Estudo da distribuição de selênio em animais experimentais em função da espécie de selênio ingerida e via de administração

Abstract

Selenium is an essential micronutrient that plays an important role in many physiological processes. Its clinical significance is related to the action of selenocompounds as adjuvant in preventing diseases such as cancer and heart failure and also in improving the immunological defenses of the organism. Since the ability of selenium in acting as a protective agent depends on its form, it is important to investigate which form of selenium ingested promotes the best absorption and distribution in the organism. In this work, the methodology for selenium quantification in biological samples such as yeast, blood and tissues was evaluated. Graphite furnace atomic absorption spectrometry (GF AAS) and hydride generation atomic absorption spectrometry (HG AAS) were compared for total selenium quantification. The results showed that the HG AAS is more adequate for Se determination after acidic digestion procedure, since the interferences caused by the acids in the GF AAS measurements could not be minimized. Two different animals were used as model for studying the absorption and distribution of selenium in different organs, chicken and rabbit. Selenium was determined or in the fat extracted from the muscle of chicken meat and in the residue of this tissue after fat extraction. Fat was extracted with organic solvents (methanol:dichloromethane 1:3) and analysed by GF AAS. The muscle residue was digested with a HNO3/HClO4 mixture and the selenium reduction, before the HG measurement, carried out with NaBr/sulfamic acid. The limits of detection were 1 μg/L and 6 μg/L for GF AAS and HG AAS respectively. The results showed that selenium is distributed between fat (20%) and tissue residue (80%). The speciation of the selenoamino acids, selenomethionine (SeM) and selenocistine (SeC) was carried out by chromatography using the reagents 9-fluorenylmethyl chloroformate (FMOC) and o-phthaldehyde (OPA) with UV and fluorimetric detection, respectively. The High Performance Liquid Cromatography (HPLC) method was carried out with reversed phase and FMOC. The limits of detection were 0.50 and 0.20 mg/L for SeM and SeC respectively. In the HPLC-OPA method the amino acids were separated by ion-exchange and the limits of detection were 0.005 and 0.009 mg/L for SeM and SeC, respectively. The yeast used as selenium supplementation for the chicken analyzed in this work was characterized according to its selenium content. Total selenium, the fraction soluble in water and the selenium present in the proteins were determined by acid digestion, extraction with water, and separation of proteins by adsorption in a polyethylene powder column, respectively. The measurements were carried out by HG AAS (digested sample) and by GF AAS (aqueous extract and proteic fraction). The selenoamino acids were also determined in the aqueous extract and in the protein fraction after protein hydrolysis using the HPLC-OPA method. From the total selenium present in the aqueous extract, 6% was inorganic and 94% organic. Considering the organic part, 98% was present as SeM. Fifty eight per cent was bound to proteins, whereas 37% was found as free amino acid. Two groups of chicken were treated with selenium, one group with sodium selenite and the other with the above mentioned yeast. The animals treated with the yeast presented higher Se levels in the muscle and also in the fat. The mean value of selenium found in these chickens was 0.13 μg/g for the ones treated with sodium selenite and 0.32 μg/g for those treated with the yeast. It was also investigated the supplementation of selenium, using both forms, inorganic (sodium selenite) and organic selenium (SeM) through the parenteral via in rabbits. The administration was carried out in two different ways. In one experiment 0.2 mg/kg Se was administrated, and the species SeM and SeC were evaluated in the animal s blood after 15; 30; 60; and 120 min after the injection. In the other, both forms of selenium (0.1 mg/kg) were administrated for 6 weeks, in alternating days (sub-chronic treatment). In the fist experiment, significant changes were observed in the total Se in the serum of the animals treated with sodium selenite, whereas in the animals treated with SeM a high but constant Se level was observed. No changes were observed in the SeC level for both groups of animals after the administration of both Se forms. On the other hand, as could be expected, there was a peak of SeM just after the administration (15 min) in the group that received SeM. After the sub-chronic treatment, Se was increased in serum, urine, and also in the brain, heart, muscle, and splen of the animals treated with SeM. By contrast, Se was increased only in the liver of the animals treated with inorganic selenium. In the kidney no difference was observed either for SeM or selenite administration. The parameters for toxicological evaluation (measurement of ALA-D activity, TBARS, ascorbic acid, among others) did not show any difference between both administrated forms of selenium.