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The pharmacokinetics and metabolism of 8-methoxypsoralen (8-MOP) were measured in the catheterized rat after pretreatment for 3 days with phenobarbital (PB), beta-naphthoflavone (BNF), 8-MOP, or vehicle. After an iv injection of 10 mg/kg of [14C]8-MOP, timed blood samples were collected and analyzed using a sensitive and specific assay for [14C]8-MOP. Total body clearance of 8-MOP increased from 0.55 +/- 0.06 liter/kg/hr in control rats to 5.6 +/- 0.4, 2.7 +/- 0.4, and 1.2 +/- 0.0 liters/kg/hr in rats pretreated with BNF, PB, and 8-MOP, respectively, indicating that all three compounds are inducers of 8-MOP metabolism. The pattern of urinary metabolites was altered by the enzyme inducers. The urinary excretion of the sulfate conjugate of 5-hydroxy-8-methoxypsoralen was increased from 10 to 40% of the dose after pretreatment with PB. This intact conjugate was identified using thermospray and fast atom bombardment mass spectrometry. Pretreatment with 8-MOP and BNF increased 2- and 3-fold, respectively, the urinary excretion of a labile sulfate conjugate of 5,8-dihydroxypsoralen. Metabolism of 8-MOP was demonstrated in the 9000 g supernatant and microsomes of rat liver and shown to be inducible by pretreatment of rats with BNF, PB, and 8-MOP. 8-MOP was metabolized in incubations with liver microsomes at rates of 0.22 +/- 0.06, 0.38 +/- 0.06, 0.78 +/- 0.07, and 0.91 +/- 0.03 nmol/min/mg of protein for vehicle, 8-MOP-, PB-, and BNF-pretreated rats, respectively. Results of our investigation indicate that the success of therapy with 8-MOP may be influenced by pharmacokinetic interactions with other drugs.

Glucuronide metabolites of carbamazepine (5 H-dibenz[b,f]azepine-5-carboxamide) were identified in human urine following chromatography on XAD-2 resin, permethylation, and combined gas chromatography and mass spectrometry with an SE-30 capillary column. Eight glucuronide metabolites, previously unidentified in man, were characterized as their permethylated derivatives. These included carbamazepine N-glucuronide (M+. 482), three isomers of dihydroxycarbamazepine O-glucuronide (M+. 542), three isomers of hydroxymethoxycarbamazepine O-glucuronide (M+. 542), and one isomer of hydroxycarbamazepine O-glucuronide (M+. 512). Other glucuronide metabolites, previously identified following enzymatic hydrolysis, were characterized as the unhydrolyzed, permethylated glucuronides, 10,11-dihydro-10,11-di--hydroxy carbamazepine O-glucuronide (M+. 544), and three isomers of monohydroxycarbamazepine O-glucuronide (M+. 512).

The concentration of phenytoin (diphenylhydantoin, DPH) was measured in plasma and semen of rabbits and man. In the rabbit, a single iv injection of DPH (4.64 mg) resulted in a concentration-time curve for DPH in semen parallel to the concentration-time curve for DPH in plasma (t1/2beta = 171 +/- 29 min). A semen/plasma drug concentration ratio of 0.20 was maintained for at least 8 hr, demonstrating that DPH concentrations in semen are directly proportional to DPH concentrations in plasma. In epileptic subjects maintained on oral DPH the mean drug concentration in semen was 2.31 microgram/ml while that in plasma was 13.8 microgram/ml. The mean semen-plasma DPH concentration ratio in man was 0.17; this closely approximates the observed ratio in rabbits.

A specific high-pressure liquid chromatographic assay was developed for the measurement of the narcotic antagonist drug, naltrexone. When 10 mg of naltrexone . HCl was added to the perfusate of the isolated perfused rat liver, the concentration of the drug in the perfusate declined rapidly with an apparent half-life of about 9 min. At 30 min 90% of the unchanged drug had been removed from the perfusate; the liver at this time retained 47% of the total radioactivity, of which only 75 represented unchanged drug. The bile at 30 min contained 28% of the total radioactivity and by 120 min this had risen to 70%. Initially naltrexone glucuronide was the principal metabolite in the bile. After 60 min N-dealkylated naltrexone glucuronide was the principal metabolite in bile. A marked increase in bile flow was noted immediately after the addition of the drug to the perfusate. The choleretic effect was attributed to the osmotic effect of the metabolites of naltrexone in the bile.

The metabolism of four benzomorphan compounds was studied in the isolated perfused rat liver, and glucuronide metabolites were identified by combined gas chromatography-mass spectrometry (GC/MS). Cyclazocine, ketocyclazocine, volazocine, and pantazocine were each added to the perfusate of the isolated perfused rat liver and the bile collected for 3 hours. The residue from evaporation of the bile was derivatized with the dimethylsulfoxide anion and methyl iodide, and the permethylated glucuronide metabolites were identified by GC/MS. The four compounds were hydroxylated by the liver and excreted in the bile as phenolic glucuronides. For example, permethylated hydroxycyclazocine glucuronide had a mass spectrum with a molecular ion at m/e 533 and fragment ions at m/e 301 (aglycone), m/e 260 (loss of cyclopropyl group) and prominent ions at m/e 232, 201, 169, 141, and 101 caused by fragmentation of the permethylated glucuronic acid moiety. Perdeuteriomethylation demonstrated that pentazocine, volazocine, and cyclazocine were further metabolized by methylation of one hydroxy substituent and glucuronidation on the other. Pentazocine, cyclazocine, and ketocyclazocine were also metabolized to phenolic glucuronides of the parent drugs. N-deakylated metabolites of pentazocine, volazocine, and cyclazocine were identified both as permethylated glucuronic acid conjugates and as the trimethylsilyl derivatives of the aglycones, obtained by enzymatic hydrolysis on the conjugates in bile.

Four healthy subjects and four addicts on high daily maintenance doses of methadone each received a parenteral dose of methadone hydrochloride following an overnight fast. The concentration of methadone in blood was compared with that in the gastric juice obtained over 8 hr by continuous low-pressure suction via a nasogastric tube. The concentration in the gastric juice was 25-200 times that measured at the same time in the blood. Thus, 8 hr after the injection mean blood concentrations of 28 and 210 ng of methadone per ml were recorded in the normal subjects and the addicts, respectively. The corresponding concentrations in gastric juice were 2,200 ng/ml and 18,000 ng/ml, respectively. In the normal subjects about 2% of the administered dose was recovered in the gastric juice in 8 hr, whereas in addicts about 7% was recovered. The greater recovery of methadone from the addicts appears to be the result of the larger volume of gastric juice recovered from the latter subjects. Methadone was also excreted in the saliva of both groups of subjects. In addicts, salivary concentrations were often 10 times those recorded in the blood. The N-monodemethylated metabolite of methadone was identified in the gastric juice of addicts by gas chromatography and mass spectrometry.

5,5-Bis(4-hydroxyphenyl) hydantoin has been identified as a minor metabolite of diphenylhydantoin (DPH) in the rat and human. This metabolite was synthesized in the laboratory from 4,4'-dihydroxybenzophenone. Gas chromatographic and mass spectrometric comparison of the permethylated synthetic compound with the permethylated derivative of the metabolite obtained from biological sources showed that they were identical. The metabolite was excreted as a glucuronide and accounted for about 1% of the total hydroxylated metabolites of DPH in human urine and rat bile. When the synthetic standard was added to the recirculating perfusate of the isolated perfused rat liver, a monoglucuronide, a trihydroxy-DPH glucuronide, and a dihydroxymethoxy-DPH glucuronide were identified in the bile.

Metabolites of mephenytoin (5-ethyl-3-methyl-5-phenylhydantoin) were characterized in human urine following chromatography on XAD-2 resin, permethylation, and combined gas chromatography and mass spectrometry. Four glucuronide metabolites previously unidentified in man were characterized as their permethylated derivatives by chemical-ionization and electron-impact mass spectrometry. These metabolites included 5-ethyl-5-(hydroxyphenyl)-3-methylhydantoin O-glucuronide; 5-hydroxyethyl-3-methyl-5-phenyl-hydantoin O-glucuronide; 5-ethyl-5-(hydroxymethoxyphenyl)-3-methylhydantoin O-glucuronide; and a metabolite tentatively identified as 5-ethyl-5-phenylhydantoin N3-glucuronide in which both N-demethylation and glucuronide conjugation of the hydantoin ring have occurred. Mephenytoin, N-demethylmephenytoin, 5-ethyl-5-(hydroxyphenyl)-3-methylhydantoin, and 5-ethyl-5-(hydroxymethoxyphenyl)-3-methylhydantoin were characterized in extracts of enzymatically hydrolyzed urine.

The effects of 8-methoxypsoralen (8-MOP) on drug metabolism in vivo were studied in catheterized rats. Rats were pretreated with a single i.p. injection of 5.4 or 27 mg/kg of 8-MOP, 30 min before an i.v. injection of either caffeine (CA; 10 mg/kg), hexobarbital (HB; 40 mg/kg), phenytoin (DPH; 15 mg/kg) or 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH; 15 mg/kg). Clearance of CA, HB and DPH, respectively (drugs eliminated primarily by phase-1 biotransformation), decreased from 0.25 +/- 0.02, 2.9 +/- 0.2 and 1.6 +/- 0.1 liters/kg/hr (means +/- S.E.) in control rats to 0.062 +/- 0.006, 0.65 +/- 0.15 and 0.09 +/- 0.03 liters/kg/hr in rats pretreated once with 27 mg/kg of 8-MOP. In contrast, the clearance of HPPH, which is eliminated primarily by glucuronidation, decreased only slightly from 1.3 +/- 0.1 liters/kg/hr in controls to 0.89 +/- 0.02 liters/kg/hr in rats pretreated with a single injection of 27 mg/kg of 8-MOP. Induction of drug metabolism by 8-MOP was studied in vivo in rats pretreated with 50 mg/kg/day of 8-MOP for 3 days and given an i.v. injection of CA, HB, DPH or HPPH 24 hr after their last pretreatment. This regimen increased the clearance of CA from 0.25 +/- 0.02 liters/kg/hr in controls to 1.08 +/- 0.04 liters/kg/hr in pretreated rats but had no significant effect on the elimination of HB, DPH and HPPH. Thus, acutely, 8-MOP is a potent, nonselective inhibitor of phase-1 metabolism in vivo. In contrast, chronically, it is a specific inducer of the metabolism of CA.(ABSTRACT TRUNCATED AT 250 WORDS)

The Ca²⁺-dependent binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) stimulates the catalytic oxidation of l-arginine to nitric oxide. The CaM-dependent increase in catalytic activity is associated with an increase in the flow of electrons from the flavoprotein to the heme domain. In the presence of suboptimal arginine concentrations, uncoupled turnover of nNOS produces both nitric oxide and superoxide, reactive species which combine to form peroxynitrite. We demonstrate here that peroxynitrite and other oxidants produced by nNOS oxidize the methionine residues of CaM and show that the ability of CaM to stimulate nNOS is impaired by this oxidative modification. Of the nine Met residues, those at the C-terminus (Met-144, -145, -124, -109) are most sensitive to oxidation. Correlation of the Met oxidation pattern with ability to stimulate nNOS suggests that oxidation of Met-36 is particularly important for the stimulation of nNOS. Incubation of nNOS with suboptimal concentrations of arginine results in sulfoxidation of the CaM methionine residues. Although nitration of the tyrosine residues in CaM could also occur, this does not occur to a significant extent in the present system. The results suggest that peroxynitrite may exert a feedback effect on its own formation by oxidizing CaM and thereby decreasing its ability to stimulate the turnover of nNOS.

Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH4). In the presence of BH4, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH4-free protein is decreased by the addition of BH4 without a corresponding increase in the high-spin component. Addition of BH4 decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH4 directly modulates the heme environment. BH4-free eNOS is completely inactive, but catalytic activity is recovered when BH4 (EC50 approximately 200 nM) is added. The spectroscopically determined binding constants for L-arginine are approximately 1.9 microM in the presence and approximately 4.0 microM in the absence of BH4. The BH4-supplemented enzyme has an activity of 90-120 nmol of citrulline.min-1.mg-1 and Km values of 3 and 14 microM for L-arginine and N-hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH4-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH4-reconstituted protein. Addition of BH4, increases the percent of the dimer by only approximately 5%. The results establish that BH4 influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.

The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes. The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms. The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D. H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D. Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein. The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene. L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction. The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites.

Two polyacetylenic antibiotics, peniophorin A and B, have been isolated from a strain of Peniophora affinis. Both have antibacterial and antifungal activity, but B is 3 to 20 times more active than A. Gram-positive cocci and a strain of Proteus vulgaris were especially susceptible to these two antibiotics. Both peniophorins contained an aromatic ring; B was an acid, and A was neutral. Peniophorin B was shown to be 2-(1-oxo-2,4-pentadiynl)phenyl acetic acid. The structure of A was only partially elucidated; it is suspected to be 6-[2-(1-oxo-2,4-pentadiynl)phenyl]5-methoxy-3-oxo-4-hexene-1-ol.

A numerical investigation is presented for the axisymmetric spin-up of fluid in a partially filled cylindrical cavity. It is an extension of earlier analyses to those cases where the liquid free surface intersects one or both endwalls. Previous models of the laminar Ekman layer pumping are modified heuristically for situations where the layer(s) no longer covers the entire wall. Numerical results for a range of Reynolds number, Froude number, and fill ratio have been obtained. They clearly demonstrae that it is the bottom wall Ekman layer which is primarily responsible for spin-up.

The effects of aminohydroxybutane bisphosphonate (AHBuBP) on bone after disuse osteopenia were studied in tail-suspended rats. Male Sprague-Dawley rats (weight range, 313-352 g) randomized into four groups of eight animals received 2 ml kg-1 day-1 of either AHBuBP (0.3 mg kg-1 day-1) or normal saline (vehicle) subcutaneously on days 14 and 15 of a 28-day experiment. The groups were 1) nonsuspended, saline; 2) suspended on days 14 to 28, saline; 3) suspended on days 0 to 28, AHBuBP; and 4) suspended on days 0 to 28, saline. On days 19 and 26, all rats received 15 mg/kg (1 ml/kg) of calcein. On day 28, they were sacrificed and their tibias and femurs were analyzed in vitro for bone density, strength and stiffness. The tibias were also analyzed histomorphometrically. The tibias and femurs from AHBuBP-treated rats were as dense as those in the nonsuspended group, whereas tail suspension in the untreated rats for 14 and 28 days caused a significant decrease in bone density. However, in measurements of bone strength and stiffness, the samples from the rats that received AHBuBP were similar to those of untreated rats suspended for 14 days, suggesting the newly formed bone was weaker. In the AHBuBP group, compared with all others, static histologic measurements of the proximal tibial metaphyses showed an increased bone area and perimeter and a decreased percentage of osteoid perimeter without a difference in the percentage of eroded perimeter. Dynamic histologic studies showed a decreased bone formation rate and decreased longitudinal growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Varying concentrations of ethyl alcohol were injected either into the left main coronary artery or intravenously in anesthetized intact dogs. Effects of alcohol on intracardiac conduction (by His bundle electrogram) were examined at spontaneous and paced (atrial) heart rates. Alcohol by the intracoronary route prolonged atrioventricular node and intraventricular conduction times by approximately 5 to 15%. These changes preceded a depression of left ventricular systolic pressure and of the rate of rise of left ventricular pressure and an elevation of left ventricular end-diastolic pressure. Intracoronary injections of contrast medium (sodium diatriozoate) or iso-osmolar solutions of sucrose and injections of similar amounts of alcohol in the ascending or descending aorta did not affect intracardiac conduction. Increasing atrial pacing rates resulted in prolongation of atrioventricular nodal conduction intervals, but did not influence intraventricular conduction time. At each pacing rate, alcohol depressed both atrioventricular nodal and intraventricular conduction. The data suggest that alcohol has a direct depressant effect on intracardiac conduction.

Tricaine methanesulfonate, administered at a dose of 150 mg/kg i.p., produced a flaccid paralysis and loss of the righting reflex in a number of poikilothermic species including the frog. Leopard frogs (Rana pipiens) given 150 mg/kg i.p. regained the righting reflex at 113 +/- 28 (S.D.) minutes after injection. A similar dose administered i.p. to mice produced no apparent pharmacological response. The biological half-life (T1/2) of tricaine in frogs (R. pipiens) was about 70 minutes at temperatures of 23 and 37.5 degrees C; at 7 degrees C the T1/2 was 309 minutes. In contrast, in the mouse the drug was metabolized so rapidly that 5 minutes after i.p. administration of 5 mg of tricaine methanesulfonate (250 mg/kg) none of the unchanged drug could be recovered from the animal. It was, however, recovered quantitatively as Bratton-Marshall reacting metabolites, including m-aminobenzoic acid. Incubations of tricaine with serum from bullfrogs, mice and humans indicated that the drug was metabolized to m-aminobenzoic acid with an apparent Km of 3 X 10(-3) M for the reaction. The Vmax for incubations of the drug with bullfrog and human serum was 40 nmol/min/ml of serum, whereas in mouse serum it was 93 nmol/min/ml of serum. In vitro studies with liver homogenates showed that mouse liver metabolized tricaine 39 times more rapidly than frog liver. We conclude, therefore, that the liver is the major site of tricaine hydrolysis in mammals and that the selective toxicity of tricaine for poikilotherms is a consequence of their slower rate of hepatic biotransformation of tricaine.

The disposition of 40 mg of d-methadone HCl was studied in New Zealand White rabbits. After an intravenous infusion to male rabbits, the beta phase half-life was 106 +/- 21 min and the apparent volume of distribution was 12.6 liters/kg b.w. After an i.m. injection, the peak drug concentration in arterial blood (1.02 microgram/ml) occurred 10 min after the injection, whereas the peak concentration in semen (2.58 microgram/ml) occurred 140 min later. The semen/blood drug concentration ratio was 6.8 at 150 min after the injection. The concentration of d-methadone was measured in blood, spleen, liver, lung, prostate, seminal vesicle, testis and heart 130 minutes after an i.m. dose. All tissues had drug levels that exceeded the concentration in blood (0.42 microgram/ml). The concentrations in lung (33.5 microgram/g), kidney (15.2 microgram/g) and spleen (30.6 microgram/g) were particularly elevated. After intravaginal implantation, d-methadone was rapidly absorbed into the systemic circulation of female rabbits; the peak drug level in blood (0.94 microgram/ml) occurred 60 min after drug administration.

A specific gas chromatographic assay has been developed for measurement of valproic acid (VPA) and its major conjugated metabolites. In rats given single intravenous doses, the decline in blood concentration of VPA was dose-dependent and followed first-order kinetics only at the lowest dose. The time required for the maximum concentration of VPA, after completion of the brief distribution phase, to decline by 50% was 11.7, 41 and 125 min at doses of 15, 150 and 600 mg of NaVPA per kg, respectively. A secondary increase in drug concentration, abolished by exteriorization of the bile, was observed in all intact rats with all doses. Some 45 to 55% of the dose appeared in the bile in 5 hr as VPA glucuronide. Urinary excretion of VPA glucuronide in intact animals accounted for 23 and 51% of the 15 and 150 mg/kg doses, respectively. Tissue distribution studies in rats sacrificed 20 and 90 min after dosage with [14C] NaVPA (150 mg/kg) showed that the drug concentration was highest in blood, moderate in liver, kidney, heart and lung and low in brain, fat, testis and skeletal muscle. During this interval the drug concentration declined in all tissues, whereas the total conjugated metabolites in the small intestine increased from 7 to 28% of the administered dose. The large intestine contained 30 times as much free drug as conjugate. Reabsorption of free VPA, released by hydrolysis of conjugate in the large bowel, accounted for the secondary rise in concentration of VPA observed in blood after single doses. The major urinary metabolites, VPA-glucuronide and 2-n-propylgutaric acid, were identified by gas chromatography/chemical ionization mass spectrometry. Sodium VPA caused a dose-dependent stimulation of bile flow, the magnitude and duration of which closely followed the blood concentration of VPA.