Affiliation:
1. Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany (A.F.R., M.B.).
2. German Center for Cardiovascular Research, Berlin, Germany (A.F.R., M.B.).
3. Attoquant Diagnostics, Vienna, Austria (O.D., M.P.).
4. Charité Universitätsmedizin Berlin, Germany (M.B.).
5. Institute for Biology, University of Lübeck, Germany (M.B.).
6. Division of Pharmacology and Vascular Medicine, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, The Netherlands (A.H.J.D.).
Abstract
BACKGROUND:
Angiotensin-(1-12), measured by a self-developed, polyclonal antibody-based radioimmunoassay, has been suggested to act as an alternative precursor of angiotensin II. A more reliable detection method would be liquid chromatography-tandem mass spectrometry.
METHODS:
We set up the quantification of human and murine angiotensin-(1-12) by liquid chromatography-tandem mass spectrometry and then used this method to measure angiotensin-(1-12) in human, rat, and mouse blood samples, as well as in mouse brain, mouse kidney, and rat heart. We also verified ex vivo angiotensin-(1-12) generation and metabolism in human blood samples incubated at 37 °C.
RESULTS:
Stabilization of blood in guanidine hydrochloride was chosen for sample collection since this allowed full recovery of spiked angiotensin-(1-12). Angiotensin-(1-12) was undetectable in human blood samples when incubating nonstabilized plasma at 37 °C, while angiotensin-(1-12) added to nonstabilized human plasma disappeared within 10 minutes. Stabilized human blood samples contained angiotensin II, while angiotensin-(1-12) was undetectable. Blood, hearts, and kidneys, but not brains, of wild-type mice and rats contained detectable levels of angiotensin II, while angiotensin-(1-12) was undetectable. In renin knockout mice, all angiotensins, including angiotensin-(1-12), were undetectable at all sites, despite a 50% rise in angiotensinogen. Angiotensin-(1-12) metabolism in human blood plasma was not affected by renin inhibition. Yet, blockade of angiotensin-converting enzyme and aminopeptidase A, but not of chymase, neutral endopeptidase, or prolyl oligopeptidase, prolonged the half-life of angiotensin-(1-12), and angiotensin-converting enzyme inhibition prevented the formation of angiotensin II.
CONCLUSIONS:
We were unable to detect intact angiotensin-(1-12) in humans, rats, and mice, either in blood or tissue, suggesting that this metabolite is an unlikely source of endogenous angiotensins.
Publisher
Ovid Technologies (Wolters Kluwer Health)