Direct measurement of glycine-extended adrenomedullin in plasma and tissue using an ultrasensitive immune complex transfer enzyme immunoassay in rats

Abstract

The mature form of the vasodilator peptide adrenomedullin (AM-m) is synthesized from a glycine-extended precursor (AM-Gly) by enzymatic amidation. We have developed a highly sensitive enzyme immunoassay (Immune Complex Transfer Enzyme Immunoassay; ICTEIA) that enables us to measure levels of AM-Gly in plasma and tissue directly. The detection limit of this assay is 1 amol/assay, and the intra- and inter-assay precision are 4.5-14.1% and 9.9-20.5%, respectively. Dilution curves for plasma samples showed good linearity, and the analytical recovery was 107-116.6%. Using ICTEIA, we determined that the plasma concentration of immunoreactive AM-Gly is substantially higher than that of AM-m (5.22±2.56 vs. 1.21± 0.79 fmol/ml). In contrast, levels of AM-Gly were much lower than those of AM-m in the lung, heart, kidney, adrenal gland and liver. We also evaluated AM-Gly and AM-m levels in rats in a morbid state induced by intraperitoneal administration of lipopolysaccharide (LPS). In most tissues, levels of AM-m and AM-Gly were both increased by LPS; however, AM-Gly/AM-m ratios were not significantly affected, which suggests that AM-Gly is rapidly converted to AM-m in tissue.