Serum leptin levels are not influenced by arginine and insulin infusion and by acute changes of GH.

The aim of this study was to evaluate the relationship between GH and leptin in a group of short children and adolescents. Leptin and GH serum levels were measured before and during pharmacological stimulation tests (arginine and insulin) in a group of 45 children (30 male, 15 female), mean age 8.6+/-3.9 yr, affected by idiopathic isolated GH deficiency (GHD), and in a group of 27 children (15 male, 12 female), age 10.9+/-3.3 yr, with constitutional growth delay. Results showed that basal and peak leptin levels as well as the AUC were significantly higher in GHD patients compared to controls (p<0.05) and correlated with BMI SDS (p<0.0001) in GHD patients. No change in leptin serum levels was observed during either stimulation test. No correlation was found, however, between basal leptin serum levels and basal, peak and the AUC of GH during the tests. Moreover, no correlation was found between the acute changes of serum GH concentration during both stimulation tests and leptin serum levels. The results suggest that leptin and GH secretion is not correlated and that leptin serum levels mainly reflect the amount of fat tissue, which is higher in GHD patients.

High sensitivity simultaneous determination in hair of the major constituents of ecstasy (3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylene-dioxyethylamphetamine) by high-performance liquid chromatography with direct fluorescence detection.

A simple, but sensitive and specific high-performance liquid chromatographic assay for the simultaneous determination of the major constituents of "ecstasy" [i.e. 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDE)] with direct fluorimetric detection, particularly intended for the routine analysis of hair, is described. Hair samples (100 mg) were overnight incubated in 1 ml of 0.25 M HCl at 45 degrees C and extracted with a commercial liquid-liquid method. The dried residue reconstituted with 500 microl of 0.05 M NaH2PO4 pH 5.2 was injected. Isocratic reversed-phase liquid chromatography was carried out on a column (250x4.6 mm I.D.) packed with spherical 5-microm poly(styrene-divinylbenzene) particles; the mobile phase was composed of 0.1 M potassium phosphate (pH 3)-acetonitrile (82:18). The excitation and the emission wavelengths were set to 285 and 320 nm, respectively. Under the described conditions, MDA, MDMA and MDE eluted in symmetric peaks with an analysis time of 30 min. The limit of detection was lower than 1 ng/ml, with a signal-to-noise ratio of 5, for each compound in solution, allowing a cut-off of 0.1 ng/mg in the hair matrix to be established. The intra-day precision (n = 6) of the assay was characterised by RSDs between 1.0 and 3.0% and between 0.52 and 0.88% for concentrations of 10 and 100 ng/ml, respectively; in day-to-day precision tests (n = 6), RSDs ranged between 5.12 and 11.12%, respectively, for the same concentrations. Interferences from as many as 92 therapeutic and/or abused drugs currently in use in the population were excluded, including N-methyl-1-(3,4-methylenedioxyphenyl)-2 butanamine (MBDB).

Expression of MAGE and GAGE in high-grade brain tumors: a potential target for specific immunotherapy and diagnostic markers.

The mRNA expression of the tumor-associated antigens MAGE and GAGE was examined in 60 high-grade brain tumors. This analysis was performed by using reverse transcription-PCR, Southern blotting, and sequencing. It was demonstrated that, of the eight GAGE genes, GAGE-2 and -7 were expressed in five of seven normal brains. Four groups of tumors--adult glioblastoma multiforme (n = 20), pediatric glioblastoma multiforme (n = 9), medulloblastomas (n = 15), and ependymomas (n = 14)--were analyzed for mRNA expression. The following frequencies were observed: MAGE-1, 0, 0, 13, and 0%, respectively; MAGE-2, 5, 11, 60, and 57%; MAGE-3 & -6, 0, 0, 13, and 0%; GAGE-1, 65, 11, 13, and 43%; and GAGE-3-6 and -8: 75, 78, 47, and 93%, respectively. Two unclassified tumors expressed GAGE-3-6 and -8 only. The absence of GAGE-1 expression in normal brain, its relatively high frequency of expression in high-grade brain tumors, and its unique 3' sequence, suggest it may represent a useful target for specific immunotherapy. The detection method of reverse transcription-PCR and Southern blotting may also be useful for rapid screening of biopsy specimens both for diagnostic purposes and to determine a patient's eligibility for specific immunotherapy.

Murine Stim1 maps to distal chromosome 7 and is not imprinted.

STIM1 (GOK) maps to a region of human Chromosome (Chr) 11p15.5 that is implicated in several embryonal tumors, and some evidence indicates that STIM1 may have a growth suppressor role in rhabdomyosarcoma. In this study we have mapped the murine homolog, Stim1, to the same position as Hbb on distal mouse Chr 7. This region is separated by 20 cM from the region of distal Chr 7 that contains Igf2, H19, and other imprinted genes. Using strain-specific polymorphisms, we have shown that Stim1 is expressed from both parental alleles in fetal and neonatal mouse tissues. Similar analyses of human Wilms' tumor and normal kidney tissues demonstrated biallelic expression of STIM1 in the majority of samples. These data demonstrate that Stim1 expression is not regulated by genomic imprinting in either mouse or human tissues. Thus, if STIM1 is a tumor suppressor at 11p15.5, loss of expression is not due to imprinting effects.

Hair analysis for abused drugs by capillary zone electrophoresis with field-amplified sample stacking.

The present paper describes the methodological optimisation and validation of a capillary zone electrophoresis method for the determination of morphine, cocaine and 3,4-methylenedioxymethamphetamine (MDMA) in hair, with injection based on field-amplified sample stacking. Diode array UV absorption detection was used to improve analytical selectivity and identification power. Analytical conditions: running buffer 100 mM potassium phosphate adjusted to pH 2.5 with phosphoric acid, applied potential 10 kV, temperature 20 degrees C, injection by electromigration at 10 kV for 10 s, detection by UV absorption at the fixed wavelength of 200 nm or by recording the full spectrum between 190 and 400 nm. Injection conditions: the dried hair extracts were reconstituted with a low-conductivity solvent (0.1 mM formic acid), the injection end of the capillary was dipped in water for 5 s without applying pressure (external rinse step), then a plug of 0.1 mM phosphoric acid was loaded by applying 0.5 psi for 10 s and, finally, the sample was injected electrokinetically at 10 kV for 10 s. Under the described conditions, the limit of detection was 2 ng/ml for MDMA, 8 ng/ml for cocaine and 6 ng/ml for morphine (with a signal-to-noise ratio of 5). The lowest concentration suitable for recording interpretable spectra was about 10-20-times the limit of detection of each analyte. The intraday and day-to-day reproducibility of migration times (n = 6), with internal standardisation, was characterised by R.S.D. values < or = 0.6%; peak area R.S.D.s were better than 10% in intraday and than 15% in day-to-day experiments. Analytical linearity was good with R2 better than 0.9990 for all the analytes.

Simultaneous chiral separation of 3,4-methylenedioxymethamphet- amine, 3-4-methylenedioxyamphetamine, 3,4-methylenedioxyethylam- phetamine, ephedrine, amphetamine and methamphetamine by capillary electrophoresis in uncoated and coated capillaries with native beta-cyclodextrin as the chiral selector: preliminary application to the analysis of urine and hair.

The importance of the chiral analysis of amphetamine-related substances in both clandestine preparations and biological samples is widely recognized. For this purpose, capillary electrophoresis was successfully applied by several authors, but only few reports concerned ring-substituted amphetamines, which represent the main components of "ecstasy", a widely abused "recreational" substance. In the present work, the simultaneous chiral analysis of ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3-4-methylenedioxyamphetamine (MDA) and 3,4-methalenedioxyethylamphetamine (MDE) is reported, by using capillary electrophoresis with native beta-cyclodextrin (15 mM) as the chiral selector. After preliminary tests at different pH values (phosphate buffer 100 mM, pH 2.5-9.0) and with bare or coated fused-silica capillaries, the optimized conditions were: pH 2.5 phosphate, uncoated capillary (45 cm x 50 microm inner diameter), potential 10 kV. Detection was either by fixed wavelength (200 nm) or multiwavelength (190-400 nm) UV absorbance. Under these conditions, good resolution was obtained for all the analytes, with excellent chiral selectivity and efficiency. The sensitivity for the individual enantiomers was better than 0.2 microg/mL, analytical precision was characterized by relative standard deviation values < 0.8% (< or = 0.15% with internal standardization) for migration times intra-day and < 2.0% (< or = 0.54% with internal standardization) day-to-day; linearity, in the range 0.156-40 microg/mL, and accuracy were also satisfactory. After a simple liquid-liquid extraction, urine samples could be analyzed with a sensitivity well below the recommended NIDA cut-off of 500 ng/mL. For hair samples, it was necessary to increase the sensitivity by applying a field-amplified sample stacking procedure, which allowed the chiral determination of MDA, MDMA and MDE at concentrations occurring in real samples from ecstasy users, with the possibility of recording UV spectra of the peaks.

Optimization of a simple method for the chiral separation of phenethylamines of forensic interest based on cyclodextrin complexation capillary electrophoresis and its preliminary application to the analysis of human urine and hair.

Because of the forensic importance of the chiral analysis of amphetamine and other phenethylamines for investigating their synthetic pathways and the metabolic patterns of these compounds, a capillary electrophoresis method has been developed based on the chiral selectivity of beta-cyclodextrin. The influence of different experimental conditions, such as cyclodextrin nature and concentration, voltage, temperature and buffer concentration and pH, on analytical performance has been studied. The optimized analytical conditions are: capillary: bare fused silica, 50 microns I.D., 40 cm effective length; buffer: 150 mM phosphate pH = 2.5, 15 mM beta-cyclodextrin; voltage: 10 kV; temperature: 17.5 degrees C; detection: UV absorption at 200 nm wavelength. Under these conditions, amphetamine, methamphetamine and ephedrine have been easily separated, with baseline resolution of the respective enantiomers. Sensitivity was better than 300 ng per ml. The average precision of migration times of the three analytes was good with RSD = 0.45% and 0.58% in intra-day and day-to-day tests, respectively. Reproducibility of peak heights was also good, with RSD = 2.51% and 3.14% in intra-day and day-to-day tests, respectively. The preliminary analysis of amphetamine in human urine and hair samples, subjected to a simple work-up procedure based on liquid-liquid extraction, showed clean blank electropherograms, excellent chiral resolution and sensitivity, suitable for the analysis of real samples from amphetamine users.

Pharmacokinetics of a new nitroderivative of acetylsalicylic acid after a single dose in rats.

The pharmacokinetics of a new ASA-nitroderivative compound, NCX 4016 (ASA-NO2), was evaluated using an HPLC method. After single equimolar doses of ASA (35 mg kg(-1)) or ASA-NO2 (65 mg kg(-1)), no detectable levels of these compounds have been observed in rat plasma samples. SA peak levels were obtained at 3 h and 6 h after ASA and ASA-NO2 administration respectively. The elimination rate constants of SA were similar after ASA and ASA-NO2, suggesting a similar elimination phase of this metabolite in rats. From these data it is evident that ASA-NO2 is slowly metabolized in ASA, which is rapidly converted to SA.

Evidence for uptake1-mediated efflux of catecholamines from pulmonary endothelial cells of perfused lungs of rats.

Previous pharmacological studies have demonstrated that pulmonary endothelial cells and noradrenergic neurones possess the same transporter for inward transport of catecholamines, uptake1. In noradrenergic neurones, it has been shown that uptake1 is also involved in the carrier-mediated outward transport, or efflux, of noradrenaline and dopamine. The aim of the present study was to examine the efflux of noradrenaline and dopamine from perfused lungs of rats to determine whether uptake1, in addition to diffusion, mediates efflux of catecholamines from pulmonary vascular endothelial cells. The effects of reducing the cellular sodium gradient and of substrates and inhibitors of uptake1 on the efflux of 3H-noradrenaline and 3H-dopamine from rat lungs were measured. Isolated perfused lungs of rats (monoamine oxidase and catechol-O-methyltransferase inhibited) were loaded with 3H-(-)-noradrenaline or 3H-dopamine for 10 min followed by perfusion with either (1) a low sodium, amine-free Krebs solution, in which NaCl was replaced by either Tris.HCl or LiCl, for 15 or 10 min, respectively or (2) amine-free Krebs solution for 30 min in the absence or presence of a substrate or inhibitor of uptake1 for the last 15 min. The rate constants for spontaneous efflux of noradrenaline and dopamine from the lungs were 0.0163 min-1 and 0.0466 min-1, respectively. When NaCl was replaced by Tris.HCl during efflux, the rate constants for efflux of noradrenaline and dopamine were increased 2.5-fold and 3-fold, respectively, whereas, when NaCl was replaced by LiCl, the rate constants were increased 8-fold and 4-fold, respectively. The uptake1 substrates, dopamine (1 and 3 mumol/l) and adrenaline (40 mumol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mumol/l) had a similar effect on the efflux of dopamine. The uptake1 inhibitors, imipramine (3 and 10 mumol/l) and nisoxetine (50 nmol/l), caused small and gradual increases in the efflux of noradrenaline and dopamine from rat lungs. These results demonstrate that efflux of noradrenaline and dopamine from rat lungs is affected by alterations in the normal sodium gradient across the cell and by drugs that interact with the uptake1 transporter. Thus, it can be concluded that the spontaneous efflux of catecholamines from pulmonary vascular endothelial cells is mediated predominantly by uptake1. In addition, efflux of catecholamines from the lungs has a diffusional component, which, combined with inhibition of reuptake, accounts for the small increase in amine efflux by inhibitors of uptake1.

A kinetic investigation of the pulmonary metabolism of dopamine in rats shows marked differences compared with noradrenaline.

The aim of this study was to investigate the deamination of dopamine in the intact pulmonary circulation of isolated lungs of the rat. The first part of the study showed that dopamine is not converted to noradrenaline by dopamine-beta-hydroxylase (DBH) when dopamine is perfused through isolated lung preparations with monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited. Hence, it was not necessary to inhibit DBH in subsequent experiments. The metabolite profile for deamination of dopamine in the lungs was examined by determining whether MAO and semicarbazide-sensitive amine oxidases (SSAO) contribute to the deamination of dopamine (and noradrenaline), and by determining the activity of MAO (kMAO) for the metabolism of dopamine. Lungs were perfused with 1 nmol/l 3H-dopamine or 3H-noradrenaline with COMT inhibited and, in experiments to determine the contribution of SSAO to deamination, with MAO inhibited. Inhibition of MAO reduced the deamination of dopamine and noradrenaline by 99.8% and 98.6%, respectively, indicating that MAO, and not SSAO, was responsible for deamination of the catecholamines in the lungs. The kMAO value for deamination of dopamine was 3.89 min-1. Further experiments were carried out to determine the contributions of MAO-A and MAO-B to the deamination of dopamine in lungs perfused with 1 nmol/l 3H-dopamine and 100 nmol/l lazabemide or 300 nmol/l Ro41-1049, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)