A sensitive and specific erythropoietin immunoprecipitation assay: application to pharmacokinetic studies.
Previous pharmacokinetic studies with radiolabeled erythropoietin have relied on results of nonspecific methods to derive pharmacokinetic parameters. Dependence on nonspecific protein precipitation or total radioactivity may result in falsely high determinations of plasma radiolabeled erythropoietin and erroneous determinations of pharmacokinetic elimination and distribution parameters. In the present study pharmacokinetic parameters were derived by using a specific, sensitive, and reproducible immunoprecipitation assay for biologically active iodine 125-labeled recombinant human erythropoietin (125I-rhEp) and compared with those obtained by using nonspecific protein precipitation with trichloroacetic acid (TCA). Tracer amounts of 125I-rhEp were administered by bolus injection to six newborn lambs. Plasma-precipitable radioactivity assayed by the immunoprecipitation method became progressively lower with time relative to those observed with the TCA method. Pharmacokinetic parameters derived from the immunoprecipitation assay demonstrated significantly more rapid plasma and elimination clearances, shorter terminal half-life, shorter mean body residence time, and shorter distribution time when compared with the TCA assay (p less than 0.01). Volume of distribution was not different. Comparison of immunoprecipitation and TCA assay results from gel permeation fractions of iodinated erythropoietin demonstrated that immunoprecipitation assay results provide a better evaluation of biologically active hormone as determined by comparisons with SDS-PAGE and erythropoietin radioreceptor data. We conclude that 125I-rhEp pharmacokinetic parameters derived by using the immunoprecipitation assay more accurately reflect physiologic conditions than do those derived by using TCA precipitation.
Widness
J A
JA
Department of Pediatrics, College of Pharmacy, University of Iowa, Iowa City 52242.
Schmidt
R L
RL
Veng-Pedersen
P
P
Modi
N B
NB
Sawyer
S T
ST
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000032:analysis* / Q000378:metabolism / Q000493:pharmacokinetics*; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D011233:Precipitin Tests / Q000379:methods*; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000378:metabolism; D017467:Receptors, Erythropoietin; D012756:Sheep; D013997:Time Factors; D014238:Trichloroacetic Acid
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D014238:Trichloroacetic Acid