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PubMed1995

Factors influencing testosterone metabolism by anuran larvae.

The effects of experimental conditions--steroid concentration, temperature, treatment duration, tadpole density, and delivery mode--on testosterone (T) metabolic rates were examined in larval anurans. T uptake into tadpoles was measured by scintillation counting of [3H]T and metabolism of T was studied by thin layer chromatography in two anuran species, the African clawed frog (Xenopus laevis) and the western toad (Bufo boreas). T was extensively metabolized by both species, but the effects of each experimental condition on T metabolism varied between species. In the study of delivery mode, tadpoles injected with T dissolved in saline metabolized T faster than animals in groups where the steroid was added directly to the water. Animals injected with T dissolved in oil metabolized T the slowest, but T was short-lived in all cases. The current study demonstrates that concentration, temperature, treatment duration, tadpole density, and delivery mode may all affect steroid metabolic rates, but these effects vary between species in unpredictable ways. Since steroid metabolism may either activate or deactivate steroids, these factors are important in understanding the potency of steroids in tadpoles. A review of the literature dealing with steroid effects in anuran larvae illustrates these problems.

Analysis of Variance↗
PubMed1992

Replication and cytopathology of human parvovirus B19 in human umbilical cord blood erythroid progenitor cells.

Human parvovirus B19 productively infected erythroid progenitor (EP) cells from umbilical cord blood, in vitro as shown by an increase of viral DNA in supernatant fluid assayed by dot blot hybridization and liquid scintillation counting. Progeny virus was released into the supernatant fluid of CD34+ EP cells which had been purified by immunomagnetic separation. This supernatant fluid was infectious for bone marrow cells. Erythroid bursts infected with virus showed characteristic cytopathic effect by electron microscopy consisting of cytoplasmic vacuolization, marginated chromatin, and nuclear inclusions of lattice or crystalline arrays. Cultures of umbilical cord blood EP cells may be useful for the propagation of parvovirus B19 serological testing reagents and the study of virus-host cell interactions.

Base Sequence↗
PubMed1980

Separation of radiolabelled protein from brain and spinal cord of spinal hemisected rats on SDS polyacrylamide slab gels.

After hemisection of the spinal cord in rats, alterations in amino acid incorporation into protein have been observed in brain and spinal cord. Proteins mediating these changes have been studied in the present experiment. Male, Long-Evans hooded rats were given either a laminectomy and dura incision (sham) at spinal segment T2, a left spinal cord hemisection, or no operative procedures. One hour prior to utilization at 1, 3 and 14 days postoperation, amimals were injected subcutaneously with 200 microCi of [3H]-L-lysine and 200 microCi of [3H]-L-amino acid mixture. Samples were prepared for electrophoresis and proteins separated on linear gradient SDS polyacrylamide slab gels (7.5-20% acrylamide). The Gel slabs were cut into slices and processed for scintillation counting. In right somatomotor cortex, there was evidence for a general stimulation of amino acid incorporation at one day postoperation in animals which received surgery. At other postoperation intervals, significant increases in proteins in the regions of 90-150,000, 50-65,000 and 35-45,000 molecular weight (MW) were observed. In spinal cord, significant increases in proteins in the regions of 55-65,000, 30-45,000 and 15,000 MW were observed over postoperation time in all animals receiving surgery compared to normals. A large, significant increase in the radioactivity of proteins in the region of 18-20,000 MW were observed in the spinal cord lesion site of spinal hemisected animals alone at three days postoperation. These results indicate that increases in amino acid incorporation into the brain and spinal cord of laminectomized and spinal hemisected animals observed previously are theresult of both specific and nonspecific changes in protein incorporation of precursors.

Amino Acids↗
PubMed1981

Influence of triiodothyronine on the amino acid uptake of brain and spinal cord in normal and spinal hemisected adult rats.

The thyroid hormones triiodothyronine (T3) and L-thyroxine appear to enhance regeneration in the peripheral and central nervous system (CNS). The following experiments examine possible metabolic substrates for the action of T3 on the adult rat CNS after spinal hemisection. The protein incorporation of (3H)lysine after a left spinal hemisection (T2) or control operations was examined 1, 3, 7, and 14 days postoperation. Triiodothyronine (1 microgram/kg body weight in a bicarbonate buffer) was injected daily for the postoperation or equivalent time period. One hour prior to decapitation, animals were given a subcutaneous injection of 200 microCi of (3H)lysine. Samples of brain and spinal cord were dissolved, and the radioactivity of acid-precipitable protein and acid-soluble fractions were determined by scintillation counting. T3 treatment influenced the general levels of incorporation of all treated groups over all days postoperation. Specific effects were observed in spinal hemisected T3-treated animals. A significant hemispheric (P less than 0.05) asymmetry was present at 3 days postoperation with the right somatomotor cortex higher in protein radioactivity than the left. In spinal cord, the area of the lesion and areas just caudal to the lesion were higher in (3H)lysine incorporation in T3-treated rats relative to controls. T3 effects appear to involve an increased sensitivity of the cells of the injured nervous system to the hormone.

Amino Acids↗
PubMed1985

Coexistence of high-affinity uptake mechanisms for putative neurotransmitter molecules in chick embryo retinal neurons in purified culture.

We report here that high-affinity uptake mechanisms for two or three putative neurotransmitter molecules coexist in many of the neurons present in glia-free, purified neuronal monolayers from chick embryo retina. Replicate cultures were incubated with the tritiated forms of the amino acids gamma-aminobutyric acid (GABA), taurine (TAU) and aspartate (ASP) either individually or in binary or tertiary combinations. Incubation conditions were those typically used to analyze high-affinity uptake mechanisms. At the end of the incubation period the cultures were either lysed in water for measurement of intracellular radioactivity by liquid scintillation counting, or fixed in glutaraldehyde and prepared for autoradiography. Our results indicate that there is coexistence of uptake mechanisms for putative neurotransmitters in retinal cells. Biochemical measurements showed that, at the concentrations used in these experiments, two or more radioactive amino acids could be taken up simultaneously by the cultured populations without extensive inhibition. Moreover, the percentage of cells that appeared autoradiographically labeled in cultures exposed to two or more radioactive amino acid was less than the sum of the percentages of labeled cells when each amino acid was applied individually. Numerical analysis of the autoradiographs was carried out to determine the percentage of cells that could take up only one, only two, or the three putative amino acid neurotransmitters under investigation. This analysis showed that approximately 20% of the neurons have only the mechanism for TAU, whereas very few if any neurons have high-affinity uptake mechanisms for GABA alone or ASP alone. Our experiments have identified populations of cells that take up GABA and TAU (but not ASP) or ASP and TAU (but not GABA). Interestingly, we have not seen any neurons that can take up ASP and GABA and cannot take up TAU, although as many as 50% of the neurons can take up ASP, GABA, and TAU simultaneously. By showing simultaneous uptake of different putative neurotransmitters within individual neurons, our results emphasize the concept that the presence in a given neuron of a high-affinity uptake mechanism for a particular molecule is not a sufficient criterion to assign a neurotransmitter function to that molecule in that neuron.

Absorption↗
PubMed1992

Metabolic relationships between proteins of myelin and paranodally shedded, partially degraded myelin fragments in the rabbit CNS.

The "close-to-node" regions of myelinated nerve fibres, i.e., the paranodal end segments, are generally thought to be sites of high metabolic activity and myelin sheath turnover. Data on turnover rates of individual myelin constituents are conflicting but there exists a common belief that myelin is metabolized as independent molecules rather than as a unit. The occurrence of paranodal Marchi-positive bodies, with morphological and biochemical properties consistent with partially degraded myelin, prompted us to examine the temporal dynamics of the incorporation of radioactive precursor label in the major proteins of myelin and the Marchi-positive bodies. 3H-leucine was administered intrathecally in adult rabbits. After various survival times, the spinal cord was subfractionated by ultracentrifugation in a discontinuous two-step 0.32 M/0.85 M sucrose gradient. Myelin was collected from the interface and a floating fraction, heavily enriched in Marchi-positive bodies, was recovered on top of the 0.32 M sucrose. By scintillation counting and by gel fluorography combined with immunoblotting, a gradual appearance with time of partially degraded peptides of myelin-associated protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase was seen in the floating fraction but not in myelin. The temporal dynamics of the specific activities of these two proteins and myelin-basic protein and proteolipid protein were consistent with a typical source-product relationship between myelin and the material in the floating fraction. In conjunction with earlier morphological and biochemical findings, these data may suggest that Marchi-positive bodies appear as a consequence of myelin catabolism.

Animals↗
PubMed1991

Macrophage exposure to polymethyl methacrylate leads to mediator release and injury.

To understand further the role of macrophages in the loosening of cemented arthroplasty, several in vitro effects of polymethyl methacrylate (PMMA) particle exposure in these cells were studied. The kinetics of arachidonic acid and derived inflammatory mediator release was characterized following macrophage exposure to either PMMA or control polystyrene particles. Temporal release of radiolabeled products by [14C]arachidonate-labeled cells was determined by sequential scintillation counting. Significant dose-dependent release of arachidonic acid mediators by macrophages was observed within half an hour of exposure to either PMMA or styrene particles. Unexposed control cells incubated in media alone did not release detectable amounts of radiolabeled products. The leakage of intracellular lactate dehydrogenase (LDH), a marker of cell injury, was detected spectrophotometrically 4 h following exposure to PMMA but not styrene. PMMA-induced LDH release was dose dependent. In contrast, polystyrene exposure failed to increase LDH release above unexposed control cells. These in vitro studies reveal that macrophages rapidly released arachidonic acid and derived inflammatory mediators in response to both PMMA and styrene particles. However, cells exposed to PMMA are lethally damaged, as reflected by the subsequent leakage of their intracellular LDH. We propose that a similar sequence of events may occur when macrophages encounter PMMA particles at the bone-cement interface. This is characteristic of a foreign body granulomatous response.

Animals↗
PubMed2002

Functional characterization of monocarboxylic acid, large neutral amino acid, bile acid and peptide transporters, and P-glycoprotein in MDCK and Caco-2 cells.

Bidirectional transport studies were conducted to determine whether substrates of five intestinal transporters showed carrier-mediated asymmetric transport across MDCK (Madin-Darby canine kidney) cell monolayers grown under standard conditions. Drug concentrations were quantitated using liquid scintillation counting, liquid chromatography/mass spectrometry/mass spectrometry, or liquid chromatography/mass spectrometry. In the presence of a pH gradient, benzoic acid exhibited net apical-to-basolateral transport, with apparent permeability ratios (apical-to-basolateral permeability/basolateral-to-apical permeability) ranging from 14 to 25. The addition of valproic acid reduced the permeability ratio by 70-90%. Cephalexin transport also exhibited net absorption in the presence of a pH gradient, with apparent permeability ratios ranging from 14 to 71, depending on growth conditions. Radiolabeled phenylalanine exhibited a low level of carrier-mediated absorption with an apparent permeability ratio of 1.8 that was reduced to 1.0 in the presence of unlabeled L-phenylalanine. Taurocholic acid did not exhibit carrier-mediated absorption. Cyclosporine and fexofenadine exhibited P-glycoprotein-mediated efflux from both MDCK and Caco-2 cells, which was more sensitive to inhibition in MDCK cells. These results suggest that although MDCK cell monolayers may be a useful model for evaluating transport by the absorptive monocarboxylic acid and peptide transporters and the efflux transporter, P-glycoprotein, they are not useful for predicting large neutral amino acid or bile acid transport in the intestine.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Pharmacokinetics and pharmacological effect of lerisetron, a new 5-HT3 antagonist, in rats.

The pharmacokinetics of lerisetron, a novel 5HT(3) antagonist, are studied together with its efficacy in inhibiting the serotonin (5-HT)-evoked transient bradycardia reflex (von Bezold-Jarisch reflex) in Sprague Dawley rats. [(14)C]Lerisetron (50, 100, and 200 microg/kg) was given to rats by intravenous (iv) injection and plasma levels of unchanged (UL) and total (unchanged + changed, TL) drug were measured by liquid chromatography with radioactivity monitoring and scintillation counting, respectively. Linearity of UL and TL pharmacokinetics over the dose range was established by noncompartmental analysis. Protein binding of lerisetron was measured in vitro by ultrafiltration. The unbound fraction was 14.4 +/- 1.4%. A nonlinear mixed effects ("population") bicompartmental pharmacokinetic analysis showed that volume of distribution and clearance (CL) were high for both forms of the drug, but CL was significantly smaller for TL [(mean +/- SEM) 0.014 +/- 0.03 L/min for UL versus 0.006 +/- 0.03 L/min for TL, p < 0.05)]. Large interindividual variabilities were observed for both forms. The response to lerisetron administration (inhibition of bradycardia) was evaluated at different doses (2, 3, 5, 6, and 10 microg/kg, iv) at times 2-180 min after dose administration and related to simulated concentrations. Inhibition was 100% 5 min after the 10-microg/kg dose and, 3 h later, it was still > 10%. Response to lerisetron was dose related in the range studied. Pharmacodynamic parameters were estimated by a sigmoid E(max) naive-pooled model. The parameters were also different between the two forms: EC(50) was 0.44 ng/mL (CV = 5.9%) for UL and 0.88 ng/mL (CV = 4.9%) for TL. We conclude that UL and TL pharmacokinetics were linear and that the differences in the kinetics and dynamics between the two forms suggest the presence of at least one metabolite.

Animals↗
PubMed2005

Evaluation of dodecylmaltoside as a permeability enhancer for insulin using human carcinoma cells.

Dodecylmaltoside (DDM), an alkylglycoside showing tissue-permeability-enhancing properties, has been successful in improving nasal and ocular transport of poorly absorbed drugs. It was hypothesized that optimization of DDM concentration would improve the transport of insulin across epithelial monolayers without causing cell damage. Samples of markers and insulin were collected over a 6-h period and transepithelial electrical resistance was measured at concurrent time points to ascertain the effect of DDM on tight junctions. Samples were analyzed for lucifer yellow and insulin using reversed-phase high-performance liquid chromatography and for (3)[H]-mannitol by scintillation counting. A significant increase in the transport of markers and insulin was recorded in DDM-treated cells compared with controls. DDM enhanced the transport of markers and insulin in a concentration-dependent manner. Decreased transepithelial electrical resistance values confirmed that enhanced transport is caused by loosening of tight junctions. Cell recovery was >95% in 8 h indicating the potential of DDM as a penetration enhancer for clinical administration of insulin and other poorly absorbed drugs without causing cell damage.

Caco-2 Cells↗
PubMed1975

Maternal-fetal transfer of 14C-di-2-ethylhexyl phthalate and 14C-diethyl phthalate in rats.

14C-Di-2-ethylhexyl and 14C-diethyl phthalates were administered intraperitoneally to pregnant rats on either Day 5 or 10 of gestation. Rats were sacrificed at 24-hr intervals starting on Days 8 and 11, respectively; maternal blood, fetal tissue, amniotic fluid, and placentas (whenever possible) were obtained. The 14C-activity of each sample was determined by scintillation counting. It was found that both diesters and/or their metabolic products were present in each of these compartments throughout the gestation period, thus suggesting that the embryo-fetal toxicity and teratogenesis reported previously could be the results of a direct effect of the compound (or its metabolites) upon developing embryonic tissue. Additionally, the reduction in concentration of 14C from these tissues as a function of time was found to fit a first-order excretion curve. From this model curve, the half-life for both compounds was calculated; the average was about 2.33 days for di-2-ethylhexyl phthalate and 2.22 days for diethyl phthalate.

Amniotic Fluid↗
PubMed1976

Aqueous solubility of 14C-triamcinolone acetonide.

The blue tetrazolium colorimetric assay has often been employed in the analysis of corticosteroids. However, the reaction between blue tetrazolium and corticosteroids partly depends on the apparent pH of the system. In an earlier study of the temperature dependence of the solubility and dissolution of triamcinolone acetonide in aqueous dissolution media (distilled water and 0.2-1.0 M potassium chloride solutions), pH was not considered. To determine the reliability of the data previously reported, the solubility of 14C-triamcinolone acetonide was determined by a radioisotopic method. The results of the analyses, using a liquid scintillation counting system, indicate that the steroid solubility is generally about 20% lower than that reported previously.

Carbon Radioisotopes↗
PubMed1978

Fate of 14C-3-methylamino-1,2,3,4-tetrahydrocarbazole hydrochloride in rats and dogs.

The fate of 14C-3-methylamino-1,2,3,4-tetrahydrocarbazole hydrochloride, a potential psychotherapeutic agent, was studied in rats and dogs. Rats were given a single oral or intraperitoneal dose while dogs received the drug either orally or intravenously. Radioactivity in plasma samples and excreta was determined by liquid scintillation counting. In addition, 14C-levels in various rat tissues were estimated. The drug appeared to be well absorbed in both species. 14C-levels were highest in liver and lung and lowest in plasma. Excretion was primarily urinary and was more complete within the first 96 hr in the rat than in the dog, which suggested a longer 14C-half-life in the dog. No evidence was seen that the drug was demethylated.

Animals↗
PubMed1979

Hydrocortisone stability in human feces.

Hydrocortisone stability in human feces was studied under various conditions to determine whether stability accounts for the variable effects of hydrocortisone enemas. Recovery from feces and assay specificity were assured using dual isotopes, TLC separation, and liquid scintillation counting. Hydrocortisone degraded slightly from 7 to 26% in 24 hr when incubated in fresh human feces at 37 degrees. Less than 7% degradation occurred in feces stored at 10 degrees, and negligible degradation occurred with hydrocortisone in water at 37 degrees. Fecal bacteria may account for the observed degradation. Hydrocortisone stability in feces may contribute to local persistence and may account partly for its efficacy in ulcerative colitis treatment.

Chromatography, Thin Layer↗
PubMed1981

Pharmacokinetics of [14C]bretylium tosylate in rats.

The pharmacokinetics of bretylium tosylate were investigated in eight male Charles River rats. Each animal received an intravenous dose (10 mg/kg) of [14C)bretylium tosylate. Serial blood samples, urine, and feces were collected for up to 72 hr. Bretylium concentrations in plasma and amounts excreted in urine and feces were determined by scintillation counting. On the average, 88 and 95% of the dose were recovered in urine and feces in 24 and 72 hr, respectively. Urinary recovery accounted for 65.6 of the dose while 29.7% was excreted in the feces. Bretylium concentrations in plasma declined triexponentially and were fitted to a three-compartment open model. Bretylium has a very high apparent volume of distribution (15 liters/kg), and its beta half-life averaged 5.5 hr. Mean values of the apparent volume of the central compartment, plasma clearance, renal clearance, and excretion rate constants of bretylium in rats were 1 liter/kg, 1.93 liters/hr/kg, 1.27 liters/hr/kg, and 1.24 hr-1, respectively. The results indicate that: (a) bretylium is strongly bound to the tissues and is eliminated by active urinary secretion and by biliary excretion in rats, and (b) there are strong similarities between the pharmacokinetics of bretylium in humans and rats and that this animal model might be suitable for interaction studies with other drugs.

Animals↗
PubMed1981

Immunological studies of poisonous anacardiaceae: effect of vehicle on absorption of 3-n-pentadecylcatechol and its diacetate ester derivative after oral feeding in rats.

Tritium-labeled 3-n-pentadecylcatechol and its diacetate ester were fed to Sprague-Dawley rats. Both compounds were dissolved in ethanol and in corn oil vehicles and administered by gavage. The rats were placed in metabolic cages, and the urine and feces was determined by liquid scintillation counting, and the percentage of the administered dose was utilized as a measure of absorption. While there was no difference between the absorption of either compound, absorption was affected by the vehicle. Approximately 30% of the administered radioactivity appeared in the urine when ethanol was the vehicle, but about half that amount (14%) was excreted in the urine when the compounds were dissolved in corn oil. A subsequent bile cannulation study showed that the balance of the radioactivity found in the feces was not a result of biliary excretion. The majority of the activity recovered from urine and feces was eliminated within 48 hr after dosing. These data indicate that oil is a poor vehicle for GI absorption of urushiol components.

Administration, Oral↗
PubMed1982

Properties, stability, assay, and preliminary pharmacokinetics of the immunomodulatory 1,2-O-isopropylidene-3-O-3'(N',N'-dimethylamino-n-propyl)-D-glucofuranose hydrochloride.

1,2-O-Isopropylidene-3-O-3'(N',N'-dimethylamino-n-propyl)-D -glucofuranose hydrochloride (I) is a new agent with claimed immunomodulatory action and antiviral activity. Thin-layer chromatographic procedures and identifying tests were developed to separate the drug, its synthetic precursors, and solvolytic products, and were applied to stability studies. It is stable in 0.1 N NaOH at 60 degrees where its acid solvolysis product, 3-O-3'-(N',N'-dimethylamino-n-propyl)-D-glucose is readily degraded. The partition coefficient of I (pK'a = 9.28) between chloroform and plasma was 6.4 +/- 0.2 SEM between pH 10.5 and 11.0. Plasma and urine (0.5 ml) adjusted to pH 11.0 were extracted with 10 ml of chloroform and the extract evaporated. The reconstituted residue in 50 microliters of benzene, with the disopropylaminoethyl analog of I as an internal standard, was derivatized with 50 microliters of heptafluorobutyric anhydride at 60 degrees for 45 min and was evaporated and reconstituted in 100 microliters of benzene to be assayed for I by GLC with electron capture detection with a sensitivity of 5 ng/0.5 ml of biological fluid. The procedure was applied to pharmacokinetics in the dog and a two-compartment body model was observed with a terminal half-life of 103-130 min. At the 40-mg dose, 60-64% was excreted renally unchanged and 20-34% as unidentified metabolites. At the 200-mg dose 82-85% was excreted unchanged and 15-17% as unidentified metabolites. The respective renal clearances of I were 135 and 163 ml/min. The respective total clearances of I were 204 and 191 ml/min. These metabolites were apparently unextracted with chloroform from biological fluids at pH 11 and the liquid scintillation counting (LSC) assay of extracted radiolabeled I appeared synonomous with the GLC assay of I in such fluids.

Adjuvants, Immunologic↗
PubMed1982

Concomitant adsorption and stability of some anthracycline antibiotics.

Using liquid scintillation counting and liquid chromatographic techniques, it has been demonstrated that the anthracycline antibiotics, doxorubicin hydrochloride, N-trifluoroacetyladriamycin-14-valerate, and N-trifluoroacetyladriamycin-14-octanoate, can be strongly absorbed to the walls of containers, depending on the nature of both the container material and the solute. It also has been shown that the esters are unstable in the chemical growth media commonly used in cell culture studies. Both the adsorption and stability effects are suggested as being factors which should be carefully considered in interpretation of the in vitro and perhaps in vivo activities of the anthracycline esters.

Adsorption↗