In Vivo and in Vitro Plasma Disappearance and Metabolism of Somatostatin-28 and Somatostatin-14 in the Rat*

Abstract

The present study was designed to compare the in vivo and in vitro plasma disappearance of somatostatin-28 (S- 28) and somatostatin-14 (S-14) in the rat and to investigate the nature of the immunoreactive fragments generated by the metabolism of the two peptides. Synthetic S-28 or S-14 (1–5 μg) was given as a single iv injection into the external jugular vein of male rats, and 0.2-ml blood samples were obtained from the opposite jugular vein at 0.25, 0.5, 0.75,1, 2, 3, 5, and 10 min. The in vitro degradation of S-28 and S-14 was studied by incubating 6.0 ng peptide/ml rat plasma at 37 C for 3 h. Somatostatin-like immunoreactivity (SLI) was measured in acid-ethanol extracts of plasma samples by RIA using 1) antibody R149 directed toward the central segment of S-14, which detects both S-14 and S-28 equally, and 2) antibody S-39 directed toward the N-terminus of S-14, which is specific for S-14. After S-14 injection, the intact peptide (monitored by S-39 RIA) underwent rapid disappearance, with 50% loss within 0.15 min. By contrast, the decay of R149 SLI after S-14 injection was relatively slower (50% loss in 0.4 min). Sephadex gel chromatography of plasma extracts showed that this difference was due to the rapid generation of a fragment with a molecular weight similar to that of S-14, which reacted in the R149 but not in the S-39 RIA and which probably represented des-Ala1-SRIF. Disappearance of S-28 in vivo proceeded more slowly than that of S-14 (50% loss in 1.35 min). Sephadex G-25 gel chromatography of plasma extracts from S- 28-injected rats showed two peaks of SLI, a major peak (>90% of SLI) corresponding to S-28 and a minor peak (4–7% of SLI) which coeluted with S-14 and comprised predominantly intact S-14 (for up to 0.5 min). Both peptides were relatively slowly inactivated by plasma in vitro, with a t½ for S-28 (210 min) approximately 4 times longer than that of S-14 (57 min). Analysis by gel filtration of plasma extracts after S-28 incubation showed 36% conversion to a peak corresponding to S-14 at 30 min. It is concluded that 1) S-14 and S-28 are rapidly eliminated in vivo, 2) the t½ of S-28 is significantly longer than that of S-14, 3) the in vivo metabolism of S-14 involves very rapid cleavage of the molecule at the N-terminus to yield fragments such as des-Ala1- SRIF, as well as degradation of both intact S-14 and des-Ala1- SRIF into nonimmunoreactive fragments, 4) the main pathway for metabolism of S-28 in vivo involves rapid breakdown of the molecule into nonimmunoreactive fragments and only slight (4–7%) conversion to S-14, 5) the relative stability of S-14 and S- 28 in plasma in vitro suggests that plasma degradation is not an important physiological route for the disposal of the two peptides, and 6) S-14 is a significant product of the in vitro plasma metabolism of S-28.