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Plasma Variation of Transferrin-Iron and Phosvitin-Iron During the Laying Period in Chicken Hens
Plasma Variation of Transferrin-Iron and Phosvitin-Iron During the Laying Period in Chicken Hens M. A. LOPEZ-BERJES and J. M. RECIO Catedra de Fisiologia Animal, Facultad de Ciencias, Universidad de Valladolid, Valladolid, Spain J.PLANAS Catedra de Fisiologia Animal, Facultad de Biologia, Universidad de Barcelona, Barcelona, Spain (Received for publication April 7, 1980)
1981 Poultry Science 60:1951-1956
INTRODUCTION
The presence of two iron-carrier proteins in bird plasma is well known. The existence of a transferrin auxiliary mechanism was suggested by our studies on plasma iron in laying hens (Planas and de Castro, I960; Planas et al, 1961). Greengard et al. (1965) suggested that the iron in plasma of estrogenized chickens might be bound to a phosphoprotein (phosvitin). A demonstration of this suggestion can be seen in the works of Ali and Ramsay (1968, 1974) and Morgan (1975). Osaki et al. (1975) analyzed the Fe (II) oxidative properties of the phosvitin and the migration of iron from this phosphoprotein to transferrin and its possible significance in the iron metabolism of laying hens. However, the quantitative variation of the iron bound to both protein carriers during the course of the adult life span in females has not been studied. For that reason, the subject was studied in females during the interval extending from the immature sexual state until decreased laying due to aging. MATERIALS AND METHODS The hens used were Double Back Hybrids,
H-5, located at the installations of the poultry breeder "Hibridos Americanos de Valladolid" in Vega de Tirados, Salamanca, during the experiment. They were kept in four battery cages, and 20 hens were selected and labeled when they were 20 weeks old. Egg production of the group was recorded daily for each cage (5 hens) and checked against the production of the 6000 hens in the building. Six of the 20 experimental females, selected at random, were bled for the first time at the age of 20 weeks. They were bled once a week up to the 29th week and subsequently bled on the 41st, 62nd, and 98th weeks. Blood samples were always drawn with heparinized syringes at about 10 am from the wing vein after a 10 to 12 hr fast. Blood was centrifuged immediately to separate the plasma. Six White Rock Cornish roosters, obtained from a commercial poultry breeder, were kept in large single battery cages under natural light in the laboratory. All the birds had free access to commercial food and water. The 6 adult male chickens were treated with an intramuscular injection of |3-estradiol benzoate (Schering® vials) at dose level of 5 mg/kg body weight. The hormone was injected
1951
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
ABSTRACT The distribution of plasma iron between transferrin-iron (Tf-Fe) and phosvitin-iron (Phv-Fe) over a 98 week laying period was established in hens. Six plasma samples were collected at random from a group of 20 pullets, starting when the birds were 20 weeks old. Samples were collected weekly until the 29th week and subsequently on the 41st, 62nd, and 98th weeks. Detailed assays were carried out to differentiate Phv-Fe from Tf-Fe. Phosphoprotein (phosvitin) concentrations were also determined. At the beginning of sexual activity, (23 to 24 weeks) females showed a parallel increase of Tf-Fe and Phv-Fe. During the period of maximum egg production (29 to 41 weeks) Tf-Fe was 65% and the Phv-Fe 35% of the total plasma iron. During this period the transferrin saturation reached a maximum of 80%; both transport mechanisms worked simultaneously. Estrogen administration to male chickens (2 injections of 5 mg/kg body weight) precipitated similar variation patterns of the two plasma irons over a period of 7 days. It is suggested that the estrogens were responsible for these variations. (Key words: laying hen, plasma iron, phosvitin, transferrin, estrogen)
1952
LOPEZ-BERJES ET AL.
j Fe/l00mj pli
/•'•
20
22 23 24 25 26 27 28 29
TIBC
41
T^S^-J
62
98
FIG. 1. Plasma iron (PI), total iron binding capacity (TIBC) of the plasma, and the egg production in a lot of 20 hens 20 to 98 weeks of age.
RESULTS
Variations of the plasma iron and the TIBC of the transferrin in the females over 78 weeks are shown in Figure 1. Plasma iron variations and the distribution between transferrin and phosvitin, as well as the values of the total iron binding of the transferrin and the phosphoprotein (phosvitin) concentration, in a random lot of 6 hens (20 to 98 weeks of age) are shown in Table 1. The plasma iron designated as "total" refers to values obtained by the method of ICSH (1971); the "transferrin iron" refers to the value after magnesium carbonate fractionation (Planas et al, 1961). The difference between the two values was assumed to be the phosvitin-Fe. The TIBC obtained using Ramsay's method (1957) expressed the transferrin concentration and the saturation was calculated according to the "transferrin iron". The immature hens (20 to 22 weeks old) had very low phosvitin concentrations and plasma iron was mainly bound to the transferrin (81 to 92%). Howtver, the beginning of the laying period was followed by a parallel increase in the phosphoprotein, transferrin, and total plasma iron concentration. The maximum peak in the plasma levels of these parameters was concomitant to highest egg production (>90%) in 29-week-old hens. However, the plasma iron and the transferrin iron were not statistically significant in the case of 28 to 41-week-old hens, while the phosvitin content was significantly high in 29 and 41-week-old hens (Table 1). Saturation of the transferrin was normally low in nonlaying hens, reaching only approximately 50%. During the laying period the saturation percentage increased, reaching a maximum of 80% during the 26th to 29th weeks, at which time plasma iron values were greater than the binding capacity of the transferrin. The progressive increase in plasma iron up to the age of 29 weeks (90% egg production) was due more to the transferrin (59%) than to phosvitin (41%) (Table 1). The plasma phosvitiniron was directly related to the increase of the phosphorus of phosvitin. The ratio of phosvitin-iron to phosvitin-phosphorus was constant (.34-.38). The experimental group established by estrogenizing male chickens showed similar plasma patterns. The natural variation of
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
at time zero and again 24 hr later. Blood samples were taken using the procedure described at 48, 72, 96, and 168 hr after the initial estrogen injection. This group of roosters was used as an experimental model to show how estrogen can produce a similar variation during the short period of 1 week. Whole plasma iron was determined by the method described by the International Committee for Standardization in Hematology (ICSH, 1971). The total iron binding capacity (TIBC) was determined following the technique of Ramsay (1957). The plasma iron determination was also carried out on aliquots of plasma that had been previously fractioned with magnesium carbonate to eliminate the nontransferrin iron. This procedure was done in accordance with that of Planas et al. (1961); 1 ml plasma plus 2 ml distilled water and 200 mg magnesium carbonate were shaken for 30 min and then centrifuged. The clear supernatant contained the Tf-Fe and solid carbonate contained the iron that was bound to the phosvitin. The phosvitin determination for chicken plasma was carried out after the precipitation with HCIO4. The lipids were eliminated from the precipitate by shaking with ethanol/ether (3:1) and cloroform/ether (1:1). The lipid-free precipitate was then hydrolyzed with KOH at 37 C and the phosphate determined by the technique of Allen (1940). Statistical analysis of data was done with a simple analysis of variance using the individual values and then applied to the range test of Wilcoxon Mann Whitney.
F=69 P<.001
Simple analysis of variance
F=69 P<.001
6 13NS2 24* 15NS 22** 16NS 32** 24NS 19NS 22NS 15* 26**
F=63 P<.001
115 ± 6 117 ± IONS 166 + 16* 195 ± IONS 2 9 0 ± 15** 310±IONS 380 ± 17** 379 ± 13NS 401 ±IONS 370 ± 15NS 340 + H N S 256 ± 19**
Tf-Fe
Plasma iron (/ig %)
+ ± ± + + ± ± + ± ± + + 3 3NS 10** 6NS 9* 6NS 15** 12NS IONS IONS 5* 8**
F=68 P<.001
11 16 67 80 112 127 202 201 210 206 170 103
Phv-Fe
(Tf-Fe), phosvitin-iron (Phv-Fe), total iron binding in blood plasma of females1
(T
± ± ± ± ± ± ± ± ± ± ± ±
F=22 P<.0
230 257 272 309 400 390 475 554 514 524 457 416
TIBC (Mg %
capacity
N u m b e r of specimens = 6. Mean + SE. Range test Wilcoxon Mann Whitney Egg p r o d u c t i o n is calculated from th
N S , nonsignificant.
**P<.01.
*P<.05.
2
1
125 133 233 275 402 437 582 580 610 576 510 355
0 0 15 25 40 60 75 90 >90 >90 55 35
20 20 23 24 25 26 27 28 29 41 62 98
± ± ± ± ± ± + ± ± ± ± ±
Total
Egg production (%)
Age (weeks)
T A B L E 1. Total iron, transferrin-iron
from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
F=167 P<.001
Simple analysis of variance
± J** + 23** ± 18* + 8**
9*
6
± 6** + 19** + 19NS ± 7* *
F=82 P<.001
186 362 323 97
143 ±
117 ±
Tf-Fe
Plasma iron (jug %)
**P<.01.
3
± 6NS ± 15** ± IONS ± 3**
F=53 P<.001
67 196 165 20
50 ± 10**
19 ±
Phv-Fe
Mean + SE. Number of specimens = 6. Range test of Wilcoxon Mann Whitney.
NS, nonsignificant.
*P<,05.
2
1
253 558 488 117
7
193 + 1 3 * *
136 +
Total
0 Injection 24 Injection 48 72 96 168
Time (hr)
± 1 ± 2 ± ± 1
F=33 P<.001
288 453 404 227
262 ± 1
251 ± 1
TIBC (Mg %)
TABLE 2. Total iron, transferrin-iron (Tf-Fe), phosvitin-iron (Phv-Fe), total iron binding c phosphoprotein (Phv) levels in blood plasma of male chickens treated w (5 mg estradiol benzoate/kg BW)1
d from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
TRANSFERRIN AND PHOSVITIN-IRON LAYING HENS
plasma iron observed in laying hens was simulated, quite well, by this experimental model after the short period of only 168 hr (Table 2). DISCUSSION
In earlier studies, the nontransferrin Fe in laying hens was found to be 50%, and there were no differences in transferrin content between laying and nonlaying hens (Planas and de Castro, I960; Planas et al, 1961). The fact that data were segregated by age and reproductive status in the current study, as opposed to a random sampling in the earlier studies, could explain the differences. Also, in the more detailed current study, it appears that the transferrin in laying hens was not necessarily saturated with iron, as had been previously suggested by Planas et al. (1961) and
Morgan (1975). So, it could be that both transport mechanisms work simultaneously from the beginning of sexual activity (Table 1). Data obtained with estrogenized males were similar to those of laying hens. Therefore, we could assume that estrogens are the direct causative agent of the response. In birds, estrogen effects iron mobilization for egg production and simultaneously provides an additional transport mechanism for iron distribution. This should be interpreted as an adaptative response. It seems that the physiological destination of iron bound to each carrier is not the same. According to Morgan (1975), the phosvitin-Fe goes to the ovaries to be incorporated into the yolk and the hematopoietic organs receive the transferrin-iron. Further experimental work would be necessary to establish support for these two separate roles of plasma iron in birds. ACKNOWLEDGMENT
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
Transferrin is the universal iron carrier in vertebrate plasma. However, in aves a plasma phosphoprotein (the phosvitin) also contributes to this work. The phosvitin concentration depends on the estrogen levels for its synthesis and can also be induced in males after estrogen administration. In laying hens, as opposed to immature pullets, the increased levels of transferrin and phosvitin were probably due to changes in plasma estrogen levels. Radioimmunoassays for estrogen during the ovulatory cycle in hens (Peterson and Common, 1972) and under different reproductive conditions (Senior, 1974) support this assertion. Estrogen enhances iron metabolism in birds. The laying period presents a rise in plasma iron valus (Ramsay and Campbell, 1954; Planas et al., 1961) and an increase in the plasma iron turnover (Planas and Balasch, 1970; Balasch and Planas, 1972). At the same time, estrogens stimulate phosvitin synthesis in the chicken liver (Heald and McLachlan, 1963; Beuving and Gruber, 1971; Greengard et al., 1964); it was first suggested by Greengard et al. (1965) that phosvitin might participate in plasma iron transport. This fact was clearly established in the papers of Ali and Ramsay (1968, 1974) and Morgan (1975). However, the beginning and the quantitative participation of phosvitin in plasma iron transport was still unknown. The present data (Table 1) show that during the laying period phosvitin carried from 29 to 36% of the plasma iron. Transferrin at this time was near the saturation level of 63 to 80% and the phosvitin acted as an auxiliary transport mechanism.
1955
The authors are indebted to J. Gallego of the Department of Statistics, University of Valladolid, for his expertise and guidance in the statistical and computer analysis of the data. REFERENCES
Allen, R.J.L., 1940. The estimation of phosphorus. Biochem.J. 34:858-865. Ali, K. E., and W.N.M. Ramsay, 1968. Phosphoprotein-bound iron in the plasma of the hying hen. Biochem.J. 110:36. Ali, K. E., and W.N.M. Ramsay, 1974. Phosphoprotein-bound iron in the blood of plasma of the laying hen. Q. J. Exp. Physiol. 59:159-165. Balasch, J., and J. Planas, 1972. Iron metabolism in duck and turkey. Rev. Espan. Fisiol. 28:125— 128. Beuving, G., and M. Gruber, 1971. Induction of phosvitin synthesis in roosters by estradiol injection. Biochem. Biophys. Acta 232:529-536. Greengard, O., M. Gordon, M. A. Smith, and G. Acs, 1964. Studies on the mechanism of diethylstilbesterol-induced formation of phosphoprotein in male chickens. J. Biol. Chem. 329:20792082. Greengard, O., N. Mendelsohn, and M. Gordon, 1965. Iron accumulation in cockerel plasma after estrogen relation to induced phosphoprotein synthesis. Science 147:1571-1572. Heald, P. J., and P. M. McLachlan, 1963. Isolation of phosvitin from the plasma of the laying hen. Biochem. J. 87:571-576. International Committee for Standardization in Hematology, 1971. Brit. J. Haematol. 20:451-453. Morgan, E. H., 1975. Plasma iron transport during egg laying and after oestrogen administration in domestic fowl (Gallus domesticus). Q. J. Exp. Physiol. 60:233-247.
1956
LOPEZ-BERJES ET AL.
Osaki, S., R. C. Sexton, E. Pascual, and E. Frieden, 1975. Iron oxidation and transferrin formation by phosvitin. Biochem. J. 151:519-525. Peterson, A. J., and R. H. Common, 1972. Estrone and estradiol concentrations in peripheral plasma of laying hens as determined by radioimmunoassay. Can. J. Zool. 50:395—404. Planas, J., and J. Balasch, 1970. Blood iron metabolism in fowl and rabbit. Rev. Espan. Fisiol. 26: 307-314. Planas, J., and S. de Castro, 1960. El transporte del hierro serico en las gallinas en relaci6n con la puesta. Rev. Espan. Fisiol. 16:197—205. Planas, J., S. de Castro, and J. M. Recio, 1961. Serum
iron and its transport mechanism in fowl. Nature 189:668-669. Ramsay, W.N.M., 1957. Determination of the total iron binding capacity of serum. Clin. Chim. Acta 2:221-226. Ramsay, W.N.M., and E. A. Campbell, 1954. Iron metabolism in the laying hen. Biochem. J. 58: 313-317. Senior, B. E., 1974. Radioimmunoassay of the oestrone and oestradiol in the peripheral plasma of the domestic fowl in various physiological states and on hypophysectomized and ovariectomized fowls. Acta Endocrinol. 75: 133-140.
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
1981 Poultry Science 60:1951-1956
INTRODUCTION
The presence of two iron-carrier proteins in bird plasma is well known. The existence of a transferrin auxiliary mechanism was suggested by our studies on plasma iron in laying hens (Planas and de Castro, I960; Planas et al, 1961). Greengard et al. (1965) suggested that the iron in plasma of estrogenized chickens might be bound to a phosphoprotein (phosvitin). A demonstration of this suggestion can be seen in the works of Ali and Ramsay (1968, 1974) and Morgan (1975). Osaki et al. (1975) analyzed the Fe (II) oxidative properties of the phosvitin and the migration of iron from this phosphoprotein to transferrin and its possible significance in the iron metabolism of laying hens. However, the quantitative variation of the iron bound to both protein carriers during the course of the adult life span in females has not been studied. For that reason, the subject was studied in females during the interval extending from the immature sexual state until decreased laying due to aging. MATERIALS AND METHODS The hens used were Double Back Hybrids,
H-5, located at the installations of the poultry breeder "Hibridos Americanos de Valladolid" in Vega de Tirados, Salamanca, during the experiment. They were kept in four battery cages, and 20 hens were selected and labeled when they were 20 weeks old. Egg production of the group was recorded daily for each cage (5 hens) and checked against the production of the 6000 hens in the building. Six of the 20 experimental females, selected at random, were bled for the first time at the age of 20 weeks. They were bled once a week up to the 29th week and subsequently bled on the 41st, 62nd, and 98th weeks. Blood samples were always drawn with heparinized syringes at about 10 am from the wing vein after a 10 to 12 hr fast. Blood was centrifuged immediately to separate the plasma. Six White Rock Cornish roosters, obtained from a commercial poultry breeder, were kept in large single battery cages under natural light in the laboratory. All the birds had free access to commercial food and water. The 6 adult male chickens were treated with an intramuscular injection of |3-estradiol benzoate (Schering® vials) at dose level of 5 mg/kg body weight. The hormone was injected
1951
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
ABSTRACT The distribution of plasma iron between transferrin-iron (Tf-Fe) and phosvitin-iron (Phv-Fe) over a 98 week laying period was established in hens. Six plasma samples were collected at random from a group of 20 pullets, starting when the birds were 20 weeks old. Samples were collected weekly until the 29th week and subsequently on the 41st, 62nd, and 98th weeks. Detailed assays were carried out to differentiate Phv-Fe from Tf-Fe. Phosphoprotein (phosvitin) concentrations were also determined. At the beginning of sexual activity, (23 to 24 weeks) females showed a parallel increase of Tf-Fe and Phv-Fe. During the period of maximum egg production (29 to 41 weeks) Tf-Fe was 65% and the Phv-Fe 35% of the total plasma iron. During this period the transferrin saturation reached a maximum of 80%; both transport mechanisms worked simultaneously. Estrogen administration to male chickens (2 injections of 5 mg/kg body weight) precipitated similar variation patterns of the two plasma irons over a period of 7 days. It is suggested that the estrogens were responsible for these variations. (Key words: laying hen, plasma iron, phosvitin, transferrin, estrogen)
1952
LOPEZ-BERJES ET AL.
j Fe/l00mj pli
/•'•
20
22 23 24 25 26 27 28 29
TIBC
41
T^S^-J
62
98
FIG. 1. Plasma iron (PI), total iron binding capacity (TIBC) of the plasma, and the egg production in a lot of 20 hens 20 to 98 weeks of age.
RESULTS
Variations of the plasma iron and the TIBC of the transferrin in the females over 78 weeks are shown in Figure 1. Plasma iron variations and the distribution between transferrin and phosvitin, as well as the values of the total iron binding of the transferrin and the phosphoprotein (phosvitin) concentration, in a random lot of 6 hens (20 to 98 weeks of age) are shown in Table 1. The plasma iron designated as "total" refers to values obtained by the method of ICSH (1971); the "transferrin iron" refers to the value after magnesium carbonate fractionation (Planas et al, 1961). The difference between the two values was assumed to be the phosvitin-Fe. The TIBC obtained using Ramsay's method (1957) expressed the transferrin concentration and the saturation was calculated according to the "transferrin iron". The immature hens (20 to 22 weeks old) had very low phosvitin concentrations and plasma iron was mainly bound to the transferrin (81 to 92%). Howtver, the beginning of the laying period was followed by a parallel increase in the phosphoprotein, transferrin, and total plasma iron concentration. The maximum peak in the plasma levels of these parameters was concomitant to highest egg production (>90%) in 29-week-old hens. However, the plasma iron and the transferrin iron were not statistically significant in the case of 28 to 41-week-old hens, while the phosvitin content was significantly high in 29 and 41-week-old hens (Table 1). Saturation of the transferrin was normally low in nonlaying hens, reaching only approximately 50%. During the laying period the saturation percentage increased, reaching a maximum of 80% during the 26th to 29th weeks, at which time plasma iron values were greater than the binding capacity of the transferrin. The progressive increase in plasma iron up to the age of 29 weeks (90% egg production) was due more to the transferrin (59%) than to phosvitin (41%) (Table 1). The plasma phosvitiniron was directly related to the increase of the phosphorus of phosvitin. The ratio of phosvitin-iron to phosvitin-phosphorus was constant (.34-.38). The experimental group established by estrogenizing male chickens showed similar plasma patterns. The natural variation of
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
at time zero and again 24 hr later. Blood samples were taken using the procedure described at 48, 72, 96, and 168 hr after the initial estrogen injection. This group of roosters was used as an experimental model to show how estrogen can produce a similar variation during the short period of 1 week. Whole plasma iron was determined by the method described by the International Committee for Standardization in Hematology (ICSH, 1971). The total iron binding capacity (TIBC) was determined following the technique of Ramsay (1957). The plasma iron determination was also carried out on aliquots of plasma that had been previously fractioned with magnesium carbonate to eliminate the nontransferrin iron. This procedure was done in accordance with that of Planas et al. (1961); 1 ml plasma plus 2 ml distilled water and 200 mg magnesium carbonate were shaken for 30 min and then centrifuged. The clear supernatant contained the Tf-Fe and solid carbonate contained the iron that was bound to the phosvitin. The phosvitin determination for chicken plasma was carried out after the precipitation with HCIO4. The lipids were eliminated from the precipitate by shaking with ethanol/ether (3:1) and cloroform/ether (1:1). The lipid-free precipitate was then hydrolyzed with KOH at 37 C and the phosphate determined by the technique of Allen (1940). Statistical analysis of data was done with a simple analysis of variance using the individual values and then applied to the range test of Wilcoxon Mann Whitney.
F=69 P<.001
Simple analysis of variance
F=69 P<.001
6 13NS2 24* 15NS 22** 16NS 32** 24NS 19NS 22NS 15* 26**
F=63 P<.001
115 ± 6 117 ± IONS 166 + 16* 195 ± IONS 2 9 0 ± 15** 310±IONS 380 ± 17** 379 ± 13NS 401 ±IONS 370 ± 15NS 340 + H N S 256 ± 19**
Tf-Fe
Plasma iron (/ig %)
+ ± ± + + ± ± + ± ± + + 3 3NS 10** 6NS 9* 6NS 15** 12NS IONS IONS 5* 8**
F=68 P<.001
11 16 67 80 112 127 202 201 210 206 170 103
Phv-Fe
(Tf-Fe), phosvitin-iron (Phv-Fe), total iron binding in blood plasma of females1
(T
± ± ± ± ± ± ± ± ± ± ± ±
F=22 P<.0
230 257 272 309 400 390 475 554 514 524 457 416
TIBC (Mg %
capacity
N u m b e r of specimens = 6. Mean + SE. Range test Wilcoxon Mann Whitney Egg p r o d u c t i o n is calculated from th
N S , nonsignificant.
**P<.01.
*P<.05.
2
1
125 133 233 275 402 437 582 580 610 576 510 355
0 0 15 25 40 60 75 90 >90 >90 55 35
20 20 23 24 25 26 27 28 29 41 62 98
± ± ± ± ± ± + ± ± ± ± ±
Total
Egg production (%)
Age (weeks)
T A B L E 1. Total iron, transferrin-iron
from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
F=167 P<.001
Simple analysis of variance
± J** + 23** ± 18* + 8**
9*
6
± 6** + 19** + 19NS ± 7* *
F=82 P<.001
186 362 323 97
143 ±
117 ±
Tf-Fe
Plasma iron (jug %)
**P<.01.
3
± 6NS ± 15** ± IONS ± 3**
F=53 P<.001
67 196 165 20
50 ± 10**
19 ±
Phv-Fe
Mean + SE. Number of specimens = 6. Range test of Wilcoxon Mann Whitney.
NS, nonsignificant.
*P<,05.
2
1
253 558 488 117
7
193 + 1 3 * *
136 +
Total
0 Injection 24 Injection 48 72 96 168
Time (hr)
± 1 ± 2 ± ± 1
F=33 P<.001
288 453 404 227
262 ± 1
251 ± 1
TIBC (Mg %)
TABLE 2. Total iron, transferrin-iron (Tf-Fe), phosvitin-iron (Phv-Fe), total iron binding c phosphoprotein (Phv) levels in blood plasma of male chickens treated w (5 mg estradiol benzoate/kg BW)1
d from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
TRANSFERRIN AND PHOSVITIN-IRON LAYING HENS
plasma iron observed in laying hens was simulated, quite well, by this experimental model after the short period of only 168 hr (Table 2). DISCUSSION
In earlier studies, the nontransferrin Fe in laying hens was found to be 50%, and there were no differences in transferrin content between laying and nonlaying hens (Planas and de Castro, I960; Planas et al, 1961). The fact that data were segregated by age and reproductive status in the current study, as opposed to a random sampling in the earlier studies, could explain the differences. Also, in the more detailed current study, it appears that the transferrin in laying hens was not necessarily saturated with iron, as had been previously suggested by Planas et al. (1961) and
Morgan (1975). So, it could be that both transport mechanisms work simultaneously from the beginning of sexual activity (Table 1). Data obtained with estrogenized males were similar to those of laying hens. Therefore, we could assume that estrogens are the direct causative agent of the response. In birds, estrogen effects iron mobilization for egg production and simultaneously provides an additional transport mechanism for iron distribution. This should be interpreted as an adaptative response. It seems that the physiological destination of iron bound to each carrier is not the same. According to Morgan (1975), the phosvitin-Fe goes to the ovaries to be incorporated into the yolk and the hematopoietic organs receive the transferrin-iron. Further experimental work would be necessary to establish support for these two separate roles of plasma iron in birds. ACKNOWLEDGMENT
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 16, 2015
Transferrin is the universal iron carrier in vertebrate plasma. However, in aves a plasma phosphoprotein (the phosvitin) also contributes to this work. The phosvitin concentration depends on the estrogen levels for its synthesis and can also be induced in males after estrogen administration. In laying hens, as opposed to immature pullets, the increased levels of transferrin and phosvitin were probably due to changes in plasma estrogen levels. Radioimmunoassays for estrogen during the ovulatory cycle in hens (Peterson and Common, 1972) and under different reproductive conditions (Senior, 1974) support this assertion. Estrogen enhances iron metabolism in birds. The laying period presents a rise in plasma iron valus (Ramsay and Campbell, 1954; Planas et al., 1961) and an increase in the plasma iron turnover (Planas and Balasch, 1970; Balasch and Planas, 1972). At the same time, estrogens stimulate phosvitin synthesis in the chicken liver (Heald and McLachlan, 1963; Beuving and Gruber, 1971; Greengard et al., 1964); it was first suggested by Greengard et al. (1965) that phosvitin might participate in plasma iron transport. This fact was clearly established in the papers of Ali and Ramsay (1968, 1974) and Morgan (1975). However, the beginning and the quantitative participation of phosvitin in plasma iron transport was still unknown. The present data (Table 1) show that during the laying period phosvitin carried from 29 to 36% of the plasma iron. Transferrin at this time was near the saturation level of 63 to 80% and the phosvitin acted as an auxiliary transport mechanism.
1955
The authors are indebted to J. Gallego of the Department of Statistics, University of Valladolid, for his expertise and guidance in the statistical and computer analysis of the data. REFERENCES
Allen, R.J.L., 1940. The estimation of phosphorus. Biochem.J. 34:858-865. Ali, K. E., and W.N.M. Ramsay, 1968. Phosphoprotein-bound iron in the plasma of the hying hen. Biochem.J. 110:36. Ali, K. E., and W.N.M. Ramsay, 1974. Phosphoprotein-bound iron in the blood of plasma of the laying hen. Q. J. Exp. Physiol. 59:159-165. Balasch, J., and J. Planas, 1972. Iron metabolism in duck and turkey. Rev. Espan. Fisiol. 28:125— 128. Beuving, G., and M. Gruber, 1971. Induction of phosvitin synthesis in roosters by estradiol injection. Biochem. Biophys. Acta 232:529-536. Greengard, O., M. Gordon, M. A. Smith, and G. Acs, 1964. Studies on the mechanism of diethylstilbesterol-induced formation of phosphoprotein in male chickens. J. Biol. Chem. 329:20792082. Greengard, O., N. Mendelsohn, and M. Gordon, 1965. Iron accumulation in cockerel plasma after estrogen relation to induced phosphoprotein synthesis. Science 147:1571-1572. Heald, P. J., and P. M. McLachlan, 1963. Isolation of phosvitin from the plasma of the laying hen. Biochem. J. 87:571-576. International Committee for Standardization in Hematology, 1971. Brit. J. Haematol. 20:451-453. Morgan, E. H., 1975. Plasma iron transport during egg laying and after oestrogen administration in domestic fowl (Gallus domesticus). Q. J. Exp. Physiol. 60:233-247.
1956
LOPEZ-BERJES ET AL.
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