Mechanism
of Hypotension
Following Rapid Infusion Anesthetized Dogs
of Protamine
Sulfate in
Nguyen D. Kien, PhD, Darcy D. Quam, DVM, John A. Reitan, MD, and David A. White, MD Protamine sulfate (PS), used to neutralize the anticoagulant effect of heparin, is often associated with systemic hypotension. Whether this hypotension is secondary to a depression of myocardial function is not clear. The present study tested the hypothesis that systemic hypotension was accompanied by a depression in myocardial function and examined the possible role of histamine in mediating the cardiovascular response to PS. Seven conditioned dogs were chronically instrumented with pressure and ultrasonic dimension transducers. Studies were conducted under halothane anesthesia 7 to 10 days after instrumentation. Cardiac contractility was assessed using the slope, E,,, of the linear regression of the left ventricular end-systolic pressure-diameter relationship. Intravenous infusion of PS, 5 mg/kg, when given in periods of less than 30 seconds, decreased systemic arterial pressure by 45% (from 101 f to 54 f 5 mm Hg) without change in heart rate. Cardiac output decreased by 22% from control
P
ROTAMINE SULFATE (PS) is used routinely following cardiopulmonary bypass (CPB) to neutralize the anticoagulant effect of heparin. However, PS may cause profound systemic hypotension, an effect that could be detrimental to outcome in patients with significant cardiac or cerebral vascular disease. Although this hypotensive effect has been well described,’ the exact mechanisms by which PS decreases arterial blood pressure are unclear. It has been postulated that the release of endogenous vasoactive substances, such as histamine evoked by PS, may be involved, but supporting evidence is lacking.* In addition, whether concomitant myocardial depression accompanies the systemic vasodilation remains controversial. The conflicting results from previous studies may relate to (1) the variable rates of administration of PS among studies and (2) differing methodologies used in determining the myocardial effects. The specific aims of this study were to examine the infusion rate-dependent effects of PS on myocardial performance and to evaluate the possible role of histamine in mediating the cardiovascular effects of PS. MATERIALS
AND METHODS
This study was approved by the institutional committee on animal use and care. Seven healthy adult mongrel dogs of either sex, weighing between 20 and 26 kg, were instrumented 1 to 2 weeks before experimentation. The animals were anesthetized with thiamylal (15 mg/kg), and the trachea intubated and ventilated by a positive-pressure Harvard respirator (Harvard Apparatus, South Natick, MA). Anesthesia was maintained with 1.5% halothane in oxygen. Under sterile conditions, a thoracotomy was performed at the fifth intercostal space. Inflatable hydraulic occluders were placed around both inferior and superior vena cavae for varying preload during assessment of myocardial performance. An electromagnetic flow probe (Biotronex Lab Inc., Kensington, MD) was placed around the ascending aorta. A high-fidelity micromanometer (Kanigsberg Instruments Inc., Pasadena, CA) was implanted in the left ventricular (LV) cavity via an apical stab wound for the measurement of LV pressure. This manometer was calibrated against a Statham pressure transducer (Gould Inc., Oxnard, CA) connected to a fluid-filled LV catheter
Journalof Cardiothoracic and VascularAnesthesia,
and the slope E,, decreased by 37% (from 14.5 f 1.2 to 8.7 f 1.4 mm Hg/mm). Systemic vascular resistance decreased by 34% (from 2581 f 121 to 1712 + 200 dyne-s-cme5). The cardiovascular
depression
caused by PS was transient
and could not be reproduced by a repeated dose given within a W-minute
period. Antagonists
mine and cimetidine) cardiovascular
of histamine (diphenhydra-
could not attenuate
the PS-induced
depression. This depression was independent
of preheparinization
and did not occur when PS was infused
slowly over a 2-minute period. The data clearly demonstrate negative
inotropic
and vasodilator
effects of PS following
rapid administration. These findings suggest that PS should be administered slowly to minimize or to avoid its hypotensive effect, paired. Cop y&ht
particularly
when
myocardial
function
is im-
o 1992 by W. B. Saunders Company
implanted adjacently. Pairs of ultrasonic crystals were implanted onto the LV to measure the anterior-posterior transverse diameter on the minor axis, segmental length, and wall thickness of the lateral free wall as previously described.3,4 Proper alignment of the crystals was confirmed with a high-frequency oscilloscope (Textronix, Beaverton, OR). Ventricular dimensions were monitored using a four-channel sonomicrometer (Triton Technology, San Diego, CA). Additional catheters were placed in the aorta via a carotid artery and jugular vein for blood pressure measurement and venous access, respectively. Instrument wires and catheters were tunneled subcutaneously to exit at the intrascapular region and contained in the pocket of an elastic animal jacket. The chest was then closed and the animal was returned to the kennel. Each dog received penicillin and streptomycin for 4 days after surgery. Analgesics were given as needed during the recovery period. The catheters were flushed with heparinized saline and the wound dressings were changed on a daily basis. On the day of the experiment, anesthesia was induced with thiamylal, 15 mg/kg, and maintained at 1.25% halothane in 0,. Mechanical ventilation was controlled, PCO, was maintained at 40 2 3 mm Hg. Instrument wires and catheters were connected to a monitoring panel. Hemodynamic variables were recorded on a Gould direct pen-writing oscillograph (Gould-Brush Inc, Cleveland, OH) and on FM analog tape (Ampex Corp, Redwood City, CA) for subsequent analysis. Baseline measurements were recorded after 20 minutes of hemodynamic stability. PS, 5 mg/kg, was infused intravenously over various rates ranging from 10 seconds to 5 minutes. Infusionls of PS were repeated after 90- to 120-minute intervals except when specified. Each animal received no more
From the Department of Anesthesiology, University of California School of Medicine, Davis, CA. This study was approved by the institutional committee on the care and use of laboratory animals. The use of animals was in accordance with the guidelines of Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Council (DHHS publication No [NIH] 85-23, 1985). Address reprint requests to Nguyen D. Kien, PhD, Department of Anesthesiology, TB-170, University of California, School of Medicine, Davis, CA 95616. Copyright D 1992 by U! B. Saunders Company 1053-0770/92/0602-0005$03.00/0
Vol6, No 2 (April), 1992: pp 143-147
143
144
KIEN Ei 4_
than 3 doses of PS in a single day at infusion parentheses following this experimental plan:
rates
indicated
DAY 1:
1 PS (30 seconds) 2 PS (5 minutes) 3 PS (30 seconds) (infused within ho-minute terval from second dose)
DAY 3:
1 PS (20 seconds) 2 Heparin + PS (20 seconds) 3 PS (3 minutes)
DAY 5:
1 PS (2 minutes) 2 PS ( 10 seconds) 3 Histamine antagonists
On day 1, the infusion
in-
+ PS (10 seconds)
rates of the first and second
doses of PS
were randomized. A third dose of PS was given at intervals ranging from 20 to 60 minutes from the second dose. At the end of the experiment, the animals were allowed to recover from anesthesia and returned to the kennel for a 48-hour resting period. Subsequent
experiments
rate that induced
were performed systemic
in which PS was infused
hypotension)
following
(at a
administration
of heparin, 150 W/kg, to determine the role of the PS-heparin complex. To examine the role of histamine (H), PS was infused (at a rate that induced
systemic hypotension)
following
administration
of H antagonists (diphenhydramine, 1 mgikg, cimetidine, 4 mgi kg). The effectiveness of the antagonists was tested with 5 pglkg of H before use with PS. Following the last experiment, the animals were then killed with an anesthetic overdose and intravenous injection of saturated potassium cloride. Implanting devices were retrieved and their locations were confirmed. Data were sampled over 10 to 20 seconds for assessment of contractility during volume emptying produced by transient vena caval occlusion.
Vena caval occlusion
of data was recorded tion. Paired
was released,
and the next set
after a brief period of hemodynamic
measurements
of end-systolic
LV pressure
RESULTS
in
equilibra-
The dogs were studied 1 to 2 weeks after instrumcnration. They were healthy and fully recovered from the ctfccts of surgery. Representative digital LV pressure and diamcter data from one dog arc shown in Fig 1. This plot depicts the LV pressure-diameter relationship at a control baseline. During vena caval occlusion. the pressure-diameter loop becomes smaller and shifts to the left. End-systolic points are at the upper left corner of loops. Hemodynamic values at baseline and peak pressure response at 2 minutes after the onset of PS infusion arc shown in Tables 1 and 2. Table 1 summarizes systemic effects following either (I) rapid infusion of PS (given in 2 min). When infused rapidly, PS decreased mean arterial pressure and cardiac output (CO) 45.5 -C 5.2 and 21.8 + 2.9%, respectively. However. heart rate and LV end-diastolic pressure did not change significantly. Calculated stroke volume and systemic vascular resistance (SVR) decreased by 19.0 ? 6.7 and 33.9 +- 17.5%, respectively. The decrease in systemic arterial pressure was rapid in onset (10 to 15 seconds after completion of PS infusion) and transient (4.2 lr 0.6 minutes). Data on myocardial effects of PS are included in Table 2. Myocardial contractility, indicated by the slope E,,, dccreased significantly from 13.8 2 2.4 to 7.7 * 2.3 mm Hgimm following rapid infusion of PS. Percents of systolic shortening of LV minor-axis diameter and wall thickness decreased by 14.9 ? 12.5 and 20.7 ? 5.0%, respectively, but the change in systolic shortening of the segmental length
and diame-
ter during 10 to 15 consecutive cardiac cycles with no apparent dysrhythmias were used for each regression analysis. Dysrhythmias were identified by abnormal changes in LV pressure, diameter, and electrocardiogram (ECG). The first cardiac cycle in the series for analysis was selected at the onset of LV pressure reduction so that the ranges of pressure and diameter during vena caval occlusion were minimized. For each heart beat, end-systole was defined as 20 milliseconds before the peak negative dP/dt, and end-diastole was assumed at the peak R wave of the ECG. Respiratory influence on hemodynamic measurements was eliminated by turning off the respirator momentarily during contractility assessment. The autonomic sympathetic reflex caused by preload reduction was minimized by eliminating heart beats with R-R intervals varying more than 10% from baseline. The slope, E,,, of the linear regression of
20,0
the LV end-systolic pressure-diameter relationship by the least squares method was used as an index of cardiac contractility: P,, = E,, (D,, - D,,). where P,, and D,, are LV end-systolic pressure and diameter, respectively. D, is the intercept of the diameter axis. Percent of systolic shortening was calculated as the percent of systolic dimension change divided by the end-diastolic dimension as previously described.’ Stroke volume and systemic vascular resistance were calculated using standard formulas. Results were summarized as mean f SEM. Statistical analysis included analysis of variance for repeated measures and t-test with Bonferroni correction. Significance was recognized at a probability less than 0.05.
Fig 1. Representative loops from one dog demonstrating LV end-systolic pressure-diameter relationship obtained during vena caval occlusion. Data of LV pressure (mm Hg) and minor axis (MA) diameter (mm) from 12 cardiac cycles during a control period are digitized at B-millisecond intervals. Note the regression line at the upper left corner of loops (slope E,, = 14.8 mm Hg/mm and correlation coefficient r = O.sS8).
MECHANISM
OF PROTAMINE-INDUCED
145
HYPOTENSION
Table 1. Systemic Effects of Protamine Sulfate Group
HR (beats/min)
103 f 6
102 + 7
b
112?7
109 * 8
c
109 + 9
107% 6
b
CO (L/min)
SV (mL)
LVP peak (mm Hg)
LVEDP (mm Hg)
SVR (dyne. s. cm-“)
PS
a
a MAP (mm Hg)
Baseline
97 f 5 104*
5
% Change 1.6 f 7.8 -3.7
IT 9.6
2.2 ? 9.1
58 + 5*
-45.5
? 5.2
56 -t 6*
-49.5
+ 6.7
c
93 ? 4
-7.6
+ 4.5
a
3.0 2 0.3
2.4 + 0.5’
86 r 6
-21.8
2 2.9
b
3.1 ? 0.4
2.5 + 0.6*
-18.7
+ 3.6
c
2.9 ? 0.5
3.1 2 0.7
a
28.9 + 2.6
22.8 2 2.9*
-19.0
& 6.7
b
29.0 2 3.4
23.4 ? 2.7”
-17.2
2 9.7
c
27.7 2 2.3
29.3 & 3.1 89 2 7*
2.4 + 7.9
3.1 2 8.2
a
113?6
-12.7
+ 6.3
b
120 + 7
105 * 9
-9.5
2 3.2
c
118+7
115 + 6
-0.8
+ 9.0
a
7.7 * 0.5
9.1 2 0.8
21.4 2 14.9
b
6.5 + 0.6
9.6 ? 0.9’
24.3 t 10.6
c
5.8 + 0.7
7.2 k 0.6
4.0 + 2.8
a
2581 ‘- 121
1712 + 200’
-33.9
? 17.5
b
2746 -c 179
1608 + 243*
-35.6
2 24.5
c
2389 ? 147 2160 + 131
-5.6
* 10.4
NOTE. Groups are a, rapid infusion of PS (given in ~30
set); b,
histamine antagonists + rapid infusion of PS; and c, slow infusion of PS (given in > 2 min). Abbreviations:
PS, protamine sulfate; HR. heart rate; MAP, mean
arterial pressure;
CO, cardiac output; SV, stroke volume; LVP, left
ventricular pressure;
LVEDP, left ventricular end-diastolic
pressure;
SVR, systemic vascular resistance. Mean t SEM. *P < 0.05 compared with baseline.
did not reach statistical significance. Cardiovascular depression induced by rapid infusion of PS was independent of preheparinization and was not observed following a repeated dose administered within a 60-minute interval. Pretreatment with diphenhydramine and cimetidine did not attenuate the effects of rapidly infused PS. Data obtained following administration of antagonists are presented in group b of Tables 1 and 2. In contrast to rapid infusion, there were no significant changes in either systemic or cardiac responses to slow infusion of PS (given in > 2 minutes) as shown in group c of Tables 1 and 2. DISCUSSION
of the present study demonstrate that protamine causes systemic hypotension by a combination of vasodilator and myocardial depressant effects. This systemic hypotension is significantly attenuated when protamine is administered at a slow rate, suggesting an infusion rate-dependent effect on the cardiovascular system. Rapid infusion of protamine was associated with a significant decrease in arterial pressure, which was attributed in part to a decrease in SVR secondary to arterial vasodilation. This finding was consistent with observations made by other investigators in both animal@’ and humans.‘.’ Whether this vasodilation also alters venous capacitance to cause a reduction in preload and a resulting decrease in CO remains unclear. Pulmonary arterial pres-
sure was not measured in these chronically instrumented dogs, but pulmonary hypertension associated with protamine has been well documented.’ Thromboxane production during protamine reversal of heparin anticoagulation has been implicated as a cause of vasoconstriction in the pulmonary bed.” ‘The present results show that LV enddiastolic pressure did not change significantly, suggesting that the decrease in CO observed following rapid infusion of protamine may be caused in part by mechanisms other than preload reduction. In regard to myocardial contractility, previous studies reported conflicting data using LV dP/dt as an index of contractility. Because LV dP/dt correlates to the basal contractile state and is not independent of loading conditions,” this index may not provide a reliable assessment of contractility in the presence of the protamine-induced systemic hypotension. Consequently, LV dP/dt was found to either increase,‘* decrease7.‘3”4 or remain constant’5 after protamine administration. In this study, LV dimensions and pressure were measured simultaneously during vena caval occlusion for the assessment of myocardial performance. Whereas regional performance was evaluated from the percent of systolic shortening of segmental length and wall thickness of the LV, the slope, E,,, of the linear regression of the LV end-systolic pressure-diameter relationship was used as an index of global contractility. This slope has been found sensitive to inotropic changes and independent of loading conditions.‘6~‘R The hypotension induced by rapid infusion of protamine was caused in part by a reduction in CO secondary to myocardial depression. Indeed, cardiac contractility decreased significantly as indicated by decreases in both global and regional indices (Table 2). This finding confirms that the hypotension induced by protamine is not predominantly caused by systemic vasodilation. Although the exact determination of what extent myocardial depression contribTable 2. Myocardial Effects of Protamine Sulfate Group
E,, (mm Hg/mm)
% SS (MA)
The results
% ss (SL)
% SS (WT)
Baseline
PS
% Change
a
13.8 2 2.4
7.7 + 2.3’
-35.6
+ 9.8
b
14.5 * 1.2
8.7 f 1.4’
-37.4
2 11.3
C
14.7 ? 2.8
a
6.4 + 0.6
4.8 + l.O*
-14.9
b
5.7 + 0.8
4.7 * 0.9
-12.6?
C
5.1 ?I 0.9
5.3 f 1.2
a
7.0 + 0.7
6.3 + 1.1
-8.8
? 12.4
b
8.2 + 1.8
7.3 + 0.9
-4.6
2 8.6
C
9.2 k 1.2
8.9 + 0.9
-1.5
+ 4.2
a
9.6 + 1.2
7.7 + 1.2x
-20.7
+ 5.0
b
10.1 + 1.6
8.0 + 1.6*
-15.1
+ 7.3
C
10.8 2 2.0
12.6 ? 1.7
10.7 2 1.7
-3.1
-c 7.4 + 12.5 9.7
3.0 ? 7.1
0.5 + 4.7
Note. Groups are a, rapid infusion of PS (given in ~30
set); b,
histamine antagonists + rapid infusion of PS; and c, slow infusion of PS (given in > 2 min). Abbreviations: PS. protamine sulfate: E,,, index of cardiac contractility; % SS, percent of systolic shortening: MA, minor axis; SL, segmental length: WT. wall thickness. Mean IT SEM.
?? P < 0.05 compared with baseline.
146
KIEN ET Al
utes to the decrease in arterial pressure is not within the scope of this study, a decrease in myocardial contractility may be considered as a major factor accounting for reduced stroke volume and hypotension. The negative inotropic effect of protamine reported in this study is in accordance with the results of earlier studies, in which protamine was rapidly infused.‘,‘j On the other hand, it is at variance with other investigations in which similar techniques for evaluating LV performance were used.ls” These investigators reported an absence of myocardial depression following either intracoronary or intravenous infusion of protamine. The discrepancy between their results and the present ones may be related to the fact that in their studies, protamine was infused either repeatedly at short intervals or over a longer infusion time. In this study, a “refractory” period of 60 minutes was found in which repeated doses of protamine evoked little or no cardiovascular response. Other investigators also reported less pronounced hemodynamic changes in dogs given a second dose of protamine 30 minutes after the initial dose. The failure of sequential doses to elicit a cardiovascular depression for a 60-minute period after the initial dose suggests there is a release of vasoactive substances evoked by rapid protamine infusion. When intravenous infusion was longer than 2 minutes, neutralization of heparin was achieved without any cardiovascular derangement caused by protamine in this animal model (Tables 1 and 2). In vitro studies have been performed using isolated heart muscle preparation to examine the direct effect of protamine on myocardial contractility independent of loading conditions, or neural and humoral reflex mechanisms. The results demonstrated a direct negative inotropic effect of protamine in dogs,‘“,” rabbits,22.Z” and humans.2’ The reported narrow margin of safety of the protamine doseresponse curve in rabbit? may provide some explanation for the differing effects of protamine when infused at various rates. The exposure of the myocardium to toxic concentrations of protamine may occur when intravenous infusion of the drug is rapid. Although hypocalcemia may
have been responsible for the cardiac depression associated with protamine, it remains controversial whether strum level of Ca”
decreases during
infusion
of PS?”
Recent
studies show a reduction in cellular adenosinc triphosphatc and impaired oxygen use following protaminc infusion, which may contribute to the adverse effects of this drug.‘. In spite of earlier reports of H release following PS administrationg.” the present data suggest that H was not involved in the PS-induced cardiovascular depression. The cardiovascular effects of protamine were examined using chronically instrumented dogs so that repeated experimentation was possible. The dog seems to be sensitive to protamine toxicity,h and may exhibit a more severe cardiac depression than that observed in humans. Because of the species-dependent variation in response to protamine, the results of this study should not be extrapolated directly into clinical situations. Nevertheless, in the dog, cardiovascular depression is predictable following rapid infusion of protamine, which makes this animal an excellent subject for studying the mechanism of action of protamine. In summary, the present study demonstrated that rapid infusion of 5 mg/kg of protamine substantially decreased systemic arterial pressure. This hypotension was accompanied by a decrease in LV contractility. The antagonists of H did not attenuate the cardiovascular depression induced by rapid infusion of protamine, suggesting that H was not involved in mediating the effects of protamine. Furthermore, the cardiovascular depression induced by protaminc was not caused by a complementary effect with heparin. It could have been prevented when protamine was infused at a slower rate. The results suggest that protamine should be administered slowly, particularly when myocardial function is impaired.
ACKNOWLEDGMENT The authors their technical script.
thank Richard Martucci and Collette LaRocque for support, and Lynn Gall for preparing the manu-
REFERENCES 1. Horrow JC: Protamine Allergy. J Cardiothorac Anesth 2:225242,198s 2. Jastrzebski J, Hilgard P, Sykes K: Pulmonary vasoconstriction produced by protamine and protamine-heparin complex in the isolated cat lung perfused with blood or dextran. Cardiovasc Res 9X591-696,1975 3. Vatner SF, Franklin D, Higgins CB, et al: Left ventricular response to severe exertion in untethered dogs. J Clin Invest 51:3052-3060,1972 4. Gallager KP, Matsuzaki M, Osakada G, et al: Effect of exercise on the relationship between myocardial blood flow and systolic wall thickening in dogs with acute coronary stenosis. Circ Res 52:716-729,1983 5. Kien ND, White DA, Reitan JA, et al: Cardiovascular function during controlled hypotension induced by adenosine triphosphate or sodium nitroprusside in the anesthetized dog. Anesth Analg 66:103-110, 1987 6. Jaques LB: A study of toxicity of protamine salmine. Br J Pharmacol4:135-144, 1949
7. Goldman BS, Joison J, Austen WG: Cardiovascular protamine sulfate. Ann Thorac Surg 7:459-471, 1969 8. Shapira N, Schaff HV, Piehler effects of protamine sulfate in man. 84:505-514, 1982
effects of
JM, et al: Cardiovascular J Thorac Cardiovasc Surg
9. Frater RWM, Oka Y, Hong Y, et al: Protamine-induced circulatory changes. J Thorac Cardiovasc Surg 87:687-692, 1984 10. Morel DR, Lowenstein E, Nguyen TD, et al: Acute pulmonary vasoconstriction and thromboxane release during protamine reversal of heparin anticoagulation in awake sheep: Evidence for the role of reactive oxygen metabolites following nonimmunologicat complement activation. Circ Res 62:905-915,198s 11. Mason DT: Usefulness and limitations of the rate of rise of intraventricular pressure (dP/dt) in the evaluation of myocardial contractility in man. Am J Cardiol23:516-527,1969 12. Gourin A, Streisand RL, Breineder JK, et al: Protamine sulfate administration and the cardiovascular system. J Thorac Cardiovasc Surg 62:193-204, 1971
MECHANISM
OF PROTAMINE-INDUCED
HYPOTENSION
13. Fadali MA, Ledbetter M, Papacostas C, et al: Mechanism responsible for the cardiovascular depressant effect of protamine sulfate. Ann Surg 180:232-235,1974 14. Marin-Neto JA, Sykes MK, Marin JLB, et al: Effect of heparin and protamine on left ventricular performance in the dog. Cardiovasc Res 13:254-259,1979 15. Green CE, Higgins CB, Kelley MJ, et al: Cardiovascular effects of protamine sulfate. Invest Radio1 16:324-329,198l 16. Van Trigt P, Christian CC, Fagraeus L, et al: Myocardial depression by anesthetic agents (halothane, enflurane and nitrous oxide): Quantitation based on end-systolic pressure-dimension relations. Am J Cardiol53:243-247,1984 17. Mahler F, Cove11 JW, Ross J, Jr: Systolic power-diameter relations in normal conscious dog. Cardiovasc Res 9:447-455, 1979 18. Little WC, Freeman GL, O’Rourke RA: Simultaneous determination of left ventricular end-systolic pressure-volume and pressure-dimension relationships in closed-chest dogs. Circulation 71:1301-1308,1985 19. Taylor RL, Little WC, Freeman GL, et al: Comparison of the cardiovascular effects of intravenous and intraaortic protamine in the conscious and anesthetized dog. Ann Thorac Surg 42:22-26, 1986 20. Iwatsuki N, Matsukawa S, Iwatsuki K: A weak negative
147
inotropic effect of protamine sulfate upon the isolated canine heart muscle. Anesth Analg 59:100-102,198O 21. Lin CI, Luk HN, Wei J, et al: Electromechanical effects of protamine in isolated human atria1 and canine ventricular tissues. Anesth Analg 68:479-485, 1989 22. Caplan RA, Su JY: Differences in threshold for protamine toxicity in isolated atria1 and ventricular tissue. Anesth Analg 63:1111-1115,1984 23. Lee T, Lin Y: Protamine: A cardiotoxic agent with negative inotropism. Anesthesiology 74:635, 1991 24. Johnston CL Jr, Grinnan EL, Wilson HC, et al: Protamineinduced hypocalcemia. Endocrinology 87:211-217, 1970 25. Nahrwold ML, Denlinger JK: The effects of heparin and protamine infusion on serum ionized calcium in anesthetized man. Anesthesiol Rev 312425,1976 26. Conahan TJ, Andrews RW, MacVaugh H: Cardiovascular effects of protamine sulfate in man. Anesth Analg 60:33-36, 1981 27. Wakefield TW, Ucros I, Kresowik TF, et al: Decreased oxygen consumption as a toxic manifestation of protamine sulfate reversal of heparin anticoagulation. J Vast Surg 9:772-777,1989 28. Schnitzler S, Renner H, Pfiiller: Histamine release from rat mast cells induced by protamine sulfate and polyethylene imine. Agents Actions 11:74-75, 1981
of Hypotension
Following Rapid Infusion Anesthetized Dogs
of Protamine
Sulfate in
Nguyen D. Kien, PhD, Darcy D. Quam, DVM, John A. Reitan, MD, and David A. White, MD Protamine sulfate (PS), used to neutralize the anticoagulant effect of heparin, is often associated with systemic hypotension. Whether this hypotension is secondary to a depression of myocardial function is not clear. The present study tested the hypothesis that systemic hypotension was accompanied by a depression in myocardial function and examined the possible role of histamine in mediating the cardiovascular response to PS. Seven conditioned dogs were chronically instrumented with pressure and ultrasonic dimension transducers. Studies were conducted under halothane anesthesia 7 to 10 days after instrumentation. Cardiac contractility was assessed using the slope, E,,, of the linear regression of the left ventricular end-systolic pressure-diameter relationship. Intravenous infusion of PS, 5 mg/kg, when given in periods of less than 30 seconds, decreased systemic arterial pressure by 45% (from 101 f to 54 f 5 mm Hg) without change in heart rate. Cardiac output decreased by 22% from control
P
ROTAMINE SULFATE (PS) is used routinely following cardiopulmonary bypass (CPB) to neutralize the anticoagulant effect of heparin. However, PS may cause profound systemic hypotension, an effect that could be detrimental to outcome in patients with significant cardiac or cerebral vascular disease. Although this hypotensive effect has been well described,’ the exact mechanisms by which PS decreases arterial blood pressure are unclear. It has been postulated that the release of endogenous vasoactive substances, such as histamine evoked by PS, may be involved, but supporting evidence is lacking.* In addition, whether concomitant myocardial depression accompanies the systemic vasodilation remains controversial. The conflicting results from previous studies may relate to (1) the variable rates of administration of PS among studies and (2) differing methodologies used in determining the myocardial effects. The specific aims of this study were to examine the infusion rate-dependent effects of PS on myocardial performance and to evaluate the possible role of histamine in mediating the cardiovascular effects of PS. MATERIALS
AND METHODS
This study was approved by the institutional committee on animal use and care. Seven healthy adult mongrel dogs of either sex, weighing between 20 and 26 kg, were instrumented 1 to 2 weeks before experimentation. The animals were anesthetized with thiamylal (15 mg/kg), and the trachea intubated and ventilated by a positive-pressure Harvard respirator (Harvard Apparatus, South Natick, MA). Anesthesia was maintained with 1.5% halothane in oxygen. Under sterile conditions, a thoracotomy was performed at the fifth intercostal space. Inflatable hydraulic occluders were placed around both inferior and superior vena cavae for varying preload during assessment of myocardial performance. An electromagnetic flow probe (Biotronex Lab Inc., Kensington, MD) was placed around the ascending aorta. A high-fidelity micromanometer (Kanigsberg Instruments Inc., Pasadena, CA) was implanted in the left ventricular (LV) cavity via an apical stab wound for the measurement of LV pressure. This manometer was calibrated against a Statham pressure transducer (Gould Inc., Oxnard, CA) connected to a fluid-filled LV catheter
Journalof Cardiothoracic and VascularAnesthesia,
and the slope E,, decreased by 37% (from 14.5 f 1.2 to 8.7 f 1.4 mm Hg/mm). Systemic vascular resistance decreased by 34% (from 2581 f 121 to 1712 + 200 dyne-s-cme5). The cardiovascular
depression
caused by PS was transient
and could not be reproduced by a repeated dose given within a W-minute
period. Antagonists
mine and cimetidine) cardiovascular
of histamine (diphenhydra-
could not attenuate
the PS-induced
depression. This depression was independent
of preheparinization
and did not occur when PS was infused
slowly over a 2-minute period. The data clearly demonstrate negative
inotropic
and vasodilator
effects of PS following
rapid administration. These findings suggest that PS should be administered slowly to minimize or to avoid its hypotensive effect, paired. Cop y&ht
particularly
when
myocardial
function
is im-
o 1992 by W. B. Saunders Company
implanted adjacently. Pairs of ultrasonic crystals were implanted onto the LV to measure the anterior-posterior transverse diameter on the minor axis, segmental length, and wall thickness of the lateral free wall as previously described.3,4 Proper alignment of the crystals was confirmed with a high-frequency oscilloscope (Textronix, Beaverton, OR). Ventricular dimensions were monitored using a four-channel sonomicrometer (Triton Technology, San Diego, CA). Additional catheters were placed in the aorta via a carotid artery and jugular vein for blood pressure measurement and venous access, respectively. Instrument wires and catheters were tunneled subcutaneously to exit at the intrascapular region and contained in the pocket of an elastic animal jacket. The chest was then closed and the animal was returned to the kennel. Each dog received penicillin and streptomycin for 4 days after surgery. Analgesics were given as needed during the recovery period. The catheters were flushed with heparinized saline and the wound dressings were changed on a daily basis. On the day of the experiment, anesthesia was induced with thiamylal, 15 mg/kg, and maintained at 1.25% halothane in 0,. Mechanical ventilation was controlled, PCO, was maintained at 40 2 3 mm Hg. Instrument wires and catheters were connected to a monitoring panel. Hemodynamic variables were recorded on a Gould direct pen-writing oscillograph (Gould-Brush Inc, Cleveland, OH) and on FM analog tape (Ampex Corp, Redwood City, CA) for subsequent analysis. Baseline measurements were recorded after 20 minutes of hemodynamic stability. PS, 5 mg/kg, was infused intravenously over various rates ranging from 10 seconds to 5 minutes. Infusionls of PS were repeated after 90- to 120-minute intervals except when specified. Each animal received no more
From the Department of Anesthesiology, University of California School of Medicine, Davis, CA. This study was approved by the institutional committee on the care and use of laboratory animals. The use of animals was in accordance with the guidelines of Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Council (DHHS publication No [NIH] 85-23, 1985). Address reprint requests to Nguyen D. Kien, PhD, Department of Anesthesiology, TB-170, University of California, School of Medicine, Davis, CA 95616. Copyright D 1992 by U! B. Saunders Company 1053-0770/92/0602-0005$03.00/0
Vol6, No 2 (April), 1992: pp 143-147
143
144
KIEN Ei 4_
than 3 doses of PS in a single day at infusion parentheses following this experimental plan:
rates
indicated
DAY 1:
1 PS (30 seconds) 2 PS (5 minutes) 3 PS (30 seconds) (infused within ho-minute terval from second dose)
DAY 3:
1 PS (20 seconds) 2 Heparin + PS (20 seconds) 3 PS (3 minutes)
DAY 5:
1 PS (2 minutes) 2 PS ( 10 seconds) 3 Histamine antagonists
On day 1, the infusion
in-
+ PS (10 seconds)
rates of the first and second
doses of PS
were randomized. A third dose of PS was given at intervals ranging from 20 to 60 minutes from the second dose. At the end of the experiment, the animals were allowed to recover from anesthesia and returned to the kennel for a 48-hour resting period. Subsequent
experiments
rate that induced
were performed systemic
in which PS was infused
hypotension)
following
(at a
administration
of heparin, 150 W/kg, to determine the role of the PS-heparin complex. To examine the role of histamine (H), PS was infused (at a rate that induced
systemic hypotension)
following
administration
of H antagonists (diphenhydramine, 1 mgikg, cimetidine, 4 mgi kg). The effectiveness of the antagonists was tested with 5 pglkg of H before use with PS. Following the last experiment, the animals were then killed with an anesthetic overdose and intravenous injection of saturated potassium cloride. Implanting devices were retrieved and their locations were confirmed. Data were sampled over 10 to 20 seconds for assessment of contractility during volume emptying produced by transient vena caval occlusion.
Vena caval occlusion
of data was recorded tion. Paired
was released,
and the next set
after a brief period of hemodynamic
measurements
of end-systolic
LV pressure
RESULTS
in
equilibra-
The dogs were studied 1 to 2 weeks after instrumcnration. They were healthy and fully recovered from the ctfccts of surgery. Representative digital LV pressure and diamcter data from one dog arc shown in Fig 1. This plot depicts the LV pressure-diameter relationship at a control baseline. During vena caval occlusion. the pressure-diameter loop becomes smaller and shifts to the left. End-systolic points are at the upper left corner of loops. Hemodynamic values at baseline and peak pressure response at 2 minutes after the onset of PS infusion arc shown in Tables 1 and 2. Table 1 summarizes systemic effects following either (I) rapid infusion of PS (given in 2 min). When infused rapidly, PS decreased mean arterial pressure and cardiac output (CO) 45.5 -C 5.2 and 21.8 + 2.9%, respectively. However. heart rate and LV end-diastolic pressure did not change significantly. Calculated stroke volume and systemic vascular resistance (SVR) decreased by 19.0 ? 6.7 and 33.9 +- 17.5%, respectively. The decrease in systemic arterial pressure was rapid in onset (10 to 15 seconds after completion of PS infusion) and transient (4.2 lr 0.6 minutes). Data on myocardial effects of PS are included in Table 2. Myocardial contractility, indicated by the slope E,,, dccreased significantly from 13.8 2 2.4 to 7.7 * 2.3 mm Hgimm following rapid infusion of PS. Percents of systolic shortening of LV minor-axis diameter and wall thickness decreased by 14.9 ? 12.5 and 20.7 ? 5.0%, respectively, but the change in systolic shortening of the segmental length
and diame-
ter during 10 to 15 consecutive cardiac cycles with no apparent dysrhythmias were used for each regression analysis. Dysrhythmias were identified by abnormal changes in LV pressure, diameter, and electrocardiogram (ECG). The first cardiac cycle in the series for analysis was selected at the onset of LV pressure reduction so that the ranges of pressure and diameter during vena caval occlusion were minimized. For each heart beat, end-systole was defined as 20 milliseconds before the peak negative dP/dt, and end-diastole was assumed at the peak R wave of the ECG. Respiratory influence on hemodynamic measurements was eliminated by turning off the respirator momentarily during contractility assessment. The autonomic sympathetic reflex caused by preload reduction was minimized by eliminating heart beats with R-R intervals varying more than 10% from baseline. The slope, E,,, of the linear regression of
20,0
the LV end-systolic pressure-diameter relationship by the least squares method was used as an index of cardiac contractility: P,, = E,, (D,, - D,,). where P,, and D,, are LV end-systolic pressure and diameter, respectively. D, is the intercept of the diameter axis. Percent of systolic shortening was calculated as the percent of systolic dimension change divided by the end-diastolic dimension as previously described.’ Stroke volume and systemic vascular resistance were calculated using standard formulas. Results were summarized as mean f SEM. Statistical analysis included analysis of variance for repeated measures and t-test with Bonferroni correction. Significance was recognized at a probability less than 0.05.
Fig 1. Representative loops from one dog demonstrating LV end-systolic pressure-diameter relationship obtained during vena caval occlusion. Data of LV pressure (mm Hg) and minor axis (MA) diameter (mm) from 12 cardiac cycles during a control period are digitized at B-millisecond intervals. Note the regression line at the upper left corner of loops (slope E,, = 14.8 mm Hg/mm and correlation coefficient r = O.sS8).
MECHANISM
OF PROTAMINE-INDUCED
145
HYPOTENSION
Table 1. Systemic Effects of Protamine Sulfate Group
HR (beats/min)
103 f 6
102 + 7
b
112?7
109 * 8
c
109 + 9
107% 6
b
CO (L/min)
SV (mL)
LVP peak (mm Hg)
LVEDP (mm Hg)
SVR (dyne. s. cm-“)
PS
a
a MAP (mm Hg)
Baseline
97 f 5 104*
5
% Change 1.6 f 7.8 -3.7
IT 9.6
2.2 ? 9.1
58 + 5*
-45.5
? 5.2
56 -t 6*
-49.5
+ 6.7
c
93 ? 4
-7.6
+ 4.5
a
3.0 2 0.3
2.4 + 0.5’
86 r 6
-21.8
2 2.9
b
3.1 ? 0.4
2.5 + 0.6*
-18.7
+ 3.6
c
2.9 ? 0.5
3.1 2 0.7
a
28.9 + 2.6
22.8 2 2.9*
-19.0
& 6.7
b
29.0 2 3.4
23.4 ? 2.7”
-17.2
2 9.7
c
27.7 2 2.3
29.3 & 3.1 89 2 7*
2.4 + 7.9
3.1 2 8.2
a
113?6
-12.7
+ 6.3
b
120 + 7
105 * 9
-9.5
2 3.2
c
118+7
115 + 6
-0.8
+ 9.0
a
7.7 * 0.5
9.1 2 0.8
21.4 2 14.9
b
6.5 + 0.6
9.6 ? 0.9’
24.3 t 10.6
c
5.8 + 0.7
7.2 k 0.6
4.0 + 2.8
a
2581 ‘- 121
1712 + 200’
-33.9
? 17.5
b
2746 -c 179
1608 + 243*
-35.6
2 24.5
c
2389 ? 147 2160 + 131
-5.6
* 10.4
NOTE. Groups are a, rapid infusion of PS (given in ~30
set); b,
histamine antagonists + rapid infusion of PS; and c, slow infusion of PS (given in > 2 min). Abbreviations:
PS, protamine sulfate; HR. heart rate; MAP, mean
arterial pressure;
CO, cardiac output; SV, stroke volume; LVP, left
ventricular pressure;
LVEDP, left ventricular end-diastolic
pressure;
SVR, systemic vascular resistance. Mean t SEM. *P < 0.05 compared with baseline.
did not reach statistical significance. Cardiovascular depression induced by rapid infusion of PS was independent of preheparinization and was not observed following a repeated dose administered within a 60-minute interval. Pretreatment with diphenhydramine and cimetidine did not attenuate the effects of rapidly infused PS. Data obtained following administration of antagonists are presented in group b of Tables 1 and 2. In contrast to rapid infusion, there were no significant changes in either systemic or cardiac responses to slow infusion of PS (given in > 2 minutes) as shown in group c of Tables 1 and 2. DISCUSSION
of the present study demonstrate that protamine causes systemic hypotension by a combination of vasodilator and myocardial depressant effects. This systemic hypotension is significantly attenuated when protamine is administered at a slow rate, suggesting an infusion rate-dependent effect on the cardiovascular system. Rapid infusion of protamine was associated with a significant decrease in arterial pressure, which was attributed in part to a decrease in SVR secondary to arterial vasodilation. This finding was consistent with observations made by other investigators in both animal@’ and humans.‘.’ Whether this vasodilation also alters venous capacitance to cause a reduction in preload and a resulting decrease in CO remains unclear. Pulmonary arterial pres-
sure was not measured in these chronically instrumented dogs, but pulmonary hypertension associated with protamine has been well documented.’ Thromboxane production during protamine reversal of heparin anticoagulation has been implicated as a cause of vasoconstriction in the pulmonary bed.” ‘The present results show that LV enddiastolic pressure did not change significantly, suggesting that the decrease in CO observed following rapid infusion of protamine may be caused in part by mechanisms other than preload reduction. In regard to myocardial contractility, previous studies reported conflicting data using LV dP/dt as an index of contractility. Because LV dP/dt correlates to the basal contractile state and is not independent of loading conditions,” this index may not provide a reliable assessment of contractility in the presence of the protamine-induced systemic hypotension. Consequently, LV dP/dt was found to either increase,‘* decrease7.‘3”4 or remain constant’5 after protamine administration. In this study, LV dimensions and pressure were measured simultaneously during vena caval occlusion for the assessment of myocardial performance. Whereas regional performance was evaluated from the percent of systolic shortening of segmental length and wall thickness of the LV, the slope, E,,, of the linear regression of the LV end-systolic pressure-diameter relationship was used as an index of global contractility. This slope has been found sensitive to inotropic changes and independent of loading conditions.‘6~‘R The hypotension induced by rapid infusion of protamine was caused in part by a reduction in CO secondary to myocardial depression. Indeed, cardiac contractility decreased significantly as indicated by decreases in both global and regional indices (Table 2). This finding confirms that the hypotension induced by protamine is not predominantly caused by systemic vasodilation. Although the exact determination of what extent myocardial depression contribTable 2. Myocardial Effects of Protamine Sulfate Group
E,, (mm Hg/mm)
% SS (MA)
The results
% ss (SL)
% SS (WT)
Baseline
PS
% Change
a
13.8 2 2.4
7.7 + 2.3’
-35.6
+ 9.8
b
14.5 * 1.2
8.7 f 1.4’
-37.4
2 11.3
C
14.7 ? 2.8
a
6.4 + 0.6
4.8 + l.O*
-14.9
b
5.7 + 0.8
4.7 * 0.9
-12.6?
C
5.1 ?I 0.9
5.3 f 1.2
a
7.0 + 0.7
6.3 + 1.1
-8.8
? 12.4
b
8.2 + 1.8
7.3 + 0.9
-4.6
2 8.6
C
9.2 k 1.2
8.9 + 0.9
-1.5
+ 4.2
a
9.6 + 1.2
7.7 + 1.2x
-20.7
+ 5.0
b
10.1 + 1.6
8.0 + 1.6*
-15.1
+ 7.3
C
10.8 2 2.0
12.6 ? 1.7
10.7 2 1.7
-3.1
-c 7.4 + 12.5 9.7
3.0 ? 7.1
0.5 + 4.7
Note. Groups are a, rapid infusion of PS (given in ~30
set); b,
histamine antagonists + rapid infusion of PS; and c, slow infusion of PS (given in > 2 min). Abbreviations: PS. protamine sulfate: E,,, index of cardiac contractility; % SS, percent of systolic shortening: MA, minor axis; SL, segmental length: WT. wall thickness. Mean IT SEM.
?? P < 0.05 compared with baseline.
146
KIEN ET Al
utes to the decrease in arterial pressure is not within the scope of this study, a decrease in myocardial contractility may be considered as a major factor accounting for reduced stroke volume and hypotension. The negative inotropic effect of protamine reported in this study is in accordance with the results of earlier studies, in which protamine was rapidly infused.‘,‘j On the other hand, it is at variance with other investigations in which similar techniques for evaluating LV performance were used.ls” These investigators reported an absence of myocardial depression following either intracoronary or intravenous infusion of protamine. The discrepancy between their results and the present ones may be related to the fact that in their studies, protamine was infused either repeatedly at short intervals or over a longer infusion time. In this study, a “refractory” period of 60 minutes was found in which repeated doses of protamine evoked little or no cardiovascular response. Other investigators also reported less pronounced hemodynamic changes in dogs given a second dose of protamine 30 minutes after the initial dose. The failure of sequential doses to elicit a cardiovascular depression for a 60-minute period after the initial dose suggests there is a release of vasoactive substances evoked by rapid protamine infusion. When intravenous infusion was longer than 2 minutes, neutralization of heparin was achieved without any cardiovascular derangement caused by protamine in this animal model (Tables 1 and 2). In vitro studies have been performed using isolated heart muscle preparation to examine the direct effect of protamine on myocardial contractility independent of loading conditions, or neural and humoral reflex mechanisms. The results demonstrated a direct negative inotropic effect of protamine in dogs,‘“,” rabbits,22.Z” and humans.2’ The reported narrow margin of safety of the protamine doseresponse curve in rabbit? may provide some explanation for the differing effects of protamine when infused at various rates. The exposure of the myocardium to toxic concentrations of protamine may occur when intravenous infusion of the drug is rapid. Although hypocalcemia may
have been responsible for the cardiac depression associated with protamine, it remains controversial whether strum level of Ca”
decreases during
infusion
of PS?”
Recent
studies show a reduction in cellular adenosinc triphosphatc and impaired oxygen use following protaminc infusion, which may contribute to the adverse effects of this drug.‘. In spite of earlier reports of H release following PS administrationg.” the present data suggest that H was not involved in the PS-induced cardiovascular depression. The cardiovascular effects of protamine were examined using chronically instrumented dogs so that repeated experimentation was possible. The dog seems to be sensitive to protamine toxicity,h and may exhibit a more severe cardiac depression than that observed in humans. Because of the species-dependent variation in response to protamine, the results of this study should not be extrapolated directly into clinical situations. Nevertheless, in the dog, cardiovascular depression is predictable following rapid infusion of protamine, which makes this animal an excellent subject for studying the mechanism of action of protamine. In summary, the present study demonstrated that rapid infusion of 5 mg/kg of protamine substantially decreased systemic arterial pressure. This hypotension was accompanied by a decrease in LV contractility. The antagonists of H did not attenuate the cardiovascular depression induced by rapid infusion of protamine, suggesting that H was not involved in mediating the effects of protamine. Furthermore, the cardiovascular depression induced by protaminc was not caused by a complementary effect with heparin. It could have been prevented when protamine was infused at a slower rate. The results suggest that protamine should be administered slowly, particularly when myocardial function is impaired.
ACKNOWLEDGMENT The authors their technical script.
thank Richard Martucci and Collette LaRocque for support, and Lynn Gall for preparing the manu-
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