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J K Nicholson

Publications and source records attributed to J K Nicholson.

At least 127 records · Page 7Linked to original sources

Uroscopy in the 21st century: high-field NMR spectroscopy.

From the experiments described, it can be seen that there are different research approaches that can be taken and these are summarized in Table 1. Whereas much scientific research is principally hypothesis led, there remains, nevertheless, an important place for exploratory research. High resolution NMR can measure, directly and simultaneously, a wide range of endogenous metabolites in biological fluids and has the unique capability of providing structural information on the metabolites detected. It has proved to be a powerful research tool with which to study inherited metabolic diseases, renal disease, drug metabolism, and toxicity, and can be used to monitor the effects of drug therapy. For instance, by using a library of experimental toxins one can map the metabolic profile of site-specific nephron injury. With this approach in man one could eventually take an unknown disease such as Balkan nephropathy and predict the initial site of tubular injury, the mode of injury and therefore the kind of toxin capable of producing that injury. NMR spectroscopic techniques are still advancing rapidly, with ever increasing sensitivity and sophistication of NMR pulse sequences to enhance structural elucidation in complex mixtures. Given the advances in directly coupled HPLC-NMR and even HPLC-NMR-mass spectroscopy it is likely that these technologies in conjunction with pattern recognition will make major contribution to our understanding of renal processes and provide new diagnostic insights in the 21st century.

Forecasting↗

Evaluation of a method for counting absolute numbers of cells with a flow cytometer.

We evaluated a method for performing absolute cell counts of lymphocyte populations with a flow cytometer. In this method, TruCount, test tubes that contain a known number of brightly fluorescent polystyrene beads are provided by the manufacturer. Whole anticoagulated blood is accurately pipetted into the tubes and mixed with fluorochrome-labeled monoclonal antibodies, the erythrocytes are lysed, and this mixture is analyzed on the flow cytometer. Absolute counts of lymphocyte subsets are calculated by determining the ratio of beads to the cell population of interest and then multiplying this ratio by the number of beads in the tube. We found this method to be reproducible. The values we obtained by the TruCount method were 5 to 10% higher than those obtained by conventional methods (flow cytometry and automated hematology) used to determine absolute numbers of cells. We believe that these differences are due to the methods of determining absolute cell counts and not to faulty identification of lymphocyte subsets.

Adolescent↗

Urinary proton magnetic resonance studies of early ifosfamide-induced nephrotoxicity and encephalopathy.

Ifosfamide is an oxazophosphorine widely used in the treatment of cancer in children and adults. Nephrotoxicity and neurotoxicity are major side effects. The aim of this study was to use high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy of urine to identify novel biochemical markers of ifosfamide-induced toxicity. Urine samples were collected from 10 nonencephalopathic patients (who had not previously received nephrotoxic chemotherapy) immediately prior to the first ifosfamide dose and at timed intervals for up to four treatment cycles. The findings were compared with those for urine samples collected from five patients during acute encephalopathic episodes. 1H NMR urinalysis identified a series of characteristic time-related changes in the excretion profiles of low molecular weight endogenous metabolites during ifosfamide therapy. These changes included a decreased excretion of hippurate and an increased excretion of glycine, histidine, glucose, lactate, and trimethylamine-N-oxide. Two nonencephalopathic patients had marked but transient glutaric or adipic aciduria during the second cycle of ifosfamide treatment. Urinary retinol-binding protein rose acutely after each treatment cycle but usually returned to baseline levels. Maximum renal toxicity was observed by the fourth treatment cycle. The ratio of the urinary excretion of the uroprotectant mesna (active form) to dimesna (inactive form) correlated with the degree of renal toxicity. For the encephalopathic patients, the ifosfamide-induced changes in the urinary low molecular weight metabolite profile were similar to those for the nonencephalopathic group. In contrast to previous reports, none of the encephalopathic group developed glutaric aciduria, and i.v. methylene blue did not reverse neurotoxicity in the two patients who received it. The results suggest that ifosfamide nephrotoxicity involves both cortical and medullary regions of the nephron and that the urinary mesna:dimesna ratio may be important in assessing the degree of cytoprotection. This study demonstrates that 1H NMR can provide novel biochemical information on ifosfamide-induced toxicity and will be of value in the optimization of ifosfamide therapy.

Adult↗

Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine.

The direct coupling of HPLC with NMR spectroscopy has been extended by splitting the HPLC eluent after conventional UV detection and sending part to a NMR spectrometer and part to an ion-trap mass spectrometer in a "triplehyphenated" HPLC-NMR-MS system. Combined UV, 1H NMR, and positive-ion electrospray MS detection was achieved in the continuous-flow mode using whole human urine from a subject dosed with acetaminophen. By means of HPLC-NMR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirmation of the identity of the acetaminophen glucuronide and sulfate metabolites, together with a number of endogenous metabolites. In particular, the HPLC-NMR-MS approach allowed the unequivocal identification of phenylacetylglutamine in human urine, an endogenous metabolite not previously observed in 1H NMR spectra of urine because of extensive overlap with resonances from other metabolites. The analytical advantages and complementarity of NMR and MS techniques in direct hyphenation with HPLC are discussed. The new technique of HPLC-NMR-MS will provide the scope for more comprehensive and fully automated analysis of biofluids and other complex mixtures than was previously available from single hyphenation of these instruments.

Acetaminophen↗

Development of a simple liquid chromatographic method for the separation of mixtures of positional isomers and anomers of synthetic 2-, 3- and 4-fluorobenzoic acid glucuronides formed via acyl migration reactions.

Many drugs containing carboxylate groups form beta-1-O-acyl glucuronides as their major phase II metabolites in vivo. These ester glucuronides are potentially reactive due to the susceptibility of the acyl group to nucleophilic reactions resulting in hydrolysis, acyl migration or covalent adduct formation. In the present study, a number of synthetic fluorobenzoic acid glucuronide conjugates were chosen as models for chromatographic studies. A high-performance liquid chromatography method is presented for the simultaneous determination of the 1-, 2-, 3- and 4-positional isomers of the acyl glucuronides, and their alpha- and beta-anomers for the 2-, 3- and 4-fluorobenzoic acids as well as each aglycone formed as a result of hydrolysis. The same elution order was found for the acyl migrated glucuronide isomers of the three fluorobenzoic acids in their equilibrium mixtures. The alpha-4-O-acyl isomer eluted first followed by the beta-4-O-acyl isomer, then the beta-1-O-acyl, the beta-3-O-acyl, the alpha-3-O-acyl, the alpha-2-O-acyl and finally the beta-2-O-acyl isomer eluted. The method was used to determine the overall degradation rates, the acyl migration rates and the hydrolysis rates of 1-O-(2-fluorobenzoyl)-beta-D-glucopyranuronic acid 1-O-(3-fluorobenzoyl)-beta-D-glucopyranuronic acid and 1-O-(4-flurobenzoyl)-beta-D-glucopyranuronic acid in a buffer system pH 7.4 at 25 degrees C. It was found that the order of beta-1-glucuronide acyl migration rates was 2-fluorobenzoyl > 3-fluorobenzoyl > 4-fluorobenzoyl. Both the acyl migration rates and the elution order were interpreted in terms of electronic effect of the fluorine substituent on the carbonyl carbon.

Acylation↗

High-resolution diffusion and relaxation edited one- and two-dimensional 1H NMR spectroscopy of biological fluids.

A new approach to the characterization of biomolecules in whole biological fluids is presented based on simplification of 1H NMR spectra by utilizing differences in molecular diffusion coefficients alone and combinations of relaxation and diffusion parameters. New NMR pulse sequences incorporating both spectral editing features together with solvent water resonance elimination are presented. The methods are exemplified using whole human blood plasma, and it is shown that it is possible to obtain NMR spectra of the slowly diffusing species (generally large molecules) by diffusion editing, the slowly relaxing species (generally small molecules) by spin relaxation editing, or spectra showing any range of molecular mobility using a combination of the two methods. The diffusion-based editing methods are also applicable to the selection of resonances in two-dimensional NMR spectroscopy of biofluids, and we show this for the first time by the production of 1H-1H diffusion-edited TOCSY spectra of human blood plasma where the resonance intensities are weighted according to the molecular diffusion coefficient. In this case, by measuring a diffusion-edited 1H-1H TOCSY NMR spectrum of plasma, it is possible to obtain signals from only the macromolecular components, and this may be of benefit in the analysis of blood lipoproteins. In complex biofluids, the combination of diffusion and relaxation editing brings about considerable spectral simplification leading to an easier resonance assignment process. We also demonstrate the production of 1H NMR spectra with intensities corresponding to diffusion coefficient rather than number of protons, and this opens up new possibilities for pattern recognition classification of samples based on altered molecular mobility features of biofluid components.

Blood↗

Direct characterization of drug glucuronide isomers in human urine by HPLC-NMR spectroscopy: application to the positional isomers of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid glucuronide.

In this work, 400 and 600 MHz 1H HPLC-NMR spectroscopic methods were developed and applied to separate and identify the positional glucuronide isomers and anomers of the model nonsteroidal antiinflammatory drug, 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid, in whole human urine. The HPLC methods utilized either an isocratic system, comprising 30% acetonitrile in water at pH 2.5, or a gradient elution system increasing from 30% to 60% acetonitrile, in order to achieve improved separation of the 2-, 3-, and 4-O-acylglucuronide isomers from the faster eluting endogenous urinary metabolites. Directly coupled stop-flow 1H HPLC-NMR spectroscopic measurements were made at the retention times indicated by the UV-monitored chromatographic peaks. The glucuronide isomers were identified from the 1H NMR spectra on the basis of their chemical shifts and spin-spin coupling patterns. The elution order was 4-O-acyl-, 3-O-acyl-, and finally 2-O-acylglucuronide, with tR values of 10.04, 11.68, and 12.64 min, respectively. Although the alpha- and beta-anomers of each of the positional isomers could not be separated in these solvent systems, they could be identified in the individual 1H NMR spectra. This work shows for the first time that directly coupled HPLC-NMR spectroscopy can be used directly to isolate and characterize acyl-migrated isomers of drug glucuronides in whole urine. This approach will be of value in the study of glucuronide acyl migration reactions of nonsteroidal antiinflammatory drugs and other xenobiotic ester glucuronides in whole biofluids.

Acetates↗

Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry.

The light-scatter characteristics of lymphocytes are commonly used to gate lymphocytes for further analysis in a lysed whole-blood assay. Because lymphocytes can be identified by antigens that they possess, a light-scatter gate can be validated by measuring parameters other than light scatter. When a specimen possesses poor light scatter (usually from contaminating nonlymphocytes within the light-scatter gate for lymphocytes), the quality of the gate and, thus, the analyses of lymphocyte subsets can be compromised. We present data to demonstrate the use of CD45 fluorescence combined with side scatter (SSC) for analyzing lysed whole-blood specimens. When we compared CD45/SSC to light scatter (forward and side scatter) for validating a lymphocyte gate, both methods performed similarly in recovering as many lymphocytes as possible in the gate (lymphocyte recovery); however, the CD45/SSC gate had fewer contaminants within the gate (lymphocyte purity). Replicate CD3 values from the CD45/SSC gate were less variable than those from the light-scatter gate, confirming that most of the variability in a light-scatter gate is due to nonlymphocyte contaminants in the gate. We propose that lymphocytes be gated using CD45 fluorescence as well as side-scattering properties and that CD3 also be included in each data analysis tube for quality control.

Acquired Immunodeficiency Syndrome↗

750 MHz HPLC-NMR spectroscopic studies on the separation and characterization of the positional isomers of the glucuronides of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid.

Ester glucuronides (beta-1-O-acyl-D-glucopyranuronates) of many drugs can undergo a series of acyl migration reactions, resulting in positional isomers and anomers which can react with serum proteins with possible toxicological consequences. We have investigated the acyl migration of the ester glucuronides of the model drug 6,-11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid in pH 7.4 buffer using directly coupled 750 MHz stopped-flow HPLC-NMR spectroscopy. Using a reversed phase isocratic HPLC method with 21% acetonitrile and 79% D2O in the mobile phase, it was possible to separate and hence identify the individual positional isomers of the model drug glucuronide by 750 MHz HPLC-NMR. The order of elution of the isomers from the C18 column was 4alpha-, 4beta-, aglycon, 1beta-, 3beta-, 3alpha-, 2alpha-, 2beta- (alpha- and beta- referring to the anomerization state at C1 on the glucuronide ring and the numbers referring to the carbon number on the glucuronide ring to which the drug moiety has migrated). It is shown that directly coupled ultra-high-field HPLC-NMR spectroscopy offers a unique analytical advantage for obtaining structural information of interconverting compounds in equilibrium mixtures, and this method will be of value in the study of reactive drug glucuronides of toxicological importance.

Acetates↗

Aspergillosis of the sphenoid sinus: presentation as a pituitary mass and postoperative gallium-67 imaging.

Invasive aspergillosis of the paranasal sinuses is an uncommon, but well-recognized, form of fungal infection. We describe an elderly female patient with invasive aspergillosis of the sphenoid sinus and associated osteomyelitis of the skull base, with clinical presentation as a pituitary mass. Postoperative gallium scan showed intense uptake in the sphenoid sinus, which resolved after treatment with amphotericin-B. This case demonstrates some of the pathologic and clinical features of invasive aspergillosis and suggests a potential role for gallium-67 imagining in monitoring clinical response to therapy.

Aged↗

Comparative biochemical effects of low doses of mercury II chloride in the F344 rat and the multimammate mouse (Mastomys natalensis).

The biochemical effects and comparative nephroxicity of mercury II chloride (HgCl2) dosed at 0.75 mg/kg i.p. was investigated in the Fisher 344 rat (F344) and Mastomys natalensis using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy of urine, histopathology and clinical chemical techniques. The effects of HgCl2 treatment were followed for up to 4 days post-dosing (p.d.). In F344 rats there was extensive proximal tubular damage and renal cortical necrosis together with elevated levels of urinary gamma-glutamyl transpeptidase (gamma GT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The 1H NMR spectra of urine obtained from Hg-treated F344 rats also showed increased levels of glucose, alanine, lactate, valine and hippurate (0-48h p.d.) with decreased levels of citrate, succinate and 2-oxoglutarate (24-48h p.d.). Mastomys were found to be highly resistant to HgCl2 toxicity at 0.75 mg/kg and the histological appearance of the renal cortex of treated animals was virtually identical to controls. There were no elevations in urinary ALP, gamma GT and LDH activities in HgCl2-treated Mastomys and there were no biochemical abnormalities in low MW components of Mastomys urine following HgCl2-treatment, as shown by 1H NMR spectroscopy. Urinary gamma GT activity was found to be much higher in F344 rats than Mastomys. Since gamma GT activity is involved in the tubular reabsorption of Hg2+, the lower levels of gamma GT in Mastomys might partially account for the lower toxicity of Hg2+ in this species.

Animals↗

NMR spectroscopic and theoretical chemistry studies on the internal acyl migration reactions of the 1-O-acyl-beta-D-glucopyranuronate conjugates of 2-, 3-, and 4-(trifluoromethyl) benzoic acids.

High resolution 19F NMR spectroscopy has been used to investigate the kinetics of internal acyl migration and hydrolysis of the synthetic beta -1-O-acyl-D-glucopyranuronates of 2-, 3-, and 4-(trifluoromethyl) benzoic acids (TFMBAs) in phosphate buffer solutions at 30 degrees C as models of drug ester glucuronides. Apparent first-order degradation of the 1-O-acyl glucuronide and the sequential appearance of 2-, 3-, and 4-O-acyl isomers as both alpha- and beta-anomeric forms were observed for each TFMBA isomer. The overall degradation rate constants of the 2-, 3-, and 4-TFMBA 1-O-acyl isomers were 0.065 h-1, 0.25 h-1, and 0.52 h-1. In order to probe the reasons for these differences in reactivity, theoretical structural and electronic parameters for the beta-anomers of the 1-O-acyl glucuronides, their beta-2-O-acyl isomers, and both structures of the postulated ortho-acid ester intermediate were computed using semiempirical molecular orbital (AM1 and PM3) methods. The distinction between the slowly reacting 2-TFMBA glucuronide and the much faster reacting 3- and 4-TFMBA glucuronides could be observed by calculation of the relative bond order of the C-O bonds in the ortho-acid ester intermediates. The slow internal acyl migration rate of the 2-TFMBA isomer was also partly attributed to the high degree of steric hindrance of the trifluoromethyl group obstructing attack by the glucuronic acid 2-hydroxy group on the carbonyl carbon to form the ortho-acid ester intermediate. Some calculated molecular orbital properties, namely, dipole moment, energy of the lowest unoccupied molecular orbital (LUMO), LUMO density, and nucleophilic frontier density on the carbonyl carbon, were also shown to be related to the measured half-lives. This work gives insight into the molecular physicochemical properties that influence the acyl migration kinetics of simple model drug glucuronides and is of potential importance in understanding more complex drug glucuronide acyl migration reactions of toxicological interest.

Acylation↗

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.

Animals↗

Mechanisms of human immunodeficiency virus Type 1 (HIV-1) neutralization: irreversible inactivation of infectivity by anti-HIV-1 antibody.

An assay for the neutralization of human immunodeficiency virus type 1 (HIV-1) is described in which the reduction in infectious titer of HIV-1 after preincubation at 37 degrees C with antibody-positive serum is the measure of neutralization. The assay format and its controls allow several experimental manipulations that, taken together, indicate an effect of antibody on HIV-1 infectivity that occurs before or independently of HIV-1 attachment. The direct inactivation of HIV-1 infectivity by antibody is irreversible and temperature dependent, requires a bivalent antibody directed against accessible envelope determinants, and does not require a heat-labile or (Ca2+)- or (Mg2+)-dependent cofactor. The mechanism of inactivation cannot be explained by agglutination of virus, nor is it associated with disruption or dissociation of envelope protein from virions. Rather, the antibody is likely to perturb some metastable property of the envelope that is required for entry. Laboratory-adapted HIV-1 isolates were more sensitive to the inactivating effects of sera than were primary patient isolates. The latter were particularly resistant to inactivation by contemporary autologous sera, a feature not explained by blocking antibodies. Additional studies showed a weak relationship between disease course and serum inactivation of the reference LAI laboratory strain of HIV-1. Heteroduplex analysis and autologous inactivation assays of sequential specimens from individual patients indicate that over time, the viral quasispecies that emerge and dominate are resistant to the inactivating effects of earlier sera.

Animals↗

Quantitative structure-metabolism relationships for substituted benzoic acids in the rabbit: prediction of urinary excretion of glycine and glucuronide conjugates.

1. Quantitative relationships between molecular physicochemical properties of 24 substituted benzoic acids and their metabolic fate in the rabbit have been investigated using computational chemistry and multivariate statistical methods. 2. A total of 34 molecular properties were calculated for each compound using computational chemistry and were related statistically to the % molar recovery of glycine and glucuronide conjugates excreted in the urine of rabbits for the 24 compounds. 3. Compounds were successfully classified according to their dominant metabolic fate based on urinary excretion data, where stepwise linear regression analysis of the theoretical property data achieved good predictive fits for observed versus predicted % molar urinary recovery as glucuronide conjugates (r2 = 0.79) and % molar urinary recovery as glycine conjugates (r2 = 0.66). 4. Quantitative prediction of the urinary excretion of glucuronide and glycine conjugates of the parent compounds was achieved based on a statistical model using calculated molecular physicochemical parameters. Interpretation of the molecular properties, which are important for predicting the metabolic class, should give new insights into basic mechanisms of drug metabolism and underlying molecular recognition events that determine disposition and metabolism.

Animals↗

1H and 19F-nmr spectroscopic studies on the metabolism and urinary excretion of mono- and disubstituted phenols in the rat.

1. 1H and 19F-nmr spectroscopy was used to investigate quantitatively the urinary excretion of the metabolites of 15 substituted phenols in the rat. The compounds studied were: 2-, 3-, and 4-fluorophenols; 2-, 3-, and 4-trifluoromethylphenol; 2,4-, 2,6- and 3,4-difluorophenol; 2-fluoro-5-trifluoromethylphenol, 3-fluoro-5-trifluoromethylphenol, 2-trifluoromethyl-4-fluorophenol; 3-chloro-4-fluorophenol, 3-fluoro-4-chlorophenol, and 3-methyl-4-fluorophenol. All compounds were dosed to the Sprague-Dawley rat (10 mg/kg i.p.) and urine was collected over the periods 0-8, 8-24 and 24-48 h post-dosing and analyzed using nmr spectroscopy. 2. The compounds were excreted in the urine mainly as glucuronide or sulphate conjugates or as the unchanged parent compound. There was considerable variation in the urinary excretion of the compounds over 48 h ranging from 22.1 to 93.6% of the dose. There was no apparent relationship between the molecular weight of compounds or their metabolites and the percentage molar recovery of each in the urine. 3. Ortho-substituted phenols in general showed a greater propensity for glucuronidation than did either meta- or para-substituted compounds, irrespective of the substituent group. The molar glucuronide-to-sulphate ratio for ortho-substituted compounds was found to be 2.2 +/- 0.9 whereas the ratio for both meta- and para-substituted compounds was 0.8 +/- 0.2 (p < 0.0001). 4. There were characteristic substituent effects of phenolic glucuronidation or sulphation on the 19F-nmr chemical shifts for both F- and CF3-substituted phemols and these substituent effects were a useful aid to metabolite signal assignment. 5. These studies show that nmr spectroscopy provides a rapid and convenient approach to the construction of metabolic databases of simple xenobiotics for the investigation of structure-metabolism relationships.

Animals↗