Eight weeks of nitrate supplementation improves blood flow and reduces the exaggerated pressor response during forearm exercise in peripheral artery disease

Abstract

Peripheral artery disease (PAD) is characterized by a reduced blood flow (BF) and an elevated blood pressure (pressor) response during lower extremity exercise. Although PAD is evident in the upper extremities, no studies have determined BF and pressor responses during upper extremity exercise in PAD. Emerging evidence suggests that inorganic nitrate supplementation may serve as an alternative dietary strategy to boost nitric oxide bioavailability, improving exercising BF and pressor responses during exercise. The present study investigated 1) BF and pressor responses to forearm exercise in patients with PAD (n = 21) relative to healthy age-matched control subjects (n = 16) and 2) whether 8 wk of NaNO₃ supplementation influenced BF and pressor responses to forearm exercise in patients with PAD. Patients with moderate to severe PAD were randomly assigned to a NaNO₃ (1 g/day, n = 13)-treated group or a placebo (microcrystalline cellulose, n = 8)-treated group. Brachial artery forearm BF (FBF; via Doppler) and blood pressure (via finger plethysmography) were measured during mild-intensity (~3.5-kg) and moderate-intensity (~7-kg) handgrip exercise. The absolute change (from baseline) in FBF was reduced (except in the 3.5-kg condition) and BP responses were increased in patients with PAD compared with healthy control subjects in 3.5- and 7-kg conditions (all P < 0.05). Plasma nitrate and nitrite were elevated, exercising (7-kg) ΔFBF was improved (from 141 ± 17 to 172 ± 20 ml/min), and mean arterial pressure response was reduced (from 13 ± 1 to 9 ± 1 mmHg, P < 0.05) in patients with PAD that received NaNO₃ supplementation for 8 wk relative to those that received placebo. These results suggest that the BF limitation and exaggerated pressor response to moderate-intensity forearm exercise in patients with PAD are improved with 8 wk of NaNO₃ supplementation.

NEW & NOTEWORTHY Peripheral artery disease (PAD) results in an exaggerated pressor response and reduced blood flow during lower limb exercise; however, the effect of PAD in the upper limbs has remained unknown. These results suggest that 8 wk of inorganic nitrate supplementation improves the blood flow limitation and exaggerated pressor response to moderate-intensity forearm exercise in PAD.

INTRODUCTION

Peripheral artery disease (PAD), a progressive atherosclerotic disease of the upper and lower extremities, is closely linked to cardiovascular and cerebrovascular mortality (9, 14). Several characteristics are associated with PAD, including flow-limiting atherosclerotic stenosis in conduit vessels and reduced blood flow to the periphery, which is mechanistically attributed to endothelial dysfunction (17, 30), impaired skeletal muscle metabolism (27), and a heightened exercise pressor reflex (27, 40). Blood flow responses are impaired in the exercising lower limbs of patients with PAD, leading to reductions in functional work capacity (25, 26). However, PAD of the upper extremity is not rare (31, 34, 39) and, when present, can manifest as an impaired endothelial function in the brachial artery (4, 33). Although there is little evidence of impaired brachial artery endothelium-mediated vasodilation at rest in PAD (4, 33), the hyperemic and vasodilatory responses to upper extremity exercise in PAD have not been studied. Given that endothelium-mediated vasodilation is an important mechanism for increasing local skeletal blood flow during exercise, it is possible that flow and vasodilation are impaired during upper extremity exercise in PAD. Furthermore, although it is well understood that patients with PAD have an augmented blood pressure (i.e., pressor) response to dynamic exercise of the lower extremities (relative to healthy control subjects) (19, 29, 40, 43), it remains uncertain whether these differences can be extrapolated to other limbs (i.e., upper body) during dynamic exercise in PAD.

Several lines of evidence suggest that patients with PAD have severely compromised endothelial function in the upper and lower limbs (30); this is likely due, in part, to an impaired ability of endothelial cells to release/produce nitric oxide (NO) (8, 42). More specifically, NO generated by the l-arginine-NO synthase (NOS) pathway is a robust vasodilator and key regulator of vascular integrity (11). Accumulating evidence suggests that inorganic nitrate (NO₃) may be reduced to nitrite (NO₂) and, subsequently, NO (i.e., the NO₃-NO₂-NO pathway), representing an alternative and differentially regulated system for NO generation that operates in parallel with the classic l-arginine-NOS pathway in low-Po₂ environments (5, 22, 23). Moreover, PAD is a condition of hypoxia in the peripheral tissues; therefore, any intervention that improves blood flow and oxygenation to these areas could improve physical function and may serve as a significant, alternative treatment option to medications with known negative side effects. Along these lines, recent evidence in experimental animals suggests that chronic dietary NO₃ supplementation increases blood flow and vasodilation during exercise (10). Therefore, increasing plasma NO₃ and NO₂ levels in patients with PAD via long-term ingestion of inorganic NO₃ supplementation may enhance NO bioavailability and, subsequently, the hyperemic and vasodilator responses to forearm exercise.

The primary aims of this investigation were to examine 1) the acute hyperemic, vasodilatory, and blood pressure responses to forearm exercise in patients with PAD relative to healthy control subjects and 2) whether 8 wk of inorganic NO₃ supplementation in the form of NaNO₃ influenced the hyperemic, vasodilatory, and pressor responses to forearm exercise in PAD. We hypothesized that exercising forearm blood flow (FBF) and forearm vascular conductance (FVC) would be reduced in patients with PAD compared with healthy control subjects. We further hypothesized that 1) blood pressure responses to forearm exercise would be elevated in patients with PAD (relative to healthy control subjects) and 2) 8 wk of NaNO₃ supplementation would elevate plasma levels of NO₃ and NO₂, leading to increased FBF and FVC and reduced blood pressure responses during forearm exercise in PAD.

METHODS

Subjects

Twenty-one older adults (12 men and 9 women, 72 ± 2 yr old) with documented, moderate to severe PAD were recruited into the study. Exclusion criteria were nonatherosclerotic vascular disease, critical limb ischemia, active ischemic ulceration, recent (within 6 mo) revascularization, symptomatic coronary artery disease or heart failure, renal disease, hypotension (resting systolic blood pressure < 90 mmHg), smoking or history of smoking within the past year, and use of phosphodiesterase 5 inhibitor drugs. Hemodynamic responses of patients with PAD were compared with those of a healthy age-matched control group (7 men and 9 women) whose data were derived from a database of subjects that participated in previous studies in our laboratory. All healthy control subjects completed a general health history screening and were generally healthy, free of diagnosed cardiovascular or metabolic complications, nonobese (body mass index ≤ 30 kg/m²), nonsmokers, and not taking any vasoactive medications. All women enrolled in the study were postmenopausal and not receiving hormone therapy. All participants (patients with PAD and age-matched control subjects) were considered sedentary to recreationally active. Before participation, all participants provided written informed consent approved by the University of Iowa’s Institutional Review Board.

Experimental Design

Protocol 1: effect of forearm exercise on hemodynamics in patients with PAD compared with healthy control subjects.

Protocol 1 was a cross-sectional study to assess whether patients with PAD exhibit an impaired hyperemic and heightened pressor response during forearm exercise. Forearm hemodynamic responses in a cohort of patients with PAD were compared with responses of healthy age-matched control subjects. Accordingly, healthy control subjects and patients with PAD performed the same protocol: mild-intensity (3.5-kg) and moderate-intensity (7-kg) handgrip exercise.

Protocol 2: effect of NaNO₃ supplementation on hemodynamics in PAD.

Protocol 2, the interventional portion of the study, was a randomized, double-blind, placebo-controlled design, such that subjects were randomly assigned to a NaNO₃ (1 g/day)-treated group or a placebo (microcrystalline cellulose)-treated group for 8 wk. Pre- and postsupplement assessments were identical for both visits within each respective intervention of the investigation. Subjects were instructed to follow a low-NO₃ diet 72 h before each study visit. For each experimental visit, all subjects arrived at the laboratory after an overnight fast and abstained from exercise as well as alcohol and caffeine consumption for 24 h before all study visits. Additionally, they did not use prescription medication on the morning of all study visits. Brachial artery blood velocity and mean arterial pressure (MAP) responses to forearm exercise were recorded in a 60-s period (baseline) before and during 3 min of continuous dynamic forearm contractions.

Experimental Measurements

Venous blood sampling.

Venous blood was sampled only for protocol 2 and was obtained from an antecubital vein using aseptic techniques for the determination of NO metabolites [$N{O}_3^{-}$ concentration ([$N{O}_3^{-}$]) and $N{O}_2^{-}$ concentration ([$N{O}_2^{-}$])]. Blood was collected in tubes containing EDTA and immediately centrifuged at 3,000 rpm for 15 min. Plasma was divided into aliquots, added to Eppendorf tubes, and immediately frozen at −80°C for future analysis. Plasma was thawed, and [$N{O}_3^{-}$] and [$N{O}_2^{-}$] were measured within 30 min using a chemiluminescence NO analyzer (model NOA 280i, Sievers Instruments, Boulder, CO). Plasma [$N{O}_3^{-}$] and [$N{O}_2^{-}$] were determined by addition to vanadium III chloride in hydrochloric acid at 90°C and addition to potassium iodide in acetic acid at room temperature, respectively.

Forearm exercise.

Rhythmic, dynamic forearm exercise (completed with the left hand) consisted of squeezing and releasing two handles together 4 cm apart on a custom-built, handgrip device attached to a simple pulley device at two absolute workloads. We selected a mild-intensity (3.5-kg) and a moderate-intensity (7-kg) absolute handgrip exercise workload that could be performed over a 3-min duration by both groups of participants. Each forearm exercise trial was separated by 10 min of quiet rest. Forearm contractions, which were completed at 20 min⁻¹ for 3 min, were controlled by a metronome (1:2-s duty cycle). Accordingly, for each duty cycle, subjects were cued to quickly contract with their forearm muscles, bringing the pillars together and then rapidly releasing the handles (all within 1 s); this was followed by a 2-s period of complete relaxation. Thus, the calculated blood flow during steady state included the contraction and relaxation phases. Subjects were cued to contract at the sound of the beat of a metronome and release the weight (without the intent to contract upon descent) when the metronome went silent during the relaxation phase.

Measurement of FBF and blood pressure.

Brachial artery diameter and blood velocity were determined with a 12-MHz linear-array Doppler probe (model M12L, Vivid 7, General Electric, Milwaukee, WI) with the insonation angle kept at 60°. Velocity waveforms were synchronized to a data-acquisition system (WinDaq, DATAQ Instruments, Akron, OH) through a Doppler audio transformer (13). End-diastolic brachial artery diameter measurements were obtained at rest and at the end of steady-state conditions. FBF (in ml/min) was calculated as the product of mean blood velocity (in cm/s) and brachial artery cross-sectional area (in cm²). Brachial artery pressure was measured in duplicate using an automated cuff (Cardiocap/5, Datex-Ohmeda, Louisville, CO) after 20 min of rest, before the initiation of experimental testing. For measurement of beat-by-beat arterial pressure by finger plethsymography (Nexfin, Edward Lifesciences, Irvine, CA), a finger cuff was placed around the middle phalanx of the third digit on the nonexercising hand to assess blood pressure changes throughout each exercise protocol.

Data Acquisition and Analysis

Data were collected at 250 Hz, stored on a computer, and analyzed offline with signal-processing software (WinDaq). MAP was determined from the finger artery pressure waveforms from the Nexfin device. All hemodynamic measurements within each trial were determined by averaging values during the last 30 s of rest and steady-state exercise (third min of exercise). FVC (in ml·min⁻¹·100 mmHg⁻¹) was calculated using the quotient of FBF and MAP. To account for potential baseline differences in FBF and FVC (between groups or after the intervention period) the change (Δ) in FBF and FVC during exercise was adjusted (exercise – baseline values) and expressed as ΔFBF and ΔFVC.

Statistical Analyses

All statistical analyses were completed using SigmaPlot version 11.0 (Systat Software, San Jose, CA). For the cross-sectional comparisons (protocol 1) between patients with PAD and healthy control subjects, differences in subject demographics as well as exercising FBF, FVC, and MAP responses between groups were assessed using an independent-samples t-test. For the intervention portion of the study (protocol 2), two-way repeated-measures ANOVA was used to compare group (NaNO₃ vs. placebo) × time (pre vs. post) differences before and after 8 wk of supplementation. If a significant interaction existed, pair-wise comparisons were made using Tukey’s post hoc analysis. Significance was set at P < 0.05. Values are means ± SE.

RESULTS

Protocol 1

Subject characteristics for the PAD and control groups are shown in Table 1. Compared with healthy age-matched control subjects, patients with PAD exhibited greater weight, body mass index, and resting systolic blood pressure (P < 0.05). True baseline values for FBF (58 ± 11 vs. 50 ± 9 ml/min) and FVC (63 ± 12 vs. 54 ± 11 ml·min⁻¹·100 mmHg⁻¹) before 3.5-kg forearm exercise for PAD and control groups were not significantly different from one another. Furthermore, baseline FBF and FVC did not differ between 3.5- and 7-kg forearm exercise within either group, respectively.

Table 1.

Characteristics of patients with PAD and healthy control subjects at rest

Characteristics	Patients with PAD	Control Subjects
Number of subjects	21	16
Age, yr	72 ± 2	66 ± 1
Height, m	1.70 ± 0.02	1.68 ± 0.02
Weight, kg	82.9 ± 2.5*	70.7 ± 2.5
Body mass index, kg/m2	28.8 ± 1.1*	25.1 ± 0.7
Systolic blood pressure, mmHg	135 ± 3*	123 ± 4
Diastolic blood pressure, mmHg	74 ± 3	75 ± 2
Mean arterial pressure, mmHg	94 ± 2	91 ± 2
Ankle brachial index	0.78 ± 0.04
Previous revascularization, n	18 (86)
Coronary artery disease, n	3 (14)
Type 2 diabetes mellitus, n	6 (29)
Medication use, n
Statin	19 (91)
Angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker	8 (38)
β-Blocker	9 (43)
Ca2+ channel blocker	7 (33)
Blood thinner	8 (38)
Insulin	3 (14)

Values are means ± SE. Values in parentheses denote percentages of patients with peripheral arterial disease (PAD) with each specific condition or taking each respective drug.

^*P < 0.05 vs. control subjects.

Comparisons of hemodynamic responses of the PAD and control groups during exercise are shown in Figs. 1 and 2. At rest, there were no differences in FBF and FVC between the PAD and control groups. Changes in FBF and FVC during forearm exercise were less in the PAD group than in the control group during 7-kg forearm exercise (all P < 0.05; Fig. 1). There was a trend for a decrease in FBF (P = 0.08) and FVC (P = 0.06) responses to mild-intensity (3.5-kg) exercise in the PAD group compared with the control group; however, these changes were not statistically significant. The absolute change in MAP in response to forearm exercise was increased in the PAD group during 3.5- and 7-kg workloads compared with the control group (both P < 0.05; Fig. 2).

Fig. 1.

Change (Δ) in forearm blood flow (A) and forearm vascular conductance (B) during mild-intensity (3.5-kg) and moderate-intensity (7-kg) forearm exercise in patients with peripheral artery disease (PAD) and healthy age-matched control subjects (protocol 1). Values are means ± SE; n = 21 patients with PAD and 16 control subjects. *P < 0.05 vs. control (by independent t-test).

Fig. 2.

Change (Δ) in mean arterial pressure during mild-intensity (3.5-kg) and moderate-intensity (7-kg) forearm exercise in patients with peripheral artery disease (PAD) and healthy age-matched control subjects (protocol 1). Values are means ± SE; n = 21 patients with PAD and 16 control subjects. *P < 0.05 vs. control (by independent t-test).

Protocol 2

Subject characteristics among the PAD groups (NaNO₃ vs. placebo) are shown in Table 2. No differences in age, body mass index, and resting systolic and diastolic blood pressures were observed between the NaNO₃- and placebo-treated groups. Systolic blood pressure was reduced after NaNO₃ (from 136 ± 4 to 129 ± 5 mmHg, P < 0.05) but not placebo (from 132 ± 5 to 132 ± 4 mmHg) supplementation. Diastolic blood pressure and MAP were unchanged in both groups. Furthermore, the relative percentage of subjects in each experimental group did not differ for medication use and/or clinical history.

Table 2.

Characteristics of NaNO₃- and placebo-treated patients with peripheral artery disease at rest

Characteristics	NaNO3	Placebo
Sex, men/women	6/7	6/2
Age, yr	74 ± 3	69 ± 4
Body mass index, kg/m2	29.2 ± 1.6	28.1 ± 1.3
Systolic blood pressure, mmHg	136 ± 4	132 ± 5
Diastolic blood pressure, mmHg	72 ± 3	77 ± 4
Mean arterial pressure, mmHg	94 ± 3	95 ± 4

Values are means ± SE.

Plasma [$N{O}_3^{-}$] and [$N{O}_2^{-}$].

Eight weeks of inorganic NO₃ supplementation increased plasma [$N{O}_3^{-}$] from 32.3 ± 5.6 to 379.8 ± 56.7 µM (P < 0.05) and [$N{O}_2^{-}$] from 192.2 ± 15.0 to 353.1 ± 38.7 nM (P < 0.05). Placebo supplementation did not change plasma [$N{O}_3^{-}$] (34.3 ± 6.6 to 77.5 ± 24.7 µM, P = 0.23) or [$N{O}_2^{-}$] (249.7 ± 12.6 to 230.3 ± 28.9 nM, P = 0.47). Subsequently, plasma [$N{O}_3^{-}$] and [$N{O}_2^{-}$] were higher after 8 wk of inorganic NO₃ than placebo supplementation (P < 0.05 for both).

Effect of dietary NaNO₃ supplementation on hemodynamics.

Baseline (resting) FBF and FVC values were not different after NaNO₃ versus placebo before each exercise trial (3.5 and 7 kg). Eight weeks of inorganic NO₃ supplementation increased FBF and FVC during 7-kg forearm exercise (group × time interaction, both P < 0.05; Fig. 3), whereas there was no interaction within the 3.5-kg condition (NaNO₃ vs. placebo); however, there was a main effect of time for FVC (pre- vs. postsupplementation). Similarly, 8 wk of inorganic NO₃ supplementation attenuated the pressor (MAP) response (pre- vs. postsupplementation, P < 0.05) during 7-kg forearm exercise (Fig. 4); however, the pressor (MAP) response during 3.5-kg forearm exercise (Fig. 4), while it qualitatively showed a decrease within the NaNO₃-treated group, did not reach statistical significance (group × time interaction, P = 0.08), but there was a main effect of time for MAP (pre- vs. postsupplementation), respectively.

Fig. 3.

Change (Δ) in forearm blood flow (FBF; A) and forearm vascular conductance (B) during mild-intensity (3.5-kg) and moderate-intensity (7-kg) forearm exercise in NaNO₃-supplemented and placebo-treated groups (protocol 2). Values are means ± SE; n = 13 NaNO₃-supplemented and 8 placebo-treated subjects. There was no interaction for FBF (P = 0.07) and FVC (P = 0.08) before and after NaNO₃ (or placebo) supplementation during 3.5-kg forearm exercise. There was an interaction (two-way repeated-measures ANOVA) and a significant increase in FBF and FVC during 7-kg forearm exercise before versus after NaNO₃ supplementation. *P < 0.05 vs. Pre.

Fig. 4.

Change (Δ) in mean arterial pressure (MAP) during mild-intensity (3.5-kg) and moderate-intensity (7-kg) forearm exercise in NaNO₃-supplemented and placebo-treated groups (protocol 2). Values are means ± SE; n = 13 NaNO₃-supplemented and 8 placebo-treated subjects. There was no interaction (two-way repeated-measures ANOVA) for MAP (P = 0.08) during 3.5-kg forearm exercise; however, there was an interaction during 7-kg forearm exercise, such that NaNO₃ supplementation reduced MAP (Pre vs. Post). *P < 0.05 vs. Pre.

DISCUSSION

This is the first study to examine hyperemic, vasodilatory, and pressor responses to acute forearm exercise in patients with PAD and whether 8 wk of inorganic NO₃ supplementation (in the form of NaNO₃) improves these hemodynamic responses. Our primary findings are as follows: 1) steady-state FBF and FVC during moderate-intensity forearm exercise are impaired in patients with PAD relative to their healthy age-matched counterparts, 2) 8 wk of inorganic NO₃ supplementation effectively improves FBF and FVC during moderate-intensity forearm exercise in patients with PAD, and 3) the heightened pressor response during acute forearm exercise in PAD is improved (reduced) with 8 wk of inorganic NO₃ supplementation. Collectively, our findings suggest that 1) inorganic NO₃ supplementation represents a potent and effective therapeutic nutritional strategy to improve blood flow and vascular conductance in the exercising forearm of patients with diagnosed PAD and 2) the exaggerated pressor responses during exercise in PAD can be improved with inorganic NO₃ supplementation.

Forearm Exercise Blood Flow and Vasodilation Are Reduced in PAD

PAD is typically characterized by atherosclerotic plaque formation in the large arteries, most notably, within the lower extremities, resulting in reduced blood flow and perfusion to the working muscle (32). While PAD has been widely investigated in the lower extremities, it is considered to be a diffuse disease, affecting both lower and upper extremities (9), subsequently resulting in upper and lower extremity weakness. Although previous research has shown that flow-mediated dilation in the brachial artery (endothelium-dependent function) is impaired in patients with PAD (4, 33), information related to exercise blood flow in the upper extremities in PAD was lacking. The present study, therefore, presents the first data demonstrating that blood flow and vasodilation are impaired during moderate-intensity (7-kg) forearm exercise in patients with PAD. Our finding in the forearm is in agreement with previous evidence of reduced exercising blood flow in the lower extremities of patients with PAD (25, 26). Furthermore, our findings suggest that the hyperemic and vasodilatory responses during mild-intensity (3.5-kg) forearm exercise, while approaching statistical significance (P = 0.06–0.08), appear to remain similar to those of healthy age-matched control subjects. Collectively, these results are interpreted to suggest that the FBF and vasodilatory responses during mild-intensity handgrip exercise are likely negligible in PAD. However, as exercise intensity increases (i.e., moderate intensity), the ability to increase blood flow and, presumably, O₂ delivery is decreased in the face of increasing metabolic demand in PAD.

Forearm Exercise Blood Flow and Vasodilation Are Improved With 8 wk of Inorganic NO₃ Supplementation in PAD

To our knowledge, this is the first study to examine the effect of 8 wk of inorganic NO₃ supplementation on upper extremity hemodynamics in PAD. One manifestation of PAD is limb ischemia resulting in claudication within the peripheral tissues, which creates a hypoxic environment. Therefore, any dietary intervention that improves blood flow and oxygenation under such instances may serve as a significant, alternative treatment option to medications with known negative side effects. Along these lines, inorganic NO₃ acts as a targeted supplement, whereby the resulting NO₂ is reduced to NO in tissues with a low Po₂, which may facilitate better overall blood flow response and allow for greater O₂ extraction, especially in individuals with an impaired hyperemic response to exercise (i.e., PAD) (15, 18, 41). Additionally, NO₂ and/or NO signaling increases blood flow and targets areas of hypoxia (7, 24), most notably related to type II muscle fibers (10). Therefore, increasing circulating NO₂ may preferentially increase peripheral blood flow where it is needed and possibly improve physical function through increased blood flow and tissue oxygenation in PAD.

In support of this hypothesis, acute (2–3 h) inorganic NO₃ ingestion in the form of beetroot juice ameliorates the impaired forearm exercising blood flow responses in older adults under hypoxic conditions (6) and improves muscle oxygenation in patients with PAD (16). In addition, Ferguson et al. (10) recently demonstrated that inorganic NO₃ supplementation improves vascular control and elevates skeletal muscle O₂ delivery during exercise predominantly in glycolytic type II muscles in isolated animal preparations, thus providing a potential mechanism by which NO₃ supplementation improves metabolic control during higher-intensity exercise. Interestingly, the impaired hyperemic responses of the present study were observed during moderate-intensity (7-kg) but not mild-intensity (3.5-kg) exercise in PAD. Thus, our findings could be interpreted to suggest that, in moderate-intensity exercise conditions, which facilitate a lower-Po₂ environment, typified by a greater recruitment of type II muscle fibers, there is a greater hyperemic impairment in patients with PAD. Importantly, 8 wk of NaNO₃ supplementation, which boosts NO bioavailability (based on elevations in its plasma metabolites), mitigates the PAD-associated hyperemic impairment during moderate-intensity forearm exercise.

Exercise Pressor Response in PAD

It is well known that patients with PAD have an exaggerated blood pressure response to lower body dynamic exercise (1, 2, 20, 29) due, in part, to an augmented muscle mechano- and metaboreflex compared with healthy age-matched control subjects (21, 27, 40). This exaggerated pressor response during exercise may be a normal compensatory mechanism in the face of reduced peripheral perfusion associated with PAD; however, sustained high afterload/pulse pressure may damage the vasculature and tissues of the heart and brain over time (29). The present study adds to preexisting evidence in the legs (29, 40) by demonstrating an exaggerated pressor response during mild- to moderate-intensity handgrip exercise in patients with PAD relative to healthy control subjects. Furthermore, 8 wk of inorganic NO₃ supplementation ameliorates the exaggerated pressor response during moderate-intensity handgrip exercise in patients with PAD, similar to levels in healthy age-matched control subjects. Therefore, because dynamic exercise is the primary stimulus that provokes symptoms in PAD (e.g., intermittent claudication) and because patients with PAD have larger pressor responses to higher-intensity exercise (2, 12), our findings are novel and important, as they demonstrate that the exaggerated pressor response to forearm contractions can be improved (attenuated) by 8 wk of inorganic NO₃ supplementation.

Experimental Considerations

There are a few experimental considerations that warrant mentioning. First, absolute, rather than relative, intensity forearm exercise was examined in the present study. Therefore, it may be speculated that differences between patients with PAD and control subjects (before NaNO₃ supplementation) are a result of the exercise stimulus, inasmuch as performing an absolute exercise intensity may yield differences in relative intensity and, consequently, differences in exercising blood flow. However, evidence suggests that hand function/strength remains unchanged until the age of 65 yr, after which it declines gradually (36). Accordingly, patients with PAD and control subjects in the present study were ~65 yr of age, and these groups did not differ with age. Furthermore, it has recently been shown that maximum voluntary contraction of the forearm and ratings of perceived exertion during forearm exercise are similar between PAD and control groups (35). Collectively, this evidence strongly supports the premise that patients with PAD and control subjects in the present study likely exhibited similar maximum voluntary contractions, and, therefore, absolute workloads were performed at a very similar relative intensity between groups. Second, although impairment in forearm hyperemia and FVC was observed in the present study, it may have minimal clinical significance, as the functional limitation and, most importantly, the deterioration in the quality of life of patients with PAD can be largely ascribed to leg symptoms. In addition, different patients with PAD were included in the NaNO₃- and placebo-supplemented groups; therefore, this study design may not be statistically as “strong” as a study design in which one group of patients with PAD underwent both interventions separately. Finally, although previous work showed that ~75% of patients exhibit atherosclerotic lesions within the arms, it cannot be definitively concluded that the patients with PAD in the present study had atherosclerotic lesions within the arm, as only documented atherosclerotic lesions within the legs were used for clinical diagnosis of PAD. Accordingly, the results of the present study have merely attempted to elucidate whether abnormalities of vascular function, which may occur in the absence (and potentially before the development) of critical atherosclerotic lesions, exist within the vessels of the arm. Future work, therefore, may benefit from delineating the patients with PAD with atherosclerotic lesions within the arm from those who do not on the exercise pressor, hyperemic, and vasodilator responses in PAD, respectively.

Perspectives and Significance

PAD is considered a diffuse disease whereby atherosclerotic lesions are evident in both the upper and lower extremities. Furthermore, individuals with PAD may not exhibit claudication in the upper limbs, but the impairment in hyperemia and conductance could contribute to impairments in functional work capacity and/or exercise tolerance. Therefore, mechanistic information related to blood flow and vasodilation within the exercising forearms of patients with PAD warrants appropriate attention. Interestingly, over 20 yr ago, Sorensen and colleagues (39) reported a very high (75%) prevalence of atherosclerosis in the brachial arteries of autopsied subjects, suggesting that the brachial circulation may serve as a reasonable “surrogate” for studying atherosclerosis in humans. Despite this information, almost all available evidence on resting and exercising blood flow in PAD has been demonstrated in the lower extremities, and until this study, there was no information regarding circulatory function within the arms during exercise. Therefore, our findings are novel and relevant, as we present the first evidence of upper extremity impairment in exercising blood flow in PAD. Importantly, this study shows that 8 wk of inorganic NO₃ supplementation increases brachial artery blood flow during upper extremity exercise in PAD. Furthermore, from a clinical and practical perspective, an exaggerated pressor response increases the likelihood of adverse cardiovascular events during or after exercise in “at-risk” individuals (37, 38) and apparently healthy adults (3). Therefore, the capacity to reduce the exaggerated blood pressure response to exercise via dietary NaNO₃ supplementation has important clinical value in terms of the potential to reduce future cardiovascular events in patients with PAD.

Conclusions

This study demonstrates that, in the moderate-intensity exercise condition, FBF and FVC are lower and pressor responses are exaggerated (i.e., increased) in patients with PAD compared with healthy control subjects. Furthermore, 8 wk of inorganic NO₃ supplementation improved brachial artery blood flow and vasodilation and reduced the exaggerated pressor responses during moderate-intensity forearm exercise in patients with PAD. Collectively, our findings suggest that the impaired hyperemic and exaggerated pressor responses to upper extremity exercise in PAD can be improved by 8 wk of inorganic NO₃ supplementation.

GRANTS

This work was supported by American Heart Association Grant 13GRNT16490002 (to D. P. Casey) and National Institutes of Health Clinical and Translational Science Award U54-TR-001356.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.T.K. and D.P.C. analyzed data; N.T.K., W.E.H., and D.P.C. interpreted results of experiments; N.T.K. prepared figures; N.T.K. drafted manuscript; N.T.K., K.U., W.E.H., and D.P.C. edited and revised manuscript; N.T.K., K.U., W.E.H., and D.P.C. approved final version of manuscript; K.U., W.E.H., and D.P.C. performed experiments; D.P.C. conceived and designed research.