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Serum levels of digoxin in infants and children
566
September, 1972 T h e Journal of P E D I A T R I C S
Serum leMs of digoxin in infants and children Serum digoxin levels during maintenance therapy in children of three different age-weight groups were determined by radioimmunoassay. During the postabsorptive interval (1 to 2 hours after an oral dose), infants in the 5 to 9 Ks. group were found to have significantly higher serum digoxin levels than children in the 9 to 18 Kg. group, who in turn had higher levels than older children. Serum levels were comparable for all groups during equilibrium (5 to 9 hours after an oral dose). Since digoxin is primarily eliminated unchanged in the urine, our data suggest that infants and young children may have relatively more rapid rates of excretion than do older children and adults.
Richard W. Krasula, B.S., Piero A. Pellegrino, M.D., Alols R. Hastreiter, M.D., and Lester F. Soyka, M.D., "~ Chicago, Ill.
T H E D I S C O V E R Y of a m e t h o d for producing specific antibodies to digoxin 1 has provided a means for the development of a sensitive, specific radioimmunoassay3 We have independently developed a similar assay and have measured serum levels during the postabsorptive (1 to 2 hours) and the plateau (5 to 9 hours) phases following an oral dose of digoxin during maintenance therapy. This report will present data validating the methodology employed and will summarize our preliminary findings. From the Department of Pharmacology and Division of Pediatric Cardiology, Abraham Lincoln School of Medicine, University of Illinois Medical Center. Supported in part by United States Public Health Service Grants HD-05362 and GM-81, a Faculty Dev'elopment Award in Clinical Pharmacology from the Pharmaceutical Manufacturers Association Foundation, and the Goodenberger Medical Research Fund of the University of Illinois Medical Foundation. "~Reprlnt address: University o[ Illinois Medical Center, Department o[ Pharmacology, 901 & Wolcott St., Chicago, Ill. 60612.
Vol. 81, No. 3, pp. 566-569
METHODS Assay procedure. A conjugate of digoxin and bovine serum albumin (BSA) was prepared according to the method of Butler and Chen. 1 Antisera were raised in rabbits by injection of the conjugate in complete Freund's adjuvant at weekly intervals for four weeks. By four months, the antibody titer was such that 0.1 ml. of 1:10,000 dilution of the serum bound 50 per cent of a tracer dose of tritiated digoxin (9.0 curies per millimole; New England Nuclear Corp., Boston, Mass.). Assays were run in duplicate with each tube containing 0.1 ml. of pooled normal human serum, 0.1 ml. of a standard, 0.1 ml. of a 5 ng. per milliliter dilution of tritiated digoxin, 0.1 ml. of antisera, and sufficient phosphate-BSA buffer to make a final volume of 0.8 ml. All tubes were incubated at 25 ~ C. for 15 minutes. Free and bound digoxin were separated
Volume 81 Number 3
Serum levels o[ digoxin
567
8.0
PresentStudy .~ ~~
6.0
(Schw;rz-Mann)
lIB 4.0
(BurroughsWellcomeCo.)
I
2.0 !
I
I
I
I
1.0
2.0 .3.0 4-.0 Digoxin (ng/ml) Fig. 1. Comparison of standard curves obtained from: 9 9 present study, x. x Butler,6 @ @ commercial radioimmunoassay kit (Schwarz-Mann), O O antisera (Burroughs Wellcome & Co.). Values on the ordinate (l/B) are the reciprocals of :the fraction of antibody-bound digoxin. by adding 0.2 ml. of a BSA-charcoal suspension and centrifuging at 2,000 g for ten minutes. A 0.7 ml. aliquot of the supernate was transferred to a counting vial, 10 ml. of scintillation "cocktail" added, and the vial counted in a Packard Tri-Carb spectrometer. A standard curve was prepared by plotting a regression line, obtained by the method of least squares, of the reciprocal of the fraction bound digoxin ( l / B ) versus the concentration of the digoxin standard (D). Complete details are available from the authors upon request. Patients. All patients had congenital heart disease and were studied while hospitalized. All received digoxin (Lanoxin, Burroughs Wellcome & Co., New York) orally in two equal doses at 9 A.M. and 9 P.M. Children under five years of age received the elixir, and older children were given tablets. The patients were divided into three groups consistent with the dosage regimen used in our hospital, which is based on body weight. The maintenance dosages (25 per cent of the total digitalization dose) were as follows:
Group 1, 5 to 9 Kg., 0.022 rag. per kilogram (0.01 rag. per pound) ; Group 2, 9 to 18 Kg., 0.016 rag. per kilogram (0.0075 rag. per pound) ; and Group 3, greater than 18 kilograms, 0.011 rag. per kilogram (0.005 nag. per pound). All patients in Group 1 were six months to two years of age, those in Group 2 ranged from two to five years, and those in Group 3 were five years and older. Data were included only from those patients who were normokalemic, euthyroid, and had normal blood urea nitrogen values. All were judged to be adequately digitalized, and none had clinical or electrocardiographic signs of toxicity. Blood was obtained by venipuncture at 1 to 2 and 5 to 9 hours after administration of digoxin. These times reflect peak and equilibrium serum levelsY, 4 RESULTS
Precision. When plotted as 1/B versus D, the standard curve having the steepest slope indicates the most sensitive assayJ The standard curve obtained with our radioimmunoassay procedure had the same slope as that
56 8
Krasula et al.
The Journal o[ Pediatrics September 1972
Table I. Mean serum digoxin levels Postabsorptive mean serum digoxin (ng./ml.)
Plateau mean serum digoxin (ng./ml.)
Group
Weight (Kg.)
Age (yr.)
Mean maintenance dose (mg./Kg./day)
1
5- 9
~-2
0.020 + 0.001
3.2 + 0.3 (4;6)
1.0 + 0.2 (9;13)
2
9- 18
2-5
0.016 + 0.001
2.2 + 0.3* (4;7)
1.4 + 0.2 (11;13)
3
> la
> 5
0.009 + 0.001
1.6 -+ OAt (4;5)
0.9 -+ 0.2 (6;7)
Mean • S.E.M. (figures in parentheses denote number of patients; number of determinations). *Group 2 is significantly different from Group 1 (P < 0.05). "~Group 3 is significantly different from Group 1 (P < 0.005).
published by Butler, 6 and as that obtained with materials supplied in a commercially available kit (Schwarz-Mann, New York) (Fig. 1). The slope of the line was steeper than that found with an antiserum donated by the Burroughs Wellcome & Co. (Greenville, N. C.). While the y-intercept and slope of the standard curve varied slightly from day to day, eight replications of a 2 ng. standard over a 2 week period showed little deviation (S.E.M. + 0.04 ng.). Specificity. Eight plasma samples containing no digoxin were assayed: All gave results of less than 0.2 ng. per milliliter. When concentrations of 10-2 to 10-5 M of physiologically active steroids (progesterone, estradiol17fl, testosterone, cortisol, and cholesterol) were substituted for digoxin, no interference was found?, 2, Serum levels. At 1 to 2 hours after administration, the mean serum digoxin levels were found to decrease with increasing age, being 3.2 (S.E.M. + 0.3) ng. per milliliter in Group 1, 2.2 _+ 0.3 ng. per milliliter in Group 2, and 1.6 + 0.4 ng. per milliliter in Group 3 (Table I ) . T h e level in Group 1 differed significantly from that in either Group 2 (P < 0.05) or Group 3 (P < 0.005). At equilibrium, mean levels were comparable for all groups, being 1.0 + 0.2, 1.4 + 0.2,' and 0.9 + 0.2 rig. per milliliter, respectively. DISCUSSION
Recommendations in regard to dosage for infants and children are two to three times greater than for adults, whether based on
body weight or surface area. s, 9 Within the pediatric age group, doses generally employed are inversely proportional to age, except in the case of premature infants. Thus the dosage schedule employed in this study was that in actual practice in our hospital. The data indicate that, despite the relatively larger doses, the serum levels at equilibrium were the same for each age group and equivalent to those reported in adults3, 3, 7, 10 The postabsorptive levels were directly related to dose; e.g., Group 1 received twice the dose of Group 3 and the initial serum levels were twice as high (Table I ) . Thus poor absorption does not appear to be present. Similarly, Hernandez and associates 11 found no impairment of dixogin absorption in infants. Age-related differences in digoxin metabolism may exist but seem an unlikely explanation for our data. Less than 10 per cent of an oral dose undergoes biotransformation in adults, 12 and no metabolites have been found in the urine of infants? 1 Our finding of lesser rates of fall in serum levels between sampling periods with increasing age is consistent with a decreasing rate of urinary excretion. We are presently investigating whether more rapid excretion or differences in distribution are responsible for the age-related decrease in dosage needed to maintain serum levels believed to be within the therapeutic range. The technical assistance of Miss Diana Lipton, and the efforts of Dr. W. Meier in obtaining serum samples are greatly appreciated.
Volume 81 Number 3
Serum levels o[ digoxin
REFERENCES
I. Butler, V. P., Jr., and Chen, J. P.: Digoxinspecific antibodies, Proc. Natl. Acad. Sci. 57: 71, 1967. 2. Smith, T. W., Butler, V. P., Jr., and Haber, E.: Determination of therapeutic and toxic serum digoxin concentrations by radioimmunoassay, N. Engl. J. Med. 281:12121 1969. 3. White, R. J., Chamberlain, D. A., Howard, M., and Smith, T. W.: Plasma concentrations of digoxin after oral administration in the fasting postprandial state, Br. Med. J. 1: 380, 1971. 4. Mirkin, B. L., and Larese, R.: Immunoassay of serum digoxin levels in children: therapeutic implications, Proc. Soc. Pediatr. Res., Forty-first Annual Meeting, Atlantic City, N. J., Apr. 28- May 1, 1971. 5. Berson, S. A., and Yalow, R. S.: General principles of radioimmunoassay, Clin. Chim. Acta 22: 51, 1968. 6. Butler, V. P., Jr.: Digoxin: Immunologic ap-
7. 8.
9. 10. 11.
12.
5 69
proaches to measurement and reversal of toxicity, N. Engl. J. Med. 283: 1150, 1970. Oliver, G. C., Parker, B. M., and Parker, C. W.: Radioimmunoassay for digoxin, Am. J. Med. 51: 186, 1971. Goodman, L. S., and Gilman, A.: The pharmacologic basis of therapeutics, ed. 4, New York, 1970, The Macmillan Company, p. 700. Shirkey, H. C.: Pediatric dosage handbook, Washington, D. C., 1970. American Pharmaceutical Association, p. 75. Belier, G. A., Smith, T. W., Abelmann, W. H., Haber, E., and Hood, W. B., Jr.: Digitalis intoxication, N. Engl. J. Med. 284: 989, 1971. Hernandez, A., Burton, R . M., Pagtakhan, R. D., and Goldring, D.: Pharmacodynamics of SH-digoxin in infants, Pediatrics 44: 418, 1969. Doherty, J. E., Hall, W. H., Murphy, M. L., and Beard, O. W.: New information regarding digitalis metabolism, Chest 59: 433, 1971.
September, 1972 T h e Journal of P E D I A T R I C S
Serum leMs of digoxin in infants and children Serum digoxin levels during maintenance therapy in children of three different age-weight groups were determined by radioimmunoassay. During the postabsorptive interval (1 to 2 hours after an oral dose), infants in the 5 to 9 Ks. group were found to have significantly higher serum digoxin levels than children in the 9 to 18 Kg. group, who in turn had higher levels than older children. Serum levels were comparable for all groups during equilibrium (5 to 9 hours after an oral dose). Since digoxin is primarily eliminated unchanged in the urine, our data suggest that infants and young children may have relatively more rapid rates of excretion than do older children and adults.
Richard W. Krasula, B.S., Piero A. Pellegrino, M.D., Alols R. Hastreiter, M.D., and Lester F. Soyka, M.D., "~ Chicago, Ill.
T H E D I S C O V E R Y of a m e t h o d for producing specific antibodies to digoxin 1 has provided a means for the development of a sensitive, specific radioimmunoassay3 We have independently developed a similar assay and have measured serum levels during the postabsorptive (1 to 2 hours) and the plateau (5 to 9 hours) phases following an oral dose of digoxin during maintenance therapy. This report will present data validating the methodology employed and will summarize our preliminary findings. From the Department of Pharmacology and Division of Pediatric Cardiology, Abraham Lincoln School of Medicine, University of Illinois Medical Center. Supported in part by United States Public Health Service Grants HD-05362 and GM-81, a Faculty Dev'elopment Award in Clinical Pharmacology from the Pharmaceutical Manufacturers Association Foundation, and the Goodenberger Medical Research Fund of the University of Illinois Medical Foundation. "~Reprlnt address: University o[ Illinois Medical Center, Department o[ Pharmacology, 901 & Wolcott St., Chicago, Ill. 60612.
Vol. 81, No. 3, pp. 566-569
METHODS Assay procedure. A conjugate of digoxin and bovine serum albumin (BSA) was prepared according to the method of Butler and Chen. 1 Antisera were raised in rabbits by injection of the conjugate in complete Freund's adjuvant at weekly intervals for four weeks. By four months, the antibody titer was such that 0.1 ml. of 1:10,000 dilution of the serum bound 50 per cent of a tracer dose of tritiated digoxin (9.0 curies per millimole; New England Nuclear Corp., Boston, Mass.). Assays were run in duplicate with each tube containing 0.1 ml. of pooled normal human serum, 0.1 ml. of a standard, 0.1 ml. of a 5 ng. per milliliter dilution of tritiated digoxin, 0.1 ml. of antisera, and sufficient phosphate-BSA buffer to make a final volume of 0.8 ml. All tubes were incubated at 25 ~ C. for 15 minutes. Free and bound digoxin were separated
Volume 81 Number 3
Serum levels o[ digoxin
567
8.0
PresentStudy .~ ~~
6.0
(Schw;rz-Mann)
lIB 4.0
(BurroughsWellcomeCo.)
I
2.0 !
I
I
I
I
1.0
2.0 .3.0 4-.0 Digoxin (ng/ml) Fig. 1. Comparison of standard curves obtained from: 9 9 present study, x. x Butler,6 @ @ commercial radioimmunoassay kit (Schwarz-Mann), O O antisera (Burroughs Wellcome & Co.). Values on the ordinate (l/B) are the reciprocals of :the fraction of antibody-bound digoxin. by adding 0.2 ml. of a BSA-charcoal suspension and centrifuging at 2,000 g for ten minutes. A 0.7 ml. aliquot of the supernate was transferred to a counting vial, 10 ml. of scintillation "cocktail" added, and the vial counted in a Packard Tri-Carb spectrometer. A standard curve was prepared by plotting a regression line, obtained by the method of least squares, of the reciprocal of the fraction bound digoxin ( l / B ) versus the concentration of the digoxin standard (D). Complete details are available from the authors upon request. Patients. All patients had congenital heart disease and were studied while hospitalized. All received digoxin (Lanoxin, Burroughs Wellcome & Co., New York) orally in two equal doses at 9 A.M. and 9 P.M. Children under five years of age received the elixir, and older children were given tablets. The patients were divided into three groups consistent with the dosage regimen used in our hospital, which is based on body weight. The maintenance dosages (25 per cent of the total digitalization dose) were as follows:
Group 1, 5 to 9 Kg., 0.022 rag. per kilogram (0.01 rag. per pound) ; Group 2, 9 to 18 Kg., 0.016 rag. per kilogram (0.0075 rag. per pound) ; and Group 3, greater than 18 kilograms, 0.011 rag. per kilogram (0.005 nag. per pound). All patients in Group 1 were six months to two years of age, those in Group 2 ranged from two to five years, and those in Group 3 were five years and older. Data were included only from those patients who were normokalemic, euthyroid, and had normal blood urea nitrogen values. All were judged to be adequately digitalized, and none had clinical or electrocardiographic signs of toxicity. Blood was obtained by venipuncture at 1 to 2 and 5 to 9 hours after administration of digoxin. These times reflect peak and equilibrium serum levelsY, 4 RESULTS
Precision. When plotted as 1/B versus D, the standard curve having the steepest slope indicates the most sensitive assayJ The standard curve obtained with our radioimmunoassay procedure had the same slope as that
56 8
Krasula et al.
The Journal o[ Pediatrics September 1972
Table I. Mean serum digoxin levels Postabsorptive mean serum digoxin (ng./ml.)
Plateau mean serum digoxin (ng./ml.)
Group
Weight (Kg.)
Age (yr.)
Mean maintenance dose (mg./Kg./day)
1
5- 9
~-2
0.020 + 0.001
3.2 + 0.3 (4;6)
1.0 + 0.2 (9;13)
2
9- 18
2-5
0.016 + 0.001
2.2 + 0.3* (4;7)
1.4 + 0.2 (11;13)
3
> la
> 5
0.009 + 0.001
1.6 -+ OAt (4;5)
0.9 -+ 0.2 (6;7)
Mean • S.E.M. (figures in parentheses denote number of patients; number of determinations). *Group 2 is significantly different from Group 1 (P < 0.05). "~Group 3 is significantly different from Group 1 (P < 0.005).
published by Butler, 6 and as that obtained with materials supplied in a commercially available kit (Schwarz-Mann, New York) (Fig. 1). The slope of the line was steeper than that found with an antiserum donated by the Burroughs Wellcome & Co. (Greenville, N. C.). While the y-intercept and slope of the standard curve varied slightly from day to day, eight replications of a 2 ng. standard over a 2 week period showed little deviation (S.E.M. + 0.04 ng.). Specificity. Eight plasma samples containing no digoxin were assayed: All gave results of less than 0.2 ng. per milliliter. When concentrations of 10-2 to 10-5 M of physiologically active steroids (progesterone, estradiol17fl, testosterone, cortisol, and cholesterol) were substituted for digoxin, no interference was found?, 2, Serum levels. At 1 to 2 hours after administration, the mean serum digoxin levels were found to decrease with increasing age, being 3.2 (S.E.M. + 0.3) ng. per milliliter in Group 1, 2.2 _+ 0.3 ng. per milliliter in Group 2, and 1.6 + 0.4 ng. per milliliter in Group 3 (Table I ) . T h e level in Group 1 differed significantly from that in either Group 2 (P < 0.05) or Group 3 (P < 0.005). At equilibrium, mean levels were comparable for all groups, being 1.0 + 0.2, 1.4 + 0.2,' and 0.9 + 0.2 rig. per milliliter, respectively. DISCUSSION
Recommendations in regard to dosage for infants and children are two to three times greater than for adults, whether based on
body weight or surface area. s, 9 Within the pediatric age group, doses generally employed are inversely proportional to age, except in the case of premature infants. Thus the dosage schedule employed in this study was that in actual practice in our hospital. The data indicate that, despite the relatively larger doses, the serum levels at equilibrium were the same for each age group and equivalent to those reported in adults3, 3, 7, 10 The postabsorptive levels were directly related to dose; e.g., Group 1 received twice the dose of Group 3 and the initial serum levels were twice as high (Table I ) . Thus poor absorption does not appear to be present. Similarly, Hernandez and associates 11 found no impairment of dixogin absorption in infants. Age-related differences in digoxin metabolism may exist but seem an unlikely explanation for our data. Less than 10 per cent of an oral dose undergoes biotransformation in adults, 12 and no metabolites have been found in the urine of infants? 1 Our finding of lesser rates of fall in serum levels between sampling periods with increasing age is consistent with a decreasing rate of urinary excretion. We are presently investigating whether more rapid excretion or differences in distribution are responsible for the age-related decrease in dosage needed to maintain serum levels believed to be within the therapeutic range. The technical assistance of Miss Diana Lipton, and the efforts of Dr. W. Meier in obtaining serum samples are greatly appreciated.
Volume 81 Number 3
Serum levels o[ digoxin
REFERENCES
I. Butler, V. P., Jr., and Chen, J. P.: Digoxinspecific antibodies, Proc. Natl. Acad. Sci. 57: 71, 1967. 2. Smith, T. W., Butler, V. P., Jr., and Haber, E.: Determination of therapeutic and toxic serum digoxin concentrations by radioimmunoassay, N. Engl. J. Med. 281:12121 1969. 3. White, R. J., Chamberlain, D. A., Howard, M., and Smith, T. W.: Plasma concentrations of digoxin after oral administration in the fasting postprandial state, Br. Med. J. 1: 380, 1971. 4. Mirkin, B. L., and Larese, R.: Immunoassay of serum digoxin levels in children: therapeutic implications, Proc. Soc. Pediatr. Res., Forty-first Annual Meeting, Atlantic City, N. J., Apr. 28- May 1, 1971. 5. Berson, S. A., and Yalow, R. S.: General principles of radioimmunoassay, Clin. Chim. Acta 22: 51, 1968. 6. Butler, V. P., Jr.: Digoxin: Immunologic ap-
7. 8.
9. 10. 11.
12.
5 69
proaches to measurement and reversal of toxicity, N. Engl. J. Med. 283: 1150, 1970. Oliver, G. C., Parker, B. M., and Parker, C. W.: Radioimmunoassay for digoxin, Am. J. Med. 51: 186, 1971. Goodman, L. S., and Gilman, A.: The pharmacologic basis of therapeutics, ed. 4, New York, 1970, The Macmillan Company, p. 700. Shirkey, H. C.: Pediatric dosage handbook, Washington, D. C., 1970. American Pharmaceutical Association, p. 75. Belier, G. A., Smith, T. W., Abelmann, W. H., Haber, E., and Hood, W. B., Jr.: Digitalis intoxication, N. Engl. J. Med. 284: 989, 1971. Hernandez, A., Burton, R . M., Pagtakhan, R. D., and Goldring, D.: Pharmacodynamics of SH-digoxin in infants, Pediatrics 44: 418, 1969. Doherty, J. E., Hall, W. H., Murphy, M. L., and Beard, O. W.: New information regarding digitalis metabolism, Chest 59: 433, 1971.