Biologic variation of copper, ceruloplasmin and copper/ceruloplasmin ratio (Cu:Cp) in serum.

Diagnostic algorithms for Wilson's disease (WD) recommend the determination of serum ceruloplasmin in addition to the slit-lamp examination required to identify Kayser–Fleischer rings as clues in order to decide on or decline further testing, while measurements of total serum copper and/or ceruloplasmin-unbound (“free”) copper are of value in monitoring pharmacotherapy [1,2]. The direct measurement of “free” copper is, however, difficult and not routinely available [3]. To overcome this issue, equations have been proposed for assessing “free” copper fraction in serum; however, they can produce biologically implausible negative results in a significant number of patients [4]. The use of copper/ceruloplasmin ratio (Cu:Cp) has been proposed to overcome such problems [5]. Despite their clinical role, aspects related to biologic variation (BV) of total copper and ceruloplasmin in serum have not received enough attention, while information on “free” copper BV is totally lacking. We could retrieve only one published study carefully determining the BV of total copper using a well designed protocol [6]. Here we performed an assessment of BV components of total copper and ceruloplasmin concentrations in serum, and derived Cu:Cp, in the same cohort of subjects by an accurately designed protocol. We collected five blood specimens from each of 19 healthy volunteers (10 men and 9 women; age range, 23–48 years) on the same day, every twoweeks for twomonths. Ostensibly healthy subjects were studied to ensure that any copper and ceruloplasmin fluctuation in serum could truly reflect biology and not modifications due to pathologic (e.g., inflammatory) processes. In accordance with the Helsinki II Declaration, the study design was explained thoroughly to the subjects, and informed consent was obtained. None of subjects took any medication or consumed substantial (>10 g/day) quantities of alcohol. Women had regular menstrual cycle and did not use hormonal contraceptives. Venous blood was obtained at 0900 from subjects who had fasted for 12 h and had not smoked or exercised in thatmorning. Sampleswere collected by the same phlebotomistwith minimal stasis using vacuum collection tubes. After centrifugation, serum specimens were stored at−80 °C until analysis. When all specimens were available, they were thawed and analyzed in a single run in duplicate in random order. Copper and ceruloplasmin were determined on Roche Cobas c501 analyzer using a colorimetric and an immunoturbidimetric assay, respectively. Cu:Cp was calculated by the