A fluorometric assay to determine labile copper(II) ions in serum

Labile copper(II) ions (Cu²⁺) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu²⁺ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu²⁺ subset. This study introduces a fluorometric high throughput assay using the novel Cu²⁺ binding fluoresceine-peptide sensor FP4 (Kd of the Cu²⁺-FP4-complex 0.38 pM) to determine labile Cu²⁺ in human and rat serum. Using 96 human serum samples, labile Cu²⁺was measured to be 0.14 ± 0.05 pM, showing no correlation with age or other serum trace elements. No sex-specific differences in labile Cu²⁺ concentrations were noted, in contrast to the total copper levels in serum. Analysis of the effect of drug therapy on labile Cu²⁺ in the sera of 19 patients with WD showed a significant decrease in labile Cu²⁺ following copper chelation therapy, suggesting that labile Cu²⁺ may be a specific marker of disease status and that the assay could be suitable for monitoring treatment progress.