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Oxidative Changes Associated with ß-Carotene and -Tocopherol Enrichment of Human Low-Density Lipoproteins

Department of Nutrition, University of Nevada, Reno, Nevada

Address reprint requests to: Stanley T. Omaye, Department of Nutrition, Mail Stop 142, University of Nevada, Reno, NV 89557

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Objective: To determine what effects enrichment of human low-density lipoprotein (LDL) with combinations of -tocopherol and ß-carotene would exert on LDL oxidation and attempt to define the nature of the effects.

Methods: Human plasma was pooled and -tocopherol and ß-carotene was added in a four-by-four design resulting in the enrichment of LDL with -tocopherol and ß-carotene in varying concentrations. Enriched and control LDL was oxidized in Cu²⁺ mediated oxidation system and resistance of LDL to oxidation was determined by lag time, thiobarbituric acid reactive substances (TBARS) activity, and rate of oxidation.

Results: Increasing LDL -tocopherol concentration had a linear relationship with lag time and a negative correlation with rate of oxidation. LDL ß-carotene concentration was linearly correlated with the rate of LDL oxidation and ß-carotene loss, and exponentially related to TBARS concentration.

Conclusions: These results support earlier findings for the protective effect of -tocopherol against LDL oxidation, and suggest that ß-carotene participates as a prooxidant in the oxidative degradation of LDL under these conditions. Since high levels of -tocopherol did not mitigate the prooxidative effect of ß-carotene, these result indicate that increased LDL ß-carotene may cancel the protective qualities of -tocopherol.

Key words: ß-carotene, -tocopherol, low-density lipoproteins, lipid peroxidation, toxicity

	INTRODUCTION

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Although it has been known for some time that elevated plasma low-density lipoprotein (LDL) is strongly associated with increased risk of cardiovascular disease, the possible mechanisms by which elevated LDL mediates atherosclerosis remain unclear. A series of in vivo and in vitro studies have provided the background for the development of a current theory of atherogenesis which implicates oxidative modified LDL in atheroma formation and progression [1–3]. Evidence supporting in vivo oxidation of LDL is provided by a variety of biochemical, histochemical, pathologic, and immunocytological studies. For example, products of lipid peroxidation have been found associated with plasma LDL [4] and trace amounts of oxidized LDL have been found in human and animal plasma [5,6]. Plasma of humans and rabbits has been shown to contain autoantibodies against several forms of oxidized LDL [7,8], and, in certain subjects, oxidized LDL and/or peroxide levels are elevated [9–11]. The presence of epitopes oxidized LDL [7,8,12], as well as oxidized LDL itself [13], have been demonstrated in arterial lesions in humans and rabbits [14–16].

Cardiovascular diseases resulting from oxidative damage may be prevented and/or mitigated by dietary antioxidants [17]. Thus, optimization of plasma antioxidant levels, and hence LDL content of antioxidants, may prevent atherosclerotic cardiovascular diseases. For the most part, research efforts have focused almost exclusively on the effect of single dietary antioxidants on the resistance of LDL to in vitro oxidation. Increasing awareness of the complex interactions among the enzymes, proteins, and water and fat-soluble dietary compounds that constitute the body’s antioxidant protection system have led in recent years to investigations which examine the effects that combinations of dietary antioxidants have on the susceptibility of LDL to oxidative modification [18].

The protective effect of -tocopherol against oxidation of LDL in both cell-mediated and cell-free in vitro systems has been repeatedly confirmed [19–23]. Ascorbate has been shown to be an effective water-soluble antioxidant for LDL by itself [13,24], as well as playing a synergistic role with -tocopherol [25]. Interest in ß-carotene and other carotenoids reached a peak in the early 1990’s in response to epidemiological data indicating that a diet high in fruits and vegetables and a high plasma level of ß-carotene associated with decreased risk of cardiovascular diseases and cancer [26,27]. ß-Carotene may act additively and synergistically with -tocopherol to protect tissues in vivo [28], perhaps, in part, by protection of ß-carotene by -tocopherol [25,29]. ß-Carotene was reported to be 20 times more potent on a molar basis than -tocopherol as an antioxidant for LDL in vitro [30]. Paradoxically, other reports indicate that ß-carotene has prooxidant effects which are dependent on oxygen tension [31].

The goal of the present project was to establish what effects enrichment of LDL with various combinations of -tocopherol and ß-carotene would produce on the process of LDL oxidation and to attempt to define the nature of the effects.

	MATERIALS AND METHODS

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Subjects
A total of 239 students, between the ages of 18 and 25 at the University of Nevada, Reno, volunteered to participate in this study which spanned 1 year. They reported themselves to be in good health, with no history of heart disease. The purpose and procedures were explained and written consent was obtained from all participants. The research proposal was approved by the Biomedical Human Subjects Review Committee of the University of Nevada, Reno.

Sample Collection and Preparation
Non-fasting blood samples were obtained by venipuncture and collected into Vacutainer brand evacuated tubes (Becton Dickinson, Rutherfood, NJ) containing 15% EDTA. All samples were collected in subdued light and immediately placed on ice in the dark. Samples were centrifuged at 1,900 xg for 20 minutes to separate the plasma fraction. Plasma from all subjects was pooled prior to enrichment experiments.

In vitro -Tocopherol and ß-Carotene Enrichment
D, L.--tocopherol, Type V, 67% and ß-carotene was synthetic, all-trans, type I, ß-carotene, 97%, both purchased from Sigma Chemical Company, St Louis, MO. These synthetic forms are commonly found in a variety of vitamin or nutrient supplements. Foods contain different, but related compounds, predominantly -tocopherol and carotenoids [32,33].

Experiment 1.
Five mg of ß-carotene was added to a 10 ml volumetric flask and the volume adjusted with dimethyl sulfoxide, purged with nitrogen, sealed tightly and wrapped in foil. The flask was placed on an Adams Nutator Mixer (Clay Adams, Div. Becton, Dickinson, and Co., Parsippany, NJ) and mixed gently for 2 hours, then allowed to stand overnight. Prior to enrichment experiments, the solution was filtered with Whatman #1 filter paper to remove undissolved ß-carotene, and the concentration of ß-carotene was determined by HPLC as described below.

Plasma was supplemented with -tocopherol or ß-carotene according to a modification described by Esterbauer et al [13]. Pooled plasma was divided equally into 50 ml plastic centrifuge tubes. An aliquot of a solution containing 1 mg/ml -tocopherol dissolved in dimethyl sulfoxide (DMSO) was added to reach a final concentration of 0, 30, 40 and 50 µg -tocopherol/ml plasma. Final concentrations of 4, 8, and 16 µg ß-carotene were adjusted, respectively, per ml plasma. Tubes were incubated in a 37°C water bath under nitrogen, and in the dark, for 6 hours. Samples were mixed by vortexing briefly every 30 minutes.

Experiment 2 and 3.
For -tocopherol in experiment 2, procedures were followed according to the protocol described in Experiment 1; however, final concentrations were adjusted to 50, 100, and 200 µg -tocopherol/ml plasma.

For ß-carotene in experiment 3, the plasma concentration were adjusted as described by Gaziano et al [34]. ß-Carotene was dissolved in tetrahydrofuran (THF) and added to plasma to reach a final concentration of 200, 300 and 500 µg ß-carotene, respectively, per ml plasma.

The samples were vortexed briefly and incubated in a 37°C water bath under nitrogen and in the dark. After 30 minutes of incubation, all samples were removed and were stored overnight at 4°C in the dark.

Isolation of LDL
LDL was isolated as described by Chung et al [35]. Enriched plasma was adjusted to a density of 1.21 g/ml by adding 0.3265 g of KI per mol plasma and layered under phosphate buffered saline (0.01 M phosphate and 0.15 M NaCl) in Quick-seal centrifuge tubes (Beckman). LDL was isolated by single vertical spin density gradient ultracentrifugation at 4°C and 300,000 xg for 3 hours using a fixed-angle Beckman 70.1 T1 rotor (Beckman Instruments, Palo Alto, CA) in a Beckman L2-85 ultracentrifuge (Beckman Instruments, Palo Alto, CA). The LDL fraction was collected by inserting the needle of a syringe into the centrifuge tube directly beneath the LDL band and aspirating the band. LDL samples were stored under nitrogen at 4°C in the dark and used within a week for experiments. The LDL fraction obtained was identified by modification of the electrophoretic techniques described by Kane (1973).

Oxidation of LDL
Prior to oxidation of LDL by incubation (0 to 4 hours) with Cu²⁺, LDL was filtered through PD-10 columns (Pharmacia, Uppsala, Sweden) to remove the EDTA. Protein was determined using the BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as the standard. A solution of 1 µl of 40 mM Cu²⁺ was added for every 100 mg protein per ml sample, shaken to oxygenate, placed in a 37°C water bath and covered with foil to protect against photo degradation of the antioxidants.

Aliquots were taken for initial -tocopherol and ß-carotene content, and assessment of lipid peroxidation by thiobarbituric acid reactive substances (TBARS) [36], and at regular intervals thereafter. Oxidation was arrested by pipetting samples into glass culture tubes containing 200 µM EDTA and immediately placing on ice.

LDL -Tocopherol and ß-Carotene Content
-Tocopherol was extracted from LDL and analyzed by using the method of Kahlon et al [37]. For ß-carotene, 1 ml of LDL was deproteinated with 1 ml ethanol containing 0.125% BHT. The ß-carotene was extracted into 2.0 ml hexane/0.125% BHT by vortexing for 3 minutes. After a 10 minute centrifugation at 1,900 xg, 1 ml of the hexane layer was removed and evaporated to dryness in a vacuum sample concentrator (Savant Instruments, Inc, Faringdale, NY). Dried samples were stored under nitrogen at -70°C until analysis. LDL ß-carotene concentrations were quantified by reverse phase HPLC with detection at 450 nm according to a modification of the method described by Ringer et al [38]. Evaporated samples were resuspended in 200 µl mobile phase and injected into a 0.46x15 cm Utrasphere 5 µm C-18 ODS reverse phase HPLC column (Beckamn Instruments, Inc., San Ramon, CA).

Analysis of Data and Statistical Methods
The length of the lag phase for TBARS was obtained by finding the point of intersection of the slopes of the propagation and lag phases and extrapolating it to the horizontal axis [22]. The lag time is the interval between 0 minutes and the point of intersection. Maximum change in TBARS was calculated by subtracting TBARS concentration (nmol/mg LDL protein) at the end of the lag phase from the TBARS concentration at the end of the propagation phase. The rate of oxidation was determined by dividing the maximum change in TBARS by the length of the propagation phase. The length of the propagation phase is determined by subtracting time of the lag phase from the time at which the propagation phase ends.

Determination of the statistical significance of antioxidant incorporation into LDL at each level of enrichment was made by Mann-Whitney nonparametric one-tail test, except for Experiment 3 in which a two sample t-test was used. Repeated measures ANOVA was used to compare entire time course curves of LDL oxidation. Post-tests between the time course curves of all pairs of supplementation levels were done using a paired T-test. Kinetics data including lag time, maximum TBARS, and oxidation rate related to baseline antioxidant concentration and percent ß-carotene related to baseline -tocopherol concentration was analyzed using the Pearson r. Antioxidant incorporation, TBARS and LDL antioxidant concentration are expressed as the mean±SD [39]. Coefficient of determination=r².

	RESULTS

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Incorporation of Antioxidants into Low-Density Lipoprotein
The LDL band isolated by single vertical spin density ultracentrifugation was characterized by an orange color, and was located just above the center of the ultracentrifuge tube. When subjected to polyacrilamide gel electrophoresis and compared to published photographic data [40], the band was identifiable as LDL.

In Experiment 1, supplementation of plasma with antioxidants prior to isolation of LDL resulted in significant (p<0.05) enrichment of LDL in both -tocopherol and ß-carotene at each of the three concentrations of supplementation. The following LDL concentrations of -tocopherol (µg/mg LDL protein) were found for each respective level of -tocopherol (µg/ml plasma) enrichment: 5.85±0.34 for the 0 level; 7.56±0.4 for the 30 level; 10.8±0.41 for the 40 level; 14.2±0.36 for the 50 level. The following LDL concentrations of ß-carotene (µg/mg LDL protein) were found for each respective level of ß-carotene (µg/ml plasma) enrichment: 0.25±0.05 for the 0 level; 0.35±0.06 for the 4 level; 0.58±0.05 for the 8 level; 0.97±0.3 for the 16 level.

In Experiment 2, supplementation of plasma with antioxidants prior to isolation of LDL resulted in significant (p<0.05) enrichment of LDL in -tocopherol at each of the three concentrations of supplementation. Enrichment of LDL with ß-carotene was significant between the 0 and 200 µg levels, and between the 300 and 500 µg levels. The following LDL concentrations of -tocopherol (µg/mg LDL protein) were found for each respective level of -tocopherol (µg/ml plasma) enrichment: 5.20±0.40 for the 0 level; 19.60±0.4.8 for the 50 level; 41.0±7.47 for the 100 level; 72.4±9.75 for the 200 level. The following LDL concentrations of ß-carotene (µg/mg LDL protein) were found for each respective level of ß-carotene (µg/ml plasma) enrichment: 0.17±0.04 for the 0 level; 3.20±1.70 for the 200 level; 4.80±1.60 for the 300 level; 10.0±2.26 for the 500 level.

In Experiment 3, supplementation of plasma with antioxidant prior to isolation of LDL resulted in significant (p<0.01) enrichment of LDL in both -tocopherol and ß-carotene at each of two concentrations of supplementation except for the 300 µg ß-carotene supplementation which was significant at p<0.05. The following LDL concentrations of -tocopherol (µg/mg LDL protein) were found for each respective level of -tocopherol (µg/ml plasma) enrichment: 7.57±0.19 for the 0 level; 34.60±3.508 for the 100 level; 71.1±6.40 for the 200 level. The following LDL concentrations of ß-carotene (µg/mg LDL protein) were found for each respective level of ß-carotene (µg/ml plasma) enrichment: 0.14±0.14 for the 0 level; 3.80±0.14 for the 300 level; 8.00±0.12 for the 500 level.

Protection of LDL by Added Antioxidants
Table 1 summarizes the effect of LDL antioxidant concentration on oxidation kinetics for Experiment 1. The rate of oxidation for the unsupplemented (0 level of -tocopherol supplementation) LDL was nearly twice as high as the rate for -tocopherol supplemented LDL. At the 16 µg ß-carotene level, the unsupplemented -tocopherol LDL had an oxidation rate similar to the supplemented LDL. Lag times had a linear relationship with -tocopherol concentration for all sets of curves but the 4 µg level of ß-carotene supplementation. TBARS activity was highest for the unsupplemented LDL (0 -tocopherol supplementation) in all sets except for the 16 µg ß-carotene level.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Relationship between -tocopherol and a) length of lag phase (n=16, r2=0.49, p<0.01); b) rate of LDL oxidation (n=16, r2=0.325, p<0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Relationship between ß-carotene and maximum TBARS activity (n=16, r2=0.563, p<0.001).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Relationship between initial LDL ß-carotene concentration and amount of ß-carotene oxidized in 60 minutes (r2=0.990, p<0.0001).

In this experiment there was an inverse relationship between baseline ß-carotene concentration and lag phase (r²=0.608), a direct correlation between TBARS activity (r²=0.828), and rate of LDL oxidation (r²=0.8940). None of these relationships were significant, however, due to the small sample size.

Table 4 summarizes the effect of LDL antioxidant concentration on oxidation kinetics for Experiment 3. No significant differences were observed among the kinetics parameters in the time course of LDL supplemented with ß-carotene. Significant differences were observed when comparing unsupplemented plasma with each concentration of -tocopherol supplemented plasma and significant differences were observed between each concentration of -tocopherol supplemented plasma when compared to all other concentrations. Both LDL -tocopherol (r²=0.960) and ß-carotene (r²=0.792) concentrations had a positive association with lag times. A negative association was observed between -tocopherol and maximum TBARS activity (r=-0.99) and -tocopherol and rate of oxidation (r=-0.72), whereas LDL ß-carotene concentrations exhibited a positive association with TBARS activity (r=0.86) and rate of oxidation (r=0.83).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Relationship between LDL -tocopherol and length of lag phase for all experiments (n=19, r2=0.819, p<0.0001).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Relationship between LDL ß-carotene concentration and a) rate of oxidation (n=22, r2=0.372, p<0.005); b) TBARS concentration for all experiments (n=22, r2=0.649, p<0.05).

	DISCUSSION

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The length of the lag phase had a strong linear correlation with LDL -tocopherol content in these experiments. Using the slope of the relationship between LDL -tocopherol concentration and the lag phase determined in these experiments, the results suggest that for every microgram increase of LDL -tocopherol content, there is a corresponding increase in lag time of 1.55 minutes under these conditions.

A negative association between baseline LDL -tocopherol concentration and the rate of oxidation was observed in these experiments. Although the relationship between -tocopherol concentration and TBARS was not significant, the combined effects of a slight decrease in TBARS activity and a longer propagation phase resulted in the observed decreased rate of oxidation. These results suggest that increased concentration of -tocopherol not only prolongs the time period before autocatalytic oxidation begins, but slows the autooxidative reactions once the propagation phase begins, and, ultimately, decreases the final concentration of oxidative products for up to 4 hours.

A number of studies which examined the effect of structural and compositional characteristics of LDL related to its oxidative susceptibility concluded that these characteristics determined LDL’s resistance to oxidation. By pooling a large number of individual samples that, theoretically, had a wide range of structural and compositional characteristics, it was possible to separate the proposed effect of these different characteristics from the effect of -tocopherol and ß-carotene concentrations. It appears that LDL -tocopherol concentration is indeed an independent variable in the determination of lag phase length.

The observations: 1) -tocopherol concentration was related to a slowing of the rate of the lipid peroxidation in the propagation phase, and 2) a decrease in the final concentration of TBARS, can be explained in terms of the amount of peroxyl radicals scavenged by -tocopherol. The more molecules of -tocopherol that are present in each LDL particle, the greater the ratio of -tocopherol to unsaturated fatty acids. An increased ratio of -tocopherol to unsaturated fatty acids corresponds to increased peroxyl radical-scavenging, therefore, decreased production of degradative products of lipid peroxidation. This equates to increased lag time and formation rate of TBARS.

In all experiments, increased LDL concentrations of ß-carotene were correlated with rate and TBARS concentration. The high level of TBARS generated with higher concentrations of ß-carotene very likely accounts for the increase in rate of oxidation observed with the higher concentrations of ß-carotene, because the length of the lag phase was not related to ß-carotene concentration. Three observations related to ß-carotene concentration in these experiments are of particular interest. Firstly, with increasing LDL concentrations of ß-carotene there is a corresponding increase in ß-carotene oxidation, so that for every microgram increase of LDL ß-carotene, there is a loss of between 0.7 and 0.8 µg of ß-carotene under the conditions of these experiments. Secondly, there appears to be an exponential relationship between TBARS concentration and ß-carotene concentration. Thirdly, when data from all experiments is analyzed together, there is a positive association between the length of the lag phase and ß-carotene concentration. These results do not find support in published studies to date. In the only study published previous to this work in which similar conditions were used, Gaziano et al [34] found that, although there was increased oxidative susceptibility of LDL from men who had taken ß-carotene supplements, LDL loaded with ß-carotene in vitro did not display increased susceptibility to Cu²⁺-mediated oxidation.

Our observations do support the suggestion that the carbon-centered radical (LOO-ß-carotene radical) can, under ambient to high oxygen tension, react with oxygen to form peroxyl radicals (LOO-ß-carotene-OO · ), therefore participating in the propagating reaction [43]. The trend toward increased lag phase with increasing ß-carotene concentrations may be the initial result of ß-carotene’s radical scavenging properties. However, the initial antioxidant action is followed by an amplification of oxidative reactions with the lipid peroxyl-ß-carotene-peroxyl radical participating in the chain reaction. This explains the nearly 1 to 1 relationship between ß-carotene concentration and ß-carotene oxidation, and the exponential increase in TBARS with increasing ß-carotene concentration observed in this study.

	CONCLUSIONS

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There are limitations in extrapolating from our findings to describe what may occur in vivo. However, in this study, it was observed that increased LDL -tocopherol concentration corresponds to an increased lag phase. This support observation from previous studies [10,13,20,23,41]. Unlike previous studies, however, the pooled samples used in these experiments allowed the generalization to be made that, under these conditions, for every microgram increase from baseline LDL -tocopherol concentration there is a corresponding increase in lag phase of about 1.5 minutes.

It is apparent from the exponential relationship observed between LDL ß-carotene concentration and the concentration of TBARS that ß-carotene, after an initial antioxidant action, participates in the autocatalytic chain reaction of fatty acid peroxidation in the propagation phase of LDL oxidation. It appears that this occurs despite high concentration of -tocopherol. These data suggest that increased amounts of ß-carotene in the LDL may counteract any protective effect that -tocopherol may exert.

The results obtained in this set of experiments are not surprising considering the proposed mechanisms of antioxidant action for -tocopherol and ß-carotene. To date, however, this study is the first to report these particular effects of -tocopherol and ß-carotene in the kinetics of LDL oxidation using this type of system.

	ACKNOWLEDGMENTS

 
This work was supported in part by a Nevada Agricultural Station Hatch Research Grant, #1106-152-0717. Authors thank Mr. Ronald Ota for his technical assistance.

	FOOTNOTES

 
Research was done to partially fulfill thesis requirements of HT Bowen.

Received July 1, 1997. Accepted October 1, 1997.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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