximal walking time) in patients with PAD. Secondary aims included investigation on plasma nitrate and nitrite concentrations, muscle oxygenation (assessed with near-infrared spectroscopy [NIRS]), blood pressure, and arterial stiffness (as measured with pulse wave velocity [PWV]). We hypothesized that elevation in plasma nitrate and nitrite concentrations improves exercise tolerance, muscle oxygenation, and cardiovascular function in patients with PAD, with a similar effect when enhancing nitrate-rich vegetable (NRV) intake compared with supplementation.

Methods

Study Population

Eleven male and seven female (n = 18) participants with an ankle brachial index (ABI) in the incident leg of <0.9 were included and completed all visits of this study (Figure 1). ABI is the ratio of blood pressure in the lower legs to the blood pressure in the arms; a ratio of <0.9 is considered a diagnosis of PAD (Aboyans et al., 2012). All participants had a history of stable intermittent claudication for >3 months (classified as Fontaine Stage IIA-III, Rutherford 1–4) and were recruited between June and November 2018 from vascular clinics by physician referral. This study was approved by the Medical Ethical Committee and conforms to the principles for use of human subjects and tissue outlined in the declaration of Helsinki.

Figure 1
Figure 1

—Flow diagram illustrating the movement of participants through the crossover study, investigating the effects of high nitrate intakes in patients with PAD, which was conducted between July 2018 and December 2018. PAD = peripheral arterial disease.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 31, 5; 10.1123/ijsnem.2021-0054

Prior to participation, patients were informed of the experimental procedures, associated risks, and potential benefits of participation both in writing and verbally and gave their written informed consent to participate. All patients performed a screening treadmill test to maximum claudication, aimed at familiarizing them with all the testing apparatus and exercise protocols. All participants were receiving antiplatelet and lipid lowering therapy unless medically contraindicated by their physician. There were no changes in participants’ medication regimens throughout the experiments. Exclusions were based on past medical history of endovascular or surgical intervention for claudication within the last 12 months, chronic kidney disease and/or insulin-dependent diabetes, severe peripheral neuropathy, any condition other than PAD that limits walking, and/or use of isosorbide dinitrate/mononitrate and/or contraindicated sildenafil, tadalafil, or vardenafil.

Study Protocol

Participants were instructed to visit the University medical center on three separate occasions with 7–14 days washout to prevent any carryover effect. Upon arrival, participants rested for 10 min, followed by vascular measurements (blood pressure and PWV) and a venous blood draw.

Patients were randomly assigned to consume a standardized breakfast including 150 g NRV, 70 ml of NO3 rich concentrated BRJ (Beet-it Sport®, 70 mL; James White Drinks Ltd), or 70 ml of NO3 depleted BRJ (placebo; nitrate depleted Beet-it Sport®) stratified by sex. Due to the three experimental arms of crossover design, participants were randomly assigned to each of the six possible sequences of conditions, and every individual served as their own control. Based on the order in which participants entered the study, a unique participant code was assigned. These participant codes were linked to the order of treatments using a web-based random number generator. All randomization and blinding procedures were executed by an independent technician. Supplement containers that the participants received were coded with the unique participant codes and an independent technician ensured participants received supplements in the correct order (with the Code A or B on the bottles). Participants were left to rest in a sitting position for 150 min (Jonvik et al., 2016a; Webb et al., 2008) and were asked to remain seated as much as possible (with the exception of going to the bathroom). After 150 min, measurements were repeated (i.e., vascular function and blood sample). Directly after 180 min, participants started the treadmill test with NIRS monitoring of the oxygenation status of the gastrocnemius muscle. Following the exercise test (10 min into recovery), blood pressure was measured and a blood sample was taken. All measurements were performed in a temperature-controlled room (at 21–22 °C). A schematic of the study visits is shown in Figure 2.

Figure 2
Figure 2

—Schematic representation of the study design. In a randomized crossover design, 18 patients with PAD followed a nitrate intake protocol (∼6.5 mmol) through the consumption of NRV, via BRJ, and nitrate-depleted BRJ (placebo). Blood samples were taken; blood pressure and arterial stiffness were measured in fasted state and 150 min after dietary intervention. Each intervention was followed by a maximal walking exercise test to determine COT and PWT. Gastrocnemius oxygenation during exercise was measured by NIRS. In addition, blood samples were taken and blood pressure was measured 10 min after exercise. BRJ = beetroot juice; NRV = nitrate-rich vegetable; PAD = peripheral arterial disease; COT = claudication onset time; PWT = peak walking time; NIRS = near-infrared spectroscopy; PWV = pulse wave velocity.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 31, 5; 10.1123/ijsnem.2021-0054

Plasma Nitrate and Nitrite Concentrations

Blood samples (5 ml) were collected in fasted state, at rest (before breakfast/pre), before exercise testing (150 min after breakfast/post), and 10 min into recovery, for the analysis of plasma nitrate and nitrite concentrations. Each sample was collected in Lithium–Heparin containing tubes, for analysis of nitrite and nitrate. To minimize any nitrate/nitrite conversion, the tubes were immediately centrifuged at 4 °C and 3,000 rpm (1,000g) for 5 min after which the remaining plasma (∼3.5 ml) was aliquoted (600 μl aliquots) and frozen in liquid nitrogen and stored at −80 °C until analysis. Analysis of plasma nitrate and nitrite concentrations was performed using the chemiluminescence technique, which has been described in previous studies (Bailey et al., 2010; Wylie et al., 2013).

Exercise Tolerance

Participants walked on the treadmill, at an initial workload of 3.2 km/hr, 0% grade for 2 min. Subsequent stages increase 2% in grade every 2 min (maximal Stage 9). The speed was set constant (3.2 km/hr) throughout the test. This protocol (Gardner protocol) is specifically designed for a claudication-limited PAD population (Nicolaï et al., 2009). Peak walking time (PWT; in seconds), defined as the walking time at which ambulation could not continue due to maximal pain, was recorded when the patient refused to continue the test. Additionally, COT, defined as the walking time at which the patient first experienced pain, was registered. A validated 1–4 claudication pain rating scale was used to monitor progression of claudication pain during the test, with 1 = minimal discomfort, 2 = moderate pain, 3 = intense pain, and 4 = unbearable pain (Pescatello et al., 2014). A score of 2 was defined as COT (in seconds). Patients were blinded for the distance/time walked by covering the display of the treadmill.

Muscle Oxygenation

The oxygenation status of the gastrocnemius muscle of the leg with the worst PAD symptoms and lowest ABI was monitored at rest and during exercise using a commercially available (PortaMon3) NIRS system (Artinis Medical Systems B.V., Elst Gelderland, The Netherlands). The system consists of a small portable unit that contains a light source, which emits near-infrared wavelengths of 850 and 764 nm and a detection probe (30 mm away) to measure the returning signals. A differential path length factor of 4 was used (Duncan et al., 1995). The intensity of incident and transmitted light is transmitted in real time via Bluetooth to a computer and recorded continuously at 10 Hz and used to estimate concentration changes from the resting baseline for deoxygenated hemoglobin (HHb) and oxygenated hemoglobin (HbO2). Therefore, the NIRS data represent a relative change based on the optical density from the initial measure.

Skinfold thickness at the site of application of the NIRS sensor was measured using Harpenden skinfold calipers (British Indicators Ltd., St. Albans, Herts, United Kingdom). The skin was cleaned and shaved. NIRS was placed and secured with tape along the medial aspect of the calf (medial gastrocnemius muscle), which was tight enough to prevent probe movement but not to restrict leg blood flow or venous return. Thereafter, NIRS was wrapped in a black, light-absorbing cloth to minimize the possibility that extraneous light could influence the signal. A permanent marker was used to mark the sensor placement for accurate repositioning during total study period. The NIRS measurements were collected continuously at rest, during arterial occlusion (before exercise testing), and for the entire duration of the walking test. During NIRS measurements at rest, patients were asked to remain silent and motionless and to avoid muscle contractions.

The tissue saturation index (TSI) is an absolute measure of HbO2, which can specify the extent to which blood is oxygenated. Normal physiological TSI (%) readings within skeletal muscle would be in the region of ∼60% to 80%. Tissue saturation index was calculated as previously described in the literature (Jones et al., 2014). Additionally, reoxygenation rate (ΔO2Hb) was determined. Reoxygenation rate reflects the velocity at which the recovery (i.e., the resaturation of HHb/myoglobin) starts off after release of ischemia and/or exercise and is, therefore, directly related to microvascular function (Van Beekvelt et al., 2001). The reoxygenation rate (in μM O2Hb/s) was calculated as the rate of increase in O2Hb during the initial 3 s after cessation of occlusion and exercise.

Estimates of muscle oxygen consumption (mVO2) were calculated as the rate of change in the Hb difference signal ([HbDif] = [HbO2] − [HHb]) during arterial occlusion, analyzed using simple linear regression as previously described (Ryan et al., 2012). Briefly, the tourniquet was rapidly (0.5 s) inflated to a suprasystolic pressure (250–300 mmHg) to occlude both venous outflow and arterial inflow, completely arresting blood flow, resulting in an increase of [HHb] and simultaneous decrease in [HbO2] as oxygen was released from hemoglobin and consumed by the surrounding muscle tissue (Van Beekvelt et al., 2001). During walking test, estimates of mVO2 were calculated as the rate of change in the Hb difference during the first stage. After correcting for changes in blood volume (Ryan et al., 2012), the slope of the [HbDif] signal for both arterial occlusions was averaged and converted into milliliters of O2 per minute per 100 g of tissue (mV O2 [ml O2´min´100 g] = abs [HbDif/2 × 60]/p10 × 1.04] × 4 × 22.4/1,000), assuming 22.4 L for the volume of gas (temperature, pressure, dry) and 1.04 kg for muscle density (Van Beekvelt et al., 2001).

Vascular Function

Blood pressure and heart rate

After a 10 min supine rest period, blood pressure and heart rate were taken four times, right sided and in supine position, using an automated cuff (Omron Healthcare Inc., Lake Forest, IL), with the last three measurements averaged to obtain mean systolic blood pressure, mean diastolic blood pressure, and mean arterial blood pressure. Blood pressure and heart rate were measured before breakfast, 150 min after breakfast consumption, and 10 min after exercise, into recovery.

Arterial stiffness

Pulse wave velocity was measured as a marker for arterial stiffness using a three-lead electrocardiogram and an Echo-Doppler ultrasound machine (Waki Doppler; Atys Medical,  Soucieu-en-Jarrest, France) after 10 min of supine rest before breakfast and 150 min after breakfast. For central PWV, tonometry measures were taken at the right carotid and common femoral artery. For peripheral PWV, measures were taken at the right femoral and posterior tibial artery. Both measures were calculated as: distance (in meters)/pulse wave transit time (in seconds) as previously described (Maessen et al., 2017). At least five cardiac cycles were recorded for the analysis.

Dietary Intervention

Patients were asked to keep a 3-day food and exercise record prior to all test days in order to get an impression of their habitual dietary intake and physical activity. All diaries were checked by a dietitian especially trained for this task using standardized procedures including a standardized weight and portion book. Diaries were analyzed using “Compl-eat™,” a web-based program built by Wageningen University  (Wageningen, The Netherlands). Compl-eat™ is based on the five-step multiple pass method, a validated technique to increase accuracy (Conway et al., 2003). The program includes a wide selection of foods commonly used in a Dutch food pattern and has previously been used to assess dietary nitrate intake, with nitrate contents of food included in the program’s software (Jonvik et al., 2016b). Participants refrained from caffeine and alcohol intake in the 12 and 24 hr preceding a test day, respectively (Breese et al., 2013; Wilkerson et al., 2012). Patients were provided with a standardized diner to be consumed in the evening before test days (∼10 kcal/kg). To prevent any attenuation in the reduction of nitrate in the oral cavity by commensal bacteria, participants refrained from using any antibacterial mouthwash/toothpaste, chewing gum, and tongue scraping throughout the experimental period (Govoni et al., 2008).

On the test days the participants consumed a standardized breakfast, including two slices whole grain bread (70 g) with low-fat butter (5 g) and young aged cheese (30 g), one glass low-fat milk (175 ml), and 70 ml concentrated red BRJ (Beet-it Sport®) or placebo (nitrate depleted Beet-it Sport®; placebo). The NRV breakfast consisted of a green smoothie (200 ml) and two beetroot waffles (200 g). The ingredients of the green smoothie were mango (50 g), arugula (17 g), zucchini (73 g), orange juice (70 ml), and rhubarb compote (40 g), and the ingredients of the beetroot waffles were an egg, whole meal flour (100 g), low-fat milk (60 ml), and beetroots (100 g). The vegetables were bought fresh from one producer. Energy intake (∼420 kcal) and the amount of protein (∼29 g), fat (∼15 g), and carbohydrate (∼43 g) were kept similar in all meals and provided ∼400 mg (6.5 mmol) of dietary nitrate. Arugula, rhubarb, beetroot, and zucchini were chosen as they are the vegetables most suitable for this study, based on the nitrate content. Besides, in our previous study (van der Avoort et al., 2020) and a pilot study in patients with PAD, we found that these vegetables are favorable and relatively “easy” to incorporate into a breakfast recipe. We expected this amount of dietary nitrate to be feasible, since this clinical population generally has low vegetable intake (Gardner et al., 2011). Based on previous analyses performed in our laboratory across the season, we calculated the expected amount of raw vegetables needed per participant to ensure a dose of 400 mg nitrate. Nitrate content of vegetables is influenced by environmental factors (humidity, temperature, water content, and exposure to sunlight) as well as food preparation (washing, peeling, and cooking; Du et al., 2007). In order to control for this as much as possible, cooking procedures were kept similar during all test days and the amount of raw vegetables was adjusted to the season (i.e., during summer/fall (July–September) a larger amount of raw vegetables was prepared).

Statistical Analysis

A power calculation was performed with difference in the increase in exercise tolerance (PWT in seconds) as the primary outcome measure. The sample size (N) was calculated with a power of 80% (1 − β = 0.80) and a significance level of 5% (α = .05). Based on data from a previous acute BRJ supplementation study (Kenjale et al., 2011), we expected the SD of the intervention difference (BRJ vs. placebo) to be approximately 65 s (13%). The final number of participants to be included after screening was 18.

The data were checked for normality using the Shapiro–Wilk test and analyzed using a two-factor (Time × Intervention) repeated-measures analysis of variance. In cases of significant interactions, paired-sample t tests were performed to detect differences between before and after intervention data for each intervention separately. If a significant main effect of intervention was observed, Bonferroni corrected post hoc tests were used to locate the differences. Data were analyzed with SPSS (version 25.0; IBM Corp., Armonk, NY) and presented as mean ± SD. Statistical significance was set at p < .05.

Results

General characteristics of all participants are reported in Table 1. Body weight and habitual level of physical activity did not change throughout the experimental period (3–4 weeks; data not shown). Consumption of the NRV, BRJ, and placebo was well tolerated by all participants and no adverse events were reported. Reported dietary intake before all test days is presented in Supplemental Table S1 (available online).

Table 1

Characteristics of All Participants (N = 18) With PAD

Characteristics
Male/female11/7
Age (years)73 ± 8
Body mass index (kg/m2)25 ± 3
Calf skinfold (mm)12.2 ± 0.7
ABI0.73 ± 0.10
Systolic blood pressure (mmHg)159 ± 16
Diastolic blood pressure (mmHg)70 ± 13
Mean arterial pressure (mmHg)100 ± 12
Smoking status
 Current smoker5
 Ex-smoker12
 Nonsmoker1
Type 2 diabetes mellitus4
Pharmacology (n)
 PPI6
 Antihypertensive18
 Cholesterol lowering drugs16
 Antidiabetic drugs4
 Antithrombotic drugs17

Note. PAD = peripheral arterial disease; ABI = ankle brachial index; PPI = proton pump inhibitors.

Plasma Nitrate and Nitrite Concentrations

Fasting plasma nitrate concentrations did not differ between test days (NRV: 49 ± 25 μmol/L, BRJ: 47 ± 20 μmol/L, and placebo: 42 ± 16 μmol/L; p = .529). A Time × Intervention interaction (p <.001) was observed, with separate analyses showing an increase in plasma nitrate at 2-hr time point for NRV (202 ±89 μmol/L; p < .001), BRJ (494 ± 110 μmol/L; p < .001), and placebo intervention (80 ± 19 μmol/L; p < .001). The increase with BRJ supplementation was found to be highest (p < .001). After exercise, plasma nitrate concentrations did not change compared with the 2-hr time point after breakfast (Figure 3a). Likewise, fasting plasma nitrite concentrations did not differ between testing days (NRV: 172 ± 79 nmol/L, BRJ: 192 ± 63 nmol/L, placebo: 197 ± 120 nmol/L; p = .771). A Time × Intervention interaction (p < .001) was observed, with separate analyses showing an increase at 2-hr time point following NRV (430 ± 472 nmol/L; p = .002), BRJ (1,353 ± 1,316 nmol/L; p = .001), and placebo (560 ± 733 nmol/L; p = .008). The increase with BRJ supplementation was found to be highest (p = .001). After exercise, plasma nitrite concentrations did not change compared with the 2-hr time point after breakfast (Figure 3b).

Figure 3
Figure 3

—Mean plasma nitrate (a) and nitrite (b) concentrations prior to intervention (before) and following consumption of NRV (○), BRJ (▪), or placebo (♦). The 150 min indicates the time point 150 min following consumption, which was at the onset of the exercise test. Recovery was 10 min after maximal walking exercise. Values are group mean ± SD. NO3 = nitrate; NO2 = nitrite; NRV = nitrate-rich vegetable; BRJ = beetroot juice. *Significantly different from before, p < .001. #Significantly different from placebo, p < .001.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 31, 5; 10.1123/ijsnem.2021-0054

Exercise Tolerance

The group mean and individual COT (Figure 4a) and PWT (Figure 4b) responses following NRV, BRJ, and placebo are shown in Figure 4. Mean COT and PWT did not differ between NRV consumption (413 ± 187 s and 745 ± 220 s, respectively), BRJ supplementation (392 ± 154 s and 746 ± 176 s), and placebo (403 ± 176 s and 696 ± 222 s; p = .762 and p = .165, respectively).

Figure 4
Figure 4

—Mean (a) COT (in seconds) and (b) PWT (in seconds) during the Gardner walking exercise protocol, following NRV, BRJ, and placebo. Values are group mean ± SD and individual scores. NRV = nitrate-rich vegetable; BRJ = beetroot juice; COT = claudication onset time; PWT = peak walking time.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 31, 5; 10.1123/ijsnem.2021-0054

Muscle Oxygenation

Near-infrared spectroscopy–derived parameters during ischemia and a maximal graded exercise test following NRV, BRJ supplementation, and placebo are shown in Table 2. HHb, HbO2, and Hbtot values were measured during the exercise test at baseline, Stage 1 (100 s), Stage 2 (220 s), and at amplitudes of COT and PWT. The average data of the total group are shown up to 220 s, as this is the point at which some patients reached PWT and had to stop. Overall for HHb and Hbtot, significant Time × Intervention interactions were observed (both; p < .001). Analyses for every stage separately showed a tendency toward significantly higher Hbtot values at Stages 1 and 2 after NRV consumption, compared with BRJ supplementation (p = .071 and p = .084, respectively). Average HbO2 changed during exercise (time; p < .001), but this effect was not significantly different between interventions.

Table 2

Near-Infrared Spectroscopy–Derived Dynamics During Ischemia and a Maximal Graded Exercise Test Following the Consumption of NRV, BRJ, or Placebo in 18 Patients With PAD

p value
Muscle oxygenationNRVBRJPlaceboTimeTime × Intervention
HHb
 Baseline (AU)0.29 ± 1.000.08 ± 0.420.02 ± 0.23<.001<.001
 Stage 1 (AU)10.55 ± 5.847.71 ± 5.008.98 ± 6.80
 Stage 2 (AU)7.55 ± 14.4611.46 ± 7.3012.89 ± 8.49
 Amplitudes at COT (AU)16.95 ± 9.2814.20 ± 8.5415.57 ± 7.70
 Amplitudes at PWT (AU)19.05 ± 8.7716.53 ± 7.1017.67 ± 9.21
HbO2
 Baseline (AU)0.61 ± 1.350.12 ± 0.510.12 ± 0.32<.001.774
 Stage 1 (AU)−9.41 ± 7.02−10.16 ± 7.12−10.63 ± 8.80
 Stage 2 (AU)−10.63 ± 8.06−11.89 ± 9.09−11.67 ± 9.48
 Amplitudes at COT (AU)−9.76 ± 8.74−11.46 ± 8.99−11.70 ± 9.14
 Amplitudes at PWT (AU)−7.41 ± 9.01−9.86 ± 9.29−9.34 ± 8.09
Hbtot
 Baseline (AU)0.90 ± 2.320.20 ± 0.640.14 ± 0.49<.001<.001
 Stage 1 (AU)1.13 ± 6.88a−2.45 ± 5.12−1.12 ± 4.41
 Stage 2 (AU)3.82 ± 7.87a−0.42 ± 6.401.21 ± 4.47
 Amplitudes at COT (AU)7.20 ± 9.012.73 ± 5.423.87 ± 3.68
 Amplitudes at PWT (AU)11.64 ± 9.206.66 ± 5.848.31 ± 5.14
p value
TSI in rest (%)60 ± 360 ± 360 ± 3.957
Reoxygenation rate after ischemia 
 μM O2Hb/s0.54 ± 0.350.68 ± 0.330.57 ± 0.26.171
 Percentage28 ± 2225 ± 1630 ± 29.898
Reoxygenation rate after exercise 
 μM O2Hb/s1.80 ± 0.59b1.51 ± 0.431.16 ± 0.47<.001
 Percentage40 ± 5521 ± 1719 ± 22.164
mVO2 during ischemia (ml O2·min−1·100 g−1)0.73 ± 0.570.78 ± 0.680.70 ± 0.57.895
mVO2 during exercise (ml O2·min−1·100 g−1)−0.77 ± 1.66−1.12 ± 1.38−1.03 ± 1.42.643

Note. Values are mean ± SD. The p values for differences between the three groups were derived by repeated-measures analysis of variance. Bold values indicate p value <.05. HHb = deoxygenated hemoglobin concentration; HbO2 = oxyhemoglobin concentration; Hbtot = total hemoglobin concentration; AU = arbitrary units; mVO2 = muscle oxygen consumption; COT = claudication onset time (in seconds); TSI = tissue saturation index; PAD = peripheral arterial disease; PWT = peak walking time (in seconds); BRJ = beetroot juice; NRV = nitrate-rich vegetable.

aTendency toward significantly difference from BRJ, p = .071 (Stage 1) and p = .084 (Stage 2). bSignificantly different from placebo, p < .05.

The average TSI at rest (i.e., before exercise) was identical after all interventions (all, 60 ± 3%; p = .957). Individual TSI ranged from 55% to 64%. The reoxygenation rate (ΔO2Hb) after occlusion did not significantly differ between interventions (p = .171). After exercise, there was an overall significant effect of intervention (p < .001). Post hoc analyses revealed that reoxygenation rate after exercise was significantly higher after consuming the NRV compared with the placebo breakfast (1.8 ± 0.6 vs. 1.2 ± 0.5 AU; p < .001). Muscle oxygen consumption (mVO2) during ischemia and during exercise did not differ between interventions (p = .857 and p = .643, respectively).

Vascular Function

Blood pressure

The mean systolic and diastolic blood pressure, mean arterial pressure, and heart rate responses for all interventions throughout the visits are shown in Table 3. Systolic blood pressure, diastolic blood pressure, mean arterial blood pressure, and HR did not change during test days and no differences were observed between interventions.

Table 3

Measurements of Vascular Function (Blood Pressure and PWV) in a Fasted State (Before); 150 Min Following the Consumption of NRV, BRJ, or Placebo Intervention; and After Exercise (Recovery) in 18 Patients With PAD

p values
Blood pressureNRVBRJPlaceboTimeTime × Intervention
Systolic blood pressure (mmHg)
 Before148 ± 16148 ± 14145 ± 15.131.192
 150 min141 ± 10138 ± 17136 ± 10
 Recovery162 ± 16154 ± 18161 ± 19
Diastolic blood pressure (mmHg)
 Before69 ± 969 ± 969 ± 8.333.544
 150 min65 ± 1062 ± 1062 ± 9
 Recovery71 ± 1267 ± 1472 ± 24
Mean arterial pressure (mmHg)
 Before95 ± 995 ± 794 ± 9.213.945
 150 min90 ± 887 ± 1086 ± 8
 Recovery101 ± 1099 ± 1599 ± 11
Heart rate
 Before61 ± 1360 ± 1162 ± 12.363.586
 150 min60 ± 1060 ± 1060 ± 11
 Recovery71 ± 1272 ± 1374 ± 15
PWV
 Central PWV (m/s)
  Before7.8 ± 3.09.4 ± 4.210.0 ± 5.3.195.207
  150 min10.0 ± 4.18.3 ± 3.210.2 ± 2.8
 Peripheral PWV (m/s)
  Before10.0 ± 2.610.9 ± 3.911.3 ± 3.0.279.575
  150 min11.0 ± 2.510.0 ± 3.911.2 ± 3.6

Note. Values are mean ± SD. The p values for differences between the three groups were derived by repeated-measures analysis of variance. PWV = pulse wave velocity; PAD = peripheral arterial disease; NRV = nitrate-rich vegetable; BRJ = beetroot juice.

Arterial stiffness

The mean central and peripheral PWV (in meters per second) at baseline and after the interventions are shown in Table 3. Both central PWV and peripheral PWV did not change (time effects; p = .195 and p = .279, respectively) and no differences were observed between interventions (Time × Intervention effects; p = .207 and p = .570, respectively).

Discussion

In this randomized controlled crossover study, the consumption of ∼6.5 mmol dietary nitrate in the form of whole NRV and BRJ significantly increased plasma nitrate and nitrite concentrations in patients with PAD, although to a greater extent with BRJ supplementation compared with NRV consumption. Despite the greater circulating plasma nitrate and nitrite concentrations, both dietary strategies did not acutely affect COT, maximal walking time, gastrocnemius muscle oxygenation, blood pressure, or arterial stiffness. Accordingly, our results did not support the hypothesis that a single bolus of dietary nitrate intake improves exercise tolerance in patients with PAD, which may call into question its potential therapeutic benefit within this population.

Once dietary nitrate is ingested, nitrate is rapidly absorbed through the gastrointestinal tract (i.e., small intestine) and can be reduced to nitrite by oral commensal bacteria under the tongue or in the stomach, and further reduced to NO through nonenzymatic synthesis (Lundberg et al., 2004). The current study is the first to investigate whether the intake of NRV improves exercise tolerance and elicits cardiovascular effects in a patient group. We found on average a approximately fourfold (NRV) and approximately 10-fold (BRJ) increase in circulating plasma nitrate (Figure 3a). Plasma nitrite concentrations increased approximately fourfold after NRV consumption and approximately eightfold after BRJ supplementation (Figure 3b), indicating NO bioavailability was likely enhanced. These increases are similar in magnitude to other well-controlled studies investigating BRJ supplementation in patients (Eggebeen et al., 2016; Kenjale et al., 2011) and to those reported in previous studies in healthy populations where consumption of green leafy vegetables was pursued (Ashworth et al., 2015; Bondonno et al., 2014). The increases in plasma nitrate and nitrite concentrations were significantly greater with the BRJ compared with the NRV intervention. This may be explained by a difference in the time to reach peak plasma values between BRJ (∼120 min) and NRV (∼180 min), as found in previous studies directly comparing both intervention strategies (Jonvik et al., 2016b; van der Avoort et al., 2020). The potentially longer time needed to reach peak plasma nitrate concentrations following ingestion of vegetables can be related to the higher fiber content and the larger volume of food ingested, likely resulting in a slower gastric emptying and digestion (Schneeman, 2002). The total amount of nitrate, provided through NRV, was determined on the basis of previous analyses performed in our laboratory and was not measured regularly throughout the study period. Although cooking procedures were kept similar during all test days and the amount of raw vegetables was adjusted to the season (i.e., during summer/fall [July–September] a larger amount of raw vegetables was prepared), variation in dietary nitrate content could also explain differences in plasma nitrate and nitrite responses.

Surprisingly, we observed an increase in plasma nitrate and nitrite concentrations (approximately twofold and fourfold, respectively) following the placebo intervention (i.e., breakfast with nitrate-depleted BRJ). The “standard” breakfast (i.e., placebo intervention) included dairy products (cheese and milk), which could contain a small amount of L-arginine. L-arginine is an amino acid and is a contributor to plasma nitrite production through the L-arginine/nitric oxide synthase [NOS]/NO/nitrite pathway (Madigan & Zuckerbraun, 2013). Although this might have influenced current plasma nitrite concentrations, L-arginine has not been convincingly shown to increase NO synthesis or to alter physiological responses to exercise in previous studies (Alvares et al., 2011). The elevated concentration in individuals with PAD in the placebo condition may be associated with habitual upregulation of inducible NOS, which is endemic in this population, due to inflammation or oxidative stress (Smith et al., 2011; Pautz, 2010, p. 518). The elevated plasma concentrations may also be explained by the use of antihypertensive and cholesterol-lowering medication during test day, upregulating endothelial NOS activity (Andrade et al., 2013). In any case, the elevated plasma nitrate and nitrite concentration in the placebo condition is likely related to cardiovascular disease pathology (Ramesh et al., 1999) and could have mitigated the attainment of the benefits of higher intakes of dietary nitrate that have been reported in other populations.

In patients with PAD, walking is hampered by insufficient arterial blood flow to the lower extremities, resulting in ischemia-induced claudication pain (Hamburg & Creager, 2017). Since upregulation of the nitrate–nitrite–NO pathway has been previously shown to facilitate (hypoxic) vasodilation in healthy participants (Horiuchi et al., 2017), we expected an improvement in walking capacity following nitrate intake in patients with PAD. However, exercise capacity, reflected by the onset of claudication pain and maximal walking time, was not improved (Figure 4). Others have found no improvement in exercise capacity after nitrate supplementation in patients with heart failure (Hirai et al., 2016), chronic obstructive pulmonary disease (Beijers et al., 2018), or Type 2 diabetes mellitus (Shepherd et al., 2015). These and our results contrast with other clinical studies showing an improvement in exercise performance in heart failure (Eggebeen et al., 2016; Zamani et al., 2015) and in patients with chronic obstructive pulmonary disease (Berry et al., 2015). To date, only one study investigated the acute effects of BRJ supplementation (∼9 mmol) in a small group of patients with PAD, finding an increase in both COT and PWT during a maximal treadmill test (Kenjale et al., 2011). Although the exact reasons for these discrepancies with our findings remain unclear, it is likely that the various applications of supplementation regimes underlie the differences in exercise tolerance-enhancing effects. It may be that chronic supplementation protocols and/or higher doses of BRJ are required to increase muscle contractile function (i.e., muscle speed and power) in older diseased individuals (Gallardo et al., 2020). Thus, while a single meal containing ∼6.5 mmol of dietary nitrate is not effective to enhance exercise tolerance in patients with PAD, it can be suggested that a chronic “dosing” strategy provides a greater likelihood of achieving ergogenic benefits. Still, we believe an increase in the habitual intake of NRV may represent a “healthier” and more sustainable alternative to nitrate supplementation. Such an approach would have the additional benefit of providing a high diversity of vitamins, trace minerals, dietary fibers, and many more (yet undefined) compounds. However, more work is needed to assess the effectiveness of long-term, sustained compliance to a diet with such high amounts of NRV in clinical populations. From a previous study, it is suggested that cigarette smokers may not derive the same vascular benefits, compared with nonsmoking controls, if they increase dietary nitrate intake (Bailey et al., 2016). Given that a number of the participants in current study were smokers (5/18), and even more were ex-smokers (11/18), is it possible that this influenced our results. However, it was not possible to directly compare our results with other studies in clinical populations, since smoking status was not consistently reported. Since all patients served as their own control, smoking status did not have a substantial impact on the primary aim of current study (i.e., comparison of BRJ supplementation vs. NRV). Nevertheless, we believe that future research should consider the effects of cigarette smoking on dietary nitrate metabolism more carefully. Another possible reason for discrepancies with our findings could be differences in disease severity. Kenjale et al. included patients with more severe PAD symptoms (i.e., claudication pain; ABI: 0.64 ± 0.20) compared with our patient group (ABI: 0.73 ± 0.10), and may therefore have presented lower COT (183 ± 84 s) and PWT (467 ± 223 s) after nitrate supplementation (Kenjale et al., 2011). Our group of patients with PAD showed improvements, albeit not significant ones, in PWT after both nitrate interventions (∼50 s). This could indicate that exercise tolerance effects are more pronounced in patients with severe PAD symptoms. Nitric oxide is also known as an important mediator in blood pressure regulation (Woessner et al., 2017). In the present study, we found no effect of acute dietary nitrate consumption on blood pressure and arterial stiffness. The findings of a recent review indicated mixed results for the effects of acute and short-term dietary nitrate supplementation on blood pressure in older adults (Stanaway et al., 2017). Our results may be influenced by current use of antihypertensive medications, which could affect NO metabolism (Gilchrist et al., 2013), suggesting that an additional reduction in blood pressure might not occur in a group of patients whose blood pressure is already controlled. In the current study, all patients were on antihypertensive medication and 16 patients were taking low-dose statins as well, which may improve endothelial NOS expression and improve baseline NO bioavailability (Omar et al., 2016). Another possible explanation for the lack of changes in blood pressure might be the vascular aging process in which the capacity to convert nitrate to NO is possibly reduced and the sensitivity of vascular smooth muscle cells to the vasodilatory and endothelial effects of NO might be diminished (Siervo et al., 2015). This might also be the case in patients with PAD and it would therefore be worthwhile for future studies to investigate if higher or more prolonged intake of nitrate is required to stimulate vasodilatation and induce structural changes of the arterial wall (Kim et al., 2019).

In line with the lack of significant performance and blood pressure regulation effects, there were no changes in muscle oxygenation between interventions, as measured with NIRS (i.e., HHb; HbO2; Table 2). The change in HHb is considered to appropriately reflect the balance between O2 delivery and extraction in the microvascular bed of skeletal muscle (Ryan et al., 2012). The absence of difference in HHb between the interventions indicates that local muscle oxygenation was not increased with dietary nitrate consumption during walking exercise. The HHbtot data are the sum of both HHb and HbO2 data and have previously been used to evaluate changes in microvascular blood volume (Bailey et al., 2009). Our results indicate a trend toward a higher increase in blood volume to the gastrocnemius muscle, which may indicate local vasodilation and higher perfusion following NRV compared with BRJ and placebo. In addition, NRV consumption resulted in a higher reoxygenation rate directly after exercise. These advantages in local blood flow did not translate into enhanced functional capacity in patients with PAD, suggesting that coexisting pathologies may contribute to impairment in exercise tolerance (Szuba et al., 2006).

In conclusion, consumption of an NRV meal or a single bolus of concentrated BRJ (both ∼6.5 mmol dietary nitrate) substantially increased plasma nitrate and nitrite concentrations in patients with PAD. The greater plasma nitrate and nitrite levels did not translate into improvement in exercise tolerance in these patients. In addition, muscle oxygenation and cardiovascular function were not affected. More research is warranted to evaluate the potential clinical benefits of more prolonged dietary nitrate intake on functional capacity and vascular function in these patients.

Acknowledgments

The authors thank all the participants of the study. This trial was registered at www.trialregister.nl as NL7110 (NTR7315), 2018-06-19. The authors’ responsibilities were as follows: C.M.T. van der Avoort, L.J.C. van Loon, L.B. Verdijk, P.P.C. Poyck, and M.T.E. Hopman conceived and designed the study. C.M.T. van der Avoort performed the data collection and the statistical analysis. C.M.T. van der Avoort, L.J.C. van Loon, L.B. Verdijk, D.T.J. Thijssen, and M.T.E. Hopman interpreted the data. C.M.T. van der Avoort drafted the manuscript, with contribution of L.J.C. van Loon, L.B. Verdijk, D.T.J. Thijssen, and M.T.E. Hopman. C.M.T. Van der Avoort had primary responsibility for final content. All authors critically read the draft and approved the final version of the manuscript submitted for publication. None of the authors declared a conflict of interest. The results of the present study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation, and do not constitute endorsement by the American College of Sports Medicine. This study was part of the EAT2MOVE project and supported by a grant from the Province of Gelderland.

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van der Avoort, van Loon, and Verdijk are with the NUTRIM, the School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre, Maastricht, The Netherlands. van der Avoort and van Loon are also with the Institute of Sport and Exercise Studies, HAN University of Applied Sciences, Nijmegen, Gelderland, The Netherlands. van der Avoort, Thijssen, and Hopman are also with the Department of Physiology, Institute for Health Sciences, Radboud University Medical Center, Nijmegen, Gelderland, The Netherlands. Poyck is with the Department of Vascular and Transplant Surgery, Radboudumc, Nijmegen, Gelderland, The Netherlands. Thijssen is also with the Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United Kingdom.

van der Avoort (cindy.vanderavoort@han.nl) is corresponding author.

Supplementary Materials

  • View in gallery

    —Flow diagram illustrating the movement of participants through the crossover study, investigating the effects of high nitrate intakes in patients with PAD, which was conducted between July 2018 and December 2018. PAD = peripheral arterial disease.

  • View in gallery

    —Schematic representation of the study design. In a randomized crossover design, 18 patients with PAD followed a nitrate intake protocol (∼6.5 mmol) through the consumption of NRV, via BRJ, and nitrate-depleted BRJ (placebo). Blood samples were taken; blood pressure and arterial stiffness were measured in fasted state and 150 min after dietary intervention. Each intervention was followed by a maximal walking exercise test to determine COT and PWT. Gastrocnemius oxygenation during exercise was measured by NIRS. In addition, blood samples were taken and blood pressure was measured 10 min after exercise. BRJ = beetroot juice; NRV = nitrate-rich vegetable; PAD = peripheral arterial disease; COT = claudication onset time; PWT = peak walking time; NIRS = near-infrared spectroscopy; PWV = pulse wave velocity.

  • View in gallery

    —Mean plasma nitrate (a) and nitrite (b) concentrations prior to intervention (before) and following consumption of NRV (○), BRJ (▪), or placebo (♦). The 150 min indicates the time point 150 min following consumption, which was at the onset of the exercise test. Recovery was 10 min after maximal walking exercise. Values are group mean ± SD. NO3 = nitrate; NO2 = nitrite; NRV = nitrate-rich vegetable; BRJ = beetroot juice. *Significantly different from before, p < .001. #Significantly different from placebo, p < .001.

  • View in gallery

    —Mean (a) COT (in seconds) and (b) PWT (in seconds) during the Gardner walking exercise protocol, following NRV, BRJ, and placebo. Values are group mean ± SD and individual scores. NRV = nitrate-rich vegetable; BRJ = beetroot juice; COT = claudication onset time; PWT = peak walking time.

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