Oral swab

Samples were obtained with an oral swab (Sarstedt, Newton, NC) placed under the tongue for 120 sec, to minimize variations in volume and analytes. Adolescents provided four samples at −80, 0, 30, and 50 minutes relative to film administration. Cortisol was measured using an enzyme immunoassay (Siemens Immulite 1000; sensitivity 60 ng/dL, intra- and inter-assay CVs 5.8-11.2%). Salivary cortisol reflects circulating free plasma cortisol (physiologically active cortisol). Cortisol area under the curve with respect to ground (AUC_g) and to increase (AUC_i) were calculated using formulas by Pruessner et al.^{17 (link)} Because cortisol was collected during a laboratory stressor and meal paradigm, cortisol output was assumed to reflect a combination of cortisol response to food intake and the film.

A standardized laboratory cold-pressor stress test (CPT) paradigm, widely used in youth (39 (link)), evaluated adolescents’ cortisol reactivity. At approximately 3:00 pm, adolescents were seated in a quiet room and instructed to relax and engage in restful activities. At 4:00 pm, the cold-pressor stress test was administered. A bath of ice water was maintained at a consistent temperature of 10°C monitored by a thermometer. Each participant was instructed to keep her arm in cold water (−10°C) for as long as possible. Participants were instructed to remove their hand at any time if it became too uncomfortable for her. Salivary samples to measure cortisol were obtained with an oral swab (Sarstedt, Newton, North Carolina) placed under the tongue for 120 seconds immediately before and just after CPT and at 20, 40 and 60 minutes after the test. Intra-individual peak cortisol reactivity was assessed as the highest cortisol measurement 20–60 minutes after the test. Cortisol was measured using an enzyme immunoassay (Siemens Immulite 1000; sensitivity=60 ng/dL, intra- and inter-assay coefficients of variability=5.8–11.2%).

Post-awakening cortisol and diurnal cortisol rhythm are assessed with salivary samples collected in the participant’s home environment on one weekday and one weekend day overlapping with the same at-home collection period of devices to measures activity and sleep. Samples are obtained with an oral swab (Sarstedt, Newton, NC) placed under the tongue for 120 seconds upon awakening, 15, 30, and 45 minutes after awakening, 4:00 pm, and bedtime. Participants are instructed to refrain from eating, drinking (other than water), or brushing their teeth until after completion of the 45-minute post-wake sample and for 15-minutes prior to afternoon and bedtime samples. Completion of a participant log is used to encourage adherence, and we also use screw-top bottles that automatically, electronically record opening times to improve accuracy of collection timing, given its high importance. Participants are also reimbursed $5 per day for completion of samples. Salivary cortisol is a well-accepted non-invasive indicator of circulating physiologically active free plasma cortisol [73 (link)]. Latent growth models will be used to express non-linear change in cortisol across two days as awakening response and diurnal rhythm.