STUDIES OF'THE EFFECT OF Gian-m .CARDiOTONIC AGENTS ON THE CARDIAC - OUTPUT m DOGS AS DETERMINED BY ' THE DYE DILUTiON‘METHOD Thesis for the Degree of M. _S. MICHIGAN STATE COLLEGE Jack R. Schmid 1954' jut-3:5 .— This is to certify that the thesis entitled STUDIES Oi“ THIS EFFEE'I Ob‘ CERTAIN Cf-ILDIUI‘ONIC AGEL‘JTS ON THE CANDIAC OUTPUT IN DOGS 11.8 DnlL‘hEd’iIIx'bD BY 'I'Huj DYE DILUTION I‘lb‘I‘h'OD presented by Jack R. Schmid has. been accepted towards fulfillment of the requirements for MASTER Or' SCIENCE degree in Department of Physiology and PharmaCOIOgy /%/CQ%J% o ){ajor professor Date Iaugust 5, 19514 STUDIES OF 1H3 EFFECT OF CERTAIN CARDIOrONIC AGENTS ON THL CAmUInC OUTPUT IN DOGS AS DdTnflManD DY THE DIE DILUTION.mEiHOD By deck R. Sghmid AN ABSLRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of PJSLER OF SCIuNCE Department of PhysiolOgy and Pharmacology Year l95h Approved Jack R. Schmid THESIS ABSTRACT The Stewart-Hamilton injection method for determining the cardiac output in the intact pentobarbitalized dOg was explored as a.possible method by which to study the effect of drugs e.g., Strephanthus or epinephrine, on the cardiac output. Thirty healthy mongrel male and female dogs were used in this investigation. The dogs were deeply anesthetized with pentobarbital sodium; the common carotid artery was exposed and cannulated to facilitate the collection of blood samples needed to determine the cardiac output; the external jugular vein was made accessible for injection of the dye T-182h and for the drugs used for experimentation. Three milligrams of the dye were used for each determination. A second determination was carried out thirty minutes after the first, and at this time a drug was introduced so that its effect could be measured and compared with the first. The samples of blood containing the dye were collected at two and three second intervals from the carotid artery. The plasma dye concen- trations were determined using a fisher- electrophotometer and the values were plotted on a logarithmic ordinate against a linear time abscissa. The curve was then extrapolated to the base line and delineated. A linear replot was then.made and the area inscribed by the curve was measured. The ordinate dividing the curve into two equal halves is the average concentration from which the cardiac output can.be determined. -1- n (rm-4C“! 51" QJL}‘ij«J Jack R. Schmid The results obtained from six dogs in which two dye injections were made thirty minutes apart were not significantly different and it was assumed that the method was feasible for showing the effect of a drug on the cardiac output. A commercial epinephrine solution was tested and found to give a significant increase in cardiac output in five dogs. Norepinephrine was next tested and it was shown that there was no sig- nificant difference in cardiac output, but that there was a trend toward a decrease in cardiac output. Serveroside, a glycoside of StrOphanthus, produced a profound decrease in cardiac output in some cases and a small but not significant decrease in others. The results depended on the time of the arterial sampling after injection of the drug. -2- STUDIES OF THE EFFECT OF CERTAIN CARDIOTONIC AGENTS ON THE CARDIAC OUTPUT IN DOGS AS DETERMleD BY THE DIE DILUTION METHOD By Jack R. Schmid A TnESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology and PharmaCOIOgy 19Sh AC mMEDcamNTS The author would like to express his sincere thanks to Dr. W. D. Collings for his kind assistance during the course of this research and in the preparation of the manuscript. To Dr. B. V. filfredson for his encouragement, suggestions and the use of the facilities of the department. he is also greatly indebted to Miss Louise Feng and to Mr. Clarence Decker for their assistance in all the experi- ments performed to make this thesis possible. Grateful aclmowledgment is also due to Mr. Jack Monroe for the care of the experimental animals, to Mr. Howard hardy for technical assistance, to Mr. Robert Cornwall for his many helpful suggestions and , finally, his fellow students for their encouragement and kind interest in the problem. WWHH~¥§ as" ' asset—x- TABLE OF CONTENTS Page INIRODUCTION..................................................... 1 REVIEW OF THE LITERATURE......................................... Dilution Principle............................................ The Application of the Dilution Principle to the he asurement of Cardiac Output.......................................... DeveIOpment of the hamilton Method. ... ... .... ....... ....... Objections to the Dye Dilution Method... ....... ..... ....... (ION? w W EXPERIMENTAL-0.00.0.0.0000.00.000000000000000.00000.0.0000000000. 12 General Procedm‘eOOOOOOOOO0.0.0.0....0...-......OOOCOOOOOOOIOO l2 ReSLllts.O......OOOOOOO000......00.000.00.00.......OOOOCOOOO... 19 (Eel-1P OnBOOO'OOOOOIOO0.0.0.000.........OIOO.......OOOOOOOIOO 19 GrOIJ‘p TWOCOOOOOOOOOOOO0.0.00.0...I0.0.0..........OOOOOOOOOO 20 Group ThreGOOOQ000000000000000000000000.0000.000.000.00.000 22 Group Four000000o.coco-.0000000000000000...o.oooooooo-oooo. 23 Group Fiveoooooooooooo.ooooooooooooooooo0.0000000000000000. 2h DISCUSSIONOOOO......OOOOOO...00.0.0.0.OO...OOOOOOOOOOOOOOIOOOOOO. 31 SUMMARX AND CONCLUSIONSOOOOOO......OOOOOOOOOOO0....00.00.000.000. 38 BIBLIOGRAPHY..................................................... ho APP‘EEJDIXOO......OOOOOQOOOOOOOOOOOO0......OOOOOOOOOOOOOOOOOOOOO... SO INTRODUCTION During recent years there has been considerable investigation of the measurement of cardiac output by the dye dilution method. In 1932,‘ Hamilton reported the use of this method in the study of mechanisms in- volved in the regulation of the circulation; in this study he reported the effects of epinephrine on the output of the heart using the dye Brilliant Vital Red as indicator. His procedure was to determine the cardiac output before and after injection of the drug using two successive injections of the dye. however, he presented.no evidence that determining the cardiac output in two successive experiments on the same animal would give comparable results and thereby establish the validity of such a procedure. Surtshin (1950), using T-lBZh, found that the plasma.dye con— centration curves following two dye injections, given h9 to Zhl minutes apart, were not significantly different. These data suggest that if the validity of such a procedure could be established it would have the earmarks of a feasible pharmacological method for determining the effects of various cardiovascular agents on the output of the heart. ‘With a few additional simultaneous measurements e.g., heart rate, hematocrit and electrocardiogram, it would be possible to obtain.manw other important data concerning the cardiovascular status of the subject under the influence of these agents. This technique presented an Opportunity to gain insight to some of the actions and mechanisms involved in phamacodynamics. The selection of the cardiotonic agents to be employed in this study presented the usual problems encountered in most studies of this nature i.e., mode of administration, an effective dose and the time of onset of action. Epinephrine and norepinephrine,1 because of their opposite effects on the total peripheral resistance, were chosen because it was thought that these two sympathomimetic amines would show some interesting effects on the cardiac output, which is dependent to some extent on the resistance to flow. Strephanthus is known to increase the cardiac output of the failing heart, but the normal heart responds to this glycoside in another fashion resulting in a decrease in cardiac output. It seemed worth-while to investigate the effects of Strephanthus on the cardiac output of the dog deeply anesthetized with pentobarbital sodium. The particular form of Strophanthus used in these experiments was sarveroside,2 a short acting glycoside with one-half the activity and toxicity of ouabain . 1Courtesy of Dr. A. M. Lands, Sterling-WinthrOp Research institute. 2Courtesy of Dr. M. J. Vander Brook, The Upjohn Company. REVIEW OF THh LITERATURE Dilution Principle The importance of the dilution principle as a means of studying the composition of the body was reviewed.by Edelman in 1952. The extent to which a substance is diluted in a solvent constitutes a measure of the volume of the solvent . . . . This simple relationship may be expressed mathematically by the following equation: 01"; Ca V3. where C1 and V; are, respectively, the concentration and volume of the solute before dilution, and C, and V; are the concentration and volume after dilution. . . . This equation is derived from the simple consideration that the product of the concentration and volume has the dimension of weight or mass, and within a closed system the mass of the solute is constant regardless of the extent of its dilution i.e., Clvl . Gav; ' The concentration and volume of the solute before dilution are known, and the concentration after dilution is experi~ mentally measured. The only unknown in the equation then is V; which is easily computed. . . . In applying the dilution principle to animal studies it is assumed that the cardiovascular system is a closed system, which of course is not the case at all. A very small amount of liquid is being added or removed all the time so that the dilution curve is not flat but is slep- ing constantly until the tracer is completely removed. The Application_gf the Dilution Principle to the.Measurement of Cardiac Output In 1897, Stewart showed that it was possible to estimate the quantity of blood put out by the heart of a dOg from the dilution of a known amount of injected foreign substance by the blood, which passes through the heart and lungs during a known period of time. Stewart devised two methods to obtain cardiac output which were both'based on the assumption that none of the injected material had time to recircup late through the systemic cardiovascular bed before the sampling was completed. The first method is referred to as the "constant infusion method". In this method the indicator is injected into the blood stream at a constant rate; after a few seconds the indicator was said to have reached a constant concentration in the arterial blood which indicated that it had'been diluted quantitatively by the aortic stream. This is said to occur prior to recirculation and reaches a "concentration plateau" from which cardiac output may be calculated. The following equation gives the relationship between the factors involved in the constant infusion method and the cardiac output: 1. r-._.i__ C where‘s is the flow in.L. per min., i is the rate of injection in mg. per min., and g is the concentration of the indicator at the height of the plateau in mg. per L. The‘ggggndfmethod is really a technical simplification of the first and is often referred to as the "rapid injection method". A known amount of indicator is rapidly injected. The time required for collection of a sample of blood representing the average concentration of the injected substance in the arterial stream is necessary to determine the flow. If the rate of sampling is con- stant, the flow is equal to the amount of injected substance, divided by the product of the average concentration and the duration of sampl- ing during the first circulation of the indicator: where f - flow (L./min.) 60 I I - dye injected (mg.) 2° f ' c t c - average concentration (mg./L.) t - time of first circulation (sec.) It is not clear from Stewart's description of these two methods if he had considered the possibility that the samples taken contained, in part, twice-circulated indicator. hamilton and Remington (19h?) observed while using the method that the indicator begins to recirculate before a “concentration plateau" has been established and that this invalidates a method of this type. Howard, 33 gl_. (1953), confirmed this viewpoint and further pointed out that spurious plateaus are frequently encountered that are unrelated to a valid estimate of flow, and that one cause of these plateaus is fluctuations in venous inflow as often observed in changes during the respiratory cycle. Rashkind and.Morton (19h9) and Wiggers (19%) found the constant infusion method entirely satisfactory in their hands. However, the basic soundness of the Stewart principles is recognized in their adoption for hydraulic measurements (Dow; gt_al., l9h6), in which the flow of water in pipes and rivers is accurately determined. It may be noted that this was independently develOped by engineers (Hamilton, 19h5). Development of the Hamilton Method In 1928, Hamilton, gt al., revived and extended the method by using nonpdiffusing and proteinpbinding dyes. They began a series of studies using Stewart's second method for the estimation of the output of the heart. Injection of the indicator was made rapidly into the jugular vein and serial samples were removed by cardiac puncture from the left ventricle under local anesthetic (novacaine) in dOgs. Samples were collected into small tubes mounted on a revolving kymograph of‘ known speed. The samples were analyzed for dye concentration, colori- metrically against standards, in the Bausch and Lamb microcolorimeter. The cardiac output was calculated in liters per minute using Stewart's second equation: In order to prove the validity of the method, the Hamilton group, under the direction of Kinsman (1929), proceeded to test it in artificial glass models in which recirculation was allowed to occur. They found that it was necessary to find some means of mathematically prolonging the primary curve so that all the dye during its first circulation could be accounted for. The fact that the time of recovery of all the dye approaches infinity suggested to them a 10garithmic scale for con- centration plotted against the time on a linear scale. Such a semi- logarithmic plot made the descending limb of the curve a straight line to the point of recirculation and by extrapolation of this line to the base line the time for complete removal of the dye could be determined, and from this information'an accurate measurement of the average con- centration dm'ing this ”wash out" period could be made by integration of the enclosed curve when replotted on a linear scale. From these recirculation ezqaeriments in glass models the average error was found to be 44.8 per cent. This same group, under the leadership of Moore (1929), next pro- ceeded to test the validity of the method by comparing itwith an accepted method, the direct Fick, to show that in actual practice it gives output values that check. It was found that the average differ- ence between the calculated values for cardiac output by the two methods was only h.7 per cent in six dOg experiments indicating that it was a valid method. When applied to human beings, the method gave values for the cardiac output which were much too high in comparison with those obtained by the Grollman acetylene technique which was then very popular; as a result the method fell into disrepute. When the direct Fick method of estimating blood flow became applicable for human experi- ments by venous catheterization of. the heart, it was found that the values obtained for cardiac output by this method were considerably higher than those obtained by the acetylene method. hamilton, gt _a_l_.. (19h8) , once again investigated the validity of the dye method by com- paring it with the direct Fick in man. The two methods showed satis- factory agreement in forty-two cases with no evidence of systematic differences in the determination of cardiac output. This was confirmed by Doyle, gt 3;. (1953), Nahas, gt 5;. (1953), and by Werko, 31; 3;. (l9h9). Shore (19h5) showed that samples taken from the right auricle or right ventricle may be in considerable error as a result of obtain- ing a nonprepresentative sample of mixed venous blood. The dye method has been compared with the isotope dilution method by Dow, it 11. . (191m) and Lawson, gt El. (1952b), and it.was found that the curves for the dye and the tagged cells were practically identical, but there were indications of a.more rapid transit of the cells in some part of the circuit. When the Hamilton dye method was compared with the cuvette and earpiece oximeter dye methods the average differences of cardiac output was of the order of 3.5 per cent (Ring, 1952). Objections to the de Dilution Method The Stewartedamilton method was criticized on the grounds that some of the dye was being retained in the lungs, and that this was re- sponsible for the high values obtained for cardiac output. Hamilton, _e_t_ 5;. (193a), affirmed that the dye he used in earlier studies, phenol- tetraiodépthalein sodium, was not a non-diffusible dye, and that it should not be used in this method; at the same time they showed, by perfusion of the lungs and the left heart, that the dye "brilliant vital red" did not diffuse from the pulmonary vascular bed during its first passage through; and that all the dye injected could be recovered. Gregersen and.Rawson (19h3) found that the dye T-182h was so firmly bound to the albumin that its disappearance rate, during the first hour after injection, was a measure of the rate of escape of the circulating albumin. Dow and.Hahn (19h6), and later Lawson, gt a1. (1952), found no preferential retention of dye in or on the vessels of the lesser circulation. It was concluded by Dow and Hahn that the high cardiac outputs obtained by the dye injection method were not in error as the result of retention of dye in the lungs. Hamilton, 25.3io (l9h8), defended the use of large vessel hematocrit, in preference to whole body hematocrit, on the grounds that the flow is measured from the great veins to the great arteries, each of which has the same hematocrit, The trapping of plasma among the cells has been shown by Gregersen, Gibson and Stead (1935) to leave four per cent of T-182h unaccounted. for in the plasma above the cells. This cannot be dismissed as a possible source of error in cardiac output studies on the assumption that all the samples are likewise affected and therefore the error cancels. The areas inscribed in the linear time concentration curves, using samples which have and have not been corrected for trapped plasma, may not be the same and therefore not yield the same cardiac outputs. The factor used to correct for trapped plasma is considered.by Reeve (19%) to be 0.95 while Gregersen (1951) maintains it is 0.96. This difference, although small in appearance, is one of an aggregate which could determine the accuracy of the method.as Ring, 22.5i- (1952), pointed out. Hamilton and Remington (19h?) observed that when injection of dye was made into the left ventricle it was practically cleared from the stream before recirculation occurred. In general, the dilution curves resulting from the more central injection sites describe a smaller area 10 than do the more peripheral injection curves. The values obtained for the cardiac output from the more central curves were shown to be smaller, by hetzel and Swan (1953), and laggeg by Coe, Best and Lawson (1950) and Lawson, 33 31. (195h), than the peripheral curves. It was pointed out by werko, gt £1, (l9h9), that dilution curves derived from injection into the pulmonary artery nearly approach zero concentration and thus require extrapolation of a.smaller part of the curve, which is most desirable. The variations in results obtained due to the different sites of injection of the dye could account for some of the inconsist- encies reported. While the basic principle for the dilution method is quite simple, its application to research on the cardiovascular system has proved to be more complicated. The accuracy of the dye method seems to rest upon the ability of the investigator to delineate the primary dilution curve (Lagerlof, gt g1., 19b9; Dow and Hamilton, 1950; Nahas,'gt'§l., 1953; and Schreiner, 22 al., 1953). The part played by the state of the subject, e.g., anesthesia, voluntary movement, metabolism, extrinsic innervation of the heart, etc., must be considered. Stewart realized the influence of the many factors which control the output of the heart and discussed them in his treatise on the cardiac output back in 1921. werko, gt 51. (19h9), have further pointed out that the dye method covers the cardiac output during a short interval of time, and it is thus possible that the cardiac output determined by this method is more influenced by the phases of respiration. Perhaps the most undesirable part of the Stewart-Hamilton method is the inconvenience of collecting the many blood samples required, and the marw hours of tedious colorimetry, graphing, and calculating necessary to obtain a single cardiac output (White, 19117, and Lewis, 1953). 12 EXPERDIIENTAL general Procedure Healthy male and female mongrel dogs in a post nutritive state were deeply anesthetized with pentobarbital sodium (approximately 30 mg./kg.) so that the respiratory rate was controlled between ten and fourteen ventilations per minute, and the palpebral reflex was abolished. A mid- line incision was made on the ventral surface of the neck and the external jugular vein on one side and common carotid artery on the other side were isolated by blunt dissection. The vago-sympathetic trunk was carefully separated from the carotid artery and the artery was cannulated with a piece of polyethylene tubing of the preper size, and approximately twenty centimeters long. Electrocardiograms were taken in all the experiments using either a Sanborn "Poltuiso" recorder or a.Cardiotron. It was necessary to obtain the heart rates just prior to the cardiac output determination and at the time when the dilution curve was being formed. This was carried out inmost experiments for both first and second determinations. Approximately three mg. of T4821;1 were carefully drawn into a calibrated syringe and the needle was inserted into the lumen of the exposed external jugular vein. The cannula was checked for flow and 1An amount necessary to insure adequate Optical density in the plasma samples. 13 a sample of blood was drawn for the hematocrit and the blank. The signals for injections of the dye and drug, as well as the signals for the arterial sampling procedure, were accurately recorded on.magnetic tape. The tape recorder and electrocardiograph were started and at the preper signal the dye was injected as quickly as possible into the jugular vein. Serial samples were collected in three m1. collecting tubes, each containing one drop of heparin sodium (10 mg./ml.), from the cannulated carotid artery at two second intervals up to twelve seconds, then every three seconds to twentyaseven seconds at which time the sampling was terminated. Two Wintrobel hematocrit tubes were filled, capped, and centrifuged for forty minutes at 2500 rpm. The samples were centrifuged for twenty minutes and set aside until the experiment was completed. ' The second determination on each dog was made thirty minutes after the first injection of dye. It was carried out in much the same manner as the first determination, the difference being that a drug was generally injected, at a specified time, prior to the injection of the second sample of dye. In order to insure patency of the cannula during the relatively long interval of time between determinations, approxi- mately three to five ml. of heparinized saline solution (10 mg./200 ml.) were flushed through the cannula and this solution was allowed to remain in the cannula until just prior to the beginning of the second determin- ation. Samples for the plasma volume determinations were taken from 1Capacity 1 ml., 3 mm. bore and graduated from 0-100, both up and down the hematocrit tube. 1b The carotid artery at an interval of eight minutes after the injection of the dye for each reapective determination. One ml. samples of plasma, containing the dye, were drawn from the tOps of the sample tubes using clean pipettes. They were diluted to a total volume of three ml. with physiological saline solution in three ml. "Fisher" micro cells. Their optical densities were determined by a Fisher ElectrOphotometer using the 650 mu red filter and subtracting the value obtained for the blank from each sample. The Optical densities Of the blank samples were generally values between 1.5-3.0 indicating a very small amount of lipoid material and hemoglobin present which was not significant enough to interfere with the colorimetry. Due to the fact that T-l82h, at tie concentration desired, did not strictly conform to beer's Law a plotted calibration curvel was employed to obtain the individual sample concentrations. Strict analytical procedure was followed in preparing the series of standard solutions used in plotting the calibration curve. The concentration values of the plasma.aamples were corrected for dilution and expressed in terms of mg. per ml.; and then further adjusted for any variations due to body weight by dividing by mg. dye injected per gram body weight. These values were then plotted on a logarithmic .Ordinate against a linear time abscissa and the dye concentration curve was drawn (Figure 1). There were generally three points in a straight 1Optical density for the ordinate, and concentration in mg. per ml. for the abscicca. This curve was determined by plotting the Optical densities of serial samples Of T-182h diluted to a final volume of three ml. i.e., one ml. of a standard dye solu- tion, one ml. pooled dogs' plasma and one ml. physiological saline solution. 1&0 130 120 ..l;_. 100 Flow 80 mgjml . ’40 mg.iism. 20 l_lli_ " A #4 f‘ l l l 1 s 10 15 20 25 Time in Seconds Fig. 1. Semi-lOgarithmic Plot of Time Concentration Curve. Experiment 68. 15 16 line on the descending limb of the curve so that extrapolation to the base line was quite accurate in most cases. After delineation of the curve a linear plot of the curve was made using one second values from the semi-logarithmic curve (Figure 2). The area inscribed in the linear curve was determined using a heuffel and Esser planimeter, and the ordinate that divides the inscribed curve into equal halves is the average value for reciprocal of flow which is used to determine the cardiac output} The cardiac Output, or flow, is calculated using the following equation: where Fp . plasma flow (ml./gm./min.) c - reciprocal of flow (gms./ml.) t - clearance time” Of dye (seconds) (1) Fp - ENS? The plasma flow may be converted to a whole blood value using the eQuation: where Fp - plasma flow (ml ./gm./min.) Fb - whole blood flow (ml ./gn.) Hct hematocrit Fp 1 .OO-dict (2) Fb - Stroke volumes were calculated from: SV - .2115 where SV - stroke volume (m1./beat) F - whole blood flow (ml ./dog/min.) hR = heart rate (beats/min.) 11c the process of correcting for any variation due to the body weight the average concentration is not obtained; instead a value is obtained which is the reciprocal of flow in terms Of body weight. aTheoretical time in which one central circulation would be cleared of dye if no recirculation occurred. __.1._. Flow m.ml. mg.k. gm. 130 120 110 100 80 70 so b0 30 20 10 17 Area = 16.0 sq. in. Av. value for = 32.0 gms./cc. Flow Clearance time a 20.8 seconds Cardiac output a 90.1 cc ./kg./min. I l I T I t I I 2 h 6 81012 M16 1820 22 2h Time in Seconds Fig. 2. Linear Replot of Time Concentration Curve. Eneriment 613. 18 Plasma volumes were calculated from the eight minute samples using the equation: c where PV -=plasma.volume (ml.) PV-ET . c - dye inJected (mg.) c' - concentration of dye of eight minute sample (mg./ml.) Slood volumes were obtained from the plasma. volumes and the hematocrit as follows: PV 1 .00~Hct BV - where BV - blood volume (101.) Surface areas were calculated from the Rubner formula: S A - 0.107 x wa/a where S A = body surface area (sq. M.) w =- weight of dog (kg.) Cardiac indexes were calculated so that the flow could be expressed in terms of the body surface area: C I . EE— where C I = cardiac index F - flow (L./min.) Stroke indexes were also calculated to express the stroke volume in terms of surface area: S I - g X where S I - stroke index (ml./beat/sq. M.) 19 Results Group One Before the dye method for determining the cardiac output could be accepted as a suitable one for testing the effect of a drug, it was necessary to compare the results obtained from a series of two successive determinations of cardiac output onifluasame dog, and to show that there was no significant difference in their values. Six dogs weighing from eleven to nineteen kilograms were used in this group. The dogs received no drug treatment during the course of the experiments. They were anesthetized with pentobarbital sodium (30 mg./kg.). The Operative procedure was carried out as quickly and carefully as possible to minimize any deterioration of the dog. _A period of approximately thirty minutes were allowed for recovery from the surgery before the experiment was continued.1 Table I presents the data which were collected and computed for this series of experiments. Part A contains the values obtained from the first dye injection. This serves as the control for the values obtained from the second dye injection in part B. Values for the hemato- crit remained unchanged from the first run to the second. Plasma volumes remained the same in dogs two, four and six; Experiment number five shows an increase in plasma volume from the first run of about fourteen per cent. Eightdminute samples were not taken for part B of lThese preliminary steps and precautions were followed throughout theremaining experiments whenever possible. 20 experiments one and three in this group. The plasma volumes could not be computed for these dogs. The cardiac.indexes varied from a dif- ference of eighteen per cent in dog number two, to a difference of less than one per cent in dog number five; the difference between the means of parts A and B was only two per cent. There was little change in clearance time for dogs one, two and four, while dog number six showed a substantial change of 7.3 seconds, which is well over one-half the initial value for clearance of the dye in run A. Dogs three and five showed differences of comparable magnitude but Opposite in di- rection. It should be noted that when the clearance time is either increased or decreased for a particular dog in this group, the cardiac index for this same dog is also changed but always Opposite in direction. This point will be discussed in detail later. There was no significant difference shown for cardiac output (t -=0.hh). Group Two A commercial epinephrine solution was injected rapidly into the external jugular vein of seven dogs, ranging in weight from 7.6 to 25.0 kgms., prior to the determination Of cardiac output. Some of these dogs had been used in a previous experiment in which the stroke index was measured With a strain gauge and/or a hamilton Optical manometer system within two hours prior to this investigation, i.e., dogs three, four and five. The dose and the time from drug injection to blood sampling were varied in an effort to establish a suitable treatment for future experiments. The total volume of epinephrine injected was one 21 milliliter. The dose varied from 1.5 to 2.6 micrOgrams per kilogram (Table 11o) . Table II shows the data collected and calculated for all experi- ments in which the commercial epinephrine was injected with, or just prior to, the injection of the dye. Dogs one and two should be considered in a separate group due to the mode of administration of the drug, i.e., mixed in the dye solution. It is not known if epine- phrine is fully active when mixed with the dye. Furthermore, different time factors in drug action did not warrant including these two types of experiments in the same group. Epinephrine has a very short onset of action and duration which depends, in part, on the rate of its oxidation (Sollmann, 1950). DOgs one and two do not show any change worthy of discussion other than an increase in plasma volume over control of twenty-five per cent for dog one; also an increase of clearance time of 5.6 seconds or thirty three per cent over the control value for the same dog. An examination of the data for the last five dogs in this group present some very interesting changes. hematocrit values were increased slightly in dogs five and seven (7 and 8% reSpectively). In all five remaining experiments of the group the plasma volumes were increased substantially. Cardiac indexes were increased in all dogs with an average of thirty seven per cent. This was significant at the five per cent level (t - 2.h9). Stroke indexes were also increased in every experhment with an average increase of twenty per cent. This was significant at the one per cent level (t = h.28). When the clearance 22 time decreased, i.e., dOgs three and six, the cardiac index and stroke index increased.more than when clearance time increased or remained unchanged. It appears that the heart rates were affected in these experhments. However, due to inadequate measurement of the pre—drug heart rate it is necessary to omit a comparison. Group5Three Valuable information concerning dose and time of onset of action was obtained from the previous experiments using a commercial epinephrine preparation. It was shown that a dose of approximately two micrograms per kilogram body weight, given about five seconds prior to injection of the dye solution, altered cardiac output significantly. This dosage and time schedule was substantiated by Brown (l95h) and supplemented by him with additional facts concerning the use of another sympathomimetic amine, l-norepinephrine. Preliminary dose-reaponse experiments showed that a dose of two micrograms per kilogram body weight of l-norepinephrine bitartrate mono- hydrate, administered rapidly into the external jugular vein, gave a substantial pressor reSponse in the intact anesthetized dOg. The time of onset of the pressor reaponse varied in several experiments on two dogs between 7.5 and 9.5 seconds. It was the purpose of the following experiments to measure the cardiac output, after injecting this drug, and at a time just prior to and during the pressor reSponse. The injection time for l-norepinephrine was set at 5.5 seconds prior to injection of the dye. 23 The results of seven experiments are shown in Table IiI in which the drug is compared to control values. In experiment five the hematocrit increased from 0.37 to O.hl, an increase of eleven per cent; the other six experiments showed no change. Plasma volume increased slightly in five out of seven cases. There is no significant difference in cardiac indexes at the five per cent level (t - 1.38) but attention should be called to the fact that in five of the seven dogs tested the cardiac index fell. Stroke indexes fell to a.much lower level and are significant at the five per cent level (t - 2.h7). Clearance time was prolonged in every experiment, and eSpecially in experiment four where the difference is 29.3 seconds or a lhl per cent increase from the control value. Other experiments, i.e., two and seven, gave values for clearance time substantially increased. Group Four Sarveroside is a glycoside of StrOphanthus with one-half the activity and toxicity of ouabain. The latency in the heart-lung preparation was very short (one to two minutes) and maximum effect was seen in ten minutes (Vander Brook, l95h). Meyers (l95h) found in acute experiments, that two normal dogs responded similarly to intravenous ouabain (0.0h mg./kg.). The cardiac output was reduced thirty per cent after twenty minutes in these two experiments. Preliminary experiments on four normal dogs showed that a dose of sarveroside (0.08 mg./kg.) injected at a constant rate for twenty seconds produced a.mild bradycardia and a slight increase in blood pressure. The blood pressure returned to normal in approximately five minutes. 2h It was concluded from the results of these experiments that at an interval of one and one-half minutes after the beginning of injection of the drug, a substantial change had occurred in the cardiovascular system; and that the determination of the cardiac output at this time would probably show a significant change from the control value. Table IV lists the data obtained on five degs ranging in weight from 9.1 to 11.5 kgms. and treated using the procedure outlined above. The hematocrit fell slightly in dOgs two, three and four. Changes in plasma volume were as follows; it decreased in dogs one, three and four, and increased in dogs two and five. The changes seen in dogs two and three were large (21 and 27% reSpectively), but in the Opposite direction. The cardiac indexes in all experiments were decreased from control values, with a.mean decrease of forty-six per cent. This was signifi- cant at the one—tenth per cent level (t - 13.6h). Stroke indexes also decreased in all experiments with a.mean decrease of forty-two per cent. This was also significant at the one-tenth per cent level (t - 6.02). Clearance times were increased in all experiments. The magnitude and particularly the direction of the change in clearance time very Obviously indicate a relationship between clearance time and cardiac output. Group Five The procedure was the same as for the previous group with the exe ception of the time of determination of cardiac output after injection of the drug. The preliminary dose-reSponse experiments indicated that 25 the effect of the drug had changed considerably at the end of five minutes in the direction of normalcy. Sarveroside was injected five minutes before the determination of cardiac output in five dOgs ranging in weight from 8.5 to 9.8 kgms. Table V presents the data for this group. The hematocrit values, with one exception, increased in this group in contrast to Group Four in which all decreased slightly. Dog one shows an increase of seventeen per cent over the control, and dOg three also shows a definite increase. Plasma volume demonstrates the same variation as in Group Four. It appears that the trend is for a mild decrease in.plasma volume after Sarveroside. Cardiac indexes were generally decreased, but d0g one demonstrated a substantial increase. Although the average difference between the control and drug values was twenty-nine per cent, this was not significant at the five per cent level (t = 1.23). It appears that if more dogs would have been used there would have been a significant decrease in cardiac output. Stroke indexes decreased in four out of five cases. Dog one showed an increase which once again interfered with the possible significance of these values at the five per cent level (t - 1.51). As was expected, the clearance time increased for dogs demonstrating a decrease in cardiac output, and decreased for the one exception, d0g number one. 26 TABLE I CARDlAC OUTPUT* FnOM TWO SUCCESSIVE lNJbCTIONS OF T-182h Controls Experi- Surface Plasma. Cardiac Cardiac Clearance ment weight Area hemato- Volume Output index Time No. (kg.) (M2) crit (00.) (cc./min.) (L/min./h2) ksec.) A. First Dye lnjegtion 1A 19.3 0.77 0.h6 627 h396 5.71 13.0 2A 12.1 0.56 0.51 596 3506 6.26 11.1 3A 1h.0 0.62 0.37 689 2117 3.h1 22.3 hA 11.0 0.52 0.h2 6h9 1516 2.92 22.2 5A 13.6 0.61 0.u6 632 2569 b.21 19.0 6A 11.0 0.52 0.h3 5&5 2092 h.00 13.5 Mean h.b2 S.E. i 0.53 B. Second Dye Injection--Thirty Minutes Later 15 19.3 0.77 0.h6 -- h969 6.h5 12.3 28 12.1 0.56 0.51 596 hl2h 7.36 12.2 38 lh.0 0.62 0.36 -- 1823 2.9h 18.3 be 11.0 0.52 0.h3 6u1 1&6? 2.82 21.5 55 13.6 0.61 0.h6 719 2501 b.23 23.9 68 11.0 0.52 0.hh 551 1770 3.b0 20.0 Mean 14 .53 S.E. i 0.95 t - differences 0.hh a Derived from plasma dye concentration. «...-"f. .v-.-.— I- , a _ . l 9 o . C n _. _ . . . _ _ r . . a s . n _ _ , . U c . . u . i , a . a, ,. . n ,. A . . , _ V 6 , v _ 9 v a c a O a _ . . / _ c u s . a . . .‘HJ 5-! V .QUI- - r~—. «-.. 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