Procedure for Blood Glutathione Peroxidase Determination in Cattle and Swine
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From the State Veterinary Serum Laboratory, Copenhagen, Denmark.
PROCEDURE FOR BLOOD GLUTATHIONE PEROXIDASE DETERMINATION IN CATTLE AND SWINE By N. Agcrgaard and P. Thode Jensen
AGERGAARD. N. and P. THODE JENSEN : Procedure for blood glutathione peroxidase determination in cattle and swine. Acta vet. scand. 1982, 2:1, 515-527. - An improved testing system has been developed for direct measurement of glutathione peroxidase activity in heparinized whole blood at 37 °C. Without loss in net yield the consumption of has been found to be considerably lower with the new technique than with previously described techniques. Within the range 0-700 mKat/1 the GSH-Px activity in red cells may be measured with a high degree of accuracy and reproducibility without preceding separation and washing. The stability of the enzyme in bovine and porcine whole blood at 22°C, 4°C, and -20°C was determined . g l u tat hi 0 n e per 0 x ida s e; s e len i u m s tat us; met h dolo g y; s tor age; cat tIe ; s win e.
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On account of the regularly observed correlation between glutathione peroxidase GSH-Px (EC 1.11.1.9) activity and selenium status (cf. Underwood 1977, Van Vleet 1980, Sunde & Hoekstra 1980) the determination of GSH-Px levels in blood and tissue has been adapted as a useful procedure for detecting selenium deficiency. Most of the hitherto described methods for GSH-Px determination in domestic animals involve laborious separation and washing procedures. The present work was carried out in order to optimize the determination of GSH-Px activity directly on heparinized whole blood. Through systematic adjustment of each of the component reactions the technique has been optimized at 37°C in respect
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of the concentration of donor and receptor substrates, ionic strength, and pH. Moreover, the GSH-Px stability in bovine and porcine whole blood has been determined at different temperatures. MATERIAL AND METHODS Heparinized blood samples (143 USP units of Sodium-heparinate/TO ml, Venoject® tubes) have been used for examination of the GSH-Px reaction kinetics. To examine the stability of the enzyme, samples of whole blood have been stored at 22°C, 4°C and -20°C. The enzyme activity determination is based upon measurement of the rate of glutathione [GSH] oxidation by tert. butyl hydroperoxide [TBH] = [R-OOH] as catalyzed by the GSH-Px present in a hemolysate or plasma sample (Giinzler et al, 1974). Because of the coupled test system there will be no progressive loss of GSH, its concentration being maintained at a constant level by the addition of exo genous glutathione reductase [GR J and NADPH, which will immediately convert any oxidiced glutathione [GSSG] produced to the reduced form GSH : H-UOH \
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2 GSII \
GSII··Px
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NADPII
The rate of GSH formation is monitored through the NADPH consumption as recorded at 366 nm. Two minutes after the reaction is started by the addition of TBH, the NADPH consumption rate is recorded over a period of 60 s. The difference in co
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