332

Biochimica et Biophysica Acta, 583 (1979) 332--343 © Elsevier/North-Holland Biomedical Press

BBA 28828

IN VITRO METABOLISM OF SELENITE IN SHEEP BLOOD FACTORS C O N T R O L L I N G THE DISTRIBUTION OF SELENIUM AND THE LABELLING OF PLASMA P R O T E I N

CECIL H. McMURRAY and W. BRIAN DAVIDSON Biochemistry Department, Veterinary Research Laboratories, Stoney Road, Stormont, Belfast (U.K.)

(Received June 29th, 1978) Key words: Selenite metabolism; Selenium distribution; Plasma protein labelling; (Sheep blood)

Summary Some factors controlling the distribution of Na27SSeO3 in sheep blood were studied in vitro. After centrifuging Na]SSeO3-incubated blood most of the radioactivity was found in the plasma. The labelling of plasma protein by 7SSe was dependent on the presence of erythrocytes. The degree of labelling of plasma protein increased with e r y t h r o c y t e concentration. When phosphatebuffered saline-washed erythrocytes were suspended in phosphate-buffered saline and incubated with Na~SSeO3 the majority of the 7SSe was detected in the erythrocytes. On incubating these labelled erythrocytes with unlabelled plasma there was a transfer of radioactivity to the plasma. The calculated activation energy for the labelling o f plasma was 107.52 kJ/mol. Albumin was shown not to be a principal acceptor of 7SSe from the erythrocytes by ammonium sulphate precipitation of radioactive plasma. Addition of Na2SeO3 to the labelled blood resulted in the transfer of 7SSe from plasma to the erythrocytes. Radioactive plasma incubated at 37°C was thermolabile with respect to its 7SSe content whereas in whole blood the degree of 7SSe binding to plasma protein did not vary suggesting that a recycling of selenium was occurring in blood. From the results presented an in vitro model of selenium metabolism in blood is postulated. Introduction Schwarz and Foltz [1] first showed selenium to be an essential trace element as a c o m p o n e n t of 'Factor 3', a complex which prevented liver necrosis inde-

333 pendent of vitamin E in the rat. Rotruck et al. [2], demonstrated in seleniumsupplemented animals that selenium helped prevent oxidative damage to rat erythrocytes incubated with glucose, which in the absence of selenium caused increased haemolysis and increased haemoglobin oxidation. In contrast vitamin E only protected against haemolysis suggesting that the selenium effect was distinct from that of vitamin E. This confirmed the earlier work o f Schwarz and Foltz [1], and further work b y Thompson and Scott [3], that selenium had properties distinct from those of vitamin E. l~otruck et al. [4] and Flohe et al. [5], independently demonstrated that selenium was an integral part of the enzyme glutathione peroxidase. This enzyme had earlier been shown to be responsible for the destruction of lipid peroxides in cells [6,7]. Several groups of workers have carried o u t experiments in various species to investigate the mechanism of selenium transport in whole blood, using either in vivo or in vitro experiments. Studies have been carried o u t in the sheep [8], the human [9--11], the dog [12], the mouse [13,14], the rat [15], the chick [16], and the c o w [17]. Lee et al. [10] in an in vitro study using human blood and Sandholm [13] in an in vivo study with mice demonstrated that 50--70% of the radioactive sodium selenite (Na~SSeO3) used accumulated inside the erythrocytes within 1 min. Thereafter there was a gradual expulsion of the 7SSe from the cell. Sandholm [13] further demonstrated that as 7SSe is expelled from the erythrocyte, radioactivity in the plasma increases. These experiments have also shown that plasma proteins do n o t bind selenium introduced as selenite unless erythrocytes are present, suggesting that a transformation of selenite occurs within the erythrocytes. Several groups of workers have carried o u t studies on the kinetics of selenium disappearance after intravenous injection [18,10,13], while others have carried o u t preliminary studies to characterise the selenium binding proteins [12,14,18--20]. McConnell et al. [12] suggested that albumin might be the initial acceptor for 7SSe after selenite administration with transfer taking place to more stable proteins with time. However others [16] have concluded that albumin only binds selenium when supraphysiological levels of selenite are employed. Based on paper electrophoresis Sandholm [13] also concluded a role of albumin in selenium binding. It is difficult to rationalize the results of different workers into a generalized model for selenite metabolism in blood due to the use of different species and the variety of selenium specific activities employed in different experimental conditions. In this report, a variety of conditions are investigated relating to the in vitro metabolism of selenite. These studies have enabled an initial unified model of the primary steps of selenium metabolism in blood to be postulated for the sheep. Materials and Methods The chemicals were the purest available from British Drug House Poole, Dorset, England, with the exception of ovalbumin which was purchased from Sigma Ltd., Surrey, England. Isotopes were obtained from the Radiochemical Centre Amersham, Buckinghamshire, England. Na~SSeO3 had a specific activity

334 of 5--20 Ci/g Se while that of the ~25I-labelled human albumin was 0.2 Ci/g protein. Sephadex G-25 was obtained from Pharmacia Fine Chemicals. Erythrocytes were counted using a Coulter Counter (Model ZF). Inorganic selenium was assayed b y the method of Hall and Gupta [21]. Gamma isotopes were measured by gamma scintillation spectroscopy in a Nuclear Enterprises Gamma Scintillation Counter (Model No 8312), using 2 inches diameter Sodium Iodide Crystal. The efficiency of detection of radioactivity was 80%. Albumin was estimated at pH 4.0 by formation of a coloured complex with bromocresol green as described b y Doumas et al. [22]. Glutathione peroxidase activity was assayed by the method of Thompson et al. [23] using cumenehydroperoxide as substrate. This method is a modification of the spectrophotometric assay of Beutler [24]. Ammonium sulphate precipitation was carried o u t in 10% (w/v) saturation steps. After salt addition to labelled plasma precipitates were allowed to form for 15 rain at 4°C. Precipitates were collected b y centrifugation at 10 000 Xg and taken up in phosphate-buffered saline. Sephadex G-25 chromatography was used to separate protein-bound selenium compounds. The sheep used in this study were maintained indoors on a diet consisting of hay and concentrates containing 0.03 and 3.0 ppm selenium, respectively. The mean selenium and glutathione peroxidase values of the sheep were 1.00 ~g/ml whole blood and 250 I.U./g haemoglobin, respectively. Blood samples were obtained by jugular puncture and collected in lithium heparin tubes (Searle and Co Ltd). Plasma was separated from blood by centrifugation at 2000 × g for 15 min at 20°C. In experiments requiring more rapid separation centrifugation was carried out at 4000 )

In vitro metabolism of selenite in sheep blood: factors controlling the distribution of selenium and the labelling of plasma protein.

332 Biochimica et Biophysica Acta, 583 (1979) 332--343 © Elsevier/North-Holland Biomedical Press BBA 28828 IN VITRO METABOLISM OF SELENITE IN SHEEP...
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