1. Introduction
Acute and/or sub-chronic exogenous nitrate (NO
) ingestion has demonstrated potential for improved sub-maximal exercise time-to-exhaustion (TTE) [
1], time-trial performance [
2,
3], and graded exercise testing (GXT) performance [
4]. However, others report NO
ingestion has limited or no effect on performance outcomes [
5,
6,
7]. Specificity of study designs with varying time-course supplementation (i.e., acute vs. chronic), NO
sources, diverse bioactive phytochemicals, or enzymatic changes from enterosalivary circulation may contribute to variable bioavailability and production of nitric oxide (NO) and, thus, influence the observed outcomes [
8]. Wylie and colleagues [
9] recently characterized responses to acute ingestion of 4.2, 8.4, and 16.8 mmol of NO
from beetroot juice. In that study, plasma (all instances of plasma nitrate and nitrite noted in brackets) [NO
] and [NO
] increased dose-dependently with peaks observed 2–4 h post-ingestion with the 6.8 mmol dose compared to 1–2 h post-ingestion with the lower doses. Moreover, arterial blood pressure dose-dependently decreased post-ingestion, but a significant effect was observed for the lowest dose (4.2 mmol) at only the 1-h post-ingestion time point, further illustrating a more transient response to lower doses of NO
. Thus, the timing of NO
-mediated effects may depend upon the dose delivered.
An acute 2000 mg dose of red spinach extract (RSE) delivering ~180 mg (~2.9 mmol) of NO
has been shown to increase plasma [NO
] and [NO
] to peaks of 253 μmol/L (ca. a four-fold change) and 0.56 μmol/L (~1.8-fold change), respectively [
10]. Interestingly, these peak concentrations are similar to, or exceed, those observed with acute ingestion of relatively higher NO
doses from beetroot juice [
9,
11]. Moreover, with acute RSE ingestion plasma [NO
] peaked at 1 h, although plasma [NO
] was highly variable between consecutive 15 min time points with the largest spikes between 30–180 min post-ingestion [
10]. Collectively, the data suggest differential pharmacokinetics from those observed with beetroot. The majority of inorganic nitrate supplementation studies regarding exercise performance have utilized beetroot. Remarkably, limited studies have evaluated NO
rich leafy vegetable and, more specifically, RSE on exercise performance [
12]. Importantly, not only is RSE rich in NO
, potassium (>10% by weight), and anti-oxidant polyphenols (e.g., amaranthine), but it is also devoid of sugar and oxalates.
Given the aforementioned more transient elevations in plasma [NO
] and [NO
] with lower doses of NO
, as well as previous work illustrating peak plasma [NO
] occurring between 45–90 min post-ingestion of 2000 mg of this RSE [
10], we chose to evaluate the effects on physical performance at 65–75 min post-RSE/placebo (PBO) consumption. This RSE is included in numerous multi-ingredient ‘pre-workout’ and ‘energy’ supplements (as Oxystorm
®) with doses ranging from ~500–2000 mg. Thus, we chose to use a ‘medial’ dose of 1000 mg to determine whether ingestion of the same RSE nitrate source previously characterized [
10] would increase plasma [NO
] and [NO
] and affect GXT performance. We hypothesized the acute medial dose of RSE would increase exercise economy and improve oxygen utilization during GXT.
4. Discussion
The primary findings are that a single 1000 mg dose of RSE compared to PBO (1) significantly increased plasma [NO], but not [NO]; and (2) significantly increased the VT during GXT commencing at 65–75 min post-ingestion.
Previously, we reported a three-fold increase in plasma [NO
] from BL 65–75 min post-ingestion of a single 1000 mg dose of RSE [
15]. However, in that study, mean plasma [NO
] was reported to increase from 12 to 36 µM. Herein, using ozone-based chemiluminescence [NO
] was found to increase from 40 to 184 µM, representing a 4.6-fold change. The markedly different [NO
] concentrations and fold-changes likely represent the quantitative limitations associated with [NO
] and [NO
] measurement using Griess-based assays [
24]. Indeed, the peak plasma [NO
] response to a single 1000 mg dose of RSE herein was ~73% of that observed in another study utilizing a single 2000 mg dose of RSE [
10]. Thus, given the greater sensitivity of the ozone-based chemiluminescence method [
17] and a dose-response that appears to be consistent with previous RSE literature, from a quantitative standpoint our current findings are likely more accurate than those previously reported.
Despite utilizing methods with greater sensitivity for detecting plasma [NO
], no statistically significant effect of RSE ingestion on plasma [NO
] was observed. Moreover, the moderate effect sizes suggesting an increase in [NO
] at both the PRE and POST time points with RSE ingestion were not greater than their respective 95% confidence interval. The lack of a significant alteration in plasma [NO
] with RSE ingestion could be due the ingested NO
dose. Following un-blinding, 1000 mg of RSE was diluted in nitrate-free water and tested for [NO
] using the ozone-based chemiluminescence methods described to better characterize the ingested dose. We found ~11.5% NO
by weight, which equates to the delivery of ~115 mg of NO
. It has been previously shown that acute KNO
3 supplementation, with a NO
dose of twice that given herein, resulted in a 1.4-fold peak increase in plasma [NO
] at ~1 h post-ingestion [
25]. Moreover, in an investigation specific to RSE, a dose of twice that delivered herein resulted in ~1.5–2-fold increases in plasma [NO
] at 30 and 90 min post-ingestion [
10]. The plasma [NO
] fold-change observed in the present study (~1.1) was considerably less than expected, particularly given that we observed peak plasma [NO
] levels near 200 μM which have been previously associated with significant (greater than two-fold) increases in plasma [NO
] with beetroot-derived NO
[
9]. Thus, the present results suggest that the pharmacokinetics associated with RSE may differ significantly from other exogenous NO
sources and that a greater increase in plasma [NO
] from RSE may be required to affect plasma [NO
].
Regarding GXT performance, no significant alteration in TTE or VO
2 peak was observed, though previous reports have indicated that exogenous NO
supplementation may actually decrease VO
2 peak [
4], or have little [
26] to no effect [
27] on these outcomes. However, VT during GXT was significantly higher during the RSE condition, suggesting a delayed onset of significant anaerobic metabolism. Given the acute nature of our study, more immediate and plausible proposed mechanisms by which NO
supplementation may influence oxygen uptake and utilization could include a reduction of NO
to NO, directly influencing mitochondrial efficiency [
28], and vascular tone [
8] and/or tissue oxygenation [
11,
29]. However, the support for these mechanisms hinges on the appearance of NO
in the plasma and we did not observe a significant effect of acute RSE ingestion on plasma [NO
]. Though it is plausible that peak plasma [NO
] could have occurred later due to the lag time associated with NO
appearance [
30,
31], the point remains that a marked increase of plasma [NO
] was not observed at time points before or after the GXT protocol.
Previous studies have mostly evaluated exercise performance based upon a pharmacokinetic profile consistent with higher doses of NO that demonstrate peak plasma [NO] occurring ~2.5 h post-ingestion. Herein, we evaluated the effects of RSE on exercise performance at a considerably earlier time point (i.e., 65–75 min post-ingestion) due to the aforementioned plasma [NO] and [NO] responses to lower doses of exogenous NO, and specifically RSE. Thus, the timing of exercise performance measures post-ingestion of exogenous NO may warrant further consideration, particularly as it relates to lower doses. In addition, beyond NO, it remains possible that other phytochemicals and/or non-elucidated mechanisms contributed to our observations with submaximal exercise performance and RSE.
Though alternative, non-NO
mediated mechanisms that could affect VT without changes in VO
2 peak is beyond the scope of this study, a few points warrant consideration. First, we have previously reported an increase in lower limb resistance vessel reactivity ~1 h following acute ingestion of a 1000 mg dose of RSE [
15] that may improve oxygen delivery/utilization in skeletal muscle during exercise. Moreover, RSE is rich in potassium, which is strongly associated with ventilation and may contribute to the exercise induced hyperemic response [
32]. Secondly, it is noteworthy that RSE is devoid of sugar (in contrast to beetroot juice), which may affect the metabolic response to exercise. Third, although an antioxidant profile is not exclusive to RSE, some polyphenols are unique in RSE (e.g., amaranthine) and the relative concentrations of constituents can differ markedly from other exogenous NO
sources, which may affect exercise performance (e.g., quercetin). However, the ergogenic potential and pharmacodynamics associated with potassium and specific polyphenols should be investigated. Finally, given that we only verbally confirmed adherence with dietary guidelines we cannot rule out the possibility that nutrition in the days leading up to exercise trials affected the observed outcomes.
Practical Applications
Practically, although we did not observe an alteration in the TTE or VO
2 peak with GXT, the results presented herein regarding acute RSE ingestions and exercise are intriguing given that the VT is highly associated with the lactate threshold and endurance exercise performance [
18]. If the observed alteration in the VT with RSE ingestion is, in fact, associated with significant alteration of the anaerobic threshold, it is plausible that a 1000 mg dose of RSE may improve sustained sub-maximal exercise performance and/or potentiate higher sub-anaerobic training intensities. However, steady-state submaximal exercise was not investigated herein with participants experiencing a range of submaximal efforts for only brief periods (i.e., 3 min). Further investigations to characterize the effects of RSE on prolonged submaximal exercise performance as well as potential mechanisms for action beyond NO
are warranted. Future investigations should specifically evaluate the effects of RSE on more precise markers of the anaerobic threshold, including local markers of skeletal muscle metabolism.