Biological effects and metabolic interactions after chronic and acute administration of 4-methylpyrazole and ethanol to rats

4-Methylpyrazole in a dose producing an inhibition of alcohol dehydrogenase of about 60% was given alone or in combination with ethanol (10%) as sole drinking fluid to growing rats in periods up to 38 weeks. No effects were observed on the weight curves. Hematologic analyses showed normal values for blood and bone marrow. Studies of liver function with transaminase, bilirubin and albumin did not reveal any functional changes. Kidney function was normal as judged by creatinine and normal electrolytes. Electronmicroscopy of liver, kidney, and heart did not reveal any changes related to treatment. Combined treatment of ethanol and 4-methylpyrazole caused an increase of the microsomal drug-metabolizing activity. Chronic administration of ethanol and 4-methylpyrazole indicated that there is a mutual interaction in the metabolism of ethanol and 4-methylpyrazole, leading to a higher concentration of both ethanol and 4-methylpyrazole in the blood. Acute experiments, where alcohol dehydrogenase is saturated with ethanol, indicated a much slower elimination of 4-methylpyrazole. Administration of ethanol and 4-methylpyrazole in acute experiments showed a lower concentration of 4-hydroxymethylpyrazole in the blood indicating that ethanol interferes with the 4-methylpyrazole- and/or 4-hydroxymethyl-pyrazole-metabolizing enzymes. The present investigation has shown that the acute and chronic toxicity of 4-methylpyrazole alone or in combination with ethanol is minimal at doses that are effective in blocking ethanol metabolism in the rat. Because of its low toxicity and powerful inhibitory capacity, 4-methylpyrazole should be a potential tool for experimental clinical investigation of alcohol metabolism and its effects. 4-Methylpyrazole is also a potential therapeutic agent in methanol or ethylene glycol poisoning.     

Age characteristics of cadmium content in the organism of poisoned rats with experimental metabolic acidosis

The paper demonstrates age peculiarities of cadmium content in the liver, kidneys and spleen of 3-month and 18-month rats poisoned by cadmium sulfate. It was registered that the content of cadmium is 266.7 times higher in 3-month old rats, and 141.8 times in 18 months rats in comparison with intact ones. A biological model of introducing 3-month and 18-month rats into the state of metabolic acidosis before and after poisoning with cadmium sulfate was developed. The result of the research showed that changes in acid-base equilibrium of poisoned rats towards metabolic acidosis may influence a decrease of cadmium content in their organisms. Thus, when modeling of metabolic acidosis before the poisoning with cadmium, the content of cadmium decreases in 3-month rats' liver by 21%, in kidneys by 53%; in metabolic acidosis modeling after cadmium poisoning its content decreases, accordingly, by 44% and by 56.5% in comparison with the just poisoned ones. In metabolic experimental acidosis modeling the content of cadmium in 3-month poisoned rats' spleen decreased by 36.5% before and after poisoning. Such changes were also registered in 18-month old rats but to lower extent. Thus, the results of the researches showed more effective correction of cadmium intoxication decrease in the organism of 3-month rats when using the model of acid-base equilibrium change in the organisms of poisoned animals.     

Ethanol and Methanol Can Improve Huperzine A Production from Endophytic Colletotrichum gloeosporioides ES026

Huperzine A (HupA) is a plant alkaloid that is of great interest as a therapeutic candidate for the treatment of Alzheimer''s disease. However, the current production of HupA from plants in large quantity is unsustainable because the plant resource is scarce and the content of HupA in plants is extremely low. Surprisingly, this compound was recently found to be produced by various endophytic fungi, which are much more controllable than the plants due to simpler genetics and ease of manipulation. However, it might be due to the innate properties of endophytic symbiosis, that production of this chemical in large quantity from endophytes has not yet been put into practice. Endophytic Colletotrichum gloeosporioides ES026 was previously isolated from a HupA producing plant and the fungi also proved to produce HupA. In this study, various fermentation conditions were tried to optimize the production of HupA from C. gloeosporioides ES026. Optimization of these parameters resulted in a 25.58% increase in HupA yield. Potato extracts supplemented with glucose or sucrose but not maltose facilitated HupA producing from the fungi. A final concentration of 0.5–2% ethanol stimulated the growth of fungi while methanol with the same treatment slightly inhibited the growth. However, both methanol and ethanol greatly increased the HupA production with the highest yield of HupA (51.89% increment) coming from ethanol treatment. Further analysis showed that both ethanol and methanol were strong inducers of HupA production, while ethanol was partially used as a carbon source during fermentation. It was noticed that the color of that ethanol treated mycelia gradually became dark while methanol treated ones stayed grey during fermentation. The present study sheds light on the importance of optimizing the fermentation process, which, combined with effective inducers, maximizes production of chemicals of important economic interest from endophytic fungi.     

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.     

Acute Paracetamol Poisoning

Of 41 cases of acute paracetamol poisoning one died of gastrointestinal haemorrhage and acute massive necrosis of the liver, three became jaundiced, and 13 others had biochemical evidence of hepatocellular damage. Liver damage is a toxic effect which is present in most patients who ingest more than 15 g. of paracetamol. One patient with liver damage survived renal failure due to acute tubular necrosis. It is suggested that the renal lesion was also the result of paracetamol overdosage.Profound hypoglycaemia and metabolic acidosis may also complicate severe poisoning. Plasma levels of para-aminophenol fall rapidly, and procedures currently used to enhance the elimination of the drug cannot be expected to prevent development of hepatic damage.     

Effect of selected alcohol dehydrogenase inhibitors on the human heart lactate dehydrogenase activity--an in vitro study

Metabolic acidosis complicates methanol, ethylene glycol and other alcohol intoxications. It is caused firstly by acid metabolites and secondly by the lactate elevation. The aim of the study was to evaluate the effect of alcohol dehydrogenase (ADH; EC 1.1.1.1) inhibitors and substrates: 4-methylpyrazole (4-MP), cimetidine, EDTA, ethanol and methanol on lactate dehydrogenase (LDH; EC 1.1.1.27) activity. The activity of LDH was determined spectrophotometrically in in vitro human heart homogenates with the mentioned compounds at 0.01, 0.1, 1.0 mM concentrations of 4-MP, cimetidine, EDTA, and 12.5, 25.0, 50.0 mM of ethanol and methanol. The LDH activity was significantly inhibited by 0.1 mM (p<0.05) and 1.0 mM (p<0.01) 4-MP and 1.00 mM EDTA (p<0.05). Higher LDH activity vs. control was observed in the samples incubated with all studied ethanol and methanol concentrations but these differences were not statistically significant. Thus, 4-MP was found to be the most effective inhibitor of LDH of all compounds tested. Therefore, such effect of 4-MP seems to be an additional advantage in methanol and ethylene glycol intoxications.     

Metabolic profiles and genetic diversity of denitrifying communities in activated sludge after addition of methanol or ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Bilateral peripheral facial nerve palsy in acute poisoning with ethylene glycol

Acute poisonings with polyethylene glycol become more and more frequent. The acute poisoning with polyethylene glycol leads to a considerable metabolic acidosis and acute renal failure. Usually there is no peripheral nervous system involvement. Two cases of the bilateral facial nerve palsy in patients with renal failure due polyethylene glycol poisoning are presented. It seems that it is the first report on the lesions to facial nerve involvement in the course of the acute polyethylene glycol poisoning.     

Toxicity of short-chain alcohols to the nematode Caenorhabditis elegans: a comparison of endpoints

The toxicities of 4 short-chain alcohols--namely methanol, ethanol, iso-propanol and iso-butanol--were compared in the nematode Caenorhabditis elegans using several different ecotoxicological endpoints. Range-finding tests were conducted using transgenic PC161 worms carrying a double reporter construct (GFP plus lacZ) linked to the stress-inducible hsp16-1 promoter. These tests showed little response from the GFP reporter, but gave good dose-response curves for the lacZ reporter--showing clear induction at 0.5% v/v ethanol in an overnight assay, but only at 4% in a shorter 6-h assay. Comparison of the short-term dose-response curves shows a confusing pattern of differences between the four alcohols tested, although dose-dependence is evident across at least part of the concentration range. Feeding inhibition assays are somewhat inconclusive with regard to alcohol type, although iso-butanol and iso-propanol appear more toxic than ethanol, while methanol is least toxic. To resolve some of the remaining ambiguities, we also used a fecundity assay to show that iso-propanol is more toxic than ethanol, and a lethality assay to show that iso-butanol is more toxic than iso-propanol. Most of the endpoints studied are consistent with the following order of toxicity: iso-butanol > iso-propanol > ethanol > or = methanol.     

Acute drug poisoning in the adult

In a prospective study of 349 patients with acute poisoning treated at The Montreal General Hospital in 1972 benzodiazepines and non-barbiturate hypnotics were found to be the most frequent putative drugs. Of the 108 patients admitted to hospital 37% had taken an overdose of a drug prescribed for them by their psychiatrist or other physician; 48% had formerly taken an overdose of drugs and 44% had had previous psychiatric treatment. Unconsciousness, respiratory depression, metabolic acidosis and acidemia, and hypokalemia were the most frequent clinical abnormalities observed. Treatment was supportive. There were six deaths. The average duration of coma was short; only five surviving patients remained unconscious for more than 24 hours. Respiratory complications were frequent.It is recommended that more attention be paid to recognizing patients whose behaviour pattern might include such an impulsive gesture, and that alternatives be found for barbiturate and non-barbiturate hypnotics.     

Characterization of an inhibitor of hepatic cholesterogenesis present in hepatic microsomes.

An inhibitor of hepatic cholesterol synthesis present in hepatic microsomes can be solubilized either by an acetone or an ethanol powder preparation. Other methods such as methanol and chloroform:methanol powder preparations and treatment with EDTA do not solubilize the factor. The factor appears to be proteinaceous since its activity is lost after exposure to proteolytic enzymes and heat treatment. In addition, the inhibitor does not require a phospholipid for activity. 3this inhibitor is stable for long periods (60 hrs.) at room temperature and can be isolated in good yield from liver maintained at 4 degrees C for 8 hours postmortem.     

On the cytochemical demonstration of glycogen in neutrophil granulocytes: periodic acid-Schiff reaction and diastase (amylase) digestion test (author's transl)]

The results of the present investigation indicate clearly that treatment of blood smears with diastase resp. amylase is unsuitable to identify glycogen in neutrophil granulocytes. This may be attributed to the contamination with proteases of commonly used preparations of diastase resp. amylase. Thus strong PAS-reactive material which presents most probably not glycogen but PAS-positive glycoproteins may be eliminated by the proteolytic activity of the contaminants. - In detail it has been shown that susceptibility resp. resistance of the PAS-positive material against treatment with diastase resp. amylase is highly dependent on both type of fixation and fixation time: Fixation with formol free absolute alcohol (ethanol, methanol), leads also after prolonged fixation time to a complete loss of PAS-staining after preliminary treatment with diastase resp. amylase. On the other side after fixation with formol containing fixatives (for example formol/ethanol and acetic acid/formol/ethanol) only after short term fixation practically a complete loss of PAS-staining material is observed. However, after long term fixation more or less complete resistance of the PAS-stainable material against treatment with diastase resp. amylase has been found.     

The biooxidation of methanol,ethanol and isopropanol by a defined co-culture at elevated temperatures

The introduction of more imaginative enrichment and isolation procedures has permitted the isolation of pure cultures of thermotolerant methylotrophic bacteria, a group that was previously unknown. One potential application for such bacteria is in the aerobic biotreatment of petrochemical industry wastewaters at elevated temperatures. Here, the growth and biooxidation characteristics of one such bacterium, Bacillus sp. NCIB 12522, in co-culture with a Gram-negative thermophilic non-methylotrophic solvent utilizing bacterium, NA 17, on a mixture of methanol, ethanol and isopropanol, under both steady and transient state continuous flow culture conditions are reported. The results indicate that at dilution rates <0.2 h^–1 effective biooxidation can be achieved at temperatures between 50° and 57 °C. As a result of step increases in bioreactor feed concentrations, the fraction of the methylotroph present in the co-culture changed according to whether the methanol or the ethanol concentrations were increased, but when isopropanol was increased, no change in the methylotroph fraction occurred between the initial and final steady states.     

Evaluation of the feasibility of alcohols serving as external carbon sources for biological phosphorus removal induced by the oxic/extended‐idle regime

Recently, a novel operational regime (i.e., the oxic/extended‐idle [OEI] regime) has been reported to successfully achieve enhanced biological phosphorus removal (EBPR) when employing glucose and volatile fatty acids as the sole substrate. In the OEI regime, polyphosphate accumulating organisms (PAOs) could get a selective advantage over other populations during the extended‐idle period where polyphosphate released but polyhydroxyalkanoates and glycogen transformations were negligible/low, thus energy requirements for maintenance purposes in the period could be covered by polyphosphate release. This study further evaluated the feasibility of alcohols as external carbon sources for EBPR induced by the OEI regime, as the available substrate in the raw wastewater is often deficient. First, phosphorus removal in the OEI process was compared, respectively, with methanol and ethanol as the sole substrate. The results showed that the ethanol‐reactor achieved 90.8 ± 2.3% of phosphorus removal, which was approximate twofold than the methanol‐reactor. Further studies displayed that the cells in the ethanol‐reactor contained more PAOs, and had higher activities of exopolyphosphatase and polyphosphate kinase than those in the methanol‐reactor. Also, the aerobic transformations of polyhydroxyalkanoates and glycogen in the ethanol‐reactor were, respectively, higher and lower than those in the methanol‐reactor, which were consistent with the reactors performances. Then, the feasibility of using ethanol as external substrate to enhance EBPR in the OEI process was confirmed for a municipal wastewater. Finally, EBPR performance and metabolic transformation values between the OEI and the anaerobic/oxic (A/O) regimes with ethanol as the sole substrate were compared. The results showed that EBPR in the ethanol‐OEI reactor was higher than that in the ethanol‐A/O reactor. All the above results proved that ethanol was a favorable external substrate to the OEI regime for EBPR enhancement. Biotechnol. Bioeng. 2013; 110: 827–837. © 2012 Wiley Periodicals, Inc.     

甲醇营养型酵母高密度培养过程中甲醇和乙醇的GC快速检测

采用气相色谱法（GC）快速检测甲醇营养型酵母发酵液中的甲醇、乙醇含量,具有样品处理简便,测定时间较短,结果重视性好的特点。在1－10mg/mL范围内具有很好的线性关系。在毕赤酵母高密度表达发酵过程中应用此法对甲醇和乙醇进行实时监空,细胞终密度超过300g/L（干重）。本方法为甲醇营养型酵母工程菌的发酵中试工艺研究提供了重要 的发酵生化参数。     

Metabolic Profiles and Genetic Diversity of Denitrifying Communities in Activated Sludge after Addition of Methanol or Ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Characterization of an Inhibitor of Hepatic Cholesterogenesis Present in Hepatic Microsomes

An inhibitor of hepatic cholesterol synthesis present in hepatic microsomes can be solubilized either by an acetone or an ethanol powder preparation. Other methods such as methanol and chloroform:methanol powder preparations and treatment with EDTA do not solubilize the factor.

The factor appears to be proteinaceous since its activity is lost after exposure to proteolytic enzymes and heat treatment. In addition, the inhibitor does not require a phospholipid for activity.

This inhibitor is stable for long periods (60 hrs.) at room temperature and can be isolated in good yield from liver maintained at 4°C for 8 hours postmortem.     

The self-diffusion and hydrogen bond interaction in neat liquid alkanols: a molecular dynamic simulation study

Self-diffusion of methanol, ethanol, 1-propanol and 2-propanol has been studied by molecular dynamics simulation in the temperature range between the melting pressure curve and 478 K at pressures up to 300 MPa. The simulation results on self-diffusion of methanol, ethanol and 2-propanol (for 2-propanol, at high temperatures) agree well with experiment, which suggests that the simulation method is a powerful tool to obtain self-diffusion coefficients over wide range of temperature and pressure, under which it is rather difficult for experiments. The local structures of methanol, ethanol and 2-propanol are investigated by calculating the radial distribution functions, H-bond numbers, coordination numbers and the ratios of H-bond number divided by coordination number. The correlation between self-diffusion and structural properties, and the influence of temperature and pressure on them are discussed. The degree of forming H-bond space network in methanol, ethanol and water is higher than that in 2-propanol, and they are all higher than those in ammonia and methylamine. The simulation results demonstrate that the effect of hydrogen bonding on the translational dynamics in methanol and ethanol is more pronounced than that in 2-propanol.     

Lactic acidosis secondary to metformin overdose: a case report

ABSTRACT: INTRODUCTION: Metformin is a commonly used treatment modality in type 2 diabetes mellitus, with a welldocumented side effect of lactic acidosis. In the intensive care setting lactate and pH levelsare regularly used as a useful predictor of poor prognosis. In this article we highlight howhigh lactate levels are not an accurate predictor of mortality in deliberate metforminoverdose. CASE PRESENTATION: We present the case of a 70-year-old Caucasian man who took a deliberate metforminoverdose of unknown quantity. He had a profound lactic acidosis at presentation with a pH of6.93 and a lactate level of more than 20mmol/L. These figures would normally correspondwith a mortality of more than 80%; however, with appropriate management this patient'scondition improved. CONCLUSION: We provide evidence that the decision to treat severe lactic acidosis in deliberate metforminoverdose should not be based on arterial lactate and pH levels, as would be the case in otheroverdoses. We also demonstrate that appropriate treatment with hemodiafiltration and 8.4%sodium bicarbonate, even in patients with a very high lactate and low pH, can be successful.     

The time course of miniature endplate currents and its modification by receptor blockade and ethanol

Miniature endplate currents (MEPCs) recorded from mouse diaphragms with a point voltage clamp, without inhibition of acetylcholinesterase (AChE) and in the absence of any drug, showed in their decay phase consistent deviations from an exponential time course, consisting of (a) "curvature," a progressive increase of decay rate during most of the decay phase, followed by (b) "late" tails. Both phenomena persisted when MEPCs (and channel lifetime) were prolonged by ethanol. Curvature was increased by muscle fiber depolarization and decreased by hyperpolarization. Receptor blockade by (+)-tubocurarine, alpha-bungarotoxin, hexamethonium, or myasthenic IgG accelerated the decay of the main part of MEPCs and eliminated curvature; the time constant of MEPCs became close to the channel time constant. We conclude that curvature arises from repeated action of ACh with cooperativity in ACh-receptor interaction; the voltage sensitivity of curvature follows from the voltage sensitivity of channel closing. Ethanol, in addition to its effect to prolong channel lifetime, enhances the tendency of ACh to act more than once to open channels before being lost to the system. Analysis of the rising phase of the MEPC, in terms of driving functions, also indicated that ethanol promotes channel opening by ACh; this action can account for a substantial increase of MEPC height by ethanol when MEPCs are made small by receptor blockade. Driving functions were also voltage sensitive, in a manner indicating acceleration of channel opening, but reduction of channel conductance, with hyperpolarization. Poisoning or inhibition of AChE prolonged MEPCs without altering the duration of ionic channels. Since ethanol caused further prolongation of MEPCs after poisoning of AChE, with little change in MEPC height, we conclude that the extension of mean channel lifetime by ethanol is accompanied by a similar extension of ACh binding to receptors. After poisoning of AChE, MEPCs became very variable in time course and the decay rate (tau-1) was correlated with MEPC height with a slope of log tau vs. log height of 0.77 for MEPCs of greater than 60% mean size. This slope is larger than expected from cooperativity in ACh-receptor interaction. Correlation of tau and height of MEPCs also exists when AChE is intact; the slope of log tau vs. log height was 0.12 with or without prolongation of MEPCs by ethanol.