Cosupplementation with vitamin E and coenzyme Q10 reduces circulating markers of inflammation in baboons

¹ From the Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX (XLW, DLR, and MCM); the Division of Cardiothoracic Surgery, Michael E DeBakey Department of Surgery, Baylor College of Medicine, Houston (XLW); the Southwest National Primate Research Center, San Antonio, TX (MCM); and the Centre for Vascular Research, School of Medical Sciences, University of New South Wales, and the Department of Haematology, Prince of Wales Hospital, Sydney, Australia (RS)

² Supported by NIH grants P01 HL028972 and P51 RR013986; by NIH grants R01 HL066053 and R01 HL071608 (to XLW); and by the the National Health & Medical Research Council of Australia (to RS). XLW is an American Heart Association Established Investigator (0440001N).

³ Reprints not available. Address correspondence to XL Wang, MS NAB 2010, One Baylor Plaza, Houston, TX 77030. E-mail: xlwang{at}bcm.tmc.edu.

ABSTRACT  
Background: Inflammation and oxidative stress are processes that mark early metabolic abnormalities in vascular diseases.

Objectives: We explored the effects of a high-fat, high-cholesterol (HFHC) diet on vascular responses in baboons and the potential response-attenuating effects of vitamin E and coenzyme Q₁₀ (CoQ₁₀) supplementation.

Design: We used a longitudinal design by subjecting 21 baboons (Papio hamadryas) to sequential dietary challenges.

Results: After being maintained for 3 mo on a baseline diet (low in fat and cholesterol), 21 baboons were challenged with an HFHC diet for 7 wk. The serum C-reactive protein (CRP) concentrations did not change. Subsequent supplementation of the HFHC diet with the antioxidant vitamin E (250, 500, or 1000 IU/kg diet) for 2 wk reduced serum CRP concentrations from 0.91 ± 0.02 to 0.43 ± 0.06 mg/dL. Additional supplementation with CoQ₁₀ (2 g/kg diet) further reduced serum CRP to 30% of baseline (0.28 ± 0.03 mg/dL; P = 0.036 compared with the HFHC diet). Introduction of the HFHC diet itself significantly decreased serum P-selectin (from 48.8 ± 7.2 to 32.9 ± 3.7 ng/dL, P = 0.02) and von Willebrand factor (from 187.0 ± 10.1 to 161.9 ± 9.0%, P = 0.02) concentrations. However, neither vitamin E alone nor vitamin E plus CoQ₁₀ significantly altered the serum concentrations of P-selectin or von Willebrand factor.

Conclusions: Dietary supplementation with vitamin E alone reduces the baseline inflammatory status that is indicated by the CRP concentration in healthy adult baboons. Cosupplementation with CoQ₁₀, however, significantly enhances this antiinflammatory effect of vitamin E.

Key Words: Atherosclerosis • oxygen radicals • cytokines • endothelial function • antioxidants

INTRODUCTION  
It is now widely accepted that inflammation and oxidative stress are 2 important processes integrally involved in the development and progression of vascular diseases. It has been postulated that a high-fat, high-cholesterol (HFHC) diet can induce inflammatory changes in vascular systems that result in oxidative stress and endothelial cell injury (1–3). Reactive oxygen species are produced by all cells and have the potential to react with biological molecules, forming a variety of stable and unstable intermediates. Oxidative damage—the accumulation of oxidatively modified biomolecules—has been associated with aging (4, 5), neurodegenerative diseases (6), memory impairment (7), cancer (8–10), and cardiovascular disease (CVD; 11–13).

Cardiovascular responses to oxidative stress, especially the oxidation of LDL and the resultant effects on vessel wall biology, have been studied intensively (14). Oxidized LDL is thought to be trapped in the subendothelial space of the artery wall and thus may initiate an inflammatory response that contributes to atherogenesis. Vascular cells including neutrophils, monocytes, and endothelial and smooth muscle cells generate reactive species such as superoxide anion, hydrogen peroxide, hypochlorite, and peroxynitrite in response to different stimuli, including infectious agents, mechanical stress, environmental factors, the peptide angiotensin II, cytokines, oxidized LDL, and transition metals (15).

While thought to act as signaling molecules involved in the cellular response to triggers such as tumor necrosis factor , some of these reactive species have the potential to directly initiate an inflammatory response (16–18). Thus, antioxidants not only scavenge reactive species but also have the potential to reduce inflammatory responses and the damage that they cause. Therefore, administration of anti-inflammatory agents may also minimize oxidative burden.

C-reactive protein (CRP) is a well-established marker of inflammation and is classified as an acute phase reactant (17, 19–21). Often showing coordinated responses with interleukin 6, elevated serum concentrations of CRP are associated with increased risks of myocardial infarction and sudden cardiac death in apparently healthy persons and of macrovascular complications in persons with type 2 diabetes. P-selectin, a platelet -granule membrane protein that redistributes to the plasma membrane during platelet activation and degranulation, is often used as a marker of platelet activation (22, 23). Because of its role in leukocyte-mediated inflammation, P-selectin is hypothesized to play a role in the initiation of atherosclerosis and thrombosis (23–25). Von Willebrand factor (vWF) is a well-recognized marker for endothelial cells, and endothelial dysfunction and increased risk for CVD are associated with elevated serum concentrations of vWF (26, 27).

In the current study, we investigated the effects of different diets on circulating markers of inflammation and endothelial dysfunction—specifically CRP, P-selectin, and vWF—and on a general marker of serum antioxidant status. For this investigation, we sequentially challenged baboons (Papio hamadryas) with an HFHC diet, an HFHC diet supplemented with vitamin E, and an HFHC diet supplemented with vitamin E plus coenzyme Q₁₀ (CoQ₁₀), with the aim of establishing a model of oxidative stress and inflammation that may facilitate future interventions and drug trials in humans. The use of a nonhuman primate model has clear advantages over the use of small-animal models, because the nonhuman primates more closely resemble humans in many aspects, including metabolism, anatomy, physiology, and pathology. Detection of the effects of vitamin E and CoQ₁₀ supplementation on circulating inflammatory, vascular, and redox markers in these pedigreed baboons would enable the use of the baboon model in future studies to search for, detect, localize, and characterize the genes responsible for variations in susceptibility to the effects of dietary supplementation with these compounds.

SUBJECTS AND METHODS  
Animals
Twenty-one 3-y-old baboons (13 males and 8 females) that were members of an extended pedigreed colony were selected for this experiment. Some of the baboons were related to each other, although the average kinship coefficient was 0.0176 (range, 0.000-0.3203). For 3 mo, the baboons were consuming a baseline diet (Harlan-Teklad, Madison, WI) that was low in fat (7% of energy) and cholesterol (0.02 mg/g) before they were assigned to a series of consecutive HFHC diets containing 41% of the energy as fat (by the addition of lard) and 6.37 mg cholesterol/g. The fatty acid composition of the baseline and HFHC diets is shown in Table 1. As can be seen, compared with the baseline diet, the HFHC diet contains a substantially lower ratio of polyunsaturated fatty acids (PUFAs) to saturated fatty acids (SFAs) and a higher ratio of monounsaturated fatty acids (MUFAs) to PUFAs. The sequence of HFHC diets used is indicated in Table 2: HFHC alone for 7 wk, HFHC plus variable amounts of vitamin E (HFHC-E) for 2 wk, and HFHC-E plus CoQ₁₀ (2 g/kg; HFHC-EQ) for 2 wk. Supplemental vitamin E (DL-alpha tocopheryl acetate; Bio-Serv, Frenchtown, NJ) was added at 4 amounts to subgroups of baboons: subgroup A, 0 IU/kg diet (n = 6); subgroup B, 250 IU/kg (n = 5); subgroup C, 500 IU/kg (n = 5); and subgroup D, 1000 IU/kg (n = 5). The vitamin E content in the baseline diet was 40 IU/kg diet. The CoQ₁₀ was donated by Kaneka Corporation (Osaka, Japan). The HFHC diets were formulated by adding fat and vitamins to a defatted monkey chow meal preparation. The diets were then pelletted, bagged, and stored at –20 °C until the day of use. Baboons were maintained in a group cage and fed ad libitum during the experiment. Blood samples were obtained at the end of each diet phase from overnight-fasted animals. Plasma and serum were obtained by low-speed centrifugation and frozen at –80 °C until use. Experimental procedures were approved by the Institutional Animal Care and Use Committee at the Southwest Foundation for Biomedical Research, which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care International.

View this table:
TABLE 1. Measured composition of the diets¹

 

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TABLE 2. Sampling scheme with vitamin E (IU/kg diet) and CoQ₁₀ (g/kg diet) supplementation¹

 
Blood biochemistry
Cholesterol concentrations in serum samples were measured enzymatically with the use of reagents purchased from Boehringer Mannheim Diagnostics (Roche Diagnostics USA, Indianapolis) and a Ciba-Corning Express Plus clinical chemistry analyzer (Bakersfield, CA). HDL cholesterol was measured in the supernatant fluid after precipitation of apolipoprotein B-containing lipoproteins in tubes containing heparin and Mn²⁺, and non-HDL cholesterol was calculated as the difference between total cholesterol and HDL cholesterol; CVs were 2.3% for total and 6.5% for HDL cholesterol. Total antioxidant status (TAS) measures the overall antioxidant capacity of serum. TAS was defined as the ability of serum antioxidants to prevent oxidation of 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) induced by metmyoglobin plus hydrogen peroxide, and it was measured by using a TAS kit (Calbiochem, San Diego; 28). CRP concentrations were measured with the use of a high-sensitivity assay kit (Kamiya Biomedical, Seattle) by using a latex particle-enhanced immunoturbidometric method. Soluble P-selectin was quantified in serum by using a sandwich-style enzyme-linked immunoassay kit (R&D Systems, Minneapolis). The vWF was quantified in serum by using a sandwich-style enzyme-linked immunoassay kit (Diagnostica Stago, Parsippany, NJ), with units expressed as a percentage of an international standard value. CVs for control products in these biochemical assays were 4.0% for TAS, 5.0% for CRP, 4.5% for P-selectin, and 7.0% for vWF. Plasma concentrations of -tocopherol and total CoQ₁₀ (defined as ubiquinone-10 plus ubiquinol-10) were measured by using HPLC as described previously with interassay CVs of 15% and 11%, respectively (29, 30). Plasma -tocopherol concentrations were expressed in relation to cholesterol concentrations because lipids affect the concentration of the vitamin, and the plasma cholesterol concentrations were changed by the dietary intervention. Thus, plasma -tocopherol concentrations were calculated by multiplying the individual -tocopherol value (µmol/L) by 1000 and then divided that figure by the corresponding value for unesterified cholesterol as measured by HPLC. We also measured concentrations of cholesteryl arachidonate (20:4) and linoleate (18:2) in all samples by HPLC. All buffers were filtered and argon-flushed immediately before use. Organic solvents and all other chemicals used were of the highest quality available.

Statistical analyses
Results are presented as mean ± SEM and compared by repeated-measures analysis of variance (ANOVA). We initially explored the effects of diet and vitamin supplementation on circulating variables by using a general linear model of a repeated-measures ANOVA, in which we modeled (incorporated) diet as a categorical factor, vitamin E dose (0, 250, 500, or 1000 IU/kg diet) as a continuous variable, and the interaction between diet and vitamin E dose. For variables for which no significant diet x vitamin E dose interaction was detected, we subsequently combined the data for the HFHC-E and HFHC-EQ diets so that we could examine the main effects of vitamin supplementation and interaction between vitamin E dose and CoQ₁₀ supplementation by ANOVA. Finally, to minimize potential confounding effects due to relatedness among the animals from which these samples were obtained, we used a paired t test to compare the effects of the HFHC diet with those of the baseline and vitamin-supplemented diets. The effect of vitamin E supplementation was compared with the measurements during the HFHC diet only because it is the baseline diet for vitamin supplementation. Paired t tests were also used to augment observations made by means of ANOVA regarding the effect of additional CoQ₁₀ supplement by comparing the HFHC-EQ diet to both the HFHC and HFHC-E diets. After Bonferroni’s correction of resultant P values (standard P values multiplied by the number of variables compared), two-tailed P < 0.05 was regarded as statistically significant.

RESULTS  
Dietary challenge and vascular profiles
Our initial two-factor repeated-measures ANOVAs detected a diet x vitamin E dose interaction effect for only one of the traits examined: plasma -tocopherol (P < 0.001); vitamin E dose alone also exerted a significant effect on the variance in this trait (P < 0.001), but the categorical dietary composition factor itself did not. Therefore, we could validly combine data from the HFHC-E and HFHC-EQ diets for subsequent analyses of the effects of vitamin E on remaining traits (for which no significant diet x vitamin E interactions were detected). Because there was no dose-dependent effect by vitamin E supplementation or interactive effect of vitamin E and the HFHC diet on any of the measured vascular profiles except plasma -tocopherol concentrations, we combined animals supplemented with vitamin E at 250, 500, and 1000 IU/kg diet into a single group when we assessed the effects of vitamin E on vascular profiles.

Effect of HFHC diet on vascular profiles
As observed previously (31), the HFHC diet substantially increased serum total cholesterol concentrations (Table 3), and the increases in HDL cholesterol and non-HDL cholesterol were similar. Concentrations of CRP and TAS were not changed by the HFHC diet, but this diet significantly decreased the concentrations of P-selectin and vWF (Table 3, P = 0.0013). In addition, total CoQ₁₀ concentrations increased as a result of the HFHC diet (Table 3, P = 0.0013). In response to the HFHC diet, the plasma PUFA cholesteryl arachidonate (20:4) and linoleate (18:2) concentrations were also increased (Table 3). The lipid-standardized -tocopherol concentrations were decreased by the HFHC diet and correlated significantly with the concentrations of unesterified cholesterol (r = 0.825, P = 0.0001), cholesteryl arachidonate (r = 0.628, P = 0.002), and linoleate (r = 0.696, P = 0.0001). The same relations were also observed for samples collected during the baseline diet (P < 0.05 for all). There were also significant correlations between plasma CRP concentrations and cholesteryl arachidonate (r = 0.551, P = 0.01) and linoleate (r = 0.589, P = 0.005). However, the significant correlations were observed only for the HFHC diet samples. There were no significant correlations between concentrations of P-selectin, vWF, TAS, and arachidonate or linoleate or the ratio of HDL to non-HDL cholesterol before or after the HFHC diet challenge.

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TABLE 3. Effect of a high-fat, high-cholesterol (HFHC) diet for 7 wk on blood biochemistry in all 21 baboons¹

 
Interactive effects of vitamin E and CoQ₁₀ supplementation on plasma vitamin concentrations
As shown in Table 4, supplementation with vitamin E achieved a significant increase in plasma -tocopherol concentrations in baboons. As expected, supplementation with 1000 IU vitamin E/kg diet resulted in slightly higher plasma lipid-standardized -tocopherol concentrations (23.0 ± 2.4 µmol/L) than did supplementation with 500 IU/kg diet (22.0 ± 1.1 µmol/L) or 250 IU/kg diet (15.6 ± 1.9 µmol/L). Although absolute concentrations of -tocopherol showed an apparent dose-dependent increase, the actual increases were similar for all 3 doses (1.9-, 2.3-, and 2.3-fold, respectively) because of differences in the baseline concentrations. We further noted that vitamin E supplementation tended to reduce plasma total CoQ₁₀ concentrations and, conversely, that CoQ₁₀ supplementation tended to reduce plasma -tocopherol concentrations (Table 4).

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TABLE 4. Effect of a high-fat, high-cholesterol (HFHC) diet and antioxidant supplements on plasma -tocopherol and coenzyme Q₁₀ (CoQ₁₀) concentrations¹

 
Effects of vitamin E and CoQ₁₀ supplementation on serum markers of inflammation, antioxidant status, and endothelial cell function
Because the repeated-measures ANOVA showed no dose-dependent effect of vitamin E supplementation on any of the measured vascular factors and because proportional increases in plasma -tocopherol were similar for all 3 amounts of vitamin E supplementation, we pooled the 3 vitamin E groups (n = 15) into a single group. In these 15 baboons, vitamin E supplementation reduced the CRP concentrations from 0.91 ± 0.21 to 0.43 ± 0.06 mg/dL—a 53% reduction. Additional dietary supplementation with 2 g CoQ₁₀/kg diet further reduced the serum CRP concentration to 0.28 ± 0.03 mg/dL (P = 0.036 compared with the HFHC diet)—a decrease of nearly 70%. Using the same linear model of ANOVA, we detected a significant effect on CRP concentrations of supplementation with vitamin E (P = 0.008) and with vitamin E plus CoQ₁₀ (n = 15, P = 0.018). It was shown previously that cosupplementation with vitamin E and CoQ₁₀ reduced the extent of lipoprotein lipid oxidation in the vessel wall of apolipoprotein E-deficient mice (29), which provided evidence for the notion that, in addition to increasing the antioxidant status, the 2 antioxidants in combination can reduce oxidative stress and damage in vivo. The effect of cosupplementation with vitamin E and CoQ10 on plasma concentrations of F2-isoprostanes, a commonly used marker of in vivo oxidative stress (32), is not known at present. TAS concentrations were significantly increased by vitamin E supplementation (from 1.16 to 1.24 mmol/L, n = 15; P = 0.004). The addition of CoQ₁₀ (Table 5) caused a further increase to 1.26 mmol/L, a 9% increase from HFHC diet concentrations (P = 0.004). However, the difference in TAS concentrations between vitamin E alone and vitamin E plus CoQ₁₀ supplementation was not statistically significant (P = 0.088).

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TABLE 5. Effects of antioxidant supplement on serum inflammation, antioxidant status, and endothelial markers among 15 baboons fed different doses of vitamin E with or without coenzyme Q₁₀ (CoQ₁₀)¹

 
The HFHC diet significantly reduced serum concentrations of P-selectin and vWF. Supplementing this diet with vitamin E achieved further reduction only in the concentrations of vWF (P = 0.015 in the linear ANOVA model). Cosupplementation with vitamin E plus CoQ₁₀ did not make additional alterations to these measurements of endothelial function (Table 5).

DISCUSSION  
A key finding of our current study is the dramatic effect of vitamin E and CoQ₁₀ cosupplementation on markers of vascular inflammation, as indicated by serum concentrations of CRP. Dietary vitamin E at a dose as low as 250 IU/kg diet, which corresponds to a human supplementation of 200 IU vitamin E/d, had already achieved a reduction by nearly 50% in serum CRP concentrations. The human estimation is based on the fact that a moderately active baboon consumes 500 g diet/d (1500 kcal at a caloric density of 3 kcal/g); the 125 IU vitamin E/d (250 IU/kg diet) consumed by a baboon is then translated to 200 IU vitamin E/d for a human subject (on the basis of a human diet of 2500 kcal/d). An additional 20% reduction was achieved by cosupplementation with 2 g CoQ₁₀/kg diet. This result is remarkable given that the baboons used did not have any inflammatory condition at the time of the study, and the 2-wk HFHC diet did not initiate a significant inflammatory response. In light of the fact that elevated CRP has been associated with vascular dysfunction (33), which in turn is associated with CVD (34), the results from our longitudinal study suggest an anti-inflammatory benefit of dietary supplementation with vitamin E plus CoQ₁₀.

The therapeutic effects of vitamin E supplementation on CVD outcome are still disputed (35, 36), and animal experiments examining the effect of vitamin E supplements on atherosclerosis generally show positive, neutral, and negative results (37). However, we are aware of only 2 studies in nonhuman primates, and both of these reported a beneficial effect of vitamin E on atherosclerosis (38, 39). In addition, where examined, cosupplementation with vitamin E and CoQ₁₀ was shown to be more antiatherogenic in a mouse model of atherosclerosis than was supplementation with the vitamin alone (29). Our finding that, in addition to its antioxidant capacity, vitamin E reduces circulating concentrations of CRP suggests that an anti-inflammatory mechanism involving vitamin E could offer additional protection, as suggested by Jialal et al (17) and Devaraj and Jialal (40). However, some studies reported a lack of effect of vitamin E on plasma inflammatory markers (41–44). Although such inconsistency remains to be resolved, those studies typically used low doses of vitamin E cosupplemented with vitamin C (41–43) or retrospective correlation analyses (44). What is new in the present study, however, is that cosupplementation with vitamin E plus CoQ₁₀ appeared to be more anti-inflammatory than did supplementation with vitamin E alone, as judged by the circulating concentrations of CRP (P = 0.019). It can be extrapolated that cosupplementation with CoQ₁₀ may achieve better protection against inflammation-related vascular diseases.

An interesting observation is the apparently reducing effects of HFHC diet on serum P-selectin and vWF. This potentially antiatherogenic reduction of P-selectin and vWF is not consistent with the increased atherogenicity associated with elevations of dietary fat and cholesterol (25). Whereas the mechanisms for the reduction in P-selectin and vWF concentrations in response to the HFHC diet are not clear, studies by Thomsen et al (45) and Rasmussen et al (46) showed that the consumption of MUFAs reduced vWF concentrations more than did the consumption of PUFAs. The HFHC diet in this study indeed had 60% more MUFAs but 70% less PUFAs than did the baseline diet (Table 1). However, it should be recognized that the absolute concentrations of all classes of fatty acids are higher in the HFHC diet. Alternatively, the lower P-selectin and vWF concentrations in the baboons could be related to increased HDL-cholesterol concentrations when they were fed the HFHC diet. Furthermore, neither vitamin E alone nor vitamin E plus CoQ₁₀ brought any change to serum P-selectin and vWF concentrations. This appears at odds with findings in human subjects, in whom vitamin E supplement was found to reduce the concentrations of endothelial adhesion molecules (47–49). However, most of those studies employed subjects with various metabolic disorders. The baboons in our study were all healthy individuals and had only a short-term HFHC diet load.

We also noted that vitamin E supplementation tended to reduce the concentrations of plasma CoQ₁₀ and that CoQ₁₀ supplementation tended to reduce -tocopherol concentrations. The reason or reasons for this tendency are not clear at present. One previous study also showed that cosupplementation of apolipoprotein E-deficient mice with vitamin E and CoQ₁₀ reduced plasma vitamin E concentrations more than did supplementation with vitamin E alone (29), although in that case, vitamin E supplements did not reduce plasma CoQ₁₀ concentrations. A possible explanation for these differences is that one lipid-soluble antioxidant may displace the other, given their similar distribution in plasma lipoproteins. This could be particularly relevant when HDL, which is small in relation to LDL, is a major carrier of lipid-soluble antioxidants, as appears to be the case in baboons, according to the ratio of total to HDL cholesterol.

A limitation of the current study is the small number of animals used in each subgroup under different doses of vitamin E supplement, which reduces our statistical power to detect dose-dependent responses. Another limitation is the extrapolation of our findings in baboons to human subjects, who may have different metabolic profiles. For example, there are some differences in lipid profiles between baboons and humans: eg, baboons tend to have higher concentrations of HDL cholesterol than of non-HDL cholesterol (50).

In summary, our study shows that dietary supplementation with vitamin E has anti-inflammatory effects in baboons, as judged by circulating concentrations of CRP. Cosupplementation with vitamin E and CoQ₁₀ significantly enhanced both anti-inflammatory and antioxidant protection. These effects may in turn help protect against vascular diseases. The significant effects of and interindividual variation in these effects in baboons show that the pedigreed baboon may be a reasonable model in which to study the genetic basis for regulating responses to dietary antioxidant supplementation.

ACKNOWLEDGMENTS  
We thank Perry H Moore Jr, Israel O Gamboa, Jane F VandeBerg, Catherine Jett, and Katherine Choy for technical support.

XLW initiated the concept of the project, analyzed and interpreted the data, and wrote the manuscript. DLR participated in the development of the project; supervised the execution of the animal experiment, some laboratory analyses, and data collection; and contributed to writing the manuscript. MCM participated in the study design, data analyses, and manuscript writing. RS participated in the development of the project and supervised some laboratory assays, results interpretation, and manuscript writings. None of the authors had any personal or financial conflicts of interest.

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