Abstract
Background/Objectives: Beta-hydroxybutyrate (BHB) exists as two enantiomers with potentially distinct biological activities. While D-BHB is the physiological form produced during ketogenesis, L-BHB is present in equal amounts in racemic supplements, yet its biological effects remain poorly understood. Additionally, the ketone precursor 1,3-butanediol (BD) is used in some formulations despite limited safety data. Methods: We investigated acute (single gavage, 2-h time course) and short-term (daily gavage for 8 days) hepatic effects of D-BHB, L-BHB, and 1,3-butanediol compared to a vehicle control in male C57BL/6 mice. Acute studies assessed hepatic ATP dynamics and lipid peroxidation (MDA) at multiple timepoints. Eight-day protocols evaluated mitochondrial function (oxygen consumption, Complex II activity, SDH activity), lipid accumulation (triglycerides), and inflammatory markers (IL-1β, TNF-α, CRP). Results: Acute ATP responses differed markedly among treatments. Compared to the baseline and the control, L- and D-BHB elicited significant increases in ATP, while BD caused sustained ATP depletion. Over this same time, oxidative stress markers remained stable in the control and both BHB groups but increased dramatically with BD. After 8 days, the mitochondrial effects of BD were more apparent with a significant reduction in complex II-supported respiration and activity. Both forms of BHB maintained control levels of inflammation and BD showed significant effects on all inflammatory markers. Hepatic triglycerides increased only with BD treatment. Conclusions: This study reveals striking hepatic effects of various ketone supplements. In contrast to the positive or inert effects of BHB enantiomers, 1,3-butanediol induces significant hepatic stress. These findings have implications for ketone supplement formulation and highlight the therapeutic potential of D- and L-BHB.
Keywords: D-beta-hydroxybutyrate; L-beta-hydroxybutyrate; 1,3-butanediol; hepatic metabolism; mitochondrial function; inflammation; oxidative stress; ketones; enantiomers
1. Introduction
Beta-hydroxybutyrate (BHB), the primary ketone body produced during states of carbohydrate restriction, exists as two enantiomers with distinct metabolic fates. D-BHB (R-3-hydroxybutyrate) represents the physiological form produced endogenously during ketogenesis and serves as an efficient energy substrate via BHB dehydrogenase-mediated oxidation [1]. L-BHB (S-3-hydroxybutyrate), while not produced in significant quantities endogenously from the liver, is produced significantly in the heart [2]. Moreover, L-BHB comprises half of racemic BHB supplements and exhibits altered clearance with potentially unique signaling properties [3,4].
Emerging evidence indicates that BHB enantiomers may exert distinct physiological effects. Cardiovascular studies have demonstrated enantiomer-specific effects on cardiac output and vascular function, with L-BHB showing distinct hemodynamic properties compared to D-BHB [5]. Furthermore, L-BHB has been reported to be oxidized more slowly than D-BHB and to persist longer in circulation, suggesting distinct metabolic handling compared to the D-enantiomer [6]. However, the hepatic responses to pure enantiomers remain largely unexplored despite the liver’s central role in ketone metabolism.
1,3-Butanediol (BD) offers an alternative approach to elevating blood ketones, serving as a precursor that undergoes conversion to BHB via alcohol and aldehyde dehydrogenases [7]. Because this metabolic pathway overlaps with ethanol oxidation, concerns arise regarding potential hepatotoxicity, NAD+ depletion, and oxidative stress, particularly with repeated administration [8]. Previous studies have shown that BD can induce CNS depression and even physical dependence similar to ethanol [9], yet it continues to be used in some commercial ketone formulations.
The liver’s response to exogenous ketones extends beyond simple substrate metabolism. Acute ketone exposure can rapidly alter hepatic energy status, mitochondrial dynamics, and inflammatory signaling [10]. These immediate responses may differ substantially from adaptations to sustained supplementation, where enzyme induction, metabolic remodeling, or cumulative stress may emerge [11]. Understanding both temporal dynamics is essential for optimizing therapeutic ketone interventions.
This study provides a comprehensive evaluation of hepatic responses to D-BHB, L-BHB, and 1,3-butanediol through both acute time-course analysis and 8-day supplementation protocols. We hypothesized that L-BHB would demonstrate anti-inflammatory properties distinct from D-BHB’s metabolic effects, while BD would induce hepatic stress through its alcohol-like metabolism. By including a vehicle control group, we aimed to determine whether these compounds maintain, enhance, or compromise hepatic homeostasis.
2. Materials and Methods
2.1. Animals and Study Design
Male C57BL/6J mice (10–12 weeks old) were housed under controlled conditions (12 h light/dark cycle, 22 ± 2 °C, ad libitum access to standard chow and water). Animals were randomly assigned to four treatment groups: Control (water), D-BHB, L-BHB, or 1,3-butanediol. All compounds were administered via oral gavage at 1624 mg/kg body weight in a volume of 0.1 mL per 10 g body weight. D-β-hydroxybutyric acid (D-BHB) and L-β-hydroxybutyric acid (L-BHB) were obtained from Ketone Labs (Salt Lake City, UT, USA) and racemic 1,3-butanediol from Sigma-Aldrich (B84785; St. Louis, MO, USA). Control animals received equivalent volumes of water. This dosing protocol was selected to enable direct comparison with prior investigations of hepatic ATP responses to BHB enantiomers and precursors.
Two experimental protocols were conducted. The acute protocol involved a single oral gavage with liver tissue collection at 15, 30, 60, and 120 min post-administration (n = 7 per timepoint per group). The eight-day protocol consisted of daily oral gavage for 8 consecutive days with terminal tissue collection 2 h after the final dose (n = 6–8 per group). Animal health was monitored via body weight, which did not differ between groups. All procedures were approved by the Institutional Animal Care and Use Committee at Brigham Young University (022524; 25 February 2024) in accordance with NIH guidelines.
2.2. Tissue Collection and Sample Preparation
Mice were euthanized by cervical dislocation under deep isoflurane anesthesia. The liver was rapidly excised, weighed, and divided. Portions were snap-frozen in liquid nitrogen within 30 s for biochemical assays, and adjacent pieces were placed in ice-cold BIOPS relaxation buffer (10 mM Ca-EGTA, 0.1 μM free calcium, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM DTT, 6.56 mM MgCl2, 5.77 mM ATP, 15 mM phosphocreatine, pH 7.1) for immediate mitochondrial respirometry. Frozen tissue was homogenized (1:10 w/v) in the appropriate ice-cold buffer using a bead mill (Qiagen TissueLyser II, 30 Hz, 2 × 2 min; Germantown, MD, USA). Homogenates were centrifuged at 12,000× g for 10 min at 4 °C, and supernatants were used immediately or stored at −80 °C.
2.3. Acute Hepatic ATP and Oxidative Stress
Hepatic ATP content was quantified in perchloric acid-neutralized extracts using the Roche ATP Bioluminescence Assay Kit HS II (cat. 11699709001; Indianapolis, IN, USA) according to the manufacturer’s instructions. Results are expressed as µmol/g wet tissue weight.
Lipid peroxidation was determined as malondialdehyde (MDA) equivalents using the Abcam TBARS (TCA method) Assay Kit (ab118970; Waltham, MA, USA). Results are expressed as nmol/g wet tissue.
2.4. Mitochondrial Function
High-resolution respirometry was performed in saponin-permeabilized liver tissue using an Oroboros Oxygraph-2k at 37 °C in MiR05 respiration medium (Oroboros Instruments, Innsbruck, Austria). The substrate–inhibitor protocol was as follows: glutamate (10 mM) + malate (2 mM) (GM), followed by ADP (5 mM) for Complex I-supported State 3 respiration, and then succinate (10 mM) for maximal ETS capacity. Oxygen consumption rates are expressed as ml O2/g protein/s. Complex II factor was calculated as the ratio of succinate-supported State 3 respiration to glutamate/malate-supported State 3 respiration in the presence of ADP (arbitrary units). Succinate dehydrogenase activity was measured via succinate dehydrogenase activity (ab228560) on liver homogenate supernatant. As an additional outcome, we scrutinized ATP and oxidative stress production as a function of oxygen consumption.
2.5. Hepatic Triglycerides and Inflammatory Cytokines
Hepatic triglycerides were extracted by the Folch method and quantified using the Fujifilm Wako L-Type TG M enzymatic kit (Greenwood, SC, USA). Results are expressed as mg/g wet liver weight. Liver inflammatory cytokines (IL-1β, TNF-α, and C-reactive protein) were measured in RIPA homogenates using the Milliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (MCYTOMAG-70K; Austin, TX, USA) on a Luminex MAGPIX instrument (Austin, TX, USA). Concentrations were normalized to total protein content (BCA assay) and expressed as pg/mg total protein.
2.6. Statistical Analysis
Data were analyzed using GraphPad Prism version 10. Acute time-course data were analyzed by two-way repeated-measures ANOVA, followed by Tukey’s post hoc test. The area under the curve (AUC) was calculated using the trapezoidal rule. Chronic data were analyzed by a one-way ANOVA, followed by Dunnett’s multiple-comparison test against the control group. All data are presented as mean ± SD. Statistical significance is denoted as * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CON; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. BHB when indicated.
3. Results
3.1. Acute Hepatic Energy and Oxidative Stress Responses
3.1.1. Enantiomer-Specific ATP Dynamics
Hepatic ATP levels following acute gavage revealed distinct patterns among treatments (Figure 1A). Control (CON) animals maintained stable ATP levels throughout the 2 h observation period. D-BHB treatment resulted in ATP dynamics that were significantly elevated due to a 60 min and later elevation. L-BHB also induced significant ATP elevation over the time course, albeit with an earlier elevation of 15–30 min. In striking contrast, BD treatment caused severe ATP depletion, reaching −1.2 μmol/g by 30 min with only partial recovery by 120 min. The area under the curve analysis (Figure 1B) confirmed these differences, with BD showing significantly greater ATP depletion compared to all other groups (p < 0.001).
Figure 1.
Acute hepatic energy and oxidative stress responses to BHB enantiomers and 1,3-butanediol. Group identifiers (line graphs): Control (▼), D-BHB (●), L-BHB (■), and BD (▲). Group identifiers (bar graphs): (A) Time-course changes in hepatic ATP content (µmol/g) over 15, 30, 60, and 120 min following a single oral gavage of control, D-BHB, L-BHB, or BD. (B) ATP AUC analysis demonstrating significantly greater ATP depletion with BD compared to all groups, while D-BHB and L-BHB exhibited increased levels of ATP. (C) Hepatic malondialdehyde (MDA) levels (nmol/g) over the same time course. (D) MDA AUC indicating significantly elevated lipid peroxidation with BD, with D-BHB and L-BHB shifting lower than control. Data are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON; ## p < 0.01, #### p < 0.001 vs. BHB.
3.1.2. BD-Induced Oxidative Stress
Lipid peroxidation, measured as MDA levels, was reduced in D-BHB and L-BHB groups throughout the time course compared to the baseline (Figure 1C,D). However, BD treatment induced progressive and sustained MDA accumulation, reaching levels approximately 3-fold higher than control by 120 min. AUC analysis (Figure 1D) demonstrated that BD-induced oxidative stress was significantly greater than all other groups (p < 0.001), while BHB forms significantly reduced MDA, more so in L-BHB than D-BHB.
3.1.3. Increased Mitochondrial Efficiency with BHB
Within the same protocol, a sample of liver tissues was processed for a single measurement of mitochondrial respiration with glutamate + malate + ADP + succinate (Figure 2A). This was used as a baseline respiration rate to enable a rough metric to determine the degree to which the liver produced ATP (Figure 2B) or reactive oxygen species (ROS; Figure 2C) per unit oxygen consumed.
Figure 2.
Mitochondrial respiratory function, ATP:Oxygen consumption ratio and ROS:Oxygen consumption with acute treatment. (A) Mitochondrial oxygen consumption (ml O2/g protein/s) in permeabilized liver tissue following 1 hr exposure. Respiration was measured under glutamate/malate/ADP/succinate. Peak ATP (B) and MDA (ROS; (C)) levels were compared with respiration rates. Data are mean ± SD. ** p < 0.01., *** p < 0.001 vs. CON; #### p < 0.0001 vs. BHB.
3.1.4. 1,3-Butanediol Reduces Mitochondrial Function
After 8 days of daily supplementation, mitochondrial oxygen consumption revealed striking treatment effects (Figure 3A). We detected no changes across the different titrations to test mitochondrial respiration in either BHB condition compared with CON, though L-BHB trended towards greater respiration with the addition of succinate (S). However, BD supplementation significantly, impacted this same state. These findings were further supported by measuring complex II-supported respiration (S-D; CII Factor; Figure 3B) and succinate dehydrogenase (SDH) activity (Figure 3C).
Figure 3.
Mitochondrial respiratory function, Complex II activity, and SDH activity after 8-day supplementation. (A) Mitochondrial oxygen consumption (ml O2/g protein/s) in permeabilized liver tissue following 8-day supplementation. Respiration was measured under glutamate/malate (GM), ADP-stimulated State 3 (D), and succinate-supported (S) conditions. (B) Complex II factor was determined as the difference between S and D states and (C) Succinate dehydrogenase (SDH) activity.Data are mean ± SD. * p < 0.05, ** p < 0.01. vs. CON; ## p < 0.01, vs. BHB.
3.2. Lipid Accumulation and Inflammatory Responses
3.2.1. 1,3-Butanediol Increases Hepatic Inflammatory Cytokines
Inflammatory cytokine analysis revealed remarkable treatment-specific effects (Figure 4A). Control animals established a baseline inflammatory tone. Whereas BHB forms elicited no change, BD elicited a significant increase in all of the cytokines. The magnitude of BD’s effects was robust, with a roughly sixfold increase in TNFα levels vs. BHB.
Figure 4.
Hepatic inflammatory cytokine expression and triglyceride accumulation after 8-day supplementation. (A) Hepatic inflammatory cytokines (IL-1β, TNF-α, and CRP) following 8-day supplementation, with BD trending toward increased inflammatory signaling. (B) Hepatic triglyceride content (mg/g). Triglycerides were significantly elevated only in the BD group, while the control, D-BHB, and L-BHB maintained comparable levels. Data are mean ± SD. * p < 0.05 vs. all other conditions.
3.2.2. BD-Specific Triglyceride Accumulation
Due to a similar metabolism of BD to ethanol, we sought to understand hepatic lipid content. Hepatic triglyceride content after 8 days (Figure 4B) was significantly elevated only in the BD group compared to the control (p < 0.05). Both D-BHB and L-BHB maintained triglyceride levels similar to the control.
4. Discussion
This study provides a comprehensive evaluation of hepatic responses to exogenous ketone supplements, revealing distinct metabolic profiles among BHB enantiomers and 1,3-butanediol. Our findings demonstrate that D-BHB and L-BHB exert either beneficial or neutral effects on hepatic energy status, mitochondrial function, and inflammation, while BD induces significant hepatic stress characterized by ATP depletion, oxidative stress, and lipid accumulation. These results align with and extend recent findings by Ari and D’Agostino demonstrating formulation-dependent hepatic outcomes of chronic ketone supplementation, where ketone salts preserved liver health while BD-based ketone esters and precursors drove inflammation and steatosis [12].
4.1. Acute Hepatic Energy Dynamics
The acute ATP responses observed in this study reveal fundamentally different metabolic fates for these compounds. Both D-BHB and L-BHB elevated hepatic ATP levels, suggesting efficient energy provision without metabolic cost. D-BHB enters the canonical ketone oxidation pathway through BHB dehydrogenase, generating acetyl-CoA for TCA cycle oxidation [1]. The stable ATP elevation with D-BHB reflects its integration as a readily oxidizable fuel substrate, consistent with its established role as an efficient energy carrier during fasting and carbohydrate restriction [10].
The ATP-elevating effect of L-BHB is particularly noteworthy, given its distinct metabolic pathway. Unlike D-BHB, L-BHB is not efficiently oxidized through traditional ketolytic enzymes and exhibits slower clearance from circulation [4,13]. Recent pharmacokinetic studies demonstrate that L-BHB accumulates extensively in tissues, including the liver, the brain, the heart, and muscle, following oral supplementation, suggesting tissue-specific metabolic or signaling roles beyond simple oxidation [7]. The ATP elevation observed with L-BHB may reflect reduced energy expenditure through anti-inflammatory signaling, rather than direct oxidative contribution.
In stark contrast, BD induced severe and sustained ATP depletion. This finding aligns with BD’s metabolic pathway through alcohol and aldehyde dehydrogenases, which consumes NAD+ and generates toxic intermediates [14]. The similarity to ethanol metabolism raises significant concerns, as chronic NAD+ depletion is a hallmark of alcoholic liver disease and contributes to impaired mitochondrial function [15]. Previous studies have reported that BD administration causes metabolic acidosis and hepatic sinusoidal dilation at concentrations required to achieve therapeutic ketosis [16], and our ATP data provide mechanistic insight into the hepatic energy burden imposed by this compound. Notably, recent work has demonstrated that chronic BD administration induced the most severe hepatic alterations among ketone formulations tested, including widespread fat deposits, hepatocyte swelling, dense RBC accumulation, and sinusoidal congestion [12]. Our acute ATP depletion findings provide a mechanistic basis for these chronic histopathological outcomes.
4.2. Oxidative Stress and Redox Balance
The divergent effects on lipid peroxidation further distinguish these compounds. Both BHB enantiomers reduced MDA levels below the control baseline, suggesting active antioxidant properties, rather than the mere absence of oxidative insult. BHB has been shown to enhance the expression of oxidative stress resistance genes through histone acetylation modifications, particularly via the inhibition of class I histone deacetylases [16]. Additionally, BHB metabolism spares cytoplasmic NAD+ relative to glucose oxidation, potentially supporting cellular redox homeostasis [11].
The pronounced oxidative stress induced by BD reflects the production of reactive intermediates during its hepatic metabolism. The aldehyde intermediate generated during BD oxidation can directly damage cellular macromolecules and deplete glutathione reserves [8]. This oxidative burden, combined with NAD+ depletion, creates a metabolically stressed hepatic environment that may predispose to lipid peroxidation and cellular injury.
4.3. Mitochondrial Adaptation Following Repeated Supplementation
The eight-day supplementation protocol revealed important differences in mitochondrial adaptation. While neither BHB enantiomer significantly altered mitochondrial respiration compared to the control, L-BHB showed a trend toward enhanced succinate-supported respiration. This is consistent with previous findings that BHB enhances complex II (succinate dehydrogenase) activity in various tissues [17,18]. The mechanism may involve BHB-mediated changes in succinyl-CoA availability, protein succinylation, or direct effects on electron transport chain components [11].
BD supplementation significantly impaired complex II-supported respiration and SDH activity. This finding has important implications, given the dual role of complex II in the electron transport chain and the TCA cycle. Impaired complex II function compromises both ATP synthesis and metabolic flexibility, potentially creating a feed-forward cycle of mitochondrial dysfunction.
The mechanism by which BD decreases SDH activity likely involves altered metabolic pathways through ketone production, rather than direct enzyme inhibition. BD is metabolized to β-hydroxybutyrate and acetoacetate, altering cellular redox state and substrate availability for the TCA cycle [7]. This shift in metabolic fuel preference away from succinate oxidation could reduce flux through SDH, effectively decreasing its activity through substrate competition. Studies demonstrate that BD treatment alters citric acid cycle intermediates, with D-BD increasing citrate levels, suggesting upstream metabolic changes that create an environment less favorable for SDH activity by reducing flux through later portions of the TCA cycle [19]. The observed impairment may also reflect normal metabolic adaptation to alternative fuel availability. When BD provides abundant ketone bodies as an alternative energy source, cells may downregulate SDH activity as part of physiological metabolic flexibility [20,21]. The loss of SDH activity results in dependency on alternative anaplerotic pathways, particularly pyruvate carboxylation, to maintain cellular anabolism [22]. Previous work in perfused livers demonstrated that BD metabolism diverts carbon toward lipid synthesis, rather than complete oxidation [7], consistent with the metabolic inefficiency reflected in our mitochondrial function data and supporting reduced reliance on SDH-dependent energy production.
4.4. Inflammatory Responses and Hepatoprotection
A central finding of this study is the differential inflammatory profile among treatments. Both BHB enantiomers maintained control-level inflammatory markers, while BD induced significant elevations in IL-1β, TNF-α, and CRP. These findings are strongly corroborated by recent work that demonstrated that BD, ketone esters, and MCT all significantly elevated TNF-α expression compared to the control, while ketone salts (containing D,L-BHB) actually reduced TNF-α below control levels [12]. Their immunohistochemical analysis revealed that ketone salt-treated animals exhibited TNF-α-positive areas of only 28.4% compared to 43.5% in the BD group and 48.2% in the ketone ester group [12].
The anti-inflammatory properties of BHB are well established and involve multiple mechanisms, including NLRP3 inflammasome inhibition [23], GPR109A receptor activation [24], and NF-κB pathway modulation [25]. Importantly, the seminal work by Youm et al. demonstrated that NLRP3 inflammasome inhibition by BHB is independent of chirality, with both D-BHB and L-BHB (S-BHB) efficiently blocking inflammasome activation [23]. This may explain why both enantiomers maintained favorable inflammatory profiles in our study and why racemic ketone salts demonstrated anti-inflammatory effects [12].
The inflammatory response to BD likely reflects the hepatic stress induced by its ethanol-like metabolism. BD requires oxidation by alcohol dehydrogenase, similar to ethanol metabolism, producing BHB through sequential conversion to β-hydroxybutyraldehyde. This metabolic pathway imposes additional redox stress on hepatocytes by depleting cellular NAD+, contributing to the pronounced steatosis and vascular congestion observed with BD treatment. NAD+ depletion and oxidative stress activate inflammatory cascades, and the accumulation of metabolic intermediates can directly stimulate pattern recognition receptors [26]. The approximately sixfold increase in TNF-α with BD treatment observed in our study is particularly concerning, given the role of this cytokine in hepatic insulin resistance and steatohepatitis progression [27].
4.5. Hepatic Lipid Accumulation
The selective accumulation of triglycerides with BD treatment parallels the patho-physiology of alcoholic fatty liver disease. When 1,3-butanediol is metabolized to β-hydroxybutyrate, it undergoes sequential oxidation that consumes NAD+ and generates NADH, thereby contributing to NAD+ depletion and further shifting the hepatic redox state toward a more reduced environment, favoring fatty acid synthesis over oxidation [14]. Previous work in rats demonstrated that BD feeding increases hepatic β-hydroxybutyrate levels and the β-hydroxybutyrate: acetoacetate ratio, while also increasing the lactate: pyruvate ratio, consistent with an elevated hepatic cytoplasmic NADH/NAD+ ratio that would favor lipogenesis [28]. In addition, the observed reductions in complex II-supported respiration and SDH activity in the present study suggest impaired mitochondrial oxidative capacity, which may further limit β-oxidation and contribute to hepatic triglyceride retention.
Our triglyceride findings are consistent with the comprehensive histological analysis by Ari and D’Agostino, who reported that BD induced pronounced macrovesicular steatosis with widespread fat deposits, while ketone salts showed no fat deposits and preserved hepatic structure [12]. Their quantitative analysis revealed that the ketone ester and MCT groups exhibited significantly greater fat droplet area and count compared to the control, whereas ketone salts had the lowest fat droplet counts overall. Importantly, combining ketone salts with MCT appeared to buffer the fat-accumulating effects observed with MCT alone, suggesting potential synergistic metabolic benefits [12].
The absence of triglyceride accumulation with BHB enantiomers is reassuring and consistent with evidence that ketone bodies may protect against hepatic steatosis. Impaired hepatic ketogenesis has been causally linked to NAFLD development [29], and ketone body supplementation may help dispose of excess hepatic fatty acids through ketogenic pathways. The BHB activation of GPR109A also exerts anti-lipolytic effects that reduce fatty acid flux to the liver [24]. The preserved hepatic architecture observed with ketone salt supplementation in both our study and previous work suggests that direct BHB administration, particularly as racemic D,L-BHB salts, may represent the most hepatoprotective approach to exogenous ketosis [12].
4.6. Clinical Implications
These findings have immediate relevance for the growing market of exogenous ketone supplements and the potential use in clinical applications. Our data, combined with the recent chronic supplementation study by Ari and D’Agostino [12], provide converging evidence that formulation choice critically determines hepatic safety outcomes. Racemic BHB supplements containing equal proportions of D- and L-BHB appear safe based on both our acute/short-term data and the 4-week chronic administration data from Ari and D’Agostino, who concluded that ketone salts demonstrated “the most favorable safety profile under the tested conditions, maintaining normal hepatic structure” [12].
Pure D-BHB supplements would provide an efficient energy substrate without hepatic stress, while formulations enriched in L-BHB may offer enhanced anti-inflammatory and signaling benefits, given its longer tissue residence time and accumulation in metabolically active organs [4]. The enantiomer-specific tissue distribution studies demonstrate that L-BHB concentrations increase extensively in the brain, the heart, the liver, and muscle following racemic supplementation, with potentially distinct signaling roles [4].
The concerning profile of BD warrants a serious reconsideration of its use in ketone supplementation, particularly for chronic administration. While BD effectively raises circulating ketone levels [3], both our data and prior work demonstrate this occurs at a significant hepatic cost. Furthermore, early studies demonstrated that BD can depress CNS activity and induce physical dependence similar to ethanol [9], raising additional safety concerns beyond hepatic effects.
Individuals with existing liver disease, metabolic syndrome, or those consuming alcohol should especially avoid BD-containing products. The hepatic stress induced by BD may outweigh any benefits from elevated ketone levels, particularly when safer alternatives in the form of direct BHB salt supplementation exist. As Ari and D’Agostino emphasize, “formulation choice is critical for the safe long-term use of exogenous ketones” [12], especially in the context of high-dose chronic use in older individuals who are more sensitive to BD-induced narcotic effects and toxicity.
4.7. Limitations
Several limitations merit consideration. First, the use of only male mice limits generalizability, given known sex differences in hepatic metabolism and inflammatory responses. Females typically exhibit greater hepatic antioxidant capacity and attenuated inflammatory signaling, which could influence both the magnitude of BD-induced hepatic stress and the hepatoprotective effects of BHB enantiomers. Second, the single dose tested may not represent optimal therapeutic dosing, and dose-response relationships remain to be established. Third, the eight-day supplementation period, while revealing important adaptive responses, may not capture longer-term effects or potential adverse consequences. Whether the hepatic alterations observed with BD persist, worsen, or adapt over longer supplementation periods remains unknown. Longer-term studies are necessary to determine whether compensatory mechanisms emerge or whether metabolic stress progresses with sustained exposure. Fourth, circulating triglyceride and D- and L-β-hydroxybutyrate concentrations were not measured, limiting the assessment of systemic lipid handling and precluding a direct correlation of hepatic outcomes with circulating ketone exposure. Finally, molecular mechanism studies examining specific signaling pathways activated by each compound would strengthen mechanistic interpretation.
Additionally, while the single dose of 1624 mg/kg enabled a direct comparison across formulations, this approach does not account for the distinct metabolic processing of each compound. D-BHB and L-BHB enter circulation directly as ketone bodies, whereas BD undergoes extensive hepatic metabolism involving NAD+ consumption and aldehyde intermediate generation. Consequently, equimolar dosing does not necessarily produce equivalent systemic ketone exposure or hepatic metabolic burden across compounds. Dose-response studies would strengthen our conclusions and better inform human-relevant exposure recommendations for different ketone supplement formulations.
While the mechanistic findings presented here are supported by direct biochemical and functional measurements, broader safety implications regarding long-term human use require cautious interpretation and further clinical validation.
5. Conclusions
This comprehensive evaluation reveals that BHB enantiomers and 1,3-butanediol exert fundamentally distinct effects on hepatic metabolism. Both D-BHB and L-BHB demonstrate favorable hepatic profiles, elevating ATP levels acutely while maintaining control-level mitochondrial function, inflammatory markers, and lipid content following repeated administration. In contrast, 1,3-butanediol induces significant hepatic stress characterized by ATP depletion, oxidative damage, mitochondrial impairment, inflammation, and triglyceride accumulation. Beyond our findings and those noted earlier, previous work has detailed a wide range of considerations from BD [30,31,32], including developmental complications in offspring [33].
Our findings align with and extend recent work by Ari and D’Agostino demonstrating that chronic hepatic responses to exogenous ketones are highly formulation-dependent [12]. Their comprehensive histological, immunohistochemical, and biochemical analyses over 4 weeks of chronic administration corroborate our shorter-term findings, with ketone salts (D,L-BHB) preserving normal hepatic morphology, while BD and ketone esters induced steatosis, vascular congestion, and elevated inflammatory markers. Together, these complementary studies provide strong evidence that direct BHB supplementation, particularly as racemic salts, offers a favorable hepatic safety profile, while BD-based formulations pose significant hepatic risks.
These findings have important implications for ketone supplement formulation and clinical application. While BD effectively elevates circulating ketones, its hepatic burden may outweigh benefits, and it raises safety concerns for chronic use, particularly in aged or health-compromised individuals [12]. BHB enantiomers, whether administered as racemic mixtures or pure forms, appear to offer safe and potentially beneficial approaches to exogenous ketosis. The distinct tissue distribution and signaling properties of D- and L-BHB suggest that formulation optimization may further enhance therapeutic outcomes. As interest in ketone therapeutics expands beyond neurological applications to metabolic and cardiovascular disease, understanding enantiomer-specific effects and formulation-dependent safety profiles will be critical for developing safe and effective interventions.
Abbreviations
The following abbreviations are used in this manuscript:
| BHB | beta-hydroxybutyrate |
| D-BHB | R-3-hydroxybutyrate |
| L-BHB | S-3-hydroxybutyrate |
| BD | 1,3-butanediol |
| NAD+ | nicotinamide adenine dinucleotide |
| CNS | central nervous system |
| C57BL/6 | Cold Spring Harbor ‘57’, Black ‘6’ |
| NIH | National Institutes of Health |
| BIOPS | biopsy preservation solution |
| Ca-EGTA | calcium ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid |
| K-MES | potassium 2-Morpholinoethanesulfonic acid |
| DTT | dithiothreitol |
| ATP | adenosine triphosphate |
| MDA | malondialdehyde |
| TBARS | thiobarbituric acid reactive substances |
| TCA (method) | trichloroacetic acid |
| MiR05 | mitochondrial respiration medium 05 |
| GM | glutamate + malate |
| ADP | adenosine diphosphate |
| ETS | electron transfer system |
| DCPIP | 2,6-dichlorophenolindophenol |
| TG | triglyceride |
| IL-1β | interleukin-1 beta |
| TNF-α | tumor necrosis factor-alpha |
| CRP | C-reactive protein |
| RIPA | radioimmunoprecipitation assay |
| MAP | mouse antibody production |
| BCA | bicinchoninic acid |
| ANOVA | analysis of variance |
| AUC | area under curve |
| CON | control |
| AU | arbitrary units |
| SDH | succinate dehydrogenase |
| TCA | tricarboxylic acid cycle |
| NLRP3 | NLR family pyrin domain containing 3 |
| NF-κB | nuclear factor kappa-light-chain-enhancer of activated B cells |
| NAFLD | non-alcoholic fatty liver disease |
| acetyl-CoA | acetyl-coenzyme A |
Author Contributions
Conceptualization, B.T.B., R.R.P. and P.R.R.; methodology, T.J.M., M.D.M., G.P., M.K.B., R.R., T.L.S., T.S.P., D.S.N., B.T.B. and D.P.D.; software, B.T.B., R.R.P. and P.R.R.; formal analysis, T.J.M., M.D.M., A.J.P., J.R.H., E.J.R., D.P.D., J.A.A., R.R.P., P.R.R. and B.T.B. resources, B.T.B. and P.R.R.; data curation, B.T.B., R.R.P. and P.R.R.; writing—original draft preparation, T.J.M., M.D.M. and B.T.B.; writing—review and editing, All authors; visualization, B.T.B. and P.R.R.; supervision, B.T.B. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
The animal study protocol was approved by the Institutional Review Board of Brigham Young University (Protocol code 022524; 25 February 2024).
Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.
Conflicts of Interest
B.T.B. is an advisor for Unicity International and receives royalties from the sale of a book about insulin resistance. D.P.D. is an inventor of ketone (BHB) and 1,3-butanediol-related patents owned by the University of South Florida: USPTO #9,801,903, 9,675,577, 9,795,580, 10,980,764, 10,792,268, 10,064,6462, 10,842,767, 10,980,764, 10,945,975, 11,452,704, 11,596,616, 11,766,417, 11,974,973, and 12,310,938. D.P.D. is a co-owner of the company Ketone Technologies LLC. These interests have been reviewed and managed by the university in accordance with its Institutional and Individual Conflict of Interest policies. All authors declare that there are no additional conflicts of interest.
Funding Statement
This research was funded by internal support provided by Brigham Young University.
Footnotes
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Data Availability Statement
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