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Am J Physiol Endocrinol Metab 280: E31–E39, 2001. An in vivo study of ovine placental transport of essential amino acids CINZIA L. PAOLINI,1 GIACOMO MESCHIA,2 PAUL V. FENNESSEY,2 ADRIAN W. PIKE,2 CECILIA TENG,2 FREDERICK C. BATTAGLIA,2 AND RANDALL B. WILKENING2 1 Department of Obstetrics and Gynecology, Dipartimento di Medicina Chirurgia e Odontoiatria San Paolo, University of Milano, 20142 Milano, Italy; and 2Departments of Pediatrics, Physiology, and Mass Spectroscopy, Division of Perinatal Medicine, University of Colorado School of Medicine, Denver, Colorado 80045 Received 31 March 2000; accepted in final form 12 September 2000 transport; sheep; placenta; stable isotopes UNDER NORMAL PHYSIOLOGICAL CONDITIONS, essential amino acids are transported from mother to fetus at different rates (4, 10, 11). One of the functional meanings of this observation is that, to meet requirements for protein, accretion mandates different supply rates of amino acids. For example, whole body homogenates of the fetal lamb contain 2.4 times more leucine than phenylalanine (12). As a consequence, fetal growth requires a 2.4-fold greater accretion rate of leucine than of phenylalanine. The umbilical uptake of each essential amino acid exceeds its fetal accretion (4), because the fetus has a relatively high rate of amino acid catabo- Address for reprint requests and other correspondence: F. C. Battaglia, Fitzsimons, Bldg. 260, POB 6508, MS F441, Aurora, CO 80045-0508 (E-mail: [email protected]). http://www.ajpendo.org lism (1, 2, 8, 13). Nevertheless, umbilical uptake and fetal accretion are well correlated. In a recent study (4), the correlation coefficient between uptake and accretion of essential amino acids was 0.88. The mechanisms that determine transport rates of amino acids from the maternal to the umbilical circulation include the expression of several amino acid transport systems on both the maternal and fetal surfaces of the trophoblast (14, 16), as well as the regulation of maternal and fetal plasma amino acid concentrations. This knowledge suggests the hypothesis that differences in amino acid affinities for the transport systems that each amino acid utilizes combine with differences in maternal plasma amino acid concentrations to produce the range of placental transport rates that are required by normal fetal development. The present study was designed to explore the relationship between transplacental flux and maternal plasma concentration for all of the essential amino acids. Isotopically labeled leucine, isoleucine, valine, phenylalanine, lysine, methionine, threonine, histidine, and tryptophan were injected simultaneously into the maternal circulation of pregnant sheep. Measurements of amino acid concentrations, isotopic molar percent enrichments in maternal and fetal plasma, and umbilical plasma flow were used to calculate the transplacental flux and clearance of each amino acid. MATERIALS AND METHODS Surgery and Animal Care All animal experimentation was performed according to the Helsinki Convention for the use and care of animals and was approved by the local institutional animal care and use committee. Seven pregnant Columbia-Rambouillet sheep were studied. Animals underwent surgery at 122 ⫾ 1.3 days of gestation. Average body weight was 38.1 ⫾ 1.6 kg. After 48 h of fasting, the animals were sedated with intravenous pentobarbital sodium (10 mg/kg as initial dose) and given pontocaine hydrochloride spinal anesthesia (6 mg). The The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society E31 Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 Paolini, Cinzia L., Giacomo Meschia, Paul V. Fennessey, Adrian W. Pike, Cecilia Teng, Frederick C. Battaglia, and Randall B. Wilkening. An in vivo study of ovine placental transport of essential amino acids. Am J Physiol Endocrinol Metab 280: E31–E39, 2001.—Under normal physiological conditions, essential amino acids (EA) are transported from mother to fetus at different rates. The mechanisms underlying these differences include the expression of several amino acid transport systems in the placenta and the regulation of EA concentrations in maternal and fetal plasma. To study the relation of EA transplacental flux to maternal plasma concentration, isotopes of EA were injected into the circulation of pregnant ewes. Measurements of concentration and molar enrichment in maternal and fetal plasma and of umbilical plasma flow were used to calculate the ratio of transplacental pulse flux to maternal concentration (clearance) for each EA. Five EA (Met, Phe, Leu, Ile, and Val) had relatively high and similar clearances and were followed, in order of decreasing clearance, by Trp, Thr, His, and Lys. The five high-clearance EA showed strong correlation (r2 ⫽ 0.98) between the pulse flux and maternal concentration. The study suggests that five of the nine EA have similar affinity for a rate-limiting placental transport system that mediates rapid flux from mother to fetus, and that differences in transport rates within this group of EA are determined primarily by differences in maternal plasma concentration. E32 PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO Study Protocol Each animal was studied on two consecutive days, with the exception of one animal that was studied only once because of catheter failure. Each study day the animal was injected with one of two infusates (infusate A or B, Table 1). To check that results were comparable on both study days, L-[1-13C]leucine was included in both infusates. To prepare the infusates, amino acids labeled with stable isotopes (98–99 atom % pure) were obtained from Cambridge Isotope Laboratories and dissolved in 20 ml of sterile isotonic saline. At least one day before the studies, the oxygenation of mother and fetus was checked by measurements of O2 content and O2 saturation in both circulations to verify that it was within normal range. At the onset of each study, 25 ml of maternal blood were transfused into the fetal circulation to expand fetal blood volume as a replacement for fetal sampling. This represents ⬍10% expansion of fetal blood volume. Then, a solution of 3 H2O (325 Ci/20 ml) was infused at 3 ml/h in the fetal pedal vein after a 4-ml priming dose. The 3H2O infusion was used to calculate umbilical blood flow by the steady-state diffusion method (15). Approximately 60 min after the start of the 3 H2O infusion, four sets of 1-ml blood samples were drawn from the maternal femoral artery (A), uterine vein (V), umbilical vein (␥), and fetal artery (␣) for analysis of the amino acid concentrations and enrichments, fetal and maternal blood O2 content and hematocrit, and 3H2O concentration (for the steady-state period). After completion of this sampling, the amino acid infusate of 20 ml (see Table 1) was injected as a bolus over a 1.39 ⫾ 0.03-min period in the maternal femoral vein. During and after the bolus, blood samples (0.9 ml) were drawn simulta- neously from the four vessels (i.e., A, V, ␣, and ␥). The time sequence for sampling was 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10, 15, 20, 30, 40, 50, 60, 90, and 120 min after the bolus. This sampling sequence was based on a previous study that utilized a similar design (7). At 30 and 90 min, blood was also withdrawn for 3H2O and hematocrit measurements. At the end of the first study, antibiotics (ampicillin 500 mg) were injected into the amniotic catheter, and a second 25-ml blood transfusion was given to the fetus. The next day the study was repeated with the second infusate. In two studies, infusate A was given first, and in five studies, infusate B was given first. Within 1 h after completion of the second study, the mother and fetus were injected intravenously with a euthanasia solution (12 ml of 26% pentobarbital sodium). At autopsy, fetal and placental weights were measured separately, and catheter positions were verified. Biochemical Analysis Samples drawn for the measurement of hemoglobin concentration and oxygen saturation were collected into heparinized capillary syringes. Hemoglobin concentration (expressed as oxygen capacity in mM), oxyhemoglobin saturation (%), and blood oxygen content (mM) were immediately measured in duplicate in a hemoximeter (OSM-2, Radiometer, Copenhagen, Denmark). For 3H2O whole blood analysis, 0.1-ml plasma samples were solubilized in 1.0 ml of Soluene-350 (quaternary ammonium hydroxide in toluene) and subsequently mixed with 15 ml of Hionic-Fluor. Blood 3H2O concentrations were calculated by using the relation of blood water content to hematocrit (15). The radioactivity was counted in a Packard TriCarb 460 C liquid scintillation counter. Blood samples for measurement of plasma amino acid concentrations and stable isotopic enrichments were collected in syringes coated with EDTA and were centrifuged to obtain plasma, which was stored at ⫺70°C until analysis. Amino acid concentrations were measured by HPLC. Plasma was quickly thawed, deproteinized with a solution of 10% sulfosalicylic acid, with L-norleucine added as internal standard, and buffered with LiOH to pH 2.2. Samples were then centrifuged at 14,000 rpm for 10 min, and the supernatant fraction was filtered through a millipore filter and loaded into a Dionex HPLC (Sunnyvale, CA) with refrigerated autosampler. The samples were analyzed by cation exchange column with gradiant change from three different buffers isothermally. Ninhydrin was used as color reagent, and a dual wavelength spectrophotometer with wavelengths of 440 and 570 nm were used for concentration determinations. The column, ninhydrin reagent, and buffer were purchased from Pickering Laboratories (Mountain View, CA). All instrument operation and data processing were controlled by Dionex A1–450 software. Table 1. Composition of the two infusates Infusate A Infusate B Amino acid Infusate, M Amino acid Infusate, M L-[1- C]leucine 13 L-[1- C]isoleucine 13 L-[1- C]phenylalanine 13 L-[1- C]lysine 2 L-[indole- H5]tryptophan 62.1 ⫾ 12.9 44.1 ⫾ 9.5 20.2 ⫾ 4.7 97.4 ⫾ 6.3 15.5 ⫾ 2.7 L-[1- C]leucine 13 L-[1- C]valine 2 L-[methyl- H3]methionine 13 L-[1- C]threonine 13 L-[ring 2- C]histidine 67.8 ⫾ 14.3 113.9 ⫾ 22.4 28.0 ⫾ 2.0 232.4 ⫾ 1.9 25.4 ⫾ 0.9 13 13 Values are means ⫾ SE. For some amino acids, the infusate had a large SE because their concentrations were reduced after analysis of the results of the first experiment. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 uterus was exposed through a midline abdominal incision. After the fetus was exposed through a 6-cm incision of the uterine wall, polyvinyl catheters (1.4 mm OD) were placed in the fetal pedal artery and vein and in the umbilical vein. One catheter was placed in the amniotic cavity to give intraamniotic antibiotic prophylaxis to the fetus (ampicillin 500 mg). The maternal femoral artery and vein were catheterized last. All catheters were tunneled subcutaneously to a plastic pouch secured to the animal’s flank. Sheep were standing within 6 h after surgery in individual carts and had free access to water, food (alfalfa pellets), and a mineral block. Ampicillin (500 mg) was injected daily into the amniotic cavity for the first 3 days after surgery. Animals were allowed to recover from laparotomy for 7 days before study. They were fed alfalfa pellets and water ad libitum. Their average food intake was 30 ⫾ 4 g per kg body weight. Catheters were maintained patent with daily heparinized saline flushes (35 U heparin/ml). Six out of seven animals were singleton; one carried a twin pregnancy, and in that case only one fetus was studied. E33 PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO Stable isotopic enrichments were analyzed with a gas chromatograph-mass spectrometer in the electron impact mode, in 250 l of acidified plasma with 50 nM of L-norleucine as internal standard. The amino acid fraction was separated from other fractions on a 1.5-cm cation-exchange resin column (50W ⫻ 8, 100–200 mesh, hydrogen form) eluted with 1 ml of 15% NH4OH. Percentage recoveries of basic, neutral, and acidic amino acids were tested with corresponding radiolabeled amino acids and varied from 80 to 92%. Samples were dried, derivatized with 15% tert-butyldimethylsilyl, or TBDMS, and injected in triplicate on a gas chromatograph (Hewlett-Packard 5890) equipped with a 30-m DB-1 0.25-mm-ID 0.25-m film thickness, with helium as the carrier gas, and a HP 5970 Mass Selective Device. Enrichments of plasma amino acids were calculated by comparing the difference in peak area ratios between enriched and unenriched samples according to the formula ⫻ 共ratio of enriched sample ⫺ ratio of unenriched sample兲/ 关共ratio of enriched sample ⫺ ratio of unenriched sample兲 ⫹ 1兴 where MPE expresses the relative abundance of the enriched tracer in excess of that occurring naturally. Peaks were read at mass-to-charge ratios (m/z) 303/302 for 13 13 13 L-[1- C]valine, L-[1- C]leucine, and L-[1- C]isoleucine, at m/z 337/336 for L-[1-13C]phenylalanine, at m/z 323/320 for 2 13 L-[methyl- H3]methionine, at m/z 301/300 for L-[1- C]lysine, at m/z 405/404 for L-[1-13C]threonine, at m/z 441/440 for 13 L-[ring 2- C]histidine, and at m/z 380/375 for L-[indole2 H5]tryptophan. Amino acids were monitored in selective ion monitoring, or SIM, in the same run for each infusate. Calculations Flows and O2 consumption. Umbilical blood flow (UBF) was calculated as previously described by use of the tritiated water infusion rate, the fetal tritiated water accumulation rate, and the difference in umbilical venoarterial concentrations (15). Umbilical plasma flow (UPF) was calculated according to the formula UPF共ml 䡠 min ⫺ 1 䡠 kg ⫺ 1兲 ⫽ UBF共ml 䡠 min ⫺ 1 䡠 kg ⫺ 1兲 䡠 共1 ⫺ fetal fractional hematocrit兲 Fetal oxygen consumption (mol 䡠 min⫺1 䡠 kg⫺1) was calculated as the product of UBF (mol 䡠 min⫺1 䡠 kg⫺1) and the venoarterial difference in O2 content (mol/ml) across the umbilical circulation. Uptakes and clearance. The steady-state umbilical uptake of each amino acid (mol 䡠 min⫺1 䡠 kg⫺1) was calculated as the product of UPF (ml 䡠 min⫺1 䡠 kg⫺1) and the umbilical venoarterial concentration difference (mol/ml) of the amino acid. The isotope concentration of each amino acid (M) was estimated as the product of (MPE/100 ⫺ MPE) and the amino acid concentration (M). The umbilical isotope uptake (mol 䡠 min⫺1 䡠 kg⫺1) was estimated as the product of UPF (ml 䡠 min⫺1 䡠 kg⫺1) and the integral of the umbilical venoarterial difference in the isotope concentration (mol 䡠 ml⫺1 䡠 min⫺1) divided by the time of integration (min). The isotope clearance (ml 䡠 min⫺1 䡠 kg⫺1) was calculated as [umbilical isotope uptake (mol 䡠 min⫺1 䡠 kg⫺1) divided by the integral of the maternal arterial enrichment (in mol 䡠 ml⫺1 䡠 min)] divided by the time of integration (min). Data Analysis and Statistics All data are presented as means ⫾ SE. The significance of the difference among amino acids was calculated by the F-test and the Mann-Whitney U-test. RESULTS Table 2 presents data for mean gestational age at surgery and at autopsy; maternal, fetal, and placental weight; number of cotyledons; UBF; UPF; and fetal oxygen consumption. There were no significant differences in umbilical or uterine blood flow or in fetal O2 saturations between the control (time 0) samples and the 120-min samples. As shown in Fig. 1, the maternal plasma MPE of all essential amino acids increased rapidly after injection of labeled amino acids: we were able to detect all of the isotopes infused into the maternal circulation within 30 s from the beginning of the bolus. For all of the amino acids, the peak in both the maternal artery and uterine vein enrichments occurred in the sample taken at 60 s from the start of the bolus. After the peak, there was an exponential decay that was similar among most amino acids but slower for lysine and threonine. The enrichments of all of the essential amino acids increased in the umbilical circulation, giving evidence of transplacental transport. To observe placental transport for each amino acid, a large amount of each isotope was injected. As a consequence, the total amino acid concentrations (i.e., the sum of labeled and unlabeled molecules) increased from 2 to 6 times in the maternal arterial plasma during the first 10 min after the bolus (Fig. 2). This increase produced an increase in the umbilical venous concentration of the corresponding amino acid. However, the rise in umbilical concentration was much less than in the mother and differed among amino acids (Fig. 2). For each amino acid, the umbilical venous peak of MPE was delayed with respect to the 1-min maternal peak. Time to peak was ⬃4 min for phenylalanine, isoleucine, and leucine and occurred much later for Table 2. Age, weight, flow, and O2 consumption data Type of Pregnancy Gestational age, days At surgery At autopsy (day 2 of study) Weight Maternal, kg Fetal, g Placental, g Number of cotyledons Umbilical blood flow, ml 䡠 min⫺1 䡠 kg⫺1 Umbilical plasma flow, ml 䡠 min⫺1 䡠 kg⫺1 Fetal O2 consumption, mol 䡠 min⫺1 䡠 kg⫺1 Values are means ⫾ SE. 6 Singleton ⫹ 1 Twin 122.0 ⫾ 1 130.0 ⫾ 1 38.1 ⫾ 1.6 2,753.5 ⫾ 168 335.3 ⫾ 16.2 83.4 ⫾ 7.3 207.3 ⫾ 12.0 137.2 ⫾ 9.2 342.2 ⫾ 10.8 Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 molar percent enrichment 共MPE兲 共%兲 ⫽ 100 The transplacental pulse flux of each amino acid (mol 䡠 min⫺1 䡠 kg⫺1) was calculated as the product of isotope clearance (ml 䡠 min⫺1 䡠 kg⫺1) and maternal arterial concentration (mol/ml) at steady state. E34 PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 Fig. 1. Amino acid enrichments for the 1st (A) and 2nd (B) infusate in maternal artery (‚), uterine vein (Œ), umbilical vein (F), and fetal artery (E). Time 0 is the start of isotope bolus injection. histidine (14.2 ⫾ 1.0 min) and threonine (22.6 ⫾ 2.5 min; Table 3). The data were further analyzed by recognizing that the infused amino acids have widely different concentrations in maternal and fetal plasma at time 0 (Table 4). Also, maternal isotopic concentration changes are not immediately translated into the fetal circulation because of transport and accumulation in the placenta. The fetal uptake at any point in time reflects the combined effects of maternal and placental concentration changes over a period of time preceding the fetal measurement. To produce directly comparable results, the following procedure was adopted. First, the MPE and concentration data were used to calculate isotope concentrations in maternal and fetal plasma. Second, the maternal arterial isotope concentration was integrated to produce an average maternal isotope concentration over the time of integration. Third, the isotope concentrations in fetal plasma were expressed as ratios of fetal isotope concentration to the average maternal arterial isotope concentration. Figure 3 presents isotope concentrations in the umbilical vein and artery normalized for the average maternal arterial isotope concentration in the first 10 min after the infusion. Despite the fact that fetal isotope concentrations are expressed in comparable units, it is clear that there are marked differences in the rate of concentration change in fetal plasma. The E35 PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO Table 3. MPE time to peak in umbilical venous plasma Amino Acids 13 L-[1- C]phenylalanine 13 L-[1- C]isoleucine 13 L-[1- C]leucine (A) 13 L-[1- C]leucine (B) 2 L-[methyl- H3]methionine 2 L-[indole- H5]tryptophan 13 L-[1- C]lysine 13 L-[1- C]valine L-[ring 13 L-[1- 2-13C]histidine C]threonine n Table 4. Plasma amino acid concentrations before the bolus Time to Peak, min 7 7 7 6 3.9 ⫾ 0.3 3.9 ⫾ 0.3 4.0 ⫾ 0.4 4.0 ⫾ 0.4 6 7 7 6 5.8 ⫾ 0.5 6.3 ⫾ 0.5 6.4 ⫾ 0.7 7.6 ⫾ 1.1 6 14.2 ⫾ 1.5 6 22.6 ⫾ 2.5 Values are means ⫾ SE; n, no. of experiments. Leucine Isoleucine Phenylalanine Lysine Tryptophan Valine Methionine Threonine Histidine Maternal Artery Umbilical Vein Fetal Artery 212.19 ⫾ 20.3 143.74 ⫾ 16.7 64.44 ⫾ 6.8 136.35 ⫾ 17.5 41.84 ⫾ 2.9 351.18 ⫾ 54.2 28.54 ⫾ 2.0 199.68 ⫾ 35.2 48.30 ⫾ 1.6 212.8 ⫾ 16.8 130.75 ⫾ 11.5 123.0 ⫾ 9.1 67.02 ⫾ 7.1 50.65 ⫾ 6.5 603.04 ⫾ 69.4 71.84 ⫾ 8.52 330.11 ⫾ 66.1 57.88 ⫾ 7.17 178.16 ⫾ 15.3 109.68 ⫾ 11.7 111.7 ⫾ 9.0 49.25 ⫾ 4.6 42.96 ⫾ 6.03 571.39 ⫾ 63.2 66.50 ⫾ 10.4 314.48 ⫾ 65.2 55.01 ⫾ 8.0 Values are means ⫾ SE expressed in M. The leucine concentration presented is the average of leucine concentrations measured before first and second infusions, because these concentrations were not significantly different. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 Fig. 2. Total plasma amino acid concentrations for the 1st (A) and 2nd (B) infusate in maternal artery (‚) and umbilical vein (F). Time 0 is the start of the isotope bolus injection. E36 PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 Fig. 3. Umbilical venous (F) and arterial (E) isotope concentrations expressed as a fraction of average maternal arterial isotope concentration over the first 10 min after isotope bolus injection. most important information is provided by umbilical venous plasma concentrations, because they represent the fetal plasma draining the placenta. In the first minute after infusion, changes in umbilical vein concentration provide information about placental transport that is virtually unaffected by fetal metabolism. According to Fig. 3, there are five amino acids (leucine, isoleucine, valine, phenylalanine, and methionine) that show rapid increases in the umbilical vein after the maternal bolus. On the other hand, threonine, lysine, and histidine demonstrate smaller and less rapid concentration changes. The data in Fig. 3 were used in conjunction with UPF to calculate isotope clearances for the first 10 min after the bolus. Analogous data were used to calculate clearances for Table 5. Isotope transplacental clearances calculated over the first 10 and 20 min after bolus injection Isotope Clearance, ml 䡠 min⫺1 䡠 kg⫺1 Amino Acids 10 min 20 min Methionine Phenylalanine Leucine (A) Leucine (B) Isoleucine Valine Tryptophan Threonine Histidine Lysine 17.09 ⫾ 2.96 15.63 ⫾ 1.80 14.19 ⫾ 1.35 13.57 ⫾ 1.32 14.36 ⫾ 2.09 12.51 ⫾ 1.99 7.03 ⫾ 2.39 4.07 ⫾ 0.82 3.70 ⫾ 0.72 2.88 ⫾ 0.56 17.69 ⫾ 3.52 14.31 ⫾ 2.40 13.56 ⫾ 1.31 11.54 ⫾ 1.32 13.90 ⫾ 2.34 11.97 ⫾ 2.73 9.21 ⫾ 3.65 6.53 ⫾ 1.29 4.64 ⫾ 0.78 3.44 ⫾ 0.74 Values are means ⫾ SE. E37 PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO Table 6. Umbilical steady-state uptakes and transplacental pulse fluxes for the 9 essential amino acids 10 min 20 min Amino Acid Steady-State Uptake, mol 䡠 min⫺1 䡠 kg⫺1 Pulse flux, mol 䡠 min⫺1 䡠 kg⫺1 Pulse flux/steady-state uptake ratio Pulse flux, mol 䡠 min⫺1 䡠 kg⫺1 Pulse flux/steady-state uptake ratio Methionine Phenylalanine Leucine (A) Leucine (B) Isoleucine Valine Tryptophan Threonine Histidine Lysine 0.73 ⫾ 0.33 1.55 ⫾ 0.21 4.75 ⫾ 0.35 4.76 ⫾ 0.64 2.89 ⫾ 0.14 4.34 ⫾ 1.18 1.05 ⫾ 0.41 2.14 ⫾ 0.75 0.39 ⫾ 0.60 2.44 ⫾ 0.25 0.51 ⫾ 0.09 0.97 ⫾ 0.11 2.80 ⫾ 0.32 3.06 ⫾ 0.48 1.96 ⫾ 0.33 4.12 ⫾ 0.58 0.28 ⫾ 0.09 0.73 ⫾ 0.11 0.18 ⫾ 0.04 0.35 ⫾ 0.04 0.69 0.63 0.59 0.64 0.68 0.95 0.27 0.34 0.45 0.14 0.53 ⫾ 0.11 0.88 ⫾ 0.14 2.53 ⫾ 0.26 2.66 ⫾ 0.53 1.88 ⫾ 0.37 3.96 ⫾ 0.85 0.36 ⫾ 0.14 1.16 ⫾ 0.16 0.22 ⫾ 0.04 0.41 ⫾ 0.06 0.72 0.57 0.53 0.56 0.65 0.91 0.34 0.54 0.56 0.17 Values are means ⫾ SE or ratios. DISCUSSION equal values of maternal isotope concentration and for a small fetal compared with maternal arterial isotope concentration. The latter condition minimizes isotope back flux from fetus to placenta so that the clearance approximates unidirectional transport of the maternal amino acid. Five of the nine essential amino acids, i.e., the branched-chain amino acids leucine, isoleucine, and valine, plus methionine and phenylalanine, demonstrated similar and relatively fast clearances. All five are zwitterions with hydrophobic side chains. This evidence suggests that the five amino acids have similar affinity for a rate-limiting transport system that mediates rapid flux from mother to fetus. Placental amino acid transport utilizes two sets of transport systems located, respectively, on the maternal and fetal surface of the trophoblast (14). A study on the transport of nonmetabolizable amino acids by the ovine placenta in vivo has led to the hypothesis that the neutral amino acids with the most rapid flux from mother to fetus have relatively high affinity for a system of exchange transporters that is located on the fetal surface of the placenta and has properties similar to the Na-independent “ᐉ” system (9). In agreement with this hypothesis, the branched-chain amino acids The present study confirms that, under normal physiological conditions, essential amino acids have different umbilical uptakes. Table 6 shows leucine and valine having steady-state uptakes in the 5–4 mol 䡠 min⫺1 䡠 kg fetus⫺1 range, followed in order of decreasing values by isoleucine, lysine, threonine, and phenylalanine (range of 3–1.5) and tryptophan, methionine, and histidine (1 or lower). The present study is the first in which tryptophan umbilical uptake has been measured. Uptake data for the other amino acids are in agreement with previous measurements (4). Pulse fluxes of essential amino acids into the umbilical circulation also demonstrate a wide range in values (Table 6). To account for the contribution of differences in maternal plasma amino acid concentration to this variability, we calculated placental isotope clearances. Clearances measured in the first 10 min after bolus injection represent fluxes across the placenta for Fig. 4. Pulse flux calculated over the 1st 10 min after the bolus vs. maternal plasma arterial concentration for the 5 essential amino acids that have high and similar pulse flux clearances. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 the first 20 min after the bolus. The results of these calculations are shown in Table 5. Five amino acids (methionine, phenylalanine, leucine, isoleucine, and valine) show relatively high isotope clearances, which do not differ significantly within the group and which range between 17 and 12.5 ml 䡠 min⫺1 䡠 kg⫺1 in the first 10 min. The remaining four amino acids (tryptophan, threonine, histidine, and lysine) show 10-min clearances that are smaller (range between 7 and 2.9 ml 䡠 min⫺1 䡠 kg⫺1) than those of the first group (P ⬍ 0.01). There were no significant differences between the 10- and 20-min clearances, indicating that the results of the 10-min clearance calculation were not critically dependent on the selection of the time interval. Separation of the nine essential amino acids into two broad groups was evident also by comparing initial pulse fluxes and umbilical steady-state uptakes (Table 6). The 10-min pulse flux-to-steady-state uptake ratio was greater for methionine, phenylalanine, leucine, isoleucine, and valine than for tryptophan, threonine, histidine, and lysine. For lysine, the pulse flux was considerably smaller than steady-state uptake over both the 10- and 20-min periods (Table 6). E38 PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO tions may create a large difference between pulse flux and steady-state direct flux across the placenta. The steep increase in maternal concentration of the test isotope may decrease its transplacental clearance to below normal if the rate-limiting transport system is near saturation. Furthermore, the simultaneous increase in concentration of the coinfused isotopes may alter the clearance via interaction with a shared transport system. Although the simultaneous infusion of several amino acids has the advantage of providing comparative data that are not biased by interanimal variability, it introduces the potential for competition among the amino acids. Normal, steady-state direct fluxes across the sheep placenta have been measured only for leucine (13) and threonine (1). The leucine direct flux was 3.4 ⫾ 0.4 mol 䡠 min⫺1 䡠 kg fetus⫺1 and 0.87 of umbilical uptake, and its transplacental clearance was 15.5 ml 䡠 min⫺1 䡠 kg fetus⫺1 (13). For threonine, direct flux was 2.2 ⫾ 0.2 mol 䡠 min⫺1 䡠 kg fetus⫺1 and 0.52 of umbilical uptake, and the transplacental clearance was 8.2 ml 䡠 min⫺1 䡠 kg fetus⫺1 (1). The leucine steady-state flux values are similar to the leucine pulse flux data in Tables 5 and 6, so that there is no clear evidence that the conditions of the pulse flux experiment inhibited the tranplacental leucine flux. The comparison of threonine pulse and steady-state data suggests that the threonine pulse flux and clearance might have been less than the analogous steady-state values, but again the discrepancy is not sufficiently large to draw any firm conclusion. For the other essential amino acids, the pulse flux-tosteady-state umbilical uptake ratios listed in Table 6 are the only available clue about the relationship of the measured pulse flux to the normal steady-state direct transplacental flux. Inspection of Table 6 shows that the pulse flux of lysine was exceptionally small compared with its umbilical uptake, suggesting that the transplacental flux of lysine measured in the present study might have been much less than the normal steady-state direct mother-to-fetus lysine flux. Further studies are needed to investigate whether placental efflux of lysine is near a saturated maximum under normal physiological conditions and whether increases in the maternal concentration of other amino acids interfere with lysine transport to the fetus. In conclusion, the present study supports the hypothesis (9) that the amino acids that are transported most rapidly from the ovine placenta into the fetal circulation are zwitterions with relatively high affinity for the Na-independent “ᐉ” system exchange transporters. In addition, it shows that there are significant differences in transplacental clearance among the essential amino acids. These differences are likely to be related to differences in affinity and to competition for shared transporters and to the utilization of separate transport systems. This work was supported by Fondo “Amalia Griffini” 1998, held by Provincia di Varese, Italy, and National Institute of Child Health and Human Development Grant PO1-HD-20761. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017 methionine and phenylalanine have been shown to be substrates of the “ᐉ” system (3). An important implication of the finding that five essential amino acids have similar placental clearances is that, among these amino acids, differences in transport rate from mother to fetus depend upon differences in maternal concentration (Fig. 4). For example, both pulse flux and umbilical uptake were three times greater for leucine than for phenylalanine, and these differences correlated with a threefold difference in maternal plasma concentration. It would appear that, for five of the nine essential amino acids, the regulatory mechanisms that set maternal concentration play a major role in controlling the supply rate to the fetus. The importance of factors other than maternal concentration in determining a wide range of transplacental fluxes is demonstrated by comparing threonine, lysine, and histidine with the other essential amino acids. In the first 10 min after maternal injection, the threonine, lysine, and histidine isotopes showed much slower increases in umbilical concentration and significantly smaller clearances then the leucine, isoleucine, valine, methionine, and phenylalanine isotopes. The behavior of tryptophan was intermediate. Threonine is a zwitterion with a hydrophilic side chain and is a substrate for neutral amino acid transporters present in the placenta (5). Its sluggish appearance in the umbilical circulation indicates relatively low affinity for the transporters that mediate rapid flux of neutral amino acids from placenta to fetus. Lysine and histidine are basic amino acids. Three sodiumindependent transport systems for basic amino acids, named y⫹, y⫹L, and b°,⫹, have been shown to be present in the human trophoblast (6). Such information is not available for the ovine trophoblast. The b°,⫹ transporter is an exchanger of basic and neutral amino acids and is located on the fetal surface only, suggesting the hypothesis that it promotes the efflux of basic amino acids from placenta to fetus in exchange for a neutral amino acid flux in the opposite direction (6). It is apparent that, under the conditions of our study, the specialized transport systems for basic amino acids produced relatively small lysine and histidine transplacental clearances. The relation between umbilical uptake and motherto-fetus pulse flux of any given essential amino acid is complex. Steady-state umbilical uptake is the algebraic sum of three fluxes: 1) direct flux from maternal to fetal circulation, 2) flux into the umbilical circulation from the pool of placental amino acids released by placental protein turnover, and 3) back flux from the umbilical circulation into the placenta (1, 13). These fluxes occur while the maternal, placental, and fetal concentrations of all the amino acids are virtually constant and set at normal physiological levels. By contrast, each pulse flux measured in the present study represents direct mother-to-fetus transport at the same time that there is a transient and large increase in the maternal concentration of the test isotope and of the four coinfused isotopes. These experimental condi- PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO REFERENCES 9. Jozwik M, Teng C, Timmerman M, Chung M, Meschia G, and Battaglia FC. 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