BIOLOGICAL TRACE ELEMENT RESEARCH 2, 255r267
Intestinal Iron Absorption in Chickens I. Experimental Conditions M. T. MARTI,M. P. SAIZ, M. T. MITJAVILA, AND J. PLANAS*
Department of Animal Physiology, Faculty of Biology, University of Barcelona, Barcelona, Spain Received February 29, 1980; Accepted April 2, 1980
Abstract Intestinal iron absorption in chickens was studied in vivo, using an intestinal perfusion technique in closed circuit. The results obtained show that iron absorption, at 30 min intervals, is a linear function of test solution iron concentrations of up to 776/.t g Fe/20 mL. At higher concentrations, iron saturation occurs. The mucosal epithelial cells seem to be less a limiting factor than in rats. However, in chickens, the binding capacity of plasma might play an important role in the regulation of iron absorption. Iron absorption versus time was analyzed in 15, 30, 60, and 120 min periods for the iron concentration of 14 p g Fe/20 mL. Intestinal iron absorption showed a linear relationship between these two parameters. A period of perfusion of either 30 or 60 min by a solution of 14/.tg Fe/20 mL appears suitable since no interference by a saturation process can then occur. Index Entries: Intestinal iron absorption, in chickens; perfusion, of intestines by Fe; iron, intestinal absorption in chickens; mucosal epithelial cells, and intestinal Fe absorption; transferrin, and intestinal Fe absorption; chickens, intestinal Fe absorption in.
Introduction The iron diet requirements of (1-5) and its metabolism in (6-9) birds have been the object of many studies, and it is known that iron is mobilized during the laying period, which in chickens implies the loss of I mg of this metal per 9 1980 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163 4984/80/12004)255502.60
255
256
MARTI ET AL.
egg (I0). In an early paper of Ramsay and Campbell (11) on iron metabolism in laying hens, and later in our own studies on this species, as well as others (6-8) the importance of the intestinal iron absorption in providing for the great iron needs during the laying period has been suggested. However, until now, nobody has investigated whether laying birds present an increase in the iron absorption rate during this state. Knowledge about intestinal iron absorption in chickens is nil, and the optimal conditions for the process are unknown, just as are its effects on several physiological variables (age, race, sex, laying). It is well known that, in mammals, ferrous salts are better absorbed than ferric ones (12), unless the iron is given in ionized form (13, 14). The addition of both forms of iron salts to the diet has not produced consistent differences in chickens or in rats (15). The chicken's duodenal pH ranges between 6 and 7, and ferric ions could be expected to precipitate, blocking the intestinal absorption. To avoid this, Van Campen (16, 17) used ascorbic acid and histidine in rats; the first worked as a reducing and chelating agent, and the second only as chelator. In the present paper, we have tried to analyze and establish the best experimental conditions in which to study the in vivo intestinal iron absorption in chickens, using a peffusion technique in a closed circuit as the first step for later studies on the influence of the physiological parameters referred to above.
Methods Female New Hampshire chickens, approximately 6 weeks old, were used for all experiments. The animals were fed for 3-4 weeks prior to the experiments with a commercial diet to ensure constant iron conditions. The diet was analyzed by atomic absorption spectroscopy (18) and was found to contain 187 ppm Fe. Radioactive iron (59Fe) was obtained from the Radiochemical Center (Amersham) as ferric chloride in 0. IM HCI, with a specific activity of l0 mCi/mg Fe. Ascorbic acid and histidine were obtained from Merck. The iron test solution was a modified Ringer-Locke buffer, to which the cold iron (FeC13) was added in different concentrations (14, 70, 156, 348, 776, and 1740 # g Fe/20 m L perfusion fluid, equivalent to 12.5, 63,140, 312, 695, and 1559/.t M Fe). L-Histidine, glucose, and 59FeCl3 were added to the constant concentrations of 0. l M, 55 mM, and 0.2/,tCi/mL, respectively. Finally ascorbic acid was added to 10-fold iron concentration.Calcium was omitted from the Ringer-Locke buffer because it seemed to decrease both the iron absorption by the intestine (19) and the net transfer in concentrations that do not normally inhibit the mucosal uptake (20). The different solutions obtained were adjusted to pH 6.2 since this is the normal value of chicken duodenal pH. By increasing histidine concentration in the
INTESTINAL IRON ABSORPTION IN CHICKENS
257
same proportion as iron, the osmolarity of the test solution could be increased. We therefore decided to keep histidine at the constant rate of 0.1 M. Under these conditions, the osmolarity of our solutions increased to a maximum of 100 mOsmol, compared to the original Ringer-Locke glucose solution. According to Thomson and Valberg (21) a variation from 8 to 408 mOsmol does not significantly affect intestinal iron transport in rats. The chickens were anesthetised by an intramuscular injection of chloral hydrate with a dose of 0.3g/kg body weight and an incision was made under the sternal keel. An inflow cannula was inserted at the beginning of the duodenum and was held in place by a ligature. At a distance of 12-20 cm, and always before the common bile duct, an outflow cannula was inserted and a ligature tightened around it. The cannulated segment remained in situ and care was taken not to impair its own innervation and blood supply. The intestinal lumen was rinsed with 10 mL of Ringer-Locke solution and was then connected to a fluid pump (Gilson Minipuls) which continuously recycled the perfusion liquid, using a graduated cylinder as a reservoir, through the lumen of the cannulated intestinal loop at a rate of 3.0 mL/min and with an hydrostatic pressure of 14 cm. The animal and the perfusion system, including the liquid, were placed in a thermostated vitrine at 42~ C, the usual rectal temperature of the animals studied. A 30-min perfusion was carried out with 20 mL of Ringer-Locke buffer, and subsequently a second perfusion was carried out with the same volume of the test solution. Before the experiment, 3 mL of blood were withdrawn from the radial vein, with an heparinized syringe, in order to determine the plasma iron, using the Ramsay's method (22); the hemoglobin concentration was determined by Drabkin's reagent (23), and the hematocrit by using a microcentrifuge. At the end of the experiment, 1 mL of heparinized blood was withdrawn from the radial vein in order to determine the amount of radioactivity and the same was done with 1 mL of the perfused solution. Also, 1 mL of the initial test solution was counted. The animals were desanguinated. The liver and the intestinal loop were immediately removed from the carcass. Liver uptake of iron was determined by counting the radioactivity in this organ. Net transfer of iron was calculated by the addition of the radioactivity present in the intestinal loop to the residual radioactivity of the perfused solution and the subtraction of this value from the radioactivity of the initial test solution. The intestinal segment was opened, washed twice with 20 mL of 10mM EDTA-NaC1 (0.9%) for 10 min to remove the unabsorbed iron. Subsequently, the radioactivity taken up by the intestinal wall, which is the mucosal uptake, was counted. Total iron absorption was determined as the sum of the mucosal uptake and net transfer. Results were expressed as mean + S E M in # g or % Fe/g of intestine; as # g F e / m L of blood and as # g Fe/g of liver. The differences between the groups were determined by the analysis of the variance.
258
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Intestinal Iron Absorption in Chickens I. Experimental Conditions M. T. MARTI,M. P. SAIZ, M. T. MITJAVILA, AND J. PLANAS*
Department of Animal Physiology, Faculty of Biology, University of Barcelona, Barcelona, Spain Received February 29, 1980; Accepted April 2, 1980
Abstract Intestinal iron absorption in chickens was studied in vivo, using an intestinal perfusion technique in closed circuit. The results obtained show that iron absorption, at 30 min intervals, is a linear function of test solution iron concentrations of up to 776/.t g Fe/20 mL. At higher concentrations, iron saturation occurs. The mucosal epithelial cells seem to be less a limiting factor than in rats. However, in chickens, the binding capacity of plasma might play an important role in the regulation of iron absorption. Iron absorption versus time was analyzed in 15, 30, 60, and 120 min periods for the iron concentration of 14 p g Fe/20 mL. Intestinal iron absorption showed a linear relationship between these two parameters. A period of perfusion of either 30 or 60 min by a solution of 14/.tg Fe/20 mL appears suitable since no interference by a saturation process can then occur. Index Entries: Intestinal iron absorption, in chickens; perfusion, of intestines by Fe; iron, intestinal absorption in chickens; mucosal epithelial cells, and intestinal Fe absorption; transferrin, and intestinal Fe absorption; chickens, intestinal Fe absorption in.
Introduction The iron diet requirements of (1-5) and its metabolism in (6-9) birds have been the object of many studies, and it is known that iron is mobilized during the laying period, which in chickens implies the loss of I mg of this metal per 9 1980 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163 4984/80/12004)255502.60
255
256
MARTI ET AL.
egg (I0). In an early paper of Ramsay and Campbell (11) on iron metabolism in laying hens, and later in our own studies on this species, as well as others (6-8) the importance of the intestinal iron absorption in providing for the great iron needs during the laying period has been suggested. However, until now, nobody has investigated whether laying birds present an increase in the iron absorption rate during this state. Knowledge about intestinal iron absorption in chickens is nil, and the optimal conditions for the process are unknown, just as are its effects on several physiological variables (age, race, sex, laying). It is well known that, in mammals, ferrous salts are better absorbed than ferric ones (12), unless the iron is given in ionized form (13, 14). The addition of both forms of iron salts to the diet has not produced consistent differences in chickens or in rats (15). The chicken's duodenal pH ranges between 6 and 7, and ferric ions could be expected to precipitate, blocking the intestinal absorption. To avoid this, Van Campen (16, 17) used ascorbic acid and histidine in rats; the first worked as a reducing and chelating agent, and the second only as chelator. In the present paper, we have tried to analyze and establish the best experimental conditions in which to study the in vivo intestinal iron absorption in chickens, using a peffusion technique in a closed circuit as the first step for later studies on the influence of the physiological parameters referred to above.
Methods Female New Hampshire chickens, approximately 6 weeks old, were used for all experiments. The animals were fed for 3-4 weeks prior to the experiments with a commercial diet to ensure constant iron conditions. The diet was analyzed by atomic absorption spectroscopy (18) and was found to contain 187 ppm Fe. Radioactive iron (59Fe) was obtained from the Radiochemical Center (Amersham) as ferric chloride in 0. IM HCI, with a specific activity of l0 mCi/mg Fe. Ascorbic acid and histidine were obtained from Merck. The iron test solution was a modified Ringer-Locke buffer, to which the cold iron (FeC13) was added in different concentrations (14, 70, 156, 348, 776, and 1740 # g Fe/20 m L perfusion fluid, equivalent to 12.5, 63,140, 312, 695, and 1559/.t M Fe). L-Histidine, glucose, and 59FeCl3 were added to the constant concentrations of 0. l M, 55 mM, and 0.2/,tCi/mL, respectively. Finally ascorbic acid was added to 10-fold iron concentration.Calcium was omitted from the Ringer-Locke buffer because it seemed to decrease both the iron absorption by the intestine (19) and the net transfer in concentrations that do not normally inhibit the mucosal uptake (20). The different solutions obtained were adjusted to pH 6.2 since this is the normal value of chicken duodenal pH. By increasing histidine concentration in the
INTESTINAL IRON ABSORPTION IN CHICKENS
257
same proportion as iron, the osmolarity of the test solution could be increased. We therefore decided to keep histidine at the constant rate of 0.1 M. Under these conditions, the osmolarity of our solutions increased to a maximum of 100 mOsmol, compared to the original Ringer-Locke glucose solution. According to Thomson and Valberg (21) a variation from 8 to 408 mOsmol does not significantly affect intestinal iron transport in rats. The chickens were anesthetised by an intramuscular injection of chloral hydrate with a dose of 0.3g/kg body weight and an incision was made under the sternal keel. An inflow cannula was inserted at the beginning of the duodenum and was held in place by a ligature. At a distance of 12-20 cm, and always before the common bile duct, an outflow cannula was inserted and a ligature tightened around it. The cannulated segment remained in situ and care was taken not to impair its own innervation and blood supply. The intestinal lumen was rinsed with 10 mL of Ringer-Locke solution and was then connected to a fluid pump (Gilson Minipuls) which continuously recycled the perfusion liquid, using a graduated cylinder as a reservoir, through the lumen of the cannulated intestinal loop at a rate of 3.0 mL/min and with an hydrostatic pressure of 14 cm. The animal and the perfusion system, including the liquid, were placed in a thermostated vitrine at 42~ C, the usual rectal temperature of the animals studied. A 30-min perfusion was carried out with 20 mL of Ringer-Locke buffer, and subsequently a second perfusion was carried out with the same volume of the test solution. Before the experiment, 3 mL of blood were withdrawn from the radial vein, with an heparinized syringe, in order to determine the plasma iron, using the Ramsay's method (22); the hemoglobin concentration was determined by Drabkin's reagent (23), and the hematocrit by using a microcentrifuge. At the end of the experiment, 1 mL of heparinized blood was withdrawn from the radial vein in order to determine the amount of radioactivity and the same was done with 1 mL of the perfused solution. Also, 1 mL of the initial test solution was counted. The animals were desanguinated. The liver and the intestinal loop were immediately removed from the carcass. Liver uptake of iron was determined by counting the radioactivity in this organ. Net transfer of iron was calculated by the addition of the radioactivity present in the intestinal loop to the residual radioactivity of the perfused solution and the subtraction of this value from the radioactivity of the initial test solution. The intestinal segment was opened, washed twice with 20 mL of 10mM EDTA-NaC1 (0.9%) for 10 min to remove the unabsorbed iron. Subsequently, the radioactivity taken up by the intestinal wall, which is the mucosal uptake, was counted. Total iron absorption was determined as the sum of the mucosal uptake and net transfer. Results were expressed as mean + S E M in # g or % Fe/g of intestine; as # g F e / m L of blood and as # g Fe/g of liver. The differences between the groups were determined by the analysis of the variance.
258
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