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J Am Coll Cardiol, 2000; 36:94-102
© 2000 by the American College of Cardiology Foundation
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CLINICAL STUDIES: ENDOTHELIAL FUNCTION

Vitamin E supplementation improves endothelial function in type I diabetes mellitus: a randomized, placebo-controlled study

R. Andrew P. Skyrme-Jones, BSc, MB, ChB*,1, Richard C. O’Brien, MBBS, PhD{dagger}, Karen L. Berry, BSc* and Ian T. Meredith, MBBS, PhD*

* Centre for Heart and Chest Research, Monash Medical Centre and Monash University, Melbourne, Australia
{dagger} Department of Medicine, Monash Medical Centre and Monash University, Melbourne, Australia

Manuscript received August 20, 1999; revised manuscript received January 18, 2000, accepted March 27, 2000.

Reprint requests and correspondence: Dr. Ian T. Meredith, Cardiovascular Centre, Monash Medical Centre, 246 Clayton Road, Clayton, Melbourne, Victoria, 3168, Australia
ian.meredith{at}med.monash.edu.au


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
OBJECTIVES

We sought to determine, in a double-blind, placebo-controlled, randomized study, whether vitamin E supplementation (1,000 IU for three months) would improve impaired conduit and resistance vessel endothelial vasodilator function (EVF) and systemic arterial compliance (SAC) in type I diabetes mellitus (DM).

BACKGROUND

Oxidative stress is thought to be important in the pathogenesis of impaired EVF. Consistent with this hypothesis, we have recently shown that impaired EVF is related to low density lipoprotein (LDL) vitamin E content (VEC) in young subjects with type 1 DM.

METHODS

We assessed EVF in the brachial artery (using noninvasive ultrasound, flow-mediated vasodilation [FMD]; n = 41) and in the forearm resistance vessels (by flow responses to intrabrachial acetylcholine [ACh]; n = 21) and measured SAC (simultaneous aortic blood flow and carotid pressure measurements; n = 41) before and after active or placebo therapy.

RESULTS

The LDL VEC was increased by 127% after supplementation, resulting in a significant reduction in the oxidative susceptibility of LDL. There was no time-dependent change in FMD or in the response to ACh or SAC in the placebo group. A significant improvement in FMD (2.6 ± 0.6% to 7.0 ± 0.7%, p < 0.005) and the dose response to ACh (p < 0.05) were observed in those randomized to vitamin E therapy. Systemic arterial compliance was not affected by vitamin E (0.41 ± 0.03 vs. 0.49 ± 0.06 arbitrary compliance units, p = NS). The change in FMD was related to the change in LDL VEC (r = 0.42, p < 0.05) and the change in the oxidative susceptibility of LDL (r = 0.64, p < 0.0001).

CONCLUSIONS

Short-term daily oral supplementation with vitamin E improves EVF in both the conduit and resistance vessels of young subjects with type I DM.

Abbreviations and Acronyms
  ACh = acetylcholine
  ANOVA = analysis of variance
  DM = diabetes mellitus
  EVF = endothelial vasodilator function
  FMD = flow-mediated vasodilation
  LDL = low density lipoprotein
  NTG = nitroglycerin
  PKC = protein kinase C
  SAC = systemic arterial compliance
  VEC = vitamin E content

Impaired endothelial vasodilator function (EVF) of the conduit (1) and resistance (2–4) vessels, together with impaired arterial compliance (5,6), is a feature of type I diabetes mellitus (DM), even in the absence of clinically detectable complications. These and other abnormalities of the arterial circulation are promoted in part by lipid abnormalities (7), hyperglycemia (8) and hyperinsulinemia (9). In turn, these factors may produce their pathogenic effects by glycooxidation of proteins and lipids (10) and activation of the polyol pathway (11), with increased free radical production (10), as well as activation of protein kinase C (PKC) (12).

The observation that vitamin E can decrease the susceptibility of low density lipoprotein (LDL) to oxidation (13), quench free radicals and reduce activation of PKC (14), among a number of other actions, has raised the possibility of an antiatherogenic role for this lipid-soluble antioxidant. Indeed, consistent with this, epidemiologic studies have demonstrated an inverse relation between vitamin E and cardiovascular disease (15,16), as well as the severity of coronary artery disease (17).

We have previously shown that plasma and LDL vitamin E is reduced in young diabetic patients with impaired EVF and that there is a direct relation between LDL vitamin E and endothelial function in this group (18). Several (19–22), but not all (23), experimental studies have found that vitamin E supplementation can improve endothelial function in hypercholesterolemic and diabetic models. This effect of vitamin E has not been replicated in most clinical studies (24–26), including type II DM (27), although improvement in arterial compliance (28) and carotid intima– media thickness (29) has been noted. The effect of vitamin E supplementation on endothelial function in type I DM has not been assessed. However, we have previously shown that LDL vitamin E content (VEC) is reduced in type I DM (18) and that EVF in the forearm is related to LDL VEC (18). This has also recently been demonstrated in the coronary circulation (30). In view of our findings, we undertook a randomized, placebo-controlled trial in young patients with type I DM to examine the effect of three months of therapy with oral vitamin E (1,000 IU) on conduit and resistance vessel EVF and systemic arterial compliance (SAC), as this may in part be endothelium-dependent.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Subjects.   Forty-one subjects with type I DM were recruited by advertisement and participated in this study, which was approved by the Human Research Ethics Committee of Monash Medical Centre. All subjects gave written, informed consent. None of the subjects were taking any vasoactive medications. All of the diabetics were treated with insulin. The average daily total insulin dose was 70 ± 27 U (mean ± SD). The duration of disease was on average 110 ± 75 months (mean ± SD). None of the diabetics had clinical evidence of hypertension, retinopathy or neuropathy, and albumin excretion rates were normal (<20 µg/min) in all of them.

Experimental design.   The 41 diabetic subjects were randomized in a double-blind manner to receive three months of treatment with 1,000 IU/day of oral vitamin E (all-rac-alpha-tocopherol) (Hellay Laboratories, Victoria, Australia) or placebo (Hellay Laboratories, Victoria, Australia). Twenty-one subjects were randomized to receive placebo and 20 to active therapy. All diabetic subjects had flow-mediated vasodilation (FMD), brachial artery responses to nitroglycerin (NTG) and SAC measured before and after therapy. In addition, a subset of 20 diabetics (10 on placebo and 10 on active therapy) underwent studies of the resistance vessels using venous occlusion plethysmography before and after three months of therapy. The subjects were reviewed at six weeks to check glycemic control and to monitor for any adverse events.

General procedure.   Subjects attended the laboratory in fasted condition, having refrained from aspirin and nonsteroidal anti-inflammatory drugs for at least five days before the study and caffeine-containing beverages for 12 h, as described previously (2). Blood glucose levels were documented at the time of brachial artery ultrasound and twice during venous occlusion plethysmography (Table 1).


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  		Table 1 Baseline Morphometric Characteristics and Glycemic Control of the Diabetic Subgroups Randomized to Placebo or Vitamin E Therapy

 
Brachial artery ultrasound.   Endothelium-dependent and -independent vasodilation was assessed in the brachial artery using transcutaneous ultrasound, as described by our group previously (31), using a high resolution ultrasound machine (ATL, HDI Ultramark 9, Seattle, Washington) with a 7- to 10-MHz linear-array transducer. Endothelium-dependent FMD was assessed as the percent change in arterial diameter in response to reactive hyperemia associated with 5 min of ischemia. Endothelium-independent vasodilation was assessed in response to a single tablet of sublingual NTG.

Venous occlusion plethysmography.   Forearm blood flow (milliliters per 100 ml forearm tissue per min) measurement was achieved by this well-validated technique (32), as described by this group previously (2,33). A 20-gauge, 5-cm polyethylene catheter (Cook, Brisbane, Australia) was introduced into the brachial artery of the nondominant forearm under local anesthesia under aseptic conditions. The arterial line was used for on-line measurement of blood pressure and for direct intra-arterial drug infusions.

Drug infusion protocol
All drugs were infused for 3 min at a rate of 0.4 ml/min before commencing measurement of forearm blood flow. Forearm blood flow responses were then measured continuously for 2 min while continuing infusion of each dose of drug. Endothelium-dependent vasodilation was assessed in response to acetylcholine (ACh) chloride (Miochol, Iolab Pharmaceuticals, Sydney, Australia) at doses of 2.7, 9 and 27 µg/min cumulatively. Endothelium-independent vasodilation was assessed in response to sodium nitroprusside (Faulding, Melbourne, Australia) at a dose of 9 µg/min.

Systemic arterial compliance.   The assessment of SAC was carried out noninvasively using the area method of calculation as described by Liu et al. (34) and previously by our group (6). This technique relies on the simultaneous measurement of aortic flow volume and associated driving pressure, together with aortic root dimensions. Systemic arterial compliance is then calculated from the following formula: SAC = Ad/(R[Ps – Pd]), where Ad = diastolic area; R = total peripheral resistance; and Ps and Pd = end-systolic and end-diastolic blood pressure, respectively (6,35). Flow velocity of the ascending aorta was measured from the suprasternal notch using a continuous wave hand-held Doppler flow velocimeter (Multi Dopplex II, Huntleigh Technology, Cardiff, United Kingdom) (6,35). Aortic root dimensions were assessed by two-dimensional echocardiography (ATL, HDI Ultramark 9, Seattle, Washington), with measurements taken from the point of insertion of the aortic valve leaflets. Aortic root pressure was estimated by noninvasive applanation tonometry of the right carotid artery using a Millar Mikro-Tip pressure transducer (model SSD-713, Millar Instruments, Houston, Texas).

Biochemical techniques.   Isolation of LDL and assessment of LDL particle size
Using density gradient ultracentrifugation, LDL cholesterol was isolated from ethylenediamine tetra-acetic acid (EDTA) plasma, as previously described (36). The LDL particle diameter was assessed by nondenaturing polyacrylamide gradient gel electrophoresis (Gradipore, N.S.W., Australia), as previously described (37).

Assessment of plasma and LDL VEC
The measurement of plasma vitamin E was performed using reverse phase high performance liquid chromatography using an ultraviolet absorption detector, as described previously (38). The VEC of LDL was measured on LDL previously isolated by ultracentrifugation, using high performance liquid chromatography with a Spherisorb ODS-2, 5-µm analytic column (Alltech Associates, Baulkham Hills, NSW, Australia) and with standard purchased from Sigma Chemicals (St. Louis, Missouri) and expressed as a ratio of LDL vitamin E to LDL cholesterol in µmol/mmol LDL.

Assessment of LDL oxidative susceptibility
Low density lipoprotein was isolated by density gradient ultracentrifugation as described earlier. Assessment of oxidative susceptibility was performed using a technique originally described by Esterbauer et al. (39) and modified by McDowell et al. (40). Copper (5 µmol) was added to isolated LDL diluted to 150 µmol/liter cholesterol in phosphate-buffered saline. After the generation of products resulting from the oxidation of lipids, absorbance was measured at 234 nm. The lag time was calculated as the time intercept between the line of maximal slope of the propagation phase of this reaction and the baseline absorbance at time = 0.

Power calculation.   On the basis of previous interventional studies using FMD, we performed a power calculation expecting to observe a 4% to 5% difference in FMD, resulting from active treatment. Thus, with 90% power at p < 0.01, we calculated that between 16 and 20 patients would be required in each of the placebo and active therapy subgroups.

Statistical analysis.   The clinical characteristics are expressed as the mean value ± SD. Data are expressed as the mean value ± SEM. Statistical analysis was performed using the Statview 4.5 software program. The differences between groups, with respect to clinical characteristics, FMD, NTG-induced dilation and SAC at baseline, were analyzed using the unpaired t test. Two-way repeated measures analysis of variance (ANOVA) was used to compare the effects of treatment (vitamin E or placebo) on FMD, NTG, SAC and the vasodilator response to sodium nitroprusside and ACh with post hoc testing using the Scheffé F test. The data for which ANOVA was used are presented in each case as follows: p value for treatment by period interaction effect, p value for treatment effect and p value for period effect. Linear regression analyses were performed to examine the relations between the change in oxidative susceptibility of LDL, LDL VEC and physiologic variables, FMD, the response to ACh and arterial compliance. Statistical significance was accepted at p < 0.05.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Baseline biochemical, clinical and morphometric characteristics.   Baseline clinical, morphometric and biochemical characteristics of the diabetic subgroups randomized to placebo or vitamin E are shown in Tables 1 and 2. There were no significant differences in these variables between the two groups, with glycosylated hemoglobin levels indicating moderate glycemic control. There was no significant difference in the mean lipid levels of the diabetic group and a matched group of healthy control subjects from our laboratory, although six of these subjects had a cholesterol level >5.5 mmol/liter but were not on lipid-lowering therapy (equal numbers in placebo and vitamin E groups). In addition, four diabetics smoked (equal numbers in placebo and vitamin E groups). Plasma vitamin E was not different at baseline between the placebo and vitamin E–treated subgroups. After active therapy, plasma vitamin E increased by 105%, from 19.7 ± 1.4 to 40.3 ± 3.9 µmol/liter. This was accompanied by a 127% increase in LDL VEC, from 2.2 ± 0.2 to 5.0 ± 0.3 µmol/mmol (Fig. 1). Vitamin E supplementation did not affect any lipid or glycemic variables.


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  		Table 2 Lipid Profiles and Vitamin E Concentrations Before Treatment in the Placebo and Active Therapy Subgroups

 


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  		Figure 1 a, Plasma VEC in placebo subgroup (striped bars at baseline; solid bar after treatment) and vitamin E–treated subgroup (open bar at baseline; solid bar after treatment). b, Low density lipoprotein VEC in placebo subgroup (striped bars at baseline; solid bar after treatment) and vitamin E–treated subgroup (open bar at baseline; solid bar after treatment).

 
Conduit vessel responses.   Endothelium-dependent vasodilation in the brachial artery (FMD)
Baseline brachial artery diameter and FMD were not different between the placebo and active therapy diabetic subgroups (4.3 ± 0.2 vs. 4.2 ± 0.1 mm, p = NS and 2.4 ± 0.7% vs. 2.6 ± 0.6%, p = NS, respectively) (Fig 2a). There was no time-dependent change in FMD in the diabetic subgroup randomized to placebo therapy (2.4 ± 0.7% before therapy vs. 2.5 ± 0.6% after therapy, p = NS) (Fig. 2a). However, in the active therapy subgroup, there was a significant improvement in FMD in response to vitamin E supplementation (2.6 ± 0.6% vs. 7.0 ± 0.7%; ANOVA: p < 0.0001 for treatment by period interaction effect, p < 0.0084 for treatment effect and p < 0.0001 for period effect).



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  		Figure 2 a, Flow-mediated dilation in placebo subgroup (striped bars at baseline; solid bar after treatment) and vitamin E–treated subgroup (open bar at baseline; solid bar after treatment). Flow-mediated dilation was not altered in the placebo group, but showed a significant increase after vitamin E therapy. b, Nitroglycerin-induced vasodilation. After vitamin E supplementation, there was a trend toward an improvement in the NTG response.

 
For the study cohort as a whole, baseline FMD was a function of LDL VEC (r = 0.38, p < 0.05), but not plasma vitamin E. Furthermore, there was a correlation between the change in FMD at the end of three months of treatment and the change in LDL VEC (r = 0.42, p < 0.05) (Fig. 3a).



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  		Figure 3 a, Relation between the change ({Delta}) in FMD and the change in LDL VEC at follow-up in the cohort. As the {Delta}LDL vitamin E increases, so does the magnitude of {Delta}FMD (r = 0.42, p < 0.05). b, Relation between {Delta}FMD and the change in lag time to oxidation of LDL at follow-up. Reduced oxidative susceptibility after vitamin E supplementation is associated with improved FMD (r = 0.64, p < 0.0001).

 
There was no relation between FMD after vitamin E therapy and LDL particle size or other variables of the lipid profile. Ambient glucose at the time of FMD measurement was similar both before and after treatment in both the active therapy and placebo subgroups.

Endothelium-independent vasodilation in the brachial artery (NTG)
There was no difference in baseline brachial artery diameter before the assessment of NTG between the placebo and active therapy subgroups (4.3 ± 0.2 vs. 4.2 ± 0.2 mm, p = NS). There was no time-dependent effect on the response to NTG in the placebo subgroup (13.8 ± 1.3% vs. 13.6 ± 1.2%, p = NS) (Fig. 2b). In the subgroup receiving active therapy, there was a trend toward an increase in NTG after treatment (13.2 ± 1.3% vs. 16.2 ± 1.1%; ANOVA: p = 0.08 for treatment by period interaction effect, p = 0.62 for treatment effect and p = 0.16 for period effect (Fig. 2b). There was no relation between NTG and LDL VEC or LDL particle size.

Resistance vessel responses.   Rest forearm blood flow
There was no time-dependent change in basal flow in either the placebo subgroup (2.8 ± 0.5 vs. 2.7 ± 0.3 ml/min per 100 ml forearm tissue; p = NS by ANOVA) and no effect of vitamin E on rest flow (2.8 ± 0.5 vs. 2.7 ± 0.3 ml/min per 100 ml forearm tissue; p = NS by ANOVA).

Endothelium-dependent vasodilation
Intra-arterial infusion of ACh produced a dose-dependent increase in forearm blood flow in both diabetic subgroups (p < 0.005). There was no difference in the response to the graded infusion of ACh between the two subgroups before treatment. There was no time-dependent change in the dose-response to ACh in the placebo subgroup (Fig. 4). However, there was augmentation in the responses to ACh in the active vitamin E–treated subgroup (ANOVA: p = 0.04 for treatment by period interaction effect, p < 0.01 for treatment effect, and p < 0.0001 for period effect). At the highest dose of ACh, the maximal vasodilator response increased from 9.6 ± 1.3 to 15.3 ± 1.2 ml/min per 100 ml forearm tissue (p < 0.0005) in the active therapy subgroup. The slope of the dose-response curve increased from 0.30 ± 0.04 to 0.46 ± 0.04 (p < 0.0005).



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  		Figure 4 Absolute forearm blood flow (a) and absolute forearm vascular resistance (b) during graded intra-arterial infusion of ACh before (open circles) and after (solid circles) therapy with vitamin E and before (open squares) and after (solid squares) placebo. Absolute forearm blood flow and vascular resistance were augmented after vitamin E therapy, but there was no change in the response in the placebo group (ANOVA, p < 0.05).

 
Endothelium-independent vasodilation
There was no difference in the baseline response to sodium nitroprusside between the diabetic subgroups randomized to active therapy or placebo (4.2 ± 0.4 vs. 4.9 ± 0.8 ml/min per 100 ml forearm tissue; p = NS). After three months of placebo therapy, the endothelium-independent response to sodium nitroprusside was not different (4.2 ± 1.3 vs. 3.6 ± 0.6 ml/min per 100 ml forearm tissue; p = NS). There was no difference in the magnitude of change in forearm blood flow between the placebo and active treatment subgroups after therapy (3.6 ± 0.2 vs. 3.7 ± 0.3 ml/min per 100 ml forearm tissue; p = NS).

Systemic arterial compliance.   Systolic, diastolic, pulse, mean arterial pressure and heart rate were similar in the two diabetic subgroups. There was no difference in SAC between the two groups before treatment (0.41 ± 0.03 vs. 0.46 ± 0.04 acu, p = NS). There was no time-dependent change in SAC in the placebo subgroup (0.41 ± 0.04 before placebo vs. 0.47 ± 0.04 acu after placebo; p = NS by ANOVA). Furthermore, SAC was not altered by active therapy with vitamin E (0.46 ± 0.04 vs. 0.53 ± 0.06 arbitrary compliance units; p = NS by ANOVA).

Oxidative susceptibility of LDL and endothelial vasodilator function.   The lag time to oxidation of LDL was similar at baseline in the placebo and vitamin E subgroups (76.2 ± 2.8 vs. 76.7 ± 2.8 min, p = NS). There was no time-dependent change in oxidative susceptibility of LDL in the placebo subgroup (76.2 ± 2.8 vs. 74.7 ± 2.9 min, p = NS). After three months of vitamin E therapy, the lag time to oxidation of LDL increased by 28% (76.7 ± 2.8 vs. 98.2 ± 4.2 min; ANOVA: p < 0.0001 for treatment by period interaction effect, p < 0.05 for treatment effect and p < 0.0001 for period effect). The change in oxidative susceptibility of LDL was related to LDL VEC (r = 0.57, p < 0.001). In turn, the change in FMD was related to the change in oxidative susceptibility of LDL after either placebo administration or treatment with vitamin E (r = 0.64, p < 0.0001) (Fig. 3b).


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
We have previously shown that plasma vitamin E and LDL VEC are reduced in young subjects with type I DM and that LDL VEC correlates with the integrity of endothelial function (18). We therefore hypothesized that vitamin E supplementation might restore endothelial function in this group. A double-blind, placebo-controlled study design was used to assess the effect of vitamin E on 1) EVF in both the conduit and resistance vessels; and 2) SAC in a group of young subjects with type I DM and no evidence of macrovascular or microvascular disease. We observed that short-term (three months) treatment with 1,000 IU of oral all-rac-alpha-tocopherol improves conduit and resistance vessel EVF, but not SAC, in type I DM. Responses to FMD and the graded intrabrachial infusions of the endothelium-dependent vasodilator ACh were improved in the diabetic subgroup randomized to active therapy. Furthermore, the change in FMD after treatment was directly related to the change in LDL VEC and the change in the oxidative susceptibility of LDL.

There were no differences in the baseline characteristics between the diabetic groups randomized to placebo or active therapy, indicating successful randomization into two comparable groups. Importantly, we observed no time-dependent effects on vascular reactivity or biochemical measurements in the placebo group, thus providing a stable set of subjects with which to compare the effects of vitamin E supplementation.

The aim of supplementation with vitamin E was to increase LDL VEC levels and thereby reduce the oxidative susceptibility of LDL. One thousand IU of vitamin E was specifically chosen to at least double LDL VEC. This was achieved with a 127% increase and a corresponding 28% increase in the lag time to oxidation of LDL. The change in lag time of LDL was directly related to the change in LDL VEC.

Previous studies.   Vitamin E has been shown to improve endothelium-dependent relaxation in experimental models of diabetes (19,20). To the best of our knowledge, this is the first study in diabetic subjects to demonstrate improved endothelial function with this antioxidant, although a recent study showed no benefit from eight weeks of vitamin E therapy (1,600 IU) in subjects with type II DM. Intra-arterial infusion of the water-soluble antioxidant vitamin C, however, has been shown to improve resistance vessel endothelial function in subjects with both type I (41) and type II DM (42), suggesting that oxygen-derived free radicals contribute to abnormal vascular reactivity in diabetes, probably by quenching nitric oxide. Our study is the first to investigate the effect of vitamin E therapy on endothelial function and arterial compliance in subjects with type I DM, and it highlights the fact that impaired EVF can be improved in a relatively short period with appropriate interventions.

Mechanisms of action of vitamin E.   The improvement in EVF observed in this study appears to be related to an improvement in the oxidative susceptibility of LDL, in view of the direct relation between these two variables. Consistent with this, a relation between improvement in EVF and a reduction in measures of oxidation of LDL has been observed in other studies after vitamin E supplementation (43–45). There is evidence that a reduction in oxidized LDL would be expected to increase nitric oxide bioavailability (46–48) and thus improve endothelium-dependent vasodilation.

Activation of PKC occurs in diabetes (14) as a result of hyperglycemia (49) and oxidized LDL (50). This activation of PKC also reduces nitric oxide bioavailability through a number of mechanisms, including downregulation of nitric oxide synthase activity (51). The fact that vitamin E is an efficient inhibitor of PKC (50) provides a further mechanism by which this compound may improve EVF, which is distinct from its antioxidant action.

Long-term administration of vitamin E has been shown to improve insulin sensitivity (52), which itself may improve endothelial function. In this study, we did not measure insulin action, but there was no effect of vitamin E on glycemic control subjects, as measured by glycosylated hemoglobin levels. Moreover, the effect of vitamin E cannot be simply explained on the basis of a change in the lipid profile per se, as has been seen in animal models. Total, LDL and high density lipoprotein cholesterol and LDL particle size were not affected by vitamin E, and are therefore unlikely to account for the improvement in endothelial function we observed. Vitamin E also inhibits leukocyte adhesion to endothelial cells (53) by inhibiting the expression of adhesion molecules (54), as well as monocyte transmigration (55). These multiple effects on the vascular system probably underlie the epidemiologic (15,16) and clinical studies (17,56) demonstrating a benefit with regard to vascular disease from vitamin E.

Nitroglycerin response.   We observed a trend toward an improvement in the brachial artery response to sublingual NTG in the diabetic subgroup randomized to active therapy. Consistent with this, a recent study in hypercholesterolemic subjects demonstrated an improvement in endothelium-independent vasodilation after vitamin E therapy (57). The response to NTG is partly flow-mediated, consequent on some increase in forearm blood flow. Thus, restoration of FMD might also improve the vasodilator response to NTG. Nitroglycerin is, of course, biotransformed to nitric oxide. The brachial artery responses to NTG may also have improved as a result of the effects of vitamin E on the cyclic guanosine monophosphate signal cascade in smooth muscle cells or simply through reduced deactivation of exogenous nitric oxide.

Improvement in the NTG response may have been more apparent if we had studied a larger number of diabetic subjects. We therefore cannot be certain that vitamin E did not cause a more generalized improvement in vascular reactivity which accompanied an improvement in endothelium-dependent relaxation, although there was no improvement in the response to sodium nitroprusside in the resistance circulation.

Systemic arterial compliance.   We and other investigators have previously documented a reduction in SAC in subjects with type I DM (5,6). This is most likely due to a combination of genetic, metabolic and hormonal alterations (58) leading to structural alterations of the arterial wall. However, there is some evidence that the endothelium, through the generation of vasoactive mediators such as nitric oxide or endothelin, may influence arterial stiffness (59). Moreover, data in older subjects without vascular disease suggest that vitamin E can improve impaired arterial compliance (28). This effect might be the result of improved EVF, a reduction in activated PKC (60) or perhaps an effect on vascular smooth muscle cell proliferation (61). In this study, however, we did not observe any change in SAC in patients randomized to active therapy. This suggests that endothelium-dependent vascular reactivity may be altered more readily by vitamin E, as compared with abnormalities of the arterial wall structure in early diabetic vascular disease.

Discrepancies in response to vitamin E.   The effect of vitamin E therapy on EVF has been studied in hypercholesterolemic patients, with some (57,62) but not all (24–26) studies showing a beneficial effect. The lack of consistency in the results with hypercholesterolemic patients may possibly be explained by concomitant lipid-lowering therapy (26), combination therapy with antioxidants (24), lower doses of vitamin E (25), a shorter duration of treatment (24–26), smaller numbers of patients (63) or more advanced disease (25). Differences in the pathophysiology of vascular dysfunction in type I DM and hypercholesterolemia may be another alternative to explain the inconsistency in the results of antioxidant studies. In this regard, activation of PKC in diabetes might be expected to promote the development of vascular disease to a greater extent than in hypercholesterolemia. Reversal of PKC activation by vitamin E (14) could therefore have a greater effect on vascular reactivity in diabetic subjects. It is not clear, however, why no benefit from vitamin E therapy was seen in subjects with type II DM recently, although a shorter duration of therapy, older subjects, a different dose of vitamin E or higher baseline levels of vitamin E may be relevant (27).

Potential limitations.   It is possible that we would have seen an increase in the vasodilator response to sodium nitroprusside after vitamin E therapy if we had used more than one dose. For this reason, a nonspecific improvement in vascular function in the resistance circulation cannot be excluded, as is the case in the brachial artery.

Concern has been raised over potential pro-oxidant effects of vitamin E (64), which could be an explanation for some of the negative findings in subjects with hypercholesterolemia. However, a key study in a rabbit model, which further raised these concerns, demonstrated adverse effects on endothelial function at much higher comparative doses than those used in our study (22). It is also notable that one of the studies in hypercholesterolemic subjects which did show a benefit with vitamin E also used a dose of 1,000 IU/day (62). This suggests that vitamin E at this dose is unlikely to have physiologically important pro-oxidant effects in humans.

Vitamin E is a generic term that includes all entities that exhibit the biologic activity of alpha-tocopherol. In nature, eight substances (comprising the tocopherols and tocotrienols) exhibit biologic activity. d-Alpha-tocopherol is the predominant form and has the highest biopotency. Synthetic vitamin E may be a mixture of stereoisomers of alpha-tocopherol, but is usually either d-alpha-tocopherol or all-rac-alpha-tocopherol. In this study, we used all-rac-alpha-tocopherol. There has been controversy in the published data regarding the relative biologic potencies of these stereoisomers. This may have considerable relevance with respect to the conflicting results of epidemiologic and interventional studies that relate to natural or synthetic vitamin E. Thus, possible differences in the biopotency of vitamin E need to be borne in mind when interpreting study results. It is possible in this study that a different stereoisomer may have improved endothelium-independent vasodilation to a greater extent or may have improved arterial compliance. This requires further study.

Conclusions.   We have shown, for the first time to our knowledge, in a randomized, double-blind, placebo-controlled study, that vitamin E therapy can improve both conduit and resistance vessel EVF in young subjects with type I DM in a relatively short period. Moreover, this improvement in EVF was directly related to increased LDL VEC and reduced oxidative susceptibility of LDL. Systemic arterial compliance, however, was not affected. These findings suggest that vitamin E may have a role in the management of early vascular disease in subjects with type I DM.


   Acknowledgments
 
We thank the doctors in the Young Adult Diabetic Clinic at Monash Medical Centre for permission to study their patients. We also thank all of the diabetic and control subjects for their time and effort toward completion of this study.


   Footnotes
 
This study was supported by a grant from Diabetes Australia Research Trust.

1 Dr. Skyrme-Jones is supported by a medical postgraduate research scholarship from the Cardiac Society of Australia and New Zealand. Back


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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