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Human placental oxygen transfer and consumption. Dissociation by cooling or use of respiratory enzyme inhibitors
Europ. J. Obstet. Gynec. 0 Elsevier/North-Holland
reprod.
Biol.,
Biomedical
1980, 10/2, Press
83-98
83
HUMAN PLACENTAL OXYGEN TRANSFER AND CONSUMPTION. DISSOCIATION BY COOLING OR USE OF RESPIRATORY ENZYME INHIBITORS
A. GUIET-BARA Department
of Reproductive
Accepted
for publication
GUIET-BARA, tion by cooling Biol.,
*
10/Z,
Biology,
University
of Paris VI, Paris, France
29 July 1979
A. (1979): Human placental or use of respiratory enzyme
oxygen transfer and consumption. Dissociainhibitors. Europ. J. Obstet. Gynec. reprod.
83-98.
The regulation of oxygen placental transfer is studied during the in vitro perfusion of normal postpartum human placental lobules. Oxygen consumption of the placental tissue is measured during the perfusion of the fetal placental circulation alone (single perfusions). To evaluate the transplacental diffusion of oxygen, fetal placental and uteroplacental circulations are simultaneously perfused (dual perfusions). The results obtained at various temperatures (37 and 4’C) and after use of respiratory enzyme inhibitors (fluorodinitrobenzene and oligomycin) prove that oxygen consumption is related to trophoblast physiological activity, while oxygen transfer relies on a simple diffusion mechanism. placental
perfusion;
trophoblast
metabolic
activity;
oxygen
transfer
mechanism
INTRODUCTION
This research project was carried out in order to dissociate placental oxygen consumption from fetal maternal oxygen diffusion through the placenta (Guiet-Bara, 1978). Oxygen consumption seems to correspond to the trophoblast physiological activity, while placental transfer of oxygen relies on a physiological mechanism, which has to be defined. Physical methods have been employed for this study: change in temperature (cooling) during the placental perfusion, as well as biochemical techniques: use of respiratory enzyme inhibitors (such as fluorodinitrobenzene and oligomycin).
* Reprint
Reproduction France.
requests
to: Dr. A. Guiet-Bara, Universite Pierre et Marie Curie, Biologie de la (Pr. M. Panigel), Bt A, 7, Quai Saint Bernard, 75230 Paris Cedex 05,
84 MATERIAL
27 placed room lobule
AND METHODS
normal term human placentas, taken after spontaneous delivery, were in heparinized Earle’s medium and then put in a constant temperature (either 37 or 4°C) adjacent to the labor room. An intact placental was immediately perfused for about 1.5-2 h.
Placental perfusion technique (Figs. 1 and 2) (1) Fetal placental blood vessel catheterization The allantochorial arterial branch, which supplies fetal blood to the placental lobule, is cannulated on the chorial plate. The plastic tubing is advanced until it reaches the beginning of the cotyledonary artery, and it is then kept in place by two ligatures. All the neighboring arterial branches are
+i
Fig. 1. Perfusion system. A: gas mixture with 5% 02, 5% CO2 and 90% N,; B: gas mixture with 95% O2 and 5% C02. C,, Cz;’ C3, Cd, two-way stop-cocks; Dt;gas dispersion tube; Foe. per, res., fetal perfusion reservoirs; Fm, flowmeter; Man, manometer; Mat. per. res., maternal perfusion reservoir.
85
Fig, 2. The placental lobule in the perfusion chamber. CP, center piece; IAA, perfusion inflow to allantochorial artery; ISA, perfusion inflow to the uteroplacental spiral artery; L, perfused lobule; LC, lower chamber; OAV, perfusion outflow from allantochorial vein; OUV, perfusion outflow from uteroplacental veins; UC, upper chamber.
then obliterated by a ligature. A physiological solution is then perfused to identify the vessel through which the venous outflow of perfusate takes place. This chorioallantoic vein is cannulated in the same way as described for the artery. (2) The perfusion chamber and the u teroplacen tal artery cannulation The perfusion medium washes residual fetal blood out of the cotyledonary vascular tree; the cotyledon whitens, showing its borders clearly. This area is then excised out of the placental disk. The lower part of the perfusion chamber is filled with the physiological medium used to perfuse the fetal cotyledonary circulation. The isolated lobule is placed upside down on the surface of the physiological liquid, with its basal plate up. The placental tissue and the fetal membranes covering the perfused lobule are held in place by the middle piece of the artificial uterus, which is then screwed onto the lower part of the chamber. On the basal plate surface, the end of a uteroplacental spiral artery (or only its opening) is looked for to insert a fine glass cannula 200 pm in diameter. This glass cannula is connected to the maternal perfusion circuit through a silicone tubing, which is attached to the Plexiglas tube. This tube opens into the top of the upper chamber, which is then screwed onto the middle piece of the artificial uterus and filled with physiological medium (the same one used to perfuse the uteroplacental circulation). Perfusion of the intervillous space is initiated, and the perfusate
86
flows back into the upper chamber on the basal plate.
through
the numerous
venous
openings
Perfusion outfit (Fig. 1). The fetal perfusion circuit will carry the perfusion liquid from a fetal perfusion reservoir into the cannulated allantochorial artery on one ‘side and, on the other side, the perfusate flows back through the allantochorial vein to a collecting flask. This type of perfusion is said to be an open circuit: the perfusion medium is constantly renewed and circulates only once. The maternal perfusion circuit is connected in the same way as the fetal one. The perfusion medium is propelled through the circuit by a Lillehei digital pump, T6S model (Sigmamotor). The hydrostatic pressure at the entrance of each circuit is measured using Ludwig mercury manometers; it varies from 40 to 90 mm Hg. The flows are controlled at the entrance and exit of each circuit, to detect a possible loss of perfusion medium due to edema or tearing of the fetal vasculature. Brooks flowmeters are used for this purpose. The perfusion is carried out at a flow rate of 6-12 ml/min for the fetal circulation and at a flow rate of 9-13 ml/min for the maternal circulation. Perfusion medium. The perfusion media used are either Earle’s physiological salt solution, or the same Earle’s solution, to which a respiration inhibitor (FDNB or oligomycin) is added. The l-fluoro-2,4_dinitrobenzene (Merck) is dissolved in Earle’s at 0.38 or 8 mM/l dilution. Oligomycin (Sigma) is a mixture of 15% oligomycin A and 85% oligomycin B; it is dissolved in Earle’s at a concentration ranging from 1.25 to 10 mg oligomycin/l perfusion medium, The perfusion media are equilibrated with one of the following gaseous mixtures: A (5% O2 + 5% CO* + 90% N,) or B (95% O2 + 5% CO*). Mixture A, poor in oxygen, simulates the oxygen concentration in the fetal blood coming from the umbilical artery; mixture B, rich in oxygen, simulates the oxygen concentration in the maternal blood flowing in the placenta from the uterine artery. To evaluate oxygen consumption by the placental Experimental protocol. tissue, the isolated lobule fetal placental circulation alone is perfused (single perfusion) with Earle’s solution equilibrated either with gaseous mixture A or B. To measure the diffusion of oxygen from uteroplacental to fetal placental circulation, the dual perfusion is used. The fetal cotyledonary circulation is perfused with Earle’s equilibrated with A, while the uteroplacental circulation is perfused with Earle’s equilibrated with B to establish a transplacental concentration gradient for oxygen. The perfusion medium is sampled every 15 min at the entrance and exit of each perfusion circuit; pH, PO* and PCOZ are determined using a Beckman physiological gas analyzer (model 160). To measure the amount of Statement and interpretation of the results. oxygen dissolved in the physiological salt solution (Earle’s), we aPPb Henry’s law.
a7 Mean values (+ to.os SD) are given. Student’s different mean values.
t-test is used to compare
the
RESULTS
Oxygen consumption
inhibition
The results are presented
by cooling placental
tissue
in Table I.
(1) POz changes in the fetal placental
circulation
(a) Single perfusion. The fetal side of the placenta is perfused at 4°C using Earle’s solution equilibrated with A (5% 0,). The venous cotyledonary PO2 (PvO,) is the same as the cotyledonary arterial PO2 (PaO,). No tissue respiration takes place in the placenta. After beginning the perfusion of the fetal placental (b) Dual perfusion. circulation using Earle’s + 5% 02, the uteroplacental circulation is simultaneously perfused using Earle’s + 95% Oz. The venous cotyledonary PO* becomes higher than the arterial POz; the percentage of venous PO, increase compared to the arterial PO, averages 38%. Lowering the temperature to 4°C does not hinder an important diffusion of oxygen from the maternal to the fetal side of the placenta. (2) Amount of oxygen diffusing from the uteroplacental to the fetal placental circulation during the dual perfusion This amount is calculated as the product of the arteriovenous concentration gradient by the cotyledonary perfusion flow. If this amount is related to one kilo placental weight, it reaches an average of 0.97 f. 0.06 ml/min/kg. This value corresponds to the one measured during dual perfusions at physiological temperature (37°C) (Guiet-Bara, 1978). Placental (FDNB)
oxygen
(I) Single perfusion
consumption
inhibition
by
l-fluoro-2,4-dinitrobenzene
(Table II)
(a) PO2 changes in the cotyledonary circulation. The fetal placental circulation is perfused using Earle’s solution equilibrated either with mixture A (5% 0,) or with mixture B (95% 0,). A POz arteriovenous gradient appears due to placental tissue respiration: (APIOz). The placenta is then perfused with Earle’s + FDNB + the same gaseous mixtures as stated above. The arteriovenous cotyledonary PO* gradient is then measured in each case: APzOz < AP,Oz. If a t-test is done for paired values, the average of (AP102AP202) is very significantly different from 0 (P < 0.01). This fact points to FDNB being an inhibitor of placental respiration.
arterial PO2 ; PvOz = cotyledonary
venous POZ.
142 138 126 124 140 131 133.5 f 3.3
0 0 0.04 0.05 0 0.05 0.02 * 0.01
148 130 120 115 145 125 130.5 i: 5.7
148 130 122 117 145 128 131.7 + 5.3
11.8 7.8 6.9 8.4 9.3 10.2 9.1 -c 0.8
Pa02 = cotyledonary
1 2 3 4 5 6 Mean * to.05 SD
Pa02 (mm Hg)
02 consumption (ml/min/kg)
Pa02 (mm Hg) Pvoz (mm Hg)
Dual perfusion
Single perfusion
190 195 180 171 192 177 184.2 + 4.1
33.8 41.3 42.5 37.9 37.2 35.3 38.0 f 1.3
Pvq increase compared to Pa& due to 02 transfer (%I
LOBULES AT 4OC
PVOZ (mm Hg)
DURING THE PERFUSION OF PLACENTAL
Cotyledonary flow (ml/min)
AND CONSUMPTION OF OXYGEN
Placenta no.
TRANSFER
TABLE I
-
0.76 1.17 1.12 0.98 0.92 0.87 0.97 + 0.06
01 transfer (ml/min/kg)
-
II
119
5
Pa02
= cotyledonary
arterial PO2
; PvO2 = cotyledonary
venous
* 4.0
33 32 28 31.0
253 306 284 281 f 38
377 450 395 407 + 55
95 95 95
12 13 14 Mean f to.05 SD P02.
8 8 8
0.38 0.38 0.38 0.38 * 1.5
8
62 27 24 28 25 26.0
(mM/II
(%)
FDNB
Concentration of FDNB in the solution
After
UTILIZATION
Oxygen utilization coefficient
288 308 274 323 298 + 17
Hg)
395 405 380 430 403 + 17
45
PVlOZ
(mm
Hg)
Pal02
(mm
FDNB
02 % in the gas mixture
Before
OXYGEN
95 95 95 95
no.
PO* AND
8 9 10 11 Mean * lo.o5 SD
7
Placenta
ARTERIAL AND VENOUS COTYLEDONARY BEFORE AND AFTER THE USE OF FDNB
TABLE
Hg)
370 455 403 409 f 62
410 395 390 426 405 * 13
116
(mm
Pa202
COEFFICIENT
Hg)
333 423 375 377 + 65
336 336 335 354 340 + 7
116
(mm
pv202
DURING
10 7 7 8.0 t 2.5
18 15 14 17 16.0 f 1.5
0
(%I
Oxygen utilization coefficient
A SIMPLE
70 78 75 75 + 6
34 38 50 28 38 + 7
100
(%I
Decrease of oxygen utilization coefficient after FDNB
PERFUSION
90
(b) Oxygen oxygen content
utilization
coefficient
in the cotyledonary oxygen
content
artery - oxygen
content
in the cotyledonary
in the cotyledonary
vein x 100.
artery
For one placenta, when FDNB 8 mM/l Earle is perfused equilibrated with mixture A (5% 0,), the oxygen utilization coefficient decreases to zero. This coefficient reached 62% before the inhibitor was used. For 7 other placentas, the perfusion solution is equilibrated with mixture B (95% O,), and two different concentrations of FDNB are used (0.38 mM/l or 8 mM/l). In 4 cases, FDNB (0.38 mM/l) decreases the oxygen utilization coefficient from 26 to 16% average, this difference being significant (P< 0.05). In 3 cases, FDNB (8 mM/l) decreases the oxygen utilization coefficient from 31 to 8% average, this difference being very significant (P < 0.02). On the other hand, the percentage of decrease of the oxygen utilization coefficient averages 38% when FDNB of 0.38 mM/l is used, and 75% using FDNB of 8 mM/l. It is thus twice as important with the more concentrated solution. This difference is significant (P < 0.05). (c) Oxygen consumption of placental tissue. For each placenta, a decrease in oxygen consumption is noticed during the perfusion when pure Earle’s solution is replaced by FDNB in Earle. The percentage decrease in the amount of oxygen consumed after FDNB is administered reaches 63% with the concentration 0.38 mM/l and 68% with 8 mM/l, when the perfusion solution is equilibrated with mixture B (95% 0,). (2) Effect of FDNB during the dual perfusion
of the placental
lobule
(a) PO2 changes in the cotyledonary circulation (Table III). The fetal placental circulation is first perfused using Earle equilibrated with the gas mixture A (5% 0,). We can thus measure the difference of the cotyledonary arteriovenous PO* due to the placental tissue respiration (APOOZ). Uteroplacental circulation perfusion is then initiated using Earle equilibrated with mixture B (95% 0,). The cotyledonary arteriovenous PO* is then measured (AP,O*). Taking into account the tissue respiration, it is possible to infer the cotyledonary venous POZ increase as compared to the cotyledonary arterial PO* due to oxygen diffusion (AP’,O,). Lastly, the cotyledonary circulation is perfused with Earle + FDNB (0.38 mM/l or 8 mM/l) equilibrated with gas mixture A (5% 0,). Two kinds of results are thus obtained: -When placental respiration is weak compared to maternal fetal oxygen transfer (Table III, placentas 15-19), the increase of the cotyledonary venous POZ compared to the cotyledonary arterial POZ due to fetal maternal oxygen diffusion is less important after the use of FDNB. This increase averages 108 + 23 mm Hg before the use of FDNB and 46.4 f 7.0 mm Hg after the use of the inhibitor, this difference being significant (P = 0.01). - When the placental tissue respiration is important compared to maternal
III
no.
SD 116 105 110.5
t 3.7
74 44 59
? 4.2
t8
+ 5.1
44 51 47.5
127 130 113 120 124 122.8
13 32 24 19 28 23.2
119 96 95 109 81 100
132 128 119 128 109 123.2
118 95 106.5
Pa 10, (mm Hg)
AP”O, (mm Hg)
PveO* (mm Hg)
PaeOz (mm Hg)
45.5
3 24 13.5
+ 23
+ 22
+ 26 71 20
60 67 126 108
-36 -48 -98 -85
45 85 65
130 155
-117 --125
Hg)
AP’,O, (mm Hg)
244 255 149 168 222 208
AP,O2
FDNB
(mm
before
115 102 113.5
+2
125 124 121 122 116 122
Paz02 ( mm Hg)
74 91 82.5
161 153 136 132 142 145 +7
I%202 (mm Hg)
after
41 11 26
--36 -29 -15 -10 --26 -23.2 * 5.8
AP*O* (mm Hg)
FDNB
PO,! DURING USE OF FDNB
Dual perfusion
ARTERIAL COTYLEDONARY BEFORE AND AFTER THE
( mmHg)
pv,o2
Dual perfusion
Single perfusion
IN THE VENOUS COTYLEDONARY PO2 COMPARED TO AND DURING DUAL PERFUSION OF PLACENTAL LOBULES
_
33 33 33
49 61 39 29 54 46.4 + 7.0
AP’, 0, (mmHg)
SIMPLE
Pa02 = cotyledonary arterial PO,; PvOz = cotyledonary venous PO,; APeOz = PaeOz-PveOz = arteriovenous cotyledonary PO2 difference during a simple perfusion; API02 = PaiOT-Pv102 = arteriovenous cotyledonary PO2 difference during a dual perfusion before the use of FDNB; AP’iO2 = (PaeOz-Pve02) - (PaiOz-Pv102) = PvO:! increase compared to Pa02 due to 02 transfer before the use of FDNB, taking into account the tissue respiration; API02 = Pa202-Pv202 = arteriovenous cotyledonary PO2 difference during a dual perfusion after the use of FDNB; APi = (Pa&-PvaOz) - (Pa202-Pvz02) = PvOz increase compared to Pa02 due to 02 transfer after the use of FDNB, taking into account the tissue respiration.
20 21 Mean
15 16 17 18 19 Mean _+to.05
Placenta
VARIATIONS PERFUSION
TABLE
IV
no.
15 17 18 19 Mean k t0.~5 SD
Placenta
0.22 0.42 0.45 0.52 0.40 + 0.10
02 consumption (ml/min/kg)
COTYLEDONARY PERFUSION PLACENTAL CIRCUIT DURING
TABLE
FDNB
12 6.5 13.2 6.1 9.5 f 2.9
Cotyledonary flow (mllmin)
Before
1.90 0.88 1.48 1.57 1.46 + 0.34
FDNB
4.8 5.1 2.0 3.6 3.88 t 1.12
Cotyledonary flow (ml/min)
After
OF OXYGEN TRANSFERRED OF PLACENTAL LOBULES,
02 transfer (ml/min/kg)
FLOW AND AMOUNT THE DUAL PERFUSION
0.84 0.57 0.49 0.73 0.66
+ 0.13
02 transfer (ml/min/kg)
60 22 85 41 52 f 21
(%)
Decrease in flow rate after FDNB
-56 35 67 54 53 + 11
(a)
Decrease in amount of oxygen transferred after FDNB
FROM UTEROPLACENTAL TO FETAL BEFORE AND AFTER THE USE OF FDNB
93
fetal oxygen transfer (Table III, placentas 20 and 21), the increase of the cotyledonary venous PO* compared to the cotyledonary arterial POz due to fetal maternal oxygen diffusion is more important after the use of FDNB. The amount of oxygen (b) Transplacental oxygen diffusion (Table IV). transferred from the uteroplacental to the fetal placental circulation decreases during the course of dual perfusions (4 experiments) when the cotyledonary vascular tree is perfused with FDNB in Earle. This could be explained by an important oxygen diffusion as compared to the placental tissue oxygen consumption. The quantity of oxygen transferred before FDNB is administered averages 1.46 + 0.34 ml/min/kg placenta. This amount decreases to 0.66 + 0.13 ml/min/kg placenta when FDNB is added to the perfusion medium. The difference between the two cases is significant (P < 0.01). The decrease in the amount of oxygen which is transferred reaches more than 50%. In one case, the amount of oxygen transferred increased after FDNB was added. This could be explained by a weak oxygen diffusion as compared to the placental tissue oxygen consumption. Inhibition
of placental
oxygen
consumption
by oligomycin
It has been observed that oligomycin dissolved in the Earle’s physiological solution which is perfused in the fetal placental circulation inhibits placental oxygen consumption. We have mainly studied the effect of oligomycin during dual perfusions of human placental lobules (Table V). (1) Changes of the PO2 in the cotyledonary circulation of fetal placental and uteroplacental circulations
during dual perfusion
The cotyledonary circulation alone has first been perfused using Earle’s equilibrated with the 5% O2 gaseous mixture to determine the decrease of the cotyledonary venous POz compared to the cotyledonary arterial PO, due to placental tissue respiration. Then, the uteroplacental circulation has been simultaneously perfused with Earle’s equilibrated with the 95% O2 gaseous mixture to determine the increase of cotyledonary venous POz as compared to cotyledonary arterial PO* due to oxygen diffusion from the maternal to the fetal side of the placenta. At last, oligomycin is added to the fetal perfusion medium at 1.25 to 10 mg/l concentration. After 15 min dual perfusion, in each case, the increase of the cotyledonary venous PO, is more important after the use of oligomycin. With the t-test for paired values this difference appears hi.ghly significant (P < 0.01). (2) Transplacental diffusion of oxygen The amount of oxygen transferred is determined during dual perfusions with Earle’s and when oligomycin is added to the perfusion medium on the fetal side of the placenta. An increase reaching about 17% is observed for the amount of oxygen which is transferred through the placenta, and this fact may be due to a lower rate of tissue respiration in the placenta.
V
no.
22 23 24 25 26 27 Mean + to.05 SD
Placenta
11.8 6.4 7.2 9.5 10.2. 8.9 9*1
Cotyledonary flow(ml/min) 48 68 85 55 76 71 67.2 + 5.8
(mm Hd
(mm Hd
107 111 122 95 103 133 112 t 6
PVOQ?
Pa002
Single perfusion
0.916 0.483 0.389 0,435 0.453 0.968 0.607
* 0.112
(QZ;min/kg)
130 121 133 100 112 128 120.7
+ 5.4
(mm Hd
Pal02
Dual perfusion
141 128 165 114 118 143 134.8
jr 8.1
AP;O2
68 50 69 54 33 77 58.5
+ 6.9
(mm Hd
0.332 0.099 0.318 0.165 0.122 0.224 0.210
+ 0.042
Q102 (mI/min/kg)
AND AMOUNT OF OXYGEN DURING THE DUAL PERFU-
oligomycin
(mm Hd
PVlOZ
before
OXYGEN CONSUMPTION DURING THE PERFUSION OF THE COTYLEDONARY CIRCULATION TRANSFERRED FROM THE UTEROPLACENTAL TO THE FETAL PLACENTAL CIRCULATION SION OF PLACENTAL LOBULES BEFORE AND AFTER THE USE OF OLIGOMYCIN
TABLE
no.
130 119 zk6
1.192 0.817 f 0.132
93 61 85 67 41
88 72.5 f 8.5
156 147 +8
Hg)
144 153 174 129 127
AP;02 (mm
pv202
(mm
oligomycin
Pa202
Hg)
after
(mm
Hg)
Dual perfusion
110 135 126 102 113
--
1.248 0.582 0.707 0.600 0.575
(ml/mm/kg) (Dual perfusion before oligomycin)
Q’,O,
1.435 0.672 0.831 0.732 0.688
1.365 0.954 + 0.228
0.397 0.347 f 0.055
Q;O2 (ml/min/kg)
0.519 0.189 0.442 0.297 0.235
Q202 (ml/min/kg)
11 14 + 3
25 II 16 13 8
AP;O,AP; 0, (mm Hg)
-
17.5 22.0 19.7 14.5 17.4 + 1.3
15.0 15.5
(S)
Increaw in amount of oxygen transferred after oligomycin
Pa02 = cotyledonary arterial POg; PvOz = cotyledonary venous P02; AP;Oz = (PaaOz-PvaOz) - (Palo*-PvrO,) = Pv02 increase compared to Pa02 due to 02 transfer before the use of oligomycin, taking into account the tissue respiration; APi = (Pa”Oz-PvaOz) (PazO2-Pv202) = PvOz increase compared to Pa02 due to 02 transfer after the use of ohgomycin, taking into account the tissue respiration; QO2 = 02 consumption; Q,02 = amount of oxygen transferred before the use of oligomycin; Q’,O, = Qr02 + QO, = amount of oxgen transferred before the use of oligomycin, taking into account the tissue respiration; Q 2 0 2 = amount of oxygen transferred after the use of oligomycin; QkO, = Q202 + QO, = amount of oxygen transferred after the use of oligomycin, taking into account the tissue respiration.
22 23 24 25 26 27 Mean * 10,~~ SD
Placenta
96 DISCUSSION
The human placenta metabolic activity is difficult to study in utero. An experimental model allows an in vitro study of placental respiration in order to prove the value of the dual perfusion technique used (Panigel, 1968). A previous work has determined placental tissue oxygen consumption and transplacental oxygen diffusion (Guiet-Bara, 1978). In this paper, the effect of cold temperature and of ATPase inhibitors (FDNB or oligomycin) on oxygen placental consumption and oxygen maternal fetal diffusion will be considered. Effect of FDNB. FDNB added to the medium perfused in the fetal placental circulation leads to a decrease of the oxygen utilization coefficient and of the placental oxygen consumption. These results confirm Infante and Davies’ (1965) observations obtained with frog muscle. As regards oxygen diffusion from the uteroplacental to the fetal placental circulation, two different types of observations can be made: - When placental tissue respiration is weak as compared to oxygen transfer, the increase of the cotyledonary venous PO, compared to the cotyledonary arterial PO* is less important after the use of FDNB. - When placental tissue respiration is important compared to oxygen transfer, the increase of the cotyledonary venous POZ compared to the cotyledonary arterial POZ is more important after the use of FDNB. The effect of FDNB could be partly related to the inhibition of placental tissue respiration. Moreover, FDNB, in most cases, causes a decrease in the amount of oxygen transferred through the placenta; this fact can be explained by the decrease in cotyledonary perfusion flow observed after FDNB is added due to the increase in viscosity of the perfusion medium. If the average percentage decrease in flow, as well as the average percentage decrease in oxygen transfer, when FDNB is added are calculated, values near 52-53s are obtained. During placental lobule dual perfusion, oligomycin Effect of oligomycin. added to the medium circulating on the fetal side of the placenta increases the amount of oxygen transferred by 16%. This increase may be due to partial inhibition of placental oxygen consumption. This fact confirms the observations of Van Rossum (1962), showing a 20% inhibition of liver slice respiration after the addition of oligomycin, and those of Whittam et al. (1964), Wu (1964) and Tobin and Slater (1965), who related a 30% inhibition of respiration due to the action of oligomycin on rat and rabbit kidney slices, frog muscle, rat diaphragm and brain tissues. Contrary to FDNB and oligomycin, The action of cold temperatures. cooling the placenta down to 4°C during perfusion experiments permit.s the complete dissociation of t.issue oxygen consumption from oxygen diffusion from the uteroplacental to the fetal placental circulation. During
97
hypothermia, at temperatures varying from 25 to 5”C, a 32-98s decrease in oxygen metabolism has been observed in different tissues and organs (Fuhrman and Field, 1942; Bergstrand and Sterky, 1954; Levy, 1959; Semb et al., 1960; Holobut et al., 1969). These facts confirm our observations: i.e., no placental tissue oxygen consumption can be measured at 4°C. However, the diffusion of oxygen from the uteroplacental to the fetal placental circulation remains the same as when the temperature is near 37°C. These observations tend to prove that the oxygen consumption observed within the first 2 h of placental perfusion has to be related to the living trophoblast metabolic activity. We have already discussed this point in a previous publication (Guiet-Bara, 1978), and compared the results obtained in our perfusion experiments to those given by other authors when placental slices and homogenates have been used, or when umbilical vessels were perfused (Nyberg and Westin, 1957; Friedman and Sachtleben, 1960). In this work, trophoblast oxygen consumption was measured from fetal circuit perfusions. One has to remember that trophoblast mainly derives its oxygenation from intervillous space blood (Panigel, 1973). Moreover, our present in vitro experiments have to be perfected by adding oxygen carriers to the perfusion media to allow a better understanding of physiological oxygen transfer through the perfused placenta. However, it can already be pointed out that when one evaluates maternal fetal transfer of oxygen using Fick’s equation, and compares this value to the one measured in these experiments, it becomes clear that there is no significant difference between the two figures (P < 0.05). Diffusion of oxygen in the model used at present seems to take place through simple diffusion, as suggested by Bartels (1970). REFERENCES Bartels, H. (1970): Prenatal Respiration, pp. 61-67. Editors: A. Neuberger and E.L. Tatum. North-Holland Publishing Company, Amsterdam. Bergstrand, A. and Sterky, G. (1954): Renal function in hypothermia. Acta physiol. stand.,
31,
13-21.
Friedman, E.A. and Sachtleben, M.R. (1960): Placental oxygen consumption in vitro. 1. Baseline studies. Amer. J. Obstet. Gynec., 79, 1058-1069. Fuhrman, F.A. and Field, J. (1942): Influence of temperature on the stimulation of oxygen consumption of isolated brain and kidney by 2,4_dinitrophenol. J. Pharmacol. exp.
Ther.,
75, 58-63.
Guiet-Bara, A. (1978): Oxygen consumption and diffusion in isolated human term placental lobules perfused in vitro. In: Oxygen Transport To Tissue, Vol. 3, pp. 479484. Editors: I.A. Silver, M. Erecinska and H.I. Bicher. Plenum Publishing Corporation, New York. Holobut, W., Modrzejewski, E. and Stazka, W. (1969): Irrigation sanguine et consommation d’oxygene de divers organes en temperature normale et en hypothermie. J. Physiol. (Paris),
61, 507-517.
Infante, A.A. and Davies, R.E. (1965): The effect of 2,4-dinitrofluorobenzene activity of striated muscle. J. biol. Chem., 240, 3996-4001. Levy, M.N. (1959): Oxygen consumption and blood flow in the hypothermic, kidney.Amer. J. Physiol., 197, 1111-1114. Nyberg, R. and Westin, B. (1957): The influence of oxygen tension and some human placental vessels. Acta physiol. &and., 39, 216-227.
on the perfused drugs on
98
Panigel, M. (1968): Placental perfusion. In: Fetal Homeostasis, Vol. 4, pp. 15-Z Editor: R.M. Wynn. Appleton-Century-Crofts, New York. Panigel, M. (1973): Experimental models for in vivo and in vitro investigations on place tal hemodynamics and oxygen supply to the fetus. In: Oxygen Transport To Tissr, pp. 1061-1065. Editors: D.F. Bruley and H.I. Bicher. Plenum Publishing Corporatia New York. Semb, C., Krog, J. and Johansen, K. (1960): Renal metabolism and blood flow duri local hypothermia studied by means of renal perfusion in situ. Actn chir. scam Suppl. 253, 196-202. Tobin, R.B. and Slater, E.C. (1965): The effect of oligomycin on the respiration of tiss slices. Biochim. biophys. Acta (Amst.), 105, 214-220. Van Rossum, G.D.V. (1962): The effect of oligomycin on cation transport in slices of I liver. Biochem. J., 84, 35-36P. Whittam, R., Wheeler, K.P. and Blake, A. (1964): Oligomycin and active transport res tions in cell membranes. Nature (Lond.), 203, 720-724. Wu, R. (1964): The effect of azide and oligomycin on inorganic phosphate transport slices of rat kidney. Biochim. biophys. A& (Amst.), 82, 212-215.
reprod.
Biol.,
Biomedical
1980, 10/2, Press
83-98
83
HUMAN PLACENTAL OXYGEN TRANSFER AND CONSUMPTION. DISSOCIATION BY COOLING OR USE OF RESPIRATORY ENZYME INHIBITORS
A. GUIET-BARA Department
of Reproductive
Accepted
for publication
GUIET-BARA, tion by cooling Biol.,
*
10/Z,
Biology,
University
of Paris VI, Paris, France
29 July 1979
A. (1979): Human placental or use of respiratory enzyme
oxygen transfer and consumption. Dissociainhibitors. Europ. J. Obstet. Gynec. reprod.
83-98.
The regulation of oxygen placental transfer is studied during the in vitro perfusion of normal postpartum human placental lobules. Oxygen consumption of the placental tissue is measured during the perfusion of the fetal placental circulation alone (single perfusions). To evaluate the transplacental diffusion of oxygen, fetal placental and uteroplacental circulations are simultaneously perfused (dual perfusions). The results obtained at various temperatures (37 and 4’C) and after use of respiratory enzyme inhibitors (fluorodinitrobenzene and oligomycin) prove that oxygen consumption is related to trophoblast physiological activity, while oxygen transfer relies on a simple diffusion mechanism. placental
perfusion;
trophoblast
metabolic
activity;
oxygen
transfer
mechanism
INTRODUCTION
This research project was carried out in order to dissociate placental oxygen consumption from fetal maternal oxygen diffusion through the placenta (Guiet-Bara, 1978). Oxygen consumption seems to correspond to the trophoblast physiological activity, while placental transfer of oxygen relies on a physiological mechanism, which has to be defined. Physical methods have been employed for this study: change in temperature (cooling) during the placental perfusion, as well as biochemical techniques: use of respiratory enzyme inhibitors (such as fluorodinitrobenzene and oligomycin).
* Reprint
Reproduction France.
requests
to: Dr. A. Guiet-Bara, Universite Pierre et Marie Curie, Biologie de la (Pr. M. Panigel), Bt A, 7, Quai Saint Bernard, 75230 Paris Cedex 05,
84 MATERIAL
27 placed room lobule
AND METHODS
normal term human placentas, taken after spontaneous delivery, were in heparinized Earle’s medium and then put in a constant temperature (either 37 or 4°C) adjacent to the labor room. An intact placental was immediately perfused for about 1.5-2 h.
Placental perfusion technique (Figs. 1 and 2) (1) Fetal placental blood vessel catheterization The allantochorial arterial branch, which supplies fetal blood to the placental lobule, is cannulated on the chorial plate. The plastic tubing is advanced until it reaches the beginning of the cotyledonary artery, and it is then kept in place by two ligatures. All the neighboring arterial branches are
+i
Fig. 1. Perfusion system. A: gas mixture with 5% 02, 5% CO2 and 90% N,; B: gas mixture with 95% O2 and 5% C02. C,, Cz;’ C3, Cd, two-way stop-cocks; Dt;gas dispersion tube; Foe. per, res., fetal perfusion reservoirs; Fm, flowmeter; Man, manometer; Mat. per. res., maternal perfusion reservoir.
85
Fig, 2. The placental lobule in the perfusion chamber. CP, center piece; IAA, perfusion inflow to allantochorial artery; ISA, perfusion inflow to the uteroplacental spiral artery; L, perfused lobule; LC, lower chamber; OAV, perfusion outflow from allantochorial vein; OUV, perfusion outflow from uteroplacental veins; UC, upper chamber.
then obliterated by a ligature. A physiological solution is then perfused to identify the vessel through which the venous outflow of perfusate takes place. This chorioallantoic vein is cannulated in the same way as described for the artery. (2) The perfusion chamber and the u teroplacen tal artery cannulation The perfusion medium washes residual fetal blood out of the cotyledonary vascular tree; the cotyledon whitens, showing its borders clearly. This area is then excised out of the placental disk. The lower part of the perfusion chamber is filled with the physiological medium used to perfuse the fetal cotyledonary circulation. The isolated lobule is placed upside down on the surface of the physiological liquid, with its basal plate up. The placental tissue and the fetal membranes covering the perfused lobule are held in place by the middle piece of the artificial uterus, which is then screwed onto the lower part of the chamber. On the basal plate surface, the end of a uteroplacental spiral artery (or only its opening) is looked for to insert a fine glass cannula 200 pm in diameter. This glass cannula is connected to the maternal perfusion circuit through a silicone tubing, which is attached to the Plexiglas tube. This tube opens into the top of the upper chamber, which is then screwed onto the middle piece of the artificial uterus and filled with physiological medium (the same one used to perfuse the uteroplacental circulation). Perfusion of the intervillous space is initiated, and the perfusate
86
flows back into the upper chamber on the basal plate.
through
the numerous
venous
openings
Perfusion outfit (Fig. 1). The fetal perfusion circuit will carry the perfusion liquid from a fetal perfusion reservoir into the cannulated allantochorial artery on one ‘side and, on the other side, the perfusate flows back through the allantochorial vein to a collecting flask. This type of perfusion is said to be an open circuit: the perfusion medium is constantly renewed and circulates only once. The maternal perfusion circuit is connected in the same way as the fetal one. The perfusion medium is propelled through the circuit by a Lillehei digital pump, T6S model (Sigmamotor). The hydrostatic pressure at the entrance of each circuit is measured using Ludwig mercury manometers; it varies from 40 to 90 mm Hg. The flows are controlled at the entrance and exit of each circuit, to detect a possible loss of perfusion medium due to edema or tearing of the fetal vasculature. Brooks flowmeters are used for this purpose. The perfusion is carried out at a flow rate of 6-12 ml/min for the fetal circulation and at a flow rate of 9-13 ml/min for the maternal circulation. Perfusion medium. The perfusion media used are either Earle’s physiological salt solution, or the same Earle’s solution, to which a respiration inhibitor (FDNB or oligomycin) is added. The l-fluoro-2,4_dinitrobenzene (Merck) is dissolved in Earle’s at 0.38 or 8 mM/l dilution. Oligomycin (Sigma) is a mixture of 15% oligomycin A and 85% oligomycin B; it is dissolved in Earle’s at a concentration ranging from 1.25 to 10 mg oligomycin/l perfusion medium, The perfusion media are equilibrated with one of the following gaseous mixtures: A (5% O2 + 5% CO* + 90% N,) or B (95% O2 + 5% CO*). Mixture A, poor in oxygen, simulates the oxygen concentration in the fetal blood coming from the umbilical artery; mixture B, rich in oxygen, simulates the oxygen concentration in the maternal blood flowing in the placenta from the uterine artery. To evaluate oxygen consumption by the placental Experimental protocol. tissue, the isolated lobule fetal placental circulation alone is perfused (single perfusion) with Earle’s solution equilibrated either with gaseous mixture A or B. To measure the diffusion of oxygen from uteroplacental to fetal placental circulation, the dual perfusion is used. The fetal cotyledonary circulation is perfused with Earle’s equilibrated with A, while the uteroplacental circulation is perfused with Earle’s equilibrated with B to establish a transplacental concentration gradient for oxygen. The perfusion medium is sampled every 15 min at the entrance and exit of each perfusion circuit; pH, PO* and PCOZ are determined using a Beckman physiological gas analyzer (model 160). To measure the amount of Statement and interpretation of the results. oxygen dissolved in the physiological salt solution (Earle’s), we aPPb Henry’s law.
a7 Mean values (+ to.os SD) are given. Student’s different mean values.
t-test is used to compare
the
RESULTS
Oxygen consumption
inhibition
The results are presented
by cooling placental
tissue
in Table I.
(1) POz changes in the fetal placental
circulation
(a) Single perfusion. The fetal side of the placenta is perfused at 4°C using Earle’s solution equilibrated with A (5% 0,). The venous cotyledonary PO2 (PvO,) is the same as the cotyledonary arterial PO2 (PaO,). No tissue respiration takes place in the placenta. After beginning the perfusion of the fetal placental (b) Dual perfusion. circulation using Earle’s + 5% 02, the uteroplacental circulation is simultaneously perfused using Earle’s + 95% Oz. The venous cotyledonary PO* becomes higher than the arterial POz; the percentage of venous PO, increase compared to the arterial PO, averages 38%. Lowering the temperature to 4°C does not hinder an important diffusion of oxygen from the maternal to the fetal side of the placenta. (2) Amount of oxygen diffusing from the uteroplacental to the fetal placental circulation during the dual perfusion This amount is calculated as the product of the arteriovenous concentration gradient by the cotyledonary perfusion flow. If this amount is related to one kilo placental weight, it reaches an average of 0.97 f. 0.06 ml/min/kg. This value corresponds to the one measured during dual perfusions at physiological temperature (37°C) (Guiet-Bara, 1978). Placental (FDNB)
oxygen
(I) Single perfusion
consumption
inhibition
by
l-fluoro-2,4-dinitrobenzene
(Table II)
(a) PO2 changes in the cotyledonary circulation. The fetal placental circulation is perfused using Earle’s solution equilibrated either with mixture A (5% 0,) or with mixture B (95% 0,). A POz arteriovenous gradient appears due to placental tissue respiration: (APIOz). The placenta is then perfused with Earle’s + FDNB + the same gaseous mixtures as stated above. The arteriovenous cotyledonary PO* gradient is then measured in each case: APzOz < AP,Oz. If a t-test is done for paired values, the average of (AP102AP202) is very significantly different from 0 (P < 0.01). This fact points to FDNB being an inhibitor of placental respiration.
arterial PO2 ; PvOz = cotyledonary
venous POZ.
142 138 126 124 140 131 133.5 f 3.3
0 0 0.04 0.05 0 0.05 0.02 * 0.01
148 130 120 115 145 125 130.5 i: 5.7
148 130 122 117 145 128 131.7 + 5.3
11.8 7.8 6.9 8.4 9.3 10.2 9.1 -c 0.8
Pa02 = cotyledonary
1 2 3 4 5 6 Mean * to.05 SD
Pa02 (mm Hg)
02 consumption (ml/min/kg)
Pa02 (mm Hg) Pvoz (mm Hg)
Dual perfusion
Single perfusion
190 195 180 171 192 177 184.2 + 4.1
33.8 41.3 42.5 37.9 37.2 35.3 38.0 f 1.3
Pvq increase compared to Pa& due to 02 transfer (%I
LOBULES AT 4OC
PVOZ (mm Hg)
DURING THE PERFUSION OF PLACENTAL
Cotyledonary flow (ml/min)
AND CONSUMPTION OF OXYGEN
Placenta no.
TRANSFER
TABLE I
-
0.76 1.17 1.12 0.98 0.92 0.87 0.97 + 0.06
01 transfer (ml/min/kg)
-
II
119
5
Pa02
= cotyledonary
arterial PO2
; PvO2 = cotyledonary
venous
* 4.0
33 32 28 31.0
253 306 284 281 f 38
377 450 395 407 + 55
95 95 95
12 13 14 Mean f to.05 SD P02.
8 8 8
0.38 0.38 0.38 0.38 * 1.5
8
62 27 24 28 25 26.0
(mM/II
(%)
FDNB
Concentration of FDNB in the solution
After
UTILIZATION
Oxygen utilization coefficient
288 308 274 323 298 + 17
Hg)
395 405 380 430 403 + 17
45
PVlOZ
(mm
Hg)
Pal02
(mm
FDNB
02 % in the gas mixture
Before
OXYGEN
95 95 95 95
no.
PO* AND
8 9 10 11 Mean * lo.o5 SD
7
Placenta
ARTERIAL AND VENOUS COTYLEDONARY BEFORE AND AFTER THE USE OF FDNB
TABLE
Hg)
370 455 403 409 f 62
410 395 390 426 405 * 13
116
(mm
Pa202
COEFFICIENT
Hg)
333 423 375 377 + 65
336 336 335 354 340 + 7
116
(mm
pv202
DURING
10 7 7 8.0 t 2.5
18 15 14 17 16.0 f 1.5
0
(%I
Oxygen utilization coefficient
A SIMPLE
70 78 75 75 + 6
34 38 50 28 38 + 7
100
(%I
Decrease of oxygen utilization coefficient after FDNB
PERFUSION
90
(b) Oxygen oxygen content
utilization
coefficient
in the cotyledonary oxygen
content
artery - oxygen
content
in the cotyledonary
in the cotyledonary
vein x 100.
artery
For one placenta, when FDNB 8 mM/l Earle is perfused equilibrated with mixture A (5% 0,), the oxygen utilization coefficient decreases to zero. This coefficient reached 62% before the inhibitor was used. For 7 other placentas, the perfusion solution is equilibrated with mixture B (95% O,), and two different concentrations of FDNB are used (0.38 mM/l or 8 mM/l). In 4 cases, FDNB (0.38 mM/l) decreases the oxygen utilization coefficient from 26 to 16% average, this difference being significant (P< 0.05). In 3 cases, FDNB (8 mM/l) decreases the oxygen utilization coefficient from 31 to 8% average, this difference being very significant (P < 0.02). On the other hand, the percentage of decrease of the oxygen utilization coefficient averages 38% when FDNB of 0.38 mM/l is used, and 75% using FDNB of 8 mM/l. It is thus twice as important with the more concentrated solution. This difference is significant (P < 0.05). (c) Oxygen consumption of placental tissue. For each placenta, a decrease in oxygen consumption is noticed during the perfusion when pure Earle’s solution is replaced by FDNB in Earle. The percentage decrease in the amount of oxygen consumed after FDNB is administered reaches 63% with the concentration 0.38 mM/l and 68% with 8 mM/l, when the perfusion solution is equilibrated with mixture B (95% 0,). (2) Effect of FDNB during the dual perfusion
of the placental
lobule
(a) PO2 changes in the cotyledonary circulation (Table III). The fetal placental circulation is first perfused using Earle equilibrated with the gas mixture A (5% 0,). We can thus measure the difference of the cotyledonary arteriovenous PO* due to the placental tissue respiration (APOOZ). Uteroplacental circulation perfusion is then initiated using Earle equilibrated with mixture B (95% 0,). The cotyledonary arteriovenous PO* is then measured (AP,O*). Taking into account the tissue respiration, it is possible to infer the cotyledonary venous POZ increase as compared to the cotyledonary arterial PO* due to oxygen diffusion (AP’,O,). Lastly, the cotyledonary circulation is perfused with Earle + FDNB (0.38 mM/l or 8 mM/l) equilibrated with gas mixture A (5% 0,). Two kinds of results are thus obtained: -When placental respiration is weak compared to maternal fetal oxygen transfer (Table III, placentas 15-19), the increase of the cotyledonary venous POZ compared to the cotyledonary arterial POZ due to fetal maternal oxygen diffusion is less important after the use of FDNB. This increase averages 108 + 23 mm Hg before the use of FDNB and 46.4 f 7.0 mm Hg after the use of the inhibitor, this difference being significant (P = 0.01). - When the placental tissue respiration is important compared to maternal
III
no.
SD 116 105 110.5
t 3.7
74 44 59
? 4.2
t8
+ 5.1
44 51 47.5
127 130 113 120 124 122.8
13 32 24 19 28 23.2
119 96 95 109 81 100
132 128 119 128 109 123.2
118 95 106.5
Pa 10, (mm Hg)
AP”O, (mm Hg)
PveO* (mm Hg)
PaeOz (mm Hg)
45.5
3 24 13.5
+ 23
+ 22
+ 26 71 20
60 67 126 108
-36 -48 -98 -85
45 85 65
130 155
-117 --125
Hg)
AP’,O, (mm Hg)
244 255 149 168 222 208
AP,O2
FDNB
(mm
before
115 102 113.5
+2
125 124 121 122 116 122
Paz02 ( mm Hg)
74 91 82.5
161 153 136 132 142 145 +7
I%202 (mm Hg)
after
41 11 26
--36 -29 -15 -10 --26 -23.2 * 5.8
AP*O* (mm Hg)
FDNB
PO,! DURING USE OF FDNB
Dual perfusion
ARTERIAL COTYLEDONARY BEFORE AND AFTER THE
( mmHg)
pv,o2
Dual perfusion
Single perfusion
IN THE VENOUS COTYLEDONARY PO2 COMPARED TO AND DURING DUAL PERFUSION OF PLACENTAL LOBULES
_
33 33 33
49 61 39 29 54 46.4 + 7.0
AP’, 0, (mmHg)
SIMPLE
Pa02 = cotyledonary arterial PO,; PvOz = cotyledonary venous PO,; APeOz = PaeOz-PveOz = arteriovenous cotyledonary PO2 difference during a simple perfusion; API02 = PaiOT-Pv102 = arteriovenous cotyledonary PO2 difference during a dual perfusion before the use of FDNB; AP’iO2 = (PaeOz-Pve02) - (PaiOz-Pv102) = PvO:! increase compared to Pa02 due to 02 transfer before the use of FDNB, taking into account the tissue respiration; API02 = Pa202-Pv202 = arteriovenous cotyledonary PO2 difference during a dual perfusion after the use of FDNB; APi = (Pa&-PvaOz) - (Pa202-Pvz02) = PvOz increase compared to Pa02 due to 02 transfer after the use of FDNB, taking into account the tissue respiration.
20 21 Mean
15 16 17 18 19 Mean _+to.05
Placenta
VARIATIONS PERFUSION
TABLE
IV
no.
15 17 18 19 Mean k t0.~5 SD
Placenta
0.22 0.42 0.45 0.52 0.40 + 0.10
02 consumption (ml/min/kg)
COTYLEDONARY PERFUSION PLACENTAL CIRCUIT DURING
TABLE
FDNB
12 6.5 13.2 6.1 9.5 f 2.9
Cotyledonary flow (mllmin)
Before
1.90 0.88 1.48 1.57 1.46 + 0.34
FDNB
4.8 5.1 2.0 3.6 3.88 t 1.12
Cotyledonary flow (ml/min)
After
OF OXYGEN TRANSFERRED OF PLACENTAL LOBULES,
02 transfer (ml/min/kg)
FLOW AND AMOUNT THE DUAL PERFUSION
0.84 0.57 0.49 0.73 0.66
+ 0.13
02 transfer (ml/min/kg)
60 22 85 41 52 f 21
(%)
Decrease in flow rate after FDNB
-56 35 67 54 53 + 11
(a)
Decrease in amount of oxygen transferred after FDNB
FROM UTEROPLACENTAL TO FETAL BEFORE AND AFTER THE USE OF FDNB
93
fetal oxygen transfer (Table III, placentas 20 and 21), the increase of the cotyledonary venous PO* compared to the cotyledonary arterial POz due to fetal maternal oxygen diffusion is more important after the use of FDNB. The amount of oxygen (b) Transplacental oxygen diffusion (Table IV). transferred from the uteroplacental to the fetal placental circulation decreases during the course of dual perfusions (4 experiments) when the cotyledonary vascular tree is perfused with FDNB in Earle. This could be explained by an important oxygen diffusion as compared to the placental tissue oxygen consumption. The quantity of oxygen transferred before FDNB is administered averages 1.46 + 0.34 ml/min/kg placenta. This amount decreases to 0.66 + 0.13 ml/min/kg placenta when FDNB is added to the perfusion medium. The difference between the two cases is significant (P < 0.01). The decrease in the amount of oxygen which is transferred reaches more than 50%. In one case, the amount of oxygen transferred increased after FDNB was added. This could be explained by a weak oxygen diffusion as compared to the placental tissue oxygen consumption. Inhibition
of placental
oxygen
consumption
by oligomycin
It has been observed that oligomycin dissolved in the Earle’s physiological solution which is perfused in the fetal placental circulation inhibits placental oxygen consumption. We have mainly studied the effect of oligomycin during dual perfusions of human placental lobules (Table V). (1) Changes of the PO2 in the cotyledonary circulation of fetal placental and uteroplacental circulations
during dual perfusion
The cotyledonary circulation alone has first been perfused using Earle’s equilibrated with the 5% O2 gaseous mixture to determine the decrease of the cotyledonary venous POz compared to the cotyledonary arterial PO, due to placental tissue respiration. Then, the uteroplacental circulation has been simultaneously perfused with Earle’s equilibrated with the 95% O2 gaseous mixture to determine the increase of cotyledonary venous POz as compared to cotyledonary arterial PO* due to oxygen diffusion from the maternal to the fetal side of the placenta. At last, oligomycin is added to the fetal perfusion medium at 1.25 to 10 mg/l concentration. After 15 min dual perfusion, in each case, the increase of the cotyledonary venous PO, is more important after the use of oligomycin. With the t-test for paired values this difference appears hi.ghly significant (P < 0.01). (2) Transplacental diffusion of oxygen The amount of oxygen transferred is determined during dual perfusions with Earle’s and when oligomycin is added to the perfusion medium on the fetal side of the placenta. An increase reaching about 17% is observed for the amount of oxygen which is transferred through the placenta, and this fact may be due to a lower rate of tissue respiration in the placenta.
V
no.
22 23 24 25 26 27 Mean + to.05 SD
Placenta
11.8 6.4 7.2 9.5 10.2. 8.9 9*1
Cotyledonary flow(ml/min) 48 68 85 55 76 71 67.2 + 5.8
(mm Hd
(mm Hd
107 111 122 95 103 133 112 t 6
PVOQ?
Pa002
Single perfusion
0.916 0.483 0.389 0,435 0.453 0.968 0.607
* 0.112
(QZ;min/kg)
130 121 133 100 112 128 120.7
+ 5.4
(mm Hd
Pal02
Dual perfusion
141 128 165 114 118 143 134.8
jr 8.1
AP;O2
68 50 69 54 33 77 58.5
+ 6.9
(mm Hd
0.332 0.099 0.318 0.165 0.122 0.224 0.210
+ 0.042
Q102 (mI/min/kg)
AND AMOUNT OF OXYGEN DURING THE DUAL PERFU-
oligomycin
(mm Hd
PVlOZ
before
OXYGEN CONSUMPTION DURING THE PERFUSION OF THE COTYLEDONARY CIRCULATION TRANSFERRED FROM THE UTEROPLACENTAL TO THE FETAL PLACENTAL CIRCULATION SION OF PLACENTAL LOBULES BEFORE AND AFTER THE USE OF OLIGOMYCIN
TABLE
no.
130 119 zk6
1.192 0.817 f 0.132
93 61 85 67 41
88 72.5 f 8.5
156 147 +8
Hg)
144 153 174 129 127
AP;02 (mm
pv202
(mm
oligomycin
Pa202
Hg)
after
(mm
Hg)
Dual perfusion
110 135 126 102 113
--
1.248 0.582 0.707 0.600 0.575
(ml/mm/kg) (Dual perfusion before oligomycin)
Q’,O,
1.435 0.672 0.831 0.732 0.688
1.365 0.954 + 0.228
0.397 0.347 f 0.055
Q;O2 (ml/min/kg)
0.519 0.189 0.442 0.297 0.235
Q202 (ml/min/kg)
11 14 + 3
25 II 16 13 8
AP;O,AP; 0, (mm Hg)
-
17.5 22.0 19.7 14.5 17.4 + 1.3
15.0 15.5
(S)
Increaw in amount of oxygen transferred after oligomycin
Pa02 = cotyledonary arterial POg; PvOz = cotyledonary venous P02; AP;Oz = (PaaOz-PvaOz) - (Palo*-PvrO,) = Pv02 increase compared to Pa02 due to 02 transfer before the use of oligomycin, taking into account the tissue respiration; APi = (Pa”Oz-PvaOz) (PazO2-Pv202) = PvOz increase compared to Pa02 due to 02 transfer after the use of ohgomycin, taking into account the tissue respiration; QO2 = 02 consumption; Q,02 = amount of oxygen transferred before the use of oligomycin; Q’,O, = Qr02 + QO, = amount of oxgen transferred before the use of oligomycin, taking into account the tissue respiration; Q 2 0 2 = amount of oxygen transferred after the use of oligomycin; QkO, = Q202 + QO, = amount of oxygen transferred after the use of oligomycin, taking into account the tissue respiration.
22 23 24 25 26 27 Mean * 10,~~ SD
Placenta
96 DISCUSSION
The human placenta metabolic activity is difficult to study in utero. An experimental model allows an in vitro study of placental respiration in order to prove the value of the dual perfusion technique used (Panigel, 1968). A previous work has determined placental tissue oxygen consumption and transplacental oxygen diffusion (Guiet-Bara, 1978). In this paper, the effect of cold temperature and of ATPase inhibitors (FDNB or oligomycin) on oxygen placental consumption and oxygen maternal fetal diffusion will be considered. Effect of FDNB. FDNB added to the medium perfused in the fetal placental circulation leads to a decrease of the oxygen utilization coefficient and of the placental oxygen consumption. These results confirm Infante and Davies’ (1965) observations obtained with frog muscle. As regards oxygen diffusion from the uteroplacental to the fetal placental circulation, two different types of observations can be made: - When placental tissue respiration is weak as compared to oxygen transfer, the increase of the cotyledonary venous PO, compared to the cotyledonary arterial PO* is less important after the use of FDNB. - When placental tissue respiration is important compared to oxygen transfer, the increase of the cotyledonary venous POZ compared to the cotyledonary arterial POZ is more important after the use of FDNB. The effect of FDNB could be partly related to the inhibition of placental tissue respiration. Moreover, FDNB, in most cases, causes a decrease in the amount of oxygen transferred through the placenta; this fact can be explained by the decrease in cotyledonary perfusion flow observed after FDNB is added due to the increase in viscosity of the perfusion medium. If the average percentage decrease in flow, as well as the average percentage decrease in oxygen transfer, when FDNB is added are calculated, values near 52-53s are obtained. During placental lobule dual perfusion, oligomycin Effect of oligomycin. added to the medium circulating on the fetal side of the placenta increases the amount of oxygen transferred by 16%. This increase may be due to partial inhibition of placental oxygen consumption. This fact confirms the observations of Van Rossum (1962), showing a 20% inhibition of liver slice respiration after the addition of oligomycin, and those of Whittam et al. (1964), Wu (1964) and Tobin and Slater (1965), who related a 30% inhibition of respiration due to the action of oligomycin on rat and rabbit kidney slices, frog muscle, rat diaphragm and brain tissues. Contrary to FDNB and oligomycin, The action of cold temperatures. cooling the placenta down to 4°C during perfusion experiments permit.s the complete dissociation of t.issue oxygen consumption from oxygen diffusion from the uteroplacental to the fetal placental circulation. During
97
hypothermia, at temperatures varying from 25 to 5”C, a 32-98s decrease in oxygen metabolism has been observed in different tissues and organs (Fuhrman and Field, 1942; Bergstrand and Sterky, 1954; Levy, 1959; Semb et al., 1960; Holobut et al., 1969). These facts confirm our observations: i.e., no placental tissue oxygen consumption can be measured at 4°C. However, the diffusion of oxygen from the uteroplacental to the fetal placental circulation remains the same as when the temperature is near 37°C. These observations tend to prove that the oxygen consumption observed within the first 2 h of placental perfusion has to be related to the living trophoblast metabolic activity. We have already discussed this point in a previous publication (Guiet-Bara, 1978), and compared the results obtained in our perfusion experiments to those given by other authors when placental slices and homogenates have been used, or when umbilical vessels were perfused (Nyberg and Westin, 1957; Friedman and Sachtleben, 1960). In this work, trophoblast oxygen consumption was measured from fetal circuit perfusions. One has to remember that trophoblast mainly derives its oxygenation from intervillous space blood (Panigel, 1973). Moreover, our present in vitro experiments have to be perfected by adding oxygen carriers to the perfusion media to allow a better understanding of physiological oxygen transfer through the perfused placenta. However, it can already be pointed out that when one evaluates maternal fetal transfer of oxygen using Fick’s equation, and compares this value to the one measured in these experiments, it becomes clear that there is no significant difference between the two figures (P < 0.05). Diffusion of oxygen in the model used at present seems to take place through simple diffusion, as suggested by Bartels (1970). REFERENCES Bartels, H. (1970): Prenatal Respiration, pp. 61-67. Editors: A. Neuberger and E.L. Tatum. North-Holland Publishing Company, Amsterdam. Bergstrand, A. and Sterky, G. (1954): Renal function in hypothermia. Acta physiol. stand.,
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