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Measuring Angiotensin-I Converting Enzyme Inhibitory Activity by Micro Plate Assays: Comparison Using Marine Cryptides and Tentative Threshold Determinations with Captopril and Losartan
- Henda, Yesmine
Ben, Labidi, Anis, Arnaudin, Ingrid, Bridiau, Nicolas, Delatouche, Régis, Maugard, Thierry, Piot, Jean-Marie, Sannier, Frédéric, Thiéry, Valérie, Bordenave-Juchereau, Stéphanie
- Journal of agricultural and food chemistry 2013 v.61 no.45 pp. 10685-10690
- angiotensin I, angiotensin II, antagonists, hypertension, inhibitory concentration 50, peptidyl-dipeptidase A
- To determine the angiotensin-I converting enzyme (ACE) inhibitory activity of marine cryptides, different methods were tested. ACE inhibition was measured using two synthetic substrates, (N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) and N-hippuryl-His-Leu hydrate salt (HHL)), and a natural one, angiotensin-I. The IC₅₀ value (defined as the concentration of inhibitory molecule needed to inhibit 50% of the ACE activity) of the reference synthetic inhibitor captopril was in the nanomolar range (1.79–15.1 nM) when synthetic substrates were used, whereas it exhibited IC₅₀ of micromolar range (16.71 μM) with angiotensin-I. We chose losartan, an antagonist of angiotensin-II receptor as negative control for the ACE inhibition. Losartan was also able to inhibit ACE whatever the substrate tested, with IC₅₀ of micromolar range (17.13–146 μM). We defined this value as a limit above which molecules are not showing in vitro ACE inhibitory activity. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK), and Val-Ala-Pro (VAP) were tested as inhibitors of ACE with synthetic and natural substrates. IC₅₀ displayed were substrate-dependent. With FAPGG as substrate, IW, VAP, KY, IY, AP, AKK, and VIY show IC₅₀ values over the IC₅₀ value of losartan and should not be considered as inhibitors of ACE. VY, VW, KW, and LKP exhibited IC₅₀ value lower than the IC₅₀ value of losartan for all substrates tested and were thus considered as good candidates for effectively decreasing hypertension. It appears that the comparison of IC₅₀ is not consistent when IC₅₀ values are obtained with different substrates and different methods. In vitro ACE inhibitory activity assays should always include various ACE substrates and references such as captopril and a negative control to obtain data reliable to discriminate ACE inhibitory peptides.