Download An in vivo study of ovine placental transport of essential amino acids

Am J Physiol Endocrinol Metab
280: E31–E39, 2001.
An in vivo study of ovine placental transport
of essential amino acids
CINZIA L. PAOLINI,1 GIACOMO MESCHIA,2 PAUL V. FENNESSEY,2 ADRIAN W. PIKE,2
CECILIA TENG,2 FREDERICK C. BATTAGLIA,2 AND RANDALL B. WILKENING2
1
Department of Obstetrics and Gynecology, Dipartimento di Medicina Chirurgia e Odontoiatria
San Paolo, University of Milano, 20142 Milano, Italy; and 2Departments of Pediatrics, Physiology,
and Mass Spectroscopy, Division of Perinatal Medicine, University of Colorado
School of Medicine, Denver, Colorado 80045
Received 31 March 2000; accepted in final form 12 September 2000
transport; sheep; placenta; stable isotopes
UNDER NORMAL PHYSIOLOGICAL CONDITIONS,
essential amino
acids are transported from mother to fetus at different
rates (4, 10, 11). One of the functional meanings of this
observation is that, to meet requirements for protein,
accretion mandates different supply rates of amino
acids. For example, whole body homogenates of the
fetal lamb contain 2.4 times more leucine than phenylalanine (12). As a consequence, fetal growth requires a
2.4-fold greater accretion rate of leucine than of phenylalanine. The umbilical uptake of each essential
amino acid exceeds its fetal accretion (4), because the
fetus has a relatively high rate of amino acid catabo-
Address for reprint requests and other correspondence: F. C.
Battaglia, Fitzsimons, Bldg. 260, POB 6508, MS F441, Aurora, CO
80045-0508 (E-mail: [email protected]).
http://www.ajpendo.org
lism (1, 2, 8, 13). Nevertheless, umbilical uptake and
fetal accretion are well correlated. In a recent study (4),
the correlation coefficient between uptake and accretion of essential amino acids was 0.88.
The mechanisms that determine transport rates of
amino acids from the maternal to the umbilical circulation include the expression of several amino acid
transport systems on both the maternal and fetal surfaces of the trophoblast (14, 16), as well as the regulation of maternal and fetal plasma amino acid concentrations. This knowledge suggests the hypothesis that
differences in amino acid affinities for the transport
systems that each amino acid utilizes combine with
differences in maternal plasma amino acid concentrations to produce the range of placental transport rates
that are required by normal fetal development.
The present study was designed to explore the relationship between transplacental flux and maternal
plasma concentration for all of the essential amino
acids. Isotopically labeled leucine, isoleucine, valine,
phenylalanine, lysine, methionine, threonine, histidine, and tryptophan were injected simultaneously
into the maternal circulation of pregnant sheep. Measurements of amino acid concentrations, isotopic molar
percent enrichments in maternal and fetal plasma, and
umbilical plasma flow were used to calculate the transplacental flux and clearance of each amino acid.
MATERIALS AND METHODS
Surgery and Animal Care
All animal experimentation was performed according to
the Helsinki Convention for the use and care of animals and
was approved by the local institutional animal care and use
committee. Seven pregnant Columbia-Rambouillet sheep
were studied. Animals underwent surgery at 122 ⫾ 1.3 days
of gestation. Average body weight was 38.1 ⫾ 1.6 kg. After
48 h of fasting, the animals were sedated with intravenous
pentobarbital sodium (10 mg/kg as initial dose) and given
pontocaine hydrochloride spinal anesthesia (6 mg). The
The costs of publication of this article were defrayed in part by the
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0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
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Paolini, Cinzia L., Giacomo Meschia, Paul V. Fennessey, Adrian W. Pike, Cecilia Teng, Frederick C. Battaglia, and Randall B. Wilkening. An in vivo study of ovine
placental transport of essential amino acids. Am J Physiol
Endocrinol Metab 280: E31–E39, 2001.—Under normal
physiological conditions, essential amino acids (EA) are
transported from mother to fetus at different rates. The
mechanisms underlying these differences include the expression of several amino acid transport systems in the placenta
and the regulation of EA concentrations in maternal and
fetal plasma. To study the relation of EA transplacental flux
to maternal plasma concentration, isotopes of EA were injected into the circulation of pregnant ewes. Measurements of
concentration and molar enrichment in maternal and fetal
plasma and of umbilical plasma flow were used to calculate
the ratio of transplacental pulse flux to maternal concentration (clearance) for each EA. Five EA (Met, Phe, Leu, Ile, and
Val) had relatively high and similar clearances and were
followed, in order of decreasing clearance, by Trp, Thr, His,
and Lys. The five high-clearance EA showed strong correlation (r2 ⫽ 0.98) between the pulse flux and maternal concentration. The study suggests that five of the nine EA have
similar affinity for a rate-limiting placental transport system
that mediates rapid flux from mother to fetus, and that
differences in transport rates within this group of EA are
determined primarily by differences in maternal plasma concentration.
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PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO
Study Protocol
Each animal was studied on two consecutive days, with the
exception of one animal that was studied only once because of
catheter failure. Each study day the animal was injected with
one of two infusates (infusate A or B, Table 1). To check that
results were comparable on both study days, L-[1-13C]leucine was included in both infusates. To prepare the infusates, amino acids labeled with stable isotopes (98–99 atom
% pure) were obtained from Cambridge Isotope Laboratories
and dissolved in 20 ml of sterile isotonic saline. At least one
day before the studies, the oxygenation of mother and fetus
was checked by measurements of O2 content and O2 saturation in both circulations to verify that it was within normal
range.
At the onset of each study, 25 ml of maternal blood were
transfused into the fetal circulation to expand fetal blood
volume as a replacement for fetal sampling. This represents
⬍10% expansion of fetal blood volume. Then, a solution of
3
H2O (325 ␮Ci/20 ml) was infused at 3 ml/h in the fetal pedal
vein after a 4-ml priming dose. The 3H2O infusion was used
to calculate umbilical blood flow by the steady-state diffusion
method (15). Approximately 60 min after the start of the
3
H2O infusion, four sets of 1-ml blood samples were drawn
from the maternal femoral artery (A), uterine vein (V), umbilical vein (␥), and fetal artery (␣) for analysis of the amino
acid concentrations and enrichments, fetal and maternal
blood O2 content and hematocrit, and 3H2O concentration
(for the steady-state period).
After completion of this sampling, the amino acid infusate
of 20 ml (see Table 1) was injected as a bolus over a 1.39 ⫾
0.03-min period in the maternal femoral vein. During and
after the bolus, blood samples (0.9 ml) were drawn simulta-
neously from the four vessels (i.e., A, V, ␣, and ␥). The time
sequence for sampling was 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0,
6.0, 8.0, 10, 15, 20, 30, 40, 50, 60, 90, and 120 min after the
bolus. This sampling sequence was based on a previous study
that utilized a similar design (7).
At 30 and 90 min, blood was also withdrawn for 3H2O and
hematocrit measurements. At the end of the first study,
antibiotics (ampicillin 500 mg) were injected into the amniotic catheter, and a second 25-ml blood transfusion was given
to the fetus. The next day the study was repeated with the
second infusate. In two studies, infusate A was given first,
and in five studies, infusate B was given first. Within 1 h
after completion of the second study, the mother and fetus
were injected intravenously with a euthanasia solution (12
ml of 26% pentobarbital sodium). At autopsy, fetal and placental weights were measured separately, and catheter positions were verified.
Biochemical Analysis
Samples drawn for the measurement of hemoglobin concentration and oxygen saturation were collected into heparinized capillary syringes. Hemoglobin concentration (expressed as oxygen capacity in mM), oxyhemoglobin saturation
(%), and blood oxygen content (mM) were immediately measured in duplicate in a hemoximeter (OSM-2, Radiometer,
Copenhagen, Denmark).
For 3H2O whole blood analysis, 0.1-ml plasma samples
were solubilized in 1.0 ml of Soluene-350 (quaternary ammonium hydroxide in toluene) and subsequently mixed with 15
ml of Hionic-Fluor. Blood 3H2O concentrations were calculated by using the relation of blood water content to hematocrit (15). The radioactivity was counted in a Packard TriCarb 460 C liquid scintillation counter.
Blood samples for measurement of plasma amino acid
concentrations and stable isotopic enrichments were collected in syringes coated with EDTA and were centrifuged to
obtain plasma, which was stored at ⫺70°C until analysis.
Amino acid concentrations were measured by HPLC. Plasma
was quickly thawed, deproteinized with a solution of 10%
sulfosalicylic acid, with L-norleucine added as internal standard, and buffered with LiOH to pH 2.2. Samples were then
centrifuged at 14,000 rpm for 10 min, and the supernatant
fraction was filtered through a millipore filter and loaded into
a Dionex HPLC (Sunnyvale, CA) with refrigerated autosampler. The samples were analyzed by cation exchange column
with gradiant change from three different buffers isothermally. Ninhydrin was used as color reagent, and a dual
wavelength spectrophotometer with wavelengths of 440 and
570 nm were used for concentration determinations. The
column, ninhydrin reagent, and buffer were purchased from
Pickering Laboratories (Mountain View, CA). All instrument
operation and data processing were controlled by Dionex
A1–450 software.
Table 1. Composition of the two infusates
Infusate A
Infusate B
Amino acid
Infusate, ␮M
Amino acid
Infusate, ␮M
L-[1- C]leucine
13
L-[1- C]isoleucine
13
L-[1- C]phenylalanine
13
L-[1- C]lysine
2
L-[indole- H5]tryptophan
62.1 ⫾ 12.9
44.1 ⫾ 9.5
20.2 ⫾ 4.7
97.4 ⫾ 6.3
15.5 ⫾ 2.7
L-[1- C]leucine
13
L-[1- C]valine
2
L-[methyl- H3]methionine
13
L-[1- C]threonine
13
L-[ring 2- C]histidine
67.8 ⫾ 14.3
113.9 ⫾ 22.4
28.0 ⫾ 2.0
232.4 ⫾ 1.9
25.4 ⫾ 0.9
13
13
Values are means ⫾ SE. For some amino acids, the infusate had a large SE because their concentrations were reduced after analysis of
the results of the first experiment.
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uterus was exposed through a midline abdominal incision.
After the fetus was exposed through a 6-cm incision of the
uterine wall, polyvinyl catheters (1.4 mm OD) were placed in
the fetal pedal artery and vein and in the umbilical vein. One
catheter was placed in the amniotic cavity to give intraamniotic antibiotic prophylaxis to the fetus (ampicillin 500
mg). The maternal femoral artery and vein were catheterized
last. All catheters were tunneled subcutaneously to a plastic
pouch secured to the animal’s flank. Sheep were standing
within 6 h after surgery in individual carts and had free
access to water, food (alfalfa pellets), and a mineral block.
Ampicillin (500 mg) was injected daily into the amniotic
cavity for the first 3 days after surgery. Animals were allowed to recover from laparotomy for 7 days before study.
They were fed alfalfa pellets and water ad libitum. Their
average food intake was 30 ⫾ 4 g per kg body weight.
Catheters were maintained patent with daily heparinized
saline flushes (35 U heparin/ml).
Six out of seven animals were singleton; one carried a twin
pregnancy, and in that case only one fetus was studied.
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PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO
Stable isotopic enrichments were analyzed with a gas
chromatograph-mass spectrometer in the electron impact
mode, in 250 ␮l of acidified plasma with 50 nM of L-norleucine as internal standard. The amino acid fraction was
separated from other fractions on a 1.5-cm cation-exchange
resin column (50W ⫻ 8, 100–200 mesh, hydrogen form)
eluted with 1 ml of 15% NH4OH. Percentage recoveries of
basic, neutral, and acidic amino acids were tested with corresponding radiolabeled amino acids and varied from 80 to
92%. Samples were dried, derivatized with 15% tert-butyldimethylsilyl, or TBDMS, and injected in triplicate on a
gas chromatograph (Hewlett-Packard 5890) equipped with a
30-m DB-1 0.25-mm-ID 0.25-␮m film thickness, with helium
as the carrier gas, and a HP 5970 Mass Selective Device.
Enrichments of plasma amino acids were calculated by comparing the difference in peak area ratios between enriched
and unenriched samples according to the formula
⫻ 共ratio of enriched sample ⫺ ratio of unenriched sample兲/
关共ratio of enriched sample ⫺ ratio of unenriched sample兲 ⫹ 1兴
where MPE expresses the relative abundance of the enriched
tracer in excess of that occurring naturally.
Peaks were read at mass-to-charge ratios (m/z) 303/302 for
13
13
13
L-[1- C]valine, L-[1- C]leucine, and L-[1- C]isoleucine, at
m/z 337/336 for L-[1-13C]phenylalanine, at m/z 323/320 for
2
13
L-[methyl- H3]methionine, at m/z 301/300 for L-[1- C]lysine,
at m/z 405/404 for L-[1-13C]threonine, at m/z 441/440 for
13
L-[ring 2- C]histidine, and at m/z 380/375 for L-[indole2
H5]tryptophan. Amino acids were monitored in selective ion
monitoring, or SIM, in the same run for each infusate.
Calculations
Flows and O2 consumption. Umbilical blood flow (UBF)
was calculated as previously described by use of the tritiated
water infusion rate, the fetal tritiated water accumulation
rate, and the difference in umbilical venoarterial concentrations (15).
Umbilical plasma flow (UPF) was calculated according to
the formula
UPF共ml 䡠 min ⫺ 1 䡠 kg ⫺ 1兲 ⫽ UBF共ml 䡠 min ⫺ 1 䡠 kg ⫺ 1兲
䡠 共1 ⫺ fetal fractional hematocrit兲
Fetal oxygen consumption (␮mol 䡠 min⫺1 䡠 kg⫺1) was calculated as the product of UBF (␮mol 䡠 min⫺1 䡠 kg⫺1) and the
venoarterial difference in O2 content (␮mol/ml) across the
umbilical circulation.
Uptakes and clearance. The steady-state umbilical uptake
of each amino acid (␮mol 䡠 min⫺1 䡠 kg⫺1) was calculated as the
product of UPF (ml 䡠 min⫺1 䡠 kg⫺1) and the umbilical venoarterial concentration difference (␮mol/ml) of the amino acid.
The isotope concentration of each amino acid (␮M) was
estimated as the product of (MPE/100 ⫺ MPE) and the amino
acid concentration (␮M).
The umbilical isotope uptake (␮mol 䡠 min⫺1 䡠 kg⫺1) was estimated as the product of UPF (ml 䡠 min⫺1 䡠 kg⫺1) and the
integral of the umbilical venoarterial difference in the isotope
concentration (␮mol 䡠 ml⫺1 䡠 min⫺1) divided by the time of integration (min).
The isotope clearance (ml 䡠 min⫺1 䡠 kg⫺1) was calculated as
[umbilical isotope uptake (␮mol 䡠 min⫺1 䡠 kg⫺1) divided by the
integral of the maternal arterial enrichment (in ␮mol 䡠
ml⫺1 䡠 min)] divided by the time of integration (min).
Data Analysis and Statistics
All data are presented as means ⫾ SE. The significance of
the difference among amino acids was calculated by the
F-test and the Mann-Whitney U-test.
RESULTS
Table 2 presents data for mean gestational age at
surgery and at autopsy; maternal, fetal, and placental
weight; number of cotyledons; UBF; UPF; and fetal
oxygen consumption. There were no significant differences in umbilical or uterine blood flow or in fetal O2
saturations between the control (time 0) samples and
the 120-min samples.
As shown in Fig. 1, the maternal plasma MPE of all
essential amino acids increased rapidly after injection
of labeled amino acids: we were able to detect all of the
isotopes infused into the maternal circulation within
30 s from the beginning of the bolus. For all of the
amino acids, the peak in both the maternal artery and
uterine vein enrichments occurred in the sample taken
at 60 s from the start of the bolus. After the peak, there
was an exponential decay that was similar among most
amino acids but slower for lysine and threonine. The
enrichments of all of the essential amino acids increased in the umbilical circulation, giving evidence of
transplacental transport.
To observe placental transport for each amino acid, a
large amount of each isotope was injected. As a consequence, the total amino acid concentrations (i.e., the
sum of labeled and unlabeled molecules) increased
from 2 to 6 times in the maternal arterial plasma
during the first 10 min after the bolus (Fig. 2). This
increase produced an increase in the umbilical venous
concentration of the corresponding amino acid. However, the rise in umbilical concentration was much less
than in the mother and differed among amino acids
(Fig. 2).
For each amino acid, the umbilical venous peak of
MPE was delayed with respect to the 1-min maternal
peak. Time to peak was ⬃4 min for phenylalanine,
isoleucine, and leucine and occurred much later for
Table 2. Age, weight, flow, and O2 consumption data
Type of Pregnancy
Gestational age, days
At surgery
At autopsy (day 2 of study)
Weight
Maternal, kg
Fetal, g
Placental, g
Number of cotyledons
Umbilical blood flow, ml 䡠 min⫺1 䡠 kg⫺1
Umbilical plasma flow, ml 䡠 min⫺1 䡠 kg⫺1
Fetal O2 consumption, ␮mol 䡠 min⫺1 䡠 kg⫺1
Values are means ⫾ SE.
6 Singleton ⫹ 1 Twin
122.0 ⫾ 1
130.0 ⫾ 1
38.1 ⫾ 1.6
2,753.5 ⫾ 168
335.3 ⫾ 16.2
83.4 ⫾ 7.3
207.3 ⫾ 12.0
137.2 ⫾ 9.2
342.2 ⫾ 10.8
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molar percent enrichment 共MPE兲 共%兲 ⫽ 100
The transplacental pulse flux of each amino acid
(␮mol 䡠 min⫺1 䡠 kg⫺1) was calculated as the product of isotope
clearance (ml 䡠 min⫺1 䡠 kg⫺1) and maternal arterial concentration (␮mol/ml) at steady state.
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PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO
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Fig. 1. Amino acid enrichments for the
1st (A) and 2nd (B) infusate in maternal artery (‚), uterine vein (Œ), umbilical vein (F), and fetal artery (E). Time
0 is the start of isotope bolus injection.
histidine (14.2 ⫾ 1.0 min) and threonine (22.6 ⫾ 2.5
min; Table 3).
The data were further analyzed by recognizing that
the infused amino acids have widely different concentrations in maternal and fetal plasma at time 0 (Table
4). Also, maternal isotopic concentration changes are
not immediately translated into the fetal circulation
because of transport and accumulation in the placenta.
The fetal uptake at any point in time reflects the
combined effects of maternal and placental concentration changes over a period of time preceding the fetal
measurement. To produce directly comparable results,
the following procedure was adopted. First, the MPE
and concentration data were used to calculate isotope
concentrations in maternal and fetal plasma. Second,
the maternal arterial isotope concentration was integrated to produce an average maternal isotope concentration over the time of integration. Third, the isotope
concentrations in fetal plasma were expressed as ratios
of fetal isotope concentration to the average maternal
arterial isotope concentration.
Figure 3 presents isotope concentrations in the
umbilical vein and artery normalized for the average
maternal arterial isotope concentration in the first
10 min after the infusion. Despite the fact that fetal
isotope concentrations are expressed in comparable
units, it is clear that there are marked differences in
the rate of concentration change in fetal plasma. The
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PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO
Table 3. MPE time to peak in umbilical
venous plasma
Amino Acids
13
L-[1- C]phenylalanine
13
L-[1- C]isoleucine
13
L-[1- C]leucine (A)
13
L-[1- C]leucine (B)
2
L-[methyl- H3]methionine
2
L-[indole- H5]tryptophan
13
L-[1- C]lysine
13
L-[1- C]valine
L-[ring
13
L-[1-
2-13C]histidine
C]threonine
n
Table 4. Plasma amino acid concentrations
before the bolus
Time to Peak, min
7
7
7
6
3.9 ⫾ 0.3
3.9 ⫾ 0.3
4.0 ⫾ 0.4
4.0 ⫾ 0.4
6
7
7
6
5.8 ⫾ 0.5
6.3 ⫾ 0.5
6.4 ⫾ 0.7
7.6 ⫾ 1.1
6
14.2 ⫾ 1.5
6
22.6 ⫾ 2.5
Values are means ⫾ SE; n, no. of experiments.
Leucine
Isoleucine
Phenylalanine
Lysine
Tryptophan
Valine
Methionine
Threonine
Histidine
Maternal Artery
Umbilical Vein
Fetal Artery
212.19 ⫾ 20.3
143.74 ⫾ 16.7
64.44 ⫾ 6.8
136.35 ⫾ 17.5
41.84 ⫾ 2.9
351.18 ⫾ 54.2
28.54 ⫾ 2.0
199.68 ⫾ 35.2
48.30 ⫾ 1.6
212.8 ⫾ 16.8
130.75 ⫾ 11.5
123.0 ⫾ 9.1
67.02 ⫾ 7.1
50.65 ⫾ 6.5
603.04 ⫾ 69.4
71.84 ⫾ 8.52
330.11 ⫾ 66.1
57.88 ⫾ 7.17
178.16 ⫾ 15.3
109.68 ⫾ 11.7
111.7 ⫾ 9.0
49.25 ⫾ 4.6
42.96 ⫾ 6.03
571.39 ⫾ 63.2
66.50 ⫾ 10.4
314.48 ⫾ 65.2
55.01 ⫾ 8.0
Values are means ⫾ SE expressed in ␮M. The leucine concentration presented is the average of leucine concentrations measured
before first and second infusions, because these concentrations were
not significantly different.
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Fig. 2. Total plasma amino acid concentrations for the 1st (A) and 2nd (B) infusate in maternal artery (‚) and umbilical
vein (F). Time 0 is the start of the isotope
bolus injection.
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PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO
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Fig. 3. Umbilical venous (F) and arterial (E) isotope concentrations expressed as a fraction of average maternal arterial isotope concentration over
the first 10 min after isotope bolus injection.
most important information is provided by umbilical
venous plasma concentrations, because they represent the fetal plasma draining the placenta. In the
first minute after infusion, changes in umbilical vein
concentration provide information about placental
transport that is virtually unaffected by fetal metabolism. According to Fig. 3, there are five amino acids
(leucine, isoleucine, valine, phenylalanine, and methionine) that show rapid increases in the umbilical
vein after the maternal bolus. On the other hand,
threonine, lysine, and histidine demonstrate smaller
and less rapid concentration changes. The data in
Fig. 3 were used in conjunction with UPF to calculate
isotope clearances for the first 10 min after the bolus.
Analogous data were used to calculate clearances for
Table 5. Isotope transplacental clearances calculated
over the first 10 and 20 min after bolus injection
Isotope Clearance, ml 䡠 min⫺1 䡠 kg⫺1
Amino Acids
10 min
20 min
Methionine
Phenylalanine
Leucine (A)
Leucine (B)
Isoleucine
Valine
Tryptophan
Threonine
Histidine
Lysine
17.09 ⫾ 2.96
15.63 ⫾ 1.80
14.19 ⫾ 1.35
13.57 ⫾ 1.32
14.36 ⫾ 2.09
12.51 ⫾ 1.99
7.03 ⫾ 2.39
4.07 ⫾ 0.82
3.70 ⫾ 0.72
2.88 ⫾ 0.56
17.69 ⫾ 3.52
14.31 ⫾ 2.40
13.56 ⫾ 1.31
11.54 ⫾ 1.32
13.90 ⫾ 2.34
11.97 ⫾ 2.73
9.21 ⫾ 3.65
6.53 ⫾ 1.29
4.64 ⫾ 0.78
3.44 ⫾ 0.74
Values are means ⫾ SE.
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Table 6. Umbilical steady-state uptakes and transplacental pulse fluxes for the 9 essential amino acids
10 min
20 min
Amino Acid
Steady-State Uptake,
␮mol 䡠 min⫺1 䡠 kg⫺1
Pulse flux,
␮mol 䡠 min⫺1 䡠 kg⫺1
Pulse flux/steady-state
uptake ratio
Pulse flux,
␮mol 䡠 min⫺1 䡠 kg⫺1
Pulse flux/steady-state
uptake ratio
Methionine
Phenylalanine
Leucine (A)
Leucine (B)
Isoleucine
Valine
Tryptophan
Threonine
Histidine
Lysine
0.73 ⫾ 0.33
1.55 ⫾ 0.21
4.75 ⫾ 0.35
4.76 ⫾ 0.64
2.89 ⫾ 0.14
4.34 ⫾ 1.18
1.05 ⫾ 0.41
2.14 ⫾ 0.75
0.39 ⫾ 0.60
2.44 ⫾ 0.25
0.51 ⫾ 0.09
0.97 ⫾ 0.11
2.80 ⫾ 0.32
3.06 ⫾ 0.48
1.96 ⫾ 0.33
4.12 ⫾ 0.58
0.28 ⫾ 0.09
0.73 ⫾ 0.11
0.18 ⫾ 0.04
0.35 ⫾ 0.04
0.69
0.63
0.59
0.64
0.68
0.95
0.27
0.34
0.45
0.14
0.53 ⫾ 0.11
0.88 ⫾ 0.14
2.53 ⫾ 0.26
2.66 ⫾ 0.53
1.88 ⫾ 0.37
3.96 ⫾ 0.85
0.36 ⫾ 0.14
1.16 ⫾ 0.16
0.22 ⫾ 0.04
0.41 ⫾ 0.06
0.72
0.57
0.53
0.56
0.65
0.91
0.34
0.54
0.56
0.17
Values are means ⫾ SE or ratios.
DISCUSSION
equal values of maternal isotope concentration and for
a small fetal compared with maternal arterial isotope
concentration. The latter condition minimizes isotope
back flux from fetus to placenta so that the clearance
approximates unidirectional transport of the maternal
amino acid.
Five of the nine essential amino acids, i.e., the
branched-chain amino acids leucine, isoleucine, and
valine, plus methionine and phenylalanine, demonstrated similar and relatively fast clearances. All five
are zwitterions with hydrophobic side chains. This
evidence suggests that the five amino acids have similar affinity for a rate-limiting transport system that
mediates rapid flux from mother to fetus. Placental
amino acid transport utilizes two sets of transport
systems located, respectively, on the maternal and
fetal surface of the trophoblast (14). A study on the
transport of nonmetabolizable amino acids by the ovine
placenta in vivo has led to the hypothesis that the
neutral amino acids with the most rapid flux from
mother to fetus have relatively high affinity for a system of exchange transporters that is located on the
fetal surface of the placenta and has properties similar
to the Na-independent “ᐉ” system (9). In agreement
with this hypothesis, the branched-chain amino acids
The present study confirms that, under normal physiological conditions, essential amino acids have different umbilical uptakes. Table 6 shows leucine and valine having steady-state uptakes in the 5–4 ␮mol 䡠 min⫺1 䡠
kg fetus⫺1 range, followed in order of decreasing values
by isoleucine, lysine, threonine, and phenylalanine
(range of 3–1.5) and tryptophan, methionine, and histidine (1 or lower). The present study is the first in
which tryptophan umbilical uptake has been measured.
Uptake data for the other amino acids are in agreement
with previous measurements (4).
Pulse fluxes of essential amino acids into the umbilical circulation also demonstrate a wide range in values (Table 6). To account for the contribution of differences in maternal plasma amino acid concentration to
this variability, we calculated placental isotope clearances. Clearances measured in the first 10 min after
bolus injection represent fluxes across the placenta for
Fig. 4. Pulse flux calculated over the 1st 10 min after the bolus vs.
maternal plasma arterial concentration for the 5 essential amino
acids that have high and similar pulse flux clearances.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017
the first 20 min after the bolus. The results of these
calculations are shown in Table 5. Five amino acids
(methionine, phenylalanine, leucine, isoleucine, and
valine) show relatively high isotope clearances,
which do not differ significantly within the group and
which range between 17 and 12.5 ml 䡠 min⫺1 䡠 kg⫺1 in
the first 10 min. The remaining four amino acids
(tryptophan, threonine, histidine, and lysine) show
10-min clearances that are smaller (range between 7
and 2.9 ml 䡠 min⫺1 䡠 kg⫺1) than those of the first group
(P ⬍ 0.01). There were no significant differences
between the 10- and 20-min clearances, indicating
that the results of the 10-min clearance calculation
were not critically dependent on the selection of the
time interval.
Separation of the nine essential amino acids into two
broad groups was evident also by comparing initial
pulse fluxes and umbilical steady-state uptakes (Table
6). The 10-min pulse flux-to-steady-state uptake ratio
was greater for methionine, phenylalanine, leucine,
isoleucine, and valine than for tryptophan, threonine,
histidine, and lysine. For lysine, the pulse flux was
considerably smaller than steady-state uptake over
both the 10- and 20-min periods (Table 6).
E38
PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO
tions may create a large difference between pulse flux
and steady-state direct flux across the placenta. The
steep increase in maternal concentration of the test
isotope may decrease its transplacental clearance to
below normal if the rate-limiting transport system is
near saturation. Furthermore, the simultaneous increase in concentration of the coinfused isotopes may
alter the clearance via interaction with a shared transport system. Although the simultaneous infusion of
several amino acids has the advantage of providing
comparative data that are not biased by interanimal
variability, it introduces the potential for competition
among the amino acids.
Normal, steady-state direct fluxes across the sheep
placenta have been measured only for leucine (13) and
threonine (1). The leucine direct flux was 3.4 ⫾ 0.4
␮mol 䡠 min⫺1 䡠 kg fetus⫺1 and 0.87 of umbilical uptake,
and its transplacental clearance was 15.5 ml 䡠 min⫺1 䡠 kg
fetus⫺1 (13). For threonine, direct flux was 2.2 ⫾ 0.2
␮mol 䡠 min⫺1 䡠 kg fetus⫺1 and 0.52 of umbilical uptake,
and the transplacental clearance was 8.2 ml 䡠 min⫺1 䡠 kg
fetus⫺1 (1).
The leucine steady-state flux values are similar to
the leucine pulse flux data in Tables 5 and 6, so that
there is no clear evidence that the conditions of the
pulse flux experiment inhibited the tranplacental
leucine flux. The comparison of threonine pulse and
steady-state data suggests that the threonine pulse
flux and clearance might have been less than the analogous steady-state values, but again the discrepancy is
not sufficiently large to draw any firm conclusion. For
the other essential amino acids, the pulse flux-tosteady-state umbilical uptake ratios listed in Table 6
are the only available clue about the relationship of the
measured pulse flux to the normal steady-state direct
transplacental flux. Inspection of Table 6 shows that
the pulse flux of lysine was exceptionally small compared with its umbilical uptake, suggesting that the
transplacental flux of lysine measured in the present
study might have been much less than the normal
steady-state direct mother-to-fetus lysine flux. Further
studies are needed to investigate whether placental
efflux of lysine is near a saturated maximum under
normal physiological conditions and whether increases
in the maternal concentration of other amino acids
interfere with lysine transport to the fetus.
In conclusion, the present study supports the hypothesis (9) that the amino acids that are transported
most rapidly from the ovine placenta into the fetal
circulation are zwitterions with relatively high affinity
for the Na-independent “ᐉ” system exchange transporters. In addition, it shows that there are significant
differences in transplacental clearance among the essential amino acids. These differences are likely to be
related to differences in affinity and to competition for
shared transporters and to the utilization of separate
transport systems.
This work was supported by Fondo “Amalia Griffini” 1998, held by
Provincia di Varese, Italy, and National Institute of Child Health
and Human Development Grant PO1-HD-20761.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 12, 2017
methionine and phenylalanine have been shown to be
substrates of the “ᐉ” system (3).
An important implication of the finding that five
essential amino acids have similar placental clearances is that, among these amino acids, differences in
transport rate from mother to fetus depend upon differences in maternal concentration (Fig. 4). For example, both pulse flux and umbilical uptake were three
times greater for leucine than for phenylalanine, and
these differences correlated with a threefold difference
in maternal plasma concentration. It would appear
that, for five of the nine essential amino acids, the
regulatory mechanisms that set maternal concentration play a major role in controlling the supply rate to
the fetus.
The importance of factors other than maternal concentration in determining a wide range of transplacental fluxes is demonstrated by comparing threonine,
lysine, and histidine with the other essential amino
acids. In the first 10 min after maternal injection, the
threonine, lysine, and histidine isotopes showed much
slower increases in umbilical concentration and significantly smaller clearances then the leucine, isoleucine,
valine, methionine, and phenylalanine isotopes. The
behavior of tryptophan was intermediate.
Threonine is a zwitterion with a hydrophilic side
chain and is a substrate for neutral amino acid transporters present in the placenta (5). Its sluggish appearance in the umbilical circulation indicates relatively
low affinity for the transporters that mediate rapid flux
of neutral amino acids from placenta to fetus. Lysine
and histidine are basic amino acids. Three sodiumindependent transport systems for basic amino acids,
named y⫹, y⫹L, and b°,⫹, have been shown to be
present in the human trophoblast (6). Such information is not available for the ovine trophoblast. The b°,⫹
transporter is an exchanger of basic and neutral amino
acids and is located on the fetal surface only, suggesting the hypothesis that it promotes the efflux of basic
amino acids from placenta to fetus in exchange for a
neutral amino acid flux in the opposite direction (6). It
is apparent that, under the conditions of our study, the
specialized transport systems for basic amino acids
produced relatively small lysine and histidine transplacental clearances.
The relation between umbilical uptake and motherto-fetus pulse flux of any given essential amino acid is
complex. Steady-state umbilical uptake is the algebraic sum of three fluxes: 1) direct flux from maternal
to fetal circulation, 2) flux into the umbilical circulation
from the pool of placental amino acids released by
placental protein turnover, and 3) back flux from the
umbilical circulation into the placenta (1, 13). These
fluxes occur while the maternal, placental, and fetal
concentrations of all the amino acids are virtually
constant and set at normal physiological levels. By
contrast, each pulse flux measured in the present study
represents direct mother-to-fetus transport at the
same time that there is a transient and large increase
in the maternal concentration of the test isotope and of
the four coinfused isotopes. These experimental condi-
PLACENTAL TRANSPORT OF ESSENTIAL AMINO ACIDS IN VIVO
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