Quantification, isolation and structural determination of bradykinin and hydroxyprolyl-bradykinin in tumor ascites

The presence of kinins in ascitic tumor fluids from rodents and human patients was identified and quantified. In bioassay, kinin content was found to be 1 to 40 ng/ml, and by enzyme immunoassay, 0.6 to 2.5 ng/ml. In particular, a high kinin content, 40 ng/ml, was found in the ascites of a gastric cancer patient by bioassay. Purification of this kinin in the ascites from the gastric cancer patient was performed by ethanol precipitation, gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two peaks (peak A and peak B) showed kinin activity. Peak A did not correspond to either bradykinin or other known kinins, such as lysyl-bradykinin and T-kinin, whereas peak B corresponded to bradykinin. Peak A contained 8 amino acid residues from bradykinin minus one proline plus an additional hydroxyproline. Sequence analysis of peak A showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of peak A on reversed-phase HPLC was exactly the same as that of synthetic hydroxyprolyl3-bradykinin (Hyp3-bradykinin) but was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of Hyp3-bradykinin in mammalian system.