Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3255-3262
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3255-3262.)
© 1997 American Heart Association, Inc.
Tetradecylthioacetic Acid Inhibits the Oxidative Modification of Low Density Lipoprotein and 8-Hydroxydeoxyguanosine Formation In Vitro
Ziad A. Muna;
Khaled Doudin;
Jon Songstad;
Rune J. Ulvik;
;
Rolf K. Berge
From the Department of Clinical Biology, Division of Biochemistry,
University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway
(Z.A.M., R.J.U., R.K.B) and the Department of Chemistry, University of Bergen,
N-5007 Bergen, Norway (K.D., J.S.).
Correspondence to Ziad A. Muna, Department of Clinical Biology, Division of Biochemistry, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. E-mail ziad.muna{at}ikb.uib.no
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Abstract
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Abstract Oxidative modification of low-density
lipoprotein (LDL)
is thought to play a key role in the formation of
foam cells
and in initiation and progression of atherosclerotic plaque.
The
hypolipidemic 3-thia fatty acids contain a sulfur atom and might
therefore
possess reducing (antioxidant) properties. Consequently, the
effects
of 3-thia fatty acids on the susceptibility of LDL particles
to
undergo oxidative modification in vitro were studied.
Tetradecylthioacetic
acid (TTA), incorporated into the LDL particle and
increased
the lag time of copper ion induced LDL oxidation in a
dose-dependent
manner. 80 µmol/L TTA reduced the generation of
lipid
peroxides during copper ion induced LDL oxidation (for 2 hours)
by
100%, 2,2'-azobis-(2,4-dimethylvaleronitrile) induced LDL oxidation
by
64%, and 2,2'-azobis-(2-amidinopropane hydrochloride) induced
LDL
oxidation (for 6 hours) by 21%. The electrophoretic mobility
of the
oxidized LDL was reduced by TTA in both copper ion and
azo-compounds
initiated oxidation. This fatty acid analogue
was effectively able to
reduce in a dose dependent manner the
formation of
8-hydroxydeoxyguanosine from 2-deoxyguanosine with
ascorbic acid as the
radical producer. TTA bound copper(II)
ions and did not reduce
copper(II) to copper(I). It failed to
scavenge the
1.1-diphenyl-2-picrylhydrazyl radicals. The results
suggest that the
modification of LDL in the lipid and protein
moieties can be
significantly reduced by TTA. This acid may
exert its antioxidant
effect partially through metal ion binding
and through free
radical scavenging.
Key Words: tetradecylthioacetic acid • 3-thia fatty acids • oxidized LDL • atherosclerosis • lipid peroxidation • copper reduction.
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Introduction
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Excess
of triacylglycerol and cholesterol in
the blood accelerates
the development of atherosclerotic
coronary heart disease.
1 Reduction of high blood
concentrations of lipids by drugs or
by diet has therefore for several
years been accepted as a routine
therapeutic measure for people at
risk. In addition, a new therapeutic
strategy based on the use of
antioxidants seems to get increasing
support.
2 Antioxidants
may reduce the susceptibility of LDL
to oxidative modification and thus
inhibit the appearance of
foam cells and the formation of fatty
streaks, which are the
first steps in the atherosclerotic
process.
3 A possible role
for redox reactions in the
pathophysiology of atherosclerosis
in vivo has for
instance been suggested by the inhibiting effect
of antioxidant therapy
on the progression of atherosclerosis
in WHHL
rabbits.
4,5 The significance of LDL-oxidation as an
early
event in the pathogenesis of
atherosclerosis,
6 has stimulated
the search
for new agents with antioxidant properties and the
hope of developing
new and better drugs for treating coronary
heart disease and
other diseases caused by vascular atherosclerosis.
Recently, the effect of sulfur substituted fatty acid analogues on
lipid metabolism and blood lipids has been extensively
studied.7 Long-chain thia fatty acids are activated
to their CoA thioesters in the endoplasmic reticulum (ER).8
They cannot be ß-oxidized but are metabolized by extra mitochondrial
ß-oxidation and sulfur oxidation in the ER followed by peroxisomal
ß-oxidation to short sulfinyl dicarboxylic acids.9 In
vivo, long-chain 3-thia fatty acids are incorporated into
phospholipids,10 particularly in heart and
hepatocytes. Among plasma lipoproteins, they were found
within VLDL and LDL.11 TTA has been reported as one of the
most potent lipid lowering of the monocarboxylic 3-thia fatty acids,
causing reduction in both cholesterol (data to be
published) and triacylglycerol in
rats.12 TTA induces proliferation of peroxisomes and
mitochondria7,13–15 that may lead to increased oxidative
stress in the body. For instance, when rats were treated with
peroxisome proliferators (ciprofibrate and perfluorodecanoic acid)
there was an increase in 8-hydroxy-deoxyguanosine (8-OH-dG), which is a
marker of oxidative damage of DNA.16–18 In order to reveal
redox properties of TTA, we tested the compound in two quite different
experimental models: the effect of TTA on oxidative modification of LDL
caused by copper and nonmetal oxidants, and its effect on the ascorbate
induced oxidation of the DNA-fragment 2-deoxyguanosine. In both systems
TTA behaved as an antioxidant.
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Methods
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Chemicals
2,2'-azobis-(2-amidinopropane hydrochloride) (AAPH) and
2,2'-azobis-(2,4-dimethylvaleronitrile)
(AMVN) were purchased from
Polysciences Inc. All other chemicals
were from common commercial
sources and were of reagent grade.
Fatty Acid Analogues
The different 3-thia fatty acids were synthesized as previously
described.19 These were dissolved in ethanol or in
potassium hydroxide and added in the indicated concentrations
simultaneously with the oxidative agent, or as
indicated.
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Synthesis of 1-[14C]-Tetradecylthioacetic
Acid
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1-Tetradecanethiol was distilled at 84°C to
86°C/0.01
mm Hg and [
14C]-bromoacetic acid (1
mCi bromoacetic acid (55
mCi/mmol)) was distilled at 117°C to
118°C/15 mm Hg.
All other chemicals were flushed with
argon. 42 mg tetradecanethiol
were placed in a round bottom flask. 2 mL
methanol were added
and the mixture was stirred under argon to prevent
oxidation
of the sulfides. After 15 minutes 1.70 mL of 0.75
mol/L KOH
in methanol were carefully added. The reaction mixture
was then
stirred for 20 minutes. 19 mg [
14C]-bromoacetic
acid were dissolved
in 10 mL methanol. The solution was allowed to
evaporate to
5 mL under a flush of nitrogen after which 1.68 mL of
757 mmol/L
KOH were added. The reaction mixture was bubbled
through with
argon and boiled for 24 hours. 48 mg of 37% HCl in 10 mL
water
were then added. This led to precipitation of the product.
Before
the mixture was set at 4°C to continue precipitation, pH
was
measured and seen to have a value of 4 to 5.
1-[
14C]-tetradecylthioacetic
acid was transferred to a
G-4 filter, washed with distilled
water, and recrystalized from
dichloromethane in 10% pentane.
It had a melting point of 63°C and
the yield was 78%.
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LDL Preparation and Storage Time
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LDL was prepared from fresh human plasma obtained from healthy
volunteers
by sequential ultracentrifugation, as
described in detail elsewhere.
20,21 Briefly, LDL was taken
as the 1.019 to 1.063 density fraction.
The LDL fractions were dialyzed
extensively against 150 mmol/L
sodium chloride, 16
mmol/L sodium phosphate and 4 mmol/L potassium
phosphate,
pH 7.4, bubbled with nitrogen. Protein was measured by the
Bio-Rad
Protein assay (Bio-Rad Laboratory) using bovine serum
albumin
as a standard. The purity of the LDL preparation was
evaluated
with agarose gel electrophoresis, using the Beckman, Paragon
system.
After being dialyzed it was necessary to test the stability of the LDL
under storage conditions. Four different LDL preparations obtained from
4 different healthy donors were subjected to copper ion oxidation after
different storage intervals (0 to 6 days, 0 to 4°C and 7 days,
-80°C). The lag time, the half-time for the maximum diene
production (T1/2), the rate of propagation, and the
maximum diene production were monitored. The results showed
that the LDL stored at 0 to 4°C gave values that were within the
experimental error of those obtained with freshly prepared LDL, while
the oxidation parameters of LDL stored at -80°C were
outside experimental error (data not shown). Therefore, we used LDL
stored at 4°C and within 6 days after it was dialyzed.
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Copper Ion–Induced LDL Oxidation and Monitoring of Conjugated
Dienes
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The kinetics of LDL oxidation and the conjugated diene method
have
been described by others.
22,23 However, we optimized
the experimental
conditions in our own laboratory with the following
main results.
The increase in LDL protein concentration caused an
increase
in the lag time and T
1/2, and a delay and higher
maximum of
diene production. When the copper ion concentration
was increased,
the lag time decreased, however, not beyond a minimum
level.
The initial and the maximum diene concentrations remained
virtually
constant for a fixed LDL protein concentration (data not
shown).
25 µg/mL LDL protein was enough to obtain sufficient
analytical
sensitivity with a change in the absorbance at 234 nm of
about
1. 10 µmol/L Copper ion was the corresponding
optimal
concentration, and 37°C was the optimal reaction temperature.
The
oxidation was initiated as described earlier.
24
Basically, 25
µg LDL-protein were placed in 1 mL quartz cuvette to
which
calcium magnesium free phosphate buffered solution (PBS) at
pH
7.4 was added. TTA and the other compounds tested, were added
as
specified. The oxidation was initiated by adding freshly
prepared
CuCl
2X2H
2O solution to a final concentration of
10
µmol/L. Oxidation was performed at 37°C in a
single-beam
UV-spectrophotometer (Shimadzu MPS-2000), with a capacity
of
measuring 6 samples simultaneously. Absorbance was
recorded
every 2 minutes up to 3 hours. The initial absorbance was
set
to an arbitrary value and then the increase was recorded over
the
time period.
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Preincubation
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25 µg/mL LDL protein and 20 µmol/L TTA
(final concentrations)
were preincubated at 37°C for 1 hour before
oxidation was
initiated by the addition of 10 µmol/L
copper ions. The
reaction was monitored by measuring the change in
absorbance
at 234 nm.
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Electrophoretic Mobility
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Oxidation of LDL was initiated by either copper ions or the
azo-compounds,
AAPH and AMVN, being water- and lipid-soluble,
respectively.
Azo compounds thermally decompose to peroxyl radicals at
constant
rates in the water or lipid phases.
25,26 The
oxidation of LDL
(50 µg/mL) was carried out at 37°C for
different time
intervals in the presence of 5 µmol/L
copper ions, or
for 6 hours in the presence of 4 mmol/L
AAPH or 1 mmol/L AMVN,
(final concentration, dissolved in PBS or
methanol, respectively),
in the absence or presence of TTA. The
reaction was arrested
after appropriate time intervals by cooling to
4°C and by
addition of EDTA (200 µmol/L final
concentration) and
BHT (40 µmol/L final concentration).
Aliquots were assayed
by agarose gel electrophoresis (Paragon, Beckman
Instruments,
Inc.) in 0.05 mol/L barbital buffer, pH 8.6, and
stained with
sudan black B. REM was calculated as the mobility of
oxidized
LDL relative to that of native LDL. The intra-assay
coefficient
of variation was 1.5% (n=9 copper-oxidized LDL).
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Measurement of Lipid Peroxides
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The same aliquots analyzed for electrophoretic mobility
were
analyzed for their lipid peroxides content by a
colorimetric
method in which hemoglobin catalyzes the
reaction of hydroperoxides
with the methylene blue derivative, forming
an equimolar concentration
of methylene blue with absorbance maximum at
675 nm. The assay
was performed using a kit purchased from Kamiya
Biomedical Company.
The intra-assay coefficient of variation was 2.0%
(n=8 copper-oxidized
LDL).
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Comparing Methods for Detecting Oxidation of LDL
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The oxidation of 25 µg/mL LDL protein by 10
µmol/L
copper ions was monitored over time by the three
methods; conjugated
dienes, lipid peroxides production, and
electrophoresis. Electrophoretic
mobility was the first
parameter to be affected followed by
conjugated dienes and
lipid peroxides (data not shown).
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Analysis of 8-OH-dG
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Formation of 8-OH-dG was obtained as described
elsewhere.
27 Briefly, 3 mmol/L ascorbic acid
was added to 167 µmol/L
2-deoxyguanosine, in 100
mmol/L phosphate buffer of pH 8.1 at
37°C. The fatty acids
tested were dissolved in 1% Triton
X-100 and added at the indicated
concentration simultaneously
with the oxidative agent, and
incubated for several time intervals
as specified in each experiment.
Separation of 8-OH-dG by high
performance liquid
chromatography (HPLC) was performed by using
4.6 x
150 mm Supelcosil C-8 column protected by a 2 x 20 mm
Perisorb
RP-18 precolumn in a HPLC system from Spectra-Physics. The
mobile
phase was 8% aqueous methanol containing 10 mmol/L
NaH
2PO
4X2H
2O
at a flow rate of 1 mL
per minute. 8-OH-dG was detected using
an electrochemical detector (Esa
Coulochem 2) equipped with
a 5011 high-sensitivity analytical cell. The
potentials for
the electrodes 1 and 2 were adjusted to 150 and 430 mV,
respectively.
A 5020 guard cell was set at a potential of 450 mV. The
8-OH-dG
standard was a generous gift from Dr Aarsaether.
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Scavenging of DPPH Radical
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Scavenging of 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radicals
was
measured as described previously.
28 Briefly, a 0.03 mL
of the
stock solution of the tested compounds (0.1 mmol/L)
was mixed
with 3 mL of 0.1 mmol/L DPPH solution (in
ethanol) in a cuvette
and the time course of the optical density change
was determined
at 517 nm for 20 minutes.
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Wavelength Spectra Measurement
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The UV and the visible spectra of copper ion in the presence
and
absence of TTA or palmitic acid, were measured as
described
29 using Cary 1 Varian spectrophotometer. The pH
was about 6, the
copper salts and the various fatty acids were
dissolved in ethanol
and the experiment was run at room temperature.
The absorbance
curves of the visible (400 to 850 nm) and the UV (250 to
400
nm) wavelength ranges were recorded.
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The Effect of TTA on the Oxidation State of Copper
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In order to check the oxidation state of the copper after mixing
TTA
and copper(II) to form a complex, color-specific redox reagents
were
used. Bathocuproine sulfonate that gives an orange-red color
with
copper(I), but no color with copper(II), was used as described
elsewhere.
30 Briefly, 10 mL of 0.1 mol/L
Cu(ClO
4)
2·6H
2O were prepared
in
water and slowly added to 10 mL of 0.2 mol/L solution of
fatty
acid in ethanol. The mixture was stirred for 30 minutes
at room
temperature. A precipitate appeared and the reaction
mixture was
filtered and the residue was washed with water and
ethanol to remove
unreacted copper(II) perchlorate and fatty
acid respectively. The pale
blue crystals were dried. The presence
of copper(I) in the TTA-copper
complex, was examined by adding
1 mL of 10 mmol/L
bathocuproine sulfonate solved in 10% ammonium
acetate as well as 1 mL
water to 1 mL of ethanol solution (10
mL) containing 16.3 mg of the
complex. As a control the same
experiment was performed with either
2.9 mmol/L of
Cu(ClO
4)
2.6H
2O,
or 9.7 mg palmitic
acid solved in ethanol (10 mL). Further,
the experiments were repeated
with 1 mL hydroxylamine ·
HCl (10%) instead of water.
Hydroxylamine reduces copper(II)
to copper(I).
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Interactions Between LDL and TTA
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To test whether TTA can bind to the LDL particle in vitro, we
added
different concentrations of freshly prepared LDL to a constant
concentration
of radio labeled TTA (10 µmol/L final
concentration).
After 1 hour incubation at room temperature, 6%
perichloric
acid (final concentration) was added and the mixture was
centrifuged
at 12000 rpm for 12 minutes. The radioactivity in
both the supernatant
and the pellet was measured.
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Results
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The Effect of TTA on Copper Ion Induced LDL Oxidation
Diene Production
The 3-thia fatty acids had different effect on the kinetics
of LDL
oxidation (Fig 1
). Palmitic acid that was
used as a control
and 3-thia fatty acids of chain length from
octylthioacetic
acid to hexadecylthioacetic acid increased the lag
time, but
not as significant as TTA. Shorter and longer 3-thia fatty
acids
were less effective. TTA increased the lag time and the half
time
in a dose-dependent manner (Fig 2
),
whereas the rate of
oxidation and maximal diene production
remained unchanged compared
to the control (Table 1
). Palmitic acid had no significant
effect
on oxidative modification of LDL.
-Tocopherol and reduced glutathione
increased the lag
time while addition of the polyunsaturated
fatty acid,
eicosapentaenoic acid (20:5 n-3) reduced the
lag
time (data not shown). No changes in the lag time of LDL oxidation
were
obtained by the addition of the sulfinyl or sulfonyl forms of
TTA,
tetradecyloxyacetic acid, and 3, 14-dithiahexadecanedioic
acid (Table 1
).
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Table 1. Change in Percentage of Some Indexes of Copper
Ion-Induced LDL Oxidation due to Addition of 20 µmol/L of
Different Sulfur and Oxygen Substituted Fatty Acids
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Lipid Peroxidation
TTA reduced the production of lipid peroxides originating
from LDL oxidation, being almost blocked when 80 µmol/L
of TTA was added and incubated for 1 or 2 hours (Fig 3). However, the antioxidant capacity of
TTA was less effective in inhibiting lipid peroxide production
after 3 hours.
Electrophoretic Mobility
The changed electrophoretic mobility of LDL oxidized for
various time intervals is shown in Fig 4.
Addition of TTA reduced the REM of oxidized LDL in a dose-dependent
manner. Indeed, the REM of the copper ion-treated LDL incubated for 1
and 2 hours in the presence of 80 µmol/L of TTA was
similar to native LDL.
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Figure 4. The effect of various TTA concentrations on copper
ion induced LDL oxidation as assessed by agarose gel electrophoresis.
Data shown are from one representative
experiment.
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To test the effect of TTA and palmitic acid on the
electrophoresis method, increasing concentrations of TTA and palmitic
acid (dissolved in KOH) were added to LDL (50 µg/ml) which had
been oxidized for six hours by 4 mmol/L AAPH at 37°C.
Addition of TTA at a concentration of more than 160
µmol/L, was found to increase the electrophoretic mobility,
probably explained by an increase in the net negative charge caused by
the sulfur atom. Palmitic acid up to 320 µmol/L, had no
effect on the electrophoretic mobility (data not shown).
The Effect of TTA on Ascorbic Acid-Induced 2-Deoxyguanosine
Modification
Fig 5 shows the effects of TTA on
ascorbic acid-induced formation of 8-OH-dG from 2-deoxyguanosine
incubated for different time intervals. This 3-thia fatty acid reduced
in a dose-dependent manner the 8-OH-dG formation at all time intervals.
More than 50% reduction in ascorbic acid-induced formation of 8-OH-dG
was observed in the presence of 100 µmol/L TTA. No
inhibition of 8-OH-dG generation was observed when the sulfonyl form of
TTA or tetradecyloxyacetic acid was present (Table 2).
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Table 2. Amount of 8-OH-dG Produced by Ascorbate After
Incubation with 100 µmol/L of Different Fatty Acid Analogues for
Different Time Intervals
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Mechanisms of Action
Binding of Copper Ion to TTA
In order to verify the mechanisms by which TTA inhibits oxidation
of the LDL, its effect on copper ion binding was tested. This was done
by measuring the UV and the visible absorbance spectra of TTA in the
presence and absence of copper ion. The results were compared with
those obtained by adding palmitic acid that was used as a control. The
visible absorbance spectra measure the d-d transition for copper from
nondegenerated t2g-orbitals (which make the band very broad)
to dx2-y2-orbitals where the
separation increased with the stability of the complex, ie, the more
stable the complex is, the smaller the
max.31 This means that copper in
H2O (max 821 nm) are less stable than in
palmitic acid (max 814 nm), which again are less stable
than in TTA (max 794 nm) (Fig 6A).
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Figure 6. The absorbance of (1) copper ions, (2) palmitic
acid and copper ions, (3) TTA and copper ions and (4) TTA in ethanol
scanned at (A) visible spectra, (B) ultraviolet spectra.
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The UV spectra (Fig 6B
) of the TTA-copper complex showed two main bands
at 275 nm and 332 nm indicating that there are two different
transitions in the TTA-copper complex. In comparison, the palmitic
acid-copper complex had only a single band, at 275 nm. The common
absorbance peak at 275 nm, is due to charge-transfer transition from
oxygen nonbonding electrons to the antibonding (t2g*)
orbitals of the copper ion. The band at 332 nm in the TTA-copper
complex is assumed to be due to the charge-transfer transition of the
sulfur nonbonding electrons to the antibonding (t2g*) of the
copper ion.32 This suggests that in the palmitic
acid-copper complex there is an oxygen-copper bond, while in the
TTA-copper complex there are both sulfur-copper and oxygen-copper
bonds. The shift of max to longer wavelengths results
from weakening the copper-ligand bond.31 Apparently, the
copper-oxygen bond is stronger than the copper-sulfur one.
The Effect of TTA on the Oxidation State of Copper
Table 3 shows that neither palmitic
acid nor TTA has any effect on the oxidation state of copper, ie,
copper (II) was not reduced to copper (I).
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Table 3. Absorbance at 479 nm of Different Solutions Tested
for Copper(I) and Copper(II) with the Use of Bathocuproine Sulfonate
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LDL Oxidation Initiated by Azo Compounds
To examine possible alternative mechanisms, LDL was subjected to
copper ion-independent oxidation, initiated by the lipid and water
soluble azo compounds AMVN and AAPH, respectively. The effect of TTA
and palmitic acid on the AAPH and AMVN induced LDL oxidation as
assessed by electrophoresis is shown in Table 4. With increased amounts of TTA, the REM
of the LDL particle decreased. It should be emphasized that this method
underscores the real effect due to the interference of TTA with the
method (see above). Nevertheless, TTA decreased the REM as compared to
palmitic acid. When AMVN was used, TTA inhibited LDL oxidation in a
dose-dependent manner. 160 µmol/L TTA reduced the content
of lipid peroxides by 84% and the REM by 28%. In comparison with AAPH
as the oxidative agent, the lipid peroxides were reduced by 35% and
the REM by 9% (Table 4).
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Table 4. Effect of Different Concentrations of TTA and
Palmitic Acid on Both the Relative Electrophoretic Mobility and the
Lipid Peroxides Production of Both AMVN and AAPH-Induced LDL
Oxidation for Six Hours
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DPPH Radical Scavenging
We also tested the capacity of TTA to scavenge the DPPH free
radical and compared the effect with that of probucol, a synthetic
antioxidant, and -tocopherol, a natural antioxidant. As
shown in Fig 7, TTA failed to scavenge
the DPPH radicals.
Interactions Between LDL and TTA
Radioactive 14C labeled TTA was incorporated into the
LDL particle under the in vitro conditions (Fig 8). Preincubation of TTA with the LDL
particle had almost the same preventive effect on the oxidizability of
the LDL particle (Table 5) as when TTA
was added simultaneously with copper ions (Table 1).
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Figure 8. The interaction between C14 labeled TTA
and different LDL protein concentrations. The values are ratio active
counts in the pellet part. Data are shown as mean±SD (n=3).
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Discussion
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This study presents evidence that besides being a
hypolipidemic
agent TTA also possesses antioxidant properties as
assessed
in two different experimental systems and by several methods
of
detecting molecular oxidative damage. This is of interest not
only
because delay of LDL-oxidation may be part of the anti-atherosclerotic
mechanism
of TTA, but also because inhibition of 8-OH-dG formation may
indicate
a more omnipotent antioxidant role for TTA.
TTA not only reduced the peroxidation of lipids in LDL, but
obviously also protected the protein moiety of the LDL particle against
oxidation as is evident from its beneficial effect on the
electrophoretic mobility (Table 4).
Under the present experimental conditions TTA was incorporated into
the LDL particle (Fig 8), and obviously very fast as deduced from the
finding that the protection of LDL oxidation was almost the same
whether TTA was preincubated with LDL before oxidation, or added
simultaneously with the pro-oxidant. Our finding is
supported by others who found TTA in the LDL fraction of the TTA
treated hamsters.11
Earlier, it has been reported that 2 hours after a single dose (150
mg/kg body weight) of TTA to rats, plasma concentration was
about 100 µmol/L.33 Thus, the TTA
concentrations used in this study were kept well within a range which
may be of biological relevance (Fig 2). This may also be true for using
copper as a pro-oxidant, since its in vivo involvement in
atherosclerosis is becoming more evident through
several lines of studies.34–36
An intriguing question concerns the mechanism by means of which TTA
behaves as an antioxidant. TTA did not change the oxidation state of
copper ions (Table 3), implying that it was kept in the copper (II)
state. Therefore, the antioxidant effect of TTA might be due to
attenuating the redox cycle between the copper (II) and copper (I)
redox states. This cycle has been proposed as the mechanism of action
by which copper confer its catalytic role, and that copper (I) rapidly
forms the ultimate initiating radical, that is capable of abstracting a
hydrogen atom from a polyunsaturated fatty acid.22,37
Binding of copper on the other hand could not be the full explanation
for several reasons. First, TTA was effective also when metal
independent oxidants like azo compounds (Table 4), and ascorbate were
used (Table 2). Second, the fact that copper induced LDL oxidation was
delayed, and not prevented by TTA, contradicts that the effect of TTA
could be explained only by the formation of an inactive TTA-copper
complex. The introduction of an extended lag period into the oxidation
process by TTA (Fig 1) is typical for an antioxidant that acts by
radical scavenging. In contrast, metal ion-binding antioxidants are
expected to give a constant inhibition through the
reaction.39 Moreover, the results from the experiments with
the azo compounds, AMVN and AAPH, suggest that TTA was able to scavenge
the peroxyl radicals, with higher capacity to scavenge the peroxyl
radicals generated in the lipid phase than in the water phase (Table 4). This is consistent with our finding that TTA is
incorporated into the LDL particle and most probably is located in the
lipid phase. It seems plausible that TTA mainly incorporated in the
lipid phase as radiolabeled TTA injected intravenously into
rats was found in the triacylglycerol fraction of
the LDL (data to be published).
Nevertheless, the change of the spectra of copper caused by TTA and
palmitic acid, is consistent with the formation of a
copper-fatty acid complex (Figs 6A & 6B). In agreement with this
assumption is the finding of Balasubramanian et al38 that
nonesterified fatty acids such as palmitic acid, myristic acid and
lauric acid inhibit iron-dependent lipid peroxidation.
The negative result obtained with TTA in the classical DPPH radical
scavenging test (Fig 7), indicates that the conditions required by TTA
to take part in redox reactions are different from that of
-tocopherol and probucol. On the other hand, the
substantial power of TTA to attenuate the effect of radicals generated
by ascorbate and protect DNA-fragments from oxidation, is of
considerable biological interest (Fig 5). If a prediction of the free
radical reactions is to be made, one should consider the essential
physico-chemical factors like the one electron reduction potential
which is a key thermodynamic property,40 and the rate
constant for the reaction which is another important factor. Thus, it
must be kept in mind that a reaction that is in principle
thermodynamically possible may not be kinetically feasible, ie, the
rate constant for the reaction is too small to be biologically
significant.41 Given that the key elements involved in LDL
oxidation are not totally identified and that considerable variation in
the physico-chemical factors results from different incubation
conditions when different compounds are questioned, it is not
surprising to find that TTA behaves differently in different radical
generating systems.
Although the data of the present study cannot be translated
directly into the in vivo situation, one may point out that when rats
were treated with TTA for 12 weeks, there was a significant increase in
the plasma monounsaturated fatty acids. This was
mainly accounted for by an increased level of oleic acid (18:1 n-9) and
a decrease in the polyunsaturated fatty acids accompanied with reduced
levels of linoleic acid (18:2 n-6).42 It has been reported
that the LDL content of 18:2 n-6 strongly correlates with either the
rate of oxidation or the extent of oxidation, the percent of 18:1 n-9
in LDL correlate well with the delay before LDL oxidation and the
reduction in the content of polyunsaturated fatty acids in LDL
particles will decrease the substrate being available to be
oxidized.43 The in vivo effects of TTA that have been
reported in the above mentioned study affecting the lipid
metabolism and thus, LDL lipid composition and structure
indicate further antioxidant properties.
In summary, this study suggests that 3-thia fatty acids exert
antioxidant properties with TTA being the most potent among them. It
appears that charge and fatty acid chain length play an important role
in altering the oxidative resistance that might be mediated through the
lipid solubility. We have for the first time described intrinsic
antioxidant properties of TTA that may be of biopharmacological
interest both in the prevention of atherosclerosis and
in the protection of DNA against oxidative damage. The underlying redox
mechanism seems to be a combination of metal ion binding and radical
scavenging. Further studies are in progress to clarify molecular
mechanisms and biological relevance.
|
Selected Abbreviations and Acronyms
|
|---|
| TTA |
= |
Tetradecylthioacetic
acid=CH3-CH2-(CH2)12-S-CH2-COOH |
| Tetradecylsulfinylacetic acid |
= |
CH3-CH2-(CH2)12-SO-CH2-COOH |
| Tetradecylsulfonylacetic acid |
= |
CH3-CH2-(CH2)12-SO2-CH2-COOH |
| Tetradecyloxyacetic acid |
= |
CH3-CH2-(CH2)12-O-CH2-COOH |
| 3, 14-Dioxyhexadecanedioic acid |
= |
HOOC-CH2-O-(CH2)10-O-CH2-COOH |
| 3, 14-Dithiohexadecanedioic acid |
= |
HOOC-CH2-S-(CH2)10-S-CH2-COOH |
| LDL |
= |
low-density lipoprotein |
| ER |
= |
endoplasmic reticulum |
| PBS |
= |
phosphate-buffered solution |
| HPLC |
= |
high-performance liquid chromatography |
| UV |
= |
ultraviolet |
| REM |
= |
relative electrophoretic mobility |
|
|
Acknowledgments
|
|---|
The authors are grateful to Kari Williams and Hans Henriksen
for
excellent technical assistance. We are also grateful to
Dr Niels
Aarsaether (University of Bergen) for providing us
with the 8-OH-dG.
The work was supported by a grant from the
University of
Bergen.
Received December 12, 1996;
accepted July 31, 1997.
|
References
|
|---|
Top
Abstract
Introduction
) no addition, (
) TTA,
(x) palmitic acid, (
) octylthioacetic acid, (
)
undecylthioacetic acid, (
) pentadecylthioacetic acid and (
)
hexadecylthioacetic acid.
) 80 µmol/L. The oxidation time were: 0 (native LDL), 1, 2
and 3 hours. Data shown are from one representative
experiment.
