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Ouabain-induced ventricular arrhythmia in rabbit: Influence of antibodies
Lüe Sciences Vol. 10, Part I, pp. 341-345, 1971 . Printed in Great Britain
OüABAIN-II~UCED VSNfRICULAR ARRHYTHHIA IN RABBIT :
Pergamon Press
LNFLUSNCE OF ANTIBODIEB
Frank Ciofalo and Hilary Ashe* IIniveraity of California, Irvine, California Collage of Medicine, Irvine, California 92664 ; *Reference Laboratory, North Hollywood, California 91609
(Received in final form 8 February 1971)
OIIABAIN vas chemically coupled to bovine serum albumin (BSA) and rabbits mere
immunised pith the cable:. Sach animal vas bled 2 or 3 times over a 4 reek period before being boosted 7 days prior to challenge with a single toxic dose of owbain (150 pg/kg) . Sera free all immunised animals contained antibodies to the antigen (Ag) . Of the 5 animals challenged pith owbain, 2 animals exhibited toxic aymptans and died within the sane tine period u It controls, vhereu 3 animals exhibited o0 ouabain-induced toxic symptoms . vas found that the nonprntected rabbits formed AH siainly toward the BSA sioiety vhereaa protected rabbits had forted AB directed mainly toward the owbain moiety of the complex. Recently, Butler and Chan (1) reported the induction of antibody foroation to digoxin in rabbits .
Since we have been interested in digitalis toxicity in
rabbits, it vas felt that a study of the relationship between antibody fozoa rive to a cardiac glycoside and the toxicity of that glycoside would be of interest .
Furthermore, the use of such antibodies night be of value in the
specific treatment of digitalis overdosage (keeping in mind the enormous possibility of precipitating serum sickness in the patient due to the injection of a foreign protein) .
The present i~estigation uses ouabain as the cardiac
glycoside because of its coon wage is animal ta:icity studies . Materials and Methods Ouabain was chemically coupled to bovine serum albumin (BSA) as described by Butler and Chen (1) .
Their preparations contained 2 .7 and 5 digozin
residues/mole bovine serum albumin (BSA) as determined spectrophotooetrically . Our two preparations, designated A and B, contained 9 and 12 residues of owbain/hole BSA as determined spectrophotometrically at 550 nm .
Bovine serum
albumin ws also carried through the coupling procedure without the ouabain, since it vas felt that the procedure might modify the BSA to the extent that
341
342
Rabbit Anti-Ouabain Antiserum
Vol . 10, No. B
notval BSA would not be useful in the determination of antibody specificity . Albino rabbits of the New Zealand strain were immunised by injections into the toepads of 0 .4 ml of 1 mg/ml ouabain-BSA conjugate in complete Freund'a adjuvant .
The ouabain-BSA conjugate was injected into the rabbit three times
over a four week period .
Single booster shots were given intramuscularly one
week prior to bleeding and the bleedings were three weeks apart . from each bleeding was kept separate and frozen st -LSo C .
The serum
Control (sham)
immunised rabbits were prepared by immunising 4 rabbits with the modified BSA, using the same immunisation procedure as above .
Five of twelve experimental
animals survived four booster shots and bleedings, whereupon 24 hours following the final booster, the animals were prepared for challenge with ouabain . Three of the four rabbits immunised with modified BSA also survived two booster shots and 24 hours after the second booster were prepared for challenge with ouabain .
The anímala were anesthetised with Dial-urethane (Cabs), bi-
laterally vagotomised and connected to a polygraph for recording of the electrocardiogram (lead II) .
A single toxic dose of ouabain (150 pg/kg iv) was
administered and note was taken of the time to development of ventricular irregularities and death (when occurring) . bleedout was performed .
At the time of death, a terminal
All samples were assayed for antibody by the micro-
comples~ent fization technique of Wasserman and Levine (2) . Results Figure 1 depicts the results obtained using the microcomplement fization technique and the serum from a bleeding of an animal which was later found not to be protected against the toxic concentration of ouabain .
This figure ie
a plot of complement fixed versus concentration of the antigen with and without the presence of two inhibitors (ouabain and modified BSA - called BSA II) .
When
ouabain is used as the inhibitor, there is very little change in complement fixed .
Hwever, when BSA II is used, ít seems to be a good inhibitor of com-
plement fixation, indicating that this animal Foamed antibodies, but mainly towards the BSA moiety .
Rab}ait Az~i-Ouabain Az~iserum
Vol . 10, No . 6
343
FIG . 1 Plots of complement fixed versus antigen concentration in the absence and in the presence of inhibitors, ouabain or modified BSA (BSA II) . A decrease in complement fixed when an inhibitor is preaent indicates the presence of antibodies tward that particular compound . (3Aß2 ~ Second bleeding of animal #3 immunised with conjugate A . Serum dilution ~ 1/900 .) Figure 2 shows the results, under the same conditions, with serum of the third bleeding of another animal .
100 v
ao
c
a0 E n 40 E a U t0 >< 0 02
LO
Antioaa
(iYmq
SA
FIG . 2 Plots of complement fixed versus antigen concentration . See Fig . 1 Cor details . (2Aß3 - Third bleeding of animal #2 immunized with conjugate A . Serum dilution = 1/900 .)
Rabbit Anti-Ouabain Antiserum
344
Vol. 10, No. ß
In contrast to the animal used in Figure 1, this animal formed antibodies mainly toward the ouabain portion of the conjugate, although antibodies are also present against the BSA moiety .
This animal vas later found to be
protected against ouabain toxicity . The results of the ouabain challenge in ten control (non imswniaed), three control immunized and ín the five experimental immunized animals are presented in Table 1 . TABLE 1 Ouabain-induced Toxicity in Immunized, Sham Immunized and Non immunised Rabbits Ouabain was administered as a single intravenous injection (150 F+g/kg) .
Animal s)
Time to Development of : Ventric lar Ventricular extrasystole tachycardia Death (min) (min) (min)
Controls (10)
0 .89 + 0 .16
2 .59 + 0 .57
5 .90 + 0 .60
Controls (3) (sham immun .)
1 .05 + 0 .21
2 .85 + 0 .70
5 .25 + 0 .51
Antibodies to ouabain
2 A
none
none
none
yes
1 B
none
none
none
yes
2 B
none
none
none
? (anticomplementary)
3 A
1 .5
2 .0
5 .0
Very little or none
6 A
2 .5
3 .0
5 .5
no
The non-immunized and sham immunised animals all exhibited ventricular eztrasystole, ventricular tachycardia and died in ventricular fibrillation .
At
this dose of ouabain, antiarrhythmic agents such as propranolol and diphenylhydantoin prolong the time to development of these symptoms, but do not reduce the incidence significantly (3) .
Of the five immunised animals tested, three
Vol . 10, No. 8
RabMt A~i-Ouatiain An#iserum
did not even exhibit a single ouabain-induced toxic sysiptaa .
945 The other two
animals ezhibited all of the tonic sysptom and the tine for developsunt of these symptoms was not significantly different than seen with control animals . The two unprotected animals had antibodies but they were mainly directed toward the BSA moiety of the code: .
1Wo of the three protected animals
definitely had antibodies to ouabain, whereas the third may have had antibodies to ouabain but the sers~ vas too anticompleaentary to be tested with the technique used . Discussion The results of the present study indicate that in
vivo,
antibodies to a
cardiac glycoside have the ability to decrease the toxicity of that glycoside . The antibody titer found in these imunised animals ws too low, however, to warrant a purification attmpt .
An attempt has been rade to wke the anti-
body pore specific by coupling ouabain to rabbit serum albumin for use as the antigen .
The results have been less than satisfactory and the probable reason
for this is that the conjugate foxed is not different enough from normal rabbit serum albumin to induce ~ch antibody formation .
The present study
indicates it may be possible to use antibodies to decrease the toxicity of cardiac glycosides, but high antibody titers and good antibody specificity would be required before a purification attempt would be warranted . Ackoarledgenent-The authors wish to acknowledge the excellent technical assist ance of Mrs . Carol Gunther, Mr . Alezander Marston and firs . Judy Gale . Re ferences 1.
0 . BVtT.ES and J . CHBN, Proc . Nat . Aced . Sci . 57, 71 (1967) .
2.
E . RASSSRMAN and L . LEVINE, J . Immun . 87, 290 (1960) .
3.
F . CIOFALO, Eur . J . Pharmacol . 9, 281 (1970) .
OüABAIN-II~UCED VSNfRICULAR ARRHYTHHIA IN RABBIT :
Pergamon Press
LNFLUSNCE OF ANTIBODIEB
Frank Ciofalo and Hilary Ashe* IIniveraity of California, Irvine, California Collage of Medicine, Irvine, California 92664 ; *Reference Laboratory, North Hollywood, California 91609
(Received in final form 8 February 1971)
OIIABAIN vas chemically coupled to bovine serum albumin (BSA) and rabbits mere
immunised pith the cable:. Sach animal vas bled 2 or 3 times over a 4 reek period before being boosted 7 days prior to challenge with a single toxic dose of owbain (150 pg/kg) . Sera free all immunised animals contained antibodies to the antigen (Ag) . Of the 5 animals challenged pith owbain, 2 animals exhibited toxic aymptans and died within the sane tine period u It controls, vhereu 3 animals exhibited o0 ouabain-induced toxic symptoms . vas found that the nonprntected rabbits formed AH siainly toward the BSA sioiety vhereaa protected rabbits had forted AB directed mainly toward the owbain moiety of the complex. Recently, Butler and Chan (1) reported the induction of antibody foroation to digoxin in rabbits .
Since we have been interested in digitalis toxicity in
rabbits, it vas felt that a study of the relationship between antibody fozoa rive to a cardiac glycoside and the toxicity of that glycoside would be of interest .
Furthermore, the use of such antibodies night be of value in the
specific treatment of digitalis overdosage (keeping in mind the enormous possibility of precipitating serum sickness in the patient due to the injection of a foreign protein) .
The present i~estigation uses ouabain as the cardiac
glycoside because of its coon wage is animal ta:icity studies . Materials and Methods Ouabain was chemically coupled to bovine serum albumin (BSA) as described by Butler and Chen (1) .
Their preparations contained 2 .7 and 5 digozin
residues/mole bovine serum albumin (BSA) as determined spectrophotooetrically . Our two preparations, designated A and B, contained 9 and 12 residues of owbain/hole BSA as determined spectrophotometrically at 550 nm .
Bovine serum
albumin ws also carried through the coupling procedure without the ouabain, since it vas felt that the procedure might modify the BSA to the extent that
341
342
Rabbit Anti-Ouabain Antiserum
Vol . 10, No. B
notval BSA would not be useful in the determination of antibody specificity . Albino rabbits of the New Zealand strain were immunised by injections into the toepads of 0 .4 ml of 1 mg/ml ouabain-BSA conjugate in complete Freund'a adjuvant .
The ouabain-BSA conjugate was injected into the rabbit three times
over a four week period .
Single booster shots were given intramuscularly one
week prior to bleeding and the bleedings were three weeks apart . from each bleeding was kept separate and frozen st -LSo C .
The serum
Control (sham)
immunised rabbits were prepared by immunising 4 rabbits with the modified BSA, using the same immunisation procedure as above .
Five of twelve experimental
animals survived four booster shots and bleedings, whereupon 24 hours following the final booster, the animals were prepared for challenge with ouabain . Three of the four rabbits immunised with modified BSA also survived two booster shots and 24 hours after the second booster were prepared for challenge with ouabain .
The anímala were anesthetised with Dial-urethane (Cabs), bi-
laterally vagotomised and connected to a polygraph for recording of the electrocardiogram (lead II) .
A single toxic dose of ouabain (150 pg/kg iv) was
administered and note was taken of the time to development of ventricular irregularities and death (when occurring) . bleedout was performed .
At the time of death, a terminal
All samples were assayed for antibody by the micro-
comples~ent fization technique of Wasserman and Levine (2) . Results Figure 1 depicts the results obtained using the microcomplement fization technique and the serum from a bleeding of an animal which was later found not to be protected against the toxic concentration of ouabain .
This figure ie
a plot of complement fixed versus concentration of the antigen with and without the presence of two inhibitors (ouabain and modified BSA - called BSA II) .
When
ouabain is used as the inhibitor, there is very little change in complement fixed .
Hwever, when BSA II is used, ít seems to be a good inhibitor of com-
plement fixation, indicating that this animal Foamed antibodies, but mainly towards the BSA moiety .
Rab}ait Az~i-Ouabain Az~iserum
Vol . 10, No . 6
343
FIG . 1 Plots of complement fixed versus antigen concentration in the absence and in the presence of inhibitors, ouabain or modified BSA (BSA II) . A decrease in complement fixed when an inhibitor is preaent indicates the presence of antibodies tward that particular compound . (3Aß2 ~ Second bleeding of animal #3 immunised with conjugate A . Serum dilution ~ 1/900 .) Figure 2 shows the results, under the same conditions, with serum of the third bleeding of another animal .
100 v
ao
c
a0 E n 40 E a U t0 >< 0 02
LO
Antioaa
(iYmq
SA
FIG . 2 Plots of complement fixed versus antigen concentration . See Fig . 1 Cor details . (2Aß3 - Third bleeding of animal #2 immunized with conjugate A . Serum dilution = 1/900 .)
Rabbit Anti-Ouabain Antiserum
344
Vol. 10, No. ß
In contrast to the animal used in Figure 1, this animal formed antibodies mainly toward the ouabain portion of the conjugate, although antibodies are also present against the BSA moiety .
This animal vas later found to be
protected against ouabain toxicity . The results of the ouabain challenge in ten control (non imswniaed), three control immunized and ín the five experimental immunized animals are presented in Table 1 . TABLE 1 Ouabain-induced Toxicity in Immunized, Sham Immunized and Non immunised Rabbits Ouabain was administered as a single intravenous injection (150 F+g/kg) .
Animal s)
Time to Development of : Ventric lar Ventricular extrasystole tachycardia Death (min) (min) (min)
Controls (10)
0 .89 + 0 .16
2 .59 + 0 .57
5 .90 + 0 .60
Controls (3) (sham immun .)
1 .05 + 0 .21
2 .85 + 0 .70
5 .25 + 0 .51
Antibodies to ouabain
2 A
none
none
none
yes
1 B
none
none
none
yes
2 B
none
none
none
? (anticomplementary)
3 A
1 .5
2 .0
5 .0
Very little or none
6 A
2 .5
3 .0
5 .5
no
The non-immunized and sham immunised animals all exhibited ventricular eztrasystole, ventricular tachycardia and died in ventricular fibrillation .
At
this dose of ouabain, antiarrhythmic agents such as propranolol and diphenylhydantoin prolong the time to development of these symptoms, but do not reduce the incidence significantly (3) .
Of the five immunised animals tested, three
Vol . 10, No. 8
RabMt A~i-Ouatiain An#iserum
did not even exhibit a single ouabain-induced toxic sysiptaa .
945 The other two
animals ezhibited all of the tonic sysptom and the tine for developsunt of these symptoms was not significantly different than seen with control animals . The two unprotected animals had antibodies but they were mainly directed toward the BSA moiety of the code: .
1Wo of the three protected animals
definitely had antibodies to ouabain, whereas the third may have had antibodies to ouabain but the sers~ vas too anticompleaentary to be tested with the technique used . Discussion The results of the present study indicate that in
vivo,
antibodies to a
cardiac glycoside have the ability to decrease the toxicity of that glycoside . The antibody titer found in these imunised animals ws too low, however, to warrant a purification attmpt .
An attempt has been rade to wke the anti-
body pore specific by coupling ouabain to rabbit serum albumin for use as the antigen .
The results have been less than satisfactory and the probable reason
for this is that the conjugate foxed is not different enough from normal rabbit serum albumin to induce ~ch antibody formation .
The present study
indicates it may be possible to use antibodies to decrease the toxicity of cardiac glycosides, but high antibody titers and good antibody specificity would be required before a purification attempt would be warranted . Ackoarledgenent-The authors wish to acknowledge the excellent technical assist ance of Mrs . Carol Gunther, Mr . Alezander Marston and firs . Judy Gale . Re ferences 1.
0 . BVtT.ES and J . CHBN, Proc . Nat . Aced . Sci . 57, 71 (1967) .
2.
E . RASSSRMAN and L . LEVINE, J . Immun . 87, 290 (1960) .
3.
F . CIOFALO, Eur . J . Pharmacol . 9, 281 (1970) .