Spatial and Temporal Pattern Analyses of Esca Grapevine Disease in Vineyards in France.

To assess the capacity of esca to spread within vineyards of the Bordeaux region, over 8 years of annual records, containing between 1,200 and 2,300 contiguous Cabernet Sauvignon vines from 15 mature vineyards, were used for spatial statistical analyses. A group of nonparametric tests, based on join count statistics and on permutation methods, was developed to characterize the spatial structure of esca-symptomatic vines in terms of spread in any direction or within-row only. Among vineyards, a large range of spatial patterns, from random to strongly structured, associated with various prevalence rates that increased over time were observed. In four vineyards, the complex esca distribution pattern indicated different levels of clustering. By contrast, in other vineyards, only small clusters of two adjacent symptomatic vines were observed, and they were localized along rows, without enlargement over time, except in one vineyard. An analysis of spatial dependence between previously and newly symptomatic vines within k-order neighborhoods (k = 1 to 5), showed, for 5 of the 15 vineyards, that the newly symptomatic vines were located close to previously infected vines, without a favored orientation or neighbor order. All the results together suggested a limited potential for secondary local spread from neighboring symptomatic vines.

Requirement for omega and (omega;-1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11.

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of omega and (omega;-1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A. Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the alkyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were omega and (omega;-1)-hydroxylated with an efficiency at least similar to palmitic acid. The (omega;-1)/omega ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly omega-hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for omega and (omega;-1)-hydroxylation inside the access channel of active site.

Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of omega- and (omega-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes.

In order to characterize the nature of the active site of cytochrome P450 2E1, the metabolism of various fatty acids with cis/trans geometric configurations has been investigated. A system coupling atmospheric pressure chemical ionization-mass spectrometry detection with HPLC separation was developed as an alternative method for the characterization of hydroxylated metabolites of oleic and elaidic acids in rat and human liver microsomes. Oxidation of oleic and elaidic acids led to the formation of two main metabolites which were identified by LC-MS and GC-MS as omega and (omega-1)-hydroxylated (or 17-OH and 18-OH) fatty acids, on the basis of their pseudo-molecular mass and their fragmentation. The assay was accurate and reproducible, with a detection limit of 25 ng per injection, a linear range from 25 to 1128 ng per injection, no recorded interference, intra-day and inter-day precision with variation coefficients <14%. This LC-MS method was validated with oleic acid by using both radiometric and mass spectrometric detections. A significant correlation was found between the two methods in human (r=0.86 and 0.94 with P<0.05 and 0.01) and rat liver microsomes (r=0.90 and 0.85 with P<0.01 and 0.05) for 17-OH and 18-OH metabolites, respectively. HPLC coupled to mass spectrometry for the analysis of hydroxylated metabolites of elaidic acid offers considerable advantages since the method does not require use of a radioactive molecule, completely separates the two hydroxymetabolites, confirms the identification of each metabolite, and is as sensitive as the radiometric analysis method. This method allowed the comparative study of oleic and elaidic acid hydroxylations by both human and rat liver microsomal preparations.

Involvement of cytochrome P450 2E1 in the (omega-1)-hydroxylation of oleic acid in human and rat liver microsomes.

In vitro techniques have been used to investigate the nature of microsomal cytochrome P450 involved in the metabolism of oleic acid, a physiological monounsaturated fatty acid. Like lauric acid, which is currently used as a model substrate of fatty acid metabolism, the alkyl chain of oleic acid is hydroxylated on its omega and (omega-1) carbons. The identity of these hydroxylated metabolites was ascertained by GC/MS and LC/MS. The omega/omega-1 ratio of oleic acid metabolites (1.22+/-0.01) was found to be similar to that obtained with lauric acid in rat liver microsomes (1.10+/-0.02), while in human liver microsomes this ratio was 0.75+/-0.5 for lauric acid and 5.2+/-2.6 for oleic acid. After treatment of rats with ethanol or clofibrate, inducers of CYP2E1 and CYP4A, respectively, the hydroxylations of oleic acid were shown to be less inducible than those of lauric acid. Five in vitro approaches were used to identify the P450 isoform(s) responsible for the microsomal (omega-1)-hydroxylation of oleic acid: effect of various inducers in rats, correlation studies between specific P450 catalytic activities in a panel of 25 human liver microsomes, chemical inhibitions, immuno-inhibitions and metabolism by cDNA-expressed human P450 enzymes. From the above results, it can be ascertained that P450 2E1 is the main enzyme involved in the (omega-1)-hydroxylation of oleic acid. Furthermore, the omega-hydroxylation of oleic acid was shown to be mainly catalyzed by P450 4A enzymes in human liver microsomes. The turnover number of (omega-1)-hydroxylation of lauric and oleic acids decreased from 7.8 to 1.5 min(-1), respectively, suggesting that the dodecane alkyl chain allows optimal binding to the active site of CYP2E1.

Effect of pyrazole and dexamethasone administration on cytochrome P450 2E1 and 3A isoforms in rat liver and kidney: lack of specificity of p-nitrophenol as a substrate of P450 2E1.

The induction effects of pyrazole and dexamethasone (known to be specific to P450 2E1 and 3A enzymes, respectively), given alone or simultaneously, were studied in rat liver and kidney microsomes. Pyrazole treatment induced the catalytic activity and the amount of P450 2E1 enzyme in both organs. Immunoreactive P450 2E1 and 4-nitrophenol 2-hydroxylation increased 8- and 13-fold, respectively (versus control), in the kidney, but only 2.4- and 2.7-fold (versus control) in the liver after pyrazole treatment. As assessed by nifedipine oxidation activity, dexamethasone treatment increased the P450 3A catalytic activity approximately 4-fold (versus control) in the liver, but not in the kidney, suggesting that P450 3A was not inducible in the kidney. Pyrazole decreased P450 3A activity in the liver but did not modify it in the kidney. A combination of both chemicals induced both enzymes, but to a lesser extent than treatment with each single chemical compound. Furthermore, the 2-hydroxylation of p-nitrophenol, considered one of the most specific substrates for monitoring the level of P450 2E1, was mediated also by P450 3A, at least in dexamethasone-treated rats. Finally, this experimental work demonstrated that P450 3A induction is organ-specific, and it also demonstrated the lack of specificity of p-nitrophenol as a P450 2E1 substrate.

In vitro interactions between fluoxetine or fluvoxamine and methadone or buprenorphine.

Methadone and buprenorphine, widely used in the treatment of opioid abuse, are metabolized by cytochrome P450 3A4, while fluoxetine and fluvoxamine, both selective serotonin reuptake inhibitors, are known to be P450 2D6 and 3A4 inhibitors in vitro. This study deals with the in vitro interactions between methadone or buprenorphine and fluoxetine or fluvoxamine. Fluoxetine inhibited methadone N-demethylation (Ki = 55 microM), but conversely did not inhibit buprenorphine dealkylation. Norfluoxetine inhibited the metabolism of both methadone and buprenorphine metabolisms (Ki 13 and 100 microM, respectively). Fluvoxamine inhibited methadone N-demethylation with a Ki of 7 microM and buprenorphine dealkylation, uncompetitively, with a Ki of 260 microM. Finally, these results suggest that care should be taken when selective serotonin reuptake inhibitors are administered in the treatment of drug craving. This is particularly true in the case of fluvoxamine which is more potent than fluoxetine in inhibiting methadone and buprenorphine metabolism.

Inhibition of methadone and buprenorphine N-dealkylations by three HIV-1 protease inhibitors.

Ritonavir, indinavir, and saquinavir, all human immunodeficiency virus-1 protease inhibitors with a potent antiviral effect during triple therapy, are extensively metabolized by liver cytochrome P450 3A4. As this P450 isoform is involved in the metabolism of about 50% of drugs, coadministration of protease inhibitors with other drugs may lead to serious effects due to enzyme inhibition. Among these drugs, methadone and buprenorphine, both metabolized by P450 3A4, are potential candidates to drug interactions. In this study, metabolic interactions between these protease inhibitors and methadone or buprenorphine were studied in vitro in a panel of 13 human liver microsomes. Ritonavir was the most potent competitive inhibitor with Ki about 50 and 20 nM for methadone and buprenorphine metabolisms, respectively. Indinavir and saquinavir also inhibited methadone N-demethylation (Ki about 3 and 15 microM, respectively) and buprenorphine N-dealkylation (Ki about 0.8 and 7 microM, respectively). The rank order of inhibition potency against metabolism of methadone and buprenorphine was ritonavir > indinavir > saquinavir. There is obvious potential for clinically significant drug interactions, particularly with ritonavir. In brief, caution should be advised if human immunodeficiency virus-1 protease inhibitors are coadministered with methadone and buprenorphine.

Involvement of cytochrome P450 3A4 in N-dealkylation of buprenorphine in human liver microsomes.

Buprenorphine is a long acting analgesic of the opiate family. Recently, it has been proposed for the opioid dependency treatment at a large scale. The drug is extensively metabolized by the hepatic cytochrome P450 in man, yielding a N-dealkylated metabolite, norbuprenorphine. The specific forms of P450 involved in this oxidative N-demethylation were examined in a panel of 18 human liver microsomal preparations previously characterized with respect to their P450 contents. Buprenorphine was N-dealkylated with an apparent Km of 89 +/- 45 microM (n = 3). The metabolic rates were 3.46 +/- 0.43 nmol/(min x mg of protein). This metabolic pathway was strongly correlated with 6 catalytic activities specific to P450 3A4 and with the immunodetectable P450 3A content of liver microsomal samples (r = 0.87). Buprenorphine metabolism was 62-71% inhibited by three mechanism-based inhibitors (TAO, erythralosamine, gestodene), by nifedipine as competitive inhibitor (Ki = 129 microM) and by ketoconazole 0.6 microM (25% residual activity), all these inhibitors specific to P450 3A. Among 10 heterologously expressed P450s tested, only P450 3A4 was able to dealkylate buprenorphine with a turnover number of 9.6 min(-1). Morever, this catalytic activity was inhibited up to 80% (vs control) by anti-rat P450 3A antibody. Taken together, all these data demonstrate that P450 3A4 is the major enzyme involved in hepatic buprenorphine N-dealkylation.

Both cytochromes P450 2E1 and 3A are involved in the O-hydroxylation of p-nitrophenol, a catalytic activity known to be specific for P450 2E1.

4-Nitrophenol 2-hydroxylation activity was previously shown to be mainly catalyzed by P450 2E1 in animal species and humans. As this chemical compound is widely used as an in vitro probe for P450 2E1, this study was carried out to test its catalytic specificity. First, experiments were carried out on liver microsomes and hepatocyte cultures of rat treated with different inducers. Liver microsomes from pyrazole- and dexamethasone-treated rats hydroxylated p-nitrophenol with a metabolic rate increased by 2.5- and 2.7-fold vs control. Dexamethasone treatment increased the hepatic content of P450 3A but not that of P450 2E1. Two specific inhibitors of P450 3A catalytic activities, namely, ketoconazole and troleandomycin (TAO), inhibited up to 50% of 4-nitrophenol hydroxylation in dexamethasone-treated rats but not in controls. Hepatocyte cultures from dexamethasone-treated rats transformed p-nitrophenol into 4-nitrocatechol 7.8 times more than controls. This catalytic activity was inhibited by TAO. Similarly, hepatocyte cultures from pyrazole-treated rats hydroxylated p-nitrophenol with a metabolic ratio increased by about 8-fold vs control. This reaction was inhibited by diethyl dithiocarbamate and dimethyl sulfoxide, both inhibitors of P450 2E1. Second, the capability of human P450s other than P450 2E1 to catalyze the formation of 4-nitrocatechol was examined in a panel of 13 human liver microsomes. Diethyl dithiocarbamate and ketoconazole reduced 4-nitrophenol hydroxylase activity by 77% (+/- 11) and 13% (+/- 16), respectively. Furthermore, the residual activity following diethyl dithiocarbamate inhibition was significantly correlated with seven P450 3A4 catalytic activities. Finally, the use of human cell lines genetically engineered for expression of human P450s demonstrated that P450 2E1 and 3A4 hydroxylated 4-nitrophenol with turnovers of 19.5 and 1.65 min-1, respectively. In conclusion, P450 3A may make a significant contribution to 4-nitrophenol hydroxylase activity in man and rat.

Involvement of cytochrome P450 3A4 enzyme in the N-demethylation of methadone in human liver microsomes.

Methadone has become one of the most widely used drugs for opiate dependency treatment. This drug is extensively metabolized by the cytochrome P450 hepatic enzyme family in man, yielding an N-demethylated metabolite that cyclizes spontaneously into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. The specific forms of cytochrome P450 involved in this oxidative N-demethylation were examined in a panel of 20 human liver microsomal preparations previously characterized with respect to their P450 enzyme contents. Methadone was demethylated with an apparent Km of 545 +/- 258 microM (n = 3). The metabolic rates were 745 +/- 574 pmol/(min.mg of protein). This metabolic pathway was strongly correlated with estradiol 2-hydroxylation, testosterone 6 beta-hydroxylation, nifedipine oxidation, erythromycin N-demethylation, and toremifene N-demethylation, all of these monooxygenase activities being supported by P450 3A4. Furthermore, the total P450 3A content of liver microsomal samples, determined by immuno-quantification using a monoclonal anti-human P450 3A4 antibody, was correlated with methadone demethylation (r = 0.72; p < 0.003). Methadone metabolism was 60-72% inhibited either by three mechanism-based inhibitors of P450 3A4 (gestodene, TAO, and erythralosamine) or by four reversible inhibitors of P450 3A (ketoconazole, dihydroergotamine, quercetin, and diazepam with an apparent Ki of 50 microM) and by two nonspecific inhibitors (metyrapone and SKF-525A). Conversely, quinidine (inhibitor of P450 2D6), 7,8-benzoflavone (inhibitor of P450 1A), or sulfaphenazole (inhibitor of P450 2C) did not significantly inhibit, and may even have activated, methadone metabolism. Four heterologously expressed P450 proteins were able to catalyze the N-demethylation of methadone, namely, P450 2C8, P450 2C18, P450 2D6, and P450 3A4. However, referring to their relative liver content, it can be asserted that P450 3A4 is the major enzyme involved in the N-demethylation of methadone on average. Accordingly, caution should be advised in the clinical use of methadone when other drugs are also administered that induce or inhibit P450 3A4, such as rifampicin or diazepam, respectively.

Comparison of caffeine metabolism by slices, microsomes and hepatocyte cultures from adult human liver.

1. Caffeine was used as a molecular probe for hepatic monooxygenase activity in three in vitro models from human livers: slices, microsomes and hepatocyte cultures. 2. A h.p.l.c. method for determination of all possible metabolites of caffeine (16 compounds) is described. 3. Caffeine biotransformation by these three in vitro systems was low. However, metabolite formation was shown to proceed at a rate close to that calculated from in vivo caffeine elimination half-life. 4. Metabolic profiles were the same whatever the in vitro system used. The ratio of primary demethylated metabolites was similar to that measured by in vivo studies, i.e. the selectivity for caffeine N-3 demethylation to paraxanthine was retained. 5. Significant correlation (p less than 0.001) between 7-ethoxyresorufin O-deethylation and caffeine demethylations was demonstrated for the 12 human livers studied.

A program for parenteral and combined parenteral and enteral nutrition of neonates and children in an intensive care unit.

A computer program for parenteral and combined parenteral and enteral nutrition of children in intensive care unit is described. It acts as a guide for the prescribing physician by using informative messages, food composition tables and alarms when mistakes or uncommon prescription values are input from the keyboard.

Determination of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol in human plasma by selected ion monitoring.

5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) were measured in human peripheral plasma by gas chromatography-selected ion monitoring as their t-butyl dimethylsilyl ethers. The purification of the plasma extract involved a Sep-Pak C18 cartridge and high-performance liquid chromatography. The concentrations (mean +/- SD) of the two diols in four normal males are: 213 +/- 111 pg/ml for the 3 alpha-diol and 246 +/- 133 pg/ml for the 3 beta-diol.

Testosterone metabolism in the uropygial gland of the quail.

The metabolism of testosterone in the uropygial gland of the quail principally results in the production of 17 alpha, 5 beta derivatives. Moreover, an unusually small amount of testosterone is converted to 5 alpha-dihydrotestosterone. These results question the role played by intracellular 5 alpha-reduction in the response of the gland to testosterone stimulation.

Use of chemical ionization in gas chromatographic/mass spectrometric screening of human urine for disaccharides containing inositol.

Gas chromatography/mass spectrometry (GC/MS) in the positive chemical ionization (CI) mode was used to screen normal urine for inositol-containing disaccharides in the form of permethylated derivatives, after borodeuteride reduction of the reducing saccharides. Ammonia was the reactant gas. The results revealed the existence of deoxyhexosyl-inositol and hexosyl-inositol disaccharides, and of a new compound, N-acetylhexosaminyl-inositol disaccharide. Up to four isomers of deoxyhexosyl-inositol could be present in the same sample even though only one of them has so far been fully characterized in man. As regards the hexosyl-inositols, one to three isomers were present in the same sample and probably corresponded to the three isomers of galactosyl-inositol recently described in man. N-Acetylhexosaminyl-inositol (identified elsewhere by us as N-acetylgalactosaminyl-alpha (1-1)-myo-inositol) was seen in only a few samples. No relationship can be found between the excretion of all these inositol-containing disaccharides on the one hand, ABO(H) blood group and 'Secretor' status (Se or sese) of the donors on the other. The gas chromatographic CI mass spectrometric technique used here with various ammonia pressures can be applied to the screening of other biological fluids or tissues for inositol glycosides.

Glycosylated haemoglobin: high-performance liquid chromatographic determination of 5-(hydroxymethyl)-2-furfuraldehyde after haemoglobin hydrolysis.

A specific and accurate method for the quantitation of the azomethine linkage present in non-enzymatically glycosylated haemoglobin is described. This protein is hydrolysed for 5 h in 1 M oxalic acid at 100 degrees C to yield 5-(hydroxymethyl)-2-furfur-aldehyde (5-HMF), known as a specific degradation product of hexoses linked to the protein. 5-HMF is then purified through a Sep-Pak C18 cartridge and measured by its absorption at 280 nm after separation on a C18 reversed-phase silica column. Quantitation is made accurate by using 1-methylxanthine as internal standard throughout the whole procedure. The identity and the purity of the 5-HMF chromatographic peak was ascertained by UV spectroscopy, gas chromatography on a glass capillary column and mass spectrometry. The method has been successfully used for 5-HMF determinations in monitoring diabetes mellitus patients. The mean values, expressed as nmol of 5-HMF per mg of haemoglobin were 0.64 +/- 0.13 (S.D.) for 27 controls and 1.32 +/- 0.39 for 78 diabetic patients. Unlike the usually employed thiobarbituric acid assay, the present procedure is truly specific for the 5-HMF determination.

Transformations of zearalenone and alpha-zearalanol by homogenates of human prostate glands.

The present study applied high-pressure liquid chromatography, gas chromatography/mass spectrometry and crystallizations to constant specific activity for identification of the radiometabolites of tritiated zearalenone and alpha-zearalanol in homogenates of human prostate glands. Radiolabelled zearalenone or alpha-zearalanol (6.3 microM) was incubated for 30 min at 37 degrees C with prostatic homogenate in 5 ml 0.067 M phosphate buffer (pH 7.4) containing 0.5 mM oxidized or reduced NAD or NADP. Reduction of zearalenone occurred only with NADPH while oxidation of alpha-zearalanol was favoured both by NADP+ and NAD+. Separation of the substrates from their metabolites by high-pressure liquid chromatography showed the radioactivity of the metabolite of zearalenone to be associated with the alpha-zearalenol carrier while that of the alpha-zearalanol metabolite was found with the zearalanone carrier. Crystallizations to constant specific activity after isotopic dilution and gas chromatography/mass spectrometry confirmed the identity and the radiochemical purity of the alpha-zearalanol substrate and of its zearalanone metabolite. In contrast, the poor radiochemical purity of the zearalenone substrate and of its alpha-zearalenol metabolite was explained by the presence of large quantities of cis-isomer contaminants which were identified by gas chromatography/mass spectrometry. Formation of the cis-isomer of zearalenone was shown to occur spontaneously in incubations with boiled homogenates. The other stereospecific reductions or oxidations of the substrates were due to the prostatic enzymes which could be those involved in the C19-steroid hormone metabolism.

[Dosage of the pentanoic, propyl 2 acid (valproic acid). Utilization of esters of heavy alcohols (author's transl)]. / Dosage de l'acide propyl-2, pentanoïque (acide valproïque). Utilisation d'esters d'alcools lourds.

The authors describe a method of dosage of valproic acid by gas chromatography. The derivation consists of the synthesis of esters of the valproic acid using heavy alcohols. This method of derivation can be applied to other carboxylic acids.