Journal of Pharmacokinetics and Biopharmaceutics, Vol. 3, No. 5, 1975
RIA vs. C P B for Prednisolone W . A. Colburn 1 and R. H . Buller ~
Received Feb. 25, 1975--Final June 16, 1975
Comparisons of the disadvantages and advantages of radioimmunoassay in competitive protein binding for prednisolone are presented. KEY WORDS: radioimmunoassay ; competitive protein binding; prednisolone.
In the April 1974 issue of this Journal (Vol. 2, No. 2), Leclercq and Copinschi introduced a report entitled "Patterns of Plasma Levels of Prednisolone After Oral Administration in Man" (1). In the article, the authors use a competitive protein binding assay (CPBA) for the estimation of plasma prednisolone levels. Also, in their article the authors make reference to the prednisolone radioimmunoassay (RIA) developed in this laboratory (2). The authors, in assessing the radioimmunoassay procedure as applied in our laboratories, came to some conclusions which we feel must be challenged. Moreover, we feel the authors failed to point up some of the potential problems inherent in the CPBA system. We would like to clarify some of the comments by the authors relative to the RIA procedure. These may well have arisen because we failed to sufficiently describe our technique. Simultaneously, we would like to point up some of the predominant difficulties inherent in the use of CPBA, with specific reference to its manner of application by Leclercq and Copinschi. I. RIA A. The authors claim that the RIA is more difficult than the competitive protein binding assay, since one has to prepare a specific antibody for RIA, whereas corticosteroid binding globulin (CBG) is easily available for CPBA. It is true that the production of a specific antibody is tedious and time consuming, but with lThe Upjohn Company, Biological Control, 7832-41-2, Kalamazoo, Michigan 49001. 329 9 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 100I 1. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
330
Colburn and Buller
the formation of a specific antibody the assay becomes much simpler to implement than any other quantitative assay procedure now available. No extraction or purification of plasma samples is required for the RIA because the affinity of the steroid prednisolone for the antibody is much greater than the affinity for plasma proteins. No extraction of the sample is needed; therefore, no evaporation of solvent is required. Finally, only one 4~ incubation step is required for the RIA, whereas the CPBA requires two, an initial 45~ incubation period followed by an overnight 4~ incubation period. B. The authors state that the sensitivity of their assay is "sufficient for the levels measured." Their system requires the extraction of 200 pl of plasma for each analysis, whereas the RIA procedure requires only 2/~1 of plasma for each analysis. No extraction is necessary for the latter. C. Our report indicates 10~o cross-reactivity of the prednisolone antibody with cortisol. Leclercq and Copinschi refer to this statement as a significant deterrent to the use of RIA for the measurement of prednisolone in plasma. However, it must be noted that cross-reactivity in itself is not of primary importance. Of primary importance is the interference of these compounds in relation to their respective concentrations in the system. Since only 2/~1 of plasma is required for analysis in RIA, the interference from cortisol is negligible. We pointed this out in the discussion section of our article. D. Leclercq and Copinschi claim that cortisol interference was eliminated from our RIA by arbitrarily correcting all samples for the 0-hr concentration. This is not true. We corrected all 0-hr samples to zero because minor fluctuations of cortisol in the baseline occurs with time, not because of cross-reactivity with prednisolone. Using the controls which are built into the RIA, it is possible to correct for these fluctuations, thereby reducing or eliminating potential errors. II. CPBA A. The CPBA has been used for the estimation of prednisolone in plasma prior to and since the article of Leclercq and Copinschi (3,4). B. The specificity of the CPBA assay was determined by Leclercq and Copinschi for a few endogenous steroids. The authors did not investigate the potential interference of various metabolites and/or degradation products in their assay system (zV7). As has been disclosed in the literature, the principal plasma metabolite
RIA vs. CPB for Prednlso|one
331[
(prednisone) interferes approximately 10-25~. Other known metabolites such as 20fi-dihydroprednisolone were not examined for their possible interference in the assay. These interferences can be eliminated in the CPBA only by chromatography. C. The accuracy of the CPB assay procedure was not disclosed in the report. However, a coefficient of variation in excess of 13 ~/o is revealed in the reproducibility section of their article; moreover, the uncorrected recovery data approximated only 82%. These data show that the accuracy of the method does not compare favorably to RIA. D. In summary, the CPBA provides a possible means of measurement of plasma steroids following the administration of prednisolone (or prednisone). Leclercq and Copinschi did not include data in their publication to substantiate that they were truly measuring only the intact prednisolone molecule. The literature indicates that in all probability they were measuring prednisolone, its major metabolite prednisone, and to various degrees all other metabolites (8-10). We believe the criticisms made against the RIA procedure by these authors is unwarranted. Cross-reactivity with cortisol does exist, but, because of the increased sensitivity of the RIA, cortisol interference is insignificant in the assay system. We agree that raising antibodies in animals is time consuming; however, after antibodies have been elicited, the RIA procedure is much simpler to perform on a routine basis. In order to regain the perspective of both RIA and CPBA with respect to their use in biopharmaceutical studies, it is well to consider the handicaps of both systems. The CPBA can be used without chromatography for the gross estimation of plasma and/or urine concentrations of drug. In combination with chromatography, it can be used to measure specific compounds of interest. However, when CPBA is applied with chromatography plus the necessary extraction processes the assay becomes a cumbersome procedure which is unsuitable for the routine analysis of large numbers of samples. The RIA requires production of a conjugate and subsequent elicitation of antibodies in a suitable animal species. It is time-consuming. Nonetheless, RIA, when properly investigated and characterized, can be a specific, accurate, and precise method which does not require the use of extraction or chromatography and which provides a means of measuring minute quantities of drug in hundreds of samples in a relatively short period of time. Neither CPBA nor RIA is infallible, but with due care in delineating their characteristics both can serve as very useful tools in the study of biopharmaceutics and pharmacokinetics of drugs.
332
Colburn and Buller
REFERENCES l. R. Leclercq and G. Copinschi. Patterns of plasma levels of prednisolone after oral administration in man. J. Pharmacokin. Biopharm. 2:175-187 (1974). 2. W. A. Colburn and R. H. Buller. Radioimmunoassay for prednisolone. Steroids" 21:833846 (1973). 3. D. H. Sandberg, C. Z. Bacallao, and W. W. Cleveland. Measurement of plasma prednisolone by competitive protein-binding assay. Biochem. Med. 4: 383 (1970). 4. A. K. Turner, C. J. Carroll, J. L. Pinkers, D. Charles, and S. C. Chattoraj. Simultaneous competitive protein binding assay for cortisol, cortisone, and prednisolone in plasma, and itg clinical application. Clin. Chem. 19:731-736 (1973). 5. C. H. Gray, M. A. S. Green, N. J. Holness, and J. B. Lunnon. Urinary metabolic products of prednisone and prednisolone. J. Endocrinol. 14:146-154 (1956). 6. E. Caspi and M. M. Pechet. Metabolism of 1-dehydrosteroids in man. I. J. Biol. Chem. 230:843 851 (1958). 7. A. Vermeulen and E. Caspi. Metabolism of t-dehydrosteroids in man. II. J. Biol. Chem. 234:2295-2297 (1959). 8. A. Vermeulen. The metabolism of 4-14C-prednisolone. J. Endocrinol. 18:278-291 (1959). 9. H. G. Morris, G. Dekodre, S. M. Winkler, and C. M. Caro. Effect of oral prednisone on the measurement of plasma steroid concentrations by competitive protein binding radioassay. J. Pediat. 85:248-253 (1974). 10. H. Millar, M. Pearson, and M. H. Briggs. Steroid interference with plasma corticosteroid measurements. Med. J. Aust. 1 : 1047-1048 (I 974).
RIA vs. C P B for Prednisolone W . A. Colburn 1 and R. H . Buller ~
Received Feb. 25, 1975--Final June 16, 1975
Comparisons of the disadvantages and advantages of radioimmunoassay in competitive protein binding for prednisolone are presented. KEY WORDS: radioimmunoassay ; competitive protein binding; prednisolone.
In the April 1974 issue of this Journal (Vol. 2, No. 2), Leclercq and Copinschi introduced a report entitled "Patterns of Plasma Levels of Prednisolone After Oral Administration in Man" (1). In the article, the authors use a competitive protein binding assay (CPBA) for the estimation of plasma prednisolone levels. Also, in their article the authors make reference to the prednisolone radioimmunoassay (RIA) developed in this laboratory (2). The authors, in assessing the radioimmunoassay procedure as applied in our laboratories, came to some conclusions which we feel must be challenged. Moreover, we feel the authors failed to point up some of the potential problems inherent in the CPBA system. We would like to clarify some of the comments by the authors relative to the RIA procedure. These may well have arisen because we failed to sufficiently describe our technique. Simultaneously, we would like to point up some of the predominant difficulties inherent in the use of CPBA, with specific reference to its manner of application by Leclercq and Copinschi. I. RIA A. The authors claim that the RIA is more difficult than the competitive protein binding assay, since one has to prepare a specific antibody for RIA, whereas corticosteroid binding globulin (CBG) is easily available for CPBA. It is true that the production of a specific antibody is tedious and time consuming, but with lThe Upjohn Company, Biological Control, 7832-41-2, Kalamazoo, Michigan 49001. 329 9 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 100I 1. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
330
Colburn and Buller
the formation of a specific antibody the assay becomes much simpler to implement than any other quantitative assay procedure now available. No extraction or purification of plasma samples is required for the RIA because the affinity of the steroid prednisolone for the antibody is much greater than the affinity for plasma proteins. No extraction of the sample is needed; therefore, no evaporation of solvent is required. Finally, only one 4~ incubation step is required for the RIA, whereas the CPBA requires two, an initial 45~ incubation period followed by an overnight 4~ incubation period. B. The authors state that the sensitivity of their assay is "sufficient for the levels measured." Their system requires the extraction of 200 pl of plasma for each analysis, whereas the RIA procedure requires only 2/~1 of plasma for each analysis. No extraction is necessary for the latter. C. Our report indicates 10~o cross-reactivity of the prednisolone antibody with cortisol. Leclercq and Copinschi refer to this statement as a significant deterrent to the use of RIA for the measurement of prednisolone in plasma. However, it must be noted that cross-reactivity in itself is not of primary importance. Of primary importance is the interference of these compounds in relation to their respective concentrations in the system. Since only 2/~1 of plasma is required for analysis in RIA, the interference from cortisol is negligible. We pointed this out in the discussion section of our article. D. Leclercq and Copinschi claim that cortisol interference was eliminated from our RIA by arbitrarily correcting all samples for the 0-hr concentration. This is not true. We corrected all 0-hr samples to zero because minor fluctuations of cortisol in the baseline occurs with time, not because of cross-reactivity with prednisolone. Using the controls which are built into the RIA, it is possible to correct for these fluctuations, thereby reducing or eliminating potential errors. II. CPBA A. The CPBA has been used for the estimation of prednisolone in plasma prior to and since the article of Leclercq and Copinschi (3,4). B. The specificity of the CPBA assay was determined by Leclercq and Copinschi for a few endogenous steroids. The authors did not investigate the potential interference of various metabolites and/or degradation products in their assay system (zV7). As has been disclosed in the literature, the principal plasma metabolite
RIA vs. CPB for Prednlso|one
331[
(prednisone) interferes approximately 10-25~. Other known metabolites such as 20fi-dihydroprednisolone were not examined for their possible interference in the assay. These interferences can be eliminated in the CPBA only by chromatography. C. The accuracy of the CPB assay procedure was not disclosed in the report. However, a coefficient of variation in excess of 13 ~/o is revealed in the reproducibility section of their article; moreover, the uncorrected recovery data approximated only 82%. These data show that the accuracy of the method does not compare favorably to RIA. D. In summary, the CPBA provides a possible means of measurement of plasma steroids following the administration of prednisolone (or prednisone). Leclercq and Copinschi did not include data in their publication to substantiate that they were truly measuring only the intact prednisolone molecule. The literature indicates that in all probability they were measuring prednisolone, its major metabolite prednisone, and to various degrees all other metabolites (8-10). We believe the criticisms made against the RIA procedure by these authors is unwarranted. Cross-reactivity with cortisol does exist, but, because of the increased sensitivity of the RIA, cortisol interference is insignificant in the assay system. We agree that raising antibodies in animals is time consuming; however, after antibodies have been elicited, the RIA procedure is much simpler to perform on a routine basis. In order to regain the perspective of both RIA and CPBA with respect to their use in biopharmaceutical studies, it is well to consider the handicaps of both systems. The CPBA can be used without chromatography for the gross estimation of plasma and/or urine concentrations of drug. In combination with chromatography, it can be used to measure specific compounds of interest. However, when CPBA is applied with chromatography plus the necessary extraction processes the assay becomes a cumbersome procedure which is unsuitable for the routine analysis of large numbers of samples. The RIA requires production of a conjugate and subsequent elicitation of antibodies in a suitable animal species. It is time-consuming. Nonetheless, RIA, when properly investigated and characterized, can be a specific, accurate, and precise method which does not require the use of extraction or chromatography and which provides a means of measuring minute quantities of drug in hundreds of samples in a relatively short period of time. Neither CPBA nor RIA is infallible, but with due care in delineating their characteristics both can serve as very useful tools in the study of biopharmaceutics and pharmacokinetics of drugs.
332
Colburn and Buller
REFERENCES l. R. Leclercq and G. Copinschi. Patterns of plasma levels of prednisolone after oral administration in man. J. Pharmacokin. Biopharm. 2:175-187 (1974). 2. W. A. Colburn and R. H. Buller. Radioimmunoassay for prednisolone. Steroids" 21:833846 (1973). 3. D. H. Sandberg, C. Z. Bacallao, and W. W. Cleveland. Measurement of plasma prednisolone by competitive protein-binding assay. Biochem. Med. 4: 383 (1970). 4. A. K. Turner, C. J. Carroll, J. L. Pinkers, D. Charles, and S. C. Chattoraj. Simultaneous competitive protein binding assay for cortisol, cortisone, and prednisolone in plasma, and itg clinical application. Clin. Chem. 19:731-736 (1973). 5. C. H. Gray, M. A. S. Green, N. J. Holness, and J. B. Lunnon. Urinary metabolic products of prednisone and prednisolone. J. Endocrinol. 14:146-154 (1956). 6. E. Caspi and M. M. Pechet. Metabolism of 1-dehydrosteroids in man. I. J. Biol. Chem. 230:843 851 (1958). 7. A. Vermeulen and E. Caspi. Metabolism of t-dehydrosteroids in man. II. J. Biol. Chem. 234:2295-2297 (1959). 8. A. Vermeulen. The metabolism of 4-14C-prednisolone. J. Endocrinol. 18:278-291 (1959). 9. H. G. Morris, G. Dekodre, S. M. Winkler, and C. M. Caro. Effect of oral prednisone on the measurement of plasma steroid concentrations by competitive protein binding radioassay. J. Pediat. 85:248-253 (1974). 10. H. Millar, M. Pearson, and M. H. Briggs. Steroid interference with plasma corticosteroid measurements. Med. J. Aust. 1 : 1047-1048 (I 974).
