Changes in the Metabolic Clearance of Vasopressin and in PlasmaVasopressinase throughout Human PregnancyJohn M. Davison, Elizabeth A. Sheills, William M. Barron,* Alan G. Robinson,* and Marshall D. LindheimertMedical Research Council (MRC) HumanReproduction Group, Princess Mary Maternity Hospital, University of Newcastle, Newcastleupon Tyne, NE2 3BD United Kingdom; *Departments of Medicine, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania15261; and tDepartments of Obstetrics and Gynecology and Medicine, University of Chicago, Chicago, Illinois 60637

Abstract

Metabolic clearance rates (MCR) of arginine vasopressin(AVP) were measured serially in five women starting beforeconception, during gestational weeks 7-8 (early), 22-24 (mid-dle), and 36-38 (late pregnancy), and again 10-12 wk postpar-tum. Hormonal disposal rates were determined after waterloading to suppress endogenous AVPrelease using a constantinfusion method designed to achieve three different steady-state concentrations of plasma AVP (PAvp) on each test occa-sion. Dose schedules were altered in mid- and late pregnancy toobtain comparable AVP levels at each stage of the protocol.Prehydration decreased plasma osmolality sufficiently to sup-press AVP release, as circulating AVP-neurophysin measuredserially in three of the womenwas undetectable. The MCRofAVP was similar before conception (0.75±0.31, 0.79±0.34,and 0.76±0.28 liters/min at PAVP of 2.6±1.9, 4.7±2.4, and8.3±3.9 pg/ml), in early pregnancy (0.89±0.34, 0.97±0.04,and 0.95±0.40 liters/min at PAVP of 2.2±2.1, 3.9±3.2, and7.9±3.4 pg/ml), and postpartum (0.70±0.21, 0.69±0.24, and0.75±0.20 liters/min at PAVP 3.5±1.8, 5.1±3.7, and 9.1±4.2pg/ml). Values at mid-pregnancy (2.8±1.3, 3.0±1.2, and2.7±1.2 liters/min at PAVP 2.3±2.2, 4.0±3.6, and 7.7±3.9pg/ml) and late pregnancy (3.2±1.4, 3.3±1.4, and 2.9±1.2liters/min at PAVP 1.9±2.0, 3.8±2.6, and 7.4±4.1 pg/ml) in-creased 3-4-fold (all P < 0.01). Plasma vasopressinase, unde-tectable at 7-8 gestational wk, increased markedly by mid- andslightly more by late gestation. Finally, relationships betweenPAVP and urine osmolality were similar before, during, andafter pregnancy. Weconclude that marked increments in theMCRof AVP occur between gestational weeks 7 and 8 andmid-pregnancy, which parallel the period of greatest rise inboth trophoblastic mass and plasma vasopressinase. Therewas no evidence of a renal resistance to AVPduring gestation.

Introduction

Human gestation is characterized by profound alterations inosmoregulation. Plasma osmolality (Posmoi)' and osmotic

Reprint requests from Great Britain and the Eastern hemisphereshould be addressed to Dr. Davison, MRCReproduction Group,Princess Mary Maternity Hospital, University of Newcastle, Newcastleupon Tyne, NE2 3BD, UK. Reprint requests from the Western hemi-sphere should be addressed to Dr. Lindheimer, Department of Medi-cine, Section of Nephrology, Box 453, 5841 South Maryland Avenue,Chicago, IL 60637.

Received for publication 22 August 1988 and in revised form 18November 1988.

1. Abbreviations used in this paper: DDAVP, 1-desamino-[8-D-argi-nine] vasopressin; DI, diabetes insipidus; MCR, metabolic clearance

thresholds for thirst and arginine vasopressin (AVP) releasedecrease - 10 mosmol/kg during the first trimester. Thesedecrements are maintained till term ( 1-3). Another interestingosmoregulatory change is that the rise in circulating AVPlevels provoked by increases in body tonicity (APAVP/APmO1),unaltered early in gestation, is markedly decreased in the thirdtrimester (3). This latter event could represent a reduced se-cretory response to osmotic stimuli, but might also reflect anincrease in hormonal metabolic clearance rates (MCR). In-deed, in a preliminary report we have noted that the MCRareincreased fourfold during the third trimester compared withmeasurements postpartum (4). The present study extends thislatter observation. MCRof AVP were measured serially involunteers, starting before conception, continuing throughoutpregnancy, and again 10-12 wk postpartum. This study wasdesigned to determine hormonal disposal rates at three differ-ent steady-state plasma concentrations during each test. Fur-thermore, by manipulating exogenous AVP infusion rates wesucceeded in duplicating these levels and were able to comparetheir influence both on the MCRand urine osmolality (Uosmoi)first before, three times during, and again after pregnancy.Results were also correlated with circulating levels of cystine-aminopeptidase (EC 3.4.11.3; vasopressinase), an enzyme re-cently implicated in the etiology of transient vasopressin-resis-tant diabetes insipidus (DI) of pregnancy (5).

Methods

SubjectsSerial studies were performed on five normotensive healthy subjectsonce before conception, between gestational weeks 7 and 8, 22 and 24,36 and 38 (designated as early, mid-, and late pregnancy), and again10-12 wk postpartum, when none were breast feeding or ingesting oralcontraceptives. In addition, two women with a single kidney, onesubject carrying twins and another triplets, were studied but only inlate pregnancy and postpartum. All volunteers gave informed consentin writing to protocols approved by both the Ethical Committee ofNewcastle Health Authority and the Clinical Investigation Committeeof the University of Chicago Pritzker School of Medicine. All subjectshad successful pregnancies.

ProtocolsFig. 1 is a schema of the protocol. Each study began at 9:00 a.m. withthe volunteers seated comfortably. Control blood samples were ob-tained and an infusion of normal saline (0.03 ml/kg per min) wasstarted, after which the subject drank 20 ml/kg of tap water. Clearancemeasurements began 1 h later and after U,,,mol had decreased below 100mosmol/kg. Three AVP(Argipressin; Parke-Davis, Detroit, MI) deliv-ery rates, each preceded by a priming dose, were used during each test(Fig. 1 ).2 Each infusion was maintained for 75 min, urine collected at

rates; PAVP, plasma arginine vasopressin; POSmo0, plasma osmolality;Uosmoi, urine osmolality.2. Dose schedules, altered in mid- and late pregnancy to obtain compa-

rable levels at each stage of the protocol, were based on preliminary

Metabolic Clearance of Vasopressin in Pregnancy 1313

J. Clin. Invest.C) The American Society for Clinical Investigation, Inc.0021-9738/89/04/1313/06 $2.00Volume 83, April 1989, 1313-1318

(1) (2) (3) (4)1 i ~~~~~~II I I I I

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(2) nonpregnant; early pregnancymidgestation; late pregnancy

3)non pregnant: early pregnancymidgestation: late pregnancy

4)nonpregnant: early pregnancymidgestation: late pregnancy

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15-mmn intervals during the last 45 min, and blood samples obtained at45, 60, and 75 min, respectively (Fig. 1). Constancy of the delivery ratewas verified at frequent intervals using a calibrated burette within theintravenous set-up, while Posmot were maintained at 270 mosmol/kgduring pregnancy and 275 mosmol/kg preconception and postpartum-,by periodic ingestion of water. These tonicities should be sufficientlylow to ensure suppression of endogenous AVP release, but to confirmthis, plasma AVP-neurophysin levels were determined in three of thesubjects on 15 test occasions. Finally, vasopressinase levels were mon-itored starting with the initial test, and then every 2-3 wk until thepostpartum study.

Analytical proceduresBlood for osmolality and vasopressinase activity was collected inchilled hepariniized tubes, small portions were immediately drawn formicrohematocrit determination, and the remainder was centrifuged at40C. Osmolality was determined by freezing point depression on anosmometer (model 3DII; Advanced Instruments, Inc., NeedhamHeights, MA) on freshly separated plasma, and the remaining samplewas stored at -20'C until used. Blood for AVP determination wascollected in a Il0-ml syringe containing 0.1I ml phenanthroline (1 -20-phenanthroline monohydrate; Sigma Chemical Co., St. Louis, MO)solution (60 -mg/mil) and then rapidly added to chilled heparinizedtubes, centrifuged in the cold, and extracted within 3 h of venipunc-ture. Validation of this technique, which immediately inactivates thehigh concentrations of cystine aminopeptidase (vasopressinase) pres-ent in the plasma of gravidas, is detailed elsewhere (2).

RIA of A VP. Details of 'our extraction technique and assay proce-dures have been published (2). Briefly, plasma is extracted by a modifi-cation of the acetone method and 200-,gl samples are assayed by anonequilibrium method. The tracer has a high specific activity(> 1,000 Ci X ug'1); highly purified AVP (potency, 400 U/mtg, lot800207; Ferring Arzneimittial GbhG, Wittland, FRG) is used for iodin-ation and standards, and our antiserum (10169) is quite specific andsensitive. Assay sensitivity is 0.1I pg/assay tube, while its 50% displace-ment is 1.6 pg/assay tube.

Vasopressinase activity. An index derived from the rapidity' bywhich the enzyme degraded the '251-AVP tracer was used to monitorvasopressinase activity during and after pregnancy (5, 6). Phenanthro-line-free plasma (1.0 ml) was incubated at 220C with tracer (2 X l05cpm). 50-jil aliquots were drawn before and 5, 10, 15, 30, and 60 minafter incubation, and immediately diluted in 1 ml chilled 40C assay

data (4), as we'll as unpublished observations, which indicated that thedelivery of substantially more hormone would be necessary to obtainmeasurable PAvp as gestation progressed.

buffer containing phenanthroline (60 mg/mi), a step that inactivatedthe enzyme (2). Degradation of the iodinated tracer was measured byloss of binding to excess AVP antiserum and residual activity (at 60min) expressed as percentage of bound tracer from the aliquot ob-tained before incubation (usually > 95%). Sensitivity of this index wascompared with that of a standard photometric fluorometric assay thatuses 5-benzyl-L-cystine-p-nitroanilide as a substrate (7).

A VP-neurophysin. Preparation of -the antiserum, iodination oftracer, and RIA for AVP (nicotine-stimulated) neurophysin are de-scribed in detail elsewhere (8, 9). Briefly, 50,gl of plasma are assayed bya nonequilibrium technique, the minimal detectable level of neuro-physin being 0.5 ng/ml.

Cakculations and statistical analysis. MCRwas measured by astandard formula: infusion rate (calibrated during each study) multi-plied by infusate AVPconcentration (verified by RIA), divided by PAvp(mean of three separate determinations obtained after reaching equi-librium). To assess if a steady-state AVP level had been achieved dur-ing each of the three constant infusions of AVP per study, a test ofpiecewise linear trend was used (10, 1 1). To determine whether or notthe MCRwas significantly different at the three infusion rates, thevalues at each rate of infusion within each subject or each of the fivetest occasions were analyzed using an analysis of variance (ANOVA).From the significance of the F ratio from this analysis', comparison wasperformed using the t test (1 1). Paired comparisons between test occa-sions were performed using t test. For all analyses, values ofFP. 0.05were considered significant. All data are given as M'ean+-SD.

Results

Table I and Fig. 2 summarize results in all five subjects.- Alter-ing infusion rates produced similar PAvp levels at each stage ofthe serial study. PAvp during the final 30 min of all infusionswere quite constant, demonstrating that steady-state levels hadindeed been achieved by this time. This constancy is illUstratedin Table IL, which summarizes the mean variation and its SDfor PAvp and P,,,mol at each infusion rate throughout the study.Not shown coefficients of variation's Were consistently below10%. These data again underscore the adequacy of steady-statevalues used to calculate the MCRin this investigation.

Of particular interest, the MCRin mid- and late pregnancy(3-4 liters/mmn) were fourfold those measured before concep-tion, early in gestation, or postpartum (all P < 0.0 1; Table Iand Fig. 2). Small increases in hormonal disposal rates werenoted during the first trimester (when osmotic secretorythresholds are decreasing or have just reached their lowestvalues, but the AVP response to Osmotic Stimuli [APAvp/APosmoil is similar to values in the nongravid state [3]), butthese changes were not significant. Similarly, the MCRin lategestation were slightly higher than those in mid-pregnancy, butagain these increments did not reach significance. In essence,the greatest changes took place between gestational weeks 6and 8 and 22 and 24.

Note also that the MCRat any stage was unaltered despitevirtually tripling circulating hormone concentrations betweenthe first and final infusion rates of each test. Table I and Fig. 2depict relationships between PAvp and Uosmoi during eachphase of the study. Similar Uosmoi were recorded when PAVPwas comparable before, during, and after gestation.

Table I also contains AVP-neurop hysin levels measured inthree volunteers. Values before water loading were similar tothose reported in the literature (8, 9) and became undetectable(< 0.5 ng/ml) after hydration.

The MCRin the women with a single kidney and thosecarrying twins and triplets are summarized in Table III. These

1314 Davison', Sheills, Barron, Robinson, and Lindheimer

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Fig. 3 compares vasopressinase activity derived by our iso-tope index and a standard substrate method. Activity was de-tected earlier in pregnancy using the tracer method (7-8 wk)and then the substrate technique (9-10 wk). Enzyme activityincreased 40-fold by mid- and 50-fold by late pregnancy usingeither assay (P < 0.00 1), while vasopressinase remained detect-able for a longer period after gestation using the tracer (5-6wk) compared with the substrate (4 wk) assay. Not shown onthe figure, enzyme levels in the women carrying twins andtriplets were consistently at the upper 1 SD boundary, theirfinal values near term being 270 and 281 mIU/ml, respec-tively.

Discussion

Wemonitored clearances of exogenously infused AVP in fivewomen starting before conception, continuing throughoutpregnancy, and 8-10 wk postpartum. The MCRat mid- andlate pregnancy was markedly increased compared with valuesin the first trimester as well as when the women were notpregnant. Hormonal disposal rates at any phase of the studywere uninfluenced by the circulating levels of AVP and theincrements observed during mid- and late gestation seemed tocorrelate with striking increases in vasopressinase activity pres-ent at these times. Wealso measured basal AVP in these sub-jects, noting that levels were similar throughout pregnancy aswell as the nonpregnant state. Thus, hormonal productionrates must also have increased by mid-gestation.

The observation that infused AVP is cleared more rapidlyduring gestation confirms and extends a recent report in whichwe suggested that the MCRof endogenously secreted hormonewas increased in late pregnancy (12). This was based on obser-vations during and after pregnancy in a protocol where PAVPhad been increased secondarily to increments in Posmol inducedby hypertonic saline infusion after which the subjects drankwater, a maneuver that rapidly suppresses endogenous hor-mone release by an oropharyngeal-neuroendocrine reflex,even in the face of sustained hyperosmolality (12). Withinminutes of drinking, AVP levels started to decrease and thehormonal t1/2 in late gestation was half that in early pregnancyor postpartum. However, as noted in that report, peak AVPlevels attained with hypertonic saline were too low (usuallybetween 5 and 10 pg/ml), and post-drinking blood sampleswith detectable PAVPtoo few, to accurately assess the t/2. Nev-ertheless, these previous observations add confidence to theconclusion that our present infusion clearance data reflect theMCRof endogenously secreted hormone. This is not a moot

x point for in pregnancy it is conceivable that the intravenousinfusion of AVP might activate or increase plasma vasopres-sinase. In this respect circulating vasopressinase activity mea-

+1 sured before, during, or after an infusion was similar in a givensubject on any test occasion (Sheills, E. A., and J. M. Davison,unpublished observations).

in The reason for the striking increments in MCRin latex: gestation is not apparent from our data and one can only

Metabolic Clearance of Vasopressin in Pregnancy 1315

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speculate concerning causality. Renal and hepatic blood flowincrease during gestation and the kidney and liver are organswhere considerable AVP degradation takes place. Renal he-modynamics, however, increase quite early in pregnancy (at atime when the MCRis hardly altered), and the combinedincrements in flow to both organs (controversial for the liverand 30-50% for the kidney) are far less than the increase inhormonal disposal recorded in mid- and late gestation (13).Also, the percent rise in MCRnoted in the two gravidas withsingle kidneys was similar to that in the normal volunteers.

The placenta, the mass of which increases throughout ges-tation, may inactivate substantial quantities of AVPin situ. Inpreliminary studies we perfused 25I-AVP at rates comparableto in vivo conditions (14). The preparation perfused on thematernal side was capable of inactivating - 1 ng/min oftracer, while perfusion of the fetal side was without effect.

Table II. Variation of PAVPand Posmot Measured at 15-minIntervals during Final 45 Min of Each Infusion during 225Measurements of MCRPerformed on Five Patients

Variation of

Infusion PAVP Pd

pg/kg/min pg/ml mosmol/kg

Prepregnancy 24 0.52±0.29 1.3±0.742 0.44±0.28 0.9±0.772 0.57±0.26 0.9±0.9

Early pregnancy 24 0.42±0.31 1.2±0.942 0.63±0.34 1.1±0.872 0.50±0.29 0.8±0.4

Mid-pregnancy 84 0.64±0.29 0.9±0.9144 0.61±0.26 1.4±0.6246 0.43±0.27 1.2±0.6

Late pregnancy 84 0.29±0.19 0.9±0.9144 0.41±0.20 1.3±0.8246 0.34±0.21 1.1±0.8

Postpartum 24 0.56±0.34 1.1±0.742 0.61±0.32 1.1±0.772 0.53±0.34 1.5±0.6

Mean±SD; n = 5 patients.

Figure 2. Summary of PAVP, UOSm01and MCRAVP,(±SD) measured seri-ally in all five womenstarting be-fore conception, then during gesta-tional weeks 6-8, 22-24, and36-38, as well as 10-12 wk postpar-tum. The three infusion rates aredetailed in Fig. 1.

Thus, the placenta too may account for the increased MCRobserved on these studies. Of interest, also, is that estimatedtrophoblastic mass increases 1,000-fold between gestationalweeks 6 and 24, plateauing thereafter (15). Uteroplacentalblood flow follows a similar pattern of change, and at term hasbeen estimated to be about 500 ml/min blood flow (16). Inthese respects the MCRof AVPwas almost maximal by 22-24wk, increasing slightly but not significantly near term.

The placenta is also the source of circulating vasopressin-ase, an enzyme capable of inactivating large quantities of AVPin vitro. Whether or not this enzyme is active in vivo, however,is unclear, but it is of interest that alterations in the hormonalMCRcorrelated best with increments in plasma vasopressin-ase in the present study. Of further interest, pregnant sheep,whose placentae produce no detectable vasopressinase, do nothave an increased MCRof AVP during their gestation (17).Currently we are measuring the MCRof l-desamino-[8-D-ar-ginine]vasopressin (DDAVP), an AVP analogue resistant toenzymatic inactivation by cystine-aminopeptidase (18). If va-sopressinase does play a role in the increased MCRof AVPduring mid- and late gestation, any increments in the disposalrate of infused DDAVPought to be considerably less thanthose observed for the native hormone.

Oxytocin, which differs from AVPby just two amino acids,is also inactivated in vitro by cystine-aminopeptidase, and thusmight be expected to be metabolized in a manner similar tovasopressin in pregnant women. Surprisingly, however, Amicoet al. observed no differences in the MCRof infused oxytocininto term pregnant subjects compared with nonpregnant vol-

Table III. MCRof A VP in Third Trimester and Postpartumin One Womanwith Twins, Another with Triplets, and TwoSubjects with a Single Kidney

Third trimester Postpartum

Infusion (pg/kg per min) 84 144 42 72MCR

Twins (mIl/min) 3,417 2,994 981 818Triplets (ml/min) 2,627 2,588 812 729One kidney (ml/min) 1,895 2,806 339 550One kidney (ml/min) 2,318 2,103 553 621

1316 Davison, Sheills, Barron, Robinson, and Lindheimer

0 , -. 300 Figure 3. Plasma vaso-pressinase activity mea-

N5 ~~~~~~~~sured serially in the five200 womenwho underwent

vamopressunm _ Nie|1 | iVasopessainf the metabolic clearance%ini~rstia ty150 30 studies. Measurements ofimmunore(ikity ("its) Pivredfrom started before

100 conception and were re-peated at 2-3-wk inter-vals until postpartumweeks 10- 12. Two indi-

1020 30 40 5 10 ~~~~~~~~~~~~~~ceswere used, one de-Pregnancy (weeks) Post~rtum rived from the rapidity by(weeks)

which the enzyme de-graded '25I-AVP tracer (vertical lines) and the other by photometry using 5-benzyl-L-CYstine-p-nitroanilide as substrate (speckled area). Solidand dashed lines enclosing the vertical lines and speckled area represent ± 1 SD.

unteers (19). However, there is evidence that oxytocin may bemore resistant to enzymatic degradation in vivo, especially inthe central nervous system (20). Also, and in contrast withobservations reported by Amico et al. (19), we have prelimi-nary data that suggest that the MCRof infused oxytocin isincreased in a manner similar to that of AVPat term (21).

The increased MCRof AVP in pregnancy may explaincertain other changes in osmoregulation during gestation. Asnoted previously, the rise in circulating hormone provoked byincreasing body tonicity (APAvp/APosmo) decreases in the thirdtrimester. Initially we considered that this might reflect a re-duced secretory response to osmotic stimuli, as occurs in cer-tain hypervolemic states such as primary aldosteronism (22),for indeed extracellular volume increases markedly in preg-nancy (23). However, the osmotic sensitivity to thirst (Athirstintensity/APOSmOj; see reference 3) would also be expected to bedecreased in such circumstances (24) and it was not (3). An-other possibility considered was that an increase in hormonaldisposal rate would explain reductions in the ratioAPAVP/APosmol with little influence on the relationship betweenthirst intensity and Posmoi. This led to the current study anddata supporting the latter hypothesis.

Several problems in water handling known to occur inpregnancy may also relate to our observations. For example,some women with central DI may require more AVPduringgestation. There is a syndrome labeled transient DI of preg-nancy that usually presents during the second half of gestationand remits postpartum (25). Some of the women with thiscomplication may have subclinical lesions of central DIbrought to the fore by the increased hormonal MCRof preg-nancy (25-27). Still other patients have been described withtransient vasopressin-resistant DI and high circulating levels ofvasopressinase, and one of them described by Durr et al. (5)responded to DDAVPafter large doses of AVPhad failed toeffect UOsmoi.

Performance of a serial study also allowed us to evaluatethe influence of pregnancy on renal effects of AVP. Somehavesuggested resistance to the hydroosmotic effects of AVPuponthe kidney during gestation, both in humans and in two ani-mal species (28-31). Several reasons, including incrementsduring pregnancy in PGE2excretion, a hormone that opposesthe effects of AVP at the tubular level, have been offered toexplain this resistance. Review of the literature, however, re-veals that such claims have been based solely on the influenceof exogenously administered hormone on Uosmol, without not-

ing if such maneuvers achieve similar PAvp levels during thepregnant and nonpregnant states. For example, administra-tion of similar quantities of AVP during the nongravid andpregnant states would be expected to produce lower PAVPvalues in the latter if either the MCRor volume of distributionwere increased in pregnant and control subjects or animals.Our experiments permitted comparison of Uomol at similarAVP levels, measured during steady-state conditions. Similarlevels of PAVP produced approximately the same UOSmoi inearly, mid-, and late gestation as well as in the nonpregnantstate (Table I and Fig. 2).

Finally, our observations in postpartum subjects are con-sistent with the MCRfor AVPmeasured in nonpregnant pop-ulations and reported by Robertson et al. (32), Beardwell et al.(33), and Moses and Steciak (34), who used constant-infusiontechniques, and Engel et al. (35), who performed a single-in-jection protocol. Moses and Steciak, however, observed incre-ments in the MCRas PAvp increased, but we did not. Thisdiscrepancy may be explained by the fact that we measuredPAVP at three levels within the physiological range, while eventhe lowest concentrations observed by Moses and Steciak (34)were supraphysiological.

In conclusion, these data demonstrate that the MCRofAVP similar to nonpregnant values during gestational weeks7-8 measures fourfold by weeks 22-24 and remains at theseelevated levels through term. These rises seem to parallel theperiods of rapid increase in both trophoblastic mass and circu-lating vasopressinase. The increase in hormonal disposal ratesmay explain the appearance of certain polyuric syndromes inlate gestation.

Acknowledgments

Wethank Ms. P. Papadatos and Ms. I. White for excellent secretarialassistance.

This work was supported by the Medical Research Council, GreatBritain, National Institutes of Health grants HD-5572, 24498, andRR-55, and a Clinical Investigator Award (HL-01 145) to William M.Barron.

References

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Metabolic Clearance of Vasopressin in Pregnancy 1317

2. Davison, J. M., E. A. Gilmore, J. Dfirr, G. L. Robertson, andM. D. Lindheimer. 1984. Altered osmotic thresholds for vasopressinsecretion and thirst in pregnancy. Am. J. Physiol. 246:F105-F109.

3. Davison, J. M., E. A. Shiells, P. R. Philips, and M. D. Lind-heimer. 1988. Serial evaluation of vasopressin release and thirst inhuman pregnancy. Role of human chorionic gonadotrophin in theosmoregulatory changes of gestation. J. Clin. Invest. 81:798-806.

4. Davison, J. M., W. M. Barron, and M. D. Lindheimer. 1987.Metabolic clearance rates of vasopressin increase markedly in lategestation: a possible cause of polyuria in pregnant women. Trans.Assoc. Am. Phys. 100:91-98.

5. Durr, J. A., J. G. Hoggard, J. M. Hunt, and R. W. Schrier. 1987.Diabetes insipidus in pregnancy associated with abnormally high cir-culating vasopressinase activity. N. Engl. J. Med. 316:1070-1074.

6. Durr, J. A., R. W. Schrier, and M. D. Lindheimer. 1985. A newmethod for measuring vasopressinase activity in plasma. Clin. Res.33:26A. (Abstr.)

7. Small, C. W., and W. B. Watkins. 1974. S-benzyl-L-cystine-p-nitroanilide: a new substrate for the determination of oxytocinase withimproved specificity. Biochem. Med. 9:103-112.

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1318 Davison, Sheills, Barron, Robinson, and Lindheimer