title: Sample dilution: a methodological pitfall in the measurement of tissue but not serum ace-activity.
abstract: Many tissue ACE-assays suffer from underestimation of the ACE-activity at low sample dilutions. However, measurement of ACE-activity as the amount of hippuric acid produced by cleavage of the commonly used substrate hippuryl-histidyl-leucine might circumvent this problem. In this study, we investigated whether sample dilution affects the measurement of ACE-activity in rat tissue and serum. We found that serum ACE-activity was not affected by sample dilution. In homogenates of aorta, kidney, left ventricle, and lung, however, ACE-activity increased 1.6-2.8 times with increasing sample dilution until, ultimately, a plateau was reached at dilution factor 100, 50, 20, and 100, respectively. In addition, tissue homogenates inhibited the activity of exogenous ACE, whereas serum did not. These data suggest that the dilution effect probably results from interactions of inhibitory substances from the homogenates with ACE. The implications of these findings are that tissue ACE-activity measurements by any assay should be performed using sample dilution at the plateau. In many studies in the literature, specifications of sample dilution are lacking. Our findings demonstrate that caution is warranted in the interpretation of these studies.
authors.affiliation: Groningen Institute for Drug Studies, Department of Medicine, State University, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Koiter
authors.forename: G
authors.initials: G
authors.lastname: Navis
authors.forename: P E
authors.initials: PE
authors.lastname: de Jong
authors.forename: W H
authors.initials: WH
authors.lastname: van Gilst
authors.forename: D
authors.initials: D
authors.lastname: de Zeeuw
mesh_terms: D000818:Animals; D001011:Aorta / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006352:Heart Ventricles / Q000201:enzymology; D006626:Hippurates / Q000032:analysis; D007668:Kidney / Q000201:enzymology*; D008168:Lung / Q000201:enzymology*; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D007703:Peptidyl-Dipeptidase A / Q000032:analysis* / Q000097:blood; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results
chemical_list: D006626:Hippurates; D007703:Peptidyl-Dipeptidase A; C030514:hippuric acid