Biomedical subjects

J C Lindon

Publications and source records attributed to J C Lindon.

At least 19 recordsLinked to original sources

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↗

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

Investigation of the metabolite variation in control rat urine using (1)H NMR spectroscopy.

An exploratory statistical analysis has been undertaken of 640 (1)H NMR spectra of rat urine, obtained from predose and control animals during the course of eight separate toxicology studies. The aim was to determine the degree and type of variation between (1)H NMR spectra from such control animals and to investigate the variations in the spectral descriptors based on averaged peak intensities. The results showed that many of the spectral descriptors had skew and/or multimodal distributions, and that it was possible to distinguish between samples of urine collected at different times of day with a success rate of (89%) and to classify 90% of the predose spectra into their correct study group using principal component and linear discriminant analyses. The results show that successful classification can be achieved of NMR spectra of control rat urine, which exhibit more subtle changes than those previously reported when treated and control animals were compared. The results presented here suggest that it will be possible to identify very subtle toxicological changes if care is taken to standardize the experimental conditions used during toxicity screens.

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↗

PubMed2001

Application of directly coupled HPLC NMR to separation and characterization of lipoproteins from human serum.

Disorders in lipoprotein metabolism are critical in the etiology of several disease states such as coronary heart disease and atherosclerosis. Thus, there is considerable interest in the development of novel methods for the analysis of lipoprotein complexes. We report here a simple chromatographic method for the separation of high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein from intact serum or plasma. The separation was achieved using a hydroxyapatite column and elution with pH 7.4 phosphate buffer with 100-microL injections of whole plasma. Coelution of HDL with plasma proteins such as albumin occurred, and this clearly limits quantitation of that species by HPLC peak integration. We also show, for the first time, the application of directly coupled HPLC 1H NMR spectroscopy to confirm the identification of the three major lipoproteins. The full chromatographic run time was 90 min with stopped-flow 600-MHz NMR spectra of each lipoprotein being collected using 128 scans, in 7 min. The 1H NMR chemical shifts of lipid signals were identical to conventional NMR spectra of freshly prepared lipoprotein standards, confirming that the lipoproteins were not degraded by the HPLC separation and that their gross supramolecular organization was intact.

Chromatography, High Pressure Liquid↗

PubMed2001

Recovery of underwater resonances by magnetization transferred NMR spectroscopy (RECUR-NMR).

A method for detecting small molecule NMR resonances under a water peak in biological samples is presented. After high-efficiency solvent suppression using double WATERGATE, either a TOCSY- or ROESY-based coherence transfer sequence is applied to reestablish the resonances close to, or under, water through magnetization transfer using scalar or dipolar coupling, respectively. The use of the TOCSY and ROESY methods ensures an in-phase magnetization transfer, which makes the new approach readily extended for the measurement of transverse relaxation times, internuclear ROEs, and ROE buildup rates. An extension of the new approach for J-resolved spectroscopy is also presented and tested using a sample of human blood plasma.

Blood Glucose↗

PubMed2001

LC-1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems.

The reactive metabolite S-naproxen-beta-1-O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC-1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-beta-1-O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase 1 (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4-O-acyl isomers, beta-1-O-acyl glucuronide, 3-O-acyl isomers, 2-O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: alpha/beta-4-O-acyl isomers, S-naproxen aglycone, beta-1-O-acyl glucuronide, 3-O-acyl isomers, and alpha/beta-2-O-acyl isomers. In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-1H NMR (U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113-122]. The alpha-1-O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-beta-1-O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37 degrees C. In both HPLC systems it eluted just before the beta-1-O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a alpha-1-O-acyl isomer when studying glucuronide reactivity and degradation.

Chromatography, High Pressure Liquid↗

PubMed2001

Directly coupled HPLC-NMR and HPLC-MS approaches for the rapid characterisation of drug metabolites in urine: application to the human metabolism of naproxen.

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool for the structural identification of xenobiotic metabolites in complex biological matrices such as plasma, urine and bile. However, these fluids are dominated by thousands of signals resulting from endogenous metabolites and it is advantageous when investigating drug metabolites in such matrices to simplify the spectra by including a separation step in the experiment by directly-coupling HPLC and NMR. Naproxen (6-methoxy-alpha-methyl-2-naphthyl acetic acid) is administered as the S-enantiomer and is metabolised in vivo to form its demethylated metabolite which is subsequently conjugated with beta-D-glucuronic acid as well as with sulfate. Naproxen is also metabolised by phase II metabolism directly to form a glycine conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the present investigation, the metabolism of naproxen was investigated in urine samples with a very simple sample preparation using a combination of directly-coupled HPLC-1H NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer system was developed which allows the same chromatographic method to be used for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these methods is complementary in information content since the NMR spectra provide evidence to distinguish isomers such as the type of glucuronides formed, and the HPLC-MS data allow identification of molecules containing NMR-silent fragments such as occur in the sulfate ester.

Buffers↗

PubMed2001

A rapid and facile method for the dereplication of purified natural products.

A new approach to the use of commercial databases for the dereplication of purified natural products has been developed. This is based on searching a text file that links each structure with its molecular weight and an exact count of the number of methyl, methylene, and methine groups it contains. Analysis of such a text file, constructed from a database containing more than 126,000 natural product structures, revealed that these data, readily measured using MS and NMR spectroscopy, are highly discriminating. The identification of an alkaloid and a sesquiterpene using this new approach is described.

Alkaloids↗

PubMed2001

Metabonomic investigations into hydrazine toxicity in the rat.

The systemic biochemical effects of oral hydrazine administration (dosed at 75, 90, and 120 mg/kg) have been investigated in male Han Wistar rats using metabonomic analysis of (1)H NMR spectra of urine and plasma, conventional clinical chemistry, and liver histopathology. Plasma samples were collected both pre- and 24 h postdose, while urine was collected predose and daily over a 7 day postdose period. (1)H NMR spectra of the biofluids were analyzed visually and via pattern recognition using principal component analysis. The latter showed that there was a dose-dependent biochemical effect of hydrazine treatment on the levels of a range of low molecular weight compounds in urine and plasma, which was correlated with the severity of the hydrazine induced liver lesions. In plasma, increases in the levels of free glycine, alanine, isoleucine, valine, lysine, arginine, tyrosine, citrulline, 3-D-hydroxybutyrate, creatine, histidine, and threonine were observed. Urinary excretion of hippurate, citrate, succinate, 2-oxoglutarate, trimethylamine-N-oxide, fumarate and creatinine were decreased following hydrazine dosing, whereas taurine, creatine, threonine, N-methylnicotinic acid, tyrosine, beta-alanine, citrulline, Nalpha-acetylcitrulline and argininosuccinate excretion was increased. Moreover, the most notable effect was the appearance in urine and plasma of 2-aminoadipate, which has previously been shown to lead to neurological effects in rats. High urinary levels of 2-aminoadipate may explain the hitherto poorly understood neurological effects of hydrazine. Metabonomic analysis of high-resolution (1)H NMR spectra of biofluids has provided a means of monitoring the progression of toxicity and recovery, while also allowing the identification of novel biomarkers of development and regression of the lesion.

Administration, Oral↗

PubMed2001

Investigation of water environments in a C18 bonded silica phase using 1H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy.

High resolution 1H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra have been obtained on typical C18 bonded silicas used in chromatographic solid-phase extraction separations. It has been shown for the first time that water molecules distributed in distinct physico-chemical environments within the chromatographic system can be detected directly using a simple 1H MAS NMR measurement. The resonances assigned to water protons in differing physico-chemical environments have distinct chemical shifts, line widths, relaxation times (T1 and T2) and also exhibit temperature dependent coalescence behaviour. This novel MAS approach may lead to a better understanding of the environments of other analytes in mixtures during such separations.

Journal Article↗

PubMed2001

The potential of 19F NMR spectroscopy for rapid screening of cell cultures for models of mammalian drug metabolism.

The use of microbial cultures as a complementary model for mammalian drug metabolism has been well established previously. Here is a preliminary investigation into the potential of 19F NMR spectroscopy as a rapid screening tool to quantify the biotransformations of fluorine-containing model drugs. Biotransformations of three model drugs in 48 taxonomically diverse organisms were measured by acquiring 19F NMR spectra at 376 MHz. The presence of fluorine in the molecules allowed rapid, simultaneous detection of over 20 biotransformation products without sample pretreatment, chromatography, mass spectrometric techniques or the use of radiolabelled substrates. The detection limit at 376 MHz using 5 mm NMR tubes was ca. 0.3 microg ml(-1) using a typical analysis time of 20 min per sample. With the recent advent of flow injection NMR technology, analysis time of 5 min could be achieved with less sample. This approach may be used to develop fast small-scale microbial screens for the biosynthesis of metabolite standards and production of novel drug analogues, whilst also having a role in reducing animal experiments needed to identify animal and human metabolites of fluorinated xenobiotics.

Animals↗