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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2273-2279.)
© 1997 American Heart Association, Inc.

Articles

Dose-Response Comparison of RRR--Tocopherol and All-Racemic -Tocopherol on LDL Oxidation

S. Devaraj; B. Adams-Huet; C.J. Fuller; ; I. Jialal

From the Center for Human Nutrition (C.J.F., I.J.) and Departments of Internal Medicine (B.A-H., I.J.) and Pathology (S.D., I.J.), University of Texas Southwestern Medical Center, Dallas, Tex.

Correspondence to I. Jialal, MD, PhD, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9072. E-mail jialal.i{at}pathology.swmed.edu

	Abstract

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Abstract Much data have accrued in support of the concept that oxidation of LDL is a key early step in atherogenesis. The most consistent data with respect to micronutrient antioxidants and atherosclerosis appear to relate to -tocopherol (AT), the predominant lipid-soluble antioxidant in LDL. There are scant data on the direct comparison of RRR-AT and all-racemic (rac)-AT on LDL oxidizability. Hence, the aim of the present study was to examine the relative effects of RRR-AT and all-rac-AT on plasma antioxidant levels and LDL oxidation in healthy persons in a dose-response study. The effect of RRR-AT and all-rac-AT at doses of 100, 200, 400, and 800 IU/d on plasma and LDL AT levels and LDL oxidation was tested in a randomized, placebo-controlled study of 79 healthy subjects. Copper-catalyzed oxidation of LDL was monitored by measuring the formation of conjugated dienes and lipid peroxides over an 8-hour time course at baseline and again after 8 weeks. Plasma AT, lipid-standardized AT, and LDL AT levels rose in a dose-dependent fashion in both the RRR-AT and all-rac-AT groups compared with baseline. There were no significant differences in plasma, lipid-standardized, and LDL AT levels between RRR-AT and all-rac-AT supplementation at any dose comparison. The lag phases of oxidation were significantly prolonged with doses 400 IU/d of RRR-AT and all-rac-AT, as measured by conjugated-dienes assay and at 400 IU/d of RRR-AT and 800 IU/d of both forms of AT by lipid peroxide assay. Again, there were no significant differences in the lag phase of oxidation at each dose for RRR-AT when compared with all-rac-AT. Also, there were no significant differences in LDL oxidation after in vitro enrichment of LDL with RRR-AT and all-rac-AT. Thus, supplementation with either RRR-AT or all-rac-AT resulted in similar increases in plasma and LDL AT levels at equivalent IU doses, and the degree of protection against copper-catalyzed LDL oxidation was only evident at doses 400 IU/d for both forms.

Key Words: RRR--tocopherol • all-racemic -tocopherol • LDL oxidation • -tocopherol

	Introduction

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The oxidative modification of LDL has been proposed as a key early step in atherogenesis. Oxidized LDL is taken up by the macrophage scavenger receptor, which is not regulated by intracellular cholesterol,¹ and thus may result in foam cell formation. Oxidized LDL has numerous effects on arterial wall cells that could facilitate atherosclerosis, such as cytotoxicity to the endothelium, stimulation of adhesion molecule expression, monocyte chemotaxis, and inhibition of NO production.^{2 3 4 5} Furthermore, several lines of evidence support the existence of oxidized LDL in-vivo.^{6 7} The most persuasive evidence for the in vivo existence of oxidized LDL comes from animal studies in which dietary antioxidant supplementation inhibited the progression of atherosclerosis.^{8 9 10}

-Tocopherol (AT), the most active form of vitamin E, is the predominant lipophilic antioxidant in LDL.¹¹ The most consistent data with respect to micronutrient antioxidants and atherosclerosis appear to relate to AT. Several lines of evidence support an inverse relation between AT and atherogenesis. Low levels of AT have been shown in epidemiological studies to be related to an increased frequency of cardiovascular mortality.^{12 13} Case-control studies have shown that patients with angina have lower levels of AT than do normal control subjects,¹⁴ and both men and women in the highest compared with the lowest quintile have a significantly decreased risk of coronary artery disease.^{15 16} Pharmacological doses of AT have been shown to reduce LDL oxidizability in healthy volunteers^{17 18 19 20 21} and in individuals with diabetes.^{22 23} In addition, recent research indicates that AT supplementation in patients with cardiovascular disease can reduce lesion progression and coronary events.^{24 25}

The natural source AT consists of only one isomer (RRR-AT), whereas synthetic AT is an equimolar mixture of eight isomers arising from the three chiral centers on the phytyl tail (all-racemic [rac] AT), one eighth of it being RRR-AT. In the liver, RRR-AT is preferentially incorporated into nascent VLDL particle by the 30-kDa tocopherol-transfer protein.^{26 27} AT supplementation results in its enrichment in LDL, allowing one to assess its biological effect as an antioxidant.^{17 18 19 20 21} However, there have been few direct comparisons of RRR-AT and all-rac-AT on LDL oxidizability. Reaven and Witztum²⁸ showed that there were no differences between RRR- and all-rac-AT supplementation in hyperlipidemic volunteers on the oxidative susceptibility of LDL. However, these investigators used a single pharmacological dose of 1600 mg/d and acknowledged that differences could be manifested at lower doses. Thus, it is unknown whether lower doses would show any differences in LDL oxidative susceptibility. RRR-AT and all-rac-AT are available for therapeutic purposes as international units (IU). Hence, the objective of the present study was to examine the relative effects of equivalent IU doses of RRR- and all-rac-AT on plasma and LDL AT levels and on LDL oxidation in healthy volunteers at doses ranging from 100 to 800 IU/d.

	Methods

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The study design was a randomized, placebo-controlled trial. A total of 79 healthy subjects (60 male, 19 female) were included in the study if they fulfilled the following inclusion criteria: nonsmoker; not taking vitamin supplements, oral contraceptives, thyroxine, or lipid-lowering drugs; alcohol intake <1 oz/d; normal plasma glucose, hepatic, and renal function tests; no gastrointestinal disorders; no acute medical conditions at least 3 months prior to entry into the study; and, if female, not pregnant. All subjects gave informed consent, and the study protocol was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas.

The study subjects were randomly assigned to take either placebo (soybean oil), RRR-AT, or all-rac-AT capsules at dosages of 100, 200, 400, or 800 IU/d for 8 weeks. The capsules were provided by Henkel Corp and prepared after taking into account that the relative activity of RRR-AT is 1.36 times that of all-rac-AT. While the IU dose and milligram equivalents were identical for all-rac-AT, the respective milligram equivalents for RRR-AT for 100, 200, 400, and 800 IU were 73.5, 154, 294, and 588, respectively. A fasting blood sample (180 mL) was obtained at baseline and after 8 weeks of placebo, RRR-AT, or all-rac-AT supplementation for plasma lipid and lipoprotein profiles; plasma AT, ß-carotene, and ascorbate levels; and LDL isolation. Samples for LDL isolation were collected on ice, and plasma was separated by low-speed centrifugation at 4°C. Samples for plasma ascorbate were deproteinized with ice-cold 10% metaphosphoric acid, and the supernatant was purged with N₂ and stored at -20°C.²¹ Plasma lipid and lipoprotein levels were assayed by the Lipid Research Clinics methodology; cholesterol and triglyceride levels were determined enzymatically.²¹ AT concentrations in plasma and LDL and ß-carotene concentrations in plasma were measured after extraction by reversed-phase high-performance liquid chromatography.²⁹ Plasma levels of AT and ß-carotene were lipid standardized as described previously,²¹ whereas LDL antioxidant concentrations were expressed per milligram protein. Plasma ascorbate levels were measured spectrophotometrically after derivatization with 2,4-dinitrophenylhydrazine.²¹ Plasma and LDL fatty acid levels (14:0, 16:0, 18:0, 18:1, 18:2, 18:3, and 20:4) were measured by gas chromatography after extraction and transmethylation.²¹

LDL (d=1.019 to 1.063 g/mL) was isolated by preparative ultracentrifugation in NaBr-NaCl solutions containing 1 mg/mL EDTA as described previously.²¹ The isolated LDL was extensively dialyzed against three exchanges (4, 4, and 2 L) of saline-EDTA at 4°C for 24 hours, after which the LDL was filtered and protein content measured by the method of Lowry et al.³⁰ After overnight dialysis against metal-free PBS, pH 7.4 (treated with Chelex 100 resin), copper-catalyzed LDL oxidation was undertaken. LDL (200 µg protein per milliliter) was oxidized in a cell-free system with 5 µmol/L copper in PBS at 37°C, and the time course of oxidation was followed for 8 hours.²¹ At 0.5, 1, 1.5, 2, 3, 4, 5, and 8 hours, oxidation was arrested by refrigeration and addition of 200 µmol/L EDTA and 40 µmol/L butytated hydroxytoluene.

The indices of oxidation used in this study included measurement of conjugated dienes and lipid peroxides. The amount of conjugated dienes formed during oxidation was determined by measuring the absorbance of LDL against a PBS blank at 234 nm after a 1:4 dilution of the samples in PBS. Data are expressed as the increase in conjugated dienes over baseline (A234 nm).²¹ The lipid peroxide content of the LDL formed during oxidation was measured by a modified iodometric method.³¹ In brief, 100 µL of LDL was added to 900 µL of CHOD-iodide reagent and kept at room temperature in the dark for 60 minutes, after which absorbance was read at 365 nm against a reagent blank.

Oxidation kinetics (lag phase, oxidation rate, and maximum amount of oxidation) were determined for both measures of LDL oxidation. The rate of LDL oxidation was determined from the propagation phase of the time-course curve by using a spline function. The lag phase was obtained by drawing a tangent to the slope of the propagation phase and extrapolating it to the x axis: the lag time constitutes the time interval from zero time to the intersection point.²¹ The interassay variability for lag phase for conjugated dienes and lipid peroxides for subjects at baseline and after 8 weeks of supplementation with placebo in this laboratory was 8.5% (n=25) and 9.4% (n=17), respectively.

Statistics
Repeated-measures ANOVA models with grouping factors for AT type and dose were used to assess the response of the parameter of interest. Because interactions between dose and other factors were present, a two-factor repeated-measures ANOVA with one grouping factor (AT type) and one repeated factor (0 versus 8 weeks) was performed at each dose. Differences in responses from week 0 to week 8 between the two types of AT were assessed with the typexweek interaction factor (which is equivalent to two-sample t tests of ). The level of significance for ANOVA was.05. Multiple comparisons were performed with paired t tests to compare baseline and 8-week measurements within the RRR-AT or all-rac-AT groups by using the.025 level of significance to adjust for multiplicity of testing (Bonferroni correction). Statistical analysis was performed using bmdp (SPSS Inc). Data are presented as mean±SD unless otherwise indicated.

	Results

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Salient characteristics of the subjects and their lipid profiles at baseline and 8 weeks are listed in Table 1. Both age and body mass index were similar among the nine groups. There were no changes in diet and activity level of the subjects during the course of the study. Also, none of the subjects reported any adverse effects of supplementation with either RRR-AT or all-rac-AT. There were no significant differences in plasma lipid and lipoprotein levels between baseline and 8 weeks in any group or between RRR-AT and all-rac-AT. Plasma fatty acid levels for the subjects at baseline and after 8 weeks of supplementation with RRR-AT or all-rac-AT are shown in Table 2. The concentrations of fatty acids at 8 weeks compared with baseline values or between RRR-AT and all-rac-AT were not significantly different at any dose of RRR-AT or all-rac-AT.

View this table: [in this window] [in a new window]   Table 1. Group Characteristics and Lipid and Lipoprotein Profile

View this table: [in this window] [in a new window]   Table 2. Plasma Fatty Acid Levels Before and After -Tocopherol Supplementation

Table 3 shows the effect of various doses of RRR-AT or all-rac-AT supplementation on plasma and LDL antioxidant levels. Supplementation had no effect on plasma ascorbate and ß-carotene levels in any group. There were no significant increases in plasma and LDL AT levels in the placebo group, in accordance with previous findings.^{21 28} After supplementation with RRR-AT or all-rac-AT, there were significant increases in plasma AT levels and lipid-standardized AT levels compared with baseline at all doses. Plasma AT levels rose progressively in both the RRR-AT and all rac-AT groups: in the RRR-AT group, the median increment over baseline at 100 IU/d was 68.5% and at 800 IU/d, 175%, while in the all-rac-AT group, the increments at 100 IU/d and 800 IU/d were 37.8% and 151%, respectively. Also, no significant differences were seen in either plasma or lipid-standardized AT levels between RRR-AT and all-rac-AT supplementation at any dose comparison (P>.1). LDL AT levels rose in a similar fashion (Table 3). However, there was no significant difference between LDL AT levels in the RRR-AT–compared with the all-rac-AT–supplemented group at any dose. When the plasma, lipid-standardized, and LDL AT levels for the entire group that received RRR-AT (100 to 800 IU/d) was compared with the entire group that received all-rac-AT (100 to 800 IU/d), there were no significant differences between the two groups for both the absolute increments and the percent change (data not shown).

View this table: [in this window] [in a new window]   Table 3. Effect of -Tocopherol Supplementation on Plasma and LDL Antioxidant Levels

The time course of copper-catalyzed LDL oxidation was monitored by measurement of conjugated dienes and lipid peroxides. From the time-course curves, the lag phase of oxidation was derived. It has previously been shown that there is increased LDL oxidizability, as evidenced by shortening of the lag phase, with major risk factors for accelerated atherosclerosis, such as diabetes, hypertension, and chronic renal disease^{32 33 34} and in subjects with a preponderance of small, dense LDL who are also more prone to premature atherosclerosis.³⁵ Also, a significant inverse relation has been shown between the lag phase of oxidation and clinical atherosclerosis.^{36 37} Thus, the lag phase appears to be a relevant measure of LDL oxidizability. However, while the lag phase confirms antioxidant activity with AT enrichment of LDL, it provides no information on the role of aqueous antioxidants in-vivo or the relevant oxidizing species in vivo. In Table 4 are shown the lag phases for the various groups, as measured by conjugated dienes and lipid peroxide assays. In the groups that received placebo, 100 or 200 IU RRR-AT, or all-rac-AT, there was no significant change in the lag phase at 8 weeks compared with baseline (Table 4). For the conjugated-dienes assay, there were significant increases in the lag phase at 8 weeks for both the RRR-AT and the all-rac-AT groups that received 400 and 800 IU/d of AT. The lag phase as measured by lipid peroxide assay showed a similar pattern. There were significant increases in lag phase at 800 IU/d for both RRR-AT and all-rac-AT groups. While there was also a significant increase in lag phase with 400 IU/d of RRR-AT (P=.01), the increase in lag phase with 400 IU/d of all-rac-AT was not significant (P=.04). Also, there were no significant differences in response to supplementation in the lag phases of oxidation as measured by conjugated dienes and lipid peroxide assays at each dose for RRR-AT compared with the all-rac-AT–supplemented group (Table 4). However, a two-way ANOVA of the percent changes for lag phase for conjugated dienes from baseline to 8 weeks showed a significant increase in the RRR-AT group compared with the all-rac-AT group (P=.035). There was no significant difference in percent change for lag phase for lipid peroxides between the RRR-AT and all-rac-AT groups. Also, no significant difference was seen in maximum oxidation or oxidation rate for conjugated dienes and lipid peroxide assays between RRR-AT and all-rac-AT–supplemented groups (data not shown).

View this table: [in this window] [in a new window]   Table 4. Effect of -Tocopherol Supplementation on LDL Lag Phase

Although the antioxidant activity of AT resides on the chroman ring, we wanted to determine whether the configuration of the phytyl side chain had any influence on its antioxidant effect. Thus, the effect of RRR-AT and all-rac-AT on LDL oxidation was assessed in vitro after enrichment of plasma with AT and isolation of LDL. As shown in Table 5, there were no significant differences in antioxidant effects of RRR-AT and all-rac-AT at both concentrations, as determined by measurement of the lag phase.

View this table: [in this window] [in a new window]   Table 5. Effect of In Vitro Enrichment of Plasma With RRR-AT and All-rac-AT on LDL AT Levels and LDL Oxidation

	Discussion

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Low circulating levels of AT are related to an increased incidence of cardiovascular disease, and increased intake of AT appears to be protective against cardiovascular disease.^{15 16 25} AT supplementation has been shown to reduce the progression of atherosclerosis in some animal models^{38 39 40} and has also been shown to decrease the oxidative susceptibility of LDL after AT supplementation in human volunteers.^{17 18 19 20 21 22 23} In addition, AT has other antiatherogenic effects, such as inhibition of smooth muscle proliferation, monocyte-endothelial adhesion, and platelet aggregation and the preservation of endothelium-dependent vasodilation.^{41 42 43 44} Recently, we have shown in a randomized, placebo-controlled study that AT supplementation significantly inhibited the release of reactive oxygen species, interleukin-1ß, monocyte-endothelial adhesion, and lipid oxidation by human monocytes.⁴⁵ With regard to its antiatherogenic effect on LDL oxidation, we have shown in an earlier dose response study (60 to 1200 IU/d of all-rac-AT for 8 weeks) that only doses 400 IU/d of all-rac-AT significantly decreases the oxidative susceptibility of LDL.²¹ It is interesting to note that a recent clinical trial showed that RRR-AT in doses 400 IU/d resulted in a significant reduction in nonfatal myocardial infarction.²⁵ The main objective of this study was to compare the effects of supplementation with equivalent IU doses (the therapeutically available form) of RRR-AT and all-rac-AT on antioxidant levels and the susceptibility of LDL to oxidation to ascertain whether both forms of AT had similar effects.

None of the subjects entered into the study reported any side effects with either RRR-AT or all-rac-AT. Several double-blind, controlled supplementation studies with doses ranging from 200 to 1600 IU/d of AT have shown no consistent adverse effects.^{19 23 28 46} Also, there were no significant differences in plasma lipids, lipoproteins, and fatty acids after 8 weeks of supplementation with RRR-AT or all-rac-AT. In addition, AT supplementation with RRR-AT or all-rac-AT at any of the doses studied did not have any effect on the circulating concentrations of micronutrient antioxidants such as ascorbate and ß-carotene. All of the above findings are in agreement with the literature.^{17 18 19 20 21 22 23}

Plasma, lipid-standardized, and LDL AT levels rose progressively after supplementation with RRR-AT and all-rac-AT. However, there were no significant differences in AT levels between RRR-AT and all-rac-AT supplementation at equivalent IU dosages. This finding is in agreement with the earlier studies of Reaven and Witztum²⁸ and Winklhofer-Roob et al.⁴⁷ In the former study, the authors showed no significant differences in AT levels and LDL oxidative susceptibility between RRR-AT (n=7) and all-rac-AT (n=8) supplementation (1600 mg/d for 2 months) in 15 mildly hyperlipidemic (10 female and 5 male) volunteers. However, the high dosage of AT used in that study may have "inundated" the physiological system, thereby negating any differences that might have been seen with lower doses. In the latter study, there were no significant differences in plasma AT levels after 6-week supplementation of 31 young cystic fibrosis patients (18 male and 13 female) with 400 IU/d with RRR-AT (n=10) or all-rac-AT in either fat-soluble (n=10) or water-miscible (n=9) forms. However, these authors did not measure LDL AT levels and also did not study any critical antioxidant end points.

The natural form of AT, RRR-AT, is known to be more biologically active than the synthetic all-rac form, with a relative activity 1.36 times that of all-rac-AT as derived from animal bioassays such as the resorption-gestation test in rats.^{26 48} It appears that the RRR configuration of the phytyl tail is optimal for maximum biopotency. RRR-AT is preferentially secreted into VLDL,²⁶ and this is possibly the function of the hepatic tocopherol-transfer protein. While all-rac-AT is a mixture of eight stereoisomers, the tocopherol-transfer protein in the liver seems to recognize mainly the R conformation of the phytyl tail at the C-2 position. It has previously been shown that the bioavailability of RRR-AT is 1.5 times greater than the all-rac form.⁴⁹ Based on the above considerations, it appears that those subjects who consumed 100 IU/d of RRR-AT had ingested only 74 mg (100/1.36); for those who were supplemented with 100 IU/d (100 mg) of all-rac-AT, the bioavailability of the ingested AT equated to 66 mg (100/1.5) of RRR-AT. Based on the bioavailability data, the differences between RRR-AT and all-rac-AT at 100, 200, 400, and 800 IU/d in bioavailable milligram equivalents ranged between 10% and 12%. Thus, it is not surprising that while there was trend to higher levels of plasma and LDL AT with RRR-AT than all-rac-AT, given their similar milligram doses based on the bioavailability data, these differences are not significant. In this study, the effects of both RRR-AT and all-rac-AT supplements was similar, but a type II error cannot be ruled-out due to the small sample size. To further explore the direct comparisons of RRR-AT and all-rac-AT, a more powerful study with a sufficiently large number of subjects to detect a small effect is necessary. Conversions of IU to milligrams of ingested AT are difficult to perform, since at a given dose it is unknown how much of each isomer of all-rac-AT appears in plasma. Further, the methodology to measure various chiral forms of AT (gas chromatography–mass spectrometry) is tedious, requires relatively large concentrations of AT, and is available in only very few laboratories. Given the large group of subjects studied in the present report (n=79), it would have been difficult to study this aspect in depth. However, it would be reasonable for future studies to be directed to providing this information.

To gain insight into the effect of RRR-AT and all-rac-AT on the oxidative susceptibility of LDL, the time course of copper-catalyzed oxidation was performed, and the lag phase, maximum oxidation, and oxidation rate were computed from the data. This study confirmed our earlier dose-response study, in that doses 400 IU/d resulted in significant prolongation of the lag phase of oxidation as determined by the conjugated-dienes assay. Lag phase, as determined by lipid peroxide assay, was also significantly increased after supplementation with 800 IU/d in both groups and 400 IU/d of RRR-AT. However, the 400 IU/d all-rac-AT group also showed a significant trend (P=.04) to increased lag phases after supplementation when compared with baseline. There were no significant differences in lag phases of oxidation between RRR-AT and all-rac-AT groups at any of the doses studied. This is in agreement with the findings of Reaven and Witztum,²⁸ who showed that LDL isolated from groups that received 1600 mg RRR-AT and all-rac-AT were equally resistant to oxidation as measured by the formation of conjugated dienes and lipid peroxides. However, in that study, the pharmacological dose of AT may have obliterated any differences in beneficial effects that might have been seen with lower doses of RRR-AT compared with all-rac-AT.

Also, there appears to be no significant difference between RRR-AT and all-rac-AT on LDL oxidation in vitro as evidenced by the lag phase. Thus, it appears that the major function of the phytyl chain is to "anchor" the AT molecule to LDL and biomembranes.⁵⁰ Niki et al⁵¹ arrived at similar conclusions by using the oxidation of methyl linoleate and soybean phosphatidylcholine as substrates for oxidation.

In conclusion, this study has shown that in healthy individuals, supplementation with either RRR-AT or all-rac-AT resulted in similar increases in plasma and LDL AT levels at equivalent IU doses. Also, the degree of protection by RRR-AT and all-rac-AT against copper-catalyzed LDL oxidation was similar. This study has again reiterated that the threshold for efficacy against LDL oxidation is 400 IU/d of RRR-AT or all-rac-AT. Thus, our study suggests that future clinical trials designed to test the antioxidant effect of AT may use either RRR-AT or all-rac-AT at doses 400 IU/d.

	Acknowledgments

 
This work was supported by a research grant from the Henkel Corp and by National Institutes of Health grant M01-RR-00633. We thank Dr S.M. Grundy for support, Dr Jim Clark for critical discussions, Khanh Vu and Payam S. Mazidi for technical assistance, and Elizabeth Thurston for manuscript preparation.

Received December 8, 1996; accepted March 28, 1997.

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