@Copyright 1986 by The Humana Press Inc. All fights of any nature whatsoever reserved. 016,3-4984/86/1002--0079502.20
Selenium-Mediated Biochemical Changes in Japanese Quail I. Formulation of Semipurified Low-Selenium Diet and Effect on Glutathione Peroxidase VASANTHY NARAYANASWAMI, 1 R. PADMA BAI, 2 MARY BABU, 2 AND K. LALITHA*'1
IDepartrnent of Chemistry, Indian Institute of Technology, ~adras-600 036, India; and 2Central Leather Research Institute, Adyar, ~adras-600 020, India Received November 1, 1985; Accepted November 8, 1985
ABSTRACT Essentiality of selenium (Se) for Japanese quail, Coturnix coturnix japonica, was confirmed using a formulated semipurified low-Se diet (basal) (0.05 ppm). Selenium-deficiency symptoms appeared in quails on this diet within 15 d, which corresponded to low levels of hemolysate glutathione peroxidase (GSH-Px) activity. Selenium administration at 0.05 and 2.0 ppm levels resulted in an increase of hemolysate GSH-Px activity by 64 and 116%, respectively, in both short- and long-term experiments. Growth over a 2-mo period increased the hemolysate GSH-Px activity by 120% at each level of dietary Se. A differential response was exhibited by hepatic mitochondrial and soluble GSH-Px activity to Se supplementation, the former increasing progressively with increments of Se at 0.05, 2.0, and 4.0 ppm by 45, 70, and 150%, respectively. The soluble GSH-Px activities of tissues, such as liver, kidney, and testis, and RBC membrane-bound activity remained unchanged in long-term studies at different levels of Se. Replenishment of Se to quails maintained on low-Se diets reflected no change in RBC membrane-bound and liversoluble GSH-Px activities, although the activity in hemolysate in*Author to whom all correspondence and reprint requests should be addressed. Biological Trace ElementResearch
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creased consistently with Se. The GSH-Px activity in hemolysate was restored to the levels comparable to those of long-term studies only at Se administration at the 2.0-ppm level. The differential response of mitochondrial and soluble GSH-Px activities to Se and other related observations on mitochondrial functions suggest an additional role for Se in mitochondrial membrane processes and glutathione-related metabolic regulations. Index Entries: Selenium, and related biochemical changes; selenium deficiency in the Japanese quail, Coturnix coturnix japonica; selenium and experimental deficiency and repletion-biochemical studies; selenium and metabolic regulation; glutathione peroxidase in selenium deficiency in quails, uncomplicated; glutathione peroxidase activities in tissues of Japanese quail; glutathione peroxidase and growth-related changes; glutathione peroxidase, differential responses to selenium at subcellular level.
INTRODUCTION Essentiality of Selenium (Se) in biological systems has b e e n recognized since Se-deficient diets (< 0.04 ppm) have been k n o w n to produce nutritional disorders to varying extent in different species, including rats (1), chicks (2), turkeys (3), pigs (4), and other farm animals and livestock (5); these disorders are responsive to Se supplementation in trace amounts. The paradoxical nature of Se is n o w being understood, and the importance of Se in cellular metabolism has been attributed to its function as an antioxidant and as a constituent of enzymes (6,7), microsomal electron transport (8), and redox centers (9). A few selenoproteins, such as rat testis cytosol selenoprotein (10), from mammalian systems have also been reported, though their functions are not yet defined. Other interesting aspects of Se that deserve mention are its anticarcinogenic effect (11,12) and preventive role in certain diseases (5,13), such as neuronal ceroid lipofuschinosis and myocardial lesions. In mammals, one of the prime attributes of Se is its role in the enzyme glutathione peroxidase (GSH-Px) (glutathione H202 oxidoreductase, EC 1.11.1.9), the only reported mammalian selenoenzyme, which was established about a decade ago (7). Glutathione peroxidase has been purified from a variety of sources, including bovine (14), ovine (15), and h u m a n erythrocytes (16), and rat liver (17). Extensive work has been carried out on the e n z y m e from bovine erythrocytes (13,18), and its selenocysteine active site has been studied in detail (19,20). The erythrocyte GSH-Px activity varies directly with the dietary Se level and is indicative of the Se status of the system (21,22). Torula-yeast- and casein-based diets, earlier used for Se-deficiency studies in chicks, cause severe disorders resulting from extremely low levels of Se (23), and, thus, do not permit long-term studies. Also, in trial experiments in our laboratory, such diets were found to be unsatisfactory Biological Trace Element Research
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for Se-deficiency studies with quails and necessitated the formulation of a semipurified, low-Se diet suitable for long-term studies relating to the effect of Se in tissues of quails. The formulation of such a low-Se diet and the results of short- and long-term studies using this diet in quails are reported here.
MATERIALS AND METHODS Glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase, hydrogen peroxide, vitamins, methionine, and choline chloride were purchased from Sigma Chemical Co., USA. Other reagents used were of AR grade. Deionized water, containing no detectable Se, was used throughout, unless otherwise specified.
GSH-Px Assay The GSH-Px activity was assayed by a modification of the method of Paglia and Valentine (24), as adapted by Burk et al. (25), using 0.25 mM H202 as substrate. All assays were carried out in duplicate. The GSH-Px activity is expressed as nmol NADPH oxidized/min/mg protein or g tissue at 37~ Protein was determined by the method of Lowry et al. (26). Selenium was estimated by the fluorimetric method, using 2,3-diaminonaphthalene (27) after wet digestion of sample. Statistical analysis was done by Student's t-test. TABLE 1 Level of Se in Various Dietary Constituents Dietary constituents Soybean meal Maize Ragi Peanut oil cake Peanut oil Gingely oil Sunflower oil Mineral mix Wheat bran Deoiled rice bran Rice polish Tap water Distilled water Deionized water ~
Level of Se, ~g/g 0.02 0.12 0.10 0.13 0.15 0.37 0.40 0.10 0.26 0.47 0.11 0.16 0.07 ND~
not detectable.
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Experimental Design After an a s s e s s m e n t of the Se c o n t e n t in normally u s e d dietary constituents (Table 1), a suitable, low-Se diet was f o r m u l a t e d (Table 2), a n d will be referred to as basal diet in the text. Selenium was s u p p l e m e n t e d as s o d i u m selenite at the required levels. The f o r m u l a t e d semipurified diet, w i t h no a d d e d Se, h a d an intrinsic Se c o n t e n t of 0.054 p p m .
Short-Term Studies The short-term e x p e r i m e n t s were aimed at s t u d y i n g the effect of low-Se diet d u r i n g a 15-d period. Day-old chicks of Japanese quail, w e i g h i n g 7-8 g, were obtained from the Poultry Research Station, Madras, India. The quails were divided at r a n d o m into four g r o u p s of eight a n d m a i n t a i n e d on basal, 0.05-, 1.0-, a n d 2.0-ppm S e - s u p p l e m e n t e d diets. Deionized water was given ad libitum. At the e n d of 15 d, the quails were sacrificed.
TABLE 2 Composition of Formulated Low-Se Semipurified Diet Constituents
Wt%
Soybean meal Maize Ragi Peanut oil cake Mineral mi:c~ Vitamin mixb Choline chloride Methionine Peanut oil ADE oilC Corn starch Level of selenium~---0.054ppm
50.0 10.0 10.0 8.0 4.0 1.0 0.25 0.20 5.0 1.0 11.0
~ mix (g/kg mix): NaC1, 30.0; KCI, 187.0; MgCO3, 60.5; Na2HPO4, 190.0; CaCO3, 520.0; MnSO4, 4.0; FeSO4 9 7H20, 6.4; ZnCO3, 1.4; CuSO4 95H20, 0.5; COCO3, 0.005; KI, 0.08; NaMoO4.5H20, 0.025; HBO3, 0.075. bVitamin mix (g/kg mix in corn starch): nicotinamide, 4.0; thiamine HC1, 0.4; riboflavin, 0.8; D-Ca-pantothenate, 1.6; biotin, 0.024; folic acid, 0.32; pyridoxine, 0.8; menadione, 0.045; vitamin B12, 0.004. cADE oil (IU/kg diet): retinyl acetate, 10,000 IU; cholecalciferol, 3000 IU; tocopheryl acetate, 20 IU. dSelenium analysis by fluorimetric method
(27). Biological Trace Element Research
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Long-Term Studies The long-term studies were carried out on similar lines, and the four groups of quails were maintained on basal, 0.05-, 1.0-, and 2.0-ppm Sesupplemented diets for 50 d, after which they were sacrificed. Se-Repletion Studies Sixty 1-d-old quails were maintained on basal diet for 35 d, after which they were divided at random into four groups: one group continued on the basal diet; Se was replenished to the other three groups at levels of 0.05, 1.0, and 2.0 ppm in the diet for a period of 25 d. Preparation of Subcellular Fractions The quails were anesthetized by ether at the end of the experimental period. Blood was collected in heparinized tubes by decapitation. The red blood cells (RBC) were isolated by centifugation of blood at 3000g for 10 min. The plasma and buffy coat layer were removed and the RBCs hemolyzed with 2 vol of distilled water at 4~ for 24 h. The RBC membrane was separated from the hemolysate by centrifugation at 3000g for 10 m/n, washed thoroughly, and dispersed in buffer. The membrane fractions were incubated with Triton X-100 for 3 h at 4~ to release the membrane-bound enzymes and centrifuged. The supematant was used for studies on membrane-bound GSH-Px activities. Tissues, such as liver, kidney, and testis, were quickly excised, washed thoroughly in ice-cold sucrose solution (0.25M), dried between folds of filter paper, weighed, minced, and hontogenized in 0.25M sucrose (1:10 w/v) in a Potter-Elvehjem-type glass homogenizer, with Teflon pestle. The homogenate was centrifuged at 600g for 5 min, and the cell debris and nuclear fraction were removed. The supernatant was further centrifuged at 10,000g for 30 min to separate the mitochondria. The washed mitochondrial fraction was suspended uniformly in 0.25M sucrose to give 1 mL suspension/g tissue. The mitochondrial and postmitochondrial supematant fractions were used for enzymatic studies. The enzyme assays were conducted immediately after preparation of samples, and the remaining fractions were stored at -20~ for subsequent analysis.
RESULTS AND DISCUSSION A semipurified, low-Se diet was formulated after screening the Se content of the ingredients normally used for quail diet and choosing those constituents with low levels of Se. Trial experiments were conducted by using proportionate mixtures of ingredients with low levels of Se and sufficient levels of other major dietary factors, such as proteins, fats, and carbohydrates. A satisfactory dietary composition was thus achieved; the final Se level of the formulation was sufficiently low (0.054 ppm, confirmed by fluorimetric analysis of the formulated diet), to cause Biological Trace Element Research
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deficiency symptoms in quails while adequately providing the rest of the nutrients of a normal diet. Selenium levels lower than 0.05 ppm resulted in high mortality rates. Selenium was added to this diet at various levels for the studies. The choice of ingredients of normally used cereals and pulses (broken grains) was made to avoid severe and complicated deficiency symptoms that arise during the usage of synthetic purified diets, using casein, amino acid mixtures, and so on (23), which are unsuitable for long-term studies. Unidentified nutritional factors in these cereals and pulses are thus indicated in preventing undesirable complications. The basal diet was compared with the standard quail diet used in the Poultry Research Station during trial experiments. The low-Se semipurified diet resulted in manifestations of typical Se-deficiency symptoms, such as feather-loss, weakness, marked skin changes, and the like, even within 15 d, which did not appear when Se was supplemented adequately to this formulation at levels from 0.1 to 2.0 ppm. Selenium deficiency in quails fed this diet was further confirmed by the low levels of activity of GSH-Px in the hemolysate when compared to those of quails fed Se-supplemented diets. Also, there was increased uptake of Se in studies with radiotracers by the birds fed the formulated basal diets (28). Addition of Se to basal diet had a marked effect on the growth pattern of quails. The weight gain was maximal in groups fed 0.05 and 2.0 ppm Se in short- and long-term studies, followed by the group fed basal diet, whereas minimal increase was observed in the group fed 4.0 ppm Se. Also, the quails in the 4-ppm Se group suffered poor health and feather loss, with drastic deteriorative skin changes. Similar symptoms to a lesser extent of severity were observed for quails maintained on basal diets as well. In replenishment studies in which Se was supplemented at levels of 0.05, 1.0, and 2.0 ppm to quails previously maintained on basal diet, a general improvement in health and feather growth was observed, especially at levels of 2.0 ppm Se.
GSH-Px ActMty in Short-Term Studies The GSH-Px activity in the hemolysate of quails maintained on basal and Se-supplemented diets for 15 d is represented in Table 3. Quails fed basal diet had the lowest activity of GSH-Px, 101.86 U. At 0.05 and 2.0 ppm Se, the GSH-Px activity increased significantly by 63.8 and 116.8%, respectively. Similar results have been reported in chicks and rats (21,22), wherein the GSH-Px activity varied proportionately with the Se status of the system. The GSH-Px activity in the hepatic mitochondrial and postmitochondrial supernatant fractions of quails is represented in Table 4. Selenium supplementation up to a level of 2.0 ppm did not result in any significant increase in activity of the soluble enzyme, whereas a level of 4.0 ppm resulted in a moderate increase in activity of about 25%. The Biological Trace Edement Research
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TABLE 3 Effect of Dietary Se on GSH-Px Activity in Hemolysate of Quails (Short Term) Level of Se ppm Basal 0.05 2.00 4.00
GSH-Px activity in hemolysate, U/rag protein* 101.86 166.80 220.80 250.30
+ 2.31 a + 4.53 b + 9.80 c _+ 4.93 c
*GSH-Px activity: 1 U represents 1 nmol NADPH oxidized/min at 37~ Values are expressed as average -+ SEM. Means bearing the same superscript do not vary significantly (P < 0.001). m i t o c h o n d r i a l GSH-Px registered a t r e n d of significant increase in activity w i t h i n c r e a s i n g d i e t a r y Se. The d i e t a r y Se h a d a m o r e p r o n o u n c e d effect o n the GSH-Px activity in RBC h e m o l y s a t e a n d liver m i t o c h o n d r i a t h a n in t h e liver s u p e r n a t a n t . S t u d i e s f r o m o u r l a b o r a t o r y h a v e also s h o w n t h a t a n increase in the i n g e s t e d Se results in p r o n o u n c e d e n h a n c e m e n t of m i t o c h o n d r i a l functional efficiency (28). Such a n increase in o x y g e n u p t a k e is likely to accelerate the g e n e r a t i o n of s u p e r o x i d e a n i o n s , w h i c h in t u r n leads to inc r e a s e d s u b s t r a t e availability for GSH-Px. T h e s e related s e q u e n c e s of reactions w o u l d e v e n t u a l l y reflect u p o n t h e o b s e r v e d increase in t h e m i t o c h o n d r i a l GSH-PX activity. Similar cases of oxidative stress h a v e also b e e n r e p o r t e d in rats (29). A possible role has b e e n i m p l i e d for GSH-Px in this organelle in r e g u l a t i o n of m i t o c h o n d r i a l - s u b s t r a t e oxidations (30,31). TABLE 4 Effect of Dietary Se on GSH-Px Activities in Hepatic Mitochondrial and Postmitochondrial Supernatant Fractions of Quails (Short Term) I'2 GSH-Px Activity~ Liver mitochondria
Liver supernatant Level of Se pprn Basal 0.05 2.00 4.00
U/mg protein
U/g tissue x 10 .-3
304.1 + 7.52 a 36.5 + 0.67 c 305.4 + 3.52 a 37.5 +__ 0.22 c 307.8_+ 8.88 a 35:0-+ 1.14 ~ 380.9 ___ 23.7 b 43.0 _+ 1.49 d
U/mg protein 101.8 147.3 173.4 255.0
_+ 11.52 e + 16.6 f -+ 19:93 ~ • 25.5 h
U/g tissue X 2.26 3.30 3:60 5.96
------+
10 -3
0.42 0.36 0.70 0.77
'Values are expressed as average _+ SEM. 2In a vertical column, means bearing the same superscript do not vary significantly: P < 0.02 between a and b, and e and g; P < 0.01 between c and d, and e and h. 3GSH-Px activity, units as defined in Table 3. Biological Trace Element Research
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From the results presented, the distribution of the liver-soluble and mitochondrial GSH-Px in 15-d-old quails is as follows: 75:25 for quails maintained on basal diet; 66:33 at 0.05-ppm Se level; 60:40 at 2.0-ppm Se; and 63:37 at 4.0-ppm Se. Increase in the mitochondrial form of GSH-Px with Se is probably a result of the scavenging of toxic c o m p o u n d s of increased oxidative stress. The distribution of GSH-Px in rat liver has been reported to be in the ratio of 70:30 (19). There is considerable variation a m o n g different species regarding distribution of GSH-Px in various organs. Even within a given species, the organelle distribution of Sed e p e n d e n t and - i n d e p e n d e n t GSH-Px differs widely (32). The lack of response of the liver soluble GSH-Px to dietary Se is indicative of the presence of a S e - i n d e p e n d e n t e n z y m e (33), to a major extent.
GSH-Px Activity in Long-Term Studies The effect of Se at levels of 0.05 and 2.0 p p m on GSH-Px and the variation of GSH-Px u p o n aging were studied in long-term experiments. The toxic s y m p t o m s at 4.0-ppm Se, apparent even within 15 d, precluded long-term studies at this level. In the hemolysate of quails, the increase in GSH-Px activity was about 63 a n d 116% at dietary Se levels of 0.05 and 2.0 p p m , respectively, w h e n c o m p a r e d to the basal group (Table 5). These results are consistent with those of short-term studies. The GSH-Px activity in the RBC m e m brane does not s h o w any response to dietary Se variation (Table 5). At a dietary Se level of 2.0 p p m , the increase in GSH-Px activity was reflected to a larger extent in the hemolysate w h e n c o m p a r e d to the differences observed in the hepatic soluble fractions (Table 6). The GSH-Px of the soluble fractions of kidney and testis is relatively unaffected by Se supplementation in long-term studies. Rat testes have been s h o w n to contain a selenoprotein other than GSH-Px (10); also, the testes tend to
TABLE 5 Effect of Dietary Se on GSH-Px Activities in Hemolysate and RBC Membranes of Quails (Long Term) 1'2 GSH-Px Activity~ Level of Se ppm Basal 0.05 2.0
Hemolysate, U/mg protein
RBC membrane, u/mg protein
228.6 + 6.80~ 374.5 ___ 6.43 b 495.0 + 23.58 c
156.3 + 4.18 167.0 + 4.48 145.6 +_ 7.42
~Values are e x p r e s s e d as a v e r a g e + SEM. ZMeans b e a r i n g the s a m e superscript, do not v a r y significantly: P < 0.001 b e t w e e n a a n d b, a n d a a n d c; P < 0.01 b e t w e e n b a n d c. ~GSH-Px activity, units as d e f i n e d in Table 3. Biological Trace Element Research
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TABLE 6 Effect of Dietary Se on GSH-Px Activities the Postmitochondrial Supernatant Fractions of Liver, Kidney, and Testis of Quails (Long Term) ~'2 GSH-Px Activity~ Liver Level of Se ppm Basal 0.05 2.0
U/mg protein 290.5 _+ 12.1a 294.7 ___ 9.8 a 352.8 ___ 10.2b
U/g tissue X
Kidney 10 - 3
26.3 ___ 1.4 27.9 _+ 1.99 32.1 + 2.2
U/g tissue x
10 - 3
5.83 + 0.12 5.63 --- 0.28 5.43 _ 0.87
Testis U/g tissue x
10 - 3
7.34 _+ 0.87 6.98 ___ 0.74 5.64 -4-_ 0.49
~Values are expressed as average + SEM. :Means bearing the same superscript, do not vary significantly (P < 0.01). 3GSH-Px activity, units as defined in Table 3.
accumulate Se w h e n administered in small doses (28), which is indicative of other functions for Se. In the early stages of growth in quails, the GSH-Px activity is higher in the liver than in the RBCs, with only the RBC activity increasing u p o n growth. The increase in activity of GSH-Px in hemolysate of quails during growth, up to a period of 2 mo, is about 124% at each level of dietary Se. In rats, the liver GSH-Px activity has been reported to increase with the age of the animal (34,35). The differential response of GSH-Px in hepatic and RBC fractions to Se could be attributed to the presence of a Se-independent GSH-Px, in addition to a selenoenzyme. These activities are present to an equal extent in the liver of several species, including rats (32). Seleniumi n d e p e n d e n t GSH-Px activity is at least partially caused by one or more glutathione-S-transferases (36).
Se-Replenishment Studies Quails maintained on low-Se basal diet for 35 d, w h e n replenished with different levels of Se for 25 d s h o w e d increased levels of GSH-Px activity in erythrocytes (Table 7). The response was maximal at a Se level of 2.0 p p m , with an increase in activity of about 134% w h e n c o m p a r e d to the basal group. On the other hand, the m e m b r a n e - b o u n d e n z y m e in the RBCs s h o w e d no significant increase in activity u p o n Se replenishment. Selenium replenishment at levels of 1.0 and 2.0 p p m increased the GSH-Px activity marginally in the soluble fractions of the liver (Table 7). Selenium administration to quails on a low-Se diet, at 2.0 p p m level for 25 d, replenished the GSH-Px activity in the hemolysate to a level comparable to the activity in quails fed 2.0 p p m Se for a long term of 50 d. Replenishment of Se at 0.05- and 1.0-ppm levels did not correspondingly restore the hemolysate GSH-Px activities to long-term levels. Varied responses of GSH-Px activities to Se status have been reported for different species. Individual variations b e t w e e n species and Biological Trace Element Research
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Narayanaswami et al. TABLE 7 Effect of Se Replenishment on GSH-Px Activities in Hemolysate, RBC Membranes, and Hepatic Postmitochondrial Supernatant Fraction of Quails Maintained on Low-Selenium Diet2 '2 GSH-Px Activity~ Hemolysate
Level of Se ppm Basal 0.05 1.00 2.00
RBC membrane
U/rag protein 195.2 249.9 273.3 483.2
+_ + + +
14.8a 11.2b 19.4b 4.67*
U/rag protein 119.2 _+ 9.67 139.2 + 15.6 136.4 +_ 7.34 149.6 ___ 12.9
Liver supernatant U/rag protein 334.0 341.8 346.5 354.2
+_ 5.06 --- 7.47 _+ 6.4 + 44.2
U/rag tissue x 10 -3 33.35 35.14 35.47 42.3
_ 2.88 + 2.15 ___ 0.41 _+ 0.55
~Values are expressed as average -+ SEM. 2Means bearing the same superscript, do not vary significantly (P < 0.001). 3GSH-Px activity, units as defined in Table 3.
within the species a m o n g different organs have also been s h o w n (32). Results of investigations reported here suggest that the GSH-Px in the h e m o l y s a t e of quails is a fairly reliable indicator of the Se status of the system. The response of the mitochondrial GSH-Px to Se implies both a direct and indirect involvement of this element. Selenium plays a direct role by being an i n h e r e n t c o m p o n e n t of the s e l e n o e n z y m e GSH-Px a n d an indirect role by contributing to increased mitochondrial oxidations. Thus, Se m a y have a regulatory role not only in sulfhydryl metabolism, but also in the mitochondrial oxygen metabolism. Studies on the exact nature of interaction and extent of involvement of Se in the various cellular reactions, especially in the mitochondria, are in progress in our laboratory.
ACKNOWLEDGMENT The authors are grateful to Dr. K. Thomas Joseph, Central Leather Research Institute, for his keen interest, constant e n c o u r a g e m e n t , and m a n y helpful suggestions during the course of this work.
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Vol. 10, 1986
Selenium-Mediated Biochemical Changes in Japanese Quail I. Formulation of Semipurified Low-Selenium Diet and Effect on Glutathione Peroxidase VASANTHY NARAYANASWAMI, 1 R. PADMA BAI, 2 MARY BABU, 2 AND K. LALITHA*'1
IDepartrnent of Chemistry, Indian Institute of Technology, ~adras-600 036, India; and 2Central Leather Research Institute, Adyar, ~adras-600 020, India Received November 1, 1985; Accepted November 8, 1985
ABSTRACT Essentiality of selenium (Se) for Japanese quail, Coturnix coturnix japonica, was confirmed using a formulated semipurified low-Se diet (basal) (0.05 ppm). Selenium-deficiency symptoms appeared in quails on this diet within 15 d, which corresponded to low levels of hemolysate glutathione peroxidase (GSH-Px) activity. Selenium administration at 0.05 and 2.0 ppm levels resulted in an increase of hemolysate GSH-Px activity by 64 and 116%, respectively, in both short- and long-term experiments. Growth over a 2-mo period increased the hemolysate GSH-Px activity by 120% at each level of dietary Se. A differential response was exhibited by hepatic mitochondrial and soluble GSH-Px activity to Se supplementation, the former increasing progressively with increments of Se at 0.05, 2.0, and 4.0 ppm by 45, 70, and 150%, respectively. The soluble GSH-Px activities of tissues, such as liver, kidney, and testis, and RBC membrane-bound activity remained unchanged in long-term studies at different levels of Se. Replenishment of Se to quails maintained on low-Se diets reflected no change in RBC membrane-bound and liversoluble GSH-Px activities, although the activity in hemolysate in*Author to whom all correspondence and reprint requests should be addressed. Biological Trace ElementResearch
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creased consistently with Se. The GSH-Px activity in hemolysate was restored to the levels comparable to those of long-term studies only at Se administration at the 2.0-ppm level. The differential response of mitochondrial and soluble GSH-Px activities to Se and other related observations on mitochondrial functions suggest an additional role for Se in mitochondrial membrane processes and glutathione-related metabolic regulations. Index Entries: Selenium, and related biochemical changes; selenium deficiency in the Japanese quail, Coturnix coturnix japonica; selenium and experimental deficiency and repletion-biochemical studies; selenium and metabolic regulation; glutathione peroxidase in selenium deficiency in quails, uncomplicated; glutathione peroxidase activities in tissues of Japanese quail; glutathione peroxidase and growth-related changes; glutathione peroxidase, differential responses to selenium at subcellular level.
INTRODUCTION Essentiality of Selenium (Se) in biological systems has b e e n recognized since Se-deficient diets (< 0.04 ppm) have been k n o w n to produce nutritional disorders to varying extent in different species, including rats (1), chicks (2), turkeys (3), pigs (4), and other farm animals and livestock (5); these disorders are responsive to Se supplementation in trace amounts. The paradoxical nature of Se is n o w being understood, and the importance of Se in cellular metabolism has been attributed to its function as an antioxidant and as a constituent of enzymes (6,7), microsomal electron transport (8), and redox centers (9). A few selenoproteins, such as rat testis cytosol selenoprotein (10), from mammalian systems have also been reported, though their functions are not yet defined. Other interesting aspects of Se that deserve mention are its anticarcinogenic effect (11,12) and preventive role in certain diseases (5,13), such as neuronal ceroid lipofuschinosis and myocardial lesions. In mammals, one of the prime attributes of Se is its role in the enzyme glutathione peroxidase (GSH-Px) (glutathione H202 oxidoreductase, EC 1.11.1.9), the only reported mammalian selenoenzyme, which was established about a decade ago (7). Glutathione peroxidase has been purified from a variety of sources, including bovine (14), ovine (15), and h u m a n erythrocytes (16), and rat liver (17). Extensive work has been carried out on the e n z y m e from bovine erythrocytes (13,18), and its selenocysteine active site has been studied in detail (19,20). The erythrocyte GSH-Px activity varies directly with the dietary Se level and is indicative of the Se status of the system (21,22). Torula-yeast- and casein-based diets, earlier used for Se-deficiency studies in chicks, cause severe disorders resulting from extremely low levels of Se (23), and, thus, do not permit long-term studies. Also, in trial experiments in our laboratory, such diets were found to be unsatisfactory Biological Trace Element Research
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for Se-deficiency studies with quails and necessitated the formulation of a semipurified, low-Se diet suitable for long-term studies relating to the effect of Se in tissues of quails. The formulation of such a low-Se diet and the results of short- and long-term studies using this diet in quails are reported here.
MATERIALS AND METHODS Glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase, hydrogen peroxide, vitamins, methionine, and choline chloride were purchased from Sigma Chemical Co., USA. Other reagents used were of AR grade. Deionized water, containing no detectable Se, was used throughout, unless otherwise specified.
GSH-Px Assay The GSH-Px activity was assayed by a modification of the method of Paglia and Valentine (24), as adapted by Burk et al. (25), using 0.25 mM H202 as substrate. All assays were carried out in duplicate. The GSH-Px activity is expressed as nmol NADPH oxidized/min/mg protein or g tissue at 37~ Protein was determined by the method of Lowry et al. (26). Selenium was estimated by the fluorimetric method, using 2,3-diaminonaphthalene (27) after wet digestion of sample. Statistical analysis was done by Student's t-test. TABLE 1 Level of Se in Various Dietary Constituents Dietary constituents Soybean meal Maize Ragi Peanut oil cake Peanut oil Gingely oil Sunflower oil Mineral mix Wheat bran Deoiled rice bran Rice polish Tap water Distilled water Deionized water ~
Level of Se, ~g/g 0.02 0.12 0.10 0.13 0.15 0.37 0.40 0.10 0.26 0.47 0.11 0.16 0.07 ND~
not detectable.
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Experimental Design After an a s s e s s m e n t of the Se c o n t e n t in normally u s e d dietary constituents (Table 1), a suitable, low-Se diet was f o r m u l a t e d (Table 2), a n d will be referred to as basal diet in the text. Selenium was s u p p l e m e n t e d as s o d i u m selenite at the required levels. The f o r m u l a t e d semipurified diet, w i t h no a d d e d Se, h a d an intrinsic Se c o n t e n t of 0.054 p p m .
Short-Term Studies The short-term e x p e r i m e n t s were aimed at s t u d y i n g the effect of low-Se diet d u r i n g a 15-d period. Day-old chicks of Japanese quail, w e i g h i n g 7-8 g, were obtained from the Poultry Research Station, Madras, India. The quails were divided at r a n d o m into four g r o u p s of eight a n d m a i n t a i n e d on basal, 0.05-, 1.0-, a n d 2.0-ppm S e - s u p p l e m e n t e d diets. Deionized water was given ad libitum. At the e n d of 15 d, the quails were sacrificed.
TABLE 2 Composition of Formulated Low-Se Semipurified Diet Constituents
Wt%
Soybean meal Maize Ragi Peanut oil cake Mineral mi:c~ Vitamin mixb Choline chloride Methionine Peanut oil ADE oilC Corn starch Level of selenium~---0.054ppm
50.0 10.0 10.0 8.0 4.0 1.0 0.25 0.20 5.0 1.0 11.0
~ mix (g/kg mix): NaC1, 30.0; KCI, 187.0; MgCO3, 60.5; Na2HPO4, 190.0; CaCO3, 520.0; MnSO4, 4.0; FeSO4 9 7H20, 6.4; ZnCO3, 1.4; CuSO4 95H20, 0.5; COCO3, 0.005; KI, 0.08; NaMoO4.5H20, 0.025; HBO3, 0.075. bVitamin mix (g/kg mix in corn starch): nicotinamide, 4.0; thiamine HC1, 0.4; riboflavin, 0.8; D-Ca-pantothenate, 1.6; biotin, 0.024; folic acid, 0.32; pyridoxine, 0.8; menadione, 0.045; vitamin B12, 0.004. cADE oil (IU/kg diet): retinyl acetate, 10,000 IU; cholecalciferol, 3000 IU; tocopheryl acetate, 20 IU. dSelenium analysis by fluorimetric method
(27). Biological Trace Element Research
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Long-Term Studies The long-term studies were carried out on similar lines, and the four groups of quails were maintained on basal, 0.05-, 1.0-, and 2.0-ppm Sesupplemented diets for 50 d, after which they were sacrificed. Se-Repletion Studies Sixty 1-d-old quails were maintained on basal diet for 35 d, after which they were divided at random into four groups: one group continued on the basal diet; Se was replenished to the other three groups at levels of 0.05, 1.0, and 2.0 ppm in the diet for a period of 25 d. Preparation of Subcellular Fractions The quails were anesthetized by ether at the end of the experimental period. Blood was collected in heparinized tubes by decapitation. The red blood cells (RBC) were isolated by centifugation of blood at 3000g for 10 min. The plasma and buffy coat layer were removed and the RBCs hemolyzed with 2 vol of distilled water at 4~ for 24 h. The RBC membrane was separated from the hemolysate by centrifugation at 3000g for 10 m/n, washed thoroughly, and dispersed in buffer. The membrane fractions were incubated with Triton X-100 for 3 h at 4~ to release the membrane-bound enzymes and centrifuged. The supematant was used for studies on membrane-bound GSH-Px activities. Tissues, such as liver, kidney, and testis, were quickly excised, washed thoroughly in ice-cold sucrose solution (0.25M), dried between folds of filter paper, weighed, minced, and hontogenized in 0.25M sucrose (1:10 w/v) in a Potter-Elvehjem-type glass homogenizer, with Teflon pestle. The homogenate was centrifuged at 600g for 5 min, and the cell debris and nuclear fraction were removed. The supernatant was further centrifuged at 10,000g for 30 min to separate the mitochondria. The washed mitochondrial fraction was suspended uniformly in 0.25M sucrose to give 1 mL suspension/g tissue. The mitochondrial and postmitochondrial supematant fractions were used for enzymatic studies. The enzyme assays were conducted immediately after preparation of samples, and the remaining fractions were stored at -20~ for subsequent analysis.
RESULTS AND DISCUSSION A semipurified, low-Se diet was formulated after screening the Se content of the ingredients normally used for quail diet and choosing those constituents with low levels of Se. Trial experiments were conducted by using proportionate mixtures of ingredients with low levels of Se and sufficient levels of other major dietary factors, such as proteins, fats, and carbohydrates. A satisfactory dietary composition was thus achieved; the final Se level of the formulation was sufficiently low (0.054 ppm, confirmed by fluorimetric analysis of the formulated diet), to cause Biological Trace Element Research
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deficiency symptoms in quails while adequately providing the rest of the nutrients of a normal diet. Selenium levels lower than 0.05 ppm resulted in high mortality rates. Selenium was added to this diet at various levels for the studies. The choice of ingredients of normally used cereals and pulses (broken grains) was made to avoid severe and complicated deficiency symptoms that arise during the usage of synthetic purified diets, using casein, amino acid mixtures, and so on (23), which are unsuitable for long-term studies. Unidentified nutritional factors in these cereals and pulses are thus indicated in preventing undesirable complications. The basal diet was compared with the standard quail diet used in the Poultry Research Station during trial experiments. The low-Se semipurified diet resulted in manifestations of typical Se-deficiency symptoms, such as feather-loss, weakness, marked skin changes, and the like, even within 15 d, which did not appear when Se was supplemented adequately to this formulation at levels from 0.1 to 2.0 ppm. Selenium deficiency in quails fed this diet was further confirmed by the low levels of activity of GSH-Px in the hemolysate when compared to those of quails fed Se-supplemented diets. Also, there was increased uptake of Se in studies with radiotracers by the birds fed the formulated basal diets (28). Addition of Se to basal diet had a marked effect on the growth pattern of quails. The weight gain was maximal in groups fed 0.05 and 2.0 ppm Se in short- and long-term studies, followed by the group fed basal diet, whereas minimal increase was observed in the group fed 4.0 ppm Se. Also, the quails in the 4-ppm Se group suffered poor health and feather loss, with drastic deteriorative skin changes. Similar symptoms to a lesser extent of severity were observed for quails maintained on basal diets as well. In replenishment studies in which Se was supplemented at levels of 0.05, 1.0, and 2.0 ppm to quails previously maintained on basal diet, a general improvement in health and feather growth was observed, especially at levels of 2.0 ppm Se.
GSH-Px ActMty in Short-Term Studies The GSH-Px activity in the hemolysate of quails maintained on basal and Se-supplemented diets for 15 d is represented in Table 3. Quails fed basal diet had the lowest activity of GSH-Px, 101.86 U. At 0.05 and 2.0 ppm Se, the GSH-Px activity increased significantly by 63.8 and 116.8%, respectively. Similar results have been reported in chicks and rats (21,22), wherein the GSH-Px activity varied proportionately with the Se status of the system. The GSH-Px activity in the hepatic mitochondrial and postmitochondrial supernatant fractions of quails is represented in Table 4. Selenium supplementation up to a level of 2.0 ppm did not result in any significant increase in activity of the soluble enzyme, whereas a level of 4.0 ppm resulted in a moderate increase in activity of about 25%. The Biological Trace Edement Research
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TABLE 3 Effect of Dietary Se on GSH-Px Activity in Hemolysate of Quails (Short Term) Level of Se ppm Basal 0.05 2.00 4.00
GSH-Px activity in hemolysate, U/rag protein* 101.86 166.80 220.80 250.30
+ 2.31 a + 4.53 b + 9.80 c _+ 4.93 c
*GSH-Px activity: 1 U represents 1 nmol NADPH oxidized/min at 37~ Values are expressed as average -+ SEM. Means bearing the same superscript do not vary significantly (P < 0.001). m i t o c h o n d r i a l GSH-Px registered a t r e n d of significant increase in activity w i t h i n c r e a s i n g d i e t a r y Se. The d i e t a r y Se h a d a m o r e p r o n o u n c e d effect o n the GSH-Px activity in RBC h e m o l y s a t e a n d liver m i t o c h o n d r i a t h a n in t h e liver s u p e r n a t a n t . S t u d i e s f r o m o u r l a b o r a t o r y h a v e also s h o w n t h a t a n increase in the i n g e s t e d Se results in p r o n o u n c e d e n h a n c e m e n t of m i t o c h o n d r i a l functional efficiency (28). Such a n increase in o x y g e n u p t a k e is likely to accelerate the g e n e r a t i o n of s u p e r o x i d e a n i o n s , w h i c h in t u r n leads to inc r e a s e d s u b s t r a t e availability for GSH-Px. T h e s e related s e q u e n c e s of reactions w o u l d e v e n t u a l l y reflect u p o n t h e o b s e r v e d increase in t h e m i t o c h o n d r i a l GSH-PX activity. Similar cases of oxidative stress h a v e also b e e n r e p o r t e d in rats (29). A possible role has b e e n i m p l i e d for GSH-Px in this organelle in r e g u l a t i o n of m i t o c h o n d r i a l - s u b s t r a t e oxidations (30,31). TABLE 4 Effect of Dietary Se on GSH-Px Activities in Hepatic Mitochondrial and Postmitochondrial Supernatant Fractions of Quails (Short Term) I'2 GSH-Px Activity~ Liver mitochondria
Liver supernatant Level of Se pprn Basal 0.05 2.00 4.00
U/mg protein
U/g tissue x 10 .-3
304.1 + 7.52 a 36.5 + 0.67 c 305.4 + 3.52 a 37.5 +__ 0.22 c 307.8_+ 8.88 a 35:0-+ 1.14 ~ 380.9 ___ 23.7 b 43.0 _+ 1.49 d
U/mg protein 101.8 147.3 173.4 255.0
_+ 11.52 e + 16.6 f -+ 19:93 ~ • 25.5 h
U/g tissue X 2.26 3.30 3:60 5.96
------+
10 -3
0.42 0.36 0.70 0.77
'Values are expressed as average _+ SEM. 2In a vertical column, means bearing the same superscript do not vary significantly: P < 0.02 between a and b, and e and g; P < 0.01 between c and d, and e and h. 3GSH-Px activity, units as defined in Table 3. Biological Trace Element Research
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From the results presented, the distribution of the liver-soluble and mitochondrial GSH-Px in 15-d-old quails is as follows: 75:25 for quails maintained on basal diet; 66:33 at 0.05-ppm Se level; 60:40 at 2.0-ppm Se; and 63:37 at 4.0-ppm Se. Increase in the mitochondrial form of GSH-Px with Se is probably a result of the scavenging of toxic c o m p o u n d s of increased oxidative stress. The distribution of GSH-Px in rat liver has been reported to be in the ratio of 70:30 (19). There is considerable variation a m o n g different species regarding distribution of GSH-Px in various organs. Even within a given species, the organelle distribution of Sed e p e n d e n t and - i n d e p e n d e n t GSH-Px differs widely (32). The lack of response of the liver soluble GSH-Px to dietary Se is indicative of the presence of a S e - i n d e p e n d e n t e n z y m e (33), to a major extent.
GSH-Px Activity in Long-Term Studies The effect of Se at levels of 0.05 and 2.0 p p m on GSH-Px and the variation of GSH-Px u p o n aging were studied in long-term experiments. The toxic s y m p t o m s at 4.0-ppm Se, apparent even within 15 d, precluded long-term studies at this level. In the hemolysate of quails, the increase in GSH-Px activity was about 63 a n d 116% at dietary Se levels of 0.05 and 2.0 p p m , respectively, w h e n c o m p a r e d to the basal group (Table 5). These results are consistent with those of short-term studies. The GSH-Px activity in the RBC m e m brane does not s h o w any response to dietary Se variation (Table 5). At a dietary Se level of 2.0 p p m , the increase in GSH-Px activity was reflected to a larger extent in the hemolysate w h e n c o m p a r e d to the differences observed in the hepatic soluble fractions (Table 6). The GSH-Px of the soluble fractions of kidney and testis is relatively unaffected by Se supplementation in long-term studies. Rat testes have been s h o w n to contain a selenoprotein other than GSH-Px (10); also, the testes tend to
TABLE 5 Effect of Dietary Se on GSH-Px Activities in Hemolysate and RBC Membranes of Quails (Long Term) 1'2 GSH-Px Activity~ Level of Se ppm Basal 0.05 2.0
Hemolysate, U/mg protein
RBC membrane, u/mg protein
228.6 + 6.80~ 374.5 ___ 6.43 b 495.0 + 23.58 c
156.3 + 4.18 167.0 + 4.48 145.6 +_ 7.42
~Values are e x p r e s s e d as a v e r a g e + SEM. ZMeans b e a r i n g the s a m e superscript, do not v a r y significantly: P < 0.001 b e t w e e n a a n d b, a n d a a n d c; P < 0.01 b e t w e e n b a n d c. ~GSH-Px activity, units as d e f i n e d in Table 3. Biological Trace Element Research
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TABLE 6 Effect of Dietary Se on GSH-Px Activities the Postmitochondrial Supernatant Fractions of Liver, Kidney, and Testis of Quails (Long Term) ~'2 GSH-Px Activity~ Liver Level of Se ppm Basal 0.05 2.0
U/mg protein 290.5 _+ 12.1a 294.7 ___ 9.8 a 352.8 ___ 10.2b
U/g tissue X
Kidney 10 - 3
26.3 ___ 1.4 27.9 _+ 1.99 32.1 + 2.2
U/g tissue x
10 - 3
5.83 + 0.12 5.63 --- 0.28 5.43 _ 0.87
Testis U/g tissue x
10 - 3
7.34 _+ 0.87 6.98 ___ 0.74 5.64 -4-_ 0.49
~Values are expressed as average + SEM. :Means bearing the same superscript, do not vary significantly (P < 0.01). 3GSH-Px activity, units as defined in Table 3.
accumulate Se w h e n administered in small doses (28), which is indicative of other functions for Se. In the early stages of growth in quails, the GSH-Px activity is higher in the liver than in the RBCs, with only the RBC activity increasing u p o n growth. The increase in activity of GSH-Px in hemolysate of quails during growth, up to a period of 2 mo, is about 124% at each level of dietary Se. In rats, the liver GSH-Px activity has been reported to increase with the age of the animal (34,35). The differential response of GSH-Px in hepatic and RBC fractions to Se could be attributed to the presence of a Se-independent GSH-Px, in addition to a selenoenzyme. These activities are present to an equal extent in the liver of several species, including rats (32). Seleniumi n d e p e n d e n t GSH-Px activity is at least partially caused by one or more glutathione-S-transferases (36).
Se-Replenishment Studies Quails maintained on low-Se basal diet for 35 d, w h e n replenished with different levels of Se for 25 d s h o w e d increased levels of GSH-Px activity in erythrocytes (Table 7). The response was maximal at a Se level of 2.0 p p m , with an increase in activity of about 134% w h e n c o m p a r e d to the basal group. On the other hand, the m e m b r a n e - b o u n d e n z y m e in the RBCs s h o w e d no significant increase in activity u p o n Se replenishment. Selenium replenishment at levels of 1.0 and 2.0 p p m increased the GSH-Px activity marginally in the soluble fractions of the liver (Table 7). Selenium administration to quails on a low-Se diet, at 2.0 p p m level for 25 d, replenished the GSH-Px activity in the hemolysate to a level comparable to the activity in quails fed 2.0 p p m Se for a long term of 50 d. Replenishment of Se at 0.05- and 1.0-ppm levels did not correspondingly restore the hemolysate GSH-Px activities to long-term levels. Varied responses of GSH-Px activities to Se status have been reported for different species. Individual variations b e t w e e n species and Biological Trace Element Research
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Narayanaswami et al. TABLE 7 Effect of Se Replenishment on GSH-Px Activities in Hemolysate, RBC Membranes, and Hepatic Postmitochondrial Supernatant Fraction of Quails Maintained on Low-Selenium Diet2 '2 GSH-Px Activity~ Hemolysate
Level of Se ppm Basal 0.05 1.00 2.00
RBC membrane
U/rag protein 195.2 249.9 273.3 483.2
+_ + + +
14.8a 11.2b 19.4b 4.67*
U/rag protein 119.2 _+ 9.67 139.2 + 15.6 136.4 +_ 7.34 149.6 ___ 12.9
Liver supernatant U/rag protein 334.0 341.8 346.5 354.2
+_ 5.06 --- 7.47 _+ 6.4 + 44.2
U/rag tissue x 10 -3 33.35 35.14 35.47 42.3
_ 2.88 + 2.15 ___ 0.41 _+ 0.55
~Values are expressed as average -+ SEM. 2Means bearing the same superscript, do not vary significantly (P < 0.001). 3GSH-Px activity, units as defined in Table 3.
within the species a m o n g different organs have also been s h o w n (32). Results of investigations reported here suggest that the GSH-Px in the h e m o l y s a t e of quails is a fairly reliable indicator of the Se status of the system. The response of the mitochondrial GSH-Px to Se implies both a direct and indirect involvement of this element. Selenium plays a direct role by being an i n h e r e n t c o m p o n e n t of the s e l e n o e n z y m e GSH-Px a n d an indirect role by contributing to increased mitochondrial oxidations. Thus, Se m a y have a regulatory role not only in sulfhydryl metabolism, but also in the mitochondrial oxygen metabolism. Studies on the exact nature of interaction and extent of involvement of Se in the various cellular reactions, especially in the mitochondria, are in progress in our laboratory.
ACKNOWLEDGMENT The authors are grateful to Dr. K. Thomas Joseph, Central Leather Research Institute, for his keen interest, constant e n c o u r a g e m e n t , and m a n y helpful suggestions during the course of this work.
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