Hajime Tsutsumi

Identification of saliva stains by determination of the specific activity of amylase

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.

Identification of human urinary stains by enzyme-linked immunosorbent assay for human uromucoid

An enzyme-linked immunosorbent assay (ELISA) of the sandwich type for identification of human urinary stains using commercially available anti-human uromucoid was developed. When experimentally prepared urinary stains of humans and animals, 2 by 2 cm in area, were subjected to analysis, human stains could be differentiated from animal ones except chimpanzee and Old World monkey ones. Stains of other human body fluids showed negative reactions. The reactions did not decrease when human urinary stains were stored at room temperature for three months. The present ELISA provides a useful presumptive test for urinary stains of human origin.

Determination of total hemoglobin in forensic blood samples with special reference to carboxyhemoglobin analysis.

For the determination of total hemoglobin (Hb) in blood containing elevated carboxyhemoglobin (COHb), a newly developed reagent containing a 100-fold concentration of ferricyanide (20 g/l) and a 2-fold concentration of Sterox SE was compared with a standard reagent (0.2 g/l ferricyanide), the reagent of van Kampen and Zijlstra, using forensic blood samples and experimentally heated blood samples. There were no significant differences between the spectra of hemiglobincyanide (HiCN) solution produced with our reagent and the van Kampen and Zijlstra reagent using experimentally heated blood samples. Although the spectra of HiCN changed gradually with increased heating time and with the passage of time after mixing, the absorbance at 540 nm (A540) did not change until at least 120 min for both the reagents. When forensic blood samples containing elevated COHb were mixed with the van Kampen and Zijlstra reagent, total-Hb concentrations determined 5 min after mixing were 10-20% higher than those determined after 180 min. The overestimates of total Hb determined after 5 min resulted in comparable underestimates of percentage saturation of COHb (COHb%) when COHb% was obtained from the ratio of COHb content, determined by gas chromatogrpahy, to total-Hb concentration in blood. However, there was an extremely good correlation between the values of total Hb in forensic blood samples determined with the van Kampen and Zijlstra reagent after 180 min and those determined with our reagent after 5 min. From the results obtained, our reagent proved to be suitable for the determination of total Hb in forensic science practice.

Storage of blood for methemoglobin determination: Comparison of storage with a cryoprotectant at −30°C and without any additions at −80°C or −196°C

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood or mixtures of blood and a cryoprotectant at refrigerated or various freezing temperatures were examined using blood samples from nitrite-administered rats and from autopsy cadavers. When whole blood was stored at 3 degrees C, Met-Hb reduction was observed in blood samples from nitrite-administered rats and in the blood from a victim poisoned by a weed killer containing some oxidant. When samples were stored at -30 degrees C, Met-Hb formation by autoxidation was inevitably observed in blood samples stored as whole blood, whereas addition of a cryoprotectant to whole blood could prevent Met-Hb formation in all the blood samples. When whole blood was stored at -80 degrees C or -196 degrees C, Met-Hb concentrations were practically stable until at least 30 days regardless of the initial values except in the control rat blood samples stored at -80 degrees C which showed slight formation of Met-Hb. From the results obtained, both the storage with a cryoprotectant at -30 degrees C and that without any additions at -80 degrees C or -196 degrees C proved to be suitable for long-term storage of blood samples from autopsy cadavers for Met-Hb determination.

Identification of human urinary stains by the quotient uric acid/urea nitrogen

Uric acid (UA) and urea nitrogen (UN) were determined in urinary stains and the UA/UN x 20 values were calculated. The values in human urinary stains were 1.11-4.21, while those in other mammals except some of chimpanzees, were under 0.7, and those in fecal stains of birds were over 80. Most of the stains of other human body fluids or plant juices tested contained neither UA nor UN, and some contained one, but never the other. Ascorbic acid (AS) of up to 100 mg/dl in urine did not interfere with UA determination when dried human urinary stains were analyzed. It was also found that the contents of UA were very low at the peripheral parts of urinary stains. The present results indicate that the quotient UA/UN is useful for identification of human urinary stains in forensic practice provided that the peripheral part of the stain is not used.

Phosphoglucomutase1and 6-Phosphogluconate Dehydrogenase Types in Human Skin and Adipose Tissue

Attempts were made to detect phenotypes of the enzymes phosphoglucomutase 1(PGM1) and phosphogluconate dehydrogenase (PGD) in human skin and adipose tissues. Both enzymes could be typed using approximately 3 mg wet weight of tissue. Phenotypes could be distinguished after up to 15 days of aging for PGM1 and ten days of aging for PGD. Analysis of isoenzymes is potentially useful for mediolegal identification of human skin and adipose tissue.

Identification of Fetal Bloodstains by Enzyme-Linked Immunosorbent Assay for Human α-Fetoprotein

A rapid and highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of human alpha-fetoprotein (AFP) using commercially available reagents was devised and applied to identification of fetal bloodstains. When experimentally prepared bloodstains, 1 by 2 mm in area, were submitted to analysis, only fetal bloodstains showed positive reactions in the present ELISA. The reactions did not change significantly when these bloodstains were stored at room temperature for one week. The present ELISA seems to be suitable for forensic science practice.

Forensic study on stains of blood and saliva in a chimpanzee bite case

A case of a boy bitten by a chimpanzee is reported. Forensic investigations were carried out on his T-shirt which had been torn open at the right armpit. By presumptive tests using chemical reagents and definitive tests using antisera against human hemoglobin A (HbAo), human serum and human saliva amylase, it was demonstrated that stains scattered around the torn portion of the shirt consisted of saliva or both blood and saliva. Though the blood was immunologically assumed to be human, the saliva could not be identified as human or chimpanzee. The mixed stains and the saliva stains were grouped A and secretor. By treatment of the mixed stains with a mixture of two volumes of chloroform and one volume of methanol (CM solution), the water-insoluble blood substance derived from blood was typed as O and the water-soluble substance derived from saliva was typed as A and secretor. The former was the victims blood type and the latter were the chimpanzee saliva types.

Comparative immunology of mammalian uricase using anti-hog liver uricase

1. Uricase was partially purified from livers of five mammals (dog, bovine, horse, house musk shrew and guinea pig), and its immunological properties were studied using anti-hog liver uricase prepared in rabbits. 2. Results obtained by immunodiffusion suggest that hog uricase has four sites of partial antigens (a, b, c, d), while the enzyme of other mammals has only part of them, and a cladogram of these six mammals is presented showing development of partial antigens. 3. Results obtained by immunoelectrophoresis suggest that the protein structure of house musk shrew uricase is considerably different from the other five mammals tested.

Comparative studies on an antigenicity of plasma proteins from humans and apes by ELISA: A close relationship of chimpanzee and human

1. Antigenic differences between human and ape plasma proteins were quantitatively investigated by enzyme-linked immunosorbent assay (ELISA) using antisera against human and chimpanzee plasmas. 2. With anti-human plasma serum, both the chimpanzee and gorilla were very close to the human, although the chimpanzee was slightly closer to the human than to the gorilla; relative immunological distance (relative ID) of the chimpanzee was 71, while that of the gorilla was 74. 3. With anti-chimpanzee plasma serum, the chimpanzee was found to be closely related to the human; relative ID of the chimpanzee was 58, while that of the gorilla was 75. 4. From these a molecular phylogeny for humans and apes was deduced; among living apes, the chimpanzee is the most closely related species to the human.