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.     

Relationships between the deinstitutionalization of healthcare for patients with mental disorder, substance abuse, and isolated death

Isolated death (ID) (i.e., dying alone without anyone noticing for several days) has been suggested to be related to social isolation, mental disorder, and alcohol and/or drug abuse. A major transfer of patients with a mental disorder and/or alcohol and/or drug abuse from institutionalized care to treatment as outpatients has been enacted in Sweden during the past decade. On the basis of the assumption that such deinstitutionalization is likely to result in increased social isolation, our working hypothesis was that the incidence of ID among patients belonging to these categories has increased in Sweden. The present study involved all deaths subjected to a medicolegal examination in Stockholm County (with a population of approximately 1.9 million people) during the period 1992-2000. The pattern of ID (defined as cases involving a postmortem delay between death and discovery of at least 1 week), as well as the incidence of fatalities subjected to medicolegal examination with a record of mental disorder and/or alcohol and/or drug abuse was evaluated. Throughout this period, the proportion of the deceased with a record of a mental disorder was high among all the cases examined and higher still among the cases of ID, especially among those younger than 65 years of age. There was a rather limited increase in the incidence of ID and a much more pronounced increase in the number of former psychiatric patients whose deaths were subjected to medicolegal examination, but did not satisfy the criteria for ID. A record of alcohol and/or drug abuse was more common than a diagnosis of mental disorder among both the males and females who died at an age of less than 65, with a clear difference between the cases of ID and non-ID in the case of men. There was no significant increase in incidence over the course of this study. Thus, this study reveals a slight increase in the number of IDs and a more pronounced increase in the number of medicolegal examination of non-IDs of individuals with a record of a mental disorder.     

Medicolegal aspects of witnessed suicide due to gunshot to the head. II. Legal medicine aspects and examination of the firing hand

The second part of the paper on suicides by gunshots to the head in the presence of witnesses focuses on relevant morphological autopsy findings such as entrance site, signs of close range or contact shots, bullet path etc. and also discusses selected aspects of ascertaining gunshot residues. For the identification of the shooter an integral medicolegal assessment of all the facts including the investigation results and the autopsy findings is essential. However, the morphological findings alone do not allow safe diagnosis, as for example in a homicide the temporal region, which was affected in all our cases, may have been deliberately chosen by the perpetrator as a localization typical of suicide. Thus methods to ascertain gunshot residues on the firing hand (by means of adhesive films and the polyvinyl-alcohol collection method--PVAL) are of great practical importance. In seven cases adhesive films and/or the polyvinyl-alcohol collection method were used. In one case the gunshot residues (GSR) were analysed by means of tape lifts and subsequent scanning electron microscopy (SEM). It was found that especially the combined application of topographical (adhesive tape/PVAL) and cumulative (SEM) methods allowed for the doubtless identification of the shooter, thus usually confirming the suicide. By the example of one case it is demonstrated that without the immediate collection of evidence at the scene objective reconstruction of the event becomes impossible. On the basis of the reported cases recommendations are finally given for a differentiated approach in the medicolegal evaluation of alleged witnessed suicide by gunshot (to the head).     

A laboratory incubation method for determining the rate of microbiological degradation of skeletal muscle tissue in soil

A controlled laboratory experiment is described, in principle and practice, which can be used for the of determination the rate of tissue decomposition in soil. By way of example, an experiment was conducted to determine the effect of temperature (12 degrees, 22 degrees C) on the aerobic decomposition of skeletal muscle tissue (Organic Texel x Suffolk lamb (Ovis aries)) in a sandy loam soil. Measurements of decomposition processes included muscle tissue mass loss, microbial CO2 respiration, and muscle tissue carbon (C) and nitrogen (N). Muscle tissue mass loss at 22 degrees C always was greater than at 12 degrees C (p < 0.001). Microbial respiration was greater in samples incubated at 22 degrees C for the initial 21 days of burial (p < 0.01). All buried muscle tissue samples demonstrated changes in C and N content at the end of the experiment. A significant correlation (p < 0.001) was demonstrated between the loss of muscle tissue-derived C (Ct) and microbially-respired C (Cm) demonstrating CO2 respiration may be used to predict mass loss and hence biodegradation. In this experiment Q10 (12 degrees C-22 degrees C) = 2.0. This method is recommended as a useful tool in determing the effect of environmental variables on the rate of decomposition of various tissues and associated materials.