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PubMed2004

Contamination of surface soils, river water and sediments by trace metals from copper processing industry in the Churnet River Valley, Staffordshire, UK.

The impacts of a functional and a demolished copper processing works on the aquatic and terrestrial environment in the vicinity of the works was investigated by determining the levels of selected trace metals in river water, river sediments, channel margin sediments and overbank soils. Samples were taken at five sites within an area of the Churnet Valley in Staffordshire, where the River Churnet flows through the two works. Analysis of river water samples by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) has shown that only copper is present above background levels considered to typify uncontaminated rivers. Analysis of river sediments, channel margin sediments and overbank soils by nitric-perchloric acid digestion and Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) analysis has indicated contamination by arsenic, cadmium and copper in the vicinity of both works. Arsenic and copper are deposited primarily within the aquatic environment, although some contamination of the terrestrial environment by copper is also observed. Cadmium is deposited primarily within the terrestrial environment. The deposition of arsenic and copper in river and channel margin sediments respectively is also related to current and historical contamination.

Arsenic↗
PubMed2004

Using 31P NMR spectroscopy at 14.1 Tesla to investigate PARP-1 associated energy failure and metabolic rescue in cerebrocortical slices.

PARP-1 activation by H(2)O(2) in an acute preparation of superfused, respiring, neonatal cerebrocortical slices was assessed from PAR-polymer formation detected with immunohistochemistry and Western blotting. (31)P NMR spectroscopy at 14.1 Tesla of perchloric acid slice extracts was used to assess energy failure in a 1-h H(2)O(2) exposure as well as in a subsequent 4-h recovery period where the superfusate had no H(2)O(2) and specifically chosen metabolic substrates. Although more data are needed to fully characterize different bioenergetic responses, a high NMR spectral resolution (PCr full-width at half-max approximately.01 ppm) and narrow widths for most metabolites (<.2 ppm) permitted accurate quantifications of spectrally resolved resonances for ADP, ATP, NAD(+)/NADH, and other high energy phosphates. It appears possible to use brain slices to quantitatively study PARP-related, NAD-associated energy failure, and rescue with TCA metabolites.

Animals↗
PubMed2003

Accumulation of methylglyoxal in the gingival crevicular fluid of chronic periodontitis patients.

BACKGROUND, AIMS: Methylglyoxal (MG), a toxic product of cellular metabolism, is elevated in tissues and fluids in a number of human diseases. A cross-sectional study was undertaken to determine whether MG accumulates in the gingival crevicular fluid (GCF) of chronic periodontitis patients. METHODS: GCF samples were collected for 30 s each from three teeth with pocket depths greater than 3 mm (DD sites), from 14 chronic periodontitis patients. Control samples were taken from three healthy sites (DH sites) in the same patients, as well as from seven subjects who were periodontally healthy (HH sites). Fluid volumes were determined and the strips were placed in 0.5 N perchloric acid. Subsequently, samples were derivatized with o-phenylenediamine and the resulting methylquinoxaline was assayed by high-performance liquid chromatography on Lichrospher(R)-100 RP-18, with UV detection. RESULTS: Mean pocket depths were 5.7+/-0.7, 2.7+/-0.6 and 2.7+/-0.5 mm (mean+/-SD) for the DD, DH and HH sites, respectively. Mean MG levels were found to be 208.7+/-241.7 and 142.9+/-235.7 pmol/site in the GCF from DD and DH sites, respectively (p=0.0023), but only 11.5+/-4.4 pmol/site for the HH sites. Bacteroides forsythus has been found to accumulate high levels of MG in culture (unpublished data) and, consistent with this, the sampled diseased sites contained higher levels of B. forsythus than the corresponding healthy sites (2.7+/-4.2 x 10(5) versus 0.7+/-1.1 x 10(5), respectively; p=0.022). Total "red complex" microorganisms were significantly elevated in the DD sites. CONCLUSIONS: In view of the known protein- and DNA-modifying effects of MG, the finding of elevated levels of MG in the GCF from chronic periodontitis patients supports the hypothesis that MG may contribute to destructive tissue damage in this disease.

Bacteroides↗
PubMed1981

Somatic-cell hybrids producing antibodies against CEA.

Monoclonal antibodies to carcinoembryonic antigen (CEA) promise improved specificity for the measurement of this widely expressed human cancer antigen. A mouse monoclonal antibody binds weakly to CEA in perchloric acid extracts of tumour but strongly to CEA similarly isolated from serum, and its spectrum of cancer detection differs from conventional antisera.

Animals↗
PubMed1982

Ability of CEA blood levels to reflect tumour burden: a study in a human xenograft model.

The relationship of serum carcinoembryonic antigen (CEA) levels to tumour size and antigen content was studied in artificially immune-deprived mice bearing human colonic, breast and lung tumour xenografts. Size was measured as in vivo volume and tumour weight at post-mortem. A multiple implant technique combined with early harvest was used to minimize centrilobular tumour necrosis. CEA was extracted from resected tumours with perchloric acid. A radioimmunoassay using chemical precipitation was used to estimate CEA in blood samples. A correlation was found between CEA blood levels and tumour size in half the tumour lines, in contrast to a recent report (Lewis & Keep, 1981). The CEA content was found to be constant for one tumour line but not another. The possibility that central necrosis in xenograft tumours may account for the discrepancies is discussed. There may be serious limitations for the use of xenograft tumour models for studying the biology of CEA.

Animals↗
PubMed1983

Immunological comparison of carcinoembryonic antigen (CEA) extracted from tumours of various organs: their use in radioimmunological CEA determinations.

CEA was extracted by the perchloric acid method from primary adenocarcinomas of the colon and the ovary, from ascitic and pleural fluids from patients with pancreatic, lung and breast cancer, and from the cyst fluid of a benign ovarian cystadenoma. Further purification included gel filtration and affinity chromatography. Antisera against CEA from colon, breast, ovary, lung and pancreatic cancer were produced in rabbits. In double diffusion experiments, all these CEA samples showed a reaction of complete immunological identity with all the anti-CEA sera, whatever their origin. CEA from colon, breast, pancreas and ovary were labelled with 125I and used in radioimmunological experiments. In a radioimmunological system where the tracer and the antiserum were constant, all the CEA used as standards gave parallel inhibition curves, having nearly identical slopes. This was another criterion of immunological identity. Sera of numerous cancer patients were assayed in several RIA systems, one of them being the classical system with colonic CEA as tracer and anti-colonic CEA as antiserum, the others being "organ specific" systems. The values obtained in these assays were found to be highly correlated: the rank coefficient of correlation between colonic and breast cancer RIA systems was rs = 0.96, that between colonic and ovarian RIA systems, 0.92, that between colonic and pancreatic RIA systems, 0.97 and that between colonic and lung RIA systems 0.96. It is thus concluded that by use of different organ-derived CEA preparations and their corresponding polyclonal antisera, no significative differences in serum CEA levels may be expected. No evidence of organ specificity of serum CEA was found.

Carcinoembryonic Antigen↗
PubMed1991

Purification and characterisation of a breast-cancer-associated glycoprotein not expressed in normal breast and identified by monoclonal antibody 83D4.

Monoclonal antibody (mAb) 83D4 was generated using formol-fixed paraffin-embedded human breast carcinoma tissue as the immunogen. Previous studies demonstrated that it was reactive with breast carcinoma tissues, but not with normal breast. The antigen identified by mAb 83D4 was detected, using ELISA, in MCF7 breast carcinoma cell line membrane extracts, in primary breast and colon carcinoma tissue extracts and in pleural effusion fluid from patients with metastatic breast cancer. No reactivity with 83D4 was found in either human milk fat globule membranes or skimmed milk. 83D4 reactive antigen was found to be a heterogeneous high molecular weight (MW) protein (apparent Mr:300-400 to over 1000 kDa) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The antigen was purified from MCF7 cells, breast and colon carcinomas and effusion fluid, by perchloric acid solubilisation followed by immunoaffinity chromatography with 83D4. The immunopurified antigen from MCF7 cells and pleural effusion fluid was further analysed by gel filtration and ion-exchange chromatography, which confirmed the high MW and indicated the charge heterogeneity of the reactive molecules. The 83D4 reactive antigen strongly bound to wheat-germ agglutinin and weakly to peanut lectin. No binding was found with lentil lectin or concanavalin A. Antigenic activity was strongly reduced by trypsin and subtilysin digestion and by treatment with sodium periodate, but it was not affected by neuraminidase. These results imply the glycoprotein nature of the 83D4-defined antigen and the involvement of carbohydrate, but probably not sialic acid, in the epitope. Purified 83D4 antigen did not display reactivity for mAb HMFG-1, directed against a polymorphic epithelial mucin, PEM, using ELISA, but bound mAb CC49 and weakly mAb B72.3, antibodies which define a tumour associated glycoprotein, TAG-72. Moreover CC49 and 83D4 showed similar reactivity pattern in immunoblotting assays. A double determinant radioimmunoassay confirmed that 83D4 antigen carries epitopes for mAb B72.3 and CC49. Competition radioimmunoassays clearly distinguished the 83D4 defined epitope from those recognised by B72.3 and CC49, demonstrating that antibody 83D4 identifies a unique epitope. It is suggested that the antigens identified by mAb 83D4 and by mAb B72.3 and CC49 may form part of the same family of carcinoma associated glycoproteins.

Antibodies, Monoclonal↗
PubMed1997

Metabolic studies of human primitive neuroectodermal tumour cells by proton nuclear magnetic resonance spectroscopy.

Well-characterized cell lines established from primitive neuroectodermal tumours (PNETs) were examined by proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic analysis of perchloric acid extracts, following amplification in cell culture. A characteristic 1H-NMR spectroscopic metabolite pattern was found for medulloblastoma cell lines, which clearly discriminates these cells from PNETs of other locations in the central nervous system (CNS), on the basis of their N-acetyl aspartate (NAA) and aspartate expression. Medulloblastoma cell lines were heterogeneous in respect of their metabolite expression, possibly owing to the heterogeneity in their differentiation along lineages of the CNS. All PNET spectra displayed similar features, including decreased NAA and creatine peaks and increased signals from choline compounds (Cho) compared with normal cerebellum. The expression of NAA by the medulloblastoma lines was in the opposite order to the extent of neuronal differentiation, which may indicate their origin from a progenitor cell with the phenotype of an oligodendrocyte-type-2 astrocyte cell.

Aspartic Acid↗
PubMed1998

Nitrogen mustard up-regulates Bcl-2 and GSH and increases NTP and PCr in HT-29 colon cancer cells.

We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment of tumours by DNA-damaging agents were related to chemotherapy-induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated HT-29 cells with 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) (an important defence against DNA damage from alkylating agents). After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained 31P MRS phenomenon: increased NTP after chemotherapy.

Adenosine Triphosphate↗
PubMed1992

Cerebral lactate turnover after electroshock: in vivo measurements by 1H/13C magnetic resonance spectroscopy.

We reported earlier that brain activation by 10 s of cortical electroshock caused prolonged elevation of brain lactate without significant change in intracellular pH, brain high-energy phosphorylated metabolites, or blood gases. The metabolic state of the elevated lactate has been investigated in further experiments using combined, in vivo 1H-observed 13C-edited nuclear magnetic resonance spectroscopy (NMRS), homonuclear J-edited 1H-NMRS, and high-resolution 1H-NMRS of perchloric acid extracts to monitor concentrations and 13C-isotopic fractions of brain and blood lactate and glucose. We now report that electroshock-elevated lactate pool in rabbit brain approaches equilibrium with blood glucose within 1 h. There was nearly complete turnover of the raised lactate pool in brain; any pool of metabolically inactive lactate could not have been > 5% of the total. In the same experiments, blood lactate underwent < 50% turnover in 1 h. The new 1H-spectroscopic methods used for these experiments are readily adaptable for the study of human brain and may be useful in characterizing the metabolic state of elevated lactate pools associated with epilepsy, stroke, trauma, tumors, and other pathological conditions.

Animals↗
PubMed2003

Non-invasive 19F MR spectroscopy of 5-fluorocytosine to 5-fluorouracil conversion by recombinant Salmonella in tumours.

The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD). The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase.

Animals↗
PubMed1999

An inhibitor of poly (ADP-ribose) synthetase activity reduces contractile dysfunction and preserves high energy phosphate levels during reperfusion of the ischaemic rat heart.

The cardioprotective properties of inhibition of poly (ADP-ribose) synthetase (PARS) were investigated in the isolated perfused heart of the rat. Hearts were perfused in the Langendorff mode and subjected to 23 min total global ischaemia and reperfused for 60 min. Left ventricular function was assessed by means of an intra-ventricular balloon. High energy phosphates were measured by 31P-NMR spectroscopy. Intracellular levels of NAD were measured by capillary electrophoresis of perchloric acid extracts of hearts at the end of reperfusion. Reperfusion in the presence of the PARS inhibitor 1,5 didroxyisoquinoline (ISO, 100 microM) attenuated the mechanical dysfunction observed following 1 h of reperfusion; 27+/-13 and 65+/-8% recovery of preischaemic rate pressure product for control and 100 microM ISO, respectively. This cardioprotection was accompanied by a preservation of intracellular high-energy phosphates during reperfusion; 38+/-2 vs 58+/-4% (P<0.05) of preischaemic levels of phosphocreatine (PCr) for control and 100 microM ISO respectively and 23+/-1 vs 31+/-3% (P < 0.05) of preischaemic levels of ATP for control and 100 microM ISO respectively. Cellular levels of NAD were higher in ISO treated hearts at the end of reperfusion; 2.56+/-0.45 vs 4.76+/-1.12 micromoles g(-1) dry weight (P<0.05) for control and ISO treated. These results demonstrate that the cardioprotection afforded by inhibition of PARS activity with ISO is accompanied by a preservation of high-energy phosphates and cellular NAD levels and suggest that the mechanism responsible for this cardioprotection may involve prevention of intracellular ATP depletion.

Adenosine Triphosphate↗
PubMed1991

Evaluation of microwave digestion and solvent extraction for the determination of trace amounts of selenium in feeds and plant and animal tissues by electrothermal atomic absorption spectrometry.

A sensitive method for the accurate determination of Se in agricultural products at sub-ppm levels is described. The proposed procedure involves the wet oxidation of samples by using a mixture of nitric, sulphuric and perchloric acids, co-extraction of Se and added Pd with diethylammonium N,N-diethyldithiocarbamate in chloroform, and electrothermal atomic absorption spectrometric determination of Se in the organic extract. Atomization and extraction conditions are discussed. Special attention is given to the wet oxidation step, and its advantages in speed and simplicity over conventional heating have been evaluated using an automated microwave digestion system. The results reported, obtained from several reference materials, confirm the accuracy of the method with which a detection limit of 0.002 micrograms g-1 of Se can be achieved.

Animal Feed↗
PubMed1992

Mercury determination by cold vapour atomic absorption spectrometry in several biological indicators from Lake Maracaibo, Venezuela.

Two sampling sessions were carried out in Lake Maracaibo and eight sites selected in four pairs one in front of the other (El Moján-Ancón de iturre, Santa Cruz de Mara-Punta de Palmas, Maracaibo-Punta de Leiva and San Francisco-La Rita). Specimens of Cynosción acoupa Maracaiboencis (curvina, n = 5), Oligoplites palometa (palometa, n = 5), Penaeus schmitti (shrimp, n = 20), and Polymesoda arctata (mussels, n = 20) were collected. For the curvina and palometa, muscle, brain, kidney, gill, liver and heart were analysed. Shrimp and mussels were analysed whole, without the shells. Samples were lyophilized and cold digested in a mixture of sulfuric, nitric and perchloric acids, potassium permanganate and hydrogen peroxide. Spectrometric determination was carried out using cold vapour atomic absorption spectrometry and sodium borohydride as reducing agent. Accuracy was tested with a National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) RM50 Albacore Tuna, and against another method. The detection limit for Hg was 53 ng l-1. Good agreement was found between results for the analysis of SRM RM50 (Hg = 0.95 +/- 0.14 mg kg-1) and the assigned value furnished by NIST (Hg = 0.95 +/- 0.10 mg kg-1). Precision and the interference were also evaluated. Mercury concentrations in brain, kidney and heart tissues of the curvina and palometa were found to be higher than those in the muscle tissue.

Animals↗
PubMed1996

Improved molecular fluorescence method for the determination of selenium in biological samples.

A procedure for the determination of selenium in whole blood and urine has been improved by optimization of the digestion and derivatization procedures. An overnight pre-digestion step with 4 + 1 concentrated nitric-perchloric acids reduced the time of mineralization at 170 degrees C from 4 h to 30-45 min. Conversion of all the selenium to selenite (SeIV) was optimized by addition of 1 ml of concentrated hydrochloric acid, with heating to 100 degrees C for 30 min. The rate of formation of the 2,3-diaminonaphthalene (DAN) complex of selenium was improved by heating to 70-100 degrees C for a minimum of 30 min. Co-addition of hexane during derivatization simplified the extraction procedure. The modified method was applied successfully to the analysis of Seronorm quality control whole blood and urine (83 and 24 micrograms l-1 Se, respectively). Samples from 12 healthy adults, gave results in expected ranges (mean concentrations of 75 +/- 8 micrograms l-1 in blood and 25 +/- 8 micrograms l-1 in urine). The structure of the Se-DAN complex was investigated using elemental analysis, FTIR spectrometry, 1H and 13C NMR spectroscopy, and FAB MS. The information obtained indicates that the selenodiazo group does not contain an Se-O bond or protonated nitrogens, as proposed in other studies.

Adult↗
PubMed2001

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation.

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.

Biomarkers↗
PubMed2002

Flash photolytic generation and study of 5-methoxy-o-quinone alpha-phenylethidet in aqueous solution: comparison of cis and trans isomer reactivity.

Cis and trans isomeric 5-methoxy-o-quinone alpha-phenylethides were generated by flash photolysis of o-hydroxybenzyl alcohol and o-hydroxystyrene precursors and their rates of decay were measured in aqueous solutions of perchloric acid, sodium hydroxide, and biphosphate (dihydrogen phosphate) ion and carbonate ion buffers. The data so obtained gave two parallel rate profiles displaced from one another by about an order of magnitude, the faster of which could be assigned to the cis isomer and the slower to the trans isomer. Both rate profiles showed acid catalyzed, uncatalyzed, and hydroxide ion catalyzed portions, with acid catalysis becoming saturated at the high acidity end of the range investigated. This saturation implies a pre-equilibrium mechanism involving quinone ethide protonation on carbonyl oxygen for the acid catalyzed process, and analysis of the data shows the trans isomer to be a slightly stronger base than the cis isomer. The fact that two separate rate profiles are observed implies that the cationic intermediates formed by substrate protonation in these acid catalyzed processes do not interconvert on the time scale of the quinone ethide decay reactions, and that in turn indicates that the former ethide bonds of the cationic intermediates have substantial double-bond character; a lower limit of 10 kcal mol(-1) may be estimated for the barrier to rotation about this bond.

Journal Article↗
PubMed2002

Waveguide surface plasmon resonance studies of surface reactions on gold electrodes.

We describe the fabrication and characterisation of gold-coated graded index channel waveguide sensors designed for simultaneous electrochemical and surface plasmon resonance studies. The active sensing electrode area is a thin gold film between 0.5 and 5 mm in length and 200 microm wide deposited on top of a 3 microm wide waveguide which forms one arm of a Y-junction while the other arm of the Y-junction serves as a reference. Using these devices we have measured simultaneously the changes in transmittance through the device whilst carrying out cyclic voltammetry in either sulfuric or perchloric acid solution or during the deposition of an UPD layer of copper at the gold surface. In all cases we obtain stable and reproducible results which demonstrate the very high sensitivity of the devices to sub-monolayer changes occurring at the gold electrode surface. The response of these integrated optoelectrochemical devices is discussed in terms of a numerical model for the propagation of light within the waveguide structure.

Journal Article↗