ALTERATION OF THE PHARMACOKINETICS OF SMALL PROTEINS BY IODINATION

The pharmacokinetics of several proteins were investigated using two d ifferent assays. A 23 kDa recombinant protein fragment of bactericidal /permeability-increasing protein (rBPI(23)) was radiolabeled with I-12 5 using Iodo-beads(R) and administered to rats. plasma samples were co llected and assayed for I-125-rBPI(23) by radioactivity. In a separate experiment, rBPI(23) was administered to rats and plasma samples were assayed for rBPI(23) by ELISA. The clearance determined from plasma c oncentrations of I-125-rBPI(23) measured by radioactivity was about 2. 5-fold lower than that of rBPI(23) determined by ELISA. In addition, t he steady state volumes of distribution and mean residence times of I- 125-rBPI(23) measured by radioactivity were four-fold and 10-fold grea ter, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were differen t when assayed by radioactivity or ELISA, but those of proteins with m olecular weights of at least 80 kDA revealed only minor differences. T o determine which assay method yielded the correct plasma pharmacokine tic profile, rBPI(23) was metabolically labeled with S-35-methionine a nd administered to rats, and plasma samples were assayed by radioactiv ity. The concentration-time profile assessed by this method was very c lose to that determined by ELISA. Exposing rBPI(23) to chloramine-T (t he oxidant used in the iodination process) and measuring its plasma co ncentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeli ng rBPI(23) using iodinated Bolton-Hunter reagent (which avoids exposi ng the protein to oxidant), and measuring I-125-rBPI(23) by radioactiv ity, yielded pharmacokinetics that were similar, although not identica l, to the pharmacokinetics of rBPI(23) measured by ELISA. Thus, our da ta suggest that directly iodinating low-molecular-weight proteins by o xidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.