Relationship of fetal alanine uptake and placental alanine
metabolism to maternal plasma alanine concentration
Michelle
Timmerman1,
Misoo
Chung2,
Randall B.
Wilkening2,
Paul V.
Fennessey2,
Frederick C.
Battaglia2, and
Giacomo
Meschia2
1 Department of Obstetrics and
Gynecology, Erasmus University, Rotterdam, The Netherlands 3000 DR; and 2 Division of
Perinatal Medicine, Departments of Pediatrics, Pharmacology and
Physiology, University of Colorado Health Sciences Center, Denver,
Colorado 80262
 |
ABSTRACT |
Uterine and umbilical uptakes of alanine (Ala)
were measured in 10 ewes before (control) and during intravenous
infusion of Ala, which increased maternal arterial Ala concentration
from 115 ± 14 to 629 ± 78 µM
(P < 0.001). In 8 of these
ewes, placental Ala fluxes were traced by constant intravenous infusion
of
L-[3,3,3-2H3]Ala
in the mother and
L-[1-13C]Ala
in the fetus. Rates are reported as micromoles per minute per kilogram
fetus. Ala infusion increased uterine uptake (2.5 ± 0.6 to 15.6 ± 3.1, P < 0.001), umbilical uptake
(3.1 ± 0.5 to 6.9 ± 0.8, P < 0.001), and net uteroplacental utilization (
0.7 ± 0.8 to 8.6 ± 2.7, P < 0.01) of Ala. Control
Ala flux to fetus from mother
(Rf,m) was much
less than the Ala flux to fetus from placenta
(Rf,p) (0.17 ± 0.04 vs. 5.0 ± 0.6). Two additional studies utilizing
L-[U-13C]Ala
as the maternal tracer confirmed the small relative contribution of
Rf,m to
Rf,p. During
maternal Ala infusion,
Rf,m increased significantly (P < 0.02) but
remained a small fraction of
Rf,p (0.71 ± 0.2 vs. 7.3 ± 1.3). We conclude that maternal Ala entering the
placenta is metabolized and exchanged for placental Ala, so that most
of the Ala delivered to the fetus is produced within the placenta. An
increase in maternal Ala concentration increases placental Ala
utilization and the fetal uptake of both maternal and placental Ala.
alanine turnover; amino acids; umbilical uptake; alanine
fluxes
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INTRODUCTION |
PLACENTAL TRANSPORT of amino acids is a complex process
in which specificity, activity, and location of amino acid transporters within the placenta (17) and placental amino acid metabolism play a
role in determining which amino acids are supplied to the fetus and at
what rates. The importance of placental metabolism in this context has
become apparent in the comparison of serine and glycine transport by
the ovine placenta. The placenta takes up serine from the maternal
circulation but does not release serine into fetal blood. Maternal
serine is used by the placenta for the production of glycine, which is
then taken up by the fetus via the umbilical circulation (12).
There are no studies on the role of placental metabolism in determining
the supply of alanine to the fetus. In sheep, alanine is transported to
the pregnant uterus (3, 7) and from the placenta to the fetus (2, 6,
10). Although this suggests alanine transport across the placenta, it
is not known whether the alanine delivered to the fetus by the placenta
is derived directly from the maternal circulation or represents a
product of placental alanine turnover. Transport of maternal alanine to the fetus has been suggested by a study in which the intravenous infusion in pregnant sheep of a solution of several amino acids, including alanine, caused a significant increase in fetal plasma alanine concentration (9). However, the increase in fetal concentration was only 12% of the increase in maternal concentration, and there was
no attempt to demonstrate that uterine and umbilical alanine uptakes
had actually increased.
The present study was designed to address two questions concerning
alanine transport by the ovine placenta. First, what fraction of the
alanine flux from placenta to fetus represents direct alanine transport
from the maternal to the fetal circulation? Second, what is the effect
of an increase in maternal plasma alanine concentration on placental
and fetal alanine uptake and utilization?
 |
METHODS |
Biological Preparation
Twelve Columbia-Rambouillet ewes pregnant with a singleton fetus were
studied. Surgery was performed at 120-128 days of gestation after
a 48-h fast with free access to water. Anesthesia and surgery were
performed as previously described (14). Polyvinyl 20-gauge catheters
were inserted in the maternal femoral artery and vein and in the
uterine veins draining each uterine horn, fetal pedal artery and vein,
and the common umbilical vein. An amniotic catheter was also placed for
the injection of antibiotics. All catheters were tunneled
subcutaneously to a pouch on the ewe's flank.
Antibiotics were given pre- and postoperatively, and analgesics were
given during the first postoperative day. All catheters were flushed
daily with a solution of heparinized saline.
At least five days were allowed for full recovery, as assessed by
normal O2 content and glucose
concentration in the fetal circulation and normal food intake. The ewes
were provided with water, alfalfa pellets, and salt ad libitum.
Experimental Design
The following experimental protocol was used to study 8 sheep at
127-134 days of gestation. Maternal and fetal blood samples were
drawn for tritium and alanine enrichment blanks. Then, a fetal infusion
of
L-[1-13C]alanine
(0.83 ± 0.09 µmol · min
1 · kg
fetus
1) and tritiated
water (0.22 µCi · min
1 · kg
fetus
1) was started via
the pedal vein. Simultaneously, a maternal infusion of
L-[3,3,3-2H3]alanine
(CIL, Andover, MA; 0.17 ± 0.02 µmol · min
1 · kg
mother
1) was started.
At steady state, four sets of samples were collected simultaneously
from the maternal artery, uterine veins, fetal artery, and the common
umbilical vein at ~120, 150, 180, and 210 min from the start of the
infusions. Each sampling represented the loss of ~10 ml of fetal
blood. Fetal blood loss was corrected by transfusing the fetus, between
sampling sets, with an equal amount of blood from a donor sheep. This
part of the experiment is referred to as the control period. Then, the
maternal infusate was replaced by one containing a mixture of
L-[3,3,3-2H3]alanine
and reagent-grade L-alanine
(1,000 µmol/ml) to raise maternal alanine concentration four- to
fivefold. At steady state, 2 h after the start of the new maternal
infusate, four sets of blood samples were collected at ~330, 360, 390, and 420 min from the beginning of the study by following the same
procedure used in collecting the control samples. This part of the
experiment is referred to as the experimental period. All samples were
analyzed for hemoglobin, hematocrit,
O2 saturation, glucose, lactate,
tritiated water, amino acid concentrations, and plasma alanine
enrichments. Maternal and fetal arterial samples were analyzed for
plasma insulin.
After collection of the last set of samples, the ewe and fetus were
euthanized by intravenous injection (Sleepaway, Fort Dodge, IA).
Necropsy was performed to obtain fetal, placental, and uterine weights.
To help in the interpretation of the tracer data generated by these
experiments, four additional animals were studied. In two of these, an
identical protocol was followed, with the exception that
L-[3,3,3-2H3]alanine
was infused into the fetus. In the other two animals, the study was
limited to the control period, and the
L-[1-13C]alanine
infusion into the fetus was combined with the infusion of
L-[U-13C]alanine
into the mother.
Analytic Methods
Hemoglobin concentration and O2
saturation were measured spectrophotometrically (OSM-3, Radiometer,
Copenhagen, Denmark). The blood O2
content was calculated from the hemoglobin concentration expressed as
O2 capacity and multiplied by the
O2 saturation. Glucose and lactate
concentrations were measured in duplicate with a glucose/lactate
analyzer (YSI model 2700 Select and Dual Standard). Plasma
3H2O
was measured on triplicate aliquots in a scintillation counter and
converted to blood
3H2O
on the basis of the hematocrit measurement (19). Plasma insulin
concentrations were determined using the RAT insulin RIA kit (Linco
Research, St. Charles, MO). Plasma samples for amino acid
concentrations were stored at
70°C until the day of
analysis. At that time, the samples were thawed quickly and
deproteinized with 15% sulfosalicylic acid containing O.3 µM
norleucine as internal standard. The pH was adjusted to 2.2 with 1.5 N
LiOH. After centrifugation, the supernatant was analyzed with a Dionex
HPLC amino acid analyzer (Dionex, Sunnyvale, CA). The same column was
used for all samples from an individual animal. Reproducibility within
the same column had a mean value of ±2%. Samples from all vessels
drawn simultaneously were loaded to run within 12 h. Amino acid
absorbances were measured after reaction with ninhydrin at 570 nm
except glutamate, which was measured at a wavelength of 440 nm.
For mass spectrometry, amino acids were first separated on 0.2-ml AG50
cation exchange resin (Bio-Rad Mesch 100-200). Plasma (0.2 ml) was
mixed with 300 µl 30% acetic acid (Fisher Scientific, Pittsburgh,
PA) to form zwitter ions and was applied to the column. After the resin
was washed with 2 ml of distilled water, the amino acids were eluted
with 750 µl NH4OH and
lyophilized. Tri-t-butyldimethylsilyl derivatives were formed with 200 µl of acetonitrile containing 15%
N-methyl-N(t-butyldimethylsilyl)trifloroacetamide
and 1.5% t-butyldimethylchlorosilane
(Aldrich Chemicals, Milwaukee, WI) at 100°C for 30 min. Tandem gas
chromatography-mass spectrometry was performed on a Hewlett-Packard
HP-5790 gas chromatographer with a 30-m DB-17 0.025-mm-ID 0.25 µm
film thickness capillary column (J and W Scientific, Folsom, CA) with
helium as the carrier gas. The selected condition was 200°C initial
port temperature, an initial column temperature of 120°C with a
5°C/min ramp to 150°C, resulting in an alanine peak at ~7
min. The ion clusters for the alanine M-57 peak were monitored at
mass-to-charge ratios 260, 261, 262, and 263.
Calculations
Blood flows and uptakes.
Umbilical and uterine blood flows
(Qf and
Qm, respectively) were calculated
by the steady-state transplacental diffusion method with tritiated
water (19). The uterine and umbilical uptakes of
O2, glucose, and lactate were
calculated by application of the Fick principle
|
(1)
|
|
(2)
|
where
a, v,
, and
refer to maternal arterial, uterine
venous, umbilical venous, and umbilical arterial concentrations, respectively.
The uterine and umbilical uptakes of alanine were similarly calculated
using plasma flows and plasma alanine concentrations
|
(3)
|
|
(4)
|
where
Hm and
Hf represent the maternal and
fetal fractional hematocrits, respectively. Equations
3 and 4 are based on
the assumption that, in sheep, the rapid amino acid exchange between circulating blood and body tissues is virtually limited to an exchange
between the tissues and the plasma compartment. This observation is
supported by observations in vivo (8) and by measurements of amino acid
fluxes between red cells and plasma in vitro (21).
The alanine molar percent enrichments (MPE) were calculated using
steady-state (rS) and blank
(rO) ion abundance ratios
|
(5)
|
Disposal rates.
The maternal plasma alanine disposal rate
(DRm) was calculated as follows
|
(6)
|
where
mMPEa
is the maternal arterial plasma enrichment of the maternally infused
tracer at steady state, mC is the
concentration of the tracer in the maternal infusate (µmol/ml), and
mI is the infusion rate of the
maternal infusate (ml/min).
The fetal plasma alanine disposal rate
(DRf) was similarly calculated
|
(7)
|
where
fMPE is the steady-state fetal
arterial plasma enrichment of the tracer infused into the fetus,
fC is the concentration of the
tracer in the fetal infusate (µmol/ml), and
fI is the infusion rate of the
fetal infusate (ml/min). We note that Eqs.
6 and 7 do not include
the disposal rate of the naturally occurring isotopes (13).
Because the DRm and
DRf calculations are based on
steady-state measurements, they also represent estimates of the entry
rate (i.e., rate of appearance) of alanine in maternal and fetal
plasma, respectively.
Tracer alanine concentrations.
Plasma tracer alanine concentrations were calculated as total plasma
concentrations times MPE divided by 100.
Placental alanine fluxes.
The maternal tracer concentration differences across the uterine
circulation [(a-v)mat.
tracer] and the MPE of the maternal tracer in
maternal arterial plasma
(mMPEa)
were used to calculate the flux of maternal alanine into the
uteroplacenta from the maternal circulation
(RUP,m)
|
(8)
|
The fetal tracer concentration differences across the
umbilical circulation, [(
-
)fetal
tracer] and the MPE of the fetal tracer in
umbilical arterial plasma
(fMPE
),
were used to calculate the flux of fetal alanine into the placenta from
the fetal circulation
(RP,f)
|
(9)
|
Fetal alanine disposal rate and the fetal-to-maternal MPE
ratio of the maternal tracer
(mMPE
/mMPEa)
were used to calculate the direct flux of maternal alanine into the
fetal systemic circulation
(Rf,m)
|
(10)
|
The
flux of alanine to the fetus from the placenta
(Rf,P) was
calculated as the sum of
RP,f and
umbilical uptake
|
(11)
|
Statistics
The data were analyzed using the Statistical Analysis System program
(SAS Institute, Cary, NC). Each sheep provided two averaged data
points, control and experimental, for each variable. All data presented
in the tables are expressed as sample means ± SE for both study
periods. Differences between study periods were tested using Student's
t-test for paired samples. Two-sided
P values were considered significant
at P < 0.05. Because of paired measurements for the same sheep, control and experimental, a general linear regression model could not be applied to the variables of
interest. Instead, the paired lines representing control and experimental data for each sheep were analyzed. Student's
t-test was applied to each slope of
the paired lines to detect statistical significance between control and
experimental periods. In addition, a general linear model program for
repeated measures was applied to the data (22). This program gave the
same average line as the line that was calculated by averaging each
slope and intercept for all paired lines.
 |
RESULTS |
Table 1 presents mean gestational age,
fetal and placental weights, blood flows, oxygen, glucose, and lactate
uptakes for the 10 sheep infused with alanine. In response to the
alanine infusion, umbilical glucose uptake increased significantly and was associated with significant increases in maternal and fetal glucose
concentrations.
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Table 1.
Maternal and fetal hematocrits, O2 capacities, arterial
O2 saturations, glucose and lactate concentrations, uterine
and umbilical blood flows, and O2, glucose, and
lactate uptakes in 10 ewes before (control) and during
(experimental) maternal alanine infusion
|
|
Alanine Concentrations and Uptakes
The infusion of alanine into the maternal circulation elevated maternal
plasma alanine concentration approximately fourfold and increased fetal
plasma alanine concentration 36% (Table
2). These concentration changes were
associated with a mean sixfold increase in uterine alanine uptake and a
twofold increase in umbilical alanine uptake. The analysis of
individual changes in uterine and umbilical uptakes showed that the two
changes were significantly correlated to the changes in maternal
concentration (Fig. 1). Uterine alanine
uptake was similar to umbilical uptake in the control period (2.5 vs.
3.1 µmol · min
1 · kg
fetus
1) but became
significantly greater than umbilical uptake during the alanine infusion
(15.5 vs. 6.9 µmol · min
1 · kg
fetus
1,
P < 0.01). Therefore, net
utilization of alanine by the uteroplacenta increased markedly in
response to maternal alanine infusion, from
0.6 to 8.6 µmol · min
1 · kg
fetus
1
(P < 0.01).

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Fig. 1.
Umbilical and uterine alanine uptakes
(A and
B) and uteroplacental alanine
utilization (C) vs. maternal alanine
concentration.
|
|
Alanine Disposal Rates and Placental Fluxes
Maternal and fetal plasma alanine enrichments approximated steady-state
conditions in both the control and experimental periods for each
alanine isotopomer (Fig. 2).
Maternal alanine disposal rate increased significantly in response to
the alanine infusion (Table 2). The increase in maternal disposal rate
was approximately equal to the alanine infusion rate (9.9 ± 1.1 vs.
8.4 ± 1.0 µmol · min
1 · kg
mother
1,
P = 0.325). The enrichment of maternal
tracer was significantly less in the uterine vein than in the maternal
artery (P < 0.001; Fig. 2). This
observation, coupled with the uterine uptake data, demonstrated
bidirectional alanine fluxes between the maternal circulation and the
pregnant uterus. Similarly, the fetal alanine tracer data demonstrated
bidirectional alanine exchange between placenta and fetus (Table 2 and
Fig. 2). The flux of alanine to the fetus from the placenta
(Rf,P)
was 26% of fetal plasma alanine entry rate in the control period
(i.e., 5.0 vs. 19.6) and 33% in the experimental period (i.e., 7.3 vs.
22.0). However, a very small portion of
Rf,P
represented direct flux of maternal alanine into the fetus
(Rf,m).
In the control period,
Rf,m
was 0.17 ± 0.04 µmol · min
1 · kg
fetus
1. This flux was
~3% of Rf,P
(0.17 vs. 5.0) and 0.9% of the fetal plasma alanine entry rate (0.17 vs. 19.6). In the experimental period, the direct flux of maternal
alanine into the fetus increased significantly to 0.71 ± 0.2 µmol · min
1 · kg
fetus
1 but remained small
compared with the other alanine fluxes, ~10% of
Rf,P (0.71 vs.
7.3) and 3% of the fetal plasma alanine entry rate (0.71 vs. 22.0).
Fetal alanine DR increased significantly in the experimental period
(Table 2) and by a value that was virtually equal to the increase
in Rf,P (2.4 vs.
2.3 µmol · min
1 · kg
fetus
1).

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Fig. 2.
Plasma enrichments for both
L-[3,3,3-2H3]alanine
(maternal tracer, A) and
L-[1-13C]alanine
(fetal tracer, B) in control and
experimental periods are molar percent enrichments (MPE, means ± SE)
plotted against time. , umbilical artery; , umbilical vein; a,
maternal artery; v, uterine vein.
|
|
The increase in
Rf,m caused by
maternal alanine infusion was significantly correlated to the increase
in maternal alanine flux to the uteroplacenta
(Rup,m)
(P < 0.01). However, the
slope of the regression line relating
Rf,m to
Rup,m was about
one-tenth of the slope relating umbilical and uterine uptakes (Fig.
3). The increase in umbilical uptake was
~29% of the increase in uterine uptake, whereas the increase in
direct alanine flux to the fetus from the mother was only 2.2% of the
increase in alanine flux into the pregnant uterus.

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Fig. 3.
Relation of umbilical (Umb) alanine uptake to uterine (Utn) alanine
uptake and flux of maternal alanine into the fetus
(Rf,m) to flux
of maternal alanine into the uteroplacenta
(RUP,m).
|
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Dependence of Results on the Choice of Tracers
Recycling of alanine through pyruvate and lactate pools makes the
disposal rate of deuterium-labeled alanine more rapid than the disposal
rate of 13C-labeled alanine,
because the deuterium label is selectively lost in the recycling (20).
This characteristic of the deuterium label requires validation of the
assumption that in estimating Rf,m we could use
the fetal disposal rate of
L-[1-13C]alanine
to calculate the fetal disposal rate of maternal
L-[3,3,3-2H3]alanine
(Eq. 10 in
Calculations). Two fetal sheep
infused with L-[3,3,3-2H3]alanine
yielded fetal plasma alanine disposal rates equal to 19.5 and 31.3 µmol · min
1 · kg
fetus
1, respectively.
Compared with the fetal disposal rates measured with
L-[1-13C]alanine
(mean 19.6, range 14.6-23.6), these data indicate that the
disposal rates of the two tracers are sufficiently similar for the
purpose of estimating the contribution of
Rf,m to placental or fetal alanine fluxes.
A second issue related to the metabolism of alanine tracers is that
rapid alanine recycling within the placenta would cause the
transplacental flux of deuterium-labeled maternal alanine to be less
than the transplacental flux of
13C-labeled maternal alanine. In
other words, 13C labeling of
maternal alanine would include in the calculation of the
Rf,m flux
maternal alanine molecules that had undergone deamination and
reamination within the placenta. To explore the magnitude of this
effect, we performed two studies by use of maternal infusion of
L-[U-13C]alanine.
The relevant results are summarized in Table
3. In this table, the fetal-to-maternal MPE
ratios of maternal tracer and the
Rf,m flux are all
greater than the corresponding mean values in Table 2 by more than four
standard deviations, thus indicating a greater transplacental flux of
the 13C-labeled tracer. Even so,
the labeling of maternal alanine with 13C confirms that the direct flux
of maternal alanine into the fetus is a small fraction of the alanine
flux to the fetus from the placenta. In the two animals of Table 3, the
direct flux of maternal alanine across the placenta was only 9 and
15%, respectively, of the alanine flux from placenta to fetus.
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Table 3.
Summary of results in 2 sheep infused iv with
L-[U-13C]alanine in the mother and
L-[1-13C]alanine in the fetus
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Effect of Alanine Infusion on Other Amino Acids
In response to the alanine infusion, the increase in maternal plasma
alanine concentration was accompanied by a significant increase in the
maternal concentrations of glutamate and glutamine and a significant
decrease in the concentrations of most other amino acids (Fig.
4). In fetal plasma, the increase in
alanine concentration was accompanied by a significant increase in the concentrations of glutamine and serine and a small decrease in the
concentrations of several amino acids. This decrease was significant only for leucine, tyrosine, and methionine. Concomitant with these changes in amino acid concentrations, there were significant increases in maternal plasma insulin (from 22.5 ± 3.9 to 28.2 ± 4.2 µU/ml, P < 0.001) and fetal plasma insulin
(from 18.1 ± 2.3 to 21.1 ± 2.8 µU/ml,
P < 0.05). The uterine and
umbilical uptakes of amino acids other than alanine did not show any
detectable change, with the exception of a significant decrease in the
uterine uptake of leucine (from 5.7 ± 0.8 to 4.4 ± 0.7 µmol · min
1 · kg
fetus
1,
P < 0.05).

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Fig. 4.
Comparison of maternal (A) and fetal
(B) neutral and acidic amino acid
concentrations between control (filled bars) and experimental (open
bars) study periods. Values are means ± SE. Significant changes
between 2 periods: * P < 0.05, ** P < 0.01, *** P < 0.001.
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|
 |
DISCUSSION |
The present study is relevant to the question of whether it is possible
to increase fetal amino acid uptake by increasing the concentration of
amino acids in maternal plasma. This question has both practical and
theoretical interest. From the practical point of view, it is important
to know whether the nutrition of a growth-restricted fetus could be
improved by increasing the concentration of nutrients in the maternal
circulation. Basic understanding of placental amino acid transport and
metabolism requires experimental data about placental and fetal amino
acid uptake as a function of maternal amino acid concentration.
Tracing the flux of maternal alanine into the fetal circulation yields
a range of flux values, depending on the type of tracers that are used.
The infusion of deuterium-labeled alanine into the maternal circulation
results in an umbilical arterial enrichment that is ~1% maternal
arterial enrichment. The normal transplacental maternal alanine flux
estimated on the basis of this extremely low fetal enrichment is ~0.2
µmol · min
1 · kg
fetus
1 (Table 2, control
data). The infusion of 13C-labeled
alanine into the maternal circulation results in an umbilical arterial
enrichment that is ~4% of the maternal arterial enrichment and an
estimated normal maternal alanine flux into the fetal circulation of
~0.8
µmol · min
1 · kg
fetus
1 (Table 3). Before
the methodological issue that is raised by this observation is
addressed, it is important to note that the experiments with either
tracer agree in showing that, under normal physiological conditions,
the flux of maternal alanine into the fetal circulation
(Rf,m) is a
relatively small fraction of the alanine flux to the fetus from the
placenta
(Rf,p). The
estimated value of the
Rf,m-to-Rf,p
fraction ranged between 0.034 (Table 2, control data) and 0.15 (Table
3). This finding implies that most of the alanine flux from placenta to
fetus represents alanine produced within the placenta.
If no other information were available, one might infer that there is
virtually no alanine flux from the maternal plasma into the placental
alanine pool supplying alanine to the fetus. However, during maternal
alanine infusion, the increase in maternal plasma alanine concentration
had the effect of doubling the fetal uptake of placental alanine. This
observation shows that the placental alanine pool delivering alanine to
the fetus has two inputs, i.e., alanine produced within the placenta
and alanine entering the placenta from the maternal circulation.
Because the amount of maternal alanine escaping into the fetus is
relatively small, the placental alanine pool must have a high turnover
rate compared with the influx of maternal alanine. Maternal alanine
transported from the maternal to the fetal surface of the placenta is
diluted by mixing with a large flux of unlabeled alanine produced
within the placenta.
The rate of placental alanine turnover is a function of placental
protein turnover and transamination reactions. The turnover of
placental proteins utilizes and releases amino acids at a rapid rate.
This has been demonstrated by the evidence that approximately one-half
of the leucine flux to the fetus from the placenta represents leucine
produced within the placenta (14). Because leucine is an essential
amino acid, protein turnover is virtually the only source of placental
leucine production. In addition to protein turnover, interconversion of
alanine and pyruvate via transamination is likely to be the second most
important mechanism for placental alanine turnover. Rapid alanine
transamination within the placenta may explain why the estimate of
maternal alanine flux into the fetus depends in part on the choice of
tracer that is used in the labeling of maternal alanine. The
interconversion of alanine and pyruvate would cause the placental
disposal rate of deuterium-labeled alanine to be more rapid than the
disposal rate of 13C-labeled
alanine, because the deuterium label is selectively lost in this
process. Therefore, the labeling of maternal alanine with
13C would yield a greater flux of
maternal alanine into the fetus than the deuterium labeling,
because the flux traced by
13C-labeled alanine
includes maternal alanine molecules that underwent reversible
transamination within the placenta. On the other hand, we cannot
exclude that other mechanisms contributed to the discrepancy in the
transplacental flux of the two tracers. For example, some of the
deuterium may have been removed within the placenta via an exchange
with water that did not involve enzymatic reactions.
The evidence presented in this study points to the conclusion that
alanine transport across the placenta depends on the interaction between placental alanine metabolism and the activity of placental amino acid transporters. The flux of amino acids into the placenta from
the maternal circulation is controlled by amino acid transporters located on the maternal surface of the organ (17). The relatively large
increase in uterine uptake in response to an increase in maternal
alanine concentration suggests that transport of maternal alanine into
the placenta was not an important factor in limiting the increase in
fetal alanine uptake. Because maternal alanine infusion decreased the
concentration of several neutral amino acids in maternal plasma, the
increased uterine alanine uptake may have been the result, at least in
part, of reduced competition in the sharing of transporters. Therefore,
the observed increase in uterine uptake induced by maternal alanine
infusion may not be predictive of uptake when alanine is infused
together with other amino acids. The flux of amino acids from placenta
to fetus is controlled by exchange transporters located on the fetal
surface of the placenta (4, 17). Placental sodium-independent (17) and
sodium-dependent (5) exchange transporters have been described. An
important role for these transporters in limiting fetal alanine uptake
is suggested by the observation that, in response to the increase in
maternal alanine concentration, the increase in umbilical alanine
uptake was only approximately one-third of the increase in uterine
uptake. The partitioning of alanine between placental utilization and
transport to the fetus depends on the availability of both pathways for
placental alanine metabolism and transport pathways that allow the
escape of alanine into the umbilical circulation.
In agreement with previous studies (11), there was a significant
lactate output by the uteroplacental tissues in the control period.
This output did not increase significantly in the experimental period
despite the increased uteroplacental alanine utilization. However,
there was an increase in umbilical glucose uptake, which suggests that
glucose was diverted from placental glucose utilization to fetal
glucose uptake. The dephosphorylation of
phosphoenolpyruvate to pyruvate is
inhibited by high alanine concentrations (18). Thus an increase of
alanine concentration within the placenta during the experimental
period may have decreased the entry rate of glucose into the glycolytic
pathway and prevented a large increase in pyruvate and lactate
concentrations via its inhibitory effect on pyruvate kinase. Figure
5 summarizes the changes in metabolic substrate fluxes associated with the maternal alanine infusion.

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Fig. 5.
Summary balance of measured substrate fluxes between fetal, placental,
and maternal compartments during control and experimental periods. Mean
fluxes are nos. representing µmol of
carbon · min 1 · kg
fetus 1.
|
|
The increased placental utilization of alanine during the experimental
period presents a problem of nitrogen excretion for the placenta. This
problem could be solved in a variety of ways, e.g., by
1) increasing ammonia excretion,
2) increasing amidation of glutamate
to glutamine, and 3) decreasing
deamination of branched-chain amino acids. In the present study, there
was a trend toward increased glutamine umbilical uptake and decreased
placental utilization of branched-chain amino acids. However, the only
trend that attained significance was a decrease in placental leucine
utilization (P = 0.04). Both maternal
and fetal glutamine concentrations increased in the experimental period
(P = 0.002), suggesting
increased maternal and fetal glutamine production. Ruderman and
co-workers (15, 16) have shown that, in the rat hindlimb, the rate of
glutamine release can be increased by increasing the supply of alanine
via the perfusate.
In the control period, fetal plasma alanine disposal rate was much
greater than the flux of alanine into the fetus from the placenta (19.6 vs. 5.0 µmol · min
1 · kg
fetus
1). In the
experimental period, the alanine disposal rate increased ~12% and by
a value that was virtually equal to the increased flux from the
placenta. These observations add to previous evidence (2) showing that
the ovine fetus has a high rate of alanine production compared with
umbilical alanine uptake. Thus a doubling of the umbilical uptake has a
relatively small effect on fetal plasma alanine turnover.
It is likely that, in all mammals, placental alanine metabolism is one
of the factors that limits the direct flux of alanine from mother to
fetus. There may be, however, quantitative differences in the degree of
limitation. Gilfillan et al. (1) reported that the umbilical venous and
arterial plasma enrichments of
13C-labeled tracer alanine infused
at a constant rate into the maternal circulation were ~44 and 25%,
respectively, of maternal peripheral venous enrichment in term human
pregnancies studied at the time of cesarean section. These ratios are
greater than the one observed in the present study for the maternally
infused [13C]alanine
(~6 and 4%) and suggest a larger contribution of the maternal
alanine flux across the human placenta to fetal alanine turnover. The
larger contribution may be the expression of a difference in the rate
of placental alanine metabolism as well as differences in placental
alanine transport and fetal alanine production rates.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Child Health and
Human Development Grants R37 HD-01866, RO1 HD-29188, and P50 HD-20761.
M. Timmerman was supported by a Fulbright Scholarship and the Ter
Meulen Fund, Royal Dutch Academy of Arts and Sciences.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: F. C. Battaglia, Univ. of Colorado Health
Sciences Center, Dept. of Pediatrics, Fitzsimons, Bldg. 260, Mail Stop
F441, PO Box 6508, Aurora, CO 80045-0508.
Received 27 February 1998; accepted in final form 11 August 1998.
 |
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