ABSTRACTCircadian clocks in the body drive daily cycles in physiology and behavior. A master clock in the brain maintains synchrony with the environmental day–night cycle and uses internal signals to keep clocks in other tissues aligned. Work in cell cultures uncovered cyclic changes in tissue oxygenation that may serve to reset and synchronize circadian clocks. Here we show in healthy humans, following a randomized controlled single‐blind counterbalanced crossover study design, that one‐time exposure to moderate ambient hypoxia (FiO2 ~15%, normobaric) for ~6.5 h during the early night advances the dim‐light onset of melatonin secretion by 9 min (95% CI: 1–16 min). Exposure to moderate hypoxia may thus be strong enough to entrain circadian clocks to a 24‐h cycle in the absence of other entraining cues. Together, the results provide direct evidence for an interaction between the body's hypoxia‐sensing pathway and circadian clocks. The finding offers a mechanism through which behaviors that change tissue oxygenation (e.g., exercise and fasting/eating) can affect circadian timing and through which hypoxia‐related diseases (e.g., obstructive sleep apnea and chronic obstructive pulmonary disease) can result in circadian misalignment and associated pathologies.Trial Registration: Registration number: DRKS00023387; German Clinical Trials Register: http://www.drks.de

Aerospace medicine required controlled terrestrial models to investigate influences of altered atmosphere conditions, such as hypoxia, on human health and performance. These models could potentially be expanded to encompass disease conditions or treatment targets regulated through hypoxia or hypercapnia. Hypoxia, a condition in which the body is deprived of adequate oxygen supply, profoundly affects human physiology at multiple levels and contributes to the pathogenesis of various diseases. Experimental exposure to hypoxic conditions has gained recognition as a model for studying diseases such as pulmonary hypertension, chronic obstructive pulmonary disease, obstructive sleep apnoea, migraine and kidney disease. This approach may be particularly useful in mechanism-oriented early-stage clinical studies. This review discusses the ability of hypoxia models from space medicine research to mimic or induce these conditions in a controlled laboratory setting as a tool for testing the efficacy and safety of new pharmaceutical interventions.

Introduction:The ability to metabolize fructose to bypass the glucose pathway in near-anaerobic conditions appears to contribute to the extreme hypoxia tolerance of the naked-mole rats. Therefore, we hypothesized that exogenous fructose could improve endurance capacity and cognitive performance in humans exposed to hypoxia.Methods:In a randomized, double-blind, crossover study, 26 healthy adults (9 women, 17 men; 28.8 ± 8.1 (SD) years) ingested 75 g fructose, 82.5 g glucose, or placebo during acute hypoxia exposure (13% oxygen in a normobaric hypoxia chamber, corresponding to oxygen partial pressure at altitude of ~3,800 m) on separate days. We measured exercise duration, heart rate, SpO2, blood gasses, and perceived exertion during a 30-min incremental load test followed by Farnsworth-Munsell 100 Hue (FM-100) color vision testing and the unstable tracking task (UTT) to probe eye-hand coordination performance.Results:Exercise duration in hypoxia was 21.13 ± 0.29 (SEM) min on fructose, 21.35 ± 0.29 min on glucose, and 21.35 ± 0.29 min on placebo (p = 0.86). Heart rate responses and perceived exertion did not differ between treatments. Total error score (TES) during the FM-100 was 47.1 ± 8.0 on fructose, 45.6 ± 7.6 on glucose and 53.3 ± 9.6 on placebo (p = 0.35) and root mean square error (RMSE) during the UTT was 15.1 ± 1.0, 15.1 ± 1.0 and 15.3 ± 0.9 (p = 0.87).Discussion:We conclude that oral fructose intake in non-acclimatized healthy humans does not acutely improve exercise performance and cognitive performance during moderate hypoxia. Thus, hypoxia tolerance in naked mole-rats resulting from oxygen-conserving fructose utilization, cannot be easily reproduced in humans.

Acute exposure to hypoxia can lead to cognitive impairment. Therefore, hypoxia may become a safety concern for occupational or recreational settings at altitude. Cognitive tests are used as a tool to assess the degree to which hypoxia affects cognitive performance. However, so many different cognitive tests are used that comparing studies is challenging. This structured literature evaluation provides an overview of the different cognitive tests used to assess the effects of acute hypoxia on cognitive performance in healthy volunteers. Less frequently used similar cognitive tests were clustered and classified into domains. Subsequently, the different cognitive test clusters were compared for sensitivity to different levels of oxygen saturation. A total of 38 articles complied with the selection criteria, covering 86 different cognitive tests. The tests and clusters showed that the most consistent effects of acute hypoxia were found with the Stroop test (where 42% of studies demonstrated significant abnormalities). The most sensitive clusters were auditory/verbal memory: delayed recognition (83%); evoked potentials (60%); visual/spatial delayed recognition (50%); and sustained attention (47%). Attention tasks were not particularly sensitive to acute hypoxia (impairments in 0%–47% of studies). A significant hypoxia level-response relationship was found for the Stroop test (p = 0.001), as well as three clusters in the executive domain: inhibition (p = 0.034), reasoning/association (p = 0.019), and working memory (p = 0.024). This relationship shows a higher test sensitivity at more severe levels of hypoxia, predominantly below 80% saturation. No significant influence of barometric pressure could be identified in the limited number of studies where this was varied. This review suggests that complex and executive functions are particularly sensitive to hypoxia. Moreover, this literature evaluation provides the first step towards standardization of cognitive testing, which is crucial for a better understanding of the effects of acute hypoxia on cognition.

Space medicine has developed controlled terrestrial models to investigate the impacts on human health and performance, and their application should be expanded to encompass disease conditions involving hypoxia and other factors, in order to make valuable contributions to clinical drug development. Hypoxia, a condition in which the body is deprived of adequate oxygen supply, profoundly affects human physiology at multiple levels and contributes to the pathogenesis of various diseases. Experimental exposure to hypoxic conditions has gained recognition as a valuable model for studying diseases like pulmonary hypertension, chronic obstructive pulmonary disease (COPD), obstructive sleep apnea (OSA), migraine, and kidney disease. The approach may be particularly useful in mechanism-oriented early-stage clinical studies. This review will discuss the ability to mimic or induce these conditions in a controlled laboratory setting using hypoxia, making it a valuable tool for testing the efficacy and safety of new pharmaceutical interventions.

Recombinant human erythropoietin (rHuEPO) is used as doping a substance. Anti-doping efforts include urine and blood testing and monitoring the athlete biological passport (ABP). As data on the performance of these methods are incomplete, this study aimed to evaluate the performance of two common urine assays and the ABP. In a randomized, double-blinded, placebo-controlled trial, 48 trained cyclists received a mean dose of 6000 IU rHuEPO (epoetin β) or placebo by weekly injection for eight weeks. Seven timed urine and blood samples were collected per subject. Urine samples were analyzed by sarcosyl-PAGE and isoelectric focusing methods in the accredited DoCoLab in Ghent. A selection of samples, including any with false presumptive findings, underwent a second sarcosyl-PAGE confirmation analysis. Hematological parameters were used to construct a module similar to the ABP and analyzed by two evaluators from an Athlete Passport Management Unit. Sensitivity of the sarcosyl-PAGE and isoelectric focusing assays for the detection of erythropoietin abuse were 63.8% and 58.6%, respectively, with a false presumptive finding rate of 4.3% and 6%. None of the false presumptive findings tested positive in the confirmation analysis. Sensitivity was highest between 2 and 6 days after dosing, and dropped rapidly outside this window. Sensitivity of the ABP was 91.3%. Specificity of the urine assays was high; however, the detection window of rHuEPO was narrow, leading to questionable sensitivity. The ABP, integrating longitudinal data, is more sensitive, but there are still subjects that evade detection. Combining these methods might improve performance, but will not resolve all observed shortcomings.

Recombinant human erythropoietin (rHuEPO) has been used as a performance-enhancing agent by athletes in a variety of sports. The resulting increase in hematocrit levels leads to increased blood viscosity and can affect blood flow, potentially increasing the athlete's risk of developing health complications. However, the actual effects of using rHuEPO on microvascular blood flow and post-occlusive reactive hyperemia are currently unknown. We therefore evaluated the effect of rHuEPO on the cutaneous microcirculation in well-trained cyclists using laser speckle contrast imaging (LSCI). This study was part of a randomized, double-blind, placebo-controlled, parallel trial designed to investigate the effects of rHuEPO in 47 well-trained adult cyclists (age 18-50 years). Subjects received a weekly dose of either rHuEPO or placebo for 8 weeks, and LSCI was performed at baseline, after a maximal exercise test in week 6, and before maximal exercise in week 8. Endpoints included basal blood flux, maximum post-occlusion reperfiision, and time to return to baseline. Despite an increase in hematocrit levels in the rHuEPO-treated group, we found no statistically significant difference in microvascular function measured between the rHuEPO-treated group and the placebo group. Our results suggest that the increased hematocrit levels in rHuEPO-treated well-trained cyclists are not associated with changes in microvascular blood flow or post-occlusive re-active hyperemia measured using LSCI.

BACKGROUND:Substances that potentially enhance performance (eg, recombinant human erythropoietin [rHuEPO]) are considered doping and are therefore forbidden in sports; however, the scientific evidence behind doping is frequently weak. We aimed to determine the effects of rHuEPO treatment in well trained cyclists on maximal, submaximal, and race performance and on safety, and to present a model clinical study for doping research on other substances. METHODS:We did this double-blind, randomised, placebo-controlled trial at the Centre for Human Drug Research in Leiden (Netherlands). We enrolled healthy, well trained but non-professional male cyclists aged 18-50 years and randomly allocated (1:1) them to receive abdominal subcutaneous injections of rHuEPO (epoetin β; mean dose 6000 IU per week) or placebo (0·9% NaCl) for 8 weeks. Randomisation was stratified by age groups (18-34 years and 35-50 years), with a code generated by a statistician who was not masked to the study. The primary outcome was exercise performance, measured as maximal power output (Pmax), maximal oxygen consumption VO2 max, and gross efficiency in maximal exercise tests with 25 W increments per 5 min, as lactate threshold and ventilatory threshold 1 (VT1) and 2 (VT2) at submaximal levels during the maximal exercise test, and as mean power, VO2, and heart rate in the submaximal exercise tests at the highest mean power output for 45 min in a laboratory setting and in a race to the Mont Ventoux (France) summit, using intention-to-treat analyses. The trial is registered with the Dutch Trial Registry (Nederlands Trial Register), number NTR5643. FINDINGS:Between March 7, 2016, and April 13, 2016, we randomly assigned 48 participants to the rHuEPO group (n=24) or the placebo group (n=24). Mean haemoglobin concentration (9·6 mmol/L vs 9·0 mmol/L [estimated difference 0·6, 95% CI 0·4 to 0·8]) and maximal power output (351·55 W vs 341·23 W [10·32, 3·47 to 17·17]), and VO2 max (60·121 mL/min per kg vs 57·415 mL/min per kg [2·707, 0·911 to 4·503]) in a maximal exercise test were higher in the rHuEPO group compared with the placebo group. Submaximal exercise test parameters mean power output (283·18 W vs 277·28 W [5·90, -0·87 to 12·67]) and VO2 (50·288 mL/min per kg vs 49·642 mL/min per kg [0·646, -1·307 to 2·600]) at day 46, and Mont Ventoux race times (1 h 40 min 32 s vs 1 h 40 min 15 s [0·3%, -8·3 to 9·6]) did not differ between groups. All adverse events were grade 1-2 and were similar between both groups. No events of grade 3 or worse were observed. INTERPRETATION:Although rHuEPO treatment improved a laboratory test of maximal exercise, the more clinically relevant submaximal exercise test performance and road race performance were not affected. This study shows that clinical studies with doping substances can be done adequately and safely and are relevant in determining effects of alleged performance-enhancing drugs. FUNDING:Centre for Human Drug Research, Leiden.

The stability of colistin methanesulfonate (CMS) was determined in quadruplicate in elastomeric home infusion pumps containing 1, 2 or 3 MU CMS and in infusion bags with 2 MU CMS all in 100 mL normal saline. Infusions were stored at room temperature (20 degrees C-24 degrees C) with or without exposure to natural light or refrigerated (4 degrees C-8 degrees C) and protected from light up to 2 weeks. In the initial solution of 2 MU CMS in 100 mL saline sampled immediately after reconstitution and dilution, 1.5% of CMS was hydrolysed to colistin. When stored at room temperature and exposed to natural light, colistin concentration in elastomeric infusion pumps increased to 2.6% in 8 days and to 2.1% when stored at 4 degrees C. CMS stability increases at lower temperatures and higher concentrations. Based on the current data, chemical stability of CMS infusion solution is sufficient for a shelf life of 7 days refrigerated plus 1 day at room temperature.