APPOINTMENT 2

Due to the nature of the testing participants were required to be fasted prior to the testing and so this appointment had to take place first thing in the morning. The participant could therefore choose for this appointment to either be at QMU or at a location convenient to them. The participants were required to be fasted to enable resting energy expenditure to be measured. This was used to confirm the dietary analysis and to check for potential over or under-reporting.

2.5.1 Energy Expenditure

Total energy expenditure (TEE) comprises of 3 main components, resting energy expenditure (REE) which accounts for 60-70% of TEE, dietary induced thermogenesis, which accounts for approximately 10% of TEE and physical activity levels (PAL) which accounts for 20-40% of TEE in healthy adults. As the activPAL^TM accelerometers provide data on physical activity levels including MET, measuring REE enabled the prediction of TEE for each participant. This was then utilised in conjunction with dietary intake to determine energy balance and give an indication of the accuracy of the diet diaries.

REE is an expression of the rate at which oxygen is used by body cells or the calculated equivalent heat production by the body, in a fasting subject at complete rest (Levine, 2005). The conditions required to accurately measure REE are for a person to be completely rested (before and during measurements), lying down but fully awake, fasted for at least 10 – 12 hours, in a thermo-neutral environment (22-26^o) and free from emotional stress (Levine, 2005). Fulfilling all these criteria is however difficult. These conditions were however adhered to where possible.

Participants were asked to fast overnight drinking only water in the morning and they were asked to rest prior to testing. Testing took place either at QMU or in the participants own home shortly after they had wakened. As participants had to be rested the preferred location was the participants own home to prevent an increase in energy expenditure as a result of travelling to QMU. The appointment time was chosen by the participant to allow them to remain fasted without too much discomfort.

REE was measured using a Fitmate Calorimeter (Cosmed Company, Rome) which was calibrated prior to the start of the study. The calorimeter is a small (20 x 24 cm) metabolic analyser designed for measurement of oxygen consumption and energy expenditure during rest and exercise. It uses a turbine flow meter for measuring ventilation, a galvanic fuel cell oxygen sensor for analysing the fraction of oxygen in expired gases, and incorporates an innovative sampling technology that allows the FitMate to retain the performance of a metabolic cart with a standard mixing chamber. REE is calculated from oxygen consumption using a fixed respiratory quotient (RQ) of 0.85, and estimated grams of urinary nitrogen using a modified Weir equation. The Weir equation measures REE in the following way:

REE = (O₂ consumed (litre) × 3.941 + produced CO₂ (litre) ×1.11) ×1440 min/d

Participants were asked to lie in a supine position and wear a mask which covered their nose and mouth and were asked to breathe normally. The mask enabled oxygen consumption and carbon dioxide production to be measured. To enable the participants to fully relax and for breathing to stabilise the calorimeter measured breathing (including number and depth of breath), oxygen consumption and carbon dioxide production for five minutes initially and then these measurements were discarded. Following this, the calorimeter measured oxygen consumption and carbon dioxide production for a further 15 minutes and these 15 minutes of testing was used to determine REE. This timeframe has been established to be adequate to determine a reliable measure of REE whilst minimising participant burden (Compher et al., 2006). If participants felt uncomfortable they were informed that they could stop the test at any time by letting the researcher know that they wished to halt the test.

During the REE measurements the researcher downloaded the data from the activPAL^TM to ensure seven consecutive days of activity had been recorded. This also provided an opportunity to compare the activity diary data with the data from the accelerometer and to identify any inconsistencies which could then be discussed with the participant following completion of the testing should this be required.

As the participants were rested for measuring REE at the end of this test blood pressure was then measured.

2.5.2 Blood pressure

Raised blood pressure provides an indication of cardiovascular disease (CVD) risk.

As CVD is linked to ageing and increased visceral adiposity, measuring blood pressure alongside markers of nutritional and functional status provides additional

information to determine the health status of a person. To determine normal blood pressure this should be measured when a person is at rest. Blood pressure was measured following European Society of Hypertension recommendations (O’Brien et al., 2003). An Omron M10-IT upper arm automated sphygmomanometer was used.

Positioning of the participant for blood pressure monitoring followed guidance of the American Heart Association (Pickering et al., 2005).

Participants were asked to remove clothing which covered the location of cuff placement and they were asked to sit in a chair with their legs uncrossed and with their back supported. Their left elbow and forearm rested on a table with the blood pressure monitor cuff at the level of the right atrium of the heart i.e. the midpoint of the sternum or the fourth intercostal space. A standard cuff with a bladder measuring 12 x 26 cm was placed around the upper arm and 1 - 2 cm above the antecubital fossa in line with the brachial artery. The cuff was tightened to the point where 2 fingers could still be inserted comfortably between the cuff and the participant’s arm. The display on the blood pressure monitor was placed facing away from the participant’s line of sight to prevent the reading causing sudden changes in blood pressure.

The participant was instructed to relax as much as possible and not to talk during the measurement procedure with the first measurements taken 5 minutes after positioning. The monitor was then switched on and as the sphygmomanometer was automated the cuff continued to inflate until the brachial artery was occluded.

Systolic and diastolic measurements were then recorded automatically by the blood pressure monitor. Blood pressure was then measured twice more with at least one minute between each measurement. In line with British Hypertension Society guidance (Williams et al., 2004) the first measurement was discarded and an

average of the two subsequent systolic and diastolic measurements was then taken and recorded as the participant’s normal blood pressure.

Fasting blood samples were required for the participants and so whilst the participants were fasted they were asked to provide two blood samples. The researcher was trained in phlebotomy as part of the NHS Lothian training programme and these procedures were followed. To collect the blood samples each participant was asked to sit in an upright position with their chosen arm outstretched and supported by a cushion. A 21G (Greiner Bio-One, Brunel Way, Stroudwater Business Park, Stonehouse, GL10 3SX) hypodermic needle was inserted into a suitable vein in the antecubital fossa. Blood (approx. 10 ml) was withdrawn into an Ethylenediaminetetraacetic acid (EDTA) vacuette (Greiner Bio-One) and a further blood sample (approx. 10 ml) was withdrawn into a lithium heparin (LH) vacuette (Greiner Bio-One). The blood samples were inverted several times in the respective vacuettes to ensure anticoagulation of the blood and were then processed in preparation for analysis (see later).

Following the measurement of REE, blood pressure measurements and provision of blood samples participants were permitted to eat and drink if they wished.

Participants were then asked to complete the questionnaire to measure levels of fatigue.

2.5.3 Fatigue

Fatigue was measured using the Multi-dimensional Fatigue Inventory (MFI) (Smets et al., 1995) which is a 20 point questionnaire to measure fatigue. It was initially developed to measure fatigue in a cancer population but it has since been validated

in a healthy population (Lin et al., 2009). Permission to use the questionnaire was sought from and granted by the developer.

The questionnaire is self-administered and measures five domains of fatigue:

physical fatigue, mental fatigue, reduced motivation, reduced activity and general fatigue. Each domain is measured by a separate subscale within the questionnaire with each subscale consisting of 4 questions all of which use a five-point Likert scale. General fatigue includes general statements about fatigue and decreased functioning and was designed to encompass both physical and psychological aspects of fatigue. Physical fatigue concerns physical sensations related to fatigue.

Mental fatigue pertains to cognitive functioning, including difficulty concentrating.

Reduced activity refers to the influence of physical and psychological factors on levels of activity. Reduced motivation relates to lack of motivation for starting any activity. Scores on each subscale range from 4 - 20, with higher scores indicating greater levels of fatigue.