Role of transferrin in the placental transfer of iron in the rabbit

Role of transferrin in the placental. transfer of iron in the rabbit T.

A.

J.

P.

R.

WRIGHT

Glasgow,

DOUGLAS RENTON

Scotland

ThiP role of transferrin in the transfer of iron from mother to fetus was investigated in rabbits in the last third of gestation. The rabbits were injected intravenously with 1311’-transferrin which had been saturated into 5eFe chloride. The radioactivity of the different isotopes was estimated in both maternal and fetal blood samples. 5eFe was shown to be cafiable of crossing the rabbit placenta at a different rate from that of transferrin. Increasing amounts of transferrin cross the rabbit placenta with advancing gestation. Maternal transferrin itself appears to play little part in the transfer of iron from mother to fetus in the rabbit.

Material

serum, iron iS attached to the protein transferrin. Morgan” reported that transferrin molecules are capable of crossing the rabbit placenta, although in much smaller amounts than molecules of either globulin or albumin. However, the role played by transferrin in the placental transfer of iron in the rabbit is as yet not fully understood. There are two ways in which iron could cross the placental barrier and reach the fetus: ( 1) by the iron molecule becoming detached from the maternal transferrin, crossing the placenta either in the free ionic form or attached to some other compound, or (2) by the iron remaining attached to the maternal transferrin, crossing the placental barrier and in this form reaching the fetal circulation. In order to distinguish between these possible mechanisms, 1311-labeled transferrin saturated with a mixture of 59FeC1, was injected intravenously into rabbits. The quantities of each of the isotopes were then measured in the serum of the fetuses. 1 N

T H E

M A T E R N A L

and

methods

Rabbit transferrin was obtained from Mann Chemicals, Ltd. Since chromatographic analysis showed that it contained albumin, it was purified before use by column chromatography. The crude transferrin was dissolved in 0.2M phosphate buffer pH 6.6, applied to a column of DEAE Sephadex A50, and eluted with 0.2M phosphate buffer pH 6.6 with increasing concentrations of NaCl. Three milliliter fractions were collected and the protein concentration of each was determined by measuring the extinction at 280 rnp.l The samples containing transferrin were pooled, dialyzed against ammonium bicarbonate, and subsequently freeze dried. The resulting material was now free of contaminating albumin. Iodination of the transferrin with 1311 was carried out by the method of McFarlane.5 Unbound Is11 was removed by dialysis against frequent changes of physiologic saline over a period of 24 hours. Evidence that free 1311 had been removed was obtained by passing a sample of the labeled transferrin through G25 Sephadex. The iodinated transferrin was then saturated with a mixture of

From the Departments of Biochemistry and Veterinary Surgery/Reproduction, University of Glasgow. 1169

i 170

Douglas, Renton, and

Decembw Am. J. Obst.

Wright

5gFeCl,, and the excess iron was removed by dialysis against physiologic saline. Finally, a sample of the double-labeled protein was passed through a column of G25 Sephadex to confirm that all the radioactivity was protein bound. Rabbits. The rabbits used were of no de& nite breed. Three separate experiments were carried out. In the first one, rabbits were killed after 20 and 26 days of gestation. In the second, a rabbit 29 days pregnant was used, and in the third experiment, a rabbit 25 days pregnant was used. Injection of 69Fe- and 1311-labeled transferrin. In each instance 2 ml. of a solution of the double-labeled protein was injected into a marginal ear vein. In the first experiment 2 rabbits (20 and 27 days pregnant) were killed one hour after the administration of the labeled transferrin by the intravenous injection of Euthatal (May & Baker). The remaining 2 rabbits were killed 2 hours after administration of the transferrin. The rabbits used in Experiments 2 and 3 were killed 2 hours after the administration of the radioactive protein. In every case a sample of maternal blood was obtained prior to death and fetal bIood was obtained within 5 minutes of the mother’s death. Plasma was separated from each of the blood samples and 0.1 ml. of each sample was diluted to 3 ml. with physiologic saline for estimation of

15, 1x8 & Oynec.

radioactivity. A portion of the original transferrin solution, containing 6gFe and lS1.I, was also suitably diluted for radioactivity assay. Two methods of measuring radioactivity were employed : Method 1. The radioactivity of each of the samples, prepared as described above, was measured in a well-type scintillation counter immediately after the rabbits were killed and again 24, 48, and 72 hours later. Method 2. The radioactivity was assayed in a well-type scintillation counter with a pulse height analyzer and the optimum conditions for estimating 59Fe and 1311 independentIy obtained. Calculations were then made to eliminate the interference by 5sFe at the optimum for 1311. It was then possible to differentiate between the activity due to 59Fe and 1311 in each of the samples examined. This allowed estimations to be made of the activity due to the individual isotopes present in each of the samples, i.e., maternal blood, fetal blood, and in the original radioactive injection. Results

Table I shows the results obtained from the 4 rabbits in Experiment 1. The differences are due entirely to radioactive decay. When these decay factors are compared with those for the pure isotope (Table IX), it will be seen that the radioactivity present

Table I. Radioactive decay of 5gFeand IS11in transferrin, maternal, and fetal blood

0 24 48 72

Rabbit 23,427 20,994 19,624 17,789

I* 100 89.6 83.7 75.9

Rabbit 31,574 30,760 30,281 29,577

1% 100 97.4 95.9 93.6

216,403 201,294 189,903 181,265

100 93.0 87.7 83.8

Rabbit 30,067 27,422 25,188 23,336

2” 100 91.0 83.8 77.6

Rabbit 21,180 20,826 20,298 19,657

2” 100 9s.3 95.8 92.8

0 24 48 72

Rabbit 14,787 13,567 12,602 11,565

3f 100 91.8 85.2 78.2

Rabbit a,595 8,200 7,966 7,647

3f 100 95.4 92.7 88.9

216,403 201,294 189,903 181,265

100 93.0 87.6 83.8

Rabbit 19,000 17,480 16,305 15,309

4+ 100 92.0 85.8 80.6

Rabbit 16,476 16,059 15,715 15,309 --

4f 150 97.4 95.3 92.9

“Rabbits fRabbits istration.

1 and 2, 26 days mated, maternal 3 and 4 (Rabbit 3, 20 days mated;

and fetal blood removed Rabbit 4, 26 days mated),

1 hour maternal

after and

administration. fetal blood removed

2 hours

after

admin-

Volllme Number

102 8

Role

in all the samples of the maternal blood is due to 1311 and in the case of the fetal blood almost entirely to radioactive iron. These results suggest that the “Fe of the doublelabeled transferrin has left the maternal circulation and crossed the placenta. The results obtained with Method 2 are shown in Tables III, IV, and V. Table III

Table II. Percentage over 72 hours* Decay ;ina”,

decay

1 (cXZs/ ~ % re100 sec.) making

0 24 48 72

602,502 588,797 578,460 564,803

“ssFe

half-life,

45.1

1 (ciZ!ts/ 1 % re100 sec.) maining

155,794 144,107 128,953 119,238

97.7 96.0 93.7 days:

of 5sFe and 1311

19

half-life,

8.04

92.5 82.7 76.5 days.

Table III. Results obtained when counting samples of 5gFe and 1311 separately at the respective optimum settings* A

(counts/ IO0 sec.)

the optimum conditions conditions for estimating

in placental

4,768 0

for estimating SSFe.

13lI;

Table V. Comparison B, the

of ratio of 59Fe and from maternal and

Ji,~;~.,

Maternal

Rabbit Rabbit Rabbit Rabbit Fetal

Rabbit Rabbit Rabbit Rabbit

21,850

la11 in transferrin blood in a rabbit

1 2 3 4

380 230 840 560

38,910

0.56/l

6,780 4,520 4,240 8,720

0.056/l 0.05/l 0.2/l 0.06/l

Radioactivity (counts/l00

and 1311 Original solution injected Maternal blood Fetal blood s9Fe

1 2 3 4

6,270 1,460 3,250 4,040

e9Fe

1,588 4 32

sec.) I.911

52,047 1,854 131

Ratio Fe/I

0.03/l 0.002/l 0.2/l

Table VI. Comparison 1311 in transferrin blood in a rabbit Substance e9Fe and

blood

No. No. No. No.

of ratio of 5QFe and from maternal and fetal 29 days pregnant

1 gy.

blood

No. No. No. No.

1171

(counts/

Table IV. Comparison

5eFe and jslI Original solut.ion injected

of iron

shows results when separate samples of 5QFeC13 and 1311 were assayed under optimum comnditions for each of the isotopes. It will be seen that when measuring 5QFe no interference from 1311 is encountered. However, when measuring 1311 some interference by the 59Fe occurs. Tables IV, V, and VI show the activity due independently to 5QFeC13 and 1311 in each of the samples examined. It will be seen that in each case there is evidence of some 1311 in the fetal samples. This would suggest that some maternal transferrin has crossed the placenta. Taking into account the different stages of gestation in the different rabbits used, it would appear that the amounts of transferrin crossing the placenta increased with advancing gestation. In each instance the fetal blood contains radioactive iron. Furthermore, the ratio between the 5QFeC1, and 1311 in the fetal blood is greatly altered, there being much more iron relative to iodine in the fetal blood than in the original mixture. These findings are in agreement with the 59FeC13/131,1 ratio found in the

Substance

1311 in transferrin fetal blood

transfer

100 sec.)

1,898 2,623

59Fe

1311 ‘A, optimum

B

of transferrin

900 575 370 680

6.9/l

2.5/l 8.8/l 5.9/l

IslI

Original solution injected Maternal blood Fetal bloo’d

of ratio of sgFe and from maternal and fetal 25 days pregnant Radioactivity (counts/l 00 sec.) IS11 5eFe

1,130 369 318

4,123 6,385 222

Ratio Fe/Z

0.27/l 0.057/l 1.4/l

1172

Douglas,

Renton, and

Wright

maternal blood and confirm the results found with Method 1 in the first experiment.

It is apparent from the above results that in the rabbit during the last third of gestation, iron can cross from mother to fetus unaccompanied by maternal transferrin. Therefore, the part played by transferrin in the placental transfer of i.ron in this species would seem to be that of transporting the iron in the maternal blood to the placental cells where it then becomes detached and crosses the placental barrier either in ionic form or attached to some other substance, and once across the placenta it becomes transported within the

fetal blood reattached to transferrin.l The movement of small amounts of transferrin across the placenta might have been achieved in one of two ways : (a) by crossing via the yolk sac placenta or (b) by crossing the chorioallantoic placenta. The previous work of Brambell, Hemmings, and Henderson3 demonstrated that y-globulins crossed from mother to fetal rabbit via the yolk sac placenta. In the experiments described the transferrin could have reached the fetus by either of these routes. The

authors wish to acknowledge the AgriResearch Council, which supported the work throughout. cultural

~~F~~~~CE~

1. Bevan, C. H., and Holiday, E. R.: Advances Protein Chem. 7: 320, 1952. 2. Bothwell, T. H., Pribilla, W. F., Mebust, W., and Finch, C. A.: Am. J. Physiol. 193: 615, 1958.

3. BrambeI1, F. W., Hemmings, W. Henderson, N. T.: Antibodies and London, 1951, University of London 4. Morgan, E. H.: J. Physiol. 171: 35, 5. McFarlane, A. S.: Nature 182: 53,

H., and Embryos, Press. 1964. 1958.