The Measurement of Digitoxin in

Human Serum by Radioimmunoassay

GEORGEC. OLIVER, JR., Biu~r M. PARKER, DANIEL L. BRASFIELD, andCHARLESW. PARKER

From the Divisions of Cardiology and Immunology, Department of Internal Medicine,Washington University School of Medicine, St. Louis, Missouri 63110

A B S T RA C T A sensitive, specific, and relativelysimple immunoassay permitting measurement ofpharmacological levels of digitoxin in humanserum has been developed. The assay involvesbinding of 125I-labeled tyrosine-digitoxigenin(specific activity > 400 mc/mg) by rabbit anti-body to digitoxin. Antibody-bound radioactivity isprecipitated by addition of a second antibody (goatanti-rabbit gammaglobulin), and precipitate radio-activity is measured. Unlabeled digitoxin can bedetermined by the extent to which it competeswith 125I-labeled digitoxigenin and thus reducesprecipitation of radioactivity. Before the assay,unlabeled digitoxin is extracted from serum withchloroform, and the chloroform solution is evapo-rated to dryness. Quantitation is accomplished byreference to a standard curve in which knownamounts of digitoxin are added to normal serum.As little as 1 mug of digitoxin per ml of serumproduces significant reduction in precipitate radio-activity.

The sera of 5 patients were analyzed before andafter digitalization. A highly significant reductionin precipitate counts in the postdigitalization serawas observed (P < 0.001). Serum digitalis levelswere measured in 19 patients receiving no digi-talis and in 19 patients taking digitoxin or digitalis

This work was presented at the 39th annual meetingof the Central Society for Clinical Investigation and ab-stracted in 1966 J. Lab Clin. Med. 68: 1002 and in 1967J. Clin. Invest. 46: 1101.

Charles W. Parker is a recipient of a Research CareerDevelopment Award of the National Institute of Allergyand Infectious Disease.

Received for publication 16 August 1967 and in revisedform 21 December 1967.

leaf. Little of no digitalis-like activity was detectedin control sera, whereas serum levels averaged 27mpg/ml in those on digitalis (range 4-60 mpg/ml,P < 0.001). Patients judged clinically to showdigitals toxicity in general had higher levels thanthose without signs of toxicity. Patients receivingdigoxin had little or no detectable digitalis in theirserum with this method.

In addition to the assay itself, other potentialuses of the antidigitalis antibody include treatmentof digitalis toxicity and studies on the tissuelocalization of digitalis.

INTRODUCTIONDespite extensive use of digitalis for nearly twocenturies, no method of measuring serum levels ofthe drug has been developed that is entirely satis-factory. Previously described methods have beeneither too cumbersome, too insensitive, or toononspecific to be suitable for routine clinical use.

Stimulated by the success of radioimmunoassaysin measuring minute levels of substances such as

insulin and growth hormone, we have investigatedthe use of such a technique for digitalis assay.With this method, we have been able to developa convenient means for the measurement of phar-macological levels of digitoxin in human sera.

METHODS

Paper chromatographyUnless otherwise stated, paper chromatography was

performed in the following manner: Whatman No. 1 pa-per was impregnated with formamide by passing the paperthrough a solution of ethanol-formamide (1: 1) and al-

The Journal of Clinical Investigation Volume 47 1968 1035

lowing it to air-dry in a vertical position for 15-30 min.After materials were spotted, the paper was developedin the ascending manner with a solution of methyl ethylketone-xylene (1: 1). When the chloroform-tetrahydro-furan-formamide system is referred to, the only changeconsists of replacing the methyl ethyl ketone-xylene solu-tion with chloroform-tetrahydrofuran-formamide (100:100: 13).

Digitalis products were located by the m-dinitroben-zene-KOH reaction (1) which indicates the presence ofan intact butenolide ring. The tyrosine group was lo-cated by the 1-nitroso-2-naphthol reaction (2).

Synthesis of 3-0-succinyl digitoxigenin (SDG)The method of Yamada was used (3). 860 mg of suc-

cinic anhydride 1 and 442 mg of digitoxigenin 2 were dis-solved in 13.4 ml of pyridine. The solution was protectedfrom light and allowed to react at room temperature for3 months. The solution was poured into 75 ml of cold2 N H2SO4. The solid product was isolated by filtrationand washed with cold water. It was then dissolved in 150ml of chloroform-methanol (2: 1). The chloroform-methanol solution was washed once with 25 ml of 1 NH2SO4, and three times with water. 25 ml of methanolwas added after each washing. The organic phase wasdried over anhydrous sodium sulfate and taken to drynesson a rotary evaporator. The residue was dissolved in 15ml of hot ethanol, and hot water was added to turbidity.The solution was allowed to cool to room temperatureand then left at 40C for 48 hr. The resultant crystals wereisolated by filtration and washed three times with coldethanol-water (3: 2). The final product was a whitepowder, with melting point of 220-227'C. The yield variedfrom 60-75 %.

Preparation of antigenSDGwas coupled by two different methods to human

serum albumin and bovine serum albumin.Carbodiimide method. 50 mg of human serum albu-

min 3 was dissolved in 25 ml of water (pH 5.5). Thesolution was filtered, and 30 mg of EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] 4 wasadded. 20 mg of SDGdissolved in 2 ml of dimethylforma-mide was then added dropwise while stirring, and the pHwas adjusted to 5.5 with dilute HCl or NaOH. After 10min, an additional 10 mg of EDC was added. The solu-tion was stirred at room temperature for 18 hr. The re-action mixture was then dialyzed exhaustively (1 wk)against water with frequent changes of the outside solu-tion. The suspension was cleared by centrifugation, andthe soluble protein fraction was lyophilized. The productcontained at least five digitoxigenin molecules per mole-cule of protein [m-dinitrobenzene assay (4)].

Isobutvlchloroformate method. A modification of themethod described by Erlanger, Borek, Beiser, and Lieber-

1 Eastman Kodak Co., Rochester, N. Y.2 Aldrich Chemical Co., Inc., Milwaukee, Wis.3 Pentex, Inc., Kankakee, Ill.4 Ott Chemical Co., Muskegon, Mich.

man (5) was used. 0.15 ml of tributylamine and 280 mgof SDG were added to 5.85 ml of dioxane. After thesolution had been cooled to 10°C, 0.08 ml of isobutyl-chloroformate5 was added. The solution was stirred for20 min with gradual cooling to 4°C. The mixture wasthen added rapidly to a well-stirred and cooled solution of840 mg of bovine serum albumin 3 in a mixture of 22.0ml of water, 22.0 ml of dioxane, and 0.84 ml of 1 NNaOH. After 1 hr another 0.4 ml of 1 N NaOH wasadded. The solution was stirred at 4°C overnight. Theproduct was purified by dialysis as described above. Itcontained at least 13 molecules of digitoxigenin per mole-cule of protein (m-dinitrobenzene assay). The syntheticsteps are summarized in Fig. 1

Synthesis of 3-0-succinyl digitoxigenin tyro-sine methyl ester (SDG-TME)100 mg of SDG was dissolved in 2.14 ml of 0.255%o

(v/v) mixture of tributylamine in dioxane. The SDGsolution was cooled to 10°C, and 0.03 ml of isobutyl-chloroformate was added. The solution was stirred for20 min, whereupon a cooled solution of tyrosine methylester in dioxane-H20 was added rapidly. The latter con-tained 49 mg of tyrosine methyl ester hydrochloride dis-solved in a mixture of 7.85 ml of water, 7.85 ml ofdioxane, and 0.3 ml of 1 N NaOH. Stirring wascontinued at 100C for 1 hr after which 0.14 ml of1 N NaOH was added. The solution was stirredovernight in an ice bath that gradually reached roomtemperature. The following morning the solution wascooled to 4°C, and 185 ml of cold water was added. Theprecipitate that formed was isolated by centrifugationand washed four times with 30 ml of water, 30 ml of1 N HCl, 40 ml of 1 M NaHCO3, and 40 ml of water(yield 50%, melting point 99°C). By paper chromatogra-phy (methyl ethyl ketone-xylene and chloroform-tetra-hydrofuran-formamide systems) the product gave a singledinitrobenzene positive, 1-nitroso-2-naphthol positive spot.Chemical analysis6 for C37H49N109; calculated: C 68.18,H 7.58, N 2.15: found C 66.75, H 7.6, N 2.2. The struc-tural formula is given in Fig. 2.

Preparation of radioactive hapten (SDG-TME-I *

A radioactive hapten was prepared by iodinating SDG-TME by a modification of the method of Hunter andGreenwood (6). 30 ,ul of a 10 ,g/ml solution of SDG-TME was mixed with 80 Al of 0.5 M phosphate buffer(pH 7.4). Approximately 20 yl of carrier-free 'I (asNa1'"I in NaOH, representing 2 mc) was added, and,exactly 2 min later, 20 ul of a freshly prepared aqueoussolution of chloramine-T (7 mg/ml) was added. 2 minlater, 20 Al of a freshly prepared aqueous solution ofsodium metabisulfite (7 mg/ml) was added. After anadditional 2 min, 0.5 ml of dimethylformamide and 0.3 ml

5 K & K Laboratories, Inc., Plainview, N. Y.6 Schwarzkopf Microanalytical Laboratories Inc.,

Woodside, N. Y.

1036 Oliver, Parker, Brasfield, and Parker

(Digitoxose)3- (

DIGITOXIN

OH

DIGITOXIGENIN

ISuccinic Anhydride

CH3

C.OCH2- CH2- C-0

OH3-0-SUCCINYL

Protein - NH2(Carbodiimide)

Digitoxigenin-C-CH2-CH2-C-N-ProteinIt II0 0OH

DIGITOXIGENIN

Tyrosine Methyl EsterIsobutyl Chloroformote)

H

Digitoxigenin-C-CH2 -CH2-C-N- C- C-O-CH3II II II0 O H CH20

**OH

SUCCINYL DIGITOXIGENIN TYROSINE METHYL ESTER

FIGURE 1 The structural formulas of the digitoxin and the aglycone, digitoxigenin, are shown. The synthetic stepsleading to antigen (digitoxigenin-protein complex) and ligand are shown. See Methods section for further details.The asterisks indicate the probable position of the radioactive iodine molecule after iodination.

of H20 were added, and the product was immediatelypurified by serial transfer through seven tubes containinga small amount (80-100 mg) of Amberlite IRA 400,chloride form, (Rohm & Haas Co., Philadelphia, Pa.).The final product was 96% pure by chromatography(methyl ethyl ketone-xylene) and had a minimum specificactivity of > 400 mc/mg. In equilibrium dialysis experi-ments, as much as 98%o of the counts could be bound by

rabbit antidigitoxigenin antibody, and there was very littlenonspecific binding to nonimmune rabbit gamma globulin(< 1%).

Immunological proceduresRandomly bred rabbits were immunized with 3.2 mg of

human serum albumin (or bovine serum albumin)-SDGcomplex in complete Freund's adjuvant (0.4 ml/per foot-

The Measurement of Digitoxin in Human Serum by Radioimmunoassay 1037

OH3-0- Succinyl Digitoxigenin Tyrosine Methyl Ester

FIGURE 2 The structural formula of the succinyl tyrosinederivative of digitoxigenin is shown.

pad). Animals were boosted with 1 mg of antigen incomplete adj uvant at 1 month and every few monthsthereafter. Sera were obtained by cardiac puncture 5-7days after booster injections. "Normal" rabbit gammaglobulin and rabbit gamma globulin containing antidigi-toxigenin antibody were purified by precipitation withammonium sulfate at 50%o of saturation followed bydiethylaminoethyl (DEAE) chromatography. The DEAEcellulose and the globulin fractions were equilibrated with0.02 M phosphate, pH 7.0. The initial protein fractionseluted from the column were pooled on the basis of 280m,u of absorbance. Gammaglobulin concentrations werebased on 280 mu of absorbance, and 1.5 was used as thevalue for a 1 mg/ml solution (1 cm light path).

Goat antibody to rabbit gamma globulin was obtainedas follows: 50 mg of rabbit gammaglobulin 3 in 10 ml ofcomplete Freund's adjuvant was injected subcutaneouslyinto multiple sites on the back and thighs of the goat.The animal was bled at 3 wk and at intervals thereafter.A crude globulin fraction was obtained by precipitationwith ammonium sulfate at 20%o of saturation and labeled"goat anti-rabbit gamma globulin."

Collection of patient blood samplesVenous blood was collected in glass tubes containing

no anticoagulant and allowed to clot at room temperature.Serum was separated by centrifugation at 4°C and imme-diately analyzed or frozen at -20°C for further analysis.Repeated analysis of samples showed no loss of digitalisactivity after as long as 3 months of storage.

Extraction of digitalis from serumAs will be discussed later, serum could not be analyzed

directly in the assay system. Digitalis was extracted fromserum and an analysis performed on the extract. 5 mlof serum was pipetted into a 15 ml glass stoppered centri-

fuge tube, and 5 ml of reagent grade chloroform wasadded. The tubes were gently inverted 10 times andallowed to settle for 10 min. The inversion and settlingprocess was repeated twice. After the third series ofinversions, the tubes were centrifuged for 30 min ina refrigerated centrifuge (40C, 1200 g). The upper serumlayer and a milky precipitate layer at the interface wereremoved by aspiration, and the chloroform layer wasfiltered. 1 ml aliquots of the filtrate were transferred toeach of the two assay tubes and evaporated to drynessin a water bath (<90'C). The assay was subsequentlycarried out in these tubes. The efficiency of the extractionwas tested by adding tritiated digitoxin7 to serum andcounting the chloroform extract in a liquid scintillationcounter.8 The efficiency of extraction was 53.6 + 5.6%.

Assay procedurePreliminary experiments were run to determine the

dilution of rabbit antibody that resulted in the precipita-tion of approximately 50% of the radioactive hapten inthe presence of excess goat anti-rabbit gamma globulinsera (see below). 20 dul of a mixture of the appropriateamount of rabbit antibody to digitoxigenin (usually a dilu-tion of about 1: 300) and carrier rabbit gamma globulin(adjusted to give a total protein concentration of 0.5mg/ml) were added to each assay tube. 50 ,ul of freshlycentrifuged radioactive hapten (SDG-TME-I*) in 2.5%egg albumin was next added. The radioactive hapten wasdiluted so that 50 1d yielded approximately 10,000 cpm.9Phosphate saline buffer (0.15 M NaCl, 0.008 M K2PO4,0.002 M NaH2PO4, pH 7.4) was added to bring thevolume of each tube to 200 ,ul. The tubes were mixed welland allowed to incubate for 1 hr at room temperature,after which they were left at 4VC overnight. The followingmorning 200 ,ul of goat anti-rabbit gamma globulin (inthe region of antibody excess) was added to each tube.After the tubes were mixed, they were allowed to incu-bate for 1 hr at room temperature, during which timetotal counts were measured. They were then incubatedat 4VC for 4 hr and centrifuged at 4VC (1000 g) for 30min. The supernatant solution was removed by carefulaspiration. The precipitate was washed once with 1 mlof cold phosphate saline, and counts were then measured.All samples were run in duplicate, and the per cent

counts in the precipitate were calculated for each tube.

Precision and reproducibilityThe precision of the method was tested by an analysis

of the per cent counts in the precipitate of 51 duplicatetubes. The coefficient of variation was ±6.7%.

A similar analysis was performed on repeated measure-

7 New England Nuclear Corp., Boston, Mass.8 Packard Tri-Carb Liquid Scintillation Spectrometer,

Model 3375, Packard Instrument Co., Inc., DownersGrove, Ill.

9 Packard Model 410A autogamma spectrophotometer,well-type, Packard Instrument Co., Inc., Downers Grove,Ill.

1038 Oliver, Parker, Brasfield, and Parker

ment of serum digitoxin levels. A total of 29 measure-ments was made on the serum from 10 patients. For serumlevels in the range 0-10 mug/ml, the coefficient of varia-tion was ± 31.7%; in the range 10-20 m~Ag/ml, the coeffi-cient of variation was ± 19%; and in the range 20-45m/g/ml, coefficient of variation was 11%

RESULTS

Several different techniques were used to demon-strate the presence of rabbit antibody to digitoxin.First, positive interfacial precipitation tests wereobtained with rabbit anti-SDG-human serum al-bumin (HSA) as the antibody and SDGbovineserum albumin (BSA) as the antigen. This result,while not conclusive, suggests the presence ofrabbit antibody. Secondly, in equilibrium dialysisexperiments when SDG-TME-I* as hapten wasused, as much as 98% of the radioactivity couldbe bound to the antibody with essentially no non-specific binding (< 1%) to "nonspecific" rabbitgamma globulin. Finally, the radioimmunoassayitself gave clear evidence that antidigitoxin anti-body had been produced. The precipitation ofradioactivity was markedly increased in the pres-ence of specific antibody as compared with "non-specific" gamma globulin. As little as 1-2 m~ugof digitoxin resulted in easily detectable reductionin the specific precipitation of radioactivity (Fig.3). The limited amount of nonspecific precipitationof radioactivity that occurred in the presence of"nonspecific" gamma globulin was not inhibitedby digitoxin.

60k 0

40

K30

- 20

4 010 t 0

0 Q02 0.2 2[

20 200 2. IAwn

AMOUNTOF DIGITALIS

FIGURE 3 The results of a typical calibration curve areshown. In this assay, as little as 0.2 m/sg of digitoxin perassay tube produced detectable reduction in precipitateradioactivity. In the range of 0.2-200 mpg, when resultsare plotted on a semilogarithmic scale (as is shown),the curve is linear.

Digitalis in human serum could be measured byits ability to inhibit the precipitation of SDG-TME-I* in the double antibody assay. Chloroformextracts of human serum containing no digitaliscaused about a 10% reduction in precipitatecounts. Therefore, in establishing calibrationcurves, known amounts of digitalis were addedto "normal" human serum and extracted withchloroform. The sera of 19 persons receivingdigitoxin or digitalis leaf have been analyzed.In every instance significant inhibition of precipita-tion over control sera was observed (P < 0.001).The serum levels of digitoxin as judged by thistechnique were in the range of 4-60 mug/ml(Fig. 4). The sera of five patients were analyzedbefore and after digitalization. In each instancemarked inhibition was found after digitalization[P <0.001 (Fig. 5)].

In contrast to digitoxin and digitalis leaf, seraof patients taking digoxin showed in general verylittle inhibition. This finding is not due entirelyto the lower levels of digoxin found in the seraof patients on digoxin (7) but is due at least inpart to a reduced ability of digoxin to compete forantibody sites. Digoxin in amounts of 5-12 mtugin saline caused little inhibition, and in general inour assay system, on an equimolar basis, digoxinwas about one tenth as active as digitoxin(Table I).

TABLE IPer Cent of Inhibition

Amount Digitoxin Digoxin

mpg2 21.75 36.0 9.7

10 53.2 18.025 66.0 31.650 79.8 40.1

Comparison between digitoxin and digoxin. For a givendegree of inhibition of precipitate radioactivity, roughlyten times more digoxin than digitoxin is needed.

The results of analysis of 19 "normal" sera indi-cated that nonspecific serum inhibition did notvary to any major extent from individual to indi-vidual. Efforts to demonstrate that the seruminhibitor is a circulating steroid have been incon-clusive to date. Cholesterol caused moderate inhi-bition at a level of 150 jug, but hyperlipemic seradid not exhibit more inhibition than normal sera.

The Measurement of Digitoxin in Human Serum by Radioimmunoassay

: L0

i

0

)U9

1039

60r

50

40

E 30

E 20

o0

CNo

Digitalis

I

Dig itoxin Digitoxin? Toxic

Digoxin

FIGURE 4 Serum digitalis levels in controls (on no digitalis), patientsmaintained on digitoxin or digitalis leaf, and patients maintained ondigoxin. In addition, data are presented on patients suspected clinically ofshowing signs of digitalis toxicity (? Toxic). With only two exceptions,essentially no digitalis-like activity was detected in the sera of controlpatients, and, in these two cases, the amount was small. In contrast,every patient on maintenance digitoxin or digitalis had detectable levels.In general, those patients who were suspected of having digitalis toxicityhad higher levels than those not toxic, but overlap was present. SeeResults section for further discussion. Patients on digoxin had in generalvery low serum levels. The one exception was a patient who had beenacutely digitalized with digoxin.

6O0

b 50

Q 40

30

*! 20

e 10

BeforeDigitalis

FIGURE 5 Results from five patients befordigitalization with digitoxin. Control and pation sera were compared in the same immun(trol studies indicated that prolonged storagein the presence of serum at - 20'C did notinhibitory activity in the immunoassay. Inpostdigitalization sera caused more inhibitiontate radoactivity than predigitalization seraThe variation in predigitalization serum valtthe graph (from 25 to 60%o precipitation of rreflects the fact that the immunoassays wereat different times under somewhat differenIn each instance, other normal sera evaluatedtime gave comparable values. Despite somecontrol serum values from assay to assay repsis of digitalis-containing sera gave reprodu.

Physiological levels of hydrocortisone, androster-one, and dehydroepiandrosterone produced little,if any, inhibition. Progesterone and testosterone inlarge amounts (500 mpg) caused moderate in-hibition.

Sera from five patients who were receivingdigitalis currently or who had taken digitalis in thepast have been screened for antidigitoxin anti-bodies. Such antibodies have been found in a pa-tient who developed thrombocytopenia (8). Un-

After diluted sera were incubated with SDG-TME-I*Digitalis for 1 hr at 370C, and proteins were separated by

re and after electrophoresis (oxoid strips). The strips wereostdigitaliza- then cut into 1 cm sections and counted in aQassay. Con->of digitalis gamma counter. Radioactivity was found at thediminish its origin and with the albumin fraction. In no in-every case, stance was radioactivity found in the gamma

n of precipi- globulin band. Thus there was no evidence for thePes shown in presence of antidigitoxin antibodies. Studies on a

adioactivity) more extended series of sera are in progress.carried out

It conditions. DISCUSSIONat the same The radioimmunoassay described in this papervariation in ..

)eated analy- provides a simple, sensitive, and specific means

icible results. of measurement of serum digitoxin levels in man.

1040 Oliver, Parker, Brasfield, and Parker

.

AS

011

Patients receiving therapeutic doses of digitoxinusually had serum levels in the 5-40 mjig/mlrange by radioimmunoassay. This finding confirmsthe results of Lukas and Peterson (9) who foundlevels of 10-50 mjug/ml by an independent method.In general, the highest serum levels of digitoxinwere in patients who were suspected of havingdigitalis toxicity clinically, a fact which supportsthe data of Doherty and Perkins (10) who founda significant correlation between blood and myo-cardial digitalis levels in dogs. We suspect thatthe relationship between blood levels and digitalistoxicity may be better than that shown in Fig. 4.In the two patients who had the lowest serum lev-els of digitalis there was some uncertainty as to theclinical diagnosis of toxicity.

The radioimmunoassay discussed herein appearsto possess significant advantages over other meth-ods for measuring serum levels of digitalis. Bio-assay methods such as those with the duck embryoheart are too laborious for routine use (11). Therubidium uptake technique of Lowenstein (7)lacks specificity. In his first publication (7) henoted considerable overlap between patients ondigitalis and those not, and a number of falsepositive tests occurred. In a subsequent publica-tion, Lowenstein and Corrill (12) stated they hadeliminated the false positives by extracting plasmawith methylene dichloride. This technique ap-peared to improve the sensitivity of their assay,but they presented no new data on the overlapproblem-. Lukas and Peterson (9) have recentlydescribed a double isotope assay for digitoxin thatappears to be quite sensitive and specific. How-ever, this technique is lengthy and laborious andis unsuitable for routine clinical use. The studiesof Okita, Talso, Curry, Smith, and Geiling (13)and Doherty and Perkins (14) in which radio-active digitalis preparations were administered tohuman patients have added to our knowledge ofthe metabolism of digitalis preparations. However,these studies can be used only for acute studies and,are not useful for measuring serum levels in aproblem patient.

At present we can measure serum levels ofdigitoxin in patients receiving digitoxin or digitalisleaf. The specificity of the radioimmunoassay issuch that digoxin, which differs from digitoxinonly in the presence of a hydroxyl group at the

C-12 position, is bound only about one tenth aswell as digitoxin. In view of the marked structuralsimilarity between digoxin and digitoxin the sub-stantial reduction in digoxin binding implies thatthere is a tight fit between digitoxin and antibodyat this position on the steroid ring. It is of interestthat Butler and Chen (15) using antidigoxin anti-body found that binding of the homologous ligand,digoxin, was favored over that of digitoxin byabout the same order of magnitude. The extent towhich other structural analogues of digitoxinwould cross-react immunologically is not known atpresent. On purely structural grounds one wouldpredict that any digitalis preparation containingthe digitoxigenin moiety would be bound well bythe anti-succinyl digitoxigenin antibody. Becauseof the relatively weak binding of digoxin and thelow serum levels of this drug, serum digoxinlevels cannot be measured with the current immu-noassay system. Work is in progress to prepareantidigoxin antibodies and 125I-labeled digoxin sothat the radioimmunoassay can be extended topatients receiving digoxin.

With the present assay system, results are notobtained before 36 hr. Work in progress indicatesthat it should be possible to shorten the time ofthe assay to 4 or 5 hr permitting the measurementof levels of digitalis in serum in acute therapeuticsituations. Encouraging results have been obtainedwith ammonium sulfate at 33% of saturation toseparate antibody-bound and free SDG-TME-I*.The extent of nonspecific precipitation of radio-activity with ammonium sulfate is relatively high,but with recent modifications nonspecific bindinghas been reduced to 10%o of the total radioactivehapten present.

As previously mentioned, in a parallel inde-pendent study Butler and Chen (15) recently haveprepared rabbit antidigoxin antibodies. Theirmethod of immunization was similar in principleto our own in that the immunizing antigen was aconjugate of digitalis with protein. Their conjuga-tion procedure involved the oxidation of digoxinwith sodium periodate followed by conjugation toprotein. They demonstrated the presence of anti-body by equilibrium dialysis with 3H-labeled di-goxin as the ligand. However, they have not beenable to measure serum levels of digitalis, and itappears that the sensitivity of their system would

The Measurement of Digitoxin in Human Serum by Radioimmunoassay 1041

have to be increased by at least two to threeorders of magnitude in order for this to be possible.The much greater sensitivity of the radioimmuno-assay described in this paper is due at least in partto the use of a labeled digitalis molecule with highspecific activity. Additional factors may include therelative affinities of the antidigitalis antibodies in-volved and the precise nature of the immunoassayprocedure.

The chloroform extraction step in the radio-immunoassay is necessary in order to reduce non-specific interference with the assay by normalserum. Conceivably antidigitalis antibodies presentin the serum of patients receiving the drug mightinterfere with the extraction. However, prelimi-nary data have failed to reveal significant levels ofthe antibody. During the extraction, steroids andother nonpolar substances normally present inserum enter the organic phase. Cholesterol, testos-terone, and progesterone are capable of inhibitingthe precipitation of radioactivity in the immuno-assay, but high concentrations are required. Atpresent, there is no indication that physiologicalvariations in the serum levels of these substanceswould influence the immunoassay to any majorextent. However, the possibility remains that highhormonal levels achieved pharmacologically mightexert a digitalis-like effect in the assay system.

Other uses of the antidigitalis antibody are pos-sible. For instance, it is possible that antidigitalisantibody could be administered to patients withdigitalis intoxication to reverse the toxic processby combining with serum or tissue digitalis. Weare currently investigating this possibility. An-other potential use for the antibody would be tohelp ascertain the location of digitalis in heartmuscle with fluorescein or ferritin labeling. Webelieve, however, that the main usefulness of theantibody will be its ability in the assay to afforda relatively rapid assessment of digitalis levels andhence of the need for more or less drug.

ACKNOWLEDGMENTS

Wewish to thank Barbara Bloss and Shirley Godt fortheir valuable technical assistance.

This work was supported by grants lRO1 HE 11233-01,HE-11034, AI 04646, and AI 00219 of the U. S. Public

Health Service. Dr. Brasfield was supported by StudentResearch Training Grant T5-GM 1608-10.

REFERENCES1. Jung, Z. 1963. Steroid glycosides and their aglycones.

In Paper Chromatography. I. M. Hais and K. Macek,editors. Academic Press Inc., New York. 402.

2. Hais, I. M. 1963. Amino acids. In Paper Chroma-tography. I. M. Hais and K. Macek, editors. AcademicPress Inc., New York. 449.

3. Yamada, A. 1960. Synthesis of succinoyl and phthaloylderivatives of digitoxigenin, gitoxigenin and oleandri-genin. Chem. Abstr. 54: 6807.

4. Wright, S. E. 1960. The Metabolism of CardiacGlycosides. Charles C Thomas, Springfield, Ill. 22.

5. Erlanger, B. F., F. Borek, S. M. Beiser, and S.Lieberman. 1957. Steroid-protein conjugates. I. Prepa-ration and characterization of conjugates of bovineserum albumin with testosterone and with cortisone.J. Biol. Chem. 228: 713.

6. Hunter, W. M., and F. C. Greenwood. 1962. Prepara-tion of Iodine-131 labeled human growth hormone ofhigh specific activity. Nature. 194: 495.

7. Lowenstein, J. M. 1965. A method for measuringplasma levels of digitalis glycosides. Circulation.31: 228.

8. Young, R. C., R. L. Nachman, and H. I. Horowitz.1966. Thrombocytopenia due to digitoxin. Am. J.Med. 41: 605.

9. Lukas, D. S., and R. E. Peterson. 1966. Doubleisotope dilution derivative assay of digitoxin inplasma, urine, and stool of patients maintained onthe drug. J. Clin. Invest. 45: 782.

10. Doherty, J. E., W. H. Perkins. 1966. Tissue con-centration and turnover of tritiated digoxin in dogs.Am. J. Cardiol. 17: 47.

11. Friedman, M., and R. Bine, Jr. 1947. Employmentof the embryonic duck heart for the detection ofminute amounts of a digitalis glycoside (lanatosideC). Proc. Soc. Exptl. Biol. Med. 64: 162.

12. Lowenstein, J. M., and E. M. Corrill. 1966. Animproved method for measuring plasma and tissueconcentrations of digitalis glycosides. J. Lab. Clin.Med. 67: 1048.

13. Okita, G. T., P. J. Talso, J. H. Curry, Jr., F. D.Smith, Jr., and E. M. K. Geiling. 1955. Metabolicfate of radioactive digitoxin in human subjects. J.Pharmacol. Exptl. Therap. 115: 371.

14. Doherty, J. E., and W. H. Perkins. 1962. Studieswith tritiated digoxin in human subjects after intra-venous administration. Am. Heart J. 63: 528.

15. Butler, V. P., Jr., and J. P. Chen. 1967. Digoxin-specific antibodies. Proc. Natl. Acad. Sci. U. S. 57:71.

1042 Oliver, Parker, Brasfield, and Parker