Reliable, sensitive, rapid and quantitative enzyme-based assay for gamma-hydroxybutyric acid (GHB)

Several assays for gamma-hydroxybutyrate (4-hydroxybutyrate, GHB) have been developed based on the enzyme gamma-hydroxybutyrate dehydrogenase (GHB-DH). Enzymatic oxidation of GHB by NAD+ is coupled to diaphorase-mediated reduction of pro-dye to yield colored product. GHB-DH from Ralstonia eutropha was cloned and expressed as a stable fusion protein easily purified by affinity chromatography. Quantitative initial velocity and endpoint versions of the assay in solution are described. Michaelis-Menten parameters for oxidation of GHB and ethanol were estimated. A semi-quantitative "dipstick" version of the assay on paper also is described. Both solution endpoint and "dipstick" assays are sensitive to about 0.05 mg GHB/mL using 10 microL of sample. Ethanol at concentrations possible in urine and agents used to stabilize physiological fluids for forensics analysis do not interfere significantly. The "dipstick" assay also allows detection of GHB in alcoholic beverages after evaporation of about one-fourth drop of beverage before testing. The enzymatic assay for GHB is reliable, sensitive, inexpensive and rapid.     

Postmortem citalopram concentrations: alone or along with other compounds

Citalopram, an antidepressant whose use has become more widespread in Spain in recent years participates directly and indirectly in the lethal mechanism in voluntary and involuntary poisonings. There were 30 cases of autopsies in the Madrid region where citalopram and other psychoactive substances (psychotropic drugs, alcohol, opiates) were detected in the corpses. The postmortem citalopram levels in relation to the manner and mechanism of death were evaluated, and a significant difference between the toxic and nontoxic cases (p < 0.01) was found. We studied the citalopram blood levels alone and along with other psychoactive products, and these cases were then further divided into those where the compounds were at deadly levels and those which were not. We found a range of citalopram levels between 0.37 and 0.83 microg/mL in which some cases were associated with citalopram toxicity and others were not. Citalopram blood levels of less than 0.35 microg/mL did not lead to fatal poisoning when it was the sole substance detected.     

The significance of pulmonary interstitial emphysema in live birth determination

The utility of pulmonary interstitial emphysema (PIE) in live birth determination is not well established. Because the distinction between live birth and stillbirth may be important in criminal proceedings, we undertook this study to investigate the relationship between the histologic finding of PIE and live birth. Sixty-six cases of infant death were retrieved and compared with 21 stillborn infants. Histologic sections of the lungs were characterized as "florid PIE," "equivocal PIE," or "absent PIE." Sixteen cases of florid PIE were identified, all in live born infants. Forty-seven cases of equivocal PIE were found in 36 live born and 11 stillborn infants. In 24 cases (14 live born infants and 10 stillborns), no PIE was identified. We examined the relationship between florid PIE in infants with sudden infant death syndrome (SIDS) or "sudden unexpected death in infancy, manner undetermined" (SUDI), and also its relationship to other variables. No association was found. The presence of equivocal PIE may be an artifact of tissue processing. Florid PIE is found only in live born infants. No correlation between the presence of florid PIE and cause of death could be determined.     

Interpretation of a 3,4-methylenedioxymethamphetamine (MDMA) blood level: discussion by means of a distribution study in two fatalities

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy" is a currently used or abused designer drug and fatalities are frequently encountered in forensic practice. However, the question remains open whether an MDMA blood level can be toxic or even potentially lethal. In order to provide insight in the interpretation of a detected MDMA concentration, the distribution of MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) in various body fluids and tissues was studied and discussed in two different fatalities. Apart from peripheral blood samples (such as femoral and subclavian blood), various blood samples obtained centrally in the human body and several body fluids (such as vitreous humour) were examined. In addition, various tissues such as cardiac muscle, lungs, liver, kidneys, and brain lobes were analysed. In contrast to the peripheral blood levels, high MDMA and MDA levels were found in cardiac blood and the majority of the organs, except for the abdominal adipose tissue. The high concentrations observed in all lung lobes, the liver and stomach contents indicate that post-mortem redistribution of MDMA and MDA into cardiac blood can occur and, as a result, blood sampled centrally in the body should be avoided. Therefore, our data confirm that peripheral blood sampling remains "the golden standard". In addition, a distinct difference in peripheral blood MDMA concentrations in our two overdose cases was established (namely 0.271 and 13.508 microg/ml, respectively). Furthermore, our results suggest that, if a peripheral blood sample is not available and when putrefaction is not too pronounced, vitreous humour and iliopsoas muscle can be valuable specimens for toxicological analysis. Finally, referring to the various mechanisms of death following amphetamine intake, which can result in different survival times (e.g. cardiopulmonary complications versus hyperthermia), the anatomo-pathological findings and the toxicological results should be considered as a whole in arriving at a conclusion.     

Serum cannabinoid levels 24 to 48 hours after cannabis smoking

Low concentrations of THC and 11-hydroxy-THC in serum samples are often claimed not to result from recent cannabis use. Prediction of time of exposure is difficult, especially if distinctive features of drug use could not be observed. Therefore, the aim of the study was to investigate the presence of THC and 11-hydroxy-THC in serum samples as well as to obtain preliminary data on the analyte profile for a time window of 24-48 hours after discontinuation of cannabis smoking. Serum samples from heavy (n = 12, > 1 joint/day), moderate (n = 11, < or = 1 joint/day) and light (n = 6, < 1 joint/week) smokers of cannabis were analyzed for THC, 11-hydroxy-THC and free THC-COOH by GC/MS as well as for glucuronidated THC-COOH by LC/MS-MS. The blood samples were collected 24-48 hours after abstaining from cannabis use. Additionally, 8 specimens were obtained from persons after discontinuation of the drug for more than 48 hours. During collection of the blood samples, distinctive effects due to drug use could not be observed. For heavy users of cannabis, THC was detectable in 8 samples, and in 5 cases both biologically active compounds, THC and 11-hydroxy-THC, were present (1.3-6.4 ng THC/mL serum, 0.5-2.4 ng 11-hydroxy-THC/mL serum). Among moderate users, in 1 sample 1.8 ng THC/mL serum and 1.3 ng 11-hydroxy-THC/mL serum were determined, and another sample was tested positive with low concentrations close to the limit of detection. In serum samples of light users both analytes could not be detected, indicating that in those persons a positive finding of THC and 11-hydroxy-THC may rather result from recent consumption than from cannabis use 1 or 2 days prior to blood sampling. The concentrations of THC-COOH and its glucuronide covered a wide range in all groups of cannabis users. However, there was a trend to higher concentrations in heavy users compared to moderate users, and the mean concentration was smaller in light smokers than in moderate smokers. Overall, the findings indicated that data from pharmacokinetic studies should be supplemented by data obtained from "real-life" samples.     

An alternative method to screen for pepper spray residue

A method was developed to screen for pepper spray residue using instruments and methods other than those techniques commonly employed to analyze chemical residue (i.e.. gas chromatography mass spectrometry-GCMS or liquid chromatography mass spectrometry-LCMS). The method employed gas chromatography (GC), thin layer chromatography (TLC), and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) to screen for dried pepper spray stains. Pepper sprays from nine different manufacturers were investigated. Capsaicin and dihydrocapsaicin were identified and unique IR reflectance spectra are presented. An additional five compounds were presumptively found. Results showed that a particular stain could be characterized as a pepper-based stain.