Both antioxidant supplementation and exercise training have been identified as interventions which may reduce oxidative stress and thus improve cardiovascular health, but the interaction of these interventions on arterial BP (blood pressure) and vascular function has not been studied in older humans. Thus in six older (71±2 years) mildly hypertensive men, arterial BP was evaluated non-invasively at rest and during small muscle mass (knee-extensor) exercise with and without a pharmacological dose of oral antioxidants (vitamins C and E, and α-lipoic acid). The efficacy of the antioxidant intervention to decrease the plasma free radical concentration was verified via EPR (electron paramagnetic resonance) spectroscopy, while changes in endothelial function in response to exercise training and antioxidant administration were evaluated via FMD (flow-mediated vasodilation). Subjects were re-evaluated after a 6-week aerobic exercise training programme. Prior to training, acute antioxidant administration did not change resting arterial BP or FMD. Six weeks of knee-extensor exercise training reduced systolic BP (from 150±8 mmHg at pre-training to 138±3 mmHg at post-training) and diastolic BP (from 91±5 mmHg at pre-training to 79±3 mmHg at post-training), and improved FMD (1.5±1 to 4.9±1% for pre- and post-training respectively). However, antioxidant administration after exercise training negated these improvements, returning subjects to a hypertensive state and blunting training-induced improvements in FMD. In conclusion, the paradoxical effects of these interventions suggest a need for caution when exercise and acute antioxidant supplementation are combined in elderly mildly hypertensive individuals.
- blood pressure
- elderly population
- free radical
- nitric oxide
Maintaining a favourable balance between pro-oxidants and antioxidants becomes increasingly difficult with age, as free radicals generated from natural metabolic processes begin to overwhelm endogenous antioxidant defences, amounting to an increase in ‘oxidative stress’ that may contribute to the development of cardiovascular disease [1–3]. Both regular physical activity and antioxidant supplementation have been identified as potential non-invasive means of controlling oxidative stress with advancing age, although evidence for the efficacy of these interventions for disease prevention remains somewhat equivocal, apparently being highly dependent on intensity and dose [4–7].
Central to the exercise-training-induced improvement in vascular health is the regular increase in shear stress, which raises production of endothelial-derived NOS (NO synthase) and the subsequent release of NO, a potent vasodilator that is tightly linked to BP (blood pressure) control [8,9] and endothelial health . Somewhat paradoxically, acute exercise increases free radicals [11,12] with potentially detrimental vascular effects, as exercise-induced ROS (reactive oxygen species) are known to decrease NO bioavailability through the conversion of NO into ONOO− (peroxynitrite). However, when acute exercise is repeated as part of a regular exercise training regimen, activity and production of endogenous antioxidant enzymes, such as the SODs (superoxide dismutases), are up-regulated as an adaptation to restore pro- and anti-oxidant balance .
Like exercise training, oral antioxidant supplements are thought to evoke positive vascular effects, and some evidence exists for the ability of a pharmacological dose of vitamin C (ascorbic acid) to reduce vascular tone  and improve NO bioavailability . Although this decrease in free radical concentration appears beneficial for vascular health, it is noteworthy that the products of free radical generation such as H2O2 and ONOO− may also act as potent vasodilators [16,17] and even increase NO bioavailability . As such, any antioxidant intervention which decreases free radical levels will concomitantly protect NO levels, but reduce vasodilation initiated by other free radicals, the balance of which will ultimately determine the impact of the intervention on systemic vascular tone and arterial BP. Indeed, we have recently demonstrated the negative effect of acute oral antioxidant administration on exercise-induced vasodilation in younger individuals , suggesting that free radicals may be an important part of the vasodilatory response during exercise. However, whether an acute oral antioxidant intervention has a similar negative effect on the vasculature in a population with a propensity for elevated oxidative stress, such as the elderly, is unknown.
To our knowledge, the combined effect of acute exercise, acute antioxidant supplementation and exercise training on arterial BP has not been studied in older humans. Therefore we hypothesized: (i) that acute oral antioxidant supplementation would attenuate free radical levels, leading to an increase in NO bioavailability, which would reduce arterial BP and improve endothelial function in older subjects, both at rest and during acute exercise; (ii) that small muscle mass KE (knee-extensor) exercise training would also lead to a reduction in arterial BP and improved FMD (flow-mediated vasodilation); and (iii) superimposed upon the exercise-trained state, acute oral antioxidant supplementation would decrease arterial BP further and improve FMD responses.
MATERIALS AND METHODS
Subjects and general procedures
Six older (age, 71±2 years; height, 172±4 cm; weight, 79±2 kg) healthy men participated in the present study. These subjects were normally active, non-smokers, with Stage 1 (mild) hypertension . Subjects were screened using a standard medical history questionnaire, and those with multiple risk factors or those taking regular medication (including antioxidant supplements) were excluded. Thus all subjects were deemed to be in good overall cardiovascular health based upon health history. Ethical approval and written informed consent was obtained in accordance with the University of California San Diego Human Subjects Protection Program requirements and in compliance with the Declaration of Helsinki. Subjects visited the laboratory on seven occasions for testing: a preliminary visit, two visits (placebo and antioxidant) for determination of antioxidant efficacy, and four visits (placebo and antioxidant) for determination of cardiovascular responses to acute exercise prior to and following the exercise training programme. In addition, all subjects visited the laboratory three times each week for 6 weeks to complete the exercise training.
Acute antioxidant administration
All subjects received both the antioxidant cocktail and placebo in a double-blind, balanced, cross-over design. Supplements were taken in two doses separated by 30 min, with the first dose ingested 2 h before initiation of the experiment. The first dose consisted of 300 mg of α-lipoic acid, 500 mg of vitamin C and 200 I.U. (international units) of vitamin E, and the second dose was 300 mg of α-lipoic acid, 500 mg of vitamin C and 400 I.U. of vitamin E. Placebo microcrystalline cellulose capsules were of similar taste and appearance, and were likewise consumed in two doses. We have previously documented the efficacy of this antioxidant cocktail to acutely reduce plasma free radical concentration in young volunteers , and in the present study have determined efficacy in an elderly cohort (see Figure 1).
Subjects reported to the laboratory on a preliminary testing day to complete health histories, physical examinations and perform a WRmax (maximal work rate) test on the KE ergometer, as described previously .
Experimental days 1 and 2: antioxidant efficacy
Subjects reported to the laboratory in a fasted state approx. 2 h after consumption of either placebo or antioxidant cocktail. Venous blood samples from the antecubital fossa were collected at rest and within 5 s following the end of a graded maximal cycle exercise test (duration of 10–12 min). All subjects completed this exercise test on two days separated by 24 h, with and without antioxidant supplementation. Spin trapping and EPR (electron paramagnetic resonance) spectroscopy (see below) were performed on these blood samples to establish the efficacy of the antioxidant cocktail in reducing free radical load.
Experimental days 3–6
Subjects reported to the laboratory in a fasted state approx. 2 h after the consumption of either placebo or antioxidant cocktail. After 30 min of rest, the FMD procedure was performed. This was followed by KE exercise at 20, 40 and 60% of WRmax. All subjects crossed over, returning 24 h later to repeat the procedure after either placebo or antioxidant cocktail consumption, based upon the previous study. This procedure (FMD and KE exercise) was repeated following the 6-week exercise training programme (see below).
After the initial test days, subjects reported to the laboratory three times each week for 6 weeks to complete individually monitored single leg KE exercise training, which consisted of varied 1 h protocols (ranging from 30–90% of WRmax), a protocol which has previously resulted in a significant improvement in V̇O2max (maximal oxygen consumption) in young and old healthy subjects . The exercise regime combined short high-intensity (5–10 min at 70–95% of WRmax) intervals with longer low-intensity (15–45 min at 40–65% of WRmax) work bouts. Graded KE exercise tests were performed to re-evaluate WRmax after weeks 2, 4 and 6 of the 6-week training protocol, with relative training work rates adjusted as improvements in WRmax were achieved.
EPR was performed on venous blood samples. Briefly, 4.5 ml of venous blood was collected into a Vacutainer® that contained 1.5 ml of the spin-trap PBN (N-tert-butyl-α-phenylnitrone; 0.140 mol/l). After centrifugation, the PBN adduct was extracted from the serum supernatant with toluene, and the adduct (200 μl) was pipetted into a precision-bore quartz EPR sample tube (Wilmad) that had been flushed with compressed N2. EPR was performed at 21 °C in an EMX X-band spectrometer (Bruker) using commercially available software (version 2.11; Bruker Win EPR System), with data processing blinded to the experimental condition.
Arterial BP was measured using automated radial tonometry (Medwave Vasotrac APM205A; BioPac). This non-invasive BP device has been validated clinically and found to be well correlated with direct radial arterial measurements [24–26], and has proven reliable in studies from our group utilizing an experimental paradigm similar to the present study [27,28]. The arm was placed at heart level during both rest (supine) and exercise (semi-recumbent) trials, with one measurement made every 8–10 s. BP readings were thus taken several times over the course of 1 min and the mean values are reported. In the case of baseline, three or four readings were taken, whereas, during exercise two or three measurements were made during the last minute of each exercise intensity. The processing of the BP data was blinded to experimental condition.
Brachial artery vasodilation following ischaemic cuff occlusion was determined by ultrasound Doppler (Logiq 7; GE Medical Systems), as described previously [29,30]. Briefly, subjects were positioned supine and a pneumatic cuff was positioned on the arm below the elbow, distal to the site of the ultrasound Doppler probe. After baseline measurements were made, the arm cuff was inflated to suprasystolic pressure (>250 mmHg) for 5 min. Brachial artery diameter and blood velocity were measured at baseline and 70–90 s after cuff occlusion, when peak vasodilation was observed. All ultrasound Doppler measurements and analyses were performed by a single sonographer (blinded to the condition during analyses), who demonstrated equal or better reproducibility in comparison with manual measurements of vessel diameter published previously . Inter-day variability for FMD testing in our laboratory is approx. 2% (≈20% coefficient of variation) .
Statistics were performed with the use of commercially available software (SigmaStat 3.10; Systat Software). Paired Student t tests and two-way repeated measure analyses were performed, with the Bonferroni test used for post-hoc analysis when a significant main effect was found. All group data are expressed as means±S.E.M. Significance was established at P <0.05 for all tests.
A clear and characteristic ‘triplet of doublets’ EPR signal proportional to the free radical concentration was detected in all subjects at rest [6453±1097 AU (arbitrary units)] in the PBN spin-trapped venous blood, and this signal was significantly attenuated (1852±169 AU) after ingestion of the antioxidant cocktail (Figure 1). Maximal cycling exercise increased the concentration of the PBN adduct, revealing a significantly greater free radical signal (9804±1593 AU; Figure 1). As with rest, consumption of the antioxidant cocktail before exercise markedly attenuated the free radical signal following exercise (2928±83 AU; Figure 1). The dominant signal had hyperfine coupling constants of aN=13.7G and abH=1.9G, consistent with published values for an oxygen-centred alkoxyl species (PBN-LO•) using similar extraction solvents .
Antioxidants and arterial BP at rest and during exercise (pre-training)
Before exercise training, antioxidant supplementation did not significantly lower resting or exercising arterial BP, although a downward trend (P=0.07) in resting SBP (systolic BP) was apparent (Figure 2). Likewise, antioxidant supplementation did not significantly lower SBP, DBP (diastolic BP) or MAP (mean arterial BP) at any exercise intensity.
Antioxidants and arterial BP at rest and during exercise (post-training)
Six weeks of KE exercise training significantly reduced resting SBP (from 150±8 mm at pre-training to 138±3 mmHg at post-training) and DBP (from 91±5 mmHg at pre-training to 79±3 mmHg at post-training), with a clear trend towards a decrease in MAP (from 111±5 mmHg at pre-training to 99±3 mmHg at post-training; P=0.07) (Figure 2). Training did not significantly alter resting heart rate (60±2 and 61±3 beats/min for pre- and post-training respectively) or body weight. During acute exercise, SBP and MAP were significantly lower after exercise training, with no reduction in exercising DBP (Figure 3). After training, antioxidant administration increased resting SBP (+16±6 mmHg), DBP (+14±3 mmHg) and MAP (+15±4 mmHg) at rest (Figure 2), and this hypertensive effect was still evident during acute KE exercise (Figure 4).
Antioxidants and endothelial function
Prior to exercise training, FMD following 5 min of lower arm cuff occlusion was similar following consumption of placebo (0.54±0.03 to 0.55±0.02 mm; ≈1%) and antioxidant cocktail (0.54±0.02 to 0.55±0.02 mm; ≈1%) (Figure 5). Following 6 weeks of exercise training, FMD was significantly greater on the placebo day (0.54±0.02 to 0.57±0.02 mm; ≈5%), but this improve-ment was blunted after acute antioxidant administration (0.54±0.02 to 0.56±0.02 mm; ≈3%) (Figure 5). Hyperaemia following cuff release was not altered as a consequence of training or antioxidant consumption, and thus vasodilation was not normalized for shear rate . The effect of training on FMD in the elderly has been reported elsewhere , and is included here for comparison with the antioxidant intervention.
In the present study, we have identified for the first time a clinically significant paradoxical cardiovascular response to exercise training and antioxidant supplementation in the elderly. Small muscle mass exercise training resulted in a significant reduction in resting and exercising arterial BP and an improvement in endothelium-dependent FMD, confirming the efficacy of this low-stress non-invasive intervention on vascular health. Before exercise training, acute oral antioxidant administration did not significantly reduce arterial BP or influence FMD. In contrast, and contrary to our hypothesis, after exercise training, acute antioxidant administration increased arterial BP to pre-training values and significantly decreased FMD, transiently negating the beneficial effects of exercise training. This negative outcome following the combination of two potentially beneficial interventions emphasizes the complex nature of oxidative stress in vivo, where pro- and anti-oxidant forces interact to maintain appropriate vasomotor tone. These findings suggest that, although exercise training alone evokes an appropriate adaptation to the increase in oxidative stress, the acute decrease in free radical concentration following antioxidant administration may have removed ROS which possesses some beneficial vasoactive properties in the exercise-trained state.
Exercise, arterial BP and endothelial function
Cardiovascular adaptations to habitual whole-body exercise training have been well described, and include a significant reduction in resting and exercising arterial BP in both young and old subjects . However, few studies have evaluated adaptations to exercise training of a small muscle mass, which allows training of muscle groups in the leg without central cardiovascular limitations [35,36]. By employing the single leg KE exercise training paradigm in the present study, we have demonstrated a clinically significant reduction in both resting and exercising arterial BP following 6 weeks of small muscle mass training (Figures 2 and 3). In terms of arterial BP, these findings not only emphasize the importance of peripheral vascular adaptations as a result of habitual exercise, but also reveal that even a relatively low-stress small muscle mass exercise regime can provide a significant improvement in mildly hypertensive individuals.
Exercise training also improved brachial artery FMD (Figure 5), a response which is thought to be related to the training-induced increase in systemic NO bioavailability . Indeed, others have demonstrated that lower limb exercise alters the pattern of shear stress in nonexercising limbs, which may lead to improvement in systemic NO availability and augmented endothelial-mediated vasodilation in vessels outside the exercised limb [38,39]. Furthermore, although the present results do not address this possibility, improved FMD in the arm of leg-trained individuals may represent involvement of the nitrate/nitrite/NO pathway, a mechanism proposed to provide a circulating source of NO . Direct determination of circulating nitrite levels in the vasculature of active and non-exercised limbs, and the contribution of this pathway to augmentation of local NO bioavailability, represents an interesting prospect which awaits further study.
Clinically, these findings are of importance in terms of exercise adherence for the general population, which classically is poor when whole-body high-stress exercise is prescribed . Additionally, these observed cardiovascular benefits of a small muscle mass exercise modality may be of great benefit to patients with central cardiopulmonary limitations, such as COPD (chronic obstructive pulmonary disease) and CHF (congestive heart failure), where whole-body exercise prescription is limited by both disease symptoms and patient compliance [42,43].
Oxidative stress, arterial BP and endothelial function
Little consensus exists in the literature with regard to the efficacy of oral antioxidants on arterial BP in humans, with some support for , but the majority of evidence against [45–48], a significant hypotensive effect in healthy subjects. The reason for these equivocal results of oral antioxidant use is unclear, but may have been due to antioxidant dosing regimens that did not effectively reduced oxidative stress . Recent work from our group  has verified the efficacy of an oral antioxidant cocktail to increase plasma total antioxidant capacity and reduce plasma alkoxyl free radical concentration in young subjects by documenting a decrease in the EPR signal intensity of PBN adducts. The present EPR results extend these observations to an elderly cohort, verifying further the ability of this antioxidant cocktail to decrease oxidative stress both at rest and at the end of maximal exercise in older individuals (Figure 1). To our knowledge, the present study is the first in humans to evaluate changes in arterial BP and endothelial function with a pharmacological antioxidant dose directly proven to decrease plasma free radical content. Despite this marked decrease in oxidative stress, we observed only a tendency towards a reduction in resting arterial BP and no effect during acute exercise following oral antioxidant administration prior to exercise training (Figure 2).
Likewise, FMD was not changed by acute antioxidant administration prior to exercise training (Figure 5), suggesting that this decrease in oxidative stress does not affect vascular function as assessed by this experimental model of endothelial-mediated vasodilation. In contrast with the present findings, others have demonstrated that high-dose infusions of ascorbic acid acutely improves endothelium-dependent vasodilation in healthy elderly subjects  and hypertensive patients , which may be attributed to a decrease in plasma free radicals, such as O2− (superoxide anion), known to decrease NO bioavailability . This disparity may be explained by the relatively low antioxidant doses administered in the present study. By design, this dose was chosen to determine the efficacy of a typical over-the-counter oral supplement that clearly decreases alcoxyl and alkyl plasma free radicals (Figure 1). The present FMD results thus suggest that this antioxidant cocktail does not improve brachial artery reactivity, in agreement with other studies which have failed to identify the efficacy of long-term oral antioxidant supplementation to improve endothelial function [49,52,53].
Age, exercise training and oxidative stress: tipping the balance
Healthy aging is associated with an increase in free radical production  and progressively increasing arterial BP [55,56]. Others have identified exercise training and antioxidant supplementation as efficacious non-invasive interventions to improve cardiovascular health in older subjects [4,6]. Our present findings of a tendency for antioxidants to reduce MAP at rest (Figure 2), and the observed reduction in arterial BP (Figure 3) and improved FMD (Figure 5) following exercise training support these studies. However, contrary to our hypothesis, the combination of exercise training and acute oral antioxidant administration did not interact to produce an additive beneficial outcome, but in fact reversed the training-induced improvements in both resting and exercising arterial BP (Figures 2 and 3) and endothelial function (Figure 5).
By design, the present study focused upon the clinical outcome of the interventions and as such does not address the mechanisms responsible for the deleterious combined effect of exercise training and acute antioxidant administration on arterial BP and vascular function. Oxidative stress is typically regarded as an unwanted by-product of cellular oxidation and is seen as a negative risk factor for cellular and vascular health . However, the downstream consequences of free radicals, such as H2O2 and ROS (i.e. ONOO−), may act as potent vasodilators [16,17] and, as such, possess the capacity to affect vascular tone. Thus it is tempting to speculate that, although exercise training alone evokes an appropriate antioxidant adaptation to the increase in oxidative stress, the even greater decrease in free radical concentration following antioxidant administration may have removed oxidative species that possess some beneficial vasoactive properties.
Indeed, Peluso et al.  have demonstrated a reduction in arterial BP in rats following acute exposure to peroxyl radicals, which was reversed following subsequent exposure to acute antioxidants. Likewise, our group has recently identified the negative impact of acute antioxidant consumption (utilizing the same dose as in the present study) on brachial artery vasodilation during handgrip exercise , suggesting an important role of free radicals in promoting exercise-induced vasodilation. Thus, although oxidative stress remains an independent cardiovascular risk factor , from these studies it appears one must carefully consider the interaction of antioxidants and free radicals, the balance of which will apparently dictate achievement of optimal vascular function. Results from the present study support this concept, suggesting that multiple interventions which elevate antioxidant levels may ultimately tip this balance, resulting in detrimental changes in arterial BP regulation and endothelial function.
Any laboratory-based longitudinal exercise training study with multiple experimental days inherently limits sample size, but in return allows a tightly controlled and supervised intervention, the advantage of multiple measurements on the same individual, and effectively allows each subject to serve as their own control. In the present study, a sufficient number of subjects were enrolled to achieve adequate statistical power (β ≥ 0.8) in the major variables, while ensuring compliance of all subjects for the duration of the training regimen. Additionally, although the present study identified a significant treatment effect on arterial BP and FMD after training, we recognize the lack of results concerning the time course for changes in arterial BP and the long-term effect of the antioxidant treatment following exercise training. The experimental design of the present study did not include a time control group, due to the relatively short training period, which we contend minimized the risk of a significant time effect. We also wish to reiterate that the exercise modality utilized for experiments involving EPR analysis (graded maximal cycling) differed from that of the exercise training programme (KE exercise). Finally, it should be noted that the finding of improved FMD following exercise training in the elderly has been reported previously , and is included here only for comparison with the antioxidant intervention.
In the present study, we have reported a negative outcome on arterial BP and endothelial function after administration of an acute oral antioxidant supplement in older subjects following exercise training. The paradoxical effect of these interventions suggests a need for caution when exercise and acute antioxidant supplementation are combined in elderly mildly hypertensive individuals. Further studies are needed to elucidate the mechanisms which govern the seemingly complex balancing act between pro- and anti-oxidant forces with advancing age.
This work was supported, in part, by the National Institutes of Health Heart, Lung, and Blood Institute [grant number HL-17731 (to R. S. R.)]; the Sam and Rose Stein Institute for Research on Aging (to R. S. R.); the Tobacco-Related Disease Research Program [grant number 15RT-0100 (to R. S. R.)]; a Parker B. Francis Fellowship from the Francis Family Foundation (to D. W. W.); and a Scientist Development Grant from the American Heart Association [grant number AHA 0835209N (to D. W. W.)].
Abbreviations: AU, arbitrary units; BP, blood pressure; DBP, diastolic BP; FMD, flow-mediated vasodilation; I.U., international units; KE, knee-extensor; MAP, mean arterial BP; ONOO−, peroxynitrite; PBN, N-tert-butyl-α-phenylnitrone; ROS, reactive oxygen species; SBP, systolic BP; WRmax, maximal work rate
- © The Authors Journal compilation © 2009 Biochemical Society