**3. Results**

72 Macro to Nano Spectroscopy

Paraffin-embedded liver tissue usually stored in Pathology Laboratories may be used for analysis when fresh tissue is not available. Some hepatic diseases present a severe imbalance in the metabolism of metals, for example the excess of copper in Wilson's disease and iron in hemochromatosis. Recently, Wortmann and colleagues have validated an analytical method similar to ours for hepatic iron quantification, following the guidelines recommended by the International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use (Wortmann et al., 2007). Due to the fact that zinc is a protective metal to human health, the assessment of zinc status has a great importance in clinical investigation. Since there is only a few data available in the literature about methods to determine zinc concentration in hepatic tissue, the assay proposed can be helpful to this analysis. Therefore, the purpose of this study was to compare zinc concentrations in fresh and deparaffinized tissues and to determine whether the concentration of this metal in liver specimens is influenced by tissue processing in paraffin

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

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

This study was approved by the Ethics in Research Committee of the Research and

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).

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

Graduate Studies, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.

blocks.

**2.1 Group 1** 

**2.2 Group 2** 

**2. Material and methods** 

concentrations (127 ± 16µg/g dry weight).

was divided in half, and two groups of samples were formed.
