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Effects of [alpha]-tocopherol and mixed tocopherol supplementation on markers of oxidative stress and inflammation in type 2 diabetes.

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 [F.sub.2]-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) [3] 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 [F.sub.2]-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 [E.sub.2] (PG[E.sub.2]) 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 PG[E.sub.2] as well as another eicosanoid, leukotriene [B.sub.4] (LT[B.sub.4]) (12), whereas [alpha]T had no effect. Arachidonic acid derived eicosanoids such as PG[E.sub.2] and LT[B.sub.4] 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

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 [F.sub.2] 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-Pague (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.sup.+], as measured by flow cytometry. Neutrophils were isolated from the neutrophil-erythrocyte pellet from the Ficoll-Pague 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 CELLDYN[TM] 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 [degrees]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 LT[B.sub.4] GENERATION

Neutrophils were stimulated with calcium ionophore A23187 (Sigma-Aldrich) to induce synthesis of LT[B.sub.4] and its metabolites according to previously published methods (20). All samples were prepared in duplicate and stored at -80 [degrees]C until analysis by RP-HPLC (21). The method allows the simultaneous determination of LT[B.sub.4] and its [omega]-oxidized metabolites using prostaglandin [B.sub.2] (Cayman Chemical) as the internal standard. The interassay CV for total LT[B.sub.4] 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 (bade 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 infra- and interassay CVs of <5% (n = 6).

WHOLE BLOOD CYCLOOXYGENASE-2 ACTIVITY ASSAY

The impact of tocopherol supplementation on PG[E.sub.2] 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:[B.sub.4], Sigma-Aldrich) was then added (final concentration 1 mg/L), and the blood incubated at 37 [degrees]C for 24 h. Plasma was separated and stored at -80 [degrees]C until analysis for PG[E.sub.2] by enzyme immunosorbent assay (EIA, Cayman Chemical). The interassay CV for PG[E.sub.2] was 14%.

[F.sub.2] ISOPROSTANES AND ERYTHROCYTE ANTIOXIDANT ENZYME ACTIVITIES

Plasma and 24 h urinary [F.sub.2]-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 infra and interassay CVs were 11.9% and 13.7% for the SOD assay, whereas the GPx assay had infra- 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 (HbA1[degrees]), 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 square] 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

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).

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).

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 [F.sub.2]-isoprostanes compared with the placebo group (P <0.001 and P = 0.001, respectively; Table 2). Neither treatment affected urinary [F.sub.2]-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.

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 PG[E.sub.2] synthesis. Unless stimulated by calcium ionophore, LT[B.sub.4] synthesis by neutrophils was undetectable. Postsupplementation, there was a significant decrease in stimulated neutrophil LT[B.sub.4] 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 LT[B.sub.4] synthesis with [alpha]T supplementation was not significant (P = 0.15).

IN VITRO SUPPLEMENTATION OF NEUTROPHILS WITH TOCOPHEROLS ALSO REDUCED LT[B.sub.4] SYNTHESIS

To gain some insights regarding the effects of individual tocopherol isomer's ability to inhibit LT[B.sub.4] 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. 1). Treatment of cells with ethanol vehicle did not cause inhibition of LT[B.sub.4] synthesis compared with controls (results not shown). At 50 and 25 [micro]mol/L, both [alpha]T and [gamma]T significantly (all P values [less than or equal to]0.006) inhibited LT[B.sub.4] 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 [micro]mol/L of each) there was a trend for increased inhibition of LT[B.sub.4] synthesis compared with either [gamma]T or [alpha]T alone (paired samples t-test, P = 0.03 and 0.14, respectively; Fig. 2B).

Discussion

EFFECT OF TOCOPHEROL SUPPLEMENTATION ON MARKERS OF OXIDATIVE STRESS

Our study is in agreement with previous reports that [alpha]T supplementation reduced [F.sub.2]-isoprostanes in persons with type 2 diabetes (9). Although we observed a decrease in total plasma [F.sub.2]-isoprostanes, urinary [F.sub.2]-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 [F.sub.2]-isoprostanes in persons with type 2 diabetes, as well as smokers (26, 27). It has been suggested that urinary [F.sub.2]-isoprostanes may partly be derived from local production in the kidney (27), and therefore data concerning urinary [F.sub.2]-isoprostanes as a marker of systemic oxidative stress must be interpreted with caution (27).

[FIGURE 1 OMITTED]

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 [greater than or equal to]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 HbA1~ 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 sup plementation 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).

[FIGURE 2 OMITTED]

We chose an ex vivo whole blood stimulation assay to test the hypothesis that [gamma]T is a better inhibitor of PG[E.sub.2] synthesis than [alpha]T in certain cell types (11). With LPS as a stimulant, PG[E.sub.2] production in this assay reflects cyclooxygenase-2 activation in peripheral monocytes (23). We found that neither [alpha]T nor mixed tocopherol supplementation affected PG[E.sub.2] 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 PG[E.sub.2] 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 PG[E.sub.2], 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 PG[E.sub.2] production.

Stimulated neutrophil LT[B.sub.4] 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 LT[B.sub.4] production. It is likely that at least some of the inhibitory activities of [alpha]T on LT[B.sub.4] synthesis could be explained by its inhibition of 5-lipoxygenase (5-LO), a key enzyme in the biosynthesis of LT[B.sub.4] (38). In contrast, there is limited information on the ability of other tocopherol isomers to inhibit 5-LO and LT[B.sub.4] 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 LT[B.sub.4] 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 [micro]mol/L [alpha]T or [gamma]T does not affect [Ca.sup.2+] ionophore-induced LT[B.sub.4] synthesis (results not shown). Future in vitro experiments need to further test doses and combinations of tocopherol isomers for the optimal inhibition of LT[B.sub.4] 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 LT[B.sub.4].

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.

Received July 20, 2006; accepted December 29, 2006. Previously published online at DOI: 10.1373/clinchem.2006.076992

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[3] Nonstandard abbreviafions: [alpha]T, [alpha]-tocopherol; [gamma]T, [gamma]-tocopherol; PG[E.sub.2] prostaglandin [E.sub.2]; LT[B.sub.4] leukotriene [B.sub.4]; TNF-[alpha], tumor necrosis factor-a; [delta]T, [delta]-tocopherol; CRP, C-reactive protein; RP-HPLC, reverse-phase HPLC; Hs-CRP, high-sensifivity C-reacfive protein; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; MPO, myeloperoxidase; LPS, lipopolysaccharide; SOD, superoxide dismutase; GPx, glutathione peroxidase; Hb[A1.sub.c], glycohemoglobin; GFR, glomerular filtration rate; 5-LO, 5-lipoxygenase.

JASON H.Y. WU, [1] NATALIE C. WARD, [1] ADELINE P. INDRAWAN, [1] CORAL-ANN ALMEIDA, [2] JONATHAN M. HODGSON, [1] JULIE M. PROUDFOOT, [1] IAN B. PUDDEY, [1] and KEVIN D. CROFT [1] *

[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.

* Address 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 61-5-9224-0246; e-mail kcroft@cyllene.uwa.edu.au.
Table 1. Baseline characteristics of the study participants. (a)

 [alpha]T Mixed
 group, tocopherol
 n = 18 group, n = 19

Age, years 64 (7) 58 (4)
Sex, male/female 13/5 12/7
Body mass index, kg/[m.sup.2] 29.2 (2.0) 27.7 (2.8)
Fasting plasma glucose, mmol/L 6.7 (2.4) 7.5 (3.0)
[HbA1.sub.c], % 6.4 (1.1) 6.6 (1.2)

Cholesterol, mmol/L
Total 4.71 (0.23) 4.70 (0.25)
LDL 2.67 (0.17) 2.64 (0.23)
HDL 1.27 (0.08) 1.36 (0.07)
Total triglycerides, mmol/L 1.70 (0.16) 1.51 (0.16)
Total leukocyte count, x [10.sup.9]/L 6.23 (0.35) 6.51 (0.35)
Absolute neutrophil count, x [10.sup.9]/L 3.52 (0.24) 3.88 (0.24)
Glomerular filtration rate, mL/min 107 (6.6) 106 (5.3)
Serum cystatin c, mg/L 0.84 (0.05) 0.82 (0.03)
Serum alpha tocopherol, [micro]mol/L (c) 33.9 (1.90) 32.0 (1.80)
Serum gamma tocopherol, [micro]mol/L (c) 2.2 (0.20) 2.2 (0.20)

 Placebo Pb
 group,
 n = 18

Age, years 62 (7) 0.025
Sex, male/female 16/2 0.192
Body mass index, kg/[m.sup.2] 27.6 (4.3) 0.250
Fasting plasma glucose, mmol/L 7.5 (2.6) 0.617
[HbA1.sub.c], % 6.7 (1.1) 0.706

Cholesterol, mmol/L
Total 4.62 (0.16) 0.947
LDL 2.63 (0.18) 0.988
HDL 1.35 (0.11) 0.694
Total triglycerides, mmol/L 1.38 (0.18) 0.412
Total leukocyte count, x [10.sup.9]/L 6.73 (0.31) 0.568
Absolute neutrophil count, x [10.sup.9]/L 3.86 (0.22) 0.485
Glomerular filtration rate, mL/min 107 (7.7) 0.994
Serum cystatin c, mg/L 0.84 (0.04) 0.901
Serum alpha tocopherol, [micro]mol/L (c) 31.9 (1.30) 0.636
Serum gamma tocopherol, [micro]mol/L (c) 2.1 (0.22) 0.855

(a) Values are mean (SE) for continuous variables.

(b) Group differences for continuous variables evaluated by ANOVA;
categorical variables by [chi square] test.

(c) Previously published data (17).

Table 2. Effect of treatment on [F.sub.2]-isoprostanes and
antioxidant enzyme activities. (a)

 Treatment

 [alpha]T group

Plasma [F.sub.2]-isoprostanes,
 pmol/L (b)
Baseline 1694 (1337-2145)
Postintervention 1306 (1040-1640) (c)

Urinary [F.sub.2]-isoprostanes,
 nmol/mol creatinine (b,e)
Baseline 227 (133-386)
Postintervention 171 (107-272)

Erythrocyte SOD activity,
 kU/g hemoglobin (f)
Baseline 8.59 (0.35)
Postintervention 9.60 (0.44)

Erythrocyte GPx activity,
 kU/ g hemoglobin (f)
Baseline 9.26 (0.65)
Postintervention 9.02 (0.54)

 Mixed tocopherol group

Plasma [F.sub.2]-isoprostanes,
 pmol/L (b)
Baseline 1467 (1109-1940)
Postintervention 1262 (952-1673) (d)

Urinary [F.sub.2]-isoprostanes,
 nmol/mol creatinine (b,e)
Baseline 201 (135-301)
Postintervention 232 (146-369)

Erythrocyte SOD activity,
 kU/g hemoglobin (f)
Baseline 8.80 (0.27)
Postintervention 9.63 (0.39)

Erythrocyte GPx activity,
 kU/ g hemoglobin (f)
Baseline 8.71 (0.41)
Postintervention 8.83 (0.36)

 Placebo group

Plasma [F.sub.2]-isoprostanes,
 pmol/L (b)
Baseline 1439 (1113-1859)
Postintervention 1572 (1221-2023)

Urinary [F.sub.2]-isoprostanes,
 nmol/mol creatinine (b,e)
Baseline 160 (115-224)
Postintervention 160 (111-231)

Erythrocyte SOD activity,
 kU/g hemoglobin (f)
Baseline 8.48 (0.37)
Postintervention 9.20 (0.44)

Erythrocyte GPx activity,
 kU/ g hemoglobin (f)
Baseline 9.95 (0.50)
Postintervention 8.97 (0.61)

(a) Data are presented as mean (SE) except where indicated. There
were no significant baseline differences for any of the variables
between groups by ANOVA. Significant P values presented are baseline
adjusted postintervention differences compared to the placebo group
analyzed using general linear models.

(b) Geometric mean (95% confidence interval).

(c) P <0.001.

(d) P = 0.001.

(e) Urinary [F.sub.2]-isoprostane values are expressed normalized to
creatinine excretion.

(f) Erythrocyte antioxidant enzyme activities are expressed as
unit/mg hemoglobin.

Table 3. Effect of treatment on inflammatory markers and eicosanoid
synthesis. (a)

 Treatment

 [alpha]T group Mixed tocopherol
 group

Plasma Hs-CRP, mg/L (b)
Baseline 1.64 (0.95-2.82) 1.60 (1.11-2.31)
Postintervention 1.66 (0.93-2.96) 1.39 (0.99-1.97)

Plasma IL-6, ng/L (b)
Baseline 1.98 (1.55-2.52) 1.31 (1.05-1.62)
Postintervention 1.65 (1.25-2.20) 1.36 (1.06-1.74)

Plasma TNF-[alpha], ng/L
Baseline 1.13 (0.06) 0.94 (0.05)
Postintervention 1.16 (0.06) 0.96 (0.05)

Plasma MCP-1, ng/L
Baseline 98.2 (6.7) 83.1 (4.9)
Postintervention 90.2 (6.1) 85.2 (4.0)

Blood MPO activity,
 kU/L blood (b,c)
Baseline 7.3 (5.1-10.3) 9.0 (6.5-12.5)
Postintervention 7.0 (5.3-9.3) 9.4 (6.4-13.8)
g/Lb

Whole blood [PGE.sub.2],
 [micro]g/L (b)
Baseline 10.64 (6.29-17.99) 9.64 (5.24-17.74)
Postintervention 11.58 (7.16-18.75) 6.81 (3.89-11.92)
6cellsd

Neutrophil total [LTB.sub.4],
 ng/[10.sup.6] cells (d)
Baseline 30.6 (1.9) 30.2 (2.3)
Postintervention 27.2 (1.6) 25.1 (2.3) (e)

 Placebo group

Plasma Hs-CRP, mg/L (b)
Baseline 1.40 (0.67-2.93)
Postintervention 1.20 (0.59-2.42)

Plasma IL-6, ng/L (b)
Baseline 1.60 (1.15-2.23)
Postintervention 1.75 (1.32-2.32)

Plasma TNF-[alpha], ng/L
Baseline 1.07 (0.08)
Postintervention 1.10 (0.06)

Plasma MCP-1, ng/L
Baseline 88.8 (7.0)
Postintervention 95.3 (6.7)

Blood MPO activity,
 kU/L blood (b,c)
Baseline 8.3 (6.4-10.8)
Postintervention 9.1 (6.8-12.2)
g/Lb

Whole blood [PGE.sub.2],
 [micro]g/L (b)
Baseline 11.37 (7.05-18.34)
Postintervention 8.16 (5.78-11.52)
6cellsd

Neutrophil total [LTB.sub.4],
 ng/[10.sup.6] cells (d)
Baseline 31.3 (1.7)
Postintervention 30.8 (1.4)

(a) Data are presented as mean (SE) except where indicated.
There were no significant baseline differences for any of the
variables between groups by ANOVA.

(b) Geometric mean (95% confidence interval).

(c) MPO activity is reported normalized per ml of blood, calculated
by multiplying MPO activity per neutrophil by the absolute neutrophil
count (22).

(d) Total [LTB.sub.4] is calculated as the sum of [LTB.sub.4],
20-hydroxy-[LTB.sub.4] (20-OH-[LTB.sub.4]), and 20-carboxy-[LTB.sub.4]
(20-COOH-[LTB.sub.4]) and is expressed per [10.sup.6] cells.

(e) Significant P values presented are baseline adjusted
postintervention differences compared to the placebo group analyzed
using general linear models: P = 0.02.
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Article Details
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Title Annotation:Nutrition
Author:Wu, Jason H.Y.; Ward, Natalie C.; Indrawan, Adeline P.; Almeida, Coral-Ann; Hodgson, Jonathan M.; Pr
Publication:Clinical Chemistry
Date:Mar 1, 2007
Words:6292
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