Determination of Substance P and Neurokinins by a Combined HPLC/RIA Procedure

Abstract

Problems in the measurement by radioimmunoassay (RIA) of the mammalian tachykinins (substance P, neurokinin A, neurokinin B) arise from (1) lack of immunogenicity, particularly for neurokinin B, (2) non-specificity of antisera raised against an individual tachykinin, and (3) molecular heterogeneity derived from the presence of biosynthetic precursors, catabolites and components with oxidized methionine residues. These problems have been overcome by separating the immunoreactive components by reverse-phase (RP) HPLC followed by quantitation using antisera of defined regional specificity.

RIA’s have been devised for substance P using antisera directed against the COOH-terminal and NH₂-terminal regions with >0.4% cross-reactivity towards the neurokinins. For the neurokinins an antiserum raised against A that shows 22% cross-reactivity with B but only 0.6% towards substance P has been used. With a C-18 column and gradient elution, various peptides — cf. (3) above — were resolved. Post-mortem formation of oxidized forms is unavoidable, but mercaptoethanol or di-thiothreitol addition to extracting solvents is helpful.

Substance P-like and neurokinin A-like immunoreactivities were high in extracts of metastases from human mid-gut tumours. HPLC has disclosed components corresponding chromatographically and immuno-chemically to substance P and neurokinin A and their oxidized forms, to a metabolite that may be [pGlu⁵] substance P (5–11), and to an MALI component more hydrophobic than neurokinin A but distinct from neurokinin B. Neurokinin A and substance P immunoreactivities are below the RIA detection limits in normal peripheral plasma, but were high in patients with carcinoid tumours.