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Dietary nitrate supplementation improves reaction time in type 2 diabetes: development
and application of a novel nitrate-depleted beetroot juice placebo
Mark Gilchrist
Paul G. Winyard
Jon Fulford
Christine Anning
Angela Shore
Nigel Benjamin
NIHR Exeter Clinical Research Facility and Institute of Biomedical and Clinical
Science, University of Exeter Medical School (previously Peninsula College of
Medicine and Dentistry), University of Exeter, Exeter EX2 5AX, UK
Manuscript including references: 6839
Abstract: 300 words
Number of tables and figures: 5
Corresponding author:
Mark Gilchrist
Clinical Lecturer in Renal Medicine
NIHR Exeter Clinical Research Facility,
University of Exeter Medical School,
Royal Devon & Exeter Foundation NHS Trust,
Barrack Road,
EX2 5AX
01392 403059
m.gilchrist@exeter.ac.uk
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1
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Abstract
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Background
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In this substudy of the effect of dietary nitrate on blood pressure, endothelial function,
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and insulin sensitivity in type 2 diabetes, we report the development of a novel nitrate
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depleted beetroot juice for use clinical trials and determine if dietary nitrate
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supplementation improved cognitive function in patients with type 2 diabetes mellitus.
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Methods
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Beetroot juice was treated with the anion exchange resin Purolite A520e. UV-vis-
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spectrophotometry, and a blind taste test were performed along with determination of
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sugar content, measurement of ascorbate and dehydroascorbate, the ionic composition
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of juice and Proton NMR. Subsequently, 27 patients, age 67.2 +/-4.9 years, (18 male)
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were recruited for a double blind, randomised, placebo-controlled crossover trial.
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Participants were randomised to begin in either order beetroot juice (nitrate content
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7.5mmol per 250mls) or placebo (nitrate depleted beetroot juice nitrate content
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0.002mmol per 250mls). At the end of each 2 week supplementation period cognitive
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function was assessed using E-prime, E-Studio software with 5 separate tests being
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performed. The tests utilised in the present study have been adapted from the
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Cambridge Neuropsychological Test Automated Battery (CANTAB)
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Results
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The differences in the UV-vis spectra were comparable to the natural variation found in
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differing cultivars. There were no discernable differences in taste, sugar content, or
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Proton NMR. Ascorbate and dehydroascorbate were undetectable in either juice. After2
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weeks of beetroot juiceSimple reaction time was significantly quicker in the active arm
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at 327±40ms versus 341.8±52.7ms in the placebo arm, mean difference 13.9±25.6ms
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(95% CI 3.8 to 24.0 ms), p=0.009. No other measures of cognitive function differed
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between treatment arms.
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Conclusion
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We have developed an effective placebo beetroot juice for use in trials of
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supplementation of dietary nitrate. Two weeks supplementation of the diet with 7.5
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mmoles of nitrate per day caused a significant improvement in simple reaction time in
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individuals with T2DM.
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Introduction
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Individuals with Type 2 Diabetes have measurable deficits in cognitive function when
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compared to age and sex matched populations[1]. The risk of developing dementia
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may be twice as high in individuals with diabetes compared with the background
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population[2]. This accelerated decline in cognitive function may have several potential
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mediators: hyperglycaemia, dyslipidaemia, genetic factors, and microangiopathy [3].
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Improvements in glycaemic control have produced mixed results. One small trial in
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elderly subjects with type 2 diabetes, two weeks intensive management of glycaemic
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control resulted in a non-significant trend to improvement in reaction time and significant
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improvements in other cognitive measures[4]. Ryan et al’ [5] found an improvement in
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working memory following significant reductions in fasting plasma glucose levels
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following a 24 week intervention with either glyburide or rosiglitazone but no differences
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in reaction time, spatial memory or rapid processing tests. In the largest study of the
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effect of glycaemic control on cognitive function a subset of the participants from the
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ACCORD trial underwent cognitive assessment and MRI scanning to determine total
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brain volume at baseline, at 20 months and 40 months [6]. While there was a significant
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reduction in the amount of total brain volume lost at 40 months in the intensive
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glycaemic control group compared with the standard glycaemic control group there was
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no difference between groups in cognitive performance.
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Vascular dysfunction is thought to be an aetiopathogenic factor in Alzheimer’s disease
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as well as vascular dementia [7]. The plasma nitrite concentration is becoming
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established as a marker of endothelial nitric oxide (NO) production and therefore
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indicative of vascular health [8]. Thus plasma nitrite may be a sensitive marker for risk
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of cognitive decline. The potential relationship between plasma nitrate and nitrite and
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cognitive function has been little studied. Two groups have found plasma NOx (total
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nitrate plus nitrite) was reduced in patients with dementia (Alzheimer’s, vascular, and
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mixed) [9] or Alzheimer’s dementia compared with healthy controls [10]. Another group
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[11] found a small but statistically significant increase in plasma NOx in patients with
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Alzheimer’s dementia compared to healthy controls
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Endothelial function determined by brachial artery flow mediated dilation is impaired in
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individuals with Alzheimer’s disease[7]. This impairment in endothelial function is
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present in younger individuals with vascular risk factors and is associated with poorer
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cognitive performance in the absence of overt clinically detectable cognitive impairment
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[12].
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High levels of vegetable consumption and in particular green leafy vegetables appear to
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slow the rate of age related cognitive decline[13]. These beneficial effects are often
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attributed to a variety of anti-oxidants or vitamins. However in a group with
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cardiovascular disease or risk factors for cardiovascular disease supplementation of
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vitamin E, C, or beta-carotene did not alter the rate of slowing of cognitive function over
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time [14]. It has been postulated that it may be the vascular actions of inorganic nitrate
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in green leafy vegetables which mediate this protective effect [15; 16].
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Inorganic nitrate from the diet is involved in a complex cycle with nitrite and nitric oxide.
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Briefly, nitrate is rapidly and completely absorbed from the stomach and small intestine
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[17]. From the circulation, it is then concentrated in the salivary glands and secreted
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into the mouth. Bacteria residing in crypts on the dorsum of the tongue use nitrate
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rather than oxygen as an alternative electron acceptor [18]. In the process nitrate is
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reduced to nitrite, which is again swallowed. Some of this nitrite will further be reduced
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to nitric oxide (NO) in the acidic environment of the stomach with important localised
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effects[19]. Some nitrite will be absorbed into the circulation where, via a number of
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mechanisms, it is reduced to NO in the vasculature[20]. This may modulate vascular
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tone and a number of cell signalling pathways. NO generated in this way and from the
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nitric oxide synthase family of enzymes which utilise L-arginine and O2 as substrates is
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rapidly oxidised to nitrate by oxyhaemoglobin. This nitrate is thought to act as a stable
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storage reservoir of NO’s bioactivity [15].
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Dietary nitrate supplementation has previously been shown to lower blood pressure
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[21; 22], improve endothelial function [21], improve exercise tolerance[23], enhance
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muscle contractile efficiency and force production [24; 25], modulate gastric blood flow
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[26], protect against ischaemia reperfusion injury[27], inhibit platelet aggregation [21],
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and potentially play a key role in host defence [28]. For review see [29]. There is some
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evidence that dietary nitrate supplementation may alter cerebral blood flow [30].
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Presley et al’s study [30] suggested that acute dietary nitrate supplementation increased
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blood flow in cerebral white matter, notably in the dorso-lateral prefrontal cortex and
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anterior cingulate cortex, areas associated with executive function. There was no
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change in overall cerebral blood flow. In the central nervous system neuronal activity is
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tightly coupled to local blood flow. This association is the cornerstone of investigational
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imaging techniques such as fMRI [31]. Nitric oxide, whether derived from eNOS or
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nNOS, is one of the key mediators of this relationship [32]. In a rat model, Piknova and
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colleagues [31] showed inorganic nitrite can restore neurovascular coupling where it
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has been disrupted by inhibitors of nNOS. Further evidence that dietary nitrate
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supplementation may be neuro-protective comes from a series of in vitro experiments
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by Gladwin’s group [33] which have demonstrated that neuroglobin may function as a
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redox sensitive nitrite reductase which may protect neurons during oxidative stress.
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We tested the hypothesis that supplementation of the diet with inorganic nitrate via
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sequential reduction to nitrite and NO would augment cognitive performance in subjects
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with T2DM.
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Materials and Methods
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The data presented are from a sub-study of a trial examining the effect of dietary nitrate
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on blood pressure, endothelial function and insulin sensitivity in type 2 diabetes, where
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additional details on methods can be found[34].
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This randomised double blind placebo controlled crossover trial was approved by the
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Devon and Torbay Research Ethics Committee (study no. 09/H0202/43). All studies
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were conducted in accordance with the Declaration of Helsinki.
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Participants were identified from the Exeter 10000 (EXTEND) bio-resource. This is a
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large cohort of well characterised individuals who have consented to being contacted
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about biomedical research projects. Twenty seven participants (9F:18M) with T2DM as
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defined by WHO, of at least five years duration were recruited.
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Using a double blind, randomised placebo-control crossover design, participants were
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randomised to begin in either order beetroot juice (nitrate content 7.5mmol per 250mls)
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or placebo (nitrate depleted beetroot juice nitrate content 0.002mmol per 250mls).
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Subjects were instructed to consume one 250ml bottle per day, for 14 days with
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cognitive function testing occurring at the end of the supplementation period. Subjects
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then entered a four week washout period before entering the opposing arm of the study.
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Throughout the study patients were asked to maintain their normal diet apart from the
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juices given and not to change any other lifestyle factors. Participants continued their
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usual antihypertensive medication and their usual hypoglycaemic medications.
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Placebo Production
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The placebo juice was produced by passing beetroot juice through a column containing
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the anion exchange resin Purolite a520e which exchanges nitrate for chloride. As it is
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widely used in the treatment of water for human consumption, it was considered that the
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resin may offer a methodology suitable for the depletion of nitrate in beetroot juice
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Nitrate concentration was determined by ozone chemiluminescence as per for plasma
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samples.
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UV-vis spectrophotometry
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Relative absorbance in the UV-vis light spectrum was determined
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spectrophotometrically using a Varian Cary 300 spectrophotometer. Samples were
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diluted 1/10 for analysis.
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Taste test
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A convenience sample of 10 members of staff from our institute agreed to participate in
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a blind taste test. They were randomly assigned to consume both juices in either order
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with a one week gap to simulate the conditions of the larger trial. Subjects were asked
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to determine the order of the juices they received.
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Sugar content
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The predominant carbohydrate in beetroot juice is sucrose [35]( Sucrose concentration
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was determined by Armanda Pinhoe, James White Drinks, Ashbocking UK using a
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Bellingham and Stanley 45-03 refractometer and expressed as oBx (degrees Brix
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equals g solute per 100ml). Refractometery is widely used to determine sugar content of
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food stuffs, including beetroot juice [35; 36]
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Measurement of ascorbate and dehydroascorbate
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Ascorbate and dehydroascorbate content were measured by reverse phase HPLC
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performed on a Dionex DX500 HPLC system (Dionex, Sunnyvale, USA). The limit of
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detection for this assay is 20μM.
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Ionic composition of juice
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The sodium, potassium, calcium, magnesium and chloride ion concentrations of the two
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juices were determined using ion specific electrodes (Roche modular ISE unit).
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Proton NMR
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AV600 spectrometer (Bruker corporation, Ma, USA) at Queen Mary University of
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London. Before analysis samples were subjected to three cycles of freeze drying and
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re-suspension in D2O. Spectra were analysed using ACD/labs ACD/NMR Processor
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Academic Edition (Advanced Chemistry Development, Canada).
Proton NMR spectra of the placebo and active juices were obtained using a Bruker
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All human studies took part in a temperature controlled (21.5-22.5oC) laboratory with the
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subjects in the seated position. Patients arrived at 09.00 having completed an overnight
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fast. The last bottle of beetroot juice was consumed with the patient’s evening meal
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(typically between 18.00 and 20.00) the day prior to the study. No oral hypoglycaemic
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agents or insulin were taken on the morning of the study. Any other regular medications
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including antihypertensives were taken as usual.
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Upon arrival at the laboratory blood samples were collected from the participants in
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Lithium-Heparin tubes (7.5mL Monovette Lithium Heparin, Sarstedt Ltd., Leicester, UK).
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Samples were centrifuged immediately in a pre-chilled centrifuge (4oC) at 3600 rpm for
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10 minutes. The plasma was immediately separated into 1ml aliquots, flash frozen in
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liquid nitrogen before transfer to a -80oC freezer.
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Following blood sampling the cognitive tests were undertaken. The subject was seated
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comfortably. Care was taken to ensure that the keyboard was within easy reach and
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the monitor was at a distance that permitted the subject to see the screen comfortably.
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If the subject normally used glasses they were permitted to do so for the study.
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Subjects who were unfamiliar with a standard QWERTY keyboard were given a trial run
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of a modified version of the study tests at the initial screening visit in order to familiarize
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them with the keyboard. The only keys used are the “space bar” and number keys 1-4.
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All cognitive tests were undertaken using E-prime, E-Studio software (Version 1.2,
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Psychology Software Tools, Inc, Sharpsburg, PA) with five separate tests being
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performed. The tests utilised in the present study have been adapted from the
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Cambridge Neuropsychological Test Automated Battery (CANTAB) [37] which have
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been shown to be sensitive to subtle cognitive deficits in neurological and psychiatric
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disorders [38]. The subject followed the on-screen instructions, moving through them at
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their own pace. Verbal clarification of the instructions was given by the investigator if
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requested by the participant. The cognitive tasks undertaken were as follows:
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Reaction time
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The subject was presented with a black screen. A white square appeared at random
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locations on the screen at variable time intervals. The subject was instructed to press
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the “space bar” as quickly as they could on seeing the white square. The time interval
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between the square appearing and the subject’s acknowledgement was recorded
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automatically. Thirty two events were analysed per visit.
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Decision reaction time
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The subject was presented with a black screen. White arrows pointing left or right
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appeared on screen in random locations. Only one arrow was present on the screen at
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any time. The sequence of left and right was random. The subject was instructed to
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press the number “1” if the arrow pointed left and “2” if it pointed right. Accuracy and
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time to depression of the key were recorded. Forty events were analysed per visit.
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Rapid processing
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The subject was presented with a three digit sequence, for example 4, 7, and 2. They
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are asked to remember that sequence. Numbers between 1 and 9 appear on the
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screen and were replaced by another number 300 milliseconds later. The subject was
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instructed to depress the space bar when the previously described sequence of
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numbers appeared, completely, in the order described, with no other numbers in
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between. The percentage of correctly identified occurrences was recorded. Three sets
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of seven occurrences of the number pattern were analysed per visit.
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Shape memory
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The subject was presented with a square composed of four smaller squares coloured
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red, blue, yellow and green. Every large square comprised one red, one blue, one
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yellow and one green square. The positioning of the squares within the square
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changed from trial to trial. Three seconds after it first appeared the large square was
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then hidden from view. There followed a variable time interval of between 1 and 5
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seconds before the subject was asked to identify which of the four options presented
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matches the square seen previously. Accuracy and time to decision were recorded.
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Twenty five trials were analysed per visit.
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Spatial memory
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Subjects were presented with a black screen. A white square would appear on the
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screen in a random location. The white box would appear in that location for 2.5
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seconds. This would disappear and another white box would appear in another random
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location. This would happen once more such that a total of three boxes had appeared.
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A screen inviting the subjects to move to the next screen by pressing the space bar
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appeared. The subject was then presented with a black screen containing two squares
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of the same size as those seen previously. One square was located in the same
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position as one of the squares seen previously. The other was located immediately
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adjacent. The squares are labelled “1” and “2”. The subject has to determine whether
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square “1” or square “2” is in the position of one previously.
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They indicated this by pressing the corresponding number key. Five sets of three are
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performed. Accuracy and time to answer were recorded.
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Plasma nitrate and nitrite analysis
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Plasma nitrate and nitrite concentrations were determined using a Sievers nitric oxide
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analyser (Sievers NOA 280, Analytix Ltd, Durham, UK), and both the methods and
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results for these determinations have been described previously[34].
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Statistical analysis
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Data were tested for normality. Paired t tests were applied to data where the differences
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in the parameter of interest between the active and the placebo conditions were
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normally distributed with data presented as mean±SD. Wilcoxon signed rank test was
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used for nonparametric data with data presented as median and IQR. Statistical
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significance was accepted at P<0.05.
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Results
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Placebo production
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Following treatment with Purolite a520e the nitrate concentration of the juice was 0.002
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mM. The nitrate concentration of the juice in the active arm of the study was 30.75mM.
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Taste test
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Five subjects correctly guessed the order and five could not discern a difference or
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guessed the wrong order. Kappa statistic for agreement was 0, that is no agreement,
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with p=1. This is essential to the validity of the placebo.
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Ascorbate and dehydroascorbate
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Both the untreated and the placebo juice had ascorbate and dehydroascorbate
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concentrations below the limits of detection for the HPLC assay (<20μM). Mean plasma
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ascorbate concentrations in European populations are approximately 50µM[39]. Thus
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there is unlikely to be any physiologically relevant change in ascorbate concentration
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from consumption of beetroot juice in the present study.
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The sucrose concentration determined by ascertaining Brix levels was unchanged pre
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and post resin treatment at 10oBx. There appears to be no substantial change in the
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concentrations of sodium, potassium or magnesium ions (table 1). The calcium ion
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concentration appears to undergo a modest fall and the chloride concentration more
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than doubles which is expected given the fact that nitrate concentration is lowered by
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anion exchange
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UV-Vis Spectroscopy
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λmax was 487 nm of with an absorbance of 0.570 for the active and 0.416 for the placebo
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were noted, thus the absorbance of the placebo juice is 73% of that of the active juice.
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These peaks correspond to the absorbance spectra of betaxanthins known to be
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present in beetroot juice[40]. Juice obtained from 2 cultivars that are used in
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commercially available beetroot juice shots were also analysed with a peak absorbance
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of 0.556 in the Gesar cultivar and 0.449 in the Bayer cultivar. It should be noted that.
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λmax was at 489 nm for Gesar and 485 nm and Bayer. Thus there is a subtle variation in
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colour between cultivars of untreated juice that is similar in nature to any variation
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between resin treated juice and untreated juice (Figure 1)
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Proton NMR
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The spectra for active and placebo juices appear remarkably similar (Figures 2 and 3).
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The peaks are likely be predominantly arising from protons related to sucrose, the most
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abundant sugar in beetroot juice [35].
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Both spectra show several well resolved doublet resonances in the region 4.8-5.5 ppm
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which correspond to the α anomeric proton of sugars [41]. Consistent with this the
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doublets have small coupling constants ofc J1, 2 <4Hz. The β anomeric protons of
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sugars have larger coupling constants (J1,2~9Hz) and appear at higher fields in the
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region 4.1-4.7 ppm [41]. Some of the signals in this region may also be due to low field
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ring proton resonances in sugar.
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All 27 subjects, 18 (66.7%) males and 9 (33.3%) females, mean age 67.2±4.9 years,
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successfully completed the study. Though previous studies have shown a blood
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pressure lowering effect with dietary nitrate supplementation, no such effect was evident
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in the current cohort. Full details of participant characteristics are in table two,
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published previously[34].
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The 30 point mini mental state examination (MMSE) was performed on enrolment to
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screen for significant cognitive impairment. MMSE score was 29.2±1.2 in the study
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cohort.
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HbA1c did not change between treatment arms at 7.61±1.12% in the placebo arm and
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7.52±0.93 in the active arm. Subjects who monitored capillary blood glucose at home
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reported no change. Fasting capillary blood glucose was not different between the two
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study arms at 8.15±2.03 mmol/l in the placebo arm and 7.87±1.79 mmol/l in the active
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arm, (mean difference 0.26±1.77 mmol/l, p=0.45, 95%CI -0.4764 to 1.044)
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Reaction time was significantly quicker in the active arm at 327±40ms compared with
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341.8±52.7ms in the placebo arm, mean difference 13.9±25.6 (95% CI 3.8 to 24.0 ms)
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p=0.009 (paired t-test). This remained significant after applying the Bonferroni
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correction for the use of multiple tests with statistical significance accepted at <0.01.No
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other measure produced a significant difference between supplementation arms (table
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3). There were no significant differences between visit 1 and visit 2 confirming the
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absence of a training effect.
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Plasma Nitrate and Nitrite
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Two weeks of ingestion of nitrate-rich beetroot juice increased median plasma nitrate
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and nitrite concentrations compared to placebo. As described previously for this cohort
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of subjects, the median plasma nitrate concentration rose from 31 µM (interquartile
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range: 19.8-41.6) in the placebo arm to 150 µM (IQR: 122.7-200.0) in the active arm
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(p<0.001). The median plasma nitrite concentration rose from 232 nM (IQR 200-265)
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in the placebo arm to 390 nM (median, IQR; 312-537) in the active arm, p<0.001[34].
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Neither change in plasma nitrite concentration nor absolute nitrite concentration were
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correlated with reaction time (data not shown).
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Recent data have shown a possible difference in platelet activation in response to
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inorganic nitrate supplementation between males and females[42] . In the present
408
study females had a significantly higher plasma nitrite concentration in the placebo arm
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at 263 nM (IQR 229-351) than males, plasma nitrite concentration 225 nM (IQR 184-
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257), p=0.045. There was no significant difference between the sexes in nitrite
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concentration after supplementation. Plasma nitrate concentration did not differ pre or
412
post supplementation between males and females. In line with this there were no
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significant differences between the sexes in performance in cognitive tests following
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nitrate supplementation (data not shown).
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Discussion
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The placebo described in the present trial has been used in multiple trials reported
418
previously, both by our group and by others. In terms of taste test, critical to its validity
419
as a placebo for use in blinded trials, we have demonstrated that it is indistinguishable
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from the high nitrate juice. There are small differences in the ionic compositions of the
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two juices but set in the context of typical daily intakes are unlikely to be of physiological
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relevance. There is some loss of pigment in the production of the placebo, however in
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the beetroot shot and placebo produced commercially this is not apparent to the naked
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eye. Furthermore the UV-vis spectroscopy data suggest that the extent of the
425
difference between the juice and its placebo is not dissimilar to the difference that can
426
be observed between two commercially available cultivars.
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This is the first trial showing dietary nitrate supplementation may improve cognitive
429
function. With the present data it is not possible to elucidate the mechanism
430
underpinning the improvement in simple reaction time. Two broad considerations that
431
must be made are whether it is due to a genuine improvement in cognition or whether
432
cognition remains unaffected but the reaction time decreases due to more rapid muscle
433
contraction [25], or indeed both. Certainly there is growing evidence for improvements
434
in physical performance following dietary nitrate supplementation [23; 24; 25].
19
435
Regardless of the mechanism this finding may have important implications in a large
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section of the elderly population.
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People with T2DM have slower reaction times than age matched healthy controls to a
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variety of stimuli [43; 44]. Reaction time appears to be predictive of overall cognitive
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performance with ageing[45]. In an elderly population, a slower reaction time is
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associated with an increased risk of falls[46]. As falls are associated with increased
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mortality in elderly populations the improvement in reaction time finding may have direct
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clinical implications[47].
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In an intervention trial where subjects with T2DM were given a balance training program
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a statistically significant improvement in simple reaction time of 7ms was achieved [43].
448
This was associated with a significant reduction in the risk of falling as determined by
449
the long-form physiological profile assessment. Thus the 13.9ms improvement we
450
observed may be clinically significant. It seems likely in Morrison’s trial that the
451
improvement in reaction time was due to an improvement in physical or musculoskeletal
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performance rather than having a cognitive component.
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A number of trials investigating the effects of dietary nitrate supplementation assess the
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outcome measures at around the time of the peak in plasma nitrite which typically
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occurs at around 3 hours post nitrate supplementation[21; 29]. It is worthy of note that
457
the improvement in reaction time occurred more than 12 hours following the last dose of
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458
nitrate. This may, if anything, lead to an underestimate of the effect size particularly if
459
cerebral blood flow is a key mediator[30].
460
461
Dietary intervention with ginseng panax has been studied to determine whether this can
462
improve cognitive function. In a trial in young healthy volunteers no change in simple
463
reaction time was seen[48]. Interestingly there was a 13.98ms difference in choice
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reaction time between ginseng and placebo in contrast to the lack of difference in our
465
cohort. It should be noted that the Cochrane group has found no consistent effect of
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ginseng on cognition [49].
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If the fall in simple reaction time seen in our study represents a true enhancement of
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cognitive processing speed possible underlying mechanisms include improvements in
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cerebral perfusion [30], better neurovascular coupling [31], or other as yet unidentified
471
improvements in cellular efficiency analogous to the improved but incompletely
472
understood increase in muscle contractile efficiency seen during exercise following
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dietary nitrate supplementation [24; 25]
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Nitrite’s potential role as a vasodilator in the systemic circulation is well described [50].
476
Presley et al’s study would suggest that dietary nitrate supplementation can alter
477
cerebral blood flow [30]. This was however a small study and multiple dietary
478
constituents other than nitrate were altered. Work from our own group, however found
479
no such alteration in cerebral blood flow in a group of healthy older subjects, mean age
480
64±2.7 [51]. Conahey and colleagues work in new born lambs showed that while nitrite
21
481
crossed the blood brain barrier, CSF cGMP concentration did not change despite a
482
doubling of plasma cGMP concentration [52]. This would suggest systemic changes in
483
nitrite do not affect the tone of cerebral vasculature.
484
485
In the spatial and shape memory trials where reaction time was measured in addition to
486
the primary parameter of task accuracy the lack of difference between treatment arms
487
may be due to the greater variance in response times brought about by the increased
488
difficulty of the task. A larger study would be required to elucidate whether reaction time
489
improved on these measures.
490
491
The effect of dietary nitrate supplementation on cognitive function in healthy individuals
492
is not completely understood. Kelly’s study used three measures of cognitive function
493
(serial subtractions, rapid visual information processing, number recall) in an acute
494
supplementation trial with no difference found between active and placebo arms[51].
495
For both healthy subjects and patients with diabetes the hypothesis that dietary nitrate
496
may be behind the retardation of progression of cognitive decline with aging seen in
497
studies reporting a protective effect from green leafy vegetables [13] needs to be tested
498
with a longer term prospective intervention study.
499
500
It could be argued that two weeks of an intervention is insufficient time in which to see
501
an improvement in cognitive performance. If cognitive changes were to arise as a result
502
of improvements in cellular efficiency analogous to the improved but incompletely
503
understood increase in muscle contractile efficiency or changes in blood flow then two
22
504
weeks is more than sufficient to procure these effects. Furthermore, in a small parallel
505
groups study two weeks intensive management of glycaemic control resulted in a non
506
significant trend to improvement in reaction time and significant improvements in other
507
cognitive measures in elderly subjects with type 2 diabetes[4].
508
509
510
The assessment of cognitive function in the current study was not exhaustive. Testing
511
other cognitive domains may have produced different results. For example, in Ryan et
512
al’s [5] study where an improvement in working memory was demonstrated following
513
significant reductions in fasting plasma glucose levels following a 24 week intervention
514
with either glyburide or rosiglitazone there were no differences in reaction time, spatial
515
memory or rapid processing tests all of which were tested with a similar protocol to the
516
present study.
517
518
Though this is the largest study examining the effect of inorganic nitrate
519
supplementation in a group at high risk of developing cognitive impairment it is the
520
nevertheless small. As mentioned above for those tests with larger intra-subject
521
variance larger trials would be required. It is also likely that the study is underpowered
522
to find correlations between changes in plasma nitrite concentration and changes in
523
cognitive function tests. However the present study will inform power calculations for
524
future studies.
525
23
526
Determination of dietary nitrate’s possible effect on cognitive function in multiple
527
domains, acutely and in the longer term is worthy of further consideration and should be
528
the subject of further investigation.
529
530
531
532
533
Acknowledgements
534
given in this paper are those of the authors and do not necessarily represent those of
535
NIHR, the NHS or the Department of Health. We thank all the study volunteers. The
536
authors wish to thank Dr Tim McDonald for assistance with analysis of the ionic
537
composition of the juices, Dr Michael Page and Prof Nick Smirnoff for assistance with
538
the ascorbate/dehydroascorbate analysis, and Dr Steve Simpson for assistance with the
539
proton NMR. We are grateful to James White Drinks Ltd. for the donation of juices
540
used in the study and in particular Armanda Pinhoe at James White Drinks and David
541
Upson of Stoke Farm Orchards for assistance in the production of the placebo juice.
This project was supported by the NIHR Exeter Clinical Research Facility. The views
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717
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Legends
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727
Table1. Ionic composition of juice pre and post resin treatment in context of typical daily
728
intake.
729
730
Table 2:Participant characteristics reproduced with permission from [34]
731
732
Table 3: Effect of dietary nitrate supplementation on selected aspects of cognitive
733
function in subjects with T2DM. * P=0.009
734
Figure 1: UV-vis spectroscopy of A active juice with absorbance peak at 487nM of 0.570
735
and 0.416 for the placebo and B Juice from the Gesar and Bayer cultivars with
736
absorbances of 0.556 in and 0.449 respectively.
737
738
Figure 2: (A) Proton NMR spectra of active juice in the study, (B) Proton NMR spectra
739
for placebo juice used in the study. Both spectra have been normalised to the largest
740
peak at 3.7ppm.
741
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746
747
29
748
Tables
749
750
Table 1.
Source
Placebo
Typical Daily
Intake
Sodium
17.7 mM
16.4 mM
150mmol
Potassium
76.6 mM
73.0 mM
71mmol
Calcium
0.48 mM
0.38 mM
20mmol
Magnesium
7.4 mM
7.7 mM
7mmol
Chloride
27.3 mM
60.5 mM
150mmol
751
752
Table 2
Age (years)
Mean
67.2
Std. Deviation
4.9
Duration of diabetes (years)
13.6
8.1
Mean office systolic blood pressure (mm Hg)
142.9
13.9
Mean office diastolic blood pressure (mm Hg)
81.1
9.2
BMI (Kg/m2)
30.8
3.2
HbA1c (%)
7.6
1.1
Serum Creatinine (mmol/l)
88.2
27.9
Total number of antihypertensives
2.1
1.1
Retinopathy
5 (18.5%)
Neuropathy
8 (29.6%)
Nephropathy
1 (3.7%)
753
30
754
Table 3:
Placebo
Active
Reaction Time (ms)
341.8±52.7
327.9±40*
Decision Reaction Time (ms)
716.6±161.3
721.3±169.4
Rapid Processing (accuracy %)
Shape Memory (accuracy %)
71.8±11.1
94.8±6.9
72±13.2
95±4.5
Spatial Memory (accuracy %)
80.4±9.4
78.8±12.4
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
31
774
Figures
775
776
Figure1:
777
Active
Placebo
778
Bayer
Gesar
779
780
781
782
Wavelength
Wavelength
783
784
785
786
787
788
789
790
791
792
793
794
795
796
32
797
798
799
Figure 2:
A
800
801
802
803
804
805
806
807
B
33