R Davies Dissertation 2015

The Effect of Acute Dietary Nitrate Supplementation on Resistance-Exercise Induced Oxidative Stress in Healthy Adults

Sodium Nitrate Supplementation and Oxidative Stress in Resistance-Exercise

ContentsThe Effect of Acute Dietary Nitrate Supplementation on Resistance-Exercise Induced Oxidative Stress in Healthy Adults...................................................................................................................................3

Abstract.............................................................................................................................................3

Introduction.......................................................................................................................................4

Methodology.....................................................................................................................................7

Results.............................................................................................................................................11

Discussion/Conclusion.....................................................................................................................21

Strengths/Limitations......................................................................................................................24

Acknowledgments...........................................................................................................................25

References.......................................................................................................................................25

Appendix 1: Fitness Activity Questionnaire.....................................................................................35

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The Effect of Acute Dietary Nitrate Supplementation on Resistance-Exercise Induced Oxidative Stress in Healthy Adults

AbstractPurpose Supplementation with ergogenic aids such as beetroot juice has been widely

reported to enhance exercise performance. There is limited evidence exploring the effects

of oxidative stress (OS) on resistance-exercise, and even little encompassing dietary nitrate

which is metabolised to nitric oxide (NO) a free radical. Exercise induces OS and enables the

increased production of reactive oxygen species (ROS). The purpose of this study was to

investigate whether NaNO3 had any impact on OS induced in resistance-exercise. Method

This randomised placebo-controlled double-blind crossover study of 10 participants used

knee-extensions to induce OS while supplementing with 5mg/kg bodyweight (BW) sodium

nitrate (NaNO3) or a 3.3mg/kgBW sodium chloride placebo. Malondialdehyde (MDA) and

ascorbate were used as biomarkers to assess OS pre-exercise, 2hrs post-exercise, and 24hrs

post-exercise. Dietary analysis was accounted for in a food frequency questionnaire (FFQ)

and 24hr dietary recalls. Results No statistically significant results were found that indicated

whether NaNO3 had any influence on OS biomarkers. No difference was found amongst

gender, but a significant statistical difference was seen in males in the placebo group at

24hrs post-exercise (p=0.046) Conclusion The findings indicate that compared to a sodium

chloride placebo, no statistically significant changes were observed in the OS in resistance-

exercise except for in males at 24hrs post-exercise taking the placebo. These results suggest

that dietary nitrate supplementation with 5mg/kgBW dosage does not adversely affect OS

biomarkers, contrary to established belief, and the use of NaNO3 may be beneficial

therapeutically in the dietetic management of chronic obstructive pulmonary disease

(COPD) and cardiovascular health.

Key words: sodium nitrate, oxidative stress, resistance-exercise, malondialdehyde,

ascorbate

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Introduction Nitrate is metabolised to nitrite and subsequently to NO which exerts cardiovascular

benefits by promoting vasodilation, increased blood flow and oxygen delivery to muscles

and tissues (Machha and Schechter, 2011; Kapil et al, 2014); once ingested plasma nitrate

peaks at 1.5hrs and plasma nitrite at 2.5-3hrs (Webb et al, 2008). Accordingly, evidence

supporting dietary nitrate’s role in a range of beneficial vascular effects is accumulating

(Lidder and Webb, 2012; Clements et al, 2014). Many studies investigating the beneficial

effect of nitrate supplementation, commonly in the form of nitrate-rich beetroot juice, on

exercise performance (Zafeiridis, 2014) in a variety of ages and populations but the

underlying mechanisms remain obfuscated (Bailey et al, 2010; Lansley et al, 2011; Larsen et

al, 2011; Clements et al, 2014).

Regular physical exercise has been linked to many health benefits including reduced risk of

obesity, cardiovascular disease, diabetes, osteoporosis, and some cancers, and in

ameliorating mental health status (Blair et al, 2001; Warburton et al, 2006; Zschucke et al,

2013). However, exercise induces OS but the underlying mechanisms have yet to be

established (Fisher-Wellman and Bloomer, 2009). It has been thought that mitochondria in

muscle cells are capable of generating ROS via electron leakage (Jackson, 2000) and

superoxide production at complexes I and III in the electron-transport chain (Muller et al,

2004) however, there is increasing evidence demonstrating that this is not as detrimental as

was once thought (Powers et al, 2011). Furthermore, muscle oxygen consumption and

oxidative phosphorylation increase in exercise, which may relate to higher

ROS and superoxide production (Urso and Clarkson, 2003), but Vollaard et al (2005) suggest

it is unlikely that an increase in oxygen consumption is the only cause for increases in ROS

production during exercise, and other possible mechanisms include muscle damage,

ischemia/reperfusion and the associated activation of endothelial xanthine oxidase,

inflammatory responses, and the autooxidation of catecholamines (Gomes et al, 2012).

Powers and Jackson (2008) have suggested numerous potential mechanisms resulting in

exercise-induced OS such as contracting skeletal muscles generating reactive nitrogen

species from NO, and superoxide anions, which react together to form peroxynitrite, a more

powerful oxidant, which modifies metalloproteins, damages DNA, and induces lipid

peroxidation (Pacher et al, 2007). NO can also exert adverse physiological effects by binding

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with iron (Valko et al, 2007) and resulting in methaemoglobinaemia (Kapil et al, 2014).

Therefore, it can be surmised that an increase of NO would lead to oxidative damage.

Conversely, it has been proposed that exercise-induced ROS may be produced in a

controlled and regulated manner and this process is crucial in allowing optimal skeletal

muscle adaptations to occur (Jackson, 2008; Radak et al, 2008; Fisher-Wellman and

Bloomer, 2009; Powers et al, 2011).

OS occurs when the production of ROS exceeds the antioxidant defence mechanisms

enabling oxidative damage to cellular proteins, lipids, DNA, and other molecules which may

lead to abnormal cellular function (Halliwell, 2007). OS has been implicated in the

pathogenesis of a number of chronic-progressive diseases (West, 2001; Lakshmi et al, 2009;

de Lemos et al, 2012; Pitocco et al, 2013; Sosa et al, 2013), and is characterised by increases

in oxidants and oxidation products, and decreases in anti-oxidant agents (Powers and

Jackson, 2008). Measurements of OS in exercise studies have assessed the impact of ROS on

molecular products such as MDA, a lipid peroxide end product, and on the human

antioxidant defence system by measuring redox changes in endogenous antioxidants which

includes serum ascorbate (Fisher-Wellman and Bloomer, 2009), and ascorbate has been

shown to protect against lipid peroxidation and protein oxidation in human plasma (Jomova

and Valko, 2011). Assessing OS in exercise is problematic as the onset of exercise-induced

OS may result from multiple factors: intensity and duration of exercise, age, training status,

and dietary intake (Fisher-Wellman and Bloomer, 2009), and results are variable (Bailey et

al, 2004; El Abed et al, 2011; Farney et al, 2012; Bouzid et al, 2014).

Exercise-induced OS is evident in both aerobic and anaerobic exercise (Bloomer and Smith,

2009). However, the degree of oxidative damage appears to be dependent on the type,

intensity, and duration of exercise, and the nutritional status of the participant (Bloomer

and Goldfarb, 2004). OS response in exercise has been primarily investigated in aerobic-

exercise with fewer studies reporting on resistance-exercise (Fisher-Wellman and Bloomer,

2009). Evidence indicates that high-intensity resistance-exercise produces a measurable

increase in OS (Bloomer et al, 2006; Bloomer et al, 2007; Hudson et al, 2008; Fisher-

Wellman and Bloomer, 2009). The amount and intensity of resistance-exercise necessary to

induce acute OS is unclear. Some studies employed three or four sets (Hudson et al, 2008)

while others used up to 30 sets (Beaton et al, 2002) of repetitions; intensity varies from 50%

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1 repetition maximum (RM) to moderate (60-65%), (Beaton et al, 2002) to 80% (Liu et al,

2005).

Despite the recent popularity for beetroot juice as an ergogenic aid, evidence for dietary

nitrate’s effects on OS, particularly in humans, is limited (Ujawska et al, 2009; Dreißigacker

et al 2010; Carlström et al, 2011; Krajka-Kuźniak et al, 2012; Singh et al, 2012; Ashmore et al,

2014 Larsen et al, 2014). Beetroot has a high antioxidant capacity and contains many other

components besides nitrate (Vulic et al, 2012) therefore, it is not possible to differentiate

betweeen nitrate or any of the other components in beetroot on OS in execise.

Consequently, this study aims to investigate the effect of acute dietary nitrate

supplementation on OS induced in resistance-exercise in young healthy physically active

adults by measuring MDA and ascorbate levels. The research strategy for this study involved

reviewing relevant literature using Google Scholar, PubMed, Cochrane, and the University of

Plymouth’s Library Primo databases for articles in English with the following key words used

in multiple combinations: beetroot, beetroot juice, dietary nitrate, exercise, anaerobic

exercise, resistant-exercise, oxidative stress, MDA, and ascorbate. The bibliographies of

identified articles with these key words were searched for additional references. It was

necessary to collate relevant studies examining the effect of OS and exercise (Bloomer and

Fisher-Wellman, 2008; Panza et al, 2008) with those of OS and dietary nitrate (Larsen et al,

2014), and dietary nitrate/beetroot juice and exercise (Larsen et al, 2007, 2010, 2011, 2014;

Bailey et al, 2009; Bescos et al, 2011, 2012; Lansley et al, 2011; Vanhatalo et al, 2011;

Murphy et al, 2012; Muggeridge et al, 2013; Wylie et al, 2013): participant range=8-131;

median=10. Therefore, this study aimed for a convenience sample of 15 to coincide with

previous studies and to allow for possible non-compliance

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Methodology

Design

This study utilised a randomised, placebo-controlled, double-blind crossover design.

Participants were randomised to treatment A (NaNO3) or B (placebo) by the project

supervisor prior to the initial visit.

Recruitment

Due to time constraints, and the exploratory nature of the study, convenience sampling, in

the form of informative oral and e-mail requests, was used to recruit participants from the

student population of Plymouth University (Endacott and Botti, 2007). Participants recruited

numbered 16 of which 10 completed the study. Six participants failed to complete the

study: one unable to complete the exercise test; one unable to commit to the test time-

period; two unable to give blood samples; and two withdrew due to side effects to nitrate

supplementation. Dose of NaNO3 (A) was 10mg/kgBW and placebo (B) 7mg/kgBW for three

participants. These participants reported side effects including nausea and feelings of

general unwellness. Therefore, the remaining participants were supplemented with

5mg/kgBW NaNO3and 3.5mg/kgBW placebo. A further participant reported side-effects on

this NaNO3dose and attended hospital services on advice from medical staff. No adverse

effects were found upon medical examination however, this participant was withdrawn

from the study due to ethical considerations.

Participants were restricted to the ages of 18-40 years based on OS and aging theories

(Finkel and Holbrook, 2000) and gender was not an exclusion criterion (Fisher-Wellman and

Bloomer, 2009). Physical exercise status was assessed by a questionnaire used in Bloomer

and Fisher-Wellman (2008) to ascertain exercise status and to categorise participants to

allow any outliers to be excluded (appendix 1). Those who smoke (Takajo et al, 2001;

Bloomer et al, 2008) have diabetes (West, 2001; de Lemos et al, 2012; Pitocco et al,

2013), neurodegenerative disorders (Uttara et al, 2009; Melo et al, 2011), cancer (Sosa et al,

2013), cardiovascular disease (Lakshmi et al, 2009), and rheumatoid arthritis (Valko et al,

2007) were also excluded due to the incumbent presence of disease-associated OS.

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Exercise protocol

Participants were asked to perform 10 sets of 10 single-leg extensions (or until exhaustion)

at submaximal load (SML), calculated at 80% of their individual 1RM on a leg-extension

machine determined by initially starting on the highest weight and moving down each

weight until the participant was able to do one repetition at full leg extension (motion range

set at 100%). Leg used was randomised by project supervisor. All subjects were familiarised

on proper usage of the equipment prior to participating in this study. The configuration of

the leg-extension machine was adapted to each participant as per the manufacturer’s on-

machine instructions. Participants were asked to do a light warm-up of 5mins brisk walking

on the treadmill to prevent muscle injury (Deminice, 2010). This study utilised single-leg

extensions to isolate a muscle group to induce OS whilst allowing the other leg to be used in

the subsequent repeated exercise test (Nikolaidis et al, 2007; Paschalis et al, 2007). This

allowed for a relatively short washout period for the exercise test as any muscle damage

induced would have limited effects on the following test. Exercise tests were separated by

one week (Hudson et al, 2008). A limit of 30s was given to complete a repetition before

exercises were discontinued. Verbal encouragement was given throughout. As per Hudson

et al (2008) and Deminice et al (2010), we allowed 90s rest period between sets.

Participants were instructed to return to the laboratory to repeat the protocol using the

alternate leg and supplement the following week. This provided a minimum 3-day washout

period for nitrate supplementation and a minimum 6-day washout period between exercise

protocols (Paschalis et al, 2007).

A Visual Analogue Scale (VAS) was utilised to assess muscle pain subjectively immediately

before and after exercise, and 2hrs post-exercise in both groups which participants rated on

a scale 1-10 (1=no pain, 10=worst pain). This provided a subjective evaluation of exercising-

muscle intensity and multiple measurements intra-individually aided reliability (Vautier,

2011).

Supplements

Supplements were labelled A for NaNO3 and B for placebo. Three participants were provided

with either 4 doses of NaNO3 10mg/kg bodyweight (BW) supplement or placebo

(7mg/kgBW) (Larsen et al, 2011; Bescos et al, 2011, 2012; Larsen et al, 2014) to be ingested

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singly 48hrs, 24hrs, and 2.5hrs prior to the exercise protocol and 2.5hrs prior to the 24hrs

post-exercise blood test dissolved in liquid of the participants choosing. However, these

participants reported side-effects including nausea, vomiting, and general unwellness

therefore, the remaining participants were given NaNO3 5mg/kgBW and 3.5mg/kgBW

placebo.

Nutritional Assessment

Participants arrived at the laboratory in a non-fasted state, having avoided specified items

(table 1), and strenuous exercise 24hrs prior to testing and to the 24hrs post-exercise blood

test. Participants were required to complete a modified FFQ to assess average intake of

nutrients that may affect OS status. The FFQ was modified from the validated FFQ used in

the EPIC-Norfolk study (Riboli and Kaaks, 1997; Slimani et al, 2002) to reflect the research

purposes of this study (Cade et al, 2001; Shim et al, 2014). The FFQ used in this study

covered meat and fish, dairy products and fats, fruit, vegetables, and tea/coffee. This data

was analysed using the same FETA (2014) software used to analyse the data from the EPIC-

Norfolk study (Mulligan et al, 2014). 24hr dietary recalls were conducted by members of the

research team and were used to assess dietary intakes that may affect OS prior to the

exercise test and prior to the 24hr post-exercise blood sample. A 24hr recall limits study-

burden and is similarly effective as a weighed record (Bingham et al, 1994, 1997; Brunner et

al, 2001). This data was analysed using Nutritics (2011) software. Both FFQ and dietary

recalls were used to assess dietary intakes of energy in kcal (Merry, 2006), vitamins A, C, and

E, and copper, manganese, selenium, and zinc (Evans and Halliwell, 2001; Wernerman,

2012), and iron (Evans and Halliwell, 2001; Fang et al, 2002) to indicate consistency and

allow potential explanations of nutritional impact on OS status.

Participants were asked to avoid antibacterial mouthwash due to Govoni et al (2008)

reporting a reduction in the formation of systemic nitrite on removal of oral bacteria.

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Table 1 Items to avoid

Dietary sources Corresponding evidence

Rocket, spinach, lettuce, radish, beetroot, Chinese cabbage

Lidder and Webb, 2012

Alcohol Das and Vasudevan, 2007

Caffeinated beveragesCoimbra et al, 2006; Olcina et al,

2006, 2008; Tsai et al, 2006; Arent et

al, 2008; Panza et al, 2008

Dietary supplements Urso and Clarkson, 2003

Biochemical Analyses

Four 10ml blood samples were taken intravenously from the forearm cubital region by a

trained phlebotomist at baseline (to allow for later nitrate/nitrite analysis, if deemed

appropriate), immediately before commencing exercise, 2hrs post-exercise, and at 24hrs

post-exercise for each supplement group. Blood samples were labelled with participant

code, and centrifuged at 3500rpm for 5mins and then stored at -80˚C for the following

analyses: total:reduced ascorbate as per Sato et al (2010) and Mitton and Trevithick

(1994b); and MDA as per Agarwal and Chase (2002).

Ethics

This study received ethical approval from the School of Health Professionals Bachelor’s

Degree Ethics Subcommittee and a risk assessment was performed to minimise harm.

Participants were fully briefed on the study protocol and informed consent was obtained.

Participants were informed of their right to withdraw at any time. Blood samples were

treated according to the Human Tissue Act (2004) and stored anonymously. This study

complied with data handling in accordance with the Data Protection Act (1998).

Statistics

Due to the small sample size (n=10), all data were tested non-parametrically (Redmond,

2002). Statistical differences were assessed using Wilcoxon signed rank test and Spearman

rho for correlations. All data are presented as medians (min/max). Statistical analysis was

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performed on IBM SPSS software version 21.0 (Chicago, IL, USA). Statistical significance was

accepted when p ≤ 0.05 as per convention.

Results Data collection was conducted in May and June 2015 at the University of Plymouth’s Link-

Lab. Baseline characteristics are presented in table2.

Table 2 Baseline characteristics

Total number Median Min/Max

Gender Male 6

Female 4

Age (yrs) 21 20-34

BMI (kg/m2) 22.3 19.1-25.6

Average Nutrient Intake (FFQ)

Vitamin A - (retinol equivalents) (µg/day) 920 480-2364

Vitamin C - (mg/day) 88 35-156

Vitamin E - (a-tocopherol equivalents) (µg/day)

7 5-21

Copper (mg/day) 0.7 0.3-1.2

Iron (mg/day) 5.8 4.4-12.4

Manganese (mg/day) 2 1-4

Selenium (µg/day) 40 16-76

Zinc (mg/day) 6 3-9

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Table 3 Medians (min/max) biomarkers at exercise intervals

A B P-valueMedian Min/Max Median Min/max

Total Ascorbate (μM)

Pre-exercise 68.40 17.95 - 98.86 67.01 33.27 - 92.34 0.3862hrs post-exercise 70.17 17.95 - 101.24 69.26 36.88 - 92.89 0.85924hrs post-exercise 79.18 37.96 - 93.80 64.44 33.27 - 101.24 0.285

Reduced Ascorbate (μM)

Pre-exercise 54.64 17.68 - 91.97 68.77 21.82 - 89.05 0.7672hrs post-exercise 61.46 19.89 - 117.66 67.26 23.20 - 98.86 0.51524hrs post-exercise 66.19 17.68 - 94.16 65.15 21.54 - 92.88 0.414

% Reduced Ascorbate

Pre-exercise 89.86 41.18 - 102.78 91.97 44.96 - 120.31 0.1392hrs post-exercise 83.15 40.51 - 167.65 92.94 69.11 - 121.92 0.85924hrs post-exercise 97.75 43.75 - 118.97 96.85 43.41 - 125.76 0.386∆ pre:2hr post exercise 1.18 -33.79 - 22.82 -1.43 -31.16 - 66.93 0.889∆ pre:24hr post exercise 6.29 -41.03 - 24.04 2.30 -7.87 - 15.54 0.515

MDA (μM)Pre-exercise 3.75 1.16 - 21.84 3.96 0.87 - 20.23 0.9592hrs post-exercise 3.69 -0.93 - 21.16 4.24 1.73 - 18.61 0.59424hrs post-exercise 3.90 2.83 - 12.41 4.24 2.01 - 25.18 0.139∆ pre:2hr post 0.33 -3.35 - 8.71 0.24 -2.85 - 13.54 0.374∆ pre:24hr post 0.18 -17.52 - 7.03 0.45 -1.42 - 6.02 0.678

∆=change

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Males A B P-valueMedian Min/Max Median Min/max


Pre-exercise 55.31 17.96-76.07 52.74 33.27-80.51 0.7532hrs post-exercise 60.86 17.95-79.21 56.48 36.88-83.77 0.91724hrs post-exercise 62.28 37.96-80.56 57.53 33.27-68.99 0.249



% Reduced Ascorbate

Pre-exercise 83.03 41.03-102.78 85.69 44.96-118.31 0.2492hrs post-exercise 77.87 40.51-110 86.45 69.11-121.92 0.17324hrs post-exercise 81.46 43.46-118.97 94.26 43.41-125.76 0.463∆ pre:2hr post exercise -3.69 -33.79-11.43 0.95 -31.16-66.93 0.345∆ pre:24hr post exercise 10.94 -41.03-24.04 5.5 -7.87-15.54 0.917

MDA (μM)Pre-exercise 4.05 2.42-21.83 4.15 0.87-20.23 0.7532hrs post-exercise 3.62 -0.93-21.16 7.85 1.73-18.61 0.91724hrs post-exercise 3.77 2.83-12.41 5.36 3.17-25.18 0.046*∆ pre:2hr post -0.43 -3.35-7.67 -0.10 -2.85-13.54 0.917∆ pre:24hr post -0.28 -9.43-7.03 2.36 -1.41-6.02 0.600

* significance indicated at p-value ≤0.05

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Females A B P-valueMedian Min/Max Median Min/max





% Reduced Ascorbate

Pre-exercise 90.12 86.25-96.92 105.79 83.57-120.31 1.0002hrs post-exercise 112.94 89.63-167.65 97.88 82-113.79 0.10924hrs post-exercise 100.19 92.54-112.5 102.91 81.71-125 0.715∆ pre:2hr post exercise 13.1 3.38-22.82 -4.05 -14.6- -1.24 0.180∆ pre:24hr post exercise 6.29 3.47-9.88 -0.41 -6.82-4.69 0.109

MDA (μM)Pre-exercise 3.54 1.16-21.26 3.76 2.50-5.02 0.7152hrs post-exercise 5.07 2.75-12.23 3.88 2.54-5.30 0.10924hrs post-exercise 4.69 3.01-8.50 3.47 2.01-5.58 0.068∆ pre:2hr post 1.59 1.51-8.71 0.31 -0.77-0.70 0.109∆ pre:24hr post 1.96 -17.52-4.98 -0.02 -1.02-0.56 0.109

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Table 6 Intra-group supplementation

A (p value) B (p value)

Total ascorbate Pre-exercise vs 2hrs post-exercise 1.000 0.059

2hrs post-exercise vs 24hrs post-exercise 0.374 0.508

Pre-exercise vs 24hrs post-exercise 0.878 0.953

Reduced ascorbate Pre-exercise vs 2hrs post-exercise 0.674 0.575



% Reduced ascorbate Pre-exercise vs 2hrs post-exercise 0.779 0.508



MDA Pre-exercise vs 2hrs post-exercise 0.441 0.799



VAS scores

Participants subjectively rated pain intensity out of 10 in A: pre-exercise 0 (0-2), post-

exercise 4.5 (1-8), and 24hrs post-exercise 1 (0-6); in B: pre-exercise 0 (0-3.5), post-exercise

4 (2-9), and 24hrs post-exercise 1 (0-7).

Biochemical Analyses

Samples were analysed for MDA and total:reduced ascorbate except for one sample which

was haemolysed at 2hrs post-exercise in supplement A and another sample did not yield

sufficient plasma for the analysis of reduced ascorbate at pre-exercise in supplement A.

Wilcoxon signed-ranks test was performed on all data collected due to dependent variables

and related samples design employed (Morgan et al, 2013). There were no statistically

significant results for MDA nor total or reduced ascorbate biomarkers in exercise tests

comparing A and B supplementations (table3) except for in males at 24hrs post-exercise in

supplement B (p=0.046) (table4). Neither were there statistically significant results for MDA

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and ascorbate biomarkers in exercise tests within the same supplementation group (table

6). The variability of data is highlighted in boxplots for MDA (figure1) and percent of

reduced ascorbate (figure2) with additional information in table3. The boxplots graphically

demonstrate that the medians across exercise intervals are similar for MDA and percent

reduced ascorbate. They also indicate that there is an increase in variability in MDA values

post-exercise in A and B which decreases at the 24hr post-exercise time-point. This increase

in variability is not seen in percent reduced ascorbate for neither group. There are high

outliers for MDA: two participants in pre-exercise A, the same participant in pre-exercise B

and 24hrs post-exercise B. For percent reduced ascorbate there is one low outlier (one

participant) in pre-exercise A, and one high outlier (one participant) in 2hrs post-exercise A.

Spearman rho correlations were performed for MDA and reduced ascorbate associations in

exercise intervals which showed no statistically significant associations (figure3). The

correlations computed for MDA and reduced ascorbate in supplement A indicate the effect

size is small (r<0.1) and negatively correlated for pre- (r=-0.033; p=0.932) and 24hrs post-

exercise (r=-0.018; p=0.96), but the effect size is small to medium (0.1<r<0.3) and positive in

2hrs post-exercise (r=0.2; p=0.606). In supplement B, the correlations computed for MDA

and reduced ascorbate indicate no effect size in pre-exercise (r=0.006; p=0.987), but a larger

than typical effect size (r<0.5) is seen at 2hrs (r=-0.576; p=0.082) and 24hrs post-exercise

(r=-0.527; p=0.117) with a negative correlation suggesting reduced ascorbate is inversely

associated with MDA. In OS biomarkers across gender (table4 and table5), no statistical

significance was identified. Individual results demonstrate that a few participants may have

skewed the overall results by experiencing greater changes in biochemical markers than the

majority of other participants (figure5). From the graphs in figure5, it is possible to discern

that values at pre-exercise in both groups for both MDA and percent reduced ascorbate

were variable but the majority of participants were within the range 0-5μM MDA and 80-

100μM for percent reduced ascorbate.

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Dietary Assessment

The median values derived from the FFQ are detailed in table2. The median values for

nutrients analysed in 24hr dietary recalls are detailed in table7: no statistically significant

differences between nutrient intakes across supplement groups with the exceptions of

energy pre-exercise (p=0.028), copper (p=0.005) and manganese (p=0.017) 24hrs post-

exercise.

Table 7 Medians (min/max) of 24hr dietary recalls

A

Median (min-max)

B

Median (min-max)

P value

Energy (kcal) Pre 1963 (1027-2503) 2110 (1630-4249) 0.028*

Post 2632 (1516-3047) 2143 (1254-2759) 0.093

Vitamin A (retinol equivalents) (μg)

Pre 530 (330-3102) 893 (158-5752) 0.333

Post 710 (182-2612) 449 (179-2030) 0.241

Vitamin C (mg) Pre 82 (33-233) 144 (21-334) 0.139

Post 114 (28-321) 89 (9-220) 0.445

Vitamin E (mg) Pre 9 (2-15) 11 (4-23) 0.114

Post 15 (1-24) 11 (3-17) 0.139

Iron (mg) Pre 14.1 (5.0-24.3) 14.4 (7.9-33.8) 0.508

Post 14.8 (6.0-25.6) 12.2 (8.2-27.9) 0.169

Zinc (mg) Pre 9 (5-16) 10 (6-24) 0.799

Post 13 (6-16) 9 (4-14) 0.386

Copper (mg) Pre 1 (1-2) 1 (1-3) 0.114

Post 2 (1-3) 1 (1-2) 0.005*

Manganese (mg) Pre 3 (2-6) 3 (2-12) 0.169

Post 4 (3-6) 3 (2-5) 0.017*

Selenium (μg) Pre 44 (15-176) 69 (24-188) 0.721

Post 79 (26-198) 69 (24-188) 0.575

* significance indicated at p-value ≤0.05

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Figure 1 Pre-exercise A (n=10; 2h post-exercise A (n=9); 24h post-exercise A (n=10), and pre-exercise B (n=10); 2h post-exercise (n=10); 24h post exercise (n=10).

Figure 2 Pre-exercise A (n=9; 2h post-exercise A (n=9); 24h post-exercise A (n=10), and pre-exercise B (n=10); 2h post-exercise (n=10); 24h post exercise (n=10).

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Figure 3 Spearman rho correlations for MDA and reduced ascorbate in supplementation A (left) and B (right) across exercise intervals. Supplement A pre-exercise (MDA n=10, reduced ascorbate n=9; r=-0.033; p=0.932), 2hrs post-exercise (n=9; r=0.2; p=0.606), 24hrs post-exercise (n=10; r=-0.018; p=0.96); in supplement B pre-exercise (n=10; r=0.006; p=0.987), 2hrs post-exercise (n=10; r=-0.576; p=0.082), 24hrs post-exercise (n=10; r=-0.527; p=0.117).

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Discussion/Conclusion This randomised placebo-controlled double-blind crossover study of 10 participants sought

to investigate the effects of dietary nitrate supplementation on OS induced in resistance-

exercise. The findings indicate that compared to placebo, no statistically significant changes

were observed in OS in resistance-exercise except for in males at 24hrs post-exercise taking

the placebo, suggesting that dietary nitrate supplementation does not adversely affect OS

biomarkers, contrary to established belief. This study initially used 10mg/kgBW NaNO3 but

participants experienced side-effects which included nausea, and general feelings of

unwellness. Therefore, in the interest of participant wellbeing and ethics, the dose was

reduced to 5mg/kgBW. This is the first study to report such side-effects and in Bescos et al

22

Figure 4 Matrix of graphs showing participants’ MDA and % reduced ascorbate values at pre-exercise and 24hrs post-exercise in Supplement A (left) and supplement B (right). MDA, Malondialdehyde; numbers correspond to participant code


(2011;2012) where 10mg/kgBW was also used, no side-effects were reported. There has

been controversy concerning intakes of nitrates due to assumptions that it may be

physiologically harmful and potentially carcinogenic (Kapil et al, 2014), and there is concern

that unintentional intakes of nitrite supplements to improve exercise performance rather

than nitrate and nitrate-rich foods can be toxic in large doses (Lundberg et al, 2011; Jones,

2014). Long-term effects of NaNO3 supplementation are unknown but Hezel et al, (2015)

examined nitrate supplementation in healthy rats for approximately 18 months and

reported no adverse effects on 350mg/day dose, and in humans, Bondonno et al, (2015)

reported no adverse effects during a seven day high nitrate diet (range: 225 to 570 mg/day).

Lethal doses of nitrate in humans have been reported to occur at 330mg/kgBW (Alexander

at al, 2008). Researchers referred to in this study have investigated nitrate influence on OS

in exercise through food sources namely beetroot which contains a mean of 1.88mmol/80g

nitrate where 1mmol of nitrate is equivalent to 62mg (Lidder and Webb, 2013).

MDA levels in this study were not statistically significant except for males 24hrs post-

exercise in supplement B (p=0.0046) and in females there was a trend to significance at the

same juncture (p=0.068) which would warrant further investigation. However, median

values for MDA in this study are higher than reported in other studies. Bloomer and Fisher-

Wellman (2008) investigated resting OS biomarkers in exercise trained and untrained young

men and women, and it was found that MDA was lower in women (trained=0.43μmol/L

(±0.06); untrained=0.71μmol/L (±0.05)) than in men ((trained=0.70μmol/L (±0.06);

untrained=0.77μmol/L (±0.10)), and MDA was lower in trained compared with untrained

participants. Few studies have assessed OS in resistance-exercise but those that measured

levels of MDA post-exercise have reported conflicting results (Bloomer and Goldfarb, 2004).

Indeed, the impact of resistance-exercise on OS differs between individuals and can be

influenced by a multitude of endogenous (pathophysiology, cellular metabolism, anxiety)

and exogenous (diet, pathogens, drugs, pollutants) sources (Rahal et al, 2014). The

assessment of MDA is problematic as it is the high variable between participants. Nielsen et

al (1997) generated parametric estimate reference MDA intervals for men 0.41-1.29μmol/L

and women 0.33-1.22μmol/L aged 20-79yrs, but these intervals were derived from a sample

that included smokers and alcohol consumers which would limit the applicability of these

values to this study’s sample which excluded smokers and restricted alcohol consumption.

23


Reference intervals provided in a review by Lykkesfeldt (2007) indicate a range of 0.1-

4.0μmol/L for healthy non-smokers, and 0.1-4.1μmol/L in smokers, but no specific ranges

were provided for gender. The MDA levels in this study are within the ranges reported by

Lykkesfeldt (2007) and show very little difference from pre-exercise to 24hrs post-exercise in

both A and B groups. Furthermore, lipid peroxidation can be related to behavioural and

physiological conditions such as body fat composition and smoking (Block et al, 2002), and

MDA production can be enzymatic or non-enzymatically induced under stress conditions

(Ayala et al, 2014) therefore, a wider interpretation and appreciation for individual factors

should be utilised in future studies employing MDA as a biomarker for OS in resistant-

exercise.

Ascorbate levels in this study were not statistically significant but there is a trend to

significance in supplement B for total ascorbate between pre-exercise and 2hrs post-

exercise (p=0.059). Very few studies have investigated ascorbate levels in OS and resistance-

exercise, and the results for ascorbate in this study are far higher than those reported

elsewhere. Rietjens et al, (2007) used leg-extensions in eight healthy males with serum

vitamin C concentrations at 15.3±0.6μM pre-exercise, raising to 16.4±0.7μM post-exercise,

and returning to 15.3±0.5μM at 24hrs post-exercise. Bailey et al (2004) investigated

incremental knee-extension exercises in five men and found no change in ascorbate levels in

75% exercise intensity in arterial, 54.3±18.1μmol/L, and in venous, 56.9±20.7μmol/L, blood

samples, and at 100% intensity in arterial, 56.4±17.9μmol/L, and venous 58.2±20.5μmol/L

blood samples.

This study used a subjective pain scale to gauge oxidative damage but an assessment of

protein kinase levels as a measure of muscle damage and of protein carbonyls for OS which

could have more beneficial and these are considered to be more appropriate measures in

resistance-exercise protocols (Lee et al, 2002; Nikolaidis et al, 2007; Paschalis et al, 2007;

Hudson et al, 2010; Margaritelis et al, 2014). A further improvement could be to extend the

study and perform additional blood tests two, three, and four days after the exercise tests

because protein carbonyls and MDA levels have been seen to increase and return to

baseline status a number of days post-exercise (Goldfarb et al, 2005; Nikolaidis et al, 2007;

Paschalis et al, 2007).

24


Strengths/LimitationsThis study is one of few examining the role of dietary nitrate on OS induced in resistance-

exercise that informs the evidence base. A randomised placebo-controlled double-blind

crossover design was employed to enhance robustness and provide objective assessment of

biomedical parameters within an exercise protocol. This study accounted for dietary intakes

that may affect OS status by utilising a validated FFQ and undertook 24hr dietary recalls to

increase validity (Byers, 2001; Krystal et al, 2005). However, there are limitations which

hinder reliability and validity. The nature of the cross-over design may affect measurable

outcomes by the first phase over-arching and affecting the second (Bate and Jones, 2008)

which may not be adequately accounted for by the washout period. This study used a small

convenience sample therefore, the results of this study are indicative of a small group of

young healthy physically active adults and not representative of a wider population leading

to low external validity. The FFQ used only looked at food groups for energy and

micronutrient intakes and excluded cereals, therefore, a large proportion of carbohydrate-

based and fortified foods were not included limiting the effectiveness of the FFQ data. The

dietary recalls were conducted unsystematically by all researchers at different time-periods

which may have resulted in different approaches to data collection yielding potentially

misreported results. The exercise protocol may not have been sufficient to induce OS either

due to the type, intensity or duration of the exercise. The means to assess OS may not have

been sufficient due to the biomarkers chosen and sampling time-intervals. Biochemical

analyses could be limited due to pipetting and assay errors, malfunctioning equipment,

miss-labelling, and human error. Although efforts were made to ensure participants adhered

to the study protocol outside of the laboratory, it is possible that some may have smoked,

drunk alcohol, engaged in strenuous physical activity, not taken the supplements on time or

at all, and some participants may have underlying pathophysiological diseases unknown to

themselves and the researchers at the time of the study.

There are avenues of development for nitrate supplementation within dietetics in the

management of those with chronic obstructive pulmonary disease where exercise and diet

are core therapeutics (Berry et al, 2015), and in the management of pulmonary

hypertension and ventilator-induced lung injury in critical care, and in conditions involving

ischemia and reperfusion (Lundberg et al, 2009), and in the recommendation to increase

25


vegetable intake as advised in the DASH diet to proffer cardiovascular health benefits

(Harnden et al, 2010) which may be achievable with dietary sources of nitrate.

Acknowledgments We are grateful to the EPIC-Norfolk Study team for the use of the EPICFFQ software. EPIC-

Norfolk is supported by programme grants from the Medical Research Council UK

(G9502233, G0300128) and Cancer Research UK (C865/A2883). We are also grateful to Raul

Bescos and Desley White for their guidance in this study. We are also thankful to Liz Preston

and staff at the Link Lab, Plymouth University for allowing us to conduct our research there.

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Appendix 1: Fitness Activity Questionnaire

Please describe your current participation in the following types of exercise:

1. Aerobic (aerobic classes, walking, jogging, stair climbing, hiking, cycling, etc.)Frequency (# of days per week): Duration (time spent per session): minutesIntensity (difficulty level): light somewhat hard hard very hardHow long have you been participating in aerobic activity as described above? Years

2. Anaerobic (weight training, sprinting, etc.)Frequency (# of days per week): Duration (time spent per session): minutesIntensity (difficulty level): light somewhat hard hard very hardHow long have you been participating in anaerobic activity as described above? Years

3. Organized or Recreational sportsType of sport(s): Frequency (# of days per week): Duration (time spent per session): minutesIntensity (difficulty level): light somewhat hard hard very hardHow long have you been participating in sports activity as described above? Years

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R Davies Dissertation 2015

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