Simple and rapid determination of the acetaldehyde dehydrogenase (ALDH2) genotypes by nonradioactive single-strand conformation polymorphism analysis.

The genotyping of mitochondrial acetaldehyde dehydrogenase (ALDH2) is very important in alcohol studies. We describe an ALDH2 genotyping method based on nonradioactive single-strand conformation polymorphism (SSCP) analysis on mini-gels following amplification with a mutated primer set. The three ALDH2 genotypes were clearly and unambiguously distinguished. This method was applied to the ALDH2 genotyping of 129 unrelated Japanese. The allele frequency of ALDH2*2 was calculated to be 0.271, which was consistent with the previous data. The method proved to be simple, rapid and reliable, and dispensed with isotopic reagent and expensive restriction enzymes and equipment. The SSCP method described here is valuable in routine ALDH2 genotyping.

Analysis of bile acids in urine specimens from healthy humans: determination of several bile acids with beta-hydroxyl and carbonyl groups.

Urinary bile acids of 39 healthy male undergraduates were analyzed by capillary gas chromatography and capillary gas chromatography-mass spectrometry. 3 alpha-Hydroxy-12-oxo-5 beta-cholanoic acid, 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid, 3 beta, 7 alpha-dihydroxy-5 beta-cholanoic acid, and 3 alpha, 7 alpha, 12 beta-trihydroxy-5 beta-cholanoic acid, in addition to known bile acids, were identified and then quantified. The major part of the urinary bile acids was occupied by secondary bile acids. Every 7 beta-hydroxylated bile acid species was found in more than 80% of the subjects. The bile acid detected in the largest amount was 3 alpha-hydroxy-12-oxo-5 beta-cholanoic acid. The metabolites of cholic acid were quantitatively more predominant than those of chenodeoxycholic acid. These results indicate that bile acids with beta-hydroxyl and carbonyl groups at the C-3,7 and/or 12 positions are usual bile acids usually found in the urine of healthy humans. It is concluded that the occurrence of these bile acids is an effect of the intestinal bacterial flora and living conditions.

[Toxicological findings in a death involving hydrogen sulfide].

A case of fatal hydrogen sulfide poisoning in a sewage disposal plant is described. Hydrogen sulfide levels were determined in several postmortem body tissues and fluids using gas chromatography with a flame photometric detection (GC-FPD). The concentrations of hydrogen sulfide in the blood, muscle, liver, lung, kidney, heart and brain were 0.305, 0.690, 1.089, 1.338, 1.170, 1.366 and 0.875 (micrograms/g or ml), respectively. The results are discussed in the light of the existing literature on the toxicological significance of hydrogen sulfide concentrations.

Toxicological index of paraquat: a new strategy for assessment of severity of paraquat poisoning in 128 patients.

A new assessment of the severity of paraquat poisoning in 128 patients has been developed. It involves toxicological index of paraquat and discriminant function score. This system not only allows a more accurate assessment of severity of the poisoning, but also provides a more reliable prediction of the outcome in an early stage for the purpose of forensic and clinical toxicology.

Potential bioactivated neurotoxicants, N-methylated beta-carbolinium ions, are present in human brain.

Potential bioactivated neurotoxicants, 2-N-methyl-beta-carbolinium and 2,9-N,N'-dimethyl-beta-carbolinium ions, as well as N-methylation activities which form these charged species, were analyzed for the first time in the parietal association cortex and the substantia nigra of human brain using GC/MS and HPLC. The brains were taken during forensic autopsies from corpses without obvious degeneration of substantia nigra. In the cortex, 2-methyl-norharmanium ion (2-MeNH) and 2,9-dimethyl-norharmanium ion (2,9-Me2NH) were detected in almost all samples. 2-Methyl-harmanium ions (2-MeHA) and 2,9-dimethyl-harmanium ions (2,9-Me2HA) were detectable in only two samples. In substantia nigra samples pooled from 3 or 4 brains for analysis, 2-MeNH and 2,9-Me2NH levels were higher than those in the cortex, whereas 2-MeHA and 2,9-Me2HA were below detection limits. Their precursors, norharman (NH) and harman (HA), were also measured using HPLC/fluorescence detection. In both regions, NH and HA were present in almost all samples; levels of NH and HA were also significantly higher in the nigra than in the cortex. Using 9-methyl-NH and 2-MeNH as substrates, in vitro N-methylation of the 2[beta] and 9[indole] nitrogens toward beta-carbolines was measured both in the cortex and in the nigra. 2[beta]-N-Methylation activity was significantly higher than 9[indole]-N-methylation activity in both regions. Recent studies show that beta-carbolinium ions resemble the synthetic parkinsonian toxicant, MPP+, with respect to structure and neurotoxic activity. Such 'bioactivated' carbolinium ions could be endogenous causative factors in Parkinson's disease.

Decreased superoxide dismutase activity in erythrocyte in Parkinson's disease.

Superoxide dismutase (SOD) activities in Parkinson's disease (PD) were significantly lower than those in controls, especially in a treated PD group. However, SOD activities in an untreated PD group did not decrease. There was a significant correlation between SOD activities and the duration of illness in the treated PD group (p < 0.05). There was a significant correlation between SOD activities and the present doses of L-DOPA/carbidopa in the treated PD group.

Orosomucoid (ORM) typing by isoelectric focusing: evidence for several new variants including ORM1 and ORM2 silent alleles.

Human orosomucoid (ORM) is composed of two tightly linked loci, ORM1 and ORM2, located on chromosome 9q. Some haplotypes contain duplicated ORM1 or ORM2. The present study using isoelectric focusing has revealed seven ORM haplotypes including new variants and/or silent alleles detected in eight Japanese families. ORM1*5.2-ORM2*15 in combination with common ORM1*1-ORM2*1 produced a five-band pattern. ORM1*5-ORM2*1 was observed in three families. ORM2*9 was coexistent with ORM1*1 or ORM1*2.1. ORM1*Q0 was linked to ORM2*1 or ORM2*14. ORM2*Q0 was suggested to be in association with ORM1*2. The ORM haplotypes with a silent gene must have arisen from unequal crossing-over between the tandemly duplicated genes of ORM.

Orosomucoid (ORM) typing by isoelectric focusing: an analysis of ORM haplotypes.

A new separator isoelectric focusing method for typing of orosomucoid (ORM) was developed. This method provided a superior resolution of ORM patterns: two close bands of ORM1*5.2 products were clearly separated. A total of 364 subjects from Okinawa (Japan) were classified into 21 ORM phenotypes determined by 6 ORM1 and 7 ORM2 alleles including a polymorphic silent allele, ORM2*QO, and 2 new rare variants, ORM2*18 and ORM2*19. These phenotypes were also explained by 12 ORM haplotypes, half of which were polymorphic.

A rapid and sensitive method for the determination of paraquat in plasma and urine by thin-layer chromatography with flame ionization detection.

A new method for the determination of paraquat in biological fluids has been developed. It involves thin-layer chromatography with flame ionization detection (TLC/FID) and simple solid-phase extraction with a disposable octadecylsilane column. This procedure allows direct loading of the samples on the disposable column. When Chromarod SII is used as the stationary phase and methanol-2N hydrochloric acid (2:3, v/v) as the mobile phase, recovery levels of paraquat in spiked materials are from 87.5 to 98.0%. This method can be used for samples of paraquat at levels as low as 50 ng.

PI Mtoyoura: a new PI M subtype found by separator and hybrid isoelectric focusing.

In the course of a population study of alpha 1-antitrypsin polymorphism by separator isoelectric focusing, a variant phenotype having a somewhat narrower spacing than PI M1M3 was observed in a Japanese blood donor. Family studies by hybrid isoelectric focusing in a carrier ampholyte-supplemented immobilized pH gradient from 4.35-4.65 revealed that the products of the responsible gene, PI*Mtoyoura, were extremely close but slightly cathodal to those of PI*M1. The difference in isoelectric point between them corresponded to the resolving limit of isoelectric focusing. For this reason, although the propositus' father was deduced to have the genotype PI*M1/PI*Mtoyoura, the products of these two genes failed to form a double band pattern. Thus, the gene frequency for PI*Mtoyoura was unknown. These findings, however, indicate that a further microheterogeneity in the PI M subtype exists at least in the Japanese.

Genetic polymorphism of alpha 2HS-glycoprotein in a French population. Description of two new rare variants.

A French population was investigated for genetic polymorphism of alpha 2HS-glycoprotein (A2HS; nomenclature according to Human Gene Mapping 7, Los Angeles, 1983) using isoelectric focusing and immunoblotting. Three variants were observed together with two common alleles A2HS 1 and A2HS 2, whose frequencies were significantly different from the data in Canadians and Egyptians. An anodal variant to A2HS 1 was identical to a variant with two different nomenclatures reported by three different groups, indicating that there is a confusion in the A2HS nomenclature. The others were new variants with cathodal isoelectric points to A2HS 2 in the native state.

Evaluation of paraquat concentrations in paraquat poisoning.

The toxicological significance of paraquat concentrations in paraquat poisonings was evaluated by means of multivariate analysis methods. Paraquat could be determined by a newly developed procedure, which involved thin-layer chromatography with flame ionization detector (TLC-FID) and solid-phase extraction with a disposable octadecylsilane cartridge. This new method proved to be simple, rapid and reliable for the analysis of paraquat in our seven cases of suicidal poisoning. The relationship between plasma paraquat concentration (C) and time from ingestion (T) could be best described by the following functions. The regression equation of fatal cases was ln[ln(C X 1000)] = 2.5453 - 0.2114 lnT. The regression equation of survivors was ln[ln(C X 1000)] = 2.1041 - 0.2826 lnT. The discriminant function (D) to separate the fatal and survival cases was D = 1.3114 - 0.1617 lnT - 0.5408 [ln(C X 1000)] (fatal cases: D less than 0, survivors: D greater than 0). The discriminant function was demonstrated to have a high reliability for the toxicological significance in our seven poisoned patients. The significant correlation between plasma paraquat concentration and urine paraquat concentration (C') in our cases was obtained. The regression equation was lnC' = 0.953 lnC + 1.409. This also indicated that urinary concentrations are 3.3 - 4.5 times greater than plasma concentrations. The multiple regression equation among plasma paraquat concentration, time from ingestion, and the ingested volume (V) of Gramoxone (trade name of paraquat), was lnC = 0.009V - 0.232T + 3.612. It is suggested that the determination of paraquat is of great value, and that these data are useful in assessing the severity and predicting the outcome of poisoning for forensic and clinical purposes.

Gas chromatographic and gas chromatographic-mass spectrometric determination of gasoline in a case of gasoline vapor and alcohol poisoning.

A case of fatal poisoning due to the combined effect of alcohol and gasoline following an automobile accident is described. Toxicological analyses by means of gas chromatography and gas chromatography-mass spectrometry permitted the identification and quantitation of alcohol and several hydrocarbons in the heart blood and in the gas in the lung. Great variation was found in the estimates of blood gasoline concentration, depending on which of six constituents of gasoline was chosen for quantitation. The cause of this variation is discussed, together with the possible mechanisms leading to death.

Phosphoglucomutase-1 subtypes: polymorphic occurrence of PGM1*7+ and geographical variation in Japan.

The distribution of gene frequencies in the phosphoglucomutase-1 (PGM1) system was investigated in two Japanese populations from Yamaguchi (Western Japan) and Okinawa (Southern Japan) using an improved isoelectric focusing method permitting the successful detection of the most anodal variant PGM1 3+. PGM1*7+ occurred with a polymorphic frequency of 0.012-0.021. A difference in the gene frequency was observed between the two populations. In comparison with neighboring populations, the Yamaguchi population was similar to Mongolians and Koreans in North China, and Okinawa to Zhuang in South China.

Distribution of Hp, Tf, Gc and Pi polymorphisms in a Nepalese population.

Hp typing and Tf, Gc and Pi subtypings were performed on 144 serum samples from a Nepalese population in the Katmandu Valley, Nepal. The obtained allele frequencies are as follows: Hp1 = 0.1771, Hp2 = 0.8229; TfC1 = 0.7222, TfC2 = 0.2500, TfC3 = 0.0174, TfCnepal (TfC9) = 0.0104; Gc1F = 0.2448, Gc1S = 0.4825, Gc2 = 0.2727; PiM1 = 0.6076, PiM2 = 0.2118, PiM3 = 0.1806. The relationship between this sample population and the Indian population is discussed.