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A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS-MS) procedure was developed for the determination of iodide (I(-)). A gold (Au) and I(-) complex was formed immediately after the addition of the chelating agent NaAuCl(4) to I(-) solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI-MS-MS instrument. I(-) quantification was performed by selecting reaction monitoring of the product ion I(-) at m/z 127 derived from the precursor ion (197)AuI(2)(-) at m/z 451. I(-) concentration was measured in the quantification range from 10(-7) to 10(-5) M using 50 microL of solution within 10 min. Iodate was reduced to I(-) with ascorbic acid and determined. I(-) concentration in reference urine 2670a was measured after treatments.

A very simple and sensitive method for the simultaneous analysis of naphthalene and p-dichlorobenzene in human whole blood and urine by headspace capillary gas chromatography-mass spectrometry (GC-MS) is presented. The advantages of the method were that as much as 1 mL of headspace vapor could be injected into a GC port in the splitless mode, and that the addition of deuterated naphthalene and p-dichlorobenzene as internal standards resulted in much better headspace extraction efficiencies, which resulted in high sensitivity. The detection limits for both naphthalene and p-dichlorobenzene were 1 ng mL(-1) for whole blood and 0.5 ng mL(-1) for urine. Validation data, such as the linearity of calibration curves, reproducibility and recovery rates, were all satisfactory. Using this method, both compounds could actually be detected from whole blood samples of a male volunteer after the inhalation of each gas of the compounds.

A very simple method for the analysis of methamphetamine and amphetamine in human whole blood and urine by headspace gas chromatography (GC) has been presented. It neither needs solid-phase microextraction nor cryogenic trapping devices, but only a conventional capillary GC instrument with flame ionization detection (FID). The two special points to be mentioned in this method are the in-matrix derivatization of amphetamines for vaporization and the capability of injection of as large as 5 mL of the headspace vapor into a GC instrument in the splitless mode for sensitive detection. After heating a whole blood or urine sample containing amphetamines, α-methylbenzylamine (internal standard, IS) and heptafluoro-n-butyryl chloride under alkaline conditions in a 7.0-mL vial at 90 °C for 20 min, 5 mL of the headspace vapor was drawn with a glass syringe and injected into the gas chromatograph. During injection the column was at 40 °C to trap the analytes, and then the oven temperature was programmed up to 320 °C. Sharp peaks were obtained for each analyte and IS, and only a relatively small number of background impurity peaks for the whole blood and urine samples. The detection limits for each amphetamine were estimated to be 0.1 μg mL−1 for whole blood and 0.03 μg mL−1 for urine. Precision and linearity were also tested to confirm the reliability. Methamphetamine and amphetamine could be determined from whole blood and urine obtained at autopsy in three methamphetamine poisoning cases. The identity of each peak appearing in the gas chromatograms was confirmed by GC/mass spectrometry.

柳田國男は、周知のように日本民俗学の創設者であり、日本近代の学問形成に大きな影響力を及ぼしたひとである。小田内通敏は、日本の近代人文地理学の成立に大きく貢献したひとりである。小田内は、人文地理学の確立をもとめて柳田農政学の門をたたいた。かれが師と仰ぐ新渡戸稲造と柳田によって創設された「郷土会」は、「土地と人の結びつき」から郷土、地方の形態・基盤の研究を行っていた。郷土会の活動を通して柳田は民俗学の、そして小田内は人文地理学の確立を目指したのである。柳田はやがて郷土会のあり方に不満を持ち、「郷土研究」をより総合的な学問に位置づけ、その「総論」として民間伝承論・民俗学の確立を目指した。それは郷土（村）の生活を生活の外形、生活の解説、生活の意識などの三つの資料から総合的に捉えようとするものであった。柳田は、各郷土、地方の調査研究から得られえたこれらの結果を比較総合することによって日本の民族に固有の思想と信仰と感情を明らかにすることを最終目標としたのである。郷土研究はそのための「手段」と位置づけられた。したがって柳田においては、郷土の個性を追求することではなく、それらを貫く、一般性、共通性を追求する科学研究が目指されていた。一方小田内は、郷土、地域の特性、個性を追求することをかれの学問―人文地理学の目標においていた。彼らの方法に基づいて行われた研究が、柳田民俗学を結集した日本の山村・海村生活の調査・研究であり、また小田内指導下の文部省郷土教育の綜合郷土研究であった。前者では村をひとつの有機体として捉えていない点に、後者では郷土の綜合、個性を捉えていない点に問題があった。いずれにしても二人の「郷土研究」の方法の違いはかれらの学問観・科学観の違いによるものであった。

Inorganic arsenite (As3+) and arsenate (As5+) are well-known poisons, and the toxicity of As3+ is about ten times that of As5+. In this study, a simple, rapid, and sensitive method was developed for As3+ in plasma using electrospray ionization (ESI) tandem mass spectrometry (MS-MS). After washing plasma with trichloroethylene (TCE), As3+ in the aqueous layer was reacted with pyrrolidinedithiocarbamate (PDC, C4H8NCSS-), and the produced As(PDC)3 was extracted with methyl isobutyl ketone (MIBK); a 1-µl aliquot of the MIBK layer containing As(PDC)3 was introduced into the MS-MS instrument in the direct-flow injection mode. Other arsenic compounds such as As5+, monomethyl arsonic acid, dimethyl arsinic acid, arsenobetaine, arsenocholine, and tetramethyl arsonium did not produce As(PDC)3. Therefore, without liquid chromatographic separation, As3+ alone could be detected after washing with TCE followed by solvent extraction of As(PDC)3 with MIBK. Thus, inorganic As5+ was reduced to As3+ with thiosulfate, and then the total inorganic As was quantifi ed as As3+; As5+could be calculated by subtracting As3+from the total inorganic As. The MS-MS quantification was performed by selected reaction monitoring using a peak at m/z 114 of a product ion (C4H8NCS)+ formed by collision-induced dissociation from the precursor ion As(PDC)2 + at m/z 367. The mass spectral identification on MS-MS spectrum was possible even at 1 ng As3+/ml plasma. The calibration curve for As3+ showed linearity from 0.5 to 100 ng/ml plasma. The limits of detection by selected reaction monitoring were 0.3 ng/ml in water and 0.2 ng/ml in plasma. The analysis could be completed in less than 15 min, because chromatographic separation was not necessary before the MS-MS detection.

A simple, rapid and sensitive method using tandem mass spectrometry (MS-MS) has been developed for the determination of chromate Cr6+ in urine. Cr6+ is a substantial component of Urine Luck, which is used to conceal the presence of drugs in urine. Cr6+ was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. Then a 1-μl aliquot of isoamyl alcohol containing Cr-DDC complex was directly injected into an MS-MS instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 363.8 of product ion CrO(DDC)2 + obtained by collision-induced dissociation from the precursor ion, CrOH(DDC)3 + at m/z 513.1. This method was validated with the analysis urine samples obtained from volunteers. A linear calibration curve could be obtained in the range of 0.18–100 ng/ml. The limits of detection and quantification of Cr6+ were 0.05 and 0.18 ng/ml, respectively, using only 10 μl of urine. Results could be obtained in less than 10 min for a sample. After oxidation of Cr3+ to Cr6+, near 100% recovery was confirmed using standard reference materials such as SRM 2670a (low level and high level) and SRM 1643e. The most outstanding advantage of this ESI-MS-MS method is that it gives excellent product ion mass spectra for identification of Cr6+.

A simple, rapid, and sensitive method has been developed for determination of platinum (Pt) in blood and urine by tandem mass spectrometry (MS-MS). Pt4+ in wet-ashed blood or acid-treated urine was complexed with diethyldithiocarbamate (DDC), extracted with isoamyl alcohol, and acidified with oxalic acid; a 1-μl aliquot of the isoamyl alcohol layer containing the Pt-DDC complex was directly injected into the MS-MS instrument without chromatographic separation. The quantitation was performed using selected reaction monitoring at m/z 491 of the product ion Pt(DDC)2+, which was produced by collision-induced dissociation from the precursor ion Pt(DDC)3+ at m/z 639. This method was validated for the analysis of blood and urine samples; the limits of quantitation were about 0.3 and 0.1 ng/ml for blood and urine, respectively, using only 10 μl of sample. The calibration curves for Pt in urine and blood showed linearity from 0.1 to 30 ng/ml. Because chromatographic separation is not required before MS-MS detection, the analysis can be completed in less than 10 min.

We experienced an autopsy case in which the cause of death was judged as poisoning by multiple new psychoactive substances, including AB-CHMINACA, 5-fluoro-AMB and diphenidine [Forensic Toxicol. 33 (2015): 45-53]. Although unchanged AB-CHMINACA could be detected from 8 solid tissues, it could neither be detected from blood nor urine specimens. In this article, we obtained eight kinds of reference standards of AB-CHMINACA metabolites from a commercial source. The AB-CHMINACA metabolites from the urine specimen of the abuser were extracted by a modified QuEChERS method and analyzed by liquid chromatography-tandem mass spectrometry before and after hydrolysis with β-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, the concentrations of the two metabolites were not increased, suggesting that the metabolites were not in the conjugated forms. The metabolites detected were 4-hydroxycyclohexylmethyl AB-CHMINACA (M1), followed by N-[[1-(cyclohexylmethyl)-1H-indazol-3-yl]carbonyl]-l-valine (M3). Their concentrations were 52.8 ± 3.44 and 41.3 ± 5.04 ng/ml (n=10) for M1 and M3, respectively. Although there is one preceding report showing the estimations of metabolism of AB-CHMINACA without reference standards, this is the first report dealing with exact identification using reference standards, and quantification of M1 and M3 in an authentic urine specimen.

Platinum (Pt) levels were determined in various tissues and body fluids obtained from a patient who died 181 days after cisplatin overdosing. The symptoms of cisplatin overdose, however, might have almost disappeared by day 40, and the patient’s death was ascribed to the recurrence of malignant lymphoma. Determination of Pt derived from cisplatin was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as internal standard. Pt and Ag complexed with diethyldithiocarbamate (DDC) in wetashed blood, and tissue solutions were extracted into isoamyl alcohol, and then acidified with oxalic acid. By injecting an aliquot of the isoamyl alcohol layer into a mass spectrometer in the direct flow injection mode, the quantitation was performed using the signals of Pt(DDC)3 + and Ag(DDC)2 + at m/z 639 and 403, respectively. The Pt levels ranged from 25ng/ml in blood to 2050ng/g wet weight in the liver of the patient, indicating that Pt remained at high levels in tissues, even after a period as long as 181 days after cisplatin overdosing.

Ishikawa Sanshiro was a representative Japanese anarchist who ranked with Kotoku Shusui and Osugi Sakae. He defected to Europe in 1913 and remained there for 8 years, develping a close relation ship with Paul Reclus, in whose vegetable garden he worked in Dordogne, France. Paul Reclus was the nephew of Elisee Reclus, the greatest geographer and anarchist theorist in 19th- century France. His exile in Europe gave him the opportunity to form a friendship with Edward Carpenter, “discover” Elisee Reclus, and live an agricultural life. After coming back to Japan in 1920, Ishikawa took up residence in Chitose in the suburbs of Tokyo and studied Elisee Reclus, cultivating his garden as at Domme in Dordogne. He published many articles concerning Elisee Reclus in his personal periodical Dynamique and presided over a small circle for the “Study of Elisee Reclus” to which he delivered lectures once a month. In those days, he translated and published the first volume of the monumental work of Reclus' later years, L'Homme et la Terre, Man and the Earth. In 1933, Ishikawa published two important books, Kinsei Domin Tetsugaku (Philosophy of the Anarchist Democracy) and Rekishi Tetsugaku Joron (Introduction to the Philosophy of History), which were his original anarchist theories. They are based on Elisee Reclus' ideas, and especially in the latter, he referred frequently to Reclus' L'Homme et la Terre. He criticized the theories of evolution and dialectical historical materialism, which were the academic mainstream theories in those days in Japan Although. Ishikawa held that this was also Reclus'opinion, the author does not agree. Reclus did not take a critical stance toward the theories of evolution without acknowledging the struggle for existence and of dialectical historical materialism. Ishikawa's anti-evolutionism and anti-Marxism come from his nihinlism and Edward Carpenter's anticivilisation ideology. Although Ishikawa has the same ideas as Reclus on the view of the world and on the universe, he had a different view of humans in the universe. While Ishikawa Sanshiro had a pessimistic view of life, Reclus believed in progress and hoped for solidarity amony human beings.

A simple and modern method for simultaneous analysis of nitrite and nitrate in whole blood has been devised by ion chromatography with an autosuppressor and a conductivity detector. To our knowledge, this is the first report for such analysis dealing with whole blood as a specimen. A 1 mL volume of whole blood was mixed with 2 mL of Milli Q water, vortex‐mixed for hemolysis, and ultrafiltered to obtain a clear extract solution. It was passed through double connected mini-cartridges of silver resin and a silver absorbing material to remove a high concentration of chloride ion usually present in whole blood. An aliquot of the filtrate was analyzed on a Dionex DX‐500 instrument with a microbore IonPac® AS9‐HC column (2×250 mm). The present method gave very low backgrounds for blank whole blood at locations where nitrite should appear, and around the endogenous peak of nitrate. The spiked nitrite markedly decreased in whole blood, probably by the action of hemoglobin in vitro; it was only 10–20% 2 h ...

A very simple method for the analysis of methamphetamine and amphetamine in human whole blood and urine by headspace gas chromatography (GC) has been presented. It neither needs solid-phase microextraction nor cryogenic trapping devices, but only a conventional capillary GC instrument with flame ionization detection (FID). The two special points to be mentioned in this method are the in-matrix derivatization of amphetamines for vaporization and the capability of injection of as large as 5 mL of the headspace vapor into a GC instrument in the splitless mode for sensitive detection. After heating a whole blood or urine sample containing amphetamines, α-methylbenzylamine (internal standard, IS) and heptafluoro-n-butyryl chloride under alkaline conditions in a 7.0-mL vial at 90 °C for 20 min, 5 mL of the headspace vapor was drawn with a glass syringe and injected into the gas chromatograph. During injection the column was at 40 °C to trap the analytes, and then the oven temperature was programmed up to 320 °C. Sharp peaks were obtained for each analyte and IS, and only a relatively small number of background impurity peaks for the whole blood and urine samples. The detection limits for each amphetamine were estimated to be 0.1 μg mL−1 for whole blood and 0.03 μg mL−1 for urine. Precision and linearity were also tested to confirm the reliability. Methamphetamine and amphetamine could be determined from whole blood and urine obtained at autopsy in three methamphetamine poisoning cases. The identity of each peak appearing in the gas chromatograms was confirmed by GC/mass spectrometry.

A 50-year-old male was hit by a high-speed golf ball on the left lateral side of his neck. He collapsed immediately and was sent to hospital, where he was pronounced dead. The autopsy showed an extensive basal subarachnoid haemorrhage. Careful gross and histological examinations disclosed a rupture of the right vertebral artery at a site very close to the bifurcation. It was estimated that the impact of the golf ball on the left side of his neck resulted in the rupture of the contralateral vertebral artery, owing to its hyperextension. Although there are many reports on traumatic basal subarachnoid haemorrhage, the present type of trauma seems rare to our knowledge.

A rapid and sensitive method was developed for the determination of benzodiazepines and benzodiazepine-like substances (BZDs) by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of BZDs. Determination of 8 BZDs (with two of their metabolites) belonging to top 12 medical drugs detected in poisonous cases in Japan, was performed using diazepam-d5 as the internal standard. The limit of detection of zolpidem was 0.07ng/ml with its quantification range of 0.2-20ng/ml in blood, in the best case, and the limit of detection of flunitrazepam was 2ng/ml with its quantification range of 6-200ng/ml in blood, in the worst case. The spectra of zopiclone in MALDI-MS and MS/MS were different from those in electrospray ionization MS and MS/MS. Present method provides a simple and high throughput method for the screening of these BZDs using only 20μl of blood. The developed method was successfully used for the determination of BZDs in biological fluids obtained from two victims.

We experienced an autopsy case of a 21-year-old male Caucasian, in which the direct cause of his death was judged as subarachnoid hemorrhage. There was cerebral arteriovenous malformation, which seemed related to the subarachnoid hemorrhage. The postmortem interval was estimated to be about 2days. By our drug screening test using gas chromatography-mass spectrometry, we could identify α-pyrrolidinobutiophenone (α-PBP) in his urine specimen, which led us to investigate the postmortem distribution of α-PBP in this deceased. The specimens dealt with were right heart blood, left heart blood, femoral vein blood, cerebrospinal fluid, urine, stomach contents and five solid tissues. The extraction of α-PBP and α-pyrrolidinovalerophenone (α-PVP, internal standard) was performed by a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) method, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices, we used the standard addition method to overcome the matrix effects. The highest concentration was found in urine, followed by stomach contents, the kidney, lung, spleen, pancreas and liver. The blood concentrations were about halves of those of the solid tissues. The high concentrations of α-PBP in urine and the kidney suggest that the drug tends to be rapidly excreted into urine via the kidney after its absorption into the blood stream. The urine specimen is of the best choice for analysis. This is the first report describing the postmortem distribution of α-PBP in a human to our knowledge.

The typing of nuclear DNA from hair shafts has often been unsuccessful to date. We tried to type one of the nuclear DNA loci, HLA-DQA1, from hair shafts, using an efficient cetyl-trimethyl ammonium bromide (CTAB) precipitation for DNA purification and a sensitive semi-nested PCR. After thorough washing with ethanol and water, hair shafts were digested by proteinase K in the presence of dithiothreitol, followed by a purification step including CTAB-DNA precipitation. The specific region of HLA-DQA1 gene was amplified by the semi-nested PCR, and the amplified products were cloned and sequenced. The HLA-DQA1 genotype was determined by comparing the sequence to the known sequence of each allele. All genotypes of HLA-DQA1 were successfully typed with hair shafts from six known heterozygotes, although one of them showed the predominant appearance of one allele. For correct typing, a template DNA equivalent to a hair shaft of 5 or 10 cm in length was necessary. Without the CTAB-DNA precipitation step, DNA extract from such hair shafts inevitably contains enough melanin to inhibit PCR. The present results suggest that hair shafts can be used for the typing of nuclear DNA loci.

Urine Luck, the main component of which is pyridinium chlorochromate (PCC), is a popular adulterant used to conceal drugs present in urine samples. Electrospray ionization mass spectrometry with direct flow injection was employed to identify and quantitate PCC after complex formation of chromium (Cr6+) in PCC with diethyldithiocarbamate and its extraction with isoamyl alcohol. The quantitation was performed by selected ion monitoring at m/z 513, and the detection limit was 20 pg PCC (equivalent to 5pg Cr6+) in 1 µl of an injected volume. The integrated area of the mass chromatogram was proportional to the amount up to 2000 pg PCC with the limit of quantitation of 60 pg PCC in an injected volume. The recoveries of PCC from 50µl of urine spiked at 10−7 and 10−6M were 93% and 95%, respectively. The present method for analysis of Urine Luck is recommended for use in forensic analysis because of its speed, high sensitivity, and high specificity.

A 59-year-old male was driving a car on the road and was involved in a traffic accident, colliding with a tanker and a big lorry. When an ambulance arrived at the scene, he was in a state of cardiopulmonary arrest and was bleeding profusely from his right nostril. He was confirmed dead at hospital. The autopsy showed a laceration of the right upper lip extending to the right nostril. In the basal skull there was a notable perforation at the ethmoid bone together with the central part of the sphenoid bone including the sella turcica. In accordance with the basal skull bone fractures, there were pronounced contusion injuries at the brain stem and a contusion injury was also observed in the right part of the cerebellum. After careful investigation of a causative stick-like item that was present inside the car, it was concluded that a severe movement of the man's body, as a result of the traffic collision, caused the gear stick which was fixed to the steering wheel to become impaled in the man's right nostril. The gear stick passed through the nasal cavity and into the basal skull bones, resulting in fatal brain stem injuries.

A rapid determination method is presented for gold (Au3+) and platinum (Pt4+) in tissues using matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF-MS). Au and Pt ions in wet-ashed tissue solution were reacted with diethyldithiocarbamate (DDC), and the resulting chelate complex ions Au(DDC)2 + and Pt(DDC)3 + were detected by MALDI-Q-TOF-MS using α-cyano-4-hydroxycinnamic acid as a matrix. The limit of detection (LOD) was 0.8 ng/g tissue and the quantification range was 2–400 ng/g for Au, and the LOD was 6 ng/g tissue and the quantification range was 20–4,000 ng/g for Pt. The Pt levels detected by MALDI-Q-TOF-MS in several tissues of a patient overdosed with cisplatin were nearly the same as those detected by flow-injection electrospray ionization mass spectrometry. The LODs of Au and Pt were 0.04 pg per well (sample spot) and 0.3 pg per well, respectively. To our knowledge, this is the first attempt to quantify Au3+ and Pt4+ ions in tissues by MALDI-Q-TOF-MS.

We report a rare case of the death of a stunt man who jumped into the sea driving a motorcycle on location for a TV drama. A special ramp had been constructed for the jump. The stunt man and a passenger drove up the ramp at a speed of 60–70 km/h and jumped into the sea from a height of about five metres. Both men swam up, but the driver fell unconscious at the surface. He was sent to hospital, but did not recover. The autopsy revealed a deep laceration of the right axilla with complete rupture of the axillary artery, bone fractures of the right ribs and the right pelvis, and a small laceration of the upper lobe of the right lung. Other visceral organs were not injured. We finally judged that his cause of death was exsanguination due to rupture of the right axillary artery on the basis of the presence of a small amount of blood inside the heart and ischemic organs. All injuries were probably inflicted by a collision with the motorcycle upon reaching the water surface; the axillary injury might be due to the left handlebar piercing into the part.

A rapid and sensitive electrospray ionization tandem mass spectrometric (ESI-MS-MS) procedure was developed for the determination of cyanide (CN(-)). CN(-) in biological fluids was reacted with NaAuCl(4) to produce dicyanogold, Au(CN)(2)(-), which was extracted with methyl isobutyl ketone (MIBK). One microliter of the extract was injected directly into an ESI-MS-MS instrument. Quantification of CN(-) was performed by selected reaction monitoring of the product ion CN(-) at m/z 26 that derived from precursor ion Au(CN)(2)(-) at m/z 249. CN(-) could be measured in the quantification range of 10(-7) to 5x10(-5) M with the limit of detection at 4x10(-8) M using 10 microL of urine within 10 min. A victim's urine and gastric content were diluted with water to 4-fold and 500-fold and measured, respectively.

Determination of platinum (Pt) derived from cisplatin in tissues was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as the internal standard. Pt and Ag reacted with diethyldithiocarbamate (DDC), and were extracted using isoamylalcohol and acidified with oxalic acid. The compounds were termed Pt(DDC)(3)(+) and Ag(DDC)(2)(+), based on their m/z values exhibiting the highest peaks at m/z 639 and m/z 405, respectively. The limit of detection was 30 pg and the quantitation range was from 100 to 10,000 pg using 5 mg tissue. The present method allowed the determination of Pt in wet-ashed tissue in 10 min.

A 59-year-old male was driving a car on the road and was involved in a traffic accident, colliding with a tanker and a big lorry. When an ambulance arrived at the scene, he was in a state of cardiopulmonary arrest and was bleeding profusely from his right nostril. He was confirmed dead at hospital. The autopsy showed a laceration of the right upper lip extending to the right nostril. In the basal skull there was a notable perforation at the ethmoid bone together with the central part of the sphenoid bone including the sella turcica. In accordance with the basal skull bone fractures, there were pronounced contusion injuries at the brain stem and a contusion injury was also observed in the right part of the cerebellum. After careful investigation of a causative stick-like item that was present inside the car, it was concluded that a severe movement of the man's body, as a result of the traffic collision, caused the gear stick which was fixed to the steering wheel to become impaled in the man's right nostril. The gear stick passed through the nasal cavity and into the basal skull bones, resulting in fatal brain stem injuries.

We encountered a patient with hepatocellular carcinoma (HCC), with adrenal gland metastasis, in whom splenic metastasis was diagnosed histopathologically. A 59-year-old man visited our hospital in May 2001 with chief complaints of abdominal distension and pretibial pitting edema. Multiple HCCs associated with HCV-positive liver cirrhosis were detected. Transarterial embolization (TAE) was performed a total of 4 times for HCCs. A left adrenal gland metastatic lesion was detected and it was found to increase in diameter from 3 cm to 6 cm over a four-month period; left adrenalectomy was performed in June 2002. Because of marked splenomegaly and findings of hypersplenism, the spleen was also resected. Although no metastatic lesions were evident on macroscopic examination of the spleen, a small metastatic lesion from moderately differentiated HCC, approximately 0.5 mm in diameter, was detected histopathologically. Splenic metastasis from HCC is rare, usually occurring with metastases involving other organs. Our patient also had adrenal gland metastasis. Therefore, hematogenous metastasis to the congested spleen via the systemic circulation was suspected.

This study compares the effect of the restriction of Mg with that of all-minerals in the diet on the toxicity of paraquat. To compare the severity of the toxicity, several biological values were examined; kininogen in plasma, thiobarbituric acid reactive substances in liver, Ca level in kidney, and Mg levels in liver and kidney. Osteogenic disorder Shionogi rats that cannot synthesize vitamin C like humans did not display paraquat symptoms after receiving minute amounts of paraquat dichloride, i.e. 125 ppm in the diet for 8 days, and those biological values remained the same as those of the control. Rats fed with Mg at half of the recommended amounts also did not show any changes in those levels. The dosage of 125 ppm paraquat under the restriction of Mg, however, induced paraquat intoxication and increased those levels greatly. This result arises a question whether the intoxication is due to the imbalance of Ca and Mg or due to the shortage of Mg itself, because imbalance of Ca and Mg sometimes induces more serious effects than the shortage of Mg itself. Therefore, we fed rats an all-mineral restricted diet where the balance of Ca and Mg was maintained. The dosage of paraquat under all-mineral restriction, however, induced much more serious intoxication than that under Mg restriction. In conclusion, the shortage of Mg itself seems to be responsible for the induction of paraquat intoxication.

Minisatellite variant repeat (MVR) mapping using polymerase chain reaction (PCR) was applied to a practical case of paternity testing to evaluate the paternity probability. In order to obtain single allele mapping by allele-specific MVR-PCR, three flanking polymorphic sites for each of the MS31A and MS32 loci were investigated and all three individuals were typed as heterozygous for at least one flanking polymorphic site at each locus. Allele-specific MVR-PCR was then performed using genomic DNA. It was confirmed that one allele in the child was identical to that from the mother and the other one in the child was identical to that from the alleged father. Mapped allele codes were also compared with those in the database by dot-matrix analysis, and no identical allele was found although some motifs were shared with Japanese alleles. The paternity index and the probability of paternity exclusion in the case at these two MVR loci were calculated using the presumed values of the allele frequencies. These studies seem to illustrate the practical value of MVR mapping of MS31A and MS32 loci in paternity testing.

We report a rare case of death due to rescue using a power shovel. A 41-year-old female was accidentally buried alive by a landslide of the earth and sand upon working at a construction site. One of her colleagues started to save her using a power shovel. However, she was dug out dead at the spot about 10 min after the accident with marked head and face injuries. The autopsy disclosed that there was extensive laceration across the face and head with marked skull bone fractures. Around these injuries, extensive hemorrhage could be observed as a vital reaction. Asphyxial death had to be taken into consideration, because she was buried under the earth and sand for about 10 min; but we finally judged that the cause of her death was head injury by the power shovel inflicted during the attempted rescue.

Unidentified skeletal remains with almost every part of a single body were found in a closed shed; it wore a jacket, pants and underwears. It was brought to Hamamatsu University School of Medicine for autopsy. We estimated the victim to be male, 50–65 years old, 164–168cm high and at 5–10 years after death. The cause of his death was not clear. During the careful examination, we noticed that there was an extra vertebra (vertebra X), at the thoraco-lumbar region. The vertebra X was located between T12 and L1, and showed intermediate properties of T12 and L1. The presence of numerical variations in the thoraco-lumbar vertebrae should be more well-known for forensic scientists; the knowledge seems very important for the judgment whether the remains are of a single or multiple individuals.

A 50-year-old male was hit by a high-speed golf ball on the left lateral side of his neck. He collapsed immediately and was sent to hospital, where he was pronounced dead. The autopsy showed an extensive basal subarachnoid haemorrhage. Careful gross and histological examinations disclosed a rupture of the right vertebral artery at a site very close to the bifurcation. It was estimated that the impact of the golf ball on the left side of his neck resulted in the rupture of the contralateral vertebral artery, owing to its hyperextension. Although there are many reports on traumatic basal subarachnoid haemorrhage, the present type of trauma seems rare to our knowledge.

We report a rare case of the death of a stunt man who jumped into the sea driving a motorcycle on location for a TV drama. A special ramp had been constructed for the jump. The stunt man and a passenger drove up the ramp at a speed of 60-70 km/h and jumped into the sea from a height of about five metres. Both men swam up, but the driver fell unconscious at the surface. He was sent to hospital, but did not recover. The autopsy revealed a deep laceration of the right axilla with complete rupture of the axillary artery, bone fractures of the right ribs and the right pelvis, and a small laceration of the upper lobe of the right lung. Other visceral organs were not injured. We finally judged that his cause of death was exsanguination due to rupture of the right axillary artery on the basis of the presence of a small amount of blood inside the heart and ischemic organs. All injuries were probably inflicted by a collision with the motorcycle upon reaching the water surface; the axillary injury might be due to the left handlebar piercing into the part.

We investigated the usefulness of a toothbrush as a source of DNA for an unidentified cadaver. Ten toothbrushes were obtained from ten individuals along with their peripheral blood. We recovered from 10 to 430 ng of DNA from all but one of the toothbrushes. All ten toothbrushes, including the one containing no detectable DNA by fluorometry, were typed correctly at all of the loci tested, including nine STRs. Three toothbrushes obtained in two actual deaths also identified two victims and one suspect. Therefore, toothbrushes seem to be useful as a source of evidential DNA for personal identification.

The tetrameric short tandem repeat (STR) locus (D7S809) has been evaluated in the Japanese population. In order to detect the alleles, PCR was carried out using primers, one of which was end labelled with 32 P, and PCR products were separated by electrophoresis on a denaturing polyacrylamide gel. Using this method, accurate genotypes could be determined from as little as 0.5 ng of genomic DNA. Thirteen different alleles were identified on 256 chromosomes tested. All alleles differed in size by one (4 bp) repeat unit, and no ‘interalleles’ were found. The estimated heterozygosity and the polymorphism information content (PIC) were 0.86 and 0.83, respectively. We observed 42 of the 91 possible different genotypes. The power of discrimination (PD) was 0.96, and no significant deviations from the Hardy-Weinberg equilibrium were found. We retyped all apparently homozygous samples using an alternative pair of flanking primers in order to confirm homozygosity. We also demonstrated a typing result involving sexual assault. D7S809 appears to be a very useful STR locus for forensic practice in Japanese.

Summary To sample the diversity of allelic structures at the D7S21 (MS31A) locus in the Japanese, allele-specific minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) was performed on genomic DNA from a number of Japanese individuals. Three polymorphic positions in the MS31A 5′ flanking DNA were typed from 214 un-related Japanese, and the distribution of haplotypes was analysed. Allele-specific MVR-PCR, using primers that discriminate between different alleles at these polymorphic positions in heterozygous individuals, allows single alleles to be mapped from genomic DNA in approximately 80% of Japanese. 149 Japanese alleles have been mapped to date and all of them, except for two pairs of indistinguishable alleles, have different internal structures. More than half of the mapped alleles showed similar regions of internal structure to other alleles and were classified into groups on this basis.

Nine tetranucleotide short tandem repeat (STR) loci, D3S1358, vWA, FGA TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820, were analyzed in the Japanese population with a newly released kit for personal identification using multiplex PCR with fluorescent-labeled primers following capillary electrophoresis. The observed heterozygosities were 0.67, 0.77, 0.82, 0.61, 0.62, 0.73, 0.78, 0.81 and 0.74, respectively, and the combined discrimination power of the nineplex was 0.9999999991. None of the nine loci deviated from Hardy-Weinberg equilibrium expectations using the chi-square test, homozygosity test, likelihood ratio test and exact test after the grouping of the alleles. The nine STR loci allele frequencies were significantly different from those of other ethnic populations.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to a practical forensic case of an infant and placenta found in an incinerator. They were thought to be left for a few days postmortem, and the infant was severely burnt when found. DNA was extracted from the infantile muscle and maternal placental hematoma. MVR-PCR analysis as well as other common DNA typing (D1S80, HLA-DQA1) were performed on both DNA samples. Both MVR diploid codes were matched although some extra faint bands in the ladder were observed from the maternal placental sample, which probably indicated superimposing of an allele derived only from the mother, and not the infant. In order to detect the original maternal alleles, three flanking polymorphic sites were typed and allele-specific MVR-PCR was performed. Finally, one maternal allele not inherited by the infant and two alleles from the infant were typed. Two alleles suggested the infant and/or mother was Japanese. The two diploid codes including one possibly from the mother were deduced and compared with other codes in the databases for evaluating the discriminating power.