**1. Introduction**

70 Macro to Nano Spectroscopy

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Atomic absorption spectroscopy (AAS) is a reliable method to determine metal concentrations. Zinc is a fundamental trace element because of its role in several essential biochemical functions. It is a component or co-factor of several enzymes, such as alcohol dehydrogenase and superoxide dismutase. It is of fundamental importance in cell division, genetic expression, and physiological processes, such as growth and development, immunity and wound healing; also, it plays a structural role in stabilizing biomembranes (Hambidge, 2000; Kruse-Jarres, 2001).

The importance of the determination of the hepatic concentration of certain metals is clearly established in the investigation of hereditary hemochromatosis and Wilson's disease (Pietrangelo, 2003, Roberts et al; 2003). As well, some interesting studies were published on the importance of zinc related to hepatic diseases. Decreases in plasma (Halifeoglu et al., 2004; Schneider et al., 2009; Pereira et al., 2011) or serum (Hamed et al., 2008, Matsuoka et al., 2009) zinc concentrations have been described in patients with chronic liver diseases. Authors that measured zinc in the liver parenchyma of adults and children with liver cirrhosis found low zinc levels (Milman et al., 1986; Göksu & Özsoylu, 1986; Sharda & Bhandari, 1986; Kollmeier et al., 1992; Adams et al., 1994). In patients with alcoholic cirrhosis, studies found abnormal zinc concentrations not only in the liver parenchyma (Rodriguez-Moreno et al., 1997), but also in subcellular fractions of the liver (Bode et al., 1988). In other diseases, such as biliary atresia (Bayliss et al., 1995; Sato et al., 2005), Indian childhood cirrhosis (Bhardwaj et al., 1980; Sharda & Bhandari, 1986), and chronic hepatitis B (Gür et al., 1998), also were found low liver zinc concentrations.

<sup>\*</sup> Corresponding Author

An Assay for Determination of

**2.3 Statistical analysis** 

two groups (p = 0.057).

*Samples* 

**3.1 Group 1 – Analysis of fresh liver tissue** 

**3.2 Group 2 – Liver tissue analyzed after deparaffinization** 

**3.3 Comparison between analysis of fresh and deparaffinized samples** 

1 213.3 246.6 2 178.5 148.0 3 169.5 163.3 4 241.2 153.8 5 154.3 201.8 6 154.0 201.4 7 179.4 171.6 8 169.2 193.7 9 170.8 185.1

paraffin, deparaffinized, lyophilized and then analyzed.

*Concentration fresh liver*

*(*μ

**3. Results** 

Hepatic Zinc by AAS – Comparison of Fresh and Deparaffinized Tissue 73

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.

The software Microsoft Excel for Windows® and the Statistical Package for Social Sciences® 12.0 (SPSS) were used to create a database and to conduct statistical analysis. Measures of central tendency and dispersion were used to describe data, with means and standard deviations for quantitative variables. The Student t test for paired samples was used for

Zinc concentration in group 1 was 173.6 ± 37.9 μg/g dry tissue (mean ± standard deviation). Table 1 shows the results for the 27 fresh bovine liver samples analyzed after lyophilization.

Zinc concentration in group 2 was 220.2 ± 127.0 μg/g dry tissue (mean ± standard deviation). Table 1 shows the results of 27 bovine liver samples that were embedded in

Liver zinc concentrations in fresh and deparaffinized samples were compared by Student t test for paired samples, and there were no statistically significant differences between these

*g/g dry tissue) Concentration deparaffinized liver* 

*(*μ

*g/g dry tissue)* 

Reagents were analyzed to assess contamination by zinc, which was negligible.

comparisons between groups. The level of significance was established at p<0.05.

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 blocks.
