PLASMAPROTEIN SYNTHESIS IN THE HUMANFETUS ANDPLACENTA'

By JOSEPHDANCIS,2 NANCYBRAVERMAN,AND JOHN LIND

(From the Department of Pediatrics and of Obstetrics and Gynecology, New York University,Bellevue Medical Center, New York, N. Y.; the Wenner-Gren Cardiovascular Re-

search Laboratory, and the Department of Pharmacology, Royal VeterinaryCollege, Stockholm, Sweden)

(Submitted for publication August 16, 1956; accepted November 16, 1956)

Electrophoretic studies of human fetal plasmahave demonstrated a protein pattern in which allmajor fractions are present (1). The site of ori-gin of these plasma proteins is not known. Pre-sumably they may be synthesized by the motherand merely circulate in the fetus as the result ofplacental transfer, or they may be synthesized bythe placenta or the fetus.

The gammaglobulin level is high at birth andfalls during the first weeks of life (2). This led tothe assumption that the gammaglobulin found atbirth is derived from the mother by transferthrough the placenta, and that the newborn infantdoes not synthesize it. Because gammaglobulinlevels (2) and antibody levels (3) are often higherin cord blood than in maternal blood there has beenspeculation that the placenta may be the site ofsynthesis. The problem of plasma protein syn-thesis in the placenta has been approached bycomparing levels of the various fractions in bloodfrom the umbilical artery and vein and from themother (4). It was concluded that the placentasynthesizes albumin.

The present experiments were designed to in-vestigate the synthesis of plasma protein by pla-centa and fetal liver. Term placenta was obtainedat normal deliveries. Liver and placental tissueswere also taken from three fetuses of 3 to 4months gestation (Table I). For purposes of com-parison fetal heart was similarly investigated. Thefetuses were delivered surgically for psychiatricand social indications and were presumably nor-mal. Tissue slices were incubated with glycine-2-

1 This investigation was supported by a research grantfrom the Association for the Aid to Crippled Childrenand by Public Health Service Research Grant No. 4024,National Institutes of Health.

2John and Mary R. Markle Scholar in Medical Science.A large part of the work was done while Dr. Dancis wasa Lowell Palmer Senior Research Fellow.

C14, and the incorporation of radioactivity intoplasma proteins was studied. The proteins wereisolated and identified electrophoretically and byimmunological techniques.

PROCEDURE

Incubation

Tissues were procured immediately after delivery anddropped into chilled isotonic saline. Slices about 0.5 mm.thick were prepared with a Stadie-Riggs slicer. Onegram of tissue was added to 2.5 ml. of Krebs-Ringer-bicarbonate buffer (5) in a 25-ml. flask to which hadbeen added 5 micrograms of Aureomycin and about onemillion counts per minute of glycine-2-C,4 (Tracerlab).Radioactivity was determined as described below. Theslices were incubated with constant shaking for 18 hoursin a water bath at 370 C.

ElectrophoresisAt the end of incubation, the tissue slices were centri-

fuged down and discarded. Incubation media from eachtype of tissue were pooled and dialyzed against threechanges of isotonic saline during 24 hours at 50 C. Thebag contents were lyophilized and redissolved in 1 ml.isotonic saline to reduce to volumes suitable for electro-phoresis. The samples were dialyzed for three hoursagainst veronal buffer, pH 8.6, and then separated bystarch electrophoresis and the protein pattern developedas described by Kunkel and Slater (6). One ml. of hu-man plasma was run in parallel on the same block toserve as marker for the plasma protein fractions.

To determine the radioactivity curve, the protein wasprecipitated from an aliquot of each eluted fraction bythe addition of equal volumes of 10 per cent trichloraceticacid (TCA). The precipitate was washed with 5 percent TCA and extracted with ether to remove the TCA.The precipitate was redissolved in 0.05 N NaOH, trans-ferred to a planchette, and the radioactivity determined.

Immunological identification of radioactive proteins

Liver. Carrier precipitates were made in eluates fromthe starch electrophoresis by adding various plasma frac-tions and their specific antisera prepared in rabbits.Fractions 5 to 12 (Figure 1), representing the alpha andbeta globulins, were pooled, divided into five aliquots, and

398

PLASMA PROTEIN SYNTHESIS IN FETUS AND PLACENTA

TABLE I

Data on fetuses

Gestation* GestationtWeight in days in days

Fetus (grams) (history) (from weight)

I 150 130 120II 140 195 120

III 240 130 130

* Calculated from menstrual history.t Estimated from weight according to graph by Widdas

(18).

a specific precipitate of about 2 mg. was made in eachaliquot. A preliminary titration was done in a smallsample of each aliquot, so that appropriate amounts ofantigen and antibody were added to produce the precipi-tate at or near the equivalence point. Precipitates wereprepared with the following fractions: human albumin,guinea pig albumin, human beta globulin, beta lipopro-tein, and metal-combining globulin. Similar precipitateswere prepared in the albumin fraction (17 and 18, Fig-ure 1). All of the specific precipitates were made in ali-quots of the eluates containing approximately the sameamount of radioactivity. This permits comparison of theamount of radioactivity incorporated in each specificprecipitate.

The fibrinogen was located in fractions 12 to 14 byadding thrombin to the concurrently separated humanplasma. A precipitate was then formed in fractions 12 to14 of the incubating medium by adding carrier fibrinogenand the specific antiserum. A similar control precipitatewas made in fractions 16 to 18.

The precipitates were washed with saline, dissolved inconcentrated ammonium hydroxide, and transferred toplanchettes for determination of radioactivity.

Heart. Because of insufficient material, only electro-phoretic separation could be done in the first experiment.In the second experiment, a preliminary separation wasperformed by dialysis against 1.75 M ammonium sulfate(7). The precipitate, containing all the gamma globu-lin and a fraction of the other globulins, but little albumin,was redissolved in saline. Immunological precipitateswere made in the "albumin" fraction (supernatant) andthe "globulin" fraction.

Placenta. After electrophoretic separation, it wasdemonstrated that the radioactive proteins were distributedin the alpha and beta globulin fractions. These fractionswere separated (Figure 3, fractions 5 to 9 and 10 to 14,respectively), and specific precipitates were made in eachfraction.

In earlier experiments with term placentas the tech-nique described by Keston and Katchen was used (8).After incubation the tissues were homogenized and themixture centrifuged. The supernatant was dialyzedagainst saline and specific precipitates were made in ali-quots of the supernatant without preliminary electropho-resis. To provide an estimate of non-specific adsorptionby the protein precipitates, specific precipitates were made

zwa

-J

0

8 10 12FRACTIONS

w

z

,CoI--z

0

FIG. 1. SYNTHESIS OF PROTEINS BY FETAL LIVERA: Electrophoretic pattern of human plasma proteins.B: Total radioactivity of proteins in the incubating me-

dium. Note similarity to A.C: Pattern of proteins in incubation medium.

with physically similar but heterologous proteins (oval-bumin, guinea pig albumin, and bovine gamma globulin).

Preparation of specific antiseraAntisera were prepared in rabbits by the injection of

plasma protein fractions with Freund's adjuvant (9).Most of the antisera were prepared by injecting inter-digitally 3 to 5 mg. of antigen emulsified in 0.5 ml. Falba,1 ml. Bayol F and 1 mg. of killed tubercle bacilli. Someof the antisera prepared early in the study required 50 mg.of antigen in 4 ml. of adjuvant, and the injections wereintramuscular.

The human gamma globulin and fibrinogen were ob-tained from the Department of Biophysical Chemistry,Harvard University. The gamma globulin was electro-phoretically pure. The antiserum to fibrinogen was puri-fied by absorption with human serum.

The antisera against beta lipoprotein and metal-com-bining globulin were generously supplied by Dr. Leon-hard Korngold of the Sloan-Kettering Institute forCancer Research. The preparation of the former andthe tests for specificity have been described (10). Thelatter was prepared by injection of the crystalline antigenand absorption of the antiserum with human gammaglobulin and albumin.

The human albumin was supplied by Dr. T. W. Greenof Cutter Laboratories, Berkeley, California. It wasstated to be 98 per cent electrophoretically pure with themajor contaminant alpha globulins. Bovine gammaglobulin was obtained from Armour and ovalbumin fromDr. Bernard Katchen.

The beta globulins were prepared by starch electro-

399

JOSEPH DANCIS, NANCYBRAVERMAN,AND JOHN LIND

phoresis of human plasma. Antisera were preparedagainst alpha and beta globulin fractions individuallybut were found to be immunologically indistinguishableby the techniques used in this study. They were thenused interchangeably. The guinea pig albumin and anti-serum were prepared in similar fashion.

Determination of radioactivity

The protein precipitates were transferred in solutionto aluminum planchettes covered with filter paper toproduce even distribution, and dried under an infra-redlamp. Radioactivity was determined in a D-46A Nu-clear Flow Gas Counter, and the counts were correctedto infinite thinness.

RESULTS

The electrophoretic pattern of normal plasmaproteins (Figure 1 A) is paralleled strikingly bythe total radioactivity of the proteins in the mediumused for the incubation of liver slices (Figure 1 B),except that there is no incorporation of radioac-tivity into proteins with gammaglobulin mobility.The electrophoretic pattern of the proteins in theincubation medium (Figure 1 C), probably repre-senting primarily liver proteins, is quite dissimilar.

Carrier precipitates were made by adding vari-ous plasma proteins and their specific antisera tofractions of the electrophoretically separated pro-teins, as previously described. The results are pre-

TABLE II

Incorporation of radioactivity into proteins by fetal liver(counts per minute) *

Experiment II Experiment III

Globulin Albumin Globulin AlbuminPrecipitates fraction fraction fraction fraction

Total proteint 1594 1854 1210 915Albumin (human) 95 1130 51 980Beta globulin 670 44 598 13Metal-combining 171 10 148 2Lipoprotein 239 32 279 18Gammaglobulin 688 566 560 169Albumin 58 26 27 50

(guinea pig)

Fibrin- Fibrin- Fibrin- Fibrin-ogen ogen ogen ogen

fraction control fraction control

Total proteint 574 890 474 572Fibrinogen 186 78 201 105

* Carrier precipitates were formed by adding the proteinand its specific antiserum to appropriate electrophoreticfractions (e.g., albumin precipitate in albumin fraction)and, as a control, in inappropriate fractions (e.g., albuminin globulin fraction). Guinea pig albumin precipitates arefurther controls.

t TCA precipitable radioactivity.

sented in Table II. It is evident that a significantamount of radioactivity is carried down by a spe-cific precipitate prepared in the appropriate frac-tion (e.g., the human albumin precipitate in thealbumin fraction), and that it is considerably lessin the inappropriate portions of the curve (e.g.,the albumin precipitate in the globulin fraction).Precipitates prepared with guinea pig albumin andits specific antiserum, to give an index of non-specific adsorption, also contain much less radio-activity. The gammaglobulin precipitates seemedto give non-specific results. This suspicion wasverified by demonstrating extensive cross-reactionbetween human albumin and our gammaglobulinantiserum. The gammaglobulin precipitates alsoincorporated as much radioactivity as the betaglobulin precipitates even though the radioactiveproteins had an electrophoretic mobility corre-sponding to the latter.

The results of the experiments with fetal heartare presented in Figure 2 and Table III. Theelectrophoretic pattern of the radioactive proteinsin the incubation medium (Figure 2 B) resemblesmore closely that of the presumed tissue proteins(C) than the plasma proteins (A). The amountof radioactivity incorporated is much less than inthe liver experiment (note the difference in scalein Figures 1 and 2).

Unfortunately there was insufficient materialafter electrophoretic separation to permit immu-nological identification. Accordingly, in a secondexperiment, preliminary separation was done withammonium sulfate. There is a significant amountof radioactivity in the beta globulin and albuminprecipitates made in the supernatant (Table III).However, the results with guinea pig albumin in-

TABLE III

Incorporation of radioactivity into specific precipitatesby fetal heart (counts per minute) *

Globulin Albuminfraction fraction

Total protein 107 124Albumin (human) 0 56Beta globulin 10 40Gammaglobulin 8Albumin (guinea pig) 38

* Incubation medium proteins were separated by am-monium sulfate precipitation into two fractions and specificprecipitates made in each fraction.

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PLASMA PROTEIN SYNTHESIS IN FETUS AND PLACENTA

I--

z

J-I

so2I-.a.0

FIG. 2. SYNTHESIS OF PROTEINS BY FETAL HEARTA: Electrophoretic pattern of human plasma proteins.B: Total radioactivity of proteins in the incubating

medium.C: Pattern of proteins in incubation medium. Note

similarity of B and C in this figure.

dicate that this is probably because of non-specificadsorption.

The results of an experiment with term pla-centa are given in Figure 3. The total radioac-tivity of the proteins in the incubating medium (B)parallels the protein pattern (C) more closelythan that of the plasma proteins (A). There isno radioactivity in proteins with albumin or gammaglobulin mobilities. It is presumed that the albu-min peak in C is derived from blood in the tissueslices since the placenta apparently does not syn-thesize proteins with this mobility (B).

The results of similar experiments with pla-centas from 3 to 4-month pregnancies are not re-produced here because there are no important dif-ferences. The radioactive proteins moved pri-marily in the beta and alpha globulin zones. Therewere no radioactive proteins with albumin mo-bility, but a small amount overlapped the fastermoving gammaglobulins. As with term placentathe pattern resembled more closely that of theproteins of the incubating medium than that ofplasma proteins.

Carrier precipitates were made by adding plasmaproteins and the specific antisera to the alpha andbeta globulin fractions (Figure 1, fractions 5 to 9and 10 to 14, respectively). In the early placenta,the amount, of radioactivity in the precipitates was

zhiI-

z

0I-

2 4 6 8 10 12 14 16 18FRACTIONS

FIG. 3. SYNTHESIS OF PROTEINS BY TERMPLACENTAA: Electrophoretic pattern of human plasma.B: Total radioactivity of proteins in the incubating

medium.C: Pattern of proteins in incubation medium.

about the same -as that obtained with guinea pigalbumin, and probably represented non-specificabsorption. However, in term placentas there wassignificantly greater radioactivity in the precipi-tates prepared with plasma globulins (Table IV).

In earlier experiments using immunological iso-lation of proteins without preliminary electropho-retic separation (Table V), there is more radio-activity in the gamma globulin precipitate thanin the control precipitates. In the type of experi-ment presented in Table V A, the slices were ho-mogenized in the incubating medium and the im-munological precipitates made in the supernatant.In Table V B, the precipitates were made in the in-cubating medium without preliminary homogeniza-tion of the tissue. The reduction in non-specificadsorption is evident.

DISCUSSION

This investigation was originally undertakento determine if the placenta could synthesizeplasma proteins. The albumin and gammaglobu-lin fractions were first investigated because re-ports from other workers had suggested that theplacenta may synthesize these fractions. The

401

JOSEPH DANCIS, NANCYBRAVERMAN,AND JOHN LIND

TABLE IV

Incorporation of radioactivity into proteinsby placenta (counts per minute)

Early placenta* Late placenta*

Beta Alpha Beta AlphaProtein globulins globulins globulins globulins

Total protein 213 260 368 443Albumin (human) 7 7 0 25Alpha globulin 30 105Beta globulin 4 21 36 125Gammaglobulin 19 19 43 124Albumin (egg) 8 2Albumin (guinea pig) 18 14

* Early placenta was from a 3 to 4 months gestation;late placenta was at term. Carrier precipitates weremade by adding the plasma protein fraction and its specificantiserum to aliquots after electrophoretic separation.Total protein refers to TCA precipitable radioactivity.

technique described by Keston and Katchen (8)(see Procedure) seemed suitable, and in TableV A are presented the results of a typical experi-ment with term placenta. The amount of radio-activity incorporated into the gammaglobulin pre-cipitate appeared significant; however, the amountof radioactivity in the control precipitates was alsoappreciable. In a second series of experiments(Table V B), the specific precipitates were made inthe incubating medium without' preliminary ho-mogenization of the tissue, since it seemed reason-able that the plasma proteins would diffuse out ofthe cell. This reduced the amount of radioac-tivity brought down in the control precipitates,verifying the negative results with albumin andmaking more significant the results with gammaglobulin. Cross-reaction between bovine and hu-man gammaglobulin ( 11 ) probably accounted forthe intermediate amount of radioactivity in theformer precipitates. These results have beenpreviously presented (12).

The technique of electrophoretic separation fol-lowed by immunological precipitation was resortedto in an attempt to better identify the proteins.The data obtained with fetal liver and heart willbe discussed first, since they add to the interpreta-tion of the results with placenta.

The experiments with liver give clear cut evi-dence that the fetus is already capable of synthesiz-ing plasma proteins by 3 to 4 months gestation.The electrophoretic separations indicate that theliver is capable of synthesizing all of the electro-phoretically identifiable proteins except gammaglobulin. These results conform with those of

TABLE V

Incorporation of radioactivity into specific precipitatesby term placenta (counts per minute)

A* Bt

Egg albumin 75 6Humanalbumin 77 4Bovine gammaglobulin 115 30Humangammaglobulin 355 72

*A: The tissue slices were homogenized in the incu-bating medium and carrier precipitates prepared in thesupernatant.

t B: Carrier precipitates were made in the incubatingmedium without preliminary homogenization of tissues.

perfusion experiments with adult rat liver (13)and slice experiments with adult guinea pig liver(14). The immunological studies confirmed theidentity of the proteins and gave further informa-tion about sub-fractions of the beta globulins.

The gammaglobulin precipitates gave puzzlingresults. The radioactivity carried down in thealbumin fraction was probably the result of cross-reaction. Welater demonstrated extensive cross-reaction between our gamma globulin antiserumand albumin. However, the gammaglobulin pre-cipitates were just as effective as the /globulinprecipitates in bringing down radioactivity amongproteins with beta and alpha globulin mobility.This was also true in experiments with term pla-centa, as will be discussed below. It is well knownthat the plasma globulins are closely related im-munologically (15). The question may be raisedas to whether immunological differentiation is evenless distinct at this early stage of development.

The results with fetal heart are clearly differentfrom those obtained with liver, and conform withwhat might be expected from an organ that syn-thesizes proteins for its own use. The radioactiveproteins that have diffused into the medium aresmall in amount and the electrophoretic patternresembles that of the non-radioactive proteins inthe medium, probably representing tissue proteins.

Because of the small amount of tissue availablefrom fetal heart, and the low level of incorporationof radioactivity into proteins, there was insufficientmaterial after electrophoretic separation to permitthe preparation of immunological precipitates inthe various fractions. In the second experiment,ammonium sulfate, precipitation was employed toseparate roughly the proteins. The albumin andbeta globulin precipitates made in the supernatant,

402

PLASMA PROTEIN SYNTHESIS IN FETUS AND PLACENTA

after ammonium sulfate precipitation, had radio-activity. Since this fraction might be expected tocontain the albumin and a large proportion of thebeta globulins, and since there was also radioac-tivity in these fractions as determined electro-phoretically, this finding might have been con-sidered significant. However, a heterologousprecipitate indicated that the radioactivity was in-corporated by non-specific adsorption. The pro-portion of radioactivity brought down with theguinea pig albumin precipitate in this experimentmay be contrasted with that in the fetal liver(Tables II and III).

In the experiments with term placenta, electro-phoretic identification of the radioactive proteinsdemonstrated that the placenta did not synthesizeproteins with either albumin or gamma globulinmobility (Figure 3 B). The proteins moved intothe alpha and beta globulin area and appeared tobe related immunologically to all three globulinfractions in that each carrier precipitate broughtdown approximately the same amount of radio-activity (Table IV).

Particular pains were taken with the gammaglobulin precipitates because of the paradoxicalresults with electrophoretic separation. In a se-ries of experiments, different antigens and differ-ent antisera were used. In one experiment thegammaglobulin antigen was prepared by precipi-tating diphtheria toxoid with immune human se-rum, the precipitate being largely composed of thespecific anti-diphtheria gammaglobulin. The re-sults in all experiments were essentially the same.

It would be of interest to know if the globulinssynthesized by the placenta have functional simi-larities to plasma globulins. Good and Zak havepresented an interesting report (16) of a womanwith agammaglobulinemia who was immunizedrepeatedly during pregnancy. Antibodies weredetected in the mother in the last months of preg-nancy and circumstantial evidence indicated thatthe placenta might be the source of the antibodies.Since some antibodies have electrophoretic mo-bilities similar to those of the radioactive proteinsin our experiments with placenta (17), we madeattempts to demonstrate antibody synthesis usingthe Keston and Katchen technique with placentasfrom mothers immunized against tetanus anddiphtheria. The attempts were unsuccessful. Thisdoes not exclude the possibility that the placenta

synthesizes antibodies in amounts too small to bedetected by this technique, or that it may synthe-size antibodies under abnormal conditions (forexample, agammaglobulinemia in the mother).

It is not likely that under normal circumstancesthe placenta contributes significantly to the plasmaproteins of the fetus. The placenta from preg-nancies of 3 to 4 months duration does not synthe-size any plasma proteins detectable by this tech-nique (Table IV). By this stage of pregnancy,the fetal liver is already actively synthesizing allplasma protein fractions except the gammaglobu-lin. The gamma globulins as identified electro-phoretically are probably supplied by the mother.

SUMMARY

1. Liver slices from human fetuses of 3 to 4months gestation were incubated with glycine-2-C14. The proteins were separated electropho-retically and immunologically, and the incorpora-tion of radioactivity was determined. It was con-cluded that the liver at this stage of development isalready capable of synthesizing plasma proteins,exclusive of gammaglobulin.

2. Similar studies were conducted using humanfetal heart for purposes of comparison. The dif-ferences were striking and conform with the pic-ture of an organ synthesizing proteins for its ownuse.

3. Experiments with human placenta from preg-nancies of 3 to 4 months duration have demon-strated that the placenta will incorporate glycine-2-C14 into proteins with alpha and beta globulinmobilities. These proteins are immunologicallyunrelated to plasma proteins.

4. Similar experiments with term placentas re-vealed incorporation of radioactivity into proteinswith similar electrophoretic mobility. However,these proteins are related immunologically toplasma globulins.

5. At neither stage of pregnancy does the pla-centa synthesize proteins that are electrophoreti-cally identifiable as albumin or gammaglobulin.

6. It is concluded that under normal circum-stances the placenta does not contribute signifi-cantly to the plasma proteins of the fetus. Begin-ning early in gestation, the fetal liver is capableof synthesizing all plasma proteins except gammaglobulin. The gamma globulin is probably de-rived from the mother.

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JOSEPH DANCIS, NANCYBRAVERMAN,AND JOHN LIND

ACKNOWLEDGMENT

We are indebted to Dr. A. M. Pappenheimer, Jr. ofthe Department of Immunology and Dr. Milton Levy ofthe Department of Biochemistry for many helpful dis-cussions. Dr. Bernard Katchen assisted with the pre-liminary experiments.

REFERENCES

1. Moore, D. H., DuPan, R. M., and Buxton, C. L., Anelectrophoretic study of maternal, fetal, and infantsera. Am. J. Obst. & Gynec., 1949, 57, 312.

2. Orlandini, T. O., Sass-Kortsak, A., and Ebbs, J. H.,Serum gamma globulin levels in normal infants.Pediatrics, 1955, 16, 575.

3. Osborn, J. J., Dancis, J., and Rosenberg, B. V., Stud-ies of the immunology of the newborn infant. III.Permeability of the placenta to maternal antibodyduring fetal life. Pediatrics, 1952, 10, 450.

4. Ewerbeck, H., von, and Levens, H. E., tUber diebildung der Serumeiweisskorper in der Placentasowie ihren beitrag am Fettstoffwechsel des Feten.Ztschr. f. Kinderh., 1951, 99, 297.

5. Umbreit, W. W., Burris, R. H., and Stauffer, J. F.,Manometric Techniques and Tissue Metabolism.Minneapolis, Burgess Publishing Co., 1949.

6. Kunkel, H. G., and Slater, R. J., Zone electrophore-sis in a starch supporting medium. Proc. Soc.Exper. Biol. & Med., 1952, 80, 42.

7. Eisen, H. N., Carsten, M. E., and Belman, S., Stud-ies of hypersensitivity to low molecular weightsubstances. III. The 2,4-dinitrophenyl group as a

determinant in the precipitin reaction. J. Im-munol., 1954, 73, 296.

8. Keston, A. S., and Katchen, B., Incorporation of gly-cine-2-C14 into homologous antibody by rabbittissue slices. J. Immunol., 1956, 76, 253.

9. Freund, J., and Bonanto, M. V., The effect of paraffinoil, lanolin-like substances and killed tuberclebacilli on immunization with diphtheric toxoid andBact. typhosum. J. Immunol., 1944, 48, 325.

10. Korngold, L., and Lipari, R., Immunochemical stud-ies of human plasma beta lipoprotein. Science,1955, 121, 170.

11. Maurer, P. H., The cross reactions between albuminsof different species and gamma globulins of differ-ent species. J. Immunol., 1954, 72, 119.

12. Dancis, J., Braverman, N., and Katchen, B., Thesynthesis of serum proteins by human placenta.Am. J. Dis. Child., 1955, 90, 558.

13. Miller, L. L., and Bale, W. F., Synthesis of all plasmaprotein fractions except gamma globulins by liver.The use of zone electrophoresis and lysine-E-C'4 todefine the plasma proteins synthesized by the iso-lated perfused liver. J. Exper. Med., 1954, 99,125.

14. Dancis, J., and Braverman, N., Unpublished data.15. Cohn, M., Deutsch, H. F., and Wetter, L. R., Bio-

physical studies of blood plasma proteins. XIII.Analysis of immunological heterogeneity of humangamma globulin fractions. J. Immunol., 1950, 64,381.

16. Good, R. A., and Zak, S. J., Disturbances in gammaglobulin synthesis as "experiments of nature."Pediatrics, 1956, 18, 109.

17. Slater, R. J., The serum gamma-globulins defined byelectrophoretic and immunologic analyses. Arch.Biochem. & Biophys., 1955, 59, 33.

18. Widdas, W. F., Some aspects of foetal and placentalphysiology. ttudes Neonatales, 1954, III, 165.

Taken from data by Friedenthal, H., Allgemeine undspezielle Physiologie des Menschenwachstums furAnthropologen, Physiologen, Anatomen, und Arztedargestellt. Berlin, Springer, 1914.

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