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In vitro study of hormone degradation by heart-lung machine with bubble oxygenator
69
Resuecitation, ll(1964) 69-77 Elsevier Scientific Publishers Ireland Ltd.
IN VITRO STUDY OF HORMONE DEGRADATION BY HEART-LUNG MACHINE WITH BUBBLE OXYGENATOR
J. MALATINSKP, M. VIGAEjb, D. VGANSKP”, and DANA JEiOVAb
R. KVETrjANSKkb,
JANA JLJR~O&OVAb
“Postgraduate Medical School, Kramdre, Limbovd 5, 633 03 Bratislava and bInstitute of Experimental Endocrinology, Slovak Academy of Sciences, Kmmcire, 809 36, Bmtislava (Czechoslovakia) (Received February
34
1963)
SUMMARY
In the present experiment with heart-lung machine set in a closed system in vitro, the blood containing increased levels of hormones was circulated at 30°C for 90 min and at 37°C for 30min; a fraction of the priming perfusate was studied in parallel at the same temperatures in an incubator. The level of growth hormone decreased gradually to a mean of 76% at 30°C and no further decrease was found at 37°C. The mean insulin level fell within 30 min to 32% and no substantial further changes were observed during the remaining period of study; re-warming failed to produce significant additional changes. Cortisol did not change appreciably. However, the oxygenator altered the level of catecholamines markedly. At 30°C the initial level of noradrenaline fell precipitously to 9% within 30 min and this excessively low level was sustained throughout the study. Adrenaline showed even more pronounced changes. Denaturation of dopamine was less marked, falling to about 70% within 30 min. The present experiment revealed that the levels of hormones respond differently to artificial oxygenation during extracorporeal circulation. Cortisol resisted degradation by the oxygenator, while growth hormone and insulin were denatured significantly. The moderate degradation of growth hormone by the machine may not play an important role during open heart surgery. However, a marked oxidation denaturation of catecholamines by 90% and 70% and denaturation of insulin by 70%, might prove relevant during surgery.
Addtess
all correspondence: Dr. Jozef Malatinskjl,
Hospital,
KramBre, 633 05 Bratislava,
CSc.,
Postgrad.
Czechoslovakia.
0300-9572/64/$03.00 @ 1964 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland Ltd.
Medical
School,
Derer’s
70 INTRODUCTION
During its passage through the heart-lung machine for open heart surgery, biologically active substituents of blood may be damaged by chemical or mechanical factors. Chemical impairment may result from toxic substances leaching out of plastic tubing (Duke and Vane, 1968) or from the residual effects of improper sterilization. Mechanical damage is the result of rough surfaces in the inside of the circuit, or of turbulent flow at sites where flow velocity and pressure gradients are high (Clement, 1971). This model experiment with the standard equipment used routinely for clinical purposes of open heart surgery was designed to ascertain, whether or not inherent physical and chemical factors interfere significantly with the integrity of some hormones and, therefore, whether the concentrations measured during open heart surgery are influenced appreciably by the performance of heart-lung machine. By analogy with chemical and mechanical damage to other constituents of circulating blood (Lee, Krumhaar, Fonkalsrud, Schjeide and Manney, 19611, certain physico-chemical degradation processes and/or physical adsorption were expected to occur in the apparatus. In particular, partial destruction was expected with hormones exposed to the effects of a higher oxygen tension, while the blood is passing through the bubble oxygenator. However, of great interest were also possible specific differences in the artificially produced reduction of individual hormone levels (growth hormone, insulin, cortisol, catecholamines and thyroxine).
METHODS
Extracorporeal circulation circuit For the present model experiment, the routinely used circuit was employed comprising a modification of Lillehei-De Wall apparatus Premacard 2 produced by Prema, in conjunction with the disposable Bentley Temptrol Adult Type Q-100 bubble oxygenator. The blood drained from the superior and inferior venae cavae, owing to gravitational force, flows via the venous line into the oxygenator placed 60 cm below the level of the right atrium. The oxygenated blood then passes into the reservoir of arterial blood with changeable level of its position. By elevating or depressing the level of the reservoir, an optimal amount of blood is maintained in the oxygenator. A 46 ~_~rn microemboli blood filter is inserted in the arterial reservoir before the line reaches the arterial pump. The two arterial membrane pumps pump the blood from the apparatus into the patient (arterial line). The machine is equipped with a heat exchanger for thermoregulation of the blood. When setting the heart-lung machine into motion, the arterial pumps are first switched on and afterwards the clamps from venae cavae are removed. A reverse procedure is used at the completion of the perfusion. Oxygen is
71 delivered into the oxygenator in a quantity equal to three to four times the minute output of the pump. The standard’ machine Premacard 2 (Fig. la) is equipped with two membrane pumps, connected together in parallel; these are designed so that at the same time as one of them is expelling, the other is sucking in blood, and vice versa. This results in a continuous blood flow from the arterial
-lVC
fig. 1. Schematicdrawing of the heart-lung machine: (a) used for open heart surgery; (b) employed for the experiment as a closed system; (A + B) Oxygenator Temptrol Q-100 adult type (produced by Bentley Lab. Inc.); (A) Oxygenator consisting of oxygenation and defoaming parts, and arterial 40 pm blood filter; (B) Arterial reservoir with integral heat exchanger; (C) Two membrane pumps; (D)Watertemperatureregulationsupply;03)Arterial cannula; 0 Arterial blood sampling outlet; (G) Venous blood sampling outlet; 0 Coronary suction pump; (I) Coronary suction; (J) Sensor for measuring arterial pre~eure; (K) Reservoir and coronary suction filter; (al) Arterial line; (vl) Venous line; (Ao) Aorta; and (SVC, IVC) Superior and inferior venae cavae.
72 cannula. For the purpose of the model experiment, the arterial “patient” line was terminated by an arterial cannula having a 4mm internal diameter, sufficiently large to afford an adequate flow with a small pressure gradient, comparable with the clinical conditions of extracorporeal circulation (Fig. lb). Priming The circuit was primed with a volume of 2550 ml, containing 950 ml of whole blood and 900 ml of packed RBC (with the addition of: 0.6g CaClz (10%); 3000 IU of heparin/500 ml of blood); 430 ml of Hartmann’s solution; 100 ml of human albumin (20%); 80 ml of mannitol (10%); 50 ml of sodium bicarbonate (4.2%), and 40 ml of glucose (40%). Thus, at the outset of extracorporeal circulation a haematocrit of 26% was achieved. Prior to starting the closed circuit re-circulation, adequate anticoagulation was achieved by perfusion with 2500 IU of heparin/lOOO ml of the total solution except for the blood. The initial value of haemolysis was 131.95 mg/lOO ml and the value during the perfusion was 267.70 mg/lOO ml. The haematocrit decreased to a value of 23%. Hormones Hormones were added into the perfusate in quantities sufficient to raise the concentrations to those that could be expected during surgery with cardiopulmonary bypass or corresponding to upper normal physiological values. The amounts added were as follows: cortisol, 200 ng . ml-l; insulin 100 PU * ml-‘; human growth hormone, 20 ng . ml-‘; adrenaline 2 ng . ml-‘; noradrenaline, 2 ng - ml-‘. The hormones, added initially to the Hartmann’s solution, were thoroughly mixed with the solution and, on completing the priming, the solutions were shaken gently for 20 min. Afterwards, 300 ml of the priming volume was poured off into an infusion bottle for a blind incubator experiment and the bulk was put into the extracorporeal circuit. Two control samples were taken at 5-min intervals from both of these separate fractions of the priming volume. Circulation The extracorporeal circulation in vitro was instituted at a flow rate of 3800 ml/min, corresponding to 2.3 l/m2 per min during cardiac surgery. A closed system was employed, i.e. the arterial and venous lines were connected together to establish continuous recirculation (Fig. lb). The circulation of the priming volume was continued for 90min at a temperature of 3O”C, and subsequently the perfusate was re-warmed and set in operation at 37°C for 30 min. Sampling On starting the model extracorporeal circulation with the functional bubble oxygenator, the blood samples were drawn from the blood outlet of the
73 bubble oxygenator at 30,60 and 90 min, while the blood was at a temperature of 30°C; after rising to 37°C the circulation was run for another 30min, when the last sample was withdrawn. A parallel sampling was performed with the blind incubator experiment, following similar time intervals and temperatures. Laboratory methods The blood glucose level was determined by the glucose oxidase technique (Huggett and Nixon, 1957). Radioimmunoassay of plasma immunoreactive insulin (IRI) was performed by the method of Herbert, Lau, Gottlieb and Bleisher (1965). Plasma cortisol was estimated by a modified method of competitive protein binding (Murphy, 1967). Human growth hormone OIGH) was estimated by coated charcoal immunoassay (Lau, Gottlieb and Herbert, 1966). Plasma levels of adrenaline, noradrenaline and dopamine were measured by a simultaneous radioenzymatic assay (Da Prada and Zurcher, 1976; Peuler and Johnson, 1977). Thyroxine was estimated by radioimmunoassay using polyethyleneglycol separation technique (Fiildes, HrEka, Veleminsky, Kokesova, Langer and Listiakova, 19781.
RESULTS
Growth hormone (Fig. 2) During the working in vitro of the heart-lung machine, the human growth hormone (hGHl concentration in the blood taken from the oxygenator outflow, compared with the blood in the incubator, decreased moderately to a mean of 76% of the control value. The decline was gradual, reaching the lowest value after the circulation was continued for 90 min at a temperature of 30°C. The depressed level remained unchanged even after rewarming to 37°C. Imm unoreactive insulin The concentration of insulin in the blood coming from the oxygenator, after being circulated for 30min at 30°C fell precipitously to 32% of the control value; in contrast to hGH, the insulin level was reduced rapidly within 30 min (Fig. 2). This finding differed from that in the blood resting in the incubator at the same temperature. Approximately the same depressed concentrations of 28435% were sustained for 90 min. Rewarming to 37°C provoked no further significant change of concentration in the blood withdrawn from the circuit; however, under control incubator conditions a somewhat more marked decrease in concentration of immunoreactive insulin (IRI) was observed.
cortisoz
Cortisol levels remained unaffected by artificial effects of the heart-lung
74 machine and, in particular, of the bubble oxygenator. No significant changes from control values were noted. Thyroxine The thyroxine concentration did not show any significant changes due to the heart-lung machine, even as a result of the change in temperature (Fig. 2). Cutecholumines (Fig. 3) Adrenaline, noradrenaline and dopamine were substantially degraded by the bubble oxygenator and the extracorporeal circulation circuit functioning for 30 min. As compared with control values at 30°C the mean concentration of noradrenaline dropped precipitously to 9% within 30min, and this extremely low concentration persisted throughout the experiment, including the rewarming to 37°C. Adrenaline showed an even more pronounced fall. Dopamine destru&ion was relatively milder at normal temperature, as well as at the lower temperature. NA 100 % =I697
p9ml’
hGH 100 % = 2L n9mi’
DA 100 % = 263,l
p9ml”
OJ A 100 % =187L 0 i THYROXINE %
100 60
i
100 % =35,6rgm?
d OXYG min OXYG INCUBATOR
Fig. 2.
I
p9mt
~~-----~..._______o
% ‘!
0 JTZMm ----
1;
‘OXYGL T:30’C + C ----
T:37’C 1 30
0 60
+ 90
? 30 min
OXYG INCUBATOR
Fig. 3.
Fig. 2. Percentage change in human growth hormone BGH) (100% = 24.0 ng . ml-‘), immunoreactive insulin (IRI) (100% = 79.3 pI.J . ml-‘) and thyroxine levels during r-e-circulationin the heart-lung machine compared with levels in uncirculated fluid in the incubator at 30°C and 37°C. Fig. 3. Percentage changes in adrenaline (100% = 1874 pg. ml-‘), noradrenaline (100% = 1897 pg. ml-‘) and dopamine levels during r-e-circulationin the heart-lung machine compared with levels in uncirculated fluid in the incubator at 30°C and 37°C.
75 DISCUSSION
During cardiac surgery, after starting cardiopulmonary bypass (CPB), the blood levels of hormones usually decline, or, their tendency to increase slows down markedly (Taylor, Bain, Maxted, Hutton, McNab and Caves, 19781. The primary causal factor appears to be haemodilution, as well as a decrease in endocrine secretion due to induced hypothermia and hypoperfusion. Nevertheless, the decline in hormone levels caused by denaturation in the heartlung machine may also contribute to the cessation or attenuation of the rise in hormone concentrations during severe surgical stress. Our experiment in vitro shows that individual hormones react differently to oxygenation, extracorporeal circulation and other inherent factors. The concentrations of cortisol and thyroxine remained unchanged when the blood was re-circulated for 2 h in the machine producing non-pulsatile flow. Growth hormone showed a more marked degradation only after l-hour recirculation and, in spite of it being a relatively large molecule, its concentration, failed to change very markedly. Despite the careful mixing of blood in the incubator, insulin was degraded to some extent; the cause of this change might be a haemolysis accompanied by a release from red blood cells of the enzyme glutathion-insulin-transhydrogenase, splitting the insulin molecule into A and B chains (Varandani, 1974). A number of other factors have also been proposed to be responsible for the destruction of these hormones, which is enhanced markedly in the machine. The denaturation of the plasma proteins and hormones has been ascribed primarily to the presence of blood-gas interfaces in the oxygenator (Clement, 1971). Mechanical factors come also into play, although continuous blood flow produced by the pump used is less traumatic to the blood than pulsatile blood flow. The size of arterial cannula is important as the cannula represents the narrowest point in the arterial line and imposes a considerable resistance, when flow rates are high; big swings of pressure proximal to this point and high velocity jets through the cannula result in significant damage to the blood and plasma proteins, i.e. also to the human growth hormone and insulin. It has also been suggested that adverse effects may arise from the tubing made of plastic materials. Some kinds of tubing made from PVC may lead to haemolysis of the bMod being perfused. Meyler, Willebrands and Durrer (1960) reported that the toxicity of some PVC tubing was caused by the presence of organic tin compounds. Haberman, Raker and Arndts (1968) observed deleterious eff ects of some plastic materials and their components on immune reactions including antibodies and serum proteins. Toxic substances were found to be released from PVC tubing by a leaching process, starting upon the addition of fresh plasma, the amount of leaching activity being dependent on the particular plasma protein concentration. The most striking changes were observed in the concentration of adrenaline and noradrenaline and, to a lesser degree, in the level of dopamine. Within the first 30 min about 90% of the amount added was degraded in the
76 machine. The presumed cause of degradation of catecholamines appears to be oxidation by oxygen. This process has been utilized with some techniques of catecholamine determination, when catecholamines are oxidized (von Euler and Lishajko, 1961). The difference between the degree of degradation under control conditions in the incubator and in the experimental circulation including the oxygenator, which differ mainly by the bubbling oxygen, supports the proposed mechanism. The importance of temperature for the degradation of hormones under the present experimental conditions cannot be determined exactly. The increase in temperature up to 37°C during the last 30 min of the experiment seems not to have been of sufficiently long duration to increase the rate of degradation under control conditions in the incubator. A similar level was found in the blood passing through the oxygenator, although catecholamine and insulin concentrations at the end of the experiment were already lowered substantially. The significance of the results obtained for evaluating changes of hormone levels studied during cardiopulmonary bypass cannot be interpreted unambiguously. The fall of cortisol or the arrest of the rising trend following the onset of bypass showed no degradation in the oxygenator. On the other hand, the growth hormone and insulin denaturated during blood oxygenation in the oxygenator, rose significantly during bypass. The resultant net level of hormones during bypass is dependent mainly on the rate of secretion. The degree of degradation may modify the level, however, its proportion is difficult to assess during a parallel release of the hormones from the glands. A moderate degradation in the oxygenator, as was found, e.g. for human growth hormone, may not play a significant part in the blood level of the hormone under conditions of surgery. In contrast to this, a marked oxidation of catecholamines or the degradation of insulin, which were destroyed in the experiment in vitro within 30 min, by 90% and 70%, respectively, might also be important during surgery. To sustain elevating blood concentrations, a much higher rate of secretion may be necessary than would be expected from increments in the concentration. ACKNOWLEDGEMENTS
The authors wish to express their appreciation to Mrs M. Masarykovd and Mrs E. Andelovi for skilled technical assistance. REFERENCES Clement, A.J. (1971) The physiological background of and apparatus used for extracorporeal circulation. Br. J. Anaeeth., 43, 233-243. da Prada, M. and Zurcher, G. (1976) Simultaneous radioenzymatic determination6 of plasma and tissue adrenaline, noradrenaline and dopamine within the fentomole range. Life Sci., 19, 1161-1174.
77 Duke, H.N. and Vane, J.R. (1968) An adverse effect of polyvinylchloride tubing used in extracorporeal circulation. Lancet, 2 (No 7568), 21-23. Foldes, O., Hrcka, R., Veleminsky, J., Kok&vl, H., Langer, P. and Liatiakova, M. (1978) A sensitive radio-immunoassay of L-thyroxine in the serum by means of own specific highly effective antibodies. B&hem. Clin. Bohemoslov., 7, 143-151. Haberman, E., Raker, K.O. and Amdta, D. (1968) Radio-Retentionselektrophorese: ein quantitatives und spezifisches Verfahren zur Bestimmung einzelner Proteine im Nanogrammbereich. Klin. Wschenschr., 46, 4647. Herbert, V., Lau, KS., Gottlieb, C.W. and Bleisher, G.J. (1965) Coated charcoal immunoassay of insulin. J. Clin. Endocrinol. Metab., 25, 1375-1379. Huggett, A.St.G. and Nixon, D.A. (1957) Use of glucose oxidase, proxidase and odianosidine in determination of blood and urinary glucose. Lance& ii, 368-369. Lau, K.S., Gottlieb, C.W. and Herbert, V. (1966) Preliminary report on coated charcoal immunoassay of human chorionic “growth hormone-prolactin” and growth hormone. Proc. Sot. Exp. Biol. Med., 123, 126131. Lee, W.H., Krumhaar, D., Fonkalsrud, E.W., Schjeide, O.A. and Manney, J.V. (1961) Destruction of plasma proteins as a cause of morbidity and death after intracardiac operations. Surgery, 50, 29. Meyler, F.L., Willebrands, A.F. and Durrer, D. (1966) The influence of polyvinylchloride (P.V.C.) tubing on the isolated perfused rat’s heart. Circul. Res., 8, 44-46. Murphy, B.E.P. (1967) Some studies of the protein-binding of steroid and their application to the routine micro and ultra micro measurement of various steroids in the bcdy fluids by competitive protein-binding radioassay. J. Clin. Endocrinol. Metab., 27, 973-996. Peuler, J. and Johnson, G.A. (1977) Simultaneous single isotope radioenxymatic assay of plasma norepinephrine and dopamine. Life Sci., 21, 625-636. Taylor, K.M., Bain, W.H., Maxted, K.J., Hutton, M.M., McNab, W.Y. and Caves, P.K. (1978) Comparative studies of pulsatile and non-pulsatile flow during cardiopulmonary bypass. J. Thorac. Cardiovasc. Surg., 75, 569-573. Varandani, P.T. (1974) Insulin degradation in insulinoma: evidence for the occurrence of an inactive form of glutathione-insulin-transhydrogenase and for the absence of insulin A and B chains degrading protease( Biochem. Biophys. Res. Commun., 60, 1119-1126. von Euler, U.S. and Lisbajko, S. (1961) Improved technique for the fluorimetric estimation of catecholamines. Acta Physiol. Scan., 51, 348.
Resuecitation, ll(1964) 69-77 Elsevier Scientific Publishers Ireland Ltd.
IN VITRO STUDY OF HORMONE DEGRADATION BY HEART-LUNG MACHINE WITH BUBBLE OXYGENATOR
J. MALATINSKP, M. VIGAEjb, D. VGANSKP”, and DANA JEiOVAb
R. KVETrjANSKkb,
JANA JLJR~O&OVAb
“Postgraduate Medical School, Kramdre, Limbovd 5, 633 03 Bratislava and bInstitute of Experimental Endocrinology, Slovak Academy of Sciences, Kmmcire, 809 36, Bmtislava (Czechoslovakia) (Received February
34
1963)
SUMMARY
In the present experiment with heart-lung machine set in a closed system in vitro, the blood containing increased levels of hormones was circulated at 30°C for 90 min and at 37°C for 30min; a fraction of the priming perfusate was studied in parallel at the same temperatures in an incubator. The level of growth hormone decreased gradually to a mean of 76% at 30°C and no further decrease was found at 37°C. The mean insulin level fell within 30 min to 32% and no substantial further changes were observed during the remaining period of study; re-warming failed to produce significant additional changes. Cortisol did not change appreciably. However, the oxygenator altered the level of catecholamines markedly. At 30°C the initial level of noradrenaline fell precipitously to 9% within 30 min and this excessively low level was sustained throughout the study. Adrenaline showed even more pronounced changes. Denaturation of dopamine was less marked, falling to about 70% within 30 min. The present experiment revealed that the levels of hormones respond differently to artificial oxygenation during extracorporeal circulation. Cortisol resisted degradation by the oxygenator, while growth hormone and insulin were denatured significantly. The moderate degradation of growth hormone by the machine may not play an important role during open heart surgery. However, a marked oxidation denaturation of catecholamines by 90% and 70% and denaturation of insulin by 70%, might prove relevant during surgery.
Addtess
all correspondence: Dr. Jozef Malatinskjl,
Hospital,
KramBre, 633 05 Bratislava,
CSc.,
Postgrad.
Czechoslovakia.
0300-9572/64/$03.00 @ 1964 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland Ltd.
Medical
School,
Derer’s
70 INTRODUCTION
During its passage through the heart-lung machine for open heart surgery, biologically active substituents of blood may be damaged by chemical or mechanical factors. Chemical impairment may result from toxic substances leaching out of plastic tubing (Duke and Vane, 1968) or from the residual effects of improper sterilization. Mechanical damage is the result of rough surfaces in the inside of the circuit, or of turbulent flow at sites where flow velocity and pressure gradients are high (Clement, 1971). This model experiment with the standard equipment used routinely for clinical purposes of open heart surgery was designed to ascertain, whether or not inherent physical and chemical factors interfere significantly with the integrity of some hormones and, therefore, whether the concentrations measured during open heart surgery are influenced appreciably by the performance of heart-lung machine. By analogy with chemical and mechanical damage to other constituents of circulating blood (Lee, Krumhaar, Fonkalsrud, Schjeide and Manney, 19611, certain physico-chemical degradation processes and/or physical adsorption were expected to occur in the apparatus. In particular, partial destruction was expected with hormones exposed to the effects of a higher oxygen tension, while the blood is passing through the bubble oxygenator. However, of great interest were also possible specific differences in the artificially produced reduction of individual hormone levels (growth hormone, insulin, cortisol, catecholamines and thyroxine).
METHODS
Extracorporeal circulation circuit For the present model experiment, the routinely used circuit was employed comprising a modification of Lillehei-De Wall apparatus Premacard 2 produced by Prema, in conjunction with the disposable Bentley Temptrol Adult Type Q-100 bubble oxygenator. The blood drained from the superior and inferior venae cavae, owing to gravitational force, flows via the venous line into the oxygenator placed 60 cm below the level of the right atrium. The oxygenated blood then passes into the reservoir of arterial blood with changeable level of its position. By elevating or depressing the level of the reservoir, an optimal amount of blood is maintained in the oxygenator. A 46 ~_~rn microemboli blood filter is inserted in the arterial reservoir before the line reaches the arterial pump. The two arterial membrane pumps pump the blood from the apparatus into the patient (arterial line). The machine is equipped with a heat exchanger for thermoregulation of the blood. When setting the heart-lung machine into motion, the arterial pumps are first switched on and afterwards the clamps from venae cavae are removed. A reverse procedure is used at the completion of the perfusion. Oxygen is
71 delivered into the oxygenator in a quantity equal to three to four times the minute output of the pump. The standard’ machine Premacard 2 (Fig. la) is equipped with two membrane pumps, connected together in parallel; these are designed so that at the same time as one of them is expelling, the other is sucking in blood, and vice versa. This results in a continuous blood flow from the arterial
-lVC
fig. 1. Schematicdrawing of the heart-lung machine: (a) used for open heart surgery; (b) employed for the experiment as a closed system; (A + B) Oxygenator Temptrol Q-100 adult type (produced by Bentley Lab. Inc.); (A) Oxygenator consisting of oxygenation and defoaming parts, and arterial 40 pm blood filter; (B) Arterial reservoir with integral heat exchanger; (C) Two membrane pumps; (D)Watertemperatureregulationsupply;03)Arterial cannula; 0 Arterial blood sampling outlet; (G) Venous blood sampling outlet; 0 Coronary suction pump; (I) Coronary suction; (J) Sensor for measuring arterial pre~eure; (K) Reservoir and coronary suction filter; (al) Arterial line; (vl) Venous line; (Ao) Aorta; and (SVC, IVC) Superior and inferior venae cavae.
72 cannula. For the purpose of the model experiment, the arterial “patient” line was terminated by an arterial cannula having a 4mm internal diameter, sufficiently large to afford an adequate flow with a small pressure gradient, comparable with the clinical conditions of extracorporeal circulation (Fig. lb). Priming The circuit was primed with a volume of 2550 ml, containing 950 ml of whole blood and 900 ml of packed RBC (with the addition of: 0.6g CaClz (10%); 3000 IU of heparin/500 ml of blood); 430 ml of Hartmann’s solution; 100 ml of human albumin (20%); 80 ml of mannitol (10%); 50 ml of sodium bicarbonate (4.2%), and 40 ml of glucose (40%). Thus, at the outset of extracorporeal circulation a haematocrit of 26% was achieved. Prior to starting the closed circuit re-circulation, adequate anticoagulation was achieved by perfusion with 2500 IU of heparin/lOOO ml of the total solution except for the blood. The initial value of haemolysis was 131.95 mg/lOO ml and the value during the perfusion was 267.70 mg/lOO ml. The haematocrit decreased to a value of 23%. Hormones Hormones were added into the perfusate in quantities sufficient to raise the concentrations to those that could be expected during surgery with cardiopulmonary bypass or corresponding to upper normal physiological values. The amounts added were as follows: cortisol, 200 ng . ml-l; insulin 100 PU * ml-‘; human growth hormone, 20 ng . ml-‘; adrenaline 2 ng . ml-‘; noradrenaline, 2 ng - ml-‘. The hormones, added initially to the Hartmann’s solution, were thoroughly mixed with the solution and, on completing the priming, the solutions were shaken gently for 20 min. Afterwards, 300 ml of the priming volume was poured off into an infusion bottle for a blind incubator experiment and the bulk was put into the extracorporeal circuit. Two control samples were taken at 5-min intervals from both of these separate fractions of the priming volume. Circulation The extracorporeal circulation in vitro was instituted at a flow rate of 3800 ml/min, corresponding to 2.3 l/m2 per min during cardiac surgery. A closed system was employed, i.e. the arterial and venous lines were connected together to establish continuous recirculation (Fig. lb). The circulation of the priming volume was continued for 90min at a temperature of 3O”C, and subsequently the perfusate was re-warmed and set in operation at 37°C for 30 min. Sampling On starting the model extracorporeal circulation with the functional bubble oxygenator, the blood samples were drawn from the blood outlet of the
73 bubble oxygenator at 30,60 and 90 min, while the blood was at a temperature of 30°C; after rising to 37°C the circulation was run for another 30min, when the last sample was withdrawn. A parallel sampling was performed with the blind incubator experiment, following similar time intervals and temperatures. Laboratory methods The blood glucose level was determined by the glucose oxidase technique (Huggett and Nixon, 1957). Radioimmunoassay of plasma immunoreactive insulin (IRI) was performed by the method of Herbert, Lau, Gottlieb and Bleisher (1965). Plasma cortisol was estimated by a modified method of competitive protein binding (Murphy, 1967). Human growth hormone OIGH) was estimated by coated charcoal immunoassay (Lau, Gottlieb and Herbert, 1966). Plasma levels of adrenaline, noradrenaline and dopamine were measured by a simultaneous radioenzymatic assay (Da Prada and Zurcher, 1976; Peuler and Johnson, 1977). Thyroxine was estimated by radioimmunoassay using polyethyleneglycol separation technique (Fiildes, HrEka, Veleminsky, Kokesova, Langer and Listiakova, 19781.
RESULTS
Growth hormone (Fig. 2) During the working in vitro of the heart-lung machine, the human growth hormone (hGHl concentration in the blood taken from the oxygenator outflow, compared with the blood in the incubator, decreased moderately to a mean of 76% of the control value. The decline was gradual, reaching the lowest value after the circulation was continued for 90 min at a temperature of 30°C. The depressed level remained unchanged even after rewarming to 37°C. Imm unoreactive insulin The concentration of insulin in the blood coming from the oxygenator, after being circulated for 30min at 30°C fell precipitously to 32% of the control value; in contrast to hGH, the insulin level was reduced rapidly within 30 min (Fig. 2). This finding differed from that in the blood resting in the incubator at the same temperature. Approximately the same depressed concentrations of 28435% were sustained for 90 min. Rewarming to 37°C provoked no further significant change of concentration in the blood withdrawn from the circuit; however, under control incubator conditions a somewhat more marked decrease in concentration of immunoreactive insulin (IRI) was observed.
cortisoz
Cortisol levels remained unaffected by artificial effects of the heart-lung
74 machine and, in particular, of the bubble oxygenator. No significant changes from control values were noted. Thyroxine The thyroxine concentration did not show any significant changes due to the heart-lung machine, even as a result of the change in temperature (Fig. 2). Cutecholumines (Fig. 3) Adrenaline, noradrenaline and dopamine were substantially degraded by the bubble oxygenator and the extracorporeal circulation circuit functioning for 30 min. As compared with control values at 30°C the mean concentration of noradrenaline dropped precipitously to 9% within 30min, and this extremely low concentration persisted throughout the experiment, including the rewarming to 37°C. Adrenaline showed an even more pronounced fall. Dopamine destru&ion was relatively milder at normal temperature, as well as at the lower temperature. NA 100 % =I697
p9ml’
hGH 100 % = 2L n9mi’
DA 100 % = 263,l
p9ml”
OJ A 100 % =187L 0 i THYROXINE %
100 60
i
100 % =35,6rgm?
d OXYG min OXYG INCUBATOR
Fig. 2.
I
p9mt
~~-----~..._______o
% ‘!
0 JTZMm ----
1;
‘OXYGL T:30’C + C ----
T:37’C 1 30
0 60
+ 90
? 30 min
OXYG INCUBATOR
Fig. 3.
Fig. 2. Percentage change in human growth hormone BGH) (100% = 24.0 ng . ml-‘), immunoreactive insulin (IRI) (100% = 79.3 pI.J . ml-‘) and thyroxine levels during r-e-circulationin the heart-lung machine compared with levels in uncirculated fluid in the incubator at 30°C and 37°C. Fig. 3. Percentage changes in adrenaline (100% = 1874 pg. ml-‘), noradrenaline (100% = 1897 pg. ml-‘) and dopamine levels during r-e-circulationin the heart-lung machine compared with levels in uncirculated fluid in the incubator at 30°C and 37°C.
75 DISCUSSION
During cardiac surgery, after starting cardiopulmonary bypass (CPB), the blood levels of hormones usually decline, or, their tendency to increase slows down markedly (Taylor, Bain, Maxted, Hutton, McNab and Caves, 19781. The primary causal factor appears to be haemodilution, as well as a decrease in endocrine secretion due to induced hypothermia and hypoperfusion. Nevertheless, the decline in hormone levels caused by denaturation in the heartlung machine may also contribute to the cessation or attenuation of the rise in hormone concentrations during severe surgical stress. Our experiment in vitro shows that individual hormones react differently to oxygenation, extracorporeal circulation and other inherent factors. The concentrations of cortisol and thyroxine remained unchanged when the blood was re-circulated for 2 h in the machine producing non-pulsatile flow. Growth hormone showed a more marked degradation only after l-hour recirculation and, in spite of it being a relatively large molecule, its concentration, failed to change very markedly. Despite the careful mixing of blood in the incubator, insulin was degraded to some extent; the cause of this change might be a haemolysis accompanied by a release from red blood cells of the enzyme glutathion-insulin-transhydrogenase, splitting the insulin molecule into A and B chains (Varandani, 1974). A number of other factors have also been proposed to be responsible for the destruction of these hormones, which is enhanced markedly in the machine. The denaturation of the plasma proteins and hormones has been ascribed primarily to the presence of blood-gas interfaces in the oxygenator (Clement, 1971). Mechanical factors come also into play, although continuous blood flow produced by the pump used is less traumatic to the blood than pulsatile blood flow. The size of arterial cannula is important as the cannula represents the narrowest point in the arterial line and imposes a considerable resistance, when flow rates are high; big swings of pressure proximal to this point and high velocity jets through the cannula result in significant damage to the blood and plasma proteins, i.e. also to the human growth hormone and insulin. It has also been suggested that adverse effects may arise from the tubing made of plastic materials. Some kinds of tubing made from PVC may lead to haemolysis of the bMod being perfused. Meyler, Willebrands and Durrer (1960) reported that the toxicity of some PVC tubing was caused by the presence of organic tin compounds. Haberman, Raker and Arndts (1968) observed deleterious eff ects of some plastic materials and their components on immune reactions including antibodies and serum proteins. Toxic substances were found to be released from PVC tubing by a leaching process, starting upon the addition of fresh plasma, the amount of leaching activity being dependent on the particular plasma protein concentration. The most striking changes were observed in the concentration of adrenaline and noradrenaline and, to a lesser degree, in the level of dopamine. Within the first 30 min about 90% of the amount added was degraded in the
76 machine. The presumed cause of degradation of catecholamines appears to be oxidation by oxygen. This process has been utilized with some techniques of catecholamine determination, when catecholamines are oxidized (von Euler and Lishajko, 1961). The difference between the degree of degradation under control conditions in the incubator and in the experimental circulation including the oxygenator, which differ mainly by the bubbling oxygen, supports the proposed mechanism. The importance of temperature for the degradation of hormones under the present experimental conditions cannot be determined exactly. The increase in temperature up to 37°C during the last 30 min of the experiment seems not to have been of sufficiently long duration to increase the rate of degradation under control conditions in the incubator. A similar level was found in the blood passing through the oxygenator, although catecholamine and insulin concentrations at the end of the experiment were already lowered substantially. The significance of the results obtained for evaluating changes of hormone levels studied during cardiopulmonary bypass cannot be interpreted unambiguously. The fall of cortisol or the arrest of the rising trend following the onset of bypass showed no degradation in the oxygenator. On the other hand, the growth hormone and insulin denaturated during blood oxygenation in the oxygenator, rose significantly during bypass. The resultant net level of hormones during bypass is dependent mainly on the rate of secretion. The degree of degradation may modify the level, however, its proportion is difficult to assess during a parallel release of the hormones from the glands. A moderate degradation in the oxygenator, as was found, e.g. for human growth hormone, may not play a significant part in the blood level of the hormone under conditions of surgery. In contrast to this, a marked oxidation of catecholamines or the degradation of insulin, which were destroyed in the experiment in vitro within 30 min, by 90% and 70%, respectively, might also be important during surgery. To sustain elevating blood concentrations, a much higher rate of secretion may be necessary than would be expected from increments in the concentration. ACKNOWLEDGEMENTS
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