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Biomedical subjects

S A Barker

Publications and source records attributed to S A Barker.

At least 19 recordsLinked to original sources
PubMed2003

An investigation into the structure and bioavailability of alpha-tocopherol dispersions in Gelucire 44/14.

In this investigation we describe the preparation, physical characterisation and in vivo behaviour of solid dispersions of a liquid nutraceutical, alpha-tocopherol, in Gelucire 44/14 with a view to establishing whether dispersion in this matrix may provide a means of formulating a liquid drug in a solid dosage form while also improving the oral bioavailability. Using Vitamin E Preparation USP as the source of alpha-tocopherol, dispersions were prepared using a melt-fusion method with active loadings up to 50% (w/w) and characterised using differential scanning calorimetry and optical microscopy. Capsules containing 300 IU alpha-tocopherol were manufactured and the absorption profiles compared to a commercial soft gelatin capsule preparation in healthy human volunteers. Confocal laser scanning microscopy (CLSM) studies were performed in order to elucidate the mechanism by which drug release may be occurring. Differential scanning calorimetry studies indicated that the presence of the active had a negligible effect on the melting profile of the carrier, indicating limited miscibility between the two components, a conclusion supported by the microscopy studies. Similarly, the dispersions were shown to exhibit a glass transition corresponding to the incorporated drug, indicating molecular cooperativity and hence phase separation from the lipid base. Despite the phase separation, it was noted that capsules stored for 18 months under ambient conditions showed no evidence of leakage. Bioavailability studies in six healthy male volunteers indicated that the Gelucire 44/14 formulation showed an approximately two-fold increase in total alpha-tocopherol absorption compared to the commercial preparation. Confocal laser scanning microscopy studies indicated that, on contact with water, the dispersions formed two interfacial layers, from which the Gelucire 44/14 disperses in the liquid medium as small particles. Furthermore, evidence was obtained for the dispersed material becoming incorporated into the hydrated lipid. In conclusion, the dispersion of the liquid drug in Gelucire 44/14 appears to allow the dual advantages of the preparation of a solid formulation and improved bioavailability of this material.

Adult↗
PubMed2001

Distribution of the hallucinogens N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine in rat brain following intraperitoneal injection: application of a new solid-phase extraction LC-APcI-MS-MS-isotope dilution method.

A method for the solid-phase extraction (SPE) and liquid chromatographic-atmospheric pressure chemical ionization-mass spectrometric-mass spectrometric-isotope dilution (LC-APcI-MS-MS-ID) analysis of the indole hallucinogens N,N-dimethyltryptamine (DMT) and 5-methoxy DMT (or O-methyl bufotenin, OMB) from rat brain tissue is reported. Rats were administered DMT or OMB by the intraperitoneal route at a dose of 5 mg/kg and sacrificed 15 min post treatment. Brains were dissected into discrete areas and analyzed by the methods described as a demonstration of the procedure's applicability. The synthesis and use of two new deuterated internal standards for these purposes are also reported.

Animals↗
PubMed2001

Low frequency dielectric investigations into the relaxation behavior of frozen polyvinylpyrrolidone-water systems.

The low frequency dielectric response of aqueous solutions containing 0, 1, 5, and 10% w/v polyvinylpyrrolidone (PVP) was studied to characterize the low temperature relaxation behavior of these systems. Complementary modulated temperature differential scanning calorimetry (MTDSC) studies allowed measurement of the glass transition temperature for these materials, corresponding to the behavior of the nonfrozen phase. Dielectric investigations in the frequency range of 10(6) to 10(-2) Hz were performed on the systems in the liquid state, with a Maxwell-Wagner response noted for both the PVP solutions and water. The solid-phase responses were studied over a range of temperatures down to -70 degrees C, with a relaxation peak observed for the PVP systems in the kilohertz region. The spectra were modeled using the Havriliak-Negami equation and the corresponding relaxation times were calculated, with a satisfactory fit to the Arrhenius equation noted. The calculated activation energies were similar to literature values for the dielectric relaxation of water. It is suggested that the dielectric response is primarily a reflection of the relaxation behavior of the water molecules in the nonfrozen fraction, thereby indicating that the dielectric technique may yield insights into specific components of frozen aqueous systems.

Calorimetry, Differential Scanning↗
PubMed2001

RGSZ1 and Ret RGS: two of several splice variants from the gene RGS20.

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for G alpha(z). We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans approximately 107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3' untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5' end of the RGSZ1 ORF contains several in-frame ATG codons (3-5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5' exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple G(z)-selective GAPs in different tissues.

3' Untranslated Regions↗
PubMed2001

Combustion products of 1,3-butadiene are cytotoxic and genotoxic to human bronchial epithelial cells.

Adverse health effects of airborne toxicants, especially small respirable particles and their associated adsorbed chemicals, are of growing concern to health professionals, governmental agencies, and the general public. Areas rich in petrochemical processing facilities (e.g., eastern Texas and southern California) chronically have poor air quality. Atmospheric releases of products of incomplete combustion (e.g., soot) from these facilities are not subject to rigorous regulatory enforcement. Although soot can include respirable particles and carcinogens, the toxicologic and epidemiologic consequences of exposure to environmentally relevant complex soots have not been well investigated. Here we continue our physico-chemical analysis of butadiene soot and report effects of exposure to this soot on putative targets, normal human bronchial epithelial (NHBE) cells. We examined organic extracts of butadiene soot by gas chromatography-mass spectrometry (GC-MS), probe distillation MS, and liquid chromatography (LC)-MS-MS. Hundreds of aromatic hydrocarbons and polycyclic aromatic hydrocarbons with molecular mass as high as 1,000 atomic mass units were detected, including known and suspected human carcinogens (e.g., benzo(a)pyrene). Butadiene soot particles also had strong, solid-state free-radical character in electron spin resonance analysis. Spin-trapping studies indicated that fresh butadiene soot in a buffered aqueous solution containing dimethylsulfoxide (DMSO) oxidized the DMSO, leading to CH(3)* radical formation. Butadiene soot DMSO extract (BSDE)-exposed NHBE cells displayed extranuclear fluorescence within 4 hr of exposure. BSDE was cytotoxic to > 20% of the cells at 72 hr. Morphologic alterations, including cell swelling and membrane blebbing, were apparent within 24 hr of exposure. These alterations are characteristic of oncosis, an ischemia-induced form of cell death. BSDE treatment also produced significant genotoxicity, as indicated by binucleated cell formation. The combination of moderate cytotoxicity and genotoxicity, as occurred here, can be pro-carcinogenic.

Apoptosis↗
PubMed2001

Quantitation of adenine nucleotides in equine colonic mucosal tissue using high performance liquid chromatography.

The objectives were to use high performance liquid chromatography (HPLC) to validate an established method for adenine nucleotide separation in equine colonic mucosal tissue, to determine the inherent variability in the tissue and extraction method, and to determine the stability of ATP, ADP, and AMP in the tissue with time. Equine colonic mucosal tissue obtained from a single horse was immediately submersed in liquid nitrogen, and stored at -70 degrees C. Samples were lyophilized, extracted, and separated by HPLC. The limit of quantitation was 0.05 microg/mL. The coefficient of variation for the instrument was less than 10% for all nucleotides measured. When the tissue was not homogenized prior to sampling, there were significant differences in adenine nucleotide content between samples. However, when the tissue was homogenized prior to analysis, these differences were no longer significant. There was no significant decrease in ATP, ADP, or AMP content over a 54-day analysis period.

Adenine Nucleotides↗
PubMed2000

Matrix solid-phase dispersion.

Matrix solid-phase dispersion (MSPD) is a patented process, first reported in 1989, for conducting simultaneous disruption and extraction of solid and semi-solid samples. MSPD permits complete fractionation of the sample matrix components as well as the ability to selectively elute a single compound or several classes of compounds from the same sample. The method has been applied to the isolation of drugs in food animal tissues but has also found wide application in the analysis of herbicides, pesticides and pollutants from animal tissues, fruits, vegetables and other matrices. The present article provides a review of MSPD applications in these and related fields and discusses the factors known to affect MSPD methods. Both the practical and theoretical aspects of MSPD are also presented.

Animals↗
PubMed2000

Applications of matrix solid-phase dispersion in food analysis.

Matrix solid-phase dispersion (MSPD), a patented process for the simultaneous disruption and extraction of solid and semi-solid samples, was first reported in 1989. Since that time, MSPD has found application in numerous fields, but has proven to be particularly applicable for the analysis of drugs, pollutants, pesticides and other components in foods. The present article provides a review of these and related applications and discusses both the practical and theoretical aspects for the use of MSPD in sample processing.

Food Analysis↗
PubMed2000

An investigation into the use of stepwise isothermal high sensitivity DSC as a means of detecting drug-excipient incompatibility.

The use of stepwise isothermal high sensitivity differential scanning calorimetry (HSDSC) as a novel means of detecting excipient incompatibility is described using aspirin mixes with magnesium stearate and stearic acid as model systems. Aspirin, magnesium stearate and stearic acid alone and as mixes were studied in scanning mode using conventional DSC and were then subjected to a stepwise heating programme using HSDSC, whereby the samples were heated to temperatures between 45 and 70 degrees C and held for 1 h, during which the heat flow to or from the sample was measured. The data indicated that while no thermal events were detected for the individual components or mixes with stearic acid other than melting of stearic acid, 50% w/w mixes of magnesium stearate showed a marked endothermic response at temperatures above 55 degrees C. The data were fitted to an adaptation of an existing kinetic model for the degradation process and a reasonable correlation found. Mixes of the drug with the two excipients were then studied at 60 degrees C over 6 h at concentrations between 1 and 50% w/w. Incompatibilities with magnesium stearate concentrations as low as 1% w/w could be detected using this approach. Compacts of magnesium stearate and aspirin were also studied, with considerably more pronounced thermal events taking place compared to the powder mixes. It is concluded from these studies that while the study has highlighted certain limitations of the approach, stepwise isothermal DSC represents a potentially highly useful means of detecting excipient incompatibilities.

Aspirin↗
PubMed2000

An investigation into the low temperature thermal behaviour of vitamin E preparation USP using differential scanning calorimetry and low frequency dielectric analysis.

The thermal and dielectric responses of Vitamin E Preparation USP have been examined to further understand the melting and solidification of this material. A TA Instruments 2920 Differential Scanning Calorimeter was used to examine the thermal response of the sample at a range of scanning speeds. Isothermal dielectric studies were performed using a Novocontrol Dielectric Spectrometer over a range of temperatures down to -70 degrees C and a frequency range of 10(6)-10(-2) Hz. The differential scanning calorimetry (DSC) studies showed an anomalous response whereby at slow heating rates (2 degrees C min(-1)) a small exotherm followed immediately by an endotherm was observed. This response was considerably diminished in magnitude at higher rates (5 degrees C min(-1)) and was not observed at the fastest heating rate of 10 degrees C min(-1). No thermal events were seen on cooling the sample to -60 degrees C. It was suggested that the material formed a glass on cooling, with a predicted transition temperature of approximately -100 degrees C. Further studies using a liquid nitrogen cooling system indicated that the system did indeed exhibit a glass transition, albeit at a higher temperature than predicted (ca -63 degrees C). Low frequency dielectric analysis showed a clear relaxation peak in the loss component, from which the relaxation time could be calculated using the Havriliak-Negami model. The relationship between the relaxation time and the temperature was studied and was found to follow the Vogel-Tammann-Fulcher (VTF) modification of the Arrhenius equation. It is therefore concluded that Vitamin E Preparation USP is a glass-forming material that exhibits kinetically-hindered recrystallisation and melting behaviour. The study has also indicated that DSC and low frequency dielectric analysis may be powerful complementary tools in the study of the low temperature behaviour of pharmaceuticals.

Calorimetry, Differential Scanning↗
PubMed1999

Multiple roles for PI 3-kinase in the regulation of PLCgamma activity and Ca2+ mobilization in antigen-stimulated mast cells.

Cross-linking the IgE-bound FcepsilonRI with polyvalent antigen leads to Ca2+-dependent degranulation from mast cells and basophils, initiating the allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of FcepsilonRI-mediated tyrosine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLCgamma activity and is demonstrated by a dramatic inhibition of FcepsilonRI-induced Ins(1,4,5)P3 production after treatment of RBL-2H3 cells with wortmannin, a PI 3-kinase inhibitor. We show that PI 3-kinase lipid products support Ins(1,4,5)P3 production in at least two ways: by promoting translocation and phosphorylation of PLCgamma1 and by direct stimulation of both PLCgamma isoforms. In vitro stimulation of PLCgamma activity by PtdIns(3,4,5)P3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca2+ influx. Compared with control cells, Ca2+ responses are markedly diminished in antigen-stimulated cells after wortmannin pretreatment. Differences include both a longer lag time to the initial elevation in Ca2+ after antigen and an inhibition of the sustained Ca2+ influx phase. However, thapsigargin challenge during the sustained phase demonstrates no difference in the state of the Ca2+ stores in antigen-stimulated cells in the presence or absence of wortmannin. These data suggest that sufficient Ins(1,4,5)P3 is synthesized in wortmannin-treated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca2+ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca2+ influx in antigen-stimulated RBL-2H3 cells, one that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase.

Animals↗
PubMed1998

Analysis of DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione, by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

Sensitive and specific isotope dilution liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods were developed for the detection and quantitative analysis of S-[2-(N7-guanyl)ethyl]glutathione as a DNA adduct formed upon exposure of animals to carcinogenic 1,2-dihaloethanes. Separation and analysis were performed using microbore HPLC coupled in-line to an electrospray ionization triple quadrupole mass spectrometer. S-[2-(N7-guanyl)[2H4]-ethyl] glutathione was synthesized and used as internal standard. These methods provide structural confirmation of the adduct as well as quantitative analysis with the accuracy and precision necessary to measure biologically relevant levels in small tissue sample sizes (< 1 g). The sample detection limits in in vivo tissue extracts were 100 pg and 5 pg on-column for LC/MS and LC/MS/MS methods, respectively. Selected-ion monitoring mode was used to monitor the product ions of the doubly charged molecular ion. The application of these methods was demonstrated by measuring the DNA adduct levels in rat and fish samples after exposure to 1,2-dihaloethanes. The method has application in studies of DNA adduct formation as a biological marker of exposure to carcinogens and for environmental monitoring of 1,2-dihaloethanes.

Animals↗
PubMed1998

Wortmannin-sensitive phosphorylation, translocation, and activation of PLCgamma1, but not PLCgamma2, in antigen-stimulated RBL-2H3 mast cells.

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcepsilonRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcepsilonRI cross-linking activates both PLCgamma1 and PLCgamma2. Activation is accompanied by the increased phosphorylation of both PLCgamma isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCgamma isoforms have distinct subcellular localizations. PLCgamma1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCgamma1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCgamma2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcepsilonRI cross-linking. The activation of PLCgamma1, but not of PLCgamma2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCgamma1 with little inhibition of PLCgamma2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCgamma1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCgamma1. Although less abundant than PLCgamma2, activated PLCgamma1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

Androstadienes↗
PubMed1997

Analysis of toxic wastes in tissues from aquatic species. Applications of matrix solid-phase dispersion.

One of the difficult tasks in studying toxic wastes in the environment is the process involved in their extraction from biological matrices. This review addresses the regulatory aspects which mandate subsequent analyses in aquatic species and the studies which have addressed these problems from a variety of analytical perspectives. In this regard, the tissue extraction process known as matrix solid-phase dispersion is also reviewed and data are presented indicating that it may provide a generic process for the extraction and subsequent analysis of pesticides, polynuclear aromatic hydrocarbons and polychlorinated biphenyls.

Animals↗
PubMed1996

Fenbendazole-related drug residues in milk from treated dairy cows.

Oral administration of [14C] fenbendazole (FBZ) at a dose of 5.0 mg/kg leads to the presence of radiolabel in the milk of lactating dairy cows. However, the maximum mean concentration of total FBZ equivalents quantitated to one-third of the recommended safe concentration in milk (1.67 micrograms/mL). The label is equally distributed to the fat and aqueous portions of the milk. The maximum level, in general, is attained approximately 24-36 h after drug administration, with the highest levels ranging from 24 to 48 h after administration. The residues rapidly deplete, attaining levels of 10-20 ng/mL by day 5, and are essentially undetectable by radiolabel monitoring by day 6. Extraction of the milk by matrix solid phase dispersion indicated that the label was distributed between traces of the parent drug, FBZ, and predominantly, the FBZ sulphoxide (SO) and sulphone (SO2) metabolites. No other radiolabelled peaks were observed. Based on these data the metabolites of FBZ, FBZ-sulphone and FBZ-sulphoxide, could be used as marker residues for monitoring the administration of FBZ to lactating dairy cows.

Administration, Oral↗
PubMed1995

Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.

Actins↗