Intermittent Exogenous Ketosis (IEK) at High Altitude

Altitude-related hypoxia decreases human functional capacity, especially during exercise. Even with prolonged acclimatization, the physiological adaptations are insufficient to preserve exercise capacity, especially at higher altitudes completely. Consequently, there has been an ongoing search for various interventions to mitigate the negative effects of hypoxia on human performance and functional capacity. Interestingly, early data in rodents and humans indicate that intermittent exogenous ketosis (IEK) by ketone ester intake improves hypoxic tolerance, i.e.by facilitating muscular and neuronal energy homeostasis and reducing oxidative stress. Furthermore, there is evidence to indicate that hypoxia elevates the contribution of ketone bodies to adenosine-triphosphate (ATP) generation, substituting glucose and becoming a priority fuel for the brain. Nevertheless, it is reasonable to postulate that ketone bodies might also facilitate long-term acclimation to hypoxia by upregulation of both hypoxia-inducible factor-1α and stimulation of erythropoietin production. The present project aims to comprehensively investigate the effects of intermittent exogenous ketosis on physiological, cognitive, and functional responses to acute and sub-acute exposure to altitude/hypoxia during rest, exercise, and sleep in healthy adults. Specifically, we aim to elucidate 1) the effects of acute exogenous ketosis during submaximal and maximal intensity exercise in hypoxia, 2) the effects of exogenous ketosis on sleep architecture and quality in hypoxia, and 3) the effects of exogenous ketosis on hypoxic tolerance and sub-acute high-altitude adaptation. For this purpose, a placebo-controlled clinical trial (CT) in hypobaric hypoxia (real high altitude) corresponding to 3375 m a.s.l. (Rifugio Torino, Courmayeur, Italy) will be performed with healthy individuals to investigate both the functional effects of the tested interventions and elucidate the exact physiological, cellular, and molecular mechanisms involved in acute and chronic adaptation to hypoxia. The generated output will not only provide novel insight into the role of ketone bodies under hypoxic conditions but will also be of applied value for mountaineers and athletes competing at altitude as well as for multiple clinical diseases associated with hypoxia.

Acclimatization, Altitude, Cardiorespiratory, Cerebrovascular, Exercise, Ketone, Acute Mountain Sickness, Sleep, Cognitive

Ketone ester: A total of 300g ketone ester supplementation will be provided in one of the 72h experimental sessions in order to establish intermittent exogenous ketosis. Sucralose (5% w/w) is added to the ketone ester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate Hypobaric hypoxia: 72 hours experimental protocol conducted at terrestrial altitude

Placebo: Water, 5% sucralose (w/w), octaacetate (1 mM) Hypobaric hypoxia: 72 hours experimental protocol conducted at terrestrial altitude

Subjects will breathe 4 min 3% CO2 and 4 min 6% CO2 separated by 4 min of breathing ambient air. The middle cerebral artery will be continuously recorded by transcranial Doppler.

Cognitive function will be assessed by the computerized psychometric test battery: The Psychology Experiment Building Language (PEBL). The following cognitive tests will be used: The color-stroop test (measures attention, processing speed, and inhibitory control; the time it takes to complete the task and the accuracy of the responses; the number of correct and incorrect responses), the digit-span test (measures an individual's working memory capacity and short-term memory; the score of correctly remembered digit span), the ppvt test (measures the reaction time, attention, concentration; the time to react on the visual signal) the fitts test (measures the hand-eye coordination, fine motor skills, concentration; time to position the target) and the timewall test (measures the reasoning, calculating, reaction time, strategy and problem-solving; estimate the time when a moving target will reach a location behind a wall).

On Day 1 at sea-level (in normoxia). On Day 0 and Day 2 (4 hours and 48 hours) after exposure to hypobaric hypoxia, respectively.

Acute Mountain Sickness (AMS) will be assessed by the Lake Louise scale. The symptoms measured on the test include headache, gastrointestinal upset, fatigue/weakness, dizziness/light-headedness, and sleep disturbance. These are rated with an intensity level from 0 (the lowest) to 3 (the highest). A total score that is ≥3, including a headache, is indicative of AMS.

Every day at 9.00 p.m. (before sleep) and at 6.15 a.m. (upon waking) in normoxia and hypobaric hypoxia, respectively.

Change in lung function estimating forced vital capacity (FVC) and forced expiratory volume in 1st second (FEV1).

Heart rate (HR, bpm) will be continuously monitored during different exercise bouts of a variety of intensities (moderate and heavy intensities will be used).

Every day during each 20-90 min long exercise bout performed between 9 a.m. and 6 p.m.. On Day 0 and Day 1 in normoxia. On Day 0, Day 1, Day 2, and Day 3 in hypobaric hypoxia.

Oxygen consumption (VO2, L/min and mL/min/kg) will be continuously monitored during different exercise bouts of variety intensities (moderate and heavy intensities will be used).

Every day during each 20-90 min long exercise bout performed between 9 a.m. and 6 p.m.. On Day 0 and Day 1 in normoxia. On Day 0, Day 1, Day 2, and Day 3 in hypobaric hypoxia.

Muscle oxygenation/deoxygenation will be continuously recorded during each exercise bout by Near Infra-Red Spectroscopy (NIRS) placed on the vastus lateralis. NIRS measure the quantity of oxygenated and deoxygenated haemoglobin and myoglobin (microM) in the investigated areas (vastus lateralis).

Every day during each 20-90 min long exercise bout performed between 9 a.m. and 6 p.m.. On Day 0 and Day 1 in normoxia. On Day 0, Day 1, Day 2, and Day 3 in hypobaric hypoxia.

Brain oxygenation/deoxygenation will be continuously recorded during each exercise bout by Near Infra-Red Spectroscopy (NIRS) placed at the frontal levels. NIRS measure the quantity of oxygenated and deoxygenated haemoglobin (microM) in the investigated areas (prefrontal cortex).

Every day during each 20-90 min long exercise bout performed between 9 a.m. and 6 p.m.. On Day 0 and Day 1 in normoxia. On Day 0, Day 1, Day 2, and Day 3 in hypobaric hypoxia.

Muscle oxygen consumption will be assessed using a previously validated protocol. Briefly, a Near Infra-Red Spectroscopy (NIRS) optode will be placed on the vastus lateralis muscle. Before the protocol, an ischemic calibration will be performed to normalize the NIRS signals by inflating the blood pressure cuff to 250-300 mmHg for a maximum of 5 min. Resting mV#O2 will be assessed from the decrease in muscle oxygenation which accompanies the arterial occlusion.Then, each subject will perform a 3 x 6 minutes moderate-intensity exercise, 8 minutes heavy-intensity exercise and graded exercise test. To measure the recovery of oxygen consumption after exercise, subject will have a series of arterial occlusion as follows: 5 occlusions 5sec on-5sec off, 5 occlusions 5sec on-5sec off, and 5 occlusions 10 sec on-20 sec off.

Every day before each 20-90 min long exercise bout performed between 9 a.m. and 6 p.m.. On Day 0 and Day 1 in normoxia. On Day 0, Day 1, Day 2, and Day 3 in hypobaric hypoxia.

Throughout the entire duration of the night, up to 8 hours after individual bedtime (between 10 p.m. and 6 a.m.). On Day 0 in normoxia. On Day 0 and Day 2 in hypobaric hypoxia.

Blood samples will be collected on Day 1 in normoxia and Day 1, Day 2 and Day 3 in hypobaric hypoxia at 6 a.m. (upon waking).

Saliva samples will be collected on Day 1 in normoxia and Day 1, Day 2 and Day 3 in hypobaric hypoxia at 6 a.m. (upon waking).

Urine samples will be collected on Day 1 in normoxia and Day 1, Day 2 and Day 3 in hypobaric hypoxia at 6 a.m. (upon waking).

At sea level: subjects will breath 6 min normal ambient air (21% O2, 0.03% CO2), 6 hypoxic normocapnic (13.8% O2, 0.03% CO2), and 6 min normoxic hypercapnic (21% O2, 3% CO2) air. At high altitude: subjects will breath 6 min hypobaric hypoxic (21% O2, 0.03% CO2), hypobaric normoxic (32% O2, 0.03% CO2), hypobaric normoxic hypercapnic (32% O2, 3% CO2) air.

Throughout the entire duration of the night, up to 8 hours after individual bedtime (between 10 p.m. and 6 a.m.). On Day 0 in normoxia. On Day 0 and Day 2 in hypobaric hypoxia.

Measured using ELISA of collected nocturnal urine. Subjects empty bladder before sleep and urine will be collected throughout the entire duration of the night, up to 8 hours. Up to 8 hours from 10 p.m. to 6 a.m. on Day 0 in normoxia and Day 0, Day 1 and Day 2 in hypobaric hypoxia.

Inclusion Criteria: Males or females between 18 and 35 years old Body Mass Index (BMI) between 18 and 25 Physically fit and regularly involved in physical activity (2-5 exercise sessions of > 30min per week) Good health status confirmed by a medical screening Non smoking Exclusion Criteria: Any kind of injury/pathology that is a contra-indication for hypoxic exposure and/or to perform high-intensity exercise Intake of any medication or nutritional supplement that is known to affect exercise, performance or sleep Intake of analgesics, anti-inflammatory agents, or supplementary antioxidants, from 2 weeks prior to the start of the study. Recent residence or training under hypoxia; more than 7 days exposure to altitude > 2000m during a period of 3 months preceding the study. Night-shifts or travel across time zones in the month preceding the study Blood donation within 3 months prior to the start of the study Smoking More than 3 alcoholic beverages per day Involvement in elite athletic training at a semi-professional or professional level Any other argument to believe that the subject is unlikely to successfully complete the full study protocol