Effect of pregnancy on the metabolic clearance rate and the volume of distribution of oxytocin in the baboon

Wlodzimierz B. Kowalski1, Lubomir Diveky1, Ramkrishna Mehendale1, Michael Parsons2, and Laird Wilson Jr.1

1 Departments of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois 60612; and 2 University of South Florida, Tampa, Florida 33606

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pharmacokinetic parameters of oxytocin (OT) metabolism were determined during the last third of pregnancy and again 4-8 wk after delivery in the baboon. Animals were placed on a tether system with venous and arterial access and a continuous monitoring of uterine contractions during gestation. Two methods of determining OT pharmacokinetics were utilized (bolus injection vs. continuous infusion). The metabolic clearance rate of OT as determined during the bolus trials (n = 7) was 22.2 ± 1.5 ml · min-1 · kg-1 in pregnancy and 16.3 ± 1.4 ml · min-1 · kg-1 postpartum (P < 0.05), respectively, and 23.7 ± 2.8 vs. 16.9 ± 3.7 ml · min-1 · kg-1 (P < 0.05), respectively, as determined during the 1-h infusion trials (n = 4). The initial dilution volume and the volume of distribution at steady state of OT after administration did not differ between pregnant and postpartum animals (P > 0.05). The mean residence time (MRT) of OT was shorter during pregnancy, 7.7 ± 0.8 vs. 10.8 ± 1.2 min postpartum (P < 0.05). In summary, OT metabolism during pregnancy in the baboon is characterized by 1) increased clearance rate (1.4-fold), 2) accelerated turnover due to the shorter MRT, and 3) unaltered distribution.

metabolism; volume of distribution; pharmacokinetics

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

WHETHER OXYTOCIN (OT) clearance is increased during gestation has been a subject of debate over the past years (15). Most, if not all, studies have focused on humans. Initial investigations in women failed to find differences in metabolic clearance rate (MCR) of OT during gestation (2, 10). However, more recently, Thornton et al. (15) reported a four- to fivefold increase in OT clearance in pregnant compared with nonpregnant women. Humans appear to be unique due to the presence in blood of "oxytocinase," more appropriately referred to as cystine aminopeptidase (CAP; EC 3.4.11.3), a placental enzyme secreted into the blood that is capable of degrading OT and vasopressin (16). Metabolism of OT occurs primarily in the kidneys, liver, and placenta (3). Because MCRs by individual organs are additive, the MCR of OT would be expected to increase during gestation, supporting the report of Thornton et al.

The physiological role of circulating OT is unequivocal during lactation because it is necessary for the milk ejection reflex to occur (8, 11, 12). In pregnancy, OT along with other factors promotes uterine contractions (7), but its precise role in the initiation and progress of parturition is not fully understood. In the pregnant baboon, an OT antagonist inhibits spontaneous uterine contractions (18). In the rhesus monkey, OT antagonists prolong gestation (5). These studies clearly indicated that, in those subhuman primates, OT plays an important role in the regulation of uterine contractility during gestation. In addition, before parturition, the number of myometrial OT receptors dramatically increases, correlating with increased contractile responsiveness to OT at the end of pregnancy (3, 14, 17). On the other hand, availability of systemic (i.e., circulating in blood) OT for receptor binding represents another level of regulation. This can occur through changes in OT secretion, MCR, and distribution, all three of which determine instantaneous levels of circulating hormone available for the receptor binding. In the present study, we addressed the question of whether OT clearance is increased during pregnancy in the baboon, a surrogate model for human pregnancy. Metabolic parameters were determined during the last third of gestation and 4-8 wk after delivery in the same baboon to minimize confounding effects of interanimal variation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals

Baboons (Papio anubis) with timed pregnancies from the breeding colony of the Biological Resource Laboratory at the University of Illinois, weighing 16-20 kg, were used. The animals were housed in cages individually and were maintained under a controlled photoperiod (lights on from 0600 to 1800) and temperature (21°C) and fed food and water ad libitum. All experimental procedures were approved by the Animal Care Committee of the University of Illinois.

At 115-125 days of gestation (term = 184 ± 2 days), baboons were put on a tether system under 1% isoflurane anesthesia as previously described (19). Four vinyl cannulas (0.86 × 1.32 mm) were inserted: one each in the aorta and vena cava via the femoral artery and vein, respectively (total length from animal to infusion pump = 4.9 m), and two in the amniotic fluid (total length from animal to transducer = 3.7 m), the latter for the continuous monitoring of intrauterine pressure changes with a Gould model P23id pressure transducer, as detailed previously (19). A nylon jacket was placed on the animals, and the cannulas were led outside the cage via a tether attached to the jacket and to the adjacent room where all experimental procedures were performed. Vascular cannulas were connected to syringes on a Harvard infusion pump, and heparinized saline (100 U/ml) was infused at a rate of 0.582 ml/h to keep them patent. Volume of the dead space of vascular cannulas was 2 ml. After delivery of an infant, amniotic fluid cannulas were removed. All experiments commenced at least 2 wk after surgery or 4 wk after delivery.

Experimental Procedures

Trial 1: Bolus intravenous infusion of OT. To determine the effect of pregnancy on OT metabolism and distribution, seven baboons received an intravenous bolus injection of 500 mU of OT (Pitocin; Parke-Davis; 1 mU = 2 pmol) at 1300: one time during pregnancy (between days 140 and 170) and one time 4-8 wk after delivery. Two basal samples of arterial blood (3 ml) were collected before OT injection at -10 and -5 min, and then blood was collected at +0.5, 1.5, and 2.5 min (1 ml each) and at 5, 7.5, 10, 15, 20, 25, and 30 min (3 ml each).

Trial 2: Continuous intravenous infusion of OT. Four baboons were used to determine the MCR of OT using a continuous hormone infusion technique at a constant rate. A 1-h test of intravenous OT (Pitocin) infusion was done in each animal starting at 1300, one time during pregnancy (between days 140 and 170) and one time between 4 and 8 wk postpartum. OT was infused with Harvard infusion pumps at incremental doses of 10, 20, and 40 mU/min, each for 20-min infusion segments. Incremental doses were tested to determine if the concentration of OT would alter the results. Arterial blood samples (3 ml each) for the OT RIA were collected before infusion was started at -10 and -5 min, then every 10 min for the time of infusion (i.e., in the middle and at the end of each infusion segment), and for 30 min thereafter, at +2.5, 5, 7.5, 10, 15, 20, 25, and 30 min after the infusion was stopped.

Sample Collection

Two milliliters of saline from the cannula dead space were discarded and 1 or 3 ml of blood, as appropriate, were transferred from the syringe to the plastic tubes kept on ice. The cannula was flushed with 3-5 ml of heparinized saline after each withdrawal (i.e., 1-3 ml for the isovolumetric replacement of lost blood and 2 ml to fill the dead space of the cannula). Blood samples were centrifuged, and the plasma was stored at -70°C until assayed for OT. Withdrawal of the first milliliter of blood (as assessed by the volume of withdrawn saline in the syringe) was routinely accomplished within 5 s.

Blood collection was carried out in the room adjacent to the animal housing facility. Three of seven baboons lactated after delivery (in the 4 nonlactating animals, the fetuses died before or during delivery or were rejected by the mother). The lactating animals were discouraged from breastfeeding during OT administration by the presence of the investigator in the room near the cage. The presence of the investigator appeared not to affect the results, as determined by comparing the data between the postpartum lactating and nonlactating animals (visual inspection revealed overlapping data).

OT RIA

Plasma OT was measured by RIA after extraction with petroleum ether/acetone, essentially as described by Amico et al. (1). Volume of the plasma used for the extraction was 0.5 ml. All samples from one animal were analyzed in one batch. The intra- and interassay coefficients of variation were 13 and 16%, respectively. The assay sensitivity was 0.6 pg/tube. All results were corrected for extraction efficiency, based on [3H]arginine vasopressin recovery in each sample and on the average ratio of [3H]OT to [3H]arginine vasopressin recoveries of 1.2. The average efficiency of OT extraction so determined was 74%. The detection limit of the assay for 0.5-ml samples of the plasma was therefore 4.0 pg/ml.

Data Analysis

Data were analyzed after subtracting baseline values of OT levels at -10 and -5 min when OT was detected in these samples.

Trial 1: Bolus infusion of OT. The disappearance of OT from blood after bolus infusion was modeled by the weighted least squares method for two compartments using the PCNONLIN program (SCI Software, Lexington, KY). The weight used was (concentration)-1. The following parameters of OT metabolism were calculated (13): 1) the MCR of OT = OT dose/AUCOT, where AUCOT is the area under the curve of OT concentrations vs. time from zero to infinity; 2) the volume of distribution at steady state (VSS) of OT = OT dose × AUMCOT/AUC2OT, where AUMCOT is the area under the first moment curve of OT concentrations vs. time, extrapolated to infinity; 3) the initial dilution volume (VD) of OT = OT dose/Ca + Cb, where Ca and Cb are time 0 intercepts for the initial and terminal exponential terms, respectively; and 4) the mean residence time (MRT) of OT = VSS/MCR. Half-lives for the two phases of OT disappearance, T1/2alpha for the initial phase and T1/2beta for the terminal phase, were estimated by the computer program. The fraction of OT elimination associated with the terminal phase (fb) was calculated as (0.693 × Cb)/(T1/2beta  × AUCOT), and the fraction of OT elimination associated with the initial phase (fa) was determined as 1 - fb (13).

Trial 2: Continuous infusion of OT. For each infusion rate (i.e., 10, 20, or 40 mU/min), the MCR of OT = infusion rate/CSS, where CSS is the steady-state (plateau) concentration of OT, attainable at a given rate. The fraction of the CSS achieved at the end of each of the 20-min infusion segments (F20) was calculated as (13) fa(1 - e-at) + fb(1 - e-bt), where a = 0.693/T1/2alpha , b = 0.693/T1/2beta , and t = 20 min (duration of OT infusion at each rate). Arithmetic means of parameters a, b, fa, and fb were determined using results of the bolus trials, separately for the pregnancy (n = 7) and for the postpartum period (n = 7), and two so computed values of the F20 were further used. The CSS of OT for each infusion rate in each trial was thus determined as C20min/F20, where C20min is the OT level at the end of each 20-min infusion segment.

Statistics

All statistical analyses were performed using SPSS 6.1 for personal computers (Chicago, IL). Data for the MCR, the VSS, and the VD were expressed per kilogram body weight. Due to heterogeneity of variance, log transformation was used for the MRT data before statistical analysis. ANOVA with repeated measures on two main factors, OT dose and pregnancy status, was performed to test for a possible dose-dependent effect of OT on its clearance. Results were considered significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Baseline levels of OT in most of the trials ranged from less than detectable (i.e., <4 pg/ml) to 10 pg/ml. No spontaneous uterine contractions were observed in any of the pregnant animals in the afternoon on the day of a study or the afternoons immediately preceding a study.

Trial 1: Bolus Infusion of OT

The two-compartmental system model for OT disappearance showed a good fit with correlation coefficients of 96-99%. Examples of the disappearance of OT from the blood from the same animal during pregnancy and after delivery are shown in Fig. 1. MCR, VSS, and VD in pregnant vs. postpartum animals are shown in Fig. 2. The MCR was significantly higher (P < 0.05) in the pregnant vs. postpartum animals (Fig. 2A). In contrast, no difference (P > 0.05) was found in VSS and VD between pregnant and postpartum animals (Fig. 2B). Of the other quantitative parameters measured (MRT, T1/2alpha , T1/2beta , fa, and fb), only MRT was lower (P < 0.05) in pregnant vs. postpartum animals (Table 1).


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Fig. 1.   Typical disappearance curve of oxytocin from blood after bolus infusion in a baboon (no. 5962) postpartum (A) and during the pregnant state (B). The curve represents results of 2-compartmental modeling (1 pg = 1 fmol).


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Fig. 2.   Effect of pregnancy on oxytocin (OT) pharmacokinetics as determined by bolus infusion of OT. A illustrates that pregnancy increased the metabolic clearance rate (MCR) of OT ~40% compared with nonpregnant animals (* P < 0.05). B: in contrast to MCR, pregnancy had no effect on the initial dilution volume (VD) or the distribution at steady state (VSS) of OT compared with postpartum animals (P > 0.05). Means ± SE; n = 7.

                              
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Table 1.   Secondary (dependent) parameters of OT disposition determined during bolus trials in pregnant vs. postpartum baboons

Trial 2: Continuous Intravenous Infusion of OT

Because pregnancy status, but not dose effect (means ± SE for 10, 20, and 40 mU/min were 18.5 ± 2.2, 21.6 ± 3.4, and 21.3 ± 2.3, respectively; P > 0.05), was significant, only the MCR of OT for pregnancy status is presented. The MCR of OT was significantly higher during pregnancy than postpartum and was similar to the values determined from the bolus infusion study of trial 1 (P < 0.05; Fig. 3).


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Fig. 3.   MCR of OT determined after continuous infusion of OT in pregnant and postpartum animals. Three different doses of OT were infused (dose effect) in pregnant and postpartum animals (pregnancy status), but only the main effect of pregnancy status was significant (* P < 0.05), and thus only this effect is presented. The values are similar to those determined after bolus infusion of OT (see Fig. 2). Means ± SE; n = 4.

Linear correlation between OT clearance rates measured by two techniques, the bolus administration vs. the continuous infusion, was r = 0.75 (P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although investigators initially failed to find a difference in MCR in pregnant compared with nonpregnant humans (2, 10), a recent report by Thornton et al. (15) showed a four- to fivefold increase during pregnancy. It is somewhat surprising that the earlier studies found no effect of pregnancy on OT MCR, since an additional organ for OT metabolism is present, the placenta, and it releases the enzyme oxytocinase, or CAP, into the blood. The presence of CAP in blood of humans may explain the much higher rate of OT MCR compared with the 40% increase detected in the baboon studies. Circulating oxytocinase is either very low or absent in baboon blood, as we recently reported (9).

Distribution of OT (VSS or VD) was not affected by the pregnancy in the baboon. Reassuringly, the two volumes of distribution of OT determined in this study, VSS and the VD, were of the order of physiological spaces, the extracellular fluid and the plasma volumes, respectively. Such distribution is consistent with the two-compartmental model.

Half-lives (T1/2alpha and T1/2beta ) of OT disappearance from the circulation were not affected by pregnancy. However, the MRT of OT, which is a statistical concept defined as the mean time that each molecule of a hormone resides in the body and which is determined by the ratio of the VSS and the MCR (13), was shorter during gestation. This finding indicates that, in addition to the increased clearance rate of OT from the circulation, which affects hormone levels, the turnover rate of OT in the whole body is increased as well in pregnant animals. Intuitively, one might expect that, if MCR is significant, the T1/2 and MRT should also be significant. However, the equations that determine these values utilize different data, and thus they do not always correlate with each other as was the situation in this study.

One might be critical of determining pharamacokinetic parameters by the administration of unlabeled hormone as was done in the present study. The accurate determination of a hormone's clearance by the administration of an unlabeled hormone requires a constant rate of endogenous secretion or a very low secretion rate compared with the administered dose. This is the case for OT in this study. Neurohypophysial OT secretion and uterine contractions exhibit diurnal variation during primate gestation. Peak values of both are observed at night. Daytime OT levels in blood are usually less than detectable when no myometrial contractile activity is observed (6, 19). In the present study, no significant uterine contractions were present before OT bolus or continuous infusions had been administered in any of the trials, and all of the infusions were done during the daytime. On the other hand, lactational OT release associated with nipple stimulation in the baboon is pulsatile, with hormone levels oscillating between 0 and 20 pg/ml. In our study, none of the three lactating baboons was allowed to breastfeed before or during OT administration, and the baseline values of OT were close to the detection limit of the RIA in those cases. In addition, because maximal OT levels achieved during bolus trials (time 0 intercepts) were 500-1,000 pg/ml and those at the end of the 1-h infusion were 200-400 pg/ml, the highest admixture of endogenous OT resulting from an OT pulse during the lactation period would be of the order of 2-4% after bolus and 5-10% during or after infusion.

The physiological significance of enhanced OT clearance during pregnancy is only speculative. Enhanced clearance could protect the uterus from spurious releases of OT and thus contribute to uterine quiescence throughout pregnancy. However, what happens in the evening during nocturnal uterine contractions or at term during labor has not been addressed in this study. One might speculate that the clearance rate diminishes in the evening or during labor to enhance systemic OT levels and contribute to uterine activity via this mechanism. Further studies are needed to address these issues.

In summary, our data indicate that, in the baboon, 1) OT clearance after bolus administration can be modeled by a two-compartmental system; 2) the half-life of OT in the initial phase of distribution is very short (1 min), followed by a more prolonged terminal elimination half-life of ~10 min, but neither the initial nor terminal phase half-lives are affected by pregnancy; 3) OT clearance is increased during gestation by 40%, whereas OT distribution is unaffected; and 4) the MRT of OT was significantly shorter during pregnancy, indicating a more rapid turnover rate of the hormone.

    ACKNOWLEDGEMENTS

This work was supported in part by NIH Grant HD-25888 to L. Wilson, Jr. and by a Fulbright Fellowship to L. Diveky.

    FOOTNOTES

Address for reprint requests: L. Wilson Jr., Dept. of Obstetrics and Gynecology, Univ. of Illinois at Chicago, M/C 808, 820 S. Wood St., Chicago, IL 60612.

Received 11 December 1997; accepted in final form 16 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Amico, J. A, S. M. Seif, and A. G. Robinson. Oxytocin in human plasma: correlation with neurophysin and stimulation with estrogen. J. Clin. Endocrinol. Metab. 52: 988-993, 1981[Abstract].

2.   Amico, J. A., J. Seitchik, and A. G. Robinson. Studies on oxytocin in plasma of women during hypocontractile labor. J. Clin. Endocrinol. Metab. 58: 274-279, 1984[Abstract].

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5.   Giussani, D. A., S. L. Jenkins, C. A. Mecenas, J. R. Owiny, R. A. Wentworth, J. A. Winter, J. B. Derks, M. Honnebier, and P. W. Nathanielsz. The oxytocin antagonist Atosiban prolongs gestation in the rhesus monkey (Abstract). J. Soc. Gynecol. Invest. 2: 265, 1995.

6.   Hirst, J. J., G. J. Haluska, M. J. Cook, and M. J. Novy. Plasma oxytocin and nocturnal uterine activity: maternal but not fetal concentrations increase progressively during late pregnancy and delivery in rhesus monkeys. Am. J. Obstet. Gynecol. 169: 415-422, 1993[Medline].

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9.  Kowalski, W., S. C. Pak, M. Parsons, and L. Wilson, Jr. Apparent lack of circulating oxytocinase activity in the pregnant baboon (Abstract). Biol. Reprod. 54, Suppl. 1: 124, 1996.

10.   Leake, R. D., R. E. Weitzman, and D. A. Fisher. Pharmacokinetics of oxytocin in the human subject. Obstet. Gynecol. 56: 701-704, 1980[Abstract].

11.   Lincoln, D. W., A. Hill, and J. B. Wakerley. The milk-ejection reflex of the rat: an intermittent function not abolished by surgical levels of anaesthesia. J. Endocrinol. 57: 459-476, 1973[Medline].

12.   Meyer, C., M. J. Freund-Mercier, Y. Guerne, and P. H. Richard. Relationship between oxytocin release and amplitude of oxytocin cell neurosecretory bursts during suckling in the rat. J. Endocrinol. 114: 263-270, 1987[Abstract].

13.   Rowland, M., and T. N. Tozer. Distribution kinetics. In: Clinical Pharmacokinetics: Concepts and Applications (3rd ed.). Baltimore, MD: Williams & Wilkins, 1995, p. 313-328.

14.   Soloff, M. S. The role of oxytocin in the initiation of labor, and oxytocin-prostaglandin interactions. In: The Onset of Labor: Cellular and Integrative Mechanisms, edited by D. McNellis, J. R. G. Challis, P. C. MacDonald, P. W. Nathanielsz, and J. M. Roberts. Ithaca, NY: Perinatology, 1988, p. 87-123.

15.   Thornton, S., J. M. Davison, and P. H. Baylis. Effect of human pregnancy on metabolic clearance rate of oxytocin. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R21-R24, 1990[Abstract/Free Full Text].

16.   Walter, R., and W. H. Simmons. Metabolism of neurohypophyseal hormones: considerations from a molecular viewpoint. In: Neurohypophysis, edited by A. M. Moses, and L. Share. Basel, Switzerland: Karger, 1977, p. 167-188.

17.   Wilson, L., Jr., and M. T. Parsons. Endocrinology of human gestation. In: Reproductive Endocrinology, Surgery and Technology, edited by E. Y. Adashi, J. A. Rock, and Z. Rosenwaks. Philadelphia, PA: Lippincott-Raven, 1996, p. 452-475.

18.   Wilson, L., Jr., M. T. Parsons, and G. Flouret. Inhibition of spontaneous uterine contractions during the last trimester in pregnant baboons by an oxytocin antagonist. Am. J. Obstet. Gynecol. 163: 1875-1882, 1990[Medline].

19.   Wilson, L., Jr., M. T. Parsons, and G. Flouret. Forward shift in the initiation of the nocturnal estradiol surge in the pregnant baboon: is this the genesis of labor? Am. J. Obstet. Gynecol. 165: 1487-1498, 1991[Medline].


AJP Endocrinol Metab 274(5):E791-E795
0193-1849/98 $5.00 Copyright © 1998 the American Physiological Society




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