The effect of a dietary ketone on immunity, tolerability and performance in well-trained, healthy male cyclists.

Pre-experimental tests and time trial familiarisation: Participants will arrive at the laboratory at a time of convenience and having abstained from caffeine, alcohol and strenuous exercise for the previous 24 h. Participants will be required to fast for at least four hours prior to arrival. Participants will be asked to void their bladder before their body mass (shorts only), height and sum of 8 skinfolds are measured by an International Society for the Advancement of Kinanthropometry Level 1 Accredited Anthropometrist. To determine VO2peak, participants complete a continuous incremental cycling protocol originally described by Achten and colleagues (2002) and adapted by Achten and colleagues (2003), requiring participants to start cycling at 95W with increments of 35W every 3 min until either; 1) volitional exhaustion or 2) cadence cannot be maintained >60 RPM. If the participants VO2peak is >60ml/kg/min, they will be deemed eligible for the study. Participants will re-mount the cycle ergometer, which will be switched from hyperbolic mode to linear mode, and precisely on 15 min of rest they will commence a time trial equivalent to 7kJ/kg (~25-35 min). Compared to hyperbolic mode, which allows the cycle ergometer to produce a set power output independent of cadence, the power output in linear mode is cadence (RPM) dependant, with power (W) calculated according to the formula: W = L x (RPM)2 Where RPM is the cadence and L is a (constant) linear factor. L will be calculated to elicit a power output of 70% Wmax at an RPM of 90. Maximal power output (Wmax) will be calculated according to the formula Wmax = Wfinal + (t/T) · Winc Where Wfinal is the power output (W) of the final completed stage, t is the time achieved in the final uncompleted stage (s), T is the duration of each stage (180 s) and Winc is the workload increment (35 W). Pre-experimental, familiarisation and experimental tests / trials will be completed on an electromagnetically braked cycle ergometer (Lode Excalibur Sport, Groningen, Netherlands). The expired gas will be collected and analysed continuously using a computerised metabolic system with mixing chamber (Parvo Medics TrueOne 2400, Salt Lake City, Utah, USA). Ventilatory thresholds (VT1 and VT2) will be estimated during the continuous incremental cycling protocol. VT1 will be inferred by the criteria of the first marked increase in VE/VO2 with no concomitant increase in VE/VCO2. VT2 will be inferred by the criteria of an increase in both VE/VO2 and VE/VCO2 and a decrease in PETCO2. Ratings of perceived exertion (RPE) (Borg scale) will be noted for each stage and heart rate will be continuously measured using short-range telemetry. The work rate equivalent to 85% VT2 will be inferred for use in the experimental procedures for performance trials. Familiarisation trial: Three to seven days after the pre-experimental test, participants will return to the laboratory between 07:00 and 08:00h having fasted from 23:00 h the previous day. Participants will we be asked to consume a high carbohydrate diet (6g/kg) during the previous 24 hours to standardise nutritional status. Participants will be provided with dietary guidelines and asked to record their intake via written and photographic documentation. If certified, participants will be asked to repeat their dietary protocol prior to each subsequent experimental procedure. Participants will also be advised to consume 500 ml of water upon wakening to standardise hydration status. They will then provide a saliva sample (practice sample). Participants will mount the cycle ergometer and cycle for 85 min at the power output equivalent to 85% of their VT2 as determined in the pre-experimental test. Every 20 min, participants will have a ventilatory mask fitted for 3 min for gas analysis. Following completion, they will be allowed 5 min to rest and use the lavatory. Participants will re-mount the cycle ergometer, which will be switched from hyperbolic mode to linear mode, and precisely on 5 min of rest they will commence a time trial equivalent to 7kJk/g (~25-35 min). Participants will be able to consume water ad-libitum throughout the familiarisation test. Participants will then provide a saliva sample (practice sample) following completion, after which, they will be reminded of the requirements of the experimental tests. Experimental procedure: Five to ten days after the familiarisation test, participants will arrive at the laboratory between 06:00 and 08:00 h having fasted from 23:00 h the previous day and refrained from caffeine and alcohol for the previous 24 hours and strenuous exercise for the previous 48 h. Participants will be advised to consume 500 ml of water upon waking to standardise hydration status. Upon arrival at the lab, participants will have a cannula inserted into their antecubital vein and asked to rest quietly for ~10 min prior to initial blood and saliva samples, ketone levels and tolerability scores being collected (baseline measures). Participants will be asked to void their bladder before their body mass (shorts only) measured. Participants will then consume a citrus flavoured drink (2ml/kg) (30 min before commencing exercise). The drink will be formulated to provide 0.35g/kg of 1,3-BD or placebo. Participants will be blinded to the inclusion of 1,3-BD. Twenty-five minutes following the intake of 1,3-BD and immediately prior to commencing exercise, blood and saliva samples, blood ketone, lactate and glucose levels will be collected. Participants will then mount the cycle ergometer and cycle 85 min 85% VT2 power output. Every 20 min, participants will have a ventilatory mask fitted for 3 min for gas analysis and RPE will be recorded. Every 30 min, blood ketones and glucose levels will be measured (capillary finger prick). After 60 min of cycling (i.e. 90 minutes following the initial intake of the citrus flavoured drink), participants will either consume an additional 2mlkg-1 citrus flavoured drink containing 0.35gkg-1 of 1,3-BD or placebo. After 85 min of cycling, participants will be allowed 5 min to rest. During this time, blood samples, blood ketone, lactate and glucose levels will be collected. Precisely on 5 min, participants will re-mount the cycle ergometer, which will be switched from hyperbolic mode to linear mode, and they will complete a TT equivalent to 7kJ/kg (~25 – 35 min) from a stationary start. Participants will be restricted to 2mlkg-1 every 15 min during exercise. Before leaving the cycle ergometer, blood samples, blood ketone, lactate and glucose levels will be collected. Immediately afterwards, saliva samples, and tolerability scores will be collected. Participants will then remove wet clothing, towel dry themselves and have their body mass measured (shorts only) to estimate fluid loss. Participants will then be provided with 5ml/kg of water and asked to rest until 1 h post time trial when blood and saliva samples, blood ketone and glucose and tolerability scores will be collected. Following completion of the trial, participants will be provided with 400 – 600 ml of a carbohydrate drink and asked to return to the laboratory 5 – 10 days later to repeat the protocol with the alternative dietary intervention (i.e. placebo or 1,3-BD). Safety monitoring: Participant tolerability, side-effects and abnormal haematological values will be immediately reported to a medical doctor. This study will be immediately terminated when advised by the medical doctor or following intractable symptoms after 1,3-BD consumption in 2 trial arms (i.e. 10% of trials). Participants will be followed up 3 days and 1 week after the consumption of 1,3-BD to ascertain if any adverse effects occurred. Participants will also be encouraged to report any symptoms following consumption of 1,3-BD by email, phone or text. Common to Part A and B Capillary blood samples for ketone, gucose and lactate monitoring: Samples will be collected from participants’ fingers using standardised techniques. The site of puncture will be cleaned with an alcohol swab then allowed to air dry. Skin puncture will occur in one quick, deliberate strike of the lancet. The first drop of blood will be wiped away, then subsequent accumulation of blood will be used in analysis. Afterwards, pressure and/or a plaster will be applied to stop further bleeding. Blood beta-hydroxybutyrate (ketone) and glucose will be measured using Freestyle Optium Neo (Abbott Diabetes Care, Victoria, Australia) and blood lactate will be measured using the Lactate 2 Pro (Akray, Japan). Tolerability questionnaire: The questionnaire is organised in three sections as previously described by Pfeiffer et al. (2009) and adapted to include additional symptoms. Section 1 addresses upper abdominal problems; Section 2, lower abdominal problems; and Section 3, systemic problems. Each participant will be assessed on the 29 items on a 10-point scale ranging from 0, no problem at all, to 9, the worst it has ever been. Salivary sampling and analysis: Unstimulated saliva samples will be collected. Participants will be instructed to be in a seated position, leaning forward with their head tilted down and asked to empty their mouth of any residual saliva before the timed sample collection begins. Each saliva collection will last 3 min, during which time participants will be requested to minimise orofacial movement and passively dribble into a pre-weighed vial. Samples will be weighed to estimate saliva volume (the density will be assumed to be 1.0g/ml and micro-centrifuged to remove cells and insoluble matter before storing at -20oC for later analysis. The saliva flow rate (ml/min) will be determined by dividing the volume of saliva by collection time. Saliva samples will be analysed for salivary immunoglobulin A using a commercially available ELISA kit (DRG, Germany). Blood sampling: All blood samples will be collected via cannulation of the antecubital vein into three evacuated vacutainers, two containing tripotassium ethylene diamine tetraacetic acid (K3EDTA) and one containing no additive to obtain serum. All vacutainers will be centrifuged for 10 min at 1500 x g and 4oC. Plasma and serum will be aliquoted and stored at -80C for later analysis. Blood Analysis Blood cell counts: Blood collected into the K3EDTA vacutainer will be used to analyse for total and differential leukocyte counts, haematocrit and haemoglobin contents using a haematology analyser (AC.TTM 5diff analyser, Beckman Coulter, UK). All cell counts will be adjusted for plasma volume changes, with changes being estimated from haemoglobin and haematocrit values according to Dill & Costill (1974). Whole blood culture: 2 ml of whole blood obtained from an K3EDTA vacutainer will be added to 2 ml of RPMI medium with no added stimulant (unstimulated) or stimulant (stimulated) at a concentration designed to elicit maximal lymphocyte response (final stimulant concentration will be determined with prior titration work). The stimulant that will be used will be Staphylococcal enterotoxin B from Staphylococcus aureus. The whole blood cultures will then be incubated for 24 h at 37 °C, 5 % CO2 (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Unstimulated and antigen-stimulated lymphocyte subset cell counts and activation: Whole blood cultures will be used to analyse lymphocyte cell subsets and their activation (CD69+) according to their cell surface markers CD4+, CD8+ CD25+ (T cells) via the Muse (Millipore, Darmstadt, Germany). Unstimulated and antigen-stimulated whole blood cytokine production: Blood cultures will be centrifuged for 4 min at 13,000g in a microcentrifuge. Supernatants will be collected and stored frozen at -80 °C until analysis. Cytokines interleukin (IL)-1, IL-1ra, IL-2, IL-4, IL-8, IL-10, interferon (IFN)-? and tumour necrosis factor (TNF)-a) will be determined using a multiplex kit (Millipore, NZ) on the MAGPIX. Unstimulated and antigen-stimulated peripheral blood mononuclear cells IFN- ?, IL-4 and IL-10 gene expression: A gene expression assay will be used to measure the expression of genes coding for IFN-y, IL-4 and IL-10 in peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMC) will be isolated from the whole blood culture and total RNA will be extracted. PBMC RNA will be treated with DNase to remove contaminated genomic DNA.  Purified RNA will be reversed transcribed by a one-step real time PCR assay (Roche Light Cycler instruments (2.0 & 480)) to generate cDNA and specific PCR products for IFN-y, IL-4 and IL-10 genes. In addition to these genes, a reference gene will be used to normalize results. Plasma cortisol: Plasma concentrations of cortisol will be measured using ELISA kits (DRG, Germany). Blood glucose: Blood glucose will be measured using the COBAS analyser. Substrate utilisation calculations: The application of indirect calorimetry (stoichiometry) will be used to estimate in vivo substrate utilisation (respiratory quotient (RQ)) from gaseous exchange measurements (respiratory exchange ratio (RER)). The stoichiometric equations (see below) originally published by Frayn and colleagues (1983), which assumes the contribution of protein is negligible, has been validated at exercise intensities up to 80-85% VO2max where a steady state has been achieved. CHO oxidation (g/min) = 4.585 VCO2 – 3.226 VO2 Fat oxidation (g/min) = 1.695 VO2 – 1.701 VCO2