Factors determining human chorion laeve permeability in vitro

Factors determining human chorion laeve permeability in vitro A.

ELMORE

BRADLEY ANN

SEEDS, C.

STOLEE,

EICHHORST,

M.D. B.S.

B.A.

Minneapolis, Minnesota An increased mean daffwion permeability across human chorion laeue in vitro was measured for meperidine (D = 5.26 x 1 O-’ cm.= sec.-‘) and diarepam (D = 4.51 x 1 Om6 cm.’ sec.-l). These values corresponded to large chloroform-buffer partition coefficients (49 and 29) measured for these two compounds. Dtff usion permeability values of 3.98 and 2.18 x 1 Oa6 cm.’ SK-~ measured for urea and glucose corresponded to their relative insolubility in lipid, as indicated by chloroform-buffer partition coefjcients of 0.05 and 0.004, respectively. An increase in placental permeability in vitro to the weak organic acid 5,5-dimethyl, 2,4-oxazalidinedione at lower pHs corresponded to an increase in the fat-soluble nonionized fraction of this compound. These data support the concept that this tissue is most permeable to compounds of relatively small molecular size andlor with a high level of lipid solubility. The large diff usion permeability values measured for meperidine and diuzepam suggest that these compounds will dijfim rapidly between mother andfetus at a maximal rate limited only by uterine bloodjow. (AM. J. OBSTET. GYNECOL. 128: 13, 1977.)

A MAJORITY OF placental transfer in the human being occurs by simple diffusion between mother and fetus. In a previous study, we reported data that suggested several pathways for diffusion across human placental tissue.’ Water-soluble, lipid-insoluble compounds most likely cross between the cells through large water-filled extracellular channels. The major factor influencing the permeation of these water-soluble compounds is their molecular size. Lipid-soluble compounds appear to cross this tissue by solubility in and diffusion through cell lipid or by means of transcellular penetration in addition to that fraction crossing through extracellular spaces. This study represents additional observations on human placental permeability in vitro to several different solutes comprising a wide range of molecular

weights and lipid solubilities, in an effort to define further the influence of such characteristics on the mechanism, pathway, and rate of diffusion of these compounds. This study also intends to provide some indication of the transport of these compounds between mother and fetus in vivo. Methods

Tissues (human chorion laeve) used for these studies were obtained within 10 minutes of delivery and mounted in an in vitro diffusion chamber (Fig. l), as 95 previously described ‘, 3: A gas mixture containing per cent oxygen and 5 per cent carbon dioxide is bubbled through No. 22 spinal needles into the base of both chambers, which are divided by the mounted membrane. The gas provides oxygen requirements, mixing and maintaining the pH of a 20 mM. bicarbonate solution between 7.4 and 7.5. The bathing medium also contains physiologic quantities of other electrolytes and 50 mg per cent glucose. These experiments were performed between 4 and 38” C, by suspension of the diffusion chambers in a constant-temperature water bath. Concentration differences across the mounted tissue layer were created by the addition of 0.1 &i of C14-meperidine (Demerol), 0.5 &i of H3-diazepam

From the Department of Obstetrics and Gynecology, University of Minnesota Medical School. Presented by invitation at the Eighty-seventh Annual Meeting of the American Association of Obstetricians and Gynecologists,Hot Springs, Virginia, September 9-l 1, 1976. Reprint requests: Dr. A. Elnwre Seeds, Department of Obstetrics and Gynecology Universiiy of Cincinnati, College of Medicine, 231 Bethesda Ave., Cincinnati, Ohio 45267. 13

14

Seeds,

Eichhorst,

and Stolee

Fig. 1. Diagram of diffusion chamber of Battaglia and coworkers’ used for these studies. (Valium), 10 mg. per cent 5,5-dimethyl, 2,4-oxazalidinedione (DMO), 10 mg. per cent urea, 100 mg. per cent glucose, or 10 mg. per cent para-amino hippurate (PAH) to one side of the system. Each solute was studied individually in separate experiments. From serial measurements of changes in solute concentration on both sides of the chamber over 90 minutes, permeability constants (cm. sec.-‘) were calculated. Diffusion coefficients (cm.’ sec.-l) were estimated with the weight of a known area of membrane and an assumed specific gravity of 1.0. Such flux experiments and the necessary calculations have been described previously.” The effect of temperature on the diffusion of these solutes was studied, and activation energies for diffusion were calculated from an Arrhenius plot of the solute diffusion rate versus the reciprocal of the absolute temperature. Large activation energies for the diffusion of lipid-soluble compounds across the placenta and other body membranes have been interpreted previously as evidence that these compounds are crossing this tissue by solubility in and diffusion across cells, in addition to diffusion through extracellular channels.” -) Smaller activation energies, similar to values obtained for the free diffusion of these solutes in a solution of water, have been consistent with the in-

terpretation that these solutes cross the placental tissue through aqueous channels that possess the physical characteristics of bulk water. Chorion laeve permeability to DMO was measured at pH 5.8 with 28 mM, phosphate buffer and IO0 per cent oxygen bubbled through the chamber. The ditfusion of DMO was simultaneously compared to PAH, a similar-sized lipid-insoluble compound whose pcrmc;tbility characteristics do not change with differing pll. Partition coefficients for separation of‘ solute bctween chloroform and the buffer solutions used in these diffusion experiments were measured ah follows: 2 ml. of buffer solution containing a known amount ot solute was shaken with 2 ml. of chloroform until quilibrium concentrations of solute in the two sohcnt phases was achieved. No further changes in solute concentration were observed in anv sample after 10 minutes. The suspension was then centrifuged, the bufftil solution was separated. and solute analysis NX performed again in triplicate. C14-meperidine and H”-diazepam activity wt’rc counted in a polyether 61 1 solution in a Beckman licluid scintillation counter.” PAH concentration 1,as determined calorimetrically by means of a clia~oti/ation reaction, described by Bratton and Marshall. DMO and urea were also measured colorirnetrically .‘I I\ hilt glucose concentration was measured enzymatic-ally.

Results The diffusion of meperidine, diazepam, III-ea. and glucose across term human chorion laeve between 4” and 38” C. is summarized in Table I. The highest mean permeability value at body temperature l+as measured for meperidine (molecular tvcight 28.5; D = 5.26 x 10.’ cm.’ per sec.-‘). Placental tissue \\as least permeable to glucose (molecular weight 184; D = 2.18 x IO--’ cm.’ per SKY’). The differing permeabilities of these two relatively similar-sized compounds corresponded to their widely separate tipicl solubilities, as indicated by chloroform-buffer partitioning coefficients. Partition values of 39 \\.ere mcasured for meperidine and 0.004 for glucose. An intermediate permeability value (D = 3.98 x IO-” cm.’ prr sec.) was measured for the diffusion of the smallrr urea molecule (molecular weight 60) across placental tissue at 38” C. Although urea was relatively insoluble in lipids, as indicated by a chloroform buffer partition coefficient of 0.02, it penetrated this tissue at an intc’rmediate rate because of its small size. The diffusion across placental tissue of two larger compounds with a high lipid solubility, meperidine and diazepam, showed a large temperature dependence. while the variation in diffusion permeability ber\teran 1

Volume Number

Chorion laeve permeability

128 1

15

meperidine 7.8 Kc01/mole

0.8 K

diazepom 6.6 Kcal /mole

Fig. 2. Arrhenius glucose. Table

I. Diffusion

of human

Temperature Merperidine (molecular weight 284) Diazepam (molecular weight 285) Urea (molecular weight 60) Glucose (molecular weight 184) DMO (molecular weight 129) PAH (molecular weight 196)

3.4 ‘/r x IO3

3.3

fC.)

38” 20” 4” 38 20” 4 38” 20” 4” 38 20” 4” 38” 38”

chorion . Partition CHCL,

3.6

to diazepam, meperidine,

urea, and

laeve coejjcient

buffer(pH

29 0.05 0.004 (pH (pH (pH (pH

of 7.4)

P

X

105cm.

D X IO6 cm2 sec.-’

sec.-’

10.84 + 0.99 7.49 f 0.80 1.74 f 0.25 9.82. -L 1.05 6.04 f 0.55 2.07 f 0.35 8.89 ” 1.58 8.32 t 0.67 3.10 * 0.34 4.70 rt_0.23 4.12 f 0.20 1.68 f 0.11

49

0.03 0.35 0.01 0.02

I

3.5

plot of human chorion laeve permeability

permeability

Compound

I

I

I

I

3.2

(8) (8) (4) (8) (6) (6) (8) (6) (4) (16) (14) (12)

5.26 3.13 1.14 4.51 3.62 1.24 3.98 3.41 2.05 2.18 1.82 1.01 3.20 3.97 2.63 2.40

7.4) 5.8) 7.4) 5.8)

5 0.48 -c 0.19 + 0.16 f 0.25 2 0.32 f 0.12 +- 0.29 f 0.22 2 6.24 f 0.18 ?z 0.12 * 0.11 2 0.28 + 0.57 2 0.20 r 0.34

(8) (8) (4) (8) (6) (6) (8) (6) (4) (16) (14) (12) (6) (pH (6) (pH (6) (pH (6) (pH

7.4) 5.8) 7.4) 5.8)

Results are given as means 2 standard errors. The number of experiments is in parentheses. and 38” C. for lipid-insoluble compounds, such as urea and glucose, was small. Activation energies (AE) were computed for diffusion of meperidine (7.8 Kcal. per mole), diazepam (6.6 Kcal. per mole), glucose (3.9 Kcal. per mole), and urea (3.4 Kcal. per mole) from a plot of their diffusion rates versus the reciprocal of the absolute temperature (Fig. 2). In vitro permeability of chorion laeve to the weak organic acid DMO (molecular weight 129; pK 6.13) was measured at pH 5.8 and 7.4 in separate groups of experiments. In each group, the permeability to DMO was simultaneously measured and compared to the permeability of PAH, a compound whose lipid insolubility changes very little over a wide pH range. An increased ability of the DMO to cross the placental tissue at the lower pH is indicated by a mean D value of 3.97 X 10m6 cm.* per sec.-‘, compared to 3.20 at a

Table II. Chorion laeve permeability water-soluble compounds. I Molecular Solute

Urea DMO Glucose PAH

weight

60 129 184 190

of D x IO’ cm.a sec.-’

3.98 3.20 2.18 2.63

pH of 7.4. The increased permeability at the lower pH corresponded to increased chloroform buffer partitioning, measured at the lower pH. The chloroform buffer partition coefficient for this compound at pH 5.8 was 0.35 compared to 0.03 at pH 7.4. In all of these experiments, solute transfer was linear with gradient and independent of membrane orientation.

16

Seeds,

Table

Eichhorst,

and Stolee

III. Permeability

of chorion

laeve to DMO

and PAH at pH 5.8 and pH 7.4

PH

Partition coejjicient of CHC13 buffer (70)

DMO ( ‘% in CHC13)

5.8 7.4

0.35 0.03

26 3

Nonionized fraction 82 5

DMO (75)

D mm*

DP.M*

3.97 3.20

2.40 2.63

D o.w-Dr..,2 1.57 0.57

*D is given in units of 1O-6 cm.* sec.-’ Table

IV. Comparison

of permeability

data

I;, Water Meperidine Diazepam Antipyrine Urea DMO PAH Salicylate Glucose

18

6.9

285 284

49 29

180

23

60

129 194 136 184

0.05 0.03

0.01 0.02 0.004

5.3 4.5 4.5 4.0 3.2 2.6 2.0 2.2

24

24 6

*From Battaglia, F. C., Behrman, R. E., Meschia, G., Seeds, A. E., and Bruns, P. D.: Clearance of inert molecules, Na, and Cl ions across the primate placenta, AM. J. OBSTET. GYNECOL. 102: 1135, 1968.

Comment Permeability of human placental tissue in vitro to the compounds used in this study was consistent with previous observations suggesting that molecular size and lipid solubility are major factors in determining the transfer of substances across this membrane. The highest permeability values were measured for the highly lipid-soluble analgesic agents meperidine and diazepam. The rather large temperature dependence and activation energies (6.6 to 7.8 Kcal. per mole), calculated for the diffusion of these compounds, suggests that these substances not orily are crossing this tissue through extracellular water-filled channels but also are diffusing across the ceils by solubility in and diffusion through cell lipid. The decreased ability of glucose to penetrate this tissue corresponded to the low lipid solubility and small activation energy for diffusion (3.9 Kcal. per mole) measured for this solute. Such data suggest that glucose is diffusing only through the water-filled extracellular spaces, since the small activation energies measured in these studies are similar to values previously measured for the diffusion of a similar-sized compound in a solution of water.7 Thus, this solute is probably diffusing through large extracellular channels in which the structure of solvent water is unchanged. There was no evidence in these studies in vitro to suggest the facilitated diffusion of glucose, as

has been described across other body membranes. Although the lipid-insoluble urea (chloroform buffer partition coefficient 0.02) crossed this tissue much more readily than glucose (with a D value of 3.98), the relatively low temperature dependence of this diffusion process (activation energy for diffusion 3.3 Kcal. per mole) suggested that this solute was also diffusing by means of the extracelluIar water-filled pathways and that the increased permeability of this compound was due to its smaller molecular size. Placental permeability to the weak organic acid DMO also corresponds to its relatively small molecular size and lipid insolubility at pH 7.4. When the diffusion permeability for the water-soluble, lipid-insoluble compounds included in this study is arranged according to molecular size (Table II), it can be seen that the ability of these solutes to penetrate this tissue corresponds very closely to their relative molecular size, with the smaller compounds crossing more rapidly. Table III shows that the proportion of nonionized DMO (pK 6.13) changes significantly within the pH range of 5.8 to 7.4 and that the increase in the nonionized fraction at the lower pH significantly increases the lipid solubility or the chloroform-buffer partition coefficient of this compound. An increased diffusion permeability for DMO was measured at pH 5.8 (3.97 at pH 5.8 versus 3.20 at pH 7.4). This higher permeability at the lower pH corresponds to the increased nonionized and lipid-soluble fraction of this compound. The increase in permeability to DMO caking place at a lower pH can be better appreciated by comparing DMO diffusion with the diffusion of the lipid-insoluble compound PAH. The lipid insolubilit) of PAH and placental tissue permeability to this cornpound does not change over this pH range; thus, the penetration of this compound served as a marker or reference measurement at both pH’s. Since PAH crosses this tissue principally through extracellular waterfilled spaces, the DMO permeability minus the PAH permeability (DIjMO - DPAH) can be used to estimate the proportion of DMO crossing the membrane by the transcellular pathway, by means of solubility in and diffusion through cell lipid. A DDMt, - DpAIs of 1.57 at pH 5.8 represents a significant increase from the DDM,, - DPAH of 0.57 measured at pH 7.4. Such data

Volume Number

Chorion

128 1

support the concept that decreasing the pH and thus increasing the nonionized lipid-soluble fraction of weak acid such as DMO increases the placental diffusion of such compounds by adding a transcellular permeation route through cell lipid in addition to that portion already diffusing between the cells. Table IV compares permeability data obtained in the present study with previously reported data from our laboratory on in vitro permeability of human chorion laeve to other solutes. The clinical significance of such transfer data is best illustrated by comparing the permeability measured in vitro for these solutes with that of several other‘marker molecules. In vivo diffusion clearance studies performed in sheep and the rhesus monkey by Battaglia and coworkers* and Meschia and associates’ have indicated that the transfer of antipyrine between mother and fetus represents a maximal or perfusion-limited clearance. Thus, any solute shown to diffuse in vitro at a similar or greater rate than antipyrine could be expected to cross the placenta in vivo at a maximal or flow-limited rate. Table IV shows the in vitro permeability of meperidine and diazepam to be similar to or greater than that measured for antipyrine. The large diffusion permeability values measured for meperidine and diazepam correspond to their respective high lipid solubilities as given by chloroform-buffer partition coefficients. These values indicate that these compounds will diffuse between mother and fetus at a

laeve

permeability

17

rapid or maximal rate, limited only by uterine blood how. Such a hypothesis corresponds to the rapid appearance of these compounds in fetal circulation in vivo after administration to the mother.“* ‘I Urea crosses the primate placenta in vivo at a slower or intermediate rate, partially flow limited and partially membrane or diffusion limited. When the lower in vitro permeabilities of glucose and DMO are compared to the permeability of urea, one could predict that the diffusion of these lipid-insoluble solutes in vivo would also be partially membrane limited. Thus, the transfer of these compounds between mother and fetus would occur at an intermediate rate, significantly below that observed for flow-limited diffusion. Demonstration of the increased permeability of placental tissue in vitro to the nonionized, lipid-soluble DMO at lower pH’s is consistent with previous work carried out in this laboratory” and suggests the possibility that pH changes in vivo may have a significant effect on placental permeability to weak organic acids with a pK in the physiologic range. In conclusion. this study reports the permeability of placental tissue in vitro to a variety of lipid-soluble and insoluble compounds. It demonstrates that placental tissue is most permeable to compounds of a relatively small molecular size and/or with increased lipid solubility. The increase in placental permeability to the weak organic acid .DMO (pK 6.1) at a lower pH, corresponding to an increase in the fat-soluble, nonionized fraction of this compound, is also demonstrated.

REFERENCES

1. Seeds, A. E., Schruefer, J. J., Reinhardt, J. A., and Garlid, K. D.: Diffusion mechanisms across human placental tissue, Gynecol. Invest. 4: 31, 1973. 2. Battaglia, F. C., Hellegers, A. E., Meschia, G., and Barron, D. H.: In udtro investigations of the human chorion as a membrane system, Nature 196: 1061, 1962. 3. Lloyd, S. J., Garlid, K. D., Reba, R. C., and Seeds, A. E.: Permeability of different layers of the human placenta to isotopic water, J. Appl. Physiol. 26: 274, 1969. 4. Hays, R. M., and Leaf, A.: The state of water in the isolated toad bladder in the presence and absence of vasonressin, 1. Gen. Phvsiol. 45: 933. 1962. 5. ‘Bratton,-A. C., ani Marshall, E.’ K., Jr.: A new coupling component for sulfanilamide determination, J. Biol. Chem. 128: 537, 1939. 6. Butler, T. C.: Quantitative studies of the demethylation of trimethadione (tridione), J. Pharmacol. Exp. Ther. 108: 11, 1953.

Discussion St. John’s, Newfoundland, Canada. Transfer studies from the maternal organism to the fetus have been numerous but are far from complete or specific. Drugs which affect the fetus must DR.

DAVID

CHARLES,

7. Longsworth,

L. G.: Temperature dependence of diffusion in aqueous solutions, J. Physiol. Chem. 58: 770,1954. 8. Battaglia, F. C., Behrman, R. E., Meschia, G., Seeds, A. E., and Bruns, P. D.: Clearance of inert molecules, Na, and Cl’ions across the primate placenta, AM. J. OBSTET. GYNECOL. 102: 1135, 1968. 9. Meschia, G., Battaglia, F. C., and Bruns, P. D.: Theoretical and experimental study of transplacental diffusion, J. Appl. Physiol. 22: 1171, 1967. 10. Shier, R. W., Sprague, A. D., and Dilts, P. V., Jr.: Placental transfer of meperidine HCl. Part II, AM. J. OBSTET. GYNECOL. 115: 555, 1973. 11. Erkola, R., Kangas, L.. and Pekkarinen, A.: The transfer of diazepam across the placenta during labor, Acta.

Obstet. Gynecal. Stand. 52: 167, 1973. 12. Schruefer, J. J., Haburchak, D. R., Garlid, K. D., and Seeds, A. E.: Non-ionic diffusion of pentobarbital across human chorion laeve, Biol. Neonate 27: 251, 1975.

traverse the placenta or fetal membranes unless they are administered by intrauterine fetal transfusion. Most transport mechanisms known to be operational in other biological systems have been observed in the placenta or have been postulated. By the application of

18

Seeds,

Eichhorst,

and Stolee

more refined procedures involving autoradiography, in combination with light and electron microscopy, one can expect advances in studies pertaining to placental and membrane transport. There is no question that conventional electron microscopy, so gainfully employed in the past, holds great promise in future exploration concerning the transfer of substances from mother to fetus. More definitive information concerning diffusion of substances across membranes will be available in the future by the use of spin-labeled molecules and the determination of the nuclear magnetic resonance relaxation times of the water and lipid phases in the membrane. The fact that substances may reach the fetus by routes other than the placenta has been well documented. The in vitro studies by Battaglia and his associates’ suggest that exchange of water and some solids might, under certain circumstances, take place across the fetal membranes in the human being. This work has been confirmed by Levitt and his coworkers,’ who studied the extraplacental transfer of steroid hormones between fetus and mother. From such studies it can be concluded that the fetal membranes are permeable to steroid hormones present in the amniotic sac, but, on the other hand, there is only scant information concerning the mechanism of transfer in vitro for other pharmacologic agents. Dr. Seeds has continued his studies with a modified Ussing chamber to indicate that the “smooth chorion” is capable of penetration by many substances. In previous studies, he has demonstrated that urea, D-arabinose, and sucrose cross the chorion laeve without much difficulty. He noted that although these substances do not penetrate as rapidly as water molecules they do exert an osmotic effect. Such studies have established the fact that the “smooth chorion” is quite porous with extracellular diffusion channels. The transfer of drugs is determined not only by the size but also by the molecular geometry of the compound. The rate of entry of the compound is inversely proportional to its molecular weight and degree of ionization but proportional to its lipid solubility. To ascertain the diffusion capabilities of a membrane separating two circulations functioning at different pressures in vivo, it is necessary to equate the relative directions of flow. On account of the complexity of the placenta, only approximations of the diffusing capacity can be obtained by making such assumptions as constant permeability in all areas and uniform flow patterns. Therefore, in extrapolating membrane transport from the chorion laeve to clinical problems, one is dealing with the amniotic fluid and not directly with the fetal circulation. A further point is that antipyrine is not completely inert, as it does, to some degree, enter into ionic reactions with other molecules in tissues.

Similarly, lipid-soluble molecules pass freely through lipid membranes. Therefore, lipid solubility and the degree of ionization of a drug play an important role in transfer. Another aspect which must be considered is the metabolic activity of the cell. The rate of entry of a drug is primarily controlled by the lipid solubility of the nonionized drug molecule. The latter pertains particularly to placental transfer. In general, nonionized drugs with high levels of- fat solubility are transferred rapidly across the placental barrier. The present study indicates that solutes traverse multicellular membranes through aqueous channels and/or across the plasma membrane of the cellular layer. Man\ pharmacologic agents. by virtue of their molecular &e and chemical nature, are bound to various protein molecules in the plasma or other body compartments. ‘l‘he interaction with protein ser\‘es the useful purpose of- providing a depot since the bound portion of the drug is in rquilibrium with the free form. Diazepam is lipid soluble and has a small molecular weight; therefore. it readily penetrates hiological membranes. One would be interested to know whether Dr. Seeds has used serum as a perfilsate because the over-all transfer of a substance may be related to its water solubility and protein binding. Furthermore, one must differentiate the terms dittilsion and transport as in the latter case there are energy requirements. In Fig. 2, which depicts the permeability to diazcpam, meperidinc, urea. and glucose, one is surprised to see a series of‘ exactly straight lines when one might at least expect borne phase transition of tissue at the higher temperatures. It would have been nice to have checked and (ompared the diffusion of a hexose similar to glucose, specifically one which wt)ultf be less likely to undergo metabolism. Furthermore, as diffusion is time dependent, one wonders whether there is any difference in the results if the membrane is reversed in the diffusion chamber. In conclusion. what is the relationship of this in vitro system to the changes which occur during late pregnancy and labor? REFERENCES

1. Battaglia. F. C.. Hellegers, A. E., Meschia, C., and Barron. D, 11.: In vitro investigations of the human chorion as a membrane svstem, Nature 196: 1061, 1962. 2. Levitz, M., &ndon, C. P.. Dancis. J.. Tillinger, K. G.. Wiquist, N., and Diczfalusy, E.: The fate of intra-amniotic estrone-C’” sulfate in human pregnancy, presented at the Forty-fourth Meeting of the Endocrine Society, Chicago. 1962. DR. in the amply branes

RALPH WYNN, Chicago, Illinois. Recent reports obstetric and anatomic literature have attested to the importance of the paraplacental memin maternofetal transfer. The significance of

Volume Number

Chorion laeve permeability

128 1

Fig. 1. Human (X15,000.)

chorion

Fig. 2. Human amnion terpreted as intracelluiar

laeve

at 28 weeks’

at term, showing huge vacuoles. (X 15,000.)

gestation,

intercellular

showing

lateral

intercellular

spaces (arrows),

which

spaces

have been

(arrow).

misin-

19

20

Seeds,

Eichhorst,

and Stoke

the amnion, chorion, and yolk sac in rodents and lagomorphs has long been recognized, but analogous functions have been identified recently for primates as well, including man. Much more attention has been devoted to the amnion than to the nonplacental chorion, except perhaps in the rabbit. However, results of experiments involving the true chorion of the rabbit cannot be transferred readily to man. In the rabbit, the true chorion remains primarily avascular, whereas in man the chorion laeve, after initial vascularization by allantoic vessels, becomes secondarily avascular. These structures, though perhaps analogous, are not homologous. Dr. Seeds’ studies of the human chorion are thus timely, if not overdue. Although he refers to the results of his experiments in terms of placental transfer, he is more appropriately referring to a paraplacental, or nonplacental, route of maternofetal exchange. Dr. Seeds’ data are properly obtained and clearly presented. Inasmuch as they confirm the well-known direct relation of transfer across membranes to lipid solubility and the inverse relation to molecular size, they provide little basis for disagreement. Furthermore, they point out the major role of the intercellular spaces in the transfer of water-soluble compounds. In the case of lipid-soluble compounds, transcellular penetration is a major additional route. Our unpublished ultrastructural studies of the epithelium (trophoblast) of the chorion laeve illustrate these lateral intercellular spaces (Fig. 1), which, although prominent, do not reach the size of those found between epithelial cells in the amnion (Fig. 2). These huge amnionic spaces have been misinterpreted, on the basis of light microscopy, to be intracellular vacuoles.’ Dr. Seeds’ interesting experiments stimulate a few suggestions and questions. He states that there is no evidence of facilitated diffusion of glucose, which occurs in other tissues, but he fails to rule out this possibility. In order to do so, the system must be saturated by high levels of glucose, whereas he used only a single low concentration. A recent study by Bedford and co-workers’ describes the “saturation behavior” that is characteristic of facilitated diffusion. Further evidence for a glucose pump could be provided by comparing the transfer rates of stereoisomers of this compound. In Table I of the manuscript, permeability constants and diffusion coefficients are given for meperidine, diazepam, urea, and glucose, but only permeability constants are given for DMO and PAH. These values of 3.20 and 2.63 for DMO and PAH, respectively, then appear in Table II as diffusion coefficients, which must be different numerically from the permeability constants. Are these tables simply constructed incorrectly? In Table IV the diffusions of nine substances are compared. It is clear that transfer in vivo depends upon such additional factors as maternal and fetal

blood flows and concentration gradients. Even in vitro, however, protein binding significantly affects the maternofetal passage of drugs. The comparison of diazepam and meperidine, which are highly bound to protein, with antipyrine. which is essentially unbound, thus does not appear valid. I hope that Dr. Seeds will continue to pursue this important work even in this era of increasing obstacles to basic research. REFERENCES I. Wynn, R. M.. and French, G. L.: Comparative ultrastructure of the mammalian amnion, Obstet. Gynecol. 31: 759, 1968. 2. Bedford, M. R.. Larson, K, B., and Raichle, M. E.: In vivo measurement of blood-brain transport of glucose, Physiologist 19: 122, 1976. DR. JACK N. BLECHNER, Farmington, Connecticut. Dr. Seeds, I wonder if you would comment on something that Dr. Wynn’s electron micrographs bring up. It seems that the relative magnitude of transfer across the membrane for a compound that diffuses very rapidly and one that diffuses slowly is not explained by the size of the pores between cells compared to the total surface of the membrane. The pores that are shown on the electron micrographs represent a small fraction of the total surface or volume through which a molecule would have to flop’. If most of the transfer of the non-lipid-soluble substance occurs through thkse small pores, it would appear that the difference between a highly lipid-soluble substance and a water-soluble substance should be enormously different. To put it another way, the model that is presented has to be refined before the physiology and the anatomy come into focus in my own mind. DR. SEEDS (Closing). Dr. Charles pointed out the fact that chorion laeve, which was studied here, separates the amniotic fluid from the maternal compartment and is not the tissue layer across which in vivo placental transfer takes place, and that is quite true. However, there is merit in studying the chorion laeve in vitro. It has been thought that the permeability of this tissue corresponded to permeability measured across chorion frondosum in in vivo studies. At the same time, it is very difficult to study solute transfer across chorion frondosum in vitro. In my opinion, there is no in vitro preparation where you can keep all the variables constant and study transfer across chorion frondosum as accurately as this can be done with chorion laeve. This study has not addressed itself to protein binding. We cannot answer any of the questions on protein-binding problems because we were looking at the free or unbound form of the solutes here. The chemical potential gradient resulting in diffusion in these studies was established by the free or unbound molecule; therefore. the permeability values reported here are for unbound solute.

Volume Number

128 1

Increase in diffusion permeability at the higher temperatures is greater for the lipid-soluble compounds. As Dr. Charles pointed out, the slopes representing the activation energies are a straight line. Three data points were involved between 4” and 38” C., and we thought the data from these studies represented a straight line. This is an approximation then of the activation energy, and the data do not allow us to go beyond that approximation. Similar studies on solute transfer across other multicellular membranes such as the toad bladder and frog skin have also reported changes in diffusion permeabilities as a straight line in an Arrhenius plot. It has been shown in past studies with this preparation that permeability is constant over at least a four- to six-hour period. That is, there is no permeability change with time in this diffusion chamber. In addition, normal oxygen and glucose uptakes and no evidence of abnormal lactate production have been reported for these membranes under the conditions of these experiments. With respect to Dr. Wynn’s question, we did not claim to have ruled out facilitated diffusion as a mechanism of glucose transfer in these experiments. We stated that there was no evidence from these data to indicate facilitated diffusion. We did not use extremely high concentration gradients in these experiments. However, the similar diffusion rate for glucose

Chorion laeve permeability

21

compared to that of other similar-sized compounds, the linearity of transfer rate with gradient over the concentration range of these experiments, and the lack of difference in membrane permeability to glucose regardless of membrane orientation (whether diffusion was taking place in a direction from fetal to maternal or maternal to fetal surface) do not suggest facilitated diffusion as the mechanism by which glucose is transported here but are compatible with a simple diffusion process. In the tables that Dr. Wynn mentioned, DMO values were expressed as diffusion constants, not as permeability constants. Dr. Blechner points out that histologic observations indicate the pores are very small and comprise a proportionately small fraction of the total surface area of the membrane. I do not know that this is really incompatible with our permeability data in that whatever the surface area represented by these extracellular channels this appears to be the pathway by which the water-soluble compounds are crossing this tissue and that the only difference in the lipid-soluble compounds is their ability to cross the remainder of the membrane surface area away from these pores through cell lipid, resulting in a significantly increased membrane permeability to these compounds.