Strategies to Augment Ketosis: Optimization of Ketone Delivery Strategies

BHB Monoester, C8 Diester, and (R) 1,3 Butanediol are commercially available in the products 'deltaG' (TdeltaS Global, FL, USA), 'Cognitive Switch' (Juvenescence Ltd, NJ, USA) and 'Avela' (Genomatica, CA, USA), respectively. The AcAc Diester is commercially available and is currently being used in clinical studies of Angelman's Disease. Therefore, we will standardize KE dosing to LBM (assessed using DXA) for all trials at 180 and 360 mg/kg LBM, which for a participant with 70 kg of lean mass corresponds to ~12.5 and 25 g, respectively. These doses are representative of typical commercial serving sizes and are expected to elevate blood BHB in the range of 1.5 - 2 mM39,61. All KEs will be consumed in their finished commercial form. As the KEs have different delivery matrices and flavorings (water-based beverage, emulsion beverage, gel capsules), we will not blind participants to each condition as this will not alter the metabolic outcomes of interest. Screening Visit: Participants that meet the initial qualifying criteria will visit the study center for a screening meeting in a private office to discuss the informed consent form with research team. The informed consent form will be provided to the participant for their review, the study will be described in full detail and any questions the interested participant has will be encouraged and responded to. The participant will be informed that even if they have signed the consent form, their participation in the study is dependent on anthropometric measures and diet and medical questionnaire answers to determine if they meet the study criteria. If the participant provides consent, they will be provided with questionnaires including Automated Self-administered 24-hour Dietary Assessment Tool (ASA24®),and medical history. All collected samples and data will be coded to maintain participant anonymity. We will give the participants a small volume of Study Product to screen for tolerance of the bitter tasting Study Products. We will also measure height, weight, BMI and body composition using a DXA scanner. If the participant is eligible for the study and is still interested in participating then they will be randomized to a study product order and scheduled to return to the study center for the first testing visit. Eligible participants will report to the study center in compliance with pre-test instructions (fasted > 10h, no alcohol >24h, no exercise >24h, consumed pre-test food). Upon testing day, participants will complete a baseline Beverage Tolerability Questionnaire (BTQ) and satiety visual analogue scale. Participants will be asked to completely void their bladder and a sample will be analyzed for hydration status. Bluetooth heart rate monitor chest strap will be administered. A study team member will assist the participant with application of a continuous ketone meter into the back of the arm, this will be removed and replaced with a fresh sensor at Test Days at ~2-week intervals; the sensor will be removed 24 hours after the cessation of the last in lab testing bout. Participants will be given written instructions on how to remove and dispose of monitor. A trained member of the study team will insert an IV cannula into a vein in the antecubital fossa to allow for repeated blood sampling. The cannula will be flushed with a small volume of saline after each sample withdrawal to maintain patency. We will collect 56mL of blood (7 x 8mL tubes) per testing day, which will be about 504mL of blood for the entire study. This is about 2.1 cups of blood. We will also collect capillary blood samples from a finger for real-time analysis of blood BHB and glucose concentration, using lancing device, commercially available test strips and a handheld monitor (KetoMojo, CA, USA). Participants will wear a fitted facemask connected to a metabolic cart for 10 minutes to collect measures of respiratory gas exchange. Participants will exhale once into a commercially available handheld breath acetone analyzer (Readout, MI, USA). Baseline blood sample, baseline respiratory gas measures and baseline breath acetone will be collected. Participants will then consume the Study Product that they were randomly allocated for that Test Day (details of Study Products attached in other files). Time of ingestion should be +/- 60 minutes from the time established at Test Day 1; they will be given 5 minutes to consume the Product. After Study Product consumption, they will remain at the study center for ~4 hours, repeating the aforementioned tests at 30min, 60 min, 90min, 180min & 240min. Participants will be asked to minimize ambulatory movement during the Test Day. Non caloric beverages (i.e.,water) will be permitted ad libitum and intake volumes will be recorded. At the end of each Test Day, the heart rate monitor and IV cannula will be removed and a dressing will be applied to the cannula site. Participants will be given a snack to consume before leaving the site.

Inclusion criteria

• Male
• BMI between 18 and 29 kg/m2
• Aged 20 - 30 years
• Participant is willing and able to comply with all study procedures including the following prior to Test Days: fasting (>10 h; water only), no alcohol (>24 h), no exercise (>24 h), no acute illness and controlled feeding before each Test Day, maintain diet, exercise, medication, and supplement habits throughout the study.
• Participant has no health conditions that would prevent completion of the study requirements as judged by the Investigator based on health history.
• Participant understands the study procedures and signs forms providing informed consent to participate in the study and authorizes the release of relevant protected health information to the Investigator.

Exclusion criteria

• Participant follows a low-carbohydrate diet (<30% energy from carbohydrate) or have used exogenous ketone supplements within 4-months of study participation.
• Participant has a Primary Care Physician diagnosed history or presence of uncontrolled and/or clinically important hypertension (blood pressure >150/95 mmHg), pulmonary, cardiac, hepatic, renal, endocrine (including type 1 and 2 diabetes), hematologic, immunologic, neurologic (e.g., Alzheimer's or Parkinson's diseases), psychiatric (including unstable depression and/or anxiety disorders) or biliary disorders.
• Participant has a known allergy, intolerance, or sensitivity to any of the ingredients in the study beverages, including soy and milk protein, wheat, shellfish, fin fish, eggs, tree nuts or peanuts (production facility handles nuts).
• Participant has unstable use of a medication or supplement that the Investigator considers may affect the outcomes of the trial.
• Consumption of alcohol more than 3 drinks per day or more than 18 drinks per week.
• Consumption of tobacco.
• Consumption of cannabis.
• Participant is currently in another research study or has been in the 14 days before screening.
• Participant has had a blood draw or donation in the last 8 weeks.
• Participant has a clinically important gastrointestinal (GI) condition that would potentially interfere with the evaluation of the study beverage [e.g., inflammatory bowel disease, irritable bowel syndrome, chronic constipation, severe constipation (in the opinion of the Investigator), history of frequent diarrhea, history of surgery for weight loss, gastroparesis, systemic disease that might affect gut motility according to the Investigator, medication managed reflux and/or clinically important lactose intolerance].
• Participant has a condition the Investigator believes would interfere with his ability to provide informed consent, comply with the study protocol, which might confound the interpretation of the study results, or put the participant at undue risk.