Youkichi Ohno

A new view of alcohol metabolism and alcoholism--role of the high-Km Class III alcohol dehydrogenase (ADH3).

The conventional view is that alcohol metabolism is carried out by ADH1 (Class I) in the liver. However, it has been suggested that another pathway plays an important role in alcohol metabolism, especially when the level of blood ethanol is high or when drinking is chronic. Over the past three decades, vigorous attempts to identify the enzyme responsible for the non-ADH1 pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. Recently, using ADH3-null mutant mice, we demonstrated that ADH3 (Class III), which has a high Km and is a ubiquitous enzyme of ancient origin, contributes to systemic alcohol metabolism in a dose-dependent manner, thereby diminishing acute alcohol intoxication. Although the activity of ADH3 toward ethanol is usually low in vitro due to its very high Km, the catalytic efficiency (kcat/Km) is markedly enhanced when the solution hydrophobicity of the reaction medium increases. Activation of ADH3 by increasing hydrophobicity should also occur in liver cells; a cytoplasmic solution of mouse liver cells was shown to be much more hydrophobic than a buffer solution when using Nile red as a hydrophobicity probe. When various doses of ethanol are administered to mice, liver ADH3 activity is dynamically regulated through induction or kinetic activation, while ADH1 activity is markedly lower at high doses (3–5 g/kg). These data suggest that ADH3 plays a dynamic role in alcohol metabolism, either collaborating with ADH1 or compensating for the reduced role of ADH1. A complex two-ADH model that ascribes total liver ADH activity to both ADH1 and ADH3 explains the dose-dependent changes in the pharmacokinetic parameters (β, CLT, AUC) of blood ethanol very well, suggesting that alcohol metabolism in mice is primarily governed by these two ADHs. In patients with alcoholic liver disease, liver ADH3 activity increases, while ADH1 activity decreases, as alcohol intake increases. Furthermore, ADH3 is induced in damaged cells that have greater hydrophobicity, whereas ADH1 activity is lower when there is severe liver disease. These data suggest that chronic binge drinking and the resulting liver disease shifts the key enzyme in alcohol metabolism from low-Km ADH1 to high-Km ADH3, thereby reducing the rate of alcohol metabolism. The interdependent increase in the ADH3/ADH1 activity ratio and AUC may be a factor in the development of alcoholic liver disease. However, the adaptive increase in ADH3 sustains alcohol metabolism, even in patients with alcoholic liver cirrhosis, which makes it possible for them to drink themselves to death. Thus, the regulation of ADH3 activity may be important in preventing alcoholism development.

A column-switching LC/MS/ESI method for detecting tetrodotoxin and Aconitum alkaloids in serum

A liquid chromatography-mass spectrometry-electrospray ionization (LC/MS/ESI) method coupled with a column-switching technique has been developed for the determination of tetrodotoxin (TTX) and Aconitum alkaloids and their metabolites, such as aconitine, mesaconitine, hypaconitine, jesaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine and 14-anisoylaconine, in serum. An on-column column-switching technique was employed to analyze TTX and Aconitum alkaloids and their metabolites without pretreatment of the serum. Combination of a multimode column with reversed phases and cation exchange for TTX, and use of a multimode column with reversed phases and a hydrophobic polymer column for Aconitum alkaloids and their metabolites provided successful separation and MS determination in ESI positive mode. A 100 microl serum sample was directly injected into a precolumn. For TTX monitored at m/z 320.1 in the selected ion monitoring mode, the calibration curve was linear within the range 0.1-100 ng/ml and the limit of detection was 0.1 ng/ml. For aconitine, mesaconitine, hypaconitine and jesaconitine, linear calibration curves were obtained up to 500 ng/ml and the limit of detection ranged from 0.2 to 1 ng/ml. For benzoylaconine, benzoylmesaconine, benzoylhypaconine and 14-anisoylaconine, linear calibration curves were obtained up to 500 ng/ml and the limit of detection ranged from 2 to 50 ng/ml. Recoveries from serum samples were within the range 78-119% for all the compounds studied.

The Experimental Approach to the Murder Case of Aconite Poisoning

AbstractAconites (wolfsbane) are the plants in the genus Aconitum (the Ranunculaceae family), containing highly toxic alkaloids such as aconitine, mesaconitine. hypaconitine, and jesaconitine in all parts of the plant. In 1986 a case of sudden death of a woman traveler was autopsied in Okinawa by the author. The cause of her death was revealed to be aconite poisoning in 1987 by detecting the Aconitum alkaloids in her blood by the gas chromatography / selected ion monitoring (GC/SIM) method, newly developed for this case. The husband was suspected of the murder by the fact that he insured her life for 185 million yen. In 1991 he was arrested and indicted on a charge of murder of the wife. However, the time lag between the time of her death and of her separation from the husband was still unexplainable. Just before the indictment the police disclosed the fact that tetrodotoxin, the pufferfish toxin, was also detected in the victims blood. Then, we executed the animal experiments to clarify the toxicity of ...

Pattern Recognition Analysis of Proton Nuclear Magnetic Resonance Spectra of Brain Tissue Extracts from Rats Anesthetized with Propofol or Isoflurane

Background General anesthesia is routinely used as a surgical procedure and its safety has been endorsed by clinical outcomes; however, its effects at the molecular level have not been elucidated. General anesthetics influence glucose metabolism in the brain. However, the effects of anesthetics on brain metabolites other than those related to glucose have not been well characterized. We used a pattern recognition analysis of proton nuclear magnetic resonance spectra to visualize the changes in holistic brain metabolic phenotypes in response to the widely used intravenous anesthetic propofol and the volatile anesthetic isoflurane. Methodology/Principal Findings Rats were randomized into five groups (n = 7 each group). Propofol and isoflurane were administered to two groups each, for 2 or 6 h. The control group received no anesthesia. Brains were removed directly after anesthesia. Hydrophilic compounds were extracted from excised whole brains and measured by proton nuclear magnetic resonance spectroscopy. All spectral data were processed and analyzed by principal component analysis for comparison of the metabolite profiles. Data were visualized by plotting principal component (PC) scores. In the plots, each point represents an individual sample. The propofol and isoflurane groups were clustered separately on the plots, and this separation was especially pronounced when comparing the 6-h groups. The PC scores of the propofol group were clearly distinct from those of the control group, particularly in the 6-h group, whereas the difference in PC scores was more subtle in the isoflurane group and control groups. Conclusions/Significance The results of the present study showed that propofol and isoflurane exerted differential effects on holistic brain metabolism under anesthesia.

Experimental estimation of postmortem interval using multivariate analysis of proton NMR metabolomic data

Nuclear magnetic resonance (NMR) spectroscopy has recently been applied to metabolic studies. In particular, metabolic profiles of tissues or of the whole body can easily be acquired through multivariate analysis of NMR spectra. The present study investigates metabolic changes after death in rat femoral muscles using pattern recognition of proton NMR spectra. Rats were killed by suffocation, cocaine overdose and induced respiratory failure, and then low molecular weight metabolites extracted using perchlorate from excised tissues were measured using proton NMR. All spectral data were processed and assessed by multivariate analysis to obtain metabolic profiles of the tissues. The results of principal component analysis (PCA) score plots soon after death showed that the metabolic profiles of the tissues differed according to the mode of death. The principal component (PC) scores of the data varied hourly and correlated with postmortem interval. The present results showed that NMR-based metabolic profiling could provide useful information with which to estimate postmortem intervals and causes of death.

Dose and time changes in liver alcohol dehydrogenase (ADH) activity during acute alcohol intoxication involve not only class I but also class III ADH and govern elimination rate of blood ethanol

BACKGROUND The elimination rate of blood ethanol usually depends on the activity of liver alcohol dehydrogenase (ADH). During acute alcohol intoxication, however, it is unclear how liver ADH activity changes with dose and time and what the involvement is of the two major isozymes of liver ADH: the classically known class I ADH and the very high Km class III ADH. We investigated dose- and time-wise changes in liver ADH activity and the contents of both ADHs by administering ethanol to mice, and analyzed the relationship among these ADH parameters to assess the contributions of these ADHs to liver ADH activity and ethanol metabolism in vivo. METHODS Mice were given ethanol doses of 0, 1, 3 or 5 g/kg body weight and killed 0.5, 1, 2, 4, 8 or 12 h after administration. The elimination rate of blood ethanol was calculated from the regression line fitted to the blood ethanol curve. The liver ADH activity of crude extract was conventionally measured with 15 mM ethanol as a substrate. The liver class I and class III ADH contents were determined by enzyme immunoassay. These three ADH parameters were statistically analyzed. RESULTS The change in liver ADH activity depended on both dose and time (P<0.001 by two-way ANOVA, n=74), but the change in the class I content depended on dose alone (P<0.0001). The class III content depended on both dose and time (P<0.001) with a time course similar to that of liver ADH activity for each dose. The sum of the class I and class III contents exhibited a higher correlation with liver ADH activity (r=0.882, P<0.0001) than the class I content alone did (r=0.825). The mean liver ADH activity during ethanol metabolism for each dose correlated significantly with the elimination rate of blood ethanol (r=0.970, P<0.0001). CONCLUSION Liver ADH activity changes dose and time dependently during acute alcohol intoxication and governs the elimination rate of blood ethanol through the involvement not only of class I but also of class III ADH.

An accidental case of aconite poisoning due to Kampo herbal medicine ingestion

An accidental case of aconite intoxication occurred after a patient took a therapeutic dose of Kampo herbal medicine containing Aconiti tuber, Uzu but had used the wrong decoction procedure. The poisoning was likely caused by an increased level of Aconitum alkaloids in the decoction; the patient developed aconite intoxication due to incomplete decoction. Aconitum alkaloid levels in the leftover solution which the patient had drunk and in the decoction extracted from 3g Uzu were determined. It was found that decoction makes the medicine safer to drink. Older individuals, especially those with dementia, have a higher risk of aconite poisoning because they sometimes do not boil the medicine appropriately.

Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I) to ADH 3 (Class III) in Mouse

ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CLT (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its V max⁡/mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3.

Time-of-flight mass spectrometry (TOF-MS) exact mass database for benzodiazepine screening

Time-of-flight mass spectrometry coupled with liquid chromatography (TOF-MS) has been developed for screening and determination of benzodiazepines with an exact mass database. Benzodiazepines display similar chemical structures and molecular weights, and thus show similar mass spectra and protonated molecule ions. Discrimination of mass spectrometry at low resolving power using single liquid chromatography mass spectrometry (LC-MS) is commonly difficult. TOF-MS analysis was performed using a 1100 TOF (Agilent Technologies) equipped with a Zorbax C18 Extend column. Purine and fluorine compound solution was always introduced into the ion source, and real-time mass adjusting was performed. Specimens were prepared utilizing the liquid-liquid extraction procedure with 1-chlorobutane. Benzodiazepines are widely used in medical practice in Japan, and data acquired from TOF-MS measurements of 41 benzodiazepines, including active metabolites, were used to create an exact mass database. This database comprised molecular formulae, calculated exact masses and retention times. Calibrations were also included in a database. Precision for the 41 drugs was considered sufficient for quantitative analysis. In analysis of samples from patient who had taken > or =2 benzodiazepines, selectivity was improved using the TOF-MS exact mass database. TOF-MS is effective for forensic toxicology in discriminating between benzodiazepines with similar structure and metabolites.

Phytophenols in whisky lower blood acetaldehyde level by depressing alcohol metabolism through inhibition of alcohol dehydrogenase 1 (class I) in mice

We recently reported that the maturation of whisky prolongs the exposure of the body to a given dose of alcohol by reducing the rate of alcohol metabolism and thus lowers the blood acetaldehyde level (Alcohol Clin Exp Res. 2007;31:77s-82s). In this study, administration of the nonvolatile fraction of whisky was found to lower the concentration of acetaldehyde in the blood of mice by depressing alcohol metabolism through the inhibition of liver alcohol dehydrogenase (ADH). Four of the 12 phenolic compounds detected in the nonvolatile fraction (caffeic acid, vanillin, syringaldehyde, ellagic acid), the amounts of which increase during the maturation of whisky, were found to strongly inhibit mouse ADH 1 (class I). Their inhibition constant values for ADH 1 were 0.08, 7.9, 15.6, and 22.0 mumol/L, respectively, whereas that for pyrazole, a well-known ADH inhibitor, was 5.1 mumol/L. The 2 phenolic aldehydes and ellagic acid exhibited a mixed type of inhibition, whereas caffeic acid showed the competitive type. When individually administered to mice together with ethanol, each of these phytophenols depressed the elimination of ethanol, thereby lowering the acetaldehyde concentration of blood. Thus, it was demonstrated that the enhanced inhibition of liver ADH 1 due to the increased amounts of these phytophenols in mature whisky caused the depression of alcohol metabolism and a consequent lowering of blood acetaldehyde level. These substances are commonly found in various food plants and act as antioxidants and/or anticarcinogens. Therefore, the intake of foods rich in them together with alcohol may not only diminish the metabolic toxicity of alcohol by reducing both the blood acetaldehyde level and oxidative stress, but also help limit the amount of alcohol a person drinks by depressing alcohol metabolism.