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Neutral Amino Acid Uptake by the Microvillous Plasma Membrane of the Human Placenta Is Inversely Related to Fetal Size at Birth in Normal Pregnancy¹

Medical Research Council Environmental Epidemiology Unit, University of Southampton, Southampton General Hospital (K.M.G., N.M., C.W.), Southampton, United Kingdom SO16 6YD; the Department of Child Health and School of Biological Sciences, University of Manchester, St. Mary’s Hospital (J.G., C.P.S.), Manchester, United Kingdom M13 OJH; and the Institute of Human Nutrition, University of Southampton (A.J.), Southampton, United Kingdom SO9 3TU

Address all correspondence and requests for reprints to: Dr. Keith Godfrey, Medical Research Council Environmental Epidemiology Unit, University of Southampton, Southampton General Hospital, Southampton, United Kingdom SO16 6YD. E-mail: kmg{at}mrc.soton.ac.uk

	Abstract

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Understanding the physiological regulation of fetal growth is important, as normal variations in size at birth relate to differences in neonatal and adult health. Although fetal growth directly reflects net placental transfer, little is known about how normal fetal growth relates to the transfer capabilities of the placental epithelium, the syncytiotrophoblast. The Na⁺-dependent and Na⁺-independent uptakes of methylaminoisobutyric acid (MeAIB) by vesicles prepared from the syncytiotrophoblast microvillous plasma membrane give measurements of system A neutral amino acid transporter activity and diffusive permeability, respectively. In 62 normal pregnancies, we related vesicle MeAIB uptakes to neonatal anthropometry. Smaller babies with a lower abdominal circumference had higher placental system A activity per mg membrane protein (P = 0.004); activity rose from 0.020 to 0.043 nmol/30 sec/mg protein as abdominal circumference fell from 34.6 cm or more to 32.0 cm or less. Within the normal range of fetal and placental size, this may reflect a tendency toward compensatory up-regulation of the placental system A transporter in smaller babies. Babies with a lower abdominal circumference also had higher Na⁺-independent MeAIB uptakes (P = 0.0005); this could reflect important compositional changes in the microvillous plasma membrane, leading in vivo to increased back-diffusion of amino acids out of the syncytiotrophoblast.

	Introduction

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UNDERSTANDING the influences that determine fetal size and tissue composition at birth is important, as these are major determinants of the baby’s subsequent health in postnatal life. Infants who were either growth retarded or macrosomic at birth are at increased risk of mortality and morbidity during the neonatal period (1, 2), and follow-up studies have shown that normal variations in weight, thinness, and abdominal circumference at birth are associated with differences in the rates of coronary heart disease, hypertension, and noninsulin dependent diabetes in adult life (3, 4, 5, 6, 7).

Fetal size and tissue composition at birth reflect the net transfer of solute and water across the placenta during pregnancy, as this organ provides the major pathway for materno-fetal exchange. The net transfer of small lipophilic solutes, such as O₂ and CO₂, is primarily dependent on uterine and umbilical blood flow (8), and impairment of the uteroplacental and/or fetoplacental circulations is associated with restricted fetal growth. For hydrophilic solutes, alterations in materno-fetal exchange are determined by changes in the forces driving transfer, such as electrochemical gradients between plasma in maternal and fetal placental microvasculature, and by changes in the transporting epithelium of the placenta, the syncytiotrophoblast. A variety of mechanisms mediate solute transfer across the syncytiotrophoblast (8, 9); although few of these have been related to fetal size, some information is available for one amino acid-transporting mechanism.

Amino acid transfer across the syncytiotrophoblast is mediated by transcellular mechanisms using transport proteins in its microvillous (maternal facing) and basal (fetal facing) plasma membranes (8); each transport protein is specific for a particular class of amino acid. The activity in the microvillous plasma membrane of one of these, the system A transporter, specific for short side-chain neutral amino acids, is lower per mg membrane protein in placentas from growth-retarded babies than in placentas from appropriately grown babies (10, 11). Although this suggests a direct relationship between system A transporter activity and fetal growth, system A activity is also lower in the placentas of macrosomic babies born to diabetic women than in placentas of appropriately grown babies from uncomplicated pregnancies (12). Either or both of these findings may, however, have been confounded by placental pathology at extremes of abnormality. Consequently, here we examined the relationship between the activity of the system A transporter in the syncytiotrophoblast microvillous plasma membrane and anthropometric measurements of fetal size at birth in a group of women with normal pregnancies. The vesicle technique used (11, 13) also gave a measure of the diffusive permeability of the microvillous plasma membrane to the nonmetabolizable amino acid analog methylaminoisobutyric acid (MeAIB), and this was also related to neonatal anthropometry.

	Subjects and Methods

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This study was approved by the local ethics committee. The subjects, who gave informed consent, were drawn from a parallel study of fetal growth rates among 562 white Caucasian women, aged 16 yr or older, with singleton pregnancies and known menstrual dates who attended the antenatal booking clinic at the Princess Anne Hospital (Southampton, UK) before 17 weeks gestation. This represented 93% of a group of 604 consecutive referrals who fulfilled the above entry criteria and delivered within the district. Four infants were stillborn or died during the neonatal period, 3 had major congenital abnormalities, and 15 were delivered before 252 days (36 weeks) gestation, leaving 540 infants without major congenital abnormalities born after 36 weeks gestation who survived the neonatal period.

The women were visited by a trained research nurse in early pregnancy (median gestation, 14.7 weeks) and were interviewed again around 28 weeks. They were asked about their menstrual and obstetric histories and current smoking habits and were requested to ask their family about their own birth weight. Body mass index was derived as weight/height², using the woman’s first recorded weight in pregnancy and her height measured in the antenatal clinic. Social class was allocated according to the current or last occupation of the baby’s father (14).

After delivery, the baby and trimmed placenta were weighed using digital scales, and the baby’s crown-heel length and head, abdominal, and mid-upper arm circumferences were measured as described for a previous study (15). Tests of repeatability have shown that discrepancies in measurements between fieldworkers were small in comparison with the overall between-subject SD (15). The ponderal index (birth weight/length³) and the ratio of head to abdominal circumference were derived as measures of thinness and disproportion at birth. Duration of gestation at birth was calculated from the woman’s menstrual dates (16) or from early pregnancy ultrasound data (16) if this indicated that the menstrual dates were unlikely to be correct.

Preparation of microvillous plasma membrane vesicles

The initial preparation of microvillous plasma membrane vesicles took 6 h per placenta and had to commence soon after delivery; subjects were therefore selected on the basis of having delivered in the morning. The 62 women studied were shorter than other women in the cohort (mean height, 1.62 vs. 1.64 m; P = 0.05) as they included an excess delivered by cesarean section (48% vs. 12%); otherwise, their characteristics and those of their babies (Table 1), were similar to those of pregnancies from whom vesicles were not prepared.

Measurement of Na⁺-dependent and Na⁺-independent MeAIB uptakes

MeAIB uptake into vesicles was measured at room temperature in the presence and absence of an inwardly directed Na⁺ gradient, as previously described (11). As the system A transporter is Na⁺ dependent, its activity can be measured as the initial rate of Na⁺-dependent MeAIB uptake, as this is transported relatively specifically by system A (19). Uptake was initiated by the addition of vesicles (protein concentration, 10–22 mg/mL) to extravesicular buffer containing 145 mmol/L NaCl or KCl (11) and was stopped at 30 sec (approximating to the initial rate) (11, 12) or at 24 h (equilibrium) (11, 12) by the addition of ice-cold stop buffer (11). The resulting solution was applied to a 0.45-µm Millipore filter (Millipore Corp., Bedford, MA) under vacuum and washed with ice-cold stop buffer. Filters were dissolved in 2-ethoxyethanol and counted in an LKB Wallac MiniBeta liquid scintillation counter after the addition of Optiphase Hisafe II scintillation fluid (Wallac, Milton Keynes, UK). Counts were corrected for counts retained on the filters in the absence of protein. Radioisotopes were obtained from New England Nuclear (Stevenage, UK), and chemicals were purchased from Sigma (Poole, UK) or Merck (Lutterworth, UK).

Statistical analysis

We related MeAIB uptakes to the methodological variables and then to the womens’ characteristics and neonatal anthropometry using the tabulation of means, two-sample t tests, and linear regression treating measurements as continuous variables. Measurements whose distributions were skewed were transformed by taking square roots or logarithms to satisfy assumptions of normality inherent in the statistical analyses. For clarity of presentation, back-transformed values are shown in the tables. Adjustments for alkaline phosphatase enrichment and room temperature were made by inclusion of the enrichment factor and indicator variables for room temperature and the absence of a temperature reading in multiple regression analyses.

	Results

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Measurements of alkaline phosphatase activity in microvillous plasma membrane vesicles showed satisfactory enrichment in all 62 preparations (enrichment factor range, 10.5–37.7; median, 22.9-fold). The enrichment factor was not related to placental weight or to any of the measures of fetal size. As in our previous studies (11, 12), experiments measuring vesicle MeAIB uptake at frequent intervals showed that this was linear up to 30 sec; equilibrium uptake was achieved by 24 h (data not shown).

Table 1 shows the characteristics of the 62 women and infants together with the initial (30 sec) Na⁺-independent MeAIB uptake (with K⁺ alone in the extravesicular buffer) and system A activity (30 sec Na⁺-dependent MeAIB uptake (uptake with Na⁺ in the extravesicular buffer minus that with K⁺ alone)). Uptakes were positively skewed, and statistical analyses used square root transformed values. System A activity was highly correlated with Na⁺-independent MeAIB uptake (r = 0.77; P < 0.001), reflecting a very strong correlation between the MeAIB uptake in extravesicular buffers containing Na⁺ and K⁺ (r = 0.88; P < 0.001).

Effect of time delay, room temperature, and vesicle enrichment and storage

The median delay between delivery of the placenta and the start of vesicle preparation was 70 min (inter-quartile range, 47–86 min; range, 20–199 min) and was unrelated to alkaline phosphatase enrichment or MeAIB uptakes. Ambient room temperature was recorded for all except the first four preparations; it ranged from 18–26 C and was not related to alkaline phosphatase enrichment. System A activity and Na⁺-independent MeAIB uptake rose with increasing room temperature (P = 0.04 and P = 0.005 respectively); both also rose with increasing alkaline phosphatase enrichment, particularly after taking account of room temperature (adjusted P values of 0.05 and 0.0002 for system A activity and Na⁺-independent MeAIB uptake, respectively; Table 2).

Maternal and neonatal characteristics

Although system A activity was not related to maternal smoking or body mass index in early pregnancy, Na⁺-independent MeAIB uptake was lower in women who smoked, particularly in those who smoked at 28 weeks gestation; independently of maternal smoking, Na⁺-independent MeAIB uptake was also lower in women with a higher body mass index in early pregnancy (Table 2). System A activity, but not Na⁺-independent MeAIB uptake, tended to be lower in vesicles prepared from the placentas of higher social class women (P = 0.14 and P = 1.0, respectively). Taking account of room temperature and vesicle enrichment had little effect on the associations between Na⁺-independent MeAIB uptake and maternal smoking and body mass index, but did strengthen the association between system A activity and social class (Table 2). Neither system A activity nor Na⁺-independent MeAIB uptake was related to the woman’s parity, height, own birth weight, age, or mode of delivery (vaginal vs. cesarean section; data not shown).

Of the 62 infants, 32 were boys and 30 were girls; birth weight ranged from 2580–4820 g. The infant’s gender and duration of gestation at delivery were not related to MeAIB uptakes. Table 3 shows that both system A activity and Na⁺-independent MeAIB uptake tended to increase with decreasing placental weight and size at birth and with an increasing ratio of head to abdominal circumference; uptakes were not related to ponderal index. The strongest associations, before and after adjustment for room temperature and vesicle enrichment, were with abdominal circumference; as this fell from 34.6 cm or more to 32.0 cm or less, system A activity rose from 0.020 to 0.043 nmol/30 sec/mg protein, and Na⁺-independent MeAIB uptake rose from 0.012 to 0.019 (P = 0.004 and P = 0.0005, respectively, for linear trends treating measurements as continuous variables and adjusting for room temperature and vesicle enrichment). After taking account of abdominal circumference, no additional variance in system A activity or Na⁺-independent MeAIB uptake was explained by other neonatal anthropometric measurements. Regression analyses showed that these associations were independent of the vesicle storage conditions, maternal height, and infant’s sex and duration of gestation and were similar in women that had vaginal and cesarean section deliveries. The magnitude and statistical significance of the associations of Na⁺-independent MeAIB uptake with neonatal abdominal circumference and maternal smoking and body mass index were little changed by simultaneously taking all three variables into account.

The median inter-quartile range equilibrium MeAIB uptake (at 24 h in the presence of extravesicular Na⁺) was 1.35 (0.44–2.58) nmol/mg protein; values were transformed by taking logarithms to satisfy assumptions of normality. Equilibrium uptake was not related to room temperature or vesicle enrichment, but was higher in vesicles frozen before the measurements (0.95 vs. 2.03 nmol/mg protein; P = 0.02). Equilibrium MeAIB uptake was not related to any of the maternal, placental, or fetal variables, except for tending to be higher in primiparous vs. multiparous pregnancies (P = 0.05, unadjusted; P = 0.1, adjusted for vesicle freezing) and in lower weight placentas (P = 0.006, unadjusted; P = 0.01, adjusted for vesicle freezing).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
To study how normal fetal growth relates to the transfer capabilities of the maternal-facing microvillous plasma membrane of the syncytiotrophoblast, we have related a specific component of placental amino acid transport (system A activity) to fetal size and proportions at birth in 62 normal pregnancies. The vesicle technique used also provided measurements of the Na⁺-independent uptake of MeAIB, which is thought to give a measure of diffusive permeability to this solute (see below). The placentas studied included an excess from shorter women delivered by cesarean section. System A activity and Na⁺-independent MeAIB uptake were not, however, related to the woman’s height or mode of delivery, and the associations that we found were independent of height and mode of delivery. It seems unlikely that our findings resulted from biases associated with selection of the women.

Vesicle integrity, purity, and transporter activity

We studied placentas from women recruited in early pregnancy who often delivered within hours of each other and had to accept a variable delay between delivery and the start of vesicle preparation. Neither this delay nor the mode of delivery was related to the alkaline phosphatase enrichment (a measure of vesicle purity) or to MeAIB uptakes, and the distributions of enrichment and MeAIB uptakes were similar to those in previous reports (10, 11, 12). This suggests that the activities measured are stable within the microvillous plasma membrane. Although the effect of room temperature on MeAIB uptakes indicates that our findings should be interpreted cautiously, adjustment for this variable tended to strengthen the associations found. Correlations between MeAIB uptakes and vesicle enrichment are to be expected and emphasize the need to check this in comparisons of MeAIB uptake rates (11, 12).

All activity data are normalized per mg membrane protein. Little is known about the quantitative relationship between vesicle lipid, nontransporter protein, and transporter protein contents, and the associations found could represent differences in overall vesicle composition rather than in transporter activity. The steep rise in system A activity with increasing ambient temperature is, however, as expected for a solute/transporter protein reaction; the shallower rise in Na⁺-independent MeAIB uptake with increasing temperature is consistent with diffusion (20).

System A activity and maternal and neonatal characteristics

Previous reports have suggested that maternal smoking might affect Na⁺-dependent amino acid transport by the placenta (21, 22). We found that system A activity was not related to maternal smoking or to the woman’s parity, age, height, or body mass index; this suggests that the effects of these influences on fetal growth (23) are not mediated by alterations in placental amino acid transporter activity. The weak relation between lower social class and higher system A activity could reflect the effects of maternal nutrition.

System A activity was inversely related to placental weight and measures of size at birth, particularly neonatal abdominal circumference. This is consistent with our observation that system A activity is reduced in placentas from macrosomic babies born to diabetic women (12), but it is opposite that expected from the observation that system A activity is lower in placentas from growth-retarded fetuses (10, 11). Whereas placentas from growth-retarded fetuses are morphologically grossly abnormal (24), those from well controlled diabetic women appear to be relatively little affected (25). We therefore postulate that the normal relationship between system A activity per mg membrane protein (taken to be equivalent to the number of transporters per unit microvillous plasma membrane) and fetal and placental sizes is the inverse one, tending to keep the total number of transporters per fetus and placenta approximately constant. In fetal growth retardation, we propose that placental pathology may lead to an abnormal reduction in transporter activity per mg membrane protein that, combined with smaller placental size, may compromise provision of the neutral amino acids carried on system A, including glycine, alanine, and serine. This might be particularly important, as maternal intakes and endogenous production of glycine may not always be sufficient to meet the demand in pregnancy (26, 27).

Equilibrium uptake gives a measure of vesicle volume, which, because of surface area/volume ratio considerations, might influence transport activity measurements. The relation between equilibrium uptake and placental weight suggests that changes in surface area/volume ratio may have influenced the relations between system A activity and Na⁺-independent MeAIB uptake and placental weight. Equilibrium uptake was not, however, related to fetal size at birth.

Na⁺-independent MeAIB uptake

Studies of syncytiotrophoblast plasma membranes suggest that Na⁺-independent MeAIB uptake is either via a low affinity Na⁺-independent transporter or by diffusion and is not mediated by a transport protein (28, 29). The latter is supported by observations that other inert hydrophilic solutes of similar size or larger than MeAIB can diffuse across the lipid bilayer of the microvillous plasma membrane (30) and by the relatively shallow rise in Na⁺-independent MeAIB uptake with increasing ambient temperature. In vivo, a diffusional pathway would result in net efflux of amino acid out of the syncytiotrophoblast, as intracellular concentrations are higher than those in maternal plasma (31).

Na⁺-independent MeAIB uptake was lower in women who smoked and in those with a high body mass index. These findings need replication in further studies, but could reflect the effects of maternal smoking and fatness on the profile and peroxidation of lipids in the microvillous plasma membrane (32). Counter to expectations based on the lower Na⁺-independent MeAIB uptake of vesicles from placentas of growth-retarded babies (11), Na⁺-independent uptake was inversely associated with fetal size at birth; the associations tended to be stronger than those for system A activity.

Physiological implications

Although our data cannot explain why system A activity and Na⁺-independent MeAIB uptake are highly correlated with each other and are both associated with neonatal abdominal circumference, some speculation as to the possibilities is instructive. 1) Changes in plasma membrane protein content or lipid composition may be of importance, as our measurements are normalized per mg membrane protein (see above), and lipid composition can affect both transport protein activity and solute diffusion (33). 2) A lower abdominal circumference reflects a smaller fetal liver. This may impair the metabolic activity of the fetus and lead to alterations in placental composition and metabolism. Recent studies in sheep have, for example, shown amino acid cycling between the fetal liver and the placenta; transport of maternal glycine by the placental system A transporter is supplemented by substantial glycine production in the placenta from serine originating in the fetal liver, and a proportion of the glycine from these two sources is then cycled back to serine in the fetal liver (34). Babies with a small abdominal circumference could increase placental system A activity as an adaptation to impaired amino acid cycling by the fetal liver. 3) There could be a direct biological connection between system A activity and the diffusional pathway represented by Na⁺-independent MeAIB uptake. For example, a higher microvillous plasma membrane permeability in smaller babies might increase back-diffusion of amino acids from the syncytiotrophoblast to maternal blood, lowering cytosolic amino acid concentrations and either reducing transinhibition (35) or up-regulating expression of the transporter.

Although it is energetically disadvantageous for the microvillous plasma membrane to have pathways for both active uptake and diffusional loss of amino acid, the latter may be an unavoidable property of the lipid bilayer. In vivo the flux through each pathway will be influenced by the prevailing electrochemical gradients, by the activity of the Na⁺ pump maintaining a Na⁺ gradient for amino acid uptake on system A, and probably by hormonal effects (36). Although insulin and insulin-like growth factor I acutely increase cytotrophoblast system A activity in vitro (37, 38), the effects of these hormones cannot explain our findings, as their plasma concentrations are lower in smaller fetuses (15, 39).

Control of the activity of transport moieties in the plasma membranes of the syncytiotrophoblast requires much further study. Our data suggest that such control together with compositional changes in the microvillous plasma membrane could exert effects of fundamental importance in the physiological regulation of fetal nutrient transfer, with consequent short and long term effects on the fetus.

	Acknowledgments

 
We thank the mothers who gave us their time; the antenatal clinic, labor, and postnatal ward staff for their assistance; Mr. T. Wheeler, Prof. E. J. Thomas, and Mr. P. Gillibrand for allowing us to include their patients; Mr. S. Grant for performing ultrasound scans; and Drs. I. Campbell and G. Phillips for technical assistance. Fieldwork was carried out by J. Hammond, V. Davill, L. Greenaway, and C. Sloan. We also thank Prof. Robert Boyd for his helpful comments.

	Footnotes

 
¹ This work was supported by the Wellcome Trust and the Wessex Medical Trust; subjects in this study were drawn from a cohort study funded by WellBeing and the Medical Research Council.

Received February 13, 1998.

Revised April 29, 1998.

Accepted June 16, 1998.

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