Methods for the quantitation of abscisic acid and its precursors from plant tissues.

Methods are given for the quantitation of the plant stress hormone, abscisic acid (ABA), and its two metabolic precursors, ketone and enolate, that are applicable to all species tested so far. The plant extract is homogenized at neutral pH, hexane-soluble neutrals are extracted and discarded, and then the free ABA and other organic acids are extracted as ion pairs. The remaining aqueous phase is acidified, allowed to stand, neutralized, and extracted to give the ABA ex ketone fraction and then the aqueous phase is treated with base and again extracted to give the ABA ex enolate fraction. Each of these three fractions, free ABA, ABA ex ketone, and ABA ex enolate, along with a deuteriated internal standard, [side-chain-(2)H(4)]ABA, is then derivatized with pentafluorobenzyl bromide and purified on an automated sample preparation system. The resulting pentafluorobenzyl abscisate samples are then quantified using electron capture negative ionization mass spectrometry with methane as the reagent gas. Using these procedures free ABA, and ABA from its precursors, can be quantified at the level of 100 fg on column. If a large volume injector is used so that the total sample is injected it should be possible to quantify ABA and its precursors in the parts per billion range on a few milligrams of plant tissue.

Development of tolerance to kava in mice.

1. The development of tolerance to the aqueous extract of kava, and to the lipid soluble extract (kava resin) was tested in mice. 2. Tolerance to the unknown pharmacologically active ingredient(s) developed very rapidly, given parenterally, in the aqueous extract. A minimally effective daily dose (50 mg/kg) of the aqueous extract for 3 days was sufficient to produce tolerance to a test dose of 150 mg/kg, which is close to the ED50. As tolerance was evident at the first test period it can be assumed to be physiological tolerance. 3. Kava resin decreased spontaneous motility and caused a loss of muscle control. A minimally effective daily dose of kava resin (100 mg/kg) did not produce tolerance to the above effects of a weekly test dose of kava resin (166 mg/kg) within 7 weeks. In a further experiment the dose was raised to 150 mg/kg twice daily and this schedule caused partial tolerance to occur within 3 weeks, but very little further tolerance developed over the ensuing 2-week period. 4. To try to induce learned (behaviourally acquired) tolerance a dose of 166 mg/kg kava resin was injected daily and animals were tested each day while under the influence of the drug. However, even under these conditions, there was no tolerance evident within 3 weeks, when the experiment was terminated. 5. It appears difficult to induce the development of physiological or learned tolerance to kava resin in mice.

The antinociceptive actions of kava components in mice.

1. The antinociceptive properties of the aqueous extract of the intoxicating beverage kava, and of the lipid soluble extract (kava resin) were tested in mice, by the tail immersion and abdominal constriction methods. Both extracts showed analgesic effects in both tests. 2. Eight purified pyrones from the lipid soluble extract were also tested for activity in the tail immersion test, and kawain, dihydrokawain, methysticin and dihydromethysticin were found to be very effective in producing analgesia. Using the tail immersion test the time course of action of the extracts of the four effective pyrones of kava were studied. 3. Naloxone, in doses which inhibited morphine-induced analgesia in both tests, was completely ineffective in reversing the antinociceptive activities of the kava extracts, showing that analgesia produced by kava occurs via non-opiate pathways.

Positive interaction of ethanol and kava resin in mice.

1. The lipid soluble extract of the psychoactive beverage kava has hypnosedative properties which can be measured by the length of time that the righting reflex is lost. 2. Ethanol and the lipid soluble extract (kava resin) have been shown greatly to increase each others hypnotic action in mice. Ethanol also increases the toxicity of kava markedly. 3. This interaction of kava and alcohol has important clinical and social consequences since, in contrast to traditional usage, kava is now often taken in conjunction with alcoholic drinks.

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.

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.

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).

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.

Evidence that alpha-methyl-p-tyramine is implicated in behavioural augmentation to amphetamine.

Behavioural studies showed that administration of alpha-methyl-p-tyramine (AMT; 10 mg/kg i.p.) to rats 24 hr before treatment with d-amphetamine (AMPHET; 4 mg/kg i.p.) resulted in augmentation of AMPHET-induced stereotype activity. Parallel experiments involving electro-chemical estimation of dopamine metabolites in the striatum showed that the decrease in the concentration of homovanillic acid (HVA) produced by AMPHET (4 mg/kg) was enhanced in AMT (10 mg/kg) pretreated animals. These findings suggest that AMT derived from previous doses of AMPHET may play a role in the phenomena of behavioural augmentation observed after chronic administration of AMPHET.

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.

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.

Plasma quantitation of warfarin and warfarin alcohol by gas chromatography chemical ionization mass spectrometry in patients on warfarin maintenance therapy.

A quantitative method has been developed to measure plasma concentrations of warfarin and warfarin alcohol. The analytical procedure uses deuterated analogues as internal standards, and the technique of selected ion monitoring following gas chromatography methane chemical ionization mass spectrometry of the 4'-methyl ethers of warfarin and warfarin alcohol. Concentrations of warfarin and warfarin alcohol have been measured in plasma samples from 43 patients maintained on chronic warfarin therapy and compared with the 'apparent warfarin' concentration as measured by a fluorometric procedure. The study demonstrated a high degree of correlation between the gas chromatography mass spectrometric derived sum of the individual concentrations of warfarin and warfarin alcohol, and the 'apparent warfarin' concentration determined from a spectrofluorometric assay.

Analysis of the venom of the Sydney funnel-web spider, Atrax robustus using gas chromatography mass spectrometry.

Thirteen compounds have been identified using gas chromatography mass spectrometry in the venom of the Sydney funnel-web spider, Atrax robustus. The compounds were identified as their trimethylsilyl or pentafluoropropionate derivatives and were citric acid, lactic acid, phosphoric acid, glycerol, urea, glucose, gamma-aminobutyric acid, glycine, spermidine, spermine, tyramine and octopamine. Female venom contained trace quantities of 5-methyoxytryptamine which was not detected in male venom. Quantitative determination of tyramine and octopamine was achieved using chemical ionization (CH4) gas chromatography mass spectrometry and deuterated internal standards.

Quantitation of plasma warfarin levels by gas chromatography chemical ionization mass spectrometry.

A quantitative assay for plasma concentrations of warfarin has been developed using a deuterated warfarin analogue as internal standard. The method employs methylation (diazomethane) of warfarin to its 4'-methoxy derivative and gas chromatography chemical ionization mass spectrometry. Selected ion monitoring was used to relate the response of warfarin methyl ether and the internal standard. This technique can measure warfarin plasma concentrations five days after a single oral (25 mg) dose. Warfarin half-lives were determined in 12 healthy male volunteers. Each plasma analysis required six minutes of instrument time.