Eur J Appl Physiol (2006) 97: 347–354 DOI 10.1007/s00421-006-0190-0
OR I G I NA L A RT I C L E
Buddy Walser · Rose M. Giordano · Charles L. Stebbins
Supplementation with omega-3 polyunsaturated fatty acids augments brachial artery dilation and blood flow during forearm contraction Accepted: 20 March 2006 / Published online: 25 April 2006 © Springer-Verlag 2006
Abstract Omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have beneWcial eVects on the heart and vasculature. We tested the hypothesis that 6 weeks of dietary supplementation with DHA (2.0 g/day) and EPA (3.0 g/ day) enhances exercise-induced increases in brachial artery diameter and blood Xow during rhythmic exercise. In seven healthy subjects, blood pressure, heart rate and brachial artery diameter, blood Xow, and conductance were assessed before and during the last 30 s of 90 s of rhythmic handgrip exercise (30% of maximal handgrip tension). Blood pressure (MAP), heart rate (HR), and brachial artery vascular conductance were also determined. This paradigm was also performed in six other healthy subjects who received 6 weeks of placebo (saZower oil). Placebo treatment had no eVect on any variable. DHA and EPA supplementation enhanced contraction-induced increases in brachial artery diameter (0.28 § 0.04 vs. 0.14 § 0.03 mm), blood Xow (367 § 65 vs. 293 § 55 ml min¡1) and conductance (3.86 § 0.71 vs. 2.89 § 0.61 ml min¡1 mmHg¡1) (P < 0.05). MAP and HR were unchanged. Results indicate that treatment with DHA and EPA enhances brachial artery blood Xow and conductance during exercise. These Wndings may have implications for individuals with cardiovascular disease and exercise intolerance (e.g., heart failure) Keywords Docosahexaenoic acid · Eicosapentaenoic acid · Brachial artery blood Xow · Brachial artery conductance · Brachial artery dilation · Exercise
B. Walser · R. M. Giordano · C. L. Stebbins (&) Department of Internal Medicine, Division of Cardiovascular Medicine, TB 172, University of California, Davis, CA 95616-8634, USA E-mail:
[email protected] Tel.: +1-530-7524714 Fax: +1-530-7523264 C. L. Stebbins Department of Physiology and Membrane Biology, University of California, Davis, CA 95616-8634, USA
Introduction Dietary supplementation with the omega-3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), or consumption of Wsh with high concentrations of these PUFAs has been found to have beneWcial eVects on the cardiovascular system. These eVects include reductions in oxidative stress and endothelial cell damage, and improvements in endothelial-dependent vascular function (Fleischhauer et al. 1993; Mehta et al. 1988; Tagawa et al. 1999, 2002). Dietary supplementation with DHA or EPA has speciWcally been shown to enhance acetylcholine-induced coronary and forearm vasodilation (Fleischhauer et al. 1993; Tagawa et al. 1999). EPA has also been reported to increase forearm reactive hyperemia immediately after vascular occlusion evoked by static handgrip exercise (Tagawa et al. 2002). These results raise the possibility that dietary supplementation with DHA and EPA might augment increases in blood Xow during exercise. On the other hand, DHA + EPA can enhance muscle sympathetic nerve activity during forearm contraction (Monahan et al. 2004), which might evoke vasoconstriction that oVsets their potential beneWcial eVects on blood Xow. Thus, the net eVect of DHA and EPA on blood Xow to skeletal muscle during exercise is unclear. Consequently, we chose to test the hypotheses that; (1) 6 weeks of dietary supplementation with DHA and EPA enhances brachial artery dilation, but not blood pressure, during forearm contraction, and (2) this enhanced dilation is associated with increases in brachial artery blood Xow and conductance.
Materials and methods All procedures and protocols were approved by the Human Subjects in Research Committee, University of California, Davis, and all subjects gave written, informed consent. Thirteen healthy subjects were studied in this
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single-blind, placebo-control study. Seven subjects (Wve males, two females; age range 28–57 years) were placed in the experimental group and received DHA and EPA supplements. The other six made up the control group (four males, two females, age range 26–55 years) and received saZower oil. Pretreatment baseline characteristics of these two groups are presented in Table 1. All subjects completed a health and activity questionnaire to determine their eligibility for the study. Individuals were excluded if they were obese (body mass index > 27 kg m¡2), hypertensive, current or recent smokers, diabetic, or had cardiovascular disease, known hyperlipidemia, or any limitations to exercise. They were also excluded if they consumed dietary supplements containing omega-3 fatty acids, ate Wsh more than once a week, or were taking medications for any preexisting medical condition. Participation in regular exercise was allowed with the exception of athletes training for endurance related competition. Experimental protocol Each subject visited the lab two or three times. During the initial visit, the subjects performed a one-time maximal voluntary contraction (MVC) with their dominant hand using a handgrip dynamometer. This value was used to determine each subject’s contraction intensity for the exercise protocol, which was set at 30% of MVC. After a rest period of at least 15 min, a control exercise protocol was performed. The exercise protocol consisted of two 90 s periods of rhythmic handgrip exercise at 30% of the subject’s MVC and a rate of one contraction /sec. Handgrip was performed with the subject in the supine position. A metronome provided an auditory cue for the subjects, beeping two diVerent tones, alternating each half-second. The subjects were instructed to contract on one tone and relax their hand completely on the other. A strip chart recorder connected to the handgrip dynamometer provided a visual feedback so that the proper peak tension could be maintained. In addition, the subject was given verbal feed back when peak tension deviated from the prescribed level. Prior to exercise, a three lead ECG was attached to the torso, and a sphygmomanometer cuV was placed
on the non-dominant arm. Then, the subject rested for 5–10 min and blood pressure and heart rate were recorded. Brachial artery diameter and blood velocity were measured immediately prior to the onset of exercise. All four of these variables were measured again during the last 30–45 s of the contraction period. At the end of contraction, the subject rested for 15 min and then repeated the protocol. After treatment with DHA and EPA or saZower oil, all subjects returned to the laboratory at a similar same time of day and repeated the protocol at the same absolute work intensity. Dietary supplementation with DHA and EPA Each subject was given soft gel capsules containing either Wsh oils or a placebo. Each capsule of Wsh oil contained »600 mg of PUFAs, of which »200 mg was DHA and »300 mg was EPA (J.R. Carlson Laboratories, Inc., Arlington Heights, IL, USA). Each capsule also contained 10 mg of vitamin E that was added as a stabilizer to prevent rancidity. Subjects were instructed to take ten capsules/day for 42 days (6 weeks). The total daily dose was »2,000 mg of DHA and »3,000 mg of EPA. The placebo group received saZower oil (Solgar, Leonia, NJ), which consisted of the omega-6 polyunsaturated fatty acids linoleic acid (»800 mg/capsule), oleic acid (»160 mg/capsule), and palmitic acid (»100 mg/capsule), and these subjects were asked to take nine capsules/day. Each control subject also took 100 mg of vitamin E/day. The dose of saZower oil was selected because its number of calories was similar to that provided by the daily dose of Wsh oil (»80 cal). All subjects were instructed not to change their diet or begin or end an exercise program. They were also asked to record any deviations in their daily consumption of capsules and note potential side eVects such as diarrhea or intestinal distress. Compliance was veriWed by counting the number of capsules returned at the end of the study and reviewing the subjects’ notes. Subjects were contacted after 2–3 weeks of supplementation to assess and encourage their compliance. The subjects were given enough capsules to last 7 weeks in case there were delays or conXicts in scheduling their return visits. Measurement of brachial artery diameter and blood Xow
Table 1 Baseline age, height, weight, systolic blood pressure (SAP), diastolic blood pressure (DBP), body mass index (BMI), and cholesterol values for the placebo and DHA + EPA groups prior to treatment Variable
Placebo group
DHA + EPA group
Age (years) Height (cm) Weight (kg) SAP (mmHg) DBP (mmHg) BMI (kg m¡2) Cholesterol (mmol l¡1)
45 § 6 174 § 5 73 § 4 117 § 5 71 § 4 24.2 § 1.3 4.5 § 0.5
43 § 4 181 § 5 75 § 5 122 § 4 71 § 4 23.6 § 0.9 4.6 § 0.7
Brachial artery diameters and blood velocities in the exercising arm were recorded using pulsed Doppler/ ultrasound (Sonos 5500, Phillips Medical Systems) and a 7.5 MHz linear probe. The angle of insonation was 60° and the sample depth was 4 cm. All measurements were made with the subject in the supine position. At rest, the brachial artery was located and then a mark made on the subject’s skin at the distal end of the transducer. This was done so that the distal end of the probe could be repositioned at the same point on the arm (2.5–3.5 cm proximal to the antecubital fossa) during contraction and subsequent measurements throughout the protocol. This site
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was measured and recorded at the end of the experiment in order to use a similar anatomical site for brachial artery measurements during the post-treatment protocol. Ultrasound images were recorded to videotape for later analysis, starting during the last 2 min of each rest period. The image of the brachial artery was obtained for at least ten heart beats, and at least Wve velocity waves were recorded. Brachial artery images and blood velocity tracings were taken during the last 30 s of the 90 s contraction. Once the exercise protocol was complete, brachial artery diameters and blood velocities were measured from the videotape. Brachial artery images were selected when they occurred near the end of diastole, and where interfaces between the lumen and proximal and distal walls of the vessel were clear enough to allow diameter measurements. Brachial artery diameters were measured at three points in any given image, and the mean of these three measurements was taken as the vessel diameter. Mean velocity was measured for at least Wve beats during each period, and the mean of those Wve measurements was taken as the mean blood velocity for that period. Brachial artery blood Xow was calculated from vessel diameter and mean blood velocity using the following formula: V £ d2/4 £ 60, where V is the mean blood velocity, and d is the brachial artery diameter. To determine if brachial artery dilation during exercise was greater after supplementation with DHA and EPA than could be explained by an increase in blood Xow alone, changes in diameter were normalized for changes in Xow ( brachial artery diameter/ brachial artery blood Xow expressed as mm ml¡1 min¡1 £ 1,000). Blood pressure and heart rate measurements Blood pressure and heart rate were measured in the nonworking arm using an Omron HEM 907-XL blood pressure monitor. During the rest periods, blood pressure was measured every 2–3 min during the 10 min period immediately prior to the onset of exercise to determine that the subject’s blood pressure was within a normal range for healthy subjects. Due to a lag time for inXation of the pressure cuV, the device took about 45 s to measure blood pressure. Thus, during exercise, measurements were initiated at the 45 s point in order to ensure that the measurement was completed by the end of the exercise period. Exclusion of subjects In addition to the seven subjects who received DHA and EPA treatment, and the six who received saZower oil, three other subjects, who began the study, were excluded from data analysis. In two of these subjects, we were unable to obtain clear images of the brachial artery. As a result, we could not accurately or reliably measure brachial artery diameter. A third subject was removed from the study when it was discovered that he was consuming at least 1.4 kg of salmon/week.
Data analysis All data are expressed as mean § SEM. SigniWcant diVerences in changes from rest to exercise in blood pressure, heart rate, peak developed tension, brachial artery diameter, percent change in brachial artery diameter, brachial artery blood Xow and brachial artery conductance between pre-and post-Wsh oil treatment and preand post-placebo supplementation were determined using the two-way Student’s paired t-test. This test was also used to detect diVerences in resting values of these same variables between conditions. The exercise-induced change in the mean of each variable from control to post-placebo conditions was compared to the same change from control to post-DHA and EPA conditions using the unpaired Student’s t-test. Statistically signiWcant diVerences were accepted at P < 0.05.
Results Mean arterial pressure, heart rate, and peak developed tension Compared to control conditions, 6 weeks of treatment with placebo (saZower oil) or DHA and EPA had no eVects on resting blood pressure or heart rate. Increases in these variables in response to 90 s of rhythmic contraction were also unaVected by either intervention (Fig. 1). Average peak developed tension during the control contraction in each group was similar to that seen after supplementation with placebo or DHA and EPA (Fig. 1). Brachial artery diameter Brachial artery diameter at rest was not altered by either placebo or DHA and EPA supplementation (Fig. 2). Placebo treatment had no eVects on absolute or percent increases in brachial artery diameter during rhythmic contraction (Fig. 2). Conversely, the absolute increase in brachial diameter caused by contraction was signiWcantly elevated following supplementation with DHA and EPA (Fig. 2). This was also the case for the percent change in diameter (Fig. 2). Contraction-induced changes in mean absolute and percent increases in brachial artery diameter from control to post-DHA and EPA treatment were signiWcantly greater (P < 0.05) than those that occurred from control to postplacebo treatment (0.14 § 0.05 vs. ¡0.02 § 0.01 mm and 4.70 § 1.00 vs. ¡0.20 § 0.06%, respectively). The diVerence in the change in normalized brachial artery diameter during exercise between the pre- and postDHA and EPA supplementation conditions (0.53 § 0.14 vs. 1.06 § 0.31 mm ml¡1 min¡1 £ 1,000) did not quite reach statistical signiWcance (P = 0.06). This was also the case in the placebo treated group (0.55 § 0.13 vs. 0.41 § 0.19 mm ml¡1 min¡1 £ 1,000). However, a trend
350 Fig. 1 Mean changes in mean arterial blood pressure (MAP) heart rate (HR) and peak tension development ( tension) in response to 90 s of intermittent forearm contraction (30% MVC) before, and 6 weeks after dietary supplementation with either saZower oil (placebo) (N = 6) or with DHA (2.0 g/ day) + EPA (3.0 g/day) (N = 7). Numbers beneath histograms are pre-exercise values
for an eVect of Wsh oil treatment is suggested by the fact that six of seven subjects in the DHA + EPA group demonstrated an augmentation of this change, compared to only one of six subjects in the placebo group. Brachial artery blood Xow and conductance Neither placebo nor DHA and EPA treatment had any eVect on resting brachial artery blood Xow or conductance (Fig. 3). Contraction-induced increases in these two variables were also not aVected by placebo treatment (Fig. 3). Supplementation with DHA and EPA, however, caused a 30 § 6% increase in brachial artery blood Xow during exercise and a 55 § 21% increase in brachial artery conductance (Fig 3).
Exercise-evoked changes in mean brachial artery blood Xow and conductance from control to post-DHA and EPA treatment conditions were signiWcantly greater than those observed from the control to post-placebo treatment conditions (74 § 18 vs. 14 § 18 ml min¡1 and 0.93 § 0.22 vs. 0.17 § 0.17 ml min¡1 mmHg¡1, respectively).
Discussion We found that 6 weeks of dietary supplementation with DHA and EPA enhanced brachial artery dilation and blood Xow during rhythmic contractions of the forearm. Previous studies, using venous occlusion plethysmography,
351 Fig. 2 Mean absolute increases in brachial artery diameter (BAD) and per cent increases in BAD (BAD%) during 90 s of intermittent forearm contraction (30% MVC) before and 6 weeks after dietary supplementation with either saZower oil (N = 6) or DHA + EPA (N = 7). Numbers beneath histograms are pre-exercise values. Pre-exercise values were not diVerent between conditions. *P < 0.05, vs. pre-dietary supplementation DHA + EPA
have shown that supplementation with EPA in patients with coronary artery disease and endothelial dysfunction can enhance reactive hyperemia in the forearm vasculature following static contraction-induced arterial occlusion and augment pharmacological-induced activation of endothelial-dependent forearm vasodilation (Tagawa et al. 1999; Tagawa et al. 2002). Our investigation is the Wrst to demonstrate that DHA and EPA increase brachial artery blood Xow during rhythmic exercise and that this increase is, in part, associated with concomitant increases in arterial diameter. The combined doses of DHA (2 g/day) and EPA (3 g/ day) given to our subjects were selected because previous studies have found that they can improve acetylcholineinduced, endothelium-dependent coronary vasodilation (Fleischhauer et al. 1993) and reduce norepinephrineand angiotensin II-evoked attenuations in skeletal muscle blood Xow after 3–6 weeks of treatment (Chin et al. 1993). While we felt that these doses of DHA and EPA would optimize the chances of enhancing brachial artery blood Xow and conductance during exercise, it is possible that lower doses also could have beneWcial eVects. Each capsule of DHA and EPA contained 10 I.U. of vitamin E, which was added by the manufacturer to prevent oxidation of these oils. This may have been problematic because there is some evidence that vitamin E supplementation can improve endothelial function and
forearm blood Xow under certain conditions (Visioli 2001). Thus, vitamin E might have contributed to the augmentations in exercise-induced increases in brachial artery diameter and blood Xow that we observed after DHA and EPA supplementation. This outcome is unlikely, however, because subjects that received the placebo oil were also given the same dose of vitamin E (100 I.U./day), and they demonstrated no changes in brachial artery diameter or blood Xow. Our contraction paradigm was chosen to optimize accuracy in assessing brachial artery diameter and blood velocity by minimizing arm movement during exercise. Dynamic forearm contractions, producing muscle tension of this intensity (30–33% of maximum), cause 2–4 fold increases in brachial artery blood Xow that can be sustained for 2–3 min without causing fatigue (Stebbins et al. 2002). This paradigm was also selected because it evokes increases in cardiac output and blood pressure that are relatively small compared to concomitant increases in brachial artery blood Xow (Stebbins et al. 2002). Thus, changes in blood Xow under these conditions should reXect the action of local factors (e.g., endothelial factors) more than central cardiovascular eVects (e.g., large changes in cardiac output). Prior to supplementation with DHA and EPA, changes in blood Xow during contraction were smaller than those observed in our previous study (»300 vs. »450 ml min¡1)
352 Fig. 3 Mean changes in brachial artery blood Xow (BABF) and conductance (BAC) in response to 90 s of intermittent forearm contraction before and 6 weeks after dietary supplementation with saZower oil (N = 6) or DHA + EPA (N = 7). Numbers beneath histograms are pre-exercise values. Pre-exercise values were not diVerent between conditions. *P < 0.05, vs. pre-dietary supplementation with DHA + EPA
(Stebbins et al. 2002). This discrepancy was likely due to diVerences in body position. Subjects were in the supine position in the present study and upright in the earlier study (Stebbins et al. 2002). This diVerence is relevant because Xow mediated vasodilation in the brachial artery increases as the body is progressively moved from the supine to upright position (Guazzi et al. 2004). The validity of our Wndings is dependent on the repeatability of assessments of brachial artery diameter and blood Xow between conditions over time. Otherwise, the changes we observed might have been due to variability in our measurements. Although we do not have conclusive evidence of repeatability of our measurements, comparison of pre-and post-placebo conditions revealed no diVerences in exercise-evoked changes in brachial artery diameter or blood Xow. Since no diVerences were expected in this group, these observations provide some indication that our assessments were repeatable.
Possible mechanisms of action of DHA and EPA During exercise, increases in skeletal muscle blood Xow are believed to be initiated, in part, by byproducts of metabolism that evoke dilation in pre-capillary terminal arterioles (Folkow et al. 1971). Dilation then progresses proximally upward through feed arteries and conduit vessels (e.g., brachial artery) that are external to the tissue (Segal 1992). To a large extent, vasodilation in these external arteries is induced by Xow-mediated dilation evoked by increases in shear stress on the endothelium (Koller and Kaley 1991). The fact that terminal arteries are conWgured in series, with progressively larger feed arteries, underlies this response. We measured blood Xow in a conduit vessel (i.e., the brachial artery). Our Wndings that DHA and EPA supplementation tended to augment exercise-induced increases in normalized brachial artery dilation suggest that endothelial function was enhanced in addition to any potential
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changes in metabolic-induced vasodilation of terminal arterioles. Such an eVect could be caused, in part, by the action of DHA and EPA on Xow mediated release of vasodilator substances from the endothelium. Although somewhat controversial, it is believed that Xow mediated dilation in the brachial artery is dependent, in part, on nitric oxide (Green 2005), whose production can be elevated in response to shear stress (Rubanyi et al. 1996). We found that treatment of cultured endothelial cells with physiological concentrations of DHA augments expression of endothelial nitric oxide synthase (eNOS) (Stebbins et al. 2004), the primary enzyme responsible for the synthesis of NO from L-arginine. This eVect, in turn, could raise production of this potent vasodilator during eNOS activation (e.g., shear stress). Similar eVects of DHA and EPA on the endothelium of terminal arterioles might enhance metabolic-induced dilation and shear stress that originates in these vessels. Interestingly, neuronal NOS (nNOS) is expressed in large quantities in skeletal muscle (Frandsen et al. 1996), and blockade of this enzyme attenuates exercise hyperemia (Hickner et al. 1997). If DHA enhances nNOS expression, as it does eNOS, an increase in NO production and consequent dilation of terminal arteries during contraction would not be surprising. The vascular endothelium is also a major source of eicosanoids (Cannon 1984) and the release of these substances can be facilitated by an increase in vascular shear stress (Grabowski et al. 1985). Given that Wsh oils have been found to augment endothelial release of the vasodilator prostaglandins PGI2 and PGI3 (Hishinuma et al. 1985), they also may be capable of facilitating Xow mediated vasodilation. These potential actions of DHA and EPA are relevant because combined eVects of prostaglandins and nitric oxide have been shown to contribute to increases in brachial artery blood Xow during exercise (Schrage et al. 2004). Consequently, DHA- and EPAinduced potentiation of NO and vasodilator prostaglandin release could reasonably be expected to enhance brachial artery diameter and blood Xow during exercise. Metabolic eVects of Wsh oils on skeletal muscle have also been reported. These include reduced stimulation of plasma glucose Xuxes during exercise (Delarue et al. 2003), increases in insulin-induced glucose transport (Sohal et al. 1992), and potential increases in peripheral insulin sensitivity (D’Alessandro et al. 2002). Such eVects might modulate muscle metabolism and changes in metabolic-induced dilation of terminal arteries and shear stress during exercise that aVect concomitant increases in brachial artery blood Xow. Another mechanism of action by which DHA and EPA may have enhanced brachial artery blood Xow during exercise involves attenuations of sympathetic-induced vasoconstriction in skeletal muscle resistance vessels. As mentioned previously, this vasoconstriction may actually be enhanced by dietary supplementation with DHA and EPA (Monahan et al. 2004). Thus, the ability of DHA and EPA to enhance increases in brachial artery diameter and blood Xow in active muscle occurs in spite of a con-
comitant increase in muscle sympathetic nerve activity (which would tend to attenuate Xow mediated vasodilation). This apparent paradox may be explained, in part, by the potential for these Wsh oils to enhance NO production in skeletal muscle, possibly via increased expression of nNOS. NO generated by this enzyme appears to play an important role in the muscle blood Xow response to exercise via attenuation of -adrenergic constriction in resistance arterioles (Sander et al. 2000) and by inducing exercise-evoked increases in blood Xow (Hickner et al. 1997). These outcomes imply that eVects of DHA and EPA on active muscle blood Xow are likely expressed locally in skeletal muscle and/or its vasculature and not by central modulation of sympathetic outXow. Limitations Findings from this investigation are limited to eVects of DHA and EPA on blood pressure, vasodilation and blood Xow during acute dynamic contractions of a small muscle mass. Prolonged exercise, involving a larger muscle mass, might cause diVerent eVects. For example, during long term exercise with a larger muscle mass (e.g., running), these eVects of DHA and EPA may reduce overall systemic vascular resistance such that blood pressure is also reduced. Perspectives Our observations suggest that dietary supplementation with DHA and EPA can enhance blood Xow and oxygen delivery to exercising skeletal muscle and increase vascular conductance at a given work intensity. Thus, it is tempting to contend that treatment with these PUFAs could delay the onset of fatigue and allow individuals to work at higher workloads for longer periods of time. However, studies involving greater muscle mass and a higher intensity of exercise will be necessary to determine if this is actually the case. From a clinical viewpoint, DHA and EPA supplementation could beneWt patients who exhibit endothelial dysfunction and elevated systemic vascular resistance during exercise. These abnormalities contribute to reductions in blood Xow and oxygen delivery to active skeletal muscle and/or increases in afterload that are associated with exercise intolerance in conditions such as heart failure, hypertension, and diabetes (Clark et al. 1996; Kingwell et al. 2003; Lim et al. 1996). If DHA and EPA treatment could enhance skeletal muscle blood Xow and conductance in these patients, as it did in our healthy subjects, then their functional capacity and quality of life might also be improved.
Conclusions Our results indicate that 6 weeks of dietary supplementation with the DHA and EPA augment brachial artery
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blood Xow and conductance during short term rhythmic forearm contraction. EVects were unrelated to increases in perfusion pressure and likely occurred in spite of DHA-and EPA-induced increases in muscle sympathetic nerve activity. These Wndings have implications for increasing oxygen delivery to active skeletal muscle and improving functional capacity, especially in pathological conditions where endothelial dysfunction and exercise intolerance occur (e.g., coronary artery disease, heart failure, diabetes). Acknowledgement This work was supported by the UC Davis Clinical Nutrition Research Unit, NIH P30-DK35747.
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