Effect of human pregnancy rate of oxytocin S. THORNTON,
J. M. DAVISON,
on metabolic clearance
AND
P. H. BAYLIS
Department of Obstetrics and Gynaecology, Princess Mary Maternity Hospital, Newcastle upon Tyne NE2 3BD; and Endocrine Unit, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom
S., J. M. DAVISON, AND P. H. BAYLIS. Effect of pregnancy on metabolic clearance rate of oxytocin. Am.
THORNTON,
human
J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): 1990.-The metabolic clearance rate (MCR) of oxytocin (OT) was determined by use of constant infusion techniques to achieve low and high plasma OT concentrations in 10 women in late pregnancy and again 8-10 wk postpartum (mean plasma oxytocinase activity was 2.1 IU/ml plasma at term and ~0.1 IU/ml plasma 8-10 wk postpartum). At the lower plasma OT concentrations (5.0 and 5.2 pg/ml, pregnant and postpartum, respectively) produced by infusion of 17.9 ng/ min in pregnancy and 4.3 ng/min postpartum, mean MCR of OT was increased fourfold during pregnancy (5.7 t 0.6 and 1.3 t 0.1 l/min, pregnant and postpartum, respectively; P < 0.001). At the higher plasma OT concentrations (8.0 and 8.0 pg/ml, pregnant and postpartum, respectively) produced by infusion of 35.7 ng/min in pregnancy and 8.5 ng/min postpartum, mean MCR of OT was likewise markedly increased during pregnancy compared with postpartum values (7.1 t 1.9 and 1.4 t 0.1 1/ min, respectively; P < 0.01). The MCR of OT was independent of plasma concentration (between 5 and 8 pg/ml) during pregnancy and in the postpartum period. It is concluded that the MCR of OT is increased markedly during human pregnancy. This may be due to concomitant increases in in vivo cystine aminopeptidase activity or other less specific pregnancy-associated metabolic changes. F21-F24,
cystine aminopeptidase; constant infusion; radioimmunoassay
METABOLIC CLEARANCE RATE (MCR) of oxytocin (OT) has been determined by a variety of protocols that rely on the measurement of OT either by bioassay (9) or radioimmunoassay (1, 2, 8, 12). The MCR of OT has been reported to be remarkably similar for a wide range of plasma hormone concentrations and to be similar in pregnant and nonpregnant women (1,12). This is particularly surprising since, in addition to pregnancy-associated cardiovascular changes that could influence renal and/or hepatic blood flow, a circulating aminopeptidase (oxytocinase) is produced by the human placenta, which rapidly degrades OT in vitro. It has been suggested that oxytocinase has no activity in vivo (1, 12), but these studies used OT assays where there may have been failure to completely inhibit oxytocinase activity. This causes OT degradation in patient samples or, in the absence of extraction, degradation of labeled OT in the radioimmunoassay (6), leading to spurious results. No studies to date have determined the MCR of OT in the THE
0363-6119/90
$1.50
Copyright
same women, pregnant and nonpregnant, using an OT radioimmunoassay adequately validated for use in pregnancy. We have therefore combined techniques of complete oxytocinase inhibition (4, 15) with a solid-phase radioimmunoassay (4, 5) to measure the MCR of OT at two plasma hormone concentrations at term during induction of labor and in the same women 8-10 wk postpartum. The results are correlated with the plasma oxytocinase activity (14). PATIENTS
AND
METHODS
Ten healthy women (age range 20-37 yr) were studied. All required induction of labor at term for obstetric indications. Informed consent was obtained for a protocol approved by the Ethical Committee of the Newcastle Health Authority. A 19-gauge indwelling intravenous cannula (BOC; Helsingborg, Sweden) fitted with a three-way tap was inserted into a vein in each forearm 10 min before the onset of the study. Samples of blood (5 ml) were withdrawn at 60-s intervals (n = 6) before infusing synthetic OT. In addition, a single 2-ml sample was taken for determination of oxytocinase activity. Synthetic 02‘ (Syntocinon; Sandoz, UK; 1 mIU = 2 pg) was infused at a constant rate (17.9 ng/min in pregnancy and 4.3 ng/min postpartum). Thirty minutes after the start of infusion further samples of blood (n = 6) were withdrawn at 60-s intervals. The infusion rate was increased (35.7 ng/min pregnancy and 8.5 ng/min postpartum), and further samples (n = 6) were taken after 30 min. In agreement with other workers (l), we have demonstrated that a steady-state plasma concentration was achieved 30 min after commencing a constant OT infusion. The OT concentration of the infusate was accurately determined by direct assay of a suitable dilution, and the result was used for the calculation of MCR. Oxytocinase inhibition. All samples for OT measurement (including those from postpartum patients) were taken into chilled syringes preloaded with 50 ~1 of o-phenanthroline (125 mmol/l) and 50 ~1 of EDTA (1 mol/l) and immediately transferred to a chilled lithium heparin tube. Extraction was commenced within 60 min (Fig. 1). Lower concentrations of inhibitor or delayed extraction result in degradation of OT. Extraction. Plasma samples (2 ml) were extracted by a modification of florisil-acetone technique (3, 4, 13).
0 1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpregu at Macquarie Univ (137.111.162.020) on February 14, 2019.
R21
R22
METABOLIC
CLEARANCE
14
OF OXYTOCIN
RESULTS
1 T
A
0
C
1. Inhibition of oxytocinase activity by o-phenanthroline and EDTA. Synthetic oxytocin was added to pooled pregnancy plasma (n = 5 for each group). o-Phenanthroline (10 rl; 125 mmol/l) and EDTA (1 mol/l) were added per ml of blood. Recovery of OT is shown. A: immediate inhibition and extraction. B: immediate inhibition, extraction at 60 min. C: inhibition at 60 min followed by immediate extraction. FIG.
Recovery was 77, 70, and 76% for 2, 5, and 10 pg added OT/ml plasma, respectively. Radioimmunoassay of OT. Plasma OT was measured in triplicate using a solid-phase radioimmunoassay (5), whereby the antibody was coupled to activated cellulose (17) and the OT/OT-antibody cellulose complex was separated at 1,500 g. Radiolabeled OT was produced by a modification of the technique described by Greenwood (10). The mean binding of labeled hormone in the absence of unlabeled hormone was 12.6 +- 0.3%, nonspecific binding was 0.2 + O.Ol%, and 50% displacement of radiolabeled OT occurred at 3.9 + 0.1 pg of unlabeled OT (n = 120). The assay detection limit was < 0.7 pg/ tube, which corresponds to 1.4 pg OT/ml of the original plasma sample. The intra-assay and interassay coefficients of variation were 9 and 14.8%, respectively, at 10 pg OT/ml plasma. Cross-reactivity with neurohypophysial hormones and OT analogues was minimal (5); the relative displacement of 50% ‘251-labeled oxytocin as a percentage of OT required to produce the same displacement was as follows: oxytocin 100%; arginine vasopressin 0.05%; lysine vasopressin 0.01%; arginine vasotocin 0.06%; tocinoic acid 1.2%; mesotocin 0.5%; and isotocin 0.3%. Measurement
RATE
Rate of infusion. The actual rates of infusion were, by design, different in the pregnant and postpartum states to achieve similar plasma OT concentrations. The mean rates were 17.9 + 1.4 and 35.7 f 1.4 ng/min during pregnancy and 4.3 rt 0.3 and 8.5 + 0.5 ng/min postpartum. Plasma OT concentrations. The mean basal plasma OT concentration was 1.5 + 0.3 and 1.7 + 0.5 pg/ml during pregnancy and postpartum, respectively (P > 0.05). The mean plasma OT concentration at the lower infusion rate was 5.0 k 0.5 pg/ml in pregnancy and 5.2 + 0.4 pg/ ml postpartum (P > 0.05). At the higher infusion rate the mean plasma OT concentration was 8.0 + 0.9 pg/ml in pregnancy and 8.0 + 0.3 pg/ml postpartum (P > 0.05; Fig. 2). MCR of OT at different plasma concentrations. The MCR of OT was independent of the plasma hormone concentration. During pregnancy the mean MCR was 5.7 + 0.6 and 7.1 + 1.9 l/min at plasma 01‘ concentrations of 5.0 and 8.0 pg/ml, respectively (P > 0.05). Postpartum the mean MCR was 1.3 + 0.1 and 1.4 k 0.1 l/min at p>o 05 I
1
Limit of
Detection A
B
C
D
2. Plasma oxytocin concentration during infusion. A: pregnant, low infusion rate. B: nonpregnant, low infusion rate. C: pregnant, high infusion rate. D: nonpregnant, high infusion rate. Hatched bars, results from pregnant women, and black bars, results from nonpregnant women. FIG.
pco.01 I
Oxytocinase activity determined by S-benzyl-L-cysteine-4p-nitroanilide radation (14). The change in absorbance at 410 nm determined at 37°C for 10 min. Clearance calculations. The MCR was calculated cording to of oxytocinase.
MCR (ml/min)
=
was degwas
p 0.05).
CLEARANCE
of 5.2 and 8.0 pg/ml, respec-
Comparison of MCR during pregnancy and postpartum.
At the lower plasma OT concentrations the MCR of OT was markedly increased during pregnancy in the same subjects (5.7 t 0.6 and 1.3t 0.1 l/min during pregnancy and postpartum, respectively; P c 0.001).This difference was also apparent at the higher plasma OT concentration (7.1 t 1.9 and 1.4 t 0.1 l/min during pregnancy and postpartum, respectively; P < 0.01;Fig. 3). Plasma oxytocinase activity. The mean plasma oxytocinase activity was high during pregnancy at term (2.1t 0.2 IU/ml plasma) and extremely low (C 0.1 IU/ml plasma) in the postpartum study. Correlation
between
MCR
and oxytocinase
activity.
There was a significant correlation within all subjects between MCR and oxytocinase activity during and after pregnancy (r = 0.63; P C 0.001). There was no significant correlation between MCR and oxytocinase activity during pregnancy so that individuals with high oxytocinase levels did not necessarily have higher MCRs. DISCUSSION
Previous workers (1, 12) have failed to document an increase in MCR during gestation compared with nonpregnant patients because of inadequate oxytocinase inhibition during sample collection and the use of unmatched groups of subjects. For instance, Amico and coworkers (1) compared the MCR of OT in pregnant women at term with male volunteers and for oxytocinase inhibition utilized o-phenanthroline and EDTA (5 and 375 pg/ml blood, respectively) at concentrations that were much lower than in this study (250 pg/ml blood and 3.72 mg/ml blood, respectively). Indeed, we have been unable to demonstrate complete oxytocinase inhibition with reduced inhibitor concentrations (4). Similarly, unmatched groups of pregnant, nonpregnant, and male volunteers were studied by Leake et al. (12). Oxytocinase inhibition was dependent on chilling during plasma separation and the addition of acetone, both of which we have found to be inadequate. Oxytocinase is almost exclusively a pregnancy enzyme, and its inhibition is therefore not necessary in nonpregnant individuals. Reassuringly, the reported (1, 2, 8, 12) MCRs of OT in men and nonpregnant women (1.1-1.9 l/min, assuming a mean body weight of 70 kg) are remarkably similar to values in postpartum women in our study (1.3-1.4l/min). Furthermore, the observation that the MCR of OT is independent of the plasma hormone concentration (within therapeutic levels) is also supported by our results. In marked contrast to other studies, however, we have demonstrated a profound increase in the MCR of OT during pregnancy. The reason for the increment is not apparent and we can only speculate on the cause. It may be due to in vivo OT degradation by oxytocinase and/or pregnancy-associated changes in hepatic or renal function. The former is more likely, since an increase in oxytocinase activity occurred in the same patients during
RATE
OF
R23
OXYTOCIN
pregnancy. Of interest, we have demonstrated’ that the MCR of OT is not increased at 14-16 wk gestation, a time when gestational hepatic and renal adaptations are maximal but increments in oxytocinase activity are not evident. Moreover, the increase in MCR is unlikely to be caused solely by an increase in hepatic and renal function, since the pregnancy-associated increase in blood flow to these organs (controversial for liver and 30-50% for kidney) is much less than the proportional increase in MCR. Arginine vasopressin (AVP) differs from OT by two amino acids and is rapidly degraded in vitro by the same group of cystine aminopeptidases (vasopressinase) as OT. Davison and co-workers (7), utilizing similar techniques for complete vasopressinase inhibition, have demonstrated three- to fourfold increases in the MCR of AVP during pregnancy, again possibly due to in vivo degradation by cystine aminopeptidase produced by the human placenta, which also has a considerable capacity to degrade vasopressin in situ (11). The accurate determination of MCR by an infusion technique relies on a constant rate of endogenous OT release. Increases in endogenous release result in an apparent decrease in the MCR. As our previous work (16) demonstrated low plasma OT concentrations throughout the first stage of labor, it is extremely unlikely that decreases in endogenous OT production in the present study caused an increase in the MCR. In conclusion, we have demonstrated that the MCR of OT is significantly increased during gestation when oxytocinase activity is high and that, within the range studied, the MCR of OT is independent of the plasma OT concentration. We thank the consultant and nursing staff of the Princess Mary Maternity Hospital, Newcastle upon Tyne, for allowing us to study patients under their care, the patients who cooperated in this study, and the department of Statistics, University of Newcastle upon Tyne, for their assistance. We are grateful for financial support from the Medical Research Council (UK) and the Scientific and Research Committee, Newcastle Health Authority. Address for reprint requests: S. Thornton, Princess Mary Maternity Hospital, Great North Road, Newcastle upon Tyne NE2 3BD, UK. Received
6 October
1989; accepted
in final
form
23 February
1990.
REFERENCES 1. AMICO, J. A., J. SEITCHIK, AND A. G. ROBINSON. Studies of oxytocin in plasma of women during hypocontractile labor. J. Clin. Endocrinol. Metab. 58: 274-279, 1984. 2. AMICO, J. A., J. S. ULBRECHT, AND A. G. ROBINSON. Clearance studies of oxytocin in humans using radioimmunoassay measurements of the hormone in plasma and urine. J. Clin. Endocrinol. Metab. 64: 340-345, 1987. 3. BEARDWELL, C. G. Radioimmunoassay of arginine vasopressin in human plasma. J. Clin. Endocrinol. Metab. 33: 254-260, 1971. 4. BURD, J. M., J. M. DAVISON, D. R. WEIGHTMAN, AND P. H. ’ The MCR of OT was determined in two women admitted for midtrimester termination of pregnancy. The experimental protocol was identical to that used in term pregnancy, but subjects were not invited to return for nonpregnant determination of MCR. The OT infusion rate was 32.4 and 64.8 ng/min, producing a mean plasma OT concentration of 21 and 37.4 pg/ml, respectively. The mean MCR of OT was 1.7 and 1.8 l/min at the low and high rates of infusion, respectively. Plasma oxytocinase activity was 0.11 IU/ml plasma.
Downloaded from www.physiology.org/journal/ajpregu at Macquarie Univ (137.111.162.020) on February 14, 2019.
R24
5. 6. 7.
8.
9.
10.
METABOLIC
CLEARANCE
BAYLIS. Evaluation of enzyme inhibitors of pregnancy associated oxytocinase: application to the measurement of plasma immunoreactive oxytocin during human labour. Actu Endocrinol. 114: 458464,1987. BURD, J. M., D. R. WEIGHTMAN, AND P. H. BAYLIS. Solid phase radioimmunoassay for the direct measurement of human plasma oxytocin. J. Immunoassay 6: 227-243,1985. CHARD, T. The radioimmunoassay of vasopressin and oxytocin. J. Endocrinol. 58: 143-160, 1973. DAVISON, J. M., E. A. SHEILLS, W. M. BARRON, A. G. ROBINSON, AND M. D. LINDHEIMER. Changes in the metabolic clearance of vasopressin and in plasma vasopressinase throughout human pregnancy. J. Clin. Invest. 83: 1313-1318, 1989. DAWOOD, M. Y., 0. YLIKORKALA, D. TRIVEDI, AND R. GUPTA. Oxytocin levels and disappearance rate and plasma follicle-stimulating hormone and Luteinizing hormone after oxytocin infusion in men. J. Clin. Endocrinol. Metab. 50: 397-400, 1980. FABIAN, M., M. L. FORSLING, J. J. JONES, AND J. S. PRYOR. The clearance and antidiuretic potency of neurohypophysial hormones in man, and their plasma binding and stability. J. Physiol. Lond. 204:653-668,1969. GREENWOOD. F. C.. W. M. HUNTER. AND J. S. GLOVER. The - -_---
RATE
11.
12.
13*
14 . 15 ’ 16. 17.
OF OXYTOCIN
preparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem. J. 89: 114, 1963. LANDON, M. J., D. K. COPAS, E. A. SHEILLS, AND J. M. DAVISON. Degradation of radiolabelled arginine vasopressin (‘“?-AVP) by the human placenta perfused in vitro. Br. J. Obstet. Gynuecol. 95: 488-492,1988. LEAKE, R. D., R. E. WEITZMAN, AND D. A. FISHER. Pharmacokinetics of oxytocin in the human subject. Obstet. Gynecol. 56: 701704,198O. ROOKE, P., AND P. H. BAYLIS. A new and sensitive radioimmunoassay for plasma arginine vasopressin. J. Immunoassay 3: 115-131, 1982. SMALL, C. W., AND W. B. WATKINS. S-benzyl-1-cysteine-4p-nitroanilide. A new substrate for the determination of oxytocinase with improved specificity. Biochem. Med. 9: 103-112,1974. THORNTON, S., J. M. DAVISON, AND P. H. BAYLIS. A novel strategy for inhibition of oxytocinase activity during frequent blood sampling (Abstract). Clin. Sci. Lond. 73, Suppl. 17: 13P, 1987. THORNTON, S., J. M. DAVISON, AND P. H. BAYLIS. The progress of labour is not dependent upon plasma oxytocin. In: Eicosanoids and Fatty Acids (Band 4). Vienna, Austria: Facultas, 1988, p. 149. WIDE, L. Radioimmunoassays employing immunosorbents. Actu Endocrinol. Suppl. 142: 207-221,1969.
Downloaded from www.physiology.org/journal/ajpregu at Macquarie Univ (137.111.162.020) on February 14, 2019.
J. M. DAVISON,
on metabolic clearance
AND
P. H. BAYLIS
Department of Obstetrics and Gynaecology, Princess Mary Maternity Hospital, Newcastle upon Tyne NE2 3BD; and Endocrine Unit, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom
S., J. M. DAVISON, AND P. H. BAYLIS. Effect of pregnancy on metabolic clearance rate of oxytocin. Am.
THORNTON,
human
J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): 1990.-The metabolic clearance rate (MCR) of oxytocin (OT) was determined by use of constant infusion techniques to achieve low and high plasma OT concentrations in 10 women in late pregnancy and again 8-10 wk postpartum (mean plasma oxytocinase activity was 2.1 IU/ml plasma at term and ~0.1 IU/ml plasma 8-10 wk postpartum). At the lower plasma OT concentrations (5.0 and 5.2 pg/ml, pregnant and postpartum, respectively) produced by infusion of 17.9 ng/ min in pregnancy and 4.3 ng/min postpartum, mean MCR of OT was increased fourfold during pregnancy (5.7 t 0.6 and 1.3 t 0.1 l/min, pregnant and postpartum, respectively; P < 0.001). At the higher plasma OT concentrations (8.0 and 8.0 pg/ml, pregnant and postpartum, respectively) produced by infusion of 35.7 ng/min in pregnancy and 8.5 ng/min postpartum, mean MCR of OT was likewise markedly increased during pregnancy compared with postpartum values (7.1 t 1.9 and 1.4 t 0.1 1/ min, respectively; P < 0.01). The MCR of OT was independent of plasma concentration (between 5 and 8 pg/ml) during pregnancy and in the postpartum period. It is concluded that the MCR of OT is increased markedly during human pregnancy. This may be due to concomitant increases in in vivo cystine aminopeptidase activity or other less specific pregnancy-associated metabolic changes. F21-F24,
cystine aminopeptidase; constant infusion; radioimmunoassay
METABOLIC CLEARANCE RATE (MCR) of oxytocin (OT) has been determined by a variety of protocols that rely on the measurement of OT either by bioassay (9) or radioimmunoassay (1, 2, 8, 12). The MCR of OT has been reported to be remarkably similar for a wide range of plasma hormone concentrations and to be similar in pregnant and nonpregnant women (1,12). This is particularly surprising since, in addition to pregnancy-associated cardiovascular changes that could influence renal and/or hepatic blood flow, a circulating aminopeptidase (oxytocinase) is produced by the human placenta, which rapidly degrades OT in vitro. It has been suggested that oxytocinase has no activity in vivo (1, 12), but these studies used OT assays where there may have been failure to completely inhibit oxytocinase activity. This causes OT degradation in patient samples or, in the absence of extraction, degradation of labeled OT in the radioimmunoassay (6), leading to spurious results. No studies to date have determined the MCR of OT in the THE
0363-6119/90
$1.50
Copyright
same women, pregnant and nonpregnant, using an OT radioimmunoassay adequately validated for use in pregnancy. We have therefore combined techniques of complete oxytocinase inhibition (4, 15) with a solid-phase radioimmunoassay (4, 5) to measure the MCR of OT at two plasma hormone concentrations at term during induction of labor and in the same women 8-10 wk postpartum. The results are correlated with the plasma oxytocinase activity (14). PATIENTS
AND
METHODS
Ten healthy women (age range 20-37 yr) were studied. All required induction of labor at term for obstetric indications. Informed consent was obtained for a protocol approved by the Ethical Committee of the Newcastle Health Authority. A 19-gauge indwelling intravenous cannula (BOC; Helsingborg, Sweden) fitted with a three-way tap was inserted into a vein in each forearm 10 min before the onset of the study. Samples of blood (5 ml) were withdrawn at 60-s intervals (n = 6) before infusing synthetic OT. In addition, a single 2-ml sample was taken for determination of oxytocinase activity. Synthetic 02‘ (Syntocinon; Sandoz, UK; 1 mIU = 2 pg) was infused at a constant rate (17.9 ng/min in pregnancy and 4.3 ng/min postpartum). Thirty minutes after the start of infusion further samples of blood (n = 6) were withdrawn at 60-s intervals. The infusion rate was increased (35.7 ng/min pregnancy and 8.5 ng/min postpartum), and further samples (n = 6) were taken after 30 min. In agreement with other workers (l), we have demonstrated that a steady-state plasma concentration was achieved 30 min after commencing a constant OT infusion. The OT concentration of the infusate was accurately determined by direct assay of a suitable dilution, and the result was used for the calculation of MCR. Oxytocinase inhibition. All samples for OT measurement (including those from postpartum patients) were taken into chilled syringes preloaded with 50 ~1 of o-phenanthroline (125 mmol/l) and 50 ~1 of EDTA (1 mol/l) and immediately transferred to a chilled lithium heparin tube. Extraction was commenced within 60 min (Fig. 1). Lower concentrations of inhibitor or delayed extraction result in degradation of OT. Extraction. Plasma samples (2 ml) were extracted by a modification of florisil-acetone technique (3, 4, 13).
0 1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpregu at Macquarie Univ (137.111.162.020) on February 14, 2019.
R21
R22
METABOLIC
CLEARANCE
14
OF OXYTOCIN
RESULTS
1 T
A
0
C
1. Inhibition of oxytocinase activity by o-phenanthroline and EDTA. Synthetic oxytocin was added to pooled pregnancy plasma (n = 5 for each group). o-Phenanthroline (10 rl; 125 mmol/l) and EDTA (1 mol/l) were added per ml of blood. Recovery of OT is shown. A: immediate inhibition and extraction. B: immediate inhibition, extraction at 60 min. C: inhibition at 60 min followed by immediate extraction. FIG.
Recovery was 77, 70, and 76% for 2, 5, and 10 pg added OT/ml plasma, respectively. Radioimmunoassay of OT. Plasma OT was measured in triplicate using a solid-phase radioimmunoassay (5), whereby the antibody was coupled to activated cellulose (17) and the OT/OT-antibody cellulose complex was separated at 1,500 g. Radiolabeled OT was produced by a modification of the technique described by Greenwood (10). The mean binding of labeled hormone in the absence of unlabeled hormone was 12.6 +- 0.3%, nonspecific binding was 0.2 + O.Ol%, and 50% displacement of radiolabeled OT occurred at 3.9 + 0.1 pg of unlabeled OT (n = 120). The assay detection limit was < 0.7 pg/ tube, which corresponds to 1.4 pg OT/ml of the original plasma sample. The intra-assay and interassay coefficients of variation were 9 and 14.8%, respectively, at 10 pg OT/ml plasma. Cross-reactivity with neurohypophysial hormones and OT analogues was minimal (5); the relative displacement of 50% ‘251-labeled oxytocin as a percentage of OT required to produce the same displacement was as follows: oxytocin 100%; arginine vasopressin 0.05%; lysine vasopressin 0.01%; arginine vasotocin 0.06%; tocinoic acid 1.2%; mesotocin 0.5%; and isotocin 0.3%. Measurement
RATE
Rate of infusion. The actual rates of infusion were, by design, different in the pregnant and postpartum states to achieve similar plasma OT concentrations. The mean rates were 17.9 + 1.4 and 35.7 f 1.4 ng/min during pregnancy and 4.3 rt 0.3 and 8.5 + 0.5 ng/min postpartum. Plasma OT concentrations. The mean basal plasma OT concentration was 1.5 + 0.3 and 1.7 + 0.5 pg/ml during pregnancy and postpartum, respectively (P > 0.05). The mean plasma OT concentration at the lower infusion rate was 5.0 k 0.5 pg/ml in pregnancy and 5.2 + 0.4 pg/ ml postpartum (P > 0.05). At the higher infusion rate the mean plasma OT concentration was 8.0 + 0.9 pg/ml in pregnancy and 8.0 + 0.3 pg/ml postpartum (P > 0.05; Fig. 2). MCR of OT at different plasma concentrations. The MCR of OT was independent of the plasma hormone concentration. During pregnancy the mean MCR was 5.7 + 0.6 and 7.1 + 1.9 l/min at plasma 01‘ concentrations of 5.0 and 8.0 pg/ml, respectively (P > 0.05). Postpartum the mean MCR was 1.3 + 0.1 and 1.4 k 0.1 l/min at p>o 05 I
1
Limit of
Detection A
B
C
D
2. Plasma oxytocin concentration during infusion. A: pregnant, low infusion rate. B: nonpregnant, low infusion rate. C: pregnant, high infusion rate. D: nonpregnant, high infusion rate. Hatched bars, results from pregnant women, and black bars, results from nonpregnant women. FIG.
pco.01 I
Oxytocinase activity determined by S-benzyl-L-cysteine-4p-nitroanilide radation (14). The change in absorbance at 410 nm determined at 37°C for 10 min. Clearance calculations. The MCR was calculated cording to of oxytocinase.
MCR (ml/min)
=
was degwas
p 0.05).
CLEARANCE
of 5.2 and 8.0 pg/ml, respec-
Comparison of MCR during pregnancy and postpartum.
At the lower plasma OT concentrations the MCR of OT was markedly increased during pregnancy in the same subjects (5.7 t 0.6 and 1.3t 0.1 l/min during pregnancy and postpartum, respectively; P c 0.001).This difference was also apparent at the higher plasma OT concentration (7.1 t 1.9 and 1.4 t 0.1 l/min during pregnancy and postpartum, respectively; P < 0.01;Fig. 3). Plasma oxytocinase activity. The mean plasma oxytocinase activity was high during pregnancy at term (2.1t 0.2 IU/ml plasma) and extremely low (C 0.1 IU/ml plasma) in the postpartum study. Correlation
between
MCR
and oxytocinase
activity.
There was a significant correlation within all subjects between MCR and oxytocinase activity during and after pregnancy (r = 0.63; P C 0.001). There was no significant correlation between MCR and oxytocinase activity during pregnancy so that individuals with high oxytocinase levels did not necessarily have higher MCRs. DISCUSSION
Previous workers (1, 12) have failed to document an increase in MCR during gestation compared with nonpregnant patients because of inadequate oxytocinase inhibition during sample collection and the use of unmatched groups of subjects. For instance, Amico and coworkers (1) compared the MCR of OT in pregnant women at term with male volunteers and for oxytocinase inhibition utilized o-phenanthroline and EDTA (5 and 375 pg/ml blood, respectively) at concentrations that were much lower than in this study (250 pg/ml blood and 3.72 mg/ml blood, respectively). Indeed, we have been unable to demonstrate complete oxytocinase inhibition with reduced inhibitor concentrations (4). Similarly, unmatched groups of pregnant, nonpregnant, and male volunteers were studied by Leake et al. (12). Oxytocinase inhibition was dependent on chilling during plasma separation and the addition of acetone, both of which we have found to be inadequate. Oxytocinase is almost exclusively a pregnancy enzyme, and its inhibition is therefore not necessary in nonpregnant individuals. Reassuringly, the reported (1, 2, 8, 12) MCRs of OT in men and nonpregnant women (1.1-1.9 l/min, assuming a mean body weight of 70 kg) are remarkably similar to values in postpartum women in our study (1.3-1.4l/min). Furthermore, the observation that the MCR of OT is independent of the plasma hormone concentration (within therapeutic levels) is also supported by our results. In marked contrast to other studies, however, we have demonstrated a profound increase in the MCR of OT during pregnancy. The reason for the increment is not apparent and we can only speculate on the cause. It may be due to in vivo OT degradation by oxytocinase and/or pregnancy-associated changes in hepatic or renal function. The former is more likely, since an increase in oxytocinase activity occurred in the same patients during
RATE
OF
R23
OXYTOCIN
pregnancy. Of interest, we have demonstrated’ that the MCR of OT is not increased at 14-16 wk gestation, a time when gestational hepatic and renal adaptations are maximal but increments in oxytocinase activity are not evident. Moreover, the increase in MCR is unlikely to be caused solely by an increase in hepatic and renal function, since the pregnancy-associated increase in blood flow to these organs (controversial for liver and 30-50% for kidney) is much less than the proportional increase in MCR. Arginine vasopressin (AVP) differs from OT by two amino acids and is rapidly degraded in vitro by the same group of cystine aminopeptidases (vasopressinase) as OT. Davison and co-workers (7), utilizing similar techniques for complete vasopressinase inhibition, have demonstrated three- to fourfold increases in the MCR of AVP during pregnancy, again possibly due to in vivo degradation by cystine aminopeptidase produced by the human placenta, which also has a considerable capacity to degrade vasopressin in situ (11). The accurate determination of MCR by an infusion technique relies on a constant rate of endogenous OT release. Increases in endogenous release result in an apparent decrease in the MCR. As our previous work (16) demonstrated low plasma OT concentrations throughout the first stage of labor, it is extremely unlikely that decreases in endogenous OT production in the present study caused an increase in the MCR. In conclusion, we have demonstrated that the MCR of OT is significantly increased during gestation when oxytocinase activity is high and that, within the range studied, the MCR of OT is independent of the plasma OT concentration. We thank the consultant and nursing staff of the Princess Mary Maternity Hospital, Newcastle upon Tyne, for allowing us to study patients under their care, the patients who cooperated in this study, and the department of Statistics, University of Newcastle upon Tyne, for their assistance. We are grateful for financial support from the Medical Research Council (UK) and the Scientific and Research Committee, Newcastle Health Authority. Address for reprint requests: S. Thornton, Princess Mary Maternity Hospital, Great North Road, Newcastle upon Tyne NE2 3BD, UK. Received
6 October
1989; accepted
in final
form
23 February
1990.
REFERENCES 1. AMICO, J. A., J. SEITCHIK, AND A. G. ROBINSON. Studies of oxytocin in plasma of women during hypocontractile labor. J. Clin. Endocrinol. Metab. 58: 274-279, 1984. 2. AMICO, J. A., J. S. ULBRECHT, AND A. G. ROBINSON. Clearance studies of oxytocin in humans using radioimmunoassay measurements of the hormone in plasma and urine. J. Clin. Endocrinol. Metab. 64: 340-345, 1987. 3. BEARDWELL, C. G. Radioimmunoassay of arginine vasopressin in human plasma. J. Clin. Endocrinol. Metab. 33: 254-260, 1971. 4. BURD, J. M., J. M. DAVISON, D. R. WEIGHTMAN, AND P. H. ’ The MCR of OT was determined in two women admitted for midtrimester termination of pregnancy. The experimental protocol was identical to that used in term pregnancy, but subjects were not invited to return for nonpregnant determination of MCR. The OT infusion rate was 32.4 and 64.8 ng/min, producing a mean plasma OT concentration of 21 and 37.4 pg/ml, respectively. The mean MCR of OT was 1.7 and 1.8 l/min at the low and high rates of infusion, respectively. Plasma oxytocinase activity was 0.11 IU/ml plasma.
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5. 6. 7.
8.
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METABOLIC
CLEARANCE
BAYLIS. Evaluation of enzyme inhibitors of pregnancy associated oxytocinase: application to the measurement of plasma immunoreactive oxytocin during human labour. Actu Endocrinol. 114: 458464,1987. BURD, J. M., D. R. WEIGHTMAN, AND P. H. BAYLIS. Solid phase radioimmunoassay for the direct measurement of human plasma oxytocin. J. Immunoassay 6: 227-243,1985. CHARD, T. The radioimmunoassay of vasopressin and oxytocin. J. Endocrinol. 58: 143-160, 1973. DAVISON, J. M., E. A. SHEILLS, W. M. BARRON, A. G. ROBINSON, AND M. D. LINDHEIMER. Changes in the metabolic clearance of vasopressin and in plasma vasopressinase throughout human pregnancy. J. Clin. Invest. 83: 1313-1318, 1989. DAWOOD, M. Y., 0. YLIKORKALA, D. TRIVEDI, AND R. GUPTA. Oxytocin levels and disappearance rate and plasma follicle-stimulating hormone and Luteinizing hormone after oxytocin infusion in men. J. Clin. Endocrinol. Metab. 50: 397-400, 1980. FABIAN, M., M. L. FORSLING, J. J. JONES, AND J. S. PRYOR. The clearance and antidiuretic potency of neurohypophysial hormones in man, and their plasma binding and stability. J. Physiol. Lond. 204:653-668,1969. GREENWOOD. F. C.. W. M. HUNTER. AND J. S. GLOVER. The - -_---
RATE
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13*
14 . 15 ’ 16. 17.
OF OXYTOCIN
preparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem. J. 89: 114, 1963. LANDON, M. J., D. K. COPAS, E. A. SHEILLS, AND J. M. DAVISON. Degradation of radiolabelled arginine vasopressin (‘“?-AVP) by the human placenta perfused in vitro. Br. J. Obstet. Gynuecol. 95: 488-492,1988. LEAKE, R. D., R. E. WEITZMAN, AND D. A. FISHER. Pharmacokinetics of oxytocin in the human subject. Obstet. Gynecol. 56: 701704,198O. ROOKE, P., AND P. H. BAYLIS. A new and sensitive radioimmunoassay for plasma arginine vasopressin. J. Immunoassay 3: 115-131, 1982. SMALL, C. W., AND W. B. WATKINS. S-benzyl-1-cysteine-4p-nitroanilide. A new substrate for the determination of oxytocinase with improved specificity. Biochem. Med. 9: 103-112,1974. THORNTON, S., J. M. DAVISON, AND P. H. BAYLIS. A novel strategy for inhibition of oxytocinase activity during frequent blood sampling (Abstract). Clin. Sci. Lond. 73, Suppl. 17: 13P, 1987. THORNTON, S., J. M. DAVISON, AND P. H. BAYLIS. The progress of labour is not dependent upon plasma oxytocin. In: Eicosanoids and Fatty Acids (Band 4). Vienna, Austria: Facultas, 1988, p. 149. WIDE, L. Radioimmunoassays employing immunosorbents. Actu Endocrinol. Suppl. 142: 207-221,1969.
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