Ketone Body 3-hydroxybutyrate Research Articles

Targeted Quantitative Plasma Metabolomics Identifies Metabolite Signatures that Distinguish Heart Failure with Reduced and Preserved Ejection Fraction.

Two general phenotypes of heart failure (HF) are recognized: HF with reduced ejection fraction (HFrEF) and with preserved EF (HFpEF). To develop HF disease phenotype-specific approaches to define and guide treatment, distinguishing biomarkers are needed. The goal of this study was to utilize quantitative metabolomics on a large, diverse population to replicate and extend existing knowledge of the plasma metabolic signatures in human HF. Quantitative, targeted LC/MS plasma metabolomics was conducted on 787 samples collected by the Penn Medicine BioBank from subjects with HFrEF (n=219), HFpEF (n=357), and matched non-failing Controls (n=211). A total of 90 metabolites were analyzed, comprising 28 amino acids, 8 organic acids, and 54 acylcarnitines. 733 of these samples were also processed via an OLINK protein panel for proteomic profiling. Consistent with previous studies, unsaturated forms of medium/long chain acylcarnitines were elevated in the HFrEF group to a greater extent than the HFpEF group compared to Controls. A number of amino acid derivatives, including 1- and 3-methylhistidine, homocitrulline, and symmetric (SDMA) and asymmetric (ADMA) dimethylarginine were elevated in HF, with ADMA elevated uniquely in HFpEF. Plasma branched-chain amino acids (BCAA) were not different across the groups; however, short-chain acylcarnitine species indicative of BCAA catabolism were significantly elevated in both HF groups. The ketone body 3-hydroxybutyrate (3-HBA) and its metabolite C4-OH carnitine were uniquely elevated in the HFrEF group. Linear regression models demonstrated a significant correlation between plasma 3-HBA and NT-proBNP in both forms of HF, stronger in HFrEF. These results identify plasma signatures that are shared as well as potentially distinguish between HFrEF and HFpEF. Metabolite markers for ketogenic metabolic re-programming in extra-cardiac tissues were identified as unique signatures in the HFrEF group, possibly related to the lipolytic action of increased levels of BNP. Future studies will be necessary to further validate these metabolites as HF biosignatures that may guide phenotype-specific therapeutics and provide insight into the systemic metabolic responses to HFpEF and HFrEF. What Is New?: "Real world" targeted metabolomic profiling on wide range of metabolites in a diverse population of patients with HFrEF and HFpEF.Levels of 3-hydroxybutyrate and its metabolite C4OH-carnitine were uniquely increased in the HFrEF group and correlated with levels of plasma NT-proBNP in both the heart failure groups, indicating the possibility of a heart-adipose-liver axis.Asymmetric dimethylarginine, a known inhibitor of nitric oxide synthase, was uniquely upregulated in HFpEF suggesting that there may also be an underlying component of vascular dysregulation contributing to HFpEF pathophysiology.What Are the Clinical Implications?: The plasma metabolomic changes seen in the heart failure cohorts support the existing theory of metabolic reprogramming, providing further rationale for the pursuit of therapeutic targets for the treatment of heart failure.Quantitative metabolomic profiling shows promise for guiding therapeutic decisions in HFrEF and HFpEF.Modulation of natriuretic peptides may enhance the delivery of ketone and fatty acids to the "fuel starved" failing heart.

Hemodynamic effects of treatment with the ketone body 3-hydroxybutyrate in cardiogenic shock - a randomized, controlled, assessor-blinded porcine trial

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Novo Nordisk Foundation Independent Research Fund DK. Background Cardiogenic shock (CS) is a life-threatening disease with limited evidence-based treatment options. Treatment with the ketone body 3-hydroxybutyrate (3-OHB) induces favorable hemodynamic effects in patients with heart failure and CS. It increases cardiac output (CO) and left ventricular (LV) ejection fraction (LVEF) while reducing cardiac filling pressures. Yet, whether treatment with 3-OHB demonstrates beneficial hemodynamic effects during acute ischemic LV stunning in CS must be fully elucidated. This study aimed to examine the acute hemodynamic effects and underlying cardiovascular mechanisms of 3-OHB infusion in the acute phase of LV stunning in a porcine model of ischemic CS. Methods Sixteen 60-kg-pigs were included in a randomised, controlled, assessor-blinded crossover study. CS was achieved by repeated, slow injections of microspheres into the left main coronary artery. CS was defined as ≥30% reduction in CO and/or mixed venous saturation (SVO2). Upon the onset of CS, following a one-hour no-touch period, the pigs received an infusion with 3-OHB and isoosmolar, isovolumic saline (control) for two hours each in random order. Infusion periods were separated by a one-hour washout period. CO (primary endpoint), SVO2, and pulmonary pressures were measured by right heart catheterization. A pressure-volume admittance catheter was placed in the LV to measure LVEF, contractility (the slope of end systolic pressure-volume relationship, denoded as Ees), afterload (arterial elastance, Ea, and end-systolic pressure, ESP), preload (end-diastolic volume, EDV), and work-related parameters. Results CS was successfully achieved in all randomized animals. CO decreased by 36% from 4.5±1.0 L/min to 2.9±0.5 L/min (p<0.001) and SVO2 decreased by 31% from 55.0±8.1% to 37.7±5.8% (p<0.001). During 3-OHB infusion, CO increased by 0.9 L/min (95% CI: 0.4 to 1.3 L/min) compared with the control infusion. In parallel, Ees increased by 0.22 mmHg/mL (95% CI: 0.08 to 0.37 mmHg/mL), LVEF improved by 7 percentage points (95%CI: 1 to 13 percentage points) and cardiac efficiency increased by 6 percentage points (95%CI: 1 to 11 percentage points). Systemic vascular resistance was decreased by 479 dyn*cm5 (95% CI: 736 to 221 dyn*cm5); Ea and ESP remained similar. Heart rate was increased during 3-OHB infusion by 21 bpm (95% CI: 11 to 32 bpm) while no significant change was observed in stroke volume (p=0.6). Conclusion Treatment with 3-OHB improved CO, LVEF, and cardiac efficiency in CS. The increase was mediated through increased ventricular contractility and decreased peripheral resistance accompanied by increased heart rate.CO, Ees and cardiac efficiencyEndpoint parameters during 3-OHB

Cardiovascular Effects of Oral Ketone Ester Treatment in Patients With Heart Failure With Reduced Ejection Fraction: A Randomized, Controlled, Double-Blind Trial.

Heart failure triggers a shift in myocardial metabolic substrate utilization, favoring the ketone body 3-hydroxybutyrate as energy source. We hypothesized that 14-day treatment with ketone ester (KE) would improve resting and exercise hemodynamics and exercise capacity in patients with heart failure with reduced ejection fraction. In a randomized, double-blind cross-over study, nondiabetic patients with heart failure with reduced ejection fraction received 14-day KE and 14-day isocaloric non-KE comparator regimens of 4 daily doses separated by a 14-day washout period. After each treatment period, participants underwent right heart catheterization, echocardiography, and blood sampling at plasma trough levels and after dosing. Participants underwent an exercise hemodynamic assessment after a second dosing. The primary end point was resting cardiac output (CO). Secondary end points included resting and exercise pulmonary capillary wedge pressure and peak exercise CO and metabolic equivalents. We included 24 patients with heart failure with reduced ejection fraction (17 men; 65±9 years of age; all White). Resting CO at trough levels was higher after KE compared with isocaloric comparator (5.2±1.1 L/min versus 5.0±1.1 L/min; difference, 0.3 L/min [95% CI, 0.1-0.5), and pulmonary capillary wedge pressure was lower (8±3 mm Hg versus 11±3 mm Hg; difference, -2 mm Hg [95% CI, -4 to -1]). These changes were amplified after KE dosing. Across all exercise intensities, KE treatment was associated with lower mean exercise pulmonary capillary wedge pressure (-3 mm Hg [95% CI, -5 to -1] ) and higher mean CO (0.5 L/min [95% CI, 0.1-0.8]), significantly different at low to moderate steady-state exercise but not at peak. Metabolic equivalents remained similar between treatments. In exploratory analyses, KE treatment was associated with 18% lower NT-proBNP (N-terminal pro-B-type natriuretic peptide; difference, -98 ng/L [95% CI, -185 to -23]), higher left ventricular ejection fraction (37±5 versus 34±5%; P=0.01), and lower left atrial and ventricular volumes. KE treatment for 14 days was associated with higher CO at rest and lower filling pressures, cardiac volumes, and NT-proBNP levels compared with isocaloric comparator. These changes persisted during exercise and were achieved on top of optimal medical therapy. Sustained modulation of circulating ketone bodies is a potential treatment principle in patients with heart failure with reduced ejection fraction. URL: https://www.clinicaltrials.gov; Unique identifier: NCT05161650.

Circulating 3-hydroxy butyrate predicts mortality in patients with chronic heart failure with reduced ejection fraction.

In patients with chronic heart failure with reduced ejection fraction (HFrEF), myocardial ketone metabolism is increased and short-term treatment with the ketone body 3-hydroxy butyrate (3-OHB) has beneficial haemodynamic effects. In patients with HFrEF, we investigated whether the level of circulating 3-OHB predicted all-cause mortality and sought to identify correlations between patient characteristics and circulating 3-OHB levels. We conducted a cohort study in 218 patients with HFrEF. Plasma 3-OHB levels were measured using high-performance liquid chromatography tandem mass spectrometry. Data on all-cause mortality were obtained by reviewing the patients' medical records, which are linked to the national 'Central Person Registry' that registers the timing of all deaths in the country. Mean left ventricular ejection fraction was 35±8.6%, mean age was 67±10years, 54% were New York Heart Association II, and 27% had type 2 diabetes mellitus. Median follow-up time was 7.3 (interquartile range 6.3-8.4) years. We observed large variations in 3-OHB levels between patients (median 59μM, range: 14-694μM). Patients with 3-OHB levels above the median displayed a markedly increased risk of death compared with those with low levels {hazard ratio [HR]: 2.1 [95% confidence interval (CI): 1.3-3.5], P=0.003}. In a multivariate analysis, 3-OHB predicted mortality independently of known chronic heart failure risk factors [HR: 1.004 (95% CI: 1.001-1.007), P=0.02] and with a similar statistical strength as N-terminal pro-brain natriuretic peptide (NT-proBNP) [HR: 1.0005 (95% CI: 1.000-1.001), P=0.02]. For every 100μmol increase in plasma 3-OHB, the hazard of death increased by 49%. The following factors significantly predicted 3-OHB levels in the univariate analysis: free fatty acids (FFAs) [β: 238 (95% CI: 185-292), P<0.0001], age [β: 2.43 (95% CI: 1.14-3.72), P<0.0001], plasma insulin {β: -0.28 [95% CI: -0.54-(-0.02)], P=0.036}, body mass index {β: -3.15 [95% CI: -5.26-(-0.05)], P=0.046}, diabetes [β: 44.49 (95% CI: 14.84-74.14), P=0.003], glycosylated haemoglobin [β: 1.92 (95% CI: 0.24-3.59), P=0.025], New York Heart Association class [β: 26.86 (95% CI: 5.99-47.72), P=0.012], and NT-proBNP [β: 0.03 (95% CI: 0.01-0.04), P=0.001]. In a multivariate analysis, only FFAs predicted 3-OHB levels [β: 216 (95% CI: 165-268), P>0.001]. In patients with HFrEF, circulating 3-OHB was a strong predictor of all-cause mortality independently of NT-proBNP. Circulating FFAs were the best predictor of 3-OHB levels.

Effects of ketone body 3-hydroxybutyrate on cardiac and mitochondrial function during donation after circulatory death heart transplantation

Normothermic regional perfusion (NRP) allows assessment of therapeutic interventions prior to donation after circulatory death transplantation. Sodium-3-hydroxybutyrate (3-OHB) increases cardiac output in heart failure patients and diminishes ischemia–reperfusion injury, presumably by improving mitochondrial metabolism. We investigated effects of 3-OHB on cardiac and mitochondrial function in transplanted hearts and in cardiac organoids. Donor pigs (n = 14) underwent circulatory death followed by NRP. Following static cold storage, hearts were transplanted into recipient pigs. 3-OHB or Ringer’s acetate infusions were initiated during NRP and after transplantation. We evaluated hemodynamics and mitochondrial function. 3-OHB mediated effects on contractility, relaxation, calcium, and conduction were tested in cardiac organoids from human pluripotent stem cells. Following NRP, 3-OHB increased cardiac output (P < 0.0001) by increasing stroke volume (P = 0.006), dP/dt (P = 0.02) and reducing arterial elastance (P = 0.02). Following transplantation, infusion of 3-OHB maintained mitochondrial respiration (P = 0.009) but caused inotropy-resistant vasoplegia that prevented weaning. In cardiac organoids, 3-OHB increased contraction amplitude (P = 0.002) and shortened contraction duration (P = 0.013) without affecting calcium handling or conduction velocity. 3-OHB had beneficial cardiac effects and may have a potential to secure cardiac function during heart transplantation. Further studies are needed to optimize administration practice in donors and recipients and to validate the effect on mitochondrial function.

Abstract 13255: Cardiac and Generalized SGLT2 Deficiency Does Not Protect Against Development of Heart Failure With Reduced Ejection Fraction in Mice

Sodium-glucose cotransporter 2 inhibitors (SGLT2i), originally designed for the treatment of diabetes, exert profound cardiovascular benefit and are now the standard-of-care in all forms of heart failure (HF). The mechanism of their benefit in HF is unknown. We hypothesized SGLT2i act via an on-target, extra-cardiac mechanism to promote ketosis and improve myocardial energetics. Generalized- (gKO) and cardiac-specific (csKO) SGLT2 knockout mice were generated by breeding SGLT2 floxed mice with EIIa or αMHC Cre recombinase lines, respectively. While csKO grew and developed normally, gKO mice were slightly smaller compared to WT littermates. There was no baseline cardiac phenotype in either group. The gKO mice exhibited profound glucosuria, decreased fasting serum glucose and augmented fasting ketosis compared to controls, and comparable in magnitude to the metabolic effects of 14 days of treatment with empagliflozin (Empa, 50mg/kg/day orally) in WT mice. Targeted metabolomic analysis of gKO and Empa-treated WT mice identified increased circulating and myocardial levels of the ketone body 3-hydroxybutyrate (1.9-2.3x, p&lt;0.01), along with an increase in a subset of tricarboxylic acid cycle intermediates and C4-OH, a ketone oxidation by-product. Empa treatment in WT mice following trans-aortic constriction with apical myocardial infarction (TAC/MI) improved LV dysfunction and pathological remodeling at 6 weeks (left ventricular ejection fraction (EF) 36±6 vs 28±9, p&lt;0.03; tibia length-normalized biventricular weight 9.6±0.4 vs 11.3±1.6, p&lt;0.001). In contrast, csKO and gKO mice subjected to TAC/MI had no difference in EF, strain, ventricular hypertrophy or dilation compared to WT controls. In conclusion, generalized SGLT2 deficiency phenocopies the metabolic effects of SGLT2i treatment but neither gKO nor csKO afford cardiac protection in an established HF model. These results suggest an off-target mechanism for beneficial effects of SGLT2i in HF.

Bioenergetic functions of mitochondria in liver, pancreatic acinar cells, and sperm cells of rats fed short-term high-fat or high-fat high-sugar diets

An unhealthy diet often is a cause of obesity, chronic inflammation, and metabolic disruption in multiple organs. However, the direct influence of elevated lipid or sugar consumption on liver, pancreatic, and sperm mitochondria is not well understood. The aim of the study was to investigate the functional activity of mitochondria of liver, pancreatic acinar cells, and sperm cells in rats on a short-term (7 weeks) diet with high fat or high fat and high sugar content. Male Wistar rats were on a basic, high-fat or high-fat high-sugar diet for 7 weeks. At the end of the experiment, visceral fat mass, blood glucose and lipids were measured. Mitochondrial functional activity was evaluated with oxygen consumption assay. In isolated pancreatic acinar cells, NAD(P)H autofluorescence and mitochondrial membrane potential were also studied. No difference in body mass was observed between the 3 groups at the end of the experiment. Visceral fat mass was slightly but significantly elevated in rats on a high-fat high-sugar diet. Both diets did not affect plasma glucose or triglyceride levels but caused a modest elevation of total plasma cholesterol. Respiration and oxidative phosphorylation of isolated liver mitochondria were not affected by any experimental diet. In pancreatic acinar cells, a high-fat diet caused a significant decrease of basal respiration by ~15%, but no effects were observed on the maximal rate of uncoupled respiration, mitochondrial membrane potential, or NAD(P)H autofluorescence. In these cells, a ketone body 3-hydroxybutyrate caused elevation of uncoupled respiration and NAD(P)H level irrespectively of the diet. Diets did not cause any change in sperm concentration, viability or motility. Surprisingly, in animals on a high-fat high-sugar diet, a significant increase in both basal and maximal respiration of sperm cells was observed. Collectively, these data show that while the elevated fat and sugar content in the diet does not cause significant obesity, no detrimental effects on mitochondria of the liver, pancreas, and sperm cells are observed. Keywords: diet, liver, mitochondria, pancreatic acinar cells, sperm

Ketone body 3-hydroxybutyrate elevates cardiac output through peripheral vasorelaxation and enhanced cardiac contractility

The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output and myocardial perfusion without affecting blood pressure in humans, but the cardiovascular sites of action remain obscure. Here, we test the hypothesis in rats that 3-OHB acts directly on the heart to increase cardiac contractility and directly on blood vessels to lower systemic vascular resistance. We investigate effects of 3-OHB on (a) in vivo hemodynamics using echocardiography and invasive blood pressure measurements, (b) isolated perfused hearts in Langendorff systems, and (c) isolated arteries and veins in isometric myographs. We compare Na-3-OHB to equimolar NaCl added to physiological buffers or injection solutions. At plasma concentrations of 2–4 mM in vivo, 3-OHB increases cardiac output (by 28.3±7.8%), stroke volume (by 22.4±6.0%), left ventricular ejection fraction (by 13.3±4.6%), and arterial dP/dtmax (by 31.9±11.2%) and lowers systemic vascular resistance (by 30.6±11.2%) without substantially affecting heart rate or blood pressure. Applied to isolated perfused hearts at 3–10 mM, 3-OHB increases left ventricular developed pressure by up to 26.3±7.4 mmHg and coronary perfusion by up to 20.2±9.5%. Beginning at 1–3 mM, 3-OHB relaxes isolated coronary (EC50=12.4 mM), cerebral, femoral, mesenteric, and renal arteries as well as brachial, femoral, and mesenteric veins by up to 60% of pre-contraction within the pathophysiological concentration range. Of the two enantiomers that constitute racemic 3-OHB, D-3-OHB dominates endogenously; but tested separately, the enantiomers induce similar vasorelaxation. We conclude that increased cardiac contractility and generalized systemic vasorelaxation can explain the elevated cardiac output during 3-OHB administration. These actions strengthen the therapeutic rationale for 3-OHB in heart failure management.

Beneficial Effects of Ketone Ester in Patients With Cardiogenic Shock: A Randomized, Controlled, Double-Blind Trial

BackgroundCardiogenic shock (CS) is a life-threatening condition with sparse treatment options. The ketone body 3-hydroxybutyrate has favorable hemodynamic effects in patients with stable chronic heart failure. Yet, the hemodynamic effects of exogenous ketone ester (KE) in patients with CS remain unknown. ObjectivesThe authors aimed to assess the hemodynamic effects of single-dose enteral treatment with KE in patients with CS. MethodsIn a double-blind, crossover study, 12 patients with CS were randomized to an enteral bolus of KE and isocaloric, isovolumic placebo containing maltodextrin. Patients were assessed with pulmonary artery catheterization, arterial blood samples, echocardiography, and near-infrared spectroscopy for 3 hours following each intervention separated by a 3-hour washout period. ResultsKE increased circulating 3-hydroxybutyrate (2.9 ± 0.3 mmol/L vs 0.2 ± 0.3 mmol/L, P < 0.001) and was associated with augmented cardiac output (area under the curve of relative change: 61 ± 22 L vs 1 ± 18 L, P = 0.044). Also, KE increased cardiac power output (0.07 W [95% CI: 0.01-0.14]; P = 0.037), mixed venous saturation (3 percentage points [95% CI: 1-5 percentage points]; P = 0.010), and forearm perfusion (3 percentage points [95% CI: 0-6 percentage points]; P = 0.026). Right (P = 0.048) and left (P = 0.017) ventricular filling pressures were reduced whereas heart rate and mean arterial and pulmonary arterial pressures remained similar. Left ventricular ejection fraction improved by 4 percentage points (95% CI: 2-6 percentage points; P = 0.005). Glucose levels decreased by 2.6 mmol/L (95% CI: −5.2 to 0.0; P = 0.047) whereas insulin levels remained unaltered. ConclusionsTreatment with KE improved cardiac output, biventricular function, tissue oxygenation, and glycemic control in patients with CS (Treatment With the Ketone Body 3-hydroxybutyrate in Patients With Cardiogenic Shock [KETO-SHOCK1]; NCT04642768)

Hemodynamic Effects of Ketone Bodies in Patients With Pulmonary Hypertension.

Background Pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension (CTEPH) are debilitating diseases with a high mortality. Despite emerging treatments, pulmonary vascular resistance frequently remains elevated. However, the ketone body 3-hydroxybutyrate (3-OHB) may reduce pulmonary vascular resistance in these patients. Hence, the aim was to assess the hemodynamic effects of 3-OHB in patients with PAH or CTEPH. Methods and Results We enrolled patients with PAH (n=10) or CTEPH (n=10) and residual pulmonary hypertension. They received 3-OHB infusion and placebo (saline) for 2 hours in a randomized crossover study. Invasive hemodynamic and echocardiography measurements were performed. Furthermore, we investigated the effects of 3-OHB on the right ventricle of isolated hearts and isolated pulmonary arteries from Sprague-Dawley rats. Ketone body infusion increased circulating 3-OHB levels from 0.5±0.5 to 3.4±0.7 mmol/L (P<0.001). Cardiac output improved by 1.2±0.1 L/min (27±3%, P<0.001), and right ventricular annular systolic velocity increased by 1.4±0.4 cm/s (13±4%, P=0.002). Pulmonary vascular resistance decreased by 1.3±0.3 Wood units (18%±4%, P<0.001) with no significant difference in response between patients with PAH and CTEPH. In the rat studies, 3-OHB administration was associated with decreased pulmonary arterial tension compared with saline administration (maximal relative tension difference: 12±2%, P<0.001) and had no effect on right ventricular systolic pressures (P=0.63), whereas pressures rose at a slower pace (dP/dtmax, P=0.02). Conclusions In patients with PAH or CTEPH, ketone body infusion improves cardiac output and decreases pulmonary vascular resistance. Experimental rat studies support that ketone bodies relax pulmonary arteries. Long-term studies are warranted to assess the clinical role of hyperketonemia. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT04615754.

Cerebrospinal Fluid Metabolomics: Pilot Study of Using Metabolomics to Assess Diet and Metabolic Interventions in Alzheimer's Disease and Mild Cognitive Impairment.

Brain glucose hypometabolism is an early sign of Alzheimer's disease (AD), and interventions which offset this deficit, such as ketogenic diets, show promise as AD therapeutics. Conversely, high-fat feeding may exacerbate AD risk. We analyzed the metabolomic profile of cerebrospinal fluid (CSF) in a pilot study of older adults who underwent saline and triglyceride (TG) infusions. Older adults (12 cognitively normal (CN), age 65.3 ± 8.1, and 9 with cognitive impairment (CI), age 70.9 ± 8.6) underwent a 5 h TG or saline infusion on different days using a random crossover design; CSF was collected at the end of infusion. Aqueous metabolites were measured using a targeted mass spectroscopy (MS) platform focusing on 215 metabolites from over 35 different metabolic pathways. Data were analyzed using MetaboAnalyst 4.0 and SAS. Of the 215 targeted metabolites, 99 were detectable in CSF. Only one metabolite significantly differed by treatment: the ketone body 3-hydroxybutyrate (HBA). Post hoc analyses showed that HBA levels were associated with age and markers of metabolic syndrome and demonstrated different correlation patterns for the two treatments. When analyzed by cognitive diagnosis group, TG-induced increases in HBA were over 3 times higher for those with cognitive impairment (change score CN +9.8 uM ± 8.3, CI +32.4 ± 7.4, p = 0.0191). Interestingly, individuals with cognitive impairment had higher HBA levels after TG infusion than those with normal cognition. These results suggest that interventions that increase plasma ketones may lead to higher brain ketones in groups at risk for AD and should be confirmed in larger intervention studies.

Amperometric Biosensor Strip With Carbon Nanotube and Ketone Body 3-Hydroxybutyrate Dehydrogenase

We propose an amperometric biosensor strip for the detection of ketone body 3-hydroxybutyrate (3HB) in serum. The biosensor strip has a small volume ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\approx 2 ~\mu \text{L}$ </tex-math></inline-formula> ) capillary chamber. The working electrode consists of carbon nanotubes (CNTs) and 3HB dehydrogenase (3HBDH). The CNTs are dispersed in aqueous media with sodium cholate (SC) surfactant and mixed with HBDH solution. Ultraviolet–visible, electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) demonstrated that the CNTs were well dispersed in aqueous solution. Any type of CNT enhances the current owing to an enzymatic reaction without an electron transfer mediator. A small amount of HBDH/CNT solution is dipped into a small-area (1.5 mm2) electrode and sealed with a coversheet to make the reaction chamber. The sensor can detect 3HB in human serum within the range of 0.05–4.4 mM, which can cover physiological use. The response time is 15 s.

Abstract 15256: Treatment With Ketone Ester in Patients With Cardiogenic Shock Improves Cardiac Performance

Introduction: Cardiogenic shock is a life-threatening condition but no medical therapy has been documented to improve outcome. Recent evidence suggests that the ketone body 3-hydroxybutyrate (3-OHB) has favorable hemodynamic effects in patients with stable chronic heart failure. However, the hemodynamic effects of exogenous ketone supplements to patients with cardiogenic shock remains unknown. Hypothesis: In patients hospitalized with cardiogenic shock, acute treatment with a ketone ester has beneficial hemodynamic effects. Methods: In a randomized, double-blind, crossover study, we included 12 patients with cardiogenic shock who were treated with inotropes and/or vasopressors. In random order, patients received an enteral bolus of 500 mg/kg ketone ester (KetoneAid Inc.) and isocaloric, isovolemic placebo containing maltodextrin. Each study period lasted for three hours and was separated by a 3-hour washout period. The primary endpoint was change in cardiac output expressed as area under curve (AUC). Secondary endpoints were recorded at the end of each study period and included mixed venous saturation (SVO 2 ), pulmonary capillary wedge pressure, and echocardiographic measures of contractile function. Results: The ketone ester increased circulating 3-OHB significantly vs. placebo (peak: 3.7±0.5 mmol/L vs. 0.3±0.1 mmol/L, AUC: 6.0±0.9 mmol/L x hour vs. 0.3±0.1 mmol/L x hour, all p&lt;0.001). This was accompanied by an increase in cardiac output (5.1±0.3 L/min vs. 4.4±0.3 L/min, p=0.043; AUC of relative change: 61±22 L vs. 1±18 L, p=0.044), primarily due to a higher stroke volume (53±4 mL vs. 47±4 mL, p=0.048). Ketone ester increased SVO 2 vs. placebo (61±2% vs. 57±3%, p=0.035) whereas heart rate, mean arterial, pulmonary arterial, and pulmonary capillary wedge pressures did not differ between study periods. Ketone ester increased left ventricular ejection fraction (32±3% vs. 28±3%, p=0.005) and global longitudinal strain (-7.3±0.7% vs. -6.5±0.6%, p=0.01) as compared with placebo. Conclusions: Treatment with ketone ester has beneficial hemodynamic effects in patients with cardiogenic shock by increasing cardiac output and left ventricular function.

Influence of biological sex and exercise on murine cardiac metabolism

Although the structural and functional effects of exercise on the heart are well established, the metabolic changes that occur in the heart during and after exercise remain unclear. In this study, we used metabolomics to assess time-dependent changes in the murine cardiac metabolome following 1 session of treadmill exercise. After the exercise bout, we also recorded blood lactate, glucose, and ketone body levels and measured cardiac mitochondrial respiration. In both male and female mice, moderate- and high-intensity exercise acutely increased blood lactate levels. In both sexes, low- and moderate-intensity exercise augmented circulating 3-hydroxybutryrate levels immediately after the exercise bout; however, only in female mice did high-intensity exercise increase 3-hydroxybutyrate levels, with significant increases occurring 1 h after the exercise session. Untargeted metabolomics analyses of sedentary female and male hearts suggest considerable sex-dependent differences in basal cardiac metabolite levels, with female hearts characterized by higher levels of pantothenate, pyridoxamine, homoarginine, tryptophan, and several glycerophospholipid and sphingomyelin species and lower levels of numerous metabolites, including acetyl coenzyme A, glucuronate, gulonate, hydroxyproline, prolyl-hydroxyproline, carnosine, anserine, and carnitinylated and glycinated species, as compared with male hearts. Immediately after a bout of treadmill exercise, both male and female hearts had higher levels of corticosterone; however, female mice showed more extensive exercise-induced changes in the cardiac metabolome, characterized by significant, time-dependent changes in amino acids (e.g., serine, alanine, tyrosine, tryptophan, branched-chain amino acids) and the ketone body 3-hydroxybutyrate. Results from experiments using isolated cardiac mitochondria suggest that high-intensity treadmill exercise does not acutely affect respiration or mitochondrial coupling; however, female cardiac mitochondria demonstrate generally higher adenosine diphosphate sensitivity compared with male cardiac mitochondria. Collectively, these findings in mice reveal key sex-dependent differences in cardiac metabolism and suggest that the metabolic network in the female heart is more responsive to physiological stress caused by exercise.

Defects in the Proteome and Metabolome in Human Hypertrophic Cardiomyopathy.

Defects in energetics are thought to be central to the pathophysiology of hypertrophic cardiomyopathy (HCM); yet, the determinants of ATP availability are not known. The purpose of this study is to ascertain the nature and extent of metabolic reprogramming in human HCM, and its potential impact on contractile function. We conducted proteomic and targeted, quantitative metabolomic analyses on heart tissue from patients with HCM and from nonfailing control human hearts. In the proteomic analysis, the greatest differences observed in HCM samples compared with controls were increased abundances of extracellular matrix and intermediate filament proteins and decreased abundances of muscle creatine kinase and mitochondrial proteins involved in fatty acid oxidation. These differences in protein abundance were coupled with marked reductions in acyl carnitines, byproducts of fatty acid oxidation, in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, branched chain amino acids, and their breakdown products, were all significantly increased in HCM hearts. ATP content, phosphocreatine, nicotinamide adenine dinucleotide and its phosphate derivatives, NADP and NADPH, and acetyl CoA were also severely reduced in HCM compared with control hearts. Functional assays performed on human skinned myocardial fibers demonstrated that the magnitude of observed reduction in ATP content in the HCM samples would be expected to decrease the rate of cross-bridge detachment. Moreover, left atrial size, an indicator of diastolic compliance, was inversely correlated with ATP content in hearts from patients with HCM. HCM hearts display profound deficits in nucleotide availability with markedly reduced capacity for fatty acid oxidation and increases in ketone bodies and branched chain amino acids. These results have important therapeutic implications for the future design of metabolic modulators to treat HCM.

Altered cholesterol homeostasis in critical illness-induced muscle weakness: effect of exogenous 3-hydroxybutyrate

BackgroundMuscle weakness is a complication of critical illness which hampers recovery. In critically ill mice, supplementation with the ketone body 3-hydroxybutyrate has been shown to improve muscle force and to normalize illness-induced hypocholesterolemia. We hypothesized that altered cholesterol homeostasis is involved in development of critical illness-induced muscle weakness and that this pathway can be affected by 3-hydroxybutyrate.MethodsIn both human critically ill patients and septic mice, the association between circulating cholesterol concentrations and muscle weakness was assessed. In septic mice, the impact of 3-hydroxybutyrate supplementation on cholesterol homeostasis was evaluated with use of tracer technology and through analysis of markers of cholesterol metabolism and downstream pathways.ResultsSerum cholesterol concentrations were lower in weak than in non-weak critically ill patients, and in multivariable analysis adjusting for baseline risk factors, serum cholesterol was inversely correlated with weakness. In septic mice, plasma cholesterol correlated positively with muscle force. In septic mice, exogenous 3-hydroxybutyrate increased plasma cholesterol and altered cholesterol homeostasis, by normalization of plasma mevalonate and elevation of muscular, but not hepatic, expression of cholesterol synthesis genes. In septic mice, tracer technology revealed that 3-hydroxybutyrate was preferentially taken up by muscle and metabolized into cholesterol precursor mevalonate, rather than TCA metabolites. The 3-hydroxybutyrate protection against weakness was not related to ubiquinone or downstream myofiber mitochondrial function, whereas cholesterol content in myofibers was increased.ConclusionsThese findings point to a role for low cholesterol in critical illness-induced muscle weakness and to a protective mechanism-of-action for 3-hydroxybutyrate supplementation.

Ketone body 3-hydroxybutyrate as a biomarker of aggression

Human aggression is a complex behaviour, the biological underpinnings of which remain poorly known. To gain insights into aggression biology, we studied relationships with aggression of 11 low-molecular-weight metabolites (amino acids, ketone bodies), processed using 1H nuclear magnetic resonance spectroscopy. We used a discovery sample of young adults and an independent adult replication sample. We studied 725 young adults from a population-based Finnish twin cohort born 1983–1987, with aggression levels rated in adolescence (ages 12, 14, 17) by multiple raters and blood plasma samples at age 22. Linear regression models specified metabolites as the response variable and aggression ratings as predictor variables, and included several potential confounders. All metabolites showed low correlations with aggression, with only one—3-hydroxybutyrate, a ketone body produced during fasting—showing significant (negative) associations with aggression. Effect sizes for different raters were generally similar in magnitude, while teacher-rated (age 12) and self-rated (age 14) aggression were both significant predictors of 3-hydroxybutyrate in multi-rater models. In an independent replication sample of 960 adults from the Netherlands Twin Register, higher aggression (self-rated) was also related to lower levels of 3-hydroxybutyrate. These exploratory epidemiologic results warrant further studies on the role of ketone metabolism in aggression.

Ketone Body 3-Hydroxybutyrate Ameliorates Atherosclerosis via Receptor Gpr109a-Mediated Calcium Influx.

Atherosclerosis is a chronic inflammatory disease that can cause acute cardiovascular events. Activation of the NOD‐like receptor family, pyrin domain containing protein 3 (NLRP3) inflammasome enhances atherogenesis, which links lipid metabolism to sterile inflammation. This study examines the impact of an endogenous metabolite, namely ketone body 3‐hydroxybutyrate (3‐HB), on a mouse model of atherosclerosis. It is found that daily oral administration of 3‐HB can significantly ameliorate atherosclerosis. Mechanistically, 3‐HB is found to reduce the M1 macrophage proportion and promote cholesterol efflux by acting on macrophages through its receptor G‐protein‐coupled receptor 109a (Gpr109a). 3‐HB–Gpr109a signaling promotes extracellular calcium (Ca2+) influx. The elevation of intracellular Ca2+ level reduces the release of Ca2+ from the endothelium reticulum (ER) to mitochondria, thus inhibits ER stress triggered by ER Ca2+ store depletion. As NLRP3 inflammasome can be activated by ER stress, 3‐HB can inhibit the activation of NLRP3 inflammasome, which triggers the increase of M1 macrophage proportion and the inhibition of cholesterol efflux. It is concluded that daily nutritional supplementation of 3‐HB attenuates atherosclerosis in mice.

Dietary resistant starch preserved through mild extrusion of grain alters fecal microbiome metabolism of dietary macronutrients while increasing immunoglobulin A in the cat.

Dietary digestion-resistant starch (RS) provides health benefits to the host via gut microbiome-mediated metabolism. The degree to which cats manifest beneficial changes in response to RS intake was examined. Healthy cats (N = 36) were fed identically formulated foods processed under high (n = 17) or low (n = 19) shear extrusion conditions (low and high RS levels [LRS and HRS], respectively). Fecal samples collected after 3 and 6 weeks' feeding were assayed for stool firmness score, short-chain fatty acids, ammonia, and changes to the global metabolome and microbiome; fecal immunoglobulin A (IgA) was analyzed at week 6. Few differences were seen in proximate analyses of the foods; stool firmness scores did not differ. In cats consuming HRS food, concentrations of fecal butyrate and the straight chain:branched chain fatty acid ratio were significantly greater in feces at both weeks 3 and 6, while fecal ammonia was reduced at week 6 relative to feces from LRS-fed cats. Fecal IgA concentrations were significantly higher at week 6 with HRS food. RS consumption altered 47% of the fecal metabolome; RS-derived sugars and metabolites associated with greater gut health, including indoles and polyamines, increased in the cats consuming HRS food relative to those fed the LS food, while endocannabinoid N-acylethanolamines decreased. Consumption of HRS food increased concentrations of the ketone body 3-hydroxybutyrate in feces and elevated concentrations of reduced members of NADH-coupled redox congeners and NADH precursors. At the microbiome genus-level, 21% of operational taxonomic units were significantly different between food types; many involved taxa with known saccharolytic or proteolytic proclivities. Microbiome taxa richness and Shannon and Simpson alpha diversity were significantly higher in the HRS group at both weeks. These data show that feline consumption of grain-derived RS produces potentially beneficial shifts in microbiota-mediated metabolism and increases IgA production.

SUN-548 Ketone Bodies in Critical Illness Alter Cholesterol Synthesis in Skeletal Muscle, Interlinked with Protection Against Weakness

Background: Critically ill patients often develop muscle weakness, which hampers recovery. In septic mice, supplementing parenteral nutrition (PN) with ketone body 3-hydroxybutyrate (3HB) attenuated muscle weakness, but also normalized sepsis-induced low cholesterol1. As 3HB can be metabolized into cholesterol, we hypothesized that improved muscle function with 3HB was related to altered cholesterol metabolism. Methods: In a catheterized, fluid-resuscitated, antibiotics-treated mouse model of prolonged sepsis (cecal ligation and puncture), septic mice received PN supplemented with either D,L-3HB sodium salt (PN+3HB; 150 mg/day) or isocaloric glucose (PN+gluc) and healthy pair-fed mice served as controls (n=15-17 mice/group). After 5 days, ex vivo muscle force and markers of cholesterol metabolism were assessed. In 600 weak and non-weak human critically ill patients (weakness assessed on day 8±1 in ICU by MRC sum score), serum total cholesterol concentration was measured on ICU day 3 or last day for shorter stayers. Results: In mice, PN+3HB counteracted the sepsis-induced lowering of plasma cholesterol (p=0.04), which correlated positively with absolute muscle force (R2=0.19, p=0.002). Plasma mevalonate concentration, a surrogate marker of cholesterol synthesis, was reduced by sepsis (p=0.03 vs. controls), but normalized by PN+3HB (p=0.001 vs. PN+gluc). Skeletal muscle expression of cholesterol synthesis genes Srebf2, Hmgcr and Hmgcs1 was higher in PN+3HB than in PN+gluc septic mice (p≤0.01). Expression of cholesterol uptake receptor Ldlr was also increased in PN+3HB septic mice (p=0.02 vs. PN+gluc), whereas PN+3HB did not affect cholesterol efflux transporters. In contrast, PN+3HB did not alter sepsis-induced alterations in markers of hepatic cholesterol metabolism. Plasma concentration of ubiquinone, a central co-factor of the mitochondrial respiratory chain derived from mevalonate, was increased by sepsis, irrespective of PN+3HB (p<0.0001 vs. controls) and PN+3HB could not counteract sepsis-induced muscular mitochondrial dysfunction (p≤0.0009 vs. controls). This excludes the involvement of ubiquinone in muscle weakness attenuation by 3HB supplementation. However, higher muscular Nceh1 expression was observed with PN+3HB (p≤0.04 vs. controls and PN+gluc), suggesting enhanced shuttling of newly formed free cholesterol to the membranes. In human ICU patients, lower serum cholesterol concentration was observed in weak vs. non-weak patients (p=0.0002). In a multivariate model adjusted for baseline risk factors, low serum cholesterol concentrations were independently associated with muscle weakness (p=0.05). Conclusion: 3HB supplementation of PN enhanced muscle cholesterol synthesis and increased plasma cholesterol, which appeared to independently protect against sepsis-induced muscle weakness. 1 Goossens et al. (2019). Crit Care. 23: 236.