Microvascular Research 98 (2015) 48–53
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Short-term dietary nitrate supplementation augments cutaneous vasodilatation and reduces mean arterial pressure in healthy humans Jeremy T. Keen a, Erica L. Levitt a, Gary J. Hodges c, Brett J. Wong a,b,⁎ a b c
Department of Kinesiology, Kansas State University, Manhattan, KS, USA Department of Kinesiology & Health, Georgia State University, Atlanta, GA, USA Department of Kinesiology, Brock University, St. Catharines, ON L2S 3A1, Canada
a r t i c l e
i n f o
Article history: Accepted 21 December 2014 Available online 30 December 2014 Keywords: Nitric oxide Microvasculature Blood pressure Microdialysis Beetroot juice
a b s t r a c t Nitrate supplementation in the form of beetroot juice has been shown to increase nitric oxide (NO) where nitrate can be reduced to nitrite and, subsequently, to NO through both nitric oxide synthase (NOS)-dependent and -independent pathways. We tested the hypothesis that nitrate supplementation would augment the NO component of the cutaneous vasodilatation to local skin heating in young, healthy humans. Participants reported to the lab for pre- and post-supplement local heating protocols. Nitrate supplementation consisted of one shot (70 ml) of beetroot juice (0.45 g nitrate; 5 mM) for three days. Six participants were equipped with two microdialysis fibers on the ventral forearm and randomly assigned to lactated Ringer's (control) or continuous infusion of 20 mM L-NAME (NOS inhibitor). The control site was subsequently perfused with L-NAME once a plateau in skin blood flow was achieved to quantify NOS-dependent cutaneous vasodilatation. Skin blood flow via laser-Doppler flowmetry (LDF) and mean arterial pressure (MAP) were measured; cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to %CVCmax. Beetroot juice reduced MAP (Pre: 90 ± 1 mm Hg vs. Post: 83 ± 1 mm Hg) and DBP (Pre: 74 ± 2 mm Hg vs. Post: 62 ± 3 mm Hg) (P b 0.05). The plateau phase of the local heating response at control sites was augmented post-beetroot juice (91 ± 5%CVCmax) compared to pre-beetroot juice (79 ± 2%CVCmax) (P b 0.05). There was no difference in the %NOS-dependent vasodilatation from pre- to post-beetroot juice. These data suggest that nitrate supplementation via beetroot juice can reduce MAP and DBP as well as augment NOS-independent vasodilatation to local heating in the cutaneous vasculature of healthy humans. © 2014 Elsevier Inc. All rights reserved.
Introduction Local heating of the skin results in a robust increase in cutaneous blood flow, termed cutaneous thermal hyperemia. Cutaneous thermal hyperemia in response to rapid, non-painful local heating exhibits a reproducible biphasic increase in skin blood flow: an initial transient increase in skin blood flow punctuated by a brief nadir followed by a prolonged secondary plateau phase. The initial peak is predominantly mediated by a sensory nerve axon reflex mechanism, possibly involving the neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP) (Brain et al., 1986; Wong and Minson, 2011) and activation of transient receptor potential vanilloid type 1 (TRPV-1) channels (Wong and Fieger, 2010). The prolonged secondary plateau is mediated largely (~60%) by nitric oxide (NO) (Bruning et al., 2012; Kellogg et al., 1999, 2008, 2009; Minson et al., 2001, 2002) via endothelial NO synthase (eNOS) (Bruning et al., 2012; Kellogg et al., 2008, 2009), where inhibition of NO synthase (NOS) significantly reduces the secondary plateau ⁎ Corresponding author at: Department of Kinesiology & Health, P.O. Box 3975, Georgia State University, Atlanta, GA 30302-3975, USA. Fax: +1 404 413 8053. E-mail address: [email protected] (B.J. Wong).
http://dx.doi.org/10.1016/j.mvr.2014.12.002 0026-2862/© 2014 Elsevier Inc. All rights reserved.
phase of cutaneous thermal hyperemia by ~ 60–70% (Hodges and Sparks, 2014; Kellogg et al., 1999, 2008, 2009; Minson et al., 2001). Dietary nitrate supplementation via beetroot juice has been shown to improve measures of health (Kapil et al., 2010; Webb et al., 2008b), increase blood flow to contracting skeletal muscle (Ferguson et al., 2013), and enhance peripheral vasodilatation and endothelial-derived NO in peripheral arterial disease patients (Kenjale et al., 2011). The increase in NO bioavailability from nitrate supplementation has been shown to occur through both NOS-independent (Lundberg and Weitzberg, 2009; Lundberg et al., 2008) and NOS-dependent (Vanin et al., 2007; Webb et al., 2008a) pathways. Studies have demonstrated that reduction of nitrate to nitrite and, subsequently, to NO, can provide a valuable pathway in which the body can utilize NO through a NOS-independent pathway unlike the traditional L-arginine pathway that is reliant on the NOS enzyme and cofactors (e.g., Ca2 + and BH4). Whether it is through NOS-independent or NOS-dependent pathways, nitrate supplementation may provide a means by which to improve NO-mediated vasodilatation. The purpose of this study was to investigate the effect of dietary nitrate supplementation via beetroot juice on the skin blood flow response to local heating in healthy humans. We tested the hypothesis that nitrate supplementation would augment the contribution of NO
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to cutaneous thermal hyperemia in healthy humans primarily by increasing NOS-dependent vasodilatation. Methods Ethical approval The Institutional Review Board at the Kansas State University approved all protocols of this study. A written informed consent was reviewed and signed by each subject prior to participation. All procedures and protocols were performed in conjunction with the standards set forth by the Declaration of Helsinki. Subjects
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Mean arterial pressure was monitored beat-by-beat via photoplethysmography (NexfinHD; BMEYE, Amsterdam, The Netherlands) from a finger on the right (non-experimental) hand and verified every 5 min via automated brachial oscillometry (S/5 Light Monitor; GE Healthcare; Madison, WI, USA) from the right (non-experimental) arm. All blood pressure measurements were made with the subject in the supine position. Resting blood pressures were taken after the subject had been resting in the supine position for at least 10 min. Heart rate was recorded from the beat-by-beat arterial blood pressure waveform. Red blood cell (RBC) flux was used as an index of skin blood flow via laser-Doppler flowmetry (LDF) (PeriFlux 5010 laser-Doppler perfusion monitor; Perimed; Järfälla, Sweden). Local heating units (PF5020 local heating units and PeriFlux 5020 Temperature Unit; Perimed; Järfälla, Sweden) were placed on the skin directly over each microdialysis membrane, and an integrated laser-Doppler probe (Probe 413; Perimed; Järfälla, Sweden) was placed in the center of each local heating unit to measure RBC flux directly over each microdialysis site.
Six male subjects (age 24 ± 1 yr, height 177 ± 6 cm, body mass 80 ± 13 kg, BMI 26 ± 3 kg/m2) participated in this study. All subjects were healthy, non-smokers, taking no medications and were free of cardiovascular, respiratory, and metabolic diseases as determined by a medical history questionnaire. Subjects were asked to refrain from exercise, consumption of abnormal amounts (N300 g/day) of leafy green vegetables (e.g., spinach), consumption of alcohol, and the use of mouthwash products containing alcohol during the three day beetroot juice supplementation period. Exercise and consumption of large quantities of leafy green vegetables can increase systemic nitrates while alcohol and alcohol-based mouthwash can kill the bacteria required to reduce nitrate to nitrite and subsequently to nitric oxide (Bailey et al., 2009; Govoni et al., 2008). Subjects were asked to refrain from caffeine at least 12 h prior to the study. Testing was administered in a thermoneutral environment with a room temperature of ~23 °C.
Lactated Ringer's solution was administered at the control site. A 20 mM concentration of L-NAME was used as a non-selective NOS inhibitor (Bruning et al., 2012; Brunt et al., 2013; Hodges et al., 2006; Smith et al., 2011). At the end of the local heating protocol, a 54 mM concentration of sodium nitroprusside (SNP) was administered while simultaneously locally heating the skin to 43 °C to elicit maximal vasodilatation. All drugs were infused through the microdialysis fibers at a rate of 2 μl·min−1 via microinfusion pumps (Bee Hive controller and Baby Bee Syringe Pumps; Bioanalytical Systems, West Lafayette, IN, USA) to each randomly assigned site.
Dietary intervention
Local heating protocol
Upon completion of pre-supplement testing, subjects received three days of nitrate rich beetroot juice (70 ml shots; ~5 mM/day; 0.45 g of nitrates) (Beet It, James White Drinks, Ipswich, UK) (Bailey et al., 2010; Vanhatalo et al., 2010). Subjects were instructed to drink one 70 ml shot for three consecutive days with the last shot consumed in the laboratory approximately 1.5–2 h prior to beginning the local heating protocol. Peak circulating plasma level of nitrites (approximate range: 200–600 nM) has been shown to occur approximately 1.5–2 h after consumption of nitrates (Bailey et al., 2009; Vanhatalo et al., 2010; Webb et al., 2008a). We chose a three day supplementation period as this appears to be the minimum short-term duration that results in sustained reductions in blood pressure (Larsen et al., 2006, 2007).
Baseline skin blood flow, blood pressure, and mean arterial pressure were collected for approximately 5–10 min after the trauma resolution period. Subsequently, one of two treatments was infused at random to each microdialysis site: 1) lactated Ringer's solution (control site) or 2) 20 mM L-NAME (non-selective NOS inhibitor). After a period of at least 45 min of drug infusion, the temperature of the local heating units was increased from 33 °C to 42 °C at a rate of 1 °C every 10 s until a steady-state was achieved for approximately 20–30 min (Minson et al., 2001; Wong and Fieger, 2010). Once a plateau was established and maintained, the control site was then infused with 20 mM L-NAME in order to quantify NOS-dependent vasodilatation during local heating. A 5–10 min plateau to L-NAME infusion at the control site was established (post-L-NAME drop) whereupon each site was infused with 54 mM SNP and heated to 43 °C to achieve maximal vasodilatation.
Subject instrumentation Testing was conducted with the subject resting in the supine position with the experimental arm at heart level. To avoid the use of anesthetics, ice was used to numb the desired location prior to placement of microdialysis fibers (Hodges et al., 2009). Fiber placement was approximately 3–5 cm apart. Microdialysis fibers which had a 10 mm long semi-permeable membrane with a 55-kDa molecular mass cutoff (CMA 31 Linear Probe; CMA Microdialysis, Kista, Sweden) (Fieger and Wong, 2010, 2012; Wong, 2013; Wong and Fieger, 2010, 2012) were placed by first threading a 23-gauge needle through the intradermal layer of the skin on the ventral aspect of the left forearm. A fiber was then threaded through the lumen of the needle, and the needle was then removed, leaving the membrane in place. Approximately 45–90 min was allowed for resolution of the trauma response following microdialysis fiber placement before the continuation of the protocol. During this time, all fibers were perfused with lactated Ringer's solution at a rate of 2 μl·min−1.
Drugs administered
Data collection and analysis Data were digitized and stored at 100 Hz on a personal computer and analyzed offline using signal-processing software (Windaq; Data Instruments, Akron, OH, USA). Blood flow measurements were normalized to mean arterial pressure and expressed as cutaneous vascular conductance (CVC), calculated as the ratio of RBC flux to mean arterial pressure (i.e., RBC flux/MAP). CVC values were normalized as a percentage of maximal vasodilation (%CVCmax) via SNP infusion and local heating to 43 °C. Data analysis was performed by one of the authors who was blinded to the microdialysis treatments (control and L-NAME) and supplement (BRJ). To determine the effect of nitrate supplementation on resting blood pressure variables, we analyzed all blood pressure variables during the baseline data collection period after the subject had been in the supine position for at least 10 min.
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Table 1 Blood pressure data for pre- and post-beetroot juice (nitrate) supplementation periods.
Pre-beetroot juice Post-beetroot juice
SBP (mm Hg)
DBP (mm Hg)
MAP (mm Hg)
122 ± 2 123 ± 1
74 ± 2 62 ± 3⁎
90 ± 1 83 ± 1⁎
Data are presented as mean ± SEM. SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure. ⁎ P b 0.05 vs. pre-beetroot juice.
All data were analyzed via SPSS 20 (IBM Corporation) and P-values b 0.05 were considered to be significant. All data are presented as mean ± SEM. Data were analyzed using two-way repeated measures ANOVA and paired t tests as appropriate. Pairwise comparisons were performed via paired t test to determine where significant differences occurred and a Bonferroni correction factor for multiple comparisons was used as appropriate. Laser-Doppler flux values are reported as arbitrary units (AU) and percent changes from baseline were calculated. The percent NOS-dependent vasodilation was calculated at the control sites as: ½ðLH control plateau–post L‐NAME dropÞ=LH plateau 100:
Results Blood pressure and heart rate data Blood pressure data pre- and post-beetroot juice (i.e., nitrate supplementation) is summarized in Table 1. There was no effect of nitrate supplementation on systolic blood pressure (pre-beetroot juice: 122 ± 2 vs. post-beetroot juice: 123 ± 1 mm Hg; P = 0.414). Diastolic blood pressure was significantly reduced from pre-beetroot juice (74 ± 2 mm Hg) to post-beetroot juice (62 ± 3 mm Hg; P = 0.004). Mean arterial pressure was significantly reduced from pre-beetroot juice (90 ± 1 mm Hg) to post-beetroot juice (83 ± 1 mm Hg; P b 0.001). There was no effect of beetroot juice supplementation on resting heart rate (pre-beetroot juice: 59 ± 4 beat min− 1 vs. post-beetroot juice: 59 ± 3 beat min−1; P = 0.831).
Flux values at the plateau of the local heating response at control sites (Pre: 160 ± 11 AU and Post: 153 ± 16 AU) were attenuated by L-NAME (Pre: 97 ± 6 AU and Post: 96 ± 8 AU; P b 0.05 for all conditions) for both pre- and post-beetroot juice supplementation. There was no effect of beetroot juice supplementation on plateau flux values at the control (P = 1.000) or L-NAME sites (P = 1.000). Plateau flux data are shown in Table 2. Also shown in Table 2 is the percent increase in flux from baseline. The percent increase in flux from baseline pre-beetroot juice at control sites (925 ± 88%) was significantly greater compared to L-NAME sites (596 ± 94%; P b 0.05). There was no difference in the percent increase from baseline post-beetroot juice between control (714 ± 103%) and L-NAME sites (415 ± 36%; P = 0.200). Beetroot juice supplementation had no effect on percent increase at control or L-NAME sites (P = 1.000 for both conditions).
Cutaneous vascular conductance data There was no significant difference in absolute maximal CVC values within or between control sites (pre-beetroot juice: 1.81 ± 0.51 vs. post-beetroot juice: 1.78 ± 0.15; P = 0.418) or L-NAME sites (pre-beetroot juice: 1.99 ± 0.37 vs. post-beetroot juice: 1.70 ± 0.44; P = 0.375). Fig. 1 shows the group mean data for the initial peak of the thermal hyperemic response to local heating for pre-and post-beetroot juice at control (solid bars) and L-NAME (gray bars) sites. Although L-NAME significantly attenuated (P b 0.001) the initial peak compared to control sites for both pre-beetroot juice (control: 74 ± 3%CVCmax vs. L-NAME: 59 ± 5%CVCmax) and post-beetroot juice (control: 75 ± 4%CVCmax vs. L-NAME:
59 ± 5%CVCmax) conditions, there was no main effect of beetroot juice on the initial peak. Fig. 2 shows the group mean data for the sustained plateau phase of the thermal hyperemic response to local heating for pre-and postbeetroot juice at control (solid bars) and L-NAME (gray bars) sites. The secondary plateau was attenuated at L-NAME sites compared to control sites for both pre- and post-beetroot juice (P b 0.001). Three days of beetroot juice augmented the secondary plateau at control sites (prebeetroot juice: 79 ± 2%CVCmax vs. post-beetroot juice: 91 ± 5%CVCmax, P = 0.002) and at L-NAME sites (pre-beetroot juice: 38 ± 4%CVCmax vs. post-beetroot juice: 52 ± 3%CVCmax, P b 0.01). The individual responses
Baseline laser-Doppler flux values at the control (16 ± 4 AU) and L-NAME (15 ± 5 AU) sites for the pre-beetroot juice condition were not significant from each other (P = 1.000). Similarly, post-beetroot juice baseline flux values were not different between control (20 ± 5 AU) and L-NAME (19 ± 4 AU; P = 1.000) sites. Baseline flux values at control sites (P = 0.337) and L-NAME sites (P = 1.000) were not different between pre- and post-beetroot juice. Baseline flux data are shown in Table 2.
Table 2 Flux values (arbitrary units; AU) for baseline and the plateau phase of thermal hyperemia and the percent increase in flux values from baseline for pre- and post-beetroot juice.
Control Pre-BRJ Post-BRJ
Baseline
Plateau
% increase from baseline
16 ± 4 20 ± 5
160 ± 11 153 ± 6
925 ± 88% 714 ± 103%
15 ± 5 19 ± 4
97 ± 6⁎ 96 ± 8⁎
596 ± 95%⁎ 415 ± 36%
L-NAME
Pre-BRJ Post-BRJ
Values for flux values are mean AU ± SEM; units are arbitrary flux units. ⁎ P b 0.05 vs. control within same condition.
Cutaneous Vascular Conductance (% Maximal)
Laser-Doppler flux data 100 Pre-beetroot juice Post-beetroot juice
80
*
*
60
40
20
0 Control
L-NAME
Fig. 1. Effect of nitrate supplementation on the initial peak response to local heating of the skin. Three days of nitrate supplementation had no effect on the initial peak response of cutaneous thermal hyperemia at either the control or L-NAME sites. Inhibition of NOS (L-NAME sites) significantly attenuated the initial peak compared to control for both pre- and post-beetroot juice periods. *P b 0.05 vs. control.
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100 *
100
Pre-beetroot juice Post-beetroot juice
80
*#
60
*#
40
20
0 Control
L-NAME
Fig. 2. Effect of nitrate supplementation on the plateau phase of cutaneous thermal hyperemia. The plateau phase of cutaneous thermal hyperemia was significantly increased at both the control and L-NAME sites following three days of beetroot juice compared to pre-beetroot juice. L-NAME attenuated the plateau compared to control for both preand post-beetroot juice periods. *P b 0.05 vs. pre-beetroot juice. #P b 0.05 vs. control sites.
for the plateau phase pre- and post-beetroot juice supplementation are shown in Fig. 3A for the control site and Fig. 3B for the L-NAME site. Fig. 4 shows group mean data for the percent NOS-dependent contribution to the plateau phase of the cutaneous thermal hyperemic response to local heating. Following three days of beetroot juice, there was no significant difference in the percent NOS-dependent contribution to the plateau of cutaneous thermal hyperemia (pre-beetroot
Cutaneous Vascular Conductance (% Maximal)
100
A
90
80
70
Cutaneous Vascular Conductance (% Maximal)
60
70
Pre-Beetroot Juice
Post-Beetroot Juice
Pre-Beetroot Juice
Post-Beetroot Juice
B
60
50
40
30
20
Fig. 3. Individual responses for the plateau phase pre- and post-beetroot juice supplementation. A) control site, and B) L-NAME site. Solid gray lines are the responses for each participant. The thick dashed black line is the group mean response.
% NOS-Dependent Vasodilatation
Cutaneous Vascular Conductance (% Maximal)
J.T. Keen et al. / Microvascular Research 98 (2015) 48–53
80
60
40
20
0 Pre-beetroot juice
Post-beetroot juice
Fig. 4. Effect of nitrate supplementation on the %NOS-dependent vasodilatation. Nitrate supplementation had no effect on the calculated %NOS-dependent vasodilatation compared to pre-supplementation.
juice: 60 ± 4%NOS-dependent vasodilatation vs. post-beetroot juice: 63 ± 3%NOS-dependent vasodilatation; P = 0.427). Discussion The primary finding of this study is that nitrate supplementation modestly increases NOS-independent vasodilatation in the cutaneous vasculature of healthy humans. This conclusion was supported by the observation of an increase in the plateau phase of cutaneous thermal hyperemia at both the control sites and at sites where L-NAME was continuously perfused to inhibit NOS. In addition to the increase in NOS-independent vasodilatation, we also observed a significant decrease in both diastolic blood pressure and mean arterial pressure, findings that are consistent with previous reports (Kapil et al., 2010; Webb et al., 2008b). Contrary to our hypothesis, we did not observe an increase in NOS-dependent vasodilatation. Nitrate supplementation had no effect on the magnitude of the decrease in CVC when L-NAME was perfused during the plateau phase and, thus, had no effect on the calculated %NOS contribution to the plateau phase of thermal hyperemia. Our data therefore suggest that three days of dietary nitrate supplementation can augment cutaneous vasodilatation and reduce blood pressure in healthy humans. The reduction in diastolic and mean arterial blood pressure ultimately results in an increase in cutaneous vascular conductance. The data from the present study demonstrating a decrease in diastolic blood pressure is in agreement with previous observations that nitrate supplementation via beetroot juice can reduce blood pressure even in normotensive subjects (Kapil et al., 2010; Vanhatalo et al., 2010; Webb et al., 2008b). Most studies have observed a significant reduction in systolic pressure; however, Larsen et al. (2006) observed no change in systolic blood pressure but significant reductions in diastolic and mean blood pressures following three days of inorganic nitrate supplementation. It is unclear why we did not observe a decrease in systolic blood pressure but it may be related to dosage and duration of nitrate supplementation as other studies have used four to six days of supplementation (Vanhatalo et al., 2010). In the present study we used a three day supplementation period as this appears to be the minimal short-term duration that results in sustained effects on blood pressure (Larsen et al., 2006). Although we observed no effect of nitrate supplementation on systolic blood pressure, we did observe significant reductions in both diastolic blood pressure and mean arterial blood pressure of ~12 mm Hg and ~7 mm Hg, respectively. It has been suggested that the observed decreases in diastolic and mean arterial pressure are related to increases in bioavailable NO (Kapil et al., 2010; Lundberg et al.,
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2011; Webb et al., 2008b), which would ultimately reduce vascular resistance. Nitrate supplementation was effective at reducing diastolic and mean arterial blood pressures but it had no effect on HR, which is consistent with previous nitrate literature in animal and human experiments (Ferguson et al., 2013; Webb et al., 2008b). Inasmuch as the calculation of cutaneous vascular conductance is dependent on mean arterial pressure, the decrease in mean arterial pressure may largely explain the observed augmentation of the plateau phase of cutaneous thermal hyperemia. A decrease in mean arterial pressure following nitrate supplementation would result in a calculated increase in vascular conductance. Since nitrate supplementation had no effect on baseline flux values or flux values during the plateau of thermal hyperemia, the calculated percent increase in flux from baseline was unaffected by nitrate supplementation, suggesting that nitrate supplementation had no direct effect on increasing skin blood flow (flux values; Table 2). It would thus appear that the increase in the plateau phase of thermal hyperemia was due largely to a decrease in MAP. Whether the augmented plateau is due to an increase in NOS-independent bioavailable NO, a decrease in mean arterial pressure, or some combined effect, both the augmented plateau and decrease in mean arterial pressure are consistent with the postulation that nitrate supplementation exerts positive effects on the cardiovascular system, in this case improved microvascular function and a decrease in vascular resistance. Our present data support the growing body of evidence that suggests that nitrate supplementation can improve vascular function independent of NOS. This conclusion is based on our finding that the plateau phase of cutaneous thermal hyperemia was augmented at both control and NOS-inhibited (L-NAME) sites. The continuous inhibition of NOS at these sites would effectively inhibit NO generation via NOS and, as such, any increase in the plateau phase would have to be independent of NOS activity. Nevertheless, we cannot rule out that the augmented plateau following nitrate supplementation at either the control or L-NAME sites was due to upregulation of other known mediators of the plateau phase, such as potassium channels or EDHFs (Brunt et al., 2013) or that nitrates had some effect on local sympathetic nerve activity and its related peptides (Hodges et al., 2008; Hodges and Sparks, 2013; Houghton et al., 2006); however, it is, to our knowledge, unknown how, or if, nitrates may interact with potassium channels and/or EDHFs in the vasculature. Studies aimed at investigating how nitrates interact with known contributors to thermal hyperemia would help resolve some of these questions. Although we observed a modest increase in NOS-independent vasodilatation, it is possible that we did not observe greater effects due to a ‘ceiling effect’. For example, Brunt and colleagues (Brunt et al., 2011) found that estradiol supplementation augments the initial peak but not the plateau phase of thermal hyperemia. Because the plateau phase of thermal hyperemia averaged ~90%CVCmax, these authors postulated that there may be a ceiling effect and minimal room to increase NO-dependent vasodilatation, despite the known ability of estrogen to upregulate NO. Choi et al. (2014) recently considered this potential limitation of a ceiling effect and reported that local heating to 39 °C increases cutaneous vascular conductance to ~40% of maximal yet is approximately 80% NO-dependent (Choi et al., 2014). In the present study, it is therefore possible that greater increases in NOS-dependent and independent vasodilatation may be observed if the skin is heated to a lower temperature (Choi et al., 2014) or in populations with reduced responses, such as aging, hypercholesterolemia, or hypertension (Holowatz et al., 2011; Minson et al., 2002; Smith et al., 2011). Experimental considerations & limitations It is important to note that this study specifically incorporated male subjects in order to reduce skin blood flow variability due to changes in sex hormone levels across the menstrual cycle and oral contraceptive phase in female subjects (Charkoudian et al., 1999). It is thus possible
that reproductive hormones could affect NO bioavailability. To our knowledge there have been no studies investigating the relationship between reproductive hormones and dietary nitrates. The potential influence of female sex hormones and nitrate supplementation warrants future investigation. Conclusion To our knowledge, this is the first study to investigate the effects of nitrate supplementation via beetroot juice on cutaneous thermal hyperemia during local heating in healthy subjects. Data from the present study suggest that nitrate supplementation provides a modest improvement of NOS-independent vasodilatation as well as reductions in diastolic and mean arterial blood pressures. Our data suggest that the improved thermal hyperemic response is due primarily to a reduction in mean arterial blood pressure resulting in an increased calculated cutaneous vascular conductance and not due to a direct increase in skin blood flow (flux values). Future research is needed to better understand the effects of dietary nitrate supplementation on the skin blood flow response during local heating in not only healthy humans, but also patient populations where nitrate supplementation may have more marked effects on NO-mediated vasodilatation. Conflict of interest None. Author contributions This study was conducted in the Department of Kinesiology at the Kansas State University by Jeremy T. Keen in partial fulfillment of the requirements for the degree of Master of Science. Jeremy T. Keen was responsible for the data collection and data analysis, interpretation of the data, and drafting the manuscript. Erica L. Levitt was responsible for assisting with the data collection and analysis, interpretation of the data, and editing all drafts of the manuscript. Gary J. Hodges was responsible for the data analysis, interpretation of the data, and editing all drafts of the manuscript. Brett J. Wong was responsible for the experimental design, data analysis and interpretation, and editing all drafts of the manuscript. All authors approved the final version of the manuscript. Acknowledgments The authors would like to thank all the subjects who participated in this study; your time and effort are very much appreciated. The authors appreciate the work of Jena K. Eder and for her assistance with the data collection. References Bailey, S., et al., 2009. Dietary nitrate supplementation reduces the O2 cost of lowintensity exercise and enhances tolerance to high-intensity exercise in humans. J. Appl. Physiol. 107, 1144–1155. Bailey, S., et al., 2010. Dietary nitrate supplementation enhances muscle contractile efficiency during knee-extensor exercise in humans. J. Appl. Physiol. 109, 135–148. Brain, S.D., et al., 1986. Potent vasodilator activity of calcitonin gene-related peptide in human skin. J. Invest. Dermatol. 87, 533–536. Bruning, R., et al., 2012. Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin. J. Appl. Physiol. 112, 2019–2026. Brunt, V., et al., 2011. 17β-Estradiol and progesterone independently augment cutaneous thermal hyperemia but not reactive hyperemia. Microcirculation 18, 347–355. Brunt, V.E., et al., 2013. No independent, but an interactive, role of calcium-activated potassium channels in human cutaneous active vasodilation. J. Appl. Physiol. 115, 1290–1296. Charkoudian, N., et al., 1999. Influence of female reproductive hormones on local thermal control of skin blood flow. J. Appl. Physiol. 87, 1719–1723. Choi, P.J., et al., 2014. New Approach to Measure Cutaneous Microvascular Function: An Improved Test of NO-mediated Vasodilation by Thermal Hyperemia. Ferguson, S.K., et al., 2013. Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control in rats. J. Physiol. 591, 547–557.
J.T. Keen et al. / Microvascular Research 98 (2015) 48–53 Fieger, S.M., Wong, B.J., 2010. Adenosine receptor inhibition with theophylline attenuates the skin blood flow response to local heating in humans. Exp. Physiol. 95, 946–954. Fieger, S.M., Wong, B.J., 2012. No direct role for A1/A2 adenosine receptor activation to reflex cutaneous vasodilatation during whole-body heat stress in humans. Acta Physiol. 205, 403–410. Govoni, M., et al., 2008. The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide 19, 333–337. Hodges, G.J., Sparks, P.A., 2013. Contributions of endothelial nitric oxide synthase, noradrenaline, and neuropeptide Y to local warming-induced cutaneous vasodilatation in men. Microvasc. Res. 90, 128–134. Hodges, G., Sparks, P., 2014. Noradrenaline and neuropeptide Y contribute to initial, but not sustained, vasodilatation in response to local skin warming in humans. Exp. Physiol. 99, 381–392. Hodges, G.J., et al., 2006. The involvement of nitric oxide in the cutaneous vasoconstrictor response to local cooling in humans. J. Physiol. 574, 849–857. Hodges, G.J., et al., 2008. The involvement of norepinephrine, neuropeptide Y, and nitric oxide in the cutaneous vasodilator response to local heating in humans. J. Appl. Physiol. 105, 233–240. Hodges, G.J., et al., 2009. The effect of microdialysis needle trauma on cutaneous vascular responses in humans. J. Appl. Physiol. 106, 1112–1118. Holowatz, L.A., et al., 2011. Oral atorvastatin therapy restores cutaneous microvascular function by decreasing arginase activity in hypercholesterolaemic humans. J. Physiol. 589, 2093–2103. Houghton, B.L., et al., 2006. Nitric oxide and noradrenaline contribute to the temperature threshold of the axon reflex response to gradual local heating in human skin. J. Physiol. 572, 811–820. Kapil, V., et al., 2010. Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension 56, 274–281. Kellogg, D., Liu, Y., Kosiba, I.F., O'Donnell, D., 1999. Role of nitric oxide in the vascular effects of local warming of the skin in humans. J. Appl. Physiol. 86, 1185–1190. Kellogg Jr., D.L., et al., 2008. Endothelial nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo. Am. J. Physiol. Heart Circ. Physiol. 295, H123–H129. Kellogg, D., et al., 2009. Roles of nitric oxide synthase isoforms in cutaneous vasodilation induced by local warming of the skin and whole body heat stress in humans. J. Appl. Physiol. 107, 1438–1444. Kenjale, A.A., et al., 2011. Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease. J. Appl. Physiol. 110, 1582–1591.
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Larsen, F.J., et al., 2006. Effects of dietary nitrate on blood pressure in healthy volunteers. N. Engl. J. Med. 355, 2792–2793. Larsen, F.J., et al., 2007. Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol. 191, 59–66. Lundberg, J., Weitzberg, E., 2009. NO generation from inorganic nitrate and nitrite: role in physiology, nutrition and therapeutics. Arch. Pharm. Res. 32, 1119–1126. Lundberg, J., et al., 2008. The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156–167. Lundberg, J.O., et al., 2011. Roles of dietary inorganic nitrate in cardiovascular health and disease. Cardiovasc. Res. 89, 525–532. Minson, C.T., et al., 2001. Nitric oxide and neurally mediated regulation of skin blood flow during local heating. J. Appl. Physiol. 91, 1619–1626. Minson, C.T., et al., 2002. Decreased nitric oxide- and axon reflex-mediated cutaneous vasodilation with age during local heating. J. Appl. Physiol. 93, 1644–1649. Smith, C., et al., 2011. Upregulation of inducible nitric oxide synthase contributes to attenuated cutaneous vasodilation in essential hypertensive humans. Hypertension 58, 935–942. Vanhatalo, A., et al., 2010. Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, R1121–R1131. Vanin, A.F., et al., 2007. Nitric oxide synthase reduces nitrite to NO under anoxia. Cell. Mol. Life Sci. 64, 96–103. Webb, A., et al., 2008a. Mechanisms underlying erythrocyte and endothelial nitrite reduction to nitric oxide in hypoxia: role for xanthine oxidoreductase and endothelial nitric oxide synthase. Circ. Res. 103, 957–964. Webb, A., et al., 2008b. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension 51, 784–790. Wong, B., 2013. Sensory nerves and nitric oxide contribute to reflex cutaneous vasodilation in humans. Am. J. Physiol. Regul. Integr. Comp. Physiol. 304, R651–R656. Wong, B.J., Fieger, S.M., 2010. Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans. J. Physiol. 588, 4317–4326. Wong, B.J., Fieger, S.M., 2012. Transient receptor potential vanilloid type 1 channels contribute to reflex cutaneous vasodilation in humans. J. Appl. Physiol. 112, 2037–2042. Wong, B.J., Minson, C.T., 2011. Altered thermal hyperaemia in human skin by prior desensitization of neurokinin-1 receptors. Exp. Physiol. 96, 599–609.
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Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
Short-term dietary nitrate supplementation augments cutaneous vasodilatation and reduces mean arterial pressure in healthy humans Jeremy T. Keen a, Erica L. Levitt a, Gary J. Hodges c, Brett J. Wong a,b,⁎ a b c
Department of Kinesiology, Kansas State University, Manhattan, KS, USA Department of Kinesiology & Health, Georgia State University, Atlanta, GA, USA Department of Kinesiology, Brock University, St. Catharines, ON L2S 3A1, Canada
a r t i c l e
i n f o
Article history: Accepted 21 December 2014 Available online 30 December 2014 Keywords: Nitric oxide Microvasculature Blood pressure Microdialysis Beetroot juice
a b s t r a c t Nitrate supplementation in the form of beetroot juice has been shown to increase nitric oxide (NO) where nitrate can be reduced to nitrite and, subsequently, to NO through both nitric oxide synthase (NOS)-dependent and -independent pathways. We tested the hypothesis that nitrate supplementation would augment the NO component of the cutaneous vasodilatation to local skin heating in young, healthy humans. Participants reported to the lab for pre- and post-supplement local heating protocols. Nitrate supplementation consisted of one shot (70 ml) of beetroot juice (0.45 g nitrate; 5 mM) for three days. Six participants were equipped with two microdialysis fibers on the ventral forearm and randomly assigned to lactated Ringer's (control) or continuous infusion of 20 mM L-NAME (NOS inhibitor). The control site was subsequently perfused with L-NAME once a plateau in skin blood flow was achieved to quantify NOS-dependent cutaneous vasodilatation. Skin blood flow via laser-Doppler flowmetry (LDF) and mean arterial pressure (MAP) were measured; cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to %CVCmax. Beetroot juice reduced MAP (Pre: 90 ± 1 mm Hg vs. Post: 83 ± 1 mm Hg) and DBP (Pre: 74 ± 2 mm Hg vs. Post: 62 ± 3 mm Hg) (P b 0.05). The plateau phase of the local heating response at control sites was augmented post-beetroot juice (91 ± 5%CVCmax) compared to pre-beetroot juice (79 ± 2%CVCmax) (P b 0.05). There was no difference in the %NOS-dependent vasodilatation from pre- to post-beetroot juice. These data suggest that nitrate supplementation via beetroot juice can reduce MAP and DBP as well as augment NOS-independent vasodilatation to local heating in the cutaneous vasculature of healthy humans. © 2014 Elsevier Inc. All rights reserved.
Introduction Local heating of the skin results in a robust increase in cutaneous blood flow, termed cutaneous thermal hyperemia. Cutaneous thermal hyperemia in response to rapid, non-painful local heating exhibits a reproducible biphasic increase in skin blood flow: an initial transient increase in skin blood flow punctuated by a brief nadir followed by a prolonged secondary plateau phase. The initial peak is predominantly mediated by a sensory nerve axon reflex mechanism, possibly involving the neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP) (Brain et al., 1986; Wong and Minson, 2011) and activation of transient receptor potential vanilloid type 1 (TRPV-1) channels (Wong and Fieger, 2010). The prolonged secondary plateau is mediated largely (~60%) by nitric oxide (NO) (Bruning et al., 2012; Kellogg et al., 1999, 2008, 2009; Minson et al., 2001, 2002) via endothelial NO synthase (eNOS) (Bruning et al., 2012; Kellogg et al., 2008, 2009), where inhibition of NO synthase (NOS) significantly reduces the secondary plateau ⁎ Corresponding author at: Department of Kinesiology & Health, P.O. Box 3975, Georgia State University, Atlanta, GA 30302-3975, USA. Fax: +1 404 413 8053. E-mail address: [email protected] (B.J. Wong).
http://dx.doi.org/10.1016/j.mvr.2014.12.002 0026-2862/© 2014 Elsevier Inc. All rights reserved.
phase of cutaneous thermal hyperemia by ~ 60–70% (Hodges and Sparks, 2014; Kellogg et al., 1999, 2008, 2009; Minson et al., 2001). Dietary nitrate supplementation via beetroot juice has been shown to improve measures of health (Kapil et al., 2010; Webb et al., 2008b), increase blood flow to contracting skeletal muscle (Ferguson et al., 2013), and enhance peripheral vasodilatation and endothelial-derived NO in peripheral arterial disease patients (Kenjale et al., 2011). The increase in NO bioavailability from nitrate supplementation has been shown to occur through both NOS-independent (Lundberg and Weitzberg, 2009; Lundberg et al., 2008) and NOS-dependent (Vanin et al., 2007; Webb et al., 2008a) pathways. Studies have demonstrated that reduction of nitrate to nitrite and, subsequently, to NO, can provide a valuable pathway in which the body can utilize NO through a NOS-independent pathway unlike the traditional L-arginine pathway that is reliant on the NOS enzyme and cofactors (e.g., Ca2 + and BH4). Whether it is through NOS-independent or NOS-dependent pathways, nitrate supplementation may provide a means by which to improve NO-mediated vasodilatation. The purpose of this study was to investigate the effect of dietary nitrate supplementation via beetroot juice on the skin blood flow response to local heating in healthy humans. We tested the hypothesis that nitrate supplementation would augment the contribution of NO
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to cutaneous thermal hyperemia in healthy humans primarily by increasing NOS-dependent vasodilatation. Methods Ethical approval The Institutional Review Board at the Kansas State University approved all protocols of this study. A written informed consent was reviewed and signed by each subject prior to participation. All procedures and protocols were performed in conjunction with the standards set forth by the Declaration of Helsinki. Subjects
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Mean arterial pressure was monitored beat-by-beat via photoplethysmography (NexfinHD; BMEYE, Amsterdam, The Netherlands) from a finger on the right (non-experimental) hand and verified every 5 min via automated brachial oscillometry (S/5 Light Monitor; GE Healthcare; Madison, WI, USA) from the right (non-experimental) arm. All blood pressure measurements were made with the subject in the supine position. Resting blood pressures were taken after the subject had been resting in the supine position for at least 10 min. Heart rate was recorded from the beat-by-beat arterial blood pressure waveform. Red blood cell (RBC) flux was used as an index of skin blood flow via laser-Doppler flowmetry (LDF) (PeriFlux 5010 laser-Doppler perfusion monitor; Perimed; Järfälla, Sweden). Local heating units (PF5020 local heating units and PeriFlux 5020 Temperature Unit; Perimed; Järfälla, Sweden) were placed on the skin directly over each microdialysis membrane, and an integrated laser-Doppler probe (Probe 413; Perimed; Järfälla, Sweden) was placed in the center of each local heating unit to measure RBC flux directly over each microdialysis site.
Six male subjects (age 24 ± 1 yr, height 177 ± 6 cm, body mass 80 ± 13 kg, BMI 26 ± 3 kg/m2) participated in this study. All subjects were healthy, non-smokers, taking no medications and were free of cardiovascular, respiratory, and metabolic diseases as determined by a medical history questionnaire. Subjects were asked to refrain from exercise, consumption of abnormal amounts (N300 g/day) of leafy green vegetables (e.g., spinach), consumption of alcohol, and the use of mouthwash products containing alcohol during the three day beetroot juice supplementation period. Exercise and consumption of large quantities of leafy green vegetables can increase systemic nitrates while alcohol and alcohol-based mouthwash can kill the bacteria required to reduce nitrate to nitrite and subsequently to nitric oxide (Bailey et al., 2009; Govoni et al., 2008). Subjects were asked to refrain from caffeine at least 12 h prior to the study. Testing was administered in a thermoneutral environment with a room temperature of ~23 °C.
Lactated Ringer's solution was administered at the control site. A 20 mM concentration of L-NAME was used as a non-selective NOS inhibitor (Bruning et al., 2012; Brunt et al., 2013; Hodges et al., 2006; Smith et al., 2011). At the end of the local heating protocol, a 54 mM concentration of sodium nitroprusside (SNP) was administered while simultaneously locally heating the skin to 43 °C to elicit maximal vasodilatation. All drugs were infused through the microdialysis fibers at a rate of 2 μl·min−1 via microinfusion pumps (Bee Hive controller and Baby Bee Syringe Pumps; Bioanalytical Systems, West Lafayette, IN, USA) to each randomly assigned site.
Dietary intervention
Local heating protocol
Upon completion of pre-supplement testing, subjects received three days of nitrate rich beetroot juice (70 ml shots; ~5 mM/day; 0.45 g of nitrates) (Beet It, James White Drinks, Ipswich, UK) (Bailey et al., 2010; Vanhatalo et al., 2010). Subjects were instructed to drink one 70 ml shot for three consecutive days with the last shot consumed in the laboratory approximately 1.5–2 h prior to beginning the local heating protocol. Peak circulating plasma level of nitrites (approximate range: 200–600 nM) has been shown to occur approximately 1.5–2 h after consumption of nitrates (Bailey et al., 2009; Vanhatalo et al., 2010; Webb et al., 2008a). We chose a three day supplementation period as this appears to be the minimum short-term duration that results in sustained reductions in blood pressure (Larsen et al., 2006, 2007).
Baseline skin blood flow, blood pressure, and mean arterial pressure were collected for approximately 5–10 min after the trauma resolution period. Subsequently, one of two treatments was infused at random to each microdialysis site: 1) lactated Ringer's solution (control site) or 2) 20 mM L-NAME (non-selective NOS inhibitor). After a period of at least 45 min of drug infusion, the temperature of the local heating units was increased from 33 °C to 42 °C at a rate of 1 °C every 10 s until a steady-state was achieved for approximately 20–30 min (Minson et al., 2001; Wong and Fieger, 2010). Once a plateau was established and maintained, the control site was then infused with 20 mM L-NAME in order to quantify NOS-dependent vasodilatation during local heating. A 5–10 min plateau to L-NAME infusion at the control site was established (post-L-NAME drop) whereupon each site was infused with 54 mM SNP and heated to 43 °C to achieve maximal vasodilatation.
Subject instrumentation Testing was conducted with the subject resting in the supine position with the experimental arm at heart level. To avoid the use of anesthetics, ice was used to numb the desired location prior to placement of microdialysis fibers (Hodges et al., 2009). Fiber placement was approximately 3–5 cm apart. Microdialysis fibers which had a 10 mm long semi-permeable membrane with a 55-kDa molecular mass cutoff (CMA 31 Linear Probe; CMA Microdialysis, Kista, Sweden) (Fieger and Wong, 2010, 2012; Wong, 2013; Wong and Fieger, 2010, 2012) were placed by first threading a 23-gauge needle through the intradermal layer of the skin on the ventral aspect of the left forearm. A fiber was then threaded through the lumen of the needle, and the needle was then removed, leaving the membrane in place. Approximately 45–90 min was allowed for resolution of the trauma response following microdialysis fiber placement before the continuation of the protocol. During this time, all fibers were perfused with lactated Ringer's solution at a rate of 2 μl·min−1.
Drugs administered
Data collection and analysis Data were digitized and stored at 100 Hz on a personal computer and analyzed offline using signal-processing software (Windaq; Data Instruments, Akron, OH, USA). Blood flow measurements were normalized to mean arterial pressure and expressed as cutaneous vascular conductance (CVC), calculated as the ratio of RBC flux to mean arterial pressure (i.e., RBC flux/MAP). CVC values were normalized as a percentage of maximal vasodilation (%CVCmax) via SNP infusion and local heating to 43 °C. Data analysis was performed by one of the authors who was blinded to the microdialysis treatments (control and L-NAME) and supplement (BRJ). To determine the effect of nitrate supplementation on resting blood pressure variables, we analyzed all blood pressure variables during the baseline data collection period after the subject had been in the supine position for at least 10 min.
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Table 1 Blood pressure data for pre- and post-beetroot juice (nitrate) supplementation periods.
Pre-beetroot juice Post-beetroot juice
SBP (mm Hg)
DBP (mm Hg)
MAP (mm Hg)
122 ± 2 123 ± 1
74 ± 2 62 ± 3⁎
90 ± 1 83 ± 1⁎
Data are presented as mean ± SEM. SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure. ⁎ P b 0.05 vs. pre-beetroot juice.
All data were analyzed via SPSS 20 (IBM Corporation) and P-values b 0.05 were considered to be significant. All data are presented as mean ± SEM. Data were analyzed using two-way repeated measures ANOVA and paired t tests as appropriate. Pairwise comparisons were performed via paired t test to determine where significant differences occurred and a Bonferroni correction factor for multiple comparisons was used as appropriate. Laser-Doppler flux values are reported as arbitrary units (AU) and percent changes from baseline were calculated. The percent NOS-dependent vasodilation was calculated at the control sites as: ½ðLH control plateau–post L‐NAME dropÞ=LH plateau 100:
Results Blood pressure and heart rate data Blood pressure data pre- and post-beetroot juice (i.e., nitrate supplementation) is summarized in Table 1. There was no effect of nitrate supplementation on systolic blood pressure (pre-beetroot juice: 122 ± 2 vs. post-beetroot juice: 123 ± 1 mm Hg; P = 0.414). Diastolic blood pressure was significantly reduced from pre-beetroot juice (74 ± 2 mm Hg) to post-beetroot juice (62 ± 3 mm Hg; P = 0.004). Mean arterial pressure was significantly reduced from pre-beetroot juice (90 ± 1 mm Hg) to post-beetroot juice (83 ± 1 mm Hg; P b 0.001). There was no effect of beetroot juice supplementation on resting heart rate (pre-beetroot juice: 59 ± 4 beat min− 1 vs. post-beetroot juice: 59 ± 3 beat min−1; P = 0.831).
Flux values at the plateau of the local heating response at control sites (Pre: 160 ± 11 AU and Post: 153 ± 16 AU) were attenuated by L-NAME (Pre: 97 ± 6 AU and Post: 96 ± 8 AU; P b 0.05 for all conditions) for both pre- and post-beetroot juice supplementation. There was no effect of beetroot juice supplementation on plateau flux values at the control (P = 1.000) or L-NAME sites (P = 1.000). Plateau flux data are shown in Table 2. Also shown in Table 2 is the percent increase in flux from baseline. The percent increase in flux from baseline pre-beetroot juice at control sites (925 ± 88%) was significantly greater compared to L-NAME sites (596 ± 94%; P b 0.05). There was no difference in the percent increase from baseline post-beetroot juice between control (714 ± 103%) and L-NAME sites (415 ± 36%; P = 0.200). Beetroot juice supplementation had no effect on percent increase at control or L-NAME sites (P = 1.000 for both conditions).
Cutaneous vascular conductance data There was no significant difference in absolute maximal CVC values within or between control sites (pre-beetroot juice: 1.81 ± 0.51 vs. post-beetroot juice: 1.78 ± 0.15; P = 0.418) or L-NAME sites (pre-beetroot juice: 1.99 ± 0.37 vs. post-beetroot juice: 1.70 ± 0.44; P = 0.375). Fig. 1 shows the group mean data for the initial peak of the thermal hyperemic response to local heating for pre-and post-beetroot juice at control (solid bars) and L-NAME (gray bars) sites. Although L-NAME significantly attenuated (P b 0.001) the initial peak compared to control sites for both pre-beetroot juice (control: 74 ± 3%CVCmax vs. L-NAME: 59 ± 5%CVCmax) and post-beetroot juice (control: 75 ± 4%CVCmax vs. L-NAME:
59 ± 5%CVCmax) conditions, there was no main effect of beetroot juice on the initial peak. Fig. 2 shows the group mean data for the sustained plateau phase of the thermal hyperemic response to local heating for pre-and postbeetroot juice at control (solid bars) and L-NAME (gray bars) sites. The secondary plateau was attenuated at L-NAME sites compared to control sites for both pre- and post-beetroot juice (P b 0.001). Three days of beetroot juice augmented the secondary plateau at control sites (prebeetroot juice: 79 ± 2%CVCmax vs. post-beetroot juice: 91 ± 5%CVCmax, P = 0.002) and at L-NAME sites (pre-beetroot juice: 38 ± 4%CVCmax vs. post-beetroot juice: 52 ± 3%CVCmax, P b 0.01). The individual responses
Baseline laser-Doppler flux values at the control (16 ± 4 AU) and L-NAME (15 ± 5 AU) sites for the pre-beetroot juice condition were not significant from each other (P = 1.000). Similarly, post-beetroot juice baseline flux values were not different between control (20 ± 5 AU) and L-NAME (19 ± 4 AU; P = 1.000) sites. Baseline flux values at control sites (P = 0.337) and L-NAME sites (P = 1.000) were not different between pre- and post-beetroot juice. Baseline flux data are shown in Table 2.
Table 2 Flux values (arbitrary units; AU) for baseline and the plateau phase of thermal hyperemia and the percent increase in flux values from baseline for pre- and post-beetroot juice.
Control Pre-BRJ Post-BRJ
Baseline
Plateau
% increase from baseline
16 ± 4 20 ± 5
160 ± 11 153 ± 6
925 ± 88% 714 ± 103%
15 ± 5 19 ± 4
97 ± 6⁎ 96 ± 8⁎
596 ± 95%⁎ 415 ± 36%
L-NAME
Pre-BRJ Post-BRJ
Values for flux values are mean AU ± SEM; units are arbitrary flux units. ⁎ P b 0.05 vs. control within same condition.
Cutaneous Vascular Conductance (% Maximal)
Laser-Doppler flux data 100 Pre-beetroot juice Post-beetroot juice
80
*
*
60
40
20
0 Control
L-NAME
Fig. 1. Effect of nitrate supplementation on the initial peak response to local heating of the skin. Three days of nitrate supplementation had no effect on the initial peak response of cutaneous thermal hyperemia at either the control or L-NAME sites. Inhibition of NOS (L-NAME sites) significantly attenuated the initial peak compared to control for both pre- and post-beetroot juice periods. *P b 0.05 vs. control.
51
100 *
100
Pre-beetroot juice Post-beetroot juice
80
*#
60
*#
40
20
0 Control
L-NAME
Fig. 2. Effect of nitrate supplementation on the plateau phase of cutaneous thermal hyperemia. The plateau phase of cutaneous thermal hyperemia was significantly increased at both the control and L-NAME sites following three days of beetroot juice compared to pre-beetroot juice. L-NAME attenuated the plateau compared to control for both preand post-beetroot juice periods. *P b 0.05 vs. pre-beetroot juice. #P b 0.05 vs. control sites.
for the plateau phase pre- and post-beetroot juice supplementation are shown in Fig. 3A for the control site and Fig. 3B for the L-NAME site. Fig. 4 shows group mean data for the percent NOS-dependent contribution to the plateau phase of the cutaneous thermal hyperemic response to local heating. Following three days of beetroot juice, there was no significant difference in the percent NOS-dependent contribution to the plateau of cutaneous thermal hyperemia (pre-beetroot
Cutaneous Vascular Conductance (% Maximal)
100
A
90
80
70
Cutaneous Vascular Conductance (% Maximal)
60
70
Pre-Beetroot Juice
Post-Beetroot Juice
Pre-Beetroot Juice
Post-Beetroot Juice
B
60
50
40
30
20
Fig. 3. Individual responses for the plateau phase pre- and post-beetroot juice supplementation. A) control site, and B) L-NAME site. Solid gray lines are the responses for each participant. The thick dashed black line is the group mean response.
% NOS-Dependent Vasodilatation
Cutaneous Vascular Conductance (% Maximal)
J.T. Keen et al. / Microvascular Research 98 (2015) 48–53
80
60
40
20
0 Pre-beetroot juice
Post-beetroot juice
Fig. 4. Effect of nitrate supplementation on the %NOS-dependent vasodilatation. Nitrate supplementation had no effect on the calculated %NOS-dependent vasodilatation compared to pre-supplementation.
juice: 60 ± 4%NOS-dependent vasodilatation vs. post-beetroot juice: 63 ± 3%NOS-dependent vasodilatation; P = 0.427). Discussion The primary finding of this study is that nitrate supplementation modestly increases NOS-independent vasodilatation in the cutaneous vasculature of healthy humans. This conclusion was supported by the observation of an increase in the plateau phase of cutaneous thermal hyperemia at both the control sites and at sites where L-NAME was continuously perfused to inhibit NOS. In addition to the increase in NOS-independent vasodilatation, we also observed a significant decrease in both diastolic blood pressure and mean arterial pressure, findings that are consistent with previous reports (Kapil et al., 2010; Webb et al., 2008b). Contrary to our hypothesis, we did not observe an increase in NOS-dependent vasodilatation. Nitrate supplementation had no effect on the magnitude of the decrease in CVC when L-NAME was perfused during the plateau phase and, thus, had no effect on the calculated %NOS contribution to the plateau phase of thermal hyperemia. Our data therefore suggest that three days of dietary nitrate supplementation can augment cutaneous vasodilatation and reduce blood pressure in healthy humans. The reduction in diastolic and mean arterial blood pressure ultimately results in an increase in cutaneous vascular conductance. The data from the present study demonstrating a decrease in diastolic blood pressure is in agreement with previous observations that nitrate supplementation via beetroot juice can reduce blood pressure even in normotensive subjects (Kapil et al., 2010; Vanhatalo et al., 2010; Webb et al., 2008b). Most studies have observed a significant reduction in systolic pressure; however, Larsen et al. (2006) observed no change in systolic blood pressure but significant reductions in diastolic and mean blood pressures following three days of inorganic nitrate supplementation. It is unclear why we did not observe a decrease in systolic blood pressure but it may be related to dosage and duration of nitrate supplementation as other studies have used four to six days of supplementation (Vanhatalo et al., 2010). In the present study we used a three day supplementation period as this appears to be the minimal short-term duration that results in sustained effects on blood pressure (Larsen et al., 2006). Although we observed no effect of nitrate supplementation on systolic blood pressure, we did observe significant reductions in both diastolic blood pressure and mean arterial blood pressure of ~12 mm Hg and ~7 mm Hg, respectively. It has been suggested that the observed decreases in diastolic and mean arterial pressure are related to increases in bioavailable NO (Kapil et al., 2010; Lundberg et al.,
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2011; Webb et al., 2008b), which would ultimately reduce vascular resistance. Nitrate supplementation was effective at reducing diastolic and mean arterial blood pressures but it had no effect on HR, which is consistent with previous nitrate literature in animal and human experiments (Ferguson et al., 2013; Webb et al., 2008b). Inasmuch as the calculation of cutaneous vascular conductance is dependent on mean arterial pressure, the decrease in mean arterial pressure may largely explain the observed augmentation of the plateau phase of cutaneous thermal hyperemia. A decrease in mean arterial pressure following nitrate supplementation would result in a calculated increase in vascular conductance. Since nitrate supplementation had no effect on baseline flux values or flux values during the plateau of thermal hyperemia, the calculated percent increase in flux from baseline was unaffected by nitrate supplementation, suggesting that nitrate supplementation had no direct effect on increasing skin blood flow (flux values; Table 2). It would thus appear that the increase in the plateau phase of thermal hyperemia was due largely to a decrease in MAP. Whether the augmented plateau is due to an increase in NOS-independent bioavailable NO, a decrease in mean arterial pressure, or some combined effect, both the augmented plateau and decrease in mean arterial pressure are consistent with the postulation that nitrate supplementation exerts positive effects on the cardiovascular system, in this case improved microvascular function and a decrease in vascular resistance. Our present data support the growing body of evidence that suggests that nitrate supplementation can improve vascular function independent of NOS. This conclusion is based on our finding that the plateau phase of cutaneous thermal hyperemia was augmented at both control and NOS-inhibited (L-NAME) sites. The continuous inhibition of NOS at these sites would effectively inhibit NO generation via NOS and, as such, any increase in the plateau phase would have to be independent of NOS activity. Nevertheless, we cannot rule out that the augmented plateau following nitrate supplementation at either the control or L-NAME sites was due to upregulation of other known mediators of the plateau phase, such as potassium channels or EDHFs (Brunt et al., 2013) or that nitrates had some effect on local sympathetic nerve activity and its related peptides (Hodges et al., 2008; Hodges and Sparks, 2013; Houghton et al., 2006); however, it is, to our knowledge, unknown how, or if, nitrates may interact with potassium channels and/or EDHFs in the vasculature. Studies aimed at investigating how nitrates interact with known contributors to thermal hyperemia would help resolve some of these questions. Although we observed a modest increase in NOS-independent vasodilatation, it is possible that we did not observe greater effects due to a ‘ceiling effect’. For example, Brunt and colleagues (Brunt et al., 2011) found that estradiol supplementation augments the initial peak but not the plateau phase of thermal hyperemia. Because the plateau phase of thermal hyperemia averaged ~90%CVCmax, these authors postulated that there may be a ceiling effect and minimal room to increase NO-dependent vasodilatation, despite the known ability of estrogen to upregulate NO. Choi et al. (2014) recently considered this potential limitation of a ceiling effect and reported that local heating to 39 °C increases cutaneous vascular conductance to ~40% of maximal yet is approximately 80% NO-dependent (Choi et al., 2014). In the present study, it is therefore possible that greater increases in NOS-dependent and independent vasodilatation may be observed if the skin is heated to a lower temperature (Choi et al., 2014) or in populations with reduced responses, such as aging, hypercholesterolemia, or hypertension (Holowatz et al., 2011; Minson et al., 2002; Smith et al., 2011). Experimental considerations & limitations It is important to note that this study specifically incorporated male subjects in order to reduce skin blood flow variability due to changes in sex hormone levels across the menstrual cycle and oral contraceptive phase in female subjects (Charkoudian et al., 1999). It is thus possible
that reproductive hormones could affect NO bioavailability. To our knowledge there have been no studies investigating the relationship between reproductive hormones and dietary nitrates. The potential influence of female sex hormones and nitrate supplementation warrants future investigation. Conclusion To our knowledge, this is the first study to investigate the effects of nitrate supplementation via beetroot juice on cutaneous thermal hyperemia during local heating in healthy subjects. Data from the present study suggest that nitrate supplementation provides a modest improvement of NOS-independent vasodilatation as well as reductions in diastolic and mean arterial blood pressures. Our data suggest that the improved thermal hyperemic response is due primarily to a reduction in mean arterial blood pressure resulting in an increased calculated cutaneous vascular conductance and not due to a direct increase in skin blood flow (flux values). Future research is needed to better understand the effects of dietary nitrate supplementation on the skin blood flow response during local heating in not only healthy humans, but also patient populations where nitrate supplementation may have more marked effects on NO-mediated vasodilatation. Conflict of interest None. Author contributions This study was conducted in the Department of Kinesiology at the Kansas State University by Jeremy T. Keen in partial fulfillment of the requirements for the degree of Master of Science. Jeremy T. Keen was responsible for the data collection and data analysis, interpretation of the data, and drafting the manuscript. Erica L. Levitt was responsible for assisting with the data collection and analysis, interpretation of the data, and editing all drafts of the manuscript. Gary J. Hodges was responsible for the data analysis, interpretation of the data, and editing all drafts of the manuscript. Brett J. Wong was responsible for the experimental design, data analysis and interpretation, and editing all drafts of the manuscript. All authors approved the final version of the manuscript. Acknowledgments The authors would like to thank all the subjects who participated in this study; your time and effort are very much appreciated. The authors appreciate the work of Jena K. Eder and for her assistance with the data collection. References Bailey, S., et al., 2009. Dietary nitrate supplementation reduces the O2 cost of lowintensity exercise and enhances tolerance to high-intensity exercise in humans. J. Appl. Physiol. 107, 1144–1155. Bailey, S., et al., 2010. Dietary nitrate supplementation enhances muscle contractile efficiency during knee-extensor exercise in humans. J. Appl. Physiol. 109, 135–148. Brain, S.D., et al., 1986. Potent vasodilator activity of calcitonin gene-related peptide in human skin. J. Invest. Dermatol. 87, 533–536. Bruning, R., et al., 2012. Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin. J. Appl. Physiol. 112, 2019–2026. Brunt, V., et al., 2011. 17β-Estradiol and progesterone independently augment cutaneous thermal hyperemia but not reactive hyperemia. Microcirculation 18, 347–355. Brunt, V.E., et al., 2013. No independent, but an interactive, role of calcium-activated potassium channels in human cutaneous active vasodilation. J. Appl. Physiol. 115, 1290–1296. Charkoudian, N., et al., 1999. Influence of female reproductive hormones on local thermal control of skin blood flow. J. Appl. Physiol. 87, 1719–1723. Choi, P.J., et al., 2014. New Approach to Measure Cutaneous Microvascular Function: An Improved Test of NO-mediated Vasodilation by Thermal Hyperemia. Ferguson, S.K., et al., 2013. Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control in rats. J. Physiol. 591, 547–557.
J.T. Keen et al. / Microvascular Research 98 (2015) 48–53 Fieger, S.M., Wong, B.J., 2010. Adenosine receptor inhibition with theophylline attenuates the skin blood flow response to local heating in humans. Exp. Physiol. 95, 946–954. Fieger, S.M., Wong, B.J., 2012. No direct role for A1/A2 adenosine receptor activation to reflex cutaneous vasodilatation during whole-body heat stress in humans. Acta Physiol. 205, 403–410. Govoni, M., et al., 2008. The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide 19, 333–337. Hodges, G.J., Sparks, P.A., 2013. Contributions of endothelial nitric oxide synthase, noradrenaline, and neuropeptide Y to local warming-induced cutaneous vasodilatation in men. Microvasc. Res. 90, 128–134. Hodges, G., Sparks, P., 2014. Noradrenaline and neuropeptide Y contribute to initial, but not sustained, vasodilatation in response to local skin warming in humans. Exp. Physiol. 99, 381–392. Hodges, G.J., et al., 2006. The involvement of nitric oxide in the cutaneous vasoconstrictor response to local cooling in humans. J. Physiol. 574, 849–857. Hodges, G.J., et al., 2008. The involvement of norepinephrine, neuropeptide Y, and nitric oxide in the cutaneous vasodilator response to local heating in humans. J. Appl. Physiol. 105, 233–240. Hodges, G.J., et al., 2009. The effect of microdialysis needle trauma on cutaneous vascular responses in humans. J. Appl. Physiol. 106, 1112–1118. Holowatz, L.A., et al., 2011. Oral atorvastatin therapy restores cutaneous microvascular function by decreasing arginase activity in hypercholesterolaemic humans. J. Physiol. 589, 2093–2103. Houghton, B.L., et al., 2006. Nitric oxide and noradrenaline contribute to the temperature threshold of the axon reflex response to gradual local heating in human skin. J. Physiol. 572, 811–820. Kapil, V., et al., 2010. Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension 56, 274–281. Kellogg, D., Liu, Y., Kosiba, I.F., O'Donnell, D., 1999. Role of nitric oxide in the vascular effects of local warming of the skin in humans. J. Appl. Physiol. 86, 1185–1190. Kellogg Jr., D.L., et al., 2008. Endothelial nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo. Am. J. Physiol. Heart Circ. Physiol. 295, H123–H129. Kellogg, D., et al., 2009. Roles of nitric oxide synthase isoforms in cutaneous vasodilation induced by local warming of the skin and whole body heat stress in humans. J. Appl. Physiol. 107, 1438–1444. Kenjale, A.A., et al., 2011. Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease. J. Appl. Physiol. 110, 1582–1591.
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Larsen, F.J., et al., 2006. Effects of dietary nitrate on blood pressure in healthy volunteers. N. Engl. J. Med. 355, 2792–2793. Larsen, F.J., et al., 2007. Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol. 191, 59–66. Lundberg, J., Weitzberg, E., 2009. NO generation from inorganic nitrate and nitrite: role in physiology, nutrition and therapeutics. Arch. Pharm. Res. 32, 1119–1126. Lundberg, J., et al., 2008. The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156–167. Lundberg, J.O., et al., 2011. Roles of dietary inorganic nitrate in cardiovascular health and disease. Cardiovasc. Res. 89, 525–532. Minson, C.T., et al., 2001. Nitric oxide and neurally mediated regulation of skin blood flow during local heating. J. Appl. Physiol. 91, 1619–1626. Minson, C.T., et al., 2002. Decreased nitric oxide- and axon reflex-mediated cutaneous vasodilation with age during local heating. J. Appl. Physiol. 93, 1644–1649. Smith, C., et al., 2011. Upregulation of inducible nitric oxide synthase contributes to attenuated cutaneous vasodilation in essential hypertensive humans. Hypertension 58, 935–942. Vanhatalo, A., et al., 2010. Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, R1121–R1131. Vanin, A.F., et al., 2007. Nitric oxide synthase reduces nitrite to NO under anoxia. Cell. Mol. Life Sci. 64, 96–103. Webb, A., et al., 2008a. Mechanisms underlying erythrocyte and endothelial nitrite reduction to nitric oxide in hypoxia: role for xanthine oxidoreductase and endothelial nitric oxide synthase. Circ. Res. 103, 957–964. Webb, A., et al., 2008b. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension 51, 784–790. Wong, B., 2013. Sensory nerves and nitric oxide contribute to reflex cutaneous vasodilation in humans. Am. J. Physiol. Regul. Integr. Comp. Physiol. 304, R651–R656. Wong, B.J., Fieger, S.M., 2010. Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans. J. Physiol. 588, 4317–4326. Wong, B.J., Fieger, S.M., 2012. Transient receptor potential vanilloid type 1 channels contribute to reflex cutaneous vasodilation in humans. J. Appl. Physiol. 112, 2037–2042. Wong, B.J., Minson, C.T., 2011. Altered thermal hyperaemia in human skin by prior desensitization of neurokinin-1 receptors. Exp. Physiol. 96, 599–609.
