title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2).
abstract: Angiotensin-converting enzyme 2 (ACE2) is a recently described member of the renin-angiotensin system that hydrolyzes angiotensin (Ang) II to Ang-(1-7), and may thereby protect against cardiovascular and renal diseases. ACE2 is a type 1 integral membrane protein and contains a catalytically active ectodomain that can be shed from the cell surface into the extracellular space, via cleavage by a disintegrin and metalloproteinase-17 (ADAM-17). ACE2 enzymatic activity and protein can be detected in biological fluids, including urine, plasma, and conditioned cell culture media. We present a detailed method for measurement of ACE2 activity in biological fluids, using hydrolysis of an intramolecularly quenched fluorogenic ACE2 substrate, in the absence or presence of the ACE2 inhibitors MLN-4760 or DX600. Recombinant human or mouse ACE2 is used to generate standard curves for this assay, with ACE2 detection ranging from 1.56 to 50 ng/ml. While MLN-4760 potently inhibits the activity of both human and mouse ACE2, DX600 (linear form) only effectively blocks human ACE2 activity in this assay. In biological samples of human and mouse urine, cell culture medium from mouse proximal tubular cells, and mouse plasma, the mean intra- and inter-assay coefficients of variation (CVs) of the assay range from 1.43 to 4.39 %, and from 7.01 to 13.17 %, respectively. We present data on the time and substrate concentration dependence of the assay, and show that exogenous D -glucose, creatinine, urea, and albumin do not interfere with its performance. In biological fluids, this assay is a simple and reliable method to study the role of ACE2 and its shed fragments in cardiovascular and renal diseases.
authors.affiliation: Division of Nephrology, Department of Medicine, Ottawa Hospital Research Institute, Kidney Research Centre, University of Ottawa, Ottawa, ON, Canada.
authors.forename: Fengxia
authors.initials: F
authors.lastname: Xiao
authors.affiliation: Division of Nephrology, Department of Medicine, Ottawa Hospital Research Institute, Kidney Research Centre, University of Ottawa, Ottawa, ON, Canada. kburns@ottawahospital.on.ca.|Division of Nephrology, Department of Medicine, Ottawa Hospital Research Institute, Kidney Research Centre, University of Ottawa, 1967 Riverside Dr., Rm. 535, Ottawa, ON, Canada, K1H 7W9. kburns@ottawahospital.on.ca.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Burns
mesh_terms: D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002318:Cardiovascular Diseases / Q000097:blood / Q000378:metabolism / Q000652:urine; D003404:Creatinine / Q000494:pharmacology; D005453:Fluorescence; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007674:Kidney Diseases / Q000097:blood / Q000378:metabolism / Q000652:urine; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D010455:Peptides / Q000494:pharmacology; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism* / Q000652:urine; D012084:Renin-Angiotensin System / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
chemical_list: C486469:2-(1-carboxy-2-(3-(3,5-dichlorobenzyl)-3H-imidazol-4-yl)ethylamino)-4-methylpentanoic acid; C556151:DX600 peptide; D007093:Imidazoles; D010455:Peptides; D014508:Urea; D003404:Creatinine; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; C000705328:Ace2 protein, mouse; D000085962:Angiotensin-Converting Enzyme 2; D007930:Leucine; D005947:Glucose
keywords: Angiotensin-converting enzyme 2; Fluorescence; Plasma; Proximal tubule; Substrate; Urine