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Iron transfer across the perfused rabbit placenta
Life Sciences Vol. 9, Part II, Printed in Great Britain
pp . 765-772, 1970 .
Pergamon Press
IRON TRPNSFER ACROSS THE PERFUSED RABBIT PLACENTA Erica Baker and E .H . Morqan Department of Physiolo3y, University of Western Australia Nedlands, W .A . 6009 (Received 28 February 1970 ; in final form 14 May 1970) The transfer of iron from mother to foetus in the rabbit, and probably in other species, involves uptake of iron by the chorioallantoic placenta from the maternal plasma iron-carrying protein, transferrin and transfer to the foetal plasma where it is bound by transfetrin and carried to foatal tissues l-3 . The importance of foetal transferrin in this process is uncertain . It was therefore decided to investigate the problem by perfusion of the placenta from the foetal side through the umbilical vessels . The perfusion fluid could be altered so as to be free of transferrin, or to contain iron-unsaturated or iron-saturated transferrfn . The results showed that foatal transferrin did not play an essential role in placental transfer of iron, but in the absence of the foetus the transfer process was less efficient than with intact fo~to-placental units . Materials and Methods The experiments were performed oa pregnant New Zealand rabbits 28-30 days after mating . They were anaesthetized with pentothal sodium . The umbilical vessels of one foetus were cannulated by the method of Faber and Hart4 , the foetus was removed and the incision in the uterine wall closed by a purse-string suture . Perfusion was performed into an umbilical artery using a peristaltic pump which maintained a flour rate of I .5-2 .0 ml/min . The effluent perfusion fluid from the umbilical vein was collected into weighed test tubes which were reweighed to determine the volume . In different experiments the following 785
7gg
Vol. 9, No . 13
IRON TRANSFER
perfusion fluids were used : heparinized rabbit plasma in which the transferrin was 30-45% saturated with iron, rabbit plasma with the transferrin saturated with iron by the addition of ferrous sulphate, Hanks and Wallace buffered salt solution s , 5% dextran in Hanks and Wallace solution, and 0 .15 M NaCl . As soon as the placental perfusion was established transferrin was injected by a marginal ear vein .
s 9 Fe
bound to plasma
In two experiments the injection
solution also contained 125 1-labelled rabbit transferrin prepared and labelled as previously described s . imately 5 minutes .
Perfusion samples were collected at intervals of approx-
Maternal blood samples were also collected at 10-15 minute
intervals from üie marginal vein of the other ear to the one injected . was perfused for 20-30 minutes with each perfusion fluid .
The placenta
Radioactivity was
measured in each sample of perfusion fluid and in the serum from the blood samples using a well-type scintillatiol detector .
In the experiments in which 125 I_trans-
feirin was used as well as s9 Fe the two isotopes were counted in each sample by use of a pulse-height analyzer, and protein-bound 1251 was determined by precipstating the proteins with 10% trichloracetic acid (after the addition of isotope-free serum to protein-free perfusate samples) .
At the end of each perfusion radioactiv-
sty was also measured in all of the placentas and foetuses remaining in the uterus using a whole body radioactivity counter . Four experiments were performed in which perfusion was maintained for 100180 minutes (Table 1) .
In the first two the sequence of perfusion fluids was plasma
(unsaturated with iron) , 0 .15 M NaCl, plasma (unsaturated) , plasma (iron-saturated) and Hanks and Wallace solution .
In the other two experiments (3 and 4) the sequence
was Hanks and Wallace solution, plasma (unsaturated), plasma (iron-saturated), Hanks and Wallace solution, plasma (unsaturated), and Hanks and Wallace solution . Another variation between the first and second two experiments was that in the latter several of the foetuses were removed (foetectomy) leaving the placentas in situ 3
Vol. 9, No. 13
IRON TRANSFER
prior to cannulatinq another placenta .
787
In addition to these 4 experiments 8 other
perfusions were performed in which leaks or blockages developed within 90 min utes of cannulation .
The results obtained from them, although limited, were
entirely consistent with the data reported below . TABLE 1 Details of 4 pregnant rabbits in which one placenta was perfused by the umbilical vessels . Rabbit
Days pregnant
Number of Plasma iron foetuses concentration (wq/100 ml)
Plasma 59 Fe half-time (minutes)
Duration of perfusion (minutes)
1
28
4
107
18
180
2
28
10
49
24
100
3
30
7
61
17
170
4
28
8
102
23
120
Results In all of the experiments the radioactive iron disappeared initially from the plasma in an exponential manner with a short half-time (Table 1) .
It appeared in
the placental perfusion solution within 1 minute of intravenous injection, increased 1n concentration during the next 5-10 minutes and then decreased as the perfusion was continued .
This pattern was not affected by the nature of the perfusion solution .
Altholgh the radioactivity transferred to the perfusion fluid varied somewhat from sample to sample the variation was not related to the presence or absence of transferrin in the perfusion or whether the transferrin was iron-saturated or not . This is illustrated in Fiq . 1 for which the rate of iron transferred from maternal plasma to the perfusion solution has been calculated from the specific activity of maternal plasma iron at the mid-point of each collection time interval and the radioactivity of the perfusate sample .
788
IRON TRANSFER
Vol. 9, No . 13
+~oo
.
.
RATE Of
.
IRON TRANSFER o-10
.
.
"
,
.
"
. . " .
~lA1YA
fAIIN!
f- iAiYA 70Yrl~
q0~ rP
i
NANRi RX~ION
007
FIG . 1 Rate of iron transfer from maternal plasma to placental perfusion fluid in rabbit I . In the two rabbits given 125 1-transferrin as well as S9 Fe the rate of placental transfer of tranafeain, as determined by perfusate protein-bound 125I, was several hundred times less than that of S9 Fe (Fig . 2) . affected by the nature of the perfusion solution .
It too, was not
Hence iron transfer from
maternal plasma to perfusion solution cannot have occurred by passage of the iron-transferrin complex . The efficiency of iron transfer across the perfused placentas was evaluated by comparing the total S9 Fe content of the pooled perfusate and the perfused placenta at the end of each experiment with that in the foetus and placenta of in tact foeto-placental units (Table 2) .
In all experiments the radioiron content of
the perfused placenta was greater than that of placentas from units in the same animal, and the a.nount of iron in the perfusate was less than that of the intact foeto-placental units . In the case of foetectomized placentas (rabbits 3 & 4), the S9Fe in the placentas was very close to that of the above two totals, but was greater than tit+e S9Fe of intact or perfused placentas (Table 2) .
Vol . 9, No. 13
IRON TRANSFER
789 ~` fe~uw
lo
^~ - ~fana~n -.y ff11YY
ro
ol
oa
'°°~°~~Yf77n r
.0
b
170
40
700
171NYTFf
FIG . 2
Changes in maternal serum radioactivity and cumulative transfer of 59 Fe ( ~ ) and protein bound 1251 ( o ) to perfusion fluid in rabbit 3 . The vertical lines indicate the times of changing perfusion fluids . TABLE 2 Radiofron content of perfused, intact and foetectomized placentas and of perfusate and foetuses of 4 pregnant rabbits injected with 59 Fe . The results are given as the mean and range, with number of measurements in brackets . Rabbit
59 Fe (% injected dose) Perfused placenta
Perfusate
Intact placenta
Foetus
1
9 .9 (1)
7 .4 (I)
2
7. .2 (1)
1 .2 (1)
3
5 .6 (1)
2 .5 (1)
4
1 .6 (1)
3 .2 (1)
5 .6 (3) 2 .8-8 .8 0 .5 (9) 0 .2-1 .0 0 .4 ;2) 0 .3-0 .5 0 .8 (1)
9 .9 (3) 8 .7-11 .2 5 .0 (9) 0 .3-11 .1 7 .7 (2) 6 .2-9 .1 4 .3 (1)
Foetectomized placenta 9 8 4 3
.5 (4) .2-10 .4 .8 (6) .8-6 .6
770
IRON TRANSFER
Vol . 9, No . 13
An attempt was made to determine the chemical nature of the radioactive iron in the Hanks and Wallace perfusate in rabbits 3 and 4, by determining whether the 59 Fe was dialysable, or precipitated with cold ethanol, and by subjecting the perfusates to gel filtration .
It was found that 15-80%
of the radioiron in different transferrin-free perfusate samples was dialysable against 0 .1 M sodium acetate pH 7 .0 over 3 days at 4 ° C, while only 1 .72 .1% of the radioactivity of plasma perfusate samples dialyzed under the same conditions .
Treatment with ethanol (-20 ° C,
final concentration 80%) complete-
ly precipitated the 59Fe from plasma perfusate samples, but also precipitated 85-98% of the activity of transferrin-free samples .
Gel filtration was performed
with .Sephadex GI O, G25 and G100 and with Biogel P100, using 0 .15 M NaCl as the elution solution .
When transferrin-free samples of perfusion fluid were
used the 59Fe adsorbed to the columns and did not appear as peaks indicate of certain molecular sizes, but when plasma perfusion samples were used the 59Fe eluted quantitatively as a single peak at the position expected for tran s ferrin . Discussion The present results show clearly that the presence of transferrin in the fluid perfusiog the umbilical vessels of the placenta is not essential for iron transfer to that fluid .
There was no evidence that changing the per
fusion solution from plasma to Hanks and Wallace solution or NaCl,
or from
iron-unsaturated to iron-saturated plasma affected the rate of iron transfer . This does not mean, however, that the composition of the umbilical blood does not affect iron transfer .
Indeed the fact that radioiron transfer to ~e
perfusate was less than that to intact foetuses in the same uterus strongly suggests that the presence of the foetus does in some way influence iron
Vol . 9, No . 13
transfer .
IRON TRANSFER
77 1
The mechanism for this is uncertain, but it may provide a means
of altering iron transfer to keep pace with the rate of foetal growth .
The
accumulation of 59 Fe by the foetectomized placentas confirms previous reports of this in the rabbit 1 " 3 and rat 8 .
It illustrates the autonomous role which
the placenta plays in iron exchange between mother and foatus . The results of the experiments in which 59 Fe and 125 I_transferrin were used show that iron does not pass from maternal to foetal blood bound to maternal transferrin .
These results also show that very little exchange of
transferrin from matemal to foetal blood occurs across the chorioallantoic placenta, altho;~gh transfer via the yolk sac " which is the route for immunoglobulin transfer 9 cannot be ruled out . However, previous experiments with intact pregnant rabbits have shown that little matemo-foetal exchange of trays ferrin does o~cur in the rabbit 10 . The chemical form of the radioactive iron in the transferrin-free perfusate samples remains uncertain .
It was almost certainly in a small mole-
cular fo-m because at least part was dialysable .
This would be expected in
order to allow the iron to pass through the endothelium of foetal capillaries in the placenta at the rate observed .
However, the results obtained so far
do not distinguish between the three possibilities which were considered in this work, viz .
ionic iron or iron chelated to fructose or to glycine .
Acknowledgement-We would like to thank Miss M . Struthers for skilled technical assistance . The work was supported by a grant from the Australian Research Grants Committee and was undertaken during tenure of a C .S .I .R .O . Senior Postgraduate Studentship by E . Baker . References 1.
T .H . BOTHWELL, W .F . PRIBILIA, W . j . Physiol . 193, 615 (1958) .
MEBUST and C .A . FINCH, Amer_ .
2.
J . DAVIES, E . B . BROWN, D . STEWART, C .W . TERRY and J . SISSON, Amer . T . Physiol . 197, 87 (1959) .
772
IRON TRANSFER
Vol . 9, No. 13
3.
E . BAKER and E .H .
4.
J.J.
5.
J .H . HANKS and R .E . WALLACE, (1949) .
6.
E.
7.
G .T . WARNER and R . OLIVER, Brit . J . Radiol . 35,
8.
S .R . GLASS ER, C . WRIGHT and R .M . HEYSSEL, Amer . 1 . Physiol . _215, 205 (1968) .
9.
F .W .R . BRAMBELL, Biol . Rev . a 33, 488 (1958) .
10.
MORGAN, Ouart . 1 . Exp . Physiol .
FABER and F . M . HART, Circulation Res .
BAKER and E .H .
MORGAN,
E .H . MORGAN, J . Physiol .
26 (1964) .
173 (1969) .
816 (1966) .
Proc . Soc . Exp .
Biochemistry 8,
171,
19,
54,
1133
Biol . Med . s 71,
196
(1969) . 349 (1962) .
pp . 765-772, 1970 .
Pergamon Press
IRON TRPNSFER ACROSS THE PERFUSED RABBIT PLACENTA Erica Baker and E .H . Morqan Department of Physiolo3y, University of Western Australia Nedlands, W .A . 6009 (Received 28 February 1970 ; in final form 14 May 1970) The transfer of iron from mother to foetus in the rabbit, and probably in other species, involves uptake of iron by the chorioallantoic placenta from the maternal plasma iron-carrying protein, transferrin and transfer to the foetal plasma where it is bound by transfetrin and carried to foatal tissues l-3 . The importance of foetal transferrin in this process is uncertain . It was therefore decided to investigate the problem by perfusion of the placenta from the foetal side through the umbilical vessels . The perfusion fluid could be altered so as to be free of transferrin, or to contain iron-unsaturated or iron-saturated transferrfn . The results showed that foatal transferrin did not play an essential role in placental transfer of iron, but in the absence of the foetus the transfer process was less efficient than with intact fo~to-placental units . Materials and Methods The experiments were performed oa pregnant New Zealand rabbits 28-30 days after mating . They were anaesthetized with pentothal sodium . The umbilical vessels of one foetus were cannulated by the method of Faber and Hart4 , the foetus was removed and the incision in the uterine wall closed by a purse-string suture . Perfusion was performed into an umbilical artery using a peristaltic pump which maintained a flour rate of I .5-2 .0 ml/min . The effluent perfusion fluid from the umbilical vein was collected into weighed test tubes which were reweighed to determine the volume . In different experiments the following 785
7gg
Vol. 9, No . 13
IRON TRANSFER
perfusion fluids were used : heparinized rabbit plasma in which the transferrin was 30-45% saturated with iron, rabbit plasma with the transferrin saturated with iron by the addition of ferrous sulphate, Hanks and Wallace buffered salt solution s , 5% dextran in Hanks and Wallace solution, and 0 .15 M NaCl . As soon as the placental perfusion was established transferrin was injected by a marginal ear vein .
s 9 Fe
bound to plasma
In two experiments the injection
solution also contained 125 1-labelled rabbit transferrin prepared and labelled as previously described s . imately 5 minutes .
Perfusion samples were collected at intervals of approx-
Maternal blood samples were also collected at 10-15 minute
intervals from üie marginal vein of the other ear to the one injected . was perfused for 20-30 minutes with each perfusion fluid .
The placenta
Radioactivity was
measured in each sample of perfusion fluid and in the serum from the blood samples using a well-type scintillatiol detector .
In the experiments in which 125 I_trans-
feirin was used as well as s9 Fe the two isotopes were counted in each sample by use of a pulse-height analyzer, and protein-bound 1251 was determined by precipstating the proteins with 10% trichloracetic acid (after the addition of isotope-free serum to protein-free perfusate samples) .
At the end of each perfusion radioactiv-
sty was also measured in all of the placentas and foetuses remaining in the uterus using a whole body radioactivity counter . Four experiments were performed in which perfusion was maintained for 100180 minutes (Table 1) .
In the first two the sequence of perfusion fluids was plasma
(unsaturated with iron) , 0 .15 M NaCl, plasma (unsaturated) , plasma (iron-saturated) and Hanks and Wallace solution .
In the other two experiments (3 and 4) the sequence
was Hanks and Wallace solution, plasma (unsaturated), plasma (iron-saturated), Hanks and Wallace solution, plasma (unsaturated), and Hanks and Wallace solution . Another variation between the first and second two experiments was that in the latter several of the foetuses were removed (foetectomy) leaving the placentas in situ 3
Vol. 9, No. 13
IRON TRANSFER
prior to cannulatinq another placenta .
787
In addition to these 4 experiments 8 other
perfusions were performed in which leaks or blockages developed within 90 min utes of cannulation .
The results obtained from them, although limited, were
entirely consistent with the data reported below . TABLE 1 Details of 4 pregnant rabbits in which one placenta was perfused by the umbilical vessels . Rabbit
Days pregnant
Number of Plasma iron foetuses concentration (wq/100 ml)
Plasma 59 Fe half-time (minutes)
Duration of perfusion (minutes)
1
28
4
107
18
180
2
28
10
49
24
100
3
30
7
61
17
170
4
28
8
102
23
120
Results In all of the experiments the radioactive iron disappeared initially from the plasma in an exponential manner with a short half-time (Table 1) .
It appeared in
the placental perfusion solution within 1 minute of intravenous injection, increased 1n concentration during the next 5-10 minutes and then decreased as the perfusion was continued .
This pattern was not affected by the nature of the perfusion solution .
Altholgh the radioactivity transferred to the perfusion fluid varied somewhat from sample to sample the variation was not related to the presence or absence of transferrin in the perfusion or whether the transferrin was iron-saturated or not . This is illustrated in Fiq . 1 for which the rate of iron transferred from maternal plasma to the perfusion solution has been calculated from the specific activity of maternal plasma iron at the mid-point of each collection time interval and the radioactivity of the perfusate sample .
788
IRON TRANSFER
Vol. 9, No . 13
+~oo
.
.
RATE Of
.
IRON TRANSFER o-10
.
.
"
,
.
"
. . " .
~lA1YA
fAIIN!
f- iAiYA 70Yrl~
q0~ rP
i
NANRi RX~ION
007
FIG . 1 Rate of iron transfer from maternal plasma to placental perfusion fluid in rabbit I . In the two rabbits given 125 1-transferrin as well as S9 Fe the rate of placental transfer of tranafeain, as determined by perfusate protein-bound 125I, was several hundred times less than that of S9 Fe (Fig . 2) . affected by the nature of the perfusion solution .
It too, was not
Hence iron transfer from
maternal plasma to perfusion solution cannot have occurred by passage of the iron-transferrin complex . The efficiency of iron transfer across the perfused placentas was evaluated by comparing the total S9 Fe content of the pooled perfusate and the perfused placenta at the end of each experiment with that in the foetus and placenta of in tact foeto-placental units (Table 2) .
In all experiments the radioiron content of
the perfused placenta was greater than that of placentas from units in the same animal, and the a.nount of iron in the perfusate was less than that of the intact foeto-placental units . In the case of foetectomized placentas (rabbits 3 & 4), the S9Fe in the placentas was very close to that of the above two totals, but was greater than tit+e S9Fe of intact or perfused placentas (Table 2) .
Vol . 9, No. 13
IRON TRANSFER
789 ~` fe~uw
lo
^~ - ~fana~n -.y ff11YY
ro
ol
oa
'°°~°~~Yf77n r
.0
b
170
40
700
171NYTFf
FIG . 2
Changes in maternal serum radioactivity and cumulative transfer of 59 Fe ( ~ ) and protein bound 1251 ( o ) to perfusion fluid in rabbit 3 . The vertical lines indicate the times of changing perfusion fluids . TABLE 2 Radiofron content of perfused, intact and foetectomized placentas and of perfusate and foetuses of 4 pregnant rabbits injected with 59 Fe . The results are given as the mean and range, with number of measurements in brackets . Rabbit
59 Fe (% injected dose) Perfused placenta
Perfusate
Intact placenta
Foetus
1
9 .9 (1)
7 .4 (I)
2
7. .2 (1)
1 .2 (1)
3
5 .6 (1)
2 .5 (1)
4
1 .6 (1)
3 .2 (1)
5 .6 (3) 2 .8-8 .8 0 .5 (9) 0 .2-1 .0 0 .4 ;2) 0 .3-0 .5 0 .8 (1)
9 .9 (3) 8 .7-11 .2 5 .0 (9) 0 .3-11 .1 7 .7 (2) 6 .2-9 .1 4 .3 (1)
Foetectomized placenta 9 8 4 3
.5 (4) .2-10 .4 .8 (6) .8-6 .6
770
IRON TRANSFER
Vol . 9, No . 13
An attempt was made to determine the chemical nature of the radioactive iron in the Hanks and Wallace perfusate in rabbits 3 and 4, by determining whether the 59 Fe was dialysable, or precipitated with cold ethanol, and by subjecting the perfusates to gel filtration .
It was found that 15-80%
of the radioiron in different transferrin-free perfusate samples was dialysable against 0 .1 M sodium acetate pH 7 .0 over 3 days at 4 ° C, while only 1 .72 .1% of the radioactivity of plasma perfusate samples dialyzed under the same conditions .
Treatment with ethanol (-20 ° C,
final concentration 80%) complete-
ly precipitated the 59Fe from plasma perfusate samples, but also precipitated 85-98% of the activity of transferrin-free samples .
Gel filtration was performed
with .Sephadex GI O, G25 and G100 and with Biogel P100, using 0 .15 M NaCl as the elution solution .
When transferrin-free samples of perfusion fluid were
used the 59Fe adsorbed to the columns and did not appear as peaks indicate of certain molecular sizes, but when plasma perfusion samples were used the 59Fe eluted quantitatively as a single peak at the position expected for tran s ferrin . Discussion The present results show clearly that the presence of transferrin in the fluid perfusiog the umbilical vessels of the placenta is not essential for iron transfer to that fluid .
There was no evidence that changing the per
fusion solution from plasma to Hanks and Wallace solution or NaCl,
or from
iron-unsaturated to iron-saturated plasma affected the rate of iron transfer . This does not mean, however, that the composition of the umbilical blood does not affect iron transfer .
Indeed the fact that radioiron transfer to ~e
perfusate was less than that to intact foetuses in the same uterus strongly suggests that the presence of the foetus does in some way influence iron
Vol . 9, No . 13
transfer .
IRON TRANSFER
77 1
The mechanism for this is uncertain, but it may provide a means
of altering iron transfer to keep pace with the rate of foetal growth .
The
accumulation of 59 Fe by the foetectomized placentas confirms previous reports of this in the rabbit 1 " 3 and rat 8 .
It illustrates the autonomous role which
the placenta plays in iron exchange between mother and foatus . The results of the experiments in which 59 Fe and 125 I_transferrin were used show that iron does not pass from maternal to foetal blood bound to maternal transferrin .
These results also show that very little exchange of
transferrin from matemal to foetal blood occurs across the chorioallantoic placenta, altho;~gh transfer via the yolk sac " which is the route for immunoglobulin transfer 9 cannot be ruled out . However, previous experiments with intact pregnant rabbits have shown that little matemo-foetal exchange of trays ferrin does o~cur in the rabbit 10 . The chemical form of the radioactive iron in the transferrin-free perfusate samples remains uncertain .
It was almost certainly in a small mole-
cular fo-m because at least part was dialysable .
This would be expected in
order to allow the iron to pass through the endothelium of foetal capillaries in the placenta at the rate observed .
However, the results obtained so far
do not distinguish between the three possibilities which were considered in this work, viz .
ionic iron or iron chelated to fructose or to glycine .
Acknowledgement-We would like to thank Miss M . Struthers for skilled technical assistance . The work was supported by a grant from the Australian Research Grants Committee and was undertaken during tenure of a C .S .I .R .O . Senior Postgraduate Studentship by E . Baker . References 1.
T .H . BOTHWELL, W .F . PRIBILIA, W . j . Physiol . 193, 615 (1958) .
MEBUST and C .A . FINCH, Amer_ .
2.
J . DAVIES, E . B . BROWN, D . STEWART, C .W . TERRY and J . SISSON, Amer . T . Physiol . 197, 87 (1959) .
772
IRON TRANSFER
Vol . 9, No. 13
3.
E . BAKER and E .H .
4.
J.J.
5.
J .H . HANKS and R .E . WALLACE, (1949) .
6.
E.
7.
G .T . WARNER and R . OLIVER, Brit . J . Radiol . 35,
8.
S .R . GLASS ER, C . WRIGHT and R .M . HEYSSEL, Amer . 1 . Physiol . _215, 205 (1968) .
9.
F .W .R . BRAMBELL, Biol . Rev . a 33, 488 (1958) .
10.
MORGAN, Ouart . 1 . Exp . Physiol .
FABER and F . M . HART, Circulation Res .
BAKER and E .H .
MORGAN,
E .H . MORGAN, J . Physiol .
26 (1964) .
173 (1969) .
816 (1966) .
Proc . Soc . Exp .
Biochemistry 8,
171,
19,
54,
1133
Biol . Med . s 71,
196
(1969) . 349 (1962) .