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IUGR pregnancies represent a serious obstetrical problem, both because of their frequency and because of the mortality and morbidity they cause. IUGR is associated with many complications of pregnancy, including pregnancy-induced hypertension, chronic vascular disease, nutritional and metabolic diseases including diabetes, and with maternal drug abuse(1). In addition, it is associated with increased fetal mortality and morbidity during pregnancy and with increased neonatal mortality and morbidity after delivery(2). It has also been associated with long-term effects into adulthood(3,4). Because of these problems, it has stimulated research into techniques to assess clinical severity(5–10).

In an ovine model of severe IUGR, two studies have shown that the fetal and placental fluxes of two essential amino acids, leucine and threonine, are altered in IUGR fetuses. Specifically, the transplacental transport of L-[1-13C]-leucine and of L-[1-13C]-threonine are significantly reduced(11,12). As a consequence, the fetal enrichment of the stable isoptomers of both amino acids is lower with respect to maternal steady state enrichments, compared with normal pregnancies(11,12).

The present study was undertaken to verify whether, in human pregnancies complicated by IUGR, the transplacental flux of leucine is modified compared with normal pregnancies. It also tests whether the magnitude of the change in steady state fetal/maternal leucine enrichments is correlated with the clinical severity of IUGR determined by fetal heart rate and fetal arterial velocimetry data.

METHODS

The study was performed in the Department of Obstetrics and Gynecology of the San Paolo Institute of Biomedical Sciences in Milan, Italy. The protocol of the study was approved by the San Paolo Ethical Committee and by the Colorado Multiple Institute Review Board of the University of Colorado Health Sciences Center. Informed consent was obtained from all patients.

Patients. The clinical data are presented in Table 1. Complete maternal and umbilical venous data were obtained from 4 of 6 AGA and 14 IUGR, between 26.6 and 38.3 weeks, at the time of in utero FBS. All pregnancies included in the study were scheduled for fetal blood sampling for clinical indications. FBS was performed under ultrasonographic guidance, and fetal blood was sampled from the umbilical vein, as described by Marconi et al.(13). Two additional normal pregnancies were studied. In one of six normal pregnancies, the umbilical artery was sampled because of the fetal position in utero; in another pregnancy, no fetal blood sample was obtained. In all pregnancies, gestational age was determined by the last menstrual period and was confirmed by ultrasound at 18-21 weeks of gestation.

Table 1 Clinical data on pregnancies
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In AGA fetuses, the indication for FBS was maternal thrombocytopenia. Fetal platelet counts were within the normal range for all fetuses included in the study. No other pathologic conditions were present in AGA pregnancies. 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 the Italian standards for birth weight and gestational age(14).

In IUGR fetuses, FBS 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 FHR was recorded before FBS. IUGR pregnancies were classified into three groups by clinical severity based on a classification previously proposed(6). The PI of the umbilical artery was measured by Doppler velocimetry: a coaxial pulsed Doppler velocimeter with a sample volume of 5 mm and a high-pass filter set at 100 Hz were used with the lowest possible settings for energy output. For each reading, three consecutive waveforms were measured on hard copies by means of a computerized planimeter. The pulsatility index was measured according to the Gosling formula (systolic velocity minus diastolic velocity divided by mean velocity); the mean velocity was calculated by dividing the area of the maximal velocity by the length of the cycle(15). PI values were considered abnormal if more than two standard deviations above the mean of the normal fetuses. The reference values were those obtained in our laboratory from a cross-sectional study of 440 normal fetuses(16).

FHR was recorded by a cardiotocography (HP series 50A); FHR was considered abnormal if at least one of the following patterns was present: (1) less than two accelerations of the heart rate to an amplitude of ≥10 beats/min lasting ≥15 sec during a period of at least 30 min; (2) variability of ≤5 beats/min during a period of at least 60 min; (3) U-shaped (late) decelerations in the heart rate after Braxton Hicks contractions(6).

IUGR patients were classified as follows: (Table) The percent reductions of the abdominal circumference for IUGR fetuses are presented in Table 1.

Protocol of the study. L-[1-13C]-leucine (98.8 atom % pure) was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). The tracer was dissolved in sterile isotonic saline for i.v. administration and tested for sterility and pyrogenicity. 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.

A prime (1.03 ± 0.2 µmol · kg-1) followed by a constant infusion (0.165 ± 0.07 µmol · kg-1 · min-1) of L-[1-13C]-leucine was given into a peripheral vein to the mother. Mean infusion time was 125.7 ± 43 min. Maternal samples from the brachial vein were "arterialized" as described by Sonnenberg and Keller(18) and were obtained at time 0 and every 20 min, starting 60 min after the infusion began. The "arterialization" of the brachial venous samples was obtained by placing the arm between two heated thermophores whose temperature was adjusted until the maternal peripheral blood oxygen saturation was maintained >90%: the sampling site of the brachial vein was proximal to the wrist. Many metabolic studies have demonstrated that amino acid samples obtained from a heated dorsal hand vein approximate those obtained simultaneously from arterial blood. Similar results have been obtained for plasma isotopic enrichments of [13C]leucine and [13C]KIC(19).

Fetal blood sampling was performed after 114 ± 42 min. Blood for all analyses was collected into heparinized syringes and immediately stored on ice. pH, Po2, and Pco2 were measured with an ABL 330; Hb concentration and O2 saturation were measured with a Radiometer OSM3 Oximeter. Glucose and lactate concentration were measured in duplicate with a Yellow Springs Analyzer. All analyses were completed within 10 min after sampling. O2 content was calculated according to the formula: O2 cont [mmol·l-1] = Hb conc [g·l-1]·O2 sat·0.05982 After centrifugation, the plasma was kept at -18°C until analysis by HPLC amino acid chromatography and by gas chromatography-mass spectroscopy, using a HP 5972A mass spectrometer equipped with a HP 5890 gas chromatograph.

Analytical method and calculation. Plasma leucine and KIC enrichments were determined by gas chromatography-mass spectroscopy: tert-butyldimethylsilyl derivatives of leucine were prepared and analyzed in triplicate. KIC was prepared as quinoxalinol TMS derivatives and analyzed in triplicate(20,21). Plasma leucine and KIC enrichments (MPE) were calculated by using the difference in peak area ratios between enriched and unenriched samples. Peaks were read at 303 and 302 m/z and at 233 and 232 m/z, respectively.

Plasma leucine concentrations were measured by ion-exchange chromatography on an automated amino acid analyzer (Kontron Chromakon 500). Tracer and tracee concentrations were calculated according to the following formulas: Tracer conc = total conc·mpe·0.01 Tracee conc = total conc - tracer conc As the MPE can be written as tracer/(tracer + tracee), the tracer/tracee ratio is equal to: Tracer/tracee = mpe/(1 - mpe)

The F/M MPE ratio was calculated as the ratio between fetal plasma leucine enrichment and the maternal plasma leucine enrichment at steady state, the latter calculated as the mean of the three or four samples taken over the last hour of infusion. The maternal leucine DR was calculated as: DR (µmol·kg-1·min-1) = Inf. Rate (µmol·kg-1·min-1)·[(MPEi/MPEp) - 1] where MPEi = enrichment of the infusate; MPEp = enrichment of the maternal plasma at steady state. For the 10 patients in whom [13C]-KIC enrichments were determined, the maternal leucine DRKIC was calculated where MPEp = KIC enrichment in maternal plasma. The two normal pregnancies from which there were no fetal umbilical venous samples were included only for the calculation of the maternal plasma leucine disposal rate.

Statistics. All data are expressed as mean ± SEM. The significance of the difference among groups was calculated with a two-tailed unpaired t test. Regression analysis was performed by the least-squares method.

RESULTS

Table 2 presents the mean oxygenation and acid base data for AGA and IUGR groups at the time of in utero blood sampling. In each patient, the maternal plasma enrichments were plotted versus time to verify maternal steady state enrichments. Leucine concentrations were only slightly higher in the umbilical vein than in the maternal "arterialized" vein both in AGA (0.133 ± 0.04 versus. 0.124 ± 0.02) and in IUGR pregnancies (0.126 ± 0.03 versus 0.118 ± 0.01), but there were no significant differences between normal versus IUGR pregnancies.

Table 2 Oxygenation and pH at the time of FBS (mean ± SEM)
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Table 3 presents the maternal and fetal L-[1-13C]-leucine-enrichment values and the F/M leucine-enrichment ratio at steady state for each pregnancy. The F/M leucine-enrichment ratio was 0.89 ± 0.04 in normal pregnancies. This ratio was significantly lower in IUGR pregnancies (0.7 ± 0.08) than in AGA pregnancies (p < 0.001). Furthermore, the F/M ratio in IUGR pregnancies decreased significantly from group 1 to group 3, i.e., with increasing clinical severity. Figure 1 presents the mean F/M leucine MPE ratios for AGA and for the three groups of IUGR pregnancies. The results are similar when the calculated tracer/tracee ratios are used to compare groups (Fig. 2).

Table 3 Maternal and fetal plasma leucine enrichments at steady state
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Figure 1
figure 1

Leucine F/M enrichment ratio in AGA and IUGR pregnancies at FBS.

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Figure 2
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Fetomaternal tracer/tracee leucine ratio in AGA and IUGR pregnancies at FBS.

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To further compare the F/M leucine-enrichment ratio with indexes of clinical severity, this ratio was examined versus umbilical venous oxygenation and lactacidemia. There was a significant correlation between F/M plasma leucine-enrichment ratio and umbilical venous oxygen content (F/M leu MPE ratio = 0.054 UVO2 cont + 0.44; p < 0.002, r2 = 0.47; Fig. 3a), and between F/M plasma leucine-enrichment ratio and umbilical venous lactate concentration (F/M leu MPE ratio = -0.013 UVlac conc + 0.89; p < 0.001, r2 = 0.53; Fig. 3b). In contrast, no significant correlations were found when the F/M leucine MPE ratio was compared with either umbilical venous pH or base excess. Maternal leucine disposal rate calculated by using plasma leucine-enrichment was not different in AGA and IUGR mothers (1.73 ± 0.4 versus 1.54 ± 0.5 µmol · kg-1 · min-1, respectively) with a significant positive relationship (Fig. 4) between maternal plasma leucine DR and maternal leucine concentration according to the equation: Leu DR (µmol·kg-1·min-1) = 0.36 + 11.2 · Leu conc (µM); r2 = 0.33; p < 0.01

Figure 3
figure 3

Leucine F/M enrichment ratio vs. umbilical venous oxygen content (a) and vs. umbilical venous lactate concentration (b) in AGA and IUGR fetuses at FBS.

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Figure 4
figure 4

Maternal leucine plasma disposal rate vs. maternal leucine plasma concentration in AGA and IUGR.

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The KIC concentration and enrichments were not measured in all IUGR patients (in only 40%); therefore, comparisons of KIC between AGA and IUGR were not possible. However, in normal patients, the KIC concentration averaged 8.8% of the maternal leucine concentration, and the KIC/leucine ratio is within the range previously presented in the literature. The plasma KIC enrichments in both the maternal and fetal circulations averaged, respectively, 94 ± 2 and 95 ± 2% of the plasma leucine enrichment. Given the fact that KIC enrichments were 94% of leucine enrichments, the maternal plasma DR using plasma KIC enrichments (2.32 ± 0.3 versus 1.73 ± 0.4 µmol · kg-1 · min-1) was significantly higher (p < 0.05) than the maternal plasma disposal rate calculated with the maternal leucine enrichments.

DISCUSSION

The study presents the first data describing the normal steady state fetal-to-maternal plasma enrichments for leucine. There have been descriptions of this relationship in pregnant sheep, but the only previous study in humans was performed at the time of cesarean section.

Chien et al.(22) infused L-[1-13C]-leucine and L-[15N]-phenylalanine into pregnant women at the time of cesarean section. Maternal and fetal plasma enrichments and fetal fluxes for the two amino acids were reported. From these data, one can calculate a F/M MPE ratio for leucine of approximately 0.70 in appropriately grown fetuses. This value is 20% lower than the one we present for AGA fetuses and similar to that in group 2 IUGR fetuses. However, in our study the umbilical venous sample was obtained under relatively undisturbed conditions, whereas in Chien's study the samples were taken at cesarean section under anesthesia and after a period of time in which the fetus is exteriorized for flow measurements-conditions which could significantly alter placental perfusion. There have been several studies that have examined maternal plasma leucine disposal rate in human pregnancies; this literature is summarized in Table 4. Given minor differences among the studies, the values are in reasonable agreement among the authors.

Table 4 Maternal leucine plasma DR in the literature and in the present study
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In studies of normal and IUGR pregnant sheep, it has been shown that the F/M enrichment ratio is significantly reduced in IUGR pregnancies, both for L-[1-13C]-leucine and for L-[1-13C]-threonine. The values for leucine were 0.46 ± 0.05 in the controls and 0.30 ± 0.03 (p < 0.05) in the IUGR(11). The same IUGR model was studied for threonine, and the fetal/maternal enrichment ratio was 0.23 ± 0.01 in the control and 0.19 ± 0.01 in the IUGR pregnancies, (p < 0.01)(12). In these studies it was possible to measure directly fetal plasma disposal rate of each of the two amino acids, as well as the transplacental flux from the maternal circulation into the fetal circulation. In both studies, the decrease in the fetal/maternal enrichment ratio could be attributed to a reduction in the transplacental flux, rather than to a significant increase in protein breakdown. Several important deductions can be drawn from these animal studies compared with the data obtained in the present study. The significantly lower F/M enrichment ratio found for IUGR human pregnancies in the present study could be due to increased protein catabolism in the placental and fetal tissue proteins, or it could be due to a decrease in the transplacental leucine flux. The animal studies suggest that the latter is the more likely explanation. In addition, there is a major difference in the two species, even in normal pregnancies. The present study demonstrates that almost 90% of fetal plasma leucine is derived from the maternal circulation (89 ± 0.04%). In a recent paper, we have investigated the placental transfer of leucine in normal pregnancies at the time of FBS, infusing L-[1-13C]-leucine as a bolus into the mother(25). In the previous study, the F/M leucine ratio after 300 sec from the bolus injection (0.82) was a bit lower than the value of 0.89 for AGA in the present study. This observation suggests that in the human fetus, a much smaller component of fetal plasma leucine is contributed by leucine entry from protein breakdown in the placental and fetal tissues, compared with the ovine fetus, where only 46% of the fetal leucine is derived from the maternal circulation(11). Furthermore, a recent in vitro study has shown that the system A amino acid transporter activity in the microvillous plasma membrane of placentas from IUGR fetuses is significantly lower, compared with AGA(26), and this reduction is related to the severity of growth restriction. Comparable data were also found in a rodent model of IUGR(27).

The present study further strengthens the approach to define clinical severity in IUGR pregnancies by fetal arterial velocimetry and FHR data. Defining more homogeneous subsets of IUGR pregnancies is a necessary step if any clinical intervention protocols are to be attempted. It is encouraging that these data for fetal and maternal leucine enrichments correlate with the data obtained from fetal arterial velocimetry and computerized fetal heart monitoring. We have previously demonstrated that data for fetal acid-base status, lactate concentrations, and fetal oxygen contents correlate with the same classification of clinical severity(6). The data we present are in agreement with our previous report: IUGR fetuses of groups 2 and 3 had significantly lower O2 saturations and higher lactate concentration than control AGA pregnancies.

Also, measurements of F/M glucose concentration differences have shown that the magnitude of this glucose concentration difference increases stepwise from group 1 through group 3(28). Thus, diverse biochemical and physiologic data are all pointing to the conclusion that, functionally, there is a graded increase in clinical severity from group 1 through group 3 patients.

The data in the present report further clarify the issue of whether IUGR babies in group 1 should be considered as "normal small," a term proposed by Chard et al.(29) for term small-for-gestational-age babies. These fetuses have normal umbilical arterial PI and FHR, normal oxygenation, and acid base values, as well as normal F/M glucose concentration differences(6,28). Despite these findings, IUGR pregnancies of group 1 (as well as of groups 2 and 3) have F/M amino acid concentration differences that are significantly lower than AGA pregnancies(30). The present study demonstrates that the F/M leucine-enrichment ratio is also significantly reduced in IUGR fetuses of group 1, compared with normal fetuses, confirming the presence of an impairment of leucine metabolism and/or transport. Thus, these fetuses should not be considered "normal small."

Finally, the observation that the leucine F/M enrichment ratio correlates with the umbilical venous O2 content and lactate concentration rather than with the umbilical venous pH or base deficit is in agreement with data coming from a number of laboratories which contend that the latter measurements are of limited usefulness in assessing fetal well being in IUGR pregnancies(5,6), compared with measurements of O2 content and lactate concentrations, which are independent of gestational age and, thus, more useful clinically.

Table 5
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