Search PubMed⌕ Search

Biomedical subjects

M S Roberts

Publications and source records attributed to M S Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1997

Tissue and perfusate pharmacokinetics of melphalan in isolated perfused rat hindlimb.

Melphalan is commonly used as a cytotoxic agent in isolated limb perfusion for locally recurrent malignant melanoma. The time course of melphalan concentrations in perfusate and tissues during a 60-min melphalan perfusion and 30-min drug-free washout in the single-pass perfused rat hindlimb was examined using a physiologically based pharmacokinetic model. The rat hindlimbs were perfused with Krebs-Heinseleit buffer containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 at a constant rate of 3.8 ml/min. The concentration of melphalan in perfusate and tissues was determined by high-performance liquid chromatography. The tissue concentrations of melphalan were significantly higher with the perfusate containing dextran than BSA during the 60-min perfusion. During the washout period, the melphalan concentration in the perfusates decreased rapidly in first few minutes, followed by a slower monoexponential decline. The estimated half life (t1/2) for melphalan removal from skin and fat was 59 +/- 2 min for both BSA and dextran perfusates. However, the estimated t1/2 for melphalan removal from muscle was 79 and 96 min for BSA and dextran washout perfusates, respectively. The predicted concentration-time profiles obtained for melphalan with BSA and dextran perfusates appear to correspond closely to the observed data. This study showed that the uptake of melphalan into perfused tissues is impaired by the use of perfusates in which melphalan is highly bound. Melphalan washout from muscle, but not skin and fat, was facilitated by the use of perfusates in which melphalan is highly protein bound.

Adipose Tissue↗
PubMed1996

High-performance liquid chromatographic assay for common sunscreening agents in cosmetic products, bovine serum albumin solution and human plasma.

This paper reports the development of a reversed-phase high-performance liquid chromatographic assay for quantifying five of the most common sunscreen agents, namely 2-ethylhexyl-p-dimethyl aminobenzoate (Escalol 507), 2-ethylhexyl-p-methoxycinnamate (Parsol MCX); 4-tert.-butyl-4'-methoxydibenzoylmethane (Parsol 1789), 2-hydroxy-4-methoxybenzophenone-3 (oxybenzone) and 2-ethylhexyl-salicylate (octylsalicylate). The assay permits analysis of the sunscreen agents in formulations and in biological fluids, including bovine serum albumin (BSA) solution, a common additive to in vitro skin diffusion cell receptor fluids, as well as human plasma. Separation was achieved using an ODS C154 column with a methanol-water (88:12) mobile phase. The analytes were detected by ultraviolet light absorption at a wavelength of 315 nm. The assay was linear with minimum detectable limits, calculated as greater than 3-times the baseline noise level: for oxybenzone and Escalol 507, 0.05 microgram/ml; for Parsol 1789 and Parsol MCX, 0.1 microgram/ml; for octylsalicylate, 1 microgram/ml. Recoveries from both plasma and 2% BSA were within the range 89-107%. The inter- and intra-day coefficients of variation for the five agents were not more than 4% at the upper end of the linear range and not more than 10% at the lower end. Preliminary stability studies of the sunscreen agents in a commercial product and in two diffusion cell receptor fluids were also conducted.

4-Aminobenzoic Acid↗
PubMed1996

High-performance liquid chromatographic determination of phenol, 4-nitrophenol, beta-naphthol and a number of their glucuronide and sulphate conjugates in organ perfusate.

This paper describes a simple and more sensitive reversed-phase HPLC method for the quantification of phenol, 4-nitrophenol and beta-naphthol and some of their glucuronide and sulphate conjugates in aqueous solution and liver perfusate buffer. Methanol-water mobile phases with ion-pairing agents for each phenolic group are detailed. The assay showed good recovery, accuracy and precision and is suitable for the quantification of these phenolic compounds in liver perfusion experiments.

Animals↗
PubMed1996

An optimized model for rat liver perfusion studies.

Conditions which influence the viability, integrity, and extraction efficiency of the isolated perfused rat liver were examined to establish optimal conditions for subsequent work in reperfusion injury studies including the choice of buffer, use of oncotic agents, hematocrit, perfusion flow rate, and pressure. Rat livers were perfused with MOPS-buffered Ringer solution with or without erythrocytes. Perfusates were collected and analyzed for blood gases, electrolytes, enzymes, radioactivity in MID studies, and lignocaine in extraction studies. Liver tissue was sampled for histological examinations, and wet:dry weight of the liver was also determined. MOPS-buffered Ringer solution was found to be superior to Krebs bicarbonate buffer, in terms of pH control and buffering capacity, especially during any prolonged period of liver perfusion. A pH of 7. 2 is chosen for perfusion since this is the physiological pH of the portal blood. The presence of albumin was important as an oncotic agent, particularly when erythrocytes were used in the perfusate. Perfusion pressure, resistance, and vascular volume are flow-dependent and the inclusion of erythrocytes in the perfusate substantially altered the flow characteristics for perfusion pressure and resistance but not vascular volume. Lignocaine extraction was relatively flow-independent. Perfusion injury as defined by enzyme release and tissue fine structure was closely related to the supply of O2. The optimal conditions for liver perfusion depend upon an adequate supply of oxygen. This can be achieved by using either erythrocyte-free perfusate at a flow rate greater than 6 ml/min/g liver or a 20% erythrocyte-containing perfusate at 2 ml/min/g.

Animals↗
PubMed1996

Tissue distribution kinetics as determinant of transit time dispersion of drugs in organs: application of a stochastic model to the rat hindlimb.

A stochastic theory of drug transport in a random capillary network with permeation across the endothelial barrier is coupled with a model of tissue residence time of drugs assuming radial intratissue diffusion. Axial diffusion is neglected both in tissue as well as in the radially well-mixed vascular phase. The convective transport through the microcirculatory network is characterized by an experimentally determined transit time distribution of a nonpermeating vascular indicator. This information is used to identify three adjustable model parameters characterizing permeation, diffusion, and steady-state distribution into tissue. Predictions are made for the influence of distribution volume, capillary permeability, and tissue diffusion on transit time distributions. The role of convection (through the random capillary network), permeation, and diffusion as determinants of the relative dispersion of organ transit times has been examined. The relationship to previously proposed models of capillary exchange is discussed. Results obtained for lidocaine in the isolated perfused hindleg in rats indicate that although the contribution of intratissue diffusion to the dispersion process is relatively small in quantitative terms, it has a pronounced influence on the shape of the impulse response curve. The theory suggests that the rate of diffusion in muscle tissue is about two orders of magnitude slower than in water.

Anesthetics, Local↗
PubMed1996

Modeling solute sorption into plastic tubing during organ perfusion and intravenous infusions.

The uptake of solutes into plastic infusion and perfusion tubing has been well documented, but the kinetics of the uptake process is not well-defined. Three mathematical models have been developed to describe the outflow fraction concentration--time profiles for solutes sorbed into the plastic tubing during infusion and perfusions. The models are referred to as model 1, convection--diffusion; model 2, convention-- interfacial resistance--diffusion; ad model 3, convection--interfacial resistance--infinite sink models. In each model, plug flow is assumed and, in order to minimize the number of variables required, solutions are limited to early times when the plastic behaves as an infinite sink. Initial conditions of (i) no solution in the tubing and (ii) a preloading of tubing with drug solution are considered for each of the three models. Two parameters, one being the transit time of solution through tubing (tmin) and the other a measure of the affinity and diffusivity of the solute in the plastic (SN), are sufficient to describe the outflow concentration--time profiles for solutes with sorption into tubing being limited by diffusion in the plastic (model 1). A single parameter, which is the effective interfacial permeability coefficient (H), is sufficient to describe the outflow concentration--time profiles for solutes with sorption into tubing being limited by an aqueous--plastic interfacial barrier (model 3). The three parameters (tmin, SN, and H) are required when uptake into tubing is limited by a combination of diffusion into plastic and an interfacial resistance (model 3). Each model has a characteristic outflow concentration--time profile determined by the relative magnitude of diffusivity of the solute in the plastic to that across the interfacial barrier. The sorption of nitroglycerin and isosorbide dinitrate are adequately described by the convection--diffusion model (model 1 (ii)) whereas the convection--interfacial resistance--diffusion model (model 2 (ii)) is required to describe the sorption of diazepam and chlorpromazine.

Adsorption↗
PubMed1996

In vitro and in vivo evaluation in rabbits of a controlled release 5-fluorouracil subconjunctival implant based on poly(D,L-lactide-co-glycolide).

PURPOSE: To design a controlled release 5-fluorouracil (5-FU) implant to provide prolonged antifibroblast concentrations of 5-FU in the subconjunctival tissues but low concentrations of 5-FU in other ocular tissues. METHOD: Implants (5 mg; 2.5 mm diameter x 1.2 mm thickness) of 5-FU or 9:1, 8:2, 7:3 5-FU to polymer mass ratios were made by compression. Poly(D,L-lactide-co-glycolide) polymers with 50:50 and 75:25 lactide to glycolide ratios were used. In vitro release characteristics of four types of implants were studied: 5-FU alone (CT), 5-FU/ polymer matrices (MT), coated 5-FU/polymer matrices with a central hole drilled through the matrix and coating (CM1), and with a central hole in the coating (CM2). MT and CM1 (9:1 drug/polymer) were selected for subconjunctival implantation in rabbits. 14C-5-FU levels in various ocular tissues and retrieved pellets were monitored. RESULTS: First-order release was observed from CT, MT and CM1 implants. Zero-order release profiles were observed from CM2 implants. Drug release was retarded by formulating 5-FU in a matrix comprising poly(D,L-lactide-co-glycolide) which in turn could be modified by the lactide/glycolide ratio of the polymer, the drug to polymer ratio, coating, and hole dimensions. First-order release kinetics were observed for MT and CM1 implants in the in vivo study in rabbits. A correlation between in vitro and in vivo release was established which allowed in vivo release to be predicted from in vitro release data. For CM1, therapeutic tissue concentrations of 35.2 micrograms/g (conjunctiva) and 17.7 micrograms/g (sclera) were found at the implantation site up to 200 hours post-implantation. Tracer levels were undetectable in other ocular tissues. CONCLUSIONS: The CM1 implant maintained steady antifibroblast levels in target tissues and minimized levels in nontarget tissues.

Animals↗
PubMed1996

Bacillus vallismortis sp. nov., a close relative of Bacillus subtilis, isolated from soil in Death Valley, California.

Five Bacillus strains isolated from Death Valley soil were shown to belong to a previously unidentified species, for which we propose the name Bacillus vallismortis. The type strain is strain DV1-F-3 (= NRRL B-14890). On the basis of previously published restriction digestion data, B. vallismortis is most closely related to Bacillus subtilis. At this time B. vallismortis can be distinguished from B. subtilis only by differences in whole-cell fatty acid compositions, DNA sequences, and levels of reassociation of genomic DNA.

Bacillus subtilis↗
PubMed1996

The disposition of aspirin and salicylic acid in the isolated perfused rat liver: the effect of normal and retrograde flow on availability and mean transit time.

The effect of changing the direction of perfusate flow from anterograde to retrograde on the disposition of acetylsalicylic acid (aspirin) and salicylic acid was studied in the single pass in-situ perfused rat liver. Mixtures of aspirin, [14C]salicylic acid and the inert reference solute [3H]sucrose were administered as boluses into the liver using red blood cell and albumin-free perfusate media at a flow rate of 30 mL min-1/liver. Hepatic availability (F), mean transit time (MTT) and normalized variance (CV2) for aspirin, preformed [14C]salicylic acid, salicylic acid produced from aspirin in the liver and [3H]sucrose were deduced from the outflow concentration profiles using statistical moment analysis. The values for F, MTT and CV2 for the solutes under anterograde perfusion were: aspirin (0.73 +/- 0.04, 15.13 +/- 2.01 s, 0.33 +/- 0.09, n = 5), preformed [14C]salicylic acid (1.05 +/- 0.06, n = 12, 43.19 +/- 2.21 s, 1.08 +/- 0.08, n = 5), salicylic acid from aspirin (0.33 +/- 0.05, 42.82 +/- 9.16 s, 0.73 +/- 0.10, n = 5) and [3H]sucrose (1.05 +/- 0.05, 16.88 +/- 0.77 s, 0.74 +/- 0.10, n = 5). The corresponding values for retrograde perfusions were: aspirin (0.73 +/- 0.02, 17.41 +/- 3.06 s, 0.32 +/- 0.09, n = 5), preformed [14C]salicylic acid (1.14 +/- 0.02, 44.42 +/- 3.16 s, 0.95 +/- 0.07, n = 5), salicylic acid from aspirin (0.33 +/- 0.09, 36.47 +/- 10.28 s, 0.58 +/- 0.05, n = 5) and sucrose (1.01 +/- 0.04, 18.08 +/- 1.61 s, 0.76 +/- 0.15, n = 5). No significant differences in F or MTT were apparent between anterograde and retrograde perfusions for all solutes. The MTT and CV2 data for [14C]salicylic acid and salicylic acid produced from aspirin is suggestive of a permeability limitation for salicylic acid transport.

Animals↗
PubMed1996

Distribution of systemically administered ampicillin, benzylpenicillin, and flucloxacillin in excisional wounds in diabetic and normal rats and effects of local topical vasodilator treatment.

The present study assessed the suitability of the streptozotocin-treated diabetic rat as a model for the study of diabetes-impaired wound healing. The distribution of three antibiotics, ampicillin, benzylpenicillin, and flucloxacillin, in wound and adjacent tissue sites on the abdomens and legs of normal and diabetic rats was determined 30 min after intravenous administration of a single bolus containing 50 mg of all three antibiotics per kg of body weight. Tissue/plasma ratios showed that antibiotic tissue penetration appeared to be related to protein binding. The treatment of wound sites with vasodilators (1% solution) to increase local blood flow and antibiotic delivery to the site was then determined and appeared to be more effective with endothelium-independent sodium nitroprusside than with endothelium-dependent acetylcholine in diabetic rats. These results suggest that coadministration of topical vasodilators to wound sites in neuropathic diabetic patients undergoing antibiotic therapy for infected ulcers could increase antibiotic delivery to wound tissue sites.

Acetylcholine↗
PubMed1996

Maternal to fetal thyroxine transmission in the human term placenta is limited by inner ring deiodination.

Placental deiodination of T4 to rT3 has been proposed as the factor controlling materno-fetal transmission of T4. We investigated T4 transfer in the isolated perfused human placental lobule with and without addition of the deiodinase inhibitor, iopanoic acid. T4 (150 nmol/L) in protein-free medium was added to the maternal circuit. Without iopanoic acid, the appearance of T4 in the fetal circuit was very low, with fetal T4 levels reaching only 4.1 +/- 0.84 pmol/L at 6 h. Levels of rT3 rose progressively in both circuits, reaching 28.8 +/- 5.5 nmol/L in the maternal and 12.4 +/- 3.2 nmol/L in the fetal circuit by 6 h. No T3 could be measured in either circuit. Addition of 0.5 nmol/L iopanoic acid to maternal perfusate, however, resulted in significant reduction in the appearance of rT3 [maternal levels, 0.58 +/- 0.06 nmol/L (2% of control values); fetal levels, 0.33 +/- 0.03 nmol/L (2.7% of control values)] and a major (approximately 2700-fold) increase in T4 appearance in the fetal circuit, with fetal T4 levels reaching 10.1 +/- 3.4 nmol/L at 6 h. These results support the hypothesis that placental inner ring (type III) deiodination is a major factor controlling placental transmission of maternal T4.

Female↗
PubMed1996

The effect of protein binding on the deep tissue penetration and efflux of dermally applied salicylic acid, lidocaine and diazepam in the perfused rat hindlimb.

Doses of radiolabeled water, salicylic acid, lidocaine and diazepam were applied in 2 ml of phosphate buffer saline to the exposed dermis on the thigh of a perfused rat hindlimb. Hindlimbs were perfused with Krebs-Heinseleit buffer at 37 degrees C containing 4% albumin or 2.5% dextran 40 at a constant rate of 4 ml/min. Clearance of solutes from dermal sites was monitored by frequent sampling at the dermal site and of the outflowing perfusate. In addition, the concentration of solutes in the various tissues below the treated and contralateral sites were determined at the end of a perfusion after dissection, solubilization and scintillation counting of the individual tissues. Estimates of the absorption rate constant from the dermis and the elimination rate constant from the perfused limb were made by the simultaneous fitting of absorption and efflux data with a one-compartmental pharmacokinetic model. Diazepam was cleared fastest through the dermis, followed by water, salicylic acid and lidocaine. The efflux of diazepam and salicylic acid from the hindlimb was significantly enhanced by the presence of albumin in the perfusate relative to no albumin (P < .05), whereas the efflux of water and lidocaine were unaffected by changes in albumin content. Analysis of individual tissues at the end of the perfusion showed that water had the highest relative tissue concentrations with diazepam the least, high concentrations appearing to partition into and remain associated with the dermis. The presence of albumin in the perfusate significantly increased the concentration of diazepam found in contralateral skin samples (P < .05) and showed the same trend for salicylic acid and lidocaine. These findings show that protein binding significantly affects the deep tissue penetration and distribution of dermally applied solutes and that this effect is more prominent for highly protein-bound solutes.

Administration, Cutaneous↗
PubMed1996

Local deep tissue penetration of compounds after dermal application: structure-tissue penetration relationships.

Topically applied compounds can penetrate directly into deeper underlying tissues. In this report, an attempt has been made to establish structure-deep tissue penetration relationships for a variety of solutes with diverse physicochemical properties. Stepwise multiple linear regression analysis was performed with the concentration in the immediately overlying tissue, the solute molecular size and the octanol/water partition coefficient as independent variables. During the initial period of direct penetration, the concentration of any solute in a given tissue was dependent on the concentration in the preceding tissue. The presence of molecular weight and lipophilicity terms as independent variables improved the regressions for some tissues. The solute concentration in the deeper tissues of sacrificed animals was higher for the smaller solutes. Due to the dominance of solute clearance by blood perfusing the tissues, the dependence of solute concentrations in anesthetized animal tissues on size was less than observed for sacrificed animals. The predictions from these regression analyses yielded predictions similar to those based on a physiological pharmacokinetic model. However, only about 50% of the data was explained by both models. Based on the preliminary qualitative and quantitative analysis, deep tissue penetration of solutes after application, as aqueous solutions, to the epidermis is greater for smaller solutes with adequate lipophilicity.

Administration, Cutaneous↗
PubMed1995

High-performance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in perfusate and plasma and melphalan in tissues from human and rat isolated limb perfusions.

A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 micrograms ml-1) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma were all 100 +/- 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8 - 56.8 micrograms ml-1, for DOH in the concentration range 0.5 - 30.0 micrograms ml-1 and for MOH in the range 1.4-25.1 micrograms ml-1, in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C.V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (fu) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb.

Adipose Tissue↗
PubMed1995

Nucleosomes reconstituted in vitro on mouse mammary tumor virus B region DNA occupy multiple translational and rotational frames.

The mouse mammary tumor virus acquires a highly reproducible chromatin structure when integrated into cellular DNA. Previous studies have suggested that the LTR is arranged as a series of six phased nucleosomes, that occupy specific positions on the LTR. On the basis of nucleosome reconstitution studies using DNA from the B region of the LTR, it has been argued that this sequence directs a uniquely positioned nucleosome. Here we demonstrate in vitro that reconstituted B region nucleosomes adopt at least five distinct translational positions in two rotational frames on a 206 bp fragment of DNA. We have resolved an initial reconstitute into its component species using nondenaturing gel electrophoresis, and precisely mapped the positions of each species using a hydroxyl radical footprinting assay. To confirm the nucleosome positions determined with the hydroxyl radical assay, nucleosome boundaries were mapped using exonuclease III. Comparison of the results from the hydroxyl radical footprinting and exonuclease III assays revealed a symmetrical pattern of overdigestion by exonuclease III which made unequivocal determination of nucleosome boundaries dubious. We conclude that the general use of exonuclease III to map the positions of nucleosomes may lead to incorrect assignment of position, and that assignment of position through the determination of the nucleosome pseudo-dyad from hydroxyl radical footprinting data represents a superior method of analysis.

Animals↗
PubMed1995

Nucleosome positioning on the MMTV LTR results from the frequency-biased occupancy of multiple frames.

The translational positions of nucleosomes in the promoter region of the mouse mammary tumor virus (MMTV) were defined at high resolution. Nucleosome boundaries were determined in primer extension assays using full-length single-stranded mononucleosomal DNA prepared from cells treated with formaldehyde, a reversible protein-DNA cross-linking agent. Multiple boundaries were observed in both the nucleosome A (Nuc-A) and Nuc-B region of the promoter, indicating multiple nucleosome translational frames. The different nucleosome frames in both the Nuc-A and Nuc-B regions were occupied unequally. The most frequently occupied frames were found clustered within 50-60 bases of each other, resulting in a distribution centered in the positions defined previously at low resolution for Nuc-A and Nuc-B. The most abundant 5' ends of the frames in the B region were found between -235 and -187, and the 3' ends between -86 and -36, whereas in the A region the most abundant 5' ends were between -22 and +42, and the 3' ends between +121 and +186. Although frames in the Nuc-B region of the LTR extend at a low frequency in the 5' direction toward the Nuc-C region, there is a sharp discontinuity in the 3' direction toward Nuc-A, suggesting the presence of a boundary constraint in the A-B linker. The positions and relative occupancies of nucleosome frames, in either the B or the A region, did not change when the promoter was activated with dexamethasone.

Animals↗
PubMed1995

Importance of dermal blood supply and epidermis on the transdermal iontophoretic delivery of monovalent cations.

The effect of dermal blood flow on the transdermal iontophoresis of five monovalent cationic solutes has been investigated using an in vivo rat model. The iontophoretic flux of solutes from topical application sites was shown, using anesthetized and sacrificed rats, to be independent of the dermal blood supply. The presence of a viable blood supply significantly affected the extent of penetration of solutes into deeper underlying tissues during iontophoresis. Higher tissue concentrations of solutes were found in the upper tissue layers of sacrificed rats, with no blood supply, compared to those in anesthetized rats. In all animals the highest concentration of solute after 2 h iontophoresis was found in the epidermis. Iontophoretic application of solutes to skin with the epidermis removed resulted in significantly lower fluxes than from sites where the skin was intact. These findings suggest that local blood flow determines systemic and underlying tissue solute absorption but not epidermal penetration fluxes during iontophoretic delivery. Finally, the dependence of iontophoretic transport of a solute on the solute's size in vivo was similar to relationships previously reported for excised human skin studies.

Administration, Cutaneous↗