Pro-oxidant effect of α-tocopherol in patients with Type 2 Diabetes after an oral glucose tolerance test

1475-2840-6-8 1475-2840 Original investigation Pro-oxidant effect of α-tocopherol in patients with Type 2 Diabetes after an oral glucose tolerance test – a randomised controlled trial Winterbone S Mark mark.winterbone@bbsrc.ac.uk Sampson J Mike mike.sampson@nnuh.nhs.uk Saha Shikha shikha.saha@bbsrc.ac.uk Hughes C Jackie jackie.brown@bbsrc.ac.uk Hughes A David davida.hughes@bbsrc.ac.uk

Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, UK

Bertram Diabetes Research Unit, Norfolk & Norwich University Hospital NHS Trust, Norwich, NR4 7UY, UK

Cardiovascular Diabetology 1475-2840 2007 6 1 8 http://www.cardiab.com/content/6/1/8 1731642910.1186/1475-2840-6-8 19 2 2007 22 2 2007 22 2 2007 2007 Winterbone et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

As a part of a larger study investigating the effects of α-tocopherol on gene expression in type 2 diabetics we observed a pro-oxidant effect of α-tocopherol which we believe may be useful in interpreting outcomes of large intervention trials of α-tocopherol.

Methods

19 type 2 diabetes subjects were randomised into two groups taking either 1200 IU/day of α-tocopherol or a matched placebo for 4 weeks. On day 0 and 29 of this study oxidative DNA damage was assessed in mononuclear cells from fasted blood samples and following a 2 h glucose tolerance test (GTT).

Results

On day 0 there was no significant difference in oxidative DNA damage between the two groups or following a GTT. On day 29 there was no significant difference in oxidative DNA damage in fasted blood samples, however following a GTT there was a significant increase in oxidative DNA damage in the α-tocopherol treatment group.

Conclusion

High dose supplementation with α-tocopherol primes mononuclear cells from patients with type 2 diabetes for a potentially damaging response to acute hyperglycaemia.

Background

Type 2 diabetes is associated with an increased risk of atherosclerosis. Increased oxidative stress and damage to lipoproteins, cell membrane components and chromosomal DNA may play a role in this increased risk of atherosclerosis 12. Increased susceptibility to oxidative DNA damage has been reported in type 2 diabetes 34, and we have shown recently an inverse relationship between oxidative DNA damage and telomere length in blood monocytes from patients with type 2 diabetes 5. The potential role of oxidative stress in atherogenesis made antioxidant interventions appealing as a vascular risk reduction strategy, but there has subsequently been a lack of evidence of improved vascular outcomes in large scale antioxidant clinical trials 67. A recent meta-analysis has also suggested an increased risk of all-cause mortality from vitamin E supplementation 8. Potential reasons for this lack of benefit have been reviewed 9, as has the possible pro-oxidant effect of antioxidants in disease processes with existing high background levels of oxidative stress 10.

In this report we show an increase in oxidative DNA damage in mononuclear cells from patients with type 2 diabetes who had been supplemented with 1200 IU/d α-tocopherol, following a glucose tolerance test. This level of supplementation was chosen as we previously have shown no effect on DNA strand breaks or oxidisability with a lower dose of 400 IU α-tocopherol daily 3, but higher doses have shown a reduction in DNA single strand breaks using the comet assay 11.

Subjects and Methods

Subjects

All subjects gave written informed consent which was approved by the local ethics committee. We studied 19 subjects with type 2 diabetes, all Caucasian males between 50 and 65 years, who were recruited if they were non smokers, not taking dietary supplements, had never received gliclazide, antihypertensives, or angiotensin converting enzyme inhibitors, which have antioxidant or anti-inflammatory properties. Subjects were treated with diet alone (n = 5), metformin alone (n = 3), sulphonylureas alone (n = 4), metformin and sulphonylureas in combination (n = 3), and insulin alone or in combination with metformin (n = 4). Thirteen of the 19 subjects were taking an HMG CoA reductase inhibitor ('statin'). The volunteers were randomised into two groups, taking either 1200 IU α-tocopherol/d (n = 10) or matching placebo (n = 9) for 4 weeks. Compliance was monitored by pill count and plasma α-tocopherol concentrations. Table 1 summarises the clinical features of the two groups.

Table 1

Clinical and biochemical data groups in type 2 diabetes treated with 1200 IU α-tocopherol/day or placebo for 4 weeks

α-tocopherol Group (n = 10)

Placebo Group (n = 9)

Day 0

Day 29

Day 0

Day 29

Age (Y)

62.7 (1.81)

61.9 (1.92)

Diabetes duration

11.1 (2.52)

2.6 (0.4)^a

BMI

29.9 (1.2)

29.4 (1.3)

WHR

0.92 (0.026)

0.94 (0.021)

Fasting plasma glucose (mmol/L)

10.7 (0.94)

10.2 (0.86)

7.6 (0.39)^a

7.7 (0.41)^b

Fasting plasma insulin (mU/L)

49.6 (6.67)

56.2 (9.91)

55.5 (7.81)

54.5 (6.87)

Post-GTT plasma glucose (mmol/L)

20.2 (1.25)^c

19.1 (1.26)^c

15.1 (0.94)^c

15.0 (1.32)^c

Post-GTT plasma insulin (mU/L)

135 (37.1)^d

124 (31.9)^d

218 (47.7)^c

261 (87.0)^d

HbA1c (%)

8.4 (0.6)

7.2 (0.3)

Plasma α-tocopherol (μmol/L)

28.9 (1.82)

66.5 (7.26)^e

24.4 (1.71)

25.0 (1.78)

8-oxoquanine fluorescence (MFI)

462 (28.0)

456 (19.0)

506 (27.1)

495 (15.2)

Data shown are means (SE)

^ap < 0.01 placebo v α-tocopherol; ^bp < 0.05 placebo v α-tocopherol;

^cp < 0.01 Fasting v post-GTT; ^dp < 0.05 Fasting v post-GTT

^ep < 0.01 Day 0 v Day 29

Materials

On day 0 and day 29 of the study fasting blood samples were collected into vacutainer CPT tubes (Becton Dickinson, Oxford, UK). Volunteers were given a standard oral 75 g glucose tolerance test (GTT), and a further blood sample taken after 2 h. Mononuclear cells were separated by centrifugation. Oxidative DNA damage was assessed by measuring 8-oxoguanine (8-OG) using a Biotrin OxyDNA test kit (Biotrin International, Dublin, Ireland), as we have previously described 5. In brief, 1 × 10⁶mononuclear cells were incubated with 1% paraformaldeyde for 15 min on ice, washed once with PBS, resuspended with 70% ethanol and kept at -20°C until analysed. Cells were washed with PBS then incubated with blocking buffer at 37°C for 1 h, washed twice, then incubated with FITC-labelled 8-OG probe for 1 h. The cells were washed twice and analysed by flow cytometry. Plasma insulin was measured using a human insulin-specific (no cross-reactivity with proinsulin) ELISA (Dako Cytomation, Ely, UK) and glucose by the glucose oxidase method. Plasma α-tocopherol was measured by HPLC as previously described 3.

Data are expressed as mean and one standard error (SE) or 95% confidence intervals (CI). Differences between groups were analysed by paired or unpaired two-tailed t-tests and significance taken as p < 0.05.

Results

Both subject groups were matched for age, BMI and plasma insulin. However, by chance, the subjects allocated to the α-tocopherol treatment group had a significantly longer duration of diabetes (p < 0.01) and a higher fasting plasma glucose concentration than the placebo treatment group (p < 0.01).

Baseline data

At the start of the study no differences were apparent in DNA damage, as assessed by 8-OG mean fluorescence intensity (MFI), in the mononuclear cells of the two groups (Table 1).

Day 0, post GTT (GTT1)

No significant change in the level of DNA damage was detected following a GTT in the mononuclear cells of either group (fig 1).

Figure 1

Change in oxidative DNA damage, assessed by 8-oxoguanine fluorescence, in mononuclear cells isolated from type 2 diabetes patients before (GTT1) and after (GTT2) taking 1200 IU/day α-tocopherol or matched placebo for 4 weeks, following a glucose tolerance test. Data shown are means; error bars represent 95% confidence intervals. AT: α-tocopherol, GTT: Glucose Tolerance Test

Day 29, fasting sample

Following the 4 week intervention, plasma α-tocopherol was significantly increased (p < 0.01) in the α-tocopherol supplemented group but there was no significant change in mononuclear cell DNA damage in either treatment group (Table 1).

Day 29, post GTT (GTT2)

There was a significant increase in DNA damage, assessed as a 13.6% (95% CI: 6.3–20.9) increase in 8-OG fluorescence, in the mononuclear cells from the α-tocopherol supplemented group following GTT (Fig 1).

A correlation (R = 0.649, p = 0.045), using simple linear regression, was observed between duration of diabetes and the percent change in 8-OG florescence following a GTT in the α-tocopherol supplemented group on day 29.

Discussion

This study shows that after a relatively high dose of α-tocopherol for 4 weeks there was no change in oxidative DNA damage in mononuclear cells from subjects with type 2 diabetes, as we have reported previously 3. However, following a glucose load with increased oxidative stress 2 the level of oxidative DNA damage increased significantly in α-tocopherol-supplemented type 2 diabetes patients which correlated with the duration of diabetes. This increase in oxidative DNA damage was not apparent in the placebo group. These data suggest that the high dose α-tocopherol treatment has primed a damaging response to acute hyperglycaemia in type 2 diabetes and may be related to the duration of the disease.

The ability of α-tocopherol to act as a pro-oxidant and increase peroxidation of lipids has been long known in vitro 1213 and increased DNA damage, attributed to α-tocopherol, in cultured cells has been described following an insult capable of generating reactive oxygen species (ROS) 1415. Upon encountering ROS, α-tocopherol within lipid becomes oxidised forming its own radical, which requires co-antioxidants (e.g ascorbic acid) in order for the α-tocopherol to be regenerated. If the tocopherol radical is not eliminated there is an increase in lipid peroxidation, a process known as tocopherol-mediated peroxidation (TMP) 16. These peroxidised lipids can produce a range of ROS which are able to damage DNA. H₂O₂, generated by TMP, while not damaging to DNA directly, is able to cross membranes and can react with transition metals (Fe, Cu) associated with DNA to generate hydroxyl radicals (^•OH), by the Fenton reaction, to cause damage to DNA 1718. In addition Cu,Zn-Superoxide dismutase has been reported to release free copper when it is oxidatively damaged 1920 which could lead to increased generation of ^•OH.

Alpha-tocopherol has been reported to reduce γ-tocopherol concentrations in blood plasma 21 which could have a disadvantageous effect. Gamma-tocopherol is a potent scavenger of reactive nitrile species (RNS) such as nitric oxide (^•NO) and peroxynitrate (ONOO-) 22, which may also damage biomolecules such as DNA. Peroxynitrate can also oxidise tetrahydrobioptrein (BH4), a co-factor of nitric oxide synthase (NOS) which causes uncoupling of NOS, resulting in the generation of superoxide (^•O₂) instead of nitric oxide (^•NO) 23.

The timing of ingestion of supplements relative to meal times has been shown to effect markers of inflammation and may have an effect on oxidative stress 24, but the effects were seen following a single dose of vitamin E, whilst subjects on the current study, although only instructed to take supplements daily, had sustained elevated plasma levels of α-tocopherol. Other factors which may affect the outcomes of vitamin E supplementation have been discussed in a recent review 25.

High α-tocopherol intakes, at least without co-supplementation with other antioxidants such as vitamin C, which can reduce the α-tocopherol-induced lipid peroxidation observed in vitro 26, may result in amplification of ROS generated in response to an increase in oxidative stress and increased RNS due to suppression of γ-tocopherol bioavailability. High dose vitamin C, used in EDTA chelation therapy, has been shown to have a pro-oxidant effect 27 and ceruloplasmin, a copper containing metalloenzyme, has been suggested to have a pro-oxidant effect in conditions of increased oxidative stress, such as diabetes, by the disruption of copper binding 28. A recent report 29 has suggested that the optimal serum concentration of α-tocopherol to reduce mortality from cardiovascular disease and cancer is 30–33 μmol/L. The mean plasma α-tocopherol concentration achieved in the supplemented group in this study was 66.5 μmol/L which may be high enough to be detrimental rather than beneficial.

In a recent review of oxidative stress and antioxidant use, Johansen et al 30 point out that most clinical trials conducted to date were not designed to specifically assess the effects of antioxidant use in diabetic patients, who experience a high level of oxidative stress, and that endpoints measured did not include specific markers of oxidative stress. However, in the current study we measured levels of 8-oxoguanine, a specific marker of oxidative DNA damage formed during free radical damage to DNA.

Limitations

This study does have some limitations. First, the patient numbers are too low to form definitive conclusions. Second, the patients are taking different pharmacological treatments, although only subjects who had never received gliclazide, antihypertensives or ACE inhibitors, which have anti-oxidant or anti-inflammatory properties, were included in this study. Thirdly, there is a mismatch in duration of diabetes and plasma glucose between the two groups, which could affect the results. However HbA1c was not significantly different between each group and neither group differed significantly in their fasting levels of mononuclear cell oxidative damage either at the beginning or end of the study, nor showed any significant change following a GTT prior to supplementation.

Conclusion

This report is to our knowledge the first to show a pro-oxidant action of α-tocopherol associated with increased DNA damage in patients with type 2 diabetes. Although this is a small sample these preliminary findings, given the observed correlation between duration of diabetes and increase in oxidative damage, suggest the possibility that high dose vitamin E is potentially more damaging in patients with longer duration of disease and needs further investigation. This data may be useful in interpreting negative vascular outcomes in large α-tocopherol intervention trials in subjects with increased basal levels of oxidative stress such as type 2 diabetes or atherosclerosis.

Abbreviations

8-OG: 8-oxoguanine

AT: α-tocopherol

GTT: Glucose tolerance test

ROS: Reactive oxygen species

RNS: Reactive nitrile species

TMP: tocopherol-mediated peroxidation

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

MSW measured 8-oxoguanine fluorescence and drafted the manuscript

MJS and DAH conceived and planned the study

SS measured serum α-tocopherol levels

JCH performed immunoassays

Acknowledgements

This work was funded by a Core Strategic Grant from the Biotechnology & Biological Sciences Research Council, U.K. and the Norwich & Norfolk Diabetes Trust.

Oxidative stress and diabetic cardiovascular complications Jay D Hitomi H Griendling KK Free Radical Biology and Medicine 2006 40 183 192 10.1016/j.freeradbiomed.2005.06.018 Plasma F2 Isoprostanes: Direct evidence of increased free radical damage during acute hyperglycemia in type 2 diabetes Sampson MJ Gopaul N Davies IR Hughes DA Carrier MJ Diabetes Care 2002 25 537 541 Increased DNA oxidative susceptibility without increased plasma LDL oxidizability in Type II diabetes: effects of a-tocopherol supplementation M.J S S A S R G W I.R D DA H S S Clinical Science 2001 101 235 241 10.1042/CS20010112 Oxidative damage to DNA in diabetes mellitus Dandona P Thusu K Cook S Snyder B Makowski J Armstrong D Nicotera T The Lancet 1996 347 444 445 10.1016/S0140-6736(96)90013-6 Monocyte Telomere Shortening and Oxidative DNA Damage in Type 2 Diabetes Sampson MJ Winterbone MS Hughes JC Dozio N Hughes DA Diabetes Care 2006 29 283 289 10.2337/diacare.29.02.06.dc05-1715 Effects of Long-term Vitamin E Supplementation on Cardiovascular Events and Cancer: A Randomized Controlled Trial The HOPE and HOPE-TOO Trial Investigators JAMA 2005 293 1338 1347 10.1001/jama.293.11.1338 MRC/BHF Heart Protection Study of antioxidant vitamin supplementation in 20 536 high-risk individuals: a randomised placebo-controlled trial Heart Protection Study Collaborative G The Lancet 2002 360 23 33 10.1016/S0140-6736(02)09328-5 Meta-Analysis: High-Dosage Vitamin E Supplementation May Increase All-Cause Mortality Miller ER III Pastor-Barriuso R Dalal D Riemersma RA Appel LJ Guallar E Ann Intern Med 2005 142 37 46 Is the Oxidative Modification Hypothesis Relevant to Human Atherosclerosis?: Do the Antioxidant Trials Conducted to Date Refute the Hypothesis? Steinberg D Witztum JL Circulation 2002 105 2107 2111 10.1161/01.CIR.0000014762.06201.06 The antioxidant paradox Halliwell B The Lancet 2000 355 1179 1180 10.1016/S0140-6736(00)02075-4 Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation Sardas S Yilmaz M Oztok U Cakir N Karakaya AE Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2001 490 123 129 10.1016/S1383-5718(00)00157-1 Vitamin E in human low-density lipoprotein. When and how this antioxidant becomes a pro-oxidant. Bowry VM Igold S Stocker R Biochemical Journal 1992 288 341 344 Paradoxical actions of antioxidants in the oxidation of low density lipoprotein by peroxidases. Santanam N Parthasarathy S J Clin Invest 1995 95 2594 2600 http://www.pubmedcentral.com/articlerender.fcgi?artid=295942 Exacerbating Effect of Vitamin E Supplementation on DNA Damage Induced in Cultured Human Normal Fibroblasts by UVA Radiation. Nocentini S Guggiari M Rouillard D Surgis S Photochemistry and Photobiology 2001 73 370 377 10.1562/0031-8655(2001)073<0370:EEOVES>2.0.CO;2 Genotoxicity of idarubicin and its modulation by vitamins C and E and amifostine Blasiak J Gloc E Wozniak K Mlynarski W Stolarska M Skorski T Majsterek I Chemico-Biological Interactions 2002 140 1 18 10.1016/S0009-2797(02)00012-1 The ambivalence of vitamin E in atherogenesis Stocker R Trends in Biochemical Sciences 1999 24 219 223 10.1016/S0968-0004(99)01404-8 Tocopherol Induces Oxidative Damage to DNA in the Presence of Copper(II) Ions Yamashita N Murata M Inoue S Burkitt MJ Milne L Kawanishi S Chem Res Toxicol 1998 11 855 862 10.1021/tx970129v Oxidative Damage to DNA Constituents by Iron-mediated Fenton Reactions. THE DEOXYGUANOSINE FAMILY Henle ES Luo Y Gassmann W Linn S J Biol Chem 1996 271 21177 21186 10.1074/jbc.271.35.21167 Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme Sato K Akaike T Kohno M Ando M Maeda H J Biol Chem 1992 267 25371 25377 Copper, zinc superoxide dismutase enhances DNA damage and mutagenicity induced by cysteine/iron Yoon SJ Koh YH Floyd RA Park JW Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 2000 448 97 104 10.1016/S0027-5107(00)00005-1 How an Increased Intake of Alpha-Tocopherol Can Suppress the Bioavailability of Gamma-Tocopherol Wolf G Nutrition Reviews 2006 64 295 299 10.1301/nr.2006.jun.295-299 {gamma}-Tocopherol, the major form of vitamin E in the US diet, deserves more attention Jiang Q Christen S Shigenaga MK Ames BN Am J Clin Nutr 2001 74 714 722 Endothelial Regulation of Vasomotion in ApoE-Deficient Mice : Implications for Interactions Between Peroxynitrite and Tetrahydrobiopterin Laursen JB Somers M Kurz S McCann L Warnholtz A Freeman BA Tarpey M Fukai T Harrison DG Circulation 2001 103 1282 1288 Timing of Antioxidant Vitamin Ingestion Alters Postprandial Proatherogenic Serum Markers Carroll MF Schade DS Circulation 2003 108 24 31 10.1161/01.CIR.0000074221.68903.77 Vitamin E in Humans: An Explanation of Clincal Trial Failure. Robinson I de Serna DG Gutierrez A Schade DS Endocrine Practice 2007 12 576 582 Tocopherol-mediated peroxidation. The prooxidant effect of vitamin E on the radical-initiated oxidation of human low-density lipoprotein Bowry VW Stocker R Journal of the American Chemical Society 1993 115 6029 6044 10.1021/ja00067a019 Acute prooxidant effects of vitamin C in EDTA chelation therapy and long-term antioxidant benefits of therapy Hininger I Waters R Osman M Garrel C Fernholz K Roussel AM Anderson RA Free Radical Biology and Medicine 2005 38 1565 1570 10.1016/j.freeradbiomed.2005.02.016 Does oxidative stress change ceruloplasmin from a protective to a vasculopathic factor? Shukla N Maher J Masters J Angelini GD Jeremy JY Atherosclerosis 2006 187 238 250 10.1016/j.atherosclerosis.2005.11.035 Higher baseline serum concentrations of vitamin E are associated with lower total and cause-specific mortality in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study Wright ME Lawson KA Weinstein SJ Pietinen P Taylor PR Virtamo J Albanes D Am J Clin Nutr 2006 84 1200 1207 Oxidative stress and the use of antioxidants in diabetes: Linking basic science to clinical practice Johansen JS Harris AK Rychly DJ Adviye E Cardiovascular Diabetology 2005 4:5