Direct measurement of glycine-extended adrenomedullin in plasma and tissue using an ultrasensitive immune complex transfer enzyme immunoassay in rats.
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.
Yamaga
Junichi
J
First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Hashida
Seiichi
S
Kitamura
Kazuo
K
Tokashiki
Mariko
M
Aoki
Toshihiro
T
Inatsu
Haruhiko
H
Ishikawa
Naoto
N
Kangawa
Kenji
K
Morishita
Kazuhiro
K
Eto
Tanenao
T
D000311:Adrenal Glands / Q000737:chemistry; D053607:Adrenomedullin; D000818:Animals; D000936:Antigen-Antibody Complex; D002138:Calibration; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005998:Glycine; D007201:Indicator Dilution Techniques; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008297:Male; D009206:Myocardium / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000097:blood* / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000936:Antigen-Antibody Complex; D010455:Peptides; D053607:Adrenomedullin; D005998:Glycine