Uterine blood flow, oxygen and glucose uptakes at mid-gestation in the sheep

In early ovine fetal development, the placenta grows more rapidly than the fetus so that at mid-gestation the aggregate weight of placental cotyledons exceeds fetal weight. The purpose of this study was to compare two separate methods of measuring uterine blood flow and glucose and oxygen uptakes in seven mid-gestation ewes, each carrying a single fetus. Uterine blood flow to both uterine horns was measured by microsphere and by tritiated water steady-state diffusion methodology. Calculations of tritiated water blood flows and oxygen and glucose uptakes were based on measurements of arteriovenous concentration differences across each uterine horn. The distribution of blood flow and oxygen uptake between the two uterine horns was strongly correlated with placental mass distribution. The two methods gave comparable results for uterine blood flow (457 +/- 35 vs 476 +/- 35 ml/min), oxygen uptake (457 +/- 35 vs 476 +/- 35 mumol/min), and glucose uptake (63 +/- 8 vs 64 +/- 6 mumol/min). Uterine blood flow was approximately 38% of the late gestation value and 56.1 +/- 1 times higher than umbilical blood flow. Uteroplacental oxygen consumption was about 58% of late gestation measurements and 3.9 +/- 0.5 times higher than fetal oxygen uptake. We confirm that the large placental mass of mid-gestation is associated with high levels of maternal placental blood flow and placental oxidative metabolism.     

On the relative contribution of viscous flow vs. diffusional (frictional) flow to the stationary state flow of water through a "tight" membrane

The practice of calculating the diffusion contribution to the total pressure-driven flow of water through a tight membrane by using the self-diffusion coefficient for tritiated water is examined by a theoretical analysis. Equations of motion for water and membrane in pressure-driven water flow and water, membrane, and tritiated water in self-diffusion of tritiated water are adapted from Bearman and Kirkwood (1958). These equations of motion are used to develop an equation for the pressure-driven flow of water. Because of the lack of specific information about the detailed structure of most membranes, as well as considerations of the need to eliminate some of the mathematical difficulties, an "equivalent capillary" model is used to find a solution to the equation of motion. The use of the equivalent capillary model and possible ambiguities in distinctions between diffusion and hydrodynamic flow are discussed     

Matching of maternal and fetal flow ratios in the sheep placenta

Local interaction of maternal and fetal placental blood flows was studied in two groups of unanaesthetized near-term sheep. Five sheep were exposed to a simulated dive to 100 feet of seawater (4.03 atmospheres) for 25 min. Six fetuses received an infusion of noradrenaline (6.8 micrograms/[kg x min]). Radioactive microspheres were administered simultaneously to mother and fetus before (control) and after (test) the experimental manipulation. Maternal and fetal relative activities, defined as % of total placental radioactivity divided by % of total placental weight, were calculated for 1-g pieces of cotyledonary tissue under control and test conditions. Pieces of cotyledons were defined as matched if the direction of change in relative activity from control to test was the same for mother and fetus. In the absence of an interaction between the maternal and fetal placental circulations, the probability of a piece of cotyledon being matched is 0.5. In each series of experiments the proportion of all cotyledon pieces having maternal and fetal relative activities that changed in the same direction was significantly greater than 0.5. Thus, the majority of the placental mass responds to a physical or chemical perturbation of the fetus in such a way that changes in relative perfusion are qualitatively matched in the adjacent maternal and fetal placental circulations.     

Urea and amino acid transport by an isolated human placenta]

A method of bilateral perfusion of the isolated human placenta was used to study the urea transport from the fetal placental stream into the maternal one, and of amino acid transport in the opposite direction. Experiments demonstrated that the method provided a sufficiently full perfusion of the intervillous space and offered possibilities for studying the placental transport. With equal amino nitrogen concentration in both circulations, its content in the fetal stream increased during the experiment. This elevation was more expressed when amino acid was added to the maternal circulation. The idea of amino acid "secretion" by the trophoblast cell elements into the fetal circulation was confirmed by the above experiments.     

The relationships between fetal and maternal placental blood flows.

Individual maternal and fetal flows to 706 placental cotyledons obtained from 9 chronically catheterized pregnant ewes and their fetuses (gestation age 123-141 days) were measured. The larger the cotyledon the greater the maternal and fetal blood flow to it. Both fetal and maternal flows to larger cotyledons, however, tended to be lower when corrected for the weight of the cotyledon perfused. Changes in fetal placental flow (dfgc, ml/min/g) occurring within 15 min of administration of 15 mg i.v. of captopril to the ewe were dependent on changes in fetal placental vascular resistance (dcotfr) and maternal flow (dmgc) according to the equation dfgc = 0.123 + 0.185 dmgc - 0.026 dcotfr. Changes in maternal placental flow occurring within 15 min of administration of 15 mg i.v. of captopril to the ewe were dependent on changes in maternal placental vascular resistance (dcotmr) and changes in fetal flow according to the equation dmgc = 0.483 + 0.496 dfgc - 0.0198 dcotmr. The changes in fetal flow over the next 1.5h of treatment with captopril at 6 mg/h were dependent on neither changes in fetal placental vascular resistance nor maternal placental flow. changes in maternal placental flows over the same time were no longer related to changes in fetal flow and depended only to a minimal extent on changes in maternal placental resistance. These analyses suggest that treatment of the pregnant ewe with captopril may have disturbed the normal relationships between maternal and fetal placental circulations.     

Maternal DDAVP-induced hyponatremia preserves fetal urine flow during acute fetal hemorrhage

Maternal administration of DDAVP induces maternal and fetal plasma hyponatremia, accentuates fetal urine flow, and increases amniotic fluid volume. Fetal hemorrhage represents an acute stress that results in fetal AVP secretion and reduced urine flow rate. In view of the potential therapeutic use of DDAVP for pregnancies with reduced amniotic fluid volume, we sought to examine the impact of maternal hypotonicity during acute fetal hemorrhage. Chronically catheterized pregnant ewes (130 +/- 2 days) were allocated to control or to DDAVP-induced hyponatremia groups. In the latter group, tap water (2,000 ml) was administered intragastrically to the ewe followed by DDAVP (20 microg bolus, 4 microg/h) and a maintenance intravenous infusion of 5% dextrose water for 4 h to achieve maternal hyponatremia of 10-12 meq/l. Thereafter, ovine fetuses from both groups were continuously hemorrhaged to 30% of estimated blood volume over a 60-min period. DDAVP caused similar degree of reductions in plasma sodium and osmolality in pregnant ewes and their fetuses. In response to hemorrhage, DDAVP fetuses showed greater reduction in hematocrit than control fetuses (14 vs. 10%). Both groups of fetuses demonstrated similar increases in plasma AVP concentration. However, the AVP-hemorrhage threshold was greater in DDAVP fetuses (22.5%) than in control (17.5%). Hemorrhage had no significant impact on plasma osmolality, electrolyte levels, or cardiovascular responses in either group of fetuses. Despite similar increases in plasma AVP, DDAVP fetuses preserved fetal urine flow rates, with values threefold those of control fetuses. These results suggest that under conditions of acute fetal stress of hemorrhage, maternal DDAVP may preserve fetal urine flow and amniotic fluid volume.     

The role of fetal urinary excretion in the transfer of ethanol into amniotic fluid after maternal administration of ethanol to the near-term pregnant ewe

The objective of this study was to determine whether fetal urinary excretion is a major route of ethanol transfer into the amniotic fluid surrounding the fetus following maternal administration of ethanol. Conscious instrumented pregnant ewes between 130 and 137 days' gestation (term, 147 days) with (n = 3) or without (n = 3) a catheter in the fetal bladder were administered 1 g ethanol/kg maternal body weight as a 1-h maternal intravenous infusion. Maternal blood, fetal blood, and amniotic fluid samples were collected at selected times, and fetal urine was collected continuously from the bladder-cannulated fetus during the 14-h study for the determination of ethanol concentrations. Fetal urinary excretion of ethanol occurred, and the total amount of ethanol excreted represented 0.30 +/- 0.07 (SD)% of the maternal ethanol dose. The renal clearance of ethanol by the fetus was 0.43 +/- 0.06 mL/min. The pharmacokinetics of ethanol in the maternal-fetal unit and the amniotic fluid for the bladder-cannulated fetal preparation were similar to the data for the nonbladder-cannulated preparation. The data indicate that fetal urinary excretion of ethanol is a secondary route of ethanol transfer into the amniotic fluid. It would appear that diffusion of ethanol across membranes from the maternal and fetal circulations is a major route of ethanol transfer into this intrauterine compartment.     

A model of diffusion and mass flow of water in cylindrical membrane systems with application to plant roots

Summary Measurements of the radial diffusion of tritiated water, combined with axial and radial flow in an artificial cylindrical membrane, are examined with the aid of a mathematical model. The results are used to assess how far measurement of diffusion of labeled water in plant roots may throw light on pathways of movement of water and on barriers to flow.     

Movement of Water and Solutes in Sieve Tubes of Willow in Response to Puncture by Aphid Stylets. Evidence against a mass flow of solution

Experiments are described in which bark strips of willow were sealed to polythene tubes having two compartments. This allowed investigations to be made of the transport along the sieve tubes of tritiated water, ¹⁴C-labelled sugars, and ³²P-phosphates from one compartment, towards a stylet situated in the bark over the other compartment. Although activity from both ¹⁴C and ³²p was detected in the stylet exudate usually within 1 hour from isotope application, tritium activity was never detected even after a period of 8 hours in most experiments, though in certain cases, very low activities were detected after 4 hours. Subsequent experiments in which stylets were sited over both compartments showed that tritium activity moved laterally into the punctured sieve element more rapidly than either ¹⁴C or ³²P. Experiments using both live and dead bark in which stylets were not employed, showed that within 4 hours tritium activity had moved by diffusion along the whole length of a bark strip, therefore after this time tritium activity could have moved into the stylet exudate by a diffusional process. The lack of rapid longitudinal movement of tritiated water along the sieve tubes, indicates that the transport process is unlikely to be a mass flow of solution.     

Determination of the membrane permeability coefficient and the reflection coefficient by the two-dimensional laminar flow model for intestinal perfusion experiments

We performed single perfusion experiments in the small intestine of rats in order to prove that the two-dimensional laminar flow model is suitable to determine the membrane permeability coefficient and the reflection coefficient. We used progesterone as an aqueous-diffusion-limited drug, urea as a membrane transport-limited drug and the tritiated water as an intermediate substance. The membrane permeability coefficient for progesterone was calculated to be 3.6 X 10(-4) cm/s. This value did not change when the thickness of the aqueous diffusion layer was altered by increasing the perfusion rate 10-fold. It was directly demonstrated that the two-dimensional laminar flow model was suitable to analyze the data of intestinal perfusion experiments. Membrane permeability coefficients for urea and tritiated water were determined to be 3.4 X 10(-5) cm/s and 8.9 X 10(-5) cm/s, respectively. In the presence of water absorption with the hypotonic perfusion solution, the reflection coefficient for urea was 0.84. This value is thought to be theoretically reasonable, suggesting the usefullness of the two-dimensional laminar flow model to obtain the reflection coefficient in the intestinal membrane.     

Electric potential differences in the isolated guinea-pig placenta

Knowledge of the magnitude of the electric potential differences between the maternal and fetal circulations and the trophoblast is necessary to describe transport of ions into and out of the trophoblast as it occurs in placental transfer of charged molecules. The value of the electric potential difference is also of significance in describing the transport of neutral molecules when their transport is coupled to electrogenic co-transport systems. We developed a method to obtain the values of these potential differences, in the isolated guinea-pig placenta perfused on both sides with an artificial medium. A positively charged ion that carries a radioactive label is allowed to equilibrate between the trophoblast and its circulations. The intracellular equilibrium concentration can be calculated and, because the extracellular concentration is known, the potential difference can be obtained with the Nernst equation. Rapid equilibrium is obtained by charging the trophoblast by means of perfusion of the placenta with the ion at a high concentration, followed by reduction of the concentration in the medium until equilibrium is observed. This is done in both a continuous and discontinuous manner. In addition to measurements of the potential differences, their origin was investigated. It was shown that at least part of the potential difference is generated by the action of transcellular Na--K exchange, because depolarization could always be obtained by decreasing the transmembrane Na and K gradients. Mean values obtained were delta psi F = 71 +/- 21 mV (+/- SD) for the potential difference between the fetal circulation and the trophoblast and delta psi m = 64 +/- 16 mV for the potential difference between the maternal side and the trophoblast with the cell interior negative.     

Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate

Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (3H-test and 14C-extracellular marker) technique. [3H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible 3H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [3H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [3H]folate or [3H]methotrexate in either direction was not detectable within 5-6 min. At the brush-border side (maternal) uptake of [3H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells.     

The effect of maternal fluid intake on the volume and composition of fetal urine

The effects on fetal renal function of restricting maternal water intake to 1 l/day for 6 days was investigated in 7 chronically-catheterized fetuses (gestation age 118-131 days). Restriction of water intake caused a significant decrease in maternal urine flow rate and significant increases in maternal plasma and urinary osmolality. Fetal renal function was investigated on the third and sixth days of the period of restricted maternal intake of water. Urine flow rate from the fetus was depressed significantly, and urinary osmolality increased significantly. The glomerular filtration rate remained unchanged, and free water clearance was decreased. These changes indicate increased water reabsorption in the distal parts of the nephron, probably consequent upon increased circulating levels of antidiuretic hormone. In 3 fetuses whose mothers subsequently had free access to water, these changes in urine flow rate and free water clearance that occurred during water restriction were reversed. There was an inverse correlation between maternal plasma osmolality and fetal free water clearance corrected for glomerular filtration rate. It is concluded that when water intake by a pregnant animal is restricted, the availability of water to the fetus is reduced and fetal sheep respond by producing a concentrated urine.     

Transplacental carbohydrate and sugar alcohol concentrations and their uptakes in ovine pregnancy

The concentrations of glucose, fructose, sorbitol, glycerol, and myo-inositol in sheep blood and tissues have been reported previously (1--5). However, the other polyols that are at low concentrations have not been investigated in pregnant sheep due to technical difficulties. By using HPLC and gas chromatography-mass spectrometry, seven polyols (myo-inositol, glycerol, erythritol, arabitol, sorbitol, ribitol, and mannitol) and three hexoses (mannose, glucose, and fructose) were identified and quantified in four blood vessels supplying and draining the placenta (maternal artery, uterine vein, fetal artery, and umbilical vein). Uterine and umbilical blood flows were measured, and uptakes of all the polyols and hexoses in both maternal and fetal circulations were calculated. There was a significant net placental release of sorbitol to both maternal and fetal circulations. Fructose was also taken up significantly by the uterine circulation. Maternal plasma mannose concentrations were higher than fetal concentrations, and there was a net umbilical uptake of mannose, characteristics that are similar to those of glucose. Myo-inositol and erythritol had relatively high concentrations in fetal plasma (697.8 plus minus 53 microM and 463.8 plus minus 27 microM, respectively). The ratios of fetal/maternal plasma arterial concentrations were very high for most polyols. The concentrations of myo-inositol, glycerol, and sorbitol were also high in sheep placental tissue (2489 plus minus 125 microM/kg wet tissue, 2119 plus minus 193 microM/kg wet tissue, and 3910 plus minus 369 microM/kg wet tissue), an indication that these polyols could be made within the placenta.     

Plasma levels of vitamin D metabolites in fetal and pregnant ewes

The plasma concentrations of calcium; inorganic phosphorus; 25-hydroxyvitamin D; 24,25-dihydroxyvitamin D; and 1,25-dihydroxyvitamin D were determined in sheep maternal and fetal arterial circulations. In addition, plasma concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were determined simultaneously across the uterine and umbilical circulations. Fetal arterial levels of calcium (r = 0.560); inorganic phosphorus (r = -0.095); and 1,25-dihydroxyvitamin D (r = 0.040) were significantly higher than and did not correlate with maternal arterial levels. Maternal levels of 25-hydroxyvitamin D were significantly higher than and correlated (r = 0.693) with fetal 25-hydroxyvitamin D levels. No significant difference existed between maternal and fetal arterial levels of 24,25-dihydroxyvitamin D. No significant difference was detected in the concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D across the uterine or umbilical circulations.     

Distinct composition of human fetal HDL attenuates its anti-oxidative capacity

In human high-density lipoprotein (HDL) represents the major cholesterol carrying lipoprotein class in cord blood, while cholesterol is mainly carried by low-density lipoprotein in maternal serum. Additionally, to carrying cholesterol, HDL also associates with a range of proteins as cargo. We tested the hypothesis that fetal HDL carries proteins qualitatively and quantitatively different from maternal HDL. These differences then contribute to distinct HDL functionality in both circulations. Shotgun proteomics and biochemical analyses were used to assess composition/function of fetal and maternal HDL isolated from uncomplicated human pregnancies at term of gestation. The pattern of analyzed proteins that were statistically elevated in fetal HDL (apoE, proteins involved in coagulation, transport processes) suggests a particle characteristic for the light HDL₂ sub-fraction. In contrast, proteins that were enriched in maternal HDL (apoL, apoF, PON1, apoD, apoCs) have been described almost exclusively in the dense HDL₃ fraction and relevant to its anti-oxidative function and role in innate immunity. Strikingly, PON1 mass and activity were 5-fold lower (p < 0.01) in the fetus, which was accompanied by attenuation of anti-oxidant capacity of fetal HDL. Despite almost equal quantity of CETP in maternal and fetal HDL, its enzymatic activity was 55% lower (p < 0.001) in the fetal circulation, whereas LCAT activity was not altered. These findings indicate that maternally derived HDL differs from fetal HDL with respect to its proteome, size and function. Absence of apoA-1, apoL and PON1 on fetal HDL is associated with decreased anti-oxidative properties together with deficiency in innate immunity collectively indicating distinct HDLs in fetuses.     

Corticosteroid-binding globulin status at the fetomaternal interface during human term pregnancy

The status of the corticosteroid-binding globulin (CBG) at the fetomaternal interface, especially in the maternal intervillous blood space (I), was investigated and compared to that of CBG in the maternal (M) and fetal (umbilical arteries [A] and vein [V]) peripheral circulations at term. Immunoquantitation of plasma CBG showed that the CBG concentration in I was 30% less than that in M (P < 0.001) and threefold higher than that in umbilical cord blood (P < 0.001). The microheterogeneity of CBG studied by immunoaffinoelectrophoresis in the presence of concanavalin A and Western blotting indicated that the CBG in I was mainly of maternal origin and different from fetal CBG. A CBG mRNA, but no classic 50- to 59-kDa CBG, was found in isolated term trophoblastic cells. The steroid environment of the CBG in I differed greatly from that in the peripheral maternal and fetal circulations, because the progesterone:cortisol molar ratio in I was 75-fold higher than that in M and 7- to 10-fold higher than that in the fetal circulation. Binding studies revealed that the affinity constants of CBG for cortisol in I, A, and V were significantly lower than that in M plasma (P < 0.02) in their respective hormonal contexts. The binding parameters for I-CBG stripped of endogenous steroids and lipids were close to those for M-CBG but different from those of fetal CBG (P < 0.001). These data reflect the physiological relevance of the CBG-steroid interaction, especially with very CBG-loaded progesterone at the fetomaternal interface during late pregnancy.     

Fetal perfusion with maternal blood; a possible sequel to fetal catheterization in the pig

Plasma gamma-globulin concentrations were increased in three catheterized pig fetuses in which the packed red cell volume rose rapidly after surgery, but were not increased in one fetus in which the packed cell volume remained normal. The increases in packed cell volume and gamma-globulin concentration were closely correlated. The source of the gamma-globulin was investigated in one sow with catheterized fetuses. Maternal proteins, identified by [125I]-labelling and gel diffusion, crossed to the circulation of the one catheterized fetus with an elevated packed cell volume but not to the other, in which packed cell volume remained normal, nor to two unoperated fetuses. The results suggest that vascular anastmosis between fetal and maternal circulations is a possible sequel to cannulation in this species.     

Expression of members of the multidrug resistance protein family in human term placenta

The placenta serves, in part, as a barrier to exclude noxious substances from the fetus. In humans, a single-layered syncytium of polarized trophoblast cells and the fetal capillary endothelium separate the maternal and fetal circulations. P-glycoprotein is present in the syncytiotrophoblast throughout gestation, consistent with a protective role that limits exposure of the fetus to hydrophobic and cationic xenobiotics. We have examined whether members of the multidrug resistance protein (MRP) family are expressed in term placenta. After screening a placenta cDNA library, partial clones of MRP1, MRP2, and MRP3 were identified. Immunofluorescence and immunoblotting studies demonstrated that MRP2 was localized to the apical syncytiotrophoblast membrane. MRP1 and MRP3 were predominantly expressed in blood vessel endothelia with some evidence for expression in the apical syncytiotrophoblast. ATP-dependent transport of the anionic substrates dinitrophenyl-glutathione and estradiol-17-beta-glucuronide was also demonstrated in apical syncytiotrophoblast membranes. Given the cellular distribution of these transporters, we hypothesize that MRP isoforms serve to protect fetal blood from entry of organic anions and to promote the excretion of glutathione/glucuronide metabolites in the maternal circulation.