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D Perrett

Publications and source records attributed to D Perrett.

At least 55 records · Page 3Linked to original sources
PubMed1995

Analysis of internucleosomal DNA fragmentation in apoptotic thymocytes by dynamic sieving capillary electrophoresis.

The analysis, by slab gel electrophoresis, of internucleosomal DNA cleavage or laddering, characteristic of apoptosis in many cell systems, is labour intensive, difficult to automate and at best only semi-quantitative. In this report we show that CE, using dilute solutions of hydroxyethylcellulose as a replaceable sieving matrix, can be applied to the relatively rapid analysis of DNA laddering in whole digests of apoptotic rat thymocytes. Also, using the sensitivity of laser-induced fluorescence detection and the highly sensitive nuclei acid stain YO-PRO-1, the CE method reported here can use 1000-2000 fold fewer cells than needed for traditional slab gel methods.

Animals↗
PubMed1995

Rapid determination of drugs in biofluids by capillary electrophoresis. Measurement of antipyrine in saliva for pharmacokinetic studies.

A micellar electrokinetic capillary chromatography method was developed that permitted the resolution of antipyrine from endogenous compounds and its quantitation in neat saliva in as little as 1 min. Final conditions were: SpectraPhoresis 1000, 30(23) cm x 50 microns silica capillary, 50 mM sodium phosphate pH 9.6, 50 mM SDS, 10 s hydrodynamic load, detection scanning 200-300 nm or 260 nm, run 25 kV. To overcome the effects of Joule heating the capillary was cooled to 15 degrees C. Sensitivity was < 10 microM and linearity extended to 350 microM. Comparison with an HPLC assay demonstrated that hydrodynamic injection gave a loading bias unless samples and standards were of equal viscosity. For 75 samples from five subjects the correlation of CE vs. HPLC was then r = 0.99.

Antipyrine↗
PubMed1993

Separation and tryptic digest mapping of normal and variant haemoglobins by capillary electrophoresis.

Characterization of haemoglobin (Hb) through whole protein separations and tryptic digest mapping allows the identification of structural Hb variants which result in haemoglobinopathies. Tryptic digest mapping by conventional two-dimensional paper chromatograph-electrophoresis provides high resolution but requires 48 h, while gradient elution reversed-phase high-performance liquid chromatography (HPLC) is faster (1.5 h), there is decreased resolution. CE analysis provides a fast separation with high resolution. We have used CE to optimise the tryptic digestion of globin purified from normal human haemoglobin A and to analyze tryptic digests from normal Hb. The separations were optimised and peak identification performed using UV scanning detection. In the optimised tryptic digest separation up to 28 peaks could be resolved in < 20 min. These peaks were identified as far as possible and a high-resolution map of the digest was constructed. The optimised analytical conditions were used to observe the separation pattern obtained from normal adult haemoglobin (HbA), common variant haemoglobins and some rarer haemoglobin variants.

Adult↗
PubMed1993

Optimized separation of purine bases and nucleosides in human cord plasma by capillary zone electrophoresis.

An optimized separation of the main purine compounds of human serum by capillary zone electrophoresis is presented. Separations were performed in an uncoated silica capillary (44 cm x 75 microns I.D., 37 cm to window) on a SpectraPhoresis 1000 system with UV detection. The separation of adenine (Ade), adenosine (Ado), guanine (Gua), guanosine (Guo), hypoxanthine (Hyp), inosine (Ino), xanthine (Xan) and uric acid (UA) was optimized with respect to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.4), 37 degrees C, 20 kV and 9 s load and detection at 260 nm. Linearity extended from 1 to 125 microM. The sensitivity of the method was 0.5 microM, which is adequate for measuring Ade, Gua, Hyp and UA in plasma samples. Plasma samples from newborns were precipitated with an equal volume of perchloric acid (7%, v/v), the supernatant was adjusted to neutral pH with potassium carbonate and, before injection, the sample was alkalized with sodium hydroxide. The method presented here allows the determination of Ade, Guo, Hyp and UA. The levels of the determined purines were compared in samples from control newborns, preterm babies and newborns with asphyxia or acidic serum pH values.

Calibration↗
PubMed1993

Assay of pyridinium crosslinks in serum using narrow-bore ion-paired reversed-phase high-performance liquid chromatography.

The pyridinium crosslinks are important biomarkers of mature hard tissue collagen degradation. This paper describes an isocratic ion-paired reversed-phase high-performance chromatographic assay using narrow-bore columns and high-sensitivity fluorescence detection to enable for the first time the determination of pyridinium crosslinks in both serum and synovial fluid samples. Extracted freeze-dried acid hydrolysates were re-suspended in 20 mM pentafluoropropionic acid (PFPA). Separations were carried out using an Exsil 100 5-microns ODS2 column (100 mm x 2.1 mm I.D.) eluted with 10 mM PFPA in water at 0.15 ml/min and detected using a Jasco 821-FP detector (xenon lamp: excitation 290 nm, emission 400 nm). Fluorescent response was linear from 269 to 8620 fmol for pyridinoline (Pyr) and 85 to 2710 fmol for deoxypyridinoline (dPyr). The limits of detection were 28 and 57 fmol, respectively. The coefficient of variation for extraction and analysis of normal serum was 7.96% for Pyr and 6.30% for dPyr (n = 6). The mean +/- S.D. concentration of Pyr in normal serum was 3.26 +/- 0.83 nM.

Biomarkers↗
PubMed1993

D-lysine reduces the non-enzymatic glycation of proteins in experimental diabetes mellitus in rats.

D-Lysine, the non-physiological isomer of L-lysine, can competitively reduce protein non-enzymatic glycation in vitro. To study the effect of D-lysine in vivo, 6-8-week old Sprague-Dawley rats with streptozotocin-induced diabetes mellitus were treated from diagnosis for 45 days with two daily subcutaneous injections of D-lysine (0.5 g.ml-1.day-1). Another group of diabetic rats was only injected with equal volumes of physiological saline (0.9% NaCl). Glycated haemoglobin was measured by ion exchange chromatography, and glycated serum and lens proteins by boronate affinity gel chromatography. Serum and urinary creatinine concentrations were evaluated by the alkaline-picrate reaction. Urinary lysine concentrations at mid- and end-study were evaluated by cation exchange chromatography. Blood glucose concentrations, serum creatinine levels and creatinine clearances, measured at the end of the study, were similar in both diabetic groups (> 22.0 mmol/l, < or = 106 mumol/l and approximately 0.02 ml/s, respectively). Urinary lysine concentration in D-lysine-treated diabetic animals was more than 50-fold higher than in placebo-treated diabetic rats. In D-lysine-treated vs placebo-treated diabetic animals, a statistically significant reduction was found in the levels of glycated haemoglobin (stable HbA1; mean +/- SD = 3.00 +/- 0.74% vs 4.02 +/- 0.46%, p < 0.05; labile HbA1 = 3.92 +/- 0.89% vs 5.84 +/- 0.61%, p < 0.005), glycated serum proteins (1.40 +/- 0.47% vs 2.52 +/- 1.15%, p < 0.05) and glycated lens proteins (4.90 +/- 0.96% vs 5.98 +/- 0.65%, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Increased levels of urinary collagen crosslinks in females with rheumatoid arthritis.

Bone loss is a feature of RA, but the exact mechanisms involved are not clear. The collagen crosslinks deoxypyridinoline (DPYR) and pyridinoline (PYR) are specific indices of 'mature' collagen breakdown and reflect increased bone turnover. The aims of the study were to examine crosslink levels in RA and their association with disease activity and the effect of steroids. Urinary crosslinks corrected for creatinine were measured on morning fasting samples by HPLC in 70 postmenopausal women with rheumatoid arthritis (RA) aged 45-65 and compared with 169 postmenopausal healthy age-matched controls from the population. Mean levels of PYR were significantly higher in RA cases than in controls (52.4 versus 37.5 nmols/mmolCr) although mean levels of DPYR did not differ significantly. A weak correlation was found with ESR and PYR (r = 0.35) but not with other markers of disease activity. Thirteen of the RA cases were current steroid users and their levels of DPYR and PYR even with low doses, were significantly elevated above those of non-users, ex-users and controls. The finding of raised urinary PYR but not the bone specific DPYR in nonsteroid using RA cases suggests that the increased collagen breakdown does not primarily come from bone but from other sources such as cartilage and synovium. The large increases in collagen excretion in low dose steroid users, may reflect the higher risk of osteoporosis in this group.

Aged↗
PubMed1993

The long-term use of D-penicillamine for treating rheumatoid arthritis: is continuous therapy necessary?

Patients with RA often fail to comply with their therapy. We investigated the extent of non-compliance with D-penicillamine therapy and also whether patients needed continuous treatment after they had shown a therapeutic response. We developed a simple urinary test, measuring cysteine-penicillamine mixed disulphide by a high performance liquid chromatography technique, as an indicator of compliance. Using this method we evaluated compliance in 59 consecutive RA patients attending rheumatology outpatients for monitoring of D-penicillamine therapy. Evidence of poor compliance was shown in 39%. There was no relationship between poor compliance and disease activity. A possible explanation for this paradox emerged from a therapeutic study of the efficacy of intermittent treatment in patients in partial clinical remission on long-term D-penicillamine. In 14 randomly selected patients the daily dose of D-penicillamine was reduced in frequency over 6 months from 250-750 mg daily to the same dose taken 1 week out of every 4. The patients were compared to matched controls who remained on continuous therapy. After 30 months both the intermittent and continuous therapy groups had similar clinical and laboratory scores for disease activity. Our results suggest that many patients may not comply with their prescribed D-penicillamine regime, but such variation in compliance may not be clinically important since intermittent treatment may sustain response in the longer term.

Adult↗
PubMed1993

Plasma and 24 h-urinary catecholamine concentrations in normal and patient populations.

Using a single HPLC-ECD methodology, plasma catecholamine data were compiled from 545 patients and 144 healthy subjects, and 24 h urinary free catecholamine data from 106 patients. From these data, normal reference ranges were derived for the clinical investigation of suspected phaeochromocytomas, specifically to address the question as to when a result is normal and when further investigation may be required. We have shown that noradrenaline and adrenaline concentrations in plasma and urine are not normally distributed, and basal plasma noradrenaline is significantly lower in normal volunteers than in hospital patients. We have also demonstrated that a reference range which does not take account of these factors will give a significant number of false-positive results. Age and sex did not appear to be significant variables for either plasma or urinary catecholamines. In the investigation of phaeochromocytoma, 95% confidence limits may be used as a warning to repeat sampling and 99% confidence limits as rendering the diagnosis extremely probable. In patients with phaeochromocytomas, urinary catecholamines had a 99.5% specificity and 100% sensitivity in the diagnosis, and thus provide for a rapid and efficient diagnostic screen.

Adolescent↗
PubMed1992

Increased DNA strand breaks in mononuclear cells from patients with rheumatoid arthritis.

Immune dysfunction is linked with lymphocyte DNA metabolism. In particular, DNA damage may impair lymphocyte function and induce increased cell turnover; such changes are of relevance to the pathogenesis of rheumatoid arthritis. The rate of DNA unwinding in alkaline solution was used as a measure of the number of DNA strand breaks in mononuclear cells freshly isolated from peripheral blood. The rate of DNA unwinding was significantly increased in cells from patients with rheumatoid arthritis compared with those from healthy subjects and from patients with other autoimmune and connective tissue diseases. These findings support the hypothesis that DNA damage is increased in patients with rheumatoid arthritis and it is one factor contributing to immune dysfunction in this disease.

Adolescent↗
PubMed1992

Sulphydryl reactivity of the HLA-B27 epitope: accessibility of the free cysteine studied by flow cytometry.

HLA-B27 has an unpaired cysteine on or near its serologically defined spondylitis associated epitope, and it has been argued that its sulphydryl side chain may be chemically reactive. In a previous study it was shown that chemical treatment of HLA-B27 cells with the sulphydryl binding agent p-chloromercuriphenylsulphonic acid (pCMPSA) specifically reduced binding of antibodies to HLA-B27 by up to 80%, as measured in a cellular enzyme linked immunosorbent assay (CELISA). The effect of sulphydryl blockade on intact B27 cells was investigated using flow cytometry. Compared with the CELISA, inhibition required higher concentrations of pCMPSA, and the degree of inhibition produced by a greater than or equal to 30 microM solution of pCMPSA as measured by flow cytometry (median 28.9%) was significantly lower than that measured by CELISA (median 73.6%; p = 1.6 x 10(-6)). Analysis of unfixed, cell surface HLA-B27 by flow cytometry suggests that on most B27 molecules the unpaired sulphydryl site is not available. On the basis of this evidence for modification after translation, a new 'altered self' hypothesis is proposed for the part which HLA-B27 plays in inflammatory disease.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Comparative performance of ion exchange and ion-paired reversed phase high performance liquid chromatography for the determination of nucleotides in biological samples.

We have compared anion exchange chromatography on APS-Hypersil (4.6 x 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS-Hypersil (4.6 x 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange systems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.

Animals↗