Pro-oxidant effect of α-tocopherol in patients with Type 2 Diabetes after an oral glucose tolerance test – a randomised controlled trial

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 [1, 2]. Increased susceptibility to oxidative DNA damage has been reported in type 2 diabetes [3, 4], 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 [6, 7]. 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].

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).

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 [12, 13] and increased DNA damage, attributed to α-tocopherol, in cultured cells has been described following an insult capable of generating reactive oxygen species (ROS) [14, 15]. 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 [17, 18]. In addition Cu,Zn-Superoxide dismutase has been reported to release free copper when it is oxidatively damaged [19, 20] 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.

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.

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.