Acta Clinica Belgica International Journal of Clinical and Laboratory Medicine
ISSN: 1784-3286 (Print) 2295-3337 (Online) Journal homepage: http://www.tandfonline.com/loi/yacb20
Radioimmunoassay Of Digitalis Glycosides F.M. Belpaire & M.G. Bogaert To cite this article: F.M. Belpaire & M.G. Bogaert (1976) Radioimmunoassay Of Digitalis Glycosides, Acta Clinica Belgica, 31:4, 212-221, DOI: 10.1080/17843286.1976.11717089 To link to this article: http://dx.doi.org/10.1080/17843286.1976.11717089
Published online: 17 May 2016.
Submit your article to this journal
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yacb20 Download by: [Australian Catholic University]
Date: 12 August 2017, At: 18:06
212
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
F.M. BELPAIRE and M.G. BOGAERT
INTRODUCTION
Several methods for quantitation of levels of cardiac glycoside3 in plasma (or serum) have been described: the double isotope dilution derivative assay (17), the inhibition of red blood cell Rb.. uptake (l6), the NA-K ATPase inhibition method (3), and the radioimmunoassay (5). Most of these methods are tim-- consuming, the radioimmunoassay (RIA) is not : whole serum or plasma can be used and no extraction step is needed. Because of its simplicity, rapidity and sensitivity, RIA of cardiac glycosides is commonly performed in the clinical laboratory, mainly since assay kits are commercially avaiJable. As discussed in the subsequent pap r (2). the interpretation of plasma levels of digitalis glycosides is not easy, and needs to be done in conjunction with the clinical observation of the patient. But the technique in itself can be criticised for lack of precision and accuracy, and for the existence of interfering factors. The purpose of this paper is to decrib-- our own experience with the technique, using commercially available RIA kits as well as antibodies produced in our own laboratory, and to stress different pitfalls of the procedure. This
H eymans Instituut voor Farmakologie - A.Z., De PinteJaan 135, 9000 Gent.
Acta Clinica Belgica, 31, 4 (1976)
discussion will primarely focus on digoxin , but digitoxin and other glycosides will also be mentioned. .PRODUCIJO
OF ANTIBODIES
Antibodies against digitalis glycosides that are available commercially, are obtained in the rabbit. For the preparation of the antigen we foJJowed the procedure described by Smith et al. (20) : digoxin is coupled to ~uman ser u~ . albumin and this conjugate is unmunogemc m rabbits (5). The rabbits were immunised by intradermic injection of 0.8 ml of antigen preparation once w ekly during four weeks, followed by intramuscular .injection of 0.4 ml every week or every 2 weeks. Afte~ f~ur weeks of immunisation, only 307o bmdmg of lab ..Jed digoxin was obtained for a serum dilution of 1/10. The final serum dilutions for obtaining 507o binding of 'H-digoxin to the antibody in function of time of immunisation are shown in Figure 1. The results of five different rabbits are given : after 22 weeks, a titer of about 240 000 is obtained in three rabbits, and a titer of about 160.000 in two rabbits. The percent binding of 'H-digoxin and 'H-digitoxin on the antiserum in the five rabbits in function of the final dilution of the antiserum is shown in igure 2. The antiserum was obtained after 19 weeks of immunisation. he final serum dilution for obtaining 507o binding of digoxin is about 9 times higher than for 507o binding of digitoxin.
213
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
300,000
Final dilution
X 0
A
200,000
X
0
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
•
X
e
100,000
•
0
10
12
14
\6
18
20
22
Weeks
Fig. I - Final dilution of 0.1 ml antiserum for obtaining 50% binding of 'H-digoxin (0.5 ng), at different times after immunisation in five rabbits. Each rabbit is indicated by a different symbol. GEN RAL PRO EDURE FOR PERF ORMING THE RIA
RIA can be p rformed dir ctly on serum, whereas analysi of urine or tissue samples requires an extraction step, e.g. with dichlormethane. The principl of the RIA procedure can b-- ummaris d as follows. An aliquot of serum (or an extract of urine or tissue) containing the glycoside to b measured, is mixed with an appropriate quantity of lab led glycoside; tritium-labvled or '"'!-labeled glycosid can b.. . used. The use of '"I-Iab Jed glycosid (Y-emitter) has the advantage over the use of tritium-labvled product that th counting procedure is fast and cheap ; furth rmore the problems of ample quenching ar eliminated. On th other hand, tritium-lab I d glycoside (fJemitter) ba the advantage that its half-life
(12.3 years) is much longer than for the Y-emitter (60 days). Antiserum is then added in such amount that in the absence of non lab.Jed glycoside (i.e. for blank serum) ,50 7o of the labeled glycoside is bound to the antibody. After incubation, the unbound glycoside is adsorbed on charcoal. After centrifugation to remove the charcoal, the sup rnatant containing the antibody bound labvled glycoside, is measured in a scintillation spvctrometer ; the amount of labvled glycoside bound is invers ly related to the amount of unlabvled glycoside that was present in the serum. Standard curves are made by adding known amounts of glycoside to blank serum and runnin g these samples through the whole procedure. ach commercial kit carries complete instructions for its use. When working with « home made » antiboActa Clinica Belgica, 31, 4 (1976)
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
214
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
dies, for the assay of digoxin (patients samples in the range of 0-4 ng/ml), we used the solutions and procedure described for the « Lanoxitest f3 » kit of Wellcome (see the manufacturers instructions for details). For the assay of digitoxin (patient samples in the range of 5- 40 ng/ ml), the' same procedure is followed as for digoxin, except that labeled (0.01 p.C, spec. act. 12.2 Ci/ mmol) and non labeled digitoxin were used instead of labeled and non labeled digoxin for setting up the standard curves. An amount of antiserum which, in the absence of non labeled glycosides, binds approximately 50 % of 'H-digoxin, respectively 'H-digitoxin, was used. As can been seen in Figure 2, the amount of antiserum needed is much larger for digitoxin as for digoxin, since the affinity for digoxin is much higher than that for digitoxin ; the concentration of antibodies against digitoxin is perhaps smaller.
Each time a calibration curve was set up, a blank was obtained by incubating a sample of standard serum with 'H-digoxin and buffer, without antiserum.
CALCULATION OF THE RESULTS OF RIA
After substraction of the CPM of the blank from the CPM of the antibody bound H' digmdn of each sample, the percent binding of the labeled glycoside can be calculated. A calibration line is obtained by calculating the percent binding for standard concentrations of cold glycoside added; however, by plotting the reciprocal value of the CPM of each sample against standard concentrations of the cold glycoside, a straight line is obtained (Figure 3). The glycoside content of the unknown samples can then be calculated by means of the regression equation of this straight line.
% binding of 3H-glycoside
70 DIGITOXIN
60 50
:~
~~ •
40 30
20
·~
10
I
0 8.10 3
8.10 4 Final dilution of antiserum
Fig. 2- Percent binding of 'H-digoxin and 'H-digitoxin as a function of final antiserum dilution in five rabbits, 19 weeks after immunisation. The same rabbits (and the same symbols) as in Figure I were used. Acta Clinica Belgica, 31, 4 (1976)
215
RAD!OIMMU OASSAY OF DIGITALIS GLYCOSIDES
·sol%
b inding of 3H-digoxin
• 1.0
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
30
4 Reciprocal counts per min ( x10- )
8 7
\.
•
\.
20
6
~-
5
~.
I. 3 2
10
B
A 0
0
I. 3 2 Digoxin concen tration ( ng /ml)
2
3
4
Digoxin concentration ( ng/ml )
Fig. 3 - Standard curve for RlA of digoxin. A. Binding of 'H-digoxin to antiserum, as a function of digoxin concentration. B. R eciprocal counts per minute of 'H-digoxin bound to antiserum, as a function of digoxin concentration. REPRODUClBILITY OF THE METHOD
The reproducibili ty of the method was expressed by the coefficient of variation (SD I mean x 100). A patient sample was analysed for digoxin , 10 times «within run» and 10 times « b tween run », using the «home made » antibodies. The coefficient of vari ation was 8.5tfo «within», and 10.4 'fo « between » runs, for an av rag measurement of 1.29 ng / ml digoxin. These data are in agreem nt with those of other authors (6, 11, 12). SPECIFICITY
Specificity wa d fin d by comparing the inhibition of a ntibody binding of labeled digoxin or labeled digitoxin by closely related compound uch as lanato ide C, ,8-me-
thyldigoxin, acetyldigoxin, desacetyllanatoside C, acetyldigoxin and gitaloxin. The structure of these compounds is shown in Figure 4. As th e antibodi s in the anti-digitalis sera are directed larg ly towards the aglycon portion of the molecule, most antisera to one cardiac glycoside cross-react to some extent with other glycosides. The results for 'H-digoxin and 'H-digitoxin binding in the presence of these different compounds are presented in Figure 5. From the results it is clear that the digoxin antibody has approximately the same affinity for the different substances with the same steroid nucleus as digoxin, i.e. with the h ydroxyl group in the 12-po ition on th nucleus. For oth r substances. such as digitoxin and formylgitoxin, the affinity is much lower. The digoxin assay system can thus bv Acta Clinica Belgica, 31, 4 (1976)
RADIOIMMUNOASSAY OF DIGITALIS GLYCOS!DES
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
216
used for th e determina tion of ,8-methyldi- toxin in the presence of different amounts of their respective metabolites is shown in goxin , lana toside C. desacetyllana toside and 6. All the metabolites studied were Figure acetyldigoxin. The digitoxin assay on the displace 'H-digoxin or 'H-digitoxin to able determination for used other hand can be from the antibody to approximately the saof acetyldigitoxin. These results are in agreeme extent and thus interfere in the determent with those of Larbig and Kochsiek mination of di goxin or digitoxin . The two (14) . This cross-reactivity can give problems genines however are slightly less active in if it is not known which glycoside a patient displacing 'H-digoxin or 'H-digitoxin than is receiving. the pa rent molecule or than the other meFrom Figure 5, it can also be seen that, tabolites. Thi s is in agreement with the reif one has to measure digitalis levels in a sults of others (14, 19, 21) . It should be nopatient treated with digitoxin , it is not adted tha t the metabolites of di goxin and divisable to use 'H-digoxin and cold digitoxin gitoxin are cardio-active to varying degrees for makin g the standard curve. Indeed , digi(1 8), so that measuring as well the parent toxin is metabolised to digoxin for about molecule as the metabolites could be an in10% . and the interference of the digoxin dex of the total cardioactive material prepresent in th e serum mak es th at falsely high values for digitoxin will b.., obtained. In contrast, when digitoxin is determin ed using 'H-digitoxin , the interference by di goxin present in the sample is much smaller as shown in F igure 5 B. The affinity of antidigoxin antibodies for gitaloxin , is much lower than for digoxin and digitoxin . However antigitaloxin antibodi es were prepared with hi gher affinity for gitaloxin than for digoxin (15). The binding of 'H-digoxin and of 'H-digi• I~
Fig. 4 -· Structural f ormulas of differ ent cardiac glycosides.
R, 3 digitoxose
+ acetyl + glucose 3 d ig itoxose + acetyl 3 digitoxose + glucose 3 dig itoxose + met hyl 3 dig itoxose +
3 dig itoxose
R,= H R,=H
R,= OH R,= H
R,= H R,= OH
dig itoxin lanato ide A
d igoxin lanatoside C (CedilanideR) acetyld igoxi n (Cedigoci neR) desacetyll ana to ide-C (Desacen) ,8- methyld igoxin (LanitopR)
gitoxin
acetyld igitox in (AcylanideR)
formyl
Acta Clinica Belgica, 31, 4 (1 976)
form ylgitoxin (Cristalox ineR)
217
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
50
l
% binding of 3H-digoxin
. ..
.•
'
40
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
50 1% binding of 3H-digitoxin
40
30
30
20
20
10
10
A 2
3
4
5
6
pmoles glycoside
• A
\ \. B
·~'·- .
10 20 ·3o 40 5o 60 pmoles glycoside
Fig. 5 - A. Binding of 'H-digoxin in the presence of increasing concentrations of various cardiac glycosides : digoxin • acetyldigoxin 6. /3-methy fdigoxin + digitoxin • lanatoside C X desacetyllanatoside C 0 formylgitoxin A B. Binding of 'H-digitoxin in the presence of increasing concentrations of various cardiac glycosides: digitoxin • acetyldigitoxin t:. gitaloxin A digoxin •
sent. In the case of digoxin, metabolisation is only of minor importance (7) ; for digitoxin, however, metabolic degradation is extensive but the contribution of inactive metabolites to the serum concentrations of digitoxin measured is not clear. It is thus clearly impossible to rely on a
plasma level of a glycoside when two or more glycosides are administered at the same time, or when there has been a recent change from one glycoside to another. It bas been demonstrated that some human steroids interfere in the RIA of digitalis glycosides (10, 23). The presence of Acta Clinica Belgica, 31, 4 (1976)
218
RADIOIMMUNOASSAY OF DIGI T A LIS GLYCOSIDES
prednisone e.g. interferes only when high doses of this product are used ; spironolactone however interferes even when the usual therapeutic doses are given; e.g. in patients receiving no digitalis but receiving 100, 300 or 400 mg spironolacton'e/day, an apparent digoxin plasma level of respectively 0.4, 0.7 and 0.9 ng/ml was measured
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
(10).
50
l
% binding of 3H-digoxin
COMPARISON OF DIFFERENT COMMER· CIALLY AVAlLABLE KITS
Only few laboratories produce their own antisera, and most of them use the commercially available RIA-kits. Recently different comparative studies of these kits have been published. In these studies large discrepancies were found in the analysis of serums of patients. In a study of Kubasik et al. (11, 12) using 50
l
% b;nd;ng of 3H-d;g;tox;n
• 40
30
30
20
20
10
10
A 2
3 4 5 6 7 pmoles glycoside
10
20 30 40 50 60 70 pmoles glycoside
Fig. 6 - A . Binding of 'H-digoxin in the presence of different concentrations of metabolites: digoxin • digoxigenin-bis-digitoxoside 0 digoxigenin-mono-digitoxoside ~ digoxigenin X B. Binding of 'H-digitoxin in the presence of different concentrations of metabolites: digitoxin • digitoxigenin-bis-digitoxoside 0 digitoxigenin-mono-digitoxoside ~ digitoxigenin X Acta Clinica Belgica, 31, 4 (1976)
219
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
tritiated or iodinated digoxin, the plasma values of digoxin analysed with five different kits ran ged from 0.8 to 1.7 ng/ ml in a sample of one patient, from 2.4 to 4.6 ng/ ml in a sample of another patient. D etermination of digoxin added to control sera yielded reproducible results. In the study of Voshall et al. (22) , using "'I-labeled digoxin , some kits gave systematica11y higher values than other kits. A patient sample analysed with four different kits gave the fo11owin g results : 5.8, 3.5, 2.0, 1.2 ng/ml. The same authors also found that digoxinfree sera gave positive results with some kits. e.g. 0.0, 0.6, 1.8, 2.2 ng/ m1. We have compared samples from patients with renal insufficiency who were receiving di goxin , using 6 different kits (1). In some of these samples divergent results were obtained (e.g. for one sample: 0.64, 0.78, 1.11. 1.38, 3.30, 3.13 ng/ ml). Digoxin-free sera gave values around zero with the 6 kits. The evaluation of digoxin in blank sera to which digoxin was added, was reproducible. These data show that some problems may arise in the RIA of di gitalis glycosides of some samples. The reason for these discrenancies is not always known. Some factors that could affect RIA of digitalis glycosides will b di scussed in the followin g paragraph.
FACTORS A FF CTlNG RIA OF GLYCOSIDES
Different factors affecting the RIA of digitalis glycosides have been described : 1. It is necessary to test the labeled and the unlabeled glycosides used by thin layer chromatography, since the purity of different commercial supplies varies consid rably. 2. The pati nt samples must be free of radioactivity, e.g. the radioactivity introduced by diagnostic tests. 3. Adequate quench correction is very important when tritiated glycosid s are used ; indeed some serum samples contain
hemoglobin or bile pigments which may qu ench . Quench correction can be done by adding an internal standard or b y use of an ex ternal standard, or by using the channel ratio method. 4. Incubation time and incubation temnerature of the antigen-antibody reaction, a nd the duration of contact with charcoal must be well sta nda rdized and optimal conditions must b ~ defined. Kuno-Sakai and Saka i (13) described the effect of varying the incubation time of the antigen-antibody reaction and of varyin g the duration of adsorption on dex tran coated charcoal, for different commercial RIA kits. In all cases the di goxin concentration measured increa~ed lin e!'!.rly with adsorption time, but the denree of increase varied from kit to kit. 5. In samples of uremic patients chemiluminesence of the serum can sometimes be observed, so that higher counts of the sample . and consequentl y a lower value of digoxin are measured . For such samples it is necessa rv to correct for this chemilumin~ sence (4). 6. Some authors (9 , 22) stated that the albumin content of a serum sample can influ nee the RIA of digoxin : with sera from patients with hypoalbuminaemia, bincling of the labeled glycoside was enhanced, and this resulted in a lower digoxin value measured. The exact mechanism by which albumin interferes with the binding of labt>led digoxin to the antibody remains unclear; in our hands, changes in albumin concentration did not explain di screpancies in the results observed. ONCLUSION
Although RIA of cardiac glycosides has become a routine technique, the procedure is not entirely free from pitfalls. M easurement of cardiac glycosides should never be performed without du e attention to the possibility that, even if the assay is done with optimal technical care, smaller or larger inaccuracies are present. Acta Clinica Belgica, 31, 4 (1976)
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
220
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
SUMMARY
SAMENVATIING
In this paper, several aspects of the radioimmu noassay (RIA) method for digitalis glycosides are discussed. Although this method is used routinely, the results are not always accurate. Kits for RIA of digitalis glycosides are commercial ly availabl e, but antibodies, e.g. against digoxin, can easily be obtained in rabbits : high titers are reached within a few months . The general procedure is described; results can be calculated from a calibration line. The coefficient of variation «within run :1> and « between run » for the assay of digox in in patient serum is a pproximately 10%. The RIA method has a limited specificity, and cross-reaction between different glycosides is present ; this makes i.t possible to measure several glycosides, including digitoxin , using the antibodies against digoxin. M etabo·lites of digoxin and digi.toxin ·interfere with the determination of th e paren t molecules. Important discrepancies exist between the results obtained with the different commercial kits: literature data and perso nal experience are cited. Tn a final paragraph, known sources of error are discussed. When performing RIA of digitalis glycosides, on e should never forget the possibility that small er or larger errors of measurement are possible.
In dit artikel worden verschillende aspekten van de radio-immunologische bepalingsmethode voor digitalisglycosi den besprok en. Kits voo r deze bepaling zijn in de handel beschikbaar, maar men kan ook zelf gemakkelijk antiJichamen, bijvoorbeel d tegenover digoxine, opwekken bij het konijn : na enkele maanden reeds worden hoge titers bereikt. De algemene procedure wordt beschJreven ; bij elk e bepaling is een ijklijn nodig om de resuJ taten te berekenen . De variatiekoefficienten ''within run" en " between run" voor de bepaling van digoxine in het serum van patienten liggen rond de 10%. De radio-immunologische bepalingsmethode heeft slechts een beperkte specificiteit en er bestaat kruisreaktie tussen de verschiUende glycosiden: daardoor is het mogelijk, gebru.ik makend van de antilichamen tegen digoxine, verschill ende glycosiden, waaronder digitoxine, te meten . De metabolieten van digoxine en digitoxine interfereren met de bepaling van de moedermolekules. Er bestaan belangrijke afwijkingen tussen d e resultaten verkregen met de verschillende kom merciele kits: hieromtrent worden literatuurgegegevens en persoonlijke ervaring geci terd . Ten slotte worden gekende oorzaken van fouten op deze methode bediskuteerd.
RESUME
KEY WORDS
Lffl auteurs discutent plu sieurs aspects de Ia methode radioimmunologique de determination des digitaliques. Differents kits sont commercialises; on peut aussi obtenir facilement des anticorps chez le lapin, qui developpe apres qu elques mois deja, des titres eleves. La m ethode elle-meme est decrite : chaque determination necessite Une COUI!'be d'etaJonnage. Les coefficients de variation "within run" et " between run" pour Ia determination de taux plasmatiques de di,goxine. atteignent environ 10% . La speci ficifi te de Ia methode radio-immuno logioue est limitee. II y a reaction croisee entre lee; differents glycosides. ce qui perm et de d eterminer p1usieurs glyco.sides en employant 1es anticoros contre 1a digoxine. De meme, lorsqu'on fait le dosage pl asmatique de Ia digitoxin e ou de la d igoxine, il y a interference des metabolites de ces substances. Les resultats de d eterm in ations obtenues par differents kits commerciali ses montrent des variations importantes. Les donn ees de Ia literature ainsi que !'experien ce des auteur sont rap portees. Enfin , les auteurs d iscutent les sourc d'erreurs dont i1 faut teni!l" compte dans ]'interpretation des resultats de ces dosages.
Digitalis glycosides - Radioimmunoassay
Acta Clinica Belgica, 31, 4 (1976)
REFERENCES 1. BELPATRE, F .M., BOGAERT, M .G . and DE BROE. M .E. (1975) - Radioimmunoassay of digox in : a comparison of different comm ercial kits in renal failure . Clin . Chim. Acta, 62, 255. 2. BOGAERT, M .G ., BELPAIR . F.M ., MUS SCHE, M .M . and VAN DURM , J.P. (1976) -Use of radioimmunoassay of digi.tallis glycoides in man . Acta Clin . Belg. 3 1, 222. 3. BURNETT, G.H. and CONKLIN, R .L. (1968) - The enzymatic a say of pla rna di gitoxin levels. J . Lab. Clin . Med., 71, 1 40. 4. BUTLER, V.P. (1971) - Digoxin radioimmu noassay. Lancet, i, 186 . 5. BUTLER , V.P . and HEN, J .P. (1967) Digoxin-soecific a.ntibodies. Proc. Nat. Acad . ci., 57, 71. 6. HAMB RS , R . . (1974) - Digoxin ~ra dio immunoassay : improved precision with iodinated tracer. lin . him . Acta, 57, 19 1. 7. DOH RTY, J . . (1968) - Th e clinical pha rmacology of digitalis glycosid es : a revi ew. Amer. J. M ed. Sci., 255, 382.
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
221 8. DOHERTY, J .E. (1973) - Digitalis glycosi· des : Pharmacokinetics and their clinical im plications. Ann. Intern. Med., 79, 229. 9. HOLTZMAN, J.L., SHAFER, R.B. and ERICKSON, R.R . (1974) - Methodological causes of discrepancies in radioirrununoassay for digoxin in human serum. C lio . Chern., 20, 1194. 10. HUFFMAN, D .H. (1974) - The effect of spironolactone and canrenone on the digoxin radioimmunoassay. Res. Comm. Chern. Path . Pharmacol., 9, 787. II. KUBASIK , .P. , NORKUS, N.S . and SINE, H.E. (1974a) - Comparison of commercial kits for radioimmunoassay : the radioim munoa ay of serum digoxin using iodinated tracer. Clin. Biocherr.., 7, 307. 12. KUBASIK, N .P., SCHAUS lL, S. and SINE, H.E. (1974b) - Comparison of commercial kits for radioimmunoassay of erum dilin . Biochem., goxin using tritium tracer. 7, 206. 13 . KU 0 -SAKAI, H. and SAKAI, H. (1975) Effects on radioimmunoassay of digoxin of varying incubation periods for antigen-an tibody J'eaction and varying periods of adsorption by dextran -coated charcoal. Clin. hem., 2/, 277. 14. LARBIG, D . and KOCHSIEK, K. (1972) Zur radioimmunchemischen B timmung von Digoxi n und Digoxin derivaten . Deutsch Med . Wschr., 97, 1310. 15. LESNE, M. and DOLPHEN, R . (1975) Preparation et caracteristiques d'un anticorps antigitaloxigenine pour !'etude de Ia pharma · cocinetique de Ia gitaloxine et de Ia p enta-
16.
17.
18. 19.
20.
21.
22.
23.
formy1gitoxine. Arch. Int. Physiol. Biochem., 83. 136. LOWENSTEIN, J .M . and CORRlLL, E.M. (1966) - An improved method for measuring plasma and tissue concentration of digitalis glycosides. J . Lab. Clin. Med ., 67. 1048 . LUKAS , D .S. and PETERSON, R.E. (1966) - Double isotope dilution derivative assay of digitoxin in plasma, urine and blood of patients maintained on the drug. J . Clin. In vest., 45, 782. LULLMANN, H. and PETERS, Th. (1971) - The cardioactivity of digitoxin metabolites. urop. J. Pharmacol., 14, 204. PETERS, U ., HAUSAMEN, T .U. and GROSSE-BROCKHOFF, F. (1974) - Therapie mit Digoxin unter Kontrolle des Serumdigitoxinspiegels . Deutsch. Med . W schr., 99. 1701. SMITH, T.W., BUTLER, V .P. and HABER , E. (1970) - Characterization of antibodies of rugh affinity and specificity for the digitalis glycoside digoxin. Biochem., 9, 331. STOLL, R.G., CHRISTENSEN, M.S ., SAKMAR, E. and WAGNER, J .G. (1972) - The spocificity of the digoxin radioimmunoassay procedure. Res. Comm. Chern. Path. Pharmacol., 4, 503. YOSHALL, D.L., HUNTER, L. and GRA · DY, H.J. (1975) - Effect of albumin on serum digoxin radioimmunoassay. Clio . Chern ., 2/. 402. ZEEGERS, J.J .W ., MAA , A .H.J., WILL BRANDS, A.F., KRUYSWIJK, H.H. and JAMBROES, G. (1973) - The radioimmunoa ay of plasma-digoxin. Clin . Crum. Acta, 44, 109.
Acta
linica Belgica. 31, 4 (1976)
ISSN: 1784-3286 (Print) 2295-3337 (Online) Journal homepage: http://www.tandfonline.com/loi/yacb20
Radioimmunoassay Of Digitalis Glycosides F.M. Belpaire & M.G. Bogaert To cite this article: F.M. Belpaire & M.G. Bogaert (1976) Radioimmunoassay Of Digitalis Glycosides, Acta Clinica Belgica, 31:4, 212-221, DOI: 10.1080/17843286.1976.11717089 To link to this article: http://dx.doi.org/10.1080/17843286.1976.11717089
Published online: 17 May 2016.
Submit your article to this journal
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yacb20 Download by: [Australian Catholic University]
Date: 12 August 2017, At: 18:06
212
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
F.M. BELPAIRE and M.G. BOGAERT
INTRODUCTION
Several methods for quantitation of levels of cardiac glycoside3 in plasma (or serum) have been described: the double isotope dilution derivative assay (17), the inhibition of red blood cell Rb.. uptake (l6), the NA-K ATPase inhibition method (3), and the radioimmunoassay (5). Most of these methods are tim-- consuming, the radioimmunoassay (RIA) is not : whole serum or plasma can be used and no extraction step is needed. Because of its simplicity, rapidity and sensitivity, RIA of cardiac glycosides is commonly performed in the clinical laboratory, mainly since assay kits are commercially avaiJable. As discussed in the subsequent pap r (2). the interpretation of plasma levels of digitalis glycosides is not easy, and needs to be done in conjunction with the clinical observation of the patient. But the technique in itself can be criticised for lack of precision and accuracy, and for the existence of interfering factors. The purpose of this paper is to decrib-- our own experience with the technique, using commercially available RIA kits as well as antibodies produced in our own laboratory, and to stress different pitfalls of the procedure. This
H eymans Instituut voor Farmakologie - A.Z., De PinteJaan 135, 9000 Gent.
Acta Clinica Belgica, 31, 4 (1976)
discussion will primarely focus on digoxin , but digitoxin and other glycosides will also be mentioned. .PRODUCIJO
OF ANTIBODIES
Antibodies against digitalis glycosides that are available commercially, are obtained in the rabbit. For the preparation of the antigen we foJJowed the procedure described by Smith et al. (20) : digoxin is coupled to ~uman ser u~ . albumin and this conjugate is unmunogemc m rabbits (5). The rabbits were immunised by intradermic injection of 0.8 ml of antigen preparation once w ekly during four weeks, followed by intramuscular .injection of 0.4 ml every week or every 2 weeks. Afte~ f~ur weeks of immunisation, only 307o bmdmg of lab ..Jed digoxin was obtained for a serum dilution of 1/10. The final serum dilutions for obtaining 507o binding of 'H-digoxin to the antibody in function of time of immunisation are shown in Figure 1. The results of five different rabbits are given : after 22 weeks, a titer of about 240 000 is obtained in three rabbits, and a titer of about 160.000 in two rabbits. The percent binding of 'H-digoxin and 'H-digitoxin on the antiserum in the five rabbits in function of the final dilution of the antiserum is shown in igure 2. The antiserum was obtained after 19 weeks of immunisation. he final serum dilution for obtaining 507o binding of digoxin is about 9 times higher than for 507o binding of digitoxin.
213
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
300,000
Final dilution
X 0
A
200,000
X
0
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
•
X
e
100,000
•
0
10
12
14
\6
18
20
22
Weeks
Fig. I - Final dilution of 0.1 ml antiserum for obtaining 50% binding of 'H-digoxin (0.5 ng), at different times after immunisation in five rabbits. Each rabbit is indicated by a different symbol. GEN RAL PRO EDURE FOR PERF ORMING THE RIA
RIA can be p rformed dir ctly on serum, whereas analysi of urine or tissue samples requires an extraction step, e.g. with dichlormethane. The principl of the RIA procedure can b-- ummaris d as follows. An aliquot of serum (or an extract of urine or tissue) containing the glycoside to b measured, is mixed with an appropriate quantity of lab led glycoside; tritium-labvled or '"'!-labeled glycosid can b.. . used. The use of '"I-Iab Jed glycosid (Y-emitter) has the advantage over the use of tritium-labvled product that th counting procedure is fast and cheap ; furth rmore the problems of ample quenching ar eliminated. On th other hand, tritium-lab I d glycoside (fJemitter) ba the advantage that its half-life
(12.3 years) is much longer than for the Y-emitter (60 days). Antiserum is then added in such amount that in the absence of non lab.Jed glycoside (i.e. for blank serum) ,50 7o of the labeled glycoside is bound to the antibody. After incubation, the unbound glycoside is adsorbed on charcoal. After centrifugation to remove the charcoal, the sup rnatant containing the antibody bound labvled glycoside, is measured in a scintillation spvctrometer ; the amount of labvled glycoside bound is invers ly related to the amount of unlabvled glycoside that was present in the serum. Standard curves are made by adding known amounts of glycoside to blank serum and runnin g these samples through the whole procedure. ach commercial kit carries complete instructions for its use. When working with « home made » antiboActa Clinica Belgica, 31, 4 (1976)
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
214
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
dies, for the assay of digoxin (patients samples in the range of 0-4 ng/ml), we used the solutions and procedure described for the « Lanoxitest f3 » kit of Wellcome (see the manufacturers instructions for details). For the assay of digitoxin (patient samples in the range of 5- 40 ng/ ml), the' same procedure is followed as for digoxin, except that labeled (0.01 p.C, spec. act. 12.2 Ci/ mmol) and non labeled digitoxin were used instead of labeled and non labeled digoxin for setting up the standard curves. An amount of antiserum which, in the absence of non labeled glycosides, binds approximately 50 % of 'H-digoxin, respectively 'H-digitoxin, was used. As can been seen in Figure 2, the amount of antiserum needed is much larger for digitoxin as for digoxin, since the affinity for digoxin is much higher than that for digitoxin ; the concentration of antibodies against digitoxin is perhaps smaller.
Each time a calibration curve was set up, a blank was obtained by incubating a sample of standard serum with 'H-digoxin and buffer, without antiserum.
CALCULATION OF THE RESULTS OF RIA
After substraction of the CPM of the blank from the CPM of the antibody bound H' digmdn of each sample, the percent binding of the labeled glycoside can be calculated. A calibration line is obtained by calculating the percent binding for standard concentrations of cold glycoside added; however, by plotting the reciprocal value of the CPM of each sample against standard concentrations of the cold glycoside, a straight line is obtained (Figure 3). The glycoside content of the unknown samples can then be calculated by means of the regression equation of this straight line.
% binding of 3H-glycoside
70 DIGITOXIN
60 50
:~
~~ •
40 30
20
·~
10
I
0 8.10 3
8.10 4 Final dilution of antiserum
Fig. 2- Percent binding of 'H-digoxin and 'H-digitoxin as a function of final antiserum dilution in five rabbits, 19 weeks after immunisation. The same rabbits (and the same symbols) as in Figure I were used. Acta Clinica Belgica, 31, 4 (1976)
215
RAD!OIMMU OASSAY OF DIGITALIS GLYCOSIDES
·sol%
b inding of 3H-digoxin
• 1.0
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
30
4 Reciprocal counts per min ( x10- )
8 7
\.
•
\.
20
6
~-
5
~.
I. 3 2
10
B
A 0
0
I. 3 2 Digoxin concen tration ( ng /ml)
2
3
4
Digoxin concentration ( ng/ml )
Fig. 3 - Standard curve for RlA of digoxin. A. Binding of 'H-digoxin to antiserum, as a function of digoxin concentration. B. R eciprocal counts per minute of 'H-digoxin bound to antiserum, as a function of digoxin concentration. REPRODUClBILITY OF THE METHOD
The reproducibili ty of the method was expressed by the coefficient of variation (SD I mean x 100). A patient sample was analysed for digoxin , 10 times «within run» and 10 times « b tween run », using the «home made » antibodies. The coefficient of vari ation was 8.5tfo «within», and 10.4 'fo « between » runs, for an av rag measurement of 1.29 ng / ml digoxin. These data are in agreem nt with those of other authors (6, 11, 12). SPECIFICITY
Specificity wa d fin d by comparing the inhibition of a ntibody binding of labeled digoxin or labeled digitoxin by closely related compound uch as lanato ide C, ,8-me-
thyldigoxin, acetyldigoxin, desacetyllanatoside C, acetyldigoxin and gitaloxin. The structure of these compounds is shown in Figure 4. As th e antibodi s in the anti-digitalis sera are directed larg ly towards the aglycon portion of the molecule, most antisera to one cardiac glycoside cross-react to some extent with other glycosides. The results for 'H-digoxin and 'H-digitoxin binding in the presence of these different compounds are presented in Figure 5. From the results it is clear that the digoxin antibody has approximately the same affinity for the different substances with the same steroid nucleus as digoxin, i.e. with the h ydroxyl group in the 12-po ition on th nucleus. For oth r substances. such as digitoxin and formylgitoxin, the affinity is much lower. The digoxin assay system can thus bv Acta Clinica Belgica, 31, 4 (1976)
RADIOIMMUNOASSAY OF DIGITALIS GLYCOS!DES
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
216
used for th e determina tion of ,8-methyldi- toxin in the presence of different amounts of their respective metabolites is shown in goxin , lana toside C. desacetyllana toside and 6. All the metabolites studied were Figure acetyldigoxin. The digitoxin assay on the displace 'H-digoxin or 'H-digitoxin to able determination for used other hand can be from the antibody to approximately the saof acetyldigitoxin. These results are in agreeme extent and thus interfere in the determent with those of Larbig and Kochsiek mination of di goxin or digitoxin . The two (14) . This cross-reactivity can give problems genines however are slightly less active in if it is not known which glycoside a patient displacing 'H-digoxin or 'H-digitoxin than is receiving. the pa rent molecule or than the other meFrom Figure 5, it can also be seen that, tabolites. Thi s is in agreement with the reif one has to measure digitalis levels in a sults of others (14, 19, 21) . It should be nopatient treated with digitoxin , it is not adted tha t the metabolites of di goxin and divisable to use 'H-digoxin and cold digitoxin gitoxin are cardio-active to varying degrees for makin g the standard curve. Indeed , digi(1 8), so that measuring as well the parent toxin is metabolised to digoxin for about molecule as the metabolites could be an in10% . and the interference of the digoxin dex of the total cardioactive material prepresent in th e serum mak es th at falsely high values for digitoxin will b.., obtained. In contrast, when digitoxin is determin ed using 'H-digitoxin , the interference by di goxin present in the sample is much smaller as shown in F igure 5 B. The affinity of antidigoxin antibodies for gitaloxin , is much lower than for digoxin and digitoxin . However antigitaloxin antibodi es were prepared with hi gher affinity for gitaloxin than for digoxin (15). The binding of 'H-digoxin and of 'H-digi• I~
Fig. 4 -· Structural f ormulas of differ ent cardiac glycosides.
R, 3 digitoxose
+ acetyl + glucose 3 d ig itoxose + acetyl 3 digitoxose + glucose 3 dig itoxose + met hyl 3 dig itoxose +
3 dig itoxose
R,= H R,=H
R,= OH R,= H
R,= H R,= OH
dig itoxin lanato ide A
d igoxin lanatoside C (CedilanideR) acetyld igoxi n (Cedigoci neR) desacetyll ana to ide-C (Desacen) ,8- methyld igoxin (LanitopR)
gitoxin
acetyld igitox in (AcylanideR)
formyl
Acta Clinica Belgica, 31, 4 (1 976)
form ylgitoxin (Cristalox ineR)
217
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
50
l
% binding of 3H-digoxin
. ..
.•
'
40
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
50 1% binding of 3H-digitoxin
40
30
30
20
20
10
10
A 2
3
4
5
6
pmoles glycoside
• A
\ \. B
·~'·- .
10 20 ·3o 40 5o 60 pmoles glycoside
Fig. 5 - A. Binding of 'H-digoxin in the presence of increasing concentrations of various cardiac glycosides : digoxin • acetyldigoxin 6. /3-methy fdigoxin + digitoxin • lanatoside C X desacetyllanatoside C 0 formylgitoxin A B. Binding of 'H-digitoxin in the presence of increasing concentrations of various cardiac glycosides: digitoxin • acetyldigitoxin t:. gitaloxin A digoxin •
sent. In the case of digoxin, metabolisation is only of minor importance (7) ; for digitoxin, however, metabolic degradation is extensive but the contribution of inactive metabolites to the serum concentrations of digitoxin measured is not clear. It is thus clearly impossible to rely on a
plasma level of a glycoside when two or more glycosides are administered at the same time, or when there has been a recent change from one glycoside to another. It bas been demonstrated that some human steroids interfere in the RIA of digitalis glycosides (10, 23). The presence of Acta Clinica Belgica, 31, 4 (1976)
218
RADIOIMMUNOASSAY OF DIGI T A LIS GLYCOSIDES
prednisone e.g. interferes only when high doses of this product are used ; spironolactone however interferes even when the usual therapeutic doses are given; e.g. in patients receiving no digitalis but receiving 100, 300 or 400 mg spironolacton'e/day, an apparent digoxin plasma level of respectively 0.4, 0.7 and 0.9 ng/ml was measured
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
(10).
50
l
% binding of 3H-digoxin
COMPARISON OF DIFFERENT COMMER· CIALLY AVAlLABLE KITS
Only few laboratories produce their own antisera, and most of them use the commercially available RIA-kits. Recently different comparative studies of these kits have been published. In these studies large discrepancies were found in the analysis of serums of patients. In a study of Kubasik et al. (11, 12) using 50
l
% b;nd;ng of 3H-d;g;tox;n
• 40
30
30
20
20
10
10
A 2
3 4 5 6 7 pmoles glycoside
10
20 30 40 50 60 70 pmoles glycoside
Fig. 6 - A . Binding of 'H-digoxin in the presence of different concentrations of metabolites: digoxin • digoxigenin-bis-digitoxoside 0 digoxigenin-mono-digitoxoside ~ digoxigenin X B. Binding of 'H-digitoxin in the presence of different concentrations of metabolites: digitoxin • digitoxigenin-bis-digitoxoside 0 digitoxigenin-mono-digitoxoside ~ digitoxigenin X Acta Clinica Belgica, 31, 4 (1976)
219
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
tritiated or iodinated digoxin, the plasma values of digoxin analysed with five different kits ran ged from 0.8 to 1.7 ng/ ml in a sample of one patient, from 2.4 to 4.6 ng/ ml in a sample of another patient. D etermination of digoxin added to control sera yielded reproducible results. In the study of Voshall et al. (22) , using "'I-labeled digoxin , some kits gave systematica11y higher values than other kits. A patient sample analysed with four different kits gave the fo11owin g results : 5.8, 3.5, 2.0, 1.2 ng/ml. The same authors also found that digoxinfree sera gave positive results with some kits. e.g. 0.0, 0.6, 1.8, 2.2 ng/ m1. We have compared samples from patients with renal insufficiency who were receiving di goxin , using 6 different kits (1). In some of these samples divergent results were obtained (e.g. for one sample: 0.64, 0.78, 1.11. 1.38, 3.30, 3.13 ng/ ml). Digoxin-free sera gave values around zero with the 6 kits. The evaluation of digoxin in blank sera to which digoxin was added, was reproducible. These data show that some problems may arise in the RIA of di gitalis glycosides of some samples. The reason for these discrenancies is not always known. Some factors that could affect RIA of digitalis glycosides will b di scussed in the followin g paragraph.
FACTORS A FF CTlNG RIA OF GLYCOSIDES
Different factors affecting the RIA of digitalis glycosides have been described : 1. It is necessary to test the labeled and the unlabeled glycosides used by thin layer chromatography, since the purity of different commercial supplies varies consid rably. 2. The pati nt samples must be free of radioactivity, e.g. the radioactivity introduced by diagnostic tests. 3. Adequate quench correction is very important when tritiated glycosid s are used ; indeed some serum samples contain
hemoglobin or bile pigments which may qu ench . Quench correction can be done by adding an internal standard or b y use of an ex ternal standard, or by using the channel ratio method. 4. Incubation time and incubation temnerature of the antigen-antibody reaction, a nd the duration of contact with charcoal must be well sta nda rdized and optimal conditions must b ~ defined. Kuno-Sakai and Saka i (13) described the effect of varying the incubation time of the antigen-antibody reaction and of varyin g the duration of adsorption on dex tran coated charcoal, for different commercial RIA kits. In all cases the di goxin concentration measured increa~ed lin e!'!.rly with adsorption time, but the denree of increase varied from kit to kit. 5. In samples of uremic patients chemiluminesence of the serum can sometimes be observed, so that higher counts of the sample . and consequentl y a lower value of digoxin are measured . For such samples it is necessa rv to correct for this chemilumin~ sence (4). 6. Some authors (9 , 22) stated that the albumin content of a serum sample can influ nee the RIA of digoxin : with sera from patients with hypoalbuminaemia, bincling of the labeled glycoside was enhanced, and this resulted in a lower digoxin value measured. The exact mechanism by which albumin interferes with the binding of labt>led digoxin to the antibody remains unclear; in our hands, changes in albumin concentration did not explain di screpancies in the results observed. ONCLUSION
Although RIA of cardiac glycosides has become a routine technique, the procedure is not entirely free from pitfalls. M easurement of cardiac glycosides should never be performed without du e attention to the possibility that, even if the assay is done with optimal technical care, smaller or larger inaccuracies are present. Acta Clinica Belgica, 31, 4 (1976)
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
220
RADIOIMMUNOASSAY OF DIGITALIS GLYCOSIDES
SUMMARY
SAMENVATIING
In this paper, several aspects of the radioimmu noassay (RIA) method for digitalis glycosides are discussed. Although this method is used routinely, the results are not always accurate. Kits for RIA of digitalis glycosides are commercial ly availabl e, but antibodies, e.g. against digoxin, can easily be obtained in rabbits : high titers are reached within a few months . The general procedure is described; results can be calculated from a calibration line. The coefficient of variation «within run :1> and « between run » for the assay of digox in in patient serum is a pproximately 10%. The RIA method has a limited specificity, and cross-reaction between different glycosides is present ; this makes i.t possible to measure several glycosides, including digitoxin , using the antibodies against digoxin. M etabo·lites of digoxin and digi.toxin ·interfere with the determination of th e paren t molecules. Important discrepancies exist between the results obtained with the different commercial kits: literature data and perso nal experience are cited. Tn a final paragraph, known sources of error are discussed. When performing RIA of digitalis glycosides, on e should never forget the possibility that small er or larger errors of measurement are possible.
In dit artikel worden verschillende aspekten van de radio-immunologische bepalingsmethode voor digitalisglycosi den besprok en. Kits voo r deze bepaling zijn in de handel beschikbaar, maar men kan ook zelf gemakkelijk antiJichamen, bijvoorbeel d tegenover digoxine, opwekken bij het konijn : na enkele maanden reeds worden hoge titers bereikt. De algemene procedure wordt beschJreven ; bij elk e bepaling is een ijklijn nodig om de resuJ taten te berekenen . De variatiekoefficienten ''within run" en " between run" voor de bepaling van digoxine in het serum van patienten liggen rond de 10%. De radio-immunologische bepalingsmethode heeft slechts een beperkte specificiteit en er bestaat kruisreaktie tussen de verschiUende glycosiden: daardoor is het mogelijk, gebru.ik makend van de antilichamen tegen digoxine, verschill ende glycosiden, waaronder digitoxine, te meten . De metabolieten van digoxine en digitoxine interfereren met de bepaling van de moedermolekules. Er bestaan belangrijke afwijkingen tussen d e resultaten verkregen met de verschillende kom merciele kits: hieromtrent worden literatuurgegegevens en persoonlijke ervaring geci terd . Ten slotte worden gekende oorzaken van fouten op deze methode bediskuteerd.
RESUME
KEY WORDS
Lffl auteurs discutent plu sieurs aspects de Ia methode radioimmunologique de determination des digitaliques. Differents kits sont commercialises; on peut aussi obtenir facilement des anticorps chez le lapin, qui developpe apres qu elques mois deja, des titres eleves. La m ethode elle-meme est decrite : chaque determination necessite Une COUI!'be d'etaJonnage. Les coefficients de variation "within run" et " between run" pour Ia determination de taux plasmatiques de di,goxine. atteignent environ 10% . La speci ficifi te de Ia methode radio-immuno logioue est limitee. II y a reaction croisee entre lee; differents glycosides. ce qui perm et de d eterminer p1usieurs glyco.sides en employant 1es anticoros contre 1a digoxine. De meme, lorsqu'on fait le dosage pl asmatique de Ia digitoxin e ou de la d igoxine, il y a interference des metabolites de ces substances. Les resultats de d eterm in ations obtenues par differents kits commerciali ses montrent des variations importantes. Les donn ees de Ia literature ainsi que !'experien ce des auteur sont rap portees. Enfin , les auteurs d iscutent les sourc d'erreurs dont i1 faut teni!l" compte dans ]'interpretation des resultats de ces dosages.
Digitalis glycosides - Radioimmunoassay
Acta Clinica Belgica, 31, 4 (1976)
REFERENCES 1. BELPATRE, F .M., BOGAERT, M .G . and DE BROE. M .E. (1975) - Radioimmunoassay of digox in : a comparison of different comm ercial kits in renal failure . Clin . Chim. Acta, 62, 255. 2. BOGAERT, M .G ., BELPAIR . F.M ., MUS SCHE, M .M . and VAN DURM , J.P. (1976) -Use of radioimmunoassay of digi.tallis glycoides in man . Acta Clin . Belg. 3 1, 222. 3. BURNETT, G.H. and CONKLIN, R .L. (1968) - The enzymatic a say of pla rna di gitoxin levels. J . Lab. Clin . Med., 71, 1 40. 4. BUTLER, V.P. (1971) - Digoxin radioimmu noassay. Lancet, i, 186 . 5. BUTLER , V.P . and HEN, J .P. (1967) Digoxin-soecific a.ntibodies. Proc. Nat. Acad . ci., 57, 71. 6. HAMB RS , R . . (1974) - Digoxin ~ra dio immunoassay : improved precision with iodinated tracer. lin . him . Acta, 57, 19 1. 7. DOH RTY, J . . (1968) - Th e clinical pha rmacology of digitalis glycosid es : a revi ew. Amer. J. M ed. Sci., 255, 382.
Downloaded by [Australian Catholic University] at 18:06 12 August 2017
221 8. DOHERTY, J .E. (1973) - Digitalis glycosi· des : Pharmacokinetics and their clinical im plications. Ann. Intern. Med., 79, 229. 9. HOLTZMAN, J.L., SHAFER, R.B. and ERICKSON, R.R . (1974) - Methodological causes of discrepancies in radioirrununoassay for digoxin in human serum. C lio . Chern., 20, 1194. 10. HUFFMAN, D .H. (1974) - The effect of spironolactone and canrenone on the digoxin radioimmunoassay. Res. Comm. Chern. Path . Pharmacol., 9, 787. II. KUBASIK , .P. , NORKUS, N.S . and SINE, H.E. (1974a) - Comparison of commercial kits for radioimmunoassay : the radioim munoa ay of serum digoxin using iodinated tracer. Clin. Biocherr.., 7, 307. 12. KUBASIK, N .P., SCHAUS lL, S. and SINE, H.E. (1974b) - Comparison of commercial kits for radioimmunoassay of erum dilin . Biochem., goxin using tritium tracer. 7, 206. 13 . KU 0 -SAKAI, H. and SAKAI, H. (1975) Effects on radioimmunoassay of digoxin of varying incubation periods for antigen-an tibody J'eaction and varying periods of adsorption by dextran -coated charcoal. Clin. hem., 2/, 277. 14. LARBIG, D . and KOCHSIEK, K. (1972) Zur radioimmunchemischen B timmung von Digoxi n und Digoxin derivaten . Deutsch Med . Wschr., 97, 1310. 15. LESNE, M. and DOLPHEN, R . (1975) Preparation et caracteristiques d'un anticorps antigitaloxigenine pour !'etude de Ia pharma · cocinetique de Ia gitaloxine et de Ia p enta-
16.
17.
18. 19.
20.
21.
22.
23.
formy1gitoxine. Arch. Int. Physiol. Biochem., 83. 136. LOWENSTEIN, J .M . and CORRlLL, E.M. (1966) - An improved method for measuring plasma and tissue concentration of digitalis glycosides. J . Lab. Clin. Med ., 67. 1048 . LUKAS , D .S. and PETERSON, R.E. (1966) - Double isotope dilution derivative assay of digitoxin in plasma, urine and blood of patients maintained on the drug. J . Clin. In vest., 45, 782. LULLMANN, H. and PETERS, Th. (1971) - The cardioactivity of digitoxin metabolites. urop. J. Pharmacol., 14, 204. PETERS, U ., HAUSAMEN, T .U. and GROSSE-BROCKHOFF, F. (1974) - Therapie mit Digoxin unter Kontrolle des Serumdigitoxinspiegels . Deutsch. Med . W schr., 99. 1701. SMITH, T.W., BUTLER, V .P. and HABER , E. (1970) - Characterization of antibodies of rugh affinity and specificity for the digitalis glycoside digoxin. Biochem., 9, 331. STOLL, R.G., CHRISTENSEN, M.S ., SAKMAR, E. and WAGNER, J .G. (1972) - The spocificity of the digoxin radioimmunoassay procedure. Res. Comm. Chern. Path. Pharmacol., 4, 503. YOSHALL, D.L., HUNTER, L. and GRA · DY, H.J. (1975) - Effect of albumin on serum digoxin radioimmunoassay. Clio . Chern ., 2/. 402. ZEEGERS, J.J .W ., MAA , A .H.J., WILL BRANDS, A.F., KRUYSWIJK, H.H. and JAMBROES, G. (1973) - The radioimmunoa ay of plasma-digoxin. Clin . Crum. Acta, 44, 109.
Acta
linica Belgica. 31, 4 (1976)
