The testis is the principal organ of male fertility, responsible for the production of spermatozoa and their maturation into sperm. However, the underlying biochemistry of the testis is relatively understudied. The fluidic and homogeneous nature of the testis makes it an ideal organ for high resolution magic angle spinning (MAS) 1 H NMR spectroscopy. In this study we have catalogued the low molecular weight metabolites. The testis contains large amounts of creatine, of which a substantial proportion was shown to be extracellular using bipolar gradients to measure apparent diffusion coefficients. The tissue also contained relatively high amounts of uridine. ß

1. The products arising from intramolecular acyl migration reactions of drug ester glucuronides are reactive towards cellular proteins and can potentially cause toxic side-effects. The relationship between molecular structure and the degradation rates (kd) of 1beta-O-acyl glucuronides were investigated systematically using a series of model compounds based on 4-substituted benzoic acids. 2. A rational method for selecting suitable compounds for inclusion was used and 10 glucuronide esters, predicted to produce a wide range of transacylation rates, were synthesized via a simple "one-pot" method using an imidazolide intermediate. The 10 substituents, where X = NO2, CN, I, Br, F, H, nPr, Et, OMe, O-nPr, had degradation rate half-lives (t1/2 = loge(2)/kd) ranging from 0.9 to 106.6 h. The reactions resulted in mixtures, which predominantly consisted of the desired 1beta-O-acyl glucuronides. 3. It was demonstrated that further purification was unnecessary for determination of kd of the synthetic 1beta-O-acyl glucuronides. Degradation rates (kd) were calculated by following the disappearance of the 1H-NMR signal from the 1beta-anomeric proton of the glucuronic acid moiety as the reaction progressed in pH 7.4 buffer inside an nuclear magnetic resonance tube. Each measured degradation rate represents a pseudo-first-order rate constant, which is a combination of the transacylation rate (1beta to 2beta isomer) and the hydrolysis rate. 4. Degradation rates show a clear relationship with substituent properties, with half-life increasing as the substituent becomes more electron-donating, e.g. 4-nitro t1/2 = 0.9 h and 4-propoxy t1/2 = 106.6 h.

High-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy is ideal for monitoring the metabolic environment within tissues, particularly when spectra are weighted by physical properties such as T 1 and T 2 relaxation times and apparent diffusion coefficients (ADCs). In this study, spectral-editing using T 1 and T 2 relaxation times and ADCs at variable diffusion times was used in conjunction with HRMAS 1 H NMR spectroscopy at 14.1 T in liver tissue. To enhance the sensitivity of ADC measurements to low molecular weight metabolites a T 2 spin echo was included in a standard stimulated gradient spin-echo sequence. Fatty liver induced in rats by chronic orotic acid feeding was investigated using this modified sequence. An increase in the combined ADC for the co-resonant peaks glucose, betaine, and TMAO during fatty liver disease was de- Key words: apparent diffusion coefficient; fatty liver; spectraleditingThe observation of metabolites within intact tissue using 1 H NMR spectroscopy is confounded by a number of physical factors which broaden spectral resonances, including dipolar couplings, chemical shift anisotropy, and bulk magnetic susceptibility differences. High-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy negates much of the impact of these phenomena (1). This is further aided by the liquid nature of the cytosol and small susceptibility effects across tissues, producing spectral linewidths comparable to the solution-state for tissue samples (Յ1 Hz) (2-4).However, resonances from one metabolite may be coresonant with those from another, making assignments and quantification difficult, despite the resolution improvements given by HRMAS, especially for tissues with high lipid content (3,5,6). For metabolites that are coresonant with intense lipid signals, spectral editing is needed either making use of the molecular environment (7) or using selective pulse sequences (8).Spectra may be edited by attenuating 1 H resonances with relatively short T 2 relaxation times using spin echo delays in a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. As T 2 relaxation is primarily influenced by rotational correlation times, the CPMG pulse sequence reduces the intensities of resonances of large immobile species, such as lipids, relatively more than low molecular weight mobile metabolites. However, the disadvantages of using long T 2 spin echo delays include heating of the sample and J-modulation of strongly coupled resonances. Conversely, the resonance contributions of fast-moving molecules can be reduced using diffusion ordered spectroscopy (DOSY) (3,9), relying on the application of field gradients across a sample so that signal intensity is attenuated according to the diffusion rate of the molecule. Apparent diffusion coefficients (ADCs) are not only determined by molecular size, but also by cellular environment, being influenced by viscosity of the surrounding medium and the size of the subcellular compartment in which the metabolite is located. Furthermore, Pfeuffer et al. (10) have elegantly s...

A widely held view in drug metabolism and pharmacokinetic studies is that the initial 1-isomer to 2-isomer step in the intramolecular acyl migration of drug ester glucuronides is irreversible, and that alpha-1-O-acyl isomers do not occur under physiological conditions. We investigated this hypothesis using high-performance liquid chromatography directly coupled to proton nuclear magnetic resonance spectroscopy (HPLC/1H NMR) and mass spectrometry (LC/MS) to probe the migration reactions of S-naproxen beta-1-O-acyl glucuronide, in phosphate buffer at pH 7.4, 37 degrees C. We report the first direct observation of the alpha-1-O-acyl isomer of a drug ester glucuronide (S-naproxen) formed in a biosystem via the facile acyl migration of the corresponding pure beta-1-O-acyl glucuronide. The unequivocal identification of the reactive product was achieved using stopped-flow one-dimensional HPLC/1H NMR and two-dimensional 1H-1H total correlation spectroscopy (1H-1H TOCSY). Parallel LC/ion-trap mass spectrometry yielded the confirmatory glucuronide masses. Moreover, "dynamic" stopped-flow HPLC/1H NMR experiments revealed transacylation of the isolated alpha-1-O-acyl isomer to a mixture of alpha/beta-2-O-acyl isomers; the reverse reaction from the isolated alpha/beta-2-O-acyl isomers to the alpha-1-O-acyl isomer was also clearly demonstrated. This application of "dynamic" stopped-flow HPLC/1H NMR allows key kinetic data to be obtained on a reactive metabolite that would otherwise be difficult to follow by conventional HPLC and NMR methods where sample preparation and off-line separations are necessary. These data challenge the widely held view that the alpha-1-O-acyl isomers of drug ester glucuronides do not occur under physiological conditions. Furthermore, the similar formation of alpha-1-O-acyl isomers from zomepirac and diflunisal beta-1-O-acyl glucuronides has recently been confirmed (Corcoran et al., unpublished results). Such reactions are also likely to be widespread for other drugs that form ester glucuronides in biological systems. Ultimately, the presence of significant quantities of the kinetically labile alpha-1-O-acyl glucuronide isomer may also have toxicological implications in terms of reactivity toward cellular proteins.

The novel application of magic angle spinning 1 H NMR spectroscopy, coupled with pattern recognition techniques, has identified biochemical changes in lipid and glutamate metabolism that precede classical nephrotoxicity. These changes occurred in the bank vole (Clethrionomys glareolus) after chronic dosing, at a low level of exposure and at a renal Cd 2+ concentration (8.4 W Wg/g dry wt) that was nearly two orders of magnitude below the WHO critical organ concentration (200 W Wg/g wet wt). These early stage effects of Cd 2+ on the biochemistry of renal tissue may reflect adaptation mechanisms to the toxic insult or the preliminary stages of the toxicological cascade. ß