",r 1986 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163498418611004q)317S02.00
Selenium-Mediated Biochemical Changes in Japanese Quails II. Preliminary Studies on Glutathione Peroxidase Activity and Collagen Characteristics in the Skin MARY BABU, 1 PADMA BAI,' VASANTHY NARAYANASWAMI, 2 K. LALITHA, z* AND J O S E P H K. T H O M A S '
'Biopolyrner Laboratory, Central Leather Research Institute, Adyar, /Hadras 600 020, India; and 2Department of Chemistry, Indian Institute of Technology, M a d r a s 600 036, India Received April 21, 1986; Accepted May 8, 1986
ABSTRACT The effect of selenium (Se) on collagen characteristics and glutathione peroxidase (GSH-Px) activity in the skin of Japanese quails Coturnix coturnix japonica fed a formulated, semipurified, low-Se diet (basal) (0.05 ppm) was investigated. The quails exhibited severe Se-deficiency symptoms and significant reduction in skin GSH-Px activity at the end of 30 d. Selenium supplementation at a 2-ppm level restored the normal skin conditions and enhanced skin GSH-Px activity significantly. But a dietary Se level of 0.1 ppm was found to be inadequate in restoring the general skin conditions and GSH-Px activity. A markedly low total collagen content of about 23% was observed in the skin of quails fed the basal diet, compared to 39% of total collagen content in the skin of the 2-ppm Se-supplemented group. Molecular organization of skin collagen of quails on the basal and 0.l-ppm Se diet showed an abundance of monomeric forms with less crosslinks, compared to the presence of polymeric forms with more crosslinks, indicating enhanced stability in the skin collagen of quails on the 2-ppm diet. The delay in the in vitro fibril formation of collagen from the basal and 0.1-ppm Se groups, compared to a relatively faster rate in the case of the 2-ppm Se group, indicates a dis*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Resea[ch
31 7
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Babu et at turbance in the aggregation phenomenon of collagen. The increase in skin GSH-Px activity and concurrent increase in polymeric collagen on increasing the dietary Se level suggest a possible role for Se in collagen metabolism. Index Entries: Selenium, deficiency in the Japanese quails, Coturnix coturnix japonica; skin glutathione peroxidase and response to selenium level; selenium deficiency, and growth-related changes; selenium deficiency, and collagen metabolism; collagen, characteristics in selenium deficiency; collagen, metabolism and response to selenium level.
INTRODUCTION Selenium (Se) has gained sufficient importance in trace-element metabolism after the establishment of its essentiality about a decade ago (1). Since then, the biological interactions of Se have been the subject of intensive research. Deficiency of Se causes a variety of diseases, such as liver necrosis, exudative diathesis, nutritional muscular dystrophy, and so on, in farm animals, rats, chicks, and turkeys (2-4). These diseases have been found to be responsive to subsequent supplementation of Se (5). So far the essentiality of Se has been attributed to its role in the enzyme glutathione peroxidase (GSH-Px) (glutathione H202 oxidoreductase, EC 1.11.1.9), the only reported mammalian selenoenzyme (6). High-resolution X-ray crystallographic studies indicate the presence of selenocysteine at the active site of the enzyme (7). Glutathione peroxidase plays an important role in cellular detoxification mechanisms by removing hydrogen peroxide and other peroxides that are formed during various metabolic processes (8). Failure to remove these toxic intermediates would result in severe, free-radical-mediated damage to the cellular components (9). An attempt has been made to seek other possible roles for this element by studying the effect of Se on collagen metabolism. It has been shown that corn-soybean-based diets containing 0.06 ppm Se caused Se deficiency in ducks (10), with a decrease in collagen content of the tendons, which was attributed to degenerative changes in the fibroblast. This paper describes the studies of the effects of Se on collagen characteristics and GSH-Px in the skin of Japanese quails Coturnix coturnix japonica, wherein Se deficiency was created by the usage of a semipurified, low-Se diet (0.054 ppm) (11).
MATERIALS AND METHODS Glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase, M-ethyl maleimide, phenylmethyl Biological Dace Element Research
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sulphonyl fluoride, hydrogen peroxide, vitamins, methionine, choline chloride, acrylamide, methylene bis-acrylamide, ammonium persulphate, sodium selenite, N,N,N',N'-tetramethylethylenediamine, and pepsin were purchased from Sigma Chemical Co., USA. Other reagents used were of analar grade. Deionized water, containing no detectable Se, was used throughout, unless otherwise specified.
Experimental Design The semipurified, low-Se diet (basal) was prepared as described in our earlier paper (11), the Se content was 0.054-ppm, as determined by fluorimetric analysis (12). Selenium was supplemented as sodium selenite at dietary levels of 0.1 and 2.0 ppm. One-day-old chicks of Japanese quails, weighing 7-8 g, were obtained from the Poultry Research Station, Madras, India and divided at random into three groups of 10 and maintained on basal and 0.1- and 2.00-ppm Se-supplemented diets. Deionized water was given ad libitum. At the end of 30 d, the quails were sacrificed and the skins removed for collagen extraction; GSH-Px activity was determined in the homogenates of skin of the different groups of quail.
GSH-Px Assay Glutathione Peroxidase activity was assayed by a modification of the method of Paglia and Valentine (13), as adapted by Burk et al. (14), using 0.25 mM H202 as substrate. All assays were carried out in duplicate. Glutathione peroxidase activity was expressed as nmol NADPH oxidized/min/mg protein or/g tissue at 37~ Protein was determined by the method of Lowry et al. (15). Selenium was estimated by the fluorimetric method, using 2,3-diamino naphthalene (12). Statistical analysis was done by Student's t-test.
Isolation and Characterization of Collagen Sequential extraction of collagen from the skin samples was carried out (16) using neutral buffer 0.05M Tris-HCl, pH 7.5, containing 1M sodium chloride, in the presence of enzyme inhibitors, such as EDTA (20.00 mM), N-ethyl maleimide (2.00 mM), and phenyl methyl sulfonyl fluoride (1.0 raM) for 48 h. The residue was further extracted with 0.5M acetic acid for 48 h. The residue left after acid extraction was solubilized with pepsin in the ratio of 1:100 (enzyme:protein). The soluble fractions from salt, acid, and pepsin sequential extractions were subjected to the purification procedure of Miller and Rhodes (16) to obtain collagen; purified collagen from these fractions were lyophilized and stored at -20~ All operations were carried out at O--4~ Collagen content of the skin and other solubilized fractions were estimated by measuring the hydroxyproline content (17). Biological Trace Element Research
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Acid-extracted native collagen (0.2%) in 0.01M sodium phosphate buffer at pH 7.4, containing urea (2M) and sodium dodecyl sulfate (0.1%), was heated at 60~ for 15 min for controlled denaturation. The d e n a t u r e d collagen (200 tJ-g) was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (18), and the proportion of collagen subunit r forms and their conjugates ([3 and ~/) were densitometrically scanned at 660 nm. The rate of fibril formation was monitored at 30~ turbidimetrically at 540 nm (19).
RESULTS AND DISCUSSION Quail chicks fed the formulated, Iow-Se, semipurified diet manifested typical Se-deficiency symptoms, such as poor growth, feather loss, weak gait, and drastic deteriorative skin changes, whereas quails fed the Se-supplemented diets exhibited normal growth and feathering. An Se level of 0.1 ppm was found to be insufficient for the dietary requirement of quails that exhibited fragile skin and edema. Trial experiments have indicated that dietary Se levels less than 0.05 ppm resulted in weakness and poor growth in quails, sometimes leading to death. The semipurified diet used in the present studies was sufficiently low in Se content (0.054 ppm) to cause deteriorative skin changes, enabling the study of collagen characteristics in the skin of quails. Glutathione peroxidase activity in the skin of quails maintained on basal and Se-supplemented diets is represented in Table 1. Minimal activity of GSH-Px of7.8 U was observed in quails fed the basal diet. At 0.1and 2.00-ppm dietary Se levels, the skin GSH-Px activity increased to 9.8 and 15.0 U, respectively. A two-fold increase in GSH-Px activity at a dietary Se level of 2.00 ppm was observed. A dietary Se level of 0.1 ppm does not appear to meet the nutritional requirements for quails, as indicated by growth and general skin conditions of the quails as well as the GSH-Px activity in the skin. TABLE 1 Effect of Dietary Se on GSH-Px Activity and Collagen Content of the Skin of Japanese Quails '-' Dietary level of Se, pm Basal 0.1 2.0
GSH-Px activity, U/rng protein'
Collagen content, g/100 g tissue
7.8 + 0.75a 8.56 _+ 0.29~ 15.1 + 0.10 b
23.86 _+ 0.4 " 24.6 +_ 0.53a 39.2 _+ 0.46~'
'Values are expressed as average _+ SEM. :In a vertical column, means bearing the same superscript do not vary significantly; p < 0.001 between a and b. ~GSH-Px activity: 1 U represents 1 nmol NADPH oxidized/rain at 37~ Biolc~ical Trace Element Research
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The total collagen content of the skin of the quails fed various dietary levels of Se is indicated in Table 2. The collagen contents of quails fed the basal and 2.00-ppm Se diets were 23.8 and 39.2 g/100 g tissue, respectively. The low levels of collagen (25.6%) in the 0.1-ppm Se group clearly indicate that this dietary level of Se is inadequate for maintaining normal and healthy skin in quails. Preliminary investigations revealed that quails fed the commercial diet had a skin collagen content of about 40%. An Se level of 2,00 ppm appears to meet the nutritional requirements of quails for normal collagen metabolism in the skin. The pronounced reduction in total collagen content in the skin of the Se-deficient quails reflects yet another possible role for Se in collagen metabolism. This is further supported by the results of sequential extraction of collagen from the skin of quails maintained at different dietary Se levels (Table 2). Neutral salt extraction of skin solubilizes (20) the recently synthesized collagen in the monomeric form and the residue when subjected to treatment with 0.5M acetic acid extracts polymeric collagen in addition to monomeric forms. Subsequent treatment of the residue with pepsin solubilizes aggregated and highly crosslinked polymeric collagen by cleaving crosslinks present in the nonhelical regions of collagen. The insoluble residue left behind after these treatments consists of collagen that is crosslinked to a very large extent and in organized association with other macromolecules of connective tissue. The results of such sequential extraction indicate a significantly higher content of 8% of neutral salt-soluble collagen in the skin of deficient quails w h e n compared to 3.43% in the 2.00-ppm Se group, although no significant change was registered in the 0.1-ppm Se group. The acid-soluble collagen content in the 2.00-ppm Se group was about 80% of that in the deficient and 0.1-ppm Se group. On the contrary, the pepsin-soluble collagen content and the collagen content of insoluble residue in the 2.00-ppm Se group had increased by 34 and 66%, respectively. These changes clearly indicate that Se at a level of 2.00 ppm is essential for the process of collagen aggregation and maturation. This process is severely impaired not only in the deficient group, but also in TABLE 2 Relative Distribution of Collagen Fractions ~ in the Skin of Japanese Quails Dietary level of Se, ppm Basal 0.1 2.0
Neutral salt fraction
Acid soluble fraction
8.06 +_ 0.30~ 53.5 _+ 0.56" 7.38 _+ 0.35" 52.48 _+ 0.45" 3.43 +_ 0.33I' 42.9 _+ 0.43I'
Pepsin soluble fraction
Insoluble fraction
31.9 _+ 0.59~ 6.2 _+ 0.52~ 32.7 +- 0.48~ 6.4 _+ 0.34~ 41.66 _+ 0.40I' 10.56 _+ 0.44I'
'Values are expressed in percentages. Values are expressed as average _+ SEM. In a vertical column, means bearing the same superscript do not vary significantly; p < 0.001 between a and b. Biological Trace Element Research
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Babu et aL
the 0.1-ppm Se group. Further confirmation of these observations were obtained from the analysis of SDS-PAGE studies of acid-soluble collagen from the skin of quails from the basal and 2.00-ppm Se groups. The relative proportions of the subunit 0t and the aggregates 13and "7 are indicative of a corroborating abundance of monomeric or polymeric forms of collagen in the tissue. A highly crosslinked polymer of soluble collagen is less amenable to denaturation, resulting in more of the [~ and ~/units, with a much less et content. On the contrary, a soluble collagen with lesser extent of crosslinking will readily be denatured to individual e~ chains. A predominantly monomeric form of collagen in the skin of the low-Se group and a highly crosslinked polymeric collagen in the 2.00-ppm Se group are indicated by the results presented in Fig. 1. Relative proportions of the subunit forms ed and or2, represented as e~, and the aggregates 13and ~/, were calculated from densitometric scanning of the gel and shown in Table 3. The relative proportion of the e~ subunit and the aggregates in the low-Se group is 45:55, whereas in the 2.00-ppm Se group there is a drastic reduction in a ec subunit, the proportion being 17:83. Aggregation of the collagen molecule is evaluated by the rate of fibril formation. A delay in the fibril formation would indicate a disturbance in the aggregation phenomenon of collagen molecules. The progressive polymerization of the collagen molecule, which is monitored turbidimetrically as a function of time, is depicted in Fig. 2. The rate of collagen fibril formation in the low-Se group was much slower, with a t0.5 of 4.0 min, as against a t05 of 2.2 min observed for the 2.00-ppm SE group, which is comparable to that of the commercial-diet-fed quail skin collagen, with a t0.5 of 2.0 min. The low skin collagen content, GSH-Px activity, and pepsin-soluble collagen content and slow rate of aggregation of molecules in the low-Se basal group are clearly indicative of a role for Se in the metabolic processing of collagen, which is grossly altered in Se-deficiency conditions. Similar disturbances in the organization of collagen have also been reported in the tendons of Se-deficient ducks (10). Intra- and intermolecular crosslinks of collagen are fundamental prerequisites for strength and mechanical properties of the skin (21); the fragile skin of quails fed low-Se and 0.1-ppm dietary Se can thus be attributed to defects in the formation of such crosslinks. The increase in skin GSH-Px and the concurrent increase in polymeric collagen level, with an increasing dietary Se level, could have interesting implications regarding a role for Se and involvement of GSH-Px in the assembly of the collagenous framework. Protection of membranes by removal of peroxides is one of the well-known roles of GSH-Px (8). From the present studies a role for Se is indicated in the overall metabolism of collagen. That Se is contributing to normal aggregation and
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crosslinking of collagen is evidenced by these results. The decrease in the collagen content in Se deficiency could probably be a result of decreased collagen synthesis or increased degradation. As to the exact nature of events in Se deficiency and specific role for Se, more detailed studies are required.
9
A
.i! "if..~;
, *~iiJ B
I'l QI ~D
A
9
I
B
A
b
&
Fig. 1. Polyacrylamide gel electrophoretic profile of quail skin collagen and its densitometric scanning pattern: (1) 2.0-ppm Se-supplemented quails; (2) Basal diet fed quails.
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TABLE 3 Relative Proportions of Collagen Subunits c~, [3, and ~/ in Acid-Soluble Fraction of the Skin of Japanese Quails '-~ Relative proportions of subunits in percentages
Dietary level of Se, ppm Basal 2.0
~
[3
"y
45.4 _+ 0.39~ 16.75 • 0.38 I'
22.41 _+ 0.44" 38.51 _+ 0.37 t'
31.5 .+_ 0.44" 46.36 _+ 0.34 l'
' V a l u e s are e x p r e s s e d in p e r c e n t a g e s . ' V a l u e s are e x p r e s s e d as a v e r a g e _+ SEM; p < 0.001 b e t w e e n a a n b.
1.2
o
o.8
2 0.4
0
0
I z.
I 8
I 12
I 16
20
TIME ( m i n )
Fig. 2. Fibril formation of skin collagen of quails, in phosphate buffer at neutral pH at 30~ monitored at 540 nm: (1) Basal diet; (2) 0.1-ppm Se; (3) 2.0 ppm Se.
SO/vt/vL.MY In the p r e s e n t investigation, a f o r m u l a t e d semipurified c o r n - s o y bean meal c o n t a i n i n g a low a m o u n t of Se (0.05 p p m ) was f o u n d to create Se-deficiency s y m p t o m s in J a p a n e s e quails. In addition, a significant red u c t i o n in their skin GSH-Px activity a n d l o w e r e d total collagen content, p r e d o m i n a n c e of m o n o m e r i c forms of collagen with r e d u c e d crosslink c o n t e n t , a n d a slow rate of aggregation of their skin collagen w e r e observed. S u p p l e m e n t a t i o n of Se at a 2 - p p m level in the diet increased GSH-Px activity as well as total collagen c o n t e n t in the skin, w h i c h was f o u n d to contain m o r e p o l y m e r i c collagen a n d h a d a faster rate of aggreBiological Trace ElementResearch
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gation, indicating greater stability. S u p p l e m e n t a t i o n of Se at the 0.1-ppm level was s h o w n to be insufficient to restore the above normal conditions. The results suggest a possible role for Se in the metabolism of collagen.
ACKNOWLEDGMENT The a u t h o r s wish to thank Dr. G. Thyagarajan, Director of the Central Leather Research Institute, Madras, India, for his kind permission to publish this paper.
REFERENCES 1. T. C. Stadtman in Advances in Enzymoloqy, vol. 48, A. Meister, ed., John Wiley and Sons, New York, 1979, pp 1-128. 2. C. K. Chow and A. L. Tappel, ]. Nutr. 104, 444 (1974). 3. S. T. Omaye and A. L. Tappel, ]. Nutr. 104, 747 (1974). 4. S. tt. Oh, A. L. Pope, and W. G. Hoekstra, ]. Anita. Sci. 42, 984 (1976). 5. M. L. Scott, Nutr. Abstr. Rev. 32, 1 (1962). 6. J. 1". Rotruck, A. L. Pope, H. E. Ganther, D. G. Hafeman, and W. G. Hoekstra, Science 179, 588 (1973). 7. O. Epp. R. Ladenstein, and A. Wendel, Eur. J. Biochem. 133, 51 (1983). 8. C. K. Chow and A. L. Tappel, Lipids 7, 518 (1972). 9. L. Flohe, in Free Radicals in Biology, vol. 5 Pryor, W. A., ed., A. P., New York 1982, pp 223--249. 10. R. G. Brown, R. R. Sweeney, and E. T. Moran Jr., Comp. Biochem. Physiol. 72A, 383 (1982). 11. N. Vasanthy, R. Padma Bai, Mary Babu and K. Lalitha, Biol. Trace Element Res., 1985 (in press). 12. D. M. Ihnat, ]. Assoc. Off. Anal. Chem. 57, 368 (1974). 13. D. E. Paglia and W. N. Valentine, ]. Lab. Clin. Med. 70, 158 (1967). 14. R. R. Lawrence and R. F. Burk, Biochem. Biophys. Res. Commun. 71, 952 (1976). 15. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 16. E. J. Miller and R. K. Rhodes, Methods in Enzymology 82, 33 (1982). 17. R. E. Newman and M. A. Logan, ]. Biol. Chem. 184, 299 (1950). 18. H. Furthmayr and R. Timpl, Anal. Biochem. 41, 510 (1967). 19. J. Bello and M. R. Bello, Biochim. Biophys. Acta 147, 272 (1967). 20. K. A. Piez, in Treatise on Collagen, vol. 1, GN Ramachadran, ed., Academic, New York, 1967, pp 207-248. 21. N. D. Light and A. J. Bailey, in Biology of Collagen A. Viidik and J. Vuust, eds., Academic, New York, 1980, pp. 15--38.
Biological Trace Element Research
VoL 10. 1986
Selenium-Mediated Biochemical Changes in Japanese Quails II. Preliminary Studies on Glutathione Peroxidase Activity and Collagen Characteristics in the Skin MARY BABU, 1 PADMA BAI,' VASANTHY NARAYANASWAMI, 2 K. LALITHA, z* AND J O S E P H K. T H O M A S '
'Biopolyrner Laboratory, Central Leather Research Institute, Adyar, /Hadras 600 020, India; and 2Department of Chemistry, Indian Institute of Technology, M a d r a s 600 036, India Received April 21, 1986; Accepted May 8, 1986
ABSTRACT The effect of selenium (Se) on collagen characteristics and glutathione peroxidase (GSH-Px) activity in the skin of Japanese quails Coturnix coturnix japonica fed a formulated, semipurified, low-Se diet (basal) (0.05 ppm) was investigated. The quails exhibited severe Se-deficiency symptoms and significant reduction in skin GSH-Px activity at the end of 30 d. Selenium supplementation at a 2-ppm level restored the normal skin conditions and enhanced skin GSH-Px activity significantly. But a dietary Se level of 0.1 ppm was found to be inadequate in restoring the general skin conditions and GSH-Px activity. A markedly low total collagen content of about 23% was observed in the skin of quails fed the basal diet, compared to 39% of total collagen content in the skin of the 2-ppm Se-supplemented group. Molecular organization of skin collagen of quails on the basal and 0.l-ppm Se diet showed an abundance of monomeric forms with less crosslinks, compared to the presence of polymeric forms with more crosslinks, indicating enhanced stability in the skin collagen of quails on the 2-ppm diet. The delay in the in vitro fibril formation of collagen from the basal and 0.1-ppm Se groups, compared to a relatively faster rate in the case of the 2-ppm Se group, indicates a dis*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Resea[ch
31 7
Vol. l O, JgB6
318
Babu et at turbance in the aggregation phenomenon of collagen. The increase in skin GSH-Px activity and concurrent increase in polymeric collagen on increasing the dietary Se level suggest a possible role for Se in collagen metabolism. Index Entries: Selenium, deficiency in the Japanese quails, Coturnix coturnix japonica; skin glutathione peroxidase and response to selenium level; selenium deficiency, and growth-related changes; selenium deficiency, and collagen metabolism; collagen, characteristics in selenium deficiency; collagen, metabolism and response to selenium level.
INTRODUCTION Selenium (Se) has gained sufficient importance in trace-element metabolism after the establishment of its essentiality about a decade ago (1). Since then, the biological interactions of Se have been the subject of intensive research. Deficiency of Se causes a variety of diseases, such as liver necrosis, exudative diathesis, nutritional muscular dystrophy, and so on, in farm animals, rats, chicks, and turkeys (2-4). These diseases have been found to be responsive to subsequent supplementation of Se (5). So far the essentiality of Se has been attributed to its role in the enzyme glutathione peroxidase (GSH-Px) (glutathione H202 oxidoreductase, EC 1.11.1.9), the only reported mammalian selenoenzyme (6). High-resolution X-ray crystallographic studies indicate the presence of selenocysteine at the active site of the enzyme (7). Glutathione peroxidase plays an important role in cellular detoxification mechanisms by removing hydrogen peroxide and other peroxides that are formed during various metabolic processes (8). Failure to remove these toxic intermediates would result in severe, free-radical-mediated damage to the cellular components (9). An attempt has been made to seek other possible roles for this element by studying the effect of Se on collagen metabolism. It has been shown that corn-soybean-based diets containing 0.06 ppm Se caused Se deficiency in ducks (10), with a decrease in collagen content of the tendons, which was attributed to degenerative changes in the fibroblast. This paper describes the studies of the effects of Se on collagen characteristics and GSH-Px in the skin of Japanese quails Coturnix coturnix japonica, wherein Se deficiency was created by the usage of a semipurified, low-Se diet (0.054 ppm) (11).
MATERIALS AND METHODS Glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase, M-ethyl maleimide, phenylmethyl Biological Dace Element Research
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319
sulphonyl fluoride, hydrogen peroxide, vitamins, methionine, choline chloride, acrylamide, methylene bis-acrylamide, ammonium persulphate, sodium selenite, N,N,N',N'-tetramethylethylenediamine, and pepsin were purchased from Sigma Chemical Co., USA. Other reagents used were of analar grade. Deionized water, containing no detectable Se, was used throughout, unless otherwise specified.
Experimental Design The semipurified, low-Se diet (basal) was prepared as described in our earlier paper (11), the Se content was 0.054-ppm, as determined by fluorimetric analysis (12). Selenium was supplemented as sodium selenite at dietary levels of 0.1 and 2.0 ppm. One-day-old chicks of Japanese quails, weighing 7-8 g, were obtained from the Poultry Research Station, Madras, India and divided at random into three groups of 10 and maintained on basal and 0.1- and 2.00-ppm Se-supplemented diets. Deionized water was given ad libitum. At the end of 30 d, the quails were sacrificed and the skins removed for collagen extraction; GSH-Px activity was determined in the homogenates of skin of the different groups of quail.
GSH-Px Assay Glutathione Peroxidase activity was assayed by a modification of the method of Paglia and Valentine (13), as adapted by Burk et al. (14), using 0.25 mM H202 as substrate. All assays were carried out in duplicate. Glutathione peroxidase activity was expressed as nmol NADPH oxidized/min/mg protein or/g tissue at 37~ Protein was determined by the method of Lowry et al. (15). Selenium was estimated by the fluorimetric method, using 2,3-diamino naphthalene (12). Statistical analysis was done by Student's t-test.
Isolation and Characterization of Collagen Sequential extraction of collagen from the skin samples was carried out (16) using neutral buffer 0.05M Tris-HCl, pH 7.5, containing 1M sodium chloride, in the presence of enzyme inhibitors, such as EDTA (20.00 mM), N-ethyl maleimide (2.00 mM), and phenyl methyl sulfonyl fluoride (1.0 raM) for 48 h. The residue was further extracted with 0.5M acetic acid for 48 h. The residue left after acid extraction was solubilized with pepsin in the ratio of 1:100 (enzyme:protein). The soluble fractions from salt, acid, and pepsin sequential extractions were subjected to the purification procedure of Miller and Rhodes (16) to obtain collagen; purified collagen from these fractions were lyophilized and stored at -20~ All operations were carried out at O--4~ Collagen content of the skin and other solubilized fractions were estimated by measuring the hydroxyproline content (17). Biological Trace Element Research
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Acid-extracted native collagen (0.2%) in 0.01M sodium phosphate buffer at pH 7.4, containing urea (2M) and sodium dodecyl sulfate (0.1%), was heated at 60~ for 15 min for controlled denaturation. The d e n a t u r e d collagen (200 tJ-g) was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (18), and the proportion of collagen subunit r forms and their conjugates ([3 and ~/) were densitometrically scanned at 660 nm. The rate of fibril formation was monitored at 30~ turbidimetrically at 540 nm (19).
RESULTS AND DISCUSSION Quail chicks fed the formulated, Iow-Se, semipurified diet manifested typical Se-deficiency symptoms, such as poor growth, feather loss, weak gait, and drastic deteriorative skin changes, whereas quails fed the Se-supplemented diets exhibited normal growth and feathering. An Se level of 0.1 ppm was found to be insufficient for the dietary requirement of quails that exhibited fragile skin and edema. Trial experiments have indicated that dietary Se levels less than 0.05 ppm resulted in weakness and poor growth in quails, sometimes leading to death. The semipurified diet used in the present studies was sufficiently low in Se content (0.054 ppm) to cause deteriorative skin changes, enabling the study of collagen characteristics in the skin of quails. Glutathione peroxidase activity in the skin of quails maintained on basal and Se-supplemented diets is represented in Table 1. Minimal activity of GSH-Px of7.8 U was observed in quails fed the basal diet. At 0.1and 2.00-ppm dietary Se levels, the skin GSH-Px activity increased to 9.8 and 15.0 U, respectively. A two-fold increase in GSH-Px activity at a dietary Se level of 2.00 ppm was observed. A dietary Se level of 0.1 ppm does not appear to meet the nutritional requirements for quails, as indicated by growth and general skin conditions of the quails as well as the GSH-Px activity in the skin. TABLE 1 Effect of Dietary Se on GSH-Px Activity and Collagen Content of the Skin of Japanese Quails '-' Dietary level of Se, pm Basal 0.1 2.0
GSH-Px activity, U/rng protein'
Collagen content, g/100 g tissue
7.8 + 0.75a 8.56 _+ 0.29~ 15.1 + 0.10 b
23.86 _+ 0.4 " 24.6 +_ 0.53a 39.2 _+ 0.46~'
'Values are expressed as average _+ SEM. :In a vertical column, means bearing the same superscript do not vary significantly; p < 0.001 between a and b. ~GSH-Px activity: 1 U represents 1 nmol NADPH oxidized/rain at 37~ Biolc~ical Trace Element Research
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The total collagen content of the skin of the quails fed various dietary levels of Se is indicated in Table 2. The collagen contents of quails fed the basal and 2.00-ppm Se diets were 23.8 and 39.2 g/100 g tissue, respectively. The low levels of collagen (25.6%) in the 0.1-ppm Se group clearly indicate that this dietary level of Se is inadequate for maintaining normal and healthy skin in quails. Preliminary investigations revealed that quails fed the commercial diet had a skin collagen content of about 40%. An Se level of 2,00 ppm appears to meet the nutritional requirements of quails for normal collagen metabolism in the skin. The pronounced reduction in total collagen content in the skin of the Se-deficient quails reflects yet another possible role for Se in collagen metabolism. This is further supported by the results of sequential extraction of collagen from the skin of quails maintained at different dietary Se levels (Table 2). Neutral salt extraction of skin solubilizes (20) the recently synthesized collagen in the monomeric form and the residue when subjected to treatment with 0.5M acetic acid extracts polymeric collagen in addition to monomeric forms. Subsequent treatment of the residue with pepsin solubilizes aggregated and highly crosslinked polymeric collagen by cleaving crosslinks present in the nonhelical regions of collagen. The insoluble residue left behind after these treatments consists of collagen that is crosslinked to a very large extent and in organized association with other macromolecules of connective tissue. The results of such sequential extraction indicate a significantly higher content of 8% of neutral salt-soluble collagen in the skin of deficient quails w h e n compared to 3.43% in the 2.00-ppm Se group, although no significant change was registered in the 0.1-ppm Se group. The acid-soluble collagen content in the 2.00-ppm Se group was about 80% of that in the deficient and 0.1-ppm Se group. On the contrary, the pepsin-soluble collagen content and the collagen content of insoluble residue in the 2.00-ppm Se group had increased by 34 and 66%, respectively. These changes clearly indicate that Se at a level of 2.00 ppm is essential for the process of collagen aggregation and maturation. This process is severely impaired not only in the deficient group, but also in TABLE 2 Relative Distribution of Collagen Fractions ~ in the Skin of Japanese Quails Dietary level of Se, ppm Basal 0.1 2.0
Neutral salt fraction
Acid soluble fraction
8.06 +_ 0.30~ 53.5 _+ 0.56" 7.38 _+ 0.35" 52.48 _+ 0.45" 3.43 +_ 0.33I' 42.9 _+ 0.43I'
Pepsin soluble fraction
Insoluble fraction
31.9 _+ 0.59~ 6.2 _+ 0.52~ 32.7 +- 0.48~ 6.4 _+ 0.34~ 41.66 _+ 0.40I' 10.56 _+ 0.44I'
'Values are expressed in percentages. Values are expressed as average _+ SEM. In a vertical column, means bearing the same superscript do not vary significantly; p < 0.001 between a and b. Biological Trace Element Research
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Babu et aL
the 0.1-ppm Se group. Further confirmation of these observations were obtained from the analysis of SDS-PAGE studies of acid-soluble collagen from the skin of quails from the basal and 2.00-ppm Se groups. The relative proportions of the subunit 0t and the aggregates 13and "7 are indicative of a corroborating abundance of monomeric or polymeric forms of collagen in the tissue. A highly crosslinked polymer of soluble collagen is less amenable to denaturation, resulting in more of the [~ and ~/units, with a much less et content. On the contrary, a soluble collagen with lesser extent of crosslinking will readily be denatured to individual e~ chains. A predominantly monomeric form of collagen in the skin of the low-Se group and a highly crosslinked polymeric collagen in the 2.00-ppm Se group are indicated by the results presented in Fig. 1. Relative proportions of the subunit forms ed and or2, represented as e~, and the aggregates 13and ~/, were calculated from densitometric scanning of the gel and shown in Table 3. The relative proportion of the e~ subunit and the aggregates in the low-Se group is 45:55, whereas in the 2.00-ppm Se group there is a drastic reduction in a ec subunit, the proportion being 17:83. Aggregation of the collagen molecule is evaluated by the rate of fibril formation. A delay in the fibril formation would indicate a disturbance in the aggregation phenomenon of collagen molecules. The progressive polymerization of the collagen molecule, which is monitored turbidimetrically as a function of time, is depicted in Fig. 2. The rate of collagen fibril formation in the low-Se group was much slower, with a t0.5 of 4.0 min, as against a t05 of 2.2 min observed for the 2.00-ppm SE group, which is comparable to that of the commercial-diet-fed quail skin collagen, with a t0.5 of 2.0 min. The low skin collagen content, GSH-Px activity, and pepsin-soluble collagen content and slow rate of aggregation of molecules in the low-Se basal group are clearly indicative of a role for Se in the metabolic processing of collagen, which is grossly altered in Se-deficiency conditions. Similar disturbances in the organization of collagen have also been reported in the tendons of Se-deficient ducks (10). Intra- and intermolecular crosslinks of collagen are fundamental prerequisites for strength and mechanical properties of the skin (21); the fragile skin of quails fed low-Se and 0.1-ppm dietary Se can thus be attributed to defects in the formation of such crosslinks. The increase in skin GSH-Px and the concurrent increase in polymeric collagen level, with an increasing dietary Se level, could have interesting implications regarding a role for Se and involvement of GSH-Px in the assembly of the collagenous framework. Protection of membranes by removal of peroxides is one of the well-known roles of GSH-Px (8). From the present studies a role for Se is indicated in the overall metabolism of collagen. That Se is contributing to normal aggregation and
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crosslinking of collagen is evidenced by these results. The decrease in the collagen content in Se deficiency could probably be a result of decreased collagen synthesis or increased degradation. As to the exact nature of events in Se deficiency and specific role for Se, more detailed studies are required.
9
A
.i! "if..~;
, *~iiJ B
I'l QI ~D
A
9
I
B
A
b
&
Fig. 1. Polyacrylamide gel electrophoretic profile of quail skin collagen and its densitometric scanning pattern: (1) 2.0-ppm Se-supplemented quails; (2) Basal diet fed quails.
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TABLE 3 Relative Proportions of Collagen Subunits c~, [3, and ~/ in Acid-Soluble Fraction of the Skin of Japanese Quails '-~ Relative proportions of subunits in percentages
Dietary level of Se, ppm Basal 2.0
~
[3
"y
45.4 _+ 0.39~ 16.75 • 0.38 I'
22.41 _+ 0.44" 38.51 _+ 0.37 t'
31.5 .+_ 0.44" 46.36 _+ 0.34 l'
' V a l u e s are e x p r e s s e d in p e r c e n t a g e s . ' V a l u e s are e x p r e s s e d as a v e r a g e _+ SEM; p < 0.001 b e t w e e n a a n b.
1.2
o
o.8
2 0.4
0
0
I z.
I 8
I 12
I 16
20
TIME ( m i n )
Fig. 2. Fibril formation of skin collagen of quails, in phosphate buffer at neutral pH at 30~ monitored at 540 nm: (1) Basal diet; (2) 0.1-ppm Se; (3) 2.0 ppm Se.
SO/vt/vL.MY In the p r e s e n t investigation, a f o r m u l a t e d semipurified c o r n - s o y bean meal c o n t a i n i n g a low a m o u n t of Se (0.05 p p m ) was f o u n d to create Se-deficiency s y m p t o m s in J a p a n e s e quails. In addition, a significant red u c t i o n in their skin GSH-Px activity a n d l o w e r e d total collagen content, p r e d o m i n a n c e of m o n o m e r i c forms of collagen with r e d u c e d crosslink c o n t e n t , a n d a slow rate of aggregation of their skin collagen w e r e observed. S u p p l e m e n t a t i o n of Se at a 2 - p p m level in the diet increased GSH-Px activity as well as total collagen c o n t e n t in the skin, w h i c h was f o u n d to contain m o r e p o l y m e r i c collagen a n d h a d a faster rate of aggreBiological Trace ElementResearch
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gation, indicating greater stability. S u p p l e m e n t a t i o n of Se at the 0.1-ppm level was s h o w n to be insufficient to restore the above normal conditions. The results suggest a possible role for Se in the metabolism of collagen.
ACKNOWLEDGMENT The a u t h o r s wish to thank Dr. G. Thyagarajan, Director of the Central Leather Research Institute, Madras, India, for his kind permission to publish this paper.
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