| Abstract: |
Wilson’s disease (WD) is a genetic disorder of copper metabolism and is characterised by the accumulation of this metal in various body tissues, mostly liver, brain and the cornea of the eye. This overload is caused by mutations in the Cu-transporter gene (ATP7B gene) that participates in Cu metabolism and its removal from the body. An early diagnosis of WD is crucial to prevent the progression of the disease that could lead to irreversible hepatic, neurological and psychiatric damages.
A combination of tests are currently used in the diagnosis of WD: ATP7B mutation testing, clinical diagnosis (e.g. Kayser-Fleischer Ring) and biochemical tests such as serum ceruloplasmin, 24h urinary copper, serum free copper and calculated non-ceruloplasmin copper (NCC) or exchangeable Cu (CuEXC). CuEXC corresponds to the labile fraction of Cu in serum that is not bound to ceruloplasmin (CER). It should include Cu bound to proteins, mostly Albumin (ALB) and, to a lower extent, alpha-2-macroglobulin, low molecular weight Cu species and free Cu. EDTA is used as Cu chelating agent able to complex loosely-bound Cu from ALB and alpha-2-macroglobulin, while preserving Cu bound to CER. Calculated NCC or CuEXC, which is currently determined by subtracting Cu-CER from total serum copper, is considered so far the best guide to WD’s treatment efficacy and follow-up1. However, negative values have been obtained using this indirect approach and no reference methodology for the accurate quantification of Cu-CER has been reported so far. Therefore, there is an increasing need for reliable methods, enabling quantification of CuEXC to underpin measurements in the clinic.
The aim of this work was to evaluate the performance of speciation methodology to determine CuEXC as a direct method for WD diagnosis. An anion-exchange HPLC-ICP-MS method was developed for the first time for the fractionation of Cu associated with proteins in serum under the frame of the EU ReMiND project. It was evaluated for the detection of Cu bound to Cu-containing species after EDTA treatment in control human and WD patient serum samples. Since Cu released from ALB was found to be the main contributor to CuEXC, further method development focused on the accurate quantification of Cu bound to ALB by using double matching species-specific isotope dilution analysis. The reference methodology developed here will be invaluable to provide CuEXC reference values and Cu speciation data to support clinical measurements. It will also help monitoring efficiency of Cu chelating drugs, aiming at finding more effective therapies. |
