Norepinephrine kinetics and cardiac output during nonhypotensive lower body negative pressure ROBERT G. BAILY, URS LEUENBERGER, GRETCHEN DAVID SILBER, AND LAWRENCE I. SINOWAY
LEAMAN,
Division of Cardiology, The Milton S. Hershey Medical Center, The Pennsylvania College of Medicine, Hershey, Pennsylvania 17033
BAILY, ROBERT G., URS LEUENBERGER, GRETCHEN LEAMAN, DAVID SILBER, AND LAWRENCE I. SINOWAY. Norepinephrine kinetics and cardiac output during nonhypotensive lower body negativepressure. Am. J. Physiol. 260 (Heart Circ. Physiol.
29): H1708-Hl712,1991.-Recently we have shown that arterial norepinephrine (NE) concentration increases significantly during lower body negative pressure (LBNP) of -15 mmHg. Interestingly, the increase was found to be related predominantly to a decrease in arterial NE clearance. We postulated that this reduction in clearance would be related to a reduction in cardiac output. Accordingly, we measured both cardiac output (2dimensional echocardiographic/Doppler technique) and arterial NE kinetics ( [3H]NE continuous infusion radiotracer technique) during LBNP of -15 mmHg. These measures of cardiac output and arterial NE spillover and clearance were obtained in 12 normal subjects at baseline, 5 and 10 min (Early) and 25 and 30 min (Late) of LBNP. We found that arterial NE concentration increased significantly, by 25% Early and 22% Late (P = 0.001). Spillover, however, did not change (P = 0.258), whereas clearance decreased by 12% Early and 19% Late (P = 0.014), and cardiac output decreased by 15% Early and 19% Late (P = 0.001). These reductions in clearance and cardiac output correlated significantly (r = 0.61, P = 0.001). No correlation was noted between spillover and cardiac output (r = 0.027, P = 0.874). We conclude that the increases in arterial NE concentration during nonhypotensive LBNP are predominantly due to decreased cardiac output with resultant decreases in systemic clearance of NE. These findings suggest that the ability to clear NE from the circulation is linked to the level of cardiac output and that low cardiac output states by themselves may lead to an elevation in arterial plasma NE concentrations. microneurography; cardiopulmonary baroreceptors; echocardiographic/Doppler cardiac output LOWER BODY NEGATIVE PRESSURE (LBNP) of -15 mmHg results in selective unloading of low-pressure baroreceptors (2). We have recently completed studies demonstrating that cardiopulmonary baroreceptor unloading results in sympathetic nervous system (SNS) activation that is predominantly localized to skeletal muscle. This conclusion was based on the fact that we noted a 67% increase in muscle sympathetic nerve activity, a 23% increase in forearm venous norepinephrine (NE) spillover (representing skin and muscle), no change in skin blood flow, and no change in arterial NE spillover (representing generalized SNS activation) (2). Interestingly, however, despite the relatively localized
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nature of the neural response, we found that arterial and forearm venous plasma plasma N E concentration NE concentration increased similarly during isolated low-pressure baroreceptor unloading with -15 mmHg LBNP. This suggested that the increase in arterial plasma NE concentration during -15 mmHg LBNP was dependent on factor(s) other than SNS activation. In fact, we found that reductions in systemic clearance of NE appeared to be primarily responsible for the significant increases in arterial plasma NE concentration in our subjects. Best and Halter (4) have shown that intravenous infusions of propranolol prompt an increase in arterial NE concentration, a decrease in arterial NE clearance and no change in arterial NE spillover at a time when blood pressure is unchanged and heart rate is decreasing. The authors speculated that a decrease in blood flow was responsible for the increase in arterial NE concentration secondary to a reduction in systemic clearance of NE, rather than an increase in generalized neural activation. Based on the findings of Best and Halter and our previous study (2), we postulated that LBNP of -15 mmHg would result in reduced arterial NE clearance secondary to decreases in cardiac output. This in turn would result in an increase in arterial NE concentration that would not necessarily be related to generalized activation of the SNS. In this study we used noninvasive two-dimensional (2D) echocardiographic/Doppler measures of cardiac output and continuous infusion radiotracer measures of arterial NE spillover and clearance to assess the potential relationship between decreased cardiac output and arterial NE clearance during isolated cardiopulmonary baroreceptor unloading. METHODS
Subjects. Twelve subjects with a mean age of 25 t 0.7 (*SE) yr participated in NE kinetic studies and of those 12,7 had cardiac output measurements made at the same time as the plasma samples were taken. The other five subjects had cardiac output measured on a separate day under the same experimental conditions. The protocol was reviewed and approved by the Clinical Investigation Committee of the Milton S. Hershey Medical Center. Informed consent was obtained from each individual. No subject enrolled in the study had a history of heart
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disease, hypertension, or thyroid disease, and no subject was taking medications or substances that might have altered SNS activity. All subjects were familiarized with LBNP before the study. Experimental protocol. Studies were performed in a quiet, partially darkened temperature-controlled room. Subjects were comfortably positioned in a partial left lateral decubitus position in an LBNP chamber. The partial left lateral decubitus position provided an optimal imaging window for 2-D echocardiographic/Doppler measurements of cardiac output. Polyethylene wrap was placed around the subject’s torso at the level of the iliac crests to obtain an airtight seal. Subjects were instrumented with a U-in. al-gauge antecubital venous cannula for infusion of[3H]NE in the right arm, and a left radial artery cannula was inserted for continuous hemodynamic monitoring as well as arterial blood sampling. Respirations were monitored via pneumograph. Arterial blood samples were obtained to determine baseline (Base) plasma NE and to provide plasma for determination of [3H]NE recovery by aluminum oxide (alumina) adsorption (1). The subjects received an initial bolus of [3H]NE (15 &i/m”) over 5 min, followed by continuous infusion of 0.35 PCi. mine1 . me2 at an infusion rate of 0.2 ml/min for 20 min. Blood samples were obtained from the left radial artery at 15 and 20 min of the initial continuous infusion period. In addition, simultaneous measures of cardiac output were obtained by 2-D echocardiographic/Doppler techniques in seven subjects and on a separate day in five subjects under the same experimental conditions. After the initial 20 min of continuous infusion, LBNP of -15 mmHg was initiated. This level of LBNP has been shown to decrease central venous pressure without associated alterations in mean arterial pressure (MAP), aortic pulse pressure, or heart rate, thereby avoiding significant unloading of arterial (carotid and aortic) baroreceptors (15, 24). Blood samples were again obtained at 5 and 10 min (Early) and again at 25 and 30 min (Late). Measures of cardiac output by 2-D echocardiographic/Doppler techniques were obtained during the 5-min Base, Early, and Late measurement periods. Analysis of NE and r3H]NE. Plasma NE concentrations were determined by high-performance liquid chromatography with electrochemical detection after alumina adsorption and extraction with perchloric acid (2). The E3H]NE concentration was determined by alumina adsorption as previously described (2, 6, 7) NE kinetic calculations. Norepinephrine clearance (1 g min-‘. mw2) and spillover (nmol . min-l mw2) were determined by the technique described by Esler et al. (8,9) in which a constant infusion of [3H]NE was administered until steady state was achieved. At steady state, the inference was made that NE clearance is equal to the steady-state [ 3H] NE infusion rate (disintegrations/min) divided by the actual plasma [3H]NE concentration (disintegrations min-’ 1-l) (2, 6, 7) l
l
l
NE clearance =
[3H] NE infusion rate plasma [3H]NE
Plasma NE spillover was then calculated
from the [3H]-
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NE infusion rate and the plasma [3H]NE specific activity mine1 . nmol total NE-‘) (disintegrations l
NE spillover =
[ 3H] NE infusion rate [ 3H] NE specific activity
Norepinephrine rerelease may occur at sympathetic nerve terminals; the contribution to the overall pool for our purposes, however, was assumed to be negligible. Echocardiographic/Doppler-obtained
cardiac
output.
Echocardiographic/Doppler assessments of cardiac output changes during LBNP were performed to determine whether decreases in cardiac output could be implicated in explaining the progressive decreases in arterial NE clearance n .oted in our subjects. These measurements provide an accu rate noninvasive mean s of assessing stroke volume and cardiac output in humans (3, 14, 17, 19-21) and have been used to evaluate cardiac output during LBNP (3). Echocardiographic/Doppler-derived cardiac outputs were measured by imaging from a standard parasternal long axis view to obtain the left ventricular outflow tract (LVOT) diameter (21) and from the apical long axis view to measure pulsed Doppler flow velocity integrals (17) (Fig. l), using an Excel 3 echo/Doppler system (Interspec, Conshohocken, PA) with a 3.5 MHz transducer. Cardiac outputs for each of the measurement periods, Base, Early, and Late, were made by averaging five representative LVOT diameters and five Doppler flow velocities during each measurement period. Assuming the LVOT is circular, the cross-sectional area was determined (17, 21) and the modified Bernoulli equation was used to determine beat-to-beat cardiac cycle variations in stroke volume (3, 17, 19, 21). Statistics and data analysis. The NE kinetic data defined as Base represents the average of the 15- and 20min resting period blood samples. NE kinetic data defined as Early and Late are averages of the 5- and lomin and 25- and 30-min blood samples, respectively. Statistical analyses of data including arterial NE concentration, spillover, clearance, and cardiac output were made using a within-subject one- ,way analysis of variance for repeated measures (22). If a significant F value was found, determination of significance between measurement time periods was accomplished using the StudentNewman-Keuls post hoc analysis (23). Linear regression analyses were performed to determine the presence of significant correlations between arterial NE clearance and cardiac output and . between arterial NE spillover and cardiac output (23). All data are expressed as means t SE. RESULTS
Complete results of this study are shown in Table 1. We noted no significant change in heart rate or MAP in response to LBNP. Arterial plasma NE concentrations increased significantly Early by 25% and by 22% Late (P = O.OOl), as expected. Arterial NE spillover did not change significantly (P = 0.258), but arterial NE clearance decreased significantly from Base to Early by 12%, and from Base to Late by 19% (P = 0.0140; Fig. 2). Similarly, nonin-
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FI(:. 1. Two-dimensional echocardiographic images of the heart during systole showing the parasternal long axis view (top /c/t), which demonstrates where the left ventricular outflow tract diameter is measured (white lure with arrows), and showmg the apical long axis view (top r&t), which demonstrates the position of the left ventricular outflow velocity sample volume (SV) immediately proximal to the aortic valve. Doppler recording of left ventricular outflow velocity (bottom I+) before lower body negative pressure (LBNP) and at 25 mm of -15 mmHg LBNP (bottom qh() m I subject. LV, left ventricle; AO, ascendmg aorta; LA, left atrium; RV, right ventricle; AV, aortic valve. The flow velocity integral (FVI) is derived by digitizing the velocity spectral envelope from onset of flow to aortic valve rloxure. TABLE
1. Study results RWhle
Norepmephrme kinetics data Arterial norepinephrine, pg/ml Arterial clearance, 1. mu-‘. mmi Arterial spillover, nmol. min-’ m-i Cardiac output data Cardiac output, ml/min Hemodynamic parameters Mean arterial pressure, mmHg Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Heart rate, beats/mm Values are means + SE; n = 12 subjects. 0.05 and t I’ < 0.01 indicate sigmficance analysis, after determmation of significant
244k-29 l.lOf0.09 1.59&0.18
Early
306+29t 0.97+0.07 1.78kO.15
298t30t 0.89+0.03” 1.61cO.17
5,095+267
4,315+165t
4,102*153t
77*4 119+4 60*2 58&Z
76f2 119?5 62-r-2 62?3
77f2 119&5 63?2 61&3
See text for definitions of Base, Early, between each time period and basehne F values by one-way analysis of variance
vasively measured cardiac output decreased by 15% from Base to Early and by 19% from Base to Late (P = 0.001; Fig. 2). Arterial NE clearance and cardiac output correlated positively (r = 0.61, P = 0.001; Fig. 3), whereas there was found to be no correlation between cardiac output and arterial NE spillover (r = 0.027, P = 0.874; Fig. 4).
df
F ratlo
2 and 22 2 and 22 2 and 22
14.562 5.21% 1.442
0.0001 0.014 0.258
2 and 22
32.201
0.0001
Late
2 2 2 2
and Late measurement values as determined for repeated measures.
and and and and
22 22 22 22
1.453 0.100 2.950 0.356
I’
0.255 0.906 0.074 0.704
periods; df, degrees of freedom. * I’ i by Student-Newman-Keuls post hoc
DISCUSSION
A recent report from our laboratory demonstrated an increase in arterial NE concentration with LBNP of -15 mmHg (2). This increase in arterial NE concentration was associated with a reduction in arterial NE clearance and no statistically significant change in arterial NE
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0
A/*o
NE CLEARANCE CARDIAC
OUTPUT
n A
-10
-20
-30 L
BASE EARLY LATE 2, Percentage change in cardiac output and arterial norepinephrine (NE) clearance measured at baseline (Base), at 5 and 10 min (Early) and at 25 and 30 min (Late) during continuous lower body negative pressure at -15 mmHg pressure. Data (means k SE) are presented in Table 1. FIG.
r = 0.61, p
3.0
4.0
5.0
6.0
q
0.001
7.0
8.0
CARDIAC OUTPUT (lmin.) 3. Graph showing significant correlation between arterial norepinephrine (NE) clearance and cardiac output measurements obtained during the experimental period. FIG.
r = 0.027, p = 0.874
I we
3.0
4.0
I
5.0
I
I
6.0
7.0
8.0
CARDIAC OUTPUT (Vmin.) Graph showing absence of significant correlation between arterial norepinephrine (NE) spillover and cardiac output measurements obtained during the experimental period. FIG.
4.
spillover. Similarly, Best and Halter (4) have shown a prompt increase in arterial NE concentration, a decrease in arterial NE clearance and no change in arterial NE spillover after intravenous infusion of propranolol at a time when blood pressure was unchanged and heart rate was decreasing. They suggested a potential link between reductions in cardiac output and . reductions in . arterial NE clearance. Based on their findings and our recent data, we postulated a similar link. These findings contrast with our values nreviouslv obtained from the fore-
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arm venous circulation, where a statistically significant increase in forearm venous NE spillover and no change in NE clearance was demonstrated during -15 mmHg LBNP. We observed a 67% increase in muscle sympathetic nerve activity and no change in skin blood flow. These results suggested that the effects of sympathetic nerve activation were predominantly isolated to the human limb muscle vasculature. The present report expands on these findings by confirming our postulate that a relationship exists between the reduction in arterial NE clearance and the reduction in cardiac output. Moreover, our prior observation that arterial NE spillover does not increase with -15 mmHg LBNP was reconfirmed. A limitation of this study is our inability to evaluate regional sympathetic neural activity from potentially important vascular beds such as the splanchnic bed and the lung. Prior studies by Johnson et al. (15) and Hirsch et al. (13) have demonstrated that LBNP of less than -20 mmHg can result in a vasoconstrictor response in the splanchnic bed with an estimated decrease in local blood flow of -10%. This, in conjunction with the increase in skeletal muscle vascular resistance, should theoretically be associated with an increase in arterial NE spillover. Although we found no increase in arterial NE spillover in this or our previous study, it must be remembered that arterial NE spillover reflects the sum of changes in spillover across multiple regional vascular beds. In addition, it must be remembered that the degree of NE spillover can be significantly influenced by factors aside from increased sympathetic outflow. These include local mechanisms such as neural junctional cleft width, presynaptic a2-receptor activity, pre- and postsynaptic NE uptake, and local blood flow (11). Theoretically, for the same degree of neural activation a reduction in blood flow will decrease NE spillover. Thus it is possible that LBNP of -15 mmHg led to reductions in blood flow to certain organs that may have resulted in an underestimation of generalized neural activity. In addition to the above-mentioned concerns regarding the importance of differences in regional vascular NE spillover, the regional clearance of NE from plasma can also be significantly affected by local junctional mechanisms that we are unable to assess. The present study also does not localize the site of reduced NE clearance, although several possibilities exist. The lungs under normal circumstances are an important contributor to clearance of NE, and the entire cardiac output passes through this vascular bed (10). Thus a 19% reduction in arterial NE clearance and cardiac output would not be surprising if this organ were the major contributor to the response we have described. The splanchnic circulation is also an important system that clears NE. However, during low levels of LBNP, splanchnic blood flow has been shown to fall 40% and this vascular bed receives only -20% of the total cardiac output (13). Thus we believe it is unlikely that reduced splanchnic clearance of NE alone could entirely explain our results. A more plausible explanation would be that cardiac output is reduced to multiple vascular beds resulting in a generalized reduction in NE clearance. Future studies will be necessary to clarify this issue. Potential implications. The finding that arterial NE
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clearance decreased significantly and correlated directly with decreases in cardiac output during orthostatic maneuvers that are known to result in decreased stroke volume and cardiac output holds implications in other low-flow states such as heart failure (CHF). We have previously shown that patients with severe CHF demonstrate a significant decrease in forearm venous NE clearance supine and in response to orthostatic stress (6, 7). The present findings expand on this observation by suggesting that reductions in NE clearance correlate with changes in cardiac output. Thus we speculate that in a disease such as CHF the elevated level of plasma NE is influenced not only by the degree of neural activation, which is clearly enhanced and the major determinant of the plasma NE level, but also by the ability to clear NE, which is linked to the level of cardiac output (6, 7, 12). Thus NE is likely to be an important index of prognosis in CHF (5) because it reflects not only neural activation but the resting level of cardiac output. Future experiments examining this relationship will be necessary to confirm this postulate. Conclusions. In conclusion, the present study uses noninvasive 2-D echocardiographic/Doppler techniques to measure cardiac output and continuous infusion [3H]NE radiotracer methodology to study cardiopulmonary baroreceptor function in humans. Our data suggest that during -15 mmHg LBNP, the ability to clear NE from the circulation is linked to the level of cardiac output. By inference, we suggest that NE clearance has an important modulating influence on plasma NE concentrations under a variety of stressful situations. We gratefully acknowledge the technical assistance of Kristin Gray, Danuta Strzelecka, and Susan Deiling and the secretarial assistance of Verna Ebersole. This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-30691; L. I. Sinoway is a recipient of Clinical Investigator Award 1 K08 HL-01744 and First Award 1 R29 HL-44667. Address for reprint requests: R. G. Baily, The Milton S. Hershey Medical Center, The Pennsylvania State University Dept. of Medicine, Division of Cardiology, PO Box 850, Hershey, PA 17033. Received 9 October 1990; accepted in final form 14 January 1991. REFERENCES
1. ANTON, A. H., AND D. F. SAYRE. A study of factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther 138: 360-375, 1962. 2. BAILY, R. G., S. A. PROPHET, J. S. SHENBERGER, R. ZELIS, AND L. I. SINOWAY. Direct neurohumoral evidence for isolated sympathetic nervous system activation to skeletal muscle in response to cardiopulmonary baroreceptor unloading. Circ. Res. 66: 1720-1728, 1990. 3. BERK, M. R., J. EVANS, C. KNAPP, M. R. HARRISON, T. KOTCHEN, AND A. N. DEMARIA. Influence of alterations in loading produced by lower body negative pressure on aortic blood flow acceleration. J. Am. Coil. Cardiol. 15: 1069-1074, 1990. 4. BEST, J. D., AND J. B. HALTER. Blood pressure and norepinephrine spillover during propranolol infusion in humans. Am. J. Physiol. 248 (Regulatory Integrative Comp. Physiol. 17): R400-R406,1985. 5. COHN, J. N., T. B. LEVINE, M. T. OLIVARI, V. GARBERG, D. LURA, G. S. FRANCIS. A. B. SIMON. AND T. RECTOR. Plasma noreninenh-
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rine as a guide to prognosis in patients with chronic congestive heart failure. N. Engl. J. Med. 311: 819-823, 1984. 6. DAVIS, D., R. BAILY, AND R. ZELIS. Abnormalities in systemic norepinephrine kinetics in human congestive heart failure. Am. J. Physiol. 254 (Endocrinol. Metab. 17): E760-E766,1988. 7. DAVIS, D., L. I. SINOWAY, J. ROBISON, J. R. MINOITI, F. P. DAY, R. BAILY, AND R. ZELIS. Norepinephrine kinetics during orthostatic stress in congestive heart failure. Circ. Res. 61, Suppl. I: I87-I-90, 1987. 8. ESLER, M. Assessment of sympathetic nervous function in humans from noradrenaline plasma kinetics. Clin. Sci. 62: 247-254, 1982. 9. ESLER, M., G. JACKMAN, A. BOBIK, D. KELLEHER, G. JENNINGS, P. LEONARD, H. SKEWS, AND P. KORNER. Determination of norepinephrine apparent release rate and clearance in humans. Life Sci. 25: 1461-1470,1979. P. LEONARD, N. SACHARIAS, F. BURKE, 10. ESLER, M., G. JENNINGS, J. JOHNS, AND P. BLOMBERY. Contribution of individual organs to total noradrenaline release in humans. Acta Physiol. Stand. 527, Suppl. : ll-16,1984. 11. ESLER, M. D., G. J. HASKING, I. R. WILLETT, P. W. LEONARD, AND G. L. JENNINGS. Noradrenaline release and sympathetic nervous system activity. J. Hypertens. 3: 117-129, 1985. 12. HASKING, G. J., M. D. ESLER, G. L. JENNINGS, D. BURTON, AND P. I. KORNER. Norepinephrine spillover to plasma in patients with congestive heart failure: evidence of increased overall and cardiorenal sympathetic nervous activity. Circulation 73: 615-621,1986. S. S. CUTLER, V. J. DZAU, AND 13. HIRSCH, A. T., D. J. LEVENSON, M. A. CREAGER. Regional vascular responses to prolonged lower body negative pressure in normal subjects. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H219-H225,1989. L. L., D. K. STEWART, S. R. BARNES, S. B. FRANKLIN, 14. HUNTSMAN, J. S. COLOCOUSIS, AND E. A. HESSEL. Non-invasive Doppler determination of cardiac output in men. Circulation 67: 593-602, 1983. 15. JOHNSON, J. M., L. B. ROWELL, M. NIEDERBERGER, AND M. M. EISMAN. Human splanchnic and forearm vasoconstrictor responses to reductions of right atria1 and aortic pressures. Circ. Res. 34: 515524,1974. 16. LEIMBACH, W. N., B. G. WALLIN, R. G. VICTOR, P. E. AYLWARD, G. SUNDLOF, AND A. L. MARK. Direct evidence from intraneural recordings for increased central sympathetic outflow in patients with heart failure. Circulation 73: 913-919, 1986. 17. LEWIS, J. F., L. C. Kuo, J. G. NELSON, M. C. LIMACHER, AND M. A. QUINONES. Pulsed Doppler echocardiographic determination of stroke volume and cardiac output: clinical validation of two methods using the apical window. Circulation 70: 425-431,1984. 18. LOEPPKY, J. A., E. R. GREENE, D. E. HOEKENGA, A. CAPRIHAN, AND U. C. LUFT. Beat-by-beat stroke volume assessment by pulsed Doppler in upright and supine exercise. J. Appl. Physiol. 50: 11731182,198l. 19. LOEPPKY, J. A., D.‘E. HOEKENGA, E. R. GREENE, AND U. C. LUFT. Comparison of noninvasive pulsed Doppler and Fick measurements of stroke volume in cardiac patients. Am. Heart J. 107: 339-346, 1984. 20. NISHIMURA, R. A., M. J. CALLAHAN, H. V. SCHAFF, D. M. ILSTRUP, F. A. MILLER, AND A. J. TAJIK. Noninvasive measurement of cardiac output by continuous-wave Doppler echocardiography: initial experience and review of the literature. Mayo Clin. Proc. 59: 484-489,1984. 21. PASQUALE, M. J., T. E. NOONAN, AND W. R. DAVIDSON, JR. Does aortic valve area remain constant under varying hemodynamic conditions in man? (Abstract). Circulation 78: 11-350, 1988. 22. SCHEFLER, W. C. Statistics for Health Professionals. Menlo Park, CA: Addison-Wesley, 1984, p. 214-216. 23. SNEDECOR, G. W., AND W. G. COCHRAN. Statistical Methods (7th ed.). Ames: Iowa State Univ. Press, 1980, p. 235 and 237. 24. ZOLLER, R. P., A. L. MARK, F. M. ABBOUD, P. G. SCHMID, AND D. D. HEISTAD. The role of low pressure baroreceptors in reflex vasoconstrictor responses in man. J. Clin. Invest. 51: 2967-2972, 1972.
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LEAMAN,
Division of Cardiology, The Milton S. Hershey Medical Center, The Pennsylvania College of Medicine, Hershey, Pennsylvania 17033
BAILY, ROBERT G., URS LEUENBERGER, GRETCHEN LEAMAN, DAVID SILBER, AND LAWRENCE I. SINOWAY. Norepinephrine kinetics and cardiac output during nonhypotensive lower body negativepressure. Am. J. Physiol. 260 (Heart Circ. Physiol.
29): H1708-Hl712,1991.-Recently we have shown that arterial norepinephrine (NE) concentration increases significantly during lower body negative pressure (LBNP) of -15 mmHg. Interestingly, the increase was found to be related predominantly to a decrease in arterial NE clearance. We postulated that this reduction in clearance would be related to a reduction in cardiac output. Accordingly, we measured both cardiac output (2dimensional echocardiographic/Doppler technique) and arterial NE kinetics ( [3H]NE continuous infusion radiotracer technique) during LBNP of -15 mmHg. These measures of cardiac output and arterial NE spillover and clearance were obtained in 12 normal subjects at baseline, 5 and 10 min (Early) and 25 and 30 min (Late) of LBNP. We found that arterial NE concentration increased significantly, by 25% Early and 22% Late (P = 0.001). Spillover, however, did not change (P = 0.258), whereas clearance decreased by 12% Early and 19% Late (P = 0.014), and cardiac output decreased by 15% Early and 19% Late (P = 0.001). These reductions in clearance and cardiac output correlated significantly (r = 0.61, P = 0.001). No correlation was noted between spillover and cardiac output (r = 0.027, P = 0.874). We conclude that the increases in arterial NE concentration during nonhypotensive LBNP are predominantly due to decreased cardiac output with resultant decreases in systemic clearance of NE. These findings suggest that the ability to clear NE from the circulation is linked to the level of cardiac output and that low cardiac output states by themselves may lead to an elevation in arterial plasma NE concentrations. microneurography; cardiopulmonary baroreceptors; echocardiographic/Doppler cardiac output LOWER BODY NEGATIVE PRESSURE (LBNP) of -15 mmHg results in selective unloading of low-pressure baroreceptors (2). We have recently completed studies demonstrating that cardiopulmonary baroreceptor unloading results in sympathetic nervous system (SNS) activation that is predominantly localized to skeletal muscle. This conclusion was based on the fact that we noted a 67% increase in muscle sympathetic nerve activity, a 23% increase in forearm venous norepinephrine (NE) spillover (representing skin and muscle), no change in skin blood flow, and no change in arterial NE spillover (representing generalized SNS activation) (2). Interestingly, however, despite the relatively localized
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nature of the neural response, we found that arterial and forearm venous plasma plasma N E concentration NE concentration increased similarly during isolated low-pressure baroreceptor unloading with -15 mmHg LBNP. This suggested that the increase in arterial plasma NE concentration during -15 mmHg LBNP was dependent on factor(s) other than SNS activation. In fact, we found that reductions in systemic clearance of NE appeared to be primarily responsible for the significant increases in arterial plasma NE concentration in our subjects. Best and Halter (4) have shown that intravenous infusions of propranolol prompt an increase in arterial NE concentration, a decrease in arterial NE clearance and no change in arterial NE spillover at a time when blood pressure is unchanged and heart rate is decreasing. The authors speculated that a decrease in blood flow was responsible for the increase in arterial NE concentration secondary to a reduction in systemic clearance of NE, rather than an increase in generalized neural activation. Based on the findings of Best and Halter and our previous study (2), we postulated that LBNP of -15 mmHg would result in reduced arterial NE clearance secondary to decreases in cardiac output. This in turn would result in an increase in arterial NE concentration that would not necessarily be related to generalized activation of the SNS. In this study we used noninvasive two-dimensional (2D) echocardiographic/Doppler measures of cardiac output and continuous infusion radiotracer measures of arterial NE spillover and clearance to assess the potential relationship between decreased cardiac output and arterial NE clearance during isolated cardiopulmonary baroreceptor unloading. METHODS
Subjects. Twelve subjects with a mean age of 25 t 0.7 (*SE) yr participated in NE kinetic studies and of those 12,7 had cardiac output measurements made at the same time as the plasma samples were taken. The other five subjects had cardiac output measured on a separate day under the same experimental conditions. The protocol was reviewed and approved by the Clinical Investigation Committee of the Milton S. Hershey Medical Center. Informed consent was obtained from each individual. No subject enrolled in the study had a history of heart
$1.50 Copyright 0 1991 the American Physiological
Society
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disease, hypertension, or thyroid disease, and no subject was taking medications or substances that might have altered SNS activity. All subjects were familiarized with LBNP before the study. Experimental protocol. Studies were performed in a quiet, partially darkened temperature-controlled room. Subjects were comfortably positioned in a partial left lateral decubitus position in an LBNP chamber. The partial left lateral decubitus position provided an optimal imaging window for 2-D echocardiographic/Doppler measurements of cardiac output. Polyethylene wrap was placed around the subject’s torso at the level of the iliac crests to obtain an airtight seal. Subjects were instrumented with a U-in. al-gauge antecubital venous cannula for infusion of[3H]NE in the right arm, and a left radial artery cannula was inserted for continuous hemodynamic monitoring as well as arterial blood sampling. Respirations were monitored via pneumograph. Arterial blood samples were obtained to determine baseline (Base) plasma NE and to provide plasma for determination of [3H]NE recovery by aluminum oxide (alumina) adsorption (1). The subjects received an initial bolus of [3H]NE (15 &i/m”) over 5 min, followed by continuous infusion of 0.35 PCi. mine1 . me2 at an infusion rate of 0.2 ml/min for 20 min. Blood samples were obtained from the left radial artery at 15 and 20 min of the initial continuous infusion period. In addition, simultaneous measures of cardiac output were obtained by 2-D echocardiographic/Doppler techniques in seven subjects and on a separate day in five subjects under the same experimental conditions. After the initial 20 min of continuous infusion, LBNP of -15 mmHg was initiated. This level of LBNP has been shown to decrease central venous pressure without associated alterations in mean arterial pressure (MAP), aortic pulse pressure, or heart rate, thereby avoiding significant unloading of arterial (carotid and aortic) baroreceptors (15, 24). Blood samples were again obtained at 5 and 10 min (Early) and again at 25 and 30 min (Late). Measures of cardiac output by 2-D echocardiographic/Doppler techniques were obtained during the 5-min Base, Early, and Late measurement periods. Analysis of NE and r3H]NE. Plasma NE concentrations were determined by high-performance liquid chromatography with electrochemical detection after alumina adsorption and extraction with perchloric acid (2). The E3H]NE concentration was determined by alumina adsorption as previously described (2, 6, 7) NE kinetic calculations. Norepinephrine clearance (1 g min-‘. mw2) and spillover (nmol . min-l mw2) were determined by the technique described by Esler et al. (8,9) in which a constant infusion of [3H]NE was administered until steady state was achieved. At steady state, the inference was made that NE clearance is equal to the steady-state [ 3H] NE infusion rate (disintegrations/min) divided by the actual plasma [3H]NE concentration (disintegrations min-’ 1-l) (2, 6, 7) l
l
l
NE clearance =
[3H] NE infusion rate plasma [3H]NE
Plasma NE spillover was then calculated
from the [3H]-
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NE infusion rate and the plasma [3H]NE specific activity mine1 . nmol total NE-‘) (disintegrations l
NE spillover =
[ 3H] NE infusion rate [ 3H] NE specific activity
Norepinephrine rerelease may occur at sympathetic nerve terminals; the contribution to the overall pool for our purposes, however, was assumed to be negligible. Echocardiographic/Doppler-obtained
cardiac
output.
Echocardiographic/Doppler assessments of cardiac output changes during LBNP were performed to determine whether decreases in cardiac output could be implicated in explaining the progressive decreases in arterial NE clearance n .oted in our subjects. These measurements provide an accu rate noninvasive mean s of assessing stroke volume and cardiac output in humans (3, 14, 17, 19-21) and have been used to evaluate cardiac output during LBNP (3). Echocardiographic/Doppler-derived cardiac outputs were measured by imaging from a standard parasternal long axis view to obtain the left ventricular outflow tract (LVOT) diameter (21) and from the apical long axis view to measure pulsed Doppler flow velocity integrals (17) (Fig. l), using an Excel 3 echo/Doppler system (Interspec, Conshohocken, PA) with a 3.5 MHz transducer. Cardiac outputs for each of the measurement periods, Base, Early, and Late, were made by averaging five representative LVOT diameters and five Doppler flow velocities during each measurement period. Assuming the LVOT is circular, the cross-sectional area was determined (17, 21) and the modified Bernoulli equation was used to determine beat-to-beat cardiac cycle variations in stroke volume (3, 17, 19, 21). Statistics and data analysis. The NE kinetic data defined as Base represents the average of the 15- and 20min resting period blood samples. NE kinetic data defined as Early and Late are averages of the 5- and lomin and 25- and 30-min blood samples, respectively. Statistical analyses of data including arterial NE concentration, spillover, clearance, and cardiac output were made using a within-subject one- ,way analysis of variance for repeated measures (22). If a significant F value was found, determination of significance between measurement time periods was accomplished using the StudentNewman-Keuls post hoc analysis (23). Linear regression analyses were performed to determine the presence of significant correlations between arterial NE clearance and cardiac output and . between arterial NE spillover and cardiac output (23). All data are expressed as means t SE. RESULTS
Complete results of this study are shown in Table 1. We noted no significant change in heart rate or MAP in response to LBNP. Arterial plasma NE concentrations increased significantly Early by 25% and by 22% Late (P = O.OOl), as expected. Arterial NE spillover did not change significantly (P = 0.258), but arterial NE clearance decreased significantly from Base to Early by 12%, and from Base to Late by 19% (P = 0.0140; Fig. 2). Similarly, nonin-
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FI(:. 1. Two-dimensional echocardiographic images of the heart during systole showing the parasternal long axis view (top /c/t), which demonstrates where the left ventricular outflow tract diameter is measured (white lure with arrows), and showmg the apical long axis view (top r&t), which demonstrates the position of the left ventricular outflow velocity sample volume (SV) immediately proximal to the aortic valve. Doppler recording of left ventricular outflow velocity (bottom I+) before lower body negative pressure (LBNP) and at 25 mm of -15 mmHg LBNP (bottom qh() m I subject. LV, left ventricle; AO, ascendmg aorta; LA, left atrium; RV, right ventricle; AV, aortic valve. The flow velocity integral (FVI) is derived by digitizing the velocity spectral envelope from onset of flow to aortic valve rloxure. TABLE
1. Study results RWhle
Norepmephrme kinetics data Arterial norepinephrine, pg/ml Arterial clearance, 1. mu-‘. mmi Arterial spillover, nmol. min-’ m-i Cardiac output data Cardiac output, ml/min Hemodynamic parameters Mean arterial pressure, mmHg Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Heart rate, beats/mm Values are means + SE; n = 12 subjects. 0.05 and t I’ < 0.01 indicate sigmficance analysis, after determmation of significant
244k-29 l.lOf0.09 1.59&0.18
Early
306+29t 0.97+0.07 1.78kO.15
298t30t 0.89+0.03” 1.61cO.17
5,095+267
4,315+165t
4,102*153t
77*4 119+4 60*2 58&Z
76f2 119?5 62-r-2 62?3
77f2 119&5 63?2 61&3
See text for definitions of Base, Early, between each time period and basehne F values by one-way analysis of variance
vasively measured cardiac output decreased by 15% from Base to Early and by 19% from Base to Late (P = 0.001; Fig. 2). Arterial NE clearance and cardiac output correlated positively (r = 0.61, P = 0.001; Fig. 3), whereas there was found to be no correlation between cardiac output and arterial NE spillover (r = 0.027, P = 0.874; Fig. 4).
df
F ratlo
2 and 22 2 and 22 2 and 22
14.562 5.21% 1.442
0.0001 0.014 0.258
2 and 22
32.201
0.0001
Late
2 2 2 2
and Late measurement values as determined for repeated measures.
and and and and
22 22 22 22
1.453 0.100 2.950 0.356
I’
0.255 0.906 0.074 0.704
periods; df, degrees of freedom. * I’ i by Student-Newman-Keuls post hoc
DISCUSSION
A recent report from our laboratory demonstrated an increase in arterial NE concentration with LBNP of -15 mmHg (2). This increase in arterial NE concentration was associated with a reduction in arterial NE clearance and no statistically significant change in arterial NE
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0
A/*o
NE CLEARANCE CARDIAC
OUTPUT
n A
-10
-20
-30 L
BASE EARLY LATE 2, Percentage change in cardiac output and arterial norepinephrine (NE) clearance measured at baseline (Base), at 5 and 10 min (Early) and at 25 and 30 min (Late) during continuous lower body negative pressure at -15 mmHg pressure. Data (means k SE) are presented in Table 1. FIG.
r = 0.61, p
3.0
4.0
5.0
6.0
q
0.001
7.0
8.0
CARDIAC OUTPUT (lmin.) 3. Graph showing significant correlation between arterial norepinephrine (NE) clearance and cardiac output measurements obtained during the experimental period. FIG.
r = 0.027, p = 0.874
I we
3.0
4.0
I
5.0
I
I
6.0
7.0
8.0
CARDIAC OUTPUT (Vmin.) Graph showing absence of significant correlation between arterial norepinephrine (NE) spillover and cardiac output measurements obtained during the experimental period. FIG.
4.
spillover. Similarly, Best and Halter (4) have shown a prompt increase in arterial NE concentration, a decrease in arterial NE clearance and no change in arterial NE spillover after intravenous infusion of propranolol at a time when blood pressure was unchanged and heart rate was decreasing. They suggested a potential link between reductions in cardiac output and . reductions in . arterial NE clearance. Based on their findings and our recent data, we postulated a similar link. These findings contrast with our values nreviouslv obtained from the fore-
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arm venous circulation, where a statistically significant increase in forearm venous NE spillover and no change in NE clearance was demonstrated during -15 mmHg LBNP. We observed a 67% increase in muscle sympathetic nerve activity and no change in skin blood flow. These results suggested that the effects of sympathetic nerve activation were predominantly isolated to the human limb muscle vasculature. The present report expands on these findings by confirming our postulate that a relationship exists between the reduction in arterial NE clearance and the reduction in cardiac output. Moreover, our prior observation that arterial NE spillover does not increase with -15 mmHg LBNP was reconfirmed. A limitation of this study is our inability to evaluate regional sympathetic neural activity from potentially important vascular beds such as the splanchnic bed and the lung. Prior studies by Johnson et al. (15) and Hirsch et al. (13) have demonstrated that LBNP of less than -20 mmHg can result in a vasoconstrictor response in the splanchnic bed with an estimated decrease in local blood flow of -10%. This, in conjunction with the increase in skeletal muscle vascular resistance, should theoretically be associated with an increase in arterial NE spillover. Although we found no increase in arterial NE spillover in this or our previous study, it must be remembered that arterial NE spillover reflects the sum of changes in spillover across multiple regional vascular beds. In addition, it must be remembered that the degree of NE spillover can be significantly influenced by factors aside from increased sympathetic outflow. These include local mechanisms such as neural junctional cleft width, presynaptic a2-receptor activity, pre- and postsynaptic NE uptake, and local blood flow (11). Theoretically, for the same degree of neural activation a reduction in blood flow will decrease NE spillover. Thus it is possible that LBNP of -15 mmHg led to reductions in blood flow to certain organs that may have resulted in an underestimation of generalized neural activity. In addition to the above-mentioned concerns regarding the importance of differences in regional vascular NE spillover, the regional clearance of NE from plasma can also be significantly affected by local junctional mechanisms that we are unable to assess. The present study also does not localize the site of reduced NE clearance, although several possibilities exist. The lungs under normal circumstances are an important contributor to clearance of NE, and the entire cardiac output passes through this vascular bed (10). Thus a 19% reduction in arterial NE clearance and cardiac output would not be surprising if this organ were the major contributor to the response we have described. The splanchnic circulation is also an important system that clears NE. However, during low levels of LBNP, splanchnic blood flow has been shown to fall 40% and this vascular bed receives only -20% of the total cardiac output (13). Thus we believe it is unlikely that reduced splanchnic clearance of NE alone could entirely explain our results. A more plausible explanation would be that cardiac output is reduced to multiple vascular beds resulting in a generalized reduction in NE clearance. Future studies will be necessary to clarify this issue. Potential implications. The finding that arterial NE
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clearance decreased significantly and correlated directly with decreases in cardiac output during orthostatic maneuvers that are known to result in decreased stroke volume and cardiac output holds implications in other low-flow states such as heart failure (CHF). We have previously shown that patients with severe CHF demonstrate a significant decrease in forearm venous NE clearance supine and in response to orthostatic stress (6, 7). The present findings expand on this observation by suggesting that reductions in NE clearance correlate with changes in cardiac output. Thus we speculate that in a disease such as CHF the elevated level of plasma NE is influenced not only by the degree of neural activation, which is clearly enhanced and the major determinant of the plasma NE level, but also by the ability to clear NE, which is linked to the level of cardiac output (6, 7, 12). Thus NE is likely to be an important index of prognosis in CHF (5) because it reflects not only neural activation but the resting level of cardiac output. Future experiments examining this relationship will be necessary to confirm this postulate. Conclusions. In conclusion, the present study uses noninvasive 2-D echocardiographic/Doppler techniques to measure cardiac output and continuous infusion [3H]NE radiotracer methodology to study cardiopulmonary baroreceptor function in humans. Our data suggest that during -15 mmHg LBNP, the ability to clear NE from the circulation is linked to the level of cardiac output. By inference, we suggest that NE clearance has an important modulating influence on plasma NE concentrations under a variety of stressful situations. We gratefully acknowledge the technical assistance of Kristin Gray, Danuta Strzelecka, and Susan Deiling and the secretarial assistance of Verna Ebersole. This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-30691; L. I. Sinoway is a recipient of Clinical Investigator Award 1 K08 HL-01744 and First Award 1 R29 HL-44667. Address for reprint requests: R. G. Baily, The Milton S. Hershey Medical Center, The Pennsylvania State University Dept. of Medicine, Division of Cardiology, PO Box 850, Hershey, PA 17033. Received 9 October 1990; accepted in final form 14 January 1991. REFERENCES
1. ANTON, A. H., AND D. F. SAYRE. A study of factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther 138: 360-375, 1962. 2. BAILY, R. G., S. A. PROPHET, J. S. SHENBERGER, R. ZELIS, AND L. I. SINOWAY. Direct neurohumoral evidence for isolated sympathetic nervous system activation to skeletal muscle in response to cardiopulmonary baroreceptor unloading. Circ. Res. 66: 1720-1728, 1990. 3. BERK, M. R., J. EVANS, C. KNAPP, M. R. HARRISON, T. KOTCHEN, AND A. N. DEMARIA. Influence of alterations in loading produced by lower body negative pressure on aortic blood flow acceleration. J. Am. Coil. Cardiol. 15: 1069-1074, 1990. 4. BEST, J. D., AND J. B. HALTER. Blood pressure and norepinephrine spillover during propranolol infusion in humans. Am. J. Physiol. 248 (Regulatory Integrative Comp. Physiol. 17): R400-R406,1985. 5. COHN, J. N., T. B. LEVINE, M. T. OLIVARI, V. GARBERG, D. LURA, G. S. FRANCIS. A. B. SIMON. AND T. RECTOR. Plasma noreninenh-
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rine as a guide to prognosis in patients with chronic congestive heart failure. N. Engl. J. Med. 311: 819-823, 1984. 6. DAVIS, D., R. BAILY, AND R. ZELIS. Abnormalities in systemic norepinephrine kinetics in human congestive heart failure. Am. J. Physiol. 254 (Endocrinol. Metab. 17): E760-E766,1988. 7. DAVIS, D., L. I. SINOWAY, J. ROBISON, J. R. MINOITI, F. P. DAY, R. BAILY, AND R. ZELIS. Norepinephrine kinetics during orthostatic stress in congestive heart failure. Circ. Res. 61, Suppl. I: I87-I-90, 1987. 8. ESLER, M. Assessment of sympathetic nervous function in humans from noradrenaline plasma kinetics. Clin. Sci. 62: 247-254, 1982. 9. ESLER, M., G. JACKMAN, A. BOBIK, D. KELLEHER, G. JENNINGS, P. LEONARD, H. SKEWS, AND P. KORNER. Determination of norepinephrine apparent release rate and clearance in humans. Life Sci. 25: 1461-1470,1979. P. LEONARD, N. SACHARIAS, F. BURKE, 10. ESLER, M., G. JENNINGS, J. JOHNS, AND P. BLOMBERY. Contribution of individual organs to total noradrenaline release in humans. Acta Physiol. Stand. 527, Suppl. : ll-16,1984. 11. ESLER, M. D., G. J. HASKING, I. R. WILLETT, P. W. LEONARD, AND G. L. JENNINGS. Noradrenaline release and sympathetic nervous system activity. J. Hypertens. 3: 117-129, 1985. 12. HASKING, G. J., M. D. ESLER, G. L. JENNINGS, D. BURTON, AND P. I. KORNER. Norepinephrine spillover to plasma in patients with congestive heart failure: evidence of increased overall and cardiorenal sympathetic nervous activity. Circulation 73: 615-621,1986. S. S. CUTLER, V. J. DZAU, AND 13. HIRSCH, A. T., D. J. LEVENSON, M. A. CREAGER. Regional vascular responses to prolonged lower body negative pressure in normal subjects. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H219-H225,1989. L. L., D. K. STEWART, S. R. BARNES, S. B. FRANKLIN, 14. HUNTSMAN, J. S. COLOCOUSIS, AND E. A. HESSEL. Non-invasive Doppler determination of cardiac output in men. Circulation 67: 593-602, 1983. 15. JOHNSON, J. M., L. B. ROWELL, M. NIEDERBERGER, AND M. M. EISMAN. Human splanchnic and forearm vasoconstrictor responses to reductions of right atria1 and aortic pressures. Circ. Res. 34: 515524,1974. 16. LEIMBACH, W. N., B. G. WALLIN, R. G. VICTOR, P. E. AYLWARD, G. SUNDLOF, AND A. L. MARK. Direct evidence from intraneural recordings for increased central sympathetic outflow in patients with heart failure. Circulation 73: 913-919, 1986. 17. LEWIS, J. F., L. C. Kuo, J. G. NELSON, M. C. LIMACHER, AND M. A. QUINONES. Pulsed Doppler echocardiographic determination of stroke volume and cardiac output: clinical validation of two methods using the apical window. Circulation 70: 425-431,1984. 18. LOEPPKY, J. A., E. R. GREENE, D. E. HOEKENGA, A. CAPRIHAN, AND U. C. LUFT. Beat-by-beat stroke volume assessment by pulsed Doppler in upright and supine exercise. J. Appl. Physiol. 50: 11731182,198l. 19. LOEPPKY, J. A., D.‘E. HOEKENGA, E. R. GREENE, AND U. C. LUFT. Comparison of noninvasive pulsed Doppler and Fick measurements of stroke volume in cardiac patients. Am. Heart J. 107: 339-346, 1984. 20. NISHIMURA, R. A., M. J. CALLAHAN, H. V. SCHAFF, D. M. ILSTRUP, F. A. MILLER, AND A. J. TAJIK. Noninvasive measurement of cardiac output by continuous-wave Doppler echocardiography: initial experience and review of the literature. Mayo Clin. Proc. 59: 484-489,1984. 21. PASQUALE, M. J., T. E. NOONAN, AND W. R. DAVIDSON, JR. Does aortic valve area remain constant under varying hemodynamic conditions in man? (Abstract). Circulation 78: 11-350, 1988. 22. SCHEFLER, W. C. Statistics for Health Professionals. Menlo Park, CA: Addison-Wesley, 1984, p. 214-216. 23. SNEDECOR, G. W., AND W. G. COCHRAN. Statistical Methods (7th ed.). Ames: Iowa State Univ. Press, 1980, p. 235 and 237. 24. ZOLLER, R. P., A. L. MARK, F. M. ABBOUD, P. G. SCHMID, AND D. D. HEISTAD. The role of low pressure baroreceptors in reflex vasoconstrictor responses in man. J. Clin. Invest. 51: 2967-2972, 1972.
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