**2. Material and methods**

This study was conducted after the validation of the graphite furnace AAS method to determine zinc concentration in bovine liver tissue (Fröehlich et al., 2006), in accordance with the guidelines established by ICH, FDA and ANVISA (ICH, 2005; FDA, 2001; ANVISA, 2003). We used a standard zinc solution (1 mg/ml) and standard bovine liver material from the National Institute of Standards and Technology (NIST, SRM 1577b) with known zinc concentrations (127 ± 16µg/g dry weight).

A steel scalpel was used to obtain 29 wedge biopsies from the same bovine liver obtained from a local market. Each specimen measured about 4 x 2 cm, and 2 were excluded from each group due to contamination and loss of material. Each of the 27 remaining specimens was divided in half, and two groups of samples were formed.

This study was approved by the Ethics in Research Committee of the Research and Graduate Studies, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.

### **2.1 Group 1**

The samples in group 1 were placed in eppendorf tubes previously decontaminated with 10% nitric acid (HNO3). Samples were lyophilized, using a lyophilizer (Micro moduli 97, Edwards) for 72 hours. After lyophilization, 500 µL of concentrated HNO3 (twice distilled, Zn concentration < 0.5 µg/g) was added to each sample, and sonication was applied for 1 hour. The samples were then placed in an incubator (303, Biomatic) at 60º C for 1 hour to complete digestion of organic matter. From each of the 27 solutions prepared, 50 µl were poured into automatic sampler vials, and 950 µl of pure water (Milli-Q Plus, Millipore®) was added. Concentrations were then determined with a graphite furnace (HGA 800, Perkin-Elmer) AAS (AAnalyst-3000, Perkin-Elmer) and an automatic sampler (AS-72).

#### **2.2 Group 2**

Samples in the second group were fixed in formalin, embedded in paraffin, and later deparaffinized. The material was kept in paraffin blocks for about 7 days. The samples were deparaffinized by placing them in an incubator at 60º C for about 30 minutes to dissolve the paraffin block, and then in an average of two xylene baths (about 30 minutes each) alternating with 4 baths of alcohol (99%) and distilled water until the paraffin was totally removed. The samples were lyophilized for 72 h and concentrations were determined using AAS, following the same procedure described for samples in group 1. Mean dry weight of the 27 samples after lyophilization was 39.9 mg, and, after deparaffinization, 38 mg. Reagents were analyzed to assess contamination by zinc, which was negligible.
