A new individualization marker of semen: deoxyribonuclease I (DNase I) polymorphism.

We describe a method for obtaining specific and reproducible deoxyribonuclease I (DNase I) typing from liquid semen. Isoelectric focusing of the enzymes on polyacrylamide gel (IEF-PAGE, pH 3.5-5) was accomplished using a 0.5-mm thick gel. The separated isozymes were visualized by a new activity staining method, dried agarose film-overlay (DAFO). Pretreatment of semen samples with neuraminidase markedly enhanced the isozyme-band resolution and sensitivity. The method was simple and reliable, with high resolution and sensitivity. The DNase I types in semen samples were correlated with the types found in corresponding blood and urine samples. DNase I typing could therefore provide an additional discriminant characteristic in the forensic examination of semen.     

Methamphetamine impurity profiling using a 0.32 mm i.d. nonpolar capillary column

Classification of seized methamphetamine by impurity profiling can provide very useful information in criminal investigations of drug traffic routes, sources of supply and relationships between seizures. The aim of this study is to improve and develop an analytical method for detecting impurities such as starting materials and by-products in illegally prepared methamphetamine.HCl samples. A 50mg sample of methamphetamine.HCl was dissolved in 1 ml of buffer solution (four parts 0.1M phosphate buffer pH 7.0 and one part 10% Na2CO3). Impurities were extracted with 0.5 ml of ethyl acetate containing four internal standards (ISs) (n-decane, n-pentadecane, n-nonadecane and n-hexacosane) and analyzed by gas chromatography (GC) using a flame ionization detector (FID) on a DB-5 capillary column (0.32 mmi.d. x 30 m, film thickness 1.0 microm). The use of a middle-bore column offered better separation of the impurity peaks. The correction of the retention times of impurity peaks with four ISs made peak identification very accurate for subsequent data processing. Twenty-four characteristic peaks were selected for comparison and similarity and/or dissimilarity between samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. The results indicate that the present method would be useful for methamphetamine impurity profiling.     

A novel multiplex PCR system consisting of Y-STRs DYS441, DYS442, DYS443, DYS444, and DYS445

We have developed a new sensitive multiplex PCR system consisting of five male-specific and polymorphic tetranucleotide STRs--DYS441 (GDB: 10013873), DYS442 (GDB: 10030304), DYS443 (GDB: 10807127), DYS444 (GDB: 10807128), and DYS445 (GDB: 10807129) on the Y chromosome. Fifty pg DNA per 10 microL reaction volume was required for the correct typing of five STRs. Using this system, the five Y-STRs were correctly typed from blood and semen stains that had been stored for several years at room temperature.     

Transferrin polymorphism detected in human urine using isoelectric focusing followed by immunoblotting

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF*C1 = 0.7265, TF*C2 = 0.2624, TF*D = 0.0083 and TF*B = 0.0028.     

Immunochemical studies on blood group A substance from human hair

Blood group A-active substance was extracted from urea-treated human hair uith methanol-ethyl ether 1:1, v/v) or chloroform-methanol (1:1, v/v). The serological activity of blood group A substance in the hair was destroyed by A-decomposing enzyme from Clostridium tertium with concomitant development of blood group H activity. It is concluded therefore that the extract from the hair of group A contained blood group A-active glycolipid with N-acetylgalactosamine as the non-reducing sugar.     

New detection method for uropepsinogen (PGA) using isoelectric focusing and immunoblotting techniques

Uropepsinogen (PGA) was isolated and purified from human urine using a column chromatography series. The purified PGA was injected into a rabbit and a PGA-specific antibody was obtained. PGA isozymogen in human urine could be detected reproducibly by immunoblotting using this antibody after isoelectric focusing electrophoresis (IEF) on polyacrylamide gels. This technique may prove to be useful in the genetic study of PGA polymorphism.     

GC polymorphism detected in human urine by isoelectric focusing and immunoblotting

Genetic polymorphism of GC (vitamin D-binding protein) in human urine was revealed by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels containing 2 M urea. Urine samples from 530 unrelated Japanese from the Fukui district, being only 1-2 ml of original urine, were examined, and correct GC typing was achieved by comparison with the results of direct grouping using plasma. Six common and twelve rare phenotypes were observed. The frequencies of the genes were 0.473 for GC*1F, 0.241 for GC*1S, 0.254 for GC*2, and 0.032 for the total of six rare alleles.     

A study of the metabolism of methamphetamine and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in isolated rat hepatocytes

The metabolism of methamphetamine (MA) and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in freshly isolated rat hepatocytes was investigated, and compared with in vivo results. A suspended hepatocyte culture, established from male Wistar rats using a collagenase perfusion technique, was incubated in the presence of MA or 2C-B. After enzymatic hydrolysis of the conjugated forms, the metabolites were extracted by liquid-liquid partition and analyzed by gas chromatography/mass spectrometry (GC/MS). Amphetamine, p-hydroxymethamphetamine and p-hydroxyamphetamine were detected in the culture fluids of the rat hepatocytes inoculated with MA. The alcohol derivative, carboxylic acid derivative, 2-O-desmethyl-2C-B, 2-O-desmethyl-N-acetyl-2C-B and 5-O-desmethyl-N-acetyl-2C-B were detected in the case of 2C-B. The major metabolite of MA in rat hepatocytes was p-hydroxymethamphetamine. This is in good agreement with the urinary excretion profile for rats that were fed MA. 2-O-Desmethyl-2C-B and the carboxylic acid derivative were the major recovered metabolites of 2C-B in the rat hepatocyte culture, a slight deviation from the in vivo findings, in which 5-O-desmethyl-N-acetyl-2C-B was found to be the main component. Metabolites with a hydroxy group were largely present in their conjugated forms in the culture fluids, except for 2-O-desmethyl-2C-B. Taking these results into consideration, a primary hepatocyte culture system has the potential to provide a quick and handy method for estimating the in vivo metabolic fate of abused drugs.     

Identification of impurities and the statistical classification of methamphetamine using headspace solid phase microextraction and gas chromatography-mass spectrometry

The profiling of impurities in methamphetamine (MA) using headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) is described. The extraction of the impurities with an SPME fiber was examined under varying conditions. Optimal chromatograms were obtained when a 50 mg MA sample at 85 degrees C for 30 min was extracted using a fiber coated with divinylbenzene/carboxen/polydimethylsiloxane. MA samples from nine different origins were analyzed under optimized extraction conditions. Compounds related to MA such as benzaldehyde, benzyl alcohol, amphetamine, benzyl methyl ketone, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, N-acetylamphetamine, N-acetylmethamphetamine and N-formylmethamphetamine were detected in the chromatograms. Trace amounts of ethanol, diethyl ether and acetic acid were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different, depending on the sample. After the areas of the eight principal peaks were converted to their square root and logarithm, similarities among the samples were evaluated by Euclidian distance, cosine distance and correlation coefficient. The results showed that a combination of logarithmic conversion and cosine distance was the most suitable for discriminating and classifying the samples. HS-SPME/GC-MS is a simple and effective method for the extraction and identification of impurities. The present method, in combination with an appropriate statistical analysis, would be useful for developing a profile of impurities in MA.     

Analysis of hallucinogenic constituents in Amanita mushrooms circulated in Japan

The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of <10-2845ppm/46-1052ppm in the cap of A. muscaria and 188-269ppm/1554-1880ppm in the cap of A. pantherina. In the caps, these compounds had a tendency to be more concentrated in the flesh than in the cuticle. On the other hand, the IBO/MUS contents in the stem were far lower than in the caps. In the "extracts purported to contain A. muscaria" products, IBO/MUS were detected below the lower limit of calibration curve (<10ppm/<25ppm) or not detected. However, these samples contained other psychoactive compounds, such as psychoactive tryptamines (5-methoxy-N,N-diisopropyltryptamine and 5-methoxy-N,N-dimethyltryptamine), reversible monoamine oxidase inhibitors (harmine and harmaline) and tropane alkaloids (atropine and scopolamine), which were not quantified. This is the first report of the chemical analysis of Amanita mushrooms that are circulated in the drug market.