Determination of artemether and its metabolite, dihydroartemisinin, in plasma by high-performance liquid chromatography and electrochemical detection in the reductive mode.

An analytical method for the determination of artemether (A) and its metabolite dihydroartemisinin (DHA) in human plasma has been developed and validated. The method is based on high-performance liquid chromatography (HPLC) and electrochemical detection in the reductive mode. A, DHA and artemisinin, the internal standard (I.S.), were extracted from plasma (1 ml) with 1-chlorobutane-isooctane (55:45, v/v). The solvent was transferred, evaporated to dryness under nitrogen and the residue dissolved in 600 microliters of water-ethyl alcohol (50:50, v/v). Chromatography was performed on a Nova-Pak CN, 4 microns analytical column (150 mm x 3.9 mm I.D.) at 35 degrees C. The mobile phase consisted of pH 5 acetate-acetonitrile (85:15, v/v) at a flow-rate of 1 ml/min. The analytes were detected by electrochemical detection in the reductive mode at a potential of -1.0 V. Intra-day accuracy and precision were assessed from the relative recoveries (found concentration in % of the nominal value) of spiked samples analysed on the same day (concentration range 10.9 to 202 ng/ml of A and 11.2 to 206 ng/ml of DHA in plasma). The mean recoveries over the entire concentration range were from 96 to 100% for A with C.V. from 6 to 13%, from 92% to 100% for DHA (alpha-tautomer) with C.V. from 4 to 16%. For A, the mean recovery was 96% at the limit of quantitation (LOQ) of 10.9 ng/ml with a C.V. of 13%. For DHA, the mean recovery was 100% at the LOQ of 11.2 ng/ml with a C.V. of 16%.

Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection.

A methodology is described for the quantitation of 7-alkyl- and O6-alkylguanine in DNA isolated from experimental animals exposed to alkylating agents. Following purification, the DNA is hydrolysed under acid conditions after which 7-alkyl- and O6-alkylguanine are separated from unmodified bases by HPLC using a strong cation exchange column. The fractions containing the methylated purines are subsequently analyzed by HPLC using a reverse phase column coupled to an electrochemical detector (amperometric). This method allows the detection of 10-20 fmoles 7-alkyl- and O6-alkylguanine, when pure markers are analyzed. In practice, the detection limit is 0.5 adducts per 10(6) nucleotides for the methylated and 1 adduct per 10(6) nucleotides for the ethylated form of 7-alkyl- and O6-alkylguanine using 25 micrograms DNA.

4-[123I]iodospiperone as a ligand for dopamine DA receptors: in vitro and in vivo experiments in a rat model.

Radioiodinated spiperone is of interest for dopamine (DA) receptor studies in the living human brain by single photon emission computed tomography (SPECT). Stimulated by data obtained with [11C]-N-methyl-spiperone we synthesized 4-[123I]iodospiperone and investigated the in vitro binding characteristics of this ligand to the striatal membrane of the rat and the in vivo distribution over various rat brain regions. The in vitro binding experiments showed that this radioligand displays about 10 times less affinity for the DA receptor than spiperone and specific binding, as shown with [3H]spiperone, was not observed. Displacement by butaclamol was not observed. The in vivo studies demonstrated that both 4-[123I]iodospiperone and [3H]spiperone concentrate in striatal tissue, respectively, 1.9 and 3.5 times as high as in cerebellar tissue. Haloperidol pretreatment largely prevented this accumulation. In view of the obtained target-to-non-target ratios we believe, however, that this accumulation in brain areas rich in DA-receptors does not offer prospects for clinical receptor imaging with SPECT.

The use of microwave irradiation with low formalin concentrations to enhance the conversion of dopamine into norsalsolinol in rat brain: a pilot study.

The fixation of the neurotransmitter dopamine in the central nervous system by perfusion with formalin solutions seems to take place mainly via the formalin-induced condensation product norsalsolinol. In the present investigation the influence of microwave irradiation of the formalin-induced condensation of dopamine was studied in vitro and in vivo by making use of different, relatively low, formalin concentrations. It appeared that in vitro and in vivo the dopamine conversion was complete with 4% formalin and no influence of microwaves was noted. However, by making use of much lower formalin concentrations (0.2% and 0.4%) the condensation of dopamine was strongly augmented, in vitro (200%) and in vivo (at least 500%) using microwave techniques. There was a considerable loss in non-microwaved tissue (30%) after perfusion in vivo. This was lower (10%) in microwaved tissue. In experiments with perfused brain tissue which allowed a more complete calculation, a loss was found. This might be caused by a strong binding of dopamine and/or norsalsolinol to tissue components or to side reactions that could not be traced by the present experimental techniques.

Repeated administration of HA-966 and haloperidol to rats: similar tolerance to striatal dopamine accumulation after HA-966 challenge, but dissimilar effects on striatal [3H]spiperone binding.

Repeated administration of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) to rats induces tolerance, as shown by a decreased, drug-stimulated accumulation of dopamine (DA) in the striatum. In the present study we compared the adaptive response of the striatal dopaminergic system to repeated administration of HA-966 with the adaptive response observed after repeated haloperidol. These treatments deprive dopamine (DA) receptors from their agonist and cause a blockade of DA receptors, respectively. Tolerance to HA-966 was not accompanied by a change in the specific binding of [3H]spiperone to striatal membranes. This is in contrast to the well-documented up-regulation of DA receptors that occurs with tolerance to haloperidol. Repeated haloperidol pretreatment also diminished DA accumulation following a challenge dose of HA-966, to a similar extent as that caused by repeated pretreatment with HA-966. These similar effects of pretreatment with HA-966 or haloperidol on the response to the HA-966 challenge are in line with, and strengthen, the idea that an increased sensitivity of presynaptic DA receptors is responsible for the decreasing effect of HA-966 after its repeated administration. Haloperidol and HA-966 clearly have different effects on postsynaptic DA receptors, as is shown by their differential effects on striatal [3H]spiperone binding.

Tritium isotope effect in reversed-phase high-performance liquid chromatographic analysis of dopamine and dihydroxyphenylacetic acid.

A tritium isotope effect has been demonstrated in the high-performance liquid chromatographic analysis of dopamine and its acidic metabolite dihydroxyphenylacetic acid. The chromatographic system consisted of tributyl-n-phosphate, bound to a ChromSpher C8 column, as stationary phase, and a citrate buffer, containing the ion-pairing agent perchlorate, as the mobile phase. For detection we used continuous electrochemical monitoring (for the total amount of solutes) and discontinuous liquid scintillation counting (for radiolabelled molecules) of the column effluent. [3H]Dopamine and [3H]dihydroxyphenylacetic acid were biosynthesized by incubation of homogenates of striatal tissue from rat brains with 3H-labelled L-tyrosine. The tritium-labelled compounds were eluted before the corresponding unlabelled analogues. The capacity factor reduction increased with the number of tritium atoms incorporated in the molecules: for single, double and triple tritium-labelled dopamine the separation factors amounted to 1.015, 1.028 and 1.033, respectively. No isotope separation was observed for 7-14C-labelled dopamine and dihydroxyphenylacetic acid. The isotope effect observed is ascribed to a decrease in lipophilicity following tritium substitution for hydrogen.

Monitoring the specific activities of dopamine and its metabolites in striatum and olfactory tubercle after intravenous administration of l-[(3)H]tyrosine: Complex relations, indicating more than two compartments.

It is still a matter of debate whether in dopaminergic nerve endings dopamine (DA) is present in different functional and/or metabolic compartments. To investigate this, DA metabolism was studied in vivo by measuring the specific activity of DA and its metabolites after intravenous administration of l-[3,5-(3)H]tyrosine (200 ?Ci/rat) to freely moving animals. The incorporation of (3)H into DA and metabolites was determined in striatum and olfactory tubercle at 5, 10, 20, 40, 60 and 80 min after [(3)H]tyrosine administration. In both structures the level of [(3)H]tyrosine initially declined monoexponentially, but deviated from that pattern later on. The curves representing the formation in time of [(3)H]DA and [(3)H]metabolites were very similar in both structures, although as a whole, the levels in the olfactory tubercle were higher. The relative patterns of the specific activities of DA and those of its metabolites, a possible clue to DA compartmentation, neither indicated a clearcut metabolic one-compartment, nor a two-compartment system. The flow of radioactivity through DA metabolism could in fact only be explained by assuming more complex metabolic relations.

Forty-year follow-up of United States prisoners of war.

The authors performed structured psychiatric examinations of 188 former prisoners of war (POWs). Sixty-seven percent had had posttraumatic stress disorder. Of those affected, 29% had fully recovered, 39% still reported mild symptoms, 24% had improved but had moderate residual symptoms, and 8% had had no recovery or had deteriorated. Presence of posttraumatic stress disorder was not significantly correlated with other mental disorders. Delayed onset was not seen. The findings confirm the DSM-III concept of and criteria for posttraumatic stress disorder.

The relationship of histrionic personality disorder to antisocial personality and somatization disorders.

The authors examined the association of antisocial personality disorder, somatization disorder, and histrionic personality disorder, both within individuals and within families, in 250 patients. All three disorders overlapped considerably within individuals; the strongest relationship was between antisocial personality and histrionic personality. A high prevalence of antisocial personality was reported in the families of patients with somatization disorder but not in the families of patients with histrionic personality. The authors suggest that histrionic individuals develop antisocial personality if they are male and somatization disorder if female; moreover, all three conditions may represent alternative manifestations or different stages of the same underlying diathesis.

Effects of morphine on dopamine metabolism in rat striatum and limbic structures in relation to the activity of dopaminergic neurones.

Administration of morphine results in efflux of dopamine provided that the nerve impulse flow of the dopaminergic neurones is impaired. In the present study we investigated whether the morphine-induced increase in dopamine metabolite levels is related to impulse flow in a similar way. Pretreatment with gamma-butyrolactone to impair nerve impulse flow, abolished the effect of morphine on the concentrations of dihydroxyphenylacetic acid and homovanillic acid. Pretreatment with apomorphine had a similar effect, as well as combined pretreatment with gamma-butyrolactone and apomorphine. Since gamma-butyrolactone and apomorphine both reduce nerve impulse flow, but gamma-butyrolactone increases while apomorphine decreases dopamine biosynthesis, it would appear that the antagonism of morphine-induced increases in dopamine metabolites is due to the common property of impulse flow reduction. It was also shown, however, that pretreatment with alpha-methyl-paratyrosine, which inhibits dopamine biosynthesis, resulted in antagonism of morphine's effect on dopamine metabolite levels. It is concluded, therefore, that morphine-induced dopamine efflux is observed under conditions when no effect on dopamine metabolism is observed, and vice versa. Three effects of morphine on dopaminergic neurones can be distinguished: an increase in impulse flow in nigrostriatal dopaminergic neurones, increased dopamine biosynthesis and catabolism, and efflux of dopamine. The first effect probably is effected in the cell body areas, while the latter two effects may be produced at the level of the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Depressed women with panic attacks.

Of a group of 288 depressed female inpatients, 43 (15%) had secondary panic attacks. Compared to other depressives, the subgroup with panic attacks had significantly higher frequencies of anorexia, weight loss, gastrointestinal disturbances, hypochondriasis, and psychomotor agitation, and significantly lower frequencies of melancholic symptoms, including loss of interest in usual activities, guilt feelings, delusional thinking, psychomotor retardation, and orientation or memory impairment. Patients with panic attacks were less likely to have a depressed parent and were more likely to be described as having been nervous, worrisome, sensitive, and sexually dysfunctional before the onset of depression. Phenomenologically, they resembled "anxious depressives" as described by other authors.

Chronic depressions. Part 2. Sleep EEG differentiation of primary dysthymic disorders from anxious depressions.

In this clinical, psychometric and polysomnographic study, primary dysthymics (N = 20) were compared with anxious depressives (N = 22), and non-psychiatric controls (N = 11). Beck and MMPI depression scores were similar in the two affective groups. Prominent insomnia occurred in 82% of the anxious group; hypersomnia was more characteristic of the dysthymic group. On night 1, the anxious group had the poorest sleep efficiency (P less than 0.001), while dysthymics had the highest REM% (P less than 0.05) and shortest REM latency (P less than 0.01). On night 2, differences tended to be minimized, although the number of awakenings was still high (P less than 0.05) in the anxious group, and REM% was highest (P less than 0.01) and REM latency shortest (P less than 0.01) in the dysthymics. These findings suggest that patients with primary anxiety disorders experience greater sleep continuity difficulties on the adaptation night. Despite significant clinical overlap in depressive symptomatology between the two groups, REM% and REM latency appear as sturdy psychophysiological markers in differentiating primary dysthymics and anxious depressives on both nights. These data suggest that distinct anxious depressive and subaffective dysthymic subtypes can be distinguished within the universe of the atypical depressions.

A procedure to measure the specific activities of dopamine and its metabolites in rat striatum, based on HPLC, electrochemical detection and liquid scintillation counting.

A procedure to determine the specific activities (s.a.) of the putative neurotransmitter dopamine (DA) and its metabolites in rat striatum is described. For this purpose 200 mu Ci L-[3,5-3H]tyrosine was injected via a jugular vein cannula into freely moving rats. Contents and radioactivity of striatal tyrosine, DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) were determined by means of HPLC, electrochemical detection (ECD) and liquid scintillation counting. In the phase system applied DA, DOPAC, 3-MT and HVA can be separated in a single run after direct injection of striatal supernatants. The selectivity of the phase system was sufficient to analyse the supernatant of two rat striata over a 150 X 4.6 mm column, even when DA turnover was strongly stimulated. Tyrosine levels and radioactivity were measured by re-analysis of the front peak with divergent mobile phase parameters. In order to demonstrate the applicability of the present procedure, the s.a. of DA and its metabolites, as found 20 min after [3H]tyrosine administration, and the effect of haloperidol thereupon, are presented.

Analysis of serotonin and derivatives by reversed-phase ion-pair partition chromatography with fluorometric and electrochemical detection.

A flexible and efficient system is described for reversed-phase ion-pair partition-chromatographic analysis of serotonin, its precursors tryptophan and 5-hydroxytryptophan, its main metabolite 5-hydroxyindoleacetic acid, and tryptamine. The chromatographic system consists of tri-n-butylphosphate as stationary phase and buffered water-methanol mixtures, containing perchlorate, as mobile phases. Retention can be selectively influenced by means of the pH, the perchlorate concentration, and the methanol content of the mobile phase, as well as the temperature of the phase system. The compounds of interest can be separated within 10 min and no interference from catecholamines and derivatives was observed. Compared with electrochemical detection, fluorometric detection yielded more favourable detection limits and was more selective when supernatants of brain tissue homogenates were directly injected. Both detection systems showed inadequate selectivity if urine samples were directly injected, but 5-hydroxyindoleacetic acid could readily be assayed.

Determination of dopamine and its acidic metabolites in brain tissue by HPLC with electrochemical detection in a single run after minimal sample pretreatment.

A method, based on reverse-phase liquid-liquid chromatography, has been developed for the determination, in a single run, of dopamine (DA) and its acidic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), combined with electrochemical detection (ECD). If applied to brain tissue, sample pretreatment can be reduced to centrifugation, filtration and adjustment of pH and perchlorate concentration prior to introduction into the liquid chromatograph. The relation between the perchlorate (counterion) concentration of the mobile phase and the retention (k') of the amines is linear, as is the relation between the H+ concentration of the mobile phase and the retention of the acidic metabolites. This flexible phase system, combined with a simple and therefore reproducible sample pretreatment, warrants a high throughput of samples. The procedure offers good possibilities for routine analysis of catecholamines and their acidic metabolites in the picogram range. Some typical examples of the behaviour of this phase system and the electrochemical detector are presented and discussed.

Dopaminergic and adrenergic effects on plasma LH levels in rats with hypothalamic islands.

In female rats hypothalamic islands were prepared by making a cut around the mediobasal hypothalamus. Three weeks later the rats were ovariectomized. Two weeks after ovariectomy, part of the animals showed elevated blood LH levels with oscillations, although the mean LH levels were significantly lower than in sham-operated ovariectomized animals. There was a significant correlation between the occurrence of elevated blood LH levels after ovariectomy and the inclusion of at least part of the suprachiasmatic nucleus within the islands. Nor-adrenaline content of the hypothalamus was decreased significantly, but there was no significant change in hypothalamic dopamine content. Injection of doses of apomorphine, ranging form 2.5 microgram/kg to 25 mg/kg sc into island-bearing and sham-operated animals caused a dose-dependent decrease of plasma LH. However, the magnitude of the decrease was smaller in rats with hypothalamic islands than in controls. Even the highest doses of apomorphine did not decrease plasma LH further than to about 100 ng of LH-RP-1/ml. Phenoxybenzamine (10 and 40 mg/kg ip) and phentolamine (20 mg/kg ip) caused a decrease of plasma LH in ovariectomized control rats, but in ovariectomized rats with hypothalamic islands these drugs caused an increase. Clonidine (100 microgram/kg sc followed by 100 microgram/kg ip) had no effect in ovariectomized control rats, but in ovariectomized rats with a hypothalamic island an increase was induced. These results may have been caused by an increased sensitivity of hypothalamic alpha-receptors resulting from severing afferent noradrenergic fibres.