High-performance liquid chromatography-fluorescence detection method for endogenous gamma-aminobutyric acid validated by mass spectrometric and gas chromatographic techniques.
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Animals↗
Biomedical subjects
J M Tusell
Publications and source records attributed to J M Tusell.
Regional concentrations of GABA, serotonin and noradrenaline in brain at onset of seizures induced by lindane (gamma-hexachlorocyclohexane).
The main objective of this study was to determine the modifications induced by the pesticide lindane (gamma-hexachlorocyclohexane) in the regional concentration of neurotransmitters in brain, taking the tonic-clonic seizure as the main sign of its neurotoxic action. The animals were given lindane (150 mg/kg p.o. in olive oil) and killed at the onset of the first seizure (mean latency time: 18.3 +/- 5.5 min, n = 16). The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and the biogenic amines noradrenaline (NA) and serotonin (5-HT), as well as its corresponding acidic metabolite, were determined by high performance liquid chromatography with fluorimetric or electrochemical detection in different areas of the brain: hippocampus, mesencephalon, colliculi, frontal cortex, parietal cortex, striatum and thalamus. The concentration of GABA in whole tissue was only significantly decreased in the colliculi. The concentration of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were decreased in the colliculi, striatum and frontal cortex, suggesting a decreased synthesis and release of 5-HT, but increased in the parietal cortex. The concentration of NA was significantly decreased in the parietal cortex. Thus, the results indicate that lindane induces some modifications of the concentration of neurotransmitters in cortical structures, basal nuclei, midbrain and colliculi. These changes may be consequent upon the proposed interaction of lindane with the GABAergic system, although a direct action of lindane on other neurotransmitter systems cannot be excluded.
Animals↗
Convulsant effect of lindane and regional brain concentration of GABA and dopamine.
Lindane (gamma-hexachlorocyclohexane) is an organochlorine insecticide with known neurotoxic effects. Its mechanism of action is not well understood although it has been proposed that lindane acts as a non-competitive antagonist at the gamma-aminobutyric acid (GABA)-A receptor. We studied the effect of lindane (150 mg/kg) on the GABAergic and dopaminergic systems by measuring the concentration of GABA, dopamine and its metabolites in 7 brain areas at the onset of seizures. All animals suffered tonic convulsions at 18.3 +/- 1.4 min after lindane administration. The concentration of GABA was only slightly but significantly decreased in the colliculi without modifications in the other areas. The concentration of dopamine was increased in the mesencephalon and that of its metabolite DOPAC was also increased in the mesencephalon and the striatum.
3,4-Dihydroxyphenylacetic Acid↗
Effect of lindane at repeated low doses.
The relationship between brain concentration of lindane and its convulsant effect have been studied in rats after repeated low doses of lindane. The mean brain plateau concentration was achieved in 5-8 days. The doses administered (5, 12 and 20 mg/kg) and the brain concentration of lindane at the plateau were highly correlated. The incidence rate of response (percentage of rats with tonic convulsions) was also highly correlated with the log of concentration of lindane in brain for all doses and days studied. A decrease in brain concentration of lindane was observed after 12 days of daily administration at doses of 5 and 12 mg/kg but not at the highest dose (20 mg/kg).
Animals↗
Relationship between lindane concentration in blood and brain and convulsant response in rats after oral or intraperitoneal administration.
The relationships between brain and blood concentrations of lindane and its convulsant effects have been studied in rats after different po and ip doses. A good correlation was observed between dose and the frequency and time to onset of tonic seizures, the intensity of the response and lethality. An oral ED50 (84 mg/kg) and an ip ED50 (131 mg/kg) for the half-maximal incidence rate of lindane-induced tonic seizures was determined. The incidence of response was directly proportional to the log of lindane concentration in brain and blood, with an EC50 of 5.3 micrograms/g and 1.5 micrograms/ml, respectively. The threshold concentration of lindane eliciting tonic seizures was estimated to be approximately 5 micrograms/g in brain and approximately 1.5 micrograms/ml in blood. The kinetics of lindane in brain and blood were examined after a single 60 mg/kg po or ip dose over a period from 5 min to 1 week. Concentrations of lindane in brain and blood were found to be highly correlated.
Adipose Tissue↗
Comparative ontogenesis of brain tryptamine, serotonin, and tryptophan.
The pattern of ontogenetic development of tryptophan (TP), tryptamine (T), indole-3-acetic acid (IAA), 5-hydroxytryptamine (5-HT; serotonin), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the brains of rats aged 1-45 days is presented. Analysis of the five components in each brain allows the calculation of the acid/amine and amine/amino acid ratios. These metabolic indexes are a useful tool to study and compare the metabolic origins and fates of both amines. The ontogenetic patterns of TP, T, and IAA are very similar, especially during the first week postpartum. The highest and lowest levels found for T were 2.2 ng/g and 0.1 ng/g at the 1st and 5th day, respectively. The temporal relationship between the T/TP and IAA/T ratios suggests the existence of mechanisms protecting T against monoamine oxidase (MAO) which develop in parallel to synaptogenesis. Significant correlations were found between TP and IAA during the whole period studied and between TP and T during the first week after birth. The 5-HT peak found during the first postpartum week could be due to a non-neuronal pool of 5-HT protected against MAO and possibly contained in mast cells. Preliminary determinations on leptomeningeal membranes suggest the existence of such a pool.
Animals↗
Comparison of high-performance liquid chromatography and gas chromatography-mass spectrometry for the analysis of indole-3-acetic acid in brain tissue.
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Animals↗
Determination of tryptamine in brain tissue by capillary gas chromatography mass spectrometry (selected ion monitoring).
The development of an analytical method for the determination of brain tryptamine using capillary gas chromatography mass spectrometry (selected ion monitoring) is presented. The method involves solvent extraction of brain homogenates and further derivatization (pentafluoropropionyl derivatives) of the dried extracts. Gas chromatographic analysis is performed using a bonded phase silica fused capillary column. Selected monitoring of m/z 289 and 292 (isotope dilution technique) allows the determination of tryptamine in adult rat brain (450 +/- 73 pg g-1) (means +/- SEM), n = 6.
Animals↗
Improved liquid-chromatographic determination of 5-hydroxyindoles.
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Ascorbic Acid↗
On the metabolic origin of plasmatic indole-3-acetic acid in the rat.
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Animals↗
Liquid-chromatographic determination of indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human plasma.
We describe a "high-performance" reversed-phase liquid-chromatographic method for determination of indole-3-acetic acid (I) and 5-hydroxyindole-3-acetic acid (II) in human plasma. I is eluted at 1.0 mL/min with a mixture of 1-pentanesulfonic acid (pH 3.1), methanol, and water. It is detected by fluorometry. A mixture of citric acid/sodium phosphate solution (pH 4.8) and methanol, at 1.5 mL/min, is used to elute II, which is detected with an electrochemical cell. Platelet-poor plasma samples were pretreated with HCl, perchloric acid, and trichloroacetic acid for protein precipitation. Best results were obtained with the last (protein precipitation is incomplete with HCl, while recoveries of I are concentration dependent with perchloric acid). Analytical recoveries were 58% (SD 3.1%, CV 5.3%, n = 12) and 79% (SD 3.3%, CV 5.3%, n = 9) for I and II, respectively. Concentrations of I and II in plasma ranged from 0.61 to 3.32 (mean 1.54, SD 0.59, n = 15) mumol/L and from 33.0 to 102.6 (mean 51.8, SD 20.1, n = 16) nmol/L, respectively.
Adult↗
Study of leucine-enkephalin in rat brain by a new high-performance liquid chromatographic method.
A specific method is presented for the assay of leucine-enkephalin (Leu-Enk) and its metabolites by reversed-phase high-performance liquid chromatography on a muBondapak C18 column with a mobile phase of methanol-water and 0.005 M tetrabutylammonium phosphate. The four substances are resolved by a linear program from 8 to 70% methanol in 25 min. Detection is achieved by monitoring the absorbance at 280 nm. Time of analysis can be reduced by means of a new high-speed liquid chromatography package. This method allows the study of the effect of Phe-Ala on the cerebral metabolism of Leu-Enk. For this purpose, membrane preparations from rat striatum were incubated in the presence of [3H]Leu-Enk with different concentrations of Phe-Ala from 10(-7) to 10(-3) M during 1 h at 37 degrees C. Collected eluates were counted by liquid scintillation. The results suggest the presence of three membrane enzymes which generate the three metabolites, Tyr, Tyr-Gly-Gly and Tyr-Gly, in order of abundance. Maximum inhibition of [3H]Leu-Enk degradation is obtained at a concentration of 10(-3) M Phe-Ala.
Animals↗
Prostaglandins E and F, and 19-hydroxylated E and F (series I and II) in semen of fertile men. Gas and liquid chromatographic separation with selected ion detection.
Low concentrations of prostaglandins (PG) could be related to male clinical infertility althought relevant experimental data are scarce. The aim of this work is to establish reliable seminal PG levels in fertile men by rigorous sample control, to prevent degradation, and by rapid and simple extraction and assay procedures. Single semen samples from healthy fertile men were immediately centrifuged (within 30 min of ejaculation) adding PGF2 alpha D4 to the seminal plasma as internal standard. The samples were next ultrafiltered and the PGs in the ultrafiltrate were derivatized with a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and piperidine (1:1) at 60 degrees for 30 min. Optimum gas-liquid chromatographic separation of all of the peaks of interest was achieved on 4 m x 1/4 in. I.D. Dexsil 300 packed columns at 280 degrees. The detection and quantitation of all the peaks of interest depends on the selected ion monitoring of specific masses. The values obtained (in micrograms/ml, range in parentheses) were: PGEs, 63.5 +/- 49.3 (9--164); PGFs, 2.6 +/- 1.92 (0.95--6.63); 19-OH PGEs, 592.6 +/- 312.5 (142.1--1047); and 19-OH PGFs, 12.66 +/- 5.21 (4--19). Individual values for members of both series I and II are also presented. The sample collection and extraction procedures were further checked by high-performance liquid chromatography on a muPorasil column, with individual isolation and collection of all of the PGs, including the 19-OH PGs not previously separted by liquid chromatography.
Adult↗
A simple extraction procedure for prostaglandins in human seminal plasma with specific detection by selected ion monitoring.
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Chromatography, Gas↗
Mass spectrometric determination of new prostaglandin derivatives (series A and E).
The reaction of prostaglandins E1, E2, A1 and A2 with a 1:1 mixture of N,O-bis-trimethylsilyltrifluoroacetamide and piperidine favours the quantitative enolization of the 9-keto group of prostaglandins E, thus giving the corresponding tetrakis-TMS derivatives with molecular ions at m/e 642 and m/e 640 for prostaglandins E1 and E2, respectively, and also gives a new type of prostaglandin A-piperidyl-TMS derivative characterized by very strong base peaks at m/e 464 and 462, with molecular ions at m/e 637 and 635 for prostaglandins A1 and A2, respectively. The probable reaction mechanisms prevailing in both cases are discussed in accordance with the low and high resolution mass spectral data presented.
Chemical Phenomena↗
Effects of lindane on spontaneous behavior of rats analyzed by multivariate statistics.
The effects of subconvulsant doses (10, 15, 30 mg/kg) of the organochlorine insecticide gamma-hexachlorocyclohexane (lindane) on the spontaneous activity of male Wistar rats were assessed in an automated wheel-shaped activity monitor in 4 hr sessions. Most of the activity variables defined on the raw data showed statistically significant differences among treatment groups. These data can be interpreted as a lindane-induced change in the temporal distribution of the activity. The multivariate statistical technique principal component analysis was used to obtain a composite variable that measured this change. The variable obtained was found to be well correlated with dose levels and brain concentration of lindane and was used to estimate a minimal effective dose (MED) of 1.85 mg/kg and a upper limit for a minimal effective concentration (MEC) in brain of 0.84 micrograms/g by a log-linear regression approach. The results obtained with this approach further support the conclusion that the behavioral effects of lindane can be significant at exposure levels far below its LD50.
Analysis of Variance↗
Lindane inhibition of [35S]TBPS binding to the GABAA receptor in rat brain.
The inhibition of [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) binding to the GABAA receptor by the insecticide gamma-hexachlorocyclohexane, lindane, was studied in several brain regions and using different membrane preparation methods, both in vitro and after dosing the animals with the chemical. In the latter studies, the amount of lindane remaining in the membrane suspensions used for binding assays was determined. In vitro data showed values of IC50 from 150 to 1675 nM, varying in function of the membrane preparation method used. This may account for the discrepancies in IC50 values found in the literature. IC50 values within the range of 150-250 nM were determined using extensively washed membranes from several brain regions, so no evidence arose for brain regional differences in the affinity of lindane for the TBPS binding site. After different schedules of acute treatment with lindane, we found a manifest relationship between the extent of the observable inhibition of [35S]TBPS binding and the lindane amount remaining in the membrane suspensions used for binding assays. This relationship was in good agreement with the in vitro data, so no support for an in vivo acute regulation of the binding site was obtained.
Animals↗
On the effects of lindane on the plus-maze model of anxiety.
The behavioral effects of an acute subconvulsant dose of the insecticide and ectoparasiticide gamma-hexachlorocyclohexane, lindane, were compared in the plus-maze ((+)-maze) animal model of anxiety to those elicited by the anxiogenic GABAA-antagonist pentylenetetrazole (PTZ) and the anxiolytic benzodiazepine diazepam. The effects of the coadministration of diazepam and lindane, and of diazepam and PTZ were also studied. The results from the analysis of the data with the multivariate statistical technique principal component analysis indicated that the effects of subconvulsant doses of lindane and PTZ on the behavior of rats in the (+)-maze are similar, and opposite to those elicited by diazepam, and that the administration of the convulsants would antagonize the effects of the benzodiazepine. The putative anxiogenic properties of the insecticide and the utility of the (+)-maze model in neurotoxicological research are discussed.
Animals↗