Effects of sodium bicarbonate and sodium chloride on the elimination of etorphine in equine urine.

The combination of large doses of sodium bicarbonate and the potent narcotic, etorphine, has reportedly been given to racehorses in attempts to improve their performance and also to "mask" the presence of etorphine in urine samples. The increased urinary output and pH associated with sodium bicarbonate (approximately 500 g) administration may reduce the urinary concentration of etorphine, making it more difficult to detect. Our experiment was designed to examine the effects of this combination. Six Thoroughbred horses were used in a latin-square design with three horse pairs and three treatments consisting of the following: etorphine (20 micrograms), etorphine (20 micrograms) plus sodium bicarbonate (1.0 g/kg), and etorphine (20 micrograms) plus sodium chloride (0.7 g/kg). Sodium chloride was used to distinguish between the urinary alkalinizing effects of sodium bicarbonate and the diuretic effects associated with the large electrolyte load. Venous blood and urine samples were collected prior to and for 24 h post-treatment. Sodium bicarbonate produced a significant metabolic alkalosis and an increase in urine pH. Both sodium bicarbonate and sodium chloride produced a profound diuresis. After sodium bicarbonate and sodium chloride treatments, the urinary concentration of etorphine, measured by radioimmunoassay (RIA), was reduced and in some cases could not be detected. Extraction of the urine samples, prior to RIA analysis, increased the sensitivity of the assay and in most cases gave a positive result. We conclude that the coadministration of etorphine and sodium bicarbonate or sodium chloride can make the detection of etorphine more difficult because of the dilutional effects associated with the administration of a large electrolyte load.

Occurrence of (E)-4-hydroxy-2-nonenal in plasma and synovial fluid of patients with rheumatoid arthritis and osteoarthritis.

(E)-4-Hydroxy-2-nonenal (HNE), a cytotoxic propagation product of lipid peroxidation, is present in the synovial fluid (0.54 (0.19) mumol/l; mean (SE), n = 9) and plasma (0.34 (0.09) mumol/l, n = 9) of patients with rheumatoid arthritis. This compound was also found in the synovial fluid (0.24 (0.19) mumol/l, n = 9) and plasma (0.09 (0.03) mumol/l, n = 9) of patients with osteoarthritis. The concentration of HNE in the plasma of patients with rheumatoid arthritis was significantly greater than in patients with osteoarthritis.

Gas chromatographic mass-specific investigation of dextromoramide (Palfium) metabolism in the horse.

Dextromoramide (Palfium) was given by intravenous injection to a Thoroughbred horse at a dosage of 20 mg and urine was collected 2, 4, 6 and 8 h after drug administration. Enzymatic hydrolysis of the urine followed by solvent extraction gave a residue which was back-extracted into 0.1 M sulphuric acid. After basification to pH 9 and solvent extraction, the resulting residue was submitted to gas chromatographic-mass spectrometric analysis. Both electron-impact and ammonia chemical-ionization mass spectra were recorded and, based on the observed fragmentation patterns, the principal metabolites in horse urine were shown to be 2,2-diphenyl-3-methyl-4-morpholinobutyramide (compound 2) and the product of hydroxylation of one phenyl ring in dextromoramide (compound 3), respectively. The electron-impact mass spectra of compounds 2 and 3, and of their derivatisation products from oncolumn methylation in the gas chromatograph, are reported.

Effect of aqueous and lipid-soluble extracts of kava on the conditioned avoidance response in rats.

The aqueous, pyrone-free extract from kava (Piper methysticum) and the lipid-soluble extract (kava resin) were tested for their effect on amphetamine-induced hypermotility in mice and on conditioned avoidance response behavior in rats in a shelf-jump apparatus. Both kava extracts reduced amphetamine-induced hypermotility. Aqueous kava extract in i.p. doses of 30 mg/kg to 500 mg/kg had no effect on conditioned avoidance responses. At or below 100 mg/kg i.p. kava resin also failed to modify the number of conditioned avoidance responses obtained. However, 125 mg/kg of resin significantly reduced the number of conditioned avoidance responses by 18%. Increasing the dose of kava to 150 mg/kg caused ataxia and sedation which was so marked that a modified protocol was necessary. Only a marginally greater effect on conditioned avoidance response was obtained under these conditions. The effect of kava extract was slight compared to that of the standard antipsychotic drugs chlorpromazine and haloperidol in our procedure.

Comparison of the central nervous system activity of the aqueous and lipid extract of kava (Piper methysticum).

The central nervous activity of the aqueous extract of kava was examined in mice, and compared to the effect of the lipid-soluble extract. The aqueous extract caused a loss of spontaneous activity without loss of muscle tone. No hypnotic effect was seen, but some analgesia was produced. The anticonvulsant effect against strychnine was very slight and there was no evidence of local anesthetic action. There was a slight anti-apomorphine effect and tetrabenazine-induced ptosis was decreased. The lipid-soluble extract (kava resin) also decreased spontaneous motility, together with a marked reduction of motor control. Hypnosis, determined by loss of righting reflex, was produced, analgesia was marked, and a local anesthetic action evident. Kava resin also decreased apomorphine-induced hyperreactivity and partially reversed tetrabenazine-induced ptosis. Kava resin produces a greater range of pharmacological actions than the aqueous extract, and the latter is orally inactive in mice and rats. The pharmacological effects of kava ingestion appear to be due to the activity of the compounds present in the lipid-soluble fraction.

Identification of some human urinary metabolites of the intoxicating beverage kava.

Methane chemical ionization (CI) gas chromatography-mass spectrometry (GC-MS) has been used to identify some of the human urinary metabolites of the kava lactones following ingestion of kava prepared by the traditional method of aqueous extraction of Piper methysticum. All seven major, and several minor, kava lactones were identified in human urine. Observed metabolic transformations include the reduction of the 3,4-double bond and/or demethylation of the 4-methoxyl group of the alpha-pyrone ring system. Demethylation of the 12-methoxy substituent in yangonin (or alternatively hydroxylation at C-12 of desmethoxyyangonin) was also recognised. This product was isolated by high-performance liquid chromatographic analysis of crude urine extracts and characterised by methane CI GC-MS. In contrast to the situation prevailing in the rat no dihydroxylated metabolites of the kava lactones, or products from ring opening of the 2-pyrone ring system, were identified in human urine. GC-MS analysis of urine can be readily utilised to determine whether donors have recently consumed kava.

Uptake into mouse brain of four compounds present in the psychoactive beverage kava.

A technique using gas chromatography-mass spectrometry and deuterated internal standards is described for the quantitation in brain tissue of four constituents of the intoxicating beverage kava. Dihydrokawain, kawain, desmethoxyyangonin, and yangonin were administered ip to mice at a dosage of 100 mg/kg. At specific time intervals (5, 15, 30, and 45 min), the mice were sacrificed and the brain concentrations of these four compounds determined. After 5 min, dihydrokawain and kawain attained maximum concentrations of 64.7 +/- 13.1 and 29.3 +/- 0.8 ng/mg wet brain tissue, respectively, and were rapidly eliminated. In contrast, desmethoxyyangonin and yangonin had poorly defined maxima corresponding to concentrations of 10.4 +/- 1.5 and 1.2 +/- 0.3 ng/mg wet brain tissue, respectively, and these compounds were more slowly eliminated from brain tissue. When crude kava resin was administered ip at a dosage of 120 mg/kg, the concentration in brain of kawain and yangonin markedly increased (2 and 20 times, respectively) relative to the values measured from their individual injection. In contrast, dihydrokawain and desmethoxyyangonin, after the administration of crude resin, remained at the percentage incorporation into brain tissue established for their individual ip injection.

Identification by methane chemical ionization gas chromatography/mass spectrometry of the products obtained by steam distillation and aqueous acid extraction of commercial Piper methysticum.

Bornyl cinnamate has been identified as a constituent of kava resin and of the steam distillate of Piper methysticum. 5-Hydroxydihydrokawain was identified in commercial samples of P. methysticum originating from Vanuatu provided an initial aqueous extraction was employed. Commercial preparations, and fresh samples of the root of this plant from Fiji, lacked this compound. Two previously described N-cinnamoyl pyrrolidine alkaloids were also observed along with stigmasterol in kava resin from Fiji and Vanuatu. The products derived from aqueous 2 M hydrochloric acid extraction of P. methysticum were determined from methane chemical ionization gas chromatography/mass spectrometry analysis which identified a series of hydroxylated compounds (15a-d) derived from formal decarbonylation of the parent kava lactones. The products (13a-c) of dehydration of these compounds were also observed. The efficiency of kava resin extraction from plant material by water (the traditional method of preparation of the kava beverage) was typically 5-10% of that recovered by direct extraction with an organic solvent.

Distribution and chemical composition of the toxic skin secretions from trunkfish (family Ostraciidae).

The components of the mucus skin secretions from eight species of trunkfish found in the coastal waters of Australia were analyzed by combined chemical ionization-gas chromatography mass spectrometry. The species investigated were Anoplacapros lenticularis, Aracana aurita, Aracana ornata, Lactoria fornasini, Ostracion cubicus, Rhinesomas reipublicae, Strophiurichthys inermis and Strophiurichthys robustus. The beta-substituted choline chloride esters (mainly acetoxy, but with some species having butyryloxy, valeryloxy and one species with caproyloxy) of palmitic acid were the predominant components in almost all cases. High concentrations of monounsaturated palmitic acid were present in S. inermis and S. robustus. Trace quantities of C14, C17 and C18 choline chloride esters were also detected as were compounds where the choline moiety was modified by addition of one extra carbon.

Stereoselective accumulation of hydroxylated metabolites of amphetamine in rat striatum and hypothalamus.

The stereoselective accumulation of alpha-methyl-p-tyramine (AMPT) and alpha-methyl-p-octopamine (AMPO) in rat striatum and hypothalamus after acute and chronic administration of the (+)- and (-)-isomers of amphetamine (Amphet) and the acute administration of (+)- and (-)-AMPT has been investigated by chemical ionization gas chromatography mass spectrometry (c.i.g.c.m.s.). Two h after the administration of (+)- or (-)-AMPT (5 mg kg-1 i.p.), the concentrations of the isomers in striatal tissue were approximately equal; 18 h later, the concentration of the (+)-isomer was 10 times that of the (-)-isomer. The concentrations of AMPO in the striatum and hypothalamus 20 h after administration of (+)-AMPT were 68 ng g-1 and 484 ng g-1 respectively. After the administration of the (-)-isomer of AMPT, small quantities of AMPO were detected in both brain areas. Twenty h after the last of 7 daily injections of (+)-Amphet (5 mg kg-1, i.p.), the concentration of AMPO in the hypothalamus was 5.4 times the concentration at 20 h after one injection. In the striatum, the corresponding ratio for AMPO was 3.5 and for AMPT was 2.5. These data indicate that, although both isomers of AMPT formed from Amphet administered systemically, cross the blood brain barrier, the (+)-isomers AMPT and AMPO are preferentially stored in striatal and hypothalamic aminergic nerve terminals. The accumulations of AMPT and AMPO in rat striatum and hypothalamus after chronic administration of Amphet demonstrates that these metabolites persist in neuronal storage in these brain areas for days after administration. The half-lives of (+)-AMPT and (+)-AMPO in striatal neuronal storage, calculated from this data, were 1.5 days and 2.5 days, respectively. The corresponding half-life for hypothalamic (+)-AMPO was 7 days. 7. These findings suggest the involvement of accumulated AMPT and AMPO in the development of behavioural augmentation to repeated injections of Amphet (Randrup & Munkvad, 1970).

The concentration in brain of octopamine and tyramine after portal-systemic bypass in rats: neuroamine concentrations determined simultaneously by methane chemical ionization gas chromatography mass spectrometry.

The concentration in brain of both octopamine (OCT) and tyramine (TYR) was significantly increased in rats 8 weeks after portal-systemic bypass. This suggests that the increase in OCT is secondary to increased decarboxylation of tyrosine to TYR. However, the role these neuroamines, particularly OCT, play in the development of hepatic encephalopathy remains controversial.

Effect of chlordimeform and clonidine on the turnover of P-octopamine in rat hypothalamus and striatum.

The effect of the invertebrate octopamine agonists chlordimeform and clonidine on the concentration and turnover of p-octopamine and m- and p-tyramine was determined in rat hypothalamus and striatum. Clonidine (0.25 mg/Kg, s.c.) did not alter the concentration of p-octopamine in the hypothalamus or p-tyramine in the striatum. Administration of chlordimeform (50 mg/Kg, i.p.) resulted in an increase in p- and m-tyramine concentrations in the striatum but not that of p-octopamine in the hypothalamus. This increase in the tyramine isomers is consistent with the ability of chlordimeform and its metabolite, demethylchlordimeform, to inhibit monoamine oxidase (MAO). The concurrent administration of chlordimeform (50 mg/Kg, i.p.) and pargyline (75 mg/Kg, i.p.) produced a significant decrease in the accumulation of octopamine in the hypothalamus but not in the striatum. In contrast, the concurrent administration of clonidine (0.25 mg/Kg, s.c.) and pargyline (75 mg/Kg, i.p.) caused a significant decrease in the accumulation of octopamine in the striatum but not hypothalamus. These results show that the turnover of octopamine in the hypothalamus and striatum is decreased by chlordimeform and clonidine, respectively. Further, clonidine is known to modulate the turnover of amines in mammalian noradrenergic nerve terminals by an action at presynaptic adrenergic receptors. These data suggest that two mechanisms, one involving presynaptic adrenergic receptors in the striatum, and the other involving as yet unidentified receptors in the hypothalamus, modulate the turnover of octopamine in the mammalian brain.

The effects of (+)-amphetamine, alpha-methyltyrosine, and alpha-methylphenylalanine on the concentrations of m-tyramine and alpha-methyl-m-tyramine in rat striatum.

The concentration in rat striatum of the meta and para isomers of tyramine and alpha-methyltyramine, after the administration of (+)-amphetamine, alpha-methyl-p-tyrosine (AMPT) and alpha-methylphenylalanine (AMPA) has been determined using chemical ionization gas chromatography mass spectrometry (c.i.g.c.m.s.). Twenty hours after the last of 7 daily injections of (+)-amphetamine (5 mg kg-1 i.p.) the concentration of alpha-methyl-p-tyramine in striatal tissue increased twofold compared to the concentration 20 h after a single injection. In contrast the concentration of alpha-methyl-m-tyramine did not change. alpha-Methyl-m-tyramine and alpha-methyldopamine were found in the striatum at concentrations of 42 ng g-1 and 13.5 ng g-1 respectively after treatment of rats 20 h before with AMPA (100 mg kg-1 i.p.). After treatment with AMPT (100 mg kg-1, 20 h before decapitation) only the para isomer of alpha-methyltyramine could be detected (13.7 ng g-1) although the striatal concentration of alpha-methyldopamine was 274 ng g-1, a level 20 times greater than that observed after AMPA treatment. The combined administration of both AMPT and AMPA (100 mg kg-1 each, 20 h) resulted in a reduction of the striatal concentration of alpha-methyl-m-tyramine but not alpha-methyl-p-tyramine. These data suggest that alpha-methyl-m-tyramine in rat striatum is formed by the enzyme tyrosine hydroxylase on substrate AMPA, rather than by ring dehydroxylation of alpha-methyldopa and alpha-methyldopamine. Significant reductions in the striatal concentrations of m-tyramine 2 h after the administration of AMPT, suggest that tyrosine hydroxylase is involved similarly in the production of m-tyramine.

Analysis of some commercial preparations for migraine treatment using ion-pair high-performance liquid chromatography with addition of salts to the mobile phase.

When appropriate salts are added to the mobile phase in ion-pair high-performance liquid chromatography (HPLC) it is possible to arrive at an isocratic solvent with which complex mixtures of nitrogenous compounds of different pKa values and lipophilic characteristics may be separated. Selectivity in the manipulation of solute retention depends on the salt type, its concentration, the percentage of organic modifier in the mobile solvent and the solute itself. In addition to a dramatic reduction in analysis time, the use of salts can also improve the resolution of closely eluted peaks. With judicious control of the pH of the mobile solvent, the addition of salts to the mobile phase can cause the retention of compounds of different pKa values to alter in a contrasting manner. Under typical ion-pair HPLC conditions, an increase in salt concentration in the mobile solvent enhances the retention of neutral compounds and reduces the retention of ionized compounds. An inverse log-log relationship between the capacity factor of a solute and the salt concentration in the mobile phase was found. Examples are given of the use of salts in mobile solvents for ion-pair HPLC of a number of pharmaceutical preparations employed for the treatment of migraine.

Absence of alpha-methyldopamine in rat striatum after chronic administration of d-amphetamine.

Direct measurement by gas chromatography methane chemical ionization mass spectrometry of alpha-methyldopamine and alpha-methylnorepinephrine in rat striatum has shown the failure of these compounds to be accumulated in vivo after chronic administration of d-amphetamine despite the accumulation of alpha-methyltyramine, an immediate in vitro precursor. Further, both alpha-methyldopamine and alpha-methyltyramine accumulate in rat striatum after administration of alpha-methyltyrosine. These data suggest that, after administration of alpha-methyltyrosine, alpha-methyldopamine is formed via decarboxylation of alpha-methyldopa and not from hydroxylation of alpha-methyltyramine. Finally, our results indicate that alpha-methyldopamine does not play a role in the development of tolerance to d-amphetamine.

A chemical ionization gas chromatographic mass spectrometric assay for octopamine and tyramine in rat brain.

A method has been developed for the quantitation of the putative phenolamine neurotransmitter octopamine, and its precursor tyramine, in brain tissue. The procedure employs methane chemical ionization of the pentafluoropropionate derivatives of octopamine and tyramine together with the use of deuterated internal standards and selected ion monitoring. Deuterated analogues of octopamine and tyramine are added to brain homogenates in aqueous perchloric acid and ion exchange is used to isolate the brain amines. The method is capable of measuring 20 pg of octopamine and tyramine. The measured concentration (ng g-1 wet tissue) of octopamine and tyramine in rat brain was as follows: whole brain (less cerebellum) (0.6 and 2.2); hypothalamus (3.2 and tyramine value not statistically significant); striatum (0.5 and 11.8) and cortex (0.6 and 1.0). Administration of pargyline resulted in an increase (around ten-fold) in octopamine and tyramine concentration in all the above brain regions. In contrast alpha-methyltyrosine produced only a small increase (50%) in the concentration of tyramine in the striatum.

Uridine and inosine: pressor nucleosides from man, rat and dog.

Short-acting pressor compounds isolated from rat kidney, brain, heart and spleen have been identified as inosine and uridine by gas chromatography, mass spectrometry, high pressure liquid chromatography and analysis of ultraviolet spectra. Inosine was further identified by nuclear magnetic resonance. These compounds have also been found in kidneys from hypertensive man, rejected renal transplants and dog and beef kidney. Tissues were extracted by acid ethanol extraction followed by gel filtration and high voltage paper electrophoresis. Compounds found to be pressor in the anaesthetised rat resisted proteolytic enzymes, boiling for 10 min, extremes of pH and incubation with plasma from the source species for up to 20 min. The pressor effects of bolus injections of active gel filtration fractions, uridine and inosine were short-lived with a maximum effect at 5-6 s. Intravenous (I.V.) infusions of extracts gave a sustained pressor response without a concurrent change in heart rate. The effect on blood pressure was not accompanied by increased heart rate and persisted when the pressor effects of angiotensin II, noradrenaline and 5-hydroxy-tryptamine were blocked pharmacologically.

Identification of minor metabolites of 5-fluorocytosine in man by chemical ionization gas chromatography mass spectrometry.

A new minor metabolite of 5-fluorocytosine, 5-hydroxy-5-fluorocytosine, was found in the urinary gravel excreted by a patient being treated with this drug. A metabolite of 5-fluorocytosine in animals, alpha-fluoro-beta-alanine, was identified and quantitated in the urine of nine patients. In general, alpha-fluoro-beta-alanine represented 0.05--0.25% of the daily dose of 5-fluorocytosine, both for orally and intravenously administered drug. Minor metabolites have been implicated in the occasional toxicity observed in patients receiving 5-fluorocytosine.

Identification of new urinary metabolites in man of quinine using methane chemical ionization gas chromatography-mass spectrometry.

1. Six new metabolites of quinine have been identified in urine of man by methane chemical ionization g.l.c.--mass spectrometry. 2. Metabolites identified were: unchanged quinine and dihydroquinine, 3-hydroxyquinine, 3-hydroxydihydroquinine, 6'-hydroxycinchonidine, 6'-hydroxydihydrocinchonidine, quinine-10,11-epoxide and quinine-10,11-dihydrodiol. 3. 10-Chloro-11-hydroxydihydroquinine was identified as an artifact of the isolation procedure. 4. Chloroquine and desethylchloroquine (artifacts) were identified in the urine, 17 days after completion of a 48 h treatment with chloroquine.