Pattern of Change in Renal Function Following Radical Nephrectomy for Renal Cell Carcinoma.

Radical nephrectomy (RN) is an independent risk factor for the development of chronic kidney disease (CKD) in those with renal cell carcinoma (RCC). We aimed to examine the pattern of change in post-operative renal function in patients who underwent RN for RCC over a 3 year period at our institution. We performed a retrospective review of histological and biochemical findings in patients undergoing RN for RCC over a 38 month period. Estimated glomerular filtration rate (eGFR) was recorded pre- and post-operatively and at follow-up. We analysed data on 131 patients (median follow-up 24 months). The proportion of patients with advanced CKD increased significantly at follow-up with 48 (85.7%) patients, classified as having stage 2 CKD pre-operatively, being re-classified as stage 3-5. Mean eGFR was significantly lower pre-operatively (76.6 mL/min/1.73 m2) compared to hospital discharge (61 mL/min/1.73 m2, p < 0.001) and follow-up (55.5 mL/min/1.73 m2, p < 0.001). Those with pT1 tumours sustained a significantly greater decline in eGFR compared to other stages. In conclusion, patients with pT1 a and pT1 b tumours sustain a disproportionate decline in renal function and may benefit the most from NSS.

High-resolution magic-angle spinning 13C NMR spectroscopy of cerebral tissue.

Monitoring the metabolism of (13)C-labelled substrates by biological tissues allows both the rate of metabolism and the relative importance of metabolic pathways to be determined. In this study high-resolution magic-angle spinning (HRMAS) (13)C NMR spectroscopy is assessed as a technique for determining the labelling of metabolites in brain slices. Freshly prepared rat brain slices were superfused in isotonic salt solution containing [1-(13)C] glucose. HRMAS (1)H and (13)C NMR spectra were acquired of the slices ( approximately 10 mg) at 3 degrees C. Using (1)H NMR spectroscopy it was demonstrated that the concentration of key metabolites indicative of metabolic degradation, including N-acetyl aspartate and lactate, did not change significantly across the approximately 11 h time period required for (13)C NMR spectra. The approach produced high-resolution spectra of intact tissue with the labelling patterns of tissues being indicative of both labelling via pyruvate dehydrogenase found in both neuronal and glial cells, and pyruvate carboxylase, found only within glial cells. This approach is a versatile tool for monitoring the compartmentation of metabolites directly, and will also allow the investigation of aqueous and lipid metabolites simultaneously.

Advancing NMR sensitivity for LC-NMR-MS using a cryoflow probe: application to the analysis of acetaminophen metabolites in urine.

Cryogenic cooling of the NMR radio frequency coils and electronics to give greatly enhanced sensitivity is arguably the most significant recent advance in NMR spectroscopy. Here we report the first cryogenic probe built in flow configuration and demonstrate the application to LC-NMR-MS studies. This probe provides superior sensitivity over conventional noncryogenic flow NMR probes, allowing the use of 100 microL of untreated urine (40% less material than previous studies that required preconcentration) and yet revealing drug metabolites hitherto undetected by LC-NMR-MS at 500 MHz. Besides the known sulfate and glucuronide metabolites, previously undetected metabolites of acetaminophen were directly observable in a 15-min on-flow experiment. Simultaneous MS data also provided knowledge on the NMR-silent functional moieties. Further, stop-flow LC-NMR-MS experiments were conducted for greater signal-to-noise ratios on minor metabolites. The cryoflow probe enables the NMR analysis of lower concentrations of metabolites than was previously possible for untreated biofluids. This strategy is generally applicable for samples containing mass-limited analytes, such as those from drug metabolism studies, biomarker and toxicity profiling, impurity analysis, and natural product analysis.

HPLC/1H NMR spectroscopic studies of the reactive alpha-1-O-acyl isomer formed during acyl migration of S-naproxen beta-1-O-acyl glucuronide.

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.

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.

S-naproxen-beta-1-O-acyl glucuronide degradation kinetic studies by stopped-flow high-performance liquid chromatography-1H NMR and high-performance liquid chromatography-UV.

Acyl-migrated isomers of drug beta-1-O-acyl glucuronides have been implicated in drug toxicity because they can bind to proteins. The acyl migration and hydrolysis of S-naproxen-beta-1-O-acyl glucuronide (S-nap-g) was followed by dynamic stopped-flow HPLC-1H NMR and HPLC methods. Nine first order rate constants in the chemical equilibrium between six species (S-nap-g, its alpha/beta-2-O-acyl, alpha/beta-3-O-acyl, alpha/beta-4-O-acyl, and alpha-1-O-acyl-migration isomers, and S-naproxen aglycone) were determined by HPLC-UV studies in 25 mM potassium phosphate buffer, pH 7.40, 25 mM potassium phosphate buffer in D2O pD 7.40, and 25 mM potassium phosphate buffer in D2O pD 7.40/MeCN 80:20 v/v (HPLC-1H NMR mobile phase). In the 25 mM potassium phosphate buffer (pH 7.40) the acyl-migration rate constants (h(-1)) were 0.18 (S-nap-g-alpha/beta-2-O-acyl isomer), 0.23 (alpha/beta-2-O-acyl-alpha-1-O-acyl), 2.6 (alpha-1-O-acyl-alpha/beta-2-O-acyl), 0.12 (alpha/beta-2-O-acyl-alpha/beta-3-O-acyl), 0.048 (alpha/beta-3-O-acyl-alpha/beta-2-O-acyl), 0.059 (alpha/beta-3-O-acyl-alpha/beta-4-O-acyl), and 0.085 (alpha/beta-4-O-acyl-alpha/beta-3-O-acyl). The hydrolysis rate constants (h(-1)) were 0.025 (hydrolysis of S-nap-g) and 0.0058 (hydrolysis of all acyl-migrated isomers). D2O and MeCN decreased the magnitude of all nine kinetic rate constants by up to 80%. The kinetic rate constants for the degradation of S-nap-g in the mobile phase used for HPLC-1H NMR determined using HPLC-UV could predict the results obtained by the dynamic stopped-flow HPLC-1H NMR experiments of the individual acyl-migrated isomers. It is therefore recommended that beta-1-O-acyl glucuronide degradation kinetics be investigated by HPLC-UV methods once the identification and elution order of the isomers have been established by HPLC-1H NMR.

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.

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.

Directly coupled liquid chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the identification of drug metabolites in urine: application to diclofenac using chlorine and sulfur detection.

We report the application of high-performance liquid chromatography (HPLC) linked to inductively coupled plasma mass spectrometry (ICPMS) and orthogonal acceleration time-of-flight mass spectrometry (oa-TOFMS) for the identification of phase I and II urinary metabolites of diclofenac. The metabolites were separated by reversed-phase HPLC monitored with a UV diode array detector (UV-DAD) after which 90% of the eluent was directed to an ICPMS source, with the remainder going to an oa-TOF mass spectrometer. Compounds containing (35)Cl, (37)Cl and (32)S were detected specifically using ICPMS and identified by oa-TOFMS. The metabolites detected and identified in this way included glucuronic acid and sulfate conjugates, mono- and dihydroxylated and free diclofenac. In addition a previously unreported in vivo metabolite, an N-acetylcysteinyl conjugate of diclofenac, was also characterised. This is the first application of the combination of HPLC/UV-DAD/ICPMS/oa-TOFMS for the investigation of the metabolic fate of chlorinated xenobiotics by direct biofluid analysis.

750 MHz HPLC-NMR spectroscopic identification of rat microsomal metabolites of phenoxypyridines.

Directly coupled 750 MHz HPLC-1H NMR spectroscopy has been applied to the characterisation of low level metabolites of 3-amino-2-(2-fluorophenoxy)pyridine (AP) and 3-nitro-2-(2-fluorophenoxy)pyridine (NP) in rat microsomes. In stop-flow HPLC-NMR mode, the direct injection of microsomal extracts enabled the separation and characterisation of minor metabolites. NP is converted into AP to an extent of 93.4% and this is further metabolised to 4- and 6-hydroxy-AP (6 and 0.6% respectively). Unequivocal identification of these metabolites was achieved without the use of a radiolabel or synthetic standards and thus demonstrates the applicability of directly coupled HPLC-NMR to metabolite identification in in vitro systems. The potential exists for HPLC-NMR and HPLC-NMR-MS to provide rapid metabolic information within the timescale of high throughput lead optimisation exercises in drug discovery.