Reducing occupational stress with a B-vitamin focussed intervention: a randomized clinical trial: study protocol
Reducing occupational stress with a B-vitamin focussed intervention: a randomized clinical trial: study protocol
Con Stough 0
Tamara Simpson 0
Justine Lomas 0
Grace McPhee 0
Clare Billings 0
Stephen Myers 2
Chris Oliver 2
Luke A Downey 0 1
0 Centre for Human Psychopharmacology, Swinburne University , PO Box 218, HawthornVictoria, Melbourne , Australia
1 Department of Psychology, Swansea University , Swansea, Wales , UK
2 NatMed Research Unit, Southern Cross University , Lismore, NSW , Australia
Background: Workplace stress in Australia and other western countries has been steadily increasing over the past decade. It can be observed not only in terms of increased compensation claims but also costs due to absenteeism, loss of productivity at work and reduced psychological and physiological health and well-being. Given the cost and pervasive effects of stress in the modern workforce, time efficient and cost-effective interventions capable of reducing occupational stress (or strain) and burnout are urgently required for the improved well-being of stressed employees. One intervention gaining scientific traction is supplementation with nutritional interventions, particularly the B group vitamins. Methods: This study was developed to examine the effects of B group vitamins on workplace stress and mood variables with a sample of full-time employed older adults who subjectively report feeling stressed. The study is a randomized, double-blind, placebo-controlled, parallel-groups clinical trial where 200 (N = 100/group) participants will be randomized to receive Blackmores® Executive B Stress Formula or placebo daily for a period of 6 months. Participants will be tested at baseline and 6 months post-randomization on workplace stress, cognitive, personality and mood measures, cardiovascular (brachial and aortic systolic and diastolic blood pressures as well as arterial stiffness), biochemical (assays to measure inflammation and safety) as well as genetic assessments (to assess stress processing) and neuroimaging measures (to investigate in vivo mechanisms of action of B vitamins). In addition to this pre- and post- supplementation testing, participants will also complete a battery of self-report questionnaires online to assess their stress and mood once a month for the duration of the study. The primary aim of the study is to investigate the effects of B vitamin supplementation on work related stress. The secondary aims are to explore the mechanisms underpinning any changes in mood or workplace stress due to the B vitamin intervention by examining relationships between cognitive, biological, neuroimaging and cardiovascular variables over 6 months. A subset of 40 participants (N = 20/group) will undergo neuroimaging at baseline and at 6 months using functional magnetic resonance imaging (fMRI) and magnetic resonance spectroscopy (MRS) in order to further explore in vivo mechanisms of action of B vitamins. Trial registration: Australia and New Zealand Clinical Trials Register (ANZCTR):ACTRN12613000294752
B vitamins; Stress; Cognition; Genetics; Homocysteine; Occupational stress; Work-related stress; MRI; fMRI; MRS
Background and rationale
Stress in the workplace is an increasing problem in
Australia and most Western countries. Stress related
claims have steadily risen each year in all states of
Australia from 1997 to 2006 . Additionally, it has been
estimated that occupational stress costs the Australian
economy $14.81 billion per year in stress related
workplace inactivity and absenteeism at work . The World
Health Organization (WHO) has stated that the direct
cost of occupational stress in 1992 in the USA was US$42
billion . The cost of high levels of Occupational Stress
can be seen at personal, organizational and societal levels.
Workers who experience high occupational stress are
significantly more likely to make a stress-related work cover
claim, use significantly more medical resources and
contribute less to an organization through increased
absenteeism and lost productivity. Prolonged experience of
high levels of stress and not being able to meet workplace
demands can lead to ‘burnout’. Workers experiencing
greater mental and physical health issues are costly to treat
whether the symptoms are chronic or acute, reducing the
worker’s ability to return to the workplace. The WHO has
claimed that in the USA some US$300 billion is lost in
productivity, absenteeism and staff turn-over each year due
to high levels of occupational stress (amounting to
approximately 2.6% of US GDP). These data highlight
the need for time and cost effective interventions to
reduce occupational stress.
What is occupational stress?
The US National Institute for Occupational Health
and Safety (NIOSH) has argued that Occupational
stress occurs when there is a discrepancy between
the demands of the environment/workplace and an
individual’s ability to carry out and complete these
demands. Increased workplace demands diminish the
ability of our body to deal with stress . Additionally,
poorer diets because of longer hours spent at work
also result in a diminished capacity for our body to
deal with stress.
Dietary vitamins in the central nervous system
The central nervous system is dependent upon the
nutrients supplied through a good, varied, healthy
diet. Good nutrition is imperative, particularly for
maintaining the brain’s structure and normal cognitive
function . Vitamins and micronutrients play a
significant biochemical role in maintaining cognitive
processes within the brain. The role of antioxidants
and B vitamins contained in food and their relationship to
maintaining cognitive processes and general health is
Fresh fruits, green vegetables and berries contain a
rich source of vitamins A, C and E. These food sources
contain dietary antioxidants that can prevent, inhibit or
repair damage caused by oxidative stress [5,6].
Oxidative stress is representative of an imbalance or
disruption in the redox state (oxidation/reduction
reactions) within cells causing impaired signalling and
regulation, resulting in impaired functioning . The
maintenance of redox homeostasis is essential for
healthy cellular function. Antioxidant vitamin C levels
are particularly high in the brain and are needed for the
transformation of dopamine into noradrenalin as well as
the production of some neurotransmitters . Vitamin E
is a lipid soluble antioxidant which has neuroprotective
effects against free radical damage, preventing cellular
injuries to the brain related to oxidative stress .
Vitamins E and A protect against lipid peroxidation, a
damaging process affecting the permeability of cell
membranes . Vitamin C works synergistically with B
group vitamins and is vital for the metabolism and
utilization of folate/folic acid .
B group vitamins can be found in a wide range of
foods like whole grains, bananas, beans and meats.
Folate/folic acid, B6 and B12 represent some of the B
group vitamins. Folate, B6 and B12 vitamins are known
to contribute to the regulation of healthy levels of the
amino acid HCy, and share a synergistic role in the
maintenance of cardiovascular and neural health, and
are also vital for energy metabolism [5,8,9]. Cross
sectional and prospective studies have identified that
elevated plasma HCy levels is associated with the
promotion of spontaneous cell death (apoptosis),
incidence of stroke, brain atrophy, Alzheimer’s disease,
bone fracture and is considered a risk factor for
cerebrovascular disease [11,12].
Folate/folic acid, B6 and B12 vitamins are essential for
the methylation of HCy to methionine in the central
nervous system. Methionine plays a crucial role in
one-carbon metabolism; biological processes for DNA
synthesis, repair and other methylation reactions . If
HCy is not sufficiently converted back to methionine, the
methylation process will be inhibited resulting in a
buildup of HCy. An elevated level of HCy increases the
likelihood for oxidative stress, leading to negative events
like mitochondrial membrane damage and DNA strand
breakage [14,15]. The role of HCy in disease pathogenesis
remains unclear. HCy could play a direct role in the
disease process or be simply a marker of
folate, B6 and or B12 vitamin deficiency. However,
research has identified that chronic stress depletes
vitamin B6  while supplementation with B6
vitamins could be a therapeutic strategy in reducing
stress . Therefore, one mechanism by which B group
vitamin intervention may aid the reduction
of stress and fatigue ratings of those in full time
employment is through the uninhibited clearance of HCy.
In addition, neuroimaging studies suggest that high
levels of plasma HCy and low levels of B vitamins are
linked to a higher incidence of brain atrophy and
degeneration [18,19]. This type of data suggests that
HCy plays a role in brain ageing, by contributing to
subclinical brain changes in what we would believe to
be an otherwise healthy population.
Vitamin supplementation interventions for work
One potential pharmacological intervention to assist
our bodies to cope with increased levels of stress that has
received some preliminary support recently is vitamin B
supplementation. Dietary deficiencies in micronutrients
have been implicated in altered mood states (including
work stress and psychiatric symptoms) in otherwise
healthy individuals . Supplementation with
micronutrients to overcome these dietary deficiencies has been
observed to improve perceived stress, mild psychiatric
symptoms, and some aspects of everyday mood in a
recent meta-analysis of studies examining short-term
multivitamin supplementation . These positive effects
have been postulated to have occurred through alteration
of biochemical processes affected by poorer dietary
status through supplementation. Specifically, high doses
of B vitamins have been suggested to be effective in
improving mood states in both clinical and non-clinical
Previous studies examining the relationship
between B vitamins and stress
Work stress is the result of an imbalance between
the assessment of environmental demands and an
individual’s resources and skills . Despite the widespread use
of vitamins to compensate for the busy lifestyle and
irregular eating patterns that often accompany stress,
there are few controlled trials directly investigating
the relationship between multivitamins and psychological
strain . Despite strong advertising campaigns from
many Vitamin companies, many of the claims do not have
strong evidence. Therefore more studies are urgently
required to help inform the public about the efficacy
of vitamins on states such as stress which are often
targeted by vitamin companies. There are a few smaller
studies on this topic. Two studies using a multivitamin
formula observed a reduction in stress symptoms
after supplementation of just 28 and 30 days [24,25].
Schlebusch and colleagues used a well-designed protocol,
and screened for a highly stressed sample . After
30 days of supplementation, significant treatment effects
were evident, with the multivitamin reducing the level of
anxiety and stress and improving psychological well-being.
Carroll and colleagues  used a similar design with a
smaller sample but employed more stringent exclusion
criterion. Eighty male participants were supplemented
over 28 days. They demonstrated significant reductions in
anxiety and perceived stress in the multivitamin group in
comparison to the placebo group. These participants also
reported being less tired and having greater concentration
compared to the placebo group. Our research centre has
recently examined whether a popular multivitamin
supplement available in Australia, (Blackmore’s Executive
B Stress Formula) containing a complex of mostly B group
vitamins, improved work related stress (the only study
to address work stress variables) . The duration of
administration of the multivitamin in this study was
90 days, a significant increase in comparison to previous
studies of 30 days of multivitamin administration [24-26].
Sixty participants, recruited from the community, completed
the 3-month, double-blind, randomized, placebo-controlled
trial in which personality , work demands, mood, anxiety
and strain were assessed. The primary analysis revealed that
the Vitamin B group reported significant reduction in
Personal Strain (p = 0.02), from weeks 4 (M 92.10, SE 2.44)
to week 12 (M 85.54, SE 2.27), while the placebo group
showed a significant increase in levels of strain from week 4
(M 88.84, SE 4.33) to week 12 (M 93.44, SE 4.03).
B vitamins and cognition
Cross sectional and prospective studies in the elderly
have revealed a positive relationship between cognitive
performance and B vitamin intake, and a negative
relationship between cognitive performance and B vitamin
deficiency as evidenced through plasma HCy levels .
It is through the process of methylation reactions that B
Vitamins are believed to influence cognitive performance
. It has therefore been postulated that the HCy
lowering properties of B vitamins, could potentially
mitigate the effects of cognitive decline [30,31].
Recently, studies have emerged that have identified
a link between brain atrophy, cognitive ability and B
vitamin levels. For example, Smith and colleagues 
investigated vitamin B6, B12 and folic acid
supplementation with an older sample that were
experiencing mild cognitive impairment, and observed that
the rate of brain atrophy declined in those who consumed
vitamins compared to placebo. They also observed that
high brain atrophy was associated with lower cognitive
test scores. Similar results were obtained by de Jager and
colleagues , where vitamin B supplementation
significantly slowed cognitive impairment over 2 years. This was
especially the case in those who had high levels of plasma
HCy. Promising results have also been detected with folic
acid supplementation alone, where 3 years of
supplementation significantly improved cognitive facets relating to
processing speed, memory and attention . It must be
noted, however, that these studies have focussed on
participants who are elderly and therefore experience cognitive
decline more readily than younger individuals. It also
highlights the need for better vitamin B nutrition in those
who are older and working, where cognitive decline is
both prevalent and occupationally detrimental.
Few randomised controlled trials have investigated the
cognitive benefits of vitamin intake in younger, healthy
cohorts. However, a randomized controlled study
conducted by Pipingas and colleagues  investigated
the effects of 16 weeks supplementation of a multivitamin
with 138 participants aged between 20 and 50 years.
While these researchers did not observe any significant
improvements in cognitive ability, they noted that plasma
folate, B6 and B12 levels were increased and HCy levels
decreased after multivitamin supplementation.
The contribution of B vitamins to cardiovascular
and neural health
Healthy vitamin and mineral status is paramount for
healthy neurocognitive function and well-being .
During pregnancy it is an accepted practice for women to
supplement their diet with folate. Early studies established
that dietary supplementation of folic acid reduced the risk
of babies being born with neural tube defects such as
spina bifida and anencephaly . Folic acid intake is
therefore paramount during pregnancy to assist the
proper closure of the foetal neural plate which under rapid
development forms the spinal cord and skull.
A wide variety of neurological symptoms manifest in
humans as a result of vitamin B12 deficiency. Numbness
of the skin, hands or feet, muscle weakness and disjointed
motor coordination are some of the symptoms indicative
of sensory and motor disorders as a result of the
degeneration of the spinal cord due to inadequate myelin .
Whereas confusion, apathy, depression and dementia like
symptoms are indicative of cerebral disorders that can
vary in severity and can include memory and judgement
impairments . When folate or vitamin B12 is
insufficient, an accumulation of HCy occurs which is understood
to be highly toxic to neurons and believed to be the cause
of these neurological, sensory and motor symptoms
described . Therefore, it is suggested that
consumption of vitamin B12 will address the accumulation of HCy,
and in turn, alleviate the associated neurological, sensory
and motor system symptoms.
Folate, B6 and B12 vitamins are the key vitamins
that have a direct effect on mood and
neurotransmitter regulation. Methionine is required in the synthesis
of S-adenosylmethionine (SAM), solely responsible for
methylation reactions in the brain . The products
of these reactions include the neurotransmitters related to
psychological well-being such as dopamine, serotonin and
norepinephrine in addition to phospholipids, proteins,
DNA and myelin . Furthermore, SAM is essential for
the maintenance of choline as well as the production of
acetylcholine in the central nervous system (important for
memory and mood, skeletal muscle control, heart rate and
breathing and transforming cysteine into the most
abundant antioxidant in the body, glutathione) . Research
has established the efficacy of SAM as an anti-depressant
treatment for those suffering from depression with recent
reviews detailing that supplementation with SAM for four
weeks or more is as effective as treatment with tricyclic
antidepressants for clinical depression [41,42]. In terms of
cognitive effects, B12 vitamin and folate deficiency may
cause a disruption to the SAM pathway, leading to a
reduction in the production of neurotransmitters. Therefore,
deficiency may have a direct effect on mood and cognitive
function by virtue of these vitamins in the production of
A clear association exists between HCy levels and
cardiovascular health . An increased level of HCy
has a harmful effect on the cardiovascular system
through actions that promote blood clotting with
platelets and the release of growth factors for vascular
endothelium . A reduction in the bioavailability of
a powerful vasodilator, endothelial nitric oxide, may
be related to vascular damage . Kang and
colleagues  reported a relationship between
jobrelated stress, plasma HCy levels and cardiovascular
risk factors in a group of 152 workers. Similarly,
other researchers  reported a significant
relationship between acute psychological stress and elevated
plasma HCy levels, blood pressure and heart rate.
They concluded that one mechanism, high HCy, through
psychological stress may contribute to the initiation and
progression of vascular disease. These findings suggest
that the interrelationship between B vitamins and folate in
the regulation of HCy may affect cognitive function
via direct effects on the brain or indirectly via
mechanisms working on the cardiovascular system. As such,
examination of the effect of B-vitamin
supplementation upon stress, mood, and cognitive performance
alongside measurement of probable biological and
neurological mechanisms of action of B vitamins should help
clarify the therapeutic role of these commonly available
Study protocol: reducing occupational stress with
a B-vitamin focussed intervention
Given the growing incidence, cost and pervasive effects
of stress in the modern workforce, the primary objective
of this research is to determine whether administra
tion of B-vitamins reduces occupational stress (or strain),
burnout, and the cost of absenteeism in a population of
older workers with high levels of occupational stress. We
will examine the relationship between B-vitamin
supplementation, workplace stress, cognitive, personality and
mood measures, cardiovascular (brachial and aortic
systolic and diastolic blood pressures as well as arterial
stiffness), biochemical (assays to measure inflammation
and safety) as well as genetic assessments (to assess stress
processing) and neuroimaging measures (to investigate
in vivo mechanisms of action of B vitamins) to identify the
mechanisms through which B-vitamin supplementation
may improve the well-being of Australian workers.
Aims and study hypotheses
The primary aim of this study is to investigate 6 month
effects of a B vitamin formulation (Blackmores® Executive B
Stress Formula) on mood and workplace stress in a sample
of full time, healthy employees. The secondary aim is to
examine the mechanisms underpinning any mood or
workplace stress enhancing actions of B vitamins by examining
relationships between cognitive, biological (biochemical,
genetic and brain metabolites) and cardiovascular variables
over 6 months. By examining the interrelationship between
inflammation, cardiovascular health and cognitive
performance, the current study aims to identify modifiable
risk factors for workplace stress that can be targeted by
supplementation. It is hypothesised that 6 month
supplementation with Executive B Stress Formula will improve
measures of workplace stress, relative to placebo with the
greatest effects observed at 6 months. It is also expected
that there will be an improvement in general health
factors, cognitive performance, biological variables
and cardiovascular health. The primary psychological
outcome measure is the difference between the B-vitamin
groups and placebo group over time on the total stress/
strain score of the PSQ from the OSI-R. The primary
physiological outcomes will be the differences levels of
HCy in blood plasma. The secondary outcome measures
are the differences between the B vitamin group and
placebo group in mean changes over time on: the
other OSI-R Questionnaires and their subscales; the
GHQ-12, the state-trait anxiety and mood measured
by the Profile of Moods Scale (POMS).
The study will be conducted at the Centre for Human
Psychopharmacology, Swinburne University, Melbourne.
Additional recruitment will occur at NatMed Research
Unit, Southern Cross University, NSW.
The participant group will consist of 200 full time
employees aged between 30–65 years, who report
feeling stressed in the workplace. Participants will be
randomised to receive Blackmores® Executive B Stress
Formula or placebo. Participants will be excluded from
the study if they have a psychiatric or neurological disease,
significant endocrine, renal, pulmonary, gastrointestinal or
cardiovascular disorder, other disorder affecting food
metabolism, a diagnosis of diabetes (type 1 or type 2)
recent history (past 5 years) of chronic/severe illness
(longer than 12 months), current regular alcohol use
exceeding 14 standard drinks per week for women or
28 standard drinks per week for men, vision that is not
corrected to normal. Participants will also be excluded
from the study if they are currently pregnant, breastfeeding
or planning to become pregnant in the next 6 months. To
be eligible, participants cannot be taking anticoagulant
medication (e.g. Warfarin), psychoactive medication
including antidepressants, antipsychotics, anxiolytics,
illicit drugs or significant cognitive enhancing drugs
(e.g. chronic intake of substances such as Gingko).
For the neuroimaging component of the study current
smokers will not be included. To be eligible on the
study day visits, participants must adhere to the study
day restrictions. These restrictions include not drinking
alcohol for 24 hours prior to the scheduled study day, and
not drinking caffeine 12 hours prior to the study day.
Participants are also asked to fast for 12 hours prior
to the study day appointment so that biochemical
markers can be assayed from obtaining fasting blood
The study was ethically approved by the Swinburne
University Research Ethics Committee (project number
2012/293) and all participants will provide written
informed consent. The trial has been registered with the
Australian and New Zealand Clinical Trials Registry
Eligible participants are required to attend two testing
sessions and commit to completing five online
questionnaires. An overview of the testing sessions is provided in
the clinical trial flow chart (Figure 1). Participants are
initially screened over the telephone for eligibility prior
to their first visit. During the first visit participants are
again screened for eligibility and for compliance to
the study day restrictions which were described above.
A detailed medical history is also acquired. Voluntary
written informed consent is obtained from all participants.
Those who are eligible are then asked to give blood and
Figure 1 Protocol flow diagram.
complete a series of workplace stress, personality, mood
and general health questionnaires. A light breakfast is
provided to participants prior to completing a cognitive
test battery. At the conclusion of the session participants
are randomised to their treatment group and provided with
enough supplements for 6 months. As outlined in Figure 1,
participants will have to complete online assessments
relating to workplace stress, mood and general health
from the first to the fifth month post randomization.
At visit two, the 6 month assessment, participants will
undergo the same procedure from visit 1. Participants
will be asked to return any remaining supplements,
enabling the investigators to estimate compliance to
treatment by counting the remaining amount.
Participants involved in the neuroimaging sub-study will
be required for an additional 2 hours on the initial and
last visit to the Centre for Human Psychopharmacology
(see Figure 1). Additional screening for eligibility for
these participants regarding any metallic implants is
conducted to ensure their safety to undergo Magnetic
Resonance Imaging (MRI) scans. Participants complete a
practice version of the N-back task being used in this
component of the study, to familiarise themselves with
the task and to minimise practice effects. Cognitive data
obtained during the training session will not be included
in statistical analysis. On the first visit both “trait” and
“state” assessments of the Spielbeger State-Trait Anxiety
Inventory  will be administered prior to the MRI
scans to determine a baseline anxiety level. The “state”
version of the questionnaire will be administered again,
after the MRI scan to ensure the experience of the MRI
has not impacted on the participant’s responses. This
scale is a widely used instrument for measuring fluctuating
levels of anxiety. Additionally, participants will complete
the vocabulary and matrix reasoning subsets of the
Wechsler Abbreviated Scale of Intelligence (WASI)
 on visit one, which is a reliable measure of general
intelligence for use in clinical and research settings.
Participants will lie in the MRI scanner whereby different
imaging techniques will be employed to determine brain
structure, blood flow, brain metabolite levels, resting state
connectivity as well as completing a cognitive task
(N-back) to assess functional connectivity. At the second
and final visit, six months post treatment, the same
procedure from visit one will be implemented and
participants will be asked to complete the “state” version
of the STAI before and after the MRI scan.
The sample size of the study is 200 participants, 100
participants in each arm. Power analysis was conducted
using G*Power 3.1.2. For a repeated measures design with
two groups (treatment vs placebo) and three time points
(baseline, three months and six months), previous
research conducted at Swinburne University of Technology
identified a treatment related improvement of 19% in
ratings of psychological distress after three months
supplementation with Blackmores® Executive Stress
The Executive B Stress Formulation is manufactured by
Blackmores®, Australia. The ingredients and dose of each
ingredient of the Executive B Stress Formulation is listed
in Table 1. The inert placebo tablets provided by
Blackmores® are matched in size and colour to the
Executive B tablets and include a small amount of
riboflavin (2 mg) so as to provide a similar urine
colouration effect. The participants will be instructed to
take two tablets, one at breakfast and one at lunch
time daily, for the duration of 24 weeks (6 months).
To prevent potentially acute supplementation effects,
the participants are asked not to take their tablets on
the final study visit.
Randomization and safety
Randomization of participants to treatment groups will
be determined by random allocation. Each participant
number will be assigned to a treatment group (active
Vitamin B1 (thiamine hydrochloride)
Vitamin B5 (pantothenic acid from calcium
pantothenate 75 mg)
Vitamin B6 (pyridoxine hydrochloride)
Vitamin B12 (cyanocobalamin)
Ascorbic acid (total vitamin C 250 mg)
Vitamin E (D-alpha-tocopheryl acid succinate 41.3 mg)
Potassium phosphate monobasic
Avena sativa (Oats) extract equiv. to dry seed
or placebo) using a computerized random number
generator by a disinterested third party ensuring all
other parties remain blind to the treatment allocation.
Eligible, recruited participants will be assigned a
participant number. Participant numbers will be allocated
sequentially and once assigned will not be re-used.
Randomization codes will be kept in a password
protected computer file and will only be accessed in case
of emergency. All potential adverse events will be
monitored throughout the trial, with oversight from
the Swinburne University of Technology Human Research
The primary study outcome will be the effect of Executive
B formula on work related stress as measured by a well
validated work related stress measure. The Occupational
Stress Inventory-Revised (OSI-R) measures three
dimensions of occupational adjustment: occupational stress,
psychological strain and coping resources . Within
each of these areas a number of subscales provide more
detailed information. The instrument yields scores on
14 different scales ranging from “Role Overload” and
“Interpersonal Strain” to “Self-Care”. High scores on the
subscales measuring “occupational roles” and “personal
strain” and low scores on the subscales measuring
“personal resources” indicate high psychological stress and
poor occupational adjustment. In a previous investigation
of occupational stress, this measure identified a reduction
in personal strain and depressed mood in response to
90 days of B vitamin supplementation .
A range of cognitive, stress, mood, health, personality,
cardiovascular, biochemical, genetic and neuroimaging
measures will be collected at different time points as
part of the study. These are described below and listed
in Table 2.
To investigate the effect of supplementation on
cognitive performance the Swinburne University Computerised
Cognitive Assessment Battery (SUCCAB) will be
implemented at visit one (baseline) and 6 months post
supplementation. The SUCCAB is a validated battery of 8 tests
assessing a range of cognitive functions that decline
with age . Cognitive enhancing effects of multivitamin
supplementation in middle aged  and elderly
individuals  have been identified using this cognitive battery
Stress, mood, health and dietary habits
Several self-report questionnaires will be used to assess
stress, mood and general health. Symptoms of stress will
Table 2 Summary of the secondary outcomes
implemented in Executive B across all time points
B Vitamins (B6, B12)
B Vitamins (B6, B12)
N-Back working memory
COMT = Catechol-O-methyltransferase gene, SLC6A4 = Serotonin-transporter
gene-linked polymorphic region, FKBP5 = Glucocorticoid receptor-regulating
co-chaperone of stress proteins, NPY = Neuropeptide Y, ADCYAP1R1 = Pituitary
adenylate cyclase-activating polypeptide receptor, MTHFR =
Methylenetetrahydrofolate reductase, CPSI = Carbamoyl phosphate synthetase
1, CBS and DPEP1 = Cystathionine –β-synthase and Dipeptidase 1, FFQ = Food
Frequency Questionnaire, BDI-II = Beck Depression Inventory II, POMS = Profile
of Mood States, GHQ = General Health Questionnaire, PSS = Perceived Stress
Scale, NEO PI-R = NEO-Personality Inventory Revised, WASI = Wechsler
Abbreviated Scale of Intelligence.
be measured with the Perceived Stress Scale (PSS) .
Depressive symptoms and state-trait anxiety will be
measured with the Beck Depression Inventory II
(BDI-II)  and the Spielberger State-Trait Anxiety
Inventory  respectively. Further assessment of
mood will be conducted with the Profile of Mood
States . Pre-existing general health and general
health for the duration of the trial will be assessed
with the General Health Questionnaire (GHQ) .
Dietary habits will be inferred from an in-house Food
Participants will be asked to complete the NEO PI-R
. The NEO PI-R is the most widely used
questionnaire used to assess personality traits. Consisting of
240 items, this questionnaire assesses the Five Factor
Model of personality by scoring participants across
personality dimensions and related facets of Openness,
Conscientiousness, Extroversion, Agreeableness and
Neuroticism. As personality traits are considered stable
across time, participants will only complete this
questionnaire once at the beginning of the trial. The research
investigators will examine how different personality types
respond to the intervention.
The results of a meta-analysis predicted that occupational
stress increases the risk of heart disease by 50% .
Additionally, evidence suggests that a contributing
factor in cognitive decline is an increase in arterial
stiffness with ageing . Given the interrelationship
identified between B group vitamins and HCy with
cognition and cardiovascular health, it could be argued
that chronic supplementation of B vitamins may have a
positive influence on cardiovascular function. Therefore,
cardiovascular measures in the current study will enable
exploration of whether improvements to cardiovascular
function are a mechanism by which B group vitamins
improve cognitive ability.
Brachial blood pressure measuring standard brachial
systolic and diastolic pressure alone with a clinically
validated automated sphygmomanometer may not be a
true assessment of central aortic events . Including
measurements of aortic pressures (PWA: Pulse Wave
Analysis; central aortic pressures) and carotid-femoral
Pulse Wave Velocity (PWV; aspects of arterial stiffness)
provides a reliable measurement of cardiovascular events
. Measuring PWV between the carotid and femoral
artery sites is considered a gold standard in the assessment
of artery stiffness .
PWA to measure participant’s central pulse pressure,
augmented pressure and augmentation index will be
obtained using a SphygmoCor® device (Model EM4C,
AtCor Medical, Sydney, Australia). Participants will be
supine and have an arm cuff placed around their brachial
artery on their left side with measurements obtained
after 10 minutes of rest. To ensure the accuracy of the
assessment, blood pressure will be taken three times,
the first reading will be discarded and the second two
will be averaged.
The same device will be used with participants to
measure PWV between the carotid and femoral
arteries to obtain measurements of arterial stiffness. The
averaged brachial systolic and diastolic pressures from
the first assessment will be entered into the computer
software (Version 18.104.22.168) to assist in measuring arterial
stiffness. The distance between the carotid and femoral
artery and femoral artery to cuff sites will be measured
using a flexible tape measure and entered into the
software program whereby the length of the aorta is estimated
by the computer program. A femoral cuff will be placed
over the participant’s femoral artery and a tonometer
pressure sensor applanating the carotid artery, to capture
the carotid waveform, will be applied to obtain
measurements of aortic stiffness.
Blood sampling is to be conducted via venepuncture by
a registered nurse or qualified phlebotomist on each of the
testing days, (visit 1 and visit 2) following a period of fasting
from the night before. Biochemical markers of Vitamin
B12, Vitamin B 6, Folate and HCy will be obtained.
Additionally, safety profiling will be measured through
a full blood count, high sensitivity C-reactive Protein
and biochemical Liver Function Tests.
Blood samples via venepuncture will be used to provide
specific genetic information which has been shown to
be implicated in stress processing. In a review of current
genetic work, five candidate gene polymorphisms were
identified as being illustrative of individual differences
in emotion processing . They are:
catechol-Omethyltransferase (COMT), serotonin transporter (SLC6A4),
neuropeptide Y (NPY), a glucocorticoid receptor-regulating
co-chaperone of stress proteins (FKBP5) and pituitary
adenylate cyclase-activating polypeptide receptor
(ADCYAP1R1). Emotional processing could be implicated
in stress processing, and any change in expression due to
vitamin supplementation. Processing of emotions is
critical to most aspects of human behaviour, and individual
differences in the processing of emotional stimuli exist as
functions of personality (Neuroticism), neuropsychiatric
disorders, and with specific reference to this proposal,
Neuroimaging will be conducted in a subset of 40
participants in order to explore the in vivo mechanisms of action
of Executive B formula in the brain to elucidate
underlying cognitive and health effects of B group vitamins.
Previous imaging studies looking at vitamin supplementation
have been conducted using electroencephalography (EEG;
e.g., Macpherson H, Ellis KA, Sali A and Pipingas A ).
Few studies have investigated the in vivo effects of
supplements on brain metabolites as measured with magnetic
resonance spectroscopy (MRS) and functional magnetic
resonance imaging (fMRI). The technique of MRS has
been used to study alterations in brain metabolites of
people with clinical disorders like depression  and
schizophrenia , or Alzheimer’s disease and mild
cognitive impairment [65,66]. However, as far as we
understand, no neuroimaging studies have been
conducted using the technique of MRS to investigate the
relationship of brain metabolic function, cognition
and cardiovascular health in response to B vitamin
Structural and functional MRI (fMRI) scans will be
acquired using a Siemens 3 Tesla Tim Trio MRI
scanner (Erlargen, Germany), located at the Centre for Human
Psychopharmacology, Swinburne University of
Technology. A structural scan will be obtained of each participant
and used as a reference point for further functional scans.
Scanning in a resting state will occur in order to assess
activity in the default mode network (DMN) for
approximately 6 minutes. Following DMN there will be scanning
for brain metabolite levels in three regions of interest
using the technique of MRS for approximately 35 minutes.
Following MRS an arterial spin labelled perfusion
sequence will be conducted for approximately 6 minutes
to investigate the brain’s tissue perfusion. Finally, changes
in the blood oxygenation-level dependent (BOLD) signal
will be analysed while participants compete an in-scanner
version of the N-Back working memory task (approximately
10 minutes) developed for The Human Connectome
The primary analysis will investigate the effect of treatment
on workplace stress levels over the course of the study
using Repeated Measures Analysis of Variance (ANOVA)
techniques (with linear mixed modelling used to account
for any missing data). Secondary outcome variables will be
analysed using similar statistical techniques. Pearson’s
correlation coefficients or the non-parametric equivalent,
Spearman’s R will be used to investigate relationships
between other variables of interest such as stress,
biochemical, cardiovascular or mood/health factors collected
at baseline. Results will be considered statistically
significant at an alpha level of P < 0.05 corrected for multiple
Analysis of brain metabolites from MRS as well as
the fMRI data during N-Back working memory task will
be conducted using a region of interest (ROI) approach.
Between group (Executive B Formula versus inert placebo)
functional differences in predefined brain regions will
be statistically analysed. The ROIs that will be analysed in
the N-Back working memory task will include the
dorsolateral, ventrolateral and medial prefrontal cortex,
anterior cingulate, parietal cortex and sensorimotor cortex
. The MRS ROIs that will be analysed will include the
dorsolateral prefrontal cortex, posterior cingulate, and
amygdala for their well-established association with stress
and cognitive factors [65,69,70].
Occupational stress is a multibillion dollar problem.
Effective strategies reducing occupational stress are
urgently required. Dietary supplementation with B
group vitamins may be an economically viable and
sustainable intervention. Reducing occupational stress will
have enormous benefits for decreasing stress claims,
absenteeism, and increasing work productivity. Vitamin B
supplementation is also likely to have additional health
and quality of life benefits.
ANOVA: Analysis of Variance; ANZCTR: Australia and New Zealand Clinical
Trials Registry; BDI-II: Beck Depression Inventory II; BOLD: Blood oxygenation
level dependent; BP: Blood pressure; DMN: Default Mode Network;
EEG: Electroencephalography; Exec B: Executive B Study; FFQ: Food
frequency questionnaire; fMRI: Functional magnetic resonance imaging;
GHQ: General health questionnaire; HCy: Homocysteine; MRS: Magnetic
resonance spectroscopy; ROI: Region of Interest; NEO PI-R: NEO
personality-inventory revised; OSI-R: Occupational stress inventory-revised;
POMS: Profile of Mood States; PSS: Perceived stress scale; SAM:
S-adenosylmethionine, SUCCAB, Swinburne University Computerized Cognitive
Assessment Battery; WASI: Wechsler abbreviated scale of intelligence.
CS and LD conceived the study. CS and LD were applicants for funding.
All authors were involved in designing the study and drafting the protocol.
All authors read and approved the final protocol.
This study is partly funded by Blackmores® Australia who is providing
Blackmores® Executive B Stress Formulation and the inert placebo for the
study. Dr Luke Downey is supported by a National Health and Medical
Research Council (NH&MRC) biomedical fellowship (APP1054279). Tamara
Simpson is funded by an Australian Postgraduate Award scholarship. We
thank Dr Matthew Pase for his comments related to the cardiovascular
section of the manuscript.
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