~)Copyright 1986 by Tile t iumana Press Inc. All rights of any nature whatsoever reserved. 0163-4984186/1100qX)51 $02.80
Effects of Dietary Selenite, Copper, and Zinc on Tissue Trace Mineral Levels in Chicks G. F.
COMBS,JR.,'.* Q. Su, 2 C. H. LIu,2 AND S. B. COMBS'
'Department of Poultry and Avian Sciences, and Division of Nutritional Sciences, 306 Rice Hall, Come# University, Ithaca, NY 14853; and qnstitute of Animal Science, Chinese Academy of Agricultural Sciences
Received June 3, 19862~ccepted July 22, 1986 ABSTRACT Studies were conducted to determine whether nutritional selenium (Se) status affects the nutritional status of the chick with respect to other trace elements, particularly copper (Cu) and Zinc (Zn). Severe Se deficiency was produced in chicks by the use of diets that contained exceedingly low contents (less than 0.010 ppm) of Se, but contained adequate amounts of all other known essential nutrients. This diet was based upon corn and soybean meal produced in areas of China with endemic Se deficiency of geobotanical origin. A level of at least 0.10 ppm Se was found to be required to maintain normal Se status of chicks fed this diet, and Se deficiency resulted in decreased levels of Cu, Zn, and molybdenum in the pancreas (liver and plasma levels were not affected). High dietary supplementation of Zn nor Cu did not affect the short-term utilization of Se, as indicated by the 18-h responses of Se-dependent glutathione peroxidase in plasma and liver. Index Entries: Selenium, effects of on trace mineral levels; glutathione peroxidase; zinc, effects of on trace mineral levels; copper, effects of on trace mineral levels; iron; chick.
*Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
5]
Vol. I 1, 1986
52
Combs et aL
INTRODUCTION Interactions of selenium (Se) and other essential dietary minerals have been demonstrated; however, the metabolic bases for these effects, as well as their implications in situations of marginal Se status, are unclear. Chmielnicka et al. (1) found that oral administration of Na2SeO3 to rats nearly doubled the tissue retention of injected zinc (Zn). The administration of Se with Zn enhanced hepatic levels of Zn-metallothionein and increased the deposition of Zn in soluble high-molecular-weight proteins in kidney, also indicating improved utilization of Zn by Se. Studies by Naganuma et al. (2), however, indicated that parenterally administered Zn can alter the tissue disposition of Se, reducing the uptake of Se from a parenteral dose of Na2SeO3 by heart and liver. Near-toxic levels of dietary Se (e.g., 4 ppm) were shown to reduce liver Zn levels and to increase kidney Zn levels, suggesting that the utilization of Zn was reduced under such conditions. Amer et al. (3) found that oral Se therapy (Na2SeO3) of marginal-Se calves resulted in a rapid drop in plasma copper (Cu) and ceruloplasmin levels that was not prevented by feeding higher levels (up to 200 ppm) of Cu. Similar evidence of competitive effects of Se and Cu was reported by Thomson and Lawson (4), who observed that Se treatment of lambs born to ewes pastured on Se- and Cu-deficient fields reduced the efficacy of oral Cu supplements for the improvement of tissue Cu levels. Decreases in tissue activities of Se-dependent glutathione peroxidase (SeGSHpx) caused by high levels of Cu may explain the enhanced lipid peroxidation apparent in vivo (as indicated by exhalation of volatile alkanes) in the Cuintoxicated rat (5). It has been well established that when either Se or Cu is in excess, the opposite mineral can alleviate signs of toxicity. These studies, which have been conducted with chicks (6,7) ponies (8), and rats (9), show that Cu levels increase in Se intoxication, and that the lethality of dietary Se is reduced by high dietary levels of Cu. This suggests that when each is present in excess, the two elements become less metabolically available, perhaps because of the formation of Se-Cu adducts or by competing in some way (presumably via sulfhydryl binding) for binding to carriers and/or storage proteins. Jenkinson et al. (10) found that Cu deficiency in the rat reduced both the hepatic activity of SeGSHpx and the response of that enzyme activity to dietary supplements of Na2SeO3. Copper deficiency also reduced the tissue disposition of Se in such a way as to mimic nutritional Se deficiency. Therefore, both deficiencies and excesses of Cu have been reported to affect Se status. In view of these known interactions of Se with Zn and Cu, it is of interest to know whether the utilizations of Cu and/or Zn are altered under conditions of nutritional Se deficiency, and whether excesses of Cu or Zn can affect the utilization of dietary Se. in addition to being of fundamental interest in understanding the metabolic bases for the clinical manBiological Trace Element Research
Vol. 11, 1986
Selenium Mineral Interactions in Chicks
53
ifestations of Se-deficiency syndromes in animals, knowledge of these potential interactions is of practical value in dealing with likely mineral imblances that may occur in animal production in several parts of the developing world.
MATERIALS AND METHODS
Animals and Diets Male, Single Comb White Leghorn chicks were used in all experiments. The Se experiments were conducted in Beijing, PRC, and the chicks were obtained from a local hatchery. Chicks were installed at 1 d of age in stainless-steel, wire-floored, cages in a room maintained at 33-35~ with a 24-h day, as described previously (ll). In each experiment, four replicate groups of 10 or 14 chicks each were assigned randomly to each dietary treatment. The corn-soy-based diet used previously to produce uncomplicated Se deficiency in the chick was used in these studies; its composition and special origin were described by Combs et al. (11). It was found to contain 0.010 ppm Se; it was supplemented with 100 IU all-rac-alpha-tocopheryl acetate/kg. The basal diet also contained (by actual analysis) 4.75 ppm Cu, 21.1 ppm Zn, 69.6 ppm Fe, 13.5 ppm Mn, 1.57 ppm Mo, and 0.07 ppm Cd. Diets were provided ad libitum.
Preparation of Samples Blood was obtained by anterior cardiac puncture, using a heparinized syringe; plasma was prepared by centrifugation at 1000g for 10 rain at room temperature. Other tissues were excised immediately after chicks were killed by cervical dislocation. Tissues were minced and homogenized in 4 vol of 50 mM Tris, 154 mM KCI, pH 7.4, buffer using a motor-driven Teflon-glass Potter-Elvejhem type tissue grinder operated at 4~ Soybean trypsin inhibitor (Sigma Chemical Co., St. Louis, MO) was added (1 mg/mL) to the homogenizing medium for pancreas. Plasma and homogenates were analyzed immediately for enzyme activities, iron (Fe), and/or Se, but were held at -20~ for analysis of othe minerals. JVlineral Analyses
Selenium was determined by the fluorometric procedure of Olson et al. (12) using diaminonapthalene. In Expts. I and 2, Zn, Cu, Mn, and Mo were determined by inductively coupled argon plasma atomic emission spectrometry (using a Jarrell-Ash ICP 9000 instrument, Fisher Scientific Instruments, Springfield, NJ). Iron was determined by the chemical method of Weiser et al. (13), i.e., plasma total iron-binding capacity was determined by measuring the acid-precipitable iron after the addition of an excess of ferric ion to the sample. Biological Trace Element Research
VoL 11, 1986
54
Combs et al.
Measurement of Enzyme Activities Selenium-dependent glutathione peroxidase was measured by the glutathione reductase-coupled assay of Paglia and Valentine (14), as modified by Lawrence and Burk (15), using 0.25 mM H202 as substrate. Superoxide dismutase was determined by the inhibition of the spontaneous oxidation of epinephrine at pH 10.2, according to the method of Misra and Fridovich (16). Cytochrome c oxidase was determined by the method of Prohaska and Wells (17). Amylase was determined by the method of Bernfeld (18). Protein was determined by the method of Lowry et al. (19).
Histological Methods For histological studies, the duodenal loop and pancreas were exposed via an abdominal incision made immediately after killing the chick by cervical dislocation. A section of the pancreas, ca. I cm in length, with the attached duodenal loop, was quickly excised and fixed in 10% neutral-buffered formalin. The remaining sections of the pancreas were then removed and prepared for biochemical studies as described above. Histological sections of fixed pancreas specimens were mounted and stained with hematoxylin and eosin. They were examined by light microscopy; histological condition was scored according to the system described by Cantor et al. (20), in which a score of 1.0 represents completely normal pancreatic histology and a score of 5.0 represents complete acinar degeneration and advanced periacinar fibrosis.
Expedments Experiment 1 was conducted to determine the dietary Se requirement of the chick fed the corn-soy-based diet and whether changes in nutritional Se status affect the chick's status with respect to other trace elements. This was accomplished by feeding chicks for 40 d the basal diet supplemented with Na2SeO3 to achieve 0.010, 0.025, 0.050, 0.100, or 0.200 ppm total Se. Chicks were killed at 0, 5, 12, 20, 27, 34, and 40 d for determinations of SeGSHpx in plasma, liver, and pancreas and for determination of amylase in plasma. Experiments 2 and 3 were conducted to determine whether the utilization of dietary Se by chicks fed the practical diet was affected by high dietary supplements of either Cu or Zn. In Expt. 2, chicks were fed the practical diet supplemented with Se (as Na2SeO3) to provide a nutritionally marginal level of Se (0.025 ppm total Se) and with graded levels of either Cu (5, 100, 250, 500, or 1000 ppm total Cu achieved by adding CuSO4"SH20) or Zn (40, 250, 500, 1000, or 2500 ppm total Zn achieved by adding ZnO). In Expt. 3, chicks were fed the basal diet or the same diet supplemented with a high level (1000 ppm) of either Cu (as CuSO45H20) or Zn (as ZnO) for 20 d. At that time, five chicks per treatment Biological Trace Element Research
Vol. I I, 1986
Selenium Mineral Interactions in Chicks
55
were killed for determination of liver minerals. The remaining chicks in each treatment group were assigned randomly to either of two treatments: 2 p,g Se administered in 0.5 mL of an aqueous solution of Na2SeO3 by crop intubation; or a control (no Se) treatment consisting of 0.5 mL distilled water administered in the same way. Chicks were killed after 18 h; three composite samples from three chicks each were prepared for measurements of SeGSHpx in plasma and liver and five composite samples from three chicks each were prepared for measurements of Se in liver.
Statistical Analyses All data were evaluated statistically by Analysis of Variance. When significant (p < 0.05) effects were detected, post hoc analyses, using a Duncan's multiple range test, were conducted to rank treatment means. These tests were conducted using Systat 2.1 (a statistical analytical system configured for microcomputers, Systal, Inc., Evanston IL).
RESULTS Supplementation of the Se-deficient practical diet with Na2SeO3 did not consistently affect chick growth (Fig. 1), but produced increases in the primary parameters of nutritional Se status, i.e., the activities of SeGSHpx in plasma, liver, and pancreas (Table 1). These activities were greatest among chicks fed the highest level (0.20 ppm) of Se, in which there were two orders of magnitude greater than the enzyme activities of chicks fed the unsupplemented basal diet. At hatching, plasma activities of SeGSHpx were found to be 8.76 _+ 0.28 nmol NADPH/min/mg protein (X + SEM, n = 5). The 0.10-ppm dietary level of Se prevented any decline in SeGSHpx activity in plasma or liver (Figs. 2 and 3). At 40 d of age, signs of pancreatic atrophy were observed among chicks fed less than 0.10 ppm Se; this is indicated by the significantly elevated pancreas histological scores of those treatment groups (Table 1). Elevated pancreas scores were associated with elevated activities of amylase in plasma, also indicative of pancreatic dysfunction. The activities of superoxide dismutase in liver and pancreas were not significantly affected by dietary Se level (Table 2). In contrast, hepatic cytochrome c oxidase activities were significantly reduced in chicks fed less than 0.10 ppm Se; however, no significant effects were noted on this activity in pancreas. Tissue Se levels were significantly and directly related to the dietary concentration of the mineral; but this was not true for Cu, Zn, Fe, and Mo in plasma, or for these minerals as well as Mn in liver (Table 3). However, chicks fed the lower levels of Se showed significant reductions of Cu (by 12%), Zn (by 10%) and Mo (by ca. 24%) in pancreas. Alterations in Fe metabolism were also evident in Se-deficient chicks, which showed slight elevations in Biological Trace Element Research
VoL I 1, 1986
56
Combs et aL I I Dietary Se key 500-1I 9 .010 ppm i 9 .025 ppm II D .050 ppm I * .100 ppm 4001 9 .2DO ppm i
~* ~ / p l l
i t
/"
3OO
10
;
1;
2"0
1o
4"o
Days of age
Fig. 1. Growth of vitamin E-adequate chicks fed graded levels of Se; the effect of supplemental Se was not significant (p > 0.05) (n = 4, based on peraverage body weight at each day indicated. As chicks were removed for biochemical analyses, the numbers of chicks per pen decreased by 2 from the original number, 14, at 0 and 6 d). plasma Fe and concomitant depressions in plasma total-Fe-binding capacity (Table 4). In Expts. 2 and 3, supplementation of the marginaI-Se practical diet (containing 0.025 p p m Se) with high levels of Cu or Zn did not affect hepatic Se levels (Table 5). However, hepatic levels of Mn and Mo tended to be reduced at the higher levels of supplementation of each nutrient, which, in this experiment, did not affect either growth rate or feed intake. The short-term (18-h) utilization of Se for SeGSHpx, as indicated by the Se-induced increases in the activities of this enzyme in liver and plasma, was not significantly affected by high levels (1000 ppm) of either Cu or Zn (Table 6).
DISCUSSION The present study undertook to determine whether the utilization of supplemental Se (as Na2SeO3) by chicks fed a low-Se practical diet is affected by excesses of Cu and Zn, and whether Se-deficiency produced by this diet alters the utilization of other trace elements. In order to accomplish this, it was also necessary to determine the dietary Se requirement of vitamin E-adequate chick for Se for the maintenance of normal pancreBiological Trace Element Research
Vol. 11, 1986
Selenium/~ineral Interactions in Chicks
b~
57
+1 +1 +1 +1 +1 ,.,D t'o ,,O b-.. ',,o
g. 8o ~
t% ~
"..D t'~ cq ~-q r +l +l
+1 +1 +1
',,O oO ",D ',.D t'4 ,,-k
u~ .~_ +1 +1 +1 +1 +1 OL'-,
t".. b , - ' , ~
-;
Effects of Dietary Selenite, Copper, and Zinc on Tissue Trace Mineral Levels in Chicks G. F.
COMBS,JR.,'.* Q. Su, 2 C. H. LIu,2 AND S. B. COMBS'
'Department of Poultry and Avian Sciences, and Division of Nutritional Sciences, 306 Rice Hall, Come# University, Ithaca, NY 14853; and qnstitute of Animal Science, Chinese Academy of Agricultural Sciences
Received June 3, 19862~ccepted July 22, 1986 ABSTRACT Studies were conducted to determine whether nutritional selenium (Se) status affects the nutritional status of the chick with respect to other trace elements, particularly copper (Cu) and Zinc (Zn). Severe Se deficiency was produced in chicks by the use of diets that contained exceedingly low contents (less than 0.010 ppm) of Se, but contained adequate amounts of all other known essential nutrients. This diet was based upon corn and soybean meal produced in areas of China with endemic Se deficiency of geobotanical origin. A level of at least 0.10 ppm Se was found to be required to maintain normal Se status of chicks fed this diet, and Se deficiency resulted in decreased levels of Cu, Zn, and molybdenum in the pancreas (liver and plasma levels were not affected). High dietary supplementation of Zn nor Cu did not affect the short-term utilization of Se, as indicated by the 18-h responses of Se-dependent glutathione peroxidase in plasma and liver. Index Entries: Selenium, effects of on trace mineral levels; glutathione peroxidase; zinc, effects of on trace mineral levels; copper, effects of on trace mineral levels; iron; chick.
*Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
5]
Vol. I 1, 1986
52
Combs et aL
INTRODUCTION Interactions of selenium (Se) and other essential dietary minerals have been demonstrated; however, the metabolic bases for these effects, as well as their implications in situations of marginal Se status, are unclear. Chmielnicka et al. (1) found that oral administration of Na2SeO3 to rats nearly doubled the tissue retention of injected zinc (Zn). The administration of Se with Zn enhanced hepatic levels of Zn-metallothionein and increased the deposition of Zn in soluble high-molecular-weight proteins in kidney, also indicating improved utilization of Zn by Se. Studies by Naganuma et al. (2), however, indicated that parenterally administered Zn can alter the tissue disposition of Se, reducing the uptake of Se from a parenteral dose of Na2SeO3 by heart and liver. Near-toxic levels of dietary Se (e.g., 4 ppm) were shown to reduce liver Zn levels and to increase kidney Zn levels, suggesting that the utilization of Zn was reduced under such conditions. Amer et al. (3) found that oral Se therapy (Na2SeO3) of marginal-Se calves resulted in a rapid drop in plasma copper (Cu) and ceruloplasmin levels that was not prevented by feeding higher levels (up to 200 ppm) of Cu. Similar evidence of competitive effects of Se and Cu was reported by Thomson and Lawson (4), who observed that Se treatment of lambs born to ewes pastured on Se- and Cu-deficient fields reduced the efficacy of oral Cu supplements for the improvement of tissue Cu levels. Decreases in tissue activities of Se-dependent glutathione peroxidase (SeGSHpx) caused by high levels of Cu may explain the enhanced lipid peroxidation apparent in vivo (as indicated by exhalation of volatile alkanes) in the Cuintoxicated rat (5). It has been well established that when either Se or Cu is in excess, the opposite mineral can alleviate signs of toxicity. These studies, which have been conducted with chicks (6,7) ponies (8), and rats (9), show that Cu levels increase in Se intoxication, and that the lethality of dietary Se is reduced by high dietary levels of Cu. This suggests that when each is present in excess, the two elements become less metabolically available, perhaps because of the formation of Se-Cu adducts or by competing in some way (presumably via sulfhydryl binding) for binding to carriers and/or storage proteins. Jenkinson et al. (10) found that Cu deficiency in the rat reduced both the hepatic activity of SeGSHpx and the response of that enzyme activity to dietary supplements of Na2SeO3. Copper deficiency also reduced the tissue disposition of Se in such a way as to mimic nutritional Se deficiency. Therefore, both deficiencies and excesses of Cu have been reported to affect Se status. In view of these known interactions of Se with Zn and Cu, it is of interest to know whether the utilizations of Cu and/or Zn are altered under conditions of nutritional Se deficiency, and whether excesses of Cu or Zn can affect the utilization of dietary Se. in addition to being of fundamental interest in understanding the metabolic bases for the clinical manBiological Trace Element Research
Vol. 11, 1986
Selenium Mineral Interactions in Chicks
53
ifestations of Se-deficiency syndromes in animals, knowledge of these potential interactions is of practical value in dealing with likely mineral imblances that may occur in animal production in several parts of the developing world.
MATERIALS AND METHODS
Animals and Diets Male, Single Comb White Leghorn chicks were used in all experiments. The Se experiments were conducted in Beijing, PRC, and the chicks were obtained from a local hatchery. Chicks were installed at 1 d of age in stainless-steel, wire-floored, cages in a room maintained at 33-35~ with a 24-h day, as described previously (ll). In each experiment, four replicate groups of 10 or 14 chicks each were assigned randomly to each dietary treatment. The corn-soy-based diet used previously to produce uncomplicated Se deficiency in the chick was used in these studies; its composition and special origin were described by Combs et al. (11). It was found to contain 0.010 ppm Se; it was supplemented with 100 IU all-rac-alpha-tocopheryl acetate/kg. The basal diet also contained (by actual analysis) 4.75 ppm Cu, 21.1 ppm Zn, 69.6 ppm Fe, 13.5 ppm Mn, 1.57 ppm Mo, and 0.07 ppm Cd. Diets were provided ad libitum.
Preparation of Samples Blood was obtained by anterior cardiac puncture, using a heparinized syringe; plasma was prepared by centrifugation at 1000g for 10 rain at room temperature. Other tissues were excised immediately after chicks were killed by cervical dislocation. Tissues were minced and homogenized in 4 vol of 50 mM Tris, 154 mM KCI, pH 7.4, buffer using a motor-driven Teflon-glass Potter-Elvejhem type tissue grinder operated at 4~ Soybean trypsin inhibitor (Sigma Chemical Co., St. Louis, MO) was added (1 mg/mL) to the homogenizing medium for pancreas. Plasma and homogenates were analyzed immediately for enzyme activities, iron (Fe), and/or Se, but were held at -20~ for analysis of othe minerals. JVlineral Analyses
Selenium was determined by the fluorometric procedure of Olson et al. (12) using diaminonapthalene. In Expts. I and 2, Zn, Cu, Mn, and Mo were determined by inductively coupled argon plasma atomic emission spectrometry (using a Jarrell-Ash ICP 9000 instrument, Fisher Scientific Instruments, Springfield, NJ). Iron was determined by the chemical method of Weiser et al. (13), i.e., plasma total iron-binding capacity was determined by measuring the acid-precipitable iron after the addition of an excess of ferric ion to the sample. Biological Trace Element Research
VoL 11, 1986
54
Combs et al.
Measurement of Enzyme Activities Selenium-dependent glutathione peroxidase was measured by the glutathione reductase-coupled assay of Paglia and Valentine (14), as modified by Lawrence and Burk (15), using 0.25 mM H202 as substrate. Superoxide dismutase was determined by the inhibition of the spontaneous oxidation of epinephrine at pH 10.2, according to the method of Misra and Fridovich (16). Cytochrome c oxidase was determined by the method of Prohaska and Wells (17). Amylase was determined by the method of Bernfeld (18). Protein was determined by the method of Lowry et al. (19).
Histological Methods For histological studies, the duodenal loop and pancreas were exposed via an abdominal incision made immediately after killing the chick by cervical dislocation. A section of the pancreas, ca. I cm in length, with the attached duodenal loop, was quickly excised and fixed in 10% neutral-buffered formalin. The remaining sections of the pancreas were then removed and prepared for biochemical studies as described above. Histological sections of fixed pancreas specimens were mounted and stained with hematoxylin and eosin. They were examined by light microscopy; histological condition was scored according to the system described by Cantor et al. (20), in which a score of 1.0 represents completely normal pancreatic histology and a score of 5.0 represents complete acinar degeneration and advanced periacinar fibrosis.
Expedments Experiment 1 was conducted to determine the dietary Se requirement of the chick fed the corn-soy-based diet and whether changes in nutritional Se status affect the chick's status with respect to other trace elements. This was accomplished by feeding chicks for 40 d the basal diet supplemented with Na2SeO3 to achieve 0.010, 0.025, 0.050, 0.100, or 0.200 ppm total Se. Chicks were killed at 0, 5, 12, 20, 27, 34, and 40 d for determinations of SeGSHpx in plasma, liver, and pancreas and for determination of amylase in plasma. Experiments 2 and 3 were conducted to determine whether the utilization of dietary Se by chicks fed the practical diet was affected by high dietary supplements of either Cu or Zn. In Expt. 2, chicks were fed the practical diet supplemented with Se (as Na2SeO3) to provide a nutritionally marginal level of Se (0.025 ppm total Se) and with graded levels of either Cu (5, 100, 250, 500, or 1000 ppm total Cu achieved by adding CuSO4"SH20) or Zn (40, 250, 500, 1000, or 2500 ppm total Zn achieved by adding ZnO). In Expt. 3, chicks were fed the basal diet or the same diet supplemented with a high level (1000 ppm) of either Cu (as CuSO45H20) or Zn (as ZnO) for 20 d. At that time, five chicks per treatment Biological Trace Element Research
Vol. I I, 1986
Selenium Mineral Interactions in Chicks
55
were killed for determination of liver minerals. The remaining chicks in each treatment group were assigned randomly to either of two treatments: 2 p,g Se administered in 0.5 mL of an aqueous solution of Na2SeO3 by crop intubation; or a control (no Se) treatment consisting of 0.5 mL distilled water administered in the same way. Chicks were killed after 18 h; three composite samples from three chicks each were prepared for measurements of SeGSHpx in plasma and liver and five composite samples from three chicks each were prepared for measurements of Se in liver.
Statistical Analyses All data were evaluated statistically by Analysis of Variance. When significant (p < 0.05) effects were detected, post hoc analyses, using a Duncan's multiple range test, were conducted to rank treatment means. These tests were conducted using Systat 2.1 (a statistical analytical system configured for microcomputers, Systal, Inc., Evanston IL).
RESULTS Supplementation of the Se-deficient practical diet with Na2SeO3 did not consistently affect chick growth (Fig. 1), but produced increases in the primary parameters of nutritional Se status, i.e., the activities of SeGSHpx in plasma, liver, and pancreas (Table 1). These activities were greatest among chicks fed the highest level (0.20 ppm) of Se, in which there were two orders of magnitude greater than the enzyme activities of chicks fed the unsupplemented basal diet. At hatching, plasma activities of SeGSHpx were found to be 8.76 _+ 0.28 nmol NADPH/min/mg protein (X + SEM, n = 5). The 0.10-ppm dietary level of Se prevented any decline in SeGSHpx activity in plasma or liver (Figs. 2 and 3). At 40 d of age, signs of pancreatic atrophy were observed among chicks fed less than 0.10 ppm Se; this is indicated by the significantly elevated pancreas histological scores of those treatment groups (Table 1). Elevated pancreas scores were associated with elevated activities of amylase in plasma, also indicative of pancreatic dysfunction. The activities of superoxide dismutase in liver and pancreas were not significantly affected by dietary Se level (Table 2). In contrast, hepatic cytochrome c oxidase activities were significantly reduced in chicks fed less than 0.10 ppm Se; however, no significant effects were noted on this activity in pancreas. Tissue Se levels were significantly and directly related to the dietary concentration of the mineral; but this was not true for Cu, Zn, Fe, and Mo in plasma, or for these minerals as well as Mn in liver (Table 3). However, chicks fed the lower levels of Se showed significant reductions of Cu (by 12%), Zn (by 10%) and Mo (by ca. 24%) in pancreas. Alterations in Fe metabolism were also evident in Se-deficient chicks, which showed slight elevations in Biological Trace Element Research
VoL I 1, 1986
56
Combs et aL I I Dietary Se key 500-1I 9 .010 ppm i 9 .025 ppm II D .050 ppm I * .100 ppm 4001 9 .2DO ppm i
~* ~ / p l l
i t
/"
3OO
10
;
1;
2"0
1o
4"o
Days of age
Fig. 1. Growth of vitamin E-adequate chicks fed graded levels of Se; the effect of supplemental Se was not significant (p > 0.05) (n = 4, based on peraverage body weight at each day indicated. As chicks were removed for biochemical analyses, the numbers of chicks per pen decreased by 2 from the original number, 14, at 0 and 6 d). plasma Fe and concomitant depressions in plasma total-Fe-binding capacity (Table 4). In Expts. 2 and 3, supplementation of the marginaI-Se practical diet (containing 0.025 p p m Se) with high levels of Cu or Zn did not affect hepatic Se levels (Table 5). However, hepatic levels of Mn and Mo tended to be reduced at the higher levels of supplementation of each nutrient, which, in this experiment, did not affect either growth rate or feed intake. The short-term (18-h) utilization of Se for SeGSHpx, as indicated by the Se-induced increases in the activities of this enzyme in liver and plasma, was not significantly affected by high levels (1000 ppm) of either Cu or Zn (Table 6).
DISCUSSION The present study undertook to determine whether the utilization of supplemental Se (as Na2SeO3) by chicks fed a low-Se practical diet is affected by excesses of Cu and Zn, and whether Se-deficiency produced by this diet alters the utilization of other trace elements. In order to accomplish this, it was also necessary to determine the dietary Se requirement of vitamin E-adequate chick for Se for the maintenance of normal pancreBiological Trace Element Research
Vol. 11, 1986
Selenium/~ineral Interactions in Chicks
b~
57
+1 +1 +1 +1 +1 ,.,D t'o ,,O b-.. ',,o
g. 8o ~
t% ~
"..D t'~ cq ~-q r +l +l
+1 +1 +1
',,O oO ",D ',.D t'4 ,,-k
u~ .~_ +1 +1 +1 +1 +1 OL'-,
t".. b , - ' , ~
-;
