@Copyright 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3201-3----0415 $02.00
The Effect of Barium Selenate Injection on Selenium Concentration and Giutathione Peroxidase Activity in Blood of Pregnant Ewes Fed Selenium-Deficient Diet B. & ZACHARA, *'~ U. TRAF1KOWSKA, 1 M. KAPTUR, 2 C. KIMBER, 2 AND H. LEJMAN 3
~Department of Biochemistry, and 3Diagnostic Laboratory, Medical School, Bydgoszcz; and 2Stock-farm, Sarnokleski, Bydgoszcz, Poland Received January 31, 1991; Accepted March 21, 1991
ABSTRACT Selenium (Se) levels in whole blood and plasma, and glutathione peroxidase (GSH-Px) activities in red cells and plasma were measured in ewes fed an Se-deficient diet injected with barium selenate before breeding season. Highly significant increases in Se levels and GSH-Px activities (P < 0.001) were observed throughout the gestation period and during lactation. In the control group, Se levels and GSH-Px activities decreased significantly (P < 0.001), and were at critically low levels during lambing and lactation periods. Index Entries: Selenium; barium selenate; glutathione peroxidase; whole blood; plasma; erythrocytes; ewes; pregnancy.
INTRODUCTION Diseases of domestic animals caused by selenium (Se) deficiency are w i d e s p r e a d t h r o u g h o u t the world. In order to correct the deficiencies, the supply of Se must be supplemented either orally or parenterally (1,2). *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
4] 5
VoL 32, 1992
416
Zachara et al.
Deposel preparation containing barium selenate has recently been developed (3). It combines practical acceptability and efficacy with a good safety margin. Deposel has a very low solubility, and after injection slowly releases Se, having a prolonged effect. Deposel injection to ruminants had the ability to sustain elevated GSH-Px activity in whole blood for extended periods of time (1-4). The purpose of the present experiment was to study the effect of Deposel injection on Se concentrations and GSH-Px activities in blood components of pregnant and lactating ewes.
IV TERIALS AND METHODS In 1989, 100 Merino ewes (mean weight 59.8 kg), 13-14 mo old, were divided into two groups of 50 each. They were fed with a locally grown Se-deficient diet (0.03-0.04 mg Se/kg D.M.). Three weeks before the breeding season (end of November), one group was injected subcutaneously with Deposel (1 mg Se/kg body wt; Robert Young & Co. Ltd., Glasgow, Scotland) in the neck region. The other group was left untreated. From 15 ewes of each group, blood samples were taken at the commencement of the study, at the time of Deposel injection, and subsequently during gestation on days 19, 53, 98, 140, and 166 after Se injection, as well as 14 and 42 d after lambing (190 and 218 d after injection). Se concentrations in whole blood and plasma were determined by the fluorometric method of Watkinson (5). GSH-Px activities in red cells and plasma were measured by the coupled method of Paglia and Valentine (6). A detailed description of precision, accuracy (IAEA blood standard samples), and statistical analysis has been published earlier (7).
RESULTS AND DISCUSSION In the untreated group, whole blood Se concentration significantly decreased (Table 1) within the first month of pregnancy (P < 0.01). In the further gestation period, both whole blood and plasma Se concentrations decreased linearly, reaching the severely deficient levels during lambing and lactation. The injection of Deposel induced a significant increase in whole blood and plasma Se concentrations in ewes within 19 d (both P < 0.0001). In whole blood, a further, almost linear increase was observed up to the end of pregnancy. The highest level (2.3 x the initial value) was noted within 20-24 wk after Se injection. In the initial period of lactation, whole blood Se slightly decreased, but was still about 2 x higher than the initial value (P < 0.0001). Similar observations were presented by Overnes et al. (8). They noted that the injection of Deposel in ewes induced an adequate increase in blood Se within 8 wk, and a further increase to approx 300 ng/mL was noted within 16-20 wk. This level was maintained throughout the lambing season and throughout the Biological Trace Element Research
Vol. 32, 1992
Effect of Barium Selenate
417
Table 1 Effect of Deposel Injection on Whole Blood and Plasma Selenium Concentration (ng/mL) in Ewes During Pregnancy and Lactation D a ys postlambing
Days p os t i nj e c t i on 0
19
Material, e x p e r i m e n t a l g r o u p , n = 15 W h o l e blood 124 178 • 34 • 27 Plasma 33 116 • 16 _+ 11 Control g r o u p , n = 15 W h o l e blood Plasma
110 _+ 27 24 • 8
n . m '~ n.m. a
53
98
206 37 119 • 20
249 +_ 36 97 + 21
86 _+ 15 22 _+ 3
52 _+ 17 16 • 3
•
140 288 46 109 • 15
•
• •
45 11 20 7
166 282 43 105 + 18 •
31 12 8 _+_ 2 •
14 264 44 78 • 10 •
• •
27 7 10 4
42 230 48 82 • 15 •
• •
24 6 8 4
~n.m.: Not measured.
following summer. In our trial, plasma Se reached a plateau after 19 d of Deposel injection (3.5 x the initial value), and this level was maintained throughout the whole period of pregnancy. Several authors (3,8) reported that the injection Deposel in sheep maintained adequate Se level for at least 6 mo. In each blood sample of the Se-treated group, mean red cell and plasma GSH-Px activities (Table 2) were significantly higher than in the control group (P < 0.02-0.0001). In the experimental group, 19 d after Se injection, the red cell GSH-Px activity was slightly, but not significantly, higher than the initial value. At this time, the enzyme activity in the plasma increased significantly (P < 0.0001). The highest red cell GSH-Px activity (3.9 x the initial value) was noted after 20 wk of Deposel injection. In the later period, the activity decreased to a certain degree, but 6 wk after lambing, it was still 2.4 x higher compared to the initial value (P < 0.0001). Elevated (not less than 2 x ) plasma GSH-Px activities were maintained throughout the whole study period (P < 0.001-0.0001). Many authors have shown that Se administration to ruminants increased the synthesis of GSH-Px in the red cells and in various tissues (2,9-12). This study shows that the red cell GSH-Px activity increased more slowly, when compared to the plasma enzyme of Se-treated ewes. This is because red cell GSH-Px reflects the animal's Se status at the time of erythropoiesis, and therefore, a less immediate influence of Se treatment could be expected on red cells compared to tissues with a higher turnover rate (10). The average life-span of an erythrocyte in sheep is about 4 mo (13). Our results confirm this: The highest whole blood Se concentrations as well as the red cell GSH-Px activities in the Se-treated ewes were noted during the fourth month after Deposel injection. In the studies performed by Cawley and McPhee (3) and by Finch and Turner (4), the Deposel injected into ewes induced also the highest GSH-Px activity in whole blood within 3-5 mo. When the Deposel was used in pregnant Biological Trace Element Research
Vol. 32, 1992
Zachara et aL
418
Table 2 Effect of Deposel Injection on GSH-Px Activity in Red Cells (U/g Hb) and Plasma (U/L) in Ewes During Pregnancy and Lactation Days postlambing
D a y s postinjection Material
0
Experimental g r o u p , n = 15 Red cells
178 61 163 • 50
• Plasma
Control g r o u p , W h o l e blood
n
=
19 209 65 293 • 79
•
53
•
376 85 542
•
98 440 77 300 • 88
•
140
166
687 • •
526 •
288 73
14 529 •
360 •
42
• 296
•
436 67 392
•
15 152 44 163 • 37
n.m."
• Plasma
144 35 140 • 41 •
n.mf
• •
79 31 74 20
• •
63 33 60 20
41 10 47 • 15 •
• •
49 19 48 23
• •
36 11 44 17
"n.m.: Not measured.
ewes (3), not only were the ewes protected against Se deficiency, but their lambs were also well protected. In the control group, red cell and plasma GSH-Px activities decreased almost linearly until lambing season and were maintained thereafter at critically tow levels (more than 3 x lower than the initial values; P < 0.0001). It has been shown (14) that, in sheep peripheral blood, GSHPx activity in erythrocytes is about 99 x that in plasma. Beilstein and Whanger (15) have also demonstrated that, in sheep erythrocytes, 79% of Se is incorporated into GSH-Px, whereas in plasma, only 25% of the element was found in this enzyme. Therefore, erythrocyte GSH-Px activity correlates well with whole blood Se concentration in sheep and in some other animals. At the commencement of the trial, the correlation coefficient (r) between whole blood Se concentration and red cell GSH-Px activity for all ewes (n = 30) was 0.464 (P < 0.01), whereas at all other times during the study taken together, high correlations were obtained for experimental (r = 0.829; P < 0.0001) as well as for the control group (r = 0.844; P < 0.0001). The correlations we found in this trial correspond well with other studies (2,11,12,16). The close relationship and simplicity of determination should enable red cell GSH-Px activity to be used to evaluate blood Se concentration in sheep. The regression equation derived for the control group in this study is: GSH-Px (U/g Hb) -- 1.32 x (Se) + 9.82. The two principal conclusions from this study are: (1) Deposel injection seems to be suitable and safe for protecting ewes against Se deficiency during pregnancy and lactation, and (2) it also has the ability to sustain elevated GSH-Px activities in the blood of ewes.
ACKNOWLEDGMENTS We are grateful to Robert Young & Co. Ltd., Glasgow for donating the Deposel preparation used in this study. The author (B. A. Z.) kindly Biological Trace Element Research
Vol. 32, 1992
Effect of Barium Selenate
419
appreciates the assistance and financial support of Labcatal, Montrouge, France for the participation in the Chromium-Trace Elements Congress. This research was supported, in part, by the Polish Central Research Programme, CPBR 10.17/IV.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16.
A. MacPherson, and J. S. Chalmers, Vet. Rec. 115, 544 (1984). C. G. Mackintosh, J. Gill, and K. Turner, New Zeal. Vet. ]. 37, 143 (1989). G. D. Cawley, and I. McPhee, 114, 565 (1984). J. M. Finch, and R. J. Turner, 119, 430 (1986). J. H. Watkinson, Anal. Chem. 38, 92 (1966). D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med. 70, 158 (1967). W. Wasowicz, and B. A. Zachara, J. Clin. Chem. Clin. Biochem. 25, 409 (1987). G. Overnes, K. Moksnes, and A. Froslie Acta Vet. Scand. 26, 164 (1985). L. D. Koller, P. J. South, J. H. Exon, and G. A. Whitbeck, Cornell Vet. 73, 323 (1983). A. M. Smith, and M. F. Picciano, J, Nutr. 116, 1068 (1986). B. A. Zachara, U. Trafikowska, H. Labgdzka, A Sosnowski, and R. Kanarkowski, Biomed. Biochim. Acta 49, S186 (1990). K. R. Millar, W. J. Meads, A. T. A|byt, A. D. Sheppard, and B. G. Scahill, New Zeal. Vet. I. 36, 59 (1988). F. Atroshi, S. Sankari, S. Osterberg, and M. Sandholm, Res. Vet. Sci. 31,267 (1981). A. D. Sheppard, and K. R. Millar, New Zeal. Vet. ]. 29, 77 (1981). M. A. Beilstein, and P. D. Whanger, J. Nutr. 113, 2t38 (1983). G. H. M. Counotte and J. Hartmans, Vet. Quart. 11, 155 (1989).
Biological Trace Element Research
Vol. 32, 1992
The Effect of Barium Selenate Injection on Selenium Concentration and Giutathione Peroxidase Activity in Blood of Pregnant Ewes Fed Selenium-Deficient Diet B. & ZACHARA, *'~ U. TRAF1KOWSKA, 1 M. KAPTUR, 2 C. KIMBER, 2 AND H. LEJMAN 3
~Department of Biochemistry, and 3Diagnostic Laboratory, Medical School, Bydgoszcz; and 2Stock-farm, Sarnokleski, Bydgoszcz, Poland Received January 31, 1991; Accepted March 21, 1991
ABSTRACT Selenium (Se) levels in whole blood and plasma, and glutathione peroxidase (GSH-Px) activities in red cells and plasma were measured in ewes fed an Se-deficient diet injected with barium selenate before breeding season. Highly significant increases in Se levels and GSH-Px activities (P < 0.001) were observed throughout the gestation period and during lactation. In the control group, Se levels and GSH-Px activities decreased significantly (P < 0.001), and were at critically low levels during lambing and lactation periods. Index Entries: Selenium; barium selenate; glutathione peroxidase; whole blood; plasma; erythrocytes; ewes; pregnancy.
INTRODUCTION Diseases of domestic animals caused by selenium (Se) deficiency are w i d e s p r e a d t h r o u g h o u t the world. In order to correct the deficiencies, the supply of Se must be supplemented either orally or parenterally (1,2). *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
4] 5
VoL 32, 1992
416
Zachara et al.
Deposel preparation containing barium selenate has recently been developed (3). It combines practical acceptability and efficacy with a good safety margin. Deposel has a very low solubility, and after injection slowly releases Se, having a prolonged effect. Deposel injection to ruminants had the ability to sustain elevated GSH-Px activity in whole blood for extended periods of time (1-4). The purpose of the present experiment was to study the effect of Deposel injection on Se concentrations and GSH-Px activities in blood components of pregnant and lactating ewes.
IV TERIALS AND METHODS In 1989, 100 Merino ewes (mean weight 59.8 kg), 13-14 mo old, were divided into two groups of 50 each. They were fed with a locally grown Se-deficient diet (0.03-0.04 mg Se/kg D.M.). Three weeks before the breeding season (end of November), one group was injected subcutaneously with Deposel (1 mg Se/kg body wt; Robert Young & Co. Ltd., Glasgow, Scotland) in the neck region. The other group was left untreated. From 15 ewes of each group, blood samples were taken at the commencement of the study, at the time of Deposel injection, and subsequently during gestation on days 19, 53, 98, 140, and 166 after Se injection, as well as 14 and 42 d after lambing (190 and 218 d after injection). Se concentrations in whole blood and plasma were determined by the fluorometric method of Watkinson (5). GSH-Px activities in red cells and plasma were measured by the coupled method of Paglia and Valentine (6). A detailed description of precision, accuracy (IAEA blood standard samples), and statistical analysis has been published earlier (7).
RESULTS AND DISCUSSION In the untreated group, whole blood Se concentration significantly decreased (Table 1) within the first month of pregnancy (P < 0.01). In the further gestation period, both whole blood and plasma Se concentrations decreased linearly, reaching the severely deficient levels during lambing and lactation. The injection of Deposel induced a significant increase in whole blood and plasma Se concentrations in ewes within 19 d (both P < 0.0001). In whole blood, a further, almost linear increase was observed up to the end of pregnancy. The highest level (2.3 x the initial value) was noted within 20-24 wk after Se injection. In the initial period of lactation, whole blood Se slightly decreased, but was still about 2 x higher than the initial value (P < 0.0001). Similar observations were presented by Overnes et al. (8). They noted that the injection of Deposel in ewes induced an adequate increase in blood Se within 8 wk, and a further increase to approx 300 ng/mL was noted within 16-20 wk. This level was maintained throughout the lambing season and throughout the Biological Trace Element Research
Vol. 32, 1992
Effect of Barium Selenate
417
Table 1 Effect of Deposel Injection on Whole Blood and Plasma Selenium Concentration (ng/mL) in Ewes During Pregnancy and Lactation D a ys postlambing
Days p os t i nj e c t i on 0
19
Material, e x p e r i m e n t a l g r o u p , n = 15 W h o l e blood 124 178 • 34 • 27 Plasma 33 116 • 16 _+ 11 Control g r o u p , n = 15 W h o l e blood Plasma
110 _+ 27 24 • 8
n . m '~ n.m. a
53
98
206 37 119 • 20
249 +_ 36 97 + 21
86 _+ 15 22 _+ 3
52 _+ 17 16 • 3
•
140 288 46 109 • 15
•
• •
45 11 20 7
166 282 43 105 + 18 •
31 12 8 _+_ 2 •
14 264 44 78 • 10 •
• •
27 7 10 4
42 230 48 82 • 15 •
• •
24 6 8 4
~n.m.: Not measured.
following summer. In our trial, plasma Se reached a plateau after 19 d of Deposel injection (3.5 x the initial value), and this level was maintained throughout the whole period of pregnancy. Several authors (3,8) reported that the injection Deposel in sheep maintained adequate Se level for at least 6 mo. In each blood sample of the Se-treated group, mean red cell and plasma GSH-Px activities (Table 2) were significantly higher than in the control group (P < 0.02-0.0001). In the experimental group, 19 d after Se injection, the red cell GSH-Px activity was slightly, but not significantly, higher than the initial value. At this time, the enzyme activity in the plasma increased significantly (P < 0.0001). The highest red cell GSH-Px activity (3.9 x the initial value) was noted after 20 wk of Deposel injection. In the later period, the activity decreased to a certain degree, but 6 wk after lambing, it was still 2.4 x higher compared to the initial value (P < 0.0001). Elevated (not less than 2 x ) plasma GSH-Px activities were maintained throughout the whole study period (P < 0.001-0.0001). Many authors have shown that Se administration to ruminants increased the synthesis of GSH-Px in the red cells and in various tissues (2,9-12). This study shows that the red cell GSH-Px activity increased more slowly, when compared to the plasma enzyme of Se-treated ewes. This is because red cell GSH-Px reflects the animal's Se status at the time of erythropoiesis, and therefore, a less immediate influence of Se treatment could be expected on red cells compared to tissues with a higher turnover rate (10). The average life-span of an erythrocyte in sheep is about 4 mo (13). Our results confirm this: The highest whole blood Se concentrations as well as the red cell GSH-Px activities in the Se-treated ewes were noted during the fourth month after Deposel injection. In the studies performed by Cawley and McPhee (3) and by Finch and Turner (4), the Deposel injected into ewes induced also the highest GSH-Px activity in whole blood within 3-5 mo. When the Deposel was used in pregnant Biological Trace Element Research
Vol. 32, 1992
Zachara et aL
418
Table 2 Effect of Deposel Injection on GSH-Px Activity in Red Cells (U/g Hb) and Plasma (U/L) in Ewes During Pregnancy and Lactation Days postlambing
D a y s postinjection Material
0
Experimental g r o u p , n = 15 Red cells
178 61 163 • 50
• Plasma
Control g r o u p , W h o l e blood
n
=
19 209 65 293 • 79
•
53
•
376 85 542
•
98 440 77 300 • 88
•
140
166
687 • •
526 •
288 73
14 529 •
360 •
42
• 296
•
436 67 392
•
15 152 44 163 • 37
n.m."
• Plasma
144 35 140 • 41 •
n.mf
• •
79 31 74 20
• •
63 33 60 20
41 10 47 • 15 •
• •
49 19 48 23
• •
36 11 44 17
"n.m.: Not measured.
ewes (3), not only were the ewes protected against Se deficiency, but their lambs were also well protected. In the control group, red cell and plasma GSH-Px activities decreased almost linearly until lambing season and were maintained thereafter at critically tow levels (more than 3 x lower than the initial values; P < 0.0001). It has been shown (14) that, in sheep peripheral blood, GSHPx activity in erythrocytes is about 99 x that in plasma. Beilstein and Whanger (15) have also demonstrated that, in sheep erythrocytes, 79% of Se is incorporated into GSH-Px, whereas in plasma, only 25% of the element was found in this enzyme. Therefore, erythrocyte GSH-Px activity correlates well with whole blood Se concentration in sheep and in some other animals. At the commencement of the trial, the correlation coefficient (r) between whole blood Se concentration and red cell GSH-Px activity for all ewes (n = 30) was 0.464 (P < 0.01), whereas at all other times during the study taken together, high correlations were obtained for experimental (r = 0.829; P < 0.0001) as well as for the control group (r = 0.844; P < 0.0001). The correlations we found in this trial correspond well with other studies (2,11,12,16). The close relationship and simplicity of determination should enable red cell GSH-Px activity to be used to evaluate blood Se concentration in sheep. The regression equation derived for the control group in this study is: GSH-Px (U/g Hb) -- 1.32 x (Se) + 9.82. The two principal conclusions from this study are: (1) Deposel injection seems to be suitable and safe for protecting ewes against Se deficiency during pregnancy and lactation, and (2) it also has the ability to sustain elevated GSH-Px activities in the blood of ewes.
ACKNOWLEDGMENTS We are grateful to Robert Young & Co. Ltd., Glasgow for donating the Deposel preparation used in this study. The author (B. A. Z.) kindly Biological Trace Element Research
Vol. 32, 1992
Effect of Barium Selenate
419
appreciates the assistance and financial support of Labcatal, Montrouge, France for the participation in the Chromium-Trace Elements Congress. This research was supported, in part, by the Polish Central Research Programme, CPBR 10.17/IV.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16.
A. MacPherson, and J. S. Chalmers, Vet. Rec. 115, 544 (1984). C. G. Mackintosh, J. Gill, and K. Turner, New Zeal. Vet. ]. 37, 143 (1989). G. D. Cawley, and I. McPhee, 114, 565 (1984). J. M. Finch, and R. J. Turner, 119, 430 (1986). J. H. Watkinson, Anal. Chem. 38, 92 (1966). D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med. 70, 158 (1967). W. Wasowicz, and B. A. Zachara, J. Clin. Chem. Clin. Biochem. 25, 409 (1987). G. Overnes, K. Moksnes, and A. Froslie Acta Vet. Scand. 26, 164 (1985). L. D. Koller, P. J. South, J. H. Exon, and G. A. Whitbeck, Cornell Vet. 73, 323 (1983). A. M. Smith, and M. F. Picciano, J, Nutr. 116, 1068 (1986). B. A. Zachara, U. Trafikowska, H. Labgdzka, A Sosnowski, and R. Kanarkowski, Biomed. Biochim. Acta 49, S186 (1990). K. R. Millar, W. J. Meads, A. T. A|byt, A. D. Sheppard, and B. G. Scahill, New Zeal. Vet. I. 36, 59 (1988). F. Atroshi, S. Sankari, S. Osterberg, and M. Sandholm, Res. Vet. Sci. 31,267 (1981). A. D. Sheppard, and K. R. Millar, New Zeal. Vet. ]. 29, 77 (1981). M. A. Beilstein, and P. D. Whanger, J. Nutr. 113, 2t38 (1983). G. H. M. Counotte and J. Hartmans, Vet. Quart. 11, 155 (1989).
Biological Trace Element Research
Vol. 32, 1992
