Liquid chromatography-tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone.

A liquid chromatography tandem mass spectrometry assay for serum testosterone (T) and trideuterated testosterone (d(3)T) was developed in order to support clinical research studies that determine the pharmacokinetics, production rate, and clearance of testosterone by administration of trideuterated testosterone. After adding 19-nortestosterone as the internal standard (I.S.), sodium acetate buffer, and ether, to a serum aliquot, the mixture was shaken and centrifuged, and the ether was dried. The extract was reconstituted in methanol and 15 microl was injected into a liquid chromatograph equipped with an autosampler and Applied Biosystems-Sciex API 300 triple quadrupole mass spectrometer operated in the positive ion mode. T, d(3)T, and I.S. were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0-20 ng/ml for T and 0-2.0 ng/ml for d(3)T. The LOQs for T and d(3)T were 0.5 ng/ml and 0.05 ng/ml. The recoveries for T and d(3)T were 91.5 and 96.4%. For T at 1.25 ng/ml and 4.0 ng/ml, the intra-day precision (RSD, %) was 3.9 and 4.3% and intra-day accuracy 0.01 and 4.5%, respectively. The inter-day precision at these levels was 5.3 and 5.4% and inter-day accuracy was 1.9 and 0.3%. For d(3)T at 0.125 ng/ml and 0.4 ng/ml, the intra-day precision (RSD, %) was 2.8 and 8.3% and intra-day accuracy was 1.8 and 5.6%. The inter-day precision at these levels was 10.0 and 7.6% and inter-day accuracy was 5.7 and 3.4%. The concentrations of T in the 38 healthy subjects ranged from 2.5 to 14.0 ng/ml (mean 6.2 ng/ml).

Estradiol-induced IP(3) mediates the estrogen receptor activity expressed in human cells.

The recent findings that estradiol-induced IP(3)/PKC-alpha signalling pathway triggers DNA synthesis in HepG2 cells, containing estrogen receptor unable to stimulate gene transactivation, raises the hypothesis that this pathway represents an alternative signalling present when the amount of estrogen receptor (ER) is insufficient to mediate genomic effects. beta-estradiol-stimulated DNA synthesis and target gene expression have been studied in HepG2 and, ER-alpha or ER-beta negative, HeLa cells. We also examined whether either receptor is required for rapid effects of estrogen on DNA synthesis. Finally, the consequences of increased ER expression on estrogen-induced DNA synthesis and synthetic target gene expression have been evaluated. Our data indicate that the E2-induced IP(3) production is dependent on expression of either ER-alpha or ER-beta in both HepG2 and HeLa cells. Moreover, inhibition of the IP(3) second messenger pathway blocks E2-induced cellular actions suggesting that this second messenger is responsible for estrogen's rapid, non-genomic effects on both DNA synthesis and gene expression.

Activation of IP(3)-protein kinase C-alpha signal transduction pathway precedes the changes of plasma cholesterol, hepatic lipid metabolism and induction of low-density lipoprotein receptor expression in 17-beta-oestradiol-treated rats.

The intracellular concentration of cholesterol is regulated by the balance between endogenous synthesis and exogenous uptake. Oestrogens have been reported to be involved in the physiological regulation of cellular cholesterol content. Relevant reports have focused on long-term responses and there is a lack of information about the relationship between the timing of the oestrogen effect and the regulation of cholesterol homeostasis. The aim of this work has been to set up a systematic picture of the short-term effects induced by oestrogen on hepatic lipid metabolism in vivo and the involvement of some relevant signal transduction pathways. At intervals after oestrogen administration (30 min to 6 h), oestrogen receptor expression and changes in liver cAMP, IP(3) and protein kinase C-alpha (PKC-alpha) were followed. Changes in the expression of the low density lipoprotein receptor at mRNA and protein levels, and of hydroxy-methyl-glutaryl-CoA reductase activity have been verified. At the same time, the content of hepatic cholesterol, ubiquinone and dolichol and of plasma cholesterol have been determined. Changes of rab 5 and rab 8, small GTP-binding prenylated proteins involved in the transfer of neosynthesised proteins through the cell, have been also checked. In vivo treatment with oestradiol produced no change in cyclic AMP but a rapid increase in IP(3), increased PKC-alpha localisation on the membranes and enhanced expression of the low density lipoprotein receptor in the liver occurred. PKC inhibition completely prevented any increase in low density lipoprotein receptor mRNA in isolated and perfused rat liver. Early changes of ubiquinone and dolichol content and a later reduction in hepatic hydroxy-methyl-glutaryl-CoA reductase activity and plasma cholesterol content were also detectable. A functional role of the IP(3) -protein kinase C-alpha pathway in the induction of the low density lipoprotein receptor is suggested. Experimental Physiology (2001) 86.1, 39-45.

beta-estradiol stimulation of DNA synthesis requires different PKC isoforms in HepG2 and MCF7 cells.

The role exerted by protein kinase C (PKC) on estrogen-induced DNA synthesis has been investigated in hepatic and mammary gland cells, HepG2 and MCF7. 17-beta-estradiol stimulated DNA synthesis in HepG2 and MCF7 cells, maximal effect occurring at 10 nM. DNA synthesis stimulation was prevented by anti-estrogen ICI 182,780 and by inhibitor of PKC, Ro 31-8220. The rapid estradiol effects in MCF7 cells were determined by following the inositol trisphosphate (IP(3)) production and PKC-alpha membrane translocation. After estradiol treatment the increase of IP(3) production, prevented by anti-estrogen or by phospholipase C (PLC) inhibitor (neomycin), was present in MCF7 cells. In MDA cells, devoid of estrogen receptor, no effect was observed. The PKC-alpha presence on the membranes appeared unchanged in MCF7 cells. The PLC inhibitors, neomycin and U73,122, and PKC-alpha down regulator, phorbol 12-myristate 13-acetate (PMA), were able to prevent estradiol-induced DNA synthesis in hepatoma cells, but ineffective in mammary cells; wortmannin, an inhibitor of phosphoinositide 3-kinases (PI3-K), blocked DNA synthesis in both cell lines. These data show that beta-estradiol, via an estrogen receptor-mediated mechanism, activates more signal transduction pathways, and consequently different PKC isoforms in two responsive cell lines. In both cell lines PI3-K/PKC pathway is functional to the estrogen regulation of DNA synthesis, whereas in HepG2 cells the parallel involvement of the PLC/PKC-alpha pathway is present. The reported results indicate that the DNA synthesis stimulation by beta-estradiol requires the estrogen receptor and utilises one or more activated pathways in dependence on the cell equipment.

Selective mechanism-based inactivation of rat CYP2D by 4-allyloxymethamphetamine.

The high selectivity of amphetamine and its derivatives for CYP2D-mediated oxidations suggested the use of the phenylisopropylamine skeleton as a template for a selective inhibitor of this important enzyme. Accordingly, 4-allyloxymethamphetamine-amine (ALLMA) was synthesized and its ability to selectively inactivate CYP2D was investigated both in in vitro and in vivo experiments. Incubation studies with rat liver microsomes demonstrated that this compound suppressed the CYP2D-mediated methylenedioxymethamphetamine (MDMA) demethylation in time- and dose-dependent manner and that the inhibition required the presence of NADPH. The development of irreversible inhibition was associated with oxidation at position 4 of the aromatic ring, the common site of CYP2D-mediated oxidation of this group of compounds. In in vivo studies doses of ALLMA (1-10 mg/kg) were administered to adult male Sprague-Dawley rats and liver microsomes were obtained 3 hr later. Methamphetamine p-hydroxylation and low Km MDMA demethylation activities, both mediated by CYP2D, were reduced by more than 80% after a dose of 10 mg/kg. Cytochrome P-450 reactions attributed to P-450s other than CYP2D, such as aniline p-hydroxylation, the high Km system of MDMA demethylation and the N-demethylation of methamphetamine, benzphetamine, aminopyrine and erythromycin, all appeared to be minimally affected. The importance of aromatic ring oxidation in the metabolism is such that inhibition of CYP2D would be expected to cause a significant change in the pharmacokinetics of these compounds. The kinetics of MDMA metabolic activity in microsomes from ALLMA-pretreated rats were comparable to those from female Dark-Agouti rats, an animal model for CYP2D1 deficiency.

Disposition of methylenedioxymethamphetamine and three metabolites in the brains of different rat strains and their possible roles in acute serotonin depletion.

3,4-Methylenedioxymethamphetamine (MDMA) affects both dopamine and serotonin (5-HT) systems. One of its acute actions is to cause a reversible fall in steady-state brain 5-HT concentrations. To investigate the chemical basis of this acute effect, the brain levels of the parent compound and three major metabolites, 3,4- 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (DHMA) and 6-hydroxy-3,4-methylenedioxymethamphetamine (6-OHMDMA), were monitored, together with 5-HT levels, over a period of 6 hr in male Sprague-Dawley (SD) rats. The temporal relationships between drug concentrations of both stereoisomers and depletions were evaluated first. There was no correlation between the concentrations of the compounds measured and the extent of 5-HT depletion. Brain levels of MDMA and MDA were higher than plasma levels and exhibited a stereoselectivity in that (-)-MDMA and (+)-MDA levels were higher than those of enantiomers. The relationship between the dose of ((+)-MDMA and reduction in 5-HT levels was next investigated in SD male, SD female, and Dark Agouti (DA) female rats. These animals exhibit different capabilities of MDMA metabolism. There is a lower level of MDA, the N-demethylated metabolite of MDMA, in female SD rats than in males. Female DA rats are deficient in CYP2D isozymes, one of the enzymes responsible for demethylenation of MDMA to DHMA at pharmacological concentrations of substrate. there was a significant accuulation of MDMA in the brain and plasma of DA rats, but their 5-HT depletion was somewhat attenuated. The results indicated that MDMA ++ was apparently not the single, causative agent for the acute 5-HT depletion, which may also involve a metabolite formed by CYP2D.

Pharmacokinetic and pharmacodynamic analysis of the actions of D-amphetamine and D-methamphetamine on the dopamine terminal.

To establish whether the actions of D-amphetamine (Amp) and D-methamphetamine (MeAmp) on the striatal dopamine system were equipotent, pharmacokinetic profiles of each drug were applied to an analysis of their respective induced dopamine efflux profiles. Amp or MeAmp (1 and 5 mg/kg i.v.) was administered to chloral hydrate-anesthetized rats; plasma and brain kinetics were then assessed from 5 to 60 min. Dose-dependent increases in Amp and MeAmp plasma levels resulted in proportional increases in striatum levels that were equivalent for both drugs; elimination rates also were similar and were characterized by a first-order decay process. After MeAmp administration, low levels of brain MeAmp metabolites were detected throughout the 1-hr time period; relative to MeAmp, Amp and p-hydroxy-MeAmp levels were less than 10 and 1%, respectively. The drug-induced dopamine efflux profiles in the striatum were characterized by microdialysis; Amp and MeAmp (1, 2.5 and 5 mg/kg i.v.) effected equivalent, dose-dependent increases in extracellular dopamine levels. For both drugs at 5- and 10-min postinjection, increases in drug striatum levels preceded increases in dopamine efflux. In contrast, from the time of the peak dopamine responses observed at 10 to 20 min until the end of the study at 90 min, changes in striatal drug levels were correlated with extracellular dopamine levels; this correlation was similar for both drugs. These results indicate that Amp and MeAmp pharmacokinetics and their subsequent dopamine responses in the striatum are equivalent. The pharmacokinetic analysis can be extended to the interpretation of other comparative studies that assess effects of Amp and MeAmp.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P4502D isozymes catalyze the 4-hydroxylation of methamphetamine enantiomers.

The 4-hydroxylation of S(+)- and R(-)-methamphetamine by rat liver microsomes was examined in Sprague-Dawley and Dark Agouti strains to determine the role of cytochrome P4502D (CYP2D) subfamily isozymes in catalyzing the reaction. In the study, anti-P450-BTL IgG, bufuralol, and quinine, a substrate and inhibitors of CYP2D isozymes, respectively, were found to block approximately 90% of the reaction as catalyzed by microsomes from Sprague-Dawley rats. Reconstituted systems of CYP2D isozymes purified from rat liver microsomes also mediated the reaction. These observations and the minimal activity found in microsomes from Dark Agouti rats support the notion that methamphetamine, like other phenylisopropylamine compounds, is oxidized on the 4-position of the aromatic ring by CYP2D isozymes.

Inactivation of constitutive hepatic cytochromes P450 by phencyclidine in the rat.

The purpose of this study was to determine whether phencyclidine (PCP) inhibits constitutive hepatic cytochrome P450 (CYP) isozymes when administered to naive adult male Sprague-Dawley rats. Animals were pretreated with PCP (25 mg/kg/day for 2 days), killed 3 and 16 hr after the last dose, and liver microsomes prepared. The washed microsomes were then assayed for benzphetamine, methamphetamine (MA), and methylenedioxymethamphetamine (MDMA) N-demethylation together with MDMA demethylenation and MA 4-hydroxylation activities. MDMA demethylenation (low substrate concentration), MA 4-hydroxylation, and metoprolol alpha-hydroxylation reactions, which are catalyzed by CYP2D isozymes, were reduced > 74% 3 hr after the last PCP dose and were only partially restored 13 hr later. Benzphetamine and (-)-MDMA N-demethylation activities were restored to control values 16 hr after the last dose. These results indicate that PCP suppresses constitutive isozymes, including CYP2C11 and members of the CYP2D subfamily. The suppression of cytochromes P450 activity by PCP in vivo is consistent with its in vitro actions found in this and other studies, and demonstrates that alteration of CYP activity is another pharmacological effect of this compound.

A pharmacokinetic analysis of 3,4-methylenedioxymethamphetamine effects on monoamine concentrations in brain dialysates.

Interpretation of the in vivo actions of 3,4-methylenedioxymethamphetamine (MDMA) is complicated by the formation of the active metabolite, 3,4-methylenedioxyamphetamine (MDA). This study evaluates the role of MDA in the dopamine releasing actions of (+)-MDMA. In the study, rats were given subcutaneous doses of (+)-MDMA and concentrations of monoamines and their metabolites in striatal dialysate were measured at 15 min intervals. In parallel experiments, plasma concentrations of (+)- and (-)-MDMA and MDA were determined by GC/MS procedures. The time course of MDMA levels was comparable for the two isomers as were their bioavailabilities. In contrast, the plasma levels of MDA were about three times higher after (+)-MDMA. (+)-MDMA caused a rapid increase in striatal dialysate levels of dopamine and decreased extracellular levels of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). There was a significant correlation between dopamine concentration in striatal dialysate and plasma MDMA concentration, but not with plasma MDA. These results indicate that MDMA itself has stereoselective actions on dopamine neurons. However, the higher plasma MDA levels after (+)-MDMA may account for part of the enantiomeric differences in the behavioral and neurotoxicological effects of MDMA.

Stereochemical differences in the metabolism of 3,4-methylenedioxymethamphetamine in vivo and in vitro: a pharmacokinetic analysis.

The in vivo N-demethylation of (+) and (-)3,4-methylenedioxymethamphetamine (MDMA) to 3,4-methylenedioxyamphetamine (MDA) was determined and the pharmacokinetic relationship between the two compounds calculated. The levels of MDA formed after iv administration of (+)MDMA to male rats were about 3 times greater than those for (-)MDMA, although the plasma levels of the parent drugs were comparable. Plasma MDA concentrations were lower in phenobarbital-pretreated rats, but SKF 525-A pretreatment, at the dose used, had minimal effects. In liver microsome experiments conducted with microM concentrations of (+)MDMA, 3,4-dihydroxymethamphetamine (N-methyl-alpha-methyldopamine) was shown to be the major metabolite. MDA was also formed in vitro, but the enantioselectivity was the opposite of that found in vivo, pointing out the difficulties in extrapolation of in vitro observations to in vivo disposition. The high levels of MDA observed after administration of (+)MDMA to intact animals suggest that this active metabolite could be important in the overall effects of (+)MDMA.

The alpha carbon oxidation of some phencyclidine analogues by rat tissue and its pharmacological implications.

1. The metabolism of phencyclidine (PCP) and three congeners, differing in the structure of the amine moiety, by liver microsomes from phenobarbital-pretreated rats, was determined. 2. The metabolites generated by sequential oxidation of the two carbons alpha to the nitrogen were measured for PCP and its diethyl analogue (PCDE). 3. Alpha hydroxylation was a dominant metabolic pathway for PCDE, but less so for PCP. 4. Evaluation of affinities for the N-methyl-D-aspartate (NMDA) and sigma receptors in vitro showed that the product of alpha-hydroxylation of PCDE, phenylcyclohexylethylamine (PCE), was very potent. 5. Therefore, the in vivo actions of PCDE could include a significant contribution by PCE. 6. All congeners formed phenylcyclohexylamine (PCA), the product of a second alpha-hydroxylation, with PCDE and the pyrrolidine analogue generating the largest proportion.

Mentally retarded and normal children's performance on gross motor reaction- and movement-time tasks with varying degrees of uncertainty of movement.

This study evaluated the effect of uncertainty of movement, as a source of environmental stimuli, on the RT and movement time of 8- and 10-yr.-old mildly retarded and normal boys and girls during a 4.6-m run. With uncertainty of movement, retarded subjects (6 boys, 9 girls) displayed significant increases in both RT and movement time. For normal children (10 boys, 10 girls), no change in performance was noted as the task was simple. These results highlighted the notion of skill constancy and the importance of controlling potential stimuli during skill acquisition.

The effect of movement uncertainty on reaction and movement times of learning disabled and normal boys.

This study examined the effect of potential stimuli, as a source of movement uncertainty, on the reaction time (RT) and movement time (MT) of learning disabled (LD) children during a simple running task. The ability of LD children to attend to a process stimuli is known to be inferior. Such a notion is relevant to motor skill development as it imposes on the child's ability to develop from movement consistencies to movement constancies. When confronted with movement uncertainty, LD children showed significant increases in RT and MT over a 4.6 m. run. The performance of normal controls remained constant, thus emphasizing the simplicity of the task. The importance of skill consistency was highlighted, as was the consideration of inferior psychological processes of LD children while implementing movement activities.

The metabolism of phencyclidine by rabbit liver preparations.

The in vitro metabolism of phencyclidine by rabbit liver 9000g supernatant fraction produces primarily three known hydroxylated metabolites-namely, 4-phenyl-4-piperidinocyclohexanol, 4-(4'-hydroxypiperdino)-4phenylcyclohexanol, and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine-plus a new metabolite formed by oxidative scission of the piperidine ring yielding an aminoalcohol, and much smaller amounts of five unidentified metabolites. Incubation with 50% deuterium-labeled PCP indicates that these compounds are metabolites by monitoring the resulting doublets found in their mass spectra. The time, cofactor, and protein-dependent formation of these compounds confirms that they are indeed, metabolites. DPEA inhibits the production of the four quantitated metabolites with maximal I50 values of approximately 50 microM, implying the involvement of cytochrome P-450 in these reactions.

Combined treatment of pediatric medulloblastoma. A review of an integrated program )two-arm chemotherapy trial).

29 out of 34 consecutive patients of pediatric age, operated upon for posterior fossa medulloblastoma, were divided into 2 groups according to the chemotherapeutic treatment (intrathecal methotrexate or intravenous cyclophosphamide) received at random after surgery and radiation treatment. The modalities of irradiation and chemotherapy are described. 9 patients have not yet shown a local recurrence and are alive at varous intervals after surgery. Only 1 patient with local recurrence is still alive 31 months after the primary operation. The mean actuarial survival of the whole series of patients is about 38 months. Differences between the two groups, concerning either the survival rate till local recurrence, or the general and neurologic conditions of survival, are not statistically significant. Hematologic toxicity was more pronounced in the group treated with cyclophosphamide, whereas late neurologic sequelae were a more prominent feature of the intrathecal methotrexate trial.