CH, CH,

Fig. 1. Structure of digoxin

R2 CH3 21

�11�8�4

R1O 8 OH15

4H6

Compound � at 3 position

Di goxi n

Digi toxi ri

Deslanoside

Lanatoside C

OH tridigitoxose

H tridigitoxose

OH tridigitoxose - glucose

OH didigitoxose - acetyldigitoxose-

glucose

Uuabain: has only I sugar attached to Ci.

viz. b-deoxy-u-uxannose.

The C19 position contains the -CH2OH

rauical instead of -CR3. While a-OH

group is on Cl, Cl, and Cli.

Fig.2. Structure of key cardiac glycosides

CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986 5

CLIN. CHEM. 32/1, 5-12 (1986)

Digoxin-Issues and ControversiesSteven J. Soldin

This review deals briefly with the clinical pharmacology of

digoxin and reviews in some depth the problems inherent inmethods of digoxin measurement. Digoxin-like factors (mate-rials that cross react in digoxin immunoassays) are dis-cussed, and some current evidence suggesting the existenceof a new hormone, endoxin (endogenous digoxin), is summa-rized.

The year 1985 marks the bicentenary of the publication ofa classic in medical history, William Withering’s An Ac-count of the Foxglove, and Some of its Medical Uses; withPractical Remarks on Dropsy, and Other Diseases. Reported-ly (1), Withering first concerned himself with the foxglove in1775 when his opinion was asked about “a family recipe forthe cure of dropsy ... kept a secret by an old woman inShropshire.” The medicine is purported to have been com-posed of 20 or more herbs, but he was quick to recognize thatthe foxglove was the active component. Withering’s (2)detailed description of its toxic effects-”sickness, vomiting,purging, giddiness, confused vision, objects appearing greenand yellow; increased secretion of urine, with frequentmotions top� with it, and sometimes inability to retain it;slow pulse, even as slow as 35 in a minute. cold sweats,convulsions, syncope and death” (1)-as well as his record-ing of its uses in the treatment of dropsy (edema) and itsdiuretic effects are a milestone in medical history. In theearly 20th century it became accepted that the primaryeffect of digitalis was on the heart, and that the digitalisglycosides were useful as anti-arrhythmics for treatment ofatrial fibrillation and flutter, and to improve the function ofthe heart as a pump (i.e., enhance the contractility ofmyocardial muscular tissue) in the treatment of cardiacfailure.

Digitalis may have played a role in the illness of VincentVan Gogh, and his use of yellow and green color swirlsperhaps was generated by his memory of toxic visualsymptoms (3). Digitalis toxicity has in fact plagued themedical profession for over 200 years. According to Burchell(4): “The situation qualified as a professional disgrace on thebasis of 3 items: the situation persists, physicians are oftenslow to recognize it and over the decades, writers have beenharsh in their denunciation of fellow physicians whentoxicity has occurred. The Index Medicus, in the codesDigitalis Toxicity and Digitalis Poisoning lists about 20papers per year.” Withering’s account estimates the inci-dence of toxicity between 1780 and 1784 as 18%, whilestudies among patients being admitted to hospital in the

Departments of Clinical Biochemistry and Pharmacology, Uni-versity of Toronto, and Research Institute, Hospital for Sick Chil-dren, Toronto, Ontario M5G 1X8, Canada.

Received September 1, 1985; accepted September 5, 1985.

1970s quote figures of 20 to 30% (5). Are we doing littlebetter today than Withering was in his time?

“Digitalis” and “digitalis compounds” are terms thatencompass the entire group of cardiac glycoside inotropicdrugs (those influencing the contractility of myocardialmuscular tissue) and chronotropic drugs (those affecting therate of rhythmic movements such as the heart beat). Cardi-ac glycosides of medicinal importance are obtained fromD�gitalis purpurea Linne (Fam. Scrophulariaceae) (digitox-in, digitalis, gitalin), from Digitalis lanata Ehrhart (Fam.Scrophulariaceae) (digoxin, digitoxin, lanatoside C, deslano-side, acetyldigitoxin), Strophanthus gratus (ouabain), andAcokanthera schimperi (ouabain) (6-9).

These compounds have a characteristic ring structure(aglycone or genin) to which is coupled one or more sugars.The aglycone portion of the glycoside consists of a steroidnucleus and an aj3-unsaturated five- or six-membered lac-tone ring at the C17 position of the steroid nucleus (6-8).The hydroxyl groups at C3 and 14 are in the /3-configura-tion. The sugars are attached usually through the C3hydroxyl (Figures 1 and 2).

0.75 mg DIGOXIN IV.

20

00

80

E so

0)C

z0

4

I-

z‘U

‘-I

z0Uz2<

0t)

a

08

06

hi

TISSUI DIGOXIN

0 0 2 4 a 8 20 22 Xi

HOURS

Fig. 3. Relationship between tissue and plasma digoxin concentrationsafter administration of an intravenous dose of the drug

6 CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986

Brief Summary of the Clinical Pharmacology ofDigoxin

Digoxin is one of the drugs most widely used in currenthealth care delivery and is the fifth most commonly pre-scribed drug in North America. The main pharmacologicalproperty of the cardiac glycosides is their ability to increasethe force and velocity of myocardial systolic contraction-positive inotropic action (10). The currently accepted mecha-nism of action of digoxin is its inhibitory action on theNa�/K�-transporting ATPase (EC 3.6.1.37) transmembranepump of the sarcolemina (11, 12). The inhibition results inthe accumulation of intracellular sodium ions, which arethought to cause displacement of bound calcium ions. Theresulting free calcium ions cause a positive inotropic effect,resulting in a more forceful contraction of the myocardium(13). This is undoubtedly an oversimplification, because theway in which digitalis affects movements of calcium ions inheart muscle is not yet fully understood. Extracellularcalcium ions are probably very important in myocardialcontraction, as evidenced by the pronounced changes incontractile force brought about by changes in the extracellu-lar concentration of calcium ions. Intracellular calcium ionscan increase as a result of either an increased influx fromthe extracellular environment (14) or enhanced release ofbound intracellular calcium ions from sites such as thesarcoplasmic reticulum (15).

In patients with congestive heart failure, increased myo-cardial contractility and cardiac output reduce sympathetictone, thus slowing increased heart rate and causing diuresisin edematous patients (6, 7, 16, 17). Glycoside-inducedslowing of heart rate in patients without congestive heartfailure is negligible and is primarily attributable to vagal(cholinergic) and sympatholytic effects on the sinoatrialnode (17, 18), but with toxic doses it is caused by directdepression of sinoatrial node automaticity (19, 20). Themajor indication for digoxin use is the combination ofcongestive heart failure and atrial fibrillation. Acute left-ventricular failure is usually treated with load reductionbefore therapy with digitalis is instituted. Another indica-tion for use of this drug is chronic congestive failure withsinus rhythm that has failed to respond to diuretics alone.The amount of digoxin available for binding to Na�/K�-ATPase is influenced by several factors: dose, bioavailabilityof the preparation used, apparent volume of distribution ofthe drug, degree of metabolism to phannacologically activeand inactive metabolites, and the rate at which the drug iseliminated.

Factors influencing myocardial sensitivity to digoxin in-clude hypo- and hyperkalemia, hypercalcemia, hypo- andhypermagnesemia, acid-base disorders, myocardial isch-emia, hypoxemia, underlying heart disease, and pulmonarydisease (13).

Bioavailability: The bioavailability or completeness ofabsorption of digoxin varies markedly (55 to 85%) from onepreparation to the next, and many reports of toxic digoxinconcentrations can be attributed to a patient’s changingfrom one formulation to another (21-23). Intestinal motilityaffects the absorption of digoxin tablets. For example, mal-absorption syndromes that are characterized by intestinalhypermotility and motility-stimulating drugs such as meto-clopramide decrease absorption. Drugs that retard gastroin-testinal motility, such as the anti-cholinergics, tend toincrease bioavailability (24, 25).

Digoxin distribution: Figure 3 shows the time course ofdigoxin concentration in serum after an intravenous ii�jec-tion or infusion. The distribution of digoxin is aptly de-scribed by a two-compartment open pharmacokinetic model(26). The first very rapid decrease in concentration, mainly a

result of dilution in blood, takes a few minutes. During thedistribution or a phase the drug equilibrates between thecentral and peripheral compartment (27). The central com-partment may be thought to represent plasma and thehighly perfused organs such as liver and kidneys; theperipheral compartment represents the deeper tissues, par-ticularly skeletal muscle and myocardium. Skeletal muscleforms the largest storage depot of digoxin. Nevertheless, thedigoxin concentration during maintenance therapy is muchlower in skeletal muscle than in myocardium. The ratio ofthe concentrations in plasma and heart reportedly is be-tween 1:30 and 1:200 (28-30). The apparent volume ofdistribution (Vd) of digoxin can be defined as “the volume ofbody water which would be required to contain the amountof drug in the body if it were uniformly present in the sameconcentrations in which it is in blood.” Vd is highly variableand ranges from 3 to 10 L./kg body weight (31). This large Vdis a further indication of digoxin’s significant binding totissues. Vd can be markedly altered by factors that alter thisdigoxin tissue binding. Only 20 to 25% of digoxin is bound toplasma proteins (32, 33).

Digoxin elimination and metabolism: The eliminationhalf-life of digoxin in healthy test subjects reportedly variesbetween 26 and 45 h (32), although a significantly longer t�

has been reported for patients with reduced renal function(34). Renal clearance of digoxin occurs by glomerular ifitra-tion, tubular reabsorption, and tubular secretion, with thelast accounting for half the digoxin in the urine (35).Digoxin is distributed across the placenta, and some issecreted into breast milk (36,37). Digoxin and its metabo-lites are also present in bile. The small intestine is a major

site of absorption of digoxin, so enterohepatic recycling maybe a significant factor in the pharmacokinetics. An excellentdiscussion of digoxin pharmacokinetics can be found inseveral reviews (13, 32, 38, 39), and Table 1 summarizessome of the pharmacokinetic variables for digoxin in man.

Some years ago it was believed that relatively polarglycosides such as digoxin, deslanoside, and ouabain werenot metabolized appreciably (40,41,42). Today it is appreci-ated that metabolism can be extensive and can involvereduction of the lactone ring to form dihydrodigoxin and thestepwise removal of sugar molecules, followed by epimeriza-tion of the 3/3-hydroxyl to the 3a-(epi) position and conjuga-tion to give the polar metabolites 3-epi-glucuronide and 3-epi-sulfate (43-46). Figure 4 shows the pathways involved.Gault et al. (46) studied digoxin biotransformation in pa-tients with normal renal function or with advanced renalfailure. Some patients were found to have extensive bio-transformation, with polar metabolites predominating (47).

55-85%20-25%

26-45 h11-50 h1.5-5.0 h

7-11 days2-10 days3-10 LJkg1.0-2.5 nmol/L> 2.5 nmol/L

conjugation

Dihydrodigoxigenin products

2-62

4-216677

Ref erenee no.

57, 5858

57, 585959

CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986 7

Table 1. Some Pharmacoklnetic Variables forDlgoxin

Oral dose absorbedProtein boundHalf-lifeAdultsChildren

lime to peak plasma concn.Time to steady state

AdultsChildren

Volume of distributionEffective blood concns.Toxic blood concns.

uigoxin ) Dihydrodigoxin

Digoxigenio

bts-digltoxlsicle

(2 sugars) I

Digoxigenin

mono-digitoxiside I(1 sugar)

�1f 4,Digoxigeni n

Epidigoxigenin(no sugars)..��..�

Conjugation

products

Fig. 4. Pathways of digoxin metabolism

These authors developed a liquid-chromatographic proce-dure for separating all of the digoxigenin bis-digitoxiside,the monodigitoxiside, digoxigenin, the 3-epidigoxigemn, the3-keto-digoxigenin, and the polar metabolites of digoxin(45). [3HlDigoxin-12a was administered to individuals withnormal and poor renal function. Six hours after the dose,only 58% and 64% of the radioactivity present in the serumsamples was associated (co-chromatographed) with digoxin(46) in those individuals with impaired and normal renalfunction, respectively. And 28% and 22% of the radioactivityin these respective two groups was associated with the so-called polar metabolites. The dihydro derivatives co-chro-matographed with the unreduced precursor compounds inthe study described above. The percentage of the labelpresent as digoxin 6 h after the dose will therefore be evensmaller than the figures given by these authors. In thisregard, Clark and Kalman (48) reported urinary dihydrodi-goxin to account for 7 to 47% of the labeled digoxin productspresent, while Greenwood et al. (49) found that an averageof 16.4% of the oral digoxin dose was excreted as dihydrodi-goxin. In a study in which digoxin was administered orally,Gault et al. (50) reported that <2% of the byproducts in theurine were dihydro derivatives. It is postulated that thedihydro derivatives are formed in the intestine by thebacterial flora (51-53); consequently, the route of adminis-tration will govern the extent of conversion to these com-pounds. In a recent study (54)19 patients’ sera were studied,6 h after they had ingested 12a-[6H}digoxin, by both a liquidchromatography/RIA procedure and a regular RIA method.Metabolites accounted for 1 to 99% of the total plasmaradioactivity, averaging 40%. it is therefore imperative thatany reliable procedure for the measurement of digoxin bespecific. Studies assessing the degree of cross reactivity withdigoxin metabolites and digoxin-like factors are also essen-tial. Unfortunately, few if any of the commercially availableantibodies now meet these requirements for specificity.

Gault et al. (45) found that readily extractable metabolites,such as the mono -and bis-digitoxisides of digoxigenin,digoxigenin itself, and 3-keto-digoxigenin cross react, on theaverage, by 90% or more, confirming earlier reports of lackof specificity of digoxin antibodies (55, 56).

Table 2 gives the percentage cardioactivity of digoxin

metabolites. Stepwise removal of the sugar residues leads toa stepwise loss of cardioactivity, while reduction of thelactone ring results in almost total loss of pharmacologicalactivity (57-59).

Treatment of digoxin toxicity with Fab antibody frag-ments: The concept of using hapten-specific antibodies toreverse the toxic effects of a drug has been advanced (60-63). More recently, digoxin-specific Fab antibody fragmentshave been purified and used to treat patients with advanced,life-threatening digoxin toxicity (64-66). Although in suchcircumstances such an immunological approach is feasible,practical, can be life saving, and has been used experimen-tally for over 10 years, the antibody fragments are not freelyavailable to institutions in North America, an issue whichshould be of some concern to us all.

Immunoassays for Digoxin

Digoxin is routinely measured in serum by immunoassay,an antibody to digoxin being raised against a conjugate ofbovine serum albumin and digoxin, which is prepared byperiodate oxidation of the vicinal hydroxyl groups of theterminal sugar and then coupling of the resulting aldehydegroups to the amino groups of the bovine serum albumin(67). Thus the conjugate linkage is through the carbohy-drate moiety of digoxin. It is not surprising, therefore, thatthe antibodies generated against this conjugate are directedfor the most part against the steroid moiety of digoxin. As aconsequence, digoxigemn bis- and monodigitoxiside anddigoxigenin itself all react with the antibodies, whereasdihydrodigoxigenin and dihydrodigoxin metabolites-inwhich C22 is reduced-show little or no cross reactivity. Thecross reactivity of the metabolites of digoxin with the anti-digoxin antibodies is well known and is usually stated formost commercial antisera. What has not been appreciatedpreviously is that in many patients (approximately two outof three) digoxin undergoes considerable to extensive metab-olism, and measurement of “digoxin” concentrations inserum with nonspecific antibodies 6 to 24 h after a doseclearly can lead to problems in interpreting results. This isprobably responsible fo�r the lack of a consensus as to thecorrelation of serum digoxin concentration with either itstherapeutic or its toxic effects. Some investigators havefound a good relationship between the serum digoxin con-centration and indices of improved contractility (68-71);

others have not (72-75).

The relationship between serum digoxin concentration asmeasured by use of currently available nonspecific antibod-ies and the reduction of ventricular rate in patients withatrial fibrillation is also poor (76-78). The rationale for thetherapeutic monitoring of a particular drug requires thatthe drug meet certain established criteria:

Table 2. Percentage Cardioactlvity of DigoxinMetabolites

ActIvity relativeMetabolite to digoxln, %

DihydrodigoxinDihydrodigoxigeninDigoxigeninDigoxigenin mono-digitoxisideDigoxigenin bis-digitoxiside

Table 3. SpecifIcity of New Dlgltoxose-SpecificAntibody for Dlgoxln Measurement (79)

Dlgoxln concn, mol/L, as measured by

New

Metebolite Digoxin antibody

Compound

Psychosine

a-Hydroxymyristic acid

a-Hydroxylaunc acid

Cerebrosides Type II

Ceramide

L-a-MOnopalmitoyl-lec-

ithin

L-a-Monomyristoyl-lOc-Ithin

1 -AIkyl-2-hydroxyphos-phatidylcholine

Monolysocardiolipin

Cerebrosides Type I

Sulfatides

DigoxigeninMonodigitoxisideBisdigitoxisidoDlgftoxigenDigoxigenin,

monodigitoxiside,bisdigitoxiside,and dihydrodigoxin

10.27.76.1

10.50.64 of

each

00002.56

00.70.802.56

FPIA

13.810.0

6.059.65.2

1000500

1000500

1000500

1000500

1000500

1000

1Fluorescence polarization immunoassay.

<0.7<0.2

0.30.81.31.40.6

9.7<0.2

0.30.71.50.30.5

0.4 0.70.2 <0.20.3 0.40.5 0.90.8 2.00.4 0.30.5 0.5

Fluorescence polarization immunoaseay.

8 CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986

#{149}a specific and reliable method must be available for itsmeasurement

#{149}there must be a well-defined therapeutic and toxic range,and the therapeutic index must be small

#{149}a poor correlation must exist between dose and serumconcentration -

#{149}a good correlation must exist between concentration inserum and pharmacological effect

Clearly, digoxin does not meet many of these criteria. Noreliable specific routine method currently exists for itsmeasurement. To attempt to correlate the concentrations ofdigoxin in serum by�ising the currently available nonspecif.ic antisera with therapeutic efficacy and toxicity is anexercise in frustration. To realize that this problem exists isthe first step in finding a solution. The digitoxose sugars arevery specific, and clearly there would be advantages toraising antibodies to the carbohydrate component of themolecule. Could digoxin be linked to albumin, perhaps viathe steroid ring, in such a way that the sugars remainedintact, thereby allowing the digitoxose sugars to act asantigenic determinants? This proved possible, and recentlyour group (79) successfl.illy synthesized a novel digoxin-bovine serum albumin conjugate via the reductive ozonoly-sis of the lactone ring. Antibodies raised against this conju-gate show minimal cross reactivity to digoxigenin bis-digitoxiside, monodigitoxiside, digoxigenin, and digitoxin.With this antibody, digoxin can be measured in the presence

of all these metabolites (Table 3). Unfortunately, dihydrodi-goxin and its metabolites, which usually comprise about12% of the end products after a dose of digoxin (51), crossreact with the new antibody. However, it now becomesfeasible to consider a double-antibody approach: one anti-body screening out the cross reactivity for dihydrodigoxinand its metabolites while the other permits only the mea-surement of the steroid ring attached to the three digitoxosesugars. Once a specific procedure for measuring digoxin isavailable, valid studies can be done on the correlationbetween “true digoxin concentration” and its pharmacologi-cal efficacy. Reference procedures incorporating a liquid-chromatography step to separate digoxin from its metabo-lites before the immunoassay with nonspecific antibodieswould provide reliable results. Many studies have beenreported on drug/drug interactions with digoxin-e.g., spir-onolactone, amiloride, amiodarone, verapamil, and quini-dine (80-85). Hitherto, the methods used to measure digox-in rarely have included a chromatographic separation beforethe nonspecific immunoassay. Many of the antibodies avail-able today for measurement of digoxin will give considera-bly greater values for apparent digoxin on an osmolar basiswhen its metabolites are present than when digoxin itself ispresent (79, 86). Some of the conclusions of drug/druginteractions for digoxin may need to be reevaluated once a

specific immunoassay is available, because these drugsconceivably could alter the extent to which digoxin ismetabolized via a particular pathway, thereby altering the

metabolite concentration and affecting quantification whennonspecific assays are used.

In current immunoassays for digoxin there is cross reac-tion not only with many of the digoxin metabolites but alsowith many apparently less-related or even structurallyunrelated compounds (55, 87), among them a group ofsteroids that includes progesterone, cortisone, 11-hydroxy-progesterone, 21-deoxycortisone, and others (55,87,88,89).Table 4 lists a group of structurally unrelated substancesthat also were found to cross react in the immunoassay

systems studied (55). The studies of steroids and other lipidscausing increased digoxin readings were performed on bothaqueous media vs aqueous digoxin standards and serum vsdigoxin standards dissolved in serum (Table 5) (55). Becauseof the cross reactivity demonstrated by some of the com-pounds listed, we wondered whether some commonly useddrugs, injected into laboratory animals, would give false-positive readings for digoxin. These drugs themselves do notinterfere in the immunoassay systems studied. However,they might be converted to compounds or else elicit releaseof substances that do interfere in the immunoassay systemsstudied. Hydrocortisone and epinephrine were injected into

a sheep or dog in doses commonly used for humans. Consid-erable digoxin immunoreactivity was generated in sampleswithdrawn from the injection site 2 and 5 mm later (Table6).

During the past 15 years, digoxin in body fluids has beenroutinely measured almost exclusively through the use ofvarious immunoassay procedures (90-96). While liquid-chromatographic (97-100) and gas chromatographic-massspectrometric (101) procedures have been described, theyhave not been widely applied in the routine clinical chemis-

Table 4. Cross Reactivity of Lipids In Dlgoxlnlmmunoassays

Measured dlgoxln nmol/L

Concn In buffer Buffer Serumor serum

tested,mg/L FPIA AlA FPIA RIA

500 1.1 <0.2 0.5 <0.21000 1.6 <0.2 0.8 0.3

500 0.5 <0.2 0.2 <0.21000 0.7 <0.2 0.4 <0.2

500 0.6 <0.2 0.3 <0.21000 1.0 <0.2 0.4 <0.2

500 0.5 <0.2 0.4 <0.21000 0.5 <0.2 0.4 0.2

550 0.3 <0.2 <0.2 <0.21100 0.3 <0.2 0.2 <0.2

500 0.5 1.7 <0.2 0.2

0.41.9

5.1 0.30.6 0.5

0.6<0.2

2.3<0.7

2.1 0.82.9 0.2

1.10.2

Table 5. Cross ReactIvity in Digoxinimmunoassays

Measured digoxln, nmol/L

Concn,mg/L

2550

Stds. In buffer

FPIA RIA

6.4 5.3>6.4 >6.4

Stds. In serum

FPIA RIA

5.0 1.05.6 1.7

CompoundsdIssolved In buffer

11 a-Hydroxy-pro-

gesterone

Cortisone

Progesterone

L-a-MonomyristOyl-lecithin

Sulfatides

25 1.8 0.6 4.8 1.450 3.3 1.4 >6.4 3.3

25 2.6 2.2 2.7 0.850 3.8 2.2 3.9 1.0

500 0.4 1.8 1.9 0.61000 0 6.1 2.3 2.1

Table 6. False-PosItIve Dlgoxln Measurementsafter Injection of Eplnephrine and Hydrocortlsone

Into Sheep and Dogs

Drug & dose

Hydrocortisone, 100 mg

BodyweIght, kg

Dog

13.6

Sheep

49.0 Hydr000rtisone, 500 mg

50.5 Epinephrine, 0.5 mg

review these atrial natriuretic factor(s); good reviews areavailable (124-126). Atrial natriuretic factors cause a natri-uresis, diuresis, and drop in blood pressure, but they neither

_________________ cross react with digoxin antibodies nor inhibit Na�fK�-

ATPase. In contrast, the postulated natriuretic hormone hasbeen isolated from plasma (106, 114, 127), placenta (128),

and brain (129); it cross reacts with digoxm antibodies,inhibits Na�/K�-ATPase, and also causes a natriuresis anddiuresis. Studies by Gault etal. (127), Hamblyn et al. (130),and Devynck et al. (106) lend credence to the probableexistence of an endogenous natriuretic hormone (endoxin), aconcept previously postulated by LaBella (131) and others.Gault’s group showed in a preliminary study that theconcentration of digitalis-like factor(s) in plasma more than

doubled in response to both an intravenous salt load and ahigh-salt diet, in patients with mild hypertension. Hamblynet al. (130), using a kinetic Na�/K�-ATPase assay, demon-

500 0.6 1.1 1.4 0.3 strated a significant correlation between concentrations of1000 0.6 1.7 0.6 0.5 an inhibitor of Na�/K�-ATPase activity in plasma and the

mean arterial blood pressure of normotensive and hyperten-sive individuals. Devynck et al. (106) found that in two-thirds of untreated hypertensives and several of the normo-tensive subjects with a family history of hypertension, thepotency of the digitalis-like compound, as measured by its

interference with ouabain binding to the erythrocyte, was

lime mln significantly greater than in the controls.Recent fast-atom bombardment mass-spectrometric and

Assay 0 2 5 nuclear magnetic resonance data obtained in our laboratory

on digoxin-like factors purified from placenta (unpublished)indicated the possible presence of mono- and diglycerides.We already knew that similar compounds could cross reactin digoxin immunoassays (55), but we had not evaluated

their ability to inhibit ssRb uptake by the erythrocyte. Thisexperiment revealed that some of these compounds possessconsiderable ability, in micromolar concentrations, to inhib-it seRb uptake. Data recently published by Tamura et al.(132) indicate that linoleic and oleic acids act as endogenousNa47K4-ATPase inhibitors. These compounds were isolatedand identified from porcine plasma after the pigs hadundergone volume-expansion experiments. Is endogenousdigoxin a family of monoacyl and diacyl glycerols, and dothese compounds play some role as endogenous regulators ofthe ubiquitous enzyme Na4iK�-ATPase?

The etiology of essential hypertension, a disease prevalent

in cultured societies, is unknown. The discovery of two newhormones, one of which has already been structurallyidentified and which affect the body’s handling of salt andwater, is encouraging and gives some hope that the patho-genesis of essential hypertension may begin to be unrav-elled in the years ahead.

FPIA <0.4 aRIA <0.4

1.6 2.03.2 4.2

FPIA <0.4 2.4RIA <0.4 12.2FPIA <0.4 <0.4 <0.4RIA <0.4 1.0 1.8

CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986 9

a Digoxin equivalents, nmoLfL.

try laboratory, owing to their marked lack of sensitivity,which would necessitate lengthy sample preparation andlarge sample size. The lack of specificity of present immuno-assays for digoxin is a serious shortcoming to the rationalapplication of the principles of therapeutic drug monitoringto the improvement of patient care in this area.

Digoxin-Like lmmunoreactive Substances and theConcept of Endogenous Digoxin

For several years digoxin immunoreactivity has beenreported in dog and human plasma and urine from individ-uals known never to have received the drug (87, 102 -116).Considerable interest has been generated by these findings,with many investigators believing that we are on the brinkof discovering a natriuretic hormone. The term “endoxin”has been coined by Gruber et al. (114) because the factor ofinterest binds to digoxin antibodies. The postulated nab-i-uretic hormone (endodoxin) should not be confused withatrial natriuretic factor. In 1959, Bompiani et al. (117)reported histological studies of granular structures betweenthe myocytes of rat-heart atria. In 1979, de Bold (118)

reported that the number of these granules changed duringwater deprivation. He and others later reported (119) thatintravenous injection of rat atrial extracts caused a strikingnatriuresis and diuresis in rats. This discovery resulted inatrial natriuretic factor becoming the focus of attention inmany laboratories, and a number of peptides-variouslynamed “atriopeptins,” “auriculins,” “cardionatrins,” and“atrial natriuretic factors” have recently been purified andsequenced (120-123). It is beyond the scope of this article to

References1. Wilkins MR. Kendall MJ, Wade OL. William Withering anddigitalis, 1785 to 1985. Br Med J 290, 7-8 (1985).

2. Withering W. An account of the foxglove and some its medicaluses with practical remarks on dropsy and other diseases. InClassics of Cardiology, FA Willius, TE Keys, Eds., Henry Schwnan,Inc., New York, NY, 1941, pp 231-252.3. Lee TC. Van Gogh’s vision, digitalis intoxication. J Am MedAssoc 245, 727-729 (1981).

4. Burchell HB. Digitalis poisoning Historical and forensic aspects.JAm Coil Cardiol 1,502-516 (1983).

5. George CF. Digitalis intoxication: A new approach to an oldproblem. Br Med J 286, 1533-1534 (1983).

6. Smith TW. Digitalis glycosides, Part I. NEngi J Med 283, 719-722 (1973).

7. Goodman LS, Gilman A. The Pharmacological Basis of Thera-peutics, 5th ed., The Macmillan Co., New York, NY, 1975, pp 653-679, 692-693.

10 CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986

8. Wilson CO. Gisvold 0, Doerge RF. Textbook of Organic, Medici-nal and Pharmaceutical Chemistiy, 7th ed., Lippincott, Philadel-phia, PA, 1977, pp 797-809.

9. Wade A. Martindale-the Extra Pharmacopoeia, 27th ed., ThePharmaceutical Press, London, England, 1977, pp 485-498.

10. Cattell M, Gold H. The influence of digitalis glycosides on theforce of contraction of mammalian cardiac muscle. JPharmacolExpTher 62, 116-125 (1938).

11. Smith TW, Haber E. Digitalis (first of four parts). NEnglJMed289, 945-1010 (1973).

12. Marks BH. Factors that affect the accumulation of digitalisglycosides by the heart. In Basic and Clinical Pharmacology ofDigitalis, BH Marks, AM Weissler, Eds., Charles C Thomas,Springfield, IL, 1972, pp 69-93.

13. Davis TD, Vanderveen RL, Nienhuis M. Digoxin. In AppliedClinical Pharmacokinetics, D Mungall, Ed., Raven Press, NY, 1983,pp 79-101.

14. Longer GA. Ionic basis of myocardial contractility. Ann RevMed 28, 13-20 (1977).

15. Klaus W, Lee KS. Influence of cardiac glycosides on calciumbinding in muscle subcellular components. J Pharmacol Exp Ther166, 68-76 (1969).

16. Gill MA, Miscia VF, Gourley DR. The treatment of commoncardiac arrhythmias. JAm Pharmaceut Assoc 16, 20-29 (1976).17. Rosen MB, Wit AL, Hoffman BF. Electrophysiology and phar-macology of cardiac arrhythmias, Part IV: Cardiac antiarrhythmicand toxic effects of digitalis. Am Heart J 89, 391-399 (1975).

18. Doherty JE, Kane JJ. Clinical pharmacology and therapeuticuse of digitalis glycosides. Drugs 6, 182-221 (1973).

19. Dreifus LS, Watanabe Y. Clinical correlates of the electrophysi-ologic action of digitalis on the heart. Semin Drug Treat 2, 179-201(1972).

20. Smith 1W, Haber E. thg�talis, Part IV. N Engi J Med 289,1125-1129 (1973).

21. Lindenbaum J, Mellow MH, Blackstone MO, Butler VP Jr.Variation in biologic availability of digoxin from four preparations.N Engi J Med 285, 1344-1347 (1971).

22. Greenblatt D.J, Duhme DW, Koch-Weser J, Smith TW. Evalua-tion of digoxin bioavailability in single-dose studies. N Engi J Med289, 651-654 (1975).

23. Huffman DH, Azarnoff DL. Absorption of orally given digoxinpreparations. JAm Med Assoc 222, 957-960 (1972).

24. Manninen V, Melin J, Apajalahti A, Karesoja M. Alteredabsorption of digoxin in patients given propantheline and metoclo-pramide. Lancet i, 398-400 (1973).

25. Heizer WD, Smith TW, Goldflnger SE. Absorption of digoxin inpatients with malabsorption syndromes. N Engi J Med 285, 257-259 (1971).

26. Reuning RH, Sams RA, Natari RE. Role of pharmacokinetics indrug dosage adjustment. I. Pharmacologic effects, kinetics andapparent volume of distribution of digoxin. J Clin Pharmacol 13,127-41 (1973).

27. Soldin SJ. Therapeutic drug monitoring. In Pediatric ClinicalChemistry, JM Hicks, RL Boeckx, Eds., Saunders, Philadelphia,PA, 1984, pp 601-621.

28. Lichey J, Havestatt CII, Weinmann J, et al. Human myocardi-um and plasma digoxin concentration in patients on long-termdigoxin therapy. mt � Clin Pharmacol 16, 460-462 (1978).

29. Andersson K-E, Bertler A, Wettrell G. Post-mortem distribu-tion and tissue concentrations of digoxin in infants and adults. ActaPaediatr Scand 64, 497-504 (1975).

30. Doherty JE, Perkins WH, Flanigan WJ. The distribution andconcentration of tritiated digoxin in human tissues. Ann Intern Med66, 116-124 (1967).

31. Koup JR, Greenblatt D.J, Jusko WJ, et al. Pharmacokinetics ofdigoxin in normal subjects after intravenous bolus and infusiondoses. J Pharmacoki net Biopharm 3, 18 1-192 (1975).

32. Lisalo E. Clinical pharmacokinetics of digoxin. Cliii Pharmaco-kinet 2, 1-16 (1977).

33. Weintraub M. Interpretation of the serum digoxin concentra-tion. Clin Pharmacoki net 2, 205-219 (1977).

34. Koup JR, Jusko WJ, Elwood CM, Kohl RK. Digoxin pharmaco-kinetics: Role of renal failure in dosage regimen design. ClinPharmacol Ther 18, 9-21 (1975).

35. Steiness E. Renal tubular secretion of digoxin. Circulation 50,103-107 (1974).

36. Padeletti L, Porciani MC, Scimone G. Placental transfer ofdigoxin (beta-methyl-digoxin) in man. ml J Clin Pharmacol Bio-pharmacol 17, 82-83 (1979):

37. Finley JP, Waxman MD, Wong PY, Lickrish GM. Digoxinexcretion in human milk. J Pediatr 94, 339-340 (1979).

38. Smith ‘1W, Antman EM, Friedman PL, et al. Digitalis glyco-sides: Mechanisms and manifestations of toxicity. Part L ProgCard iovasc Dis 26, 413-458 (1984).

39. Smith 1W, Antman EM, Friedman PL, et al. Digitalis glyco-sides: Mechanisms and manifestations of toxicity. Part II. ProgCardiovasc Di.s 26, 495-540 (1984).

40. Seldon R, Margolies MN, Smith TW. Renal. and gastrointesti-nal excretion of ouabain in dog and man. J Pharmacol Exp Ther.188, 615-623 (1974).

41. Doherty JE. Digitalis glycosides: Pharmacokinetics and theirclinical implications. Ann Intern Med 79, 229-238 (1973).

42. Marks BH, Dutta S, Gautheir J, et al. Distribution in plasmauptake by the heart and excretion of ouabain-H3 in human subjects.J Pharmacol Exp Ther 145, 351-356 (1964).

43. Luchi RJ, Gruber JW. Unusually large digitalis requirements:A study of altered digoxin metabolism. Am J Med 45, 322-328(1968).

44. Doherty JE. Metabolism of digitalis in man. In ref. 12, pp 11-20.45. Gault MB, Kaira J, Longerich L, Dawe M. Digoxigenin bio-transformation. Clin Pharmacol Ther 31, 695-704 (1982).

46. Gault MB, Longerich L, Leo JCK, et al. Digoxin biotransforma-tion. Clin Pharmacol Ther 35, 74-82 (1984).

47. Longerich L, Loo J, Dawe M, et al. Serum digoxin and metabo.lites in patients with renal failure. Clin Invest Med 5, 42B (1982).

48. Clark DB, Kalman SM. Dihydrodigoxin: A common metaboliteof digoxin in man. Drug Metab Dispos 2,148-150(1974).

49. Greenwood H, Snedden W, Howard RP, London J. The mea-surement of urinary digoxin and dihydrodigoxin by radioim-munoassay and by mass spectrometry. Clin Chim Acta 62,213-214(1975).

50. Gault MH, Sugden D, Maloney C, et al. Biotransformation andelimination of digoxin with normal and minimal renal function.Cliii Pharmacol Ther 25, 499-513 (1979).

51. Peters U, Falk LC, Kairnan SM. Digoxin metabolism in pa-tients. Arch Intern Med 138, 1074-1076 (1978).

52. Lindenbaum J, Rund D, Butler VP, et al. Inactivation ofdigoxin by the gut flora: Reversal by antibiotic therapy. N Engi JMed 305, 789-794 (1981).

53. Magnusson JO. Metabolism of digoxin after oral and intra-jejunal administration. Br J Clin Pharmacol 16, 741-742 (1983).

54. Gault MB, Longerich L, Dawe M, Vasdev SC. Combined liquidchromatography/radioimmunoassay with improved specificity forserum digoxin. Clin Chem 31, 1272-1277 (1985).

55. Soldin SJ, Papanastasiou-Diamandi A, Heyes J, et al. Areinununoassays for digoxin reliable? Cliii Biochem 17, 317-320(1984).

56. Stoll RG, Christensen MS, Sakmar E, Wagner JG. The specific-ity of the digoxin radioiminunoassay procedure. Res CommunChem Pathol Pharmacol 4, 503-.fl10 (1972).

57. Bach F.J, Reiter M. The differences in velocity between thelethal and inotropic action of dihydrodigoxin. Arch Exp PatholPharmakol 248, 437-4.49 (1964).

58. Brown BT, Stafford A, Wright SE. Chemical structure andpharmacologic activity of some derivatives of digitoxigenin anddigoxigenin. Br J Pharmacol 18, 311-324 (1962).

59. Lage GL, Spratt JL. Structure-activity correlation of the lethal-ity and central effects of selected glycosides. JPharmacolExp Ther152, 501-508 (1966).

60. Smith TW, Butler VP Jr, Haber B. Cardiac glycoside-specillcantibodies in the treatment of digitalis intoxication. In Antibodiesin Human Diagnosis and Therapy, E Haber, R Krause, Eds., RavenPress, New York, NY, 1977, pp 365-389.

61. Schmidt DH, Butler VP Jr. Reversal of digoxin toxicity withspecific antibodies. J Cliii Invest 50, 1738-1744 (1971).

62. Butler VP Jr, Chen JP. Digoxin-specific antibodies. Proc NailAcad Sci USA 57, 7 1-78 (1967).

CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986 11

63. Curd J, Smith TW, Jaton J-C, et al. The isolation of digoxm-specific antibody and its use in reversing the effects of digoxin. ProcNat! Acad Sci USA 68, 2401-2406 (1971).

64 Smith TW, Butler VP Jr, Haber E, et al. Treatment of life-threatening digitalis intoxication with digoxin-specific Fab anti-body fragments. N Engi J Med 307, 1357-1362 (1982).

65. Antman EM, Smith TW. Digitalis toxicity. Ann Rev Med 36,357-367 (1985).

66. Doherty JE. Digoxin antibodies and digitalis intoxication. NEngi J Med 307, 1398-1399 (1982).

67. Butler VP, Chen JP. Digoxin specific antibodies. Proc Nat!Acad Sci USA 57, 7 1-78 (1967).

68. Waldorf 5, Hansen PB, Kjaegard H, et al. Amiloride-inducedchanges in digoxin dynamics and kinetics: Abolition of digoxin-induced inotropism with amiloride. Clin Pharmacol Ther 30, 172-176 (1981).

69. Kim Yl, Noble RJ, Zipes DP. Dissociation of the isotropic effectof digitalis from its effect on atrioventricular conduction. Am JCardiol 36, 459-467 (1975).

70. Shapin W, Narahara K, Taubert K. Relationship of plasmadigitoxin and digoxin to cardiac response following intravenousdigitalization in man. Circulation 42, 1065-1072 (1970).

71. Steiness E, Waldorf S, Hansen P, et al. Reduction of digoxin-induced inotropism during quinidine administration. Cliii Pharma-col Ther 27, 791-795 (1980).

72. Ford AR, Aronson JK, Grahame-Smith DG, Carver JG.Changes in cardiac receptor sites, 86-rubidium uptake and intracel-lular sodium concentrations in the erythrocytes of patients receiv-ing digoxin during the early phases of treatment of cardiac failurein regular rhythm and of atrial fibrillation. Br J Cliii Pharmacol 8,125-134 (1979).

73. Jogestrand T, Ericsson R, Sundquist K. Skeletal muscle digoxinconcentration during digitalization and during withdrawal of digox-in treatment. Eur J Clin Pharinacol 19, 97-105 (1981).

74. Belz GO, Aust PE, Schneider B. Time course of the effects ofsingle intravenous doses of digitoxin or digoxin in normal volun-teers. J Cardiovasc Pharmacol 3, 1116-1125 (1981).

75. Chamberlain DA. The relationship of clinical effect to plasmadigoxin concentration. Postgrad Med J 50, 29-35 (1974).

76. Redfors A. Plasma digoxin concentration-its relation to digoxindosage and clinical effects in patients with atrial fibrillation. BrHeart J 34, 383-391 (1972).

77. Chamberlain DA, White RJ, Howard MB, Smith TW. Plasmadigoxin concentrations in patients with atrial fibrillation. Br Med Jin, 429-432 (1970).

78. Goldman S, Probst P, Seizer A, Cohn K. Inefficacy of”therapeu-tic” serum levels of digoxin in controlling the ventricular rate inatrial fibrillation. Am J Cardiol 35, 651-655 (1975).

79. Thong B, Soldin SJ, Lingwood CA. Lack of specificity of currentanti-digoxin antibodies, and preparation of a new, specific, polyclo-nal antibody that recognizes the carbohydrate moiety of digoxin.Clin Chem 31, 1625-1631 (1985).

80. Waldorff 5, Hansen PB, Kjaegard H, et al. Aniiloride-inducedchanges in digoxin dynamics and kinetics: Abolition of digoxin-induced inotropism with amiloride. Cliii Pharmacol Ther 30, 172-176 (1981).

81. Belz (1(1, Aust PE, Munkes R. Digoxin plasma concentrationsand nifedipine. Lancet i, 844-845 (1981).

82. Klein HO, Lang R, Segni ED, Kaplinsky E. Verapainil-digoxininteraction. N Engl J Med 303, 160 (1980).

83. Moysey JO, Jaggarav NSV, Grundy EN, Chamberlain DA.Amiodarone increases plasma digoxin concentrations. Br Med J282, 272 (1981).84. Steiness E. Renal tubular secretion of digoxin. Circulation 50,103-107 (1974).

85. Waldorf 5, Andersen J, Heeboll-Nielsen N, et al. Spironolac-tone-induced changes in digoxin kinetics. Cliii Pharmacol Ther 24,162-167 (1978).

86. Valdes R Jr, Brown BA, Graves SW. Variable cross-reactivityof digoxin metabolites in digoxin imnumoassays. Am J Clin Pathol82, 210-213 (1984).

87. Hicks JM, Brett EM. Falsely increased digoxin concentrationsin samples from neonates and infants. Ther Drug Monitor 6,461-464 (1984).

88. Schreiber V, Stepan J, Starka L. Digoxin-like immunoreactiv-ity of certain steroids and other hormones. Physiol Bohemoslov 30,569-571 (1981).89. LaBella FS, Bihler I, Kim R-S. Progesterone derivatives thatbind to the digitalis receptor Effects on 86Rb uptake. Can JPhysiolPhEzrmacol 62, 1057-1064 (1984).

90. Glassman A, Tilden R, Bruno F. Radioimmunoassay of digoxin.South Med J 63, 1367 (1970).

91. Cerceo E, Ello8o CA. Factors affecting the radioimmunoassay ofdigoxin. Clin Chem 18, 539-543 (1972).

92. Rumley AG, Trope A, Rowe D.JF, Hainsworth IR. Comparisonof digoxin analysis by. EMIT with Immunophase and Dac-Celradioimmunoassay. Ann Cliii Biochem 17, 315-318 (1980).

93. Rawal N, Leung FY, Henderson AR. Fluorescence polarizationimmunoassay of serum digoxin: Comparison of the Abbott immuno-assay with the Ainerlex radioimmunoassay. Clin Chem 29, 586(1983). Letter.

94. Schermarin JM, Bourdon R. Effect of deproteinization on deter-mination of serum digoxin by fluorescence polarization immunoas-say. Clin Chem 30, 337-338 (1984). Letter.

95. Smith TW, Butler VP, Haber E. Characterization of antibodiesof high affinity and specificity for the digitalis glycoside digoxin.Biochemistry 9, 331 (1970).

96. Smith TW, Butler VP, Haber E. Determination of therapeuticand toxic serum digoxun concentration by radioimmunoassay. NEngI J Med 281, 12 12-1216 (1969).

97. Kaizuka H, Takahashi K. High-performance liquid chromato-graphic system for a wide range of naturally occurring glycosides. JChromatogr 258, 135-146 (1983).

98. Castle MC. Isolation and quantitation of picomole quantities ofdigoxin, digitoxin and their metabolites by high-pressure liquidchromatography. J Chromatogr 115, 437-445 (1975).

99. Fujii Y, Fujii H, Yamazaki M. Separation and determinationsof cardiac glycosides in Digitalis purpurea leaves by micro highperformance liquid chromatography. J Chromatogr 258, 147-153(1983).

100. Jinno K. Correlation between the retention of cardiac glyco-sides in reversed-phase high-performance liquid chromatographywith a diphenysilyl stationary phase, their structure and biologicalactivity. J Chromatogr 264, 485-486 (1983).

101. Watson E, Clark DR. Kalman SM. Identification by gaschromatography-mass spectroscopy of dihydrodigoxin, a metaboliteof digoxin in man. J Pharmacol Exp Ther 184, 424-431 (1973).

102. Danzer LA, Pratt HW, Lewis M, Chandramouli B. Falsepositive digoxin results in infant sera with some commercial RIAmethodologies. Clin Biochem 16, 362 (1983).

103. Valdes R, Graves SW, Brown BA, Landt M. Endogenoussubstance in newborn infants causing false positive digoxin mea-surements. J Pediatr 102, 947-950 (1983).

104. Balzan S, Clerico A, Brazia del Chicca M, et al. Digoxin-likeimmunoreactivity in normal hwnan plasma and urine as detectedby solid phase radioimmunoassay. Cliii Chem 30, 450-451 (1984).

105. Graves SW, Valdes R, Brown BA, et al. Endogenous digoxin-immunoreactive substance in human pregnancies. J Cliii Endo-crinol Metab 58, 748-751 (1984).

106. Devynck M-A, Pernollet MG, Rosenfeld JB, Meyer P. Mea-surements of digitalis-like compound in plasma: Application instudies of essential hypertension. Br Med J 287, 631-634 (1983).

107. Hicks JM, Brett EM. Falsely positive digoxin results in serumspecimens from acute care infants. Cliii Chem 29, 1249 (1983).Abstract.

108. Vasdev S, Fernandez PG, Prabhakaran V, et al. Exploratorystudies of urinary digitalis-like factor(s) (DLF) in healthy andhypertensive subjects. Clin Invest Med 6, 58 (1983).

109. Whitmer KR, Eppa D, Schwartz A. Endogenous inhibitor ofNa, K-ATP-ase: “Endodigin”. Curr Top Membr Transp 19,945-950(1983).110. Gault MB, Vasdev SC, Longerich LL, at al. Plasma digitalis-like factor(s) increase with salt loading. N Engi J Med 309, 1459(1984).111. Soldin SJ, Papanastasiou-Diainandi A, Lingwood C, KuksisA. Extraction and purification of digoxin immunoreactive materialsfrom urine of normal adults by liquid chromatography. Clin Chem6, 1013 (1984). Abstract.

12 CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986

112. Koren G, Farine D, Mareksy D, at al. Significance of theendogenous digoxin-like substance in infants and mothers. ClinPharrnacol Ther 36, 759-764 (1984).

113. Pudek MR., Seccombe DW, Whitefleld MF. Digoxin-like im-munoreactivity in premature and full-term infants not receivingdigoxin therapy. N Engi J Med 308,904-905(1983).

114. Gruber KA, Whitaker JM, Buckalew VM Jr. Endogenousdigitalis-like substance in plasma of volume-expanded dogs. Nature(London) 287, 743-745 (1980).

115. Gruber KA, Rudel LL, Bullock BC. Increased circulatinglevels of an endogenous digoxin-like factor in hypertensive mon-keys. Hypeiiensioii 4,348-354(1982).

116. Besch HR., Hufferd S, Lake M, et al. False elevation ofapparent digoxin levels in plasma of premature infants. Cliii Chem12. 1168 (1976). Abstract.

117. Bompiani GD, Rouiller C, Hatt PY. La tissu de conduction ducoeur chez le rat. Etude au microscope electromque. Arch MalCoeur 52, 1257 (1959).118. de Bold AJ. Heart atria granularity effects changes in water-electrolyte balance. Proc Soc Exp Biol Med 161, 508-511 (1979).

119. de Bold AJ, Borenstein RB, Veresa AT, Sonnenberg H. Arapid and potent natriuretic response to intravenous injection ofatrial myocardial extracts in rats. Life Sci 28,89-94(1981).120. Atlas SA, Kleinert ND, Camargo MJ, et al. Purification,sequencing and synthesis of natriuretic and vasoactive rat atrialpeptide. Nature (London) 309, 717-719 (1984).

121. Oikawa S. Imai M, Ueno A, et al. Cloning and sequenceanalysis of cDNA encoding a precursor for human atrial natriureticpolypeptide. Nature (London) 309, 724-726 (1984).

122. Yamanaka M, Greenberg B, Johnson L, et al. Cloning and

sequence analysis of the cDNA for the rat atrial natriuretic factorprecursor. Nature (London) 309, 719-722 (1984).

123. Maki M, Takayanagi R, Misono KS, et al. Structure of ratatrial natriuretic factor precursor deduced from cDNA sequence.Nature (London) 309, 722-724(1984).

124. Palluk R, Gaida W, Hoefke W. Atrial natriuretic factor. LifeSci 36, 1415-1425 (1985). Review.

125. Granthain JJ, Edwards RM. Natriuretic hormones: At last,bottled in bond? J Lab Clin Med 103, 333-336 (1984).

126. Sagnella GA. MacGregor GA. Cardiac peptides and the con-trol of sodium excretion. Nature (London) 309,666-667(1984).

127. Gault MB, Vasdev SC, Longerich LL, et al. Plasma digitalis-like factor(s) increase with salt loading. N Engi J Med 309,1459(1984).

128. Diamandis EP, Papanastasiou-Diamandi A, Soldin SJ. Digox-in immunoreactivity in cord and maternal serum and placentalextracts. Partial characterization of immunoreactive substances byhigh-performance liquid chromatography and inhibition ofNa�,K’-ATPase. Cliii Biochem 18, 48-55 (1985).

129. Fishman MC. Endogenoua digitalis-like activity in mRmmali-an brain. Proc NatI Acad Sci USA 76, 4661-4663 (1979).

130. Haxnblyn JM, Ringel R, Schaefer J, et al. A circulatinginhibitor of (Na�/KiATPase associated with essential hyperten-sion. Nature (London) 300, 650-652 (1982).

131. LaBella FA. Is there an endogenous digitalis? Trends Phar-macol Sci 3, 334-335 (1982).132. Tamura M, Kuwano H, Kinoshita T, Inagami T. Identificationof linoleic acid and oleic acids as endogenous Na�/V-ATPaseinhibitors from acute volume-expanded hog plasma. J Biol Chem260, 9672-9677 (1985).