Transvascular fluid flux from the pulmonary vasculature at rest and during exercise in horses
1 Veterinary Faculty, University of Ljubljana, Ljubljana SI-1115, PO Box 3425, Slovenia
2 Department of Clinical Studies, University of Guelph, Ontario, Canada N1G 2W1
3 Department of Medicine, McMaster University Medical Centre Hamilton, Ontario, Canada L8N 3Z5
Abstract
Exercise causes changes in pulmonary haemodynamics through redistribution of blood flow, increase in the pulmonary surface area, and increase in pulmonary vascular pressures. These changes contribute to the increase in fluid exchange across the alveolar–capillary barrier. To determine the extent of the fluid exchange across the alveolar–capillary barrier at rest and during exercise, six horses were exercised on a high-speed treadmill until fatigue. Arterial and mixed venous blood were sampled at rest and during exercise and recovery. Blood volume changes across the lung (BV; measured in percentage) were calculated from changes in plasma protein and haemoglobin concentration, and haematocrit. Cardiac output (Q) was calculated using the Fick equation. Fluid flux (JV–A; measured in l min–1) across the alveolar–capillary barrier was then quantified based on Q and BV. At rest, no fluid movement occurred across the pulmonary vasculature (0.6 ± 0.6 l min–1). During exercise, the amount of fluid moved from the pulmonary circulation was 8.3 ± 1.3 l min–1 at 1 min, 6.4 ± 2.9 l min–1 at 2 min, 10.1 ± 1.0 l min–1 at 3 min, 12.9 ± 2.5 l min–1 at 4 and 9.6 ± 1.5 l min–1 at fatigue (all P < 0.0001). Erythrocyte volume decreased by 6% (P < 0.01) across the lungs, which decreased the colloid osmotic gradient in the pulmonary vasculature. Decrease colloid osmotic gradient along with increased hydrostatic forces in the pulmonary vasculature would enhance displacement of fluid into the pulmonary interstitium. In conclusion, exercise caused large increases in transpulmonary fluid fluxes in horses. Here, we present a simple method to calculate transpulmonary fluid fluxes in different species, which can be used to elucidate mechanisms of lung fluid balance in vivo.
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Introduction
Maximal exercise resulted in a marked increase in cardiac output (Q) with a less prominent increase in pulmonary macro- and microvascular pressures (Wagner et al. 1986; Groves et al. 1987; Schaffartzik et al. 1993; Newman et al. 1993). Such adaptations coexist with redistribution of blood flow across the lung through capillary recruitment and increase in the pulmonary surface area (Bake et al. 1968; Hlastala et al. 1996). Changes in pulmonary haemodynamics during exercise increase fluid and solute movement across the alveolar–capillary barrier (Dexter et al. 1951; Johnson et al. 1960). A possible consequence of such adaptations is the development of pulmonary oedema (McKechnie et al. 1979) or, more commonly, perivascular oedema and/or parenchymal interstitial oedema that worsens the pulmonary gas exchange in dogs (Younes et al. 1987), small ruminants (Coates et al. 1984), pigs (Schaffartzik et al. 1993), humans (Schaffartzik et al. 1992; McKenzie et al. 2005) and, to a lesser extent, horses (West et al. 1993).
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Healthy animals and humans are capable of eliminating excessive lung fluid during strenuous exercise even when the pulmonary capillary hydrostatic pressure rises above the oedemagenic threshold (Wagner et al. 1986; Groves et al. 1987; Newman et al. 1988, 1993; Erickson et al. 1992; Manohar, 1993). Most of the excessive lung fluid is eliminated via the pulmonary lymph system (Staub et al. 1967; Mitzner & Sylvester, 1986). In sheep, and probably in other species, during strenuous exercise the lung lymphatic flow rises several-fold (Coates et al. 1984; Newman et al. 1988, 1993). Pulmonary lymph flow correlates with the rate of filtration from the pulmonary vasculature even though pulmonary lymph flow is mixed with lymph flow from non-pulmonary tissues (Demling & Gunther, 1982; Drake et al. 1986) and the conducting airways (Wagner et al. 1998).
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Although the increase in lymph flow correlates with increased exchange of fluid across pulmonary capillaries, quantification of fluid movement from the pulmonary circulation at rest and during exercise through lymph-flow analysis is not possible. Nor can it be calculated using Starling's equation (Starling, 1896), because forces governing these events in lung compartments cannot be defined and water transport across the membrane is also regulated independently of solute transport (aquaporins) (King et al. 2004).
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We conducted a study where fluid movement across the alveolar–capillary barrier (across the lung) was quantitatively assessed by comparison of blood volume differences between mixed venous blood and arterial blood in horses at rest and during exercise. Horses were chosen because their increase in pulmonary arterial pressure (PPA) during exercise, in contrast to other species (Slonim et al. 1954; Elkins & Milnor, 1971; Wagner et al. 1986; Younes et al. 1987; Newman et al. 1993), is substantial (Wagner et al. 1989; Erickson et al. 1992; Manohar, 1993; Wilkins et al. 2001), which should translate into a relatively high pulmonary capillary pressure (Sinha et al. 1996). In addition, in horses (Wilkins et al. 2001), as well as in humans (McKenzie et al. 2005), the lung water has been reported to increase during exertion. Based on that, we hypothesized that exercising horses develop detectable transvascular fluid flux from the pulmonary vasculature into the pulmonary interstitium.
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Methods
Six race-fit standardbred horses (2 female, 4 geldings), with a mean age of 5.7 years (range, 5–6 years), mean weight of 450 kg (range, 402–497 kg) and mean peak O2 uptake of 159.9 ml kg–1 min–1 (range, 141.1–179.4 ml kg–1 min–1) were used. Horses were recruited with owner/trainer consent. The study protocols were approved by the Animal Care Committee according to the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, Ontario). All horses were healthy when returned to their owners, and resumed their normal training and racing activities.
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Pre-experimental protocol
Five days prior to the experiment, each horse was familiarized with the treadmill. During the first 3 days, horses were given repeated walking exercise on the treadmill for 20 min daily (15 min walk, 5 min slow pace) at 10% treadmill inclination, followed by 2 days of exercise with the respiratory mask fitted on their nose. Before every experiment, horses were weighed and fitted with a safety harness, hobbles and heart rate meter (Equistat Model HR-8 A, EQB Inc., Unionville, PA, USA).
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On day 6, peak O2 uptake was determined for each horse, during three treadmill exercise periods: warm-up, incremental exercise and recovery. During the warm-up period the horses were walked on a horizontal treadmill with no inclination for 5 min at 2–3 m s–1 and then trotted for 5 min at 4–5 m s–1. At the end of this 10 min warming-up period, the treadmill was inclined to 10% and the speed brought to 8 m s–1. The incremental exercise consisted of a stepwise increase of velocity of 1 m s–1 every 60 s. An open flow-through system was used for collection of pulmonary gases throughout the entire exercise protocol. Peak O2 uptake was determined as the point at which no further increase in occurred, despite an increase in speed, or a level of exercise where the horse could no longer maintain pace with the treadmill speed.
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Experimental protocol
Experiments were carried out 72 h after the determination of . During this time, horses rested and were hand walked for 15 min daily. The skin at the site of catheter insertion was clipped, desensitized with EMLA cream (lidocaine 2.5% and prilocaine 2.5%; AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA) and aseptically prepared. Pulmonary Swan-Ganz catheter (Baxter Healthcare Corp., Irvine, CA, USA) and a 150-cm long central venous polyethylene blood catheter (#240) were placed aseptically via the left and right jugular vein into the pulmonary artery for mixed central venous blood sampling and core body temperature measurements. Correct catheter placement was assured by observing characteristic pressure waveforms on an oscilloscope (Criticare 1100, Criticare Systems Inc., Waukesha, WI, USA). A 20-gauge catheter (Insyte-W, Infusion Therapy Systems Inc., Sandy, UT, USA) was inserted into the facial or transverse facial artery. A 30-cm long extension tubing with a three-way stopcock was connected to intravenous and intra-arterial catheters. Catheters and extension tubing were all sutured securely to the skin. Horses then rested on the treadmill until their heart rate returned to resting values.
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The high intensity exercise experiment followed; this consisted of a warm-up phase as outlined for the testing in the pre-experimental protocol. The subsequent treadmill velocity was set to produce 80% of the previously determined . Horses were exercised until fatigue. During recovery, horses were walked on the treadmill at 2–3 m s–1.
Pulmonary gas collection
Pulmonary gas exchange was measured at rest, during exercise and during the recovery period. Before exercise, the respiratory mask was fitted on the horse's nose. An open flow-through system was used for collection of expired gases throughout the entire exercise protocol (Wagner et al. 1989). The expired gas was drawn into the O2 analyser (Ametek, Model S-3 A/1, Pittsburgh, PA, USA), which measured the concentration of inspired O2. Inspired concentration of O2 was recorded in 10-s periods throughout the experiment. For analysis, the average of three measurements in a 30-s period was used; this coincided with blood-sampling intervals.
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Blood sampling and blood analysis
Resting arterial and mixed venous blood were collected simultaneously under anaerobic conditions twice in a 5-min interval. Further sampling was performed in 60-s intervals during the exercise period until fatigue. During the recovery period, sampling was performed starting after the treadmill was stopped (0 min) and then 1, 2, 3, 5, 10 and 15 min into the recovery period. Prior to each sampling, 10 ml blood was withdrawn from catheters and discarded. Blood samples were collected into lithium-heparinized syringes (S-Monovette, Sarstedt AG & Co, Nümbrecht, Germany), stored on ice, and analysed in duplicate with the Stat Profile M Analyser (Nova Biomedical Corporation, Waltham, MA, USA) immediately after the treadmill protocol ended. Stat Profile M Analyser uses conductivity for measurement of haematocrit (Hct) (coefficient of variance (CV) = 2.4%), and conductivity/reflectance for haemoglobin (Hb) (CV = 1.8%) and O2 saturation (CV = 0.7%) analysis. Blood O2 content was calculated from the O2 saturation and the Hb concentration using standard equations. Total plasma protein (CV = 0.8%) was measured using a clinical refractometer (Attago, Tokyo, Japan).
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Calculations
Plasma volume changes across the lung (PVv–a) were calculated from changes in plasma protein concentration [PP] at the same time point from central venous to arterial blood (across the lung) (Dill & Costill, 1974):
(1)
where [PPv] is the plasma protein concentration in venous blood and [PPa] the plasma protein concentration in arterial blood. To account for changes in plasma volume relative to Hct, eqn (1) was adjusted for changes in the Hct across the lungs:
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(2)
where %HctPVv–a is the plasma volume change relative to Hct changes from central venous (Hctv) to arterial (Hcta) blood.
Changes in erythrocyte volume (EVv–a) across the lungs were calculated from changes in haemoglobin concentration ([Hb]) and haematocrit (Hct) in venous ([Hbv] and Hctv) and arterial blood ([Hba] and Hcta) (Costill et al. 1974):
(3)
For calculation of the blood volume (BV) the %EVv–a was adjusted for Hct changes across the lung:
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(4)
Blood volume changes across the lung were then measured from HctPVv–a and HctEVv–a:
(5)
Cardiac output (l min–1) was calculated using Fick's principle using and blood O2 content from central venous and arterial blood. Fluid flux (JV–A; measured in l min–1) across the lung was then quantified based on Q and %BV:
(6)
Statistical analysis
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Exercise and recovery parameters were compared to resting values using one-way repeated-measures ANOVA. A significant F ratio was further analysed using Dunnett's post hoc analysis. Pearson's correlation analysis was used to determine whether there was a significant association between the cardiac output and fluid exchange. A statistical significance level of P < 0.05 was used and data expressed as means ±S.E.M.
Results
Horses were exercised at the mean velocity of 9.1 ± 0.15 m s–1. The mean duration of exercise to fatigue was 4.7 ± 0.2 min.
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Respiratory changes
increased progressively during exercise. Horses reached of 151.2 ± 5.9 ml kg–1 min–1. VO2 decreased immediately after the treadmill was stopped due to fatigue and remained elevated compared to rest throughout the recovery (P < 0.0001) (Table 1).
Arterial and venous blood O2 pressures decreased during exercise (P < 0.0001) (Table 1).
Oxygen saturation in venous blood decreased substantially during exercise from 67.2 ± 14% at rest to below 10% during exercise (P < 0.0001). It remained decreased until min 10 of recovery (P 0.02) and achieved resting value at min 15 of recovery (Table 1). Oxygen saturation in arterial blood (SaO2) decreased (P < 0.0001) from 98.4 ± 0.4% to 90.9 ± 0.4% by min 2 of exercise with no further decline during the exercise period (P < 0.0001). It returned to the resting value immediately after cessation of exercise.
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Exercise resulted in an immediate decrease of the venous blood oxygen content (CvO2) below 3 ml dl–1 (P < 0.0001). During recovery it increased above the resting value until min 3 of recovery and then returned to the resting value. Arterial blood oxygen content (CaO2) increased immediately after the start of exercise to above 22 ml dl–1 and then progressively increased during exercise to 23.8 ± 0.5 ml dl–1 at fatigue (P < 0.0001). It remained increased throughout recovery (P 0.03) (Table 1).
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Haematocrit, haemoglobin and plasma protein
Haematocrit and Hb level increased (P < 0.0001) during exercise. Both slowly declined during recovery; however, they remained elevated above the resting value at the end of recovery. Plasma protein concentration did not change significantly during exercise. Haematocrit and levels of haemoglobin and plasma protein did not change across the lung (Table 2).
Erythrocyte, plasma and blood volume changes
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At rest EV did not change across the lung (0.3 ± 1.5%). By the minute first of exercise EV decreased rapidly, continued to decrease throughout the exercise (P 0.01) and returned to the resting value after the first minute of recovery (Table 3 and Fig. 1).
All values are means ±S.E.M. (n= 6). Ftg, fatigue.
Plasma volume did not change across the lung during the rest, exercise or recovery period of the experiment (Table 3 and Fig. 1).
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At rest, there was no change in BV across the lung (1.3 ± 0.8%). During exercise, loss of BV across the lung was small and ranged from 2.7 ± 0.3% min 1 of exercise to 4.2 ± 0.7% at min 4 of exercise. During recovery, BV changes decreased to the resting value (Table 3 and Fig. 1).
Fluid exchange between the pulmonary vasculature and pulmonary interstitium
Q increased from 74.7 ± 19.3 l min–1 at rest to over 300 l min–1 during exercise (P < 0.0001) and was not significantly different from the resting value by the first minute of recovery (Table 3).
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At rest, 0.6 ± 0.6 l min–1 of fluid moved between the pulmonary vasculature and the pulmonary interstitium. During exercise, however, 8.3 ± 1.3 (l fluid) min–1 moved from the pulmonary vasculature at the first min and increased to 12.9 ± 2.5 l min–1 at min 4 of exercise (P < 0.0001). The fluid loss from the pulmonary circulation decreased immediately after cessation of exercise to 6.8 ± 0.4 l min–1 (P < 0.0001), then by the first minute of recovery to 4.2 ± 0.5 l min–1 (P < 0.0008) and was not significantly different from the resting value thereafter (Table 3 and Fig. 2).
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Values are means ±S.E.M. (n= 6). different from rest (P < 0.05). BL, base line – resting period. Ftg, fatigue.
A positive association was found between Q and the JV–A (P < 0.0001, r-Sq = 0.44, r = 0.668) (Fig. 3).
Values are means ±S.E.M. (n= 6).
Discussion
This is the first study that has quantified the transvascular flux in lungs during exercise based on BV changes and Q. Horses have great athletic capabilities, which make them suitable subjects to study circulatory effects on lung fluid dynamics at rest and during exercise. We exercised horses based on their predetermined to minimize the variable effect of the level of fitness of individual horses. Transvascular fluxes in lungs of horses during exercise reached approximately 4% of Q or 12 l min–1. EV decreased by 6% across the lung, which counterbalanced the effect of oncotic pressures in plasma and enhanced fluid movement from the pulmonary vasculature.
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Previous studies have shown increased fluid movement across the pulmonary tissue during exertion (Coates et al. 1984; Newman et al. 1988). The increase of fluid movement has been shown to be caused by: (1) pulmonary capillary recruitment and/or dilatation coupled with the increase in the pulmonary surface area (Bake et al. 1968; Hlastala et al. 1996); (2) increased pulmonary microvascular pressures (Sinha et al. 1996); and (3) changes in the pulmonary transcapillary gradients (Bland & McMillan, 1977).
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Coates et al. (1984), using Q as an index of the increase in pulmonary surface area and capillary recruitment, reported a significant linear correlation between the pulmonary lymph flow and Q (r= 0.87, P < 0.01) during various levels of steady-state exercise. We have also found a positive, although less strong, association between Q and JV–A (r= 0.668, P < 0.0001, R-Sq = 0.44) (Fig. 3). The difference can be attributed to a non-steady-state transition to steady exercise in this experiment and to the ability of horses to develop a very high PPA. PPA determines the rise in pulmonary capillary pressure and consequent increase in the transvascular filtration across the lung (Sinha et al. 1996). Mean PPA in horses during exertion may reach 80 mmHg (Erickson et al. 1990). This is in contrast to other species studied where mean PPA during exertion rarely exceeds 40 mmHg (Slonim et al. 1954; Elkins & Milnor, 1971; Wagner et al. 1986; Younes et al. 1987; Newman et al. 1993). The reason for the difference between horses and other species is still speculative. It is possible that the increase in haematocrit and consequent rheological changes during exercise (Davis & Manohar, 1988; Fedde & Wood, 1993; Sinha et al. 1996) may contribute to the discrepancy.
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Hansen (1961) hypothesized that erythrocytes may play an important role in the regulation of oncotic pressure in the pulmonary capillaries. In the present study, the decrease in EV was an important contributor to the total fluid flux from the pulmonary vasculature (Table 3). Pulmonary transvascular fluxes increase as a function of the net transvascular driving pressure: hydrostatic pressure gradient minus protein osmotic pressure gradient (Starling, 1896; Bland & McMillan, 1977). The equilibrium between fluid and its solutes during exercise became altered with active fluid release from erythrocytes. The dilution of plasma protein reduced the plasma colloid osmotic pressure gradient, which, assisted by the increased hydrostatic pressure, favoured the movement of fluid out of the pulmonary vasculature thus restoring the transvascular gradient to its equilibrium state. Therefore, oncotic forces have an important function along with hydrostatic forces in the extent of fluid movement from the pulmonary vasculature during exercise.
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The decrease in EV across the lung during this experiment is in agreement with previous erythrocyte morphology studies (Van Beaumont et al. 1981; Honess et al. 1996; Speake et al. 1997; Gibson et al. 2000; Aliberti et al. 2001). In peripheral tissues in deoxygenated blood, Cl– (and water) is exchanged for HCO3– across the erythrocyte plasma membrane (Band 3) (Bretcher, 1971). Na+–H+ exchange, coupled to Cl––HCO3– exchange via the membrane anion exchanger AE1 and stimulated by low PO2, increased [H+], decreased cell volume, and (exercise-derived) -adrenergic stimuli, all contribute to net influx of NaCl (and water) into erythrocyte, increasing the cell volume. Na+–K+–2 Cl– cotransport is activated by similar stimuli and contributes to solute concentration in the erythrocyte. The Na+–K+-ATPase remains active, decreasing the erythrocyte [Na+]; however, low PO2 inhibits K+–Cl– cotransport resulting in an increase in EV. When erythrocytes enter the lung capillary bed, these processes are reversed and would account for the decrease in EV observed in this study (Honess et al. 1996; Speake et al. 1997; Gibson et al. 2000).
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In the present study, fluid flux returned to normal by min 2 of recovery, which is in agreement with experiments in the lung lymphatic system (Coates et al. 1984; Newman et al. 1988, 1993; Schaffartzik et al. 1993). In human athletes, post-exercise clearance of accumulated pulmonary fluid to pre-exercise levels is achieved within 2 h (Hanel et al. 2003). In contrast to our study, where horses were allowed to cool down actively, Hanel et al. (2003) measured post-exercise fluid dynamics in lungs of athletes positioned in a supine position, which probably prolonged lung fluid clearance.
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Methodological considerations
In the present study we have measured total transvascular fluxes from the pulmonary circulation, which include fluid that contributes to changes in lung lymph flow and total lung water. Lung lymph flow studies not only measure transpulmonary fluid dynamics but also include lymph flow from non-pulmonary tissues (Demling & Gunther, 1982; Drake et al. 1986) and the conducting airways (Wagner et al. 1998). Gravimetric lung-fluid dynamic studies only detect variations in total lung water and are unable to account for alterations when changes are to be contributed to the vascular, interstitial and/or cellular compartments in lungs (Lin et al. 1998). Methods in the present study provide accurate results acutely in an exercising subject, which can be applied to other animals and humans, in contrast to gravimetric lung studies, which require stagnant experimental conditions, or lymphatic studies, which require extensive surgery and chronic instrumentation.
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Methods for calculation of BV changes used in this study are simple and have been validated previously (Dill & Costill, 1974; Costill et al. 1974; Harrison, 1985). Hct, plasma protein and Hb levels can be measured very precisely with the CV for duplicate measures varying from 0.7 to 2.4%. The precision of these measurements allow small changes in the variables to be detected from mixed venous to arterial blood.
Conclusion
, http://www.100md.com Fluid moves from the pulmonary circulation during exertion in horses. This is the result of changing transvascular gradients between the hydrostatic and osmotic pressures influenced by increased microvascular pressures and EV regulation across the lung. Lesser influence of the erythrocyte fluid release on the fluid flux from the pulmonary vasculature should be expected in species other than equides that do not possess the equivalent erythrocyte storage and release capability. Horses are probably capable of accommodating higher pressures in the pulmonary microvasculature during exercise and aggravate ventilation–perfusion inequality to a lesser extent compared to other species where other factors, such as lymph-flow dynamics and pulmonary macro- or microvascular permeability, may be of greater importance for accommodating local stresses of exertion. Nevertheless, the method presented herein can be used clinically in cases of abnormal fluid balance in the lungs, or perhaps other organs, and to study fluid dynamics in high-level athletes.
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Younes M, Bshouty Z & Ali J (1987). Longitudinal distribution of pulmonary vascular resistance with very high pulmonary blood flow. J Appl Physiol 62, 344–358., 百拇医药(Modest Vengust, Henry Staempfli, Laurent)
2 Department of Clinical Studies, University of Guelph, Ontario, Canada N1G 2W1
3 Department of Medicine, McMaster University Medical Centre Hamilton, Ontario, Canada L8N 3Z5
Abstract
Exercise causes changes in pulmonary haemodynamics through redistribution of blood flow, increase in the pulmonary surface area, and increase in pulmonary vascular pressures. These changes contribute to the increase in fluid exchange across the alveolar–capillary barrier. To determine the extent of the fluid exchange across the alveolar–capillary barrier at rest and during exercise, six horses were exercised on a high-speed treadmill until fatigue. Arterial and mixed venous blood were sampled at rest and during exercise and recovery. Blood volume changes across the lung (BV; measured in percentage) were calculated from changes in plasma protein and haemoglobin concentration, and haematocrit. Cardiac output (Q) was calculated using the Fick equation. Fluid flux (JV–A; measured in l min–1) across the alveolar–capillary barrier was then quantified based on Q and BV. At rest, no fluid movement occurred across the pulmonary vasculature (0.6 ± 0.6 l min–1). During exercise, the amount of fluid moved from the pulmonary circulation was 8.3 ± 1.3 l min–1 at 1 min, 6.4 ± 2.9 l min–1 at 2 min, 10.1 ± 1.0 l min–1 at 3 min, 12.9 ± 2.5 l min–1 at 4 and 9.6 ± 1.5 l min–1 at fatigue (all P < 0.0001). Erythrocyte volume decreased by 6% (P < 0.01) across the lungs, which decreased the colloid osmotic gradient in the pulmonary vasculature. Decrease colloid osmotic gradient along with increased hydrostatic forces in the pulmonary vasculature would enhance displacement of fluid into the pulmonary interstitium. In conclusion, exercise caused large increases in transpulmonary fluid fluxes in horses. Here, we present a simple method to calculate transpulmonary fluid fluxes in different species, which can be used to elucidate mechanisms of lung fluid balance in vivo.
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Introduction
Maximal exercise resulted in a marked increase in cardiac output (Q) with a less prominent increase in pulmonary macro- and microvascular pressures (Wagner et al. 1986; Groves et al. 1987; Schaffartzik et al. 1993; Newman et al. 1993). Such adaptations coexist with redistribution of blood flow across the lung through capillary recruitment and increase in the pulmonary surface area (Bake et al. 1968; Hlastala et al. 1996). Changes in pulmonary haemodynamics during exercise increase fluid and solute movement across the alveolar–capillary barrier (Dexter et al. 1951; Johnson et al. 1960). A possible consequence of such adaptations is the development of pulmonary oedema (McKechnie et al. 1979) or, more commonly, perivascular oedema and/or parenchymal interstitial oedema that worsens the pulmonary gas exchange in dogs (Younes et al. 1987), small ruminants (Coates et al. 1984), pigs (Schaffartzik et al. 1993), humans (Schaffartzik et al. 1992; McKenzie et al. 2005) and, to a lesser extent, horses (West et al. 1993).
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Healthy animals and humans are capable of eliminating excessive lung fluid during strenuous exercise even when the pulmonary capillary hydrostatic pressure rises above the oedemagenic threshold (Wagner et al. 1986; Groves et al. 1987; Newman et al. 1988, 1993; Erickson et al. 1992; Manohar, 1993). Most of the excessive lung fluid is eliminated via the pulmonary lymph system (Staub et al. 1967; Mitzner & Sylvester, 1986). In sheep, and probably in other species, during strenuous exercise the lung lymphatic flow rises several-fold (Coates et al. 1984; Newman et al. 1988, 1993). Pulmonary lymph flow correlates with the rate of filtration from the pulmonary vasculature even though pulmonary lymph flow is mixed with lymph flow from non-pulmonary tissues (Demling & Gunther, 1982; Drake et al. 1986) and the conducting airways (Wagner et al. 1998).
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Although the increase in lymph flow correlates with increased exchange of fluid across pulmonary capillaries, quantification of fluid movement from the pulmonary circulation at rest and during exercise through lymph-flow analysis is not possible. Nor can it be calculated using Starling's equation (Starling, 1896), because forces governing these events in lung compartments cannot be defined and water transport across the membrane is also regulated independently of solute transport (aquaporins) (King et al. 2004).
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We conducted a study where fluid movement across the alveolar–capillary barrier (across the lung) was quantitatively assessed by comparison of blood volume differences between mixed venous blood and arterial blood in horses at rest and during exercise. Horses were chosen because their increase in pulmonary arterial pressure (PPA) during exercise, in contrast to other species (Slonim et al. 1954; Elkins & Milnor, 1971; Wagner et al. 1986; Younes et al. 1987; Newman et al. 1993), is substantial (Wagner et al. 1989; Erickson et al. 1992; Manohar, 1993; Wilkins et al. 2001), which should translate into a relatively high pulmonary capillary pressure (Sinha et al. 1996). In addition, in horses (Wilkins et al. 2001), as well as in humans (McKenzie et al. 2005), the lung water has been reported to increase during exertion. Based on that, we hypothesized that exercising horses develop detectable transvascular fluid flux from the pulmonary vasculature into the pulmonary interstitium.
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Methods
Six race-fit standardbred horses (2 female, 4 geldings), with a mean age of 5.7 years (range, 5–6 years), mean weight of 450 kg (range, 402–497 kg) and mean peak O2 uptake of 159.9 ml kg–1 min–1 (range, 141.1–179.4 ml kg–1 min–1) were used. Horses were recruited with owner/trainer consent. The study protocols were approved by the Animal Care Committee according to the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, Ontario). All horses were healthy when returned to their owners, and resumed their normal training and racing activities.
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Pre-experimental protocol
Five days prior to the experiment, each horse was familiarized with the treadmill. During the first 3 days, horses were given repeated walking exercise on the treadmill for 20 min daily (15 min walk, 5 min slow pace) at 10% treadmill inclination, followed by 2 days of exercise with the respiratory mask fitted on their nose. Before every experiment, horses were weighed and fitted with a safety harness, hobbles and heart rate meter (Equistat Model HR-8 A, EQB Inc., Unionville, PA, USA).
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On day 6, peak O2 uptake was determined for each horse, during three treadmill exercise periods: warm-up, incremental exercise and recovery. During the warm-up period the horses were walked on a horizontal treadmill with no inclination for 5 min at 2–3 m s–1 and then trotted for 5 min at 4–5 m s–1. At the end of this 10 min warming-up period, the treadmill was inclined to 10% and the speed brought to 8 m s–1. The incremental exercise consisted of a stepwise increase of velocity of 1 m s–1 every 60 s. An open flow-through system was used for collection of pulmonary gases throughout the entire exercise protocol. Peak O2 uptake was determined as the point at which no further increase in occurred, despite an increase in speed, or a level of exercise where the horse could no longer maintain pace with the treadmill speed.
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Experimental protocol
Experiments were carried out 72 h after the determination of . During this time, horses rested and were hand walked for 15 min daily. The skin at the site of catheter insertion was clipped, desensitized with EMLA cream (lidocaine 2.5% and prilocaine 2.5%; AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA) and aseptically prepared. Pulmonary Swan-Ganz catheter (Baxter Healthcare Corp., Irvine, CA, USA) and a 150-cm long central venous polyethylene blood catheter (#240) were placed aseptically via the left and right jugular vein into the pulmonary artery for mixed central venous blood sampling and core body temperature measurements. Correct catheter placement was assured by observing characteristic pressure waveforms on an oscilloscope (Criticare 1100, Criticare Systems Inc., Waukesha, WI, USA). A 20-gauge catheter (Insyte-W, Infusion Therapy Systems Inc., Sandy, UT, USA) was inserted into the facial or transverse facial artery. A 30-cm long extension tubing with a three-way stopcock was connected to intravenous and intra-arterial catheters. Catheters and extension tubing were all sutured securely to the skin. Horses then rested on the treadmill until their heart rate returned to resting values.
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The high intensity exercise experiment followed; this consisted of a warm-up phase as outlined for the testing in the pre-experimental protocol. The subsequent treadmill velocity was set to produce 80% of the previously determined . Horses were exercised until fatigue. During recovery, horses were walked on the treadmill at 2–3 m s–1.
Pulmonary gas collection
Pulmonary gas exchange was measured at rest, during exercise and during the recovery period. Before exercise, the respiratory mask was fitted on the horse's nose. An open flow-through system was used for collection of expired gases throughout the entire exercise protocol (Wagner et al. 1989). The expired gas was drawn into the O2 analyser (Ametek, Model S-3 A/1, Pittsburgh, PA, USA), which measured the concentration of inspired O2. Inspired concentration of O2 was recorded in 10-s periods throughout the experiment. For analysis, the average of three measurements in a 30-s period was used; this coincided with blood-sampling intervals.
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Blood sampling and blood analysis
Resting arterial and mixed venous blood were collected simultaneously under anaerobic conditions twice in a 5-min interval. Further sampling was performed in 60-s intervals during the exercise period until fatigue. During the recovery period, sampling was performed starting after the treadmill was stopped (0 min) and then 1, 2, 3, 5, 10 and 15 min into the recovery period. Prior to each sampling, 10 ml blood was withdrawn from catheters and discarded. Blood samples were collected into lithium-heparinized syringes (S-Monovette, Sarstedt AG & Co, Nümbrecht, Germany), stored on ice, and analysed in duplicate with the Stat Profile M Analyser (Nova Biomedical Corporation, Waltham, MA, USA) immediately after the treadmill protocol ended. Stat Profile M Analyser uses conductivity for measurement of haematocrit (Hct) (coefficient of variance (CV) = 2.4%), and conductivity/reflectance for haemoglobin (Hb) (CV = 1.8%) and O2 saturation (CV = 0.7%) analysis. Blood O2 content was calculated from the O2 saturation and the Hb concentration using standard equations. Total plasma protein (CV = 0.8%) was measured using a clinical refractometer (Attago, Tokyo, Japan).
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Calculations
Plasma volume changes across the lung (PVv–a) were calculated from changes in plasma protein concentration [PP] at the same time point from central venous to arterial blood (across the lung) (Dill & Costill, 1974):
(1)
where [PPv] is the plasma protein concentration in venous blood and [PPa] the plasma protein concentration in arterial blood. To account for changes in plasma volume relative to Hct, eqn (1) was adjusted for changes in the Hct across the lungs:
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(2)
where %HctPVv–a is the plasma volume change relative to Hct changes from central venous (Hctv) to arterial (Hcta) blood.
Changes in erythrocyte volume (EVv–a) across the lungs were calculated from changes in haemoglobin concentration ([Hb]) and haematocrit (Hct) in venous ([Hbv] and Hctv) and arterial blood ([Hba] and Hcta) (Costill et al. 1974):
(3)
For calculation of the blood volume (BV) the %EVv–a was adjusted for Hct changes across the lung:
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(4)
Blood volume changes across the lung were then measured from HctPVv–a and HctEVv–a:
(5)
Cardiac output (l min–1) was calculated using Fick's principle using and blood O2 content from central venous and arterial blood. Fluid flux (JV–A; measured in l min–1) across the lung was then quantified based on Q and %BV:
(6)
Statistical analysis
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Exercise and recovery parameters were compared to resting values using one-way repeated-measures ANOVA. A significant F ratio was further analysed using Dunnett's post hoc analysis. Pearson's correlation analysis was used to determine whether there was a significant association between the cardiac output and fluid exchange. A statistical significance level of P < 0.05 was used and data expressed as means ±S.E.M.
Results
Horses were exercised at the mean velocity of 9.1 ± 0.15 m s–1. The mean duration of exercise to fatigue was 4.7 ± 0.2 min.
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Respiratory changes
increased progressively during exercise. Horses reached of 151.2 ± 5.9 ml kg–1 min–1. VO2 decreased immediately after the treadmill was stopped due to fatigue and remained elevated compared to rest throughout the recovery (P < 0.0001) (Table 1).
Arterial and venous blood O2 pressures decreased during exercise (P < 0.0001) (Table 1).
Oxygen saturation in venous blood decreased substantially during exercise from 67.2 ± 14% at rest to below 10% during exercise (P < 0.0001). It remained decreased until min 10 of recovery (P 0.02) and achieved resting value at min 15 of recovery (Table 1). Oxygen saturation in arterial blood (SaO2) decreased (P < 0.0001) from 98.4 ± 0.4% to 90.9 ± 0.4% by min 2 of exercise with no further decline during the exercise period (P < 0.0001). It returned to the resting value immediately after cessation of exercise.
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Exercise resulted in an immediate decrease of the venous blood oxygen content (CvO2) below 3 ml dl–1 (P < 0.0001). During recovery it increased above the resting value until min 3 of recovery and then returned to the resting value. Arterial blood oxygen content (CaO2) increased immediately after the start of exercise to above 22 ml dl–1 and then progressively increased during exercise to 23.8 ± 0.5 ml dl–1 at fatigue (P < 0.0001). It remained increased throughout recovery (P 0.03) (Table 1).
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Haematocrit, haemoglobin and plasma protein
Haematocrit and Hb level increased (P < 0.0001) during exercise. Both slowly declined during recovery; however, they remained elevated above the resting value at the end of recovery. Plasma protein concentration did not change significantly during exercise. Haematocrit and levels of haemoglobin and plasma protein did not change across the lung (Table 2).
Erythrocyte, plasma and blood volume changes
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At rest EV did not change across the lung (0.3 ± 1.5%). By the minute first of exercise EV decreased rapidly, continued to decrease throughout the exercise (P 0.01) and returned to the resting value after the first minute of recovery (Table 3 and Fig. 1).
All values are means ±S.E.M. (n= 6). Ftg, fatigue.
Plasma volume did not change across the lung during the rest, exercise or recovery period of the experiment (Table 3 and Fig. 1).
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At rest, there was no change in BV across the lung (1.3 ± 0.8%). During exercise, loss of BV across the lung was small and ranged from 2.7 ± 0.3% min 1 of exercise to 4.2 ± 0.7% at min 4 of exercise. During recovery, BV changes decreased to the resting value (Table 3 and Fig. 1).
Fluid exchange between the pulmonary vasculature and pulmonary interstitium
Q increased from 74.7 ± 19.3 l min–1 at rest to over 300 l min–1 during exercise (P < 0.0001) and was not significantly different from the resting value by the first minute of recovery (Table 3).
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At rest, 0.6 ± 0.6 l min–1 of fluid moved between the pulmonary vasculature and the pulmonary interstitium. During exercise, however, 8.3 ± 1.3 (l fluid) min–1 moved from the pulmonary vasculature at the first min and increased to 12.9 ± 2.5 l min–1 at min 4 of exercise (P < 0.0001). The fluid loss from the pulmonary circulation decreased immediately after cessation of exercise to 6.8 ± 0.4 l min–1 (P < 0.0001), then by the first minute of recovery to 4.2 ± 0.5 l min–1 (P < 0.0008) and was not significantly different from the resting value thereafter (Table 3 and Fig. 2).
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Values are means ±S.E.M. (n= 6). different from rest (P < 0.05). BL, base line – resting period. Ftg, fatigue.
A positive association was found between Q and the JV–A (P < 0.0001, r-Sq = 0.44, r = 0.668) (Fig. 3).
Values are means ±S.E.M. (n= 6).
Discussion
This is the first study that has quantified the transvascular flux in lungs during exercise based on BV changes and Q. Horses have great athletic capabilities, which make them suitable subjects to study circulatory effects on lung fluid dynamics at rest and during exercise. We exercised horses based on their predetermined to minimize the variable effect of the level of fitness of individual horses. Transvascular fluxes in lungs of horses during exercise reached approximately 4% of Q or 12 l min–1. EV decreased by 6% across the lung, which counterbalanced the effect of oncotic pressures in plasma and enhanced fluid movement from the pulmonary vasculature.
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Previous studies have shown increased fluid movement across the pulmonary tissue during exertion (Coates et al. 1984; Newman et al. 1988). The increase of fluid movement has been shown to be caused by: (1) pulmonary capillary recruitment and/or dilatation coupled with the increase in the pulmonary surface area (Bake et al. 1968; Hlastala et al. 1996); (2) increased pulmonary microvascular pressures (Sinha et al. 1996); and (3) changes in the pulmonary transcapillary gradients (Bland & McMillan, 1977).
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Coates et al. (1984), using Q as an index of the increase in pulmonary surface area and capillary recruitment, reported a significant linear correlation between the pulmonary lymph flow and Q (r= 0.87, P < 0.01) during various levels of steady-state exercise. We have also found a positive, although less strong, association between Q and JV–A (r= 0.668, P < 0.0001, R-Sq = 0.44) (Fig. 3). The difference can be attributed to a non-steady-state transition to steady exercise in this experiment and to the ability of horses to develop a very high PPA. PPA determines the rise in pulmonary capillary pressure and consequent increase in the transvascular filtration across the lung (Sinha et al. 1996). Mean PPA in horses during exertion may reach 80 mmHg (Erickson et al. 1990). This is in contrast to other species studied where mean PPA during exertion rarely exceeds 40 mmHg (Slonim et al. 1954; Elkins & Milnor, 1971; Wagner et al. 1986; Younes et al. 1987; Newman et al. 1993). The reason for the difference between horses and other species is still speculative. It is possible that the increase in haematocrit and consequent rheological changes during exercise (Davis & Manohar, 1988; Fedde & Wood, 1993; Sinha et al. 1996) may contribute to the discrepancy.
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Hansen (1961) hypothesized that erythrocytes may play an important role in the regulation of oncotic pressure in the pulmonary capillaries. In the present study, the decrease in EV was an important contributor to the total fluid flux from the pulmonary vasculature (Table 3). Pulmonary transvascular fluxes increase as a function of the net transvascular driving pressure: hydrostatic pressure gradient minus protein osmotic pressure gradient (Starling, 1896; Bland & McMillan, 1977). The equilibrium between fluid and its solutes during exercise became altered with active fluid release from erythrocytes. The dilution of plasma protein reduced the plasma colloid osmotic pressure gradient, which, assisted by the increased hydrostatic pressure, favoured the movement of fluid out of the pulmonary vasculature thus restoring the transvascular gradient to its equilibrium state. Therefore, oncotic forces have an important function along with hydrostatic forces in the extent of fluid movement from the pulmonary vasculature during exercise.
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The decrease in EV across the lung during this experiment is in agreement with previous erythrocyte morphology studies (Van Beaumont et al. 1981; Honess et al. 1996; Speake et al. 1997; Gibson et al. 2000; Aliberti et al. 2001). In peripheral tissues in deoxygenated blood, Cl– (and water) is exchanged for HCO3– across the erythrocyte plasma membrane (Band 3) (Bretcher, 1971). Na+–H+ exchange, coupled to Cl––HCO3– exchange via the membrane anion exchanger AE1 and stimulated by low PO2, increased [H+], decreased cell volume, and (exercise-derived) -adrenergic stimuli, all contribute to net influx of NaCl (and water) into erythrocyte, increasing the cell volume. Na+–K+–2 Cl– cotransport is activated by similar stimuli and contributes to solute concentration in the erythrocyte. The Na+–K+-ATPase remains active, decreasing the erythrocyte [Na+]; however, low PO2 inhibits K+–Cl– cotransport resulting in an increase in EV. When erythrocytes enter the lung capillary bed, these processes are reversed and would account for the decrease in EV observed in this study (Honess et al. 1996; Speake et al. 1997; Gibson et al. 2000).
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In the present study, fluid flux returned to normal by min 2 of recovery, which is in agreement with experiments in the lung lymphatic system (Coates et al. 1984; Newman et al. 1988, 1993; Schaffartzik et al. 1993). In human athletes, post-exercise clearance of accumulated pulmonary fluid to pre-exercise levels is achieved within 2 h (Hanel et al. 2003). In contrast to our study, where horses were allowed to cool down actively, Hanel et al. (2003) measured post-exercise fluid dynamics in lungs of athletes positioned in a supine position, which probably prolonged lung fluid clearance.
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Methodological considerations
In the present study we have measured total transvascular fluxes from the pulmonary circulation, which include fluid that contributes to changes in lung lymph flow and total lung water. Lung lymph flow studies not only measure transpulmonary fluid dynamics but also include lymph flow from non-pulmonary tissues (Demling & Gunther, 1982; Drake et al. 1986) and the conducting airways (Wagner et al. 1998). Gravimetric lung-fluid dynamic studies only detect variations in total lung water and are unable to account for alterations when changes are to be contributed to the vascular, interstitial and/or cellular compartments in lungs (Lin et al. 1998). Methods in the present study provide accurate results acutely in an exercising subject, which can be applied to other animals and humans, in contrast to gravimetric lung studies, which require stagnant experimental conditions, or lymphatic studies, which require extensive surgery and chronic instrumentation.
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Methods for calculation of BV changes used in this study are simple and have been validated previously (Dill & Costill, 1974; Costill et al. 1974; Harrison, 1985). Hct, plasma protein and Hb levels can be measured very precisely with the CV for duplicate measures varying from 0.7 to 2.4%. The precision of these measurements allow small changes in the variables to be detected from mixed venous to arterial blood.
Conclusion
, http://www.100md.com Fluid moves from the pulmonary circulation during exertion in horses. This is the result of changing transvascular gradients between the hydrostatic and osmotic pressures influenced by increased microvascular pressures and EV regulation across the lung. Lesser influence of the erythrocyte fluid release on the fluid flux from the pulmonary vasculature should be expected in species other than equides that do not possess the equivalent erythrocyte storage and release capability. Horses are probably capable of accommodating higher pressures in the pulmonary microvasculature during exercise and aggravate ventilation–perfusion inequality to a lesser extent compared to other species where other factors, such as lymph-flow dynamics and pulmonary macro- or microvascular permeability, may be of greater importance for accommodating local stresses of exertion. Nevertheless, the method presented herein can be used clinically in cases of abnormal fluid balance in the lungs, or perhaps other organs, and to study fluid dynamics in high-level athletes.
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