Protein binding of calcium and strontium in guinea pig maternal and fetal blood plasma

CURRENT INVESTIGATION

Protein binding of calcium and strontium in guinea pig maternal and fetal blood plasma A.

ROBERT

ERIC M. J.

TWARDOCK,

YUNG-HUE1 K. R.

Urbana,

AUSTIN,

PH.D.

D.V.M., KUO,

B.S.,

PH.D.

M.S.

HOPKINS,

B.S.

Illinois

In I3 pregnant guinea pigs the average concentrations of total Ca and ultrafilterable Ca (UF Ca) were 1.6 and 0.3 mg. per 100 ml. higher in fetal plasma than in maternal Qlasrna, respectively. The direction of the UF Ca gradient was from fetus to dam in 9 animals and from dam to fetus in 4 animals. The average UF Ca gradient was lower than the average total Ca concentration differences because more fetal plasma Ca was protein bound (34.8 per cent) than maternal plasma Ca (24.8 per cent), even though total plasma protein concentrations were the same in both plasmas (4.3 Gm. per 100 ml.). The greater Ca-binding capacity of fetal plasma protein was attributed to its higher albumin content (2.71 Cm. per 100 ml.) than that of maternal plasma protein (2.16 Gm. Qer 100 ml.). After in vivo labeling, h5Ca was slightly less ultrafilterable (4.3 to 8.1 per cent) than stable Ca in both maternal and fetal plasma, with individual values as much as 30 per cent lower. The UF 85Sr/UF W?a values for male, nonpregnant female, pregnant female, and fetal plasmas after in vitro labeling were 1.09, 1 .OS, 0.99, and 1.06, respectively.

ionic calcium is known to be the physiologically active component of the total plasma calcium. Sr, like Ca, is an element of Group 2 in the periodic table, and various studies1have shown that its metabolism is qualitatively similar to Ca metabolism, but that quantitative differences exist. However, little is known about the comparative distribution of the two ions between the protein-bound fraction and the ultrafilterable fraction in blood plasma. Investigators have found a higher concentration of total Ca in fetal blood plasma than in maternal plasma in several mammalian species.2-5 Some have thus suggested

EXISTS inthebloodplasma in three different forms: ( 1) ionic calcium, (2) calcium which is reversibly combined with citrate, phosphate, and other small complexes, and (3) calcium which is reversibly combined with plasma protein. The first two fractions are diffusible or ultrafilterable through semipermeable membranes. The CALCIUM

This research was supported National Institute of Child

by the Health and

%&$z..7$7eveloQment, Grant No.

Submitted, in part, by E. Y. Kuo and J. R. Hopkins in partial fulfillment of the requirements for the MS. degree, University of Illinois at UrbanaChampaign. 1008

Volume 110 Number 7

Protein binding

that active transport across the placental membranes must occur in the movement of Ca fmm dam to fetus in order to maintain such a difference. Considering that only the diffusible (ultrafilterable) fraction of plasma Ca is transferable through a membrane, it is essentialthat the gradient of diffusible Ca between dam and fetus be studied when trying to determine whether active transport exists. In the present study, the amounts of ultrafilterable (UF) Ca and Sr in the blood plasma of pregnant guinea pigs and their fetuses were measured with the objectives of determining the actual concentration gradient of LJF Ca and of assessingthe role of protein binding in the so-called “discrimination” against Sr movement by the placenta.6-8

phore&.* Ten microliter samples were aPplied to strips run at 2.6 to 3.4 volts Per centimeter (5.4 ma.) for 16 hours. Verona1 buffer solution with pH 8.6 and 0.075M ionic strength was used. Measurements of pH were made with a Beckman Model 76A meter and Model 39030 combination electrode. Plasma coBection and preparation. The pregnant female was anesthetized with intraperitoneafly injected pentobarbital Na (40 mg. per kilogram of body weight), sun@+mented with inhaled halothane when necessary. For the in vivo labeling experiments, the maternal carotid artery was cannulated. Following hysterotomy, the gravid uterus was exteriorized in a pan containing isotonic saline solution at 37O C., while attempting not to disturb the uterine blood supply. Fetuses were exposed one by one, with care taken not to disrupt the placental attachment or the umbilical vessels. Fetal blood was drawn from the umbilical vein and pooled. After collection of fetal blood, m.zternal blood was obtained by cardiac punclure or from the maternal carotid artery catheter; blood was also taken from the uterine vein. Blood samples from maIe adults and nonpregnant female adults were obtained by cardiac puncture. To prevent CO, lossfrom the samples, the heparinized syringes were coated with mineral oil prior to blood cohection, and the blood was immediate]) centrifuged under mineral oil to obtain plasma. Radionuclide labeling and a.~,+ For in vitro labeling of plasma, approximately I

Materials and methods The comparative binding of *%a and 85Sr by blood plasma protein after in vitro labeling was studied in 4 adult male, 4 adult female, and 5 pregnant guinea pigs and their fetuses. The ultrafilterabihties of d5Ca and stable Ca in plasma after in viva labeling, as related to plasma protein and pH, were measured in 13 pregnant adults and their fetuses. The pregnant animals were between 56 and 65 days’ gestation. Stable Ca concentrations of plasmas and ultrafiltrates were measured by atomic absorption spectrometry.” Total protein analysis was done by the method of Lowry and associates.0Percentagesof albumin and globulin were measured by paper strip electro“JamIt-Ash 83-000 series.

Company,

W&ham,

Massachusetts,

Table I. Comparative ultrafilterability

Number UF 4%3 of(%)animais UF UF

*?Sr (%) 85Sr/UF

45Ca

2 7.: 59.4 64.4

+ (0.05)+@ 6.6

1.09 N.S. = not significant. *Mean 2 standard deviation. Levels and percentage of UF % in individual

and strontium

1009

Model

of 4”Ca and s5Srin guinea pig plasma Nonpregnant

Male

of calcium

Pregnant

-~

female

Maternal

4 65.0 + 2.4 (0.01) 70.0 -+ 1.8 1.08

5 56.6 + 6.9 (N.S.) 55.8 _+ 7.6 0.99

of significance for pafred t tests on means of differences samples areshown in parentheses.

female Feral 46.8

t 7.6 (0.05) 49.5 ?: 7.4 1.06

between percentagt:

of lJF 4JC!a

1010 Twardock

et al.

pc of 45ca and 0.5 pz of Y3r (in chloride form) was added to 4 ml. of plasma. The labeled plasma was mixed thoroughly and allowed to stand for at least 30 minutes to allow exchange between radioactive ions and protein-bound stable ions. For in vivo labeling, approximately 20 ,QCof 45Ca was injected via the maternal jugular vein. Thirty to forty minutes were allowed to elapse between 45Ca injection and the start of maternal and fetai blood collection. Radioactivity assays were carried out on both the labeled whole plasma and its ultrafiltrate. 45Ca concentrations were measured by Geiger-Mueller or liquid-scintillation counting, and Y4r was measured by a sodium iodide crystal detector. The equation for calculating ultrafilterable *5Ca or “Sr was as follows : % ultrafilterable %a

or *sSr =

c.p.m./O.l ml. ultrafiltrate -

c.p.m./O.l ml. labeled plasma

x 100.

Ultrafiltration. The simple ultrafiltration method described by Prasad and FlinklO was modified for this study. A piece of cellulose dialyzing tubing (s/4 inch inflated diameter, approximately 25 cm. long) was opened, and one end of the piece was closed by knotting. Three milliliters of labeled plasma was quickly delivered into the tubing through the open end which then was also knotted. The tubing was bent into a “U” shape and wrapped with a double layer of narrowmeshed white gauze, 5 by 5 inches. The tubing and gauze were then inserted into a 15 ml. centrifuge tube with the two knots and the outer edge of the gauze protruding above the mouth of the centrifuge tube. Then the tube was stoppered tightly, holding the tubing knots and the gauze edge as a firm support during centrifugation. After centrifuging for 80 minutes (380 g at room temperature), 0.5 to 0.7 ml. of crystal-clear protein-freeI’ ultrafiltrate was collected, To determine whether the cellulose tubing contained Ca which could have been washed into the ultrafiltrate, distilled water was

for& through several pieces of tubing by the centrifugation method and its Ca concmtration was measured before and after tentrifugation. Results

CQmparatiVe 45Ca and %r binding. ReSUh from the in vitro labeling e.Vpebments are shown in Table I. It is evident that 85Sr was slightIy more ultrafilterable than Q5Cain Plasma from male, nonpregnant female, and fetal guinea pigs, whereas they were almost equally ultrafilterable in maternal plasma. The highest ultrafilterabilities were found in nonpregnant female plasma, and the lowest was in fetal plasma. Paired t tes@ on the differences between UF Wa and 85Sr for individual samplesshowed significant differencesin male (5 per cent level), nonpregnant female (1 per cent level), and fetal plasma (5 per cent level). Binding by maternal plasma was not significantly different. Calcium ultrafilterability and protein composition of maternal and fetal plasma. Results for 13 dams and their fetusesare given in Tables II to IV. The average total Ca of maternal artery plasma (7.7 mg. per 100 ml.) was 1.6 mg. per 100 ml. lower than the average fetal plasma Ca concentration. The average UF Ca of maternal plasma (5.8 mg. per 100 ml.) was also lower than fetal plasma UF Ca, but only by 0.3 mg. per 100 ml. Protein-bound (PB) Ca was, therefore, considerably higher in fetal than in maternal plasma. These differences are reflected in the fact that Ca was 10 per cent lessultrafilterable in fetal (65.2 per cent) than maternal plasma (75.2 per cent). Of the 13 animals, the UF Ca gradient (downhill) was from fetus to dam in 9 and from dam to fetus in 4. Uterine vein plasma Ca averaged 6.8 mg. per 100 ml., a decrease of 0.9 mg. per 100 ml. from arterial plasma. The decrease occurred in the UF Ca fraction with no change in the average fetal plasma PB Ca. Results of t tests on meansof differences between paired samples showed the abovementioned differences to be significant at a probability of 0.02 or better, except for the difference between UF Ca in maternal carot-

Volume Number

110 7

Protein

Table II. Calcium 13 guinea pigs -- --- .----

and protein

composition

of maternal

Total Ca (mg./lOO ml.) UF Ca (mg./lOO ml.) PR Ca (mg./lOO ml.) UF Ca (%) UF 45Ca (%) PI-1 Albumin (% of protein) Globulin (% of protein) Albumin/globulin Total protein (Gm./lOO Albumin (Gm./lOO ml.) Globulin f Gm./lOO ml.) *Ten tMean

of the

13 animals

? standard

7.7 5.8 1.9 75.2 69.5 7.4 50.5 49.5 1.02 4.28 2.16 2.12

ml.)

were

sampled

from

Uterine

+ 0.9+ + 0.7

6.8 4.9 1.9 72.4 68.1 7.3

c 0.9 + 0.7

three

were

4.0 8.2 0.1 2.0 2.0 0.08 0.52

the maternal

calcium

and

plasma

carotid

artery:

1011

in

Fetal plasma umbitical vein

vein

9.3 6.1 3.2 65.2 57.1 7.2 63.4 36.6 1.73 4.26 2.71 1.55

+ 3.4 k 6.4 5 0.09

sampled

strontium

by heart

t t

0.4 0.6

_+ 6.1 + 12.1 r 0.09 i 3.2 ?. 2.2 5 0.16 -f l.fll

puncturr

deviation.

total and ultrafilterable

Total calcium

-.

and fetal blood

artery*

f t. f + 2 A A

Table III. Means of differences between in maternal and fetal blood plasma - --.

Maternal Uterine Maternal

of

plasma

Maternal Carotid

binding

carotid-umbilical vein-umbilical carotid-uterine

vein vein vein

-1.63 -2.44 +0.81

Ca concentrations

plasma (ma./1 00 ml.)

+ 0.86

UF calcium

(0.001)” (0.001) (0.001)

+ 0.94 + 0.36

-0.30 -1.14 +0.85

plasma (mp./lOO

+ 0.90 2 1.05 + 0.46

ml.)

(N.S.) (0.001; (0.001: -I-.

..__--

N.S. = not significant. “Mean of the ences are significant

differences as calculated

between by the

paired values from 13 animals “t” test are shown in parentheses.

Table IV. Means of differences between ultrafilterable 4oCa and 45Ca in maternal and fetal blood plasma

/5%UF Maternal Uterine Umbilical

carotid vein vein

artery

+5.69 +4.94 +8.10

40Ca - 5% UF 45Ca + 7.04 2 6.00 -t 8.43

(0.02)' (0.02) 10.021

*Mean of the differences between paired values animals rt standard deviation. Probability values fe~ences are significant as calculated by the “t” shewn in parentheses.

from 13 that diftest are

id and umbilical vein plasma (Tables III and IV). TotaI protein concentrations were essentially the same in maternal and fetal plasma, but the distribution between albumin and globulin was considerably different. Maternal plasma protein was composed almost equally of albumin and globulin, whereas fetal plasma protein contained considerably more albumin (2.71 Gm. per 100 ml.) than

f

standard

drviation.

Probability

v&es

that

differ-

globulin (1.55 Gm. per 100 ml.). Plasma pH decreased progressively from maternal artery (7.4) to vein (7.3) to fetal umbilical vein (7.2). There was a small but consistent difference between the ultrafilterability of stable Ca and %a, and the percentage of UF 4SCa was about twice as variable as the percentage of UF stable Ca, as shown bv the standard deviations (Tables II and IV,), The difference was not caused by Ca washing out of the cellulose tubing. The Ca concentration of distilled water after passage through 6 different sections of tubing by the centrifugation method was increased no more than 0.2 mg. per 100 ml., and by an average of 0.1 mg. per 100 ml. Since data for individual animals are not given, it should be mentioned that most of the increased UF 45Ca variability was a result of low values from 6 animals. In -1 animals fetal plasma percentage af UF 45Ca

1012

Twardock

et al.

Amer.

was lower than the percentage of UF stable Ca by 30.1, 17.6, 15.6, and 10.7, with no other differences greater than 7 per cent. Uterine vein differences in 2 animals were 23.4 and 8.7 per cent, with no others greater than 6 per cent. Carotid plasma differences in 3 animals were 25.7, 11.5, and 7.3 per cent, with no others greater than 6 per cent. None of the 6 dams had unusually low UF 45Ca in all 3 plasmas; 3 had low values in 2 plasma but there was no consistency as to which plasmaswere low. In no instance was the percentage of UF 45Cagreater than the percentage of UF stable Ca. Comment

Our results agree with the well known fact that the concentration of Ca in fetal plasma is higher than in maternal plasma. They are also in agreement with the findings of Delivoria-Papadopoulos and colleagues*in human beings and sheep and Burnette and co-workers3 in guinea pigs that the average UF Ca of fetal plasma in a group of animals is higher than in their maternal plasma. However, the average UF Ca gradient* of 0.3 mg. per 100 ml. in our guinea pigs was much smaller than the gradients of 4.2 mg. per 100 ml. in sheepand 1.8 mg. per 100 ml. reported in man.* Burnette and co-workers3 reported UF Ca differences of 0.9 and 0.7 mg. per 100 ml. between fetal and maternal plasma in groups of 20 and 10 guinea pigs, with the fetal fraction higher than the maternal fraction in all animals. Our findings indicate a smaller average gradient and variation from animal to animal in the direction of the gradient. The fact that the gradient was from dam to fetus in 4 of our 13 guinea pigs suggeststhat active transport may not always be required for Ca movement to the fetus. When Burnette and co-workers3 gave vitamin D and parathyroid extract to 10 pregnant guinea pigs, maternal plasma total Ca and UF Ca increased while fetal Ca did not, resulting in average concentration differences of 0.5 mg. per 100 ml. of total

from

*Gradient a higher

will be used to indicate to a lower concentration.

movement

downhii

August J. Obstet.

1, 1971 Gym.

Ca and 0.2 mg. per 100 ml. of UF Ca from dam to fetus. Variations between individual animals were not mentioned. Burnette and co-workers3 also found that guinea pig plasma Ca was slightly more than 80 per cent ultrafiltrable in both dam and fetus, whereas we observed a distinct difference between percentage of UF Ca in the dam (75.2) and fetus (65.2). We are in agreement that guinea pig maternal plasma Ca is more ultrafilterable than in other species, but the fetal plasma percentage of UF Ca of our guinea pigs was considerably lower than that of Burnett and co-workers and more like that of the sheep and human being.4 Our results also differ from those of Delivoria-Papadopoulos and colleagues4who found fetal plasma Ca to be approximately 6 per cent more ultrafilterable than maternal plasma Ca in both the sheep and human being. Reasonsfor such differences probably include speciesand ultrafiltration techniques, as well as the diet and stage of gestation of our guinea pigs. Differences from other species should not be overemphasized since the guinea pig is somewhat unique in terms of the amount of Ca the dam must provide her fetuses near term. She must transfer approximately 400 per cent of her total plasma Ca per hour in comparison to 7 per cent per hour in the human being, 50 per cent in the bovine species,and 100 per cent in the rat and rabbit.13-15 Reasonsfor the stable Ca ultrafilterability differences in maternal and fetal plasma can be found in their protein composition. Maternal and fetal plasma protein concentrations were essentially the samein our guinea pigs, whereas Delivoria-Papadopoulos and colleagues4found that the protein concentration in fetal plasma was lower than in maternal plasma in both humans and sheep. They also found that Ca binding per gram of protein was equal in human maternal and fetal plasma, but higher in sheep fetal than maternal plasma. They explained their observations on the basisof the relative distributions of protein fractions in maternal and fetal plasmasof the two species,plasma proteins being essentially the samein the human

Volume Number

110 7

mother and fetus while sheep fetal plasma is known to have higher albumin-to-globulin (A/G) ratios than maternal plasma. However, MacklB reported higher A/G values in human cord blood than maternal blood at delivery. Our data show that the guinea pig dam and fetus also have different plasma protein compositions; their average maternal and fetal A/G’s were 1.02 and 1.73, respectively (Table II). Since albumin is known to have a much smaller molecular weight than other plasma proteins (albumin 70,000 to 80,000; globulins 150,000 to 1,300,000), the average molecular weight of fetal plasma proteins is lower than that of the maternal plasma. Therefore, fetal plasma has more moles of protein than maternal plasma in each unit weight of protein and thus has a larger number of anionic binding sites for calcium. This interpretation is supported by the finding that plasma albumin binds 10.5 moles of Ca per lo5 Gm. of protein, whereas plasma globulin binds 5.6 moles of Ca per lo5 Gm. of protein .I7 Consequently, more Ca was bound by guinea pig fetal plasma proteins due to their high A/G value even though total plasma protein concentrations were the same in dam and fetus. This occurred in spite of the slightly lower pH of fetal plasma (7.2) which would cause an increased UF Ca relative to maternal plasma IJH (7.4). The per cent s5Sr/per cent 45Ca ratio of the ultrafiltrate provides an estimate of the relative binding of 45Ca and s5Sr by the blood plasma proteins. When this ratio exceeds 1, the plasma proteins bind a higher percentage of 45Ca than they do 85Sr, thus allowing more 85Sr than 45Ca to be ultrafiltered. In previous studies with human serunP8p la and dog plasma,8 the per cent 85Sr /per cent 45Ca ratios of ultrafiltrates were between 1.20 and 1.30. Szymendera and MadajewiczZO reported that there was no difference in the binding of Ca and Sr by human blood plasma. Ultrafiltration of cow and goat blood plasma’ revealed 85Sr to be 10 to 20 per cent more ultrafilterable than 45Ca. Results from the present study show that plasma proteins of male, nonpregnant female, and

Protein

binding

of calcium

and

strontium

1013

fetal guinea pigs bound greater percentages of 45Ca than they did of 86Sr, as seen from the per cent UF 85Sr/per cent UF YL~ ratios ( 1.Ol to 1.17). In contrast, the plasma proteins of female guinea pigs in late pregnancy exhibited approximately equal affinity for 45Ca and 85Sr (ratios of 0.96 to 1.01) . The role of protein binding in Ca-Sr discrimination has been investigated in the case of the kidney81 ?a,?l and mammary gla.nd,22 but this approach has not been applied to the placenta in which it is known that the passage of Sr relative to Ca is labout half.69 1&tlj Our results show that fetal plasma proteins bind more Ca than Sr while maternal plasma proteins do not, which suggests that after passing into the fetal circulation Sr might diffuse back to the maternal side more readily than Ca. If proteins in other fetal tissues have similar Ca-Sr binding properties, placental Ca-Sr discrimination might be at least partially explained. However, protein binding cannot be the entire answer. Twardock and associatesZ3 perfused the fetal circulation of the placenta with male guinea pig plasma while the maternal circulation was intact and observed Ca-Sr discrimination equal to that which occurs in the intact pregnant animal. It is interesting to note that 4”Ca was less ultrafilterable than stable Ca after in \,ivo Iabeling. The average differences in mnternal and fetal plasma were small (4.3 to 8.1 per cent) but individual animals had differences as great as 30 per cent. There have been numerous reports of specific activity (SA) differences between blood Ca and its excretory and secretory products, urine and milk. Urine Ca SA’s both higher and lower than those of blood plasma have been observrd in several species and under a wide variety of experimental conditions including lasting, route of isotope and anesthetic administration, age of subject, and Ca assay method.81 24-16 Visek and colleague?’ observed a higher Ca SA in milk than in blood which could not be explained by a time lag between secretion and sampling. It is difficult to arrive at a single explanation for such obsrrvations because of their heterogeneity. Proposed

1014

Twardock

et al.

mechanisms include slowly exchanging plasma Ca pools14l 24v25 and tissue Ca pools which contribute low SA Ca under certain conditions.26-27 Although most attempts to demonstrate the presence of nonuniform SA fractions in plasma Ca have failed,25-27 Briscoe and Raganz4 showed that the SA of serum Ca taken from elderly human patients after intravenous 47Ca injection could be decreased 19 per cent by dialysis. The SA of urine Ca was greater than that of serum Ca in the same patients for a period as long as 4 days after injection. Our results show that the reverse

REFERENCES

Comar, C. L.: Some over-all aspects of strontium-calcium discrimination, in Wasserman, R. H., editor: The Transfer of Calcium and Strontium Across Biological Membranes, New York, 1963, Academic Press, Inc., pp. 405-417. J. W., Wolkoff, A. S., and Flowers, 2. Bawden, C. E.: Obstet. Gynec. 25: 548, 1965. 3. Burnette, J. C., Simpson, S. M., Chandler, D. C., Jr., and Bawden, J. W.: J. Dent. Res. 47: 444, 1968. 4. Delivoria-Papadopoulos, M., Battaglia, F. C., Bruns, P. D., and Meschia, G.: Amer. J. Physiol. 213: 363, 1967. 5. Hallman, N., and Salmi, L.: Acta Paediat. 42: 126, 1953. 6. Comar, C. L., Whitney, I. B., and Lengemann, F. W.: Proc. Sot. Exp. Biol. Med. 88: 232, 1955. 7. Twardock, A. R.: Studies on the movement of calcium and strontium across the bovine mammary gland, in Wasserman, R. H., editor: The Transfer of Calcium and Strontium Across Biological Membranes, New York, 1963, Academic Press, Inc., pp. 327-339. M., and Robinson, B. H. B.: Renal 8. Walser, excretion and tubular reabsorption of calcium and strontium, in Wasserman, R. H., editor: The Transfer of Calcium and Strontium Across Biological Membranes, New York, 1963, Academic Press, Inc., pp. 305-326. 0. H., Rosenbrough, N. J., Farr, A. 9. Lowry, L., and Randall, R. J.: J. Biol. Chem. 193: 265, 1951. 10. Prasad, A. S., and Flink, E. B.: J. Appl. Physiol. 10: 103, 1957. P. S., Jr., and Neuman, W. F.: Amer. 11. Chen, J. Physiol. 180: 623, 1955. in Research, Ames, Iowa, 12. Ostle, B.: Statistics 1964, Iowa State College Press, p. 121.

.imer.

August J. Obstet.

1, 1971 Gynec.

situation exists in the pregnant guinea pig. UF Ca having a lower SA than PB Ca. A possible common denominator in cases of nonuniform SA pools is stress involving Ca homeostasis, excretion, and secretion. Instances in which SA differences have been suspected or demonstrated have involved starvation or fasting,25 use of elderly patients,21 reduced renal blood flo~,“~ and lactation. During late gestation, the guinea pig fits this pattern because of the severe drain on maternal Ca reserves caused by fetal calcification.

13.

1.

14. 15.

16.

17.

18. 19. 20. 21. 22. 23.

24. 25. 26. 27.

Comar, C. L.: Ann. N. Y. Acad. Sci. 64: 281, 1956. Twardock, A. R.: Amer. J. Physiol. 213: 837, 1967. Wasserman, R. H., Comar, C. L., Nold, M. M., and Lengemann, F. W.: Amer. J. Physiol. 189: 91, 1957. Mack, H. C.: The Plasma Proteins in Pregnancy, Springfield, Illinois, 1955, Charles C Thomas, Pub&her, pp. 95-108. Simkiss. K.: Calcium in Renroductive Phvsiology, A Comparative Study of Vertebrates, New York, 1967, Reinhold Publishing Corp., p. 31. Kara, M., Samachson, J., and Spencer, H.: T. Clin. Endocr. 23: 981. 1963. Samachson, J., and Ledkrer, H.: Proc. Sot. Exp. Biol. Med. 98: 867, 1958. Szymendera, J., and Madajewicz, S.: Nature 217: 968, 1968. Samachson, J., and Laszlo, H. S.: J. Appl. Physiol. 17: 525, 1962. Twardock, A. R., Prinz, W. H., and Comar, C. L.: Arch. B&hem. 89: 309. 1960. Twardock, A. R., Downey, H. F., Kirk, E. S., Austin, M, K.: Comparative transfer of calcium and strontium and of potassium and cesium in the guinea pig placenta, Symposium on the Radiation Biology of the Fetal and Juvenile Animal, Richland, Washington, May, 1969, Oak Ridge, 1969, United States Atomic Energy Commission Division of Technical Information Extension. Briscoe, A. M., and Ragan, C.: J. Appl. Physiol. 20: 453, 1965. Giese, W., and Comar, C. L.: Nature 202: 31, 1964. Miller, E. J., Neuman, W. F., and Bazerque, P. M.: Amer. T. Phvsiol. 206: 755. 1964. Visek, W. J., Monroe, R. A., Swanson, E. W., and Comar, C. L.: J. Dairy Sci. 36: 373, 1953. 1

,