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Inhibition of pulmonary surfactant by plasma from normal adults and from patients having cardiopulmonary bypass
J
THORAC CARDIOVASC SURG
91:248-251, 1986
Inhibition of pulmonary surfactant by plasma from normal adults and from patients having cardiopulmonary bypass Plasma from normal adults and from children and adults having cardiopulmonary bypass inhibited the ability of pulmonary surfactant to reach low surface tension in proportion to the amount of protein added. No increase in the extent of inhibition of surfactant action per milligram of protein was seen in plasma taken before or after bypass in adults or children.
P. Terence Phang, M.D., and Kevin M. W. Keough, Ph.D., St. John's, Newfoundland, Canada
Lung dysfunction after cardiopulmonary bypass (CPB) ranges in severity from minor atelectasis to respiratory distress syndrome (RDS). In elective cardiac operations in adults today, the majority of patients exhibit minor atelectasis and slight loss of lung volumes. However, RDS is still seen with significant frequency in cases of prolonged bypass and in children. The literature I·? presents confusing evidence regarding surfactant inhibition by normal blood, plasma proteins, blood exposed to a CPB circuit, and the timing of the surfactant dysfunction in relation to the lung injury. This study investigated the hypothesis that the plasma components that enter the air spaces through leaking lung membranes" could inhibit surfactant from proper function and that some component(s) may be increased by CPB. Surfactant dysfunction would lead to atelectasis and an increase in transalveolar hydrostatic forces that would increase pulmonary edema. The study is, to
From the Departments of Biochemistry, Surgery, and Pediatrics, Memorial University of Newfoundland, St. John's, Newfoundland, Canada Al B 3X9. Supported by the Department of Surgery and the Research Fund of the Faculty of Medicine, Memorial University of Newfoundland, the Newfoundland Lung Association, and the Medical Research Council of Canada. Received for publication Aug. 27, 1984. Accepted for publication April 9, 1985. Address for reprints: Dr. K. M. W. Keough, Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A I B 3X9.
248
our knowledge, the first to determine if the CPB procedure in patients has an influence on the levels of putative surfactant inhibitors. Methods Testing conditions. Surface tension measurements were performed on a pretreated? surface balance in a humid environment chamber described before," with air temperature at 37° ± 2° C and subphase temperature at 33° ± 2° C. A constant amount of surfactant prepared by a modification of previous techniques II , 12 was mixed with varying amounts of plasma by vortexing and by passing the mixture into and out of a 50 ~l syringe seven times. After 5 minutes (there was no difference between an interaction time of 5 and 30 minutes) the mixture was layered on the balance. After 4 minutes (there were no differences in isotherms obtained from mixtures layered for 1 to 10 minutes) compressions were begun. The minimum surface tension (gamma-min) on the first compression was used to indicate the level of surfactant function. The difference between the gamma-min of the mixture of surfactant and plasma and the gamma-min of surfactant alone (delta gamma-min) was used to indicate the effect of the plasma. Control dispersions of surfactant were analyzed before and after each series of measurements. Tests were done with two standard dispersions which, when loaded on the surface, gave 7.4 ~g of phospholipid (SAM E) and 6.5 ~g of phospholipid (SAM F), as determine by phosphorus assay." Protein in plasma was determined by the biuret method." Test groups. Approval for the study was obtained
Volume 91 Number 2 February, 1986
Pulmonary surfactant
25
Table I. Summary of comparisons
n Value
Comparison Adult CPB plasma, SAM E, groups J to 4 Adult CPB plasma, SAM F, groups I to 4 Child CPB plasma, SAM F, prepump vs. postpump Adult CPB-2 vs. child prepump, plasma, SAM F Adult CPB-3 vs. child prepump, plasma, SAM F
249
No No No
>0.25 >0.25 >0.25
20 0
E E
15
0
Yes
<0.01
No
>0.05
O'l
.2 Qi
Lrgrnd: cpa. Cardiopulmonary bypass, SAM E, 7.4 I'g of phospholipid. SAM I,. 1>,5 I'g of phospholipid. For cpa groups, sec text.
10
"0
5
o -+,---t:_-_---,--..,...----.---r----l
o
from the Human Investigation Committee of Memorial University of Newfoundland and the hospitals and surgeons involved. Informed verbal consent was obtained before blood was taken. The groups tested were normal adults and adult patients and pediatric patients undergoing elective CPB operations. Blood samples from the adult patients were obtained preoperatively on insertion of an indwelling arterial cannula (CPB-I group), after induction with the general anesthetic before CPB was started (CPB-2 group), immediately after bypass (CPB-3 group), and 20 to 24 hours postoperatively (CPB-4 group). In the children, blood was collected once the child was anesthetized before bypass (prepump group) and immediately after bypass (postpump group). CPB was performed with hemodilution. In most instances bubble oxygenators were employed, but in some membrane oxygenators were used. Blood was collected in glass syringes and placed into glass tubes in the presence of heparin. Testing was done within 10 hours of sampling. Statistical analysis was by analysis of variance and an overall F test of the lines of best fit. Results The effect of plasma on surfactant. Plasma prepared from normal adults prevented the surfactant from reaching low surface tension (data not shown). There was a linear relationship between delta gamma-min and the amount of protein interacted with the surfactant. The effect of CPB on surfactant inhibitors in plasma. The plasma from the adult and pediatric patients inhibited surfactant in a direct relation between delta gamma-min and the ratio of total protein/ phospholipid (Figs. I and 2). Analysis of variance of the data for the effects of plasma taken from all of the adult CPB groups 1 to 4 tested with SAM E or with SAM F (different patients from those with SAM E) showed that
0.2
0.4
0.6
0.8
1.2
1.4
total protein/pi (mg/I'g x '0)
Fig. 1. Comparison of adult CPB plasma using SAM E. pl. phospholipid, ~ . , ... , Data and regression line from CPB-l (n = 5). 0 - - - - , Data and regression line for CPB-2 (n = 4). 0 - ... - , Data and regression line for CPB-3 (n = 5). 0 - . -, Data and regression line for CPB-4 (n = 5). For explanation, see text.
there was no difference between these groups (Fig. I, Table I). No difference was found for the effects of plasma from the children before and after bypass (Fig. 2, Table I). There was a significant difference between the plasma taken from the adult CPB-2 (prepump) group and that taken from the child prepump group when tested with SAM F (Table I). Plasma from the adult prepump group showed slightly greater inhibition than that from the child prepump group, the difference being due mainly to a greater elevation (Y intercept) and not to the slope. There was no significant difference between the plasma in adult CPB-3 (postpump) group and the child postpump group (SAM F). There was no significant difference between the effect of human albumin alone and that of the pump priming solution that was circulated in a CPB circuit. This indicated that there was no effect of heparin or plasticizing agents that could be introduced by the priming solution and leaching of the tubing during pumping. RDS did not develop in any of the adult patients entered in the study. The length of time on CPB ranged from 60 to 120 minutes. The effect of prolonged bypass was examined in one child who had RDS postoperatively and who subsequently died of cardiac and pulmonary complications. There was no obvious difference between samples taken before bypass, at a pump time of 1'h
The Journal of Thoracic and Cardiovascular Surgery
2 5 0 Phangand Keough
25
l:>
20 0
E E
o
0
l))
.E Qi
'v · .~ yo
l:>
15
DO
10
iJ
l:>
ODD 0
~'
~,/l:> t:J.,' t:J.
A'~
5
0 0
0
0
l:>
0
0.2
0.4
,
0.6
0.8
1.2
1.4
total protein/pi (mg/~g x
10)
Fig. 2. Comparison of pediatric CPB plasma using SAM F. pi, phospholipid. .:l _ ... - , Data and regression line for prepump plasma (n = 5). 0 - . -, Data and regression line for postpump plasma (n = 5). For explanation see text.
hours, and at a pump time of 3 hours (data not shown). No difference was seen between prepump and postpump groups in adults or children. This suggests that CPB does not enhance the production of surfactant inhibitors in plasma, at least those that could be detected in this study. Increased inhibitors may have been produced by the CPB and may not have been detected in this study, possibly because they decayed before assay or because of their presence in the airways but not in plasma. The plasma of CPB patients was hemodiluted with a solution containing albumin. Thus, it would have been richer in albumin than in other plasma proteins. Since albumin is less inhibitory than other protein fractions (data not shown), there is some possibility that inhibitors were actually produced in small amounts by the bypass procedure. Further testing would be required to confirm or exclude this possibility. Comparisons were made between the plasma of normal adults and that of patients before CPB (CPB-l or CPB-2). No conclusive evidence for a difference between the normal and patient groups was obtained.
Discussion In studies by Gardner, Finley, and Tooley' and Mandelbaum and Giarnmona,' blood pumped in a CPB circuit inhibited surfactant. They used whole blood to prime the CPB circuit, older oxygenators, and no
filtering devices. Mandelbaum and Giammona' reported that inhibitors present in dog blood exposed in vitro to oxygenator circuits were not present if the animal was included in the circuit. To our knowledge, this study is the only one in which the effect of CPB on surfactant inhibitors in patients has been investigated. It would appear that in modern CPB with hemodilution techniques and short bypass times, there is little indication of an increase in surfactant inhibitor production per milligram of protein in plasma which is caused by the procedure itself. The data indicated the possibility that plasma components entering the air spaces through leaking lung membranes may inhibit surfactant from proper function and may contribute to lung collapse and edema seen in the adult RDS of any cause. CPB did not significantly increase the levels of inhibition per milligram of protein in the assay used in this study. We wish to thank the surgeons, Dr. G. Cornel, Dr. V. Aldrete, and Dr. K. Melvin; and the cardiac pump team, G. Walsh and W. O'Reilly. We are also grateful for technical assistance and advise from N. Kariel and Dr. A. Lee and for the statistical consultations with A. Cornish, Ph.D., Department of Mathematics and Statistics, Memorial University of Newfoundland. REFERENCES Gardner RE, Finley TN, Tooley WH: The effect of cardiopulmonary bypass on surface activity of lung extracts. Bull Soc Internat Chir 21:542-551, 1962 2 Tierney OF, Johnson RP: Altered surface tension of lung extracts and lung mechanics. J Appl Physiol 20: 12531260, 1965 3 Mandelbaum I, Giammona ST: Extracorporeal circula-
tion, pulmonary compliance, and pulmonary surfactant. J THORAC CARDIOVASC SURG 48:881-889, 1964 4 Taylor FB, Abrams ME: Effect of surface activelipoprotein on clotting and fibrinolysis, and of fibrinogen on surface tension of surface active lipoprotein. Am J Moo 40:346-350, 1966
5 Hepps SA, Roe BB, Wright RR, Gardner RE: Amelioration of the pulmonary post-perfusion syndrome with hemodilution and low molecular weight dextran. Surgery 54:232-243, 1963
6 Camishion RC, Fraimow W, Kelsey OM, Tokunga K, Davies AL, Joshi P, Cathcart RT, Pierucci L: Effect of partial and total cardiopulmonary bypass with whole blood or hemodilution primingon pulmonary surfactant activity. Surg Res 8: 1-6, 1968 7 Panossian A, Hagstrom JWC, Nehlsen SL, Veith FJ: Secondary nature of surfactant changes in postperfusion pulmonarydamage. J THoRAc CARDIOVASC SURG 57:628634, 1969
Volume 91 Number 2 February, 1986
8 Royston D, Catley DM, Higenbottam T, Wallwork J, Minty BD: Changes in alveolar capillary barrier (ACB) function associated with cardiopulmonary bypass (CPB). Br J Anaesth 55:917P, 1983 9 Goerke J, Gonzales J: Temperature dependence in dipalmitoyl phosphatidylcholine monolayer stability. J Appl PhysioI51:1108-11l4,1981 IO Hawco MW, Davis PJ, Keough KMW: Lipid fluidity in lung surfactant. Monolayers of saturated and unsaturated lecithins. J Appl Physiol 51:509-515,1981 11 King RJ, Clements JA: Surface active materials from dog
Pulmonary surfactant
251
lung 1. Method of isolation. Am Physiol 223:707-714, 1972 12 Yu S, Harding P, Smith N, Possmayer F: Bovine pulmonary surfactant. Chemical composition and physical properties. Lipids 18:522-529, 1983 13 Bartlett GR: Phosphorus assay in column chromatography. J Bioi Chern 234:466-478, 1959 14 Gornall AG, Bardawill CJ, David MM: Determination of serum proteins by means of the biuret reaction. J Biol Chern 177:751-766, 1949
THORAC CARDIOVASC SURG
91:248-251, 1986
Inhibition of pulmonary surfactant by plasma from normal adults and from patients having cardiopulmonary bypass Plasma from normal adults and from children and adults having cardiopulmonary bypass inhibited the ability of pulmonary surfactant to reach low surface tension in proportion to the amount of protein added. No increase in the extent of inhibition of surfactant action per milligram of protein was seen in plasma taken before or after bypass in adults or children.
P. Terence Phang, M.D., and Kevin M. W. Keough, Ph.D., St. John's, Newfoundland, Canada
Lung dysfunction after cardiopulmonary bypass (CPB) ranges in severity from minor atelectasis to respiratory distress syndrome (RDS). In elective cardiac operations in adults today, the majority of patients exhibit minor atelectasis and slight loss of lung volumes. However, RDS is still seen with significant frequency in cases of prolonged bypass and in children. The literature I·? presents confusing evidence regarding surfactant inhibition by normal blood, plasma proteins, blood exposed to a CPB circuit, and the timing of the surfactant dysfunction in relation to the lung injury. This study investigated the hypothesis that the plasma components that enter the air spaces through leaking lung membranes" could inhibit surfactant from proper function and that some component(s) may be increased by CPB. Surfactant dysfunction would lead to atelectasis and an increase in transalveolar hydrostatic forces that would increase pulmonary edema. The study is, to
From the Departments of Biochemistry, Surgery, and Pediatrics, Memorial University of Newfoundland, St. John's, Newfoundland, Canada Al B 3X9. Supported by the Department of Surgery and the Research Fund of the Faculty of Medicine, Memorial University of Newfoundland, the Newfoundland Lung Association, and the Medical Research Council of Canada. Received for publication Aug. 27, 1984. Accepted for publication April 9, 1985. Address for reprints: Dr. K. M. W. Keough, Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A I B 3X9.
248
our knowledge, the first to determine if the CPB procedure in patients has an influence on the levels of putative surfactant inhibitors. Methods Testing conditions. Surface tension measurements were performed on a pretreated? surface balance in a humid environment chamber described before," with air temperature at 37° ± 2° C and subphase temperature at 33° ± 2° C. A constant amount of surfactant prepared by a modification of previous techniques II , 12 was mixed with varying amounts of plasma by vortexing and by passing the mixture into and out of a 50 ~l syringe seven times. After 5 minutes (there was no difference between an interaction time of 5 and 30 minutes) the mixture was layered on the balance. After 4 minutes (there were no differences in isotherms obtained from mixtures layered for 1 to 10 minutes) compressions were begun. The minimum surface tension (gamma-min) on the first compression was used to indicate the level of surfactant function. The difference between the gamma-min of the mixture of surfactant and plasma and the gamma-min of surfactant alone (delta gamma-min) was used to indicate the effect of the plasma. Control dispersions of surfactant were analyzed before and after each series of measurements. Tests were done with two standard dispersions which, when loaded on the surface, gave 7.4 ~g of phospholipid (SAM E) and 6.5 ~g of phospholipid (SAM F), as determine by phosphorus assay." Protein in plasma was determined by the biuret method." Test groups. Approval for the study was obtained
Volume 91 Number 2 February, 1986
Pulmonary surfactant
25
Table I. Summary of comparisons
n Value
Comparison Adult CPB plasma, SAM E, groups J to 4 Adult CPB plasma, SAM F, groups I to 4 Child CPB plasma, SAM F, prepump vs. postpump Adult CPB-2 vs. child prepump, plasma, SAM F Adult CPB-3 vs. child prepump, plasma, SAM F
249
No No No
>0.25 >0.25 >0.25
20 0
E E
15
0
Yes
<0.01
No
>0.05
O'l
.2 Qi
Lrgrnd: cpa. Cardiopulmonary bypass, SAM E, 7.4 I'g of phospholipid. SAM I,. 1>,5 I'g of phospholipid. For cpa groups, sec text.
10
"0
5
o -+,---t:_-_---,--..,...----.---r----l
o
from the Human Investigation Committee of Memorial University of Newfoundland and the hospitals and surgeons involved. Informed verbal consent was obtained before blood was taken. The groups tested were normal adults and adult patients and pediatric patients undergoing elective CPB operations. Blood samples from the adult patients were obtained preoperatively on insertion of an indwelling arterial cannula (CPB-I group), after induction with the general anesthetic before CPB was started (CPB-2 group), immediately after bypass (CPB-3 group), and 20 to 24 hours postoperatively (CPB-4 group). In the children, blood was collected once the child was anesthetized before bypass (prepump group) and immediately after bypass (postpump group). CPB was performed with hemodilution. In most instances bubble oxygenators were employed, but in some membrane oxygenators were used. Blood was collected in glass syringes and placed into glass tubes in the presence of heparin. Testing was done within 10 hours of sampling. Statistical analysis was by analysis of variance and an overall F test of the lines of best fit. Results The effect of plasma on surfactant. Plasma prepared from normal adults prevented the surfactant from reaching low surface tension (data not shown). There was a linear relationship between delta gamma-min and the amount of protein interacted with the surfactant. The effect of CPB on surfactant inhibitors in plasma. The plasma from the adult and pediatric patients inhibited surfactant in a direct relation between delta gamma-min and the ratio of total protein/ phospholipid (Figs. I and 2). Analysis of variance of the data for the effects of plasma taken from all of the adult CPB groups 1 to 4 tested with SAM E or with SAM F (different patients from those with SAM E) showed that
0.2
0.4
0.6
0.8
1.2
1.4
total protein/pi (mg/I'g x '0)
Fig. 1. Comparison of adult CPB plasma using SAM E. pl. phospholipid, ~ . , ... , Data and regression line from CPB-l (n = 5). 0 - - - - , Data and regression line for CPB-2 (n = 4). 0 - ... - , Data and regression line for CPB-3 (n = 5). 0 - . -, Data and regression line for CPB-4 (n = 5). For explanation, see text.
there was no difference between these groups (Fig. I, Table I). No difference was found for the effects of plasma from the children before and after bypass (Fig. 2, Table I). There was a significant difference between the plasma taken from the adult CPB-2 (prepump) group and that taken from the child prepump group when tested with SAM F (Table I). Plasma from the adult prepump group showed slightly greater inhibition than that from the child prepump group, the difference being due mainly to a greater elevation (Y intercept) and not to the slope. There was no significant difference between the plasma in adult CPB-3 (postpump) group and the child postpump group (SAM F). There was no significant difference between the effect of human albumin alone and that of the pump priming solution that was circulated in a CPB circuit. This indicated that there was no effect of heparin or plasticizing agents that could be introduced by the priming solution and leaching of the tubing during pumping. RDS did not develop in any of the adult patients entered in the study. The length of time on CPB ranged from 60 to 120 minutes. The effect of prolonged bypass was examined in one child who had RDS postoperatively and who subsequently died of cardiac and pulmonary complications. There was no obvious difference between samples taken before bypass, at a pump time of 1'h
The Journal of Thoracic and Cardiovascular Surgery
2 5 0 Phangand Keough
25
l:>
20 0
E E
o
0
l))
.E Qi
'v · .~ yo
l:>
15
DO
10
iJ
l:>
ODD 0
~'
~,/l:> t:J.,' t:J.
A'~
5
0 0
0
0
l:>
0
0.2
0.4
,
0.6
0.8
1.2
1.4
total protein/pi (mg/~g x
10)
Fig. 2. Comparison of pediatric CPB plasma using SAM F. pi, phospholipid. .:l _ ... - , Data and regression line for prepump plasma (n = 5). 0 - . -, Data and regression line for postpump plasma (n = 5). For explanation see text.
hours, and at a pump time of 3 hours (data not shown). No difference was seen between prepump and postpump groups in adults or children. This suggests that CPB does not enhance the production of surfactant inhibitors in plasma, at least those that could be detected in this study. Increased inhibitors may have been produced by the CPB and may not have been detected in this study, possibly because they decayed before assay or because of their presence in the airways but not in plasma. The plasma of CPB patients was hemodiluted with a solution containing albumin. Thus, it would have been richer in albumin than in other plasma proteins. Since albumin is less inhibitory than other protein fractions (data not shown), there is some possibility that inhibitors were actually produced in small amounts by the bypass procedure. Further testing would be required to confirm or exclude this possibility. Comparisons were made between the plasma of normal adults and that of patients before CPB (CPB-l or CPB-2). No conclusive evidence for a difference between the normal and patient groups was obtained.
Discussion In studies by Gardner, Finley, and Tooley' and Mandelbaum and Giarnmona,' blood pumped in a CPB circuit inhibited surfactant. They used whole blood to prime the CPB circuit, older oxygenators, and no
filtering devices. Mandelbaum and Giammona' reported that inhibitors present in dog blood exposed in vitro to oxygenator circuits were not present if the animal was included in the circuit. To our knowledge, this study is the only one in which the effect of CPB on surfactant inhibitors in patients has been investigated. It would appear that in modern CPB with hemodilution techniques and short bypass times, there is little indication of an increase in surfactant inhibitor production per milligram of protein in plasma which is caused by the procedure itself. The data indicated the possibility that plasma components entering the air spaces through leaking lung membranes may inhibit surfactant from proper function and may contribute to lung collapse and edema seen in the adult RDS of any cause. CPB did not significantly increase the levels of inhibition per milligram of protein in the assay used in this study. We wish to thank the surgeons, Dr. G. Cornel, Dr. V. Aldrete, and Dr. K. Melvin; and the cardiac pump team, G. Walsh and W. O'Reilly. We are also grateful for technical assistance and advise from N. Kariel and Dr. A. Lee and for the statistical consultations with A. Cornish, Ph.D., Department of Mathematics and Statistics, Memorial University of Newfoundland. REFERENCES Gardner RE, Finley TN, Tooley WH: The effect of cardiopulmonary bypass on surface activity of lung extracts. Bull Soc Internat Chir 21:542-551, 1962 2 Tierney OF, Johnson RP: Altered surface tension of lung extracts and lung mechanics. J Appl Physiol 20: 12531260, 1965 3 Mandelbaum I, Giammona ST: Extracorporeal circula-
tion, pulmonary compliance, and pulmonary surfactant. J THORAC CARDIOVASC SURG 48:881-889, 1964 4 Taylor FB, Abrams ME: Effect of surface activelipoprotein on clotting and fibrinolysis, and of fibrinogen on surface tension of surface active lipoprotein. Am J Moo 40:346-350, 1966
5 Hepps SA, Roe BB, Wright RR, Gardner RE: Amelioration of the pulmonary post-perfusion syndrome with hemodilution and low molecular weight dextran. Surgery 54:232-243, 1963
6 Camishion RC, Fraimow W, Kelsey OM, Tokunga K, Davies AL, Joshi P, Cathcart RT, Pierucci L: Effect of partial and total cardiopulmonary bypass with whole blood or hemodilution primingon pulmonary surfactant activity. Surg Res 8: 1-6, 1968 7 Panossian A, Hagstrom JWC, Nehlsen SL, Veith FJ: Secondary nature of surfactant changes in postperfusion pulmonarydamage. J THoRAc CARDIOVASC SURG 57:628634, 1969
Volume 91 Number 2 February, 1986
8 Royston D, Catley DM, Higenbottam T, Wallwork J, Minty BD: Changes in alveolar capillary barrier (ACB) function associated with cardiopulmonary bypass (CPB). Br J Anaesth 55:917P, 1983 9 Goerke J, Gonzales J: Temperature dependence in dipalmitoyl phosphatidylcholine monolayer stability. J Appl PhysioI51:1108-11l4,1981 IO Hawco MW, Davis PJ, Keough KMW: Lipid fluidity in lung surfactant. Monolayers of saturated and unsaturated lecithins. J Appl Physiol 51:509-515,1981 11 King RJ, Clements JA: Surface active materials from dog
Pulmonary surfactant
251
lung 1. Method of isolation. Am Physiol 223:707-714, 1972 12 Yu S, Harding P, Smith N, Possmayer F: Bovine pulmonary surfactant. Chemical composition and physical properties. Lipids 18:522-529, 1983 13 Bartlett GR: Phosphorus assay in column chromatography. J Bioi Chern 234:466-478, 1959 14 Gornall AG, Bardawill CJ, David MM: Determination of serum proteins by means of the biuret reaction. J Biol Chern 177:751-766, 1949