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In vitro inhibitory effect of quinolinic acid on aldehyde oxidase activity of
guinea pig liver: a proposed mechanism*
Mohamed A. Al-Omar, PhD.
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia
E-mail: [email protected]
Web-site: www.dr-alomar.com
* This manuscript has been accepted as an abstract in 40th IUPAC congress in China
2005.
1
ABSTRACT
Objective: The aim of the present study was to investigate the interaction of quinolinic
acid (QA) with partially purified guinea pig liver aldehyde oxidase in terms of superoxide
anion production (O2.), hydrogen peroxide (H2O2) formation and the overall substrate
oxidation. Due to the structural similarity of QA to some aldehyde oxidase substrates,
such as 2-pyrimidinone, the effect of QA on aldehyde oxidase activity has been
investigated in the present study.
Methods: The interaction between QA and aldehyde oxidase has been measured by
spectophotometerically and fluorimetrically methods using phthalazine (a classical
heterocyclic substrate) and indole-3-aldehyde (an excellent aldehyde substrate).
Results: The inhibitory effects of QA on indole-3-aldehyde and phthalazine oxidation,
superoxide anion production and hydrogen peroxide formation were found to be
competitive inhibition in all three cases (Ki = 77-106 µM, r = 0.995, p<0.005).
Conclusion: QA inhibitory effect on aldehyde oxidase suggests that it may play a role in
inhibition of initial rates of superoxide anion formation but may increase overall
production of this radical by aldehyde oxidase. QA had a dual effect on superoxide anion
production from the two substrates; initial rates were reduced but after 5-8 minutes
reaction rates were enhanced. Both effects were concentration dependent.
Keywords:
Quinolinic acid, aldehyde oxidase, reactive oxygen species, superoxide
anion, hydrogen peroxide.
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Introduction
Quinolinic acid (Figure 1, QA) is a physiologically active neurotoxic metabolite of the
L-tryptophan-kynurenine pathway (1). In the brain, QA is an endogenous excitotoxic
agonist of the N-methyl-D-aspartate (NMDA) receptor (2). It can modulate the effects of
excitotoxins in central nervous system, therefore, attention has been recently focused on
the metabolic role of QA. Immune stimulation accelerates the entire enzymatic cascade,
resulting in enhanced intracellular NAD+ production from QA biotransformation (2).
Figure 1
Aldehyde oxidase (EC 1.2.3.1) catalyses nucleophilic attack at an electron-deficient
carbon atom adjacent to a ring nitrogen atom in N-heterocyclic compounds which are
oxidised to cyclic lactams (3, 4). Studies on aldehyde oxidase and xanthine oxidase (EC
1.1.3.22) have shown that modulation of enzyme activities, cofactor availability,
substrate concentration and oxygen tension can all affect rates of intracellular reactive
oxygen species (ROS) production (3, 4).
Reduction of oxygen, during substrate turnover, leads to the formation of superoxide
anion and hydrogen peroxide as ROS. This capacity has attracted attention to the possible
role of aldehyde oxidase as a source of ROS. For example, excessive oxygen radicals
have been implicated in neuronal injury (5). In vivo, it seems that aldehyde oxidase
together with cytochrome P450 are, quantitatively, the most important cellular sources
for ROS (6). Although the liver is the main site for aldehyde oxidase, this enzyme has
also been reported in kidney, lung, muscle, spleen, stomach, heart, and brain (4).
Excessive production of ROS which react with various cell components such as lipids,
proteins or nucleic acids results in cell damage. Consequently, aldehyde oxidase has been
implicated in pathophysiology of alcohol liver injury, visual processes, synthesis of
retinoic acid and reperfusion tissue injury (6). Retinoic acid is an important hormone in
the differentiation and development of neurons and glia (see conclusion) as well as cellcell signaling in the central nervous system (7). Recently, it has been shown that altered
retinoic acid synthesis could be implicated in the etiology of Parkinson’s disease and
3
schizophrenia (8, 9). Alternatively, aldehyde oxidase is a source of oxygen radicals,
which may contribute to these diseases (10). Although xanthine oxidase generates ROS,
it should be noted that in vivo, the enzyme exists predominantly as dehydrogenase (EC
1.1.1.204), reacting with NAD+, whereas aldehyde oxidase reacts exclusively with
oxygen (11).
The present study investigated the interaction of QA with hepatic aldehyde oxidase
in terms of ROS production and the overall substrate oxidation. In fact, guinea pig liver
aldehyde oxidase has been shown to be an excellent model for the human liver enzyme,
therefore it has been used throughout this study (12). The kinetic properties for QA were
compared in terms of mode of inhibition and inhibitor affinity, Ki, with molybdenum-site
inhibitor, chlorpromazine (13). Using the fluorimetric method, that has been developed
recently, it has been possible to compare the kinetics of hydrogen peroxide production
with that of substrate oxidation and superoxide anion production which have been
measured using optimized spectrophotometric assays.
Methods
Reagents and Chemicals:
All reagents and solvents are of analytical grade. All chemicals and reagents were
purchased from Sigma-Aldrich Chemical Company Ltd. (Gillingham-Dorset, SP8 4XT,
UK). QA and hydrogen peroxide-30% (w/w) have been purchased from Sigma-Aldrich
Chemical Company Ltd (Louis, MO 633178, USA). Cytochrome-c (from horse heart),
horseradish peroxidase (HRP, type I, EC 1.11.1.7) and superoxide dismutase (SOD, from
bovine liver suspension in 3.8 M (NH4)2SO4, pH 7.0, 3,000 unit/mg protein) were also
purchased from Sigma Chemical Company Ltd. (Gillingham-Dorset, SP8 4XT, UK).
Sorenson’s phosphate buffer (67 mM) was made from two separate solutions containing
either 9.511 g/L Na2HPO4 or 9.118 g/L KH2PO4 in distilled water (14).
Preparation of partially purified molybdenum hydroxylases from guinea pig liver:
Aldehyde oxidase was partially purified from liver homogenate of mature
Dunkin-Hartley guinea pigs following a published methodology (14). The animals were
killed by cervical dislocation between 10:00 am and 11:00 am daily. Freshly excised liver
4
from Dunkin-Hartley guinea pigs was placed in ice-cold isotonic KCl (1.15%),
containing 100 µM EDTA and the gall bladder and any excess fat were removed before
homogenized using a Ystral® D-79282 homogenizer for 2-3 minutes. Aliquots of the
homogenate were transferred to 50 ml polycarbonate centrifuge tubes. The centrifuge
tubes were equalized in weight and centrifuged at 15,000 g for 45 minutes at 4°C using
an MSE Fisons Hi-spin 21 centrifuge fitted with an angle rotor 8 x 50 ml head. Partially
purified enzyme was stored in liquid N2 until needed.
Determination aldehyde oxidase activities:
Enzyme activity was determined spectrophotometrically using a Cary 50 UV/VIS
spectrophotometer (Varian Australia Pty Ltd., Mulgrave/Victoria, Australia), which was
linked to a cell temperature control unit. With the exception of enzyme, which was kept
on ice until mixing with other components, all solutions were pre-warmed to 37 °C. The
spectrophotometer was computer-controlled by Carry WinUV® spectroscopy software
package with additional kinetics software (2002).
Aldehyde oxidase activity in partially purified molybdenum hydroxylase fractions
was monitored, at 37 °C, using 100 µM 2-pyrimidinone, 50 µM phenanthridine, 100 µM
phthalazine (enzyme fraction was diluted, 1:10) and 100 µM indole-3-aldehyde (1:40
dilution) as substrates in 67 mM Sorenson’s phosphate buffer, pH 7.0, containing 100 µM
EDTA. Enzyme activity of guinea pig liver molybdenum hydroxylase fractions was also
measured in the presence of 1, 10, 50 and 100 µM QA (Figure 2). The initial velocity for
substrate oxidation was determined by measuring the change in absorbance/minute and
calculating enzyme activities in µmol/min/mg protein in the presence and absence of QA.
Determination of superoxide anion:
Reduction of cytochrome c by partially purified molybdenum hydroxylase
fractions at 550 nm was followed using phthalazine, indole-3-aldehyde, 2-pyrimidinone,
phenanthridine and xanthine as substrates. Superoxide anion reacts with ferricytochromec (76 µM) reducing Fe(III) to Fe(II).
5
Determination of hydrogen peroxide formation:
Hydrogen peroxide formation was monitored fluorimetrically, using an MPF-3
Fluorescence Spectrophotometer with the excitation wavelength set at 395 nm and the
emission wavelength set at 470 nm. The slit width was fixed at 6 mm for both excitation
and emission, using a 150-watt Xenon Lamp as the light source. Hydrogen peroxide
generated during molybdenum hydroxylase-catalysed oxidation was reacted with
horseradish peroxidase to form a complex (compound I). The complex causes the
oxidation of the fluorophore, Scopoletin, resulting in a decrease in fluorescence, which is
proportional to original hydrogen peroxide content as described previously (15).
Protein determination.:
A Pierce Bicinchoninic acid (BCA®) protein reagent assay kit was used. This
technique depends on the production of Cu (I) from the reaction of protein with Cu (II) in
an alkaline medium as described by Smith et al (16).
Statistical analysis:
The presented data are mean ± SD. The significance of difference between the
means in the presence and absence of QA was computed using student’s T-test and P
value less than 0.05 was considered significant.
Results
Effect of QA and other typical inhibitors on substrate oxidation:
Aldehyde oxidase and xanthine oxidase are both present in guinea pig partially
purified molybdenum hydroxylase fractions; accordingly specific enzyme inhibitors were
used to confirm the specificity of the spectrophotometric assay. Chlorpromazine and
menadione were used as specific aldehyde oxidase inhibitors (4, 17). Under the
conditions used in this study, these inhibitors usually inhibit substrate oxidation by 9899% (17). In the present study, 100 µM of chlorpromazine and menadione decreased
initial oxidation rates of 100 µM phthalazine, indole-3-aldehyde, 2-pyrimidinone and 50
µM phenanthridine by 97-99% (Table 1, p<0.001). In contrast, 100 µM allopurinol, a
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xanthine oxidase inhibitor, caused a negligible reduction in substrate oxidation (2-4%)
(18). As it has been shown that allopurinol is slowly converted by aldehyde oxidase to
oxipurinol, it though that allopurinol is a competitive substrate of aldehyde oxidase (19).
QA was tested as an inhibitor for aldehyde oxidase activity and found to be a moderate,
but significant, inhibitor. The effect of QA on substrate oxidation has been compared to
those of traditional aldehyde oxidase inhibitors (Table 1). By using 1-tailed student’s ttest, it has been shown that 100 µM QA caused a significant inhibition (p<0.005).
Figure 2
In the absence of substrate, QA was incubated with the enzyme preparation using
oxygen as electron acceptor and the incubation mixture was monitored by repetitive
scanning between 200-700 nm for up to 10 minutes. There were no change observed in
the spectrum of QA. It was therefore concluded that QA is not a substrate for guinea pig
liver aldehyde oxidase or any component present in the preparation. However, it has been
reported that QA can interact with carbohydrate and lipid metabolism in the liver (19). In
this study it has been found that QA also inhibited xanthine oxidation in the enzyme
preparation which strongly indicate that the compound interacts with xanthine oxidase
(Table 1). However, its inhibitory effect on xanthine oxidase activity was less potent than
that on aldehyde oxidase. The inhibition of QA on indole-3-aldehyde and phthalazine
oxidation, catalyzed by guinea pig liver aldehyde oxidase, were found to be competitive
with an inhibitor constant (Ki) value of 77 ± 3.8 µM and 86 ± 4.2 µM using oxygen as
electron acceptor, respectively.
Table 1
Effect of QA and other typical inhibitors on ROS:
Electrons egress from the enzyme can be followed using electron acceptors that
interact with the enzyme at different redox centers. Potassium ferricyanide (K3Fe(CN)6)
accepts electrons from iron-sulfur center whereas cytochrome-c is reduced by superoxide
anion at FAD site. The inhibition of potassium ferricyanide was found to be equipotent to
that of cytochrome-c or oxygen reduction during indole-3-aldehyde oxidation by
aldehyde oxidase (Table 2). This may indicate that QA inhibits the enzyme at the
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molybdenum center. However, the inhibition of ROS formation may shed more light on
the specific-site of interaction.
Table 2
The effect of QA on production of ROS, hydrogen peroxide and superoxide
anion, has been compared to those of chlorpromazine and menadione during the
oxidation of 50 µM phenanthridine (Table 3). Furthermore, the inhibitory constants for
interaction of QA with substrates and ROS production are abridged in table 4. The effect
of QA on superoxide anion production (40%) was almost equipotent to that of hydrogen
peroxide formation (42%) or substrate oxidation (36%). Similarly, chlorpromazine
inhibits the superoxide anion production, hydrogen peroxide formation and substrate
oxidation, but in a more potent pattern. In contrary to chlorpromazine, menadione has
been found to be non-equipotent on the three processes. In fact, interaction of menadione
is thought to occur at the FAD site (20), which is consistent with the ability of menadione
to act as an electron acceptor of xanthine oxidase. As a result, the oxidation rates of
xanthine oxidase are enhanced in the presence of menadione (20, 21). No reaction has
been observed during the incubation of QA with cytochrome-c or potassium ferricyanide
alone, which indicates that QA has no intrinsic reaction with the oxidized form of iron.
Noteworthy, Rajagopalan et al. suggested that partially purified preparation of aldehyde
oxidase is more active than highly purified one, partly due to the presence of catalase that
render the incubation medium from hydrogen peroxide (21). Similar conclusion has been
reached during development of the novel fluorimetric method for measurement of
hydrogen peroxide (15).
Table 3
Table 4
Discussion
Aldehyde oxidase has a broad substrate specificity and thus catalyses the
oxidation of a wide range of endogenous compounds and xenobiotics. Thus, this enzyme,
8
in addition to cytochrome P450, is a major defense mechanism for the removal of
drugs/xenobiotics from the body (5,6). Consequently, as an obligate aerobe, aldehyde
oxidase is one of the most important sources of ROS. During aldehyde oxidase-catalyzed
reactions, ROS, hydrogen peroxide and superoxide anion, are produced in substantially
high amounts as reviewed recently (5, 6, 15). Furthermore, aldehyde oxidase generates
hydrogen peroxide and superoxide anion under normal physiological conditions whereas
most other sources, including xanthine oxidase, produce ROS only in certain pathological
conditions such as ischaemia. In addition, aldehyde oxidase generates both hydrogen
peroxide and superoxide anion simultaneously, whereas the majority of other systems
produce either hydrogen peroxide or superoxide anion (5, 6). Glial cells form 90% of the
cells within the nervous system (22). They are thought to support the environment around
nerve cells; for example, by regulating the concentrations of free ions in the extracellular
spaces of spinal cord, transporting materials to and from blood vessels, and providing
metabolic support for the neuronal membrane (23). Berger (24) found that aldehyde
oxidase was highly expressed in glial cells of the lateral motor column; the region of the
spinal cord which degenerates in amyotrophic lateral sclerosis (ALS). This, together with
the fact that aldehyde oxidase is selectively localized in the motor neurons of mouse brain
and spinal cord, suggest that the enzyme could play a role in the homeostasis of motor
neurons (25). In models of inflammatory neurological disorders, such as allergic
encephalitis, bacterial and viral infections, forebrain global ischaemia, spinal trauma
brain or hippocampal damage, QA levels are extremely elevated (26,27). Recent study by
Belle et al. showed that QA stimulates production of free radicals and lipid peroxidation
(28). With this respect, QA was found to have a dual effect on superoxide anion
production from the two substrates; initial rates were reduced but after 5-8 minutes
reaction rates were enhanced. Both effects were concentration dependent. This finding
may link between noxious effects of QA and aldehyde oxidase in one side and ALS in the
other side.
Heterocycles containing an amino- (29) or hydrazine (30) substituents, adjacent to
a ring nitrogen, are potent aldehyde oxidase inhibitors. In this study, QA inhibited the
oxidation of indole-3-aldehyde and phthalazine catalyzed by aldehyde oxidase. However,
it would appear that QA is a progressive inhibitor of aldehyde oxidase as inhibition was
9
more marked as the reaction proceeded. QA interaction with drug-metabolizing
molybdenum hydroxylases has not been reported previously in literature. In this study,
QA was found to be a remarkable inhibitor of guinea pig liver aldehyde oxidase with less
reactivity towards xanthine oxidase. As QA inhibits superoxide anion production,
hydrogen peroxide formation and substrate oxidation to the same level and in conjunction
with the fact that it has similar inhibitory effect on oxygen compared to that of potassium
ferricyanide and cytochrome-c, as artificial electron acceptors, the site of interaction is
thought to be the active-site (i.e. molybdenum center). In agreement, the inhibition
pattern was competitive where the inhibition decrease as the substrate concentration
increase. Chlorpromazine had similar inhibitory effect on superoxide anion production,
hydrogen peroxide formation and substrate oxidation (Table 4). Ki values for
chlorpromazine ranged from 0.86-101 µM and in each case chlorpromazine exhibited
non-competitive inhibition. Johns (17) has shown that the chlorpromazine analogue, Nmethylphenothiazine and N-methylphenazine are substrate for human aldehyde oxidase,
which indicates that the drug may react with aldehyde oxidase at molybdenum binding
site. This would be consistent with that chlorpromazine interfering with electron transfer
from the substrate thus inhibiting all three processes to the same extent. However, in view
of the non-competitive nature of chlorpromazine inhibition, it would appear that
chlorpromazine may bind to reduced molybdopterin cofactor, Mo(IV), rather than the
oxidized cofactor, Mo(VI) (5).
As a result, different concentrations of QA may modulate the activity of aldehyde
oxidase and thus affect its capacity to form ROS in a concentration dependent manner.
Further studies on the exact mechanism of interaction of QA with molybdenum
hydroxylases are currently conducted in our laboratories.
References
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Scopoletin. J Med Sci 2005;5:10-20.
16. Smith PK., Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD,
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24. Berger R. Analysis of aldehyde oxidase and xanthine dehydrogenase/oxidase as
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25. Bendotti C, Prosperini E, Kurosaki M, Garattini E, Terao M. Selective Localization
of mouse aldehyde oxidase mRNA in the choroid plexus and motor neurons. Neuro
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26. Beagles KE, Morrison PF, Heyes MP. Quinolinic acid: in vivo synthesis rates,
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Neurochem 1998;70:281-291.
27. Behan WM, McDonald M, Darlington LG, Stone TW. Oxidative stress as a
mechanism for quinolinic acid-induced hippocampal damage: protection by
melatonin and deprenyl. Br J Pharmacol 1999;128:1754-1760.
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COOH
N
COOH
Quinolinic acid
Figure 1-
Chemical structures of QA (CAS: 89-00-09)
14
% of Control l
120
100
*
*
80
*
**
**
**
60
40
20
0
100µM Phthalazine
Control
Figure 2-
1 µM QA
100µM Indole-3-aldehyde
10 µM QA
100µM 2-Pyrimidinone
50 µM QA
100 µM QA
Inhibition of phthalazine, indole-3-aldehyde and phenanthridine oxidation by
guinea pig liver aldehyde oxidase in the presence of (1, 10, 50 and 100 μM) QA
(n = 3 ± SD; *p<0.05, **p<0.01 vs. control), the control values as indicated in
table 1
15
Table 1-
Comparison of effects of QA, potent aldehyde oxidase inhibitors and xanthine
oxidase inhibitor on the oxidation of phthalazine, indole-3-aldehyde, 2pyrimidinone and phenanthridine catalyzed by partially purified guinea pig liver
molybdenum hydroxylase fractions
Inhibitors
% Inhibition*
(100 µM)
Phthalazine
Indole-3-aldehyde
2-Pyrimidinone
Phenanthridine
Xanthine
(100 µM)
(100 µM)
(100 µM)
(50 µM)
(50 µM)
Control
0.283
0.275
0.216
0.274
0.0355
Chlorpromazine
99 ± 2
98 ± 2
98 ± 2
99 ± 1
3±1
(0.003)**
(0.006)
(0.004)
(0.003)
(0.034)
98 ± 2
97 ± 2
98 ± 2
98 ± 3
1±4
(0.006)
(0.008)
(0.004)
(0.005)
(0.035)
41 ± 3
36 ± 5
38 ± 3
36 ± 3
21 ± 6
(0.167)
(0.175)
(0.133)
(0.176)
(0.028)
2±2
2±2
3±1
4±2
98 ± 3
(0.277)
(0.272)
(0.210)
(0.263)
(0.001)
Menadione
QA
Allopurinol
* Results are expressed as mean percentage inhibition ± SD (male/female guinea pigs, n= 3-4)
** Result in brackets are the mean n= 3-4 of initial rates in the present of inhibitor (µmol/min./mg protein)
16
Table 2-
Effects of QA, chlorpromazine and menadione, potent aldehyde oxidase
inhibitors, on the oxidation of indole-3-aldehyde and allopurinol, potent xanthine
oxidase inhibitor on the oxidation of xanthine catalyzed by partially purified
guinea pig liver molybdenum hydroxylase fractions using different electron
acceptors
% Inhibition of the oxidation of
Electron acceptor
indole-3-aldehyde (50 µM)
Xanthine (50 µM)
Chlorpromazine
Menadione
QA
Allopurinol
QA
Potassium ferricyanide
98 ± 3
97 ± 4
35 ± 3
97 ± 2
27 ± 2
Cytochrome c
96 ± 4
98 ± 4
37 ± 5
97 ± 4
20 ± 3
Oxygen
97 ± 2
99 ± 2
37 ± 2
99 ± 1
23 ± 3
17
Table 3-
Effects of QA, chlorpromazine and menadione on the ROS formation during
phenanthridine oxidation catalyzed by partially purified guinea pig liver
molybdenum hydroxylase fractions using different electron acceptors
% Inhibition of ROS production during the oxidation of phenanthridine (50 µM)*
ROS
Chlorpromazine (100 µM)
Menadione (100 µM)
QA (100 µM)
Superoxide anion
99 ± 2**
96 ± 3
40 ± 1
Hydrogen peroxide
99 ± 3
81 ± 3
42 ± 3
* The results are expressed as mean percentage inhibition ± SD (male/female guinea pigs, n = 4)
** The control rates for superoxide anion and hydrogen peroxide formation are 0.048 and 0.175
µmol/min./mg protein
18
Table 4-
The inhibitory constants (Ki) for interaction of QA with the oxidation of
phthalazine and indole-3-aldehyde and their conjunct ROS (mean ± SD, n =3).
Kinetic constants
Ki values (µM)
Phthalazine
Indole-3-aldehyde
QA
QA
Chlorpromazine
(100 µM)
(100 µM)
(1 µM)
Substrate
86 ± 4.2
77 ± 3.8
0.86 ± 0.03
Superoxide anion
94 ± 3.5
106 ± 3.1
0.95 ± 0.12
Hydrogen peroxide
88 ± 4.7
85 ± 5.2
1.1 ± 0.2
19