Production and utilization of amino acids by ovine placenta in vivo

Misoo Chung, Cecilia Teng, Michelle Timmerman, Giacomo Meschia, and Frederick C. Battaglia

Division of Perinatal Medicine, Departments of Physiology, Obstetrics-Gynecology and Pediatrics, University of Colorado School of Medicine, Denver, Colorado 80262

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Uterine and umbilical uptakes of plasma amino acids were measured simultaneously in eighteen singleton pregnant ewes at 130 ± 1 days gestation for the purpose of establishing which amino acids are produced or used by the uteroplacenta under normal physiological conditions and at what rates. The branched-chain amino acids (BCAA) had uterine uptakes significantly greater than umbilical uptakes. Net uteroplacental BCAA utilization was 8.0 ± 2.5 µmol · kg fetus-1 · min-1 (P < 0.005) and represented 42% of the total BCAA utilization by fetus plus uteroplacenta. There was placental uptake of fetal glutamate (4.2 ± 0.3 µmol · kg fetus-1 · min-1, P < 0.001) and no uterine uptake of maternal glutamate. Umbilical uptake of glutamine was ~61% greater than uterine uptake, thus demonstrating net uteroplacental glutamine production of 2.2 ± 0.9 µmol · kg fetus-1 · min-1 (P < 0.021). In conjunction with other evidence, these data indicate rapid placental metabolism of glutamate, which is in part supplied by the fetus and in part produced locally via BCAA transamination. Most of the glutamate is oxidized, and some is used to synthesize glutamine, which is delivered to the fetus. There was net uteroplacental utilization of maternal serine and umbilical uptake of glycine produced by the placenta. Maternal serine utilization and glycine umbilical uptake were virtually equal (3.14 ± 0.50 vs. 3.10 ± 0.46 µmol · kg fetus-1 · min-1). This evidence supports the conclusion that the ovine placenta converts large quantities of maternal serine into fetal glycine.

fetus; uptakes; placental metabolism; branched-chain amino acids; serine; glycine; glutamate; glutamine

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AMINO ACID UPTAKE by the pregnant uterus and by the fetus is a vital aspect of maternal and fetal nutrition. Uterine uptake defines amino acid supply to both the placenta and fetus by the maternal organism, whereas umbilical uptake defines amino acid supply to the fetus. Therefore, the comparison of uterine and umbilical amino acid uptakes provides basic information about uteroplacental amino acid metabolism in vivo. Uterine and umbilical uptakes of individual amino acids are not necessarily equal, because amino acids can be produced or used by the uteroplacental tissues. Although these tissues include the myometrium, there is evidence that their metabolic activity represents primarily placental metabolism, since at least 80% of uteroplacental glucose utilization is by tissues that exchange glucose with the umbilical circulation (20).

A test of the hypothesis that placental metabolism plays a physiologically important role in determining which amino acids are supplied to the fetus and at what rates requires simultaneous measurements of uterine and umbilical blood flows and of amino acid concentration differences across the uterine and umbilical circulations. Although such measurements have been feasible in sheep for more than a decade, there has been only one previous study attempting to quantify uterine and umbilical amino acid uptakes simultaneously in a chronic sheep preparation (15). This and other studies in which uterine (11, 23) and umbilical (4, 13, 14) amino acid uptakes were measured in separate animals have shown that whole blood amino acid concentration differences across the uterine and umbilical circulations are relatively small, hence difficult to measure. An additional difficulty is that fetal sheep blood contains some compounds, e.g., methionine sulfone and N-methyl-lysine, which are in high concentration and may interfere with the chromatographic separation of physiologically important amino acids. Thus, in the previous comparison of uterine and umbilical uptakes (15), glycine concentrations were not measured despite the significant contribution that glycine makes to umbilical uptake of amino acids (7).

One reason for the relatively small whole blood concentration differences of amino acids across the uterine circulation is that the amino acids carried by adult ovine red blood cells do not contribute significantly to the rapid amino acid exchange between body organs and circulation (16). The exchange is virtually limited to the plasma compartment. Therefore, the accuracy of uterine amino acid uptake estimate can be improved by calculating uterine uptake as the product of uterine arteriovenous plasma concentration differences times uterine plasma flow, provided that plasma is separated from red blood cells within a few minutes of sampling. At the onset of this study, we had evidence that the umbilical uptake of glutamate is best estimated as the product of umbilical venoarterial plasma concentration difference times umbilical plasma flow (22), but it was not clear whether this method of calculation could be extended to other fetal amino acids.

In the first part of this study, we present in vitro evidence that the exchange of amino acids between fetal red blood cells and plasma is sufficiently slow to allow the use of plasma concentrations and plasma flows for estimating umbilical amino acid uptakes. In the second part, we present the results of simultaneous measurements of uterine and umbilical amino acid uptakes in 18 pregnant sheep, using the plasma concentration difference times plasma flow calculation. These measurements demonstrate significant placental metabolism of several amino acids.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In Vitro Study

This in vitro study was done to determine whether a decrease in plasma amino acid concentrations causes a net flux of amino acids from red blood cells to plasma. Fetal sheep blood (131 days gestation) was drawn in a syringe containing EDTA. The hematocrit was measured and used to calculate how much plasma should be withdrawn from the blood to increase its hematocrit to ~50%. The blood was centrifuged briefly, the calculated amount of plasma was taken out, and the red blood cells were mixed again with the remaining plasma. Duplicate 3-ml samples of this high-hematocrit blood were incubated at two different temperatures, 0 and 38°C. At time 0, each blood sample was mixed with 1 ml of isotonic saline having the same temperature as the sample. The saline contained known quantities of [1-14C]glutamic acid and Evans blue dye as two internal indicators for calculating plasma volume before and after dilution. One-milliliter aliquots of the diluted blood were removed at 0.5, 2, and 5 min, stored in ice, and then centrifuged for 10 min in a refrigerated centrifuge. The diluted plasma samples were used to measure the concentrations of [1-14C]glutamic acid, Evans blue dye, and amino acids. Three-milliliter undiluted plasma samples were carried through the same incubation routines as the blood samples and were used to measure the initial amino acid concentrations. These concentrations and the changes in plasma volume were used to calculate expected amino acid plasma concentration changes in the absence of any appreciable amino acid efflux from the red blood cells.

In Vivo Study

Surgery and animal care. Eighteen pregnant Columbia-Rambouillet sheep were studied. Each carried a single fetus. Animals underwent laparotomy at 120-127 days of gestation after a 48-h fast with free access to water. Surgery was performed under a combination of tetracaine spinal anesthesia and pentobarbital sodium sedation. Polyvinyl catheters were placed in the uterine veins draining each uterine horn. After the uterus was opened, catheters were inserted in the fetal pedal artery and vein and in the common umbilical vein. An amniotic catheter was also placed for the injection of antibiotics in the amniotic cavity. The uterus and the abdominal incisions were closed, after which catheters were placed in the maternal femoral artery and vein. All catheters were tunneled subcutaneously to a pouch on the ewe's flank. Animals received perioperative antibiotics, 500 mg of ampicillin and 500 mg of gentamycin, intramuscularly. The day of surgery and the first 3 days after surgery, 500 mg of ampicillin were administered into the amniotic cavity via the amniotic catheter. Analgesics were given to the ewe 2 days postoperatively. All catheters were flushed daily with a solution of heparin in normal saline. The ewes were allowed to recover until the intake of alfalfa pellets and water was normal (at least 5 days). All studies were approved by the University of Colorado Health Sciences Center Animal Care and Use Committee.

Study design. Studies were performed at 125-134 days of gestation. Blood samples were drawn from the maternal artery, uterine veins, fetal pedal artery, and umbilical vein for tritiated water concentration, oxygen saturation, glucose concentration, lactate concentration, and amino acid concentrations as baseline data. Then, a constant tritiated water infusion was started into the fetal pedal vein. Sixty minutes after the start of the infusion, at steady state, four sets of samples were collected simultaneously from the maternal artery, uterine veins, fetal pedal artery, and the common umbilical vein. All samples were analyzed for tritiated water, hemoglobin, hematocrit, O2 saturation, glucose, lactate, and amino acid concentrations. At the end of the experiment, the ewe and fetus were euthanized by intravenous injection (Sleepaway, Fort Dodge, IA). Autopsy was performed to obtain fetal, placental, and uterine weights.

Analytic methods. Hemoglobin and O2 saturation were measured spectrophotometrically (OSM-3, Radiometer, Copenhagen, Denmark). The blood O2 content was calculated from the hemoglobin content expressed as O2 capacity multiplied by the O2 saturation. Plasma 3H2O was measured on triplicate aliquots in a scintillation counter and converted to blood 3H2O on the basis of hematocrit measurement. Glucose and lactate concentrations were measured in duplicate by means of a glucose-lactate analyzer (Yellow Springs Instruments model 2700 Select and Dual Standard). Plasma samples for amino acid concentrations were frozen at -70°C within 5 min until the day of analysis, at which time they were thawed quickly and deproteinized with 15% sulfosalicylic acid containing 0.3 µmol/l norleucine as internal standard. The pH was adjusted to 2.2 with 1.5 N LiOH. After centrifugation, the supernatant was analyzed with a Dionex high-performance liquid chromatography (HPLC) amino acid analyzer (Dionex, Sunnyvale, CA). The same HPLC column was used for all samples from an individual animal. Reproducibility within the same column had a mean value of ±2%. Samples from all vessels drawn simultaneously were loaded to run within 12 h. Amino acid concentrations were measured after reaction with ninhydrin at 570 nm except for proline, which was measured at a wavelength of 440 nm.

Calculations

Blood flows, uptakes, and accretion rate. Umbilical (bfumb) and uterine (bfutn) blood flows were calculated using the steady-state transplacental diffusion method, with tritiated water as the blood flow indicator (20). Umbilical amino acid uptakes were calculated as the product of umbilical plasma flow [pfumb = bfumb × (1 - fractional fetal hematocrit)] and the plasma concentration differences of each amino acid (Delta aa) between umbilical vein (gamma ) and umbilical artery (alpha ). Similarly, uterine uptakes were calculated using uterine plasma flow [pfutn = bfutn × (1 - fractional maternal hematocrit)] and the plasma concentration differences (Delta aa) between maternal artery (a) and uterine vein (v)
Umbilical uptake = pf<SUB>umb</SUB> × (&Dgr;aa)<SUB>&ggr;-&agr;</SUB>
Uterine uptake = pf<SUB>utn</SUB> × (&Dgr;aa)<SUB>a-v</SUB>
The umbilical uptake of each amino acid was compared with its normal fetal accretion rate. The fetal accretion rate was calculated from data on amino acid concentration per gram of nitrogen of the fetal carcass (19) and an estimated fetal nitrogen accretion rate of 848 mg N · kg fetus-1 · day-1. This estimate is the product of N content per kilogram of fetus (21 g/kg) and fractional rate of N accretion (0.0404 day-1) at 130 days of gestation. Both N content and fractional accretion rate were derived from measurements of fetal weight and body composition at different gestational ages (3). Note that the N fractional accretion rate is greater than the fractional fetal growth rate (~0.032) because N content per unit wet weight increases with gestation (3).

Statistics

All data are expressed as means ± SE. Differences between two groups (maternal vs. fetal, uterine vs. umbilical, and so forth) are tested using Student's t-test for paired samples. Two-tailed values were considered significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In Vitro Study

The degrees of plasma dilution calculated on the basis of [1-14C]glutamate and Evans blue dye were virtually identical and were expected to cause a decrease in plasma amino acid concentrations to 62.6% of initial value. Initial and postdilution average hematocrits were 44 and 33%, respectively. With the exception of alanine, the observed concentrations did not deviate significantly from expected and showed no detectable change between the 0.5-, 2-, and 5-min samples. Furthermore, the observed concentrations at two different temperatures were not significantly different from one another. The alanine data indicated a significant (P < 0.02) efflux of this amino acid from red blood cells at 38°C. The expected alanine concentration change was 192 µM (514-322 µM). At 38°C, the observed change was 188 µM at 2 min and 174 µM at 5 min. This is not a rapid efflux. On the assumption that the rate of efflux is proportional to the plasma concentration change, failure to cool and centrifuge the blood for 5 min after sampling would cause an approximate 10% underestimate in the umbilical uptake of alanine. On the basis of these results, we decided to calculate all amino acid uptakes as the product of plasma concentration differences times plasma flows.

In Vivo Study

The eighteen animals had normal values of fetal and placental weight, uterine and umbilical blood flows, hematocrit, hemoglobin, O2 saturations, maternal and fetal glucose and lactate concentrations, and uterine and umbilical uptakes of O2, glucose, and lactate (3). These data are summarized in Tables 1 and 2.

                              
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Table 1.   Gestational age, fetal and placental weights, and uterine and umbilical blood flows

                              
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Table 2.   Data on oxygen, glucose, and lactate metabolism

Mean concentrations of amino acids in maternal arterial, uterine venous, umbilical arterial, and umbilical venous plasma are presented in Table 3. The table also presents the mean percent concentration changes across the uterine and umbilical circulations for each amino acid. Each change was calculated using the highest value of the arteriovenous difference as the reference value. For example, the umbilical percent change of glutamine was calculated as 100(gamma  - alpha )/gamma and the percent change of glutamate as 100(gamma  - alpha )/alpha . All uterine concentration changes were <11%. Changes across the umbilical circulation were more variable. Extraction of fetal plasma glutamate by the placenta created a 72% concentration change across the umbilical circulation. Figure 1 compares the maternal and fetal arterial plasma amino acid concentrations. Among the essential amino acids, valine, threonine, phenylalanine, and methionine had significantly higher fetal concentrations. Among the nonessential amino acids, fetal serine concentration was nine times higher than maternal concentration.

                              
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Table 3.   Amino acid concentrations in maternal arterial, uterine venous, umbilical arterial, and umbilical venous plasma


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Fig. 1.   Comparison between maternal and fetal arterial amino acid plasma concentrations. Bar graphs are means ± SE. Statistically significant differences between maternal and fetal concentrations: * P < 0.05, ** P < 0.01, *** P < 0.001. P values were determined by Student's t-test for paired samples.

Figure 2 presents the uterine and umbilical uptakes expressed as micromoles per kilogram of fetus per minute. Both uptakes are also expressed in nitrogen equivalents (mg N · kg fetus-1 · day-1) in Table 4. Both the uterine and umbilical uptakes of all essential amino acids were significantly greater than zero. The umbilical uptakes of glutamate and serine were negative, i.e., there were uptakes of fetal glutamate and serine by the placenta. There was a small efflux of glutamate into the uterine circulation that was of borderline significance (P = 0.046). The uterine uptake of glycine was not significantly different from zero. Uterine uptakes normalized for maternal arterial concentration, i.e., expressed as plasma clearances, are shown in Fig. 3. The data are grouped into neutral, basic, and acidic amino acids to emphasize the role of amino acid transporters in uterine uptake (see DISCUSSION).


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Fig. 2.   Comparison between uterine and umbilical uptakes of amino acids. Bar graphs are means ± SE. Statistically significant differences between uterine and umbilical uptakes: * P < 0.05, ** P < 0.01, *** P < 0.001. P values were determined by Student's t-test for paired samples.

                              
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Table 4.   Amino acid nitrogen uptakes and food nitrogen intake


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Fig. 3.   Bar graphs are means ± SE. Statistically significant differences from 0: * P < 0.05, ** P < 0.01, *** P < 0.001. 

Eight amino acids demonstrated a significant difference between uterine and umbilical uptakes. The net uteroplacental utilization rates of these amino acids, normalized for placental weight, are presented in Fig. 4. Serine had the highest net utilization rate and glycine the highest net production rate. Most of the serine used was maternal serine, and most of the glycine produced was delivered into the umbilical circulation. There was net uteroplacental utilization of the branched-chain amino acids (BCAA), i.e., valine, leucine, and isoleucine taken up from the maternal circulation, and of the glutamate taken up from the umbilical circulation. Uteroplacental utilization of BCAA represented ~40% of their uterine uptake. Concomitant with BCAA and glutamate utilization, there was net glutamine production, so that umbilical glutamine uptake included glutamine synthesized by the placenta. Finally, there was statistical evidence for a relatively small uteroplacental production of methionine. However, the significance of this evidence was not strong (P = 0.047).


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Fig. 4.   Uteroplacental utilizations that were significantly different from zero, expressed per 100 g placenta and ranked by magnitude (negative utilization = production). Bar graphs are means ± SE. Levels of statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001. P values were determined by Student's t-test for paired samples.

Umbilical uptake of each essential amino acid was in excess of its estimated normal fetal accretion rate (Fig. 5). Among the nonessentials, umbilical arginine uptake was remarkably large. It was almost three times the estimated accretion and made the largest contribution to the umbilical uptake of amino acid nitrogen (Table 4). The combined glutamine and arginine uptakes represented ~45% of the total amino acid nitrogen delivered by the placenta to the fetus.


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Fig. 5.   Comparison between mean umbilical uptakes and estimated normal fetal accretions of amino acids. For pairs of amino acids (glutamine + glutamate and asparagine + aspartate) individual accretion rates are not known.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This discussion addresses two separate but related issues. The first is the contribution of red blood cells to the exchange of amino acids between the placenta and the uterine and the umbilical circulations. The second issue, which is the primary focus of the study, addresses the conclusions that can be drawn about placental amino acid metabolism from the simultaneous measurement of uterine and umbilical amino acid uptakes.

Plasma-Red Blood Cell Exchange

Normal adult sheep erythrocytes have a sodium-dependent transport system for neutral and basic amino acids (31). Although this transport system is important for erythrocyte viability (31), it transports amino acids quite slowly in comparison with the rate of amino acid exchange between blood and tissues. The characteristics of erythrocyte amino acid transport change during development, as shown by the finding that reticulocytes possess sodium-dependent glycine transporters, which are lost during maturation (5). Nevertheless, fetal and adult erythrocytes do share some properties, such as their impermeability to glutamate (28). The present study shows that a large decrease in the concentration of fetal plasma amino acids is not followed by rapid, net transport of neutral and basic amino acids from red blood cells to plasma. This in vitro evidence indicates that there is no mechanism in either adult or fetal red blood cells for the fast loading and unloading of amino acids that would make them suitable for amino acid shuttling among organs.

An alternative approach to the study of the role of erythrocytes in amino acid exchange is a comparison of plasma uptakes (i.e., plasma concentration differences × plasma flow) with blood uptakes (i.e., whole blood concentration differences × blood flow) across the circulation of different organs. If erythrocytes do not contribute to the exchange, plasma and blood uptakes should be equal. According to a recent study, there is no significant difference between plasma and blood uptakes of most amino acids across the splanchnic and hepatic circulations of sheep. These data support the conclusion that there is no major involvement of erythrocytes in the amino acid exchange between blood and tissues (16). In vivo data do not lend themselves to a more precise conclusion because the comparison of plasma and blood uptakes depends on the accuracy of comparing small percent changes in concentration.

A consequence of amino acid uptake being limited primarily to plasma amino acids is that, at normal hematocrits, plasma concentration differences across organs are ~1.5 times the whole blood concentration differences. For this reason plasma differences can be determined more precisely. An additional consideration is that the transporter molecules located on the maternal and fetal surfaces of the placenta are exposed to plasma amino acid concentrations. Plasma concentration measurements allow one to estimate transport rates in relationship to the concentrations that are relevant to transport. A theoretical possibility that has been discussed in the physiological literature (16) is direct amino acid exchange between erythrocytes and the intracellular amino acid pools of some organs. However, evidence for such an exchange has been difficult to confirm. It presupposes a close tissue-red blood cell interaction for which there is no known mechanism. Another theoretical concern is that amino acid uptake calculations assume no production of free amino acids within the blood as it circulates through the organ. This assumption would not be the case if the organ stimulated rapid hydrolysis of blood proteins and peptides. In practical terms, measurements of uterine and umbilical plasma amino acid uptakes provide the best quantitative evidence about rates of placental amino acid transport and metabolism in vivo. Given the complexity of the placental system with its perfusion by two different circulations, this evidence needs to be considered in the light of all the available information.

Net Placental, Uterine, and Umbilical Uptakes

Uterine and umbilical uptakes of amino acids can be examined with emphasis on either metabolic pathways or transport systems. From the metabolic viewpoint, an obviously important distinction is between essential and nonessential amino acids. In the present study, all the essentials demonstrated both uterine and umbilical uptakes, with each umbilical uptake being ~1.8-3.3 times greater than the estimated normal accretion rate. This finding establishes significant fetal catabolism of all the essential amino acids. Fetal oxidation of leucine (17, 25), threonine (1), and lysine (18) has been verified by tracer methodology. Among the essentials, the three BCAA form a unique group. In the present study, they had the highest uterine uptake and demonstrated net uteroplacental utilization. Uteroplacental utilization of BCAA had been demonstrated in the previous study in which uterine and umbilical blood uptakes were compared (15). The ovine placenta releases into the fetal and maternal circulations the keto acids, which are formed in BCAA deamination (17, 26). Because BCAA deamination results in the formation of glutamate via transamination with alpha -ketoglutarate, it is likely that placental uptake of fetal glutamate and uteroplacental BCAA utilization represent two separate mechanisms for supplying glutamate to the placenta. Previous studies from our laboratory have demonstrated that ~70% of the fetal glutamate taken up by the placenta is rapidly oxidized (22) and that the placenta excretes ammonia (4, 10). The present study shows for the first time significant net glutamine production by the placenta, in agreement with the evidence that glutamine synthetase is present in this organ (24). Some of the glutamine delivered to the fetus by the placenta is converted back to glutamate by the fetal liver, which produces most of the glutamate consumed by the placenta (28). This establishes a glutamate-glutamine shuttle, which promotes oxidation of glutamate in the placenta and fetal hepatic utilization of the amide group of glutamine. We may infer from the present set of observations that the stoichiometry of placental glutamate supply and utilization, under normal physiological conditions, is roughly as follows. BCAA deamination and placental uptake of fetal glutamate supply the placenta with ~11.2 µmol · kg fetus-1 · min-1 of glutamate (8.0 from BCAA deamination and 3.2 from glutamate net uptake). Approximately 2.2 µmol of the glutamate combine with ammonia to produce glutamine, which is delivered to the fetus. The remainder (~9 µmol) becomes available for oxidation and provides the placenta with ~6.8 µmol of ammonia nitrogen (i.e., 9.0-2.2), which can be channeled into either synthetic processes via additional glutamine formation or excretion. From separate measurements of uterine (10) and umbilical (4) ammonia uptakes, it can be estimated that total ammonia excretion by the late gestation ovine placenta is ~10 µmol · kg fetus-1 · min-1, with most of the excretion being into the uterine circulation. Late gestation umbilical uptake of ammonia nitrogen is ~2 µmol · kg fetus-1 · min-1 (4). Placental glutamate oxidation is likely to serve several functions. Glutamate output by the fetal liver allows the fetus to uncouple the oxidation of glucogenic amino acids from gluconeogenesis (22). Glutamate oxidation by placental mitochondria generates NADPH and therefore plays a role in biosynthetic reactions such as steroidogenesis (12). The role of BCAA in placental glutamate metabolism is outlined in Fig. 6.


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Fig. 6.   Schematic diagram of contribution of branched-chain amino acid (BCAA) deamination and fetal glutamate uptake to placental glutamate (GLU). Oxidation of placental glutamate produces ammonia, which can be used either in synthesis of glutamine (GLN) or excreted into uterine and umbilical circulations. Values are in µmol · kg fetus-1 · min-1. alpha KG, alpha -ketoglutarate; alpha KA, branched-chain keto acids. Fetal uptake of placental glutamine (2.2 µmol · kg fetus-1 · min-1) is likely to underestimate placental glutamine synthetic rate because placenta is likely to use amide group of glutamine.

Placental conversion of maternal and fetal serine into glycine has been demonstrated by experiments of serine tracer infusion into the maternal (21) and fetal (8) circulations. In the present study, umbilical glycine uptake was represented entirely by glycine produced within the placenta; all of the serine taken up by the pregnant uterus was used by the uteroplacental mass. Uterine serine uptake could account for most of the placental glycine production. In conjunction with the tracer studies, these data point to the conclusion that one of the major metabolic functions of the ovine placenta is glycine production from serine and that the major source of placental glycine is maternal serine. The conversion of serine to glycine channels the beta -carbon of serine via the production of methylenetetrahydrofolate into synthetic reactions that require activated one-carbon units, such as purine synthesis. Because purine synthesis also requires the utilization of glycine and the amide group of glutamine, it represents an aspect of placental metabolism that could link together in a common function all the major findings about placental amino acid production and utilization. Measurements of placental nucleotide metabolic rates in vivo are needed to assess the quantitative importance of this linkage.

An unexpected finding was a small but statistically significant production of methionine, an essential amino acid, by the placenta. Sheep plasma contains methionine sulfone and methionine sulfoxide, which suggests that reduction of methionine sulfoxide to methionine within the ovine placenta may be a source of fetal methionine. Methionine sulfoxide can be used instead of methionine to sustain the growth of weanling rats (2).

It is worth pointing out that amino acids may participate in important metabolic reactions within the placenta without showing net placental utilization or production, either because the reaction rates are not sufficiently rapid or because utilization and production go on simultaneously. For example, the placenta is capable of producing nitric oxide and citrulline from arginine (6). One possible outcome of this reaction would be net arginine utilization and net citrulline production similar to that described for the small bowel (30). However, uterine and umbilical uptakes of arginine were virtually equal. The net uteroplacental utilization of citrulline that was found was of borderline significance (P = 0.06). This suggests that there may be approximately equal rates of citrulline production from arginine and arginine synthesis from citrulline, a hypothesis that requires tracer studies for confirmation.

The catabolism of amino acids in both the placenta and fetus make the nitrogen requirements of the pregnant uterus considerably greater than the requirements estimated from fetal accretion. In the present study, the nitrogen uptake represented by the combined uptake of all the measured amino acids was ~1,568 mg N · kg fetus-1 · day-1 for the uterus and 1,191 mg N · kg fetus-1 · day-1 for the fetus. These uptakes are ~84 and 40% greater than the estimated 848 mg N · kg fetus-1 · day-1 fetal accretion rate. Uterine nitrogen uptake was also a large fraction, approximately one-third, of the maternal nitrogen dietary intake. The 377 mg N · kg fetus-1 · day-1 difference between uterine and umbilical amino acid nitrogen uptakes is somewhat higher than the uteroplacental ammonia excretion of ~200 mg N · kg fetus- · day-1, estimated from measurements of uterine (10) and umbilical (4) ammonia uptakes. It is uncertain whether this discrepancy represents mostly experimental error or as yet unidentified placental utilization of amino acid nitrogen.

Transport mechanisms interact with metabolic activity in providing the amino acid uptakes that are measured in vivo. The relationship between amino acid transport by the maternal circulation and uterine amino acid uptake is defined by the percent concentration changes of amino acids across the uterine circulation, which represent the uptake-to-circulatory supply ratios. As demonstrated in Table 3, these ratios were quite small for each amino acid, including those with the highest uptakes. Therefore, under normal physiological conditions, transport by the maternal circulation is not a limiting factor for the uterine uptake of amino acids. The uterine clearance of neutral amino acids was variable. This variability is probably related to the presence of several transport systems for neutral amino acids on the maternal surface of the placenta, each having highest affinity for different amino acids (27). The virtual absence of glycine uptake by the pregnant uterus, despite a very high maternal plasma glycine concentration, suggests that the sodium-dependent glycine system is not expressed in the ovine placenta.

Placental transport systems for cationic amino acids have not been studied in detail. The sodium-independent y+ system, which does not discriminate between arginine and lysine, has been found on the basal membrane of the human trophoblast (27). The present study shows relatively high and similar uterine clearances of ornithine, arginine, and lysine, with transport of arginine and lysine into the fetal circulation.

The absence of glutamate uptake by the ovine pregnant uterus is puzzling because a sodium-dependent transporter for glutamate on the maternal surface of the human trophoblast has been reported (27). This led to the prediction that there is placental uptake of maternal glutamate. The finding that the ovine placenta does not verify this prediction may reflect a species difference. Another possibility is that, within the population of cells exchanging glutamate between pregnant uterus and maternal blood, uptake by one group is masked by efflux from another group.

The placental uptake of glutamate and serine from the fetal circulation presupposes the presence on the fetal surface of the placenta of transporters, which pump these amino acids into the placenta from the umbilical circulation. A sodium-dependent, high-affinity, electrogenic transporter for anionic amino acids, which resembles the X<SUP>−</SUP><SUB>AG</SUB> system, has been shown to be present on the basal surface of the human trophoblast (27), which is consistent with the in vivo data. Its electrogenic property suggests that transport of fetal glutamate into the placenta may serve to regulate intracellular Ca+ as has been reported for glutamate transport in pituitary cells (29). Placental uptake of fetal serine is more difficult to explain than glutamate uptake because serine shares transport systems with other neutral amino acids that have net flux from placenta to fetus. The placental uptake of fetal serine results from bidirectional serine fluxes between placenta and fetus (8). Similarly, the umbilical uptakes of leucine (25) and alanine (9) result from bidirectional fluxes, but, in contrast to serine, there is a net umbilical uptake of these amino acids. The neutral amino acid transporters on the fetal surface of the placental epithelium include sodium-dependent transport systems that promote the flux of fetal neutral amino acids into the placenta (27). The in vivo data highlight the difficulty of attempting to predict the direction of net amino acid uptake from the study of transporters in isolated placental membrane vesicles. One possible explanation consistent with both in vivo and in vitro data is that the neutral amino acid exchange between placenta and fetus involves transport systems that mediate fluxes of opposite sign and that serine interacts strongly with one or more of the sodium-dependent transport systems on the fetal surface of the placenta. The finding that the sodium-dependent ASC transport system, for which serine has relatively high affinity, is present on the fetal surface of the human trophoblast (27) supports this hypothesis. In addition, the high serine concentration of fetal plasma may promote placental uptake of fetal serine by sodium-independent exchangers via counter-transport with other amino acids.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Child Health and Human Development Grants 5-R37 HD-01866 and 5-RO1 HD-29374.

    FOOTNOTES

Address for reprint requests: F. C. Battaglia, Univ. of Colorado Health Sciences Center, Dept. of Pediatrics, 4200 East Ninth Ave., B199, Denver, CO 80262.

Received 27 May 1997; accepted in final form 4 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Anderson, A. H., P. V. Fennessey, G. Meschia, R. B. Wilkening, and F. C. Battaglia. Placental transport of threonine and its utilization in the normal and growth-restricted fetus. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E892-E900, 1997[Abstract/Free Full Text].

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3.   Battaglia, F. C., and G. Meschia. An Introduction to Fetal Physiology. Orlando, FL: Academic, 1986.

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13.   Lemons, J. A., E. W. Adcock III, M. D. Jones, Jr., M. A. Naughton, G. Meschia, and F. C. Battaglia. Umbilical uptake of amino acids in the unstressed fetal lamb. J. Clin. Invest. 58: 1428-1434, 1976[Medline].

14.   Lemons, J. A., and R. L. Schreiner. Metabolic balance of the ovine fetus during the fed and fasted states. Ann. Nutr. Metab. 28: 268-280, 1984[Medline].

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19.   Meier, P., C. Teng, F. C. Battaglia, and G. Meschia. The rate of amino acid nitrogen and total nitrogen accumulation in the fetal lamb. Proc. Soc. Exp. Biol. Med. 167: 463-468, 1981.

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21.   Moores, R. R., Jr., C. C. Rietberg, F. C. Battaglia, P. V. Fennessey, and G. Meschia. Metabolism and transport of maternal serine by the ovine placenta: glycine production and absence of serine transport into the fetus. Pediatr. Res. 33: 590-594, 1993[Abstract].

22.   Moores, R. R., Jr., P. R. Vaughn, F. C. Battaglia, P. V. Fennessey, R. B. Wilkening, and G. Meschia. Glutamate metabolism in the fetus and placenta of late-gestation sheep. Am. J. Physiol. 267 (Regulatory Integrative Comp. Physiol. 36): R89-R96, 1994[Abstract/Free Full Text].

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25.   Ross, J. C., P. V. Fennessey, R. B. Wilkening, F. C. Battaglia, and G. Meschia. Placental transport and fetal utilization of leucine in a model of fetal growth retardation. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E491-E503, 1996[Abstract/Free Full Text].

26.   Smeaton, T. C., J. A. Owens, K. L. Kind, and J. S. Robinson. The placenta releases branched-chain keto acids into the umbilical and uterine circulations in the pregnant sheep. J. Dev. Physiol. (Eynsham) 12: 95-99, 1989[Medline].

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28.   Vaughn, P. R., C. Lobo, F. C. Battaglia, P. V. Fennessey, R. B. Wilkening, and G. Meschia. Glutamine-glutamate exchange between placenta and fetal liver. Am. J. Physiol. 268 (Endocrinol. Metab. 31): E705-E711, 1995[Abstract/Free Full Text].

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31.   Young, J. A., C. Ellory, and E. M. Tucker. Amino acid transport in normal and glutathione-deficient sheep erythrocytes. Biochem. J. 154: 43-48, 1976[Medline].


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