Complementary Therapies in Medicine br obtain informed conse
Complementary Therapies in Medicine 42 (2019) 12–18
obtain informed consent, provide instructions on the collection of saliva samples, and supply participants with labeled salivettes. Participants were asked to collect five saliva samples on two consecutive days at the following times: at awakening, 30 min after waking, 12 PM, 4 PM, and 9 PM. Commensurate with recommended protocols,42 participants were instructed to rinse their mouth 10 min before saliva collection in order to avoid sample dilution, to place the salivette directly under their tongue for 3 min, and to store the salivettes in the refrigerator until delivery to the laboratory for the second visit (approximately 1 week after their first visit). Further, participants were asked to refrain from smoking and drinking alcohol 24 h prior to saliva collection, as well as avoid caﬀeine products and exercising 1 h before saliva collection. Upon arrival at the Laboratory, the saliva samples were transferred to Eppendorf tubes and stored in a freezer at −80 °C until processed for cortisol.
The second visit lasted approximately 2 h and served to assess participants’ reactive Cucurbitacin I responses. During this visit, participants were subjected to the Trier Social Stress Test (TSST; see description below)43 at which time seven saliva samples were collected. The first sample was collected upon arrival at the laboratory (“arrival” sample), the second and third during the TSST (i.e., “anticipation” sample which occurred between the speech preparation and the speech delivery; “arithmetic” sample which occurred upon completion of the arithmetic task). After completing the TSST, participants were taken to a quiet room for 1 h to complete a questionnaire assessing sociodemographic characteristics and aerobic PA. Four additional saliva samples were collected during this time: 10, 20, 40, and 60 min after the TSST. Second visits were conducted between 3 PM and 5 PM in order to co-incide with the time at which cortisol levels are near their lowest and most stable daytime values.44
Cortisol was assessed using commercially available highly-sensitive enzyme-linked immunosorbent assay (ELISA). The assay kits and the protocols was provided by Salimetrics, State College, PA, USA.42 For diurnal cortisol, cortisol concentrations were assessed on two con-secutive days and the means of each of the five time points were sub-sequently computed for each participant. For reactive cortisol, cortisol concentrations were assessed before, during, and after the TSST as described previously. The TSST is a two-task protocol that has been designed to induce a physiological stress response.45,46 A detailed overview of the TSST protocol has previously been published.43 Briefly, the task consists of a 5-min mock interview and a 5-min arithmetic subtraction task in front of three confederate evaluator judges. Parti-cipants were told that the session would be video-taped for later review. The TSST has successfully been shown to induce subjective stress, cardiovascular changes, and endocrine responses.45,46
A single item was used to assess participants’ engagement in aerobic PA. Participants reported how often they participated in cardiovascular activities per week. Cardiovascular activity was described to partici-pants as any exercise, regardless of its intensity, that raises heart rate. Response options were: once per week or less, 2–3 times per week, 4–5 times per week, and 6–7 times per week, and more than 7 times per week. Responses were then dichotomized into ‘no/low PA’ and ‘moderate/ high PA’ levels based upon two diﬀerent methods. For the first di-chotomization (PA1), participants who reported engaging in aerobic PA once per week or less were placed in the ‘no/low PA’ group and all other participants in the ‘moderate/high PA’ group. For the second dichotomization (PA2), participants who reported engaging in aerobic PA three times per week or less were placed in the ‘no/low PA’ group and other participants in the ‘moderate/high PA’ group. We divided PA frequency both ways to determine if the groups would be distinguished
M. Lambert et al.
by diﬀerence in PA frequency, i.e., none or almost none vs. irregular PA (PA1) and none, almost none, or irregular vs. regular PA (PA2). The sample sizes were insuﬃcient to analyze the data based on a tertiary scheme reflecting low, moderate, and high PA frequencies.
2.4. Data preparation and analyses
Once collected from participants, the saliva samples were trans-ferred to Eppendorf tubes and stored in a −80 °C freezer; they were processed in duplicate within 3 months at the University of Ottawa Stress, Immunocompetence, and Health Laboratory using commercially available highly-sensitive enzyme linked immunosorbent assay kits and the protocol designed by Salimetrics, State College, PA, USA. For diurnal cortisol, the means and standard deviations were computed for samples taken at the same time across both days, yielding five scores in mol/L per participant, which were then used to determine the area under the curve to increase (AUCi) using trapezoidal integration methods47 to represent participants’ overall daily cortisol secretion level. For reactive cortisol, the seven saliva samples collected during the TSST protocol were used to compute the AUCi representing partici-pants’ individual overall cortisol response to stress.