title: Development and clinical application of an enzyme immunoassay for the determination of midregional proadrenomedullin.
abstract: Adrenomedullin (ADM) is a 52-amino acid peptide with a variety of physiologic functions such as immunomodulating activity, direct bactericidal activity, maintenance of renal homeostasis, and vasodilatory activity. Midregional proADM (MR-proADM) is derived from a larger 185-amino acid precursor peptide, prepro-adrenomedulin (preproADM), by posttranslational processing. It is suggested to be co-synthesized with ADM in equimolar amounts and has the advantages over ADM in having a longer half-life, no bioactivity, and no binding to protein. Therefore, MR-proADM serves as a surrogate for ADM secretion. In this study, we attempted to develop an enzyme immunoassay (EIA) for quantifying MR-proADM-like immunoreactive substance (IS), which is applicable for monitoring plasma MR-proADM levels. By using β-d-galactosidase-labeled preproADM(83-94) as a marker antigen, anti-rabbit IgG-coated immunoplate as a bound/free separator, and 4-methylumbelliferyl-β-d-galactopyranoside as a fluorogenic substrate, a sensitive and specific EIA was developed for the quantification of MR-proADM-IS in human plasma. The lower limit of quantification was 0.032 pmol/well, and the steep competitive inhibition EIA calibration curve obtained was linear between 0.16 and 10 nmol/L. By using human plasma samples containing 0.2 and 2.0 nmol/L of MR-proADM, the interassay coefficients of variation (reproducibility) were 10.78% and 8.83%, respectively, and intraassay coefficients were 3.91% and 7.81%. Plasma MR-proADM-IS level was significantly higher in patients with chronic renal failure (1.39 ± 0.50 nmol/L) compared with healthy subjects (0.19 ± 0.07 nmol/L). These results suggest that our EIA may be useful to evaluate plasma MR-proADM levels as a biomarker in various clinical settings.
authors.affiliation: Department of Clinical Pharmacy, Oita University Hospital, Hasama-machi, Oita, 879-5593, Japan. y-suzuki@oita-u.ac.jp
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Hiroki
authors.initials: H
authors.lastname: Itoh
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Katagiri
authors.forename: Fuminori
authors.initials: F
authors.lastname: Sato
authors.forename: Yukie
authors.initials: Y
authors.lastname: Sato
authors.forename: Kanako
authors.initials: K
authors.lastname: Kawasaki
authors.forename: Yuhki
authors.initials: Y
authors.lastname: Sato
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Mimata
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takeyama
mesh_terms: D053607:Adrenomedullin / Q000737:chemistry / Q000378:metabolism*; D000328:Adult; D000595:Amino Acid Sequence; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007676:Kidney Failure, Chronic / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011498:Protein Precursors; C088633:proadrenomedullin; D053607:Adrenomedullin