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Plasma oxytocin levels during human pregnancy and labor as determined by radioimmunoassay
Plasma oxytocin levels during human pregnancy and labor as determined by radioimmunoassay P.
KUMARESAN,
P.
B.
W.
D.V.M.,
ANANDARANGAM,
DIANZON,
ALOIS Brooklyn,
D.V.M. M.D.
VASICKA, New
PH.D.*
M.D.
York
Plasma oxytocin concentrations were measured by radioimmunoassay with the use of unextracted plasma in 285 pregnant patients. In nonpregnant women the mean plasma oxytocin level was
A L T H o u G H oxytocin has been used extensively for the induction of labor in human subjects, information about physiologic levels during human pregnancy and labor is lacking. Oxytocin is known to be a potent stimulant of uterine activity. The hypothesis that oxytocin release occurs as a normal component of the process of parturition remains to be proved. In order to study factors which
initiate labor, and the causes of abnormal labor and parturition, circulating blood levels of oxytocin must be studied. Bioassay techniques have been utilized for these purposes, but most bioassays are relatively insensitive. Radioimmunoassay offers more precise means of measuring oxytocin levels. The successful production of antisera to oxytocin has been reported.‘, * The development of a radioimmunoassay for oxytocin has been described3-6 but detailed results of studies on oxytocin levels in an unexpected human plasma have not been reported. Levels of radioimmunoassayable oxytocin in goats’ and pregnant rats8 have been reported. Oxytocin levels of 12Op.U per milliliter have been reported in goats’ plasma during labor. In pregnant rats the mean oxytocin levels were 24p.U per milliliter on day 18 of pregnancy and 118 and 106pU per milliliter, respectively, on days 20 and 21. Four hours after delivery the oxytocin level fell abruptly in maternal plasma and pituitary. When oxytocin was extracted from
From the State University of New York, Downstate Medical Center, and The Department of Obstetrics and Gynecology of the Coney Island Hospital afiliated with the Maimonides Medical Center. Received Revised Accepted
for publication September October
May
24, 1973.
21, 1973. 3, 1973.
Reprint requests: Dr. Hormonal Laboratories Coney Island Hospital, New York 11235.
P.
Kumaresan,
of Obs./Gyn., Brooklyn,
*Supported by grants from The Population Council of New York and The National Institutes of Child Health and Human Development USPHS HD 04984-01. 215
216
Kumaresan
et al.
the plasma of women during labor and measured by radioimmunoassay, only 19 per cent showed detectable levels, with a range of 2 to 18pU per milliliter.” A very sensitive and specific antibody for oxytocin has been produced in this laboratory, enabling us to develop a radioimmunoassay for oxytocin in whole plasma. Oxytocin antibody has been tested for the dissociation of immunologic activity,“’ for the neutralization of oxytocin in \-iv0 in lactating rats,” and for effect upon litter size in rats.” The objective of this study was to investigate the measurement of oxytocin levels in plasma throughout human pregnancy and random sampling during labor by this radioimmunoassay technique. It had been previously reported by us that oxytocin was detectable in all pregnancy plasmas tested so far.‘:‘. 11 Materials
and
methods
Patient study. Individual samples were obtained between weeks 4 and 40 of pregnancy in 280 consecutive patients. In addition, multiple random samples were collected during active labor in five patients at term. The samples were taken without regard to complications of pregnancy such as toxemia, diabetes, hypertension, and others. Serial samples were also obtained before and after oxytocin infusion of 100 mU. per minute in women who were admitted for saline abortions in the second trimester of pregnancy (weeks 16 to 20). Blood collection. Polyethylene test tubes containing 372p.g of ethylene diamine tetraacetate disodium (EDTA) (Fisher) and 5Fg of phenanthroline ( I, lo-phenanthrolinei (Merck) per milliliter of blood, respectively. were kept in an ice-cold water bath. The addition of these two chemicals and the chilling of the blood prevent the destruction of oxytocin by oxytocinase. In our laboratory 8-hydroxyquinoline (Merck) did not prevent oxytocinase activity in plasma. EDTA and phenanthroline were dissolved in distilled water and 50~1 were added in each test tube. The phenanthroline does not dissolve in aqueous fluid, so lOOp.1
May Am. J. Obstet.
15, 1974 Gynecol.
of ethyl alcohol has been used to dissolve 10 to 15 mg. of phenanthroline and then rinsed with aqueous fluid before transferring from the test tube to the stock solution or incubating buffer. A 5 ml. quantity of blood was collected from each antecubital vein into a dried heparinized syringe: these were immediately transferred to the polyethylene test tubes which were mixed gently a few times and transferred to an ice bath. The blood kvas centrifuged (International Kefrigerated Centrifuge Model No. B-20) at 4’ C. (4,000 r.p.m., 10 minutes), and the plasma was separated and kept at -20’ C. until it was assayed. Buffer. Phosphate buffer was used (O.O5M, pH 7.51 containing 372pg of EDTA, 48hLg of cystine, and 5pg of phenanthroline per milliliter to prevent oxytocinase activity in the assay system. Higher concentrations of phenanthroline interfere with the antigenantibody reaction, while lower concentrations may not be sufficient to prevent the enzyme activity. In addition, 5,J of human serum albumin (30 per cent solution) and 5pl of normal rabbit serum were added to each milliliter of buffer in order to prevent adsorption of antibodies onto the glassware and destran and also albumin and rabbit serum were added for the purpose of helping to form a thick precipitate to facilitate separation of precipitate bound and free hormone. Merthiolate ( 1OOpg per milliliter) was added to prevent the growth of molds in the buffer. This buffer is hereafter referred to as “incubating buffer.” The incubating buffer \vas used for purification of labelled hormone on the Sephadex column, for dilution of the standard and labeled hormone, and for incubation of the plasma samples at a 1:5 dilution. Phosphate buffer with or without the additives was used for washing the precipitate. Oxytocin standard. Synthetic oxytocin (Lot 73 11 Y 92 B Parke-Davis), with a biologic potency of about 500 I.U. per Inilliproduction, gram, was used for antibody radioiodination, and standards. Antibody production. Antibodies to synthetic oxytocin were produced in rabbits bj
Volume Number
119 2
injecting oxytocin coupled to bovine serum albumin by glutaraldehyde as previously described.l* The most sensitive antibody obtained showed high affinity and specificity bound one half of tracer oxytocin at a final dilution of 1: 50,000. Hormone labeling and purification. Labeling at a specific activity of 100 mc. per milligram was achieved by the method of Hunter and Greenwood’” with the use of either lnlI or lz51 (Union Carbide Corporation, Tuxedo, New York). The hormone was dissolved in distilled water (pH3) and was stored for many months at -20’ C. without deterioration. Phosphate buffer was used for iodination (0.25M, pH 7.5). Chloramine T. (3.42 mg. per milliliter) was dissolved immediately before use in phosphate buffer (0.25M, pH 7.5). The reagents were rapidly added to a 12 by 75 mm. plastic test tube in the following order: (1) 20~1 of buffer, 13lI or 125 I in an amount adequate to (2) provide a specific activity of 100 mc. per milligram, (3) 10~1 ( 1Opg) of oxytocin solution, (4) 20~11 of chloramine T. solution, and (5 ) 50~1 of human serum albumin. Oxytocin is sensitive to reduction by sodium metabisulfite and this step was omitted.16 The radioactive mixture was applied to a Sephadex G-25 column (Medium beads) (1.2 by 25 cm.) together with 100~1of normal plasma containing bromphenol blue as marker. Incubation buffer was used for elution of the labeled hormone. Successive 1 ml. fractions were collected. Two major peaks appeared from the column purification. High molecular weight material, consisting for the most part of damaged, aggregated, and proteinbound oxytocin, was eluted first, followed by intact oxytocin.“, “j The oxytocin peak is inhomogenous, with progressive increase in iodination and specific activity. The selective binding to the dextran of iodinated hormone may be used to advantage in selecting a hormone of high specific activity. On the other hand, the selection of a very retarded fraction, which has been overiodinated, results in a preparation with significant impairment of binding to antibody. In practice a rapid screening test is performed with various
Plasma oxytocin
during
pregnancy
and labor
217
oxytocin 1311or lz51 fractions from the second peak. Free iodide was removed from labeled hormone fractions by adding about 100 mg. per milliliter of 3403 Amberlite (ion-exchange resin) IRA-400 CP (Mallinckrodt, New York) and mixing on a vortex mixer for less than a minute. The amberlite is allowed to settle and the supernatant is used for the screening test. Tracer quantities of each fraction are incubated for several hours with antibody, and the reaction giving the highest antibody bound/free ratio (B/F) is used for the assay purposes. The best fraction is kept at -20° C. until assay, which usually was carried out during the same week. Standard curve. Dilution of unlabeled and labeled oxytocin and antibody was made in incubating buffer. Unlabeled standard hormone was used at concentrations of 0.25 to 2OOpU per milliliter in the buffer containing 1:5 male plasma. The final incubation mixture of 1 ml. contained antibody at a 1:50,000 dilution, and tracer of oxytocin 1311or lz51 of approximately I-2p.U (2 to 4 pg.) . The tubes were incubated for six days for the first antibody reaction and for 12 to 18 hours for the second antibody reaction. Reproducibility of this assayhas been previously reported. I” Corrections were made for nonbindable radioactivity by adding excess first antibody into two tracer tubes on the fifth day of incubation, incubating for a day, followed by the addition of second antibody. All assay standards and samples were set up in duplicate. Plasma was assayed at a final dilution of 1: 5 in an incubation volume of 1 ml. Incubation. Incubation was carried out in 10 by 75 mm. disposable glass test tubes. The first antibody reaction was allowed to proceed for six days and the second antibody reaction for 12 to 18 hours at 4O C. Separation of bound and free labeled hormone. Separation was carried out by one of
several methods: ( 1) double antibody precipitation of the bound hormone, (2) supersaturated ammonium sulfate precipitation of the bound hormone, (3) Carbowax 6,000 precipitation of the bound hormone, (4)
218
Kumaresan
May 15, 1974 Am. J. Obstet. Gynecol.
et al.
Anttbody Tracer lnltlal Bindable Double
RabbII F-10 ‘1 50.000 = l-2 )JU ;2-4 pg’/ml B/F ratlo :093 :005’mean!se radloacitvlty’ 83 IO 01 O/o anttbody technique
N =
0
’
I 25
u 75
50
Synthetic
I 100
Oxytocin
16
125
150
Concentration.
Fig. 1. Standard curve for the radioimmunoassay value of 16 replicate determinations.
oxytocin.
Female
plasma
standard
0
’
1 25
, 50
Synthetic
Fig.
2. Comparison
1 75 Oxytocln
of immunoreactivity
paper chromatoelectrophoresis, or (5 ) talc. Methods 4 and 5 were not satisfactory in our laboratory. The double antibody method was preferred by us and was the one used in these studies. The anti-rabbit gamma globulin antiserum was produced in sheep and used as second antibody. After incubation, the free and bound fractions were
s 200
jJU/ml
of synthetic
I {A) male
175
Each
point
is the mean
plasma
1: 5
curve
I 100
125
Concentration.
between
synthetic
I50
175
200
VU/ml.
oxytocin
and plasma
oxytocin.
separated by centrifuging at 4’ C. (4,000 r.p.m., 10 minutes) and the supernatant was aspirated with disposable pipettes and transferred into disposable plastic test tubes (17 by 100 mm.). The precipitate was washed with 1 ml. of phosphate buffer and recentrifuged. The first and second supernatants were mixed together and counted for one
Volume Number
119 2
Plasma
oxytocin
during
pregnancy
and
labor
219
I :
oxylocin rate
I
iv. intuslon = 100 mU /min.
= / I M +-+
0
10
20
30
40 time
50 in
60
70
e-e Z-2
S.J. -1 .u. FL W.D.
A-A
T.E.
80
90
100
minutes
Fig. 3. Oxytocin infusion and recovery study during human pregnancy.
minute (Picker autowell II). The precipitate was counted separately for another minute. Results
Antiserum specificity. The specificity of the antigen-antibody reaction is rather high. Arginine and lysine vasopressin, for example, cross-react poorly in the assay system. The cross-reactivity of these antibodies to vasopressin is about 1,000 fold less than oxytocin. It has been reported that a variety of structural analogues of oxytocin varied in their cross-reactivity from almost zero to a reactivity actually exceeding that of oxytocin itself? Standard curve. Fig. 1 demonstrates a mean dose-response curve for synthetic oxytocin (N = 16). Complete standard curves were run in all assays. The mean of 83 t 0.01 (S.E.M.) per cent (range, 73 to 90 per cent) of the labeled oxytocin was immunologically bindable in the presence of excess oxytocin antibody. Nonspecifically
trapped labeled oxytocin in the precipitate was 2 + 0.002 per cent (range, 0 to 3). Corrections were made in all assay tubes for the nonbindable radioactivity’s contribution to the “free” counts and for the trapped radioactivity’s contribution to the “bound” counts. The mean initial B/F ratio was 0.93 + 0.05 (range, 0.65 to 1.12). In the presence of unlabeled oxytocin the change of B/F ratio was expressed as per cent of initial B/F ratio and plotted on the vertical axis VS. synthetic oxytocin concentration on the horizontal axis.
Demonstration of parallelism of immunoreactivity. Comparison of immunoreactivity between the synthetic standard oxytocin and human plasma oxytocin is depicted in Fig. 2. The standard curve was set up in 1:5 male plasma and compared to oxytocin in female plasma in three dilutions-l :20, and 1: 10, and 1:5. The results indicate that ( 1) the standard curve in male plasma at, 1:5 dilution is not significantly different from that of the mean standard curve in btiffer
220
Kumaresan
May 15, 1974 Am. J. Obstet. Gynrcol.
et al.
n=
Ii \ 3 a
450
’
400
-
350
-
300
-
250
-
10
17
21
total
22
28
pregnant
18
patient8
31
=
43
45
55
280
.e St
.Y. .. .. ..
.*.
50
0’ 1
49
.: *
0
4-8
.., Q-12
13-18
17-20
weeks
Fig. 4. Plasma
oxytocin
concentrations
only (Fig. 1) , (2) five samples of female plasma (three from patients in labor at term and two from patients in midpregnancy who had intravenous oxytocin infusion) show that the oxytocin content is inversely proportional to the dilution. and (3) there is a clear parallelism between the standard curve in male plasma and the oxytocin level in five samples of diluted female plasma. These data indicate that endogenous plasma oxytocin and synthetic oxytocin react identically in this assay system. Oxytocin infusion-serial samples. Oxytocin in the rate of 100 mu. per minute was infused during midpregnancy in five pregnant patients, and blood was collected every five minutes during and 1, 2, 5, 10. 20, 30, and 40 minutes after cessation of infusion (Fig. 3). From the onset of infusion plasma oxytocin increased markedly with levels of 300 to 85OpU per milliliter at 15 to 20 minutes. After 20 minutes of infusion, the level of plasma oxytocin began to fluctuate. They remained high, however, within
of
during
.:
::
:
21-24
25-28
20-32
*; .. ... .. .. :: ‘.’ ‘:. .
*.
33-W
3740
gestation different
stages of pregnancy.
the 60 minutes of oxytocin infusion. Infusion was stopped after 60 minutes and this was followed by a uniform and orderly decline of oxytocin within 10 to 20 minutes. The levels of oxytocin during and after the infusion of oxytocin represent further evidence that the immunoassayable oxytocin is measured. Oxytocin levels during pregnancy-individual samples. The results of pregnancy samples from 280 patients in various stages of pregnancy are shown in Figs. 4 and 5 and Tables I and II. The mean range of plasma oxytocin levels betlveen weeks 4 and 40 of pregnancy was 66 to 165pU per milliliter, with gradually increasing levels from 25 weeks to term. Fig. 5 illustrates the linear regression between plasma oxytocin level and weeks of gestation in human pregnancy. The correlation coefficient (r) is 0.89. The significance of the correlation coefficient (P) is
Volume Number
119 2
Plasma
oxytocin
during
pregnancy
and
labor
to calculate the linear regression coefficient (P), and 95 per cent
(I’), confi-
221
Y 180
i
r = 0.89
I P c 0.01
1
140 E >
‘20 -
a E
100 -
i
80-
’
60-
E 2 a
4017
20 -
95%
confidence
llmlts , 40
0 0
4
8
12
16 weeks
20
24
28
were analyzed on a computer programmed correlation coefficient (r), significance of the correlation dence limits of the linear regression (shaded area).
were obtained from five patients during active labor, totaling 79 samples. The mean oxytocin level was 181 + 1OpU per milliliter with a range of 35 to 435pU per milliliter. Marked fluctuations of plasma oxytocin were encountered during active labor. Because of random samplings during labor, this information is not sufficient to answer the question whether or not there is more oxytocin secreted during labor than in pregnancy. Comment Beyond the general agreement that oxytocin is secreted during parturitionI and lactation,” the quantitative aspects are not clear. All the bioassays in current use have serious defects with respect to specificity and sensitivity. These difficulties are particularly troublesome when measurement of hormone concentration in the plasma is attempted. “Oxytocin-like” activity has been detected in human blood during normal labor as determined by bioassay.“’ I9 It has been suggested that oxytocin level increased 1OOpU per milliliter or even more. In large animal studies blood concentration of oxytocin was increased during labor and reached a maximum at the time of delivery.20s “I The current status of bioassays for oxytocin has been
36
x
geslatton
Fig. 5. Results
samples. Samples
32
Table I. Mean during
oxytocin
level in women
1 ~z!s
I :Gi
pregnancy
Week-s of gestation Nonpregnant 4-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40
15 17 21 22 28 18 31 43 45 55
Table II. Mean during
pregnancy
38
Mean
69.7
S.D. S.E.M.
53.8 10.4
1.0 66 73 60 88 70 93 117 151 165
*17 214 + 7 513 + 12 2 11 512 + 13 + 14
O-10 3-250 O-275 lo-110 O-250 lo-205 10-235 lo-320 10-335 20-420
oxytocin level in women as tabulated by trimester
1 A,$-;2 No.
<
I K,.,,:
)“,;;:“IC,;Z:““I”,~ 68 74.2 53.5 6.5
119 123.7 79.9 7.3
55 164.7 106.3 14.4
StuAvs. Avs. A vs. B vs. Bvs. C vs. SD.
= One
1 dent’s t I 0.4 4.2 5.2 5.0 5.6 2.4
B C D C D D standard
deviation.
P value < < < < < <
0.5 0.001 0.001 0.001 0.001 0.01
222
Kumaresan
May 15, 1974 Am. J. Obstet. Gynecol.
et al.
carefully summarized by Sawyer.‘” Clearly the results of bioassays cannot be used for comparison with the results of radioimmunoassay. Kadioimmunoassayable oxytocin has been measured in goats’ plasma during labor’ and in pregnant rats-the highest on days 20 and 21 of gestation.” Using radioimmunoassay, Chard and associates!’ measured oxytocin levels in extracted human plasma and reported that the oxytocin was undetectable during labor in 81 per cent of the samples assayed. In the 19 per cent of their samples showing positive results the values ranged from 2 to 18pU per milliliter. They found, though, measurable oxytocin in 76 per cent of 38 samples of umbilical arterial plasma at delivery. They concluded that the baby may be the major source of osytocin for its own delivery. Our study reported here is limited to findings of oxytocin individual samples of 280 nonselected patients in various stages of pregnancy and in 79 random samples of another group of five patients who were in active labor. No data about radioimmunoassayable oxytocin in rnaternal plasma throughout pregnancy are available for comparison with this study. Our findings of oxytocin during labor agree with similar findings of Chard and associates!’ in that the osytocin during labor fluctuates in a matter of minutes (personal communication). They are in variance, hovvever, in the levels and the quantity of oxytocin found. Chard and associates” measured oxytocin levels in the range of 2 to 18pU per milliliter in labor but our findings of plasma oxytocin are considerably higher. The quantitative differences in clinical results could be explained by the differences in our and Chard’s methods, namely: (1 ) sensitive and specific antibody for osytocin. (2) high
purification of labeled hormone, (3) quantity of labeled hormone used as a tracer, (4) duration of incubation at 4’ C., (5) method of blood collection, and (6) extraction of the plasma oxytocin vs. the use of whole plasma. Moreover, the quantitative results and especially the detection of small quantities, such as between 0-1Op.U per milliliter, entirely depend on the quality of the standard curve. The standard curve must be set up for each assay. If the per cent B/F ratio on vertical does not fall gradually and evenly in relation to quantity of oxytocin on horizontal, as an example, 1OpU per milliliter may be easily missed and a false reading could be obtained. The data reported here must be considered preliminary because of marked scatter of results for individual weeks of pregnancy and because of individual samples without regard to pregnancy complications. Moreover, no conclusion could be drawn concerning the question whether or not there is more oxytocin secreted during labor than in pregnancy simply because this study has not been designed to follow individual patients throughout pregnancy and labor and because marked fluctuations of oxytocin were found during labor. The radioimmunoassayable oxytocin level in plasma in this study increased progressively from 66 to 1651~U per milliliter between weeks 4 and 40 of pregnancy. These data are consistent with the theoretical concepts of oxytocin secretion and with osytocinase increasing progressively in the blood of normal pregnant women.z:‘-9” Appreciation is expressed to Dr. Seymour M. Glick for suggestions, guidance, and criticism of this work illustrations.
from the Parke-Davis
REFERENCES
1. Gilliland, P. F., and Prout, T. E.: Metabolism 14: 918, 1965. 2. Gusdon, J. P.: AM. J. OBSTET. GYNECOL.. 98: 526, 1967. 3. Glick, S. M., Kumaresan, P., Kagan, A., and Wheeler. M.: Radioimmunoassay of oxytocin,
and to Dr. Cassian Li The synthetic oxytocin
for medical was a gift
Company.
in Margoulies, M., editor: Protein and peptide Hormones, Amsterdam, 1968, 4. 5.
PolyEx-
cerpta Medica Foundation, pp. 81-83. Chard, T., Kitau, M. J., and Landon, J.: J. Endocrinol. 46: 269 1970. Bashore, R. A.: AM. ‘J. OBSTET. GYNECOL. 113: 488, 1972.
Volume Number
6.
7.
8.
9.
10. 11. 12. 13.
14.
15. 16.
119 2
Seppala, M., Aho, I., Tissari, A., and Rouslahti, E.: AM. J. OBSTET. GYNECOL. 114: 791, 1972. Chard, T., Boyd, N. R. H., Forsling, M. L.: McNeily, A. S., and Landon, J.: J. Endocrinol. 48: 223, 1970. Kumaresan, P., Subramanian, M., Anandarangam, P. B., and Kumaresan, M.: In Program of Fifty-fifth Annual Meeting of The Endocrine Society (Abst. 381), 1973. Chard, T., Hudson, C. N., Edwards, C. R. W., and Boyd, N. R. N.: Nature 234: 352, 1971. Kumaresan, P., Kagan, A., and Glick, S. M.: Science 166: 1160, 1969. Kumaresan, P., Kagan, A., and Glick, S. M.: Nature (London) 230: 468, 1971. Kumaresan, P.: AM. J. OBSTET. GYNECOL. 118: 68, 1974. Kumaresan, P., Anandarangam, P. B., and Vasicka, A.: Paper presented at IV International Congress of Endocrinology, Washington, D. C. (Abst. 516), 1972. Kumaresan, P., Anandarangam, P. B., and at VII World Vasicka, A. : Paper presented Congress of Obstet. Gynecol. Moscow, U. S. S. R. (Abst. 102), 1973. Hunter, W. M., and Greenwood, F. Cl.: Nature (London) 194: 495, 1962. Glick, S. M., Wheeler, M., Kagan, A., and
Plasma
17.
18.
19.
20.
21.
22.
23. 24. 25.
oxytocin
during
pregnancy
and
labor
223
Kumaresan, P. : Pharmacology of Hormonal Polypeptide and Proteins, New York, 1968, Plenum Press, p. 93. Coch, J. A., Brovelto, J., Cabor, H. M., Fielitz, C. A., and Caldeyro-Barcia, R.: AM. J. OBSTET. GYNECOL. 91: 10, 1965. Coch, J. A., Fieltiz, C., Brovelto, J., Cabor, H. M., Coda, H., and Fraga, A.: J. Endocrinol. 40: 137, 1968. Hawker, R. W., Walmsley, C. F., Roberts, V. S., Blackshawe, J. K., and Downes, J. S.: J. Clin. Endocrinol. 21: 985, 1961. Folley, S. J., and Knaggs, G. S.: In Pinkerton, J. H. M., editor: Advances in Oxytocin Research, New York, 1965, Pergamon Press, p. 37. Fitzpartick, R. J., and Walmsley, C. F.: In Pinkerton, J. H. M., editor: Advances in Oxytocin Research, New York, 1965, Pergamon Press, p. 51. Sawyer, W. H.: In Harris, G. W., and Donovan, B. T., editors: The Pituitary Gland, Los Angeles, 1966, University of California Press, p. 288. and Nesvadba, H.: Mh Chem. TUPPY, I-L 88: 977, 1957. Antes, I. G.: AM. J. OBSTET. GYNECOL. 113: 291, 1972. Floyd, W. S., Margulis, R. R., and Woods, C.: Obstet. Gynecol. 41: 553, 1973.
KUMARESAN,
P.
B.
W.
D.V.M.,
ANANDARANGAM,
DIANZON,
ALOIS Brooklyn,
D.V.M. M.D.
VASICKA, New
PH.D.*
M.D.
York
Plasma oxytocin concentrations were measured by radioimmunoassay with the use of unextracted plasma in 285 pregnant patients. In nonpregnant women the mean plasma oxytocin level was
A L T H o u G H oxytocin has been used extensively for the induction of labor in human subjects, information about physiologic levels during human pregnancy and labor is lacking. Oxytocin is known to be a potent stimulant of uterine activity. The hypothesis that oxytocin release occurs as a normal component of the process of parturition remains to be proved. In order to study factors which
initiate labor, and the causes of abnormal labor and parturition, circulating blood levels of oxytocin must be studied. Bioassay techniques have been utilized for these purposes, but most bioassays are relatively insensitive. Radioimmunoassay offers more precise means of measuring oxytocin levels. The successful production of antisera to oxytocin has been reported.‘, * The development of a radioimmunoassay for oxytocin has been described3-6 but detailed results of studies on oxytocin levels in an unexpected human plasma have not been reported. Levels of radioimmunoassayable oxytocin in goats’ and pregnant rats8 have been reported. Oxytocin levels of 12Op.U per milliliter have been reported in goats’ plasma during labor. In pregnant rats the mean oxytocin levels were 24p.U per milliliter on day 18 of pregnancy and 118 and 106pU per milliliter, respectively, on days 20 and 21. Four hours after delivery the oxytocin level fell abruptly in maternal plasma and pituitary. When oxytocin was extracted from
From the State University of New York, Downstate Medical Center, and The Department of Obstetrics and Gynecology of the Coney Island Hospital afiliated with the Maimonides Medical Center. Received Revised Accepted
for publication September October
May
24, 1973.
21, 1973. 3, 1973.
Reprint requests: Dr. Hormonal Laboratories Coney Island Hospital, New York 11235.
P.
Kumaresan,
of Obs./Gyn., Brooklyn,
*Supported by grants from The Population Council of New York and The National Institutes of Child Health and Human Development USPHS HD 04984-01. 215
216
Kumaresan
et al.
the plasma of women during labor and measured by radioimmunoassay, only 19 per cent showed detectable levels, with a range of 2 to 18pU per milliliter.” A very sensitive and specific antibody for oxytocin has been produced in this laboratory, enabling us to develop a radioimmunoassay for oxytocin in whole plasma. Oxytocin antibody has been tested for the dissociation of immunologic activity,“’ for the neutralization of oxytocin in \-iv0 in lactating rats,” and for effect upon litter size in rats.” The objective of this study was to investigate the measurement of oxytocin levels in plasma throughout human pregnancy and random sampling during labor by this radioimmunoassay technique. It had been previously reported by us that oxytocin was detectable in all pregnancy plasmas tested so far.‘:‘. 11 Materials
and
methods
Patient study. Individual samples were obtained between weeks 4 and 40 of pregnancy in 280 consecutive patients. In addition, multiple random samples were collected during active labor in five patients at term. The samples were taken without regard to complications of pregnancy such as toxemia, diabetes, hypertension, and others. Serial samples were also obtained before and after oxytocin infusion of 100 mU. per minute in women who were admitted for saline abortions in the second trimester of pregnancy (weeks 16 to 20). Blood collection. Polyethylene test tubes containing 372p.g of ethylene diamine tetraacetate disodium (EDTA) (Fisher) and 5Fg of phenanthroline ( I, lo-phenanthrolinei (Merck) per milliliter of blood, respectively. were kept in an ice-cold water bath. The addition of these two chemicals and the chilling of the blood prevent the destruction of oxytocin by oxytocinase. In our laboratory 8-hydroxyquinoline (Merck) did not prevent oxytocinase activity in plasma. EDTA and phenanthroline were dissolved in distilled water and 50~1 were added in each test tube. The phenanthroline does not dissolve in aqueous fluid, so lOOp.1
May Am. J. Obstet.
15, 1974 Gynecol.
of ethyl alcohol has been used to dissolve 10 to 15 mg. of phenanthroline and then rinsed with aqueous fluid before transferring from the test tube to the stock solution or incubating buffer. A 5 ml. quantity of blood was collected from each antecubital vein into a dried heparinized syringe: these were immediately transferred to the polyethylene test tubes which were mixed gently a few times and transferred to an ice bath. The blood kvas centrifuged (International Kefrigerated Centrifuge Model No. B-20) at 4’ C. (4,000 r.p.m., 10 minutes), and the plasma was separated and kept at -20’ C. until it was assayed. Buffer. Phosphate buffer was used (O.O5M, pH 7.51 containing 372pg of EDTA, 48hLg of cystine, and 5pg of phenanthroline per milliliter to prevent oxytocinase activity in the assay system. Higher concentrations of phenanthroline interfere with the antigenantibody reaction, while lower concentrations may not be sufficient to prevent the enzyme activity. In addition, 5,J of human serum albumin (30 per cent solution) and 5pl of normal rabbit serum were added to each milliliter of buffer in order to prevent adsorption of antibodies onto the glassware and destran and also albumin and rabbit serum were added for the purpose of helping to form a thick precipitate to facilitate separation of precipitate bound and free hormone. Merthiolate ( 1OOpg per milliliter) was added to prevent the growth of molds in the buffer. This buffer is hereafter referred to as “incubating buffer.” The incubating buffer \vas used for purification of labelled hormone on the Sephadex column, for dilution of the standard and labeled hormone, and for incubation of the plasma samples at a 1:5 dilution. Phosphate buffer with or without the additives was used for washing the precipitate. Oxytocin standard. Synthetic oxytocin (Lot 73 11 Y 92 B Parke-Davis), with a biologic potency of about 500 I.U. per Inilliproduction, gram, was used for antibody radioiodination, and standards. Antibody production. Antibodies to synthetic oxytocin were produced in rabbits bj
Volume Number
119 2
injecting oxytocin coupled to bovine serum albumin by glutaraldehyde as previously described.l* The most sensitive antibody obtained showed high affinity and specificity bound one half of tracer oxytocin at a final dilution of 1: 50,000. Hormone labeling and purification. Labeling at a specific activity of 100 mc. per milligram was achieved by the method of Hunter and Greenwood’” with the use of either lnlI or lz51 (Union Carbide Corporation, Tuxedo, New York). The hormone was dissolved in distilled water (pH3) and was stored for many months at -20’ C. without deterioration. Phosphate buffer was used for iodination (0.25M, pH 7.5). Chloramine T. (3.42 mg. per milliliter) was dissolved immediately before use in phosphate buffer (0.25M, pH 7.5). The reagents were rapidly added to a 12 by 75 mm. plastic test tube in the following order: (1) 20~1 of buffer, 13lI or 125 I in an amount adequate to (2) provide a specific activity of 100 mc. per milligram, (3) 10~1 ( 1Opg) of oxytocin solution, (4) 20~11 of chloramine T. solution, and (5 ) 50~1 of human serum albumin. Oxytocin is sensitive to reduction by sodium metabisulfite and this step was omitted.16 The radioactive mixture was applied to a Sephadex G-25 column (Medium beads) (1.2 by 25 cm.) together with 100~1of normal plasma containing bromphenol blue as marker. Incubation buffer was used for elution of the labeled hormone. Successive 1 ml. fractions were collected. Two major peaks appeared from the column purification. High molecular weight material, consisting for the most part of damaged, aggregated, and proteinbound oxytocin, was eluted first, followed by intact oxytocin.“, “j The oxytocin peak is inhomogenous, with progressive increase in iodination and specific activity. The selective binding to the dextran of iodinated hormone may be used to advantage in selecting a hormone of high specific activity. On the other hand, the selection of a very retarded fraction, which has been overiodinated, results in a preparation with significant impairment of binding to antibody. In practice a rapid screening test is performed with various
Plasma oxytocin
during
pregnancy
and labor
217
oxytocin 1311or lz51 fractions from the second peak. Free iodide was removed from labeled hormone fractions by adding about 100 mg. per milliliter of 3403 Amberlite (ion-exchange resin) IRA-400 CP (Mallinckrodt, New York) and mixing on a vortex mixer for less than a minute. The amberlite is allowed to settle and the supernatant is used for the screening test. Tracer quantities of each fraction are incubated for several hours with antibody, and the reaction giving the highest antibody bound/free ratio (B/F) is used for the assay purposes. The best fraction is kept at -20° C. until assay, which usually was carried out during the same week. Standard curve. Dilution of unlabeled and labeled oxytocin and antibody was made in incubating buffer. Unlabeled standard hormone was used at concentrations of 0.25 to 2OOpU per milliliter in the buffer containing 1:5 male plasma. The final incubation mixture of 1 ml. contained antibody at a 1:50,000 dilution, and tracer of oxytocin 1311or lz51 of approximately I-2p.U (2 to 4 pg.) . The tubes were incubated for six days for the first antibody reaction and for 12 to 18 hours for the second antibody reaction. Reproducibility of this assayhas been previously reported. I” Corrections were made for nonbindable radioactivity by adding excess first antibody into two tracer tubes on the fifth day of incubation, incubating for a day, followed by the addition of second antibody. All assay standards and samples were set up in duplicate. Plasma was assayed at a final dilution of 1: 5 in an incubation volume of 1 ml. Incubation. Incubation was carried out in 10 by 75 mm. disposable glass test tubes. The first antibody reaction was allowed to proceed for six days and the second antibody reaction for 12 to 18 hours at 4O C. Separation of bound and free labeled hormone. Separation was carried out by one of
several methods: ( 1) double antibody precipitation of the bound hormone, (2) supersaturated ammonium sulfate precipitation of the bound hormone, (3) Carbowax 6,000 precipitation of the bound hormone, (4)
218
Kumaresan
May 15, 1974 Am. J. Obstet. Gynecol.
et al.
Anttbody Tracer lnltlal Bindable Double
RabbII F-10 ‘1 50.000 = l-2 )JU ;2-4 pg’/ml B/F ratlo :093 :005’mean!se radloacitvlty’ 83 IO 01 O/o anttbody technique
N =
0
’
I 25
u 75
50
Synthetic
I 100
Oxytocin
16
125
150
Concentration.
Fig. 1. Standard curve for the radioimmunoassay value of 16 replicate determinations.
oxytocin.
Female
plasma
standard
0
’
1 25
, 50
Synthetic
Fig.
2. Comparison
1 75 Oxytocln
of immunoreactivity
paper chromatoelectrophoresis, or (5 ) talc. Methods 4 and 5 were not satisfactory in our laboratory. The double antibody method was preferred by us and was the one used in these studies. The anti-rabbit gamma globulin antiserum was produced in sheep and used as second antibody. After incubation, the free and bound fractions were
s 200
jJU/ml
of synthetic
I {A) male
175
Each
point
is the mean
plasma
1: 5
curve
I 100
125
Concentration.
between
synthetic
I50
175
200
VU/ml.
oxytocin
and plasma
oxytocin.
separated by centrifuging at 4’ C. (4,000 r.p.m., 10 minutes) and the supernatant was aspirated with disposable pipettes and transferred into disposable plastic test tubes (17 by 100 mm.). The precipitate was washed with 1 ml. of phosphate buffer and recentrifuged. The first and second supernatants were mixed together and counted for one
Volume Number
119 2
Plasma
oxytocin
during
pregnancy
and
labor
219
I :
oxylocin rate
I
iv. intuslon = 100 mU /min.
= / I M +-+
0
10
20
30
40 time
50 in
60
70
e-e Z-2
S.J. -1 .u. FL W.D.
A-A
T.E.
80
90
100
minutes
Fig. 3. Oxytocin infusion and recovery study during human pregnancy.
minute (Picker autowell II). The precipitate was counted separately for another minute. Results
Antiserum specificity. The specificity of the antigen-antibody reaction is rather high. Arginine and lysine vasopressin, for example, cross-react poorly in the assay system. The cross-reactivity of these antibodies to vasopressin is about 1,000 fold less than oxytocin. It has been reported that a variety of structural analogues of oxytocin varied in their cross-reactivity from almost zero to a reactivity actually exceeding that of oxytocin itself? Standard curve. Fig. 1 demonstrates a mean dose-response curve for synthetic oxytocin (N = 16). Complete standard curves were run in all assays. The mean of 83 t 0.01 (S.E.M.) per cent (range, 73 to 90 per cent) of the labeled oxytocin was immunologically bindable in the presence of excess oxytocin antibody. Nonspecifically
trapped labeled oxytocin in the precipitate was 2 + 0.002 per cent (range, 0 to 3). Corrections were made in all assay tubes for the nonbindable radioactivity’s contribution to the “free” counts and for the trapped radioactivity’s contribution to the “bound” counts. The mean initial B/F ratio was 0.93 + 0.05 (range, 0.65 to 1.12). In the presence of unlabeled oxytocin the change of B/F ratio was expressed as per cent of initial B/F ratio and plotted on the vertical axis VS. synthetic oxytocin concentration on the horizontal axis.
Demonstration of parallelism of immunoreactivity. Comparison of immunoreactivity between the synthetic standard oxytocin and human plasma oxytocin is depicted in Fig. 2. The standard curve was set up in 1:5 male plasma and compared to oxytocin in female plasma in three dilutions-l :20, and 1: 10, and 1:5. The results indicate that ( 1) the standard curve in male plasma at, 1:5 dilution is not significantly different from that of the mean standard curve in btiffer
220
Kumaresan
May 15, 1974 Am. J. Obstet. Gynrcol.
et al.
n=
Ii \ 3 a
450
’
400
-
350
-
300
-
250
-
10
17
21
total
22
28
pregnant
18
patient8
31
=
43
45
55
280
.e St
.Y. .. .. ..
.*.
50
0’ 1
49
.: *
0
4-8
.., Q-12
13-18
17-20
weeks
Fig. 4. Plasma
oxytocin
concentrations
only (Fig. 1) , (2) five samples of female plasma (three from patients in labor at term and two from patients in midpregnancy who had intravenous oxytocin infusion) show that the oxytocin content is inversely proportional to the dilution. and (3) there is a clear parallelism between the standard curve in male plasma and the oxytocin level in five samples of diluted female plasma. These data indicate that endogenous plasma oxytocin and synthetic oxytocin react identically in this assay system. Oxytocin infusion-serial samples. Oxytocin in the rate of 100 mu. per minute was infused during midpregnancy in five pregnant patients, and blood was collected every five minutes during and 1, 2, 5, 10. 20, 30, and 40 minutes after cessation of infusion (Fig. 3). From the onset of infusion plasma oxytocin increased markedly with levels of 300 to 85OpU per milliliter at 15 to 20 minutes. After 20 minutes of infusion, the level of plasma oxytocin began to fluctuate. They remained high, however, within
of
during
.:
::
:
21-24
25-28
20-32
*; .. ... .. .. :: ‘.’ ‘:. .
*.
33-W
3740
gestation different
stages of pregnancy.
the 60 minutes of oxytocin infusion. Infusion was stopped after 60 minutes and this was followed by a uniform and orderly decline of oxytocin within 10 to 20 minutes. The levels of oxytocin during and after the infusion of oxytocin represent further evidence that the immunoassayable oxytocin is measured. Oxytocin levels during pregnancy-individual samples. The results of pregnancy samples from 280 patients in various stages of pregnancy are shown in Figs. 4 and 5 and Tables I and II. The mean range of plasma oxytocin levels betlveen weeks 4 and 40 of pregnancy was 66 to 165pU per milliliter, with gradually increasing levels from 25 weeks to term. Fig. 5 illustrates the linear regression between plasma oxytocin level and weeks of gestation in human pregnancy. The correlation coefficient (r) is 0.89. The significance of the correlation coefficient (P) is
Volume Number
119 2
Plasma
oxytocin
during
pregnancy
and
labor
to calculate the linear regression coefficient (P), and 95 per cent
(I’), confi-
221
Y 180
i
r = 0.89
I P c 0.01
1
140 E >
‘20 -
a E
100 -
i
80-
’
60-
E 2 a
4017
20 -
95%
confidence
llmlts , 40
0 0
4
8
12
16 weeks
20
24
28
were analyzed on a computer programmed correlation coefficient (r), significance of the correlation dence limits of the linear regression (shaded area).
were obtained from five patients during active labor, totaling 79 samples. The mean oxytocin level was 181 + 1OpU per milliliter with a range of 35 to 435pU per milliliter. Marked fluctuations of plasma oxytocin were encountered during active labor. Because of random samplings during labor, this information is not sufficient to answer the question whether or not there is more oxytocin secreted during labor than in pregnancy. Comment Beyond the general agreement that oxytocin is secreted during parturitionI and lactation,” the quantitative aspects are not clear. All the bioassays in current use have serious defects with respect to specificity and sensitivity. These difficulties are particularly troublesome when measurement of hormone concentration in the plasma is attempted. “Oxytocin-like” activity has been detected in human blood during normal labor as determined by bioassay.“’ I9 It has been suggested that oxytocin level increased 1OOpU per milliliter or even more. In large animal studies blood concentration of oxytocin was increased during labor and reached a maximum at the time of delivery.20s “I The current status of bioassays for oxytocin has been
36
x
geslatton
Fig. 5. Results
samples. Samples
32
Table I. Mean during
oxytocin
level in women
1 ~z!s
I :Gi
pregnancy
Week-s of gestation Nonpregnant 4-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40
15 17 21 22 28 18 31 43 45 55
Table II. Mean during
pregnancy
38
Mean
69.7
S.D. S.E.M.
53.8 10.4
1.0 66 73 60 88 70 93 117 151 165
*17 214 + 7 513 + 12 2 11 512 + 13 + 14
O-10 3-250 O-275 lo-110 O-250 lo-205 10-235 lo-320 10-335 20-420
oxytocin level in women as tabulated by trimester
1 A,$-;2 No.
<
I K,.,,:
)“,;;:“IC,;Z:““I”,~ 68 74.2 53.5 6.5
119 123.7 79.9 7.3
55 164.7 106.3 14.4
StuAvs. Avs. A vs. B vs. Bvs. C vs. SD.
= One
1 dent’s t I 0.4 4.2 5.2 5.0 5.6 2.4
B C D C D D standard
deviation.
P value < < < < < <
0.5 0.001 0.001 0.001 0.001 0.01
222
Kumaresan
May 15, 1974 Am. J. Obstet. Gynecol.
et al.
carefully summarized by Sawyer.‘” Clearly the results of bioassays cannot be used for comparison with the results of radioimmunoassay. Kadioimmunoassayable oxytocin has been measured in goats’ plasma during labor’ and in pregnant rats-the highest on days 20 and 21 of gestation.” Using radioimmunoassay, Chard and associates!’ measured oxytocin levels in extracted human plasma and reported that the oxytocin was undetectable during labor in 81 per cent of the samples assayed. In the 19 per cent of their samples showing positive results the values ranged from 2 to 18pU per milliliter. They found, though, measurable oxytocin in 76 per cent of 38 samples of umbilical arterial plasma at delivery. They concluded that the baby may be the major source of osytocin for its own delivery. Our study reported here is limited to findings of oxytocin individual samples of 280 nonselected patients in various stages of pregnancy and in 79 random samples of another group of five patients who were in active labor. No data about radioimmunoassayable oxytocin in rnaternal plasma throughout pregnancy are available for comparison with this study. Our findings of oxytocin during labor agree with similar findings of Chard and associates!’ in that the osytocin during labor fluctuates in a matter of minutes (personal communication). They are in variance, hovvever, in the levels and the quantity of oxytocin found. Chard and associates” measured oxytocin levels in the range of 2 to 18pU per milliliter in labor but our findings of plasma oxytocin are considerably higher. The quantitative differences in clinical results could be explained by the differences in our and Chard’s methods, namely: (1 ) sensitive and specific antibody for osytocin. (2) high
purification of labeled hormone, (3) quantity of labeled hormone used as a tracer, (4) duration of incubation at 4’ C., (5) method of blood collection, and (6) extraction of the plasma oxytocin vs. the use of whole plasma. Moreover, the quantitative results and especially the detection of small quantities, such as between 0-1Op.U per milliliter, entirely depend on the quality of the standard curve. The standard curve must be set up for each assay. If the per cent B/F ratio on vertical does not fall gradually and evenly in relation to quantity of oxytocin on horizontal, as an example, 1OpU per milliliter may be easily missed and a false reading could be obtained. The data reported here must be considered preliminary because of marked scatter of results for individual weeks of pregnancy and because of individual samples without regard to pregnancy complications. Moreover, no conclusion could be drawn concerning the question whether or not there is more oxytocin secreted during labor than in pregnancy simply because this study has not been designed to follow individual patients throughout pregnancy and labor and because marked fluctuations of oxytocin were found during labor. The radioimmunoassayable oxytocin level in plasma in this study increased progressively from 66 to 1651~U per milliliter between weeks 4 and 40 of pregnancy. These data are consistent with the theoretical concepts of oxytocin secretion and with osytocinase increasing progressively in the blood of normal pregnant women.z:‘-9” Appreciation is expressed to Dr. Seymour M. Glick for suggestions, guidance, and criticism of this work illustrations.
from the Parke-Davis
REFERENCES
1. Gilliland, P. F., and Prout, T. E.: Metabolism 14: 918, 1965. 2. Gusdon, J. P.: AM. J. OBSTET. GYNECOL.. 98: 526, 1967. 3. Glick, S. M., Kumaresan, P., Kagan, A., and Wheeler. M.: Radioimmunoassay of oxytocin,
and to Dr. Cassian Li The synthetic oxytocin
for medical was a gift
Company.
in Margoulies, M., editor: Protein and peptide Hormones, Amsterdam, 1968, 4. 5.
PolyEx-
cerpta Medica Foundation, pp. 81-83. Chard, T., Kitau, M. J., and Landon, J.: J. Endocrinol. 46: 269 1970. Bashore, R. A.: AM. ‘J. OBSTET. GYNECOL. 113: 488, 1972.
Volume Number
6.
7.
8.
9.
10. 11. 12. 13.
14.
15. 16.
119 2
Seppala, M., Aho, I., Tissari, A., and Rouslahti, E.: AM. J. OBSTET. GYNECOL. 114: 791, 1972. Chard, T., Boyd, N. R. H., Forsling, M. L.: McNeily, A. S., and Landon, J.: J. Endocrinol. 48: 223, 1970. Kumaresan, P., Subramanian, M., Anandarangam, P. B., and Kumaresan, M.: In Program of Fifty-fifth Annual Meeting of The Endocrine Society (Abst. 381), 1973. Chard, T., Hudson, C. N., Edwards, C. R. W., and Boyd, N. R. N.: Nature 234: 352, 1971. Kumaresan, P., Kagan, A., and Glick, S. M.: Science 166: 1160, 1969. Kumaresan, P., Kagan, A., and Glick, S. M.: Nature (London) 230: 468, 1971. Kumaresan, P.: AM. J. OBSTET. GYNECOL. 118: 68, 1974. Kumaresan, P., Anandarangam, P. B., and Vasicka, A.: Paper presented at IV International Congress of Endocrinology, Washington, D. C. (Abst. 516), 1972. Kumaresan, P., Anandarangam, P. B., and at VII World Vasicka, A. : Paper presented Congress of Obstet. Gynecol. Moscow, U. S. S. R. (Abst. 102), 1973. Hunter, W. M., and Greenwood, F. Cl.: Nature (London) 194: 495, 1962. Glick, S. M., Wheeler, M., Kagan, A., and
Plasma
17.
18.
19.
20.
21.
22.
23. 24. 25.
oxytocin
during
pregnancy
and
labor
223
Kumaresan, P. : Pharmacology of Hormonal Polypeptide and Proteins, New York, 1968, Plenum Press, p. 93. Coch, J. A., Brovelto, J., Cabor, H. M., Fielitz, C. A., and Caldeyro-Barcia, R.: AM. J. OBSTET. GYNECOL. 91: 10, 1965. Coch, J. A., Fieltiz, C., Brovelto, J., Cabor, H. M., Coda, H., and Fraga, A.: J. Endocrinol. 40: 137, 1968. Hawker, R. W., Walmsley, C. F., Roberts, V. S., Blackshawe, J. K., and Downes, J. S.: J. Clin. Endocrinol. 21: 985, 1961. Folley, S. J., and Knaggs, G. S.: In Pinkerton, J. H. M., editor: Advances in Oxytocin Research, New York, 1965, Pergamon Press, p. 37. Fitzpartick, R. J., and Walmsley, C. F.: In Pinkerton, J. H. M., editor: Advances in Oxytocin Research, New York, 1965, Pergamon Press, p. 51. Sawyer, W. H.: In Harris, G. W., and Donovan, B. T., editors: The Pituitary Gland, Los Angeles, 1966, University of California Press, p. 288. and Nesvadba, H.: Mh Chem. TUPPY, I-L 88: 977, 1957. Antes, I. G.: AM. J. OBSTET. GYNECOL. 113: 291, 1972. Floyd, W. S., Margulis, R. R., and Woods, C.: Obstet. Gynecol. 41: 553, 1973.