There is strong evidence to support a beneficial effect of exercise training on endothelial function, as measured by endothelium-dependent vasodilation [5
]. On the other hand, although reduced NO production and bioactivity is a major contributor to endothelial dysfunction, less is known regarding the effects of exercise training on NOx levels. Cross-sectional studies generally show that urinary and plasma NOx levels are higher in trained vs. untrained persons [24
]. In the few intervention studies that exist, NOx levels were increased after 3 to 12 weeks of exercise training [6
]. In contrast, the major finding of the present study is that plasma NOx levels are unchanged after 24 weeks of aerobic exercise training in older adults. In addition, long-term exercise training had no effect on vasomotor function, as assessed by changes in the FBF hyperemic response.
Endothelial dysfunction can be assessed in plasma and urine samples by measuring circulating markers of NO metabolism (i.e. NOx) or markers of endothelial injury, such as soluble adhesion molecules [1
]. Alternatively, functional methods can be used to measure NO-related vasomotion in response to pharmacological or physiological stimuli. NO regulates various functions of the cardiovascular system, including vascular tone, platelet activation, and cell proliferation. As such, decreased NO bioactivity may be one of the earliest detectable findings during the development of cardiovascular diseases [1
]. It has been suggested that because plasma NOx levels may not reflect biologically active NO and are not solely endothelium-dependent, these measurements should be made in combination with functional assessments. In the present study we examined both plasma NOx levels and FBF at rest and during reactive hyperemia, and data from both measures indicate that long-term training does not improve endothelial function in older adults.
Our study examined 82 older (mean age = 58 yrs), overweight (mean BMI = 28 kg/m2
) men and women with CVD risk factors including dyslipidemia and hypertension. Mean baseline plasma NOx levels were ~19 μmol/L and were unchanged after dietary stabilization and a 24-week exercise intervention consisting of aerobic exercise at 70% VO2
max for 40 min/day, 3–4 days/wk. In a previous study, Roberts et al. reported that a 3-week diet and exercise intervention increased urinary NOx levels in 11 men with CVD risk factors [26
]. The study population, which included 7 hypertensives and 2 diabetics, were 38 to 72 yrs old and predominately obese (mean BMI = 37.6 kg/m2
). The subjects followed a low-fat (~10% of calories), high-carbohydrate (~70–75% of calories) diet and walked daily for 45–60 min at 70–85% VO2
max. With such an intensive dietary intervention, it is not clear if the improvement in NOx levels was due primarily to the dietary intake, the exercise training, or both. It is also not clear if age had any effect on the training-induced changes in NOx levels. Moreover, the measurement of NOx in plasma vs. urine may reflect different aspects of NO metabolism [27
]. Another study reported that 8 weeks of aerobic exercise training increased plasma NOx levels by 58% in 8 young healthy subjects (mean age = 20 yrs) who exercised at 70% VO2
max for 1 hr/day, 3–4 days/week [7
]. Differences in the age and health status of the study population may account for some of the varying results. Thus, while previous studies examined only a small number of subjects after a short-term training intervention, this study provides the first examination of changes in plasma NOx levels after long-term training in an older population.
Changes in NO levels after exercise not only depend on the duration of the training program, but may also be influenced by age. Recently, Lauer et al. reported that among healthy subjects with no CVD risk factors, older persons (mean age = 58 yrs) had an impaired capacity to increase plasma nitrite levels after acute exercise compared to younger persons (mean age = 25 yrs) [13
]. Consistent with this, Gomes et al. found that cycling for 45 min/day, 3 days/week, for 12 weeks at a heart rate corresponding to the anaerobic threshold did not improve plasma nitrite levels in 18 patients with metabolic syndrome (mean age = 46 yrs) [28
]. On the other hand, 12 weeks of cycling at ~50% VO2
max for 30 min/day, 5 days/week increased plasma NOx levels by 54% (from ~28 to 43 μmol/L) in 10 elderly women (mean age = 63 yrs) who were normotensive, non-obese, and not on any medications including HRT. Besides the difference in health status, differences in the measurement of NO (nitrite only vs. nitrite + nitrate) make it difficult to compare these results. However, in the present study subjects exercised not only for a longer duration, but also at a higher intensity. It is possible that the higher exercise intensity in our study negatively affected the plasma NOx response to exercise, as 12 weeks of aerobic exercise training at 75% VO2
max has been shown to impair training-induced improvements in endothelial function in young healthy men (mean age = 25 yrs), most likely due to increased oxidative stress [29
]. In contrast, training at 50% VO2
max augmented endothelial function through increased NO production. Older individuals may be particularly vulnerable to the adverse effects of high-intensity training. Taken together, these studies highlight the need to clarify the effect of aerobic exercise training on NO levels in older adults. Although in our study age was not associated with changes in plasma NOx levels with training, it is likely that the aging process per se
alters NO-related adaptations to long-term exercise training.
To our knowledge, only 3 other studies have examined endothelial vasomotor function after long-term training in humans. Hambrecht et al. found that in chronic heart failure patients (mean age = 56 yrs), 24 weeks of exercise training at 70% VO2
max for 40–60 min/day, 5–6 days/week significantly increased acetylcholine-induced blood flow in the femoral artery [11
]. In addition, 16 weeks of aerobic exercise training enhanced flow-mediated dilation in the brachial artery and blood flow in ocular resistance vessels in diabetics (mean age = 42 yrs) [9
]. In the third study, 4 weeks of a hospital-based exercise training program (at 80% VO2
max for 60 min/day, 6 days/week), followed by 20 weeks of a home-based program (at 80% VO2
max for 20 min/day, 7 days/week), significantly improved coronary blood flow in patients with coronary artery disease (mean age = 60 yrs) [10
]. In our population of older men and women free of clinical CVD and diabetes, there was no improvement in blood flow in forearm resistance vessels with exercise training. Differences in the study population, the vascular bed examined, and the measurement of endothelial function may account for the differences in our results.
As mentioned previously, most studies demonstrate that training programs of 12 weeks or less elicit significant improvements in NO-related vasomotor function. However, these adaptations appear to be transient and may disappear in the longer term [5
]. Improvements in endothelial function in the short term are largely due to shear stress-induced changes in endothelial NO synthase expression, phosphorylation status, and enzyme activity [5
]. In contrast, prolonged exercise training may lead to structural enlargement of blood vessels [30
], and over time an increase in arterial diameter may occur that reduces the shear stress signal associated with a given exercise-induced elevation in blood flow and allows NO bioactivity to return towards pre-training levels [33
]. These structural changes are consistent with the restoration of endothelial NO synthase expression, NO production, and endothelial-dependent vasodilation to control levels in the fully trained state [11
]. While these findings are supported by both animal and human data, more studies are needed to identify factors that may influence long-term training adaptations, such as characteristics related to the study population (i.e. age, gender, and health status) and the training program (i.e. exercise mode, frequency, intensity, and duration).
Although the number of subjects in this study with plasma NOx levels was larger than in previous studies, we had limited power to detect changes with training for the FBF variables. In addition, because we did not have a non-exercise control group, we cannot rule out the possibility that exercise training simply prevented a decline in NOx levels and FBF. Similarly, because this study only included 50–75 yr-olds, we do not know if our exercise intervention would have improved plasma NOx levels and FBF in younger persons. We also cannot determine whether an increase in plasma NOx levels occurred earlier in the intervention (i.e. within the first 12 weeks of training). Finally, because we did not distinguish between endothelial-dependent and endothelial-independent vasodilation, we cannot determine whether the role of NO in the reactive hyperemic response is altered by exercise training. Nevertheless, measuring reactive hyperemia using venous occlusion plethysmography is a commonly used method to assess endothelial function in resistance arteries and correlates well with more invasive measures using intra-arterial drug infusions [38
Summary and Conclusion
This is the first study to demonstrate that 24 weeks of aerobic exercise training does not increase plasma NOx levels or augment FBF in older men and women. Despite the limitations mentioned above, participants in our study were carefully monitored to keep their diet, medications, and body weight constant during the progressive, well-standardized exercise training program. We also followed standard procedures to ensure that blood samples for the measurement of NOx were drawn in the morning after an overnight fast and within 24–36 hrs after exercise. In addition, plasma samples were deproteinized before analysis to minimize experimental artifacts. While our results are consistent with the idea that improvements in NO production and endothelial function with exercise are transient and may be lost over time, more long-term training studies with repeated assessments of NO production and endothelial function are needed to better characterize the time course of vascular adaptations to exercise training. In addition, future studies will need to clarify the effect of aging on pathways involved in these adaptive responses.