Estimating the time between drinking and death from tissue distribution patterns of ethanol.

To establish a method for estimating the time between the last consumption of alcohol and death, we examined the ethanol levels in body fluids and tissues of rats that had been orally administered 1 g/kg ethanol. We observed the following relationships between ethanol levels in the cardiac blood (blood in the heart itself), vitreous humor, and urine: cardiac blood &#62; vitreous humor &#62; urine at 10 min (early absorption stage); vitreous humor &#62; cardiac blood &#62; urine from 20 to 50 min (late absorption stage); vitreous humor &#62; urine &#62; cardiac blood from 60 to 120 min (distribution stage); and urine &#62; vitreous humor &#62; cardiac blood at 180 min (excretion stage). It was also observed that, in cases of death immediately following drinking, ethanol levels in the stomach contents are very high, and the following ratios of ethanol levels were observed: skeletal muscle to cardiac blood--less than 1; liver to cardiac blood--around 1. buccal mucosa to cardiac blood-greater than 1. These ratios at equilibrium after drinking were around 1, lower than 1 and around 1, respectively. We also measured alcohol levels in the cardiac blood, urine, vitreous humor and stomach contents of nine cadavers who had consumed alcohol prior to death. The relationships between the time since last consumption of alcohol and relative ethanol levels in these specimens were in good accordance with the results of the animal experiments. </p

Role of adenohypophyseal mixed cell-follicles in age estimation.

In this study we used paraffin-embedded human pituitary obtained from 248 autopsy cases and identified mixed cell follicles by the immunohistochemical method. We examined the number and size of the mixed cell follicles, and the ratio of each component cell of these follicles, in the anterior pituitary at various age groups. The number of follicles increased with age, and the size of the follicles also tended to enlarge with age. Statistical analysis showed that a high correlation existed between age and the number or the size of the mixed cell-follicles formed by various adenohypophyseal cells. In addition, when the proportions of the different cell types that formed the follicles were examined, sex differences were observed with aging for the GH cells, the PRL cells, and the gonadotroph (GTH) cells, while no changes were observed with aging in both men and women for the ACTH cells and TSH cells. These results indicate that the number, size, and ratio of each component cell of follicles in the anterior pituitary are adequately applicable for the purpose of age estimation in routine forensic medicine.</p

Increase of S-100 protein-positive stellate cells in the anterior pituitary of chronic alcoholic patients with fatty liver or fatty cirrhosis.

Healthy subjects 40 years old were used as controls in a study of stellate cells (S-100 protein-containing cells, or S-100 cells) in subjects with chronic alcoholism and fatty liver or fatty cirrhosis. S-100 cells were sparsely found in the adenohypophysis of control subjects, and these cells sometimes formed small clusters. However, in chronic alcoholics with fatty liver or fatty cirrhosis, the number of stellate cells in the anterior pituitary tended to be 17 times higher than it was in the control group. No increase in the number of S-100 positive cells that constitute the large and small follicles in the intermediate pituitary. The physiological function of the S-100 protein has not yet been identified. The fact that an increase in prolactin-secreting and growth hormone-secreting cells, as well as a decrease in gonadotrophs were observed in the hypophysis of alcoholics suggests that the function of stellate cells may be closely related to these phenomena. Our results also imply that the stellate cells found in the anterior and intermediate pituitary differ in function although they both produce S-100 proteins.</p

Effects of antineoplastics, antibiotics and antidiabetics on acetaldehyde metabolism after alcohol ingestion.

The main purpose of this study was to evaluate the inhibitory effects of 5-fluorouracil antineoplastics, cephem antibiotics containing the methyltetrazolylthiol (MTT) group and antidiabetics on aldehyde dehydrogenase (ALDH) activity in vivo and in vitro. In in vivo experiments, rats were given a 100 mg/kg dose of drugs (10 mg/kg for glibenclamide) orally or intraperitoneally. When each drug was administered singly immediately after an oral administration of 1.5 g/kg ethanol, only carmofur, an antineoplastic, produced marked increases in blood acetaldehyde concentrations. This action was also noted when ethanol was ingested 15 h after administration. The remaining drugs did not increase blood acetaldehyde concentrations. When rats were treated with carmofur at 12 h intervals for 3 consecutive days and were given 1.5 g/kg ethanol after the final treatment, blood acetaldehyde concentrations were elevated more significantly than with a single administration of carmofur. Furthermore, daily administration of cephem antibiotics containing the MTT group, latamoxef, cefamandole, cefoperazone and cefbuperazone, significantly increased blood acetaldehyde concentrations. Daily administration of sulfonylurea antidiabetics, chlorpropamide and acetohexamide, slightly increased blood acetaldehyde concentrations. Drugs causing increases in blood acetaldehyde concentrations when administration was combined with ethanol ingestion also inhibited ALDH activity in vitro. The results of the in vitro experiments roughly correlated with those of the in vivo experiments. The inhibitory effects of drugs on ALDH activity were in the following order: carmofur &#62;&#62; cephem antibiotics containing the MTT group &#62; sulfonylurea antidiabetics.</p

IgA2 genotyping by polymerase chain reaction (PCR) using allele-specific amplification primers.

A method of genotyping IgA2 alleles in the human immunoglobulin alpha 2 heavy chain constant region (C alpha 2 gene) was developed by using the polymerase chain reaction (PCR). By this method, the genotype was determined by discriminating base substitution in the 3'-flanking region of alleles, A2m*1 and A2m*2, which manifest A2m serum types, by nested PCR using allele-specific primers. Three types, IgA2*1/IgA2*1, IgA2*2/IgA2*1, and IgA2*2/IgA2*2, were detected from DNA extracted from lymphocytes. Genotyping was possible from 100 pg of DNA by this method. The estimated allele frequency in 318 Japanese subjects was 0.561 for IgA2*1 and 0.439 for IgA2*2. Analysis of 29 cases of paternity tests suggested that the data follow Mendel's law of inheritance. This genotype could also be detected in whole blood, blood stains, saliva stains, and various organs and tissues. These results suggest the usefulness of the present method for paternity testing and individual identification in forensic medicine.</p

A new HLA-DRB1 genotyping method using single nucleotide polymorphism (SNP) analysis with multiplex primer extension reactions and its application to mixed samples.

We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.</p

Haptoglobin genotyping by allele-specific polymerase chain reaction amplification

We performed haptoglobin (Hp) genotyping by polymerase chain reaction using allele-specific primer-pairs. The major six genotypes of Hp were identified using this method. Among Japanese individuals living in Ehime and Okayama Prefectures, the allele frequencies were estimated to be Hp2 = 0.723 and Hp1s = 0.277. Genotyping of Hp was possible with 0.3 ng of DNA and with 0.125 microliter of blood. It was also possible with whole blood left at room temperature for a month and also with the bloodstains left at room temperature for three years. In the heated blood samples, both alleles, Hp2 and Hp1s, were detected in those heated at 100 degrees C for 2 h. In bloodstains, Hp2 and Hp1s were detected in samples heated at 100 degrees C for 2 h and 120 degrees C for 30 min. In addition, the genotype could be detected in samples other than blood such as saliva, hair roots, tissue sections and dental pulps. The present method for Hp genotyping is expected to become a useful method in forensic analysis.</p

Age Estimation Using S-100 Protein-Positive Stellate Cells in Anterior Pituitary

We examined the embryonic development as well as postnatal development of S-100 protein-positive stellate cells in the anterior lobe of human hypophysis using immunohistochemical method, and investigated the possibility of using the frequency of the stellate cells for age estimation. A definite positive correlation was observed between the proportion of the stellate cell and age, in both males (r = 0.987) and females (r = 0.986). The linear regression equation was y = 0.206x - 1.82 for males and y = 0.239x - 2.22 for females (x: age, y: percentage of 5-100 protein-positive stellate cells). Although the reason for an increase in stellate cells with age remains unknown, the present results indicate that the proportion of stellate cells in the anterior pituitary can be applied reliably to estimate age

Comparison of German and Japanese general practitioners' awareness of suicide and attitudes toward patients with suicidal ideation.

The authors designed a questionnaire to investigate the differences in German and Japanese general practitioners? (GP) awareness of suicide and attitudes toward patients with suicidal ideation in their respective societies. The purpose of this study was to obtain insights leading to a better means of suicide prevention in primary care in Japan. The background for conducting the study was declining suicides in the past 20 years and the lower suicide rate in Germany compared with the present situation in Japan, where the number of suicides has in recent years continued to exceed 30,000, resulting in a suicide rate approximately 2 times higher than that in Germany. The questionnaire was randomly mailed to GPs in Okayama-Prefecture (western Japan) and Hamburg-State (northern Germany) and was collected in the same way. The patterns of answers were compared between the 2 countries, and the differences were statistically analyzed. Japanese GPs seem to have a lower will to prevent suicide in daily practice compared to German GPs and a great lack of knowledge about treatment of suicidal patients. These observations suggest that improving GPs? interest in the problem of suicide and providing training programs for the treatment of patients with suicidal intentions might be a means of achieving better suicide prevention in Japan