Strategies to Augment Ketosis: Optimization of Ketone Delivery Strategies

One important difference between KE compounds is the ketone-promoting components, which determines the circulating ratio of blood ketone bodies, BHB and AcAc, and may in turn lead to important metabolic and signaling differences. Whereas some actions of the ketone bodies BHB and AcAc are shared, R-BHB has a broad range of signaling functions that are distinct from AcAc, some of which are shared by the non-circulating, non-oxidizable enantiomer, S-BHB. AcAc also has metabolic and signaling actions that are independent of BHB and is selectively oxidized in some cells that cannot oxidize BHB. Furthermore, responses to different ketone bodies vary between tissue types. A second difference between KE arises from the balance between direct delivery of ketones compared to indirectly elevating ketone concentration via metabolism of non-classical or classical ketogenic precursors. Classical ketogenesis itself may drive adaptation and some of the functional benefits associated with ketosis. BDO is included in all of the KE compounds, but it is currently unknown how consumption of BDO alone, and its metabolism via non-classical ketogenesis acutely affects metabolism. Additionally, ketogenesis is now understood to occur in certain cells outside the liver with important local biological effects, for example ketogenesis driven by medium chain fatty acids has been reported in astrocytes in vitro. Provision of systemic BHB by a KE may elicit different biological effects in some tissues such as the brain versus promoting in situ ketogenesis in that tissue. Overall, not only are functional effects of KE incompletely defined, but also it is unknown which effects are common to all KE versus which are specific to an individual KE compound (i.e., BHB Monoester vs AcAc Diester) or which may be attributable to the BDO precursor common to all of the KE. This study will be the first comparative full crossover study of all available KE and the precursor BDO at two serving sizes. Outcomes will focus on established effects of the BHB Monoester (including the effects on ketones, glucose and acid-base balance) and compare these with the effects of the AcAc Diester, C8 Ketonef Diester and BDO.

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.