Effect of antioxidant protection by p-coumaric acid
on low-density lipoprotein cholesterol oxidation
Lun-Yi
Zang1,
Greg
Cosma2,
Henry
Gardner3,
Xianglin
Shi1,
Vince
Castranova1, and
Val
Vallyathan1
1 Pathology and Physiology Research Branch, Health Effects
Laboratory Division, National Institute for Occupational Safety and
Health, Morgantown, West Virginia 26505-2888; 2 Department of
Environmental Health, Colorado State University, Fort Collins, Colorado
80523-1676; 3 U.S. Army Center for Environmental Health
Research, Fort Detrick, Maryland 21701-5010
 |
ABSTRACT |
Mechanisms in
which p-coumaric acid (CA) acts as an antioxidant are not
well understood. This study investigated whether CA can act as a direct
scavenger of reactive oxygen species (ROS) and whether it minimizes the
oxidation of low-density lipoprotein (LDL). Rats were administered CA
in drinking water at low or high doses for 10, 21, and 30 days (uptakes
were 29 and 317 mg/day, respectively). Blood levels of
8-epiprostaglandin F2
were monitored as a marker of LDL
oxidation. Oral administration of CA (317 mg/day) for 30 days
significantly inhibited LDL oxidation. CA also reduced LDL cholesterol
levels in serum but had no effect on levels of high-density lipoprotein
cholesterol. In vitro studies that used electron spin resonance in
combination with spin trapping techniques were used to determine the
ability of CA to scavenge ROS and alter LDL oxidation. CA effectively
scavenged ·OH in a dose-dependent manner. IC50 and
maximum velocity for CA scavenging of ·OH were 4.72 µM and 1.2 µM/s, respectively, with a rate constant of 1.8 × 1011 M
1 · s
1. Our
studies suggest that the antioxidant properties of CA may involve the
direct scavenging of ROS such as ·OH.
hydroxyl radical; lipid peroxidation; reactive oxygen
species
 |
INTRODUCTION |
MANY STUDIES HAVE
ATTEMPTED to determine whether antioxidants prevent oxidation of
low-density lipoprotein (LDL) and slow the progression of
atherosclerosis (9, 22, 24). In this respect, an
increasing awareness and importance are given to nutrition and
specifically to plant products in the diet. p-Coumaric acid (CA) widely exists in fruits, such as apples and pears, and in vegetables and plant products, such as beans, potatoes, tomatoes, and
tea. It is an intermediate product of the phenylpropanoid pathway in
plants. CA has been suggested to exhibit antioxidant properties
(3, 4, 12-14). It was reported that CA in vitro can
provide antioxidant protection to LDL as a result of the chain-breaking activity of CA (3). Diet supplementation with a crude
extract of CA isolated from pulses resulted in the reduction of ester cholesterol, providing a protective mechanism against the development of atherosclerosis (23). The ability of CA to prevent
excessive lipid peroxidation on the basis of its chain-breaking
activity of
-tocopherol oxidation has also been demonstrated
(12). More recently, Castelluccio et al. (3)
reported that CA was effective in enhancing the resistance of LDL to
oxidation. If CA is an efficient antioxidant for LDL, it may play a key
role in the purported effect of oxidized lipoprotein on platelet
activity to inhibit atherogenesis. In addition, the dehydrogenation
polymer of CA was reported to have anti-human immunodeficiency virus
activity (26). However, from these studies, the mechanism
of action of CA is not fully understood, although it has been proposed
that the antioxidant properties of CA are due to its ability to
directly scavenge reactive oxygen species (ROS) (10, 11).
To address whether CA can act as a direct scavenger of ROS and thereby
modify the age-related changes in blood lipid profiles, we have
investigated the effects of CA administered through drinking water in
rats fed a diet containing 5% fat. At days 0,
10, 21, and 30, we monitored the
oxidative by-product of lipid peroxidation, 8-epiprostaglandin
F2
(8-EPI), and cholesterol levels in serum of rats
consuming a low or high dose of CA. To investigate further the
scavenging efficiency of CA, we studied the effects of CA on ROS
generation with the use of electron spin resonance (ESR) techniques. If
CA scavenges ROS, the characteristic ESR signals of trapped ROS
generated in a given reaction system should decrease with the addition
of CA. We also report new experimental data showing that CA is capable
of scavenging ·OH, inhibiting lipid peroxidation in vivo,
and thereby reducing serum LDL cholesterol levels.
 |
MATERIALS AND METHODS |
Hydrogen peroxide (H2O2) was obtained
from Fisher Scientific (Pittsburgh, PA). CA, high-density lipoprotein
(HDL) cholesterol reagent (PTA/MgCl2), cholesterol
diagnostic kits, cholesterol calibrator,
5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 3-cyano-proxyl free
radical, and FeSO4 were purchased from Sigma Chemical (St. Louis, MO). 8-EPI immunoassay kit EA-84 was obtained from Oxford Biomedical Research (Oxford, MI).
Animal treatment.
Adult (60 days old) Sprague-Dawley male rats weighing 500-510 g
were obtained from Hill Top Farms (Scottsdale, PA). Rats were fed a
diet of standard Purina chow containing 5% fat and water ad libitum.
Animals were housed in single cages and were given a low dose (1 mg/ml)
or high dose (10 mg/ml) of CA in water. CA uptake was estimated from
the daily consumption of water per 500 g body wt. The CA uptakes
were 29 ± 3.6 and 317 ± 33.8 mg/day for the low and high
doses, respectively. On a daily basis, there was an ~10-15%
variability in the uptake of CA between animals. The animals were
placed into four groups (6 in each group), based on CA treatment, and
killed at predesignated times (0, 10, 21, and 30 days). All animals
receiving 29 or 317 mg/day CA and all control (sham) rats were killed
by an overdose of pentobarbital sodium (1.5 ml). Blood, ~15 ml from
each rat, was collected directly from the heart. Blood was then allowed
to clot at room temperature and then centrifuged at 600 g
for 5 min, and the serum was collected. The serum samples were divided
into several aliquots and stored at
70°C until used for the assays.
Measurement of lipid peroxidation.
8-EPI, a product of lipoprotein peroxidation, is a potent
vasoconstrictor in rats and rabbits (16-19). This
lipoprotein by-product can be used as a marker of oxidative stress in
atherosclerosis and carcinogenesis (17-19). The
method is based on a competitive enzyme-linked immunoassay for the
determination of 8-EPI in biological samples (16). 8-EPI
in the samples competes with 8-EPI conjugated to horseradish peroxidase
for binding to the antibody coated on the plate. The peroxidase
activity results in color development in the substrate, with color
intensity being proportional to the amount of unconjugated 8-EPI in the
samples. Unless otherwise stated, the reaction mixture consisted of 100 µl of rat serum and 100 µl of diluted 8-EPI horseradish enzyme
conjugate in each well (antibody-coated plate). The plates were allowed
to stand in the dark at room temperature for 2 h. They were then
inverted, and the contents emptied by being patted dry upside down on
lint-free towels. Each well was washed four times with 400 µl of wash
buffer, and 200 µl of substrate were added to each well. After an
incubation of 20 min, 50 µl of 1 M sulfuric acid were added to each
well, and the plates were read at 450 nm with the use of a Spectromax 250 (Molecular Devices, Sunnyvale, CA). The quantitative values for
8-EPI were calculated from a standard curve produced using the same
reagents. All estimations were carried out in duplicate, using groups
with and without spiked samples and negative controls. Butyl hydroxy
toluene was used to inhibit lipid peroxidation in negative controls.
Assays of cholesterol.
Measurements of total cholesterol and HDL cholesterol in the rat serum
were made according to the standard procedures established by the Sigma
diagnostics kits (nos. 352 and 352-4) using a Beckman DU 650 spectrophotometer (Beckman Instruments, Columbia, MD). LDL cholesterol
values were calculated from the total and HDL cholesterol results.
Spin trapping and ESR measurements.
The typical spin-trapping reaction mixture consisted of DMPO (250 µM), H2O2 (1 mM), and FeSO4 (100 µM) in air-saturated phosphate buffer (10 mM, pH 7.4) in the absence
or presence of CA. We used a low concentration of DMPO (250 µM)
because of the competition reaction between DMPO and CA for ·OH. The
concentration of CA in stock phosphate buffer (pH 7.4) was measured by
ultraviolet spectroscopy at 286 nm with an extinction coefficient of
1.9 × 104
M
1 · cm
1 (1) and then
adjusted to the desired final concentration. The reaction was initiated
by mixing 20 µl of 5 mM FeSO4 stock solution (prepared in
0.01 N H2SO4 because Fe2+ can be
oxidized at pH values higher than 3.0), 0.25 ml of 1 mM DMPO, 0.25 ml
of 4 mM H2O2, and 0.48 ml (or 0.50 ml) of
phosphate buffer (pH 7.4) in the presence or absence of various
concentrations of CA stock solutions (20 µl) in different
concentrations. An ESR flat cell was calibrated and fixed in the ESR
cavity. Immediately after samples were mixed, they were siphoned into
the ESR flat cell assembly (60 × 8.5 × 0.25 mm ID), and the
ESR spectrum was recorded at 1 min of reaction time at room
temperature. The ESR signal of the DMPO-·OH adduct produced
by the standard reaction of H2O2 with
Fe2+ was monitored for at least 15 min at room temperature,
and its decay rate was found to be ~2%/min. Because the Fenton
reaction is complete within seconds, commercially available
3-cyano-proxyl free radical was used as a nitroxide standard for
determining relative concentrations of the spin adduct of DMPO with
·OH. The relative ·OH radical concentration was quantitated by
measuring heights of the first peak of the DMPO-·OH signals. The
maximum concentration of DMPO-·OH was calculated to be ~70 µM.
The recordings of ESR spectra were performed with a Varian 109 ESR
spectrometer. Unless otherwise stated, the ESR parameters were set at
100 kHz, X-band, microwave frequency of 9.73 GHz, microwave power of 20 mW, modulation amplitude of 0.63 G, time constant of 0.25 s, scan time of 8 min, scan width of 200 G, and receiver gain of 2 × 104.
Statistical analysis.
The data presented are means ± SD of three in vivo experiments
for each time interval and a minimum of five or more in vitro experiments in duplicate. A Student's t-test was used to
determine differences between control groups and animals treated with
CA. A probability value of 0.05 or smaller was considered significant. Data analyses were made using Sigmastat (Jandel Scientific, San Rafael, CA).
 |
RESULTS |
Effect of CA on rat serum lipid peroxidation.
8-EPI was used as a marker for the estimation of lipid peroxidation
products resulting from oxidative injury (16-19).
Figure 1 shows the in vivo effect of CA
consumption on the formation of 8-EPI in rat serum. The accumulation of
lipid peroxidation by-products in serum from control animals not
receiving CA was found to increase with increasing age of the rats. At
day 0, the average 8-EPI level in control group was 0.125 ng/ml. At day 30, the average 8-EPI level in the control
group was 0.238 ng/ml. This age-related increase in 8-EPI after 30 days
was significantly greater (1.9-fold) compared with the 8-EPI level
(0.125 ng/ml) at day 0. In contrast, CA was found to inhibit
significantly the formation of the lipid peroxidation product, 8-EPI.
In animals administered 29 mg CA/day for 30 days, there was a modest
inhibition of 8-EPI formation (~8.4%), whereas a significant
inhibition of 8-EPI formation (~60%) was noted after administration
of 317 mg CA/day for 30 days. A modest inhibition of 8-EPI production
was also observed at day 10 (~2%) and at day
21 (~2.5%) in animals administered 317 mg CA/day. To examine
the possibility of CA interaction with 8-EPI, standards containing
different concentrations of 8-EPI were mixed with CA and evaluated
using the same protocol. There was no significant effect on absorbance
at 450 nm, indicating that CA did not react with 8-EPI and that it was
an effective inhibitor against lipid peroxidation.

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Fig. 1.
Inhibition of lipid peroxide formation in the blood serum
of rats administered p-coumaric acid (CA) in drinking water
at low (29 mg/day) or high (317 mg/day) levels for 30 days. The amount
of CA intake by rat was calculated from the daily consumption of water
containing CA. Data are means ± SD of 3 experiments in
duplicates. Each assay sample contained 100 µl of serum and the
amount of reagents indicated in MATERIALS AND METHODS.
* Value is significantly different from day 0 (P = 0.006). ** Value is significantly different
from day 30 control (P = 0.002). 8-EPI,
8-epiprostaglandin F2 ; NS, not significant; brackets
indicate concentration.
|
|
Effect of CA on rat serum cholesterol.
Atherogenesis is closely associated with circulating levels of LDL
cholesterol and resulting reactions leading to injury
(28). Elevated LDL has been associated with a higher
incidence of atherosclerosis (28, 27). LDL cholesterol
levels in serum of control groups not receiving CA were found to
increase with increasing age of the rats. As shown in Fig.
2, at day 30, the total serum
cholesterol increased ~40% compared with day 0 and LDL
cholesterol level increased ~100%. In contrast, HDL cholesterol
levels were not found to change significantly (data not shown). For
rats treated with 29 mg CA/day, CA reduced total cholesterol by 12%
and LDL cholesterol by 30% in 30 days. At 317 mg CA/day, CA inhibited
total cholesterol by ~17% and LDL cholesterol by ~33% in 30 days.
Animals administered 317 mg CA/day for 10 and 21 days also showed a
remarkable decline in LDL cholesterol levels (~75 and 68%,
respectively). There was no significant effect on HDL cholesterol in
CA-treated animals at any of these time periods. These data indicate
that CA is capable of decreasing LDL cholesterol levels in circulating
blood of animals administered CA at all three time intervals monitored.

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Fig. 2.
Effect of CA supplementation on the levels of total serum
cholesterol and low-density lipoprotein (LDL) cholesterol. Data are
means ± SD of values obtained from 3 rats administered CA in the
drinking water (0, 29, or 317 mg/day). Each assay sample contains 50 µl of serum and the amount of reagents indicated in MATERIALS
AND METHODS. * Value is significantly different from day
0. ** Value is significantly different from the day
30 controls. Total (TDL) cholesterol day 0 vs.
day 30: P = 0.02; day 30 vs. day
30 CA: P = 0.01. LDL cholesterol day 0 vs. day 30: P = 0.02; day 30 vs.
day 30, 29 mg CA/day: P = 0.05; day
30 vs. day 30, 317 mg CA/day: P = 0.04.
|
|
ESR spectra of the hydroxyl radical spin adduct.
In in vitro studies, the Fenton reaction (Fe2+ + H2O2
Fe3+ + ·OH + OH
) was used as a source for ·OH generation (6,
30). Figure 3 shows the ESR
spectra of the DMPO-·OH adduct obtained in the reaction mixtures of
H2O2 (1 mM) and FeSO4 (100 µM)
containing DMPO (250 µM) in the absence or presence of the indicated
amount of CA. The ESR spectrum in Fig. 3a was obtained in a
reaction mixture in the absence of CA. This spectrum exhibited four
splitting lines with an intensity ratio of 1:2:2:1, which typically
results from the interactions of an uncoupled electron with a primary nitrogen atom along with a secondary
-proton. The hyperfine
splitting constants of the signal (aN = aH = 14.9 G) are consistent with previously
reported values for DMPO-·OH (29-31), indicating
that the signal results from trapped ·OH. CA was found to
significantly inhibit the formation of the DMPO-·OH adduct. As shown
in Fig. 3, b-d, at concentrations as low as
2.5 µM, CA inhibited ~40% of the EPR signal intensity and, at 20 µM, CA inhibited ~86% of the EPR signal intensity. To examine the
possibility of CA interaction with DMPO-·OH, 20 µM CA was added to
the reaction mixture of DMPO (250 µM), H2O2
(1 mM), and FeSO4 (100 µM) 1 min after initiation of the
reaction, i.e., after DMPO-·OH adduct formation. There was no
observed effect on ESR signal intensity (data not shown), indicating
that CA is an effective scavenger for ·OH.

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Fig. 3.
Electron spin resonance (ESR) spectra of spin adducts of
hydroxyl radical observed during the reaction of FeSO4 (100 µM), H2O2 (1 mM), and 250 µM
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in air-saturated
phosphate buffer, pH 7.4, in the presence of 0 (a), 2.5 µM
(b), 5.0 µM (c), 10 µM (d), and 20 µM (e) CA. ESR spectroscopy settings were as described in
MATERIALS AND METHODS.
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|
Effect of CA on ESR signal intensity.
The dose dependence of CA inhibition of the DMPO-·OH adduct ESR
signal intensity is depicted in Fig. 4.
Its IC50 value, i.e., the concentration of CA required to
cause 50% inhibition of ESR signal intensity, was estimated to be
~4.7 µM. To obtain a more precise value, the percentage of ESR
signal intensity was plotted against the log of the CA concentration,
which resulted in a linear curve with an equation y =
52.783log(x) + 85.576 (Fig. 4, inset). Therefore, when y = 50, the IC50 value was
calculated to be 4.72 µM, which is consistent with our roughly
estimated value of CA concentration that caused 50% inhibition of ESR
signal intensity. No inhibition of ESR signal was observed in the
reaction mixture incubated under identical conditions in the absence of
CA, indicating that inhibition of the ESR signal of DMPO-·OH is
entirely dependent on the presence of CA.

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Fig. 4.
Dose-dependent inhibition of DMPO-·OH signal intensity
by CA. Reaction mixture was same as in Fig. 3 in the presence of the
indicated concentration of CA. Values are means ± 5 units SD of 5 determinations. Inset: data displayed as the percentage of
ESR signal intensity against log[CA]. ESR spectroscopy settings were
the same as described in Fig. 3. , Time constant; , ratio of
slope to intercept; Vmax, maximum velocity;
kCA, rate constant of CA scavenging
·OH.
|
|
To quantitatively confirm the elimination of ·OH by CA in this
system, we used the methods reported by Buettner et al.
(2). If we assume that the velocity of CA scavenging ·OH
is equal to the rate of formation of spin adducts, when the ESR signal
intensity of DMPO-·OH is suppressed by 50%, then we can obtain the
following equation
|
(1)
|
where brackets indicate concentration, kCA
is the rate constant of CA scavenging ·OH, and
kDMPO is the rate constant of DMPO trapping
·OH. Because the ·OH concentration on both sides of Eq. 1 are equivalent, we can omit this item and obtain the following equation
|
(2)
|
With the use of the IC50 value of CA described above,
4.72 µM, and kDMPO of 3.4 × 109 M
1 · s
1, i.e., the
value for DMPO trapping ·OH (7), we obtained a rate
constant of 1.8 × 1011
M
1 · s
1 for CA scavenging ·OH in
this aqueous solution (pH 7.4).
Effect of CA on the rate of hydroxyl radical elimination.
Because CA does not interact with DMPO-·OH, there exists a
competition between DMPO and CA for available ·OH. Therefore, the kinetic parameters of CA, as the competitor in the test system, can be
obtained. In a typical reaction, Fe2+ and
H2O2 generate ·OH, which in turn reacts with
DMPO to form DMPO-·OH spin adducts. CA inhibits the spin adduct
formation by scavenging ·OH. Because the maximum concentration of
DMPO-·OH generated was determined to be ~70 µM (see
MATERIALS AND METHODS) and the DMPO concentration in the
reaction system is four times larger than the ·OH concentration, we
assume that all ·OH can be trapped and that DMPO-·OH formation
reaches a maximum velocity (Vmax). Therefore,
DMPO-·OH can represent the relative concentration of ·OH. Because
one DMPO molecule can trap one ·OH molecule with a high rate constant
(3.4 × 109
M
1 · s
1), the concentration of
inhibited DMPO-·OH spin adducts should be identical to the
concentration of ·OH eliminated by CA. If the reduced ESR signal
intensity is directly proportional to the concentration of CA, a
first-order kinetic plot should yield a straight line. Under
steady-state-conditions, the decrease in concentration of ·OH could
be expressed as follows
|
(3)
|
where dV is the velocity of scavenging of ·OH by CA.
Under a given condition, dV values can be obtained at
different concentrations of CA. Figure 5
depicts the velocity of CA scavenging ·OH as a function of CA
concentration. The hyperbolic nature of the curve in Fig. 5 indicates
that CA scavenged ·OH to completion. The Lineweaver-Burk plot (Fig.
5, inset) demonstrated linearity with a correlation coefficient of 0.985. The reciprocal of the y-intercept
value of the plot, Vmax, i.e., the maximum rate
of CA scavenging ·OH in this system, was found to be 1.2 µM/s. The
ratio of the slope to intercept from Fig. 5 provides
kd/kCA (where
kd is the dissociation constant), the
-value (8, 32), which is the concentration of CA that
eliminates one-half of the ·OH. This
-value was found to be
4.78 × 10
6 M, which is similar to the
IC50 value (4.72 µM) obtained in Fig. 4. Thus, when
kCA = 1.8 × 1011
M
1 · s
1, i.e., the value obtained
above for CA scavenging ·OH, the kd value for
the decay of ·OH was calculated to be 8.6 × 105
s
1. These data strongly support the hypothesis that CA is
an efficient scavenger for ·OH.

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Fig. 5.
Rate of ·OH scavenging as a function of CA
concentration during the reaction of FeSO4 (100 µM) and
H2O2 (1 mM) in air-saturated phosphate buffer,
pH 7.4, in the presence of 250 µM DMPO. Values are means ± SD of 5 determinations. Inset: double reciprocal plot of the rate of
DMPO-·OH reduction vs. CA concentration. ESR spectroscopy settings
were the same as described in Fig. 3.
|
|
The scavenging rate constants for the reactions of several compounds
with ·OH have been reported (5, 15, 25) and are presented in Table 1. As shown in Table
1, the rate constant value obtained using CA as an antioxidant is one
order of magnitude larger than all of the listed compounds. Because all
the listed compounds were reported to have been tested at pH 7.0 at
room temperature, we believe the phenomenal scavenging property of CA
is probably also due to its phenolic structure. Phenolic compounds are
also good reductants that may contribute to this antioxidant property.
Thus it appears that CA is a powerful antioxidant with a specific
scavenging efficiency for ·OH.
 |
DISCUSSION |
Increasing evidence indicates that oxidative modification of LDL
is a major causative factor in the pathogenesis of atherosclerosis, and
endogenous antioxidant supplementation may protect against oxidation of
LDL and ameliorate the progression of atherosclerosis. The results
reported here clearly demonstrate that CA is able to directly scavenge
·OH, protect LDL from oxidation, and reduce serum cholesterol levels.
In addition, we also demonstrated that CA significantly reduced the
levels of a lipid peroxidation by-product, 8-EPI, in serum of rats
administered CA. The spin trapping-ESR techniques that we applied in
these studies also substantiated the scavenging efficiency of CA, which
was 10-fold greater than other antioxidants. It is known that other
antioxidants such as vitamin C, vitamin E,
-carotene, ubiquinol-10,
and others contribute to the protection of LDL from oxidation.
Therefore, during a propagation phase of enhanced lipid peroxidation,
supplementation with CA may prove beneficial.
Although CA has been reported to act as an antioxidant (3, 4,
12-14, 23), little direct evidence was available to support this claim. Our ESR studies demonstrate that CA is indeed an effective antioxidant with a scavenging rate for ·OH one order of magnitude greater than other common antioxidants (Table 1). The biological role
of this potent ·OH scavenger was confirmed by its abilities to
inhibit the formation of a lipid peroxidation by-product, 8-EPI, in
serum and to reduce serum cholesterol levels. Such in vivo actions may
be due to the ability of CA to scavenge ·OH, as demonstrated in vitro
using ESR techniques in which CA decreased DMPO-·OH formation (Fig.
3). Additional support for this conclusion came from the ESR kinetic
study, in which the Lineweaver-Burk plots resulted in a linear
relationship with a correlation coefficient of 0.985 (Fig. 5,
inset), indicating that there is a stoichiometric
relationship between CA concentration and scavenging of ·OH. The
value for kCA was found to be 1.8 × 1011 M
1 · s
1, which is
close to the diffusion controlled rate. Vmax of
·OH scavenged by CA was found to be 1.2 µM/s. The IC50
value of CA required to cause 50% inhibition of ESR signal intensity
was found to be 4.72 µM, which is equal to the
-value, the
concentration of CA required to scavenge one-half of the ·OH
generated in the test system. The lifetime of ·OH in aqueous solution
was found to be 1.2 µs. These data strongly demonstrate that CA is a
powerful ·OH scavenger, capable of virtually eliminating the ·OH
generated. Thus CA may block LDL peroxidation and reduce serum
cholesterol levels by scavenging ·OH.
It is known that cholesterol can be obtained from diet or it can be
synthesized in the liver. Cholesterol is transported to peripheral
tissues by lipoproteins and regulates de novo cholesterol synthesis.
LDL is the major carrier of cholesterol in blood. LDL contains a core
of about 1,500 esterified cholesterol molecules; the most common fatty
acyl chain in these esters is linoleate, a polyunsaturated fatty acid.
A shell of phospholipids and unesterified cholesterol surrounds this
highly hydrophobic core. If the LDL shell ruptures, serum cholesterol
levels will increase. ·OH is well known to be capable of extracting a
hydrogen atom from unsaturated fatty acids, causing lipid peroxidation
and initiating free radical chain reactions. As a result, free radical
chain reactions can cause massive damage to biological molecules such
as DNA, cellular membranes, and lipoproteins. Therefore, scavenging
·OH should prevent lipid peroxidation and may reduce serum
cholesterol levels as well as protect LDL against oxidation. Because CA
scavenges ·OH at a near-diffusion control rate, it can be expected to
prevent lipid peroxidation and reduce serum cholesterol levels. As
shown in Figs. 1 and 2, the administration of CA lowered the level of the lipid peroxidation by-product, 8-EPI, by 60% and reduced the LDL
cholesterol level by 33% in rat serum of animals treated with 317 mg
CA/day for 30 days. Thus our results provide a plausible explanation for the previously proposed ability of CA to prevent lipid
peroxidation, reduce serum cholesterol levels, and enhance the
resistance of LDL to oxidation (3, 12, 23).
Atherosclerosis resulting in coronary heart disease is the leading
cause of death in men after age 35. In atherosclerosis, increases in
cholesterol are closely associated with circulating levels of LDL
cholesterol and increasing morbidity. Circulating monocytes are capable
of releasing superoxide anion radicals, which can be dismuted by
superoxide dismutase and converted to ·OH, via participation of
transition metals such as iron (21). Oxidation of LDL by
ROS is currently considered to be an important pathobiological pathway
by which incremental changes occur in the endothelial cells lining the
intima in blood vessels. Repeated or continuing insults from ROS
ultimately result in their injury and development of disease
(20). Although cells contain multiple antioxidant defenses
to protect themselves against free radicals, these protective
mechanisms are kept at equilibrium and are not present in excess.
Exogenous sources of antioxidants are needed to compensate the
oxidative injury resulting from pollution, cigarette smoke, and other
toxic biochemical reactions that result in lipid peroxidation. The
water and lipid solubilities of CA, together with its substantial
efficiency to scavenge ·OH and to inhibit lipid peroxidation and
reduce LDL cholesterol levels in vivo, provide important insights for
future investigations. Dietary supplements may provide a useful
approach for the prevention of injury to vascular cell membranes, thus
preventing or delaying the onset of cardiovascular disease.
 |
ACKNOWLEDGEMENTS |
This work was partially supported by U. S. Army Center for
Environmental Health Research funding to Colorado State University.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: V. Vallyathan, Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and
Health, 1095 Willowdale Rd., Morgantown, WV 26505-2888 (E-mail: vav1{at}cdc.gov).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 July 1999; accepted in final form 13 April 2000.
 |
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