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# Circulating Beta-Hydroxybutyrate Levels in Advanced HFrEF: Determinants and Prognostic Impact

Luca Monzo, MD, PhD¹,², Jan Kovar, PhD¹, Barry A. Borlaug, MD³, Jan Benes, MD, PhD¹, Martin

Kotrc, MD¹, Katerina Kroupova, MD¹, Antonin Jabor, MD, PhD¹, Janka Franekova, MD, PhD¹,

Vojtech Melenovsky, MD, PhD¹

¹Institute for Clinical and Experimental Medicine (IKEM), Prague, Czech Republic

²Université de Lorraine, Centre d'Investigations Cliniques Plurithématique 1433 and Inserm U1116, CHRU Nancy, FCRIN INI-CRCT (Cardiovascular and Renal Clinical Trialists), Nancy, France

³Department of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota, USA

**Corresponding author:** Vojtech Melenovsky, MD, PhD. Department of Cardiology, Institute for Clinical and Experimental Medicine – IKEM. Prague, 140 21, Czech Republic. Phone: +420-739528029 - Fax: +420-261362986. E-mail: vojtech.melenovsky@ikem.cz

**Short title:** Role of β-hydroxybutyrate in HFrEF

**Words count:** 3037
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**Background** Patients with heart failure (HF) display metabolic alterations, including heightened ketogenesis, resulting in increased β-hydroxybutyrate (β-OHB) formation. We aimed to investigate the determinants and prognostic impact of circulating β-OHB levels in patients with advanced HF and reduced ejection fraction (HFrEF).

**Methods and results** A total of 867 patients with advanced HFrEF (age 57±11 years, 83% male, 45% diabetic, 60% NYHA III), underwent clinical and echocardiographic examination, circulating metabolite assessment, and right heart catheterization (n=383). The median β-OHB level was 64 (IQR: 33-161) µmol/L (normal 0-74 µmol/L). β-OHB levels correlated with increased markers of lipolysis (free fatty acids; FFA), higher natriuretic peptides, worse pulmonary hemodynamics, and lower humoral regulators of ketogenesis (insulin/glucagon ratio). During a median follow-up of 1126 (IQR: 410-1781) days, there were 512 composite events, including 324 deaths, 81 left ventricular assist device implantations and 107 urgent cardiac transplantations. In univariable Cox regression, increased β-OHB levels (T3 vs. T1: HR 1.39, [95% CI 1.13-1.72], p=0.002) and elevated FFA levels (T3 vs. T1: HR 1.39, [95% CI 1.09; 1.79], p = 0.008) were both predictors of a worse prognosis. In multivariable Cox analysis evaluating the simultaneous associations of FFA and β-OHB levels with outcomes, only FFA levels remained significantly associated with adverse outcomes.

**Conclusion** In patients with advanced HFrEF, increased plasma β-OHB correlate with FFA levels, worse RV function, greater neurohormonal activation and other markers of HF severity. The association between plasma β-OHB and adverse outcomes is eliminated after accounting for FFA levels, suggesting that increased β-OHB is a consequence reflecting heightened lipolytic state, rather than a cause of worsening HF.

**Keywords:** Ketone bodies; free fatty acid; cardiovascular disease; prognosis; heart failure; β-hydroxybutyrate
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**Introduction**

Metabolism of the failing heart is characterized by decreased flexibility and efficiency of
substrate consumption, with a switch from the preferred utilization of free fatty acids (FFA) towards
glucose and alternative energy sources, such as ketone bodies (KB).<sup>1, 2</sup> With heightened catabolism
or starvation, there is increased lipolytic activity and increased KB formation in the liver through the
conversion of free fatty acids (FFA).<sup>3, 4</sup> Beta-hydroxybutyrate (β-OHB) is the most stable and
abundant KB in the circulation.

Experimental and human observations suggest that the metabolic shift towards increased KB
utilization in the heart may be an adaptive process with potentially beneficial effects in patients with
HF, mainly through the provision of an ancillary fuel in a setting of decreased mitochondrial
function.⁴ Indeed, KB provide more energy per metabolized carbon than glucose, and require less
oxygen than free fatty acids for ATP generation.⁵ Ketone bodies have also signalling role and act
through pathways that improve cell survival and attenuate inflammation.⁴ Exogenous administration
of β-OHB has beneficial cardiovascular hemodynamic effects and prevents adverse cardiac
remodeling in experimental models.⁶ Notably, recent studies suggested that sodium-glucose
cotransporter 2 (SGLT2) inhibitors may partly exert their benefit on cardiovascular system via the
elevation of circulating β-OHB.⁷⁻⁹

On the other hand, studies in patients with chronic heart failure (HF) have shown a positive
association between elevated KB level and both disease severity<sup>10, 11</sup> and adverse prognosis.<sup>12, 13</sup>
Similarly, KB elevations predicted worse outcomes in patients with decompensated HF,<sup>14</sup> in patients
after myocardial infarction,<sup>15</sup> and on chronic hemodialysis,<sup>16</sup> as well as in the general population.<sup>17</sup>
The determinants of ketogenesis in patients with HF are poorly understood, and it remains unclear
whether ketonemia is an aggravating cause or an adaptive consequence in advanced HF. In this study,
we aimed to investigate the determinants and prognostic impact of circulating β-OHB levels in
patients with advanced HF and reduced ejection fraction (HFrEF).
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# Methods

## Study population

The study retrospectively evaluated patients with chronic (>6 months) symptomatic HFrEF (LV ejection fraction ≤40%) electively hospitalized for consideration of advanced therapies at the Institute for Clinical and Experimental Medicine (IKEM) in Prague (CZ) from January 2008 to December 2016. Patients with acute ischemia, uncontrolled cardiac arrhythmia, hemodynamic instability needing inotropes or mechanical circulatory support, reversible cardiac dysfunction, active malignancy, endocrine disease, chronic or acute infection were excluded. Patients with hypervolemia on admission (based on clinical examination) were enrolled into the study after diuresis and reaching normovolemia (central venous pressure < 10 mmHg), if possible. The local ethics committee approved the protocol. All patients signed informed consent with the procedures and with participation in this research study.

## Study Protocol

After signing informed consent, patients underwent history review, physical examination, echocardiography, electrocardiogram, Minnesota Living with Heart Failure Questionnaire (MLHFQ), blood sampling with circulating metabolite assessment. Some patients underwent right heart catheterization and body composition assessment by dual-energy X-ray absorptiometry (DEXA) and the skinfold method, if clinically indicated as part of the diagnostic work-up or for research purposes. All investigations were performed in a post-absorptive state following an overnight fasting.

Blood samples were consistently collected from a peripheral vein between 6 and 7 a.m., immediately chilled on ice, and centrifuged within 20 minutes at a force of 800×g at a temperature of 4 °C. Plasma was collected, distributed into aliquots, and frozen at -80 °C. β-OHB concentration was analysed instead of total KB, since it is the most abundant ketone in the bloodstream³ and has a key role in human metabolism and cellular signaling.¹⁸ β-OHB (normal value: 0-74 µmol/L, CV [intra-
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assay] 5%) and FFA (normal value: men: 0.1-0.6 mmol/L, women: 0.1-0.45 mmol/L, CV [intra-assay] 1.5%) were measured using a commercially available enzymatic assays (Autokit 3-HB and NEFA-HR, Wako Chemicals, USA). The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated using the formula: $[\text{glucose (nmol/L)} * \text{insulin (µU/mL)}/22.5]^{1/2}$. Fasting glucose ≥126 mg/dL or use of antidiabetic medication defined diabetes mellitus. Insulin concentration was determined using Insulin IRMA kit (Beckman Coulter, Prague, CZ; normal value: 2.1-22.0 mIU/L, CV [intra-assay] 4%) and glucagon using Glucagon RIA kit (Millipore, Billerica, MA, USA; normal value (fasting): 50-150 pg/mL, CV [intra-assay] 4-7%). Estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

Right heart catheterization was performed in a subgroup (n=383) according to current recommendations²⁰ using a 7Fr balloon-tipped triple-lumen Swan-Ganz catheter (Braun Melsungen AG, Melsungen, Germany) inserted via the right internal jugular vein. Pressure waveforms were recorded and annotated by an invasive hemodynamic module (Mac-Lab, GE Healthcare, Chicago, IL).

Bicipital, tricipital, subscapular and supra iliac skinfolds measurements were performed in 322 patients to assess the body composition using a Lange adipometer, with a constant pressure of 10 g/mm² on the contact surface and accuracy of 1 mm, with a 0–65 mm scale.¹⁹ Body density values were calculated using the older adults-specific equation of Durnin and Womersley (DWE),²¹ and converted into fat percentage by using the Siri equation: $\text{body fat percentage} = (4.95/\text{DWE}) - 4.50) \times 100.19$

## Data Analysis

Data are shown as mean ± standard deviation (SD) or median and (25th–75th interquartile range [IQR]) for continuous variables (according to distribution) and total count (n) with proportion (%) for categorical variables. One-way ANOVA and Kruskal–Wallis tests were used to compare
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continuous variables between groups depending on the normality of the distribution, and the $\chi^2$ test was used for categorical variables. Normality was assessed using the Shapiro-Wilks test. To assess the association of each variable with $\beta$-OHB plasma concentrations, separate multiple linear regression analyses adjusted for sex and age were conducted and Spearman's $\rho$ was calculated. Univariable and multivariable (adjusted for age, sex, BMI, MLHFQ score, diabetes, eGFR, HF medical treatment, LVEF, and ischemic HF aetiology) Cox proportional hazards regression were used to analyse the association of $\beta$-OHB and FFA plasma concentrations with the adverse outcome, defined as a combined endpoint of death, urgent transplantation, or left ventricular assist device (LVAD) implantation without heart transplantation. Kaplan-Meier curves with the log-rank statistics were used to illustrate the outcome. A p-value <0.05 was considered significant. All analyses were performed using JMP pro 17.0 statistical software (SAS Institute, Inc., Cary, NC).

# Results

## *Baseline characteristics*

A total of 867 consecutive patients referred for advanced HF therapies evaluation (cardiac devices implantation, cardiac transplantation or LVAD implantation) in our centre were enrolled. Median $\beta$-OHB level was 64 (IQR: 33-161) $\mu$mol/L (for normal ranges see methods). Based on the distribution in the overall population, $\beta$-OHB tertiles were created with cut-offs of: 1st tertile (T1): < 40 $\mu$mol/L (n=278); 2nd tertile (T2): 40 - 114 $\mu$mol/L (n=279) and 3rd tertile (T3): > 114 $\mu$mol/L (n=288). Patients in the highest $\beta$-OHB tertile were older, with a higher body mass index (BMI), were more congested (increased right atrial [RA] pressure and brain natriuretic peptide [BNP]), had worse kidney function and more severe right ventricular (RV) dysfunction (higher right atrial [RA]/pulmonary artery wedge pressure [PAWP] ratio). From a metabolic perspective, patients with higher $\beta$-OHB were more likely to have diabetes, accompanied by increased markers of lipolysis (FFA) and significant alterations in humoral regulators of liver ketogenesis (insulin/glucagon ratio) (Table 1).
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Determinants of β-OHB plasma concentrations

At univariable regression analysis, β-OHB plasma concentration was strongly and directly
correlated with FFA levels (β ± standard error [SE] = 1.708 ± 0.120; Spearman's ρ = 0.619, all
p<0.001), reflecting surplus delivery into the liver (Figure 1). Similarly, BNP (β ± SE = 0.197 ±
0.029; Spearman's ρ = 0.192, all p<0.001), which act as a lipolytic hormone in the adipose tissue,
showed a significant direct correlation with β-OHB levels. Right ventricular afterload (mean
pulmonary artery pressure: β ± SE = 0.016 ± 0.004, Spearman's ρ = 0.181; pulmonary vascular
resistance: β ± SE = 0.357 ± 0.083, Spearman's ρ = 0.187; all p<0.001), left ventricular filling
pressure (PAWP: β ± SE = 0.014 ± 0.006, p=0.015; Spearman's ρ = 0.107, p=0.036), and even more
RV filling pressure (RA pressure: β ± SE = 0.037 ± 0.008; Spearman's ρ = 0.227, all p<0.001) were
all positively associated with β-OHB levels. RA/PAWP ratio (β ± SE = 0.648 ± 0.216, p=0.003;
Spearman's ρ = 0.190, p<0.001), a marker of RV failure, was also directly correlated with β-OHB
levels (Figure 2). From a metabolic viewpoint, insulin/glucagon ratio (β ± SE = -1.231 ± 0.043;
Spearman's ρ = -0.185, all p<0.001) and HOMA-IR (β ± SE = -0.105 ± 0.042, p=0.012; Spearman's
ρ = -0.068, p=0.082) were inversely associated with β-OHB concentrations, while diabetic status (β
± SE = 0.177 ± 0.034; Spearman's ρ = 0.215, all p<0.001) and insulin use (β ± SE = 0.191 ± 0.051;
Spearman's ρ = 0.150, p<0.001) were positively associated. In multivariable regression analysis, FFA
concentrations remained the only independent predictor of β-OHB plasma concentrations (Table 2).

Outcomes

During a median follow-up of 1126 (IQR: 410; 1781) days, there were 512 composite events
including 324 deaths, 81 left ventricular assist device (LVAD) implantations and 107 urgent cardiac
transplantations. In Kaplan-Meier plots, the risk of the composite outcome increased with increasing
β-OHB (log-rank χ² = 10.8, overall p-value = 0.005) and FFA (log-rank χ² = 8.9, overall p-value =
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0.011) levels (Figure 3). Univariable Cox regression analysis showed that patients in the highest tertile of β-OHB had an increased risk of adverse clinical events compared to those in the lowest tertile (T3 vs. T1: HR 1.39, [95% CI 1.13; 1.72], p = 0.002). Patients with elevated FFA concentration also displayed an increased risk of the composite outcome (T3 vs. T1: HR 1.39, [95% CI 1.09; 1.79], p = 0.008) (Supplemental Figure 1). Notably, the association between higher FFA levels and the increased risk of clinical events was not significantly influenced by β-OHB levels, as showed in Cox regression analysis (FFA levels > median and β-OHB levels ≤ median vs. FFA and β-OHB levels > median: HR 0.86, [95% CI 0.60; 1.22], p = 0.404) and in Kaplan-Meier plots (Figure 4). Sensitivity analysis using as endpoint a composite of death or urgent cardiac transplantation (i.e., excluding LVAD implantation to avoid potential referral bias to our tertiary centre) showed results similar to the primary analysis (β-OHB, T3 vs. T1: HR 1.61, [95% CI 1.27; 2.03], p < 0.001; FFA, T3 vs. T1: HR 1.52, [95% CI 1.16; 2.00], p = 0.003).

In multivariable Cox analysis evaluating the simultaneous associations of FFA and β-OHB levels with outcomes, only FFA levels remained significantly associated with adverse outcomes, both as continuous variables (β-OHB, log-rank χ2 = 0.57, p-value = 0.447; FFA, log-rank χ2 = 10.9, p-value < 0.001) and, even if more borderline likely due to the reduced sample size, according to tertiles (β-OHB, T3 vs. T1: HR 1.31, [95% CI 0.96; 1.78], p = 0.084; FFA, T3 vs. T1: HR 1.39, [95% CI 1.01; 1.92], p = 0.048) (Supplemental Figure 1).

## Discussion

We explored the determinants and the prognostic impact of β-OHB in patients with advanced HFrEF. We demonstrated a close relationship between β-OHB levels and neurohumoral activation, worse hemodynamic, and more impaired RV function. Notably, we observed a direct association between β-OHB levels and an increased risk of clinical events in patients with advanced HFrEF. However, the association between β-OHB levels and adverse outcome was no longer evident after accounting for FFA concentrations, which were also associated with an increased risk.
Altered myocardial use of energy-providing substrates and impaired metabolic flexibility are the key features of HF-related metabolic derangement.<sup>22-24</sup> In patients with HFrEF, myocardial β-OHB oxidation rate is significantly increased,<sup>25</sup> with a linear β-OHB uptake in proportion to the delivery and with no upper threshold.<sup>10</sup> Based on experimental studies, the increased reliance on KB of the failing heart has been postulated to be an adaptive process,<sup>26</sup> aiming to provide the energy-starved myocardium with an extra source of substrate to decrease its energy deficit and support its contractile function.<sup>27</sup> It is therefore quite surprising that several recent observational studies<sup>11, 13, 28</sup> found a negative association between circulating β-OHB and survival in HF. Our findings reveal a more intricate connection between β-OHB and HF prognosis. Our findings enrich the existing literature, clarifying the ongoing debate about the prognostic value of KB in HF.<sup>12, 13</sup> Specifically, we demonstrated that the increase in circulating β-OHB likely reflects the severity of HF and the lipolytic overdrive triggered by the heightened neurohormonal activation. Therefore, the preference for using KB as a substrate in the failing heart might primarily stem from the increased availability of β-OHB and its cardiac uptake through concentration-dependent zero-order kinetics.<sup>10</sup>

*Determinants of β-OHB levels in HF population*

The amount of circulating β-OHB is affected by several factors. Increased β-OHB production secondary to a heightened hepatic delivery of β-OHB precursors, namely FFA, can be consequential to an increase in lipolytic hormones, or from either an absolute or a relative deficiency of insulin signalling in adipose tissue, as in diabetes. Liver ketogenesis is also activated by low insulin and increased glucagon, as during starvation.<sup>3</sup> Additionally, β-OHB level is influenced by the net rate of peripheral tissue utilization and the extent of renal reabsorption.<sup>4</sup>

Our study demonstrates that β-OHB level in advanced HF is primarily influenced by neurohumoral activation and the rate of lipolysis, along with the presence of diabetes,<sup>29</sup> while renal function is less relevant. Notably, we found that β-OHB concentration was inversely associated with insulin/glucagon ratio, an important regulator of liver ketogenesis, and directly with markers of
insulin resistance (i.e., HOMA-IR). In addition, we showed a significant association between elevated β-OHB levels and worse hemodynamic status, namely increased RA pressure, pulmonary pressure, PVR, and PAWP. β-OHB level was also inversely associated with RV function (i.e., RA/PAWP ratio), suggesting that systemic catabolism associated with ventricular failure may be reflected by β-OHB.<sup>19</sup>

The elevation of KB in HF is likely a consequence of increased release of FFA from adipose tissue and the subsequent overload of hepatocytes with beta-oxidation byproducts, which are then converted into β-OHB in order to provide an additional source of energy for other organs. The HF-related neurohormonal overdrive, characterized by elevated levels of catecholamines, angiotensin II and natriuretic peptides, results in heightened activation of adipocyte lipolysis,<sup>30, 31</sup> leading in turn to a rise in both circulating FFA and β-OHB. Consistently, high glycerol concentrations were observed in the adipose interstitial fluid of HF patients, indicating the high breakdown activity of triglycerides in this condition.<sup>31</sup> Direct in-vivo measurements documented that lipolytic actions of BNP are not attenuated even in patients with advanced HF, and BNP-driven lipolysis has been implicated in the development of cardiac cachexia.<sup>19, 32</sup>

The link between increased lipolysis, ketone production, and low body weight was recently documented even in the general population.<sup>33</sup> Not only HF, but also other stress or catabolic situations, such as ST-elevation myocardial infarction,<sup>15</sup> or chronic dialysis,<sup>16</sup> are also associated to an increased blood level of KB, likely as a direct consequence of the sympathetic stress response, systemic catecholamine surge and FFA release.<sup>15</sup>

## β-OHB levels and prognosis in HF population

In patients with HFrEF, increasing circulating KB are associated with worse prognosis.<sup>13, 23</sup>
<sup>34</sup> Our study demonstrates that FFA, the precursors of KB, are also independent predictor of adverse outcome, replacing KB in the multivariate model. Although we cannot draw definitive explanations for this finding from observational data, we can offer some potential insights. Enhanced ketogenesis
in HF seems mainly driven by neurohormonal activation, as previously demonstrated by the positive association between HF severity and β-OHB levels,²³ being in turn just the mirror of a worse clinical status. This could also be supported by an association between β-OHB levels and worsen cardiac and pulmonary hemodynamics and higher BNP in the present study. High KB levels in HF can also reflects enhanced triglyceride metabolism, and consequent FFA production. As the last product of this metabolic cascade, β-OHB may represent a surrogate marker for heightened triglyceride mobilization and FFA flux.³³

Previous observations reported that circulating FFA were associated with several comorbidities including coronary heart disease, hypertension, diabetes mellitus and atrial fibrillation that increase the risk for HF,³⁵ but also adverse outcomes such as sudden cardiac death and increased cardiovascular mortality.³⁶, ³⁷ From a mechanistic perspective, high FFA levels and their consequent preferential use as a metabolic substrate may reduce myocardial energy efficiency, through the uncoupling of mitochondrial proteins, and alter insulin signalling pathways and tissue insulin sensibility.³⁸ Moreover, the imbalance between FFA uptake and oxidation may results in intracellular accumulation of toxic lipid species that drive the cardiac lipotoxicity.³⁹ Finally, FFA have been involved in several cellular pathways causing endothelial dysfunction and apoptosis, playing a key role in accelerated atherosclerosis.⁴⁰ Altogether, these effects may provide biological plausibility for the observed increased risk of adverse outcomes in HF patients with higher FFA concentrations.

## Strengths and Limitations

The strengths of this study are its unparalleled sample size and depth of phenotyping, including invasive hemodynamics, anthropometry, and metabolic hormones, making it the most comprehensive cohort to date exploring the determinants and prognostic impact of β-OHB in HFrEF. The study has also inherent limitations. Due to its observational design, it is not possible to establish a causal link to the results obtained. Patients were referred or enrolled in a single tertiary centre, leading to a selection bias. We measured plasma FFA and β-OHB at a single time point. Whether
repeated measures and evaluation of trajectories of change in FFA and β-OHB over time may have
provided stronger associations with clinical outcomes is unknown. Our population mainly comprises
middle-age, male Caucasian individuals with advanced HFrEF, which could limit the generalization
of these findings to other HF populations, such as HFpEF. We did not directly measure peripheral
lipolysis, therefore hypothesis related to this were speculative. Due to the period of observation, the
study population was not treated with SGLT2 inhibitors; consequently, is not possible to provide any
insights about their effect on KB metabolism. The lack of granularity in our mortality outcome data,
which prevents us from specifically analysing cardiovascular deaths and excluding other causes of
death not exclusively related to HF conditions. Finally, our analysis was based on absolute levels of
metabolites, but measurements of metabolite flux (i.e. appearance and disappearance over time)
would provide more definitive answers.³³

Conclusions

In patients with advanced HFrEF, both β-OHB and FFA levels were directly associated with an increased risk of clinical events, including death, urgent transplantation, or LVAD implantation. Notably, the association between plasma β-OHB and adverse outcomes vanishes upon accounting for FFA levels, suggesting that β-OHB might merely reflects a heightened lipolytic state induced by neurohormonal overdrive due to a worse clinical status, as demonstrated by the direct association between KB level and worse hemodynamic parameters, RV dysfunction and BNP.

Funding

Agency for Healthcare research (AZV NU22-02-00161), National Institute for Research of Metabolic
and Cardiovascular Diseases funded by the European Union - Next Generation EU (Programme
EXCELES, Project No. LX22NPO5104) and by MH CZ - DRO (“Institute for Clinical and
Experimental Medicine – IKEM, IN 00023001”)
**Conflict of interests**

None.
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**Figure Legends**

**Graphical abstract** Summary of the study design and key findings of the study. In the central panel, (+) and (−) means that the variable is directly or inversely associated with β-hydroxybutyrate (β-OHB) levels, respectively. The gold arrow means that FFA levels are the only variable that remained associated with β-OHB levels in the multivariable model. Abbreviations: FFA, free fatty acids; HFrEF, heart failure with reduced ejection fraction; HOMA-IR, homeostatic model assessment for insulin resistance; LV, left ventricle; RHC, right heart catheterization; RV, right ventricle; TG, triglycerides. Parts of the figure were created using Biorender.com.

**Figure 1** Correlation between beta-hydroxybutyrate levels and free fatty acids levels.

**Figure 2** Correlation between beta-hydroxybutyrate levels and A) mean pulmonary artery pressure; B) pulmonary vascular resistance; C) right atrial (RA) pressure; D) pulmonary artery wedge pressure (PAWP); E) RA/PAWP ratio.

**Figure 3** Kaplan–Meier curves for the composite outcome of death, urgent transplantation, or left ventricular assist device implantation without heart transplantation stratified by A) beta-hydroxybutyrate (β-OHB) level tertiles and B) free fatty acids (FFA) level tertiles.

**Figure 4** Kaplan–Meier curves for the composite outcome of death, urgent transplantation, or left ventricular assist device implantation without heart transplantation stratified by beta-hydroxybutyrate (β-OHB) and free fatty acids (FFA) levels above or below the median.
**Supplemental Figure 1** Forest plot of the risk of the composite outcome in univariable and multivariable analyses by tertiles of β-hydroxybutyrate (β-OHB) and free fatty acids (FFA).
Abbreviations: CI, confidence interval; HR, hazard ratio; T1, tertile 1; T2, tertile 2; T3, tertile 3.
<table><caption>**Table 1.** Baseline characteristics according to β-hydroxybutyrate plasma concentration tertiles</caption><thead><tr><th></th><th>Tertile 1<br>(βOHB &lt; 40<br>µmol/L)</th><th>Tertile 2<br>(βOHB 40 – 114<br>µmol/L)</th><th>Tertile 3<br>(βOHB &gt; 114<br>µmol/L)</th><th>p-value</th></tr></thead><tbody><tr><td colspan="5"><strong>Clinical characteristics</strong></td></tr><tr><td>Age, years</td><td>55 ± 11</td><td>57 ± 11</td><td>60 ± 11</td><td>&lt;0.001</td></tr><tr><td>Females, n (%)</td><td>45 (16)</td><td>43 (15)</td><td>58 (20)</td><td>0.278</td></tr><tr><td>Body mass index, kg/m<sup>2</sup></td><td>27.0 ± 4.9</td><td>28.2 ± 5.2</td><td>28.3 ± 5.1</td><td>0.004</td></tr><tr><td>Body fat (skinfold method), %</td><td>25.4 ± 10.6</td><td>26.2 ± 9.6</td><td>23.8 ± 9.9</td><td>0.197</td></tr><tr><td>NYHA class, n (%): I-II; III-IV</td><td>98 (35)/157 (57)/23<br>(8)</td><td>89 (32)/176<br>(63)/14 (5)</td><td>86 (29)/176 (61)/26<br>(9)</td><td>0.429</td></tr><tr><td>MLHFQ score</td><td>43.8 ± 22.2</td><td>41.8 ± 21.6</td><td>42.6 ± 23.0</td><td>0.641</td></tr><tr><td>HF ischemic etiology, n (%)</td><td>129 (47)</td><td>135 (50)</td><td>153 (54)</td><td>0.284</td></tr><tr><td>HF duration, years</td><td>8.4 [4.6; 13.5]</td><td>8.6 [5.2; 14.4]</td><td>9.6 [5.9; 16.7]</td><td>0.023</td></tr><tr><td>Diabetes mellitus, n (%)</td><td>92 (33)</td><td>124 (44)</td><td>163 (57)</td><td>&lt;0.001</td></tr><tr><td colspan="5"><strong>Laboratory values</strong></td></tr><tr><td>Hemoglobin, g/L</td><td>142 ± 18</td><td>141 ± 16</td><td>139 ± 18</td><td>0.079</td></tr><tr><td>Sodium, mmol/L</td><td>139 ± 3</td><td>138 ± 4</td><td>139 ± 4</td><td>0.609</td></tr><tr><td>Potassium, mmol/L</td><td>4.4 ± 0.4</td><td>4.3 ± 0.4</td><td>4.3 ± 0.5</td><td>0.005</td></tr><tr><td>eGFR, mL/min/1.73 m<sup>2</sup></td><td>72 ± 21</td><td>70 ± 22</td><td>65 ± 23</td><td>0.001</td></tr><tr><td>HbA<sub>1</sub>C, mmol/mol</td><td>44 [39; 49]</td><td>45 [41; 50]</td><td>47 [41; 59]</td><td>&lt;0.001</td></tr><tr><td>HOMA-IR index</td><td>2.2 [1.4; 4.2]</td><td>2.4 [1.4; 4.3]</td><td>2.2 [1.2; 3.7]</td><td>0.378</td></tr><tr><td>Insulin/glucagon ratio</td><td>0.10 [0.06; 0.17]</td><td>0.10 [0.06; 0.16]</td><td>0.08 [0.05; 0.12]</td><td>&lt;0.001</td></tr><tr><td>Insulin, mIU/L</td><td>8.91 [5.97; 16.22]</td><td>9.89 [5.84; 15.75]</td><td>8.32 [4.72; 12.67]</td><td>0.009</td></tr><tr><td>Glucagon, pg/mL</td><td>103.5 ± 45.9</td><td>109.3 ± 44.5</td><td>111.1 ± 55.7</td><td>0.154</td></tr><tr><td>BNP, ng/L</td><td>401 (150; 906)</td><td>450 (212; 1019)</td><td>620 (268; 1301)</td><td>&lt;0.001</td></tr><tr><td>Free fatty acids, mmol/L</td><td>0.39 ± 0.26</td><td>0.57 ± 0.22</td><td>0.72 ± 0.28</td><td>&lt;0.001</td></tr><tr><td colspan="5"><strong>Echocardiography</strong></td></tr><tr><td>LV end-diastolic diameter, mm</td><td>69 ± 9</td><td>71 ± 10</td><td>69 ± 9</td><td>0.012</td></tr><tr><td>LV ejection fraction, %</td><td>24 ± 6</td><td>23 ± 6</td><td>24 ± 6</td><td>0.200</td></tr><tr><td>E/Em ratio</td><td>15 ± 8</td><td>15 ± 7</td><td>16 ± 9</td><td>0.348</td></tr><tr><td>TAPSE, mm</td><td>16 ± 5</td><td>16 ± 5</td><td>16 ± 6</td><td>0.895</td></tr><tr><td>Mitral regurgitation, n (%): 0-2<br>grade; 3-4 grade</td><td>162 (58)/115 (42)</td><td>140 (50)/138 (50)</td><td>140 (50)/141 (50)</td><td>0.072</td></tr><tr><td>Tricuspid regurgitation, n (%): 0-2<br>grade; 3-4 grade</td><td>222 (80)/56 (20)</td><td>207 (75)/70 (25)</td><td>204 (72)/78 (28)</td><td>0.107</td></tr><tr><td colspan="5"><strong>Hemodynamics</strong></td></tr><tr><td>RA pressure, mmHg</td><td>8.5 ± 5.5</td><td>9.9 ± 5.9</td><td>10.9 ± 6.0</td><td>0.007</td></tr><tr><td>RA/PAWP ratio</td><td>0.38 ± 0.20</td><td>0.41 ± 0.26</td><td>0.46 ± 0.22</td><td>0.026</td></tr><tr><td>Mean PA pressure, mmHg</td><td>32.5 ± 11.5</td><td>34.8 ± 12.0</td><td>35.9 ± 11.9</td><td>0.073</td></tr><tr><td>PCWP, mmHg</td><td>22.3 ± 8.5</td><td>23.6 ± 9.1</td><td>23.4 ± 8.4</td><td>0.376</td></tr><tr><td>Cardiac output, L/min</td><td>3.9 ± 1.2</td><td>3.8 ± 0.9</td><td>3.8 ± 1.1</td><td>0.844</td></tr><tr><td>PVR, Wood's units</td><td>2.4 [1.7; 4.0]</td><td>2.6 [1.9; 4.0]</td><td>2.9 [2.1; 4.8]</td><td>0.080</td></tr><tr><td>Systolic blood pressure, mmHg</td><td>115 ± 17</td><td>116 ± 20</td><td>118 ± 20</td><td>0.153</td></tr><tr><td>Diastolic blood pressure, mmHg</td><td>72 ± 10</td><td>74 ± 11</td><td>73 ± 12</td><td>0.159</td></tr><tr><td>Heart rate, bpm</td><td>75 ± 14</td><td>75 ± 15</td><td>77 ± 15</td><td>0.149</td></tr><tr><td colspan="5"><strong>Treatment</strong></td></tr><tr><td>Furosemide, n (%)</td><td>241 (87)</td><td>253 (91)</td><td>257 (89)</td><td>0.317</td></tr><tr><td>ACE or ARB, n (%)</td><td>217 (78)</td><td>217 (78)</td><td>230 (80)</td><td>0.852</td></tr></tbody></table>
<table><tr><td>MRA, n (%)</td><td>217 (78)</td><td>217 (78)</td><td>214 (74)</td><td>0.444</td></tr><tr><td>Betablocker, n (%)</td><td>245 (88)</td><td>243 (87)</td><td>255 (89)</td><td>0.899</td></tr><tr><td>OAD/insulin, n (%)</td><td>71 (25)/19 (7)</td><td>89 (32)/35 (12)</td><td>107 (37)/55 (19)</td><td>0.001</td></tr><tr><td>Devices, n (%): ICD; CRT</td><td>163 (60)/85 (32)</td><td>165 (62)/88 (33)</td><td>153 (56)/87 (32)</td><td>0.905</td></tr></table>

**Abbreviations:** ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; BNP, brain natriuretic peptide; βOHB, β-hydroxybutyrate; CRT, cardiac resynchronization therapy; eGFR, estimated glomerular filtration rate; HbA1C, glycated hemoglobin; HOMA-IR, homeostasis model assessment of insulin resistance; MLHFQ, Minnesota living with heart failure questionnaire; ICD, implantable cardioverter defibrillator; LV, left ventricle; MRA, mineralocorticoid receptor antagonist; NYHA, New York Heart Association; OAD, oral antidiabetic drugs; PA, pulmonary artery; PCWP, pulmonary capillary wedge pressure; PVR, pulmonary vascular resistance; RA, right atrium; TAPSE, tricuspid annular plane systolic excursion.
<table><caption>**Table 2.** Age- and sex-adjusted and multivariable linear regression analyses on plasma concentration of β-hydroxybutyrate</caption><thead><tr><th rowspan="2"></th><th colspan="2">Age- and Sex-Adjusted</th><th colspan="2">Multivariable</th></tr><tr><th>Standardized β ± SE</th><th>p-value</th><th>Standardized β ± SE</th><th>p-value</th></tr></thead><tbody><tr><td colspan="5"><strong>Clinical characteristics</strong></td></tr><tr><td>Body mass index, kg/m<sup>2</sup></td><td>0.016 ± 0.007</td><td>0.014</td><td></td><td></td></tr><tr><td>Body fat (skinfold method), %</td><td>-0.006 ± 0.006</td><td>0.331</td><td></td><td></td></tr><tr><td>NYHA class, I-IV</td><td>0.065 ± 0.057</td><td>0.253</td><td></td><td></td></tr><tr><td>MLHFQ score</td><td>-0.001 ± 0.002</td><td>0.906</td><td></td><td></td></tr><tr><td>HF ischemic etiology (yes vs. no)</td><td>-0.041 ± 0.039</td><td>0.288</td><td></td><td></td></tr><tr><td>HF duration, years</td><td>0.050 ± 0.041</td><td>0.226</td><td></td><td></td></tr><tr><td>Diabetes mellitus (yes vs. no)</td><td>0.177 ± 0.034</td><td>&lt;0.001</td><td>-</td><td>-</td></tr><tr><td colspan="5"><strong>Laboratory values</strong></td></tr><tr><td>Hemoglobin, g/L</td><td>-0.002 ± 0.002</td><td>0.193</td><td></td><td></td></tr><tr><td>eGFR, mL/min/1.73 m<sup>2</sup></td><td>-0.002 ± 0.002</td><td>0.203</td><td></td><td></td></tr><tr><td>HbA<sub>1</sub>C, mmol/mol</td><td>0.741 ± 0.138</td><td>&lt;0.001</td><td>0.342 ± 0.377</td><td>0.365</td></tr><tr><td>HOMA-IR index</td><td>-0.105 ± 0.042</td><td>0.012</td><td></td><td></td></tr><tr><td>Insulin, mIU/L</td><td>-0.008 ± 0.002</td><td>&lt;0.001</td><td>-</td><td>-</td></tr><tr><td>Glucagon, pg/mL</td><td>0.001 ± 0.001</td><td>0.048</td><td></td><td></td></tr><tr><td>Insulin/glucagon ratio</td><td>-1.231 ± 0.043</td><td>&lt;0.001</td><td>-0.137 ± 0.101</td><td>0.174</td></tr><tr><td>BNP, ng/L</td><td>0.197 ± 0.029</td><td>&lt;0.001</td><td>0.062 ± 0.087</td><td>0.478</td></tr><tr><td>Free fatty acids, mmol/L</td><td>1.708 ± 0.120</td><td>&lt;0.001</td><td>1.384 ± 0.264</td><td>&lt;0.001</td></tr><tr><td colspan="5"><strong>Echocardiography</strong></td></tr><tr><td>LV end-diastolic diameter, mm</td><td>0.010 ± 0.004</td><td>0.010</td><td></td><td></td></tr><tr><td>LV ejection fraction, %</td><td>-0.002 ± 0.006</td><td>0.716</td><td></td><td></td></tr><tr><td>E/Em ratio</td><td>0.006 ± 0.005</td><td>0.261</td><td></td><td></td></tr><tr><td colspan="5"><strong>Hemodynamics</strong></td></tr><tr><td>RA pressure, mmHg</td><td>0.037 ± 0.008</td><td>&lt;0.001</td><td>0.022 ± 0.015</td><td>0.146</td></tr><tr><td>PA mean pressure, mmHg</td><td>0.016 ± 0.004</td><td>&lt;0.001</td><td>-0.001 ± 0.008</td><td>0.985</td></tr><tr><td>PA wedge pressure - mmHg</td><td>0.014 ± 0.006</td><td>0.015</td><td></td><td></td></tr><tr><td>RA/PA wedge pressure ratio</td><td>0.648 ± 0.216</td><td>0.003</td><td>-</td><td>-</td></tr><tr><td>Cardiac output, L/m</td><td>-0.053 ± 0.047</td><td>0.266</td><td></td><td></td></tr><tr><td>PVR - Wood's unit</td><td>0.357 ± 0.083</td><td>&lt;0.001</td><td>-</td><td>-</td></tr><tr><td colspan="5"><strong>Treatment (yes vs. no)</strong></td></tr><tr><td>ACE or ARB use</td><td>-0.019 ± 0.041</td><td>0.635</td><td></td><td></td></tr><tr><td>MRA use</td><td>0.019 ± 0.040</td><td>0.624</td><td></td><td></td></tr><tr><td>Betablocker use</td><td>0.023 ± 0.053</td><td>0.659</td><td></td><td></td></tr><tr><td>Insulin use</td><td>0.191 ± 0.051</td><td>&lt;0.001</td><td>0.082 ± 0.139</td><td>0.768</td></tr></tbody></table>

In the multivariable model, dashes stand for variables not tested because of possible collinearity, meanwhile empty rows mean that variables were not tested because not strongly statistically significant (i.e., p<0.001) in the adjusted univariate analysis.

**Abbreviations:** ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; BNP, brain natriuretic peptide; eGFR, estimated glomerular filtration rate; HbA<sub>1</sub>C, glycated hemoglobin; HOMA-IR, homeostasis model assessment of insulin resistance; hs-CRP, high-sensitivity C-reactive protein; MLHFQ, Minnesota living with heart failure questionnaire; LV, left ventricle; MRA, mineralocorticoid receptor antagonist; NYHA, New York Heart Association; PA, pulmonary artery; PVR, pulmonary vascular resistance; RA, right atrium.
Graphical abstract

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Supplemental Figure 1</figcaption></figure>

<figure><img src="image_8.png" /></figure>