0021-972X/78/4705-1106$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 5 Printed in U.S.A.
Plasma Immunoreactive Relaxin Levels in Pregnant and Nonpregnant Women E. MILIKIN O'BYRNE, BYRNES T. CARRIERE, LUXURIE SORENSEN, ALBERT SEGALOFF, CHRISTIAN SCHWABE, AND BERNARD G. STEINETZ Pharma Division, CIBA-GEIGY Corporation (E.M.O'.B., B.G.S.), Ardsley, New York 10502; the Alton Ochsner Medical Foundation (B.T.C., L.S., A.S.), New Orleans, Louisiana 70121 and the Medical University of South Carolina (C.S.) Charleston, South Carolina 29401 ABSTRACT. Immumoreactive relaxin was measured in plasma samples obtained from human volunteers utilizing the RIA procedure of Sherwood et al., as modified by O'Byrne and Steinetz for heterologous plasma samples. Immunoreactive hormone was not detected in samples obtained from men, and only rarely in plasma of nonpregnant women. Immunoreactive relaxin was present as early as the fourth week of pregnancy and was detectable throughout the course of gestation. Im-
A
BOUT 40 yr ago, Pommerenke (1) and L Abramson et al. (2) reported that serum obtained from women in the first trimester of pregnancy induced "relaxation" at the public symphysis when injected into estrogen-primed guinea pigs. Two decades later, Zarrow et al. (3), also using a guinea pig bioassay method, found a relaxin-like substance present in serum throughout human gestation. Relaxin activity was not detected in sera of men or nonpregnant women (2, 3). However, much more recently, Bryant and coworkers (4-7) reported the presence of "immunoreactive relaxin" in sera of men as well as pregnant and nonpregnant women. Bryant et al. (4-7) utilized the NIH porcine relaxin extract NIH-RPl, as immunizing antigen, as standard, and also as radioligand after iodination by the method of Hunter and Greenwood (8). There are several reasons why this work arouses skepticism. 1) NIH-R-P1 is impure, having only about 15% of the biological activity of the purest preparations thus far described and consisting of about eight or nine different components by acrylamide gel electrophoresis (Refs 9, 10 and our own unpublished observations). It is thus not a suitable radioligand
munoreactive relaxin tended to be higher early in pregnancy, and there was no peak just before parturition as occurs in many other species. Our results are at variance with those of Bryant and coworkers, who reported high levels of immunoreactive relaxin in men and nonpregnant as well as pregnant women. The possible reasons for this discrepancy are presented. (J Clin Endocrinol Metab 47: 1106, 1978)
for RIA. 2) Highly purified, biologically active porcine relaxin contains no tyrosine or histidine and fails to iodinate by the method of Hunter and Greenwood (10-16). Thus, it seems possible that the inI-labeled material used by Bryant et al. (4-7) is not relaxin. 3) Our own initial RIAs of human pregnancy plasma (17) suggest that the relaxin levels (also determined with a rabbit antiporcine relaxin antiserum) may actually be 1-2 orders of magnitude lower than those reported by Bryant et al. (4-7). Because of the numerous questions raised by the foregoing, we have measured immunoreactive relaxin in plasma samples obtained from men, nonpregnant women at various stages of the menstrual cycle, women taking steroidal oral contraceptives, menopausal women, and women throughout the course of pregnancy. Relaxin was not detectable in plasma of men and only rarely was detectable in nonpregnant women. The immunoreactive hormone was present throughout pregnancy and appeared to be highest very early in gestation. Materials and Methods Collection of blood samples
Received April 20, 1977. Address requests for reprints to: Bernard G. Steinetz, CIBA-GEIGY Corporation, Ardsley, New York 10501.
Blood samples were collected by venipuncture into heparinized syringes from men, nonpregnant
1106 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 April 2016. at 06:20 For personal use only. No other uses without permission. . All rights reserved.
PLASMA RELAXIN IN PREGNANT WOMEN
4
women (once weekly), women taking norethindrone-mestranol combinations (0.5 mg-0.05 mg, Oracon, Norinyl, and Orthonovum) and women in various stages of pregnancy. Plasma was separated, lyophilized, and stored in a deep freeze until time of assay. Individual plasma samples from pregnant patients were split into aliquots and lyophilized. Samples were then assayed for immunoreactive relaxin independently in two laboratories (C-G and AOMF). Plasma samples obtained from pregnant women were assayed in 0.2- and 0.4-ml volumes in triplicate. Plasma samples from nonpregnant women were assayed in triplicate at the 0.4-ml volume only, except where immunoactivity was encountered. The latter samples were then reassayed at the 0.2and 0.4-ml volumes in triplicate. RIA
s
.
1
The RIA of relaxin was conducted by our modification (17) of the method of Sherwood et al. (13). Purified porcine relaxin was used for immunization of rabbits (13) and highly purified porcine relaxin (CM-A and B; —3000 U/mg) was used for standards and for preparation of polytyrosyl relaxin, as previously described (13, 17). In the assays reported, serum from a single rabbit bleeding (designated R67/12/74) was used. The addition of tyrosine to the relaxin molecule does not affect the biological activity (13), but is necessary for iodination (l2r>I) by the chloramine T procedure of Hunter and Greenwood (8). The porcine relaxin standards (25-1000 pg) are prepared in assay buffer (AOMF) or in buffer containing pooled male human plasma in an amount equivalent (0.2-0.4 ml) to that being assayed as unknowns (CG) as a precaution against introducing nonspecifically interacting plasma constituents. Preliminary experiments showed no detectable immunoactive relaxin in plasma from males or menopausal females. The characteristics of the heterologous RIA are similar to those described by Sherwood et al. (13) for the homologous RIA of porcine relaxin. Because the levels of immunoactive relaxin are so low in peripheral plasma, it was impossible to construct a complete concentration-response curve. However, logit-log concentration-response curves were made using extracts of luteal tissue obtained at cesarean section, and these were compared with the porcine relaxin standard curve. The mean slopes of the porcine standard and the luteal extract were, respectively, —1.420 and —1.344. The mean linear correlation coefficient (picograms of immunoactive relaxin detected per vol luteal extract) was 0.987 (four assays). Furthermore, the immunoactive relaxin concentra-
1107
TABLE 1. Immunoreactive relaxin levels at two concentrations of 40 consecutive plasma samples obtained from pregnant women Immunoreactive relaxin (ng/ml ± SE) Sample nos.
27270-27297 27300-27339
Ratio of activities
n
20 20
0.2-ml Assays
0.4-ml Assays
0.347 ± 0.038 0.360 ± 0.037
0.350 ± 0.031 0.311 ± 0.027
(0.2/0.4 X 100; 100 116
tions computed for the 0.2- and 0.4-ml volumes of plasma were in close agreement, as shown for 40 consecutively assayed samples in Table 1. The sensitivity of the heterologous assay ranged between 25-50 pg equivalents of the porcine relaxin standard (P < 0.05). The reproducibility was ±11.89% for replicate assays of the same plasma sample. The mean intraassay coefficient of variation for 20 unknowns was 7.08%, and the interassay coefficient of variation (AOMF vs. CG) was 14.73%. The exact bioequivalence of human immunoactive relaxin to the pure porcine hormone must await the isolation and purification of the former. However, in a paper published elsewhere (18), extracts of the corpora lutea obtained from women undergoing cesarean section were bioassayed by the classical guinea pig public symphysis palpation method and the results obtained were compared with the immunoactivity of the same samples in the heterologous RIA. The bioactivity was equivalent to about 1.5 /xg pure porcine relaxin/corpus luteum, whereas immunoactivity was about 67% of that value. The correspondence was well within the errors of the assays.
Results Relaxin RIAs were performed on 0.4-ml plasma samples obtained once weekly from four normal volunteers over the period of two menstrual cycles. No immunoreactive relaxin was detected at any stage of the cycle. Plasma samples were obtained weekly for two cycles from four women taking norethindrone-mestranol contraceptives. All samples from three of the women were negative in the relaxin RIA, while one woman consistently showed a relatively high level (0.67-1.18 ng equivalents to porcine relaxin/ml). The average relaxin level tended to be higher in the first trimester of pregnancy than in the second or third trimesters (Fig. 1 and Table 2). There was no tendency for relaxin levels to rise just before
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 April 2016. at 06:20 For personal use only. No other uses without permission. . All rights reserved.
O'BYRNE ET
1108
•ICK&M • 1978 Vol 47 • No 5
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Weeks of Pregnancy FIG. 1. Immunoreactive relaxin in plasma of women throughout the course of gestation. Numbers in parenthesis indicate the numbers of women sampled at each time interval. Vertical bars indicate ±SKM. • , First trimester; ©,
Vol. 47, No. 5 Printed in U.S.A.
Plasma Immunoreactive Relaxin Levels in Pregnant and Nonpregnant Women E. MILIKIN O'BYRNE, BYRNES T. CARRIERE, LUXURIE SORENSEN, ALBERT SEGALOFF, CHRISTIAN SCHWABE, AND BERNARD G. STEINETZ Pharma Division, CIBA-GEIGY Corporation (E.M.O'.B., B.G.S.), Ardsley, New York 10502; the Alton Ochsner Medical Foundation (B.T.C., L.S., A.S.), New Orleans, Louisiana 70121 and the Medical University of South Carolina (C.S.) Charleston, South Carolina 29401 ABSTRACT. Immumoreactive relaxin was measured in plasma samples obtained from human volunteers utilizing the RIA procedure of Sherwood et al., as modified by O'Byrne and Steinetz for heterologous plasma samples. Immunoreactive hormone was not detected in samples obtained from men, and only rarely in plasma of nonpregnant women. Immunoreactive relaxin was present as early as the fourth week of pregnancy and was detectable throughout the course of gestation. Im-
A
BOUT 40 yr ago, Pommerenke (1) and L Abramson et al. (2) reported that serum obtained from women in the first trimester of pregnancy induced "relaxation" at the public symphysis when injected into estrogen-primed guinea pigs. Two decades later, Zarrow et al. (3), also using a guinea pig bioassay method, found a relaxin-like substance present in serum throughout human gestation. Relaxin activity was not detected in sera of men or nonpregnant women (2, 3). However, much more recently, Bryant and coworkers (4-7) reported the presence of "immunoreactive relaxin" in sera of men as well as pregnant and nonpregnant women. Bryant et al. (4-7) utilized the NIH porcine relaxin extract NIH-RPl, as immunizing antigen, as standard, and also as radioligand after iodination by the method of Hunter and Greenwood (8). There are several reasons why this work arouses skepticism. 1) NIH-R-P1 is impure, having only about 15% of the biological activity of the purest preparations thus far described and consisting of about eight or nine different components by acrylamide gel electrophoresis (Refs 9, 10 and our own unpublished observations). It is thus not a suitable radioligand
munoreactive relaxin tended to be higher early in pregnancy, and there was no peak just before parturition as occurs in many other species. Our results are at variance with those of Bryant and coworkers, who reported high levels of immunoreactive relaxin in men and nonpregnant as well as pregnant women. The possible reasons for this discrepancy are presented. (J Clin Endocrinol Metab 47: 1106, 1978)
for RIA. 2) Highly purified, biologically active porcine relaxin contains no tyrosine or histidine and fails to iodinate by the method of Hunter and Greenwood (10-16). Thus, it seems possible that the inI-labeled material used by Bryant et al. (4-7) is not relaxin. 3) Our own initial RIAs of human pregnancy plasma (17) suggest that the relaxin levels (also determined with a rabbit antiporcine relaxin antiserum) may actually be 1-2 orders of magnitude lower than those reported by Bryant et al. (4-7). Because of the numerous questions raised by the foregoing, we have measured immunoreactive relaxin in plasma samples obtained from men, nonpregnant women at various stages of the menstrual cycle, women taking steroidal oral contraceptives, menopausal women, and women throughout the course of pregnancy. Relaxin was not detectable in plasma of men and only rarely was detectable in nonpregnant women. The immunoreactive hormone was present throughout pregnancy and appeared to be highest very early in gestation. Materials and Methods Collection of blood samples
Received April 20, 1977. Address requests for reprints to: Bernard G. Steinetz, CIBA-GEIGY Corporation, Ardsley, New York 10501.
Blood samples were collected by venipuncture into heparinized syringes from men, nonpregnant
1106 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 April 2016. at 06:20 For personal use only. No other uses without permission. . All rights reserved.
PLASMA RELAXIN IN PREGNANT WOMEN
4
women (once weekly), women taking norethindrone-mestranol combinations (0.5 mg-0.05 mg, Oracon, Norinyl, and Orthonovum) and women in various stages of pregnancy. Plasma was separated, lyophilized, and stored in a deep freeze until time of assay. Individual plasma samples from pregnant patients were split into aliquots and lyophilized. Samples were then assayed for immunoreactive relaxin independently in two laboratories (C-G and AOMF). Plasma samples obtained from pregnant women were assayed in 0.2- and 0.4-ml volumes in triplicate. Plasma samples from nonpregnant women were assayed in triplicate at the 0.4-ml volume only, except where immunoactivity was encountered. The latter samples were then reassayed at the 0.2and 0.4-ml volumes in triplicate. RIA
s
.
1
The RIA of relaxin was conducted by our modification (17) of the method of Sherwood et al. (13). Purified porcine relaxin was used for immunization of rabbits (13) and highly purified porcine relaxin (CM-A and B; —3000 U/mg) was used for standards and for preparation of polytyrosyl relaxin, as previously described (13, 17). In the assays reported, serum from a single rabbit bleeding (designated R67/12/74) was used. The addition of tyrosine to the relaxin molecule does not affect the biological activity (13), but is necessary for iodination (l2r>I) by the chloramine T procedure of Hunter and Greenwood (8). The porcine relaxin standards (25-1000 pg) are prepared in assay buffer (AOMF) or in buffer containing pooled male human plasma in an amount equivalent (0.2-0.4 ml) to that being assayed as unknowns (CG) as a precaution against introducing nonspecifically interacting plasma constituents. Preliminary experiments showed no detectable immunoactive relaxin in plasma from males or menopausal females. The characteristics of the heterologous RIA are similar to those described by Sherwood et al. (13) for the homologous RIA of porcine relaxin. Because the levels of immunoactive relaxin are so low in peripheral plasma, it was impossible to construct a complete concentration-response curve. However, logit-log concentration-response curves were made using extracts of luteal tissue obtained at cesarean section, and these were compared with the porcine relaxin standard curve. The mean slopes of the porcine standard and the luteal extract were, respectively, —1.420 and —1.344. The mean linear correlation coefficient (picograms of immunoactive relaxin detected per vol luteal extract) was 0.987 (four assays). Furthermore, the immunoactive relaxin concentra-
1107
TABLE 1. Immunoreactive relaxin levels at two concentrations of 40 consecutive plasma samples obtained from pregnant women Immunoreactive relaxin (ng/ml ± SE) Sample nos.
27270-27297 27300-27339
Ratio of activities
n
20 20
0.2-ml Assays
0.4-ml Assays
0.347 ± 0.038 0.360 ± 0.037
0.350 ± 0.031 0.311 ± 0.027
(0.2/0.4 X 100; 100 116
tions computed for the 0.2- and 0.4-ml volumes of plasma were in close agreement, as shown for 40 consecutively assayed samples in Table 1. The sensitivity of the heterologous assay ranged between 25-50 pg equivalents of the porcine relaxin standard (P < 0.05). The reproducibility was ±11.89% for replicate assays of the same plasma sample. The mean intraassay coefficient of variation for 20 unknowns was 7.08%, and the interassay coefficient of variation (AOMF vs. CG) was 14.73%. The exact bioequivalence of human immunoactive relaxin to the pure porcine hormone must await the isolation and purification of the former. However, in a paper published elsewhere (18), extracts of the corpora lutea obtained from women undergoing cesarean section were bioassayed by the classical guinea pig public symphysis palpation method and the results obtained were compared with the immunoactivity of the same samples in the heterologous RIA. The bioactivity was equivalent to about 1.5 /xg pure porcine relaxin/corpus luteum, whereas immunoactivity was about 67% of that value. The correspondence was well within the errors of the assays.
Results Relaxin RIAs were performed on 0.4-ml plasma samples obtained once weekly from four normal volunteers over the period of two menstrual cycles. No immunoreactive relaxin was detected at any stage of the cycle. Plasma samples were obtained weekly for two cycles from four women taking norethindrone-mestranol contraceptives. All samples from three of the women were negative in the relaxin RIA, while one woman consistently showed a relatively high level (0.67-1.18 ng equivalents to porcine relaxin/ml). The average relaxin level tended to be higher in the first trimester of pregnancy than in the second or third trimesters (Fig. 1 and Table 2). There was no tendency for relaxin levels to rise just before
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 April 2016. at 06:20 For personal use only. No other uses without permission. . All rights reserved.
O'BYRNE ET
1108
•ICK&M • 1978 Vol 47 • No 5
AL.
0.7-1 CO
E
tn
('6!
0.6-
Q.
E 0.5
x JO d> 01 (D
0.4—1
T
urn 1 .,
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I
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8
1 12
1
1 16
1
| 20
.
[—i 24
| 28
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| 32
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| 36
i
| 40
1
1 44
Weeks of Pregnancy FIG. 1. Immunoreactive relaxin in plasma of women throughout the course of gestation. Numbers in parenthesis indicate the numbers of women sampled at each time interval. Vertical bars indicate ±SKM. • , First trimester; ©,
