This activity is responsive to selenium intake in the diet (Paynter et al., 1979). ... protein structure of RBC glutathione peroxidase only during erythropoesis.
J. Appl. Anim. Res. 8 (1995)21-27
Selenium and Glutathione Peroxidase Correlation in Different Blood Samples in Sheep M.T.Verde, M.C.Sanz, J.J. Ramos, A. Fernandez, M.C. Marca, T. Saez. Department Patologia Animal (Patologia General) Facultad de Veterinaria, C/Miguel Servet, 177 50013 Zaragoza, Spain (Received March 24, 1995;accepted May 21, 1995)
Abstract Verde. M.T.,Sanz, M.C., Ramos, J.J., Fernande, A., Mama, M.C. and Saez, T. 1995. Selenium and glutathione peroxidase correlation in M e r e n t blood samlples in sheep. J. Appl. Anim. R~s.,8 : 21-27.
The correlations between GSH-Px in whole blood and selenium levels in different samples have been studied (serum, plasmaheparine, plasma-EDTA, whole blood-heparine and whole bloodEDTA). The highest correlation has been found with whole bloodheparin, which in 90 animals was of 0.97. Key words : Selenium, glutathione peroxidase, sheep.
Introduction Various functions of the immune system are inhibited by a selenium and vitamin E deficiency. Epidemiological studies suggest that them is an inverse relationship between the incidence of several types of cancer and Se intake in humans (Combs and Combs, 1986). Glutathione peroxidase (GSH-Px) is a selenodependent enzyme found in the blood of different
21 J. Appl. Anim. Res. 0971-2119/95/$05.008 GSP, India
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M. T. Ver& and wworkers
species of animals. The activity of this enzyme is a good sensitive measure for the selenium status in animals (Paynter et al., 1979).
GSH-Px activity is higher in the erythrocyte in comparision to other tissues. This activity is responsive to selenium intake in the diet (Paynter et al., 1979). The determination of glutathione peroxidase activity in red blood cells has been adopted as a useful procedure in detecting clinical and subclinical Se deficiencies since a linear relationship exists between the Se levels in tissues and red blood cell with the GSH- Px activity (Sankari and Athorsi, 1983). Since selenium is incorported into the protein structure of RBC glutathione peroxidase only during erythropoesis (Hafeman et al., 1974),the activity of the enzyme in the circulating red blood cell population will reflect the selenium intake of the animal. There are two types of glutathione peroxidase enzymes and only one of them is selenium dependent (Scholz et al., 1981). Erythrocytes of sheep have the selenium dependent GSH-Px only (Pehrson, 1985) which makes the assessment of this enzyme in whole blood even more convenient. The objective of this study was first t o obtain the best sample to calculate the correlation between the Se concentration in different blood samples and the activity of GSH-Fk in blood. Secondly using a total of 90 animals with Merent Se concentrations to calculate the correlation between the Se and the GSH-Fk activity in blood.
Materials and Methods First phase :Sample selection :
Animals : 21 . b a a Aragoneaa breed of sheep (2-8 years) were used. These animals were fed on a diet supplemented with block form mineral correctors including selenium, beginning six months before the experiment. Samples : Each ewe was bled from the jugular vein and the blood was distributed into three different tubes (S, H and E.) From each of the tubes, several "es were obtained and were preserved frozen at -2OC until analyaed.
-TubeS : with serum -Tube H : with heparin, divided into two parts one with the whole blood (B. hep) and the other with plasma (P. hep) -Tube E : with EDTA, one with whole blood (B. EDTA) and another with plasma (P.EDTA)
23
Correlation :Selenium and glutathione
Analysis : Selenium concentration was assessed in each case (S, R. hep, P. hep, B. EDTA, and P. EDTA) by atomic absorption spectrophotometry in graphite furnace using a Zeeman effect correction (Zeeman Atomic Absortion Spectrophotometer Model Varian 400 GFASS). The samples were not submitted to previous extraction procedures since non specific absorbances due to sample impurities are corrected by the Zeeman system (Neve and Therond, 1991).
The samples were defrosted before mading and diluted with a Triton X-100 at 1%0.The whole blood dilution was of 1:2 and the fierum and plasma dilution was of 1:l. The total quantity used for each reading was of 500 pl. The standards used t o elaborate the calibration chart were of 0,15, 100, and 200 selenium ppb Table 1 shows the reading conditions and characteristics. Blood GSH-Px activity analysis was performed by a conventional liquid spectrophotometric monotest system (WSEL-Glutathione Peroxidase. Cat. No. RS 504. W D O X Laboratories) based on the method by Paglia and Valentine (1967). Readings were made using a Perkin-Elmer Lamnda 5 spectrophotometer with a wavelength of 340 nm. Second phase :Correlation between selenium and glutathwne peroxidase :
Table 1 Conditions for Se quantification used in the Zeeman atomic absorption spectrometer program Spectrophotometer Program Alternative wavelengths (nm)
204.0
Spectral bandwidths (nm)
0.5
Peak area max. Abs.
2.20
L.D (Abs)
0.004
Furnace Parameters Maximum ash temperature
1200 c
Atomize temperature
2600 C
Gas flow (Ilmin)
3.0
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M. T.Vmak and coworkers
Animals : 31, 29, and 30 sheep from three flocks and similar to those in the first phase were used. Each of the flocks came from a different area and were run under extensive conditions. The first flock had no supplement, the second was supplemented with barley (500 g/animdday) and straw ad libitum, and the third was given a specific p a r e n t e d szlenium supplement (50 mg of barium selenate d c administered 60 days before sampling). Samples : 10 ml. of blood were drawn from the jugular vein from each of the ewes and put into heparin tubes. Samples were divided into two eppendorf tubes : one was used to obtain selenium levels in whole blood and the other was used to measuure the glutathione peroxidme. The samples were then stored at -2OC until analysis. Analysis : The same method, as described in the previous phase, was used for the selenium and blood GSH-Px analysis.
Results and Discussion The GSH-Px activity varied between 232 and 602 U/g Hb. Selenium concentrations within the different samples were of 76.94 ppb in serum, 250.69 ppb in whole blood with heparin, 85.79 ppb in plasma with heparin, 253.59 ppb in whole blood with EDTA and 85.80 ppb in EDTA plasma (Table 2). The levels of selenium in whole blood are three times higher than those in the plasma (Table 2). "his is due to the fact that most of this element (75- 85% in the ovine species) is incorporated into the erythrocyte by the GSH-Px enzyme. This varies from the human being where only 10-15% of selenium is associated to this enzyme (Beiktein and m a n g e r , 1983).
The best correlations obtained were those between the GSH-Px activity and the levels of selenium in whole blood (-0.756 using EDTA as an anticoagulant and r=0.809 using heparin). The correlations with selenium in the serum were 0.658, while in plasma were 0.617 and 0.694 when using EDTA or heparin, respectively (Table 2). These results agree with those by Kovac and Sankari (1988).One of the probable reasons for such a variation could be the fact that even minimum amount of hemolysis can affect the selenium-GSH-Fx correlation when using serum or plasma instead of whole blood, since the greatest GSH-Px activity in whole blood is due to the enzyme in the erythrocytes (Koller et al., 1984). Having selected the best correlation between selenium levels and GSH-Px activity in the whole heparinized blood, a larger ovine population
25
Correlation :Selenium and glutathiom
Table 2
GSH-Px blood levels, the levels of Se in serum, plasma, and blood (n=21)and their correlation S.D.
r
P
Parameter
Mean
GSH-F’x (U/g Hb) Se-serum (ppb) Se-blood hep (ppb) Se-plasma hep (ppb) Se-blood EDTA (ppb) Se-plasma EDTA (ppb)
458.26 95.12 76.94 24.73 0.658 0.004 250.59 68.9 0.809 0.001 85.06 25.64 0.694 0.002 253.59 54.89 0.756 0.0004 85.80 23.80 0.617 0.0038
0
50
100
150 200
250
equation of regression
y=2.543~+259.319 y=2.532~+231.598 y=1.122x+173.868 y=1.316x+121.19 y=2.456x+242.635
300
350
Fig. 1 : Correlation between the blood level of selenium and GSH-Px activity in heparinized whole blood. y = 1.964~+ 12.827, r = 0.97, (p c O.OOOl), (SE of slope = 0.05, t = 38.928)
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M. T.Ye&
and wworkers
was subjected to assessment of selenium status. The correlation of GSH-F'x activity and selenium concentration in whole blood was observed to be r=0.97,y=1.964x+12.827 (Fig. 1). This correlation is slightly higher than that obtained by other authors : Thompson et aE. (1676), e0.92, Wiener and Woolliam (1983), r=0.80, Koller et al. (1984) and Osame et aE. (19901, ~0.80 and slightly lower than Kovac and Sankari (19881, ~ 0 . 9 9 2 . Having drawn this line of regression and the fact that animals with values of Se in blood of 0.05 ppm are considered deficient (Blood et al., 1992) a GSH-Px activity lower than 111.027 L- 1.946 U/g Hb could be considered deficient.
Acknowledgements This research was supported by project 932/91 of the Comision Interministerial de Ciencia y Tecnologia (CICYT).
References Beilstein, M A and Wbanger, P.D. 1983.Distribution of selenium and glutathione peroxidase in blood hctions tiom humans, rhesus and s q u i d , monkeys, rats and sheep. J. Nub., 113 : 21382146. Blood, C.D., Radostitis, O.M., Arundel, J.H. and Gay, C.C. 1992. Medicina Vetennuria. 78 Edicion. Ed. Interamericana. McGraw-Hill. Madrid. Combs, G.F.and Combs, S.B. 1986. The role of selenium in nutrition. Academic Press, WC London.
Hafernan, D.G., Sunde, R.A. and Hoekstra, W.G.1974. Effect of dietary selenium on erythcyte and liver glutathione peroxidase in rat. J. Nutr., 104 : 680-687. Koller, L.D., South, P.J., Exon, J.H.,Whit, GA. and Maas, J. 1984. Comparision of selenium levels and glutathione peroxidase activity in bovine whole blood. Can. J. Comp. Med., 48 : 431-433. Kovac, G. and Sankari, S. 1988. Glutathione peroxidase activity, selenium concentration in blood and masticatory muscle of cattle. Folia Veter., 32 : 79-94. Neve, J. and Therond, P. 1991. Le selenium. In "Les oligoelemnts en medicine et biologie". Ed. Lavoisier, Paris : 426457. Osame, S., Ohtani, T. and Ichijo, S. 1990. Studies on serum tocopherol and selenium levels and blood glutathione peroxidase activities in lambs with white Muscle Disease. Jpn. J. Vet. Sci., 62 : 706-710.
Correlation :Selenium and glutathione
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Paglia. D.E. and Valentine, W.N. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione pemxidase. J. Lab. Clin.Med., 70(1): 168-169. Paynter, D.I., Anderson, J.W. and Mcdonald, J.W. 1979. Glutathione Peroxidase and selenium in sheep : Part 11. Aus. J. Agric. Res., 30 : 703-709. Pehrson, B. 1986. Sel"enium-dependent and non-selenium-dependent glutathione peroxidase activity in tissues from young bulls. Zbl. Vet.Med. A., 32 : 48a-491. Sankari, S. and Athrosi, F. 1983. Effect of dietary selenium on erythrocyte glutathione peroxidase and blood selenium in two types of Finnsheep genetically selected for high and low glutathione peroxidase activity. Zbl. Vet. Med. A, 30 : 452-458.
Scholz, R.W., Cook, L.S. and Todhimter, D.A. 1981. Distribution of selenium Dependent and nonselenium-Dependent Glutathione Peroxidase Activity in Tissues of Young Cattle. A J . Vet. Res., 42 : 1724-1729. Thompson, R.H., Mcmurray, C.H. and BlancMower, W.J. 1976. The level of selenium and glutathione pemxidase activity in blood of sheep, cow and pigs. Res. Vet. Sci., 20 : 229. Wiener, G. and Woolliam, S. 1983. Selenium concentration in the blood and wool and glutathione peroxidase activity in the blood of three breeds of sheep. Res. Vet. Sci., 34 : 366-366.
