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The placental transfer of orally administered iron
TOXICOLOGY
AND
The
APPLIED
6, 669-675
PH.ARhIACOLOGY
Placental
Transfer WAYNE
American
Cyattamid
Company, Received
(1964)
of H.
Orally
Administered
Iron’
LINKENHEIMER
dgricultural January
Division, ?I,
Princeton,
New
Jrrse.~
1964
Voluminous literature has resulted from the many studies of the mechanisms concerned with the regulation of absorption of orally administered iron. The principal regulatory theory emerging from these studies, and the one that has received the widest acceptance, is that of the “mucosal block,” originally proposed by Hahn and co-workers (1943) and supported in several publications by Granick (1946, 1947, 1949, 1951, 1954). Essentially, this theory implies that the mucosal epithelium of the gastrointestinal tract may accept or refuse iron depending on the requirement of the individual. More recently, however, Reissman (1955) and Heilmeyer (1958) have presented evidence that appears to be in conflict with the “mucosal block” theory; their findings suggest that iron continues to be absorbed from the intestinal tract when the concentration of protein-bound iron in the mucosa is very high. In strict terms of the “mucosal block” theory, this would not be expected since the need for iron supposedly had been fulfilled. Charley and Saltman (1960) and Saltman et ~2. (1962) have shown that low molecular weight iron-sugar chelates enhance the absorption of iron by facilitating the movement of iron through the intestinal wall, presumably independent of the “mucosal block,” if such a mechanism actually exists. The primary regulator of iron absorption under the conditions of their experiments seems to be the plasma. Furthermore, their work suggested that fructose is superior to other sugars in achieving iron absorption. The purpose of the work reported in this paper was to determine the differences, if any, in the uptake of iron by fetuses of pregnant rats fed either ferrous sulfate or ihe iron-fructose chelate described by Charley and Saltman (1960) and Saltman et al. (1962). MATERIALS
AND
METHODS
The ferrous sulfate solution was prepared by dissolving 496 mg FeS04e 7H:O and 1.0 g ascorbic acid in 5 ml of distilled water. To this was added 0.2 ml of a Fe5QS04 solution. The final volume was brought to 20 ml with distilled water. This stock solution was stored in a brown flask at 4O C. Each day, 1 ml of the stock solution was diluted to 10 ml with distilled water for oral dosing. Iron-fructose chelate was prepared by dissolving 496 mg FeSOA. 7HzO in 5 ml of distilled water, to which was added 0.2 ml of a FebQS04 solution. About 12 ml of invert sugar” was added to the ferrous sulfate solution with vigorous stirring; 6S 1 Presented in part before the Science, Chicago, Illinois, November 2 Nulomoline No. 7. 72% solids
54th Annual Meeting of the American 1962. and total inversion, Sucrest Corporation, 669
Society Sew
of Yorli.
Animal
670
WAYNE
H.
LINKENHEIMER
NaOH was added dropwise to pH 7.5. The final volume was adjusted to 20 ml with an invert sugar diluent prepared by mixing 120 ml of invert sugar with 80 ml of distilled water. The stock iron chelate solution was stored in a brown flask at 4’; dilutions were made daily with the invert sugar diluent. Triplicate samples of each of the iron preparations were taken for specific activity determinations. The specific activity per milligram iron was 89,400 cpm for the ferrous sulfate solution and 86,600 cpm for the iron-fructose chelate. The data presented are divided into two parts and hereafter will be referred to as experiment 1 and experiment 2. The rats used in experiment 1 were of the CFN strain” obtained on the third day after breeding. The rats were fed Wayne Lab Blox” for the duration of the experiment. Daily single oral doses of either ferrous sulfate or the iron-fructose chelate were given by stomach tube to each rat from the 5th through the 19th day of gestation. The daily dose of iron for individuals in both treatment groups was 100 pg, providing a total dose per rat of 1.5 mg over the 15-day period. On the twentieth day of gestation, the fetuses were surgically removed: extreme care was exercised to avoid fetal blood loss and surface contamination of the fetuses with maternal blood. Maternal blood samples were obtained by cardiac puncture. Maternal organs and fetuses were frozen and stored until counted in a well-type scintillation counter. Fetuses and maternal organs, except the maternal liver, were counted intact. The liver was weighed and an approximate l-g sample was removed from the median lobe for counting procedures. The l-g sample data were adjusted to the weight of the intact liver. Bone marrow iron uptake was based on the iron content of the intact left femur. Blood samples were centrifuged. The plasma was removed with a pipette, and the remaining cells were washed and resuspended in isotonic saline for counting procedures. Total red cell volume was calculated on the basis of 6.09 of the body weight as representative of the total blood volume; the hematocrit percentage was determined with Van Allen pipettes. Experiment 2 was designed to determine the effect of time of administration on iron uptake. Three groups of pregnant rats were given individual daily oral doses of 500 pg of iron as ferrous sulfate or the iron-fructose chelate during the 5th through the 9th day of gestation. Three to five rats from each treatment was sacrificed on the lOth, 15th, and 20th days of gestation for the determination of iron uptake by the methods previously described. Two other groups were fed either of the two forms of iron during the 10th through the 14th days of gestation and were sacrificed on the 15th and 20th days. The final groups were fed the respective iron forms during the 15th through the 19th day of gestation and sacrificed on the 20th day. Regardless of the form of iron or the days of treatment, all pregnant rats were fed by stomach tube 500 pg of elemental iron per day for 5 days for a total dose of 2.5 mg. RESULTS
AND
DISCUSSION
The in utevo fetal iron uptake data for experiment 1 are presented in Table 1. The mother rats were fed either ferrous sulfate or the iron-fructose chelate daily 3 Carworth Farms, 4 Allied Mills, Inc.,
Kew City, New Chicago, Illinois.
York.
PLACENTAL
TRANSFER
671
OF IRON
during the 5th through the 19th day of gestation. Radioactivity from Fez9 was detected in all the fetuses regardless of the iron source: this finding indicated a transport of the administered iron from the maternal to the fetal circulations. Iron uptake by the fetuses showed an average of 41.9 ug of iron per fetus for the ferrous UPTAKE
OF IRON
TABLE BY RATS in
Number Number Of of Iron
source
mothers
Ferrous sulfate Iron-fructose chelate a Level
of significance:
1 Utero:
EXPERIMENT
Iron uptake Averageuptake/offspring
offspring
ONE
(Kg) a
Total uptake/litter
5
44
41.9
(15.2-55.8)
362.9
3
30
67.9%
(51.8-77.2)
679.4
* P <
0.001.
Range
is given
in parentheses.
sulfate groups and 67.9 ug for the iron-fructose groups. This difference was significant at the 0.1% level. Comparable values calculated as total iron uptake per litter were 362.9 ug and 679.4 yg, respectively. The maternal organ iron uptake data for experiment 1 are given in Table 2. All the maternal organs examined of the ironTABLE UPTAKE
Iron
source
Number of mothers
Ferrous sulfate Iron-fructose chelate a Levels
OF IRON
of significance:
BY MATERNAL
2
ORGANS
OF
Iron Red cells
5 3 *
RATS:
EXPERIMENT
uptake
(pg)a
Plasma
Liver
Spleen
176
1 .o
383”
1.0
79 188*
1.5 5.6*
1
Uterus
Kidney
5.0
3.6 5.3*
10.0**
P < 0.05; ** P < 0.01.
fructose groups, with the exception of plasma, had greater iron uptake than the ferrous sulfate groups. The differences for the red blood cells, liver, spleen, and kidney were significant at the 5% level. The uterine difference was significant at the 1% level. The latter difference does not appear to be due to a unique mechanism, but rather to lesser variability between individual measurements. Since the total iron-binding capacity of the plasma is relatively low compared with the concentration of iron in the storage pools, maximum plasma iron concentrations are readily attained during the course of normal iron transport. Therefore, the similarity of plasma values between the two treatment groups probably represents equilibration of iron in transport. The general conclusions that may be made from the results of experiment 1 are: iron supplied to pregnant rats as iron-fructose chelate elicited higher concentrations of iron in all tissues studied except the plasma; differences varied in favor of ironfructose chelate from P < 0.05 to P < 0.0001; it appeared that iron from this source was, to a limited extent, selectively deposited in fetal tissues. The data for the in utero uptake of iron by fetuses during different periods of gestation for experiment 2 are presented in Table 3. The mothers were fed either ferrous sulfate or iron-fructose chelate at the rate of 500 ug of iron per rat per day during different periods of pregnancy as outlined in the tables.
--
Iron-fructose Iron-fructose Iron-fructose
Iron-fructose Iron-fructose Iron-fructose
5-9 5-9 s-9
10-14 10-14 15-19
‘I: P <
Ferrous Ferrous Ferrous
IO-14 10-14 1.5-19
of significance:
Ferrous Ferrous Ferrous
5-9 5-9 i-9
” Level
Iron
Days of iron administration during gestation
~~~~___
0.05. Range
chdate chdate chelate
chelate chelate chelate
sulfate sulfate sulfate
sulfate sulfate sulfate
source
is given
in parentheses.
15 20 20
10 15 20
5 4 4
4 4 .i
1 .j 20 20
10 15 20
Days of gestation at sacrifice
OF IRON
3 4 4
5 2 4
Number of mothers
UPTAKE
TABLE
38 36 29
.55 43 36
SO 23 .3x 2s .3x 34
Kumber of offspring
BY RATS
in
EXPERIMENT
38/38 36,/36 29/29
26/55 42/43 36/36
16/50 16/23 .30/38 28/28 38/38 34/34
Off spring demonstrating iron uptake (number/total)
Cfteyo:
3
0.3 (O-25) 0.8 (O-2.4) 2.9 (O-12.3)
Iron per offspring (P&9”
3.8 (0.8-6.0) 16.3* (4.9-30.3) 38.8 (30.2-53.1
0.4 (O-1.9) 2.2* (O-5.0) 8.5* (3.5-15.6)
.i.l (1.1-6.3) 9.0 (4.0-13.7) 35.4 (22.1-73.3)
2
)
146.8 585.8 1127.2
22.5 95.1 306.0
15.0 18.4 110.2 86.8 .142.8 1203.6
Total pg of iron by all offspring
5.8 23.4 45.0
0.9 3 .8 12.2
0.6 0.7 4.4 3 .J 1.3.6 48.1
Per cent of administered dose in offspring
PLACENTAL
TRANSFER
OF
IRON
673
Attention should be directed to the values indicating the number of offspring showing uptake of the iron administered to the mothers as determined by the presence or absence of radioactivity. The fetuses of the mothers fed during the 5th to 9th days of gestation and sacrificed on day 10 show partial uptake of radioactive iron, 16 of 50 in the ferrous sulfate groups and 26 of 55 for the iron-fructose chelate. If feeding is done during the same 5-9-day interval, but the sacrifice delayed until the 15th day of gestation, the radioactive iron uptake is still incomplete. However, only 16 of 23 of the ferrous sulfate groups have positive uptake whereas 42 of 43, or 98r/r, of the iron-fructose group fetuses have iron uptake from that iron source. Similarly, if sacrifice is delayed until the 20th day of gestation, iron uptake by the ferrous sulfate group is still partial but radioactive iron was detected in all of the progeny of the iron-fructose treated groups. The partial radioactive iron uptake did not appear to be related to particular litters or position of fetuses in the uterus. At all other intervals of feeding or sacrifice: all fetuses showed radioactive iron uptake from the particular source-iron fed to the mothers. The micrograms of iron per fetus show distinct differences between the two sources of iron and periods of treatment. It appears that when animals are sacrificed 1 day after cessation of treatment, no significant differences are obtained in the uptake of iron by the fetuses. However, when sacrifice is delayed until 6 or 11 days after treatment has been stopped, the fetal iron uptake differences are significant at the 57% level in favor of the iron-fructose chelate. These differences probably result from the greater storage of iron in the maternal organs of the rats receiving the iron-fructose chelate, providing more iron for release to fetal tissues and resulting in higher fetal iron concentration. These data expressed as the percentage of administered dose present in the fetuses suggest similar differences. Although, in general, the ironfructose source is superior to ferrous sulfate, it was observed that considerable iron from the ferrous sulfate preparation was found in the fetuses at various intervals. Table 4 presents the maternal iron uptake data for experiment 2. Consideration of the effects of iron source across all treatment periods reveals that the uptake of iron from the iron-fructose source was superior to ferrous sulfate. Period of treatment differences were significant only for liver and kidney. The levels of radioactive iron! regardless of source, tended to increase in the blood following withdrawal of treatment. This effect was coincident with a decrease in the iron concentration of other organs, particularly the liver. These observations are consistent with the kinetics of iron storage, mobilization, and utilization, since the absorbed iron is stored to a large extent in the liver and, as new red blood cells are formed, the stored iron is released for hemoglobin synthesis. The results of both experiments 1 and 2 indicate that iron chelated with fructose is more efficiently absorbed from the gastrointestinal tract of pregnant rats than is the iron from ferrous sulfate. The efficiency of absorption permits more iron to be stored in the maternal tissues, which may be released into the maternal circulation and transferred to the fetuses. Therefore, more iron appears in the fetuses from the iron-fructose source than from ferrous sulfate. The differences observed are probably a result of maternal concentration differences, not of unique maternal-fetal iron transfer mechanisms.
chelate chelate chelate
chelate chelate chelate
Iron-fructose Iron-fructose Iron-fructose
Iron-fructose Iron-fructose Iron-fructose
5-9 5-9 5-9 10-14 lo-14 15-19
sulfate sulfate sulfate sulfate
Ferrous Ferrous Ferrous Ferrous
5-9 lo-14 10-14 15-19
sulfate
Ferrous
Iron source Ferrous sulfate
5-9
Days of iron administration during gestation 5-9
4 4 4 4
2
Number of mothers 5
UPTAKE
TABLE
15 20 20
20
15
10
20 20
15
20
219.7 308.2 278.8
170.3 227.8 228.1
163.5 160.5 180.8 180.4
136.5
130.5
10 15
4 ORGANS
Red cells
BY MATERNAL
Days of gestation at sacrifice
OF IRON
190.5 131.8 77.8
131.8 140.4 53.2
31.2 134.9 43.6 91.1
76.1
Liver 70.9
Average
OF RATS:
iron
4.5 3.9 2.6
3.8 4.8 2.9
1.6 3.1 1.2 3.3
1.6
Spleen 2.5
uptake
EXPERIMENT
organs
9.6
10.0
12.0
8.4 14.8 6.6
4.9 6.4 5.2 8.1
4.9
Kidney 5.3
by various
2
2.3 0.2 0.8
1.8 2 .o 0.6
1.6
0.8 0.3 0.8
0.6
1 .o
Bone marrow
(ug)
1.3 0.9 0.4
0.7 OS 0.8
0 0.3 0.8 0.8
0.6
0.1
Muscle
c 2
.x
$ 5 2
PLACENTAL
TRANSFER
OF IRON
675
SUMMARY Transfer of iron from pregnant rats to fetuses was studied using ferrous sulfate or a chelate of iron and fructose as the sources of iron fed to the pregnant rats. The transfer of iron to developing fetuses took place earlier in the gestation period and was more complete in the rats receiving the iron-fructose chelate. In both the experiments described, the iron supplied as the fructose chelate produced greater concentrations of iron in all tissues studied, compared with corresponding ferrous sulfate-treated group. The differences in favor of the iron-fructose chelate varied in significance from P < 0.05 to P < 0.001. It may also be concluded that the superiority of the iron-fructose chelate over ferrous sulfate was consistent regardless of the period of administration or the time of sacrifice during gestation. Period differences noted were most marked in the blood, liver, and fetuses. The results obtained suggest that iron from the iron-fructose-fed mothers may have been selectively transferred to the fetal tissues; however, this effect is probably due to a difference in maternal iron stores rather than a unique maternal-fetal transfer mechanism. REFERENCES CHARLEY, P., and SALTMAN, P. D. (1960). The regulation of iron transport in the gut. Federation Proc. 19, 248 (Abstract). HANN, P. F., BALE, W. F., Ross, J. F., BALFAUR, W. M., and WIPPLE, G. H. (1943). Radioactive iron absorption by gastrointestinal tract. /. Exptl. Med. 78, 169. GRAKICK, S. (1946). Ferritin: its properties and significance for iron metabolism. Chem. Rev. 38, 379. GRAKICK, S. (1947). Iron and porphyrin metabolism in relation to the red blood cell. Bull. N.Y. Acad. Sci. 48, 657. GRANICK, S. (1949). Iron metabolism and hemochromatosis. Bull. N.Y. Acad. Med. 26, 403. GRANICX, S. (1951). Structure and physiologic function of ferritin. Physiol. Rev. 31, 489. GRANICX, S. (1954). Iron metabolism. Bull. N.Y. Acad. Med. 30, 81. HEILMEYER, L. (1958). Ivan in Clinical Medicine, p. 24. Univ. of California Press, Berkeley, California. RUSSMAN, K. R. (1955). Acute intestinal iron intoxication. Blood 10, 46. SALTMAX;. P. D., CHARLEY, P., and SARKAR, B. (1962). Chelation of metal ions of sugars. Federation Proc. 21, 307 (Abstract).
AND
The
APPLIED
6, 669-675
PH.ARhIACOLOGY
Placental
Transfer WAYNE
American
Cyattamid
Company, Received
(1964)
of H.
Orally
Administered
Iron’
LINKENHEIMER
dgricultural January
Division, ?I,
Princeton,
New
Jrrse.~
1964
Voluminous literature has resulted from the many studies of the mechanisms concerned with the regulation of absorption of orally administered iron. The principal regulatory theory emerging from these studies, and the one that has received the widest acceptance, is that of the “mucosal block,” originally proposed by Hahn and co-workers (1943) and supported in several publications by Granick (1946, 1947, 1949, 1951, 1954). Essentially, this theory implies that the mucosal epithelium of the gastrointestinal tract may accept or refuse iron depending on the requirement of the individual. More recently, however, Reissman (1955) and Heilmeyer (1958) have presented evidence that appears to be in conflict with the “mucosal block” theory; their findings suggest that iron continues to be absorbed from the intestinal tract when the concentration of protein-bound iron in the mucosa is very high. In strict terms of the “mucosal block” theory, this would not be expected since the need for iron supposedly had been fulfilled. Charley and Saltman (1960) and Saltman et ~2. (1962) have shown that low molecular weight iron-sugar chelates enhance the absorption of iron by facilitating the movement of iron through the intestinal wall, presumably independent of the “mucosal block,” if such a mechanism actually exists. The primary regulator of iron absorption under the conditions of their experiments seems to be the plasma. Furthermore, their work suggested that fructose is superior to other sugars in achieving iron absorption. The purpose of the work reported in this paper was to determine the differences, if any, in the uptake of iron by fetuses of pregnant rats fed either ferrous sulfate or ihe iron-fructose chelate described by Charley and Saltman (1960) and Saltman et al. (1962). MATERIALS
AND
METHODS
The ferrous sulfate solution was prepared by dissolving 496 mg FeS04e 7H:O and 1.0 g ascorbic acid in 5 ml of distilled water. To this was added 0.2 ml of a Fe5QS04 solution. The final volume was brought to 20 ml with distilled water. This stock solution was stored in a brown flask at 4O C. Each day, 1 ml of the stock solution was diluted to 10 ml with distilled water for oral dosing. Iron-fructose chelate was prepared by dissolving 496 mg FeSOA. 7HzO in 5 ml of distilled water, to which was added 0.2 ml of a FebQS04 solution. About 12 ml of invert sugar” was added to the ferrous sulfate solution with vigorous stirring; 6S 1 Presented in part before the Science, Chicago, Illinois, November 2 Nulomoline No. 7. 72% solids
54th Annual Meeting of the American 1962. and total inversion, Sucrest Corporation, 669
Society Sew
of Yorli.
Animal
670
WAYNE
H.
LINKENHEIMER
NaOH was added dropwise to pH 7.5. The final volume was adjusted to 20 ml with an invert sugar diluent prepared by mixing 120 ml of invert sugar with 80 ml of distilled water. The stock iron chelate solution was stored in a brown flask at 4’; dilutions were made daily with the invert sugar diluent. Triplicate samples of each of the iron preparations were taken for specific activity determinations. The specific activity per milligram iron was 89,400 cpm for the ferrous sulfate solution and 86,600 cpm for the iron-fructose chelate. The data presented are divided into two parts and hereafter will be referred to as experiment 1 and experiment 2. The rats used in experiment 1 were of the CFN strain” obtained on the third day after breeding. The rats were fed Wayne Lab Blox” for the duration of the experiment. Daily single oral doses of either ferrous sulfate or the iron-fructose chelate were given by stomach tube to each rat from the 5th through the 19th day of gestation. The daily dose of iron for individuals in both treatment groups was 100 pg, providing a total dose per rat of 1.5 mg over the 15-day period. On the twentieth day of gestation, the fetuses were surgically removed: extreme care was exercised to avoid fetal blood loss and surface contamination of the fetuses with maternal blood. Maternal blood samples were obtained by cardiac puncture. Maternal organs and fetuses were frozen and stored until counted in a well-type scintillation counter. Fetuses and maternal organs, except the maternal liver, were counted intact. The liver was weighed and an approximate l-g sample was removed from the median lobe for counting procedures. The l-g sample data were adjusted to the weight of the intact liver. Bone marrow iron uptake was based on the iron content of the intact left femur. Blood samples were centrifuged. The plasma was removed with a pipette, and the remaining cells were washed and resuspended in isotonic saline for counting procedures. Total red cell volume was calculated on the basis of 6.09 of the body weight as representative of the total blood volume; the hematocrit percentage was determined with Van Allen pipettes. Experiment 2 was designed to determine the effect of time of administration on iron uptake. Three groups of pregnant rats were given individual daily oral doses of 500 pg of iron as ferrous sulfate or the iron-fructose chelate during the 5th through the 9th day of gestation. Three to five rats from each treatment was sacrificed on the lOth, 15th, and 20th days of gestation for the determination of iron uptake by the methods previously described. Two other groups were fed either of the two forms of iron during the 10th through the 14th days of gestation and were sacrificed on the 15th and 20th days. The final groups were fed the respective iron forms during the 15th through the 19th day of gestation and sacrificed on the 20th day. Regardless of the form of iron or the days of treatment, all pregnant rats were fed by stomach tube 500 pg of elemental iron per day for 5 days for a total dose of 2.5 mg. RESULTS
AND
DISCUSSION
The in utevo fetal iron uptake data for experiment 1 are presented in Table 1. The mother rats were fed either ferrous sulfate or the iron-fructose chelate daily 3 Carworth Farms, 4 Allied Mills, Inc.,
Kew City, New Chicago, Illinois.
York.
PLACENTAL
TRANSFER
671
OF IRON
during the 5th through the 19th day of gestation. Radioactivity from Fez9 was detected in all the fetuses regardless of the iron source: this finding indicated a transport of the administered iron from the maternal to the fetal circulations. Iron uptake by the fetuses showed an average of 41.9 ug of iron per fetus for the ferrous UPTAKE
OF IRON
TABLE BY RATS in
Number Number Of of Iron
source
mothers
Ferrous sulfate Iron-fructose chelate a Level
of significance:
1 Utero:
EXPERIMENT
Iron uptake Averageuptake/offspring
offspring
ONE
(Kg) a
Total uptake/litter
5
44
41.9
(15.2-55.8)
362.9
3
30
67.9%
(51.8-77.2)
679.4
* P <
0.001.
Range
is given
in parentheses.
sulfate groups and 67.9 ug for the iron-fructose groups. This difference was significant at the 0.1% level. Comparable values calculated as total iron uptake per litter were 362.9 ug and 679.4 yg, respectively. The maternal organ iron uptake data for experiment 1 are given in Table 2. All the maternal organs examined of the ironTABLE UPTAKE
Iron
source
Number of mothers
Ferrous sulfate Iron-fructose chelate a Levels
OF IRON
of significance:
BY MATERNAL
2
ORGANS
OF
Iron Red cells
5 3 *
RATS:
EXPERIMENT
uptake
(pg)a
Plasma
Liver
Spleen
176
1 .o
383”
1.0
79 188*
1.5 5.6*
1
Uterus
Kidney
5.0
3.6 5.3*
10.0**
P < 0.05; ** P < 0.01.
fructose groups, with the exception of plasma, had greater iron uptake than the ferrous sulfate groups. The differences for the red blood cells, liver, spleen, and kidney were significant at the 5% level. The uterine difference was significant at the 1% level. The latter difference does not appear to be due to a unique mechanism, but rather to lesser variability between individual measurements. Since the total iron-binding capacity of the plasma is relatively low compared with the concentration of iron in the storage pools, maximum plasma iron concentrations are readily attained during the course of normal iron transport. Therefore, the similarity of plasma values between the two treatment groups probably represents equilibration of iron in transport. The general conclusions that may be made from the results of experiment 1 are: iron supplied to pregnant rats as iron-fructose chelate elicited higher concentrations of iron in all tissues studied except the plasma; differences varied in favor of ironfructose chelate from P < 0.05 to P < 0.0001; it appeared that iron from this source was, to a limited extent, selectively deposited in fetal tissues. The data for the in utero uptake of iron by fetuses during different periods of gestation for experiment 2 are presented in Table 3. The mothers were fed either ferrous sulfate or iron-fructose chelate at the rate of 500 ug of iron per rat per day during different periods of pregnancy as outlined in the tables.
--
Iron-fructose Iron-fructose Iron-fructose
Iron-fructose Iron-fructose Iron-fructose
5-9 5-9 s-9
10-14 10-14 15-19
‘I: P <
Ferrous Ferrous Ferrous
IO-14 10-14 1.5-19
of significance:
Ferrous Ferrous Ferrous
5-9 5-9 i-9
” Level
Iron
Days of iron administration during gestation
~~~~___
0.05. Range
chdate chdate chelate
chelate chelate chelate
sulfate sulfate sulfate
sulfate sulfate sulfate
source
is given
in parentheses.
15 20 20
10 15 20
5 4 4
4 4 .i
1 .j 20 20
10 15 20
Days of gestation at sacrifice
OF IRON
3 4 4
5 2 4
Number of mothers
UPTAKE
TABLE
38 36 29
.55 43 36
SO 23 .3x 2s .3x 34
Kumber of offspring
BY RATS
in
EXPERIMENT
38/38 36,/36 29/29
26/55 42/43 36/36
16/50 16/23 .30/38 28/28 38/38 34/34
Off spring demonstrating iron uptake (number/total)
Cfteyo:
3
0.3 (O-25) 0.8 (O-2.4) 2.9 (O-12.3)
Iron per offspring (P&9”
3.8 (0.8-6.0) 16.3* (4.9-30.3) 38.8 (30.2-53.1
0.4 (O-1.9) 2.2* (O-5.0) 8.5* (3.5-15.6)
.i.l (1.1-6.3) 9.0 (4.0-13.7) 35.4 (22.1-73.3)
2
)
146.8 585.8 1127.2
22.5 95.1 306.0
15.0 18.4 110.2 86.8 .142.8 1203.6
Total pg of iron by all offspring
5.8 23.4 45.0
0.9 3 .8 12.2
0.6 0.7 4.4 3 .J 1.3.6 48.1
Per cent of administered dose in offspring
PLACENTAL
TRANSFER
OF
IRON
673
Attention should be directed to the values indicating the number of offspring showing uptake of the iron administered to the mothers as determined by the presence or absence of radioactivity. The fetuses of the mothers fed during the 5th to 9th days of gestation and sacrificed on day 10 show partial uptake of radioactive iron, 16 of 50 in the ferrous sulfate groups and 26 of 55 for the iron-fructose chelate. If feeding is done during the same 5-9-day interval, but the sacrifice delayed until the 15th day of gestation, the radioactive iron uptake is still incomplete. However, only 16 of 23 of the ferrous sulfate groups have positive uptake whereas 42 of 43, or 98r/r, of the iron-fructose group fetuses have iron uptake from that iron source. Similarly, if sacrifice is delayed until the 20th day of gestation, iron uptake by the ferrous sulfate group is still partial but radioactive iron was detected in all of the progeny of the iron-fructose treated groups. The partial radioactive iron uptake did not appear to be related to particular litters or position of fetuses in the uterus. At all other intervals of feeding or sacrifice: all fetuses showed radioactive iron uptake from the particular source-iron fed to the mothers. The micrograms of iron per fetus show distinct differences between the two sources of iron and periods of treatment. It appears that when animals are sacrificed 1 day after cessation of treatment, no significant differences are obtained in the uptake of iron by the fetuses. However, when sacrifice is delayed until 6 or 11 days after treatment has been stopped, the fetal iron uptake differences are significant at the 57% level in favor of the iron-fructose chelate. These differences probably result from the greater storage of iron in the maternal organs of the rats receiving the iron-fructose chelate, providing more iron for release to fetal tissues and resulting in higher fetal iron concentration. These data expressed as the percentage of administered dose present in the fetuses suggest similar differences. Although, in general, the ironfructose source is superior to ferrous sulfate, it was observed that considerable iron from the ferrous sulfate preparation was found in the fetuses at various intervals. Table 4 presents the maternal iron uptake data for experiment 2. Consideration of the effects of iron source across all treatment periods reveals that the uptake of iron from the iron-fructose source was superior to ferrous sulfate. Period of treatment differences were significant only for liver and kidney. The levels of radioactive iron! regardless of source, tended to increase in the blood following withdrawal of treatment. This effect was coincident with a decrease in the iron concentration of other organs, particularly the liver. These observations are consistent with the kinetics of iron storage, mobilization, and utilization, since the absorbed iron is stored to a large extent in the liver and, as new red blood cells are formed, the stored iron is released for hemoglobin synthesis. The results of both experiments 1 and 2 indicate that iron chelated with fructose is more efficiently absorbed from the gastrointestinal tract of pregnant rats than is the iron from ferrous sulfate. The efficiency of absorption permits more iron to be stored in the maternal tissues, which may be released into the maternal circulation and transferred to the fetuses. Therefore, more iron appears in the fetuses from the iron-fructose source than from ferrous sulfate. The differences observed are probably a result of maternal concentration differences, not of unique maternal-fetal iron transfer mechanisms.
chelate chelate chelate
chelate chelate chelate
Iron-fructose Iron-fructose Iron-fructose
Iron-fructose Iron-fructose Iron-fructose
5-9 5-9 5-9 10-14 lo-14 15-19
sulfate sulfate sulfate sulfate
Ferrous Ferrous Ferrous Ferrous
5-9 lo-14 10-14 15-19
sulfate
Ferrous
Iron source Ferrous sulfate
5-9
Days of iron administration during gestation 5-9
4 4 4 4
2
Number of mothers 5
UPTAKE
TABLE
15 20 20
20
15
10
20 20
15
20
219.7 308.2 278.8
170.3 227.8 228.1
163.5 160.5 180.8 180.4
136.5
130.5
10 15
4 ORGANS
Red cells
BY MATERNAL
Days of gestation at sacrifice
OF IRON
190.5 131.8 77.8
131.8 140.4 53.2
31.2 134.9 43.6 91.1
76.1
Liver 70.9
Average
OF RATS:
iron
4.5 3.9 2.6
3.8 4.8 2.9
1.6 3.1 1.2 3.3
1.6
Spleen 2.5
uptake
EXPERIMENT
organs
9.6
10.0
12.0
8.4 14.8 6.6
4.9 6.4 5.2 8.1
4.9
Kidney 5.3
by various
2
2.3 0.2 0.8
1.8 2 .o 0.6
1.6
0.8 0.3 0.8
0.6
1 .o
Bone marrow
(ug)
1.3 0.9 0.4
0.7 OS 0.8
0 0.3 0.8 0.8
0.6
0.1
Muscle
c 2
.x
$ 5 2
PLACENTAL
TRANSFER
OF IRON
675
SUMMARY Transfer of iron from pregnant rats to fetuses was studied using ferrous sulfate or a chelate of iron and fructose as the sources of iron fed to the pregnant rats. The transfer of iron to developing fetuses took place earlier in the gestation period and was more complete in the rats receiving the iron-fructose chelate. In both the experiments described, the iron supplied as the fructose chelate produced greater concentrations of iron in all tissues studied, compared with corresponding ferrous sulfate-treated group. The differences in favor of the iron-fructose chelate varied in significance from P < 0.05 to P < 0.001. It may also be concluded that the superiority of the iron-fructose chelate over ferrous sulfate was consistent regardless of the period of administration or the time of sacrifice during gestation. Period differences noted were most marked in the blood, liver, and fetuses. The results obtained suggest that iron from the iron-fructose-fed mothers may have been selectively transferred to the fetal tissues; however, this effect is probably due to a difference in maternal iron stores rather than a unique maternal-fetal transfer mechanism. REFERENCES CHARLEY, P., and SALTMAN, P. D. (1960). The regulation of iron transport in the gut. Federation Proc. 19, 248 (Abstract). HANN, P. F., BALE, W. F., Ross, J. F., BALFAUR, W. M., and WIPPLE, G. H. (1943). Radioactive iron absorption by gastrointestinal tract. /. Exptl. Med. 78, 169. GRAKICK, S. (1946). Ferritin: its properties and significance for iron metabolism. Chem. Rev. 38, 379. GRAKICK, S. (1947). Iron and porphyrin metabolism in relation to the red blood cell. Bull. N.Y. Acad. Sci. 48, 657. GRANICK, S. (1949). Iron metabolism and hemochromatosis. Bull. N.Y. Acad. Med. 26, 403. GRANICX, S. (1951). Structure and physiologic function of ferritin. Physiol. Rev. 31, 489. GRANICX, S. (1954). Iron metabolism. Bull. N.Y. Acad. Med. 30, 81. HEILMEYER, L. (1958). Ivan in Clinical Medicine, p. 24. Univ. of California Press, Berkeley, California. RUSSMAN, K. R. (1955). Acute intestinal iron intoxication. Blood 10, 46. SALTMAX;. P. D., CHARLEY, P., and SARKAR, B. (1962). Chelation of metal ions of sugars. Federation Proc. 21, 307 (Abstract).