Blood and Tissue Selenium Determination by Hydride Generation Atomic Absorption Spectrophotometry

Pechová A. , L. Pavlata , J . I l lek: Blood and Tissue Selenium Determination by Hydride Generation Atomic Absorption Spectrophotometry. Acta Vet. Brno 2005, 74: 483-490. The aim of the study was to optimize the method of blood and tissue selenium determination in ruminants, and to implement it in research and diagnostic practice. A method was developed for selenium determination by hydride generation atomic absorption spectrophotometry (HG-AAS) in full blood, blood plasma and serum, the liver, skeletal muscle tissue, the myocardium, and the kidneys after wet mineralization of samples in a closed nitric acid and hydrogen peroxide system and subsequent hydrogen chloride reduction. Hydride generation was performed by 1% sodium borohydride. The resulting selenium hydride was drawn off under inert atmosphere into a flameheated quartz T-tube to atomize; absorbance at 196 nm wavelength was measured by an optic system with deuterium lamp background correction. The measurements were verified by using diverse types of reference materials with declared selenium concentration and by a method of yield measurement of calibration solution addition in mineralized samples. The results corresponded with the stated values at the 95% probability level for all reference materials used. This methodology is acceptable as to both the detection limit (0.762 μg·l-1) and the error of measurement (4.6-15%) required for authorized use in research as well as clinical and preventive livestock medicine. The practical potential of the method was documented in a group of calves supplemented with two different forms of selenium. AAS, liver, muscle, myocardium, microwave mineralization While the initial interest in selenium research focused on selenium toxicity, the activity of selenium as an essential element needed by all live organisms was discovered only later. Various selenium-deficit-induced diseases were diagnosed in different animal species. The most frequent ones are muscle dystrophy, observed in most animal species, liver necrosis (pigs, poultry, mice, and rats), microangiopathy (pigs), exudative diathesis (poultry), pancreatic fibrosis (poultry), and placental retention (ruminants). The current research is characterized by an intensive focus on selenium metabolism. With the view to objectify the selenium status in animals, at both the levels of selenium deficit and selenium toxicity, the interest in a direct selenium determination in tissues and biological fluids of various animal species has recently been increasing. There are a number of selenium determination methods (spectrophotometry, fluorometry, AAS, ICP, gas chromatography, polarography etc.). The most frequently used ones are: fluorometry (Rodriguez et al. 1994; Kuricová et al. 2003), atomic absorption spectrophotometry (Doãekalová et al. 1991; Milde et al. 2002), and hydride generation atomic absorption spectrophotometry. The last method is based on an analyte reduction producing a liquid hydride, its conversion into gas, and a subsequent atomization in the optical path of an atomic absorption spectrophotometer. The analyte is thus separated from the matrix, and a higher concentration in absorption medium compared with classical AAS arrived at. This leads to an increase in ACTA VET. BRNO 2005, 74: 483–490 Address for correspondence: Doc. MVDr. Alena Pechová, CSc. Clinic of Diseases of Ruminants Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Palackého 1-3, 612 42 Brno, Czech Republic Phone: +420 541 562 405 Fax: +420 541 562 413 http://www.vfu.cz/actavet/actavet. htmhttp://www.vfu.cz/acta-vet/actavet.htm sensitivity and a considerable reduction of interference. Optimum selenium hydride release depends on the form of the analyte and solution acidity. Tetravalent selenium (SeIV) only can hydridize, and therefore the other selenium forms (SeII, SeVI) must be converted to this form. Hydrogen chloride is most often utilized for this conversion. Sodium borohydride in an acidic medium is utilized for the hydride generation proper. The reaction is as follows: NaBH4 + HCl + 3 H2O → H3BO3 + NaCl + 8 H SeIV + 8 H → SeH4 + 2 H2 The aim of this study was to optimize the method of blood and tissue selenium determination in ruminants, and to implement the method in research and diagnostic practice. Materials and Methods Chemicals and solut ions The samples were mineralized by a mixture of H2O2 hydrogen peroxide 30% purris p.a., ACS not stabilized > 30% (Fluka) and HNO3 nitric acid purris p.a. 65% (Fluka). The mixture we used is described in more detail as part of the methodology below. The samples were reduced using HCl hydrochloric acid 35% p.a. (Carlo Erba). The hydride generation proper was done using NaBH4 sodium borohydride purum p.a. (Fluka) solution stabilized with the help of sodium hydrate p.a. (Lachema). Selenium calibration solutions were prepared by diluting the stock solution (selenium atomic spectroscopy standard solution (Fluka), concentration 1.000 g·l-1) consecutively. Verification of results was based on the following reference materials: NYC 704121, 404109 lyophilized blood (Nycomed, Norway), beef liver CRM No. 12-2-01, beef liver SRM 1577b (NIST, USA), lyophilized animal blood IAEA – A – 13 (IAEA, Vienna). Devices and equipment The selenium determination was performed using an atomic absorption spectrophotometer SOLAAR 939 (Unicam, Great Britain). Sample preparation by microwave-digestion-technique wet mineralization in a closed system was performed using an MLS-1200 device (Milestone, Italy). Analyzed samples Blood samples collected from the cows kept at the Clinic of Diseases of Ruminants, University of Veterinary and Pharmaceutical Sciences Brno, were analyzed. Blood was sampled from v. jugularis into disposable heparin test-tubes. Tissues were collected immediately after slaughter and frozen until laboratory processing. The last section of the paper presents the results of a practical application of the method in evaluating the effect of supplementation of different forms of selenium on the selenium concentrations in blood and selected tissues of calves weighing 55 – 65 kg, divided in 3 groups. The first group was controls. The calves in the second group were given inorganic selenium parenterally at the start of the experiment (Selevit inj. ad us. vet., natrii seleni anhydricus 2.2 mg, tocoferoli acetas 25 mg per 1 ml), the dose being 20 ml per animal, i.e. the total dose was 40 mg Se per animal. The calves in the third group were administered selenium orally in the form of yeast (SELPLEX, Alltech, selenium 1,000 mg·kg-1) for two months, the daily dose being 0.6 mg Se per animal, resulting in the total dose of 36 mg of selenium per animal. The animals were slaughtered at the end of the experiment and tissues were collected for analysis. Blood samples were taken and analyzed at the start of the experiment and after two months of its duration.