Edited by: Jonathan Cedernaes, Uppsala University, Sweden
Reviewed by: Jose E. Galgani, Pontifical Catholic University of Chile, Chile; Todd Hagobian, California Polytechnic State University, United States; David M. Diamond, University of South Florida, United States
This article was submitted to Nutrition and Metabolism, a section of the journal Frontiers in Nutrition
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There is growing interest in the metabolism of ketones owing to their reported benefits in neurological and more recently in cardiovascular and renal diseases. As an alternative to a very high fat ketogenic diet, ketones precursors for oral intake are being developed to achieve ketosis without the need for dietary carbohydrate restriction. Here we report that an oral D-beta-hydroxybutyrate (D-BHB) supplement is rapidly absorbed and metabolized in humans and increases blood ketones to millimolar levels. At the same dose, D-BHB is significantly more ketogenic and provides fewer calories than a racemic mixture of BHB or medium chain triglyceride. In a whole body ketone positron emission tomography pilot study, we observed that after D-BHB consumption, the ketone tracer 11C-acetoacetate is rapidly metabolized, mostly by the heart and the kidneys. Beyond brain energy rescue, this opens additional opportunities for therapeutic exploration of D-BHB supplements as a “super fuel” in cardiac and chronic kidney diseases.
The ketogenic diet is a very low carbohydrate diet that has shown therapeutic benefits in drug resistant epilepsy (
As an alternative to the ketogenic diet, exogenous ketone precursors taken orally achieve mild ketosis in the absence of dietary restriction. They can be grouped in three categories (
Exogenous production of blood ketones by three ketone precursors–MCT, KE, and D-BHB.
Once ketone precursors are absorbed and metabolized, the resulting ketones are taken up by extrahepatic tissues such as brain, heart, muscle, and kidney and metabolized to acetyl-CoA for ATP production in mitochondria (
Positron-emission tomography (PET) using the ketone tracer, 11C-AcAc, was developed initially to directly observe ketone metabolism in the brain of people developing MCI and AD (
14.1 g of pure salts of the D enantiomer (>99% enantiomeric excess) of D-BHB were used. The D-BHB supplement tested was formulated as a mixture of three salts: sodium D-beta-hydroxybutyrate (CAS Registry number 13613-65-5), magnesium (D-beta-hydroxybutyrate)2 (CAS Registry number 586976-57-0), and calcium (D-beta-hydroxybutyrate)2 (CAS Registry number 51899-07-1). Each oral serving provided 12 g D-beta-hydroxybutyric acid, 0.78 g sodium, 0.42 g magnesium, and 0.88 g calcium, citrus flavoring and sweetener (Stevia), dissolved in 150 mL of drinking water.
Chemical purity of beta-hydroxybutyric acid was determined by quantitative 1H-nuclear magnetic resonance (NMR). NMR spectra were recorded on a 600 MHz Bruker Avance III spectrometer equipped with a 5 mm TCI cryogenic probe at 300 K using a Topspin 3.5pl7 software (Bruker Biospin).
Enantiomeric purity was determined by chiral high-performance liquid chromatography (HPLC) using an HPLC-UV instrument from Agilent Technologies with a Sumichiral OA6100 (5 μm, 4.6 × 150 mm) column. The mobile phase consisted of 1 mM copper (II) sulfate in water at a flow rate of 1 mL/min. Detection of the peaks was carried out by ultraviolet detection at 254 and 210 nm. Calculation of enantiomeric excess (ee) was expressed in percentage (%) according to the following formula: ee% = [(area of D-BHB– area of L-BHB)/total area of both D and L-BHB combined] × 100.
14.5 g of an equimolar mixture of commercial D and L beta-hydroxybutyrate salt was used (KetoCaNa, KetoSports, USA). Each serving provided a mixture of 12 g D+L-Beta-hydroxybutyric acid, 1.3 g sodium, 1.2 g calcium, orange flavoring and stevia, dissolved in 150 mL of drinking water.
Fifteen grams of medium chain triglyceride (MCT) (60% caprylic C8 acid and 40% capric C10 acid) emulsified in 70 mL of a 5% aqueous milk protein solution.
The meal consisted of 2 boiled eggs, 2 pieces of toast, 1 slice of cheese, and 1 portion of fruit jam, providing a total of 423 kcal (20 g fat, 24 g protein, 32 g carbohydrate). Water was provided
The ketogenic potential of D-BHB, D+L-BHB, and MCT was tested in 3 groups of 15 participants. The 3 groups had 11 participants in common and each participant had at least a 5-day washout period between each test product intake.
The groups had a mean age range of 36–38 years, body weight of 72–74 kg, BMI of 23–24 kg/m2, fasting plasma ketones of 98–185 μM, fasting plasma glucose of 5.3–5.5 mM, and fasting plasma insulin of 6.1–7.0 mU/L. Detailed demographics for each group are reported in
After an overnight fast, participants orally consumed 150 mL of the test product at time 0. At time 30 min, a standard breakfast was provided and consumed over 15 min to mimic the real life situation and explore any interference with the test product. Blood samples (7.5 mL) were taken at regular interval over 4 h [time (min): 0, 15, 30, 45, 60, 120, 180, 240] via a venous catheter. Plasma was analyzed for total ketones and D-BHB using Autokit Total Ketone Bodies and Autokit 3-HB (Wako Diagnostics, Mountain View, CA, USA), respectively. AcAc was then calculated by subtracting total BHB from total ketones. Plasma total BHB (D+L-BHB) was analyzed by ultra-high performance liquid chromatography, tandem mass spectrometry (UHPLC-MSMS; Vantage TSQ, ThermoFischer, Germany), based on the protocol described by Zeng and Cao (
Concentration for maximum effect (Cmax) was calculated as the mean of the maximum concentration reached by each participant. Incremental area-under-the-curve (iAUC) was calculated as the mean of baseline-corrected iAUC for each individual over 4 h. Time for maximum effect (Tmax) was calculated as the mean of Tmax reached by each individual.
During the 4 h test period, gastro-intestinal tolerability was assessed with a visual analog scale (VAS; 0 to 100) for each of the following symptoms: (
This study was approved by the Ethics Committee of Canton de Vaud (Switzerland) under the generic protocol reference 2018-00503, and all participants provided written informed consent. Procedures were conducted according to the principles of the Declaration of Helsinki. This trial is registered at
The sample size was based on previous determination of the coefficient of variation of plasma BHB iAUC (SD/mean = 0.0783). With this assumption, in order to detect a 10% difference between the iAUC of two products with a power of 80% and a type 1 error rate of 5%, about 12 participants per group were needed in a complete cross over design frame. Assuming 20% of non-evaluable participants, this resulted in enrollment of 15 participants/group.
To assess a potential carry-over effect, the joint modeling of iAUC and half-life (T1/2) was estimated by including product taken, previous product taken, and interaction of product taken and previous product taken as covariates. The covariate-associated coefficients were not different from zero, supporting the assumption that product-related effects were not carried forward over visits.
Exploratory inferential results were obtained with the non-parametric Wilcoxon rank-sum exact test (
The full method for the 11C-AcAc-PET tracer experiment has been reported previously (
PET images were acquired on a PET/CT (Gemini TF, Philips Healthcare, Eindhoven, the Netherlands). On the contralateral side from the radiotracer injection, blood was arterialized by warming the forearm with a heating pad at 44°C. Blood samples were taken at 3, 6, 8, 12, 20, and 28 min post-injection.
The acquisition protocol was as follows: 370 MBq of 11C-AcAc was injected followed by a 10-min dynamic brain acquisition, in list mode, with an isotropic voxel size of 2 mm3. Immediately after the dynamic brain acquisition, three whole-body (head to mid-thigh) acquisitions were performed at 18, 25, and 35 min post-injection. The acquisition times per bed position were 30, 45, and 60 s, respectively, for the three scans. Whole body acquisitions were performed with an isotropic voxel size of 4 mm3. Finally, an 8 frame per-cycle cardiac-gated acquisition of 15 min was performed 50 min after tracer injection.
PET tracer kinetics were analyzed for the brain (PMOD Technologies Ltd., Zurich, Switzerland). Brain ketone metabolism was assessed with graphical Patlak analysis of 11C-AcAc as previously described (
To characterize the effect of D-BHB supplementation on ketone uptake in organs besides the brain, 11C-AcAc uptake by the liver, kidneys and heart were segmented on the whole-body PET/CT fusion image, and the % dose/g was calculated from the organ volume and injected dose.
Heart reorientation and cardiac function analysis were performed using the cardiac module of PMOD 3.9 to obtain the ventricular volumes, ejection fraction, and polar map (
Plasma collected during the PET scan was analyzed for D-BHB and AcAc by automated colorimetric assay on a clinical chemistry analyser (Dimension Xpand Plus; Siemens, Deerfield, IL, USA) as previously described (
This PET study was approved by the CIUSSS de l'Estrie–CHUS Research Ethics Committee.
Following intake of the D-BHB, blood ketones rapidly increased (
Blood ketone kinetics following gram-matched oral doses of D-BHB, D+L-BHB, and MCT in 15 fasted participants at rest.
Analysis of blood D-BHB and AcAc revealed a similar pattern for the three products (
As expected, oral intake of D+L-BHB resulted in a significant increase in plasma L-BHB over the first hour (
Ketone production per calorie ingested [MCT: 8.3 kcal/g; BHB: 4.6 kcal/g (42)] was significantly higher for D-BHB than for D+L-BHB and MCT (
Plasma ketone levels were between 0.9 and 1.2 mM during the first 30 min of the PET scan acquisition. The first whole-body PET/CT biodistribution of 11C-AcAc 18 min post injection of the radiotracer is illustrated in
Whole body image after supplementation with two 15 g D-BHB doses, one at −75 min and the other at −30 min prior 11C-AcAc infusion (330 MBq). An 8 min scan (30 s per bed) was acquired 18 min post-injection of the radiotracer.
11C-AcAc organ distribution after D-BHB oral intake.
From the dynamic brain scan, CMRAcAc and KAcAc could be determined for all main regions of the brain (
Despite the fact that a dynamic cardiac scan could not performed in the present study, assessment of cardiac function was still possible with the gated heart PET image (
Heart image: 15 min cardiac-gated acquisition (8 frames) obtained 50 min post-11C-AcAc injection. Left ventricle (LV), right ventricle (RV), horizontal long axis (HLA), vertical long axis (VLA) and short axis (SA) images.
To our knowledge, this is the first report of the metabolism of D-BHB in humans and its link to the organ distribution of the ketone, AcAc, by PET imaging. This study shows that D-BHB is rapidly absorbed and metabolized (
Ketone production from an exogenous dietary source has been traditionally achieved by MCT. This requires a bolus intake to saturate the liver with MCFA, producing excess acetyl-CoA which is then transformed to AcAc and BHB, which are released into systemic circulation. The Cmax achieved with MCT is usually between 300 and 600 μM, with higher values being difficult to reach due to GI side effects and liver saturation. Here we show that D-BHB, a natural and biologically active ketone isomer, raises blood ketone Cmax above 1 mM without noticeable side effects. In comparison, an equivalent dose of D+L-BHB or MCT only achieved half this ketone level, with similar Tmax at 1 h. Thus, compared to D+L-BHB, D-BHB significantly reduces the salt intake needed to achieve the same plasma ketone response.
Results from a previous study (
The present study also revealed that D-BHB conversion to AcAc provided a ~43% higher AcAc/D-BHB blood ratio (0.63) than the same dose of MCT (0.44). In this respect, KE seems to behave more like MCT, with a ratio around 0.2–0.4 (
PET has been invaluable in measuring energy metabolism at the organ level in humans. For example, with 18F-FDG, it clearly shows that glucose is the main energy substrate of the brain and that a fatty acid such as 11C-palmitate is preferentially utilized by the heart and the liver (
Most of the heart's energy requirement is normally provided by fatty acids and glucose (
The cerebral metabolic rate for ketones (CMRketones) obtained here with D-BHB (3.0 μmol/100 g/min) exceeded that obtained after MCT supplementation in mild cognitive impairment [2.49 μmol/100 g/min; (
11C-AcAc uptake by the liver (
Recent studies have shown the potential importance of ketones in cardio-metabolic health. Infusion of D+L-BHB has beneficial hemodynamic effects in adult patients with heart failure and lower ejection fraction (
This study has several limitations. First, the pharmacokinetic comparison of D-BHB, D+L-BHB and MCT was an acute, single 4 h study; the extent to which it reflects long-term differences in their metabolism remains to be determined. Second, the whole-body PET scan was done on a single person and (except for brain) was a semi-quantitative comparison across organs. The brain was chosen for the quantitative scan (CMRAcAc). Dynamic PET scanning is required in order to quantify the tracer uptake and cannot be done simultaneously on the brain as well as other organs. Cardiac gated image quality could be improved if the image was acquired sooner after tracer injection. Third, ketone production and metabolism vary depending on post-prandial metabolic status, which possibly influences organ ketone distribution. Hence, follow-up of this single observation under different feeding conditions will be needed to verify the relative differences across organs.
D-BHB appears to be a promising supplement to produce significantly higher blood ketones than D+L-BHB or MCT, and at a lower calorie intake for an equivalent dose. Moreover, exogenous D-BHB does not appear to lower the blood AcAc/D-BHB ratio, which, altogether, might make it a more effective “super fuel” compared to other ketone precursors such as MCT, D+L-BHB or KE.
The pilot 11C-AcAc-PET study clearly identifies the heart and kidney as significant consumers of exogenous ketones, in fact, more than the brain. Therefore, D-BHB supplementation could be tested in conditions such as heart failure and diabetic cardiomyopathy to improve cardiac energy efficiency and function, and in chronic kidney disease.
All datasets generated for this study are included in the article/
The studies involving human participants were reviewed and approved by the Ethics Committee of Canton de Vaud (Switzerland) under the generic protocol reference 2018-00503, Trial registration number NCT03603782 (pharmacokinetic study), and by the CIUSSS de l'Estrie, CHUS Research Ethics Committee, Sherbrooke (Canada; PET study). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
MH, EC, C-AC, SC, and BC designed the studies. Data collection was performed by MH, EC, C-AC, J-PG, and MH. C-AC and EC analyzed data. The manuscript was drafted by BC and SC and all authors discussed the results and revised critically the manuscript.
BC, MH, and J-PG are employees of Nestlé. SC has consulted for Nestlé, Bulletproof and Accera, and received research funding and/or research materials from the Alzheimer Association (USA), Mitacs, FRQS, Abitec and Nestlé. AC declares research funding from CIHR, Canadian Diabetes Association, Fonds de recherche du Quebec–Santé, Janssen, Merck, UniQure, Caprion, Eli Lilly, Novo Nordisk, GlaxoSmithKline, Novartis, Pfizer, Philips, Sanofi, Siemens, and Amgen and consulting/advisory panel participation or conference fees from Merck, Amgen, Janssen, UniQure, Servier, Novo Nordisk, and Novartis. EC, MM, and C-AC declare no competing financial interests.
We thank the Nestlé Clinical Development Unit and Metabolic Unit staff for clinical management of the pharmacokinetic study and for their help in recruiting the volunteers; and thank Isabelle Breton, Irina Monnard, Shéhérazade Corbaz, and Simona Bartova from Nestlé Research for their skillful analytical assistance for measuring the D and L-BHB.
The Supplementary Material for this article can be found online at: