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Permeability to carbohydrates of human chorion laeve in vitro
Permeability
to carbohydrates
of human
chorion laeve in vitro FREDERICK
C.
ANDRE
E.
Baltimore,
Maryland
BATTAGLIA,
M.D.*
HELLEGERS,
M.D.“”
Methods
T H E results of the in vivo studies of the permeability characteristics of the primate placenta to various carbohydrates reported thus far suggest that the transfer of carbohydrates across the placenta involves more than simple diffusion. The present in vitro studies on the human chorion laeve were carried out to determine whether specific transport systems for carbohydrates could be established for the chorion laeve. In addition, one of the more interesting questions in comparative fetal physiology is whether the differences in properties of intact placentas of different species in vivo are due to differences in permeability of the same tissue layer of different species (e.g., chorion of ungulates versus chorion of primates) or to differences in the number of tissue layers composing the different placentas. For these reasons it seemed desirable to delineate some of the in vitro permeability characteristics of the human chorion laeve to carbohydrates when studied as an isoIated tissue layer.
From the Deoartments of Pediatrics Gynecology-Obstetrics, The Johns Hopkins University and Hospital.
In each case, the chorion Iaeve studied was obtained from a placenta delivered at term after a normal uncomplicated pregnancy. In general, one placenta was used for each individual in vitro study. At each experiment the carbohydrate to be tested was always pIaced in contact with one surface of the membrane at a time. The medium used on both sides of the membrane was the bicarbonate buffered medium of Gey and Geyl containing glucose at a concentration of 50 mg. per cent. The method of obtaining and mounting the chorion laeve and the apparatus used for the in vitro studies have been described elsewllere.2 All carbohydrates used were obtained as chromatographically pure reagents from commercial sources and used without further purification. Pentoses were determined calorimetrically by the method of Roe.3 Raffinose and sucrose were determined by the method of Roe.* During a 2 hour incubation, the medium in each of the two chambers of the incubation apparatus was sampled at 30 minute intervals. Thus, the concentration of the test substance on each side of the membrane was known. The concentration gradient across the membrane for the 30 minute interval was estimated as follows:
and
This work was supported bv Research Grant No. AM-04240-02 of the National Institutes of Health, *Member, Laboratory of Perinatal Physiology, National Institutes of Neurological Diseases and Blindness, National Institutes of Health, San Puerto Rico. **Senior Kennedy,
Research Scholar, Joseph Jr., Memorial Foundation.
$ Juan,
(Cx - C,),,
where sugar ternal
P.
771
f (‘2, - CF)Q =
GradientT,-,
Cn and CF = concentrations of in chambers in contact with maand fetal surfaces of the chorion,
772
Table
Battaglia
and
I. Serial
Hellegers
determinations
0 ‘I) 40 60 80 100 120 Averace
II.
~ D-arabinose I____ I C0nc.P 1 (bM/ml.)
-~.-__(“0 n c I (PM/ml.)
I ., 1 tr,2e (min.)
Table
of single in vitro
205.6 203.1 ‘00.9 198.7 196.5 194.3 191.8
Permeability ~___
Gradient (ELM/ml.)
Table --.__.
15.3
III.
”
Permeability
Test
Carbohydrates
rate
chorion
-Transfer (pM/min.)
201.9 193.8 185.8 178.6 172.3 165.6 183.0
2.21 2.61 2.44 2.43 2.21 2.27 2.36
-.
rate
Permeability (pl/mirr.) 10.9 1 3 .5 13. I 1 .?I 6 1 ‘?.a 13.7 12.9
laeve to D-arabinose
Permeability
constant*
No. of placentas studied
(rUmin.) 18.9 14.7 20.1 10.9 16.7 13.6 12.4 14.9
3 3 1 2 2 2 1 I
3.5.
constants
for human
Gradient (PM ml.)
R&nose Sucrose Sucrose I,-xylose D-xylose L-arabinose
IV.
surface Gradient (aM/ml.)
0.10 0.43 1.46 1.06 2.80 3.05 3.43 4.91
Carbohydrates
Table
for human
Transfer (pM/min.)
5.3 29.2 72.9 97.1 16i.4 224.3 278.0 333.6 “Average,
on maternal
0.0 4.9 11.6 16.3 21.6 24.6 30.3
constants I
preparation*
28.7 28.0 337.5 27.3 27.9 33.6
of polarity
in permeability Permeability constant (@Urnin.)
chorion Transfer (yM/min.)
laeve to several rate
Permeability comtant* (rUmin.)
0.35 0.26 5.6’ 0.43 0.44 0.33
const;l:*ts
carbohydrates
~._
No. of placentrc~ studied
12.2 9.3 16.6 15.7 15.6 10.4
of human
3 2 1 1 2 3
chorion
Surface to which carbohydrate exposed
No. of placentas studied
Sucrose
12.9 9.5
Maternal Fetal
2 1
I,-arabinose
10.7 9.9
Maternal Fetal
‘I 7
D-arabinose
15.3 18.5
Maternal Fetal
15 ,&
Volume Number
89 ti
Carbohydrates
of
chorion
laeve
773
dC
Fig. 1. The average transfer rate for D-arabinose expressed in pM/min., is dQ/dt; dc is the average concentration gradient across the chorion laeve for D-arabinose during the 2 hours incubation, expressed in PM/ml. In most cases (Table II), the points represent the average of several placentas studied.
307
l
2O-.
P
0 a
I o-
1
0 0
50
I
1
100
150
1
0
200
l
250
I
I
300
350
dC Fig. 2. For this figure dc is the same as for Fig. 1. P is the average permeability constant for D-arabinose expressed in /Urnin. (data obtained from Table II).
respectively, and T, and T, = time of first and second samples, respectively. For each placenta studied, at least 4 such concentration gradients across the membrane were obtained over the 2 hour period. In Tables II, III, and IV, the average gradient represents the average value of these four determinations. Since the volume of each chamber was known, the quantity of carbohydrate transferred across the membrane could be calculated as follows :
(V) T(CT,) 1 Where V equals chamber volume in milliliters on the appearance side of the membrane (generally 10 ml.), CT1 equals concentration of sugar (pM/ml.) at T, on the appearance side of the membrane, and TX equals time of first sampling in minutes. Again, at least 4 such rates were obtained for each membrane studied and the average of 4 values is presented in Tables II, III, Transfer
rate (~Mimin.)
=
774
Battaglia
and
Hellegers
and IV as the average transfer rate for a given experiment. Finally, the permeability constant of the membrane, represented by the quantity transferred per unit time per unit concentration gradient, expressed in units of clearance by dividing the Wmin.) , was obtained transfer rate by the concentration gradient over each of the 4 time intervals. The permeability constant reported for each experiment represents the average value of the 4 determinations obtained during the 2 hour study. Results
Table I presents the data obtained on the permeability to D-arabinose during a representative single in vitro incubation of chorion. This single experiment is presented in its entirety to clarify how the average values for permeability constants and transfer rates reported in the other tables were obtained. It is clear that the mean permeability constant of 12.9 ~1 per minute determined from the 6 time intervals during which the chambers were sampled was in good agreement with the median observation of 13.3 (~1 per minute. This was true in all subsequent incubations as well, suggesting little deterioration of the membrane during the 120 minutes of observation. Table II and Fig. 1 present the mean permeability constants for D-arabinose on a series of different placentas. It is clear that the transfer rate was a linear function of the concentration gradient over the range in concentration of D-arabinose from 5 to 300 PM per milliliter. Thus, as shown in Fig. 2, the permeability constant was unchanged over this range in concentration, averaging 15.3 ~1 per minute for the entire group. Table III presents similar data on the mean permeability constants for a variety of other carbohydrates tested including the disaccharide, sucrose, and the trisaccharide, raffinose. The permeability constants for the oligosaccharides and pentoses tested were not significantly different from one another. The average permeability constant for the group as a whole (13.3 i 3.1) agreed well with
that obtained for D-arabinose alone (15.3 Ik 3.5). Table IV presents the data for three different sugars whose permeability constants were determined in both directions across the chorion. It is clear that none of the three sugars had very different permeability constants in one direction of transfer over the other. Comment
While experiments carried out with carbohydrate infusions in vivo have suggested the presence of one or more carrier systems for carbohydrates in the placenta, the present in vitro experiments provide no support for such a conclusion for the human chorion laeve. Over a wide range in concentration gradients across the membrane, the transfer of D-arabinose was proportional to the gradient, consistent with Fick’s law, governing transfer by simple diffusion. With the assumption of the general form of the equation : -dQ/dt
(1) Where
Q = D= A= dc = dx
=
=
DA
(-dc/dx)
quantity
of solute transferred (PM) diffusion coefficient (cm.?/sec.) area of membrane (cm.*) concentration gradient across chorion laeve (PM/ml.) thickness of chorion laeve (cm.) then inserting the permeability constant as defined in Methods into the above equation,
(2) permeability
constant
= 5
=
15
/#min.
= 2.5 X 10m4 ml./sec. A = 7.67 cm.? (3) Therefore
g
=
3.26
x 10-S
cm./sec.
From the derivation it can be seen that this “D/dx” represents a measure of the quantity of carbohydrate transferred across 1 cm.’ of chorion laeve per unit gradient per unit time. Therefore, if the carbohydrates tested crossed the chorion laeve by diffusion, then, for a chorion weighing 500 mg. (assuming SG = I), the following value for “D” can be calculated
Volume Number
89 G
(4)
;
Carbohydrates
III D
zrz
(2’5
x 10-4) (5 x (7.67j2
10-l) =
D = 0.21 x IO-5 cm.*/sec. The diffusion coefficient of arabinose at 20° C. and O.lM in distilled water would be 0.69 x 10-5,5 hence the resistance offered by the chorion laeve to the diffusion of these carbohydrates is not much greater than that offered by a comparable layer of water alone. In the present report, no marked differences in permeability to a wide variety of carbohydrates could be found. It is of interest that oligosaccharides crossed the membrane readily. Previous work* had shown that inulin (MW 991) did not cross the chorion. Hence, for this group of water soluble, uncharged molecules, permeability of the chorion would appear to decrease
sharply within (594-991) . The polarity across to transfer rates
of chorion
laeve
775
the molecular weight range absence of any demonstrable the membrane with respect is of some interest.
Summary
The permeability of the human chorion laeve to several carbohydrates has been studied. With a concentration gradient from 5 to 300 PM per milliliter, the quantity of D-arabinose transferred was proportional to the gradient. No differences in the permeability constants for several pentoses and oligosaccharides could be found. Sucrose and raffinose crossed the chorion laeve. The rate of carbohydrate transfer was similar regardless of direction of transfer. The diffusion coefficient of the carbohydrates was approximately 0.2 X lo-” cm.?/sec.
REFERENCES
1. Gey, G. O., and 27: 45, 1936.
2. Battaglia,
Gey,
M.
K.:
J. Cancer
4. Roe, J. H., Epstein, J. H., and Goldstein, P.: J. Biol.
F. C., Hellegers,
G., and Barron, 1962. 3. Roe, J. H., and 173: 507, 1948.
Am.
D. Rice,
H.:
A. E., Meschia,
Nature
E. W.:
196: J. Biol.
1061, Chem.
5.
Chem. 178: 839, 1949. Washburn, E. W., editor: International Critical Tables, New York, 1929, McGraw-Hill Book Co., Inc., vol. 5, p. 70.
N.
to carbohydrates
of human
chorion laeve in vitro FREDERICK
C.
ANDRE
E.
Baltimore,
Maryland
BATTAGLIA,
M.D.*
HELLEGERS,
M.D.“”
Methods
T H E results of the in vivo studies of the permeability characteristics of the primate placenta to various carbohydrates reported thus far suggest that the transfer of carbohydrates across the placenta involves more than simple diffusion. The present in vitro studies on the human chorion laeve were carried out to determine whether specific transport systems for carbohydrates could be established for the chorion laeve. In addition, one of the more interesting questions in comparative fetal physiology is whether the differences in properties of intact placentas of different species in vivo are due to differences in permeability of the same tissue layer of different species (e.g., chorion of ungulates versus chorion of primates) or to differences in the number of tissue layers composing the different placentas. For these reasons it seemed desirable to delineate some of the in vitro permeability characteristics of the human chorion laeve to carbohydrates when studied as an isoIated tissue layer.
From the Deoartments of Pediatrics Gynecology-Obstetrics, The Johns Hopkins University and Hospital.
In each case, the chorion Iaeve studied was obtained from a placenta delivered at term after a normal uncomplicated pregnancy. In general, one placenta was used for each individual in vitro study. At each experiment the carbohydrate to be tested was always pIaced in contact with one surface of the membrane at a time. The medium used on both sides of the membrane was the bicarbonate buffered medium of Gey and Geyl containing glucose at a concentration of 50 mg. per cent. The method of obtaining and mounting the chorion laeve and the apparatus used for the in vitro studies have been described elsewllere.2 All carbohydrates used were obtained as chromatographically pure reagents from commercial sources and used without further purification. Pentoses were determined calorimetrically by the method of Roe.3 Raffinose and sucrose were determined by the method of Roe.* During a 2 hour incubation, the medium in each of the two chambers of the incubation apparatus was sampled at 30 minute intervals. Thus, the concentration of the test substance on each side of the membrane was known. The concentration gradient across the membrane for the 30 minute interval was estimated as follows:
and
This work was supported bv Research Grant No. AM-04240-02 of the National Institutes of Health, *Member, Laboratory of Perinatal Physiology, National Institutes of Neurological Diseases and Blindness, National Institutes of Health, San Puerto Rico. **Senior Kennedy,
Research Scholar, Joseph Jr., Memorial Foundation.
$ Juan,
(Cx - C,),,
where sugar ternal
P.
771
f (‘2, - CF)Q =
GradientT,-,
Cn and CF = concentrations of in chambers in contact with maand fetal surfaces of the chorion,
772
Table
Battaglia
and
I. Serial
Hellegers
determinations
0 ‘I) 40 60 80 100 120 Averace
II.
~ D-arabinose I____ I C0nc.P 1 (bM/ml.)
-~.-__(“0 n c I (PM/ml.)
I ., 1 tr,2e (min.)
Table
of single in vitro
205.6 203.1 ‘00.9 198.7 196.5 194.3 191.8
Permeability ~___
Gradient (ELM/ml.)
Table --.__.
15.3
III.
”
Permeability
Test
Carbohydrates
rate
chorion
-Transfer (pM/min.)
201.9 193.8 185.8 178.6 172.3 165.6 183.0
2.21 2.61 2.44 2.43 2.21 2.27 2.36
-.
rate
Permeability (pl/mirr.) 10.9 1 3 .5 13. I 1 .?I 6 1 ‘?.a 13.7 12.9
laeve to D-arabinose
Permeability
constant*
No. of placentas studied
(rUmin.) 18.9 14.7 20.1 10.9 16.7 13.6 12.4 14.9
3 3 1 2 2 2 1 I
3.5.
constants
for human
Gradient (PM ml.)
R&nose Sucrose Sucrose I,-xylose D-xylose L-arabinose
IV.
surface Gradient (aM/ml.)
0.10 0.43 1.46 1.06 2.80 3.05 3.43 4.91
Carbohydrates
Table
for human
Transfer (pM/min.)
5.3 29.2 72.9 97.1 16i.4 224.3 278.0 333.6 “Average,
on maternal
0.0 4.9 11.6 16.3 21.6 24.6 30.3
constants I
preparation*
28.7 28.0 337.5 27.3 27.9 33.6
of polarity
in permeability Permeability constant (@Urnin.)
chorion Transfer (yM/min.)
laeve to several rate
Permeability comtant* (rUmin.)
0.35 0.26 5.6’ 0.43 0.44 0.33
const;l:*ts
carbohydrates
~._
No. of placentrc~ studied
12.2 9.3 16.6 15.7 15.6 10.4
of human
3 2 1 1 2 3
chorion
Surface to which carbohydrate exposed
No. of placentas studied
Sucrose
12.9 9.5
Maternal Fetal
2 1
I,-arabinose
10.7 9.9
Maternal Fetal
‘I 7
D-arabinose
15.3 18.5
Maternal Fetal
15 ,&
Volume Number
89 ti
Carbohydrates
of
chorion
laeve
773
dC
Fig. 1. The average transfer rate for D-arabinose expressed in pM/min., is dQ/dt; dc is the average concentration gradient across the chorion laeve for D-arabinose during the 2 hours incubation, expressed in PM/ml. In most cases (Table II), the points represent the average of several placentas studied.
307
l
2O-.
P
0 a
I o-
1
0 0
50
I
1
100
150
1
0
200
l
250
I
I
300
350
dC Fig. 2. For this figure dc is the same as for Fig. 1. P is the average permeability constant for D-arabinose expressed in /Urnin. (data obtained from Table II).
respectively, and T, and T, = time of first and second samples, respectively. For each placenta studied, at least 4 such concentration gradients across the membrane were obtained over the 2 hour period. In Tables II, III, and IV, the average gradient represents the average value of these four determinations. Since the volume of each chamber was known, the quantity of carbohydrate transferred across the membrane could be calculated as follows :
(V) T(CT,) 1 Where V equals chamber volume in milliliters on the appearance side of the membrane (generally 10 ml.), CT1 equals concentration of sugar (pM/ml.) at T, on the appearance side of the membrane, and TX equals time of first sampling in minutes. Again, at least 4 such rates were obtained for each membrane studied and the average of 4 values is presented in Tables II, III, Transfer
rate (~Mimin.)
=
774
Battaglia
and
Hellegers
and IV as the average transfer rate for a given experiment. Finally, the permeability constant of the membrane, represented by the quantity transferred per unit time per unit concentration gradient, expressed in units of clearance by dividing the Wmin.) , was obtained transfer rate by the concentration gradient over each of the 4 time intervals. The permeability constant reported for each experiment represents the average value of the 4 determinations obtained during the 2 hour study. Results
Table I presents the data obtained on the permeability to D-arabinose during a representative single in vitro incubation of chorion. This single experiment is presented in its entirety to clarify how the average values for permeability constants and transfer rates reported in the other tables were obtained. It is clear that the mean permeability constant of 12.9 ~1 per minute determined from the 6 time intervals during which the chambers were sampled was in good agreement with the median observation of 13.3 (~1 per minute. This was true in all subsequent incubations as well, suggesting little deterioration of the membrane during the 120 minutes of observation. Table II and Fig. 1 present the mean permeability constants for D-arabinose on a series of different placentas. It is clear that the transfer rate was a linear function of the concentration gradient over the range in concentration of D-arabinose from 5 to 300 PM per milliliter. Thus, as shown in Fig. 2, the permeability constant was unchanged over this range in concentration, averaging 15.3 ~1 per minute for the entire group. Table III presents similar data on the mean permeability constants for a variety of other carbohydrates tested including the disaccharide, sucrose, and the trisaccharide, raffinose. The permeability constants for the oligosaccharides and pentoses tested were not significantly different from one another. The average permeability constant for the group as a whole (13.3 i 3.1) agreed well with
that obtained for D-arabinose alone (15.3 Ik 3.5). Table IV presents the data for three different sugars whose permeability constants were determined in both directions across the chorion. It is clear that none of the three sugars had very different permeability constants in one direction of transfer over the other. Comment
While experiments carried out with carbohydrate infusions in vivo have suggested the presence of one or more carrier systems for carbohydrates in the placenta, the present in vitro experiments provide no support for such a conclusion for the human chorion laeve. Over a wide range in concentration gradients across the membrane, the transfer of D-arabinose was proportional to the gradient, consistent with Fick’s law, governing transfer by simple diffusion. With the assumption of the general form of the equation : -dQ/dt
(1) Where
Q = D= A= dc = dx
=
=
DA
(-dc/dx)
quantity
of solute transferred (PM) diffusion coefficient (cm.?/sec.) area of membrane (cm.*) concentration gradient across chorion laeve (PM/ml.) thickness of chorion laeve (cm.) then inserting the permeability constant as defined in Methods into the above equation,
(2) permeability
constant
= 5
=
15
/#min.
= 2.5 X 10m4 ml./sec. A = 7.67 cm.? (3) Therefore
g
=
3.26
x 10-S
cm./sec.
From the derivation it can be seen that this “D/dx” represents a measure of the quantity of carbohydrate transferred across 1 cm.’ of chorion laeve per unit gradient per unit time. Therefore, if the carbohydrates tested crossed the chorion laeve by diffusion, then, for a chorion weighing 500 mg. (assuming SG = I), the following value for “D” can be calculated
Volume Number
89 G
(4)
;
Carbohydrates
III D
zrz
(2’5
x 10-4) (5 x (7.67j2
10-l) =
D = 0.21 x IO-5 cm.*/sec. The diffusion coefficient of arabinose at 20° C. and O.lM in distilled water would be 0.69 x 10-5,5 hence the resistance offered by the chorion laeve to the diffusion of these carbohydrates is not much greater than that offered by a comparable layer of water alone. In the present report, no marked differences in permeability to a wide variety of carbohydrates could be found. It is of interest that oligosaccharides crossed the membrane readily. Previous work* had shown that inulin (MW 991) did not cross the chorion. Hence, for this group of water soluble, uncharged molecules, permeability of the chorion would appear to decrease
sharply within (594-991) . The polarity across to transfer rates
of chorion
laeve
775
the molecular weight range absence of any demonstrable the membrane with respect is of some interest.
Summary
The permeability of the human chorion laeve to several carbohydrates has been studied. With a concentration gradient from 5 to 300 PM per milliliter, the quantity of D-arabinose transferred was proportional to the gradient. No differences in the permeability constants for several pentoses and oligosaccharides could be found. Sucrose and raffinose crossed the chorion laeve. The rate of carbohydrate transfer was similar regardless of direction of transfer. The diffusion coefficient of the carbohydrates was approximately 0.2 X lo-” cm.?/sec.
REFERENCES
1. Gey, G. O., and 27: 45, 1936.
2. Battaglia,
Gey,
M.
K.:
J. Cancer
4. Roe, J. H., Epstein, J. H., and Goldstein, P.: J. Biol.
F. C., Hellegers,
G., and Barron, 1962. 3. Roe, J. H., and 173: 507, 1948.
Am.
D. Rice,
H.:
A. E., Meschia,
Nature
E. W.:
196: J. Biol.
1061, Chem.
5.
Chem. 178: 839, 1949. Washburn, E. W., editor: International Critical Tables, New York, 1929, McGraw-Hill Book Co., Inc., vol. 5, p. 70.
N.