Roles of Two Major Alcohol Dehydrogenases, ADH1 (Class I) and ADH3 (Class III), in the Adaptive Enhancement of Alcohol Metabolism Induced by Chronic Alcohol Consumption in Mice.

AIMS: It is still unclear which enzymes contribute to the adaptive enhancement of alcohol metabolism by chronic alcohol consumption (CAC). ADH1 (Class I) has the lowest Km for ethanol and the highest sensitivity for 4-methylpyrazole (4MP) among ADH isozymes, while ADH3 (Class III) has the highest Km and the lowest sensitivity. We investigated how these two major ADHs relate to the adaptive enhancement of alcohol metabolism. METHODS: Male mice with different ADH genotypes (WT, Adh1-/- and Adh3-/-) were subjected to CAC experiment using a 10% ethanol solution for 1 month. Alcohol elimination rate (AER) was measured after ethanol injection at a 4.0 g/kg dose. 4MP-sensitive and -insensitive AERs were measured by the simultaneous administration of 4MP at a dose of 0.5 mmol/kg in order to estimate ADH1 and non-ADH1 pathways. RESULTS: AER was enhanced by CAC in all ADH genotypes, especially more than twofold in Adh1-/- mice, with increasing ADH1 and/or ADH3 liver contents, but not CYP2E1 content. 4MP-sensitive AER was also increased by CAC in WT and Adh3-/- strains, which was greater in Adh3-/- than in WT mice. The sensitive AER was increased even in Adh1-/- mice probably due to the increase in ADH3, which is semi-sensitive for 4MP. 4MP-insensitive AER was also increased in WT and Adh1-/- by CAC, but not in Adh3-/- mice. CONCLUSION: ADH1 contributes to the enhancement of alcohol metabolism by CAC, particularly in the absence of ADH3. ADH3 also contributes to the enhancement as a non-ADH1 pathway, especially in the absence of ADH1.

Metabolic pharmacokinetics of early chronic alcohol consumption mediated by liver alcohol dehydrogenases 1 and 3 in mice.

BACKGROUND AND AIM: Alcohol dehydrogenases (ADHs) 1 and 3 are responsible for systemic alcohol metabolism. The current study investigated the contribution of liver ADH1 and ADH3 to the metabolic pharmacokinetics of chronic alcohol consumption (CAC). METHODS: The 9-week-old male mice of different ADH genotypes (wild-type [WT], Adh1-/- , and Adh3-/- ) were administered with 10% ethanol solution for 1 month, followed by acute ethanol administration (4.0 g/kg). The alcohol elimination rate (AER), area under the blood alcohol concentration curve (AUC), and the maximum blood alcohol concentration (Cmax ) were calculated. The liver content, activity, and mRNA levels of ADH were evaluated. RESULTS: Chronic alcohol consumption increased the AER and reduced the AUC in all ADH genotypes. The increased ADH1 content was correlated with AER in WT mice but not in the Adh3-/- mice. Similarly, the increased ADH3 content was also correlated with AER in both WT and Adh1-/- mice. The Cmax was significantly higher in Adh3-/- control mice than in WT control mice. It decreased in the Adh1-/- mice by CAC along with an increase in the ADH3 content. CONCLUSIONS: Alcohol dehydrogenases 1 and 3 would accomplish the pharmacokinetic adaptation to CAC in the early period. ADH1 contributes to the metabolic pharmacokinetics of CAC with a decrease in AUC in conjunction with an increase of AER by increasing the enzyme content in the presence of ADH3. ADH3 also contributes to a decrease in AUC in conjunction with not only an increase in AER but also a decrease in Cmax by increasing the enzyme content.

Visualization of different characteristics of cerebrospinal fluid with acute encephalopathy and febrile seizures using pattern recognition analysis of 1H NMR.

BACKGROUND: In acute encephalopathy, deterioration of the condition can be rapid, and early intervention is essential to prevent progression of the disease. However, in the acute period, differentiating acute encephalopathy from febrile seizures is difficult. Thus, an early diagnostic marker has been sought to enable early intervention. Proton nuclear magnetic resonance ((1)H NMR) spectroscopy is used to study the chemical characteristics of biological fluids such as cerebrospinal fluid (CSF). The purpose of this study was to ascertain if pattern recognition of (1)H NMR spectra could differentiate CSF obtained from patients with acute encephalopathy and febrile seizures. METHODS: CSF was obtained from patients with acute encephalopathy (n = 4), complex febrile seizures (n = 9), and simple febrile seizures (n = 9). RESULTS: NMR spectra of CSF did not visually differ across the three groups. Spectral data were analyzed by partial least squares discriminant analysis and visualized by plotting the partial least squares scores of each sample. The three patient groups clustered separately on the plots. CONCLUSION: In this preliminary study, we were able to visualize different characteristics of CSF obtained from patients with acute encephalopathy and simple and complex febrile seizures using pattern recognition analysis of (1)H NMR data.

[Dose effect of alcohol on sex differences in blood alcohol metabolism--cases where healthy subjects with ALDH2*1/1 genotype drunk beer with meal].

It is said that blood alcohol concentrations (BAG) are higher in female than in male due to the smaller distribution volume of alcohol in female, whereas the rate of alcohol metabolism is faster in female than in males due to a higher activity of liver alcohol dehydrogenase (ADH) in female. However, it is also known that alcohol metabolism varies depending on drinking conditions. In this study, we evaluated the dose effect of alcohol on sex differences in alcohol metabolism in daily drinking conditions, where young adults (16 males, 15 females) with ALDH2*1/1 genotype drunk beer at a dose of 0.32g or 1.0g ethanol/kg body weight with a test meal (460kcal). This study was conducted using a randomized cross-over design. In the considerable drinking condition (1.0g/kg), BAG was significantly higher in females than in males, whereas the rate of alcohol metabolism (beta) was higher in female than in male. In the moderate drinking condition (0.32g/kg), however, no sex differences in alcohol metabolism including BAG were seen. These results suggest that an increased first pass metabolism through liver ADH in female, which may be caused by the reduction of gastric emptying rate due to the meal intake, contribute to the vanishing of sex difference in BAC in the moderate drinking condition.

Simultaneous Analysis of Zolpidem, Four Hydroxyzolpidems and Two Zolpidem Carboxylic Acids in Postmortem Urine Using Liquid Chromatography--Tandem Mass Spectrometry.

Zolpidem (ZOL) is a short-acting hypnotic that is sometimes used in drug-facilitated crimes such as sexual assaults, robbery and homicides. Therefore, it is important to understand the metabolism of ZOL. This study quantified ZOL and its metabolites, including two carboxylic acids (zolpidem phenyl-4-carboxylic acid [M1] and 6-carboxylic acid [M2]) and four hydroxyzolpidems (4-(hydroxymethyl)phenyl zolpidem [M3], 6-hydroxymethyl zolpidem [M4], 7-hydroxyzolpidem [7OH] and 8-hydroxyzolpidem [8OH]) in postmortem urine using liquid chromatography--triple quadrupole mass spectrometry. The concentration of M1 was highest in all cases, followed by total 7OH in five of six samples. The concentrations of M2 and total M4 were relatively high. Most of M4 and 8OH were excreted as conjugates, whereas up to 55% of 7OH was excreted in its free form. Peaks corresponding to zolpidem dihydrodiol (ZHDH), dihydro(hydroxy)zolpidem cysteine adduct (DHZCys) and zolpidem cysteine adduct (ZCys) were also detected in all the urine samples. ZDHD was excreted as conjugates, whereas almost all DHZCys and ZCys were in their free form.

Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography-tandem mass spectrometry.

PURPOSE: Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography-triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QqTOFMS). METHODS: 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS. RESULTS: The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems. CONCLUSIONS: The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

[Alcohol metabolism at moderate drinking in healthy men. Comparison between differences of alcohol beverages, with and without meal, and genetic polymorphism].

Studies on metabolisms of alcohol and the metabolites (e.g.:acetaldehyde) after drinking give basic and important information to recognize the physiological influence of drinking to human bodies. The aims of these studies were to clarify the influences of ALDH2 genotype difference, kinds of alcohol beverages, and fasted or prandial state to alcohol metabolisms at moderate drinking. The studies were conducted by a randomized cross-over design. After overnight fast, fifteen of ALDH2*1/*1 (Experiment 1) and twenty of ALDH21/*2 (Experiment 2) in Japanese healthy men aged 40 to 59 years old drank beer or shochu at a dose of 0.32g ethanol / kg body weight with or without test meal (460 kcal). The peak of blood ethanol (C(max)) was higher with shochu than with beer in the fasted condition in both ALDH2 genotypes, however, the difference between two types of alcohol beverages went out in the prandial condition. Simultaneous ingestion of test meal with alcohol beverage significantly decreased blood ethanol concentrations and increased ethanol disappearance rate (EDR) in the both genotypes. EDR values were significantly higher in ALDH2*1/*1 type than in ALDH2*1/*2 type in the both beverages with and without meal, whereas beta values showed no significant difference between two genotypes. The concentrations of blood acetaldehyde in ALDH2*1/*2 type were higher in prandial condition than in fasted condition with shochu. These results indicate that meal modified the differences of alcohol metabolism between beer and shochu and also between ALDH2 genotypes. Thus, alcohol metabolism in daily drinking is shown to be regulated by various combinatorial drinking conditions.

Effect of Ethanol on Gene Expression in Beating Neonatal Rat Cardiomyocytes: Further Research with Ingenuity Pathway Analysis Software.

BACKGROUND: There have been no comprehensive studies of changes in heart gene expression due to ethanol exposure. Therefore, we attempted to determine gene expression in cultured neonatal rat cardiomyocytes exposed to ethanol (0, 10, 50, 100 mM) for 24 h. METHODS: The total RNA extract of beating cardiomyocytes was evaluated by DNA microarray analysis, and fold changes (FCs) in differential gene expression of ethanol-exposed cardiomyocytes were analyzed against the control using Ingenuity Pathway Analysis (IPA) software. RESULTS: The 1,394 genes with an |FC| of ≥1.8 were uploaded to IPA. IPA predicted 23 canonical pathways working in the ethanol groups. Three canonical pathways related to ethanol degradation- "Ethanol Degradation IV", "Oxidative Ethanol Degradation III", and "Ethanol Degradation II" -were inhibited in the ethanol groups. IPA predicted "ethanol" as an upregulated upstream regulator of the network with 22 downstream members for the 100 mM ethanol group only. Three members (NTRK2, TGFB3, and TLR8) were activated in all groups. Certain cellular functions were predicted to be changed dose-dependently. "Myocarditis" was dose-dependently inhibited, whereas "Cell death of heart cells" was dose-dependently activated. Several functions were inhibited in 50 mM ethanol only, eg, "Failure of heart" was enhanced in 50 mM ethanol only. Certain functions were activated in 100 mM ethanol only. "Cardiac fibrosis" was not predicted in any ethanol group. CONCLUSIONS: IPA predicted that ethanol-induced activation or inhibition of canonical pathways and functions of cardiomyocyte depended on ethanol concentration, and 3 networks related to heart functions for cardiomyocytes exposed to 3 concentrations of ethanol.

Direct and rapid PCR amplification using digested tissues for the diagnosis of drowning.

We developed a direct and rapid method for the diagnosis of death by drowning by PCR amplification of phytoplankton DNA using human tissues. The primers were designed based on the DNA sequence of the 16S ribosomal RNA gene (16S rDNA) of Cyanobacterium. Samples of lung, liver and kidney tissues were collected from 53 autopsied individuals diagnosed as death by drowning. Without DNA extraction, the tissue fragments were incubated directly in a digest buffer developed in this study, for 20 min. Using 1 microL of the tissue digest solution in PCR, the 16S rDNA was successfully amplified. The specific 16S rDNA fragment was identified from the standard picoplankton Euglena gracilis, the tissues of bodies died from drowning and water samples from the drowning scenes. On the other hand, no PCR products were found in the tissues of individuals who died from causes other than drowning. Various quantities of tissue weighing 1, 5, 10, 20 and 30 mg were tested, and the PCR amplification detected the specific 16S rDNA fragment from all the quantities of tissue tested. This method was found to be more reliable, sensitive, specific and rapid when compared to the conventional diagnosis of death by drowning using the diatom test by acid digestion method.

Cultured Neonatal Rat Cardiomyocytes Continue Beating Through Upregulation of CTGF Gene Expression.

BACKGROUND: Some cultured neonatal rat cardiomyocytes continue spontaneous beating even in serum-free medium. The present study explored the cause and genes responsible for this phenomenon. METHODS: Ingenuity Pathway Analysis (IPA) software was used to analyze fold changes in gene expression in beating neonatal rat cardiomyocytes, as compared with non-beating cardiomyocytes, which were obtained from DNA microarray data of total RNA extracts of cardiomyocytes. To confirm the involvement of the 8 genes selected by IPA prediction, cellular protein abundances were determined by Western blot. The gene expression of connective tissue growth factor (CTGF) was substantially higher in beating cardiomyocytes than in non-beating cardiomyocytes; thus, CTGF protein content released from cardiomyocytes into the culture medium was examined. RESULTS: IPA showed that the "Apelin Cardiac Fibroblast Signaling Pathway" was significantly inhibited and that microtubule dynamics and cytoskeleton organization were significantly activated. Each fluctuation in the cellular abundances of the 8 proteins in beating cardiomyocytes, as compared with non-beating cardiomyocytes, was primarily in the same direction as that of gene expression. However, the cellular CTGF protein abundance as well as CTGF content released into the medium did not substantially differ between beating and non-beating cardiomyocytes. CONCLUSIONS: The present results suggest that the large increase in CTGF gene expression in beating cardiomyocytes is not a cause but a result of beating, which may provide a putative pathway for controlling beating. Beating is sustained by developed cardiomyofibrils and directly upregulates CTGF gene expression, which is not followed by CTGF protein synthesis.

Solitary Deaths in the Tokyo Metropolis and Labor Force Status: Characteristics of Unnatural Deaths at Home among Persons Living Alone.

OBJECTIVE: To identify associations of solitary death with social determinants of health, namely, labor force status and welfare status, in Tokyo in 2015. METHODS: We obtained data on solitary deaths in 2015 in the 23 special wards of Tokyo and calculated the incidence rate and postmortem interval of solitary death in relation to sex, age, and labor force status. RESULTS: Data for 3,972 solitary deaths (2,785 males, 1,187 females) were analyzed. The non-employed rate was 79.3% among males and 89.5% among females. The incidence rate was significantly higher among non-employed persons than among employed persons in both sexes. Moreover, with the exception of women 65 years or older, the postmortem interval was significantly longer among non-employed persons than among employed persons in both sexes. CONCLUSIONS: The incidence rates of solitary death were significantly higher among non-employed persons than among employed persons in both sexes, and the postmortem interval was significantly longer for non-employed persons.

Experimental analysis of silicone leakage.

We analyzed whether gel bleed, the leakage of silicone gel from breast implants, occurs in the human body. We simulated the phenomenon with olive oil. Silicone breast implants were submerged in olive oil, and the concentration of silicone polymers in the olive oil was measured periodically with nuclear magnetic resonance spectroscopy. We found no increase in the silicone concentration. However, clinical conditions might not be adequately simulated because of the shortness of the experimental period and the lack of external stress. However, when clinical experiences and our data are considered, we think that silicone implant durability is an important factor to prevent leakage or gel bleeding.

Association between sudden unexpected deaths in bathtubs and ambient temperature among elderly Japanese adults: A time-series regression study.

BACKGROUND: Sudden unexpected deaths in bathtubs among elderly Japanese adults occur predominantly during the cold season. This study investigated the relationship between these deaths and bathing day temperature among elderly adults in Tokyo. METHODS: Data for 1408 cases of bath-related deaths from January 1 to December 31, 2015 were obtained from the Tokyo Medical Examiner's Office. We excluded 409 cases for the following reasons: criminal death, injury-related death, suicide, intoxication, non-sudden death, not bathtub-related death, out-of-bathroom death, subject aged under 65 years, undetermined bathing date, institutional housing, and bathing not at subject's home. Ultimately, 999 cases were analyzed. Daily mean temperature data were collected. A time-series regression study was performed to estimate the influence of sex, age, and bathing day temperature. Monthly changes in the population bathing in a bathtub were considered in the model. RESULTS: The relative risk (RR) of sudden unexpected death in a bathtub was 1.381 for males (95% confidence interval [CI]: 1.218-1.564) compared to females. The RRs were 4.182 (95% CI: 3.523-4.986) and 9.382 (95% CI: 7.836-11.273) among those aged 75-84 years and ≥85 years, respectively, compared to among those aged 65-74 years. The RR increased to 1.092 (95% CI: 1.082-1.102) as the daily mean temperature decreased by 1 °C. CONCLUSION: Sudden unexpected death in a bathtub correlated with bathing day temperature among elderly Japanese adults, and extremely low temperature, male sex, and older age increased the risk of such death. Our findings provide insight into preventing sudden unexpected deaths in bathtubs.

Short-time Fourier Transform of Free Induction Decays for the Analysis of Serum Using Proton Nuclear Magnetic Resonance.

Proton nuclear magnetic resonance (NMR) is useful for the analysis of biological samples such as serum. Free induction decays (FIDs) are NMR signals that follow a radio-frequency pulse applied at the resonance frequency. Short-time Fourier transform (STFT) is a basic method for time-frequency analyses. The purpose of this study was to ascertain whether the STFT of FIDs enables the sensitive detection of changes and differences in serum properties. FIDs were obtained from serum collected from young, healthy, male volunteers ≤ 40 years of age and seniors ≥ 65 years of age. Temporal changes in the instantaneous amplitudes for the time-domain analysis, fast Fourier transform for frequency-domain analysis, and STFT were applied to the FIDs. The STFT-based spectrogram represented the complex frequency components that changed dynamically over time, indicating that the spectrogram enabled the visualization of the features of an FID. Furthermore, the results of a partial least-squares discriminant analysis demonstrated that the STFT was superior to the other two methods for discriminating between serum from younger and older subjects. In conclusion, the STFT of FIDs obtained from proton NMR measurements was useful for evaluating similarities and dissimilarities in the FIDs obtained from serum samples.

Scuba-diving related deaths in Okinawa, Japan, from 1982 to 2007.

We reviewed the autopsies of scuba-diving related deaths (SDRDs) that were collected from April 1982 until March 2007. In the period under consideration, a total of 40 SDRDs were registered, out of which 34 were males and 6 females. Ages ranged from 19 to 65 years, with the average of 41.5 years (SD=12.9). Divers over the age of 40 accounted for 60% of all fatalities. The major cause of death was drowning (62.5%), followed by disease (28.5%). The average age for drowning and disease-related deaths was 38.6 (SD=12.8) and 48.7 years (SD=10.1), respectively. Of the 40 fatalities, 24 were beginners who had little or no experience. In this study, we compared SDRDs in the first term, from April 1982 to March 1995, and in the second term, from April 1995 to March 2007. The average age in the first and second terms was 35.4 and 45.2 years, respectively; the average age for the second term was 10 years older than the first. Of those in the first term, 13.3%, and of those in the second term, 40.0%, died from complications arising from already existing conditions. This study revealed that the onset of diseases during diving frequently causes fatal accidents, especially for older divers.

The Contribution of Alcohol Dehydrogenase 3 to the Development of Alcoholic Osteoporosis in Mice.

BACKGROUND: Alcohol dehydrogenase 3 (ADH3) plays major roles not only in alcohol metabolism but also in nitric oxide metabolism as S-nitrosoglutathione reductase (GSNOR). ADH3/GSNOR regulates both adipogenesis and osteogenesis through the denitrosylation of peroxisome proliferator-activated receptor γ. The current study investigated the contribution of ADH3 to the development of alcoholic osteoporosis in chronic alcohol consumption (CAC). METHODS: Nine-week-old male mice of different ADH genotypes [wild-type (WT) and Adh3-/-] were administered a 10% ethanol solution for 12 months. The femurs were evaluated by histochemical staining and computed tomography-based bone densitometry. The mRNA levels of ADH3 were evaluated in the WT mice by reverse transcription-quantitative polymerase chain reaction. RESULTS: The Adh3-/- control mice exhibited increased activities of both osteoblasts and osteoclasts and lower bone masses than the WT control mice. CAC exhibited no remarkable change in osteoblastic and osteoclastic activities, but decreased bone masses were observed in WT mice despite an increase in the mRNA levels of ADH3. Conversely, bone masses in the Adh3-/- control mice were not reduced after CAC. CONCLUSIONS: The Adh3-/- control mice exhibited a high turnover of osteoporosis since osteoclastogenesis dominated osteoblastogenesis; however, bone resorption was not enhanced after CAC. In comparison, CAC lead to alcoholic osteoporosis in WT mice, accompanied by increased mRNA levels of ADH3. Hence, ADH3 can prevent osteoporosis development in normal ADH genotypes with no alcohol ingestion. However, ADH3 contributes to the development of alcoholic osteoporosis under CAC by participating in alcohol metabolism, increasing metabolic toxicity, and lowering GSNO reducing activity.

In vivo contribution of Class III alcohol dehydrogenase (ADH3) to alcohol metabolism through activation by cytoplasmic solution hydrophobicity.

Alcohol metabolism in vivo cannot be explained solely by the action of the classical alcohol dehydrogenase, Class I ADH (ADH1). Over the past three decades, attempts to identify the metabolizing enzymes responsible for the ADH1-independent pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. In this study, we used Adh3-null mutant mice to demonstrate that Class III ADH (ADH3), a ubiquitous enzyme of ancient origin, contributes to alcohol metabolism in vivo dose-dependently resulting in a diminution of acute alcohol intoxication. Although the ethanol oxidation activity of ADH3 in vitro is low due to its very high Km, it was found to exhibit a markedly enhanced catalytic efficiency (kcat/Km) toward ethanol when the solution hydrophobicity of the reaction medium was increased with a hydrophobic substance. Confocal laser scanning microscopy with Nile red as a hydrophobic probe revealed a cytoplasmic solution of mouse liver cells to be much more hydrophobic than the buffer solution used for in vitro experiments. So, the in vivo contribution of high-Km ADH3 to alcohol metabolism is likely to involve activation in a hydrophobic solution. Thus, the present study demonstrated that ADH3 plays an important role in systemic ethanol metabolism at higher levels of blood ethanol through activation by cytoplasmic solution hydrophobicity.

Maturation of whisky changes ethanol elimination kinetics and neural effects by increasing nonvolatile congeners.

BACKGROUND: The maturation of distilled spirits is known to change constituent congeners to improve the qualities of smell and taste. However, it has been largely unknown how maturation modifies the pharmacokinetics or neuropharmacological effects of ethanol. We used single malt whiskies to investigate the effects of spirit maturation on ethanol metabolism and drunkenness. METHODS: Mice were injected with 5-year (5-y) or 20-year (20-y) aged single malt whisky with a concentration of 20% (w/v) ethanol at a dose of 3 g/kg. The concentrations of ethanol and its metabolites in the blood and the duration of loss of righting reflex (LORR) were compared between the 2 whisky groups. In addition, the effects of nonvolatile congeners in whisky on the biomedical reactivities of ethanol were investigated by administering a nonvolatile fraction added to a 20% ethanol solution, whose fraction was prepared by evaporating 16-y whisky. Liver alcohol dehydrogenase (ADH) activity was measured with whisky as the substrate or in the presence of nonvolatile congeners with ethanol as the substrate. RESULTS: The rate of ethanol elimination (mmol/kg/h) was smaller in the 20-y whisky group than in the 5-y group (p<0.01 by Fisher's protected least significant difference), which resulted in lower concentrations of blood acetaldehyde and acetate in the former group than in the latter group (p<0.01 by ANOVA). Nonvolatile congeners added to the ethanol solution also depressed the rate of ethanol elimination in mice. In vitro studies demonstrated that liver ADH activity measured with whisky as the substrate was decreased as a function of the age of the whisky, and that the activity measured with ethanol as the substrate was strongly inhibited by nonvolatile congeners. The duration of LORR was longer in the 20-y group than in the 5-y group (p<0.01). Nonvolatile congeners also prolonged the duration of ethanol-induced LORR, when administered together with ethanol. CONCLUSION: Maturation of whisky delayed ethanol metabolism to lower the level of blood acetaldehyde and acetate with increasing inhibition of liver ADH activity by nonvolatile congeners. It also prolonged drunkenness by enhancing the neurodepressive effects of ethanol, due to increases in the amount of nonvolatile congeners. These biomedical effects of whisky maturation may reduce aversive reactions and cytotoxicity due to acetaldehyde, and may also limit overdrinking with the larger neurodepression.

Pattern recognition analysis of proton nuclear magnetic resonance spectra of postmortem cerebrospinal fluid from rats with drug-induced seizure or coma.

Cerebrospinal fluid (CSF) is routinely subjected to gross evaluation in postmortem investigations; however, its use in chemical evaluations has not been fully realized. Analysis of nuclear magnetic resonance (NMR) spectra with pattern recognition methods was applied to CSF samples. Rats were treated with pentylenetetrazol (PTZ) to induce seizure or pentobarbital (PB) to induce coma, and postmortem CSF was collected after CO2 gas euthanization. Pattern recognition analysis of the NMR data was performed on individual postmortem CSF samples. The aim of this study was to determine if pattern recognition analysis of NMR data could be used to classify the rats according to their drug treatment. The applicability of NMR data with pattern recognition analysis using postmortem CSF was also assessed. Partial Least Squares-Discriminant Analysis (PLS-DA) score plots indicated that the PTZ, PB, and NS (control) groups were clustered and clearly separated. PLS-DA correlation loading plots showed respective spectral and category variances of 41% and 42% for factor 1, and 17% and 27% for factor 2. Thus, factors 1 and 2 together described 58% (41%+17%) and 69% (42%+27%) of the variation, respectively. NMR study of postmortem CSF has the potential to be utilized as both a novel forensic neurochemistry method and in the clinical setting.

Effects of chronic administrations of aconitine on body weight and rectal temperature in mice.

Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. However, aconitine has been used as one of the most popular compounds in Shino-Japanese traditional herbal medicine. Little has been reported concerned with the long-term effects of aconitine. Therefore, the authors investigated the physiological effects of chronic administrations of aconitine by determining the changes in body weight and rectal temperature of mice, compared with the concentrations of aconitine and its metabolites (benzoylaconine and aconine) in the liver and kidneys. The concentration ratio of aconitine to the total Aconitum alkaloids (from day 0 to 22; 90 min after the last administration) gradually decreased, whereas its metabolites increased until day 22. The body weight gain in aconitine-administered group was less than that of the control group until day 22. Transient rectal hypothermia occurred within 30 min after the last administration of aconitine. Then the rectal temperature gradually increased to normal level in respect to time. This study might reveal the possibilities that the drug metabolism of aconitine increased and the toxicity of aconitine decreased due to long-term administrations of aconitine.