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Conjugated estrogens in human pregnancy serum
CONJUGATED ESTROGUS IN HUM.A.N PRBGNANCY SBBUM* A. E.
RAKOFF .•
M.D., K. E.
PASCRKIS,
1\i.D., t
AND
A
0ANTAROW,
M.D.
PHILADELPHIA, PA.
(From the Depad·tmentc of Obstetric,s, Gynecology, Physiology and Medicine of fhf· Jejfers&n Medical College anrl Hospital J
well known that the estrogens in the urine of pregnant women ITareIS present almost entirely in the conjugated state. It lws been estimated1 that in the latter months of pregnancy np to 98 per cent of the urinary estrogens are in a conjugated form from which they Iilay be freed by acid hydrolysis. In the bile of pregnant women the estrogens appear to be present almost entirely as free hormone. 2 That free estrogens are present in high concentration in the serum during the last trimester of gestation is also well known; there are few data available, however, to indicate whether the estrogenic poteney of human pregnane.v blood or serum can be increased by acid hydrolysis or other methods fOl' demonstrating conjugated estrogens. The few data which are available are conflicting. The chemical nature of the estrogens in human pregnancy blood is not known and there is even a paueity of information concerning its distribution in the various components of the blood. Albrieux 3 • 4 showed that the estrogens in human pregnanq blood are equally distributed between plasma and cells, although in nonpregnant patients the cells contain twice as much estrogenic hormones as plasma per unit volume. Numerous observations which >ve have accumulated in our laboratory also indicate that the concentration of free estrogens in serum and whole blood of pregnaney are identic•al. Muhlbock" first demonstrated that a portion of the estrogen in the blood of the pregnant mare is in a combined form. Later this investigator6 reported similar findings for human pregnancy blood. He examined two specimens of placental blood obtained at the time of df•livery, two specimens from patients \Vith late toxemia of pregnancy, and one specimen from a post-partum eclamptic patient. The blood was collected in a bottle containing a}(;ohol, the latter was evaporated, and the residue extracted directly by warming with benzene, first in a neutral state to remove the .free estrogens and then after hydrolyzing twi<'c fot 6 hours with 4 per cent HCl to liherat<:' tlw (•onjugated hormone. In a preliminary report' we presented data demonstrating the presence of conjugated estrogens in serum of pregnant women. Our studies suggested that the estrogens in pregnancy serum were present in the protein fraction and were in part rather firmly ··bound'' to the protein. On the other hand, Goldberger and Frank 8 have recently reported that their attempts to increase the estrogen yield of pregnancy blood hy *Aided by a grant from the Johnson Research Fo,undat!on, New Brunswick, N. ,J. tJ. E. Mears Fellow in Physiology and Medicine.
856
ltAKOifF E'l' AL.:
ESTROGENS IN HUMAN PREGNANCY SERLTl\I
857
hydrolysis were unsuccessful. These workers used citrated blood which was laked with distilled water and hydrolyzed at pH 2.0 by boiling for 15 minutes.
Material and Methods mood for these studies was collected from normal, healthy pregnant women during the last two months of gestation. The blood was allowed to clot and the serum was separated. In each case a portion of serum was assayed directly for free estrogen. The remainder was subjec~ed to various procedures of extraction and hydrolysis. The followmg t~chniques were found to yield the best results : 1'he serum was acidified to pH 1.0 with concentrated hydrochloric acid and was heated in a boiling water bath fot· six hours. The gelatinous mass was then extracted three times by shaking; for one-half hour with four volumes of an equal mixture of ether and alcohol. The etheralcohol fractions were pooled, emporated to dryness, and taken up in corn oil to a fixed volume for biologic a.<;say. The remaining precipitate was dried and powdered; a portion of it was suspended in water and assayed similarly to determine the free hormone content of the residue. The remainder of the residue was subjected to further hydrolysis. Water was added, the pH adjusted to 1.0 and the suspension heated for six hours in the water bath, after which it was extracted with ether and ~lcohol and finally taken up in corn oil for biologic assay. In two of the experiments, the hydrolysis was carried out at pH 0.5; otherwise the technique was the same as above. Preliminary experiments showed that ether-alcohol extracts of the serum without hydrolysis had the same estrogen content as whole serum, as has also been noted by Goldberger and Frank. 8 In order, to study further the partition of estrogens in the serum, protein-free filtrates were prepared by several methods: (1) salting out with sodium sulfate ; ( 2) precipitation with sulfosalicylic acid; (3) ultrafiltration through collodion membranes. The protein fraction was extracted with ether-alcohol for free estrogens. Portions of the proteinfree filtrate and protein residues were hydrolyzed for conjugated estrogens and then re-extracted. In order to determine whether splitting of the protein by other means would increase tke estrogen yield, two specimens were subjected to tryptic digestion as follows : one part of serum was mixed with one part of 10 per cent trypsin in a buffer solution (pH 8.7) and incubated :for 16 hours. This resulted in a digestion of approximately 70 per cent of the protein. The digested material was then repeatedly extracted with ether-alcohol aHd assayed for estrogen. All specimens were assayed on adult mice of uniform size and age, seven days after castration. Pour to six dosage levels were employed with each specimen and three or more animals were injected at each dosage level. The material was injected in divided doses over a threeday period. At the end of 96 hours the animals were killed, the vaginas removed and sectioned and read according to the classification of Fluhmann.9
Results The estrogen content of the various sera and the extracts after the hydrolysis procedures are given in Table I.
bef~re
and
858
AMERICAN JOURNAL OF OBSTETRICS AND GY:t.'"ECOLOGY TABLE
I.
FREE AND COMBINED ESTROGEN IN SERUM 0~' LATE PREGNANCY RESIDUE F'R0:\1 HYDROLYSIS
80 100 100 100 90 100 120
(7)
(8)
108 120
13;} 10 100 5 110 4 100 10 Hydrolysis at pH 0.5 160 traces 200 traces After Tryptic Digestion 104 100
(j()
80 80 20 10
180
:no
44.4 42.8
108 120
0
0
It will be observed that a small increase (up to 35 per cent) occurred in several of the sera after the initial hydrolysis at pH 1.0. However, the residue from these extracts still contained some estrogenic activity, as revealed by further hydrolysis. :Moreover, small amounts could be demonstrated in water suspensions of the residue, suggesting that after initial hydrolysis a small percentage of the estrogens is ether-alcohol insoluble but water soluble. Further hydrolysis of the protein fraction resulted in considerable increase in estrogenic potency, the liberated hormone being ether-alcohol soluble. Assays of water suspensions of the residue or extracts of a third hydrolysis revealed no further increase. The specimens which were hydrolyzed at pH 0.5 gave an increase of from 60 to 67 per cent of the free estrogenic activity after the first hydrolysis. Further extraction and hydrolysis, however, yielded only a very small increment so that the total percentage of combined estrogen was of the same magnitude as obtained with the less vigorous hydrolysis. Digestion of the protein with trypsin failed materially to alter the estrogen content of the serum as shown in Table I. No estrogenic activity could be demonstrated in 8 of 9 protein-free filtrates prepared by three different methods (Table II). In one i:nstan<:e, 20 per cent of the free estrogen content was present. Hydrolysis of this fraction failed to increase the estrogenic activity. TABLE II.
ESTROGEN IN PR0TEIN·FREE FRACTION
METHOD
Sodium
BEFORE HYDROLYSIS
3 2
0 to 20
eent
AFTER HYDROLYSIS
0 0
RAKOFF ET AL. :
ESTROGENS IN HUMAN PREGNANCY SERUM
859
The protein-containing residues were extracted repeatedly with etheralcohol and yielded from 75 to 100 per cent of the free estrogenic activity. In most instances small amounts (up to 20 per cent of the free estrogenic activity) could be obtained by further extraction of the residue with water. In a number of instances, portions of this residue were subjected to further hydrolysis and yielded additional small amounts of estrogens (10 to 16 per cent of the free estrogenic activity) when extracted with ether-alcohol. Further water extraction did not increase the yield. Discussion Our results and those of Miihlbock6 indicate that combined estrogens are present in human serum of late pregnancy. Althougq the proportion of combined estrogens in the serum is not nearly as great as in the urine of such patients, as much as 50 per cent of the total estrogenic content of the serum may be present in combined or conjugated form. Rather vigorous and prolonged hydrolysis, however, is required to free the combined hormone. At a pH of 1.0 it is necessary to hydrolyze and extract twice for six hours. At a pH of 0.5 a single hydrolysis for six hours frees most of the conjugated hormone. Preliminary studies in our laboratory showed that hydrolysis for less than two hours did not result in an appreciable increase of the estrogenic content. This probably explains the negative findings of Goldberger and Frank. 8 These data indicate that the conjugated estrogens in the serum are much more resistant to hydrolysis than those in the urine, although the studies of Van Bruggen10 and of the Smiths11 suggest that under certain conditions more vigorous hydrolysis procedures will markedly increase the estrogen content of the latter also. The estrogens of pregnancy serum appear to be intimately "bound" to the protein fraction, as indicated by the fact that they do not pass through a collodion membrane, that they are precipitated with the protein fraction, that they cannot be removed completely from the latter by ether-alcohol extraction, and that vigorous and prolonged hydrolysis is necessary to liberate them completely from the latter. Tryptic digestion does not liberate the combined estrogens, suggesting that the phenomenon is not one of mere adsorption of the hormone by the protein. It is possible, however, that further digestion with other enzymes may yield different results.
Summary 1. Conjugated estrogens have been demonstrated in the serum of late pregnancy. In eight specimens examined the amount of combined hor~ mone ranged from 23 to 50 per cent of the total estrogen content. 2. Vigorous and prolonged hydrolysis is required to free the combined hormone. For complete recovery it is necessary to hydrolyze and extract twice at pH 1.0 for 6 hours; while at pH 0.5 a single hydrolysis for 6
860
.\MERICA::>; .JOI:RXAL OF OBKTETRICR AXIl GY"lF:COLOGY
hours frees most. of the conjugated hormone. Hydrolysii; fo1· lt~ss 1han 2 hours did not: result. in an appref•iable inc·rease in the Pstrogl'tl <~onh"llL 3. The free estrogen !'an he removt-d from the se!'um by extrncting with ether-alcohol. The combined hormone ean then be recovered from the residue after hydrolysis. 4. Protein-free filtrates of pregnane~' serum are prafcticall;T free of any estrogenic content, whil~e almost all of the hormone ean be recovered from the protein fraction. G. The estrogms of pregnancy serum do not pass through a 1•ollodion membrane. 6. The combinell estrogm1s of pregnanry serum are not freed hy tryptic digestion. 7. The above observations indirate that thP estrogens of preg-nnney semm nre intimately "bound" to thC' protein fraction.
References 1. Dingemanse, }J., Laquer, E., and Miihlhork, 0.: 1\fonatsehr. f. Gehurtsh. 11. Gyniik. 109: 37, 1939. 2. Canta:row, A., Rakoff; A. E., Pa;;chki~, K. E., Hansen, L. P., :md Walkling, A. A.: Proe. Soc. Exper. Bioi. & Med. 52: 256, 1943. 3. Albrieux, A. S.: Proc. S.Oe. Exper. Bioi. & Med. 47: il8l, Hl±1. 4. Albrieu:x, A. R: J. Clin. Endocrinol. 1: 983, 194 L 5. Miihlbock, 0.: Zeitsehr. f. physiol. Chern. 250: B~i, 1937. 6. Miihlbock, 0.: Lancet 1: 634, 19:~0. i. Rakoff, A. E., Paschki~, K., ani! (\mt!uow, A.: I<'ederation Proreeding.9 1: 1942. 8. Goldberger, l\f. A., and :E'rank, R. T.: A )cr. .J. OB!!:'f. & GYKEC. 4:3: 865, l!lc!2. fl. }'Iuhmann, C. P.: Endocrinology 18: 705, 1934. 10. Van Bruggen . •T. '1'. (Quoted hy Doisy, }J. A.): Biological Symposia 9: :n, 1942. 11. Smith, 0. W .. Smith, G. R., and Sf' hiller, S.: Endocrinolog·y 25: :iOO, 193!!,
RAKOFF .•
M.D., K. E.
PASCRKIS,
1\i.D., t
AND
A
0ANTAROW,
M.D.
PHILADELPHIA, PA.
(From the Depad·tmentc of Obstetric,s, Gynecology, Physiology and Medicine of fhf· Jejfers&n Medical College anrl Hospital J
well known that the estrogens in the urine of pregnant women ITareIS present almost entirely in the conjugated state. It lws been estimated1 that in the latter months of pregnancy np to 98 per cent of the urinary estrogens are in a conjugated form from which they Iilay be freed by acid hydrolysis. In the bile of pregnant women the estrogens appear to be present almost entirely as free hormone. 2 That free estrogens are present in high concentration in the serum during the last trimester of gestation is also well known; there are few data available, however, to indicate whether the estrogenic poteney of human pregnane.v blood or serum can be increased by acid hydrolysis or other methods fOl' demonstrating conjugated estrogens. The few data which are available are conflicting. The chemical nature of the estrogens in human pregnancy blood is not known and there is even a paueity of information concerning its distribution in the various components of the blood. Albrieux 3 • 4 showed that the estrogens in human pregnanq blood are equally distributed between plasma and cells, although in nonpregnant patients the cells contain twice as much estrogenic hormones as plasma per unit volume. Numerous observations which >ve have accumulated in our laboratory also indicate that the concentration of free estrogens in serum and whole blood of pregnaney are identic•al. Muhlbock" first demonstrated that a portion of the estrogen in the blood of the pregnant mare is in a combined form. Later this investigator6 reported similar findings for human pregnancy blood. He examined two specimens of placental blood obtained at the time of df•livery, two specimens from patients \Vith late toxemia of pregnancy, and one specimen from a post-partum eclamptic patient. The blood was collected in a bottle containing a}(;ohol, the latter was evaporated, and the residue extracted directly by warming with benzene, first in a neutral state to remove the .free estrogens and then after hydrolyzing twi<'c fot 6 hours with 4 per cent HCl to liherat<:' tlw (•onjugated hormone. In a preliminary report' we presented data demonstrating the presence of conjugated estrogens in serum of pregnant women. Our studies suggested that the estrogens in pregnancy serum were present in the protein fraction and were in part rather firmly ··bound'' to the protein. On the other hand, Goldberger and Frank 8 have recently reported that their attempts to increase the estrogen yield of pregnancy blood hy *Aided by a grant from the Johnson Research Fo,undat!on, New Brunswick, N. ,J. tJ. E. Mears Fellow in Physiology and Medicine.
856
ltAKOifF E'l' AL.:
ESTROGENS IN HUMAN PREGNANCY SERLTl\I
857
hydrolysis were unsuccessful. These workers used citrated blood which was laked with distilled water and hydrolyzed at pH 2.0 by boiling for 15 minutes.
Material and Methods mood for these studies was collected from normal, healthy pregnant women during the last two months of gestation. The blood was allowed to clot and the serum was separated. In each case a portion of serum was assayed directly for free estrogen. The remainder was subjec~ed to various procedures of extraction and hydrolysis. The followmg t~chniques were found to yield the best results : 1'he serum was acidified to pH 1.0 with concentrated hydrochloric acid and was heated in a boiling water bath fot· six hours. The gelatinous mass was then extracted three times by shaking; for one-half hour with four volumes of an equal mixture of ether and alcohol. The etheralcohol fractions were pooled, emporated to dryness, and taken up in corn oil to a fixed volume for biologic a.<;say. The remaining precipitate was dried and powdered; a portion of it was suspended in water and assayed similarly to determine the free hormone content of the residue. The remainder of the residue was subjected to further hydrolysis. Water was added, the pH adjusted to 1.0 and the suspension heated for six hours in the water bath, after which it was extracted with ether and ~lcohol and finally taken up in corn oil for biologic assay. In two of the experiments, the hydrolysis was carried out at pH 0.5; otherwise the technique was the same as above. Preliminary experiments showed that ether-alcohol extracts of the serum without hydrolysis had the same estrogen content as whole serum, as has also been noted by Goldberger and Frank. 8 In order, to study further the partition of estrogens in the serum, protein-free filtrates were prepared by several methods: (1) salting out with sodium sulfate ; ( 2) precipitation with sulfosalicylic acid; (3) ultrafiltration through collodion membranes. The protein fraction was extracted with ether-alcohol for free estrogens. Portions of the proteinfree filtrate and protein residues were hydrolyzed for conjugated estrogens and then re-extracted. In order to determine whether splitting of the protein by other means would increase tke estrogen yield, two specimens were subjected to tryptic digestion as follows : one part of serum was mixed with one part of 10 per cent trypsin in a buffer solution (pH 8.7) and incubated :for 16 hours. This resulted in a digestion of approximately 70 per cent of the protein. The digested material was then repeatedly extracted with ether-alcohol aHd assayed for estrogen. All specimens were assayed on adult mice of uniform size and age, seven days after castration. Pour to six dosage levels were employed with each specimen and three or more animals were injected at each dosage level. The material was injected in divided doses over a threeday period. At the end of 96 hours the animals were killed, the vaginas removed and sectioned and read according to the classification of Fluhmann.9
Results The estrogen content of the various sera and the extracts after the hydrolysis procedures are given in Table I.
bef~re
and
858
AMERICAN JOURNAL OF OBSTETRICS AND GY:t.'"ECOLOGY TABLE
I.
FREE AND COMBINED ESTROGEN IN SERUM 0~' LATE PREGNANCY RESIDUE F'R0:\1 HYDROLYSIS
80 100 100 100 90 100 120
(7)
(8)
108 120
13;} 10 100 5 110 4 100 10 Hydrolysis at pH 0.5 160 traces 200 traces After Tryptic Digestion 104 100
(j()
80 80 20 10
180
:no
44.4 42.8
108 120
0
0
It will be observed that a small increase (up to 35 per cent) occurred in several of the sera after the initial hydrolysis at pH 1.0. However, the residue from these extracts still contained some estrogenic activity, as revealed by further hydrolysis. :Moreover, small amounts could be demonstrated in water suspensions of the residue, suggesting that after initial hydrolysis a small percentage of the estrogens is ether-alcohol insoluble but water soluble. Further hydrolysis of the protein fraction resulted in considerable increase in estrogenic potency, the liberated hormone being ether-alcohol soluble. Assays of water suspensions of the residue or extracts of a third hydrolysis revealed no further increase. The specimens which were hydrolyzed at pH 0.5 gave an increase of from 60 to 67 per cent of the free estrogenic activity after the first hydrolysis. Further extraction and hydrolysis, however, yielded only a very small increment so that the total percentage of combined estrogen was of the same magnitude as obtained with the less vigorous hydrolysis. Digestion of the protein with trypsin failed materially to alter the estrogen content of the serum as shown in Table I. No estrogenic activity could be demonstrated in 8 of 9 protein-free filtrates prepared by three different methods (Table II). In one i:nstan<:e, 20 per cent of the free estrogen content was present. Hydrolysis of this fraction failed to increase the estrogenic activity. TABLE II.
ESTROGEN IN PR0TEIN·FREE FRACTION
METHOD
Sodium
BEFORE HYDROLYSIS
3 2
0 to 20
eent
AFTER HYDROLYSIS
0 0
RAKOFF ET AL. :
ESTROGENS IN HUMAN PREGNANCY SERUM
859
The protein-containing residues were extracted repeatedly with etheralcohol and yielded from 75 to 100 per cent of the free estrogenic activity. In most instances small amounts (up to 20 per cent of the free estrogenic activity) could be obtained by further extraction of the residue with water. In a number of instances, portions of this residue were subjected to further hydrolysis and yielded additional small amounts of estrogens (10 to 16 per cent of the free estrogenic activity) when extracted with ether-alcohol. Further water extraction did not increase the yield. Discussion Our results and those of Miihlbock6 indicate that combined estrogens are present in human serum of late pregnancy. Althougq the proportion of combined estrogens in the serum is not nearly as great as in the urine of such patients, as much as 50 per cent of the total estrogenic content of the serum may be present in combined or conjugated form. Rather vigorous and prolonged hydrolysis, however, is required to free the combined hormone. At a pH of 1.0 it is necessary to hydrolyze and extract twice for six hours. At a pH of 0.5 a single hydrolysis for six hours frees most of the conjugated hormone. Preliminary studies in our laboratory showed that hydrolysis for less than two hours did not result in an appreciable increase of the estrogenic content. This probably explains the negative findings of Goldberger and Frank. 8 These data indicate that the conjugated estrogens in the serum are much more resistant to hydrolysis than those in the urine, although the studies of Van Bruggen10 and of the Smiths11 suggest that under certain conditions more vigorous hydrolysis procedures will markedly increase the estrogen content of the latter also. The estrogens of pregnancy serum appear to be intimately "bound" to the protein fraction, as indicated by the fact that they do not pass through a collodion membrane, that they are precipitated with the protein fraction, that they cannot be removed completely from the latter by ether-alcohol extraction, and that vigorous and prolonged hydrolysis is necessary to liberate them completely from the latter. Tryptic digestion does not liberate the combined estrogens, suggesting that the phenomenon is not one of mere adsorption of the hormone by the protein. It is possible, however, that further digestion with other enzymes may yield different results.
Summary 1. Conjugated estrogens have been demonstrated in the serum of late pregnancy. In eight specimens examined the amount of combined hor~ mone ranged from 23 to 50 per cent of the total estrogen content. 2. Vigorous and prolonged hydrolysis is required to free the combined hormone. For complete recovery it is necessary to hydrolyze and extract twice at pH 1.0 for 6 hours; while at pH 0.5 a single hydrolysis for 6
860
.\MERICA::>; .JOI:RXAL OF OBKTETRICR AXIl GY"lF:COLOGY
hours frees most. of the conjugated hormone. Hydrolysii; fo1· lt~ss 1han 2 hours did not: result. in an appref•iable inc·rease in the Pstrogl'tl <~onh"llL 3. The free estrogen !'an he removt-d from the se!'um by extrncting with ether-alcohol. The combined hormone ean then be recovered from the residue after hydrolysis. 4. Protein-free filtrates of pregnane~' serum are prafcticall;T free of any estrogenic content, whil~e almost all of the hormone ean be recovered from the protein fraction. G. The estrogms of pregnancy serum do not pass through a 1•ollodion membrane. 6. The combinell estrogm1s of pregnanry serum are not freed hy tryptic digestion. 7. The above observations indirate that thP estrogens of preg-nnney semm nre intimately "bound" to thC' protein fraction.
References 1. Dingemanse, }J., Laquer, E., and Miihlhork, 0.: 1\fonatsehr. f. Gehurtsh. 11. Gyniik. 109: 37, 1939. 2. Canta:row, A., Rakoff; A. E., Pa;;chki~, K. E., Hansen, L. P., :md Walkling, A. A.: Proe. Soc. Exper. Bioi. & Med. 52: 256, 1943. 3. Albrieux, A. S.: Proc. S.Oe. Exper. Bioi. & Med. 47: il8l, Hl±1. 4. Albrieu:x, A. R: J. Clin. Endocrinol. 1: 983, 194 L 5. Miihlbock, 0.: Zeitsehr. f. physiol. Chern. 250: B~i, 1937. 6. Miihlbock, 0.: Lancet 1: 634, 19:~0. i. Rakoff, A. E., Paschki~, K., ani! (\mt!uow, A.: I<'ederation Proreeding.9 1: 1942. 8. Goldberger, l\f. A., and :E'rank, R. T.: A )cr. .J. OB!!:'f. & GYKEC. 4:3: 865, l!lc!2. fl. }'Iuhmann, C. P.: Endocrinology 18: 705, 1934. 10. Van Bruggen . •T. '1'. (Quoted hy Doisy, }J. A.): Biological Symposia 9: :n, 1942. 11. Smith, 0. W .. Smith, G. R., and Sf' hiller, S.: Endocrinolog·y 25: :iOO, 193!!,