Evaluation of a Simple Colorimetric Assay for Serum Angiotensin-Converting Enzyme: Comparison with a New Ion-Pair Liquid Chromatography-Assisted Assay

Abstract

SUMMARY The development and validation of two different assays for serum angiotensin-converting enzyme are reported. The first step in both analytical systems is based on enzymatic cleavage of the synthetic substrate, hippuryl-histidyl-Ieucine, under conditions advocated by Cushman and Cheung.1 The hippuric acid released is then assayed either by colorimetry following an azlactone condensation reaction with an aromatic aldehyde, or by reversed-phase ion-pair high-performance liquid chromatography (HPLC). Both procedures reveal good linearity in the range 0-80 nmol/ml per min, with an inter-assay coefficient of variation of 6· 2 %for the colorimetric assay and 4· 5 %for the HPLC-assisted assay. Recovery values measured for the colorimetric method ranged from 97 % to 102%and for the HPLC-assisted method from 98 %to 101 %. The colorimetric method is suitable for performance in small hospital laboratories since it can be carried out with simple analytical instrumentation. It is obvious nevertheless; that the HPLC-assisted assay reveals better validation criteria. The method is also simple and rapid and it has successfully been used in our laboratory for the last two years.Angiotensin-converting enzyme (ACE) (kinase II. tripeptides such as hippuryl-histidyl-Ieucine (Hip-EC 3.4.15.1), recently reviewed by Erdos,2 is an His-Leu) and hippuryl-glycyl-glycine (Hip--Glyexopeptidase which catalyses cleavage of carboxy-Gly) or on cleavage of fluorogenic substrates terminal dipeptidyl residues.P'" It catalyses cleavage consisting of a highly fluorescent group coupled to of histidyl-leucine (His-Leu) from angiotensin I to a quenching nitrophenyl moiety.l920 Enzymatic yield its more potent vasopressive octopeptide hydrolysis of these fluorogenic substrates results in angiotensin II and also inactivates bradykinin by liberation from the quenching interaction and cleavage of the Pro 7-Phe8 bond. Hence the enzyme release of fluorescence. Hydrolysis of Hip--His-Leu not only inactivates hypotensive kinins but also or Hip-Gly-Gly is followed by a wide range of activates hypertensive angiotensin. It is a mem-analytical methods. A fluorescent complex can be braneous glycoprotein constituent of the vascular formed by reaction of e-phthalaldehyde'" 22 or endothelial cells located in juxtaposition to the fluorescarnine'" with the dipeptide residues; hippuric circulating bloodstream.vP Although the lung and acid released during the assay is extracted with ethyl kidney are physiologically important sites of con-acetate and determined by spectrophotometry at version, the enzyme is also found in many other 228 nm,112 by a differential spectrophotometric organs.'! In some diseases involving pulmonary method after reaction with 2,4,6-trichloro-Schanges, such as sarcoidosis, silicosis, and triazine.i" by condensation of hippuryl azlactone asbestosis.Pv'" and also in some non-pulmonary with an aromatic aldehyde after thin-layer chromatogranulomatous diseases such as Gaucher's disease graphic separation of hippuric acid 25 or by HPLC. 26 and lepro...

The influence of the ACE (I/D) polymorphism on systemic and renal vascular responses to angiotensins in normotensive, normoalbuminuric Type 1 diabetes mellitus

“…Serum ACE activity was measured with an HPLC-assisted assay, as the generation of Hip from the substrate Hip-His-Leu. Normal values in our laboratory range from 10 to 40 U/l [22]. PRA was measured with a commercially available double-antibody RIA (Nichols Institute Diagnostics, San Juan Capistrano, Calif., USA).…”

Enhanced Responses of Blood Pressure, Renal Function, and Aldosterone to Angiotensin I in the DD Genotype Are Blunted by Low Sodium Intake

“…Plasma samples for AngII determinations were stored at Ϫ80°C. Serum ACE activity was determined with an HPLCassisted assay (30). AngII levels were determined with a RIA (Nicols Institute, San Juan Capistrano, CA).…”

Tests of activity in pulmonary tuberculosis in elderly patients: serum angiotensin converting enzyme and serum albumin

“…Serum ACE was measured using a spectrophotometric method in which free hippuric acid is formed by the dipeptidyl carboxypeptidase action of ACE on a tripeptide hippuryl‐histidylleucine (Sigma, USA). The validity and reliability of this method has been assessed by Kwarts et al 8 .…”