ouabain pharmacokinetics in and - homepage |...

Ouabain Pharmacokinetics in Dog and Man

Determination by Radioimmunoassay

BY RICHARD SELDEN, M.D., AND THOMAS W. SMITH, M.D.With the technical assistance of William Findley, B.A.

SUMMARYThe pharmacokinetics of the relatively polar and rapidly acting cardiac glycoside

ouabain (G-strophanthin) were studied in dogs and human subjects by the use of anewly devised radioimmunoassay technic. This method had high specificity and a sen-sitivity of less than 0.1 ng of ouabain per ml of plasma or urine. After administration ofa single intravenous dose, the plasma ouabain concentration fell rapidly in both dogsand humans. After 7 hours, a phase of exponential decline was reached which had ahalf-life of 18 hours in dogs and 21 hours in normal human subjects. Repeated intra-venous administration of ouabain to human subjects for 9 consecutive days resulted inthe establishment of a plateau of plasma concentration and urinary excretion after 4-5days, confirming plasma and urinary half-lives in the 19-24-hour range. The meanratio of renal clearance of ouabain to that of creatinine was 0.81 in the human subjects.The plasma half-life of ouabain determined in these studies is in good agreement withprevious observations of the half-life of dissipation of positive inotropy and of slowingof ventricular response to atrial fibrillation after ouabain administration.

Additional Indexing Words:Cardiac glycosideOuabain-specific antibody

Digitalis glycosideTritiated ouabain

G-Strophanthin

A NUMBER of useful studies reported inrecent years have defined the half-life

and metabolic fate for the clinically importantcardiac glycosides digoxin and digitoxin.' Lesscomplete data have been reported for themore polar and rapidly acting glycosideouabain (G-strophanthin). A controversialaspect of previously reported investigationshas been the half-life of the late phase ofexponential decline of serum ouabain concen-

From the Cardiac Unit, Department of Medicine,Massachusetts General Hospital and Harvard MedicalSchool, Boston, Massachusetts 02114.

Supported by the U. S. Public Health Service,National Heart and Lung Institute Grant IR01 HE14325, and Training Grant HE-5196, and by a grantfrom the Massachusetts Heart Association, NortheastChapter.

Address for reprints: Dr. Thomas W. Smith,Cardiac Unit, Massachusetts General Hospital, FruitStreet, Boston, Massachusetts 02114.

Received October 27, 1971; revision accepted forpublication January 7, 1972.

tration. Estimates of the half-life of serumradioactivity after intravenous administrationof tritiated ouabain ( 3H-ouabain) haveranged from as little as 5.5 hours in one studyof normal human volunteers2 to 50 hours inanother series of subjects with normal cardiac,renal, and liver function.3 The half-life ofdissipation of the positive inotropic effect, onthe other hand, has been estimated at 22hours.4 Slowing of the ventricular response toatrial fibrillation after a single intravenousdose of ouabain was found to be about 50%dissipated after 1 day.5The purpose of the present study was to

delineate the plasma kinetics of ouabain bymeans of a radioimmunoassay with sufficientsensitivity to measure picomolar concentra-tions of unlabeled ouabain in biologic fluids.6The plasma disappearance curve after singleintravenous doses of ouabain was determinedin dogs and normal human subjects. Plasmaconcentrations and urinary excretion were also

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OUABAIN PHARMACOKINETICS

studied in normal human subjects understeady-state conditions produced by dailyinitravenous injections of ouabain over a 9-dayperiod.

Methods

Assay Method

Details of the assay method are presentedelsewhere.6 Ouabain-specific antiserum with an

average intrinsic association constant (Ko) of1.3 x 109 M-1 and restricted heterogeneity ofbinding site affinities was obtained by serialinjections of rabbits with ouabain covalentlycoupled to human serum albumin through an

extended side arm of poly-DL-alanine. Suitableamounts of 3H-ouabain (specific activity 11.7Ci/mmole, New England Nuclear Corporation,Boston, Massachusetts) and ouabain-specific anti-body were added directly to plasma samples. Forurine assays, small aliquots of urine (10-50,uliters) were added. Following a brief period ofequilibration of labeled and unlabeled ouabainwith antibody-binding sites, activated charcoalcoated with molecular-weight 80,000 dextran was

added. Free ouabain was selectively bound to thecharcoal and centrifuged, allowing antibody-bound 3H-ouabain to be decanted into a toluene-detergent base scintillation fluid and counted in a

liquid scintillation spectrometer. Quenching vari-ation was corrected by the use of a 226radiumexternal standard. Unknown samples were esti-mated by comparison with a standard curve

constructed with known amounts of the same

ouabain preparation (Eli Lilly) used in theexperimental animals and human volunteers.These standards were prepared using plasmaobtained prior to ouabain infusion.The sensitivity of the method allowed quantita-

tion of concentrations as low as 0.05 ng/ml (fig.1). Replicate measurement of 10 identicalsamples yielded a standard deviation of 5% or

less over the range from 0.05 to 10 ng/ml.Control plasma and urine samples to whichknown amounts of ouabain had been added were

stored in the dark at 40 for 30 days to teststabilitv and recovery under these conditions.These samples yielded ouabain concentrations byradioimmunoassay which were within 5% ofvalues derived from a freshly prepared standardcurve over the range 0.1 to 10 ng/ml. Specificitystudies showed no interference from endogenoussteroid concentrations well above those occurringphysiologically. The rhamnose moiety of ouabaindid not produce measurable displacement of 3H-ouabain from the antibody-binding site whenpresent in 35,000-fold molar excess. False-positivevalues were not encountered in subjects notreceiving cardioactive drugs.Circulation, Volume XLV, June 1972

i.t.5

1Q

i.;

40

30

20

10

0.05 Q0 0Q2 0.5 2 5 10 20

UNL/YABELED 0(A1A4/N (ng/m//

Figure 1

Semilogarithmic plot of standard curve. Range ofduplicate determinations shown. The arrow on thevertical axis indicates percent antibody-bound3H-ouoabain in the absence of unlabeled ouabain. 1 ng

of 3H-ouabain tracer was used.

Canine StudiesUnder intravenous methohexital (Brevital)

anesthesia (1 mg/kg), a polyethylene catheterwas inserted into the external jugular vein ofseven 16-21-kg mongrel dogs. A control sample ofblood was obtained from each dog. Ouabain,0.025 mg/kg in 10 ml of normal saline, was theninfused into a foreleg vein over a period of 30 sec.

The jugular venous catheter, kept patent withheparinized saline, was used to obtain bloodsamples at 1/30, 1/12, 1/6, 1/4, 1/2, 1, 2, 3, 5,7, 12, 24, 30, 36, and 48 hours after the injection.The dogs were awake and ambulatory afterplacement of the jugular venous catheter.

Human StudiesThree healthy male subjects, ages 26 to 35

years, without renal, hepatic, cardiac, or otherdisease, volunteered for this study. Table 1 listsanthropometric and creatinine clearance data forthese subjects. Fully informed consent was

obtained from each subject. Each was given 0.5

Table 1Human Subjects

CreatinineAge Height Weight clearance

Subject (yr) (cm) (kg) (liters/24 hr)

1 32 190 82 196

2 35 180 73 193

3 26 168 73 146

i = mean t ronge

, I_ I. A

r.1,

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mg of ouabain intravenously via an antecubitalvein; blood samples were subsequently obtainedfrom the contralateral arm at 1/12, 1/4, 1/2, 1, 2,4, 7, 9, 12, 24, 33, and 48 hours after theinfusion. This experimental protocol was carriedout on two occasions with an interval of 2 monthsbetween studies. Ouabain and creatinine concen-trations in urine samples collected between 7 and9 hours after ouabain administration and in bloodsamples drawn at 7 and 9 hours were used tocalculate ouabain-to-creatinine renal clearanceratios. Creatinine concentrations were determinedwith an alkaline picrate assay.7

In addition to measuring plasma disappearanceafter a single dose of ouabain, experimentsdesigned to achieve a steady state were carriedout. Measurements were made during a period ofdaily intravenous administration of 0.25 mg ofouabain for 9 consecutive days. Blood sampleswere obtained just before each daily dose and at24 and 48 hours after the last intravenous dose ofouabain. All urine was collected for a period of 12days, including the 9 days of ouabain administra-tion and the 3 days subsequent to the last dose.

SamplesAll blood samples were drawn into heparinized

tubes, centrifuged, and the plasma immediatelyseparated and stored at 4°C. Urine volumes weremeasured and aliquots stored at 4° C. Ouabainconcentration determinations were carried outwithin 10 days in all cases. As noted above,control experiments showed no significant changein the concentrations measured under the storageconditions used for periods of up to 1 month.Heparinized plasma samples gave results identicalto those obtained with serum samples simulta-neously collected.

StatisticsStatistical evaluation, including least-squares

linear regression analyses, were perfoimed byconventional methods8 with the aid of an IBM360-65 time-sharing computer.

ResultsCanine Studies

Plasma ouabain concentration after intra-venous injection fell rapidly at first, thenreached a stable exponential decay phase after7 hours. Figure 2 is a semilogarithmic plot ofmean plasma ouabain concentration againsttime following the intravenous administrationof a single dose of 0.025 mg/kg of ouabain toeach of seven dogs. The initial very rapiddecline in plasma ouabain concentration had ahalf-life of about 3 min and probably

10

0.5

Meon + S.E.M.

HOIRS

Figure 2

Semilogarithmic plot of mean canine plasma ouabainconcentration vs time. Data are from seven dogs givena single intravenous dose of ouabain, 0.025 mg/kg. Thelate exponential phase has a half-life of 18 hours asdetermined by least-squares linear regression analysisof mean plasma concentrations from 7 to 48 hours(r - 0.98).

represents mixing in the intravascular com-partment as well as the initial phase of tissueuptake. This was followed by a period ofabout 6 hours characterized by a graduallydecreasing rate of ouabain disappearance.This phase was thought to represent primarilytissue uptake and distribution, with an addi-tional contribution from excretion. About 7hours after ouabain administration, a phase ofexponential decline of plasma concentrationwith a half-life of 18 hours was establishedwhich remained constant up to 48 hours.During this late phase between 7 and 48 hoursafter ouabain administration, plasma half-livesin the individual dogs ranged from 17 to 24hours.

Human StwdiesPlasma ouabain kinetics following a

single intravenous injection in normal humansubjects were very similar to the canine data.Figure 3 is a semilogarithmic plot of meanplasma ouabain concentration following theintravenous administration of a single 0.5-mgdose to each of three subjects on two

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12.0OF

10.001

t00 f Meon ± S.E.M.

200

0.0O

0.50

0.20

K_<

E0.0[

0.05L002 5 to 15 20 25 30 35 40 45 50

HOURS

Figure 3

Semilogarithmic plot of mean human plasma ouabainconcentration vs time. Mean values for six experimentsare plotted. The half-life of plasma ouabain concen-tration between 7 and 48 hours is 21 hours (r = 0.99for least-squares plot).

occasions separated by 2 months. The plasmaouabain concentrations at each interval afterouabain administration were less in thehumans than in the dogs, as would beanticipated from the relatively smaller dosesgiven on an mg/kg basis. Nevertheless, theshapes of the curves in the two species werevery similar. The half-life of mean plasmaouabain concentration for the late phase ofexponential decline as determined by least-squares linear regression analysis was 21hours. Half-lives in the individual subjectsranged from 18.4 to 25.0 hours. with a meanvalue of 21.8 + 2.0 hours (table 2).

Determination of simultaneous ouabaini andcreatinine clearances during the late phase ofexponential decline yielded a mean ouabain-to-creatinine renal clearance ratio of 0.81, witha range from 0.73 to 0.90 in individualsubjects, as shown in table 2.

In addition to these studies following asingle dose, plasma levels and urinary excre-tion of ouabain were measured in furtherexperiments designed to produce a steadystate. The same three subjects were given 0.25mg of ouabain intravenously at 24-hourintervals for 9 consecutive days. Figure 4, top,shows the mean plasma ouabain concentration

Table 2Single-Dose OuabainSubjects)

Administration (Human

Ouabain: creatininePlasma Corr (renal clearance

Subject Half-life (hr) coeff* ratio)

1 25.0 0.96 0.90

it 18.4 0.972 21.2 0.97 0.802t 23.2 0.983 22.0 0.99 0.733t 20.8 0.96

Mean 21.8 (- 2.0 SD) 0.81

*For best linear fit by least-squares linear regressionanalysis of plot of log ouabain concentration vs timebetween 12 and 48 hr.tThe second of the two studies in each subject was

carried out 2 months after the initial experiment.

immediately prior to each dose. A plateau wasestablished at 4-5 days. Mean values for 24-hour urinary excretion of ouabain during thesame period are also shown in figure 4,bottom. A rise in ouabain recovered in theurine was observed until the fourth or fifthday, when a plateau was established concomi-tant with the plasma level plateau. Mean

0.25mg OUABAIN l.V. Mean±SEM

0.6

0.5

11,1T ~~~~~~~~~~~ng/m/

0 2 3 4 5 6 7 8 9 10 11 12

DA YS

Figure 4

(Top) Mean plasma ouabain concentration immedi-ately prior to daily intravenous administration of 0.25mg to three human subjects. Vertical axis shows thepercent attainment of steady-state plateau. (Bottom)Mean 24-hour urinary ouabain excretion during thesame experiment.


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plasma concentration after establishment of asteady state (days 5-9) varied from 0.41 to 0.64ng/ml among the individual subjects (table3). Individual mean 24-hour urinary recover-ies of ouabain during the steady state variedfrom 86 to 94 jig with a mean urinaryrecovery for the group of 91 .g (37%) of the250 gg administered each day. Establishmentof a plateau of plasma concentration and ofurinary excretion occurred between the fourthand fifth day in each individual. Following thelast dose of ouabain, plasma concentration andurinary excretion declined with half-lives ofabout 20 hours.

DiscussionPrevious studies have employed tracer doses

of 3H-ouabain given to experimental animalsand human subjects.2' 3, 9-11 Renal clearancehas been found to be the predominant routeof excretion, with 45-63% of administeredradioactivity recovered in urine over the initial24 hours.2 Biliary excretion has been estimatedat only 2-8% for the initial 24 hours.2 Comparedwith normal controls, patients with renalinsufficiency have been reported to maintainhigher serum levels of the drug with prolonga-tion of the half-life of the late phase ofexponential decline.3 1 Impaired hepaticfunction, on the other hand, did not alterserum half-life or urinary excretion of 3H-ouabain.3 Hyperthyroidism shortened the se-rum half-life and increased the rate of urinaryexcretion of ouabain, while hypothyroidismhad opposite effects."

Prior estimates of plasma ouabain half-lifein man, limited by the time interval during

Table 3Plateau Values during Dailyistration of Guabain

Intravenous Admin-

Urinary excretionPlasma cone (ng/ml)* (ug/24 hr)

Subject Meant Range [ Meant Ranget

1 0.64 0.62-0.65 92 87-972 0.47 0.44-0.48 86 80-913 0.41 0.40-0.43 94 82-110

Mean 0.51 91

*24 hours after last dose.tDays 5 to 9.

which plasma radioactivity could be accurate-ly measured after administration of 3H-ouabain, have varied widely. Marks et al.2estimated the half-life at 5.5 hours in humansubjects, but cautioned that their data ex-tended for a relatively short period (about 2hours) after ouabain administration. Esti-mates of 50 hours based on reliable counts upto 6 hours3 and 13.9 hours based onmeasurements for 24 hours10 after 3H-ouabaininfusion have also been reported for normalcontrol patients and human volunteers, re-spectively. The radioimmunoassay of plasmaouabain used in our studies, with a sensitivityof less than 0.1 ng/ml, allowed accuratedetermination of plasma ouabain concentra-tion for more than 48 hours after a singlesubdigitalizing dose of 0.5 mg. This methodyielded a mean plasma ouabain disappearancecurve with a late exponential half-life of 21hours in normal human subjects, as shown infigure 3. This value was similar to the meanplasma half-life of 18 hours observed in ourcanine experiments (fig. 2). It is of interestthat a similarity between canine12-14 andhuman15 plasma digoxin half-lives has alsobeen observed.

It should be noted that plasma and urinaryconcentrations of ouabain determined by ourmethod represent maximum possible concen-trations of native ouabain present in a sample.Biotransformation, if it occurs, might yieldproducts capable of displacing 3H-ouabainfrom the antibody-binding site, thereby in-creasing the apparent ouabain concentration.A similar problem exists, of course, whenouabain concentrations are determined bymeasurement of tritium counts after adminis-tration of 3H-ouabain.

Another approach to estimating the plasmahalf-life of a cardiac glycoside (or otherdrug), the concentration of which declines inan exponential fashion, is to determine thelength of time necessary to establish a plasmaconcentration plateau with administration ofthe drug at regular intervals. The shape ofsuch a curve is described by the generalequation:

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where %0P = percent attainment of the steady-state plateau; k = the rate constant of elimina-tion; e = the natural logarithm base; andt = time.16 After one half-life, %P = 50%. Simi-larly, after two, three, four, or five half-lives,%P is 75, 87.5, 94, and 97%, respectively. Thishas been experimentally documented for 3H-digoxin in humans17 and dogs.'8 In our steady-state experiments, establishment of a plasmalevel and urinary excretion plateau after 4-5days, as shown in figure 4, indicates a half-lifeof elimination in the 19-24-hour range, in goodagreement with the mean plasma concentra-tion half-life of 21 hours observed after singledoses of ouabain.Recovery of radioimmunoassayable ouabain

from the urine of human subjects during thesteady-state plateau averaged only 37% of theintravenously administered daily dose (fig. 4).The remaining 63% presumably underwentbiotransformation to a form which interactedless strongly with the antibody-binding sitesand/or elimination from the body by anonrenal route. Further studies to clarify thisissue are underway.The plateau principle is applicable not only

to the time course of increase in plasmaconcentration or excretion of a cardiac glyco-side, but also to pharmacologic effects. Goldand DeGraff19 measured the ventricular re-sponse to atrial fibrillation during the dailyadministration of digitalis leaf and observed aplateau after about 4-5 weeks, compatible withan excretory half-life of about 6 days. Similarventricular response data for digitoxin showeda plateau at 3 weeks20 compatible with a half-life of about 4.2 days, in good agreement withthe 4.8-day half-life of plasma level declineobserved by Lukas.2'

Serial quantitation of such effects as positiveinotropy and slowing of the ventricularresponse to atrial fibrillation after single doseshas been employed to determine the effectivepharmacologic half-life of cardiac glyco-sides.4 5 Weissler et al., using systolic timeintervals from carotid pulse tracings as anindication of left ventricular contractility,determined the half-lives of dissipation of thepositive inotropic effects of digoxin andCirculation, Volume XLV, June 1972

50-

T:XZ

8 16 24 32 40 48 56HOURS

Figure 5

Semilogarithmic plot of mean change in heart rate vstime for seven patients with atrial fibrillation after asingle intravenous dose of ouabain. Data are from thestudy of Gold et al.5 The half-life of pharmacologiceffect is 23 hours.

digitoxin to be 33 and 102-112 hours, respec-tively.4 These values are in good agreementwith direct estimates of the half-lives of thesedrugs in the body.15' 21 The half-life ofouabain's inotropic effect was determined tobe 22 hours in normal human subjects byserial systolic time intervals.4 Similarly, Goldet al. observed the slowing of the ventricularresponse to atrial fibrillation after a singledose of ouabain to be about 50% dissipatedafter 1 day.5 Replotting these data5 as changein heart rate (log scale) versus time, astraight-line function with a half-life of 23hours is obtained (fig. 5). Thus the 21-hourhalf-life of plasma ouabain concentration innormal man, as determined by radioimmu-noassay, is in good agreement with priorestimates based on measurement of pharma-cologic effects.From a clinical standpoint, it is important to

note that the total amount of ouabain in thebody, and hence the risk of toxicity, in apatient placed on any regular dosage schedulewithout a loading dose (irrespective of theinterval between doses) will continue to risefor the initial 4-5 days if renal function isnormal. Accumulation will proceed for an

even longer time in the presence of a

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proloniged excretory half-time due to renialfunctional impairmenit.

References1. DOHERTY JE: The clinical pharmacology of

digitalis glycosides: A review. Amer J Med Sci255: 382, 1968

2. MARKS BH, DUTTA S, GAUTHIER J, ELLIOT D:Distribution in plasma, uptake by the heart,and excretion of H3-ouabain in humansubjects. J Pharmacol Exp Ther 145: 351,1964

3. LAHRTZ HG, REINOLD HM, VAN ZWIETEN PA:Serum concentration and urinary exeretion of3H-ouabain in patients suffering from liver orkidney diseases. Pharmacol Clin 1: 114, 1969

4. WEISSLER AM, SNYDER JR, SCHOENFELD DC,COHEN S: Assay of digitalis glycosides in man.Amer J Cardiol 17: 768, 1966

5. COLD H, MODELL W, KWIT NT, SHANE SJ,DAYRIT C, KRAMER ML, ZAHM W, OTTO HL:Comparison of ouabain with strophanthidin-3-acetate by intravenous injection in man. JPharmacol Exp Ther 94: 39, 1948

6. SMITH TW: Ouabain-specific antibodies: Irnmu-nochemical properties and reversal of Na +, K + -activated adenosine triphosphatase inhibition.J Clin Invest. In press

7. PETERS JH: The determination of creatinine andcreatine in blood and urine with the photoelec-tric colorimeter. J Biol Chem 146: 179, 1942

8. SNEDECOR GW: Statistical Methods, 5th ed. Ames,Iowa, Iowa State University Press, 1956

9. LULLMANN H, VAN ZWIETEN PA: The kineticbehavior of cardiac glycosides in vivo, mea-sured by isotope techniques. J Pharm Phar-macol 21: 1, 1969

10. KRAMER P, HORENKAMP J, WILLMS B, SCHELERF: Das Kumulations-verhalten verschiedenerHerzglykoside bei Anurie. Deutsch Med Wschr95: 444, 1970

11. EICKENBUSCH W, LAHRTZ HG, SEPPELT U, VANZWIETEN PA: Serum concentration and urinaryexcretion of 3H-ouabain and 3H-digitoxin in

patients suffering from hyperthyroidism orhypothyroidism. Klin Wschr 48: 270, 1970

12. DOHERTY JE, HARA M, LINCOLN BM, PERKINSWH: The effect of cardiopulmonary bypass onthe turnover rates of digoxin in the serum ofdogs. Amer Heart J 64: 376, 1962

13. HARRISON CE, BRANDEN-BURG RO, ONGLEY PA,ORVIS AL, OWEN CA: The distribution andexcretion of tritiated substances in experimentalanimals following the administration of digoxin-:3H. J Lab Clin Med 67: 764, 1966

14. BARR I, STMITH TW, KLEIN MD, LOWN B: Cor-relation of the electrophysiologic action ofdigoxin with serum digoxin concentration. JPharmacol Exp Ther 180: 710, 1972

15. DOHERTY JE, PERKINS WH: Studies withtritiated digoxin in human subjects afterintravenous administration. Amer Heart J 63:528, 1962

16. GOLDSTEIN A, ARONOW L, KAL-MAN SM:Principles of Drug Action. New York, Harper& Row, 1968, p 292

17. MARCUS FI, BURKHALTER L, CUCCIA C,PAVLOVICH J, KAPADIA CC: Administration oftritiated digoxin with and without a loadingdose: A metabolic study. Circulation 34: 865,1966

18. MARCUS Fl, PAVLOVICH J, BURKHALTER L,CUCCIA C: The metabolic fate of tritiateddigoxin in the dog: A comparison of digitalisadministration with and without a "loadingdose." J Pharmacol Exp Ther 156: 548, 1967

19. GoLD H, DEGRAFF AC: Studies on digitalis inambulatory cardiac patients: III. Digitalizationby a small dose method; the use of digitalis inchildren. JAMA 92: 1421, 1929

20. COLD H, CATTELL M, MODELL W, KWIT NT,KRAMER ML, ZAHM W: Clinical studies ondigitoxin (D3igitaline Nativelle): With furtherobservation on its use in the single average fulldose method of digitalization. J Pharmacol ExpTher 82: 187, 1944

21. LUKAS DS: Some aspects of the distribution anddisposition of digitoxin in man. Ann NY AcadSci 179: 338, 1971


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RICHARD SELDEN, THOMAS W. SMITH and William FindleyRadioimmunoassay

Ouabain Pharmacokinetics in Dog and Man: Determination by

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1972 American Heart Association, Inc. All rights reserved.

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