Electrochemical evidences in oxidation of acetaminophen in the presence of glutathione and N-acetylcysteine

Article abstract of DOI:10.1039/b916458h

Electrochemical oxidation of acetaminophen has been studied in the presence of glutathione and acetylcysteine. Our results indicate that N-acetyl-p-benzoquinone-imine (NAPQI) participates in a catalytic reaction with glutathione and N-acetylcysteine. Also, the observed homogeneous rate constants of the reaction of NAPQI with glutathione and acetylcysteine were estimated.

Full text of DOI:10.1039/b916458h

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Electrochemical evidences in oxidation of acetaminophen in the presence
of glutathione and N-acetylcysteinewz
Hasan Shayani-Jam and Davood Nematollahi*
Received (in Cambridge, UK) 10th August 2009, Accepted 17th November 2009
First published as an Advance Article on the web 2nd December 2009
DOI: 10.1039/b916458h
Electrochemical oxidation of acetaminophen has been studied in
the presence of glutathione and acetylcysteine. Our results
indicate that N-acetyl-p-benzoquinone-imine (NAPQI) participates
in a catalytic reaction with glutathione and N-acetylcysteine.
Also, the observed homogeneous rate constants of the reaction of
NAPQI with glutathione and acetylcysteine were estimated.
Contrary to the previous study about acetaminophen
9
3
toxicity and acetylcysteine prevention, our results show that
NAPQI consumes glutathione stores by catalytic oxidation of
it. Also, our results indicate that the rate of catalytic oxidation
of acetylcysteine by NAPQI is more than glutathione.
Cyclic voltammetry of 1 mM acetaminophen (1) shows one
anodic (A ) and the corresponding cathodic peak (C ), which
1
1
Acetaminophen (1) is used worldwide for its analgesic and
1
antipyretic properties. It is widely available and present in
correspond to the transformation of acetaminophen (1) to
NAPQI and vice versa within a quasi-reversible two-electron
1
0
many prescription and non-prescription medications. It is an
attractively alternative drug for children and people who are
sensitive to aspirin. At the recommended dosage, there are no
process (Fig. 1, curve a). A peak current ratio (IpC1/IpA1) of
nearly unity can be considered as a criterion for the stability of
the NAPQI produced at the surface of the electrode under the
experimental conditions. The oxidation of 1 in the presence of
glutathione (3a) was studied in some detail (Fig. 1, curve b).
2
side effects. However, overdoses cause liver and kidney damage.
The results of a literature survey show that approximately 5%
of a therapeutic dose of acetaminophen is metabolized by
cytochrome P450 2E1 to the reactive metabolite N-acetyl-p-
Under these conditions, the anodic peak current A
and the cathodic counterpart of it (peak C ) disappears and
increases
1
1
3
benzoquinone-imine (NAPQI) (2). NAPQI is extremely toxic
the voltammogram shows an irreversible feature.
to the liver, possibly as a result of covalent binding to proteins
4
and nucleic acids. However, under normal conditions NAPQI
More studies were performed by varying the concentration
of 3a during cyclic voltammetric experiments of 1 in the
presence of 3a. The results indicate that the current of peak
is rapidly detoxified by reaction with glutathione. But, in an
overdose, cellular glutathione (3a) is depleted resulting in the
availability of NAPQI to bind to cellular macromolecules, the
consequences of which are hepatocellular injury and cell
death. Hepatic toxicity is generally thought to occur when
glutathione (3a) stores are depleted to less than 30% of
5
normal. In most published papers, the reported pathway for
the reaction of NAPQI and glutathione (GSH) (3a) is the
3
formation of the GH-Acetaminophen adduct.
,6–8
Acetaminophen poisoning is treated with N-acetylcysteine
3b). N-Acetylcysteine prevents hepatic injury primarily by
(
restoring hepatic glutathione (3a). N-Acetylcysteine, which is
also a sulfhydryl compound, is thought to act by facilitating
hepatic glutathione (3a) synthesis or by acting as an alternate
3
substrate for the reaction with the NAPQI. Because electro-
chemical oxidation is very often parallel with the cytochrome
P450 catalyzed oxidation in liver microsomes, it was interesting
to study the anodic oxidation of acetaminophen in the
presence of glutathione (3a) in different conditions.
Faculty of Chemistry, f Bu-Ali-Sina University, Hamedan,
65178-38683, Iran. E-mail: nemat@basu.ac.ir;
Fax: +98-811-8257407
Fig. 1 Cyclic voltammograms of 1 mM acetaminophen: (a) in the
absence; (b) in the presence of 5 mM glutathione, and (c) 5 mM
glutathione in the absence of acetaminophen. Inset: Typical cyclic
voltammograms of 1 mM acetaminophen in the presence of various
concentrations of glutathione. Concentrations of glutathione from
w Electronic supplementary information (ESI) available: Experiment
procedures for synthesis of 4 and 5 and FT-IR of 4 and 5 and
simiulated cyclic voltammograms of 1 in the presence of 3a and 3b
at various concentrations. See DOI: 10.1039/b916458h
15
z Reaction equipment is described in earlier paper. All chemicals
were reagent-grade materials from E. Merck. These chemicals were
used without further purification. The homogeneous rate constants
were estimated by analyzing the cyclic voltammetric responses using
(
d) to (g) are: 0, 1, 2 and 5 mM, respectively, at a glassy carbon
electrode, in phosphate buffer solution (c = 0.2 M, pH 7.0); scan rate:
50 mV sꢀ1; t = 25 ꢁ 1 1C.
16
the DigiElchSB simulation software.
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Chem. Commun., 2010, 46, 409–411 | 409

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Fig. 2 Part I: Variation of apparent number of electrons (napp
vs. mmol of glutathione during controlled potential coulometry of
.1 mmol of acetaminophen in the presence of glutathione (0.2, 0.5, 1.0
)
Scheme 1
0
12
N-demethylation reactions. Our result rejects the results of
previously published papers which proposed the formation of
a GH-Acetaminophen adduct as the main product in oxidation
of acetaminophen (1) in the presence of glutathione (3a) and
shows that the main reaction between NAPQI (2) and 3a is a
catalytic reaction. The formation of GSSG (4), also,
previously reported by Potter and Hinson, in oxidation of
acetaminophen by Horseradish peroxidase and cytochrome
and 2.0 mmol). Eapp: 0.25 V vs. SCE. Part II: Cyclic voltammograms
of 0.1 mmol acetaminophen in the presence of 0.2 mmol glutathione,
during controlled potential coulometry at 0.25 V versus SCE. After
ꢀ1
consumption of: (a) 0 and (b) 30 C. Scan rate 100 mV s . Other
conditions are as same as Fig. 1.
A
1
(IpA1), is dependent on the concentration of 3a and
increases strongly with increasing it (Fig. 1, inset).
13
P450 and these electrochemical data confirm it.
Also, several controlled-potential coulometry experiments
were performed in aqueous solution containing 0.1 mmol of 1
in the presence of 0.2, 0.5, 1.0 and 2.0 mmols of 3a at 0.25 V
versus SCE. Cyclic voltammetric analysis carried out during
the electrolysis shows proportional to the advancement of
Such as glutathione (3a), N-acetylcysteine (3b), is a sulf-
hydryl compound, and acts as an alternate substrate for
reaction with the toxic metabolite (NAPQI). Therefore it has
been used for several decades and has proven to be the
antidote of choice in treating acetaminophen-induced
coulometry, anodic peak A
the charge consumption becomes about 33, 54, 91 and
56 coulombs, respectively. The calculated apparent number
of electrons (napp) consumed per molecule of acetaminophen
1) shows a linear relationship between it and mmols of
1
decreases and disappears when
3
hepatotoxicity. Fig. 3 (curve a) shows a typical voltammetric
curve for a 1 mM solution of 1. In this figure, curve c shows a
cyclic voltammogram recorded for 1 mM 1 in the presence of
1
2
mM N-acetylcysteine (3b). Such as glutathione case, under
(
these conditions, the anodic peak current (IpA1) increases and
glutathione (Fig. 2, part I). The continual increase of napp
0
its cathodic counterpart (peak C ) disappears. Also, under
1
under these conditions is a diagnostic criteria for the EC
1
mechanism.
these conditions, cyclic voltammetric experiments show that
the anodic peak current (IpA1) is dependent on the concentration
of 3b and increases strongly with its increase. In addition
1
On the other hand, it is shown that, proportional to the
decrease of glutathione’s concentration, the charge consumed
decreases and the Q = 19.7 C obtained when the glutathione’s
concentration becomes zero. Under this condition, napp = 2.1.
This number of electrons confirms two electrons oxidation
1
1
of 1 to NAPQI (2).
An interesting feature of the performed controlled-potential
coulometry experiments is the reappearing of peak C near the
1
end of coulometry (Fig. 2, part II). This observation also
0
reconfirms the EC (catalytic) mechanism and rejects other
mechanisms such as EC (conjugation reaction) in the electro-
chemical oxidation of 1 in the presence of glutathione (3a).
Diagnostic criteria of cyclic voltammetry and controlled
potential coulometry accompanied by the FT-IR data of the
final product (see ESIw), indicates that the reaction mechanism
of electrooxidation of acetaminophen (1) in the presence of
0
11
glutathione (3a) is EC (catalytic mechanism) (Scheme 1).
According to our results, glutathione (GSH) (3a) reacts with
electrochemically generated NAPQI (2). This reaction
converts 3a to glutathione disulfide (GSSG) (4) and reduces
the NAPQI (2) back to 1. The formation of GSSG (4)
Fig. 3 Cyclic voltammograms of 1 mM acetaminophen: (a) in the
absence; (b) in the presence of 2 mM glutathione and (c) in the
presence of 2 mM acetylcysteine. Other conditions are the same as
Fig. 1.
(see ESIw), previously reported during the oxidation of 3a by
free radical intermediates, formed during peroxidase-catalyzed
4
10 | Chem. Commun., 2010, 46, 409–411
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controlled-potential coulometry experiments were performed
homogeneous rate constants (kobs) shows that 3b reduces
NAPQI (2) about nine times more rapidly than does 3a.
Acetaminophen (1) is oxidized to N-acetyl-p-benzoquinone-
imine (NAPQI) (2). NAPQI (2) is extremely toxic to the liver,
possibly as a result of covalent binding to proteins and nucleic
acids. However, under normal conditions NAPQI (2) is
rapidly detoxified by reaction with glutathione. In most
published papers, the reported pathway for the reaction of
NAPQI (2) with glutathione (3a) is the formation of a
GH-Acetaminophen adduct. But, contrary to these reports,
our results indicate that NAPQI (2) participates in a catalytic
reaction with 3a and 3b. Also, comparison of observed
homogeneous rate constants (kobs) of NAPQI (2) with 3a
and 3b shows that 3b reduces NAPQI (2) about nine times
more rapidly than does 3a. These results are undeniable
electrochemical evidence which shows why N-acetylcysteine
(3b) can be used as an antidote in overdoses of acetaminophen
(1) and also, explains how it detoxifies acetaminophen poisoning.
in aqueous solution containing 1 and 3b and showed a linear
relationship between acetylcysteine concentration and napp
.
Also, diagnostic criteria of cyclic voltammetry and
controlled-potential coulometry accompanied by the FT-IR
data of the final product (see ESIw) indicates that the reaction
0
mechanism of electrooxidation of 1 in the presence of 3b is EC
0
(
catalytic) (Scheme 1). The existence of an EC mechanism is
supported by the following evidence: (a) Disappearing of peak
during the reverse scan. This could be indicative of the fact
C
1
that electrochemically generated NAPQI is removed by
chemical reaction with 3b. In this case, the peak current ratio
(IpC1/IpA1) strongly depends on the sweep rate and it reaches
to nearly unity in higher sweep rates. (b) Dependency of
anodic peak current (IpA1) on acetylcysteine concentration in
cyclic voltammetric experiments of 1 in the presence of 3b. (c)
Dependency of napp on acetylcysteine concentration in
controlled-potential coulometric experiments of 1 in the
presence of 3b.
Accordingly, the catalytic reaction of NAPQI (2) with
N-acetylcysteine (RSH) (3b) leads to the formation of diacetyl-
cystine (RSSR) (5). The catalytic oxidation of 3b and formation
Notes and references
1
2
3
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1
4
of RSSR uses the other redox systems previously reported.
Comparison of the current of peak A (IpA1) in the presence of
b (Fig. 3, curve c) with glutathione (3a) (Fig. 3, curve b),
1
4 D. J. Jollow, J. R. Mitchell, W. Z. Potter, D. C. Davis,
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3
1
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1
5
times) in the presence of N-acetylcysteine (3b). This indicates
that the reaction rate of 3b with NAPQI is more than 3a.
A scheme for the electrochemical oxidation of acetaminophen
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(
(
1) in the presence of glutathione (3a) and N-acetylcysteine
3b) is proposed and tested by digital simulation. On the
0
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basis of an EC mechanism, the observed homogeneous rate
constants (kobs) of reaction of NAPQI with 3a and 3b
have been estimated by comparison of the simulation results
with experimental cyclic voltammograms. The procedure is
performed based on achieving the best fit between simulated
and experimental cyclic voltammograms (see ESIw). The
method is developed for a variety of scan rates and concentra-
1
1
1
1
tions. The calculated value of the k
for 3a and 3b are
obs
ꢀ ꢀ1
1
1
530 ꢁ 22 and 13 580 ꢁ 208 M
s , respectively. Standard
3428–3434.
6 M. Rudolph, J. Electroanal. Chem., 2002, 529, 97–108. See also:
http://www.digielch.de.
deviations were obtained for four independent simulations
at various scan rates. Comparison of these observed
1
This journal is ꢂc The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 409–411 | 411