- Email: [email protected]
VASOACTIVE SUBSTANCES LIBERATED BY PROLONGED BUBBLE OXYGENATION
VASOACTIVE SUBSTANCES LIBERATED PROLONGED BUBBLE
OXYGENATION
Martin Hollenberg,
Robert Pruett,
M.D.*
and Alan Thai, M.D., Detroit,
BY
B.S.,
Mich.
T
HE bubble oxygenator has become widely accepted as a valuable tool in cardiovascular surgery, however, hypotension, pulmonary atelectasis, and renal failure may complicate prolonged cardiopulmonary bypass in man and dog. The aim of the present work was to measure and identify vasoactive substances set free as a result of bubble oxygenation of blood in an attempt to explain some of these complications. MATERIALS AND METHODS
Experiments were designed to study the effect of blood, which had previously been recirculated through a bubble oxygenator, on three biological assay preparations. The following studies were made. (1) The effect of pump-oxygenated blood on the vascular resistance of isolated canine kidneys. (2) The contractile response of strips of ox carotid artery to blood before and after bubble oxygenation. (3) The response of isolated segments of rabbit bowel to plasma from control blood and blood which had undergone prolonged pump oxygenation. Animals.—Adult mongrel dogs, weighing 10 to 30 kilograms, and marketbred albino rabbits, weighing 2 to 3 kilograms, were used. Collection and Oxygenation of Blood.—Dog blood obtained under intravenous Nembutal anesthesia (30 mg. per kilogram of body weight) by exsanguination through a polyethylene catheter placed in either the femoral or carotid arteries was collected in siliconized flasks containing 5 mg. per cent of heparin. The heparinized blood was then recirculated by an occlusive Sigmamotor pump (Model M-2) at a flow rate of 300 to 400 c.c. per minute through a bubble oxygenator for periods up to 4 hours. The oxygen flow was 6 L. per minute. The oxygenator circuit was composed of Mayon tubing containing 3 stainless steel sponges coated with Dow Corning antifoam. In most experiments, clean, but not sterile, technique was used in preparing the oxygenator. Mich.
From the Department of Surgery, Wayne State University College of Medicine, Detroit,
Supported by U. S. Public Health Service Grant No. H-6842 and a grant from the Detroit Receiving Hospital Research Corporation. Received for publication May 16, 1962. •American Heart Association Research Fellow. 402
Vol. 45, No. 3 March, 1963
L I B E R A T I O N O F VASOACTIVE SUBSTANCES
403
BUBBLE OXYGENATOR
TO BUBBLE OXYGENATOR
TO JUGULAR VEIN
Fig. 1.—Apparatus used for perfusion of the isolated canine kidney. The kidney is perfused at constant pressure and temperature alternately by blood from the oxygenator and blood issuing directly from the femoral artery of a donor dog.
The Isolated Kidney Preparation.—Kidneys, weighing 100 to 400 grams, were removed from healthy adult mongrel dogs under Nembutal anesthesia. A segment of aorta, 4 cm. in length, adjoining the renal artery was removed with each kidney, but the renal veins were excised to aid venous drainage. Following removal, the distal end of the aorta was ligated and a cannula connected to the perfusion apparatus was inserted into its proximal end. The kidneys were then immediately perfused at 37° C. and at a constant pressure of 120 mm. Hg alternately by blood from the oxygenator and blood issuing directly from a catheter in the femoral artery of a donor dog. The period of transfer during which the kidneys were without blood supply did not exceed 3 minutes. The perfusion apparatus is illustrated in Fig. 1. Blood issuing from the perfused kidneys was collected by a plastic funnel and measured with a graduated cylinder. Renal vascular resistance was calculated as the ratio of pressure difference to flow. In order to determine the importance of red cell aggregation as a cause of increased renal vascular resistance, a separate series of experiments was carried
404
HOLLENBERG, P R U E T T , T H A L
J. Thoracic and Cardiovas. Surg.
out in which plasma separated by centrifugation at 700 g for 30 minutes from the red cells of heparinized dog blood was recirculated through the bubble oxygenator. Five isolated canine kidneys were directly perfused by plasma prepared in this manner and vascular resistance was again calculated. The Ox Carotid Artery Strip Preparation.—The use of this preparation for assay of vasoactive materials has been previously described by Janeway. 1 Carotid arteries, removed from steers immediately after slaughter, were cleaned of adventitia and stored in Krebs solution at pH 7.4 to 7.6 and at 6° C. until use. Circular segments of the arteries were excised and then divided into strips measuring 3 cm. by 1 cm. by 0.2 mm. The strips were attached by a stainless steel clip to a hook at the bottom of a 20 c.c. bath of Krebs solution and were suspended at room temperature. A mixture of 95 per cent oxygen and 5 per cent carbon dioxide was slowly bubbled through the bath. Care was taken to insure that the pH of the Krebs solution did not vary from the above limits. Tension of 80 Gm. for 20 minutes was applied to the artery segments and then, by means of a slender metal wire, the strips were attached to the vertical armature of a Statham myograph at a tension of 0.4 Gm. After a period of 15 to 20 minutes, required for shortening of the strips, they were then ready for use. The sample of blood to be assayed for vasoactive material was added to the muscle bath and the response of the smooth muscle preparation was monitored by a Sanborn recording apparatus. A measure of the quantity of vasoactive material present in a sample of heparinized blood was obtained by diluting the sample until an increase in tension of the artery strip of 0.2 Gm. could no longer be elicited. A unit of vasoactive material (VAM) was thus obtained and is defined as the reciprocal of the highest dilution of blood which excites an increase in tension of 0.2 Gm. Normal values for both dog and human blood assayed by this technique did not exceed 16 units. The sensitivity of this preparation was such that it would consistently detect a wide range of vasoactive substances in minute concentration; that is, epinephrine in dilutions of 10"9 M. In addition, the response of different strips to the same dilution of epinephrine was quite consistent. The preparation was used to estimate the quantity of vasoactive material present in samples of blood removed from the pump oxygenator at intervals during recirculation. has described the effects Identification of Vasoactive Agents.—Furchgott2 of blocking agents on the contractile response of spiral strips of rabbit aorta to selected vasoactive substances. This technique was applied to the ox carotid artery preparation as a method of identifying the vasoactive agents released during bubble oxygenation. I n separate experiments (Table I) the vasoactive agents 1-epinephrine hydroehloride, 1-norepinephrine bitartrate, serotonin ereatinine sulfate, histamine dihydrochloride, and acetylcholine chloride were assayed on strips blocked by the addition to the muscle bath for a 20 minute period of dibenzyline hydroehloride, pyrilamine maleate, and D-lysergic acid diethylamide. Concentrations of these substances are expressed in moles per liter and refer to the concentration of the salt rather than the free base. In order to determine the source of the vasoactive material found in oxy-
Vol. 45, No. 3 March, 1963
L I B E K A T I O N OF VASOACTIVE SUBSTANCES
405
TABLE I . IDENTIFICATION OF VASOACTIVE SUBSTANCES*
STIMULATING AGENT
ORIGINAL SENSITIVITY
NO. OF EXPTS.
REMAINING SENSITIVITY IN PER CENT OF ORIGINALt (MEAN AND RANGE) AFTER PYRILAMINE AFTER ( 1 0 - 6 M ) AND AFTER AFTER DIBENZYPYRILAMINE LYSERGIC ACID LINE LYSERGIC (10-6M) ACID 10-6M 10-5M 10-6M
Epiuephrine 10-sM 3 100 0 100 100 Norepinephrine 10-sM 0 3 100 100 100 Histamine 10-sM 0 3 100 0 0 Serotonin 10-sM 3 0 0 100 0 Acetylcholine 10-sM 100 0 3 100 100 Dog blood oxygenated 64 units for 3 hours V.A.M. 0 3 58(25-100) 5(1-12.5) 0 •Comparison of the effects of vasoconstrictive agents and artificially oxygenated blood on strips of ox carotid artery before and after blockade with dibenzyline, pyrilamine, and lysergic acid. tRemaining sensitivity was considered to be zero if no detectable response was obtained at a 10-4M concentration of the stimulating drug.
genated blood, plasma was separated from dog blood by centrifugation at 700 g for 30 minutes to yield a platelet-rich plasma and at 30,000 g for 30 minutes to yield a plasma free of cellular elements. In each case the plasma was recirculated through the bubble oxygenator for a 3 hour period. Samples were assayed at intervals by the ox carotid strip preparation for vasoactive substances. The Rabbit Bowel Preparation.—This preparation has been previously described by Thai 3 and consists, in brief, of a saline muscle bath containing a segment of rabbit duodenum or jejunum connected to a strain gauge system so that the intraluminal pressure of the segment can be monitored. Plasma was separated from blood by centrifugation at 700 g for 30 minutes, before and after the blood had been recirculated through the bubble oxygenator for 3 hours. Each of the two samples of plasma was then added in turn to the muscle bath and the contractile response recorded. RESULTS
1. Increase in Vascular Resistance of Isolated Dog Kidneys Perfused With Blood From the Bubble Oxygenator. The studies of Halley 4 indicate that there is an increase in renal vascular resistance during cardiopulmonary bypass. To elucidate the mechanisms responsible for this change in renal blood flow the following experiment was performed. Canine kidneys were perfused alternately with blood from a bubble oxygenator and, as a control, blood from the femoral artery of a donor dog. Blood flow through the kidney under each circumstance was measured and renal vascular resistance calculated. Five identical experiments were performed and the results of a typical experiment are illustrated in Fig. 2. It is evident that the isolated kidney developed a progressive increase in vascular resistance when perfused with blood from the bubble oxygenator as the period of oxygenation increased. In this experiment the vascular resistance to the blood from the oxygenator in-
J. Thoracic and Cardiovas. Surg.
HOLLENBERG, PRTJETT, T H A L
406
creased approximately fourfold after 3y 2 hours of constant bubble oxygenation. On the other hand, during alternate intervals when the same kidney was perfused with blood directly from the femoral artery of a donor dog, the resistance fell promptly to a low and stable level. The results of the other four experiments in this group were similar. 2. Red Cell Aggregation as a Possible Cause of Increase in Renal Vascular Resistance. Since the increase in resistance may have been due to the aggregation of red cells in the renal vascular bed during perfusion,5 another series of experiments was carried out in which isolated dog kidneys were perfused directly by separated plasma which had been recirculated through the bubble oxygenator. As in the previous experiment, renal vascular resistance was calculated.
■ □
BLOOD FROM DONOR
I 12 x
BLOOD FROM OXYGENATOR
10
E" E
en
i
20
40
60 80 100 120 140 TIME (MINUTES)
160
180
200
220
F i g . 2.—Graph w h i c h c o m p a r e s t h e v a s c u l a r r e s i s t a n c e of t h e i s o l a t e d c a n i n e k i d n e y to blood flow from t h e bubble o x y g e n a t o r a n d flow from t h e f e m o r a l a r t e r y of a d o n o r dog.
The results in each of the five experiments in this group demonstrated a progressive rise in renal vascular resistance as the period of oxygenation increased. Thus it appears that red cell aggregation is not an important factor bringing about the demonstrated rise in resistance. 3. Liberation of Vasoactive Substances as a Possible Cause of Increase in Renal Vascular Resistance. The findings reported above suggested that substances were liberated during bubble oxygenation which produced a vasoconstriction of the renal arteries and their branches which yielded an increase in renal vascular resistance. Therefore, a search was made for the presence of vasoactive substances in pumpoxygenated blood. As described above, dog blood was recirculated through a bubble oxygenator for periods up to 4 hours and, at intervals during oxygenation, samples were taken and assayed quantitatively for vasoactive substances with the use of the ox carotid strip preparation. The results of a typical experi-
Vol. 45, No. 3 March, 1963
LIBERATION OF VASOACTIVE SUBSTANCES
407
ment are illustrated in Fig. 3. I t can be seen that there is a large and progressive increase in circulating vasoactive material as the period of recirculation increases. During the kidney perfusion experiments described above, a similar progressive increase in vasoactive material was detected in the perfusate from the bubble oxygenator. The rise in level of vasoactive material paralleled the rise in renal vascular resistance. 4. Identification of the Vasoactive Bubble Oxygenation.
Substances
Liberated
by
Prolonged
a. Response of the ox carotid artery strips: Previous studies by others have shown that the vasoactive substances may be broadly differentiated by the character of the smooth muscle response. It was found that artificially oxygenated blood consistently produced an immediate and rapid contraction of the bovine artery preparation. This response was similar to that elicited by a group of sub-
180
160
I 140 120
5 <
IOO
I 80 °en 60 z
40 20 1
A,
I
I
I
'
'
I
0 15 30 45 60 75 90 105 120 135 150 165180 195210 TIME OF PUMP OXYGENATION (MINUTES)
Fig;. 3.—Graph which depicts the rise in level of vasoactive material in heparinized dog blood recirculated through a bubble oxygenator.
stances among which are epinephrine, norepinephrine, serotonin, histamine, and angiotensin, but, on the other hand, different from that elicited by substances of the slow reacting type, such as bradykinin. b. Identification by means of specific blocking agents: When Furchgott's technique 2 was applied to the carotid artery preparation it was found that dibenzyline blocked the response of the strips to epinephrine, norepinephrine, histamine, serotonin, and acetylcholine but not to the polypeptides, angiotensin, and bradykinin. These results are presented in Table I. It can be seen that pyrilamine was a selective histamine antagonist, whereas D-lysergic acid blocked only serotonin. The activity of bubble-oxygenated blood was eliminated by
408
HOIXENBERG, PRUETT, THAL
J. Thoracic and Cardiovas. Surg.
dibenzyline and the latter two agents in combination. Taken together, these studies suggest that serotonin and histamine are the vasoactive agents liberated during bubble oxygenation. c. Response of rabbit jejunum to bubble-oxygenated blood: Control studies demonstrated that rabbit jejunal segments prepared in the manner described would maintain regular peristaltic activity for periods exceeding 60 minutes. It can be seen from Pig. 4 that this activity did not change when plasma removed from control heparinized blood was added to the bathing fluid. However, follow-
Reoction Of Rabbit JejunumTo Normal Dog Plasma
Reaction Of Rabbit JejunumTo Oxygenated Dog Plasma
Fig-4.—Record of peristaltic response of a rabbit jejunal segment to plasma removed from control dog blood and to plasma removed from blood previously recirculated through the bubble oxygenator.
ing the addition of plasma from blood which had previously been recirculated through the bubble oxygenator, the activity of the segment immediately increased to the point of hyperperistalsis. This result supports the impression that histamine and serotonin are released during bubble oxygenation and, in large measure, denies the possibility that adrenergic agents are present in increased quantities since catecholamines characteristically produce depression of peristalsis of the rabbit bowel segment. 5. Source of the Vasoactive Material Released. Since vasoactive substances are liberated simply by recirculation of blood through the pump oxygenator, the possibilities as to the source of the material released are limited to the constituents of blood itself, the anticoagulant heparin, and the materials used in preparing the oxygenator, such as the antifoam, Mayon tubing, etc. Platelets which are known to contain large amounts of serotonin8 formed the most likely source of the active material released. For this reason large quantities of platelet-rich and platelet-free plasma were prepared as described above and recirculated through the pump oxygenator for 3 hours. At intervals, samples were assayed quantitatively for vasoactive materials by the ox carotid strip preparation. The results showed that the vasoactivity of plasma
LIBERATION OF VASOACTIVE SUBSTANCES
Vol. 45, No 3 March, 1963
409
free of platelets did not increase from the control value of 4 units during pump oxygenation, whereas that of the platelet-rich plasma increased sixteen-fold, from 4 to 64 units. Thus it appears that the platelet fraction of blood is the source of a large amount of the vasoactive material released. 6. Liberation of Vasoactive Materials During Cardiopulmonary Bypass in Dogs and Man. A separate series of experiments was undertaken to determine the effect of a dog in the circuit on the rising levels of vasoactive material. Seven dogs were perfused by the bubble oxygenator under varying conditions. Three of the animals were perfused at low flow rates under hypothermic conditions according to the method described by Zuhdi. 7 The others were perfused under normothermic VASOACTIVITY of HYPOTHERMIC and NORMOTHERMIC TWO HOUR PERFUSIONS 128 70 60 VASOACTIVE MATERIAL (units)
50 40 30 20 10 -
LU
ill
FLOW RATE ac/lcg/min
.27
25
25
40
45
40
35
RECTAL TEMP.
29
29
28
37
37
37
37
C°
X Assay at end of 3 hour period
Pig. 5.—Graph which compares the levels of vasoactive material in the blood of dogs following extracorporeal circulation under hypothermic and normothermic conditions.
conditions at higher flow rates using the DeWall-Lillehei oxygenator. 8 I t will be seen from Pig. 5 that the rise in level of vasoactive material persisted in each case. However, it is evident that cardiopulmonary bypass under hypothermic conditions and lower flow rates liberates a much smaller quantity of vasoactive material. These results may be due to the fact that the blood undergoes less trauma when circulating through the oxygenator at lower flow rates. Finally, blood removed from the oxygenator following human perfusions, when assayed by the ox carotid strip, also has been found to contain abnormally high levels of vasoactive materials which again are proportional to the duration of the bypass. DISCUSSION
The experiments described in the present investigation present evidence that serotonin and histamine are among the vasoactive substances liberated during recirculation of dog blood through a bubble-type pump oxygenator. Dog platelets and leukocytes appear to be the source of these active substances since they con-
HOLLENBERG, P R U E T T , T H A L
410
J. Thoracic and Cardiovas. Surg.
tain both histamine and serotonin 6 and are destroyed in large numbers during extracorporeal circulation. 9 Histamine and serotonin are similar in that they may induce severe bronchoconstriction and exert a hypotensive effect following intravenous injection.10- " Serotonin has been shown to increase the vascular resistance of canine kidneys. 12 The experiments of Sir Henry Dale 13 have demonstrated that histamine by its vasoconstrictor action on the branches of the efferent hepatic veins in the dog tends to produce passive congestion of blood in the liver and splanchnic system. This phenomenon is frequently observed during prolonged canine bypass procedures. Prom the foregoing information it appears likely that the liberation of these highly vasoactive substances is responsible in part for the complications of hepatic and portal venous congestion, renal failure, atelectasis, and hypotension which not infreqently follow prolonged bypass procedures with the pump oxygenator in dogs and man. In addition, other changes in the composition of the circulating blood which occur during cardiopulmonary bypass, such as hemolysis of red cells with liberation of adenosine compounds,9 increase in acid metabolites,14 and denaturation of protein, 15 may be implicated in the pathogenesis of these complications. SUMMARY
The present study describes the liberation of vasoactive substances during prolonged recirculation of dog blood through a bubble oxygenator. The release of these active substances may be important in the pathogenesis of renal failure, atelectasis, and hypotension following cardiopulmonary bypass with the bubble oxygenator. In investigating this problem the following observations were made. 1. Heparinized dog blood recirculated through a bubble oxygenator for prolonged periods increases vascular resistance when perfused through isolated canine kidneys. Removal of red cells from the perfusate does not alter this response. 2. Artificially oxygenated dog blood induces marked smooth muscle spasm of the isolated ox carotid artery strip preparation. This response is blocked by dibenzyline and by pyrilamine and D-lysergic acid in combination, suggesting that serotonin and histamine are the substances liberated. 3. Plasma from dog blood oxygenated in this manner produces an immediate hyperperistalsis of segments of rabbit jejunum. 4. Recirculation of platelet-rich plasma through the pump oxygenator liberates vasoactive material. On the other hand, recirculation of platelet-free plasma does not. 5. Blood removed from the oxygenator following extracorporeal circulation in dog and man contains abnormally high levels of vasoactive materials. 6. Hypothermic perfusions at lower flow rates liberate less vasoactive material than normothermic perfusions at higher flow rates. The probability that the release of vasoactive substances explains some of the complications following prolonged cardiopulmonary bypass with the bubble oxygenator is discussed.
Vol. 45, No. 3 March, 1963
L I B E R A T I O N O F VASOACTIVE SUBSTANCES
411 *
x x
REFERENCES 1. Janeway, T. 0., Richardson, H . B., and Park, E . A.: Experiments on the Vasoconstrictor Action of Blood Serum, Arch. I n t . Med. 2 1 : 565, 1918. 2. Furchgott, R. F . : Dibenamine Blockade in Strips of Rabbit Aorta and I t s Use in Differentiating Receptors, J . Pharmacol. I l l : 265, 1954. 3. Thai, A. P., and Egner, W.: The Site of Action of the Staphylococcus Alpha Toxin, J . Exper. Med. 113: 67, 1961. 4. Halley, M., Reemtsma, K. and Creech, O., J r . : Hemodynamics and Metabolism of Individual Organs During Extracorporeal Circulation, Surgery 46: 1129, 1959. 5. Read, R. C , and Meyer, M.: The Role of Bed Cell Agglutination in Arteriographic Complications, S. Forum 10: 472, 1960. 6. Humphrey, I. H., and Jaques, R.: The Histamine and Serotonin Content of the Platelets and Polymorphonuclear Leukocytes of "Various Species, J . Physiol. 124: 305, 1954. 7. Zuhdi, N., McCollough, B., Carey, I., and Greer, A.: Double-Helical Reservoir HeartLung Machine, A. M. A. Arch. Surg. 82: 320, 1961. 8. DeWall, R. A., Warden, E . H., and Lillehei, C. W . : The Helix Reservoir Bubble Oxygenator and I t s Clinical Application in Extracorporeal Circulation, edited by J . G. Allen, Springfield, 111., 1958, Charles C Thomas, Publisher, p. 41. 9. DeWall, R. A., Long, D. M., Gemmill, S. H., and Lillehei, C. W.: Certain Blood Changes in Patients Undergoing Extracorporeal Circulation, J . THORACIC SURG. 37: 325, 1959. 10. Dews, P . B . : 5-Hydroxytryptamine, Effect on Organ Systems in Pharmacology in Medicine, edited by V. A. Drill, New York, 1958, McGraw-Hill Book Company, Inc., p. 316. 11. Dragstedt, C. A . : Histamine and Antihistaminics, Effects on Organ Systems in Pharmacology in Medicine, edited by V. A. Drill, New York, 1958, McGraw-Hill Book Company, Inc., p. 621. 12. Emanuel, D. A., Scott, J., Collins, R., and Haddy, F . J . : Local Effect of Serotonin in Renal "Vascular Resistance and Urine Flow Rate, Am. J . Physiol. 196: 1122, 1959. 13. Dale, H. H . : Chemical Factors in Control of Circulation, Lancet 1: 1179, 1929. 14. Paneth, M., Sellers, R., Gott, V., Weirich, W., Allen, P., Read, R. C , and Lillehei, C. W.: Physiologic Studies Upon Prolonged Cardiopulmonary Bypass With the Pump Oxygenator With Particular Reference to (1) Acid-base Balance, (2) Siphon Caval Drainage, J .
THORACIC SDRG. 34:
570,
1957.
15. Lee, W. H., Jr., and Maloney, J . V., J r . : Comparison of the Effects of Membrane and Non-Membrane Oxygenators on the Biochemical and Biophysical Characteristics of Blood, S. Forum 12: 200, 1961.
OXYGENATION
Martin Hollenberg,
Robert Pruett,
M.D.*
and Alan Thai, M.D., Detroit,
BY
B.S.,
Mich.
T
HE bubble oxygenator has become widely accepted as a valuable tool in cardiovascular surgery, however, hypotension, pulmonary atelectasis, and renal failure may complicate prolonged cardiopulmonary bypass in man and dog. The aim of the present work was to measure and identify vasoactive substances set free as a result of bubble oxygenation of blood in an attempt to explain some of these complications. MATERIALS AND METHODS
Experiments were designed to study the effect of blood, which had previously been recirculated through a bubble oxygenator, on three biological assay preparations. The following studies were made. (1) The effect of pump-oxygenated blood on the vascular resistance of isolated canine kidneys. (2) The contractile response of strips of ox carotid artery to blood before and after bubble oxygenation. (3) The response of isolated segments of rabbit bowel to plasma from control blood and blood which had undergone prolonged pump oxygenation. Animals.—Adult mongrel dogs, weighing 10 to 30 kilograms, and marketbred albino rabbits, weighing 2 to 3 kilograms, were used. Collection and Oxygenation of Blood.—Dog blood obtained under intravenous Nembutal anesthesia (30 mg. per kilogram of body weight) by exsanguination through a polyethylene catheter placed in either the femoral or carotid arteries was collected in siliconized flasks containing 5 mg. per cent of heparin. The heparinized blood was then recirculated by an occlusive Sigmamotor pump (Model M-2) at a flow rate of 300 to 400 c.c. per minute through a bubble oxygenator for periods up to 4 hours. The oxygen flow was 6 L. per minute. The oxygenator circuit was composed of Mayon tubing containing 3 stainless steel sponges coated with Dow Corning antifoam. In most experiments, clean, but not sterile, technique was used in preparing the oxygenator. Mich.
From the Department of Surgery, Wayne State University College of Medicine, Detroit,
Supported by U. S. Public Health Service Grant No. H-6842 and a grant from the Detroit Receiving Hospital Research Corporation. Received for publication May 16, 1962. •American Heart Association Research Fellow. 402
Vol. 45, No. 3 March, 1963
L I B E R A T I O N O F VASOACTIVE SUBSTANCES
403
BUBBLE OXYGENATOR
TO BUBBLE OXYGENATOR
TO JUGULAR VEIN
Fig. 1.—Apparatus used for perfusion of the isolated canine kidney. The kidney is perfused at constant pressure and temperature alternately by blood from the oxygenator and blood issuing directly from the femoral artery of a donor dog.
The Isolated Kidney Preparation.—Kidneys, weighing 100 to 400 grams, were removed from healthy adult mongrel dogs under Nembutal anesthesia. A segment of aorta, 4 cm. in length, adjoining the renal artery was removed with each kidney, but the renal veins were excised to aid venous drainage. Following removal, the distal end of the aorta was ligated and a cannula connected to the perfusion apparatus was inserted into its proximal end. The kidneys were then immediately perfused at 37° C. and at a constant pressure of 120 mm. Hg alternately by blood from the oxygenator and blood issuing directly from a catheter in the femoral artery of a donor dog. The period of transfer during which the kidneys were without blood supply did not exceed 3 minutes. The perfusion apparatus is illustrated in Fig. 1. Blood issuing from the perfused kidneys was collected by a plastic funnel and measured with a graduated cylinder. Renal vascular resistance was calculated as the ratio of pressure difference to flow. In order to determine the importance of red cell aggregation as a cause of increased renal vascular resistance, a separate series of experiments was carried
404
HOLLENBERG, P R U E T T , T H A L
J. Thoracic and Cardiovas. Surg.
out in which plasma separated by centrifugation at 700 g for 30 minutes from the red cells of heparinized dog blood was recirculated through the bubble oxygenator. Five isolated canine kidneys were directly perfused by plasma prepared in this manner and vascular resistance was again calculated. The Ox Carotid Artery Strip Preparation.—The use of this preparation for assay of vasoactive materials has been previously described by Janeway. 1 Carotid arteries, removed from steers immediately after slaughter, were cleaned of adventitia and stored in Krebs solution at pH 7.4 to 7.6 and at 6° C. until use. Circular segments of the arteries were excised and then divided into strips measuring 3 cm. by 1 cm. by 0.2 mm. The strips were attached by a stainless steel clip to a hook at the bottom of a 20 c.c. bath of Krebs solution and were suspended at room temperature. A mixture of 95 per cent oxygen and 5 per cent carbon dioxide was slowly bubbled through the bath. Care was taken to insure that the pH of the Krebs solution did not vary from the above limits. Tension of 80 Gm. for 20 minutes was applied to the artery segments and then, by means of a slender metal wire, the strips were attached to the vertical armature of a Statham myograph at a tension of 0.4 Gm. After a period of 15 to 20 minutes, required for shortening of the strips, they were then ready for use. The sample of blood to be assayed for vasoactive material was added to the muscle bath and the response of the smooth muscle preparation was monitored by a Sanborn recording apparatus. A measure of the quantity of vasoactive material present in a sample of heparinized blood was obtained by diluting the sample until an increase in tension of the artery strip of 0.2 Gm. could no longer be elicited. A unit of vasoactive material (VAM) was thus obtained and is defined as the reciprocal of the highest dilution of blood which excites an increase in tension of 0.2 Gm. Normal values for both dog and human blood assayed by this technique did not exceed 16 units. The sensitivity of this preparation was such that it would consistently detect a wide range of vasoactive substances in minute concentration; that is, epinephrine in dilutions of 10"9 M. In addition, the response of different strips to the same dilution of epinephrine was quite consistent. The preparation was used to estimate the quantity of vasoactive material present in samples of blood removed from the pump oxygenator at intervals during recirculation. has described the effects Identification of Vasoactive Agents.—Furchgott2 of blocking agents on the contractile response of spiral strips of rabbit aorta to selected vasoactive substances. This technique was applied to the ox carotid artery preparation as a method of identifying the vasoactive agents released during bubble oxygenation. I n separate experiments (Table I) the vasoactive agents 1-epinephrine hydroehloride, 1-norepinephrine bitartrate, serotonin ereatinine sulfate, histamine dihydrochloride, and acetylcholine chloride were assayed on strips blocked by the addition to the muscle bath for a 20 minute period of dibenzyline hydroehloride, pyrilamine maleate, and D-lysergic acid diethylamide. Concentrations of these substances are expressed in moles per liter and refer to the concentration of the salt rather than the free base. In order to determine the source of the vasoactive material found in oxy-
Vol. 45, No. 3 March, 1963
L I B E K A T I O N OF VASOACTIVE SUBSTANCES
405
TABLE I . IDENTIFICATION OF VASOACTIVE SUBSTANCES*
STIMULATING AGENT
ORIGINAL SENSITIVITY
NO. OF EXPTS.
REMAINING SENSITIVITY IN PER CENT OF ORIGINALt (MEAN AND RANGE) AFTER PYRILAMINE AFTER ( 1 0 - 6 M ) AND AFTER AFTER DIBENZYPYRILAMINE LYSERGIC ACID LINE LYSERGIC (10-6M) ACID 10-6M 10-5M 10-6M
Epiuephrine 10-sM 3 100 0 100 100 Norepinephrine 10-sM 0 3 100 100 100 Histamine 10-sM 0 3 100 0 0 Serotonin 10-sM 3 0 0 100 0 Acetylcholine 10-sM 100 0 3 100 100 Dog blood oxygenated 64 units for 3 hours V.A.M. 0 3 58(25-100) 5(1-12.5) 0 •Comparison of the effects of vasoconstrictive agents and artificially oxygenated blood on strips of ox carotid artery before and after blockade with dibenzyline, pyrilamine, and lysergic acid. tRemaining sensitivity was considered to be zero if no detectable response was obtained at a 10-4M concentration of the stimulating drug.
genated blood, plasma was separated from dog blood by centrifugation at 700 g for 30 minutes to yield a platelet-rich plasma and at 30,000 g for 30 minutes to yield a plasma free of cellular elements. In each case the plasma was recirculated through the bubble oxygenator for a 3 hour period. Samples were assayed at intervals by the ox carotid strip preparation for vasoactive substances. The Rabbit Bowel Preparation.—This preparation has been previously described by Thai 3 and consists, in brief, of a saline muscle bath containing a segment of rabbit duodenum or jejunum connected to a strain gauge system so that the intraluminal pressure of the segment can be monitored. Plasma was separated from blood by centrifugation at 700 g for 30 minutes, before and after the blood had been recirculated through the bubble oxygenator for 3 hours. Each of the two samples of plasma was then added in turn to the muscle bath and the contractile response recorded. RESULTS
1. Increase in Vascular Resistance of Isolated Dog Kidneys Perfused With Blood From the Bubble Oxygenator. The studies of Halley 4 indicate that there is an increase in renal vascular resistance during cardiopulmonary bypass. To elucidate the mechanisms responsible for this change in renal blood flow the following experiment was performed. Canine kidneys were perfused alternately with blood from a bubble oxygenator and, as a control, blood from the femoral artery of a donor dog. Blood flow through the kidney under each circumstance was measured and renal vascular resistance calculated. Five identical experiments were performed and the results of a typical experiment are illustrated in Fig. 2. It is evident that the isolated kidney developed a progressive increase in vascular resistance when perfused with blood from the bubble oxygenator as the period of oxygenation increased. In this experiment the vascular resistance to the blood from the oxygenator in-
J. Thoracic and Cardiovas. Surg.
HOLLENBERG, PRTJETT, T H A L
406
creased approximately fourfold after 3y 2 hours of constant bubble oxygenation. On the other hand, during alternate intervals when the same kidney was perfused with blood directly from the femoral artery of a donor dog, the resistance fell promptly to a low and stable level. The results of the other four experiments in this group were similar. 2. Red Cell Aggregation as a Possible Cause of Increase in Renal Vascular Resistance. Since the increase in resistance may have been due to the aggregation of red cells in the renal vascular bed during perfusion,5 another series of experiments was carried out in which isolated dog kidneys were perfused directly by separated plasma which had been recirculated through the bubble oxygenator. As in the previous experiment, renal vascular resistance was calculated.
■ □
BLOOD FROM DONOR
I 12 x
BLOOD FROM OXYGENATOR
10
E" E
en
i
20
40
60 80 100 120 140 TIME (MINUTES)
160
180
200
220
F i g . 2.—Graph w h i c h c o m p a r e s t h e v a s c u l a r r e s i s t a n c e of t h e i s o l a t e d c a n i n e k i d n e y to blood flow from t h e bubble o x y g e n a t o r a n d flow from t h e f e m o r a l a r t e r y of a d o n o r dog.
The results in each of the five experiments in this group demonstrated a progressive rise in renal vascular resistance as the period of oxygenation increased. Thus it appears that red cell aggregation is not an important factor bringing about the demonstrated rise in resistance. 3. Liberation of Vasoactive Substances as a Possible Cause of Increase in Renal Vascular Resistance. The findings reported above suggested that substances were liberated during bubble oxygenation which produced a vasoconstriction of the renal arteries and their branches which yielded an increase in renal vascular resistance. Therefore, a search was made for the presence of vasoactive substances in pumpoxygenated blood. As described above, dog blood was recirculated through a bubble oxygenator for periods up to 4 hours and, at intervals during oxygenation, samples were taken and assayed quantitatively for vasoactive substances with the use of the ox carotid strip preparation. The results of a typical experi-
Vol. 45, No. 3 March, 1963
LIBERATION OF VASOACTIVE SUBSTANCES
407
ment are illustrated in Fig. 3. I t can be seen that there is a large and progressive increase in circulating vasoactive material as the period of recirculation increases. During the kidney perfusion experiments described above, a similar progressive increase in vasoactive material was detected in the perfusate from the bubble oxygenator. The rise in level of vasoactive material paralleled the rise in renal vascular resistance. 4. Identification of the Vasoactive Bubble Oxygenation.
Substances
Liberated
by
Prolonged
a. Response of the ox carotid artery strips: Previous studies by others have shown that the vasoactive substances may be broadly differentiated by the character of the smooth muscle response. It was found that artificially oxygenated blood consistently produced an immediate and rapid contraction of the bovine artery preparation. This response was similar to that elicited by a group of sub-
180
160
I 140 120
5 <
IOO
I 80 °en 60 z
40 20 1
A,
I
I
I
'
'
I
0 15 30 45 60 75 90 105 120 135 150 165180 195210 TIME OF PUMP OXYGENATION (MINUTES)
Fig;. 3.—Graph which depicts the rise in level of vasoactive material in heparinized dog blood recirculated through a bubble oxygenator.
stances among which are epinephrine, norepinephrine, serotonin, histamine, and angiotensin, but, on the other hand, different from that elicited by substances of the slow reacting type, such as bradykinin. b. Identification by means of specific blocking agents: When Furchgott's technique 2 was applied to the carotid artery preparation it was found that dibenzyline blocked the response of the strips to epinephrine, norepinephrine, histamine, serotonin, and acetylcholine but not to the polypeptides, angiotensin, and bradykinin. These results are presented in Table I. It can be seen that pyrilamine was a selective histamine antagonist, whereas D-lysergic acid blocked only serotonin. The activity of bubble-oxygenated blood was eliminated by
408
HOIXENBERG, PRUETT, THAL
J. Thoracic and Cardiovas. Surg.
dibenzyline and the latter two agents in combination. Taken together, these studies suggest that serotonin and histamine are the vasoactive agents liberated during bubble oxygenation. c. Response of rabbit jejunum to bubble-oxygenated blood: Control studies demonstrated that rabbit jejunal segments prepared in the manner described would maintain regular peristaltic activity for periods exceeding 60 minutes. It can be seen from Pig. 4 that this activity did not change when plasma removed from control heparinized blood was added to the bathing fluid. However, follow-
Reoction Of Rabbit JejunumTo Normal Dog Plasma
Reaction Of Rabbit JejunumTo Oxygenated Dog Plasma
Fig-4.—Record of peristaltic response of a rabbit jejunal segment to plasma removed from control dog blood and to plasma removed from blood previously recirculated through the bubble oxygenator.
ing the addition of plasma from blood which had previously been recirculated through the bubble oxygenator, the activity of the segment immediately increased to the point of hyperperistalsis. This result supports the impression that histamine and serotonin are released during bubble oxygenation and, in large measure, denies the possibility that adrenergic agents are present in increased quantities since catecholamines characteristically produce depression of peristalsis of the rabbit bowel segment. 5. Source of the Vasoactive Material Released. Since vasoactive substances are liberated simply by recirculation of blood through the pump oxygenator, the possibilities as to the source of the material released are limited to the constituents of blood itself, the anticoagulant heparin, and the materials used in preparing the oxygenator, such as the antifoam, Mayon tubing, etc. Platelets which are known to contain large amounts of serotonin8 formed the most likely source of the active material released. For this reason large quantities of platelet-rich and platelet-free plasma were prepared as described above and recirculated through the pump oxygenator for 3 hours. At intervals, samples were assayed quantitatively for vasoactive materials by the ox carotid strip preparation. The results showed that the vasoactivity of plasma
LIBERATION OF VASOACTIVE SUBSTANCES
Vol. 45, No 3 March, 1963
409
free of platelets did not increase from the control value of 4 units during pump oxygenation, whereas that of the platelet-rich plasma increased sixteen-fold, from 4 to 64 units. Thus it appears that the platelet fraction of blood is the source of a large amount of the vasoactive material released. 6. Liberation of Vasoactive Materials During Cardiopulmonary Bypass in Dogs and Man. A separate series of experiments was undertaken to determine the effect of a dog in the circuit on the rising levels of vasoactive material. Seven dogs were perfused by the bubble oxygenator under varying conditions. Three of the animals were perfused at low flow rates under hypothermic conditions according to the method described by Zuhdi. 7 The others were perfused under normothermic VASOACTIVITY of HYPOTHERMIC and NORMOTHERMIC TWO HOUR PERFUSIONS 128 70 60 VASOACTIVE MATERIAL (units)
50 40 30 20 10 -
LU
ill
FLOW RATE ac/lcg/min
.27
25
25
40
45
40
35
RECTAL TEMP.
29
29
28
37
37
37
37
C°
X Assay at end of 3 hour period
Pig. 5.—Graph which compares the levels of vasoactive material in the blood of dogs following extracorporeal circulation under hypothermic and normothermic conditions.
conditions at higher flow rates using the DeWall-Lillehei oxygenator. 8 I t will be seen from Pig. 5 that the rise in level of vasoactive material persisted in each case. However, it is evident that cardiopulmonary bypass under hypothermic conditions and lower flow rates liberates a much smaller quantity of vasoactive material. These results may be due to the fact that the blood undergoes less trauma when circulating through the oxygenator at lower flow rates. Finally, blood removed from the oxygenator following human perfusions, when assayed by the ox carotid strip, also has been found to contain abnormally high levels of vasoactive materials which again are proportional to the duration of the bypass. DISCUSSION
The experiments described in the present investigation present evidence that serotonin and histamine are among the vasoactive substances liberated during recirculation of dog blood through a bubble-type pump oxygenator. Dog platelets and leukocytes appear to be the source of these active substances since they con-
HOLLENBERG, P R U E T T , T H A L
410
J. Thoracic and Cardiovas. Surg.
tain both histamine and serotonin 6 and are destroyed in large numbers during extracorporeal circulation. 9 Histamine and serotonin are similar in that they may induce severe bronchoconstriction and exert a hypotensive effect following intravenous injection.10- " Serotonin has been shown to increase the vascular resistance of canine kidneys. 12 The experiments of Sir Henry Dale 13 have demonstrated that histamine by its vasoconstrictor action on the branches of the efferent hepatic veins in the dog tends to produce passive congestion of blood in the liver and splanchnic system. This phenomenon is frequently observed during prolonged canine bypass procedures. Prom the foregoing information it appears likely that the liberation of these highly vasoactive substances is responsible in part for the complications of hepatic and portal venous congestion, renal failure, atelectasis, and hypotension which not infreqently follow prolonged bypass procedures with the pump oxygenator in dogs and man. In addition, other changes in the composition of the circulating blood which occur during cardiopulmonary bypass, such as hemolysis of red cells with liberation of adenosine compounds,9 increase in acid metabolites,14 and denaturation of protein, 15 may be implicated in the pathogenesis of these complications. SUMMARY
The present study describes the liberation of vasoactive substances during prolonged recirculation of dog blood through a bubble oxygenator. The release of these active substances may be important in the pathogenesis of renal failure, atelectasis, and hypotension following cardiopulmonary bypass with the bubble oxygenator. In investigating this problem the following observations were made. 1. Heparinized dog blood recirculated through a bubble oxygenator for prolonged periods increases vascular resistance when perfused through isolated canine kidneys. Removal of red cells from the perfusate does not alter this response. 2. Artificially oxygenated dog blood induces marked smooth muscle spasm of the isolated ox carotid artery strip preparation. This response is blocked by dibenzyline and by pyrilamine and D-lysergic acid in combination, suggesting that serotonin and histamine are the substances liberated. 3. Plasma from dog blood oxygenated in this manner produces an immediate hyperperistalsis of segments of rabbit jejunum. 4. Recirculation of platelet-rich plasma through the pump oxygenator liberates vasoactive material. On the other hand, recirculation of platelet-free plasma does not. 5. Blood removed from the oxygenator following extracorporeal circulation in dog and man contains abnormally high levels of vasoactive materials. 6. Hypothermic perfusions at lower flow rates liberate less vasoactive material than normothermic perfusions at higher flow rates. The probability that the release of vasoactive substances explains some of the complications following prolonged cardiopulmonary bypass with the bubble oxygenator is discussed.
Vol. 45, No. 3 March, 1963
L I B E R A T I O N O F VASOACTIVE SUBSTANCES
411 *
x x
REFERENCES 1. Janeway, T. 0., Richardson, H . B., and Park, E . A.: Experiments on the Vasoconstrictor Action of Blood Serum, Arch. I n t . Med. 2 1 : 565, 1918. 2. Furchgott, R. F . : Dibenamine Blockade in Strips of Rabbit Aorta and I t s Use in Differentiating Receptors, J . Pharmacol. I l l : 265, 1954. 3. Thai, A. P., and Egner, W.: The Site of Action of the Staphylococcus Alpha Toxin, J . Exper. Med. 113: 67, 1961. 4. Halley, M., Reemtsma, K. and Creech, O., J r . : Hemodynamics and Metabolism of Individual Organs During Extracorporeal Circulation, Surgery 46: 1129, 1959. 5. Read, R. C , and Meyer, M.: The Role of Bed Cell Agglutination in Arteriographic Complications, S. Forum 10: 472, 1960. 6. Humphrey, I. H., and Jaques, R.: The Histamine and Serotonin Content of the Platelets and Polymorphonuclear Leukocytes of "Various Species, J . Physiol. 124: 305, 1954. 7. Zuhdi, N., McCollough, B., Carey, I., and Greer, A.: Double-Helical Reservoir HeartLung Machine, A. M. A. Arch. Surg. 82: 320, 1961. 8. DeWall, R. A., Warden, E . H., and Lillehei, C. W . : The Helix Reservoir Bubble Oxygenator and I t s Clinical Application in Extracorporeal Circulation, edited by J . G. Allen, Springfield, 111., 1958, Charles C Thomas, Publisher, p. 41. 9. DeWall, R. A., Long, D. M., Gemmill, S. H., and Lillehei, C. W.: Certain Blood Changes in Patients Undergoing Extracorporeal Circulation, J . THORACIC SURG. 37: 325, 1959. 10. Dews, P . B . : 5-Hydroxytryptamine, Effect on Organ Systems in Pharmacology in Medicine, edited by V. A. Drill, New York, 1958, McGraw-Hill Book Company, Inc., p. 316. 11. Dragstedt, C. A . : Histamine and Antihistaminics, Effects on Organ Systems in Pharmacology in Medicine, edited by V. A. Drill, New York, 1958, McGraw-Hill Book Company, Inc., p. 621. 12. Emanuel, D. A., Scott, J., Collins, R., and Haddy, F . J . : Local Effect of Serotonin in Renal "Vascular Resistance and Urine Flow Rate, Am. J . Physiol. 196: 1122, 1959. 13. Dale, H. H . : Chemical Factors in Control of Circulation, Lancet 1: 1179, 1929. 14. Paneth, M., Sellers, R., Gott, V., Weirich, W., Allen, P., Read, R. C , and Lillehei, C. W.: Physiologic Studies Upon Prolonged Cardiopulmonary Bypass With the Pump Oxygenator With Particular Reference to (1) Acid-base Balance, (2) Siphon Caval Drainage, J .
THORACIC SDRG. 34:
570,
1957.
15. Lee, W. H., Jr., and Maloney, J . V., J r . : Comparison of the Effects of Membrane and Non-Membrane Oxygenators on the Biochemical and Biophysical Characteristics of Blood, S. Forum 12: 200, 1961.