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The Circulation of the Fetus in Utero

METHODS FOR STUDYING DISTRIBUTION OF BLOOD FLOW,

CARDIAC OUTPUT AND ORGAN BLOOD FLOW

By Abraham M. Rudolph, M.D., and Michael A. Heymann, M.B., B.Ch.

ABSTRACTTechniques are described for insertion of vinyl catheters into the umbilical

and limb vessels of the fetus of the sheep or the goat through small uterineincisions, with the ewes under spinal analgesia. The catheters are exteriorizedand the fetus can be studied in its normal intrauterine environment. Duringconstant infusion of antipyrine into a fetal limb vein, placental arteriovenousdifference of antipyrine was measured, and fetal umbilical blood flow wascalculated by the Fick method. "Carbonized" microspheres (50-yu, diameter)labeled with various nuclides were injected into different venous sites in thefetus. The distribution pattern of the microspheres was used to determine therelative distribution of blood flow. Experimental evidence is provided that(1) there is no significant recirculation of microspheres, (2) the distribution ofspheres is proportional to flow, and (3) circulatory physiology is not altered byinjection of spheres. Quantitative data on the distribution of umbilical venousand superior and inferior vena caval return were obtained. It was possible todetermine the actual blood flow to each of the fetal organs by relating theproportions of nuclide in each organ to that in the placenta. Total cardiacoutput was then calculable, taking into consideration the hemodynamic ar-rangement of the fetal circulation.

ADDITIONAL KEY WORDS nuclide-labeled microspheresumbilical blood flow fetal venous return ductus venosus flowforamen ovale shunt asphyxia antipyrine sheep fetusumbilical vessel catheterization differential nuclide countingelectromagnetic flowmeters

B The course of the flow of blood in thefetus has been well established by severalgroups of investigators using various tech-niques. Since the fetus of the sheep or thegoat can be removed from the uterus andmaintained with intact uteroplacental circula-tion, most of the studies have been conducted

From the Cardiovascular Research Institute andthe Department of Pediatrics, San Francisco MedicalCenter, University of California, San Francisco, Cal-ifornia 94122.

This work was initiated in the Department ofPediatrics, Albert Einstein College of Medicine,New York, New York.

The investigation was supported by the U. S.Public Health Service Grants HE 06285, HE 08378,HD 01978 and HD 02805 from the National In-stitutes of Health. The computer analysis was sup-ported in part by U. S. Public Health Service Re-search Grant FR 00122.

Accepted for publication June L, 1967.

in these animals. Although the angiographicstudies of Barclay et al. (1) graphically dem-onstrated the direction of blood flow, they didnot provide any information regarding mag-nitude of flow. Dawes et al. (2) estimated theproportions of blood distributed through thevascular shunts of the foramen ovale andductus arteriosus, based on numerous meas-urements of oxygen saturation in the greatvessels and intracardiac chambers. Morerecently Dawes et al. (3) and Assaliand Morris (4) used electromagnet flow-meters to measure flow in certain majorblood vessels such as the pulmonary artery,ascending aorta, ductus arteriosus and um-bilical vein. In all these previous studies ofblood flow there were certain major draw-backs. The fetus was exteriorized and therewas no knowledge of the effects of this ma-

CircuUtion Research, Vol. XXI, August 1967 163

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164 RUDOLPH, HEYMANN

neuver on distribution of blood flow, and ex-tensive surgical procedures involving thora-cotomy and manipulation of the major arterieshad to be performed. Further, measurementsare difficult in young fetuses because of thesmall size of the structures involved.

Our studies were initiated in an attempt tostudy the fetus in its intrauterine environ-ment. A number of different approaches wereconsidered. Meschia et al. (5) had shown thatit was possible to maintain soft vinyl cathetersin the umbilical artery and vein of the sheepfetus. We considered the possible use of theindicator dilution technique to study flow. Inview of the numerous shunting mechanismsin the fetus, the indicator dilution techniqueis not applicable to the study of flow withoutplacement of numerous sampling and injectioncatheters and making several major assump-tions. We contemplated using the methodof measuring distribution of 42K after in-travenous injection, as described by Sapir-stein (6). Since the 42K is in solution, an un-known amount of the injected nuclide wouldbe lost to the mother in the uteroplacentalcirculation. Also, since the concentrations ofthe K delivered to various fetal organswould be different because of fetal shunts,this approach was discarded.

We have examined the applicability of amethod using the injection of nuclide-labeledmicrospheres. We here describe the tech-niques we have developed and our attemptsto prove the validity of the methods. Measure-ment of fetal umbilical blood flow by an in-dependent method using antipyrine infusionin the fetus has allowed quantitation of flowto all the fetal organs. It has also been pos-sible to measure fetal cardiac output usingthis information, and the necessary calcula-tions are presented.

PREPARATION OF THE ANIMAL

Thirty-two unregistered pregnant sheep(mainly of Hampshire, Sussex, Dorset andSouthdown breeds) and 17 unregistered preg-nant goats with fetal gestational ages from 85to 150 days were studied. Several of the sheeppregnancies were dated, but in the remainderand in the goats, the gestational ages were

estimated from fetal weight and crown-rumplength, using the relationship reported byBarcroft (7). This was applicable to our ani-mals, since in those in which pregnancy wasdated, the age-weight and age-length relation-ships were comparable.

The ewes were starved for 12 to 24 hr, be-ing provided with water only. This procedurewas helpful in avoiding undue distention ofthe rumen when the animal lay on her side.Low spinal analgesia was given using a mix-ture of 2 ml of 1% tetracaine hydrochloride,0.1 ml of 1 to 1000 epinephrine and 0.5 ml of50% dextrose; the animal was blindfolded andplaced on her right side. A vinyl catheter wasplaced in a branch of the maternal femoralartery. Through a left flank incision the uter-ine horn was exposed, and vinyl catheters,passed into branches of the umbilical arteryand veins, were manipulated into major um-bilical vessels. A fetal hindlimb and forelimbwere in turn exposed through separate smallincisions within purse-string sutures, and withlocal anesthesia, vinyl catheters were insertedinto a superficial vein and, in some animals,into an artery. The limbs were replaced, andthe catheters were passed through a trocarto exit through the skin on the left side of themother's abdomen; the abdominal incisionwas closed in layers. The studies were thenconducted, usually after 2 to 3 hr, but in 3studies, the sheep was allowed to recoverfrom the spinal analgesia, and the procedureswere carried out with the ewe standing in astall, up to 3 days after the surgical pro-cedure. When the catheters were chronicallymaintained, they were filled with heparinsolution (1,000 units/ml) and sealed witha solid metal or nylon plug. They were pro-tected by a Teflon cloth sack sewn onto theskin of the mother where the cathetersemerged. This did not appear to produce anydiscomfort in the animals. The details ofthese techniques are described in the follow-ing article (pp. 185-190, this issue).

MEASUREMENT OF FETAL UMBILICAL BLOOD FLOW

Umbilical blood flow was measured by ap-plication of the Fick principle, with contin-uous infusion of antipyrine as first described

Circulation Research, Vol. XXI, August 1967



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CIRCULATION OF THE FETUS IN UTERO 165

by Meschia et al. (8). In the earlier studies,4-aminoantipyrine was used and analyzedby the method described by Huckabee (9),using the diazotization reaction. Although 4-aminoantipyrine in the blood of adult sheepcould be analyzed readily by this method,considerable difficulty was encountered withfetal blood. It became evident that the pro-tein precipitation method using trichloroaceticacid did not produce a clear filtrate. Wemodified the method by precipitating theprotein with cold perchloric acid (0.5 mlplasma in 5 ml cold 3% perchloric acid); thisproduced a clear filtrate and consistent andreliable results. In later studies, antipyrinewas used and blood samples were analyzedwith a Technicon auto-analyzer (see article onpages 185-190 of this issue).

Blood Pressure and Blood Gases.—Mater-nal arterial pressures and fetal umbilical orfemoral arterial pressure were continuouslymonitored with Statham P23D transducersand recorded on a Grass Polygraph or Off-ner direct-writing recorder. Maternal arterialand umbilical or femoral arterial and umbili-cal venous blood samples were obtained atfrequent intervals during the procedure fordetermination of pH, Pco2 and Po2. Theanalyses were performed on 0.5 to 0.75 ml ofblood using a Beckman 160 physiologicalgas analyzer, a Beckman oxygen electrode, aSeveringhaus CO2 electrode and a RadiometerpH electrode. The tensions and pH weremeasured at 38°C and corrected to maternalrectal temperature.

LABELED MICROSPHERES

The microspheres were supplied by theNuclear Products Division of the MinnesotaMining and Manufacturing Company. Theirexact composition has not yet been released,but they consist of a compound containing67% carbon and 23% oxygen. They are referredto as "carbonized microspheres." The nuclideis incorporated in the spheres and is notmerely a coating on its surface. The nuclidelabel is not lost from the spheres by solutionin surrounding fluid. This has been shownby the absence of any radioactivity count inthe supernatant fluid in which the spheres


have been suspended for several months. Thespheres we used are 50 ft in diameter with avariation of ± 5 JJL. They are suspended in asolution of 2Q% dextran and supplied in a ster-ile ampule. We have found it convenient tosuspend 2 me of the spheres in 25 ml of dex-tran. The spheres have a marked tendency toaggregate, thereby forming clumps. In orderto avoid this and to provide for even distribu-tion, one drop of polyoxyethylene (20) sorbi-tan mono-oleate (Tween) was injected intothe ampule with a no. 23 needle. This com-pletely dispels any aggregation and thespheres are quite single, as shown by micro-scopic examination.

The density of the microspheres is about1.3 to 1.6, but average density is 1.4; thisvaries somewhat with different batches ofthe same nuclide. When shaken in the dextransolution, they remain suspended for about 15to 20 sec and then slowly settle. When thespheres are drawn up into a glass-barrelsyringe, the plunger frequently jams duringinjection as some of the spheres get caughtbetween the barrel and the plunger. Dispos-able plastic syringes may be used, but largenumbers of spheres adhere to the walls of thesyringe. To facilitate injection, we have useda small glass injection chamber (Fig. 1). Softglass tubing (3 cm long, 6 mm i.d.) wasdrawn into a point at each end. A smallsegment of a no. 20 needle with the hub re-moved was annealed into each end of the

STA/NLESSSTEEL TUBE

FIGURE 1

Diagram of injection chamber. For description see text.



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STRONT/UM 3S

SCANDIUM

KEV 0

CHANNELS I

FIGURE 2

Energy spectra for four nuclides and a mixture of thefour. KEV = kiloelectron volts; channels = location ofchannel of 400-channel pulse height analyzer.

glass by heating in an open flame. The metal-glass junction was reinforced with epoxy-resin. A short length of vinyl tubing connectedto a needle and a disposable stopcock wasattached to one side of the chamber. Another

RUDOLPH, HEYMANN

short length of vinyl tubing with a needle at-tached was connected to the other side ofthe chamber. This needle was then insertedinto the ampule of microspheres, and a smallamount of the suspension was pulled intothe chamber by a disposable syringe connect-ed to the stopcock. The amount of radioactiv-ity was checked by counting the chamber,and when the appropriate amount was ob-tained, the chamber was filled with dextranor saline solution and the stopcock closed.The total radioactivity count in the chamberand tubing was again checked before injec-tion into the animal. A different chamber wasused for each of the nuclides. The amount ofradioactivity was carefully adjusted to pro-vide a total injection of about 5 X 103 to 1 X108 counts/min of each nuclide. During in-jection, the chamber was continuously shaken.The chamber and tubing were again countedafter injection to check the amount deliveredto the animal.

Presently, four nuclides with predominantlygamma emissions are being used: iodine-125(125I), chromium-51 (51Cr), strontium-85(85Sr) and scandium-46 (46Sc). The selectionof these nuclides was determined by theiravailability, their cost, their half-lives and bytheir energy spectra. 125I has a single largepeak at 35 kev and there is almost no countabove 97 kev (Fig. 2). 51Cr has a well-definedpeak at 324 kev with negligible count above415 kev (Fig. 2). 85Sr has a sharp peak at513 kev and no significant count above 600kev (Fig. 2). 46Sc has two peaks, not verysharp, at 890 and 1120 kev. Since we havemeasured the energy range of 0 to 1000 kev,only the 890 kev peak of the Sc has beenused, since all activity beyond 1000 kev hasnot been counted (Fig. 2). When the fournuclides are mixed, the characteristic peaksare well separated (Fig. 2).

MEASUREMENT OF RADIOACTIVITY ANDSEPARATION OF NUCLIDES

A. Manual Method.—All counting was donein a well-scintillation detector, using a Tracer-lab 3-inch thallium-activated sodium iodidecrystal with a well, lJs-inches in diameter




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Location of

Isotope

1251

Peak Energy

Kev

88551332435

TABLE

and Representation on1 Peak

Channel

35420513014

1

the 400-Channel

Kev

772.5-995422.5-620272.5-395

2.5-95

Analyzer for Four Nuclides

Range countedCh

AB

cD

annel groups

(309-398)(169-248)(109-158)

(1-38)

and 1% inches deep; 1100 volts was applied tothe photo multiplier tube. The output of thedetector was connected to a 400-channel an-alyzer (Technical Measurement Corporation,TMC 404C Multiple Channel Pulse HeightAnalyzer) and the whole energy spectrumwas displayed on the oscilloscope. The scin-tillation-well detector and multiple channelanalyzer system were calibrated using cesi-um-137 ( m Cs) as a standard; this nuclidehas a major peak at 662 kev. The detectionsystem was calibrated so that each of the 400channels covered a range of 2.5 kev, to givea total range of 0 to 1000 kev. The gain onthe analyzer was adjusted so that the 137Cspeak was in channel no. 264-5, at 662 kev.This was readily accomplished with a TMC522A Resolver Integrator connected to theTMC 404C Analyzer; this allowed any indi-vidual channel or group of channels to be dis-played on the oscilloscope. Using the ResolverIntegrator, it was possible to integrate all 400channels to measure total radioactivity countand, in addition, one or more groups of chan-nels. The integrated totals were then in turnprinted out on a TMC 500P Printer.

Because l25I, 51Cr, and 85Sr all have nomeasurable counts above their major peaks,calculation of the relative amounts of eachnuclide in mixtures was quite simple. Apure sample of each nuclide was countedand displayed on the oscilloscope. After in-tegrating to obtain a total count, those chan-nels which included the main peak were alsointegrated. A ratio of the counts in the peak tothe total count was obtained, and this rela-tionship is constant for the same nuclide. Theanalyzer channels and respective energyranges of the four nuclides, and the per-

Circulction Research, Vol. XXI, August 1967

centage of total count included in the peaksare shown in Table 1.

The number of counts in each of the fourgroups of channels selected was then record-ed for each of the four nuclides. Table 2shows the percentage of the total count ineach group of channels for each nuclide. Thisinformation was then used to calculate theactual activity of each individual nuclidewhen two or more nuclides were present inthe same sample. The procedure adopted tomeasure the activity of each was as follows:After counting for an appropriate period, thetotal spectral range and the four groups ofchannels are integrated. Since channels 309 to398 show activity solely due to 4(iSc, the totalscandium count can be calculated from theinformation in Table 2. The counts represent-ing scandium activity could then be subtract-d u a l ch an n elor group of channelst

After subtracting the scandium count fromchannels 169 to 248, the remainder repre-sented only strontium activity. From the rela-tionship of counts in the peak channels tototal 85Sr count, the amount of 85Sr could becalculated. The counts due to strontium activ-ity were then subtracted from the remainingtwo groups of channels and 51Cr and 12SI

TABLE 2

Percentage of Total Counts for Each Nuclide inSelected Channel Groups

1-399 A B C D

1251

i o o

100100100

30.4000

18.055.800

13.37.7

72.70

6.610.69.6

99.8

See Table 1 for channels included in A, B, C, andD.



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activity was calculated in a similar manner.Equations used for calculation of the countsfor each nuclide in a mixture based on thepercentages in A, B, C, D, here represent theintegrated counts recorded in each of thegroups of channels in Table 1.

Scandium =3.283A.Strontium = 1.7896B - 1.0592A.Chromium = 1.3761C - .1906B - .7114A.Iodine = D - .1383C - .2082B - .4011A.

To clarify the derivation of the above equa-tions for chromium and iodine, the detailedanalysis for calculation of chromium is pre-sented.

the counts obtained. A small source of gam-ma emission placed at the bottom of the welldetector produces a considerably higher countthan when it is distributed in a larger volume.This variability in count rate is extremely im-portant in separating mixtures of nuclides. Ifone nuclide is layered at the bottom of thedetector and the other near the top, a grosserror in the estimation of the relative amountsof each nuclide results. We checked the ac-curacy of the separation and the optimalgeometry in the following manner.

Small amounts of spheres labeled with eachnuclide were intimately mixed with a silicone

Cr =

C -

C -

Scandium StrontiumC-(3.283A) 0.1325- (1.7896B - 1.0592A) 0.0774

0.7267C - 0.435A - (.1385B - .082A)

0.7267

C - 0.517A - 0.1385B0.7267

Cr = 1.3761C - 0.1906B - 0.7114A.

B. Automatic Analysis.—Since the methodjust described is quite time consuming, othermethods were developed to overcome thisdifficulty. An automatic changer (Nuclear-Chicago) has been used to count up to 100samples in sequence. At the end of each count-ing period, the total spectrum recorded by themultiple-channel analyzer has been automat-ically reproduced on a paper punch tape(TMC 535). The information on the paperpunch tape has been transposed onto mag-netic tape and the type of analysis outlined inthe manual method has been performed usinga Computer IBM System 360. It has beenfound convenient to use only 100 channels ofthe analyzer for this type of analysis withoutaffecting the results obtained as comparedto the manual method. Each channel thenrepresented 10 kev.

PREPARATION OF SAMPLES FOR COUNTING

In view of the widely separated gammaemissions of the nuclides used, it was im-portant to exercise considerable care to ob-tain accurate counts in mixtures. The geom-etry of the sample has a profound effect on

elastomer (General Electric RTV 11). Afterthe silicone had hardened, it was cut up intopieces of varying size. These could be readilyhandled, and by counting a sample of eachnuclide individually and then mixing a num-ber of nuclides each with a known count rate,it was possible to determine the accuracy ofthe method for estimating the amount ofeach nuclide in a mixture. We found that themost reliable results were obtained if thesampling tube was not filled more than 2 to3 cm from the bottom. It was also very im-portant to place the different nuclides side byside and not layer them from top to bottom.When these precautions were taken, the esti-mated counts of each nuclide in a mixturewere within 1% of the actual count recordedseparately.

It is indeed fortunate that the microspheresin tissue are apparently evenly distributedand that different nuclide labels are evenlydistributed after repeated injections. Therewas no difference in the energy spectrum andtotal count rate of tissues placed in a numberof different positions in the well detector.

We have made a practice of counting thewhole animal, including the placenta andmembranes. We have been reluctant to acceptthe difference in the count rate of the injec-tion chamber before and after injection as ameasure of the total activity received by the




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animal. The count rate in the chamber beforeinjection is very high and there is a possibilitythat the well detector is inefficient at very highcount rates. After the animal was killed withsodium pentobarbital and potassium chloride,each organ was dissected and cut into piecessmall enough to reach no more than 3 cmfrom the bottom of the counting tubes. Thelarger organs and carcass cannot be handledin this way. We have therefore incineratedthe placenta and membranes; the carcasshas been minced and then also incineratedat 230°C. We have shown that a measuredamount of each nuclide does not lose any ofits activity when exposed to this temperaturefor over 24 hr.

VALIDATION OF THE MICROSPHERE TECHNIQUE ASA MEASURE OF FLOW DISTRIBUTION

Recirculation of Microspheres. — Mi-crospheres of 50-/U, diameter were selected inthe hope that all would be caught up in theperipheral precapillary vessels during the firstcirculation through the vascular system, andthat none would pass through arteriovenousshunts and thus recirculate. It was also im-portant to check that none of the micro-spheres passed from the fetal placental cir-culation to the maternal circulation, or enteredthe uterine muscle. In both adult and fetalanimals we explored the possibility of signifi-cant arteriovenous shunting in various circu-lations.

Microspheres were injected into the in-ferior vena cava in 2 guinea pigs 3 days old,1 rat and 2 mice. The various organs werecounted separately to determine how much ofthe nuclide had by-passed the pulmonary ves-sels. In the 2 mice and the guinea pigs, theradioactivity of the whole body was counted.We found that greater than 99.5% of the totalbody count was in the lungs. Small countsabove background were noted in other organs,particularly in the lower body. In the rat,only the lungs, heart, liver, kidney and in-testines were counted. There was no demon-strable count of the injected nuclide in anyorgan except the lung. In 1 mouse and 2guinea pigs, microspheres were injected intothe descending aorta below the diaphragm,


and the amount of radioactivity in the lungswas measured to assess whether microsphereshad passed through the peripheral circula-tion. No radioactivity was detected in thelungs.

Since it was possible that the vascular sys-tems in the fetus, and particularly the pla-cental circulation, may behave differentlyfrom those of the adult animal, we also at-tempted to explore the possibility of signifi-cant arteriovenous shunting in the fetal lamb.

Since right ventricular and pulmonary ar-terial blood is distributed either to the lungor through the ductus arteriosus and then tothe descending aorta, no blood ejected fromthe right ventricle should reach the heart andthe head and brain unless it has passedthrough the lung and back to the left atriumand left ventricle. We injected labeled micro-spheres into the right ventricle in 5 animals inutero and into the left pulmonary artery in 1exteriorized lamb with no lung expansion andwith placental circulation continuing. Wemeasured the proportion of total injected ra-dioactivity that was distributed to the heart,head and brain. In the Iamb in which thespheres were injected into the left pulmonaryartery, and in 3 animals in which they were in-jected into the right ventricle, there was nosignificant radioactivity in the heart, upperextremities, head or brain. In the other 2animals there was insignificant radioactivityin the heart and brain, but there was someradioactivity in the upper carcass (Table 3).Both these animals were quite asphyxiated

TABLE 3

Distribution to the Upper Body of Nuclide Injectedinto the Right Ventricle or Pulmonary Artery

Right ventricularinjection

S27G16G15S32S31

Pulmonary arteryinjectionS34

S = sheep; G =

PercentHeart

0.040.06000

0

goat.

of totalBrain

00.4000

0

counts injectedUpper carcass

6.93.8000.4

0



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at the time of the injection of spheres. Thesmall amount of nuclide passing to the uppercarcass was located mainly in the left fore-limb and head. We thought that this did notrepresent passage of spheres through thelungs but, rather, streaming from the ductusarteriosus into the brachiocephalic trunk.

The possibility of large placental arteriove-nous shunts was examined in 22 fetal animalsat various gestational ages by injection ofmicrospheres into the superior or inferior venacava. The pathways for the microspheres toreach the liver are through the hepatic ar-terial circulation, through arteriovenousshunts in the placenta via the umbilical veins,or through shunts in the gastrointestinal tractand spleen. To assess the total amount ofnuclide reaching the liver from these sources,we measured the proportion of the total in-jected count that was distributed to the liver.In 14 animals in which microspheres wereinjected into the forelimb vein, and in 17 withinjections into the hindlimb vein, 0.0 to 1.8%of the total injected nuclide was present inthe liver. The amount is so small that therecould not be any significant passage of micro-spheres across the fetal placental circulation.Furthermore, all of the spheres could havereached the liver parenchyma from the hepat-ic artery and none through placental or gas-

TABLE 4

Relationship between Flow and Nuclide Distributionin a Physical Model

Flow (ml/min)

1413376

145

65122245

9868596

Total cpm

Bloodnot measurednot measurednot measurednot measured

268,94251,10899,053

183,968Dextran542,824394,061366,36932,623

Blood collected(cpm/ml)

2824290128962810

1791183019401780

2586269425552718

cpm = counts per minute.

trointestinal arteriovenous communications.Microspheres were injected into the um-

bilical artery in 1 lamb, and no radioactivitywas detected in the liver, indicating that thespheres do not pass through arteriovenousshunts in the fetal placental circulation.

The possible passage of microspheres acrossthe fetal placenta into the adjacent uterinemuscle was examined in 5 animals by count-ing the radioactivity in the uterine musclefrom the site of attachment after removingthe cotyledons which contained microspheresinjected into the fetus.

There was no measurable radioactivity inthe uterine wall, indicating that when the pla-centa and membranes are separated from theuterus, all the spheres remain on the fetalside and that they do not enter uterine muscle.

VALIDATION OF THE USE OF MICROSPHERES TODETERMINE DISTRIBUTION OF FLOW

Circulation in a Physical Model—A rotarypump was used to perfuse a system of fourbranching tubes in each of which flow couldbe regulated. Labeled microspheres werethen injected through a needle inserted intothe tubing upstream from a mixing chamber,and the effluent was collected during, andfor several minutes after, the injection. Sixpercent dextran solution was used in one in-stance, and whole blood in two instances.Since flows were kept constant throughoutthe collection period, the proportions of ra-dioactivity in the four samples collectedshould be the same as the proportions of flow,if the microspheres were distributed accord-ing to flow rates.

The results of the three observations inthis model are shown in Table 4. In the firstexperiment, the four collecting graduated cyl-inders were well shaken, and immediatelyafter shaking, 1-ml samples were rapidlydrawn into syringes and counted. Three bloodsamples were obtained in this manner; theaverage count per milliliter of blood in eachcylinder is shown. In the other experimentwith blood and in the one using dextran, thevolume in each cylinder was recorded, andafter allowing the spheres to settle for aboutan hour, the fluid in the upper portion was




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TABLE 5

Comparison of the Ratio of Flows in the Two Umbilical Veins Measured by ElectromagneticFlowmeters and the Ratio of Nuclide Counts in the Placenta Drained by Each Vein

S7S10Sl l

S13S14S15S30

Flows

144100545266

15019290

Flowmeter(ml/min)

12980204530

12012677

Ratio

1.1311.252.701.162.201.251.531.20

12,00086,00069,500

134,00015,600

117,30059,600

115,500

Nuclide distributioncpm

10,60085,00025,800

110,0007,800

93,60038,70097,000

Ratio

1.1321.012.691.232.001.251.541.20

S = sheep; cpm = counts per minute. In sheep 11, two sets of observations were made.

suctioned off and checked for radioactivity.The total amount of nuclide collected fromeach cylinder was measured and is shown inthe second column. The third column showsthe close relationship of the counts per min-ute per milliliter of blood collected and con-firms the evenness of distribution and thefact that sphere distribution is related to flowrate.

Circulation in the Fetus.—We considered itimportant to check the validity of the methodin vivo as well as in a physical model, anddid so in two vascular systems. The mostreliable and reproducible method for meas-uring flow in vivo was the electromagneticflowmeter with cannulating flow transducers.The characteristics of the flow transducerand flowmeter systems are described in thefollowing article (pp. 185-190, this issue).

We used the electromagnetic flowmeter tocheck the proportional distributions of flowto the placenta through each umbilical artery.We had observed that there is rarely anymajor connection between the two umbilicalarteries, and only minor connections betweenthe two umbilical veins in the lamb. Injectionsof silicone elastomer into one umbilical arteryand its accompanying vein have shown thatthey are distributed to a distinct group ofcotyledons; the remaining cotyledons are sup-plied by the other artery and vein. In only 1of 9 placentas examined was there a largeinterarterial connection.

The fetus was delivered from the uterus,a saline-filled glove was placed over the head


to prevent expansion of the lungs with air,and placental circulation was allowed to con-tinue. The fetal temperature was maintainedat 37.5 to 39°C by warming pads. A mixtureof phenoxybenzamine and hexylcaine was ap-plied to the umbilical vessels. A segment ofone umbilical vein was isolated betweenclamps, and a 4- or 5-mm cannulating probe,to each side of which was attached vinyltubing of appropriate size, was inserted. Afterflow was reestablished in this vein, the pro-cedure was repeated in the other vein. Specialcare was exercised to avoid any air embolism.After flows had become stable, labeled micro-spheres were injected either into an umbilicalvein or a forelimb or hindlimb vein as pre-viously described. In some experiments, oneumbilical vein was partially or completelyclamped to modify flow, and a second nuclidewas injected. The flowmeter outputs werecontinuously recorded on an Offner recorder.Repeated mechanical zero checks were ob-tained by clamping each vein in turn. Afterthe experiment, one vein was injected withsilicone elastomer to fill the vessels all theway to the cotyledons. This identified thegroup of cotyledons drained by the vein. Thetwo groups of cotyledons were then countedseparately, and the ratios of the amount ofnuclide compared with the ratios of the flowsmeasured directly by the flowmeter.

Confirmation that the microspheres are dis-tributed in proportion to blood flow in vivois shown in Table 5. There is a good corre-lation between the ratios measured by the



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TABLE 6

Ratios of Nuclide Counts per Minute in Kidneys and Placenta when Injected Simultaneously into Different VenousSites

S18S19S20S21GlG8G3S29

Left kidney: right kidneyUpper limbvein inject.

.69

.691.19.93

1.69.82

1.091.18

Lower limbvein inject.

.81

.801.33.87

1.61.75

Umbilicalvein inject.

.901.18

RighlUpper limbvein inject.

.026

.033

.038

.029

.019

.023

.032

.050

: kidney: placentaLower limbvein inject.

.022

.034

.035

.027

.019

.024


.033

.039

LeftUpper limbvein inject.

.017

.022

.044

.026

.032

.019

.034

.059

kidney: placentaLower limbvein inject.

.018

.029

.046

.023

.029

.019


.029

.042

S = sheep; G = goat.

two methods. It is of special interest that in1 animal (not in the table), when one umbili-cal vein was completely occluded, eventhough there was still a high pressure in itsassociated umbilical artery, no nuclide wasdistributed to the portion of the placentadrained by that vein.

In 1 lamb, the flows in the two umbilicalveins and also the flow to the head and brainas measured by the microsphere distributiontechnique were compared with those meas-ured with the electromagnetic flowmeter. Inaddition to cannulating the two umbilicalveins, cannulating flow transducers were in-serted into the external jugular veins. Micro-spheres with different labels were injectedinto the forelimb and the hindlimb veins.After killing the lamb, silicone elastomerwas injected into the jugular veins to outlinethe area of venous drainage. The amountof radioactivity in each group of cotyledonsin the portion of the head and neck filledwith silicone and in the other body organs

TABLE 7

Distribution of Two Nuclides (A and B) Injected inSequence into the Umbilical Vein

BrainHeartKidneySpleen

A

19.114.33.71.0

B

20.317.84.11.1

Counts in each organ expressed as percentage ofplacenta! counts.

was measured. The flow to the head and neckwas calculated from the microsphere distri-bution and umbilical flow, as described below,and compared with the flow measured byelectromagnetic flowmeter.

The flow using the microsphere techniquewas 139 ml/min, as compared to the venousflow of 126 ml/min measured simultaneouslywith cannulating electromagnetic flow trans-ducers.

EVIDENCES OF ADEQUACY OF MIXING ANDEVENNESS OF DISTRIBUTION

Since the fetal circulation has several sitesof shunting, it was necessary to prove thatmicrospheres injected into various venous cir-culations were intimately mixed with theblood, and that there was no selective stream-ing of particles. In 5 lambs and 3 goats, wesimultaneously injected spheres labeled withdifferent nuclides into a forelimb vein andinto either a hindlimb or umbilical vein.Upper limb venous blood joins superior venacaval return, most of which passes throughthe tricuspid valve into the right ventricle, tobe distributed partly to the lung, but largelythrough the ductus arteriosus into the de-scending aorta. The portion of umbilical ve-nous blood traversing the ductus venosuspasses into the inferior vena cava, as doesblood from the hindquarters and abdominalviscera. Some of this passes into the rightventricle, to be distributed as superior venacaval blood; a larger portion passes throughthe foramen ovale and is distributed from the




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left ventricle partly to the vessels arising fromthe ascending aorta; the remainder goes tothe descending aorta. The blood in the de-scending aorta is derived from the ductusarteriosus and the arch of the aorta; if theinjected microspheres were intimately mixedin descending aortic blood, then their dis-tribution pattern to the lower body wouldbe similar, regardless of the venous site intowhich they were injected. Table 6 comparesthe ratios of nuclides in the left kidney tothose in the right kidney when two differentnuclides were injected into different sites inthe same animal, and the ratios of nuclide ineach kidney as compared to the placenta. In1 animal, two injections of spheres labeledwith different nuclides were made into theumbilical vein within 5 min. Table 7 com-pares the distribution patterns. Tables 6 and

7 show the close relationship between theratios of nuclide distributed to the organssupplied by the descending aorta when in-jected into any part of the venous system ofthe fetus or injected successively in the samevein.

Although microscopic examination of themicrosphere suspension before injectionshowed no clumping of the spheres, it wasnecessary to show that the spheres were dis-tributed evenly. Thick sections of the lambkidney showed no evidence of clumping ofspheres. The spheres were evenly distributedin the cortex, and in an occasional vesseltwo spheres were seen, one behind the other.

CALCULATION OF DISTRIBUTION OF BLOOD FLOWAND CARDIAC OUTPUT IN THE FETUS

We have combined the technique for cal-culation of distribution of flow with the simul-

HEAD

PULMONARYARTERY

oucrusART5R/0SUS

- DESCENDINGAORTA

PLACENTA

FIGURE 3

Diagrammatic representation of the fetal circulation.




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taneous measurement of umbilical blood flowby the antipyrine method to calculate cardiacoutput and organ flows in the fetus. It isimportant to take into account certain aspectsof the course of fetal blood flow to make thesecalculations. These considerations are clarifiedby referring to the diagram of the fetal circu-lation shown in Figure 3.

1. Umbilical venous blood returning to thefetus may pass to the left lobe of the liverdirectly, through the ductus venosus, or to theportal sinus to enter the right lobe of theliver. The microspheres entering the portalcirculation would be caught up in the liversinusoids, whereas those passing through theductus venosus would be mixed with inferiorvena caval blood and be distributed accord-ingly.

If microspheres are injected into an um-bilical vein, the proportion of radioactivityin the liver compared to the total radioactivityrecovered from the fetus and placenta repre-sents the proportion of umbilical venous re-turn passing through the liver. The remaindercan be assumed to have passed through theductus venosus. A small error is caused bythe microspheres which have passed throughthe ductus venosus and are then distributedto the liver through the hepatic artery. Thiswill be discussed later.

2. Inferior vena caval blood is largely di-rected through the foramen ovale into the leftatrium, but a variable proportion passes intothe right ventricle. This produces a seriousproblem if an indicator is injected into theinferior vena cava to measure its distribution.The indicator in that portion of inferior venacaval blood entering the left atrium is dilutedby pulmonary venous blood and is then dis-tributed to the upper part of the body and tothe descending aorta. The indicator in theinferior vena caval blood entering the rightventricle is diluted by superior vena caval,coronary and azygos venous return, and it isthen distributed to the lungs and throughthe ductus arteriosus to the descending aorta.

It is thus obvious that the blood beingdistributed to the tissues may have threedifferent concentrations of nuclide. Blood

passing to the heart, head and neck, brainand forelimbs from the ascending aorta hasone concentration, that passing to the lungsanother. Descending aortic blood (derivedfrom a mixture of blood containing thesetwo concentrations in variable amounts) hasa third concentration.

3. Superior vena caval blood usually passesthrough the tricuspid valve into the rightventricle, where it mixes with a certain pro-portion of inferior vena caval blood; thiswould result in a dilution of the indicator.It is then ejected into the pulmonary arteryand may pass into the pulmonary circulationor through the ductus arteriosus to the de-scending aorta. The injection of nuclide-labeled microspheres into the superior venacava is very helpful in detecting how muchsuperior vena caval blood passes through theforamen ovale. The finding of nuclide in theheart, head, brain and forelimbs providesevidence that blood has crossed the foramenovale to be distributed from the left ventricleand ascending aorta.

This analysis makes it quite clear that it isnot possible to calculate relative distributionsof blood flow on the basis of the assumptionthat the nuclide-labeled microspheres aremixed in equal concentrations in blood ar-riving at all the fetal organs. The most distalshunting site in the fetal circulation is theductus arteriosus. If it is assumed that bloodderived from the ductus arteriosus and fromthe ascending aorta mix well in the descendingaorta, then ths concentration of microsphereswould be similar in blood distributed to allorgans supplied from this vessel.

If it is assumed that the microspheres aredistributed in relation to flow, then the ratiosof microspheres in each organ are equal to theflow ratios. If the flow to any single organ isknown, then the flow to any other organ canbe estimated.

Blood Flow to Lower Body.—A measure-ment of umbilical flow by the antipyrinemethod was made just before and just afterthe injection of microspheres. The ratios ofthe amounts of radioactive material in eachof the organs supplied by the descending




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aorta to the total placental count, multipliedby umbilical (fetal placental) flow representsthe flow to that organ.

Thus for the kidney,

T1 placenta llmbilical>

where Q = flow rate and I = amount of radio-active indicator.

In a similar manner, the flows to the spleen,gastrointestinal tract, lower extremities andlower trunk can be estimated. Since all thevenous blood from these organs enters theinferior vena cava,

and the left chest wall. We thought that nosignificant error could be introduced by dis-regarding the flow from these systems.

A similar problem exists with a certainamount of blood distributed from the abdomi-nal and diaphragmatic branches of theinternal mammary artery, which are derivedfrom the ascending aorta. The flow fromthese vessels may in part return to the inferiorvena cava. We thought that this introducedno significant error either.

Upper Body Blood Flow— From the calcu-lations just described, it is possible to measurethe flow from the inferior vena cava to the

VIVC ( ^placenta + ^kidney + Qspleeu + Qcareass + Qgastrointestinal- (2)

Knowing the total inferior vena caval flowand the total amount of nuclide injected intothe inferior vena caval blood, it is now pos-sible to determine that portion of the bloodflow to each organ which is derived fromthe inferior vena cava. For example,

brain (IVC)- (3)

The ratio of nuclide in the brain to totalnuclide injected, multiplied by the total in-ferior vena caval flow, provides a measure olfcthe brain blood flow which is contributedfrom the inferior vena cava.

In this manner, the cardiac (coronary)blood flow and the pulmonary flow derivedfrom the inferior vena cava can also becalculated.

A small error in calculation of distributionof blood returning from the lower part of thebody has to be taken into account. The bloodfrom the azygos and hemiazygos venous sys-tems do not drain into the inferior vena cava,but return to the heart via the superior venacava and coronary sinus respectively.

In an attempt to assess the magnitude ofthe error involved, we injected silicone elas-tomer into the azygos venous system in 2lambs and examined the areas drained bythis system. The azygos system itself had avery small capacity and it drained the rightside of the chest wall. The hemiazygos systemhad a similarly small capacity; it drained asmall portion of the posterior abdominal wall


heart, head, brain and forelimbs. If no su-perior vena caval blood crosses the foramenovale, then the blood flow to these organs isderived totally from the inferior vena cavaand the pulmonary venous return to the leftatrium. It is thus also necessary to assesshow much of the pulmonary venous returnis distributed to the upper body. Since pul-monary venous blood mixes with inferiorvena caval blood in the left atrium, it pre-sumably will be distributed in the same pro-portions when ejected by the left ventricle.It was necessary to assess what proportionof blood ejected by the left ventricle was dis-tributed to the upper body. This was accom-plished in the following manner. In 4 lambs,a catheter was inserted into a forelimb arteryand passed retrograde to enter the left ven-tricle. The limb was replaced, and after re-covery microspheres were injected into theleft ventricle. We found that the percentagesof nuclide distributed to the ascending aortawere 29, 38, 43 and 48. We therefore assumedthat under normal conditions about one thirdof the pulmonary venous return goes to theupper body and heart. Of this total, about20 to 25% goes to the heart.

The amount of pulmonary blood flow de-rived from the inferior vena cava is knownfrom the estimate of the proportion of nuclidedistributed to the lung. However, the amountderived from the superior vena cava is notknown. In a large number of lambs studied,



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less than 5% of nuclide injected into thesuperior vena caval return was distributedto the lungs. Since about one third of pulmo-nary venous return is distributed to the upperbody, less than 1.5% of superior vena cavalreturn would be distributed to the upperbody by means of pulmonary venous return.In a full-term lamb in which superior venacaval flow is not usually above 250 ml/min,the amount of pulmonary venous blood de-rived from the superior vena cava that wouldbe distributed to the heart, head, brain andforelimbs would be less than 4 ml. We be-lieved this could be disregarded.

In most fetuses studied, only 0.2 to 2.0%of inferior vena caval nuclide was distributedto the lung. Since only one third of the pul-monary venous return is distributed to theupper body, less than 1% of total inferior venacaval flow would pass to the upper body aspart of the pulmonary venous return. Someof this would enter the coronary circulation,so that the amount passing to the forelimbs,head and brain would be considerably lessthan 1% of inferior vena caval flow. We be-lieved that this too could be disregarded inthe estimation of total superior vena cavalreturn.

In an occasional animal, the proportion ofinferior vena caval nuclide entering the lungis larger than 2% (up to 9%). Assuming thatone third of pulmonary venous return is dis-tributed to the upper body, an adjustmentcan be made in the estimate of total superiorvena caval return.

Usually no superior vena caval bloodcrosses the foramen ovale. It is, however, im-portant to document this. It can be readilydocumented by simultaneous injection ofspheres with a different nuclide label intothe superior vena caval return, thus allowinga measure of the amount of superior venacaval blood redistributed to the upper body.

In the absence of such shunting of superiorvena caval blood,

seem reasonable to disregard the pulmonaryvenous return, since it represents a verysmall amount.

When a foramen ovale shunt of superiorvena caval blood occurs, then the proportionof such shunt passing to the upper body canbe calculated and the total superior venacaval flow adjusted to correct for this.

When total superior vena caval flow hasbeen calculated, it is possible to determine theproportions of superior vena caval blood dis-tributed to each organ by using the equation

Vorgati (sve) — •y; * V?svc- 1°^

Pulmonary Blood Flow.—When nuclide hasbeen injected into both the inferior and su-perior vena caval systems, it is possible tocalculate the amount of flow to the lung con-tributed by the superior and inferior venaecavae respectively, and by addition, the totalflow.

Calculation of Cardiac Output.—The meth-ods employed allow measurement of thevenous return to the heart. Addition of su-perior and inferior vena caval returns pro-vides a measurement of cardiac output. Twocirculations are not, however, taken into ac-count, namely, coronary and pulmonary ve-nous returns. Coronary circulation returns tothe right atrium through the coronary sinus;because of the proximity of the coronarysinus to the orifice of the tricuspid valve, itis most likely that coronary venous returnenters the right ventricle to be distributedin a manner similar to that of superior venacaval blood. The pulmonary venous bloodreturns to the left atrium and is then distrib-uted by the left ventricle to the whole fetalbody.

Since the magnitudes of coronary and pul-monary flows are known, they can be addedto superior and inferior vena caval flows toprovide a measure of total cardiac output.Since the blood flow to each organ is known,

QsvC — QheniHIVO) + Ql>rai.i(IVC) + Qforelimhs (IVf:) + 0.3 Ql,,,1Es (IYC)- (4)

When less than 5% of inferior vena caval the percent of the total cardiac output dis-nuclide is distributed to the lungs, it would tributed to various organs can be calculated.




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100

8 0 -

PERCENT60

r

40-

2 0 -

• oucn/s» LIVER

100 200 300 400 500Quy(ml/min)

FIGURE 4

The percent of umbilical flow passing through theductus venosus and that to the liver are shown inrelation to total fetal umbilical venous flow (Quv)-The larger the umbilical venous flow, the greater isthe percent which passes through the ductus venosus.

All the organs of the fetus and the placentalcotyledons are weighed immediately afterdissection, allowing an estimate of the flowper unit wet weight of each organ.

DISTRIBUTION OF BLOOD FLOW

Umbilical Venous Return.—In 8 goats and5 sheep, we injected spheres into an umbilicalvein. The proportion of nuclide in the liverrepresented that portion of umbilical venousflow passing through the liver; the remainder,distributed throughout the fetal body, indi-cated the amount flowing through the ductusvenosus.

70

60

50

40

30

20

10

PERCENT

_1_ _1_ J6.8 6.9 7.0 7.1 7.2 7.3 7.4- 7.5 7.6

FIGURE 5

The percent of superior vena caval blood distributedto the upper body, and thus reflecting the shuntthrough the foramen ovale, is shown in relation toumbilical arterial pH. The lower the pH, the greateris this shunt.

Thirty-four to 91% of the umbilical venousflow passed through the ductus venosus. Therewas no obvious relationship between theblood gas status of the fetus and the propor-tion of umbilical venous return passingthrough the ductus venosus. In 9 animalswith estimated gestational ages of 100 to 130days, umbilical flow was simultaneously meas-ured by the antiyprine method; a definiterelationship was noted between the total um-bilical blood flow and the proportion passingthrough the ductus venosus; the greater thetotal flow, the larger the percentage passingthrough the shunt (Fig. 4). The number ofanimals studied was not adequate to deter-mine whether this relationship was associatedwith the lower umbilical flows in fetuses oflower gestational age.

The radioactivity counts in both the leftand right lobes of the liver were high, butoften the counts per gram of liver tissue werehigher in the left than in the right lobe.

The nuclide which passed through the duc-tus venosus was then distributed to the upperand lower portions of the fetal body in thesame manner as was that injected into thehindlimb vein or inferior vena cava.

Superior Vena Caval Blood.—We estimatedthe amount of blood returning from the headand neck and forelimbs shunted through theforamen ovale by determining the percentageof nuclide that appeared in the upper partof the body after injection into a forelimb

TABLE 8

Cardiac Output Measurements in 12 Fetuses

Gestational age(days)

95100100105105107108108115117135138

CardiacTotal

330762500435379873910

1250554

1021975554

output (ml/min)Per kg fetal weight

498600600470335700

10001080340460390280




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PERCENT

70

60

50

40

3 0 -

20

10

7.3 7.4PH

7.5J

76 0 10 15 20 25PQ_ mm HQ

FIGURE 6

The percent of inferior vena caval blood distributed to the upper body is shown in relation toumbilical arterial pH and Pos. The lower the pH and Po2, the greater is the amount ofinferior vena caval blood passing to the upper body.

PERCENT

70 —

60 —

5 0 -

40

30

20

)0 - I

2.2 16 1.6 5.4 4.9 3.9 4.6 14.9 19.9

PLACENTA KIDNEYS SHEEN UVER CUT BRAIN LUNGS HEART UPPER LOWERCARCASS CARCASS

FIGURE 7

The range and average of the percent of the cardiac output distributed to each of the fetalorgans in 8 animals are shown.

vein. When fetal umbilical arterial pH wasnormal, shunting was minimal. If, however,umbilical arterial pH was reduced by sedatingthe ewe with pentobarbital or ventilating herwith a gas mixture low in oxygen, there wasa progressive increase in the amount of shunt-ing (Fig. 5).

Inferior Vena Caval Return.—Spheres inject-ed into the veins draining into the inferiorvena cava were distributed to the whole fetal

body. When the pH and Po2 of the lambwere low, a greater proportion of inferiorvena caval blood was distributed to the upperbody (Fig. 6).

DISTRIBUTION OF CARDIAC OUTPUT

The proportions of cardiac output distrib-uted to various fetal organs are shown inFigure 7 for 8 animals studied completely.There is considerable variation in the percent

Circulation Research, Vol. XXJ, August 1967



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BLOOD FLOWml/ g /min

GUT BRAIK LUNGS HEARTPLACENTA KIDNEYS SPLEEN UVER

FIGURE 8

The range and average of the blood flows to various fetal organs in 8 animals are shown. Onepoint is missing for placenta, brain and gut because weight was not recorded. Flows arepresented as milliliters per gram of wet weight of the organ per minute.

of cardiac output delivered to the majorvascular areas. Umbilical flow was a largepart of the total cardiac output, varying from23 to 60%. The number of animals thus farstudied is not large enough to assess whetherthese large differences could be related tofetal gestational age, to variation in the bloodgas status of the fetus, or to normal physio-logical variations in fetal circulation. Theaverage and range of percentages of cardiacoutput distributed to other organs were asfollows: heart, 4.6 (1.6-10.4); lungs, 3.9 (0.2-9.0); gastrointestinal tract, 5.4 (2.0-12.5);brain, 4.9 (2.0-8.8); upper carcass, 19.9 (10.0-32.5); kidneys, 2.2 (1.4-3.8); spleen, 1.6 (0.2-3.5); and liver from hepatic artery only, 1.6(0-5.0).

Organ Blood Flows.—The total flow to eachorgan was related to the wet weight of theorgan. It should again be stressed that thesedata are unselected for fetal gestational ageand physiological status, but the averageflows in the 8 animals are shown in Figure 8.

Cardiac Output Determinations.—The car-diac output was measured in 12 animals whosegestational ages were 95 to 138 days. Totalcardiac output and output per kilogram offetal weight are shown in Table 8. Althoughthe total number of animals is small, it ap-pears that cardiac output per kilogram offetal weight is considerably higher in fetusesin earlier gestational periods.


Effects of Injection of Microspheres on theFetus.—We have estimated that we injecteda total of about 10,000 to 25,000 microsphereswith each nuclide given. Heart rate and fetalarterial blood pressure were monitored con-tinuously in all the animals during injectionof the microspheres. Fetal blood gases andumbilical blood flows were also measuredbefore and after microsphere injection. Wefound no change in any of these variables,suggesting that the number of microspheresinjected has no immediate physiological effect.Additional evidence to support the fact thatthe injection of spheres did not interfere withperipheral circulation was provided by theobservation that the distribution pattern of asecond or third injection of spheres was nodifferent from the first.

We have thus far obtained only limitedinformation regarding the chronic effects ofthe spheres. In 4 animals, we continued tomake observations for 1 to 16 days afterinjection of the spheres; there were nochanges in fetal blood pressures, blood gasesor umbilical flows attributable to the spheres.

Discussion

GENERAL CONSIDERATIONS

Traditionally, the fetal circulation has beenstudied in sheep and goats with the fetusdelivered from the uterus, but with placentalcirculation continuing; expansion of the lungs



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180 RUDOLPH. HEYMANN

with air has been prevented by covering thenose and mouth with a saline-filled bag orrubber glove. Barcroft (7) used this prepa-ration with great effectiveness to make his-toric contributions to our understanding offetal physiology, and Barclay et al. (1) exten-sively described the course of the fetal circu-lation in the same preparation, using angio-graphic techniques. The development of moresophisticated electronic instrumentation al-lowed Dawes and his group (3) and Assaliand Morris (4) to make more accurate meas-urements of blood flow in major vascularchannels, using electromagnetic flowmeters.The effects of delivery of the fetus and thesurgical procedure used to apply flow trans-ducers on the physiology of the fetal circu-lation are not known.

Sheep and goats have been considered par-ticularly suitable for studies of fetal circulationbecause the placenta is slow to separate andfetal placental circulation may be continuedfor several hours after delivery. However, itis not possible to be sure that the umbilicalblood flow does not change. In fact, obser-vation of the cotyledons often shows thatthere is some separation, even though thereis adequate flow and gas exchange to maintainthe fetus. The only published report of meas-urement of fetal umbilical blood flow withthe lamb in utero is that of Meschia et al.(8). The levels observed were 233 ±19 SEMml/min per kg. We have measured umbilicalflow by a similar method and have confirmedthe levels they reported (unpublished data);there was also no significant difference inflows per kilogram of fetal weight at differentgestational ages. Dawes and Mott (3) re-ported similar levels for exteriorized fetusesaged 87 to 95 days, but in older lambs aged137 to 141 days the level was considerablylower. This difference might be explained bythe fact that the older the fetus, the greateris the tendency for placenta] separation. Thepotential changes in placental and peripheralvascular resistance associated with deliverycould produce vast changes in distribution ofblood flow, particularly since a major portion

of the cardiac output is directed to the pla-centa.

A major advantage of the use of electro-magnetic flowmeters for recording flows isthat continuous observations can be madeover a period of several hours. This is of realvalue if the responses of the circulation tofairly acute physiological and pharmacolog-ical influences are being examined. One dif-ficulty is that the exteriorized preparationprogressively deteriorates, probably due toplacental separation. Although the studies ofthe fetus in utero that we have describedare much more physiological, the observationsof distribution of cardiac output are madeduring a single short interval of time, andfluctuations in flow would not be appreciated.The use of microspheres labeled with differ-ent nuclides to some extent overcomes thisdifficulty, since repeated observations can bemade. We have thus far used up to fournuclides in a single animal, and now haveavailable seven nuclides which may be readilyseparated in a single sample.

The technique we have described posses-ses several additional advantages. It is pos-sible to make observations on the fetus forperiods of several days or even weeks andthus study changes in fetal vascular physiol-ogy with increasing gestational age. Whereasthe utilization of various types of flowmeterhas allowed the measurement of blood flowto one lung (10) or the indirect measurementof total pulmonary blood flow by subtractingductus arteriosus flow from pulmonary arterialflow (4), we are able to calculate total pul-monary flow with our method. Assali's methodusing an electromagnetic flowmeter may becriticized on the basis that measurement ofductal flow could be quite unreliable. Theductus constricts readily and this may makerecording of flow either impossible or quiteinaccurate.

Previously it has not been possible to meas-ure certain aspects of the fetal circulation.As a result, no quantitative studies of theproportions of umbilical venous return pass-ing through the ductus venosus have been




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made. Neither has it been possible to deter-mine the proportion of superior vena cavalblood that passes across the foramen ovale.Besides being able to study the general courseof the circulation and the general patternsof distribution, it is feasible to measure bloodflow to every organ of the fetal body merelyby injecting one or two small quantities oflabeled microspheres.

VALIDATION OF THE METHOD

The distribution of blood flow to the wholebody was reported by Sapirstein using po-tassium-42 (42K) (6). This method is basedon the fact that potassium rapidly leaves thecirculation after injection and enters the tis-sues. It is held in the tissues during severalcirculations and is then only slowly releasedto the venous blood. If the 42K is evenly mixedin the arterial blood, and concentrations ofthe nuclide are equal in blood perfusing alltissues, then uptake is related to flow rate.As mentioned previously, this method is notapplicable to fetal circulatory studies. Fur-thermore, there has not, to our knowledge,been any check of the validity of this tech-nique as representing actual organ bloodflows'.

Stone et al. (11) used nuclide-labeledspheres to determine what proportion of anamount injected into the aorta passed intothe coronary circulation. They injected spherestagged with iridium-192; these spheres havethe great disadvantage that the nuclide is notmixed with the material forming the sphere,but coats its surface and is fairly readily lost.The feasibility of the use of nuclide-labeledspheres to produce local irradiation of tumorswas examined by Ya et al.(12). They injectedspheres labelel with yttrium (noY) or chro-mium (51Cr) into various circulations. Theyshowed that only minimal quantities ofspheres passed through peripheral vessels.Ring et al. (13) showed that there are noshunts in the pulmonary circuit of dogs andthat less than 1% of spheres 16 fi in diameterpass through the pulmonary vasculature.

The microspheres we used have a specificgravity considerably greater than that ofblood, and we wondered whether this mightCirculation Research, Vol. XXI, August 1967

interfere with their mixing and result in se-lective streaming. Suspension in dextran so-lution (20%) does retard their settling andmakes it easier to inject a fairly well-mixedsuspension. We have presented evidence thatthe spheres are evenly distributed and thatthere is no obvious selective streaming. Thefact that there is good mixing in the centralvascular system of spheres injected into vari-ous venous sites in the fetus is shown by theiridentical distribution patterns to the lowerpart of the body. The total number of micro-spheres injected, according to our estimates,is about 10,000 to 25,000, depending on theactivity of the sample and the particularnuclide. Simultaneous or repeated injectionsof spheres produce similar distributions, in-dicating that the lodging of microspheres inthe peripheral vascular system does not inter-fere with the distribution of blood flow. Also,we have not observed any change in umbilicalblood flow or fetal vascular pressures whenthese were followed for several days afternuclide-labeled spheres were injected into thefetus.

The confirmation that the ratio of distri-bution of nuclide-labeled spheres to variousparts of the body is related to the flow ratioshas been presented. The excellent correlationbetween head and brain flow measured di-rectly by a flowmeter and that calculatedfrom the nuclide distribution and the umbili-cal blood flow, although only measured in oneanimal, was gratifying.

Since the superior and inferior vena cavalvenous returns are distributed differently inthe fetus, any singificant recirculation of themicrospheres may markedly alter the apparentdistribution pattern. Initially, we assumedthat because the spheres are 50 ± 5 fx indiameter, very few, if any, would pass througharteriolar vessels to the venous system to re-turn to the heart for redistribution. This hasbeen confirmed by the observation that onlya minimal number of the spheres pass throughthe lungs of the adult mouse and rat and theinfant guinea pig. We have also confirmedthat there is no significant passage of spheres



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through the circulations of the lung and pla-centa in the sheep and goat fetus.

OBSERVATIONS ON VENOUS RETURN IN THE FETUS

The injection of microspheres into periph-eral venous sites has allowed us to deter-mine the patterns of distribution of bloodreturning to the heart from different circula-tions. When an injection is made into a pe-ripheral umbilical vein, the proportion of totaladministered nuclide that is not caught upin the liver represents the proportion of um-bilical venous return that passes through theductus venosus. We have had the opportunityto study this relationship in sheep fetuses inwhich there was a good acid-base and bloodgas status, as well as in some in which therewas some interference with oxygenation andblood pH. There was no obvious correlationof ductus venosus flow with umbilical venousoxygen tension or pH. In the small numberof animals in which we simultaneously meas-ured umbilical venous flow by the antipyrinemethod and umbilical vein distribution bymeans of microspheres, the greater the um-bilical venous flow, the larger was the pro-portion passing through the ductus venosus.We suggest the hypothesis that the ductusvenosus is probably a passive tube, not re-sponsive to blood gas or acid-base changes,but readily distended by an increase of um-bilical flow with, presumably, an increase inumbilical venous pressure. Since the propor-tion of umbilical venous return passingthrough the ductus venosus is lower at lowflows, there is some protection against amarked fall in the flow of well-oxygenatedblood to the liver. It was not possible fromthe small number of observations we havemade thus far to assess whether the relativelylow ductus venosus flows were related togestational ages. It appeared that if our ex-perimental preparation was not optimal, theneven in a fetus near term, less ductus venousflow occurred when umbilical flow was low.It should be stressed that we have studiedonly distribution of umbilical venous returnand the measurements do not take into ac-count portal venous return, which also may

pass either through the liver parenchyma orthrough the ductus venosus.

The course of superior vena caval bloodwas studied by injecting microspheres intoa forelimb vein. Our observations showedthat when the fetus was in a normal acid-basestatus, negligible amounts of superior venacaval blood crossed the foramen ovale, butwhen fetal asphyxia developed, the amountcrossing was larger. The more severe thefetal acidemia, the greater this shunt.

Inferior vena caval blood distribution wasexamined either by injections into the hind-limb veins or by studying distribution of thatportion of nuclide injected into the umbilicalveins which by-passed the liver. We confirmedthe findings of many previous studies thatinferior vena caval blood passed both throughthe foramen ovale into the left atrium, andthrough the tricuspid valve. We were not ableto accurately measure the proportions of in-ferior vena caval flow distributed to the rightand left sides of the heart, nor were we ableto determine the proportion of left ventricu-lar output distributed to the upper trunk andto the descending aorta, via the arch. It waspossible, however, to calculate the percentof inferior vena caval flow which was dis-tributed to the organs supplied from theascending aorta. As with superior vena cavalreturn, a larger proportion of inferior venacaval blood was distributed to the head andthe heart under conditions of fetal asphyxia.These findings suggest that under conditionsof fetal distress, blood flow to the heart andto the brain are well maintained. Since theheart is absolutely crucial in providing anadequate circulation, it seems that coronaryblood flow is protected at times of stress.Before we conducted our experiments, we hadexpected, on the basis of Barcroft's observa-tion (7), that during conditions of fetal stressthere was perhaps an increase in the differencein the upper and the lower body arterial oxy-gen tensions normally present in the fetus. Thiswould aid in providing better oxygenationto the heart and brain. Whereas the increasein the proportion of inferior vena caval bloodof relatively high oxygen tension shunted




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across the foramen ovale would tend to pro-duce this effect, there is also an increasedshunt of superior vena caval blood of lowoxygen tension. The oxygen tension in theupper body may therefore not be preferen-tially increased, but flow would probably bewell maintained.

The mechanisms responsible for these hemo-dynamic alterations are not clear. In severalof the fetuses in which severe asphyxia waspresent, we had measured umbilical bloodflow, and it was considerably decreased fromnormal levels. Although we did not havemeasurements of umbilical venous pressurein most of these, umbilical arterial pressureswere usually normal or sometimes increased.We therefore assumed that placental vascu-lar resistance was increased, due either toour manipulation or to commencing placentalseparation, particularly in those animals nearterm. This increase in placental vascular re-sistance would decrease the proportion ofleft ventricular output distributed to the pla-centa, and thus increase the amount goingto the vessels arising from the ascendingaorta. The enormous shunts of superior venacaval blood across the foramen could be re-lated to several possible mechanisms. Theright ventricular output in the fetus is partlydirected to the lung, the remainder passingthrough the ductus arteriosus to the descend-ing aorta. Since the pulmonary blood vesselsof the fetal and neonatal lung are markedlyconstricted by acidemia and hypoxemia (14,15), fetal asphyxia will cause a marked in-crease in pulmonary vascular resistance. Thiscould cause an increase in right ventricularend-diastolic pressure and an increase in rightatrial pressure, which may account for theincreased shunt across the foramen ovale. Inconditions of fetal distress related to an inter-ference in placental circulation, a rise in pla-central vascular resistance would have a simi-lar effect on the right ventricle, since theplacenta is the major circulation arising fromthe descending aorta. Another factor thatcould possibly be responsible for this shunt-ing is a change in umbilical venous flow.Since part of the inferior vena caval blood


courses directly into the left atrium, a de-crease in this flow caused by a fall in umbilicalflow could alter atrial pressure relationshipsso as to produce a shunt of superior venacaval blood into the left atrium. Any or allof these mechanisms may be involved in theasphyxial response.

CARDIAC OUTPUT AND ITS DISTRIBUTION

The figures we have obtained for cardiacoutput in the fetus are somewhat higher thanthose reported by Assali and Morris (4) andBarcroft (7). Since all previous observationshave been made on exteriorized fetuses, usu-ally associated with extensive surgery involv-ing thoracotomy on the fetus, we feel thatour results are more representative of normalphysiological conditions.

Apart from the placenta, the organs witha high flow per gram of wet weight are theheart and, surprisingly, the gut. The smallintestine receives a fairly high proportion ofcardiac output, and since it apparently is notan organ of great functional significance inthe fetus, the reason for this high flow isobscure. The liver also receives a high flow,but this is derived from the umbilical andportal venous flows; hepatic arterial flow isextremely low in the fetus.

The methods described provide the oppor-tunity to measure umbilical blood flow, car-diac output and distribution of blood flow toevery organ of the fetal body in a relativelyundisturbed fetus in utero. Although most ofour studies have been performed in sheepof 90 to 140 days gestation, we have insertedumbilical vessel catheters in a fetus of 65days gestation, and it may be possible towork on younger fetuses. The techniques wehave used should be adaptable to repeatedobservations in the same fetus with increasinggestational age.

AcknowledgmentsThe authors wish to express their gratitude to Mr.

Sidney Steinberg for his invaluable advice and as-sistance with regard to instrumentation, and toMisses Alice Lytle, Christine Mueller, Dorothy Reeseand Mrs. Marie-Elena Alcala for their skillful tech-nical assistance. They are also indebted to Dr. JonGoerke and Miss Laura Schmidt who were respon-sible for establishing the computer analysis. Mr. Ivan



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Grotenhuis of the 3M Company gave invaluableadvice and help in selecting and obtaining the ap-propriate nuclide-labeled spheres.

References1. BARCLAY, A. E., BARCBOFT, J., BARRON, D. H.,

AND FRANKLIN, K. T.: Radiographic demon-stration of the circulation through the heart inthe adult and the foetus, and the identificationof the ductus arteriosus. Brit. J. Radiol. 12:505, 1939.

2. DAWES, G. S., MOTT, J. C., AND WIDDICOMBE,

J. G.: The foetal circulation in the lamb. J.Physiol. (London) 126: 563, 1954.

3. DAWES, G. S., AND MOTT, J. C : Changes in O2

distribution and consumption in foetal lambswith variations in umbilical blood flow. J.Physiol. (London) 170: 524, 1964.

4. ASSALI, N. S., AND MORRIS, J. A.: Circulatory

and metabolic adjustments of the fetus atbirth. Biol. Neonatorum 7: 141, 1964.

5. MESCHIA, G., WOLKOFF, A. S., AND BARRON,

D. H.: Oxygen, carbon dioxide and hydrogenion concentrations in the arterial and uterinevenous bloods of pregnant sheep. Quart. J.Exptl. Physiol. 44: 333, 1959.

6. SAPIRSTEIN, L. A.: Fractionation of the cardiacoutput of rats with isotopic potassium. Circu-lation Res. 4: 689, 1956.

7. BARCROFT, J.: Researches on Pre-natal Life. Ox-ford, Blackwell, 1946.

8. MESCHIA, G., COTTER, J. R., MAKOWSKI, E. L.,

AND BARRON, D. H.: Simultaneous measure-ments of uterine and umbilical blood flowsand oxygen uptakes. Quart. J. Exptl. Physiol.52: 1, 1967.

9. HUCKABEE, W. E.: Use of 4-aminoantipyrinefor determining volume of body water avail-able for solute dilution. J. Appl. Physiol. 9:157, 1956.

10. DAWES, G. S., MOTT, J. C , WIDDICOMBE, J. G.,

AND WYATT, D. G.: Changes in the lungs ofthe new-born lamb. J. Physiol. (London) 121:141, 1953.

11. STONE, H. L., BISHOP, V. S., AND GUYTON,

A. C : Cardiac function after embolization ofcoronaries with microspheres. Am. J. Physiol.204: 16, 1963.

12. YA, P. M., GUZMAN, T., LOKEN, M. K., AND

PERRY, J. F.: Isotope localization with taggedmicrospheres. Surgery 49: 644, 1961.

13. RING, G. C , BLUM, A. S., KURBATOV, T., MOSS,

W. G., AND SMITH, W.: Size of microspherespassing through pulmonary circuit in the dog.Am. J. Physiol. 200: 1191, 1961.

14. CASSIN, S., DAWES, G. S., MOTT, J. C., Ross,

B. B., AND STRANG, L. B.: Vascular resistanceof the foetal and newly ventilated lung of thelamb. J. Physiol. (London) 171: 61, 1964.

15. RUDOLPH, A. M., AND YUAN, S.: Response of the

pulmonary vasculature to hypoxia and H + ionconcentration changes. J. Clin. Invest. 45:399, 1966.




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ABRAHAM M. RUDOLPH and MICHAEL A. HEYMANNCardiac Output And Organ Blood Flow

The Circulation of the Fetus in Utero: Methods For Studying Distribution of Blood Flow,

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1967 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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