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Clearance of inert molecules, Na, and Cl ions across the primate placenta
Clearance of inert molecules, Na, and Cl ions across the primate placenta FRENDERICK
C.
RICHARD
E.
GIA,COMO
MESCHIA
ASA. P.
E. I).
Beaverton,
BATTAGLIA
BEHRMAN
SEEDS* BRUNS Oregon,
and Denver,
Colorado
Transplacental clearances of antipyrine, tritiated water, urea, Na, and Cl ions were studied in the rhesus monkey. In order of decreasing placental permeability determined in viuo, these experiments showed the following order: tritiated water and antipyrine, urea, chloride, and sodium ions. Clearances of tritiated water and antipyrine were equal. The placental clearances of tritiated water and antipyrine were significantly different when determined across the chorion laeve in vitro. Thex observations suggest that the transplacental diffusion of these molecules is flow limited. The diffusion rates of urea, Na, and Cl ions were limited primarily by the permeability of the placental membrane. The placenta of the rhesus monkey is more permeable to Na and Cl ions than the sheep placenta. Permeability to urea per kilogram of fetal body weight is of the same order of magnitude in both species.
TRANSPLACENTAL diffusion of inert molecules (here defined as those molecules that are not metabolized by the placenta or bound to components of maternal and fetal blood and are evenly distributed between red cells and plasma), depends primarily upon three factors: uterine flow, umbilical flow, and permeability of the placental membrane.lp 2 Theoretically, the relative importance of these factors varies between the limits of 100 per cent flow-limited and 100 per cent permeability-limited diffusion2 Molecules in which diffusion is 100 per
cent flow limited. are a useful tool in studying problems of placental perfusion and in the measurement of uterine and umbilical flows by the Fick principle. Studies of substances in which diffusion is permeability limited help in characterizing the physicochemical properties of the placental barrier. Recent experiments on sheep in which the placental clearances of four substances were determined simultaneously have shown that: (a) the transplacental diffusion of antipyrine and tritiated water is primarily flow limited, (b) the diffusion of urea is limited simultaneously by flows and permeability, and (c) the diffusion of Na and Cl ions is nearly 100 per cent limited by the permeabiIity of the placental barrier.2 These same moIecules have been used in the present experiments on the placenta of a primate. The experiments had a dual purof a technique pose, I.e., the deveIopment for the measurement of uterine and umbili-
From the Division of Perinatal Medicine of the Oregon Regional Primate Research Center and the University of Colorado Medical Center. Supfrorted by United States Public Health Service Grants HD 02757, ND 00781-04, HD 01866-02, and FR 00163. *Visiting scientist, The Johns Hopkins University, Baltimore, Maryland. 1135
1136
Baitaglia
et cl/.
cal flows by the steady-state diffusion method currently used in sheep and a comparative study in placental clearance of the same substances in the sheep and in the rhesus monkey. Materials
and
methods
The following paragraphs summarize the theoretical basis of our experiments. A detailed account has been given in preceding publications.2 Experimental design. The test molecules are infused at a constant rate in a fetal vein. In a stable preparation the constant infusion produces an initial period of rapidly rising blood and tissue concentrations in the fetus and the placenta, followed by a steady state in which the concentrations and transplacental diffusion rate become nearly constant with time. At any moment during the infusion th.e transplacental diffusion rate is equal to the infusion rate minus both the rate of accumulation in the fetus and the rate of metabolism and excretion through routes other than the placenta. Substances such as antipyrine a.nd tritiated water have low rates of extraplacental excretion and metabolism. Their rate of accumulation is minimal in the steady state and can be estimated by multiplying the change of concentration in the fetal blood by the appropriate volume of distribution, Thus, when the system approaches a steady state, it is possible to calculate the transplacental diffusion rate of certain inert molecules from their infusion rate. Transplacental clearance. Analysis of mathematical models of the placenta25 3 has shown the convenience of defining a new variable which has been called the transplacental clearance (C) of a molecule.2 Transplacental clearance is defined as the ratio: transplacental diffusion rate over the concentration difference between the plasmas of umbilical and maternal arteries. Blood and plasma concentrations of urea, tritiated
tibias
=
blood plasma
water, and antipyrine are about equal; thus, either concentration can be used in calculating the clearance of these molecules. Flow-limited clearance (C&x)~ In a given physiologic preparation all inert molecules in which diffusion is 100 per cent flow limited have the same clearance. In addition, 100 per cent flow-limited clearance represents a maximum, i.e., all inert molecules in which diffusion is limited by the permeability of the placental barrier have a lower clearance. The theoretical flow-limited clearance of Na and Cl ions is smaller than that of antipyrine, labeled water, and urea because Na and Cl ions have significantly smaIIer concentrations in the red cells than in plasma. For example, maximum clearance of substances exclusively carried by plasma would be determined by plasma flows rather than blood flows. If the maximum clearance of inert substances and the ratio ((&AX) blood
concentration
plasma
of Na and Cl are de-
concentration
termined experimentally, then the maximum theoretical clearance of these ions (C*M& can be estimated as in Equation 1 at the bottom of the page. Degree of diffusion limitation. This index represents the relative increase of clearance predicted to occur if placental permeability could be increased from the actual value to infinity. For inert molecules: L
=
c,AX
D
-
c XAS
c
(Equation
2).
For _Na and Cl ions:
Thus, Ln Es of flows and ing diffusion. ability is too concentration concentration
a measure of the reEative role placental permeability in JimitIf Ln equals zero, the permehigh to have a measurabie ef-
I= &is
(Equation
I) _
Volume Number
10% 8
Clearance
of
inert
feet on transplacental diffusion; if Ln is greater than zero but significantly Iess than one, both flows and permeability play a role in limiting diffusion; as Ln approaches one, permeability becomes the only limiting factor. Diffusing capacity (K) . A comprehensive measure of placental permeability is the so-called “diffusing capacity” measurement which represents the milligrams of a given substance crossing the placenta in a minute when the concentration difference between fetal and maternal plasma is 1 mg. per milliliter. If Ln is close to zero, the diffusing capacity cannot be measured because the diffusion rate does not depend on permeability. If Ln is significantly higher than zero, the difusing capacity can be estimated by assuming a certain flow pattern in the placenta and a constant relationship between permeability and flows in all placental areas. In this paper we have assumed that the placental flows simulate a concurrent pattern and have used the following equation for the calculation of diffusing capacity of urea (symbol K) : K
=
C MAX CMAx In C MAX
- C (Equation
Animal
Clearance ______ Clearance
and Cl across
placenta
1137
Nineteen pregnant from the caged breeding colony of the Oregon Regional Primate Research Center were used in these experiments. The gestational ages at the time of the experiment ranged between 120 and 162 days, the fetal weight from 271 to 423 grams. The mothers were starved 12 hours prior to operation. The animals were restrained in the dorsal supine position on a padded frame. The trachea was intubated with the aid of nitrous oxide inhalation and atropine injection in the dose of 1 mg. per kilogram. Anesthesia was inducted with Fluothane, nitrous oxide, and oxygen and maintained with Fluothane in oxygen. The Fluothane concentration varied between 0.5 per cent and 2 per cent, depending upon the levels of maternal arterial pressure, the respiratory and cardiac rate, which were continuously monitored, and the degree of uterine relaxation during the operation. After exposing the uterus through an abdominal incision, the placenta and fetal small parts were located by palpation. A purse-string suture was placed in the myo,metrium of the uterine fundus and an incision was made within the boundaries of the suture. A fetal leg was then delivered through an area of chorion laeve, avoiding the edge of the chorion frondosum and large fetal vessels. The fetal groin was dissected and two polyethylene catheters (I.D. -0.011 inch, O.D. -0.024 inch) were placed in the femoral vein and artery, respectively. The fetal, femoral artery catheter was threaded about 2 cm. from the groin. The free end of the fetal femoral vein catheter was connected promptly to a pump (Technicon Auto-Analyzer pump) which was set to deliver 30 ~1 a minute of a solution containing the test molecules. After catheterization the fetal parts were returned to the uterus and the uterine in-
4).
rate of X rate of Y
Na,
prepa.ration.
Macaca mulatto. monkeys
The rationale for this equation has been discussed in a preceding paper.z Comparison of transplacental diffusion rates and clearances. Transplacental diffusion rates of substances which the uteroplacental mass does not produce, metabolize, or accumulate can be calculated as the product of arteriovenous (A-V) differences across the uterine or the umbilical circulation times the uterine or umbilical flow, respectively. It follows that the ratios of the diffusion rates and clearances of two molecules (X and Y) can be calculated without knowledge of the absolute values, according to the following equations : Diffusion Diffusion
molecules,
=
A-V
difference
of X
A-V
difference
of Y
of X A-V
difference
of X
x
artery
to artery
difference
of Y
of Y A-V
difference
of Y
x
artery
to artery
difference
of X
(Equation
5) .
(Equation
6).
‘Ii38
Battaglia
Decemb-- L_/ !5 i ) 6968 Am. J. Obst. & Gynec.
et a!.
cision closed around the catheters by means of the purse-string suture. One or both ovarian veins and a maternal femoral artery were then catheterized with 0.6 mm. I.D. polyvinyl catheters. After 50 minutes of infusion or longer, sets of simultaneous blood samples were obtained at 5 to 10 minute interva1.s from the maternal artery, the ovarian vein (or veins), and the fetal artery. Maternal and fetal blood amples were 0.4 and 0.2 ml., respectively. Mother and fetus were heparinized throughout the experiment. After the last set of blood samples, amniotic fluid was collected for analysis and the fetus and the placenta delivered and weighed. In some experiments blood and plasma samples were diluted and deproteinized by the Ba-ZnSOB method prior to chemical analysis. In others, analysis of antipyrine and tritiated water was performed directly on the plasma. The plasma was digested with Nuclear Chicago Solubilizer prior to liquid scintillation counting. Antipyrine and urea were analyzed calorimetrically on a Technicon Auto-Analyzer. The radioactivity of samples containing tritiated water, 9Ga, and 36C1 was measured in a Packard DualChannel liquid scintillation counter. The in vitro studies on the chorion laeve were carried out as described previously.* The calculated permeability constant for each placenta (cm. x sec.-l) represented the mean of four values calculated for each 20 minute period. The technique for separation of tissue layers from the membranous portion of the rhesus monkey placenta had been described previous1y.j
esults The infusion at constant rate into the fetus of a rhesus monkey of antipyrine, tritiated water, urea, and radioactive Na and Cl ions results in a characteristic pattern of blood concentrations, as shown in Figs. 1 and 2. These figures represent two different animals studied. In both experiments the fetuses received antipyrine, tritiated water, and urea; in addition, the fetus of Fig. 1. received ‘9Va and the fetus of Fig. 2 received 3BC1. It is
apparent that for a given artery-to-artery difference of concentration, the uterine arteriovenous differences of Na, Cl, and urea are smaller than those of antipyrine and tritiated water.
Comparison of the clearances of antipyrine and tritiated water. This comparison was carried out by two different methods. In the first method the clearance of each substance was calculated from its own transplacental diffusion rate and artery-to-artery difference and then compared. The transplacental diffusion rates of antipyrine and tritiated water were calculated by subtracting the rates of accumulation and metabolism from the infusion rates. The accumulation rate was estimated as the product of the change in concentration of the test substance per milliliter of fetal blood times the fetal weight. The rate of metabolism and extraplacental excretion was estimated on the basis of 1 ml. of fetal blood cleared of the test substance per minute per kilogram of fetus. These estimates are based on observations of antipyrine and tritiated water volumes of distribution and clearance made on viable fetuses after clamping the cord. They represent relatively small corrections. On the average, the accumulation ra,tes of antipyrine and tritiated water were 8 and 14 per cent of the infusion rate, respectively. The rate of metabolism and extraplacental excretion was about 2 per cent of the infusion rate for both molecules. The quantities of antipyrine and 3H,0 in the amniotic fluid at the end of the experiment accounted for somewhat less than half of this 2 per cent. Five animals were used for a total of 34 paired observations. The average antipyrine clearance/water clearance ratio was 1.03 i: 0.06 SD. Average values for each of the 5 animals are presented in Table I. In the second method of comparison, the
ratio
antipyrine clearance water clearance
was
calcu-
lated from the concentrations in the fetal and maternal artery and the arteriovenous differences across the uterine circulation, ac-
volume Number
102 8
Clearance
of inert
molecules,
Na,
and
Cl across
placenta
/ .
L-/*‘*\/dCENDING
AORTA
2 UTERINE
VEIN
.-*-A.-o-co-MATERNAL
m-,CC. -*~‘L-.-*-g
ARTERY
DESCENDING
UTERINE
i
AORTA
0
“E,N
0 MATERNAL
ARfEII
:
m-8 .-I-’
DESCENDING
AOdlA
,-m-
10
5 MATERNAL
ARTERY 0
c,ww,~oQ-
I DESCENDING
AGI!IA
,-‘-
10
MATERNAL --^____IIco
60
il
70
SO TIME
90
ARTRRY 1-
@
/
100
MINUTES
Fig. 1. Blood concentrations of g?Na, urea, tritiated water, and antipyrine artery, uterine vein, and maternal artery are plotted against time. Time beginning of infusion at constant rate of these substances in the fetus.
in the umbilica1 zero represents the
1139
40
Battaglia
December :5, 1968 Am. J. Obst. & Gynec.
et a[.
TIh¶E
M&KITES
Fig. 2. Blood concentrations of s%l, urea, tritiated water, and antipyrine artery, uterine vein, and maternal artery are plotted against time. Time heginning of infusion at constant rate of these substances in the fetus.
in the umbilicai zero represents the
Volume Number
102 8
Clearance
of inert
of the placental and tritiated water
Water clearance (ml. blood/mix)
A’?Ztipy?GZe
clearance (ml. blood/min.) 8.7 33.1 10.7 16.6 13.1
and
Cl across
placenta
1141
Comment
Antipyrine/ water clearance
9.0 31.4 9.7 16.3 13.2
Na,
clearance of inert molecules maximum diffusion C MAX, the index of transplacental limitation, Ln, of urea, Na, and Cl has been calculated according to Equations 2 and 3. The results of such calculations are presented in Table III. It appears that the permeability of the placental membrane is the main limiting factor in the rate of diffusion of these substances across the placenta. Urea diffusing capacity of the placental membrane. The urea diffusing capacity- of the placental membrane was estimated in 7 monkeys according to Equation 4. The results are presented in Table II. In vitro comparison of the chorion laeve permeability to antipyrine and tritiated water. Eleven placentas were studied in vitro. The mean t S.E.M. permeabilities were 1.8% i 0.22 x lOwi cm. x sec.-l, and 0.555 t 0.03 x lo-’ cm. x sec.-l for tritiated water and antipyrine, respectively. The ratio antipyrine to tritiated water permeability was 0.313 ? 0.016. This ratio is sfgnificantly different (P < 0.001) from the antipyrinetritiated water ratio obtained in vivo (1.05 k 0.04).
cording to Equation 6. In eight different experiments (Table II) the ratio was 1.03 -1: 0.04 S.D. The results of the two methods are not significantly different, and they show the transplacental clearances of antipyrine and water to be the same, within the limits of experimental error. Comparison of the cIearances of antipyrine a.nd tritiated water with those of urea, Na, and Cl ions, The comparison is shown in Table II. In these experiments the clearance of antipyrine was calculated from the transplacental diffusion rate and arteryto-artery concentration difference. All other clearances were calculated according to Equation 6. While antipyrine and tritiated water clearances appear to be the same, as already stated, the clearances of urea, Na, and CI are significantly smaller. On the assumption that the antipyrine and tritiated water clearances represent the
Table I. A comparison clearances of antipyrine in 5 rhesus monkeys
molecules,
Tritiated water diffuses about three times more rapidly than antipyrine across the chorion laeve of the primate placenta in vitro. This large difference is not surprising, because antipyrine has a molecular weight nine times greater than tritiated water and
0.97 1.05 1.10 1.02 0.99
Table II. A comparison of the placental clearances of antipyrine, water, urea, ?“Na, and 36CI in the rhesus monkey. Transplacental diffusing capacities of urea were calculated according to Equation No. 4 Diffusing Animal No. 463 232 469 097 122 190 327 275 305
(
Fez!tah
1
Anti~yrine
:Fz:
,
Urea
320 356 410 314 430 318
29.8 23.0 18.5 14.8 17.0 13.1
26.5 21.4 17.8 14.4 16.0 12.8
8.3 6.6 4.9 3.7 4.1 5.2
344 423
41.6 33.9
37.9 34.5
6.0
/
Na
(mF
capacities “y:,mi;.)
3.2 2.3 2.4 2.1
of urea Urea
2.7 3.4
9.7 7.7 5.7 4.2 4.7 6.6
4.4 5.2
6.8
-,
1142
Battaglia
Table III. limitation Experiment No. 463 232 469 097 112 190 275 305
et ai.
Index of transplacental diffusion (Ln) of urea, Na, and Cl ions /
Urea 0.732 0.725 0.743 0.772 0.761 0.620 0.771
/
Na
/
Cl
0.85 0.86 0.82 0.81 0.80 0.69 0.85 0.76
has different solubility characteristics. Thus, if placental permeability were a limiting factor in the transplacental diffusion of antipyrine, antipyrine clearance should be smaller than the clearance of tritiated water. Since the experimental evidence presented in this paper demonstrates that the clearances of antipyrine and tritiated water determined in vivo are not significantly different, it can be inferred that permeability plays a negligible role in limiting the diffusion of these molecules across the primate placenta. Therefore, this aspect of placental physiology shows no difference between monkeys and sheep.’ There are, though, quantitative differences. In a series of 29 experiments on sheep and goats under spinal the antipyrine clearance per anesthesia, kiIogram of fetus was 99 + 30 SD. ml. of blood per minute; whereas in the present series of 13 experiments the antipyrine clearance per kilogram of fetus was only 60.5 + 28 SD. ml. of blood per minute. The difference is statistically significant (P < 0.01). It is likely that part of this difference in antipyrine clearance is due to differences in anesthesia, degree of stress, and reactivity of the preparation to stress. Estimates of uterine blood flow per kilogram of tissue in anesthetized monkeys show lower rates of flow than in sheep and goats.6 Transplacental diffusion of urea is limited by placental permeability both in sheep and in monkeys. The degree of diffusion limitation is of the same magnitude in both species: the mean Lo was 0.72 (range, 0.48 - 0.87)
in a series of 10 experiments of sheep6 and 0.73 (range, 0.62 - 0.77) in the present report in monkeys. The urea diffusing capacity per kilogram of fetus measured by the same technique was 25.8 ml. per minute I 9.3 SD. in 10 sheep and 18.1 ml. per minute t 6.6 SD. in 7 monkeys. We have taken fetal body weight as our point of reference because it has been demonstrated in sheep that the urea-diffusing capacity of the placenta is directly proportional to fetal weight and has no simple correlation to placental weight. In addition, fetal body weight is a rough index of fetal metabolic rate, and it seems reasonable to assume that the properties of the placenta are related to fetal metabolic needs. Transplacental diffusion of Na and Cl ions is also limited primarily by the placental membrane: the average degree of diffusion limitation of both ions was 0.8 (range 0.76 - 0.85). The clearance per kilogram of fetus averaged 8.9 ml. per minute i- 2.9 SD., a value significantIy lower than the urea clearance per kilogram of fetus, 15.5 ml. per minute + 5.5 SD. (P < 0.02). Thus, in order of decreasing permeability, these experiments on monkeys show the following order: tritiated water and antipyrine (diffusion rate limited by flows), urea, chloride, and sodium (diffusion rate limited primarily by the permeability of the placental membrane). This is the order observed in the sheep placenta.” The major quantitative difference between the two species is in the relative magnitude of urea clearance with respect to Na and Cl ions clearance. In the monkey the clearances of the ions were about 60 per cent of the urea clearance; in sheep Na .and Cl ion clearances were less than one tenth of the urea clearance. According to Flexner and Gellhorn7 Na crosses the primate placenta more rapidly than the placenta of goats. Their standard of comparison was placental weight. Our results on the clearances of Na and Cl in sheep and monkeys do agree with Flexner and Gellhorn’s conclusion. Flexner and Gellhorn correlated these differences among species with the number of tissue layers sep-
Volume Number
102 8
Clearance
arating maternal and fetal blood. Although the correlation seems to have some validity in the case a’f Na, it should be pointed out that the over-all permeability to urea of the primate placenta is, if any, smaher than in sheep, i.e., ,the placenta with less layers seems to be ,the less permeable. In the case of antipyrine and tritiated water the placental permeabilities in both species are too large to have physiologic importance in vivo. Hence, place:ntal flows (umbilical and uterine) are the determining factors in their rate of exchange. In order to measure simultaneously uterine and umbilical flows by the Fick prin-
of
inert
molecules,
Na,
and
Cl across
placenta
1143
ciple it is necessary to know accurately the transplacental di-Fusion rate of the test substance. In addition, the test substance should give large arteriovenous differences on both circulations in order to minimize the error of measurement. Antipyrine and tritiated water fulfill these criteria. Under steadystate conditions their transplacental diffusion is about 80 to 90 per cent of the infusion rate, which can be accurately measured. The clearance of both molecules is flow limited, i.e., no inert molecules can be expected to give larger arteriovenous differences under a given set of physiologic conditions.
REFERENCES
1.
2. 3. 4.
Mesch,ia, G., Cotter, J. R., Makowski, E. L., and B#arron, D. H.: Quart. J. Exper. Physiol. 52: 1, 1967. Meschia, G., Battaglia, F. C., and Bruns, P. D.: J. AppIl. Physiol. 22: 1171, 1967. Wilkin, P.: Le placenta humain, Paris, 1958, Masson et Cie, p. 248. Battaglia, I?. C., Hellegers, A. E., Meschia, G., and Barron, D. H.: Nature 196: 1061, 1962.
5.
6.
7.
Battaglia, F. C., Bruns, P. D., Behrman, R. E., Seeds, A. E., and Hellegers, A. E.: Am. J. Physiol. 207: 500, 1964. Meschia, G., Behrman, R. E., Hellegers, A. E., Schruefer, J. J., Battaglia, F. C., and Barron, D. H.: AM. J. OBST. & GYNEC. 97: 1, 1967. Flexner, L. B., and Gellhorn, A.: AM. J. OBST. & GYNEC. 42: 965, 1942.
C.
RICHARD
E.
GIA,COMO
MESCHIA
ASA. P.
E. I).
Beaverton,
BATTAGLIA
BEHRMAN
SEEDS* BRUNS Oregon,
and Denver,
Colorado
Transplacental clearances of antipyrine, tritiated water, urea, Na, and Cl ions were studied in the rhesus monkey. In order of decreasing placental permeability determined in viuo, these experiments showed the following order: tritiated water and antipyrine, urea, chloride, and sodium ions. Clearances of tritiated water and antipyrine were equal. The placental clearances of tritiated water and antipyrine were significantly different when determined across the chorion laeve in vitro. Thex observations suggest that the transplacental diffusion of these molecules is flow limited. The diffusion rates of urea, Na, and Cl ions were limited primarily by the permeability of the placental membrane. The placenta of the rhesus monkey is more permeable to Na and Cl ions than the sheep placenta. Permeability to urea per kilogram of fetal body weight is of the same order of magnitude in both species.
TRANSPLACENTAL diffusion of inert molecules (here defined as those molecules that are not metabolized by the placenta or bound to components of maternal and fetal blood and are evenly distributed between red cells and plasma), depends primarily upon three factors: uterine flow, umbilical flow, and permeability of the placental membrane.lp 2 Theoretically, the relative importance of these factors varies between the limits of 100 per cent flow-limited and 100 per cent permeability-limited diffusion2 Molecules in which diffusion is 100 per
cent flow limited. are a useful tool in studying problems of placental perfusion and in the measurement of uterine and umbilical flows by the Fick principle. Studies of substances in which diffusion is permeability limited help in characterizing the physicochemical properties of the placental barrier. Recent experiments on sheep in which the placental clearances of four substances were determined simultaneously have shown that: (a) the transplacental diffusion of antipyrine and tritiated water is primarily flow limited, (b) the diffusion of urea is limited simultaneously by flows and permeability, and (c) the diffusion of Na and Cl ions is nearly 100 per cent limited by the permeabiIity of the placental barrier.2 These same moIecules have been used in the present experiments on the placenta of a primate. The experiments had a dual purof a technique pose, I.e., the deveIopment for the measurement of uterine and umbili-
From the Division of Perinatal Medicine of the Oregon Regional Primate Research Center and the University of Colorado Medical Center. Supfrorted by United States Public Health Service Grants HD 02757, ND 00781-04, HD 01866-02, and FR 00163. *Visiting scientist, The Johns Hopkins University, Baltimore, Maryland. 1135
1136
Baitaglia
et cl/.
cal flows by the steady-state diffusion method currently used in sheep and a comparative study in placental clearance of the same substances in the sheep and in the rhesus monkey. Materials
and
methods
The following paragraphs summarize the theoretical basis of our experiments. A detailed account has been given in preceding publications.2 Experimental design. The test molecules are infused at a constant rate in a fetal vein. In a stable preparation the constant infusion produces an initial period of rapidly rising blood and tissue concentrations in the fetus and the placenta, followed by a steady state in which the concentrations and transplacental diffusion rate become nearly constant with time. At any moment during the infusion th.e transplacental diffusion rate is equal to the infusion rate minus both the rate of accumulation in the fetus and the rate of metabolism and excretion through routes other than the placenta. Substances such as antipyrine a.nd tritiated water have low rates of extraplacental excretion and metabolism. Their rate of accumulation is minimal in the steady state and can be estimated by multiplying the change of concentration in the fetal blood by the appropriate volume of distribution, Thus, when the system approaches a steady state, it is possible to calculate the transplacental diffusion rate of certain inert molecules from their infusion rate. Transplacental clearance. Analysis of mathematical models of the placenta25 3 has shown the convenience of defining a new variable which has been called the transplacental clearance (C) of a molecule.2 Transplacental clearance is defined as the ratio: transplacental diffusion rate over the concentration difference between the plasmas of umbilical and maternal arteries. Blood and plasma concentrations of urea, tritiated
tibias
=
blood plasma
water, and antipyrine are about equal; thus, either concentration can be used in calculating the clearance of these molecules. Flow-limited clearance (C&x)~ In a given physiologic preparation all inert molecules in which diffusion is 100 per cent flow limited have the same clearance. In addition, 100 per cent flow-limited clearance represents a maximum, i.e., all inert molecules in which diffusion is limited by the permeability of the placental barrier have a lower clearance. The theoretical flow-limited clearance of Na and Cl ions is smaller than that of antipyrine, labeled water, and urea because Na and Cl ions have significantly smaIIer concentrations in the red cells than in plasma. For example, maximum clearance of substances exclusively carried by plasma would be determined by plasma flows rather than blood flows. If the maximum clearance of inert substances and the ratio ((&AX) blood
concentration
plasma
of Na and Cl are de-
concentration
termined experimentally, then the maximum theoretical clearance of these ions (C*M& can be estimated as in Equation 1 at the bottom of the page. Degree of diffusion limitation. This index represents the relative increase of clearance predicted to occur if placental permeability could be increased from the actual value to infinity. For inert molecules: L
=
c,AX
D
-
c XAS
c
(Equation
2).
For _Na and Cl ions:
Thus, Ln Es of flows and ing diffusion. ability is too concentration concentration
a measure of the reEative role placental permeability in JimitIf Ln equals zero, the permehigh to have a measurabie ef-
I= &is
(Equation
I) _
Volume Number
10% 8
Clearance
of
inert
feet on transplacental diffusion; if Ln is greater than zero but significantly Iess than one, both flows and permeability play a role in limiting diffusion; as Ln approaches one, permeability becomes the only limiting factor. Diffusing capacity (K) . A comprehensive measure of placental permeability is the so-called “diffusing capacity” measurement which represents the milligrams of a given substance crossing the placenta in a minute when the concentration difference between fetal and maternal plasma is 1 mg. per milliliter. If Ln is close to zero, the diffusing capacity cannot be measured because the diffusion rate does not depend on permeability. If Ln is significantly higher than zero, the difusing capacity can be estimated by assuming a certain flow pattern in the placenta and a constant relationship between permeability and flows in all placental areas. In this paper we have assumed that the placental flows simulate a concurrent pattern and have used the following equation for the calculation of diffusing capacity of urea (symbol K) : K
=
C MAX CMAx In C MAX
- C (Equation
Animal
Clearance ______ Clearance
and Cl across
placenta
1137
Nineteen pregnant from the caged breeding colony of the Oregon Regional Primate Research Center were used in these experiments. The gestational ages at the time of the experiment ranged between 120 and 162 days, the fetal weight from 271 to 423 grams. The mothers were starved 12 hours prior to operation. The animals were restrained in the dorsal supine position on a padded frame. The trachea was intubated with the aid of nitrous oxide inhalation and atropine injection in the dose of 1 mg. per kilogram. Anesthesia was inducted with Fluothane, nitrous oxide, and oxygen and maintained with Fluothane in oxygen. The Fluothane concentration varied between 0.5 per cent and 2 per cent, depending upon the levels of maternal arterial pressure, the respiratory and cardiac rate, which were continuously monitored, and the degree of uterine relaxation during the operation. After exposing the uterus through an abdominal incision, the placenta and fetal small parts were located by palpation. A purse-string suture was placed in the myo,metrium of the uterine fundus and an incision was made within the boundaries of the suture. A fetal leg was then delivered through an area of chorion laeve, avoiding the edge of the chorion frondosum and large fetal vessels. The fetal groin was dissected and two polyethylene catheters (I.D. -0.011 inch, O.D. -0.024 inch) were placed in the femoral vein and artery, respectively. The fetal, femoral artery catheter was threaded about 2 cm. from the groin. The free end of the fetal femoral vein catheter was connected promptly to a pump (Technicon Auto-Analyzer pump) which was set to deliver 30 ~1 a minute of a solution containing the test molecules. After catheterization the fetal parts were returned to the uterus and the uterine in-
4).
rate of X rate of Y
Na,
prepa.ration.
Macaca mulatto. monkeys
The rationale for this equation has been discussed in a preceding paper.z Comparison of transplacental diffusion rates and clearances. Transplacental diffusion rates of substances which the uteroplacental mass does not produce, metabolize, or accumulate can be calculated as the product of arteriovenous (A-V) differences across the uterine or the umbilical circulation times the uterine or umbilical flow, respectively. It follows that the ratios of the diffusion rates and clearances of two molecules (X and Y) can be calculated without knowledge of the absolute values, according to the following equations : Diffusion Diffusion
molecules,
=
A-V
difference
of X
A-V
difference
of Y
of X A-V
difference
of X
x
artery
to artery
difference
of Y
of Y A-V
difference
of Y
x
artery
to artery
difference
of X
(Equation
5) .
(Equation
6).
‘Ii38
Battaglia
Decemb-- L_/ !5 i ) 6968 Am. J. Obst. & Gynec.
et a!.
cision closed around the catheters by means of the purse-string suture. One or both ovarian veins and a maternal femoral artery were then catheterized with 0.6 mm. I.D. polyvinyl catheters. After 50 minutes of infusion or longer, sets of simultaneous blood samples were obtained at 5 to 10 minute interva1.s from the maternal artery, the ovarian vein (or veins), and the fetal artery. Maternal and fetal blood amples were 0.4 and 0.2 ml., respectively. Mother and fetus were heparinized throughout the experiment. After the last set of blood samples, amniotic fluid was collected for analysis and the fetus and the placenta delivered and weighed. In some experiments blood and plasma samples were diluted and deproteinized by the Ba-ZnSOB method prior to chemical analysis. In others, analysis of antipyrine and tritiated water was performed directly on the plasma. The plasma was digested with Nuclear Chicago Solubilizer prior to liquid scintillation counting. Antipyrine and urea were analyzed calorimetrically on a Technicon Auto-Analyzer. The radioactivity of samples containing tritiated water, 9Ga, and 36C1 was measured in a Packard DualChannel liquid scintillation counter. The in vitro studies on the chorion laeve were carried out as described previously.* The calculated permeability constant for each placenta (cm. x sec.-l) represented the mean of four values calculated for each 20 minute period. The technique for separation of tissue layers from the membranous portion of the rhesus monkey placenta had been described previous1y.j
esults The infusion at constant rate into the fetus of a rhesus monkey of antipyrine, tritiated water, urea, and radioactive Na and Cl ions results in a characteristic pattern of blood concentrations, as shown in Figs. 1 and 2. These figures represent two different animals studied. In both experiments the fetuses received antipyrine, tritiated water, and urea; in addition, the fetus of Fig. 1. received ‘9Va and the fetus of Fig. 2 received 3BC1. It is
apparent that for a given artery-to-artery difference of concentration, the uterine arteriovenous differences of Na, Cl, and urea are smaller than those of antipyrine and tritiated water.
Comparison of the clearances of antipyrine and tritiated water. This comparison was carried out by two different methods. In the first method the clearance of each substance was calculated from its own transplacental diffusion rate and artery-to-artery difference and then compared. The transplacental diffusion rates of antipyrine and tritiated water were calculated by subtracting the rates of accumulation and metabolism from the infusion rates. The accumulation rate was estimated as the product of the change in concentration of the test substance per milliliter of fetal blood times the fetal weight. The rate of metabolism and extraplacental excretion was estimated on the basis of 1 ml. of fetal blood cleared of the test substance per minute per kilogram of fetus. These estimates are based on observations of antipyrine and tritiated water volumes of distribution and clearance made on viable fetuses after clamping the cord. They represent relatively small corrections. On the average, the accumulation ra,tes of antipyrine and tritiated water were 8 and 14 per cent of the infusion rate, respectively. The rate of metabolism and extraplacental excretion was about 2 per cent of the infusion rate for both molecules. The quantities of antipyrine and 3H,0 in the amniotic fluid at the end of the experiment accounted for somewhat less than half of this 2 per cent. Five animals were used for a total of 34 paired observations. The average antipyrine clearance/water clearance ratio was 1.03 i: 0.06 SD. Average values for each of the 5 animals are presented in Table I. In the second method of comparison, the
ratio
antipyrine clearance water clearance
was
calcu-
lated from the concentrations in the fetal and maternal artery and the arteriovenous differences across the uterine circulation, ac-
volume Number
102 8
Clearance
of inert
molecules,
Na,
and
Cl across
placenta
/ .
L-/*‘*\/dCENDING
AORTA
2 UTERINE
VEIN
.-*-A.-o-co-MATERNAL
m-,CC. -*~‘L-.-*-g
ARTERY
DESCENDING
UTERINE
i
AORTA
0
“E,N
0 MATERNAL
ARfEII
:
m-8 .-I-’
DESCENDING
AOdlA
,-m-
10
5 MATERNAL
ARTERY 0
c,ww,~oQ-
I DESCENDING
AGI!IA
,-‘-
10
MATERNAL --^____IIco
60
il
70
SO TIME
90
ARTRRY 1-
@
/
100
MINUTES
Fig. 1. Blood concentrations of g?Na, urea, tritiated water, and antipyrine artery, uterine vein, and maternal artery are plotted against time. Time beginning of infusion at constant rate of these substances in the fetus.
in the umbilica1 zero represents the
1139
40
Battaglia
December :5, 1968 Am. J. Obst. & Gynec.
et a[.
TIh¶E
M&KITES
Fig. 2. Blood concentrations of s%l, urea, tritiated water, and antipyrine artery, uterine vein, and maternal artery are plotted against time. Time heginning of infusion at constant rate of these substances in the fetus.
in the umbilicai zero represents the
Volume Number
102 8
Clearance
of inert
of the placental and tritiated water
Water clearance (ml. blood/mix)
A’?Ztipy?GZe
clearance (ml. blood/min.) 8.7 33.1 10.7 16.6 13.1
and
Cl across
placenta
1141
Comment
Antipyrine/ water clearance
9.0 31.4 9.7 16.3 13.2
Na,
clearance of inert molecules maximum diffusion C MAX, the index of transplacental limitation, Ln, of urea, Na, and Cl has been calculated according to Equations 2 and 3. The results of such calculations are presented in Table III. It appears that the permeability of the placental membrane is the main limiting factor in the rate of diffusion of these substances across the placenta. Urea diffusing capacity of the placental membrane. The urea diffusing capacity- of the placental membrane was estimated in 7 monkeys according to Equation 4. The results are presented in Table II. In vitro comparison of the chorion laeve permeability to antipyrine and tritiated water. Eleven placentas were studied in vitro. The mean t S.E.M. permeabilities were 1.8% i 0.22 x lOwi cm. x sec.-l, and 0.555 t 0.03 x lo-’ cm. x sec.-l for tritiated water and antipyrine, respectively. The ratio antipyrine to tritiated water permeability was 0.313 ? 0.016. This ratio is sfgnificantly different (P < 0.001) from the antipyrinetritiated water ratio obtained in vivo (1.05 k 0.04).
cording to Equation 6. In eight different experiments (Table II) the ratio was 1.03 -1: 0.04 S.D. The results of the two methods are not significantly different, and they show the transplacental clearances of antipyrine and water to be the same, within the limits of experimental error. Comparison of the cIearances of antipyrine a.nd tritiated water with those of urea, Na, and Cl ions, The comparison is shown in Table II. In these experiments the clearance of antipyrine was calculated from the transplacental diffusion rate and arteryto-artery concentration difference. All other clearances were calculated according to Equation 6. While antipyrine and tritiated water clearances appear to be the same, as already stated, the clearances of urea, Na, and CI are significantly smaller. On the assumption that the antipyrine and tritiated water clearances represent the
Table I. A comparison clearances of antipyrine in 5 rhesus monkeys
molecules,
Tritiated water diffuses about three times more rapidly than antipyrine across the chorion laeve of the primate placenta in vitro. This large difference is not surprising, because antipyrine has a molecular weight nine times greater than tritiated water and
0.97 1.05 1.10 1.02 0.99
Table II. A comparison of the placental clearances of antipyrine, water, urea, ?“Na, and 36CI in the rhesus monkey. Transplacental diffusing capacities of urea were calculated according to Equation No. 4 Diffusing Animal No. 463 232 469 097 122 190 327 275 305
(
Fez!tah
1
Anti~yrine
:Fz:
,
Urea
320 356 410 314 430 318
29.8 23.0 18.5 14.8 17.0 13.1
26.5 21.4 17.8 14.4 16.0 12.8
8.3 6.6 4.9 3.7 4.1 5.2
344 423
41.6 33.9
37.9 34.5
6.0
/
Na
(mF
capacities “y:,mi;.)
3.2 2.3 2.4 2.1
of urea Urea
2.7 3.4
9.7 7.7 5.7 4.2 4.7 6.6
4.4 5.2
6.8
-,
1142
Battaglia
Table III. limitation Experiment No. 463 232 469 097 112 190 275 305
et ai.
Index of transplacental diffusion (Ln) of urea, Na, and Cl ions /
Urea 0.732 0.725 0.743 0.772 0.761 0.620 0.771
/
Na
/
Cl
0.85 0.86 0.82 0.81 0.80 0.69 0.85 0.76
has different solubility characteristics. Thus, if placental permeability were a limiting factor in the transplacental diffusion of antipyrine, antipyrine clearance should be smaller than the clearance of tritiated water. Since the experimental evidence presented in this paper demonstrates that the clearances of antipyrine and tritiated water determined in vivo are not significantly different, it can be inferred that permeability plays a negligible role in limiting the diffusion of these molecules across the primate placenta. Therefore, this aspect of placental physiology shows no difference between monkeys and sheep.’ There are, though, quantitative differences. In a series of 29 experiments on sheep and goats under spinal the antipyrine clearance per anesthesia, kiIogram of fetus was 99 + 30 SD. ml. of blood per minute; whereas in the present series of 13 experiments the antipyrine clearance per kilogram of fetus was only 60.5 + 28 SD. ml. of blood per minute. The difference is statistically significant (P < 0.01). It is likely that part of this difference in antipyrine clearance is due to differences in anesthesia, degree of stress, and reactivity of the preparation to stress. Estimates of uterine blood flow per kilogram of tissue in anesthetized monkeys show lower rates of flow than in sheep and goats.6 Transplacental diffusion of urea is limited by placental permeability both in sheep and in monkeys. The degree of diffusion limitation is of the same magnitude in both species: the mean Lo was 0.72 (range, 0.48 - 0.87)
in a series of 10 experiments of sheep6 and 0.73 (range, 0.62 - 0.77) in the present report in monkeys. The urea diffusing capacity per kilogram of fetus measured by the same technique was 25.8 ml. per minute I 9.3 SD. in 10 sheep and 18.1 ml. per minute t 6.6 SD. in 7 monkeys. We have taken fetal body weight as our point of reference because it has been demonstrated in sheep that the urea-diffusing capacity of the placenta is directly proportional to fetal weight and has no simple correlation to placental weight. In addition, fetal body weight is a rough index of fetal metabolic rate, and it seems reasonable to assume that the properties of the placenta are related to fetal metabolic needs. Transplacental diffusion of Na and Cl ions is also limited primarily by the placental membrane: the average degree of diffusion limitation of both ions was 0.8 (range 0.76 - 0.85). The clearance per kilogram of fetus averaged 8.9 ml. per minute i- 2.9 SD., a value significantIy lower than the urea clearance per kilogram of fetus, 15.5 ml. per minute + 5.5 SD. (P < 0.02). Thus, in order of decreasing permeability, these experiments on monkeys show the following order: tritiated water and antipyrine (diffusion rate limited by flows), urea, chloride, and sodium (diffusion rate limited primarily by the permeability of the placental membrane). This is the order observed in the sheep placenta.” The major quantitative difference between the two species is in the relative magnitude of urea clearance with respect to Na and Cl ions clearance. In the monkey the clearances of the ions were about 60 per cent of the urea clearance; in sheep Na .and Cl ion clearances were less than one tenth of the urea clearance. According to Flexner and Gellhorn7 Na crosses the primate placenta more rapidly than the placenta of goats. Their standard of comparison was placental weight. Our results on the clearances of Na and Cl in sheep and monkeys do agree with Flexner and Gellhorn’s conclusion. Flexner and Gellhorn correlated these differences among species with the number of tissue layers sep-
Volume Number
102 8
Clearance
arating maternal and fetal blood. Although the correlation seems to have some validity in the case a’f Na, it should be pointed out that the over-all permeability to urea of the primate placenta is, if any, smaher than in sheep, i.e., ,the placenta with less layers seems to be ,the less permeable. In the case of antipyrine and tritiated water the placental permeabilities in both species are too large to have physiologic importance in vivo. Hence, place:ntal flows (umbilical and uterine) are the determining factors in their rate of exchange. In order to measure simultaneously uterine and umbilical flows by the Fick prin-
of
inert
molecules,
Na,
and
Cl across
placenta
1143
ciple it is necessary to know accurately the transplacental di-Fusion rate of the test substance. In addition, the test substance should give large arteriovenous differences on both circulations in order to minimize the error of measurement. Antipyrine and tritiated water fulfill these criteria. Under steadystate conditions their transplacental diffusion is about 80 to 90 per cent of the infusion rate, which can be accurately measured. The clearance of both molecules is flow limited, i.e., no inert molecules can be expected to give larger arteriovenous differences under a given set of physiologic conditions.
REFERENCES
1.
2. 3. 4.
Mesch,ia, G., Cotter, J. R., Makowski, E. L., and B#arron, D. H.: Quart. J. Exper. Physiol. 52: 1, 1967. Meschia, G., Battaglia, F. C., and Bruns, P. D.: J. AppIl. Physiol. 22: 1171, 1967. Wilkin, P.: Le placenta humain, Paris, 1958, Masson et Cie, p. 248. Battaglia, I?. C., Hellegers, A. E., Meschia, G., and Barron, D. H.: Nature 196: 1061, 1962.
5.
6.
7.
Battaglia, F. C., Bruns, P. D., Behrman, R. E., Seeds, A. E., and Hellegers, A. E.: Am. J. Physiol. 207: 500, 1964. Meschia, G., Behrman, R. E., Hellegers, A. E., Schruefer, J. J., Battaglia, F. C., and Barron, D. H.: AM. J. OBST. & GYNEC. 97: 1, 1967. Flexner, L. B., and Gellhorn, A.: AM. J. OBST. & GYNEC. 42: 965, 1942.