the effects of acute and chronic beetroot juice ... · 234 supplementation with beetroot juice or a...
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The Effects of Acute and Chronic Beetroot Juice Supplementation on 1 Exercise Economy and Time Trial Performance in Recreationally 2
Active Females 3 4
by 5 6
Kate Aiko Wickham 7 8 9 10 11 12 13 14
A Thesis 15 Presented to 16
The University of Guelph 17 18 19 20 21 22 23
In partial fulfillment of requirements 24 for the degree of 25
Masters of Science 26 in 27
Human Health and Nutritional Sciences 28 29 30 31 32 33 34 35 36
Guelph, Ontario, Canada 37 38
© Kate Aiko Wickham, July 2018 39
i
ABSTRACT 40 41 42
THE EFFECTS OF ACUTE AND CHRONIC BEETROOT JUICE 43 SUPPLEMENTATION ON EXERCISE ECONOMY AND TIME TRIAL 44
PERFORMANCE IN RECREATIONALLY ACTIVE FEMALES 45 46 47
Kate Aiko Wickham Advisor: 48 University of Guelph, 2018 Dr. Lawrence L. Spriet 49
50
Beetroot juice (BRJ) is a supplement that reduces the oxygen cost of submaximal 51
exercise and improves performance in recreationally active males, both acutely and chronically. 52
However, evidence supporting the effects of BRJ in females is lacking. This thesis examined the 53
effects of acute and chronic BRJ supplementation in 12 recreationally active females (VO2peak: 54
40.6 + 1.2 mL O2/kg/min). Using a randomized, double-blind, placebo-controlled, crossover 55
design, subjects supplemented with 280 mL BRJ or a nitrate-free placebo for 8 days separated by 56
a 9 + 1 days washout period. On days 1 (acute) and 8 (chronic), subjects completed a 57
submaximal cycling protocol and a 4 kJ/kg time trial. Acute and chronic BRJ supplementation 58
had no effect on VO2 at 50 or 70% VO2peak, despite large increases in plasma nitrate and nitrite. 59
Additionally, there was no difference in time trial completion. To conclude, recreationally active 60
females may not benefit from acute or chronic BRJ supplementation.61
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Acknowledgements 62
63
Thank you to my parents for their unwavering support throughout my academic and life 64
journey. Dad, thank you for teaching me to persevere and to work diligently in the pursuit of my 65
dreams. Mom, thank you for teaching me to carry myself with poise and a kind heart. I owe my 66
character and my successes to you both. 67
68
Thank you to my friends, near and far, for encouraging me to lead my best life and for 69
being by my side throughout my personal growth as an academic and an individual. Your life 70
stories, and the stories we share together, have had a profound impact on my life and have helped 71
shape my values and passions. 72
73
Thank you to my MSc advisor, Dr. Lawrence Spriet, for being a steadfast role model. 74
You have taught me how powerful the combination of charisma, intelligence, tenacity, and 75
compassion can be. Thank you for the countless opportunities and challenges you have provided 76
me with. These moments have reinforced my passions and have encouraged me to continue my 77
mission to pursue a career merging the worlds of sport, academia and industry. 78
79
Thank you to my current and past Spriet Lab family including Devin McCarthy, Jamie 80
Pereira, Sebastian Jannas, Jamie Whitfield, Tyler Vermeulen, Alex Gamble, and Jessica Bigg. I 81
am thankful that I had the privilege to share the last two years of adventure with you. Thank you 82
to my HHNS family for welcoming me with open arms and providing me with an unforgettable 83
graduate school experience. 84
85
Lastly, thank you to my research participants for their dedication to my studies. Your 86
willingness to drink beetroot juice for weeks, to abstain from caffeine on several occasions, and 87
to push yourselves to your physical limits – all completed with smiles on your faces – did not go 88
unnoticed or unappreciated. 89
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Table of Contents 90
Abstract ……………………………………………………………………………… i 91
Acknowledgements ……………………………………………………………… ii 92
Table of Contents ………………………………………………………………… iii - v 93
List of Tables ……………………………………………………………………… vi 94
List of Figures …………………………………………………………………… vii - viii 95
List of Symbols and Abbreviations ………………………………………………. ix - x 96
Chapter 1: Review of Literature ………………………………………………… 1 - 39 97 98
Nitric Oxide: Roles, Origins, and Targets …………………………………………… 1 - 4 99
Beetroot Juice Pharmacokinetics, Dose, Duration …………………………………. 4 - 10 100
Dietary Nitrate Pharmacokinetics ………………………………………………………… 4 - 5 101 Dose Response: Plasma Levels, Blood Pressure, Oxygen Uptake, and Performance …… 5 - 7 102 Acute vs. Chronic Supplementation: Plasma Levels, Oxygen Uptake and Performance … 8 - 10 103
104 The Effects of Beetroot Juice Supplementation on Exercise Economy and Athletic 105 Performance ……………………………………………………………………… 10 - 20 106
107 Moderate Intensity Submaximal Exercise Economy ……………………………. 10 - 12 108
Severe and Maximal Intensity Exercise Economy ………………………………. 12 - 13 109
Time Trial Performance ………………………………………………………… 14 - 16 110
Aerobic Cycling Time Trial Performance ……………………………………. 14 - 15 111 Aerobic Running Time Trial Performance …………………………………… 15 - 16 112 Water Sport Time Trial Performance ……………………………………………… 16 113
Time to Exhaustion …………………………………………………………………………. 17 114 Sprint and Team Sport Performance …………………………………………………. 17 - 18 115 Strength Performance ………………………………………………………………………. 18 116 Performance During Hypoxia ……………………………………………………………. 19 117 Conclusion …………………………………………………………………………………. 20 118
119 Individual Responses to Beetroot Juice Supplementation ……………………… 20 - 24 120
Fitness Status …………………………………………………………………………. 21 - 23 121 Diet ………………………………………………………………………………………… 23 122 Digestive Pathway …………………………………………………………………… 23 - 24 123 124
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Beetroot Juice Improves Exercise Economy and Performance - Potential Mechanisms of 125 Action …………………………………………………………………………… 24 - 32 126
Are We Sure It's Nitrate? ……………………………………………………………… 24 - 26 127 Improved Blood Flow ………………………………………………………………… 26 128 Improved Mitochondrial Function …………………………………………………… 27 - 29 129
Reduced Oxygen Cost of Mitochondrial ATP Resynthesis ………………… 27 - 28 130 Mitochondrial Biogenesis …………………………………………… 28 - 29 131
Improved Contractile Efficiency ………………………………………………… 29 - 32 132 Reduced ATP Cost of Skeletal Muscle Force Production …………………… 29 - 31 133 Reactive Oxygen Species ……………………………………………………… 31 - 32 134 135
Women - Addressing the Gender Gap …………………………………………… 32 - 39 136 Characterization of the Menstrual Cycle ……………………………………………………. 33 137 Nitric Oxide Varies Throughout the Menstrual Cycle ………………………………… 34 - 35 138 Pharmacokinetics of Hormonal Contraceptives …………………………………………… 35 139 Previous Female Beetroot Juice Literature …………………………………………… 36 - 39 140 Conclusions ………………………………………………………………………………. 39 141
Chapter 2: Statement of the Problem …………………………………………… 40 - 42 142
Rationale ………………………………………………………………………… 40 - 41 143
Hypotheses ………………………………………………………………………. 41 - 42 144
Chapter 3: Methods ……………………………………………………………… 42 - 50 145
Subjects ………………………………………………………………………………. 42 146
Study Design ……………………………………………………………………. 42 - 43 147
Pre-Experimental Tests ………………………………………………………… 43 - 44 148
Diet, Sleep and Exercise Standardization ……………………………………………. 45 149
Experimental Exercise Protocol ……………………………………………………… 46 150
Blood Sampling and Analysis ……………………………………………………. 47 - 49 151
Compliance …………………………………………………………………………… 49 152
Statistical Analysis ………………………………………………………………. 49 - 50 153
Chapter 4: Results ………………………………………………………………… 50 - 61 154
Blood Parameters ………………………………………………………………… 50 - 52 155
Pre-Trial Characteristics ………………………………………………………………. 52 156
Environmental Characteristics ………………………………………………………… 53 157
Hydration Status ………………………………………………………………………. 53 158
Exercise Economy 50% VO2peak ………………………………………………… 53 - 54 159
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Exercise Economy 70% VO2peak ………………………………………………… 55 - 56 160
Time Trial Performance ……………………………………………………………… 57 161
Compliance, Blinding, and Adverse Events ……………………………………… 60 - 61 162
Chapter 5: Discussion …………………………………………………………… 63 - 75 163
Comparison to Previous Work in Women ………………………………………. 63 - 65 164
Nitrate Digestion …………………………………………………………………. 65 - 67 165
Nitrate and Nitrite Levels ……………………………………………………………. 67 166
Antioxidant Capacity ………………………………………………………………… 68 167
Differences in Skeletal Muscle Fiber Types and Capillarization ………………… 69 - 70 168
Mitochondrial Efficiency …………………………………………………………. 70 - 71 169
Calcium-Handling Proteins ………………………………………………………. 71 - 72 170
Individual Responders ……………………………………………………………. 72 - 73 171
Limitations ……………………………………………………………………………… 73 172
Future Directions …………………………………………………………………. 74 - 75 173
Conclusion ……………………………………………………………………………. 75 174
References …………………………………………………………………………… 76 - 87 175 176
vi
List of Tables 177
Table 1 - Respiratory variables at 50% and 70% VO2peak following acute and chronic 178 supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values reported as 179 mean + SEM. *, Chronic trials demonstrated significantly higher carbon dioxide production 180 (VCO2) compared to acute trials. **, BRJ significantly decreased ventilation (VE) compared to 181 PLA. 61 182 183 Table 2 - Heart rate (HR), rating of perceived exertion (RPE) and revolutions per minute (rpm) 184 recorded at 5 and 10 min at 50% and 70% peak oxygen consumption (VO2peak) following acute 185 and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values 186 reported as mean + SEM. 62 187
188
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List of Figures 189
Figure 1 - An illustration of the oxygen-independent and oxygen-dependent pathways for nitric 190 oxide production. 2 191 192 Figure 2 - The pharmacokinetic profile and dose response relationship of 4.2, 8.4 and 16.8 mM 193 dietary nitrate on plasma NO3- and nitrite. 194 Error! Bookmark not defined. 195 196 Figure 3 - An illustration of the effects of beetroot juice supplementation on oxygen uptake 197 kinetics from rest to moderate intensity exercise in recreationally active males. 12 198 199 Figure 4 - An illustration of the effects of beetroot juice supplementation on oxygen uptake 200 kinetics during moderate intensity exercise in simulated altitude (15% oxygen) in recreationally 201 active males. 202 Error! Bookmark not defined. 203 204 Figure 5 - Hormonal profile throughout a normal menstrual cycle with significant surges in 205 estrogen and progesterone. 206 Error! Bookmark not defined. 207 208 Figure 6 - Low estrogen and progesterone levels throughout the menstrual cycle in women 209 actively using two different forms of hormonal birth control. 35 210 211 Figure 7 - A schematic of the experimental design consisting of 7 laboratory visits. 43 212 213 Figure 8 - Experimental trial exercise protocol. 47 214
215 Figure 9 - Plasma nitrate at baseline (B) and two hours post-ingestion (2 h) of 140 mL beetroot 216 juice or nitrate-free placebo both acutely and chronically. 217 Error! Bookmark not defined. 218 219 Figure 10 - Plasma nitrite at baseline (B) and two hours post-ingestion (2 h) of 140 mL beetroot 220 juice or nitrate-free placebo both acutely and chronically. 221 Error! Bookmark not defined. 222 223 Figure 11 - Mean oxygen uptake at 50% peak oxygen uptake following acute and chronic 224 supplementation with beetroot juice or a nitrate-free placebo. 55 225 226 Figure 12 - Individual oxygen uptake at 50% peak oxygen uptake following acute and chronic 227 supplementation with beetroot juice or a nitrate-free placebo. 55 228 229 Figure 13 - Mean oxygen uptake at 70% peak oxygen uptake following acute and chronic 230 supplementation with beetroot juice or a nitrate-free placebo. 56 231 232
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Figure 14 - Individual oxygen uptake at 70% peak oxygen uptake following acute and chronic 233 supplementation with beetroot juice or a nitrate-free placebo. 57 234 235 Figure 15 - Time trial learning effect. 58 236 237 Figure 16 - Mean time trial completion. 58 238 Figure 17 - Mean time to complete each 20% split of the time trial. 59 239 240 Figure 18 - Mean heart rate at the end of each 20% split during the time trial. 59 241 242 Figure 19 - Mean rating of perceived exertion at the end of each 20% split during the time trial. 243
60 244 245 Figure 20 - Mean power output at the end of each 20% split during the time trial. 60 246
247
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List of Symbols and Abbreviations 248
ADP - Adenosine Diphosphate 249
ANT - Adenine Nucleotide Translocator 250
ATP - Adenosine Triphosphate 251
BH4 - Tetrahydrobiopterin 252
BRJ - Beetroot Juice 253
Ca2+ - Calcium 254
cGMP - Cyclic Guanosine Monophosphate 255
COXIV - Cytochrome Oxidase IV 256
CV - Coefficient of Variance 257
DBP - Diastolic Blood Pressure 258
ETC - Electron Transport Chain 259
FAD - Flavin Adenine Dinucleotide 260
FMN - Flavin Mononucleotide 261
Gc - Guanylyl Cyclase 262
H+ - Hydrogen Ion 263
HR - Heart Rate 264
KNO3 - Potassium Nitrate 265
MAP - Mean Arterial Pressure 266
NADPH - Nicotinamide-Adenine-Dinucleotide-Phosphate 267
NO3- - Nitrate 268
NaNO3 - Sodium Nitrate 269
NO - Nitric Oxide 270
x
NOS - Nitric Oxide Synthase 271
NO2- - Nitrite 272
O2 - Oxygen 273
PCr - Phosphocreatine 274
PGC1a - Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha 275
PLA - Placebo 276
PO - Power Output 277
P/O Ratio - Phosphate/Oxygen Ratio (ATP synthesis/Oxygen utilization) 278
ROS - Reactive Oxygen Species 279
RPE - Rating of Perceived Exertion 280
SBP - Systolic Blood Pressure 281
SERCA - Sarcoplasmic Reticulum Calcium ATPase 282
SOD - Superoxide Dismutase 283
TFAM - Mitochondrial Transcription Factor A 284
TT - Time Trial 285
UCP-3 - Uncoupling Protein 3 286
UVA - Ultraviolet A 287
VO2 - Oxygen Uptake 288
VO2max - Maximal Oxygen Uptake 289
VO2peak - Peak Oxygen Uptake 290
W - Watts291
1
Chapter 1: Review of the Literature 292
Nitric Oxide: Roles, Origins and Targets 293
Nitric oxide (NO) is a gaseous signalling molecule that elicits potent effects on numerous 294
biological tissues and is involved in an array of physiological processes including, but not 295
limited to: vasodilation, calcium handling, mitochondrial respiration, and neurotransmission 296
(Bailey et al. 2011). 297
NO can be produced endogenously and obtained from exogenous dietary sources. 298
Endogenously, NO is produced through the oxidation of L-arginine to L-citrulline and NO 299
(Figure 1). This reaction requires oxygen (O2) and reduced nicotinamide-adenine-dinucleotide 300
phosphate (NADPH) as a co-substrate as well as flavin adenine dinucleotide (FAD), flavin 301
mononucleotide (FMN), tetrahydrobiopterin (BH4), heme, and calmodulin as cofactors and 302
substrates and is catalyzed by the enzyme nitric oxide synthase (NOS) (Bailey et al. 2011). NOS 303
exists in several isoforms depending on its location and role within the biological system of 304
interest. These isoforms include neuronal NOS, endothelial NOS, and inducible NOS. These 305
isoforms will not be discussed in detail as they are beyond the scope of this literature review. 306
In 1994, it was acknowledged by Benjamin et al. (1994) as well as Lundberg et al. (1994) 307
that NO can be produced through an alternative exogenous pathway that does not rely on O2. 308
This anaerobic pathway involves the sequential one electron reduction of nitrate (NO3-) to nitrite 309
(NO2-) and further to NO (Figure 1). This allows the formation of NO in situations where the 310
NOS enzyme may be impaired such as hypoxia or disease. This pathway has important 311
implications for cardiovascular disease as well as exercise performance. The NO3- necessary for 312
this reaction is obtained primarily from dietary sources such as green leafy vegetables (spinach, 313
2
lettuce, kale, arugula) and root vegetables (beetroot, yams, turnips, carrots). Spinach, lettuce and 314
beetroot have the highest dietary NO3- concentrations (Hord et al. 2009). 315
Approximately 75% of ingested dietary NO3- is excreted in the urine, with only ~25% 316
entering the enterosalivary circulation (Webb et al. 2008). Dietary NO3- is swallowed and 317
subsequently absorbed from the stomach and small intestines. Once it enters the circulation, it is 318
concentrated in the salivary glands. NO3- is secreted from the salivary glands into the mouth via 319
saliva, and anaerobic bacteria facilitate the reduction of NO3- to NO2-. The NO2- is swallowed 320
again and is further reduced to NO in the acidic environment of the stomach (Bailey et al. 2011). 321
322
Figure 1: The oxygen (O2)-independent (left) and O2-dependent (right) pathways for nitric oxide 323 (NO) production. The O2-independent pathway relies on dietary nitrate (NO3-) to undergo 324 sequential reduction to nitrite (NO2-) and to NO. The O2-dependent pathway relies on a number 325 of substrates and cofactors including nicotinamide adenine dinucleotide phosphate (NADPH), 326 flavin adenine nucleotide (FAD), tetrahydrobiopterin (BH4), haem, and calmodulin as well as the 327 enzyme nitric oxide synthase (NOS). This figure also depicts an array of the putative biological 328 targets of NO (Bailey et al. 2011). 329 330
A final and newly regarded pathway by which the body generates NO is through ultra 331
violet A (UVA) exposure (Oplander et al. 2009; Liu et al. 2014; Muggeridge et al. 2015). The 332
NOS enzyme is active in a number of skin cells such as keratinocytes, melanocytes, fibroblasts 333
3
and endothelial cells (Bruch-Gerharz et al. 1998). The NO produced by skin cell NOS are 334
converted to nitroso compounds, NO2- or NO3- (Oplander et al. 2009). Remarkably, when UVA 335
rays penetrate the skin they can convert these compounds back to NO. This may help explain 336
why blood pressure is shown to be lower in countries that are closer to the equator and may 337
partially explain why blood pressure is typically lower in the summer months compared to the 338
winter (Oplander et al. 2009). 339
A plethora of nutritional supplements have been used in an attempt to augment the human 340
body’s production of NO. L-arginine is able to accumulate in the blood, but there is equivocal 341
evidence as to whether it increases NO production in healthy individuals (Bode-Böger et al. 342
1998; Liu et al. 2009; Bailey et al. 2010; Alvares et al. 2012). Furthermore, it has been shown 343
that citrulline, the co-product of NO synthesis, can be enzymatically converted back to arginine 344
to produce NO (Schwedhelm et al. 2007; Bryk et al. 2008). These supplements provide substrate 345
for the endogenous production of NO via the oxidation of L-arginine, however a greater body of 346
literature is required to support the effectiveness of these supplements. Concentrated beetroot 347
juice (BRJ) is a potent source of dietary NO3- containing 6.5 mmol (mM) or 400 mg per 70 mL 348
dose. BRJ facilitates the production of NO through the exogenous O2-independent NO3--NO2--349
NO pathway and has been shown to elevate plasma NO2- in a number of studies (Vanhatalo et al. 350
2010; Wylie et al. 2013; Bailey et al. 2015; Betteridge et al. 2016). Similarly, sodium nitrate 351
(NaNO3) and potassium nitrate (KNO3) administered as supplemental forms of NO3- can 352
augment the NO3--NO2--NO pathway. NaNO3 has been shown to elevate plasma NO2- in a 353
number of studies (Larsen et al. 2010; Bescós et al. 2012; Gasier et al. 2017), and KNO3 has also 354
been shown to increase plasma NO2- following supplementation (Kapil et al. 2010). Therefore, 355
the literature supports the use of dietary NO3- to increase plasma [NO3-] and [NO2-]. 356
4
NO is an unstable molecule and has a very short half-life in the blood of merely a few 357
milliseconds (Kelm 1999). NO is rapidly oxidized to NO2-, and this is the largest circulating pool 358
of NO3- products in the blood (Cosby et al. 2003). NO2- can be converted back to NO for cellular 359
signalling or can be further oxidized to NO3-. NO can act on several cellular targets including 360
proteins that contain transition metals or thiol groups (Tengan et al. 2013). These mechanisms 361
will be addressed in detail in a subsequent section of this literature review. 362
BRJ Pharmacokinetics, Dose and Duration 363
Dietary NO3- Pharmacokinetics 364
Pharmacokinetic profiles are essential to nutritional intervention studies as they provide a 365
framework to validate the accumulation of the treatment in the blood, demonstrate the time 366
course with which the treatment rises and peaks in the blood, and depict the rate of clearance 367
from the body. Previous research has demonstrated that plasma NO3- levels begin to rise ~30 min 368
post-ingestion and peak ~1.5 h following the ingestion of BRJ (Webb et al. 2008; Wylie et al. 369
2013) or KNO3 (Kapil et al. 2010). Plasma NO3- remains elevated for up to 24 h following a 370
single dose of dietary NO3- (Wylie et al. 2013; Kapil et al. 2010). Furthermore, plasma NO2- 371
levels begin to rise ~1.5 h post-ingestion, peaking ~2.5 h post-ingestion, and have been shown to 372
remain elevated for an additional 3 h (Webb et al. 2008; Kapil et al. 2010; Wylie et al. 2013). 373
Both plasma NO3- and NO2- have been shown to return to baseline concentrations ~24 h 374
following a large, acute dose of dietary NO3- (Webb et al. 2008; Kapil et al. 2010; Wylie et al. 375
2013). However, plasma NO2- levels have been shown to decrease by 12 h post-ingestion with a 376
moderate dose (8.4 mM) of dietary NO3- (Wylie et al. 2013). Interestingly, the slight lag time for 377
peak plasma NO2- compared to plasma NO3- likely reflects the time required for NO3- to enter the 378
enterosalivary circulation and undergo the sequential reduction from NO3- to NO2-. The results 379
5
from this study indicate that dietary NO3- should be administered every 12 h to ensure plasma 380
[NO3-] and [NO2-] remain chronically elevated above baseline concentrations. 381
The conversion in the enterosalivary circulation can be disrupted by spitting (Webb et al. 382
2008) or destruction of the anaerobic bacteria via antibacterial mouthwash (Govoni et al. 2008; 383
McDonagh et al. 2015; Woessner et al. 2016). As expected, when the enterosalivary circulation 384
is disrupted following a dose of dietary NO3- there is a concomitant termination in the reduction 385
of NO3- to NO2-, as represented by no change in plasma [NO2-] despite a significant increase in 386
plasma [NO3-]. Furthermore, since plasma [NO2-] is correlated with NO production, the 387
disruption of the enterosalivary circulation eliminates the beneficial biological effects associated 388
with dietary NO3- supplementation (Webb et al. 2008). This demonstrates that oral bacteria are 389
fundamental to the rise in plasma [NO2-] and subsequent biological effects following a dose of 390
dietary NO3-. 391
Dose Response: Plasma Levels, Blood Pressure, Oxygen Uptake (VO2) and Performance 392
To date, only two studies have investigated the dose-response relationship associated 393
with dietary NO3- supplementation. Kapil et al. (2010) investigated the dose-response associated 394
with supplementing acutely with 4, 12 and 24 mM of KNO3 in 21 healthy subjects. These 395
authors found a 7-fold increase in peak plasma [NO3-] following 4 mM KNO3, a 27-fold increase 396
following 12 mM KNO3, and a 35-fold increase following 24 mM KNO3 (Kapil et al. 2010). The 397
peak plasma [NO3-] occurred 3 h post-ingestion. In a similar manner, these authors found a 1.3-398
fold increase in peak plasma [NO2-] following 3 mM KNO3, a 2-fold increase following 12 mM 399
KNO3, and a 4-fold increase following 24 mM KNO3. The peak plasma [NO2-] occurred 2.5 h 400
post-ingestion. Interestingly, this dose-response relationship translated to dose-dependent 401
reductions in systolic blood pressure (SBP) and diastolic blood pressure (DBP). When 4 mM 402
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KNO3 was administered, the peak reduction in SBP was 2 mmHg and the peak reduction in DBP 403
was 4 mmHg. When 12 mM KNO3 was administered, the peak reduction in SBP was 6 mmHg 404
and the peak reduction in DBP was 4 mmHg. Lastly, when 24 mM KNO3 was administered, the 405
peak reduction in SBP was 6 mmHg and the peak reduction in DBP was 5 mmHg (Kapil et al. 406
2010). The results from this study demonstrate that plasma NO3- and NO2- increase in a dose-407
dependent manner which correlates with reductions in SBP and DBP. 408
Wylie et al. (2013) investigated the dose-response relationship of 4.2, 8.4, and 16.8 mM 409
of dietary NO3- administered as an acute dose of BRJ in 10 recreationally active males. These 410
authors found an 8-fold increase in peak plasma [NO3-] following 4.2 mM NO3-, a 12-fold 411
increase following 8.4 mM NO3-, and a 24-fold increase following 16.8 mM NO3- (Figure 2). 412
The peak plasma [NO3-] occurred 1 h post-ingestion following 4.2 and 8.4 mM dietary NO3- and 413
2 h post-ingestion for 16.8 mM dietary NO3-. Similarly, these authors found a 3.3-fold increase 414
in peak plasma [NO2-] following 4.2 mM NO3-, a 5-fold increase following 8.4 mM NO3-, and a 415
7.8-fold increase following 16.8 mM NO3-. The peak plasma [NO2-] occurred 2 h post-ingestion 416
following 4.2 and 8.4 mM dietary NO3- and 4 h post-ingestion for 16.8 mM dietary NO3-. 417
Similar to the results presented by Kapil et al. (2010), these authors found a dose-418
dependent relationship in the reductions of SBP, DBP, and mean arterial pressure (MAP). An 419
acute dose of 4.2 mM NO3- resulted in peak reductions of 4 mmHg in SBP, 1 mmHg in DBP, 420
and 2 mmHg in MAP. When an acute 8.4 mM NO3- dose was administered, peak reductions 421
were 8 mmHg in SBP, 3 mmHg in DBP, and 4 mmHg in MAP. Lastly, when an acute 16.8 mM 422
NO3- dose was administered, peak reductions were 10 mmHg in SBP, 4 mmHg in DBP, and peak 423
5 mmHg in MAP (Wylie et al. 2013). 424
7
The same authors also investigated the dose-response relationship with regard to the 425
reductions in baseline and end-exercise VO2 during moderate (~105 watts (W)) and severe 426
intensity (~300 W) exercise to exhaustion. There was no change in baseline VO2 with moderate 427
or severe intensity exercise. There was no change in end-exercise VO2 with an acute dose of 4.2 428
mM NO3-, however when an acute dose of 8.4 mM NO3- was administered there was a 2% 429
reduction in end-exercise VO2 during moderate intensity exercise, and the acute dose of 16.8 430
mM resulted in a 3% reduction in end-exercise VO2 with moderate intensity exercise (Wylie et 431
al. 2013). There was no effect of any dose of BRJ supplementation on end-exercise VO2 during 432
the severe exercise bout. Interestingly, an acute dose of 8.4 mM NO3- resulted in a significant 433
improvement in time to exhaustion during severe intensity exercise compared to placebo (PLA) 434
(8.4 mM: 570 + 153 vs. PLA: 498 + 113 s). Time to exhaustion was also significantly improved 435
with an acute dose of 16.8 mM NO3- (552 + 117 s) compared to PLA (493 + 114 s). The results 436
from this study indicate that an acute dose of at least 8.4 mM NO3- is required to elicit positive 437
effects on BP, the O2 cost of exercise, and to improve time to exhaustion. 438
439
8
440
Figure 2: The pharmacokinetic profile and dose response relationship of 4.2, 8.4 and 16.8 mM 441 dietary nitrate (NO3-) on plasma NO3- and nitrite (NO2-). Filled circles, control treatment; filled 442 triangles, 4.2 mM; filled squares, 8.4 mM; and filled diamonds, 16.8 mM (Wylie et al. 2013). 443 444 Acute vs. Chronic Supplementation: Plasma Levels, VO2 and Performance 445
Few studies have directly compared the effects of different durations of dietary NO3- 446
supplementation on blood parameters, health, and performance (Vanhatalo et al. 2010; Fulford et 447
al. 2013; Wylie et al. 2016). Vanhatalo et al. (2010) investigated the effects of acute and chronic 448
BRJ supplementation on BP and exercise performance in 8 healthy participants (5 males). These 449
authors found that mean plasma [NO2-] measured 2.5 h post-ingestion increased by 36% 450
following an acute dose of 5.2 mM BRJ, by 59% following 12 days of supplementation, and by 451
46% following 15 days of supplementation. SBP was lowered by ~5 mmHg with BRJ 452
supplementation at all time points, DBP was lowered by ~4 mmHg, and MAP was lowered by 453
~5 mmHg. Furthermore, moderate intensity (~90 W) end-exercise VO2 was consistently reduced 454
by ~5% following acute supplementation, 5 days of supplementation and 15 days of 455
9
supplementation. Fulford et al. (2013) explored the effects of dietary NO3- on skeletal muscle 456
force production both acutely and chronically in 8 healthy males. These authors demonstrated 457
that plasma [NO2-] measured at 2.5 h post-ingestion increased by a similar amount following an 458
acute dose of 10.2 mM NO3-, and after 5 and 15 days of supplementation. Interestingly, there 459
was no impact of acute or chronic BRJ supplementation on skeletal muscle contractile force 460
production (Fulford et al. 2013). Unfortunately, these authors did not measure VO2. 461
Wylie et al. (2016) reported the pharmacokinetic profiles and exercise effects associated 462
with different durations and doses of dietary NO3- supplementation in 34 recreationally active 463
subjects (19 males). These authors found that with daily doses of both 3 and 6 mM of dietary 464
NO3-, there was no significant difference within conditions for plasma [NO3-] or [NO2-] 465
following an acute dose, 7 days of supplementation or 30 days of supplementation. However, 466
plasma [NO3-] and [NO2-] were significantly greater in the 6 mM condition compared to the 3 467
mM condition, where plasma [NO3-] increased by 313% in the 3 mM dietary NO3- condition and 468
by 867% in the 6 mM dietary NO3- condition (Wylie et al. 2016). Plasma [NO2-] increased by 469
165% in the 3 mM condition and 579% in the 6 mM condition (Wylie et al. 2016). There was no 470
effect of the 3 mM condition on end-exercise VO2 during moderate intensity exercise (~90 W). 471
Conversely, the rise in plasma [NO2-] in the 6 mM condition was associated with a 3% reduction 472
in end-exercise VO2 during moderate intensity exercise. These authors also investigated the 473
effects of chronic supplementation without an acute dose administered 2 h prior to blood 474
sampling and exercise testing. Without the acute dose, plasma [NO3-] returned to baseline levels 475
by 24 h post-ingestion in the 3 mM condition and declined significantly in the 6 mM condition. 476
However, plasma [NO3-] was still significantly elevated above baseline in the 6 mM condition 24 477
h following the last dose of BRJ. Without the acute dose, plasma [NO2-] returned to baseline by 478
10
24 h post-ingestion in the 3 and 6 mM conditions. Similar to the chronic supplementation with 479
the acute dose administered prior to testing, there was no change in end-exercise VO2 in the 3 480
mM condition. Interestingly, when the dose was 6 mM per day the 3% reduction in VO2 was 481
conserved despite no acute dose administered 2 h prior to testing. 482
From these studies, it appears that subjects do not develop a tolerance to dietary NO3- 483
with up to 30 days of supplementation, and the effects on blood parameters, health, and exercise 484
performance are similar both acutely and chronically. Further corroborating these findings, Kelly 485
et al. (2013) had subjects visit the laboratory over 5 consecutive days during days 3 through 7 of 486
dietary NO3- supplementation and reported no difference in plasma [NO2-] measured 2.5 h post-487
ingestion across the 5 days, with BRJ consistently improving time to exhaustion to the same 488
extent (~ 13%) during severe intensity exercise tests (Kelly et al. 2013). 489
The Effects of BRJ on Exercise Economy and Athletic Performance 490
Moderate Intensity Submaximal Exercise Economy 491
For the purpose of this thesis, exercise economy is defined as a reduction in the O2 cost of 492
exercise at a fixed workload. A basic tenet of human exercise physiology states that the O2 cost 493
of submaximal exercise at a given work rate is fixed regardless of age, health and fitness status 494
(Poole and Richardson 1997). Furthermore, this tenet suggests that the O2 cost of submaximal 495
exercise is unaltered by physical, nutritional or pharmacological interventions (Poole and 496
Richardson 1997). It has been demonstrated that O2 uptake increases linearly as a function of 497
work rate, where O2 consumption is ~9 to 11 mL O2/W/min during moderate intensity exercise 498
(Poole and Richardson 1997). BRJ supplementation challenges this dogma by consistently 499
demonstrating reductions, typically between 3 to 5%, in the O2 cost of moderate intensity 500
submaximal exercise in recreationally active males as reviewed by Affourtit et al. 2015. 501
11
Moderate intensity exercise is characterized by low production and adequate clearance of 502
blood lactate, and therefore is generally classified as exercise that falls below the lactate 503
threshold (Xu and Rhodes 1999). The lactate threshold is defined as the blood lactate 504
concentration at which lactate production exceeds its clearance and accumulation occurs. The 505
onset of blood lactate accumulation occurs at an arterial lactate concentration of 4 mM/L and 506
typically occurs around 75% maximal oxygen uptake (VO2max) (Ghosh 2004). The VO2 kinetics 507
associated with moderate intensity exercise can be divided into three major components. Phase I 508
is depicted by the early and rapid rise in VO2 that occurs in the first 15 to 25 s of exercise, and 509
this has been linked to the rise in cardiac output and blood flow at the onset of exercise (Xu and 510
Rhodes 1999) (Figure 3). Phase II is represented by an exponential rise in VO2 towards steady 511
state and reflects shifts in skeletal muscle metabolism and substrate utilization. Phase III is 512
achieved after ~3 min of exercise and is defined as steady state VO2, which has been described 513
previously as ~9 to 11 mL O2/W/min and is indicated by a heavy reliance on aerobic 514
metabolism. BRJ supplementation has been shown to influence VO2 kinetics by decreasing the 515
primary VO2 amplitude and ultimately decreasing end-exercise VO2 in recreationally active 516
groups composed of predominantly male subjects (Bailey et al. 2009; Vanhatalo et al. 2010; 517
Lansley et al. 2011) (Figure 3). 518
Interestingly, these reductions in the O2 cost of exercise are present merely 2.5 h 519
following an acute dose of dietary or inorganic NO3- and persist for up to 30 days of chronic 520
supplementation. Furthermore, these effects are seen with doses ranging from 5.2 mM to 16.8 521
mM dietary or inorganic NO3-. BRJ supplementation has shown to be effective at reducing the 522
O2 cost of exercise at intensities ranging from 45 to 75% VO2peak and these reductions have been 523
elicited across a range of exercise modalities including walking (Lansley et al. 2011), running 524
12
(Lansley et al. 2011), cycling (Larsen et al. 2007; 2010; 2011; Bailey et al. 2009; Vanhatalo et al. 525
2010; Reink et al. 2015; Wylie et al. 2013; Whitfield et al. 2016) and knee extension exercise 526
(Bailey et al. 2010). 527
528 Figure 3: An illustration of the effects of beetroot juice (nitrate) supplementation on oxygen 529 uptake (VO2) kinetics from rest to moderate intensity exercise in recreationally active males. 530 Nitrate significantly reduced the oxygen cost of exercise by reducing the primary amplitude of 531 VO2 kinetics (Lansley et al. 2011). 532 533 Severe and Maximal Intensity Exercise Economy 534
Severe intensity exercise exceeds the lactate threshold. Although phase II kinetics still 535
exist in this exercise domain, VO2 kinetics are complicated by the VO2 slow component that 536
develops after a few minutes at this intensity (Xu and Rhodes 1999). During severe intensity 537
exercise, VO2 steady state (phase III) is not achieved and the slow component continues to 538
increase until task failure (Xu and Rhodes 1999). Two studies have investigated VO2 kinetics 539
during severe intensity exercise (Bailey et al. 2009; Breese et al. 2013). These groups define 540
severe intensity exercise as 70% of the difference between the gas exchange threshold (GET) and 541
peak oxygen consumption (VO2peak) (Bailey et al. 2009, ~275 W; Breese et al. 2013, ~215 W). 542
13
Bailey et al. (2009) found a significant reduction in the VO2 slow component amplitude with 6 543
days of BRJ supplementation, which translated to a 16% improvement in time to exhaustion in 8 544
healthy men. These authors also found a significantly decreased primary VO2 amplitude 545
following BRJ supplementation compared to PLA (Bailey et al. 2009). Conversely, Breese et al. 546
(2013) found no significant difference in the VO2 slow component amplitude or the primary VO2 547
amplitude following 6 days of BRJ supplementation compared to PLA in 9 healthy men. These 548
discrepancies may exist because of differing exercise protocols in which Bailey et al. (2009) 549
transitioned from very low intensity exercise to severe intensity exercise, whereas Breese et al. 550
(2013) transitioned from moderate intensity exercise to severe intensity exercise. A greater body 551
of literature is required to elucidate the VO2 kinetic profiles associated with BRJ 552
supplementation and severe intensity exercise. 553
To date, three studies have investigated the effects of BRJ supplementation on VO2max 554
(Larsen et al. 2007; 2010; Vanhatalo et al. 2010). The initial study by Larsen et al. (2007) 555
demonstrated no significant effect of 3 days of NaNO3 supplementation on VO2max in 9 well-556
trained males. However, in 2010 these authors found a significant reduction in VO2max following 557
3 days of NaNO3 supplementation in 9 healthy volunteers (7 males) (Larsen et al. 2010). A 558
reduced VO2max would likely indicate impaired exercise performance. However, Vanhatalo et al. 559
(2010) demonstrated an increase in VO2max following 15 days of BRJ supplementation in 8 560
recreationally active individuals (5 males). It is difficult to reconcile the array of outcomes seen 561
across these studies and a greater body of research is required to determine if dietary NO3- 562
substantially impacts maximal exercise performance. 563
564
565
14
Time Trial Performance 566
Time trial performance is typically measured as a set distance, set amount of work, or set 567
time allocated for exercise performance. Time trials hold the greatest real-world validity when 568
measuring exercise performance in athletes and are the most reliable and reproducible protocols 569
(Currell and Jeukendrup 2008). There are no distinguishable differences in variability between 570
time trial exercise modalities where the coefficient of variation (CV) is below 5% for cycling, 571
running, and rowing (Currell and Jeukendrup 2008). 572
Aerobic Cycling Time Trial Performance 573
A few studies have demonstrated an improvement in aerobic cycling time trial 574
performance following dietary NO3- supplementation. Although some of these studies did not 575
achieve statistical significance, they fall below the smallest worthwhile change for cycling time 576
trial performance in elite cyclists as described by Hopkins (2004). This statistical inference 577
suggests that the average CV for cycling time trials is ~ 0.6% for trained cyclists, and any change 578
that exceeds this may be deemed worthwhile (Paton and Hopkins 2006). Lansley et al. (2011) 579
found a 2.8% improvement in 4 km time trial performance, and a 2.7% improvement in 16.1 km 580
time trial performance following an acute dose of BRJ in 9 competitive male cyclists. Wilkerson 581
et al. (2012) found a 0.8% improvement on a 50 mile time trial following an acute dose of BRJ 582
in 8 trained male cyclists, and Cermak et al. (2012) found a 1.3% improvement in 10 km time 583
trial performance following 6 days of BRJ supplementation in 13 trained male cyclists and 584
triathletes. Lastly, Lee et al. (2015) found a 2.1% improvement in 20.15 km time trial 585
performance following acute supplementation with a NO containing lozenge in 16 trained 586
cyclists (8 males). All of these studies utilized a single-blinded testing protocol for the subjects 587
or a double-blinded testing protocol. However, none of these studies administered a post-testing 588
15
questionnaire to confirm if the subjects were successfully blinded to the supplementation 589
regimes. 590
Interestingly, there are also a number of studies that demonstrated no significant effect on 591
aerobic cycling time trial performance following dietary NO3- supplementation (Cermak et al. 592
2012; Lane et al. 2013; Christensen et al. 2013; Glaister et al. 2015; McQuillan et al. 2016; 593
Nyakayiru et al. 2016; Callahan et al. 2017). It is important to note that although dietary NO3- 594
supplementation may not elicit a beneficial effect on time trial performance, it has not shown to 595
be detrimental to athletic performance. Furthermore, it is important to consider that these time 596
trial studies were conducted in trained athletes, and their training status may contribute to the 597
controversial findings on athletic performance following dietary NO3- supplementation. This will 598
be addressed in a subsequent section. 599
Aerobic Running Time Trial Performance 600
There is a limited body of evidence investigating the effects of dietary NO3- 601
supplementation on aerobic running time trial performance and the outcomes are equivocal 602
(Murphy et al. 2012; Peacock et al. 2012; Boorsma et al. 2014). Murphy et al. (2012) found a 603
significant improvement in 5 km time trial performance, where running velocity was increased 604
by 5% following an acute dose of BRJ in 11 healthy individuals (5 males). Conversely, Peacock 605
et al. (2012) found no effect of an acute dose of KNO3 on 5 km running time trial performance in 606
10 elite male cross-country skiers. Similarly, Boorsma et al. (2014) found no significant effect of 607
acute or chronic BRJ supplementation on 1500 m running performance in 10 elite male distance 608
runners. The smallest worthwhile change for elite athletes in these events is a 1.1% improvement 609
in performance as described by Hopkins (2004). Neither Peacock et al. (2012) or Boorsma et al. 610
(2014) found performance benefits greater than these parameters. Again, training status may 611
16
have confounded the performance benefits associated with dietary NO3- supplementation, as 612
Peacock et al. (2012) and Boorsma et al. (2014) recruited elite runners whereas, Murphy et al. 613
(2012) recruited recreationally active individuals. 614
Water Sport Time Trial Performance 615
There is some evidence to suggest that BRJ may improve performance in water sports 616
such as rowing, kayaking, and swimming. The CV for elite rowing time trial performance is 617
noted to be ~ 0.4%, therefore any improvement greater than the CV is predicted to be a 618
worthwhile improvement (Hopkins 2004). Hoon et al. (2012) showed that acute BRJ 619
supplementation was possibly beneficial (~2 s improvement) to 2000 m rowing performance in 620
10 highly trained male rowers. Bond et al. (2012) demonstrated that 6 days of BRJ 621
supplementation was likely beneficial during 6 x 500 m rowing sprints, where the benefit was 622
almost certain in sprints 4 through 6. The CV for elite kayaking time trial performance is ~ 0.6% 623
(Hopkins 2004) and Peeling et al. (2015) found a 0.7% improvement in the distance covered 624
during a 4 min kayaking test in 6 national level male kayakers and found a 1.7% improvement in 625
500 m kayaking time trial performance in 5 international level female kayakers. However, 626
Muggeridge et al. (2013) saw no difference in 1 km kayaking time trial performance despite a 627
significantly reduced submaximal VO2 (3.1%) when kayaking for 15 min at 60% maximal 628
workload (Wmax) following an acute dose of BRJ in 8 trained male kayakers. Pospieszna et al. 629
(2016) investigated the effects of 8 days of BRJ supplementation on 6 x 50 m sprint swimming 630
performance, and 800 m endurance swimming performance in 11 female collegiate swimmers. 631
These authors noted a statistically significant improvement in sprints 4 through 6 following BRJ 632
supplementation as well as improvements in 800 m time trial performance. 633
634
17
Time to Exhaustion 635
A number of studies have reported improved time to exhaustion following BRJ 636
supplementation in healthy, recreationally active males. These improvements have been reported 637
with both acute supplementation and chronic supplementation lasting up to 15 days. 638
Furthermore, time to exhaustion has been enhanced across a range of exercise modalities 639
including: cycling (Bailey et al. 2009; Larsen et al. 2010; Wylie et al. 2013; Kelly et al. 2013; 640
Thompson et al. 2014; Bailey et al. 2015), running (Lansley et al. 2011), and knee extension 641
exercise (Bailey et al. 2010; Hoon et al. 2015). The reported improvements have been between 7 642
and 20%. 643
Sprint and Team Sport Performance 644
To date, only two studies have investigated the effects of BRJ supplementation on sprint 645
cycling performance. Rimer et al. (2016) found that acute BRJ supplementation increased peak 646
power during 4 sets of 3 – 4 s sprints, however there was no benefit of BRJ supplementation on 647
peak or mean power during a 30 second Wingate in 13 competitively trained athletes from an 648
array of sports (11 males). Conversely, after an acute dose of BRJ, Dominguez et al. (2017) 649
noted a significant increase in peak power during a 30 s Wingate, but no difference in mean 650
power in 15 recreationally active males. Since little literature exists, the results are equivocal and 651
require further investigation. 652
Due to the complexity and multifaceted nature of team sports, it is difficult to quantify 653
team sport performance in a controlled setting. One commonly employed method to bypass the 654
complexity, uncertainty, and spontaneity of stop-and-go sporting events is to create a simulated 655
environment that closely mimics gameplay. Stop-and-go team sports are typically characterized 656
by intermittent and repeated sprints, and protocols such as the running Loughborough 657
18
Intermittent Shuttle Test or the Yo-Yo Intermittent Recovery Test have been highly correlated 658
with movement patterns utilized in team sports (Currell and Jeukendrup 2008). There is mixed 659
support for the benefit of BRJ supplementation on team sport performance as assessed by 660
repeated sprint efforts. It has been shown that acute (Wylie et al. 2013) and chronic (Thompson 661
et al. 2016) BRJ supplementation can increase the distance covered during the Yo-Yo 662
Intermittent Recovery Test by ~ 4% in male recreational team-sport athletes. Another study 663
demonstrated a significant increase in mean power during 24 sets of 6 s sprints, however there 664
was no improvement during 7 sets of 30 s sprints, or 6 sets of 60 s sprints with 5 days of BRJ 665
supplementation in 34 male recreationally active team-sport athletes (Wylie et al. 2016). On the 666
contrary, Buck et al. (2015) failed to demonstrate an improvement in repeated sprint 667
performance with acute BRJ supplementation in 13 female amateur team-sport athletes. 668
Although it appears that BRJ supplementation may be beneficial for team sport performance, a 669
greater body of literature is required to validate and confirm these findings. 670
Strength Performance 671
There is minimal research published regarding the effects of BRJ supplementation on 672
strength performance. Mosher et al. (2016) demonstrated that acute supplementation with BRJ 673
improved the number of repetitions to failure and consequently the total weight lifted at failure 674
during bench press performed at 60% of 1 repetition maximum in 12 recreationally active males. 675
Conversely, Flanagan et al. (2016) found no significant effect of 3 days of BRJ supplementation 676
on the maximum number of repetitions performed at 60, 70, 80 or 90% 1 repetition maximum in 677
14 resistance-trained males. 678
679
680
19
Performance During Hypoxia 681
Exercise in simulated hypoxic environments is characterized by a reduced inspired O2 682
concentration, ultimately resulting in reduced arterial O2 content, reduced oxidative capacity of 683
contracting muscles, and impaired exercise tolerance (Vanhatalo et al. 2011; Kelly et al. 2014). 684
The reduction in inspired O2 can compromise the L-arginine pathway for NO production since it 685
relies on O2 as a substrate (Lundberg et al. 2010). Therefore, BRJ supplementation may be 686
important in hypoxic conditions because it augments the sequential reduction of NO3- to NO 687
which does not rely on O2. Most of the literature studying BRJ supplementation in hypoxia (15% 688
O2) demonstrates a profound 6 – 8% decrease in the O2 cost of exercise during moderate 689
intensity cycling (~135 – 200W) (Kelly et al. 2014; Muggeridge et al. 2014) (Figure 4), and 690
significant improvements in time to task failure (Vanhatalo et al. 2011; Kelly et al. 2014) and TT 691
performance (Muggeridge et al. 2014). Interestingly, Vanhatalo et al. (2011) found that BRJ 692
supplementation significantly attenuated the rate of metabolic perturbation associated with high 693
intensity knee extension exercise in a hypoxic environment (~15% O2), and restored exercise 694
tolerance to normoxic conditions in 9 recreationally active subjects (7 males). Specifically, after 695
60 s and 120 s of exercise, phosphocreatine (PCr) had fallen to a greater extent in the PLA 696
condition compared to the BRJ condition. Consequently, inorganic phosphate (Pi) accumulation 697
and pH reduction were greater in the PLA condition compared to the BRJ condition, ultimately 698
indicating a greater reliance on aerobic metabolism following BRJ supplementation in hypoxic 699
conditions. There was no difference in these parameters between the hypoxic BRJ condition and 700
the control normoxic condition indicating a scenario by which BRJ restored exercise tolerance. 701
702
20
703 Figure 4: An illustration of the effects of beetroot juice (BRJ) supplementation (nitrate) on 704 oxygen uptake kinetics during moderate intensity exercise (~135 W) in simulated altitude (15% 705 oxygen (O2)) in recreationally active males. There was an ~8% reduction in the O2 cost of 706 exercise with BRJ during hypoxia compared to a 3% reduction in the O2 cost of exercise with 707 BRJ during normoxia. (Kelly et al. 2014). 708 709 Conclusion 710
BRJ supplementation has been shown to elicit profound effects on exercise economy and 711
athletic performance. Specifically, BRJ supplementation has been shown to reduce the O2 cost of 712
moderate intensity exercise and improve time to exhaustion. These findings are exacerbated in 713
hypoxic situations. The effects of BRJ supplementation on time trial, sprint, team sport, and 714
strength performance are equivocal and often confounded by a lack of literature in the field. 715
Alternatively, it is often suggested that an athlete’s training status may impact their response to 716
dietary NO3- supplementation. This will be addressed in the following section. 717
Individual Responses to BRJ Supplementation 718
There is inherently individual variability in the physiological responses to nutritional 719
interventions, and the likelihood that an individual is going to respond positively to a nutritional 720
intervention is influenced by a multitude of factors. Furthermore, the magnitude of responses is 721
influenced by these very same factors and can be visualized as a continuum. When considering 722
21
the factors that influence the response to BRJ supplementation, it is important to consider 723
differences in the fitness status, dietary habits, and digestive pathway of the subjects. A “non-724
responder” is identified as an individual who consistently does not display a reduction in the O2 725
cost of exercise following BRJ supplementation. Conversely, a “responder” is an individual who 726
consistently demonstrates a reduction in the O2 cost of exercise that is greater than the coefficient 727
of variance for the measurement. 728
Interestingly, genetics may also influence an individual’s physiological responses to NO. 729
A recent study by Emdin et al. (2017) demonstrated that certain individuals may have a genetic 730
predisposition for enhanced NO signalling. This was associated with a reduced risk of coronary 731
heart disease and stroke. Interestingly, individuals who had rare genetic variants that inactivated 732
certain NO signalling genes demonstrated significantly elevated blood pressure and a 3-fold 733
increased risk of coronary heart disease (Emdin et al. 2017). Future studies should investigate the 734
possibility that individuals who have enhanced endogenous NO signalling may be less likely to 735
respond to dietary NO3- supplementation. 736
Fitness Status 737
There is substantial evidence to suggest that highly trained individuals may not respond 738
to dietary NO3- supplementation and therefore do not demonstrate a reduced O2 cost of 739
submaximal exercise (Peacock et al. 2012; Wilkerson et al. 2012; Christensen et al. 2013; 740
Boorsma et al. 2014; Lee et al. 2015; McQuillan et al. 2016; Nyakayiru et al. 2016). A study by 741
Porcelli et al. (2015) investigated the effects of chronic NaNO3 supplementation on plasma 742
[NO3-] and [NO2-], the O2 cost of exercise and time trial performance in 21 males with either 743
low, moderate, or high aerobic fitness. It was discovered that individuals with high aerobic 744
fitness had higher baseline plasma [NO3-] compared to individuals with low or moderate aerobic 745
22
fitness. Furthermore, the individuals with low or moderate aerobic fitness demonstrated the 746
greatest rises in both relative and absolute plasma [NO3-] and [NO2-] following chronic 747
supplementation with NaNO3. These authors identified that individuals with low and moderate 748
aerobic fitness elicited a 9.8% and 7.3% reduction in the O2 cost of exercise at 80% GET, 749
respectively. Interestingly, chronic NaNO3 supplementation also resulted in a significant 750
improvement in 3 km running time trial performance for individuals with low and moderate 751
aerobic fitness. However, there was no difference in the O2 cost of exercise or 3 km running time 752
trial performance for individuals with high aerobic fitness. It is possible that the individuals with 753
low and moderate aerobic fitness may experience performance benefits with dietary NO3- due to 754
the dramatic elevation in plasma [NO2-] following supplementation compared to individuals with 755
high aerobic fitness. Similar results were obtained by Carriker et al. (2016) who found a reduced 756
O2 cost of exercise at 45% (~90 W) and 60% (~150 W) VO2max in 5 males with low aerobic 757
fitness (VO2max, 42.4 + 3.2 mL/kg/min). However, these results were not found in 6 males with 758
high aerobic fitness levels (VO2max, 60.1 + 4.6 mL/kg/min) (Carriker et al. 2016). 759
Jonvik et al. (2016) collected diet records from hundreds of elite athletes to assess their 760
habitual dietary NO3- intake. These authors hypothesized that elite athletes may have a blunted 761
response to dietary NO3- supplementation because they may already have diets rich in these 762
foods. However, it was discovered that the average dietary NO3- intake was only 106 mg/d, 763
where a typical dose of BRJ is at least 400 mg of dietary NO3-. Furthermore, only 10% of the 764
athletes consumed greater than 400 mg/d of dietary NO3-. This suggests that there are other 765
physiological mechanisms at play with elite athletes and their blunted response to dietary NO3- 766
supplementation. Interestingly, Jonvik et al. (2016) reported that female athletes typically 767
ingested a larger absolute amount of dietary NO3- per day than male athletes, despite having 768
23
lower total energy intake. This was attributed to a greater consumption of vegetables by female 769
athletes. 770
Diet 771
It is possible that individuals who habitually consume high levels of dietary NO3- may not 772
respond to BRJ supplementation since they already express elevated plasma [NO3-] and [NO2-]. 773
However, it is expected that these differences would manifest as variations in individual plasma 774
[NO3-] and [NO2-]. It has been shown that traditional Japanese diets are high in dietary NO3-, and 775
adherence to this diet is associated with lower SBP and DBP (Gee and Ahluwalia 2016). 776
Furthermore, when the same Japanese population was placed on a western diet they experienced 777
significant increases in SBP and DBP (Gee and Ahluwalia 2016). It has been suggested that the 778
high dietary NO3- in the Japanese diet may be a major contributor to the population’s longevity 779
and low rates of cardiovascular disease (Gee and Ahluwalia 2016). Similarly, other authors argue 780
that individuals who follow the Dietary Approaches to Stop Hypertension (DASH) diet 781
experience reductions in blood pressure, which is partially attributed to the high leafy green, and 782
consequently high dietary NO3- content, of this diet (Ashworth et al. 2015). 783
Digestive Pathway 784
It is expected that there is interindividual variability in the way we absorb, process, and 785
store nutrients. When analyzing the potential variability in the responses to BRJ 786
supplementation, it is important to consider interindividual differences that may exist in the 787
enterosalivary circulation. First, it is possible that there are differences in the amount of NO3- 788
that is absorbed from the stomach and small intestines. This may mean that there is 789
interindividual variability in the fraction of dietary NO3- that is excreted from the body. Second, 790
the rate of and magnitude of NO3- concentration into the salivary glands may differ between 791
24
individuals. Next, there are likely differences in the amount of and activity of the bacteria that 792
reduce NO3- to NO2- inside the mouth. Lastly, there may be interindividual differences in the 793
storage, conversion and degradation of dietary NO3- that is absorbed as NO2-. There is currently 794
no literature focusing on these aspects as they are difficult to quantify and measure. However, it 795
is expected that these differences would manifest as variations in individual plasma [NO3-] and 796
[NO2-]. Interestingly, Kapil et al. (2010) found that plasma [NO2-] rose 2-fold higher in female 797
subjects (n = 12) compared to males (n = 8) following an acute dose of KNO3. This trend still 798
existed when plasma [NO2-] was normalized to body weight. These authors suggest that sex 799
differences may exist in the way nitrate loads are processed. 800
BRJ Improves Exercise Economy and Performance – Potential Mechanisms of Action 801
Are We Sure It’s Nitrate? 802
The early BRJ studies were performed before the development of a NO3--free PLA which 803
is manufactured using ion resin exchange (Lansley et al. 2011). This PLA was first introduced in 804
the BRJ literature by Lansley et al. in 2011. Up until that point, it was difficult to ascertain 805
whether the beneficial effects of BRJ supplementation were strictly from its dietary NO3- content 806
or if there were other bioactive properties in the beverage. It is now abundantly clear that the 807
main bioactive in BRJ is dietary NO3- since the beneficial effects are only reported with the NO3-808
-rich beverage compared to the NO3--free placebo. However, some authors continue to argue that 809
other bioactives within BRJ are a major contributor to its positive biological effects. A number of 810
recent publications have attributed the effects of BRJ supplementation to its high antioxidant 811
status. BRJ is high in betalain pigment, an antioxidant that gives the vegetable it’s deep red 812
colour (Clifford et al. 2016b). Specifically, BRJ is high in betanin, a subclass of the betalain 813
pigment. Clifford et al. (2016a; 2016b; 2017) have continually explored the antioxidant effects of 814
25
BRJ supplementation on muscle damage, recovery and performance in recreationally active 815
males. They have consistently shown significant reductions in muscle soreness as well as 816
attenuated performance decrements assessed using a countermovement jump following fatiguing 817
exercise with 3 days of BRJ supplementation (Clifford et al. 2016a; 2016b). However, these 818
authors have failed to show a benefit with respect to running performance or changes in 819
biomarkers of inflammation (Clifford et al. 2016b; 2017a). Furthermore, despite the high betanin 820
content of BRJ, these authors failed to demonstrate an increase in plasma betanin over an 8 h 821
period following acute BRJ supplementation suggesting that this antioxidant may not play an 822
ergogenic role (Clifford et al. 2017b). A major limitation of these studies is that they cannot 823
differentiate the potential anti-inflammatory effects of NO3- compared to betalain or betanin 824
since a NO3--free PLA was not utilized. However, in a separate set of experiments, Hoorebeke et 825
al. (2016) demonstrated that concentrated betalain supplementation improved perceived exertion 826
during submaximal treadmill running and improved 5 km time trial performance in 15 827
recreationally active males. Similarly, Montenegro et al. (2017) found that concentrated betalain 828
supplementation improved performance on a 10 km time trial, and a 5 km time trial performed 829
the next day in 22 triathletes (9 males). It is important to note, that neither of these studies 830
demonstrated a reduction in the O2 cost of exercise following supplementation suggesting 831
betalain is not responsible for this physiological response to BRJ supplementation (Hoorebeke et 832
al. 2016; Montenegro et al. 2017). Cumulatively, although it is important to recognize that there 833
are other bioactives in BRJ that may contribute to its beneficial effects, the antioxidant properties 834
associated with betalain fail to explain the consistent reduction in the O2 cost of exercise seen 835
with dietary NO3- interventions. 836
26
BRJ supplementation may improve exercise performance through a variety of 837
mechanisms. The potential mechanisms for the reduced O2 cost of exercise range from improved 838
O2 delivery and waste removal to improved contractile or mitochondrial efficiency. It has also 839
been proposed that BRJ may improve exercise performance by attenuating skeletal muscle 840
damage or facilitating mitochondrial biogenesis. 841
Improved Blood Flow 842
NO is renowned for its role as a potent vasodilator. This occurs through the interaction of 843
NO with guanylyl cyclase (Gc) which ultimately increases cyclic guanosine monophosphate 844
(cGMP) and facilitates the removal of Ca2+ from the smooth muscle cell resulting in relaxation. 845
Furthermore, NO has also been shown to improve skeletal muscle blood flow. Ferguson et al. 846
(2013a) performed a series of studies utilizing rats and BRJ supplementation. The first study 847
demonstrated a significant increase in hindlimb skeletal muscle blood flow and vascular 848
conductance during treadmill running in rats undergoing 5 days of BRJ supplementation. The 849
second study demonstrated that 5 days of BRJ supplementation attenuated the decline in O2 850
delivery/O2 utilization that occurs between contractions, meaning that the drive for O2 was 851
elevated in the periods between contractions providing enhanced delivery (Ferguson et al. 852
2013b). In humans, Richards et al. (2017) found increased forearm blood flow following acute 853
BRJ supplementation during a dynamic handgrip task in 18 healthy subjects (8 males). Although 854
this does not explain the reduced O2 cost of exercise, it may be an important factor related to the 855
performance benefits of BRJ since this may augment O2 delivery as well as CO2 and waste 856
removal. Future research needs to investigate the effects of BRJ supplementation with direct 857
measures of blood flow in human subjects. 858
859
27
Improved Mitochondrial Function 860
Reduction in the O2 Cost of Mitochondrial Adenosine Triphosphate (ATP) Resynthesis 861
The electron transport chain (ETC) is comprised of a series of complexes that use 862
electrons from NADH and FADH2 to facilitate the movement of protons (hydrogen (H+) ions) 863
from the mitochondrial matrix to the intermembrane space creating an electrochemical gradient 864
that drives the synthesis of ATP via mitochondrial membrane potential (Farrell et al. 2012). 865
Importantly, the final electron acceptor in the ETC is O2, and ATP is resynthesized from 866
adenosine diphosphate (ADP) and inorganic phosphate via the enzyme ATP synthase (Farrell et 867
al. 2012). The efficiency of ATP resynthesis depends on the rate of proton leak and slippage 868
(Farrell et al. 2012). Proton leak occurs when H+ ions are utilized via alternative pathways that 869
do not contribute to the generation of ATP such as uncoupling protein 3 (UCP-3). Slippage 870
occurs when electrons are moved without the pumping of protons, ultimately resulting in a 871
decreased membrane potential to drive ATP resynthesis (Farrell et al. 2012). The efficiency of 872
mitochondrial respiration is often expressed as the phosphate/O2 (P/O) ratio and is defined as the 873
rate of ATP synthesis divided by the rate of O2 consumption (Larsen et al. 2011). 874
It has been proposed that dietary NO3- supplementation can improve the efficiency of 875
ATP resynthesis by reducing proton leak and slippage (Clerc et al. 2007; Larsen et al. 2011). 876
Larsen et al. (2011) associated the reduced O2 cost of exercise following 3 days of NaNO3 877
supplementation with an improved P/O ratio. Specifically, these authors found a significant 878
reduction in state 4 respiration, which is characterized by availability of respiratory substrate but 879
not ADP and estimates the back leakage of protons through the inner mitochondrial membrane 880
(Larsen et al. 2011). This indicates more efficient mitochondrial ATP synthesis since a larger 881
portion of the membrane potential is contributing to ATP synthesis as opposed to uncoupling 882
28
processes. The reduction in membrane leak was attributed to a significant reduction in ANT 883
protein expression and a strong trend for a reduction in UCP-3 protein expression (Larsen et al. 884
2011). However, Whitfield et al. (2016) found dramatically different results where 7 days of BRJ 885
supplementation showed no effect on mitochondrial efficiency as expressed by no change in leak 886
respiration, UCP-3 or ANT protein content, and the P/O ratio. Although it is possible there are 887
different mechanisms of action for NaNO3 compared to BRJ supplementation, this is highly 888
unlikely and more research is required to understand these stark differences, and to elucidate the 889
effects of dietary NO3- on mitochondrial coupling and efficiency. 890
Alternatively, it has been suggested that NO is capable of reversibly inhibiting complex 891
IV of the ETC by competing with O2 at its binding site (Brown and Cooper 1994; Cleeter et al. 892
1994; Bolaños et al. 1994; Guiffrè et al. 2000). By changing the stoichiometry between O2 used 893
and ATP produced, this would result in a decrease in O2 utilization and would ultimately 894
decrease the O2 cost of exercise. 895
Mitochondrial Biogenesis 896
A few cell culture studies have identified NO as a stimulus for mitochondrial biogenesis. 897
These studies have demonstrated an increase in mitochondria DNA, mitochondrial markers such 898
as cytochrome oxidase IV (COXIV) and cytochrome C, and increased O2 consumption following 899
incubation with NO (Nisoli et al. 2003; 2004). It is thought that NO acts through Gc and cGMP 900
to activate peroxisome proliferator-activated receptor gamma coactivator a-alpha (PGC-1𝛼), the 901
master regulator of mitochondrial biogenesis (Nisoli et al. 2004; Lira et al. 2010). However, 902
these findings have not been replicated with BRJ supplementation in humans. Larsen et al. 903
(2011) found no change in mitochondrial density or mRNA content for proteins related to 904
mitochondrial biogenesis (PGC-1𝛼, mitochondrial transcription factor A (TFAM), COXIV) 905
29
following 3 days of NaNO3 supplementation. These results are not surprising as the time course 906
for significant mitochondrial biogenesis to occur is likely longer than this supplementation 907
period. Furthermore, since significant reductions in the O2 cost of exercise occur merely 2.5 h 908
following BRJ supplementation it is likely that the mechanism of action is at least partially 909
independent of increased protein content as this does not occur on this timescale. Moreover, 910
Whitfield et al. (2016) found no change in mitochondrial protein content following 7 days of 911
BRJ supplementation in recreationally active males, suggesting that BRJ supplementation may 912
not influence mitochondrial biogenesis in humans. 913
Improved Contractile Efficiency 914
Reduction in the ATP Cost of Skeletal Muscle Force Production 915
The ATP cost of skeletal muscle force production is dominated by sarcoplasmic 916
reticulum calcium ATPase (SERCA) and myosin ATPase which account for ~25 – 30% and 70% 917
of the total cost of contraction respectively (Barclay et al. 2007). Na+-K+ ATPase is shown to 918
account for ~5% of the ATP cost of skeletal muscle force production (Barclay et al. 2007). 919
Therefore, due to their robust contributions to the ATP cost of exercise, it was a logical step to 920
investigate the effect of dietary NO3- supplementation on the ATP cost associated with SERCA 921
and myosin ATPase. 922
Recently, a number of studies have contributed to the hypothesis that BRJ 923
supplementation may reduce the ATP cost of exercise. However, the exact mechanism of action 924
remains to be elucidated. Bailey et al. (2010) designed an elegant study to indirectly tease apart 925
the potential mechanisms of action associated with the reduced O2 cost of exercise following 926
BRJ supplementation. These authors used magnetic resonance spectroscopy (MRS) to determine 927
the total ATP cost of low and high intensity knee extension exercise, and estimated the relative 928
30
ATP contributions from PCr, glycolysis, and oxidative phosphorylation. These authors found a 929
significant reduction in total ATP hydrolysis during low intensity and high intensity exercise 930
with BRJ supplementation and demonstrated a 25% improvement in exercise tolerance during 931
high intensity exercise to exhaustion following BRJ supplementation. These findings suggest that 932
the O2 cost of submaximal exercise may be reduced through an improvement in skeletal muscle 933
contractile efficiency. Using a similar methodology, Fulford et al. (2013) also found a significant 934
decrease in the relative contribution of PCr to ATP hydrolysis during knee extension exercise. It 935
is important to note that these findings are based on calculations and have inherent error built 936
into them. Furthermore, Fulford et al. (2013) did not calculate the ATP contribution from aerobic 937
or glycolytic metabolism making it difficult to interpret whether BRJ supplementation decreased 938
total ATP turnover or shifted fuel utilization. 939
Other studies have used force frequency curves to assess the effects of BRJ 940
supplementation on skeletal muscle force production and contractile efficiency. Hernandez et al. 941
(2012) found a significant increase in low frequency force production at 10 Hz following 7 days 942
of NaNO3 supplementation in mice. This was accompanied by an increase in intracellular 943
calcium and an increase in the expression of calsequestrin and dihydropyridine receptor proteins, 944
both associated with calcium-handling. Haider and Folland (2014) found an increase in force 945
production at a low stimulation frequency of 10 Hz following 7 days of BRJ supplementation in 946
healthy males. Whitfield et al. (2017) reported similar findings where low stimulation frequency 947
force production was increased at 10 Hz in healthy males suggesting improved contractile 948
efficiency. However, unlike Hernandez et al. (2012), this was not associated with changes in 949
calcium-handling protein expression suggesting BRJ may influence the force produced via 950
myosin ATPase during skeletal muscle contraction. Conversely, Hoon et al. (2015) found no 951
31
effect of 4 days of BRJ supplementation on skeletal muscle force production in 19 healthy adults 952
(13 males). The discrepancies in the outcomes from these studies may be explained by the 953
variation in study model as well as the supplementation and electrical stimulation protocols, and 954
by the small magnitude of the expected changes. 955
Since dietary NO3- supplementation can reduce the oxygen cost of exercise merely 2.5 h 956
following an acute dose, it is unlikely that protein content changes in this time frame. Therefore, 957
it still remains to be elucidated if dietary NO3- supplementation results in post-translational 958
modifications that may alter the activity of calcium-handling or contractile proteins. There is 959
evidence to suggest that dietary NO3- supplementation may slow cross bridge cycling to allow 960
for greater force production per ATP hydrolyzed. Using skinned skeletal muscle fibers from 961
rabbits and differentiated myotubes from mice, Nogueira et al. (2009) demonstrated that NO was 962
able to s-nitrosylate myosin on multiple cysteine thiols. This was accomplished by adding 963
reactive nitrogen species to the reaction mixtures. These authors found that exposure to NO 964
resulted in decreased myosin ATPase activity but no change in the affinity of myosin for actin 965
suggesting a slowing of cross bridge cycling (Nogueira et al. 2009). This was the first evidence 966
that NO may play a role in increasing skeletal muscle force production via altering myosin 967
ATPase function. This work was promptly followed up by Evangelista et al. (2010) who 968
confirmed that exposure of rat skeletal muscle myosin to NO donors can s-nitrosylate cysteine 969
residues on myosin which caused a slowing of cross bridge cycling and ultimately doubled force 970
production. 971
Reactive Oxygen Species (ROS) 972
A study by Whitfield et al. (2016) demonstrated increased intermyofibrillar (IMF) 973
mitochondrial ROS production following 7 days of BRJ supplementation. These authors 974
32
suggested that increased ROS production may influence the skeletal muscle contractile 975
machinery by improving mechanical efficiency and ultimately increasing force production 976
during cross bridge cycling. Alterations in mitochondrial ROS production is an attractive theory 977
for the mechanism underlying BRJ supplementation because it helps explain the acute effects 978
and the diminished effect or lack of effect seen in elite athletic populations. These populations 979
have increased superoxide dismutase (SOD) content and activity which may buffer the 980
mitochondrial ROS production and mitigate any performance benefits of BRJ supplementation. 981
Unfortunately, these authors found no correlation between increased mitochondrial ROS 982
production and the decrease in whole body VO2 during submaximal exercise, suggesting that the 983
in vitro experimental results may not explain the whole-body effects of BRJ supplementation. 984
Clearly future work needs to investigate the in vivo effect of BRJ supplementation and 985
mitochondrial ROS production, and the effects on calcium-handling as well as skeletal muscle 986
contractile machinery. It is possible that ROS produced at the level of the mitochondria may 987
influence the ATP cost of the contractile apparatus. 988
Women – Addressing the Gender Gap 989
Throughout history women have been consistently underrepresented in sport medicine 990
and exercise research, and only recently has this gender gap begun to be attenuated. Costello et 991
al. (2014) indicated that between 2011 and 2013, women still only represented 39% of research 992
participants across 1382 studies. Furthermore, it is widely accepted that sexual dimorphism 993
exists in human physiology and metabolic responses to exercise making it extremely valuable to 994
study sex specific responses to exercise interventions. Unfortunately, a major barrier to including 995
women in exercise studies is the added complexity of the menstrual cycle and the hormonal 996
profiles associated with this process. Hormonal contraceptive use is a method used to minimize 997
33
hormonal fluctuations throughout the menstrual cycle, however this only became common 998
practice in the 1980s (Liao 2012) further contributing to the delay in active recruitment of female 999
research participants. 1000
Characterization of the Menstrual Cycle 1001
A typical female menstrual cycle is ~28 days in duration and is typically characterized by 1002
the early follicular phase (days 1 – 7), late follicular phase (days 7 – 14), early luteal phase (days 1003
14 – 21), and late luteal phase (days 21 – 28). Menses is classified as day 1 of the menstrual 1004
cycle. Estrogen and progesterone levels follow an inverse relationship throughout the menstrual 1005
cycle (Figure 5). During the early follicular and late follicular phase estrogen levels continue to 1006
rise until a peak at ovulation (day 14). Following ovulation estrogen levels decline and 1007
progesterone levels begin to rise until menses occurs again (Jonge et al. 2003). Research utilizing 1008
female participants is further complicated by abnormal menstrual cycle durations, assumption of 1009
normal hormone fluctuations, and the individual variability associated with menstrual cycle 1010
phases (Jonge et al. 2003). The most reliable method to confirm menstrual cycle phase is by 1011
measuring plasma hormone concentrations using commercially available kits (Jonge et al. 2003). 1012
1013
Figure 5: Hormonal profile throughout a normal menstrual cycle with significant surges in 1014 estrogen (estradiol) and progesterone (Sherman and Korenman 1975). 1015 1016 1017
34
NO Varies Throughout the Menstrual Cycle 1018
Some work has investigated the variation in NO production that occurs naturally 1019
throughout the menstrual cycle and there are discrepancies in the literature as to which female 1020
sex hormone, estrogen or progesterone, is the main regulator of NO production. Some authors 1021
suggested that NO metabolites are higher in the follicular phase and peak at ovulation when 1022
estrogen concentrations are at their highest (Cicinelli et al. 1996). Alternatively, it has also been 1023
proposed that NO production is inhibited by progesterone which peaks during the luteal phase 1024
(Giusti et al. 2002; Teran et al. 2002). In line with these hypotheses, Williams et al. (2001) 1025
demonstrated that brachial artery flow mediated dilatation was higher during the follicular phase, 1026
peaking near ovulation and dropping off sharply in the early luteal phase. This suggests a 1027
possible positive correlation between estrogen concentrations and NO production, as well as a 1028
potential inhibitory effect of progesterone on NO production. Lastly, when exhaled NO is 1029
measured there appears to be either no correlation of sex hormones with NO metabolites (Morris 1030
et al. 2002), or a relationship where estrogen decreases NO production and progesterone 1031
increases NO production (Mandhane et al. 2009). It is possible that these discrepancies exist 1032
because exhaled NO is a less reliable measurement of NO metabolites than blood parameters. 1033
Cell culture studies have proposed a role of estrogen in stimulating the production of NO 1034
as there are estrogen receptors located on NOS (Xia and Krukoff 2004; Townsend et al. 2011). 1035
Xia and Krukoff (2004) showed that the addition of estrogen to neuroblastoma cells resulted in 1036
the activation of eNOS and nNOS, and ultimately facilitated the production of NO. Similarly, 1037
Townsend et al. (2011) demonstrated that when human bronchial epithelial cells were exposed to 1038
estrogen there was a rapid increase in NO production, whereas administration of an estrogen 1039
receptor antagonist resulted in inhibition of NO production. These authors proposed that estrogen 1040
35
activates caveolin-1 which is able to increase intracellular Ca2+ concentrations and can ultimately 1041
activate NOS to produce NO (Townsend et al. 2011). It still remains to be elucidated whether 1042
this relationship exists in skeletal muscle. 1043
Pharmacokinetics of Hormonal Contraceptives 1044
Studies have sought to characterize the estrogen and progesterone profiles in women 1045
actively using hormonal contraceptives and it is clear that hormonal contraceptives providing 1046
synthetic estrogen and progesterone prevent ovulation by attenuating the surge in estrogen that 1047
typically occurs around day 14 of a natural menstrual cycle (Gaspard et al. 1983; Kuhl et al. 1048
1985; Jung-Hoffmann et al. 1988; Christin-Maitre et al. 2010). Hormonal contraceptive use 1049
ultimately results in stable and low estrogen levels throughout the menstrual cycle (Christin-1050
Maitre et al. 2010) (Figure 6). 1051
1052
Figure 6: Low estrogen (estradiol) and progesterone levels throughout the menstrual cycle in 1053 women actively using two different forms of hormonal birth control (Christin-Maitre et al. 1054 2010). 1055
36
Previous Female BRJ Literature 1056
There is a major gender gap in BRJ exercise research with only 5 studies that specifically 1057
investigated female populations. This is in stark comparison to the more than 45 studies with 1058
strictly male populations. Of the 5 female studies that exist, there is a diverse range of exercise 1059
modalities, fitness levels, and supplementation protocols. 1060
The first study providing insight that women may respond positively to acute and chronic 1061
dietary NO3- supplementation was performed by Vanhatalo et al. (2010) who demonstrated a 1062
~5% reduction in the O2 cost of moderate intensity exercise in a group of 5 males and 3 females 1063
following 2.5 h, 5 days and 15 days of supplementation. Although these authors do not provide 1064
the individual data points or comment on potential sex differences in the responses to BRJ 1065
supplementation, it is highly likely that most of the female subjects responded positively to this 1066
intervention. 1067
Three studies in strictly female populations have investigated the effects of BRJ 1068
supplementation on exercise performance in trained athletes. However, none of these studies 1069
measured the O2 cost of submaximal exercise. One study explored the effects of acute BRJ 1070
supplementation, sodium phosphate and the combination on repeated sprint performance and 1071
simulated game play in 13 female amateur team-sport athletes (Buck et al. 2015). The subjects 1072
ingested 6 mM dietary NO3- 3 h prior to the exercise protocol. Despite a significant rise in 1073
plasma NO3-, there was no effect of BRJ supplementation on repeated sprint performance. These 1074
authors did not measure plasma NO2- to give an indication whether NO3- was being converted to 1075
NO2- via the enterosalivary circulation. It is possible that these women did not respond to BRJ 1076
supplementation because the dose was too low to elicit a positive effect on exercise performance 1077
or the women may have been too trained. 1078
37
Similarly, Glaister et al. (2015) explored the effects of acute BRJ, caffeine, and the 1079
combination on 20 km cycling TT performance in 14 trained female cyclists. Approximately 2.5 1080
h prior to exercise, the subjects consumed ~7.3 mM dietary NO3-. Despite significant rises in 1081
plasma NO3- and NO2-, there was no effect of BRJ supplementation on TT performance. 1082
Interestingly, these authors found plasma NO2- to increase from 92 to 297 nM, whereas Lansley 1083
et al. (2011) administered an acute dose of 6.2 mM dietary NO3- and found plasma NO2- to 1084
increase from 293 to 575 nM. In contrast to the work of Glaister et al. (2015), Lansley et al. 1085
(2011) demonstrated a significant improvement in 16.1 km cycling TT performance in trained 1086
males. This may suggest that there is a plasma [NO2-] threshold that dietary NO3- 1087
supplementation must surpass in order to elicit a biological effect. 1088
Lastly, Pospieszna et al. (2016) explored the effects of chronic BRJ supplementation on 6 1089
sets of 50 m swimming sprints, and 800 m swimming performance in 11 female collegiate 1090
swimmers. The subjects participated in two, 8 day supplementation periods where they 1091
supplemented with either 0.5 L of BRJ and chokeberry juice or 0.5 L of carrot juice with added 1092
KNO3. Each supplement delivered 10.5 mM of dietary NO3- per day. A major flaw of this study 1093
was that no PLA treatment was utilized for comparison and proper blinding. Nonetheless, these 1094
authors found that both chronic BRJ and chokeberry supplementation as well as carrot juice 1095
supplementation with added KNO3 improved repeated sprint performance by 3.13% and 2.09% 1096
respectively. Specifically, both juices improved performance on sprints 4 through 6. The 1097
improvements were significantly greater with BRJ and chokeberry supplementation compared to 1098
carrot juice with added KNO3 supplementation. Furthermore, both juices significantly reduced 1099
the time to complete the 800 m swim, with greater improvements seen following carrot juice 1100
supplementation with added KNO3. It is possible that dietary NO3- elicited a positive effect on 1101
38
performance in this study since the daily dose was significantly greater than that of Buck et al. 1102
(2015) and Glaister et al. (2015) and the supplementation protocol lasted for 8 days instead of an 1103
acute dose. However, we cannot rule out the possibility of a placebo effect influencing the 1104
outcome of this study. 1105
To date, only two studies have investigated the effects of BRJ supplementation on 1106
exercise economy and performance in recreational or sedentary female populations. First, Bond 1107
Jr et al. (2014) explored the effects of acute BRJ supplementation on submaximal VO2 and blood 1108
pressure in 12 sedentary African American women (VO2peak, 26.1 + 3.3 mL/kg/min). The women 1109
were provided with either ~12 mM dietary NO3- or orange juice (negligible NO3- content) 2 h 1110
prior to blood sampling and exercise testing for 5 min each at 40, 60 and 80% VO2peak. Blood 1111
pressure was measured during exercise. These authors found significant increases in plasma NO 1112
following acute BRJ supplementation. Furthermore, there was a significant reduction in SBP at 1113
rest as well as during exercise at 40, 60 and 80% VO2peak. BRJ supplementation had no effect on 1114
VO2 at rest, but reduced VO2 by ~15% at 40, 60, and 80% VO2peak (as inferred from a figure in 1115
the manuscript). A major limitation of this study is the lack of a NO3--free PLA, and these 1116
authors failed to report the mean VO2 values associated with rest and exercise at 40, 60 and 80% 1117
VO2peak. 1118
Rienks et al. (2015) was the only other group to investigate the effects of acute BRJ 1119
supplementation on VO2 during exercise. The major outcome of this study was to determine the 1120
effects of acute BRJ supplementation on work performed during a rating of perceived exertion 1121
(RPE) clamp protocol in 10 recreationally active females (VO2peak, 36.1 + 4.7 mL/kg/min). To do 1122
this, the subjects ingested 12.9 mM dietary NO3- 2.5 h prior to cycling exercise. These authors 1123
measured VO2 during a 20 min cycling protocol where subjects exercised at a self-selected 1124
39
intensity that elicited an RPE of 13 (somewhat hard). A secondary outcome of this study was to 1125
determine the effect of acute BRJ supplementation on submaximal O2 uptake. This was assessed 1126
by 5 min at 75 W to measure constant workload VO2 following a 5 min cooldown after the 20 1127
min RPE clamp protocol. There was no effect of BRJ supplementation on the work completed or 1128
total VO2 during the 20 min RPE clamp protocol. However, acute BRJ supplementation resulted 1129
in a weak but statistically significant 4% reduction in VO2 while cycling at 75 W (p = 0.048). 1130
Interestingly, only Bond Jr et al. (2014) and Buck et al. (2015) attempted to control for 1131
menstrual cycle phase, and the selected phases differed between these two studies where Bond Jr 1132
et al. (2014) utilized the luteal phase and Buck et al. (2015) utilized the follicular phase. 1133
Furthermore, Pospieszna et al. (2016) was the only group to investigate chronic BRJ 1134
supplementation, and no study to date has combined acute and chronic supplementation into the 1135
same study protocol with strictly female participants. Lastly, only Rienks et al. (2015) recruited 1136
recreationally active subjects when the bulk of the literature suggests the reductions in VO2 are 1137
present in recreationally active males compared to their trained counterparts. Therefore, of the 5 1138
studies, three show improvement following BRJ supplementation. However, the results and 1139
validity of these studies are clouded by poor study design and poorly presented data. 1140
Conclusion 1141
Ultimately, there is very little research exploring the effects of BRJ supplementation in 1142
female populations making it difficult to draw comprehensive conclusions on exercise economy 1143
and performance. It is important to have a number of studies from a variety of laboratories to 1144
validate and support the existing research findings in sedentary, recreationally active as well as 1145
trained female subjects. Furthermore, it is important to explore the effectiveness and mechanisms 1146
behind BRJ supplementation in female populations as these have yet to be examined. 1147
40
Chapter 2: Statement of the Problem 1148
Rationale 1149
Over the last decade, a wealth of research has surfaced exploring the effects of NO3--rich 1150
BRJ on athletic performance across a multitude of exercise modalities. The vast majority of this 1151
research has been conducted on male participants, and it appears that the greatest benefits are 1152
seen in recreationally active males. Moreover, there are only 5 BRJ studies that have considered 1153
exclusively female subjects, and only 2 of those studies measured submaximal O2 uptake. Only 1154
one of these studies recruited recreationally active women, however their primary outcome was 1155
performance during a RPE clamp protocol. Furthermore, none of these studies assessed both 1156
acute and chronic BRJ supplementation in the same protocol. Therefore, the purpose of this 1157
study was to determine the effects of acute and chronic BRJ supplementation on submaximal 1158
cycling economy, and performance on a 4 kJ/kg time trial in recreationally active females. 1159
BRJ was used instead of sodium NO3- or potassium NO3- because BRJ is a commercially 1160
available product that athletes from around the globe, and across a range of calibres, use to 1161
potentially enhance their athletic performance. 1162
The subjects supplemented with BRJ and PLA both acutely and chronically to determine 1163
if short-term and long-term supplementation periods had differing effects on submaximal 1164
exercise economy and time trial performance in recreationally active women. Beneficial effects 1165
have been seen in recreationally active males both acutely and chronically. 1166
The subjects were required to ingest 13 mmol NO3- approximately 2.5 h before the 1167
exercise protocol on the mornings of the experimental trials. This dose has been shown to reduce 1168
the O2 cost of exercise and improve time trial performance in recreationally active males, 1169
whereas doses smaller than 4.2 mmol NO3- have not shown these same benefits (Wylie et al. 1170
41
2013). Secondly, pharmacokinetic profiles have demonstrated that NO3- levels peak in the blood 1171
approximately 2.5 h after consumption of BRJ (Wylie et al. 2013). On the chronic 1172
supplementation days, the subjects were instructed to consume 26 mmol NO3- as 13 mmol in the 1173
morning and 13 mmol in the evening. Following a dose of 13 mmol NO3- plasma NO3- 1174
concentrations have been shown to return to baseline 12 h post-ingestion, therefore the subject’s 1175
plasma NO3- concentrations were constantly elevated during this supplementation regime. This 1176
dose was selected as it is higher than any other female BRJ study to ensure adequate 1177
accumulation in the blood. 1178
The subjects completed a submaximal exercise protocol at wattages that elicited 50% 1179
VO2peak and 70% VO2peak to assess exercise economy at multiple submaximal workloads. 1180
Secondly, the subjects completed a 4 kJ/kg time trial as a measure of athletic performance. A 1181
time trial was used instead of a time to exhaustion protocol because of greater repeatability 1182
across trials and greater ecological validity (Currell and Jeukendrup 2008). The 4 kJ/kg time trial 1183
was selected instead of a fixed time or fixed distance time trial to normalize the subjects’ work to 1184
their body weight. 1185
Overall, the testing protocol was carefully selected to allow for a comprehensive analysis 1186
of the effects of BRJ supplementation on exercise economy and time trial performance in 1187
recreationally active females. 1188
Hypotheses 1189
1) Plasma [NO3-] and [NO2-] will be significantly elevated following acute and chronic 1190
supplementation with NO3--rich BRJ compared to baseline and PLA in recreationally active 1191
females. 1192
42
2) Acute and chronic supplementation with NO3--rich BRJ will significantly decrease the O2 cost 1193
of submaximal cycling at 50% VO2peak and 70% VO2peak in recreationally active females. 1194
3) Acute and chronic supplementation with NO3--rich BRJ will improve performance on a 4 1195
kJ/kg time trial in recreationally active females. 1196
Chapter 3: Methods 1197
Subjects 1198
Twelve recreationally active females were recruited to participate in this study (mean + 1199
SEM, age: 23 + 0.4 years; weight: 65.0 + 3.0 kg; height: 169 + 2 cm; VO2peak: 40.7 + 1.2 1200
mL/kg/min). Subjects were included in the study if they were healthy, recreationally active 1201
females, between the ages of 18 and 30, actively using hormonal contraceptives for 6 months 1202
prior to participation, and consumed less than 300 mg NO3- per day. Subjects were considered 1203
recreationally active if they were physically active 3 – 4 times per week and had a VO2peak of 1204
~ 35 – 45 mL/kg/min. Subjects reported being physical active 4 + 0.2 days per week and 1205
performed 4.5 + 1.0 h of exercise/week in the 6 months leading up to participation in this study. 1206
The subjects were informed of the study requirements and potential risks prior to providing oral 1207
and written consent. This study was approved by the University of Guelph Research Ethics 1208
Board (#REB17-02-008). 1209
Study Design 1210
This study was comprised of 7 laboratory visits (Figure 7). The first visit consisted of a 1211
VO2peak test to determine the subject’s aerobic capacity, and familiarization trials were performed 1212
during visits two and three. Then, using a double-blind, randomized, crossover design, the 1213
subjects supplemented with BRJ (6.5 mmol NO3-/70 mL; Beet It Sport, James White Drinks, 1214
Ipswitch, UK) or a NO3--free placebo (PLA, 0.006 mmol NO3-/70 mL; Beet It Sport) for 1 day 1215
43
(acute) and 8 days (chronic). The treatment conditions were separated by a washout period (9 + 1 1216
days). This time course had been previously validated to allow sufficient time for plasma NO3- 1217
and NO2- levels to return to baseline following chronic supplementation with exogenous NO3- 1218
(Nyakayiru et al. 2017). The PLA was manufactured using an ion resin exchange that selectively 1219
removed NO3- from the original product (Lansley et al. 2011). The BRJ and PLA were 1220
indistinguishable by taste, smell, appearance, and packaging. The supplementation order was 1221
counterbalanced, where 6 subjects began supplementing with BRJ and 6 began supplementing 1222
with PLA. To investigate the effects of acute and chronic BRJ supplementation on exercise 1223
performance, the subjects completed a submaximal cycling protocol and an aerobic-based time 1224
trial (TT) on days 1 and 8 of supplementation. The testing began with subjects consuming 140 1225
mL of BRJ (13 mmol NO3-) or PLA 2.5 h prior to submaximal cycling and the TT. In the 1226
evening on day 1, the subjects consumed an additional 140 mL of BRJ (13 mmol NO3-) or PLA. 1227
On days 2 – 7, the subjects consumed 280 mL of BRJ or PLA. The first dose of 140 mL was 1228
consumed in the morning with breakfast and the second 140 mL dose was consumed in the 1229
evening with dinner. 1230
1231
Figure 7: A schematic of the experimental design consisting of 7 laboratory visits. Subjects 1232 completed 2 x 8 days supplementation phases separated by a washout of 9 + 1 days. The subjects 1233 came to the laboratory on days 1 (acute trial) and 8 (chronic trial) of each supplementation phase 1234 to perform a cycling protocol consisting of submaximal cycling and an aerobic-based time trial. 1235 1236 Pre-Experimental Tests 1237
To determine VO2peak, and the cycling intensity to elicit 50% and 70% VO2peak, each 1238
subject performed an incremental exercise test to volitional exhaustion on a cycle ergometer 1239
44
(Lode Excalibur Sport, Groningen, The Netherlands). The subjects were equipped with a heart 1240
rate (HR) monitor (POLAR H10, Oulu, Finland) before warming up for 2 min at 60 W, then the 1241
power output (PO) was increased to 100 W. Once the subjects achieved steady state VO2 (~ 2 1242
min) at each stage, the PO was increased by 25 W. Steady state was defined as two x 20 s VO2 1243
measurements that were within 50 mL O2/min of each other. This process was repeated until the 1244
subjects reached volitional exhaustion (VO2peak was determined as the highest value obtained 1245
during the test). Time, HR, VO2, and PO were recorded at the end of each step increment. VO2 1246
and carbon dioxide production (VCO2) were measured (Moxus Modular VO2 System, 1247
Pittsburgh, USA) in 20 s intervals during the test. 1248
The subjects completed two familiarization trials prior to the experimental trials. The 1249
subjects arrived to the laboratory and were asked to provide a urine sample for determination of 1250
urine specific gravity (USG). Then, the subjects were weighed and equipped with a HR monitor. 1251
In the first familiarization trial the subjects were set up comfortably on the ergometer and the 1252
position was documented for subsequent trials. During the familiarization trials, the subjects 1253
completed the entire experimental exercise protocol consisting of a 5 min warm up at 60 W, 10 1254
min at 50% VO2peak, 10 min at 70% VO2peak, and a 4 kJ/kg body mass (BM) aerobic time-based 1255
TT. In the first familiarization trial the subjects consumed water ad libitum. The subjects were 1256
weighed after the trial for determination of body mass loss through sweat. If the subject lost or 1257
gained mass compared to the initial weigh in, then the volume of fluid provided during the 1258
exercise protocol was adjusted prior to the second familiarization trial. The volume of water 1259
consumed in the second familiarization trial was replicated for the subsequent experimental 1260
trials. The subjects moved to the experimental trials once there was less than 5% variability 1261
45
between the TT performances (Currell and Jeukendrup 2008). All subjects’ TT performances 1262
achieved this, therefore no subject was required to complete a third familiarization trial. 1263
1264
Diet, Sleep and Exercise Standardization 1265
The subjects recorded their dietary intake, sleep and exercise in the 48 h leading up to the 1266
familiarization and experimental trials. Subjects were asked to replicate their diet, sleep and 1267
exercise habits for the duration of the study. In the 24 h prior to the familiarization and 1268
experimental trials, the subjects were asked to refrain from intense exercise and alcohol, to 1269
abstain from caffeine in the 12 h before the trials. Subjects were not instructed to abstain from 1270
NO3- rich foods, as the pre-study screening indicated that they did not consume diets high in 1271
dietary NO3-. The use of antibacterial mouthwash and chewing gum was prohibited during the 1272
experimental trials, as there is evidence suggesting that these substances destroy the anaerobic 1273
bacteria in the mouth necessary for the conversion of NO3- to NO2- and NO (McDonagh et al. 1274
2015). Furthermore, the subjects were instructed to avoid brushing their teeth for 2 h before and 1275
after the ingestion of the BRJ or PLA. 1276
For the experimental trials, the subjects arrived to the laboratory in an overnight fasted 1277
state. A baseline blood sample (9.5 mL) was drawn for analysis of serum estrogen and 1278
progesterone as well as plasma [NO3-] and [NO2-]. The subjects were then provided with a 1279
standardized breakfast (consumed in < 15 min) that consisted of a banana, a SoLo GIâ bar, and 1280
140 mL of BRJ or PLA. This breakfast was intentionally carbohydrate-rich to ensure that 1281
carbohydrate was not limiting during exercise (79 g CHO, 16.5 g protein, 7.7 g fat). Following 1282
the standardized breakfast, the subjects were required to wait 2 h before a second blood sample 1283
was drawn (6 mL) for determination of plasma [NO3-] and [NO2-]. During this period, the 1284
46
subjects were provided with water to ensure adequate hydration prior to exercise. The volume of 1285
water consumed at rest in the first experimental trial was replicated for subsequent trials. 1286
1287
Experimental Exercise Protocol 1288
Immediately after the second blood sample, the subjects were escorted to the exercise 1289
laboratory (21.4 + 0.3oC, 56.0 + 3.0%). The subjects were asked to provide a urine sample for 1290
determination of USG and to void their bladder. They were then weighed and equipped with a 1291
HR monitor. Exercise testing occurred ~ 2.5 h post-beverage ingestion (Figure 8). The exercise 1292
protocol was designed to make VO2 measurements at two different submaximal exercise 1293
intensities, followed by an aerobic TT. The subjects completed a 5 min warmup at 60 W, 1294
followed by 10 min at 50% VO2peak (81 + 4 W) and 10 min at 70% VO2peak (131 + 5 W). The 1295
subjects were instructed to maintain a cadence of 80 revolutions per minute (rpm) throughout the 1296
submaximal cycling protocol. The subjects then rested for 3 min before completing a 4 kJ/kg BM 1297
TT (260 + 12 kJ). The ergometer was set to linear mode, and the subjects started cycling at a 1298
resistance equal to 70% VO2peak at 80 rpm. The subjects were able to increase their PO by 1299
increasing their rpms and were only shown the number of kJ completed during the TT. 1300
The rating of perceived exertion (RPE) was recorded every 5 min throughout the 1301
submaximal exercise protocol using the Borg Scale (6 – 20) (Borg 1982). Respiratory 1302
measurements were recorded over 20 s intervals for the final 4 min of each submaximal exercise 1303
intensity. Time, PO, rpm and RPE were recorded with the completion of each 20% of the TT. 1304
Verbal encouragement was provided with each 20% completed. HR was recorded continuously 1305
throughout the exercise protocol. Following the time trial, subjects completed a blinding and 1306
adverse side effects questionnaire. 1307
47
1308
1309
1310 Figure 8: Experimental trial exercise protocol. Red arrows indicate recording of rating of 1311 perceived exertion (RPE), power output (PO) and revolutions per minute (rpm) at 5 min intervals 1312 during the submaximal exercise protocol. Blue arrows indicate collection of oxygen uptake for 1313 the final 4 min of each submaximal exercise intensity. Black arrows indicate recording of RPE, 1314 PO, rpm and time elapsed with every 20% of the 4 kJ/kg body mass (BM) time trial completed. 1315 1316 Blood Sampling and Analysis 1317
On the mornings of the experimental trials, the subjects arrived to the laboratory and had 1318
a baseline blood sample drawn from the antecubital vein. This blood sample was ~9.5 mL, of 1319
which 3.5 mL was collected into a serum separator tube and 6 mL was collected into a K2EDTA-1320
coated tube. The second 6 mL blood sample was collected 2 h later into a K2EDTA-coated tube. 1321
Immediately after collection, all blood tubes were thoroughly mixed. 1322
Immediately following collection, the K2EDTA-coated tubes were centrifuged for 10 min 1323
at 2600 rpm and 4oC. The plasma was then aliquoted equally into two 1.8 mL cryovials. The 1324
serum separator tube was left for 30 min at room temperature to coagulate and centrifuged for 10 1325
min at 3000 rpm and 4oC. The serum was aliquoted equally into two 1.8 mL cryovials. All 1326
plasma and serum samples were immediately stored at -80oC for future analysis. 1327
Serum samples were analyzed for estradiol (pg/mL) and progesterone (ng/mL) 1328
concentrations using commercially available ELISA kits (Abcam, Item Nos: ab108667, 1329
ab108654, USA). For determination of serum estradiol concentrations, the serum samples were 1330
thawed on ice and vortexed for 5 s before 25 µL of sample or standard was added to the 96 well 1331
plate and 200 µL of Estradiol-horseradish peroxidase (HRP) conjugate was added to each well. 1332
48
The plate was covered and incubated for 2 hr at 37oC to allow the Estradiol-HRP to compete 1333
with the estradiol within the sample for antibody binding sites. The wells were then aspirated and 1334
washed three times with 300 µL of 1x wash solution to remove unbound material and 100 µL 1335
substrate was added to each well. The substrate was catalyzed by 3,3’,5,5’-Tetramethylbenzidine 1336
(TMB) to produce blue colouration. The plate was incubated for 30 min at room temperature 1337
before 100 µL stop solution was added to each well. The absorbance was read at 450 nm. 1338
For determination of serum progesterone concentrations, the serum samples were thawed 1339
on ice and vortexed for 5 s before 20 µL of sample or standard was added to the 96 well plate. 1340
Next, 200 µL of Progesterone-HRP conjugate was added to each well. The plate was covered 1341
and incubated for 1 hr at 37oC to allow the Progesterone-HRP to compete with the progesterone 1342
within the sample for antibody binding sites. The wells were then aspirated and washed three 1343
times with 300 µL of 1x washing solution to remove unbound material and 100 µL substrate was 1344
added to each well. The substrate was catalyzed by TMB substrate to produce blue colouration. 1345
The plate was incubated at room temperature for 15 min before 100 µL stop solution was added 1346
to each well. The absorbance was read at 450 nm. 1347
Plasma samples were analyzed for NO3- and NO2- concentrations using 1348
chemiluminescence. To quantify NO2-, the plasma sample was passed with a gastight syringe 1349
into a reduction solution containing 45 mmol/L potassium iodide and 10 mmol/L iodine in 1350
glacial acetic acid. The reduction solution was kept at 56oC and continuously bubbled with 1351
nitrogen. Sample aliquots were incubated with sulfanilamide for 15 min. Sulfanilamide forms a 1352
stable diazonium ion that traps NO2- and prevents it from further reduction to NO. The quantity 1353
of NO2- was determined by calculating the area under the curve for NO2- from the aliquots 1354
treated with sulfanilamide and subtracting this from the NO2- area under the curve for the 1355
49
untreated aliquots. Plasma NO3- was determined by reducing NO3- to NO via vanadium (III) 1356
chloride. The amount of NO3- was quantified by subtracting the previously determined NO2- 1357
concentration from the amount of NO3- reduced to NO (Lundberg and Govoni 2004). 1358
Compliance 1359
Each subject’s adherence to the chronic supplementation protocol was assessed through 1360
the return of the empty supplemental beverage containers, and through the analysis of plasma 1361
NO3- and NO2- concentrations. Subjects were instructed to return all empty supplement 1362
containers at the time of their chronic supplementation trial. 1363
Statistical Analysis 1364
Diet records were analyzed using Food Processor Nutrition Analysis Software (ESHA 1365
Research, Salem, USA). Statistical analyses were performed using Prism 7 (GraphPad Software, 1366
San Diego, USA). Statistical significance was achieved when P < 0.05. Data are presented as 1367
mean + SEM. The statistical tests for serum estradiol and progesterone, pre-trial characteristics 1368
(48 h diet and exercise record, pre-trial sleep), environmental conditions (temperature, humidity), 1369
hydration measures (pre-fluid intake, USG, during-fluid intake), submaximal exercise respiratory 1370
variables (VO2, VCO2, RER, VE, VT, and f), energy expenditure, and submaximal exercise 1371
performance (HR, RPE, and rpm) were conducted using a two-way analysis of variance 1372
(ANOVA) and Tukey’s multiple comparisons post-hoc test. 1373
Time trial learning effect was assessed with a one-way ANOVA and Tukey’s multiple 1374
comparisons post-hoc test. Time trial performance (HR, RPE, PO, rpm, and elapsed time) were 1375
analyzed using a three-way ANOVA (BRJ vs. PLA, acute vs. chronic, time) and Tukey’s 1376
multiple comparisons post hoc test. 1377
50
Responders to BRJ supplementation were arbitrarily defined as individuals who 1378
demonstrated a ³ 3% reduction in VO2 as this adheres with previous research and is greater than 1379
the coefficient of variation (CV) between the second familiarization trial, Acute PLA condition, 1380
and Chronic PLA condition. Responders to BRJ supplementation were also arbitrarily defined as 1381
individuals who demonstrated a ≥ 4.2% improvement in time trial performance as this is greater 1382
than the CV between the second familiarization trial, Acute PLA condition, and Chronic PLA 1383
condition. 1384
Chapter 4: Results 1385
Blood Parameters 1386
Estradiol and Progesterone 1387
Serum estradiol and progesterone concentrations were reported for 10 subjects. The data 1388
for 2 subjects was lost during analysis due to values below the limit of detection. Serum estradiol 1389
concentrations were not significantly different across the trials (Acute PLA, 0.01 + 0.003: 1390
Chronic PLA, 0.02 + 0.01: Acute BRJ, 0.01 + 0.01: Chronic BRJ, 0.01 + 0.01 pg/mL). Serum 1391
progesterone concentrations were not significantly different across the trials (Acute PLA, 0.98 + 1392
0.36: Chronic PLA, 0.47 + 0.13: Acute BRJ, 1.39 + 0.97: Chronic BRJ, 1.14 + 0.76 ng/mL). 1393
Nitrate (NO3-) 1394
Baseline plasma NO3- concentrations were not different in the Acute PLA (36 + 4 µM), 1395
Chronic PLA (36 + 3 µM), and Acute BRJ trials (44 + 3 µM) (Figure 9). However, plasma NO3- 1396
was significantly elevated at baseline in the Chronic BRJ trial (548 + 57 µM) compared to all 1397
other conditions (p < 0.0001). There was no significant difference in plasma NO3- 2 h post-1398
beverage ingestion in the Acute PLA (49 + 5 µM) and Chronic PLA (40 + 3 µM) trials. 1399
Conversely, there was a significant rise in plasma NO3- concentrations 2 h post-beverage 1400
51
ingestion in the Acute BRJ (776 + 33 µM) and Chronic BRJ (1125 + 65 µM) trials (p < 0.0001). 1401
Plasma NO3- concentrations were significantly greater in the Acute BRJ trial compared to Acute 1402
PLA and Chronic PLA 2 h post-beverage consumption (p < 0.0001). Similarly, 2 h post-1403
beverage consumption, plasma NO3- concentrations were significantly greater in the Chronic 1404
BRJ trial compared to Acute PLA and Chronic PLA (p < 0.0001). Interestingly, plasma NO3- 1405
concentrations were significantly greater in the Chronic BRJ compared to the Acute BRJ trial 2 h 1406
post-beverage consumption (p < 0.0001). 1407
1408
Figure 9: Plasma nitrate (NO3-) at baseline (B) and two hours post-ingestion (2 h) of 140 mL 1409 beetroot juice (BRJ) or NO3--free placebo (PLA) both acutely and chronically. Values are mean 1410 + SEM. *, significantly different than all other conditions. 1411 1412 Nitrite (NO2-) 1413
Plasma NO2- concentrations were not significantly different at baseline across all trials 1414
(Acute PLA, 231 + 28: Chronic PLA, 216 + 27: Acute BRJ, 240 + 26: Chronic BRJ, 328 + 20 1415
Acute
PLA (B)
Acute
PLA (2 h)
Chronic
PLA (B)
Chronic
PLA (2 h)
Acute
BRJ (B)
Acute
BRJ (2 h
)
Chronic
BRJ (B)
Chronic
BRJ (2 h
)0
50
100
500
800
1100
1400
NO
3- (µM
) *
*
*
52
nm) (Figure 10). There was no change in plasma NO2- concentrations 2 h post-beverage 1416
ingestion in the Acute PLA (266 + 23 nm) and Chronic PLA (280 + 24 nm) trials. The increase 1417
in plasma NO2- 2 h post-beverage ingestion was significant in both the Acute BRJ (729 + 80 nm) 1418
and Chronic BRJ (706 + 95 nm) trials (p < 0.0001). However, there was no significant difference 1419
in plasma NO2- between the Acute BRJ and Chronic BRJ trials 2 h post-beverage ingestion. 1420
1421
Figure 10: Plasma nitrite (NO2-) at baseline (B) and two hours post-ingestion (2 h) of 140 mL 1422 beetroot juice (BRJ) or nitrate-free placebo (PLA) both acutely and chronically. Values are mean 1423 + SEM. †, significantly greater than conditions with no symbol. 1424 1425 Pre-Trial Characteristics 1426
The subjects successfully maintained their dietary habits throughout the study as there 1427
was no significant difference in energy intake in the 48 h prior to the experimental trials (Acute 1428
PLA, 2447 + 210: Chronic PLA, 2150 + 104: Acute BRJ, 2155 + 190: Chronic BRJ, 2316 + 179 1429
kcal). Similarly, there was no change in physical activity in the 48 h prior to each trial. Lastly, 1430
Acute
PLA (B)
Acute
PLA (2 h)
Chronic
PLA (B)
Chronic
PLA (2 h)
Acute
BRJ (B)
Acute
BRJ (2 h
)
Chronic
BRJ (B)
Chronic
BRJ (2 h
)0
200
400
600
800
1000
NO
2- (nM
)
† †
53
there was no difference in sleep patterns the night prior to each trial (Acute PLA, 7.6 + 0.2: 1431
Chronic PLA, 7.3 + 0.2: Acute BRJ, 7.4 + 0.2: Chronic BRJ, 7.2 + 0.3 h). 1432
Environmental Characteristics 1433
The average temperature in the exercise laboratory during the exercise tests was 21.4 + 1434
0.3oC and the average relative humidity was 56.0 + 3.0%. Temperature did not differ between 1435
any of the trials (Acute PLA, 21.4 + 0.3: Chronic PLA, 21.5 + 0.4: Acute BRJ, 21.3 + 0.2: 1436
Chronic BRJ, 21.6 + 0.3oC), and there was no significant difference in relative humidity across 1437
the trials (Acute PLA, 55.3 + 2.9: Chronic PLA, 53.8 + 3.4: Acute BRJ, 58.6 + 2.8: Chronic 1438
BRJ, 56.2 + 3.1%). 1439
Hydration Status 1440
On average, the subjects consumed 665 + 49 mL of water in the 2 h prior to the exercise 1441
trial. The volume of water consumed prior to exercise did not differ between the trials (Acute 1442
PLA, 677 + 43: Chronic PLA, 674 + 47: Acute BRJ, 658 + 51: Chronic BRJ, 651 + 58 mL). The 1443
subjects arrived to the exercise laboratory very well hydrated with an average USG of 1.005 + 1444
0.0012. USG was not different across trials (Acute PLA, 1.003 + 0.001: Chronic PLA, 1.005 + 1445
0.002: Acute BRJ, 1.005 + 0.001: Chronic BRJ, 1.005 + 0.001). On average, the subjects 1446
consumed 569 + 56 mL of water throughout the exercise protocol. The volume of water 1447
consumed during the exercise protocol was unchanged across trials (Acute PLA, 576 + 56: 1448
Chronic PLA, 558 + 57: Acute BRJ, 584 + 62: Chronic BRJ, 560 + 54 mL). The subjects were 1449
successful at replenishing their sweat loss with water, as the average change in body mass was 1450
+ 0.1 + 0.1%. The percent change in BM was the same across trials (Acute PLA, + 0.1 + 0.1: 1451
Chronic PLA, + 0.2 + 0.1: Acute BRJ, + 0.2 + 0.1: Chronic BRJ, + 0.1 + 0.1%). 1452
Exercise Economy 50% VO2peak 1453
54
There was no change in mean VO2 between the trials when the subjects cycled at an 1454
intensity that elicited 50% VO2peak (Acute PLA, 1254 + 45: Chronic PLA, 1267 + 45: Acute 1455
BRJ, 1259 + 46: Chronic BRJ, 1252 + 42 mL/min O2) (Figure 11). Interestingly, 25% of the 1456
subjects were defined as responders to Acute BRJ supplementation and 42% of the subjects were 1457
defined as responders to Chronic BRJ supplementation at 50% VO2peak (Figure 12). A responder 1458
was defined as a subject who demonstrated a ≥ 3% reduction in VO2 following BRJ 1459
supplementation compared to PLA. This is in accordance with previous research (Wylie et al. 1460
2013) and outside of the calculated CV for VO2 at 50% VO2peak (2.4%). In addition, VE, VT, f, 1461
and RER were unchanged across trials (Table 1). Furthermore, there was a main effect of 1462
condition where there was a small but significant increase in VCO2 in the chronic trials (Chronic 1463
PLA, 1160 + 35: Chronic BRJ, 1172 + 36 mL/min) compared to the acute trials (Acute PLA, 1464
1147 + 33: Acute BRJ, 1155 + 43 mL/min) (p = 0.01) (Table 1). HR was not significantly 1465
different between conditions or from 5 - 10 min at 50% VO2peak (Table 2). RPE was not 1466
significantly different between conditions, however there was a main effect of time where RPE 1467
was significantly increased from 5 - 10 min at 50% VO2peak (p < 0.0001) (Table 2). The subjects 1468
successfully maintained a cadence of 80 rpm at 50% VO2peak during each of the trials (Table 2). 1469
There was no change in calculated energy expenditure between trials (Acute PLA, 62 + 2: 1470
Chronic PLA, 63 + 2: Acute BRJ, 62 + 2: Chronic BRJ, 62 + 2 kcal). 1471
1472
55
1473
Figure 11: Mean oxygen uptake (VO2) at 50% peak oxygen uptake (VO2peak) following acute and 1474 chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Data reported 1475 as mean + SEM. 1476 1477
1478 Figure 12: Individual oxygen uptake (VO2) at 50% peak oxygen uptake (VO2peak) following 1479 acute and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). 1480 1481 Exercise Economy 70% VO2peak 1482
Mean VO2 remained unchanged at 70% VO2peak between trials (Acute PLA, 1804 + 60: 1483
Chronic PLA, 1814 + 54: Acute BRJ, 1791 + 53: Chronic BRJ, 1789 + 54 mL/min O2) (Figure 1484
13). Interestingly, 25% of the subjects were defined as responders to Acute BRJ supplementation 1485
Acute
Chronic
1000
1200
1400
1600
1800
VO2 (
mL/
min
)
PLA
BRJ
56
and 33% of the subjects were defined as responders to Chronic BRJ supplementation (Figure 1486
14). A responder was defined as a subject who demonstrated a ≥ 3% reduction in VO2 following 1487
BRJ supplementation compared to PLA. This is in accordance with previous research (Wylie et 1488
al. 2013) and outside of the calculated CV for VO2 at 70% VO2peak (2.0%). Regardless of trial, 1489
VCO2, VT, f, and RER were unchanged (Table 1). However, there was a very small but 1490
significant decrease in VE in the BRJ trials (Acute BRJ, 42.4 + 1.5: Chronic BRJ, 42.6 L/min) 1491
compared to PLA (Acute PLA, 43.4 + 1.6: Chronic PLA, 44.4 + 1.4 L/min) (p = 0.002). HR was 1492
not significantly different between conditions or from 5 - 10 min at 70% VO2peak (Table 2). RPE 1493
was not significantly different between conditions, however there was a main effect of time 1494
where RPE increased from 5 - 10 min at 70% VO2peak (p < 0.0001) (Table 2). The subjects 1495
successfully maintained a cadence of 80 rpm at 70% VO2peak (Table 2). Energy expenditure was 1496
unaltered between trials (Acute PLA, 90 + 3: Chronic PLA, 90 + 3: Acute BRJ, 89 + 3: Chronic 1497
BRJ, 89 + 3 kcal). 1498
1499
Figure 13: Mean oxygen uptake (VO2) at 70% peak oxygen uptake (VO2peak) following acute and 1500 chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Data reported 1501 as mean + SEM. 1502
57
1503 Figure 14: Individual oxygen uptake (VO2) at 70% peak oxygen uptake (VO2peak) following acute 1504 and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). 1505 1506 Time Trial Performance 1507
There was no learning effect associated with time trial completion (Trial 1, 1857 + 107: 1508
Trial 2, 1873 + 112: Trial 3, 1913 + 117: Trial 4, 1912 + 120 s) (Figure 15). There was no 1509
significant difference in time trial completion across the trials (Acute PLA, 1845 + 71: Chronic 1510
PLA, 1869 + 75: Acute BRJ, 1912 + 93: Chronic BRJ, 1892 + 98 s) (Figure 16). Similarly, there 1511
was no difference in the time to complete each 20% split between trials (Figure 17). There was a 1512
main effect of time in which the final split was significantly faster than the other splits (p = 1513
0.0001). No difference was observed for HR throughout the time trial (Figure 18). However, a 1514
main effect of time was demonstrated, where HR was significantly higher in the final split 1515
compared to the rest of the time trial (p < 0.0001). There was no effect of condition on RPE, but 1516
RPE was significantly higher in the final split compared to the rest of the time trial (p < 0.0001) 1517
(Figure 19). There was no significant difference in rpm or PO between conditions during the 1518
time trial (Figure 20). However, there was a main effect of time in which rpm (p < 0.0001) and 1519
PO (p < 0.0001) were significantly increased in the final split compared to the rest of the time 1520
trial. 1521
Acute
Chronic
1400
1600
1800
2000
2200
2400VO
2 (m
L/m
in)
PLA
BRJ
58
1522
Figure 15: Time trial learning effect. Data reported as mean + SEM. 1523 1524
1525 Figure 16: Mean time trial completion. Values reported as mean + SEM. 1526 1527
59
1528 Figure 17: Mean time to complete each 20% split of the time trial. Values reported as mean + 1529 SEM. *, significantly faster than the previous splits. 1530 1531
1532 1533 Figure 18: Mean heart rate (HR) at the end of each 20% split during the time trial. Values 1534 reported as mean + SEM. *, significantly higher than previous splits. 1535 1536
20%
40%
60%
80%
100%
0
100200300
350
400
450Ti
me
(s)
Acute PLA
Acute BRJ
Chronic PLA
Chronic BRJ*
20%
40%
60%
80%
100%
150
160
170
180
190
200
HR
(bpm
)
Acute PLA
Acute BRJ
Chronic PLA
Chronic BRJ
*
60
1537 Figure 19: Mean rating of perceived exertion (RPE) at the end of each 20% split during the time 1538 trial. Values reported as mean + SEM. a, significantly greater than 20%; b, significantly greater 1539 than 20% and 40%; c, significantly greater than every other split. 1540 1541
1542 Figure 20: Mean power output at the end of each 20% split during the time trial. Values reported 1543 as mean + SEM. *, significantly higher than previous splits. 1544 1545 Compliance, Blinding and Adverse Events 1546
The subjects were 100% compliant to the supplementation protocol as assessed through 1547
the return of empty supplemental beverage containers, and plasma NO3- and NO2- analysis. The 1548
results from the blinding and adverse events questionnaire indicated that 100% of the subjects 1549
were successfully blinded to the treatments they received, and 58% of the subjects experienced 1550
mild adverse effects acutely and chronically with both treatments. Nausea was the most 1551
20%
40%
60%
80%
100%
12
14
16
18
20R
PEAcute PLA
Acute BRJ
Chronic PLA
Chronic BRJ
c
ba
a
20%
40%
60%
80%
100%
100
150
200
250
Pow
er O
utpu
t (W
)
Acute PLA
Acute BRJ
Chronic PLA
Chronic BRJ
*
61
commonly reported side effect (41%), 17% reported gastrointestinal upset, 17% reported 1552
beeturia, and 8% reported acid reflux. 1553
Table 1: Respiratory variables at 50% and 70% VO2peak following acute and chronic 1554 supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values reported as 1555 mean + SEM. *, Chronic trials demonstrated significantly higher carbon dioxide production 1556 (VCO2) compared to acute trials. **, BRJ significantly decreased ventilation (VE) compared to 1557 PLA. VT, tidal volume. 1558
50% VO2peak 70% VO2peak
VO2 (mL/min)
Acute PLA 1254 + 45 1804 + 60
Chronic PLA 1267 + 45 1814 + 54
Acute BRJ 1259 + 46 1791 + 53
Chronic BRJ 1252 + 42 1789 + 54
VCO2 (mL/min)
Acute PLA 1147 + 33 * 1697 + 46
Chronic PLA 1160 + 35 1709 + 46
Acute BRJ 1155 + 43 * 1706 + 53
Chronic BRJ 1172 + 36 1709 + 46
RER
Acute PLA 0.917 + 0.008 0.945 + 0.009
Chronic PLA 0.917 + 0.007 0.943 + 0.008
Acute BRJ 0.919 + 0.009 0.953 + 0.006
Chronic BRJ 0.937 + 0.007 0.956 + 0.008
VE (L/min)
Acute PLA 28.2 + 1.0 43.4 + 1.6
Chronic PLA 29.0 + 0.9 44.4 + 1.4
Acute BRJ 27.8 + 1.1 42.6 + 1.4 **
Chronic BRJ 28.1 + 1.0 42.4 + 1.5 **
VT (mL)
Acute PLA 1331 + 71 1588 + 79
Chronic PLA 1325 + 66 1613 + 69
Acute BRJ 1326 + 58 1581 + 63
Chronic BRJ 1319 + 55 1574 + 59
Frequency (#/min)
Acute PLA 27 + 2 34 + 2
Chronic PLA 27 + 1 34 + 2
Acute BRJ 26 + 1 33 + 2
Chronic BRJ 27 + 1 33 + 2
62
Table 2: Heart rate (HR), rating of perceived exertion (RPE) and revolutions per minute (rpm) 1559 recorded at 5 and 10 min at 50% and 70% peak oxygen consumption (VO2peak) following acute 1560 and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values 1561 reported as mean + SEM. 1562 1563
50% VO2peak 70% VO2peak 5 min 10 min 5 min 10 min
HR (bpm)
Acute PLA 132 + 14 133 + 15 160 + 15 164 + 15
Chronic PLA 133 + 10 131 + 10 160 + 10 165 + 11
Acute BRJ 133 + 11 133 + 13 160 + 13 164 + 14
Chronic BRJ 134 + 11 132 + 12 160 + 13 163 + 14
RPE
Acute PLA 9.6 + 2.0 10.1 + 1.8 12.6 + 0.8 13.5 + 0.8
Chronic PLA 9.8 + 1.9 10.7 + 0.5 12.6 + 0.9 13.8 + 0.9
Acute BRJ 10.2 + 1.7 10.2 + 1.7 12.4 + 1.4 13.5 + 1.2
Chronic BRJ 9.3 + 2.2 10.3 + 2.3 12.3 + 1.5 13.5 + 1.3
rpm
Acute PLA 82 + 0.4 81 + 0.3 80 + 0.3 80 + 0.3
Chronic PLA 81 + 0.3 81 + 0.3 81 + 0.3 80 + 0.1
Acute BRJ 81 + 0.5 80 + 0.2 81 + 0.3 81 + 0.3
Chronic BRJ 81 + 1.0 81 + 0.3 80 + 0.3 80 + 0.3
1564 1565
1566
1567
1568
1569
1570
1571
1572
1573
1574
63
Chapter 5: Discussion 1575
The principle findings of this study were that acute and chronic dietary NO3- 1576
supplementation, in the form BRJ, did not improve submaximal exercise economy or aerobic TT 1577
performance in recreationally active females. This study is novel as it is the first to investigate 1578
the effects of acute and chronic BRJ supplementation in females and is the first to address the O2 1579
cost of submaximal exercise in recreationally active females at multiple intensities. 1580
Comparison to Previous Work in Women 1581
There has been little previous BRJ literature in female populations and the results have 1582
produced equivocal findings. The first study to provide insight that women may respond 1583
positively to BRJ supplementation was performed by Vanhatalo et al. (2010). The study cohort 1584
consisted of 5 healthy males and 3 healthy females (VO2peak ~ 47 mL O2/kg/min), and these 1585
authors found a 5% reduction in the O2 cost of moderate intensity submaximal exercise on days 1586
1, 5 and 15 of supplementation with 5.2 mM dietary NO3-. Interestingly, this group demonstrated 1587
significantly elevated plasma NO2- levels prior to supplementation (~454 nM) compared to the 1588
current study (~250 nM). These baseline differences may be attributed to dietary habits or fitness 1589
status. Additionally, the subjects had a smaller rise in plasma NO2- following supplementation 1590
(~650 nM) compared to the current study (~725 nM). The differences in the rise in plasma NO2- 1591
likely reflect the differences in the BRJ doses between the studies. The dramatically different 1592
VO2 outcomes between this study and the current study may reflect the inclusion of 1593
predominantly male subjects in the study by Vanhatalo et al. (2010). 1594
To date, three studies have investigated the effects of BRJ supplementation in trained 1595
female populations. Glaister et al. (2015) found no effect of acute BRJ supplementation on 20 1596
km cycling TT performance (~35 min) in trained female cyclists. Similar to the results from the 1597
64
current study, we found no effect of BRJ supplementation on TT performance lasting ~30 min. 1598
However, it is also important to consider that Glaister et al. (2015) utilized a significantly smaller 1599
dose of BRJ (5.1 mM) which may explain the lack of effect in their study. Similar to Glaister et 1600
al. (2015), Buck et al. (2015) found no effect of acute BRJ supplementation on a 1 h exercise 1601
circuit involving repeated sprints in trained female team sport athletes. Again, it is possible that 1602
the BRJ dose was too small (6 mM) to elicit an ergogenic effect. Both Glaister et al. (2015) and 1603
Buck et al. (2015) obtained plasma samples for determination of NO3- and NO2- levels. 1604
Unsurprisingly, plasma NO3- and NO2- levels were significantly lower following 1605
supplementation compared to the current study. Lastly, Pospieszna et al. (2016) found a 1606
significant improvement in repeated sprint swimming performance as well as 800 m swimming 1607
performance in college level female swimmers with 8 days of BRJ supplementation. These 1608
authors utilized a larger dose of BRJ (10.2 mM) and this is the only study to employ a chronic 1609
supplementation protocol in female subjects. However, it is difficult to determine the validity of 1610
this study as the authors did not use a placebo supplement and no plasma samples were collected. 1611
Bond Jr et al. (2014) measured cycling exercise economy in sedentary, overweight, 1612
African American women. These authors found a significant reduction in the O2 cost of exercise 1613
at 40, 60, and 80% VO2peak following acute BRJ supplementation (~12 mM). This dose is 1614
comparable to the acute 13 mM dose administered in the current study. This may further 1615
emphasize the importance of administering a large enough dose of dietary NO3- in order to 1616
ensure a biological effect is elicited. However, these authors failed to report the VO2 data adding 1617
question to the validity of their results. Rienks et al. (2015) found no effect of acute BRJ 1618
supplementation (12.9 mM) on exercise performance during a 20 min RPE clamp protocol in 1619
recreationally active women. However, as a secondary outcome, these authors measured exercise 1620
65
economy at 75 W for 5 min immediately following the RPE clamp protocol and found a weak, 1621
but statistically significant ~4% reduction in the O2 cost of exercise (p = 0.048). The findings 1622
from these two studies are in contrast to the results from the current study, but inherent study 1623
design flaws and weak statistical significance make it difficult to be confident in the findings 1624
from these studies. Future work from across a number of laboratories is required to determine the 1625
true biological effect of dietary NO3- supplementation in female populations. 1626
Interestingly, the findings from the current study are in stark contrast to the consistent 1627
reports of a reduced O2 cost of exercise in recreationally active men following both acute and 1628
chronic BRJ supplementation (Bailey et al. 2009; Vanhatalo et al. 2010; Lansley et al. 2011; 1629
Wylie et al. 2013). Although the exact mechanism still remains to be elucidated, it is proposed 1630
that dietary NO3- supplementation improves exercise economy and TT performance by 1631
improving contractile, calcium-handling, or mitochondrial efficiency through the provision of 1632
NO (Bailey et al. 2010; Larsen et al. 2011; Hernandez et al. 2012; Whitfield et al. 2016). The 1633
results from this study suggest that there may be differences in the way women utilize dietary 1634
NO3- compared to men. It is possible that NO3- supplementation fails to elicit a response in 1635
recreationally active women because there may be differences in the way dietary NO3- is 1636
digested and handled or differences in the efficiency of NO production. Alternatively, sex 1637
differences in skeletal muscle composition, and the role of estrogen on mitochondria and 1638
contractile machinery may play an important role in determining the effectiveness of NO3- 1639
supplementation. 1640
Nitrate Digestion 1641
It is unlikely that recreationally active women have impaired function of the NO3--NO2--1642
NO pathway. A couple of insights can be drawn by comparing the data from this study to another 1643
66
study from our laboratory that utilized the same absolute dose of BRJ in recreationally active 1644
males as well as the same NO3- and NO2- analytic techniques (Whitfield et al. 2016). In this 1645
study, the relative dose of dietary NO3- was 0.40 mM/kg, whereas Whitfield et al. 2016 1646
implemented a relative dose of 0.33 mM/kg dietary NO3-. In the current study, baseline plasma 1647
NO3- and NO2- levels were 44 uM and 240 nM respectively and Whitfield et al. (2016) found 1648
baseline plasma NO3- and NO2- to be ~25 uM and ~200 nM respectively. Perhaps unsurprisingly, 1649
following acute BRJ supplementation we found plasma NO3- and NO2- levels to increase to 776 1650
uM and 729 nM respectively, whereas Whitfield et al. (2016) reported plasma NO3- and NO2- 1651
levels to increase to ~500 uM and ~500 nM respectively. This supports the notion that the greater 1652
relative dose of dietary NO3- in the current study resulted in a greater increase in plasma NO3- 1653
and NO2- and suggests that women do not have an impaired ability to convert dietary NO3- to 1654
NO2-. This is an interesting finding as 58% of the participants in this study reported mild adverse 1655
side effects associated with BRJ supplementation, with 42% of participants specifically reporting 1656
gastrointestinal (GI) upset. Beets are considered a high Fermentable Oligosaccharides 1657
Disaccharides Monosaccharides and Polyols (FODMAP) food due to their oligosaccharide 1658
content (Marcason 2012). High FODMAP foods are known to cause GI upset due to their poor 1659
digestibility (Magge and Lembo 2012). Women are more likely to be diagnosed with GI 1660
conditions such as irritable bowel syndrome (Anbardan et al. 2012; Oshima and Miwa 2015) 1661
therefore it was hypothesized that women may have more adverse responses to high FODMAP 1662
foods and may not be reaping the benefits of BRJ supplementation due to poor digestibility. 1663
However, it is important to consider that this may be confounded by the fact that young women 1664
are more likely to report GI discomfort and are more likely to consult a physician than young 1665
men (Wang et al. 2013) and based on the dramatic increases in plasma NO3- and NO2- following 1666
67
acute and chronic BRJ supplementation in this study it is unlikely that women have an impaired 1667
ability to process dietary NO3-. 1668
Nitrate and Nitrite Levels 1669
When comparing the baseline plasma NO3- and NO2- levels in this study to the findings 1670
of Whitfield et al. (2016) there are no discernable differences between the sexes. This is in 1671
contrast to findings by Kapil et al. (2010) who noted that although men and women did not differ 1672
in baseline plasma NO3-, women had significantly higher plasma NO2- levels compared to men. 1673
Independent of plasma NO3- and NO2- increases, we cannot rule out the possibility that 1674
women may rely more heavily on endogenous NO production via NOS or that women may have 1675
different dietary habits than men. First, it is possible that women rely more heavily on 1676
endogenous NO production since previous literature suggests a positive link between estrogen 1677
and endogenous NO production (Cicinelli et al. 1996). This would mean a greater reliance on 1678
endogenous NO production and decreased reliance on exogenous provision of dietary NO3-. 1679
Second, it’s possible that women have different dietary habits than men. A study by Jonvik et al. 1680
(2017) demonstrated that absolute dietary NO3- intake was higher in female athletes compared to 1681
male athletes, and when dietary NO3- intake was expressed relative to total energy intake these 1682
differences were more pronounced. However, it is unlikely that the women in this study had 1683
different dietary habits than the men in the study by Whitfield et al. (2016) as the baseline 1684
plasma NO3- and NO2- levels were similar. Furthermore, our exclusion criteria stated that 1685
participants could not consume a high dietary NO3- diet (> 300 mg dietary NO3-/day). It is 1686
unclear if Whitfield et al. (2016) controlled for dietary NO3- habits. 1687
1688
1689
68
Antioxidant Capacity 1690
It has been suggested that estrogen plays an important role in enhancing antioxidant 1691
capacity in women by regulating the activity of antioxidant enzymes. A number of studies have 1692
demonstrated that women have lower plasma levels of markers of oxidative stress than men (Ide 1693
et al. 2002; Bloomer and Fisher-Wellman 2008). Furthermore, a study by Barp et al. (2002) 1694
demonstrated that female rats have lower levels of oxidative stress in heart tissue than male rats, 1695
and this was associated with female rats have greater enzyme activity of superoxide dismutase 1696
(SOD) and glutathione peroxidase (GPx). Interestingly, it is likely that estrogen drives this 1697
relationship as it has been shown that women who undergo hysterectomies (Barp et al. 2002; 1698
Borras et al. 2003; Bellanti et al. 2013) as well as post-menopausal women (Singh et al. 2016) 1699
have reduced SOD enzyme content (Borras et al. 2003; Bellanti et al. 2013; Singh et al. 2016) 1700
and activity (Barp et al. 2002; Borras et al. 2003). 1701
It is possible that the increased antioxidant capacity seen in young, healthy women 1702
buffers the reactive nitrogen species and subsequent s-nitrosylation associated with BRJ 1703
supplementation. However, it is important to consider that the antioxidant capacity relationships 1704
described above were observed in plasma blood samples as well as heart, liver and brain tissue 1705
and may not reflect skeletal muscle. If these sex differences in antioxidant capacity exist in 1706
skeletal muscle, women may have minimized effects of BRJ supplementation on altering skeletal 1707
muscle contractile function, calcium-handling, or mitochondrial efficiency. Future work is 1708
needed to elucidate whether these differences exist at the level of the skeletal muscle and if they 1709
manifest in significant metabolic and physiological outcomes. 1710
1711
1712
69
Differences in Skeletal Muscle Fiber Types and Capillarization 1713
A number of studies have performed histochemical analysis on human skeletal muscle 1714
fibers to determine sex differences in fiber type composition. These studies consistently 1715
demonstrate that women have an ~5% greater proportion of type I oxidative muscle fibers, 1716
whereas men have a greater proportion of type II glycolytic muscle fibers (Staron et al. 2000; 1717
Haizlip et al. 2014). BRJ supplementation is purported to elicit its beneficial effects 1718
predominantly in type II muscle fibers. This is attributed to the fact that the NO3--NO2--NO 1719
pathway is an O2 independent pathway that elicits its effects in scenarios of reduced O2 delivery 1720
(Vanhatalo et al. 2011). BRJ has been suggested to facilitate the delivery of O2 to type II muscle 1721
fibers that are less oxygenated than type I muscle fibers (Wilkerson et al. 2012). Therefore, it is 1722
possible that women are less likely to respond to BRJ supplementation due to a greater 1723
proportion of type I muscle fibers, a more oxygenated skeletal muscle environment, and 1724
therefore a decreased reliance on the O2 independent NO3--NO2--NO pathway. Although this is 1725
an interesting hypothesis, it is unclear if a 5% difference in fiber type composition is a large 1726
enough difference to drive these differences in response to BRJ supplementation. 1727
However, this hypothesis is supported in two different ways. First, as previously 1728
described, there is a positive link between estrogen and NOS activity (Cicinelli et al. 1996; 1729
Townsend et al. 2011) suggesting that women may place a heavier reliance on the endogenous 1730
NO production than men, and that the exogenous NO3--NO2--NO pathway may provide a less 1731
robust contribution to NO production in women than men. Secondly, women have greater 1732
skeletal muscle capillarization (Roepstorff et al. 2006; Hoeg et al. 2009) suggesting they have 1733
enhanced O2 delivery to skeletal muscle compared to men. This would further support the notion 1734
of a decreased reliance on the NO3--NO2--NO pathway in women compared to men due to a more 1735
70
oxygenated muscle environment. Conversely, it is important to consider the fact that men have 1736
greater hemoglobin levels (Murphy 2014) and skeletal muscle mass (Janssen et al. 2000), and 1737
these are larger driving factors for O2 delivery and exercise performance than skeletal muscle 1738
capillarization and fiber type differences. Furthermore, it is unlikely that O2 delivery is hindered 1739
at 50% or 70% VO2peak making this hypothesis feasible but unlikely to explain the potential sex 1740
differences seen in this study with BRJ supplementation. 1741
Mitochondrial Efficiency 1742
There is scientific literature suggesting that women have a greater reliance on fat for fuel 1743
during submaximal exercise (Blatchford et al. 1985; Horton 1998; Tarnopolsky et al. 1990). It is 1744
possible that since women rely more heavily on oxidative metabolism, have a greater capillary 1745
density and have a greater proportion of type I muscle fibers that they may exhibit more efficient 1746
mitochondrial coupling compared to men. This notion is in part supported by evidence 1747
suggesting that the livers and brains from female rats produce less mitochondrial H2O2 compared 1748
to males indicating the potential for improved mitochondrial coupling (Borras et al. 2003). 1749
Mitochondrial H2O2 production is increased in situations of elevated electron slippage (Wong et 1750
al. 2017). Therefore, it is possible that a lower level of mitochondrial H2O2 in female rats is 1751
indicative of decreased electron slippage and ultimately improved mitochondrial coupling. 1752
Interestingly, this relationship is likely driven by estrogen as Borras et al. (2003) found increased 1753
mitochondrial H2O2 production in ovariectomized rats and that estrogen replacement therapy 1754
reduced mitochondrial H2O2 production to basal levels. Furthermore, a recent study by Acaz-1755
Fonseca et al. (2017) showed that cerebral cortex mitochondria of male mice have greater UCP-2 1756
expression compared to female mice. It is possible that this is a metabolic adaptation to deal with 1757
greater mitochondrial ROS in male mice compared to females. Although these studies did not 1758
71
investigate these relationships in skeletal muscle, it would be interesting to see if these same 1759
trends exist in this tissue. Based off of these speculations, it is possible that women have a 1760
decreased reliance on the NO3--NO2--NO pathway due to more efficient mitochondrial coupling 1761
and ultimately oxidative metabolism. Therefore, it’s possible that any effects of dietary NO3- 1762
supplementation that may affect mitochondrial efficiency may be lost in healthy women who 1763
may exhibit more efficient mitochondrial coupling compared to men. Although mitochondrial 1764
H2O2 production and UCP protein content are not the only indicators of mitochondrial coupling, 1765
they may be interesting avenues for future research to consider in skeletal muscle. 1766
A recent study by Miotto et al. (2018) investigated sex differences in mitochondrial 1767
respiratory kinetics at rest in human skeletal muscle. These authors noted that women have 1768
decreased ADP sensitivity, as well as greater sensitivity for malonyl CoA inhibition of palmitoyl 1769
CoA supported respiration compared to men. The functional role of these findings remains to be 1770
determined, but this is the first piece of evidence supporting sex differences in mitochondrial 1771
function in human skeletal muscle and therefore may support the notion of studying sex 1772
differences in mitochondrial efficiency with respect to BRJ supplementation. 1773
Calcium-Handling Proteins 1774
A final difference that may explain why women do not respond to BRJ supplementation 1775
is the possible differences that may exist in skeletal muscle calcium-handling. Intriguingly, the 1776
time to peak tension and rate of relaxation are faster in the muscle of human males compared to 1777
females (Wüst et al. 2008). Time to peak tension is used as an indirect measure of calcium 1778
release at the onset of skeletal muscle contraction and rate of relaxation is used as a proxy for the 1779
reuptake of calcium following skeletal muscle contraction. This suggests that men may have a 1780
72
greater ability to release and resequester calcium than women and may implicate sex differences 1781
in skeletal muscle calcium-handling protein content or activity. 1782
A study by Welle et al. (2008) demonstrated that healthy women have a 3.6-fold greater 1783
gene expression of RyR3 in skeletal muscle than healthy men. Although mRNA does not always 1784
translate to protein content, it is possible that women have a greater ability to release calcium via 1785
RyR in skeletal muscle. This seems counterintuitive as it would be expected that if women had 1786
greater RyR content they would have greater Ca2+ release compared to men and likely faster time 1787
to peak tension but, this theory does not account for differences in RyR protein activity or the 1788
responsiveness of female contractile elements to Ca2+ influx. However, if more Ca2+ is released 1789
via RyR with no differences in SERCA or CASQ content or activity, it may take longer to 1790
resequester this Ca2+ helping corroborate slower relaxation times seen in women. This would 1791
also result in a greater ATP cost from SERCA to pump the additional Ca2+. Perhaps most 1792
importantly, it is important to readdress the role of fiber type differences between men and 1793
women. Women have a greater proportion of type I fibers, which are known to have slower time 1794
to peak tension and half relaxation times than type II fibers (Buchthal and Schmalbruch 1970). 1795
Although these fiber type differences are around 5% they likely account for the differences in 1796
contractile properties to a greater extent than potential sex differences in skeletal muscle 1797
contractile protein content or activity. 1798
Individual Responders 1799
Although mean VO2 and time trial performance were unaltered by acute and chronic BRJ 1800
supplementation, individual responders were identified at 50% VO2peak, 70% VO2peak and for TT 1801
performance. Interestingly, 25% of the subjects were defined as responders to Acute BRJ 1802
supplementation and 42% of the subjects were defined as responders to Chronic BRJ 1803
73
supplementation at 50% VO2peak. A responder was defined as a subject who demonstrated a ≥ 3% 1804
reduction in VO2 following BRJ supplementation compared to PLA. This is in accordance with 1805
previous research (Wylie et al. 2013) and is outside of the calculated CV for VO2 at 50% VO2peak 1806
(2.4%). Furthermore, 25% of the subjects were defined as responders to Acute BRJ 1807
supplementation and 33% of the subjects were defined as responders to Chronic BRJ 1808
supplementation at 70% VO2peak. Cumulatively, 2 participants responded to BRJ 1809
supplementation in 3 of the 4 conditions, and 1 participant responded in all BRJ conditions. A 1810
TT responder was defined as a subject who demonstrated a ≥ 4.2% reduction in time to complete 1811
the 4 kJ/kg TT as this was the CV for this measurement. None of the participants demonstrated 1812
this level of improvement, however one subject who demonstrated a VO2 response both acutely 1813
and chronically to BRJ supplementation also demonstrated a 3.3% improvement in TT 1814
performance acutely and a 3.8% improvement chronically. 1815
These results are in agreement with studies by Christensen et al. (2013) and Boorsma et 1816
al. (2014) who identified individual responders to BRJ supplementation in males. In the current 1817
study, responders could not be identified based on baseline or rises in plasma NO3- and NO2- 1818
levels. This is in accordance with the findings by Boorsma et al. (2014). It is unclear why the 1819
individuals in this study responded positively to BRJ supplementation since they had similar 1820
dietary habits, exercise regimes, and fitness statuses compared to the other participants. Future 1821
research is required to elucidate the mechanisms behind individualized responses to BRJ 1822
supplementation. Nonetheless, these findings are important because despite no change in mean 1823
VO2, it is still possible for some women to respond positively to acute and chronic BRJ 1824
supplementation, and it is possible for the improvement in submaximal exercise economy to 1825
translate to slight aerobic TT performance benefits in some individuals. 1826
74
Limitations 1827
The metabolic cart used to collect the data in this study has up to 3% error in VO2 1828
measurement, therefore it is possible that the current methodology was not sensitive enough to 1829
detect small reductions in VO2 following BRJ supplementation. Typically, the reduction in VO2 1830
following BRJ supplementation is in the range of 3 – 5%. In the current study, the CV for VO2 1831
was 2.4% at 50% VO2peak and 2.0% at 70% VO2peak. Therefore, it is possible that some of the 1832
change in VO2 associated with BRJ supplementation may have been masked by inherent error 1833
associated with the metabolic cart. 1834
The present study used a 4 kJ/kg TT (~30 min) to assess exercise performance. This style 1835
of TT has been validated to have greater ecological validity compared to time to exhaustion 1836
protocols (Currell and Jeukendrup 2008). However, this may not be a valid measure of 1837
performance in recreationally active individuals who do not train for TT performance. We 1838
ensured each participant was adequately familiarized with the TT protocol twice and that there 1839
was less than 5% difference in TT completion before they graduated to the experimental trials. 1840
Interestingly, the CV for TT performance between the second familiarization trial, Acute PLA 1841
and Chronic PLA trials, where there presumably there was no effect of condition, was 4.24%, 1842
which is in agreement with Currell and Jeukendrup (2008) suggesting < 5% variability in TT 1843
performance for trained individuals. This indicates that this protocol had high repeatability 1844
despite the recreational status of the participants. 1845
Future Directions 1846
Following the completion of this study, a number of questions remain unanswered 1847
regarding sex differences that may exist with BRJ supplementation. Future work should include 1848
a study similar to that of Porcelli et al. (2015) to verify the findings from the current study and to 1849
75
determine if the effect of training status on the response to BRJ supplementation exists in 1850
women. Second, future work should directly compare the responses to acute and chronic BRJ 1851
supplementation in recreationally active male and female populations. If consistent effects are 1852
seen with VO2 and exercise performance, then muscle biopsies should be obtained to delve into 1853
the mechanisms underpinning the potential sex differences that may exist. Lastly, future work 1854
should investigate the effects of BRJ supplementation in sedentary male and female populations 1855
and should determine if improved fitness status via aerobic exercise training can remove the 1856
potential beneficial effect of BRJ supplementation. Specifically, VO2 should be measured at rest, 1857
50% and 70% VO2peak at baseline, following 4 weeks of training, and again following 10 weeks 1858
of aerobic training. Clearly, incorporating women into BRJ research is in its infancy and there 1859
are a number of avenues yet to be explored, it will be fascinating to see how the field grows over 1860
the next few years and what studies stem from the work presented in this thesis. 1861
Conclusion 1862
The main findings from the current study were that acute and chronic supplementation 1863
with NO3--rich BRJ did not improve submaximal cycling exercise economy or aerobic TT 1864
performance in recreationally active women. These findings are novel because it is the first study 1865
to investigate the effects of acute and chronic BRJ supplementation in women and is the first 1866
study to measure exercise economy at multiple exercise intensities in recreationally active 1867
women. Although we did not perform any mechanistic measures in this study, we can speculate 1868
that there may be sex differences associated with BRJ supplementation. Future research should 1869
continue to investigate the potential sex differences that may exist with dietary NO3- 1870
supplementation and should seek to determine the mechanisms underpinning these differences. 1871
1872
76
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