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-lipoic acid
1 Center for Exercise Science, University of Florida, Gainesville, Florida 32611; 2 Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; 3 Department of Physiology, University of Kuopio, Kuopio, Finland 70211; and 4 Department of Kinesiology, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of these
experiments was to examine the effects of dietary antioxidant
supplementation with vitamin E (VE) and 
-lipoic acid (-LA) on
biochemical and physiological responses to in vivo myocardial
ischemia-reperfusion (I-R) in aged rats. Male Fischer-334 rats (18 mo
old) were assigned to either 1) a control diet (CON) or
2) a VE and -LA supplemented diet (ANTIOX). After a 14-wk
feeding period, animals in each group underwent an in vivo I-R protocol
(25 min of myocardial ischemia and 15 min of reperfusion). During
reperfusion, peak arterial pressure was significantly higher
(P < 0.05) in ANTIOX animals compared with CON diet
animals. I-R resulted in a significant increase (P < 0.05) in myocardial lipid peroxidation in CON diet animals but not in
ANTIOX animals. Compared with ANTIOX animals, heart homogenates from
CON animals experienced significantly less (P < 0.05)
oxidative damage when exposed to five different in vitro radical
producing systems. These data indicate that dietary supplementation with VE and -LA protects the aged rat heart from I-R-induced lipid
peroxidation by scavenging numerous reactive oxygen species. Importantly, this protection is associated with improved cardiac performance during reperfusion.
aging; free radicals; lipid peroxidation; myocardial performance
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THERE IS MUCH INTEREST in nutritional antioxidant supplementation as a therapeutic preventative strategy against myocardial ischemia-reperfusion (I-R) injury. The interest results from findings that reactive oxygen species (ROS) contribute significantly to myocardial I-R injury (11). ROS, such as superoxide anions, hydroxyl radicals, and peroxyl radicals formed during reperfusion, can lead to lipid peroxidation within the cardiac myocyte, resulting in decreased cardiac performance (3).
It is widely believed that an optimum antioxidant supplement contains
more than one nutrient, and recently the combination of two
antioxidants, vitamin E (VE) and -lipoic acid (-LA), has
generated scientific interest. VE is the major lipid peroxidation chain-breaking antioxidant and is located in the lipid phase of the
cell. When VE quenches a ROS, a VE radical is formed that must be
recycled back to its reduced form to continue to provide antioxidant
protection. Importantly, -LA is capable of recycling VE
(26). In addition, -LA, reduced -LA (dihydrolipoic
acid), and their metabolites also function as antioxidants
(26). -LA has also been reported to be an effective
glutathione substitute, capable of increasing cellular glutathione
levels and further improving the antioxidant status of the myocardium
(14).
The combination of VE and dihydrolipoic acid (DHLA) has been shown to improve rodent cardiac performance in experiments using an in vitro I-R model (15, 16). In these studies, young adult animals were fed a high VE diet (10,000 IU/kg diet), and DHLA was added to the perfusion medium. The myocardial protection from I-R injury was reported to be due to the synergism of both of the two compounds (15), as opposed to their individual antioxidant abilities (15). At present, it is unknown whether this protection is realized in vivo.
Currently, limited information exists regarding the ability of
antioxidant supplementation to protect senescent hearts from I-R-induced injury. This is unfortunate, given that, compared with
young animals, myocardial I-R in senescent animals results in a greater
myocardial injury (21). This observation could be linked
to the fact that aging is associated with reduced myocardial antioxidant protection (32). Therefore, these experiments
examined the effects of dietary supplementation of antioxidants (VE and -LA) on I-R-induced myocardial injury in senescent animals.
Specifically, we tested the hypothesis that the dietary combination of
VE and -LA would provide protection from I-R damage in aged animals. Furthermore, on the basis of known antioxidant properties of VE and
-LA, we postulated that dietary supplements with these antioxidants would protect the heart against I-R lipid peroxidation induced by
superoxide radicals, hydroxyl radicals, hydrogen peroxide, and peroxyl radicals.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental animals and dietary supplementation.
Male Fischer-344 rats (18 mo old) were divided into two dietary groups:
1) a control diet (CON), n = 25, and
2) a VE- and -LA-supplemented diet (ANTIOX),
n = 26. CON animals were fed the AIN-93M purified diet,
which contains 75 IU DL--tocopherol acetate/kg diet.
ANTIOX animals were fed the AIN-93M purified diet with 10,000 IU
DL--tocopherol acetate/kg diet and 1.65 g -lipoic acid/kg diet. Diets were professionally prepared (Harlan Teklad, Madison, WI). Animals were fed the diet for 14 wk. Randomly selected animals from the two dietary groups were exposed to the I-R
protocol (CON, n = 15; ANTIOX, n = 14).
The remaining animals served as sham controls by undergoing the same
surgical interventions without I-R. These sham controls provided
baseline data for the myocardial levels of lipid peroxidation and
antioxidant capacity. Furthermore, ventricular tissue from sham
controls was used for the heart homogenates that were subjected to the
in vitro oxidative challenges.
Experimental protocol. The in vivo I-R procedure has been explained in detail in previous work from our laboratory (5, 27). Briefly, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was achieved by a ligature around the left coronary artery; occlusion was maintained for 25 min, followed by a 15-min period of reperfusion. During this time, cardiac performance was monitored by a catheter placed in the ascending aorta. At the completion of the experimental protocol, animals were euthanized with an overdose of pentobarbital sodium, and the heart was removed, washed in an antioxidant buffer (1 mM butylated hydroxytoluene and 100 µM EDTA in a 50 mM sodium phosphate buffer, pH = 7.4), and rapidly frozen for subsequent biochemical analyses.
Assay of tissue levels of VE. Myocardial levels of VE were determined with HPLC by use of the protocol of Cort et al. (6). Tissue preparation before VE analysis consisted of mechanical homogenization in acetone (Ultra-Turrax T25, IKA Works, Cincinnati, OH), double extraction with petroleum ether, and then reconstitution in isooctane before injection into the HPLC (ABI Analytical Spectroflow 400). Aliquots (20 µl) of the isooctane extract were injected onto a 250 × 4-mm, 10-µm LiChrosorb SI column (Baird and Tatlock, Dagenham, UK).
Assay of tissue levels of -LA.
At physiological pH, the salts of -LA dissociate and form lipoate.
Therefore, determination of -LA levels in physiological fluids or
tissues is specifically a measure of lipoate content. Tissue levels of
lipoate were measured according to Sen et al. (31), with
slight modification. Frozen left ventricle samples (0.5-0.6 g)
were ground to powder in liquid nitrogen with a mortar and pestle and
then homogenized on ice in 2 ml of 20% metaphosphoric acid with a
Teflon homogenizer. The homogenate was ultrasonicated intermittently
for 100 s (10-s periods with 10 s of rest). The sample was
extracted in hexane. After hexane evaporation under nitrogen, the
extract was solubilized in a solvent containing 50% 0.2 M
monochloroacetic acid, 30% acetonitrile, and 20% methanol. Samples
were filtered (0.22 µm) and frozen at 
80°C for HPLC analysis. Samples (0.2 ml) were separated on an Alltima C18 column
(250 × 4.6 mm, 5 µm; Alltech Associates, Deerfield, IL) by use
of a mobile phase consisting of 50% 50 mM sodium biphosphate in water, 30% acetonitrile, and 20% methanol and a flow rate of 1 ml/min. -LA was detected at a retention time of 9.5 min with a Coulechem II
multielectrode electrochemical detector (model 5100A, ESA, Chelmsford,
MA). The electrodes were set at the following potentials: electrode 1, +0.45V; electrode 2, +0.85V; and guard cell: +0.90V. Data were
collected using a PE Nelson 900 series interface [Perkin Elmer (PE),
San Jose, CA] and processed using the PE Nelson Turbochrome 4 (Perkin
Elmer) software. Racemate mixture of lipoate, used as a standard, was
provided by ASTA Medica (Frankfurt, Germany). Using this method, we
obtained a linear (r2 = 0.996) standard
curve in the range of 0.2-0.75 nmol of lipoate.
Biochemical assessment of endogenous antioxidant enzymes. To determine whether our dietary treatments altered endogenous antioxidant enzymes, a small sample of the left ventricle from all animals was assayed to measure the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). SOD and GPX activities were determined spectrophotometrically with a modification of the procedures described by Oyanagui (25) and Flohé and Günzler (8), respectively. CAT was assayed using the procedure described by Ji et al. (18).
Lipid peroxidation measurements. To determine the degree of oxidative damage in the heart, left ventricular levels of two by-products of lipid peroxidation were measured. Malonyldialdehyde (MDA) levels were determined spectrophotometrically by use of the thiobarbituric acid-reactive substance (TBARS) method previously described by Mihara and Uchiyama (24), with 1,1,3,3-tetramethoxypropane used as the standard.
Lipid hydroperoxides were quantified using the ferrous oxidation/xylenol orange technique reported by Hermes-Lima (17). Cumene hydroperoxide was used as the standard for this assay, and values were expressed as cumene hydroperoxide equivalents (CHE).In vitro measurement of tissue antioxidant capacity.
To evaluate the antioxidant potential of VE and -LA supplementation,
heart homogenates from the sham surgery animals were subjected to five
different ROS-generating systems and then analyzed for lipid
peroxidation with the TBARS assay described above. Sections of the left
ventricle from both ANTIOX and CON animals were homogenized at a
concentration of 10:1 in either 0.9% (wt/vol) saline solution (for the
aqueous generating systems) or ethanol (for the lipid phase system).
Aliquots of the homogenates were incubated at a concentration of 10 mg
protein/ml in the presence or absence of an ROS generating system. Five
ROS generating systems were used. 1) Superoxide radicals
were generated by a hypoxanthine-xanthine oxidase system, according to
the method of Fridovich (9). 2) Hydrogen
peroxide (100 µM) was added directly to heart homogenates according
to the method of Gonzalez Flecha et al. (12).
3) Hydroxyl radicals were generated in the heart homogenates
by adding 0.1 µM ferrous sulfate (FeSO4) to heart
homogenates. 4) Peroxyl radicals were generated in the
aqueous phase of the homogenate by the addition of 9.4 mM AAPH
[2,2'azobis(2-amidinopropane)-dihydrochloride] (19).
5) Peroxyl radicals were generated in lipids in the heart homogenate by thermal decomposition of 5 mM AMVN [2,2'azobis(2-4 dimethylvaleronitrile)] (19).
Statistical analysis. Biochemical data were subjected to a one-way ANOVA. Performance measures were analyzed using a two-way repeated-measures ANOVA. Scheffé's test was used post hoc, with significance established a priori at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Figure 1 contains the mean (±SE)
myocardial VE and -LA concentrations in both CON and ANTIOX diet
animals. These data indicate that our feeding protocol resulted in a
significant (P < 0.05) increase in the myocardial VE
levels in the ANTIOX animals compared with CON animals. However, no
significant differences (P > 0.05) in -LA levels
existed between the two groups.
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Cardiac performance was assessed during ischemia and reperfusion by
utilizing a fluid-filled catheter placed in the ascending aorta to
measure peak systolic pressure. The effects of the different diets on
peak arterial pressure and rate-pressure product during the
experimental protocol are shown in Fig.
2. Note that no significant differences
existed between experimental groups before and during ischemia in
either measure. However, at 2, 5, and 15 min of reperfusion, both peak
systolic pressure and rate-pressure product were significantly (P < 0.05) higher in the ANTIOX animals compared with
CON diet animals.
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Two markers of lipid peroxidation were used to determine the effects of
the different diets on cardiac damage due to I-R. Figure
3 contains the mean (±SE) values for
myocardial TBARS and CHE in both experimental groups. First, note that
similar results were obtained using the two markers of lipid
peroxidation. Also notice that, in the CON diet group, I-R surgery
resulted in a significant increase (P < 0.05) in both
myocardial TBARS and CHE levels compared with sham surgery animals.
Finally, a key finding was that antioxidant ANTIOX I-R animals had
significantly lower (P < 0.05) myocardial TBARS and
CHE levels than CON I-R animals.
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Table 1 contains mean (±SE) activities
of GPX, SOD, manganese SOD (MnSOD), copper-zinc superoxide dismutase
(Cu-ZnSOD), and CAT. Several observations are noteworthy. First,
compared with CON diet animals, myocardial GPX activity in ANTIOX
animals was significantly higher (P < 0.05) in both
the sham and the I-R surgery groups.
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Second, SOD activity was greater (P < 0.05) in
ANTIOX animals that underwent I-R surgery compared with CON diet
I-R surgery animals. Furthermore, CON diet I-R surgery animals
displayed lower (P < 0.05) total SOD activity compared
with CON diet sham surgery animals. Analysis of the different isoforms
of SOD indicated that these differences in total enzyme activity were
due to changes in the manganese isoform of the enzyme. Finally, dietary
supplementation with VE and -LA did not alter the myocardial
activity of CAT in either sham surgery or I-R surgery groups.
Figure 4A indicates that the
myocardium from ANTIOX animals was significantly (P < 0.05) better protected against lipid peroxidation in all four
aqueous radical-generating systems compared with the myocardium from
CON diet animals. Similarly, compared with CON diet animals, hearts
from antioxidant ANTIOX animals experienced less (P < 0.05) lipid peroxidation when exposed to the lipid phase (AMVN)
oxidative challenge (Fig. 4B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Overview of principal findings.
This is the first experiment to examine the effects of VE and -LA
supplementation on cardiac performance and biochemistry during in vivo
I-R in aged rats. A major finding of this study was that antioxidant
supplementation improved cardiac performance during postischemia
reperfusion. In addition, our data support the hypothesis that the
dietary combination of VE and -LA reduces myocardial lipid
peroxidation resulting from an in vivo I-R insult. Furthermore, in
vitro experiments demonstrated that this dietary antioxidant
combination protected the myocardium against lipid peroxidation induced
by five different radical-generating systems. Collectively, these data
indicate that dietary supplementation with VE and -LA protects the
senescent heart against lipid peroxidation by scavenging a variety of
ROS and that this protection is associated with improved cardiac
performance during reperfusion after myocardial ischemia.
Antioxidant supplementation improves cardiac performance during
reperfusion.
An important observation in this study was that dietary supplementation
with VE and -LA improved cardiac performance during myocardial
reperfusion after ischemia. Both peak arterial pressure and the
rate-pressure product were significantly higher during reperfusion in
ANTIOX animals compared with CON rats. Upon reoxygenation of hypoxic
heart tissue, cardiac performance generally remains depressed for
several days. The cause of this reperfusion-induced cardiac dysfunction
is believed to be due, at least in part, to an increased production of
ROS during reoxygenation of the hypoxic tissue (3, 11).
ROS have been shown to participate in numerous degenerative cellular
processes, such as membrane lipid peroxidation (13). For
example, damage to the sarcoplasmic reticulum membrane may result in
dissipation of the important transsarcolemmal calcium gradient and an
increase in the cytosolic calcium concentration (10). This
could result in a sustained contractile activation resulting in
hypercontracture, distortion of the myocardial cytoskeleton, and
diminished contractile performance (10). The present study suggests that this chain of deteriorating events might be attenuated by
preloading the myocardium with nutritional antioxidants that decrease
the ROS-induced cellular injury.
-LA.
Our findings are in agreement with previous reports regarding the
myocardial benefits of using a combination of VE and -LA by use of
an in vitro model (15, 16). In contrast, the current data
are contradictory to recent findings from our laboratory in which young
female rats were used (5). Using the same antioxidant
regimen and I-R protocol as the current experiments, we observed that
the antioxidant supplementation resulted in less I-R-induced myocardial
lipid peroxidation; nonetheless, this dietary intervention did not
improve myocardial contractile performance during reperfusion. At least
two possibilities can explain these discrepancies. First, compared with
young adult animals, senescent animals have a reduced antioxidant
capacity (32). Therefore, it is possible that dietary
antioxidant supplementation may be more beneficial in protecting
against ROS-mediated cardiac injury in older animals compared with
young animals.
A second possibility is that gender or strain differences may exist in
the responses of the animals to I-R. The previous study from our lab
used female Sprague-Dawley rats, whereas the present study used male
Fischer-344 rats. In this regard, it has been reported that enzymatic
antioxidant defenses, specifically SOD and CAT, vary with gender and
strain in rats (28). Furthermore, the female gonadotropic
hormone estrogen and its metabolites have been shown to have
antioxidant capability (33). Therefore, collectively, it
appears that age, gender, and strain differences could have contributed
to our divergent findings.
Antioxidant supplementation reduces I-R-induced myocardial lipid
peroxidation.
Lipid peroxidation is one of the most damaging processes that occurs in
the myocardium during I-R. The oxidative modification of lipids results
in alterations to the fluidity and permeability of membrane
peroxidation (13). It has been reported that lipid peroxidation of the sarcoplasmic reticulum, which occurs during I-R,
may result in altered calcium handling and subsequent contractile dysfunction (29). In the present study, two measures of
lipid peroxidation were used to determine whether dietary
supplementation reduced left ventricular lipid damage after I-R.
Compared with sham animals, I-R increased myocardial lipid peroxidation
in CON diet animals. In contrast, compared with sham, I-R did not
increase myocardial MDA and CHE in ANTIOX animals. These findings are
in agreement with studies that have reported decreased in vitro
I-R-induced lipid peroxidation with prefeeding of VE alone
(20) or in combination with -LA (5).
However, the current study is the first investigation to support the
notion that this dietary antioxidant regimen reduces I-R-induced lipid
peroxidation in the senescent heart under in vivo physiological conditions.
In vitro oxidative challenges.
To determine the mechanism by which dietary supplementation with VE and
-LA provides protection against I-R-induced myocardial lipid
peroxidation, in vitro experiments were conducted that oxidatively challenged heart homogenates from both CON and ANTIOX animals that were
not exposed to I-R. Because of the antioxidant properties of VE and
-LA, we hypothesized that heart homogenates from ANTIOX animals
would quench superoxide radicals, hydroxyl radicals, hydrogen peroxide,
and peroxyl radicals generated in vitro. Our data support this
postulate. Indeed, after exposure to five ROS generating systems, lower
levels of MDA were detected in heart homogenates from ANTIOX animals
compared with CON diet animals. Therefore, the finding that the
nutritional combination of the two antioxidants results in a myocardium
that is protected against all five ROS generating systems supports the
notion that combining aqueous and lipid phase antioxidants is
therapeutically beneficial (15).
Effects of antioxidant supplementation and I-R on myocardial antioxidant enzymes. The effect of the antioxidant supplementation on activities of myocardial enzymes important in protection against oxidative stress was also investigated. Two interesting observations warrant discussion. First, activity of GPX increased in ANTIOX animals in both the sham and I-R groups. This finding agrees with previous unpublished data from our laboratory and from other investigators (23, 30). The explanation for the increased GPX activity with VE supplementation could be the increase in cellular selenium concentrations that accompanies VE supplementation (30). Selenium is a cofactor for GPX, and higher cellular levels of VE have been shown to stimulate increased expression of the enzyme.
The second interesting finding was a significantly greater total SOD activity in the myocardium of ANTIOX animals exposed to I-R surgery compared with CON diet I-R surgery animals. Also, myocardial total SOD activity was lower in the CON diet I-R surgery animals compared with CON diet sham surgery animals. These differences in myocardial total SOD activity were due to a higher activity of the manganese isoform. This finding is in agreement with two recent studies reporting that vitamin E feeding upregulated MnSOD protein expression and expression in aortic segments of rats (23) and in rats fed a high-fructose diet (7). The mechanisms to explain these findings are unknown and warrant further investigation. The decreased activity of the mitochondrial isoform of SOD in aged animals exposed to myocardial I-R may be the result of an accumulation of hydrogen peroxide. Indeed, it has been demonstrated that hydrogen peroxide is a negative allosteric modifier of MnSOD activity (4). In the ANTIOX animals, the presence of additional antioxidants such as-LA, DHLA, or their metabolites, all of which
have been shown to quench hydrogen peroxide, may have protected MnSOD
from I-R-induced downregulation.
Limitations of the model. The male Fischer-344 rat was chosen as the experimental subject because 1) the nature of these invasive experiments precludes the use of human subjects, 2) this strain of rat is highly inbred and does not display large interanimal variations in coronary collateral circulation, and 3) the rat is a widely accepted model for the study of dietary antioxidant interventions and an accepted model for aging research (1).
At the completion of the feeding period, our animals were ~21.5 mo old. The experimental rationale for investigating this age group of animals is as follows. Our objective in these experiments was to investigate the effects of antioxidant supplementation on I-R-induced cardiac injury in old animals that were not "old age survivors" (i.e., older than the median life span of this strain). Because the median life span of male F-344 rats ranges from 22 to 29 mo, 21.5-mo-old animals are approaching senescence but are not old age survivors. The decision to use 10,000 IU of VE and 1.65 g of-LA per kg
diet was based on previous work demonstrating that these dosages of VE
(15, 16) and -LA (26) have provided
beneficial results. Also, this dietary dosage of VE results in serum
tocopherol levels in the rat of 3 mg/dl, which are similar to values
obtained in humans when large doses (600 IU/day) of -tocopherol are
consumed (22).
The surgical procedure used in these experiments has been used
successfully in our laboratory (5, 27) and has been
reported to result in both myocardial ischemia and reperfusion
(2). However, it is possible that this type of
experimental surgery could result in interanimal differences in
the magnitude of either ischemia or reperfusion. Nonetheless, we
believe that these differences are clinically relevant and better
reflect the types of I-R insults that occur in humans.
The decision not to include additional experimental groups that
consumed only VE or -LA was based on the data from Haramaki et al.
(16). These investigators reported that it was the
combination of the two antioxidants that provided protection and that
fewer benefits were observed when they were used individually.
Furthermore, in the current experiments, we chose not to include a
parallel group of younger animals. This decision was based on previous data from our laboratory indicating that dietary supplementation with
VE and -LA did not improve myocardial performance during I-R
(5). Therefore, in the absence of a parallel group of
younger animals in our current study, it is not possible to conclude
that aging results in a compromised myocardial antioxidant capacity.
Summary and conclusions.
These experiments examined the effects of dietary supplementation with
VE and -LA on myocardial physiological and biochemical responses
during in vivo I-R in the aged rat. The dietary regimen yielded a
significant increase in myocardial VE content after 14 wk of
supplementation. Dietary supplementation with these antioxidants improved cardiac performance during reperfusion after ischemia. This
improvement in recovery from ischemia appears to be due to the
supplemented hearts being protected from a wide range of ROS, resulting
in reduced lipid peroxidation. These results indicate that this
combination of antioxidant supplements provides protection against
myocardial I-R injury in old animals.
Perspectives
This is the first experiment to examine the effects of combining VE and-LE supplementation on cardiac performance and biochemistry during in vivo myocardial I-R in aged rats. The results confirm previous work using the in vitro model to support the use of this antioxidant combination as a therapeutic defense against I-R oxidative damage. A major new finding of this study was that the antioxidant supplements improved cardiac performance during
postischemia-reperfusion. In addition, our data support the hypothesis
that the dietary combination of VE and -LE reduces myocardial lipid
peroxidation resulting from an in vivo I-R insult. Furthermore, we
performed in vitro experiments indicating that this dietary antioxidant combination protected the myocardium against lipid peroxidation induced
by five different radical-generating systems. In summary, this study
provides new and important findings relative to the therapeutic role of
VE and -LE in providing protection in the senescent heart during
reperfusion after ischemia.
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ACKNOWLEDGEMENTS |
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This study was supported by research grants from the American Heart Association-Florida Affiliate (S. K. Powers) and the Finnish Ministry of Education (C. K. Sen).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Coombes, School of Human Movement Studies, Connell Bldg., Univ. of Queensland, St. Lucia, QLD 4072, Australia (E-mail: jcoombes{at}hms.uq.edu.au).
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 8 May 2000; accepted in final form 8 August 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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