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Iron kinetics in the frog (Rana montezumae)
Comp. Biochem. Physbl.,
1971,‘fr,l.QOA,pp. 517to 521. Pergamon Press. Printed in Great Britain
IRON KINETICS
(~~A ALBERT0
IN THE FROG
~~~T~ZU~A~)~ OSVALDO
CARMENAT
Instituto National de la Nutrition de Mexico Departamento de Hematologia (&%?&a
13 iWQ?& 1971)
Abstract-l. Iron kinetics was performed on Rana montezumue, originally from the Valley of Mexico (7343 ft). Iron clearance (Tt Fess) was estimated in 8 hr with a 20 per cent incorporation of Fess into erythrocytes. 2. The liver is the organ that shows most radioactivity, with a peak at 13 hr after the injection of the radioisotope. The spleen follows a similar pattern. Bone marrow presents a peak at 24 hr but lower than that of the liver. Erythroid precursors are present in the liver, spleen and bone marrow. 3. It is concluded that the liver is the main place for the production of red cell precursors in the adult frog. 4. An attempt is made to parallel erythropoiesis in intrauterine life in the human being and that observed in the frog. INTRODUCTION IN A PREVIOUSwork
(Carmena et al., 1968a) made with newborn rats, we postulated that the transition to adult erythropoiesis which occurs during the first 15-30 days of extrauterine life could be extrapolated to that happening in the intrauterine life of species whose newborn are more mature. Trying to find an animal which did not present the rapid changes which occur with the erythropoiesis of the newborn rat, we studied the erythrokinetics of the frog in order to look for analogies with the fetal erythropoiesis of mammals and especially of man. MATERIALS
AND METHODS
In a recent work (Carmena, 1970) we described in detail the conditions of housing and feeding of frogs during the work performed. Male frogs (Rana montezumae), natives of the Valley of Mexico (7343 ft), were used. The work was done during the spring and summer with minimal night temperatures of 18°C and maximal day temperatures of 27°C. Male frogs were bled by cardiac puncture under ether anesthesia. A blood pooi was made and was centrifuged for 30 min at 1500 rev/mm. Plasma was separated and incubated with Fe6* for 30 min at room temperature, mixing gently every 10 min. Intraperitoneal injections of 0.3 ml of plasma (0.3 PC of Fe6*) were administered on each animal. They were sacrificed after 30 min, 1,2,3,4,5,6,7,8 and 13 hr, and after 1,2,3,5,14,21,34,41 and 48 days. Animals * Supported by Laboratorios Farmacobiologicos, S.A. (Mexico) and Fisons, $.A. (Mexico). 7 Present address: Visiting Researcher, Instituto de Hematologfa e Inmunologla, Hospital General Docente E, Cabrera, Havana, Cuba. 517
518
ALBERT•
OSVALDOCARMENA
were bled by cardiac puncture under ether anesthesia. The plasma Fe58 incorporation was calculated on the basis of a blood volume of 8 ml/100 g of body-weight (Prosser et al., 1950), and the plasma volume was deducted from the hematocrits. The liver, the spleen, both femurs, the tibia-fibula, the ilium and the humerus + radius-ulna were dissected and the incorporation of FeSB into those organs was calculated. Fe 5g incorporation into the red blood cells was also calculated on the basis of 8 ml/100 g body-weight. Femur, liver and spleen smears were made, May-Grundwal and Giemsa staining being used, and a minimum of 500 nucleated cells (in duplicate) were counted. Hemoglobin, hematocrits, red cell counts, white cell counts and serum iron were measured by conventional techniques. Finally, liver, spleen and both femurs were weighed. RESULTS
The death rate was nearly 10 per cent. Ether anesthesia takesup to 10 min to make the animals fall asleep. However, in view of the good results obtained, no other type of anesthetic was tried. Table 1 shows the peripheral hematologic data. TABLE
~--PERIPHERAL
BLOOD
VALUES IN MALE R. montezumae
27.2 f 0.46% Hematocrit (183) Serum iron (16) 24.0 ?I 2.5 pg % Leucocytes (20) 7070 f 470 mm3 Red blood cells (20) 391,000 _+2313 mm3 Hemoglobin (41) 4.72 Z!I0.28% Figures in parentheses are the number of animals employed.
INTRAPERITONEAL
Hr 2 (13) 4(15) 6 (10) 8 (4) 13 (7) 24 (22)
Figures
in parentheses
Uptake (%) 38 f 9.0 35 f 6.0 27 f 6.0 22 + 8.0 15+5.0 14 -+3.0
+ : Standard error.
INJECTION
Days 2 (17) 3 (16)
5 (8)
14 21 34 41 48
(11) (9) (6) (5) (4)
Uptake (%) 4.0f 1.0 2.0 + 0.4 1.2 + 0.4 1.6 f 0.3 2.0 * 0.9 1.7a0.4 2.6 f 0.4 2.3 + 0.5
are the number of frogs employed.
f : Standard error.
Plasma Fe69 incorporation shown in Table 2 is from the first day up to the fortyeighth day after the injection of the radioisotope. In Table 3 Fe59 incorporation in the bone marrow is shown (the sum of all bones), and into the liver and spleen. Figure 1 shows the Fe59 uptake in each bone (ilium, tibia-fibula, humerus+ radius-ulna and both femurs). The incorporation of Fe59 to the red blood cells is depicted in Table 4. Finally, the body-weight of the animals, as well as the weight of both femurs, liver and spleen are shown in Table 5.
519
IRON KINETICS IN THEFROG
TABLE ~-PERCENTAGE OF Fe6@ UPTAKE BY THE BONR MARROW (THE SUM OF ALL BoNII.~ STUDIED), LIVER AND SPLEEN AFTER DIFFERENT HOURS FROM AN INTRAPERITONEAL INJECTION OF THE RADIOISOTOPE
Hr
Bone marrow
Liver
1 (15) 2 (13) 4 (15) 6 (10) 7 (8) 13 (7) 24 (22) 48 (17)
206 + 0.1 2.7 rf:O-2 2.5 x!Y o-1 4-2 rt O-2 4.8 + o-2 4.8 z!zO-l 10.1 * 0.7 2.8 + 0.1
1.5 50.5 7.4 If:l-3 7.1 rf:1.7 5.7 z!z3.9 9.5 z!z2-o 22.4 + 7.0 1.5*0+3*0 10.0 z!z1.4
Spleen 0.2 rt 0.04 0.3 rt 0.05 0.2 k 0.01 0‘2 rf:O-06 0*4fO*l 0.9 It 0.5 0.6 + 0.2 0.6 or0.2
Figures in parentheses are the number of animals employed.
+ : Standard error.
DISCUSSION
The peripheral hematological values found in R. montezumae agree with those reported in Rana catesbiana (Wintrobe, 1933) and in Rana temporaria (Denzer, 1950), although the number of erythrocytes is higher in three out of four Rana escdenta studied by Wells & Sutton (1915). The plasma iron values were 23-O + 2.5
I I
ooys
IO
I 20
,
If
I 50
Ordinates: percentage of Fe6n uptake. Abscissas: time after the radioisotope injection.
FIG.
/Lg[lOO ml of serum. Hemoglobin was lower in R. montezumae than in R. pipiens of the same region, although the differences were not statistically significant (Carmena & Rivera, 1968). Plasma iron clearance ( Tt FeSg), was considered from the values found 2 hr after the injection of Fe59, since at 30 and 60 min the Fe50 concentration in plasma was low, probably due to the injections via the peritoneum.
520
ALBERTOOSVALDOCARMENA
TABLE 4--Fese INCORPORATION INTO THE RED BLOOD CELLS OF R. montezumae DURING THE FIRST 4 WEEKS Days
Fess incorporation (%)
l(24) 2 (17) 4(11) 5 (8) 9 (5) 21 (9) 29 (5)
14.oIt3.0 23.0 _+4.0 25.0 + 6.0 21.0 _+l-6 19.0 _+2.6 36.0 f 7.0 22.0 + 4.0
Figures in parentheses are the number of animals employed. TABLE S-BODY
+ : Standard error.
WEIGHTAND WEIGHTS OF LIVERS,SPLEENSAND TWO FEMURS IN FIFTY FROGS Body Two femurs Liver Spleen
(50) (SO) (SO) (SO)
83.5 + 6.7 g 1*116+0.078g 2.885 f 0.309 g O-116 rt 0.026 g
Figures in parentheses are the number of frogs employed.
+ : Standard error.
In another of our unpublished works we performed the same experiment with Wistar rats, injecting plasma labelled with Fe59 intraperitoneally and intravenously. No difference in the tracing of the curves was found, but the first points were also low on those injected intraperitoneally. The Fe clearance (T, Fe59) was about 8 hr. The same values were found in R. pipiens. In the following days, the radioactivity remained in the plasma in the range 2-3 per cent up to day 48. This would be due to re-exchange with the lymphatic pool and not only to the Fe59 arriving from hemocatheresis, since the red cell survival is rather prolonged red cells is made (Carmena, 1970) : The incorporation of Fe59 into the circulating rapidly. Already at the second day 23 per cent is reached, remaining within these values during the first 4 weeks, with variations from 19 to 36 per cent with a large dispersion in several points (Table 4). Similar values were found by Cline & Waldmann (1962) at temperatures of 24-26”C, with an enormous dispersion. Fe59 incorporation into the bone marrow shows a peak after 24 hr, it being the ilium which incorporates more Fe59 and the two femurs which incorporate less (Fig. 1). The liver shows a peak of 22 per cent after 13 hr. The same happens with the spleen, although the percentage is less. If we consider the Fe59 uptake in relation to the weight of the organs (sp. act.) we find that the behavior of the liver and spleen are similar (see the weight of organs in Table 5). This agrees with the differential counts of the erythroblasts, which are 42.7 rt 2.3 per cent in the liver and 38-l+ 2.4 per cent in the spleen of ten animals studied. In the bone marrow we found 30.8 ? 4.9 per cent in four animals out of ten and fat bone marrow in the other six.
521
IRON KINETICS IN THR FROG
In conclusion we deduce from our results, that erythropoiesis in R. montexumae occurs mainly in the liver, with the participation of the bone marrow, but to a lesser degree. The spleen also mediates in this process. Extrapolating these results to humans, it could be speculated that erythrokinetics in the frog is similar to that of the human fetuses of approximately 5-6 lunar months of intrauterine life. Acknowledgements-The author is grateful to Maria Angela Rivera and Alicia Gil for their cooperation in making this work possible. REFERENCES CARMENAA. 0. (1970) Red cell survival in the frog (Runa montezumae). Comp. Biochem. Physiol., 40, 349-351. CARMENAA. O., HOWARDD. & STOHLMANF., JR. (1968a) Regulation of erythropoiesisXXII. Erythropoietin production in the newborn animal. Blood 32, 371-382. CARMENA A. 0. & RIWRA M. A. (1968b) Eritrocinesis en batracios-I. Efecto de la sangria y de la hipertrasfusion en la Rana pipiens. Rew. Invest. Clin. (Mexico) 20, 307-315. CLINE M. J. & WALDMANNT. A. (1962) Effect of temperature on erythropoiesis and red cell survival in the frog. Am.J. Physiol. 203, 401-403. DENZER H. W. (1950) Comparative altitude physiology of animals. In German Aviation Medicine, Chapt. IV-K, pp. 321-353. United States Air Force. PROSSERC. L. & WEINSTEINS. J. F. (1950) Comparison of blood volume in animals with open and closed circulatory system. Physiol. Zool. 23, 113-124. WELLS J. J. & SUTTONJ. E. (1915) Blood counts in the frog, the turtle and twelve different species of mammals. Am.J. Physiol. 39, 31-36. WINTROBEM. M. (1933) Variations in the size and hemoglobin content of erythrocytes in the blood of various vertebrates. Folia Haemat. 51, 32-49. Key
Word Index-Erythropoiesis;
iron kinetics; Rana montezumae;
FeS8; liver.
1971,‘fr,l.QOA,pp. 517to 521. Pergamon Press. Printed in Great Britain
IRON KINETICS
(~~A ALBERT0
IN THE FROG
~~~T~ZU~A~)~ OSVALDO
CARMENAT
Instituto National de la Nutrition de Mexico Departamento de Hematologia (&%?&a
13 iWQ?& 1971)
Abstract-l. Iron kinetics was performed on Rana montezumue, originally from the Valley of Mexico (7343 ft). Iron clearance (Tt Fess) was estimated in 8 hr with a 20 per cent incorporation of Fess into erythrocytes. 2. The liver is the organ that shows most radioactivity, with a peak at 13 hr after the injection of the radioisotope. The spleen follows a similar pattern. Bone marrow presents a peak at 24 hr but lower than that of the liver. Erythroid precursors are present in the liver, spleen and bone marrow. 3. It is concluded that the liver is the main place for the production of red cell precursors in the adult frog. 4. An attempt is made to parallel erythropoiesis in intrauterine life in the human being and that observed in the frog. INTRODUCTION IN A PREVIOUSwork
(Carmena et al., 1968a) made with newborn rats, we postulated that the transition to adult erythropoiesis which occurs during the first 15-30 days of extrauterine life could be extrapolated to that happening in the intrauterine life of species whose newborn are more mature. Trying to find an animal which did not present the rapid changes which occur with the erythropoiesis of the newborn rat, we studied the erythrokinetics of the frog in order to look for analogies with the fetal erythropoiesis of mammals and especially of man. MATERIALS
AND METHODS
In a recent work (Carmena, 1970) we described in detail the conditions of housing and feeding of frogs during the work performed. Male frogs (Rana montezumae), natives of the Valley of Mexico (7343 ft), were used. The work was done during the spring and summer with minimal night temperatures of 18°C and maximal day temperatures of 27°C. Male frogs were bled by cardiac puncture under ether anesthesia. A blood pooi was made and was centrifuged for 30 min at 1500 rev/mm. Plasma was separated and incubated with Fe6* for 30 min at room temperature, mixing gently every 10 min. Intraperitoneal injections of 0.3 ml of plasma (0.3 PC of Fe6*) were administered on each animal. They were sacrificed after 30 min, 1,2,3,4,5,6,7,8 and 13 hr, and after 1,2,3,5,14,21,34,41 and 48 days. Animals * Supported by Laboratorios Farmacobiologicos, S.A. (Mexico) and Fisons, $.A. (Mexico). 7 Present address: Visiting Researcher, Instituto de Hematologfa e Inmunologla, Hospital General Docente E, Cabrera, Havana, Cuba. 517
518
ALBERT•
OSVALDOCARMENA
were bled by cardiac puncture under ether anesthesia. The plasma Fe58 incorporation was calculated on the basis of a blood volume of 8 ml/100 g of body-weight (Prosser et al., 1950), and the plasma volume was deducted from the hematocrits. The liver, the spleen, both femurs, the tibia-fibula, the ilium and the humerus + radius-ulna were dissected and the incorporation of FeSB into those organs was calculated. Fe 5g incorporation into the red blood cells was also calculated on the basis of 8 ml/100 g body-weight. Femur, liver and spleen smears were made, May-Grundwal and Giemsa staining being used, and a minimum of 500 nucleated cells (in duplicate) were counted. Hemoglobin, hematocrits, red cell counts, white cell counts and serum iron were measured by conventional techniques. Finally, liver, spleen and both femurs were weighed. RESULTS
The death rate was nearly 10 per cent. Ether anesthesia takesup to 10 min to make the animals fall asleep. However, in view of the good results obtained, no other type of anesthetic was tried. Table 1 shows the peripheral hematologic data. TABLE
~--PERIPHERAL
BLOOD
VALUES IN MALE R. montezumae
27.2 f 0.46% Hematocrit (183) Serum iron (16) 24.0 ?I 2.5 pg % Leucocytes (20) 7070 f 470 mm3 Red blood cells (20) 391,000 _+2313 mm3 Hemoglobin (41) 4.72 Z!I0.28% Figures in parentheses are the number of animals employed.
INTRAPERITONEAL
Hr 2 (13) 4(15) 6 (10) 8 (4) 13 (7) 24 (22)
Figures
in parentheses
Uptake (%) 38 f 9.0 35 f 6.0 27 f 6.0 22 + 8.0 15+5.0 14 -+3.0
+ : Standard error.
INJECTION
Days 2 (17) 3 (16)
5 (8)
14 21 34 41 48
(11) (9) (6) (5) (4)
Uptake (%) 4.0f 1.0 2.0 + 0.4 1.2 + 0.4 1.6 f 0.3 2.0 * 0.9 1.7a0.4 2.6 f 0.4 2.3 + 0.5
are the number of frogs employed.
f : Standard error.
Plasma Fe69 incorporation shown in Table 2 is from the first day up to the fortyeighth day after the injection of the radioisotope. In Table 3 Fe59 incorporation in the bone marrow is shown (the sum of all bones), and into the liver and spleen. Figure 1 shows the Fe59 uptake in each bone (ilium, tibia-fibula, humerus+ radius-ulna and both femurs). The incorporation of Fe59 to the red blood cells is depicted in Table 4. Finally, the body-weight of the animals, as well as the weight of both femurs, liver and spleen are shown in Table 5.
519
IRON KINETICS IN THEFROG
TABLE ~-PERCENTAGE OF Fe6@ UPTAKE BY THE BONR MARROW (THE SUM OF ALL BoNII.~ STUDIED), LIVER AND SPLEEN AFTER DIFFERENT HOURS FROM AN INTRAPERITONEAL INJECTION OF THE RADIOISOTOPE
Hr
Bone marrow
Liver
1 (15) 2 (13) 4 (15) 6 (10) 7 (8) 13 (7) 24 (22) 48 (17)
206 + 0.1 2.7 rf:O-2 2.5 x!Y o-1 4-2 rt O-2 4.8 + o-2 4.8 z!zO-l 10.1 * 0.7 2.8 + 0.1
1.5 50.5 7.4 If:l-3 7.1 rf:1.7 5.7 z!z3.9 9.5 z!z2-o 22.4 + 7.0 1.5*0+3*0 10.0 z!z1.4
Spleen 0.2 rt 0.04 0.3 rt 0.05 0.2 k 0.01 0‘2 rf:O-06 0*4fO*l 0.9 It 0.5 0.6 + 0.2 0.6 or0.2
Figures in parentheses are the number of animals employed.
+ : Standard error.
DISCUSSION
The peripheral hematological values found in R. montezumae agree with those reported in Rana catesbiana (Wintrobe, 1933) and in Rana temporaria (Denzer, 1950), although the number of erythrocytes is higher in three out of four Rana escdenta studied by Wells & Sutton (1915). The plasma iron values were 23-O + 2.5
I I
ooys
IO
I 20
,
If
I 50
Ordinates: percentage of Fe6n uptake. Abscissas: time after the radioisotope injection.
FIG.
/Lg[lOO ml of serum. Hemoglobin was lower in R. montezumae than in R. pipiens of the same region, although the differences were not statistically significant (Carmena & Rivera, 1968). Plasma iron clearance ( Tt FeSg), was considered from the values found 2 hr after the injection of Fe59, since at 30 and 60 min the Fe50 concentration in plasma was low, probably due to the injections via the peritoneum.
520
ALBERTOOSVALDOCARMENA
TABLE 4--Fese INCORPORATION INTO THE RED BLOOD CELLS OF R. montezumae DURING THE FIRST 4 WEEKS Days
Fess incorporation (%)
l(24) 2 (17) 4(11) 5 (8) 9 (5) 21 (9) 29 (5)
14.oIt3.0 23.0 _+4.0 25.0 + 6.0 21.0 _+l-6 19.0 _+2.6 36.0 f 7.0 22.0 + 4.0
Figures in parentheses are the number of animals employed. TABLE S-BODY
+ : Standard error.
WEIGHTAND WEIGHTS OF LIVERS,SPLEENSAND TWO FEMURS IN FIFTY FROGS Body Two femurs Liver Spleen
(50) (SO) (SO) (SO)
83.5 + 6.7 g 1*116+0.078g 2.885 f 0.309 g O-116 rt 0.026 g
Figures in parentheses are the number of frogs employed.
+ : Standard error.
In another of our unpublished works we performed the same experiment with Wistar rats, injecting plasma labelled with Fe59 intraperitoneally and intravenously. No difference in the tracing of the curves was found, but the first points were also low on those injected intraperitoneally. The Fe clearance (T, Fe59) was about 8 hr. The same values were found in R. pipiens. In the following days, the radioactivity remained in the plasma in the range 2-3 per cent up to day 48. This would be due to re-exchange with the lymphatic pool and not only to the Fe59 arriving from hemocatheresis, since the red cell survival is rather prolonged red cells is made (Carmena, 1970) : The incorporation of Fe59 into the circulating rapidly. Already at the second day 23 per cent is reached, remaining within these values during the first 4 weeks, with variations from 19 to 36 per cent with a large dispersion in several points (Table 4). Similar values were found by Cline & Waldmann (1962) at temperatures of 24-26”C, with an enormous dispersion. Fe59 incorporation into the bone marrow shows a peak after 24 hr, it being the ilium which incorporates more Fe59 and the two femurs which incorporate less (Fig. 1). The liver shows a peak of 22 per cent after 13 hr. The same happens with the spleen, although the percentage is less. If we consider the Fe59 uptake in relation to the weight of the organs (sp. act.) we find that the behavior of the liver and spleen are similar (see the weight of organs in Table 5). This agrees with the differential counts of the erythroblasts, which are 42.7 rt 2.3 per cent in the liver and 38-l+ 2.4 per cent in the spleen of ten animals studied. In the bone marrow we found 30.8 ? 4.9 per cent in four animals out of ten and fat bone marrow in the other six.
521
IRON KINETICS IN THR FROG
In conclusion we deduce from our results, that erythropoiesis in R. montexumae occurs mainly in the liver, with the participation of the bone marrow, but to a lesser degree. The spleen also mediates in this process. Extrapolating these results to humans, it could be speculated that erythrokinetics in the frog is similar to that of the human fetuses of approximately 5-6 lunar months of intrauterine life. Acknowledgements-The author is grateful to Maria Angela Rivera and Alicia Gil for their cooperation in making this work possible. REFERENCES CARMENAA. 0. (1970) Red cell survival in the frog (Runa montezumae). Comp. Biochem. Physiol., 40, 349-351. CARMENAA. O., HOWARDD. & STOHLMANF., JR. (1968a) Regulation of erythropoiesisXXII. Erythropoietin production in the newborn animal. Blood 32, 371-382. CARMENA A. 0. & RIWRA M. A. (1968b) Eritrocinesis en batracios-I. Efecto de la sangria y de la hipertrasfusion en la Rana pipiens. Rew. Invest. Clin. (Mexico) 20, 307-315. CLINE M. J. & WALDMANNT. A. (1962) Effect of temperature on erythropoiesis and red cell survival in the frog. Am.J. Physiol. 203, 401-403. DENZER H. W. (1950) Comparative altitude physiology of animals. In German Aviation Medicine, Chapt. IV-K, pp. 321-353. United States Air Force. PROSSERC. L. & WEINSTEINS. J. F. (1950) Comparison of blood volume in animals with open and closed circulatory system. Physiol. Zool. 23, 113-124. WELLS J. J. & SUTTONJ. E. (1915) Blood counts in the frog, the turtle and twelve different species of mammals. Am.J. Physiol. 39, 31-36. WINTROBEM. M. (1933) Variations in the size and hemoglobin content of erythrocytes in the blood of various vertebrates. Folia Haemat. 51, 32-49. Key
Word Index-Erythropoiesis;
iron kinetics; Rana montezumae;
FeS8; liver.