Our investigations do not address the mechanisms responsible for the deleterious combined effect of exercise training and acute AO administration on exercise-induced brachial vasodilation. However, we speculate that this is likely the result of an acute disturbance in the balance between pro- and AO forces. Oxidative stress is typically regarded as an unwanted byproduct of cellular oxidation and is seen as a negative risk factor for cellular and vascular health (4). However, as previously mentioned, the downstream consequences of free radicals, such as H2O2 and other free radicals, may act as both potent vasoconstrictors and vasodilators (22) and, as such, possess the capacity to alter vascular responsiveness. Thus, it is tempting to suggest that while exercise training alone evokes an appropriate adaptation to the increase in oxidative stress, the even greater reduction in free radical concentration after acute AO administration may have removed oxidative species that possesses some beneficial vasoactive properties. Thus, this possible vasodilatory role of free radicals (25) may explain the loss of exercise-induced brachial artery vasodilation in the posttraining acute AO condition (Fig. 4, lower panel). These somewhat paradoxical findings regarding the interaction between exercise training responses and AO, albeit acute AO supplementation, do not stand in complete isolation. Indeed, there is growing evidence that exercise training, so far only in young humans, in combination with chronic AO supplementation severely attenuates exercise-induced adaptations (28). These emerging findings reinforce the concept that oxidative stress seems to be an important, if not essential, signaling process in much the same way as we have documented in terms of vascular function in young healthy people (25).
AGE, AO, AND EXERCISE
Small Muscle Mass Exercise Training, Vascular Function, and Arterial Blood Pressure
Cardiovascular adaptations to habitual, whole-body exercise training have been well described and include a significant reduction in resting and exercising arterial blood pressure in both young and old subjects (24). 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 (1,27). By using the single leg knee-extensor exercise training paradigm, we have demonstrated an increase in vascular function (Fig. 4, upper panel) as well as a clinically significant reduction in both resting and exercising arterial blood pressure after 6 wk of small muscle mass training (Figs. 5 and 6, upper panel). In terms of arterial blood pressure, 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. 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 chronic obstructive pulmonary disease and congestive heart failure, where whole-body exercise prescription is limited by both disease symptoms and patient compliance (26,30).
Oxidative Stress, Arterial Blood Pressure, and Endothelial Function
Little consensus exists in the literature with regard to the efficacy of oral AOs on arterial blood pressure in humans. Recent work from our group (25) has verified the efficacy of an acutely ingested oral AO cocktail to increase plasma total AO capacity and reduce plasma alkoxy free radical concentration in young subjects by documenting a reduction in the electron paramagnetic resonance (EPR) signal intensity of alpha-phenyl-tert-butylnitrone adducts. Our most recent EPR data extend these observations to an elderly cohort (38), further verifying the ability of this acutely ingested AO cocktail to reduce oxidative stress both at rest and at the end of maximal exercise in older individuals (Fig. 2). To our knowledge, this was the first study in humans to evaluate changes in arterial blood pressure and endothelial function with an acute pharmacological AO dose directly proven to reduce plasma free radical content. Despite the marked reduction in oxidative stress, we observed only a tendency toward a reduction in resting arterial blood pressure (Fig. 5) and no effect during acute exercise after acute oral AO administration before exercise training.
Age, Exercise Training, and Oxidative Stress - Tipping the Balance
Healthy aging is associated with an increase in free radical production (33) and progressively increasing arterial blood pressure (7). Others have identified exercise training and AO supplementation as efficacious, noninvasive interventions to improve vascular health in older subjects (17,20). Our findings of a tendency for the acutely ingested AO cocktail to reduce mean arterial blood pressure at rest (Fig. 5) and the observed reduction in arterial blood pressure (Fig. 6, upper panel) and improved exercising FMD (39) after exercise training support these studies. However, contrary to our expectations, the combination of exercise training and acute oral AO administration did not interact to produce an additive beneficial outcome but, in fact, reversed the training-induced improvements in both resting and exercising arterial blood pressure (Figs. 5 and 6, lower panel) and endothelial function assessed by FMD (38).
By design, these studies focused on the clinical outcome of the interventions and, as such, do not address the mechanisms responsible for the deleterious combined effect of exercise training and acute AO administration on arterial blood pressure and endothelial function. Oxidative stress is typically regarded as an unwanted byproduct of cellular oxidation and is seen as a negative risk factor for cellular and vascular health (4). However, as already noted, the downstream consequences of free radicals, such as H2O2 and other reactive oxygen species, may act as both potent vasoconstrictors and vasodilators (22), dependent on the paradigm, and as such, possess the capacity to affect vascular tone. Therefore, multiple interventions (exercise training and acute AO supplementation) that elevate AO levels may ultimately tip the pro- and AO balance, resulting in detrimental changes in arterial blood pressure regulation and endothelial function.
AGE, AO, EXERCISE, AND MUSCLE PERFUSION AND ENERGETICS
Novel NMR-Based Approach to the Study of Muscle Function
We have used a unique interleaved pulse sequence to acquire perfusion and 31P data every 3 s, resulting in a highly resolved set of data collected in vivo (5,13). Measurements of perfusion within the skeletal muscle tissue are made using the arterial spin labeling (ASL) method (Fig. 7A). Skeletal muscle energetics are assessed through 31P NMR spectroscopy (Fig. 7B inlay), and from these spectra, phosphocreatine (PCr) depletion and recovery kinetics are evaluated (Fig. 7B and 7D) as an index of muscle oxidative demand and capacity, respectively. Both these measurements are unique in that they allow probing of perfusion and metabolism in a noninvasive manner with very high spatial and temporal resolution and do so in a focused region of interest within the exercising muscle tissue. The simultaneous acquisition of ASL and PCr provides the truly unique opportunity to examine the real-time interplay between perfusion and metabolism in the active skeletal muscle, providing new information concerning the "matching" of these parameters in the human leg. We have recently used this NMR-based approach to examine age-related changes in perfusion and metabolism during lower leg exercise (36), which was then extended to interrogate the role of oxidative stress using a similar experimental paradigm (37).
Oxidative Stress and Skeletal Muscle Perfusion
The use of ASL has provided striking new evidence of the capacity of an acute AO intervention to improve skeletal muscle perfusion in the elderly. In the placebo condition, perfusion within the gastrocnemius and soleus muscle groups during plantar flexion exercise was attenuated by approximately 20% in older subjects (Fig. 7C) compared with young (Fig. 7A). After acute AO administration, exercise-induced perfusion and postexercise hyperemia were 30% to 40% greater in the elderly (Fig. 7C), whereas these hemodynamic parameters were unaffected by acute AO consumption in younger individuals (Fig. 7A). Viewed together, it seems that acute AO consumption effectively restores muscle perfusion to that of the younger group, ablating the peripheral hypoperfusion in the lower leg of elderly subjects.
In a similar cohort, Jablonski et al. (16) recently documented the ability of a high-dose (2 g over 20 min) intravenous infusion of ascorbic acid to improve resting leg blood flow in the elderly. Our NMR-based work extends these findings to exercise, demonstrating a similar magnitude of effect on hemodynamics but with a much lower dose, acutely ingested, over-the-counter AO cocktail. To our knowledge, this was the first study to identify the capacity of acute AO administration to restore the age-related decline in skeletal muscle perfusion during exercise. This new finding not only provided convincing evidence that oxidative stress plays a significant role in the well-documented decline of skeletal muscle blood flow with advancing age but also demonstrated an encouraging plasticity for this age-related adaptation. Whether this acute lack of vasodilation could be maintained with longer-term AO supplementation cannot be ascertained from the present findings, but this is an intriguing possibility that awaits further study.
Interestingly, acute AO administration did not improve hemodynamics in the young, which is most likely attributable to better endogenous AO capacity and therefore a lesser degree of oxidative stress in these subjects (33). In fact, despite evidence for the ability of the acutely ingested AO cocktail to reduce plasma free radical concentration both at rest and during exercise (25), end-exercise perfusion tended to be lower after AO consumption in the young group (Fig. 7A). This is in agreement with other recent work from our group identifying a negative effect of acute AO administration on exercise-induced vasodilation in young subjects (11), again adding credence to the concept that some level of free radical concentration is essential for normal function during exercise in young, healthy individuals. Again, these findings are somewhat parallel to the finding that exercise training in combination with chronic AO supplementation severely attenuates exercise-induced adaptations (28), suggesting a positive regulatory role for free radicals.
This acute positive outcome on the peripheral circulation after an acute AO-mediated reduction in vascular oxidative stress in the elderly is in contrast to the largely disappointing results from interventional trials concerning the effects of AO supplementation on many indicators of cardiovascular health (35). Such equivocal findings may be due, at least in part, to a tendency for clinical trials to focus on specific patient populations and also to a general lack of knowledge concerning in vivo AO status and efficacy. Although acute in nature, our laboratory-based studies have attempted to address both of these potentially confounding influences. Specifically, recruiting a focused cohort of subjects in good overall health with no diagnosed disease or use of prescription medication provided the opportunity to examine the impact of oxidative stress on muscle function in a "preclinical" context. Regarding efficacy, the acute AO intervention that we have used was selected based on previous studies from our group using EPR spectroscopy that have quantified the capacity of this AO cocktail to reduce plasma O2-centered free radical levels in both young (25) and older (38) subjects (Fig. 2). This previous work thus confirms the ability of the acute AO intervention to significantly attenuate vascular oxidative stress in an acute manner and lends confidence to the apparent link between reduced oxidative stress and improved muscle perfusion as measured by ASL.
Oxidative Stress and Skeletal Muscle Energetics
In the placebo condition, PCr depletion and the ratio of inorganic phosphate to PCr (Pi/PCr) were found to be 30%-40% greater in older compared with young subjects at a similar work rate, indicative of a greater "metabolic stress" during exercise in the elderly (Figs. 7B and D). In contrast to the pronounced effect of acute AO administration on perfusion in the elderly, this intervention did not alter PCr depletion or Pi/PCr, suggesting that age-related differences in muscle energetics during exercise was due to inherited differences in skeletal muscle metabolism or simply greater relative stress due to a diminished exercise capacity in the older group. To our knowledge, this was the first study to specifically examine the impact of oxidative stress on these parameters in younger and older individuals.
In contrast to the negligible effect of the acute AO supplementation on PCr depletion, PCr τ upon cessation of exercise was significantly faster in the elderly after AO consumption, suggestive of improved skeletal muscle energetics in this group (Fig. 7D). This remarkable and effect of the acute AO ingestion on skeletal muscle energetics in the elderly could be the result of either an AO-mediated change in muscle metabolism or a secondary consequence of the AO-mediated improvement in perfusion and thus O2 availability. Regarding the previous, although there is some indication that chronic AO administration may influence oxidative metabolism, the time course for the observed effect (2-3 h after consumption) in the present study makes this an unlikely mechanism for the observed improvement in PCr recovery. Alternately, we have identified the potential influence of perfusion on muscle energetics from NMR-based studies, with a clear association between O2 availability and the rate of PCr recovery at the end of exercise in healthy subjects (15). With this previous work as our basis, we speculate that the acute AO-induced increase in muscle perfusion observed in the older group augmented O2 delivery to the muscle tissue (Fig. 7C), which may be viewed as effectively improving O2 availability for oxidative metabolism within the skeletal muscle.
Evidence of Hemodynamic and Metabolic Reserve in Aging Skeletal Muscle
The observed improvement in perfusion and subsequent beneficial effect of muscle oxidative capacity after acute AO administration (Figs. 7C and D) raises the question of whether age-related changes in these parameters should be viewed as detrimental or adaptive. Theoretically, Fick's equation dictates that any decline in perfusion will decrease O2 delivery and may jeopardize O2 availability to the exercising muscle tissue. However, we have previously reported an age-related decline in limb blood flow that is accompanied by improved O2 extraction, apparently compensating for the reduction in O2 delivery and thereby maintaining O2 consumption (21). In terms of metabolism, there is little consensus regarding the impact of age on muscle energetics (29). In this NMR-based work, we did not observe an age-related difference in PCr repletion after moderate-intensity exercise in the placebo condition (Fig. 7), supporting the body of evidence against a decline in metabolic capacity in leg skeletal muscle with healthy aging. Together, these previous and current findings suggest that healthy aging is associated with a decline in exercising peripheral blood flow but that this adaptation does not result in skeletal muscle dysfunction in this cohort.
With these previous studies demonstrating an apparent maintenance of skeletal muscle function with advancing age, it is noteworthy that acute AO administration unmasked such a marked improvement in both hemodynamic and metabolic parameters (Fig. 7), which may suggest a "reserve capacity" in the elderly. In pathophysiological states, such as peripheral vascular disease characterized by chronic ischemia, the detrimental effects of reduced perfusion on muscle function and energetics are well known. In this population, peripheral blood flow improves after an acute AO intervention, which may presumably also improve muscle energetics through improved O2 supply. Although less severe, perfusion and metabolic data from our studies in healthy older subjects parallel these clinical findings (Fig. 7), suggesting that the aging skeletal muscle may operate in a functional but somewhat depressed state because of elevated oxidative stress that may be acutely restored in the presence of a more favorable, AO environment. As these NMR-based assessments of muscle metabolism were of relatively short duration (5 min), it would be interesting to examine this concept of the muscle of the elderly, working in a somewhat depressed state in the face of a more prolonged exercise challenge and subsequently greater accumulation of oxidative stress.
We have revealed an age-related pro- and AO imbalance that affects vascular function, and exercise training is capable of restoring this equilibrium. Indeed, exercise training improves vascular function such that the AO-mediated reduction in free radicals negatively affects exercise-induced brachial artery vasodilation, as previously seen in young untrained subjects. Additionally, acute AO administration after exercise training negates exercise-induced improvements in blood pressure and FMD, returning subjects to a hypertensive state and blunting FMD. Finally, measurements using NMR revealed that acute AO supplementation significantly improved end-exercise and postexercise perfusion in older subjects but did not do so in the young. Concomitantly, muscle oxidative capacity (PCr recovery) was improved with acute AO ingestion in the old, again with no effect in the young. In combination, these age-specific findings suggest an important role for oxidative stress in the differing vascular responses between young and old. However, the paradoxical effects of acute AO supplementation, when combined with exercise training, reveal an intriguing, but complex, relationship between aging, oxidative stress, and vascular function (Fig. 1).
This research was supported by the National Heart, Lung, and Blood Institute grants HL-17731 and HL-09183, the Tobacco Related Disease Research Program grant 15RT-0100, and an American Heart Association Scientist Development Grant 0835209N.
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Keywords:©2011 The American College of Sports Medicine
oxidative stress; vascular function; shear stress; blood flow; flow-mediated dilation