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Purpose In this paper, we describe six cases, where patients were falsely treated with pre-filled ropivacaine solution instead of medical saline solution for postoperative settings. Two of the patients died because of fatal concentrations of ropivacaine in blood, four survived with no further physical injury, and two of them showed typical intoxication symptoms. The collected blood and urine samples of the deceased, as well as the surviving patients, were analyzed in laboratory routine screening. Methods Ropivacaine and its main metabolite 3-OH-ropivacaine were analyzed by gas chromatography–mass spectrometry. Results All of the six cases showed positive blood results of ropivacaine. Due to the poorly secured sample material of the survived patients at hospital, the quantitative examination of 3-OH-ropivacaine could not be carried out. In one fatal case, there were ropivacaine and 3-OH-ropivacaine traceable in urine. In all cases, metamizole was found in blood and traces of piritramide in urine. The amount of metamizole was within the upper limits of therapeutical treatment values. Patients with lower blood concentrations of ropivacaine showed more physical symptoms as compared to those with higher concentrations of active substances. Conclusions In the context of symptom development and intoxication, the speed of injection was a very important factor to cause fatal ropivacaine cases. To our knowledge, these are the first reported cases of fatal intoxication with ropivacaine.

Purpose Cumyl-PEGACLONE was the first synthetic cannabinoid (SC) with a γ-carbolinone core structure detected in forensic casework and, since then, it has dominated the German SC-market. Here the first four cases of death involving its fluorinated analog, 5F-Cumyl-PEGACLONE, a recently emerged γ-carbolinone derived SC, are reported. Methods Complete postmortem examinations were performed. Postmortem samples were screened by immunoassay, gas chromatography mass spectrometry (GC–MS) or liquid chromatography tandem mass spectrometry. For quantification of SCs, the standard addition method was employed. Herbal blends were analyzed by GC–MS. In each case of death, the Toxicological Significance Score (TSS) was assigned to the compound. Results 5F-Cumyl-PEGACLONE was identified at concentrations ranging 0.09–0.45 ng/mL in postmortem femoral blood. In case 1, signs of hypothermia and kidney bleedings were noted. Despite a possible tolerance due to long term SC use, a TSS of 3 was assigned. In case 2, an acute heroin intoxication occurred and a contributory role (TSS = 1) of 5F-Cumyl-PEGACLONE was suggested. In case 3, a prisoner was found dead. GC–MS analysis of herbal blends, retrieved in his cell together with paraphernalia, confirmed the presence of 5F-Cumyl-PEGACLONE and a causative role was deemed probable (TSS = 2). In case 4, the aspiration of gastric content due to a SC-induced coma was observed (TSS = 3). Conclusions 5F-Cumyl-PEGACLONE is an emerging and extremely potent SC which raises serious public health concerns. A comprehensive analysis of circumstantial, clinical, and postmortem findings, as well as an in-depth toxicological analysis is necessary for a valid interpretation and for the assessment of the toxicological significance.

As the use and spread of ventilators continues to grow so a CMOSens Technology has established a new generation of flow sensors.

Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca2+ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca2+ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca2+ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca2+ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca2+] in mutant (YS) compared with wild-type (WT) fibers. Action potential–induced Ca2+ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca2+ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (−80 mV). Untreated WT fibers show essentially no changes in [Ca2+], whereas the Ca2+ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca2+ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca2+ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca2+ release.

Malignant hyperthermia can result from mutations in the ryanodine receptor that favor anesthetic-induced Ca2+ release. Zullo et al. find that membrane potential modulates the effect of the volatile anesthetic halothane on skeletal muscle ryanodine receptors possessing the Y524S mutation., Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca2+ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca2+ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca2+ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca2+ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca2+] in mutant (YS) compared with wild-type (WT) fibers. Action potential–induced Ca2+ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca2+ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (−80 mV). Untreated WT fibers show essentially no changes in [Ca2+], whereas the Ca2+ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca2+ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca2+ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca2+ release.

The importance of direct and indirect alcohol markers to evaluate alcohol consumption in clinical and forensic settings is increasingly recognized. While some markers are used to prove abstinence from ethanol, other markers are suitable for detection of alcohol misuse. Phosphatidyl ethanol (PEth) is ranked among the latter. There is only little information about the correlation between PEth and other currently used markers (ethyl glucuronide, ethyl sulfate, carbohydrate deficient transferrin, gamma-glutamyl transpeptidase, and methanol) and about their decline during detoxification. To get more information, 18 alcohol-dependent patients in withdrawal therapy were monitored for These parameters in blood and urine for up to 19 days. There was no correlation between the different markers. PEth showed a rapid decrease at the beginning of the intervention, a slow decline after the first few days, and could still be detected after 19 days of abstinence from ethanol.

Ethyl glucuronide, as a direct metabolite of ethanol degradation, has proven useful as a long-term marker in many forensic applications. The inability to determine ethyl glucuronide in dried blood left a missing link in many investigations. Here, we describe a new method based on mass spectrometry in a Pauli-type ion trap in order to determine this substance in dried blood samples.

A sensitive and specific method for the determination of gamma-hydroxybutyric acid (GHB) in urine and serum is described. Prior to quantification by LC/MS in a Pauli-type ion trap, the molecule is converted by a fast and simple one-step procedure into its n-butyl ester derivative. Hexa-deutero GHB has been used as internal standard.

Objective: To investigate the involvement of α1-adrenoceptors in the sympathetic regulation of glucose uptake in human adipocytes. Research Methods and Procedures: Twenty-four severely obese subjects participated in this study. The microdialysis technique was used to determine interstitial glucose concentration after stimulation of abdominal subcutaneous adipose tissue with the α1-agonist norfenefrine, the α1, 2β-agonist norepinephrine, and both agents in combination with the α1-antagonist urapidil. The effect of β-adrenoceptor stimulation was assessed by orciprenaline. Changes in local blood flow were determined using the ethanol escape technique. Results: Both norfenefrine and norepinephrine induced a concentration-dependent decrease of interstitial glucose concentration, with a greater decrease observed with norepinephrine. Preperfusion of adipose tissue with urapidil inhibited glucose decrease. The inhibition was overcome with high concentrations of norfenefrine and norepinephrine, respectively. Both adrenergic agents induced tachyphylaxia. Urapidil enhanced extracellular glucose level at high concentration. Blood flow decreased in the presence of norfenefrine and norepinephrine but increased in response to urapidil. The accelerated blood flow due to urapidil was counteracted by norepinephrine and norfenefrine. Orciprenaline decreased interstitial glucose concentration and increased nutritive blood flow. The observed changes in blood flow induced by adrenergic agents were not related to glucose uptake. Discussion: The stimulatory effect of the sympathetic nerves on glucose uptake in subcutaneous adipose tissue appears to be mediated by the α1-adrenoceptor. Norepinephrine enhances glucose entry into adipocytes independently of insulin action. In obese subjects with insulin resistance, the α1-adrenergic receptor may provide an important alternative pathway for glucose uptake.

A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Washed and cut hair segments were extracted by ultrasonication (3h, 50 degrees C) and the extracts were cleaned-up with aminopropyl SPE columns. LC-MS/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation (ESI) using a triple quadrupole mass spectrometer (Sciex API 365) with a turboionspray source and post-column addition of acetonitrile for enhanced sensitivity. The MS/MS transitions monitored were m/z 221 -->75 for EtG and 226 -->75 for D(5)-EtG as an internal standard. The method was selective and sensitive, with a detection limit of 51 pg/mg hair at a signal-to-noise ratio of 3:1. The mean recovery was 96%, with an intra- and inter-day precision of less than 11.7% at a concentration of 200 pg/mg. The linearity was assessed in the range of 25-2000 pg/mg hair, with a correlation coefficient of 0.997. The method was successfully applied to 97 human hair samples which were taken at autopsies from persons with known alcoholism or were obtained from alcoholics who were hospitalized for ethanol withdrawal, from social drinkers and from children having not consumed any alcohol. Although, approximately two-third of the alcoholics showed EtG concentrations in hair of higher than 51 pg/mg (up to >4000 pg/mg), in one-third the EtG concentration was below the detection limit. However, only in one of five hair samples of "social drinkers", the EtG concentration was above the detection limit (51 pg/mg). No EtG has been detected in the hair of children. These investigations demonstrate that heavy alcohol consumption may be but not necessarily has to be detectable by EtG analysis in hair.

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas' resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.

Background Acipimox is a hypolipidaemic agent reducing serum concentrations of triglycerides and non-esterified fatty acids. Acipimox may reduce triglyceride synthesis by decreasing non-esterified fatty acid availability from adipocytes, but this effect has yet to be demonstrated in vivo. Lipolysis after acipimox treatment was examined in subcutaneous adipose tissue of severely obese subjects with associated metabolic disorders. Methods The microdialysis technique was performed in abdominal subcutaneous adipose tissue of eight hyperinsulinaemic subjects. After oral treatment with acipimox, glycerol concentration was determined as an index of lipolysis rate. Blood flow was assessed by the ethanol escape technique. The rates of release of glycerol from human adipose tissue maximally stimulated by norepinephrine were also investigated in the presence of acipimox. Eight weight- and age-matched subjects served as a control group. Results Under acipimox treatment, basal glycerol release decreased in subcutaneous adipose tissue, whereas no effect was observed on blood flow. In stimulated adipose tissue acipimox showed no effect. Conclusion In the present study basal glycerol outflow from adipose tissue was inhibited by acipimox. The anti-lipolytic action of the agent may diminish elevated plasma concentrations of free fatty acids in subjects with severe obesity. Copyright © 2001 John Wiley & Sons, Ltd.

A marker with a specific time spectrum of detection and both high sensitivity and specificity is required to diminish the clinically as well as forensically important gap on the time axis between short- and long-term markers of alcohol consumption like ethanol and CDT, GGT or MCV, respectively. Ethyl glucuronide (EtG) is a non-volatile, water-soluble, stable upon storage, direct metabolite of ethanol with a molecular weight of 222 g/mol that can be detected in body fluids for an extended time period after the complete elimination of alcohol from the body. We investigated 107 urine and 78 serum samples of a total of 107 inpatients in 4 groups: (1) 33 inpatients in acute alcohol withdrawal and long term treatment; (2/3) 29 and 15 addicted forensic psychiatric inpatients (#64 StGB, penal code); (4) 30 recently detoxified inpatients of a station for long term treatment by LC/MS-MS with the internal standard d5-EtG and additionally in the fourth group of patients also by gas chromatography/mass spectrometry (GC/MS). In 2 out of 33 inpatients of the first group, EtG could be determined 3 days after hospitalization; in an other subject, a relapse could be detected. In 2 out of 29 and in 1 out of 15 forensic inpatients of group 2 and 3, respectively--where neither clinical impression nor routine laboratory findings gave an indication for relapse--concentrations of EtG ranged between 0.1 and 18 mg/l in urine. For the serum samples of the 30 inpatients of group 4, we could demonstrate a total agreement for the results of the GC/MS and the LC/MS-MS method as to whether a sample was found to be positive or negative for EtG. We suggest that these results strengthen our earlier findings that ethyl glucuronide is a marker of alcohol consumption in general that can be detected for an extended time period after the complete elimination of alcohol from the body and a marker for relapse control with a specific time frame of detection intermediate between short- and long-term markers.

Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies.

Summary The width of the gracilis muscle was measured before and after removal of the epimysium in 10 fresh cadavers. The average extent of muscle widening achieved by epimysium removal was over 100% (mean 112.6%; standard deviation 11.9%). This extended muscle flap enabled us to cover successfully even large soft tissue defects measuring up to 300 cm 2 . In 27 consecutive patients, soft tissue defects of the lower leg with exposed bone have been repaired by free tissue transfer of a gracilis muscle flap, covered with split skin grafts. The advantages of the gracilis muscle flap were the low donor site morbidity with almost no recognisable functional loss, the easy surgical access, the thin and flat shape of the muscle, and its adequate size after excision of the epimysium. Reconstruction with a gracilis muscle flap resulted in an inconspicuous, stable, and flat contour of the lower leg. The entire length of the vascular pedicle of the gracilis muscle was easily harvested in our patients by routinely dissecting the pedicle on both sides of the adductor longus muscle.

This study aims to show that sensitive detection of ethyl glucuronide in dried blood spotted onto various surfaces after a period of 24h is feasible. At present, there is insufficient information how tightly ethyl glucuronide (EtG) binds to various materials and how easily it can be eluted. 4ml aliquots of blood samples obtained from seven volunteers after consumption of alcoholic beverages were applied to six different surfaces. After drying and a 24h-storage at 20±2°C the samples were re-dissolved in water, and EtG was subsequently analyzed by a LC-MS Paul-type ion trap. A comparison was made between dried and corresponding fluid samples. EtG was detectable in all subjects' samples following consumption of alcohol. EtG was also detectable after a storage time of four weeks at 4°C in whole blood that had been preserved with EDTA. EtG was detectable in all samples dried on different surfaces and its concentration remained relatively constant irrespective of the particular condition of the material. Detection of EtG in blood spots from the scene may indicate recent alcohol consumption in cases where collection of blood remained undone or could not be performed.

Mechanisms leading to acute pancreatitis after a fat-enriched meal combined with excess alcohol are incompletely understood. We have studied the effects of alcohol and fat (VLDL) on pancreatic acinar cell (PAC) function, oxidative stress, and repair mechanisms by pancreatic stellate cells (PSC) leading to fibrogenesis. To do so, PAC (rat) were isolated and cultured up to 24 h. Ethanol and/or VLDL were added to PAC. We measured PAC function (amylase, lipase), injury (lactic dehydrogenase), apoptosis (TUNEL, Apo2.7, annexin V binding), oxidative stress, and lipid peroxidation (conjugated dienes, malondialdehyde, chemoluminescence); we also measured PSC proliferation (bromodeoxyuridine incorporation), matrix synthesis (immunofluorescence of collagens and fibronectin, fibronectin immunoassay), and fatty acids in PAC supernatants (gas chromatography). Within 6 h, cultured PAC degraded and hydrolyzed VLDL completely. VLDL alone (50 μg/ml) and in combination with alcohol (0.2, 0.5, and 1% vol/vol) induced PAC injury (LDL, amylase, and lipase release) within 2 h through generation of oxidative stress. Depending on the dose of VLDL and alcohol, apoptosis and/or necrosis were induced. Antioxidants (Trolox, Probucol) reduced the cytotoxic effect of alcohol and VLDL. Supernatants of alcohol/VLDL-treated PAC stimulated stellate cell proliferation and extracellular matrix synthesis. We concluded that, in the presence of lipoproteins, alcohol induces acinar cell injury. Our results provide a biochemical pathway for the clinical observation that a fat-enriched meal combined with excess alcohol consumption can induce acinar cell injury (acute pancreatitis) followed by repair mechanisms (proliferation and increased matrix synthesis in PSC).

The therapeutic opioid drug methadone (d,l-methadone hydrochloride) is the most commonly used maintenance medication for outpatient treatment of opioid dependence. In our study, we found that methadone is also a potent inducer of cell death in leukemia cells and we clarified the unknown mechanism of methadone-induced cell killing in leukemia cells. Methadone inhibited proliferation in leukemia cells and induced cell death through apoptosis induction and activated apoptosis pathways through the activation of caspase-9 and caspase-3, down-regulation of Bcl-xL and X chromosome–linked inhibitor of apoptosis, and cleavage of poly(ADP-ribose) polymerase. In addition, methadone induced cell death not only in anticancer drug–sensitive and apoptosis-sensitive leukemia cells but also in doxorubicin-resistant, multidrug-resistant, and apoptosis-resistant leukemia cells, which anticancer drugs commonly used in conventional therapies of leukemias failed to kill. Depending on caspase activation, methadone overcomes doxorubicin resistance, multidrug resistance, and apoptosis resistance in leukemia cells through activation of mitochondria. In contrast to leukemia cells, nonleukemic peripheral blood lymphocytes survived after methadone treatment. These findings show that methadone kills leukemia cells and breaks chemoresistance and apoptosis resistance. Our results suggest that methadone is a promising therapeutic approach not only for patients with opioid dependence but also for patients with leukemias and provide the foundation for new strategies using methadone as an additional anticancer drug in leukemia therapy, especially when conventional therapies are less effective. [Cancer Res 2008;68(15):6059–64]

The aim of this in vitro study was to determine the diffusion of 2-hydroxyethyl methacrylate (HEMA) released from different bonding systems (BS) through dentin. Occlusal cavities with a remaining dentin thickness (RDT) of 0.5 mm (n=90) and 0.25 mm (n=80), respectively, were prepared in dentin discs of non-carious human molars. Artificial pulp chambers were attached to the pulpal side of each dentin disc. Bonding systems were applied with (Clearfil SE Bond, OptiBond FL, OptiBond Solo Plus) or without (AdheSE, Adper Prompt L-Pop, Clearfil SE Bond, OptiBond FL, OptiBond Solo Plus Self Etch, Xeno III) prior phosphoric acid etching. HEMA was detected by gas chromatography/mass spectrometry (n=10 per BS and RDT). The highest mean HEMA concentration was found in the 0.25 mm RDT group treated with OptiBond FL (13.3 microg) and the lowest mean HEMA concentration was detected in the 0.5 mm RDT group treated with AdheSE (0.5 microg). At 0.25 mm RDT the quantities of HEMA recovered in the artificial pulp chambers were significantly higher than at 0.5 mm RDT, except for Clearfil SE Bond. Etching with phosphoric acid increased the detected HEMA quantities compared with self-etch BS. In deep cavity preparations, etching with phosphoric acid should be avoided in favor of the use of self-etch BS.

Adverse 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) effects are usually ascribed to neurotransmitter release in the central nervous system. Since clinical features such as fasciculations, muscle cramps, rapidly progressing hyperthermia, hyperkalemia, and rhabdomyolysis point to the skeletal muscle as additional target, we studied the effects of MDMA on native and cultured skeletal muscle. We addressed the question whether malignant hyperthermia (MH)-susceptible (MHS) muscle is predisposed to adverse MDMA reactions. Force measurements on muscle strips showed that 100 microM MDMA, a concentration close to that determined in some MDMA users, regularly enhanced the sensitivity of skeletal muscle to caffeine-induced contractures but did not cause contractures on its own. The left-shift of the dose-response curve induced by MDMA was greater in normal than in MHS muscle. Furthermore, MDMA did not release Ca(2+) from isolated sarcoplasmic reticulum vesicles. These findings do not support the view of an MH-triggering effect on muscle. However, MDMA induced Ca(2+) transients in myotubes and increased their acidification rate. Surprisingly, alpha-bungarotoxin, a specific antagonist of the nicotinic acetylcholine receptor (nAChR), abolished these MDMA effects. The nAChR agonistic action of MDMA was confirmed by patch-clamp measurements of ion currents on human embryonic kidney cells expressing nAChR. We conclude that the neuromuscular junction is a target of MDMA and that an activation of nAChR contributes to the muscle-related symptoms of MDMA users. The drug may be of particular risk in individuals with abundant extrajunctional nAChR such as in generalized denervation or muscle regeneration processes and may act on central nAChR.

Aims: Leptin is a cytokine-type peptide hormone, recently implicated as a putative state marker of alcohol use and in craving. Our goal was to evaluate the potential of leptin as a state and trait marker and to rule out the role of current alcohol intoxication on leptin levels. Methods: Eighteen alcohol withdrawal patients (16 males, 2 females) whose blood contained 202 mg/dl (median) of ethanol at hospitalization, who had a median age of 43.5 years and had consumed 1075 g of ethanol (median) in the last 7 days were included in the study. Leptin was determined in samples at day 1 (when still intoxicated) and day 7 of withdrawal. Expected leptin levels were calculated with a formula. For comparison, 27 blood samples of 18 abstinent persons, matched for gender, age and body mass index were used. Furthermore, mean cell volume, γ-glutamyl transferase (GGT), blood glucose, cholesterol, triglycerides and body composition (bioimpedance device) were determined. For statistical analysis, SPSS 11 was used. Results: Expected leptin levels were 1.71 ng/ml (median), leptin measured at day 1 was 2.65 ng/ml (median) and 2.85 ng/ml on day 7 for the alcohol withdrawal patients and 2.2 ng/ml (median) for the abstainers. These concentrations were not significantly different. Significant correlations were found between leptin day 1 and expected leptin levels, percentage fat body mass, cigarettes smoked per day, GGT and blood alcohol concentration. Conclusions: Our preliminary data do not support the hypothesis of leptin as a state or trait marker and suggest only a minor influence of acute intoxication on leptin levels in alcohol detoxification patients.

Ethyl glucuronide (EtG) is a promising new biological state marker of recent alcohol consumption that detects alcohol use reliably over a definite time period. Other currently available markers lack acceptable sensitivity and specificity. Our aim is to elucidate under naturalistic conditions the time course of EtG excretion in urine following alcohol consumption and to show how this can be utilized for monitoring and prognosis in patients. There are no other existing data on this issue to date. One hundred and thirty-eight urine samples from 28 male alcohol withdrawal patients were drawn every 3-24 hours for up to 94 hours after hospitalization. Breath ethanol concentration (mean) at hospitalization was 900 mg/L. Patient age in years was 40.3 (mean). Determination of urine EtG was performed by gas chromatography/mass spectrometry (GC/MS) with deuterium-labelled EtG as an internal standard. The strongest correlations (p

Background: Considerable lives and money could be saved if one could detect early stages of lapsing/ relapsing behavior in addicted persons (e.g., in safety-sensitive workplaces) and could disclose harmful drinking in social drinkers. Due to the serious public health problem of alcohol use and abuse worldwide, markers of alcohol use have been sought. Both ethyl glucuronide (EtG) and phosphatidyl ethanol (PEth) appear to have high sensitivity and specificity and a time frame of detection that may elucidate alcohol use not detected by standard testing. Our aim was to assess their potential for detecting recent covert alcohol use under controlled conditions. Methods: Thirty-five forensic psychiatric inpatients in a closed ward who had committed a substancerelated offense (§64 StGB), were followed for 12 months. The complete time spectrum of possible alcohol consumption was covered by the complementary use of breath and urinary ethanol (hours), urinary EtG (days), %carbohydrate-deficient transferrin (CDT)/PEth (weeks), and -glutamyltranspeptidase (GGT)/ mean corpuscular volume (MCV) (weeks-months). Results: Fourteen of the 146 urine samples examined were positive for EtG. In all EtG-positive cases, patients reported alcohol consumption of between 40 and 200 g of ethanol 12– 60 hr prior to testing. Urinary and breath ethanol were positive in only one case. In the blood samples, PEth was not positive in any case and %CDT did not exceed the reference value. Isoelectric focusing showed no abnormal Tf subtypes. Conclusions: The findings emphasize the diagnostic and therapeutic usefulness, specificity, and sensitivity of EtG as a marker of recent alcohol use. Such a test is needed in numerous settings, including alcohol and drug treatment (to detect lapse/relapse), in safety-sensitive work settings where use is dangerous or in other settings where use may be inappropriate (e.g., such as driving, workplace, pregnancy, or monitoring physicians or other professionals who are in recovery and working), or for testing other groups (such as children or those with medical problems) where alcohol use would be unhealthy or unsafe. The health, social and socioeconomic benefits arising from the future use of these markers is hard to overestimate.

The role of alpha(1)-adrenoceptors in lipid mobilization and blood flow was investigated in situ using microdialysis of subcutaneous adipose tissue in severely obese subjects. The lipolysis rate was assessed by determination of interstitial glycerol concentration. The alpha(1)-adrenoceptor agonist norfenefrine caused an increase in glycerol level in adipose tissue that was similar to that observed with the physiologic alpha(1,2)-beta(1)-adrenoceptor agonist norepinephrine, whereas the alpha(1)-adrenoceptor antagonist urapidil showed no effect on basal lipolysis rate. However, the enhanced glycerol concentration due to norfenefrine and norepinephrine was suppressed in the presence of urapidil. The beta-adrenoceptor antagonist propranolol showed no effect on norfenefrine-stimulated glycerol outflow. Blood flow was assessed using the ethanol escape technique. Perfusion with norfenefrine decreased blood flow, whereas urapidil enhanced blood flow significantly. Despite the increase in blood flow, the basal interstitial glycerol concentration remained unchanged. Although norfenefrine at high concentrations could inhibit the urapidil-induced increase in blood flow, the norfenefrine-induced glycerol output was not affected. These results demonstrate that alpha(1)-adrenoceptors are involved in regulation of lipolysis rate and microcirculation of adipose tissue. However, the observed changes in local blood flow were not related to glycerol output.

An improved method for the determination of ethyl glucuronide (EtG) in human serum and urine was developed using solid-phase extraction (SPE) and gas chromatography (GC) with mass spectrometric detection (MS). EtG was isolated from serum and urine using aminopropyl SPE columns after deproteination with perchloric acid and hydrochloric acid, respectively. The chromatographic separation was performed on a DB 1701 fused-silica column. At a signal-to-noise ratio of 3:1, a quantification limit of 173 and 560 ng/ml and a detection limit of 37 and 168 ng/ml could be determined for serum and urine, respectively. This indicates high specificity and sensitivity of the described method. The mean absolute recovery was approximately 85%, while intra- and inter-day precision of the assay were all less than 7.5%. The linearity of the calibration curves was satisfying as indicated by correlation coefficients of0.993. The presented method provides the basis for determination and identification of EtG in human serum and urine samples in a low-concentration range for monitoring alcohol consumption during treatment for alcohol dependence and comorbid alcohol abuse of psychotherapy patients.

In German-speaking countries, blood ethanol concentrations (BECs) are usually calculated using Widmark’s equation. The distribution factor r of this equation is a correction factor needed to obtain a reduced body mass and corresponds to the ratio of total body water and blood water content. To enhance the reliability of Widmark’s model equation, the body weight, body height, blood water content and total body water of 256 women and 273 men were measured. The ratio of body water to blood water ranged from 0.44 to 0.80 in women and from 0.60 to 0.87 in men. For both sexes equations were developed by multiple regression analysis which allow the determination of the individual, more realistic distribution factors rFI (for females) and rMI (for males) even when only body height and body weight are known. Drinking experiments revealed a clearly higher congruence of calculated and measured blood ethanol concentrations when rFI or rMI were used instead of rigid distribution factors, i.e. 0.6 for women and 0.7 for men with or without the assumption of a 10% so-called resorption deficit. Additionally, Widmark’s equation in combination with rFI or rMI allows a more accurate prediction of blood ethanol concentrations than the equations of Watson and Ulrich.

In clinical as well as in forensic practice, occupational medicine, at court, for both traffic safety and safety at workplaces biological state markers of high sensitivity and specificity, capable of monitoring those in treatment for alcohol dependence or poly-drug-abusers as well as social drinkers in risky situations (driving, workplaces) are required. The known markers can not be considered to be satisfying with regard to these parameters. Furthermore, they do not cover the whole time axis for alcohol intake — i.e. there is a gap with regard to alcohol consumption of between 1 day and 1 – 2 weeks between short- and long-term-markers like ethanol, methanol, HTOL/HIAA ratio and CDT, GGT or MCV respectively. These traditional markers are in many cases besides alcohol influenced by age, gender and a variety of substances and non-alcohol-associated diseases. Ethyl glucuronide (EtG) is a non-volatile, water-soluble, stable upon storage, direct metabolite of ethanol with a molecular weight of 222 g/mol that can be detected in body fluids, tissues and hair. With its specific time frame of detection intermediate between between short- and long term markers and a high sensitivity and specificity, ethyl glucuronide is a promising marker of alcohol consumption in general that can be detected for an extended time period after the complete elimination of alcohol from the body and a marker for relapse control that enables the therapist to intervene at an early stage of relapsing behaviour.

Ethyl glucuronide (EtG) is a non-volatile, water-soluble, stable-upon-storage, direct metabolite of ethanol and can be detected in body fluids and tissues (and also in post-mortem material) for an extended time period after the complete elimination of alcohol from the body (Alt et al. , 1997; Schmitt et al. , 1997; Seidl et al. , 1998; Wurst et al. , 1999 a , b ). The aim of the present Letter is to emphasize through our preliminary results that the detection of alcohol consumption that has taken place several weeks or months earlier, via the determination of EtG in hair, could be a useful forensic tool as well as for monitoring alcohol consumption of those in treatment for alcohol dependence. Thirty-one hair samples from four groups of subjects were analysed: (1) hair was taken post mortem from 16 subjects with a known history of alcoholism; the diagnosis was established by police investigations including …

Summary Aims Metformin has been reported to decrease the plasma concentrations of non-esterified fatty acids in Type 2 diabetic subjects. This study investigated the effects of metformin on basal and catecholamine-stimulated lipolysis in abdominal subcutaneous adipose tissue of obese, hyperinsulinaemic, hypertensive subjects. Methods Fourteen subjects with severe obesity (12 female, two male, age 35.4 ± 4 years, body mass index 48.2 ± 2 kg/m2, body fat mass 63.3 ± 5 kg) were recruited. Glycerol and lactate concentrations were determined in the presence of metformin and after administration of catecholamines using microdialysis. Simultaneously, blood flow was assessed with the ethanol escape method. Results Glycerol release was lowered by metformin during the 3-h experiment (P

We studied lipolytic activities in vivo in golden mantle ground squirrels during pre-hibernation and hibernation using microdialysis technique. Microdialysis probes were inserted into the abdominal subcutaneous adipose tissues. Baseline lipolysis were assessed by measuring glycerol concentration. Epinephrine-stimulated lipolysis was also examined. Eight squirrels (four male, four female) were studied in each of the two stages. Basal glycerol concentrations were lower in the hibernating state than in the pre-hibernation state in male squirrels (P

Ethyl glucuronide (EtG) is a non-volatile, water-soluble, direct metabolite of ethanol that can be detected in body fluids and hair. We investigated urine and serum samples from three patient groups: (1) 33 in-patients in acute alcohol withdrawal; (2) 30 detoxified in-patients (treated for at least 4 weeks) from a 'motivation station'; and (3) 43 neuro-rehabilitation patients (non-alcoholics; most of them suffering from stroke, traumatic brain injury, Parkinson's disease etc.) using gas chromatography/mass spectrometry (GC/MS) with deuterium-labelled EtG as the internal standard and additionally in the second group of patients using liquid chromatography (LC/MS-MS). We found no correlation between the concentration of EtG in urine at hospitalization and the blood-ethanol concentration (r = 0.17), the time frame of detection (r = 0.5) or the total amount of clomethiazole required for the treatment of withdrawal symptoms (r = 0.28). In four out of 30 in-patients from the 'motivation station'--where neither clinical impression nor routine laboratory findings gave indications of relapse--concentrations of EtG in urine ranged between 4.2 and 196.6 mg/l. EtG concentrations in urine of between 2.89 and 23.49 mg/l were found in seven out of 43 neuro-rehabilitation patients using GC/MS. The GC/MS and the LC/MS-MS results showed a correlation of 0.98 with Pearson's correlation test and 1.0 with Spearman's correlation test. We suggest that EtG is a marker of alcohol consumption that can be detected for an extended time period after the complete elimination of alcohol from the body. When used as a relapse marker with a specific time frame of detection intermediate between short- and long-term markers, EtG fills a clinically as well as forensically important gap. Its specificity and sensitivity exceed those of all other known ethanol markers.

Although fasciocutaneous turnover flaps are a simple and fast method for covering soft-tissue defects of the lower leg, many reconstructive surgeons have their doubts about them. They revolve around the lack of criteria for safely designing these random-pattern flaps and around the risk of donor site problems. A vertically based deep fascia turnover flap with a paratibial or parafibular pedicle is presented. Anatomic studies of 36 injected lower limbs showed the deep fascia to be supplied by a mean of 61 vessels. As musculofascial, septofascial, and periosteofascial branches, these contribute to a richly anastomosing vascular network within the deep fascia. Along the deep transverse septum at the medial tibial border, the anterior and posterior peroneal septa, and between the anterior tibial and extensor muscles, the fascia is supplied by segmental vessels in a clearly defined arrangement. Pedicled on these vessels, the deep fascia is arrangement. Pedicled on these vessels, the deep fascia is a useful candidate tissue for transversely oriented turnover flaps. These are particularly well suited for covering pretibial or prefibular soft-tissue defects. Unlike adipofascial turnover flaps, the transversely oriented deep fascia turnover flap keeps its subcutaneous layer with its intact vascular plexus so that the overlying skin is adequately perfused even in patients with sizable flaps or an extremely thin skin. Clinical experience with the vertically based paratibial or parafibular deep fascia turnover flap in six patients confirmed its usefulness for covering small to medium-sized soft tissue defects of the lower leg.

Unreliable skin perfusion has been reported frequently in the gracilis myocutaneous flap, resulting in moderately high partial flap necrosis. We modified the traditional myocutaneous operative technique by including all available regional fascia and created a myofasciocutaneous flap with increased skin viability. In addition, we defined the arterial anatomy of the flap that contributes to enhanced flap survival. In a cadaver study, blue latex was injected into the external iliac arteries of 11 cadaveric legs and the gracilis myofasciocutaneous flap dissected. Selective ink injection of the pedicle and perforating vessels also was performed in 8 legs. Two additional legs were injected with a barium-latex mixture, cut into 2-cm-thick transverse sections, and radiographed. Dissections demonstrated arterial connections between the pedicle vessel (medial femoral circumflex artery) and fasciocutaneous perforating vessels from the superficial femoral artery. Perforating vessels contribute to an axially oriented fascial network that supplies the overlying skin. Selective ink injections demonstrated the individual primary cutaneous vascular territories for each perforator. Radiographs of 2-cm-thick transverse sections confirmed the presence of arterial connections between the pedicle and the superficial femoral artery perforators. Twelve patients, previously pelvically irradiated, then underwent harvest of 18 large, longitudinally oriented (8-cm-wide, up to 30-cm-long) gracilis myofasciocutaneous flaps. All fascia beneath the skin paddle was taken in continuity with the deep fascia surrounding the gracilis muscle to minimize disturbance of any connecting vasculature held within the fascia. Patients were followed for an average of 12.1 months (range 3 to 31 months). Minor complications related to the flaps occurred in 6 of 12 patients (50 percent), i.e., seromas, mild wound infections, and a partial dehiscence; however, vascularity was excellent with no partial or complete flap necrosis. All wounds healed completely.