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Source of prostaglandin precursor in human fetal membranes: Arachidonic acid content of amnion and chorion laeve in diamnionic-dichorionic twin placentas
American Journal of
Obstetrics and Gynecology Founded volume
147
number
5
NOVEMBER
in
1920
1,1983
Papers of the Society for Gynecologic Investigation
Source of prostaglandin precursor in human fetal membranes: Arachidonic acid content of amnion and chorion laeve in diamnionic-dichorionic twin placentas Janice
R. Okita,*
Da&s,
Texas
John M. Johnston,
and Paul C. MacDonald
The lipids of the avascular human amnion and chorion laeve are known to be enriched in the essential fatty acid arachidonic acid, the obligate precursor of prostaglandins of the 2-series. The present investigation was conducted to evaluate the source of arachidonic acid that is present in human fetal membranes. To do so we determined the arachidonic acid content of the lipid fractions of amnion and chorion laeve tissues from diamnionic-dichorionic twin placentas. We found that the arachidonic acid content of amnion from twin placentas was similar to that of amnion from singleton placentas irrespective of the site of amnion sampling. However, the same was not true of chorion laeve of diamnionic-dichorionic twin placentas. The arachidonic acid content of chorion laeve that was contiguous to the chorion laeve of its twin was strikingly reduced compared to that of chorion laeve contiguous to decidua vera obtained from either singleton or twin placentas. We conclude that the arachidonic acid in amnion is derived primarily from essential fatty acids in the amniotic fluid. However, the arachidonic acid in chorion laeve may be derived from essential fatty acid in the amniotic fluid as well as in maternal plasma by way of the decidua Vera. (AM. J. OBSTET. GYNECOL. 147:477,
1983.)
From the Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Biochemistry and ObstetricsGynecology, The University of Texas Southwestenz Medical School at Dallas. This investigation was supported in part by United States Public Health Service Grant No. 5-P50-HD11149 and a Grant-in-Aid from The Robert A. Welch Foundation, Howton, Texas. Sponsored by the Society for Gynecologic Investigation. Reprint requests: John M. Johnston, Ph.D., Department of Biochemistry, The CiniversiQ of Texas Health Science Center, 5323 Har/y Hines Boulevard, Dallas, Texas 75235. *Dr. Okita was a postdoctoral trainee supported by a Grant-in-Aid from The Robert A. Welch Foundation, Houston, Texas.
It is believed that prostaglandins (PGs) serve an important role in the initiation and maintenance of human parturition. Also, there are a number of lines of evidence in support of the proposition that metabolic events that take place in amnion and cborion laeve are crucial to the generation of these prostaglandins; indeed, the following considerations seem to be pertinent to the role of the fetal membranes in human parturition: (1) The specific activity of prostaglandin synthase in amnion is greater than that in chorion laeve, decidua Vera, myometrium, or placenta.‘-” (2) The glycero-
477
478
Okita,
Johnston,
and MacDonald
plac‘enta5
Fig. I. The diamnionic-dichorionic
November Am. J. Obstet.
decidua
basalis
twin placenta
phospholipids of amnion and chorion laeve are enriched with arachidonic acid, the obligate precursor of prostaglandins of the S-series.” (3) The levels of free arachidonic acid as well as those of PGE, and PGF,, in amniotic fiuid increase during labor and as labor progresses.“-’ (4) During early labor there is a specific decrease in the arachidonic acid content of diacyl phosphatidylethanolamine and phosphatidylinositol in the amnion and chorion laeve.x (5) A phospholipase A, with relative substrate specificity for phusphatidylethanolamines with arachidonic acid in the sn-2 position is present in fetal membranes.!’ (6) Phosphatidylinositolspecific phospholipase C activity is present in human amnion and chorion laeve”): this enzyme catalyzes the hydrolysis of phosphatidylinositol to diacylglycerols. In turn there is a diacylglycerol lipase in amnion and chorion laeve that catalyzes the release of the f‘atty acid from the .rn- 1 position of diacylglycerols,” and this enzyme may be relatively specific for diacylglycerols with arachidonic acid in the sn-2 position.” In turn, there is a monoacylglycerol lipase in amnion and chorion laeve that catalyzes the release of the fatty acid in the ~71-2 position of’ monoacylglycet-ols.” There may be relative substrate specificity of this enzyme for sn-2 arachidonoylmonoacylglycerols. In this coordinated manner, by three enzymatic reactions, arachidonic acid is released from phosphatidylinositol in the fetal membranes. (7) The specific activities of phospholipases A, and C increase strikingly in human amnion late in gestation.‘” (8) Diacylglycerols, the products of the reaction catalyzed by phosphatidylinositol-specific phospholipase C, accumulate in amnion during early labor.13 (9) Nicotinamide adenine dinucleotide+-dependent 15-hydroxyprostaglandin dehydrogenase activity is not detectable in human amnion’; this enzyme catalyzes the first reaction in the inactivation of prostaglandins.‘” (I 01 Infection or hypoxia of the fetal membranes, as well as exposure to hypertonic solutions of sodium chloride, urea, or glucose, causes premature labor.‘j Therefore, an important question arises: What is the
1, I983 Gynecol.
source of the arachidonic acid that is present in amnion and chorion laeve? The crucial nature of this question is apparent when one considers that (1) human amnion and chorion laeve are avascular tissues and (2) arachidonic acid is an essential fatty acid. To address this issue, the arachidonic acid content of amnion and chorion laeve of diamnionic-dichorionic human twin placentas was determined. In such placentas there is a considerable portion of the chorion laeve tissue of each fetus that is not contiguous with decidua vera tissue but rather is contiguous with the chorion laeve tissue of its twin. Thus, in this portion of the fetal membranes neither amnion nor chorion laeve is in contact with maternal plasma, as illustrated diagramatically in Fig. I. The purpose of this study was to evaluate the roles of amniotic fluid and decidua vera (maternal plasma) as sources of the arachidonic acid in the fetal membranes.
Material and methods Diamnionic-dichorionic twin placentas were obtained immediately after cesarean section conducted either prior to or during labor and after spontaneous vaginal delivery. The tissues were immediately placed on ice and transported to the laboratory. Tissue dissections were conducted with placentas and fetal men]branes on a sheet of glass supported by ice. Sections of amnion and chorion laeve frotn each twin were taken for analysis from that portion of the membranes in which the chorion laeve was fused with the chorion laeve of its twin (hereafter referred to as the fused area) and that portion of the membranes in which the chorion laeve was contiguous to decidua vera (i.e., the nonfused area) (see Fig. 1). To separate the tissues, the amnion was first peeled away from the chorion laeve. In sections of chorion laeve contiguous to decidua Vera, the decidua vera was removed by sharp dissection by use of a glass slide as previously described.” Tissues.
Determination lipid
fractions
of of
the
amnion
arachidonic and
chorion
acid
content
laeve
of
tissues.
The methods used have been described in detail.’ Briefly, the tissues were homogenized by use of a Polytron PI 0 homogenizer (Brinkman Instruments, Inc., Westbury, New York). Lipids were extracted with chloroform: methanol (2: 1, v: v) by a modification of the extraction procedure of Folch and co-workers.‘” The extracts were filtered and washed extensively with the preequilibrated aqueous phase; the organic solvent was evaporated under a stream of nitrogen at 37” C. The residues were dissolved in chloroform : methanol (2 : I, v : v) and stored at -20” C. The glycerophospholipids were separated by two-dimensional thinlayer chromatography (TLC) as previously described.’ The fatty acid composition of the glycerophospholipids was determined by gas-liquid chromatographic analysis and quantification of methyl ester derivatives of- the
Volume Number
Prostaglandin precursor in fetal membranes
147 5
D *0.05
-
m
Singletan
q
Tui;n;\aantas
El
Twin Placsntar-Fund (n=4)
Placentor
(n=V)
- Nonfused
Area AW
&SEM by pind
f test
analysis
I-
)-
)-
PLASM PE
DIACYL PE
PI
PC
PS
NL
Fig. 2. Arachidonic acid content of lipid fractions from amnion. Arachidonic acid content is expressed as the percentage of total fatty acyl groups in each fraction. For the PLASM PE fraction, arachidonic acid is expressed as the percentage of total fatty acyl plus alkenyl groups. The p value illustrated is from the comparison of data for the fused and nonfused areas of twin placentas by paired t test analysis. The data from singleton placentas were compared with those from either fused or nonfused areas of twin placentas by t test analysis. No statistically significant differences were found. PLASM PE, Plasmalogen phosphatidylethanolamine; DIACYL PE, diacr 1 phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine; PS, phosphatidylserine; NL, total neutral lipid l-
a
q q
l-
Singleton
Piacanlae
(n=16)
T~i;~pcentas
- Nanfused
Twi;n$xntae
- Fused
Area
Area
fSEM by paired
i lsst
analysis
l-
l-
l-
PLASM PE
DIACYL PE
Fig. 3. Arachidonic acid content of lipid fractions from chorion laeve. For definition of terms see legend to Fig. 2. The p values illustrated are from the comparison of data for the fused and nonfused areas of twin placentas by paired t test analysis. The arachidonic acid content of each lipid fraction from the fused area of chorion laeve from twin placentas was found to be lower than that from chorion laeve from singleton placentas by t test analysis. By this comparison, the following p values were obtained: For PLASM PE, DIACYL PE, PI, and PC, p < 0.001, for PS, p < 0.05; and for NL, p < 0.002. By t test analysis, the arachidonic acid content of some of the lipid fractions from the nonfused area of chorion laeve from twin placentas was found to be lower than that from chorion laeve from singleton placentas. By this comparison, the following p values were obtained: For DlACYL PE and PS p < 0.05 and for PC p < 0.01,
479
480
Okita, Johnston, and MacDonald
fatty acids. The one-step scribed by Morrison and amount of heptadecanoic ple to serve as an internal quantification of the fatty
methylation reaction deSmith’? was used. A known acid was added to each samstandard for the purpose of acids.
Results The arachidonic acid composition of the lipid fractions of amnion and chorion laeve tissues of djamnionic-dichorionic human twin placentas are given in Figs. 2 and 3, respectively. The data obtained from the fused area of the fetal membranes were compared with the data from the nonfused area by paired t test analysis. The p values for these comparisons are given in Figs. 2 and 3. These data also were compared with those obtained for tissues from normal singleton pregnancies, data that have been reported previously.* The p values for these latter comparisons are given in the legends to Figs. 2 and 3. We are of the view that there are two important findings: (1) The content of arachidonic acid in the lipid fractions of amnion tissue of diamnionic-dichorionic twin placentas was similar to that of amnion tissue of singleton pregnancies irrespective of the site of amnion tissue sampling (Fig. 2). (2) In striking contrast, however, the arachidonic acid content of the lipid fractions of chorion laeve tissue from the fused area of twin placentas was markedly reduced (by approximately 66%) as compared with that from singleton pregnancies (see legend to Fig. 3). By paired t test analysis the arachidonic acid content of the chorion laeve tissue taken from the fused area of the fetal membranes was significantly lower than that from the nonfused area (Fig. 3). The data presented in Figs. 2 and 3 were obtained by analysis of fetal membranes from twin placentas obtained at cesarean section performed prior to the onset of labor. Similar analyses were conducted with fetal membranes from twin placentas obtained at cesarean section performed after the spontaneous onset of labor (n = 4) and after spontaneous vaginal delivery (n = 4). The findings of these studies (data not shown) were similar to those presented in Figs. 2 and 3 (i.e., the arachidonic acid content of chorion laeve tissue, but not amnion tissue, from the fused area was decreased as compared with that in the respective tissue from the nonfused area). To isolate chorion laeve tissue from the nonfused area of the fetal membranes, it was necessary to remove the adhering decidua vera tissue by scraping with the edge of a glass slide. Much of the cellular layer of the chorion laeve is removed by this procedure.rs It was not necessary to apply this dissection method to the chorion laeve from the fused area of the fetal membranes because in this area the chorion laeve is not contam-
November Am. J. Obstet.
I, I 983 (;yneco~.
inated by decidua. However, with several fetal membrane preparations (n = 4), analyses also were conducted on chorion Iaeve tissue from the fused area of the fetal membranes that was scraped in a manner similar to that used to remove decidua vera tissue from the chorion laeve tissue of the nonfused area. The arachidonic acid content of the lipid fractions of chorion laeve tissue from the fused area prepared both with (dara not shown) and without (data shown in Fig. 3) the scraping procedure was lower than that of chorion laeve tissue from the nonfused area.
Comment In the present investigation we found that the arachidonic acid content of the lipid fractions of amnion from diamnionic-dichorionic twin placentas was similar to that of amnion from singleton placentas. Importantly, this was true in amnion tissue irrespective of the anatomic site of sampling, i.e., amnion overlying chorion laeve that was contiguous with decidua vera or amnion overlying fused chorion laeve that was not contiguous with decidua Vera. Because amnion is an avascular tissue and because arachidonic acid is an essential fatty acid, we conclude from these findings that the arachidonic acid incorporated into the lipids of amnion in the fused area of fetal membranes must arise from essential fatty acids in the amniotic fluid. We cannot rule out the possibility that amnion in the nonfused area of fetal membranes might derive essential fatty acids from the decidua vera through the chorion laeve. However, this is not a requisite source of essential fatty acids for the amnion as evidenced by the high arachidonic acid content of amnion in the fused area of the fetal membranes. When chorion laeve from diamnionic-dichorionic twin placentas was taken at a site where the two chot-ions were fused, the arachidonic acid content of the lipid fractions of this tissue was strikingly less than that in chorion laeve continguous to decidua vera (from either singleton or twin placentas). From this finding we conclude that it is probable that there are two sources of essential fatty acids for human chorion laeve. One is amniotic Auid as evidenced by the presence of arachidonic acid in the fused area of the chorion laeve of diamnionic-dichorionic twin placentas. The arachidonic acid content of chorion laeve adjacent to decidua vera in diamnionic-dichorionic twin placentas, however, was considerably greater than that in the fused portion. Therefore, we suggest that there is a second source of essential fatty acids for chorion laeve, i.e., from maternal plasma made available to chorion laeve through the contiguous decidua Vera. Since both the amnion and chorion laeve of human fetal membranes are avascular tissues, it must be concluded that the amnion and chorion laeve in the fused
Volume Number
Prostaglandin
147 5
portion of the fetal membranes of diamnionic-dichorionic twin placentas assimilate arachidonic acid or the precursor thereof, linoleic acid, from the amniotic fluid. Arachidonic acid per se and linoleic acid, which can serve as a precursor of arachidonic acid, ultimately must be obtained from the mother since these are essential fatty acids that must be obtained from the mother’s diet. From the findings of this study alone we cannot deduce the origin of essential fatty acids in amniotic fluid. We suggest that the most likely possibilities, prior to the onset of labor, are (1) arachidonic acid bound to albumin that entered amniotic fluid from the maternal circulation and (2) secretions from the fetal lungs. We suggest that essential fatty acids from the amniotic fluid are incorporated into both amnion and chorion laeve. Further, chorion laeve is able to incorporate essential fatty acids from the contiguous decidua Vera. The following observations are supportive of this formulation of the sources of essential fatty acids for the human fetal membranes. During early labor, i.e., up to 4 cm of cervical dilatation, there is a striking loss of arachidonic acid from the diacyl phosphatidylethanolamine and the phosphatidylinositol of amnion.8 There is also a significant, but less striking, loss of arachidonic acid from these two glycerophospholipids in chorion laeve during early 1abor.s As the arachidonic acid content of amnion decreases, it is replaced by palmitic acid whereas in chorion laeve the arachidonic acid is replaced by stearic acid. The substitution of two different fatty acids for the mobilized arachidonic acid in these two adjacent tissues is supportive of the proposition that these two tissues derive fatty acids from two different sources. Near term the amniotic fluid, which we propose is the source of fatty acid for amnion, is enriched in palmitic acid because of the presence of large amounts of dipalmitoyl phosphatidylcholine, or lecithin, in surfactant. Late in the course of labor the arachidonic acid content of amnion and chorion laeve is restored to prelabor values or greater. I8 We suggest that this phenomenon comes about as follows. During labor there is a progressive increase in the free arachidonic acid content of amniotic fluid. I* Thus, as labor progresses, arachidonic acid, in greater concentrations, becomes available to be reesterified into the lipids of amnion and chorion laeve. In addition, after 4 cm of cervical dilatation is attained, the total free fatty acid levels (including arachidonic acid) in maternal plasma rise,20 possibly as the result of epinephrine-induced lipolysis and mobilization of free fatty acids. The epinephrine concentration in the maternal plasma rises during labor; however, this rise does not occur until after 3 to 4 cm of cervical dilatation has been reached.21 Thus, during labor these mechanisms may provide a means for the
precursor
in fetal membranes
replenishment of fetal membranes glandin precursor arachidonic acid. Dr. M. Linette
Casey provided
with
us with
481
the prosta-
Fig. 1.
REFERENCES 1.
2.
3. 4.
5.
6. 7. 8.
9.
Okazaki, T., Casey, M. L., Okita. J. R., MacDonald, P. C., and Johnston, J. M.: Initiation of parturition. XII. Biosynthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua, AM. J. OBSTET. GYNECOL. 139:373, 1981. Kinoshita, K., Satoh, K., and Sakamoto, S.: Biosynthesis of prostaglandin in human decidua, amnion, chorion and villa, Endocrinol. Jpn. 24:343, 1977. Mitchell, M. D.: Prostaglandins during pregnancy and the perinatal period, J. Reprod. Fertil. 62:305, 1981. Schwarz, 8. E., Schultz, F. M., MacDonald, P. C., and Johnston, J. M.: Initiation ot human parturition. III. Fetal membrane content of prostaglandin E, and F,, precursor, Obstet. Gynecol. 46:564, 1975. MacDonald, P. C., Schultz, I’. M., Duenhoelter, J. H., Gant, N. F.,Jimenez, J. M., Pritchard, J. A., Parker, J. D., and Johnston, J, M.: Initiation of human parturition. I. Mechanism of action of arachidonic acid, Obstet. Gynecol. 44:629, 1974. Keirse, M. J. N. C., and Turnl)ull, A. C.: E prostaglandins in amniotic fluid during pregnancy and labour, J. Obsret. Gynaecol. Br. Commonw. 80:970, 1973. Keirse, M. J. N. C., Flint, A. I’. F., and Turnbull, A. C.: F prostaglandins in amniotic fluid during pregnancy and labour, J. Obstet. Gynaecol. Br. Commonw. 81: 131, 1974. Okita, J. R., MacDonald, P. C., and Johnston, J. M.: Mobilization of arachidonic acid from specific glycerophosphoiipids of human fetal membranes during early labor, J. Biol. Chem. 257:14029, 1982. Okazaki, T., Okita, J. R., Mac Donald, P. C., and Johnston, J. M.: Initiation of human parturition. X. Substrate specificity of phospholipase A, in human fetal membranes. AM.
J. OBSTET.
GYNECOL.
130:432,
1978.
10. DiRenzo, G. C., Johnston, J. M., Okazaki, T., Okita, J. R., MacDonald, P. C., and Bleasdale, J. E.: Phosphatidylinositol-specific phospholipala C in fetal membranes and uterine decidua, J. clin. imest. 67:847, 1981. 11. Okazaki. T.. Sacawa. N.. Okita. 1. R.. Bleasdale. 1. E.. MacDo&ld,’ P. “c., and johnstdn: J. k.: Diacyl&$eroi metabolism and arachidonic acid release in human fetal membranes and decidua vet-a, J. Biol. Chem. 256:7316, 1981. 12. Okazaki, T., Sagawa, N., L\leasdale, J. E., Okita, Jo R., MacDonald, P. C., and Johnston, J. M.: Initiation of human aarturition. XIII. Phosoholinase C. nhosnholipase A;and diacylglycerol lipas; actiiities in’fgtal Gembranes and decidua vera tissues from early and late gestation, Biol. Reprod. 25:103, 1981. 13. Okita, J. R., MacDonald, P. C., and Johnston, J. M.: Initiation of human parturition. XIV. Increase in the diacylglycerol content of amnion during parturition, AM. J. OBSTET.
GYNECOL.
142:432,
1982.
14. Hansen, H. S.: 15-Hydroxyprostaglandin dehydrogenase, Prostaglandins 12:647, 1976. 15. MacDonald, P. C., Porter, J. C., Schwarz, B. E., and Johnston, J. M.: Initiation of parturition in rhe human female, Semin. Perinatol. 2:273, 1978. 16. Folch, J., Lees, M., and Sloane Stanley, G. H.: A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226:497, 1957. 17. Morrison, W. R., and Smith, L. R.: Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol, J. Lipid Res. 5:600, 1964. 18. Okita, J. R.: Ph.D. Dissertation, University of Texas Health Science Center, Dallas, Texas, 1981.
Okita,
Johnston,
and MacDonald
November 1. 1983 Am. J. Obstet. Gyhecol.
19. Keirse, M. J. N. C., Hicks, B. R., Mitchell, M. D., and Turnbull, A. C.: Increase of the prostaglandin precursor, arachidonic acid, in amniotic fluid during spontaneous labour, Br. J. Obstet. Gynaecol. 84:937, 1977. 20. Ogburn, P. L., Jr., Johnson, S. B., Williams, P. P., and Holman, R. T.: Levels of free fatty acids and arachidonic
acid in pregnancy and labor, J. Lab. Clin. Med. 95:943, 1980. 21. Lederman, R. P., McCann, D. S., Work, B., Jr., and Huber, M. J.: Endogenous plasma epinephrine and norepinephrine in last-trimester pregnancy and labor, AM. J. OBSTET.GYNECOL. 129:5, 1977.
Prolactin molecular heterogeneity Response t o thyrotropin-releasing hormone stimulation concanavalin A-bound and -unbound immunoassayable prolactin during human pregnancy Donna
Shoupe,
Fredrick
J. Montz,
Oscar
A. Kletzky,
and
Gere
of
S. diZerega
L0.rAn&us, Cul~forrrire Since
the milieu
of pregnancy
stimulates
physiologic
hyperprolactinemia,
we questioned
whether
prolactin
secreted during normal pregnancy contains a large-molecular weight component that binds to concanavalin A and whether this large-molecular weight prolactin contributes to the thyrotropin-releasing hormone (TRH)-releasable pool. Serum was collected from pregnant patients (n = 28) undergoing TRH stimulation tests. This serum was passed through a concanavalin A column and eluted with 0.2M a-methylmannoside. Concanavalin A-bound prolactin, as determined by radioimmunoassay, ranged from 10% to 30% of the total immunoassayable prolactin. An increase in the basal serum concentration of both concanavalin A-bound and -unbound prolactin occurred as pregnancy progressed. However, throughout gestation, only the concanavalin A-unbound prolactin increased after TRH stimulation. The concanavalin A-bound prolactin was found to have a molecular weight of 60,000 by means of Sephadex G-100 permeation chromatography. (AM. J. OBSTET. GYNECOL. 147:482, 1983.)
Molecular heterogeneity of human prolactin has been demonstrated by gel filtration of normal human serum, pregnancy serum, amniotic fluid, and serum from patients with prolactin-secreting pituitary adenomas.“” Consistently, two distinct populations of immunoassayable prolactin can be identified: (1) Little prolactin, having a molecular weight of 22,000, is reported to have a greater biologic activity and usually makes up more than 80% of the total circulating prolactin; (2) big prolactin, having a molecular weight between -10,000 and 67,000. makes up 8% to 20% of circulating prolactin. Suh and Frant? reported the highest percentage of big prolactin occurred in pregnant patients (up to 31.8%). Recently, we reported that both little and big prolac-
Avm the Department of Obstetrics and Gynecology, University qf Southern Calfornia School of Medicine. Supported in part by National Institute of Child Health and Human Deoelapment Clinical Investigator Award HD-00401. Presented at the Troenty-ninth Annuul Meeting of the Society.for Gynecologic Investigation, Dallas, Texas, March 24-27, 1982. Reprint requests: &re S. di.kegu, M.D., Women? HospitaL, Room LlOI3, 1240 N. Mission Road, Los Angeles, California 90033. 482
tin (molecular weight of 60,000) were secreted both in vivo and in vitro from prolactin-producing pituitary adenomas that bound to concanavalin A-Sepharose,’ thus suggesting that the increase in molecular weight from little to big prolactin may, in part, result from carbohydrate inclusion into the larger molecule.“. I4 Since the milieu of pregnancy stimulates physiologic hyperprolactinemia, we questioned whether prolactin secreted during normal pregnancy contains a largemolecular weight component which binds to concanavalin A and whether this large-molecular weight prolactin contributes to the thyrotropin-releasing hormone (TRH)-releasable pool.
Material and methods Twenty-eight patients with clinically normal pregnancies (first trimester, n = 7; second trimester, n = IO; third trimester, n = 11) underwent TRH stimulation (500 pg intravenously). Antecubital blood samples were collected at 0, +20, i-30, + 60, and + 120 minutes after injection and centrifuged: the serum was frozen at -20” C until processing. Samples of 1 ml of the serum were placed on a 2 ml column of concanavalin
Obstetrics and Gynecology Founded volume
147
number
5
NOVEMBER
in
1920
1,1983
Papers of the Society for Gynecologic Investigation
Source of prostaglandin precursor in human fetal membranes: Arachidonic acid content of amnion and chorion laeve in diamnionic-dichorionic twin placentas Janice
R. Okita,*
Da&s,
Texas
John M. Johnston,
and Paul C. MacDonald
The lipids of the avascular human amnion and chorion laeve are known to be enriched in the essential fatty acid arachidonic acid, the obligate precursor of prostaglandins of the 2-series. The present investigation was conducted to evaluate the source of arachidonic acid that is present in human fetal membranes. To do so we determined the arachidonic acid content of the lipid fractions of amnion and chorion laeve tissues from diamnionic-dichorionic twin placentas. We found that the arachidonic acid content of amnion from twin placentas was similar to that of amnion from singleton placentas irrespective of the site of amnion sampling. However, the same was not true of chorion laeve of diamnionic-dichorionic twin placentas. The arachidonic acid content of chorion laeve that was contiguous to the chorion laeve of its twin was strikingly reduced compared to that of chorion laeve contiguous to decidua vera obtained from either singleton or twin placentas. We conclude that the arachidonic acid in amnion is derived primarily from essential fatty acids in the amniotic fluid. However, the arachidonic acid in chorion laeve may be derived from essential fatty acid in the amniotic fluid as well as in maternal plasma by way of the decidua Vera. (AM. J. OBSTET. GYNECOL. 147:477,
1983.)
From the Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Biochemistry and ObstetricsGynecology, The University of Texas Southwestenz Medical School at Dallas. This investigation was supported in part by United States Public Health Service Grant No. 5-P50-HD11149 and a Grant-in-Aid from The Robert A. Welch Foundation, Howton, Texas. Sponsored by the Society for Gynecologic Investigation. Reprint requests: John M. Johnston, Ph.D., Department of Biochemistry, The CiniversiQ of Texas Health Science Center, 5323 Har/y Hines Boulevard, Dallas, Texas 75235. *Dr. Okita was a postdoctoral trainee supported by a Grant-in-Aid from The Robert A. Welch Foundation, Houston, Texas.
It is believed that prostaglandins (PGs) serve an important role in the initiation and maintenance of human parturition. Also, there are a number of lines of evidence in support of the proposition that metabolic events that take place in amnion and cborion laeve are crucial to the generation of these prostaglandins; indeed, the following considerations seem to be pertinent to the role of the fetal membranes in human parturition: (1) The specific activity of prostaglandin synthase in amnion is greater than that in chorion laeve, decidua Vera, myometrium, or placenta.‘-” (2) The glycero-
477
478
Okita,
Johnston,
and MacDonald
plac‘enta5
Fig. I. The diamnionic-dichorionic
November Am. J. Obstet.
decidua
basalis
twin placenta
phospholipids of amnion and chorion laeve are enriched with arachidonic acid, the obligate precursor of prostaglandins of the S-series.” (3) The levels of free arachidonic acid as well as those of PGE, and PGF,, in amniotic fiuid increase during labor and as labor progresses.“-’ (4) During early labor there is a specific decrease in the arachidonic acid content of diacyl phosphatidylethanolamine and phosphatidylinositol in the amnion and chorion laeve.x (5) A phospholipase A, with relative substrate specificity for phusphatidylethanolamines with arachidonic acid in the sn-2 position is present in fetal membranes.!’ (6) Phosphatidylinositolspecific phospholipase C activity is present in human amnion and chorion laeve”): this enzyme catalyzes the hydrolysis of phosphatidylinositol to diacylglycerols. In turn there is a diacylglycerol lipase in amnion and chorion laeve that catalyzes the release of the f‘atty acid from the .rn- 1 position of diacylglycerols,” and this enzyme may be relatively specific for diacylglycerols with arachidonic acid in the sn-2 position.” In turn, there is a monoacylglycerol lipase in amnion and chorion laeve that catalyzes the release of the fatty acid in the ~71-2 position of’ monoacylglycet-ols.” There may be relative substrate specificity of this enzyme for sn-2 arachidonoylmonoacylglycerols. In this coordinated manner, by three enzymatic reactions, arachidonic acid is released from phosphatidylinositol in the fetal membranes. (7) The specific activities of phospholipases A, and C increase strikingly in human amnion late in gestation.‘” (8) Diacylglycerols, the products of the reaction catalyzed by phosphatidylinositol-specific phospholipase C, accumulate in amnion during early labor.13 (9) Nicotinamide adenine dinucleotide+-dependent 15-hydroxyprostaglandin dehydrogenase activity is not detectable in human amnion’; this enzyme catalyzes the first reaction in the inactivation of prostaglandins.‘” (I 01 Infection or hypoxia of the fetal membranes, as well as exposure to hypertonic solutions of sodium chloride, urea, or glucose, causes premature labor.‘j Therefore, an important question arises: What is the
1, I983 Gynecol.
source of the arachidonic acid that is present in amnion and chorion laeve? The crucial nature of this question is apparent when one considers that (1) human amnion and chorion laeve are avascular tissues and (2) arachidonic acid is an essential fatty acid. To address this issue, the arachidonic acid content of amnion and chorion laeve of diamnionic-dichorionic human twin placentas was determined. In such placentas there is a considerable portion of the chorion laeve tissue of each fetus that is not contiguous with decidua vera tissue but rather is contiguous with the chorion laeve tissue of its twin. Thus, in this portion of the fetal membranes neither amnion nor chorion laeve is in contact with maternal plasma, as illustrated diagramatically in Fig. I. The purpose of this study was to evaluate the roles of amniotic fluid and decidua vera (maternal plasma) as sources of the arachidonic acid in the fetal membranes.
Material and methods Diamnionic-dichorionic twin placentas were obtained immediately after cesarean section conducted either prior to or during labor and after spontaneous vaginal delivery. The tissues were immediately placed on ice and transported to the laboratory. Tissue dissections were conducted with placentas and fetal men]branes on a sheet of glass supported by ice. Sections of amnion and chorion laeve frotn each twin were taken for analysis from that portion of the membranes in which the chorion laeve was fused with the chorion laeve of its twin (hereafter referred to as the fused area) and that portion of the membranes in which the chorion laeve was contiguous to decidua vera (i.e., the nonfused area) (see Fig. 1). To separate the tissues, the amnion was first peeled away from the chorion laeve. In sections of chorion laeve contiguous to decidua Vera, the decidua vera was removed by sharp dissection by use of a glass slide as previously described.” Tissues.
Determination lipid
fractions
of of
the
amnion
arachidonic and
chorion
acid
content
laeve
of
tissues.
The methods used have been described in detail.’ Briefly, the tissues were homogenized by use of a Polytron PI 0 homogenizer (Brinkman Instruments, Inc., Westbury, New York). Lipids were extracted with chloroform: methanol (2: 1, v: v) by a modification of the extraction procedure of Folch and co-workers.‘” The extracts were filtered and washed extensively with the preequilibrated aqueous phase; the organic solvent was evaporated under a stream of nitrogen at 37” C. The residues were dissolved in chloroform : methanol (2 : I, v : v) and stored at -20” C. The glycerophospholipids were separated by two-dimensional thinlayer chromatography (TLC) as previously described.’ The fatty acid composition of the glycerophospholipids was determined by gas-liquid chromatographic analysis and quantification of methyl ester derivatives of- the
Volume Number
Prostaglandin precursor in fetal membranes
147 5
D *0.05
-
m
Singletan
q
Tui;n;\aantas
El
Twin Placsntar-Fund (n=4)
Placentor
(n=V)
- Nonfused
Area AW
&SEM by pind
f test
analysis
I-
)-
)-
PLASM PE
DIACYL PE
PI
PC
PS
NL
Fig. 2. Arachidonic acid content of lipid fractions from amnion. Arachidonic acid content is expressed as the percentage of total fatty acyl groups in each fraction. For the PLASM PE fraction, arachidonic acid is expressed as the percentage of total fatty acyl plus alkenyl groups. The p value illustrated is from the comparison of data for the fused and nonfused areas of twin placentas by paired t test analysis. The data from singleton placentas were compared with those from either fused or nonfused areas of twin placentas by t test analysis. No statistically significant differences were found. PLASM PE, Plasmalogen phosphatidylethanolamine; DIACYL PE, diacr 1 phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine; PS, phosphatidylserine; NL, total neutral lipid l-
a
q q
l-
Singleton
Piacanlae
(n=16)
T~i;~pcentas
- Nanfused
Twi;n$xntae
- Fused
Area
Area
fSEM by paired
i lsst
analysis
l-
l-
l-
PLASM PE
DIACYL PE
Fig. 3. Arachidonic acid content of lipid fractions from chorion laeve. For definition of terms see legend to Fig. 2. The p values illustrated are from the comparison of data for the fused and nonfused areas of twin placentas by paired t test analysis. The arachidonic acid content of each lipid fraction from the fused area of chorion laeve from twin placentas was found to be lower than that from chorion laeve from singleton placentas by t test analysis. By this comparison, the following p values were obtained: For PLASM PE, DIACYL PE, PI, and PC, p < 0.001, for PS, p < 0.05; and for NL, p < 0.002. By t test analysis, the arachidonic acid content of some of the lipid fractions from the nonfused area of chorion laeve from twin placentas was found to be lower than that from chorion laeve from singleton placentas. By this comparison, the following p values were obtained: For DlACYL PE and PS p < 0.05 and for PC p < 0.01,
479
480
Okita, Johnston, and MacDonald
fatty acids. The one-step scribed by Morrison and amount of heptadecanoic ple to serve as an internal quantification of the fatty
methylation reaction deSmith’? was used. A known acid was added to each samstandard for the purpose of acids.
Results The arachidonic acid composition of the lipid fractions of amnion and chorion laeve tissues of djamnionic-dichorionic human twin placentas are given in Figs. 2 and 3, respectively. The data obtained from the fused area of the fetal membranes were compared with the data from the nonfused area by paired t test analysis. The p values for these comparisons are given in Figs. 2 and 3. These data also were compared with those obtained for tissues from normal singleton pregnancies, data that have been reported previously.* The p values for these latter comparisons are given in the legends to Figs. 2 and 3. We are of the view that there are two important findings: (1) The content of arachidonic acid in the lipid fractions of amnion tissue of diamnionic-dichorionic twin placentas was similar to that of amnion tissue of singleton pregnancies irrespective of the site of amnion tissue sampling (Fig. 2). (2) In striking contrast, however, the arachidonic acid content of the lipid fractions of chorion laeve tissue from the fused area of twin placentas was markedly reduced (by approximately 66%) as compared with that from singleton pregnancies (see legend to Fig. 3). By paired t test analysis the arachidonic acid content of the chorion laeve tissue taken from the fused area of the fetal membranes was significantly lower than that from the nonfused area (Fig. 3). The data presented in Figs. 2 and 3 were obtained by analysis of fetal membranes from twin placentas obtained at cesarean section performed prior to the onset of labor. Similar analyses were conducted with fetal membranes from twin placentas obtained at cesarean section performed after the spontaneous onset of labor (n = 4) and after spontaneous vaginal delivery (n = 4). The findings of these studies (data not shown) were similar to those presented in Figs. 2 and 3 (i.e., the arachidonic acid content of chorion laeve tissue, but not amnion tissue, from the fused area was decreased as compared with that in the respective tissue from the nonfused area). To isolate chorion laeve tissue from the nonfused area of the fetal membranes, it was necessary to remove the adhering decidua vera tissue by scraping with the edge of a glass slide. Much of the cellular layer of the chorion laeve is removed by this procedure.rs It was not necessary to apply this dissection method to the chorion laeve from the fused area of the fetal membranes because in this area the chorion laeve is not contam-
November Am. J. Obstet.
I, I 983 (;yneco~.
inated by decidua. However, with several fetal membrane preparations (n = 4), analyses also were conducted on chorion Iaeve tissue from the fused area of the fetal membranes that was scraped in a manner similar to that used to remove decidua vera tissue from the chorion laeve tissue of the nonfused area. The arachidonic acid content of the lipid fractions of chorion laeve tissue from the fused area prepared both with (dara not shown) and without (data shown in Fig. 3) the scraping procedure was lower than that of chorion laeve tissue from the nonfused area.
Comment In the present investigation we found that the arachidonic acid content of the lipid fractions of amnion from diamnionic-dichorionic twin placentas was similar to that of amnion from singleton placentas. Importantly, this was true in amnion tissue irrespective of the anatomic site of sampling, i.e., amnion overlying chorion laeve that was contiguous with decidua vera or amnion overlying fused chorion laeve that was not contiguous with decidua Vera. Because amnion is an avascular tissue and because arachidonic acid is an essential fatty acid, we conclude from these findings that the arachidonic acid incorporated into the lipids of amnion in the fused area of fetal membranes must arise from essential fatty acids in the amniotic fluid. We cannot rule out the possibility that amnion in the nonfused area of fetal membranes might derive essential fatty acids from the decidua vera through the chorion laeve. However, this is not a requisite source of essential fatty acids for the amnion as evidenced by the high arachidonic acid content of amnion in the fused area of the fetal membranes. When chorion laeve from diamnionic-dichorionic twin placentas was taken at a site where the two chot-ions were fused, the arachidonic acid content of the lipid fractions of this tissue was strikingly less than that in chorion laeve continguous to decidua vera (from either singleton or twin placentas). From this finding we conclude that it is probable that there are two sources of essential fatty acids for human chorion laeve. One is amniotic Auid as evidenced by the presence of arachidonic acid in the fused area of the chorion laeve of diamnionic-dichorionic twin placentas. The arachidonic acid content of chorion laeve adjacent to decidua vera in diamnionic-dichorionic twin placentas, however, was considerably greater than that in the fused portion. Therefore, we suggest that there is a second source of essential fatty acids for chorion laeve, i.e., from maternal plasma made available to chorion laeve through the contiguous decidua Vera. Since both the amnion and chorion laeve of human fetal membranes are avascular tissues, it must be concluded that the amnion and chorion laeve in the fused
Volume Number
Prostaglandin
147 5
portion of the fetal membranes of diamnionic-dichorionic twin placentas assimilate arachidonic acid or the precursor thereof, linoleic acid, from the amniotic fluid. Arachidonic acid per se and linoleic acid, which can serve as a precursor of arachidonic acid, ultimately must be obtained from the mother since these are essential fatty acids that must be obtained from the mother’s diet. From the findings of this study alone we cannot deduce the origin of essential fatty acids in amniotic fluid. We suggest that the most likely possibilities, prior to the onset of labor, are (1) arachidonic acid bound to albumin that entered amniotic fluid from the maternal circulation and (2) secretions from the fetal lungs. We suggest that essential fatty acids from the amniotic fluid are incorporated into both amnion and chorion laeve. Further, chorion laeve is able to incorporate essential fatty acids from the contiguous decidua Vera. The following observations are supportive of this formulation of the sources of essential fatty acids for the human fetal membranes. During early labor, i.e., up to 4 cm of cervical dilatation, there is a striking loss of arachidonic acid from the diacyl phosphatidylethanolamine and the phosphatidylinositol of amnion.8 There is also a significant, but less striking, loss of arachidonic acid from these two glycerophospholipids in chorion laeve during early 1abor.s As the arachidonic acid content of amnion decreases, it is replaced by palmitic acid whereas in chorion laeve the arachidonic acid is replaced by stearic acid. The substitution of two different fatty acids for the mobilized arachidonic acid in these two adjacent tissues is supportive of the proposition that these two tissues derive fatty acids from two different sources. Near term the amniotic fluid, which we propose is the source of fatty acid for amnion, is enriched in palmitic acid because of the presence of large amounts of dipalmitoyl phosphatidylcholine, or lecithin, in surfactant. Late in the course of labor the arachidonic acid content of amnion and chorion laeve is restored to prelabor values or greater. I8 We suggest that this phenomenon comes about as follows. During labor there is a progressive increase in the free arachidonic acid content of amniotic fluid. I* Thus, as labor progresses, arachidonic acid, in greater concentrations, becomes available to be reesterified into the lipids of amnion and chorion laeve. In addition, after 4 cm of cervical dilatation is attained, the total free fatty acid levels (including arachidonic acid) in maternal plasma rise,20 possibly as the result of epinephrine-induced lipolysis and mobilization of free fatty acids. The epinephrine concentration in the maternal plasma rises during labor; however, this rise does not occur until after 3 to 4 cm of cervical dilatation has been reached.21 Thus, during labor these mechanisms may provide a means for the
precursor
in fetal membranes
replenishment of fetal membranes glandin precursor arachidonic acid. Dr. M. Linette
Casey provided
with
us with
481
the prosta-
Fig. 1.
REFERENCES 1.
2.
3. 4.
5.
6. 7. 8.
9.
Okazaki, T., Casey, M. L., Okita. J. R., MacDonald, P. C., and Johnston, J. M.: Initiation of parturition. XII. Biosynthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua, AM. J. OBSTET. GYNECOL. 139:373, 1981. Kinoshita, K., Satoh, K., and Sakamoto, S.: Biosynthesis of prostaglandin in human decidua, amnion, chorion and villa, Endocrinol. Jpn. 24:343, 1977. Mitchell, M. D.: Prostaglandins during pregnancy and the perinatal period, J. Reprod. Fertil. 62:305, 1981. Schwarz, 8. E., Schultz, F. M., MacDonald, P. C., and Johnston, J. M.: Initiation ot human parturition. III. Fetal membrane content of prostaglandin E, and F,, precursor, Obstet. Gynecol. 46:564, 1975. MacDonald, P. C., Schultz, I’. M., Duenhoelter, J. H., Gant, N. F.,Jimenez, J. M., Pritchard, J. A., Parker, J. D., and Johnston, J, M.: Initiation of human parturition. I. Mechanism of action of arachidonic acid, Obstet. Gynecol. 44:629, 1974. Keirse, M. J. N. C., and Turnl)ull, A. C.: E prostaglandins in amniotic fluid during pregnancy and labour, J. Obsret. Gynaecol. Br. Commonw. 80:970, 1973. Keirse, M. J. N. C., Flint, A. I’. F., and Turnbull, A. C.: F prostaglandins in amniotic fluid during pregnancy and labour, J. Obstet. Gynaecol. Br. Commonw. 81: 131, 1974. Okita, J. R., MacDonald, P. C., and Johnston, J. M.: Mobilization of arachidonic acid from specific glycerophosphoiipids of human fetal membranes during early labor, J. Biol. Chem. 257:14029, 1982. Okazaki, T., Okita, J. R., Mac Donald, P. C., and Johnston, J. M.: Initiation of human parturition. X. Substrate specificity of phospholipase A, in human fetal membranes. AM.
J. OBSTET.
GYNECOL.
130:432,
1978.
10. DiRenzo, G. C., Johnston, J. M., Okazaki, T., Okita, J. R., MacDonald, P. C., and Bleasdale, J. E.: Phosphatidylinositol-specific phospholipala C in fetal membranes and uterine decidua, J. clin. imest. 67:847, 1981. 11. Okazaki. T.. Sacawa. N.. Okita. 1. R.. Bleasdale. 1. E.. MacDo&ld,’ P. “c., and johnstdn: J. k.: Diacyl&$eroi metabolism and arachidonic acid release in human fetal membranes and decidua vet-a, J. Biol. Chem. 256:7316, 1981. 12. Okazaki, T., Sagawa, N., L\leasdale, J. E., Okita, Jo R., MacDonald, P. C., and Johnston, J. M.: Initiation of human aarturition. XIII. Phosoholinase C. nhosnholipase A;and diacylglycerol lipas; actiiities in’fgtal Gembranes and decidua vera tissues from early and late gestation, Biol. Reprod. 25:103, 1981. 13. Okita, J. R., MacDonald, P. C., and Johnston, J. M.: Initiation of human parturition. XIV. Increase in the diacylglycerol content of amnion during parturition, AM. J. OBSTET.
GYNECOL.
142:432,
1982.
14. Hansen, H. S.: 15-Hydroxyprostaglandin dehydrogenase, Prostaglandins 12:647, 1976. 15. MacDonald, P. C., Porter, J. C., Schwarz, B. E., and Johnston, J. M.: Initiation of parturition in rhe human female, Semin. Perinatol. 2:273, 1978. 16. Folch, J., Lees, M., and Sloane Stanley, G. H.: A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226:497, 1957. 17. Morrison, W. R., and Smith, L. R.: Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol, J. Lipid Res. 5:600, 1964. 18. Okita, J. R.: Ph.D. Dissertation, University of Texas Health Science Center, Dallas, Texas, 1981.
Okita,
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and MacDonald
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19. Keirse, M. J. N. C., Hicks, B. R., Mitchell, M. D., and Turnbull, A. C.: Increase of the prostaglandin precursor, arachidonic acid, in amniotic fluid during spontaneous labour, Br. J. Obstet. Gynaecol. 84:937, 1977. 20. Ogburn, P. L., Jr., Johnson, S. B., Williams, P. P., and Holman, R. T.: Levels of free fatty acids and arachidonic
acid in pregnancy and labor, J. Lab. Clin. Med. 95:943, 1980. 21. Lederman, R. P., McCann, D. S., Work, B., Jr., and Huber, M. J.: Endogenous plasma epinephrine and norepinephrine in last-trimester pregnancy and labor, AM. J. OBSTET.GYNECOL. 129:5, 1977.
Prolactin molecular heterogeneity Response t o thyrotropin-releasing hormone stimulation concanavalin A-bound and -unbound immunoassayable prolactin during human pregnancy Donna
Shoupe,
Fredrick
J. Montz,
Oscar
A. Kletzky,
and
Gere
of
S. diZerega
L0.rAn&us, Cul~forrrire Since
the milieu
of pregnancy
stimulates
physiologic
hyperprolactinemia,
we questioned
whether
prolactin
secreted during normal pregnancy contains a large-molecular weight component that binds to concanavalin A and whether this large-molecular weight prolactin contributes to the thyrotropin-releasing hormone (TRH)-releasable pool. Serum was collected from pregnant patients (n = 28) undergoing TRH stimulation tests. This serum was passed through a concanavalin A column and eluted with 0.2M a-methylmannoside. Concanavalin A-bound prolactin, as determined by radioimmunoassay, ranged from 10% to 30% of the total immunoassayable prolactin. An increase in the basal serum concentration of both concanavalin A-bound and -unbound prolactin occurred as pregnancy progressed. However, throughout gestation, only the concanavalin A-unbound prolactin increased after TRH stimulation. The concanavalin A-bound prolactin was found to have a molecular weight of 60,000 by means of Sephadex G-100 permeation chromatography. (AM. J. OBSTET. GYNECOL. 147:482, 1983.)
Molecular heterogeneity of human prolactin has been demonstrated by gel filtration of normal human serum, pregnancy serum, amniotic fluid, and serum from patients with prolactin-secreting pituitary adenomas.“” Consistently, two distinct populations of immunoassayable prolactin can be identified: (1) Little prolactin, having a molecular weight of 22,000, is reported to have a greater biologic activity and usually makes up more than 80% of the total circulating prolactin; (2) big prolactin, having a molecular weight between -10,000 and 67,000. makes up 8% to 20% of circulating prolactin. Suh and Frant? reported the highest percentage of big prolactin occurred in pregnant patients (up to 31.8%). Recently, we reported that both little and big prolac-
Avm the Department of Obstetrics and Gynecology, University qf Southern Calfornia School of Medicine. Supported in part by National Institute of Child Health and Human Deoelapment Clinical Investigator Award HD-00401. Presented at the Troenty-ninth Annuul Meeting of the Society.for Gynecologic Investigation, Dallas, Texas, March 24-27, 1982. Reprint requests: &re S. di.kegu, M.D., Women? HospitaL, Room LlOI3, 1240 N. Mission Road, Los Angeles, California 90033. 482
tin (molecular weight of 60,000) were secreted both in vivo and in vitro from prolactin-producing pituitary adenomas that bound to concanavalin A-Sepharose,’ thus suggesting that the increase in molecular weight from little to big prolactin may, in part, result from carbohydrate inclusion into the larger molecule.“. I4 Since the milieu of pregnancy stimulates physiologic hyperprolactinemia, we questioned whether prolactin secreted during normal pregnancy contains a largemolecular weight component which binds to concanavalin A and whether this large-molecular weight prolactin contributes to the thyrotropin-releasing hormone (TRH)-releasable pool.
Material and methods Twenty-eight patients with clinically normal pregnancies (first trimester, n = 7; second trimester, n = IO; third trimester, n = 11) underwent TRH stimulation (500 pg intravenously). Antecubital blood samples were collected at 0, +20, i-30, + 60, and + 120 minutes after injection and centrifuged: the serum was frozen at -20” C until processing. Samples of 1 ml of the serum were placed on a 2 ml column of concanavalin