Exercise hyperemia is closely matched to metabolism in the active skeletal muscle. As such, at mild intensities, the increase in blood flow is directly related to vasodilation in the vascular bed (1), thus matching the O2 supply and demand in the contracting skeletal muscle. This response is achieved through a complex interaction of mechanical, metabolic, and neural stimuli. Although the precise mechanism(s) contributing to the increase in blood flow remains speculative, exercise hyperemia is achieved through local regulation of the microvasculature and feed arteries (39). Healthy older adults have consistently been shown to have reduced limb blood flow during exercise compared with younger adults (19,20,33,35). Increased production of reactive oxygen species (ROS) and/or decreased antioxidant defenses, as documented with normal aging, may lead to impaired vascular endothelial function (and NO bioavailability). Consistent with this theory, the antioxidant vitamin C has been successful in restoring exercise blood flow responses in older but not young individuals (19), which has been confirmed mechanistically to represent an improvement via endothelial NO availability (8). Overall, these findings suggest oxidative stress may contribute to exercise-blood flow impairments, which may be particularly beneficial in patient populations with increased ROS.
Patients with chronic obstructive pulmonary disease (COPD) have several factors that may pose a risk for reduced blood flow regulation during exercise, including endothelial dysfunction (2,11,17), increased oxidative stress (15,36), arterial stiffness (23,26), and reduced physical activity levels. Although a recent study showed improvement in endothelial function (determined by FMD) after an antioxidant intervention in COPD (17), it is unknown if these benefits would be extended to exercise hyperemia. It is alternatively possible that COPD patients may experience a reduction in exercise hyperemia because of augmented sympathetic vasoconstriction (16); however, few studies have investigated this and the role of these neural mechanisms affecting blood flow in patient populations. Supplemental oxygen may be used by COPD patients to enhance O2 delivery during dynamic exercise, but hyperoxia provides an additional source of oxidative stress that may be detrimental to exercise blood flow. Traditional views suggest hyperoxia reduces blood flow and increases vascular resistance at rest, and recently, this has been found to occur during an isolated bout of exercise (5). Hyperoxic conditions may provide an environment with high levels of ROS, thereby potentially reducing NO availability (14) and ultimately blood flow. One plausible explanation for the reduction of blood flow with hyperoxia is a ROS-mediated impairment of the vascular endothelium. Consistent with this theory, in both healthy adults (24) and patients with ischemic heart disease (27), the consequential vascular effects of hyperoxia at rest are mitigated by vitamin C administration. Most recently, findings from Ranadive et al. (37) suggest that vitamin C is only effective in preventing the hyperoxic-mediated vasoconstriction that is observed during exercise in those individuals with large reductions in blood flow. An alternative theory suggests that hyperoxia may transiently enhance muscle blood flow in young healthy individuals, secondary to carotid chemoreceptor inhibition (40). Indeed, the effect of hyperoxic inhibition of the carotid body provides an interesting parameter to evaluate in COPD that may already have elevated sympathetic activity.
Given that we expect COPD patients to have increased oxidative stress and impaired endothelial function, we hypothesized that patients with moderate COPD would have reduced forearm blood flow responses to handgrip exercise, compared with healthy age-matched controls. Because vitamin C has previously been shown to improve exercise hyperemia in healthy older adults through an endothelial-dependent mechanism (19), we used this intervention to test if indeed, an improvement in NO availability would increase hyperemia in COPD. We further hypothesized that COPD patients would exhibit the greatest increase in exercise blood flow during the combined hyperoxia + vitamin C condition. Using intravenous saline (sham-control) and vitamin C infusions (active-intervention) in all participants, we tested the aforementioned hypotheses using the following outcomes: 1) brachial blood flow during mild rhythmic handgrip exercise (normoxia and hyperoxia); and 2) vasodilator responses to flow- and nitroglycerine-mediated dilation. In addition, vitamin C, NO end products, antioxidant enzymes, and oxidative stress markers were measured to assess the effectiveness of the vitamin C intervention.
METHODS
Study Participants
A total of 20 (10 COPD [67 ± 3 yr]; 10 controls [66 ± 2 yr]) participants completed the study. COPD patients were recruited from outpatient clinics in the Calgary area, and control participants were recruited from the community. Exclusion criteria included the following: current smoker (>3 months); BMI > 35 kg·m−2; additional lung disease (including asthma); uncontrolled hypertension; current domiciliary oxygen; and history of diabetes, cerebrovascular disease, myocardial infarction, angina, arrhythmia, valvular heart disease, chronic heart failure, obstructive sleep apnea, peripheral arterial disease, chronic headaches/migraines, or blood clots/thrombosis. COPD patients were required to have a past smoking history greater than 10 pack-years and moderate-severe airflow obstruction (FEV1/FVC < 0.70; 30% ≤ FEV1 ≤ 80% predicted) evident on spirometry. COPD patients must not have had an exacerbation within previous 8 wk.
All participants provided written, informed consent, and ethical approval was obtained from the University of Calgary’s Institutional Conjoint Health Research Ethics Board and the Health Protection Branch of Health Canada before commencement.
Experimental Design
Study participants were required to visit the University of Calgary on 2 separate occasions. The first session consisted of standard pulmonary function testing, involving postbronchodilator spirometry, according to guidelines of the American Thoracic Society. Functional fitness was assessed by a 6-min walking distance test (6MWD). The main experimental session was conducted on the second visit and involved a peripheral vascular assessment. Before the second experimental session, participants were instructed to refrain from eating/drinking (6 h), caffeine (12 h), vitamin supplements (72 h), vigorous exercise (12 h), and blood pressure medication (24 h), and COPD patients were asked to refrain from short- and long-acting bronchodilators (12 and 24 h, respectively).
Upon arrival to the laboratory, subjects were seated for 10 min with their hand warmed (10 min at ∼42°C) before obtaining a capillary blood sample from the finger (ABL800 FLEX; Radiometer, Copenhagen, Denmark). An intravenous catheter was inserted in the nondominant arm for delivery of either normal saline solution or vitamin C. All physiological measurements were obtained with the participant in the supine position, resting quietly in a dimly lit room. Briefly, subjects underwent a test of FMD, followed by rhythmic handgrip exercise (HG), during infusion of saline (sham-control). After a 30-min period of rest, the protocol was repeated with the infusion of vitamin C. Lastly, nitroglycerine was administered to assess endothelial-independent dilation. Blood was drawn into EDTA and SST tubes at 3 separate times: before any experimentation (baseline; BL), immediately at the end of the saline period (saline; SAL) and vitamin C period (vitamin C; vit C). An overview of the experimental protocol is provided in Figure 1.
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FIGURE 1: Overview of main experimental protocol. All subjects completed a test of flow-mediated dilation (FMD) and handgrip exercise (HG). Participants first received intravenous saline, followed by a washout period (w/o), and repeated the tests during vitamin C infusion. Nitroglycerine-mediated dilation (NMD) was assessed after the completion of FMD and HG. Venous blood samples were obtained at baseline (BL), after sham-saline (SAL), and at the completion of vitamin C (vit C) (time of draw designated by arrows).
Measurements and Procedures
Maximal voluntary contraction
Handgrip strength was assessed in the dominant hand using a strain gauge dynamometer (Lafayette 78010, IN) and recorded as the greatest of 3 isometric contractions. Forearm volume was assessed using the water displacement method (21).
Dynamic handgrip exercise
Handgrip exercise (HG) was performed with a weighted pulley at an intensity of 10% maximal voluntary contraction (MVC) for 7 min. Audio feedback was used to coordinate a duty cycle of 20 contractions per minute (1:2 s; contraction-relaxation). A face mask was fit over the nose and mouth to collect baseline end-tidal partial pressures of O2 and CO2 (PETO2 and PETCO2, respectively) for a period of 10 min using dedicated software (Chamber v2.26; University of Oxford Laboratory of Physiology, Oxford, UK). Expired PO2 and PCO2 were analyzed at 100 Hz using mass spectrometry (AMIS 2000; Innovision, Odense, Denmark) via a fine catheter. Respiratory volumes were measured with a turbine and volume transducer (VMM-400; Interface Associates, Laguna Niguel, CA), and respiratory flow direction and timing were obtained with a pneumotachograph (RSS100-HR, Hans Rudolph, Kansas City, MO). During HG, dynamic end-tidal forcing (34) was used to prevent hyperoxia-induced alterations in PETCO2 that may have a secondary effect on brachial blood flow (43). PETCO2 and PETO2 were held near baseline conditions for the first 5 min of HG (PETCO2 = +1.5 mm Hg above rest, and PETO2 = 88.0 mm Hg), after which, isocapnic hyperoxia was administered for the final 2 min of exercise (PETO2 = 300.0 mm Hg).
Endothelial-dependent and independent dilation
The technique of FMD was used to assess endothelial-dependent dilation, according to guidelines (7,41). Briefly, a manual pneumatic cuff was rapidly inflated on the forearm to 250 mm Hg for 5 min, after which, the cuff was rapidly deflated to initiate a hyperemic blood flow response, measured in the brachial artery (BA). Physiological variables were recorded for 60 s at rest (BL), immediately before cuff inflation. Continuous measurements were resumed 30 s before cuff release and thereafter for 5 min. Nitroglycerine-mediated dilation (NMD) was assessed using sublingual nitroglycerin (0.4 mg). Baseline diameter of the brachial artery was recorded using 2D ultrasound for 60 s before drug administration and continuously for a 5-min period thereafter.
Ascorbic acid infusion
Vitamin C (ascorbic acid injection; Alveda Pharma, Canada) was diluted with normal saline and administered intravenously. A loading dose of 3 g ascorbic acid (200 mg·min−1) was administered for 15 min, followed by a maintenance dose (40 mg·min−1) during the experiment. This dosage of vitamin C has previously been shown to be effective at rapidly increasing plasma ascorbic acid concentrations more than 15 fold (12). Isotonic normal saline (0.9% NaCl) was infused at an identical flow rate to ascorbic acid.
Cardiovascular measurements
Cardiovascular variables were recorded to a personal computer at 100 Hz, and signals were integrated using a data acquisition system (PowerLab 16/35; ADInstruments Inc., Colorado Springs, CO). Data were collected and stored offline for later analysis using accompanying software (LabChart 7.0 Pro; ADInstruments). Heart rate (HR) was determined by three-lead electrocardiography (Micromon 7142B monitor; Kontron Keynes, UK), and finger pulse photoplethysmography was used to measure beat-to-beat blood pressure using the finger of the nondominant hand (Portapress; TPD Biodemical Instrumentation, Amsterdam, The Netherlands). Arterial oxyhemoglobin saturation (SpO2) was determined by finger pulse oximetry (Model 3900; Datex-Ohmeda, Louisville, CO).
Vascular Doppler ultrasound measurements
A pulsed Doppler 4-MHz probe was fixed over the brachial artery on the dominant arm, immediately superior to the antecubital fossa (Multigon Industries Inc., Yonkers, NY). Signal depth and power were adjusted accordingly to optimize the signal. The analog signal was integrated into the data acquisition system, allowing for continuous, beat-to-beat velocity measurements. A 12-MHz linear array ultrasound probe in B-mode was used to obtain brachial diameters (Vivid 7; GE, Milwaukee, WI). Video imaging was continuously recorded to a personal computer (GrabBee Software) and stored offline for later analysis.
Biological markers
Venous blood samples were collected at baseline (BL), after sham-saline (SAL), and at the completion of vitamin C infusion (Vit C) (Fig. 1). Blood was centrifuged and stored at −80°C until analysis. A complete description of biochemical assays can be found in the online supplement (see Document, Supplemental Digital Content 1, supplementary methods, https://links.lww.com/MSS/A575).
Vitamin C and antioxidant enzymes
Vitamin C concentration was assessed in serum blood (Ascorbate Assay Kit; Cayman Chemical Company, Ann Arbor, MI). Plasma superoxide dismutase (SOD) activity was determined using the method of Beauchamps and Fridovich (4), slightly modified by Oberley and Spitz (30). Plasma catalase activity was determined by the method described by Johansson and Borg (18).
Oxidative stress
Plasma malondialdehyde (MDA), a marker of lipid peroxidation, was assessed using thiobarbituric reactive substances using a modified method of Ohkawa, Ohishi, and Yagi (31). Advanced oxidation protein products (AOPP) were determined in plasma using the semi-automated method described by Witko-Sarsat et al. (44).
End products of nitric oxide metabolism (NOx)
Plasma end products of endothelium nitric oxide, nitrites, and nitrates were measured using the methods described by Misko, Schilling, Salvemini, Moore, and Currie (28). The sum of nitrite and nitrate is considered as an index of nitric oxide production.
Data Analysis
Brachial diameters were analyzed at 30 Hz, using semi-automated edge-detecting software (Brachial Analyzer; Medical Imaging Applications, Coralville, IA). Peak diameters (for FMD and NMD) were recorded as the highest 3-s average.
The mean blood flow velocity (MBV) was taken as a 60-s average to reduce variance associated with contraction-relaxation cycles during handgrip exercise. Averages were taken at the end of the fifth minute (normoxia) and at the end of the seventh minute (hyperoxia). Brachial blood flow (BBF) and brachial vascular conductance (BVC) during exercise were calculated using the following equations:
where MBV is mean brachial blood flow velocity (cm·s−1) and r is the radius (cm). Brachial vascular conductance were calculated as:
and expressed as milliliters per minute per 100 mm Hg. The associated brachial diameter was a 60-s average taken immediately preceding HG, under steady state resting conditions. The FMD was expressed as percent change in vessel diameter, compared with baseline:
NMD and absolute FMD(mm) were analyzed from the change in vessel diameter, as above.
Statistics
The Shapiro–Wilk test was used to test data normality. All dependent variables were normally distributed, except for AOPP. AOPP data were analyzed using an aligned rank transform. Statistical comparisons between groups were performed using a mixed 2 × 3 repeated-measures analysis of variance to identify any physiological differences with exercise, under baseline-sham conditions. Significant main effects and interactions were further examined using independent t tests. To assess the effect of the intervention (sham-saline vs vitamin C), a within-group 2 × 3 repeated measures was performed (baseline, HG, HG + O2). Where appropriate, dependent t tests were conducted to identify the effect of exercise (Rest vs EX), the effect of hyperoxia (EX vs EX + O2), and the combined effect of exercise + hyperoxia compared with rest (Rest vs EX + O2). The Bonferroni correction method was used to correct for multiple comparisons (i.e., significance was determined at P < 0.0167 for comparison across the 3 stages). A 2 × 2 repeated measures analysis of variance was used to assess the effect of grouping (e.g., COPD, control) and intervention (e.g., saline, vitamin C) on FMD outcomes. Where approriate, group and intervention effects were assessed using independent (between group) or dependent (within group) t tests, respectively. An independent t test was used to assess the differences in NMD between COPD and controls. Relationships between subject characteristics (e.g., disease severity, age, fitness, physical characteristics) and vascular outcomes (e.g., FMD, exercise BBF) were assessed using the Pearson correlation coefficient. All statistical analyses were performed using the statistical software SPSS version 20.0 (Chicago, IL). Data are presented as means ± SE. Significance was determined at P ≤ 0.05.
RESULTS
Study Participants
Subject characteristics of COPD patients and healthy controls are summarized in Table 1. As expected, COPD patients had reduced FEV1/FVC ratio, and moderate disease severity (FEV1 = 60% ± 5% predicted). Patients and controls were of similar age, body mass index, handgrip strength, and arm volume. One control and 5 COPD patients were being treated for hypertension (see Table, Supplemental Digital Content 2, supplementary results, https://links.lww.com/MSS/A576). Patients had a shorter 6MWD and greater smoking history than controls. Decreased 6MWD was correlated with lower FEV1 (% predicted) in COPD (r = 0.64; P < 0.05). Capillary blood measurements were not obtained in 2 participants. Two COPD patients had incomplete data for the measurements because of an adverse reaction (acute headache).
TABLE 1: Subject characteristics.
Rhythmic Handgrip Exercise
Differences between COPD and controls during sham-saline
Physiological responses to 10% handgrip exercise resulted in a similar increase in BBF, BVC, and HR between COPD patients and controls during the sham-saline condition (Fig. 2 and Table 2; P > 0.05). Expressed as a percentage, there was no difference in ΔBBF between COPD and controls (426% ± 100% vs 550% ± 82%, respectively; P > 0.05). Hyperoxia increased SpO2 and decreased HR (Table 2). There was no difference between normoxic and hyperoxic BBF or BVC during exercise, in either COPD or controls (Fig. 2 and Table 2; P > 0.05).
TABLE 2: Brachial vascular conductance and cardiovascular responses to mild, rhythmic handgrip exercise before and after vitamin C infusion in COPD patients and healthy controls.
FIGURE 2: Brachial blood flow in COPD and healthy controls at rest and during mild handgrip exercise under conditions of sham-saline and vitamin C infusion. EX, handgrip exercise under normoxic conditions; EX + O2, handgrip exercise under hyperoxic conditions. Values are means ± SE. *P ≤ 0.05 versus rest (within trial [e.g., saline or vitamin C).
Effect of vitamin C on exercise-blood flow
Resting BBF and BVC were similar between sham-saline and vitamin C conditions for both COPD and control groups (P > 0.05). Similarly, exercise-hyperemia (rest to EX) was not affected by vitamin C in either group (Fig. 2 and Table 2). Hyperoxia did not influence BBF or BVC in COPD patients or controls (Fig. 2 and Table 2).
Endothelial-Dependent and Independent Dilation
As shown in Table 3, FMD% and absolute change in brachial diameter (FMDΔ) were not different between COPD and controls during the sham-saline condition (6.0% ± 0.9% vs 5.9% ± 1.0%, respectively; P > 0.05). The vitamin C intervention significantly increased FMD% to a similar extent in both groups (P ≤ 0.05). However, baseline diameter was lower after vitamin C in both groups (P ≤ 0.05). Peak diameter reached by the brachial artery after the cuff release (FMDpeak) was comparable between COPD patients and controls during the sham-saline condition, and in both groups, vitamin C tended to reduce FMDpeak. NMD initiated similar responses between groups (COPD: 25.6% ± 1.6% vs 23.5% ± 2.3%; P > 0.05) (Table 3). Similar changes were found between groups when comparing the absolute change in brachial artery diameter with nitroglycerine administration (COPD: +0.85 ± 0.08 mm; controls: +0.85 ± 0.04 mm). No relationships were found between FMD% and exercise BBF.
TABLE 3: Vascular responses to flow-mediated and nitroglycerine-mediated dilation in COPD and healthy controls.
Biochemical Markers
Figure 3 summarizes biochemical assays of vitamin C concentration (Panel A), antioxidant enzyme activities (SOD and catalase; Panels B and C), markers of oxidative stress (MDA and AOPP; Panels D and E), and nitric oxide metabolism (NOx; Panel F). No significant differences between groups were observed at rest or in response to intravenous vitamin C (i.e., no interaction effects) (P > 0.05). Vitamin C infusion significantly increased vitamin C concentration, SOD activity, and MDA (main effect of intervention: P < 0.02).
FIGURE 3: Biochemical markers. Vitamin C (A), superoxide dismutase (SOD; B) catalase (C), malondialdehyde (MDA; D), advanced oxidation protein products (AOPP; E), and nitric oxide metabolism (NOx; F) in COPD patients and controls at baseline (BL) after sham-saline (SAL) and following vitamin C infusion (vit C). Vitamin C infusion increased the concentration of vitamin C, SOD activity, and MDA. NS = nonsignificant. Values are mean ± SE.
DISCUSSION
Main Findings
There were several main findings in this study. The novel findings were as follows: 1) moderate-COPD patients have comparable peripheral exercise blood flow to healthy older adults; 2) vitamin C infusion did not alter blood flow during exercise; 3) flow-mediated dilation in the brachial artery was not different in COPD patients and controls. Vitamin C, however, had an overall improvement on this parameter; 4) acute hyperoxia was not found to augment exercise blood flow response in either COPD or controls, suggesting that carotid chemoreceptor inhibition did not restrain blood flow (in older individuals or COPD patients) during mild, light intensity handgrip exercise. Collectively, these findings provide evidence for an overall preserved vascular response during exercise in moderate-COPD patients.
Exercise Hyperemia in COPD Patients
Exercise hyperemia under normoxic conditions
During mild, 10% MVC steady-state handgrip exercise, BBF and BVC were similar between COPD patients and older controls. Resting brachial blood flow in COPD patients was similar to resting brachial blood flow in controls (albeit higher but nonsignificant). We selected a mild EX challenge to minimize the systemic changes associated with whole-body exercise and investigate an “isolated” vascular response. Despite the modest challenge, HR significantly (∼4 bpm) increased in both groups.
Although no previous study has investigated the role of vitamin C in exercise-muscle blood flow in COPD, a previous report suggests restoration of vascular endothelial redox balance (by vitamin C) in healthy older adults, leading to increased exercise blood flow. These results were supported by a concomittent augmentation of ACh-mediated blood flow (19), which later confirmed that indeed this improvement in endothelial function was mediated by increased NO derived from the NOS pathway (8). Contrary to our hypothesis, vitamin C did not increase BBF or BVC in either COPD or control groups during exercise. These findings were unexpected, given that the results of Kirby et al. (19) indeed found vitamin C to restore forearm blood flow in healthy older individuals. Although a precise explanation for this discrepancy is unclear, it is likely explained by the difference in the route of administration (arterial vs venous). The primary role of SOD is to catalyze the conversion of superoxide (O2−) to hydrogen peroxide, therby reducing the concentration of reactive oxygen species (ROS). We expect that in the present study, the increased SOD activity would decrease O2−, thereby increasing NO bioavilability. However, despite an increase in SOD, and presumably NO, muscle blood flow was not increased. Furthermore, our measure of NOx may not adequately represent NO metabolism because it is known that one byproduct in this reaction, nitrite, can be recycled to reform NO (22).
Another important consideration of the oxidant-scavenger vitamin C (13) is the possibility of this vitamin to also exhibit pro-oxidant properties, depending on its concentration and the presence of free metal ions. By the Fenton reaction, vitamin C can reduce transition metals, leading to increased generation of free radicals. This may explain the fact that MDA increased after vitamin C administration. Along these lines, a previous study found an oral dose of 500 mg vitamin C daily for 6 wk of induced oxidation of DNA (32). Two theories exist regarding the activity of SOD. First, it is possible that SOD activity was upregulated secondary to increased superoxide production, which would explain the increase in MDA. Second, as others have suggested, SOD activity is upregulated secondary to vitamin C and is acting in the capacity of an antioxidant. In both cases, the upregulation of SOD was not enough to offset the increase in MDA. Taken together with the NOx results, our findings suggest that this did not have a negative impact on NO metabolism, as NOx would have been expected to decrease with an increase in oxidatove stress. Indeed, others have reported variation with SOD (either an increase or decrease with antioxidants), with divergent effects on oxidative stress markers (17,45).
These results provide additional evidence that the skeletal muscle blood flow response is not solely mediated by NO mechanisms. With aging, there is approximately 40% reduction in NO and complete loss of prostaglandin-mediated contribution to exercise-related vasodilation (38). In general, evidence suggests the activation of redundant pathways contributing to exercise-hyperemia. Our findings of preserved exercise hyperemia in COPD are concistent with a previous study (25), despite different study designs using different exercise challenges (i.e., mild vs maximal exercise) and vascular segments (forearm vs lower limb). It is likely that the difference in limbs specified for research study is not trivial, as important differences exist in both endothelial-dependent and independent dilation between the forearm and lower limb vasculature (29).
Exercise hyperemia under hyperoxic conditions
A secondary outcome in our study was using hyperoxia to reduce sympathetically mediated vasoconstriction, which may limit blood flow in older adults and, in particular, COPD patients. It is possible that hyperoxia exhibits diverging effects on the vasculature. Diverging opinions exists on the physiological action and precise effect of hyperoxia on blood flow and the subsequent mechanisms contributing to the neuroregulation on the vasculature. Perhaps the most traditional theory suggests that blood flow is related to O2 content, such that with hyperoxia, blood flow is reduced at rest and during exercise (5). Although we did not find that hyperoxia reduced blood flow in the present study, we also did not observe an increase in blood flow with hyperoxia, as predicted. It is possible that the duration of hyperoxia was not long enough to observe these changes. The mechanisms of action of hyperoxia during exercise are complex and likely depend on the duration, concentration, exercise intensity, and health/age status. Contrary to the traditional theories, hyperoxia has been shown to inhibit peripheral chemoreceptor outflow and transiently increase muscle blood flow in younger humans (40). Second, hyperoxia may increase the accumulation of free radicals, leading to increased oxidative stress and reduction of NO (24). During sham-saline, hyperoxia did not have a significant effect on BBF. The fraction of inspired O2 in the present study is lower than previous studies which use 100% O2. It is also likley that there would only be modest influence of the sympathetically mediated constriction during a mild (i.e., 10%), local exercise challenge, using a small muscle mass (compared with cycling, for example). The study by Stickland et al. (40) used a higher work load (∼80% of maximum) and a larger muscle mass. Moreover, as we were interested in steady-state blood flow, it is possible that the transient increase in blood flow would have occurred with the immediate onset of hyperoxic exercise (<60 s), before returning to normoxic levels, as observed in the aforementioned study (40).
Few studies have invesigated the effect of vitamin C during hyperoxic exercise. We did not observe any significant changes in blood flow during the hyperoxic exercise period during vitamin C infusion (or saline). Findings from Ranadive et al. (37) suggest that considerable variation between subjects (i.e., responders and nonresponders) and that vitamin C is only effective in preventing a decline in blood flow during hyperoxic exercise when considerable (20%) hyperoxic-mediated constriction is observed.
Endothelial Function in COPD Patients
Endothelial dysfunction is reflected by an impaired FMD response. Putative mechanisms suggest this response to be partly NO dependent (10). Increased reactive oxygen species (ROS) may decrease NO bioavailability, thereby attenuating the FMD response. We did not find COPD patients to have reduced endothelial function, as determined by FMD, when compared with controls. Contrary to our findings, the general consensus seems to support the contention that COPD patients have lower vascular endothelial function, as assessed by FMD (2,6,11,17). However, Maclay et al. (23) assessed peripheral vascular responses in COPD patients (and controls) using the technique of venous occlusion plethysmography, a measure of resistance arterial (arterioles) function. Blood flow responses to both endothelial-dependent and independent dilators were similar between COPD patients and matched controls, thereby refuting the hypothesis that COPD patients have systemic endothelial dysfunction. Collectively, these findings would suggest that heterogeneity exists within the vascular tree, as conduit artery relaxation is often found to be decreased, whereas resistance vessels remain intact.
In our study, vitamin C was found to increase FMD in COPD and controls. We did not see an interaction effect of vitamin C, suggesting that the overall pathophysiology affecting COPD is similar to healthy older adults. Indeed, a decline in FMD with “normal” aging is evident (>40–50 yr) and, similar to our findings, has been previously found to be restored with vitamin C (12,45). However, results from a recent study (17) suggest that COPD patients (FEV1 = 55%) in fact do have decreased FMD compared with controls, which is restored after an antioxidant intervention, thus suggesting a ROS-related mechanism affecting vascular function in COPD. Several plausible explanations exist for the discrepancy between our findings and those of Ives et al. (17) such as greater presence comorbidities, increased severity of lung obstruction, and increased oxidative stress (as measured by thiobarbituric acid reactive substances). Our aim was to isolate the effect of COPD, thereby excluding patients with overt comorbidities. Interestingly, the findings of Ives et al. (17) did not find that older controls increased FMD after an antioxidant intervention, as may have been expected. It is possible that the health status of the control groups differed. The healthy controls in the present study show an increase in FMD after vitamin C, suggesting oxidative stress impairment of the vascular endothelium.
Study Limitations
One technical limitation of our study was the lack of continuous arterial diameter measurements during handgrip exercise. Although we found no difference between arterial diameter measurements at rest and immediately after handgrip exercise (i.e., resting recovery), the exercise blood flow measurement relies on the assumption that arterial diameter remained constant during exercise. Previous findings (46) suggest that brachial artery diameter remains constant during handgrip below 25% MVC. A limitation of the current study is that it did not use a randomized design. To maximize volunteer retention and reduce day-to-day variability associated with the vascular measurements, we completed the sham-saline and vitamin C trials on the same day. Repeated measurements of FMD have been shown to produce reproducible results after a rest period of 5 to 10 min, or until vessel diameter returns to baseline (3). However, the effects of previous exercise on FMD responses are less certain. It is possible that the combined FMD and vitamin C trial was affected by the previous bout of exercise, which has been described as a biphasic response (i.e., decreased FMD immediately after exercise, followed by an increased FMD 24 h after aerobic exercise) as suggested by previous research (9). However, this is highly dependent on the type of exercise training used (aerobic vs resistance). Despite using a mild, rhythmic handgrip challenge (which did not evoke an increase in blood pressure), it is possible that this affected the post-vitamin C intervention FMD results. Along these lines, we found a decrease in baseline FMD diameter in the vitamin C trial. As artery size is inversely correlated with the FMD response, it is possible that this difference in artery size observed between trials may influence the functional FMD response and, therefore, the interpretation of this finding. Despite the fact that diameter decreased similarly in both groups, it may explain the overall increase in FMD after vitamin C (42). Additionally, differences in shear stress and flow patterns (i.e., antegrade vs retrograde) associated with contraction–relaxation cycles may have initiated different responses across individuals, furthermore affecting FMD. Second, because of the persisting effects of systemic vitamin C administration, we were not permitted to randomize the order of the vitamin C and saline interventions. Third, we do not have an indication of the shear rate during FMD, precluding us to make further mechanistic interpretations of our data. As shear rate is accepted as the stimulus to FMD, it is possible that shear rate was different in the study groups. However, previous findings have not found a difference in shear rate between COPD and controls, despite a difference in FMD (17). Our study is also limited by the small sample size, despite performing a sample size calculation predicting each group to have 9 subjects (19); there was greater variability than expected, thus increasing the probability of a type II error.
CONCLUSIONS
To our knowledge, this is the first study to investigate exercise hyperemia in COPD patients, compared with controls, during conditions of mild handgrip exercise. We also tested the effect of intravenous vitamin C infusion on these responses. Collectively, these results suggest that moderate-COPD patients have preserved exercise blood flow responses and endothelial function when compared with age-matched controls. Despite an increase in endothelial function after vitamin C administration in COPD and older controls, exercise blood flow did not increase, suggesting the importance of NO-independent mechanisms underlying exercise-hyperemia. These results are promising for COPD patients in that moderate patients have preserved vascular function. Caution should be applied when extending these results to other severities of COPD patients, as our patients may not be entirely representative of more severe patients or patients with comorbidities. Oxidative stress is an important factor contributing to many of the vascular impairments that are associated with COPD; however, the spectrum of severity is likely a key factor in determining the overall oxidative stress status and resultant vascular outcomes.
The authors thank the subjects for their time and enthusiasm. The authors also thank Mr. C. Dumonceaux for pulmonary function testing, Ms. L. Knox and Dr. J. Rawling for the help with medical coverage and patient recruitment.
Author contributions: Study conception/design: S. E. H., M. J. P., T. J. A., R. L.; data collection: S. E. H., X. W.; data analysis: S. E. H., X. W.; data interpretation: S. E. H., X. W., T. J. A., R. L., M. J. P.; medical supervision: T. J. A., R. L.; patient recruitment: S. E. H., R. L.; manuscript preparation: S. E. H., X. W., T. J. A., R. L., M. J. P.; fundraising and study supervision: M. J. P., T. J. A. All authors critically reviewed the manuscript and have given their final approval.
Disclosure/Conflict of Interest: None to declare.
Financial support was provided by the CIHR (M. J. P.) the Heart and Stroke Foundation of Canada (M. J. P. and T. J. A.) and Natural Sciences and Engineering Research Council of Canada (M. J. P.). S. E. H. received support from Dr. Chen Fong Doctoral Scholarship (Hotchkiss Brain Institute) and a CIHR operating grant. T. J. A. is a senior scholar of the Alberta Heritage Foundation for Medical Research. R. L. is the GSK-CIHR Professor of Inflammatory Disease. M. J. P. is the Brenda Strafford Foundation Chair in Alzheimer Research. The results of the present study do not constitute endorsement by ACSM.
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