Rat liver microsomal enzyme catalyzed oxidation of 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine

NADPH supplemented rat liver microsomal enzyme preparations catalyze the conversion of 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine to the p-hydroxyphenyl (low yield), descyclopropyl (high yield) and 2,3-dihydropyridinium and, subsequently, pyridinium (intermediary yield) metabolites. When the methine proton of the cyclopropyl group was replaced with a deuteron, a normal deuterium isotope effect (1.4) was observed on the formation of the decyclopropylated metabolite and an inverse isotope effect (0.6) on the dihydropyridinium metabolite. A larger deuterium isotope effect (3.6) was observed on the ring -carbon oxidation pathway with the 2,2,6,6-d₄ analogue as substrate. These results and the observation that the ratios of the rates of these two -carbon oxidation pathways are independent of initial substrate concentrations suggest that both pathways are catalyzed by the same active site of one form of P450. These transformations are discussed in terms of metabolic pathways that have been proposed for the cytochrome P450 catalyzed -carbon oxidation of amines.     

Reaction of benzophenone triplet with aliphatic amines. What a potent neurotoxin can tell us about the reaction mechanism

A photochemical model study of benzophenone triplet ((3)BP) with the MAO-B substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP (1)] and two of it's derivatives, 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine (2) and (±)-[trans-2-phenylcyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine (3) were performed. Literature precedent and calculations reported herein suggest that the barrier to ring opening for aminyl radical cations derived from N-cyclopropyl derivatives of tertiary amines (such as MPTP) will be low. The LFP results reported herein demonstrate that pathways for the reaction of (3)BP with 1, 2, and 3 are very similar. In each instance, disappearance of (3)BP is accompanied solely by appearance of bands corresponding to the diphenylhydroxylmethyl radical and neutral radical derived from MPTP and it's two derivatives 2 and 3. These results suggest that the reaction between benzophenone triplet and tertiary aliphatic amines proceed via a simple hydrogen atom transfer reaction. Additionally these model examinations provide evidence that oxidations of N-cyclopropyl derivatives of MPTP catalyzed by MAO-B may not be consistent with a pure SET pathway.     

Synthesis and MAO-B substrate properties of 1-methyl-4-heteroaryl-1,2,3,6-tetrahydropyridines

The parkinsonian inducing drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is bioactivated in a reaction catalyzed by the flavoenzyme monoamine oxidase B (MAO-B) to form the corresponding dihydropyridinium and subsequently pyridinium metabolites. As part of our ongoing studies to characterize the structural features responsible for this unexpected biotransformation, we have examined the MAO-B substrate properties of a variety of MPTP analogues bearing various heteroaryl groups at the 4-position of the tetrahydropyridinyl ring. The newly synthesized analogues are 4-(1-methylimidazol-2-yl)-, 4-(3-methylfuran-2-yl)-, 4-(3-methylthien-2-yl)-, 4-(3,4-dimethylpyrrol-1-yl)-, 4-(3-methylpyrrol-2-yl)-, and 4-(1,3-dimethylpyrrol-2-yl)-1-methyl-1,2,3,6-tetrahydropyridine. Except for the 4-(1-methylimidazol-2-yl) analogue, all compounds displayed good to excellent substrate properties. The 1-methyl-4-(3-methylfuran-2-yl) analogue is the most active member of this series with a kcat/Km value greater than 8,500 min(-1)mM(-1). The results of these studies are discussed in terms of catalytic pathways proposed for MAO-B.     

Chemically induced Parkinson's disease: intermediates in the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to the 1-methyl-4-phenyl-pyridinium ion

Various unstable intermediate oxidation states have been postulated in the metabolic activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to the 1-methyl-4-phenyl pyridinium ion. We now report the first direct observation of these free radical intermediates by pulse radiolysis and flash photolysis. Studies are described of various reactions of such species, in particular with dopamine whose autoxidation to dopamine quinone is reported to be potentiated by 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine.     

Oxidation Products Arising from the Action of Monoamine Oxidase B on 1-Methyl-4-Benzyl-1,2,3,6-Tetrahydropyridine, a Nonneurotoxic Analogue of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract: 1-Methyl-4-benzyl-1,2,3,6-tetrahydropyridine (MBzTP), an analogue of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, despite its rapid oxidation by monoamine oxidase B (MAOB), is not neurotoxic. The pyridinium expected to arise from the four-electron oxidation of MBzTP inhibits mitochondrial respiration and the oxidation of NADH in inner membranes and is only moderately less inhibitory than 1-methyl-4-phenylpyridinium. It is also a competitive inhibitor of dopamine uptake by the dopamine transporter and hence likely to be taken up into neurons, despite its relatively high K₁ value (K₁= 21 μM). Incubation of MBzTP with purified MAO B yields first the dihydropyridinium form, then a mixture of the pyridinium form and another unidentified product, in proportions that depend on the concentrations of MAO B and oxygen. At low MAO B concentration and moderate oxygen concentration, nonenzymatic formation, of the unidentified product predominates. The lack of neurotoxicity of MBzTP appears to be due to the oxidative destruction of the dihydropyridine and consequent failure of accumulation of 1-methyl-4-behzylpyridinium.     

1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine- and 1-Methyl-4-(2'-Ethylphenyl)-1,2,3,6-Tetrahydropyridine-Induced Toxicity in PC 12 Cells: Role of Monoamine Oxidase A

The toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), and their corresponding pyridinium species was studied in the rat pheochromocytoma PC12 cell line. MPTP and its analogues are known to be metabolized by monoamine oxidase (MAO) to dihydropyridinium intermediates which are further transformed, either enzymatically or spontaneously, into pyridinium species. MAO activity in PC12 cells is almost exclusively of the A form, and 2'Et-MPTP is a good substrate for both MAO-A and MAO-B. In contrast, MPTP is a poor substrate for MAO-A, but a good substrate for MAO-B. 2'Et-MPTP caused considerably more cell death than MPTP in the PC12 cells. However, 1-methyl-4-(2'-ethylphenyl)pyridinium and 1-methyl-4-phenylpyridinium, the corresponding pyridinium species formed from 2'Et-MPTP and MPTP, respectively, were equipotent as toxins. The toxic effects of the tetrahydropyridines and their corresponding pyridiniums were both concentration- and time-dependent. Measurements of the levels of the pyridinium species formed and the remaining tetrahydropyridine in the media indicated that 2'Et-MPTP was converted about five to seven times more readily into its toxic pyridinium species than was MPTP. There was, moreover, an excellent correlation between amount of pyridinium formed and cell death. There was also a parallel between the capacity of clorgyline and pargyline, irreversible MAO inhibitors, to decrease the formation of the pyridinium species and their capacity to protect against the toxic actions of the tetrahydropyridines. These data are consistent with the concept that the MAO-A-dependent formation of the pyridinium species from the tetrahydropyridine is a prerequisite for toxicity in PC12 cells.     

Chemically induced Parkinson's disease. II: Intermediates in the oxidation and reduction reactions of the 1-methyl-4-phenyl-2,3-dihydropyridinium ion and its deprotonated form

The one-electron reduction product of 1-methyl-4-phenyl-2,3-dihydropyridinium ion has been generated by pulse radiolysis and its absorption spectrum recorded. This radical was found to decay by second-order kinetics (2k = 9.5 x 10(8) M-1 s-1) to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion. Reactions of the above radical species and that formed by one-electron reduction of 1-methyl-4-phenylpyridinium ion, which can also be generated by one-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, with a number of molecules of biochemical interest have been studied. The one-electron reduction product of oxidised nicotinamide adenine dinucleotide efficiently reduced 1-methyl-4-phenyl-2,3-dihydropyridinium ion (k = 2.2 x 10(9) M-1 s-1). The relevance of these results in relation to redox cycling, a possible mechanism for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, is discussed.     

Catalytic biotransformation of physiologically active 1-methyl-4- aryl-1,2,3,6-tetrahydropyridines under the action of monoaminooxidase

Kinetics of monoamine oxidase (MAO) catalyzed dehydrogenation of neurotropic analogues of biogenic monoamines in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine series were studied. It is shown that methyl substitution in the phenyl ring increases significantly the enzyme-substrate affinity, but the substituent's effect on the catalytic stage largely depends upon its position in the ring. o- and m-Methyl derivatives were preferably oxidized by B type of MAO, whereas p-total derivative was oxidized by B type as well as by A type of the enzyme. In the course of the oxidation reactions MAO is irreversibly inhibited by the dihydropyridinium product of the reaction, particularly in case of methyl derivatives. The significant and structure-dependent inhibition of the enzyme might be responsible for the differences in neurotropic properties of the above substrate homologues.     

Deuterium isotope effect measurements on the interactions of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidase B

Kinetic deuterium isotope effects for the noncompetitive, intermolecular monoamine oxidase B-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the corresponding 1-methyl-4-phenyl-2,3-dihydropyridinium species MPDP+ were found to be 3.55 on Vmax and 8.01 on Vmax/Km with MPTP-6,6-d2 as the deuterated substrate. Similar values were obtained with MPTP-2,2,6-d4 and MPTP-CD3-2,2,6,6-d4. The deuterium isotope effect for the electrochemical oxidation of 1 mM MPTP-2,2,6,6-d4 was only 1.35. These results indicate that the monoamine oxidase B-catalyzed oxidation of this substrate may not proceed via a reaction pathway involving alpha-carbon deprotonation of an aminium radical intermediate. Isotope effect measurements also established that the rate of inactivation of monoamine oxidase B by MPTP is unaffected by replacement of the C-6 methylene protons with deuterons, but is retarded by replacement of the C-2 methylene protons (DKi = 1.9). The mechanism-based inactivation of monoamine oxidase B by MPTP, therefore, is likely to mediated by a species derived from the enzyme-generated 2,3-dihydropyridinium oxidation product.     

Studies on the mechanism of MPTP oxidation by human liver monoamine oxidase B

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP+) by preparations of pure human liver MAO B:monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver-Burk plots of initial reaction rates revealed that the Km,app values for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, Vmax,app values were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C-6. Deuterium substitution on the N-methyl group alone did not significantly reduce Vmax,app. These studies support the interpretation that oxidation of MPTP at the C-6 position on the tetrahydropyridine ring is a major rate-determining step in its biotransformation by MAO B.     

Evaluation of the Biological Activity of Several Analogs of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Several analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were synthesized and screened for their capacity to be oxidized by monoamine oxidase (MAO-A or MAO-B) and their capacity to produce nigrostriatal dopaminergic neurotoxicity in mice. All of the compounds were relatively weak substrates for MAO-A but many of the compounds were found to be good substrates for MAO-B. Only three of the compounds, in addition to MPTP itself, were found to be neurotoxic. These were 1-methyl-4-cyclohexyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine and 1-methyl-4-(3'-methoxyphenyl)-1,2,3,6-tetrahydropyridine. All three of these neurotoxic compounds were found to be substrates for MAO-B; in contrast no compound was found to be neurotoxic that was not oxidized by MAO-B. The capacity of the compounds studied to be oxidized by MAO-B appears to be an important aspect of the neurotoxic process.     

A novel and selective monoamine oxidase B substrate

Cyclic five- and six-membered tertiary allylamines constitute a unique class of monoamine oxidase substrates that undergo a net two-electron alpha-carbon oxidation to form the cyclic, conjugated eniminium metabolites. The corresponding saturated pyrrolidinyl and piperidinyl systems are not substrates for this flavoenzyme system. In an attempt to evaluate possible contributions that pi-orbital stabilization of the putative alpha-carbon radical intermediates may play in the catalytic pathway, we have examined the substrate properties of 3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane, the 3,4-cyclopropyl analog of the selective monoamine oxidase B substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results, which document the first reported example of a saturated, cyclic tertiary amine with monoamine oxidase substrate properties, are consistent with alpha-carbon radical stabilization as a contributing factor in the catalytic pathway.     

Quinolinic acid but not MK-801 protects the dopaminergic system from 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced toxicity in goldfish retina

The toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, intravitreally injected in goldfish eye, involves interplexiform retinal neurons and depletes tyrosine hydroxylase immunoreactivity and dopamine levels. This induced neurotoxicity was prevented by the concomitant administration in nontoxic doses (10 μg) of quinolinic acid, an endogenous structural analogue of N-methyl -aspartate with excitotoxic properties. Quinolinic acid is ineffective on the retinal degeneration induced by 1-methyl-4- phenylpyridinium ion. This fact suggests that quinolinic acid inhibits the MAO-B oxidation of 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine. MK-801, a noncompetitive antagonist of glutamate NMDA-receptors, exerts partial protective effects on MPTP-induced delayed toxicity in mammals. In the goldfish eye, MK-801, injected in low concentration, and in conjunction with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or 1-methyl-4-phenylpyridinium ion, did not prevent retinal neurodegeneration. Ten μg of MK-801 alone did not affect retinal neurons, while a higher concentration (20 μg) causes the chromatolysis of some photoreceptor nuclei.     

Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration

In the present study we demonstrate neuroprotective property of green tea extract and (-)-epigallocatechin-3-gallate in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson's disease. N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxin caused dopamine neuron loss in substantia nigra concomitant with a depletion in striatal dopamine and tyrosine hydroxylase protein levels. Pretreatment of mice with either green tea extract (0.5 and 1 mg/kg) or (-)-epigallocatechin-3-gallate (2 and 10 mg/kg) prevented these effects. In addition, the neurotoxin caused an elevation in striatal antioxidant enzymes superoxide dismutase (240%) and catalase (165%) activities, both effects being prevented by (-)-epigallocatechin-3-gallate. (-)-Epigallocatechin-3-gallate itself also increased the activities of both enzymes in the brain. The neuroprotective effects are not likely to be caused by inhibition of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine conversion to its active metabolite 1-methyl-4-phenylpyridinium by monoamine oxidase-B, as both green tea and (-)-epigallocatechin-3-gallate are very poor inhibitors of this enzyme in vitro (770 microg/mL and 660 microM, respectively). Brain penetrating property of polyphenols, as well as their antioxidant and iron-chelating properties may make such compounds an important class of drugs to be developed for treatment of neurodegenerative diseases where oxidative stress has been implicated.     

Energy-dependent uptake of N-methyl-4-phenylpyridinium, the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mitochondria

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an impurity in certain batches of illicit heroin substitutes, is known to cause parkinsonian symptoms and degeneration of the nigrostriatal cells in drug abusers and primates. Neurotoxicity depends on oxidation of MPTP by monoamine oxidase in brain cells to the dihydropyridinium form, which is further oxidized to N-methyl-4-phenylpyridinium (MPP+), the 4-electron oxidation product. The latter is widely believed to be the compound responsible for neuronal destruction and the NADH dehydrogenase of the inner membrane has been postulated to be its target. This enzyme is inhibited, however, only at very high concentrations of MPP+, while the steady-state concentration of MPP+ in the nigrostriatal cells of MPTP-treated animals is several orders of magnitude lower. This paradox has now been resolved by the discovery of an energized uptake system for MPP+ in mitochondria which rapidly concentrates MPP+ to very high concentrations in the mitochondria at micromolar external concentrations. The process is dependent on the electrical gradient of the membrane, has a Km of about 5 mM, and is completely blocked by respiratory inhibitors and uncouplers.     

Moderate chemical modifications of WAY-100635 improve the selectivity for 5-HT1A versus D4 receptors

The selectivity for 5-HT(1A) versus D(4) receptors is significantly increased when the basic side chain of WAY-100635 is replaced by a 4-phenylpiperazine (3e) or a 4-phenyl-1,2,3,6-tetrahydropyridine moiety (3i). The 4-phenyl-1,2,3,6-tetrahydropyridine compounds (3i-l) have a higher affinity for 5-HT(1A) receptors than do the corresponding unsubstituted phenylpiperazine analogues (3e-h). Compounds 3e and 3i appear to be selective for 5-HT(1A) receptors over other relevant receptors and still behave as neutral antagonists.     

Deuterium isotope effects for the oxidation of 1-methyl-3-phenyl-3-pyrrolinyl analogues by monoamine oxidase B

The parkinsonian inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a cyclic tertiary allylamine exhibiting good monoamine oxidase B (MAO-B) substrate properties. MAO-B catalyzes the ring alpha-carbon 2-electron bioactivation of MPTP to yield the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP(+)). The corresponding 5-membered ring MPTP analogue, 1-methyl-3-phenyl-3-pyrroline, also undergoes MAO-B-catalyzed oxidation to give the 2-electron oxidation product, 1-methyl-3-phenylpyrrole. Here we report the kinetic deuterium isotope effects on V(max) and V(max)/K(m) for the steady-state oxidation of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-fluorophenyl)-3-pyrroline by baboon liver MAO-B, using the corresponding pyrroline-2,2,4,5,5-d(5) analogues as the deuterated substrates. The apparent isotope effects for the two substrates were 4.29 and 3.98 on V(max), while the isotope effects on V(max)/K(m) were found to be 5.71 and 3.37, respectively. The values reported for the oxidation of MPTP by bovine liver MAO-B with MPTP-6,6-d(2), as deuterated substrate, are (D)(V(max))=3.55; (D)(V(max)/K(m))=8.01. We conclude that the mechanism of the MAO-B-catalyzed oxidation of pyrrolinyl substrates is similar to that of the tetrahydropyridinyl substrates and that a carbon-hydrogen bond cleavage step is, at least partially, rate determining.     

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.     

Analysis of γ-Aminobutyric AcidA Receptor Subunits in the Mouse Cochlea by Means of the Polymerase Chain Reaction

Abstract: Unlike 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces consistent decreases in levels of striatal dopamine (DA) with considerably smaller and more variable effects on mouse brain levels of serotonin (5-HT) and norepinephrine (NE), a novel amine-substituted MPTP analogue, 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH₂-MPTP), administered in a standard mouse dosing paradigm for MPTP (20 mg/kg X 4) did not affect striatal DA but led to marked reductions (60–70%) in levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and NE measured in frontal cortex and hippocampus 1 week after treatment. Another 2'-substituted MPTP analogue, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine, affected cortical and hippocampal 5-HT, 5-HIAA, and NE only minimally, while markedly reducing the DA content in striatum (90%), thus indicating that the substituent (-NH₂ versus -CH₃) at the 2'position is important for the differential effects of these MPTP analogues. In a replication study with a 3-week end point, hippocampal and cortical 5-HT, 5-HIAA, and NE levels remained depressed with no indication of recovery. These results suggest that 2'-NH₂-MPTP may be a novel, regionally selective neurotoxin for serotonergic and norad-renergic nerve terminals.     

Bioactivation of MPTP: reactive metabolites and possible biochemical sequelae

Expression of the selective nigrostriatal neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] requires its bioactivation by MAO B which leads to the formation of potentially reactive metabolites including the 2-electron oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium species [MPDP+] and the 4-electron oxidation product, the 1-methyl-4-phenyl pyridinium species [MPP+]. The latter metabolite accumulates in brain striatal tissues, is a substrate for dopaminergic active uptake systems and is an inhibitor of mitochondrial NADH dehydrogenase, a respiratory chain enzyme located in the inner mitochondrial membrane. In intact mitochondria this inhibition of respiration may be facilitated by active uptake of MPP+, a process dependent on the membrane electrical gradient. In considering possible mechanisms involved in the biochemical effects of MPP+, its redox cycling potential appears to be much lower than its chemical congener paraquat, based on attempted radical formation by chemical or enzymic reduction. Theoretically, a carbon-centered radical intermediate could be formed by 1-electron reduction of MPP+, or by 1-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, the free base form of MPDP+. The 1-electron reduction of such a radical could form 1-methyl-4-phenyl-1,4-dihydropyridine [DHP]. Synthetic DHP is neurotoxic in C57B mice, and its administration leads to the formation of MPP+ in the brain, presumably through rapid auto-oxidation. The hydrolysis of DHP would yield 3-phenylglutaraldehyde and methylamine. Recent studies demonstrating the formation of methylamine in brain mitochondrial preparations containing MPTP support our suggestion that DHP may be a brain metabolite of MPTP.