title: Quantification of intact plasma AGT consisting of oxidized and reduced conformations using a modified ELISA.
abstract: The pleiotropic actions of the renin-angiotensin system (RAS) depend on the availability of angiotensinogen (AGT) which generates angiotensin I (ANG I) when cleaved by renin. Thus, quantification of the intact AGT (iAGT) concentrations is important to evaluate the actual renin substrate available. The iAGT conformation exists as oxidized AGT (oxi-AGT) and reduced AGT (red-AGT) in a disulfide bond, and oxi-AGT has a higher affinity for renin, which may exacerbate RAS-associated diseases. Accordingly, we determined iAGT, oxi-AGT, and red-AGT levels in plasma from rats and mice. Blood samples were obtained by cardiac puncture and then immediately mixed with an inhibitor solution containing a renin inhibitor. Total AGT (tAGT) levels were measured by tAGT ELISA which detects both cleaved and iAGT. iAGT levels were determined by iAGT ELISA which was found to only detect red-AGT. Thus, it was necessary to treat samples with dithiothreitol, a reducing agent, to quantify total iAGT concentration. tAGT levels in rat and mouse plasma were 1,839 ± 139 and 1,082 ± 77 ng/ml, respectively. iAGT levels were 53% of tAGT in rat plasma but only 22% in mouse plasma, probably reflecting the greater plasma renin activity in mice. The ratios of oxi-AGT and red-AGT were ∼4:1 (rat) and 16:1 (mouse). Plasma iAGT consists of oxi-AGT and red-AGT, suggesting that oxidative stress can influence ANG I generation by the AGT conformation switch. Furthermore, the lower availability of plasma iAGT in mice suggests that it may serve as a limiting factor in ANG I formation in this species.
authors: Department of Physiology and Hypertension, Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana; and rsato@tulane.edu.
authors: Ryousuke
authors: 0000-0001-6505-9722
authors: R
authors: Satou
authors: Graduate School of Health Sciences, International University of Health and Welfare, Tokyo, Japan.
authors: Hiroyuki
authors: H
authors: Kobori
authors: Department of Physiology and Hypertension, Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana; and.
authors: Akemi
authors: A
authors: Katsurada
authors: Department of Physiology and Hypertension, Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana; and.
authors: Kayoko
authors: K
authors: Miyata
authors: Department of Physiology and Hypertension, Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana; and.
authors: L Gabriel
authors: LG
authors: Navar
mesh_terms: D000808:Angiotensinogen / Q000378:metabolism*; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D012084:Renin-Angiotensin System / Q000502:physiology
chemical_list: D000808:Angiotensinogen
keywords: ELISA; intact AGT; plasma RAS; renin-angiotensin system