# Oxidation of Quercetin by Myeloperoxidase

Authors: Tatjana Momić; Jasmina Savić; Vesna Vasić
Journal: Research Letters in Physical Chemistry
 (2009)

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## Abstract

Study of effect of myeloperoxidase on quercetin at pH 6.0 indicated quercetin oxidation via the formation of the oxidation product. The stability of quercetin and oxidation product was investigated as a function of time by using spectrophotometric and HPLC techniques. The apparent pseudo first-order rate constants were calculated and discussed.


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## Body

## 1. Introduction

Flavonol
quercetin (Q) (3,5,7,3′,4′-pentahydroxyflavone) (Scheme 1) due to its phenolic
structure is a strong antioxidant and free radical scavenger [1]. Oxidation of
quercetin during its antioxidative functions is usually accompanied by the
production of the quercetin radical anion, superoxide, and hydrogen peroxide [2].
Quercetin undergoes autoxidation—the nonenzymatic reaction with atmospheric
oxygen. Also, it is known that one-electron oxidation of quercetin is catalyzed
by different peroxidases like lactoperoxidase (LPO) [3] and horseradish
peroxidase (HRP) [4–7] in the presence of H2O2.Scheme 1
Oxidation of
quercetin by MPO/H2O2 system (modified from [15]).The heme enzyme myeloperoxidase (MPO) is
oxidant enzyme in the process of inflammation and atherogenesis [8]. Myeloperoxidase
is relatively nonspecific with respect to its reducing substrates. This enzyme
is able to oxidize different substrates among which anilines and phenols [9–11].
This letter deals with quercetin oxidation by myeloperoxidase-H2O2 system.


## 2. Experimental

### 2.1. Chemicals

Quercetin dihydrate (Sigma-Aldrich) of the highest quality available
(98%) was used without purification.1×10−3 M
stock solution of quercetin was prepared in methanol immediately before the
experiments. For all experiments, freshly prepared solutions of quercetin
were made by dilution of the appropriate amount of the stock solution with
phosphate buffer at pH 6.0. Myeloperoxidase was purified from human neutrophils
to a purity index (A430/A280) greater than 0.70 as
described previously [12]. Its concentration was calculated using ε430=91000M−1cm−1 per heme [13]. Hydrogen peroxide
solutions were prepared daily by diluting a stock solution, and the
concentration was determined using ε240=43.6M−1cm−1 [14]. Redistilled water was used in all experiments.


### 2.2. Oxidation of Quercetin by Myeloperoxidase

Quercetin (5×10−5 M) was incubated in 50 mM phosphate buffer, pH 6.0, with various
concentrations of MPO. Reaction was started by the addition of 50 μM H2O2,  and
UV absorbance changes were recorded. For the HPLC analysis, reaction was stopped
after 30 minutes by adding catalase (100 μg/mL). The reaction mixture was
centrifuged for 2 minutes at 10000 rpm. The clear supernatant was analyzed by HPLC.


### 2.3. HPLC Analysis

HPLC equipment consisted of an HP
1100 Series chromatograph coupled with a DAD. Chromatographic separations were
run on a C18 Pinnacle ODS column (Restek, 250 mm × 4.6 mm, 5μm) using an 80 : 20
mixture of 2 vol% H3PO4 (A)  and acetonitrile (J.T. Baker) (B) as the eluent for the first 2 minutes. The linear  gradient was
applied from 20% to 45% B from 2 to 7 minutes. An isocratic 55 : 45 mixture was
applied between 7 and 13 minutes. The eluent flow rate was 1.0 mL min−1, and the injection volume was 10 μL. The elutions were monitored with DAD at
different wavelengths between 200 and 450 nm.


## 2.1. Chemicals

Quercetin dihydrate (Sigma-Aldrich) of the highest quality available
(98%) was used without purification.1×10−3 M
stock solution of quercetin was prepared in methanol immediately before the
experiments. For all experiments, freshly prepared solutions of quercetin
were made by dilution of the appropriate amount of the stock solution with
phosphate buffer at pH 6.0. Myeloperoxidase was purified from human neutrophils
to a purity index (A430/A280) greater than 0.70 as
described previously [12]. Its concentration was calculated using ε430=91000M−1cm−1 per heme [13]. Hydrogen peroxide
solutions were prepared daily by diluting a stock solution, and the
concentration was determined using ε240=43.6M−1cm−1 [14]. Redistilled water was used in all experiments.


## 2.2. Oxidation of Quercetin by Myeloperoxidase

Quercetin (5×10−5 M) was incubated in 50 mM phosphate buffer, pH 6.0, with various
concentrations of MPO. Reaction was started by the addition of 50 μM H2O2,  and
UV absorbance changes were recorded. For the HPLC analysis, reaction was stopped
after 30 minutes by adding catalase (100 μg/mL). The reaction mixture was
centrifuged for 2 minutes at 10000 rpm. The clear supernatant was analyzed by HPLC.


## 2.3. HPLC Analysis

HPLC equipment consisted of an HP
1100 Series chromatograph coupled with a DAD. Chromatographic separations were
run on a C18 Pinnacle ODS column (Restek, 250 mm × 4.6 mm, 5μm) using an 80 : 20
mixture of 2 vol% H3PO4 (A)  and acetonitrile (J.T. Baker) (B) as the eluent for the first 2 minutes. The linear  gradient was
applied from 20% to 45% B from 2 to 7 minutes. An isocratic 55 : 45 mixture was
applied between 7 and 13 minutes. The eluent flow rate was 1.0 mL min−1, and the injection volume was 10 μL. The elutions were monitored with DAD at
different wavelengths between 200 and 450 nm.


## 2.4. UV-VIS Spectroscopic Studies

UV spectra were recorded on a
Perkin Elmer Lambda 35 UV-Vis spectrophotometer equipped with thermostatted
quartz cell. The temperature in
the cell was kept at25±0.05°C with a water-thermostatted bath.


## 3. Results and Discussion

We investigated the effect of MPO
on5×10−5 M quercetin in phosphate buffer, pH 6.0 in the presence  of
50 μM H2O2 at 25°C, spectrophotometrically and
by HPLC. The following concentrations of enzyme were used: 5, 11, 15, 20, 50,
100, 150, and 200 nM.At
the absorption spectra of samples in which concentration of MPO was ≤11 nM, two
quercetin absorption bands (254 nm and 367 nm) were observed (Figure1(a)). The
same results were yielded when MPO or H2O2 was omited
from the mixture.Absorption spectra and
HPLC chromatograms of5×10−5 M quercetin at pH 6.0 in absence (a), (b)
and presence (c), (d) of 20 nM MPO. 50 μM H2O2 was present
in all reaction solutions. Spectra were recorded over a period of 30 minutes;
arrows indicate the direction of the change. Chromatograms were recorded after
30 minutes (b) at λ367 and (d) at λ336.The absorption spectra of samples
in which concentration of MPO was ≥15 nM showed the decay of quercetin
absorption bands at 254 nm and 367 nm. At the same time simultaneous  rise of
the absorption band at 336–342 nm, depended on MPO concentration, was observed
(Figure1(c)), typical of oxidation product formation [16]. Moreover, two
well-defined  isosbestic points at 290 and 364 nm indicate that there was no
significant accumulation of intermediates in the reaction of  oxidation product
formation (Figure 1(c)). These spectral changes are analogous to those occurring
upon autoxidation of quercetin in water [16] and UV irradiation [17] as well
as oxidation with some oxidants in organic solvents [16].The
HPLC analysis of quercetin solution treated with MPO for 30 minutes (Figure1(d))
revealed that concentration of quercetin (retention time 10 minutes) was
negligible. Also, formation of major oxidation product (retention time 5.26 minutes) which was more polar than quercetin was detected (Figure 1(d)). From
literature data and the obtained spectrophotometric and HPLC results in this
work, we assume that the oxidation product detectable at 336–342 nm results
from H2O addition on the p-quinonemethide
formed by H-atom abstraction at 3-OH and 4′-OH of quercetin and subsequent
rearrangement of the central ring [16]. The complete sequence of quercetin
autoxidation induced by oxidants and catalyzed by metal ions is described
earlier [15, 16] (Scheme 1).According
to spectrophotometric and HPLC experimental data, quercetin degradation
(1) and oxidation product formation (2) as the function
of time followed the relation (1)AQ,t=A0e−kt,(2)Aoxid,t=A∞(1−e−kt), where AQ,t and Aoxid,t are the absorbances
proportional to the quercetin and its oxidation  product concentrations,
respectively, after an irradiation period of time t and k is the overall
pseudo first-order rate constant. A0 and A∞ are the absorbance proportional to the initial
concentration of quercetin and the concentration of the oxidation product on
the plateau of the kinetics curve, respectively.The
kinetic curves that describe the quercetin degradation and the formation of the
oxidation product in the presence of different MPO, concentrations are shown in
Figure2. In the presence of MPO concentration that was ≥15 nM oxidation
product was formed at the first minute of reaction (Figure 2(a)). The overall
apparent first-order rate constants for the oxidation product formation and
quercetin transformation to its degradation products were determined from the
kinetic curves and are presented in Table 1. For comparison, in Table 1 we
also presented values for these constants obtained for quercetin oxidation by
UV irradiation [17].Table 1
Rate constants of
quercetin oxidation.
k(min−1)×103pHSampleQuercetin
degradationWater adduct
formationWater adduct degradation6.0Q3.61±0.18——Q + MPO (20 nM)346.00±21.15524.60±32.302.67±0.0910.0*Non-irradiated Q8.73±0.3425.12±0.93—Irradiated Q110.00±35.2596.91±0.1210.13±0.56Reference*
[17].Change of absorbance of
quercetin in the presence of various MPO concentrations in the function of
time, at pH 6.0 (a) formation of quercetin oxidation product atλ336;
Inset: 20 nM MPO and (b) degradation of quercetin oxidation product at λ367.
(a)(b)The
rate of quercetin oxidation by MPO at pH 6.0 is 3-fold faster compared with the
rate of quercetin oxidation by UV irradiation at pH 10.0. Moreover, product
obtained for quercetin oxidation by MPO at pH 6.0 is more stable than product
of quercetin oxidation by UV irradiation at pH 10.0.



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*Source: 614362-2009-04-14.xml*