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Arm position affects calculation of posture-induced vs. cycling-induced change in plasma volume.

INTRODUCTION

Chronic aerobic exercise training (8,13,19,27) and chronic high intensity interval training (3) usually elicit plasma volume (PV) expansion. An increase in plasma volume results in increases in blood volume, venous return to the heart, and stroke volume, which together result in a decrease in heart rate (HR) at rest or at any given absolute work rate (8). Conversely, during an acute bout of exercise, PV generally decreases as exercise intensity increases (16,19,28). Additionally, PV changes with an acute change in posture. That is, PV decreases as posture becomes more upright (4,5,18). Any change in plasma volume may affect the concentration of a variety of blood constituents. Yet, few of the numerous studies reporting the PV response to an acute bout of exercise have differentiated between posture-induced versus exercise-induced changes in plasma volume ([DELTA]PV).

Cycling is the most reported mode of exercise used to examine [DELTA]PV during exercise, but only five cycling studies reported the statistical results for posture-induced versus cycling-induced [DELTA]PV. In four of these studies, PV decreased when subjects moved from supine to upright-seated rest in a thermoneutral (15,23,24) or hot (10) environment. During subsequent exercise, [DELTA]PV was dependent on intensity of exercise. The greater the intensity, the greater the decrease in PV during exercise (15,24). In a more recent study, there was no [DELTA]PV when subjects moved from the supine to the upright-seated rest posture, but during subsequent exercise, PV decreased as exercise intensity increased (29). These contrasting results may be explained by the position of the blood-sampling arm during the protocol (12,16,22).

In the study that found no [DELTA]PV when subjects moved from supine to seated rest, the blood-sampling arm was in the horizontal position, supported at the level of the heart (29). Of those studies that found a decrease in PV when subjects moved from supine to seated rest, only Pivarnik et al. (23) reported the blood-sampling arm position, resting on the handlebar of the cycle ergometer. Additionally, in each study, [DELTA]PV was determined via [DELTA] in antecubital venous hematocrit ratio (Hct) and hemoglobin concentration (Hb), which is the most common method reported in the literature for determining [DELTA]PV during an acute bout of exercise (11,16).

These observations suggest that arm position may affect both calculation of posture-induced versus exercise-induced [DELTA]PV and the concentration of a variety of blood constituents. Yet, no study has examined the effect of blood-sampling arm position on the calculation of posture-induced versus exercise-induced [DELTA]PV, when a postural control period is assigned between supine rest and an exercise bout. Therefore, the purpose of this study was to determine the influence of blood-sampling arm position on calculation of posture-induced versus cycling-induced calculation of [DELTA]PV. Change in Hct and Hb was also examined.

METHODS

Subjects

Prior to testing, 10 somewhat active (predicted [VO.sub.2] peak = 41.3 [+ or -] 7.1 mL x [kg.sup.-1] x [min.sup.-1]) male undergraduate university students provided written informed consent before the experiment began. The university's Institutional Review Board approved both the experiment and the consent form. Each subject was screened via a physical activity and medical history questionnaire. Exclusion criteria included a history of heart disease, vascular disease, hypertension, pulmonary disease, diabetes mellitus, kidney disease, liver disease, or thyroid disease, tobacco use, and present medication use.

Procedures

All data were collected in the fall season to rule out any effect of heat acclimation. Room temperature (mean = 21.8 [+ or -] 0.3 [degrees]C) and relative humidity (mean = 51.7 [+ or -] 1.8%) were maintained within a narrow range across all experiments. In order to prevent any acute effect of unaccustomed activity on the experimental result, each subject was asked not to participate in unaccustomed activity for 3 d prior to the experiment. Each subject was asked to neither eat nor drink anything, except water, for the 12 hrs preceding the test. The subjects were encouraged to drink at least one liter of water during the evening before and again on the morning of the test prior to arrival at the laboratory.

Each subject arrived at the laboratory at approximately 8:00 am and was asked to produce a 20 cc urine sample. Next, urinary specific gravity (Usg) was determined via refractometry in triplicate (Reichert--Jung TS Meter Refractometer, Model # 10400A). If the Usg reading was greater than 1.020 (2,25), the subject drank 0.5 to 1.0 liter of tap water. After ~30 min had passed, Usg was again evaluated. Each subject who had an initial Usg greater than 1.020 attained an Usg less than 1.020 after one attempt at rehydration. After the subject achieved euhydration, his height and body mass were measured. Next, the subject lay supine for 30 min with both arms supported in the horizontal position (i.e., parallel to the floor) by the examining table.

After supine rest, the subject moved to the upright-seated posture on the cycle. Upon taking the seated posture, one arm was placed on a pillow (supported by a cart), with the humerus abducted ~75[degrees] and the antecubital vein at the level of the fourth rib (horizontal arm). The arm-to-rib position was confirmed via a carpenter's level. During the transition from supine rest to seated rest, an assistant supported the horizontal arm so that the position of the antecubital vein (heart level) did not change. The other arm rested on the cycle's handle bar at ~45[degrees] of humeral flexion (pendent arm). Each arm stayed in the assigned position for the remainder of the experiment. Order for arm assignment (horizontal versus pendent) was counterbalanced for arm dominance (dominant versus non-dominant). The legs rested on the pedals during the upright-seated control period.

After 30 min of upright-seated rest, the subject began pedalling a previously calibrated cycle ergometer (Monark, Model 818 E) at a work rate of 60 W. The workrate was increased ~30 W every 2 min until the subject attained a heart rate (HR) of ~60 to 70% of his age-predicted maximal HR (220-age). After the HR reached a steady-state condition at that work rate, the work rate was further adjusted, if necessary, until the subject reached a steady-state HR of ~75% of his age-predicted maximal HR. Then, the subject cycled at that work rate for ~15 min. After completion of the exercise bout, the subject performed a 5-min active cool down, cycling at 30 W. Heart rate was recorded (Polar Heart Rate Monitor, Vantage XL) every minute of the exercise bout. Rating of perceived exertion (RPE) (7) was obtained during the 8th-min of cycling at the target HR. In addition to each arm remaining in the assigned position during seated rest and exercise, throughout the experiment, the subject was continually coached not to perform an isometric contraction or make a fist with either hand.

Blood Sampling

During supine rest, a sterile intravenous catheter was introduced into an antecubital vein of each arm and a sterile 3-way stopcock was attached to the catheter. After 30 min of supine rest, blood was drawn from each arm. The following blood-sampling procedure was carried out for each arm throughout the experiment: (a) ~2 ml of blood was withdrawn, without stasis, and discarded to account for dead space; (b) ~10 ml resting blood sample was then drawn, without stasis; and (c) sterile normal saline (1 to 2 ml) was then introduced into the catheter to keep the vein patent. Blood was drawn from the second arm ~60 sec after blood was drawn from the first arm. The order for blood sampling was counterbalanced for both arm dominance (dominant versus non-dominant) and arm position (horizontal versus pendent). In addition to the supine rest blood sample, blood was drawn after 30 min of seated rest (1,18) and after 15 min of cycling at the target HR (6,23).

Hematocrit was determined in quadruplicate via centrifuge (Clay Adams Triac Centrifuge, Model #0200) immediately after it was drawn. No correction was made to Hct for trapped plasma, Fcell ratio, or peripheral sampling. Because of the short duration of the exercise bout, it was assumed that the red cell volume remained constant (8). Hemoglobin was measured in triplicate via spectrophotometry (Milton Roy, Spectronic 20D) by use of a cyanomethemoglobin solution (Sigma Chemical # 525-A) immediately after it was drawn. Mean measures of Hct and Hb were used to calculate [DELTA]PV via the following equation (11,16):

% [DELTA] PV = {[([Hb.sub.1]) (1 - [Hct.sub.2])] / [([Hb.sub.2]) (1 - [Hct.sub.1])]} -1) x 100

[Hct.sub.1] and [Hb.sub.1] were the values before each given provocation and [Hct.sub.2] and [Hb.sub.2] were the values resulting from the given provocation (i.e., supine to seated rest, seated rest to exercise, and supine rest to exercise).

Statistical Analyses

Two-way analyses of variance with repeated measures were used to determine any significant interaction between arm position (horizontal versus pendent) and condition (posture versus exercise) on [DELTA]PV (2x2), Hct (2x3), and Hb (2x3). When Mauchley's test demonstrated that sphericity was violated, the Greenhouse-Geisser adjustment was employed to determine significance. When a significant interaction existed, post hoc paired t tests were used to determine any significant difference for the given variable between horizontal and pendent arm for each condition and effect size (ES) was calculated. When a significant effect of condition existed, post hoc Tukey's tests were made to determine any significant difference in that variable among conditions. Also, a paired t test was used to determine any significant difference between horizontal and pendent arm [DELTA]PV from supine rest through the exercise bout. Confidence intervals (CI) for all t tests are reported as the difference of the means. The SPSS Statistics 18.0 software program was used for all analyses, except the Tukey's tests and ES, which were calculated by hand. A type one error rate of a = 0.05 was set a priori to indicate statistical significance for all analyses. Pilot testing suggested a mean difference in [DELTA]PV between arm positions for each stage of the test of 10%, with a standard deviation (SD) of [+ or -] 8.0. The power to detect an effect of this size with 10 subjects and a = 0.05 was determined to be 87.5%.

RESULTS

The following were the mean (SD) descriptive characteristics for the subjects: age = 22.3 [+ or -] 2.0 yrs, height = 179.0 [+ or -] 6.7 cm, and body mass = 77.9 [+ or -] 10.4 kg. Before the supine rest period, mean Usg was 1.009 [+ or -] 0.004. The following were the mean values measured during steady-state cycling: work rate = 139.6 [+ or -] 20.0 W, HR = 153.1 [+ or -] 7.1 beats x [min.sup.-1], percentage of age-predicted maximal HR = 77.5 [+ or -] 3.8%, and RPE = 14.2 [+ or -] 1.5.

Plasma Volume

There was an interaction ([F.sub.1,9] = 9.454, P = 0.013, [[eta].sup.2] = 0.512) between condition and arm position on [DELTA]PV (Figure 1). Post hoc paired t tests revealed that [DELTA]PV was greater (t = 2.784, P = 0.021, ES = 1.29, 95% CI of difference = 2.2 to 21.6) in the pendent arm than the horizontal arm when subjects moved from supine rest to seated rest. Conversely, from seated rest through the exercise bout, the decrease in PV was greater (t = -3.137, P = 0.012, ES = 1.15, 95% CI of difference = -15.8 to -2.6) in the horizontal arm than the pendent arm Also, when [DELTA]PV from supine rest through the bout of cycling was calculated via the supine and exercise values, [DELTA]PV was similar (t = 0.871, P = 0.406, ES = 0.41, 95% CI of difference = -2.8 to 6.4) for the horizontal and pendent arm.

[FIGURE 1 OMITTED]

Hematocrit

There was an interaction ([F.sub.2,18] = 13.0, P = 0.003, [[eta].sup.2] = 0.591) between arm position and condition on Hct (Figure 2). Post hoc paired t tests revealed that, during seated rest, Hct was greater (t = -3.666, P = 0.005, ES = 0.13, 95% CI of difference = -3.8 to -0.9) in the pendent arm than in the horizontal arm. There was no difference between horizontal versus pendent arm Hct during supine rest (t = 0.527, P = 0.611, ES = 0.04, 95% CI of difference = -0.3 to 0.5) or exercise (t = 0.246, P = 0.811, ES = 0.01, 95% CI of difference = -0.4 to 0.5).

There was a main effect of condition ([F.sub.2,18] = 106.087, P<0.001, [[eta].sup.2] = 0.992) such that, when averaged across arm positions, Hct increased from supine rest through the exercise bout. A post hoc Tukey's test revealed that, when averaged across arm positions, Hct was different (P [less than or equal to] 0.05) for each condition.

[FIGURE 2 OMITTED]

Hemoglobin

There was an interaction ([F.sub.2,18] = 4.361, P = 0.029, [[eta].sup.2] = 0.326) between condition and arm position on Hb (Figure 3). Post hoc paired t tests revealed that Hb during seated rest was greater (t = -2.816, P = 0.020, ES = 0.65, 95% CI of difference = -2.0 to -0.2) in the pendent arm than in the horizontal arm. There was no difference between horizontal versus pendent arm Hb during supine rest (t = 0.137, P= 0.894, ES = 0.04, 95% CI of difference = -0.8 to 0.9) or exercise (t = -0.570, P = 0.583, ES = 0.12, 95% CI of difference = -0.9 to 0.6).

When averaged across arm positions, there was a main effect of condition ([F.sub.2,18] = 35.453, P<0.001, [[eta].sup.2] = 0.798) such that Hb increased from supine rest through the exercise bout. A post hoc Tukey's test revealed that, when averaged across arm positions, Hb was greater (P [less than or equal to] 0.05) during exercise than during supine rest and seated rest.

[FIGURE 3 OMITTED]

DISCUSSION

This study examined the influence of blood-sampling arm position on posture-induced versus exercise-induced calculation of [DELTA]PV. The position of the arm affected the evaluation of posture-induced versus exercise-induced [DELTA]PV such that [DELTA]PV was greater in the pendent arm than the horizontal arm when subjects moved from supine to seated rest, but greater in the horizontal arm than pendent arm when measured from seated rest through the exercise bout. Additionally, the decrease in PV from the end of the supine rest period through the exercise bout was similar for the horizontal and pendent arm.

Posture

When measured from supine rest through the seated rest posture, Hct and Hb were greater for the pendent arm than the horizontal arm, which is in agreement with the findings of other studies that found Hct (12) and Hb (22) greater in a pendent arm than horizontal arm when subjects moved from a supine to standing posture. The change in Hct and Hb directly affected the calculation of [DELTA]PV.

The difference in horizontal versus pendent arm [DELTA]PV during seated rest in the present study may be explained by the difference in local capillary hydrostatic forces in the two arms. Although not measured in the present study, when a limb is moved from a horizontal to a pendent position, there is an increase in regional capillary hydrostatic pressure (9), which causes an increase in local capillary filtration from the intravascular space into the interstitial space (14,17). The greater regional capillary filtration in a pendent arm causes a greater regional Hct (12) and Hb (22) compared to a horizontal arm. Concomitantly, there is a greater regional decrease in PV in a pendent limb compared to a horizontal limb (4,5), as calculated in the present study. Furthermore, Lundvall and Bjerkhoel (22) observed that, in contrast to the pendent arm antecubital venous Hb response, the horizontal arm antecubital venous Hb response was almost identical to that of the pendent arm radial arterial Hb response when subjects moved from the supine to the standing position. That is, horizontal arm antecubital venous Hb may better reflect whole-body Hb, as represented by arterial Hb, and may more accurately reflect whole-body [DELTA]PV.

Exercise

This appears to be the first study to examine the influence of arm position on [DELTA]PV during a bout of cycling. Plasma volume decreased in both the horizontal and pendent arm during exercise. This finding is in agreement with numerous studies that found a decrease in PV from rest through a bout of moderate or high intensity cycling, regardless of pre-exercise posture (16,19,28). While the decrease in PV was greater in the horizontal arm than the pendent arm from upright-seated rest period through the exercise bout in the present study, this resulted in a similar decrease in PV in the horizontal and pendent arm from supine rest period through the exercise bout. Although not measured in the present study, these results suggest that whole-body osmotic forces drive whole-body fluid shifts during exercise (16,19,20,21,26,28). That is, as exercise intensity increases, the increase in osmotically active particles (e.g., lactate) in the active muscle tissue results in a concomitant efflux of fluid from the intravascular space into the active muscle tissue (20,21,26). Lundvall (21) ascribed ~75% of transcapillary fluid flux in active muscle tissue in cats to the increase in local osmolality and ~25% to the increase in local capillary hydrostatic pressure during exercise.

Limitations of the Study

This study examined only relative [DELTA]PV, calculated via [DELTA]Hct and [DELTA]Hb in a thermoneutral environment, when cycling. Also, the sample was limited to healthy, male undergraduate students. Therefore, caution should be used when attempting to generalize these results.

CONCLUSIONS

In the present study, there was an interaction between arm position and condition such that the [DELTA]Hct, [DELTA]Hb, and calculation of [DELTA]PV from supine to upright-seated rest were greater for the pendent arm than the horizontal arm. Conversely, the [DELTA]Hct, [DELTA]Hb, and calculation of [DELTA]PV from seated rest through the cycling bout were greater for the horizontal arm than the pendent arm. That is, blood-sampling arm position affected posture-induced versus exercise-induced [DELTA]Hct, [DELTA]Hb, and calculation of [DELTA]PV. However, arm position did not affect [DELTA]Hct, [DELTA]Hb, or calculation of [DELTA]PV from supine rest through the exercise bout. Thus, when examining posture-induced versus exercise-induced Hct, Hb, or [DELTA]PV, blood-sampling arm position should be a consideration in the experimental design.

ACKNOWLEDGMENTS

The author thanks Dr. Michele Fisher for her invaluable contributions to both the statistical analysis and editing of this manuscript and Dr. Frederick Gardin and Dr. Tina Manos for their contributions in editing the manuscript. Thanks also to Marie C. Fuentes for her assistance in the data collection process.

Address for correspondence: William Sullivan, EdD, Department of Exercise Science and Physical Education, Montclair State University, Montclair, NJ, USA 07043. Phone: (973) 655-7089; Email: sullivanw@mail.montclair.edu

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William Sullivan

Department of Exercise Science & Physical Education, Montclair State University, Montclair, NJ 07043 USA
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