Mark Wefers Bettink

Chapter 6 108 Materials and Methods Subjects and ethics Data were collected from 52 healthy, nonsmoking, Dutch male volunteers of which 48 participated in an endotoxemia study registered at ClinicalTrials.gov as NCT03240497 and performed at the Radboud university medical center in Nijmegen, the Netherlands. The aim of the endotoxemia study was to investigate the effects of a training program consisting of a breathing exercise and/or exposure to cold on the inflammatory response induced by intravenous administration of LPS. A 2 by 2 design was employed in which 48 participants were randomized to 4 different groups (n=12 per group): cold exposure, breathing exercise, cold exposure and breathing exercise, and no training. All subjects in these 4 groups received LPS. Because especially the vigorous breathing exercise performed during the endotoxemia experiment may interfere with the mitochondrial measurements, only combined baseline mitoPO 2 and mitoVO 2 data (obtained before LPS administration) of the groups that were trained in cold exposure and/or the breathing exercise were used (LPS + intervention group, n=36). The untrained group is henceforth referred to as the LPS group (n=12). Details of the training procedures are described elsewhere [article in revision, attached with current submission]. Following completion of the endotoxemia study, we performed time-control experiments in 4 additional control subjects who received no LPS in a separate protocol (henceforth described as the control group). These experiments were conducted in the Erasmus Medical Center in Rotterdam, the Netherlands and registered at toetsingonline.nl as NL65767.078.18. Measurement timepoints were based on the changes in mitoPO 2 found in the LPS group (Baseline vs T1.45 hours after LPS). Both study protocols received institutional review board approval (CMO 2016-2312/ NL56686.091.16 and MEC 2018-090/NL65767.078.18). All subjects provided written informed consent and experiments were in accordance with the Declaration of Helsinki, including current revisions, and Good Clinical Practice guidelines. Subjects were screened before the start of the experiment and had a normal physical examination, electrocardiography, and routine laboratory values. Exclusion criteria were: febrile illness in the 2 weeks before the experiment, taking any prescription medication, history of spontaneous vagal collapse, participation in a drug trial or donation of blood 3 months prior to the experiment, porphyria, or participation in a previous trial where LPS was administered.