Effect of altered tissue binding on the disposition of barbital in the isolated perfused rat liver: application of the axial dispersion model.

To examine the dependence of hepatic dispersion on tissue binding, the distribution kinetics of barbital under varying conditions of barbiturate perfusate concentrations was studied in the isolated perfused rat liver preparation (n = 5). The in situ liver was perfused in a single-pass mode with protein-free Krebs bicarbonate medium (15 mL/min). During steady-state infusion with various barbiturate concentrations (barbital, 1 g/L; butethal, 0.1, 1 g/L), a bolus containing [3H]water (cellular space marker) and [14C]barbital was injected into the portal vein. The recoveries of [3H]water and [14C]barbital were complete. The mean transit time and hence the volume of distribution for barbital in the absence of bulk barbiturate concentration (56 s and 1.24 mL/g) were about 2-fold higher than those for water (29 s and 0.58 mL/g), and they decreased progressively as the perfusate barbiturate concentration increased, indicating a decrease in tissue binding. However, the relative dispersion values (CV2H) of water (0.60) and barbital (0.66) were about the same magnitude and independent of the bulk concentration of barbiturate. The one-compartment dispersion model adequately described the data of barbital with a constant DN (dispersion number) value of 0.35. The results indicate that varying the tissue binding of barbital does not change the magnitude of DN; as such it offers a new experimental approach to examine the hepatic dispersion of solutes with a large distribution volume.

Transport of various substances through human enamel and dentine.

The penetration of 14C-labelled alcohols (methanol, ethanol, n-butanol), 14C-labelled carbonic acids (formic, acidic, propionic, valerianic, octanoic, malonic, succinic and lactic acid), 14C-drugs (procain, barbital), and 14C-sugars (saccharose, xylose) into about 800 human deciduous or permanent teeth, both healthy and carious, was investigated. Dental enamel up to the cemento-enamel junction was incubated at a pH-value of 5.0 or 6.8 for 1 or 24 hours. For measurement of radioactivity, the dentine of the root was obtained by trepanation. Between intact and carious permanent teeth only slight differences were observed in case of the diffusion of methanol and ethanol (2% of the incubation medium), while n-butanol penetrated the dentine to an extent of 4.2% at a pH of 5.0. The monocarbonic acids penetrated the enamel of healthy teeth within 24 hours to an extent of 6.6-19.2% of the content of the incubation medium, while the dicarbonic (succinic and malonic) acids reached amounts of 3.6 and 9.2%, and the percentage of lactid acid which penetrated the enamel reached 2.9%, respectively. Under all conditions tested, saccharose penetration was higher in carious than in healthy teeth (3.8 vs 6.5%). The highest uptake was found in experiments with barbital; it was more pronounced in deciduous than permanent teeth (16.2 vs 12.4%). The data could be of interest in the therapy of inflammatory and other processes of the pulp.

The interaction of albumin and drugs with two haemofiltration membranes.

The sorption of phenobarbitone sodium, barbitone sodium and fluconazole onto haemofiltration membranes made from polysulphone or a co-polymer of polyamide and polyvinylpyrrolidone was investigated in the presence and absence of albumin. The sorption of albumin was also followed in the presence of phosphate-buffered saline. Drug binding to the membrane was found to be reversible. Knowledge of the lipophilicity of the drug and hydrophobic/hydrophilic nature of the membrane did not allow successful prediction of the extent of binding of all the drugs; nor did knowledge of the extent of ionization of the drug and the charge of the membrane. Albumin bound to the polysulphone membrane in a manner that suggested the surface area to which it was binding was around 10 times greater than reported. In the presence of albumin there was a larger coefficient of variation in the binding of drugs to both membranes. The presence of albumin significantly decreased the binding of fluconazole, but not the other drugs, to the polysulphone membrane; however, albumin had no effect on the binding of any of these drugs to the polyamide membrane. We conclude that the binding of drugs to haemofiltration membranes cannot be simply predicted from knowledge of the hydrophilic/hydrophobic nature or charge of the drug and membrane, nor from the protein binding of the drug.

Possible involvement of NMDA receptor-mediated transmission in barbiturate physical dependence.

1. The competitive antagonists at the N-methyl-D-aspartate (NMDA) receptor, CGP39551 and CGP37849, protected against the barbiturate withdrawal syndrome in mice, as measured by ratings of convulsive behaviour on handling. 2. The effective doses of these compounds were lower than those required to prevent seizures due to NMDA in naive animals; these were in turn lower than those needed to prevent the convulsive effects of the alpha-aminobutyric acid (GABA) antagonist, bicuculline. 3. The NMDA-receptor antagonists did not alter the increase in the incidence of convulsions due to the GABAA antagonist, bicuculline, that is seen during barbiturate withdrawal, although the latencies to these convulsions during barbital withdrawal were significantly increased after CGP39551. 4. Barbiturate withdrawal did not affect the convulsive actions of NMDA, whether measured by the incidence of convulsions or by intravenous infusion. 5. The Bmax for [3H]-dizocilpine ([3H]-MK801) binding was significantly increased by chronic barbital treatment in cerebrocortical but not in hippocampal tissues, while the Kd remained unaltered in either case. 6. At 1 h and 24 h after administration of a single dose of barbitone, the Bmax for [3H]-dizocilpine binding was unaltered in cerebrocortical tissue. Acute addition of barbitone in vitro did not alter [3H]-dizocilpine binding or the displacement of binding of thienylcyclohexylpyridine.

Tolerance to ethanol and cross-tolerance to pentobarbital and barbital in four rat strains.

Chronic ethanol treatment by gastric intubation conferred tolerance to ethanol-induced motor impairment and hypnosis in four different rat strains: Fischer 344, Long-Evans, Sprague-Dawley, and Wistar. Cross-tolerance to barbital was also observed in all strains after chronic treatment with ethanol. However, chronic ethanol treatment failed to produce cross-tolerance to pentobarbital-induced motor impairment and hypnosis in any of the four strains. The demonstration of cross-tolerance to barbital and the lack of it to pentobarbital after chronic ethanol treatment confirms and extends recent observations on the specificity of the site and/or mechanism of action of sedative-hypnotic drugs that differ in lipid solubility.

Barbital N-glucoside is not detected as a urinary excretion product of barbital in humans.

A study was undertaken to determine if humans excreted barbital N-glucoside as a urinary metabolite following oral administration of barbital. A liquid chromatography method using gradient elution was developed for detecting and quantifying barbital N-glucoside and barbital in urine. Following a single oral dose of barbital to male caucasian and oriental subjects that had previously been shown to excrete amobarbital and phenobarbital N-glucosides, no barbital N-glucoside conjugate was observed in the urine. This result indicates that N-glucosylation of barbiturates is not a general pathway for the biodisposition of barbiturates in man.

The reaction mechanism of the novel vanadium-bromoperoxidase. A steady-state kinetic analysis.

The reaction of vanadium-bromoperoxidase from the brown alga Ascophyllum nodosum with hydrogen peroxide, bromide, and 2-chlorodimedone has been subjected to an extensive steady-state kinetic analysis. Systematic variation of pH and the concentrations of these three components demonstrate that the reaction model includes four enzyme species: native bromoperoxidase, a bromoperoxidase-bromide inhibitory complex, a bromoperoxidase-hydrogen peroxide intermediate, and a bromoperoxidase-HOBr species. This latter intermediate did not display any direct interaction with the nucleophilic reagent as oxidized bromine species (Br-3, Br2, and/or HOBr) were the primary reaction products. The generation of oxidized bromine species was as fast as the bromination of 2-chlorodimedone. The enzyme did not show any specificity with regard to bromination of various organic compounds. Formation of the bromoperoxidase-bromide inhibitory complex was competitive with the reaction between hydrogen peroxide and enzyme. From the steady-state kinetic data lower limits for the second-order rate constants at various pH values were calculated for individual steps in the catalytic cycle. This pH study showed that native enzyme must be unprotonated prior to binding of hydrogen peroxide (second-order association rate constant of 2.5.10(6) M-1.s-1 at pH greater than 6). The pKa for the functional group controlling the binding of hydrogen peroxide was 5.7 and is ascribed to a histidine residue. The reaction rate between bromide and enzyme-hydrogen peroxide intermediate also depended on pH (second-order association rate constant of 1.7.10(5) M-1.s-1 at pH 4.0).

Heteroassociation of O- and N-isopropyl derivatives of barbital and phenobarbital with 9-ethyladenine.

Heteroassociation of O- and N-isopropyl derivatives of barbital and phenobarbital with 9-ethyladenine (9-EA) in CCl4 solutions were studied by infrared spectroscopy. Cyclic heterodimers of high stability (725 less than KH less than 1960 1 X mol-1) compared to the corresponding homodimers (20 less than KD less than 60 1 X mol-1) were formed. The heteroassociation constants are interpreted in terms of both the hydrogen bonding tendency of the donor and acceptor centres and the number of sites available for the formation of hydrogen bonds. Such measurements may contribute to the understanding of the interactions between barbiturates, adenosine and their receptors in the brain.

Social isolation and tolerance development to sodium barbital in mice.

The effect of the duration of isolation periods on the development of tolerance to sodium barbital was studied in mice. The HD50 values for pentobarbital-induced hypnosis were higher in all mice that were long-term treated with sodium pentobarbital, whether or not they were isolated or in groups. Diazepam HD50s were higher in mice isolated and long-term treated with sodium barbital for 38 days and in grouped animals treated with the barbiturate for 20 and 30 days. Social isolation for 8 weeks plus 23 days increased the pentobarbital hypnotic dose (HD50). These data show that social isolation increased the rate of tolerance acquisition to sodium barbital by mice.

The effects of chronic oral administration of barbital on cerebellar cyclic GMP.

Female Sprague-Dawley rats received increasing dosages of barbital mixed with ground LabBlox for 7 weeks. At the end of this period, the rats were given doses of barbital acutely, and the effects of this treatment on levels of cyclic guanosine monophosphate (cGMP) in the cerebellum were determined. Chronic administration of barbital resulted in a slight depression of cGMP compared to control values. The acute injections of barbital produced a dramatic, dose-related decrease in levels of cerebellar cGMP in both control (C) and barbital-dependent (BD) animals. This depression of cGMP is of particular interest because the barbital-dependent animals had considerably higher levels of serum barbital than matched controls. The overall effect of chronic administration of barbital was right shift in the dose-response curve for levels of cGMP following acute injections of barbital. No differences in motor activity were noted between the dependent and control animals. It is believed that these results indicate that a high degree of tolerance is developed in the cGMP system of the cerebellum after chronic oral administration of barbital. Further investigation of this neurochemical parameter may provide useful information in the study of mechanisms mediating the dependence and the abstinence syndrome to barbiturates.

The influence of cyclobarbital and diazepam on drug metabolism in vitro and their binding to cytochrome P-450.

Cyclobarbital inhibited diazepam metabolism by rat liver supernatant only at concentrations of 1 mM. Ethylmorphine N-demethylation is strongly inhibited by diazepam in a non-competitive manner whereas cyclobarbital shows a competitive inhibition type. Both cyclobarbital and oxazepam are weak inhibitors. Hexobarbital- evokes typical type I spectral changes with rat liver microsomes whereas cyclobarbital and diazepam-induced spectral changes are similar to type II or inverse type I. Barbital evoked the same type of spectral changes as diazepam and cyclobarbital. With the usual concentration in vivo the metabolism of diazepam and other drugs can scarcely be influenced by cyclobarbital.

Selective permeability of the blood-uterine lumen barrier in rats: importance of lipid solubility.

The relative abilities of three test substances ( [14C] antipyrine, [14C] barbital and [3H] mannitol) having similar molecular weights (range of 182-188) but with differing lipid solubilities (partition coefficients between chloroform and phosphate-buffered saline, pH 7.4 of 17.2, 0.23 and approximately equal to 0.002, respectively) to enter the uterine lumen from blood were examined in immature ovariectomized and nephrectomized rats treated for 3 days with progesterone alone or combined with estradiol. With [14C] antipyrine and [14C] barbital steady-state conditions for radioactivity concentrations in uterine fluid were nearly achieved by 80 min after injection. At this time, the ratios of uterine fluid to serum radioactivity concentrations for these relatively lipophilic substances were marginally less than 1.0, indicating that equilibration between serum and uterine fluid radioactivity had nearly occurred. In contrast, these ratios at 80 min ranged between 0.30 and 0.31 for the least lipophilic substance tested, [3H] mannitol. The ratios of uterine fluid to serum radioactivity concentrations at 5 min after injection in animals receiving the same hormone treatment indicated that steady-state conditions were approached at differing rates depending upon the test substance. The test substances ranked according to these ratios were [14C] antipyrine greater than [14C] barbital greater than [3H] mannitol; this ranking of compounds corresponds exactly with that of their lipid solubilities. For [14C] antipyrine and [14C] barbital, as indicated by the ratios of uterine fluid to serum radioactivity concentrations at 5 min after injection, steady-state conditions were approached more rapidly in estradiol plus progesterone-treated animals than in those receiving progesterone only.(ABSTRACT TRUNCATED AT 250 WORDS)

The thin-film adsorber hemoperfusion device: detailed mass transfer and flow characteristics.

Further testing and evaluation of the thin-film adsorber (TFA) type of hemoperfusion device is reported here to complement an earlier paper describing the development of these devices and clearance tests performed with them. The present paper describes the results of pressure drop tests, flow uniformity tests, and detailed studies of the mass transfer characteristics of the components of the TFA units. The TFA units consist of powdered activated charcoal embedded in thin films of cellulose nitrate. These films are sprinkled with small particles of granular charcoal and then wound into spools, which are then placed in a plastic housing. The use of powdered charcoal exploits the enormous rate-of-uptake advantage of powdered charcoal over the granular sorbents used in other hemoperfusion devices. The present tests showed that the pressure drops in the TFA devices are intrinsically low, but that their priming volumes are only marginally acceptable. Significant flow nonuniformities also exist. Despite this, the overall mass transfer resistance values for the TFA devices are lower than those for available commercial hemoperfusion units. Measurements of diffusion coefficients in the carbon and in the carbon-loaded polymer film showed that in the carbon-loaded film, the slowest diffusion step involves the carbon particles themselves. Other tests disclosed that the liquid external to the film (i.e., in the flow spaces) offers even greater mass transfer resistance than does the carbon-loaded film. Further evaluations of the TFA type of device are suggested, particularly concerning its thrombogenic properties.

Familial euthyroid thyroxine excess: characterization of abnormal intermediate affinity thyroxine binding to albumin.

The abnormal high capacity T4 binding site of familial euthyroid T4 excess was separable from prealbumin and T4-binding globulin but not from albumin. We therefore compared T4 binding by albumin preparations isolated from the sera of normal and affected subjects. By equilibrium dialysis, albumin from affected subjects showed an extra T4 binding site (Kd approximately 50 nM) in addition to the T4 binding sites of normal albumin (Kd approximately 4 microM). Comparison of the estimated capacity of the additional site (200 microM) with the molar concentration of albumin suggested that only about one third of albumin molecules from affected subjects contained the extra binding site. Estimates of affinity and capacity were used to derive combining powers for the diverse classes of serum T4 binding sites. From these estimates, it appears that the presence of the abnormal site accounts for the approximate doubling of normal mean total T4 (from approximately 100 nM or 7.7 micrograms/dl to approximately 200 nM or 15.5 micrograms/dl), in order to maintain a normal free T4 in the face of the increased T4 association with albumin. Studies of [125I]T4 displacement from albumin of affected subjects showed low T3 affinity and competition by barbitone. Relative molar concentrations to give equivalent displacement of [125I]T4 were: 3,3',5,5'-tetraiodothyroacetic acid, 0.4; T4, 1.0; rT3, 4; 8-anilinonaphthalene sulfonic acid, 10; T3, 80; salicylate, 200; and barbitone, 40,000. Studies with dithiothreitol suggested that disulfide bonds were critical in maintaining the T4-albumin association. These findings indicate that familial T4 excess is due to abnormal intermediate affinity, sulfhydryl-sensitive T4 binding sites that are inseparable from the albumin found in affected subjects.

Similar development of tolerance to barbital-induced inhibition of avoidance behavior and loss of righting reflex in rats.

In order to determine if tolerance develops to the inhibition of avoidance behavior by the barbiturates, the effects of barbital on avoidance were determined in rats given barbital in their sole source of drinking water for 7 or 33 days. For comparison tolerance to the loss of righting reflex was also determined in other rats at the same time. All rats were trained by one 60-min session in a one-way active avoidance task; they were then put on the chronic drug administration schedule and then tested on the appropriate day after a single IP injection of 250 mg/kg sodium barbital. To assess the degree of tolerance, the brain level of barbital found at the biological endpoint--the loss of avoidance or loss of righting reflex--was compared in the chronic barbital treated rats and controls. A similar degree of tolerance developed to both effects of the drug and it appeared to be as great after 7 as after 33 days of chronic barbital treatment.

[Effect of pyriditol on drug metabolism]. / Vliianie piriditola na metabolizm nekotorykh lekarstvennykh sredstv.

Pyriditol (encephabol, enerobol, pyrithioxin, etc.), a disulfide derivative of pyridoxin, exerts an inhibitory effect on hexobarbital and amphetamine metabolism i vivo and on ethylmorphine N-demethylation in vitro. In the latter case the inhibition proceeds according to the mixed type of action. Pyriditol potentiates the hypnotic action of hexobarbital and barbital as well as the effects of amphetamine stereotypy. The mechanism of the potentiating of hexobarbital and amphetamine effects is of combined character and is conditioned both by the physiological properties of pyriditol and its inhibitory effect on hexobarbital and amphetamine metabolism.

Interaction of thyroid hormone and hemoglobin. I. Nature of the interaction and effect of hemoglobin on thyroid hormone radioimmunoassay.

Gel filtration of human RBC lysate incubated with labeled T4 or T3 revealed co-elution of a major iodothyronine-binding fraction (R-2) and hemoglobin. Solutions of purified human hemoglobin and T3 also showed co-elution of hormone and hemoglobin. Because hematin and protoporphyrin were shown to bind labeled T3, the oxygen-binding site on hemoglobin was excluded as the site of iodothyronine-hemoglobin interaction. Analysis of hormone binding by heme and globin moieties showed T3 binding to be limited to the heme fraction. Addition of excess unlabeled T3 to hemoglobin or heme incubated with labeled T3 indicated 75% to 90% of hormone binding was poorly dissociable. These observations suggested that the presence of hemoglobin in RBC lysate or in serum could influence the measurement of T4 and T3 by specific RIA. Subsequent studies of the addition to serum of human hemoglobin revealed a significant reduction in T3 and T4 detectable by RIA in the presence of this protein. The effect was influenced by the concentration of hemoglobin and by duration and temperature of incubations of hemoglobin and serum prior to RIA. Incubated for 5 days at 4 degrees C, 14 sera containing 10 gm/dl hemoglobin showed a mean decrease in T3 concentration of 40% compared to sera incubated in the absence of hemoglobin (160.1 to 93.9 ng/dl, p less than 0.001); detectable serum T4 fell by 50% in 13 sera incubated under the same conditions (5.40 micrograms/dl without hemoglobin to 2.55 micrograms/dl in the presence of hemoglobin, p less than 0.001). Hemoglobin concentrations in serum as low as 0.1 and 0.5 gm/dl affected the RIAs significantly. Thus a major fraction of thyroid hormone binding in human RBC cytoplasm is accounted for by an interaction with hemoglobin. This interaction in serum or RBC lysates is a significant variable affecting iodothyronine determinations.