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Biomedical subjects

J K Nicholson

Publications and source records attributed to J K Nicholson.

At least 163 records · Page 9Linked to original sources

Application of the one-dimensional TOCSY pulse sequence in 750 MHz 1H-NMR spectroscopy for assignment of endogenous metabolite resonances in biofluids.

The complex 1H-NMR spectrum arising from an intact biofluid has been simplified using a one-dimensional homonuclear polarization transfer experiment (known as TOCSY or HOHAHA). This approach establishes connectivity between sequentially coupled multiplets, and the method is illustrated by the confirmation of the chemical shifts and hence resonance assignment of a number of endogenous metabolites in the 750 MHz 1H-NMR spectrum of seminal fluid. This has allowed the detection and assignment of pyroglutamate and uracil in this fluid for the first time.

Computer Simulation↗

Automatic reduction of NMR spectroscopic data for statistical and pattern recognition classification of samples.

A general method of automatically reducing NMR spectra to provide numerical descriptors of samples has been developed and investigated. These descriptors can be used as input to pattern recognition or multivariate algorithms for sample classification. The methods have been tested using 600 MHz one-dimensional 1H NMR spectra of biofluids which are complex mixtures. The approach is, in principle, applicable to multidimensional and heteronuclear NMR spectra and to other types of liquid samples such as oils and foodstuffs as well as to situations such as 1H or 31P NMR in vivo and solid state NMR in drug formulation analysis. The method relies upon apportioning the information in the spectra to individual contiguous segments and allowing specified regions of the spectra to be omitted. Three approaches, based on the number of peaks, the summed peak heights and the summed peak areas respectively in each segment, have been tested. The effect of segment width and overlap and the effects of manipulation of the NMR spectra have been evaluated in terms of the classification of the samples using principal components analysis. A simple method of generating NMR based spectral descriptors for object classification is thus proposed.

Algorithms↗

Ultra high field NMR spectroscopic studies on human seminal fluid, seminal vesicle and prostatic secretions.

Ultra high field 1H-NMR spectroscopic methods have been used to analyse the composition of seminal fluid and its component secretions, prostatic and seminal vesicle fluids from normal human subjects and those with vasal aplasia and non-obstructive infertility. The 1H-NMR spectrum of whole seminal fluid is extremely complex and many resonances are extensively overlapped in single pulse spectra even when measured at 600 or 750 MHz 1H resonance frequency. A combination of 2-D 1H-NMR methods (including J-Resolved and various 1H homonuclear correlation and 1H-13C heteronuclear correlation techniques) were applied at 600 or 750 MHz in order to extensively assign the signals from the organic components of seminal fluid. Prostatic fluid (PF) gives a much less complex metabolite profile than whole seminal fluid and can be completely analysed using 500 MHz 1H-NMR spectroscopy. The 1H-NMR spectra of prostatic fluid are dominated by signals from citrate, spermine and myo-inositol, whereas the spectra of seminal vesicle fluid (SVF) show extensively overlapped signals from complex peptide mixtures together with strong signals for glycerophosphocholine (GPC) and lactate. Whole seminal fluid is a combination of the PF and SVF constituents together with further substances that appear after mixing due to the operation of PF enzymes on SVF, e.g. peptidase activity causes rapid cleavage of peptides to amino acids and GPC is hydrolysed to choline, glycerol and inorganic phosphate. It is also shown that vasal aplasia leads to highly characteristic abnormal metabolite profiles in seminal fluid that can be readily observed in single-pulse 500 and 600 MHz 1H-NMR spectra. Measurement of the molar citrate to choline, or spermine to choline ratios in seminal fluid both show differences of 2 orders of magnitude between vasal aplasia (greater for both ratios) and non-obstructed infertile patients. This work gives an indication of the potential of high field 1H-NMR spectroscopy in the investigation and assessment of the secretory functions of the male genital tract and the evaluation of the infertile male subject.

Amino Acids↗

High resolution 1H NMR spectroscopic studies of the metabolism and excretion of ampicillin in rats and amoxycillin in rats and man.

High resolution proton nuclear magnetic resonance (1H NMR) spectroscopy has been used to investigate the metabolism and urinary excretion of the aminopenicillins, ampicillin and amoxycillin, in rats and of amoxycillin in man. 1H NMR resonances of the aminopenicillins, together with those for their 5R, 6R and 5S, 6R penicilloic acids and diketopiperazine metabolites were detected, assigned and quantified in urine samples with the aid of spin-echo NMR techniques. The dimer of amoxycillin was detected in rat urine for the first time together with novel drug-related resonances assigned to amoxycillin carbamate. Quantitative 1H NMR spectroscopic results were consistent with HPLC and microbiological data considering that only single measurements were recorded. Due to the short analysis time and simple sample preparation, NMR was particularly useful for studying the metabolism of the aminopenicillins for which sample degradation poses analytical problems. The non-invasive character of 1H NMR spectroscopic analysis of urine also provided unique information on a reversible reaction between amoxycillin and bicarbonate, an endogenous urinary metabolite.

Amoxicillin↗

Pattern recognition classification of the site of nephrotoxicity based on metabolic data derived from proton nuclear magnetic resonance spectra of urine.

The computer-based pattern recognition procedures of nonlinear mapping and principal-component analysis have been applied to analyze 1H NMR-generated metabolic data on the biochemical effects of 15 acute nephrotoxin treatments affecting the renal cortex and/or renal medulla in rats. The 1H NMR signal intensities for 16 urinary metabolites representative of several major intermediary biochemical pathways were estimated using either a simple semiquantitative scoring system or complete peak intensity quantitation. NMR-derived data were treated as input coordinates in a multidimensional metabolic space and were analyzed by pattern recognition methods through which the dimensionality was reduced for display and categorization purposes. Different nephrotoxin treatments were initially classified using semiquantitative metabolite scores on the basis of their 1H NMR-detectable biochemical effects, and a good separation of renal cortical toxin treatments from renal medullary toxin treatments was achieved. The refinement of using exact peak heights rather than metabolic data scores utilized the available metabolic information more fully and provided a unique classification of each type of toxin according to its pattern of biochemical effects and site of toxic action. Principal-component analysis provided consistently better results than did nonlinear mapping in terms of discrimination between different sites of toxicity, and maps generated from correlation matrices gave improved discrimination, compared with those based directly on the original metabolic data. A comparison between the use of an added internal quantitation standard (3-trimethylsilyl-[2,2,3,3-2H4]-1-propionate) and independently determined glucose excretion rates for scaling to the NMR-detected urinary glucose levels demonstrated that the consistent classification of site-specific nephrotoxicity was independent of the quantitation standard used. This study has provided a rigorous assessment of data processing, relative quantitation, and pattern recognition methods, and the utility of applying these methods to the classification of NMR-derived toxicological data. The considerable potential of the NMR-pattern recognition approach in the assessment of nephrotoxicity has also been confirmed with the discovery of new combinations of molecular markers of renal cellular damage.

Animals↗

Studies of the biochemical toxicology of uranyl nitrate in the rat.

High resolution 1H NMR spectroscopy of urine and plasma, conventional clinical chemical methods and histopathology have been applied to investigate the effects of uranyl nitrate (UN) on renal function and biochemistry in the Fischer 344 (F344) rat. Administration of UN (5-20 mg/kg) to male F344 rats resulted in a dose-related proximal nephropathy assessed conventionally by histopathology and urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), and related to changes in the patterns of low MW metabolites observed in 400 MHz 1H NMR spectra of urine. The changes in urinary metabolite profiles included elevations in glucose accompanied by minor elevations in certain amino acids (alanine, valine and glutamate). 1H NMR urinalysis also revealed altered excretion of low MW metabolites which are not routinely measured, such as L-lactate, acetate, citrate, succinate and 2-oxoglutarate (2-OG). In addition, the striking appearance of high concentrations of 3-D-hydroxybutyrate (HB) in the urine was noted, in the absence of acetoacetate or acetone, and it is suggested that this may provide a new marker of proximal tubular damage for certain types of nephrotoxic mechanism. Broadening of the 1H NMR signals of citrate following 10 mg/kg UN was shown to be due to a dynamic exchange process involving chelation with urinary Ca2+ and Mg2+ ions. Conventional biochemical analysis of plasma from UN-treated rats revealed dose-related increases in creatinine, urea and HB concentrations. 1H NMR-detected evidence of raised alanine amino-transferase (ALT) levels in rats administered the highest dose of UN was indicated by the partial deuteration of alanine in lyophilised plasma reconstituted in 2H2O. The degree of 1H NMR-detected abnormalities agreed well with histopathological observations and conventional biochemical indices of nephrotoxicity and more fully characterised the renal changes produced by UN. The significance of HB-uria in UN-induced proximal nephropathy is discussed in relation to biochemical observations on other proximal nephrotoxins.

Animals↗

Quality control in immunophenotyping: U.S. efforts to establish common methodology and their impact. American Society for Histocompatibility and Immunogenetics.

Several organizations in the United States have recently been involved in the quality assurance of immunophenotyping using flow cytometry. These activities include publication of guidelines for immunophenotyping, particularly in human immunodeficiency virus infection, performance evaluation and proficiency testing programs, and training and continuing education programs. Through questionnaires and results from performance evaluation programs, variability in test results has been decreasing the last few years, and more laboratories are beginning to use the same specimen processing and data analysis procedures.

Flow Cytometry↗

Selection of anticoagulants for lymphocyte immunophenotyping. Effect of specimen age on results.

In a multi-center study, whole blood specimens from 31 HIV-positive and 43 HIV-negative donors were collected in three different anticoagulants and assayed for lymphocyte subsets fresh (within 6 h), and 1 and 2 days later. Each center prepared the specimens by their routine whole blood lysis procedure, labeling with a recommended panel of two-color monoclonal antibody combinations. 1 day (up to about 30 h) after blood collection, the results obtained from blood collected in EDTA (ethylenediamine tetra-acetate), ACD (acid citrate dextrose), and heparin were similar to fresh. Up to 48 h, only ACD and heparin, not EDTA, yielded results similar to fresh specimens. These results were similar for both HIV-positive and -negative specimens.

Antibodies, Monoclonal↗

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.

Acetaminophen↗

Investigation of the human metabolism of antipyrine using coupled liquid chromatography and nuclear magnetic resonance spectroscopy of urine.

The potential of coupled high-performance liquid chromatography-nuclear magnetic resonance spectroscopy for the detection and identification of drug metabolites has been investigated by direct analysis of human urine collected following administration of antipyrine. This approach provided a rapid method of characterizing the major human urinary metabolites of this drug and promises to be of widespread value in structural studies of xenobiotic metabolites.

Antipyrine↗

Inactivation of HIV-infected H9 cells in whole blood preparations by lysing/fixing reagents used in flow cytometry.

Reagents that lyse red blood cells and fix white blood cells were tested for their ability to inactivate cell-associated human immunodeficiency virus (HIV). Whole blood was spiked with cells from an HIV-positive cell line (H9), lysed, and fixed. The cell preparations were then cocultured with T cell blasts in serial ten-fold dilutions to rescue infectious virus and measure viral titer. All commercial lysing and fixing reagents tested inactivated cell-associated HIV by 3-5 logs, while ammonium chloride had little effect. Although an additional incubation with 1% formaldehyde for 30 min did not increase the effectiveness of the commercial lysing/fixing reagents, it did inactivate cell-associated HIV in blood treated with ammonium chloride.

Cell Death↗

High-performance liquid chromatography coupled to high-field proton nuclear magnetic resonance spectroscopy: application to the urinary metabolites of ibuprofen.

The use of coupled reversed-phase high-performance liquid chromatography and high-field proton nuclear magnetic resonance spectroscopy (HPLC-NMR) for the detection and identification of the urinary metabolites of ibuprofen is described. Urine was obtained from a healthy human volunteer following a normal therapeutic dose of 400 mg of ibuprofen. Analysis was performed on both a freeze-dried urine concentrate and partially purified extracts obtained by solid-phase extraction onto C-18 bonded silica gel. Both continuous and stop-flow methods were used to obtain spectra enabling the major urinary metabolites of ibuprofen to be detected and identified.

Chromatography, High Pressure Liquid↗

Idiopathic CD4+ T-lymphocytopenia--an analysis of five patients with unexplained opportunistic infections.

BACKGROUND: Although patients with idiopathic CD4+ T-lymphocytopenia and serious opportunistic infections have been described previously, the clinical and immunologic features of this condition have not been well defined. METHODS: We studied in detail five patients with idiopathic CD4+ T-lymphocytopenia. The studies included serologic testing, culture, and polymerase chain reaction for the human immunodeficiency virus (HIV) types 1 and 2, serologic testing for the human T-cell lymphotropic virus (HTLV) types I and II, lymphocyte phenotyping, immunoglobulin quantitation, and lymphocyte-transformation assays, as well as attempts to isolate a retroviral agent. The results were compared with those in HIV-infected persons matched for CD4+ T-cell counts and with those in normal controls. We also studied the spouses of patients and the blood donors for one patient. RESULTS: In these five patients, there was no evidence of either HIV or HTLV infection. All the patients had both low percentages and low counts of CD4+ T cells, with relative increases in percentages, but not counts, of CD8+ cells. Numbers of B cells and natural killer cells were generally normal. As compared with HIV-infected persons, our patients had lower percentages and counts of CD8+ cells and more lymphopenia. CD4+ counts were relatively stable over time. Instead of the high immunoglobulin levels seen in HIV infection, these patients had normal or slightly low levels of immunoglobulins. The lymphocyte-transformation response to mitogens and antigens was depressed. Results in spouses and blood donors were normal. CONCLUSIONS: Idiopathic CD4+ T-lymphocytopenia differs from HIV infection in its immunologic characteristics and in its apparent lack of progression over time. Nothing about the immunologic or viral-culture studies performed in these patients or about their family members or blood donors suggests that a transmissible agent causes this condition.

Adult↗

CD4 T-lymphocyte determinations on whole blood specimens using a single-tube three-color assay.

We evaluated a flow cytometric method for determining the proportion of CD4-positive T lymphocytes in whole blood using a single three-color tube containing fluorochrome-labeled CD45, CD3, and CD4. Various ways of gating this sample were evaluated and results were compared with data obtained in our standard six-tube, two-color assay gated on light scatter parameters. Excellent correlation was found between the three-color analysis using a CD45/side scatter gate and the standard two-color analysis with a light scatter gate. This single-tube three-color test is a promising assay for monitoring CD4+ T-lymphocytes.

Adult↗

Studies on the effects of L(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) on 4-aminophenol-induced nephrotoxicity in the Fischer 344 rat.

4-Aminophenol (para-aminophenol; PAP) causes selective necrosis to the S3 segment of the proximal tubule in experimental animals. The mechanism of PAP nephrotoxicity has not been fully elucidated, although it has been suggested to involve glutathione (GSH)-dependent S-conjugation followed by processing by the enzyme gamma-glutamyl transpeptidase (gamma GT) to the corresponding cysteine S-conjugate. This proposed toxicity mechanism was probed further by administering L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), a potent gamma GT inhibitor, to Fischer 344 (F344) rats before treatment with PAP (100 mg/kg). AT-125 pretreatment did not appear to protect against PAP-induced nephrotoxicity as assessed by renal histopathology, clinical chemistry and proton nuclear magnetic resonance (1H NMR) spectroscopy of urine. These data suggest that renal gamma GT activity is not a prerequisite for PAP nephrotoxicity and that the generation of a cysteine S-conjugate is not a unique requirement for the induction of PAP nephrotoxicity.

Aminophenols↗

Gastro-intestinal availability of aluminium from tea.

The in vitro speciation of aluminium (Al) in black tea infusion (pH 4.8) was assessed using 3000, 10,000 and 30,000 Da cut-off ultrafilters, and the effect of adding human gastric juice (pH 2.3) and then raising the pH to 6.5 were also studied. 78% Al in the tea infusion passed through the 3000-Da ultrafilter; this percentage increased to more than 90% with the addition of gastric juice at pH 2.3, but then reduced to approximately 5% when the incubate was adjusted to pH 6.5. The breakdown of tea-derived polyphenols to low molecular weight phenols in vivo was measured using high-resolution 1H nuclear magnetic resonance spectroscopic analysis of ileostomy effluent, but there was no evidence of low molecular weight breakdown products from the polyphenols of ingested tea in this effluent. These results suggest that only a small proportion of Al in tea is potentially available for absorption throughout the small bowel. It may be misleading to estimate systemic Al absorption from tea drinking simply from total urinary aluminium excretion as has been done previously.

Aluminum↗

Raised transaminase activity of blood plasma from rats with experimentally-induced kidney damage detected by spin-echo 1H-NMR spectroscopy.

We report the application of spin-echo 1H-NMR spectroscopy to the detection of raised plasma transaminase activity in rats treated with the nephrotoxic cephalosporin antibiotic cephaloridine (CPH). Spin-echo 1H-NMR analysis of lyophilized plasma, reconstituted in H2O reveals a doublet at delta 1.48 for alanine. However when samples were reconstituted with 2H2O we noted that in samples from CPH-treated rats (but not in control samples) there was a variable degree of appearance of a singlet at delta 1.47 together with a reduction in the doublet at delta 1.48. We suggest that this is due to the release of transaminases from damaged tissue which, via a reversible conversion of alanine to pyruvate, causes selective deuteration of alanine at the alpha-hydrogen (alpha-CH) position. This observation suggests that these 1H-NMR spectral patterns are dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.

Alanine↗