Annette Thierauf

Formation of Phosphatidylethanol and Its Subsequent Elimination During an Extensive Drinking Experiment Over 5 Days

BACKGROUND For almost 30 years, phosphatidylethanol (PEth) has been known as a direct marker of alcohol consumption. This marker stands for consumption in high amounts and for a longer time period, but it has been also detected after 1 high single intake of ethanol (EtOH). The aim of this study was to obtain further information about the formation and elimination of PEth 16:0/18:1 by simulating extensive drinking. METHODS After 3 weeks of alcohol abstinence, 11 test persons drank an amount of EtOH leading to an estimated blood ethanol concentration of 1 g/kg on each of 5 successive days. After the drinking episode, they stayed abstinent for 16 days with regular blood sampling. PEth 16:0/18:1 analysis was performed using liquid chromatography-tandem mass spectrometry (high-performance liquid chromatography 1100 system and QTrap 2000 triple quadrupole linear ion trap mass spectrometer. Values of blood alcohol were obtained using a standardized method with headspace gas chromatography flame ionization detector. RESULTS Maximum measured concentrations of EtOH were 0.99 to 1.83 g/kg (mean 1.32 g/kg). These values were reached 1 to 3 hours after the start of drinking (mean 1.9 hours). For comparison, 10 of 11 volunteers had detectable PEth 16:0/18:1 values 1 hour after the start of drinking, ranging from 45 to 138 ng/ml PEth 16:0/18:1. Over the following days, concentrations of PEth 16:0/18:1 increased continuously and reached the maximum concentrations of 74 to 237 ng/ml between days 3 and 6. CONCLUSIONS This drinking experiment led to measurable PEth concentrations. However, PEth 16:0/18:1 concentrations stayed rather low compared with those of alcohol abusers from previous studies.

Urine tested positive for ethyl glucuronide after trace amounts of ethanol

AIM Ethyl glucuronide (EtG) is used commonly as a marker for the detection of non-compliance of patients in alcohol withdrawal therapy in psychiatric hospitals in Europe and in work-place monitoring programmes in the United States. With the increased use of this new marker, questions related to an unintentional uptake of ethanol resulting in detectable EtG concentrations have been discussed. The aim of this study was to determine the concentration ranges of EtG and ethyl sulphate (EtS) after the consumption of very small amounts of ethanol (1 and 3 g), which are more likely to be incidental than intended. METHODS Drinking experiments with ethanol amounts of 1 and 3 g, respectively, were performed on a total of 31 volunteers. EtG and EtS analysis in urine was performed by electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and creatinine concentration was determined using the Jaffé reaction. Furthermore, data obtained from this experimentation were then compared to data from literature. RESULTS AND CONCLUSIONS The maximum concentration of EtG normalized to creatinine after the uptake of 1 g and 3 g of ethanol was found to be 0.32 mg/l and 1.53 mg/l, respectively, and 0.15 mg/l and 1.17 mg/l for EtS; these peak concentrations are considered to be positive by many laboratories testing urine for ethanol conjugates in work-place testing progammes.

Fatal and severe codeine intoxication in 3-year-old twins—interpretation of drug and metabolite concentrations

This work presents two cases of codeine intoxication in 3-year-old monozygotic twin brothers while treated with a codeine slow-release formulation. One child had to be admitted to the hospital, whereas the other one died at home after aspiration of gastric content. The concentrations of codeine and major metabolites including morphine and corresponding glucuronide conjugates were measured by liquid chromatography–tandem mass spectrometry in serum, urine, cerebrospinal fluid, and brain tissue, respectively. A genetic polymorphism study was carried out in order to determine the ability of the children to metabolize codeine by O-demethylation. A pharmacokinetic calculation was also performed to estimate the administered dose of codeine in question. High concentrations of all substances were found in samples of both children. The pharmacokinetic estimate suggests an overdose of codeine, and the possible reasons for the high opiate concentrations are discussed. Furthermore, the postmortem distribution—during and after resuscitation—might play a major role in the interpretation of postmortem concentration levels.

Progress in monitoring alcohol consumption and alcohol abuse by phosphatidylethanol

For early diagnosis and therapy of alcohol-related disorders, alcohol biomarkers are highly valuable. Concerning specificity, indirect markers can be influenced by nonethanol-related factors, whereas direct markers are only formed after ethanol consumption. Sensitivity of the direct markers depends on cut-offs of analytical methods, material for analysis and plays an important role for their utilization in different fields of application. Until recently, the biomarker phosphatidylethanol has been used to differentiate between social drinking and alcohol abuse. After method optimization, the detection limit could be lowered and phosphatidylethanol became sensitive enough to even detect the consumption of low amounts of alcohol. This perspective gives a summary of most common alcohol biomarkers and summarizes new developments for monitoring alcohol consumption habits.

Urine tested positive for ethyl glucuronide and ethyl sulfate after the consumption of yeast and sugar

BACKGROUND To an increasing degree, EtG and EtS are routinely used for the proof of abstinence for purposes of traffic, occupational, addiction and social medicine. This routine use demands further investigations on the sensitivity and specificity of these analytes and the examination of possible genesis of positive EtG and EtS concentrations even without the consumption of ethanol. In vivo fermentation with consecutive formation of EtG and EtS was addressed by experiments with yeast products. METHODS Two experiments with bakers yeast and brewers yeast tablets were performed. The ethanol concentrations in urine of the 2 and 4 volunteers, respectively, were detected by HS-GC-FID, EtG and EtS analysis was performed by LC-ESI-MS/MS, and the creatinine concentration was determined using a method based on the Jaffé reaction. RESULTS AND CONCLUSIONS After the consumption of bakers yeast the maximum concentrations of EtG and EtS normalised to creatinine were found to be 0.67 and 1.41mg/L, respectively, and therefore clearly above the commonly applied cut-off value for the proof of abstinence of 0.1mg/L. In contrast, in this study the, uptake of yeast tablets did not result in a detection of EtG and EtS in urine.

Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death.

To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.

Influence of preservatives on the stability of ethyl glucuronide and ethyl sulphate in urine

BACKGROUND Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are specific and sensitive markers of ethanol consumption well established in monitoring withdrawal treatment in patients with chronic alcoholism. Recently, bacterial decomposition as well as in vitro and post-mortem formation of EtG was reported. The aim of this study was to investigate the influence of different preservatives on the stability of EtG and EtS concentrations in urine samples. METHODS Urine samples were doped with glucuronidase-positive Escherichia coli after sterile filtration. The preservatives used were thymol, chlorhexidine, boric acid and the combination of chlorhexidine, ethylparabene and sodium propionate. Different aliquots of urine samples were stored refrigerated (4-8 degrees C), at room temperature (18+/-1 degrees C) and in an incubator (36+/-1 degrees C) for a period of 9 days with daily sampling. EtG and EtS analyses were performed by LC-ESI-MS/MS. The number of bacteria was detected by counting the colony forming units on Columbia blood agar plates. RESULTS AND CONCLUSIONS Chlorhexidine on its own as well as in the aforementioned combination, and boric acid proved useful preservatives, while EtG degraded in samples doped with thymol. Addition of these preservatives did not interfere with the LC-MS/MS analysis.

Secondary skull fractures in head wounds inflicted by captive bolt guns: autopsy findings and experimental simulation

Apart from one article published by Rabl and Sigrist in 1992 (Rechtsmedizin 2:156–158), there are no further reports on secondary skull fractures in shots from captive bolt guns. Up to now, the pertinent literature places particular emphasis on the absence of indirect lesions away from the impact point, when dealing with the wounding capacity of slaughterer’s guns. The recent observation of two suicidal head injuries accompanied by skull fractures far away from the bolt’s path gave occasion to experimental studies using simulants (glycerin soap, balls from gelatin) and skull–brain models. As far as ballistic soap was concerned, the dimensions of the bolt’s channel were assessed by multi-slice computed tomography before cutting the blocks open. The test shots to gelatin balls and to skull–brain models were documented by means of a high-speed motion camera. As expected, the typical temporary cavity effect of bullets fired from conventional guns could not be observed when captive bolt stunners were discharged. Nevertheless, the visualized transfer of kinetic energy justifies the assumption that the secondary fractures seen in thin parts of the skull were caused by a hydraulic burst effect.

GSR deposition along the bullet path in contact shots to composite models

In contact shots, all the materials emerging from the muzzle (combustion gases, soot, powder grains, and metals from the primer) will be driven into the depth of the entrance wound and the following sections of the bullet track. The so-called “pocket” (“powder cavity”) under the skin containing soot and gunpowder particles is regarded as a significant indicator of a contact entrance wound since one would expect that the quantity of GSR deposited along the bullets path rapidly declines towards the exit hole. Nevertheless, experience has shown that soot, powder particles, and carboxyhemoglobin may be found not only in the initial part of the wound channel, but also far away from the entrance and even at the exit. In order to investigate the propagation of GSRs under standardized conditions, contact test shots were fired against composite models of pig skin and 25-cm-long gelatin blocks using 9-mm Luger pistol cartridges with two different primers (Sinoxid® and Sintox®). Subsequently, 1-cm-thick layers of the gelatin blocks were examined as to their primer element contents (lead, barium, and antimony as discharge residues of Sinoxid® as well as zinc and titanium from Sintox®) by means of X-ray fluorescence spectroscopy. As expected, the highest element concentrations were found in the initial parts of the bullet tracks, but also the distal sections contained detectable amounts of the respective primer elements. The same was true for amorphous soot and unburned/partly burned powder particles, which could be demonstrated even at the exit site. With the help of a high-speed motion camera it was shown that for a short time the temporary cavitation extends from the entrance to the exit thus facilitating the unlimited spread of discharge residues along the whole bullet path.

Experimental simulation of reentry shots using a skin-gelatine composite model.

A bullet that has passed in and out of one body segment may continue its way into another part of the body causing a second entrance (“reentry”) wound, which is often said to have an atypical and sometimes confusing appearance. To analyze this problem in a systematical approach, 9-mm Parabellum full metal-jacketed projectiles were fired at skin-gelatine composite models simulating the consecutive passage of a bullet through two parts of a body. When there was a distance between the two segments, the primary exit and the reentry skin wounds did not show any distinctive features differing from usual gunshot injuries. In the case of contact between exit and reentry site, the corresponding skin wounds resembled each other as both had central tissue defects surrounded by abrasion areas. The subsequent investigation of the simulant revealed that skin particles from both the exit and the adjacent reentry site had been displaced in the direction of the shot along the whole bullet track of the second segment. The morphological findings are presented and discussed with respect to the pertinent literature, and possible physical ballistic explanations are suggested.