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Structure Effect on Antioxidant Activity of Catecholamines toward Singlet Oxygen and Other Reactive Oxygen Species in vitro


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The reactivity of catecholamine neurotransmitters and the related metabolites were precisely investigated toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and reactive oxygen species. Catecholamines reacted immediately with DPPH radicals, their reactivity being stronger than that of ascorbic acid as a reference. Superoxide scavenging activities of catecholamines determined by WST-1 and electron spin resonance (ESR) spin trapping methods were also high. Whereas tyrosine, the dopamine precursor showed no reactivity toward superoxide. The reactivity toward singlet oxygen was evaluated by observing specific photon emission from singlet oxygen. The results revealed that reactivity of catecholamines was markedly higher than that of sodium azide, and catechin as catechol reference. The reaction of catecholamines and singlet oxygen was further studied by ESR using 55-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping reagent and rose bengal as photosensitizer. DMPO-OH signal of epinephrine was significantly small compared to other catecholamines, catechin, and 4-methylcatechol as a reference compound and was as small as that of tyrosine. The signal formation was totally dependent on singlet oxygen, and the presence of catechol compounds. These results indicated that epinephrine is the most potent singlet oxygen quencher than other catecholamines, and the secondary amino group in its alkyl side chain could play a role in unique singlet oxygen quenching property of epinephrine.
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Original Article J. Clin. Biochem. Nutr., 47, 181–190, November 2010
Advance Publication
JCBNJournal of Clinical Biochemistry and Nutrition0912-00091880-5086the Society for Free Radical Research JapanKyoto, Japanjcbn09-11210.3164/jcbn.09-112Original Article
Structure Effect on Antioxidant Activity of Catecholamines toward
Singlet Oxygen and Other Reactive Oxygen Species in vitro
Takako Shimizu1, Yuji Nakanishi1, Meiko Nakahara2, Naoki Wada1, Yoshihiko Moro-oka3,
Toru Hirano4, Tetsuya Konishi5 and Seiichi Matsugo1,*
1School of Natural System, College of Science and Engineering, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan
2Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Takeda 4-3-11, Kofu, Yamanashi 400-8511, Japan
3Emeritus Prof. of Tokyo Institute of Technology
4Hamamatsu University School of Medicine, Photon Medical Research Center,
Hamamatsu, Handayama, Higashi-ku, Hamamatsu 431-3192, Japan
5Faculty of Applied Life Sciences, Niigata University of Pharmacy & Applied Life Sciences,
Higashijima 265-1, Akiha-ku, Niigata 956-2081, Japan
1120101692010473181190Received 16.11.2009; accepted 22.12.2009
*To whom correspondence should be addressed.
Tel: +81-76-264-6219 Fax: +81-76-234-4829
Received 16 November, 2009; Accepted 22 December, 2009; Published online 16 September, 2010
Copyright © 2010 JCBN
Summary The reactivity of catecholamine neurotransmitters and the related metabolites
were precisely investigated toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and reactive
oxygen species. Catecholamines reacted immediately with DPPH radicals, their reactivity
being stronger than that of ascorbic acid as a reference. Superoxide scavenging activities of
catecholamines determined by WST-1 and electron spin resonance (ESR) spin trapping
methods were also high. Whereas tyrosine, the dopamine precursor showed no reactivity
toward superoxide. The reactivity toward singlet oxygen was evaluated by observing specific
photon emission from singlet oxygen. The results revealed that reactivity of catecholamines
was markedly higher than that of sodium azide, and catechin as catechol reference. The
reaction of catecholamines and singlet oxygen was further studied by ESR using 55-dimethyl-
1-pyrroline N-oxide (DMPO) as a spin trapping reagent and rose bengal as photosensitizer.
DMPO-OH signal of epinephrine was significantly small compared to other catecholamines,
catechin, and 4-methylcatechol as a reference compound and was as small as that of tyrosine.
The signal formation was totally dependent on singlet oxygen, and the presence of catechol
compounds. These results indicated that epinephrine is the most potent singlet oxygen
quencher than other catecholamines, and the secondary amino group in its alkyl side chain
could play a role in unique singlet oxygen quenching property of epinephrine.
Key Words:catecholamine, neurotransmitters, ROS, chemiluminescence, singlet oxygen
The catecholamines dopamine, norepinephrine, and epi-
nephrine function as neurotransmitters in the central nervous
system and also as hormones in the peripheral endocrine
system [1]. These compounds contain a characteristic cate-
chol chromophore, as indicated in their family name, and are
biosynthesized from tyrosine through an 3,4-dihydroxy-L-
phenylalanine (L-DOPA). Catecholamines affect the brain
regions associated with emotional activities such as hyper-
excitability and depression [2]. The catechol structure is
T. Shimizu et al.
J. Clin. Biochem. Nutr.
widely distributed in many naturally occurring antioxidants
and plays a role in scavenging reactive oxygen species
(ROS), typically, superoxide. Therefore, catecholamine
neurotransmitters and related metabolites are also expected
to react with ROS.
The brain is vulnerable to oxidative stress caused by high
contents of oxidizable substrates, such as polyunsaturated
fatty acid (PUFA) [3]. The brain consumes ca. 20% of
inhaled oxygen, thus, a large amount of ROS are believed to
be produced under normal metabolic and physiologic states
[4]. ROS produced in the brain have been associated with
various neurodegenerative disorders such as Parkinson’s
disease, which is characterized by the dysfunction of
dopaminergic neurons in the nigrostriatal system [5].
The external administration of antioxidant is usually not
effective to prevent brain oxidative stress because of the
presence of the blood/brain/barrier (BBB) [6]. In addition,
the activity of typical antioxidant enzymes such as super-
oxide dismutase (SOD) is lower in the brain than in other
tissues such as liver [7]. Hence, we focused our attention
on the reactivity of catecholamine neurotransmitters toward
ROS and radical including 1,1-diphenyl-2-picrylhydrazyl
(DPPH), superoxide, and singlet oxygen (Fig. 1). Superoxide
is the most abundant ROS generated under physiological
conditions and it is also generated by the auto-oxidation of
dopamine [8]. Singlet oxygen, on the other hand, has been
paid little attention on the oxidative stress compared with
other ROS, although it causes damages DNA [9], PUFA
[10], and amino acid [11] at the locus of generation. It is
produced by a photosensitized reaction (photodynamic
action) and also by enzyme-catalyzed reactions such as
myeloperoxidase-hydrogen peroxide-chloride reaction
in vivo
system [12]. It also plays a role in cellular signaling and
apoptosis [1315]. Therefore, it is necessary to study the
reactivity of singlet oxygen toward neurotransmitter mole-
cules. Although several fragmented studies have been
reported on the reactions of catecholamines toward ROS,
we have re-evaluated the scavenging activity of a series of
catecholamine neurotransmitters towards ROS, with special
focus on their reactivity toward singlet oxygen.
Materials and Methods
Dopamine hydrochloride, 3,4-dihydroxy-L-phenylalanine
(L-DOPA), L-noradrenaline (norepinephrine), L-adrenaline
(epinephrine), tyrosine, 1,1-diphenyl-2-picrylhydrazyl
(DPPH), 4-methyl catechol, hypoxanthine, and xanthine
oxidase (XOD) were purchased from Nacalai Tesque Co.
(Kyoto, Japan). Ascorbic acid and rose bengal were
purchased from Sigma-Aldrich Co. Japan (Tokyo, Japan).
benzene disulfonate sodium salt (WST-1) and 55-Dimethyl-
1-pyrroline N-oxide (DMPO) were purchased from Dojindo
Co. (Kumamoto, Japan). (+)-Catechin hydrate was purchased
from Spectrum Chemical Mfg. Corp. (Gardena, CA). Sodium
azide was purchased from Wako Co. (Osaka, Japan). DL-
Thioctic acid (lipoic acid) was purchased from Kanto chem-
ical Co. (Tokyo, Japan). Other chemicals were of the highest
grade. All reagents were used without further purification.
Water was prepared using Advantec PWU-100. For the
singlet oxygen photon counting method, water was prepared
using Yamato Scientific WG202.
UV-Vis absorption spectra were measured by JASCO
V550 UV/VIS spectrophotometer. Singlet oxygen chemilu-
minescence was detected by NIR-II Hamamatsu Photonics
KK, Japan.
DPPH radical scavenging activity evaluated by UV-vis
spectroscopic method
Dopamine and other test samples were dissolved in
25 mM phosphate buffer (pH 7.4) containing ethanol (final
33% v/v), except epinephrine. Since the solubility of
epinephrine in phosphate buffer was low, it was dissolved
in a mixture of phosphate buffer and 0.1% acetic acid.
Then, 0.5 ml of 1 mM DPPH in ethanol was added to 4.5 mL
of catecholamine solutions of various concentrations (final
ethanol 40% v/v), and the solution was stirred for 30 s. The
UV-Vis absorption spectrum of this solution was obtained in
the range from 350 nm to 700 nm. DPPH radical scavenging
activity of catecholamines was evaluated by determining
the absorbance at 526.5 nm. The reaction was also performed
in 40% methanol/phosphate buffer (pH 7.4), and the DPPH
Fig. 1. Chemical structures of catecholamine neurotransmitters
and catechin.
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
radical scavenging activity was calculated according to
equation 1 (eq. 1). In the equation, A and B indicate DPPH
absorbance in the absence and presence of the test sample,
DPPH radical scavenging activity (%) = 100 ×(AB)/A
(eq. 1)
DPPH radical scavenging activity evaluated by ESR method
The test samples were dissolved in 25 mM phosphate
buffer (pH 7.4) containing methanol (final 37.5% v/v),
except epinephrine. Epinephrine was dissolved in a mixture
of 0.4% acetic acid and phosphate buffer as described
above. Then, 40 µl of 1 mM DPPH in methanol was added
to 160 µl of test samples of defined concentrations (final
methanol 50% v/v), and the solution was stirred for 30 s.
Electron spin resonance (ESR) spectra of the solution
were obtained after 90 s, and the DPPH radical scavenging
activity was evaluated on the basis of the peak height of
DPPH radical determined after normalization with the Mn
reference peak. The conditions used for ESR measurements
(JEOL JES TE200) were as follows: temperature, 22°C;
frequency, 9.04 GHz; power, 8.00 mW; field, 341.0
10 mT
sweep time, 0.5 min; field modulation, 0.2 mT; and amplitude,
100; and time constant, 0.03.
Superoxide scavenging activity determined by WST-1 method
The test samples of various concentrations were dissolved
in 25 mM phosphate buffer (pH 7.4), and subsequently,
0.1 U/ml XOD was added to the reaction mixture (total
volume: 3 ml) containing 333 mM WST-1, 30 µM hypo-
xanthine, and phosphate buffer. Epinephrine was dissolved
1% acetic acid (60 µl) instead of phosphate buffer as
described above. The reaction was performed at room
temperature and the absorbance at 432 nm was determined
at 1 min after the addition of XOD. Superoxide scavenging
activity was evaluated by the residual amount of WST-1 as
in the following equation (eq. 2). The A and B indicate
WST-1 absorbance in the absence and presence of the test
sample, respectively.
Superoxide scavenging activity (%) = 100 ×(AB)/A
(eq. 2)
Superoxide scavenging activity evaluated by ESR spin
trapping method
Test samples of defined concentrations were dissolved
in 25 mM phosphate buffer (pH 7.4), and subsequently,
0.1 U/ml XOD was added to the reaction mixture (total
volume: 300 µl) containing 300 mM DMPO, 533 µM
hypoxanthine, and phosphate buffer. Epinephrine was
dissolved in 1% acetic acid (30 µl) and used as described
above. The ESR spectra were obtained at 50 s after the
addition of XOD, and the superoxide scavenging activity
was evaluated from the peak height of DMPO-OOH signal
(the superoxide DMPO adduct) after normalization with
the Mn reference peak [16]. The conditions used for ESR
measurement were the same as mentioned before.
Singlet oxygen scavenging activity evaluated by photon
counting method
To evaluate the singlet oxygen scavenging activity of
catecholamines, we dissolved the test samples and rose
bengal (photosensitizer) in deionized water at the concentra-
tions of 1 mM and 30 µM, respectively. The reaction mix-
ture containing test sample and 3 ml of rose bengal was
dispensed in a quartz cuvette and irradiated by YAG laser
at 532 nm (30 Hz, 40 mW) with a silicon filter (1100 nm
cut-off). Photon emission from singlet oxygen was detected
at 1268 nm using a highly sensitive near infrared (NIR)
spectrophotometer [17]. Data correction was achieved by
keeping a 5 µs time-lag after every pulse irradiation to avoid
the effect of strong artificial background fluorescence from
rose bengal. To evaluate the dose dependant activity of
catecholamines, we added 50 µl of test sample (up to 250 µl)
to the reaction mixture in a cuvette, and determined the
absorbance of the solution at each step. Singlet oxygen
scavenging activity was evaluated based on the signal
intensity of the peaks.
Reaction of catecholamines with singlet oxygen in the
presence of DMPO
The reaction mixture containing 250 µM test sample,
225 µM DMPO, and 24.5 µM rose bengal was prepared in
25 mM phosphate buffer (pH 7.4) (total volume: 200 µl).
The dose dependent activities of sodium azide and lipoic
acid were determined in the presence of catechin (250 µM)
or dopamine (250 µM), respectively. The reaction solution
was placed at 12 cm from the halogen lamp (650 W) and
irradiated by visible light for 1 min. After 100 s, the ESR
spectrum of the mixture was obtained (JES-FR30EX).
Statistical analyses
All data presented in this study were the average of at
least three times experiments and expressed as mean ±SD
values. Data were analyzed by Student’s t test.
DPPH radical scavenging activity
The DPPH radical scavenging activity of catecholamines
dissolved in aqueous ethanol revealed that the absorption
bands of DPPH radical at 526.5 nm decreased after the
addition of catecholamines in a concentration dependent
manner. The relative DPPH radical quenching ability of
catecholamines and their IC30 values (the concentrations
T. Shimizu et al.
J. Clin. Biochem. Nutr.
required for scavenging 30% of DPPH radicals) are
summarized in Table 1. The reactivity of catecholamines in
aqueous ethanol was in the following order: norepinephrine
> dopamine > epinephrine > L-DOPA > ascorbic acid.
Tyrosine did not scavenge DPPH radical in the same concen-
tration range as that of catecholamines.
The DPPH scavenging activity was also determined in
aqueous methanol. The IC30 values obtained in aqueous
methanol were significantly smaller than those obtained in
aqueous ethanol. However, in both solutions, all the examined
catecholamines showed stronger scavenging activity than
ascorbic acid.
The DPPH radical scavenging activity of catecholamines
was further determined by ESR. Since this method afforded
a clear dose response up to 80% inhibition level, the
scavenging activity of catecholamines was compared on the
basis of the IC50 values (the concentration scavenging 50%
of the original DPPH); these are presented in Table 2. The
DPPH radical scavenging activities in aqueous methanol
system obtained by ESR were similar to those determined by
UV-Vis spectroscopy as described above. The scavenging
activity of epinephrine was lower than that of other
catecholamines, as determined by both these methods. The
reactivity of catecholamines was in the following order:
dopamine > norepinephrine > L-DOPA > epinephrine >
ascorbic acid. Tyrosine did not affect the peak height of
the DPPH radical signal.
Superoxide scavenging activity
Superoxide scavenging activity of catecholamines was
determined by the WST-1 method, using tyrosine and
ascorbic acid as reference agents. The dose dependant
reactivity of catecholamines is shown in Fig. 2, and the IC30
values obtained from the reaction profile are summarized in
Table 3. The superoxide scavenging activity was in the
following order: dopamine > L-DOPA > norepinephrine >
ascorbic acid > epinephrine. Tyrosine showed no reactivity
Table 1. DPPH radical scavenging activity determined by UV-vis spectroscopy
*p<0.05, **p<0.01 compared with the indicated group using student t test.
sample ethanol methanol
IC30 (µM) IC30 (µM)
L-DOPA 14.9 ±1.0 7.75 ±1.3
dopamine 12.8 ±2.9 7.14 ±1.5
norepinephrine 12.2 ±1.0 7.69 ±1.2
epinephrine 13.3 ±1.9 10.9 ±0.7
ascorbic acid 26.3 ±0.7 15.9 ±1.0
tyrosine no reaction no reaction
** ** ** **
** ** ** ** ** ** **
Table 2. DPPH radical scavenging activity determined by ESR
*p<0.05, **p<0.01 compared with the indicated group using
student t test.
sample methanol
IC50 (µM)
L-DOPA 7.51 ±1.6
dopamine 6.33 ±0.8
norepinephrine 7.10 ±1.1
epinephrine 11.8 ±1.0
ascorbic acid 15.9 ±2.3
tyrosine no reaction
*** ** ** ***
Fig. 2. Superoxide scavenging activity determined by WST-1
method. The decrease in the absorbance at 432 nm due
to superoxide-induced formazan formation by WST-1
reduction was measured by UV-Vis spectrometer in the
presence and absence of catecholamine. Superoxide was
generated in the hypoxanthine-xanthine oxidase system.
(a) dopamine, (b) norepinephrine, (c) epinephrine, (d)
L-DOPA, (e) tyrosine, (f) ascorbic acid.
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
toward superoxide.
The superoxide scavenging activity was further evaluated
by ESR using spin trapping method. The typical ESR spectra
of DMPO-OOH signal in the presence of different concen-
trations of dopamine are shown in Fig. 3. The DMPO-OOH
signal intensity decreased in a concentration dependent
The IC50 values of different catecholamines determined
by the ESR method are listed in Table 4. The relative
activities of catecholamines determined by ESR method
were similar to those obtained by WST-1 method. The
scavenging activity of dopamine, norepinephrine, and L-
DOPA was higher than that of ascorbic acid, while the
scavenging activity of epinephrine was lower than that of
other catecholamines and ascorbic acid. The relative
activity was in the following order: dopamine > L-DOPA >
norepinephrine > ascorbic acid > epinephrine.
Singlet oxygen quenching activity
When the singlet oxygen is discharged to the ground state
oxygen, photon emission is observed at approximately
1268 nm [18]. Therefore, the photon counting of this
emission is a direct evidence of singlet oxygen generation.
In the present study, we determined the singlet oxygen
scavenging potential of catecholamines by observing this
emission using a highly sensitive NIR detection system.
The peak height of the emission spectra of singlet oxygen
decreased in the presence of dopamine in a concentration
dependent manner as shown in Fig. 4. The dose dependence
of the quenching profile shown in Fig. 5 revealed that both
Table 3. Superoxide scavenging activity determined by WST-1
*p<0.05, **p<0.01 compared with the indicated group using
student t test.
sample IC30 (µM)
L-DOPA 16.8 ±3.8
dopamine 11.6 ±0.9
norepinephrine 18.1 ±1.7
epinephrine 70.9 ±7.3
ascorbic acid 26.5 ±1.5
tyrosine no reaction
** ** *** **
Fig. 3. Dopamine dependent decrease of DMPO-OOH signal.
Superoxide was determined by ESR spin trapping method
using DMPO as the spin trap reagent and hypoxanthine-
xanthine oxidase system as superoxide generator. Precise
ESR measurement condition is given in the experimental
Table 4. Superoxide scavenging activity determined by ESR
using DMPO
*p<0.05, **p<0.01 compared with the indicated group using
student t test.
sample IC50 (µM)
L-DOPA 24.3 ±1.1
dopamine 14.6 ±2.3
norepinephrine 25.5 ±4.3
epinephrine 89.7 ±4.8
ascorbic acid 41.2 ±1.6
tyrosine no reaction
** ** ** ** **
Fig. 4. Singlet oxygen quenching activity of dopamine deter-
mined by photon counting method. Rose bengal was
used as a photosensitizer to generate singlet oxygen.
Photon emission was determined by photon counter after
laser irradiation at 532 nm in the presence and absence
of test sample. Precise reaction condition is given
experimental section.
T. Shimizu et al.
J. Clin. Biochem. Nutr.
epinephrine and norepinephrine have stronger quenching
ability than dopamine and L-DOPA. The IC50 values of test
samples are summarized in Table 5. The scavenging
activities of catecholamines were significantly higher than
those of catechin and sodium azide as typical singlet oxygen
quencher. The reactivity was in the following order:
epinephrine > norepinephrine > L-DOPA > dopamine >>
catechin > sodium azide.
The reactivity of catecholamines to singlet oxygen was
further examined by the ESR spin trapping method using
DMPO, according to a previously reported [19]. Similar to
the findings of this study, we detected the characteristic
DMPO-OH signal after irradiation in the presence of
catecholamines or catechin (Fig. 6). Simple irradiation of
rose bengal without catecholamines or catechin did not
reveal any significant radical trapped by DMPO signal.
Irradiation of catecholamines or catechin in the absence of
rose bengal did not produce any signals. Almost same signal
Fig. 5. Dose effect of catecholamines on singlet oxygen
quenching reaction. Singlet oxygen quenching activity
was measured as in Fig. 4. Precise reaction condition
is given experimental section. (a) dopamine, (b)
nephrine, (c) epinephrine, (d) L-DOPA, (e) sodium azide
(f) catechin.
Table 5. Singlet oxygen quenching activity evaluated by 1268 nm photon emission
**p<0.01 compared with the indicated group using student t test.
sample IC50 (µM)
L-DOPA 23.9 ±5.1
dopamine 29.6 ±3.2
norepinephrine 15.9 ±1.0
epinephrine 14.0 ±0.2
sodium azide 181 ±15
catechin 140 ±12
** ** ** ** **
** **
** **
Fig. 6. Singlet oxygen scavenging activity of catecholamines
evaluated by ESR spin trapping method. The reaction
mixture containing catecholamines (250 µM), rose
bengal, and DMPO was irradiated for 1 min, ESR
spectra were measured at 100 s after the irradiation.
Precise reaction condition is given experimental section.
(A) none, (B) sodium azide, (C) catechin, (D) dopamine,
(E) norepinephrine, (F) L-DOPA, (G) epinephrine.
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
intensity was observed for all test samples with catechol
structure including 4-methyl catechol, except that the signal
intensity was small in the case of epinephrine (Fig. 6,
Table 6). Sodium azide inhibited this catechol dependent
signal in a concentration dependent manner, thereby implying
that DMPO-OH formation was dependant on the presence of
singlet oxygen and that OH-adduct formation resulted from
the reaction of singlet oxygen and catecholamines (Fig. 7).
Further, DMPO-OH signal formation was reduced in the
presence of lipoic acid, but it was not completely inhibited
even at high concentrations (up to 1.25 mM) (Fig. 8).
The results in the present study revealed that cate-
cholamine neurotransmitters were highly reactive to ROS
and radical species. The DPPH and superoxide scavenging
activities of catecholamines were higher than those of
ascorbic acid and comparable to those of catechin, a flavan
having a catechol B-ring [20], although the reactivity of
epinephrine was rather weaker than those of ascorbic acid.
Further, tyrosine, which has only 1 phenolic OH group, did
not exhibit superoxide scavenging activity (Tables 3 and 4).
Ohkubo et al. [21] performed laser flash photolysis and
that cumyl peroxide reacts with neurotransmitters
norepinephrine, and epinephrine) by abstracting
the hydrogen
from the catechol OH group. The energy differ-
ence (DHT) between the original phenols and the resultant
phenoxyl radicals was several times lesser than that in
monophenols such as serotonin and tyrosine, thus, the rate of
oxidation of catecholamines was at least 10 times faster than
that of monophenols. This finding is consistent with our
present results although the ROS is not peroxyl radical but
superoxide anion radical in our present study.
Table 6. Catechol dependent DMPO-OH formation in the hoto-
dynamic singlet oxygen generating system
*p<0.05, **p<0.01 compared with the indicated group using
student t test. DMPO-OH was determined after the irradiation of
reaction mixture containing test sample, rose bengal as photo-
sensitizer and DMPO. Precise experimental condition was given
in the experimental section.
sample relative height
L-DOPA 8.9 ±0.2
dopamine 8.7 ±0.3
norepinephrine 9.1 ±0.1
epinephrine 1.5 ±0.3
4-methyl catechol 8.8 ±0.6
catechin 9.1 ±0.6
tyrosine 2.4 ±0.3
lipoic acid 1.7 ±0.3
** **
** ** **
Fig. 7. Effect of sodium azide on the catechin dependent
DMPO-OH formation. DMPO-OH formation was
measured as in Fig. 6 at various sodium azide concentra-
Fig. 8. Effect of lipoic acid on the dopamine dependant DMPO-
OH formation. DMPO-OH formation was measured as
in Fig. 6 at various lipoic acid concentrations.
T. Shimizu et al.
J. Clin. Biochem. Nutr.
Interestingly, we noted that the reaction of catecholamines
with DPPH showed a marked solvent effect. The DPPH
scavenging activities (estimated on the basis of IC values)
of catecholamines determined in aqueous methanol were
comparable between UV-Vis spectroscopy and ESR
methods, although there was a small difference among the
catecholamines. However, the IC30 values in aqueous
ethanol were markedly different from those in aqueous
methanol (Tables 1 and 2). When the relative scavenging
activities of catecholamines were compared with those of
ascorbic acid, which was used as the reference scavenging
agent, the activities of catecholamines were found to be
similar in both the solvents. Therefore, the DPPH assay is
only adaptable for the relative comparison of free radical
scavenging activities of a series of test samples, and a
common reference is required for accurate evaluation of
scavenging activity. Further, our results indicated that the
superoxide scavenging activity of epinephrine was lower
than that of other catecholamines. The same tendency was
observed for DPPH radical scavenging activity. Among all
the catecholamines used in this study, dopamine showed
the highest DPPH and superoxide scavenging activities in
all assays.
Further, the present study revealed the unique reactivity of
catecholamines toward singlet oxygen, whose reactivity was
different from that of other ROS. It is not a free radical but a
strong oxidant since it has unique electronic configuration
and 94.1 kJ/mol higher energy than the ground state oxygen
[22]. Singlet oxygen can cause damage in a wide range of
biologically important molecules [23]. The involvement of
singlet oxygen in a reaction is usually indirectly evaluated
on the basis of the presence of characteristic products, such
as “ene”-type products, or by using specific quenchers, such
as 1,4-diazabicyclo[2.2.2]octane (DABCO) or sodium azide
[24, 25]. In the present experiment, the reaction of singlet
oxygen with catecholamines and catechin were directly
examined by the photon counting method. The obtained
results revealed that the singlet oxygen quenching activity of
catecholamines was several times stronger than that of
catechin and sodium azide (Fig. 5).
The quenching of singlet oxygen proceeds via chemical
and physical processes. Sodium azide quenches singlet
oxygen mainly by the physical process [26]. It has been
reported that catechin, the green tea polyphenol, quenches
singlet oxygen mainly by the physical process and that the
catechol structure at B-ring is responsible for the physical
quenching process [27, 28]. It was also known that singlet
oxygen interacts with certain amino acids such as tryptophan
histidine, and tyrosine mainly by the chemical process. For
example, tyrosine and tyrosine derivatives that bear electron-
donating groups on their aromatic rings readily react with
singlet oxygen via the chemical process [29, 30]. In our
study, the reactivity of catecholamines toward singlet
oxygen, which was determined by evaluating the quenching
activity corresponding to the 1268 nm emission peak, was
considerably higher than that of catechin and sodium azide.
Among the catecholamines examined, norepinephrine and
epinephrine exhibited stronger quenching activities than
dopamine and L-DOPA did (Table 5). Therefore it was
expected not only the catechol structure but other factor such
as alkyl side chain property are involved in the singlet
oxygen quenching probably via both physical and chemical
In the present study, the reaction of singlet oxygen and
catecholamines was further examined by ESR using DMPO
as the spin trapping reagent. It has been reported that the
hydroxyl radical adduct of DMPO (DMPO-OH) is formed in
a photosensitized singlet oxygen generating system in the
presence of phenolic antioxidants or reduced glutathione,
thereby indicating that the hydrogen donating property of
antioxidants is essential for the formation of DMPO-OH
[31, 32]. Indeed, in our experiment, catecholamines and
catechin produced large DMPO-OH signals, however lipoic
acid, which is a strong antioxidant but is not a hydrogen
donating molecule, formed only a weak DMPO-OH signal
(Table 6). Further, catechin dependent DMPO-OH forma-
tion was inhibited by sodium azide as singlet oxygen
quencher in a concentration dependent manner (Fig. 7). The
DMPO-OH formation was therefore found to be primarily
dependent on the generation of singlet oxygen, as reported
previously [19, 31, 32]. Tyrosine did not give rise to
significant signal, although it has the same alkyl side chain
structure as L-DOPA but does not have catechol structure.
However, 4-methyl catechol, which is used as the reference
for catecholamine, formed as large DMPO-OH signal as that
of catecholamines (Table 6). Therefore, a catechol structure
is the primary requisite for DMPO-OH formation, and the
contribution of alkyl side chain to this process may not be
large. The interesting finding is that the DMPO-OH signal
of epinephrine was significantly small compared to other
catecholamines including norepinephrine, and the signal
level was as small as that of tyrosine even though it has
catechol structure.
Nishizawa et al. [31] reported the mechanism of the
DMPO-OH formation that the DMPO-singlet oxygen adducts
primarily generated is transformed into DMPO-OH in the
presence of hydrogen donating antioxidant, and they also
suggested that the free hydroxyl radical produced by
catechol oxidation is involved in DMPO-OH formation.
To evaluate the contribution of free hydroxyl radical in the
DMPO-OH formation, we studied the effect of lipoic acid on
the reaction, because lipoic acid and dihydrolipoic acid are
known to effectively scavenge hydroxyl radicals generated
from metal free system [33, 34]. Increasing concentrations
of lipoic acid inhibited catecholamine dependent DMPO-
OH formation, but this inhibitory effect was not very
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
remarkable (Fig. 8). Therefore, the free hydroxyl radicals
might not be the major source of DMPO-OH. Moreover,
epinephrine inhibited the dopamine dependant DMPO-OH
signal in a concentration dependent manner (data not
shown). Taken these discussions together, DMPO-OH signal
suppressing effect of epinephrine might be due to its higher
singlet oxygen scavenging activity. This is supported from
the data in Table 5 in that epinephrine showed the highest
reactivity toward singlet oxygen among the catecholamines.
The only structural difference between epinephrine and
norepinephrine is the alkyl side chain, the former has a
secondary amino group while the latter has the primary
amino group. Therefore, it can be presumed that owing to
the presence of the secondary amino group, epinephrine
exhibits higher activity toward singlet oxygen as compared
to other catecholamines, which has a primary amino group.
The present study showed the high reactivity of catechol
neurotransmitters (dopamine, norepinephrine, or epinephrine
and their precursor (L-DOPA) toward ROS. Therefore,
under oxidative stress conditions, catecholamine neuro-
transmitters will readily react with ROS in the brain and
oxidize dopamine to semiquinone, quinone, and dopamino-
chrome, which exert neurotoxic effects [3540]. Although
the critical role of singlet oxygen in brain disorders has not
been elucidated yet, the results of this study suggest that
further investigation to understand the significant contribu-
tion of singlet oxygen-catecholamine neurotransmitter reac-
tions in brain disorders is warranted in the future.
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... After radiation, the amount of antioxidant phospholipids in the bloodstream increases and the intensity of free-radical autooxidation of lipids in tissues decreases (25). In turn, it was found that the humoral products of activation of stressrealizing systems -catecholamines and steroid hormoneshave antioxidant activity (26,27). Their hypersecretion can be considered as a response to LPO activation, which develops through a negative feedback mechanism. ...
Full-text available
The aim of the investigation was to study the effect of acute, total gamma-irradiation in a sublethal dose on the biophysical properties of erythrocytes, the intensity of lipid peroxidation, antioxidant supply and hemocoagulating properties of erythrocytes. The experiments were carried out on 11-12-week-old age guinea pigs, males and females in equal numbers. The animals were exposed to a single total radiation at a dose of 4.5 Gy (sublethal dose, LD 50/30). The studies were carried out on the 7th day after exposure to radiation (at the height of radiation sickness). The development of radiation damage was accompanied by intense erythropoiesis and the appearance of erythrocytes with a high resistance to hemolysis and an increased sedimentation rate. After acute gamma irradiation, depletion of the antioxidant system was noted. It manifested in a decrease in the activity of superoxide dismutase of erythrocytes by 19.7% (p<0.01) and the concentration of serum ceruloplasmin by 21.5% (p<0.01). The content of thiobarbituric acid reactive substances (TBARS) and their accumulation while the incubation of erythrocytes remained within the normal range. The erythrocytes of the irradiated animals exhibited increased procoagulant and decreased antiheparin activity, which reflects conformational changes in highly radiosensitive fatty acid chains of phospholipids in their membranes. A decrease in the fibrinolytic activity of erythrocytes in irradiated animals was found.
... It is well known that wound sites could produce excessive ROS, which might result in oxidative stress [62]. Owing to the presence of catechol groups, uCNT@PDA showed excellent antioxidant and free radical scavenging activities [63]. Therefore, PCL/uCNT@PDA-0.5 and PCL/uCNT@PDA-1 nanofiber mats displayed excellent antioxidant ability, which would have great potential as wound dressing materials for wound healing and antibacterial activity. ...
Presently, efficient protection from bacterial infections is considered a potential threat owing to the raise of multidrug resistant bacterial phenotypes. Hence, there is an urgent requirement to develop alternative strategies for eliminating bacterial infections. In this regard, near infra-red (NIR) responsive photothermal agents (PTAs) has received significant attention. We report a mussel-inspired NIR-responsive unzipped CNT (uCNT)-based nanocomposite to combat against bacteria. The modification of mussel-inspired polydopamine (PDA) coating improved the NIR responsiveness and biocompatibility of uCNTs. The temperature of uCNT@PDA increased by 40°C upon exposure of 808 nm NIR light (1.0W/cm2, 10 min) showing the excellent photothermal property. PCL/uCNT@PDA nanofiber mats showed controlled photothermal heating upon low power density (0.5 W/cm2) NIR irradiation and exhibited robust mechanical strength up to 20 MPa (PCL/uCNT@PDA-1). Taking advantage of photothermal effect and ROS generation, the nanofiber displayed remarkable antibacterial efficacy against Bacillus subtilis (gram positive) and Escherichia coli (gram negative) and displayed the release of curcumin in presence of NIR light. The fabricated PCL/uCNT@PDA platform is bioactive, anti-oxidative, and non-toxic for human dermal fibroblast cells. Therefore, we anticipate that the developed uCNT@PDA is a potential PTA and the fabricated nanofiber may provide a versatile platform for eradicating bacterial contamination in medical and non-medical sectors.
... The structure of catechol is widely distributed in many natural antioxidants and plays a role in warding off free radicals. The catechetical structure of L-Dopa in methanol solvents has better DPPH radical counteracting activity than ascorbic acid (Shimizu et al., 2010). This was also evident in this study where the IC-50 value of L-Dopa isolates was smaller than IC-50 values of ascorbic acid, and that mean the L-Dopa isolates showed stronger DPPH radical counteracting activity than the ascorbic acid ( Table 5). ...
Mucuna pruriens (L.) DC (Velvet beans) is a tropical bean containing large amounts of L-Dopa (L-3,4-dihydroxyphenylalanine) and is the most important natural remedy for Parkinson’s disease. This plant has been used in Ayurveda medicine because it has a pharmacological activity, one of which is an antibacterial activity obtained from its seed extract. M. pruriens seeds fulfill the basic nutritional requirements, so they can be consumed. However, their utilization as food is limited due to the presence of some anti-nutritional factors. The objective of the research was to determine the nutritional content of seeds of M. pruriens Central Java accession by proximate analysis, isolation, and characterization of L-Dopa, determine the toxicity value (LC-50), against Artemia salina L. larvae by BSLT (brine shrimp lethality test), and determine its antibacterial activity against Streptococcus mutans ATCC 35668, and Escherichia coli ATCC 25922 by agar disk diffusion method. The isolate of L-Dopa was prepared by maceration using methanol 80%, followed by extraction using water, then, the isolated product was analyzed by analytical TLC to determine the suitable mobile phase. The sample was then run on preparative TLC. The spot that corresponds to the L-Dopa standard was collected and further purified using adsorption column chromatography. Then, the isolate was characterized by HPLC and FTIR. The antioxidant activity was determined by the DPPH radical method. Finally, anti-Parkinson's activity was determined to the rotenone-induced behavior of white rats (Rattus norvegicus) by the classic labyrinth test method. The result showed that the strongest antibacterial activity against S. mutans ATCC 35668 was shown by crude extract. Based on the HPLC analysis, the isolated L-Dopa showed a retention time of 5.345 minutes that is relatively similar to the L-Dopa standard and had a high purity. L-Dopa content in M. pruriens seed of Central Java accession was 0.49 mg/g dry weight. The L-Dopa isolate from M. pruriens Central Java accession has antioxidant activity with IC50 of 8.92 ± 0.03 ppm (highly active) and could significantly reduce the non-motoric Parkinsonism symptoms (p < 0.001) compared to the negative control.
... The following is a summary of typical antioxidants ( Table 1). Oxygen radicals scavenger (trolox [62], lutein [63], allicin [64], resveratrol [65], isoflavones [66], quercetin [67], catechin [68], theaflavin [69], curcumin [70], chlorogenic acid [71], and superoxide dismutase [72]), singlet oxygen quencher (astaxanthin, histidine [73], and lycopene [74]), hydrogen peroxide scavengers (catalase and glutathione peroxidase), and scavengers for all type of ROS (ascorbic acid [75], ergothioneine [76], L-cysteine, glutathione [77], and tocopherol [78]) are suggested to be effective in suppressing UVA-photosensitization. ...
Full-text available
Skin aging is classified into chronological aging and photoaging, involving ultraviolet radiation (UV), visible light, and others. UVA and UVA-photosensitizers (involving photocatalysis) contribute to the production of chronically induced skin damage that results in photoaging, especially wrinkles that are associated with histopathological actinic elastosis in the dermis. Hydrogen peroxide produced by the photosensitization involving photocatalysis, such as flavin, has been proposed as a risk factor for photoaging. It was also revealed that hydrogen peroxide production by UVA is amplified through the following reactions. The photosensitization of type I and type II by riboflavin as an initiator oxidizes coexisted amino acids and vitamins. The oxidized amino acids and vitamins produce reactive oxygen species (ROS), including hydrogen peroxide, through secondary UVA-photosensitization. Finally, we proposed a screening method for detecting the effects of antioxidants on UVA-photosensitization. In our previous study, histidine and other antioxidants did not inhibit UVA-photosensitized by riboflavin, even though they have been reported to scavenge singlet oxygen and superoxide. In contrast, we demonstrated that ergothioneine suppressed the production of hydrogen peroxide by UVA-photosensitization. The purpose of this report is to provide new findings for the prevention of photoaging by discussing the characteristics of UVA-photocatalysts in the skin.
... Therefore, in our model reaction of the initiated oxidation of ethylbenzene, the interaction of catecholamines with alkyl radicals is not manifested. It is possible that the high level of activity of adrenaline in aqueous micellar media can manifest itself as a result of a more complex mechanism of the inhibition of the oxidation process and an increase in the stoichiometric coefficient due to the additional inhibition by oxidation products, in contrast to the model reaction of hydrocarbon oxidation [34][35][36]. ...
Nine compounds are studied for antioxidant activity, including those from the class of catecholamines containing 3,4-hydroxyphenyl (catechol) as a common structural fragment, which imparts antioxidant properties to the compounds in the reactions of hydrocarbon substrate oxidation. The antiradical activity is determined by the chemiluminescent method by the interception of peroxyl radicals in the model reaction of the initiated oxidation of ethylbenzene (RH). The mechanism of the inhibition of chain oxidation processes by diatomic phenol compounds is provided by the presence of two active hydroxy groups with a possible intramolecular hydrogen bond, leading to a weakening of the О–Н bond and a high rate constant of hydrogen abstraction in the reaction with peroxyl radicals (kinh). This reaction is dominant and determines the inhibitory activity of antioxidants in oxidation processes. The maximum inhibitory activity is shown by 3,5- and 3,6-di-tert-butylpyrocatechins, dopamine, and epicatechin.
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Carotid atherosclerosis is a main risk factor for ischemic stroke. Plaque instability is determined by the morphological characteristics of the plaque and can be characterized by immunological biomarkers. The study aimed to examine the connection between serum levels of hs-CRP, fibrinogen, ICAM-1, VCAM-1 and carotid atherosclerosis and the different types of atherosclerotic plaques imaged by ultrasound and magnetic resonance. The study involved 120 patients with carotid atherosclerosis and 33 patients without carotid atherosclerosis. Blood samples were collected to analyze the serum level of hs-CRP, fibrinogen, ICAM-1 and VCAM-1. The ultrasound analysis included detection of atherosclerotic plaques in the internal carotid arteries, measurement of artery stenosis in percentage and determination of plaque types by the classification of Gray-Weales/Gerolacus. A small subset of 30 patients with carotid atherosclerosis performed 3T magnetic resonance imaging. Atherosclerotic plaques were classified into 8 types based on the modified MR classification of the American Heart Association. Significantly higher serum levels of hs-CRP (p <0.001) and fibrinogen (p = 0.018) were observed in patients with carotid atherosclerosis compared to patients without atherosclerosis. Criterion values for hs-CRP > 4.13mg/l and for fibrinogen > 3.6 g/l were associated with the presence of carotid plaques with accuracy of 70%. No relation was observed between the investigated biomarkers, the artery stenosis and the types of atherosclerotic plaques determined by ultrasound and magnetic resonance diagnostic methods. Hs-CRP and fibrinogen are reliable serum markers whose increased serum concentrations are connected with the presence of carotid atherosclerosis.
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While plant-derived oxidants can protect cells from oxidative damage, limited research has examined the role of dietary chlorophyll. Photoreduction of ubiquinone by chlorophyll metabolites and red light has been reported in vitro and in animal models. Herein we examined photo-oxidation and photoreduction reactions of catechols, dopamine and hydrocaffeic acid. Photo-oxidation of dopamine by methylene blue and the chlorophyll metabolites pheophorbide A, chlorin e6 and sodium copper chlorophyllin was studied by monitoring aminochrome, the cyclized product of the dopamine o-quinone with its amine. Singlet oxygen scavengers including sodium azide, ascorbate and glutathione decreased aminochrome formation by methylene blue and pheophorbide A. Addition of EDTA, a tertiary amine electron donor, to the reaction of dopamine, photosensitizer and red light decreased aminochrome formation. Photoreduction of the dopamine o-quinone produced by mushroom tyrosinase was achieved by both methylene blue and pheophorbide A only when an electron donor was included. Due to limited solubility, photo-oxidation and photoreduction reactions by pheophorbide A required 5-7.5% dimethylformamide for optimal reactivity. Catalytic photoreduction of 2,3-dimethoxy-5-methyl-p-benzoquinone by methylene blue or pheophorbide A and tertiary amine electron donors was observed. Among the chlorophyll metabolites, pheophorbide A was more effective than chlorin e6 or sodium copper chlorophyllin in photo-oxidation of dopamine and photoreduction reactions. Singlet oxygen inhibited lactate dehydrogenase A activity, and higher molecular weight protein cross-links were observed on SDS-PAGE. Hydrocaffeic acid competed with lactate dehydrogenase A for reaction with singlet oxygen produced by methylene blue; however, no protection by hydrocaffeic acid (HCA) was observed when pheophorbide A was used. Cysteine modification of lactate dehydrogenase A by the o-quinone of hydrocaffeic acid was detected using a redox cycling stain. Inclusion of an electron donor decreased protein labeling.
In vitro mechanistic research is mostly performed without taking into consideration the potential influence of cell culture media and/or their supplements and therefore, interactions between compounds of interest and medium ingredients may be overlooked. Isoproterenol (isoprenaline) is a synthetic catecholamine used as sympathomimetic drug that stimulates β-adrenergic receptors and is widely used in biomedical research. Clinical studies have shown that isoproterenol is rapidly metabolized in the human body with a plasma half-life of about 2–5 min. However, despite its use in many in vitro and ex vivo studies, the stability of isoproterenol in cell culture media has not been characterized. Our results show a decrease of isoproterenol concentration in RPMI medium but high stability of the compound in TexMACS medium. The isoproterenol oxidation product isoprenochrome forms during treatment in both media. However, isoprenochrome formation is significantly lower in TexMACS medium. The effective level of isoproterenol and the formation of oxidation products might explain the discrepancies observed in isoproterenol-induced genotoxicity and cytotoxicity.
Successful regeneration of load-bearing bone defects remains a major challenge in clinical orthopaedics. Designing biologically active 3-dimensional scaffolds possessing physiological responsiveness can potentially overcome current limitations. Here, we have described a novel approach to fabricate scaffolds with modified nanosheets reinforced on mechanically customized thermoplastic polymer-based 3D printed constructs. In this article, we have developed polydopamine-reduced graphene oxide (PD-RGO) reinforced 3D printed PLA scaffold for bone tissue construction. RGO was synthesized by reduction of GO under alkaline conditions using dopamine. 3D printed polylactic acid (PLA) scaffold with defined porosity was doped with PD-RGO. The designed scaffold was studied for its physiochemical properties and human umbilical cord-derived mesenchymal stem cell (hMSC) behaviour within the scaffold. In vitro hMSC studies revealed the influence of fibre direction and nanocoating on directional cell growth and proliferation. The fabricated scaffold showed antioxidant property along with pro-angiogenic and osteoinductive potential. The designed scaffold also successfully prevented the formation of biofilm. In vivo heterotopic implantation of the differentiated hMSC loaded scaffold confirmed the biocompatibility and bio functionality of the scaffold. In summary, the designed nanoplates doped 3D printed scaffold displays stem cell responsive, integrative and pro-regenerative multi functionalities, thereby, exhibiting potential application as bone tissue regeneration treatment alternative.
I: Biochemistry of Transmitter Molecules.- Introduction: Role of Chemical Neurotransmission in Brain Function.- References.- Classical Transmitters and Neuromodulators.- Process of Synaptic Transmission.- Classification of Synaptic Messengers.- Dale's Principle.- Definitions.- Classical Neurotransmitters.- Acetylcholine.- Amino Acids.- Monoamines.- Non-classical Transmitters.- Neuromodulators.- Characteristics of Neuromodulators.- Presynaptic Modulation.- Postsynaptic Modulation.- Conclusions.- References.- Neuropeptides.- Differences Between Peptidergic Neurones and Those Containing Classical Neurotransmitters.- One Neuropeptide Gene: Multiple Products.- Opioid Peptides.- Distribution and Functions of Neuropeptides.- Coexistence of Peptides and Classical Neurotransmitters.- Conclusions.- References.- Methods in the Mapping of Neurotransmitter Systems in the Brain.- Techniques Depending on Degeneration.- Studies Which Illustrate Complete Neurones.- Methods Which Depend on Axonal Transport.- Multiple Outputs from One Site.- Chemical Specification of Neurons.- Neurotoxins.- Immunohistochemistry.- Anterograde Tracing.- Regional Energy Metabolism.- References.- II: Function and Dysfunction.- Molecular Aspects of Central Neurotransmitter Function.- Approaches to the Study of Central Neurotransmitter Action.- Mechanisms of Neurotransmitter Action.- References.- Clinical Relevance.- The Functional Psychoses.- Degenerative Disorders and Dementia.- Conclusions.- References.- Normal and Disordered Central Neurotransmitter Function Studied through the Neuroendocrine Window of the Brain.- Basic Studies.- Clinical Studies.- Summary and Conclusions.- References.- Problems and Prospects.- Errors and Deficiencies in Concepts.- Errors in Techniques and Interpretation of Data.- Prospects.- References.
The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is frequently used to identify free radicals that are generated photochemically using dyes as photosensitizers. When oxygen is present in such systems, singlet oxygen (1O2) may be produced and can react with DMPO. We have studied the reaction of DMPO with 1O2 in aqueous solutions over a wide range of pH, using micellar Rose Bengal (pH 2−13) and anthrapyrazole (pH < 2) as photosensitizers. We found that DMPO quenches 1O2 phosphorescence (kq = 1.2 × 106 M-1 s-1), thereby initiating oxygen consumption that is slow at pH 10 but increases about 10-fold at pH < 6. This oxygen consumption is a composite process that includes efficient oxidation of both DMPO and its degradation products. The oxidation products include both products in which the DMPO pyrroline ring remains intact (DMPO/•OH and 5,5-dimethyl-2-oxo-pyrroline-1-oxyl (DMPOX) radicals) and those in which it becomes opened (nitro and nitroso products). The nitroso product itself strongly quenched 1O2 phosphorescence, while (photo)decomposition of the nitroso group, presumably to nitric oxide (NO•), produced nitrite as a minor product. We propose that 1O2 adds to the >CN(O) bond in DMPO, producing a biradical, >C(OO•)N•(O). This biradical may follow one of two pathways: (i) It may be protonated and rearrange to a strongly oxidizing nitronium-like moiety, which could be reduced to the DMPO hydroperoxide radical DMPO/•O2H while oxidizing another DMPO moiety to ultimately form DMPOX. The DMPO/•O2H could undergo further redox decomposition, e.g. via the known Fenton-like reaction, to produce both free •OH radical and the DMPO/•OH radical. (ii) The biradical >C(OO•)N•(O) may cyclize to a 1,2,3-trioxide (ozonide), which could open the pyrroline ring to form 4-methyl-4-nitropentan-1-al and 4-methyl-4-nitrosopentanoic acid. Because the oxidation of DMPO by 1O2 leads to both rapid O2 depletion and the formation of transients and products that might interfere with trapping and identification of free radicals, DMPO should be used with caution in systems where 1O2 is produced.
A variety of in vitro and in vivo studies demonstrate that dopamine is a toxic molecule that may contribute to neurodegenerative disorders such as Parkinson's disease and ischemia-induced striatal damage. While much attention has focused on the fact that the metabolism of dopamine produces reactive oxygen species (peroxide, superoxide, and hydroxyl radical), growing evidence suggests that the neurotransmitter itself may play a direct role in the neurodegenerative process. Oxidation of the dopamine molecule produces a reactive quinone moiety that is capable of covalently modifying and damaging cellular macromolecules. This quinone formation occurs spontaneously, can be accelerated by metal ions (manganese or iron), and also arises from selected enzyme-catalyzed reactions. Macromolecular damage, combined with increased oxidant stress, may trigger cellular responses that eventually lead to cell death. Reactive quinones have long been known to represent environmental toxicants and, within the context of dopamine metabolism, may also play a role in pathological processes associated with neurodegeneration. The present discussion will review the oxidative metabolism of dopamine and describe experimental evidence suggesting that dopamine quinone may contribute to the cytotoxic and genotoxic potential of this essential neurotransmitter. J. Neurosci. Res. 55:659–665, 1999. © 1999 Wiley-Liss, Inc.
Singlet oxygen (1O2) can be quenched by water, lipids, proteins, nucleic acids and other small molecules. Polyunsaturated fatty acids (PUFA) of cells principally quench 1O2 by chemical mechanisms, producing lipid hydroperoxides, while proteins physically and chemically quench 1O2. Because cell lines can have different PUFA and protein levels, we hypothesized that 1O2 toxicity will vary between cell lines. We used Photofrin® as a source of 1O2. Exposure of nine different leukemia cell lines (CEM, HEL, HL-60, K-562, KG-1, L1210, Molt-4, THP-1 and U-937) to Photofrin and light results in changes in membrane permeability (trypan blue) that vary with cell line. The greater the lipid content of the cell line, the less susceptible they are to membrane damage. When the cell media was supplemented with docosahexaenoic acid (DHA, 22:6), the overall unsaturation of cellular lipids increased. Photofrin and light resulted in increased radical formation in these supplemented cells compared to controls; however, there was no difference in membrane permeability between DHA-supplemented and control cells. Lipid-derived radical formation (electron paramagnetic resonance spin trapping) was cell line dependent; but no correlation between lipid content of cells and radical formation was found. However, we found that the greater the protein content of cells the more they were protected against membrane damage induced by Photofrin photosensitization. This suggests that cellular proteins are a key target for 1O2-mediated toxicity. A remarkable observation is that cell size correlates inversely with ability of cells to cope with a given flux of 1O2.
Proteins comprise approximately 68% of the dry weight of cells and tissues and are therefore potentially major targets for photo-oxidation. Two major types of processes can occur with proteins. The first of these involves direct photo-oxidation arising from the absorption of UV radiation by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated by the transfer of energy to ground state (triplet) molecular oxygen by either protein-bound, or other, chromophores. The basic principles behind these mechanisms of photo-oxidation of amino acids, peptides and proteins and the potential selectivity of damage are discussed. Emphasis is placed primarily on the intermediates that are generated on amino acids and proteins, and the subsequent reactions of these species, and not the identity or chemistry of the sensitizer itself, unless the sensitizing group is itself intrinsic to the protein. A particular system is then discussed – the cataractous lens – where UV photo-oxidation may play a role in the aetiology of the disease, and tryptophan-derived metabolites act as UV filters.
Levodopa is the most effective medication for Parkinson's disease (PD). In contrast, there is evidence that levodopa and its metabolites such as dopa/dopamine quinone are toxic for nigral neurons based on in vitro studies. Moreover, there is growing evidence that oxidative stress and mitochondrial dysfunction contribute the pathogenesis of PD. Thus, studies for oxidative stress give us good information for elucidating the pathogenesis of PD. In this regard, it is mandatory to develop markers such as 4-hydroxy-nonenal (HNE). HNE is a product of lipid peroxidation. Indeed, immunohistochemical studies have revealed that HNE-modified proteins accumulate within ragged red fibers (RRFs). This finding indicated that mitochondrial impairment may be linked to oxidative stress. Moreover, HNE-modified proteins accumulate in nigral neurons. In PD, mitochondrial dysfunction such as complex I deficiency has also been reported. In addition, HNE can modify alpha-synuclein (SNCA). Subsequently, this modification may trigger the aggregation of this protein. At a minimum, this modification could be associated with oligomer formation or fibrillation of SNCA.
Singlet oxygen was generated by means of rose bengal under irradiation by visible light. N(1)-acetyl-5-methoxykynuramine (AMK) was rapidly destroyed by this reactive oxygen species, whereas its formylated precursor, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), was remarkably inert. At photon fluence rates of 1400 mumol photons/m(2)s, and using 20 mum rose bengal, most of initially 0.2 mm AMK was destroyed within 2 min, whereas AFMK remained practically unchanged for much longer periods of time. Competition experiments with other scavengers revealed the following order of reactivity towards singlet oxygen: diazabicyclo-[2,2,2]-octane (DABCO) < imidazole < 4-ethylphenol < N(alpha)-acetylhistidine < histidine < melatonin < AMK, the last one being about 150 times more effective than DABCO. Contrary to the oxidation in free radical-generating systems, AMK did not form adducts with the tyrosine side chain fragment, 4-ethylphenol, under the influence of singlet oxygen. In UV-exposed cells (keratinocytes, plant cells) it is likely to be more rapidly destroyed by singlet oxygen than formed from AFMK.