Analytical validation of bovine plasma ceruloplasmin measurement by p-phenylenediamine oxidation and effect of storage time and freezing temperature.

BACKGROUND: Determination of ceruloplasmin (Cp) activity in plasma can provide an objective measure of the health of dairy cows as well as it can be used for various diagnostic purposes. The current study was designed to perform an analytical validation of a method for the determination of plasma Cp activity in dairy cows and to evaluate the influences of plasma storage times and temperatures as well as freeze-thaw cycles on the activity of this enzyme. This cohort was carried out on ten cows. For each cow, 24 aliquots of plasma, which were stored at different temperature regimes, were prepared. Both intra- and interassay coefficients of variation (CVs) were determined. The linearity was evaluated using bovine plasma Cp standard.

RESULTS: The mean values of intra- and interassay CVs were 1.08 and 2.12%, respectively. Results of linearity testing showed a high correlation coefficient (r = 0.998, P < 0.001). After 3 days of storage at room temperature and refrigeration, the plasma activity of Cp was significantly lowered (P < 0.05). Plasma samples kept at freezing for 3 months revealed insignificant changes in the activity of Cp. Repeated freeze-thaw cycles for three times had no significant influence on the activity of Cp.

CONCLUSIONS: The method is easy and may be valid at values of Cp ranging from 100 to 1000 mg/L. It seems that keeping of plasma samples at room temperature and refrigeration longer than 3 days is unsuitable for Cp measurement. In addition, Cp remains stable in plasma samples stored at freezing as well as repeat freeze-thaw cycles.