Stereochemical studies on the novel monoamine oxidase B substrates (1R,6S)- and (1S,6R)-3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane.

Previous studies have established the unexpected monoamine oxidase-B (MAO-B) substrate properties of racemic 3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane, the 3,4-cyclopropyl analog of the achiral proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The two stereocenters present in this compound provide an opportunity to examine the enantioselectivity and diastereoselectivity of the MAO-B-catalyzed ring alpha-carbon oxidation of cyclic tertiary amines to give the corresponding conjugated iminiumyl metabolites. This paper reports the results of such stereochemical studies using expressed human MAO-B as the catalyst.

The first calibration of an aminiumyl radical ion clock: why N-cyclopropylanilines may be poor mechanistic probes for single electron transfer.

Using direct and indirect electrochemical methods, the rate constant for ring opening of the radical cation generated from N-cyclopropyl-N-methylaniline was found to be 4.1 x 10(4) s(-1).

Effects of haloperidol and its pyridinium metabolite on plasma membrane permeability and fluidity in the rat brain.

The use of antipsychotic drugs is limited by their tendency to produce extrapyramidal movement disorders such as tardive dyskinesia and parkinsonism. In previous reports it was speculated that extrapyramidal side effects associated with the butyrophenone neuroleptic agent haloperidol (HP) could be caused in part by the neurotoxic effect of its pyridinium metabolite (HPP(+)). Although both HPP(+) and HP have been shown to induce neurotoxic effects such as loss of cell membrane integrity, no information exists about the difference in the neurotoxic potency, especially in the potency to induce plasma membrane damage, between these two agents. In the present study, we compared the potency of the interaction of HPP(+) and HP with the plasma membrane integrity in the rat brain. Membrane permeabilization (assessed as [(18)F]2-fluoro-2-deoxy-d-glucose-6-phosphate release from brain slices) and fluidization (assessed as the reduction in the plasma membrane anisotropy of 1,6-diphenyl 1,3,5-hexatriene) were induced by HPP(+) loading (at >or=100 microM and >or=10 microM, respectively), while comparable changes were induced only at a higher concentration of HP (=1 mM). These results suggest that HPP(+) has a higher potency to induce plasma membrane damage than HP, and these actions of HPP(+) may partly underlie the pathogenesis of HP-induced extrapyramidal side effects.

Mass spectrometric studies on 4-aryl-1-cyclopropyl-1,2-dihydropyridinyl derivatives: an examination of a novel fragmentation pathway.

Examination of the electrospray ionization product ion spectra of 1,2-dihydropyridinyl and 4-aryl-1,2-dihydropyridinyl derivatives bearing a 1-cyclopropyl or 1-trans-2-phenylcyclopropyl group has led to the characterization of unexpected fragment ions. For example, the base peak at m/z 156 present in the product ion spectrum of trans-1-(2-phenylcyclopropyl)-4-phenyl-1,2-dihydropyridine proved not to be the expected 4-phenylpyridinium species but rather the isomeric 3-phenyl-5-azoniafulvenyl species. The results of studies with a series of structural and isotopically labeled analogs require a novel fragmentation pathway to account for the formation of this and related fragment ions. One possible pathway is based on an initial 1,5-sigmatropic shift of a cyclopropylmethylene hydrogen atom that is accompanied by opening of the cyclopropyl ring. The resulting eniminium intermediates then fragment to yield the 5-azoniafulvenyl species.

Release of vesicular Zn2+ in a rat transient middle cerebral artery occlusion model.

In the brain, Zn(2+) is stored in synaptic vesicles of a subgroup of glutamatergic nerve terminals. Although it has been reported that this Zn(2+) is released upon the excitation of nerves in vitro, there has been little study of the release of Zn(2+) during ischemia in vivo. Here, using brain microdialysis, the release of vesicular Zn(2+) was investigated in vivo. When the vesicular Zn(2+) was released into the synaptic cleft by a depolarizing stimulation achieved by perfusion with Ringer's solution containing high K(+) (100mM KCl), a significant increase in the extracellular concentration of Zn(2+) could be detected by microdialysis. Then, we investigated the release of vesicular Zn(2+) in a rat transient middle cerebral artery occlusion model using microdialysis. Consequently, the extracellular Zn(2+) level in the cortex increased within 15 min of the start of occlusion and reached a peak at 30 min, which was about twice the basal level. After 30 min, it declined with time returning to the basal level 15 min after reperfusion, which was performed after 60 min of occlusion. The results suggest that vesicular Zn(2+) would be released into the synaptic cleft during brain ischemia in vivo.

Potent estrogenic metabolites of bisphenol A and bisphenol B formed by rat liver S9 fraction: their structures and estrogenic potency.

We previously demonstrated that the estrogenicity of either bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] or bisphenol B [BPB; 2,2-bis(4-hydroxyphenyl)butane] was increased several times after incubation with rat liver S9 fraction (Yoshihara et al., 2001). This metabolic activation, requiring both microsomal and cytosolic fractions, was observed with not only rat liver, but also human, monkey, and mouse liver S9 fractions. To characterize the active metabolites of BPA and BPB, we investigated the structures of the isolated active metabolites by negative mode LC/MS/MS and GC/MS. The active metabolite of BPA gave a negative mass peak at [M-H](-) 267 on LC/MS and a single daughter ion at m/z 133 on MS/MS analysis, suggesting an isopropenylphenol dimer structure. Finally, this active metabolite was confirmed to be identical with authentic 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP) by means of various instrumental analyses. The corresponding peaks of the BPB metabolite were [M-H](-) 295 and m/z 147, respectively, suggesting an isobutenylphenol dimer structure. Further, coincubation of BPA and BPB with rat liver S9 afforded an additional active metabolite(s), which gave a negative mass peak at [M-H](-) 281 and two daughter ion peaks at m/z 133 and m/z 147 on MS/MS analysis. These results strongly suggest that the active metabolite of either BPA or BPB might be formed by recombination of a radical fragment, a one-electron oxidation product of carbon-phenyl bond cleavage. It is noteworthy that the estrogenic activity of MBP, the active metabolite of BPA, is much more potent than that of the parent BPA in several assays, including two reporter assays using a recombinant yeast expressing human estrogen receptor alpha and an MCF-7-transfected firefly luciferase plasmid.

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry.

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.

Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally.

We have determined three opioidmimetics (compounds I-III) in the rat brain dialysates after intraperitoneal (i.p.) administration of compounds I-III using a liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). The dialysate samples with methanol were directly analyzed by online column-switching liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 421 of m/z 657 for compound I, m/z 421 of m/z 643 for compound II, and m/z 407 of m/z 629 for compound III) on LC-MS/MS with electrospray ionization (ESI), opioidmimetics in rat brain dialysates were determined. Calibration curves of the method showed a good linearity in the range of 10-100 ng/ml for each compound. The limit of determination was estimated to be ca. 1 ng/ml for compounds II and III, and ca. 5 ng/ml for compound I, respectively. The precision of analysis showed coefficients of variation ranging from 4.7 to 10.4% at compound III concentration (10-100 ng/ml) in Ringer's solution. As a result, the procedure proved to be very suitable for routine analysis. The method was applied to the analysis of three opioidmimetics in the brain dialysate samples from rats treated with these compounds.

Misidentification of ethyl chloride in the routine GC-FID analysis for alcohol.

UNLABELLED: GC-FID is the method of choice for alcohol screening and quantitative analysis in modern forensic medical practice. Although specific enough for routine use, some results could be misleading. In the current article we present a case of sexual asphyxia with drug and volatile substance abuse. Toxicological analysis revealed the presence of methamphetamine at a concentration of 1.3 microg/mL in blood. An ethanol-like peak was detected during our routine GC-FID test for alcohol (methylethylketone IS). Subsequent GC-MS analysis identified the peak as ethyl chloride. Levels of 0.05 mg/mL in blood and 0.01 mg/mL in urine were measured. Two facts proved misleading in our case. First: very small difference of 0.027 between the ethyl chloride and ethanol peaks in relative retention times at the GC-FID chromatograms. Second: missing evidence for the use of ethyl chloride at the scene-neither cans of the substance were found, nor such information was available otherwise. CONCLUSION: there is a substantial risk for mistaking ethyl chloride for ethanol, when ethyl chloride abuse is unanticipated. In the case of slightest uncertainty a GC-MS analysis should be employed to reliably determine the actual substance.

Purification, characterization, and mass spectrometric sequencing of a medium chain acyl-CoA synthetase from mouse liver mitochondria and comparisons with the homologues of rat and bovine.

Medium chain acyl-CoA synthetases catalyze the first reaction of amino acid conjugation of many xenobiotic carboxylic acids and fatty acid metabolism. This paper reports studies on purification, characterization, and the partial amino acid sequence of mouse liver enzyme. The medium chain acyl-CoA synthetase was isolated from mouse liver mitochondria. The purified enzyme catalyzes this reaction not only for straight medium chain fatty acids but also for aromatic and arylacetic acids. Maximal activity was found with hexanoic acid. High activities were obtained with benzoic acid having methyl, pentyl, and methoxy groups in the para- or meta-positions of the benzene ring. However, the enzyme was less active with valproic acid and ketoprofen. Salicylic acid exhibited no activity. The medium chain acyl-CoA synthetases from mouse and bovine liver mitochondria were subjected to in-gel tryptic digestion, followed by LC-MS/MS sequence analysis. The amino acid sequence of each tryptic peptide of mouse liver mitochondrial medium chain acyl-CoA synthetase differed from that from bovine liver mitochondria only in one or two amino acids. LC-MS/MS analysis provided the information about these differences in amino acid sequences. In addition, we compared the properties of this protein with the homologues from rat and bovine.

In vivo measurement of presynaptic Zn2+ release during forebrain ischemia in rats.

Previous studies have suggested that during forebrain ischemia, considerable Zn2+ is released from synaptic vesicles of gultamatergic neuronal terminals and accumulates in hippocampal CA1 pyramidal neurons, leading to delayed neuronal death. However, since a time lag exists between the accumulation of Zn2+ and the occurrence of ischemia and there are conflicting reports about the amount of Zn2+ released, the level of released Zn2+ during ischemia in vivo is still unclear. In this study, we investigated the temporal change of extracellular Zn2+ in the hippocampal CA1 area using microdialysis and the accumulation of Zn2+ in hippocampal CA1 neurons with TSQ staining in rats with a transient forebrain ischemia. The level of extracellular Zn2+ in the CA1 area increased transiently reaching a peak 15 min after occlusion, then decreased with time, returning to the basal level 15 min after reperfusion. In addition, at this peak, the level of extracellular Zn2+ was about twice the basal level. Assessment of the intracellular Zn2+ in hippocampal neurons with TSQ revealed that Zn2+ accumulate at 24 h, but not 0 and 6 h after ischemia. These results suggest that, although the synaptic vesicular Zn2+ is released into the synaptic cleft during ischemia in vivo, the amount of released Zn2+ might not be so excessive, and it does not accumulate in hippocampal CA1 pyramidal neurons immediately after ischemia.

Protective effect of zinc against ischemic neuronal injury in a middle cerebral artery occlusion model.

In this study, we investigated the effect of vesicular zinc on ischemic neuronal injury. In cultured neurons, addition of a low concentration (under 100 microM) of zinc inhibited both glutamate-induced calcium influx and neuronal death. In contrast, a higher concentration (over 150 microM) of zinc decreased neuronal viability, although calcium influx was inhibited. These results indicate that zinc exhibits biphasic effects depending on its concentration. Furthermore, in cultured neurons, co-addition of glutamate and CaEDTA, which binds extra-cellular zinc, increased glutamate-induced calcium influx and aggravated the neurotoxicity of glutamate. In a rat transient middle cerebral artery occlusion (MCAO) model, the infarction volume, which is related to the neurotoxicity of glutamate, increased rapidly on the intracerebral ventricular injection of CaEDTA 30 min prior to occlusion. These results suggest that zinc released from synaptic vesicles may provide a protective effect against ischemic neuronal injury.

Model electrochemical-mass spectrometric studies of the cytochrome P450-catalyzed oxidations of cyclic tertiary allylamines.

Single-electron transfer and hydrogen atom transfer pathways have been proposed to account for the cytochrome P450-catalyzed alpha-carbon oxidations of amines. With the aid of electrochemistry-electrospray ionization mass spectrometry, the electrochemical potentials required for the one-electron oxidations of N-methyl- and selected N-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridinyl derivatives and the chemical fates of the resulting aminyl radical cations have been investigated. Comparison of the results of these studies with those observed in the corresponding enzyme catalyzed oxidations suggests that aminyl radical cations are not obligatory intermediates in the cytochrome P450-catalyzed alpha-carbon oxidations of this class of substrates.

Analysis of medium-chain acyl-coenzyme A esters in mouse tissues by liquid chromatography-electrospray ionization mass spectrometry.

Medium-chain acyl-coenzyme A (CoA) esters are key metabolites in lipid metabolism. Liquid chromatography-electrospray ionization mass spectrometry analysis of medium-chain acyl-CoA esters is described. Eight medium-chain acyl-CoA esters were well separated on a C(8)-MS reversed-phase column using a linear gradient of ammonium acetate buffer (pH 5.3)-acetonitrile. The positive-ion mass spectra of all the saturated and unsaturated medium-chain acyl-CoA esters gave dominant [M+H](+) ions, whereas their negative-ion mass spectra showed abundant [M-H](-) and [M-2H](2-) ions. The positive-ion mode of operation was slightly less sensitive than the negative-ion detection mode. Five medium-chain acyl-CoA esters of C(6:0), C(8:0), C(8:1), C(10:0), and C(10:1) in liver, heart, kidney, and brain from the mouse were identified. The predominant acyl-CoA peaks were C(6:0), C(8:0), and C(10:0). Small amounts of medium-chain acyl-CoAs of C(8:1) and C(10:1) were detected only in heart and kidney. The analytical method is very useful for the analysis of medium-chain acyl-CoA esters in the tissues.

In vitro confirmation of selegiline N-oxidation by flavin-containing monooxygenase in rat microsome using LC-ESI MS.

In order to investigate the conversion of selegiline (SG), a drug used in the treatment of Parkinson's disease, to selegiline N-oxide (SGO) as a major metabolic pathway for SG, rat liver microsomal incubations were carried out in vitro in the presence of NADPH. SG was transformed into SGO in vitro as described in our previous human in vivo experiment. In the kinetic studies, the Vmax/Km value of the N-oxidation at pH 8 was found to be approximately four times greater than that at pH 7.4. The N-oxidation was also found to be inhibited by methimazole, an inhibitor of the flavin-containing monooxigenase (FMO) rather than by SKF 525A, an inhibitor of cytochrome P450s, and stimulated approximately two times by n-octylamine, an stimulator of FMO. Moreover, the N-oxidation activity remained almost unchanged in the presence of NADPH even after heating at 50 degrees C for a few minutes. The present data demonstrate that the N-oxidation of SG to SGO is principally mediated by FMO.