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Background/Objectives: Ketone salt (KS) containing a racemic beta-hydroxybutyrate mixture is commonly used as an alternative fuel source as it may lead to improved health and/or performance. We postulate that KS will raise acetoacetate levels and represent the effectiveness of exogenous KS as an energy source. We conducted a pilot study to quantify changes in the circulating acetoacetate following KS and to determine if any changes in acetoacetate were associated with the changes in circulating beta-hydroxybutyrate. Methods: Thirteen adults (21.6 ± 4.3 years old; seven males/six females) completed this randomized, triple-blinded, placebo-controlled, cross-over design study. Participants consumed either KS or flavor-matched placebo with a one-week washout period between supplements. Blood samples were taken before and 30 min after consuming each supplement, and plasma acetoacetate and beta-hydroxybutyrate levels were measured by gas chromatography/mass spectrometry. Results: The consumption of KS resulted in a significant increase in acetoacetate from baseline. The increase in acetoacetate after the KS supplement was significantly greater than that following the consumption of a placebo (↑ 0.57 ± 0.44 mM vs. ↑ 0.07 ± 0.23 mM, p = 0.009, d = 0.86), and significantly and strongly related to the change in blood beta-hydroxybutyrate (r = 0.757, p < 0.001). Conclusions: Our findings indicate that KS markedly increases plasma ketone body interconversion, presumably to supply peripheral tissues for ATP generation.

Terrestrial and aquatic environments contaminated with animal urine may contribute to the transmission of Leptospira , a causative agent of leptospirosis in humans and wild/domesticated animals. Although enormous amounts of work have been done decoding the ecophysiology, the factors governing the cell growth and virulence in Leptospires derived from environmental samples still remain elusive. Here, we show oxidation of a wide array of organic acids including acetoacetate by a new strain of Leptospira interrogans designated as KeTo, isolated from a sewage sample originating from a wildlife enclosure located at Mangalore, India. We further demonstrate the susceptibility of KeTo to ethyl ester of acetoacetate (ethyl acetoacetate, EA). A 4.7 Mbp genome of KeTo shared the highest relatedness to pathogenic L. interrogans RGA T (99.3%), followed by L. kirschneri 3522C T (91.3%) and other related species of Leptospira (80.8‒74.3%), and harbored genes encoding acetoacetyl-CoA synthetase and acetoacetate decarboxylase respectively involved in the acetoacetate utilization and acetone formation. In line with this, strain KeTo oxidized acetoacetate when supplied as a sole carbon. Aqueous EA suppressed biofilm formation ( p  < 0.0001) of KeTo in basal Ellinghausen–McCullough–Johnson–Harris (EMJH) medium. Similarly, significant inhibition in the growth/biofilm of Leptospira was recorded in semisolid EMJH with/without blood supplementation when exposed to volatile EA. The extent of ketone body oxidation and susceptibility to EA was found to vary with strain as evident through the analysis of L. interrogans serogroup Australis sv. Australis strain Ballico and L. interrogans serogroup Icterohaemorrhagiae sv. Lai Like strain AF61. In conclusion, our study demonstrated the ketone body metabolic ability and susceptibility to an esterified derivative of a major ketone body in the tested strains of L. interrogans . Molecular aspects governing EA-driven growth inhibition warrant further investigations to develop optimal therapeutics for leptospirosis.

Ursodeoxycholic acid (UDCA) and acetoacetate are natural compounds present in the human intestine and blood, respectively. A number of studies highlighted that besides their well-known primary biological roles, both compounds possess the ability to influence a variety of cellular processes involved in the etiology of various diseases. These reasons suggested the potential of acetoacetate–UDCA hybrids as possible therapeutic agents and prompted us to develop a synthetic strategy to selectively derivatize the hydroxyl groups of the bile acid with acetoacetyl moieties. 3α-acetoacetoxy UDCA was obtained (60% isolated yield) via the regioselective transesterification of methyl acetoacetate with UDCA promoted by the Candida antarctica lipase B (CAL-B). 3α,7β-bis-acetoacetoxy UDCA was obtained instead by thermal condensation of methyl acetoacetate and UDCA (80% isolated yield). This bis-adduct was finally converted to the 7β-acetoacetoxy UDCA (82% isolated yield) via CAL-B catalyzed regioselective alcoholysis of the ester group on the 3α position. In order to demonstrate the value of the above new hybrids as UDCA-based scaffolds, 3α-acetoacetoxy UDCA was subjected to multicomponent Biginelli reaction with benzaldehyde and urea to obtain the corresponding 4-phenyl-3,4-dihydropyrimidin-2-(1H)-one derivative in 65% isolated yield.

The metabolic ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, β-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency’s Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

The ketogenic diet (KD) is a high-fat, low-carbohydrate treatment for medically intractable epilepsy. One of the hallmark features of the KD is the production of ketone bodies which have long been believed, but not yet proven, to exert direct anti-seizure effects. The prevailing view has been that ketosis is an epiphenomenon during KD treatment, mostly due to clinical observations that blood ketone levels do not correlate well with seizure control. Nevertheless, there is increasing experimental evidence that ketone bodies alone can exert anti-seizure properties through a multiplicity of mechanisms, including but not limited to: (1) activation of inhibitory adenosine and ATP-sensitive potassium channels; (2) enhancement of mitochondrial function and reduction in oxidative stress; (3) attenuation of excitatory neurotransmission; and (4) enhancement of central γ-aminobutyric acid (GABA) synthesis. Other novel actions more recently reported include inhibition of inflammasome assembly and activation of peripheral immune cells, and epigenetic effects by decreasing the activity of histone deacetylases (HDACs). Collectively, the preclinical evidence to date suggests that ketone administration alone might afford anti-seizure benefits for patients with epilepsy. There are, however, pragmatic challenges in administering ketone bodies in humans, but prior concerns may largely be mitigated through the use of ketone esters or balanced ketone electrolyte formulations that can be given orally and induce elevated and sustained hyperketonemia to achieve therapeutic effects.The ketogenic diet (KD) is a high-fat, low-carbohydrate treatment for medically intractable epilepsy. One of the hallmark features of the KD is the production of ketone bodies which have long been believed, but not yet proven, to exert direct anti-seizure effects. The prevailing view has been that ketosis is an epiphenomenon during KD treatment, mostly due to clinical observations that blood ketone levels do not correlate well with seizure control. Nevertheless, there is increasing experimental evidence that ketone bodies alone can exert anti-seizure properties through a multiplicity of mechanisms, including but not limited to: (1) activation of inhibitory adenosine and ATP-sensitive potassium channels; (2) enhancement of mitochondrial function and reduction in oxidative stress; (3) attenuation of excitatory neurotransmission; and (4) enhancement of central γ-aminobutyric acid (GABA) synthesis. Other novel actions more recently reported include inhibition of inflammasome assembly and activation of peripheral immune cells, and epigenetic effects by decreasing the activity of histone deacetylases (HDACs). Collectively, the preclinical evidence to date suggests that ketone administration alone might afford anti-seizure benefits for patients with epilepsy. There are, however, pragmatic challenges in administering ketone bodies in humans, but prior concerns may largely be mitigated through the use of ketone esters or balanced ketone electrolyte formulations that can be given orally and induce elevated and sustained hyperketonemia to achieve therapeutic effects.

Our previous studies have shown that mangiferin decreased serum triglycerides and free fatty acids (FFAs) by increasing FFAs oxidation in both animal and cell experiments. This study sought to evaluate the effects of mangiferin on serum lipid profiles in overweight patients with hyperlipidemia. Overweight patients with hyperlipidemia (serum triglyceride ≥ 1.70 mmol/L and total cholesterol ≥ 5.2 mmol/L) were included in this double-blind randomized controlled trial. Participants were randomly allocated to groups, either receiving mangiferin (150 mg/day) or identical placebo for 12 weeks. The lipid profile and serum levels of mangiferin, glucose, L-carnitine, β-hydroxybutyrate and acetoacetate were determined at baseline and 12 weeks. A total of 97 participants completed the trial. Compared with the placebo control, mangiferin supplementation significantly decreased the serum levels of triglycerides and FFAs and insulin resistance index. Mangiferin supplementation also significantly increased the serum levels of mangiferin, high-density lipoprotein cholesterol, L-carnitine, β-hydroxybutyrate and acetoacetate and increased lipoprotein lipase activity. However, there were no differences in the serum levels of total cholesterol, low-density lipoprotein cholesterol, serum glucose and insulin between groups. Mangiferin supplementation could improve serum lipid profiles by reducing serum triglycerides and FFAs in overweight patients with hyperlipidemia, partly due to the promotion of FFAs oxidation.

Ketone bodies are metabolites that replace glucose as the main fuel of the brain in situations of glucose scarcity, including prolonged fasting, extenuating exercise, or pathological conditions such as diabetes. Beyond their role as an alternative fuel for the brain, the impact of ketone bodies on neuronal physiology has been highlighted by the use of the so-called “ketogenic diets,” which were proposed about a century ago to treat infantile seizures. These diets mimic fasting by reducing drastically the intake of carbohydrates and proteins and replacing them with fat, thus promoting ketogenesis. The fact that ketogenic diets have such a profound effect on epileptic seizures points to complex biological effects of ketone bodies in addition to their role as a source of ATP. In this review, we specifically focus on the ability of ketone bodies to regulate neuronal excitability and their effects on gene expression to respond to oxidative stress. Finally, we also discuss their capacity as signaling molecules in brain cells.

Pathway engineering is a useful technology for producing desired compounds on a large scale by modifying the biosynthetic pathways of host organisms using genetic engineering. We focused on acetoacetate esters as novel low-cost substrates and established an efficient terpenoid production system using pathway-engineered recombinant Escherichia coli. Functional analysis using recombinant E. coli proteins of 18 carboxylesterases identified from the microbial esterases and lipases database showed that the p-nitrobenzyl esterase (PnbA) from Bacillus subtilis specifically hydrolyzed two acetoacetate esters: methyl acetoacetate (MAA) and ethyl acetoacetate (EAA). We generated a plasmid (pAC-Mev/Scidi/Aacl/PnbA) co-expressing PnbA and six enzymes of the mevalonate pathway gene cluster from Streptomyces, isopentenyl diphosphate isomerase type I from Saccharomyces cerevisiae, and acetoacetyl-coenzyme A ligase from Rattus norvegicus. The plasmid pAC-Mev/Scidi/Aacl/PnbA was introduced into E. coli along with plasmid expressing carotenoid (lycopene) or sesquiterpene (β-bisabolene) biosynthesis genes, and the terpenoid production was evaluated following the addition of acetoacetate esters as substrates. These recombinant E. coli strains used MAA and EAA as substrates for the biosynthesis of terpenoids and produced almost equivalent concentrations of target compounds compared with the previous production system that used mevalonolactone and lithium acetoacetate. The findings of this study will enable the production of useful terpenoids from low-cost substrates, which may facilitate their commercial production on an industrial scale in the future.Key points• PnbA from Bacillus subtilis exhibits acetoacetate hydrolysis activity.• A plasmid enabling terpenoid synthesis from acetoacetate esters was constructed.• Acetoacetate esters as substrates enable a low-cost production of terpenoids.

α-Glycerophosphocholine (GPC) is a putative acetylcholine precursor that potentially increases growth hormone secretion through the action of acetylcholine-stimulated catecholamine. The aim of this study was to investigate acute physiologic responses to a single intake of GPC. Eight healthy male subjects (25 ± 1 y old) ingested GPC 1000 mg or a placebo in a double-blind randomized crossover study. Fasting blood samples were obtained before the administration of GPC (baseline) and 60 and 120 min after administration. All subjects repeated the identical protocol using the placebo. Plasma free choline levels significantly increased at 60 and 120 min after GPC administration. Plasma growth hormone secretion was increased significantly 60 min after taking GPC, whereas no significant change was observed with the placebo. In addition, the serum free fatty acid was increased 120 min after GPC ingestion, but no changes were seen with the placebo. Moreover, serum acetoacetate and 3-hydroxybutyrate levels, which are indices of hepatic fat oxidation, were increased at 120 min after taking GPC, whereas the placebo had no effect. These findings suggest that a single dose of GPC increases growth hormone secretion and hepatic fat oxidation, with concomitant increases in choline levels, in young adults.

The increased metabolic activity of the heart as a pump involves a high demand of mitochondrial adenosine triphosphate (ATP) production for its mechanical and electrical activities accomplished mainly via oxidative phosphorylation, supplying up to 95% of the necessary ATP production, with the rest attained by substrate-level phosphorylation in glycolysis. In the normal human heart, fatty acids provide the principal fuel (40–70%) for ATP generation, followed mainly by glucose (20–30%), and to a lesser degree (<5%) by other substrates (lactate, ketones, pyruvate and amino acids). Although ketones contribute 4–15% under normal situations, the rate of glucose use is drastically diminished in the hypertrophied and failing heart which switches to ketone bodies as an alternate fuel which are oxidized in lieu of glucose, and if adequately abundant, they reduce myocardial fat delivery and usage. Increasing cardiac ketone body oxidation appears beneficial in the context of heart failure (HF) and other pathological cardiovascular (CV) conditions. Also, an enhanced expression of genes crucial for ketone break down facilitates fat or ketone usage which averts or slows down HF, potentially by avoiding the use of glucose-derived carbon needed for anabolic processes. These issues of ketone body utilization in HF and other CV diseases are herein reviewed and pictorially illustrated.