Saliva metabolome alterations after acute stress.

Major stress has systemic effects on the body that can have adverse consequences for physical and mental health. However, the molecular basis of these damaging effects remains incompletely understood. Here we use a longitudinal approach to characterise the acute systemic impact of major psychological stress in a pig model. We perform untargeted metabolomics on non-invasively obtained saliva samples from pigs before and 24 h after transfer to the novel physical and social environment of a slaughterhouse. The main molecular changes occurring include decreases in amino acids, B-vitamins, and amino acid-derived metabolites synthesized in B-vitamin-dependent reactions, as well as yet-unidentified metabolite features. Decreased levels of several of the identified metabolites are implicated in the pathology of human psychological disorders and neurodegenerative disease, suggesting a possible neuroprotective function. Our results provide a fingerprint of the acute effect of psychological stress on the metabolome and suggest candidate biomarkers with potential roles in stress-related disorders.

RawHummus: an R Shiny app for automated raw data quality control in metabolomics.

MOTIVATION: Robust and reproducible data is essential to ensure high-quality analytical results and is particularly important for large-scale metabolomics studies where detector sensitivity drifts, retention time and mass accuracy shifts frequently occur. Therefore, raw data need to be inspected before data processing to detect measurement bias and verify system consistency. RESULTS: Here, we present RawHummus, an R Shiny app for an automated raw data quality control (QC) in metabolomics studies. It produces a comprehensive QC report, which contains interactive plots and tables, summary statistics and detailed explanations. The versatility and limitations of RawHummus are tested with 13 metabolomics/lipidomics datasets and 1 proteomics dataset obtained from 5 different liquid chromatography mass spectrometry platforms. AVAILABILITY AND IMPLEMENTATION: RawHummus is released on CRAN repository (https://cran.r-project.org/web/packages/RawHummus), with source code being available on GitHub (https://github.com/YonghuiDong/RawHummus). The web application can be executed locally from the R console using the command 'runGui()'. Alternatively, it can be freely accessed at https://bcdd.shinyapps.io/RawHummus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High throughput method for quantifying androstenone and skatole in adipose tissue from uncastrated male pigs by laser diode thermal desorption-tandem mass spectrometry.

The study aims at developing a rapid and robust mass spectrometric method capable of measuring the malodorous boar taint compounds androstenone and skatole in fat samples from male pig carcasses. The developed method is suited for use in commercial abattoirs as an at-line method to detect the presence of these compounds in carcasses or as a high-speed analysis in laboratories with high sample turnover. The chemical assay is based on salt-assisted liquid-liquid extraction and direct measurement with Laser Diode Thermal Desorption-Tandem Mass Spectrometry (LDTD-MS/MS). When fully automated as an at-line method, a single LDTD-MS/MS system will have a measuring capacity of >420 male pig carcasses per hour. The limit of quantification (LOQ) is 0.05 µg/g and 0.10 µg/g for skatole and androstenone, respectively, which is well below the expected sorting thresholds. The reproducibility of the method (%RSD) meets the industry requirement for an RSD of below 10%.

CCWeights: an R package and web application for automated evaluation and selection of weighting factors for accurate quantification using linear calibration curve.

Summary: The accuracy of any analytical method is highly dependent on the selection of an appropriate calibration model. Here, we present CCWeights, an R package for automated assessment and selection of weighting factors for accurate quantification using linear calibration curve. Additionally, CCWeights includes a web application that allows users to analyze their data using an interactive graphical user interface, without any programming requirements. The workflow and features of CCWeights are illustrated by the analyses of two datasets acquired by liquid chromatography-mass spectrometry (LC-MS). The resulting quantification table can be directly utilized for further model assessment and subsequent data analysis. Availability and implementation: CCWeights is publicly available on CRAN repository (https://cran.r-project.org/web/packages/CCWeights), with source code available on GitHub (https://github.com/YonghuiDong/CCWeights) under a GPL-3 license. The web application can be run locally from R console using a simple command "runGui()". Alternatively, the web application can be freely accessed for direct online use at https://bcdd.shinyapps.io/CCWeights/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Increased antioxidant response in medium-chain acyl-CoA dehydrogenase deficiency: does lipoic acid have a protective role?

BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is the most frequent fatty acid oxidation (FAO) defect in humans. MCAD-deficient fibroblasts are more resistant to oxidative stress-induced cell death than other FAO defects and healthy controls. METHODS: Herein we investigate the antioxidant response and mitochondrial function in fibroblasts from MCAD-deficient patients (c.985 A>G/c.985 A>G) and healthy controls. RESULTS: MCAD-deficient fibroblasts showed increased level of mitochondrial superoxide, while lipids were less oxidatively damaged, and higher amount of manganese superoxide dismutase were detected compared to healthy controls, showing forceful antioxidant system in MCADD. We showed increased maximal respiration and reserve capacity in MCAD-deficient fibroblasts compared to controls, indicating more capacity through the tricarboxylic acid (TCA) cycle and subsequently respiratory chain. This led us to study the pyruvate dehydrogenase complex (PDC), the key enzyme in the glycolysis releasing acetyl-CoA to the TCA cycle. MCAD-deficient fibroblasts displayed not only significantly increased PDC but also increased lipoylated PDC protein levels compared to healthy controls. CONCLUSIONS: Based on these findings, we raise the interesting hypothesis that increased PDC-bound lipoic acid, synthesized from accumulated octanoic acid in MCADD, may affect the cellular antioxidant pool in MCADD.

The clinical outcome of LMNA missense mutations can be associated with the amount of mutated protein in the nuclear envelope.

AIMS: Lamin A/C mutations are generally believed to be associated with a severe prognosis. The aim of this study was to investigate disease expression in three affected families carrying different LMNA missense mutations. Furthermore, the potential molecular disease mechanisms of the mutations were investigated in fibroblasts obtained from mutation carriers. METHODS AND RESULTS: A LMNA-p.Arg216Cys missense mutation was identified in a large family with 36 mutation carriers. Disease expression was unusual with a late onset and a favourable prognosis. Two smaller families with severe disease expression were shown to carry a LMNA-p.Arg471Cys and LMNA-p.Arg471His mutation, respectively. LMNA gene and protein expression was investigated in eight different mutation carriers by quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and protein mass spectrometry. The results showed that all mutation carriers incorporated mutated lamin protein into the nuclear envelope. Interestingly, the ratio of mutated to wild-type protein was only 30:70 in LMNA-p.Arg216Cys carriers with a favourable prognosis while LMNA-p.Arg471Cys and LMNA-p.Arg471His carriers with a more severe outcome expressed significantly more of the mutated protein by a ratio of 50:50. CONCLUSION: The clinical findings indicated that some LMNA mutations may be associated with a favourable prognosis and a low risk of sudden death. Protein expression studies suggested that a severe outcome was associated with the expression of high amounts of mutated protein. These findings may prove to be helpful in counselling and risk assessment of LMNA families.

Effects of a Mutation in the HSPE1 Gene Encoding the Mitochondrial Co-chaperonin HSP10 and Its Potential Association with a Neurological and Developmental Disorder.

We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spasms at 3 months of age. Clinical exome sequencing revealed heterozygosity for a HSPE1 NM_002157.2:c.217C>T de novo mutation causing replacement of leucine with phenylalanine at position 73 of the HSP10 protein. This variation has never been observed in public exome sequencing databases or the literature. To evaluate whether the mutation may be disease-associated we investigated its effects by in vitro and ex vivo studies. Our in vitro studies indicated that the purified mutant protein was functional, yet its thermal stability, spontaneous refolding propensity, and resistance to proteolytic treatment were profoundly impaired. Mass spectrometric analysis of patient fibroblasts revealed barely detectable levels of HSP10-p.Leu73Phe protein resulting in an almost 2-fold decrease of the ratio of HSP10 to HSP60 subunits. Amounts of the mitochondrial superoxide dismutase SOD2, a protein whose folding is known to strongly depend on the HSP60/HSP10 complex, were decreased to approximately 20% in patient fibroblasts in spite of unchanged SOD2 transcript levels. As a likely consequence, mitochondrial superoxide levels were increased about 2-fold. Although, we cannot exclude other causative or contributing factors, our experimental data support the notion that the HSP10-p.Leu73Phe mutation could be the cause or a strong contributing factor for the disorder in the described patient.

Citric Acid Cycle Metabolites Predict the Severity of Myocardial Stunning and Mortality in Newborn Pigs.

OBJECTIVES: Myocardial infarction and chronic heart failure induce specific metabolic changes in the neonatal myocardium that are closely correlated to outcome. The aim of this study was to examine the metabolic responses to noninfarct heart failure and inotropic treatments in the newborn heart, which so far are undetermined. DESIGN: A total of 28 newborn pigs were instrumented with a microdialysis catheter in the right ventricle, and intercellular citric acid cycle intermediates and adenosine metabolite concentrations were determined at 20-minute intervals. Stunning was induced by 10 cycles of 3 minutes of ischemia, which was performed by occluding the right coronary artery, followed by 3 minutes of reperfusion. Animals were randomized for treatment with epinephrine + milrinone, dopamine + milrinone, dobutamine, or saline. SETTING: University hospital animal laboratory. MAIN RESULTS: Ischemia-reperfusion induced right ventricular stunning and increased the concentrations of pyruvate lactate, succinate, malate, hypoxanthine, and xanthine (all, p < 0.01). During inotrope infusion, no differences in metabolite concentrations were detected between the treatment groups. In nonsurviving animals (n = 8), concentrations of succinate (p < 0.0001), malate (p = 0.009), and hypoxanthine (p = 0.04) increased compared with survivors, while contractility was significantly reduced (p = 0.03). CONCLUSIONS: Accumulation of citric acid cycle intermediates and adenosine metabolites reflects the presence of myocardial stunning and predicts mortality in acute noninfarct right ventricular heart failure in newborn pigs. This phenomenon occurs independently of the type of inotrope, suggesting that citric acid cycle intermediates represent potential markers of acute noninfarct heart failure.

Selected reaction monitoring mass spectrometry for relative quantification of proteins involved in cellular life and death processes.

Monitoring of proteins involved in cellular life and death processes is of high scientific interest since it permits the elucidation of functional changes in a variety of diseases. In this study, we have developed a nanoLC-MS/MS assay for the simultaneous detection and quantification of 24 selected proteins that are known to be important for cellular homeostasis. The Selected Reaction Monitoring (SRM) assay applies heavy-labeled peptide analogues for the relative quantification of proteins with central functions in cellular stress and metabolism, including many mitochondrial proteins. The assay includes proteins involved in the quality control of mitochondrial proteins, oxidative stress, respiratory chain, and fatty acid degradation, as well as the cytosolic glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase and ribosomal proteins. The assay can thus quantitate the balance between mitochondrial and cytosolic pathways, which is relevant in many disease states, and can be studied by comparing patient and control samples. The measured validation parameters showed satisfactory results for the proteins included in the analysis. The linear range of the monitored proteins was 0.01-20nM, with a median precision of less than 10%. The assay performed well in monitoring proteins in both cultured human skin fibroblast cells as well as in isolated peripheral blood mononuclear cells. We therefore believe that this assay is applicable for the study of cellular stress response in various types of cell defects and disease states.

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1ß (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1ß proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1ß monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.

Large-scale proteomics differentiates cholesteatoma from surrounding tissues and identifies novel proteins related to the pathogenesis.

Cholesteatoma is the growth of keratinizing squamous epithelium in the middle ear. It is associated with severe complications and has a poorly understood etiopathogenesis. Here, we present the results from extensive bioinformatics analyses of the first large-scale proteomic investigation of cholesteatoma. The purpose of this study was to take an unbiased approach to identifying alterations in protein expression and in biological processes, in order to explain the characteristic phenotype of this skin-derived tumor. Five different human tissue types (cholesteatoma, neck of cholesteatoma, tympanic membrane, external auditory canal skin, and middle ear mucosa) were analyzed. More than 2,400 unique proteins were identified using nanoLC-MS/MS based proteomics (data deposited to the ProteomeXchange), and 295 proteins were found to be differentially regulated in cholesteatoma. Validation analyses were performed by SRM mass spectrometry. Proteins found to be up- or down-regulated in cholesteatoma were analyzed using Ingenuity Pathway Analysis and clustered into functional groups, for which activation state and associations to disease processes were predicted. Cholesteatoma contained high levels of pro-inflammatory S100 proteins, such as S100A7A and S100A7. Several proteases, such as ELANE, were up-regulated, whereas extracellular matrix proteins, such as COL18A1 and NID2, were under-represented. This may lead to alterations in integrity and differentiation of the tissue (as suggested by the up-regulation of KRT4 in the cholesteatoma). The presented data on the differential protein composition in cholesteatoma corroborate previous studies, highlight novel protein functionalities involved in the pathogenesis, and identify new areas for targeted research that hold therapeutic potential for the disease.

Remote ischemic preconditioning with--but not without--metabolic support protects the neonatal porcine heart against ischemia-reperfusion injury.

BACKGROUND: While remote ischemic preconditioning (rIPC) protects the mature heart against ischemia-reperfusion (IR) injury, the effect on the neonatal heart is not known. The neonatal heart relies almost solely on carbohydrate metabolism, which is modified by rIPC in the mature heart. We hypothesized that rIPC combined with metabolic support with glucose-insulin (GI) infusion improves cardiac function and reduces infarct size after IR injury in neonatal piglets in-vivo. METHODS AND RESULTS: 32 newborn piglets were randomized into 4 groups: control, GI, GI+rIPC and rIPC. GI and GI+rIPC groups received GI infusion continuously from 40 min prior to ischemia. rIPC and GI+rIPC groups underwent four cycles of 5 min limb ischemia. Myocardial IR injury was induced by 40 min occlusion of the left anterior descending artery followed by 2 h reperfusion. Myocardial lactate concentrations were assessed in microdialysis samples analyzed by mass spectrometry. Infarct size was measured using triphenyltetrazolium chloride staining. Systolic recovery (dP/dt(max) as % of baseline) after 2 h reperfusion was 68.5±13.8% in control, 53.7±11.2% in rIPC (p<0.05), and improved in GI (83.6±18.8%, p<0.05) and GI+rIPC (87.0±15.7%, p<0.01). CONCLUSION: rIPC+GI protects the neonatal porcine heart against IR injury in-vivo. rIPC alone has detrimental metabolic and functional effects that are abrogated by simultaneous GI infusion.

Protection against myocardial ischemia-reperfusion injury at onset of type 2 diabetes in Zucker diabetic fatty rats is associated with altered glucose oxidation.

BACKGROUND: Inhibition of glucose oxidation during initial reperfusion confers protection against ischemia-reperfusion (IR) injury in the heart. Mitochondrial metabolism is altered with progression of type 2 diabetes (T2DM). We hypothesized that the metabolic alterations present at onset of T2DM induce cardioprotection by metabolic shutdown during IR, and that chronic alterations seen in late T2DM cause increased IR injury. METHODS: Isolated perfused hearts from 6 (prediabetic), 12 (onset of T2DM) and 24 (late T2DM) weeks old male Zucker diabetic fatty rats (ZDF) and their age-matched heterozygote controls were subjected to 40 min ischemia/120 min reperfusion. IR injury was assessed by TTC-staining. Myocardial glucose metabolism was evaluated by glucose tracer kinetics (glucose uptake-, glycolysis- and glucose oxidation rates), myocardial microdialysis (metabolomics) and tissue glycogen measurements. RESULTS: T2DM altered the development in sensitivity towards IR injury compared to controls. At late diabetes ZDF hearts suffered increased damage, while injury was decreased at onset of T2DM. Coincident with cardioprotection, oxidation of exogenous glucose was decreased during the initial and normalized after 5 minutes of reperfusion. Metabolomic analysis of citric acid cycle intermediates demonstrated that cardioprotection was associated with a reversible shutdown of mitochondrial glucose metabolism during ischemia and early reperfusion at onset of but not at late type 2 diabetes. CONCLUSIONS: The metabolic alterations of type 2 diabetes are associated with protection against IR injury at onset but detrimental effects in late diabetes mellitus consistent with progressive dysfunction of glucose oxidation. These findings may explain the variable efficacy of cardioprotective interventions in individuals with type 2 diabetes.

Screening analysis for medicinal drugs and drugs of abuse in whole blood using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS)--toxicological findings in cases of alleged sexual assault.

An ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method for simultaneous screening of 46 medicinal drugs and drugs of abuse in whole blood was developed and validated. The method includes most of the commonly used and abused drugs such as amphetamines, cocaine, benzodiazepines, and opioids. Chromatographic separation of the targeted drugs was achieved using a Waters ACQUITY UPLC coupled to a Waters Micromass LCT Premier XE time-of-flight mass spectrometer. The total chromatographic run time was 13.5 min injection to injection. The estimated method LOQ is in the range of 0.06-27 ng/g, which is below the therapeutic levels for each of the drugs analyzed but LSD. The extraction recovery ranged from 6% to 197% with median value 95% and mean value 82%. Matrix effect ranged from 81% suppression to 29% enhancement of the signals compared to signals obtained in the absence of biological matrix. The method was tested on 55 authentic forensic toxicology samples confirming the same positive results as found using the routine analytical procedures as well as some additional compounds. Recently there has been considerable attention paid to drug-facilitated sexual assault and the toxicological findings in these cases. As part of a pilot study to investigate the prevalence of medicinal drugs, drugs of abuse, and alcohol in victims of alleged sexual assault, biological specimens were obtained from 167 victims being examined at the Sexual Assault Center in Aarhus, Denmark. The obtained blood samples were analyzed using the novel screening method supported by additional analyses for e.g. THC and alcohol. 124 victims reported they have been drinking alcohol prior to the assault (74%). Alcohol analyses revealed 59 positive findings (48%). 35 of the cases were found positive for one or more drugs excluding alcohol (21%). 20 of the victims reported they have been subject to a drug-facilitated sexual assault (12%). For the victims suspecting drug-facilitated sexual assault, the toxicological analyses revealed four positive for alcohol and nine victims were positive for one or more drugs, with six of the victims found positive for benzodiazepines or other drugs with sedative effects. It was notable that victims tested positive for medicinal drugs and drugs of abuse as well as victims of alleged drug-facilitated sexual assault in average underwent medical examination later than the whole study population.

Fumarate is cardioprotective via activation of the Nrf2 antioxidant pathway.

The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

A UPLC-MS/MS application for profiling of intermediary energy metabolites in microdialysis samples--a method for high-throughput.

Research within the field of metabolite profiling has already illuminated our understanding of a variety of physiological and pathological processes. Microdialysis has added further refinement to previous models and has allowed the testing of new hypotheses. In the present study, a new ultra-performance liquid chromatography/electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS) method for the simultaneous detection and quantification of intermediary energy metabolites in microdialysates was developed. The targeted metabolites were mainly from the citric acid cycle in combination with pyruvic acid, lactic acid, and the ATP (adenosine triphosphate) hydrolysis product adenosine along with metabolites of adenosine. This method was successfully applied to analyze the microdialysates obtained from an experimental animal study giving insight into the hitherto unknown concentration of many interstitial energy metabolites, such as succinic acid and malic acid. With a total cycle time of 3 min, injection to injection, this method permits analysis of a much larger number of samples in comparison with conventional high performance liquid chromatography/tandem mass spectrometry HPLC-MS/MS strategies. With this novel combination where microdialysis and high sensitivity UPLC-MS/MS technique is combined within cardiologic research, new insights into the intermediary energy metabolism during ischemia-reperfusion is now feasible.

Inhibition of the malate-aspartate shuttle by pre-ischaemic aminooxyacetate loading of the heart induces cardioprotection.

AIMS: Preserved mitochondrial function is essential for protection against ischaemia-reperfusion (IR) injury. The malate-aspartate (MA) shuttle constitutes the principal pathway for transport of reducing cytosolic equivalents for mitochondrial oxidation. We hypothesized that a transient shut-down of the MA-shuttle by aminooxyacetate (AOA) during ischaemia and early reperfusion modulates IR injury by mechanisms comparable to ischaemic preconditioning (IPC). METHODS AND RESULTS: Isolated perfused rat hearts exposed to 40 min global no-flow ischaemia were studied in: (i) control, (ii) pre-ischaemic AOA (0.1 mM), (iii) IPC, and (iv) AOA+IPC hearts. IR injury was evaluated by infarct size and haemodynamic recovery. Tracer-estimated glucose oxidation and metabolic changes in glycogen, lactate, pyruvate, tricarboxylic acid (TCA) cycle intermediates, and ATP degradation products were measured. The effects of AOA on complex I respiration and reactive oxygen species (ROS) production were examined in isolated rabbit mitochondria. Treatment with AOA, IPC, or AOA+IPC induced significant infarct reduction; 28 ± 6, 30 ± 3, and 18 ± 1%, respectively, vs. 52 ± 5% of left ventricular (LV) mass for control (P < 0.01 for all). LV-developed pressure improved to 60 ± 3, 63 ± 5 and 53 ± 4 vs. 31 ± 5 mmHg (P < 0.01 for all) after 2 h reperfusion. Pre-ischaemic AOA administration inhibited glycolysis and increased glucose oxidation during post-ischaemic reperfusion similar to IPC, and suppressed complex I respiration and ROS production in the non-ischaemic heart. Changes in lactate, pyruvate, TCA intermediates, and ATP end products suggested an AOA inhibition of the MA-shuttle during late ischaemia and early reperfusion. CONCLUSION: Inhibition of the MA-shuttle during ischaemia and early reperfusion is proposed as a mechanism to reduce IR injury.

Organocatalysis with endogenous compounds: towards novel non-enzymatic reactions.

The aldol reaction of the endogeneous compounds acetone and methylglyoxal has been studied using organocatalysis in relation to biologically relevant non-enzymatic reactions. Under preparative conditions, 3-hydroxy-2,5-hexadione, known as Henze's ketol, is formed in high yield and with enantioselectivities up to 88% ee. Furthermore, Henze's ketol is also formed under simulated physiological conditions at micromolar scale, indicating that this reaction might take place in living organisms.

A fatal poisoning involving Bromo-Dragonfly.

This paper reports a fatal overdose case involving the potent hallucinogenic drug Bromo-Dragonfly (1-(8-bromobenzo[1,2-b; 4,5-b']difuran-4-yl)-2-aminopropane). In the present case, an 18-year-old woman was found dead after ingestion of a hallucinogenic liquid. A medico-legal autopsy was performed on the deceased, during which liver, blood, urine and vitreous humour were submitted for toxicological examination. Bromo-Dragonfly was identified in the liver blood using UPLC-TOFMS, and was subsequently quantified in femoral blood (0.0047 mg/kg), urine (0.033 mg/kg) and vitreous humour (0.0005 mg/kg) using LC-MS/MS. Calibration standards were prepared from Bromo-Dragonfly isolated from a bottle found next to the deceased. The structure and purity of the isolated compound were unambiguously determined from analysis of UPLC-TOFMS, GC-MS, HPLC-DAD, (1)H and (13)C NMR data and by comparison to literature data. The autopsy findings were non-specific for acute poisoning. However, based on the toxicological findings, the cause of death was determined to be a fatal overdose of Bromo-Dragonfly, as no ethanol and no therapeutics or other drugs of abuse besides Bromo-Dragonfly were detected in the liver, blood or urine samples from the deceased. To our knowledge, this is the first report of quantification of Bromo-Dragonfly in a biological specimen from a deceased person. This case caused the drug to be classified as an illegal drug in Denmark on 5th December 2007.