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Human fetal amino acid metabolism at term gestation^1,2,3

¹ From the Department of Pediatrics, Division of Neonatology, Erasmus MC–Sophia Children's Hospital, Rotterdam, Netherlands (CHPvdA, HS, KYD, and JBvG); the Department of Obstetrics and Gynecology, Division of Obstetrics and Prenatal Medicine, Erasmus MC, Rotterdam, Netherlands (EMS, JJD, and EAPS); and the Hospital Pharmacy, Erasmus MC, Rotterdam, Netherlands (AV).

² Supported by Sophia Children's Hospital Fund (grant 459), institutional grant 2006-10 (Erasmus MC–Sophia Children's Hospital, Rotterdam, Netherlands), and Nutricia Research Foundation (independent charity; Nutricia, Wageningen, Netherlands). Both grant suppliers had no involvement whatsoever in the study design, in the collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the report for publication.

³ Reprints not available. Address correspondence to JB van Goudoever, Erasmus MC–Sophia Children's Hospital, Room sp-3433, PO Box 2060, 3000 CB Rotterdam, Netherlands. E-mail: j.vangoudoever{at}erasmusmc.nl.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Knowledge on human fetal amino acid (AA) metabolism, largely lacking thus far, is pivotal in improving nutritional strategies for prematurely born infants. Phenylalanine kinetics is of special interest as is debate as to whether neonates will adequately hydroxylate phenylalanine to the semiessential AA tyrosine.

Objective: Our aim was to quantify human fetal phenylalanine and tyrosine metabolism.

Design: Eight fasted, healthy, pregnant women undergoing elective cesarean delivery at term received primed continuous stable-isotope infusions of [1-¹³C]phenylalanine and [ring-D₄]tyrosine starting before surgery. Umbilical blood flow was measured by ultrasound. Maternal and umbilical cord blood was collected and analyzed by gas chromatography–mass spectrometry for phenylalanine and tyrosine enrichments and concentrations. Data are expressed as medians (25th–75th percentile).

Results: Women were in a catabolic state for which net fetal AA uptake was responsible for 25%. Maternal and fetal hydroxylation rates were 2.6 (2.2–2.9) and 7.5 (6.2–15.5) µmol phenylalanine/(kg · h), respectively. Fetal protein synthesis rates were higher than breakdown rates: 92 (84–116) and 73 (68–87) µmol phenylalanine/(kg · h), respectively, which indicated an anabolic state. The median metabolized fraction of available phenylalanine and tyrosine in the fetus was <20% for both AAs.

Conclusions: At term gestation, fetuses still show considerable net AA uptake and AA accretion [converted to tissue 12 g/(kg · d)]. The low metabolic uptake (AA usage) implies a very large nutritional reserve capacity of nutrients delivered through the umbilical cord. Fetuses at term are quite capable of hydroxylating phenylalanine to tyrosine.

	INTRODUCTION

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These days, many premature infants survive, yet sometimes at the cost of impaired outcomes (1, 2). Inappropriate nutrition is at least partially responsible for suboptimal outcomes, because it negatively affects neonatal growth and brain development (3, 4). For several decades, the international pediatric nutritional goals have been to feed premature infants so that they grow at the same rate that they would have while in utero and thereby mimic fetal tissue composition and quality. Many infants do not reach these targets, however, because growth lags behind. Moreover, the body composition of preterm-born infants is often relatively more adipose at term-corrected age (5). Because hardly anything is known about human fetal metabolism itself, it is not surprising that fetal accretion rates are often not met. It would seem, therefore, that better mimicking of fetal growth could be achieved by putting more effort at unraveling human fetal metabolism. The obtained knowledge could then lead to improved nutritional strategies.

Current knowledge of fetal metabolism is mostly derived from animal data. Technical difficulties and ethical issues are of course causal to the lack of knowledge on human fetal metabolism. However, the use of stable isotopes to study protein metabolism during human pregnancy provides a safe research tool. The quantification of fetal phenylalanine and tyrosine kinetics is of particular importance. It does not only provide information on fetal protein breakdown and synthesis rates in general, but it also quantifies the metabolic conversion (hydroxylation) rate of the essential amino acid phenylalanine to tyrosine. Hydroxylation occurs in the liver and kidneys (6). It is important for 2 reasons: it disposes of excess phenylalanine and provides an alternative source of tyrosine if tyrosine is absent in the diet, for example, because of poor tyrosine solubility in parenteral nutrition. Parenterally fed neonates thus depend on hydroxylation for their tyrosine requirements necessary for net protein accretion. Yet, the enzymatic activity of phenylalanine hydroxylase might be suboptimal in neonates and even older infants, which makes tyrosine a conditionally essential amino acid (7). Tyrosine that is not incorporated into proteins can be degraded and oxidized through the formation of fumarate and acetoacetate. The amount of tyrosine used as a precursor of the catecholamines dopamine, norepinephrine, and adrenaline, is quantitatively negligible. In the present study, our aim was to investigate several aspects of fetal phenylalanine and tyrosine kinetics by analyzing umbilical cord blood after having infused pregnant women with stable isotopically labeled amino acids before elective cesarean delivery at term.

	SUBJECTS AND METHODS

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Setting and subjects
The study was performed at the Mother and Child Center of the Erasmus MC–Sophia Children's Hospital after approval by both the institutional medical ethical review board and the Dutch central committee on research involving human subjects (CCMO, The Hague). Pregnant women undergoing elective cesarean delivery (repeat or breech pregnancy) under spinal anesthesia at term were eligible. Exclusion criteria were maternal obesity [preconceptional body mass index (BMI; in kg/m²) > 30], preeclampsia, diabetes, severe fetal growth restriction (> –2 SD), or known fetal anomalies. Participating women gave written consent after having been fully informed about all of the study details.

Experimental design
To determine the blood flow necessary for our calculations (see below), blood flow velocity and vessel diameters were measured in the umbilical vein with an ultrasound machine (iU22; Philips Medical Systems, Eindhoven, Netherlands) as previously described (8). Ultrasound measurements were made in the late afternoon on the day preceding the cesarean delivery; the cesarean deliveries were all performed at 0800 after an overnight fast.

At least 3 h before planned surgery, the women received a priming dose of L-[1-¹³C]tyrosine (0.5 µmol/kg) that was directly followed by a primed continuous infusion of L-[1-¹³C]phenylalanine [2.5 µmol/kg; 5 µmol/(kg · h)] through a forearm vein. One hour later, a primed continuous infusion of L-[ring-2,3,5,6-D₄]tyrosine [1.5 µmol/kg; 3 µmol/(kg · h)] was started. Isotopes (all >99% enriched and tested for sterility and pyrogenicity) were bought from Buchem BV (Apeldoorn, Netherlands; a local distributor of Cambridge Isotope Laboratories, Andover, MA). Our hospital pharmacy dissolved the isotopes in 0.9% saline, and the solutions were filtered (0.2 µm) and sterilized. Tests were performed to ensure the correct identity, concentration, and a sterile and pyrogen-free product. Tracers were given with the use of Perfusor fm (B | Braun Medical BV, Oss, Netherlands) and Graseby 3000 (Graseby Medical Ltd, Watford, United Kingdom) infusion pumps for the phenylalanine and tyrosine tracers, respectively. Maternal blood was sampled before the tracer infusions started (baseline), immediately before anesthesia, and, if possible (n = 4), 20 min later just before surgery started. Fetal blood was sampled after birth from both the vein and arteries of a doubly clamped segment of the umbilical cord. The fact that there are 2 arteries in the umbilical cord did not affect our results because the concentrations of the amino acids and their enrichments in the blood of both arteries should be equal. After being collected into heparin-containing tubes, blood was centrifuged and plasma was frozen at –80°C until analyzed.

Blood sample analysis
Because calculations in a venoarterial balance model (as on the umbilical cord in the fetus, see below) largely depend on the small differences in concentration and enrichment between the vein and arteries, rather than on the absolute values, measurements must be extremely precise. To minimize the effects of potential analytic measurement errors, samples were prepared for analysis twice by using 2 different derivatization methods [propylchloroformate (PCF) and MTBSTFA; see below]. Each derivatized sample was analyzed in triplicate on 2 different gas chromatography–mass spectrometry (GCMS) devices (see below). Enrichments were calculated from the mean of all 12 analyses; concentrations were calculated from the mean of the 6 analyses by using the N-methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide (MTBSTFA) derivative only.

PCF derivatization of samples was performed by using commercial kits (EZ:Faast for hydrolysates; Phenomenex, Bester BV, Amstelveen, Netherlands) according to the enclosed protocol. As internal standards for concentration determinations, [D₈]phenylalanine and [U-¹³C₉,¹⁵N]tyrosine were added to the samples to be derivatized with MTBSTFA. Concentration calibration curves were prepared by using MTBSTFA as well. Two different enrichment calibration curves were made with either PCF or MTBSTFA derivates. Samples and calibration curves were analyzed with an MSD 5975C Agilent GCMS (Agilent Technologies, Amstelveen, Netherlands) on a VF-17ms (30 m x 0.25 mm internal diameter capillary column (Varian Inc, Middelburg, Netherlands), and a Thermo DSQ GCMS (Thermo Fisher, Breda, Netherlands) on a VF-1701 ms, 30 m x 0.25 mm internal diameter capillary column (Varian Inc).

Calculations
For the calculation of maternal whole-body phenylalanine and tyrosine kinetics, including hydroxylation rates, we used the Clarke and Bier model (9) in combination with the adjustments proposed by Thompson et al (10). To control for pregnancy, we added an extra parameter to the rate of disappearance. In our model, amino acids disappear not only through hydroxylation (or oxidation) or incorporation into protein synthesis but also through net transport to the fetus. The latter is calculated as the umbilical venoarterial concentration difference multiplied with the umbilical blood flow per kilogram maternal weight. If maternal blood was sampled twice before surgery, enrichments were averaged.

We quantified fetal whole-body kinetics using the concept of an umbilical venoarterial balance model. To do so, we rewrote the leucine arteriovenous balance model by Tessari et al (11) and the phenylalanine hydroxylation equation proposed by Nair et al (12) into a phenylalanine and tyrosine model suitable for fetal studies. The model is outlined in Figure 1, and its determinants were calculated by using the following equations, where kg in all units denotes fetal weight (birth weight):

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Schematic model of fetal phenylalanine and tyrosine metabolism. Phenylalanine and tyrosine are delivered to the fetus through the umbilical vein (Equation 1) and (Equation 8). Part of these amino acids are taken up from the fetal intravascular system into the fetal cells (Equation 4) and (Equation 11), whereas the remainder of the intravascular amino acids are transported back to the placenta through the umbilical arteries (Equation 7) and (Equation 14). Amino acids are constantly released from proteins because of proteolysis (Equation 17) and (Equation 20). Part of the available phenylalanine is hydroxylated to tyrosine (Equation 15), incorporated in proteins (Equation 16), or released into the vascular system (Equation 5). Tyrosine is either used for protein synthesis or oxidation (Equation 19) or is released into the vascular system (Equation 12). Finally, phenylalanine and tyrosine are transported back to the placenta through the umbilical arteries (Equation 2) and (Equation 9). Numbers in brackets correspond to the equations in the Methods section and to the fluxes outlined in Table 5.

where [Phe] is the total (labeled + unlabeled) phenylalanine concentration (µmol/L) and BF is umbilical blood flow [L/(kg · h)]. Subscripts indicate whether blood was sampled from the umbilical vein or the arteries (art), as below.

Rate of phenylalanine release from the fetus to the umbilical artery in µmol/(kg · h):

Fraction of phenylalanine in the umbilical vein (%) metabolized intracellularly:

where [¹³C · Phe] is the labeled phenylalanine concentration (µmol/L).

Rate of phenylalanine inflow from the umbilical vein into the intracellular compartment in µmol/(kg · h):

Rate of phenylalanine outflow from the intracellular compartment into the umbilical artery in µmol/(kg · h):

Net fetal phenylalanine uptake in µmol/(kg · h):

Rate of phenylalanine directly released from the umbilical vein to the artery without being metabolized in µmol/(kg · h):

Equations 1 through 7 can also be used for calculations of tyrosine kinetics (using tyrosine concentrations and [D₄]tyrosine enrichments), yielding Equations 8 through 14.

Rate of phenylalanine hydroxylation to tyrosine in µmol/(kg · h):

where D₄ · Tyr· E is the [D₄]tyrosine enrichment (in mole percent excess). Other enrichments are abbreviated accordingly.

Rate of intracellular phenylalanine incorporation into protein (synthesis) in µmol/(kg · h):

Rate of phenylalanine release from proteolysis (breakdown) into the intracellular space in µmol/(kg · h):

Rate of net phenylalanine accretion in µmol/(kg · h):

In our model, we could not discriminate between the 2 major intracellular pathways of tyrosine metabolism, ie, incorporation into protein and oxidation, which is why we used the sum of the latter 2 rates in µmol/(kg · h):

Rate of tyrosine release from proteolysis into the intracellular space in µmol/(kg · h):

Phenylalanine and tyrosine protein synthesis and breakdown rates can be converted from molar rates into grams of protein and grams of tissue under the assumption that 1 g protein contains 246 µmol phenylalanine and 158 µmol tyrosine (13), and new tissue contains 14% protein (14).

Hydroxylation rates in several previously performed whole-body experiments have also been calculated without tyrosine tracer infusion to measure tyrosine kinetics or proteolysis rates (10, 15). The latter are then estimated by multiplying the actual phenylalanine proteolysis rate with an average tyrosine/phenylalanine breakdown ratio (B_Tyr/B_Phe) measured in similar studies or with the theoretical tyrosine/phenylalanine molar content ratio of total-body protein. No equations were as yet available for an arteriovenous balance model; therefore, we developed them ourselves using analogous derivations to the whole-body model by Thompson et al (10). See "Supplemental Data" in the online issue for a listing of these equations, which enable a comparison of our hydroxylation rates with those of Chien et al (16), despite the fact that this group did not infuse their subjects with labeled tyrosine.

In our model, we make the following assumptions: 1) a labeled molecule will not be discriminated from an unlabeled molecule, 2) the labeled molecule will trace the movement of the unlabeled molecules, and 3) the administration of the labeled molecules will not affect the kinetics of the unlabeled molecules.

Statistics
Calculations were made with the use of Microsoft Office Excel software (version 2007; Microsoft Corp, Redmond, WA). Statistical analysis was performed by using GraphPad Prism software (version 4.0; GraphPad Software Inc, San Diego, CA). Because the number of included subjects was relatively small (n = 8), normality distribution of our data could not be determined or assumed. All results are therefore expressed as medians (25th–75th percentiles). Consequently, however, by presenting our data as medians, all rates as outlined in the table on fetal phenylalanine and tyrosine kinetics do not add up correctly as outlined in our model (Figure 1). The fluxes of each individual subject still do, nonetheless.

	RESULTS

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We included 8 fetomaternal dyads. Maternal age, preconceptional and actual BMI, and parity are shown in Table 1. Five of the cesarean sections were performed because of breech presentation; 3 because of a cesarean delivery in the patient's medical history. There were no complications during any of the cesarean sections. Visual inspection of the placentas and umbilical cords showed no abnormalities. Fetal characteristics in terms of gestational age, birth weight, birth weight z score (17), sex, umbilical blood flow, pulsatility index, umbilical arterial pH and base excess, and Apgar score are shown in Table 2. None of the infants had congenital anomalies, and all were discharged from the hospital in good health together with the mother 5 d after birth.

	DISCUSSION

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How the metabolic load of the total conceptus is handled by the gravida is not exactly known. In rats, maternal protein stores initially increase during early pregnancy and are later catabolized to sustain fetal demands necessary for rapid growth (18, 19). In humans, there is no such evidence. However, because protein intake does not seem to be substantially elevated in pregnant women, other mechanisms probably also account for the total accumulation of 925 g protein in various tissues of the fetus during pregnancy (20). Some studies showed unchanged oxidation (21–23), but others showed reduced amino acid oxidation and urea synthesis rates (24, 25) or reduced nitrogen excretion rates (22, 24, 26), probably all to spare nitrogen necessary for fetal growth (26). Maternal phenylalanine hydroxylation rates in our subjects were lower than those found in nonpregnant individuals, but were comparable with those in other pregnant women (15, 21, 27). Relating the other kinetic rates measured in this study to nonpregnant women is difficult. For one thing, the differences are probably more subtle. Moreover, changes in body weight and composition during pregnancy and the contribution of the fetoplacental compartment to maternal metabolism distort comparisons with nonpregnant women.

Whereas the net umbilical uptake of all essential amino acids was considerable in the hereafter cited studies, tyrosine uptake in the term human fetus has been shown to be small or even slightly negative (16, 28–30). During the second trimester of gestation, fetal tyrosine uptake was also found to be absent (31) or small at most (29). Although placental amino acid transporters are capable of transporting tyrosine to the fetus, the in vitro measured tyrosine influx is strongly inhibited by the presence of several other amino acids, even at physiologic concentrations (32, 33). The half-maximal inhibition (K_i) of tyrosine transport across the maternal facing trophoblastic membrane was found to be 68 ± 4.0 µmol/L with phenylalanine (32). This value does not deviate much from the observed maternal phenylalanine concentrations (Table 3). In this light, it is interesting that mothers of growth-restricted fetuses have elevated plasma amino acid concentrations, including phenylalanine (34). Whether this could result in further inhibition of fetomaternal tyrosine transport remains speculative.

Because net fetal tyrosine uptake is probably low, it thus seems that the fetus is largely dependent on endogenous tyrosine synthesis from phenylalanine. Some early in vitro studies reported substantial enzymatic activity in liver extracts from human fetuses aborted in the first or second trimester (35–37). Tyrosine formation in premature neonates immediately after death has also been described (38). One other study describes disappearing phenylalanine hydroxylase capacity during the second half of pregnancy (39), which was confirmed in a deceased premature infant (40). In addition to these in vitro studies, of which none measured renal hydroxylating capacity, only Chien et al (16) attempted to quantify in vivo phenylalanine hydroxylation rates in fetuses at term. They did not simultaneously infuse labeled tyrosine, however, and Nair et al (12) had not published their balance model by then, which resulted in a highly simplified model to determine hydroxylation rates. However, the use of their enrichment results in combination with our Equation 4.5 (provided as "Supplemental data" in the online issue) showed that the hydroxylation rates were very high [ie, 42.6 µmol/(kg · h] with the use of our median B_Tyr/B_Phe of 0.75 and 40.8 µmol/(kg · h) with the use of the theoretical breakdown ratio of 0.64 from deceased fetuses (13)]. Because these rates are much higher than the net umbilical phenylalanine uptake, they seem improbable. Hydroxylation rates in premature and term infants have been measured in several studies over the past 15 y. Mean rates in fasting premature infants receiving only glucose range from 6 to 17 µmol/(kg · h) (41–45). Some studies reported no change (41) when amino acids were also supplemented; others showed a small increase, with means ranging from 11 to 22 µmol/(kg · h) (42–44). Shortland et al (45) measured hydroxylation rates of 48 µmol/(kg · h) after amino acid supplementation (46), but these rates appear to be overestimated. Rates in healthy term infants do not seem to be different from rates in preterm infants and range from 8 to 13 µmol/(kg · h), irrespective of nutrient administration (42, 47). Our observed hydroxylation rate of 7.5 (6.2–15.5) µmol/(kg · h) agrees with the postnatal values.

Fetal growth velocities at 38 wk gestation are 8–10 g/(kg · d). Our observed growth rates [12.2 (5.4–21.3) g tissue/(kg · d)], calculated from the accretion rate of one amino acid, are not much different. Potential errors in the conversions to proteins and body weight and measurement errors are probably responsible for the small difference.

Much to our surprise, the metabolized fraction of available fetal phenylalanine and tyrosine was only 20% for both amino acids. Calculations on the data for term human fetuses by Chien et al (16) also show a metabolic uptake of 26% for phenylalanine and 36% for leucine. In ovine fetuses, we calculated from available data (48) a fraction of 25% for leucine, which does not seem to differ between normally grown and growth-restricted fetal animals. This implies that most amino acids entering the fetus through the umbilical cord remain intravascular before returning to the placenta through the umbilical arteries. Thus, it appears that the placenta provides the fetus with an enormous reserve capacity of these amino acids. Intrauterine growth restriction would, therefore, not seem to be primarily caused by a lack of amino acids.

All our concentration and enrichment measurements were done in the plasma compartment, rather than in whole blood because of analytic advantages. Many studies, however, reported rapid equilibrium between erythrocyte and plasma concentrations of various amino acids, including phenylalanine and tyrosine (49–51). The role of erythrocytes in organ amino acid delivery is thus as important as the role of the plasma compartment. Compared with normal organ balance studies, the circulation time of blood in fetal balance studies is relatively long because blood from the umbilical vein flows through the whole fetus before returning to the umbilical arteries. By then, complete mixing can be expected. Even in single organ balance studies, many groups chose to use plasma sampling in combination with whole blood flows rather than plasma flows (52–55). The latter would reduce all kinetic rates by 40% (hematocrit) and yield improbably low kinetic rates.

Whether maternal anesthesia and surgery would have any consequences on fetal metabolism remains speculative. Spinal anesthesia might result in maternal hypotension and blood flow redistribution, but these effects can be prevented by using a lateral wedge. Furthermore, blood pressure monitoring allows for prompt correction if necessary. Besides, the pulsatility index of the umbilical artery does not seem to be influenced by spinal anesthesia (56). Konje et al (57) measured flow using a transonic time flowmetry technique on a exteriorized loop of the umbilical cord during cesarean surgery. Their flow values halfway during surgery correspond well to our flow measurements. Besides, umbilical blood flow after vaginal delivery has been reported to be stable for the first 100 postnatal seconds (58). We thus assume that umbilical blood flow is fairly constant during surgery. The fetal metabolic response to maternal surgery remains speculative. A maternal noradrenaline surge after an invasive procedure did not seem to reach the human fetus (59). In mice, however, noradrenaline was suggested to have reached the placenta (60).

To conclude, we showed that the fetus at term receives considerable amounts of phenylalanine from the placenta. Nevertheless, the fetus actively uses only a relatively small part, two-thirds of which are used for net protein synthesis and one-third for hydroxylation to the semiessential amino acid tyrosine. Whether these findings would also hold true for the growth-restricted fetus or a fetus earlier in gestation remains to be elucidated.

	ACKNOWLEDGMENTS

 
We especially thank all of the participating women. We also thank Frans te Braake and Willemijn Corpeleijn. The personnel from the obstetrical and anesthesiologic departments were a great help at hand collecting all of the material and providing the facilities. The Sophia Children's Hospital Fund and the Nutricia Research Foundation had no influence on the study design, results, or publication.

The authors' responsibilities were as follows: CHPvdA, JJD, EAPS, and JBvG: participated in the design and implementation of the study, including recruitment of patients; AV: prepared and tested all intravenous stable-isotope solutions; EMS: performed the ultrasound measurements; CHPvdA: collected and prepared the blood samples for analysis; HS and KYD: provided technical supervision of the blood sample preparation and performed the mass spectrometry analyses; CHPvdA, HS, KYD, and JBvG: analyzed the data; and CHPvdA: wrote the manuscript draft. All of the authors reviewed the manuscript and approved the final version. None of the authors had a personal or financial conflict of interest.

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