Interference of carbidopa and other catechols with reactions catalyzed by peroxidases

Article abstract of DOI:10.1016/j.bbagen.2018.04.007

Background: A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been fully elucidated. Methods: Reactions of bovine myeloperoxidase with o-dianisidine, bovine lactoperoxidase with ABTS and horseradish peroxidase with 4-aminoantipyrine/phenol in the presence of carbidopa, an anti-Parkinsonian drug, and other catechols, including L-dopa, were monitored spectrophotometrically and by measuring hydrogen peroxide consumption. Results: Chromophore formation in all three enzyme/substrate systems was blocked in the presence of carbidopa and other catechols. However, the rates of hydrogen peroxide consumption were not much affected. Irreversible enzyme inhibition was also insignificant. Conclusions: Tested compounds reduced the oxidation products or intermediates of model substrates thus preventing chromophore formation. This interference may affect interpretation of results of diagnostic tests in samples from patients with Parkinson's disease treated with carbidopa and L-dopa. General significance: This mechanism allows prediction of interference in peroxidase-based diagnostic tests for other compounds, including drugs and natural products.

Full text of DOI:10.1016/j.bbagen.2018.04.007

Accepted Manuscript
Interference of carbidopa and other catechols with reactions
catalyzed by peroxidases
Beata Gąsowska-Bajger, Yuki Nishigaya, Krystyna Hirsz-
Wiktorzak, Anna Rybczyńska, Toshimasa Yamazaki, Hubert
Wojtasek
PII:
DOI:
Reference:
S0304-4165(18)30100-4
doi:10.1016/j.bbagen.2018.04.007
BBAGEN 29089
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Revised date:
Accepted date:
8 September 2017
6 April 2018
6 April 2018
Please cite this article as: Beata Gąsowska-Bajger, Yuki Nishigaya, Krystyna Hirsz-
Wiktorzak, Anna Rybczyńska, Toshimasa Yamazaki, Hubert Wojtasek , Interference of
carbidopa and other catechols with reactions catalyzed by peroxidases. The address for
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appropriate. Bbagen(2018), doi:10.1016/j.bbagen.2018.04.007
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ACCEPTED MANUSCRIPT
Interference of carbidopa and other catechols with reactions catalyzed by
peroxidases
Beata Gąsowska-Bajgera, Yuki Nishigayab, Krystyna Hirsz-Wiktorzaka, Anna
Rybczyńskaa, Toshimasa Yamazakib, and Hubert Wojtaseka*
aFaculty of Chemistry
Opole University
Ul. Oleska 48
45-052 Opole
Poland
bAdvanced Analysis Center
National Agriculture and Food Research Organization (NARO)
2-1-2 Kannondai
Tsukuba, Ibaraki, 305-8602
Japan
*To whom correspondence should be addressed:
Faculty of Chemistry
Opole University
Ul. Oleska 48
45-052 Opole
Poland
phone: +48 77 452 7122
fax: +48 77 452 7101
e-mail: Hubert.Wojtasek@uni.opole.pl
Keywords: carbidopa; catechol; hydrazine; peroxidase; enzymatic assay interference;
Parkinson’s disease
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Abstract
Background: A number of compounds, including ascorbic acid, catecholamines, flavonoids,
p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based
medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been
fully elucidated.
Methods: Reactions of bovine myeloperoxidase with o-dianisidine, bovine lactoperoxidase
with ABTS and horseradish peroxidase with 4-aminoantipyrine/phenol in the presence of
carbidopa, an anti-Parkinsonian drug, and other catechols, including L-dopa, were monitored
spectrophotometrically and by measuring hydrogen peroxide consumption.
Results: Chromophore formation in all three enzyme/substrate systems was blocked in the
presence of carbidopa and other catechols. However, the rates of hydrogen peroxide
consumption were not much affected. Irreversible enzyme inhibition was also insignificant.
Conclusions: Tested compounds reduced the oxidation products or intermediates of model
substrates thus preventing chromophore formation. This interference may affect interpretation
of results of diagnostic tests in samples from patients with Parkinson’s disease treated with
carbidopa and L-dopa.
General significance: This mechanism allows prediction of interference in peroxidase-based
diagnostic tests for other compounds, including drugs and natural products.
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1. Introduction
Peroxidases are heme-containing oxidoreductases catalyzing the oxidation of a variety
of substrates with hydrogen peroxide. Mammalian peroxidases include enzymes involved
primarily in defense reactions (myeloperoxidase, MPO; eosinophil peroxidase, EPO;
lactoperoxidase, LPO) and thyroid hormone biosynthesis (thyroid peroxidase, TPO) [1]. The
antimicrobial activity of MPO, EPO, and LPO results from their ability to convert halides,
thiocyanate, and nitrite to compounds (e.g. hypohalous acids) that are highly toxic to invading
microorganisms [2, 3]. These enzymes also participate in the metabolism of xenobiotics,
including drugs [4], and some of them, particularly MPO, have been implicated in the
pathogenesis of many diseases, such as atherosclerosis, Alzheimer’s disease, Parkinson’s
disease, and cancer [5, 6]. Therefore substantial efforts have been made to develop inhibitors
of MPO with perspectives for clinical applications [7, 8].
Hydrazine derivatives have been widely used in medicine and include the primary
anti-tuberculosis drug isoniazid, the anti-cancer agent procarbazine, the anti-Parkinsonians
carbidopa and benserazide, and the older generation anti-depressants iproniazid and
phenelzine [9]. Peroxidases oxidize hydrazine derivatives generating radical products that
generally inactivate these enzymes by covalent binding to the heme prosthetic group or the
polypeptide chain [10]. The effect of phenylhydrazine has been particularly well studied on
horseradish peroxidase (HRP) [11], LPO [12] and lacrimal-gland peroxidase [13]. Substantial
efforts have also been made to elucidate the interactions of peroxidases with isoniazid [14-17]
in attempts to understand the action of this drug on the Mycobacterium tuberculosis catalase-
peroxidase (MtCP, KatG) [18]. Inhibition of peroxidases by isoniazid also led to
investigations of these enzymes with a number of benzoic acid hydrazides [19-22].
We have recently synthesized a potential tyrosinase-activated anti-melanoma prodrug
with a hydrazine linker [23]. Before designing such prodrugs we tested the reaction of
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carbidopa ((2S)-3-(3,4-dihydroxyphenyl)-2-hydrazinyl-2-methylpropanoic acid, 1, Fig. 1) to
determine the reaction pathway following the oxidation of a compound containing both a
hydrazine group and a catechol moiety by tyrosinase [24]. However, since prodrugs with such
a structure, before reaching the target tissues, would be exposed to the oxidative systems in
the blood, we also decided to test their oxidation by mammalian peroxidases. It has been
shown that carbidopa is oxidized by horseradish peroxidase [25] but interactions of this drug
COOH
NH
COOH
NH2
HO
HO
HO
HO
HO
HO
NH2
H2N
1
2
3
CF3
COOH
COOH
H
N
HO
N
N
H
HO
HO
O
N
H
CF3
OH
OH
O
4
5
6
O
O
H
NH2
N
NH2
N
NH2
N
H2N NH2
H
H
N
7
8
9
10
Figure 1. Structures of compounds used in this study: 1 – (S)-carbidopa ((2S)-3-(3,4-
dihydroxyphenyl)-2-hydrazinyl-2-methylpropanoic acid; 2 – L-dopa ((S)-2-amino-3-(3,4-
dihydroxyphenyl)propanoic acid), 3 – dopamine (2-(3,4-dihydroxyphenyl)ethylamine);
4 – HX1 (2-(3,5-bistrifluoromethylbenzylamino)-6-oxo-1H-pyrimidine-5-carbohydroxamic
acid; 5 – 3-(3,4-dihydroxyphenyl)propanoic acid; 6 – 3,4-dihydroxyphenylacetic acid;
7 – hydrazine; 8 – phenelzine (2-phenylethylhydrazine); 9 – isoniazid (isonicotinic acid
hydrazide); 10 – benzhydrazide.
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with mammalian peroxidases have not been studied so far. Here we show that HRP, bovine
MPO and LPO oxidize carbidopa to the same products as tyrosinase. We also show that this
drug and other catechol-containing compounds (2, 3, 5, and 6, Fig. 1) interfere with the
oxidation of standard peroxidase substrates, such as o-dianisidine, ABTS, and 4-amino-
antipyrine/phenol. This effect results primarily from reduction of the products or
intermediates of the oxidation of these substrates not from reversible or irreversible inhibition
of these enzymes.
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2. Materials and Methods
2.1. Reagents
Carbidopa was obtained from Sochinaz SA (Vionnaz, Switzerland). HRP, bovine
LPO, o-dianisidine, 4-aminoantipyrine, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS), L-3,4-dihydroxyphenylalanine (L-dopa), dopamine, 3-(3,4-
dihydroxyphenyl)propanoic acid, 3,4-dihydroxyphenylacetic acid, phenelzine, isoniazid, and
HPLC grade acetonitrile were purchased from Sigma-Aldrich. Hydrogen peroxide, phenol,
hydrazine hydrate, xylenol orange, buffer components and solvents were from Avantor
Performance Materials (Gliwice, Poland), SDS was from Fluka, and benzhydrazide from Alfa
Aesar. MPO was purified from bovine spleen according to published procedures [26, 27].
Tyrosinase was isolated from mushrooms as described previously [28].
2.2. Spectrophotometric analysis of the oxidation of carbidopa by peroxidases and
tyrosinase
Solutions of carbidopa were prepared fresh daily in 1 mM phosphoric acid to prevent
autooxidation. Reactions with peroxidases were carried out at room temperature in 3 ml of
50 mM Sörensen buffer, pH 6.0, with 10-500 M carbidopa in the presence of equimolar
concentration of H2O2 and 20 to 500 nM enzymes. The concentration of the enzymes was
calculated from the absorbance of their Soret bands using their extinction coefficients:
91000 M-1×cm-1 at 430 nm for bovine MPO [21], 112000 M-1×cm-1 at 412 nm for bovine
LPO [29], and 102000 M-1×cm-1 at 403 nm for HRP [30]. Reactions were initiated by the
addition of either hydrogen peroxide or the enzymes. Reactions with tyrosinase were carried
out in 50 mM sodium phosphate buffer, pH 6.8. The concentration of tyrosinase was
determined using the Bradford assay with bovine serum albumin as a standard. UV/Vis
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spectra from 250 to 700 nm were recorded in a Jasco V-650 UV-Vis spectrophotometer
immediately after mixing the reagents and then in 1 or 2 min intervals.
2.3. HPLC Analysis
The reactions with peroxidases were carried out in 0.5 ml of 50 mM Sörensen buffer,
pH 6.0, with 500 M carbidopa in the presence of 500 M H2O2 and 50 to 500 nM enzymes.
Samples were incubated at room temperature for 0-120 min and then the enzymes were
removed from the reaction mixtures by ultrafiltration on Microcons Ultracel YM-10 (Merck
Millipore). The filtrates were then separated at 30 °C on an Accucore C18 analytical column
(100 mm length, 2.1 mm diameter, 2.6 μm particle size, Thermo Scientific) with a guard
column connected to a Dionex UltiMate 3000 HPLC instrument equipped with a UV-Vis
photodiode array detector at a flow rate of 0.2 ml/min. Samples were injected into a 5 l loop
and then eluted with the following program: 5% acetonitrile in water for 3 min, 5-50%
gradient of acetonitrile in water for 14 min, followed by 90% acetonitrile in water for 1 min.
The identity of all products was confirmed by LC-MS analysis on a Dionex UltiMate 3000
HPLC connected to a Bruker Daltonik micrOTOF-Q II mass spectrometer with ESI ionization
in the positive or negative ion mode. The mass spectrometer was calibrated with sodium
formate clusters. 6,7-Dihydroxy-3-methylcinnoline and 3-(3,4-dihydroxyphenyl)-2-
methylpropanoic acid were prepared by oxidation of carbidopa with tyrosinase in a large scale
reaction (50 mg), purified as described previously [24] and used as standards.
2.4. Oxidation of model substrates in the presence of carbidopa and other catechols
The peroxidatic activity of bovine MPO was measured with o-dianisidine. The
reaction mixture consisted of 50 mM Sörensen buffer, pH 6.0, 100 M o-dianisidine, 50 M
H2O2, 2-20 nM enzyme, and various concentrations of carbidopa, L-dopa, 3-(3,4-
dihydroxyphenyl)propanoic acid or 3,4-dihydroxyphenylacetic acid (1-40 M). Changes in
absorbance at 460 nm resulting from the oxidation of o-dianisidine were measured for 30 min.
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The peroxidatic activity of bovine LPO was measured with either o-dianisidine under
conditions identical to reactions with bovine MPO or 2,2-azino-bis(3-ethylbenzothiazoline-6
sulfonic acid) diammonium salt. The reactions with 25 M ABTS were carried out in 50 mM
Sörensen buffer, pH 6.0, with 25 μM H2O2, and 1-10 nM LPO. Absorbance of the oxidized
ABTS product (ABTS+•) at 415 nm was monitored for 30 min. The same catechols as for
MPO were applied at the same concentrations.
The activity of horseradish peroxidase was tested in 50 mM phosphate buffer, pH 7.0
with 50 μM 4-aminoantipyrine, 1 mM phenol, 50 μM H2O2, 0.5 or 1.0 μg of HRP, and various
concentrations of carbidopa, L-dopa, dopamine, 3-(3,4-dihydroxyphenyl)propanoic acid or
3,4-dihydroxyphenylacetic acid. The tested compounds were applied at 10-160 μ. Spectra
from 250 to 700 nm were recorded in a Jasco V-650 UV-Vis spectrophotometer immediately
after mixing the reagents and then in 1 min intervals for 30 min. Additionally the same
reactions were performed and the absorbance at 505 nm was measured for 10-30 min.
Hydrogen peroxide consumption was measured under the same conditions as the
reactions monitored spectrophotometrically with the modified xylenol orange method
including sorbitol [31]. Aliquots of the reaction mixtures were removed at 1 min intervals,
mixed with the xylenol orange reagent, incubated in the dark for 30 min and the absorbance
was measured at 560 nm. Under the reaction conditions (25 mM sulfuric acid) the catechols
used in this study did not interfere with this assay (Fig. S1).
2.5. Analysis of meriquinone reduction
Reduction of meriquinone, the intermediate in the oxidation of o-dianisidine by
peroxidases, was performed according to published procedures [32, 33] with slight
modifications. Reactions were carried out at room temperature in 50 mM sodium acetate
buffer, pH 4.0, with 100 μM o-dianisidine, 100 μM H2O2, 20 nM MPO in the presence of
200 μM SDS (1 mM SDS used previously in this assay with HRP [32, 33] strongly inhibited
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bovine MPO) for 10 min. After that time, when the absorbance of meriquinone at 395 nM
stabilized, the tested compounds at concentration 10 μM, 25 μM or 50 μM were added. The
following compounds were analyzed: carbidopa, L-dopa, 3-(3,4-dihydroxyphenyl)propanoic
acid, 3,4-dihydroxyphenylacetic acid, hydrazine, phenelzine, benzhydrazide, and isoniazid.
Changes in absorbance at 395 nm were then measured for 10-30 min.
2.6. Analysis of irreversible enzyme inhibition
Bovine MPO or LPO at 1.5 μM concentration were incubated with various
concentrations of carbidopa (15-1500 M) in the presence of equimolar concentration of H2O2
in 50 mM Sörensen buffer, pH 6.0 in a final volume of 0.5 mL for 1 h at room temperature
and then the reaction components were separated by dialysis overnight (20-22 h) at 4°C.
Originally the enzymes were separated from the low molecular mass components by gel
filtration on Sephadex G-25 columns. However, large part of the enzyme was retained on the
column, which indicated substantial protein cross-linking, as would be expected during
oxidation of a compound containing both a catechol and a hydrazine group. This method was
therefore abandoned. After dialysis the protein concentration was determined
spectrophotometrically with the Bradford method and the UV-Vis spectra of the enzymes in
the 250-700 nm range were also recorded. The peroxidatic activity was then measured with o-
dianisidine as described above.
2.7. Molecular modeling
Molecular docking experiments of carbidopa to human MPO (PDB ID: 1D2V) [34]
and bovine LPO (PDB ID: 3Q9K) were carried out using AutoDock Vina 1.1.2 [35] and
AutoDockTools 1.5.6 [36] with standard protocols. The crystal structures with the highest
resolutions among those deposited in the Protein Data Bank were chosen as the template
structures. A 28 × 30 × 28 Å grid box with the level of exhaustiveness set at 20 was used to
cover all the space of ligand-binding pocket around the heme. The ionized form of the ligand
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generated by the wash command of Molecular Operating Environment (MOE) software
(MOE version 2014.09; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite
#910, Montreal, QC, Canada, H3A 2R7, 2014) was used for the docking experiments. The
rotatable bonds of the small molecules were applied as default settings. The graphical
representations were prepared by PyMOL (version 1.7.0.1, Schrödinger, LLC.).
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3. Results
3.1. Oxidation of carbidopa by peroxidases and tyrosinase
Spectrophotometric analysis of mixtures of bovine MPO or LPO with carbidopa
showed slow changes of absorbance in the visible and near-UV region with a maximum of
absorption at ca. 365 nm (Fig. S2A and B), seen previously during reactions of this compound
with HRP [25] (Fig. S2C). These spectra were very similar to the spectrum of the product
obtained after oxidation of carbidopa by tyrosinase (Fig. S2D), which we have previously
identified as 6,7-dihydroxy-3-methylcinnoline [24]. The reaction rates were, however, much
smaller than in the case of tyrosinase (35-fold for bovine MPO and 16-fold for bovine LPO)
and also smaller than for HRP (11-fold for bovine MPO and 5.2-fold for bovine LPO, based
solely on the absorption at 365 nm). We also analyzed the reaction mixtures by HPLC to
determine their composition. The reaction mixture with tyrosinase contained products
identified previously [24], i.e. 6,7-dihydroxy-3-methylcinnoline, 3-(3,4-dihydroxyphenyl)-2-
methylpropanoic acid, and 3,4-dihydroxyphenylacetone (Fig. S3D). These compounds were
also present in reaction mixtures with peroxidases, although in much smaller quantities (Fig.
S3A-C), as was expected from the spectrophotometric analysis.
3.2. Spectrophotometric analysis of the influence of carbidopa on the peroxidatic activity
of bovine MPO and LPO
Since carbidopa was oxidized very slowly by the bovine enzymes, we decided to
investigate its potential to inhibit them using model substrates (o-dianisidine and ABTS). In
the spectrophotometric analysis the presence of carbidopa resulted in a dramatic decrease of
absorbance in reactions with both enzymes (Fig. 2). Essentially no absorption in the range of
the oxidation products of o-dianisidine in the case of bovine MPO and o-dianisidine or ABTS
in the case of bovine LPO was detected in the initial phase of the reactions. However, at lower
carbidopa concentrations the activity was recovered to a substantial degree after a lag period,
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as would be expected for a compound which also served as a substrate for both enzymes. At
higher concentrations the lag period was no longer proportional to concentration and was even
extended indefinitely (until the end of the measurement time - up to 120 min). These results
indicated that carbidopa may act as a suicide substrate, just as other hydrazine derivatives, and
the activity may be retained only after a limited turn-over number. This effect may have also
been caused by disruption of the peroxidase cycle. While carbidopa may have been readily
oxidized by compound I, it may have been a poor substrate for compound II leading to
accumulation of the latter, as it has been demonstrated for a number of compounds, such as
indole and tryptamine derivatives [1].
0.5
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0
100
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Time (s)
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Figure 2. Influence of carbidopa on the peroxidatic activity of bovine MPO (A) and LPO (B)
measured spectrophotometrically. The oxidation of 100 M o-dianisidine with 50 M H2O2
and 5 nM MPO was monitored at 460 nm (A) and the oxidation of 25 M ABTS with 25 M
H2O2 and 2 nM LPO was monitored at 415 nm (B) in the absence (0) or presence of carbidopa
at concentrations shown next to each curve. The concentrations of the enzymes and carbidopa
were chosen to best show the changes in the lag period.
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3.3. Irreversible inhibition of bovine peroxidases by carbidopa
Prolonged incubation of bovine MPO and LPO with carbidopa and H2O2 led to partial
loss of activity, but the concentration of carbidopa necessary to achieve this effect was quite
high. Even at the substrate to enzyme ratio equal 1000:1 (1.5 mM:1.5 M) both enzymes still

retained substantial activity (Fig. 3). The inhibitory effect was slightly stronger for bovine
MPO than for bovine LPO, confirming the results of the oxidation of carbidopa by both
enzymes (spectrophotometric and chromatographic analysis). Partition ratios [37] calculated
from these results were 1240 and 1460 for bovine MPO and bovine LPO, respectively. These
numbers are quite high when compared to other irreversible peroxidase inhibitors, including
hydrazine derivatives. For phenelzine and HRP the partition ratio was 11 [38], and for the
very effective irreversible thioxanthine inhibitor TX2 and human MPO – only 0.2 [39]. The
UV-Vis spectra of the enzymes recorded after incubation with carbidopa and dialysis have
shown little changes in the heme Soret bands (data not shown), which indicated that the
prosthetic group was not destroyed to a significant degree.
120
100
80
60
40
20
0
15
50
150
500
1500
Carbidopa concentration (M)
Figure 3. Irreversible inactivation of 1.5 M bovine MPO ( ) and bovine LPO ( )
determined after incubation with carbidopa for 1 h followed by dialysis. Enzyme activity was
monitored with o-dianisidine as a substrate. The activity was calculated with respect to a
control reaction with the enzyme which was treated in the same way in the absence of
carbidopa and H2O2.
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3.4. Molecular docking of carbidopa to human MPO and bovine LPO
The relatively inefficient irreversible inhibition of MPO and LPO by carbidopa can be
attributed to the presence of the o-diphenol moiety, which may preferentially interact with the
active site preventing the oxidation of the hydrazine group followed by covalent modification
of the enzymes’ active sites. The hydrophobic character of the access channel may lead to
preferential orientation of carbidopa with the catechol group directed towards the heme
center. Formation of 6,7-dihydroxy-3-methylcinnoline by both enzymes, which proceeds via
the o-quinone intermediate, also seemed to support this hypothesis. This mode of oxidation of
carbidopa could explain, at least in part, the lack of its reported carcinogenic effects. We
performed molecular docking experiments of carbidopa to human MPO (PDB ID: 1D2V) [34]
and bovine LPO (PDB ID: 3Q9K) using AutoDock Vina 1.1.2 [35]. Since no crystal
structures are available for bovine MPO we have used human MPO which has 87% identity
with bovine MPO. In the best-scored structures the orientation of carbidopa was indeed as we
expected – with the catechol group directed towards the heme forming a network of hydrogen
bonds (Fig. S4 and S5). While a single structure with a binding energy of -7.1 kcal×mol-1 was
obtained for human MPO, the experiments with bovine LPO provided two top-scored
structures with equal binding energies of -6.8 kcal×mol-1. The observation of the two equally
favored ligand orientations in bovine LPO suggests that the reversible interaction of carbidopa
with the heme distal side of this enzyme is weaker than with the human MPO. The detailed
results of these experiments are presented in the Supplementary Material.
3.5. Changes in H2O2 concentration during oxidation of model substrates by bovine
MPO and LPO in the presence of carbidopa
Since we excluded irreversible inhibition of MPO and LPO by carbidopa, we looked
for other mechanisms that could explain the lack of chromophore formation during reactions
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with o-dianisidine and ABTS (Fig. 2). It may have resulted from preferential oxidation of
carbidopa by peroxidases until its depletion or reduction of the oxidation products of standard
substrates. This effect was particularly likely for ABTS+•, which is frequently used to
determine antioxidant activity [40] and is readily reduced by a number of compounds,
including polyphenols [41, 42].
To determine which of these two mechanisms is involved we monitored hydrogen
peroxide consumption during the oxidation of o-dianisidine by MPO and ABTS by LPO in
the presence of carbidopa. Changes in H2O2 concentration did not correspond to changes in
absorbance. In the reactions with MPO and o-dianisidine moderate decrease in the reaction
rates was observed in the presence of carbidopa and the effect was proportional to its
concentration but no lag periods were observed (Fig. 4A). These results demonstrated that in
this case both competitive oxidation of carbidopa (at a rate lower than for o-dianisidine) and
60
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20
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25
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B
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Time (min)
Figure 4. Changes in H2O2 concentration during oxidation of o-dianisidine by bovine MPO
(A) and ABTS by bovine LPO (B) measured by the modified xylenol orange assay. Reactions
were carried out under conditions identical as in Figure 2 in the absence (●) or presence of
2 M (□), 4 M (), 6 M (■), 8 M (○), and 10 M (▲) carbidopa. The curves represent
computationally fitted trend lines. Results for LPO with all carbidopa concentrations are
presented in Figure S6.
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reduction of the oxidation product of o-dianisidine (causing lack of absorbance at 460 nm
during the lag period) took place. In reactions with LPO and ABTS a slight increase in the
reaction rate was observed in the presence of carbidopa, although no correlation with its
concentration could be seen (Fig. 4B and S6). This effect can be attributed to reduction of
ABTS+• back to ABTS, whose concentration is maintained close to the initial value until
carbidopa is consumed. Also, the final values of H2O2 concentration were lower in the
presence of carbidopa than in its absence.
3.6. Reduction of meriquinone by carbidopa and other catechols
When we oxidized ABTS with bovine LPO and added carbidopa to the reaction
mixture it was decolorized immediately. This effect could not be reproduced with o-
dianisidine, which demonstrated that it was not the final oxidation product, which was
reduced by carbidopa but one of the reaction intermediates. Such an intermediate, a charge
transfer complex between the substrate and the quinonediimine product named meriquinone
[43], may be stabilized by SDS at low pH [32]. We have therefore generated meriquinone in
reactions of o-dianisidine with MPO and tested the effect of carbidopa on its concentration.
Results of this experiment clearly demonstrated that carbidopa could reduce this intermediate
(Fig. 5). To determine the role of each of the reducing groups in carbidopa (catechol and
hydrazine) we performed this analysis with several catechols (L-dopa, 3-(3,4-
dihydroxyphenyl)propanoic acid, 3,4-dihydroxyphenylacetic acid) and with hydrazine and
several of its derivatives (phenelzine, benzhydrazide, isoniazid). The effect of all catechol-
containing compounds was similar to that of carbidopa, while the influence of hydrazine and
its derivatives was small or negligible (Fig. S7). We also analyzed the oxidation of
o-dianisidine by peroxidases in the presence of these catechols under standard assay
conditions. In all cases lack of absorbance at 460 nm in the initial phase of the reactions
followed by its appearance after a lag period was observed (Fig. S8A and B).
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1.2
0
1
0.8
10 M
0.6
25 M
0.4
50 M
0.2
0
0
100
200
300
400
500
600
Time (s)
Figure 5. Reduction of meriquinone by carbidopa. After oxidation of 100 μM o-dianisidine
with 100 μM H2O2 and 20 nM MPO in the presence of 200 μM SDS in 50 mM acetate buffer,
pH 4.0 for 10 min (time 0) the reaction was monitored at 395 nm in the absence of carbidopa
(0) or in its presence at concentrations shown next to each curve.
3.7. Influence of carbidopa and other catechols on the oxidation of 4-aminoantipyrine by
HRP
It therefore became obvious that carbidopa and the other tested catechols interfered
with bovine MPO and LPO-catalyzed reactions with both o-dianisidine and ABTS as
substrates by reducing their oxidation products or intermediates leading to changes in
absorbance which did not reflect changes in reaction rates. We have therefore decided to
study their effects on reactions catalyzed by horseradish peroxidase with 4-aminoantipyrine
and phenol as a substrate system. This reaction is applied in many medical diagnostic tests
and a number of compounds have been reported to interfere with it. Such an effect was first
discovered for tolazamide with three substrate systems: 4-aminoantipyrine, guaiacum, and
ABTS [44], and later for another hydrazine derivative – gliclazide [45]. It was also reported
for N-acetylcysteine [46], acetaminophen with MBTH used as a substrate [47], and is
particularly well documented for ascorbic acid [48-52]. Most importantly, however, such
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interference was also described for catechol-containing compounds – first for dopamine,
which interfered with creatinine measurements [49], and later for dopamine and dobutamine,
which affected detection of several analytes (cholesterol, triglycerides, and uric acid) [53, 54].
It was also reported for norepinephrine and epinephrine [50, 55], as well as for L-dopa [50].
However, it was noted that physiological and therapeutic concentrations of norepinephrine
and epinephrine are too low to cause significant interference in diagnostic tests [55].
When we carried out oxidation of 4-aminoantipyrine/phenol by HRP in the presence
of carbidopa and monitored the reactions spectrophotometrically, we obtained results very
similar to those observed for o-dianisidine/MPO and ABTS/LPO – a lag period in the increase
of absorbance of the quinoneimine chromophore and a decrease in the final absorbance
proportional to carbidopa concentration (Fig. 6). However, measurements of changes in
hydrogen peroxide concentration again have shown that the reaction rates were not much
unaffected by the presence of carbidopa (Fig. 7 and S10). These results demonstrate that in
this system (HRP/4-aminoantipyrine/phenol) carbidopa is not an effective competing
0.6
0
0.5
10 M
0.4
0.3
20 M
0.2
40 M
0.1
80 M
0
0
100
200
300
400
500
600
Time (s)
Figure 6. Changes of absorbance at 505 nm during oxidation of 50 M 4-aminoantipyrine
and 1 mM phenol with 100 M H2O2 by 0.5 g of HRP in the absence (0) or presence of
carbidopa at concentrations shown next to each curve. The concentrations of the enzyme and
carbidopa were chosen to best show the changes in the lag period.
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120
100
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
Figure 7. Changes in H2O2 concentration during oxidation of 50 M 4-aminoantipyrine and
1 mM phenol with 100 M H2O2 by 0.5 g of HRP in the absence (●) or presence of 80 M
(▲) carbidopa. The curves represent computationally fitted trend lines. Results for all
carbidopa concentrations are presented in Figure S10.
substrate as it was for MPO with o-dianisidine as a substrate (Fig. 4A) and the observed
decrease in the rate of formation and the final concentration of antipyrilquinoneimine are due
primarily to the reduction of the reaction intermediates (most likely the antipyril radical) by
this compound.
Similar results were also obtained for other catechols tested before with MPO and o-
dianisidine and LPO with o-dianisidine and ABTS as substrates (L-dopa, 3-(3,4-
dihydroxyphenyl)propanoic acid, 3,4-dihydroxyphenylacetic acid). For comparison we also
performed this analysis for dopamine, whose effects on HRP-catalyzed reactions with 4-
aminoantipyrine/phenol were previously reported [49, 50, 53-56]. Although the lag phases
were not as clear as in the case of carbidopa, the rates of increase of absorbance at 505 nm
and their final values were lower in the presence of all catechols than in their absence (Fig.
S9). The differences between the shapes of these curves can be explained by the absorption
spectra of the oxidation products of carbidopa and the other catechols. While the oxidation
products of carbidopa formed during the measurement period absorb very weakly at 505 nm,
19

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spectra of the oxidation products of the other catechols overlap in large part with the spectrum
of the antipyrilquinoneimine (Fig. S11). These data show how complex the influence of
catechols on the peroxidase catalyzed reactions may be. The value of absorbance will depend
on the measurement time, the rates of the reactions between catechols and the
intermediates/products of the oxidation of standard substrates, the rates of transformation of
the o-quinones formed (cyclization or decomposition), absorption spectra of the oxidation
products of catechols, and the ratio of absorption coefficients of the oxidation products of
standard substrates and catechols at the detection wavelength.
To illustrate the influence of some of these factors we recorded the UV-Vis spectra
during oxidation of 4-aminoantipyrine and phenol with HRP in the presence of carbidopa, L-
dopa and dopamine (Fig. 8). In reactions with carbidopa much slower increase of absorbance
at 505 nm was accompanied by an increase of absorbance at 365 nm, the maximum of
absorption for 6,7-dihydroxy-3-methylcinnoline, one of the oxidation products of carbidopa
[24] (Fig. 8B), at a rate much greater than for carbidopa alone (Fig. S11A). In the case of L-
dopa the spectra corresponded to dopachrome (maximum at 475 nm), showing that essentially
only this chromophore was formed (Fig. 8C), again at a rate much greater than for L-dopa
alone (Fig. S11B). This conclusion is supported by the maximum absorbance in this reaction
mixture (0.1734), which corresponds almost exactly to 50 M dopachrome (extinction
coefficient at 475 nm equal 3600 M-1×cm-1) showing that oxidation (indirect) of L-dopa
completely depletes hydrogen peroxide from the reaction medium (2 equivalents of H2O2 are
required for oxidation of L-dopa to dopachrome). The spectra for reactions with dopamine
indicated formation of a mixture of chromophores (dopaminochrome and
antipyrilquinoneimine) although formation of a different chromophore (e.g. an oxidized
adduct of 4-aminoantipyrine to dopamine) cannot be excluded on the basis of UV-Vis spectra
alone, since the maximum of absorption shifted to ca. 495 nm (Fig. 8D). This difference in
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behavior between L-dopa and dopamine may be explained by the difference in the pKa values
of their amino groups (8.72 for L-dopa and 10.2 for dopamine [57]), which will result in much
slower cyclization of dopaminequinone than dopaquinone (ca. 30 ×) and may allow
competing reactions (e.g. addition of 4-aminoantipyrine to dopaminequinone) to occur.
0.6
0.5
0.4
0.3
0.2
0.1
0
0.2
0.16
0.12
0.08
0.04
0
505 nm
A
B
8 and 10
6
365 nm
4
2
10
8
6
4
2
0
0
250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0.2
475 nm
0.2
0.16
0.12
0.08
0.04
0
495 nm
C
6 and 8
10
D
0.16
0.12
0.08
0.04
0
6
4
8
4
2
10
2
0
0
250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
Figure 8. UV-Vis spectra recorded during oxidation of 50 M 4-aminoantipyrine and 1 mM
phenol with 100 M H2O2 by 0.5 g of HRP in the absence of other compounds (A) and in
the presence of 80 M carbidopa (B), 80 M L-dopa (C), and 80 M dopamine (D). Spectra
shown were recorded immediately after addition of the enzyme (0) and then after 2, 4, 6, 8,
and 10 min.
Addition of any of the catechols to the formed chromophore (antipyrilquinoneimine)
caused little changes in absorbance (less than 5% decrease within 15 min), as previously
demonstrated for dopamine [56].
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4. Discussion
There are numerous reports describing the effects of polyphenols, such as flavonoids
or gallic acid and its derivatives on the activity of peroxidases [58-63]. These studies usually
rely on measuring the concentration of oxidized products of standard substrates, such as
tetramethylbenzidine, o-dianisidine or ABTS for peroxidatic activity. In the case of
chlorinating activity of myleoperoxidase chlorination of monochlorodimedone or taurine,
oxidation or chlorination of LDL is usually measured. Rarely, however, these experiments are
accompanied by monitoring the concentration of hydrogen peroxide and the slower changes
in the concentration of the oxidized products are therefore interpreted as enzyme inhibition.
However, when the tested compounds may act as antioxidants, inhibition of the enzyme
cannot be distinguished by such methods from its antioxidant effects. Inhibition of dityrosine
formation by myeloperoxidase was reported for ferulic acid, caffeic acid, curcumin and
quercetin. In this case quenching of the generated tyrosyl radical was simply excluded based
on the fact that the concentration of tyrosine (0.2 mM) was higher than the concentration of
the antioxidants (0.1 mM). The rate of tyrosine oxidation, however, was not determined [59].
Another report, however, clearly demonstrated scavenging of tyrosyl radicals by quercetin, its
derivatives and other flavonoids [64]. When the influence of flavonoids, such as rutin,
taxifolin, epicatechin, quercetin, and kaempferol, on the oxidation of LDL by MPO in the
presence of nitrite was studied, a lag period in the appearance of peroxidized lipids was
observed. This effect was very similar to the results obtained by us during oxidation of o-
dianisidine by MPO and ABTS by LPO in the presence of carbidopa and other catechols and
was attributed to the scavenging of the MPO-derived NO2 radical by flavonoids [65].
Quercetin and some of its metabolites were shown to inhibit neutrophil-mediated LDL
oxidation and this effect was also attributed to inhibition of myeloperoxidase [61].
Interestingly, modification of quercetin at either 3 or 3’ position did not affect the inhibitory
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potency, but conjugations at both positions significantly reduced this activity. We have
recently shown that the presence of a catechol in the flavonoid ring B or conjugated phenol
groups at positions 3 and 4’ enables their redox reaction with o-quinones thus leading to
apparent inhibition of tyrosinase [66]. Results obtained with myeloperoxidase [61] indicate a
similar phenomenon in this case.
The effects of polyphenols on the halogenating activity of myeloperoxidase and the
pseudohalogenating activity of lactoperoxidase vary. Several studies demonstrated that
flavonoids enhance these activities [67-70]. This effect was explained by the ability of these
compounds to reduce compound II of MPO and LPO to the native enzyme thus enabling its
participation in the (pseudo)halogenating activity. Recently, however, epigallocatechin gallate
was shown to inhibit hypochlorous acid formation by myeloperoxidase and neutrophils due to
accumulation of compound II [71].
Interference with reactions catalyzed by horseradish peroxidase is of particular
importance because of application of this enzyme in numerous commercial diagnostic tests.
Several mechanisms have been postulated to explain the effects of various compounds on
these reactions. For dopamine and dobutamine the mechanism of this interference was
initially not investigated, although it was speculated that dopamine may be oxidized by HRP
thus depleting hydrogen peroxide from the reaction medium and preventing formation of
antipyrilquinoneimine [53, 54]. Direct oxidation of catecholamines and L-dopa by HRP was
also postulated when the interference with the Trinder reaction was applied for determination
of these compounds in pharmaceutical formulations [50]. Later experiments led to a
conclusion that dopamine and 4-aminoantipyrine upon oxidation with peroxidase formed a
chromophore with an absorption coefficient smaller than antipyrilquinoneimine thus reducing
the final absorbance, while for dobutamine rapid enzymatic oxidation leading to hydrogen
peroxide depletion was postulated [56]. However, we consider the postulated mechanism of
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interference by dopamine insufficient. Although a chromophore resulting from coupling of
the oxidized 4-aminoantipyrine with dopamine may form when only these two compounds are
present in the mixture, in the presence of excess of another phenolic substrate (in our
experiments the ratio of phenol to dopamine was 12.5 to 100) such a reaction would be
marginal. Our results (Fig. 8D) indicate that under such conditions formation of
dopaminochrome (resulting from oxidation of dopamine by 4-aminoantipyril radical)
predominates and the antipyrilquinoneimine chromophore formed by coupling of oxidized 4-
aminoantipyrine with either phenol or dopamine is a minor component – it causes the shit of
the maximum of absorption from 475 nm to 495 nm but does not influence much the final
value of absorbance, which is close to the value corresponding to dopaminochrome alone. If
an adduct between dopamine and the oxidation product of 4-aminoantipyrine were formed, it
should have an absorption coefficient comparable to antipyrilquinoneimines formed with
other phenolic substrates, many of which are used in commercial diagnostic tests (e.g. phenol,
chlorophenol, 3-hydroxybenzoic acid, 2,4,6-tribromo-3-hydroxybenzoic acid) all giving
oxidation products with maxima of absorption above 500 nm and much bigger absorption
coefficients at this wavelength than dopaminochrome. To us the much greater oxidation rate
of dobutamine than dopamine and different mechanisms of interference for these two
compounds [56] are also surprising. We have shown that L-dopa and dopamine are oxidized
at comparable (very low) rates (Fig. S11B and C) and based on the structure of dobutamine
one would expect similar (smaller rather than bigger - because of the larger size of the side
chain) oxidation rate of the catechol group in this compound by HRP. We would therefore
expect for this compound reactions similar to those observed for carbidopa, L-dopa, dopamine
and other catechols, although enzymatic oxidation of the monophenolic part followed by
redox exchange with the catechol part cannot be excluded. Appropriate experiments would be
required to solve this problem.
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Reduction of the oxidation products/intermediates of standard peroxidase substrates
may also be responsible for interference in peroxidase-based diagnostic tests observed
previously for other compounds. A study of interference of several flavonoids with the
commercial assays used for monitoring free fatty acids and triglycerides demonstrated that the
concentrations of these analytes were significantly lowered in the presence of quercetin,
quercetin-3-O-glucuronide, (+)-catechin, kaempferol, and luteolin [72]. This effect was
attributed to inhibition of peroxidase by these compounds. However, lack of inhibitory effects
for genistein and naringenin, which do not contain a catechol group or two conjugated
phenolic groups in the flavonoid rings B and C, indicates that these two compounds, which
cannot form quinonoid-type products upon oxidation [66], may simply be incapable of
reducing the oxidation product of 4-aminoantipyrine. Such an effect has been shown for a
number of flavonoids and phenolic acids in the oxidation of o-dianisidine by horseradish
peroxidase [33]. All compounds showing strong interference possessed a catechol group, a
1,2,3-triol group or conjugated phenol groups in the flavonoid rings B and C and included,
among others, catechin, epigallocatechin, keampferol, morin, fisetin, myricetin, and quercetin.
For all these compounds reduction of meriquinone was demonstrated [33]. Such an effect has
also been reported for green tea polyphenols [73, 74]. In one of these reports two mechanisms
responsible for this interference have been postulated: 1) depletion of hydrogen peroxide
caused by direct oxidation of these compounds by peroxidase and 2) conversion of the
antipyrilquinoneimine back to substrates (4-aminoantipyrine and 3-hydroxybenzoic acid)
[73]. This second reaction, however, is not possible. It was postulated because formation of
the chromophore was restored when additional amount of hydrogen peroxide was added to the
reaction mixture containing green tea components after prolonged incubation with peroxidase.
However, such an effect does not require regeneration of the substrate from the final product
25

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– our results show that the substrate (4-aminoantipyrine) is regenerated from the reaction
intermediates before coupling with the phenol occurs.
Interference in commercial diagnostic tests utilizing peroxidase catalyzed reactions
was also reported for compounds containing a p-diphenol moiety. Homogentisic acid, present
at large concentration in the serum and urine of patients with alkaptonuria, interfered with
tests used for detection of uric acid [75], creatinine [76], and later several more analytes,
including cholesterol, triglycerides, and glucose [77]. Although hydrogen peroxide depletion
has been postulated as the main cause of interference by this compound, the exact mechanism
has not been elucidated. Hemostatic drugs etamsylate and calcium dobesilate (both salts of a
p-diphenol – 2,5-dihydroxybenzenesulphonic acid) were reported as agents interfering with
measurements of creatinine, uric acid, triglycerides, and cholesterol [78-80]. We suggest that
all these p-diphenols may also reduce the oxidation product of 4-aminoantipyrine, just as
catechols do. Structures of major compounds interfering with the peroxidase-based diagnostic
tests are shown in Fig. S12.
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29