Genome-wide characterization of circulating metabolic biomarkers.

Genome-wide association analyses using high-throughput metabolomics platforms have led to novel insights into the biology of human metabolism1-7. This detailed knowledge of the genetic determinants of systemic metabolism has been pivotal for uncovering how genetic pathways influence biological mechanisms and complex diseases8-11. Here we present a genome-wide association study for 233 circulating metabolic traits quantified by nuclear magnetic resonance spectroscopy in up to 136,016 participants from 33 cohorts. We identify more than 400 independent loci and assign probable causal genes at two-thirds of these using manual curation of plausible biological candidates. We highlight the importance of sample and participant characteristics that can have significant effects on genetic associations. We use detailed metabolic profiling of lipoprotein- and lipid-associated variants to better characterize how known lipid loci and novel loci affect lipoprotein metabolism at a granular level. We demonstrate the translational utility of comprehensively phenotyped molecular data, characterizing the metabolic associations of intrahepatic cholestasis of pregnancy. Finally, we observe substantial genetic pleiotropy for multiple metabolic pathways and illustrate the importance of careful instrument selection in Mendelian randomization analysis, revealing a putative causal relationship between acetone and hypertension. Our publicly available results provide a foundational resource for the community to examine the role of metabolism across diverse diseases.

Impact of heat on biological concentrations of toluene and acetone resulting from exposure by inhalation: A pilot study.

Climatic conditions raise new concerns about the potential impact of heat on the absorption and kinetics of certain chemicals. The impact of 3 temperatures (21, 25 and 30 °C WBGT) on the toxicokinetics of toluene and acetone was therefore evaluated in five human subjects during controlled exposures in an inhalation chamber. Biological samples were collected and analyzed by GC-MS/MS. Increases between 4 and 85 % were observed for solvents concentrations in blood (30 vs 21 °C) while decreases in urine samples for acetone and o-cresol were measured at the end of the exposure period (4 h). Mean blood concentrations at 4 h are well correlated with temperature. Results suggest an increased absorption and/or a decreased elimination of volatile chemicals in the presence of heat. Higher increases of blood chemical concentrations were observed in heavier individuals. Further studies should include physiologically based toxicokinetic models to help in better understanding the mechanisms involved and their respective contribution.

3D-printed smartphone-based device for fluorimetric diagnosis of ketosis by acetone-responsive dye marker and red emissive carbon dots.

A portable smartphone device is reported that uses 3D printing technology for the primary diagnosis of diseases by detecting acetone. The key part of the device consists of red carbon dots (RCDs), which are used as internal standards, and a sensing reagent (3-N,N-(diacethydrazide)-9-ethylcarbazole (2-HCA)) for acetone. With an excitation wavelength of 360 nm, the emission wavelengths of 2-HCA and RCDs are 443 nm and 619 nm, respectively. 2-HCA effectively captures acetone to form a nonfluorescent acylhydrazone via a condensation reaction occurring in aqueous solution, resulting in obvious color changes from blue-violet to dark red. The detection limit for acetone is 2.62 µM (~ 0.24 ppm). This is far lower than the ketone content in normal human blood (≤ 0.50 mM) and the acetone content in human respiratory gas (≤ 1.80 ppm). The device has good recovery rates for acetone detection in blood and exhaled breath, which are 90.56-109.98% (RSD ≤ 5.48) and 92.80-108.00% (RSD ≤ 5.07), respectively. The method designed here provides a reliable way to provide health warnings by visually detecting markers of ketosis/diabetes in blood or exhaled breath. The portable smart phone device visually detects ketosis/diabetes markers in the blood or exhaled breath through the nucleophilic addition reaction, which effectively captures acetone to form nonfluorescent acyl groups. This will be a reliable tool to warn human health.

Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites.

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Relationship between betahydroxybutyrate (BHB) and acetone concentrations in postmortem blood and cause of death.

Unexpected death caused by diabetic or alcoholic ketoacidosis is easily overlooked due to the non-specific symptoms. Although the acid betahydroxybutyrate (BHB) is the most abundant ketone body formed in conditions with ketoacidosis, routine analysis in postmortem investigations often only includes the neutral ketone body acetone. This study aims to evaluate the usefulness of implementing routine BHB analysis in postmortem cases, by investigating the relationship between BHB and acetone concentrations in postmortem blood and the main cause of death. From our database of forensic autopsy cases examined from 2012 to 2015, there were 376 cases with BHB and/or acetone detected in postmortem blood that could be paired with data from the Norwegian Cause of Death Registry. Cases were categorized into three groups based on cause of death: "Diabetes-related" (n = 38), "Alcohol-related" (n = 35) and "Other" (n = 303). Analysis of BHB in blood was performed using UHPLC-MS/MS (limit of quantification (LOQ) 52 mg/L) and of acetone using HS-GC-FID (LOQ 87 mg/L). For the purpose of the study, the acetone method was also validated for a LOQ of 23 mg/L. The median BHB concentration was significantly higher in the group of diabetes-related deaths (671 mg/L, range 68-1311 mg/L) compared to the group of alcohol-related (304 mg/L, range 65-1555 mg/L, p <0.001) and other causes of deaths (113 mg/L, range 0-1402 mg/L, p <0.001). In seven deaths (1.9%), the BHB blood concentration was above the suggested pathological threshold of 250 mg/L, without detection of acetone in blood above 23 mg/L. In 15% of deaths by other causes than diabetes or alcohol, a pathologically significant BHB blood concentration was detected. Our results indicate that BHB is a more reliable marker of pathologically significant ketoacidosis than acetone, and we suggest that BHB should be routinely analyzed in postmortem investigations.

The Ketogenic Diet: Breath Acetone Sensing Technology.

The ketogenic diet, while originally thought to treat epilepsy in children, is now used for weight loss due to increasing evidence indicating that fat is burned more rapidly when there is a low carbohydrate intake. This low carbohydrate intake can lead to elevated ketone levels in the blood and breath. Breath and blood ketones can be measured to gauge the level of ketosis and allow for adjustment of the diet to meet the user's needs. Blood ketone levels have been historically used, but now breath acetone sensors are becoming more common due to less invasiveness and convenience. New technologies are being researched in the area of acetone sensors to capitalize on the rising popularity of the diet. Current breath acetone sensors come in the form of handheld breathalyzer devices. Technologies in development mostly consist of semiconductor metal oxides in different physio-chemical formations. These current devices and future technologies are investigated here with regard to utility and efficacy. Technologies currently in development do not have extensive testing of the selectivity of the sensors including the many compounds present in human breath. While some sensors have undergone human testing, the sample sizes are very small, and the testing was not extensive. Data regarding current devices is lacking and more research needs to be done to effectively evaluate current devices if they are to have a place as medical devices. Future technologies are very promising but are still in early development stages.

Sudden Unexpected Death in MELAS Syndrome Due to Diabetic Ketoacidosis.

We present a case report of a 25 year-old man with MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes) syndrome, who died suddenly and unexpectedly from diabetic ketoacidosis. This case report illustrates why it is important for medical examiners to be familiar with the clinical and autopsy features of MELAS syndrome and to be aware of the common complications, which may lead to sudden unexpected death.

Postmortem diagnosis of ketoacidosis: Levels of beta-hydroxybutyrate, acetone and isopropanol in different causes of death.

Data from 496 autopsy cases with positive beta hydroxybutyrate (BHB), acetone or isopropanol in blood were investigated. The cases were divided into different groups according to cause of death. Cases with cause of death due to diabetic ketoacidosis (DKA, n=54) had the highest levels of BHB (median 1085mg/L) and acetone (median 330mg/L). Cases with cause of death due to alcoholic ketoacidosis (AKA, n=57) had high levels of BHB (median 500mg/L) and acetone (median 110mg/L). Cases with cause of death due to hypothermia (n=12) had similar BHB and acetone levels as the AKA group (median BHB 520mg/L and acetone 80mg/L). Cases with cause of death due to isopropanol intoxication (n=17) had high levels of isopropanol (median 430mg/L) and acetone (330mg/L), but undetected or low levels of BHB. Cases with cause of death due to other than the above mentioned (n=349) had median BHB levels of 100mg/L and median acetone levels of 20mg/L. BHB analysis is crucial for the diagnosis of postmortem ketoacidosis, since it is the main marker of ketoacidosis and helps distinguish between different causes of death. Acetone levels correlate with BHB levels in endogenous ketoacidosis, so acetone can be used as an initial screening marker to identify cases where BHB analysis should be performed, but positive acetone threshold should be maximum 20mg/L. Positive BHB is proof of endogenous ketoacidosis, whereas negative BHB indicates isopropanol intoxication or postmortem acetone/isopropanol formation by microorganisms in cases of decomposition. There is no correlation between BHB and the postmortem interval, and no sign of postmortem formation, so BHB analysis is useful even in cases of severe decomposition.

Determination of New Psychoactive Substances in Whole Blood Using Microwave Fast Derivatization and Gas Chromatography/Mass Spectrometry.

The production and consumption of new psychoactive substances (NPSs) has been raising a major concern worldwide. Due to easy access and available information, many NPSs continue to be synthesized with an alarming increase of those available to purchase, despite all the control efforts created. A new analytical method was developed and validated to determine a group of phenethylamines and synthetic cathinones: cathinone, flephedrone, buphedrone, 4-MTA, α-PVP, methylone, 2C-P, ethylone, pentylone, MDPV and bromo-dragonFLY in whole blood. A mixed-mode solid phase extraction was applied to 250 µL of sample, and the extracts were derivatized with fast microwave technique before being analyzed by gas chromatography-mass spectrometry (GC-MS). The validation procedure followed the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines with parameters that included selectivity, linearity, limits of detection and quantification, intra- and inter-day precision and accuracy, recoveries and stability. The method presented linearity between 5 and 500 ng/mL for cathinone, buphedrone, 4-MTA, methylone, 2C-P and bromo-dragonFLY, 10-500 ng/mL for flephedrone, ethylone, pentylone and MDPV, and 40-500 ng/mL for α-PVP, with determination coefficients above 0.99 for all analytes. Recoveries ranged between 70.3% and 116.6%, and regarding intra- and inter-day precision, the relative mean errors were typically lower than 8.6%. The method was successfully applied to over 100 authentic samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Sustained low-efficiency dialysis (SLED) therapy following ingestion of isopropanol in a pediatric patient.

Introduction: Cardiovascular collapse due to large ingestions of isopropanol is rare. We report a case of a pediatric patient who had severe CNS and respiratory depression and cardiovascular collapse and was not hemodynamically stable enough to undergo hemodialysis.Case report: A 14-year-old 50 kg male was initially reported to have ingested an unknown amount of HEET® gas line antifreeze about 1 h prior to emergency department (ED) arrival. Despite severe CNS and respiratory depression and cardiovascular collapse, the patient was not initially acidotic. The patient did have an elevated osmolar gap. Approximately 6 h post-ingestion relatives updated the history to reflect that the product was in fact called ISO-HEET® which contains 99% isopropanol. Based on these concerns, a serum isopropanol and acetone levels were obtained that resulted at 475 and 75 mg/dL, respectively. Nephrology was consulted and it was decided to start the patient on sustained low-efficiency dialysis (SLED) which commenced 11 h post-ingestion. Serum and ultrafiltrate concentrations for isopropanol and acetone decreased to normal range over the course of SLED therapy.Discussion: SLED was instituted in this patient primarily for the treatment of elevated serum lactate, isopropanol, and acetone concentrations. The patient's systemic clearance was calculated as 26.9 mL/min. During SLED therapy, the patient was able to clear isopropanol and acetone at 41.21 mL/min and 29.74 mL/min, respectively. SLED therapy is a viable treatment option when a patient is hemodynamically unstable and hemodialysis is not an option.

Dietary and metabolomic determinants of relapse in ulcerative colitis patients: A pilot prospective cohort study.

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.

Euglycemic Diabetic Ketoacidosis with Elevated Acetone in a Patient Taking a Sodium-Glucose Cotransporter-2 (SGLT2) Inhibitor.

BACKGROUND: Sodium-glucose cotransporter-2 (SGLT2) inhibitor medications are a class of antihyperglycemic agents that increase urinary glucose excretion by interfering with the reabsorption of glucose in the proximal renal tubules. In May of 2015, the U.S. Food and Drug Administration released a warning concerning a potential increased risk of ketoacidosis and ketosis in patients taking these medications. CASE REPORT: We present a case of a 57-year-old woman with type 2 diabetes mellitus taking a combination of canagliflozin and metformin who presented with progressive altered mental status over the previous 2 days. Her work-up demonstrated a metabolic acidosis with an anion gap of 38 and a venous serum pH of 7.08. The serum glucose was 168 mg/dL. The urinalysis showed glucose > 500 mg/dL and ketones of 80 mg/dL. Further evaluation demonstrated an elevated serum osmolality of 319 mOsm/kg and an acetone concentration of 93 mg/dL. She was treated with intravenous insulin and fluids, and the metabolic abnormalities and her altered mental status resolved within 36 h. This was the first episode of diabetic ketoacidosis (DKA) for this patient. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Diabetic patients on SGLT2 inhibitor medications are at risk for ketoacidosis. Due to the renal glucose-wasting properties of these drugs, they may present with ketoacidosis with only mild elevations in serum glucose, potentially complicating the diagnosis. Acetone is one of the three main ketone bodies formed during DKA and it may be present at considerable concentrations, contributing to the serum osmolality.

Postmortem biochemistry in suspected starvation-induced ketoacidosis.

Significantly increased blood ketone body levels can be occasionally observed in the forensic setting in situations other than exposure to cold, diabetic or alcoholic ketoacidosis. Though infrequent, these cases do occur and deserve thorough evaluation in order to establish appropriate differential diagnoses and quantify the role that hyperketonemia may play in the death process. Starvation ketoacidosis is a rare cause of metabolic acidosis and is a phenomenon that occurs normally during fasting, as the body switches from carbohydrate to lipid energy sources. The levels of ketonemia in starvation ketoacidosis is usually mild in comparison to those seen in diabetic or alcoholic ketoacidosis. In the clinical setting, several cases of starvation-induced ketoacidosis mainly associated with gastric banding, pregnancy, malnutrition and low-carbohydrate diets have been reported. However, starvation ketosis causing severe metabolic acidosis has been rarely described in the medical literature. In the realm of forensic pathology, starvation-induced hyperketonemia has been rarely described. In this paper we present the postmortem biochemical results observed in situations of suspected starvation-induced hyperketonemia that underwent medico-legal examination. In all these cases, the diagnosis of starvation induced-hyperketonemia and the subsequent ketoacidosis was established per exclusionem based on all postmortem investigation findings. A review of the literature pertaining to the clinical diagnosis of starvation ketoacidosis is also provided.

Death due to diabetic ketoacidosis: Induction by the consumption of synthetic cannabinoids?

We present a case study on a man who suffered from diabetic ketoacidosis, probably following consumption of synthetic cannabinoids. In blood from a femoral vein AB-CHMINACA, AB-FUBINACA, AM-2201, 5F-AMB, 5F-APINACA, EAM-2201, JWH-018, JWH-122, MAM-2201, STS135 and THJ 2201 could be detected by LC-MS/MS. Diagnosis of ketoacidosis as cause of death was carried out using biochemical measurements of glucose and lactate concentrations in vitreous humour (sum formula: 463 mg/dl) and cerebrospinal fluid (sum formula: 506 mg/dl), of acetone (163 mg/l in femoral venous blood) and of HbA1c (98 mmol/mol). Death due to hyperglycaemia could have been induced by skipping of insulin doses due to his intoxicated state or by the cannabinoids which were described to be able to produce hyperglycaemia themselves.

Ethylone-Related Deaths: Toxicological Findings.

Synthetic cathinones are an emerging class of designer drugs, frequently with deceptive labels and a multitude of analogs to circumvent drug control regulations. Research regarding the pharmacological effects and toxicity of these amphetamine derivatives is scarce, heightening the risk to the public health and safety. The composition of synthetic cathinone products continually changes and laboratories began to notice ethylone-positive products in late 2011. This report presents nine postmortem cases in whom ethylone was identified. Ethylone was isolated using solid-phase extraction and detected by gas chromatography-mass spectrometry. Seven of the cases had measurable concentrations of ethylone in blood, ranging from 38 to 2,572 ng/mL; ethylone was detected in the blood sample of one case with a concentration below the assay limit of quantification (25 ng/mL), and one case did not have detectable ethylone in blood. Besides ethylone, all but one case were also positive for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; seven cases had other drugs quantified in blood, including ethanol, alprazolam, benzoylecgonine, diphenhydramine, morphine and tramadol. In five cases where ethylone was present at blood concentrations >400 ng/mL, no other drugs excluding ethanol, cannabis metabolite and doxylamine (one case) were found. The assay also tested for mephedrone, methylone and three dimethoxyamphetamine analogs; no case was positive for these analytes. The present report documents postmortem blood concentrations of ethylone, a novel synthetic cathinone, along with other concurrently identified substances. The findings provide valuable information for developing analytical assays and evaluating a toxic concentration range of ethylone.

Acetone as biomarker for ketosis buildup capability--a study in healthy individuals under combined high fat and starvation diets.

BACKGROUND: Ketogenic diets are high fat and low carbohydrate or very low carbohydrate diets, which render high production of ketones upon consumption known as nutritional ketosis (NK). Ketosis is also produced during fasting periods, which is known as fasting ketosis (FK). Recently, the combinations of NK and FK, as well as NK alone, have been used as resources for weight loss management and treatment of epilepsy. METHODS: A crossover study design was applied to 11 healthy individuals, who maintained moderately sedentary lifestyle, and consumed three types of diet randomly assigned over a three-week period. All participants completed the diets in a randomized and counterbalanced fashion. Each weekly diet protocol included three phases: Phase 1 - A mixed diet with ratio of fat: (carbohydrate + protein) by mass of 0.18 or the equivalence of 29% energy from fat from Day 1 to Day 5. Phase 2- A mixed or a high-fat diet with ratio of fat: (carbohydrate + protein) by mass of approximately 0.18, 1.63, or 3.80 on Day 6 or the equivalence of 29%, 79%, or 90% energy from fat, respectively. Phase 3 - A fasting diet with no calorie intake on Day 7. Caloric intake from diets on Day 1 to Day 6 was equal to each individual's energy expenditure. On Day 7, ketone buildup from FK was measured. RESULTS: A statistically significant effect of Phase 2 (Day 6) diet was found on FK of Day 7, as indicated by repeated analysis of variance (ANOVA), F(2,20) = 6.73, p < 0.0058. Using a Fisher LDS pair-wise comparison, higher significant levels of acetone buildup were found for diets with 79% fat content and 90% fat content vs. 29% fat content (with p = 0.00159**, and 0.04435**, respectively), with no significant difference between diets with 79% fat content and 90% fat content. In addition, independent of the diet, a significantly higher ketone buildup capability of subjects with higher resting energy expenditure (R(2) = 0.92), and lower body mass index (R(2) = 0.71) was observed during FK.

Comparison of breath gases, including acetone, with blood glucose and blood ketones in children and adolescents with type 1 diabetes.

Previous studies have suggested that breath gases may be related to simultaneous blood glucose and blood ketone levels in adults with type 2 and type 1 diabetes. The aims of this study were to investigate these relationships in children and young people with type 1 diabetes in order to assess the efficacy of a simple breath test as a non-invasive means of diabetes management. Gases were collected in breath bags and measurements were compared with capillary blood glucose and ketone levels taken at the same time on a single visit to a routine hospital clinic in 113 subjects (59 male, age 7 years 11 months-18 years 3 months) with type 1 diabetes. The patients were well-controlled with relatively low concentrations of the blood ketone measured (ß hydroxybutyrate, 0-0.4 mmol l(-1)). Breath acetone levels were found to increase with blood ß hydroxybutyrate levels and a significant relationship was found between the two (Spearman's rank correlation ρ = 0.364, p < 10(-4)). A weak positive relationship was found between blood glucose and breath acetone (ρ = 0.16, p = 0.1), but led to the conclusion that single breath measurements of acetone do not provide a good measure of blood glucose levels in this cohort. This result suggests a potential to develop breath gas analysis to provide an alternative to blood testing for ketone measurement, for example to assist with the management of type 1 diabetes.