TRANSFER OF RADIOACTIVE SODIUM ACROSS THE PLACENTA OF THE CAT*

BY HERBERT A. POHL AND LOUIS B. FLEXNER

(From the Department of Embryology, Carnegie Institution of Washington, Baltimore, and the Department of Anatomy, the Johns

Hopkins University, Baltimore)

(Received for publication, January 18, 1941)

The placentae of various mammals have been classified by Grosser (1) into four groups. Differentiation among these groups is based upon the nature and num’-ler of tissue layers placed between the maternal and fetal circulations. It is the purpose of the present series of investigations to study the passage of substances across these different kinds of placentae throughout the gestation period and to correlate the findings with the morphology of the placenta. Observations with radioactive sodium have demonstrated that the use of isotopes provides the direct and relatively simple approach demanded by the problem.

The results on the placenta of the guinea pig, in which maternal blood is separated from fetal by only two tissue layers (chorionic epithelium and endothelium of fetal blood vessels; hemochorial type of placenta) have already been presented (2). The present study has to do with measurement of the rate of transfer of radio- active sodium across the placenta of the cat, in which the maternal and fetal circulations are separated by four layers (endothelium of maternal blood vessels, chorionic epithelium, chorionic mesen- thyme, and endothelium of fetal blood vessels; endotheliochorial type of placenta).

EXPERIMENTAL

Procedure

Twelve pregnant cats, which yielded forty fetuses, were used for the experiments. The pregnant animals were lightly etherized

* Aided by a grant from the Rockefeller Foundation Fluid Research Fund of the School of Medicine, the Johns Hopkins University.

163

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164 Placental Transfer of Radiosodium

and about 3 cc. of an isotonic solution, containing Na24, were injected into a foreleg vein. Anesthesia was immediately dis- continued after the injection. After a time interval fixed by the purpose of the experiment, the animal was again etherized, the fetuses delivered by cesarean section, and a sample of heart blood taken from the mother. The placentae were separated and weighed after removal of excess blood. The fetuses were weighed and then ashed at red heat with sulfuric acid. Measure- ments of the radioactivities of the fetal ash and maternal plasma were made with the pressure ionization chamber-string elec- trometer circuit previously described (2). The measured radio- activity was corrected for background, absorption by the sample, and radioactive decay in the manner already given (2).

The samples of Na24 were prepared with the high voltage genera- tor of the Department of Terrestrial Magnetism, Carnegie Institu- tion of Washington (3). The samples were not used for at least 24 hours after preparation to allow time for the disappearance of any C13*.

The amount of Na24 chosen for injection was such that samples taken from the animal would exceed a lower limit set by the precision of the measuring apparatus and would be within an upper limit fixed by the tolerance of the animal to radioactive material; i.e., less than about 1 microcurie (3.7 X lo4 P-rays per second) per cc. of animal (4-6). With the pressure ionization chamber- string electrometer, the precision of electrical measurements and convenience from point of view of the time necessary for a measure- ment required a sample strength of not less than 100 P-rays per second (2) for an over-all precision of 4 per cent in the method. The amounts of Na24 injected were chosen to meet this require- ment; they were always far below the upper limit of 1 microcurie per cc.

To obtain an adequate sample strength, the amount of Na24 taken for injection can be calculated from the relationship, total Na24 injected = the desired amount in the sample t the dilution factor. In the present experiments, since measurements of plasma samples and fetuses were made, it was necessary to refer to both of these to determine the minimal quantity of Na24 for an ex- periment .

Na24, injected intravenously, quickly comes to equilibrium

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H. A. Pohl and L. B. Flexner 165

with the extracellular fluids. It is finally distributed equally throughout a volume approximately 25 per cent of the body volume (2, 7, 8). Its equilibrium concentration in the plasma is therefore 4 times that in the body. Plasma samples of 2 gm. were taken for measurement. The dilution factor in this instance is 4 times the weight of the plasma sample (about 2 gm.) t the total weight of the animal. To obtain a plasma concentration of Na24 yielding 100 P-rays per second in a 2 gm. sample and in a cat weighing 5000 gm., the amount of Naz4 injected would be (5000 gm. f 2 gm. X 4) X 100 P-rays per second = 63,000 P-rays per second.

In the fetus at equilibrium with the mother in regard to Na24, the dilution factor is approximately the ratio of the fetal to the maternal weight (the concentration factor introduced by the difference in extracellular fluid volumes being neglected). In rate measurements on the fetus, the fetal Na24 concentration is usually about one-twelfth of the equilibrium concentration and the dilution factor is one-twelfth of the ratio of the weight of the fetus to the weight of the mother. The amount of Naz4 taken for injection (about 3 microcuries) exceeded the quantity calculated from the dilution factor by 25 to 50 per cent. This compensated for the reduction of radioactivity due to absorption of P-rays by the plasma or fetal ash (2). With fetuses of low weight, several members of a litter were pooled into a single sample in order to conserve Na24.

Before an experiment, the size of the fetuses was estimated by palpation and, when necessary, by lateral x-ray photographs; calculation of the probable weight was made from the crown-rump length, with Coronios’ data (9).

Units-The activity of the radioactive material is expressed in terms of P-particles per second. A sample of 100 P-rays per second is one that gives a deflection rate equivalent to a P-ray point source emitting 100 ,&rays per second in all directions and situated at the standard measuring position in our apparatus. 1 microcurie is about 37,000 P-rays per second in these units.

All quantities and concentrations of Naz4 found in the fetus have been corrected to unit concentration in the corresponding maternal plasma. The term “corrected” placed after a value for Na24 found in the fetus means, therefore, that the measured

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166 Placental Transfer of Radiosodium

value in the fetus has been divided by the measured value per cc. of maternal plasma; i.e., values in the fetus have been corrected to a concentration of one P-ray per second per cc. of maternal plasma.

Results

Establishment of Equilibrium between Maternal Plasma and Fetus-In order to plan experiments to determine the rate of transfer of Na24 across the placenta, it is necessary to know the shape of the curve describing the establishment of equilibrium between the maternal plasma and fetus. In these experiments

ri I!!!7 0 5 10 15 20 25

HOURS

25-

5-

0 20 40 60 00 100 120 FETAL WEIGHT IN GRAMS

FIG. 1 FIG. 2 FIG. 1. Rate of equilibration of Na24 in fetus with Naz4 in maternal

plasma. All fetuses weighed 95 gm. or more. The concentration of Na24 per gm. of fetus has been corrected to a concentration of one B-ray per cc. of maternal plasma as explained above in the section on “Units,”

FIG. 2. Variation of placental weight with change of fetal weight.

with the cat, unlike those with the guinea pig (2), no apparent error was introduced by delivering the several members of a litter at different time intervals after the injection of Na24 into the maternal circulation.

The data of Fig. 1 indicate that fetuses of 95 gm. or greater come to within 10 per cent of a limiting equilibrium value with respect to Na24 in the maternal plasma only after 12 to 18 hours. This is to be contrasted with the extracellular fluid which comes to within 10 per cent of equilibrium with the plasma in about 4 minutes (2, 10). The concentration of Na24 in the fetus appears to increase linearly for at least 5 hours after intravenous injection

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(1) (2)

gm. 130 0.031

40 0.052 10 o.osi

1 0.11 0.15 0.17

Ns?’ trans- Equilibrium Total Na Total Na F&l ferred per Daily concentra- retained in

weight gm. fetus weight tion of NaZ” Safety facto] transferred per hr. to hourly

per hr. increase per gm. fetus (corrected) * (corrected)* f&us Er~~psof

(3) (4) (5) (6) (7)

per cent I I

ml. WJ.

5.1 13.5 0.34 10.5 6.9 0.29 17.5 2.7 0.13 25 0.36 0.021

0.08

0.37 39 0.50 24

(0.55) 20 (‘3.6) 18

(0.6)

H. A. Pohl and L. B. Flexner 167

into the mother. Measurement of the increase in concentration of Na24 up to this time consequently forms a reliable criterion of the rate of transfer of Naz4 across the placenta. In view of these findings, the routine procedure for determination of placental transfer rates has been to remove fetuses 1. hour after the injection of Na24.

It is to be noted in Table I that the equilibrium concentration of Na24 is greater in small than in large fetuses (2).

Rates of Placental Transfer-The experiments have been planned to measure the rates of placental transfer at different parts of the

TABLE I

Data Necessary for Calculation of Safety Factor, Rate of Na Transfer to Fetus, and Rate of Na Accretion by Growing Fetus

The Na concentration in the maternal plasma has been assumed to be 3.3 mg. per cc. for the calculation of Columns 6 and 7. Extrapolated values are in parentheses. Underlined figures are uncertain.

* The term “corrected” is explained in the text in the section on “Units.”

gestation period. In all experiments, the placentae, following removal of excess blood, and fetuses have been weighed, and measurement has been made of the concentration of Na24 in the maternal plasma and of the total quantity of Na24 in the fetus (each sample was taken at a known interval, about 1 hour after intravenous injection into the mother). Such measurements can be expressed in terms of total transfer to a fetus per unit of time, of transfer rate per unit weight of fetus, or of transfer rate per unit weight of placenta. The data necessary for deriving these relations are presented graphically. Fig. 2 gives the data

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168 Placental Transfer of Radiosodium

on changes in placental weight with fetal weight (fetal weight can be translated into gestation age by reference to Fig. 4). The peak in the placental weight-fetal weight curve in the earlier stages of pregnancy appears to have its counterpart in the sheep (11). The change in total hourly transfer of Naz4 to the fetus with change of fetal size is presented in Fig. 3.

The data of Figs. 2 and 3 have been used to construct the curves of Figs. 4 and 5. In Fig. 4 are shown the changes in rate of transfer across a unit weight of placenta throughout as much of intrauterine life as it has been feasible to investigate. As is clear

FETAL WEIGHT IN GRAMS FETAL WEIGHT IN GRAMS FETAL WEIGHT IN GRAMS

FIG. 3 FIG. 4

FIG. 3. Variation of total hourly transfer of Naz4 to fetus with change of fetal weight.

FIG. 4. The gestation age has been estimated from the fetal weight by reference to the data of Coronios (9). The weight of the smallest fetus on the graph was 0.15 gm., corresponding to a gestation age of 15 to 20 days; the transfer rate per gm. of its placenta was 0.004 unit.

from the curve, the transfer rate per unit weight of placenta makes a striking over-all increase with age. Thus at 15 to 20 days the transfer rate is about 0.004 unit per gm. of placenta, while at 57 days it is 0.24 unit. Transfer of Na24 across a unit weight of the 57 day placenta is consequently about 60 times as rapid as across a unit weight of the 15 to 20 day placenta. As is clear from the curve, the findings indicate a decrease in transfer rate per unit weight of placenta from about the 55th day to term.

Fig. 5 gives the rate at which Na24 is supplied from the maternal plasma to each gm. of fetus as this rate varies with fetal weight. This rate is high in the early fetus and falls with increase of gesta-

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H. A. Pohl and L. B. Flexner 169

tion age. For example, the ratios of rates of transfer for gestation ages of 15 to 20 days, 40 days, and 60 days are 5.5:2.3:1. The high rate in the early fetus, in spite of a low transfer rate per gm. of placenta, is accounted for by the relatively large ratio of placental to fetal weight. Fig. 5 also shows the daily per cent weight increase of the fetus during that part of pregnancy under con- sideration. This has been calculated (2) from the data of Coronios (9). It is apparent from Fig. 5 that the transfer rate and growth rate curves run parallel.

Rate of Na Supply Compared to Fetal Need-It is possible to calculate, with knowledge of the transfer rate of Naz4, of the

f 0 20 40 60 60 100 120 140

E FETAL WEIGHT IN GRAMS

FIG. 5. Comparison of curve of daily per cent weight increase and curve of transfer rate of Naz4 per gm. of fetus during the gestation period.

concentration of Na24 in the fetus when at equilibrium with unit Na24 concentration in the maternal plasma, and of the growth rate, by what factor the Na supply to the fetus exceeds the demands of growth. This factor has been termed the safety factor (2). It has been shown that the safety factor = the transfer of Na24 per gm. of fetus per hour (corrected) X 24 X 100 + the equilibrium concentration of Na24 per gm. of fetus (corrected) X daily per cent increase in weight.

If a reasonable value for the concentration of Na in the maternal plasma is taken, the rates of Na transfer to the fetus and incor- poration of Na by the fetus may be directly calculated. Expres-

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170 Placental Transfer of Radiosodium

sions for these rates have previously been derived (2). The rate at which Na is supplied to t,he fetus is total Na24 transfer per hour (corrected) X Na,., ., where Na,.,. represents the concentration of Na in the maternal plasma. The hourly accretion of Na by the growing fetus is equilibrium concentration of Na24 per gm. of fetus (corrected) X Na,.,. X fetal weight X daily per cent increase in weight + 24 X 100.

The sodium safety factor, amount of Na transferred per hour, and the amount retained in hourly growth are given in Table I for fetuses of several sizes. It is apparent that the safety factor increases with increase of fetal age.

DISCUSSION

The placenta of the cat is adapted by changes in its weight and in transfer rate per unit weight to the requirements of the fetus during the gestation period. The more important of these two changes is in the unit transfer rate. From approximately the 17th day until term, the weight of the placenta increases 5 times, whereas the maximum change in unit transfer rate of Naz4 over the same period is of the order of 60 times.

Placental weight and unit transfer rate change in such a way that the curve describing the transfer of Na24 per unit weight of fetus, during that part of pregnancy studied, parallels the growth curve of the fetus. This is a phenomenon already noted in the guinea pig (2) and it strengthens the tentative hypothesis that the fundamental principle underlying placental transfer to a unit weight of fetus is that it shall parallel the growth rate of the fetus.

The several factors which may alter the transfer rate across the placenta have already been discussed (2). It has been pointed out that from the point of view of circulatory changes, the only suggestive evidence to account for the increase in unit transfer rate which occurs with increase of gestation age has come from studies on the sheep (11). In the sheep it has been found that the rate of blood flow through the fetal vessels of the placenta about doubles during the last half of pregnancy.

Histological changes, observed in the placenta with progress of gestation, appear, however, to be of clearer meaning. The placenta of the cat, in the classification of Grosser (l), is of the endotheliochorial type; i.e., endothelium of maternal blood vessels, chorionic epithelium, and connective tissue, and endothelium of

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H. A. Pohl and L. B. Flexner 171

fetal blood vessels are interposed between the two circulations. Aging of the placenta is accompanied by changes in the chorionic epithelium. This epithelium consists, in early forms, of two distinct layers, the outer of which is syncytial (nuclei are present but cell boundaries cannot be seen). As pregnancy advances, the inner epithelial cells progressively diminish in height and then become discontinuous, so that in many places the chorionic epithelium consists of but a single layer, the syncytium. The increase in transfer rate per unit weight of placenta can con- sequently be correlated with the thinning of the chorionic epi-

TABLE II

Comparison of Placentae of Cat and Guinea Pig

Period of gestation.

te%lof

--

0.3-0.4 0.4-0.5 0.5-0.6 0.6-0.7 0.7-0.8 0.8-0.9 0.9-1.0

Average fetal ge for indicatec

period

cat

days

21.7 27.9 34.1 40.3 46.5 52.7 58.9

( 2uinea PC

_ days

23.8 30.6 37.4 44.2 51 57.8 64.6

-

I 1

cat

om.

0.3 2 6

18 42 73

110

( 36 nea Pig - tlm.

1 2 7

17 34 64 94 -

Average pla- :ental weight

- I

cat

!Jm. gm. units units

7 1 0.004 0.18 11 1.4 0.01 0.3 14 2.2 0.024 0.85 17 2.7 0.072 1.4 13.5 3.1 0.15 0.70 13 3.7 0.23 1.85 15.5 4.7 0.21 1.85

Gui- nea Pig

Na*’ transferred per gm. placenta

per hr.

cat Zhines Pig

43 7 30 8 36 6 20 6

4.7 4 8 3.5 9 3.3

Ratio of pla- cental reights, cat to guinea

Pig

thelium which accompanies advance of pregnancy. This is suggestive evidence that changes in placental transfer rates are in considerable part due to changes in placental permeability.

The placenta of the guinea pig has previously been studied (2) with radioactive sodium in the same way as that of the cat. The gestation periods of the cat and guinea pig are of about the same duration (about 62 days for the cat and 68 days for the guinea pig). Moreover the growth rates of cat and guinea pig fetuses are not dissimilar and in Table II it can be seen that the two kinds of fetuses when of comparable age are of about the same weight. Hence it is possible to compare the two types of placentae at almost equivalent parts of pregnancy.

The guinea pig placenta, unlike that of the cat with its four

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172 Placental Transfer of Radiosodium

tissue layers, has only chorionic epithelium and endothelium of fetal blood vessels placed between the fetal and maternal blood, and in late stages the chorionic epithelium disappears. All other factors being more or less the same, it might be anticipated that the rates of transfer across unit weights of these two types of placentae would be correlated with their structural differences. This appears to be true. At all stages of gestation a unit weight of guinea pig placenta has a transfer rate many times that of the cat (Table II). Thus in the third tenth of pregnancy, transfer of Na24 across a unit weight of the guinea pig placenta is 43 times as rapid as across a unit weight of the cat placenta; this factor diminishes with the duration of gestation and reaches a value of about 9 in the last tenth of the gestation period. It is therefore clear that in these two types of placentae, large differences in unit transfer rate can be correlated with known histological differences.

It is evident from Table II that the “late” cat placenta has a unit transfer rate of the same magnitude as the “early” guinea pig pla- centa. This finding also appears to have a histological basis. A single layer of chorionic epithelium is present in the guinea pig placenta of fairly early stages; this later thins and probably disappears. The early cat placenta has two layers of chorionic epithelium, whereas in late stages there is only a single layer. From the point of view of chorionic epithelium, the early guinea pig and late cat placentae are therefore similar and this similarity appears to be reflected in the unit transfer rates.

The relatively low transfer rate per unit weight of placenta in the cat is compensated for by a relatively heavy placenta. Thus the weight of the cat placenta in early stages is 7 times, and in later stages over 3 times that of the guinea pig placenta (Table II). The compensation through increased mass of the placenta is, however, not complete. This is apparent from the total transfer rate of Na to the fetus which on the average is twice as high in the guinea pig as in the cat (Table I). In turn, the safety factor (ratio of Na retained by the fetus in growth to Na supplied the fetus) has an average value of about 25 in the cat (Table I) whereas the average in the guinea pig is 50.

We are much indebted to Dean B. Cowie of the National Cancer Institute for making the sodium bombardments with the Carnegie generator.

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H. A. Pohl and L. B. Flexner 173

SUMMARY

1. The cat fetus comes to within 10 per cent of equilibrium with Na24 in the maternal plasma only after 12 to 18 hours. This is in striking contrast to the maternal extracellular fluid which comes to within 10 per cent of equilibrium with intravenously injected Na24 in about 4 minutes.

2. Changes in the rate of placental transfer per unit weight of placenta have been measured with Na24 from a gestation age of 15 to 20 days until term. The unit transfer rate of Naz4 increases 60 times during this period.

3. The shape of the relative growth curve of the cat fetus is similar to that of the curve describing the change of rate of transfer of Na24 to a unit weight of fetus at different periods of pregnancy.

4. The fetus receives across the placenta an average of about 25 times as much Na as is incorporated in the growing tissues.

5. Differences in unit transfer rate across the cat and guinea pig placentae at comparable stages of pregnancy are analyzed on the basis of the known histological differences of the two types of placentae. It is shown that differences in transfer rate can be correlated with the histological differences.

BIBLIOGRAPHY

1. Grosser, O., Frtihentwicklung, Eihautbildung und Placentation, Munich (1927).

2. Flexner, L. B., and Pohl, H. A., Am. J. Physiol., 132, 594 (1941). 3. Flexner, L. B., and Roberts, R. B., Am. J. Physiol., 128, 154 (1939). 4. Hertz, S., Roberts, A., Means, J. H., and Evans, R. D., Am. J. Physiol.,

126, 565 (1940). 5. Crane, H. R., Physic. Rev., 68, 1243 (1939). 6. Mullins, K. J., J. Cell. and Comp. Physiol., 14, 403 (1939). 7. Griffiths, J. H. E., and Maegraith, B. G., Nature, 143, 159 (1939). 8. Greenberg, D. M., Campbell, W. W., and Murayama, M., J. BioZ.

Chem., 136, 35 (1940). 9. Coronios, J. D., Genetic Psychol. Monographs, 14, 283 (1933).

10. Hevesy, G. J., J. Chem. Xoc., 1213 (1939). Il. Barer-oft, J., and Kennedy, J. A., J. Physiol., 96, 173 (1939).

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Herbert A. Pohl and Louis B. FlexnerACROSS THE PLACENTA OF THE CATTRANSFER OF RADIOACTIVE SODIUM

1941, 139:163-173.J. Biol. Chem. 

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