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.

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.

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.

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.

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.

(1)H-Nuclear magnetic resonance-based plasma metabolic profiling of dairy cows with clinical and subclinical ketosis.

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.

Biomonitoring of volatile organic compounds and organophosphorus flame retardands in commercial aircrews after "fume and smell events".

Health risks to humans after "fume and smell events", short-term incidents on aircrafts that are accompanied by unpleasant odour or visible smoke, remain a subject of controversy. We assessed exposure to volatile organic compounds (VOC) and organophosphorus compounds (OPC) by biomonitoring in 375 aircrew members after self-reported "fume and smell events" and in 88 persons of the general population. A total of 20 parameters were analysed in blood and urine by gas chromatography and mass spectrometry. Median levels of acetone in blood and urine and 2-propanol in blood were elevated in aircrews compared to controls (p < 0.0001). Additionally, elevated peak exposures, best estimated by the 95th percentiles, were observed in aircrews for n-heptane and n-octane in blood, and acetone, 2,5-hexanedione and o-cresol in urine. Only the maximum observed levels of 2,5-hexandione in urine (768 µg/L) and toluene in blood (77 µg/L) in aircrew members were higher than the current biological exposure indices (BEI® levels) (500 and 20 µg/L, respectively) of the American Conference of Governmental Industrial Hygienists (US-ACGIH) for workers occupationally exposed to n-hexane and toluene, two well-accepted human neurotoxicants. Low-level exposures to n-hexane and toluene could be also observed in controls. The majority of OPC parameters in urine, including those of neurotoxic ortho-isomers of tricresylphosphate, were below the limit of quantitation in both aircrews and controls. Our comparative VOC and OPC analyses in biological samples of a large number of aircrew members and controls suggest that exposures are similar in both groups and generally low.

1H NMR derived metabolomic profile of neonatal asphyxia in umbilical cord serum: implications for hypoxic ischemic encephalopathy.

Neonatal hypoxic ischemic encephalopathy (HIE) is a severe consequence of perinatal asphyxia (PA) that can result in life-long neurological disability. Disease mechanisms, including the role and interaction of individual metabolic pathways, remain unclear. As hypoxia is an acute condition, aerobic energy metabolism is central to global metabolic pathways, and these metabolites are detectable using 1H NMR spectroscopy, we hypothesized that characterizing the NMR-derived umbilical cord serum metabolome would offer insight into the consequences of PA that lead to HIE. Fifty-nine at-risk infants were enrolled, together with 1:1 matched healthy controls, and stratified by disease severity (n=25, HIE; n=34, non-HIE PA). Eighteen of 37 reproducibly detectable metabolites were significantly altered between study groups. Acetone, 3-hydroxybutyrate, succinate, and glycerol were significantly differentially altered in severe HIE. Multivariate data analysis revealed a metabolite profile associated with both asphyxia and HIE. Multiple-linear regression modeling using 4 metabolites (3-hydroxybutyrate, glycerol, O-phosphocholine, and succinate) predicted HIE severity with an adjusted R2 of 0.4. Altered ketones suggest that systemic metabolism may play a critical role in preventing neurological injury, while altered succinate provides a possible explanation for hypoxia-inducible factor 1-α (HIF-1α) stabilization in HI injury.

A case of instantaneous rigor?

The question of whether instantaneous rigor mortis (IR), the hypothetic sudden occurrence of stiffening of the muscles upon death, actually exists has been controversially debated over the last 150 years. While modern German forensic literature rejects this concept, the contemporary British literature is more willing to embrace it. We present the case of a young woman who suffered from diabetes and who was found dead in an upright standing position with back and shoulders leaned against a punchbag and a cupboard. Rigor mortis was fully established, livor mortis was strong and according to the position the body was found in. After autopsy and toxicological analysis, it was stated that death most probably occurred due to a ketoacidotic coma with markedly increased values of glucose and lactate in the cerebrospinal fluid as well as acetone in blood and urine. Whereas the position of the body is most unusual, a detailed analysis revealed that it is a stable position even without rigor mortis. Therefore, this case does not further support the controversial concept of IR.

Assessment of Traub formula and ketone bodies in cause of death investigations.

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.

GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples--quantifying neonatal exposure.

A static headspace gas chromatography coupled mass spectrometry (GC-MS) method was developed and fully validated for the quantitative measurement of acetaldehyde, acetone, methanol, ethanol and acetic acid in the headspace of micro-volumes of blood using n-propanol as an internal standard. The linearity of the method was established over the range 0.2-100 mg/L (R(2) > 0.99) and the limits of detection were 0.1-0.2 mg/L and lower limits quantification 0.5-1 mg/L. Precision and accuracies fell within acceptable limits (20 % for LLOQ and 15 %) for both intra- and inter-day analyses for all compounds except acetaldehyde which had inter-day variability of ≤25 %. The method was applied to analyse blood samples from neonatal patients receiving courses of ethanol excipient containing medications. Baseline levels of acetaldehyde, acetone, methanol and ethanol could be measured in patients before dosing commenced and an increase in levels of some volatiles were observed in several neonates after receiving ethanol-containing medications.

Is the formula of Traub still up to date in antemortem blood glucose level estimation?

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.

Genetic and nongenetic variation in plasma and milk ß-hydroxybutyrate and milk acetone concentrations of early-lactation dairy cows.

This study assessed genetic variation, heritability estimates, and genetic correlations for concentrations of plasma ß-hydroxybutyrate (BHBA), milk BHBA, and milk acetone in early lactation to investigate differences between cows in susceptibility to hyperketonemia and possibilities to use test-day milk ketone bodies for genetic improvement. Blood and test-day milk samples were collected on randomly selected dairy farms in the Netherlands from cows of various parities between 5 and 60 d in milk. Plasma samples were analyzed for BHBA (reference test for hyperketonemia) and test-day milk samples were analyzed for BHBA and acetone using Fourier-transform infrared spectroscopy. The final data set consisted of plasma BHBA concentrations of 1,615 cows from 122 herds. Milk BHBA and milk acetone concentrations were determined for 1,565 cows. Genetic variation, heritability, and proportion of phenotypic variation attributable to the herd were estimated using an animal model with fixed effects for parity and season, a covariate for days in milk, and random effects for herd, animal, and error. Genetic correlations for plasma BHBA, milk BHBA, and milk acetone were estimated using bivariate analyses. The heritability estimate for plasma BHBA concentrations in early lactation was 0.17, whereas heritability estimates for milk BHBA and milk acetone were 0.16 and 0.10, respectively. This indicates that selective breeding may contribute to a lower incidence of hyperketonemia in early lactation. For the 3 traits, the proportion of variance attributable to herd was larger than the additive genetic variance, underlining the importance of on-farm feeding and management in the etiology of hyperketonemia in fresh cows. Prevention strategies for hyperketonemia can, therefore, include both feeding and management strategies at dairy farms (short-term) and genetic improvement through breeding programs (long-term). Genetic correlations between concentrations of plasma BHBA and milk BHBA (0.52) or milk acetone (0.52) were moderate. As milk ketone bodies can be routinely analyzed at test days, this may provide a practical alternative for breeding programs aimed at reducing hyperketonemia in early lactation.

Factors affecting acetone concentration in blood of dairy cows at the first stage of lactation.

The aim of this paper was to determine effects of selected genetic and environmental factors on the concentration of acetone in blood of cows in the first trimester of lactation. The study included 124 Polish Holstein-Friesian primiparous cows born, reared and managed on the same farm. The samples of blood were collected on day 2, 5, 30, 60 and 90 after calving. The highest serum acetone concentration in the animals examined was observed on day 5 after calving. The level of this compound was inversely related to the contribution of the original Holstein-Friesian breed in gene pool. Significantly higher serum acetone level was observed in cows calved in January-March period when compared to other months of the year. Besides, the level of acetone determined on day 5 after calving was associated positively with body weight determined at the same day and negatively with body weight changes between days 5 and 60 after calving.

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.

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.

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.

Rapid analysis of acetone in human plasma by derivatization desorption electrospray ionization.

Desorption electrospray ionization mass spectrometry (DESI MS) is rapidly becoming a powerful analysis tool for a wide variety of samples in the open air. By combining DESI MS and derivatization, acetone in human blood samples, one of the diabetes mellitus (DM) biomarkers, was rapidly analyzed. Prior to DESI, the plasma sample was first exposed to hydroxylamine hydrochloride and acetone in plasma was derived into fixed oxime. The reactive products were validated by DESI/MS/MS. Quantification was performed in the positive-ion electrospray ionization mode by monitoring of the product ions at m/z 74 for acetone oxime and m/z 116 for 4-methyl-2-pentanone oxime as the internal standard. The limit of detection was of the order of magnitude of 0.10 ppbv and the linear range comprised two decades of concentration, covering the relevant concentration range of acetone in plasma samples. The method has achieved fast, high-throughput analysis of acetone in human plasma with little beforehand preparation and an experiment time of only a few seconds.