Expression of oxygen-regulated protein 150 (ORP150) in skin wound healing and its application for wound age determination.

Immunohistochemical study combined with morphometry was carried out to examine the expression of oxygen-regulated protein 150 (ORP150) using 58 human skin wounds of different ages (group I, 0-12 h; II, 1-5 days; III, 7-14 days; and IV, 17-21 days). In human wound specimens aged 4-12 h, neutrophils recruited at the wound showed no positive signals for ORP150. With the increase in wound age of > or =7 days, granulation tissue and angiogenesis were observed, with the migration of macrophages and fibroblasts with ORP150-positive reactions. In semi-quantitative analysis, the average of ORP150-positive ratios in group III was highest. In group III, all samples had an ORP150-positive ratio of >40%, and 17 samples showed >50%. In group IV, three out of ten samples showed a positive ratio of 40-45% and the remaining seven cases less than 40%. Collectively, with regard to the practical applicability with forensic safety, these observations suggest that an ORP150-positive ratio of >50% strongly indicates a wound age of 7-14 days.

Determination of formoterol in rat plasma by liquid chromatography-electrospray ionisation mass spectrometry.

A sensitive method for the determination of formoterol in rat plasma is described, using high performance liquid chromatographic separation with tandem mass spectrometry. Samples were purified using liquid-liquid extraction and separated on CAPCELL PAK C18 UG120 (2.0 x 150 mm) with a mobile phase consisting of a mixture of methanol- 50 mM ammonium hydrogen carbonate (1:1 v/v). Detection was performed with a TSQ 7000 mass spectrometer using positive ion electrospray ionisation, monitoring the shift from precursor ions for formoterol at m/z 344.9 to product ions of m/z 121.0. The limit of quantitation of the method was found to be 0.1 ng/ml, when using 0.1 ml plasma. Plasma concentrations of formoterol could be quantified from 0.15 to 7.01 ng/ml, allowing the analysis of samples up to 32 h after a single oral dose of formoterol fumarate (0.25 mg) to rats.

Allylnitrile: generation from cruciferous vegetables and behavioral effects on mice of repeated exposure.

The objective of the present study was to examine the possible generation of allylnitrile from commonly consumed cruciferous vegetables, and to determine the long-term behavioral effects of its oral administration at levels comparable to or greater than human dietary exposures. On the basis of gaschromatographic and mass spectrometric analyses, allylnitrile generation was observed in eight out of twelve vegetables, broccoli, broccoli (young stems and leaves), brussels sprouts, cabbage, cauliflower, chinese cabbage, komatsuna and kaiware-daikon (young stems and leaves). The daily dietary intake of allylnitrile was estimated to be at least 0.12 micromol/kg body weight for Japanese, based on its generation from the vegetables, broccoli, cabbage, cauliflower and Chinese cabbage and their daily dietary consumption. Mice received oral doses of 2, 20, 200, 500 and 1,100 micromol/kg allylnitrile once a day, 5 days per week for 13 weeks. Mice in the lower dosage groups of 2, 20 and 200 micromol/kg exhibited no behavioral changes. Mice dosed at the level of 500 micromol/kg showed restlessness, and one of them displayed alteration in tail hanging. These abnormalities were seen around seven days following the beginning of the treatment period. Animals in the highest dosage group elicited behavioral abnormalities, and their degree increased with increasing dosage. These results suggest that allylnitrile intake levels through daily vegetable consumption is below the level producing behavioral abnormalities.

Ubiquitin expression in skin wounds and its application to forensic wound age determination.

The time-dependent expression of ubiquitin (Ub) was examined in murine skin wounds and 55 human skin wounds with different wound ages (groups I: 0-12 h, II: 1-5 days, III: 7-14 days and IV: 17-21 days). In murine skin wound specimens, neutrophils, macrophages and fibroblasts showed intensive Ub-positive reactions in the nuclei. In the human wound specimens with wound ages between 4 h and 1 day, neutrophils with strong intranuclear positive reactions for Ub were observed. With increasing wound ages, the nuclei of macrophages and fibroblasts were more intensively stained with anti-Ub antibody. Morphometrically, the intranuclear Ub-positive ratios were very low in group I. The skin wound specimens in groups II and IV showed Ub-positive ratios of >10%, and all samples in group III had Ub-positive ratios of >20%. These results suggest that a ratio of >10% for Ub indicates a wound age of at least 1 day. In contrast, Ub-positive ratios of less than 10% indicate a wound age of <1 day. Moreover, there was a significant difference in the Ub-positive ratio between group III and the other three groups. Thus, Ub-positive ratios considerably exceeding 30%, possibly indicate a wound age of 7-14 days. From the viewpoint of a forensic pathology application, the present study showed that Ub is suitable as a marker of wound age determination.

Complete transection of the pancreas due to a single stamping injury: a case report.

Complete transection of the pancreas due to a single stamping injury is reported. A 47-year-old female was stamped on in the epigastric region by her common-law husband when lying on her back. Immediately after the stamping, she complained of severe abdominal pain. In an emergency hospital, the abdominal CT (computed tomography) scan showed a large haematoma around the pancreas head. In the surgical operation that followed, a complete transection of the pancreas between the head and body was unexpectedly discovered. Furthermore, severe traumatic changes were found in the liver, mesentery, splenic artery and vein. The medico-legal autopsy revealed marked haemorrhaging in the abdominal subcutaneous adipose tissue and rectus muscle in the epigastric region. The external blunt force that acted on the abdomen had forcibly compressed the pancreas against the spinal column. Blunt pancreatic injury is relatively uncommon, but pancreatic injuries should always be considered in patients or autopsy cases after external blunt force to the abdomen.

Intratracheal gas analysis for volatile substances by gas chromatography/mass spectrometry--application to forensic autopsies.

Intratracheal gas analysis was carried out by gas chromatography/mass spectrometry (GC/MS) in 20 burned body cases (13 males and 7 females). Volatile aromatic and aliphatic hydrocarbons were detected by GC/MS using a GS-Q column with the intratracheal gas as well as the blood in 19 cases. The characteristic patterns of mass chromatograms for gasoline, kerosene (gas oil), and liquid petroleum gas could be differentiated from each other using the intratracheal gas. The burned body in one case showed no presence of volatile substances in the intratracheal gas, nor intratracheal soot, although high concentrations (1 microg/g and more) of volatile substances were detected on the clothes. The victim also had normal CO-Hb concentrations (0.1 to 0.2%) in the heart blood. The results of intratracheal gas analysis were consistent with signs of the vital reaction. In conclusion, intratracheal gas analysis provides a supportive method for diagnosing the cause of death in burned bodies, and yields for at least 48 hours valuable information on volatile hydrocarbons (being detected in deliberate or accidental fire cases) to which the body had been exposed just before death.

Rapid analysis of pesticide components, xylene, o-dichlorbenzene, cresol and dichlorvos, in blood and urine by pulse heating-gas chromatography-mass spectrometry.

Analysis of the pesticide components, xylene, o-dichlorbenzene (DCB), cresol and dichlorvos (DDVP, an organophosphate), was attempted using 3 microl of biological samples by pulse heating (Py)-gas chromatography-mass spectrometry (GC-MS) without pretreatment. Xylene, DCB, cresol and DDVP were separately detected by Py-GC-MS. The regression lines for these four substances in the blood were shown to be linear between the peak height and their concentrations, 0.1/0.2-20 microg/ml. Recoveries of DDVP, cresol, DCB and xylene at the concentration of 1.0 microg/ml by Py-GC-MS ranged from 78.8 to 89.0% in blood, and from 84.2 to 91.2% in urine. For within-run precision, the coefficient of variation values at the concentration of 1.0 microg/ml ranged from 7.9 to 10.6% in blood, and from 5.8 to 9.4% in urine. The lower detection limits of xylene, DCB, cresol and DDVP in blood were 50, 50, 50 and 150 ng/ml, respectively. In comparison with the measured value in blood that was added to these four substances, Py-GC-MS showed good accuracy similar to GC-MS after the extraction method. DDVP and xylene could be successfully determined by Py-GC-MS in blood and urine in emergency medical and autopsy cases. From these results, Py-GC-MS can be performed in a short analytical time (15 min) and is a useful method to determine pesticide components in forensic medicine and emergency medical practice.

Genetic variation and relationships at five STR loci in five distinct ethnic groups in China.

Five short tandem repeat (STR) systems of TH01, vWA, LPL, F13B and FES/FPS were investigated in five ethnic groups living in China (Tujia, Miao, Bai, Chaoxian and Han). All five loci did not deviate from the Hardy-Weinberg equilibrium (P>0.05). At the five loci of each ethnic group, the observed heterozygosity, the mean exclusion chance (MEC), and the power of discrimination (PD) ranged from 0.42 to 0.86, from 0.20 to 0.66 and from 0.61 to 0.95, respectively. For the five ethnic groups, the combined MEC and combined PD were >0. 9360 and >0.9998, respectively, suggesting that combinations of these five systems are feasible for DNA typing in forensic investigations such as personal identification or paternity testing. Furthermore, the allelic frequencies at the five loci suggested that these five ethnic groups were distinctly different communities. Judging from the phylogenetic tree constructed based on the genetic distance among the five ethnic groups, Han, Chaoxian and Tujia were involved in an identical cluster, and Miao and Bai in another. These findings indicate that each of the five groups examined is not only a distinct community, but also has a relationship with each of the others.

[Detection of methamphetamine in a severely burned cadaver--a case report].

We report an autopsy case of a severely burned cadaver in which methamphetamine (MA) and amphetamine (A) were detected. A severely burned cadaver was found in a burned-out car on the seashore. At the autopsy, the drug screening test in the urine using Triage system gave positive result for MA and/or A. Qualitative analysis by gas chromatography/mass spectrometry (GC/MS) confirmed the presence of MA and A. The concentrations of MA and A (mumol/100 g) were 1.66 in the thermo-coagulated blood, 16.3 in the urine, 4.60 in the cerebrum, 4.11 in the lung, 7.84 in the liver, 0.94 in the skeletal muscle and 0.54 in the bone marrow. From these results, we concluded that the man had been under the moderate MA and A intoxication. The cause of death was diagnosed as the death of fire on the basis of autopsy findings, and the cadaver was identified as the owner of the car by the DNA typing. This case indicated the importance of drug screening tests in autopsy cases. Furthermore, the qualitative and quantitative analysis of MA and A should be successfully performed by GC/MS even in the case of severely burned cadaver.

Determination of triazolam by GC-MS in two autopsy cases: distribution in body fluids and organs.

A detailed procedure for analysis of triazolam by GC-MS was constructed in our laboratory. At the concentration of 100 ng/ml, recoveries of triazolam in plasma and urine were 84.9 and 91.0%, respectively. The coefficients of variation in terms of its recovery were 11.5 (plasma) and 10.2% (urine). The detection limit for quantitation by the method was approximately 5 ng/g. This method was applied to two autopsy cases, giving triazolam distribution in body fluids and organs. In one case (33-year-old woman), concentrations of triazolam in the heart blood, urine, brain, lung, liver, kidney, skeletal muscles and stomach contents were 83.9, 741, 106, 165, 507, 293, 125 and 343 ng/g, respectively. From these toxicological data together with autopsy findings, her cause of death was diagnosed as triazolam poisoning. In the other case (45-year-old man), triazolam concentrations in the urine and stomach contents were 7.81 and 41.1 ng/g, respectively, but it could not be detected in the pleural blood; his cause of death was judged to be a traumatic shock, based on autopsy findings.

Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography-mass spectrometry with solid-phase extraction.

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC-MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC-MS using a DB-1 open tubular column. Their recoveries ranged from 73-95% at the target concentrations of 1.00, 10.0 and 100 microg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3-13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.

Stability of tricyclic antidepressants in formalin solutions.

We investigated the stability of the secondary amines, desipramine (DP) and nortriptyline (NRT), and the tertiary amines, imipramine (IP) and amitriptyline (AT), in formaldehyde (F) and paraformaldehyde (PF) aqueous solutions. NRT showed little instability in 0.37 to 37% F and PF solutions, but AT formation was detected and increased, up to 0.46 to 2.7%, in parallel with rising F and PF concentrations. DP was unstable and levels decreased to 74 to 96% with increasing F concentrations, and fell only to 96% in 10% PF solution. IP formation increased in the same manner as AT to 2.9 to 3.5% of the initial DP. When AT and IP were stored in F and PF solutions, concentrations of AT and IP did not change. DP in F pH 3 to 11 phosphate buffer (PB) solutions showed high recovery in the order: pH 5 > pH 7 > pH 9 > pH 3 and pH 11. DP in PF buffered solutions decreased slightly only at pH 3 (3.5%). By contrast, IP did not change at any pH (pH 3 to 11) of the F or PF solutions. During storage for 21 days at room temperature in 3.7% F and PF solutions, IP and DP degradation was accelerated when compared with the values in pH 3 and 7 PB solutions. However, IP detected in DP F or PF solution was only 0.2% of the initial DP 21 days after storage. Thus, AT, NRT, IP and DP degraded gradually in F and PF solutions during storage at room temperature. TCAs may first react nucleophilically with formaldehyde to form hemiaminals. DP in 3.7% formaldehyde aqueous solution formed little of its methylated product, IP, at room temperature.

Effect of sequence of administration on the pharmacokinetic interaction between the anticholinergic drug biperiden and [3H]quinuclidinyl benzylate or [3H]N-methylscopolamine in rats.

In rats the pharmacokinetic interactions between the anticholinergic drug biperiden and [3H]quinuclidinyl benzylate ([3H]QNB) or [3H]N-methylscopolamine ([3H]NMS) is affected by the sequence in which the drugs are administered. Drug concentrations in various tissues were determined after intravenous administration of [3H]QNB or [3H]NMS (325 ng kg(-1)). Biperiden (6.4 mg kg(-1)) was administered either 5 min before, concomitantly with or 20 min after injection of [3H]QNB or [3H]NMS. When biperiden was administered concomitantly with or before [3H]QNB, distribution of [3H]QNB among the regions of the brain and other tissues was reduced; at 4 h the ratio of the distribution of [3H]QNB for experimental animals to that for control animals ranged from 0.15 to 0.9. When biperiden was administered after [3H]QNB, the distribution of [3H]QNB in the brain and other tissues was significantly higher than for the other two treatments (P < 0.01). However, for [3H]NMS the sequence of administration had no effect on the distribution of the drug in the brain and other tissues except for the kidney. In-vitro, in crude synaptosomal membranes, the amount of [3H]QNB at 2 h relative to the control concentration at equilibrium was 87% when biperiden was added before [3H]QNB and 56% when biperiden was added after [3H]QNB. In both instances the concentration of [3H]NMS reached equilibrium within 30 min. These findings suggest that the difference between the rate constant of association and dissociation at the possible site of action gives rise to the effect of the sequence of administration on the pharmacokinetic interaction.

A transferrin D variant (DHR5) identical with DSaga by polyacrylamide gel isoelectric focusing was found in a disputed paternity case.

A transferrin (TF) variant was found in the child in a paternity case by polyacrylamide gel isoelectric focusing (PAGIF). Further analysis of serum TF disclosed that this rare TF variant was inherited from the alleged father. This TF variant was identified as TF DHR5, being identical with a variant TF DSaga, in a direct comparison with several known TF D variants by PAGIF. The isoelectric points (pIs) of holo-type (Fe(II) binding type) of TF DHR5 (= DSaga) and TF C were 5.6 and 5.4, respectively. After sialidase treatment, TF DHR5 (= DSaga) showed a single band migrating at a more basic position than TF C, thus suggesting that the pI difference between the two was due to amino acid substitution.

An autopsy case of suicidal strangulation with four looped rubber bands.

A rare case of suicidal strangulation with four looped rubber bands is reported. A 57-year-old female was found dead in the vestibule of her house, with four looped rubber bands wrapped around her neck. Her face was markedly congestive, and lots of petechial hemorrhages were seen on the face and in the palpebral conjunctive. At the medico-legal autopsy, the ligature mark was 1.7 cm in maximum width at the anterior part of the neck and accompanied by such vital reactions as linear hemorrhage, but there were no other injuries to account for her death. Many petechial hemorrhages were also found in the visceral pleurae and epicardium. There was hemocoagulum in the cardiac blood, and the other organs were as well markedly congestive. Gas-chromatographic analysis revealed ethanol; 0.83 mg/ml in the heart blood and 1.37 mg/ml in the urine. No other drugs or poisons were found with Toxi-Lab and REMEDi-HS systems. In addition, police investigation denied the possibility of homicide and disclosed her living will stating that she had suffered from a chronic heart disease. This case was of suicidal strangulation with looped rubber bands.

False identification of urinary ephedrine as methamphetamine by gas chromatography/mass spectrometry with a DB-17 column.

A 17-year-old motorcyclist was killed in a traffic accident. The urine sample collected at autopsy was screened by the Toxi-Lab system; it showed a possible presence of either methamphetamine (MA) or ephedrine (Eph). Gas chromatography-mass spectrometry (GC-MS) was carried out for a trifluoroacetyl (TFA)-derivatized sample after extraction with an Extrelut column. A peak was detected at the same retention time as that of the authentic Eph by GC-MS with a DB-17 wide-bore column; the mass spectrum of the peak was, however, very similar to that of MA. The presence of Eph and absence of MA were proved by GC-MS using a DB-1 column. The Eph in the urine was further confirmed by high performance liquid chromatography (HPLC)-MS using an ODS column. By the REMEDi HS system, Eph and methylephedrine were determined in the urine, and their concentrations (hydrochloride salt) were 0.752 and 2.13 micrograms/ml, respectively. Ethanol was detected as well in the blood (1.55 mg/ml) and urine (2.01 mg/ml) by a pulse heating method. Nicotine, caffeine and dihydrocodeine were also qualitatively detected in the urine by Toxi-Lab, HPLC-MS and REMEDi HS systems. The present case warned that urinary Eph can be misidentified as MA by GC-MS with an intermediately polar column after TFA derivatization.

Muscarinic receptor-mediated dual regulation of ADP-ribosyl cyclase in NG108-15 neuronal cell membranes.

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma x glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30-52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.

Postmortem alcohol analysis of the synovial fluid and its availability in medico-legal practices.

Alcohol analyses using the synovial fluid of the knee joint (synovial alcohol concentration, SAC) as well as blood (BAC) and urine (UAC) were performed by the pulse heating-gas chromatographic method in 12 medico-legal autopsy cases. In 11 of the 12 cases, the regression analysis showed that BAC and UAC had a fairly linear relationship with SAC, and the average ratios of the BAC/SAC and UAC/SAC were 0.76 +/- 0.12 (range 0.60-0.94) and 1.03 +/- 0.11 (range 0.90-1.21), respectively. The measured BACs showed a difference of 11.8 +/- 9.4% (0.9-27.1%) when compared with the calculated BACs by means of the average BAC/SAC ratio. Between the measured UACs and the calculated UACs, there was an average difference of 7.9 +/- 6.0% (0.3-17.8%). The present study demonstrates that the synovial fluid is one of the available biological specimens for the prediction of BAC or UAC within a range in autopsy cases in which suitable blood and/or urine specimens cannot be obtained.

Use of REMEDi HS in emergency toxicology for a rapid estimate of drug concentrations in urine, serum, and gastric samples.

The REMEDi HS is a broad spectrum drug identification system, designed for emergency toxicology screening and forensic applications. The total analysis time is about 20 min. The current library has 555 drugs and metabolites. The system has a software routine that uses an internal standard (IS) to perform quantitative analysis for target compounds when calibrators are available; further, response factors (RF) are supplied for a rapid estimate of drug concentrations when calibrators are unavailable. In the present study, The concentrations of six drugs (bromisovalum, ephedrine, hydroxyzine, diphenhydramine, ranitidine, and lidocaine) and a metabolite of lidocaine (glycinexylidide) were determined using both methods. The slopes of the regression lines between the rapid estimate method and the IS method were generally within 20% of unity, in agreement with the manufacturer's claim. Semiquantitative estimates based on RF also showed good agreement with results obtained using multipoint calibration. These estimates were sufficient for clinical differentiation of routine and toxic levels. Our study demonstrated that the REMEDi HS is particularly useful for a rapid estimate of drug concentrations in the samples from emergency cases when calibrators are not readily available. Our study also showed that this system can be used for the therapeutic monitoring of ranitidine, bromisovalum, lidocaine, and diphenhydrmine.

[Death due to cold of a 63-year-old male associated with hepatocellular carcinoma--bibliographic consideration on alcohol metabolism of the patients with liver dysfunction].

In the early morning of late December, a 63-year-old man was found dead in front of his apartment house. At medico-legal autopsy, subcutaneous hemorrhages of the occipital region and linear fractures of the occipital bone was observed, but there was no cerebral contusion leading him to death. Many white tumors, ranging from 0.5 to 2.0 cm in diameter, were observed in the liver. On sections of the liver, hepatic parenchyma was found entirely occupied by the tumors, which were histopathologically diagnosed as moderately- or well-differentiated hepatocellular carcinoma. Alcohol concentrations of the mixed intracardiac blood and the urine were determined to be 1.9 and 3.6 mg/ml, respectively. Through the police investigation, it became clarified that the male had been sleeping in front of his apartment house about at 10:30 p.m. on the day before he was found dead. The cause of his death was, therefore, considered due to cold, and the authors gave a bibliographic consideration on blood and urine alcohol concentrations and alcohol metabolism of a patient with liver dysfunction due to hepatocellular carcinoma.