Hydroethidine as a probe for measuring superoxide formation rates during air oxidation of myricetin and quercetin

Article abstract of DOI:10.1016/j.tetlet.2011.08.044

In the presence of the flavonols myricetin and quercetin, oxidation of hydroethidine (HE) by superoxide yielded ethidium (E⁺) instead of 2-hydroxyethidium (2-OH-E⁺). As a known pro-oxidant, myricetin alone was also found to be able to catalyze air oxidation of HE yielding exclusively E⁺. The reaction is inhibited by added superoxide dismutase, suggesting that superoxide is involved in the rate limiting step of the oxidation.

Full text of DOI:10.1016/j.tetlet.2011.08.044

Tetrahedron Letters 52 (2011) 5384–5387
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Tetrahedron Letters
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Hydroethidine as a probe for measuring superoxide formation rates
during air oxidation of myricetin and quercetin
⇑
Yi Ling Quek, Dejian Huang
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
In the presence of the flavonols myricetin and quercetin, oxidation of hydroethidine (HE) by superoxide
yielded ethidium (E+) instead of 2-hydroxyethidium (2-OH-E+). As a known pro-oxidant, myricetin alone
was also found to be able to catalyze air oxidation of HE yielding exclusively E+. The reaction is inhibited
by added superoxide dismutase, suggesting that superoxide is involved in the rate limiting step of the
oxidation.
Received 18 June 2011
Revised 3 August 2011
Accepted 8 August 2011
Available online 16 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Pro-oxidant
Superoxide
Myricetin
Hydroethidine
Fluorescent probe
ꢀꢁ
Fluorescent probes capable of sensing reactive oxygen species
(ROS), particularly hydrogen peroxide and superoxide, are needed
because of the critical importance of these ROS in biological sys-
tems.1,2 To delineate the mechanisms of reactive oxygen species,
the availability of selective probes targeting specific ROS is impor-
tant. For the past 40 years, 2,7-dichlorodihydrofluorescein has
been commonly used as a redox sensitive probe; its usage however
has been controversial due to its lack of selectivity.3
for measuring O2 in cell lines.9 Indeed, HE was used as the probe
ꢀꢁ
of choice for assaying O2 over the last decade. However, the dis-
covery that the oxidation product of HE was 2-OH-E+ and not E+
has put past studies that utilized HE into question.10 2-OH-E+ is
ꢀꢁ
now regarded as the sole oxidation product due to O2 and its con-
centration is suggested to be an indication of O2ꢀꢁ concentration in a
wide range of sample types including soils and animal tissues.11,12
It was later found that HE is an unsuitable probe for quantifying
intracellular superoxide due to many interfering factors.13 We re-
port herein that in the presence of polyphenolic antioxidants, the
Although there are fluorescent probes available which are
highly selective for H2O2 sensing,4,5 detection and quantification
ꢀꢁ
ꢀꢁ
of O2 has been proven, time and again, a nontrivial task. Fe(III)–
oxidation product of HE by O2 is solely E+, instead of 2-OH-E+.
cytochrome c, lucigenin and luminol as well as hydroethidine are
Our results show that E+, instead of 2-OH-E+, is a good indicator
of the superoxide activity promoted by air oxidation of myricetin.
Polyphenolic compounds are known to exert pro-oxidant activ-
ity through the generation of free radicals.14–16 Indeed, mixing
such examples. Being a mild reductant (Eo = ꢁ0.137 V),6 O2
ꢀꢁ
reduces Fe(III)–cytochrome c to its ferrous counterpart; this was
initially applied in the quantification of superoxide and is still used
as a standard assay in quantifying the activity of superoxide dis-
mutase.7 However, reductants such as polyphenolic compounds
can lead to false positive results by reducing Fe(III). Lucigenin
and luminol, the once frequently used nitro-blue tetrazolium
equimolar amounts of myricetin (50 lM) and HE in pH 7.40 phos-
phate buffer for 2 h at 37 °C, led to clean formation of E+ with no
detectable amount of 2-OH-E+, as revealed by HPLC analysis
(Fig. 1). Addition of KO2 to the HE solution under the same condi-
tions yielded mainly 2-OH-E+ with E+ as the minor product (Fig. 1).
Yet, when equal molar amounts of the flavonols, myricetin and
quercetin were mixed with HE immediately before the addition
of excess KO2, E+ was the only observed product (Scheme 1). The
progress of the reaction can be conveniently monitored by study-
ing the fluorescence signals of E+. In the presence of a large excess
of HE, the reaction was first order with respect to myricetin con-
centrations (Fig. 2). The plot of the rate of E+ formation versus
the concentration of myricetin gave a straight line and the slope
was the apparent rate constant of E+ formation. To test if
(NBT2+ 8
) and chemiluminescent probes, have similar drawbacks
and do not provide reliable results.9
ꢀꢁ
On the other hand, O2 oxidation of hydroethidine (HE) to ethi-
dium (E+) provided a seemingly attractive alternative due to there
being no interference reported from reductants often present in
the biological sample matrix. It was thus suggested to be suitable
⇑
Corresponding author. Tel.: +65 6516 8821; fax: +65 6775 7895.
E-mail address: chmhdj@nus.edu.sg (D. Huang).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.08.044

Y. L. Quek, D. Huang / Tetrahedron Letters 52 (2011) 5384–5387
5385
Figure 1. HPLC chromatograms of E+, a potassium superoxide induced oxidation product of HE and the product formed from oxidation of HE by myricetin. (A) Mixtures of HE
(50 lM) and myricetin (50 l lM) in phosphate buffer (pH 7.4). (C) Solution containing HE (50 lM) and KO2
M) in phosphate buffer (pH 7.4). (B) HPLC trace of authentic E+ (50
(excess) in phosphate buffer (pH 7.4). (D) Same as in (C) but spiked with authentic E+ (50
lM). The detector was set at 290 nm.
H2N
NH2
air saturated pH 7.4 buffer
37 °C,myricetin
N
Et
7o
C
3
,
4
.
7
H
p
E+
,
O2
flavonol
K
exclusive product
H2N
NH2
K
O2
,
OH
NH2
p
H
N
H
7
.
4
,
3
Et
7 o
C
H2N
N
HE
Et
2-OH-E+
Scheme 1. Reaction products of HE with superoxide anion in the presence or absence of myricetin or quercetin.
4.4
0.16
A
B
3.6
2.8
2.0
1.2
0.4
0.12
0.08
0.04
0.00
0
4
8
12
16
0.0
0.2
0.4
0.6
0.8
Time (min)
Myricetin (µM)
Figure 2. Kinetics of the HE/myricetin reaction in phosphate buffer (pH 7.4) monitored by fluorescence detection at kex = 485 nm, kem = 645 nm. (A) Kinetic traces with
[HE] = 25.6 M and variable myricetin concentration (j: 0.833 M). (B) Oxidation rate of HE as a function of myricetin
l
lM; ꢀ: 0.417 lM; N: 0.208 lM; ꢂ: 0.104 l
concentration in the absence (ꢀ) and presence (j) of SOD (3 U/mL). n = 3.
ꢀꢁ
O2 was involved in the oxidation, superoxide dismutase (SOD)
was first mixed with hydroethidine before myricetin was added;
in the presence of SOD the oxidation rate decreased dramatically
(Fig. 2B). This result clearly demonstrated involvement of O2ꢀꢁ. It
is also apparent from Figure 2 that the formation of E+ was much
greater with respect to the amount of myricetin added, indicating
that myricetin acted as a catalyst for the reaction. This was verified
independently by monitoring the decomposition kinetics of
myricetin using UV–vis absorbance at 376 nm (Fig. 3A). Indeed,
myricetin decomposition was slowed down in the presence of
HE, indicating that myricetin was regenerated during HE oxidation.
The best turn over number we measured was about 9 when 6.25%
(mol) of myricetin was used to catalyze the oxidation. It is known
that myricetin can generate free radicals under basic conditions
due to deprotonation of myricetin to its phenolate, which is more
sensitive to oxidation.14 We also observed that the HE oxidation
rate was highly pH dependent (Fig. 3B). The midpoint of this curve
was found at pH 7.53, which is close to the reported pKa1 of
myricetin (7.73).17 Consistently, we observed similar trends of
pH dependency on HE oxidation. The increasing pH value leads
to increasing concentrations of the phenolates, and hence, increas-
ing rates of radical formation through single electron reduction of

5386
Y. L. Quek, D. Huang / Tetrahedron Letters 52 (2011) 5384–5387
1.0
0.9
0.8
0.7
0.6
0.5
6.0
A
B
4.0
2.0
0.0
0
2
4
6
8
4.5
5.5
6.5
pH
7.5
8.5
Time (min)
Figure 3. (A) Normalized myricetin concentration at 376 nm in the absence (N) and presence (j) of HE (30
lM) in phosphate buffer (pH 7.4); initial [myricetin] = 7.5 lM. (B)
pH dependency of HE oxidation by myricetin expressed as the increase in rate of the fluorescence signal at 37 °C, buffered solution.
dissolved molecular oxygen. It is reported that the rate of polyphe-
nol autoxidation is highly sensitive to the pH of the medium stud-
ied.14 We measured the oxidation potential (Epa) of myricetin at pH
7.40 and 5.50 to be +0.030 and +0.179 V, respectively, using a
three-electrode cyclic voltametry system made up of a glassy car-
bon working electrode, a platinum counter electrode and a silver/
silver chloride reference electrode. Therefore, deprotonation of
myricetin enables it to react with dissolved molecular oxygen.
Taken together, we propose a reaction mechanism for the
myricetin-catalyzed oxidation of HE as follows (Scheme 2). The
reaction of HE with the superoxide generated from the autoxidation
of a myricetin anion leads to HEꢁ+ as suggested by Zielonka et al.18
HEꢁ+ may react with another superoxide to form 2-OH-E+.19 Alterna-
tively, a myricetin semiquinone radical may abstract a hydrogen
atom from HEꢁ+ and regenerate myricetin and form E+. The reaction
cycle stops when myricetin is irreversibly oxidized to myricetin
quinone, which does not react with HE, as we found independently.
The formation of E+ as the sole product of HE air oxidation
promoted by flavonols makes HE ideal for quantifying radical
generating pro-oxidation activity of flavonols. The dose response
relationship between HE oxidation and the flavonol concentration
provides a highly sensitive method to quantify their pro-oxidant
activity. Under the assay conditions, the HE oxidation rate is
pseudo first order with respect to the flavonol concentration; the
apparent rate constant, k0, of the reaction can be defined as the rad-
ical generating pro-oxidant activity. As shown in Table 1, the pro-
oxidant activity increases with the increasing number of hydroxy
groups on the B-ring, with myricetin being the most active. In con-
trast, galangin and kaempferol have no detectable pro-oxidant
activity. This shows that the ortho-trihydroxy moiety on the B-ring
is critical for the high radical forming pro-oxidant activity of
myricetin. Our results are consistent with the earlier study by Can-
ada et al. and this enhances the validity of HE as a probe for rapid
and convenient quantification of radical forming pro-oxidant activ-
ity.14 The pro-oxidant activity correlates with the pKa values of
myricetin and quercetin which fall in the range of 7.95–6.05 (Table
1). At pH 7.40, there are significant amounts of deprotonated flavo-
nols for all four compounds, but their reactivity with oxygen is
apparently different, as revealed by their oxidation potentials
(Epa), which increase from myricetin to galangin. The oxidation
potentials at pH 5.50 (due to phenols) are all much higher than that
at pH 7.40 (for phenolates), accounting for the lack of pro-oxidant
activity under weak acidic conditions, which is typically the case in
plant tissues where these polyphenolic compounds are normally
found. The pH switch, after these compounds enter the small intes-
tine of animals and in the bloodstream, will activate the radical
generating pro-oxidant activity of these compounds. The toxicol-
ogy effects of such reactivity switches are worthy of further study.
In summary, we have demonstrated that HE is oxidized to E+
due to the pro-oxidative effect of flavonols under physiological
conditions and is a sensitive fluorescent probe for rapid quantifica-
tion of the radical-forming activity of polyphenolic compounds.
Structurally, the number of hydroxy groups on the B-ring of
flavonols has a great impact on their pro-oxidant activity. We are
OH
OH
H2N
NH2
HO
O
OH
N
Et
OH
pH dependent
OH O
OH
myricetin
E+
O
H atom transfer
HO
O
OH
OH
O-
OH O
O2
H2N
NH2
O2
H2N
O
NH2
HO
O
N
N
H
H
OH
Et
Et
O2
OH
OH O
HE
HE
semiquinone
O2
catalyst deactivation
through dismutation
OH
NH2
O
O
H2N
HO
O
OH
N
Et
OH
OH O
2-OH-E+
Scheme 2. Proposed reaction mechanism for the oxidation of HE in the presence of myricetin.

Y. L. Quek, D. Huang / Tetrahedron Letters 52 (2011) 5384–5387
5387
Table 1
References and notes
pKa1, oxidation potential, and pseudo-first order rate constants of the oxidation of
flavonols by oxygen at 37 °C, pH 7.40
1. Pryor, W. A.; Houk, K. N.; Foote, C. S.; Fukuto, J. M.; Ignarro, L. J.; Squadrito, G. S.;
Davies, K. J. A. Am. J. Physiol Regul. Integr. Comp Physiol. 2006, 291, 491–511.
2. Fridovich, I. Science 1978, 201, 875–880.
Flavonol
k0 (minꢁ1
)
pKa1
Oxidation potential (V, vs Ag/AgCl)
3. Chen, X.; Zhong, Z.; Xu, Z.; Chen, L.; Wang, Y. Free Radical Res. 2010, 44, 587–
604.
4. Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc.
2005, 127, 16652–16659.
5. Du, L.; Li, M.; Zheng, S.; Wang, B. Tetrahedron Lett. 2010, 51, 1152–1154.
6. Petlicki, J.; van de Ven, T. G. M. J. Chem. Soc., Faraday Trans. 1998, 94, 2763–
2767.
pH 5.50
pH 7.40
Myricetin
Quercetin
Kaempferol
Galangin
0.194 0.015
0.031 0.001
Not detectable
Not detectable
7.95
6.05
6.89
7.78
0.179
0.256
0.324
0.500
0.030
0.089
0.158
0.388
7. McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049–6055.
8. Seki, H.; Ilan, Y. A.; Ilan, Y.; Stein, G. Biochim. Biophys. Acta 1976, 440, 573–586.
9. Benov, L.; Sztejnberg, L.; Fridovich, I. Free Radical Biol. Med. 1998, 25, 826–831.
10. Zhao, H.; Kalivendi, S.; Zhang, H.; Joseph, J.; Nithipatikom, K.; Vasquez-Vivar, J.;
Kalyanaraman, B. Free Radical Biol. Med. 2003, 34, 1359–1368.
11. Georgiou, C. D.; Papapostolou, I.; Grintzalis, K. Nat. Protoc. 2008, 3, 1679–1692.
12. Robinson, K. M.; Janes, M. S.; Beckman, J. S. Nat. Protoc. 2008, 3, 941–947.
13. Zielonka, J.; Kalyanaraman, B. Free Radical Biol. Med. 2010, 48, 983–1001.
14. Canada, A. T.; Giannella, E.; Nguyen, T. N.; Mason, R. P. Free Radical Biol. Med.
1990, 9, 441–449.
15. Ong, K. C.; Khoo, H.-E. Gen. Pharmacol. 1997, 29, 121–126.
16. Green, J. R.; Branch, G. E. K. J. Am. Chem. Soc. 1941, 63, 3441–3444.
17. Stöggl, W. M.; Huck, C. W.; Stecher, G.; Bonn, G. K. Electrophoresis 2006, 27,
787–792.
18. Zielonka, J.; Sarna, T.; Roberts, J. E.; Wishart, J. F.; Kalyanaraman, B. Arch.
Biochem. Biophys. 2006, 456, 39–47.
positioned to explore the application of HE as an ultrasensitive
probe in quantifying the pro-oxidant activity of common beverages
that are known to generate hydrogen peroxide as observed.20
Acknowledgments
D.H. thanks the Singapore Ministry of Education (Grant No.
R-143-000-299-112) and SERC(072-101-0015) of A⁄Star Singapore
for financial support. Y.L.Q. thanks the National University of
Singapore for research scholarship.
Supplementary data
19. Zielonka, J.; Zhao, H.; Xu, Y.; Kalyanaraman, B. Free Radical Biol. Med. 2005, 39,
853–863.
20. Long, L. H.; Halliwell, B. Free Radical Res. 2000, 32, 463–467.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.08.044.