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D Perrett

Publications and source records attributed to D Perrett.

At least 19 recordsLinked to original sources
PubMed1998

Determination of allantoin in biofluids using micellar electrokinetic capillary chromatography.

A micellar electrokinetic chromatographic method is described for the determination and quantitation of allantoin, an end-product of purine metabolism in mammals that is applicable to biofluids of different mammal species and man. The method was optimised following a study on the effect of pH and sample preparation procedure. Final conditions were 30 mM sodium tetraborate, pH 9.5, 75 mM sodium dodecyl sulphate, 20 kV and 20 degrees C. Allantoin was well resolved from endogenous compounds and could be determined in horse, dog, mouse and rabbit urine. No allantoin could be found in man. No complicated sample treatment was necessary, thus the developed method was rapid (< 5 min), sensitive (5 microM) and simple. Results from this work will permit the determination of allantoin in man as a measure of free radical generation reactions as well as its presence in the plasma and other biofluids with modification of the sample preparation procedures.

Allantoin↗
PubMed1998

Automated on-line solid-phase extraction and high-performance liquid chromatographic analysis of total and free pyridinium crosslinks in serum.

An automated on-line solid-phase extraction procedure for free and total pyridinium crosslinks in serum, with HPLC analysis, is described. The pyridinium crosslinks either following hydrolysis in 3 M HCl or free in neat serum were extracted using a Gilson Aspec XLi system onto extraction cartridges containing an octylsilane/cation exchanger sorbent. Up to 525 microliters of serum could be loaded onto the extraction cartridges. After washing, the crosslinks were eluted with 400 microliters of 100 mM sodium formate pH 5 and 380 microliters of this was concentrated on a RPB guard column eluted with 100 mM HFBA. The crosslinks were backflushed and separated on a 5-micron ODS analytical column eluted with 30 mM HFBA with 18% MeCN at 1 ml/min and detected by their native fluorescence (excitation 295 nm, emission 400 nm). The use of a high sensitivity fluorescence detector was essential. Recoveries were 95-100% with a limit of detection (S/N = 2) of 109 pmol/l (pM) for pyridinoline (Pyr) and 143 pM for deoxypyridinoline (dPyr). The inter-assay R.S.D. was 9% for pyridinoline and 10.8% for deoxypyridinoline. The throughput of the system was up to 50 samples per day.

Amino Acids↗
PubMed1998

Myocardial ischemic injury and purine metabolism in patients undergoing coronary artery bypass.

OBJECTIVES: High-energy phosphates and their catabolic products were determined in myocardium during coronary artery bypass surgery with blood cardioplegic reperfusion in order to evaluate the effects of aortic cross-clamping and reoxygenation on myocardial purine metabolism. DESIGN AND METHODS: Transmural left ventricular biopsy specimens were taken with ITu-Cut biopsy needles, before aortic cross-clamping, before cross-clamp removal and after 30' of reperfusion; perchloric extracts of the material were analyzed for nucleotide content by capillary zone electrophoresis (CZE). The CZE procedure used separates the complete spectrum of purine metabolites in myocardial extracts obtained from 0.6-8.6 mg biopsy material. RESULTS: The basal values of ATP/ADP ratio and energy charge were low, IMP content was high. After the ischemic period, ATP levels further decreased and IMP, nucleosides and bases accumulated. After reperfusion, nucleoside and base basal levels, but not energy charge, were restored to some extent. CONCLUSIONS: The study arises the problem of myocardial preservation during heart surgery. In this investigation, capillary electrophoresis was an extremely adaptable technique for the evaluation of ischemic injury and could be useful in studying the effects of cardioplegic solutions.

Aged↗
PubMed1998

Serum collagen crosslinks as markers of bone turn-over during GH replacement therapy in growth hormone deficient adults.

OBJECTIVES: Bone metabolism is an important target for GH replacement therapy. However, in adults, treatment periods exceeding 12 months are required for a positive effect of GH on bone mineral density. Thus, to detect an early effect of GH on bone, markers of bone turn-over are important. Pyridinoline (PYR) and deoxypyridinoline (DPYR) are well-defined sensitive markers of bone resorption, but to date only urinary assays have been available. We report the use of a novel assay to measure changes in serum PYR and DPYR in GH deficient (GHD) adults during GH replacement therapy. STUDY DESIGN: The study consisted of a 6-month randomized, double-blind, placebo-controlled study of the administration of GH (Genotropin) (0.25 IU/Kg/week (0.125 IU/kg/week for the first four weeks)) followed by a 6-month open phase of GH therapy. PATIENTS: Thirty-five GHD adults (17 women; mean age 39.8 years; range 21.1-59.9) on conventional hormone replacement therapy as required, were studied. MEASUREMENTS: Bone formation was analysed using serum bone alkaline phosphatase (BAP) and serum osteocalcin (OC). Bone resorption was analysed using serum pyridinoline (PYR) and serum deoxypyridinoline (DPYR). Bone mineral density (BMD) was determined by dual energy X-ray absorptiometry (DEXA). RESULTS: After 6 months placebo treatment there were no significant changes in any of the bone markers analysed, nor in BMD. In the active arm of the study there was a significant increase in serum OC, BAP, PYR and DPYR (P = 0.03, P = 0.004, P = 0.003 and P = 0.01, respectively), remaining significantly elevated over their baseline levels for the subsequent 6 months of treatment (P = 0.04, P = 0.009, P = 0.003 and P = 0.04, respectively). No changes were observed in BMD in any of the groups after 6 months GH treatment. In the active arm of the study, after 12 months GH treatment there was a significant increase in BMD at both the lumbar spine and femoral neck (P = 0.01 for both sites). CONCLUSIONS: In summary, the present study confirms that administration of GH treatment to GHD adult patients significantly activates bone remodelling, with the effect of GH both in bone formation and bone resorption markers being maximal after 6 months of treatment. The serum assay for PYR and DPYR has a number of practical and theoretical advantages over the urine assay and gave similar results to those previously reported for the urine assay.

Adult↗
PubMed1998

Postprandial plasma 5-hydroxytryptamine in diarrhoea predominant irritable bowel syndrome: a pilot study.

BACKGROUND: Increased concentrations of 5-hydroxytryptamine (5-HT) can be detected in the systemic circulation after a meal and may be involved in the physiological control of gastrointestinal motility. Abnormalities of 5-HT release after a meal might explain some of the postprandial symptoms associated with the irritable bowel syndrome (IBS). AIM: To investigate the effect of a standard meal on plasma 5-HT and urinary 5-hydroxyindole acetic acid (5-HIAA) concentrations in patients with diarrhoea predominant IBS and in healthy volunteers. METHODS: After an overnight fast, six volunteers and five patients with IBS were given a carbohydrate-rich meal. Blood and urine samples were taken before and for four hours after the meal. Platelet-poor plasma 5-HT and urinary 5-HIAA were analysed by reversed phase high performance liquid chromatography with fluorometric detection. 5-HIAA was expressed as a ratio with urinary creatinine concentration, which was measured by spectrophotometry. RESULTS: During the four hour postprandial period, 5-HT concentrations were significantly higher in patients with IBS than in healthy volunteers at 0.5 hours (p < 0.05), 2 hours (p < 0.05) and 2.5 hours (p < 0.05). 5-HT was not detected in the plasma in the fasting state in patients or volunteers. Median peak 5-HT in patients with IBS (359 (198-796) nmol/l) was significantly greater than volunteers (83 (7-190)) (p < 0.05). "Area under the curve" for 5-HT detection was greater for patients with IBS (317 (138-771)) than for healthy volunteers (51 (4-129); p < 0.05). The duration of the 5-HT peak was significantly longer in patients with IBS (3 (1-3) hours) than in the healthy volunteers (1 (1-1) hours; p < 0.01). Postprandial urinary median 5-HIAA values in controls (5.6 (5.5-5.8) mumol/mmol creatinine) and patients with IBS (3.0 (2.5-6.8) mumol/mmol creatinine) were not significantly different from preprandial values (controls: 5.9 (5.5-6.6) mumol/mmol creatinine; patients with IBS: (6.2 (2.4-9.3) mumol/mmol creatinine). CONCLUSION: These findings indicate that there may be a difference in the way that 5-HT is released in patients with diarrhoea predominant IBS, and could suggest a possible role for 5-HT in the postprandial symptoms of these patients.

Adolescent↗
PubMed1998

The relation between gastric vitamin C concentrations, mucosal histology, and CagA seropositivity in the human stomach.

BACKGROUND: Vitamin C may be protective against gastric cancer though infection with Helicobacter pylori is associated with a reduction in intragastric concentrations of vitamin C. AIMS: To examine the effects of H pylori infection, gastric juice pH, the severity and extent of gastric inflammation, and CagA antibody status of the individual on gastric juice and mucosal vitamin C concentrations. PATIENTS: One hundred and fifteen patients undergoing routine gastroscopy for investigation of dyspepsia. METHODS: High performance liquid chromatography was used to determine vitamin C concentrations. CagA antibody was detected by western blot analysis. RESULTS: Gastric juice ascorbic acid concentration was significantly lower in patients infected with H pylori compared with those uninfected (19.3 mumol/l (interquartile range (IQR) 10.7-44.5) versus 66.9 mumol/l (IQR 24.4-94.2), p = 0.003). The reduction in gastric juice ascorbic acid concentration was inversely related to the severity of gastritis (p = 0.01). CagA positive patients had significantly lower gastric juice ascorbic acid concentrations than CagA negative ones (14.8 mumol/1 (IQR 7.9-52.2) versus 39 mumol/l (IQR 19.9-142.2), p = 0.05). Decreased gastric juice dehydroascorbic acid concentrations were observed in patients with gastric atrophy and intestinal metaplasia. Mucosal ascorbic acid concentrations were also significantly lower in infected patients than uninfected patients (p = 0.04). CONCLUSIONS: The reduction in gastric vitamin C concentrations is related to gastric juice pH, the severity and extent of gastritis, the presence of H pylori, and the CagA antibody status of the individual. These findings may have implications in H pylori associated carcinogenesis.

Adult↗
PubMed1997

Direct monitoring of enzyme reactions using micellar electrokinetic capillary chromatography. Optimisation of drug glucuronide and sulfate conjugate hydrolysis.

This paper demonstrates the use of micellar electrokinetic capillary chromatography (MECC) to monitor enzyme reaction conditions. The hydrolysis reactions of model conjugated substrates (morphine and reduced flunixin glucuronides, napthyl sulfate), by proprietary beta-glucuronidase preparations, were studied under varied experimental conditions. Reactions were carried out in autosampler vials with incubation in a thermostatted CE autosampler tray. MECC was performed using borax buffer (17.5 mM, pH 9.3) modified with sodium dodecyl sulfate (70 mM). Repetitive injections were made from the sample vial throughout the course of the reactions at a frequency of up to 10 h-1. MECC provided a rapid and reproducible assay for the model substrates. Baseline interference from the enzymes prevented measurement of product increase, therefore substrate decrease was measured from the peak areas. Monitoring of reactions in this way has proved valuable in the optimisation of hydrolysis conditions used in sample preparation for drug analysis. beta-Glucuronidase preparations from Helix pomatia were found to give the best performance of those evaluated in terms of deconjugation efficiency.

Animals↗
PubMed1997

Micellar electrokinetic capillary chromatography (MECC) of UV-absorbing constituents in normal urine: a chemometric optimisation of the separation.

A micellar electrokinetic capillary chromatography (MECC) method when compared to free solution capillary electrophoresis (CZE) was shown to offer improved selectivity and resolution for the separation of UV-absorbing components of human urine. Some of the factors affecting MECC separation e.g. methanol concentration, sodium dodecyl sulphate (SDS) concentration, beta-cyclodextrin (beta-CD) concentration, voltage, pH, temperature and electrolyte additives (urea, beta-CD and Brij 35) were optimised using chemometric techniques. Three-level three-factor (3(3)) factorial designs and simplex optimisation were used to achieve optimised conditions with the goal of obtaining the maximum number of peaks in the shortest possible analysis time. Using a TSP CE2000 instrument with detection from 195-300 nm and fitted with a 75 microns x 44 cm (37 cm effective length) fused silica capillary the final optimum conditions were found to be, an electrolyte consisting of 30 mM sodium tetraborate, pH 10, containing 75 mM SDS and 10 mM beta-CD, 15 degrees C, 20 kV, 4 s hydrodynamic injection of filtered urine. These conditions were capable of separating 70 peaks from a normal human urine pool in less than 12 min. The separation of components in urine using the optimised MECC was simpler, more reproducible, faster and gave better resolution than gradient reversed-phase high performance liquid chromatography.

Analysis of Variance↗
PubMed1997

Presentation-time measures of the effects of manipulations in colour space on discrimination of famous faces.

Caricaturing the distinctive shape of famous faces can produce an advantage in reaction-time paradigms but the role of distinctive colour and intensity information in recognition of facial identity has not previously been explored. A presentation-time paradigm was developed by which stimuli could be presented for a range of brief display periods. Subjects were required to identify photo-realistic colour representations of famous faces which either were veridical, were caricatured in colour space, or had enhanced colour saturation and intensity contrast (as contrast controls). Recognition accuracy was greater when viewing the caricatured stimuli than either the veridical images or the contrast controls. The removal of colour information to produce grey-scale images also decreased accuracy of face recognition. Both results indicate that colour information aids differentiation of a class of natural stimuli with similar configurations. Thus it is demonstrated that caricaturing faces can be extended to the colour domain and, as with shape caricaturing, enhancement of distinctive information can produce a recognition advantage for famous faces.

Adult↗
PubMed1997

Analysis of urinary pseudouridine by micellar electrokinetic capillary chromatography.

We describe a capillary electrophoresis procedure, using uridine as an internal standard, for the analysis of urinary pseudouridine following solid-phase extraction. This method retains the advantages of existing chromatographic techniques but has superior resolving power and is technically less demanding. The standard curves were linear and reproducible with a detection limit of 60 fmol; chromatographic analysis was complete in under 10 min. Injection variability was < 5% and multiple independent analyses of the same urine sample for pseudouridine concentration gave coefficients of variation of < 10%. The mean (SD) urinary pseudouridine level in 18 healthy subjects was 16.1 (2.1) nmol/mumol creatinine. For a limited group of subjects where samples were taken more frequently, intra-individual variation averaged 27.5% reflecting variable excretion.

Adolescent↗
PubMed1997

Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma.

Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive-minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)-and has a working range (between-assay CV < 10%) of 180-10000 pmol/L (54-3000 pmol/L in plasma). Mean recoveries of ouabain added to plasma ranged from 90% to 100%, and plasma extracts diluted in parallel to the standard curve. Plasma OLC concentrations in 10 healthy volunteers averaged 92 pmol/L (range 55-168), assuming 100% cross-reactivity of OLC in the ouabain assay. HPLC analysis with two distinct chromatographic conditions demonstrated that endogenous human plasma OLC co-eluted with authentic ouabain. The enzyme immunoassay is rapid and easy to perform and will support further investigation of the nature of this controversial endogenous steroid.

Chromatography, High Pressure Liquid↗
PubMed1996

Determination of phase II drug metabolites in equine urine by micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MECC) using diode array detection has been investigated for the determination of phase I and phase II metabolites of drugs in biofluids. Methods were optimised for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide, normorphine, meclofenamic acid and its metabolites in equine urine. Solid-phase extraction procedure were developed to concentrate and purify the analytes from spiked and post administration urines for MECC analysis. A simple on-line procedure for monitoring the kinetics of hydrolysis of morphine-glucuronide conjugates by beta-glucuronidase was demonstrated.

Analgesics, Opioid↗
PubMed1996

Automated solid-phase extraction with high-performance liquid chromatography analysis of pyridinium crosslinks.

An automated solid-phase extraction procedure for free and total pyridinium crosslinks in urine, with on-line HPLC analysis, is described. The pyridinium crosslinks either following hydrolysis in 3 M HCl or free in urine were extracted using a Gilson Aspec system onto extraction cartridges containing an octasilane/cation-exchanger sorbent. After washing, the crosslinks were eluted with 400 microl 100 mM sodium formate, pH 5, and 50 microl was automatically injected onto the analytical HPLC column. Detection was by fluorescence (Ex 295 nm, Em 400 nm). Recoveries were 95-100% with a limit of quantitation (s/n = 5) of 1.97 nmol/liter (nM) for pyridinoline and 2.79 nM for deoxypyridinoline. The interassay coefficient of variation was 6.5% for pyridinoline and 8.2% for deoxypyridinoline. The throughput of the system was up to 70 samples per day. The method correlated well with the established manual cellulose extraction but was substantially simpler and more reliable. A good correlation with an immunoassay for deoxypyridinoline was also obtained.

Amino Acids↗
PubMed1996

Purine metabolism in HIV-1 virus-infected T lymphocyte population.

Purine nucleotide metabolism has been studied in T-lymphocyte population in healthy subjects and in AIDS bearing patients. Nucleotide content was determined by HPLC. The overall rate of purine nucleotide synthesis was measured following the incorporation of 14C-formate into the nucleotides of a T cell suspension. The authors discuss the results, which indicate interesting variations in nucleotide content and a lower nucleotide synthesis, determined by kinetic studies.

Acquired Immunodeficiency Syndrome↗
PubMed1996

5-hydroxytryptamine release into human jejunum by cholera toxin.

BACKGROUND: Cholera toxin produces intestinal secretion by activation of the adenylate cyclase complex. However animal studies have shown 5-hydroxytryptamine may be released after exposure to cholera toxin, and thereby contribute to the secretory state. AIM: To determine whether cholera toxin releases 5-hydroxytryptamine in human jejunum. SUBJECTS: Seven male subjects were given a subclinical dose of cholera toxin in a paired, controlled, randomised, double blind study. METHODS: A closed 10 cm segment of upper jejunum was exposed to 15 micrograms of cholera toxin for two hours prior to closed segment perfusion with plasma electrolyte solution containing a non-absorbable volume marker, [14C]-polyethylene glycol. 5-Hydroxytryptamine in jejunal effluent and 5-hydroxyindoleacetic acid in urine (up to seven hours after cholera toxin) were measured by high performance liquid chromatography with fluorimetric detection. RESULTS: In contrast with controls, all subjects secreted fluid in response to cholera toxin, median-2.1 ml/cm/h (interquartile range-4.1 to -0.1). During seven hours following cholera toxin, 5-hydroxytryptamine was secreted into the lumen (range 31 to 395 nmol/l) but not in control experiments. After exposure to cholera toxin median urinary 5-hydroxyindoleacetic acid was 5.7 (4.1 to 6.3), which was similar to controls 4.9 (4.1 to 6.3), which was similar to controls 4.9 (4.1 to 6.2). CONCLUSION: Thus, cholera toxin induced a secretory state and promoted the release of 5-hydroxytryptamine into the intestinal lumen, but quantitative changes in urinary 5-hydroxyindoleacetic acid were not detectable. As an intestinal secretagogue, these findings suggest that 5-hydroxytryptamine may play a part in mediating cholera toxin induced secretion in humans.

Adult↗