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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 β-hydroxybutyric acid (β-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/m 2 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 β-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 β-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., (© 2024 John Wiley & Sons Ltd.)

Ketone bodies are an energy substrate produced by the liver and used during states of low carbohydrate availability, such as fasting or prolonged exercise. High ketone concentrations can be present with insulin insufficiency and are a key finding in diabetic ketoacidosis (DKA). During states of insulin deficiency, lipolysis increases and a flood of circulating free fatty acids is converted in the liver into ketone bodies-mainly beta-hydroxybutyrate and acetoacetate. During DKA, beta-hydroxybutyrate is the predominant ketone in blood. As DKA resolves, beta-hydroxybutyrate is oxidized to acetoacetate, which is the predominant ketone in the urine. Because of this lag, a urine ketone test might be increasing even as DKA is resolving. Point-of-care tests are available for self-testing of blood ketones and urine ketones through measurement of beta-hydroxybutyrate and acetoacetate and are cleared by the US Food and Drug Administration (FDA). Acetone forms through spontaneous decarboxylation of acetoacetate and can be measured in exhaled breath, but currently no device is FDA-cleared for this purpose. Recently, technology has been announced for measuring beta-hydroxybutyrate in interstitial fluid. Measurement of ketones can be helpful to assess compliance with low carbohydrate diets; assessment of acidosis associated with alcohol use, in conjunction with SGLT2 inhibitors and immune checkpoint inhibitor therapy, both of which can increase the risk of DKA; and to identify DKA due to insulin deficiency. This article reviews the challenges and shortcomings of ketone testing in diabetes treatment and summarizes emerging trends in the measurement of ketones in the blood, urine, breath, and interstitial fluid., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: R.M.B. has received research support, consulted, or has been on a scientific advisory board for Abbott Diabetes Care, Ascensia, Bigfoot Biomedical, CeQur, Dexcom, Hygieia, Insulet, Lilly, Medtronic, Novo Nordisk, Onduo, Roche Diabetes Care, Sanofi, Tandem Diabetes Care, United Healthcare, Vertex Pharmaceutical and Zealand Pharma. His technology research is funded in part by NIH/NIDDK and Helmsley Charitable Trust. R.M.B.’s employer, nonprofit HealthPartners Institute, contracts for his services, and no personal income goes to him. K.C. receives research support provided to her institution from Dexcom, Abbott, Medtronic, Altimmune, Lilly, Novo Nordisk, and Insulet and has received consulting fees from Dexcom. E.C. is an advisory board member and consultant for Novo Nordisk, Eli Lilly, Adocia, MannKind, Lexicon, Arecor. E.C. was also a speaker for Novo Nordisk. K.D. is the chair of the Joint British Diabetes Societies for Inpatient Care and has received speaker fees, travel, or taken part in advisory boards for AstraZeneca, Sanofi Diabetes, Boehringer Ingelheim, Lilly, and Novo Nordisk. J.L.S. has conducted clinical trials for Eli Lilly, Insulet, and Medtronic and has received in-kind support for research studies from Dexcom and Medtronic. She has consulted for Eli Lilly, Lexicon, Medtronic, and Sanofi. She has been a member of advisory boards for Bigfoot Biomedical, Cecelia Health, Eli Lilly, Insulet, the T1D Fund, and Vertex. G.E.U. reports research funds to Emory University from Astra Zeneca, Abbott, and Dexcom. The support from Astra Zeneca ended in 2022. D.C.K. is a consultant to EOFlow, Fractyl Health, Integrity, Lifecare, Rockley Photonics, and Thirdwayv. J.H., A.M.Y., and I.N. have nothing to disclose.

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.

Background: An association of higher levels of β-hydroxybutyrate (β-HB) in serum with greater mortality in hemodialysis (HD) patients has been reported. This study examined the significance of arterial ketone body ratio (AcAc/β-HB), a relevant marker of energy state, in HD patients., Methods: The levels of arterial AcAc and β-HB, and AcAc/β-HB ratio were determined in 49 HD patients just before undergoing an HD session. Additionally, changes in those levels during the session were examined to investigate their associations with clinical nutritional markers., Results: Arterial β-HB, but not AcAc, was significantly higher at the baseline in 25 patients with type 2 diabetes mellitus (T2DM) as compared to 24 non-DM patients, with a significant reduction in arterial AcAc/β-HB ratio seen in those with DM. Although the arterial AcAc/β-HB ratio before the HD session was significantly higher in the non-DM group, it did not differ significantly after the session between the groups, indicating a faster rate of β-HB disappearance from circulation in non-DM HD patients during the interdialytic period. Multiple regression analysis, which included age, gender, presence/absence of DM, log HD duration, log β-HB, and log AcAc/β-HB ratio as independent variables, revealed an independent and significant association of log AcAc/ β-HB ratio, but not log β-HB, with serum albumin and uric acid., Conclusion: We found that a decreased AcAc/β-HB ratio resulting from increased β-HB, but not increased β-HB itself, was a significant factor independently associated with decreased levels of serum albumin and uric acid, known to be related to higher mortality in HD patients. Furthermore, it is possible that higher mortality in DM HD patients can be explained by reduced arterial AcAc/β-HB ratio.

Acute myocardial infarction (AMI) is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia is essential for the ischemic patient's prevention and treatment. Despite the great prevalence and incidence only a small number of studies utilize a metabolomic approach to describe AMI condition. Recent studies have shown the impact of metabolites on epigenetic changes, in these studies plasma metabolites were related to neurological outcome of the patients making metabolomic studies increasingly interesting. The aim of this study was to describe metabolomic response of an organism to ischemic stress through the changes in energetic metabolites and aminoacids in blood plasma in patients overcoming acute myocardial infarction. Blood plasma from patients in the first 12 h after onset of chest pain was collected and compared with volunteers without any history of ischemic diseases via NMR spectroscopy. Lowered plasma levels of pyruvate, alanine, glutamine and neurotransmitter precursors tyrosine and tryptophan were found. Further, we observed increased plasma levels of 3-hydroxybutyrate and acetoacetate in balance with decreased level of lipoproteins fraction, suggesting the ongoing ketonic state of an organism. Discriminatory analysis showed very promising performance where compounds: lipoproteins, alanine, pyruvate, glutamine, tryptophan and 3-hydroxybutyrate were of the highest discriminatory power with feasibility of successful statistical discrimination.

With a prevalence of up to 43 % subclinical ketosis is one of the most common diseases in dairy cows in their transition period. In itself, this may cause subsequent diseases such as clinical ketosis or lameness. Therefore, monitoring of animals in this stage is of importance. In addition to the measurement of β-hydroxybutyrate or acetoacetate in blood, milk, and urine as well as the observation of the animals, computer-assisted systems are suitable means of monitoring. Information such as animal identification and activity data are recorded on a data logger and transmitted to a computer. A change in activity may be an indication of an underlying disease days before the onset of additional clinical signs. In cases of ketosis, a decrease in activity may be observed 5 days before the clinical diagnosis is made. Thus, these data are a valuable contribution in monitoring the cattle herd's health status for both the farmer and the veterinarian. Activity measurement may also be employed for the detection of a beginning lameness. In the presence of lameness, the individual's activity decreases and periods of lying are longer. Activity measurement via transponder as a part of the herd monitoring provides important information on lameness prevalence in the herd. In the presence of a lameness a visual assessment should additionally be made. Lameness scores (Locomotion score, Gait score) have been developed for this purpose and add to determining the lameness status of the herd. This way the animals are divided into different lameness classes. Based on this classification those individuals in need of claw trimming or further treatment may be identified leading to amelioration or prevention of secondary diseases. Due to lameness and subsequent reduction of activity and feed intake, the animals may develop subclinical or clinical ketosis. Therefore, under consideration of both animal welfare and economic factors early disease detection and prophylaxis is desirable and should be a main objective of herd monitoring., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Replacing dietary saturated fatty acids (SFAs) with polyunsaturated fatty acids (PUFA) reduces the plasma low-density lipoprotein (LDL) cholesterol and subsequently the risk of cardiovascular disease. However, beyond changes in LDL cholesterol, we lack a complete understanding of the physiologic alterations that occur when improving dietary fat quality., Objectives: The aim of this study was to gain knowledge of metabolic alterations paralleling improvements in the fat quality of the diet., Methods: We recently conducted an 8-wk, double-blind, randomized controlled trial replacing SFAs with PUFAs in healthy subjects with moderate hypercholesterolemia (n = 99). In the present substudy, we performed comprehensive metabolic profiling with multiple platforms (both nuclear magnetic resonance- and mass spectrometry-based technology) (n = 99), and analyzed peripheral blood mononuclear cell gene expression (n = 95) by quantitative real-time polymerase chain reaction., Results: A large number of lipoprotein subclasses, myristoylcarnitine and palmitoylcarnitine, and kynurenine were reduced when SFAs were replaced with PUFAs. In contrast, bile acids, proprotein convertase subtilisin/kexin type 9, acetate, and acetoacetate were increased by the intervention. Some amino acids were also altered by the intervention. The mRNA levels of LXRA and LDLR were increased, in addition to several liver X receptor α target genes and genes involved in inflammation, whereas the mRNA levels of UCP2 and PPARD were decreased in peripheral blood mononuclear cells after replacing SFAs with PUFAs. Partial least squares-discriminant analysis showed that the 30 most important variables that contributed to class separation spanned all classes of biomarkers, and was in accordance with the univariate analysis., Conclusions: Applying metabolomics in randomized controlled dietary intervention trials has the potential to extend our knowledge of the biological and molecular effects of dietary fat quality. This study was registered at clinicaltrials.gov as NCT01679496., (Copyright © American Society for Nutrition 2019.)

Soft-drink diabetic ketosis, characterized by acute onset ketosis induced by excessive ingestion of sugar-containing drinks, is often seen in obese, young patients, even with undiagnosed type 2 diabetes. We herein report a 15-year-old obese patient with the apolipoprotein E4/2 phenotype, in whom eruptive xanthomas lead to a diagnosis of soft-drink diabetic ketosis. He developed multiple asymptomatic yellowish papules on the auricles, back, buttocks and the extensor surfaces of the elbows and knees. He initially visited a dermatology clinic and his blood triglyceride and HbA1c levels were found to be 6,490 mg/dL and 16.5%, respectively. He was referred to our hospital for treatment of hyperglycemia and hypertyriglyceridemia. On admission, he had ketonuria and increased blood levels of 3-hydroxybutylate and acetoacetate. He habitually drank 1-3 litters of sweet beverages daily to quench his thirst. Therefore, "soft-drink diabetic ketosis" was diagnosed. Severe hypertriglyceridemia was considered to have been a consequence of impaired insulin action and his apolipoprotein E4/2 phenotype. We treated the diabetic ketosis and hypertriglyceridemia with intensive insulin therapy and a fat-restricted diet. At discharge, he no longer required insulin therapy and his blood glucose levels were controlled with metformin and voglibose. Along with amelioration of the hyperglycemia, triglyceride levels decreased to 247 mg/dL without administration of anti-hyperlipidemia agents. The eruptive xanthoma lesions gradually diminished in size and number and eventually disappeared by 12 months. This case provides an instructive example of eruptive xanthomas serving as a sign of severe dysregulation, not only of lipid, but also glucose, metabolism.

Sodium glucose co-transporter 2 (SGLT2) inhibitors have been associated with increased serum ketone body levels in patients with type 2 diabetes mellitus (T2DM). In the present analysis we evaluated serum ketone body levels and variability in 1278 Japanese patients with T2DM treated with canagliflozin 100 or 200 mg. Similar mean increases in ketone body concentrations of ~2-fold were seen with both canagliflozin doses. The median (interquartile range) percent change from baseline was 62% (0;180) for acetoacetate and 78% (2;236) for β-hydroxybutyrate. Approximately two-thirds of the variability in each ketone measure was attributed to intra-subject variability. Intra-subject variability was higher for serum ketones than other metabolites. Patients in the lowest response tertile exhibited no increase in ketones. Those in the highest response tertile tended to be male and have higher fasting plasma glucose levels, lower insulin levels, and longer T2DM duration at baseline. Moreover, changes in serum ketones were not fully explained by changes in plasma fatty acids, suggesting downstream effects of SGLT2 inhibition on hepatic metabolism that favour ketogenesis. In summary, increases in serum ketone bodies with canagliflozin were greater and more variable than changes in other metabolic measures in Japanese patients with T2DM., (© 2018 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Background: Euglycaemic ketoacidosis has been reported after sodium-glucose cotransporter 2 (SGLT2) inhibitor treatment. However, the degree of ketonaemia and its metabolic effects have not been well investigated. Our study examined the degree of ketonaemia induced by SGLT2 inhibition and its association with metabolic profiles in type 2 diabetes mellitus (T2DM)., Methods: Biochemical parameters, including insulin, glucagon, free fatty acid (FFA), β-hydroxybutyrate (BHB) and acetoacetate (ACA) levels, were measured in 119 T2DM patients after dapagliflozin treatment for>3 months, and compared with a matched control group., Results: Levels of total ketones, BHB and ACA were significantly higher in the dapagliflozin group than in the control group: 283.7±311.0 vs 119.8±143.8μmol/L; 188.3±226.6 vs 78.0±106.7μmol/L; and 94.1±91.3 vs 41.8±39.1μmol/L, respectively (all P<0.001). After dapagliflozin treatment, BHB was higher than the upper limit of normal (>440μmol/L) in 13 (10.9%) patients who had no relevant symptoms. BHB level after dapagliflozin treatment correlated positively with HbA 1c (r=0.280), FFA levels (r=0.596) and QUICKI (r=0.238), and negatively with BMI (r=-0.222), insulin-to-glucagon ratio (r=-0.199) and HOMA-IR (r=-0.205; all P<0.05). On multivariable linear regression analysis, QUICKI was independently associated with BHB level., Conclusion: Ketone levels were higher in T2DM patients treated with dapagliflozin than in controls, but with no clinical symptoms or signs of ketonaemia. Low-grade ketonaemia after dapagliflozin treatment may also be associated with improved insulin sensitivity., (Copyright © 2017. Published by Elsevier Masson SAS.)

Therapeutic hypothermia (TH) initiated within 6 h from birth is the most effective therapeutic approach for moderate to severe hypoxic-ischemic encephalopathy (HIE). However, underlying mechanisms and effects on the human metabolism are not yet fully understood. This work aims at studying the evolution of several energy related key metabolites in newborns with HIE undergoing TH employing gas chromatography - mass spectrometry. The method was validated following stringent FDA requirements and applied to 194 samples from a subgroup of newborns with HIE (N = 61) enrolled in a multicenter clinical trial (HYPOTOP) for the determination of lactate, pyruvate, ketone bodies and several Krebs cycle metabolites at different sampling time points. The analysis of plasma samples from newborns with HIE revealed a decrease of lactate, pyruvate and β-hydroxybutyrate concentrations, whereas rising malate concentrations were observed. In healthy control newborns (N = 19) significantly lower levels of pyruvate and lactate were found in comparison to age-matched newborns with HIE undergoing TH, whereas acetoacetate and β-hydroxybutyrate levels were clearly increased. Access to a validated analytical method and a controlled cohort of newborns with HIE undergoing hypothermia treatment for the first time allowed the in-depth study of the evolution of key metabolites of metabolic junctions in this special population.

Aim: To identify demographic, clinical, metabolomic, and lifestyle related predictors of relapse in adult ulcerative colitis (UC) patients., Methods: In this prospective pilot study, UC patients in clinical remission were recruited and followed-up at 12 mo to assess a clinical relapse, or not. At baseline information on demographic and clinical parameters was collected. Serum and urine samples were collected for analysis of metabolomic assays using a combined direct infusion/liquid chromatography tandem mass spectrometry and nuclear magnetic resolution spectroscopy. Stool samples were also collected to measure fecal calprotectin (FCP). Dietary assessment was performed using a validated self-administered food frequency questionnaire., Results: Twenty patients were included (mean age: 42.7 ± 14.8 years, females: 55%). Seven patients (35%) experienced a clinical relapse during the follow-up period. While 6 patients (66.7%) with normal body weight developed a clinical relapse, 1 UC patient (9.1%) who was overweight/obese relapsed during the follow-up ( P = 0.02). At baseline, poultry intake was significantly higher in patients who were still in remission during follow-up (0.9 oz vs 0.2 oz, P = 0.002). Five patients (71.4%) with FCP > 150 μg/g and 2 patients (15.4%) with normal FCP (≤ 150 μg/g) at baseline relapsed during the follow-up ( P = 0.02). Interestingly, baseline urinary and serum metabolomic profiling of UC patients with or without clinical relapse within 12 mo showed a significant difference. The most important metabolites that were responsible for this discrimination were trans-aconitate, cystine and acetamide in urine, and 3-hydroxybutyrate, acetoacetate and acetone in serum., Conclusion: A combination of baseline dietary intake, fecal calprotectin, and metabolomic factors are associated with risk of UC clinical relapse within 12 mo., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to disclose.

We here present a conceptually novel reaction-based ELISA principle (ReactELISA) for quantitation of the carbon nucleophilic lipid metabolite acetoacetate. Key to the assay is the utilization of a highly chemoselective Friedländer reaction that captures and simultaneously stabilizes the nucleophilic metabolite directly in the biological matrix. By developing a bifunctional biotinylated capture probe, the Friedländer-acetoacetate adduct can be trapped and purified directly in streptavidin coated wells. Finally, we outline the selection and refinement of a highly selective recombinant antibody for specific adduct quantitation. The setup is very robust and, as we demonstrate via miniaturization for microplate format, amenable for screening of compounds or interventions that alter lipid metabolism in liver cell cultures. The assay-principle should be extendable to quantitation of other nucleophilic or electrophilic and perhaps even more reactive metabolites provided suitable capture probes and antibodies.

Lifestyle factors, including diet, play an important role in the survival of cancer patients. However, the molecular mechanisms underlying pathogenic links between diet and particular oncogenic mutations in human cancers remain unclear. We recently reported that the ketone body acetoacetate selectively enhances BRAF V600E mutant-dependent MEK1 activation in human cancers. Here we show that a high-fat ketogenic diet increased serum levels of acetoacetate, leading to enhanced tumor growth potential of BRAF V600E-expressing human melanoma cells in xenograft mice. Treatment with hypolipidemic agents to lower circulating acetoacetate levels or an inhibitory homolog of acetoacetate, dehydroacetic acid, to antagonize acetoacetate-BRAF V600E binding attenuated BRAF V600E tumor growth. These findings reveal a signaling basis underlying a pathogenic role of dietary fat in BRAF V600E-expressing melanoma, providing insights into the design of conceptualized "precision diets" that may prevent or delay tumor progression based on an individual's specific oncogenic mutation profile., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Aspirin and ibuprofen are the most frequently prescribed non-steroidal anti-inflammatory drugs in the world. However, both are associated with a variety of toxicities. We applied serum metabonomics and Fisher discrimination for the early diagnosis of its toxic reaction in order to help diagnose these toxicities., Methods: A total of 45 rats were randomly divided into Control group, Aspirin group, and Ibuprofen groups. The experiment groups were given intragastric aspirin (15 mg/kg) or ibuprofen (15 mg/kg) for 3 weeks. Liver function tests were performed and blood metabonomics were analyzed by gas chromatography-mass spectrometry., Results: The most important compounds altered were trihydroxybutyric acid and l-alanine in the aspirin group, and acetoacetic acid, l-alanine, and trihydroxybutyric acid in the ibuprofen group. With respect to metabolic profiles, all 3 groups were completely distinct from one another. Fisher discrimination showed that 91.1% of the original grouped cases were correctly classified by the third week. However, only 55.6% of liver function tests were able to classify grouped cases correctly., Conclusion: Trihydroxybutyric acid, l-alanine, and acetoacetic acid were the most significant indicators of altered serum metabolites following intragastric administration of aspirin and ibuprofen in rates. These metabolomic data may be used for classification of aspirin and ibuprofen toxicity., (© 2017 S. Karger AG, Basel.)

Objective Acute brain injury (ABI) is a catastrophic event, leading to disruption of the normal cerebral metabolic pathways and a subsequent cerebral energy deficit. Ketones (beta-hydroxybutyrate (BHB) and acetoacetate) may represent an alternative metabolic substrate with the potential to improve cerebral energy supply and decrease injury. The purpose of this study was to evaluate baseline ketone concentrations in the ABI population. Methods Thirty-eight patients with ABI were enrolled into the study and followed for up to 7 days. We collected arterial blood samples immediately after admission and daily to measure the levels of BHB and acetoacetate. Where possible, matching cerebrospinal fluid (CSF) specimens were also collected. Results During the study period, plasma BHB levels were increased initially but normalized by day 3 while acetoacetate levels remained within the normal range. The change in BHB was significant. There were 30 observations in 10 patients where BHB could be measured in both blood and CSF. When the data were averaged over patients there was a weak correlation between blood and CSF BHB (Spearman's ρ = 0.62, p = 0.054). Conclusion Blood ketone concentrations remain low within the ABI population. An external source of ketones will be required to increase blood concentrations to clinically relevant levels.

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been established to quantify metabolic intermediates, including lactate (Lac), pyruvate (Pyr), acetoacetate (ACAC) and 3-hydroxybutyrate (3-HB) in blood. Samples were deproteinized with methanol-acetonitrile solution, and analytes were separated on an adamantyl group-bonded reverse phase column and detected in multiple reaction monitoring mode. Total analysis time was 4 min per sample. Method validation results displayed that limits of quantification were 10.0 μmol L -1 for Lac and Pyr, and 5.0 μmol L -1 for ACAC and 3-HB. The within- and between-run coefficients of variation were in the range of 1.2-6.4% for all analytes. The recoveries were ranged from 95.6 to 111.5%. The reference values of analytes were determined for the pediatric population. Duo to instability of Lac, Pyr and ACAC in vitro, a comprehensive stability assay was performed to determine optimal conditions for sample collection, pretreatment and storage. Results showed that precipitation of protein in blood at bedside combined with low storage temperature could effectively preserve the integrity of Lac, Pyr and 3-HB, but the precipitated protein accelerated degradation of ACAC. Isolation of supernatant fluid slowed degradation of ACAC. Supernatant samples could store at -20 °C for 10 days. The use of plasma or serum to determine these intermediates was not recommended. In this study, 450 samples from patients were analyzed, and 7 patients were diagnosed as congenital lactic acidosis. With the advantages of rapid, accurate and reliable, this method is very suitable for congenital lactic acidosis screening and researches related to energy metabolism., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A complication of diabetes is diabetic ketoacidosis (DKA), which if left untreated is a life threatening condition. Prompt and accurate diagnosis of DKA is required for the commencement of life saving interventions. Measurements of ketone bodies in DKA have usually been through nitroprusside urine acetoacetate testing. The aim of this systematic review was to examine whether capillary β-hydroxybutyrate (β-OHB) testing is more accurate compared to other diagnostic methods of DKA. The following electronic databases were searched: EBSCO Host, MEDLINE, PSYCHInfo, CINAHL and Science Direct for publications from 1 January 2005 and up to and including 1 January 2016. Inclusion criteria were: Adults 18 years and over and known type 1 or type 2 diabetes. Retrospective and prospective observation studies were included. A total of nine studies met the inclusion criteria. Capillary β-OHB was found to have high sensitivity, specificity, positive predictive value and negative predictive value in identifying DKA compared to urinary ketone testing., Competing Interests: The authors declare no conflict of interest.

Diets that boost ketone production are increasingly used for treating several neurological disorders. Elevation in ketones in most cases is considered favorable, as they provide energy and are efficient in fueling the body's energy needs. Despite all the benefits from ketones, the above normal elevation in the concentration of ketones in the circulation tend to illicit various pathological complications by activating injurious pathways leading to cellular damage. Recent literature demonstrates a plausible link between elevated levels of circulating ketones and oxidative stress, linking hyperketonemia to innumerable morbid conditions. Ketone bodies are produced by the oxidation of fatty acids in the liver as a source of alternative energy that generally occurs in glucose limiting conditions. Regulation of ketogenesis and ketolysis plays an important role in dictating ketone concentrations in the blood. Hyperketonemia is a condition with elevated blood levels of acetoacetate, 3-β-hydroxybutyrate, and acetone. Several physiological and pathological triggers, such as fasting, ketogenic diet, and diabetes cause an accumulation and elevation of circulating ketones. Complications of the brain, kidney, liver, and microvasculature were found to be elevated in diabetic patients who had elevated ketones compared to those diabetics with normal ketone levels. This review summarizes the mechanisms by which hyperketonemia and ketoacidosis cause an increase in redox imbalance and thereby increase the risk of morbidity and mortality in patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: The aim of this study was to compare the ketogenic effect of the peroxisome proliferator-activated receptor-α stimulator, bezafibrate (BEZA), alone or in combination with medium-chain triacylglycerols (MCTs) in healthy adults., Methods: Eighteen healthy adults completed the study: 10 were given a therapeutic dose of BEZA (400 mg/d) for 8 wk followed by a further 4 wk of BEZA (400 mg/d) plus MCT (60 g/d). Eight other participants were given MCT alone (60 g/d) for 4 wk. All participants underwent identical metabolic study days: (a) pretreatment (the control), and after (b) BEZA combined with MCT (BEZA+MCT) or (c) an equal dose of MCT only. On the metabolic study days, a standard breakfast and lunch were given and blood samples were taken hourly to measure plasma ketones, glucose, and fatty acids., Results: The combination of BEZA+MCT increased ketones twofold during the metabolic study day. The addition of BEZA increased early ketogenic efficiency of MCT by 2.5-fold but did not result in higher peak or mean concentration of ketones during the metabolic study day. No other differences were seen in plasma metabolites or insulin during metabolic study days. On the final metabolic study day, MCT or BEZA+MCT had different effects on the plasma acetoacetate-to-β-hydroxybutyrate ratio compared with control., Conclusions: BEZA mildly potentiated the ketogenic action of MCT but did not increase peak plasma ketone concentration or overall ketone production during the metabolic study day., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 measurement is advocated for the diagnosis of diabetic ketoacidosis and assessment of its severity. Assessing the evidence base for ketone measurement in clinical practice is challenging because multiple methods are available but there is a lack of consensus about which is preferable. Evaluating the utility of ketone measurement is additionally problematic because of variability in the biochemical definition of ketoacidosis internationally and in the proposed thresholds for ketone measures. This has led to conflicting guidance from expert bodies on how ketone measurement should be used in the management of ketoacidosis. The development of point-of-care devices that can reliably measure the capillary blood ketone β-hydroxybutyrate (BOHB) has widened the spectrum of applications of ketone measurement, but whether the evidence base supporting these applications is robust enough to warrant their incorporation into routine clinical practice remains unclear. The imprecision of capillary blood ketone measures at higher values, the lack of availability of routine laboratory-based assays for BOHB and the continued cost-effectiveness of urine ketone assessment prompt further discussion on the role of capillary blood ketone assessment in ketoacidosis. In the present article, we review the various existing methods of ketone measurement, the precision of capillary blood ketone as compared with other measures, its diagnostic accuracy in predicting ketoacidosis and other clinical applications including prevention, assessment of severity and resolution of ketoacidosis., (© 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.)

Rationale: Sepsis therapeutics have a poor history of success in clinical trials, due in part to the heterogeneity of enrolled patients. Pharmacometabolomics could differentiate drug response phenotypes and permit a precision medicine approach to sepsis., Objectives: To use existing serum samples from the phase 1 clinical trial of l-carnitine treatment for severe sepsis to metabolically phenotype l-carnitine responders and nonresponders., Methods: Serum samples collected before (T0) and after completion of the infusion (T24, T48) from patients randomized to either l-carnitine (12 g) or placebo for the treatment of vasopressor-dependent septic shock were assayed by untargeted (1)H-nuclear magnetic resonance metabolomics. The normalized, quantified metabolite data sets of l-carnitine- and placebo-treated patients at each time point were compared by analysis of variance with post-hoc testing for multiple comparisons. Pathway analysis was performed to statistically rank metabolic networks., Measurements and Main Results: Thirty-eight metabolites were identified in all samples. Concentrations of 3-hydroxybutyrate, acetoacetate, and 3-hydroxyisovalerate were different at T0 and over time in l-carnitine-treated survivors versus nonsurvivors. Pathway analysis of pretreatment metabolites revealed that synthesis and degradation of ketone bodies had the greatest impact in differentiating l-carnitine treatment response. Analysis of all patients based on pretreatment 3-hydroxybutyrate concentration yielded distinct phenotypes. Using the T0 median 3-hydroxybutyrate level (153 μM), patients were categorized as either high or low ketone. l-Carnitine-treated low-ketone patients had greater use of carnitine as evidenced by lower post-treatment l-carnitine levels. The l-carnitine responders also had faster resolution of vasopressor requirement and a trend toward a greater improvement in mortality at 1 year (P = 0.038) compared with patients with higher 3-hydroxybutyrate., Conclusions: The results of this preliminary study, which were not readily apparent from the parent clinical trial, show a unique metabolite profile of l-carnitine responders and introduce pharmacometabolomics as a viable strategy for informing l-carnitine responsiveness. The approach taken in this study represents a concrete example for the application of precision medicine to sepsis therapeutics that warrants further study.

Unlabelled: The ketone body acetoacetate could be used as an alternate nutrient for the heart, and it also has the potential to improve cardiac function in an ischemic-reperfusion model or reduce the mitochondrial production of oxidative stress involved in cardiotoxicity. In this study, [(11)C]-acetoacetate was investigated as an early marker of intracellular damage in heart failure., Methods: A rat cardiotoxicity heart failure model was induced by doxorubicin, Dox(+). [(14)C]-Acetoacetate, a non-positron (β-) emitting radiotracer, was used to characterize the arterial blood input function and myocardial mitochondrial uptake. Afterward, [(11)C]-acetoacetate (β+) myocardial PET images were obtained for kinetic analysis and heart function assessment in control Dox(-) (n=15) and treated Dox(+) (n=6) rats. The uptake rate (K1) and myocardial clearance rate (k2or kmono) were extracted., Results: [(14)C]-Acetoacetate in the blood was increased in Dox(+), from 2 min post-injection until the last withdrawal point when the heart was harvested, as well as the uptake in the heart and myocardial mitochondria (unpaired t-test, p <0.05). PET kinetic analysis of [(11)C]-acetoacetate showed that rate constants K1, k2 and kmono were decreased in Dox(+) (p <0.05) combined with a reduction of 24% of the left ventricular ejection fraction (p <0.001)., Conclusion: Radioactive acetoacetate ex vivo analysis [(14)C], and in vivo kinetic [(11)C] studies provided evidence that [(11)C]-acetoacetate can assess heart failure Dox(+). Contrary to myocardial flow reserve (rest-stress protocol), [(11)C]-acetoacetate can be used to assess reduced kinetic rate constants without requirement of hyperemic stress response. The proposed [(11)C]-acetoacetate cardiac radiotracer in the investigation of heart disease is novel and paves the way to a potential role for [(11)C]-acetoacetate in cardiac pathophysiology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Plasma concentrations of 3-hydroxybutyrate (3HB) are measured more often than acetoacetate (AcAc) which may be due to the reported storage instability of AcAc. The aims of the study were to compare the storage stability of AcAc in different blood fractions over time (90days) when stored at -80°C and to determine the postprandial concentration of AcAc in whole blood, plasma and red blood cells., Methods: Blood was collected from fasting subjects (n=5): whole blood, plasma and red blood cells were isolated and deproteinised in perchloric acid, and supernatants were stored at -80°C until analysis. Postprandial concentrations of AcAc in whole blood, plasma and red blood cells were determined at regular intervals over 420min, after subjects (n=23) had consumed a mixed test meal., Results: Storing deproteinised plasma at -80°C resulted in no significant change in AcAc concentration over 60days. In contrast, whole blood AcAc concentrations significantly decreased by 51% (p=0.018) within 30days. The concentration of AcAc in fasting and postprandial plasma was notably higher than that of whole blood and red blood cells., Discussion: Our data demonstrates that plasma for AcAc analysis can be stored for longer than previously suggested provided that plasma is deproteinised and stored at -80°C., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Measurements of pyruvate and ketones bodies (acetoacetate and 3-hydroxybutyrate) are essential to the investigation of intermediary metabolism. Indeed, their blood levels reflect energy balance influenced by nutritional status. This balance can be disturbed in certain diseases such as diabetes and some inherited metabolic disorders. We have developed methods for assays on open automated biochemistry analyser, Konelab 20 XT (ThermoFischer, Whaltham USA), using kits marketed by Sobioda (Montbonnot, France) for pyruvate and Wako Chemicals GmbH (Neuss, Germany) for ketones, on deproteinised blood sample. We have validated the performance of these three quantitative methods using NF EN ISO 15189 (range B) standard criteria. We obtain satisfactory results concerning fidelity (precision measured as within and between batch CVs are respectively less than 7% and less than 6%), measuring ranges (from 7.7 to 228 μmol/L for pyruvate and from 22.6 to 650 μM for total ketone bodies), accuracy (10.4 μmol/L in physiological range for pyruvate and 7.1 μmol/L for 3-hydroxybutyrate) and comparing methods (versus manual assay with spectrophotometry on Uvikon XL). Establishment of reference ranges (35 to 74 μmol/L for pyruvate, less than 100 μM for 3-hydroxybutyrate and less than 44 μmol/L for acetoacetate) and reagents stability study (up to 12 weeks if frozen) have enabled us to finalize method validation and to add these assays to our routine laboratory repertoire.

The purpose of this study was to assess the metabolic profile of plasma samples from cows with clinical and subclinical ketosis. According to clinical signs and 3-hydroxybutyrate plasma levels, 81 multiparous Holstein cows were selected from a dairy farm 7 to 21 d after calving. The cows were divided into 3 groups: cows with clinical ketosis, cows with subclinical ketosis, and healthy control cows. (1)H-Nuclear magnetic resonance-based metabolomics was used to assess the plasma metabolic profiles of the 3 groups. The data were analyzed by principal component analysis, partial least squares discriminant analysis, and orthogonal partial least-squares discriminant analysis. The differences in metabolites among the 3 groups were assessed. The orthogonal partial least-squares discriminant analysis model differentiated the 3 groups of plasma samples. The model predicted clinical ketosis with a sensitivity of 100% and a specificity of 100%. In the case of subclinical ketosis, the model had a sensitivity of 97.0% and specificity of 95.7%. Twenty-five metabolites, including acetoacetate, acetone, lactate, glucose, choline, glutamic acid, and glutamine, were different among the 3 groups. Among the 25 metabolites, 4 were upregulated, 7 were downregulated, and 14 were both upregulated and downregulated. The results indicated that plasma (1)H-nuclear magnetic resonance-based metabolomics, coupled with pattern recognition analytical methods, not only has the sensitivity and specificity to distinguish cows with clinical and subclinical ketosis from healthy controls, but also has the potential to be developed into a clinically useful diagnostic tool that could contribute to a further understanding of the disease mechanisms., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Diabetes and alcohol abuse may cause severe metabolic disturbances that can be fatal. These may be difficult to diagnose in autopsies based solely on macroscopical and histological findings. In such cases, metabolic markers, such as postmortem glucose and ketone levels, can provide supporting information. Glucose or combined glucose and lactate, the Traub value, is often used to indicate hyperglycemia. The use of the Traub value, however, has been questioned by some, because the lactate levels are known to elevate in postmortem samples also due to other reasons than glycolysis of glucose molecules. Ketoacidosis can be detected by analyzing ketone body levels, especially beta-hydroxybutyric acid (BHB). Acetone is also elevated in severe cases of ketoacidosis. Here, we have evaluated the value of these biomarkers for postmortem determination of the metabolic disturbances. Retrospective data of 980 medico-legal autopsies performed in Finland, where glucose, lactate and ketone bodies were analyzed, was collected. Our findings show that the Traub value indicates hyperglycemia, even when glucose levels are low. For diagnosis, evaluation of complementing markers, e.g. ketone bodies and glycated hemoglobin is needed. Our results show that BHB can be used for screening and diagnosis of ketoacidosis. Acetone alone is not sufficient, since it is elevated only in the most severe cases. We also found that alcohol abuse rarely causes severe ketoacidosis. However, sporadic cases do exist where ketone body levels are extremely high. Despite this, alcoholic ketoacidosis is very rarely diagnosed as the cause of death.

Ketone bodies are produced in the liver and are utilized in other tissues in the body as an energy source when hypoglycemia occurs in the body. There are three ketone bodies: acetoacetate, beta hydroxy butyrate, and acetone. Ketone bodies are usually present in the blood, and their level increases during fasting and starvation. They are also found in the blood of neonates and pregnant women. In diabetic ketoacidosis, high levels of ketone bodies are produced in response to low insulin levels and high levels of counter-regulatory hormones.

We investigated the association of the levels of ketone bodies (KBs) with hyperglycemia and with 62 genetic risk variants regulating glucose levels or type 2 diabetes in the population-based Metabolic Syndrome in Men (METSIM) study, including 9,398 Finnish men without diabetes or newly diagnosed type 2 diabetes. Increasing fasting and 2-h plasma glucose levels were associated with elevated levels of acetoacetate (AcAc) and β-hydroxybutyrate (BHB). AcAc and BHB predicted an increase in the glucose area under the curve in an oral glucose tolerance test, and AcAc predicted the conversion to type 2 diabetes in a 5-year follow-up of the METSIM cohort. Impaired insulin secretion, but not insulin resistance, explained these findings. Of the 62 single nucleotide polymorphisms associated with the risk of type 2 diabetes or hyperglycemia, the glucose-increasing C allele of GCKR significantly associated with elevated levels of fasting BHB levels. Adipose tissue mRNA expression levels of genes involved in ketolysis were significantly associated with insulin sensitivity (Matsuda index). In conclusion, high levels of KBs predicted subsequent worsening of hyperglycemia, and a common variant of GCKR was significantly associated with BHB levels.

Objective: In humans consuming a normal diet, we investigated 1) the capacity of a medium-chain triacylglycerol (MCT) supplement to stimulate and sustain ketonemia, 2) ¹³C-β-hydroxybutyrate and ¹³C-trioctanoate metabolism, and 3) the theoretical contribution of the degree of ketonemia achieved to brain energy metabolism., Methods: Eight healthy adults (26 ± 1 y old) were given an MCT supplement for 4 wk (4 times/d; total of 20 g/d for 1 wk followed by 30 g/d for 3 wk). Ketones, glucose, triacylglycerols, cholesterol, free fatty acids, and insulin were measured over 8 h during two separate metabolic study days before and after MCT supplementation. Using isotope ratio mass spectroscopy, ¹³C-D-β-hydroxybutyrate and ¹³C-trioctanoate β-oxidation to ¹³CO₂ was measured over 12 h on the pre- and post-MCT metabolic study days., Results: On the post-MCT metabolic study day, plasma ketones (β-hydroxybutyrate plus acetoacetate) peaked at 476 μM, with a mean value throughout the study day of 290 μM. Post-MCT, ¹³C-trioctanoate β-oxidation was significantly lower 1 to 8 h later but higher 10 to 12 h later. MCT supplementation did not significantly alter ¹³C-D-β-hydroxybutyrate oxidation., Conclusions: This MCT supplementation protocol was mildly and safely ketogenic and had no side effects in healthy humans on their regular diet. This degree of ketonemia is estimated to contribute up to 8% to 9% of brain energy metabolism., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: 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., Methods: 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., Results: 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., Conclusion: 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

According to the hypothesis of Traub, also known as the 'formula of Traub', postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.

Objective: The age-related effects of fasting on lipolysis, the production of ketone bodies, and plasma insulin levels were studied in male 3-, 8-, and 32-week-old Sprague-Dawley rats., Methods: The rats were divided into fasting and control groups. The 3-, 8- and 32-week-old rats tolerated fasting for 2, 5, and 12 days, respectively., Results: Fasting markedly reduced the weights of perirenal and periepididymal white adipose tissues in rats in the three age groups. The mean rates of reduction in both these adipose tissue weights during fasting periods were higher in the order of 3 > 8 > 32-week-old rats. Fasting transiently increased plasma free fatty acid (FFA), total ketone body, β-hydroxybutyrate, and acetoacetate concentrations in the rats in the three age groups. However, plasma FFA, total ketone body, β-hydroxybutyrate, and acetoacetate concentrations in the 3-week-old rats reached maximal peak within 2 days after the onset of fasting, although these concentrations in the 8- and 32-week-old rats took more than 2 days to reach the maximal peak. By contrast, the augmentation of plasma FFA, total ketone body, β-hydroxybutyrate, and acetoacetate concentrations in the rats in the three age groups had declined at the end of each experimental period. Thus, the capacity for fat mobilization was associated with tolerance to fasting. Plasma insulin concentrations in the rats in the three age groups were dramatically reduced during fasting periods, although basal levels of insulin were higher in the order of 32 > 8 > 3 week-old rats., Conclusion: These results suggest that differences in fat metabolism patterns among rats in the three age groups during prolonged fasting were partly reflected the metabolic turnover rates, plasma insulin levels, and amounts of fat storage.

Background: Infants of diabetic mothers (IDMs) are at increased risk for metabolic complications. Type 1 and some type 2 diabetic patients have elevated levels of the ketone bodies acetoacetate (AA) and β-hydroxybutyrate (BHB)., Objective: The aim of this study was to examine how hyperketonemia in diabetic mothers affects markers of inflammation and oxidative stress in their offspring., Methods: Blood was obtained from 23 diabetic mothers and 13 healthy mothers and their infants' umbilical cords at delivery. Interleukin-8, monocyte chemotactic protein-1 (MCP-1) and protein carbonyl (protein oxidation) levels were determined by ELISA. U937 human monocyte cell culture was used to examine the effect of AA and BHB on secretion of MCP-1., Results: There was a significant increase in the levels of AA in cord blood of IDMs compared with cord blood of infants of healthy mothers. A significant increase in the levels of protein oxidation (p < 0.05) and MCP-1 levels (p < 0.05) was observed in the cord blood of IDMs. The level of MCP-1 correlated significantly (r = 0.51, p = 0.01) with the concentration of AA in the IDMs. In further experiments with cultured monocytes treated with exogenous AA (0-4 mM), a significant increase in MCP-1 secretion was observed in AA- but not BHB-treated monocytes., Conclusion: Blood levels of AA and MCP-1 are elevated in IDMs, which may contribute to the development of the metabolic complications seen in IDMs., (Copyright © 2012 S. Karger AG, Basel.)

Background: Little is known about the metabolism of acetoacetate and β-hydroxybutyrate in patients with cirrhosis and encephalopathy., Aims: We investigated the fate of ketone bodies in these conditions., Materials and Methods: We studied 18 cirrhotic patients with encephalopathy and 17 cirrhotics without. At the time of insertion of a transjugular intrahepatic portosystemic stent shunt (TIPSS) or at the time of portographical assessment of the shunt's patency, we collected blood from the internal jugular, the right atrium, the inferior vena cava, the hepatic, the portal, the splenic veins and the radial artery. We used nuclear magnetic resonance spectroscopy to measure the concentrations of acetoacetate and β-hydroxybutyrate., Results: There was no difference in the total ketone body concentrations between the two groups. The mitochondrial redox potential was significantly higher in the encephalopathics (142/54=2.63 vs 52/83=0.62) (P<0.01). β-hydroxybutyrate was significantly lower in the portal vein of encephalopathics (52 ± 4 vs 28 ± 3) (P<0.02) and in the splenic vein (48 ± 6 vs 32 ± 5) (P<0.04). Acetoacetate was significantly higher in encephalopathics in the internal jugular vein (134 ± 12 vs 92 ± 16) (P<0.03), the right atrium (112 ± 18 vs 68 ± 11) (P<0.03), the hepatic vein (162 ± 25 vs 115 ± 19) (P<0.05), the portal vein (133 ± 20 vs 81 ± 14) (P<0.02) and the splenic vein (167 ± 24 vs 122 ± 21) (P<0.04). All measurements are expressed in μmols/L., Conclusions: There are significant variations in the regional concentrations of the ketone bodies in encephalopathy., (© 2011 John Wiley & Sons A/S.)

Metabonomic characterization of the effects caused by ozone and other stressors on normal human blood was performed. Samples of blood obtained from healthy subjects were treated ex vivo with increasing concentrations of ozone and/or with UV radiation and heat. (1)H-NMR analysis of plasma samples after treatments showed the quantitative variation of some metabolites and the formation of new metabolites normally absent. Both the increment of some metabolites like formate, acetoacetate, and acetate and the decrement of pyruvate were of particular interest. Moreover, the oxidation of ascorbic acid and the transformation of uric acid into allantoin after ozonation within the therapeutic concentration range were observed. In the ozonated spectra, 2 unidentified peaks appeared at 2.82 ppm and 8.08 ppm. They are related to the direct antioxidant activity of albumin in the presence of ozone and they could be considered as specific markers of the blood ozonation.

Ketogenesis, inferred by the production of acetoacetate plus ss-hydroxybutyrate, in isolated perfused livers from 24-h fasted diabetic rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. For this purpose, alloxan-diabetic rats that received intraperitoneal regular insulin (IIH group) or saline (COG group) injection were compared. An additional group of diabetic rats which received oral glucose (gavage) (100 mg kg(-1)) 15 min after insulin administration (IIH + glucose group) was included. The studies were performed 30 min after insulin (1.0 U kg(-1)) or saline injection. The ketogenesis before octanoate infusion was diminished (p < 0.05) in livers from rats which received insulin (COG vs. IIH group) or insulin plus glucose (COG vs. IIH + glucose group). However, the liver ketogenic capacity during the infusion of octanoate (0.3 mM) was maintained (COG vs. IIH group and COG vs. IIH + glucose group). In addition, the blood concentration of ketone bodies was not influenced by the administration of insulin or insulin plus glucose. Taken together, the results showed that inspite the fact that insulin and glucose inhibits ketogenesis, livers from diabetic rats submitted to short-term IIH which received insulin or insulin plus glucose showed maintained capacity to produce acetoacetate and ss-hydroxybutyrate from octanoate., ((c) 2009 John Wiley & Sons, Ltd.)

Protein kinases are critical component in the regulation of signal transduction pathways, including neurotransmitters. Our previous studies have shown that serotonin (5-HT) altered under diabetic condition was accompanied by alterations of protein kinase C-alpha (PKC-alpha) and CaMKII, and those alterations were reversed after insulin administration. The current study showed that alloxan-induced diabetic animals revealed hyperglycemia and was associated with an increase in the content of 5-HT, PKC-alpha expression and PKC activity (P < 0.05) simultaneously in striatum (ST), midbrain (MB), pons medulla (PM), cerebellum (CB), and cerebral cortex (CCX) from 7 days to 60 days. Although the 5-HT levels in hippocampus (HC) and hypothalamus (HT) were not altered, the PKC-alpha expression and PKC activity showed increases (P < 0.05) in level in HC. Insulin administration reversed all these changes to a normal level. In contrast, the in vitro study has shown that the 5-HT levels correlated with PKC-alpha expressions as well as PKC activity (P < 0.05) only in ST, MB, and CB either after induction with phorbol 12-myristate 13-acetate (PMA) or blocking with chelerythrine, whereas PM and CCX remained elevated (P < 0.05), implying a regulatory role for PKC-alpha only in ST, MB, and CB. However, our consecutive studies have shown that the 5-HT level in PM was regulated by p38-mitogen-activated protein kinase (p38-MAPK) both in vivo and in vitro, whereas the 5-HT level in CCX was coregulated by S-100beta by protein-protein interaction with serotonin transporter (SERT) via 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (8-Br-cAMP)-induced cAMP/PKAII pathway(s)., (2008 Wiley-Liss, Inc.)

Objective: The purpose of this study was to investigate the physiologic changes of ketone bodies in patients with aneurysmal subarachnoid hemorrhage. We tested the hypothesis that the plasma ketone bodies are associated with the vasoconstrictor and lipolysis effect of circulating catecholamine., Methods: Twenty-four patients with mild aneurysmal subarachnoid hemorrhage and 18 healthy volunteers were enrolled in this study. We collected arterial blood samples immediately after admission and 30 days later to measure the levels of 3-hydroxybutyrate, acetoacetate, epinephrine and norepinephrine., Result: At the onset of aneurysmal subarachnoid hemorrhage, the plasma ketone body (3-hydroxybutyrate + acetoacetate) level and the epinephrine and norepinephrine concentrations were significantly elevated, but the arterial ketone body ratio (acetoacetate/3-hydroxybutyrate) was significantly decreased compared with that of the control group. There was a negative correlation between the plasma ketone body level and the arterial ketone body ratio. There was a positive correlation between the plasma ketone body level and epinephrine level. Thirty days after admission, the ketone body, epinephrine and norepinephrine levels, as well as the arterial ketone body ratio, showed no significant differences between the patients and controls., Conclusion: At the onset of mild aneurysmal subarachnoid hemorrhage, the plasma ketone body level was significantly increased, while the arterial ketone body ratio was significantly decreased.

Mitochondrial acetoacetyl-CoA thiolase deficiency (or beta-ketothiolase deficiency) is a rare metabolic disorder characterized by acute episodes of severe acidosis and ketosis. A case is presented of an 18-month-old boy who presented with vomiting and diarrhoea and was found to be markedly acidotic. When the acidosis persisted despite saline fluid boluses and bicarbonate correction, further investigations were undertaken. Routine biochemical investigation revealed detectable salicylate concentrations despite the parents denying its administration, which initially caused some diagnostic confusion. The results of urine organic acid analysis, however, confirmed that the diagnosis of mitochondrial acetoacetyl-CoA thiolase deficiency. The high concentrations of acetoacetate present in the patient's sample resulted in a false-positive reaction in the Trinder assay for salicylate.

Paracetamol has properties that make it useful in many kinds of drugs that are readily available to users. These drugs are often used for extensive periods of time without a doctor's supervision, which might lead to unexpected overdose. The aim of this work was to assess the extent of liver damage in rabbits after administering them with paracetamol for approximately 2 months. The results we obtained suggest that paracetamol administered for a long time causes liver function impairment. It was, however, not possible to determine the impairment by measuring the activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT) or gamma-glutamylotranspeptidase (GGTP). It was determined by measuring the concentration of ketone bodies and arterial ketone body ratio (AKBR) value. Even when taken in small doses, paracetamol causes changes in liver metabolism when taken for an extended period of time.

Purpose: To evaluate the in vivo alterations on ketone bodies metabolism after cerebral ischemia/reperfusion through an experimental model of brain ischemia induced by simple occlusion of common carotid arteries (CCAs) in Wistar rats., Methods: Forty-eight male Wistar rats were randomly distributed on two groups (S - Sham; T - Test) and further redistributed into four times sets of study. After bilateral occlusion of CCAs for 30 min, the animals of group T were allowed reperfusion for 0, 5, 10 and 15 min. Samples of cerebral tissue and systemic arterial blood were collected and the metabolites acetoacetate (ACT) and beta-hydroxybutyrate (BHB) were determined., Results: Cerebral ACT and BHB levels increased significantly in Group T after 30 min of carotid occlusion (time 0). The highest brain ketone bodies (ACT+BHB) concentration was verified at 5 min of reperfusion, decreasing after 10 min of recirculation. Systemic ketone bodies levels increased similarly between test and sham groups. Group S demonstrated a significant increase in cerebral and systemic ACT and BHB concentrations mainly after 40-45 min of study., Conclusions: The partial transient acute global brain ischemia induced by the bilateral carotid occlusion in Wistar rats triggered ketogenesis probably due to a central stimulation of catecholamine secretion. There was an increased cerebral uptake of ketone bodies following brain ischemia, reaffirming these metabolites as alternative energy substrates under conditions of cerebral metabolic stress as well as its potential role on neuroprotection. The greatest changes in ketone bodies metabolism were verified at initial minutes of recirculation as a result of the reperfusion injury phenomenon.

Keto-acidosis is usually associated with uncontrolled diabetes and typically poses few diagnostic problems when presenting as hyperglycaemia, metabolic acidosis and a high anion gap. An emaciated patient suffering from Duchenne Muscular Dystrophy and volume depletion presented with acidosis of unknown origin. Preliminary investigations appeared to rule out lactic acidosis, diabetic keto-acidosis and acidosis due to base loss. We have previously reported a technique utilizing liquid chromatography coupled to mass spectrometry (LC-MS) which can be used to characterize the underlying aetiology of acidosis and applied it to ultrafiltrate derived from a blood sample taken from this patient. The anion profile obtained on the chromatogram showed elevated levels of acetoacetate and hydroxybutyrate but no evidence of lactic acidosis, nor was the profile typical of that seen in 'unexplained' acidosis. We concluded that the patient was suffering from keto-acidosis associated with starvation and dehydration, the biochemical features being obscured by both the patient's chronic malnutrition and minimal muscle mass. A combination of enteral feeding and rehydration led to prompt resolution of the patient's metabolic acidosis., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Introduction: Acetone has been shown to have broad-spectrum anticonvulsant actions in animal seizure models and has been hypothesized to play a role in the anticonvulsant mechanism of the ketogenic diet (KD). The present study examined the ability of a KD to elevate amygdaloid afterdischarge thresholds (ADT) in fully kindled rats. The effects of the KD were studied in the presence and absence of diallyl sulfide (DAS), an inhibitor of acetone metabolism., Methods: Twenty-four adult male rats were kindled to 30 stage 5 seizures. Afterdischarge thresholds (ADT) were determined. Subjects were then administered one of the following diets: (1) KD+V (vehicle; KD+V); (2) KD+DAS; (3) control diet+V (CD+V); (4) CD+DAS. They were stimulated every second day. Blood sampling was performed every second day--on non-stimulating days--to determine levels of glucose, beta-hydroxybutyrate, acetoacetate, and acetone. After 20 days, ADTs were re-determined., Results: Blood acetone concentrations were significantly higher in the KD+DAS group as compared to the other groups, although they did not reach "therapeutic levels". None of the treatments, however, elevated ADTs., Conclusions: The KD was unable to elevate amygdaloid ADTs in fully kindled rats. Although subjects in the KD+DAS group achieved significant elevations of blood acetone, these concentrations (e.g. 0.2 mM) were much lower than those (>2.0 mM) previously shown to confer anticonvulsant activity. There appears to be large difference between humans and rats in their ability to produce elevated blood acetone levels on the KD. These data suggest that adult rats are not ideal subjects for modeling the anticonvulsant actions of the KD.

To determine the effect of nonesterified fatty acids (NEFA) on glucagon action, glucagon was infused intraportally (1.65 ng.min(-1).kg(-1)) for 3 h into 18-h-fasted, pancreatic-clamped conscious dogs in the presence [NEFA + glucagon (GGN)] or absence (GGN) of peripheral Intralipid plus heparin infusion. Additionally, hyperglycemic (HG), hyperglycemic-hyperlipidemic (NEFA + HG), and glycerol plus glucagon (GLYC + GGN) controls were studied. Arterial plasma glucagon concentrations rose equally in GGN, NEFA + GGN, and GLYC + GGN but remained basal in hyperglycemic controls. Peripheral infusions of Intralipid and heparin increased arterial plasma NEFA concentrations equally in NEFA + GGN and NEFA + HG and did not change in other protocols. After 15 min, glucagon infusion resulted in a rapid, brief increase in net hepatic glycogenolysis (NHGLY, mg.min(-1).kg(-1)) of approximately 6.0 in GGN and GLYC + GGN but only increased by 3.8 +/- 1.3 in NEFA + GGN. Thus increases in NHGLY, and consequently net hepatic glucose output (NHGO), were blunted by 40%, with no difference between the groups in the last 2.5 h of the study. NHGO and NHGLY did not significantly change in HG and NEFA + HG. Net hepatic gluconeogenic flux did not change in GGN, GLYC + GGN, or HG. However, Intralipid and heparin infusion resulted in similar increases in net hepatic gluconeogenic flux in NEFA + GGN and NEFA + HG. Thus elevated NEFA limit the initial increase in glucagon-stimulated HGO by blunting glycogenolysis, without having any effect on the gluconeogenic or glycogenolytic contributions or NHGO thereafter.

Dairy calf weaning results in blood ketone concentrations in excess of mature rates of use and can result in excretion of ketones in urine representing a loss of energy. Lasalocid is frequently supplemented as an anticoccidial agent in calf starters; however, in mature ruminants it is known to alter molar ratios of ruminal volatile fatty acids (VFA). Effects of weaning transition and postweaning ionophore supplementation on body weight, dry matter (DM) intake, average daily gain (ADG), and blood concentrations of glucose, acetoacetate, beta-hydroxybutyrate (BHBA), lactate, pyruvate, nonesterified fatty acids (NEFA), VFA, insulin, and glucagon were examined using Jersey bull calves (n = 24) over 16 wk. Calves were blocked into groups of 2 according to birth date and weight and randomly assigned to receive either a commercial pelleted starter (control), or the same diet containing lasalocid (TRT; 83 mg/kg of DM). Calves were fed milk replacer from d 3 to 34 (d 3 to 20 = 454 g/d at 12% solids; d 21 to 34 = 568 g/d at 15% solids), from d 35 to 48 calves received both replacer (d 35 to 41 = 454 g/d; d 42 to 38 = 227 g/d) and free access to control or TRT starter, and from d 49 to 112 received ad libitum control or TRT. Body weight and jugular blood metabolite concentrations were measured and recorded weekly. Postweaning DM intake, average daily gain, and feed:gain did not differ between control and TRT calves. Glucose and NEFA concentrations did not differ between control and TRT, but declined with age. Insulin and glucagon concentrations did not differ between control and TRT, but glucagon concentrations increased with weaning. Total VFA significantly increased following introduction of solid feed at d 35 in both groups with an apparent 1-wk lag in TRT VFA increases compared with control. Jugular acetate and butyrate concentrations were greater in control calves than TRT calves during wk 7. Propionate concentrations did not differ between control and TRT at any time following weaning. Blood BHBA concentrations were greater in control than TRT during wk 8 and 9. Thus, consumption of starter supplemented with lasalocid delayed peak acetate and butyrate and lowered peak BHBA concentrations. However, supplementation at concentrations currently recommended for control of coccidiosis did not appear to be sufficient to enhance growth or efficiency during the wk 7 to 16 postweaning interval for this sample size.

Although "unmeasured" anions contribute to metabolic acidosis in a variety of disease states, they are generally not measured directly but estimated from the calculation of "gaps." Among the most commonly used method, the anion gap (AG) is not only a function of "unmeasured" anions, but also it is a function of plasma non-carbonate buffers (albumin and phosphate), the plasma pH, and the method of measurement. To clarify the contribution of non-carbonate buffers to the AG, the Figge-Fencl-Waston model of human plasma was applied to laboratory values obtained from two novel populations, patients with nephrotic syndrome and patients with diabetic ketoacidosis (DKA). The model performed adequately, justifying the common clinical practice of correcting the AG for the net protein charge.

Aims: To assess the performance of a handheld bedside ketone sensor in the face of likely metabolic disturbances in diabetic ketoacidosis, namely: pH, glucose and acetoacetate., Methods: The effects of pH (7.44-6.83), glucose (5-50 mmol/l) and acetoacetate (0-5 mmol/l) were examined in venous blood to investigate the accuracy of betahydroxybutyrate measurement (0-5 mmol/l) by a handheld ketone sensor. Sensor results were compared with a reference method. Linear regression models were fitted to the difference between the methods with the concentration of metabolite as the explanatory factor., Results: Decreasing pH and increasing glucose had no effect on the accuracy of the handheld ketone sensor; the gradients of the fitted lines were -0.14 and -0.003, respectively. The 95% confidence intervals were -0.7-0.4 and -0.01-0.004, respectively (P = 0.59 and 0.4, respectively). In the acetoacetate study, a positive relationship between the sensor and reference method results was found, the gradient was 0.09. The 95% confidence interval was 0.05-0.14 (P < or = 0.001), indicating that high concentrations of acetoacetate interfere with the sensor performance., Conclusions: Acidosis and hyperglycaemia have minimal effects on the sensor performance. However, high concentrations of acetoacetate result in some overestimation of betahydroxybutyrate. This bedside ketone sensor provides useful data over a broad range of conditions likely to be encountered during moderate to severe diabetic ketoacidosis.

Objective: The ovarian stimulation increase the estradiol concentration in comparison to the one observed in the menstrual cycle. The aim of the study was to determine the influence of ovarian stimulation on serum concentrations of lipids and ketone bodies in patients undergoing IVF/ICSI treatment., Patients and Methods: 50 patients were included in this study. All patients underwent an ovarian stimulation either with recombinant follicle stimulating Hormone (Gonal-F, n = 28) or human menopausal gonadotropin (Menogon, n = 22) after pituitary down-regulation with Goserelin or Triptorelin. Five blood samples were taken: before treatment, at the beginning of ovarian stimulation, on the day of HCG injection for the ovulation induction, on the day of follicle aspiration and 14 days after embryo transfer. Afterwards, the samples were analysed with regard to the concentrations of triglyceride, total-cholesterol, HDL-/LDL-cholesterol, acetoacetate, beta-hydroxybutyrate and alpha-ketoglutarate., Results: During the ovarian stimulation, total-cholesterol andA'LDL-cholesterol decreased significantly (p(Chol) < or = 0.001; p(LDL) < or = 0.001), whereas the concentration of HDL-cholesterol increased significantly (p(HDL) < or = 0.001). Furthermore, a significant increase of beta-hydroxybutyrate and alpha-ketoglutarate has been detected (p(HBS) = 0.039; p (KG) = 0.023)., Conclusions: The observed changes in serum concentrations of lipids and ketone bodies began with the application of gonadotrophins for stimulation and persisted until the end of the treatment. Therefore, the increase of estradiol, stimulated by gonadotrophins, seems to be responsible for the changes in the serum concentrations of lipids and ketone bodies. All women undergoing ovarian stimulation should be monitored for long-term thrombogenic and atherogenic risks.