Inorganic nitrate supplementation and blood flow restricted exercise tolerance in post-menopausal women

Abstract

Exercise tolerance appears to benefit most from dietary nitrate $\left({\text{NO}}_{{\text{3}}^{-}}\right)$ supplementation when muscle oxygen (O₂) availability is low. Using a double-blind, randomized cross-over design, we tested the hypothesis that acute ${\text{NO}}_{{\text{3}}^{-}}$ supplementation would improve blood flow restricted exercise duration in post-menopausal women, a population with reduced endogenous nitric oxide bioavailability. Thirteen women (57–76 yr) performed rhythmic isometric handgrip contractions (10% MVC, 30 per min) during progressive forearm blood flow restriction (upper arm cuff gradually inflated 20 mmHg each min) on three study visits, with 7 to 10 days between visits. Approximately one week following the first (familiarization) visit, participants consumed 140 ml of ${\text{NO}}_{{\text{3}}^{-}}$ concentrated (9.7 mmol, 0.6 gm ${\text{NO}}_{{\text{3}}^{-}}$) or ${\text{NO}}_{{\text{3}}^{-}}$ depleted beetroot juice (placebo) on separate days (≥ 7 days apart), with handgrip exercise beginning 100 min post-consumption. Handgrip force recordings were analyzed to determine if ${\text{NO}}_{{\text{3}}^{-}}$ supplementation enhanced force development as blood flow restriction progressed. Nitrate supplementation increased plasma ${\text{NO}}_{{\text{3}}^{-}}$ (16.2-fold) and ${\text{NO}}_{{\text{2}}^{-}}$ (4.2-fold) and time to volitional fatigue (61.8±56.5 sec longer duration vs. placebo visit; p=0.003). Nitrate supplementation increased the rate of force development as forearm muscle ischemia progressed (p= 0.023 between 50 and 75% of time to fatigue) with non-significant effects thereafter (p=0.052). No effects of nitrate supplementation were observed for mean duration of contraction or relaxation rates (all p>0.150). These results suggest that acute ${\text{NO}}_{{\text{3}}^{-}}$ supplementation prolongs time-to-fatigue and speeds grip force development during progressive forearm muscle ischemia in postmenopausal women.

1. Introduction

During the past decade, there has been a dramatic increase in the number of studies examining the exercise performance and physiological effects of inorganic nitrate supplementation (Coggan & Peterson, 2018; McMahon et al., 2017; Woessner et al., 2018). Among less fit and recreationally active individuals, the exercise performance enhancing effects of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation have been reported across a wide range of exercise modalities, durations, and intensities (Campos et al., 2018; Hlinský et al., 2020; Senefeld et al., 2020). Less consistent ergogenic effects have been observed in highly trained individuals, which may reflect their higher baseline plasma nitrate $({\text{NO}}_{{\text{3}}^{-}})$ and nitrite $({\text{NO}}_{{\text{2}}^{-}})$ levels and/or a limit in the ability of nitrate supplementation to further enhance their cardiovascular or skeletal muscle functioning during exercise (Carriker et al., 2016; James et al., 2015; Jones et al., 2021; Peacock et al., 2012). The physiological mechanisms underlying the ergogenic effects of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation are diverse, but are thought to result from the increased production of nitric oxide (NO) via the ${\text{NO}}_{{\text{3}}^{-}}\to {\text{NO}}_{{\text{2}}^{-}}\to \mathrm{NO}$ pathway. This complementary pathway of NO generation involves the reduction of nitrate to nitrite $\left({\text{NO}}_{{\text{3}}^{-}}\to {\text{NO}}_{{\text{2}}^{-}}\right)$ by commensal bacteria within the oral cavity, followed by the conversion of ${\text{NO}}_{{\text{2}}^{-}}$ to NO within various tissues (Govoni et al., 2008; Jones et al., 2021; Larsen et al., 2007).

Importantly, the latter step in this pathway $\left({\text{NO}}_{{\text{2}}^{-}}\to \text{NO}\right)$ is greatly enhanced during conditions of lowered PO₂ and pH, driven by the elevated enzymatic activity of various nitrite reductases (deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, etc) (Cosby et al., 2003; Ghosh et al., 2013; Lundberg et al., 2009; van Faassen et al., 2009). This phenomenon is thought to explain why the exercise-enhancing effects of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation are most consistently observed during ischemic, low pH conditions and intense exercise to exhaustion i.e., when contracting muscle oxygen availability is significantly reduced or limited (Affourtit et al., 2015; Coggan & Peterson, 2018; Jones et al., 2021; Kelly et al., 2014). This pathway provides a complementary source of bioavailable NO under conditions when the classic L-Arginine/eNOS (oxygen-dependent) pathway of NO generation is limited and/or dysfunctional.

Older populations and patients with elevated cardiovascular disease (CVD) risk stand to benefit the most from ${\text{NO}}_{{\text{3}}^{-}}$ supplementation due to impairments in vascular NO production and bioavailability, deficits in muscle blood flow and oxygen supply, and exercise intolerance (Woessner et al., 2018). Indeed, there are several reports demonstrating clinically significant improvements in exercise tolerance following acute ${\text{NO}}_{{\text{3}}^{-}}$ supplementation in patients with pulmonary (Berry et al., 2015) (Kerley et al., 2015) (Kerley et al., 2019), cardiac (Coggan, Broadstreet, Mahmood, et al., 2018; Eggebeen et al., 2016; Zamani et al., 2015) and peripheral vascular (Bock et al., 2018; Kenjale et al., 2011) diseases. However, not all studies have found a beneficial effect of ${\text{NO}}_{{\text{3}}^{-}}$ on exercise tolerance in older patient populations (Ferguson et al., 2021; Hirai et al., 2017; Shepherd et al., 2015).

Postmenopausal women have reduced NO bioavailability (Bondonno et al., 2012; Taddei et al., 1996) and muscle oxygen delivery during exercise (Moore et al., 2012; Proctor & Parker, 2006), but the ergogenic effects of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation have not been investigated. We recently showed that acute ${\text{NO}}_{{\text{3}}^{-}}$ supplementation (via beetroot juice) increased plasma ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ and reduced resting blood pressures in older postmenopausal women (Kim et al., 2019). In the present report, we describe the effects of acute ${\text{NO}}_{{\text{3}}^{-}}$ supplementation on handgrip exercise tolerance in participants from that same investigation. In an attempt to maximize the conditions for conversion of ${\text{NO}}_{{\text{2}}^{-}}$ to NO, we utilized handgrip exercise combined with graded upper arm cuff occlusion (+20 mmHg per minute) to volitional fatigue. This small muscle exercise model also facilitated the quantification of rates of grip force development and relaxation (via handgrip force-time analyses) as muscle ischemia (and presumably the conversion of ${\text{NO}}_{{\text{2}}^{-}}$ to NO) progressed. We hypothesized that participants would maintain the target handgrip workload longer after consuming ${\text{NO}}_{{\text{3}}^{-}}$-rich juice, relative to placebo juice consumed on a separate visit. Moreover, based on the expected acceleration of fast-twitch muscle recruitment with progressive cuff occlusion (Krustrup et al., 2009) and studies reporting increased rates of fast-twitch muscle force development following ${\text{NO}}_{{\text{3}}^{-}}$ supplementation (Haider & Folland, 2014; Hernández et al., 2012), we hypothesized that rates of handgrip force development would be better maintained as muscle ischemia progressed following supplementation.

2. Materials and Methods

2.1. Participants

Postmenopausal women between 55 and 80 years of age were recruited from the local university community via posted flyers, newspaper ads, electronic media (e.g., e-mail messages, research participant list-serves), and by contacting former research participants who had previously given their consent to be re-contacted. Interested volunteers provided written informed consent followed by screening in the clinical research center (CRC) that included a physician-reviewed medical history, physical activity history, physical exam, height, weight, seated resting blood pressure measurements, and a venous blood draw. Individuals were excluded if they met any of the following criteria: currently participating in >3 days per week of formal exercise training (strength or aerobically-based activities), a history of major metabolic (type I or II diabetes, thyroid disorder), neuromuscular (peripheral neuropathy, etc), or cardiovascular (i.e., stage II or greater hypertension, coronary/myocardial disease, heart failure, valvular disease) disease, a body mass index < 18.5 or >35 kg/m², anemia defined by hemoglobin < 11.5 gm/dL, fasting plasma glucose > 100 mg/dL or HbA1c > 6.0%, fasting plasma LDL > 130 and/or HDL < 40 mg/dL, and impaired liver or kidney function assessed by venous blood chemistry. Volunteers were also excluded from participation if they currently smoked, were currently taking any cardiovascular medications or hormone therapy, or had donated blood or blood products in the past 3 months. Sample size for the overall investigation (Kim et al., 2019) was determined on the basis of expected nitrate supplementation-associated changes in resting aortic blood pressure. In the present analysis, handgrip time-to-fatigue was the primary outcome. This study was approved by the Office of Research Protections at Pennsylvania State University in agreement with the guidelines set forth by the Declaration of Helsinki. Study participant flow (CONSORT) is shown in Figure 1.

Figure 1:

CONSORT diagram

2.2. Study design, randomization, and blinding

This was a double blind, randomized, crossover study registered with ClinicalTrials.Gov (NCT03380000). Visit one consisted of screening procedures (as described above), peak grip strength assessment (best of 3 efforts using their right hand), and completion of handgrip exercise combined with graded upper arm cuff occlusion to volitional fatigue (described below).

Eligible volunteers returned for two beetroot study visits (visits 2 and 3), at least 7 days apart, conducted between 8AM and 1PM. Participants were instructed to avoid teeth brushing and the use of mouthwash on the morning of each visit for the purpose of preserving oral ${\text{NO}}_{{\text{3}}^{-}}$ reducing bacteria (Jones et al., 2021). They were also asked to avoid eating foods high in ${\text{NO}}_{{\text{3}}^{-}}$ (e.g., spinach, lettuce, etc.) one day prior, and to consume only water for at least 8 hours prior to arriving at the CRC.

The beetroot juice was stored in a locked cabinet located in the CRC at a temperature maintained between 22 and 25 degrees Celsius. Treatment order (nitrate-rich or nitrate-depleted beetroot juice) was randomized using an online random number generator (Randomization.com). CRC nurses dispensed the blinded juice to participants on both study visits and the juice/visit order was stored in a locked cabinet in the CRC. The CRC nurses had no role in data collection or analysis.

2.3. Blood sampling and $N{O}_{3^{-}}/N{O}_{2^{-}}$ analysis

Upon arrival at the CRC for visits 2 and 3, a venous blood sample was collected. After resting hemodynamic measures were collected (non-invasive measures of radial artery pulse waves and assessment of aortic pulse wave velocity, ~30 min; (Kim et al., 2019), participants consumed nitrate-rich beetroot juice (~9.7 mmol ${\text{NO}}_{{\text{3}}^{-}}$, BR; 140 mL Beet-It Organic, James White Juice Company) or nitrate-depleted placebo (PL, 140 mL nitrate-depleted Beet-It Organic juice, James White Juice Company). A second blood sample was drawn ~100 min after juice consumption to confirm absorption (rise in plasma ${\text{NO}}_{{\text{3}}^{-}}$ above baseline) and conversion (rise in plasma ${\text{NO}}_{{\text{2}}^{-}}$). Resting hemodynamic measures were then repeated (Kim et al., 2019). Participants were then given the opportunity to leave the room and void before returning for the graded occlusion handgrip exercise test. A third and final blood sample was collected at the end of both beetroot study visits (at least 180 min post consumption).

Venous blood samples were drawn into heparin tubes (4mL lithium heparin tubes, BD Vacutainer, Franklin Lakes, N.J., USA) and immediately centrifuged at 3200 rpm (1590 g) and 4°C for 10 min. After centrifugation, plasma was extracted and stored in −80°C freezer for later analysis of ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ concentration. A ENO-20 analyzer (EICOM, San Diego, Calif., USA) with a sensitivity of 0.1 pmol for ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ was used to measure ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ concentration in the plasma samples. Briefly, plasma was mixed with an equal volume of 100% methanol and centrifuged at 12,000 g for 10 minutes followed by sample loading into a 96-well plate. ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ were then separated via column chromatography and individually reacted with a Griess reagent, synthesizing a diazo compound. The absorbance of this red diazo compound was read at a wavelength of 540 nm using a visible light detector.

2.4. Handgrip exercise testing

Participants performed the graded occlusion handgrip testing in a semi-recumbent posture (30° head up and 20° legs up) on a padded bed with their right arm abducted 90 degrees away from their shoulder and their elbow almost fully extended. The handgrip device (Smedley hand dynamometer, Stoelting, Wood Dale, IL) was secured on a table next to the bed, level with the subject’s mid-axillary line, and adjusted to comfortably fit the participant’s hand/fingers. The subject’s elbow was supported with padding and their wrist was slightly pronated. A 10-centimeter wide pneumatic cuff connected to a Hokanson inflation system (AG101 cuff, E20 Inflator) was placed around the subject’s right upper arm.

Participants began squeezing the isometric handgrip device at a rate of 30 contractions per minute (approximately 1 sec contraction and 1 sec relaxation). The target for each contraction was 10% of the participant’s MVC. Real-time visual feedback was provided via a computer monitor with a target cursor range (9 to 11% of MVC), while the participant maintained 30 contractions per min as guided by a metronome. After 4 minutes of exercise, the upper arm cuff was inflated to 20 mmHg and gradually increased by 20 mmHg each minute thereafter. Time to fatigue was defined as the time from the start of handgrip exercise (including the initial 4 minutes of unoccluded/unrestricted exercise) to the point at which the participant stopped despite continued verbal encouragement by the investigators. Approximately every minute, participants provided a rating of perceived effort (Borg RPE 6 to 20 scale).

2.5. Handgrip force - time analysis

Forces generated during each handgrip contraction cycle were detected with a strain gauge (Stoelting, Wood Dale, IL) and were sampled in real time (400 Hz) using a data acquisition system (PowerLab, AD Instruments; Castle Hill, Australia). The data were then exported as a text file and imported to MATLAB. The force time series data were digitally filtered using a tenth-order, low-pass Butterworth filter with a 15 Hz cut-off frequency. Each trial was visually inspected, and as shown in Figure 2, four time-points were determined: (1) onset of force, (2) beginning of steady-state force, (3) end of steady-state force, and (4) offset of force. Dependent variables were calculated from the four time points via customized algorithms in MATLAB. Peak force was defined as the maximum value between points 2 and 3. Variables related to peak force included half-relaxation time (HRT: time required to reach 50% of peak); time to peak force (TPF: time of point 1 to the time of peak force); and rate of force development (RFD: peak force divided by TPF). The rate of change of force was calculated as the slope of the line between points 1 and 2 (rate of force increase), and the slope of the line between points 3 and 4 (rate of force decrease).

Figure 2.

Depiction of force variables overlaid on raw force data from an exemplar participant. Numbers one through four indicate the four time-points used to characterize each contraction: (1) force onset, (2) onset of steady-state force, (3) offset of steady-state force, and (4) offset of force. Peak force (PF) was defined as the maximum value between points 2 and 3. Time to peak force (TPF) was defined as the time from force onset to maximum force. Half-relaxation time (HRT) was defined as time from peak force until force reaches 50% of the maximum. Rate of force development (RFD) was defined as the rate (i.e., slope) of force increase from force onset to peak force. Rate of force increase was defined as the slope of the line between points 1 and 2, and, similarly, rate of force decrease was defined as the slope of the line between points 3 and 4.

To account for differences in total time to fatigue between conditions (BR vs PL) and between subjects, time was normalized to “relative rounds”: RR1 = 0–25% of total time to fatigue, RR2 = 26–50%, RR3 = 51–75%, and RR4 = 76–100%. In this way, each handgrip force variable could be compared between the BR and PL treatments at similar relative time points during the two study visits.

2.6. Statistical analysis

All results are presented as mean ± S.E.M. Time to volitional fatigue was the primary outcome variable in the present analysis, with handgrip force output variables considered secondary. To evaluate changes from pre- to post-juice consumption, paired (repeated measures) t-tests were conducted for plasma ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ concentration, time to fatigue, total number of contractions, and RPE. Handgrip force output variables were submitted to a mixed-model ANOVA to examine differences between treatment (BR, PL) and across relative time periods (RR1, RR2, RR3, RR4). Statistical tests of force output were conducted using IBM SPSS v.25 and interpreted at α = 0.05.

3. Results

3.1. Subject characteristics:

Descriptive characteristics of the 13 postmenopausal women who completed both beetroot study visits are shown in Table 1. There were no adverse events during this study. One participant withdrew after visit 1 due to a mild skin reaction (Petechiae) resulting from the ischemic handgrip protocol. Reddish-pink urine and stools (beeturia), a common reaction to beetroot juice consumption (Webb et al Hypertension 2008; Vanhatalo et al Amer J Physiol 2010; Hobbs et al Br J Nutr 2012), was anecdotally reported by several participants.

Table 1:

Subject Characteristics. Abbreviations: BMI, body mass index; HDL, high-density lipoproteins; LDL, low-density lipoproteins; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial blood pressure. Data are expressed as mean ± S.E.M and range (min – max).

	Postmenopausal women
Subjects (n)	13
Age (years)	64 ± 1.4 (57 – 76)
Height (cm)	165.5 ± 1.4 (154.9 – 172.7)
Weight (kg)	67.1 ± 3.7 (47.3 – 95.0)
BMI (kg·m2)	24.5 ± 1.3 (17.8 – 33.6)
Hematocrit (%)	41.2 ± 0.7 (38.3 – 46.5)
Total cholesterol (mg/dL)	215 ± 7 (184 – 262)
HDL (mg/dL)	73 ± 5 (49 – 111)
LDL (mg/dL)	123 ± 7 (77 – 158)
Fasting Glucose (mg/dL)	91 ± 2 (81 – 107)
SBP (mmHg)	125 ± 5 (105 – 160)
DBP (mmHg)	74 ± 3 (60 – 96)
MAP (mmHg)	91 ± 3 (76 – 110)

3.2. Plasma $N{O}_{3^{-}}$ and $N{O}_{2^{-}}$

Plasma ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ concentrations did not change following consumption of PL (${\text{NO}}_{{\text{3}}^{-}}$, −19.1 ± 11.8 μM; ${\text{NO}}_{{\text{2}}^{-}}$, 0.024 ± 0.022 μM; p>0.05 at both time points). Plasma ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ increased 16-fold (609.2 ± 40.1 μM; t (11) = 14.78, p<0.0001; Cohen’s d = 6.14) and 4-fold (0.292 ± 0.058 μM; t (11) = 4.417, p=0.0010; Cohen’s d = 1.76) above baseline, respectively, 100 min following consumption of BR (Figure 3) Plasma ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ during the BR visit remained elevated (13-fold and 6-fold, respectively) until the end of the study visit (180+ min; data not shown).

Figure 3:

Comparison of plasma nitrate (Panel A, ${\text{NO}}_{{\text{3}}^{-}}$) and plasma nitrite (panel B, ${\text{NO}}_{{\text{2}}^{-}}$) concentration changes from baseline to ~100 minutes following nitrate-rich (BR) or nitrate-depleted (placebo, PL) juice consumption (n=12). Analysis showed that ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ values did not change after PL consumption, but were significantly increased after BR consumption. Data are displayed as mean ± S.E.M. * indicates significance (p < 0.05). Data missing for 1 participant due to discomfort and difficulty obtaining the 100-minute blood sample.

3.3. Time-to-fatigue and total number of contractions

BR consumption increased ischemic handgrip exercise time-to-fatigue (61.8 ± 56.5 sec longer vs. PL visit; Figure 4A; t (12) = 3.80, p = 0.003; Cohen’s d = 0.35). This ergogenic effect was also observed with respect to the total number of contractions completed (Figure 4B: BR, 293.4 ± 82.1 contractions vs. PL, 262.3 ± 80.3 contractions; t (12) = −3.80, p = 0.003; Cohen’s d = 0.35). A positive association was observed between time-to-fatigue and plasma ${\text{NO}}_{{\text{2}}^{-}}$ at 100 minutes post BR consumption, but this did not reach statistical significance (r = 0.55; p=0.062).

Figure 4:

Time to reach volitional fatigue (panel A) and total number of handgrip contractions completed (panel B) following nitrate-rich (BR) or nitrate-depleted (placebo, PL) juice consumption. Individual responses shown at right. Analysis showed that BR consumption increased both measures of ischemic handgrip tolerance. Data are displayed as mean ± S.E.M. * indicates significant (p < 0.05) difference.

3.4. Handgrip force-time measures

The highest force produced during each contraction was submitted to a mixed-model ANOVA to examine differences between treatment (BR, PL) and across time (RR1, RR2, RR3, RR4). No main effects or interactions were observed (all p>0.105) indicating that participants completed the task as instructed until the point of fatigue.

3.4.1. Measures of handgrip force production:

Rate of force increase, TPF, and RFD were submitted to a mixed-model ANOVA to examine differences between treatment (BR, PL) and across time (RR1, RR2, RR3, RR4). No main effect of treatment was revealed for any of these outcome variables. The results demonstrated a main effect of time for rate of force increase, F(3, 36) = 8.78 N/s, p < .001, ɳ_p²= 0.423, and RFD, F(3, 36) = 4.66 %MVC/s, p = 0.007, ɳ_p² = 0.280. Main effects were interpreted using polynomials to account for the time-series nature of the data. A linear polynomial was the highest-order polynomial significant for rate of force increase, F(1, 12) = 14.85 N/s, p = 0.002, and RFD, F(1, 12) = 6.419 %MVC/s, p = 0.026.

As shown in Figure 5A, an interaction was observed for the rate of force increase, F(3, 36) = 3.17 N/s, p = 0.036, ɳ_p²= 0.209. To decompose this interaction, we first conducted two univariate fully repeated measures ANOVAs for time for BR (F(3, 36) = 8.856 N/s, p < 0.001, ɳ_p²= .425) and PL, (F(3, 36) = 5.10 N/s, p < .005, ɳ_p²= 0.298). The significant effect of time in both BR and PL conditions demonstrated that the rate of force increase became faster over time. Six paired t-tests were then conducted to further evaluate the effect of time within each condition. Specifically, paired t-tests examined RR1 v. RR2, RR2 v. RR3, and RR3 v. RR4, within each condition (BR, PL). This approach was used to determine when the rate of force increase changed within each condition. The results demonstrated that the only significant difference (between RRs) occurred between RR2 and RR3 in the BR condition.

Figure 5:

Measures of handgrip force production, expressed as a function of relative time to fatigue, following nitrate-rich (BR) or nitrate-depleted (placebo, PL) juice consumption. Analysis showed that the rate of force increase (panel A) became faster as a function of time during both treatments, with the only significant difference being observed between 50 and 75% of relative time during the BR treatment. Time to peak force (panel B) decreased as a function of time during BR, but not during the PL treatment. Data are displayed as mean ± S.E.M. * indicates significant (p < 0.05) difference.

Interactions for the other measures of force production (TPF and RFD) approached, but did not reach, statistical significance (TPF, (F(3, 36) = 2.82 s, p = .052, ɳ_p²= .191), and RFD, (F(3, 36) = 2.48 %MVC/s, p = .077, ɳ_p²= .171). These interactions were decomposed in the same way as the rate of force increase. For TPF (Figure 5B) fully repeated measures ANOVAs revealed that TPF decreased as a function of time in BR, (F(3, 36) = 5.16 s, p = .005, ɳ_p²= 0.301), but not PL (F(3, 36) = 0.80 s, p = .501, ɳ_p²= 0.063). Paired t-tests revealed that participants reached peak force faster in RR2 compared to RR1 in the BR condition. A similar trend was observed for RFD (not shown), such that RFD increased over time for BR (F(3, 36) = 5.54 %MVC/s, p = .003, ɳ_p²= .316), but not PL (F(3, 36) = 2.38 %MVC/s, p = .149, ɳ_p²= .165). The increase in RFD mirrors TPF, such that RFD was faster in RR2 compared to RR1 in the BR condition. Taken together, the results for rate of force increase, TPF, and RFD suggest that nitrate supplementation influences the rise (force production) phase of the contraction during graded cuff occlusion.

3.4.2. Measures of handgrip relaxation:

The rate of force decrease, and HRT were submitted to a mixed-model ANOVA to examine differences between treatment (BR, PL) and across time (RR1, RR2, RR3, RR4). As shown in Figure 6, no main effect of treatment, or interaction involving treatment, was detected for rate of force decrease or HRT. The results yielded a main effect of time for rate of force decrease, F(3, 36) = 11.48 %MVC/s, p < 0.001, ɳ_p²= .489, and HRT, F(3, 36) = 4.37 s, p = 0.010, ɳ_p²= 267. Main effects were interpreted using polynomials to account for the time-series nature of the data. A linear polynomial was the highest-order polynomial significant for rate of force decrease, F(1, 12) = 17.08 %MVC/s, p = 0.001, and HRT, F(1, 12) = 9.73 s, p = 0.009.

Figure 6:

Measures of handgrip relaxation, expressed as a function of relative time to fatigue, following nitrate-rich (BR) or nitrate-depleted (placebo, PL) juice consumption. Analysis showed that the rate of force decrease (panel A) and the half-relaxation time (panel B) slowed over time, but that no main effect of treatment, or interaction involving treatment, was detected for either variable. Data are displayed as mean ± S.E.M.

* indicates significant (p < 0.05) difference.

3.5. Rating of perceived effort

RPE at the end of the 4 min (unoccluded) exercise period was similar during the BR (7.8 ± 0.4) and PL (7.5 ± 0.5) visits (p = 0.71). RPE at the time of fatigue was also similar between the BR (17.3 ± 2.8) vs. PL (17.7 ± 1.7) visits (p = 0.53)

4. Discussion

The primary purpose of this study was to determine the effect of acute ${\text{NO}}_{{\text{3}}^{-}}$ supplementation on time to volitional fatigue during blood flow-restricted handgrip exercise in post-menopausal women. In these subjects, consumption of 140 ml of concentrated beetroot juice (~9.7 mmol ${\text{NO}}_{{\text{3}}^{-}}$) elevated plasma ${\text{NO}}_{{\text{2}}^{-}}$ 4- to 6-fold above that seen during the placebo juice visit. Consistent with our primary hypothesis, ${\text{NO}}_{{\text{3}}^{-}}$ supplementation significantly increased ischemic exercise endurance i.e., time to volitional fatigue. An additional novel finding was the observation that nitrate supplementation increased the rate of force development during progressive forearm muscle ischemia in these subjects. These findings appear to be the first to demonstrate an ergogenic effect of inorganic nitrate supplementation in the upper extremity muscles of older adults and during voluntary, blood flow-restricted exercise in humans.

Effect of $N{O}_{3^{-}}$ supplementation on time-to-fatigue

The significant improvement in handgrip time-to-fatigue during the ${\text{NO}}_{{\text{3}}^{-}}$ supplementation visit averaged 62 sec (~12%) longer than during the placebo visit. It is not possible to determine whether this represents a clinically or functionally meaningful change in this cohort of older women considering the low force requirement of our handgrip protocol (~10% of MVC) under the conditions of progressive blood flow restriction. There are also a limited number of published studies that have examined the ergogenic effects of nitrate supplementation in exclusively female participants (n=7 vs. n>100 studies in men; (Senefeld et al., 2020; Wickham & Spriet, 2019). Nonetheless, the 12% average improvement we observed is substantially larger than the average ergogenic effect observed across the ${\text{NO}}_{{\text{3}}^{-}}$ supplementation literature i.e., 3% across n = 80 studies (Senefeld et al., 2020). We observed a non-significant association between time-to-fatigue and plasma ${\text{NO}}_{{\text{2}}^{-}}$ following consumption of the ${\text{NO}}_{{\text{3}}^{-}}$ rich juice (p=0.062), a trend that is consistent with the enhanced utilization of the ${\text{NO}}_{{\text{3}}^{-}}\to {\text{NO}}_{{\text{2}}^{-}}\to \text{NO}$ pathway within ischemic exercising muscles (Jones et al., 2021; Lundberg et al., 2009; van Faassen et al., 2009).

To our knowledge, only two studies have previously assessed dietary ${\text{NO}}_{{\text{3}}^{-}}$ supplementation effects on handgrip exercise performance. Craig et al (Craig et al., 2018) reported faster forearm muscle VO₂ kinetics during high-intensity handgrip exercise (85% of peak power output) following ${\text{NO}}_{{\text{3}}^{-}}$ supplementation in nine young men, but reported no difference in time-to-exhaustion. A study by Bentley et al (Bentley et al., 2017) reported a ${\text{NO}}_{{\text{3}}^{-}}$ supplementation-mediated restoration of submaximal handgrip exercise capacity following an acute reduction in muscle perfusion pressure (resulting from handgrip exercise above heart level) in six young men with a previously established vasodilator limitation (i.e., reduced compensatory vasodilation). While similar to these studies with respect to ${\text{NO}}_{{\text{3}}^{-}}$ source (concentrated beetroot juice) and timing post-consumption (120 to 150 min), our study involved low intensity repetitive isometric handgrip contractions coupled with graded blood flow restriction. Our finding that ${\text{NO}}_{{\text{3}}^{-}}$ supplementation improved handgrip time-to-fatigue in a group of post-menopausal women strongly supports the long held consensus view that dietary ${\text{NO}}_{{\text{3}}^{-}}$ supplementation is most effective during conditions of low tissue oxygen supply in populations at risk of muscle O₂ delivery impairment and/or with a reduced ability to endogenously upregulate NO (James et al., 2015; Woessner et al., 2018).

Despite the robust overall influence of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation we observed, there were variable effects among individuals, with 10 subjects exhibiting increases in time-to-fatigue and three subjects showing a minimal increase or a slight decrease relative to placebo (Figure 4). This variability occurred despite administering a moderately large dose of NO₃, evidence of conversion of ${\text{NO}}_{{\text{3}}^{-}}$ to ${\text{NO}}_{{\text{2}}^{-}}$ in all 13 subjects (based on the ${\text{NO}}_{{\text{2}}^{-}}$ to ${\text{NO}}_{{\text{3}}^{-}}$ ratio), and our use of standardized pre-visit test instructions. The existence of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation responders and non-responders is commonly observed in healthy subjects (Coggan, Broadstreet, Mikhalkova, et al., 2018; Coggan et al., 2020) and in patients (James et al., 2015). Interestingly, a post-hoc (exploratory analysis) showed that the time-to-fatigue improvement with ${\text{NO}}_{{\text{3}}^{-}}$ supplementation was nearly two-fold longer in women with elevated resting blood pressure (n = 4, 88.2 ± 30.1 sec) compared to the rest of our sample (n = 9, 45.3 ± 67.8 sec). This difference, particularly in the absence of anti-hypertensive medication use (an exclusion criterion in this study), could potentially be explained by increased xanthine oxidoreductase (XOR) activity in hypertensives, which increases the ability to reduce ${\text{NO}}_{{\text{2}}^{-}}$ to NO (Ghosh et al., 2013). Further investigation into the possible modulatory influence of baseline blood pressure on the ergogenic effects of dietary ${\text{NO}}_{{\text{3}}^{-}}$ supplementation appears warranted, particularly in light of recent evidence for XOR expression in human skeletal muscle (Wylie et al., 2019).

Potential mechanisms of increased time-to-fatigue

Enhanced muscle contractility?

Our analysis of handgrip force-time dynamics provides insight into the potential mediators of the improvement in time-to-fatigue with ${\text{NO}}_{{\text{3}}^{-}}$ supplementation. As shown in Figure 5, rates of grip force development were faster as graded cuff occlusion progressed during both BR and PL visits. However, the speeding of force development was greater during the 50–75% time-to-fatigue interval (at a similar level of fatigue) on the BR visit suggestive of an improvement in muscle contractility relative to the PL visit. This potential increase in muscle contractility is in accordance with recent studies reporting increased shortening velocities and rates of force production in rodent (Hernández et al., 2012) and human (Coggan & Peterson, 2018) lower extremity limb muscles following dietary ${\text{NO}}_{{\text{3}}^{-}}$ supplementation. The faster rates of force development observed during the BR visit could reflect (1) a greater relative recruitment of type II muscle fibers (which exhibit higher cross-bridge cycling rates vs. type I fibers) in the ischemic forearm musculature of our subjects and/or (2) the type II fiber preference of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation (Hoon et al., 2015). These explanations are speculative in the absence of any measures of fiber type recruitment.

As recently reviewed (Coggan & Peterson, 2018), dietary ${\text{NO}}_{{\text{3}}^{-}}$ associated increases in skeletal muscle contractility likely reflect NO-mediated improvements in intracellular handling and cross-bridge sensitivity to calcium (Ca²⁺). Improvements in the efficiency of Ca²⁺ utilization, in turn, could explain the increased time-to-fatigue in the present study by enhancing the efficiency of force generation (i.e., reduced ATP demand and energy turnover) in the face of progressive blood flow restriction and augmented type II fiber recruitment (Krustrup et al., 2009). ${\text{NO}}_{{\text{3}}^{-}}$ mediated enhancements of muscle contractility have been most consistently observed in type II fibers of rodent models (Hernández et al., 2012), in healthy subjects performing high speed, high intensity exercises (Coggan, Broadstreet, Mikhalkova, et al., 2018; Hoon et al., 2015; Jones et al., 2021) and/or in NO-deficient (older healthy and patient) populations performing single joint, knee extensor/flexion movements (Coggan, Broadstreet, Mikhalkova, et al., 2018; Coggan & Peterson, 2018). The present study suggests ${\text{NO}}_{{\text{3}}^{-}}$ associated improvements in muscle contractility may also be observed in humans performing blood flow-restricted exercise at low exercise work rates. Further investigation into the effects of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation on fiber type recruitment and neuromuscular efficiency during different tasks in older adults and in patients with highly fatigable muscles would broaden our understanding of the ergogenic benefits, and potentially the underlying physiological mechanisms, of this supplement.

Although the rate of force decrease slowed and the HRT increased during progressive blood flow-restricted exercise, we did not identify any significant effects of ${\text{NO}}_{{\text{3}}^{-}}$ supplementation or time-by-treatment interactions for these relaxation parameters (Figure 6). These findings related to force relaxation could be due to the small sample size (coupled with increased between-subject variability for these parameters) of our study, or the fact that processes involved in muscle relaxation do not respond as readily to acute ${\text{NO}}_{{\text{3}}^{-}}$ supplementation.

Other potential mechanisms?

The possibility that ${\text{NO}}_{{\text{3}}^{-}}$ supplementation-related differences in time-to-fatigue resulted from improved muscle blood flow or local O₂ supply cannot be ruled out. However, we view these hemodynamic effects as unlikely because 1) no nitrate-supplementation studies to date have reported improvements in conduit blood flow (hyperemia) or tissue oxygenation kinetics among younger (Craig et al., 2018) or older (Casey & Bock, 2021; Casey et al., 2015) adults performing handgrip exercise and 2) we used a protocol in which blood flow to the contracting muscles was progressively and severely restricted. Improvements in metabolic efficiency (i.e., more efficient mitochondrial ATP synthesis and reduced O₂ cost of contraction) could theoretically delay perceptions of fatigue leading to extended volitional time-to-fatigue. However, ${\text{NO}}_{{\text{3}}^{-}}$ mediated reductions in O₂ cost during non-blood flow restricted handgrip exercise have not been reported using near infrared spectroscopy (Craig et al., 2018) or deep venous O₂ sampling (Richards et al., 2018), and would be challenging to detect using the present protocol i.e., during blood flow-restricted handgrip exercise at a low work rate. Further investigation of these blood flow restricted mechanisms, therefore, would benefit from ³¹P-MRS or other methods capable of quantifying intracellular metabolism (in particular intramuscular [P_i]) and ATP utilization (Meyerspeer et al., 2020)

Experimental considerations

In the present study, we did not strictly control or monitor our participants’ diet. However, participants were instructed to consume only water for 8 hours prior to each study visit and to avoid high nitrate containing food such as spinach and lettuce the day prior. Participants were asked to refrain from teeth brushing and mouthwash the day of testing, but were not asked to avoid using other antibacterial oral care products. Baseline plasma ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ did not differ on the two visits (p = 0.33 and 0.27, respectively; ${\text{NO}}_{{\text{3}}^{-}}$ vs placebo), suggesting good participant compliance with these instructions.

We did not determine the test-retest reliability of the graded blood flow restriction protocol in these participants. However, all subjects performed this protocol to volitional fatigue during the first (screening and familiarization) visit to reduce the potential for initial learning effects to confound our primary outcome (time-to-fatigue) comparison. There was also no significant order effect when comparing time-to-fatigue across the two beetroot study visits (p=0.35).

Time-to-fatigue was defined as the point at which participants were unable or unwilling (due to discomfort) to continue squeezing the handgrip device despite verbal encouragement. This criterion is more subjective than other indices of task failure such as a maximal RPE criterion or objective measures of muscle fatigue (e.g., inability to maintain a threshold of force production over a set number of contractions, etc). However, the endpoint we used resulted in high maximal RPE values across all participants i.e., at least 17 out of 20 on visit 2 and visit 3.

We used an acute dose (140 ml) of a widely used nitrate supplement and placebo (Beet-It Organic, James White Company) that resulted in large increases in plasma ${\text{NO}}_{{\text{3}}^{-}}$ and ${\text{NO}}_{{\text{2}}^{-}}$ with the active (BR) supplement in all participants (8–33 fold increase for ${\text{NO}}_{{\text{3}}^{-}}$; 2–7 fold increase for ${\text{NO}}_{{\text{2}}^{-}}$), with minimal increases observed after placebo (PL) consumption (Fig 3). Whether it would be possible to further augment time-to-fatigue with a larger ${\text{NO}}_{{\text{3}}^{-}}$ dose is unclear, but such an effect would be consistent with the dose-dependency of tissue ${\text{NO}}_{{\text{2}}^{-}}\to \text{NO}$ conversion under hypoxic conditions (Feelisch et al J Biol Chem 2008). The present findings support the use of progressive blood flow restricted handgrip exercise as a model for investigating the ${\text{NO}}_{{\text{3}}^{-}}\to {\text{NO}}_{{\text{2}}^{-}}\to \text{NO}$ pathway and potentially for identifying ${\text{NO}}_{{\text{3}}^{-}}$ supplementation responders and low-/non-responders in various populations of interest.

Translational Perspectives

The present results suggest that acute dietary ${\text{NO}}_{{\text{3}}^{-}}$ supplementation is safe and may improve ischemic small muscle exercise tolerance in older women. This adds to a small but emerging body of literature revealing positive associations between ${\text{NO}}_{{\text{3}}^{-}}$ and/or ${\text{NO}}_{{\text{2}}^{-}}$ intake and skeletal muscle (motor) function in older adults. This evidence includes higher grip strength and better functional mobility (timed up-and-go test performance) in a large cohort of older women with higher compared to lower habitual dietary ${\text{NO}}_{{\text{3}}^{-}}$ intake ((Sim et al., 2019) and improved rates of isometric force development (knee flexion and extension) and a reduction in rapid step errors (improved functional mobility) following 10 weeks of sodium ${\text{NO}}_{{\text{2}}^{-}}$ supplementation in older adults, half of whom were women (Justice et al., 2015).

The present findings also may have relevance for work-related myalgia (WRM), an upper extremity disorder involving pain and tenderness that results from the performance of repetitive tasks involving the hands for extended periods at low force levels (Green et al., 2014). A preliminary study by Green et al (Green et al., 2014) reported lower sarcoplasmic reticulum Ca²⁺ ATPase activity and Ca²⁺ uptake in extensor carpi radialis brevis (ECRB) muscle biopsies from women diagnosed with WRM vs. controls. Given the likely role of improved Ca²⁺ handling in determining muscle contractile performance benefits resulting from increased NO bioavailability (Coggan & Peterson, 2018), and the improved ischemic handgrip tolerance reported herein, it is natural to consider whether ${\text{NO}}_{{\text{3}}^{-}}$ supplementation could increase repetitive work tolerance and improve forearm muscle symptoms in persons suffering from WRM. Such investigation is warranted and could extend the potential therapeutic benefits of dietary ${\text{NO}}_{{\text{3}}^{-}}$ supplementation to workers in occupational settings.

5. Conclusions

These results suggest that acute dietary nitrate supplementation prolongs time-to-fatigue and speeds grip force development during progressive forearm muscle ischemia in postmenopausal women. These findings expand our understanding of dietary ${\text{NO}}_{{\text{3}}^{-}}$ benefits on skeletal muscle performance in general, and in postmenopausal women in particular. The mechanisms underlying these ergogenic effects, the influence of baseline blood pressure, and the occupational and clinical relevance of these findings should be investigated.

HIGHLIGHTS:

• Postmenopausal women have reduced nitric oxide bioavailability and exercise tolerance

• Acute nitrate supplementation prolonged ischemic handgrip exercise in this population

• Nitrate also increased the rate of force development as blood flow restriction progressed

• The mechanisms and clinical relevance of these findings should be explored