The world’s population is aging, with the number of people aged over 60 years expected to increase from 901 million to 1.4 billion by 2030 [1
]. Rates of disease and age-related dysfunction are increasing rapidly [2
] and are expected to cause a dramatic increase in costs in the health and disability services areas [1
]. This has led to an increased interest in the use of food-based supplements and bioactive compounds to improve or maintain health and body functions [3
Beetroot juice contains high levels of nitrate (NO3−
) which can be converted into the bioactive form, nitric oxide (NO) [4
]. Nitric oxide plays a major role in many signaling pathways and biological processes, including improving neurotransmission and blood flow, alterations in mitochondrial oxygen consumption, promotion of cognitive benefits, mood and cardiovascular function [2
]. Currently, two major pathways are known to result in the generation of NO in humans. First, through the conversion of l
-arginine to NO by the enzyme nitric oxide synthase (NOS), which is considered the dominant pathway. Secondly, via the reduction of dietary NO3−
(nitrite), which occurs in the mouth by lingual anaerobic bacteria [4
]. Nitrite is then swallowed and ultimately enters the bloodstream where it can be reduced further to NO [5
]. It is through this second, exogenous pathway of NO production that nitrate-based food supplements such as BR are likely to exert their potential health benefits [4
Acute supplementation with BR can improve physiological responses such as cardiovascular function in younger adults [7
]. Specifically, consumption of inorganic NO3−
, either in the form of sodium nitrate or BR, has been shown to result in a significant increase in plasma NO2−
levels and reduction of blood pressure in both younger (8 and 6 mmHg; systolic and diastolic blood pressure; respectively) [10
] and older [4
] (5 and 3 mmHg; systolic and diastolic blood pressure; respectively) adults. This positive effect on blood pressure has led to the suggestion that beetroot could potentially be used in medical settings as an alternative to traditional blood pressure-lowering drugs [11
Acute supplementation with BR (7.5 mmol nitrate) has been shown to improve simple reaction times in older adults [12
]. This effect may be due to an increase in blood flow to the brain which has been observed in older adults following consumption of dietary NO3−
]. Cognitive diseases have been linked to age-related reductions in cerebral blood flow (CBF) therefore nitrate supplementation may slow the process of age-related cognitive decline due to the aforementioned increased perfusion to the brain [13
]. However, other studies have shown no improvement in cognitive function or mood with acute or 3 days of NO3−
supplementation in older adults [4
]. The equivocal findings are likely due to methodological differences in the cognitive tests used, the duration of supplementation (from 2 h to 14 days), and amount of NO3−
consumed (6–12 mmol NO3−
Aging leads to a decline in body processes and functions resulting in increased blood pressure, reduced blood flow, and reduced oxygen delivery to the muscle. All of these measures have been shown to be improved following BR supplementation in both younger and older adults, however, data presented for older adults is limited [4
]. There may be greater potential for improvement in cardiovascular and cognitive function, and mood following NO3−
supplementation in older adults relative to younger adults due to age-related changes [15
]. Few studies have examined the health benefits of BR supplementation in older adults, and, to our knowledge, only one study directly compared the effects in younger versus older adults [16
], therefore further research in this area is required.
We hypothesized that acute supplementation with nitrate-rich beetroot juice would provide a greater improvement in cardiovascular responses, cognition, mood and perception in older adults (50–70 years) compared to younger adults (18–30 years). To examine this hypothesis we conducted a randomized, double-blind crossover trial to investigate and compare the effects of acute supplementation with nitrate-rich beetroot juice on plasma nitrate and nitrite concentrations, blood pressure and cognitive performance measures in older and younger adults.
2. Materials and Methods
2.1. Sample Size
A power analysis was conducted (G-Power 3.1) to calculate the sample size based on the primary outcome measure of blood pressure. Previous literature in healthy adults has shown a mean change in BP of 10 mmHg (9 SD) between BR (9.6 mmol nitrate) and control [4
]. Based on this, the required sample size was 11 per group, using an SD of 9, with a statistical power of 0.82 and α-level set at 0.05.
Twenty-four healthy, normotensive, recreationally active adults (13 younger, 18–30 years, and 11 older, 50–70 years) volunteered for this study. Participants completed a health-screening questionnaire to ensure suitability. We did not recruit tobacco smokers, users of nitrate-based dietary supplements or well-trained/elite athletes (defined as those completing >4 training sessions per week, or >6 h of intense exercise per week, or having played/playing for a national or age-equivalent sports team). Those with known diseases (e.g., dementia and cardiovascular disease) were also excluded. Prior to testing, all participants were informed of the procedures, associated risks, and the possible benefits of participation, after which written informed consent was obtained. The study was approved by the Massey University Human Ethics Committee (SOA 16/27) and registered with ACTRN (ACTRN12618001466235).
Beetroot juice, extracted from fresh washed beetroots (beta vulgaris ‘Pablo’), was blended with other fruit juices to provide a standardized beetroot juice drink with a constant soluble solid concentration (11° Brix) and nitrate concentration (10.5 mmol/150 mL). The placebo beetroot drink was produced using beetroot juice concentrate and standardized to the same constant soluble solids concentration (11° Brix) but contained a low nitrate concentration (1 mmol/150 mL). The BR and placebo juice drinks had previously been presented to a consumer sensory panel using a triangle test, with 25 participants, and were not perceived as significantly different (p
> 0.05). The nitrate concentration was analyzed using high-performance liquid chromatography (HPLC) based on the method of Cheng and Tsang [17
]. Briefly, isocratic separation was achieved using a Gracesmart C-18 column; mobile phase 0.01 M Octylammonium orthophosphate (pH 3–3.5) at a flow rate of 0.8 mL/min. Detection was carried out at 193 nm for nitrite and 213 nm for nitrate.
2.4. Study Design and Procedures
Participants were required to visit the laboratory on three separate occasions. During the first visit, they were familiarized with the testing procedure and equipment, including cognitive, perceptual and mood tests, and body weight (kg) and height (m) were measured using digital scales and a stadiometer, respectively. Participants were instructed to complete a food diary for two days prior to the second study visit and to replicate their diet and lifestyle factors for the third study visit (dietary nitrate was not restricted, in an attempt to investigate additional benefits of BR in conjunction with normal daily intake; however, the food intake diary was used to evaluate intake of dietary nitrate and correlated to baseline plasma nitrate and nitrite concentrations). Participants were asked to arrive at the laboratory in a well-rested, fasted state and to refrain from excessive exercise and alcohol consumption 24 h prior and caffeine 6 h prior to each study visit. All trials were performed in the morning at approximately the same time of day (±2 h).
The second and third visits were the supplementation trials, with each participant randomly allocated (using a random number generator) the order of consumption of the two beverages in a double-blind, crossover design. For these two study visits, participants consumed 150 mL of beetroot juice (BR; containing 10.5 mmol NO3−
) during one visit and 150 mL of a placebo (PL; containing 1 mmol NO3−
), on the other visit. A 150-mL beverage was used to obtain approximately 10.5 mmol of nitrate as this amount has been shown to be a sufficient dose to elicit beneficial effects [4
]. Drinks were consumed with a standardized, isocaloric breakfast (cereal or toast) with the same macronutrient breakdown (15 g protein, 30 g carbohydrates and 10 g fat). Participants consumed the same breakfast type for both supplementation trials. Visits two and three were separated by a one-week washout period providing sufficient time for normalization of NO3−
levels in the body [8
The protocol for visits two and three is illustrated in Figure 1
. Prior to consumption of the beverage, baseline measures of blood pressure (mean value of three measurements taken from the left arm: deluxe HEM-7130; OMRON Healthcare CO. Ltd.; Kyoto, Japan), heart rate (A1 polar chest transducer), cognitive function, perceptual and mood tests were completed followed by collection of a resting blood sample. Participants then consumed the allocated drink with breakfast, within a 10-min period. Participants were asked to remain in the laboratory for a 2.25 h absorption period while a home development show was aired for entertainment purposes (has minimal effect on cognitive stimulation or arousal levels [4
]), or participants could complete computer work or study. The 2.25 h absorption period was chosen as previous research has shown that peak plasma nitrite concentrations occur 2–3 h after beetroot juice consumption [18
]. After 2.25 h, the aforementioned measurements were repeated. Blood samples were also taken again 3.25 h post-supplementation.
2.5. Blood Measurements
Plasma nitrite and nitrate were used as biomarkers for nitric oxide availability [19
]. Six milliliter venous blood samples were taken by venepuncture, from a vein within the antecubital area, and collected into heparinized tubes. Samples were mixed, centrifuged (MF–50 Hanil Science Industrial, Incheon, South Korea) at 3500 rpm (1330 g) for 10 min, and the collected plasma aliquoted into Eppendorf tubes (0.5 mL per tube) and stored at −80 °C for later analysis of nitrite and nitrate concentrations by HPLC [20
2.6. Cognitive Measurements
Cognitive tests were completed in a quiet, isolated room using a computer placed at eye level (PsychoPy software, version 1.83.04 [21
]). The cognitive tasks included the choice reaction test (CRT), rapid visual information processing (RVIP), and Stroop tests. Each task was performed a total of four times per participant (two per trial; pre- and 2.5 h post-supplementation). These tests have been used to investigate the effects of nutritional supplementation on executive function, attention, and information processing speed [4
2.6.1. Choice Reaction Test (CRT)
The CRT task involves participants pressing the left-hand key when the word ‘LEFT’ appears and the right-hand key when the word ‘RIGHT’ appears.
2.6.2. Rapid Visual Information Processing (RVIP)
The RVIP test involves participants following a chain of single numbers from 1 to 9, which appear in a pseudo-random order at the rate of 100 per minute. The aim of the test is to correctly identify target sequences (2–4–6, 4–6–8, and 3–5–7) as quickly as possible.
2.6.3. Stroop Test
The Stroop test involves the display of different color names (‘RED’, ‘GREEN’, and ‘BLUE’) which appear on the screen, one at a time in different colored fonts (red, green or blue). The aim of this test is to press the key corresponding to the color of the font that the present word was displayed in.
2.7. Mood and Perceptual Measurements
The feeling scale (FS) was used to measure the degree of displeasure or pleasure based on an 11-point scale ranging from −5 (very bad) to +5 (very good) [23
]. The felt arousal scale (FAS) was used to measure the degree of arousal-activation based on a 6-point scale ranging from 1 (low arousal) to 6 (high arousal) [24
]. The profile of mood states (POMS) was used to evaluate mood based on ranking how one feels, on a 5-point Likert scale ranging from 0 (not at all) to 4 (extremely) for each word presented under the 7 mood states (fatigue, anger, vigor, tension, esteem, confusion, and depression) [25
]. The FS, FAS and POMS measures were all completed before and 2.5 h post-supplementation.
2.8. Statistical Analysis
Statistical analyses were completed using IBM SPSS (Version 22.0). All data except for physical characteristics and baseline measurements were analyzed using mixed-method repeated-measures analysis of variance (ANOVA) with treatment and time as within-subject factors, and age as the between-subject factor. Sphericity was tested using the Mauchly’s test to ensure the assumption of sphericity was not violated, and multivariate models were applied if these assumptions were not met. Where significant differences were found, post-hoc tests with Holm-Bonferroni correction were undertaken to assess multiple comparisons. Outliers were excluded based on the Tukey test. Relative effect sizes were calculated using partial eta squared and defined as small (η2p = 0.01), medium (η2p = 0.06), or large (η2p = 0.14). Independent Student’s t-tests were used to examine differences in physical characteristics (height, weight, age) between age groups. Data are presented as mean ± standard deviation. Statistical significance was set at p < 0.05.