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Clinical Chemistry 53: 511-519, 2007. First published February 1, 2007; 10.1373/clinchem.2006.076992
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(Clinical Chemistry. 2007;53:511-519.)
© 2007 American Association for Clinical Chemistry, Inc.

Nutrition

Effects of {alpha}-Tocopherol and Mixed Tocopherol Supplementation on Markers of Oxidative Stress and Inflammation in Type 2 Diabetes

Jason H.Y. Wu1, Natalie C. Ward1, Adeline P. Indrawan1, Coral-Ann Almeida2, Jonathan M. Hodgson1, Julie M. Proudfoot1, Ian B. Puddey1 and Kevin D. Croft1,a

1 School of Medicine and Pharmacology, University of Western Australia, Crawley, Western Australia, Australia.
2 Centre for Clinical Immunology and Biomedical Statistics, Royal Perth Hospital and Murdoch University, Perth, Western Australia, Australia.

aAddress correspondence to this author at: School of Medicine and Pharmacology, University of Western Australia, P.O. Box X2213 GPO, Perth, Western Australia 6847, Australia. Fax 61-8-9224-0246; e-mail kcroft{at}cyllene.uwa.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Vitamin E isomers may protect against atherosclerosis. The aim of this study was to compare the effects of supplementation with either {alpha}-tocopherol ({alpha}T) or mixed tocopherols rich in {gamma}-tocopherol ({gamma}T) on markers of oxidative stress and inflammation in patients with type 2 diabetes.

Methods: In a double-blind, placebo-controlled trial, 55 patients with type 2 diabetes were randomly assigned to receive (500 mg/day) (a) {alpha}T, (b) mixed tocopherols, or (c) placebo for 6 weeks. Cellular tocopherols, plasma and urine F2-isoprostanes, erythrocyte antioxidant enzyme activities, plasma inflammatory markers, and ex vivo assessment of eicosanoid synthesis were analyzed pre- and postsupplementation.

Results: Neutrophil {alpha}T and {gamma}T increased (both P <0.001) with mixed tocopherol supplementation, whereas {alpha}T (P <0.001) increased and {gamma}T decreased (P <0.005) after {alpha}T supplementation. Both {alpha}T and mixed tocopherol supplementation resulted in reduced plasma F2-isoprostanes (P <0.001 and P = 0.001, respectively) but did not affect 24-h urinary F2-isoprostanes or erythrocyte antioxidant enzyme activities. Neither {alpha}T nor mixed tocopherol supplementation affected plasma C-reactive protein, interleukin 6, tumor necrosis factor-{alpha}, or monocyte chemoattractant protein-1. Stimulated neutrophil leukotriene B4 production decreased significantly in the mixed tocopherol group (P = 0.02) but not in the {alpha}T group (P = 0.15).

Conclusions: The ability of tocopherols to reduce systemic oxidative stress suggests potential benefits of vitamin E supplementation in patients with type 2 diabetes. In populations with well-controlled type 2 diabetes, supplementation with either {alpha}T or mixed tocopherols rich in {gamma}T is unlikely to confer further benefits in reducing inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Patients with type 2 diabetes have a significantly higher risk of developing coronary heart disease and atherosclerosis (1). A possible reason for accelerated atherosclerosis in these patients is increased subclinical systemic inflammation. It is now known that inflammation plays a key role in all stages of atherosclerosis (2). Acute-phase inflammatory markers may be increased in patients with type 2 diabetes (3), and follow-up studies have suggested that increased concentrations of acute phase proteins also predict the risk of developing type 2 diabetes (4). These data support the hypothesis that activation of the innate immune system and subsequent generation of proinflammatory cytokines could be a common pathogenic feature of both type 2 diabetes and atherosclerosis (4).

Another possible mechanism for accelerated atherogenesis in type 2 diabetes is increased oxidative stress, which has been demonstrated in these patients by measuring F2-isoprostanes, a nonenzymatic peroxidation product of arachidonic acid (5). Increased oxidative stress may contribute to atherogenesis through mechanisms such as augmented lipoprotein oxidation (6). There has been an intensive search for compounds that could reduce oxidative stress, and the nutrient vitamin E has been extensively tested for this purpose. Vitamin E describes a family of compounds consisting of the tocopherols and tocotrienols (7). Past studies have focused on {alpha}-tocopherol ({alpha}T),1 because it is one of the major bioavailable forms of vitamin E consumed in the diet (7). {alpha}T can function as a chain-breaking antioxidant in vitro (8), and supplementation with {alpha}T has also been shown to reduce F2-isoprostanes in certain populations, including patients with type 2 diabetes (9).

Apart from a possible function as an antioxidant, vitamin E may also modulate inflammation (10). Although {alpha}T has been the most frequently examined vitamin E isomer (9), there is now evidence indicating that the other major dietary vitamin E isomer, {gamma}-tocopherol ({gamma}T), may have unique antiinflammatory properties not shared by {alpha}T. At physiologically relevant concentrations, {gamma}T significantly inhibited prostaglandin E2 (PGE2) synthesis in stimulated cultured murine macrophages and in human epithelial cells (11). In comparison, {alpha}T had either moderate or no effect at similar or higher concentrations. In a rat model of inflammation, supplementation with {gamma}T significantly inhibited the formation of PGE2 as well as another eicosanoid, leukotriene B4 (LTB4) (12), whereas {alpha}T had no effect. Arachidonic acid derived eicosanoids such as PGE2 and LTB4 are potential proinflammatory mediators in atherosclerosis (13)(14). In the same rat model, {gamma}T supplementation also reduced the concentration of tumor necrosis factor-{alpha} (TNF-{alpha}) (12), a proinflammatory molecule that has been shown to be associated with an increased risk of recurrent coronary events (15). Together, these studies suggest that increasing the concentrations of {gamma}T may be beneficial for protection against atherosclerosis (7).

Treating the underlying subclinical inflammation may be a useful therapeutic approach in type 2 diabetes (16). To date, there have not been any clinical studies examining the antiinflammatory potential of vitamin E isomers other than {alpha}T, in patients with type 2 diabetes. We therefore carried out a randomized, double-blind placebo-controlled intervention trial in patients with type 2 diabetes who received supplementation with either pure {alpha}T or mixed tocopherols rich in {gamma}T.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
study participants and study design
From the Perth general population we recruited 58 individuals with type 2 diabetes who had not previously used vitamin E supplements. Exclusion criteria and study design have been described previously (17). Briefly, all study participants ceased any vitamin or fish oil supplementation for at least 3 weeks before study entry and for the duration of the trial. They were randomized to 1 of 3 treatments for 6 weeks: treatment 1, RRR-{alpha}T (500 mg/day); treatment 2, mixed tocopherol enriched with {gamma}T [75 mg {alpha}T, 315 mg {gamma}T, and 110 mg {delta}-tocopherol ({delta}T)/day, all natural RRR-isomers]; and treatment 3, placebo (pure soybean oil containing <1 mg of tocopherols). The dose of {alpha}T was similar to that previously shown to reduce F2-isoprostanes and C-reactive protein (CRP) in persons with type 2 diabetes (9), and mixed tocopherols was chosen to match {alpha}T by weight. Study participants were instructed to consume two 250-mg capsules, 1 with breakfast and 1 with dinner, and to maintain their usual medication, dietary, and activity patterns. All volunteers provided fasting blood samples and a 24-h urine collection at baseline and after 6 weeks of intervention. Pre- and postintervention samples from the same individual were always analyzed in the same run. Compliance was assessed by a postintervention tablet count. The study was double-blinded, and each study participant’s grouping was revealed only after completion of all biochemical analysis. The trial was approved by the University of Western Australia Human Research Ethics Committee, and all study participants gave written informed consent.

leukocyte isolation
Peripheral blood mononuclear cells and neutrophils were isolated from venous blood collected into citrated tubes, using Ficoll-Paque (Amersham Biosciences) density centrifugation (18). Monocytes were purified from peripheral blood mononuclear cells with a magnetic activated cell sorting system, using positive selection with microbeads coated with CD14 antibody (Miltenyi Biotec). The isolated cells were at least 95% CD14+, as measured by flow cytometry. Neutrophils were isolated from the neutrophil-erythrocyte pellet from the Ficoll-Paque gradient by Dextran (Amersham Biosciences) sedimentation and removal of contaminant erythrocytes by lysis with ammonium chloride, 8.3 g/L. Typically, the percentage of neutrophils exceeded 90% (measured by CELLDYNTM Coulter counter), and cell viability was >99% (trypan blue exclusion). All cell separation procedures were carried out in <3 h, at room temperature, and under sterile conditions. Isolated cells were washed once with Hanks’ balanced salt solution (Invitrogen) and then either processed immediately for cell stimulation or frozen at –80 °C until determination of cellular tocopherol.

measurement of cellular tocopherol
Monocyte and neutrophil tocopherol content was analyzed by reverse-phase HPLC (RP-HPLC) according to previously published methods (17). Cell protein was quantified by the Bradford method (19) using bovine serum albumin (Sigma-Aldrich) as calibrator. The intra- and interassay imprecisions (CVs) for {delta}T, {gamma}T, and {alpha}T were all <15% (n = 5).

neutrophil ltb4 generation
Neutrophils were stimulated with calcium ionophore A23187 (Sigma-Aldrich) to induce synthesis of LTB4 and its metabolites according to previously published methods (20). All samples were prepared in duplicate and stored at –80 °C until analysis by RP-HPLC (21). The method allows the simultaneous determination of LTB4 and its {omega}-oxidized metabolites using prostaglandin B2 (Cayman Chemical) as the internal standard. The interassay CV for total LTB4 was 11% (n = 10).

plasma inflammation markers
High-sensitivity CRP (Hs-CRP) was assayed with the particle-enhanced immunonephelometry system on the Dade Behring BNII analyzer (Dade Behring). TNF-{alpha} and interleukin-6 (IL-6) were assayed with high-sensitivity ELISA (R&D Systems), whereas monocyte chemoattractant protein-1 (MCP-1) was measured using the human MCP-1 OptEIA reagent set (BD PharMingen). The lowest detectable concentrations were 0.15 mg/L for Hs-CRP, 0.15 ng/L for IL-6, 0.5 ng/L for TNF-{alpha}, and 30 ng/L for MCP-1. The interassay imprecisions (CVs) were 12% for MCP-1 and <8% for Hs-CRP, IL-6, and TNF-{alpha}. Neutrophil myeloperoxidase (MPO) activity was assayed according to the method of Zhang et al. (22), with intra- and interassay CVs of <5% (n = 6).

whole blood cyclooxygenase-2 activity assay
The impact of tocopherol supplementation on PGE2 synthesis was assessed in lipopolysaccharide (LPS)-stimulated whole blood (23). Briefly, fasting blood was collected into a lithium heparin-containing tube to which freshly prepared aspirin (Sigma-Aldrich) was added immediately to a final concentration of 10 mg/L. LPS (serotype 0111:B4, Sigma-Aldrich) was then added (final concentration 1 mg/L), and the blood incubated at 37 °C for 24 h. Plasma was separated and stored at –80 °C until analysis for PGE2 by enzyme immunosorbent assay (EIA, Cayman Chemical). The interassay CV for PGE2 was 14%.

f2-isoprostanes and erythrocyte antioxidant enzyme activities
Plasma and 24 h urinary F2-isoprostanes were measured by gas chromatography–mass spectrometry (24). Superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GPx; EC 1.11.1.9) activity were measured in erythrocytes by commercially available reagent sets (Cayman Chemical). The intra and interassay CVs were 11.9% and 13.7% for the SOD assay, whereas the GPx assay had intra- and interassay CVs of 4.2% and 6.3%. Hemoglobin concentration was determined by the cyanmethemoglobin method with Drabkin reagent (Sigma-Aldrich).

other biochemical measurements
Fasting plasma glucose, glycohemoglobin (HbA1c), lipids, full blood picture, serum creatinine, and cystatin C were analyzed at the Core Clinical Laboratory at Royal Perth Hospital by routine methods. Glomerular filtration rate (GFR) was estimated using the Cockcroft–Gault equation (25).

statistical analysis
All analyses used the Statistical Package for the Social Sciences (SPSS version 11.5). Nonparametric data were log transformed and results are presented as mean (SE) or geometric mean (95% confidence interval) for log-normalized data. Baseline clinical and laboratory variables were compared between randomized groups by either ANOVA for means or a {chi}2 test for proportions. Within-group changes in cellular tocopherol content were analyzed by paired samples t-test. Treatment effect of {alpha}T and mixed tocopherols compared with the placebo group was determined by general linear modeling, adjusting for baseline values and potential confounders. We used Bonferroni adjustment and accepted statistical significance at P <0.025 to adjust for multiple testing.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
characteristics of study participants
Of the 58 study participants who took part, 55 completed the study (3 withdrawing because of changes in medication). Baseline clinical details on the study participants are shown in Table 1 . The groups were well matched except that the mixed tocopherol group was younger than the {alpha}T-supplemented group (Student t-test, P = 0.009), but neither group was significantly different from the placebo group. The groups did not differ in the proportion of participants who were taking oral hypoglycemics (58%), antihypertensive treatment (51%), lipid-lowering drugs (53%), and aspirin (38%). We were careful to ensure that the medication status of all study participants remained unchanged throughout the study. Mean compliance by tablet count was 97% and was not significantly different between treatment groups. Body mass index, fasting plasma glucose, glycohemoglobin, lipids, total leukocyte, neutrophil count, GFR, and serum cystatin c were not affected by tocopherol treatments (results not shown).


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  		Table 1. Baseline characteristics of the study participants.1

cellular tocopherol enrichment by supplementation
At baseline, neutrophils contained predominantly {alpha}T followed by {gamma}T (~90% and 10% of all tocopherols, respectively; Fig. 1 ). {delta}T was undetectable in 90% of the study participants (detection sensitivity of 5 nmol/g protein, data not shown). Compared with the placebo group, mixed tocopherol supplementation led to an ~7-fold increase in {gamma}T (P <0.001) and a 40% increase in {alpha}T (P <0.01), but {alpha}T remained the major form of tocopherol present (Fig. 1 ). Mean (SD) {delta}T also increased significantly postsupplementation to 23.3 (1.9) nmol/g (P <0.001). In the {alpha}T-supplemented group, there was a 2.6-fold increase in {alpha}T (P <0.001) compared with the placebo group. {alpha}T supplementation also caused a concomitant decrease in {gamma}T compared with baseline (~30% decrease, P <0.005; Fig. 1 ). {delta}T remained undetectable in ~90% of the study participants in the {alpha}T and placebo group postsupplementation. Both {alpha}T and mixed tocopherol supplementation led to significant net increases in total tocopherol concentration in neutrophils postsupplementation (P <0.001; Fig. 1C ). The change in monocyte tocopherol concentration postsupplementation in each group was qualitatively similar to that seen in neutrophils (results not shown).


Figure 1
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  		Figure 1. Neutrophil concentration of {alpha}- (A), {gamma}- (B), and total (C) tocopherol before and after 6 weeks of supplementation with {alpha}T (black columns), mixed tocopherols (hatched columns), or placebo (white columns).

Tocopherol values are expressed as nmol/g cell protein, and results are presented as mean (SE) of each group. a, P <0.001, and b, P <0.01 compared with placebo group and adjusting for baseline values. c, P <0.005 compared with preintervention.

effect of supplementation on markers of oxidative stress and inflammation
At baseline, there were no significant differences between groups for plasma and urinary isoprostanes, as well as erythrocyte antioxidant enzyme activities analyzed by ANOVA. Treatment with either {alpha}T or mixed tocopherols significantly reduced plasma F2-isoprostanes compared with the placebo group (P <0.001 and P = 0.001, respectively; Table 2 ). Neither treatment affected urinary F2-isoprostane concentrations. There was also no significant change in erythrocyte SOD and GPx activity by {alpha}T or mixed tocopherol supplementation (Table 2 ). Adjustment for age did not change any of these results.


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  		Table 2. Effect of treatment on F2-isoprostanes and antioxidant enzyme activities.1

Neither {alpha}T nor mixed tocopherol supplement affected plasma Hs-CRP, IL-6, TNF-{alpha}, MCP-1 concentrations, or blood MPO activity (Table 3 ). Neither tocopherol treatment affected stimulated whole blood PGE2 synthesis. Unless stimulated by calcium ionophore, LTB4 synthesis by neutrophils was undetectable. Postsupplementation, there was a significant decrease in stimulated neutrophil LTB4 synthesis in the mixed tocopherol group compared with the placebo group (P = 0.02; Table 3 ). Adjustment for age did not change the result. The decrease in LTB4 synthesis with {alpha}T supplementation was not significant (P = 0.15).


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  		Table 3. Effect of treatment on inflammatory markers and eicosanoid synthesis.1

in vitro supplementation of neutrophils with tocopherols also reduced ltb4 synthesis
To gain some insights regarding the effects of individual tocopherol isomer’s ability to inhibit LTB4 synthesis by neutrophils, cells were isolated from healthy volunteers (n = 5–6) and incubated with tocopherol isomers. We examined {alpha}T and {gamma}T because these were the dominant tocopherol isomers present in neutrophils at baseline, as well as postsupplementation (Fig. 1Up ). Treatment of cells with ethanol vehicle did not cause inhibition of LTB4 synthesis compared with controls (results not shown). At 50 and 25 µmol/L, both {alpha}T and {gamma}T significantly (all P values ≤0.006) inhibited LTB4 synthesis but did not differ in their potency. In another set of experiments, when the cells were supplemented with an equal molar mixture of {alpha}T and {gamma}T (i.e., 12.5 µmol/L of each) there was a trend for increased inhibition of LTB4 synthesis compared with either {gamma}T or {alpha}T alone (paired samples t-test, P = 0.03 and 0.14, respectively; Fig. 2B ).


Figure 2
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  		Figure 2. In vitro supplementation experiments. Results are presented as mean (SE) (n = 5–6).

(A), neutrophils were isolated from fasting blood of healthy volunteers and incubated with various tocopherol isomers at 2 different concentrations for 15 min before stimulation with calcium ionophore and total LTB4 measured by RP-HPLC. (B), effect of equal molar mixed tocopherol supplementation at 25 µmol/L is compared with individual tocopherols at the same concentration. Paired sample t-tests were used to compare differences in LTB4 synthesis between vehicle (ethanol)-treated control cells and cells treated with tocopherols and also to compare the different tocopherol treatments at the same concentration. The in vitro incubation procedure did not affect cell viability at any of the doses or combinations of tocopherols used, as confirmed by trypan blue exclusion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
effect of tocopherol supplementation on markers of oxidative stress
Our study is in agreement with previous reports that {alpha}T supplementation reduced F2-isoprostanes in persons with type 2 diabetes (9). Although we observed a decrease in total plasma F2-isoprostanes, urinary F2-isoprostanes were not affected. The exact reason for this is unknown, but is unlikely to be due to altered renal function, because the study participants had GFR and serum cystatin c within the reference range. Previous studies have also shown a lack of correlation between plasma and urinary F2- isoprostanes in persons with type 2 diabetes, as well as smokers (26)(27). It has been suggested that urinary F2-isoprostanes may partly be derived from local production in the kidney (27), and therefore data concerning urinary F2-isoprostanes as a marker of systemic oxidative stress must be interpreted with caution (27).

The current observation that mixed tocopherol supplementation had an effect similar to that of {alpha}T in reducing oxidative stress is a novel finding. Apart from their potential to function as chain-breaking antioxidants, tocopherols may reduce oxidative stress through their ability to modulate cell-signaling pathways, such as inhibition of protein kinase C (28). Although most studies have focused on {alpha}T, there have been some reports indicating that supplementation with mixtures of tocopherols is as effective as {alpha}T supplementation alone (29). Using erythrocyte SOD and GPx activity as markers, we also showed that there was no perturbation of 2 of the major endogenous antioxidant defense systems. Previous studies have shown that {alpha}T supplementation at 300 mg or 600 mg/day for ≥3 months increased erythrocyte antioxidant enzyme activities in hemodialysis patients (30)(31). The difference in study populations and duration of tocopherol supplementation may account for the different observations.

effect of tocopherol supplementation on markers of systemic inflammation
Studies in persons with type 2 diabetes have shown that {alpha}T supplementation reduced CRP (32)(33). This is in contrast to our findings. We previously reported that tocopherol supplementation in our type 2 diabetic patients resulted in effective enrichment of plasma tocopherol (17), resulting in concentrations that are comparable to these previous studies (32)(33). It is therefore unlikely that the lack of impact of {alpha}T and mixed tocopherol supplementation on inflammatory markers is due to low bioavailability. Instead, the difference in results could be explained by the populations being studied. Baseline values of CRP in our study participants were much lower than those reported for the previous studies (32)(33), indicating that our study participants had very low levels of systemic inflammation. Baseline HbA1c suggests that our study participants had well-controlled diabetes, which may have contributed to the low levels of systemic inflammation, as suggested by previous studies showing that improved glycemic management in type 2 diabetes could reduce CRP (34). Additionally, unlike the previous studies (32)(33), our study did not exclude patients taking statin medication. We believe this allowed a clinically realistic approach to investigate whether supplementation with different preparations of vitamin E has antiinflammatory activities in addition to the patient’s existing therapy. Apart from their ability to reduce cholesterol synthesis, statins have been suggested to have antiinflammatory effects (35). Concentrations of inflammatory markers in our study participants were comparable to those observed in a previous study by Bruunsgaard et al. of healthy men with minor hypercholesterolemia (36). These investigators also reported a lack of any effect on these cytokines after combined {alpha}T and ascorbic acid supplementation (36). Collectively, comparison of our results to the literature suggests that tocopherol supplementation is unlikely to reduce inflammation in persons with well-controlled type 2 diabetes.

Consistent with our previous report on erythrocytes and platelets (17), we found that mixed tocopherol supplementation enriched neutrophils and monocytes with the different tocopherol isomers. The decrease in {gamma}T after {alpha}T supplementation is also in agreement with previous observations (37).

We chose an ex vivo whole blood stimulation assay to test the hypothesis that {gamma}T is a better inhibitor of PGE2 synthesis than {alpha}T in certain cell types (11). With LPS as a stimulant, PGE2 production in this assay reflects cyclooxygenase-2 activation in peripheral monocytes (23). We found that neither {alpha}T nor mixed tocopherol supplementation affected PGE2 synthesis, despite enrichment of monocytes with the tocopherol isomers. Our results are in agreement with a previous study suggesting that {alpha}T does not inhibit PGE2 synthesis in human monocytes (38). However, large within-person biological variation is evident in this assay. Our placebo control group showed a 28% decrease in PGE2, which has also been observed in a previous study (39). We may have had insufficient power to detect small effects of the tocopherol supplementation. Future studies using purified monocytes may provide more insight into the effect of {gamma}T on PGE2 production.

Stimulated neutrophil LTB4 synthesis has recently been suggested as an useful marker for assessing the leukotriene pathway in humans (40). Compared with mixed tocopherol, {alpha}T supplementation actually resulted in a greater net increase in total tocopherol content in neutrophils but caused only a nonsignificant decrease in LTB4 production. It is likely that at least some of the inhibitory activities of {alpha}T on LTB4 synthesis could be explained by its inhibition of 5-lipoxygenase (5-LO), a key enzyme in the biosynthesis of LTB4 (38). In contrast, there is limited information on the ability of other tocopherol isomers to inhibit 5-LO and LTB4 synthesis. One of the limitations of our human study is the use of a mixed tocopherol supplement. Although {gamma}T was the main isomer present in this mixture, we could not attribute the significant inhibition of LTB4 synthesis to this isomer alone. Our in vitro experiments showed no significant differences in the inhibitory activities of {alpha}T and {gamma}T and also that mixtures of tocopherols were not superior to {alpha}T alone. We also have preliminary data suggesting that in vitro incubation of human monocytes (another 5-LO expressing cell important in atherosclerosis) for up to 2 h with 50 µmol/L {alpha}T or {gamma}T does not affect Ca2+ ionophore–induced LTB4 synthesis (results not shown). Future in vitro experiments need to further test doses and combinations of tocopherol isomers for the optimal inhibition of LTB4 synthesis in difference cell types.

The ability of both pure {alpha}T and mixed tocopherol supplementation to reduce systemic lipid peroxidation in patients with type 2 diabetes suggests potential benefits of vitamin E supplementation in this population. Despite providing evidence of decreasing oxidative stress, however, our results also suggest that in populations with well-controlled type 2 diabetes, supplementation with either {alpha}T or mixed tocopherols rich in {gamma}T is unlikely to confer further benefits in reducing inflammation. Treatment of type 2 diabetes should emphasize pharmacological and lifestyle interventions to reach optimum glycemic and lipid control. Future research is warranted to investigate the ability of vitamin E isomers other than {alpha}T to alter production of inflammatory mediators such as LTB4.


   Acknowledgments
 
This study was funded by the National Health and Medical Research Council (NHMRC) of Australia (Project Grant 254568). We thank Cognis Ltd. and Cardinal Health Ltd. for providing the tocopherol capsules. We thank Dr. Valerie Burke for statistical assistance and Michael Clarke for the serum cystatin c analysis. We also thank the volunteers who took part in the study. J.H.Y.W. thanks the NHMRC for a postgraduate scholarship. N.C.W. acknowledges the assistance of a Faculty of Medicine, Dentistry and Health Sciences Fellowship from the University of Western Australia. None of the authors have a conflict of interest to disclose. This study is registered at the Australian Clinical Trials Registry (http://actr.org.au/), registration number 12605000093684.


   Footnotes
 
1 Nonstandard abbreviations: {alpha}T, {alpha}-tocopherol; {gamma}T, {gamma}-tocopherol; PGE2, prostaglandin E2; LTB4 leukotriene B4; TNF-{alpha}, tumor necrosis factor-{alpha}; {delta}T, {delta}-tocopherol; CRP, C-reactive protein; RP-HPLC, reverse-phase HPLC; Hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; MPO, myeloperoxidase; LPS, lipopolysaccharide; SOD, superoxide dismutase; GPx, glutathione peroxidase; HbA1c, glycohemoglobin; GFR, glomerular filtration rate; 5-LO, 5-lipoxygenase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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