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Department of Obstetrics and Gynecology (C.L.P., A.M.M., S.R., M.D.N., P.V.F., G.P., F.C.B.), San Paolo Department of Medicine Surgery and Dentistry, University of Milan, 20142 Milan, Italy; and Division of Perinatal Medicine, University of Colorado School of Medicine (P.V.F., F.C.B.), Denver, Colorado 80262
Address all correspondence and requests for reprints to: Anna Maria Marconi, M.D., Department of Obstetrics and Gynecology, San Paolo Department of Medicine, University of Milan, Via A. di Rudini 8, 20142 Milan, Italy. E-mail: marconi{at}enter.it
Abstract
L-[1-13C]Leucine, [1-13C]glycine, L-[1-13C]phenylalanine, and L-[1-13C]proline were infused as a bolus into the maternal circulation of seven appropriate for gestational age at 30.3 ± 3.0 wk and 7 intrauterine growth-restricted pregnancies at 26.5 ± 1.0 wk gestation to investigate placental transport in vivo. Umbilical venous samples were obtained at the time of in utero fetal blood sampling at 450 ± 74 sec from the bolus injection. In normal pregnancies the fetal/maternal (F/M) enrichment ratios for leucine (0.76 ± 0.06) and phenylalanine (0.77 ± 0.06) were higher (P < 0.01) than the F/M ratios for glycine (0.18 ± 0.04) and proline (0.22 ± 0.02). This suggests that these two essential amino acids rapidly cross the placenta in vivo. Compared with the essentials, both glycine and proline had significantly lower F/M enrichment ratios, which were not different from each other. The results support the hypothesis that amino acids with high affinity for exchange transporters cross the placenta most rapidly. In intrauterine growth-restricted pregnancies, the F/M enrichment ratio was significantly lower (P < 0.01) for L-[1-13C]leucine (0.76 ± 0.06 vs. 0.48 ± 0.07) and for L-[1-13C]phenylalanine (0.77 ± 0.06 vs. 0.46 ± 0.07) compared with appropriate for gestational age pregnancies reflecting impaired transplacental flux. The F/M enrichment ratio did not differ for [1-13C]glycine (0.18 ± 0.04 vs. 0.17 ± 0.03), and L-[1-13C]proline (0.22 ± 0.02 vs. 0.18 ± 0.04).
THE PLACENTAL TRANSPORT of amino acids in vivo has been studied in pregnant sheep using nonsteady state stable isotope methodology (1, 2, 3). Steady state protocols have also been used to compare the relative rates of transport from the maternal circulation into the fetal circulation for different amino acids (3, 4, 5). Such in vivo data are crucial because the net transport rate into the fetal circulation is a function of many variables, including placental synthesis or utilization of amino acids and their intracellular concentrations, in addition to the transport characteristics of each surface of the trophoblast, which have been extensively studied in vitro (6).
Two recent studies in sheep using nonmetabolizable tracer amino acids (7) and stable isotopes of each of the essential amino acids (2) have led to the hypothesis that the principle determinant of the rate of transplacental transport is the exit rate from the fetal surface of the trophoblast into the fetal circulation, but little is known in human pregnancy about the placental transport rates of amino acids in vivo.
The present study was designed to test the hypothesis that leucine and phenylalanine, which use exchange transporters, will cross the placenta rapidly in vivo compared with glycine and proline, which use sodium-dependent transporters primarily. Secondly, we compared in vivo the relative transport rates of these amino acids in appropriate for gestational age (AGA) and intrauterine growth-restricted (IUGR) pregnancies.
From in vivo studies across the ovine placenta and from in vitro data on human placental isolated membranes (2, 8), one would hypothesize that the transport of phenylalanine would be virtually equal to that of leucine and the transport of proline would be virtually equal to that of glycine. Furthermore, in vivo studies have shown a striking difference between these two groups of amino acids in terms of their placental transport. Both leucine and glycine transport have already been compared in terms of their rates of appearance in the fetal circulation in human pregnancies (9).
Materials and Methods
The study was performed at the Department of Obstetrics and Gynecology, San Paolo Department of Medicine, Surgery and Dentistry (Milan, Italy). The protocol of the study was approved by the San Paolo ethical committee and the Colorado Multiple Institute Review Board of the University of Colorado Health Sciences Center. Written informed consent was obtained from all patients.
Patients
We studied 7 AGA and 7 IUGR pregnancies at similar gestational
age. The clinical data are presented in Table 1
. All 14 pregnancies included in
the study were scheduled for in utero fetal blood sampling
for clinical indications. Gestational age was determined by the last
menstrual period and was confirmed by ultrasound at 18–21 wk
gestation. Intrauterine growth restriction was diagnosed in
utero when abdominal circumference was below the 10th percentile.
Growth restriction was confirmed at birth if fetal weight was below the
10th percentile according to Italian standards for birth weight and
gestational age (10).
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In IUGR fetuses, fetal blood sampling was performed for rapid karyotyping and/or for the assessment of fetal oxygenation and acid-base balance. In all cases fetal karyotype was normal, and no major malformations were present at birth. Doppler velocimetry of the umbilical artery was performed in all IUGR fetuses, and fetal heart rate was recorded before fetal blood sampling. All IUGR fetuses were classified as group 3 (pulsatility index of the umbilical artery measured by Doppler velocimetry above 2 SD; fetal heart rate abnormal) according to the classification proposed by Pardi et al. (12). Four mothers were normotensive, and three had pregnancy-induced hypertension and were receiving antihypertensive drugs (20 mg nifedipine, twice daily).
Protocol of the study
The study was performed after an overnight fast of at least 10 h. All patients were nonobese and had a normal glucose tolerance test. The patients were in the supine position at the time of the study.
L-[1-13C]Leucine, L-[1-13C]phenylalanine, [1-13C]glycine, L-[1-13C]proline (98.8 atom % pure) were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). The stable isotopes were dissolved in sterile isotonic saline for iv administration and tested for sterility and pyrogenicity. The amino acids were dissolved together in a single solution that was injected into a maternal peripheral vein over a period of 30 sec. The solution was composed of 1.12 ± 0.04 µmol/kg L-[1-13C]leucine, 1.96 ± 0.07 µmol/kg [1-13C]glycine, 1.28 ± 0.05 µmol/kg L-[1-13C]proline, and 0.89 ± 0.03 µmol/kg L-[1-13C]phenylalanine.
The time period from the administration of the bolus to the time the fetal blood sample was obtained was always more than 2 min but less than 10 min. This timing was based upon a previous study that showed that the glycine maternal plasma enrichment (MPE)/leucine MPE ratio was constant between this time interval (9).
Maternal samples were taken from a heated hand vein to represent
arterialized blood as described by Sonnenberg and Keller
(13). Two maternal samples were taken at time zero (the
time at which the bolus was begun) and every 2 min until the fetal
sample was obtained from the umbilical vein (Fig. 1).
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pH, pO2, and pCO2 were measured with an ABL 330 (Radiometer, Copenhagen, Denmark); hemoglobin concentration and O2 saturation were determined with an OSM3 Oximeter (Radiometer). Lactate concentrations were measured in duplicate with a YSI, Inc., analyzer (Yellow Springs, OH). All analyses were completed within 10 min of sampling. The O2 content was calculated according to the formula: O2 content (mmol/liter) = hemoglobin conc. (g/liter) x O2 saturation x 0.05982. After centrifugation, the plasma was kept at -180 C until analysis by HPLC amino acid chromatography and gas chromatography-mass spectroscopy using an HP 5971A mass spectrometer equipped with a HP 5890 gas chromatograph (Hewlett-Packard Co., Palo Alto, CA).
Analytical method and calculation
Plasma leucine, phenylalanine, glycine, and proline enrichments were determined by gas chromatography-mass spectrometry; tert-butyldimethylsilyl derivates of amino acids were prepared and analyzed in triplicate. Three hundred microliters of 50% acetic acid and 50 µl norleucine (1 mM) as internal standard were added to 250 µl plasma, and amino acids were separated by cation exchange columns prepared by inserting 1-cm Dowex 50 resin (hydrogen form, 100–200 mesh) in the pipettes. Amino acid fractions were collected into silanized tubes (with 10% dimethylchlorosilane in toluene) after adding 750 µl (500 plus 250 µl) 6% ammonium hydroxide. Amino acid fractions were dried and then derivatized with 150 µl 15% tert-butyldimethysilyl in acetonitrile. Standard solutions of the four amino acids were prepared in the same way as the samples. Samples and standard solutions were injected in a gas chromatograph (5890 series II, Hewlett-Packard Co.). The ions monitored were 246/247 m/z for glycine and [1-13C]glycine, 302/303 m/z for L-leucine and L-[1-13C]leucine, 286/287 m/z for L-proline and L-[1-13C]proline, and 336/337 for L-phenylalanine and L-[1-13C]phenylalanine. Plasma glycine, leucine, proline, and phenylalanine enrichments (MPE) were calculated using the difference in peak area ratios between enriched and unenriched samples. Tracer concentrations were calculated according to the formula: tracer conc. = total conc. x MPE x 0.01. The fetal/maternal (F/M) MPE ratio was calculated as the ratio between fetal plasma amino acid enrichment over the maternal plasma amino acid enrichment at the time of fetal blood sampling.
Plasma leucine, glycine, proline, and phenylalanine concentrations were measured separately by ion exchange chromatography on an automated amino acid analyzer (Chromakon 500, Kontron Instruments Ltd., Zurich, Switzerland). Plasma was thawed and deproteinized with a solution of 10% sulfosalycidic 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 Corp. filter (Bedford, MA) and loaded into a Dionex HPLC (Sunnyvale, CA) with refrigerated autosampler. Ninhydrin was used as color reagent, and a dual wavelength spectrophotometer with 440 and 570 nm wavelengths was used for concentration determinations. The column, ninhydrin reagent, and buffer were purchased from Pickering Laboratories, Inc. (Mountain View, CA). All instrument operation and data processing were controlled by Dionex A1–450 software.
Statistics
All data are expressed as the mean ± SEM. The significance of the difference between groups was calculated with two-tailed unpaired t test. Regression analysis was performed by the least squares method.
Results
Table 2 presents the oxygenation and
acid-base data as well as the plasma lactate and amino acid
concentrations for all pregnancies. Umbilical venous oxygen
saturations, oxygen contents, and pH were significantly lower, and
lactate concentration were significantly higher in IUGR fetuses
compared with AGA fetuses.
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presents the maternal plasma
enrichments for leucine as the mean ± SEM at each
time point for all patients.
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presents the maternal and fetal
plasma enrichments for the four amino acids in normal pregnancies. In
normal pregnancies, the F/M enrichment ratio was significantly higher
(P < 0.01) for leucine and phenylalanine than for
proline and glycine. There were no significant differences between the
F/M enrichment ratios and the calculated F/M tracer concentration
ratios. Significant differences between leucine-phenylalanine and
glycine-proline were also found for the F/M tracer concentration
ratios.
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presents the F/M enrichment
ratios of phenylalanine vs. leucine (left panel)
and the F/M enrichment ratios of glycine vs. proline
(right panel) in both normal and IUGR pregnancies. There was
a tight correlation (r2 = 0.96) between
phenylalanine vs. leucine enrichment ratios as well as
between proline vs. glycine ratios, suggesting similar
transport rates for these two sets of amino acids. Figure 4 presents the F/M ratios for the tracer
concentrations of the four amino acids. As for the enrichment ratios,
the regression line is calculated using all data points. There was a
highly significant correlation for the two sets of amino acids.
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).
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) in normal and IUGR pregnancies.
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From in vivo studies of the ovine placenta and from
in vitro data on human placental isolated membrane (2, 8), it was hypothesized that the transport of phenylalanine
would be approximately equal to that of leucine and that the transport
of proline would be equal to that of glycine. The strong correlation
presented in Fig. 3
between leucine and phenylalanine and between
glycine and proline suggests that these amino acids have similar
transport rates across the human placenta. The in vivo
placental transport of phenylalanine and proline in human pregnancy is
presented here for the first time.
The present study demonstrates that in human pregnancy the in vivo placental transport of amino acids that preferentially use exchange transporters (leucine and phenylalanine) is much more rapid than that of amino acids that do not (glycine and proline). This result could not be predicted from studies of isolated membranes, but was strongly suggested from in vivo studies of nonmetabolizable amino acids across the ovine placenta (7).
Placental amino acid transport uses two sets of transport systems located, respectively, on the maternal and fetal surfaces of the trophoblast (6). Previous in vivo studies have demonstrated that amino acids with the most rapid flux from mother to fetus use exchange transporters located on the fetal surface of the trophoblast that have properties similar to the sodium-independent l system (2, 7). Both branched chain amino acids and phenylalanine have been shown to use this exchange transporter (17). In contrast, methylaminoisobutyric acid, which uses the Na+-dependent A system is essentially trapped within the placenta.
In normal pregnancies the much lower F/M plasma enrichment ratio for
glycine and proline (
0.2) compared with leucine and phenylalanine
(0.8) after the maternal bolus may be due to one or more of several
factors: 1) a smaller transplacental flux, 2) a greater fetal
appearance rate from fetal production, and 3) placental production. We
cannot differentiate whether the lower appearance rate for the
nonessential amino acids is due to fetal and/or placental production of
glycine and proline, which would cause dilution of the isotope within
the fetus or placenta or to a slower transport across the placenta.
However, we know from in vitro studies that glycine and
proline have high affinity for Na+-dependent
(8), nonexchange transport systems. Given the in
vivo methylaminoisobutyric acid results mentioned above, it may
explain the reduced transport rate into the fetal circulation compared
with leucine and phenylalanine (17). The data for leucine
and glycine are in agreement with results previously obtained in AGA
pregnancies by our group (9).
The F/M enrichment ratio for leucine and phenylalanine is significantly reduced in IUGR compared with normal pregnancies. This finding is in agreement with the results of a recent study from our laboratories. This study used a steady state protocol to demonstrate that there was impaired leucine flux in IUGR compared with AGA pregnancies and that the extent of the change in leucine flux was correlated with the clinical severity within the IUGR pregnancy group (18). Surprisingly, there were no differences in the umbilical venous concentrations of the amino acids infused between AGA and IUGR. We believe however, that the number of cases is too small to detect any differences in the amino acid concentrations of the two groups, although significant differences were found in the oxygen content, lactate concentration, and amino acid enrichment ratios. This study is the first demonstration that phenylalanine placental transport in vivo is also affected in IUGR pregnancies. As leucine and phenylalanine are both essential amino acids, the reduction of the F/M enrichment ratio found in the present study is most likely due to decreased transplacental flux in IUGR pregnancies. Studies performed on isolated microvesicles of the human placenta have described defects in more than one amino acid transporter in IUGR (19, 20, 21). This observation coupled with the results of in vivo studies from our group (18) lead us to speculate that both membrane surfaces of the human trophoblast may be affected in IUGR pregnancies.
We found no significant differences for the F/M enrichment ratio of glycine and proline between AGA and IUGR pregnancies. A previous study from our group showed a reduced activity of system A in vesicles isolated from microvillous membrane of IUGR placentas (19). Both glycine and proline have been shown to interact with system A. There are two possible explanations for this discrepancy between the in vitro and in vivo work. The appearance of a labeled amino acid in the umbilical circulation is the result of the transport across the microvillous membrane, the trophoblast cytoplasm and the basal membrane. Therefore, glycine and proline could have similar rates of appearance in the fetal circulation when infused in the maternal circulation of AGA and IUGR even in the presence of different rates of placental metabolism. Further studies are needed to clarify this. However, the F/M enrichment ratio of proline and glycine is significantly correlated to the oxygen content and the lactate concentration in the umbilical vein at fetal blood sampling. This correlation is also present for leucine and phenylalanine, showing that the relative transport rates of these amino acids are related in vivo to conditions of fetal hypoxia and/or lactacidemia that accompany severe IUGR pregnancies and may further reduce amino acid transport across the placenta.
Footnotes
This work was supported by NIH Grants HD-34837 and HD-20761, March of Dimes Grant 1-FY00–216, and MURST ex 60% 2000; Progetto giovani ricercatori, University of Milan 1999.
Abbreviations: AGA, Appropriate for gestational age; F/M, fetal/maternal; IUGR, intrauterine growth-restricted; MPE, maternal plasma enrichment.
Received December 21, 2000.
Accepted May 18, 2001.
References
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