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P E Reilly

Publications and source records attributed to P E Reilly.

At least 19 recordsLinked to original sources
PubMed1992

Fluorimetric detection of serum corticosterone using high-performance liquid chromatography.

A simple, sensitive, specific and reproducible method for the determination of corticosterone concentrations in rat serum using high-performance liquid chromatography (HPLC) with fluorimetric detection is described. Corticosterone is detectable down to 0.1 ng injected onto the HPLC column. Cortisol is used as an internal standard. Ethyl acetate was used for both initial serum corticosteroid extraction and the subsequent fluorophore extraction after sulfuric acid hydrolysis; thus sulfuric acid does not enter the HPLC system. The resultant fluorophores for both corticosterone and cortisol are stable for at least two weeks at ambient temperature not requiring storage at -20 degrees C. The procedure is highly suitable for use with HPLC systems utilising automatic sample injectors. The method is specific for corticosterone; dexamethasone, cortisone and gonadal steroids are not detectable and do not interfere in this assay.

Animals↗
PubMed1992

Comparative metabolism of clinically important precursors of N-desmethyldiazepam using phenobarbitone-pretreated rat liver microsomes.

Phenobarbitone-pretreated male Sprague-Dawley rat liver microsomes were used to examine C3-hydroxylation and N-dealkylation of four clinically important benzodiazepines: diazepam (DZP), prazepam (PZP), pinazepam (PIN) and halazepam (HZP). These substrates differ only in the nature of the N-substituent of the B ring and N-desmethyldiazepam (DMD) is the N-dealkylation product in each case. C3-Hydroxylation was accordingly also studied with DMD as substrate. All monooxygenations were studied with substrates at a concentration of 10 microM, in the absence of solubilizing agents, and under conditions where the production of secondary metabolites was minimized. A 20-fold variation in the rate of C3-hydroxylation was recorded across the five substrates with HZP showing the highest rate and DMD showing the lowest rate. An almost equally large range of variation was shown for the N-dealkylation reaction, with PZP undergoing this biotransformation more than 17 times faster than DZP. Log P values (a measure of lipophilicity) for the five substrates were determined using an HPLC method and a remarkable lack of correspondence between this substrate parameter and either of the monooxygenations was noted. This suggests that multiple substrate determinants govern the relative rates of these monooxygenations. It was, however, notable that the additive rate of metabolism of these substrates by both monooxygenase routes did show an excellent correlation with substrate lipophilicity.

Animals↗
PubMed1992

Benzodiazepine metabolism in ethanol-treated male rats: use of pair-fed and age-matched controls.

The effects of chronic moderate (15%) ethanol consumption and ageing on rat hepatic cytochrome P450 monooxygenase activities were examined using diazepam, nordazepam, d-benzphetamine, erythromycin, ethylmorphine and nitrosodimethylamine (NDMA) as substrates. In addition, the effects of moderate ethanol alone on the oxidation of metoprolol, morphine and temazepam were examined. Cytochrome P450 specific content increased significantly only in the 6-week ethanol-treated rats, and no changes in percentage liver to body weights were apparent in any of the ethanol-treated animals compared with pair-fed controls. Only cytochrome P450IIIA enzyme activities displayed age-related decreases, these being identified in the pair-fed animals. C3-hydroxylation of diazepam and nordazepam (36% of controls) and N-demethylation of erythromycin and ethylmorphine (58% and 64% of controls) were decreased in 6-week ethanol-treated animals, these effects being less pronounced in the 12, 24 and 48-week ethanol-treated groups. The decrease seen for diazepam and d-benzphetamine N-demethylation caused by ethanol consumption was approximately 80% of control groups for the duration of the treatment. NDMA and morphine N-demethylations were increased to 120% of control activities and metoprolol alpha-hydroxylase was increased to 140% of control activities at 6 weeks, whilst metoprolol O-demethylase activity remained unaltered. NDMA N-demethylase activity showed a two-fold induction at 24 and 48 weeks of ethanol treatment, compared with corresponding pair-fed control groups. These results support previous findings from this laboratory showing that the same or similar P450IIIA family isozymes are involved in the C3-hydroxylation of diazepam and nordazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1991

Effects of ethinylestradiol and testosterone implants on hepatic microsomal cytochrome P450 monooxygenases of birth gonadectomized male and female Dark Agouti rats.

Monooxygenases in the cytochrome P450 IIIA subfamily are induced by a number of their xenobiotic substrates and by testosterone, an endobiotic substrate of importance in their regulation. 17 alpha-Ethinylestradiol (EE) is also metabolized by these enzymes and in this study Dark Agouti rats were used to examine the effects of subcutaneous implantation of controlled release silastic capsules containing EE to determine if this steroid also induces these enzymes. Data were compared with results obtained from equivalent groups of animals implanted with capsules containing testosterone propionate (TP). Liver microsomes prepared from male and female rats were used to identify intrinsic gender differences in the monooxygenases studied and gender differences in the responses to the implanted steroids were also determined. Effects due to imprinting of growth hormone secretion patterns were controlled by using male and female birth gonadectomized animals. Results obtained from groups with blank implants showed there were no effects due to the silastic implant material itself on the monooxygenases studied. The specific activities of erythromycin N-demethylation in liver microsomes of both EE and TP implanted male and female birth gonadectomized animals were enhanced relative to corresponding blank implanted controls consistent with both steroids having an effect to induce activity attributable to cytochrome P450 IIIA isoforms. Immunoinhibition studies using microsomes from EE treated female rats with erythromycin as substrate provided further evidence for this steroid having this induction effect. The specific activity of ethylmorphine N-demethylation was however not increased in microsomes prepared from the EE implanted female animals and was decreased in the corresponding male preparations. These findings distinguished the response to this steroid from that to TP and suggested induction by this estrogen of an isoform(s) having a more limited range of substrates than has characteristically been found in this subfamily. EE treatment also caused an increase in diazepam C3 hydroxylase consistent with an effect to induce P450 IIIA activity but this was found only in microsomes from birth gonadectomized female animals. This was in contrast to the effect of TP treatment which produced increases in this monooxygenase in both male and female animals. Another gender specific effect of EE was a striking decrease in morphine N-demethylase activity seen only in birth gonadectomized male rats. This again contrasted with the effect of TP which caused a marked increase in this activity in liver microsomes of both male and female birth gonadectomized animals consistent with the proposal that testosterone is important in the regulation of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Cimetidine: an inhibitor and an inducer of rat liver microsomal cytochrome P-450.

1. Cimetidine pretreatment of male Sprague-Dawley rats caused a significant increase in the specific content of total hepatic cytochrome P-450, supporting the hypothesis that this H2-receptor antagonist has monooxygenase induction effects. 2. Quantitative ultrastructural studies of liver of cimetidine-pretreated animals also supported this hypothesis in showing a significant proliferation of smooth endoplasmic reticulum. These ultrastructural changes were qualitatively similar to those produced by treatment of rats with phenobarbital, a well-characterized monooxygenase-inducing agent whose effects were studied for comparative purposes. 3. Competitive inhibition of metoprolol alpha-hydroxylation by cimetidine in liver microsomes prepared from untreated animals (Ki = 18.8 microM) was also demonstrated. 4. These results allowed testing of the hypothesis (Burnet et al. 1986) that inhibition of a defined monooxygenase should lead to induction of the synthesis of the relevant cytochrome P-450 isozyme. 5. The finding that metoprolol alpha-hydroxylase activity of liver microsomes was lowered, not elevated, by pretreatment of animals with cimetidine argues against the concept of a causal link between monooxygenase inhibition and induction.

Animals↗
PubMed1990

Cytochrome P450IIIA enzymes in rat liver microsomes: involvement in C3-hydroxylation of diazepam and nordazepam but not N-dealkylation of diazepam and temazepam.

Microsomes prepared from livers of male and female rats of nine inbred and outbred strains and of male Sprague-Dawley rats pretreated with monooxygenase-inducing agents were used to study N-dealkylation of diazepam and temazepam and C3-hydroxylation of diazepam and nordazepam. Both C3-hydroxylation reactions were more rapid in male than in female liver preparations, but this gender-dependent pattern was not seen with the N-dealkylation reactions. These results indicate the lack of identity of the monooxygenases responsible for the two kinds of reaction and suggest that male-specific enzyme(s) are responsible for the C3-hydroxylations. Induction studies were undertaken to further define these enzymes. To do this, liver microsomes prepared from male Sprague-Dawley rats pretreated with a variety of agents known to have different monooxygenase induction effects were used. With triacetyloleandomycin, dexamethasone, and phenobarbital pretreatment, the specific activities of the C3-hydroxylation reactions were selectively elevated over corresponding control values. These particular xenobiotics are known to enhance the abundance of cytochrome P450IIIA family enzymes, and our results strongly suggest the involvement of these enzymes in the benzodiazepine B ring monooxygenations. Formation of temazepam was also shown to be inhibited by triacetyloleandomycin. This effect was demonstrated to be equal in both saline-treated and dexamethasone-treated male Sprague-Dawley rat liver microsomes, with the antibiotic present either with diazepam throughout the entire incubation period or initially with NADPH in a preincubation mix for 15 min, following which C3-hydroxylation was initiated by the addition of diazepam. These results confirm the uniformity of the involvement of cytochrome P450IIIA family enzymes in diazepam C3-hydroxylation in untreated and inducer-treated rat liver microsomes. Recent studies with human and rabbit liver microsomal preparations have shown that orthologues of these enzymes also catalyze an equivalent hydroxylation in the B ring of midazolam. These findings, considered with the present results showing that the adjacent methyl N-substituent (absent in nordazepam but present in diazepam) did not affect the selectivity of these enzymes for the C3-hydroxylation reaction, suggest that neither steric nor electronic factors markedly influence catalysis of this monooxygenation by these enzymes.

Animals↗
PubMed1989

Massive caffeine ingestion resulting in death.

Caffeine has long been recognized as an addictive substance with numerous toxic effects. Death due to caffeine overdose is rare, due in part to its marked gastric irritation resulting in spontaneous emesis. Caffeine can be found in food, beverages, analgesics, and over-the-counter weight loss products. We report a fatality resulting from the ingestion of mail order diet pills containing caffeine as their only active ingredient. A 22-year-old female arrived in the emergency department in cardiac arrest after ingesting these diet pills. Resuscitation efforts were unsuccessful. A serum toxicology screen revealed the presence of caffeine with a level of 1,560 micrograms/ml (lethal greater than 100 micrograms/ml). To our knowledge this is the highest reported fatal caffeine level. No other drugs were identified on the screen. Based on the absence of pathology on autopsy and the high caffeine level, death was attributed to acute caffeine toxicity. This case illustrates that deaths resulting from caffeine, although rare, continue to occur.

Adult↗
PubMed1988

Differential inhibition of human liver phenacetin O-deethylation by histamine and four histamine H2-receptor antagonists.

1. The effects of histamine and four histamine H-2 receptor antagonists on phenacetin O-deethylation by microsomal preparations of four human livers was quantified by a radiometric-thin layer chromatographic method. 2. Histamine and three of these drugs, namely cimetidine, ranitidine and famotidine, were weak inhibitors of this cytochrome P-450-catalysed O-deethylation, but mifentidine was a potent competitive inhibitor with a Ki in the range 40-70 microM. 3. Cimetidine, histamine and mifentidine are all 4(5)-substituted imidazole derivatives, and the contrast between the very weak inhibitory effects of cimetidine and histamine, and the more potent effect of mifentidine, suggests that the imidazole moiety may play little role in the inhibition of phenacetin O-deethylase by mifentidine. 4. The demonstration that cimetidine, ranitidine and histamine were all poor inhibitors of phenacetin oxidation further suggests the possible lack of identity between the human liver cytochrome P-450 isoenzymes responsible for catalyzing the oxidation of metoprolol and phenacetin. This follows from recognizing that metoprolol oxidation is known, from both in vivo and in vitro studies, to be strongly inhibited by both of these H-2 receptor antagonists and from in vitro studies also to be inhibited by histamine.

Binding Sites↗
PubMed1988

Phenacetin O-deethylation by human liver microsomes: kinetics and propranolol inhibition.

1. Phenacetin O-deethylation catalysed by human liver microcomes has been examined over a substrate concentration range of 2.5 to 700 microM using preparations of eight human liver samples. Michaelis-Menten kinetics described phenacetin oxidation satisfactorily in five of these samples; apparent Km values were in the range of 17.7 to 38.4 microM. 2. In the three other livers a single rectangular hyperbolic relationship did not describe the substrate saturation data adequately; analyses in these three cases requiring two classes of catalytic site. The apparent Km value for the higher affinity class of site in these three samples was within the range quoted above, but limitations imposed by assay sensitivity and phenacetin solubility obviated accurate characterization of the lower affinity class. 3. Estimates of Vmax for the high affinity class of site in the eight livers varied eleven-fold and there was no correlation between either Km or Vmax and microsomal cytochrome P-450 specific content, NADPH cytochrome c (P-450) reductase specific activity or ethylmorphine demethylase activity. 4. Propranolol was a potent competitive inhibitor of phenacetin deethylation with apparent Ki values of 2 to 7 microM describing its effect on the higher affinity class of activity. Propranolol was also an inhibitor of the lower affinity phenacetin deethylase identified in three of the livers, however the mechanism of inhibition could not be characterized. 5. These data suggest the possibility that propranolol oxidation may be mediated in part, by one or more human liver cytochrome P-450 species catalyzing phenacetin oxidation.

Adult↗
PubMed1987

Consequences of ligand bivalency in interactions involving particulate receptors: equilibrium and kinetic studies with Sephadex-concanavalin A, butylagarose-phosphorylase b, and Fc receptor-IgG dimer interactions as model systems.

Theoretical consideration is given to the interaction of a bivalent ligand with particulate receptor sites, not only from the viewpoint of quantitatively describing the binding behavior but also from that of the kinetics of ligand release upon infinite dilution of a receptor-ligand mixture. In the latter regard, a general expression is derived that describes the time dependence of the amount of ligand bound as a function of two rate constants for the stepwise dissociation of cross-linked ligand-receptor complex and a thermodynamic parameter expressing the initial ratio of singly linked to doubly linked ligand-receptor complexes. An experimental study of the interaction between Sephadex and concanavalin A is then used to illustrate application of this recommended theoretical approach for characterizing the binding behavior and dissociation kinetics of a bivalent ligand for a system in which all ligand-receptor interactions may be described by a single intrinsic association constant. Published results on the interaction of phosphorylase b with butylagarose are also shown to comply with this simplest model of the bivalent ligand hypothesis; but those for the interaction between immunoglobulin G (IgG) dimers and Fc receptors require modification of the model by incorporation of different intrinsic association constants for the successive binding of receptor sites to the bivalent ligand. These results emphasize the need to consider ligand bivalency as a potential phenomenon in studies of interactions between protein ligands and particulate receptors and illustrate procedures by which the effects of ligand bivalency may be identified and characterized.

Animals↗
PubMed1986

The nature of microsomal monooxygenase inhibition by cimetidine.

A kinetic investigation of the inhibitory effect of cimetidine on the O-dealkylation of 7-ethoxyresorufin by rat liver microsomes has yielded linear Lineweaver-Burk plots which intersect in the second quadrant. Though technically compatible with non-competitive inhibition, the results are shown to be readily explained by the more realistic molecular concept of competitive inhibition by invoking the involvement of cytochrome P-450 isoenzymes with widely different KI values. Microsomal monooxygenase heterogeneity is also shown to provide a plausible explanation of other published results signifying the departure of chloramphenicol and phenacetin from the concept of competitive inhibition despite competition with substrate for the active-site haem group of cytochrome P-450.

Animals↗
PubMed1986

Evidence for induction of hepatic microsomal cytochrome P-450 by cimetidine: binding and kinetic studies.

The interaction of cimetidine with liver microsomes has been examined by spectral and equilibrium partition studies. First, difference spectroscopy has been used to evaluate the proportion of cytochrome P-450 in rat liver microsomes that exhibits an affinity for cimetidine in the pharmacologically relevant, low micromolar range of drug concentration. The value of 0.45 so obtained has confirmed that a substantial proportion of rat liver cytochrome P-450 has a high binding affinity for this drug. Second, a study of the binding of cimetidine to human liver microsomes by difference spectroscopy and partition equilibrium has detected a similar interaction, thus providing direct support for the postulate that the clinically observed impairment of oxidative drug metabolism may be due in part to inhibition of cytochrome P-450 monooxygenase by cimetidine. Hepatic microsomes from cimetidine-pretreated rats have been shown to exhibit elevated cytochrome P-450 specific content but a decreased proportion of sites with high affinity for the drug; this finding has been shown not to be the consequence of cimetidine-mediated, time-dependent, irreversible monooxygenase inhibition. Although cimetidine pretreatment caused enhanced specific activity of 7-ethoxyresorufin O-dealkylation, the specific activities for O-dealkylation of 7-ethoxycoumarin and 4-nitroanisole were decreased, as were those for the N-dealkylation of morphine, ethylmorphine, aminopyrine, and dimethylnitrosamine. Since cimetidine pretreatment was shown to cause no change in the Michaelis constants for oxidation of morphine or 7-ethoxyresorufin, it is argued that these results provide strong presumptive evidence for changes in the relative abundance of isoenzymes catalyzing these various oxidations. Thus, a dual role of cimetidine, acting both as inhibitor and inducer of the cytochrome P-450 system, is proposed to account for the impaired oxidative metabolism of some drugs that occurs during coadministration with this H2-receptor antagonist.

Animals↗
PubMed1986

Human-liver cytochromes P-450 involved in polymorphisms of drug oxidation.

Nine forms of cytochrome P-450 have been purified to electrophoretic homogeneity from human-liver microsomes. These include the enzymes involved in debrisoquine 4-hydroxylation, phenacetin O-deethylation and mephenytoin 4-hydroxylation, three reactions which are characterized by genetic polymorphism in humans. Evidence for the involvement of the above enzymes comes from reconstituted immunochemical inhibition studies with human-liver microsomes. These and other lines of evidence are consonant with the view that different forms of cytochrome P-450 are involved in the three reactions. The debrisoquine 4-hydroxylase has been studied most extensively in terms of its substrate specificity. In addition, an analogous rat enzyme shows some homology and serves as a useful model. The use of antibodies raised to the rat-liver enzyme in immuno-inhibition studies with human-liver microsomes provides a means of determining the extent to which this enzyme participates in other reactions. Translation of rat-liver mRNA in vitro yields the intact debrisoquine 4-hydroxylase; studies with human mRNA suggest a lower frequency than in rats. The basis for impaired catalytic activity in phenotypically poor human metabolizers appears to be an altered enzyme in all three cases, as opposed to a decreased level of a single enzyme. Using antibody screening of fusion proteins expressed in a cDNA library, it has been possible to isolate cDNA probes for all three of these cytochromes P-450 for use in screening individuals and ultimately determining the basis of these polymorphisms.

Binding Sites↗
PubMed1985

Evidence for lipid involvement in the high-affinity interaction between cimetidine and rat liver cytochrome P-450.

The spectral interaction between cimetidine and a cytochrome P-450 fraction isolated from liver of untreated rats has been shown to be markedly affected by dilauroylphosphatidylcholine. In the absence of the lipid the pigment preparation yielded a binding curve characteristic of a single isoenzyme species with low affinity for the drug, whereas its inclusion led to the observation of a much stronger interaction with a dissociation constant close to that obtained for the high-affinity component(s) of the parent microsomes; material with lower affinity was also observed. Gel chromatography and partition equilibrium studies yielded results which precluded interpretation of this finding either in terms of incomplete incorporation of the pigment into the phospholipid or of disproportionate solvation of cimetidine into the lipid phase of the reconstituted phospholipid complex. In contrast, phospholipid caused only a minor change in the strength of cimetidine binding by the predominant liver cytochrome P-450 from phenobarbitone-pretreated rats. Pronounced lipid sensitivity of cimetidine-binding affinity is thus not a general feature of the microsomal cytochrome P-450 system but rather a specific characteristic of individual isoenzyme species.

Animals↗
PubMed1985

Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism.

Two forms of cytochrome P-450 were purified to apparent homogeneity from several different preparations of human liver microsomes. One form, designated P-450DB, had relatively high catalytic activity towards the drugs debrisoquine, sparteine, bufuralol (both the (+)- and (-)-isomers), encainide, and propranolol and appears to be the enzyme involved in the polymorphic distribution of oxidative activities towards these substrates in humans. The other form, designated P-450PA, had relatively high phenacetin O-deethylase activity and appears to be involved in the variation of this activity among humans. Polyclonal antibodies raised to the two enzymes were specific for the antigens as judged by immunoelectrophoresis and immuno-inhibition studies. The two enzymes and their activities were distinguished by chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, immuno-inhibition studies, and steady-state kinetic assays. Immunochemical studies suggest that each form represents only a small fraction of the total cytochrome P-450 in human liver microsomes. These biochemical studies provide a basis for better understanding the mechanisms which underlie genetic polymorphisms involving P-450 cytochromes in humans.

Amino Acids↗