© 1997 American Heart Association, Inc.
Articles |
Paracetamol Catalyzes Myeloperoxidase-Initiated Lipid Oxidation in LDL
From the Clinical Institute of Medical and Chemical Laboratory Diagnostics (S.K.), the Department of Internal Medicine III, Division of Nephrology and Dialysis (G.S.), the Institute of Molecular Genetics (M.H.), the Institute of Tumor Biology and Cancer Research (C.S.), and the Institute of Medical Chemistry (I.H., B.M.K.G.), University of Vienna, Vienna, Austria
Correspondence to Bernhard Gmeiner, Institute of Medical Chemistry, University of Vienna, Währingerstr. 10, A-1090 Vienna, Austria.
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Abstract |
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 Top Abstract IntroductionMethodsResults and DiscussionReferences |
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Abstract The oxidative modification of LDL may play a significant role in atherogenesis. Myeloperoxidase (MPO) expressed in human atherosclerotic plaques has been suggested to be operative in vivo, making LDL atherogenic. Tyrosyl radicals generated by MPO have been shown to act as physiological pro-oxidants of lipid peroxidation in LDL. Assuming that a variety of phenolic compounds are able to form phenoxyl radicals when exposed to peroxidases, we tested the ability of paracetamol, a known analgesic drug with a tyrosine-like monophenolic structure, to act as a pro-oxidant of lipid peroxidation in LDL. Spectroscopic analyses indicated that paracetamol, similar to tyrosine, could undergo peroxidase-induced phenoxyl radical formation, which was inhibited by the radical scavenger ascorbic acid as well as by heme poisons and catalase. Measurement of conjugated dienes and lipid hydroperoxides in LDL preparations exposed to MPO/H2O2 in the absence or presence of paracetamol revealed that the drug could act as a catalyst of lipid oxidation in LDL. Similar results were found when LDL oxidation was performed with activated human neutrophils, which use MPO to promote lipid peroxidation. In conclusion, the results suggest that paracetamol could act, via a phenoxyl radical, as a catalyst of LDL oxidative modification by MPO.
Key Words: lipid peroxidation • myeloperoxidase • oxidized LDL • atherosclerosis • acetaminophen • paracetamol
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Introduction |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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Oxidatively modified LDL particles may play an important role in the development of atherosclerosis.1 This observation has led to studies focusing on the mechanisms of LDL oxidation and on the antioxidant potential of drugs or naturally occurring compounds.2 3 4 5 6 7 Lipid oxidation in LDL can be induced by transition metal ions (such as copper), hypochlorite, superoxide/nitric oxide, organic peroxyl radicals, and peroxidases.8 9 10 11 12
The oxidation reaction can be accelerated by pro-oxidants.
-Tocopherol, acting via a radical mechanism (tocopheryl
radical), was identified as such a naturally occurring pro-oxidant in
LDL by Bowry and Stocker.13 These authors showed that the
peroxyl radical-induced LDL oxidation was accelerated in the presence
of -tocopherol by the ability of the tocopheryl
radical to propagate a radical chain via its reaction with
polyunsaturated fatty acid–lipid within the lipoprotein
particle.13 This finding has brought an entirely new
aspect to the biochemistry of this otherwise known
"antioxidative" vitamin. Recently it was shown that tyrosine in
the presence of MPO or activated neutrophils could undergo
phenoxyl radical formation.14 These generated tyrosyl
radicals were able to catalyze the peroxidation of LDL,15
that is, the moderate oxidation process in the absence of the amino
acid was increased significantly when tyrosine or small
tyrosine-containing peptides were present during the reaction.
Hence, one can state that both -tocopherol and tyrosine
are converted to phenoxyl radical-like species and that these reactive
intermediates may thus initiate lipid peroxidation by similar
mechanisms.
Taking into account that various phenolic compounds have been shown to
form phenoxyl radicals in the presence of
peroxidases,16 17 18 we tested the ability of paracetamol, a
drug with a tyrosine-like monophenolic structure (see Scheme 1
), (1) to
form phenoxyl radicals and (2) to act as an LDL pro-oxidant in the
MPO-induced oxidation of LDL. Paracetamol (acetaminophen,
N-acetyl-p-aminophenol) is an effective, widely
used alternative to aspirin (acetylsalicylic acid)
as an analgesic–antipyretic agent.19 20 Therapeutic
plasma levels are in the range of 17 to 170 µmol/L;
high doses of paracetamol for longer periods of time (leading to plasma
levels of about 1 to 2 mmol/L) may cause
hepatotoxicity.21
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We found that paracetamol was able to form phenoxyl radicals and, similar to tyrosine, could catalyze the lipid peroxidation in LDL induced by MPO or activated human neutrophils.
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Methods |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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Chemicals
L-tyrosine and paracetamol (4'-hydroxyacetanilide) were from Sigma Chemical Company. MPO (EC 1.11.17), GOD (EC 1.1.3.4), and PMA were purchased from Calbiochem–Novabiochem International. HRP (grade II, EC 1.11.1.7) was from Boehringer Mannheim. All other chemicals were of analytical grade.
LDL Isolation
LDL was isolated by ultracentrifugation as
reported previously.22 The final preparation was dialyzed
against 150 mmol/L NaCl containing 0.1 mmol/L
EDTA and filter-sterilized. Protein was estimated with use of a
commercial test kit (Bio-Rad Laboratories) using bovine serum
albumin as a standard.
Isolation of Granulocytes
Isolation of human granulocytes was performed as previously
reported.23 In brief, undiluted blood anticoagulated with
heparin was layered over Ficoll Paque (1.077 g/mL). After an
initial incubation step of 45 minutes at room temperature, supernatants
were layered on 63% Percoll underlayed with 72% Percoll. Cells were
then centrifuged at 500xg for 25 minutes at room
temperature and washed twice in Hank's balanced salt solution without
Ca2+ and Mg2+, and cell pellets were
resuspended in Hank's solution without Ca2+ and
Mg2+. Cell viability in all experiments was more than 90%
as determined by trypan blue exclusion.
LDL Oxidation by MPO
LDL oxidation by MPO was carried out as reported by Savenkova et
al.15 Briefly, LDL (0.2 mg/mL) in 0.1 mol/L
phosphate buffer (pH 7.5) containing 0.1 mmol/L DTPA was
incubated in the presence of 10 µg/mL glucose, 50 ng/mL
GOD, and 3 nmol/L MPO, with or without the respective compound,
at 25°C for the indicated time. Blanks were run without GOD.
LDL Oxidation by Neutrophils
LDL oxidation by activated human neutrophils was
performed according to the methods of Savenkova et al.15
LDL (0.2 mg/mL) was incubated at 37°C up to 3.5 hours with
5x105 cells/mL in Ca2+- and
Mg2+-free Hank's balanced salt solution supplemented with
0.2 mmol/L DTPA, 1 mg/mL glucose, 1 µg/mL
aprotinin, 1 µg/mL leupeptin, and 1 µg/mL soybean
trypsin inhibitor in the absence or presence of the
respective compound. Cells were activated with 1 µmol/L PMA
dissolved in DMSO (final concentration, 0.02%).
Estimation of Lipid Oxidation Products
Conjugated Dienes
LDL lipid oxidation was analyzed by monitoring diene
conjugation as the increase in absorbance at 234 nm
(e=2.95x104/mol L-1
cm-1) according to Esterbauer et
al.24 In case of neutrophil incubations, cells were
pelleted by centrifugation (10 min at
500xg), the supernatant (0.8 mL) was extracted with
chloroform (1.5 mL), and the chloroform phase was analyzed for
dienes.
Lipid Hydroperoxides
After termination of the lipid peroxidation reaction by addition
of 300 nmol/L catalase to the incubation mixture for 30 minutes
at 25°C, total lipid hydroperoxides were measured with the
CHOD-iodide color reagent (
365=1.73x104/mol
L-1 cm-1, E. Merck)
as described by El-Saadani et al25 under the conditions of
Wallin and Camejo.26 A 0.5-mL sample was mixed with 1 mL
of CHOD-iodide reagent, and after incubation for 60 minutes at 37°C,
the absorbance was estimated at 365 nm. With the exception of cyanide,
all compounds (ie, paracetamol, tyrosine, salicylate, azide, catalase,
and methanol) were found not to interfere with the color reagent as
tested by the measurement of lipohydroperoxides of copper-oxidized
(10 µmol/L) LDL (1 mg/mL, 6 hours at 37°C) in
the presence of the respective agent. In case of cyanide-containing
incubation mixtures, lipids were extracted by the modified Dole
procedure15 27 before estimation of lipid hydroperoxides.
In brief, 0.7 mL of the samples was mixed with 1 mL of
2-propanol/heptane/2 N acetic acid (40:10:1,
vol/vol/vol), followed by the addition of 1.5 mL of
heptane. After mixing and phase separation by
centrifugation, an aliquot (0.5 mL) of the heptane
phase was dried under N2. The residues were redissolved in
200 µL of methanol, and 1 mL of color reagent was then added and
incubated as described above.
Spectroscopic Analyses
Paracetamol or tyrosine (up to 2 mmol/L)
dissolved in 0.1 mol/L phosphate buffer (pH 7.5) containing
0.1 mmol/L DTPA was treated with HRP (3
µmol/L) or MPO (3 nmol/L) in the presence of 0.1
mmol/L H2O2. After incubation at 25°C,
paracetamol and tyrosine oxidation products were
analyzed by spectrophotometry using a Perkin Elmer Lambda 2
spectrometer. Phenoxyl radical-generated diphenyl formation of the
compounds was monitored as the increase in absorbance at 320
nm.17
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Results and Discussion |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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Previous studies with both vitamin E and tyrosyl radical suggest that the reactive intermediates derived from these molecules initiate lipid peroxidation in LDL and that such reactions could occur in vivo, leading to LDL atherogenic modifications.13 15
It is well documented that peroxidases can induce phenoxyl radicals in
a variety of tyrosine-related and unrelated phenolic compounds, such as
tyramine, 2-t-butyl-4-methoxyphenol, or 2,6
di-t-butyl-4-methylphenol.16 17 18 Taking this
into consideration, we tested the analgesic drug paracetamol (see
Scheme 1
), which has a tyrosine-like monophenolic structure, for its
ability (1) to form phenoxyl radicals in the presence of peroxidases
and (2) to act as a pro-oxidant in MPO-induced LDL oxidation.
Phenoxyl Radicals Generated by Peroxidases
Phenoxyl radicals generated from monophenolic compounds in the
presence of peroxidases and hydrogen peroxide can undergo dimerization,
thus forming the respective diphenyl derivatives (eg,
dityrosine).16 17 18 Hence, phenoxyl radical formation can be
conveniently estimated by UV spectroscopy of the reaction products
absorbing near 320 nm.17 As can be seen from the spectra
in Fig 1, a and b, when paracetamol was
incubated in a system containing H2O2 and HRP
or MPO, the drug underwent phenoxyl radical formation. This was
indicated by the increase in absorbance at 320 nm and the appearance of
a dityrosine-like absorption spectrum.
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Fig 2 depicts the concentration-dependent
formation of radical-generated diphenyl derivatives during
HRP/H2O2 treatment of paracetamol. Under these
experimental conditions, paracetamol showed reaction kinetics similar
to those of tyrosine, which was run as a positive control substance
(Fig 2). Maximal dimerization products were formed at 0.4
mmol/L of compound during the 15 minutes of incubation at 25°C
(3 µmol/L HRP, 0.1 mmol
H2O2). The radical-mediated dimerization
reaction of tyrosine has been shown to be inhibited by ascorbic acid
because of the radical scavenging ability of the
vitamin.15
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) or tyrosine (
) was incubated in the presence of
HRP/H2O2 (3 µmol/L HRP, 0.1 mmol/L
H2O2) for 15 minutes at 25°, and the
oxidation products were measured as the increase in absorbance at
320 nm as described in "Methods."
Fig 3 shows that the
MPO/H2O2-induced paracetamol dimerization
(measured as the increase in absorbance at 320 nm) was also inhibited
in the presence of ascorbic acid. At a concentration of 100
µmol/L ascorbic acid, there was little diphenyl compound
formed (19% of control). This was very similar to the results obtained
with tyrosine (13% of control). At lower concentrations of ascorbic
acid (25 to 50 µmol/L), however, the tyrosine
dimerization reaction appeared to be more sensitive to the
inhibitory effect of ascorbic acid than the paracetamol
reaction (see Fig 3, a).
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In addition, the heme poisons azide and cyanide, as well as the
H2O2 scavenger catalase, inhibited the
MPO-initiated dimerization of paracetamol (see Fig 3, b).
Hence, the above results certainly indicated that paracetamol was able
to form phenoxyl radicals in presence of peroxidases (HRP or MPO) and
hydrogen peroxide, which could undergo dimerization (see Scheme 1).
LDL Oxidation by MPO
HRP-dependent oxidation of LDL has been reported by Wieland et
al.12 That this kind of LDL oxidation might play a
pathophysiological role is supported by the fact
that enzymatically active MPO has been detected in atherosclerotic
lesions.28 However, it should be stated explicitly that
HRP has an exposed heme edge and will oxidize high-molecular-weight
substrates like LDL in the absence of a low-molecular-weight catalyst.
In contrast, the active site of MPO is buried in a deep hydrophobic
cleft.29 This enzyme requires a low-molecular-weight
molecule to convey oxidizing equivalents from its active site to the
target for damage (see Savenkova et al15 and references
herein). Savenkova et al15 have shown that the MPO-induced
oxidation of LDL could be catalyzed in the presence of tyrosine. This
was due to the formation of tyrosyl radicals, which were generated
during the peroxidative process, propagating radical chain
reactions.15 Because paracetamol was able to form phenoxyl
radicals when exposed to peroxidases (see above), we tested the
hypothesis that the drug was able to stimulate LDL oxidation like
tyrosine. Therefore, LDL was subjected to peroxidase-dependent
oxidation in the absence or presence of paracetamol and tyrosine, which
was run as a positive control of phenoxyl radical-catalyzed lipid
oxidation. The increase in conjugated diene and lipid hydroperoxide
formation was taken as a parameter of LDL oxidative
modification.24 15 When LDL was treated with MPO in an
H2O2-generating system (glucose/GOD), there was
a time-dependent, slow increase in conjugated diene formation,
indicating moderate lipid oxidation. In the presence of tyrosine
(0.5 mmol/L), however, there was a considerable increase in
diene formation observed. These results were in good agreement with the
observations of Savenkova et al.15 Addition of paracetamol
(0.5 mmol/L) to the LDL-oxidizing system showed a very
similar effect of the drug on lipid oxidation. Like in the presence of
tyrosine, the rate of LDL oxidation was initially rapid, resulting in
an about threefold increase in lipid peroxidation at the end of
incubation (see Fig 4).
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indicates the control;
The formation of lipid hydroperoxides was also enhanced in the presence
of 0.5 mmol/L paracetamol (see Fig 5). Therapeutic plasma levels of 200
µmol/L paracetamol were tested using a second LDL
preparation. After a 2-hour incubation, there was a doubling of lipid
hydroperoxides compared with the control (30 versus 60 nmol/mg)
and a fourfold increase in conjugated dienes (50 versus 200
nmol/mg) in these experiments.
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It should be noted that salicylate (0.5 mmol/L), which has
been found to undergo radical formation under peroxidative
conditions,30 was also able to facilitate LDL oxidation
when tested in the MPO/H2O2 system, as
indicated by an increase in conjugated dienes and lipid hydroperoxides
(Figs 6 and 7).
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The tyrosyl radical-initiated LDL oxidation by MPO has been found to be
inhibited by the heme poisons azide and cyanide, as well as by the
H2O2 scavenger catalase.15 As seen
in Fig 8, the "paracetamol full
system" was also sensitive to these inhibitors of
peroxidase-promoted LDL oxidation.
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LDL Oxidation by Human Neutrophils
The MPO–hydrogen peroxide system of activated human
neutrophils has been shown to generate tyrosyl radical and its
dimerization product (dityrosine) when the cells are incubated in
the presence of tyrosine.14 Savenkova et al15
have also reported that the LDL oxidation reaction induced by
activated neutrophils was stimulated in the presence of
tyrosine because of the formation of phenoxyl radicals, as outlined
above. We therefore examined the ability of paracetamol to act as a
catalyst of LDL oxidation in this particular LDL-oxidizing system.
Tyrosine was run as a positive control of phenoxyl radical-facilitated
LDL oxidation. Isolated human neutrophils were activated by
phorbol ester treatment and incubated with LDL in the absence or
presence of 1.6 mmol/L paracetamol (tyrosine), and the
formation of conjugated dienes as a parameter of lipid
oxidation was monitored. All data were corrected by subtracting values
obtained from cells treated with the respective compound but without
PMA activation (Fig 9).
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There was a roughly 2.5-fold increase in lipid oxidation observed when paracetamol was present during the incubation. Tyrosine run in parallel experiments also stimulated lipid oxidation in this system, as previously reported by Savenkova et al.15 Whether these results may also have implications for a role of phagocytes in mediating tissue injury by paracetamol is not known and deserves further study.
In summary, our results indicate that paracetamol could catalyze LDL lipid oxidation initiated by MPO because of its ability to form phenoxyl radicals under peroxidative conditions. It must be noted that paracetamol has recently been found to act as an antioxidant of LDL oxidation.7 Nenseter et al7 studied lipid oxidation in an entirely different system, that is, copper ion-induced LDL oxidation. In contrast, the MPO-driven LDL oxidation is a metal ion-independent reaction. This may explain the contrary results obtained in both systems. Taking into account that MPO as well as catalytically active metal ions have been found in human atherosclerotic plaques,15 28 31 both actions (pro-oxidant and antioxidant) of the drug may be operative. It should be noted that LDL isolated from human atherosclerotic lesions exhibits elevated levels of dityrosine.32 This observation indicates that tyrosyl radical may be of physiological importance in promoting LDL oxidation in the artery wall and raises the possibility that paracetamol plays a similar role in stimulation of lipid peroxidation in vivo.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by Wissenschaftlicher Fonds des Bürgermeisters der Bundeshauptstadt Wien.
Received September 25, 1996; accepted April 15, 1997.
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References |
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