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The Journal of Nutrition Vol. 128 No. 1 January 1998,
pp. 130-135
Department of Animal Science and * Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The current dietary allowance for selenium (Se) for pigs does not consider Se requirements for expression of several newly discovered Se-dependent enzymes and has raised environmental concerns. Our objective was to determine dietary Se requirements of young pigs for the full expression of cellular (GPX1), plasma (GPX3) and phospholipid hydroperoxide (GPX4) glutathione peroxidases. In Experiment 1, 18 weanling male pigs (4 wk old) were fed a corn-soybean meal basal diet (BD, 0.03 mg Se/kg) with the addition of 0, 0.1 or 0.3 mg Se/kg (Na2SeO3). In Experiment 2, 24 weanling barrows (6 wk old) were fed a similar BD with the addition of 0, 0.2, 0.3 or 0.5 mg Se/kg. Both experiments lasted for 5 wk. Pigs fed the BD had lower (P < 0.05) tissue GPX1 and GPX4 activities, plasma GPX activity, and(or) plasma Se concentrations than those fed the Se-supplemented diets. In Experiment 1, GPX1 and GPX4 activities in liver, heart and lung were lower (P < 0.05) in pigs fed 0.1 mg Se/kg than in those fed 0.3 mg Se/kg, although no such differences existed in thyroid or pituitary. Pigs fed 0.1 mg Se/kg also had lower (P < 0.05) plasma GPX3 activity at wk 5 and higher (P < 0.05) hepatic glutathione S-transferase activity than pigs fed 0.3 mg Se/kg. In Experiment 2, GPX1 and GPX4 activities in liver and heart, GPX1 and GPX4 mRNA levels in liver and GPX3 activity in plasma exhibited plateaus at 0.2 mg Se/kg. Pigs fed the BD had greater concentrations of F2-isoprostanes (a marker of in vivo lipid peroxidation) than those fed 0.2 mg Se/kg in plasma (P < 0.03) and liver (P < 0.04). We conclude that supplemental Se at 0.2 mg Se/kg of diet is required to support the full expression of three Se-dependent glutathione peroxidases in young pigs.
KEY WORDS: glutathione peroxidase · pigs · dietary selenium · lipid peroxidation · gene expression
Nutritional essentiality of dietary selenium (Se) for mammals has been well established (Schwarz and Foltz 1957 Animals, diets, and treatments.
In Experiment 1, 18 intact male Yorkshire × Hampshire × Duroc weanling pigs (4 wk old) were divided into three groups such that weight and litter were similar among treatment groups. Pigs in Group 1 were fed a Se-deficient, corn-soybean meal basal diet (BD) as control; pigs in Groups 2 and 3 were fed the BD supplemented with 0.1 and 0.3 mg Se/kg as Na2SeO3 (Sigma, St. Louis, MO), respectively. In Experiment 2, 24 castrated Yorkshire × Hampshire × Duroc weanling pigs (6 wk old) were divided into four groups and fed a BD, similar to that of Experiment 1, supplemented with 0, 0.2, 0.3 or 0.5 mg Se/kg of diet. Both experiments lasted for 5 wk. All pigs were fed the BD for 10 d to adjust the Se status before the actual experimental period. The composition and the analyzed Se concentrations of the BD for both experiments are presented in Table 1. The BD met the requirements for all nutrients (NRC 1988), except for Se and vitamin E. Considering the possibility that dietary vitamin E might be low or destroyed during storage in swine production, we did not add vitamin E to the diets so that maximal dietary Se requirements for pigs could be determined. To prepare the Se-deficient BD with the lowest possible Se concentrations, we used slightly different sources of soybean meal in the two experiments as a result of the availability of the products and their Se concentrations. The experiments were approved by the Institutional Animal Care and Use Committee at Cornell University and conducted at the Swine Research Farm of the university. Pigs were housed individually, given free access to feed and water and checked twice daily. The experimental house was controlled at 22-25°C with a 12-h light:dark cycle. Weekly changes in individual pig body weights and daily pen feed consumption were recorded.
Sample collections.
Blood samples were taken initially and weekly in Experiment 1 or at the end of Experiment 2 from all pigs via the anterior vena cava to prepare plasma samples. Four pigs from each treatment group were slaughtered at the end of both experiments. Samples of liver, heart, muscle, lung, thyroid and pituitary were collected, frozen in liquid nitrogen, and stored at Biochemical analyses.
Activities of GPX1 and GPX4 in tissues and of GPX3 in plasma were measured by the coupled assay of NADPH oxidation as previously described (Lei et al. 1995 Statistics analysis.
Effects of dietary Se on the concentrations of F2-isoprostanes in liver or plasma were analyzed by t test; on the other measures, effects were analyzed by ANOVA as a completely randomized design with or without time-repeated measurements. The Bonferroni t test was used for multiple treatment mean comparisons. Pooled SEM and the corresponding df for errors were listed. Significance level was set as P < 0.05 unless indicated otherwise. Data were processed using SAS (1988).
Experiment 1.
The analyzed Se concentrations of the diets supplemented with 0 (BD), 0.1, and 0.3 mg Se/kg were 0.03, 0.12 and 0.34 mg Se/kg, respectively. Plasma GPX3 activity in pigs fed the BD was lower (P < 0.05) than in those fed the Se-supplemented diets at wk 2 and thereafter (Fig. 1). At the end of the experiment, plasma GPX3 activities in the two Se-supplemented groups were also different (P < 0.05). In all of the tissues assayed, GPX1 (Fig. 2) and GPX4 (Fig. 3) activities in pigs fed the BD were lower (P < 0.001) than in those fed the Se-supplemented diets. In liver, heart and lung, activities of both enzymes were each lower in pigs fed 0.1 mg Se/kg than in those fed 0.3 mg Se/kg. In muscle, only GPX4 activity was significantly different between the two Se-supplemented groups. In thyroid and pituitary, there was no further increase in GPX1 or GPX4 activity above 0.1 mg Se/kg. Heart and thyroid had relatively high GPX4 activities compared with their GPX1 activities among the assayed tissues.
Experiment 2.
The analyzed Se concentrations of the diets supplemented with 0 (BD), 0.2, 0.3 and 0.5 mg Se/kg were 0.042, 0.20, 0.29 and 0.45 mg/kg, respectively. Liver and heart GPX1 (Fig. 4) and GPX4 (Fig. 5) activities in pigs fed the BD were lower (P < 0.001) than in those fed the Se-supplemented diets, but both exhibited a plateau at 0.2 mg Se/kg. Similarly, liver GPX1 and GPX4 mRNA levels (Fig. 6) in pigs fed the BD were lower (P < 0.05) than in those fed the Se-supplemented diets and reached plateau at 0.2 mg Se/kg. Plasma GPX3 activity responded to dietary Se levels in the same way as GPX1 and GPX4 activities in liver and heart (Table 3). Plasma Se concentration in pigs fed the BD was lower (P < 0.05) than in those fed the Se-supplemented diets; in those supplemented groups, there was a difference (P < 0.05) between supplementation with 0.2 and 0.5 mg Se/kg. Overall growth performance was not affected by dietary Se concentrations. Pigs fed the BD had greater concentrations of F2-isoprostanes than those fed 0.2 mg Se/kg in plasma (1.72 vs. 1.04 nmol/L, SEM = 0.26, P < 0.03) and liver (17.2 vs. 4.3 pmol/g wet tissue, SEM = 3.6, P < 0.04).
Our data clearly indicate that supplemental Se is necessary in the Se-deficient, corn-soybean meal diets for young pigs. Pigs fed the BD had significantly lower Se concentrations and GPX3 activities in plasma, GPX1 and GPX4 mRNA levels in liver, and GPX1 and GPX4 activities in various tissues than those fed the Se-supplemented diets. In addition, pigs fed the BD also had higher hepatic GST activity than those fed the Se-supplemented diets. As a Se-independent glutathione enzyme, GST activity is induced by Se deficiency (Christensen et al. 1994 The authors are grateful to A. Attygalle for his technical help in determining F2-isoprostanes and Hoffmann-La Roche, Paramus, NJ, for providing vitamin products used in this project.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). It has been widely accepted and a common practice to supplement 0.3 mg Se/kg of diet for pigs (Meyer et al. 1981). However, recent heightened environmental concerns of potential Se pollution from animal production have questioned whether the current Se supplementation is in excess of dietary needs. In 1993, the FDA ordered that the Se supplementation be reduced to the initially approved level of 0.1 mg Se/kg. Although an amendment to the USDA Reorganization Bill was included to conditionally prohibit the FDA from implementing the cutback of Se supplementation, the potential challenge remains unless the FDA is provided with solid physiologic justification for the current level of Se supplementation in swine diets. On the other hand, the current dietary Se allowance for pigs was based on tissue Se concentrations, Se balance and the activity response of cellular glutathione peroxidase (EC 1.11.1.9, GPX14), the only known Se-dependent enzyme at that time. In fact, three other Se-dependent glutathione peroxidases: gastrointestinal (GPX2, Chu et al. 1993), extracellular or plasma (GPX3, Takahashi and Cohen 1986), phospholipid hydroperoxide (GPX4, Ursini et al. 1985) and several other selenoproteins (Sunde 1997) have been identified recently. Although effects of dietary Se on the activity of plasma GPX3, then considered to be the same enzyme as GPX1, were assessed in previous Se requirement studies, dietary Se requirements for the expression of GPX4 and other newly discovered selenoenzymes or proteins have not been studied. Relative activity distribution and age-related changes are different between GPX1 and GPX4 in pigs (Lei et al. 1997). Dietary Se requirements for the full expression of GPX4 is lower than that for GPX1 in rats (Lei et al. 1995). Recently, F2-isoprostanes have been used as a marker of in vivo lipid peroxidation (Morrow and Roberts 1994). There are no data available concerning the effect of the expression of selenoperoxidases on F2-isoprostanes in tissues of pigs deficient and adequate in Se. Thus, the objectives of this research were to determine the following: 1) the requirement of dietary Se for young pigs to express the full activities and mRNA levels of GPX1 and GPX4 in various tissues and the full activity of GPX3 in plasma; 2) the regulation of dietary Se on the expression of these three selenoperoxidases in various tissues of the pigs; 3) the effects of dietary Se supplementation on the concentration of F2-isoprostanes in plasma and liver, the Se concentration in plasma and the activity of glutathione S-transferase (EC 2.5.2.18, GST), a Se-independent glutathione peroxidase, in liver and heart of the pigs.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Composition of basal diets
80°C until analysis.
). The substrate for the activity assay of GPX1 and GPX3 was hydrogen peroxide, and for GPX4 was phosphatidylcholine hydroperoxide synthesized as outlined by Lei et al. (1995). Liver and heart GST activities were assayed using 1-chloro-2,4-dinitrobenzene as the substrate (Habig et al. 1974). Protein concentrations in tissue and plasma samples were determined by the method of Lowry et al. (1951). The enzyme unit of GPX1, GPX3, or GPX4 was defined as one nanomole of reduced glutathione oxidized and of GST as nanomoles of S-2,4-dinitrophenylglutathione formed per minute.
). The GPX1 probe was a 0.7-kb EcoRI fragment of murine GPX1 cDNA, the GPX4 probe was a 0.65-kb EcoRI/XhoI fragment of rat GPX4 and the 18S rRNA probe was an 1.4-kb BamH1 fragment of human 18S rRNA (all three were generously provided by R. A. Sunde, University of Missouri). The probes were random-primed labeled with 32P (dCTP, Du Pont, Boston, MA) by using a DNA labeling kit (Pharmacia Biotech, Piscataway, NJ) followed by G-50 column purification (Pharmacia). Levels of the GPX1 and GPX4 mRNA were determined in separate but identical blots (n = 2) by using a FuJIX BAS100 Bio-imaging Analyzer (Fuji Photo Film, Kanagawa, Japan). The relative levels of mRNA were normalized by the levels of 18S rRNA detected on the same blots after removing the GPX1 or GPX4 probe.
). Plasma 
-tocopherol concentration was determined by reverse-phase HPLC using a mobile phase of acetonitrile/tetrahydrofuran/methanol/1% ammonium acetate (68:22:7:3) and a C-18 stationary phase (McShane et al. 1991). Concentrations of F2-isoprostanes in plasma and liver of pigs fed the BD and 0.2 mg Se/kg in Experiment 2 (n = 4) were determined (Awad et al. 1994) by using gas chromatography/mass spectrometry (model HP 5890 A with a HP 5970 series mass selective ion monitoring, Hewlett-Packard, Palo Alto, CA).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (16K):
[in a new window]
Fig 1.
Effects of dietary Se concentrations on extracellular glutathione peroxidase (GPX3) activity in plasma of pigs in Experiment 1. Values are means (n = 6). At a time point, means without common letters were different (P < 0.05). The pooled SEM (df = 15) was 1.39. GSH, reduced glutathione.
View larger version (17K):
[in a new window]
Fig 2.
Effects of dietary Se concentrations on cellular glutathione peroxidase (GPX1) activity in various tissues of pigs in Experiment 1. Values are means (n = 4). Bars not sharing a common letter were different (P < 0.05) within the same tissue. The pooled SEM (df = 9) were as follows: liver, 5.0; heart, 1.49; lung, 2.56; muscle, 1.57; thyroid, 1.43; pituitary, 0.66. GSH, reduced glutathione.
View larger version (16K):
[in a new window]
Fig 3.
Effects of dietary Se concentrations on phospholipid hydroperoxide glutathione peroxidase (GPX4) activity in various tissues of pigs in Experiment 1. Values are means (n = 4). Bars not sharing a common letter were different (P < 0.05) within the same tissue. The pooled SEM (df = 9) were as follows: liver, 0.21; heart, 1.16; lung, 0.20; muscle, 0.29; thyroid, 1.16; pituitary, 0.31. GSH, reduced glutathione.
-tocopherol concentrations, were not affected by dietary Se concentrations (Table 2). At the end of the trial, plasma Se concentrations were different (P < 0.05) between any two dietary groups. Pigs fed 0.1 mg Se/kg had a higher and lower (P < 0.05) value than that of pigs fed the BD and 0.3 mg Se/kg, respectively. The same was also true of hepatic GST activity, but with an opposite trend. There was no difference in heart GST activity between pigs fed 0.1 and 0.3 mg Se/kg.
View this table:
Table 2.
Effects of dietary supplemental selenium concentrations on growth performance, selenium and -tocopherol concentrations in plasma and glutathione S-transferase activity in liver and heart of pigs in Experiment 11
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[in a new window]
Fig 4.
Effects of dietary Se concentrations on cellular glutathione peroxidase (GPX1) activity in liver and heart of pigs in Experiment 2. Values are means (n = 4). For a tissue, means not sharing common letters (liver, upper case; heart, lower case) were different (P < 0.05). The pooled SEM (df = 13) were as follows: liver, 16.4; heart, 13.0. GSH, reduced glutathione.
View larger version (13K):
[in a new window]
Fig 5.
Effects of dietary Se concentrations on phospholipid hydroperoxide glutathione peroxidase (GPX4) activity in liver and heart of pigs in Experiment 2. Values are means (n = 4). For a tissue, means not sharing common letters (liver, upper case; heart, lower case) were different (P < 0.05). The pooled SEM (df = 13) were as follows: liver, 0.69; heart, 1.95. GSH, reduced glutathione.
View larger version (13K):
[in a new window]
Fig 6.
Effects of dietary Se concentrations on relative mRNA levels of cellular glutathione peroxidase (GPX1) and phospholipid hydroperoxide glutathione peroxidase (GPX4) in liver of pigs in Experiment 2. Values are means (n = 2). Within the same mRNA, means not sharing common letters (GPX1, upper case; GPX4, lower case) were different (P < 0.05). The pooled SEM (df = 12) were as follows: GPX1, 9.8; GPX4, 9.5.
View this table:
Table 3.
Effects of dietary supplemental selenium concentrations on growth performance, plasma selenium concentration and plasma glutathione peroxidase activity of pigs in Experiments 21
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Hill et al. 1987). Thus, our results are in line with the findings by earlier researchers (Chavez 1979, Groce et al. 1971, Mahan and Parrett 1996, Meyer et al. 1981, Sharp et al. 1972, Van Vleet et al. 1973) and provide new biochemical and molecular evidence to support the Se supplementation in swine diets under the current production condition.
suggested that 0.3 mg Se/kg was required by weanling pigs based on breakpoint regression analysis of liver GPX1 and plasma GPX3 activities. As in our Experiment 1, there was no 0.2 mg Se/kg level between 0.1 and 0.3 mg Se/kg levels in their study. Thus, they found that 0.1 mg Se/kg was insufficient and 0.3 mg Se/kg was required for young pigs to express the full activity of GPX1 in liver. Another fact is that we did not add any vitamin E to the diets, and thus plasma -tocopherol concentrations of pigs were very low irrespective of dietary Se levels. Nevertheless, pigs grew normally (Meyer et al. 1981) and showed no sign of liver necrosis or cardiomyopathy at the end of the experiments (data not shown). Therefore, 0.2 mg Se/kg should be sufficient to prevent any clinical signs of Se deficiency in pigs under practical conditions in which vitamin E is often added to the diets (Ewan et al. 1969).
and 1997b). Therefore it has been questioned why maximal GPX enzyme activities should be used to determine dietary Se needs for animals and humans (Levander and Whanger 1996). Recently, F2-isoprostanes, resulting from free radical-catalyzed peroxidation of arachidonic acid, have proven to be a sensitive and reliable tool in assessing lipid peroxidation in vivo (Morrow and Roberts 1994). Our results are consistent with those of Awad et al. (1994) in Se- and vitamin E-deficient rats, although our detection was conducted under electron-impact conditions rather than the negative ion chemical ionization used in that investigation.
) and mice (Weitzel et al. 1990). Similarly, Se-deficient pigs had greater residual activities of GPX4 than GPX1, relative to the Se-adequate levels, in various tissues. Apparently, this was also true of the GPX1 and GPX4 mRNA levels in liver, although the mRNA analysis was conducted in only two pigs from each dietary group. Nevertheless, the full expression of GPX4 activities in various tissues of young pigs occurred at the same level of dietary Se (0.2 mg Se/kg) as that of GPX1. This is not consistent with the fact that less dietary Se is needed for the maximum activity expression of GPX4 than GPX1 in rats (Lei et al. 1995). Similarly, the full expression of GPX1 and GPX4 mRNA levels in the liver of these pigs also occurred at 0.2 mg Se/kg of diet. It is unclear from the results of this study whether or not pigs, similar to rats (Lei et al. 1995, Weiss et al. 1996), require less dietary Se for full expression of mRNA levels than for activities of GPX1 and GPX4. In addition, caution should be taken in consideration of the mRNA results because we used a murine GPX1 and a rat GPX4 cDNA probe to detect liver GPX1 and GPX4 mRNA levels, respectively. Sunde (1994) reported that the pig and rat GPX4 cDNA sequences were 80% identical, but there is no information, to our knowledge, on the direct sequence comparison between the pig and the mouse GPX1, although the DNA and protein sequences of GPX1 are well conserved between mice and other species (Sunde 1997).
ACKNOWLEDGMENTS
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FOOTNOTES |
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Manuscript received 2 June 1997. Initial reviews completed 31 July 1997. Revision accepted 15 September 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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structures, regulation, and functions. In: Selenium in Biology and Human Health (Burk, R. F., ed.), pp. 45-77. Springer-Verlag, New York, NY.This article has been cited by other articles:
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  J.-C. Zhou, H. Zhao, J.-G. Li, X.-J. Xia, K.-N. Wang, Y.-J. Zhang, Y. Liu, Y. Zhao, and X. G. Lei Selenoprotein Gene Expression in Thyroid and Pituitary of Young Pigs Is Not Affected by Dietary Selenium Deficiency or Excess J. Nutr., June 1, 2009; 139(6): 1061 - 1066. [Abstract] [Full Text] [PDF]
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 G. E. Lum, J. E. Rowntree, K. R. Bondioli, L. L. Southern, and C. C. Williams The influence of dietary selenium on common indicators of selenium status and liver glutathione peroxidase-1 messenger ribonucleic acid J Anim Sci, May 1, 2009; 87(5): 1739 - 1746. [Abstract] [Full Text] [PDF]
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 S. Yalcin, A Bilgili, I Onbasilar, G Eraslan, and M Ozdemir Synergistic action of sodium selenite and N-acetylcysteine in acetaminophen-induced liver damage Human and Experimental Toxicology, May 1, 2008; 27(5): 425 - 429. [Abstract] [PDF]
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C. E. Hostetler, J. Michal, M. Robison, T. L. Ott, and R. L. Kincaid Effect of selenium intake and fetal age on mRNA levels of two selenoproteins in porcine fetal and maternal liver J Anim Sci, September 1, 2006; 84(9): 2382 - 2390. [Abstract] [Full Text] [PDF]
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D. T. Shaw, D. W. Rozeboom, G. M. Hill, A. M. Booren, and J. E. Link Impact of vitamin and mineral supplement withdrawal and wheat middling inclusion on finishing pig growth performance, fecal mineral concentration, carcass characteristics, and the nutrient content and oxidative stability of pork J Anim Sci, November 1, 2002; 80(11): 2920 - 2930. [Abstract] [Full Text] [PDF]
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