J. K. Nicholson

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.

19F-NMR and directly coupled HPLC-NMR-MS investigations into the metabolism of 2-bromo-4-trifluoromethylaniline in rat : a urinary excretion balance study without the use of radiolabelling

1. The metabolic fate and urinary excretion of 2-bromo-4-trifluoromethylaniline has been studied in rat using 19F-NMR spectroscopic and directly coupled HPLC-NMR-MS methods. The compound was dosed to Sprague-Dawley rats (50 mg kg-1, i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 53.5 +/- 7.0% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-bromo-5-trifluoromethylphenylsulphate accounting for a total of 35.7 +/- 6.2% of the dose. 3. Further metabolites detected were 2-bromo-4-trifluoromethylphenylhydroxylamine-1V-glucuronide (9.7 +/- 0.2% of the dose), 2-bromo-4-trifluoromethylaniline-N-glucuronide (3.0 +/- 0.3%) and 2-amino-3-bromo-5-trifluoromethylphenylglucuronide (2-St 0-4). Minor metabolites, including 2-bromo-4-trifluoromethylphenylhydroxylamine-O-glucuronide, 2-amino-3-bromo-5-trifluoromethylphenol and 2-bromo-4-trifluoromethylphenylsulphamate, in total accounted for 2.3 +/- 0.9% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy proved to be efficient techniques for the unequivocal and rapid determination of the urinary metabolic fate and excretion balance of fluorinated xenobiotics without the need for radiolabelling.

Quantitative studies on the urinary metabolic fate of 2-chloro-4-trifluoromethylaniline in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS

1. The metabolism and urinary excretion of 2-chloro-4-trifluoromethylaniline has been studied in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS methods. The compound was dosed to three male Sprague-Dawley rats (50 mg kg(-1) i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 56.3+/-2.2% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-chloro-5-trifluoromethylphenylsulphate accounting for a total of 33.5+/-2.2% of the dose. 3. Further metabolites detected and characterized included 2-chloro-4-trifluoromethylphenylhydroxylamine glucuronide (13.2+/-0.5% of the dose), 2-amino-3-chloro-5-trifluoromethylphenylglucuronide (3.8+/-0.4% of the dose) and 2-chloro-4-trifluoromethylaniline-N-glucuronide (3.6+/-0.1% of the dose). Several minor metabolites were also found and identified, including 2-chloro-4-trifluoromethylphenylsulphamate, which together accounted for 2.1+/-0.4% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy is shown to provide an efficient approach for the unequivocal and rapid determination of the quantitative urinary metabolic fate and excretion balance of a fluorinated xenobiotic without the necessity for specific radiolabelling.

Quantitative structure-metabolism relationships (QSMR) using computational chemistry: pattern recognition analysis and statistical prediction of phase II conjugation reactions of substituted benzoic acids in the rat

1. Quantitative relationships between molecular physico-chemical properties of 22 substituted benzoic acids and the extent of excretion of their metabolites in rat urine have been investigated using computational chemistry and multivariate statistics. 2. A data set of 34 theoretically derived physico-chemical descriptors calculated was used to classify the benzoic acids according to their predominant urinary metabolic fate. 3. Quantitative structure-metabolism relationships were obtained by linear regression using combinations of physico-chemical descriptors allowing the prediction of % urinary excretion of glycine (r = 0.73) and glucuronide conjugates (r = 0.82) and % urinary excretion of the parent compound (r = 0.91).

Characterization and quantification of metabolites of racemic ketoprofen excreted in urine following oral administration to man by 1H-NMR spectroscopy, directly coupled HPLC-MS and HPLC-NMR, and circular dichroism

The identity of the human metabolites of ketoprofen (2-(3-benzoylphenyl)-propanoic acid) excreted via urine was investigated after a single oral dose of the racemic drug. Drug metabolites were concentrated and partially purified from urine using solid-phase extraction chromatography before separation and identification by directly coupled HPLC-MS and HPLC-NMR. The metabolites identified were the ester glucuronides of the parent drug and its phase I metabolites, 2-[3-(3-hydroxybenzoyl)phenyl]-propanoic acid, 2-[3-(4-hydroxybenzoyl)phenyl]-propanoic acid and 2-[3-(hydroxy(phenyl)methyl)phenyl]-propanoic acid, the latter formed by reduction of the ketone group of ketoprofen. In addition, two novel minor metabolites were identified as the ether glucuronides of 2-[3-(3-hydroxybenzoyl)phenyl]-propanoic acid and 2-[3-(4-hydroxybenzoyl)phenyl]-propanoic acid. These conjugates were all observed as diastereoisomeric pairs of unequal proportions. Purification of these metabolites by preparative chromatography allowed stereochemistry assignments. Metabolites were quantified by 1H-NMR spectroscopy after spectral simplification achieved by hydrolysis of the conjugates.

Identification of phenacetin metabolites in human urine after administration of phenacetin-C2H3: Measurement of futile metabolic deacetylation via HPLC/MS-SPE-NMR and HPLC-ToF MS

The metabolism of acetyl-labelled phenacetin-C2H3 was investigated in man following a single (150 mg) oral dose. Urine samples were collected at predose, 0–2 h and >2–4 h post-dose, and samples from each time-point were then analysed directly using 1H-nuclear magnetic resonance (NMR) spectroscopy. The phenacetin metabolites acetaminophen (paracetamol) glucuronide, sulphate and the N-acetyl-L-cysteinyl conjugate were identified by this method, and all showed clear evidence of the loss of the original 2H3-acetyl label and its replacement with 1H3 (futile deacetylation). The observed percentage futile deacetylation by 1H-NMR spectroscopy was measured as approximately 20% in each metabolite (about 2% of the recovered dose). After sample preparation by solid-phase extraction on a C18 solid-phase extraction (SPE) cartridge, further profiling was performed using high-performance liquid chromatography/mass spectrometry-solid-phase extraction-nuclear magnetic resonance (HPLC/MS-SPE-NMR) confirming futile deacetylation had taken place as indicated by NMR spectroscopy on both the isolated acetaminophen glucuronide and L-cysteinyl-metabolites. Additional analysis by high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-ToF MS) identified further phenacetin metabolites, and from these data the mean percentage of futile deacetylation was measured as 31% ± 2% for the acetylated phenacetin metabolites. A number of non-acetylated metabolites were also detected in the sample via HPLC-ToF MS. The results showed that phenacetin underwent a transient formation via a number of toxic intermediates to a much greater extent than had been observed in similar studies on acetaminophen. These results may contribute to the understanding of the analgesic nephropathy reported following chronic phenacetin consumption.

Nuclear magnetic resonance (NMR) and quantitative structure–activity relationship (QSAR) studies on the transacylation reactivity of model 1β-O-acyl glucuronides. II: QSAR modelling of the reaction using both computational and experimental NMR parameters

1. In a previously reported study, a number of 4-substituted benzoic acid acyl glucuronides were synthesized and their degradation rates determined using nuclear magnetic resonance (NMR) spectroscopy. It was shown that this reaction was strongly influenced by the nature of the substituent at the 4-position of the benzoyl moiety. 2. The overall degradation reaction rates for this series of compounds have been modelled successfully using Hammett substituent constants, computational chemistry-derived partial atomic charges and the experimentally determined carbonyl carbon 13C-NMR chemical shifts of the benzoic acids and their ethyl and glucuronide esters. 3. The primary contribution to reactivity is the scale of the electron-donating or -withdrawing effect of the substituent; however, additional contributions such as steric parameters must also be considered when modelling reactions outside a single chemical series. 4. The derived property–reactivity relationships should find utility in medicinal chemistry efforts for optimizing chemical series in pharmaceutical discovery programmes.

Studies on the metabolism of 4-fluoroaniline and 4-fluoroacetanilide in rat: formation of 4-acetamidophenol (paracetamol) and its metabolites via defluorination and N-acetylation

1. The urinary metabolic fate of 4-fluoroaniline (4-FA) and 1-[13C]-4-fluoroacetanilide (4-FAA) has been studied using NMR-based methods after 50 and 100 mg kg(-1) i.p. doses respectively to the male Sprague-Dawley rat. 2. 4-FA was both ortho- and para-hydroxylated. The major metabolite produced by ortho-hydroxylation was 2-amino-5-fluorophenylsulphate accounting for approximately 30% of the dose. Of the dose, approximately 10% was excreted via para-hydroxylation and the resulting defluorinated metabolites were N-acetylated and excreted as sulphate (major), glucuronide (minor) and N-acetyl-cysteinyl (minor) conjugates of 4-acetamidophenol (paracetamol). 3. The major route of metabolism of 1-[13C]-4-FAA was N-deacetylation and the metabolites excreted in the urine were qualitatively identical to 4-FA. The paracetamol metabolites produced via para-hydroxylation were also a product of N-deacetylation and reacetylation, as the [13C]-label was not retained. 4. These studies demonstrate the value of [13C]-labelling in understanding the contribution of N-acetylation, and futile deacetylation-reacetylation reactions, in aniline metabolism. In addition, this work sheds new light on the metabolic lability of certain aromatic fluorine substituents.

Investigation of the metabolism of 14C/13C-practolol in rat using directly coupled radio-HPLC-NMR-MS

1. The metabolic fate of 14C 13C-practolol was investigated using on-line HPLCNMR-MS following oral administration to rat. The major route of elimination for the radiolabel was via the urine with the principal biotransformation products confirmed as the 2-hydroxy- and 2-hydroxyglucronide metabolites. 2. In addition, futile deacetylation, determined by the replacement of 13C-labelled acetyl groups with endogenous 12C-acetyls accounted for ~7-10% of the urinary metabolites, corresponding to ~5% of the dose undergoing N-deacetylation. 3. Evidence for chiral metabolism was sought via NMR of isolated metabolites using beta cyclodextrin as a chiral shift agent. Practolol was excreted as a racemate. However, some enantioselective metabolism excretion had occurred as the hydroxy- and hydroxyglucuronide were not excreted as racemic mixtures. 4. Directly coupled radio-HPLC-NMR-MS is extremely effective for the identification of the metabolites of radiolabelled xenobiotics in urine samples.

Integrated HPLC-MS and 1H-NMR spectroscopic studies on acyl migration reaction kinetics of model drug ester glucuronides

Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-β-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an α,α-dimethylated ibuprofen analogue. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37°C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and 1H-NMR spectroscopy and the results compared. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, 1H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of 1H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than 1H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-β-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.