Andreas Alt

Ethyl glucuronide: a marker of recent alcohol consumption with clinical and forensic implications.

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.

Ethyl glucuronide discloses recent covert alcohol use not detected by standard testing in forensic psychiatric inpatients

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 substance-related offense ( section sign 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 gamma-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.

Ethyl glucuronide-a biological marker for recent alcohol consumption.

Ethyl glucuronide (EtG) is a non‐volatile, water‐soluble metabolite of ethanol, with a high storage stability. It can be detected in body fluids, tissues, sweat and hair for an extended time period after the elimination of ethanol from the body. EtG closes the gap between short‐term markers for alcohol consumption such as ethanol or methanol and long‐term markers for alcohol misuse such as GGT, MCV and CDT. Due to its specific time‐frame of detection and its high sensitivity and specificity, EtG is a promising marker for alcohol consumption and for relapse control that enables the therapist to intervene at an early stage of relapsing behaviour. The aim of this review is to give an overview of analytical techniques for the detection of EtG, its clinical use and remaining questions.

The calculation of blood ethanol concentrations in males and females

Abstract 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.

Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction.

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 of >0.993. 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.

Ethyl glucuronide: on the time course of excretion in urine during detoxification.

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<0.01) were found between EtG determinations in the different patient when breath ethanol concentrations (BEC) were 0 and 48 hours after BEC=0 (r=0.747), EtG 24 and 48 hours after BEC=0 (r=0.872), and in the time frame of detection (hours) of EtG and EtG 48 hours after BEC=0 (r=0.762). No significant correlation was found (Mann‐Whitney test) between EtG concentrations in urine at different time points between the groups of patients with (a) 1 or less‐2, (b) 3‐4 or more previous hospitalizations, (c) a history of seizures (yes/no) or (d) an age above or below the median (40.5). EtG excretion in urine is not random, but seems rather to follow a kinetic profile. Furthermore our preliminary data indicate, that there is no significant difference for EtG concentration in urine when correlated to group variables such as age, seizures and hospitalizations.

Methadone, Commonly Used as Maintenance Medication for Outpatient Treatment of Opioid Dependence, Kills Leukemia Cells and Overcomes Chemoresistance

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-x(L) 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.

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

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.

Metformin inhibits catecholamine-stimulated lipolysis in obese, hyperinsulinemic, hypertensive subjects in subcutaneous adipose tissue: an in situ microdialysis study.

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.

Detection of ethyl glucuronide in dried human blood using LC-MS/MS

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.