Iron-mediated generation of the neurotoxin 6-hydroxydopamine quinone by reaction of fatty acid hydroperoxides with dopamine: A possible contributory mechanism for neuronal degeneration in parkinson's disease

Article abstract of DOI:10.1021/jm970099t

Exposure of dopamine to an excess of linoleic acid 13-hydroperoxide (13- hydroperoxyoetadecadienoic acid) in the presence of ferrous ions in Tris buffer, pH 7.4, resulted in a relatively fast, oxygen-independent reaction exhibiting first-order kinetics wi

Full text of DOI:10.1021/jm970099t

J . Med. Chem. 1997, 40, 2211-2216
2211
Ir on -Med ia ted Gen er a tion of th e Neu r otoxin 6-Hyd r oxyd op a m in e Qu in on e by
Rea ction of F a tty Acid Hyd r op er oxid es w ith Dop a m in e: A P ossible
Con tr ibu tor y Mech a n ism for Neu r on a l Degen er a tion in P a r k in son ’s Disea se
†
†
†
‡
,†
Alessandro Pezzella, Marco d’Ischia, Alessandra Napolitano, Giovanna Misuraca, and Giuseppe Prota*
Department of Organic and Biological Chemistry, University of Naples “Federico II”, Via Mezzocannone 16, I-80134 Naples,
Italy, and Department of Experimental Pharmacology, University of Naples “Federico II”, Via D. Montesano 49, Naples, Italy
X
Received February 17, 1997
Exposure of dopamine to an excess of linoleic acid 13-hydroperoxide (13-hydroperoxyoctadeca-
dienoic acid) in the presence of ferrous ions in Tris buffer, pH 7.4, resulted in a relatively fast,
oxygen-independent reaction exhibiting first-order kinetics with respect to both catecholamine
and metal concentrations. Product analysis in the early stages revealed the presence of
significant amounts of the quinone of the neurotoxin 6-hydroxydopamine, together with some
aminochrome and ill-defined melanin-like material. Quinone formation required the presence
of iron, either in the ferrous or ferric form, and was unaffected by peroxidase, catalase, and
hydroxyl radical scavengers, e.g. mannitol, as well as biologically relevant antioxidants, like
ascorbate and glutathione. Hydrogen peroxide proved as effective as linoleic acid hydroperoxide
in inducing dopamine oxidation and conversion to 6-hydroxydopamine quinone. Metal chelators,
including EDTA and bipyridyl, markedly suppressed quinone formation without, however,
inhibiting dopamine oxidation. These and other results are consistent with a hydroxyl radical
independent hydroxylation/oxidation mechanism basically different from the Fenton reaction,
which involves direct interaction of the peroxide with a dopamine-Fe(III) chelate generated
during the process.
In tr od u ction
Parkinson’s disease and other severe mental disorders.5
A hint came, inter alia, from a report claiming the
formation of 1 in the caudate nucleus of the rat brain
following administration of a large dose of methyl-
amphetamine, which is known to destroy dopamine
A persisting condition of oxidative stress, with more
or less severe perturbation of the intracellular redox
equilibria and permanent deterioration of critical cell
structures, is definitely recognized as a core patho-
genetic factor underlying selective degeneration or
dysfunction of the dopaminergic neurons of the sub-
6
nerve terminals. Analytical difficulties and certain
pitfalls inherent in the methodologies for detection of 1
in vivo, however, hindered substantial progress in this
area. Moreover, the actual ability of 1 or its quinone
1
-3
stantia nigra in Parkinson’s disease.
Circumstantial
evidence accumulated over the past decades suggests
that, whatever the primary cause of injury, an intricate
cascade of intra- and extraneutronal reactions is set in
motion which, in the aftermath of the insult, may induce
(2) to induce destruction of dopaminergic nerve termi-
nals when generated in trace amounts was ques-
5
,7
tioned.
Nonetheless, the 6-hydroxydopamine hypo-
thesis of neurotoxicity in Parkinson’s disease was never
abandoned, being corroborated by model in vitro experi-
ments demonstrating that oxidation of dopamine under
conditions of physiological relevance may result in the
formation of sufficient amounts of 1 to justify its
implication in neurodegenerative diseases. Among the
oxidizing systems that have been reported to produce
a chronic oxidative diversion of dopamine metabolism
against a failing line of antioxidant defense.1b Although
the consequences of this metabolic derangement have
not been fully elucidated at the molecular level, credit
has been given to the possibility that aberrant oxidation
of dopamine could result in the generation of neurotoxic
species impairing basic metabolic functions and leading
8
eventually to neuron death.2c Arguments supporting
this view derived from the discovery by Tranzer and
some 1 from dopamine are the Fenton system, the
9
enzyme tyrosinase, and Mn(II) in the presence of
4
10
11
Thoenen in 1968 that an oxidized dopamine derivative,
oxygen, although the latter two have been disproved.
6
-hydroxydopamine (1) was able to cause selective
More recently, in a reexamination of this issue, it was
found that nucleophilic addition of hydrogen peroxide
to dopamine quinone produced, e.g. by peroxidase/
hydrogen peroxide oxidation, may lead to the formation
of 1 in relatively good yields. Since enhanced produc-
tion of hydrogen peroxide, derived in part from upregu-
destruction of peripheral catecholaminergic nerve end-
ings, possibly via spontaneous oxidative conversion to
yield potentially toxic quinone species. The consequent
burst of interest in the neurotoxic properties of 1
spurred intense research efforts aimed at demonstrating
endogenous generation of this neurotoxin as a potential
contributory mechanism in the etiopathogenesis of
11
lated monoamine oxidase activity, is well documented
1c,12
in Parkinson’s disease,
it seems conceivable that
under appropriate conditions a mechanism similar to
that observed in vitro becomes operative in vivo.
*
Address all correspondence to: Professor Giuseppe Prota, Depart-
ment of Organic and Biological Chemistry, University of Naples
Federico II”, Via Mezzocannone 16, I-80134 Naples, Italy. Phone:
As an extension of that study, we have surveyed the
reaction behavior of dopamine with a number of meta-
bolic products of oxidative stress that are putatively
formed and accumulated in the degenerating nigrostri-
“
+
39-81-7041249. Fax: +39-81-5521217. E-mail: prota@cds.unina.it.
†
Department of Organic and Biological Chemistry.
‡
Department of Experimental Pharmacology.
Abstract published in Advance ACS Abstracts, J une 1, 1997.
X
S0022-2623(97)00099-X CCC: $14.00 © 1997 American Chemical Society

2
212 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Pezzella et al.
F igu r e 2. Time course of dopamine oxidation (dashed lines,
left axis) and percent product yields (right axis) by oxidation
of 500 µM dopamine with 350 µM ferrous ions in the presence
and in the absence of 5 mM linoleic acid 13-hydroperoxide.
Dopamine decay: (b) with hydroperoxide; (O) without hydro-
peroxide. Formation of 2: (2) with hydroperoxide; (4) without
hydroperoxide. Aminochrome formation: (9) with hydroper-
oxide; (0) without hydroperoxide.
F igu r e 1. HPLC elution profile of the products formed by
oxidation of dopamine (500 µM) with linoleic acid 13-hydrop-
eroxide (5 mM) and ferrous ions (350 µM) in 0.1 M Tris buffer,
pH 7.4, at 37 °C, at 20 min reaction time. Trace A, reaction
mixture; trace B, the same as A, spiked with authentic 2.
Sch em e 1. Oxidation Chemistry of Dopamine Showing
the Competition between Aminochrome and
6-Hydroxydopamine Formation
atal tract. In this paper we report that, in addition to
hydrogen peroxide, hydroperoxides derived from oxida-
tion of polyunsaturated fatty acids, e.g. linoleic acid, are
also effective in inducing the conversion of the cat-
echolamine to 2 in the presence of ferrous ions.
Resu lts
Exposure of 500 µM dopamine to 5 mM linoleic acid
3-hydroperoxide (13-hydroperoxyoctadecadienoic acid)
1
in 0.1 M Tris buffer, pH 7.4 at 37 °C, resulted in little
or no detectable conversion of the catecholamine over
periods of time of up to 1 h. However, when ferrous
ions at 350 µM concentration were added to the incuba-
tion mixture, a relatively fast reaction occurred, indi-
cated by the rapid disappearance of the substrate and
the development of a reddish coloration denoting ami-
nochrome formation. A similar effect was also observed
with other lipid hydroperoxides, like those produced by
enzymatic oxidation of arachidonic acid. HPLC analysis
of the reaction mixture after about 20 min (Figure 1)
showed expectedly the presence of dopamine ami-
nochrome as well as of another significant component
of the oxidation mixture whose chromatographic and
spectrophotometric properties (λmax ) 495 nm) proved
min, when most of the dopamine was consumed. No
substantial change in the reaction kinetics and course
was observed under an oxygen-free atmosphere or using
Fe(III) ions in place of Fe(II). In the absence of
hydroperoxide, dopamine concentration decreased at a
much slower rate and reached a plateau at about 15%
substrate consumption after 60 min, at which time only
trace amounts of 2 could be detected.
Dopamine oxidation followed first-order kinetics for
at least 50% of the hydroperoxide/Fe(II)-promoted reac-
tion (Figure 3). The reaction showed also first-order
dependence with respect to the metal concentration, as
apparent from a logarithmic plot of the initial rates of
dopamine consumption vs Fe(II) concentration, which
gave a straight line with a slope of 1.05 (Figure 4).
No reliable information could be obtained about the
kinetic dependence of the reaction course on hydroper-
oxide concentration because of difficulties in obtaining
reproducible data under pseudo-first-order conditions.
Figure 5 shows the effect of Fe(II) concentration on
the relative yield of formation of 2 vs aminochrome in
the early phases of the hydroperoxide-promoted reac-
tion.
identical to those of 2-hydroxy-5-(2-aminoethyl)-1,4-
benzoquinone (2), the quinone of 111 (Scheme 1).
This structural assignment was confirmed by elec-
trospray-mass spectrometric (ES/MS) analysis of the
product isolated by ion-exchange chromatography and/
or preparative HPLC, exhibiting a detectable pseudo-
molecular peak at m/z 167, as well as by NaBH4
reduction, leading to a compound indistinguishable from
authentic 1. Attempts to identify other significant
products of dopamine oxidation met with failure under
a variety of conditions, nor was there evidence for other
isomeric hydroxydopamines, e.g. 5-hydroxydopamine.
Apparently, the mass balance was accounted for by
chromatographically ill-defined, melanin-like materials.
Figure 2 illustrates the time course of dopamine oxida-
tion and product formation from 500 µM dopamine and
3
50 µM ferrous ions in the presence and in the absence
of linoleic acid 13-hydroperoxide, as a control. In the
former case, substrate consumption was fast and re-
sulted in the pronounced formation of 2, peaking at 5
min and decreasing below detection limits after ca. 60
Notably, a marked increase in the yield of 2 was
observed with increasing iron concentration, the ratio

Generation of the Neurotoxin 6-Hydroxydopamine Quinone
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2213
F igu r e 3. Plot of ln dopamine concentration against time, r2
)
0.89. Slope ) 1.1 ( 0.1. Reaction conditions are as in
Figure 1.
F igu r e 6. (a) Plot of 2 (2) and aminochrome (9) concentration
left axis) and initial rate of dopamine oxidation (dashed line,
(
right axis) vs EDTA concentration. (b) Plot of 2/aminochrome
F igu r e 4. Logarithmic plot of initial rates of dopamine
ratio vs EDTA concentration. Dopamine oxidation was carried
-1
2+
2
2+
oxidation (µM s ) vs Fe concentration, r ) 0.98. Dopamine
was oxidized as detailed in Figure 1. Initial rates of dopamine
oxidation were determined after 1 min. Slope ) 1.05 ( 0.05.
out in the presence of linoleic acid 13-hydroperoxide and Fe
as detailed in Figure 1.
dopamine without forming detectable 2. No 2 was also
formed when dopamine was oxidized with tyrosinase or
periodate in the presence of hydroperoxide, but without
added metal.
Of the various antioxidants and radical scavengers
tested, mannitol and catalase were virtually inactive on
the hydroperoxide/Fe(II) system, whereas ascorbic acid
and glutathione, which are present in high concentra-
3
c
tions in brain tissues, markedly inhibited accumula-
tion of the aminochrome without, however, affecting
formation of 2 and only slightly affecting dopamine
consumption. No detectable reaction of glutathione with
F igu r e 5. Plots of 2 (2) and aminochrome (9) concentration
2
could be observed in the relevant oxidation mixture.
(
left axis) and 2/aminochrome ratio (dashed line, right axis)
2
+
against Fe concentration.
Particularly worthy of note was the effect of metal
chelators, viz. bipyridyl and EDTA. While bipyridyl,
unlike EDTA, dramatically accelerated the rate of
dopamine oxidation by the hydroperoxide/Fe(II) system,
both chelators virtually suppressed formation of 2.
In further experiments the effect of EDTA on dopam-
ine oxidation with 5 mM hydroperoxide and 350 µM
ferrous ions in Tris buffer was investigated in detail. A
plot of the initial rates of dopamine decay against EDTA
concentration (Figure 6a) showed a slight increase in
the oxidation rate with increasing chelator concentra-
tions. Analysis of the 2/aminochrome product ratio as
a function of EDTA concentration (Figure 6b) provided
confirmatory evidence for a concentration-dependent
ability of the metal chelator to suppress formation of 2.
Spectrophotometric investigation of the oxidation
mixtures revealed in the early stages the presence of a
well detectable blue chromophore with an absorption
maximum at 575 nm, due probably to a dopamine-Fe-
becoming greater than 1 when Fe(II) concentration was
about 0.7 times that of dopamine.
Table 1 shows the effects of various experimental
parameters and additives on the extent of dopamine
oxidation and product formation determined after 1 min.
Use of phosphate as a buffer caused a marked
decrease in the rate of the hydroperoxide/Fe(II)-promot-
ed reaction, compared to Tris and HEPES, with con-
comitant alteration of the product distribution toward
the aminochrome. Positively or negatively charged
surfactants like mirystyltrimethylammonium bromide
and sodium dodecyl sulfate (SDS) exerted little or no
influence on hydroperoxide/Fe(II)-promoted dopamine
oxidation.
Hydrogen peroxide proved as effective as linoleic acid
1
3-hydroperoxide in inducing dopamine oxidation and
formation of 2 in the presence of Fe(II) ions. In the
absence of Fe(II), peroxidase was unable to utilize
linoleic acid 13-hydroperoxide as a cosubstrate to oxidize
dopamine, whereas in the presence of the metal it did
not affect the reaction course. Sodium periodate, in the
presence of Fe(II), induced the rapid oxidation of
1
3
(III) chelate.
This was confirmed by a comparison
with the absorption features of dopamine solutions
containing Fe(III) salts, as well as by the lack of
significant shift of the dopamine absorption maximum
in the presence of Fe(II) under rigorously an oxygen-

2
214 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Pezzella et al.
Ta ble 1. Effect of Experimental Parameters and Additives on the Rate of Dopamine Oxidation and Product Yieldsa
buffer
(0.1 M)
initial rate
yield
aminochrome
yield (%)
-
1
b
of 2 (%)c
c
peroxide
additive (concn)
(µM s
)
none
Tris
Tris
Tris
HEPES
phosphate
Tris
nihil
f
nihil
nihil
nihil
nihil
1
1
1
1
3-HPODEd
3-HPODE
3-HPODE
3-HPODE
0.1
1.8
1.9
3.9
2.7
1.5
2.0
2.0
2.5
4.3
1.5
0.7
10
3.3
2.9
1.2
3.0
3.2
3.2
3.1
3.0
1.7
3.7
0.7
7.5
7.5
3.7
7.5
3.0
2.3
6.8
7.0
1.8
7.2
7.0
6.8
6.6
6.9
1.6
7.0
0.6
0.1
0.1
4.1
1.9
6.9
5.2
H2O2
1
1
1
1
1
1
1
3-HPODE
3-HPODE
3-HPODE
3-HPODE
3-HPODE
3-HPODE
3-HPODE
Tris
Tris
Tris
Tris
Tris
Tris
Tris
Tris
Tris
Tris
Tris
Tris
SDS (0.2 M)
MTMAB (0.2 M)
mannitol (0.2 M)
e
catalase (150 units/mL)
tyrosinase (30 units/mL)f
peroxidase (5 units/mL)
peroxidase (5 units/mL)f
NaIO4 (0.45 mM)
none
1
1
1
1
1
3-HPODE
NaIO4 (0.45 mM)f
EDTA (0.5 mM)
bipyridyl (0.9 mM)
glutathione (0.1 mM)
ascorbate (0.1 mM)
12
15
17
0.1
0.1
3-HPODE
3-HPODE
3-HPODE
3-HPODE
Tris
a
Reaction conditions as in Figure 1, unless otherwise stated. Determined at 1 min reaction time. SD e 10%. c Average of three
b
determinations, SD e 10%. Linoleic acid 13-hydroperoxide. e Myristyltrimethylammonium bromide. f No iron added.
d
of linoleic acid 13-hydroperoxide, but in the absence of
iron, i.e. under conditions where dopamine quinone is
definitely formed, failed to afford any detectable 2.
Against a significant involvement of dopamine quinone
is also the lack of inhibitory effects on the formation of
2
of glutathione and ascorbic acid, which are known to
deplete transiently generated o-quinones via either
1
6
17
nucleophilic addition or reduction mechanisms.
In fact, the observed inhibitory effect of metal chela-
tors clearly indicates that formation of 2 strictly depends
on the generation of a dopamine-Fe(III) complex and,
by implication, that the latter is the species actually
suffering hydroxylation/oxidation. In this frame, the
competition between the 6-hydroxydopamine and the
aminochrome routes would be governed by the parti-
tioning of iron between the catecholamine chelate and
other possible complexes within the reaction milieu.
Another issue concerns the mechanism of hydroxy-
lation/oxidation of the dopamine-Fe(III) complex. The
process evidently does not depend on oxygen, requires
a hydroperoxide as co-substrate, and does not involve
hydroxyl radicals as the attacking species, thus differing
significantly from Fenton-type reactions. In accord with
this interpretation are both the lack of effect of man-
nitol, a known OH radical scavenger and, by contrast,
the inhibitory effect of EDTA, a commonly used iron
chelator for Fenton reactions. Furthermore, the gen-
eration of hydroxyl radicals, which typically follows one-
electron reduction of hydrogen peroxide by ferrous ions,
is incompatible with the basic mode of decomposition
of alkyl hydroperoxides, leading mainly to alkoxyl
F igu r e 7. Plot of the absorbance at 575 nm of the dopamine-
Fe(III) chelate vs EDTA concentration in 0.1 M Tris buffer,
3
+
pH 7.4. Dopamine and Fe concentration were 500 and 350
µM.
free atmosphere. Addition of increasing concentrations
of EDTA to the dopamine-Fe(III) system caused a
proportional decrease of the blue chromophore, due
evidently to decomposition of the dopamine-Fe(III)
chelate (Figure 7).
Discu ssion
Abnormal iron accumulation3,14 and an up to 10-fold
1
5
increase in the levels of lipid hydroperoxides, along
1
with elevated production of hydrogen peroxide, figure
prominently among those pathogenetic processes which
are specifically associated with an oxidative stress
condition in the parkinsonian substantia nigra. While
these changes are indicative of toxic processes involving
crucial membrane lipid components, whether and by
what mechanism(s) they may directly affect the dopam-
ine pool in degenerating nitrostriatal neurons remains
an open issue. A novel hint is offered by the results of
the present study, which provide a chemical basis to
envisage a substantial conversion of dopamine to the
proximate oxidation product of the neurotoxin 6-hy-
droxydopamine in hydroperoxide-rich environments,
provided that iron is present in the reaction milieu.
From the chemical viewpoint, the reaction described
in this paper presents a number of peculiar features
which may be worthy of comment. Firstly, formation
of 2 does not seem to proceed via nucleophilic addition
of the hydroperoxide group onto dopamine quinone,
since chemical oxidation of dopamine in the presence
1
8
radicals and hydroxyl anions.
On this basis, a mechanism could be envisaged in
which iron chelation confers to dopamine a marked
susceptibility to attack at the 6-position of the catechol
ring by the hydroperoxide (Scheme 2). Although this
reaction seemed to be regiospecific, because of the
failure to detect other isomeric hydroxydopamines, the
poor mass balance and the different stability of isomeric
hydroxydopamines did not allow this point to be defi-
nitely assessed.
An interesting implication of our results relates to the
observed inability of glutathione and ascorbic acid to
prevent the iron/peroxide-dependent generation of 2. On

Generation of the Neurotoxin 6-Hydroxydopamine Quinone
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2215
Sch em e 2. Schematic Outline of the Proposed Mechanism of Conversion of Dopamine to 2, Highlighting Formation of
the Iron-Dopamine Chelate as the Critical Step
a chemical basis, this suggests the possible occurrence
within catecholaminergic neurons of neurotoxic pro-
cesses which defy counteraction by the primary line of
antioxidant defense in the brain. These and other
related processes, revolving around the oxidative inter-
action of dopamine with iron, may overall contribute
to progressive impairment of crucial cellular structures
and functions and, eventually, hasten the final demise
of the neuron.
37 ( 0.1 °C with circulating water. HPLC analyses were
performed with a Gilson instrument equipped with model 305
pumps and a model 317 UV detector. ES/MS spectra were
determined with a VG BIO-Q triple quadrupole mass spec-
trometer.
Ma ter ia ls. Dopamine hydrochloride, sodium periodate,
ferrous sulfate heptahydrate, ethylenediaminetetraacetic acid
disodium salt (EDTA), myristyltrimethylammonium bromide,
bipyridyl, and nonstabilized hydrogen peroxide (35% solution
in water) were from Aldrich Chemie (Steinheim, Germany).
1
9
6
-Hydroxydopamine hydrobromide, 5-hydroxydopamine hy-
Apart from the possible involvement in neuronal
degeneration, it is tempting to speculate here that the
observed peroxide/iron-induced catecholamine oxidation
may play a role in the biosynthesis of neuromelanin,
the characteristic age pigment responsible for the dark
coloration of the neuron perykaria in human substantia
drobromide, linoleic acid, arachidonic acid, ascorbic acid,
reduced glutathione, soybean lipoxygenase (linoleate:oxygen
oxidoreductase, EC 1.13.11.12, 110 000 units/mg) type IB,
mushroom tyrosinase (o-diphenol:O
1.14.18.1, 6300 units/mg), horseradish peroxidase (donor H
oxidoreductase, EC 1.11.1.7, 200 purpurogallin units/mg, E430
275 ) 2.0) type II, catalase from bovine liver (H :H
2
oxidoreductase, EC
2
O
2
/
2
0,21
E
2
O
2
2 2
O
nigra.
In nigral neurons, a mechanism may be
oxidoreductase, EC 1.11.1.6, 9740 units/mg) were purchased
from Sigma Chemicals (St. Louis, MO). Dopamine ami-
nochrome was prepared by aerial oxidation of 5,6-dihydroxy-
indoline hydrobromide11 in sodium bicarbonate buffer, pH 8.0.
6-Hydroxydopamine quinone (2) was prepared by sodium
periodate oxidation of 6-hydroxydopamine as reported previ-
ously.11
envisioned whereby basal generation of hydrogen per-
oxide and lipid peroxides in the presence of iron evokes
a slow, yet constant oxidation of a minor part of the
dopamine pool leading to the gradual intraneuronal
deposition of melanin pigments.
Con clu sion s
An a lytica l Con d ition s. HPLC was performed using a
Spherisorb S5 ODS 2 column (1 mL/min) and a 10 × 250 mm
Alltech Econosil C18 column (6 mL/min) for analytical and
preparative runs, respectively. Detection was carried out at
The present study throws light on a hitherto over-
looked hydroxyl radical independent mechanism of
conversion of dopamine to the proximate oxidation
product of the neurotoxin 6-hydroxydopamine. Al-
though basically different from the hydroxylations
induced by the peroxidase/hydrogen peroxide system
and by the Fenton reaction, the proposed reaction
process and the previous ones are not mutually exclu-
sive and may all contribute to the catecholamine
degenerative chemistry associated with dopaminergic
neuron loss.
Of course, it is too early to propose any causal
relationships between the observed chemistry and the
process of neuronal degeneration. Considering that iron
and peroxide concentrations exceed basal levels and
increase dramatically at advanced stages of substantia
nigra degeneration in Parkinson’s disease, it is conceiv-
able that formation of 2 becomes significant only after
the primary toxic processes are underway, perhaps
contributing to amplify those degenerative events that
lead eventually to dopaminergic neuron death.
2
80 nm. Mobile phase: 0.05 M phosphate buffer, pH 3.0,
containing 10 mM sodium 1-octanesulfonate-acetonitrile, 93:
7.
Syn th esis of Lin oleic Acid 13-Hyd r op er oxid e. Linoleic
acid 13-hydroperoxide was synthesized enzymatically by oxi-
dation of linoleic acid with soybean lipoxygenase. To a solution
of linoleic acid (1 mM) in 0.1 M borate buffer, pH 9.0, was
added lipoxygenase (2000 units/mL) under vigorous stirring.
The reaction was monitored spectrophotometrically at 234 nm
(conjugated diene formation), and when the absorbance pla-
teaued, it was stopped by acidification to pH 4.0 with 3 M HCl.
The mixture was extracted three times with chloroform, and
the organic layers were washed with saturated aqueous NaCl,
dried over anhydrous sodium sulfate, and evaporated to
dryness. The resultant hydroperoxide, obtained as a colorless
oil, was dissolved in methanol and stored at -20 °C. Hydro-
peroxide concentration was checked prior to each experiment
-
1
-1
using a molar extinction coefficient of 27 000 M cm at 234
nm.22
Arachidonic acid 15-hydroperoxide was prepared by a
similar procedure.
Lin oleic Acid 13-Hyd r op er oxid e-In d u ced Oxid a tion of
Dop a m in e. Kin etic Exp er im en ts. A solution of dopamine
(0.5 mM) in 0.1 M Tris buffer (unless otherwise stated), pH
7.4, was treated with appropriate volumes of a freshly pre-
pared stock solution of ferrous sulfate in water, up to the
desired concentration, and of linoleic acid 13-hydroperoxide
If supported in further studies, the mechanism de-
scribed in the present paper may contribute to create
an improved background in which to fit many of the still
unconnected observations on the biochemical pathology
of Parkinson’s disease and may yield new clues for the
development of innovative neuroprotective interventions
based on the control of peroxide and iron metabolism.
(5 mM final concentration). When required, solutions of ferric
salts or hydrogen peroxide were used in place of ferrous salts
and linoleic acid 13-hydroperoxide, respectively. Additives, e.g.
enzymes, surfactants, metal chelators, and antioxidants, were
added to the desired concentration, as indicated in the Results
section as well as in the legends to figures. The mixture was
incubated under stirring at 37 °C in a thermostated water
Exp er im en ta l Section
UV spectra were recorded on a Perkin-Elmer Lambda 7
spectrophotometer having the cell compartment controlled at

2
216 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Pezzella et al.
bath. Aliquots of the reaction mixture were periodically
withdrawn, acidified to pH 1.0 with 3 M HCl, and centrifuged
at 7000 rpm for about 15 min, and the aqueous layer was
injected into the HPLC. Identification and quantification of
dopamine, aminochrome, and 2 were carried out by comparing
retention times and integrated peak areas with external
calibration curves for authentic samples. Product identifica-
tion was also secured by coinjection of an aliquot of the reaction
mixture with the appropriate volume of a stock solution of the
relevant compound. All experiments were run at least in
triplicate. Statistical parameters were determined by linear
least-squares fitting.
(4) Tranzer, J . P.; Thoenen, H. An Electron Microscopic Study of
Selective Acute Degeneration of Sympathetic Nerve Terminals
After Administration of 6-Hydroxydopamine. Experientia 1968,
2
4, 155-156.
(
5) Langston, J . W.; Irwin, I.; Ricaurte, G. A. Neurotoxins, Parkin-
sonism and Parkinson’s Disease. Pharmacol. Ther. 1987, 32,
19-49 and references therein.
(6) Seiden, L. S.; Vosmer, G. Formation of 6-Hydroxydopamine in
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7) Evans, J . M.; Cohen, G. Can Trace Amounts of Neurotoxins
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1
29.
Isola tion a n d Ch a r a cter iza tion of 2 by F er r ou s Ion
P r om oted Oxid a tion of Dop a m in e w ith Lin oleic Acid 13-
Hyd r op er oxid e. Dopamine (10 mg, 0.5 mM) was oxidized
with linoleic acid 13-hydroperoxide (5 mM) and ferrous sulfate
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(
(0.35 mM) in Tris buffer, pH 7.4, as described above. After 1
h, or when most of the dopamine was consumed, the reaction
mixture was chromatographed on a column (15 × 1 cm) of
Dowex 50W X4 (100-200 mesh). After being washed with 0.01
M HCl (50 mL), the column was eluted with 0.1 M and 0.5 M
HCl (50 mL each, 2-mL fractions). The fractions eluted with
(
(
(
10) Garner, C. D.; Nachtman, J . P. Manganese Catalysed Auto-
Oxidation of Dopamine to 6-Hydroxydopamine in Vitro. Chem.-
Biol. Interact. 1989, 69, 345-351.
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of the Neurotoxin 6-Hydroxydopamine by Peroxidase/H
2 2
O . J .
Med. Chem. 1995, 38, 917-922.
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(13) Gerard, C.; Chehhal, H.; Hugel, R. P. Complexes of Iron (III)
with Ligands of Biological Interest: Dopamine and 8-Hydro-
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14) Ben-Shachar, D.; Riederer, P.; Youdim, M. B. H. Iron-melanin
Interaction and Lipid Peroxidation: Implications for Parkinson’s
Disease. J . Neurochem. 1991, 57, 1609-1614.
15) (a) Dexter, D. T.; Carter, C.; Agid, G.; Agid, Y.; Lees, A. J .;
J enner, P.; Marsden, C. D. Lipid Peroxidation as a Cause of
Nigral Cell Death in Parkinson’s Disease. Lancet 1986, No. 2,
0
.5 M HCl contained 2 as apparent from spectrophotometric
analysis and comparison of the HPLC elutographic properties
of the main product with those of authentic 2. The appropriate
fractions were concentrated at room temperature with a rotary
evaporator, care being taken to avoid evaporation to dryness,
and analyzed within 24 h by ES/MS.
An aliquot of the concentrated fractions containing 2 was
neutralized with crystals of sodium phosphate, treated with
an excess of sodium borohydride, and then analyzed by HPLC.
Formation of 1 was confirmed by coinjection with an authentic
sample.
(
(
6
39-640. (b) Dexter, D. T.; Wells, F. R.; Agid, G.; Agid, Y.; Lees,
A.; J enner, P.; Marsden, C. D. Increased Nigral Iron Content in
Ack n ow led gm en t. This work was supported by
grants from Ministero dell’Universit a` e della Ricerca
Scientifica e Tecnologica (Rome) and Consiglio Nazio-
nale delle Ricerche (CNR, Rome). We thank Miss
Silvana Corsani for technical assistance.
Postmortem Parkinsonian Brain. Lancet 1987, No. 2, 1219-
1
220. (c) Dexter, D. T.; Carter, C. J .; Wells, F. R.; J avoy-Agid,
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Disease. J . Neurochem. 1989, 52, 382-389. (d) Dexter, D. T.;
Wells, F. R.; Lees, A. J .; Agid, F.; Agid, Y.; J enner, P.; Marsden,
C. D. Increased Nigral Iron Content and Alterations in Other
Metal Ions Occurring in Brain in Parkinson’s Disease. J .
Neurochem. 1989, 52, 1830-1836.
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