Institutional ethics review was sought and received from the Southern Health Human Research Ethics Committee (11335A, approval date 24/1/2012). The trial was registered with the Australia and New Zealand Clinical Trial Registry (ACTRN12611001088932, registered 20/10/2011). All participants gave written informed consent prior to participation and refrained from antibacterial products including mouthwash and toothpaste during the study period. Apart from abstaining from beetroot products, no dietary restrictions were imposed. Participants’ medications remained unchanged for the duration of the trial.
BR was chosen as the source of dietary nitrate for this study for its ready commercial availability and use in prior literature. BEET-IT shots (James White Drinks, Ipswich, UK) were used for this study as the 0.3gm (4.8 mmol) dose of NO3− in each 70 mL shot approximates the 0.34-0.38gm (5.5 mmol-6.2 mmol) NO3− dose as described by prior investigators [4,5,22] without the requirement for a 500 mL daily fluid intake. Physically identical nitrate-deplete placebo shots (PL) were obtained from the manufacturer (0.00035-0.0013gm (0.0056-0.020 mmol) NO3− dose) (personal communication, Pinho, A., James White Drinks, Ipswisch, UK). The dose administered was 70 mL (0.3gm NO3−), twice daily.
Participants
Voluntary participants were recruited at a tertiary metropolitan Australian medical center either directly from the outpatient pulmonary function test laboratory or by direct referral from thoracic physicians. Inclusion criteria were Global Initiative for Chronic Obstructive Lung Disease (GOLD) class II stable COPD (‘moderate’ severity: FEV 1/FVC < 0.7, FEV 1 50-79% predicted) [1], age 45–80 and physical ability to perform an endurance shuttle walk test. GOLD II severity was chosen as a group who would be likely to experience exercise limitation but who would still be able to perform exercise testing. Exclusion criteria included a history of acute exacerbation of COPD in the preceding month, oral corticosteroid therapy, long term domiciliary O2 therapy, beta blocker therapy, ischemic heart disease or congestive cardiac failure and musculoskeletal problems limiting exercise.
Baseline measurements
At baseline (visit 1), participants underwent pre- and post-bronchodilator spirometry and measurement of carbon monoxide diffusing capacity as per American Thoracic Society/European Respiratory Society standard guidelines for pulmonary function testing [23] (Figure 1).
During the same visit, participants undertook an incremental shuttle walk test (ISWT) [24], a commonly used field walking test designed to simulate a maximal cardiopulmonary exercise test in individuals with COPD. This involved timed, accelerating shuttle walking around cones 9 m apart, forming a 10 m course. Standardized procedures were adopted as mandated by the ISWT protocol: these included uniform instructions and coaching and a repeat test to account for a learning effect. The best distance was recorded and used to estimate V̇O2max and to calculate the walking speed representing 85% of maximal exercise performance for use in the endurance shuttle walk test (ESWT) [25].
The ESWT [26] is a constant paced standardized test of endurance capacity developed for use in patients with COPD. It has face and construct validity as the exercise testing regime suitable validated for use in patients with COPD as a measure reflective of the workload undertaken by an individual with COPD in the course of their daily activities. Participants performed the walk under controlled conditions by walking at a constant predetermined rate around cones 9 m apart (10 m shuttle walk) to a constant, timed audio recording until exhaustion. The ESWT permits measurement of walking time and distance at the speed that represents their estimated 85% V̇O2max; hence it is a measure of the individual’s sub-maximal exercise endurance capacity.
Previous studies have demonstrated that it is not necessary to perform repeat determination of either the ISWT or the ESWT in this patient group [27].
Initial ESWT and safety phase
During visit 2, which occurred approximately a week after visit 1, an initial ESWT was undertaken. Immediately after the ESWT, a Modified Borg Rating Scale for Perceived Dyspnea was recorded [28]. Following this, a safety phase was performed during which participants consumed, un-blinded, active BR and underwent measurement of sitting and standing blood pressure, heart rate and pulse oximetry saturations at 0, 0.5, 1 and 4 hours post dose. The rationale for this was that BR has been previously demonstrated to decrease resting systolic blood pressure [5]. As pre-specified, if participants became symptomatic or had a >20 mmHg systolic blood pressure fall in response to the test dose, they were excluded from the remainder of the study protocol.
Double blinded randomized placebo controlled phase
Participants were randomized to receive either (active) BR or PL first in a double blind fashion by computerized allocation performed by an independent clinical trials pharmacist.
For three days prior to visit 3, participants consumed their allocated dose of juice (70 mL) twice per day. They consumed a final dose of juice on the morning of visit 3, approximately an hour prior to the performance of the ESWT. Per protocol, visits 3 and 4 occurred strictly 7 days apart.
Following visit 3, participants abstained from juice supplementation for four days, allowing a ‘wash-out’ period between dosing regimes. They then consumed whichever of BR or PL they had not previously been exposed to. This was again taken twice a day for three days prior to visit 4, with a final supplement on the morning of visit 4. Visit 4 also consisted of an ESWT.
The timing of supplementation was based on another randomized double-blind placebo-controlled crossover study [29], however washout was shortened from 10 to 7 days for logistical reasons.
Trial supplement adherence was checked by self-report and bottle count.
Sample size estimation
A recent meta-analysis of the effect of nitrate supplementation on exercise performance in healthy individuals has demonstrated that there is a significant moderate beneficial effect upon exercise performance as measured by time to exhaustion with an effect size of 0.79 (95% CI, 0.23-1.35) [7]. Eaton et al. (2006) reported that the baseline performance by moderate severity COPD patients on the ESWT was 313 meters, with a standard deviation of 193 meters, and the reported improvement in performance in response to exercise rehabilitation of 92% of baseline (302 meters). However, using the reported 92% improvement in ESWT performance in response to pulmonary rehabilitation as the minimum clinically important difference (MCID) results in an effect size of 1.6, leading to a sample size requirement of only 6–7 patients in a cross-over study in order for the study to have an 80% power at the 5% significance level. A more conservative estimate of the MCID was used instead. We chose 55% improvement on the baseline performance in the ESWT as the MCID resulting in a more conservative estimate of the required sample size: 20 patients [30].
Outcomes and statistical analysis
The pre-specified primary endpoints were distance walked (meters) and time to fatigue (minutes) on the ESWT on BR versus that on PL, with comparison by two-sided paired repeated sample t-tests. Secondary endpoints included safety phase data, Borg dyspnea scores and blood pressure. Analysis was performed using SPSS Statistics for Windows, Version 20.0 (Armonk, NY, IBM Corp, 2011). Results are presented as mean ± standard deviation, and statistical significance accepted if alpha <0.05.