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.

600 MHz 1H-NMR spectroscopy of human cerebrospinal fluid: effects of sample manipulation and assignment of resonances.

Extensive assignments of resonances in the 600 MHz 1H-NMR spectra of cerebrospinal fluid are reported. These have been achieved by the measurement of a combination of two-dimensional experiments comprising homonuclear J-resolved, COSY45, and double-quantum filtered COSY (DQCOSY) spectra. By these means the previous total of 18 endogenous metabolites, of which in general only selected resonances have been assigned, has been augmented to 46 molecules including all of the resonances of both alpha- and beta-anomers of glucose. With only a few exceptions all resonances have been assigned for all of the metabolites. In addition, the effect of freeze-drying on the 600 MHz 1H-NMR spectrum of human cerebrospinal fluid (CSF) is presented using both lyophilization with reconstitution into either H2O or D2O. Freeze-drying and reconstitution into H2O causes a significant sharpening of many small molecule resonances, including notably those of glutamate and glutamine as well as other amino acids and in addition causes the loss of volatile components, principally acetone. Further exchange of the H2O solvent by D2O causes no additional changes in the spectra.

NMR spectroscopy of human post mortem cerebrospinal fluid: distinction of Alzheimer's disease from control using pattern recognition and statistics.

1H NMR spectra have been measured at 500 and 600 MHz on 23 human cerebrospinal fluid samples obtained at autopsy from Alzheimer's disease patients and controls. The spectra at 500 MHz were quantified using 42 descriptors based on NMR peak heights and it was shown that differences between the two classes were apparent in the delta 2.4-2.9 region. Remeasured at 600 MHz a detailed examination of this chemical shift range identified citrate, aspartate, N-acetyl aspartate, methionine and glutamate in this region of the spectra. Principal components analysis showed that a separation of the two classes was possible and detailed statistics indicated that citrate level was the principal marker. Patient age and the interval between death and autopsy (parameters not closely matched between the two groups) were examined statistically to establish whether these might account for the citrate differences. Although they could possibly account for them to some extent, the relationship between citrate levels and disease state remained significant at p < 0.05. The data invite a test of the importance of citrate levels in Alzheimer's disease using samples taken ex vivo.

Quantitative structure-metabolism relationships for substituted benzoic acids in the rat. Computational chemistry, NMR spectroscopy and pattern recognition studies.

An extensive set of computed molecular properties, both steric and electronic, have been calculated using molecular orbital and empirical methods for benzoic acid (1) and a congeneric series of substituted benzoic acids, i.e. 2-, 3- and 4-fluorobenzoic acids (2-4), 2-, 3- and 4-trifluoromethyl benzoic acids (5-7), 2-, 3- and 4-methylbenzoic acids (8-10), 4-amino benzoic acid (11), 2-fluoro-4-trifluoromethyl benzoic acid (12), 4-fluoro-2-trifluoromethyl benzoic acid (13), 3-trifluoromethyl-4-fluorobenzoic acid (14). We have monitored the urinary excretion profiles and determined the metabolic fate of compounds 2-7, 12-14 in the rat using high resolution 1H and 19F NMR spectroscopy. Corresponding data for compounds 1,8-11 are taken from the literature. In all cases phase II glucuronidation or glycine conjugation reactions dominated the metabolism of these compounds. Compounds 5, 7, 12, 13 have ester glucuronides as their major metabolites; the rest primarily form glycine conjugates. Compounds (1-12) have been classified according to their calculated physicochemical properties using pattern recognition methods and principal components maps have been used as a novel type of structure-metabolism diagram. The maps of compounds in the physicochemical property space served to separate the compounds into the two major classes which related to their principal metabolic fate in vivo, namely glucuronidation versus glycine conjugation. Compounds 13 and 14 were used as further probes of the property space, and dominant metabolic fates of glucuronidation and glycine conjugation, respectively, were predicted from the previous "training set map". The metabolic fate of compounds 1-14 can thus be classified according to a simple set of physicochemical rules. Investigation of the physicochemical properties which are important in distinguishing the metabolic fate of the compounds may give insight into key features of the drug-metabolizing enzyme active sites and hence provide information on basic mechanisms of benzoate metabolism.

NMR-monitored solid-phase extraction of phenolphthalein glucuronide on phenylboronic acid and C18 bonded phases.

Preliminary studies have been undertaken to evaluate the potential of immobilized phenylboronic acid (PBA) for the solid-phase extraction (SPE) of glucuronide metabolites from urine. These studies have demonstrated that immobilized PBA can be used to specifically extract phenolphthalein glucuronide (5 mM) from urine. Urine samples were loaded onto the PBA SPE column in glycine buffer (pH 8.5) and were eluted using methanol-1% HCl (90:10, v/v). The overall recoveries of the phenolphthalein glucuronide for this procedure were high (99%), which compared well with similar studies carried out concomitantly on C18 bonded columns (93%).