Justificación

After the Loughborough University Ethical Advisory Committee had approved the study, 13 healthy, active female volunteers (aged 18 – 26 years) signed written informed consent forms to confirm their participation in the study. In order to ensure the safety of participants and to minimize confounding factors, participants had to conform to the following criteria: they had to be non-smokers; not currently taking any medication except for the combined hormone contraceptive pill (with both estradiol and progesterone); no personal history of cardiovascular disease, metabolic disease or dyslipidaemia; not pregnant (assessed by the health screen questionnaire); not taking any drugs (medical or illegal) known to affect digestion or metabolism (e.g., anabolic steroids, marijuana, amphetamines, thyroid prescription drugs); and all participants had to be able to cope with the exercise demands of the study. The physical characteristics of the participants are displayed in Table 5.1 below.

Table 5.1 Physical characteristics of the study participants Characteristics Mean ± SD Age (yrs) 21.8 ± 2.1 Height (cm) 170.0 ± 4.3 Weight (kg) 62.5 ± 6.0 BMI (kg.m-2) 21.6 ± 1.9 Body fat (%) 24.8 ± 5.1 Waist circumference (cm) 71.0 ± 4.6 Hip circumference (cm) 86.6 ± 5.5 Resting SBP (mm Hg) 122 ± 7 Resting DBP (mm Hg) 76 ± 5

VO2 max (mL.kg-1min-1) 42.5 ± 7.3

Data are displayed as mean ± SD, (n = 13). BMI = Body mass index; VO2 max = maximum oxygen uptake; SBP = systolic blood pressure; DBP = diastolic blood pressure.

5.2.2 Preliminary sessions

Before participants took part in the main trials, they were invited to attend the laboratory on two separate occasions, i.e. for preliminary sessions. On the first visit, participants undertook screening, which lasted for 2 hours. This involved participants completing physical activity and health screening questionnaires, as well as a three factor eating questionnaire which helped in assessing whether each participant was a ‘restrained’ or ‘unrestrained’ eater. Once these questionnaires were completed, a series of measurements were taken including: resting blood pressure, height and weight, waist and hip circumferences and skinfold thickness at four different sites (biceps, triceps, subscapula andsuprailiac). Once the screening phase was complete participants completed a preliminary exercise test to determine maximum oxygen uptake. This test consisted of continuous incremental cycling exercise beginning at a workload of 35 W with increments of 35 W every 3 minutes until participants reached volitional exhaustion. The exercise test was performed on a cycle ergometer (Excalibur, Lode, Groningen, The Netherlands). Expired air samples were collected using Douglas bags

for the last minute of each stage. Heart rate was measured throughout the test using short- range telemetry. Further details about this test are provided in the General Methodology (Section 3.6.3).

On the second preliminary session (familiarisation), participants were asked to familiarise themselves with the environment in which they would be tested. During this visit, participants were made aware of the protocols and were assured of the safety procedures. During this familiarisation visit, participants completed a 45-minute bicycle ride at 70% of their maximum oxygen uptake. They were also familiarised with all of the procedures to be used in the main trials.

5.2.3 Main trials

One week after the preliminary visits, participants completed two main trials with an interval of one to two weeks between each trial; each trial lasted for approximately 3 hours (Figure 5.2). Participants were advised to use public transport when travelling to the laboratory in order to minimise their physical exertion on the days of the trials if they were travelling a long distance. Participants were asked to report/make contact on the first day of bleeding, and to calculate their follicular phase (5th – 11th day) and luteal phase (18th – 23rd day). Participants then arranged a day to attend the laboratory during the luteal phase of their OCP cycle and the follicular phase of their OCP cycle.

Each trial began at 12:00 noon and lasted for nearly 3 hours (i.e. from 12:00 noon to 2:45 pm). The trials began with the participants undertaking a 45-minute cycle ride, which was predicted to elicit 70% of maximum oxygen uptake (12:00 noon to 12:45 pm). During the trial, participants were requested to complete visual analogue scales every half an hour to assess their perceptions of hunger, satisfaction, fullness and prospective food consumption (Appendix C). Four venous blood samples and three saliva samples were taken during each main trial (Figure 5.2). During each exercise stage within the trials, expired air was collected into Douglas bags. After cycling, participants rested for the remainder of the trial (sitting reading, working at computer, writing, playing video games or watching a DVD).

their second main trial. Alcohol and caffeine were prohibited on the days when participants were recording their diet. Participants were required to finish eating by 11.00 pm on the evenings before the main trials. Water was allowed ad libitum prior to and during the main trials. On the morning of each main trial participants ate breakfast between 8.00 and 9.00 am and did not consume anything else before coming to the laboratory. Participants were asked to consume identical breakfasts prior to each main trial.

One hour after the completion of exercise participants were given access to an ad libitum meal, which consisted of three types of cereals, milk, tuna, ham, chocolate bar, cereal bar, two types of muffins, orange, banana, apple, cheese, butter, margarine, mayonnaise, white and brown bread. Once the buffet was accessible, participants were told that they had 30 minutes to consume food until they were satisfied. Participants were kept in isolation throughout the buffet process in order to limit social factors from influencing food intake. Prior to the food consumption phase, all items were weighed. This was necessary in order to determine the amount of food consumed, which was the difference between the initial weight of the food and the weight remaining after food consumption. Manufacturers’ values were used to assess the energy and macronutrient content of the items consumed.

Blood samples were collected via venepuncture from an antecubital or forearm vein at four time points during the main trials as follows: 0 hours, 45 minutes, 1 hour and 45 minutes, 2 hours and 45 minutes. At each blood sampling point 4.9 mL of blood was collected. These samples were used for the measurement of plasma acylated ghrelin. Three 2 mL saliva samples were collected during each trial in sterile tubes using the passive dribble technique at time points 0 hours, 45 minutes and 1 hour and 45 minutes. Levels of progesterone and estradiol were assessed from the saliva samples. Further details about blood treatment are provided in the General Methodology (Section 3.13).

5 - Study 2 ₉₂ 5.2.4 Biochemical analysis

An enzyme immunoassay was used to determine the concentrations of plasma acylated ghrelin. In order to eliminate inter-assay, all samples from the same participant were run on the same plate. The within batch coefficient of variation for the assay was 5.0%. Further details about this test are provided in the General Methodology (Section 3.14.4).

5.2.5 Saliva analysis

Sal -estradiol were measured using commercially

available ELISA kits (Salimetrics, Newmarket, UK). The intra assay co-efficient of variation was 1.7% for progesterone and 2.2% for 17beta-estradiol. See section 3.15.1 in the General Methodology.

5.2.6 Statistical analysis

The data were analysed using Statistical Package for the Social Sciences (SPSS) software, Version 20.0 for Windows. Repeated measures two-factor ANOVA was applied in order to examine differences between the follicular and luteal trials over time in terms of appetite perceptions and acylated ghrelin. The student t-test was used to examine differences between the follicular and luteal trials with regard to baseline acylated ghrelin, baseline appetite scores, AUC values for acylated ghrelin, and AUC values for appetite. Energy and macronutrient intake and exercise responses were also examined by utilizing t-test. The Pearson Product Moment Correlation Coefficient was used to examine relationships between variables.