Serum zinc and copper measurement

The serum copper and zinc measurement was performed using an air / acetylene flame atomic absorption spectrometer (AAS) Varian model Spectr AA - 20 Victoria, Australlia [14].

The blood tubes used were immersed in a nitric acid solution (HNO₃) at 10% (v/v), following a previous day (12 h) washing in a solution of hydrochloric acid (HCl) at 10% (v/v) . They were then rinsed twice with distilled water and dried. Protein precipitation was made by diluting 1 mL of serum in 4 mL of a solution of hydrochloric acid (2 M). After homogenization according to the method of Banjoko et al. [15], each sample was allowed to settle. The clear supernatant obtained was sucked directly into the flame atomic absorption spectrophotometer at the wavelength of 324.8 nm; 213.9 nm for the copper and zinc, respectively. A multi element-standard solution 1000 ppm (Merck, USA) previously diluted at 1/500 with nitric acid-deionized water (0.03 M) was used to prepare the calibration range (0; 0.5; 1.5; 2.0; 4 ppm). The concentration measurements were performed in triplicate and adjusted against the white (HCl solution 2 M).

The normal reference values for serum copper, zinc were respectively 11.0–22.0 μmol/L, 11.5–18.5 μmol/L [9].