Serotonin as a physiological substrate for myeloperoxidase and its superoxide-dependent oxidation to cytotoxic tryptamine-4,5-dione

Article abstract of DOI:10.1042/BJ20090776

During inflammatory events, neutrophils and platelets interact to release a variety of mediators. Neutrophils generate superoxide and hydrogen peroxide, and also discharge the haem enzyme myeloperoxidase. Among numerous other mediators, platelets liberate serotonin (5-hydroxytryptamine), which is a classical neurotransmitter and vasoactive amine that has significant effects on inflammation and immunity. In the present study, we show that serotonin is a favoured substrate for myeloperoxidase because other physiological substrates for this enzyme, including chloride, did not affect its rate of oxidation. At low micromolar concentrations, serotonin enhanced hypochlorous acid production by both purified myeloperoxidase and neutrophils. At higher concentrations, it almost completely blocked the formation of hypochlorous acid. Serotonin was oxidized to a dimer by myeloperoxidase and hydrogen peroxide. It was also converted into tryptamine-4,5-dione, especially in the presence of superoxide. This toxic quinone was produced by stimulated neutrophils in a reaction that required myeloperoxidase. In plasma, stimulated human neutrophils oxidized serotonin to its dimer using the NADPH oxidase and myeloperoxidase. We propose that myeloperoxidase will oxidize serotonin at sites of inflammation. In doing so, it will impair its physiological functions and generate a toxicmetabolite that will exacerbate inflammatory tissue damage. Consequently, oxidation of serotonin by myeloperoxidase may profoundly influence inflammatory processes. The Authors Journal compilation

Full text of DOI:10.1042/BJ20090776

Biochem. J. (2010) 425, 285–293 (Printed in Great Britain) doi:10.1042/BJ20090776
285
Serotonin as a physiological substrate for myeloperoxidase and its
superoxide-dependent oxidation to cytotoxic tryptamine-4,5-dione
Valdecir F. XIMENES*, Ghassan J. MAGHZAL†, Rufus TURNER‡, Yoji KATO§, Christine C. WINTERBOURN‡ and
1
Anthony J. KETTLE‡
*Departamento de Qu´ımica, Faculdade de Cieˆncias, Universidade Estadual Paulista Ju´lio de Mesquita Filho, Bauru, SP, Brazil, †Centre for Vascular Research, School of Medical
Sciences (Pathology) and Bosch Institute, Faculty of Medicine, University of Sydney, Medical Foundation Building K25, 92-94 Parramatta Road, Camperdown, NSW 2006, Australia,
‡Free Radical Research, Department of Pathology, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand, and §School of Human Science and Environment,
University of Hyogo, Himeji 670-0092, Japan
During inflammatory events, neutrophils and platelets interact to
release a variety of mediators. Neutrophils generate superoxide
and hydrogen peroxide, and also discharge the haem enzyme
myeloperoxidase. Among numerous other mediators, platelets
liberate serotonin (5-hydroxytryptamine), which is a classical
neurotransmitter and vasoactive amine that has significant effects
on inflammation and immunity. In the present study, we show
that serotonin is a favoured substrate for myeloperoxidase
because other physiological substrates for this enzyme, including
chloride, did not affect its rate of oxidation. At low micromolar
concentrations, serotonin enhanced hypochlorous acid production
by both purified myeloperoxidase and neutrophils. At higher con-
centrations, it almost completely blocked the formation of hypo-
chlorous acid. Serotonin was oxidized to a dimer by myeloper-
oxidase and hydrogen peroxide. It was also converted into tryp-
tamine-4,5-dione, especially in the presence of superoxide. This
toxic quinone was produced by stimulated neutrophils in a reac-
tion that required myeloperoxidase. In plasma, stimulated human
neutrophils oxidized serotonin to its dimer using the NADPH
oxidase and myeloperoxidase. We propose that myeloper-
oxidase will oxidize serotonin at sites of inflammation. In doing
so, it will impair its physiological functions and generate a
toxic metabolite that will exacerbate inflammatory tissue damage.
Consequently, oxidation of serotonin by myeloperoxidase may
profoundly influence inflammatory processes.
Key words: myeloperoxidase, neutrophil, platelet, serotonin
(5-hydroxytryptamine), superoxide, tryptamine-4,5-dione.
serotonin can result in cardiovascular dysfunction [12]. Serotonin
will be available to myeloperoxidase at inflammatory sites where
platelets and neutrophils are activated [13]. This will occur
during bacterial infections, thrombosis and inflammatory events
within the respiratory system. Serotonin may also be oxidized by
myeloperoxidase in the brain, where the enzyme has been shown
to be associated with microglia and neurons [14,15].
Currently, it is not known whether serotonin competes with
other substrates of myeloperoxidase at physiologically relevant
concentrations or what products are formed when it is oxidized.
Early work by Wrona and Dryhurst [16] showed that horseradish
peroxidase could oxidize serotonin to a complex mixture of
products, which included the neurotoxin tryptamine-4,5-dione.
Furthermore, stimulated neutrophils convert serotonin into a
product that becomes bound to proteins in a reaction that requires
myeloperoxidase and hydrogen peroxide [17].
INTRODUCTION
Myeloperoxidase is a haem enzyme of neutrophils that contributes
to host defence and is also implicated in the pathogenesis of
numerous inflammatory diseases [1]. It uses hydrogen peroxide
to oxidize an array of substrates to either hypohalous acids or free
radicals [2,3]. The main reducing substrate for myeloperoxidase
is generally accepted to be chloride, which is oxidized to
hypochlorous acid. This is one of the most reactive oxidants
produced in vivo. It is produced during inflammation and has
been shown to react with proteins in atherosclerotic plaques [4],
and those in the airways of individuals with respiratory diseases
[5,6]. Under physiological conditions, thiocyanate is oxidized
as readily as chloride and is converted into the weak oxidant
hypothiocyanite [7,8]. Other substrates include bromide, tyrosine,
ascorbate, nitrite, nitric oxide and a plethora of xenobiotics
[2,3]. It is becoming increasingly apparent that production of
oxidants from these substrates contributes to the development
and progression of cardiovascular disease [9].
Serotonin (5-hydroxytryptamine) has the potential to rival
chloride and thiocyanate as a physiological substrate for
myeloperoxidase. It undergoes one-electron oxidation by the
redox intermediates of myeloperoxidase [10,11]. The rate
constants for these reactions are the highest measured for
any reducing substrate of myeloperoxidase, [11]. Oxidation
of serotonin by myeloperoxidase is physiologically important
because this neurohormonal factor is a key neurotransmitter and
has been implicated in cardiovascular function [12]. It regulates
vascular tone and stimulates cardiomyocytes and chemosensitive
nerves. Perturbation of the normal physiological concentration of
In the present study, we have investigated how readily serotonin
is oxidized by myeloperoxidase in the presence of alternative
physiological substrates. We show that serotonin is a preferred
substrate for myeloperoxidase that inhibits hypochlorous acid
production. It is converted into a dimer as well as into tryptamine-
4,5-dione.
EXPERIMENTAL
Reagents
Myeloperoxidase was purchased from Planta. Its concentration
was determined using a ε430 of 89000 M−1 · cm−1 per haem. Sero-
tonin (5-hydroxytryptamine hydrochloride), SOD (superoxide
Abbreviations used: DPI, diphenyliodonium; DTPA, diethylenetriaminepenta-acetic acid; ESI, electrospray ionization; LC, liquid chromatography; MRM,
multiple reaction monitoring; SOD, superoxide dismutase; TMB, 3,3ꢀ,5,5ꢀ-tetramethylbenzidine.
1
To whom correspondence should be addressed (email tony.kettle@otago.ac.nz).
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dismutase), catalase, creatine phosphokinase (rabbit muscle)
xanthine oxidase, PMA, DL-methionine, taurine, TMB (3,3ꢀ,5,5ꢀ-
tetramethylbenzidine), acetaldehyde, DTPA (diethylenetriamin-
epenta-acetic acid) and cytochrome c were purchased from Sigma
Chemical Co. DMSO was purchased from Merck. All of the
reagents used for buffers were of analytical grade. Blood was
obtained from healthy human volunteers with informed consent,
and with ethical approval from the Canterbury Southern Ethics
Committee.
Hydrogen peroxide solutions were prepared by diluting a 30%
stock solution (Merck) and calculating its concentration using a
ε240 of 43.6 M−1 · cm−1 [18]. Hypochlorous acid solutions were
prepared by diluting a concentrated commercial bleach solution
and calculating its concentration using a ε292 of 350 M−1 · cm−1 at
pH 12 [19]. Stock solutions of serotonin were prepared daily in
water. Tryptamine-4,5-dione was synthesized using the method
of Jiang and Dryhurst [20]. Briefly, serotonin was allowed to
react with a five molar excess of potassium nitrosodisulfonate in
water at room temperature (21◦C) for 25 min to form a dark purple
solution. The solution (500 μl) was then added to a C18 solid-phase
extraction cartridge (500 mg column; Alltech) preconditioned
with 3 ml of methanol followed by 2 ml of water. The cartridge
was then washed with water to remove salts including excess
potassium disulfonate. The purified purple product (tryptamine-
4,5-dione) was eluted with 0.1% formic acid/75% methanol and
concentrated by vacuum centrifugation. Its identity and purity
were check by infusion MS.
and hypotonic lysis of red blood cells [23]. After isolation,
neutrophils were resuspended in 10 mM phosphate buffer, pH
7.4 containing 140 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2 and
1 mg/ml glucose.
Effect of serotonin on oxidant production by stimulated neutrophils
Neutrophils (2 × 106 cells/ml) were pre-incubated at 37◦C in
PBS with 5 mM taurine and serotonin for 10 min. The reactions
were triggered by addition of 100 ng/ml PMA. Reactions were
stopped after 30 min by the addition of catalase (20 μg/ml).
Neutrophils were pelleted by centrifugation at 2000 g for 5 min
and accumulated taurine chloramine was measured in the
supernatants. Superoxide production was measured as SOD-
inhibitable reduction of ferricytochrome c [24]. Neutrophils
(2 × 106 cells/ml) were pre-incubated for 10 min at 37◦C in PBS
with catalase (20 μg/ml) and ferricytochrome c (100 μM). The
reaction was triggered by the addition of PMA (100 ng/ml) and
the rate of change in absorbance at 550 nm over the first 1 min
was measured (ε550 = 21000 M−1 · cm−1) [24].
Oxidation of serotonin by stimulated neutrophils in plasma
Neutrophils (2 × 107 cells/ml) were pre-incubated at 37◦C in
PBS and then added to three times the volume of whole plasma.
Serotonin (20 μM) was added to this suspension of cells which
was then stimulated with a combination of PMA (1 μg/ml) and
cytochalasin B (10 μg/ml). Aliquots (100 μl) were immediately
withdrawn and added to 1.5 ml microfuge tubes containing
nothing, 10 μM DPI (diphenyliodonium), 1 mM sodium azide,
catalase (100 μg/ml), SOD (20 μg/ml) or 1 mM thiocyanate.
These cells were maintained at 37◦C with repeated mixing for
30 min, then pelleted by centrifugation at 2000 g for 5 min.
The proteins in the resulting supernatants were precipitated by
adding four times the volume of ice-cold acetonitrile containing
the stable isotope of the serotonin dimer. Finally, these samples
were centrifuged to remove protein and assayed by LC (liquid
chromatography)–MS for serotonin and its dimer. To follow the
time course for the oxidation of serotonin, aliquots from the
suspension of stimulated neutrophils were removed at set time
points, and cells and proteins were precipitated as described
above.
The stable isotope of [α,α,β,β-2H4]serotonin was purchased
from Medical Isotopes. It was used to prepare the stable isotope
of the serotonin dimer by dissolving it at 5 mM in 0.01 M
phosphate buffer (pH 7.4) along with horseradish peroxidase
(5 μg/ml). Oxidation to its dimer was promoted by three additions
of hydrogen peroxide (0.5 mM). The reaction was terminated by
adding catalase (5 μg/ml). The solution was ultrafiltered using
a Vivaspin column (7500 g at 15 min) at 4◦C. The filtrate was
freeze-dried and then dissolved in an aliquot (50 μl) of water.
The sample was separated by HPLC using a Combi-RP column
(20 mm × 100 mm) with 5 mM ammonium acetate containing
1% acetic acid (solvent A). The major peak was collected, and the
column was then washed with solvent A/acetonitrile (1:1). The
fraction was freeze-dried and stored below −20◦C. The dimer
concentration was estimated by measurement of absorbance at
301 nm (ε = 8128 M−1 · cm−1) [21].
Detection of serotonin oxidation products and conjugates by HPLC
and LC–MS
Oxidation of serotonin by myeloperoxidase
Reaction products of serotonin were separated by HPLC using a
Waters 2690 Separation Module coupled to a diode array de-
tector (Waters 996) and GBC LC1250 fluorescence detector.
A Luna C18 reversed-phase column (25 mm × 4.6 mm, 5 μm
particle size) (Phenomenex) was used to separate serotonin from
its oxidation products using gradient elution. The mobile phase
consisted of eluant A (1% acetic acid and 1 mM ammonium
acetate in water), and eluant B (100% acetonitrile). The gradients
applied were 0–20 min, 100–85% eluant A; 20–25 min, 85–
50% eluant A; 25–30 min, 50% eluant A. The flow rate
was 0.8 ml/min. Serotonin oxidation products and glutathione
conjugates were also separated on a Luna C18 (2) column
(150 mm × 2.0 mm, 5 μm particle size) (Phenomenex) using
a Surveyor HPLC pump (Thermo Corp.). The column was
maintained at 30◦C, and products (20 μl injections) were eluted
at a flow rate of 0.2 ml/min using a linear gradient of two solvents:
solvent A (5 mM ammonium acetate/1% acetic acid) and solvent
B (100% acetonitrile). The gradient was as follows: 0–5 min, 0%
solvent B; 5–25 min, solvent B increased to 15%; 25–30 min,
solvent B increased to 50%; 30–35 min, solvent B maintained at
The oxidation of serotonin was followed by monitoring its
absorption spectrum between 200 and 600 nm using a Beckman
DU 7500 diode array spectrophotometer. Recordings were an
average of ten readings taken over 1 s. Initial rates of oxidation
were determined by calculating the increase in absorbance at
317 nm over the initial linear phase of the reaction. The extent of
oxidation was also determined by measuring the loss of serotonin
fluorescence (λex, 280 nm; λem, 340 nm) using a Hitachi F4500
fluorescence spectrophotometer.
Detection of hypochlorous acid
Hypochlorous acid produced by either purified myeloperoxidase
or stimulated neutrophils was trapped with 5 mM taurine and
converted into taurine chloramine. This stable oxidant was
detected by the iodide-catalysed oxidation of TMB [22].
Isolation of human neutrophils
Neutrophils were isolated from the blood by Ficoll–Hypaque
centrifugation at 2000 g for 10 min, dextran sedimentation
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50%; 35–37 min, solvent B decreased to 0%. The HPLC was
coupled to an ion-trap mass spectrometer (ThermoFinnigan LCQ
Deca XP Plus; Thermo Corp.) equipped with an ESI (electrospray
ionization) source. The mass spectrometer was operated with
positive ionization using full scan mode (scan range 100–2000
m/z). Spray voltage was set at 5.0 kV, the capillary temperature at
275◦C and the sheath gas flow at 26 units (instrument units). For
MS/MS experiments, parent ions were fragmented in the ion trap
using 28% CID (collision-induced energy).
For analysis of serotonin and its dimer in plasma, samples
were analysed using a Dionex HPLC system coupled to an
Applied Biosystems/API 4000TM QTrap triple quadrupole mass
spectrometer. Chromatography was performed using a TSKgel®
Amide 80, 5 μm column 2.0 mm × 150 mm (Tosoh). Solvent A
was 0.1% formic acid in acetonitrile and solvent B was 0.1%
formic acid in water. The initial conditions (4:1 A/B, v/v) were
held for 3 min followed by a linear gradient to 1:1 (v/v) A/B
over 7 min. These conditions were maintained for 3 min before
returning to starting conditions and re-equilibration for 7 min.
Analytes were detected using ESI in the positive-ion mode, and
data were collected using MRM (multiple reaction monitoring).
Nitrogen was used as the collision gas with a dwell time of
150 ms for each species. The mass transition used for detection
of serotonin was 159.9 to 115.1 Da. As reported previously, the
parent ion for serotonin (m/z 177 Da) fragmented in the source
to give a major ion with a m/z of 159.9 Da [25]. Mass transitions
for the serotonin dimers were monitored for the doubly charged
species; i.e. the m/z ratios were 176–158.9 Da for the serotonin
dimer and 180–162.9 Da for its stable iosotope. The electrospray
voltage and temperature were 5.5 kV and 300◦C respectively. The
declustering potential and collision energy were 26 V and 31 eV
for serotonin and 36 V and 17 eV for both serotonin dimer species.
Serotonin was quantified by comparing its peak area with those
obtained for a series of external standards in plasma. Serotonin
dimer levels were quantified by including a known concentration
of its stable isotope in the acetonitrile extract and comparing their
peak areas with those obtained for a series of external standards
of the isotope in plasma.
Figure 1 Oxidation of serotonin by myeloperoxidase
(a) Reactions were started by adding 50 μM hydrogen peroxide to 10 nM myeloperoxidase,
100 μM serotonin and 100 μM DTPA in 50 mM phosphate buffer (pH 7.4). Spectra of oxidized
compared with unoxidized serotonin were recorded at 5, 10, 40 and 120 s after starting the
reaction. The arrow indicates the direction of the spectral changes. The effects of (b) the
concentration of serotonin and (c) pH on the initial rate of serotonin oxidation was recorded
at 317 nm. Other conditions were as described in (a). Results are means S.D. for triplicate
experiments.
Reaction of serotonin with the redox intermediates of
myeloperoxidase
The absorbance spectrum of myeloperoxidase was monitored
between 350 and 700 nm during oxidation of serotonin. Reactions
were started by adding 50 μM hydrogen peroxide to 1 μM
enzyme and 100 μM serotonin in 50 mM phosphate buffer
(pH 7.4). Reaction of serotonin with compound II and compound
III were also followed by monitoring absorbance changes of these
redox intermediates [2,26]. Compound II (λmax, 456 and 630 nm)
was formed by adding 100 μM hydrogen peroxide to 1 μM
myeloperoxidase (λmax, 430 and 570 nm), followed by addition
of 10 μg/ml catalase to scavenge excess hydrogen peroxide.
Compound III (λmax, 456 and 625 nm) was formed in a similar
manner by adding 1 mM hydrogen peroxide to the enzyme.
Spectral changes of the redox intermediates were monitored upon
reducing them with 10 μM serotonin.
+
−
were no absorbance changes in the absence of myeloperoxidase,
hydrogen peroxide or serotonin (results not shown). The initial
rate of serotonin oxidation increased towards a maximum
and the concentration of serotonin that gave 50% of the
maximum velocity was approx. 250 μM (Figure 1b). The rate
of serotonin oxidation increased over physiological pH with a
3-fold enhancement at pH 8 compared with pH 5 (Figure 1c).
This latter result suggests that the phenolate is the substrate for
myeloperoxidase as is the case with tyrosine [27].
To assess whether serotonin is a potential substrate for
myeloperoxidase in vivo, we determined how readily chloride and
other physiological substrates of myeloperoxidase competed with
serotonin and inhibited its oxidation. Chloride was a poor inhibitor
of the rate of oxidation of serotonin. At concentrations between
100 and 150 mM, chloride decreased the initial rate of serotonin
oxidation by only approx. 15% (Figure 2a). Other substrates
at their physiological concentrations including bromide, urate,
nitrite, tyrosine or thiocyanate either alone or all in combination
with chloride failed to prevent oxidation of serotonin (Figure 2b).
Only ascorbate blocked oxidation.
RESULTS
Oxidation of serotonin by myeloperoxidase and hydrogen peroxide
In agreement with earlier work [10,11], serotonin was readily
oxidized by myeloperoxidase and hydrogen peroxide. The
difference spectrum of serotonin during oxidation showed two
major peaks with maxima at 238 and 317 nm. There was also
a broad shoulder between 350 and 450 nm (Figure 1a). There
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Figure 2 Effect of other alternative substrates on the oxidation of serotonin
by myeloperoxidase
Figure 3 Effect of serotonin on production of hypochlorous acid by
myeloperoxidase and human neutrophils
(a) The initial rate of serotonin oxidation was determined at various concentrations of chloride
under the reactions conditions given in Figure 1. Methionine (1 mM) was added to scavenge
hypochlorous acid. Results are representative of two separate experiments. (b) Alternative
substrates of myeloperoxidase were added to myeloperoxidase (MPO) and serotonin under
the conditions given in Figure 1. The concentrations of substrates were 100 μM bromide,
500 μM urate, 100 μM nitrite (NO2-), 100 μM tyrosine (Tyr), 50 μM ascorbate (Asc), 100 μM
thiocyanate (SCN-) or 100 mM chloride. Reactions were started by adding hydrogen peroxide.
After 10 min, the concentration of serotonin was measured by diluting the reaction mixture
100-fold in water and recording its fluorescence (λex, 280 nm; λem, 340 nm). Results are
means + S.D. for triplicate experiments. Comparisons with the control were made using
Student’s t test. *P < 0.05.
(a) Hydrogen peroxide (50 μM) was added to 10 nM myeloperoxidase in PBS containing 5 mM
taurine. After 5 min, reactions were stopped by adding 20 μg/ml catalase and the concentration
+
of accumulated taurine chloramine was measured. Results are means S.D. for triplicate
−
◦
experiments. (b) Neutrophils (2 × 106 cells/ml) were incubated at 37 C in PBS containing
5 mM taurine and various concentrations of serotoinin. They were stimulated with PMA and,
after 30 min, reactions were stopped and the amount of accumulated taurine chloramine was
+
measured. Results are means ranges for duplicate experiments and are representative of data
−
obtained for cells from two individuals.
and also reacts with radicals produced by peroxidases [29]. We
therefore wanted to determine whether superoxide influenced
the oxidation of serotonin by myeloperoxidase. Superoxide had
a marked effect on the changes of the absorption spectrum
of serotonin when it was oxidized by myeloperoxidase and a
xanthine oxidase system (Figure 4). With the complete system,
there was a loss in absorbance at approx. 280 nm and increases in
absorbance at 315 and 535 nm (Figure 4a). However, when SOD
was added to remove superoxide, the absorbance spectrum was
dominated by an increase at approx. 315 nm (Figure 4b). These
results indicated that superoxide influenced the type of products
formed by myeloperoxidase.
The products derived from serotonin were identified using
HPLC and MS (Figure 5). Two main products eluted at approx.
7 (peak 2) and 16 (peak 3) min, whereas unreacted serotonin was
detected at 15.5 min (peak 1) (Figure 5a). Glutathione (GSH) was
added to the reaction because it forms adducts with quinones.
In the presence of GSH, the product at 16 min was replaced by
another at 19 min (peak 4, Figure 5a). The earlier eluting product
(peak 2) was not affected by GSH. The products were identified
by their UV spectra and by MS. Serotonin had an [M + H+] ion
with a m/z of 177 mass units. The absorbance spectrum of peak
2 had a maximum at 317 nm and a [M+H+] ion with a m/z of
176 mass units, which would represent a doubly charged dimer of
Serotonin had dual effects on hypochlorous acid production by
purified myeloperoxidase (Figure 3a) and isolated neutrophils
(Figure 3b). Below 20 μM, it enhanced production, whereas
at higher concentrations, it progressively inhibited the enzyme.
This was not due to serotonin-scavenging hypochlorous acid
because, under the conditions of the assay, hypochlorous acid
reacted preferentially with taurine. Also, taurine chloramine
did not react with serotonin and neither did serotonin reduce
oxidized TMB (results not shown). Thus there was no artefactual
inhibition of the detection of taurine chloramine. Serotonin did
not affect production of superoxide by stimulated neutrophils
(results not shown). Consequently, in these two systems,
serotonin could have acted only by reacting with myeloperoxidase
and influencing its ability to produce hypochlorous acid.
Collectively, these results suggest that serotonin is a physiological
substrate for myeloperoxidase and should influence production of
hypochlorous acid by the enzyme.
Influence of superoxide on the oxidation of serotonin by
myeloperoxidase
Myeloperoxidase normally functions in the presence of
superoxide [2]. Superoxide is a substrate for the enzyme [28]
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Figure 4 Effect of superoxide on myeloperoxidase-dependent oxidation of
serotonin
The difference spectrum of serotonin (100 μM) was monitored after adding xanthine oxidase to
100 nM myeloperoxidase in 50 mM phosphate buffer (pH 7.4) containing 100 μM DTPA and
10 mM acetaldehyde. The rate of superoxide production was 10 μM · min−1. Reactions were
carried out either in the absence (a) or presence (b) of 20 μg/ml SOD. Scans were recorded 15,
60, 120, 180 and 240 s after adding xanthine oxidase.
serotonin (results not shown). The absorbance spectrum of peak 3
had maxima at 355 and 546 nm (results not shown). This product
had a [M + H+] ion with a m/z of 191 mass units (Figure 5b).
These features are consistent with it being tryptamine dione,
as characterized previously by Dryhurst and co-workers [20,30].
When it was fragmented in the mass spectrometer, it gave major
product ions with m/z ratios of 162 and 173 mass units (Figure 5b).
Authentic tryptamine-4,5-dione co-eluted with the product in
peak 3 and had the same mass and fragmentation pattern. The
product in the presence of glutathione (peak 4) had absorbance
maxima at 340 and 545 nm (results not shown) and a [M+H+]
ion with a m/z of 496 mass units (Figure 5c). When authentic
tryptamine-4,5-dione was reacted with GSH, it co-eluted with
peak 4, had the same absorbance spectrum and a [M + H+] ion
with the same m/z. On this basis, we propose that the product in
peak 3 is tryptamine-4,5-dione and that it reacts with GSH to form
a conjugate, identified previously by Dryhurst and co-workers as
7-S-(glutathionyl)-tryptamine-4,5-dione [20,30].
To understand the mechanism of oxidation of serotonin
by myeloperoxidase plus the xanthine oxidase system, we
determined the affects of various inhibitors on the loss of
serotonin and formation of its dimer and tryptamine-4,5-dione
(Table 1). Oxidation of serotonin to the dimer and dione required
myeloperoxidase and hydrogen peroxide because omission of this
enzyme or addition of catalase and sodium azide inhibited the
reaction. SOD had contrasting effects on the reaction. It enhanced
formation of the dimer, but inhibited formation of tryptamine-
4,5-dione. Glutathione completely prevented production of
tryptamine-4,5-dione, but it did not prevent oxidation of serotonin
or formation the dimer.
Figure 5 Identification of the oxidation products of serotonin
(a) Serotonin was oxidized by myeloperoxidase and its products were separated by
HPLC. Reactions were started by adding xanthine oxidase to 10 mM acetaldehyde, 10 nM
myeloperoxidase and 100 μM serotonin in 50 mM phosphate buffer (pH 7.4). The flux of
superoxide was 10 μM · min−1. After 5 min, 50 μl of the reaction mixtures were injected
directly into the HPLC and products were identified by their UV absorbance at 254 nm. (i)
Serotonin only, (ii) complete reaction system, and (iii) complete reaction system + 100 μM
GSH. (b) MS/MS spectrum of peak 3. The parent ion had a m/z ratio for [M + H]+ of 191 and
was fragmented with 35% collision energy. (c) Mass spectrum of the peak 4.
Reactions of serotonin with compound III of myeloperoxidase
We determined the ability of serotonin to reduce compound
III because myeloperoxidase is converted into predominantly
compound III by stimulated neutrophils [31]. When serotonin was
added to pre-formed compound III, it readily reduced the enzyme
to ferric myeloperoxidase. In contrast, over the same period,
ascorbate, which reduces compound III slowly (k = 4.0 × 102
M−1 · s−1) [32], had little affect on the spectrum of compound
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Table 1 Effects of scavengers and inhibitors of myeloperoxidase on
oxidation of serotonin
time of 5 min, it inhibited formation of tryptamine-4,5-dione by
54%. From these results, we conclude that, under physiological
conditions, neutrophils oxidized serotonin to its dimer and
tryptamine-4,5-dione using myeloperoxidase, hydrogen peroxide
and superoxide. Oxidation did not involve hypochlorous acid
because it occurred in the presence of methionine. Hence, it must
have occurred by direct reaction of serotonin with the redox forms
of myeloperoxidase to produce radical intermediates.
Serotonin (100 μM) was incubated with 10 nM myeloperoxidase (MPO) in 50 mM phosphate
buffer (pH 7.4) containing 10 mM acetaldehyde and 100 μM DTPA. Reactions were started
by adding sufficient xanthine oxidase to generate 10 μM · min−1 superoxide. Reactions were
assayed after 10 min by separating serotonin, dihydroxybitryptamine and tryptamine-4,5-dione
by HPLC. When used, catalase and SOD were present at 20 μg/ml, and the concentrations
+
of sodium azide and glutathione were 100 μM. The data are means ranges for duplicate
−
experiments. Each condition for the different variables was compared with the complete system
To assess the potential for myeloperoxidase to oxidize serotonin
in vivo, we added serotonin (20 μM) and isolated neutrophils
to plasma. The cells were then stimulated with PMA in the
presence of cytochalasin B to promote degranulation and release
of myeloperoxidase. The oxidation of serotonin and formation of
its dimer were followed using LC–MS with MRM (Figure 6a).
After approx. 10 min, serotonin dimer was detected in plasma and
it accumulated over 1 h of incubation (Figure 6b). Similar kinetics
for the loss of serotonin were also observed (results not shown).
Formation of serotonin dimer required stimulation of the cells
and was inhibited by DPI, which blocks activity of the NADPH
oxidase (Figure 6c). Dimer formation was also retarded by the
haem poison sodium azide as well as catalase and thiocyanate,
which is a substrate for myeloperoxidase. However, SOD had
no effect. Collectively, these results demonstrate that activated
neutrophils oxidize serotonin in plasma in reactions that involve
the NADPH oxidase and myeloperoxidase.
using Student’s t test. *P < 0.05.
Loss of
serotonin
(μM)
Formation of
serotonin dimer
(% of complete system)
Formation of
typtamine-4,5-dione
(% of complete system)
Reaction system
+
+
+
Complete system
−MPO
43
6
17
26
36
7
2*
6
4
5
100
7.4 0.5*
0
100
0
0
0*
−
+
−
+
−
+
−
+
−
−
−
+
−
+
−
+
+
+Catalase
+Sodium azide
+GSH
12 4*
17 3*
−
−
+
+
−
+
−
6.8 0.7*
6
0
4*
0*
−
+
97 18
−
+
+
+
+SOD
82 14
218 19*
26 8*
−
−
−
Table 2 Effects of scavengers and inhibitors of myeloperoxidase on the
oxidation of serotonin by human neutrophils
◦
Neutrophils were pre-incubated for 10 min at 37 C in PBS with 100 μM serotonin. Methionine
(1 mM) was present to scavenge hypochlorous acid. They were then stimulated with PMA. After
20 min, reactions were stopped by adding 20 μg/ml catalase and pelleting the neutrophils.
The supernatants were kept on ice until they were analysed by HPLC for serotonin,
dihydroxybitryptamine and tryptamine-4,5-dione. When used, catalase and SOD were present
at 20 μg/ml, and the concentrations of sodium azide and glutathione were 100 μM. Results
DISCUSSION
In the present study, we have shown that serotonin is a
preferred substrate for myeloperoxidase. It is readily oxidized
in the presence of physiological concentrations of other major
substrates of the enzyme. Furthermore, we have demonstrated that
human neutrophils oxidize serotonin in plasma using hydrogen
peroxide and myeloperoxidase. Consequently, serotonin and
myeloperoxidase are likely to influence each other’s activities
during inflammation when they are released by platelets and
neutrophils respectively. This interaction could either lower the
concentration of serotonin and limit its physiological effects or
shift oxidant production from hypochlorous acid to serotonin
radicals. The fate of these radicals may have a profound impact
on inflammation, particularly if they give rise to toxic tryptamine-
4,5-dione.
+
are means ranges for duplicates and are representative of results for cells from three blood
−
donors. Each condition for the different variables was compared with the complete system using
Student’s t test. *P < 0.05.
Loss of
serotonin
(μM)
Formation of
serotonin dimer
(% of complete system)
Formation of
typtamine-4,5-dione
(% of complete system)
Reaction system
+
+
+
Complete system
−Neutrophils
−PMA
39
0
5
5
100
0
6
0
0*
2*
100
0
0
0
0
0
104
0
−
+
−
+
−
+
−
+
−
+
−
+
−
−
+
−
+
−
−
+
−
+
−
+
−
+
−
+
−
+
−
0*
3*
4*
8
8
8
0*
0*
0*
0*
0*
8
+
+Catalase
+Sodium azide
+GSH
9
20 5*
−
+
17
25
63
36 6*
−
+
54 6*
−
+
+SOD
125 15
−
The reactions of serotonin with myeloperoxidase are shown in
Scheme 1. The ferric enzyme reacts with hydrogen peroxide to
form compound I, which has a formal oxidation state of 5+ [2,26].
This redox intermediate of the enzyme has a high two-electron
reduction potential of 1.16 V, which allows it to oxidize chloride
to hypochlorous acid [33]. Under physiological conditions,
chloride and thiocyanate are equally preferred substrates.
Compound I is also one of the strongest one-electron oxidants
in vivo (E0ꢀ ≈ 1.35 V) and acts on myriad substrates to produce
free radicals [34]. In this reaction, the enzyme is converted
into compound II, in which the haem has a formal oxidation
state of 4+. Dunford and Hsuanyu [11] were the first to show
that serotonin is an exceptional substrate for myeloperoxidase.
They determined its rate constants for reduction of compound
I and compound II, which are 2 × 107 and 5 × 106 M−1 · s−1
respectively. We have demonstrated that oxidation of serotonin
by myeloperoxidase is optimal at neutral to alkaline pH. At
100 μM, it competed effectively with a host of other substrates
for oxidation by myeloperoxidase. At these concentrations,
serotonin is kinetically favoured to react with compound I and, as
we found, to out-compete other substrates of myeloperoxidase.
The exception was chloride, which decreased the oxidation
III (results not shown). These results demonstrate that serotonin
is a substrate for compound III.
Oxidation of serotonin by stimulated human neutrophils
We also determined the ability of isolated neutrophils to use
myeloperoxidase to promote oxidation of serotonin. Serotonin
was incubated with neutrophils which were subsequently
stimulated with PMA. Loss of serotonin and formation of its
dimer as well as tryptamine-4,5-dione were measured by HPLC
(Table 2). Oxidation of serotonin and formation of both products
required the presence of stimulated neutrophils and was inhibited
by sodium azide and catalase. GSH, which scavenges radicals
and hypochlorous acid, prevented formation of dimer by approx.
40%. In contrast, it completely blocked production of tryptamine-
4,5-dione. Methionine, which also scavenges hypochlorous acid,
had no effect on the loss of serotonin or formation of the products.
SOD did not affect formation of tryptamine-4,5-dione over
the 30 min incubation period. However, at a shorter incubation
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Oxidation of serotonin by myeloperoxidase
291
Scheme
1
Reaction of serotonin with the redox intermediates of
myeloperoxidase
The redox intermediates of myeloperoxidase are represented by the oxidation states of the haem
iron prosthetic group where FeIII, FeV = O, FeIV = O and FeIIO2 are native myeloperoxidase,
compound I, compound II and compound III respectively. HT is 5-hydroxytryptamine or
serotonin.
At lower concentrations, we found that serotonin competed
poorly with chloride. Rather, it enhanced production of
hypochlorous acid by both purified myeloperoxidase and
neutrophils. With the isolated enzyme, serotonin would have
boosted production of hypochlorous acid by reducing compound
II back to the active ferric enzyme, as occurs with superoxide,
urate and tyrosine [36]. Enhanced turnover of compound II
fails to totally account for the ability of serotonin to increase
production of hypochlorous acid by neutrophils. This is because
superoxide reacts with myeloperoxidase to convert it into
oxymyeloperoxidase or compound III (see Scheme 1) [37], which
occurs in stimulated neutrophils [31,38]. Consequently, the rate
of production of hypochlorous acid by neutrophils is dependent
on the turnover of compound III. We found that serotonin reduced
compound III to the ferric enzyme, presumably via intermediate
formation of compound I and compound II (Scheme 1). Hence,
serotonin is likely to boost production of hypochlorous acid by
neutrophils by preventing accumulation of both compound II and
compound III.
The reactions of serotonin with compound II and compound III
are likely to be the main routes by which serotonin radicals are
produced during inflammation. Both of these redox intermediates
of myeloperoxidase will be formed in vivo. Compound II
formation is promoted by numerous physiological substrates
and compound III is the predominant form of myeloperoxidase
when neutrophils are stimulated [31]. Serotonin should compete
effectively for oxidation by compound II because most other
substrates for compound II have rate constants 100-fold less than
that of serotonin [26].
Serotonin radicals have been shown by Dryhurst and co-
workers to undergo a variety of reactions: they can dimerize to
form dihydroxybitryptamine [39], disproportionate to produce a
quinone imine [40], or couple with reactive thiols [41]. The latter
reaction produces superoxide and hydrogen peroxide, which can
drive an oxygenase activity of myeloperoxidase toward thiols
(see Scheme 2) [10]. The ability of SOD to temporarily inhibit
formation of tryptamine-4,5-dione suggests that superoxide reacts
with serotonin radical by an addition reaction to produce an
unstable hydroperoxide, which would break down to form this
toxic species (Scheme 2). The analogous reaction has been
shown to occur for tryptophan [42], melatonin [43] and tyrosine
Figure 6 Oxidation of serotonin in plasma by stimulated neutrophils
Neutrophils (5 × 106 cells/ml) were suspended in a solution of 75% plasma and 25%
◦
PBS containing 20 μM serotonin at 37 C. Cells were stimulated with PMA (1 μg/ml) and
cytochalasin B (10 μg/ml) and the conversion of serotonin into its dimer was monitored
by LC–MS. (a) A chromatogram showing serotonin (black) and co-elution of dimer (grey)
produced by stimulated neutrophils and the synthesized stable isotope of the serotonin dimer
(black). The inset shows the linear response of the peak area to the concentration of dimer.
+
(b) The time course for conversion of serotonin into its dimer. Results are means ranges for
−
duplicate experiments. (c) Effect of inhibitors on the production of serotonin dimer after 30 min
of stimulation in the presence of 10 μM DPI (n = 4), 1 mM sodium azide (n = 4), 100 μg/ml
catalase (CAT; n = 3), 20 μg/ml SOD (n = 4) or 1 mM thiocyanate (SCN; n = 4). Results are
means + S.D. for n experiments performed on two different donors and are representative of
cells obtained from three individuals. Comparisons with the stimulated control (n = 6) including
that for unstimulated cells (n = 8) were made using Student’s t test. *P < 0.05.
of serotonin at its physiological concentrations. Extracellular
concentrations of serotonin are normally at sub-micromolar
levels. However, they can reach 100 μM transiently in the
immediate vicinity of platelets when they are stimulated [35].
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292
V. F. Ximenes and others
AUTHOR CONTRIBUTION
Valdecir Ximenes undertook the majority of the experimental work and contributed to the
design and writing the paper. Ghassan Maghzal and Rufus Turner undertook investigations
using MS. Yoji Kato synthesized the sable isotope for serotonin dimer and developed
the HILIC (hydrophilic interaction liquid chromatography) method for its measurement.
Christine Winterbourne undertook experiments on reaction of superoxide with serotonin
radicals and helped to write the paper. Anthony Kettle undertook experimental work and
was responsible for designing the projects and writing the paper.
FUNDING
This work was supported by the Health Research Council of New Zealand, the New
Zealand Centre of Excellence for Growth and Development, and Conselho Nacional de
Desenvolvimento Cient´ıfico e Tecnolo´gico (CNPq) Brazil, and Fundac¸a˜o de Amparo a
Pesquisa do Estado de Sa˜o Paulo (FAPESP) Brazil.
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Received 21 May 2009/6 October 2009; accepted 14 October 2009
Published as BJ Immediate Publication 14 October 2009, doi:10.1042/BJ20090776
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