Yosuke Usumoto

Pattern of poisoning in Japan: selection of drugs and poisons for systematic toxicological analysis

Patterns of poisoning are known to be different in different countries, because of the local environmental, cultural, and religious situations. Therefore, in Japan, it is important to know the pattern of poisoning in our own country and to prepare for every poisoning case by establishing an efficient systematic toxicological analysis system in forensic practice. We conducted a retrospective study of the kinds of compounds causing poisonings and the frequency of their use based on two series of reports dealing with poisoning cases in Japan prepared by the National Research Institute of Police Science and the Japanese Society of Legal Medicine for 2003 to 2006. From these reports, 459 and 177 compounds, respectively, were extracted as poisonous compounds over the study period. After data analysis, we selected 314 drugs and poisons as important target compounds for systematic drug analysis in Japan; they included 36 volatile compounds, 14 abused drugs, 170 medical drugs, 60 pesticides, 13 natural toxins, and 21 others. This is the first study to show the toxic drugs and poisons to be analyzed in Japan based on frequency of use, and as such the list will be useful in establishing the most efficient screening system in forensic practice.

Construction of calibration-locking databases for rapid and reliable drug screening by gas chromatography-mass spectrometry

Unique calibration-locking databases were constructed for rapid and semiquantitative drug screening by gas chromatography-mass spectrometry (GCMS). In addition to the free-drug database of 127 drugs, a drug database with acetylating reagents was constructed to increase the number of detectable compounds in the analysis by GC-MS; 156 drugs, including 30 drugs of abuse, 42 hypnotics and their metabolites, 18 antipsychotic drugs, 15 antidepressants, and 12 antipyretic analgesic agents, were registered with parameters, such as the mass spectrum, retention time, qualifier ion/target ion percentage, and calibration curve using the novel GC-MS software NAGINATA. Diazepam-d5 was used as internal standard for construction of each calibration curve in the range of 0.01–5.0 μg/ml for most drugs. We examined the applicability of the constructed database to analyzing whole blood samples spiked with 40 drugs most commonly encountered in toxicological cases in Japan. The drugs in blood were extracted using enhanced polymer columns (Focus), subjected to GC-MS after incubation with acetylating reagents, and screened by the drug database. Among the 40 drugs examined, 38 and 30 drugs were successfully identifi ed at the level of 1 and 0.1 μg/ml, respectively, without using standard compounds. The time required for data analysis was less than 1 min, and semiquantitative data were also obtained simultaneously. Because new drugs and metabolites can easily be added to the databases, we can recommend them as useful tools in clinical and forensic toxicological screening.

Electrolyte analysis of pleural effusion as an indicator of drowning in seawater and freshwater

It is important for forensic pathologists to determine the diagnosis of drowning as well as the site of drowning. In a previous study, we propose that analysis of electrolytes in pleural effusion from rats may be useful for determining whether drowning has occurred in seawater or freshwater. To test this proposal, we measured the concentration of sodium, potassium and chloride ions and total protein in pleural effusion from 40 autopsy cases: 24 involving seawater drowning, 9 freshwater drowning and 7 no drowning. The concentrations of sodium and chloride ions in pleural effusion showed a significant difference between seawater drowning and freshwater drowning. The concentration of potassium ions and total protein showed no difference between each group, although they increased in proportion to the postmortem interval in cases of both seawater and freshwater drowning. These results are almost same as our previous study and, thus, the quantitative analysis of electrolytes in pleural effusion may be useful for determining whether drowning has occurred in seawater or freshwater.

Estimation of postmortem interval based on the spectrophotometric analysis of postmortem lividity

The color of postmortem lividity and control skin in 21 adult cadavers whose postmortem interval was within 72h, was measured by spectrophotometry in order to estimate the postmortem interval, objectively. The L *a *b * system, which has been widely used for the digital expression of skin color, was used and linear regression analysis was performed to determine the relationship between the postmortem interval and 31 color factors including L * (Value), a * and b * (Chroma and Hue) and C * (Chroma). The difference in Chroma between postmortem lividity and control skin (DeltaC * and DeltaC( *)/C(c)( *)) was only weakly correlated with the postmortem interval. We propose a new equation for calculating the postmortem interval using several color factors obtained by measurement of postmortem lividity, together with some autopsy findings that are known to affect the formation of postmortem lividity. The new equation makes it possible to estimate the postmortem interval within +/-4.76h.

Assessment of some problematic factors in facial image identification using a 2D/3D superimposition technique

The number of criminal cases requiring facial image identification of a suspect has been increasing because a surveillance camera is installed everywhere in the city and furthermore, the intercom with the recording function is installed in the home. In this study, we aimed to analyze the usefulness of a 2D/3D facial image superimposition system for image identification when facial aging, facial expression, and twins are under consideration. As a result, the mean values of the average distances calculated from the 16 anatomical landmarks between the 3D facial images of the 50s groups and the 2D facial images of the 20s, 30s, and 40s groups were 2.6, 2.3, and 2.2mm, respectively (facial aging). The mean values of the average distances calculated from 12 anatomical landmarks between the 3D normal facial images and four emotional expressions were 4.9 (laughter), 2.9 (anger), 2.9 (sadness), and 3.6mm (surprised), respectively (facial expressions). The average distance obtained from 11 anatomical landmarks between the same person in twins was 1.1mm, while the average distance between different person in twins was 2.0mm (twins). Facial image identification using the 2D/3D facial image superimposition system demonstrated adequate statistical power and identified an individual with high accuracy, suggesting its usefulness. However, computer technology concerning video image processing and superimpose progress, there is a need to keep familiar with the morphology and anatomy as its base.

Rapid and reliable screening method for detection of 70 pesticides in whole blood by gas chromatography–mass spectrometry using a constructed calibration-locking database

Pesticide poisoning is one of the most common causes of death by poisoning in Japan, and various kinds of pesticides including organophosphates, carbamates and pyrethroids are listed as causative substances. The purpose of our study was to develop a rapid and reliable screening method for various kinds of pesticides in whole blood by using a unique calibration-locking database and gas chromatography-mass spectrometry. A database of 70 pesticides was constructed using NAGINATA™ software with parameters such as mass spectrum, retention time and qualifier ion/target ion ratio (QT ratio) and calibration curve. Diazepam-d(5) was used as the internal standard for construction of each calibration curve within the range of 0.01-5.0 μg/ml. We examined the applicability of the constructed database by analyzing whole blood samples spiked with 70 pesticides. The pesticides in blood were extracted with hexane under acidic conditions or with an enhanced polymer column (Focus™), subjected to GC-MS, and screened by the pesticides database. Among the 70 pesticides examined, 66 and 62 were successfully identified at the level of 1 and 0.1 μg/ml, respectively, by hexane and 63 and 51 were identified by the Focus column without the use of standard compounds. The time required for data analysis was significantly reduced. Since the established method can produce qualitative and semi-quantitative data without the need for standard substances, this new screening method using NAGINATA™ should be useful for confirming the presence of pesticides in blood in future clinical and forensic cases.

Expression of material mRNA in the hypothalamus and frontal cortex in a rat model of fatal hypothermia

Diagnosis of fatal hypothermia is considered to be difficult in forensic practice because of the lack of any specific pathological findings. The mechanism that induces abnormal behavior such as undressing or hiding during the state of hypothermia has not been clarified. In order to solve these problems, we made a rat model of fatal hypothermia and investigated the expression of some mRNA within the hypothalamus and the frontal cortex. The expression of aldehyde dehydrogenase 6 family, member A1 (ALDH6A1), cocaine- and amphetamine-regulated transcript peptide (CARTPT), desmin (DES), heat shock 70kDa protein 4 (HSPA4), serotonin receptor 2A (HTR2A), opioid receptor, delta 1 (OPRD1) and transthyretin (TTR) supposedly related to fatal hypothermia was determined using quantitative real-time PCR. The expression of OPRD1 in the hypothalamus of fatal hypothermia was significantly increased, while the expression of TTR within the frontal cortex was significantly decreased compared to that in the control. These findings suggest that OPRD1 and TTR may be involved in thermoregulation at a low ambient temperature.

False negative result for amphetamines on the Triage® Drug of Abuse panel?

On-site drug screening devices are widely used today for their simple test procedures and instantaneous results. Among other devices, a Triage® Drug of Abuse panel is considered to be highly reliable for its high specificity and sensitivity of abused drugs. Although it is known that a false positive amphetamine (AMP) result may be obtained from the urine samples containing putrefactive amines or ephedrine-related compounds, no clinical false negative methamphetamine results have been reported to date. However, a false negative Triage® result was obtained from the urine of a fatal methamphetamine poisoning victim taking Vegetamine® tablets. Further experimental analyses revealed that the cross-reactivity of methamphetamine and chlorpromazine metabolites, including nor-2-chlorpromazine sulfoxide, was the cause for a false negative Triage® reaction for AMP. Forensic scientists and clinicians must be aware of the limitations of on-site drug testing devices and the need for the confirmatory laboratory tests for the precise identification and quantification of drugs in suspicious intoxication cases, as also recommended by the manufacturers.

Reliable determination of cyanide, thiocyanate and azide in human whole blood by GC–MS, and its application in NAGINATA–GC–MS screening

PurposeCyanide, its metabolite thiocyanate and azide in human biological fluids are commonly analyzed by gas chromatography–mass spectrometry (GC–MS) after derivatization with pentafluorobenzyl bromide using extractive alkylation. However, the reported methods have some drawbacks. We examined each step of these reported methods and attempted to establish a more reliable method to determine the levels of the above compounds in human whole blood. We also examined the applicability of the established method to NAGINATA–GC–MS screening.MethodsThe deproteinization method, internal standard (IS), the cause of column damage, and the effect of the addition of ascorbic acid were examined, and the best procedure was selected. The obtained data, including mass specta, retention times and calibration curves were registered to the database of NAGINATA software.ResultsThe analysis of cyanide in whole blood was possible only when the blood was deproteinized with trichloroacetic acid. A high recovery of thiocyanate and azide was obtained without the deproteinization step. K13C15N (for cyanide) and tribromobenzene (for thiocyanate and azide) were selected as ISs. The column damage caused by the phase transfer catalyst was successfully eliminated by passing the catalyst containing solution through an ethyl benzoic sulfonic silica gel column. By these improvements, a more reliable determination method was established. All anions were rapidly identified using NAGINATA software, and the approximate concentration of each compound in whole blood was obtained at the same time.ConclusionsBecause NAGINATA–GC–MS screening can rapidly identify these poisons without using toxic compounds as reference standards, it should be useful in forensic and emergency medicine laboratories.

Analysis of postmortem changes in internal organs and gases using computed tomography data

Postmortem computed tomography (PMCT) is a useful method to identify various causes of death and measure the volume of internal organs and gases. The purpose of this study was to investigate postmortem changes as measured by PMCT, and the relationship between the volume of organs and gases and postmortem interval (PMI). Forty-six cadavers (22 men, 24 women) were examined by CT before autopsy. The volumes of the lungs, intrahepatic gas, and intrarectal gas were measured by CT using a workstation. A stepwise regression analysis was used to establish a predictive equation to ascertain the measured volume using factors including sex, age, height, body mass index (BMI), body surface area (BSA), and PMI. For estimation of PMI, stepwise regression analysis was used. In the equations for each measured volume, height, diaphragmatic height, and BSA were adopted for the left lung; height and diaphragmatic height were adopted for the right lung; PMI was adopted for intrahepatic gas; and sex and PMI were adopted for intrarectal gas. In the PMI equations, left lung volume, intrahepatic gas, and intrarectal gas were adopted together with sex, weight, and BMI. Values of intrahepatic gas decreased with postmortem changes, while intrarectal gas increased. This may be useful in investigation of postmortem changes. It will be necessary to include other parts of the intestine and to analyze volume changes in gases from these parts after death.