Analytical Validation of Two Assays for Equine Ceruloplasmin Ferroxidase Activity Assessment

SIMPLE SUMMARY: Ceruloplasmin (Cp) is a glycoprotein playing many physiological roles; among them, antioxidant defense is one of the most analyzed, making it an interesting biomarker in animal welfare assessment. Usually, the assessment of this protein is based on an enzymatic assay in which substrate oxidation generates a colored solution whose intensity is related to the Cp content. However, many enzymatic assays commonly developed in humans are applied in veterinary sciences without assessing their species-specific analytical optimal responsiveness. In this paper, two Cp oxidase activity assays using different substrates were tested in the blood plasma of horses and compared on the basis of their analytical reliability. The optimization of these methods for the use in equines was carried out by varying some analytical parameters; among them, the buffer pH is the preeminent variable affecting the analytical output. Our results show that both methods are reliable for the Cp oxidase activity evaluation; nevertheless, the discrepancy observed in the analytical values expressed as Units L(−1) makes their comparison unfair. 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.