AMERICAN JOURNAL OF ~~YSIOLOOY Vol. 229, No. 5, November 1975. Prinktf
Hematocrit
in U.S.A.
of the fetal rabbit
SUSAN L. NEWCOMB AND GORDON Departments of Physiolou and Perinatal Biology, Loma Linda, California 92354
G. POWER Loma Linda University School of Medicine,
NEWCOMB, SUSAN L., AND GORDON G. POWER. Hematocrit of the rabbit placenta. Am. J. Physiol. 229(5) : 1393-1396. 1975.51Cr-labeled erythrocytes and [1251]RISA were used simultaneously to measure the fetal rabbit whole-body and placental hematocrits (Hct) and to find the ratio of placental transit times of erythrocytes and plasma. The labels were injected into the heart of 21 fetal rabbits of 26-28 days gestation, and after 60 s mixing time, the placenta and a fetal blood sample were assayed with a gamma well-type scintillation counter. Erythrocyte and plasma activity per milliliter were determined from a standard dilution of the isotopes. Large-vessel Hct was measured as the corrected packed erythrocyte percentage in capillary tubes. Large-vessel Hct was 37.3 (* 3.7 SD) %, whole-body Hct was 31.3 ( =f= 3.5) %, and the placental Hct was 25.3 (& 4.0) %. The placental/large-vessel Hct ratio was 0.676. The ratio may be estimated as 0.765 when corrected for the loss of albumin in time. The transit time of erythrocytes in the placenta was calculated to be 0.682 of that for plasma. The shorter erythrocyte transit time implies that there is less time for 0 2 and CO 2 exchange than previously thought.
fetal
erythrocyte
and plasma volumes;
transit
times; isotope dilution
THE HEMATOCRIT, the ratio of erythrocyte volume to erythrocyte volume plus plasma volume, has been extensively studied in most organs of the body. The hematocrits in small vessels of the brain ( 11) and in the splenic vascular pool (6, 8), for instance, are reported to be higher than the average value for the entire body. On the other hand, the hematocrit in most other organs (5, 6, 8, 14) including the lungs (16), kidney (15), and skeletal and cardiac muscle (6, 8), is less than the entire body. In these organs the erythrocytes circulate faster than the plasma (5, 14, IS), perhaps in part because of a tendency toward axial accumulation of erythrocytes in a laminar flow stream, and because erythrocytes may be directed preferentially into high-flow channels (7). The hematocrit of blood in the placenta has not previously been reported. The placental hematocrit is important with regard to the gas-exchanging properties of the organ for both oxygen and carbon dioxide. It is also important with regard to the viscosity of the blood on the fetal side of the placenta. In this study different labels for erythrocytes and plasma were used and data collected as to erythrocyte and plasma volume per gram of fetal and placental tissue.
METHODS Pregnant New Zealand 3,540 and 5,090 g, were of 26-28 days.
placenta
white rabbits, weighing between used with a fetal gestational age
An ear artery was cannulated under local anesthesia (Xylocaine), and 7-l 3 ml of blood were withdrawn into a syringe containing a few crystals of dry heparin. Five milliliters of this blood, together with 1 ml of ACD solution and approximately 10 &i of 51Cr (New England Nuclear Corp.) were incubated for 30 min at 25”C, whereupon 30 mg of ascorbic acid were added (to prevent further tagging). This mixture was then centrifuged and the plasma and buffy coat layers were removed. The volume was reconstituted with isotonic saline. This single washing decreased free Cr-51 to about 1 %. Next, approximately 3.6 &i of 1251-labeled serum albumin (E. R. Squibb & Sons) were added to the erythrocyte suspension. This final mixture was used for fetal injections. The maternal rabbit was anesthetized with pentobarbital drug was given as needed. C15-20 mg/kg), and additional The rabbit was placed supine and local anesthetic (XyloCaine) injected in the abdominal midline. The abdomen was opened and the distal end of a uterine horn exposed. A small uterine incision was made and the fetal head and upper torso delivered, taking great care not to stretch or in any way traumatize the umbilical cord. An incision was made through the fetal chest so that its heart was exposed, and 0.10 ml of the mixture of 51Cr- and 1251-tagged erythrocytes and plasma was injected into one of the ventricles. Next, a 60-s time interval was allowed for mixing; this was estimated to be sufficient for more than five circulatory transits of the labels through the fetal body (unpublished results). Then the umbilical cord was clamped and cut and the fetus and placenta immediately delivered. A sample of circulating fetal blood was obtained by severing the heart and letting the blood collect into a beaker containing a few crystals of dry heparin. The decidua basalis was separated from the rest of the placenta, loose membranes and large vessels were cut free, and the placenta was weighed to the nearest 0.001 g. In five instances, the entire procedure was repeated in a second fetus located in the other uterine horn, and again in a third fetus close to the vagina. In one experiment the rabbit was anesthetized with ether rather than barbiturate. In another experiment a sample of maternal blood was obtained at the end of the experiment to verify that labeled erythrocytes and plasma had not crossed the placenta in significant amounts. Samples of blood (1 .OO ml), a 1: 50 dilution of the injection isotope mixture, and entire placentas were placed in tubes. Each tube was counted for three periods of 4.0 min with a gamma well counter (Nuclear-Chicago Corp., model 1085). Hematocrits of fetal blood were determined in capillary tubes, in duplicate, averaged, and results multiplied by a factor of 0.96 to correct for trapped plasma.
1393
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1394
S. L. NEWCOMB
1. vital statistics, placental --
TABLE
Fetal
Wt
Placental
Fetal
W t
erythrocyte and plasma vohmes, and placental ml Erythrocytes g Placenta
Large-Vessel Hct+
~~
29.5 (7.0) Values TABLE Total
ml Plasma g Placenta
~~____I
3.77 (0.63) -_____ are means Z!Z SE.
37.3 (3.7)
Vol
0.027 (0.010) d for trapped
Plasma
Vol
Fetal
Erythrocyte
---
1.36 (0.30) --.-_____i~______----^. Values are means
-Placental Large-Vessel
Hct
Hct Hct
Placental Whole-Body
25.3 WO)
Hct Hct
-
0.805 (0.082) _~--___I~I__~
0.676 (0.081)
plasma.
Vol+
hematocrit Fetal
Plasma
___-~~ ___~_
______-__~Vol’
Fetal
Whole-Body
Hematocrit
Fetal Whole-Body Large-Vessel
Hct
0.839 (0.040) -~-~
-
---
Hct
_______
3.12 (0.71)
1.26 (0.29) ___-
k
G. G. POWER
-. _-___
0.080 (0.026) __~-~--
* Correcte
Total
Placental -
2. Fetal total erythrocyte and plasma volumes and whole-body _-Erythrocyte
hematocrit
AND
SE.
* Total
min us placental
_ ___-_.
injected/(counts/ml
-----~
--~
31.3 (3.5)
__~-..
volume.
Calculations. Background counts were subtracted, and counts expressed per milliliter of blood or per organ. Wr counts were then used without further corrections. The 1251 counts were used after correcting for the “‘0 overlap. The 51Cr counts per milliliter of erythrocytes were determined by dividing the counts in 1 ml of whole blood by the fractional hematocrit, corrected for trapped plasma. The 1251 counts per milliliter of plasma were determined by dividing the counts in 1 ml of whole blood by (1 corrected hematocrit). The erythrocyte content of the placenta was determined by dividing the counts in each gram of placental tissue by the counts per milliliter of erythrocytes. Similarly, the plasma content was found by dividing the 1251 counts in each gram of placenta by the counts per milliliter of plasma. The total erythrocyte dilution volume for the fetus and placenta was calculated from the 51Cr dilution: erythrocyte dilution vol = total 51Cr counts
2.79 (0.74) __-____-
erythrocyte)
From this total, the erythrocytes present in the placenta were subtracted to find the erythrocyte volume of the fetal body. Similarly, the total plasma volume was calculated from 1251 dilution. The fetal plasma volume was found by subtracting the placental plasma from the total volume. RESULTS
The mean and standard deviation of the results of 24 fetuses in 12 rabbits are summarized in Tables 1 and 2. The corrected hematocrit in large-vessel fetal blood averaged 37*3 (& 3.70 SD) %. Each gram of placental tissue contained 0.027 (A 0.010 SD) ml of fetal erythrocytes and 0.080 (& 0.026 SD) ml of plasma for an average blood volume of 0.107 ml/g. The hematocrit of blood in the placenta averaged 25.3 % (A 4.0 c/c SD) with a range from 17.5 to 34.3 %. The fetal placental hematocrit was thus 0.676 (Z/Z 0.081) of the large-vessel hematocrit. It was 0.805 (A 0.082) of the whole-body hematocrit. In the fetal body, the average erythrocyte volume was 1.26 (+ 0.29 SD) ml (or 0.0436 ml/g) and the plasma volume was 2.79 (& 0.74 SD) ml (or 0.0965 ml/g). The whole-body hematocrit was 3 1.3 % (& 3.5 SD). The ratio of whole-body hematocrit to large-vessel hematocrit was 0.839 (h 0.040 SD).
Insignificant amounts of injected 51Cr and lZ51 counts appeared in the maternal circulation at the end of the experirnent. The placental hematocrit of the fetus studied under ether anesthesia (26.8 %) was similar to the average for all other fetuses studied under barbiturate anesthesia (25.3 %) and its placental/whole-body hematocrit ratio was also similar. The placental hematocrit/circulating hematocrit ratio was not significantly different in the first, second, and third fetuses studied by using the paired-t tests. The placental hematocrit/large-vessel hematocrit ratio was independent of both placental and fetal weights. The fetal blood volume was 14.4 % of the fetal weight. This value may be compared to the adult rabbit whose blood volume ranges from 5.6 to 7.2 $6 (2) of body weigh t. Thus, a markedly larger portion of the fetus is blood. In terms of erythrocytes, 4.48 5% of the fetus and 2.84% of the adult are erythrocytes by weight. The fetal placental blood volume was 11.2 70 of placental weight. Critique of method. The results of this study must be viewed in light of its limitations. A major limitation is the effect of open-chest surgery on the fetus and its placental circulation. Figure 1 shows a rise in large-vessel hematocrit during the 2 min following injection of isotopes in an additional nine fetuses. The rising hematocrit may reflect a loss of plasma from the circulation or a release of erythrocytes into the circulation as a result of the operative procedure. The changes shown in Fig. 1 were recorded in capillary tubes
LARGE
1
TIME IN MINUTES I
2
FIG. 1. Changes in measured large-vessel hematocrit with time. Tendency for large-vessel hematocrit to rise following injection of labels at time0 is shown in an additional 9 fetuses (solid dots) and may be compared with the average data (& SD) included in this report measured after 1 min (open dot).
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PLACENTAL
1395
HEMA’IOCRIT
and are independent of any loss of isotope from the circulation. By extrapolating back in time, a corrected value for large-vessel hematocrit may be estimated prior to opening the chest. The graph suggests the observed value for largevessel hematocrit may be an overestimate by possibly 10 % but that a more precise correction probably is not justified because of considerable scatter in the data. Another limitation is that labeled albumin may have leaked into perivascular tissues during the 1-min interval following injection. To test this possibility we studied the additional nine fetuses with label mixing time intervals varying from 0.5 to 2.0 min. The results (Fig. 2) indicate that calculated placental hematocrits fall about 6 % during the 2 min following injection of labels. This is possibly because albumin may be diffusing into spaces outside of fetal vessels in the rabbit placenta. Leakage in this location would cause an underestimate in placental hematocrit and the placental/large-vessel and placental/whole-body hematocrit ratios and an overestimate in fetal whole-body hematocrit and the whole-body/large-vessel hematocrit ratios. Since sampling in the present study was completed after 1 min, these err&s may be estimated from 3 to 6 5;. The anesthetic administered to the mother may have adversely affected the fetal circulation. Since these changes, presumably, would have lowered fetal cardiac output and organ blood flow and hence minimized the degree of plasma skimming normally present, the placental hematocrits reported may be higher than would otherwise be the case. As a preventive measure we rejected any fetus that had a perceptible slow or weak heartbeat. In some rabbits in which three fetuses were studied sequentially, there was no significant difference in the hematocrits depending on the order of study, such as might have occurred if the condition of the fetuses were deteriorating rapidly. The use of maternal blood for the injections may also have altered the results if erythrocytes were removed in the fetal circulation. Since the entire study was completed within 1 min during which time the fetal heart continued to beat vigorously, and since there was no visible evidence of clotting or hemolysis, this effect, if any, was probably very small. It would have been interesting and beneficial to have conducted this experiment using a wider range of gestational age, rather than the 3-day span selected. There would have been problems, though, with very small fetuses and those closer to term whose tendency toward placental separation is greater. Using a wider range, however, a correlation between age and placental hematocrit and the degree of plasma skimming might have been possible. This is an area that deserves further study. Comparison with other organs and adult. The fetal body hematocrit/large-vessel hematocrit ratio of 0.839 (rt 0.040) is lower than the ratio in adults. In adults the ratio is typically 0.91, with a range in rats, dogs, and humans of 0.880-0.924 (1, 3, 4, 6, 9, 11, 13). This low fetal ratio might be because the fetal spleen is not as effective in sequestering large amounts of erythrocytes as it is in the adult. Differences in the liver circulation and in the bone marrow are other possibilities. All of these speculations, however, need to be verified experimentally. This low fetal ratio needs to be taken into account in calculations of
40
1I
.
3o
:
PLACENTAL HEMATOCRIT
%
.
1”
TlME
l(MlN”TES
;
FIG. 2. Changes in calculated placental hematocrits with time. Tendency for calculated hematocrit to fall is shown in results from 9 additional fetuses, studied 0.5-Z min following injection of labels. Average (+ SD) of data in this report measured after 1 min is indicated by open dot.
fetal blood volume based on the dilution of either erythrocyte or plasma markers. The placental hematocrit/large-vessel hematocrit ratio of 0.676 (zt 0.081) 1s * 1ess than the average for various other organs of the body. It is less than the spleen and lungs of dogs and rats, for example, and less than the rat uterus and human cerebral vascular pool (0.81-1.86) (6, 8, 11). There are some organs, however, with a ratio lower than the placenta, and these include the dog kidney and gastrointestinal tract and the rat ovary and bone (0.286-0.465) (6, 8). Thus, while the extent of plasma skimming in the placenta is noteworthy, it is not unique and not an exceptional adaptation of the organ. The important implications of a low placental hematocrit on gas exchange will now be discussed. Transit times. In any long-term steady state, the volume of erythrocytes and plasma flowing into the placenta will equal the volume leaving the organ. Since the hematocrit of blood in the larger vessels was greater than that in the smaller vessels within the placental tissue, it is clear that the erythrocytes must pass through the placenta more quickly than the plasma, i.e., erythrocytes must have a shorter mean transit time (tt) than the plasma. Since the placental erythrocyte (i.e., red blood cell (RBC)) volume is equal to the erythrocyte flow multiplied by the erythrocyte mean transit time, and similarly for plasma volume, then the placental hematocrit (h) may be expressed : h = while
QRBCttRBC/(Q
the large W
RBChBC
+
vessel hematocrit
(H)
=
QRBcAQRBC
Solving these equations transit times one finds: t~RBC/ttplasms
+
&asrna
is: Qplasma)
simultaneously
=
h(
1 -
H)/H(l
ttplasma)
for the ratio
-
of
h,
In the present experiments large-vessel hematocrit averaged 0.373 and placental hematocrit averaged 0.253. However, to correct for large-vessel and placental hematocrits, we used the slopes from Figs. 1 and 2 to estimate a large-vessel hematocrit of 0.34 and a placental hematocrit of 0.26 based on extrapolation of average data back to time 0. This method assumes that average data followed a time course parallel to that of the additional nine fetuses.
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1396
S. L. NEWCOMB
The transit time for erythrocytes was thus 0.682 of that of piasma. This result is important for the following reason. In placental studies it has generally been assumed that erythrocytes and plasma move together, in other words, that their mean transit times are identical. Work from this laboratory has suggested a value of about 1.7 s for them in the sheep placenta (12). If, however, the erythrocyte transit time were actually 1.26 s and the plasma transit time 1.84 s, values that would accord with the present extrapolated results, then there would be important implications in the placental transfer of a number of substances. In the case of 02, for instance, whereas maternal and fetal Paz are predicted to be closely approaching one another after 1.7 s, they are still somewhat separated and continuing to change after 1.26 s (10). In other words, the
AND
G. G. POWER
diffusion reserves of the placenta for 02 could be considered as generous as before, and a small fetal 02 difference would result from diffusional The same would possibly hold true for CO* substances whose exchange is linked to the special of the erythrocytes.
no longer maternallimitation. and other properties
We thank Tom R. Bennett for his assistance during these experiments. This work was supported in part by grants from the United Cerebral Palsy Research and Education Foundation and by National Institutes of Health Grants HD 04393 and HL 15655. G. G. Power is the recipient of Public Health Service Research Career Development Award l-K4 HD 20, 253. Received
for publication
7 October
1974.
REFERENCES 1. ALBERT, S., Y. GRAVEL, Y. TURNEL, AND C. A. ALBERT. Pitfalls in blood volume measurement. Anesthesia Analgesia, Current Res. 44 : 805-813, 1965. 2. ALTMAN, P. L. (editor). Blood and Other Body Fluids. Washington, DC. : Fed. Am. Sot. Exptl. Biol., 1961, p. 5. 3. BERSON, S. A., AND R. S. YALOW. The use of K42 or P32 labeled erythrocytes and I 131 tagged human serum albumin in simultaneous blood volume determination. J. Ciin. Invest. 31 : 572-580, 1952. 4. CHAPLIN, H., P. L. MOLLISON, AND H. VETTER. The body/venous hematocrit ratio: its constancy over a wide hematocrit range. J. Clin. Invest. 32 : 1309-l 3 16, 1956. 5. CRANE, M. G., J. E. HOLLOWAY, R. ADAMS, AND I. G. WOODWARD. The relative mean transit times of red cells and plasma in the portal circulation of the dog. J. Nuclear Med. 4: 296-305, 1963.
6. EVERETT, N. B., B. SIMMONS, AND E. P. LASHER. blood (Fes9) and plasma (Ii3i) volumes of rats liquid nitrogen freezing. Circulation Res. 4 : 419424, 7. FUNG, YUAN-CHENG. Stochastic flow in capillary Microvascular
Res.
5 : 3448,
Distribution of determined by 1956. blood vessels.
1973.
8. GIBSON, J. G. II, A. SELICMAN, W. C. PEACOCK, J. C. AUB, J. FINE, AND R. D. EVANS. The distribution of red cells and plasma in large and minute vessels of the normal dog, determined by radioactive isotopes of iron and iodine. J. Clin. Invest. 25: 848857, 1946.
9. GIBSON, J. G. II, W. C. PEACOCK, A. S. SELIGMAN, AND T. SACK Circulating red cell volume measured simultaneously by the radioactive iron and dye methods. J. Clin. Invest. 25: 838-847, 1952. 10. HILL, E. P., G. G. POWER, AND L. D. LONGO. A mathematical model of placental 02 transfer with consideration of hemoglobin reaction rates. Am. J. Physiol. 222 : 721-729, 1972. 11. LARSEN, 0. A., AND N. A. LASSEN. Cerebral hematocrit in normal man. J. Appl. Physiol. 19: 571-574, 1964. 12. LONGO, L. D., G. G. POWER, AND R. E. FORSTER II. Placental diffusing capacity for carbon monoxide at varying partial pressures of oxygen. J. A#. Physiol. 26 : 360-370, 1969. 13. NACHMAN, H. M., G. W. JAMES III, J. W. MOORE, AND E. I. EVANS. A comparative study of red cell volumes in human subjects with radioactive phosphorus tagged red cells and T-1824 dye. J. Clin. Invest. 29 : 258-264, 1950. 14. OLDENDORF, W. H., M. KITANA, S. SHIMIZU, AND S. OLDENDORF. Hematocrit of the human cranial blood pool. Circulation Res. 17 : 532-539, 1965. 15. PAPPENHEIMER, J. R., AND W. B. KINTER. Hematocrit ratio of blood within mammalian kidney and its significance for renal hemodynamics. Am. J. Physiol. 185 : 377-390, 1956. 16. RAPAPORT, E., H. KUIDA, F. W. HAYNES, AND L. DEXTER. Pulmonary red cell and plasma volumes and pulmonary hematocrit in the normal dog. Am. J. Physiol. 185 : 127-132, 1956.
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Hematocrit
in U.S.A.
of the fetal rabbit
SUSAN L. NEWCOMB AND GORDON Departments of Physiolou and Perinatal Biology, Loma Linda, California 92354
G. POWER Loma Linda University School of Medicine,
NEWCOMB, SUSAN L., AND GORDON G. POWER. Hematocrit of the rabbit placenta. Am. J. Physiol. 229(5) : 1393-1396. 1975.51Cr-labeled erythrocytes and [1251]RISA were used simultaneously to measure the fetal rabbit whole-body and placental hematocrits (Hct) and to find the ratio of placental transit times of erythrocytes and plasma. The labels were injected into the heart of 21 fetal rabbits of 26-28 days gestation, and after 60 s mixing time, the placenta and a fetal blood sample were assayed with a gamma well-type scintillation counter. Erythrocyte and plasma activity per milliliter were determined from a standard dilution of the isotopes. Large-vessel Hct was measured as the corrected packed erythrocyte percentage in capillary tubes. Large-vessel Hct was 37.3 (* 3.7 SD) %, whole-body Hct was 31.3 ( =f= 3.5) %, and the placental Hct was 25.3 (& 4.0) %. The placental/large-vessel Hct ratio was 0.676. The ratio may be estimated as 0.765 when corrected for the loss of albumin in time. The transit time of erythrocytes in the placenta was calculated to be 0.682 of that for plasma. The shorter erythrocyte transit time implies that there is less time for 0 2 and CO 2 exchange than previously thought.
fetal
erythrocyte
and plasma volumes;
transit
times; isotope dilution
THE HEMATOCRIT, the ratio of erythrocyte volume to erythrocyte volume plus plasma volume, has been extensively studied in most organs of the body. The hematocrits in small vessels of the brain ( 11) and in the splenic vascular pool (6, 8), for instance, are reported to be higher than the average value for the entire body. On the other hand, the hematocrit in most other organs (5, 6, 8, 14) including the lungs (16), kidney (15), and skeletal and cardiac muscle (6, 8), is less than the entire body. In these organs the erythrocytes circulate faster than the plasma (5, 14, IS), perhaps in part because of a tendency toward axial accumulation of erythrocytes in a laminar flow stream, and because erythrocytes may be directed preferentially into high-flow channels (7). The hematocrit of blood in the placenta has not previously been reported. The placental hematocrit is important with regard to the gas-exchanging properties of the organ for both oxygen and carbon dioxide. It is also important with regard to the viscosity of the blood on the fetal side of the placenta. In this study different labels for erythrocytes and plasma were used and data collected as to erythrocyte and plasma volume per gram of fetal and placental tissue.
METHODS Pregnant New Zealand 3,540 and 5,090 g, were of 26-28 days.
placenta
white rabbits, weighing between used with a fetal gestational age
An ear artery was cannulated under local anesthesia (Xylocaine), and 7-l 3 ml of blood were withdrawn into a syringe containing a few crystals of dry heparin. Five milliliters of this blood, together with 1 ml of ACD solution and approximately 10 &i of 51Cr (New England Nuclear Corp.) were incubated for 30 min at 25”C, whereupon 30 mg of ascorbic acid were added (to prevent further tagging). This mixture was then centrifuged and the plasma and buffy coat layers were removed. The volume was reconstituted with isotonic saline. This single washing decreased free Cr-51 to about 1 %. Next, approximately 3.6 &i of 1251-labeled serum albumin (E. R. Squibb & Sons) were added to the erythrocyte suspension. This final mixture was used for fetal injections. The maternal rabbit was anesthetized with pentobarbital drug was given as needed. C15-20 mg/kg), and additional The rabbit was placed supine and local anesthetic (XyloCaine) injected in the abdominal midline. The abdomen was opened and the distal end of a uterine horn exposed. A small uterine incision was made and the fetal head and upper torso delivered, taking great care not to stretch or in any way traumatize the umbilical cord. An incision was made through the fetal chest so that its heart was exposed, and 0.10 ml of the mixture of 51Cr- and 1251-tagged erythrocytes and plasma was injected into one of the ventricles. Next, a 60-s time interval was allowed for mixing; this was estimated to be sufficient for more than five circulatory transits of the labels through the fetal body (unpublished results). Then the umbilical cord was clamped and cut and the fetus and placenta immediately delivered. A sample of circulating fetal blood was obtained by severing the heart and letting the blood collect into a beaker containing a few crystals of dry heparin. The decidua basalis was separated from the rest of the placenta, loose membranes and large vessels were cut free, and the placenta was weighed to the nearest 0.001 g. In five instances, the entire procedure was repeated in a second fetus located in the other uterine horn, and again in a third fetus close to the vagina. In one experiment the rabbit was anesthetized with ether rather than barbiturate. In another experiment a sample of maternal blood was obtained at the end of the experiment to verify that labeled erythrocytes and plasma had not crossed the placenta in significant amounts. Samples of blood (1 .OO ml), a 1: 50 dilution of the injection isotope mixture, and entire placentas were placed in tubes. Each tube was counted for three periods of 4.0 min with a gamma well counter (Nuclear-Chicago Corp., model 1085). Hematocrits of fetal blood were determined in capillary tubes, in duplicate, averaged, and results multiplied by a factor of 0.96 to correct for trapped plasma.
1393
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1394
S. L. NEWCOMB
1. vital statistics, placental --
TABLE
Fetal
Wt
Placental
Fetal
W t
erythrocyte and plasma vohmes, and placental ml Erythrocytes g Placenta
Large-Vessel Hct+
~~
29.5 (7.0) Values TABLE Total
ml Plasma g Placenta
~~____I
3.77 (0.63) -_____ are means Z!Z SE.
37.3 (3.7)
Vol
0.027 (0.010) d for trapped
Plasma
Vol
Fetal
Erythrocyte
---
1.36 (0.30) --.-_____i~______----^. Values are means
-Placental Large-Vessel
Hct
Hct Hct
Placental Whole-Body
25.3 WO)
Hct Hct
-
0.805 (0.082) _~--___I~I__~
0.676 (0.081)
plasma.
Vol+
hematocrit Fetal
Plasma
___-~~ ___~_
______-__~Vol’
Fetal
Whole-Body
Hematocrit
Fetal Whole-Body Large-Vessel
Hct
0.839 (0.040) -~-~
-
---
Hct
_______
3.12 (0.71)
1.26 (0.29) ___-
k
G. G. POWER
-. _-___
0.080 (0.026) __~-~--
* Correcte
Total
Placental -
2. Fetal total erythrocyte and plasma volumes and whole-body _-Erythrocyte
hematocrit
AND
SE.
* Total
min us placental
_ ___-_.
injected/(counts/ml
-----~
--~
31.3 (3.5)
__~-..
volume.
Calculations. Background counts were subtracted, and counts expressed per milliliter of blood or per organ. Wr counts were then used without further corrections. The 1251 counts were used after correcting for the “‘0 overlap. The 51Cr counts per milliliter of erythrocytes were determined by dividing the counts in 1 ml of whole blood by the fractional hematocrit, corrected for trapped plasma. The 1251 counts per milliliter of plasma were determined by dividing the counts in 1 ml of whole blood by (1 corrected hematocrit). The erythrocyte content of the placenta was determined by dividing the counts in each gram of placental tissue by the counts per milliliter of erythrocytes. Similarly, the plasma content was found by dividing the 1251 counts in each gram of placenta by the counts per milliliter of plasma. The total erythrocyte dilution volume for the fetus and placenta was calculated from the 51Cr dilution: erythrocyte dilution vol = total 51Cr counts
2.79 (0.74) __-____-
erythrocyte)
From this total, the erythrocytes present in the placenta were subtracted to find the erythrocyte volume of the fetal body. Similarly, the total plasma volume was calculated from 1251 dilution. The fetal plasma volume was found by subtracting the placental plasma from the total volume. RESULTS
The mean and standard deviation of the results of 24 fetuses in 12 rabbits are summarized in Tables 1 and 2. The corrected hematocrit in large-vessel fetal blood averaged 37*3 (& 3.70 SD) %. Each gram of placental tissue contained 0.027 (A 0.010 SD) ml of fetal erythrocytes and 0.080 (& 0.026 SD) ml of plasma for an average blood volume of 0.107 ml/g. The hematocrit of blood in the placenta averaged 25.3 % (A 4.0 c/c SD) with a range from 17.5 to 34.3 %. The fetal placental hematocrit was thus 0.676 (Z/Z 0.081) of the large-vessel hematocrit. It was 0.805 (A 0.082) of the whole-body hematocrit. In the fetal body, the average erythrocyte volume was 1.26 (+ 0.29 SD) ml (or 0.0436 ml/g) and the plasma volume was 2.79 (& 0.74 SD) ml (or 0.0965 ml/g). The whole-body hematocrit was 3 1.3 % (& 3.5 SD). The ratio of whole-body hematocrit to large-vessel hematocrit was 0.839 (h 0.040 SD).
Insignificant amounts of injected 51Cr and lZ51 counts appeared in the maternal circulation at the end of the experirnent. The placental hematocrit of the fetus studied under ether anesthesia (26.8 %) was similar to the average for all other fetuses studied under barbiturate anesthesia (25.3 %) and its placental/whole-body hematocrit ratio was also similar. The placental hematocrit/circulating hematocrit ratio was not significantly different in the first, second, and third fetuses studied by using the paired-t tests. The placental hematocrit/large-vessel hematocrit ratio was independent of both placental and fetal weights. The fetal blood volume was 14.4 % of the fetal weight. This value may be compared to the adult rabbit whose blood volume ranges from 5.6 to 7.2 $6 (2) of body weigh t. Thus, a markedly larger portion of the fetus is blood. In terms of erythrocytes, 4.48 5% of the fetus and 2.84% of the adult are erythrocytes by weight. The fetal placental blood volume was 11.2 70 of placental weight. Critique of method. The results of this study must be viewed in light of its limitations. A major limitation is the effect of open-chest surgery on the fetus and its placental circulation. Figure 1 shows a rise in large-vessel hematocrit during the 2 min following injection of isotopes in an additional nine fetuses. The rising hematocrit may reflect a loss of plasma from the circulation or a release of erythrocytes into the circulation as a result of the operative procedure. The changes shown in Fig. 1 were recorded in capillary tubes
LARGE
1
TIME IN MINUTES I
2
FIG. 1. Changes in measured large-vessel hematocrit with time. Tendency for large-vessel hematocrit to rise following injection of labels at time0 is shown in an additional 9 fetuses (solid dots) and may be compared with the average data (& SD) included in this report measured after 1 min (open dot).
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PLACENTAL
1395
HEMA’IOCRIT
and are independent of any loss of isotope from the circulation. By extrapolating back in time, a corrected value for large-vessel hematocrit may be estimated prior to opening the chest. The graph suggests the observed value for largevessel hematocrit may be an overestimate by possibly 10 % but that a more precise correction probably is not justified because of considerable scatter in the data. Another limitation is that labeled albumin may have leaked into perivascular tissues during the 1-min interval following injection. To test this possibility we studied the additional nine fetuses with label mixing time intervals varying from 0.5 to 2.0 min. The results (Fig. 2) indicate that calculated placental hematocrits fall about 6 % during the 2 min following injection of labels. This is possibly because albumin may be diffusing into spaces outside of fetal vessels in the rabbit placenta. Leakage in this location would cause an underestimate in placental hematocrit and the placental/large-vessel and placental/whole-body hematocrit ratios and an overestimate in fetal whole-body hematocrit and the whole-body/large-vessel hematocrit ratios. Since sampling in the present study was completed after 1 min, these err&s may be estimated from 3 to 6 5;. The anesthetic administered to the mother may have adversely affected the fetal circulation. Since these changes, presumably, would have lowered fetal cardiac output and organ blood flow and hence minimized the degree of plasma skimming normally present, the placental hematocrits reported may be higher than would otherwise be the case. As a preventive measure we rejected any fetus that had a perceptible slow or weak heartbeat. In some rabbits in which three fetuses were studied sequentially, there was no significant difference in the hematocrits depending on the order of study, such as might have occurred if the condition of the fetuses were deteriorating rapidly. The use of maternal blood for the injections may also have altered the results if erythrocytes were removed in the fetal circulation. Since the entire study was completed within 1 min during which time the fetal heart continued to beat vigorously, and since there was no visible evidence of clotting or hemolysis, this effect, if any, was probably very small. It would have been interesting and beneficial to have conducted this experiment using a wider range of gestational age, rather than the 3-day span selected. There would have been problems, though, with very small fetuses and those closer to term whose tendency toward placental separation is greater. Using a wider range, however, a correlation between age and placental hematocrit and the degree of plasma skimming might have been possible. This is an area that deserves further study. Comparison with other organs and adult. The fetal body hematocrit/large-vessel hematocrit ratio of 0.839 (rt 0.040) is lower than the ratio in adults. In adults the ratio is typically 0.91, with a range in rats, dogs, and humans of 0.880-0.924 (1, 3, 4, 6, 9, 11, 13). This low fetal ratio might be because the fetal spleen is not as effective in sequestering large amounts of erythrocytes as it is in the adult. Differences in the liver circulation and in the bone marrow are other possibilities. All of these speculations, however, need to be verified experimentally. This low fetal ratio needs to be taken into account in calculations of
40
1I
.
3o
:
PLACENTAL HEMATOCRIT
%
.
1”
TlME
l(MlN”TES
;
FIG. 2. Changes in calculated placental hematocrits with time. Tendency for calculated hematocrit to fall is shown in results from 9 additional fetuses, studied 0.5-Z min following injection of labels. Average (+ SD) of data in this report measured after 1 min is indicated by open dot.
fetal blood volume based on the dilution of either erythrocyte or plasma markers. The placental hematocrit/large-vessel hematocrit ratio of 0.676 (zt 0.081) 1s * 1ess than the average for various other organs of the body. It is less than the spleen and lungs of dogs and rats, for example, and less than the rat uterus and human cerebral vascular pool (0.81-1.86) (6, 8, 11). There are some organs, however, with a ratio lower than the placenta, and these include the dog kidney and gastrointestinal tract and the rat ovary and bone (0.286-0.465) (6, 8). Thus, while the extent of plasma skimming in the placenta is noteworthy, it is not unique and not an exceptional adaptation of the organ. The important implications of a low placental hematocrit on gas exchange will now be discussed. Transit times. In any long-term steady state, the volume of erythrocytes and plasma flowing into the placenta will equal the volume leaving the organ. Since the hematocrit of blood in the larger vessels was greater than that in the smaller vessels within the placental tissue, it is clear that the erythrocytes must pass through the placenta more quickly than the plasma, i.e., erythrocytes must have a shorter mean transit time (tt) than the plasma. Since the placental erythrocyte (i.e., red blood cell (RBC)) volume is equal to the erythrocyte flow multiplied by the erythrocyte mean transit time, and similarly for plasma volume, then the placental hematocrit (h) may be expressed : h = while
QRBCttRBC/(Q
the large W
RBChBC
+
vessel hematocrit
(H)
=
QRBcAQRBC
Solving these equations transit times one finds: t~RBC/ttplasms
+
&asrna
is: Qplasma)
simultaneously
=
h(
1 -
H)/H(l
ttplasma)
for the ratio
-
of
h,
In the present experiments large-vessel hematocrit averaged 0.373 and placental hematocrit averaged 0.253. However, to correct for large-vessel and placental hematocrits, we used the slopes from Figs. 1 and 2 to estimate a large-vessel hematocrit of 0.34 and a placental hematocrit of 0.26 based on extrapolation of average data back to time 0. This method assumes that average data followed a time course parallel to that of the additional nine fetuses.
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1396
S. L. NEWCOMB
The transit time for erythrocytes was thus 0.682 of that of piasma. This result is important for the following reason. In placental studies it has generally been assumed that erythrocytes and plasma move together, in other words, that their mean transit times are identical. Work from this laboratory has suggested a value of about 1.7 s for them in the sheep placenta (12). If, however, the erythrocyte transit time were actually 1.26 s and the plasma transit time 1.84 s, values that would accord with the present extrapolated results, then there would be important implications in the placental transfer of a number of substances. In the case of 02, for instance, whereas maternal and fetal Paz are predicted to be closely approaching one another after 1.7 s, they are still somewhat separated and continuing to change after 1.26 s (10). In other words, the
AND
G. G. POWER
diffusion reserves of the placenta for 02 could be considered as generous as before, and a small fetal 02 difference would result from diffusional The same would possibly hold true for CO* substances whose exchange is linked to the special of the erythrocytes.
no longer maternallimitation. and other properties
We thank Tom R. Bennett for his assistance during these experiments. This work was supported in part by grants from the United Cerebral Palsy Research and Education Foundation and by National Institutes of Health Grants HD 04393 and HL 15655. G. G. Power is the recipient of Public Health Service Research Career Development Award l-K4 HD 20, 253. Received
for publication
7 October
1974.
REFERENCES 1. ALBERT, S., Y. GRAVEL, Y. TURNEL, AND C. A. ALBERT. Pitfalls in blood volume measurement. Anesthesia Analgesia, Current Res. 44 : 805-813, 1965. 2. ALTMAN, P. L. (editor). Blood and Other Body Fluids. Washington, DC. : Fed. Am. Sot. Exptl. Biol., 1961, p. 5. 3. BERSON, S. A., AND R. S. YALOW. The use of K42 or P32 labeled erythrocytes and I 131 tagged human serum albumin in simultaneous blood volume determination. J. Ciin. Invest. 31 : 572-580, 1952. 4. CHAPLIN, H., P. L. MOLLISON, AND H. VETTER. The body/venous hematocrit ratio: its constancy over a wide hematocrit range. J. Clin. Invest. 32 : 1309-l 3 16, 1956. 5. CRANE, M. G., J. E. HOLLOWAY, R. ADAMS, AND I. G. WOODWARD. The relative mean transit times of red cells and plasma in the portal circulation of the dog. J. Nuclear Med. 4: 296-305, 1963.
6. EVERETT, N. B., B. SIMMONS, AND E. P. LASHER. blood (Fes9) and plasma (Ii3i) volumes of rats liquid nitrogen freezing. Circulation Res. 4 : 419424, 7. FUNG, YUAN-CHENG. Stochastic flow in capillary Microvascular
Res.
5 : 3448,
Distribution of determined by 1956. blood vessels.
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8. GIBSON, J. G. II, A. SELICMAN, W. C. PEACOCK, J. C. AUB, J. FINE, AND R. D. EVANS. The distribution of red cells and plasma in large and minute vessels of the normal dog, determined by radioactive isotopes of iron and iodine. J. Clin. Invest. 25: 848857, 1946.
9. GIBSON, J. G. II, W. C. PEACOCK, A. S. SELIGMAN, AND T. SACK Circulating red cell volume measured simultaneously by the radioactive iron and dye methods. J. Clin. Invest. 25: 838-847, 1952. 10. HILL, E. P., G. G. POWER, AND L. D. LONGO. A mathematical model of placental 02 transfer with consideration of hemoglobin reaction rates. Am. J. Physiol. 222 : 721-729, 1972. 11. LARSEN, 0. A., AND N. A. LASSEN. Cerebral hematocrit in normal man. J. Appl. Physiol. 19: 571-574, 1964. 12. LONGO, L. D., G. G. POWER, AND R. E. FORSTER II. Placental diffusing capacity for carbon monoxide at varying partial pressures of oxygen. J. A#. Physiol. 26 : 360-370, 1969. 13. NACHMAN, H. M., G. W. JAMES III, J. W. MOORE, AND E. I. EVANS. A comparative study of red cell volumes in human subjects with radioactive phosphorus tagged red cells and T-1824 dye. J. Clin. Invest. 29 : 258-264, 1950. 14. OLDENDORF, W. H., M. KITANA, S. SHIMIZU, AND S. OLDENDORF. Hematocrit of the human cranial blood pool. Circulation Res. 17 : 532-539, 1965. 15. PAPPENHEIMER, J. R., AND W. B. KINTER. Hematocrit ratio of blood within mammalian kidney and its significance for renal hemodynamics. Am. J. Physiol. 185 : 377-390, 1956. 16. RAPAPORT, E., H. KUIDA, F. W. HAYNES, AND L. DEXTER. Pulmonary red cell and plasma volumes and pulmonary hematocrit in the normal dog. Am. J. Physiol. 185 : 127-132, 1956.
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