PERFORMANCE EFFECTS OF CITRULLINE MALATE AND BEETROOT JUICE SUPPLEMENTATION
Eric T. Trexler
A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Human Movement Science Curriculum in the Department of Allied Health Sciences in the School of Medicine.
Chapel Hill 2018
Approved by:
Abbie E. Smith-Ryan
Adam M. Persky
Eric D. Ryan
Todd A. Schwartz
Lee Stoner
© 2018 Eric T. Trexler ALL RIGHTS RESERVED
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ABSTRACT
Eric T. Trexler: Performance Effects of Citrulline Malate and Beetroot Juice Supplementation (Under the direction of Abbie E. Smith-Ryan)
The current study sought to determine the effects of citrulline malate (CitMal; 8 g) and beetroot juice (BEET; 400 mg nitrate) on blood flow, energy efficiency, and performance during leg extension exercise compared to placebo (PLA). Recreationally active males (n = 27) completed 3 testing sessions, consuming CitMal, BEET, or (PLA) 2 h prior to submaximal and maximal leg extensions tests.
Measurements included supine and standing blood pressure, superficial femoral artery diameter and blood flow (BF), vastus lateralis (VL) oxygen consumption and BF, VL cross-sectional area and echo intensity, whole-body energy expenditure and respiratory exchange ratio (RER), blood analytes (urea nitrogen, lactate, and nitrate/nitrite [NOx]), and isokinetic leg extension torque and work. For submaximal leg extension testing at 25% of maximal voluntary contraction torque, treatment had a modest effect on EI
(p = 0.04), with greater values in BEET compared to CitMal (64.9 ± 0.7 vs. 62.7 ± 0.7 arbitrary units [AU]; p = 0.04), but not PLA (63.2 ± 0.6 AU, p = 0.16). Baseline RER values differed between treatments (p =
0.01); BEET was higher than CitMal (0.78 ± 0.01 vs. 0.75 ± 0.01 AU; p = 0.01), but not PLA (0.77 ± 0.01
AU, p = 0.58). Treatment did not affect exercise RER (p = 0.64). All other submaximal measurements were unaffected by treatment (p > 0.05). For maximal exercise (5 sets of 30 repetitions at 180°∙s-1),
-1 resting NOx values were higher in BEET (233.2 ± 1.1 μmol∙L ) than CitMal (15.3 ± 1.1) and PLA (13.4 ±
1.1) at rest (p < 0.0001). Post-exercise NOx values, adjusted for resting differences, followed the same pattern (p < 0.0001). Treatment had a modest but significant effect on VL EI (p = 0.006), with BEET values higher than PLA (68.0 ± 0.7 vs. 65.5 ± 0.7 AU, p = 0.005), but not CitMal (66.3 ± 0.7, p = 0.07). No other variables were affected by treatment (p > 0.05). While BEET increased NOx, neither treatment was found to enhance performance, blood flow, metabolic efficiency, or the hormonal response to submaximal or maximal leg extension in recreationally active males.
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Dedicated to Mom and Dad.
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ACKNOWLEDGEMENTS
I would like to thank my committee members for devoting their time, attention, and expertise to my dissertation project. Without their help, this project would not have been possible. I would specifically like to thank my advisor, Dr. Abbie Smith-Ryan, for her dedication to my academic development. She has sacrificed so much to support my growth over the last six years, and her guidance has shaped my development as a scientist. The information I have learned, the skills I have developed, and the opportunities I have received are all direct results of her mentorship and generosity.
I would like to thank all of my study participants for donating so much time and effort to my dissertation project, and I would like to thank Dale Keith, Adam Lucero, Casey Greenwalt, and Shawn
Ahuja for their assistance throughout. Without the selfless efforts of these individuals, this project could not have been completed.
Finally, I would like to thank my friends, family, and colleagues for their endless support and encouragement.
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TABLE OF CONTENTS
LIST OF TABLES ...... viii
LIST OF FIGURES ...... ix
LIST OF ABBREVIATIONS...... x
CHAPTER 1: INTRODUCTION ...... 1
Purpose ...... 4
Research Questions ...... 5
Hypotheses ...... 5
Delimitations ...... 6
Limitations ...... 6
Assumptions ...... 7
Operational Definitions ...... 7
Significance of study ...... 7
CHAPTER 2: REVIEW OF LITERATURE ...... 9
Introduction ...... 9
Nitric oxide production pathways and metabolism ...... 10
Performance-Enhancing mechanisms of nitric oxide precursor supplements ...... 11
Hypertrophy-promoting mechanisms of nitric oxide ...... 15
Interventions with NOS-dependent precursor supplements ...... 16
Interventions with NOS-independent precursor supplements ...... 19
Synergistic effects with multi-ingredient formulations ...... 21
Potential Clinical Applications ...... 22
Conclusions ...... 24
CHAPTER 3: ACUTE EFFECTS OF CITRULLINE SUPPLEMENTATION ON HIGH-INTENSITY STRENGTH AND POWER PERFORMANCE: A SYSTEMATIC REVIEW AND META-ANALYSIS ...... 26
Introduction ...... 26
Methods ...... 28
vi Results ...... 33
Discussion ...... 35
Conclusions ...... 39
CHAPTER 4: EFFECTS OF CITRULLINE MALATE AND BEETROOT JUICE SUPPLEMENTATION ON BLOOD FLOW AND ENERGY METABOLISM DURING SUBMAXIMAL RESISTANCE EXERCISE ...... 41
Introduction ...... 41
Methods ...... 43
Results ...... 50
Discussion ...... 52
Conclusions ...... 55
CHAPTER 5: EFFECTS OF CITRULLINE MALATE AND BEETROOT JUICE SUPPLEMENTATION ON BLOOD FLOW, ENERGY METABOLISM, AND PERFORMANCE DURING MAXIMAL LEG EXTENSION EXERCISE ...... 57
Introduction ...... 57
Methods ...... 59
Results ...... 65
Discussion ...... 67
Conclusions ...... 71
CHAPTER 6: CONCLUSIONS ...... 73
TABLES...... 75
FIGURES ...... 80
REFERENCES ...... 90
vii LIST OF TABLES
Table 1. Characteristics of studies included in the analysis ...... 75
Table 2. Subgroup analyses ...... 77
Table 3. Participant characteristics and dietary intake information...... 78
Table 4. Resting blood pressure measurements after supplementation ...... 79
viii LIST OF FIGURES
Figure 1. PRISMA diagram ...... 80
Figure 2: Funnel plot ...... 81
Figure 3: Forest plot ...... 82
Figure 4: Timeline of each testing visit (submaximal study) ...... 83
Figure 5: Effects of treatment on muscle blood flow (mBF) and oxygen consumption (mVO2), as measured via near-infrared spectroscopy ...... 84
Figure 6: Effects of treatment on whole-body energy expenditure (EE) and respiratory exchange ratio (RER), as measured via indirect calorimetry...... 85
Figure 7: Timeline of each testing visit (maximal study) ...... 86
Figure 8: Plasma levels of blood urea nitrogen (BUN), nitrate/nitrite (NOx), and lactate ...... 87
Figure 9: Leg extension outcomes ...... 88
Figure 10: Effects of treatment on muscle cross-sectional area (CSA) and echo intensity (EI) ...... 89
ix LIST OF ABBREVIATIONS
aBF Artery blood flow aDIAM Artery diameter
ADP Adenosine diphosphate
AQP4 Aquaporin-4
ATP Adenosine triphosphate
BEET Beetroot juice
BH4 Tetrahydrobiopterin
BUN Blood urea nitrogen
CitMal Citrulline Malate
CSA Cross-sectional area cGMP Cyclic guanosine monophosphate
DEXA Dual-energy x-ray absorptiometry
EE Energy expenditure
EI Echo intensity
FAD Flavin adenine dinucleotide
GAKIC Glycine-arginine-alpha-ketoisocaproic acid
GLUT-4 Glucose transporter 4
GTP Guanosine triphosphate
Hb Hemoglobin
HHb Deoxygenated hemoglobin
HMB beta-hydroxy-beta-methylbutyrate
ICC Intra-class correlation coefficient mBF Muscle blood flow mVO2 Muscle oxygen consumption
NADPH Nicotinamide-adenine-dinucleotide phosphate
NO Nitric oxide
x NOS Nitric oxide synthase
NOx Combined nitrate and nitrite
- NO2 Nitrite
- NO3 Nitrate
NIRS Near-infrared spectroscopy
O2Hb Oxygenated hemoglobin
PLA Placebo
PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyses
RER Respiratory exchange ratio
RPE Rating of perceived exertion
RTF Repetitions to fatigue
SEM Standard error of measurement
SMD Standardized mean difference
TBW Total body water
TCA Tricarboxylic acid tHb Total hemoglobin
TTE Time to exhaustion
US Ultrasound
VL Vastus lateralis
VO2 Whole-body oxygen consumption
Yo-Yo IR1 Yo-Yo intermittent recovery level 1 test
1RM One-repetition maximum
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CHAPTER 1: INTRODUCTION
Nitric oxide (NO) is a signaling molecule with widespread effects throughout the body. While NO plays important roles in a variety of processes pertaining to neurotransmission, inflammation, immunity, and oxidative stress, NO is most widely recognized for its role as a vasodilator. By activating guanylyl cyclase, NO catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), thereby inducing relaxation of smooth muscle in vascular tissues. Nitric oxide may be formed endogenously via nitric oxide synthase (NOS)-dependent or NOS-independent pathways.
In the NOS-dependent pathway, the precursor L-arginine is converted to NO and L-citrulline. This conversion is oxygen-dependent and reliant upon tissue-specific NOS isozymes and a number of requisite cofactors [1]. Endogenous NO production is regulated in part by arginine availability [2, 3], but arginine supplementation has failed to consistently improve exercise outcomes [4], primarily due to extensive pre-systemic degradation following oral ingestion. L-citrulline can be recycled back to L-arginine by way of the urea cycle, with argininosuccinate synthase identified as the rate-limiting enzyme in this conversion [5]. L-citrulline is not subject to extensive pre-systemic degradation; as such, L-citrulline has comparatively greater oral bioavailability and improves plasma arginine levels and performance outcomes more efficiently than oral arginine supplementation [6, 7].
- - In the NOS-independent pathway, nitrate (NO3 ) is reduced to nitrite (NO2 ), which is further reduced to NO, without the need for high oxygen availability [1]. The NOS-independent pathway is stimulated by hypoxia and acidosis; as such, this pathway of NO formation may be particularly advantageous in the context of high-intensity exercise, in which anaerobic metabolism is predominant and a high degree of acidosis is observed. While there is multifactorial regulation of the relative balance
- - - between circulating levels of NO, NO2 , and NO3 [8], there is ample evidence to indicate that NO2 and
- - - NO3 serve as reservoirs for NO production, and that exogenous provision of that NO2 and NO3 confers physiological responses indicative of enhanced NO production, such as reduced blood pressure, increased vasodilation, and improved metabolic energy efficiency [9]. A number of different ingredients
1 have been used to increase NO through this pathway, with the majority of research utilizing nitrate-rich beetroot juice (BEET).
As reviewed by Bailey et al. [1], NO precursor supplements influence exercise efficiency, mitochondrial respiration, calcium handling, vasodilation, glucose uptake, and muscle fatigue. These effects have prompted great interest in the use of NO precursor supplements as a means of enhancing acute exercise performance. Numerous studies have documented performance improvements following citrulline or nitrate supplementation using aerobic exercise modalities, such as running or cycling [1, 7,
10]. Most commonly, these ingredients are found to increase resistance to fatigue, and enhance energy efficiency by reducing energy expenditure at submaximal workloads [1]. In comparison, very little research has been carried out in resistance training and other anaerobic forms of exercise, which may specifically highlight the NOS-independent pathway. Nitric oxide precursors may acutely enhance resistance training performance via favorable effects on muscle fatigue, energy efficiency, and blood flow
[1, 10], and NO plays a permissive role in muscle hypertrophy by directly promoting satellite cell activation
[11]. Collectively, evidence suggests that NO precursor supplementation may enhance acute resistance training performance and chronic training adaptations, but minimal data exist to conclusively confirm these hypotheses. Despite this lack of data, NO precursors are common ingredients in a variety of popular dietary supplements that are marketed toward, and commonly consumed by, both athletes and tactical personnel. It is important to elucidate the effects of NO precursors on strength and hypertrophy outcomes, as supplements enhancing these outcomes have high potential for applications in sport, along with clinical applications for a variety of clinical conditions and pathologies in which muscle mass or function are impaired.
Preliminary studies [12-15] have reported favorable evidence regarding the acute effects of citrulline malate (CitMal) on resistance exercise. These initial studies have documented improvements in repetitions to fatigue during multiple sets of resistance exercise in both upper body [12, 15] and lower body [13] exercises. Most recently, Glenn et al. [14] found that acute CitMal supplementation improved repetitions to fatigue for both bench press and leg press in resistance-trained females. Collectively, these studies suggest that CitMal allows for the completion of more total training volume during multiple sets of resistance exercise taken to volitional fatigue, resulting in an overall increase in total training volume [12-
2 15]. Recent meta-analyses have concluded that weekly training volume is positively associated with both strength [16] and hypertrophy [17], which may indicate that CitMal-induced increases in training volume may favorably affect chronic training adaptations. The results of these preliminary studies are promising, but the current body of CitMal research is limited; to date, only a few small studies have addressed strength outcomes. The mechanisms underlying performance benefits are still unknown, and chronic effects on performance and body composition have not been investigated. Acute performance improvements may relate to malate’s function as a tricarboxylic acid (TCA) cycle intermediate, thereby enhancing the aerobic production of adenosine triphosphate (ATP) and reducing lactate accumulation during exercise. Citrulline may also facilitate the clearance of ammonia generated during strenuous exercise by facilitating urea formation via the urea cycle, thereby minimizing the fatigue-inducing effects of ammonia accumulation. Until these potential mechanisms are sufficiently evaluated, it is not known if the effects of CitMal supplementation are directly related to blood flow or NO production. Elucidating the mechanisms underlying CitMal’s potential performance benefits is an important step in determining ideal dosing strategies and identifying other ingredients that may be co-ingested to improve outcomes via independent, complementary, or synergistic mechanisms of action.
There is abundant evidence to suggest that supplementation of dietary nitrate sources, such as
BEET, increase time to exhaustion and decrease submaximal energy expenditure during aerobic exercise
[18-20]. Emerging data also demonstrate improvements in sprint performance [21-23], but the effects of these supplements on resistance exercise are under-researched and poorly understood. To date, one study has directly evaluated the effects of BEET on traditional, multi-set, isotonic resistance training performance [24]. The crossover design involved two testing sessions in which three sets of bench press were taken to volitional fatigue, separated by two minutes of rest. Prior to exercise, participants consumed either BEET (providing 400 mg nitrate) or a placebo. Compared to placebo, BEET enhanced the number of repetitions completed and total work performed.
Despite the emergence of favorable findings in preliminary research, gaps in the literature pertaining to NO precursor supplementation persist. Studies reporting ergogenic effects have not simultaneously evaluated indices of blood flow and NO activity [12-15, 24], and there is a paucity of evidence evaluating specific mechanisms by which NO precursor supplements may enhance resistance
3 exercise performance. Furthermore, evidence supporting BEET supplementation is limited to a single study that has not been independently replicated [24]. In addition, the performance effects of NOS- dependent and NOS-independent NO precursor supplements have not been directly compared in the context of resistance exercise. Currently, these supplements are widely marketed and consumed for the purpose of enhancing resistance exercise performance, but the overall efficacy and contributing mechanisms are not conclusively known. If efficacious with regard to both vascular effects and strength performance, NO precursor supplements could significantly improve health outcomes for individuals with hypertension, ischemic conditions, sarcopenia, and a variety of muscle wasting conditions. To evaluate the current utility and future potential applications of NO precursor supplements, more research pertaining to strength outcomes and their underlying mechanisms is required.
Purpose
1. To compare the effects of acute (single-dose) CitMal, BEET, and placebo (PLA) supplementation
on arterial blood flow, whole-body energy expenditure and respiratory exchange ratio, vastus
lateralis (VL) blood flow and oxygen consumption, and VL fluid accumulation in response to
submaximal leg extension exercise
2. To compare the effects of acute (single-dose) CitMal, BEET, and placebo (PLA) supplementation
on maximal concentric isokinetic leg extension performance, including peak torque, average
torque, and total work
a. To compare the effects of acute (single-dose) CitMal, BEET, and placebo (PLA)
supplementation on arterial blood flow; whole-body energy expenditure and respiratory
exchange ratio; VL fluid accumulation; and blood biomarkers of urea cycle function, nitric
oxide metabolism, and lactate metabolism, in response to maximal leg extension
exercise
4 Research Questions
1. Does acute supplementation with CitMal or BEET influence submaximal exercise responses of
arterial blood flow, whole-body energy expenditure, respiratory exchange ratio, VL blood flow, VL
oxygen consumption, or VL fluid accumulation in comparison to PLA?
2. Does acute supplementation with CitMal or BEET influence peak torque, average torque, or total
work performed during maximal concentric isokinetic leg extension?
a. Does acute supplementation with CitMal or BEET influence maximal exercise responses
of arterial blood flow; whole-body energy expenditure; respiratory exchange ratio; VL fluid
accumulation; or blood biomarkers of urea cycle function, nitric oxide metabolism, and
lactate metabolism?
Hypotheses
1. Compared to PLA, it was hypothesized that CitMal and BEET would increase arterial blood flow,
VL blood flow, and VL fluid accumulation (cross-sectional area and echo intensity) to a similar
degree
a. Compared to PLA, it was hypothesized that CitMal and BEET would decrease whole-
body energy expenditure and VL oxygen consumption to a similar degree, with no effect
on respiratory exchange ratio
2. Compared to PLA, it was hypothesized that CitMal and BEET would acutely increase leg
extension peak torque, average torque, and total work to a similar degree
a. It was hypothesized that these performance improvements would be accompanied by
similar increases in arterial blood flow and VL fluid accumulation (cross-sectional area
and echo intensity), along with similar decreases in energy expenditure, and no effect on
respiratory exchange ratio
b. It was hypothesized that BEET would cause a marked rise in NO metabolites at rest,
whereas BEET and CitMal would both cause significant post-exercise NO metabolite
increases in comparison to PLA
5 c. It was hypothesized that CitMal, but not BEET, would reduce lactate accumulation and
increase urea accumulation during maximal exercise
Delimitations
1. Participants were males between the ages of 18-35 years old
2. Participants were recreationally active, as defined by an average of at least two hours per week
of exercise activity, including both aerobic and anaerobic forms of exercise
3. The study consisted of four total laboratory visits
4. Participants were excluded if they had a sensitivity or history of adverse reactions to any
ingredients of the experimental treatments or placebo
5. Supplementation occurred two hours prior to the onset of testing
6. Participants were randomly assigned to a treatment sequence (order), in a counter-balanced
manner
Limitations
1. Participants were primarily recruited from classes within the Department of Exercise and Sports
Science and through fliers located near fitness facilities on the campus of the University of North
Carolina at Chapel Hill (UNC-CH). Therefore, the sample was not selected in a truly random
manner
2. Results may not be applicable to females, specific clinical populations, or individuals below 18 or
above 35 years of age
3. Due to poor analyte stability, it was not feasible to directly measure nitric oxide or ammonia. As
such, these physiological parameters were indirectly assessed by measuring blood
concentrations of more stable downstream biomarkers (nitrite/nitrate and urea, respectively)
4. In order to obtain readable measurements for ultrasound and near-infrared spectroscopy
outcomes, measurements obtained immediately following the cessation of exercise were
assumed to represent hemodynamic characteristics during exercise
6 Assumptions
Theoretical
1. Participants provided accurate information on all self-reported items used in the process of
enrollment and data collection
2. Participants gave appropriate effort during exercise testing
3. Participants were honest regarding compliance with pre-testing instructions
4. Participants successfully maintained normal activity levels and nutritional habits throughout the
intervention
5. Measurements taken immediately following exercise accurately reflected hemodynamic
responses that occurred during exercise
Statistical
1. The sample of participants was representative of the population from which it was selected
2. The statistical models constructed were correctly specified
3. Model residuals were normally distributed, homoscedastic, and independent
4. Estimated treatment effects were not significantly influenced by carryover effects, sequence
effects, period × treatment interactions, or habitual nitrate × treatment interactions
Operational Definitions
1. Acute supplementation: Oral, single-dose ingestion of a dietary supplement intended to yield
effects in the hours following ingestion
2. Recreationally Active: An individual that habitually completes an average of at least two hours per
week of any type of exercise activity, including aerobic and/or anaerobic forms of exercise.
Significance of study
The current project aimed to evaluate the effects of CitMal and BEET supplementation on performance, blood flow, vascular responses, and oxygen consumption in exercising muscle. This aim was innovative due to the comprehensive set of hemodynamic parameters, metabolic biomarkers, and
7 performance outcomes to identify the effects and mechanisms of CitMal and BEET supplementation.
Strength, muscular endurance, energy expenditure and efficiency, exercise hyperemia, and downstream markers of TCA cycle and urea cycle function were collectively assessed. To our knowledge, the current study is also the first to directly compare the performance and hemodynamic effects of NOS-dependent
(CitMal) and NOS-independent (BEET) NO precursors on resistance exercise. Results of the current project enhance our understanding of the applications of NO precursor supplements for active individuals in a manner that previous research has not yet addressed.
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CHAPTER 2: REVIEW OF LITERATURE
Introduction
The use of dietary supplements is widespread among a variety of populations and continues to expand; between 2007-2011, up to 69% of US adults surveyed identified themselves as supplement users, with 53% of respondents identified as regular users [25]. While vitamin or mineral supplements were the most widely used supplement category, a substantial number of respondents also reported using specialty supplements, botanicals, and sports supplements [25]. Nitric oxide (NO) precursor supplements are a class of supplements that are widely sold and marketed toward active individuals [26,
27]. These precursors are commonly used as primary ingredients in a variety of pre-workout supplement formulas, and are increasing in popularity as health benefits from natural dietary sources of NO precursors, such as beets, spinach, and pomegranate, become more widely recognized. Nitric oxide precursor supplements are marketed based on the premise that the acute ingestion of NO precursors will transiently increase the production of NO, resulting in an enhancement of blood flow, performance, and the accumulation of intramuscular fluid [26], also known as the muscle “pump” effect [28]. If NO precursor supplements do indeed enhance strength and hypertrophy outcomes, they may have important applications in athletics and a variety of clinical populations in which blood flow, muscle strength, or muscle mass are impaired. Despite the widespread marketing of NO precursors for strength and hypertrophy outcomes, the majority of research to date on NO precursors has evaluated effects on aerobic endurance performance outcomes [10]. The purpose of the current review is to evaluate and discuss the existing literature pertaining to NO precursor supplements in relation to resistance training outcomes, and to highlight how these effects, and their underlying mechanisms, may relate to potential clinical applications.
9 Nitric oxide production pathways and metabolism
Nitric oxide is a gaseous signaling molecule with a variety of functions throughout the human body. NO is an uncharged molecule that freely permeates cell membranes, which carries out autocrine and paracrine functions following its endogenous production. Its effects as an endocrine factor are somewhat limited by its short half-life; while the exact half-life of NO varies as a function of its concentration, diffusibility, and the presence of surrounding bioreactants [8, 29], its half-life is typically estimated to be no more than a few seconds [29]. There are two pathways by which nitric oxide is formed endogenously; one pathway requires the nitric oxide synthase (NOS) enzymes (NOS-dependent pathway), while the other functions independently of this family of enzymes (NOS-independent pathway).
In the NOS-dependent pathway, the precursor arginine is converted to NO, creating citrulline as a byproduct. The conversion of arginine to NO requires the NOS enzyme, for which three isoforms exist in mammals (neuronal, nNOS; inducible, iNOS; endothelial, eNOS) [30]. Isoforms differ with regard to their distribution throughout a variety of body tissues, but all three isoforms are present within skeletal muscle, where they are believed to influence both muscle contraction and blood flow to muscle [31]. This pathway of NO production also requires sufficient oxygen availability, along with sufficient levels of several other cofactors including nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide
(FAD), tetrahydrobiopterin (BH4), haem, and calmodulin [1]. Citrulline created in this conversion is a precursor of arginine, and may be recycled accordingly to permit further production of NO. In comparison to the short half-life of NO, the half-life of arginine has been estimated at over 70 minutes [32]. Past interventions have attempted to enhance NO production via the NOS-dependent pathway by oral supplementation with arginine, the direct NO precursor, or indirectly with arginine’s precursor, citrulline
- [4]. In contrast, nitrate (NO3 ) is the primary NO precursor in the NOS-independent pathway; in this
- - pathway, NO3 is reduced to nitrite (NO2 ), which is further reduced to NO [33]. Although the first reduction
- - of NO3 to NO2 functions independently of oxygen availability and the NOS enzymes, the presence of
- facultative bacteria found in the oral cavity of humans is required. The second reduction of NO2 to NO is also independent of both oxygen and the NOS enzymes, and is stimulated by the acidosis and hypoxia
- - that accompany high-intensity exercise. The half-lives of NO3 and NO2 are substantially longer than NO, with estimated values of 5-8 hours and 1-5 minutes, respectively [34]. Soon after production, circulating
10 - - NO will readily react to directly form NO2 or NO3 , or to form intermediate compounds including nitrosylhemoglobin, S-nitrosohemoglobin, peroxynitrite, and various nitrosothiols [8]. Many of these
- - intermediate compounds are ultimately converted to NO2 or NO3 ; as such, 90% of NO in the body is
- - - ultimately converted to NO3 [35]. Serum or plasma levels of NO2 and NO3 are highly correlated with NO production, and as a result are often measured as surrogate markers of NO production [35].
Nitric oxide is a multifaceted molecule that plays important roles in immunity, inflammation, neurotransmission, gastrointestinal function, and several other biological processes [35]. NO exerts its physiological effects through both enzymatic and non-enzymatic mechanisms. The guanylate cyclase enzyme is activated by NO, which catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), thereby inducing relaxation of the smooth musculature of blood vessels, resulting in vasodilation and increased blood flow. However, NO carries out several physiological functions independently of the guanylate cyclase enzyme via posttranslational protein modifications.
Circulating NO may nitrosylate proteins, forming a reversible, covalent bond with cysteine residues that alters the structure and function of the nitrosylated protein. Nitrosylation is responsible for several cellular effects attributable to NO, as evidenced by the identification of over 3,000 nitrosylation targets in animal cells [36, 37]. In addition to nitrosylation, NO and its derivatives influence protein structure and function through other posttranslational modifications, including nitration and nitrosation [38, 39]. Posttranslational modifications appear to mediate several exercise-related effects of NO and NO-derived compounds, including glucose uptake [40-42], calcium release in the sarcoplasmic reticulum [43], contractile properties of myosin [44], and mitochondrial respiration [38, 45].
Performance-Enhancing mechanisms of nitric oxide precursor supplements
Blood Flow
Despite the variety of physiological effects induced by NO, it is most commonly associated with mechanisms related to blood flow. As such, current supplement formulas containing NO precursors are purported to enhance blood flow, which may contribute to resistance exercise performance or adaptations by enhancing cellular swelling, delivery of oxygen and energy substrates, and clearance of exercise metabolites. Nitrate has been shown to exert pronounced vasodilatory effects on the vasculature,
11 resulting in significant effects on blood pressure and blood flow [20, 46-55]. While blood flow naturally increases in response to exercise, Ferguson et al. [56] have previously shown that NO precursors can augment this blood flow response during exercise, with results demonstrating increased blood flow to the active musculature during submaximal exercise following five days of beetroot juice supplementation in rats. In addition, blood pressure and blood lactate during exercise were significantly lower in the rats receiving beetroot juice. In subsequent work [47], the same laboratory demonstrated an improvement in microvascular oxygen pressure dynamics in rats; this elevation of oxygen driving pressure reflects an improved ability to deliver oxygen delivery relative to demand, thereby improving metabolic control during exercise. More recently, Martin et al. [57] assessed the effects of a citrulline-based pre-workout supplement on exercise blood flow in humans. Results indicated that the supplement significantly increased plasma NO metabolites, which was accompanied by a significant increase in femoral artery blood flow following leg extension exercise using 80% of the one-repetition maximum load. Taken together, these studies lend support to the purported ergogenic mechanism of enhanced blood flow during exercise in response to NO precursor supplementation.
Exercise Efficiency
To date, a great deal of NO precursor supplement research has investigated potential effects on exercise efficiency. Most commonly, this parameter is assessed by measuring the energy cost of a given submaximal exercise workload. Previous studies have documented reduced oxygen consumption during walking [58], running [58], cycling [59, 60], and leg extension exercise [61]. While enhancements in blood flow and oxygen driving pressure may relate to this improvement in efficiency, changes in mitochondrial efficiency and muscular contractile efficiency may also contribute to this effect. In leg extension exercise, beetroot juice supplementation has been shown to enhance exercise capacity while attenuating exercise- induced reductions in muscle phosphocreatine concentration and increases in oxygen consumption, ADP, and inorganic phosphate, with no effect on muscle pH [61]. This finding suggests that NO supplementation may enhance the energetic efficiency of muscle contraction during exercise, although the mechanisms underlying this effect may relate to changes in both mitochondrial respiration and muscle contractile function. Larsen et al. [60] have documented reductions in ATP/ADP translocase expression in
12 human skeletal muscle mitochondria following nitrate supplementation, along with an increased ratio of
ATP produced per oxygen consumed and a reduction in state 4 respiration. Furthermore, this enhancement in oxidative phosphorylation efficiency was correlated with a reduction in oxygen consumption during exercise. These findings are in line with previous research indicating that NO and
NO-derived compounds can bind to cytochrome c oxidase [62] and mitochondrial complex I [38, 45], thereby influencing the efficiency of mitochondrial respiration.
In mice, nitrate supplementation increased the expression of calsequestrin 1 and the dihydropyridine receptor [63], which are both involved in calcium handling in the sarcoplasmic reticulum.
These morphological changes were accompanied by increased myoplasmic free calcium concentrations at stimulation frequencies ranging from 20 to 150 Hz, in addition to enhanced contractile force at frequencies ≤ 50 Hz, and increased rate of force development at 100 Hz [63]. Nitric oxide also appears to influence sarcoplasmic reticulum calcium release during muscle contraction by directly affecting ryanodine receptor activity in a dose-dependent manner, via nitrosylation or oxidation of protein thiols on the ryanodine receptor or associated regulatory proteins [64]. Further, evidence suggests that NO and associated compounds may directly alter the contractile properties of skeletal muscle by nitrosylating cysteine residues of myosin heavy chain proteins, resulting in increased force production [44]. Taken together, results suggest that NO precursor supplements enhance the amount of contractile force generated for a given amount of energy expenditure. This may allow individuals to sustain vigorous resistance exercise for a longer duration prior to fatigue and complete a greater overall training volume, thereby conferring both acute and chronic benefits to resistance exercise performance.
Glucose Utilization
Previous research has investigated the possibility that NO precursor supplementation may enhance glucose uptake, thereby increasing the efficiency with which glucose can be utilized to fuel high- intensity, glycolytic exercise. Insulin-stimulated GLUT-4 translocation and glucose uptake have previously been inhibited by pharmacological blockade of NO synthesis, and mimicked by NO-releasing drugs, through a mechanism mediated by the guanylate cyclase enzyme [65]. Additional research has shown that NO stimulates glucose uptake independently of both the insulin and contraction pathways [66], with
13 research suggesting that NO may also influence glucose uptake via post-translational protein modifications, such as nitrosylation [41]. With respect to exercise, NO appears to play a prominent role in contraction-induced skeletal muscle glucose uptake [40, 42], with studies demonstrating attenuation of contraction-induced glucose uptake when nitric oxide synthase is inhibited [67]. At rest, oral arginine has been shown to reduce glucose production without influencing insulin secretion, particularly in participants that experienced a large increase in plasma citrulline concentrations in response to arginine treatment
[68]. During exercise, arginine infusion increases glucose disposal, although it is unclear if this effect is attributable to increased insulin secretion due to equivocal effects on insulin secretion following arginine infusion [69, 70]. Notably, a slight shift toward increased carbohydrate utilization does not appear to be accompanied by an increased rate of fatigue resulting from glycolytic metabolism. For example, Larsen et al. [60] found that a slight shift toward carbohydrate utilization following nitrate supplementation was accompanied by a significant reduction in overall energy expenditure, and several studies have observed enhanced time to exhaustion during fatiguing exercise after NO precursor supplementation [7, 18, 19, 58,
61, 71].
In summary, NO induces changes in blood flow, mitochondrial function, muscle contractile function, and glucose uptake that are favorable in the context of exercise. Collectively, these effects are likely to explain previous research documenting improved energy efficiency and resistance to fatigue in response to supplementation with NO precursors. Such research has identified favorable effects of both
NOS-dependent and NOS-independent precursors on a variety of exercise outcomes including time to exhaustion (TTE) [7, 18, 19, 58, 61, 71], time trial performance [23, 72-75], and repetitions to fatigue [12,
13, 15, 24]. Enhancement of repetitions to fatigue represents an acute enhancement of resistance exercise performance, which may also allow for greater training adaptations by increasing the volume of work completed during training. These short-term, and potential long-term, effects on fatigue resistance and exercise performance may have important implications for athletic performance and a variety of clinical conditions in which muscle function is impaired.
14 Hypertrophy-promoting mechanisms of nitric oxide
Nitric oxide precursor supplements may indirectly promote muscle hypertrophy by increasing fatigue resistance and improving recovery from strenuous exercise, primarily through changes in blood flow, energy efficiency, and the contractile function of muscle. Experimental treatments containing both citrulline and dietary sources of nitrate have been shown to increase repetitions to fatigue [12, 13, 15, 24] and improve indices of recovery, including post-exercise soreness and restoration of neuromuscular function [12, 76-79]. These effects may enhance hypertrophic adaptations by increasing the total amount of volume completed over a given training period, thereby providing a more robust training stimulus for muscle growth. In addition, there is evidence to suggest that NO may play a more direct role in promoting muscle hypertrophy. Satellite cell activation, a key step in muscle repair and hypertrophy, appears to be mediated by NO; as such, experimental inhibition of the NOS enzyme attenuates satellite cell activation in response to skeletal muscle injury [11]. In line with these findings, pharmacological blockade of endogenous NO production attenuated hypertrophic adaptations to chronic muscle overloading [80], and administration of an NO-donor (isosorbide dinitrate) enhanced exercise-induced hypertrophy of the quadriceps in mice [81].
Finally, it is plausible that the cell swelling effect of NO precursor supplements, for which they are commonly marketed, may contribute to muscle hypertrophy. During resistance exercise, fluid pools in the active musculature due to the combination of increased arterial blood flow to the muscle and venous compression caused by muscle contraction; this pooling of fluid causes transient swelling of the myocyte.
As previously reviewed by Schoenfeld and Contreras [28], there are mechanistic links between this cell swelling effect and the hypertrophic adaptations associated with resistance training. Cell volume regulation is critical for proper function in a variety of cells [82]; as such, it is plausible that the transient cell swelling observed with resistance exercise may present a threat to the structural integrity of the myocyte, stimulating anabolic pathways mediated by integrin-associated volume osmosensors [83]. This theoretical link between cell swelling and hypertrophy is indirectly supported by research showing greater abundance of aquaporin-4 (AQP4) water transport channels in type II muscle fibers [84], which display a more robust hypertrophic response to resistance training [85], and the marked hypertrophy observed with venous blood flow restricted exercise, despite the use of relatively low external loads [86]. In addition,
15 muscle atrophy has been observed in AQP4-knockout mice, but this atrophy may be explained by a reduction in physical activity among mice lacking AQP4 [87]. Despite the existence of mechanistic links between NO and muscle hypertrophy, longitudinal training studies combined with NO precursor supplementation are needed to determine if these supplements enhance hypertrophy or strength adaptations in response to resistance training.
Interventions with NOS-dependent precursor supplements
As the direct precursor to NO in the classical, NOS-dependent pathway, several studies have aimed to improve exercise outcomes using oral L-arginine supplementation. As reviewed by Bescos et al.
[4], the majority of these studies have failed to identify performance benefits associated with arginine supplementation. For example, a single dose of arginine (2 grams) ingested one hour before exercise was shown to be ineffective for altering blood NO metabolite concentrations or performance on a battery of three maximal Wingate sprint tests separated by four minutes of rest [88]. Following three days of oral
L-arginine supplementation at 6 grams per day, no effects on plasma NO metabolites, lactate, ammonia, or peak and average power during intermittent sprint testing were observed [89]. After 28 days of oral L- arginine supplementation (6 grams, twice daily), Sunderland et al. [90] found no effect of arginine on maximal oxygen consumption or ventilatory threshold during a graded, maximal exercise test in trained male cyclists. In another study featuring a 4-week supplementation period [91] with arginine aspartate
(yielding 5.7 grams of arginine per dose), no benefits for maximal oxygen consumption, time to exhaustion, or any of the metabolic or endocrine parameters assessed were observed in trained male endurance athletes.
In contrast, arginine has been found to improve exercise performance outcomes when combined with other potentially bioactive components. For instance, Bailey et al. [92] found arginine supplementation to reduce oxygen cost of exercise and improve time to exhaustion; however, the supplement given provided a mixture of vitamins (including C and E) and amino acids that may have influenced the efficacy of the supplement. Similarly, Chen et al. [93] found arginine supplementation to exert favorable effects on the anaerobic threshold in elderly cyclists, but the supplement contained vitamin C, vitamin E, citrulline, and other nutrients that may have contributed to the observed findings.
16 Notably, antioxidants such as vitamin C and E increase the bioactivity of NO [94], and citrulline is a more bioavailable precursor for NO synthesis in comparison to arginine [6]. Arginine has previously been found to enhance the gas exchange threshold [95] and physical working capacity at fatigue threshold [96] in comparison to placebo when co-administered with grape seed extract; however, the results may not be directly attributable to arginine based on the bioactive compounds and high antioxidant capacity of grape seed extract. Of these studies, it is notable to consider the co-administration of antioxidants in each supplement blend, as antioxidants have been shown to enhance the bioactivity of NO [94].
In the context of strength and power outcomes in short-duration exercise tests, there are select instances in which favorable outcomes have been reported. Campbell et al. [97] found that L-arginine alpha-ketoglutarate supplementation enhanced bench press 1RM and peak power during the Wingate sprint test. Stevens et al. [98] investigated the effects of glycine-arginine-alpha-ketoisocaproic acid
(GAKIC), with results showing improvements in muscle torque, total work, and fatigue resistance during isokinetic dynamometry testing. Subsequent work evaluating GAKIC also documented an attenuation of mean power reductions during a series of five, 10-second sprints in comparison to placebo [99]. While these studies appear to provide favorable evidence for arginine-based formulations, the results are confounded by the presence of other bioactive ingredients; as such, performance effects cannot be confidently attributed to arginine. In the case of arginine-alpha-ketoglutarate, null results have been reported with regards to hemodynamic parameters (heart rate, blood pressure, and blood flow) [100] and resistance training performance (one repetition maximum, total load volume) [101], and alpha- ketoglutarate may have independent effects on metabolism, protein synthesis, and a variety of cellular processes that confound outcomes pertaining to the effects attributable to arginine [102, 103]. Similarly, there are several mechanisms by which GAKIC can influence metabolism, performance, and training adaptations in response to exercise. While the arginine component of GAKIC may indeed contribute to
NO production, GAKIC may also influence ammonia clearance, leucine regeneration, HMB synthesis,
ATP production, insulin secretion, and creatine synthesis [104]. As such, there is insufficient evidence to support the use of L-arginine, in the absence of other bioactive compounds, for the purpose of enhancing exercise performance via increased NO production.
17 While arginine supplementation has demonstrated poor efficacy in previous research pertaining to a wide variety of exercise performance outcomes, citrulline studies have shown more favorable results in comparison. These discrepant results most likely relate to differences in oral bioavailability. Arginine is subject to extensive pre-systemic degradation, resulting in oral bioavailability of approximately 60% [4,
32]. Conversely, citrulline is not subject to extensive first-pass metabolism; as a result, oral citrulline supplementation raises arginine concentrations more effectively than arginine supplementation [6]. A
1500 mg dose of citrulline increased the area under the curve of plasma arginine 46% more than a 1600 mg dose of sustained release arginine, whereas a 3000 mg dose increased it 211% more [6]. As such,
Bailey et al. [7] demonstrated that citrulline reduces blood pressure, improves oxygen kinetics, and enhances exercise performance to a greater extent that an equivalent dose of arginine. Recent studies have suggested that citrulline malate (CitMal), a combination of citrulline and malic acid, enhances resistance to fatigue during strenuous bouts of resistance exercise. Perez-Guisado and Jakeman [12] evaluated the effects of an 8gram dose of CitMal consumed prior to a 16-set resistance training workout targeting the pectoralis major muscle group. To elicit muscle fatigue, all sets of exercise were taken to the point of volitional failure, with the first four sets and last four sets of bench press used for analysis.
Results indicated that CitMal attenuated fatigue and allowed for the completion of more repetitions prior to volitional fatigue with 80% of the one-repetition maximum (1RM) load, particularly as fatigue accumulated in the later sets of the testing session. Perceived muscle soreness was also lower at 24 and 28 hours post-exercise in the CitMal condition compared to placebo.
Wax et al. [13, 15] later conducted two studies on CitMal with similar experimental designs. One such study [13] provided 8 grams of CitMal to advanced male weightlifters prior to 15 sets of lower body resistance exercise. Exercise consisted of five sets each of leg press, hack squat, and leg extension, with all five sets for each exercise using 60% of the 1RM load and taken to volitional failure. The CitMal treatment did not influence blood lactate or heart rate levels, but did improve the number of repetitions performed in comparison to the placebo treatment. In another study [15], the same group administered 8 grams of CitMal to resistance trained males prior to upper body exercise. Exercise consisted of three sets each of chin-ups, reverse chin-ups, and push-ups, with all exercises taken to volitional failure. More repetitions were performed in the CitMal condition compared to placebo, while no treatment effects were
18 observed for blood lactate. Glenn et al. [14] evaluated the effects of an 8-gram dose of CitMal before exercise in resistance trained females. Participants completed six sets of bench press and leg press to failure using 80% of the 1RM load for each; results indicated that CitMal increased repetitions completed for both upper body and lower body exercise, in addition to reducing the rating of perceived exertion
(RPE) during lower body exercise. Benefits of CitMal supplementation have also been noted in female masters athletes (mean age of 51 years) performing high-intensity exercise; in comparison to placebo, 8 grams of CitMal was shown to increase maximal grip strength, average grip strength, and both peak and explosive power during the Wingate test [105].
Although emerging evidence supports favorable effects of CitMal on muscular endurance, more research is needed to fully elucidate its mechanisms of action. The observed ergogenic effects may indeed be mediated by NO production, but may also relate to ammonia clearance resulting from citrulline’s role in the urea cycle, or aerobic ATP production resulting from malate’s role as a TCA cycle intermediate [12]. Determining the contributing mechanisms of action may have utility in formulation of future NO precursor supplements; if a significant portion of CitMal’s ergogenic effects are attributable to mechanisms unrelated to NO production, there may be potential to enhance its efficacy via combination with other NO precursors and synergistic ingredients.
Interventions with NOS-independent precursor supplements
To date, much of the research on NO precursor supplements targeting the NOS-independent pathway of NO production has evaluated effects on outcomes related to endurance exercise. As reviewed by Jones [10], there is abundant evidence indicating that beetroot juice and other sources of nitrate improve aerobic endurance exercise outcomes, including time to exhaustion and time trial performance.
There is also an emerging body of literature suggesting that beetroot juice enhances high-intensity sprint performance. Bond et al. [72] evaluated the performance effects of beetroot juice on a series of six, 500- meter rowing bouts in trained male rowers. Results indicated that 500 mL of beetroot juice, consumed daily for six days, improved sprint times, particularly in sprints 4-6 [72]. Similarly, Wylie et al. [23] found beetroot juice supplementation to improve performance on the Yo-Yo intermittent recovery level 1 (Yo-Yo
IR1) test; subsequent work by this group indicated that beetroot juice is particularly favorable for short-
19 duration sprints separated by short rest periods, with more pronounced effects on 6-second sprint performance in comparison to 30-second and 60-second sprint protocols [22]. Similar outcomes have been investigated by Thompson et al., who documented significant improvements in Yo-Yo IR1 performance [106] and a repeated sprint protocol consisting of six-second sprints [21].
Despite several studies evaluating the effects of nitrate sources on endurance exercise modalities, only one paper to date has evaluated outcomes resembling dynamic, isotonic resistance exercise [24]. Mosher et al. [24] completed a crossover trial in which bench press endurance was evaluated in 12 recreational men with at least three years of training experience. Participants completed a six-day supplementation period of consuming concentrated beetroot juice yielding 400 mg nitrate per day, or a placebo treatment. After each six-day supplementation period, participants completed three sets of bench press to volitional failure with 60% of the 1RM load, with two minutes of rest between sets. The beetroot juice treatment resulted in greater repetitions completed and more total weight lifted in comparison to the placebo condition [24]. As such, the only study to date assessing the effects of nitrate on outcomes resembling traditional resistance exercise performance has indicated a beneficial effect, and the effects of NO on blood flow and contractile function have been shown to preferentially influence type II muscle fibers responsible for high-intensity exercise with heavy external loads. For example, Ferguson et al. [48] determined that the beneficial effects of beetroot juice on blood flow and vascular conductance were greater in muscles containing a higher proportion of fast-twitch fibers, and that beetroot juice specifically increases the partial pressure of oxygen in the vasculature of fast-twitch muscle [107].
Similarly, nitrate enhances sarcoplasmic reticulum calcium handling and force production in type II muscle fibers, but not type I [63]. These fiber type-specific effects suggest preferential enhancement of contractile function in glycolytic fibers, which may translate to pronounced ergogenic effects in anaerobic, high- intensity exercise such as resistance training and short-duration sprints. While more controlled trials are needed to confirm the effects of NOS-independent NO precursor supplements on traditional resistance training performance, the available evidence indicates a favorable effect. In addition, longitudinal training studies are needed to determine if acute increases in training volume have a cumulative effect that result in an enhancement of resistance training adaptations.
20 Synergistic effects with multi-ingredient formulations
Nitric oxide is rapidly inactivated following endogenous production, resulting in a very short half- life [29]. Reaction of NO with superoxide and other reactive oxygen species is a major contributor to this rapid inactivation [8, 94]; as such, the presence of antioxidants protects NO from oxidative inactivation and enhances its availability and physiological activity [94]. Results of previous arginine studies support the purported synergistic effect between antioxidants and NO. As reviewed by Bescos et al. [4], studies investigating arginine alone typically fail to reveal performance improvements. In contrast, arginine has been efficacious when combined with grape seed extract [95, 96] or a combination of vitamins C and E
[93]. In each case, it is likely that the antioxidant properties of the complementary ingredients enhanced the efficacy of L-arginine by increasing the biological activity of NO. Even in the absence of exogenous L- arginine, grape seed extract alone has been shown to increase NO levels [108], enhance blood flow
- [109], and reduce blood pressure [110]. Many dietary sources of NO3 , such as beetroot juice and pomegranate juice, have naturally high concentrations of antioxidant compounds, which may contribute to their effects on performance and hemodynamic parameters [10, 20, 47, 54]. This is supported by the results of Flueck et al. [111], which indicate that the combination of nitrate and antioxidants in beetroot juice enhances exercise energy efficiency to a greater extent that a nitrate-matched dose of sodium nitrate. Recently, McKinley-Barnard et al. [112] investigated the effect of combining L-citrulline with reduced glutathione, an antioxidant. Results indicated that the combination of L-citrulline and glutathione raised plasma NO metabolites to a greater extent than citrulline alone or a placebo. These results provide evidence for the NO-protecting effect of antioxidants, and may suggest that dietary supplements formulated to enhance NO activity would benefit from the inclusion of NO precursors in combination with antioxidants. Furthermore, the NOS-dependent and NOS-independent NO precursors enhance NO via separate pathways; these pathways have distinct requirements with regard to substrates, enzymes, and cofactors, and are divergently inhibited and stimulated by physiological factors such as hypoxia and acidosis [1, 33]. Future research should seek to determine if NOS-dependent and NOS-independent precursors, in combination with antioxidants, exert complementary effects that enhance the stimulation of
NO production in comparison to single-ingredient formulations.
21 Potential Clinical Applications
Given the mechanisms by which NO influences blood flow, exercise capacity, and performance,
NO precursor supplements may have applications in a variety of clinical populations. Endothelial dysfunction is commonly observed with aging [113] and is associated with numerous cardiometabolic conditions including cardiovascular disease, hypertension, and diabetes [114]. Nitric oxide plays a critical role in regulation of endothelial function [115]; as such, there is much interest in the therapeutic potential for NO precursors in several pathological conditions. Supplement interventions to increase NO production have been shown to enhance blood flow [56, 57], and therefore may have applications in conditions related to ischemia, such as peripheral arterial disease (PAD) [116], ischemic stroke [117, 118], or ischemic heart disease [119]. For example, PAD is characterized by pain and exercise intolerance in response to impaired blood flow [116]. Research has demonstrated that patients with PAD have impaired endothelial NO production [120], but beetroot juice supplementation improved exercise time before the onset of claudication pain and exercise time to exhaustion in this population [116, 121]. Similarly, endothelial dysfunction is an early sign of atherosclerotic plaque formation and atherosclerotic patients exhibit reduced bioactivity of NO; as such, it has been suggested that interventions to enhance NO production and/or bioactivity may confer vasculoprotective benefits and slow disease progression [122].
Nitric oxide has also been implicated in the progression of hypertension [123]. Several trials have been conducted to determine the effects of NO precursor supplements, such as nitrate, on blood pressure in hypertensive subjects; a meta-analysis of these trials concluded that nitrate confers significant reductions in systolic blood pressure [124].
As noted previously, glucose uptake is influenced by NO [40-42, 65-67], prompting interest in potential applications in type 2 diabetes management. It is clear that irregularities in NO metabolism are associated with diabetes, as diabetic patients exhibit altered concentrations of NO metabolites [125-127], and NOS inhibition disproportionately reduces exercise-induced glucose uptake in diabetic patients compared to healthy controls [67]. Multiple research groups have implicated endogenous NO production as a viable therapeutic target for the management of type 2 diabetes and its associated complications
[126, 128, 129], but more human trials are required to evaluate the efficacy of oral NO precursor supplements in this context.
22 Sarcopenia presents a major public health concern that could be favorably affected by NO precursor supplementation, based on evidence that NO precursor supplements may enhance both muscle mass and function. Sarcopenia is defined as the age-related loss of muscle mass and function in elderly individuals [130]. As such, the very same mechanisms by which NO precursors may enhance resistance training performance and adaptations could potentially have important applications in aging individuals. Aging is associated with reduced nitrosylation of caldium-dependent proteases, which is accompanied by a loss of neuronal NOS and degradation of myofibrils [131]. Deficient NO signaling has been shown to impair the growth and function of skeletal muscle [132], and aging is associated with a shift in NOS expression that favors the inducible isoform while reducing expression of endothelial NOS expression [133]. As a result, nitric oxide metabolites [134] and endothelial function decline with age
[113], while iNOS-mediated muscle loss increases [135]. The unfavorable effects of age and inflammation on endogenous NO production, combined with the effects of NO on hypertrophy [11, 80], sarcoplasmic reticulum calcium release [43, 63, 64], contractile properties [44], and muscular endurance [12-15, 24,
105], suggest that strategies to target NO production may favorably effect outcomes in conditions related to reductions in muscle mass and function. As such, NO precursor supplementation may have benefits for sarcopenic populations that extend to other muscle wasting conditions, such as cachexia [135]. Taken together, the body of literature suggests that NO precursor supplements may have applications in a variety of conditions related to vascular health, tissue perfusion, glycemic control, and muscle wasting, but controlled interventions in humans are required to confirm their efficacy.
Potential for Adverse Outcomes
As reviewed by Clements et al. [46] and Lidder et al. [53], nitrates and nitrites have long been thought to have carcinogenic effects, with several governments and health organizations imposing upper limits for safe human consumption. Nitrates and nitrites can give rise to the production of n-nitroso compounds, which have been found to exert carcinogenic effects in animals [136]. However, more recent research has failed to substantiate links between nitrate consumption and cancer in humans [46, 53]. As it pertains to fruit and vegetable sources of nitrate and nitrite, there is also reason to believe that antioxidants, vitamins, and other components of these sources may effectively attenuate the formation of
23 n-nitroso compounds [53]. Concerns regarding high nitrate or nitrite intakes are most applicable to infants, in which excessive intakes could cause methemoglobinemia. However, this risk is due to several unique physiological characteristics that are specific to newborn [9], and are not applicable beyond the first few months of life. During exercise, blood must be strategically shunted to sustain skeletal muscle work capacity while maintaining sufficient blood pressure and blood supply to other body tissues. As such,
- - concern regarding exogenous consumption of vasodilating substances such as citrulline or NO2 /NO3 is intuitive in the context of vigorous resistance exercise. Nonetheless, the preferential shunting of blood during exercise is regulated by several redundant mechanisms to ensure that blood pressure in maintained at an appropriate level, and that sufficient blood will be distributed to tissues other than the active musculature [137]. While NO precursor supplements have been shown to enhance blood flow to active musculature during exercise [56], the current body of literature does not contain reports of adverse effects related to altered blood distribution, such as symptomatic hypotension or ergolytic effects on exercise performance. With NO precursor supplements, the most notable adverse events reported in human literature to date are minor gastrointestinal discomfort and nausea, which have been noted in a small percentage of participants following oral consumption of common dosages of citrulline malate [12], potassium nitrate [138], and beetroot juice [139]. Taken together, it would appear that neither acute nor chronic supplementation with NO precursors are unlikely to induce deleterious or ergolytic effects with respect to resistance exercise.
Conclusions
As the popularity of NO precursor supplements grows, the body of literature evaluating these ingredients has expanded in recent years. Nonetheless, gaps in the literature persist. To date, the evidence appears to clearly indicate that citrulline is a more promising NOS-dependent precursor supplement than arginine, as citrulline causes greater elevations in NO production due to marked differences in bioavailability. Several studies have indicated that citrulline malate improves fatigue resistance and repetitions performed in the context of multiple sets of strenuous resistance exercise [12-
15]. More evidence is needed to confidently determine if the ergogenic effects associated with citrulline malate are attributable to NO production; as the literature currently stands, ergogenic effects may also
24 related to ammonia clearance resulting from citrulline’s role in the urea cycle, or aerobic ATP production resulting from malate’s role as a TCA cycle intermediate. While the body of existing literature pertaining to
NOS-independent precursor supplements on aerobic endurance exercise outcomes is extensive, there are far less data pertaining to high-intensity, anaerobic exercise. Emerging studies suggest that NOS- independent precursors are efficacious in terms of high-intensity sprint exercise [21-23, 72, 106], whereas only one study has evaluated outcomes pertaining to dynamic, isotonic resistance exercise [24]. Mosher et al. [24] found that beetroot juice supplementation improved repetitions performed during multiple sets of strenuous resistance exercise, and mechanistic data suggest that NO may preferentially exert positive effects on the function of type II muscle fibers [56, 63, 107]; however, more data are needed to replicate and confirm the results of Mosher et al. [24]. Several questions remain with regard to optimizing the potential of acute oral supplementation with NO precursors, and what specific mechanisms might be contributing to observed ergogenic effects. To fill these gaps in the current literature, more research is also required to evaluate effects on long-term training adaptations, optimize supplement formulations to capitalize on potentially synergistic ingredients, and investigate applications in clinical populations. By more clearly understanding the resistance exercise-related mechanisms and effects of both NOS- dependent and NOS-independent NO precursor supplements, the potential applications for athletic and clinical populations will be more fully understood, and more efficacious interventions can be developed and implemented.
25
CHAPTER 3: ACUTE EFFECTS OF CITRULLINE SUPPLEMENTATION ON HIGH-INTENSITY STRENGTH AND POWER PERFORMANCE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Introduction
Nitric oxide (NO) is a gaseous signaling molecule with widespread effects on several physiological processes. In the context of repetitive muscle contractions, as seen in both endurance and resistance-type exercise, vasodilation and increased blood flow to the active musculature are observed
[137]. Nitric oxide plays a vital role in vasodilation, which enhances delivery of oxygen and energy substrates to active musculature [137]. The guanylyl cyclase enzyme is activated by NO, which catalyzes the conversion of guanosine triphosphate to cyclic guanosine monophosphate. Smooth muscle lining the vasculature relaxes as a result, which causes vessels to dilate and increases blood flow to exercising muscle. Vasodilation may be just one of many mechanisms by which NO levels enhance exercise performance, along with alterations in exercise efficiency, mitochondrial respiration, calcium handling in the sarcoplasmic reticulum, glucose uptake, and muscle fatigue [1]. While nitrate and nitrite may serve as precursors for NO production, the classical pathway of NO production involves the enzymatic conversion of arginine to NO via activity of the nitric oxide synthase (NOS) enzymes [1]. As such, arginine availability is a primary determinant of NO production [2, 3]. Citrulline has emerged as a promising dietary supplement to increase plasma arginine levels, thereby promoting NO production. Given the multifaceted role of NO in vasodilation and other exercise-related physiological processes, there is great interest in using citrulline supplementation to enhance high-intensity exercise performance. In the past decade, several studies have investigated the effects of citrulline supplementation on strength and power outcomes, but mixed findings have been reported [12-15, 105, 140-144]. Meta-analytic techniques can be used to elucidate the ergogenic potential of citrulline supplementation, which would have important ramifications for athletes hoping to maximize strength and power performance.
Dietary supplement consumption is prevalent among US adults, with up to 53% of this population identifying as regular users [25]. Nitric oxide precursors are a popular class of dietary supplements; given the effects of NO on a wide range of exercise-related physiological processes, NO precursor supplements
26 are commonly marketed toward athletes and other active populations engaged in high-intensity exercise
[26, 27]. As the direct precursor to NO production, preliminary studies investigated the effects of L- arginine supplementation on exercise outcomes. Select studies performed using untrained individuals showed ergogenic effects, but studies with trained participants have generally shown no significant effects [4]. For example, Liu et al. [89] studied the effect of 6 g of arginine per day for three days on intermittent cycling performance in trained judo athletes, with no ergogenic effect observed. Sunderland et al. [90] studied the effects of four weeks of L-arginine supplementation on maximal oxygen consumption (VO2 max) and ventilatory threshold in trained cyclists, with no effect of supplementation on either outcome. Notably, studies in trained athletes have shown that oral L-arginine does not significantly increase markers of systemic NO production [89, 145, 146], as bioavailability of oral L-arginine supplementation is estimated to be approximately 60% [4].
In contrast, oral supplementation with L-citrulline bypasses first-pass metabolism and enhances circulating L-arginine levels more effectively than oral L-arginine supplementation [6]. Citrulline can be recycled to produce L-arginine [4] without extensive pre-systemic degradation, thereby emerging as a promising target for NO precursor supplementation. A common form of citrulline supplementation is citrulline malate (CitMal), in which citrulline and malate are combined in ratios ranging from 1:1 to 2:1. A study in men with self-reported fatigue documented significant increases in aerobic adenosine triphosphate (ATP) production and phosphocreatine recovery during finger flexion exercise [147], while other research in trained cyclists showed an enhancement of post-exercise NO metabolite production following 6 g of CitMal supplementation [148]. In 2010, Perez-Guisado and Jakeman [12] conducted the first resistance training study with CitMal. A single, 8 g dose of CitMal consumed one hour before resistance exercise significantly enhanced the number of bench press repetitions performed over a 16-set training session.
A comprehensive review on NO precursor supplements was published by Bescos et al. [12] in
2012, with search results limited to publications from 2011 and before. At the time of its publication, citrulline research was in its infancy; only one study directly addressed the effects of citrulline supplementation on high-intensity strength or power outcomes [12], and the overall body of literature was too small to warrant a systematic review or meta-analysis. In the years since, this body of literature has
27 grown considerably. For example, Wax et al. found CitMal to improve repetitions completed across multiple sets of lower-body exercise in male weightlifters [13], and also identified an improvement in upper-body resistance training performance in resistance-trained males [15]. Similarly, Glenn et al. documented strength and power improvements in female masters tennis players following acute (single- dose) CitMal consumption [105], along with upper- and lower-body repetitions completed by resistance- trained females [14]. In contrast, several other studies have shown no benefit of citrulline-based supplements. For example, Farney et al. [143] found no effect of CitMal supplementation on leg extension peak torque or peak power following circuit training, and repetitions completed during a 10-set leg extension protocol were not improved by acute CitMal supplementation [140].
While Bescos et al. [12] thoroughly reviewed the NO precursor supplement literature available as of 2011, a substantial number of studies investigating the effects of citrulline supplements on high- intensity strength and power outcomes have emerged in the years since. The results of individual studies have been mixed, with many reporting ergogenic effects [12-15, 105] and many reporting null findings
[140-144]. Such ergogenic effects include increases in repetitions to fatigue (RTF) for bench press [12,
14], leg press [14], and multiple-exercise upper-body [15] and lower-body [13] resistance exercise protocols, in addition to improvements in handgrip strength and peak cycling power [105]. Based on the rapid emergence of several citrulline studies with equivocal findings, a systematic review to summarize the effects of citrulline supplements on strength and power outcomes is warranted. The purpose of the current manuscript was to perform a systematic review and meta-analysis of placebo-controlled trials evaluating the effects of citrulline supplementation on high-intensity exercise performance outcomes in healthy adults.
Methods
A systematic review and meta-analysis was conducted to evaluate the effects of citrulline supplementation on high-intensity exercise performance. The current systematic review was conducted and reported in accordance with guidelines outlined in the PRISMA (Preferred Reporting Items for
Systematic Reviews and Meta-Analyses) statement [149].
28 Search Strategy
To identify suitable studies for the current review, literature searches of the PubMed/Medline,
SPORTDiscus, and Web of Science databases were performed by a member of the research team (ETT).
SPORTDiscus results were refined by source type (“academic journals”), and Web of Science results were refined by document type (“article”). The literature search included published records from the inception of each database through 14 August 2018. Searches included the following keywords as search terms: “citrulline,” “citrulline malate,” or “L-citrulline”); in combination with “repetitions to fatigue,”
“resistance exercise,” “resistance training,” "strength," “strength training,” “muscle strength,” “muscular strength,” “weight training,” “weightlifting,” “weight lifting,” “muscular endurance,” “one-repetition maximum,” “one repetition maximum,” "repetitions," "sprint," or "power.”
Inclusion and Exclusion Criteria
Peer-reviewed, original research articles written in the English language were considered for inclusion; review articles and unpublished abstracts, theses, and dissertations were excluded. To be considered for inclusion, articles were required to be human experimental trials in healthy populations, in which the effects of citrulline supplementation on high-intensity strength and power performance were compared to a placebo condition. Primary outcomes included indices of high-intensity exercise performance, including strength and power variables from performance tests involving multiple repetitive muscle actions of large muscle groups, consisting of either resistance training sets or sprints lasting 30 seconds or less. Tests involving isolated actions of small muscle groups (e.g., handgrip exercise with rest periods between attempts) or isolated attempts of single-jump tasks were not included for analysis, due to differences in metabolic requirements. Fatigue index outcomes reported as a reduction from peak strength or power were not included in the absence of raw values, as such outcomes may reflect low peak values (performance impairment) or fatigue reduction (performance improvement).
Studies were excluded from consideration if they lacked a placebo condition for comparison, were carried out in clinical populations, provided a citrulline dose of less than 3 g, provided the citrulline dose less than 30 minutes prior to exercise testing (to allow for sufficient absorption [6]), or combined the citrulline ingredient with creatine, caffeine, nitrate, or other ergogenic ingredients. Citrulline treatments
29 mixed into juices containing antioxidants and other potentially bioactive phytochemicals were considered for inclusion if the study also included a comparator treatment of the same juice without citrulline added.
For studies utilizing more than two treatment arms, the current meta-analysis only included comparisons between a citrulline-supplemented treatment beverage and an identical beverage lacking added citrulline.
Text Screening
Titles and abstracts of the initial search results were independently screened for relevance by two investigators (ETT and AES), based upon a priori inclusion and exclusion criteria. Following title and abstract screening, full texts were independently screened by the same two investigators to further evaluate congruence with inclusion and exclusion criteria, and to determine which studies warranted inclusion in the analysis. Any disagreements between reviewers were discussed until a consensus decision was reached.
Data Extraction, Study Coding, and Quality Assessment
Studies were closely reviewed to extract group means, standard deviations, and sample sizes for outcome measures of interest. When values were plotted as figures, but not reported numerically in the text, values were estimated based on pixel count using calibrated images in ImageJ software (National
Institutes of Health, MD, USA). Briefly, each figure was calibrated by measuring the number of pixels between two known points on the vertical axis of the figure. Mean and standard deviation values were then estimated by measuring the pixel length of each plotted value in the figure, along with its associated error bar. For studies reporting multiple individual sets of a particular outcome, a summed overall value was calculated by summing the means of each set; an overall standard deviation was calculated by taking the square root of the summed variance from all of the individual sets. All extraction and coding was performed by ETT.
One study [142] included two experiments conducted in two separate samples; for the current meta-analysis, each sample was treated as an independent study, as discussed by Borenstein et al.
[150]. For each measured outcome meeting inclusion criteria, standardized effect sizes were calculated as Hedges’ G using the “metafor” package in R software (R Foundation for Statistical Computing, Vienna,
30 AT), yielding an effect size and an associated variance for each outcome. The SMD was used to determine the magnitude of the effect, where <0.2 was defined as trivial, 0.2–0.3 as small, 0.4–0.8 as moderate and >0.8 as large [151, 152]. Most studies reported more than one outcome meeting study inclusion criteria; the method described by Borenstein [153] was used to compute a single, aggregated effect size estimate for each study, using the “MAd” package in R software. This aggregation method requires the estimation of the within-study correlation among outcome variables; while this was not reported in the studies analyzed, Baker and Nance [154] have previously published correlations between a representative collection of variables including both strength and power outcomes of both upper- and lower-body exercises. The mean of these correlation coefficients was calculated (r = 0.70) and used as a generalized estimate of within-study correlation among the variables of interest. A sensitivity analysis was performed to assess the impact of imputing r = 0.5 or r = 1.0, to ensure that findings were robust across a range of plausible correlation values.
All studies meeting inclusion criteria were carefully reviewed to document relevant study characteristics, which were tabulated in a spreadsheet (Microsoft Excel, Microsoft Corporation, WA,
USA). Extracted information included study authors, year of publication, study design, dose and form of supplementation, timing of supplementation, participant sex, participant age, participant training status, inclusion and exclusion criteria for each trial, pre-visit guidelines, side effects, funding sources, and exercise outcomes. Exercise tasks were categorized based on type of outcome (strength or power), muscle groups utilized (upper-body or lower-body), and modality. For the purpose of categorizing training status, individuals were considered “resistance trained” (RT) if they engaged in regular resistance training at least twice a week, for at least six months preceding the trial; participants who were categorized as recreationally active, endurance-trained, or sport-trained were considered non-RT. For subgroup analyses (Table 2), all study characteristics were coded as binary variables (sex: males only vs. females included; training status: resistance trained vs. non-resistance trained; supplement form: citrulline malate vs. other [L-citrulline or L-citrulline + watermelon juice]; musculature tested: lower-body only vs. upper- body included; type of exercise outcome: strength only vs. power outcomes included; modality of exercise: resistance exercise vs. cycle ergometry; funding source: industry funded or undisclosed funding vs. other). Included studies were qualitative reviewed for risk of bias using the individual components of
31 the Cochrane Risk of Bias Tool [155]. Domains of this tool include selection bias, performance bias, detection bias, attrition bias, reporting bias, and other bias.
Meta-Analysis
A random-effects model meta-analysis was conducted using R software. Weighted estimation of standardized mean differences (SMD) across studies were pooled using the inverse variance method.
The statistical heterogeneity across different trials in meta-analysis was assessed by the I2 statistic [156], where <25% indicates low risk of heterogeneity, 25-75% indicates moderate risk of heterogeneity, and
>75% indicates considerable risk of heterogeneity [156]. The I2 statistic was calculated based upon the restricted maximum-likelihood estimator of τ2. For included studies, standard errors were plotted against
Hedges’ G values to allow for visual evaluation of potential funnel plot asymmetry. Funnel plot asymmetry was further assessed using Egger’s regression test [157], and Duval and Tweedie’s Trim and Fill method
[158]. Pooled effect point estimates are presented as SMDs, accompanied by the corresponding 95% confidence intervals (95% CIs; [Lower bound, Upper bound]).
Sensitivity analyses were conducted to assess the impact of the estimated correlation (r = 0.70) between dependent study outcomes [154]. To assess the effects of study characteristics on the pooled effect estimate, moderator effects were tested by fitting a random effects meta-regression model incorporating each coded study characteristic individually. The referent level of the moderating factor was set as the model intercept, with significance testing used to determine if the beta-coefficient corresponding to the second level of the moderating factor was significantly different from zero. Separate
SMD estimates with corresponding 95% confidence intervals were constructed for each subgroup.
Analyzed characteristics included sex of the sample, training status, citrulline form, musculature tested, type of exercise outcome tested, modality of exercise tested, and funding source, and were categorized as binary variables. All analyses were conducted by the same researcher (ETT), with all hypothesis tests conducted at the significance level of α = 0.05.
32 Results
Literature Search
The initial search yielded 181 total records, including 118 unique records and 63 duplicates. Title and abstract screening eliminated 97 irrelevant studies, resulting in 21 eligible studies for full-text screening. After full-text screening, 12 studies, consisting of 13 total independent samples (total n completing testing = 198), met the criteria for inclusion. The PRISMA flow diagram for the systematic review process is presented in Figure 1.
Studies meeting inclusion criteria are summarized in Table 1. Studies were predominantly carried out in young adult populations; all sample means were between 20 and 30 years old, with one exception of 51 ± 9 years [105]. Citrulline malate (CitMal) was the most common form of supplementation (n studies
= 10); the most common CitMal dosage was 8 g, with doses ranging from 6-12 g. Only one study using
CitMal specifically reported the ratio of citrulline to malate, but independent laboratory analysis indicated that the labeled ratio overestimated the citrulline dose and underestimated the malate dose [140]. Other supplement forms included free-form L-citrulline and L-citrulline mixed into watermelon juice, with all studies supplying a citrulline dose of at least 3 g. Two studies included female-only samples, seven included male-only samples, and four contained a mixture of males and females. Supplements were typically provided 60 minutes prior to exercise, with one study providing the supplement 40 minutes prior
[144], and another 120 minutes prior [142]. Eight studies evaluated strength outcomes only, two evaluated power outcomes only, and three evaluated both strength and power outcomes. Seven studies evaluated lower-body outcomes only, five evaluated upper-body only, and one study evaluated a combination of upper-body and lower-body tasks [14]. In all studies, supplementation was well tolerated, with one study reporting mild gastrointestinal (GI) discomfort in 15% of participants [12], and a nonsignificant trend for increased subjective ratings of GI discomfort in another study [141].
Risk of Bias
Risk of bias was generally deemed “low” for each component of the Cochrane Risk of Bias Tool.
All studies were randomized controlled trials utilizing a flavor-matched placebo and a crossover design.
All studies reported utilization of randomized sequence generation, although most lacked methodological
33 detail with regard to how the sequences were generated. All studies reported double-blinded designs with one exception [143], in which only participants were blinded; this study resulted in a small SMD (0.03), which suggests a low likelihood that this single-blinded design led to biased outcomes in favor of the supplement condition. Treatment blinding was well-documented, with placebo treatments matched with regard to flavor, smell, and appearance. Five studies further facilitated treatment concealment by requiring participants to consume the beverage while wearing nose clips to dull taste and smell sensitivity.
Two studies asked participants to identify which treatment they received at each visit [14, 105]; in both cases, hypothesis testing indicated that subjects were unable to effectively identify the treatment received. Comparatively little detail was provided with regard to blinding of testers; twelve of thirteen studies claimed to be double-blinded, with seven specifically stating that treatments were mixed and/or packaged by individuals that did not participate in testing.
Studies typically provided detailed pre-visit guidelines for participants, such as attention to dietary consistency the day before and day of testing, and abstinence from alcohol, caffeine, strenuous exercise, and other dietary supplements. Only one study lacked detail with regard to all of these factors [143], and one study instructed participants to maintain consistency with their dietary supplement intake rather than restricting supplementation altogether [140]. Of studies reporting detailed information pertaining to subject withdrawal, attrition was minimal and attributed to schedule constraints or reasons unrelated to the study.
Evidence of reporting bias was minimal; some results were presented in graphical format only without numerical values provided, and some multi-set test outcomes were reported as a cumulative sum rather than individual set-by-set data. There were isolated cases in which data pertaining to pre-visit dietary habits and/or training habits were collected and not reported, but this lack of reporting is unlikely to bias the SMD estimate of such studies. Four studies reported that no funding was obtained, and three did not disclose funding information; of those disclosing the receipt of funding, two reported industry funding, with the others (n = 4) reporting combinations of government, foundation, and/or university funding.
Pooled Effect Estimate
Statistics pertaining to among-study variance, heterogeneity, and inconsistency across studies were low (Cochrane’s Q = 6.9, p = 0.86; τ2 = 0.0 [0.0, 0.08], I2 = 0.0 [0.0, 40.0]). Visual inspection of the
34 funnel plot (Figure 2) did not reveal substantial asymmetry, and Egger’s regression test for funnel plot asymmetry yielded a nonsignificant result (z = -0.34, p = 0.73). The Duval and Tweedie Trim and Fill analysis identified no missing studies on either side of the plot.
Results of the meta-analysis identified a significant difference between citrulline and placebo treatments on measures of high-intensity strength and power performance (p = 0.036), with a small effect size (pooled SMD = 0.20 [0.01, 0.39]; Figure 3). Sensitivity analyses indicated that this finding was robust with regard to within-study correlation imputations of both r = 0.5 (SMD = 0.19 [0.02, 0.37], p = 0.029) and r = 1.0 (SMD = 0.20 [0.004, 0.405], p = 0.045) in the effect size aggregation computation.
Subgroup Analysis
Hypothesis testing yielded nonsignificant moderation effects by sex (p = 0.72), training status (p =
0.88), supplement form (p = 0.71), musculature tested (p = 0.73), type of exercise outcome (p = 0.19), modality of exercise (p = 0.82), or funding source (p = 0.77). Standardized mean differences for subgroups are presented in Table 2.
Discussion
The current systematic review and meta-analysis sought to summarize the existing literature evaluating the effects of citrulline supplementation on high-intensity strength and power outcomes.
Thirteen independent samples met inclusion criteria, with a total pooled sample size of n = 198. Results of the meta-analysis suggest that citrulline supplementation confers a significant benefit on strength and power outcomes in comparison to placebo, with a pooled standardized mean difference (Hedges’ G) of
0.20 [0.01, 0.39]. This effect size is small, but comparable to other ergogenic dietary supplements. For example, creatine has been shown to exert moderate effects on upper-body exercise (SMD = 0.42), and small effects on lower-body exercise (SMD = 0.21) [159]. Similarly, caffeine exerts effects of similar magnitude on both strength (SMD = 0.20) and power (SMD = 0.17) performance [160]. In a recent meta- analysis investigating the effects of various supplements on short-duration (45 s to 8 min) exercise tasks
[161], moderate effect sizes were reported for caffeine (SMD = 0.41) and bicarbonate (SMD = 0.40), whereas trivial effect sizes were reported for nitrate (SMD = 0.19) and beta-alanine (SMD = 0.17).
35 The current body of literature successfully navigates potential hurdles that would contraindicate meta-analytic procedures. Statistical indices related to among-study variance, heterogeneity, and inconsistency across studies were all favorable for pooled analysis, and indices related to risk of bias were generally low. Results of funnel plot analyses indicated that the body of literature did not exhibit meaningful risk of publication bias or small-study effects. Finally, the studies meeting inclusion criteria reported outcome measurements of strength and power that imposed similar physiological and metabolic demands, thereby allowing for standardization of effects via transformation to Hedges’ G values. Taken together, characteristics of the existing literature suggest that calculation of a pooled effect size point estimate is appropriate, thereby enhancing confidence in the pooled effect estimate of SMD = 0.20.
Despite the similarities between studies, a number of distinct study characteristics warrant exploration. Due to a small number of studies per subgroup, hypotheses tests of moderating effects may be underpowered and should be interpreted cautiously. Hypothesis testing identified no significant moderating effects of sex, training status, supplement form, musculature tested, type of exercise outcome, modality of exercise, or funding source. Sex-based comparisons yielded reasonably similar effect estimates between studies with male samples (n = 7) and studies with female or mixed-sex (n = 6) samples (SMD = 0.23 and 0.16, respectively). For example, Glenn et al. have documented ergogenic effects of CitMal in female masters athletes [105] and resistance-trained females [14], whereas similar results have been reported in male samples by Perez-Guisado and Jakeman [12] and Wax et al. [13, 15].
Minor differences existed between studies of varying training status, musculature tested, modality of exercise, and funding source, with SMD estimates varying by no more than 0.06 between subgroups.
Eight studies only reported strength outcomes meeting inclusion criteria, whereas five studies reported power outcomes or a mixture of strength and power outcomes. While hypothesis testing did not identify a significant moderating effect, SMD estimates differed substantially in studies including strength outcomes only (SMD = 0.30) in comparison to studies including power outcomes (SMD = 0.04). While many sports rely on sport-specific application of both strength and power, there are distinctions that separate the two constructs from one another. Strength pertains to the development of high forces, whereas power pertains to the rapid generation of force per unit time [162]. Citrulline supplements may confer ergogenic effects by either enhancing NO production or facilitating ammonia clearance [12], and
36 forms containing malate may also affect exercise via aerobic ATP production, and even systemic effects on acid-base balance [163]. Furthermore, the physiological effects of NO are multifaceted, with the potential to influence blood flow, exercise efficiency, mitochondrial respiration, calcium handling in the sarcoplasmic reticulum, glucose uptake, and muscle fatigue [1]. Of the literature included in the current analysis, strength outcomes often involved greater overall external loads (such as resistance training repetitions with 80% of one-repetition maximum [12]) in comparison to power tasks (such as cycling against a resistance of 7.5% of bodyweight [105]). In addition, strength tests often included open-ended tasks in which repetitions were completed until failure [12-15], whereas power tasks often included fixed- endpoint tasks in which individuals were challenged to complete as much work as possible in a fixed timeframe [105, 141]. Given the multifaceted mechanisms that may dictate the ergogenic effect of citrulline supplementation, distinctions pertaining to the physiological demands and testing characteristics of strength versus power tasks may contribute to the observed difference in SMD estimates.
Modest differences in SMD estimates were also observed between studies utilizing the CitMal form of supplementation in comparison to other forms of citrulline (10 studies vs. 3; SMD = 0.22 vs. 0.13).
However, given the low number of studies using alternate forms of citrulline (n = 3), these values should be interpreted cautiously. These studies reported individual SMDs of 0.43 [142], 0.11 [164], and -0.06
[142]. As such, the pooled estimate describing these studies summarized a wide range of heterogeneous effect estimates; as more studies assessing alternate forms of citrulline supplements become available, this point estimate may become more refined. Nonetheless, an independent or synergistic ergogenic effect of malate cannot be ruled out. Malate contributes to aerobic ATP production as a tricarboxylic acid
(TCA) cycle intermediate and a major component of the malate-aspartate shuttle mechanism, and may influence acid-base balance by promoting systemic alkalosis [163]. There is evidence of enhanced physical stamina following oral L-malate supplementation in mice completing a swimming task [165], but a human trial found no effect of an oral solution containing malate, succinate, and pyridoxine-alpha- ketoglutarate on cycling performance or recovery [166]. At this point in time, there is insufficient literature documenting the effects of alternate citrulline forms on strength and power outcomes to infer reduced efficacy in comparison to CitMal supplementation.
37 In addition to variability in supplement form, this body of literature features variable estimated citrulline dosages ranging from approximately 3 to 6 g. Unfortunately, the use of meta-regression or other quantitative techniques to evaluate dose-response relationships are precluded by unclear reporting. Of the available literature investigating citrulline malate, only one study clearly reported the supplement ratio of citrulline to malate within the manuscript [140]. This study also tested the observed ratio of citrulline to malate; while the product was advertised as a 2:1 ratio, analysis revealed a ratio of 1.11:1. Five separate brands of CitMal products with purported 2:1 ratios were independently tested, with observed values ranging from 1.11:1 to 1.92:1. To describe the relationship between citrulline dose and exercise response, it is critical for supplement manufacturers to consistently meet label claims, and investigators are encouraged to verify supplement content via independent analysis when possible. Investigators are also encouraged to more clearly identify funding sources, as multiple individual studies failed to disclose any statement regarding internal or external project funding. However, the observed funnel plot symmetry is not consistent with publication bias that could potentially arise from external funding pressures, and studies that included industry funding or failed to disclose funding did not report substantially different
SMD values in comparison to other studies (SMD = 0.23 vs. 0.17, respectively). Publication bias may also arise from reluctance of journals to publish null findings, but funnel plot analyses for the current body of literature are not consistent with substantial publication bias or small-study effects.
Results of the current analysis must be interpreted within the context of its limitations. When study results involve different test protocols and outcome measurements, aggregation of results requires conversion to a standardized effect size unit. This aggregation assumes that the outcomes represent effects that are similar enough to warrant combination. The current analysis mitigated this limitation by employing strict criteria to ensure that included outcomes reflected strength and power tasks with similar physiological and metabolic demands. Due to a low number of studies per subgroup, hypothesis tests evaluating moderating effects between subgroups possessed relatively low statistical power. As a result, subgroup-specific SMDs have been provided, which may inform the design of future citrulline supplementation trials and allow for readers to make preliminary inferences about how these variables may influence outcomes. Finally, the current analysis identified a statistically significant effect favoring citrulline supplementation over placebo, but it should be noted that the confidence interval of this SMD
38 ranges from 0.01 to 0.39. As more literature becomes available, this point estimate may change in magnitude and precision, and even a small shift toward the null could reverse the statistical decision to reject the null hypothesis. As such, researchers are encouraged to contribute more double-blinded, randomized, placebo-controlled interventions assessing the effects of citrulline supplements on high- intensity strength and power outcomes, with the goal of continuing to enhance the validity and precision of its effect size point estimate.
The body of citrulline supplementation research is rapidly growing, and more research is required to fully elucidate its effects on strength and power performance. More randomized trials are needed to resolve a number of research questions that persist. Notably, further research is encouraged to investigate apparently discrepant outcomes in strength versus power tasks. More studies using female samples are warranted, and studies utilizing mixed-sex samples should report sex-specific results to allow further exploration of potential sex differences. Additional studies are needed to evaluate citrulline sources other than CitMal, to determine if malate is an independent and/or synergistic contributor to ergogenic outcomes in the CitMal literature. In addition, studies should provide specific citrulline to malate ratios to allow for quantification of the citrulline dose, and verify labeled dosages with independent analysis when possible. Finally, there is a need for citrulline research in older populations. Only one study
[105] meeting inclusion criteria featured a sample with a mean age above 30 years; supplements enhancing strength and power may have important clinical applications in the management of sarcopenia.
Conclusions
The effects of citrulline supplementation on high-intensity strength and power outcomes have been studied extensively in recent years. While only one paper meeting inclusion criteria was available prior to 2015 [12], there is now sufficient published evidence to warrant meta-analytic techniques to summarize the literature. Results of the current analysis suggest that citrulline supplementation confers a significant performance benefit for high-intensity strength and power tasks in comparison to placebo, with a pooled SMD of 0.20 [0.01, 0.39]. The effect size was small (0.20), but may be relevant to high-level athletes, in which competitive outcomes are decided by small margins [161]. As the literature currently
39 stands, subgroup analysis are limited by the low number of studies per category. As such, further research is encouraged to fully elucidate the effects of potential moderating study characteristics, such as form of citrulline supplement, citrulline dose, sex, age, and strength versus power tasks.
40
CHAPTER 4: EFFECTS OF CITRULLINE MALATE AND BEETROOT JUICE SUPPLEMENTATION ON BLOOD FLOW AND ENERGY METABOLISM DURING SUBMAXIMAL RESISTANCE EXERCISE
Introduction
Nitric oxide (NO) precursor supplements, such as arginine, citrulline, and beetroot juice, are a popular class of dietary supplements, which are typically marketed toward physically active populations as ergogenic aids [26, 27]. A number of recent studies have documented performance improvements following NO precursor supplementation in a variety of exercise tasks, such as cycling [18, 73, 74], running [58], rowing [72], and resistance exercise [12, 13, 15, 24]. There are several purported mechanisms by which NO may enhance exercise performance, with evidence suggesting that NO influences vasodilation, exercise efficiency, mitochondrial respiration, calcium kinetics, and muscle fatigue
[1]. While NO precursor supplements are widely marketed and used as ergogenic aids for resistance exercise, there is currently a paucity of literature in this area, and gaps in the literature pertaining to the underlying mechanisms and performance-related effects of NO precursors on resistance exercise.
Nitric oxide induces relaxation of the vasculature by activating guanylyl cyclase, which catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) [167].
Exercise involving repetitive contractions of large muscle groups increases metabolic demand of the active musculature, thereby increasing the need for energy substrates and oxygen [137]. Increased blood flow to active musculature during exercise, known as exercise hyperemia, is therefore a critical physiological process required to enable vigorous exercise, and NO precursors may therefore influence exercise outcomes by modulating the magnitude or onset of this hyperemic response. Independent of its effects on blood flow, NO has also been shown to influence aerobic energy production and mitochondrial respiration [60]. Nitric oxide and NO-derived compounds have direct effects on mitochondria, and at varying concentrations have been shown to bind to cytochrome c oxidase [62] and mitochondrial complex
I [38, 45], and to enhance the efficiency of oxidative phosphorylation [168]. The half-life of NO is less than a few seconds [29]; as such, attempts to exploit the effects of NO on vasodilation and mitochondrial
41 efficiency via dietary supplementation are restricted to precursors of NO, such as citrulline and dietary nitrate.
Several studies have investigated the effects of beetroot juice (BEET) and other sources of nitrate on energy efficiency during aerobic exercise, as determined using various indices of oxygen consumption
(VO2) or oxygen uptake kinetics in response to exercise. Lansley et al. [58] found reductions in the oxygen cost of treadmill walking, moderate-intensity running, and high-intensity running following BEET supplementation. In two studies using sodium nitrate, improvements in energy efficiency during cycling exercise were observed in young, healthy adults [60] and young, endurance-trained men [59]. While the majority of studies evaluating nitrate-induced improvements in energy efficiency have been carried out using aerobic exercise modalities, only one has used a leg extension task similar to resistance exercise
[61]. While an enhancement of energy efficiency was observed, more research is required to replicate this finding, in order to determine if energy efficiency enhancement may contribute to previously reported improvements in resistance exercise performance following BEET supplementation [24]. For citrulline- based supplements, effects on energy efficiency during exercise are unclear. For example, Ashley et al.
[169] recently found L-citrulline supplementation to favorably effect multiple indices of VO2 kinetics in men, but not in women. During a cycling time trial, L-citrulline was not found to significantly impact VO2 response in comparison to a placebo treatment, but the ratio of power output to oxygen consumption tended to be higher in the citrulline condition [75]. While both BEET [24] and CitMal [12, 13, 15] have been found to delay fatigue during exhaustive resistance exercise, more research is needed to determine if these ingredients induce similar effects on energy efficiency, particularly with respect to resistance exercise.
Vasodilation is the most widely known physiological effect of NO, but supplementation with NO precursors has yielded mixed results on blood flow. In rodents, BEET supplementation has been shown to augment the exercise-induced increase in blood flow to the active musculature [56], but results of human studies have been comparatively less conclusive. Kim et al. [170] found no effect of BEET supplementation on brachial artery blood flow during graded handgrip exercise. In contrast, Richards et al. [171] also measured brachial artery blood flow during handgrip exercise with BEET supplementation, and found a significant increase in blood flow. One study investigating L-citrulline ingestion [172] found an
42 improvement in femoral artery blood flow during calf exercise in older men, but not in older women, whereas another study [173] reported significant increases in NO production without improvements in forearm blood flow following citrulline supplementation.
To fully understand the ergogenic potential of NO precursor supplements, more data are needed to evaluate the effects of BEET and CitMal on energy efficiency during resistance exercise, and to evaluate effects on blood flow at the level of both the conduit artery and the microvasculature of the active muscle. The purpose of the current study was to evaluate the effects of CitMal and BEET supplementation on parameters of blood flow and metabolic efficiency during submaximal leg extension exercise. It was hypothesized that CitMal and BEET would increase indices of blood flow and enhance energy efficiency in comparison to placebo (PLA).
Methods
Experimental Design
The current study evaluated the effects of citrulline malate (CitMal) and beetroot juice (BEET) supplementation in a randomized, double-blind, placebo-controlled, counterbalanced crossover design.
Participants completed a familiarization visit, in which maximal voluntary leg extension torque (MVC) was determined at a 90° knee angle. Participants completed 3 return visits, separated by 5-10 days, to assess the effects of supplementation on parameters of energy efficiency and blood flow during submaximal leg extension exercise. After 5 min of supine rest, blood pressure (BP) was measured with an automated sphygmomanometer in supine and standing positions, then a treatment beverage (CitMal, BEET, or placebo [PLA]) was consumed 2 h prior to exercise. Prior to the onset of exercise, participants laid supine for 5 min, and resting measurements were obtained for supine and standing BP, diameter (aDIAM) and blood flow (aBF) of the superficial femoral artery, cross-sectional area (CSA) and echo intensity (EI) of the vastus lateralis muscle (VL), muscle blood flow (mBF) and oxygen consumption (mVO2) of the VL; and whole-body energy expenditure (EE) and respiratory exchange ratio (RER). Exercise consisted of clusters of isotonic leg extensions, with 1 repetition every 4 s for approximately 7 min, at an intensity of 25% MVC torque. The test visit timeline is presented in Figure 4. Blood pressure, aBF, aDIAM, mBF, CSA, and EI were collectively measured to assess the effects of supplementation on the control of blood flow and fluid
43 accumulation in the active musculature. Whole-body EE and mVO2 were measured to assess the energy efficiency of exercise, and RER was measured to assess fuel utilization during exercise. These assessments of blood flow, energy expenditure, and substrate utilization were selected to evaluate several potential mechanisms by which acute NO precursor supplementation has been purported to influence resistance exercise performance.
Participants
Healthy, recreationally active, non-smoking male participants between the ages of 18-35 yrs participated in the current study. Participants were required to exercise an average of at least 2 h per week for at least 8 weeks preceding the study, and to be free from diseases or injuries that would contraindicate vigorous exercise or influence study outcomes. Participants could not have gained or lost ≥
4.5 kg within 8 weeks of enrollment, or regularly taken prescription medications or dietary supplements that would influence study outcomes within 8 weeks of enrollment. Participants were excluded if they had participated in a clinical trial involving substantial diet or exercise modifications in the previous 8 weeks, consumed more than 3 alcoholic drinks per day, or used recreational drugs within the past month. In order to obtain a clear near-infrared spectroscopy (NIRS) signal from the VL, participants were also required to have a subcutaneous adipose tissue thickness of < 12.0 mm at the site of sensor placement.
All testing visits occurred at the same time of day (±1 h), following at least 4 h of fasting.
Participants were instructed to refrain from strenuous exercise and alcohol consumption within 24 h of testing visits, and from caffeine consumption within 12 h. Participants were instructed not to smoke throughout the study, to refrain from using antibacterial mouthwash the day of testing, and to abstain from chewing gum and brushing their teeth within at least 8 h of visits [174]. Throughout the study, participants were encouraged to maintain their normal dietary and physical activity habits, and typical dietary intakes were evaluated via 3-day food logs. Diet logs were analyzed for macronutrient intake using The Food
Processor software (ESHA Research, OR, USA), and daily nitrate intake using food nitrate content estimates as previously described [175]. All participants provided written informed consent prior to participation, and all study procedures were approved by the University’s Biomedical Institutional Review
Board.
44 Familiarization visit
Prior to testing visits, participants completed a familiarization visit that began by obtaining written informed consent. Participants then completed a health history questionnaire and received instructions for pre-visit guidelines. Participants were then positioned on an isokinetic dynamometer (HUMAC NORM,
Computer Sports Medicine Inc., MA, USA). The backrest was reclined to provide a hip angle of 110° to allow for unimpeded blood flow to the leg. Dynamometer settings were recorded and replicated at all subsequent visits to ensure consistent placement and positioning. The lever arm of the dynamometer was locked in place with the right knee placed in 90° of flexion, and participants practiced 3 escalating submaximal isometric contractions of 50, 75, and 90% estimated effort to warm up. Subjects then completed 3 isometric maximal voluntary contractions (MVCs) for 3-5 s, with 1 min of rest between attempts. The highest of the 3 attempts was used to calculate the resistance for the submaximal exercise protocol (25% of highest MVC). Participants then completed leg extension familiarization, which consisted of 3 sets of isokinetic leg extension. In each set, participants completed 30 concentric leg extensions from a 90° knee angle to full leg extension at 180°∙s-1, with passive leg flexion at 90°∙s-1, and 1 min of rest between sets. Throughout the study, all leg extension tasks were completed with the arms folded across the chest, and a seatbelt tightly fastened across the chest and waist. Participants returned 2-10 days following familiarization for the first testing visit.
Height, Weight, and Blood Pressure
Upon arrival to the laboratory for testing visits, height and weight were measured in light, athletic clothing, without shoes, using a stadiometer (Perspective Enterprises PE-AIM-101, MI, USA) and calibrated electronic scale (Health-O-Meter 2101KL, IL, USA). Participants then rested quietly for 5 min in a supine position, and supine BP was measured using an automated sphygmomanometer (Omron HEM-
711DLX, IL, USA) on the left arm. Participants then assumed a standing position, and standing BP was measured with the arm fully relaxed and supported at chest-level. The supplement was provided following
BP measurement.
45 Supplementation
Treatment sequence was randomly assigned in a counterbalanced manner using Random
Allocation Software (Isfahan, Iran). Randomized treatment sequences included all permutations for the 3 treatment arms: 1) 70-mL beetroot juice beverage yielding 400 mg dietary nitrate (BEET; Beet It Sport,
James White Drinks Ltd., Ipswich, UK), 2) placebo (PLA), and 3) 8 g of unflavored citrulline malate
(CitMal; 2:1 ratio, BulkSupplements.com, NV, USA) mixed into the placebo beverage. As previously described [24], blackcurrant juice (70 mL; Ribena, Lucozade Ribena Suntory Ltd., Uxbridge, UK) was used as the placebo beverage, which is similar to BEET in taste, appearance, and macronutrient content, but has negligible nitrate content. Additional sweetener (Crystal Light; Kraft Foods, IL, USA) and lemon juice were included to further mask flavors. To maintain a double-blinded design, treatments were mixed in opaque containers by an individual that was not present for supplement ingestion or testing.
Treatments were consumed in the laboratory 2 h prior to exercise testing, as peak blood levels of NO precursors are achieved approximately 1.4-2.3 h after citrulline ingestion [6] and 2-3 h after BEET ingestion [176].
Ultrasound: Artery Blood Flow, Vastus Lateralis Imaging
Doppler ultrasonography (Logiq-e, GE Healthcare, IL, USA) was used to assess vessel diameter and blood flow through the superficial branch of the femoral artery of the right leg. The probe (12LRS, 5-
13 mhz) was positioned 1-3 cm distal to the location where the femoral artery bifurcates into the superficial and deep branches. For each image, a minimum of 4 cardiac cycles were recorded in duplex mode, with real-time imaging of the vessel and the spectral waveform velocity profile. Artery diameter
(aDIAM) and arterial blood flow (aBF) were estimated using the measure function in the device’s default software, as previously reported [54]. Velocity values for each image were averaged between up to 4 consecutive cardiac cycles; at least 2 images were obtained at each time point and averaged for analysis.
Test-retest reliability using these methods for brachial artery diameter (intraclass correlation coefficient
[ICC] = 0.82, standard error of measurement [SEM] = 0.03 cm) and blood flow (ICC = 0.86, SEM = 5.92 mL∙min-1) has been reported previously [54].
46 Brightness-mode ultrasound was also used to assess changes in VL CSA and EI due to exercise hyperemia. A panoramic scan was performed at the midpoint of the VL, and images of the VL were traced and analyzed offline by the same trained technician using ImageJ software (National Institute of Health,
MD, USA, Version 1.37). Each image was outlined using the polygon tool to capture as much of the muscle belly as possible, without capturing the epimysium. At least 2 images were captured at each time point, with values averaged for analysis. Test-retest reliability for VL CSA (ICC = 0.87, SEM = 2.12 cm2) and EI (ICC = 0.74, SEM = 4.58 arbitrary units [A.U.]) have been previously reported using this methodology [177]. Resting scans were performed following at least 5 min of supine rest. Post-exercise scans were also obtained in the supine position, with vessel scans occurring 5 min following the cessation of exercise [57].
Near-Infrared Spectroscopy
A continuous wave NIRS device (PortaLite, Artinis Medical Systems BV, The Netherlands) was used to detect relative changes in oxygenated (O2Hb), deoxygenated (HHb), and total Hb (t[Hb] = [O2Hb
+ HHb]) in the VL. The NIRS probe was fixed to the skin two-thirds from the top of the right VL, parallel to the muscle fibers and in the center of the muscle belly. The probe was covered with a custom protective shield and tape to block light and allow the probe to move with the skin during contractions. An inflatable blood pressure cuff was placed around the proximal thigh, and venous and arterial occlusions were used to estimate muscle blood flow (mBF) and muscle oxygen consumption (mVO2). For venous occlusions, the cuff was inflated to a sub-systolic pressure (60 mmHg) to occlude venous return without obstructing arterial flow. As such, venous volume increases in direct proportion to arterial flow, allowing measurement of mBF by calculating the slope of the change in t[Hb]. This slope was converted to units of mL∙min-1 per
100 mL of blood using the following equation [178]:
∆푡[퐻푏] ∙ 60 푚퐵퐹 (푚퐿 ∙ 푚푖푛−1 ∙ 100 푚퐿−1) = [ ∙ 1000] /10 [퐻푏] ∙ 1000 ( ) 4
47 To obtain a value for blood hemoglobin concentration [Hb], a venous blood sample was obtained at rest, and a droplet was analyzed using an automated hemoglobin analyzer (HemoPoint H2, Stanbio
Laboratory, TX, USA). In one instance, a resting blood sample could not be obtained; the average [Hb] from the subject’s other visits were averaged and imputed for NIRS analysis.
To assess mVO2, the cuff was inflated to a suprasystolic pressure (280 mmHg) to simultaneously occlude venous and arterial flow. This results in an increase of [HHb] and decrease in [O2Hb]; the blood
-1 volume-corrected the slope of [HHb] was converted to mLO2∙min per 100 grams of tissue [178]:
∆[퐻퐻푏] ∙ 60 푚푉푂 (푚퐿 푂 ∙ 푚푖푛−1 ∙ 100 푔−1) = 푎푏푠 [ ∙ 4] ∙ 22.4/1000 2 2 10 ∙ 1.04
Resting measures of mBF and mVO2 were taken with participants seated on the dynamometer with the leg fixed in a neutral position (150° knee angle) and the VL fully relaxed. Four, 15-second venous occlusions were applied for resting mBF measurements, with 45 s of rest between measurements. After resting mBF measurements, resting mVO2 was measured twice, using 15-30 s suprasystolic occlusions with 1-2 min of rest between measurements. Heart rhythm was continuously recorded via electrocardiography, with slope values obtained from a single cardiac cycle of each venous occlusion.
The reliability of the described method for mBF (ICC = 0.90) and mVO2 (ICC = 0.96) using leg extensions at 25% of MVC torque has been previously confirmed [178].
Indirect Calorimetry
To assess energy expenditure (EE; KCal) and RER (AU), respiratory gases were collected continuously for 15 min of rest, and throughout the submaximal leg extension test (PRE). Participants were seated on the dynamometer and connected to an indirect calorimeter (TrueOne 2400, ParvoMedics,
Inc., UT, USA) by a mouthpiece and hose. For resting values, first 5 min of data were discarded, with the final 10 minutes of rest used for analysis. Exercise values were obtained from the 8-min period in which leg extension occurred. Expired gas values were averaged every 15 s and used to calculate EE and RER by the device’s default software, and the rate of EE (KCal∙d-1) was converted to KCal burned in 8 min by
48 dividing by 180. Test-retest reliability values for resting EE (ICC = 0.94, SEM = 125.6 kcal∙d-1) and RER
(ICC = 0.83, SEM = 0.03 AU) have previously been reported [179].
Exercise protocol
Following resting measurements, participants completed the submaximal leg extension protocol.
The dynamometer was set to isotonic mode, with resistance set at 25% of MVC torque [178]. Participants completed 1 concentric repetition every 4 s, extending the leg from a 90° knee angle to a 150° knee angle, then relaxing as the leg passively returned to the flexed position (90°). Participants completed 3 min of repeated contractions to achieve a steady state. Immediately following the final repetition of this 3- min bout, the limb was supported at a 150° knee angle with the leg musculature relaxed, and a 10-s venous occlusion was used to assess mBF. Exercise was resumed for 45 s to re-establish a steady state, with participants alternating between 45-s clusters of contractions and venous occlusions until 4 mBF measurements were obtained.
Following the fourth mBF measurement, the occlusion pressure was increased to a suprasystolic pressure (280 mmHg) to allow for mVO2 measurement, and 2 more clusters of contractions (45 s) and inflations occurred. In total, the protocol consisted of six resting measurements (4 mBF, 2 mVO2) and six exercise measurements (4 mBF, 2 mVO2). Multiple measurements were taken for each outcome to allow for values to be averaged, after removal of any unclear signals. Immediately following the final NIRS measurement, the participant was laid supine on a nearby scanning table for post-exercise ultrasound measurements.
Statistical Analysis
Data were analyzed using a series of general linear mixed models. Random intercept models were fitted, with subject identified as a random effect. Preliminary models were fitted to separately confirm nonsignificance of carryover, sequence, period × treatment, and habitual nitrate × treatment interaction effects. Separate reduced models were then fitted to assess the effects of treatment on each outcome variable, with fixed effects including period, treatment, and the baseline value of the dependent variable as a covariate. Model residuals were visually assessed to confirm normality, homoscedasticity, and
49 independence. Resting blood pressure variables were compared using pre-supplementation blood pressure values as a covariate. For all other variables, outcomes measured during or immediately following the submaximal exercise bout were compared using resting values as a covariate. Data missing due to technician or equipment error were assumed to be missing completely at random, with all omnibus tests including at least 76 of 79 possible observations. In the event of a significant treatment effect, pairwise comparisons were adjusted for multiplicity using the Tukey-Kramer method. As a secondary analysis, baseline values related to blood flow and indirect calorimetry variables were compared to evaluate treatment effects on resting values. Statistical analyses were conducted using PROC MIXED
(SAS Software, NC, USA), with the a priori criterion for significance set at α = 0.05. Descriptive demographic data are presented as mean ± standard deviation, with all other data presented as adjusted least squares mean ± standard error.
Results
Participants
Thirty subjects enrolled; 3 withdrew prior to the first testing visit, and 1 withdrew after the first.
Subjects providing reasons for withdrawal either cited schedule constraints or injuries unrelated to the study; attrition did not appear to be related to any specific distinguishing characteristics. In total, 27 participants completed at least one testing visit; demographic characteristics of the sample are presented in Table 3.
Blood Pressure
Treatment did not significantly affect BP in the supine or standing posture (all p > 0.05; Table 4).
Ultrasonography
Significant differences among treatments were not observed for CSA (F[2, 48] = 1.08, p = 0.35;
CitMal: 31.7 ± 0.2, PLA: 32.1 ± 0.2, BEET: 32.0 ± 0.2 cm2). A treatment effect was observed for muscle EI
(F[2, 46.8] = 3.35, p = 0.04), with pairwise comparisons indicating significantly greater values in BEET compared to CitMal (64.9 ± 0.7 vs. 62.7 ± 0.7 AU; p = 0.04), but not PLA (63.2 ± 0.6 AU, p = 0.16). Visual
50 examination of the model residuals prompted log transformation; the log-transformed model indicated a trend for treatment (F[2, 45.6] = 3.08, p = 0.056), with pairwise comparisons yielding a similar relationship between BEET and CitMal values (64.4 ± 1.0 vs. 62.2 ± 1.0 AU; p = 0.052).
Baseline resting values did not differ among treatments for aDIAM (F[2, 47.3] = 0.30, p = 0.74) or aBF (F[2, 47.8] = 0.63, p = 0.53). For submaximal exercise responses, the effect of treatment on aDIAM was not significant (F[2, 43.4] = 0.88, p = 0.42; CitMal: 0.68 ± 0.01, PLA: 0.68 ± 0.01, BEET: 0.70 ± 0.01 cm). A significant carryover effect was observed (p = 0.05), so data were stratified by previous treatment for further analysis. No significant treatment effect was observed in the periods following CitMal or PLA provision (both p > 0.05), but aDIAM was greater in the BEET condition compared to CitMal in the period following PLA ingestion (0.71 ± 0.01 vs. 0.67 ± 0.01 cm, p = 0.02). When analyzed using data only from period 1, the treatment effect was not significant (p = 0.055), but least square means followed a similar trend, with higher values for BEET (0.69 ± 0.01 cm) in comparison to CitMal (0.66 ± 0.01 cm). For submaximal exercise responses, artery flow was not significantly affected by treatment (F[2, 48.3] = 0.75, p = 0.48; CitMal: 169.0 ± 9.6, PLA: 158.7 ± 9.6, BEET: 156.9 ± 9.6 mL∙min-1).
Near-Infrared Spectroscopy
Baseline resting values did not differ among treatments for mBF (F[2, 47.4] = 0.14, p = 0.87) or mVO2 (F[2, 47.9] = 0.65, p = 0.53). For submaximal exercise responses, a significant treatment effect was not observed for mBF (F[2, 45.5] = 0.11, p = 0.90; CitMal: 3.78 ± 0.26, PLA: 3.72 ± 0.26, BEET: 3.77 ±
-1 -1 0.26 mL∙min ∙100 ml ). Similarly, submaximal exercise mVO2 was not significantly affected by treatment
-1 -1 (F[2, 46.2] = 0.85, p = 0.44; CitMal: 1.15 ± 0.11, PLA: 1.16 ± 0.11, BEET: 1.19 ± 0.11 mLO2∙min ∙100g ).
Near-infrared spectroscopy values during submaximal exercise are presented in Figure 5.
Indirect Calorimetry
Baseline resting EE values did not differ among treatments (F[2, 47.1] = 1.40, p = 0.26). The effect of treatment on exercise EE was not significant (F[2, 47.4] = 2.10, p = 0.13; CitMal: 21.6 ± 0.5, PLA:
20.6 ± 0.5, BEET: 21.6 ± 0.5 kcal). Baseline RER values differed among treatments (F[2, 47.9] = 4.68, p =
0.01); BEET was higher than CitMal (0.78 ± 0.01 vs. 0.75 ± 0.01 AU; p = 0.01), but not PLA (0.77 ± 0.01
51 AU, p = 0.58). Treatment did not significantly affect exercise RER (F[2, 47.1] = 0.45, p = 0.64; CitMal:
0.80 ± 0.01, PLA: 0.79 ± 0.01, BEET: 0.79 ± 0.01 AU). Indirect calorimetry values during submaximal exercise are presented in Figure 6.
Discussion
Numerous studies have reported ergogenic effects on exercise tasks including time to exhaustion, time trial performance, and repetitions to fatigue from NO precursor supplements, such as
CitMal and BEET [12, 13, 15, 18, 24, 58, 72-74]. Ergogenic effects are thought to be mediated by NO, which is purported to enhance blood flow and energy efficiency during exercise. However, there is a lack of research specifically linking these mechanisms to resistance-type exercise, and more data are needed to elucidate the mechanisms underlying previously reported improvements in resistance exercise performance following NO precursor supplementation. The current findings do not suggest that acute ingestion of CitMal or BEET prior to exercise significantly alter blood flow or energy efficiency during submaximal leg extension in recreationally active males.
Parameters pertaining to blood flow were not affected by CitMal or BEET in the current study.
Neither systolic nor diastolic BP were influenced by treatment; while both CitMal and BEET have been shown to reduce blood pressure, effects are less likely to be observed in young, active, normotensive participants [13, 170], such as the current sample. Notably, NO synthesis and endothelial function often decrease with age [113, 134], which may explain age-dependent effects of NO precursor supplementation. Similarly, supplement-induced alterations of BP or blood flow are more likely to be observed in individuals with elevated BP or impaired blood flow, with underlying disruptions in NO metabolism and/or endothelial function. For example, BEET was recently shown to reduce systolic (-7.9 mmHg) and diastolic (-5.7 mmHg) BP by after acute ingestion, but mean age of the sample was 64 years, and average BP was 133/88.6 mmHg [180]. Blood pressure results of the current study are favorable for normotensive individuals interested in supplementing with NO precursors, as neither BEET nor CitMal induced resting hypotension or postural hypotension in this normotensive sample. Similarly, aDIAM, aBF, and mBF were not influenced by CitMal or BEET. While Richards et al. [171] observed approximately 12-
13% increases in brachial artery blood flow during handgrip exercise following BEET ingestion, the
52 current findings are supported by studies in which both citrulline [181] and BEET [170, 182] have failed to enhance conduit artery blood flow during or immediately following exercise. Although Richards et al. [171] evaluated a sample of young, healthy participants, the reported increase in blood flow was observed after giving a nitrate dose 3-4 times higher than the current study. Lack of significant blood flow effects in the current study may also be related, to some extent, to the use of a young, healthy sample, as BEET supplementation has been shown to increase the compensatory vasodilator response to hypoxic handgrip exercise in older adults (age: 64 ± 2 yr), but not in younger adults (age: 25 ±1 yr) [183].
While aDIAM was not affected by supplementation, a carryover effect was observed; follow-up testing revealed that aDIAM was higher in BEET than CitMal in the period following PLA ingestion. While this finding warrants further investigation, it should be noted that stratifying analysis by previous treatment is exploratory and drastically reduces the sample size of each statistical test, which increases the likelihood of a statistically significant finding being spurious [184]. With regard to mBF, the current findings are supported by previous research demonstrating no effect of citrulline on microvascular circulation
[181]. Other studies have reported significant effects of BEET on the microvasculature [185, 186], but both used higher nitrate doses than the current study, and marked methodological differences in assessment of microvascular function present challenges for making between-study comparisons. A strength of the current study is the use of intermittent venous and arterial occlusions in conjunction with
NIRS, which enhances the ability to extrapolate localized hemoglobin fluctuations with indices of mBF and mVO2. In summary, null effects on all blood flow parameters in the current study may be attributable to the use of a young, healthy, active sample in which endothelial function is unlikely to be impaired [170,
183]. These results do not support the hypothesis that alterations in blood flow are a primary mechanism explaining previous findings in which acute NO precursor supplementation has enhanced resistance exercise performance in young, healthy individuals [12, 13, 15].
The current study identified a significant effect of treatment on EI, with elevated values observed after BEET ingestion. Recent research has shown EI to increase immediately following strenuous resistance exercise [187], and 3 weeks into a resistance training program [188]. In each case, authors attribute the increase in EI to exercise-induced edema. While differences in the current study were statistically significant, the magnitude of difference was only 2.2 AU, which falls within the standard error
53 of the measurement. Furthermore, transient fluctuations in intramuscular water due to variation in hydration status, glycogen content, and exercise-induced swelling or edema have potential to bias EI measurements [189], so small EI changes should be interpreted cautiously. While the observed difference in EI could potentially reflect a modest effect of BEET on fluid accumulation in the muscle belly, this explanation is contradicted by null effects with regard to conduit artery blood flow, microvascular blood flow, and CSA. As such, it is unlikely that this small effect of BEET on EI is physiologically meaningful.
Indices of energy expenditure and oxygen consumption, measured via indirect calorimetry and
NIRS, were not influenced by treatment in the current study. A modest elevation in resting RER was observed following BEET consumption in comparison to CitMal, which indicates a shift toward increased reliance on glucose metabolism with BEET, and a shift toward fat metabolism with CitMal. A similar RER elevation following nitrate supplementation was observed during exercise by Larsen et al. [60], who noted that a change of this magnitude was of minimal physiological significance. With regard to CitMal,
Bendahan et al. [147] documented an increase in the relative contribution of aerobic metabolism following supplementation, which may result in a modest RER reduction as observed in the current study.
Furthermore, these effects on RER were observed only at rest in the current study, were not significantly different than PLA, and were not accompanied by changes in total EE, which limit the practical significance of the findings. While multi-day supplementation interventions with BEET have been shown to enhance the energetic efficiency of exercise using a variety of exercise modalities and measurement methodologies [58, 59, 61], the mechanism underlying these observations is not fully understood. Larsen et al. [60] have proposed that increased exercise efficiency is related to increased coupling of mitochondrial respiration, as evidenced by improved oxidative phosphorylation efficiency and decreased state 4 respiration. Such changes in mitochondrial efficiency are likely related to downregulation of uncoupling protein 3 (UCP-3) and adenosine nucleotide translator (ANT); alterations in protein structure or expression are more likely to be impacted by multiple-day dosing strategies in comparison to a single dose [60], which may explain the null findings in the current study. However, Whitfield et al. [190] recently found BEET to reduce whole body VO2 without altering indices of mitochondrial efficiency, which suggests that an alternative mechanism may explain previously reported improvements in exercise
54 efficiency. In summary, the current results do not support the hypothesis that alterations of energy efficiency or substrate utilization explain previously reported improvements in repetitions to fatigue following acute NO precursor supplementation [12, 13, 15].
It has been proposed that exercise efficiency improvements may relate to alterations in muscle calcium kinetics, thereby reducing the ATP cost of muscle contraction [61, 63]. Acute nitrate supplementation, particularly with doses of 8.8 mmol and above, may enhance muscle function by increasing sarcoplasmic reticulum calcium release (via nitrosylation of ryanodine receptors), and/or by increasing myofibrillar calcium sensitivity (via activation of guanylyl cyclase, and subsequent elevation of cGMP) [191]. Chronic supplementation may confer additional effects; after 7 days of nitrate supplementation, Hernandez et al. [63] observed upregulation of calcium-handling proteins in muscle
(calsequestrin 1 and dihydropyridine receptor), whereas other research has shown increased force production in the absence of changes in these proteins [192]. While more research is needed to determine the exact mechanisms by which NO may modulate exercise efficiency, these effects appear to be more reliably observed using higher and more prolonged dosing strategies than the current study.
The primary limitation of the current study is the dosing protocol, which consisted of a single dose of CitMal (8 g) or BEET (400 mg nitrate) prior to exercise. While this reflects practical application of dietary supplement use in general and athletic populations, BEET literature suggests that more pronounced findings may be observed with higher BEET dosages or more chronic, daily supplementation periods. In addition, preliminary statistical tests suggested that carryover effects were present for standing
DBP and aDIAM, despite 5-10 days of washout between testing visits. Orally ingested CitMal or BEET would be readily cleared from systemic circulation within this timeframe, but further research may be warranted to determine the time course in which CitMal or BEET may modestly influence the function of the vasculature following supplementation.
Conclusions
The current data do not support the hypothesis that a single dose of CitMal (8 g) or BEET (400 mg nitrate) enhance blood flow or energy efficiency during submaximal leg extension in young, recreationally active males. Previous studies have reported improvements in resistance exercise
55 performance, measured as repetitions to fatigue, following acute NO precursor supplementation [12, 13,
15]. Authors have speculated that such ergogenic effects may be attributable to alterations in blood flow, energy efficiency, or substrate utilization, but the current results do not support the hypothesis that these parameters are significantly altered by acute CitMal or BEET supplementation. Acute effects of NO precursor supplements on muscle contractile function, potentially mediated by alterations in sarcoplasmic reticulum calcium kinetics and myofibrillar calcium sensitivity, may warrant further research. As research demonstrating ergogenic effects of NO precursor supplements on resistance exercise performance continues to emerge [12, 13, 15, 24], more research is needed to elucidate the mechanisms underlying these effects.
56
CHAPTER 5: EFFECTS OF CITRULLINE MALATE AND BEETROOT JUICE SUPPLEMENTATION ON BLOOD FLOW, ENERGY METABOLISM, AND PERFORMANCE DURING MAXIMAL LEG EXTENSION EXERCISE
Introduction
Nitric oxide (NO) is a multifaceted signaling molecule, influencing the activity of a wide range of physiological systems in the human body. In the context of exercise, NO is thought to impact physical performance by influencing exercise efficiency, mitochondrial respiration, sarcoplasmic reticulum calcium kinetics, glucose uptake, and muscle fatigue [1]. In the nitric oxide synthase (NOS)-dependent pathway of
NO production, arginine is the direct precursor to NO, yielding citrulline as a byproduct [1]. Citrulline can then be recycled back into arginine, allowing for continuation of NO production. A second pathway of NO production functions independently of the NOS enzymes; in the NOS-independent pathway, nitrate is reduced to nitrite, which is further reduced to NO. In the past two decades, substantial research has been carried out to examine the exercise-related effects of supplementation with precursors from each NO pathway [4, 10]. Emerging studies have suggested that both citrulline malate (CitMal; NOS-dependent) and beetroot juice (BEET; NOS-independent) significantly enhance resistance exercise performance by attenuating fatigue [13, 15, 24]. However, these supplements have not been directly compared with respect to their effects on resistance exercise performance, and the underlying mechanisms by which they influence resistance exercise performance have not been fully elucidated.
Several studies have described potential mechanisms by which BEET and other sources of nitrate may enhance exercise performance via the NOS-independent pathway. Ferguson et al. [56] demonstrated that BEET supplementation increased blood flow to the active musculature during exercise in rats, and several studies have documented enhanced metabolic exercise efficiency during walking [58], running [58], cycling [59, 60], and leg extension exercise [61]. As such, BEET supplementation has been reported to enhance exercise capacity (measured as time to exhaustion) during both cycling [18] and treadmill running tests [58]. Similarly, BEET supplementation resulted in a significant improvement in
57 rowing ergometer performance in trained male rowers [72], and has also been shown to enhance cycling time trial performance in well-trained male cyclists [73, 74].
While BEET has been studied extensively in the context of endurance exercise modalities, the authors are aware of only one study to date investigating the ergogenic potential of BEET for dynamic constant external resistance exercise. Mosher et al. [24] utilized a crossover design to compare the effects of a BEET supplement containing 400 mg of nitrate on bench press repetitions completed over three sets to failure, demonstrating improved volume (total weight lifted), with no effect on blood lactate.
While this finding has yet to be replicated, evidence suggests that the ergogenic effects of BEET may be pronounced in high-intensity activities that rely on the recruitment of type II muscle fibers, such as resistance exercise. Ferguson et al. [48] found that BEET enhanced blood flow and vascular conductance to a greater degree in type II fibers than type I fibers in rodents, and later reported that BEET preferentially increases the partial pressure of oxygen in the vasculature of muscle containing higher proportions of type II fibers [107]. In other rodent-model research, sarcoplasmic reticulum calcium kinetics and force production were enhanced by 7 days of sodium nitrate-supplemented water in fast-twitch fibers, but not in slow-twitch fibers [63], and the effects of nitrate on muscle contractile properties appear to be more pronounced under conditions of acute muscular fatigue [193]. Furthermore, the NOS-independent pathway of NO production is stimulated in conditions of hypoxia and acidosis [33], as would be expected during exhaustive high-intensity sprint or resistance exercise. Despite plausible mechanistic support and promising preliminary evidence, more human research is needed to conclusively determine if BEET exerts ergogenic effects in the context of resistance exercise.
Preliminary studies have reported promising results for the ergogenic potential of citrulline-based supplements, which utilize the NOS-dependent pathway. In 2010, Perez-Guisado and Jakeman [12] evaluated the effects of CitMal (8 g) on resistance training performance. Results showed a statistically significant difference between conditions, with participants completing more repetitions prior to failure after the consumption of CitMal. In recent years, more positive findings have emerged; Wax et al. documented significant improvements in upper-body [15] and lower-body [13] repetitions to fatigue in resistance-trained males after consumption of CitMal, in comparison to a placebo treatment. Glenn et al. found acute CitMal ingestion to enhance upper- and lower-body repetitions to fatigue in resistance-trained
58 females [14], and also found CitMal to improve multiple indices of strength and power in female masters tennis players [105].
Despite these promising findings regarding the ergogenic potential of citrulline supplementation, several recent studies have also reported null findings. Chappell et al. [140] evaluated the effects of an 8 g CitMal dose in comparison to a placebo treatment, and found no ergogenic effect over ten sets of leg extension exercise. Gonzalez et al. [144] investigated the same dose, finding no significant improvement in bench press repetitions to fatigue. Furthermore, the exact mechanism driving the purported ergogenic effect of CitMal is not yet fully understood. Performance effects of CitMal may indeed be NO-mediated, but may also relate to citrulline’s role in ammonia clearance via the urea cycle [12], or malate’s role in aerobic energy production via the tricarboxylic acid (TCA) cycle [147] and malate-aspartate shuttle [165].
If substantial alterations occur in urea cycle function or aerobic energy production, such effects would likely be reflected by differences in post-exercise urea and lactate accumulation, respectively. With only one study to date assessing resistance-training outcomes following the consumption of dietary nitrate
(BEET) [24] , its effects remain uncertain in the absence of replication.
As such, more research is needed to evaluate the effects of CitMal and BEET on resistance exercise performance, and to evaluate the mechanisms that may be contributing to the purported ergogenic effect of CitMal. The purpose of the current study was to evaluate the effects of citrulline malate and beetroot juice supplementation on parameters of blood flow, metabolic efficiency, blood biomarkers (urea, lactate, and nitric oxide metabolites), and strength performance in the context of maximal leg extension exercise. It was hypothesized that CitMal and BEET would increase leg extension performance, NOx levels, blood flow, and metabolic efficiency of exercise in comparison to PLA, with no difference between treatments.
Methods
Experimental Approach to the Problem
The current study employed a randomized, double-blind, placebo-controlled, crossover design to investigate the performance effects of citrulline malate (CitMal) and beetroot juice (BEET) supplementation. Participants initially completed an enrollment visit, in which they were familiarized with
59 the leg extension exercise protocol. Between 2-10 days later, they returned to complete a series of 3 testing visits, separated by 5-10 days. At each testing visit, participants ingested 1 of 3 treatment beverages (CitMal, BEET, or placebo), followed by a 2 h rest period. Resting measurements were obtained prior to the onset of exercise, including femoral artery blood flow parameters (vessel diameter
[aDIAM] and blood flow [aBF]), vastus lateralis (VL) cross-sectional area (CSA) and echo intensity (EI), and blood analytes (plasma lactate, nitrate/nitrite [NOx], and blood urea nitrogen [BUN]). Participants completed a leg extension warmup, followed by an isokinetic leg extension protocol consisting of 5 sets of
30 maximal concentric muscle actions, for which peak torque, mean torque, and total work were measured. Indirect calorimetry was used to measure energy expenditure (EE) and respiratory exchange ratio (RER) at rest and throughout the exercise test. Following exercise, participants laid supine and post- test measurements were obtained. The testing visit timeline is presented in Figure 7. Artery blood flow, aDIAM, CSA, and EI were collectively measured to assess the effects of supplementation on the control of blood flow and fluid accumulation in the active musculature. Whole-body EE, RER, and plasma lactate were measured to assess energy efficiency and fuel utilization during exercise. Plasma NOx was measured to assess nitric oxide metabolism, and BUN was measured to assess ammonia clearance via the urea cycle. These assessments of blood flow, energy expenditure, substrate utilization, and blood analytes were selected to evaluate several potential mechanisms by which acute NO precursor supplementation may influence resistance exercise performance.
Subjects
Healthy, recreationally active, male non-smokers were recruited for the current study. A priori power analysis indicated that 21 participants would be required to ensure 80% power to detect a difference in leg extension torque between treatments with an alpha level of 0.05, assuming a correlation of 0.7 among repeated measures. To account for anticipated subject dropout and maintain a counterbalanced design, 30 total participants were enrolled. Participants were recreationally active, as defined by at least 2 h of exercise per week for at least 8 weeks preceding enrollment. Participants were free from any injuries or medical conditions that would influence blood flow, exercise tolerance, or the ability to perform leg extensions to failure. To qualify for inclusion, participants could not have used
60 medications or supplements with potential to influence study outcomes in the 8 weeks preceding enrollment. Participants were excluded if they had used recreational drugs in the past month, or if they had lost or gained ≥ 4.5 kg, participated in a clinical trial altering exercise or nutrition habits, or consumed more than 3 alcoholic drinks per day.
Participants were encouraged to maintain their normal diet and exercise habits throughout the study. Three-day diet logs were used to assess habitual dietary habits; The Food Processor software
(ESHA Research, Salem, OR, USA) was used to calculate typical macronutrient intakes, and habitual dietary nitrate intake was calculated using reference values reported by Jonvik et al. [175]. All testing visits occurred at the same time of day (± 1 h), and subjects were required to fast for at least 4 h preceding each visit. Within 24 h of testing, participants were instructed to avoid strenuous exercise and alcohol consumption, and caffeine was restricted within 12 h of test visits. Participants were instructed to avoid mouthwash use on the day of testing, and to avoid chewing gum or brushing their teeth within 8 h of visits [174]. All study procedures were approved by the University’s Biomedical Institutional Review
Board, and all participants provided informed consent prior to participation.
Familiarization Visit
A single familiarization visit was completed prior to the first testing visit. Following written informed consent, participants completed a health history questionnaire and received an explanation of pre-visit guidelines. Participants were then positioned on the isokinetic dynamometer (HUMAC NORM,
Computer Sports Medicine Inc., Stoughton, MA, USA) with a hip angle of 110°, and seat settings were recorded to enable replication at future visits. Leg extension maximal voluntary contraction (MVC) torque was measured as the highest of three attempts, with the knee fixed at 90° of flexion. Participants then completed a familiarization protocol consisting of 3 sets of 30 concentric leg extension repetitions, with 1 min of rest between sets. With each repetition, the leg was extended from a 90° knee angle to full extension at a speed of 180°∙s-1, and the leg passively returned to the flexed position at a speed of
90°∙s-1. All dynamometer testing occurred with the arms folded across the chest, with participants fastened to the chair by a seatbelt crossing the chest and waist. Participants returned for the first testing visit 2-10 days later.
61 Supplementation
Upon arrival for testing sessions, height and weight were measured using a stadiometer
(Perspective Enterprises PE-AIM-101, MI, USA) and calibrated electronic scale (Health-O-Meter 2101KL,
IL, USA). Measurements were taken in light athletic clothing, with the shoes removed. Participants then consumed 1 of 3 treatment beverages in a randomized order: a 70-mL shot of beetroot juice containing
400 mg nitrate (BEET; Beet It Sport, James White Drinks Ltd., Ipswich, UK); a placebo drink of blackcurrant juice (70 mL; Ribena, Lucozade Ribena Suntory Ltd., Uxbridge, UK); or 8 g of citrulline malate (CitMal; 2:1 ratio, BulkSupplements.com, NV, USA) mixed into the placebo beverage. To enhance the uniformity of treatments, additional lemon juice and sweetener (Crystal Light; Kraft Foods, IL, USA) were used to mask flavors. Treatments were mixed in opaque containers by an individual that was not present for supplement ingestion or testing. Peak blood levels of NO precursors are achieved approximately 1.4-2.3 h after citrulline ingestion [6] and 2-3 h after BEET ingestion [176]; as such, treatments were ingested 2 h prior to testing. Treatment sequence was randomly assigned using Random
Allocation Software (Isfahan, Iran).
Blood Analytes
A 10-mL venous blood sample was obtained from the antecubital region of the arm before (PRE) and after (POST) exercise, using lithium heparin-coated tubes. Samples were centrifuged immediately, and aliquots of plasma were frozen at -80° C for batch analysis. Blood was sampled to evaluate the effects of supplementation on urea cycle function (BUN), anaerobic metabolism (lactate), and NO activity
(nitrate/nitrite; NOx). Commercially available, enzyme-linked assays were used to quantify BUN (Kit
MBS9305638; MyBioSource.com, CA, USA), total lactate (Kit MBS755961; MyBioSource.com, CA, USA), and total NOx (Kit KGE001; R & D Systems, MN, USA). All assays were performed in duplicate and averaged for analysis. Assay coefficients of variation ranged from 3.6%-7.6%.
Ultrasound: Artery Blood Flow, Vastus Lateralis Imaging
Doppler-mode ultrasound (Logiq-e, GE Healthcare, IL, USA) was used to assess vessel diameter and blood flow. The probe (12LRS, 5-13 mhz) was placed over the superficial branch of the femoral
62 artery, 1-3 cm distal to the point at which the common femoral artery bifurcates into deep and superficial branches. Duplex mode was used to allow for simultaneous imaging of the superficial femoral artery and the spectral waveform velocity profile. A minimum of 4 cardiac cycles were recorded per captured image; a perpendicular line was drawn across the artery to measure its diameter, and the measure function within the device’s default software was used to obtain values of artery diameter and blood flow. Within each image, velocity values were averaged among up to 4 consecutive cardiac cycles; at least 2 images were captured per time point, with values averaged for analysis. Test-retest reliability values for aDIAM
(intraclass correlation coefficient [ICC] = 0.82, standard error of measurement [SEM] = 0.03 cm) and aBF
(ICC = 0.86, SEM = 5.92 mL∙min-1) have previously been reported using this methodology to assess the brachial artery [54].
Ultrasound was also used to assess vastus lateralis CSA and EI, to assess muscle belly swelling as a result of exercise hyperemia. A panoramic scan was performed at the midpoint of the VL, and images were analyzed offline using ImageJ software (National Institute of Health, MD, USA, Version
1.37). The polygon tool was used to trace the VL border to encapsulate the entire muscle, without including the surrounding fascial border. The area of the resultant polygon, and the relative brightness of its constituent pixels (EI), were then calculated. Test-retest reliability values for VL CSA (ICC = 0.87, SEM
= 2.12 cm2) and EI (ICC = 0.74, SEM = 4.58 arbitrary units [A.U.]) have previously been reported using this methodology [177]. Resting scans were obtained following at least 5 min of supine rest. Post-exercise scans were taken in the supine position, with vessel scans obtained 5 min following the conclusion of the exercise bout [57].
Indirect Calorimetry
To evaluate energy efficiency, respiratory gases were collected for 15 min prior to exercise
(PRE), and throughout the maximal leg extension test. A mouthpiece and hose were used to connect participants to an indirect calorimeter (TrueOne 2400, ParvoMedics, Inc., Sandy, UT, USA), and data were collected continuously and averaged every 15 s. Expired gases were used to calculate EE (KCal∙d-1) and RER (AU) by the device’s default software, at rest and during maximal exercise, using the following equations:
63 푘푐푎푙 퐸퐸 ( ) = [(3.9 ∗ (푉푂 (퐿 ∗ 푚푖푛−1))) + (1.1 ∗ (푉퐶푂 (퐿 ∗ 푚푖푛−1)))] ∗ 1440 푚푖푛 푑푎푦 2 2
−1 푉퐶푂2 (퐿 ∗ 푚푖푛 ) 푅퐸푅 = −1 푉푂2 (퐿 ∗ 푚푖푛 )
For resting values, the first 5 min of resting data were discarded, and the remaining 10 min were used for analysis; exercise values were calculated from gases collected during the 8-min exercise period.
The rate of EE (KCal∙d-1) was calculated for each span of time, and values for EE were then converted from KCal∙d-1 to KCal burned in 8 min by dividing by 180. Statistics pertaining to test-retest reliability for resting EE (ICC = 0.94, SEM = 125.6 kcal∙d-1) and RER (ICC = 0.83, SEM = 0.03 units) using similar methodology have been reported previously [179].
Exercise test
Prior to maximal exercise testing, a leg extension warmup was completed with the right leg.
Participants completed approximately 8 min of isotonic concentric leg extensions with 25% of MVC torque, with 1 repetition completed every 4 s, as described elsewhere (Trexler et al., in preparation).
Participants then completed a single set of 5 isokinetic leg extensions with escalating effort; the first repetition was completed at 50% effort, escalating up to 100% effort on the fifth. After the warmup, participants completed 5 sets of 30 maximal-effort, concentric leg extensions. Repetitions were completed from a 90° knee angle to full extension at a speed of 180°∙s-1, with passive leg flexion (90°∙s-1) between repetitions, and 1 min of passive rest between sets. The dynamometer’s default software was used to calculate gravity-adjusted values for peak torque, average torque, and total work for each set, with a sampling rate of 100 Hz. Peak torque was calculated as the highest torque value obtained during any single repetition during the set, whereas average torque was the average of the peak torque values obtained for all repetitions in a given set. Total work for each set was calculated as the sum of the total area under the torque-position curve for each repetition of a set. For each variable, values from all 5 sets were summed for analysis. Immediately following the final set, participants were laid supine in preparation for post-exercise measurements.
64 Statistical Analyses
A series of general linear mixed models were used for data analysis. Random intercept models were fitted, with subject identified as a random effect. Separate preliminary models were fitted to confirm that carryover, sequence, period × treatment, and habitual nitrate × treatment interaction effects were nonsignificant. Leg extension outcomes (peak torque, average torque, and total work) were assessed by fitting mixed models with fixed effects including period and treatment. All other outcomes were analyzed with fixed effects including treatment as the predictor variable, and period and resting values as covariates. Data missing due to technician or equipment error were assumed to be missing completely at random, with all omnibus tests including at least 77 of 79 possible observations. Significant treatment effects were followed by pairwise comparisons, using the Tukey-Kramer adjustment for multiplicity. Model residuals were visually assessed to confirm the absence of heteroscedasticity, correlated residuals, and non-normal distributions. As a secondary analysis, baseline NOx values were compared to assess the effects of treatment on resting NO metabolites. All analyses were performed using PROC MIXED (SAS
Software, NC, USA); the criterion for statistical significance was set a priori at α = 0.05. Descriptive demographic data are presented as mean ± standard deviation; all other data presented as adjusted least squares mean ± standard error.
Results
Participants
Thirty participants enrolled in the current study. Three withdrew prior to the first testing visit, and one withdrew after visit 1; those providing a reason for withdrawal cited schedule constraints or injuries unrelated to the study. Individuals withdrawing from the study did not appear to differ from those completing the study with regard to any identifiable distinguishing characteristics. Twenty-seven participants completed at least one testing visit (age: 22 ± 4 yrs; height: 178.4 ± 6.8 cm; weight: 78.9 ±
12.5 kg). Of the returned diet logs (n = 25), average dietary intakes were 2558 ± 699 KCal∙d-1, 280 ± 90 g∙d-1 carbohydrate, 101 ± 34 g∙d-1 fat, 131 ± 42 g∙d-1 protein, and 115 ± 98 mg∙d-1 nitrate.
65 Blood Analytes
Model residuals for BUN and NOx were not normally distributed, so log transformations were performed for both. Treatment had a significant effect on resting baseline NOx levels (F[2, 47.8] = 303.68, p < 0.0001), with higher values in BEET (233.2 ± 1.1 μmol∙L-1) compared to CitMal (15.3 ± 1.1, p <
0.0001) and PLA (13.4 ± 1.1, p < 0.0001). The effect of treatment on post-exercise NOx levels was also significant (F[2, 56.5] = 19.60, p < 0.0001), with significantly higher values in BEET (86.3 ± 1.2 μmol∙L-1) than CitMal (21.3 ± 1.1, p < 0.0001) and PLA (18.1 ± 1.1, p < 0.0001). The effect of treatment on post- exercise BUN values was not significant (F[2, 47.4] = 2.13, p = 0.13; CitMal: 1.91 ± 1.03, PLA: 1.94 ±
1.03, BEET: 1.81 ± 1.03 mmol∙L-1). One participant presented at all 3 testing visits with resting lactate values over 4 standard deviations from the group mean; all of this participant’s data were excluded from lactate analyses. The effect of treatment on post-exercise lactate was nonsignificant, but a trend was observed (F[2, 44.8] = 2.53, p = 0.09), with a trend for CitMal (0.46 ± 0.03 mmol∙L-1) to be lower than PLA
(0.54 ± 0.03 mmol∙L-1, p = 0.08), but not BEET (0.49 ± 0.03 mmol∙L-1, p = 0.62). Blood analyte outcomes are presented in Figure 8.
Leg Extension Performance
Leg extension peak torque was not significantly affected by treatment (F[2, 48.2] = 0.08, p = 0.92;
CitMal: 646.0 ± 21.6, PLA: 648.9 ± 21.6, BEET: 645.8 ± 21.6 N∙m). Average leg extension torque was also unaffected by treatment (F[2, 48.2] = 0.49, p = 0.62; CitMal: 567.8 ± 20.6, PLA: 577.4 ± 20.5, BEET:
572.4 ± 20.6 N∙m). Similarly, treatment did not have a significant effect on total leg extension work (F[2,
48] = 0.09, p = 0.91; CitMal: 12969 ± 414, PLA: 12993 ± 413, BEET: 13042 ± 414 N∙m). Leg extension outcomes are presented in Figure 9. To investigate the possibility of set-specific effects, additional models were constructed to assess the effect of supplementation at each individual set of leg extension.
These exploratory analyses confirmed that supplementation did not significantly affect peak torque, average torque, or total work for any set (all p > 0.05).
66 Ultrasonography
The effect of treatment on muscle CSA was not significant, but a trend was observed (F[2, 44.1] =
3.15, p = 0.053), with CitMal (35.4 ± 0.4 cm2) tending to be lower than PLA (36.2 ± 0.4 cm2, p = 0.06), but not BEET (36.1 ± 0.4 cm2, p = 0.13). Treatment had a significant effect on muscle EI (F[2, 45.8] = 5.78, p
= 0.006). Values for BEET were significantly higher than PLA (68.0 ± 0.7 vs. 65.5 ± 0.7 AU, p = 0.005), and tended to be higher than CitMal (66.3 ± 0.7, p = 0.07). Muscle ultrasound outcomes are presented in
Figure 10. Treatment did not significantly affect aDIAM (F[2, 44.1] = 1.67, p = 0.20; CitMal: 0.72 ± 0.01,
PLA: 0.71 ± 0.01, BEET: 0.73 ± 0.01 cm). Similarly, aBF values were not significantly affected by treatment (F[2, 46.8] = 0.71, p = 0.50; CitMal: 457 ± 34, PLA: 458 ± 34, BEET: 486 ± 34 mL∙min-1). Visual inspection of residuals suggested that log transformation may be warranted for aBF, but log transformation also yielded a nonsignificant treatment effect (p = 0.72).
Indirect Calorimetry
The effect of treatment on EE was nonsignificant, but a trend was observed (F[2, 44.6] = 2.50, p =
0.09). Values tended to be higher in BEET compared to PLA (37.8 ± 1.5 vs. 35.4 ± 1.5 kcal, p = 0.08), but not CitMal (36.2 ± 1.5 kcal, p = 0.32). Treatment did not have a significant effect on RER (F[2, 47.5] =
0.23, p = 0.80; CitMal: 1.04 ± 0.01, PLA: 1.03 ± 0.01, BEET: 1.04 ± 0.01 AU).
Discussion
Nitric oxide precursor supplements, such as CitMal and BEET, have been shown to have ergogenic effects through a number of potential mechanisms, including improvements in blood flow, energy efficiency, ammonia clearance, and aerobic ATP production [10, 12]. Although BEET increased plasma NOx, this effect did not translate to improvements in any of the blood flow, energy efficiency, or performance parameters measured. Results of the current study suggest that neither CitMal nor BEET significantly enhance leg extension strength or performance, energy efficiency, exercise hyperemia, or hormonal responses to isokinetic leg extension exercise in recreationally active males.
The most notable finding of the current study is the absence of an ergogenic effect on leg extension exercise. Citrulline malate has previously been shown to enhance bench press repetitions to
67 fatigue (RTF) in resistance trained males [12]. This finding was later supported by studies showing improved upper-body RTF [15] and lower-body RTF [13] in resistance trained males and females [14]. A single study has previously evaluated the effects of BEET on isotonic resistance exercise, with a significant increase in RTF observed in resistance trained males [24]. In contrast, the current study found no such benefit on isokinetic leg extension, despite causing a marked increase in pre-exercise plasma
NOx levels. Several methodological factors may explain the lack of ergogenic effects in the current study, such as the type of exercise test utilized, the dosing strategy of supplementation, and the characteristics of the population sampled.
Rather than open-endpoint tests of dynamic constant external resistance exercise with multi-joint exercises, the current study implemented an isokinetic, single-joint, fixed-endpoint test to allow for more precise assessment of force production. Chappell et al. [140] utilized a similar, 10-set isokinetic leg extension test, and also found no performance benefit from CitMal. Both ex vivo [63] and in vivo [194] studies have shown nitrate to enhance muscle contractile properties in a manner that increases the rate of force production and shortening velocity [191], which may be attenuated in exercise tests in which the speed of muscle actions is constrained. Coggan et al. [191] suggest that acute effects of NO precursors on muscle contractile function are attributable to increased calcium release in the sarcoplasmic reticulum and enhanced myofibrillar calcium sensitivity, thereby increasing twitch force, contraction velocity, rate of force development, and maximal power. This is supported by recent experimental findings [195], in which acute BEET supplementation enhanced leg extension torque and power at an angular velocity of 360°∙s-1, but not at velocities ranging from 90-270°∙s-1. In order to replicate these observations, future studies should investigate the effects of BEET and CitMal supplementation within a wide range of contraction velocities, with a particular emphasis on high-velocity muscle actions.
In addition, differences in dosing strategies should be noted. Peak blood levels of NO precursors are achieved approximately 1.4-2.3 h after citrulline ingestion [6] and 2-3 h after BEET ingestion [176]. As such, the current study provided treatments 2 h prior to exercise, whereas most previous citrulline studies have tested outcomes 1 h after supplement ingestion. Cutrufello et al. [142] investigated the performance effects of L-citrulline provided 1 h or 2 h before chest press testing; although neither timing strategy significantly improved performance, the 1 h condition yielded 3 additional repetitions compared to
68 placebo, whereas the 2 h condition resulted in completion of 1 less repetition than placebo. Mosher et al.
[24] provided a 6-day supplementation protocol to increase plasma NOx values over time, rather than a single, acute dose prior to exercise. Multiple-day BEET supplementation interventions enhance performance more reliably than acute, single-dose interventions [10], which may explain these discrepant findings. In addition, a dose-response relationship for BEET has been observed in other exercise modalities, with more favorable effects documented after 8.4 mmol nitrate in comparison to 4.2 mmol
[176, 196]. As the current study administered a dose of only 6.4 mmol (400 mg), further research is needed to explore the possibility that ergogenic effects may be observed at higher doses.
Given the lack of ergogenic effect in the current study, it is intuitive that indices pertaining to blood flow, energy efficiency, ammonia clearance, and lactate accumulation were largely unaffected by the experimental treatments. Previous research in adults with self-reported fatigue has indicated that CitMal enhances the efficiency of aerobic metabolism in exercising muscle after several days of supplementation
[147]. While the current study found no effect of CitMal or BEET on lactate values, the observed post- exercise lactate levels suggest that the exercise test did not elicit a large lactate response. Nonetheless, previous studies noting the ergogenic effects of BEET [24] and CitMal [13, 15] have documented increased RTF in the absence of effects on lactate, casting doubt on reduced lactate accumulation as a primary ergogenic mechanism. Similarly, post-exercise BUN values in the current study were not elevated beyond typical resting levels. Studies have shown no effect of BEET [197] on urinary urea levels, and citrulline supplementation has been shown to preserve post-race jump height performance in the absence of any effect on blood urea levels [78]. CitMal did not increase plasma NOx to a greater extent than PLA, whereas BEET caused a marked increase. Previous research has suggested that citrulline supplementation increases post-exercise changes in plasma NOx to a great extent than PLA, but this effect was not observed in the current study. This discrepancy may relate to the form and dosing strategy of supplementation; McKinley-Barnard et al. [112] provided 2 g∙d-1 of L-citrulline for 7 days, and also noted that co-ingestion of the antioxidant glutathione enhanced this effect. However, Suzuki et al. [75] used a similar dosing protocol with L-citrulline and found no significant impact on plasma NOx changes following a 4 km cycling time trial, despite observing a performance improvement.
69 Several studies have reported reduced energy cost of walking [58], running [58], cycling [59], and bilateral leg extension [61] exercise after BEET supplementation. While the exercise protocol in the current study elevated energy expenditure beyond normal resting levels, neither CitMal nor BEET significantly influenced energy expenditure during exercise. Similarly, RER was elevated in response to exercise, but was unaffected by either supplement. Notably, previous studies documenting enhanced energy efficiency of exercise have typically provided nitrate supplements for multiple (3-6) days prior to testing. Nitric oxide may enhance exercise efficiency by altering mitochondrial function via reductions in uncoupling protein-3 (UCP-3) and adenosine nucleotide translator (ANT) expression [60], but enhanced excitation-contraction efficiency via increased expression of calcium-handling proteins in muscle
(calsequestrin 1 and dihydropyridine receptor) may be an alternative mechanistic explanation [63]. As both potential mechanisms are influenced by changes in protein expression, changes are more likely to be observed with longer-term (multi-day) supplementation strategies [63]. However, it should be noted that Whitfield et al. [192] found no change in calcium-handling proteins after 1 week of BEET supplementation, despite improvements in muscle force production. There is also evidence to indicate that acute nitrate supplementation enhances contraction efficiency via nitrosylation of ryanodine receptors and/or activation of guanylyl cyclase [191], but these effects have largely been observed with nitrate doses of 8.8 mmol and above. As such, dosing strategies using longer durations of supplementation or larger acute doses appear to be warranted.
Blood flow-related outcomes in the current study (aDIAM, aBF, CSA) suggest no effect of either supplement on blood flow. The current study was carried out in a young, healthy, and relatively active sample, which may explain the absence of effect. Nitric oxide production and bioavailability decrease with age, and are increased by regular physical activity. As such, Casey et al. [183] demonstrated that BEET supplementation increases the compensatory vasodilator response to hypoxic handgrip exercise in older adults (age: 64 ± 2 yr), but not in younger adults (age: 25 ±1 yr). Interestingly, muscle EI was elevated by
BEET in comparison to PLA in the current study. Recent research has shown acute increases in CSA and
EI values immediately following fatiguing resistance exercise of the elbow flexors [187], presumably due to fluid accumulation from exercise hyperemia. As such, the small difference observed may suggest that
BEET modestly influences the accumulation of intramuscular fluid following resistance exercise, but not in
70 a magnitude substantial enough to alter CSA. However, this 2.5-unit difference in EI should be interpreted cautiously, as this magnitude of change falls within the standard error of the measurement and is of questionable physiological significance.
The primary limitation of this study is the use of single-leg, isokinetic leg extension as the exercise task. While it allows for more sensitive and precise measurement of force production in comparison to traditional resistance training, a relatively small amount of total muscle tissue is activated.
Full-body, dynamic constant external resistance exercise tests loading both eccentric and concentric muscle actions would impose a greater metabolic demand, and thereby more pronounced changes in the physiological measurements obtained. Similarly, isokinetic testing constrains the velocity of movement, which could potentially mask supplement effects related to the rate of force production or shortening velocity of muscle actions. Finally, the current study implemented a 2 h wait period following a single supplement dose. Pharmacokinetic data suggest that such a compromise is appropriate, but studies documenting the efficacy of CitMal and BEET often implement waiting periods of 1 and 2.5-3 h, respectively, and the effects of BEET appear to be more pronounced when using larger doses or multi- day dosing protocols.
Conclusions
Recent studies have reported that both CitMal and BEET increase repetitions to fatigue during dynamic constant external resistance exercise tests [13, 15, 24], but the effects of these NO precursor supplements on resistance exercise performance have not been directly compared to date. In addition, the mechanisms underlying this effect are not conclusively known, but it has been speculated that alterations in NO production, blood flow, energy efficiency, substrate utilization, lactate clearance, or ammonia clearance may contribute. Results of the current study suggest that neither 8 g CitMal nor 70 mL BEET enhance isokinetic leg extension performance in recreationally active males. Similarly, neither treatment improved indices of blood flow, metabolic efficiency, or the hormonal response to exercise to a meaningful degree. It is possible that higher or more sustained dosing strategies, or exercise tests involving more rapid muscle actions and activation of more total musculature, may be required to observe ergogenic effects of CitMal and BEET. Null effects on blood flow, metabolic efficiency, lactate clearance,
71 and plasma BUN suggest that additional focus on the direct effects of NO on muscle contractile properties [191] is warranted in future mechanistic research on acute NO precursor supplementation.
While further research is needed to investigate optimal dosing protocols and the various physiological mechanisms mediating the effects of acute NO precursor supplementation, the current data do not suggest that acute CitMal or BEET supplementation enhance isokinetic leg extension performance.
72
CHAPTER 6: CONCLUSIONS
In the past decade, numerous studies have documented improvements in aerobic exercise performance following supplementation with nitric oxide precursors, such as BEET and CitMal [4, 10].
More recently, studies investigating the effects of these supplements on strength and power outcomes have begun to emerge [12, 13, 15, 24]. In the current project, meta-analytic techniques were used to summarize the citrulline literature currently available, with results suggesting a small but significant effect of citrulline-based supplements on strength and power outcomes. Notably, only thirteen published citrulline experiments warranted inclusion in this meta-analysis, and only one study investigating the effects of BEET on outcomes resembling traditional resistance exercise has been published to date [24].
To address the paucity of research in this area, the current project also investigated hemodynamic and metabolic responses to both submaximal and maximal leg extension exercise. Resting blood pressure was not influenced by BEET and CitMal in the current project, and indices of artery blood flow, muscle blood flow, muscle fluid accumulation, whole-body energy expenditure, muscle oxygen consumption, and substrate utilization were largely unaffected during submaximal leg extension exercise.
During maximal isokinetic leg extension exercise, neither CitMal nor BEET influenced peak torque, average torque, or total work to a significant degree. Similarly, arterial blood flow, muscle fluid accumulation, whole-body energy expenditure, substrate utilization, lactate accumulation, and plasma
BUN responses to maximal exercise were largely unaffected by supplementation. Taken together, results of the current project do not support the hypothesis that previously reported ergogenic effects of NO precursor supplements on dynamic constant external resistance exercise translate to isokinetic leg extension at 180°∙s-1. Similarly, the current results do not support the hypothesis that these previously reported ergogenic effects in young, healthy participants are driven by mechanisms related to blood flow, energy expenditure, substrate utilization, lactate clearance, or ammonia clearance, as these factors were not significantly influenced by acute CitMal or BEET supplementation in the current sample of young, healthy, normotensive individuals.
73 Collectively, the body of research on BEET and CitMal supplementation appears to suggest that acute supplementation specifically enhances performance during high-velocity isokinetic actions [195,
198], or actions in which contraction velocity is not constrained [12, 13, 15, 24]. Based on the current findings, these effects do not appear to be mediated by acute effects on blood flow, energy expenditure, substrate utilization, lactate clearance, or ammonia clearance. As such, previously reported ergogenic effects may be primarily mediated by direct effects of NO on sarcoplasmic reticulum calcium release and myofibrillar calcium sensitivity, resulting in an enhancement of twitch force, shortening velocity, rate of force development, and maximal power [191]. Future research should aim to investigate which exact components of resistance exercise performance are influenced by NO precursor supplementation, how to optimize dosing protocols to affect these outcomes, and which physiological mechanisms mediate the effects of acute NO precursor supplementation on resistance exercise performance.
74
TABLES
Table 1. Characteristics of studies included in the analysis
Study Design Age Sample Sex Train- Supplement Timing Modality Strength or Upper- or Outcomes included (First author, (Mean ± size ing Form Power Lower-Body year) SD) Status Exercise Repetitions Wax, 2016 23.3 ± (multiple RDB 14 M RT 8 g CitMal 60 min RE Strength Upper [15] 1.5 exercises, multiple sets) Repetitions Wax, 2015 22.1 ± (multiple RDB 12 M RT 8 g CitMal 60 min RE Strength Lower [13] 1.4 exercises, multiple sets) Perez- Bench press 29.8 ± Guisado, RDB 41 M RT 8 g CitMal 60 min RE Strength Upper repetitions 7.6 2010 [12] (multiple sets) Martinez- 3.3 g L- Peak and mean 23.9 ± Sanchez, RDB 19 M RT Citrulline in 60 min RE Mix Lower squat force and 75 3.7
2017 [164] WMJ power
Bench press repetitions, peak Gonzalez, 21.4 ± RDB 12 M RT 8 g CitMal 40 min RE Mix Upper power, and mean 2017 [144] 1.6 power (multiple sets) Repetitions Glenn, 23.0 ± (multiple RDB 15 F RT 8 g CitMal 60 min RE Strength Mix 2017 [14] 3.0 exercises, multiple sets) Relative peak Glenn, RDB 51 ± 9.0 17 F ST/ET 8 g CitMal 60 min Cycling Power Lower power, anaerobic 2016 [105] capacity
Repetitions (multiple exercises, multiple sets), Farney, 24.1 ± RSB 12 Mix REC 8 g CitMal 60 min RE Mix Lower peak and mean 2017 [143] 3.9 leg extension torque & power before and after circuit training Repetitions (single set, da Silva, 24.0 ± RDB 9 M REC 6 g CitMal 60 min RE Strength Lower multiple 2017 [199] 3.3 exercises, multiple days) Cutrufello, 20.8 ± 6 g L- Repetitions 2015a RDB 10 Mix ST/ET 60 min RE Strength Upper 1.3 Citrulline (multiple sets) [142] Cutrufello, 20.8 ± 6 g L- Repetitions 2015b RDB 12 Mix ST/ET 120 min RE Strength Upper 1.3 Citrulline (multiple sets) 76 [142]
Peak and mean Cunniffe, 23.5 ± RDB 10 M ST/ET 12 g CitMal 60 min Cycling Power Lower power (multiple 2016 [141] 3.7 sprints) Repetitions (multiple sets); Chappell, 23.7 ± isometric, RDB 15 Mix RT 8 g CitMal 60 min RE Strength Lower 2018 [140] 2.4 concentric, and eccentric peak torque SD = Standard Deviation; RDB = Randomized, Double-Blinded; RSB = Randomized, Single-Blinded; CitMal = Citrulline Malate; M = Male; F =
Female; RT = Resistance Trained; REC = Recreationally Active; ST = Sport Trained; ET = Endurance Trained; WMJ = Watermelon Juice; min =
Minutes; RE = Resistance Exercise
Table 2. Subgroup analyses
Subgroups n studies SMD 95% CI p value
Sex
Male-only 7 0.23 [-0.01, 0.47] 0.06
Females included 6 0.16 [-0.14, 0.45] 0.29
Training Status
RT 7 0.21 [-0.02, 0.44] 0.08
non-RT 6 0.18 [-0.13, 0.49] 0.26
Citrulline Form
Citrulline malate 10 0.22 [0.01, 0.43] 0.04
Other 3 0.13 [-0.28, 0.54] 0.53
Musculature Tested
Lower-body only 7 0.17 [-0.10, 0.43] 0.21
Upper-body included 6 0.23 [-0.03, 0.49] 0.08
Type of Exercise Outcome
Strength only 8 0.30 [0.06, 0.54] 0.01
Power included 5 0.04 [-0.25, 0.34] 0.77
Modality of Exercise
Resistance exercise 11 0.21 [0.01, 0.41] 0.04
Cycle ergometry 2 0.15 [-0.35, 0.64] 0.56
Funding Source
Industry/undisclosed 5 0.23 [-0.04, 0.50] 0.10
Other 8 0.17 [-0.08, 0.43] 0.19 n studies = Number of studies; SMD = standardized mean difference (Hedges’ G); 95% CI = 95% confidence interval; RT = resistance trained
77
Table 3. Participant characteristics (n = 27) and dietary intake information (n = 25 logs returned)
Variable Mean SD Min Max
Age (yr) 22 4 18 35
Height (cm) 178.4 6.8 166.3 189.0
Weight (kg) 78.9 12.5 62.5 104.2
MVC (N∙m) 222 43 150 331
VL FT (cm) 0.5 0.2 0.3 1.1
VL MT (cm) 2.8 0.3 2.3 3.5
KCal∙d-1 2558.0 698.7 1173.0 4723.0
CHO (g∙d-1) 279.9 90.0 107.7 526.5
FAT (g∙d-1) 101.3 33.8 48.8 154.5
PRO (g∙d-1) 131.1 42.0 70.5 262.5
Nitrate (mg∙d-1) 115.1 98.1 36.1 410.9
SD = standard deviation; Min = minimum value; Max = maximum value; MVC = maximum voluntary contraction
torque; VL = vastus lateralis; FT = subcutaneous fat thickness at site of probe placement; MT = muscle
thickness at site of probe placement; KCal = kilocalories; CHO = carbohydrate; PRO = protein
78
Table 4. Resting blood pressure measurements after supplementation
Mean (mmHg) SE F (Treatment) p (Treatment)
Supine SBP F(2, 48) = 0.30 p = 0.74
CitMal 117.3 1.2
PLA 118.5 1.1
BEET 117.6 1.2
Supine DBP F(2, 49.6) = 0.74 p = 0.48
CitMal 68.4 1.1
PLA 70.0 1.1
BEET 69.9 1.1
Standing SBP F(2, 44.9) = 0.38 p = 0.68
CitMal 110.3 1.8
PLA 110.6 1.8
BEET 108.7 1.8
Standing DBP F(2, 47.9) = 0.17 p = 0.84
CitMal 66.4 1.0
PLA 66.7 1.0
BEET 67.1 1.0
SBP = systolic blood pressure; DBP = diastolic blood pressure; SE = standard error. Data
presented as least square mean ± SE. A significant carryover effect was observed for
standing DBP (p < 0.001), but analysis stratified by previous treatment revealed no significant
treatment effect in the period following the provision of CitMal, PLA, or BEET (all p > 0.05).
Similarly, analysis of data from period 1 only revealed no significant treatment effect (p =
0.42)
79
FIGURES
Figure 1. PRISMA diagram detailing systematic search and screening process
80
Figure 2. Funnel plot (standard error vs. Hedges’ G) for studies meeting inclusion criteria
81
Weight Hedges’ G (%) [95% CI]
Favors Placebo Favors Citrulline
Figure 3. Forest plot of studies meeting inclusion criteria. CI = Confidence interval; RE Model = Random
Effects Model
82
Figure 4. Timeline of each testing visit (submaximal study). CitMal = Citrulline malate; BEET = Beetroot juice; PLA = Placebo; BP = Blood pressure; FA = Femoral artery; VL = Vastus lateralis; NIRS = Near- infrared spectroscopy.
83
mVO2
1
-
1
-
∙100g
1
-
∙100 ml ∙100
1
-
∙min
2
mLO mL∙min
Figure 5. Effects of treatment on muscle blood flow (mBF) and oxygen consumption (mVO2), as measured via near-infrared spectroscopy.
84
Figure 6. Effects of treatment on whole-body energy expenditure (EE) and respiratory exchange ratio
(RER), as measured via indirect calorimetry. KCal = kilocalories; AU = arbitrary units.
85
Figure 7. Timeline of each testing visit (maximal study). CitMal = Citrulline malate; BEET = Beetroot juice;
PLA = Placebo; FA = Femoral artery; VL = Vastus lateralis.
86
NOx
*
1
1
-
-
1
-
L
L
∙
∙
L
∙
mol
μ
mmol mmol
Figure 8. Plasma levels of blood urea nitrogen (BUN), nitrate/nitrite (NOx), and lactate. *Significantly greater than CitMal and PLA (p < 0.0001).
87
N∙m
N∙m N∙m
Figure 9. Leg extension outcomes. N∙m = Newton meters; Avg = average.
88
*
2 cm
Figure 10. Effects of treatment on muscle cross-sectional area (CSA) and echo intensity (EI). AU = arbitrary units. *Significantly greater than PLA (p = 0.005).
89
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