Stoichiometric calibration of ceruloplasmin activity with copper enables molar quantification and has implications for the estimation of non-ceruloplasmin-bound copper

OBJECTIVES: Ceruloplasmin (Cp) and non-ceruloplasmin-bound copper (NCC) are measured when evaluating liver disease and disorders of copper (Cu) metabolism. Cp mass concentration is typically determined by immunoassays that detect both apo- and holoceruloplasmin. In contrast, enzymatic assays measure Cp ferroxidase activity and are therefore specific for holoceruloplasmin. Their clinical use has been limited by the absence of a method to convert enzyme activity to molar Cp concentration and to estimate NCC. METHODS: Cp ferroxidase activity and serum Cu concentrations were measured in a training dataset (n=924) to define their linear relationship and derive a conversion factor based on the stoichiometry of six Cu atoms per Cp molecule. This factor was applied to an independent validation cohort (n=12,635). Cp activity, Cp mass concentration, and serum Cu were compared, and reference intervals for Cp activity and NCC were established. RESULTS: The calibration of Cp activity assay against Cu concentration yielded a conversion factor of 0.0030 for expressing Cp activity in molar units. Reference intervals for Cp activity were 1.5-3.7 μmol/L in males and 1.6-4.3 μmol/L in females. Comparison with nephelometric Cp measurements demonstrated a positive bias and evidence of analytical interference in a subset of samples. CONCLUSIONS: We describe a method to convert Cp ferroxidase activity to molar concentration and demonstrate its application in a large clinical cohort. This approach enables estimation of NCC, although with considerable uncertainty, and establishment of reference intervals using activity-based measurements. Measurement of Cp by enzymatic activity provides a cost-effective and analytically robust alternative to immunoassays, with advantages in specificity.