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Analytical Validation of Two Assays for Equine Ceruloplasmin Ferroxidase Activity Assessment

Authors :
Stefano Cecchini Gualandi
Tommaso Di Palma
Raffaele Boni
Source :
Veterinary Sciences, Vol 10, Iss 10, p 623 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

Ceruloplasmin (Cp) assessment in biological samples exploits the oxidase activity of this enzyme against several substrates, such as p-phenylenediamine (p-P), o-dianisidine (o-D) and, most recently, ammonium iron(II) sulfate (AIS). Once developed in humans, these assays are often used in veterinary medicine without appropriately optimizing in the animal species of interest. In this study, two assays using AIS and o-D as substrates have been compared and validated for Cp oxidase activity assessment in horse’s plasma. The optimization of the assays was performed mainly by varying the buffer pH as well as the buffer and the substrate molar concentration. Under the best analytical conditions obtained, the horse blood serum samples were treated with sodium azide, a potent Cp inhibitor. In the o-D assay, 500 µM sodium azide treatment completely inhibits the enzymatic activity of Cp, whereas, using the AIS assay, a residual analytical signal was still present even at the highest (2000 µM) sodium azide concentration. Even though the analytical values obtained from these methods are well correlated, the enzymatic activity values significantly differ when expressed in Units L−1. A disagreement between these assays has also been detected with the Bland–Altman plot, showing a progressive discrepancy between methods with increasing analytical values.

Details

Language :
English
ISSN :
23067381
Volume :
10
Issue :
10
Database :
Directory of Open Access Journals
Journal :
Veterinary Sciences
Publication Type :
Academic Journal
Accession number :
edsdoj.593c17de7dd4fae8dc2f2b1537af539
Document Type :
article
Full Text :
https://doi.org/10.3390/vetsci10100623