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Home / Equine Research Bank / Vet Sci / Analytical Validation of Two Assays for Equine Ceruloplasmin...
Veterinary sciences2023; 10(10); 623; doi: 10.3390/vetsci10100623

Analytical Validation of Two Assays for Equine Ceruloplasmin Ferroxidase Activity Assessment.

Abstract: Ceruloplasmin (Cp) assessment in biological samples exploits the oxidase activity of this enzyme against several substrates, such as -phenylenediamine (-P), -dianisidine (-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 -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 -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. A disagreement between these assays has also been detected with the Bland-Altman plot, showing a progressive discrepancy between methods with increasing analytical values.
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Publication Date: 2023-10-18 PubMed ID: 37888575PubMed Central: PMC10611237DOI: 10.3390/vetsci10100623Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research investigates and compares two assays for measuring ceruloplasmin oxidase activity in horse plasma. The assays use different substrates and reveal that the enzymatic activity values can significantly vary, displaying a discrepancy with increasing analytical results.

Introduction and Methods

  • The research focuses on ceruloplasmin (Cp), an enzyme whose oxidase activity can be evaluated using different substrates like -phenylenediamine, -dianisidine, and recently, ammonium iron(II) sulfate (AIS). These assays, developed for humans, are often adopted in veterinary medicine without adequate optimization for the specific animal species.
  • In this study, two assays with AIS and -dianisidine (-D) as substrates were used, optimized, and validated for assessing Cp oxidase activity in horse plasma.
  • The optimization process involved adjusting the buffer pH and the molar concentration of the buffer and substrate.

Findings

  • Under the best analytical conditions obtained, the horse blood serum samples were treated with sodium azide, a potent inhibitor of Cp. Results showed that in the -D assay, 500 µM sodium azide treatment completely inhibits the activity of Cp, whereas in the AIS assay, some analytical signal was still present even at the highest (2000 µM) sodium azide concentration.
  • The analytical values derived from both methods were well correlated, yet the enzymatic activity values significantly differed when expressed in Units/L. This expresses the fact that the type of assay used can have a significant impact on the reported enzyme activity.

Discrepancies Detected

  • A Bland-Altman plot, a method for comparing two different ways of measuring something, was also used in this study.
  • The plot showed a noticeable discrepancy with a progressive divergence between the two methods as the analytical values increased. This further highlights the importance of assay selection, as these can significantly influence the interpretation and comparison of results within this enzyme activity context.

Cite This Article

APA
Cecchini Gualandi S, Di Palma T, Boni R. (2023). Analytical Validation of Two Assays for Equine Ceruloplasmin Ferroxidase Activity Assessment. Vet Sci, 10(10), 623. https://doi.org/10.3390/vetsci10100623

Publication

Veterinary sciences
ISSN: 2306-7381
NlmUniqueID: 101680127
Country: Switzerland
Language: English
Volume: 10
Issue: 10
PII: 623

Researcher Affiliations

Cecchini Gualandi, Stefano
  • Department of Sciences, University of Basilicata, Campus Macchia Romana, 85100 Potenza, Italy.
Di Palma, Tommaso
  • Department of Sciences, University of Basilicata, Campus Macchia Romana, 85100 Potenza, Italy.
Boni, Raffaele
  • Department of Sciences, University of Basilicata, Campus Macchia Romana, 85100 Potenza, Italy.

Conflict of Interest Statement

The authors declare no conflict of interest.

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