Statistically integrated metabonomic-proteomic studies on a human prostate cancer xenograft model in mice.

A novel statistically integrated proteometabonomic method has been developed and applied to a human tumor xenograft mouse model of prostate cancer. Parallel 2D-DIGE proteomic and 1H NMR metabolic profile data were collected on blood plasma from mice implanted with a prostate cancer (PC-3) xenograft and from matched control animals. To interpret the xenograft-induced differences in plasma profiles, multivariate statistical algorithms including orthogonal projection to latent structure (OPLS) were applied to generate models characterizing the disease profile. Two approaches to integrating metabonomic data matrices are presented based on OPLS algorithms to provide a framework for generating models relating to the specific and common sources of variation in the metabolite concentrations and protein abundances that can be directly related to the disease model. Multiple correlations between metabolites and proteins were found, including associations between serotransferrin precursor and both tyrosine and 3-D-hydroxybutyrate. Additionally, a correlation between decreased concentration of tyrosine and increased presence of gelsolin was also observed. This approach can provide enhanced recovery of combination candidate biomarkers across multi-omic platforms, thus, enhancing understanding of in vivo model systems studied by multiple omic technologies.

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.

Metabonomics, dietary influences and cultural differences: a 1H NMR-based study of urine samples obtained from healthy British and Swedish subjects.

The aim of this study was to assess the feasibility and comparability of metabonomic data in clinical studies conducted in different countries without dietary restriction. A (1)H NMR-based metabonomic analysis was performed on urine samples obtained from two separate studies, both including male and female subjects. The first was on a group of healthy British subjects (n = 120), whilst the second was on healthy subjects from two European countries (Britain and Sweden, n = 30). The subjects were asked to provide single, early morning urine samples collected on a single occasion. The (1)H NMR spectra obtained for urine samples were visually inspected and analysed chemometrically using principal components analysis (PCA). These inspections highlighted outliers within the urine samples and displayed interesting differences, revealing characteristic dietary and cultural features between the subjects of both countries, such as high trimethylamine-N-oxide (TMAO)-excretion in the Swedish population and high taurine-excretion, due to the Atkins diet. This study suggests that the endogenous urinary profile is subject to distinct cultural and severe dietary influences and that great care needs to be taken in the interpretation of 'biomarkers of disease and response to drug therapy' for diagnostic purposes.

An NMR study of the metabolic fate of 2-, 3- and 4-fluorobenzyl alcohols in the rat: detection of N-acetylcysteinyl conjugates as minor metabolites in urine.

Following the administration of 2-, 3- and 4-fluorobenzyl alcohols, the major metabolites detected in urine corresponded to the glycine conjugates of the corresponding benzoic acids. Little, or no, unchanged parent compound was detected in the samples. In addition to glycine-conjugated benzoic acids, a small proportion of the urinary metabolites for each of the fluorobenzyl alcohols was found to correspond to N-acetylcysteinyl conjugate. These were probably formed as the result of the production of a reactive sulphate ester during metabolism. The overall urinary recoveries of metabolites for the 2- and 3-fluorobenzyl alcohols were lower than that observed for the corresponding benzoic acids whilst that for 4-fluorobenzyl alcohol was similar.

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.

Spectroscopic characterisation and identification of ecdysteroids using high-performance liquid chromatography combined with on-line UV--diode array, FT-infrared and 1H-nuclear magnetic resonance spectroscopy and time of flight mass spectrometry.

A prototype multiply hyphenated reversed-phase HPLC system has been applied to the analysis of a mixture of pure ecdysteroids and an ecdysteroid-containing plant extract. Characterisation was achieved via a combination of diode array UV, 1H NMR, FT-IR spectroscopy and time of flight (TOF) mass spectrometry. This combination of spectrometers allowed the collection of UV, 1H NMR, IR and mass spectra for a mixture of pure standards enabling almost complete structural characterisation to be performed. The technique was then applied to a partially purified plant extract in which 20-hydroxyecdysone and polypodine B were identified despite incomplete chromatographic resolution and the presence of co-chromatographing interferents. The experimental difficulties in the use of such a systems for these analytes are described.

A randomized trial comparing bladder infusion with standard catheter removal after transurethral resection of the prostate.

OBJECTIVE: To examine the effect of bladder infusion before catheter removal on patients' readiness for discharge and the day of discharge after transurethral resection of the prostate (TURP). PATIENTS AND METHODS: The study comprised 75 consecutive patients undergoing TURP who were randomized to either have their catheter removed in the standard manner (38 patients), or to undergo bladder infusion before a trial of voiding (ToV) on the second day after TURP (37 patients). RESULTS: In those undergoing bladder infusion, seven (19%) patients were discharged on the same day as their ToV, compared with five (13%) in the standard group. Of the 75 patients, 15 (68%) were discharged by the third day after TURP whether or not the bladder had been filled. In the infusion group, 23 (62%) were ready for discharge on the same day as their TOV, compared with only 14 (37%) in the standard group (P < 0.05). CONCLUSION: Bladder infusion before a ToV after TURP significantly increases the rate of readiness for discharge, allowing an early decision to discharge on the second day in a large proportion of patients.

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 HPLC-NMR-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 approximately 7-10% of the urinary metabolites, corresponding to approximately 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.

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.

Bioanalysis of p-trifluoromethylmandelic acid and Mosher's acid by chiral gas chromatography and fluorine nuclear magnetic resonance to study chiral inversion: application to rat urine samples.

Methods for the nuclear magnetic resonance and gas chromatographic analysis of the enantiomers of p-trifluoromethylmandelic acid (p-TFM) and Mosher's acid (alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid) present in rat urine samples are described. Gas chromartography was performed using cyclodextrin capillary columns with both compounds analysed following derivatisation with methanolic HCl. Nuclear magnetic resonance was performed directly on the untreated urine samples following addition of beta-cyclodextrin. The methods were suitable for the determination of the individual enantiomers of the analytes in urine. Analysis of the rat urine samples indicated that the p-TFM had undergone a unidirectional enantiomeric interconversion in vivo, while the enantiomers of Mosher's acid were excreted unchanged.

Nuclear magnetic resonance and high-performance liquid chromatography-nuclear magnetic resonance studies on the toxicity and metabolism of ifosfamide.

A combination of high-resolution nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC)-NMR spectroscopic methods has been used to analyse urine from humans and rats treated with the anticancer drug ifosfamide. It was possible to detect a range of abnormal endogenous metabolites in urine after ifosfamide administration to human subjects undergoing cancer therapy and to relate the metabolic perturbations to the nephrotoxic effects of the drug. Changes observed by 1H NMR included increases in levels of urinary glucose, glycine, alanine, histidine, lactate, acetate, succinate, and trimethylamine-N-oxide and decreases in the levels of hippurate and citrate. Additional evidence was gained that ifosfamide-induced nephrotoxicity might be related to the level of oxidation of the coadministered drug mesna. By using both directly coupled continuous-flow 31P HPLC-NMR spectroscopy to determine the retention times of the phosphorus-containing metabolites and, subsequently, stop-flow 1H HPLC-NMR of the urine, it was possible to isolate and identify on-line the metabolites ifosfamide mustard, 4-hydroxy-ifosfamide, 2-dechloroethylifosfamide, and the parent compound itself. These studies illustrate the potential of combining 1H NMR spectroscopy of biofluids and HPLC-NMR spectroscopy for the investigation of drug metabolism and toxicity in humans and animals.

High resolution NMR spectroscopic studies on the metabolism and futile deacetylation of 4-hydroxyacetanilide (paracetamol) in the rat.

Paracetamol (4-hydroxyacetanilide, acetaminophen) was synthesized with the acetyl group labelled with C2H3 (paracetamol-C2H3), and dosed to rats i.p. at 25 mg/kg (N = 5) and 40 mg/kg (N = 3) body weight. Paracetamol, with a 13CH3 in the acetyl group (paracetamol-13CH3) was also synthesized and dosed to rats i.p. at 40 mg/kg (N = 3). The metabolism and excretion of the 2H-labelled compound was followed in the rat using 600 MHz 1H and 92.1 MHz 2H NMR spectroscopy of urine collected 0-8, 8-24, 24-32 and 32-48 hr post-dosing. The metabolism of paracetamol-13CH3 was also monitored using 600 MHz 1H NMR spectroscopy of urine collected 0-8, 8-24 and 24-48 hr post-dosing. For paracetamol-C2H3 the total recovery of the sulphate, glucuronide and N-acetyl cysteinyl metabolites via the urine accounted for 61.2 +/- 14.1% of the 25 mg/kg dose and 61.4 +/- 8.8% of the 40 mg/kg dose. For paracetamol-13CH3 the recovery was 102.7 +/- 3.7% indicating that the low % urinary recovery with the C2H3-labelled drug is the result of isotope effects on the disposition of paracetamol. In the case of the paracetamol-C2H3, quantitative 1H NMR analysis of urine showed that 13.3 +/- 0.5 and 10.0 +/- 1.2 mole % (25 and 40 mg/kg, respectively) of the urinary paracetamol sulphate recovered following dosing of the deuterium labelled drug had the C2H3 acetyl groups replaced by C1H3 acetyl groups from endogenous sources. In the case of the paracetamol-13CH3 8.9 +/- 0.7 mole % of the sulphate conjugate had also been transacetylated to paracetamol-12CH3. There was no significant difference between the level of futile deacetylation observed for the deuterated and 13C-labelled drug. Overall these data indicate a high level of deacetylation followed by reacetylation (i.e. futile deacetylation) prior to excretion of paracetamol via the nephrotoxic intermediate 4-aminophenol. The level of deacetylation is much higher than has previously been thought which may cast new light on the role of 4-aminophenol in the development of paracetamol induced nephrotoxicity.

Induction of 5-oxoprolinuria in the rat following chronic feeding with N-acetyl 4-aminophenol (paracetamol).

The urine of rats fed on 1% paracetamol in the diet for up to 10 weeks was analysed using 500 MHz 1H NMR spectroscopy. After 3 weeks, paracetamol-dosed rats were found to excrete massive quantities of an unknown metabolite in the urine. Using a range of 1 and 2 dimensional 1H NMR spectroscopic techniques, solid phase extraction and mass spectrometry, the metabolite was identified at 5-oxoproline (5OXP, pyroglutamic acid). Rats fed paracetamol plus methionine, which prevents the depletion of sulphur-containing amino acids, did not develop 5OXP-uria during the study period. Quantitative 1H NMR spectroscopy of whole urine showed that no 5OXP appeared in the urine in the first 2 weeks of feeding paracetamol to the animals, but urinary concentrations then rose rapidly up to 1 M in some animals. This unusually high concentration of 5OXP in the urine and its prevention by methionine indicates that chronic high level paracetamol dosing leads to severe depletion of sulphur-containing amino acids including cysteine with consequent disruption of the glutathione cycle.

Apple ripening-related cDNA clone pAP4 confers ethylene-forming ability in transformed Saccharomyces cerevisiae.

The apple ripening-related cDNA insert of clone pAP4 (G.S. Ross, M.L. Knighton, M. Lay-Yee [1992] Plant Mol Biol 19: 231-238) has previously been shown to have considerable nucleic acid and predicted amino acid sequence similarity to the insert of a tomato ripening-related cDNA clone (pTOM13) that is known to encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase (A.J. Hamilton, G.W. Lycett, D. Grierson [1990] Nature 346: 284-287; A.J. Hamilton, M. Bouzayen, D. Grierson [1991] Proc Natl Acad Sci USA 88: 7434-7437). The cDNA insert from the clone pAP4 was fused between the galactose-inducible promoter and the terminator of the yeast expression vector pYES2. Transformation of Saccharomyces cerevisiae strain F808- with this DNA construct and incubation of the yeast in the presence of D[+]-galactose allowed these cells to convert ACC to ethylene. The transformed yeast converted 1-amino-2-ethylcyclopropane-1-carboxylate isomers to 1-butene with the same 1R,2S-stereoselectivity as achieved by the native ACC oxidase from applies. Both ascorbate and Fe2+ ions stimulated the rate of the production of ethylene from ACC by the transformed yeast, whereas Cu2+ and Co2+ were strongly inhibitory; these are features of ACC oxidase. Northern analysis of the total RNA from nontransformed and transformed yeast showed that the ability to convert the ACC to ethylene was correlated with the synthesis and accumulation of a novel 1.2-kb mRNA that hybridized to the cDNA clone pAP4. We conclude that the cDNA sequence of the clone pAP4 encodes ACC oxidase.

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage.

Differential screening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7 degrees C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.

Application of high resolution 1H-NMR spectroscopy to the detection of penicillamine and its metabolites in human urine.

Preliminary studies on the use of high resolution 1H-NMR spectroscopy for the detection of the thiol drug penicillamine and its metabolites in human urine are described. The technique is rapid, simple and requires minimal sample pretreatment. Application of NMR to the qualitative analysis of penicillamine in urine is illustrated by penicillamine disulphide formation from penicillamine following spiking into human urine and the detection of penicillamine, penicillamine disulphide and penicillamine-cysteine disulphide (following oral administration of the drug to patients).

Immunohistochemical localization of IgA and secretory component in rat liver.

IgA is transported from rat blood into bile. The localization of IgA and secretory component in rat liver was studied in order to facilitate understanding of this IgA transport. Both indirect immunofluorescence and unlabeled antibody enzyme histochemical techniques were used. Immunoglobulin A was found surrounding bile canaliculi, within bile duct epithelium, on or near the plasma membranes of hepatocytes, within the cytoplasm of a few intensely stained hepatocytes, within the cytoplasm of intensely reactive cells found in or adjacent to the sinusoids, and in sparsely distributed plasma cells. Reactivity for secretory component was present surrounding bile canaliculi, within bile duct epithelium, or or near the plasma membranes of hepatocytes and in the cytoplasm of hepatocytes. These findings are compatible with the hypothesis that immunoglobulin A is transported from blood to bile through hepatocytes by a process involving secretory component. Bile duct epithelium may also have a role in immunoglobulin A transport.

Comparison of high-performance liquid chromatography and gas chromatography for the analysis of ecdysteroids.

High-performance liquid chromatography (HPLC) with ultraviolet absorption detection, and gas chromatography (GC) with an electron-capture detector have been compared for their convenience in the analysis of ecdysteroids in invertebrate tissues. Analysis by HPLC on reversed-phase materials, including C18, C22 and CN bonded phases, was explored for the separation of both polar ecdysteroids and some of their possible biosynthetic intermediates of lower polarity. The HPLC method was found to be rapid and easy to use, but less sensitive than GC with electron-capture detection. The greater sensitivity and selectivity of GC was found to be important for the more difficult analyses.