Measurement of Pro-C-Type Natriuretic Peptide in Plasma

Abstract

Accurate measurement of peptides derived from pro-C-type natriuretic peptide (proCNP) in human plasma has been difficult for several reasons. Low plasma concentrations necessitate high assay sensitivity. In addition, sufficient specificity can be difficult because of the close sequence homology of CNP with atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Cross-reacting antibodies are particularly troublesome in heart failure patients, in whom plasma concentrations of ANP and BNP are increased. Plasma extraction before measurement has also been necessary to avoid protein interference (1)(2); however, extraction can reduce assay performance because of low and inconsistent peptide recovery. The aim of the present study was to develop an RIA for measurement of proCNP in human plasma. Plasma was extracted by a simple procedure before immunoanalysis with antibodies raised against a unique epitope in proCNP. The proCNP concentrations in venous plasma from healthy individuals and heart failure patients were assessed. Additionally, proCNP concentrations were measured in plasma selectively sampled from the coronary sinus in heart failure patients. Human proCNP 1–10 extended C-terminally with 4 alanyl and 1 tyrosyl residue was custom synthesized for tracer preparation and standards. ProCNP 1–10 extended C-terminally with 4 alanyl and 1 cystyl residue was used for directional carrier coupling (Cambridge Biochemical Research Ltd.). In addition, human proCNP 1–7, proCNP 2–7, and mouse proCNP 1–10 were synthesized for specificity testing. The purities and identities of all peptides were verified by amino acid analysis and reversed-phase HPLC. Antisera against the N-terminal sequence 1–10 of human proCNP were produced by use of 10 mg of proCNP 1–10-Ala-Ala-Ala-Ala-Cys coupled to 20 mg of bovine serum albumin (3). The rabbit immunization procedure used has been described previously(4). We iodinated 5 μg (2.1 nmol) of the tyrosine-extended fragment (proCNP 1–10-Ala-Ala-Ala-Ala-Tyr), using a mild chloramine-T method (5). The iodinated peptide was …