The metabolites of rG·CSF present in the serum were investigated in rats after intravenous administration of 125I-rG·CSF at the dose of 10μg/kg, or nonlabeled-rG·CSF at the dose of 100-400μg/kg.
1. Using gel filtration on the Superose 12 column, most of the radioactivity in rat serum at 30min after administration of 125I-rG·CSF was eluted at a point corresponding to molecular weight of 19kd, this point was the same as that of intact-rG·CSF. The radioactivity was immunoreactive with anti-rG·CSF antibody and showed biological activity against NFS-60 cell lines. At 2 and 6h after administration, the radioactivity present in the serum was eluted from the column at a point corresponding to molecular weight of 46kd and 19kd. The radioactivity corresponding to 46kd showed immunoreactivity, but was biologically inactive.
2. The SDS-polyacrylamide gel electrophoresis revealed that most of the radioactivity in the serum present at 30min after administration of 125I-rG·CSF was detected at a point corresponding to intact-rG·CSF (molecular weight of 19kd). The radioactivity was also detected at a point corresponding to molecular weight of 19kd and 5kd at 2h, 19kd, 14kd and 5kd at 6h after administration.
3. The chromatofocusing using Mono-P column showed that most of the radioactivity in the serum was focused at a point corresponding to isoelectric points of 5.8 and 5.5 at 30min after administration of 125I-rG·CSF, these points were related to monosialo-rG·CSF and disialo-rG· CSF, respectively. At 2 and 6h after administration, the radioactivity showing immunoreactivity was newly found at a point corresponding to isoelectric points of 5.2 and 4.5.
4. During immunoblotting of serum samples after administration of nonlabeled-rG·CSF, the presence of a band corresponding to molecular weight of 19kd was found in all sampling times after administration. Beside the band of 19kd, only bands at a point corresponding to molecular weight of 5kd and 14kd were detected at 6h after administration.
5. Based of the fact that only an intact form of rG·CSF exists at early stage after intrave nous administration of rG·CSF, and since 2h the metabolites which have not biological activity appeared in the serum, we have concluded that an intact form mainly participate in hematologic activity of rG·CSF in vivo.
