A putative metabolite of serotonin, tryptamine-4,5-dione, is an irreversible inhibitor of tryptophan hydroxylase: possible relevance to the serotonergic neurotoxicity of methamphetamine.

Tryptamine-4,5-dione (T-4,5-D) is formed as a result of oxidation of 5-hydroxytryptamine by superoxide (O(2)(-)(*), nitric oxide (NO*), and peroxynitrite (ONOO(-)). T-4,5-D rapidly inactivates tryptophan hydroxylase (TPH), derived from rat brain, probably as a result of covalent modification of active site cysteine residues. The activity of TPH exposed to T-4,5-D cannot be restored by anaerobic reduction with dithiothreitol (DTT) and ferrous iron (Fe(2+)) indicating that the inactivation is irreversible. 7-S-Glutathionyl-tryptamine-4,5-dione, formed by the rapid reaction between T-4,5-D and glutathione, also inhibits TPH but in this case the activity is restored by anaerobic reduction with DTT/Fe(2+). The results of this investigation may be relevant to the initial reversible and subsequent irreversible inactivation of TPH evoked by methamphetamine and 3,4-methylenedioxymethamphetamine.

Tryptamine-4,5-dione, a putative endotoxic metabolite of the superoxide-mediated oxidation of serotonin, is a mitochondrial toxin: possible implications in neurodegenerative brain disorders.

The release and subsequent reuptake of 5-hydroxytryptamine (5-HT) and cytoplasmic superoxide (O2-*) generation have both been implicated as important factors associated with the degeneration of serotonergic neurons evoked by methamphetamine (MA) and cerebral ischemia-reperfusion (I-R). Such observations raise the possibility that tryptamine-4,5-dione (T-4,5-D), the major in vitro product of the O2-*-mediated oxidation of 5-HT, might be an endotoxicant that contributes to serotonergic neurodegeneration. When incubated with intact rat brain mitochondria, T-4,5-D (< or = 100 microM) uncouples respiration and inhibits state 3. Experiments with rat brain mitochondrial membrane preparations confirm that T-4,5-D evokes irreversible inhibition of NADH-coenzyme Q1 (CoQ1) reductase and cytochrome c oxidase (COX) apparently by covalently modifying key sulfhydryl (SH) residues at or close to the active sites of these respiratory enzyme complexes. Ascorbic acid blocks the inhibition of NADH-CoQ1 reductase by maintaining T-4,5-D predominantly as 4, 5-dihydroxytryptamine (4,5-DHT), thus preventing its reaction with SH residues. In contrast, ascorbic acid potentiates the irreversible inhibition of COX by T-4,5-D. This may be because the T-4,5-D-4, 5-DHT couple redox cycles in the presence of excess ascorbate and molecular oxygen to cogenerate O2-* and H2O2 that together react with trace levels of iron to form an oxo-iron complex that selectively damages COX. Thus, T-4,5-D might be an endotoxicant that, dependent on intraneuronal conditions, mediates irreversible damage to mitochondrial respiratory enzyme complexes and contributes to the serotonergic neurodegeneration evoked by MA and I-R.

Oxidation of serotonin by superoxide radical: implications to neurodegenerative brain disorders.

Many new lines of evidence implicate both superoxide anion radical (O2*-) and biogenic amine neurotransmitters in the pathological mechanisms that underlie neuronal damage caused by methamphetamine (MA), glutamate-mediated oxidative toxicity, ischemia-reperfusion, and other neurodegenerative brain disorders. In this investigation the oxidation of 5-hydroxytryptamine (5-HT, serotonin) by an O2*--generating system (xanthine/xanthine oxidase) in buffered aqueous solution at pH 7.4 has been studied. The major product of the O2*--mediated oxidation of 5-HT is tryptamine-4,5-dione (T-4, 5-D). However, O2*- and H2O2, cogenerated by the xanthine oxidase-mediated oxidation of xanthine to uric acid, together react with trace levels of iron that contaminate buffer constituents to give a chemically ill-defined oxo-iron species. This species mediates the oxidation of 5-HT to a C(4)-centered carbocation intermediate that reacts with 5-HT to give 5,5'-dihydroxy-4, 4'-bitryptamine (4,4'-D) and with uric acid to give 9-[3-(2-aminoethyl)-5-hydroxy-1H-indol-4-yl]-2,6,8-triketo-1H,3H, 7H-purine (7) as the major products. These products differ from those formed in the HO*-mediated oxidation of 5-HT under similar conditions. When the reaction is carried out in the presence of the intraneuronal nucleophile glutathione (GSH), T-4,5-D is scavenged to give 7-(S-glutathionyl)tryptamine-4,5-dione, whereas the putative carbocation intermediate is scavenged to give 4-(S-glutathionyl)-5-hydroxytryptamine. T-4,5-D also reacts with the sulfhydryl residues of a model protein, alcohol dehydrogenase, and inhibits its activity. Previous investigators have proposed that T-4, 5-D is a serotonergic neurotoxin. This raises the possibility that T-4,5-D and perhaps other putative intraneuronal metabolites formed by the O2*-/H2O2/oxo-iron-mediated oxidations of 5-HT might be endotoxins that contribute to neurodegeneration in brain regions innervated by serotonergic neurons caused by MA, ischemia-reperfusion, and other neurodegenerative brain disorders.

5-hydroxy-3-ethylamino-2-oxindole is not formed in rat brain following a neurotoxic dose of methamphetamine: evidence that methamphetamine does not induce the hydroxyl radical-mediated oxidation of serotonin.

Oxygen radicals have been implicated in the neurodegenerative and other neurobiological effects evoked by methamphetamine (MA) in the brain. It has been reported that shortly after a single large subcutaneous dose of MA to the rat, the serotonergic neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) is formed in the cortex and hippocampus. This somewhat controversial finding suggests that MA potentiates formation of the hydroxyl radical (HO.) that oxidizes 5-hydroxytryptamine (5-HT) to 5,6-DHT, which, in turn, mediates the degeneration of serotonergic terminals. A major and more stable product of the in vitro HO.-mediated oxidation of 5-HT is 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). In this investigation, a method based on HPLC with electrochemical detection (HPLC-EC) has been developed that permits measurement of very low levels of 5-HEO in rat brain tissue in the presence of biogenic amine neurotransmitters/metabolites. After intracerebroventricular administration into rat brain, 5-HEO is transformed into a single major, but unknown, metabolite that can be detected by HPLC-EC. One hour after administration of MA (100 mg/kg s.c.) to the rat, massive decrements of 5-HT were observed in all regions of the brain examined (cortex, hippocampus, medulla and pons, midbrain, and striatum). However, 5-HEO, its unidentified metabolite, or 5,6-DHT were not detected as in vivo metabolites of 5-HT. MA administration, in particular to rats pretreated with pargyline, resulted in the formation of low levels of N-acetyl-5-hydroxytryptamine (NAc-5-HT) in all brain regions examined. These results suggest that MA does not potentiate the HO.-mediated oxidation of 5-HT. Furthermore, the rapid MA-induced decrease of 5-HT might not only be related to oxidative deactivation of tryptophan hydroxylase, as demonstrated by other investigators, but also to the inhibition of tetrahydrobiopterin biosynthesis by NAc-5-HT. The massive decrements of 5-HT evoked by MA are accompanied by small or no corresponding increases in 5-hydroxyindole-3-acetic acid (5-HIAA) levels. This is due, in part, to the relatively rapid clearance of 5-HIAA from the brain and monoamine oxidase (MAO) inhibition by MA. However, the loss of 5-HT without corresponding increases in its metabolites point to other mechanisms that might deplete the neurotransmitter, such as oxidation by superoxide radical anion (O2.-), a reaction that in vitro does not generate 5-HEO or 5,6-DHT but rather another putative neurotoxin, tryptamine-4,5-dione. One hour after administration, MA evokes large depletions of norepinephrine (NE) throughout the brain but somewhat smaller decrements of dopamine (DA) that are restricted to the nigrostriatal pathway. Furthermore, MA evokes a major shift in the metabolism of both NE and DA from the pathway mediated by MAO to that mediated by catechol-O-methyltransferase. The profound and widespread effects of MA on the noradrenergic system, but more anatomically localized influence on the dopaminergic system, suggests that NE in addition to DA, or unusual metabolites of these neurotransmitters, might play roles in the neurodegenerative effects evoked by this drug.

Putative oxidative metabolites of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline of potential relevance to the addictive and neurodegenerative consequences of ethanol abuse.

Ethanol is metabolized in the brain by catalase/H2O2 to yield acetaldehyde and by an ethanol-inducible form of cytochrome P450 (P450 IIE1) in a reaction that yields oxygen radicals. Within the cytoplasm of serotonergic axon terminals these metabolic pathways together provide conditions for the endogenous synthesis of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline (1), by reaction of acetaldehyde with unbound 5-hydroxytryptamine (5-HT), and for the oxygen radical-mediated oxidation of this alkaloid. The major initial product of the hydroxyl radical (HO.)-mediated oxidation of 1 in the presence of free glutathione (GSH), a constituent of nerve terminals and axons, is 8-S-glutathionyl-1-methyl-1,2,3,4-tetrahydro-beta-carboline-5,6-dione (6). When administered into the brains of mice, 6 is a potent toxin (LD50 = 2.9 microg) and evokes episodes of hyperactivity and tremor. Compound 6 binds at the GABA(B) receptor and evokes elevated release and turnover of several neurotransmitters. Furthermore, the GABA(B) receptor antagonist phaclofen attenuates the behavioral response caused by intracerebral administration of 6. These observations suggest that 6 might be an inverse agonist at the GABA(B) receptor site. Accordingly, it is speculated that ethanol drinking might potentiate formation of 6 that contributes to elevated release of several neurotransmitters including dopamine (DA) and endogenous opioids in regions of the brain innervated by serotonergic axon terminals. Subsequent interactions of DA and opioids with their receptors might be related to the initial development of dependence on ethanol. Redox cycling of 6 (and of several putative secondary metabolites) in the presence of intraneuronal antioxidants and molecular oxygen to produce elevated fluxes of cytotoxic reduced oxygen species might contribute to the degeneration of serotonergic pathways. Low levels of 5-HT in certain brain regions of the rat predisposes these animals to drink or augments drinking. Accordingly, 6, formed as a result of ethanol metabolism in the cytoplasm of certain serotonergic axon terminals, might contribute to the initial development of dependence on ethanol, by mediating DA and opioid release, and long-term preference and addiction to the fluid as a result of the progressive degeneration of these neurons.

Potential new insights into the molecular mechanisms of methamphetamine-induced neurodegeneration.

In the event that methamphetamine evokes HO. formation within serotonergic axon terminals, the resultant oxidation of 5-HT would be expected to generate not only 5,6-DHT but also T-4,5-D, 7-S-Glu-T-4,5-D, 6, 8, and 7,7'-D (figure 1), at least three of which (T-4,5-D, 7-S-Glu-T-4,5-D, and 6) are lethal in mouse brain. Furthermore, several intermediates/products formed in the in vitro oxidation of 5-HT by HO. are readily autoxidized (4,5-DHT, 5,6-DHT, 5, 7, and 9) or redox cycled (T-4,5-D, 6, 8, 7,7'-D, 7-S-Glu-T-4,5-D) in reactions that would be expected to yield O2-. and/or H2O2 as byproducts. These byproducts, in the presence of trace levels of transition metal ion catalysts, would be readily converted into HO. (Walling 1975; Halliwell and Gutteridge 1984). Together these putative aberrant oxidative metabolites of 5-HT and HO.-forming reactions might contribute to the degeneration of serotonergic nerve terminals. Similarly, the methamphetamine-induced intraneuronal formation of HO. in dopaminergic terminals might be expected to generate not only 6-OHDA (and 2-OHDA and 5-OHDA, figure 3) but also 5,-S-CyS-DA and 5-S-Glu-DA, precursors of DHBT 17 and other more complex dihydrobenzothiazines (figure 4). DHBTs 17 to 19 are lethal in mouse brain, although at this time the biochemical/chemical mechanisms underlying this toxicity and specific neuronal systems affected are unknown. However, 5-S-CyS-DA and 17 to 19 are much more easily oxidized than DA, and the latter DHBTs appear to be capable of redox cycling reactions (Zhang and Dryhurst 1994). Thus, the HO.-mediated oxidation of DA in dopaminergic nerve terminals induced by methamphetamine might be expected to generate aberrant oxidative metabolites that (as a result of autoxidation and redox cycling reactions) potentiate formation of O2-. and/or H2O2, and then HO. and neuronal damage. A number of lines of evidence, discussed previously, suggest that aberrant metabolite(s) of DA (other than or in addition to 6-OHDA) might contribute to the methamphetamine-induced degeneration of not only dopaminergic terminals but also serotonergic terminals. Similarly, aberrant metabolite(s) of 5-HT (other than or in addition to 5,6-DHT) might be involved in the degeneration of serotonergic and dopaminergic terminals and a subpopulation of cell bodies in the somatosensory cortex. Experimental evidence indicates that some of the neurodegenerative effects evoked by methamphetamine are mediated by NMDA and GABA receptors. Thus, it will be of considerable interest to investigate the neurotoxicity of putative aberrant oxidative metabolites of 5-HT (figures 1 and 2) and DA (figures 4 and 5) towards serotonergic, dopaminergic, and other neuronal systems and their interactions with NMDA, GABA, and other brain receptors. A central question relates to mechanisms by which methamphetamine might evoke the intraneuronal formation of oxygen radicals that appear to play important roles in the overall neurodegenerative processes evoked by this drug (DeVito and Wagner 1989; Cadet et al. 1994). Once putative oxidative metabolites of 5-HT such as T-4,5-D, 7-S-Glu-T-4,5-D, 5,6-DHT, 6, 8, and 7,7'-D (figure 1) are formed intraneuronally, autoxidation/redox cycling reactions should, in principle, be capable of generating O2-. and/or H2O2, the precursors of HO.. Similarly, intraneuronal formation of 6-OHDA, 5-S-CyS-DA, and DHBTs 17 to 19 and 22 would also be expected to potentiate elevated fluxes of O2-., H2O2, and HO. as a result of the facile autoxidation/redox cycling reactions of these putative aberrant metabolites. The presence of very low concentrations of 5-S-CyS-DA in DA-rich regions of human and other mammalian brains suggest that autoxidation (Rosengren et al. 1985; Fornstedt et al. 1986, 1989, 1990) or perhaps some other form of DA oxidation is a normal reaction in vivo. Furthermore, available evidence suggests that it is cytoplasmic DA that is oxidized to give 5-S-CyS-DA (Fornstedt et al. 1989; Fornstedt and

Synthesis, redox properties, in vivo formation, and neurobehavioral effects of N-acetylcysteinyl conjugates of dopamine: possible metabolites of relevance to Parkinson's disease.

A very early event in the pathogenesis of idiopathic Parkinson's disease (PD) has been proposed to be an elevated translocation of L-cysteine (CySH) and/or glutathione (GSH) into pigmented dopaminergic cell bodies in the substantia nigra (SN) in which cytoplasmic dopamine (DA) is normally autoxidized to DA-o-quinone as the first step in a reaction leading to black neuromelanin polymer. Such an elevated influx of CySH and GSH would be expected to initially result in formation of 5-S-cysteinyldopamine (5-S-CyS-DA) and 5-S-glutathionyldopamine (5-S-Glu-DA), respectively, and might account for the massive irreversible loss of GSH and progressive depigmentation of SN cells that occurs in the Parkinsonian brain. However, 5-S-Glu-DA has not been detected in the Parkinsonian brain. Furthermore, although the 5-S-CyS-DA/DA and 5-S-CyS-DA/homovanillic acid concentration ratios increase significantly in the SN and cerebrospinal fluid, respectively, of PD patients, the absolute concentrations of 5-S-CyS-DA are extremely low and similar to those measured in age-matched control patients. One explanation for these observations is that 5-S-CyS-DA might be intraneuronally oxidized to more complex cysteinyldopamines and a number of dihydrobenzothiazines (DHBTs) and benzothiazines (BTs). Similarly, 5-S-Glu-DA might be intraneuronally oxidized to more complex glutathionyldopamines. In this investigation, however, it is demonstrated that 5-S-Glu-DA is rapidly metabolized in rat brain to 5-S-CyS-DA and 5-S-(N-acetylcysteinyl) dopamine (5) in reactions mediated by gamma-glutamyl transpeptidase (gamma-GT) and cysteine conjugate N-acetyltransferase. Similarly, 5-S-CyS-DA is metabolized to 5 in rat brain although more slowly than 5-S-Glu-DA. These reactions occur most rapidly in the midbrain, a region that contains the SN. Furthermore, 5, 2-S-(N-acetylcysteinyl)dopamine (6) and 2,5-di-S-(N-acetylcysteinyl)-dopamine (9) are toxic when administered into mouse brain having LD50 values of 14, 25, and 42 micrograms, respectively, and evoke a profound hyperactivity syndrome. These results suggest that the failure to detect 5-S-Glu-DA and the presence of only very low levels of 5-S-CyS-DA in Parkinsonian SN tissue and CSF might be related to both their intraneuronal oxidation and extraneuronal metabolism to N-acetylcysteinyl conjugates of DA. Furthermore, the toxic properties and neurobehavioral responses evoked by 5, 6, and 9 raise the possibility that these N-acetylcysteinyl conjugates of DA, in addition to certain cysteinyldopamines, DHBTs and BTs, might include endotoxins that contribute to SN cell death and other neuronal damage that occurs in PD. Methods are described for the synthesis of several N-acetylcysteinyl conjugates of DA, and their redox behaviors have been studied using cyclic voltammetry.

Hydroxyl radical-mediated oxidation of serotonin: potential insights into the neurotoxicity of methamphetamine.

When incubated with a hydroxyl radical (HO.)-generating system (ascorbic acid/Fe(2+)-EDTA/O2/H2O2), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3'-bis(2-aminoethyl)-5-hydroxy-[3,7'-bi-1H-indole]-2,4',5'- 3H-trione (7). The latter slowly cyclizes to 3'-(2-aminoethyl)-1',6',7',8'-tetrahydro-5-hydroxy-spiro[3H-indole-3,9'- [9H]pyrrolo[2,3-f]quinoline]-2,4',5' (1H)-trione (9). A minor amount of T-4,5-D dimerizes to give 7,7'-bi-(5-hydroxytryptamine-4-one) (7,7'-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO.-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO. formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO.-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO. formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO. might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.

7-S-glutathionyl-tryptamine-4,5-dione: a possible aberrant metabolite of serotonin.

Tryptamine-4,5-dione (Compound 1) is an in vitro oxidation product of 5-hydroxytryptamine (5-HT). Recent evidence has suggested that aberrant oxidations of 5-HT occur in the central nervous system of individuals with Alzheimer's disease (AD). In the event that Compound 1 is formed as a result of oxidation of 5-HT within serotonergic nerve terminals or axons, it would be expected to be rapidly conjugated by intraneuronal glutathione (GSH) to give 7-S-glutathionyl-tryptamine-4,5-dione (Compound 2). When injected into the brains of laboratory mice, Compound 2 was lethal (LD50 = 21 micrograms) and evoked hyperactivity for the first 30 min following drug administration. Particularly during this hyperactive phase Compound 2 caused a statistically significant decrease in whole brain levels of norepinephrine and 5-HT. Levels of dopamine were also decreased while whole brain concentrations of its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were increased significantly. In the presence of GSH, NADPH and ascorbic acid, Compound 2 redox cycled in reactions that catalyzed the oxidation of these cellular reductants by molecular oxygen and formed H2O2 as a byproduct. Compound 2 also reacted with molar excesses of GSH to form more structurally complex glutathionyl conjugates. Several of these conjugates have been isolated and their structures determined using spectroscopic methods. It is conceivable that one or more of these conjugates might serve as analytical markers in a search for evidence in support of the hypothesis that aberrant oxidations of 5-HT occur in the Alzheimer brain. The redox cycling properties of Compound 2 and its facile reactions with cellular nucleophiles such as GSH may represent mechanisms that contribute to the toxicity of this drug.

5,5'-Dihydroxy-4,4'-bitryptamine: a potentially aberrant, neurotoxic metabolite of serotonin.

Previous investigators have detected unknown oxidized forms of 5-hydroxytryptamine (5-HT) in the CSF of Alzheimer's disease (AD) patients. Furthermore, an unidentified autoxidation product of this neurotransmitter is an inhibitor of acetylcholinesterase (AChE), an enzyme compromised in the Alzheimer brain. In this study it is demonstrated that the major product of autoxidation of 5-HT is 5,5'-dihydroxy-4,4'-bitryptamine (DHBT). Central administration of DHBT to mice at a dose of 40 micrograms (free base) evokes profound behavioral responses, which persist until the animals die (approximately 24 h). One hour after central administration of DHBT, the levels of norepinephrine, dopamine, 5-HT, and acetylcholine and their metabolites in whole brain are greatly elevated. Disturbances to the catecholaminergic and serotonergic systems were still evident shortly before the death of animals. DHBT is also shown to be a noncompetitive inhibitor of AChE in vitro. These observations suggest that if DHBT is formed as an aberrant metabolite of 5-HT in the human brain, it could potentially be neurotoxic and contribute to the neuronal degeneration and other neurochemical and neurobiochemical changes associated with AD or perhaps other neurodegenerative diseases.

Interactions of 5-hydroxytryptamine with oxidative enzymes.

Peroxidase (EC 1.11.1.7)/H2O2, ceruloplasmin (human type X)/O2, and tyrosinase (EC 1.14.18.1)/O2 all oxidized the indolic neurotransmitter 5-hydroxytryptamine (5-HT) in the physiological pH domain. Peroxidase/H2O2 oxidized 5-HT at pH values down to about 2.5. All oxidation reactions generated complex mixtures of products which included at least one known neurotoxin, tryptamine-4,5-dione. In general, the enzymatic oxidation pathways paralleled the in vitro electrochemical oxidation of 5-HT which has permitted suggestions to be made concerning the probable mechanisms of the enzyme-mediated reactions.

Autoxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine.

The indolic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) has been widely speculated to express its neurodegenerative effects as a result of intraneuronol autoxidation. Until recently, it was believed that autoxidation led to reactive electrophilic quinone imine species which alkylated neuronal membrane proteins and that byproducts of the autoxidation reaction were cytotoxic reduced-oxygen species. This study reveals that at physiological pH carbanions of 5,7-DHT act as the primary electron-donor species to yield C(4)- and C(6)-centered free radical superoxide complexes in a 1:2 ratio. The C(4)-centered complex reacts to yield, ultimately, 5-hydroxytryptamine-4,7-dione which has been shown to be a significantly more powerful neurotoxin than 5,7-DHT. The C(6)-centered radical superoxide complexes react to give 6,6'-bis(5-hydroxytryptamine-4,7-dione). It is likely that the latter reaction yields O2.- as a cytotoxic byproduct.

Further insights into the oxidation chemistry of 5-hydroxytryptamine.

An important product of electrochemical oxidation of 5-hydroxytryptamine (5-HT) in acid solution is the purple compound tryptamine-4,5-dione (6). However, any attempt to concentrate a solution containing 6 causes it to disappear. The most important reaction of 6 is dimerization to give another purple compound 7,7'-bi-(5-hydroxytryptamine-4-one). Dione 6 can also apparently react with 2,4'-bi-5-hydroxytryptamine to give the trimer 4-[7'-(tryptamine-4,5-dione)]-2,4''-bi-5-hydroxytryptamine. Finally, 6 and other oxidation products of 5-HT react during the concentration step to yield what appears to be a trimer or perhaps a higher oligomer. This oligomer has not been identified, but it has been shown to decompose to give, in part, the neurotoxin 5-hydroxytryptamine-4,7-dione.

Oxidation of 5-hydroxytryptamine and 5,7-dihydroxytryptamine. A new oxidation pathway and formation of a novel neurotoxin.

The electrochemical oxidation of 5-hydroxytryptamine (5-HT) in acidic solution proceeds through a minor route leading first to 5,7-dihydroxytryptamine (5,7-DHT) then to 4,5,7-trihydroxytryptamine and finally to 5-hydroxytryptamine-4,7-dione. The latter compound is a major electrochemical oxidation product of 5,7-DHT at pH 2 and 7 and a major autoxidation product at pH greater than or equal to 6. Preliminary biological results indicate that 5-hydroxytryptamine-4,7-dione is a more potent central nervous system toxin than 5,7-DHT. These results show for the first time a chemical pathway from 5-HT to 5,7-DHT and suggest possible minor metabolic oxidative pathways for the neurotransmitter 5-HT to at least two powerful neurotoxins.

Investigation of the enzymic and electrochemical oxidation of uric acid derivatives.

The electrochemical oxidation of a number of N-methylated uric acids at the pyrolytic graphite and gold electrodes has been compared to their enzymic oxidation with type VIII peroxidase and H2O2. Spectral, electroanalytical and kinetic evidence supports the conclusion that for all compounds the electrochemical and enzymic reactions proceed by identical mechanisms.