Impaired glucose utilization in the brain of patients with delirium following hip fracture.

Alterations in brain energy metabolism have long been proposed as one of several neurobiological processes contributing to delirium. This is supported by previous findings of altered CSF lactate and neuron-specific enolase concentrations and decreased glucose uptake on brain-PET in patients with delirium. Despite this, there are limited data on metabolic alterations found in CSF samples, and targeted metabolic profiling of CSF metabolites involved in energy metabolism has not been performed. The aim of the study was to investigate whether metabolites related to energy metabolism in the serum and CSF of patients with hip fracture are associated with delirium. The study cohort included 406 patients with a mean age of 81 years (standard deviation 10 years), acutely admitted to hospital for surgical repair of a hip fracture. Delirium was assessed daily until the fifth postoperative day. CSF was collected from all 406 participants at the onset of spinal anaesthesia, and serum samples were drawn concurrently from 213 participants. Glucose and lactate in CSF were measured using amperometry, whereas plasma glucose was measured in the clinical laboratory using enzymatic photometry. Serum and CSF concentrations of the branched-chain amino acids, 3-hydroxyisobutyric acid, acetoacetate and ß-hydroxybutyrate were measured using gas chromatography-tandem mass spectrometry (GC-MS/MS). In total, 224 (55%) patients developed delirium pre- or postoperatively. Ketone body concentrations (acetoacetate, ß-hydroxybutyrate) and branched-chain amino acids were significantly elevated in the CSF but not in serum among patients with delirium, despite no group differences in glucose concentrations. The level of 3-hydroxyisobutyric acid was significantly elevated in both CSF and serum. An elevation of CSF lactate during delirium was explained by age and comorbidity. Our data suggest that altered glucose utilization and a shift to ketone body metabolism occurs in the brain during delirium.

Acetoacetate and d- and l-ß-hydroxybutyrate have distinct effects on basal and insulin-stimulated glucose uptake in L6 skeletal muscle cells.

Ketone bodies (acetoacetate and ß-hydroxybutyrate) are oxidized in skeletal muscle mainly during fasting as an alternative source of energy to glucose. Previous studies suggest that there is a negative relationship between increased muscle ketolysis and muscle glucose metabolism in mice with obesity and/or type 2 diabetes. Therefore, we investigated the connection between increased ketone body exposure and muscle glucose metabolism by measuring the effect of a 3-h exposure to ketone bodies on glucose uptake in differentiated L6 myotubes. We showed that exposure to acetoacetate at a typical concentration (0.2 mM) resulted in increased basal glucose uptake in L6 myotubes, which was dependent on increased membrane glucose transporter type 4 (GLUT4) translocation. Basal and insulin-stimulated glucose uptake was also increased with a concentration of acetoacetate reflective of diabetic ketoacidosis or a ketogenic diet (1 mM). We found that ß-hydroxybutyrate had a variable effect on basal glucose uptake: a racemic mixture of the two ß-hydroxybutyrate enantiomers (d and l) appeared to decrease basal glucose uptake, while 3 mM d-ß-hydroxybutyrate alone increased basal glucose uptake. However, the effects of the ketone bodies individually were not observed when acetoacetate was present in combination with ß-hydroxybutyrate. These results provide insight that will help elucidate the effect of ketone bodies in the context of specific metabolic diseases and nutritional states (e.g., type 2 diabetes and ketogenic diets).NEW & NOTEWORTHY A limited number of studies investigate the effect of ketone bodies at concentrations reflective of both typical fasting and ketoacidosis. We tested a mix of physiologically relevant concentrations of ketone bodies, which allowed us to highlight the differential effects of d- and l-ß-hydroxybutyrate and acetoacetate on skeletal muscle cell glucose uptake. Our findings will assist in better understanding the mechanisms that contribute to muscle insulin resistance and provide guidance on recommendations regarding ketogenic diets.

Pioglitazone reduces serum ketone bodies in sodium-glucose cotransporter-2 inhibitor-treated non-obese type 2 diabetes: A single-centre, randomized, crossover trial.

AIM: To examine the effects of the thiazolidinedione (TZD) pioglitazone on reducing ketone bodies in non-obese patients with T2DM treated with the sodium-glucose cotransporter-2 (SGLT2) inhibitor canagliflozin. METHODS: Crossover trials with two periods, each treatment period lasting 4 weeks, with a 4-week washout period, were conducted. Participants were randomly assigned in a 1:1 ratio to receive pioglitazone combined with canagliflozin (PIOG + CANA group) versus canagliflozin monotherapy (CANA group). The primary outcome was change (Δ) in ß-hydroxybutyric acid (ß-HBA) before and after the CANA or PIOG + CANA treatments. The secondary outcomes were Δchanges in serum acetoacetate and acetone, the rate of conversion into urinary ketones, and Δchanges in factors related to SGLT2 inhibitor-induced ketone body production including non-esterified fatty acids (NEFAs), glucagon, glucagon to insulin ratio, and noradrenaline (NA). Analyses were performed in accordance with the intention-to-treat principle. RESULTS: Twenty-five patients with a mean age of 49 ± 7.97 years and a body mass index of 25.35 ± 2.22 kg/m2 were included. One patient discontinued the study during the washout period. Analyses revealed a significant increase in the levels of serum ketone bodies and an elevation in the rate of conversion into urinary ketones after both interventions. However, differernces in levels of ketone bodies (except for acetoacetate) in the PIOG + CANA group were significantly smaller than in the CANA group (219.84 ± 80.21 µmol/L vs. 317.69 ± 83.07 µmol/L, p < 0.001 in ß-HBA; 8.98 ± 4.17 µmol/L vs. 12.29 ± 5.27 µmol/L, p = 0.018 in acetone). NEFA, glucagon, glucagon to insulin ratio, and NA were also significantly increased after both CANA and PIOG + CANA treatments; while only NEFAs demonstrated a significant difference between the two groups. Correlation analyses revealed a significant association between the difference in Δchanges in serum NEFA levels with the differences in Δchanges in ketones of ß-HBA and acetoacetate. CONCLUSION: Supplementation of pioglitazone could alleviate canagliflozin-induced ketone bodies. This benefit may be closely associated with decreased substrate NEFAs rather than other factors including glucagon, fasting insulin and NA.

Plasma metabolomics reveals the intervention mechanism of different types of exercise on chronic unpredictable mild stress-induced depression rat model.

OBJECTIVE: To study the effects of different types of exercise on the plasma metabolomics of chronic unpredictable mild stress (CUMS)-induced depressed rats based on 1H-NMR metabolomics techniques, and to explore the potential mechanisms of exercise for the treatment of depression. Rats were randomly divided into blank control group (C), CUMS control group (D), pre-exercise with CUMS group (P), CUMS with aerobic exercise group, CUMS with resistance exercise group (R), and CUMS with aerobic + resistance exercise group (E). The corresponding protocol intervention was applied to each group of rats. Body weight, sucrose preference and open field tests were performed weekly during the experiment to evaluate the extent of depression in rats. Plasma samples from each group of rats were collected at the end of the experiment, and then the plasma was analyzed by 1H-NMR metabolomics combined with multivariate statistical analysis methods to identify differential metabolites and perform metabolic pathway analysis. (1) Compared with the group D, the body weight, sucrose preference rate, and the number of crossings and standings in the different types of exercise groups were significantly improved (p < 0.05 or p < 0.01). (2) Compared to group C, a total of 15 differential metabolites associated with depression were screened in the plasma of rats in group D, involving 6 metabolic pathways. Group P can regulate the levels of 6 metabolites: valine, lactate, inositol, glucose, phosphocreatine, acetoacetic acid. Group A can regulate the levels of 6 metabolites: N-acetylglycoprotein, leucine, lactate, low density lipoprotein, glucose and acetoacetic acid. Group R can regulate the levels of 6 metabolites: choline, lactate, inositol, glucose, phosphocreatine and acetoacetic acid. Group E can regulate the levels of 5 metabolites: choline, citric acid, glucose, acetone and acetoacetic acid. The different types of exercise groups can improve the depressive symptoms in CUMS rats, and there are common metabolites and metabolic pathways for their mechanism of effects. This study provides a powerful analytical tool to study the mechanism of the antidepressant effect of exercise, and provides an important method and basis for the early diagnosis, prevention and treatment of depression.

Enzymatic Synthesis of New Acetoacetate-Ursodeoxycholic Acid Hybrids as Potential Therapeutic Agents and Useful Synthetic Scaffolds as Well.

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,7ß-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 7ß-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 lipogenic enzyme acetoacetyl-CoA synthetase and ketone body utilization for denovo lipid synthesis, a review.

Acetoacetyl-CoA synthetase (AACS) is the key enzyme in the anabolic utilization of ketone bodies (KBs) for denovo lipid synthesis, a process that bypasses citrate and ATP citrate lyase. This review shows that AACS is a highly regulated, cytosolic, and lipogenic enzyme and that many tissues can readily use KBs for denovo lipid synthesis. AACS has a low micromolar Km for acetoacetate, and supply of acetoacetate should not limit its activity in the fed state. In many tissues, AACS appears to be regulated in conjunction with the need for cholesterol, but in adipose tissue, it seems tied to fatty acid synthesis. KBs are readily utilized as substrates for lipid synthesis in lipogenic tissues, including liver, adipose tissue, lactating mammary gland, skin, intestinal mucosa, adrenals, and developing brain. In numerous studied cases, KBs served several-fold better than glucose as substrates for lipid synthesis, and when present, KBs suppressed the utilization of glucose for lipid synthesis. Here, it is hypothesized that a physiological role for the utilization of KBs for lipid synthesis is a metabolic process of lipid interconversion. Fatty acids are converted to KBs in liver, and then, the KBs are utilized to synthesize cholesterol and other long-chain fatty acids in liver and nonhepatic tissues. The conversion of fatty acids to cholesterol via the KBs may be a particularly important example of lipid interconversion. Utilizing KBs for lipid synthesis is glucose sparing and probably is important with low carbohydrate diets. Metabolic situations and tissues where this pathway may be important are discussed.

A catalytic dyad modulates conformational change in the CO2-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase.

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO2 and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H+ and CO2 to favor the metabolically preferred carboxylation product, acetoacetate.

A (S)-3-Hydroxybutyrate Dehydrogenase Belonging to the 3-Hydroxyacyl-CoA Dehydrogenase Family Facilitates Hydroxyacid Degradation in Anaerobic Bacteria.

Ketone bodies, including acetoacetate, 3-hydroxybutyrate, and acetone, are produced in the liver of animals during glucose starvation. Enzymes for the metabolism of (R)-3-hydroxybutyrate have been extensively studied, but little is known about the metabolism of its enantiomer (S)-3-hydroxybutyrate. Here, we report the characterization of a novel pathway for the degradation of (S)-3-hydroxybutyrate in anaerobic bacteria. We identify and characterize a stereospecific (S)-3-hydroxylbutyrate dehydrogenase (3SHBDH) from Desulfotomaculum ruminis, which catalyzes the reversible NAD(P)H-dependent reduction of acetoacetate to form (S)-3-hydroxybutyrate. 3SHBDH also catalyzes oxidation of d-threonine (2R, 3S) and l-allo-threonine (2S, 3S), consistent with its specificity for ß-(3S)-hydroxy acids. Isothermal calorimetry experiments support a sequential mechanism involving binding of NADH prior to (S)-3-hydroxybutyrate. Homologs of 3SHBDH are present in anaerobic fermenting and sulfite-reducing bacteria, and experiments with Clostridium pasteurianum showed that 3SHBDH, acetate CoA-transferase (YdiF), and (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) are involved together in the degradation of (S)-3-hydroxybutyrate as a carbon and energy source for growth. (S)-3-hydroxybutyrate is a human metabolic marker and a chiral precursor for chemical synthesis, suggesting potential applications of 3SHBDH in diagnostics or the chemicals industry. IMPORTANCE (R)-3-hydroxybutyrate is well studied as a component of ketone bodies produced by the liver and of bacterial polyesters. However, the biochemistry of its enantiomer (S)-3-hydroxybutyrate is poorly understood. This study describes the identification and characterization of a stereospecific (S)-3-hydroxylbutyrate dehydrogenase and its function in a metabolic pathway for the degradation of (S)-3-hydroxybutyrate as a carbon and energy source in anaerobic bacteria. (S)-3-hydroxybutyrate is a mammalian metabolic marker and a precursor for chemical synthesis and bioplastics, suggesting potential applications of these enzymes in diagnostics and biotechnology.

Longitudinal Changes in Circulating Ketone Body Levels in Patients With Acute Heart Failure: A Post Hoc Analysis of the EMPA-Response-AHF Trial.

BACKGROUND: Ketone bodies are endogenous fuels produced by the liver under conditions of metabolic or neurohormonal stress. Circulating ketone bodies are increased in patients with chronic heart failure (HF), yet little is known about the effect of acute HF on ketosis. We tested the hypothesis that ketogenesis is increased in patients with acute decompensated HF. METHODS AND RESULTS: This was a post hoc analysis of 79 patients with acute HF included in the EMPA-RESPONSE-AHF trial, which compared sodium-dependent glucose-cotransporter protein 2 inhibitor treatment with empagliflozin for 30 days with placebo in patients with acute HF [NCT03200860]. Plasma concentrations of ketone bodies acetone, ß-hydroxybutyrate, and acetoacetate were measured at baseline and 5 different timepoints. Changes in ketone bodies over time were monitored using repeated measures analysis of variance. In the total cohort, median total ketone body concentration was 251 µmol/L (interquartile range, 178-377 µmol/L) at baseline, which gradually decreased to 202 µmol/L (interquartile range, 156-240 µmol/L) at day 30 (P = .041). Acetone decreased from 60 µmol/L (interquartile range, 34-94 µmol/L) at baseline to 30 µmol/L (interquartile range, 21-42 µmol/L) ( P < .001), whereas ß-hydroxybutyrate and acetoacetate remained stable over time. Higher acetone concentrations were correlated with higher N-terminal pro brain natriuretic peptide levels (r = 0.234; P = .039). Circulating ketone bodies did not differ between patients treated with empagliflozin or placebo throughout the study period. A higher acetone concentration at baseline was univariately associated with a greater risk of the composite end point, including in-hospital worsening HF, HF rehospitalizations, and all-cause mortality after 30 days. However, after adjustment for age and sex, acetone did not remain an independent predictor for the combined end point. CONCLUSIONS: Circulating ketone body concentrations, and acetone in particular, were significantly higher during an episode of acute decompensated HF compared with after stabilization. Treatment with empagliflozin did not affect ketone body concentrations in patients with acute HF.

Acetoacetate and ß-hydroxybutyrate reduce mouse embryo viability via differential metabolic and epigenetic mechanisms.

RESEARCH QUESTION: Does the ketone acetoacetate (AcAc) alone, or combined with ß-hydroxybutyrate (ßOHB), impact mouse embryo development, metabolism, histone acetylation and viability? DESIGN: Pronucleate mouse oocytes were cultured in vitro in G1/G2 media supplemented with ketones (AcAc or AcAc + ßOHB) at concentrations representing those in maternal serum during pregnancy (0.04 mmol/l AcAc, 0.1 mmol/l ßOHB), standard diet consumption (0.1 mmol/l AcAc, 0.25 mmol/l ßOHB), ketogenic diet consumption (0.8 mmol/l AcAc, 2 mmol/l ßOHB) and diabetic ketoacidosis (2 mmol/l AcAc, 4 mmol/l ßOHB). Day 5 blastocysts were assessed for cell allocation, glucose metabolism and histone acetylation. Day 4 blastocysts exposed to 0.8 mmol/l AcAc + 2 mmol/l ßOHB were transferred to standard-fed recipient females, and E14.5 fetal and placental development assessed. RESULTS: Exposure to 2 mmol/l AcAc or 0.8 mmol/l AcAc + 2 mmol/l ßOHB did not impair blastocyst development, but significantly increased glucose consumption (P = 0.001 each), lowered glycolytic flux (P = 0.01, P < 0.001) and elevated trophectoderm (TE) histone 3 lysine 27 acetylation (H3K27ac; P < 0.001 each) compared with unexposed controls. Preimplantation AcAc + ßOHB exposure reduced post-implantation fetal development by 25% (P = 0.037), and delayed female-specific fetal limb development (P = 0.019) and estimated fetal age (P = 0.019) compared with controls. CONCLUSION: Preimplantation exposure to ketones affects underlying metabolism and histone acetylation in blastocysts that are associated with persistent, female-specific perturbations in fetal development. A periconceptional diet that elevates ketone concentrations may impair human embryonic viability.

The Catalytic Mechanism of Acetoacetate Decarboxylase: A Detailed Study of Schiff Base Formation, Protonation States, and Their Impact on Catalysis.

The enzyme acetoacetate decarboxylase (AAD) has a crucial function in the process of decarboxylating the substrate acetoacetate (AA). It has been extensively studied over the years, but its exact catalytic mechanism has remained partly unsolved due to the difficulty in assessing reaction intermediates. In this study, we combine molecular dynamics and electronic structure calculations to rediscover its catalytic mechanism. Our results show that the presence of the substrate, the acetoacetate, significantly influences the electrostatic potential of the active site. Furthermore, our simulations show that the decarboxylation reaction can take place by means of a direct proton transfer instead of via an enamine intermediate, which is thought to be strictly necessary. This work provides new insights into the role of the electrostatic interactions on the catalytic activity of AAD and for the first time connects it to the catalytic mechanism of other decarboxylases.

Identification of potential biomarkers in malnutrition children with severity by 1H-NMR-based metabolomics: a preliminary study in the Chinese population.

PURPOSE: Child malnutrition is a global public health problem, but the underlying pathophysiologic mechanisms with severity remain poorly understood, and the potential biomarkers served to the clinical diagnosis are still not available. This study aimed to identify the serum metabolic characteristics of malnourished children with severity. METHODS: Fasted overnight serum samples were collected following clinical standard procedures among 275 malnourished and 199 healthy children from the Women and Children's Hospital, Xiamen University Child Health Department from July 2020 to May 2022. Nuclear magnetic resonance (NMR)-based metabolomics strategy was applied to identify the potential serum biomarkers of malnutrition from 275 malnourished children aged 4 to 84 months with mild (Mil, 199 cases), moderate (Mod, 101 cases), and severe (Sev, 7 cases) malnutrition. RESULTS: Ten, fifteen, and fifteen differential metabolites were identified from the Mil, Mod, and Sev malnutrition groups, respectively. Eight common metabolites, including increased acetoacetate, acetone, ethanol, succinate, 3-hydroxybutyrate, and decreased alanine, methionine, and N-acetyl-glycoprotein, could be the potential biomarkers for malnourished children. The altered metabolic pathways were mainly related to energy metabolism and amino acid metabolism via the network-based pathway enrichment. CONCLUSION: Eight potential biomarkers, including acetoacetate, acetone, ethanol, succinate, 3-hydroxybutyrate, alanine, methionine, and N-acetyl-glycoprotein, could characterize the child malnutrition. Child malnutrition-induced abnormal energy metabolism, impaired nutrition utilization and the reduced nutrient availability, and more metabolic disturbance will appear with the severity. Our results are valuable for further studies on the etiology and pathogenesis of malnutrition for clinical intervention and improvement.

Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF-MEK1 Signaling.

Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a "synthetic lethal" interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild-type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.

Untargeted metabolomics elucidated biosynthesis of polyhydroxyalkanoate by mixed microbial cultures from waste activated sludge under different pH values.

Waste activated sludge has been frequently used as mixed substrate to produce polyhydroxyalkanoate (PHA). However, insufficient research on microbial metabolism has led to difficulties in regulating PHA accumulation in mixed microbial cultures (MMCs). To explore the variation of functional genes during domestication and the effect of different pH conditions on metabolic pathways during PHA accumulation, MMCs were domesticated by adding acetate and propionate with aerobic dynamic feeding strategy for 60 days. As the domestication progressed, the microbial community diversity declined and PHA-producing bacteria, Brevundimonas, Dechloromonas and Hyphomonas, were enriched. Through bacterial function prediction by PICRUSt the gene rpoE involved in starvation resistance of bacteria was enriched after the domestication. The pH value of 8.5 was the best condition for PHA accumulation in MMCs, under which a maximum PHA content reached 23.50% and hydroxybutyric (HB)/hydroxyvaleric (HV) reached 2.22. Untargeted metabolomics analysis exhibited that pH conditions of 7 and 8.5 could promote the up-regulation of significant differential metabolites, while higher alkaline conditions caused the inhibition of metabolic activity. Functional annotation showed that pH condition of 8.5 significantly affected Pyrimidine metabolism, resulting in an increase in PHA production. Regarding the pathways of PHA biosynthesis, acetoacetate was found to be significant in the metabolism of hydroxybutyric, and the alkaline condition could restrain the conversion from hydroxybutyric (HB) to the acetoacetate to protect PHB accumulation in MMCs compared with neutral condition. Taken together, the present results can advance the fundamental understanding of metabolic function in PHA accumulation under different pH conditions.

Quantitative Determination of (R)-3-Hydroxybutyl (R)-3-Hydroxybutyrate (Ketone Ester) and Its Metabolites Beta-hydroxybutyrate, 1-3-Butanediol, and Acetoacetate in Human Plasma Using LC-MS.

Ketone ester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) has gained popularity as an exogenous means to achieve ketosis. Regarding its potential as a therapeutic prodrug, it will be necessary to study its pharmacokinetic profile and its proximal metabolites (beta-hydroxybutyrate, 1,3-butanediol, and acetoacetate) in humans. Here we develop and validate two LC-MS methods for quantifying KE and its metabolites in human plasma. The first assay uses a C18 column to quantitate ketone ester, beta-hydroxybutyrate, and 1,3-butanediol, and the second assay uses a hydrophilic interaction liquid chromatography (HILIC) column for the quantitation of acetoacetate. The method was partially validated for intra- and inter-day accuracy and precision based on the ICH M10 guidelines. For both the assays, the intra- and inter-run accuracy was ±15% of the nominal concentration, and the precision (%CV) was <15% for all 4 molecules being quantified. The matrix effect for all molecules was evaluated and ranged from -62.1 to 44.4% (combined for all molecules), while the extraction recovery ranged from 65.1 to 119% (combined for all molecules). Furthermore, the metabolism of ketone ester in human plasma and human serum albumin was studied using the method. Non-saturable metabolism of ketone ester was seen in human plasma at concentrations as high as 5 mM, and human serum albumin contributed to the metabolism of ketone ester. Together, these assays can be used to track the entire kinetics of ketone ester and its proximal metabolites. The reverse-phase method was used to study the metabolic profile of KE in human plasma and the plasma protein binding of 1,3-BD.

Metabolomics profile in acute respiratory distress syndrome by nuclear magnetic resonance spectroscopy in patients with community-acquired pneumonia.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a challenging clinical problem. Discovering the potential metabolic alterations underlying the ARDS is important to identify novel therapeutic target and improve the prognosis. Serum and urine metabolites can reflect systemic and local changes and could help understanding metabolic characterization of community-acquired pneumonia (CAP) with ARDS. METHODS: Clinical data of patients with suspected CAP at the First Affiliated Hospital of Wenzhou Medical University were collected from May 2020 to February 2021. Consecutive patients with CAP were enrolled and divided into two groups: CAP with and without ARDS groups. 1H nuclear magnetic resonance-based metabolomics analyses of serum and urine samples were performed before and after treatment in CAP with ARDS (n = 43) and CAP without ARDS (n = 45) groups. Differences metabolites were identifed in CAP with ARDS. Furthermore, the receiver operating characteristic (ROC) curve was utilized to identify panels of significant metabolites for evaluating therapeutic effects on CAP with ARDS. The correlation heatmap was analyzed to further display the relationship between metabolites and clinical characteristics. RESULTS: A total of 20 and 42 metabolites were identified in the serum and urine samples, respectively. Serum metabolic changes were mainly involved in energy, lipid, and amino acid metabolisms, while urine metabolic changes were mainly involved in energy metabolism. Elevated levels of serum 3-hydroxybutyrate, lactate, acetone, acetoacetate, and decreased levels of serum leucine, choline, and urine creatine and creatinine were detected in CAP with ARDS relative to CAP without ARDS. Serum metabolites 3-hydroxybutyrate, acetone, acetoacetate, citrate, choline and urine metabolite 1-methylnicotinamide were identified as a potential biomarkers for assessing therapeutic effects on CAP with ARDS, and with AUCs of 0.866 and 0.795, respectively. Moreover, the ROC curve analysis revealed that combined characteristic serum and urine metabolites exhibited a better classification system for assessing therapeutic effects on CAP with ARDS, with a AUC value of 0.952. In addition, differential metabolites strongly correlated with clinical parameters in patients with CAP with ARDS. CONCLUSIONS: Serum- and urine-based metabolomics analyses identified characteristic metabolic alterations in CAP with ARDS and might provide promising circulatory markers for evaluating therapeutic effects on CAP with ARDS.

Synthesis of Ester-Substituted Indolizines from 2-Propargyloxypyridines and 1,3-Dicarbonyls.

Two new complementary Au(I)-catalyzed methods for the preparation of ester-substituted indolizines from easily accessible 2-propargyloxypyridines and either acetoacetates or dimethyl malonate are reported. These reactions tolerate a wide range of functionality, allowing for diversification at three distinct positions of the product (R, R1, R2). For electron-poor substrates, the highest yields are observed upon reaction with acetoacetates, while neutral and electron-rich substrates give higher yields upon treatment with dimethyl malonate.

Interference of ketone bodies on laboratory creatinine measurement in children with DKA: a call for change in testing practices.

BACKGROUND: The presence of ketone bodies (KBs) can interfere with creatinine (Cr) measurement in both enzymatic and Jaffe methods. Since a high proportion of children hospitalized for diabetic ketoacidosis (DKA) develop acute kidney injury (AKI), here we investigate whether KB interferences affect the accuracy of pediatric Cr measurement. METHODS: Residual patient plasma samples were pooled to make three Cr levels (~ 50, 100, and 250 µM). KBs (acetone, acetoacetate, and ß-hydroxybutyrate) were used to spike the pooled samples. All samples were measured for Cr by two enzymatic methods (E1 and E2), two Jaffe methods (J1 and J2), and LC-MS/MS. LC-MS/MS was considered the gold standard, and the % difference in Cr concentration was calculated for each method. RESULTS: E1 and E2 were unaffected by the presence of all three KBs. J1 and J2 were unaffected by the presence of ß-hydroxybutyrate. The presence of acetone resulted in dose-dependent positive interference in both Jaffe methods, whereas the presence of acetoacetate resulted in dose-dependent positive and negative interference in J1 and J2, respectively. CONCLUSIONS: Compared to the enzymatic methods, the Jaffe methods were much more susceptible to interference by acetone and acetoacetate, especially at lower Cr values which are commonly seen in pediatrics. Interpretation of changes in Cr concentration between different hospitals when transferring patients can become ambiguous and true kidney function unclear if different methods are used without awareness of method-specific biases. To improve DKA patient care, we recommend standardizing all of the Cr methods to an enzymatic method. A higher resolution version of the Graphical abstract is available as Supplementary information.

Converting the 3-quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4-chloroacetoacetate reductase by site-directed mutagenesis.

In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (kcat /Km ) for COBE was 51.36 s-1 ·mM-1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.

A dietary ketone ester mitigates histological outcomes of NAFLD and markers of fibrosis in high-fat diet fed mice.

Nutritional ketosis as a therapeutic tool has been extended to the treatment of metabolic diseases, including obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to determine whether dietary administration of the ketone ester (KE) R,S-1,3-butanediol diacetoacetate (BD-AcAc2) attenuates markers of hepatic stellate cell (HSC) activation and hepatic fibrosis in the context of high-fat diet (HFD)-induced obesity. Six-week-old male C57BL/6J mice were placed on a 10-wk ad libitum HFD (45% fat, 32% carbohydrates, 23% proteins). Mice were then randomized to one of three groups (n = 10 per group) for an additional 12 wk: 1) control (CON), continuous HFD; 2) pair-fed (PF) to KE, and 3) KE (HFD + 30% energy from BD-AcAc2, KE). KE feeding significantly reduced histological steatosis, inflammation, and total NAFLD activity score versus CON, beyond improvements observed for calorie restriction alone (PF). Dietary KE supplementation also reduced the protein content and gene expression of profibrotic markers (α-SMA, COL1A1, PDGF-ß, MMP9) versus CON (P < 0.05), beyond reductions observed for PF versus CON. Furthermore, KE feeding increased hepatic markers of anti-inflammatory M2 macrophages (CD163) and also reduced proinflammatory markers [tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and cellular communication network factor 1 (CCN1)] versus CON and PF (P ≤ 0.05), in the absence of changes in markers of total hepatic macrophage content (F4/80 and CD68; P > 0.05). These data highlight that the dietary ketone ester BD-AcAc2 ameliorates histological NAFLD and inflammation and reduces profibrotic and proinflammatory markers. Future studies to further explore potential mechanisms are warranted.NEW & NOTEWORTHY To our knowledge, this is the first study focusing on hepatic outcomes in response to dietary ketone ester feeding in male mice with HFD-induced NAFLD. Novel findings include that dietary ketone ester feeding ameliorates NAFLD outcomes via reductions in histological steatosis and inflammation. These improvements were beyond those observed for caloric restriction alone. Furthermore, dietary ketone ester feeding was associated with greater reductions in markers of hepatic fibrogenesis and inflammation compared with control and calorie-restricted mice.