Chapter 2: Methods
2.4 Study Assessments
Primary and secondary endpoints were pre-specified on clinicaltrials.gov. Primary measures included assessments of cognitive function and secondary outcomes included measures of muscle function. Detailed study assessments were completed at 0, 3 and 6 months, components of which are detailed in Figure 2.1.
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2.4.1 Demographics
Demographic information was collected from all participants for age, gender, marital status (married, single, cohabiting, widowed, divorced), employment history (skill level and experience in years) and education level (years of education and highest level of education achieved) (Appendix 14).
2.4.2 Nutrition
A modified food frequency questionnaire (FFQ) used by another large nutrition and ageing studies (181) was completed to gather detailed information regarding the frequency of vitamin D containing foods were collected to allow for consideration of dietary intake for all participants and to monitor any changes in dietary D intakes over the course of the study. Participants were asked whether they consumed certain food types, and if so how frequently (Appendix 15). Detailed information on portion size was not collected as this was not an outcome for this study, as there are few high
contributing food sources of vitamin D (naturally or fortified), and foods which provide good sources of vitamin D are not generally daily staple foods, it is anticipated that the contribution would be minimal. Nevertheless, important to acknowledge. Data on dietary supplement use was obtained during the telephone health screening.
The Mini Nutritional Assessment screening tool for malnutrition was included in the study design, which has been validated for use in older adult populations (182, 183).
2.4.3 Habitual vitamin D behaviour
No standard, validated sunlight questionnaires are routinely used to quantify sun exposure. Typical questions used to assess sunlight exposure are listed elsewhere and were included in our estimate of vitamin D acquired through sun exposure (184). Sun exposure contributes more to serum 25(OH)D concentrations than food sources, and due to the timing of study initiation (Spring), sun habits were therefore deemed of importance. To assess the likely contribution of sun exposure to serum D
concentrations, participants were asked multiple questions regarding sun avoidance, sun protection factor use, and sun holiday travel (Appendix 15).
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2.4.4 Cognitive sphere- assessment of cognitive function
The term “cognitive functions” refers to a variety of brain functions and processes which includes; receiving external information, processing this internally and
responding with a behaviour. It can be seen as a hierarchy, going from overall (global) to domain-specific cognitive functions. Domain-specific functions include memory, executive functioning, attention, perceptual functions, psychomotor abilities and
language skills. The importance of utilising multiple domains when assessing cognition has been highlighted in recent years (174). In order to establish overall performance within a domain it is therefore important to conduct a series of tests that will evaluate key components.
How to measure cognitive performance?
Neuropsychological performance tests are the method most commonly used in nutrition and lifestyle studies. Cognitive function can be assessed objectively by a number of validated pen-and paper or computer-based tasks, illustrated in Figure 2.2. Each test involves varying degrees of researcher involvement which requires a level of expertise to administer and extensive training is generally required. Computer based tasks
overcome this with standardised procedures which accurately measures fine details such as processing speed and reaction time.
As the evidence for an underlying link between diet and or dietary components and cognitive performance remains inconclusive, the best approach is to include a battery of cognitive tests that cover a variety of domains, this may help identify the specific cognitive processes involved. For this approach to be effective the design must also consider an appropriate test duration, considering test fatigue which may be
Figure 2. 2: Direct and indirect methods of measuring brain and cognitive function
By testing multiple cognitive domains and examining a range of cognitive abilities we could assess the effect of vitamin D supplementation and cognitive function in different brain areas. We anticipated the findings would replicate the cognitive module in
population studies to provide comparability with nationally representative (TILDA) and other longitudinal studies. During discussions at protocol development stages, a senior clinical neuropsychologist (RC) and the study Co.PI (BL), the final cognitive sphere for the present RCT included global and domain-specific measures of executive function, attention, memory and visual reasoning. Details of the assessments are detailed below.
2.4.5 Global function- Montreal Cognitive Assessment (MoCA)
The MoCA is a quick screening instrument for mild cognitive dysfunction (185). It assesses different cognitive domains, including attention and concentration, executive functions, memory, language, visuo-constructional skills, conceptual thinking,
calculations and orientation, providing an overall composite score, ranging from 0-30. Various cut-offs can be applied depending on normative data available for the study population. A score <23 is generally considered indicative of cognitive impairment, in healthy community-dwelling older adults (186). The MoCA is widely used in population studies and is administered as part of the cognitive battery in TILDA.
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2.4.6 Executive function- Trails Making Task, part B (TMT-B) and B minus A (B-
A)
Trail Making Task (187) has two components in part B the participant draws a line to join encircled numbers and letters in alternating numeric and alphabetic order (i.e. 1-A-2-B), as quick as possible, whilst instructed not to lift the pencil from the page. The numbers are placed in a somewhat random order, so the line should not overlap. Generally errors are not reported as they are reflected in the time to complete the test. Cut-offs for older adults populations for part B are 300 seconds, as this is the maximum time allowed to complete the test. Performance on TMT part B is considered a sensitive indicator of neurological impairment. Part B involves higher level cognitive skills and is more difficult due to increased visually interfering stimuli than part A (188), however, the difference in B minus A (B-A) may provide an additional measure of mental flexibility and executive functioning (189).
Studies have demonstrated activation in areas of the brain believed to be sensitive to executive functioning, motor control and cognitive flexibility (190). Sensitivity to
subcortical white matter hyper-intensities (WMH) in healthy older adults have also been shown (191). A common feature of neuropsychological instruments is the susceptibility to practice effects, short intervals of 6-weeks may reflect practice effects (192). The TMT has excellent inter-rater reliability (193).
2.4.7 Attention- Sustained Attention to Response Task
Sustained attention is measured using a computer-based Sustained Attention to Response Task (SARTfixed) (194). SART assesses executive control of behaviour. It is sensitive to transient lapses of attention, and challenges the ability to endogenously maintain an alert state (195). SART activates the sustained attention network and is sensitive to frontal lobe dysfunction (196).
It is a continuous performance reaction-time (RT) task. It requires participants to respond to digits, ‘1’ through ‘9’ which appear on screen sequentially, over a period of approximately 5 minutes. Totalling 225 digits, which vary in size but not in screen position. Participants are instructed to press the left control pad button on presentation
of each digit (GO-trial) with the exception of the 25 occasions when the digit ‘3’ (NO GO trial) appears, when they are required to withhold their responses. For each trial a digit is presented for 150ms followed by an inter-stimulus interval (ISI) (53) that varies randomly between 1000 and 1500ms. This variable interval is introduced to prevent participants from succumbing to a speed accuracy trade off that can occur when ISIs are evenly paced (53).
Commission errors (responding to NO-GO trials) reflects lapses of sustained attention, and omission errors (failure to respond to GO trials) reflects task disengagement and corresponds to lapses in attention. In older adults, fewer errors and slower mean RT represents a speed accuracy trade off, however, more SART errors accompanied by slower mean RT indicates reduced cognitive processing speed and/or lapses in sustained attention (197, 198). Greater RT variability (standard deviation [SD] of RT) is associated with attentional deficits, slower RT and increased SART errors in older adults (199).
We anticipated that those allocated vitamin D supplementation would display quicker RT, lower RT.var and less combined SART errors (SART error scores) than those allocated to placebo. The SART was selected as it has been widely used in studies in Trinity College Institute of Neuroscience (TCIN) and was used in the TILDA cognitive battery.
2.4.8 Attention- Trail Making Task, part A
In part A of the Trail Making Task the participant is asked to draw a line to connect circled numbers in a numerical sequence (i.e., 1-2-3, etc.) as rapidly in possible (200, 201). TMT-A is primarily a test of visual sustained attention, simple perceptual tracking and processing speed. Similar to TMT-B, errors are not reported as they are reflected in the time taken to complete the task.
2.4.9 Memory and Recall- Weschler Memory Scale III, Logical Memory I and II
The test assesses narrative memory under a free recall condition. Two short stories are orally presented. For older adults, one story is presented twice. The participant is asked to recall each story from memory immediately after hearing it (Logical Memory I).
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Following a minimum 20 minute interval, the participant is once again asked to retell each story from memory for a delayed recall condition (Logical Memory II). The stories administered depend on age, for ages 16-69 (Adult Battery) are story B and C; and the stories for ages 65-90 (Older Adult) are story A and B. In line with other ageing studies conducted in the Memory Research Unit (202) in TCIN, on the basis of advice from (RC), the cut off for the adult battery was 69, and the older adult version is administered to those aged 70 years and older. Final scores were then calculated from indexed scaled scores to give a comparable result irrespective of the participants’ age and version administered.
2.4.10 Visual Reasoning
The visual reasoning test from the Cambridge Mental Disorders of the Elderly
Examination (CAMDEX) battery, having also been used in TILDA and the MRU, and therefore allowing for comparability with nationally representative data was included in the cognitive battery.
Test administration: This test comprises 6 trials consisting of a grid of four boxes, three of which contain a shape, and one of which is empty. Six shapes are presented below the grid, marked A-F, and the participant is asked to select which of these options should go into the empty box in order to form a pattern.
2.4.11 Working Memory
Letter Number Sequencing (LNS) is used as an auditory working memory subtest as part of the Wechsler Adult Intelligence Scale-III (WMS-III) working memory index. An assumption in the cognitive literature is that performance on recall tasks is somewhat unaffected by ageing, whereas tasks that require the reorganisation of items prior to recall are more age-sensitive. This is presumed to be because manipulation tasks require greater prefrontal involvement than maintenance tasks, a view supported by current neuroimaging evidence (203). We decided to include this task as an exploratory cognitive component in support of the general memory section.
Test administration: A series of alternating numbers and letters are presented to the participant at a rate of 1 item per second (e.g. B, 4, P, 7), and the participant must then recall the numbers first in ascending order followed by the letters in alphabetical order.
2.4.12 Composite scores
Composite scores for each cognitive domain were calculated by obtaining standardised Z scores for each cognitive test and combining them to create total scores for; executive function (TMT-B, TMT B minus A, visual reasoning), attention (TMT A, SART RT, RT.var., SART errors), and memory (logical memory I & II and LNS working memory).
Using Z scores provides a more-reliable estimate of the effect of each domain, reduces the number of single tests reported, and allows the performance for each domain to be compared in a standardised way. Therefore an estimate of the effect size for each domain can be compared to differences in individual tests using the population standard deviation for each test.