Neurotensin in Plasma: Immunochemical and Chromatographic Character of Acid/Acetone-Soluble Material*

Abstract

Using a RIA, we have examined the character of the neurotensin-like (NT-like) peptides present in acid/acetone extracts of animal and human plasma. Plasma immunoreactivity was measured using four antisera with different specificities. Antisera which were directed against the COOH-terminus of NT measured higher levels of plasma immunoreactivity than did antisera with NH2-terminal or mixed specificity. Chromatographic fractionation of bovine plasma extracts revealed the presence of multiple substances, one of which was chromatographically and immunochemically indistinguishable from NT. We estimate the plasma level of this component to be about 15-25 fmol/ml, which is 30-50% of the measurement obtained on unfractionated extracts using antiserum HC-8. Several other components were also identified which behaved as though they were smaller than NT and seemed to share with NT four to eight of its COOH-terminal amono acids. One eluted from Sephadex G-25 in the region of the NT-like peptide previously identified in extracts of gastric mucosa. Infusion of synthetic NT into rats for 30 min did not result in the formation of these COOH-terminal relatives of NT. Our results argue strongly for the presence of NT in plasma and also indicate that other peptides, sharing COOH-terminal homologies with NT, appear in plasma, possibly from the stomach, liver, and other as yet unidentified source(s).

Elevation of Plasma Neurotensinlike Immunoreactivity after a Meal

“…Interassay coefficient of variation was 9%. The cross-reactivity of TG-1 with partial sequences was as follows: NT(1-13), 100%; NT(1-12), 120%; NT(1-11), 60%; NT(l-10), 50%; NT (1)(2)(3)(4)(5)(6)(7)(8) (Fig. 2B) was pooled, redissolved in distilled water, and injected while the column was being eluted with buffer A.…”

“…2B) was pooled, redissolved in distilled water, and injected while the column was being eluted with buffer A. A linear gradient elution program (0-30% acetonitrile in buffer A) was only partially successful; standard NT(I-11) was resolved well, but NT(1-8) and NT (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) eluted at the same time as did one of the components of NTLI. Therefore, fractions from the region containing this component were pooled, lyophilized, redissolved, and analyzed using a gradient protocol that was found to be satisfactory to separate all the available immunoreactive N-terminal partial sequences of NT.…”

“…We have developed a procedure to separate and characterize these N-terminal cross-reacting substances, and to estimate the contributions of these constitutents to plasma neurotensinlike immunoreactivity. Gel chromatography of pooled plasma extracts on Sephadex G-25 followed by reverse-phase high pressure liquid chromatography indicated that peptides coeluting with NT and its N-terminal partial sequences NT (1)(2)(3)(4)(5)(6)(7)(8) and NT (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) were present in plasma. Comparison of plasmas collected before and 1 h after a defined meal, in five experiments, demonstrated no change in C-terminal immunoreactivity and an 8-to 10-fold rise in N-terminal immunoreactivity.…”

“…Chromatographic analysis of pooled pre-and postmeal plasma in four experiments showed that essentially all of this elevation in neurotensinlike immunoreactivity measured with an N-terminal directed antiserum was due to increases in NT (1)(2)(3)(4)(5)(6)(7)(8) and NT (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), while NT itself, measured using a C-terminal directed antiserum, did not increase appreciably in peripheral plasma 1 h after the meal. Generation of tritiated substances with the same elution times as NT (1)(2)(3)(4)(5)(6)(7)(8) and NT(1-11) did occur after incubation of [3H]NT INTRODUCTION The tridecapeptide neurotensin (NT),' which has been isolated from bovine hypothalamus (1) and bovine as well as humnan small intestine (2,3), has been localized to a population of endocrinelike cells in the distal small intestinal epithelium of many avian and mammalian species, including man (4)(5)(6)(7). Plasma neurotensinlike immunoreactivity (NTLI) is composed of several immunoreactive substances, including NT, that crossreact with N-terminal and C-terminal region-specific antisera (8).…”

“…Generation of tritiated substances with the same elution times as NT (1)(2)(3)(4)(5)(6)(7)(8) and NT(1-11) did occur after incubation of [3H]NT INTRODUCTION The tridecapeptide neurotensin (NT),' which has been isolated from bovine hypothalamus (1) and bovine as well as humnan small intestine (2,3), has been localized to a population of endocrinelike cells in the distal small intestinal epithelium of many avian and mammalian species, including man (4)(5)(6)(7). Plasma neurotensinlike immunoreactivity (NTLI) is composed of several immunoreactive substances, including NT, that crossreact with N-terminal and C-terminal region-specific antisera (8). As demonstrated by gel chromatography of extracted bovine plasma, NT comprises about onethird of the C-terminal (antiserum HC-8) and one-half the N-terminal (antiserum TG-1) immunoreactivity present in the extracts.…”