Determination of two mebeverine metabolites, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma by a dual stable isotope-based gas chromatographic-mass spectrometric method

A dual stable isotope-based GC-MS method was developed for the simultaneous determination of two metabolites of mebeverine, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma. Plasma samples were treated with β-glucuronidase to cleave the glucuronide conjugates of both compounds prior to analysis. The treated plasma was prepared for analysis by solid-phase extraction using octadecylsilane cartridges. The isolated metabolites were derivatized and analyzed by GC-MS using selected-ion monitoring. Plots of peak-area ratio were linear with metabolite concentration from 2 to 200 ng/ml and the limit of detection for both metabolites was 0.5 ng/ml. The GC-MS methodology was applied to the analysis of plasma from human subjects following peroral administration of mebeverine. Pharmacokinetic parameters for both metabolites were determined and suggest that relative systemic mebeverine exposure may potentially be assessed using metabolite kinetics, if the latter subsequently are demonstrated to be linear with mebeverine dose.     

Genetic polymorphisms: impact on the risk of fetal alcohol spectrum disorders

Clinical reports on monozygotic and dizygotic twins provided the initial evidence for the involvement of genetic factors in risk vulnerability for fetal alcohol spectrum disorders (FASD) including fetal alcohol syndrome (FAS). Research with selectively bred and inbred rodents, genetic crosses of these lines and strains, and embryo culture studies have further clarified the role of both maternal and fetal genetics in the development of FASD. Research to identify specific polymorphisms contributing to FASD is still at an early stage. To date, polymorphisms of only one of the genes for the alcohol dehydrogenase enzyme family, the ADH1B, have been demonstrated to contribute to FASD vulnerability. In comparison with ADH1B*1, both maternal and fetal ADH1B*2 have been shown to reduce risk for FAS in a mixed ancestry South African population. ADH1B*3 appears to afford protection for FASD outcomes in African-American populations. Other candidate genes should be examined with respect to FASD risk, including those for the enzymes of serotonin metabolism, in particular the serotonin transporter. By its very nature, alcohol teratogenesis is the expression of the interaction of genes with environment. The study of genetic factors in FASD falls within the new field of ecogenetics. Understanding of the array of genetic factors in FASD will be enhanced by future genetic investigations, including case-control, family association, and linkage studies.     

三种一元醇从卤水中萃取硼酸的效果比较

溶剂萃取法从卤水中提取硼酸是卤水提硼较好的方法之一,萃取剂的选择是萃取操作的关键。据已有资料报道从卤水中提取硼酸效果较好的一元醇有异辛醇（又称2-乙基己醇）、异戊醇、异丁醇。本文主要通过试验对这三种一元醇的萃硼效果作一比较,并从反应机理方面对试验结果加以解释。     

Liquid chromatographic determination of ethyl alcohol in body fluids

A high-performance liquid chromatographic technique for ethyl alcohol determination in body fluids is proposed. Ethyl alcohol is quantitatively converted into acetaldehyde-phenylhydrazone by oxidation in the presence of alcohol dehydrogenase, nicotinamide–adenine dinucleotide and phenylhydrazine. The derivative is suitable for reversed-phase liquid chromatography and ultraviolet detection at 276 nm. The limits of linearity, detection and quantification as well as accuracy and reproducibility were investigated in water, serum and whole blood. Analytical responses were linear within the 0.008 to 5 g/l range, and the limit of quantification was 0.02 g/l both in aqueous standard and in biological matrix assays. Mean analytical recovery of ethyl alcohol in blood serum averaged 98.2±4.2%, imprecision (CV%) at 0.80 g/l was 2.2%, and the limit of quantification was 0.02 g/l. Serum concentrations of persons that avoided alcoholic beverages for a week were less than the limit of quantification. Ethyl alcohol concentrations in serum and whole blood compared well with those obtained by headspace gas chromatography. This simple and reliable procedure, which was also used for a urine assay, could be suitable for validation of the screening procedures used to monitor ethanol abuse.     

Polymicrogyria in fetal alcohol syndrome

BACKGROUND: Intrauterine exposure to alcohol may result in a distinct pattern of craniofacial abnormalities and central nervous system dysfunction, designated fetal alcohol syndrome (FAS). The spectrum of malformations of the brain associated with maternal alcohol abuse during pregnancy is much broader than the relatively uniform clinical phenotype of FAS. Among these malformations the most striking abnormalities involve the impairment of neuronal cell migration. However, polymicrogyria (PMG) has so far been reported only once in a human autopsy study of a child with FAS. CASE: A 16‐year‐old girl with confirmed maternal alcohol consumption during pregnancy and full phenotype of FAS presented after two generalized epileptic seizures for neurologic assessment. Cranial magnetic resonance imaging revealed bilateral PMG in the superior frontal gyrus with asymmetric distribution. History, clinical features, and genetic investigations provided no evidence for any of the known genetic or acquired causes of PMG. Therefore, we propose that prenatal alcohol exposure is the cause of PMG in this patient rather than a mere coincidence. CONCLUSION: Our observation represents only the second patient of PMG in FAS and confirms the phenotypic variability of cerebral malformations associated with maternal alcohol abuse during pregnancy. In patients with clinical features of FAS and neurologic deficits or seizures neuroimaging is recommended. Furthermore, FAS should be considered as a differential diagnosis for PMG. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Metabolite Profiling Reveals a Role for Atypical Cinnamyl Alcohol Dehydrogenase CAD1 in the Synthesis of Coniferyl Alcohol in Tobacco Xylem

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes. Sequences of NtCAD1-1 and NtCAD1-7 were deposited in GenBank under accession numbers AY911854 and AY911855, respectively.     

Efficiency of lignin biosynthesis: a quantitative analysis

Lignin is derived mainly from three alcohol monomers: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Biochemical reactions probably responsible for synthesizing these three monomers from sucrose, and then polymerizing the monomers into lignin, were analysed to estimate the amount of sucrose required to produce a unit of lignin. Included in the calculations were amounts of respiration required to provide NADPH (from NADP(+)) and ATP (from ADP) for lignin biosynthesis. Two pathways in the middle stage of monomer biosynthesis were considered: one via tyrosine (found in monocots) and the other via phenylalanine (found in all plants). If lignin biosynthesis proceeds with high efficiency via tyrosine, 76.9, 70.4 and 64.3 % of the carbon in sucrose can be retained in the fraction of lignin derived from p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, respectively. The corresponding carbon retention values for lignin biosynthesis via phenylalanine are less, at 73.2, 65.7 and 60.7 %, respectively. Energy (i.e. heat of combustion) retention during lignin biosynthesis via tyrosine could be as high as 81.6, 74.5 and 67.8 % for lignin derived from p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, respectively, with the corresponding potential energy retention values for lignin biosynthesis via phenylalanine being less, at 77.7, 69.5 and 63.9 %, respectively. Whether maximum efficiency occurs in situ is unclear, but these values are targets that can be considered in: (1) plant breeding programmes aimed at maximizing carbon or energy retention from photosynthate; (2) analyses of (minimum) metabolic costs of responding to environmental change or pest attack involving increased lignin biosynthesis; (3) understanding costs of lignification in older tissues; and (4) interpreting carbon balance measurements of organs and plants with large lignin concentrations.     

Cyclopropanol in the exploration of bacterial alcohol oxidation

Abstract Cyclopropanol selectively inhibits bacterial alcohol oxidation proceeding via NAD-independent, quinoprotein alcohol dehydrogenases. Thus, for instance, alcohol oxidation by Pseudomonas aeruginosa , grown on ethanol, was inhibited for about 50% by cyclopropanol treatment. Accordingly, cell-free extracts of untreated cells had nearly equal activities of quinoprotein and NAD-dependent alcohol dehydrogenases, whereas only the latter enzyme activity was found in cell-free extracts of cyclopropanol-treated cells. Upon incubation of Hyphomicrobium X with cyclopropanol, oxidation of alcohols was blocked while formaldehyde oxidation was not. Therefore, methanol dehydrogenase in this organism is not specifically involved in formaldehyde oxidation. The examples show that cyclopropanol-derived substrates are potential tools in revealing the physiological role of bacterial alcohol dehydrogenases.     

Evidence for a novel biosynthetic pathway that regulates the ratio of syringyl to guaiacyl residues in lignin in the differentiating xylem of Magnolia kobus DC

The biosynthetic pathways to monolignols in Magnolia kobus were investigated by feeding stems with a deuterium-labeled precursor. Pentadeutero [γ,γ-²H₂, OC²H₃] coniferyl alcohol was supplied to shoots of Magnolia kobus and the incorporation of the labeled precursor into lignin was traced by gas chromatography-mass spectrometry. In addition to the direct incorporation of the labeled precursor into guaiacyl units, we detected a significant amount of pentadeuterium-labeled syringyl units with two γ-deuterium atoms. The relative level of trideuterium-labeled syringyl monomers (the result of conversion via the cinnamic acid pathway, in which two γ-deuterium atoms are removed during enzymatic re-oxidation) was negligible. Our results provide conclusive evidence for a novel alternative pathway for generation of lignin subunits at the monolignol stage and they suggest that this new pathway might be important for regulation of the composition of lignin. Received: 21 August 1998 / Accepted: 30 September 1998     

Methylation Analysis of Fractions from the Leuconostoc mesenteroides NRRL B-1299 Dextran

Methylation analysis of five fractions of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 has shown that each fraction was a highly branched dextran with the branches being joined mainly through C-2. Detection of a small amount of 4-O-mono-methyl-d-glucose has suggested that parts of the d-glucose residues were doubly branched at both C-2 and C-3. Detection of a larger amount of 2,4,6-tri-O-methyl-d-glucose in the hydrolyzates of the methylated products of the borate insoluble fractions has shown a greater percentage of linear α-1,3-linked d-glucose residues in these fractions. It is suggested that the solubility of the dextran is closely related to the content of linear α-1,3-linked d-glucose residues.     

The effects of alcohol on fetal development

Prenatal exposure to alcohol has profound effects on many aspects of fetal development. Although alterations of somatic growth and specific minor malformations of facial structure are most characteristic, the effects of alcohol on brain development are most significant in that they lead to substantial problems with neurobehavioral development. Since the initial recognition of the fetal alcohol syndrome (FAS), a number of important observations have been made from studies involving both humans and animals. Of particular importance, a number of maternal risk factors have been identified, which may well be of relevance relative to the development of strategies for prevention of the FAS as well as intervention for those who have been affected. These include maternal age >30 years, ethnic group, lower socioeconomic status, having had a previously affected child, maternal under-nutrition, and genetic background. The purpose of this review is to discuss these issues as well as to set forth a number of questions that have not adequately been addressed relative to alcohol's effect on fetal development. Of particular importance is the critical need to identify the full spectrum of structural defects associated with the prenatal effects of alcohol as well as to establish a neurobehavioral phenotype. Appreciation of both of these issues is necessary to understand the full impact of alcohol on fetal development.     

Cofactor Recycling for Selective Enzymatic Biotransformation of Cinnamaldehyde to Cinnamyl Alcohol

The enzymatic, selective hydrogenation of cinnamaldehyde to cinnamyl alcohol is reported here. Yeast alcohol dehydrogenase was used in a substrate-coupled process with cofactor recycling. Both 100% selectivity and aldehyde conversion were achieved within 3 h. The reaction took place under very mild conditions, in the absence of toxic organic solvent. The overall process proved inexpensive and deserves further optimization studies in order to evaluate industrial applications.     

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca²⁺]_i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca²⁺ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca²⁺]_i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca²⁺]_i was observed in Ca²⁺‐free media, suggesting intracellular Ca²⁺ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP₃R), which regulates Ca²⁺ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca²⁺]_i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca²⁺‐chelator, also inhibited the alcohol‐induced surge in [Ca²⁺]_i and prevented neuronal death. In conclusion, alcohol disrupts [Ca²⁺]_i homeostasis in CGN by releasing Ca²⁺ from intracellular stores, resulting in a sustained increase in [Ca²⁺]_i. This sustained increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.     

Perillyl alcohol suppresses antigen-induced immune responses in the lung

Perillyl alcohol (POH) is an isoprenoid which inhibits farnesyl transferase and geranylgeranyl transferase, key enzymes that induce conformational and functional changes in small G proteins to conduct signal production for cell proliferation. Thus, it has been tried for the treatment of cancers. However, although it affects the proliferation of immunocytes, its influence on immune responses has been examined in only a few studies. Notably, its effect on antigen-induced immune responses has not been studied. In this study, we examined whether POH suppresses Ag-induced immune responses with a mouse model of allergic airway inflammation. POH treatment of sensitized mice suppressed proliferation and cytokine production in Ag-stimulated spleen cells or CD4⁺ T cells. Further, sensitized mice received aerosolized OVA to induce allergic airway inflammation, and some mice received POH treatment. POH significantly suppressed indicators of allergic airway inflammation such as airway eosinophilia. Cytokine production in thoracic lymph nodes was also significantly suppressed. These results demonstrate that POH suppresses antigen-induced immune responses in the lung. Considering that it exists naturally, POH could be a novel preventive or therapeutic option for immunologic lung disorders such as asthma with minimal side effects.     

Caspase-3 and calpain activities after acute and repeated ethanol administration during the rat brain growth spurt

Ethanol administration during the rat brain growth spurt triggers apoptotic neurodegeneration that appears to be mediated by caspase-3 activation. In order to gain more insight on the role of this caspase in ethanol-induced developmental neurotoxicity, we studied its expression and activity under different conditions of ethanol exposure during development. Furthermore, because of the cross-talk between caspase-3 and calpain we extended our study also at this protease. Ethanol was administered by gavage to rat pups as a single-day exposure on postnatal day (PN) 7 or from PN4 to PN10. Cleaved caspase-3 expression peaked in the cerebral cortex 12 h after ethanol treatment and returned to control values at 24 h. An identical pattern was found for caspase-3-like activity, that was increased only with the highest dose of ethanol tested (5 g/kg) and mostly in PN4. Repeated ethanol exposure, at a dose that was previously found to induce microencephaly, did not increase caspase-3 expression and activity although it decreased procaspase-3 expression and released mitochondrial cytochrome c. Repeated ethanol administration also increased calpain activity. These data show that acute and repeated ethanol administration differentially affect caspase-3 and calpain activity, suggesting that calpain activation may play a role in developmental neurotoxicity of ethanol.     

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

BACKGROUND: Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. METHODS: Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. RESULTS: The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. CONCLUSIONS: Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.     

Alcohol Dehydrogenases: Identification and Names for Gene Families

Plant gene products that have been described as `alcohol dehydrogenases' are surveyed and related to their CPGN nomenclature. Most are Zn-dependent medium chain dehydrogenases, including `classical' alcohol dehydrogenase (Adh1), glutathione-dependent formaldehyde dehydrogenase (Fdh1), cinnamyl alcohol dehydrogenase (Cad2), and benzyl alcohol dehydrogenase (Bad1). Plant gene products belonging to the short-chain dehydrogenase class should not be called alcohol dehydrogenases unless such activity is shown.     

Alcohol metabolism in Drosophila melanogaster: Uselessness of the most active aldehyde oxidase produced by the Aldox locus

Alcohol dehydrogenase is necessary for ethanol detoxification and metabolic utilization. It has been generally assumed that aldehyde oxidase (AO) produced by the Aldox locus (3–56.7) is necessary for a further transformation of acetaldehyde into acetate. We find that various mutant strains (ma-l or Aldox ⁿ) which do not produce an active enzyme show about the same tolerance to alcohol as do wild strains. This physiological paradox is probably to be explained by the discovery of another locus (not localized) which produced a small amount of AO in all tested strains. The adaptive significance of the genetically polymorphic Aldox locus is probably to be looked for in physiological pathways other than ethanol metabolism.