Lee and Yeh

6. Liebhart M. The ultrastruture of placental villi in casesof serological conflict in RH incompatibility. Pathol Eur1971;6:415.

7. Alvarez H, Sala MA, Benedetti WL. Intervillous spacereduction in the edematous placenta. AMJ OBSTET Cv­NECOL 1972;iI2:819.

8. Lee ML, Dempsey EW. Microcirculation of the rat pla­centa. AMJ OBSTET GYNECOL 1976; 126:495.

May, 1986Am J Obstet Gynecol

9. Fox H. Histological abnormalities of the placenta. In: FoxH, ed. Pathology of the placenta. London: WB Saunders,1978: 149.

10. Burger P, Chandler D, Klintworth GK. Corneal vascular­ization as studied by scanning electron microscopy of vas­cular casts. Lab Invest 1983;48: 169.

Uterine metabolism of the pregnant rabbit under chronicsteady-state conditions

Robert L. Johnson, M.D., Marc Gilbert, M.D., Stephen M. Block, M.D.,and Frederick C. Battaglia, M.D.

Denver, Colorado

The study of uterine metabolism in pregnancy under chronic steady-state conditions has been confined tolarge mammals and, more recently, to the guinea pig. The pregnant rabbit is of interest because of itsshort gestation and large litter size. We developed an indirect approach involving retrogradecatheterization of the uterine venous drainage, permitting measurement of both uterine metabolic quotients

and uterine uptakes. Radioactive microspheres were used to measure blood flow. A large lactate andammonia efflux from the uterus was found. In the fed state, ketogenic substrates were taken up in smallamounts. However, during starvation a significant increase in ketoacid uptake was observed with aconcurrent fall in acetate uptake. There was a large glucose/oxygen quotient across the uterus, but theglucose plus lactate/oxygen quotient was comparable to that found in the sheep and guinea pig(0.6 ± 0.1). It is apparent that in all three species studied under chronic steady-state conditions (sheep,

guinea pig, and rabbit) there is a large glucose uptake associated with a net lactate producton, andfuels other than glucose and lactate must be used by the uterus. (AM J OSSTET GYNECOL 1986;154:

1146-51.)

Key words: Uterine metabolism, uterine metabolic quotients, ammonia and lactateproduction, glucose and lactate quotients, placental metabolism

The metabolic demands of the developing conceptusremain an important area of interest in mammalianreproductive physiology. Metabolic requirements ofpregnancy have previously been described in largemammals' and, more recently in our laboratory, in theguinea pig." Among mammals, the variations of littersize, maturation, tissue composition, and maternal ca­loric requirements preclude simple interspecies com­parisons. Studies in conscious, unstressed sheep,' COW,3

and guinea pigs" have described glucose as the major

From the Division of Perinatal Medicine, Departments of Obstetricsand Gynecology, Pediatrics, and Physiology, University of ColoradoSchool of Medicine.

This work was supported by National Institutes of Health ProgramGrant HD-00781. R. L. Johnson and S. M. Block were supportedbyNational Institutes of Health Training Grant HD-07186.

Receivedfor publication October 4, 1985; revised January 3, 1986,acceptedJanuary 10, 1986.

Reprint requests: Frederick C. Battaglia,M.D., Professor and Chair­man, Department of Pediatrics, Box C-218, University of ColoradoSchool of Medicine, Denver, CO 80262.

1146

oxidative substrate of the uterus. A net lactate effluxfrom the uterus has also been described in all threespecies.>'

There have been few studies in small mammals car­ried out under chronic steady-state conditions. Re­cently, our laboratories have reported data on the car­diac output and uterine blood flow of the pregnantrabbit under chronic steady-state conditions, a studywhich involved catheterization of the arterial and pe­ripheral venous circulations." We now report successfulchronic catheterization of the uterine venous drainage,permitting studies of oxygen and substrate uptake bythe gravid uterus under steady-state conditions.

Material and methods

Ten New Zealand White rabbits were artificially in­seminated permitting precise pregnancy dating (within

1 day). During the last half of gestation, the animalswere anesthetized with ketamine hydrochloride, 50to 150 mg intramuscularly (Parke Davis & Co.), and

Volume 154Number 5

Table I. Summary of maternal and fetal data

Uterine matabolism of pregnant rabbit 1147

Whole uterus Left horn Uterine blood flow

Maternal Fetal Fetal Both LeftAnimal Gestational weight Weight No. of weight Weight No. of weight horns hom

No. age (days) (gm) (gm) fetuses (gm) (gm) fetuses (gm) (mllmin) (mllmin)

81-45 15-16 3980 106.3 9 42.6 69.1 7 33.3 24.41 14.2681-57 22-23 4826 246.0 10 130.2 142.9 6 80.3 37.01 21.5881-58 22-23 3838 238.4 12 125.6 98.2 5 49.2 31.89 12.7181-56 24-25 3870 245.9 10 147.2 125.7 5 73.0 29.04 16.1581-49 26-27 4090 445.4 10 339.1 220.4 5 167.9 85.49 43.1481-50 26-28 3906 367.0 10 247.5 237.4 5 172.9 32.16 23.7381-54 27-28 3920 130.3 3 65.5 88.3 2 42.7 18.59 11.0281-55 27-28 3921 236.0 10 131.1 114.7 5 59.3 19.75 10.89

Table II. Summary of metabolic data (arteriovenous differences, uptakes, and production)

Substrate

Glucose*Lactate*13-H ydroxybutyrateAcetoacetateAcetatetAmmonia*Oxygen*

Arteriovenousdifferences(umollml)

0.949 ± 0.082-1.l58 ± 0.121

0.033 ± 0.0080.016 ± 0.0030.084 ± 0.020

- 0.069 ± 0.0073.890 ± 0.214

umollmin

18.38 ± 2.78-23.74 ± 4.23

0.57 ± 0.140.25 ± 0.081.85 ± 0.40

-1.28 ± 0.1375.23 ± 8.67

Uptake

I umollminlkg

125.58 ± 8.49-151.77 ± 11.77

4.23 ± 0.912.60 ± 0.38

11.78 ± 2.50-9.15 ± 1.06

532.58 ± 38.83

Uterine uptakes calculated as product of flow to uterine horn where venous drainage wascatheterized times the arteriovenousdifferences.

*p < 0.001.tp < 0.01.

xylazine hydrochloride, 25 to 50 mg intramuscular(Bayes). Polyvinyl catheters were placed in the left ven­tricle (0.34 mm inner diameter) and distal aorta (0.23mm inner diameter). A closed-end nylon catheter (0.34mm outer diameter and 0.23 mm inner diameter) wasplaced in the left uterine vein by a retrograde tech­nique. The retrograde catheterization of the uterinevein was carried out as follows. The catheter was in­serted into the femoral vein and advanced to the junc­tion of the common iliacs. Under direct visualizationthe inferior vena cava was occluded transiently so thatthe catheter would advance up the uterine vein. Theposition of the catheter tip was confirmed by directpalpation. By this approach, the uterus need not bemanipulated at all and the catheter can be positionedto sample drainage from an entire uterine horn.

All catheters were secured with sutures and surgicaladhesive (Eastman 910) tunneled subcutaneously to anarea between the scapulas and secured in a fixed plasticcap as previously described.' The catheters were main­tained patent by flushing daily with a heparin-salinesolution (200 units/ml). The animals were given foodand water as needed and postoperative food intake wasmeasured. All animals were studied on two consecutivedays after a 3-day recovery period.

Paired arterial and venous samples were drawn si-

muitaneously for oxygen, glucose, lactate, acetate, ~­

hydroxybutyrate, acetoacetate, and ammonia deter­minations. Four sets of such samples were obtainedduring approximately 1 hour, and the mean arterio­venous difference for each substrate was used as thevalue for that day of study. After completion of sam­pling on the second day of study, all animals underwentinjection with radioactive "carbonized" microspheres(tin 113, 24 f.L in diameter) with the use of the integratedwithdrawal technique previously described." The ani­mals were put to death and the uterus of each dissectedto determine fetal, placental, and uterine weights. Ova­ries, kidneys, and adrenals were dissected to verifypaired organ flows. The calculations for uterine bloodflows were done by the reference sample method."

Two groups of animals were studied, those with die­tary intakes of >45 gm/day (previously found to be thelower limit of normal food intake)" and a second groupof starved animals with dietary intake <20 gm/day.

Chemical methods. All metabolic uptakes for oxygenand substrates across the uterine horn were calculatedby application of the Fick principle. Oxygen contentwas measured with an OSM 2 hemoximeter (Radiome­ter). The hemoximeter has the advantage of givinghighly reproducible measurements of both hemoglobin(in millimoles per liter) and oxygen saturation, from

1148 Johnson at al. May. 1986Am J Obstet Gynecol

-30.00

25.00

20.00

0

015.00

-10.00

5.00

••• •

0= LACTATE/02 QUOTIENT

1.50

2.50

1.00

2.00

0.50

3.00-.------------------------"

ClOO+---.-----,--:--""T'"---r---,.--....--r---!14.00 18.00 18.00 20.00 22.00 24.00 28.00 28.00 30.00

GEST ATIONAL AGE

Fig. 1. Gestational changes in lactate/oxygen and ammonia/oxygen quotients across the uterus.

which the oxygen content is calculated. Glucose, lactate,acetate, acetoacetate, and l3-hydroxybutyrate levelswere measured with standard enzymatic methods. 10

The ammonia content was determined according to theion-exchange method of Kurahasi et al. II The sampleswere measured in triplicate with a coefficient of vari­ation of 4%.

The average arteriovenous concentration differencesof the four sets of measurements were calculated bytaking into account each nutrient used to ascertainthe metabolic quotients for that day's study with theformula:

Average arteriovenous differenceof substrate (mmol) x k

Average arteriovenous differenceof oxygen (mmol)

where k is the number of oxygen molecules requiredfor the complete oxidative combustion of one moleculeof the substrate. This number varies from two for ac­etate to six for glucose. The mean of the four arterio­venous differences was also used to calculate the coef­ficient of extraction with the following formula:

Arteriovenous x 100 (%)

Arterial concentration

Statistical methods used were either paired or un­paired t tests, as designated. Regression lines were cal­culated by the least-squares method. All values are ex­pressed as mean ± SEM unless otherwise noted. Thesignificance of correlation coefficients was calculated byuse of hypothesis testing.

ResultsTable I presents a summary of maternal weights,

gestational age, fetal and placental weights, and uterine

blood flows for the entire uterus as well as for the in­dividual uterine horn studied. The studies covered agestational age range of 15 to 28 days.

Table II presents a summary of all metabolic dataincluding mean arteriovenous differences and uterineuptakes or production of substrates in the horn studied.The mean arteriovenous glucose difference was 0.95 ±

0.82 urnol/ml, the oxygen difference was 3.89 ± 0.21u.mol/ml, and the lactate arteriovenous differences was-1.15 ± 0.12 umol/ml. Small positive arteriovenousdifferences for ketoacids and acetate were found butdid not approach statistical significance.

Expressed per kilogram of tissue (uterus plus pla­centa plus fetus), the rabbit uterus and its contents con­sumed 125.58 ± 8.49 umol/min of glucose, 532.58 ±

38.83 urnol/min of oxygen, and 11.78 ± 2.50 urnol/min of acetate as well as small amounts of ketoacids.There was a large efflux of lactate (-151.77 ± 11.77umol/min) and of ammonia (9.15 ± 1.06 urnol/min)from the uterus.

Table III presents a summary of the metabolic quo­tients. The glucose/oxygen quotient was 1.46 ± 0.11,and the lactate/oxygen quotient was -0.91 ± 0.09.The glucose plus lactate/oxygen quotient was 0.55 ±0.064. The ketoacid/oxygen quotients were 0.04 ±

0.01 for l3-hydroxybutyrate and 0.02 ± 0.004 for ace­toacetate. Acetate/oxygen quotient was 0.04 ± 0.009.The sum of all substrate quotients was 0.65 ± 0.015.

Since ammonia and lactate are both small molecules,which we believe are produced within the placenta, itwas of interest to compare the quotients at differentstages of gestation. Fig. I illustrates the fact that thesequotients do not show a similar pattern of change. Theammonia/oxygen quotient falls while the lactate/oxy­gen quotient rises with increasing gestational age.

Table IV compares two groups of animals, one con-

Volume 154Number 5

Uterine matabolism of pregnant rabbit 1149

300

250

200

150

50-

ACETATE

pc.001

Fig. 2. The uterine uptakes of substrates by the left uterine horn in the fed and starved statesexpressed as a percentage of the fed state.

Table III. Metabolic quotients across the leftuterine horn

Table IV. Gestational age, fetal number, anduterine weight of starved versus fed animals

________1 (n ~~ 1) I--t-~a-::-e-~--

Values are mean ± SEM.

Glucose/oxygenLactate/oxygen~-H ydroxybutyrate/oxygenAcetoacetate/oxygenAcetate/oxygen

Total

*p < 0.001.tp < 0.005.

Quotient

1.46 ± 0.11*- 0.91 ± 0.09*

0.04 ± O.Olt0.02 ± 0.004*0.04 ± 0.009*

0.65 ± 0.015

Food intake/day(gm)

Fetal age (days)No. of fetusesTotal weight of left

uterine horn(gm)

127.6 ± 17.19

22.9 ± 2.115.6 ± 0.4

153.58 ± 33.0

3.25 ± 2.25

25.5 ± 1.05.0 ± 0.0

120.20 ± 5.46

suming a normal quantity of food and a subgroup ofstarved animals (animals offered food but with de­creased dietary intake). Previous observations by Gil­bert et at" defined an adequate dietary intake as a min­imum of 45 gm/day. The gestational age and litter sizewere similar in the two groups. The total uterine weightwas slightly decreased in the starved group (153.58 ±

33 gm in fed animals versus 120.20 ± 5.46 gm instarved animals), although this difference is not statis­tically significant.

Fig. 2 presents a comparison of uptakes per kilogramof left uterine horn (mean ± SEM) with uptakes instarved animals presented as a percentage of the up­takes in fed animals. The most dramatic change was inacetate uptake, which decreased 98%. Conversely, 13­hydroxybutyrate and acetoacetate uptakes increased300% and 200%, respectively. Fig. 3 demonstrates thatthere was an inverse relationship between uterine ke­toacid uptake and daily food intake.

Fig. 4 presents a comparison of the production ratesfor lactate and ammonia in starved animals expressedas a percentage of the fed state across the left uterinehorn. A significant 25% decrease in lactate production

was found. The decrease in ammonia production wasnot significant.

Comment

This study presents data describing the metabolismof the rabbit uterus under chronic steady-state condi­tions. It should be emphasized that the surgical ap­proach we have utilized for these studies differs fromthat of Gilbert et aI.,I2 who recently reported metabolicdata for the rabbit uterus. In the latter study, venousblood from a single fetoplacental unit and the sur­rounding myometrial tissue was sampled. Thus themetabolic quotients in that study reflected the metab­olism of that unit. In our approach the retrograde cath­eterization can be achieved without any direct manip­ulation of the uterus or its contents and the cathetertip can be directly localized in the main venous drainageof the horn. However, it is encouraging that the twodifferent approaches have yielded very similar esti­mates of glucose uptake and lactate production by thepregnant rabbit uterus, 127.5 versus 125.6 p.mcl/L glu­cose/min/kg in the study of Gilbert et aI.I2 versus thepresent study and 122 versus 151.8 J.1mol/L lactate/mini

1150 Johnson et al. May, 1986Am J Obstet Gynecol

2.0

.....-,cE-,Cll

!UIII: 1.0cl-ll.;:)

Cl

Uc0I-UIII:

••0125.0 250.0

FOOD INTAKE (11m)

zo;:o;:)Cl UIo =.. ...... ~w Cl.. W~ \I.

:ii .,.ID

iii

100- .

75-

50

25

0...1----......----LACTATE

P<.05

D-FED

~-STARVED

AMMONIA

N.S.

Fig. 3. Maternal food intake (grams) versus uterine ketoaciduptake (milligrams per minute per kilogram). Fig. 4. Comparison of lactate and ammonia efflux from the

left uterine horn in fed and starved states.

Table V. Comparison of chronic metabolic studies of three species in our laboratory

Coefficient of extraction (%)Gestation Glucose/oxygen Lactate/oxygen (Glucose + lactate)/

ISpecies (%) quotient quotient oxygen quotient Glucose Oxygen

RabbitGuinea pigSheep

70-9560-7580-95

1.5 ± 0.11.2 ± 0.10.8 ± 0.1

-0.9 ± 0.1-0.5 ± 0.1-0.1 ± 0.0

0.6 ± 0.10.7 ± 0.10.7 ± 0.1

19.8 ± 1.711.9 ± 1.39.0 ± 0.6

53.0 ± 3.339.0 ± 2.824.2 ± 1.1

Values are mean ± SEM.

kg, respectively. Our laboratory has previously pre­sented the results of studies on the metabolism ofsheep': 4.

5 and guinea pig uteri." Coupled with the sim­ilar studies of Comline and Silver": 13 on horse and cowuteri, five mammalian species have now been studiedunder comparable steady-state conditions.

In all five mammalian species studied thus far, uter­ine metabolism has been characterized by a very largeglucose/oxygen quotient across the uterine circulation,reflecting the high rate oflactate production within theplacenta. Table V presents the glucose plus lactate/oxy­gen quotients for the three species in which completedata are available. The quotients are significantly < 1.0and, in fact, are strikingly similar among the three spe­cies, which otherwise differ in a variety of importantways. In contrast, the coefficients of extraction for glu­cose and oxygen are quite different among these spe­cies, reflecting differences in perfusion of the uterusrelative to its metabolic rate with the three species.

Another characteristic of uterine metabolism that istrue in the rabbit, as in other species, is the efflux ofammonia. In the sheep it has been possible to dem­onstrate that the uteroplacental tissues are the sourceof the ammonia, not the fetal tissues. This was achievedby demonstrating that there was a significant entry ofammonia into both the umbilical circulation and theuterine circulation." Since the umbilical circulation or

fetal side of the placenta cannot be studied chronicallyin the rabbit, it is possible to demonstrate only thatammonia is produced within the uterus as a whole andrepresents a significant efflux of nitrogen from thepregnant uterus.

During gestation the uterine oxygen quotients forammonia and lactate do not show a similar pattern ofchange. This observation suggests that these changesare not simply a reflection of changes in the proportionof placental tissue to total uterine contents, because, inthe latter case, both quotients would show a similarpattern of change. Presumably, these changes must re­flect a basic change in metabolism of the placenta withan increasing lactate production and decreasing am­monia production relative to the metabolic rate of theuterus.

In summary, 10 pregnant rabbits were chronicallycatheterized during the last half of pregnancy. Cathe­ters were placed in the left ventricle and the distal aortaand into the main venous drainage from one pregnantuterine horn. Metabolic quotients across the uteruswere measured for glucose, lactate, ketoacids, acetateand ammonia. In addition, uterine blood flow was mea­sured by the integrated reference sample techniqueand microspheres 25 urn in diameter. A large lactateand ammonia efflux from the uterus was found. Theglucose plus lactate/oxygen quotient 'was 0.6 ± 0.1.

Volume 154Number 5

There was a small but significant uptake of ketoacidsand acetate by the uterus with the animals in the fedstate. Ketoacid uptake increased and glucose and ace­tate uptake markedly decreased when maternal foodintake was reduced. These uterine metabolic data col­lected under chronic steady-state conditions were com­pared with data collected under similar conditions forthe sheep and guinea pig. While the coefficients ofextraction differ among the species, the glucose pluslactate/oxygen quotients are remarkably similar for theuteri of all three species.

REFERENCES

I. Meschia G, Battaglia FC, Hay WW Jr, Sparks Jw. Utili­zation of substrates by the ovine placenta in vivo. Fed Proc1980;39:245-9.

2. Block SM, Sparks JW, Johnson RL, Battaglia, FC. Meta­bolic quotients of the gravid uterus of the chronically cath­eterized guinea pig. Pediatr Res 1985; 19:840-5.

3. Comline RS, Silver M. Some aspects of foetal and utero­placental metabolism in cows with indwelling umbili­cal and uterine vascular catheters. J Physiol 1976;260:571-86.

4. Burd Ll,Jones MD jr, Simmons MA, Makowski EL, Mes­chia G, Battaglia FC. Placental production and foetal uti­lization of lactate and pyruvate. Nature 1975;254:210-1.

5. Sparks JW, Hay WW Jr, Meschia G, Battaglia Fe. Parti­tion of maternal nutrients to the placenta and fetus in

Uterine matabolism of pregnant rabbit 1151

the sheep. Eur J Obstet Gynecol Reprod Bioi 1983;14:331-40.

6. Johnson RL, Gilbert M, Meschia G, Battaglia Fe. Cardiacoutput distribution and uteroplacental blood flow in thepregnant rabbit: a comparative study. AM J OBSTET GvNECOL 1985;151:682-9.

7. Sparks JW, Pegorier J -P, Girard J, Battaglia FC. Substrateconcentration changes during pregnancy in the guineapig studied under unstressed steady state conditions. Pe­diatr Res 1981;15:1340-4.

8. Makowski EL, Meschia G, Droegemueller W, BattagliaFC. Distribution of uterine blood flow in the pregnantsheep. AMJ OBSTET GYNECOL 1968;101:409-12.

9. Gilbert M, Hay WW Jr, JohnsonRL, Battaglia Fe. Someaspects of maternal metabolism throughout pregnancy inthe conscious rabbit. Pediatr Res 1984;18:854-9.

10. GirardJR, Cuendet GS, Marliss EB, et al. Fuels, hormonesand liver metabolism at term and during the early post­natal period in the rat. J Clin Invest 1973;52:3190-200.

II. Kurahasi K, Ishihara R, Uehara H. Determination of am­monia in blood plasma by an ion exchange method. ClinChim Acta 1972;42:141-6.

12. Gilbert M. Hauguel S, Bouisset M. Uterine blood flow andsubstrate uptake in conscious rabbit during late gestation.Am J Physiol 1984;247:E574-80.

13. Silver M, Comline RS. Fetal and placental 0, consumptionand the uptake of different metabolites in the ruminantand horse during late gestation. In: Reneau D, Grote J,eds. Oxygen transport to tissue. Symposium II. vol 75. NewYork: Plenum Publishing, 1976.

14. Holzman IR, Lemons JA, Meschia G, Battaglia FC. Am­monia production by the pregnant uterus. Proc Soc ExpBioi Med 1977; 156:27-30.

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