Biomedical subjects

J K Nicholson

Publications and source records attributed to J K Nicholson.

At least 37 records · Page 2Linked to original sources

PubMed2003

Detection of mono- and di-hexoses as metabolites of 4-bromoaniline using HPLC-TOF-MS/MS.

1. The metabolic fate of 4-bromoaniline (4-BrA) was investigated in rat following intraperitoneal administration at 50 mg kg(-1) using HPLC-TOF-MS/MS. 2. The sensitivity provided by the use of TOF-MS/MS, aided by the distinctive isotope pattern resulting from the presence of the bromine substituent in the molecule, enabled the detection of many previously uncharacterized metabolites in the samples. 3. Several groups of minor metabolites were detected in the urine that corresponded to a number of isomeric hexose and di-hexose-containing conjugates (possibly glucosides and diglucosides) of 4-BrA. 4. As well as hexose and di-hexose conjugates of 4-BrA, several further groups of metabolites that also contained either a sulphamate or sulphate group in addition to the sugar moieties were also detected.

Aniline Compounds↗

PubMed2003

Topics in Xenobiochemistry: do metabolic pathways exist for xenobiotics? The micro-metabolism hypothesis.

1. The relevance of the concept of 'the metabolic pathway' for the understanding of xenobiotic metabolism is discussed in the light of advances in modern analytical methods that have enabled the detection and identification of minor metabolites present at ever lower concentrations. 2. A model is suggested where the overall metabolic fate of a xenobiotic is the sum of all the possible metabolic reactions permitted by the solution chemistry of the compound modulated by factors such as the metabolizing enzyme complement of the organism, the affinity of those enzymes for the xenobiotic substrates and the probabilities of all of these processes. 3. In this probabilistic, rather than deterministic, system, the resulting proportions of particular metabolites will, therefore, depend on the sums of the probabilities of particular biotransformation reactions occurring and the stability (chemical or metabolic) of the resulting metabolite. 4. In this model, all the potential metabolic possibilities that could result for any individual xenobiotic will occur to some extent. However, in actuality, many of the resulting metabolites will be produced or excreted in such small quantities as to defy ready detection with current methods.

Animals↗

PubMed2002

Vitamin E deficiency and metabolic deficits in neuronal ceroid lipofuscinosis described by bioinformatics.

The mnd mouse, a model of neuronal ceroid lipofusinosis (NCL), has a profound vitamin E deficiency in sera and brain, associated with cerebral deterioration characteristic of NCL. In this study, the vitamin E deficiency is corrected using dietary supplementation. However, the histopathological features associated with NCL remained. With use of a bioinformatics approach based on high-resolution solid and solution state 1H-NMR spectroscopy and principal component analysis (PCA), the deficits associated with NCL are defined in terms of a metabolic phenotype. Although vitamin E supplementation reversed some of the metabolic abnormalities, in particular the concentration of phenylalanine in extracts of cerebral tissue, PCA demonstrated that metabolic deficits associated with NCL were greater than any effects produced from vitamin E supplementation. These deficits included increased glutamate and N-acetyl-L-aspartate and decreased creatine and glutamine concentrations in aqueous extracts of the cortex, as well as profound accumulation of lipid in intact cerebral tissue. This is discussed in terms of faulty production of mitochondrial-associated membranes, thought to be central to the deficits in mnd mice.

Animals↗

PubMed2002

Physiological variation in metabolic phenotyping and functional genomic studies: use of orthogonal signal correction and PLS-DA.

Metabolic phenotyping, or metabotyping, is increasingly being used as a probe in functional genomics studies. However, such profiling is subject to intrinsic physiological variation found in all animal populations. Using a nuclear magnetic resonance-based metabonomic approach, we show that diurnal variations in metabolism can obscure the interpretation of strain-related metabolic differences in two phenotypically normal mouse strains (C57BL10J and Alpk:ApfCD). To overcome this problem, diurnal-related metabolic variation was removed from these spectral data by application of orthogonal signal correction (OSC), a data filtering method. Interpretation of the removed orthogonal variation indicated that diurnal-related variation had been removed and that the AM samples contained higher levels of creatine, hippurate, trimethylamine, succinate, citrate and 2-oxo-glutarate and lower levels of taurine, trimethylamine-N-oxide, spermine and 3-hydroxy-iso-valerate relative to the PM samples. We propose OSC will have great potential removing confounding variation obscuring subtle changes in metabolism in functional genomic studies and will be of benefit to optimising interpretation of proteomic and genomic datasets.

Animals↗

PubMed2002

The metabolism of 4-trifluoromethoxyaniline and [13C]-4-trifluoromethoxyacetanilide in the rat: detection and identification of metabolites excreted in the urine by NMR and HPLC-NMR.

A combination of 19F, 1H NMR and HPLC-NMR spectroscopic approaches have been used to quantify and identify the urinary-excreted metabolites of 4-trifluoromethoxyaniline (4-TFMeA) and its [13C]-labelled acetanilide following i.p. administration at 50 mg/kg to rats. The major metabolite excreted in the urine for both compounds was a sulphated ring-hydroxylated metabolite (either 2- or 3-trifluoromethyl-5-aminosulphate) which accounted for approximately 32.3% of the dose following the administration of 4-TFMeA and approximately 29.9% following dosing of the acetanilide. The trifluoromethoxy-substituent appeared to be metabolically stable, with no evidence of O-detrifluoromethylation. There was no evidence of the excretion of N-oxanilic acids in urine, of the type seen with 4-trifluoromethylaniline.

Acetanilides↗

PubMed2002

A comparison of quantitative NMR and radiolabelling studies of the metabolism and excretion of Statil (3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-ylacetic acid) in the rat.

The identification and quantitation of the metabolites of Statil in rat bile and urine were investigated by 1H- and 19F-NMR spectroscopy and liquid scintillation counting. Male Wistar rats received a single oral dose of 100 mg/kg of radiolabelled Statil. Statil is known to produce glucuronide conjugates which are predominantly excreted into the bile in male rats. The complex multiphasic matrix of bile has been shown to make identification of the resonances by 1H-NMR spectroscopy very difficult as Statil appeared to be micellar bound giving rise to very broad signals. This not only impaired unambiguous signal characterisation but also quantification. The partial separation by SPEC-(1)H-NMR spectroscopy enabled the disruption of the micellar matrices and hence enabled the identification of Statil predominantly as aglycone, and to a lesser extent as glucuronide conjugate. In addition, minor acyl migration products of Statil glucuronide could also be detected as they were separated during the SPEC-process. 19F-NMR spectroscopic measurements on whole bile confirmed their presence as a number of overlapped signals could be observed. The selectivity, simplicity and signal dispersion characteristic of 19F-NMR spectroscopy also enabled the calculation of dose related recoveries of Statil related material in the bile and urine samples without the need for a radiolabel. The aim of this work was to investigate the usefulness and limitations of NMR spectroscopy of intact bile and urine as a means of quantifying levels of drug metabolites. The results obtained from NMR spectroscopy are compared with those obtained using scintillation techniques. Scintillation counting yields unequivocal quantification results, provided the label is preserved in metabolites as has been the case here. In general, quantification by 19F-NMR results similar to those obtained by scintillation counting (in agreement within about 20%). However, discrepancies have been observed with very small and broad 19F-NMR signals in bile. Nevertheless, 19F-NMR spectroscopy of bile is a rapid and facile method for assessing metabolite levels of fluorinated drugs.

Animals↗

PubMed2002

Identification of the urinary metabolites of 4-bromoaniline and 4-bromo-[carbonyl-13C]-acetanilide in rat.

1. The urinary excretion of 4-bromoaniline and its [carbonyl-(13)C]-labelled N-acetanilide, together with their corresponding metabolites, have been investigated in the rat following i.p. administration at 50 mg kg(-1). 2. Metabolite profiling was performed by reversed-phase HPLC with UV detection, whilst identification was performed using a combination of enzymic hydrolysis and directly coupled HPLC-NMR-MS analysis. The urinary metabolite profile was quantitatively and qualitatively similar for both compounds with little of either excreted unchanged. 3. The major metabolite present in urine was 2-amino-5-bromophenylsulphate, but, in addition, a number of metabolites with modification of the N-acetyl moiety were identified (from both the [(13)C]-acetanilide or produced following acetylation of the free bromoaniline). 4. For 4-bromoacetanilide, N-deacetylation was a major route of metabolism, but despite the detection of the acetanilide following the administration of the free aniline, there was no evidence of reacetylation (futile deacetylation). 5. Metabolites resulting from the oxidation of the acetyl group included a novel glucuronide of an N-glycolanilide, an unusual N-oxanilic acid and a novel N-acetyl cysteine conjugate.

Acetanilides↗

PubMed2002

Structure-metabolism relationships of substituted anilines: prediction of N-acetylation and N-oxanilic acid formation using computational chemistry.

1. The relationship between the in vivo metabolism of substituted anilines, in particular N-acetylation and subsequent formation of oxanilic acids, and their molecular physico-chemical properties has been investigated using computational chemistry and pattern-recognition methods. The methods revealed that the physico-chemical properties most important for N-acetylation and subsequent oxanilic acid formation were electronic descriptors based on partial atomic charges and the susceptibility of the molecules to nucleophilic attack at certain ring positions. 2. The calculated partial atom charge on the amine nitrogen was the parameter most important for predicting that an aniline would be N-acetylated. The calculated nucleophilic susceptibility of the aromatic carbon para to the amino group (NS4) was the most significant parameter for determining oxanilic acid formation following N-acetylation. Thus, highly electron-withdrawing groups substituted at this position gave higher nucleophilic susceptibilities that were related to the presence of an oxanilic acid metabolite. 3. If the parameters relating to N-acetylation were modified by other electron-withdrawing groups in the ring (particularly at the position ortho to the amino group), then acetylation and subsequent oxanilic acid formation did not occur. The introduction of groups that allow the possibility of competing oxidative metabolic pathways elsewhere in the molecule (e.g. CH(3)) also affected the production of oxanilic acids. 4. Using chemometric analysis of the computed physico-chemical properties, the result has been the generation of a model that classifies the metabolism of a number of anilines. This could be used to predict the acetylation and oxanilic formation propensity of a number of substituted anilines whose metabolism was unknown to the system, demonstrating that such techniques may be of use for predicting metabolism and hence could provide support for rational drug design.

Acetylation↗

PubMed2002

Metabolism of 4-fluoroaniline and 4-fluorobiphenyl in the earthworm Eisenia veneta characterized by high-resolution NMR spectroscopy with directly coupled HPLC-NMR and HPLC-MS.

1. Little is known about metabolism of xenobiotics by earthworms, despite their importance in soil ecotoxicity testing. Normal earthworms and earthworms treated with antibiotics to ensure inhibition of gut microflora were exposed to two model xenobiotic compounds, 4-fluoroaniline and 4-fluorobiphenyl, to determine which metabolites were produced, and whether the pattern of metabolism was affected by the presence of microbial transformation ability. 2. (19)F-NMR spectroscopy detected the number and relative proportions of metabolites and directly coupled HPLC-(1)H-NMR spectroscopy and HPLC-MS then identified the metabolites. 3. Despite uptake, no metabolism of 4-fluorobiphenyl was observed at any stage, which appears to be a consequence of the lack of oxidative Phase I metabolic activity of the earthworms towards this substrate. In contrast, 4-fluoroaniline exhibited dose-dependent metabolism. At high doses (leading to mortality within 24 h) one predominant metabolite was observed, which was identified as the N-beta-glucoside conjugate. At lower dose levels, the predominant metabolite was the gamma-glutamyl conjugate, although the glucoside and another as yet unidentified metabolite were also detected. 4. The inhibition of gut microflora did not have any influence on metabolism. The study represents the first evidence for glucoside and glutamyl conjugation as a pathway for xenobiotic metabolism in earthworms.

Aniline Compounds↗

PubMed2002

19F-NMR and directly coupled 19F/1H-HPLC-NMR spectroscopic investigations of the metabolism of the model ecotoxin 3-trifluoromethylaniline in the earthworm species Eisenia veneta.

1. The metabolic fate of the model ecotoxin 3-trifluoromethylaniline (3-TFMA) in earthworm was studied by (19)F- and directly coupled (19)F/(1)H-HPLC-NMR spectroscopy. Earthworms of Eisenia veneta spp. were subjected to the ecotoxin during a filter papercontact toxicity test at exposure levels of 1000, 100, 10, 1 and 0.1 micro g cm(-2). A metabolic profile was obtained previously by (19)F-NMR spectroscopy and metabolites were observed at all the exposure levels. 2. Identification of metabolites in individual worm extracts at the (lethal) exposure levels of 1000 and 100 micro g cm(-2) could be achieved on-line without sample preparation by (19)F/(1)H-HPLC-NMR spectroscopy. (19)F-HPLC-NMR spectroscopy was used in the continuous-flow mode, which enabled the HPLC chromatographic retention times (t(R)) of the metabolites to be established in a single analytical step. 3. In total, three (19)F-NMR signals could be detected, of which one was identified as the parent compound. Two earlier eluting metabolites were identified to be alpha- and beta-glucoside conjugates of 3-TFMA. 4. Metabolites at the lower (sublethal) exposure levels of 10, 1 and 0.1 micro g cm(-2) escaped identification by (19)F/(1)H-HPLC-NMR spectroscopy as outlined here and will require concentration prior to analysis.

Aniline Compounds↗

PubMed2002

Application of (1)H- and (19)F-NMR spectroscopy in the investigation of the urinary and biliary excretion of 3,5-, 2,4-ditrifluoromethylbenzoic and pentafluorobenzoic acids in rat.

1. The metabolism and excretion of 2,4-, 3,5-ditrifluoromethyl- and pentafluorobenzoic acids were studied in the bile-cannulated rat using (1)H- and (19)F-NMR spectroscopy following intraperitoneal administration at 50 mg kg(-1). 2. Pentafluorobenzoic acid was excreted in the urine entirely unchanged. No detectable compound or metabolites were eliminated in the bile. A total of 63.5 +/- 6.7% of the dose was recovered in the 24-h collection period. 3. In the case of 2,4-ditrifluromethyl benzoic acid, 83.9 +/- 5.2% of the dose was recovered in the 24h after administration, with about 52% being excreted in the urine and 32% in the bile. The majority of the material present in the urine was unchanged parent compound. In bile, some 60% of the compound-related material excreted was present as transacylated ester glucuronide conjugates. 4. For 3,5-ditrifluoromethylbenzoic acid, 49.6 +/- 5.3% of the dose was recovered in the 24-h collection period, with about 22% being excreted in the urine and 28% in the bile. The material excreted in both the urine and bile was a mixture of the parent acid and transacylated ester glucuronides. 5. Urinary excretion in bile-cannulated animals was similar to that found in studies using non-cannulated animals dosed at 100mg kg(-1).

Animals↗

PubMed2001

Choline containing metabolites during cell transfection: an insight into magnetic resonance spectroscopy detectable changes.

Increases in choline containing metabolites have been associated with a number of disorders, including malignant cell growth. In this study, high resolution magic angle spinning (1)H nuclear magnetic resonance spectroscopy was employed to monitor metabolite changes during cell transfection, and an increase in phosphocholine was detected. This increase appears to be correlated with cell membrane disruption associated with the insertion of plasmid DNA into cells, since the level of phosphocholine in mock transfected cells was comparable to that of control cells. These data suggest choline containing metabolite changes detected in vivo using magnetic resonance spectroscopy relate to cell membrane disruption.

Choline↗

PubMed2001

Investigations into biochemical changes due to diurnal variation and estrus cycle in female rats using high-resolution (1)H NMR spectroscopy of urine and pattern recognition.

Metabonomic methods utilizing (1)H NMR spectroscopy and pattern recognition analysis (NMR-PR) have been applied to investigate biochemical variation in a control population of female rats over time in relation to diurnal and estrus cycle fluctuations. Urine samples were collected twice daily (6 AM-6 PM and 6 PM-6 AM) from female rats (n = 10) for a period of 10 days. (1)H NMR spectroscopic analysis and PR were performed on each sample. Subtle differences in the endogenous metabolite excretion profiles of urine samples at the various stages of the estrus cycle were observed. The main inherent metabolic clustering in the principal components analysis (PCA) maps was related to interrat variation and was observed in the first two principal components (PCs), accounting for 66% of the variance in these data. Separation of urinary data according to time of sampling (day and night) was achieved in the lower PCs. Some of the differences in the urinary profiles of day and night samples causing this separation were attributed to the increase in metabolic activity of the rat during the night. Individual rat data were also mapped as a function of time, using PCA, to produce a metabolic trajectory, which in a number of cases facilitated separation of one or more stages of the estrus cycle. Several of the fluctuations observed between urine samples collected during the different stages of the estrus cycle may be related to hormone levels. Although variation in metabolite profiles relating to both diurnal and hormonal variation could be detected these perturbations were minor compared with the effects observed due to interrat variation. This is the first time that a hormonal cycle has been described for individuals based on NMR spectroscopic and multivariate analysis of metabolic data and shows the value of metabonomic methods in the investigation of physiological variation and rhythms.

Animals↗

PubMed2001

An NMR-based metabonomic approach to the investigation of coelomic fluid biochemistry in earthworms under toxic stress.

The endogenous metabolites of the coelomic fluid of the earthworm Eisenia veneta were characterised using high-resolution one-dimensional and two-dimensional 1H nuclear magnetic resonance spectroscopy. Signals from common organic acids, such as acetate, fumarate, malonate, malate, formate, and succinate, were identified together with adenosine and nicotinamide mononucleotide. The potential use of this information as a baseline dataset for future toxicological or physiological studies was demonstrated by a metabonomic analysis: a series of earthworms were dosed with the model compound 3-fluoro-4-nitrophenol, and toxic effects followed by multivariate analysis of the spectral data of the coelomic fluid. Relative concentrations of acetate and malonate were decreased in the dosed worms compared to the controls.

Acetates↗

PubMed2001

Metabolic profiling of genetic disorders: a multitissue (1)H nuclear magnetic resonance spectroscopic and pattern recognition study into dystrophic tissue.

A principal problem in understanding the functional genomics of a pathology is the wide-reaching biochemical effects that occur when the expression of a given protein is altered. To complement the information available to bioinformatics through genomic and proteomic approaches, a novel method of providing metabolite profiles for a disease is suggested, using pattern recognition coupled with (1)H NMR spectroscopy. Using this technique the mdx mouse, a model of Duchenne muscular dystrophy (DMD) was examined. Dystrophic tissue had distinct metabolic profiles not only for cardiac and other muscle tissues, but also in the cerebral cortex and cerebellum, where the role of dystrophin is still controversial. These metabolic ratios were expressed crudely as biomarker ratios to demonstrate the effectiveness of the approach at separating dystrophic from control tissue (cardiac (taurine/creatine): mdx = 2.08 +/- 0.04, control 1.55 +/- 0.04, P < 0.005; cortex (phosphocholine/taurine): mdx = 1.28 +/- 0.12, control = 0.83 +/- 0.05, P < 0.01; cerebellum (glutamate/creatine): mdx = 0.49 +/- 0.03, control = 0.34 +/- 0.03, P < 0.01). This technique produced new metabolic biomarkers for following disease progression but also demonstrated that many metabolic pathways are perturbed in dystrophic tissue.

Animals↗

PubMed2001

CCR5 and CXCR4 expression on memory and naive T cells in HIV-1 infection and response to highly active antiretroviral therapy.

OBJECTIVE: To measure CCR5 and CXCR4 chemokine receptor expression on CD4 and CD8 T cells in HIV-1 infection and to relate levels to the distribution of CD45RO memory and CD45RA-naive subsets, measures of disease activity, and response to highly active antiretroviral therapy (HAART). DESIGN: Fourteen untreated HIV-1-infected patients, 18 patients at 3-to 4-weeks after beginning HAART, and 35 uninfected control subjects were studied. METHODS: Four-color cytofluorometry with appropriate conjugated monoclonal antibodies (mAbs) was performed to define CD45RA and CD45RO subsets of CD4 and CD8 T cells and measure their expression of CCR5, CXCR4, and CD38. RESULTS: HIV-1-infected patients had higher CCR5 levels and lower CXCR4 levels on CD4 and CD8 T cells and their CD45RO/CD45RA subsets than control subjects did. However, CCR5 elevation was statistically significant only for CD4 T cells and their subsets, and CXCR4 depression was significant for CD8 T cells and their subsets (and for CD4:CD45RO cells). The elevation of CCR5 and depression of CXCR4 were not due to shifts in CD45RO/CD45RA subset proportions but to upregulation or downregulation within the subsets. CCR5 elevation on CD4 T cells was significantly restored toward normal by HAART, but the CXCR4 depression was not. CCR5 expression but not CXCR4 expression correlated with other measures of immunodeficiency (CD4 T-cell levels), active infection (viral load), and cellular activation (CD38). CONCLUSIONS: CCR5 elevation is a concomitant of immune activation and viral replication that occurs in HIV-1 infection, but the relation of CXCR4 depression to severity of infection, disease progression, and response to therapy remains undefined.

Adult↗

PubMed2001

Directly coupled high-performance liquid chromatography and nuclear magnetic resonance spectroscopic with chemometric studies on metabolic variation in Sprague--Dawley rats.

We report here the first combined use of NMR-PR (pattern recognition) analysis and directly coupled HPLC--NMR analysis to identify metabolic subpopulations in normal laboratory animals and their discriminating endogenous urinary biomarkers. Urine samples obtained from control Sprague-Dawley rats (n = 68) were analyzed using (1)H NMR spectroscopy and principal components (PC) analysis to investigate physiological variability. Two distinct subpopulations of animals were classified based on metabolite excretion profiles. Analysis of the PC loadings established the spectral regions that were responsible for classification of the subpopulations and was used to direct the identification of biomarkers using a directly coupled HPLC--NMR analysis. One population had low urinary hippurate levels together with an increased concentration of 3-(3-hydroxyphenyl)propionic acid (3-HPPA)and 3-hydroxycinnamic acid (3-HCA). The other subpopulation excreted high levels of hippurate. Thus, we report the bimodal occurrence of hippuric acid and chlorogenic acid metabolites in a genetically homogeneous population of rats maintained under identical conditions, which may have significance in relation to the understanding of the consequences of biochemical variation in animals used for drug toxicity testing.

Animals↗

PubMed2001

High-performance liquid chromatography linked to inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the simultaneous detection and identification of metabolites of 2-bromo-4-trifluoromethyl.

The use of HPLC coupled to inductively coupled plasma mass spectrometry (ICPMS) and orthogonal acceleration time-of-flight (oa-TOF) for the profiling, identification, and quantification of metabolites in rat urine following the administration of 2-bromo-4-trifluoromethylacetanilide is described. The metabolites present in the sample were separated by reversed-phase gradient chromatography with UV-diode array detection. The bulk of the eluent (90%) from the UV detector was directed to an ICPMS where bromine-containing metabolites were detected and quantified using ICPMS. The minor portion of the eluent (10%) was taken for oa-TOFMS for identification. By these means, the metabolites were identified as sulfate and glucuronide conjugates of a ring hydroxy-substituted metabolite, a N-sulfate, a N-hydroxylamine glucuronide, and N- and N-hydroxyglucuronides.

Anilides↗