Ovine fetal leucine kinetics and protein metabolism during
decreased oxygen availability
J. Ross
Milley
Division of Neonatology, Department of Pediatrics, University of
Utah School of Medicine, Salt Lake City, Utah 84132
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ABSTRACT |
The fetus depends on an uninterrupted supply of
oxygen to provide energy, not only for basal metabolism but also for
the metabolic costs of growth. By curtailing the metabolically
expensive processes of protein turnover, the fetus could conserve
energy when oxygen availability is limited. Therefore, this
investigation was performed to find whether protein synthesis and
breakdown are diminished during decreased fetal oxygen availability.
Furthermore, if these conditions reduce fetal growth, protein synthesis
should be affected more than breakdown so that protein accretion, an
important component of fetal growth, also falls. In eight chronically
prepared fetal lambs, we compared leucine kinetics (reciprocal pool
model) during control conditions with measurements made during maternal
hypoxia, a condition that limits fetal oxygen availability. Decreased
fetal oxygen availability (
43%;
P < 0.001) reduced fetal oxygen
consumption (
16%; P < 0.01),
as well as both the uptake of leucine across the placenta (
48%;
P < 0.001) and its rate of
decarboxylation (
30%; P < 0.001). Fetal protein synthesis decreased (
32%;
P < 0.001) to a greater extent than
proteolysis (
22%; P < 0.001). Consequently, fetal protein accretion, an important component of fetal growth, also decreased (
62%;
P < 0.001). We calculate that the
reduction in fetal protein synthesis and breakdown, both processes that
require intracellular expenditure of ATP, decreased fetal energy needs
sufficiently to account for most, if not all, of the decrease measured
in fetal oxygen consumption.
fetal growth; intrauterine growth retardation; protein synthesis; proteolysis
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INTRODUCTION |
NORMALLY, THE PROCESSES OF GROWTH and metabolism in the
sheep fetus are supported by a constant uptake of oxygen, glucose, lactate, and amino acids across the umbilical circulation (1). If the
availability of these substrates to the fetus is limited, fetal growth
decreases. In some instances, the availability of several substrates
that are important to fetal metabolism may be simultaneously decreased
(30). In such cases, it is impossible to distinguish which substrate
limits fetal growth. In contrast, fetal growth is also attenuated when
fetal oxygen availability is specifically limited (3, 14, 28). Because
availability of other substrates important to fetal metabolism is
normal or even increased under these conditions (20), decreased fetal oxygen availability represents an instance in which deficiency of a
single substrate limits fetal growth.
In adult muscle (5, 9), when oxygen availability is decreased, the
metabolically expensive processes of protein turnover are reduced
(i.e., both protein synthesis and protein breakdown decrease). For
protein turnover to be affected, however, oxygen availability must
decline sufficiently to decrease intracellular energy state (5), a
condition characterized by anaerobic glycolysis and consequent lactic
acid production (5). Similarly, when fetal oxygen availability is
diminished sufficiently to cause fetal lactate production, fetal
protein synthesis falls (19). Protein synthesis decreases sufficiently
to account for most of a simultaneous decrease in fetal metabolic rate.
Thus reduction of the metabolically expensive process of protein
synthesis allows conservation of limited fetal oxygen supply for other
needs.
Protein breakdown represents an additional constituent of protein
turnover that requires expenditure of intracellular energy (9).
Measurement of this variable is particularly important because fetal
protein accretion, the difference between protein synthesis and protein
breakdown, is an important component of fetal growth. Nevertheless,
there are no data indicating whether fetal proteolysis is affected by
decreased oxygen availability. Consequently, the purpose of this study
is twofold: 1) to show that
limitation of fetal oxygen availability will decrease fetal proteolysis, especially if such limitation of availability is sufficient to decrease fetal oxygen consumption; and
2) to show whether fetal protein
synthesis or fetal proteolysis is reduced to a greater extent, a
finding that shows whether changes in fetal oxygen availability affect
fetal protein accretion.
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METHODS |
Animals.
Time-dated pregnant ewes (6 singleton, 2 twin pregnancies), with
gestational ages ranging from 114 to 119 days, were obtained from
Torell Ranch (Ukiah, CA). Maternal weight averaged 55.8 ± 2.3 (SE)
kg and ranged from 49 to 68 kg. The ewes were sedated with intravenous
ketamine (10 mg/kg) and then intubated and anesthetized with inhaled
isoflurane (~2%). The uterus was exposed through a midline incision
in the maternal abdomen, and a hysterotomy was performed over the area
of the fetal neck. An incision in the fetal skin was made over a
jugular vein, and two catheters were placed in the fetal superior vena
cava. The fetal and uterine incisions were then closed, and a second
hysterotomy was performed over the fetal abdomen. The fetal skin was
incised ~2 mm from the umbilical ring, and a catheter was placed in
one umbilical vein to lie in the common umbilical vein. The fetal and
uterine incisions were again closed, and the uterus was reopened over the area of the fetal hindlimbs. Catheters were placed by way of a
fetal hindlimb artery and two hindlimb veins into the abdominal aorta
and inferior vena cava, respectively. Subsequently, all incisions,
including the maternal abdominal incision, were closed and the
catheters tunneled underneath the maternal skin to the maternal flank,
where they were stored in a pouch until needed.
Each animal received 200 mg of trimethoprim and 1,000 mg of
sulfadiazine (Di-Trim, 48% injection; Syntex Animal Health, West Des
Moines, IA) preoperatively and for 3 days postoperatively. Six days of
postoperative recovery were allowed before experiments were started to
avoid effects on the experiment due to operative stress or anesthesia.
Catheter patency was maintained by filling each catheter with saline
(0.9%) containing heparin (100 U/ml) every other day. The animals
described in this experiment all fed and drank normally during
recovery. After the experimental protocols (to be described in
Experimental design) were completed, both the fetus and ewe were killed by barbiturate overdose
(Beuthanasia-D Special; Schering, Kenilworth, NJ). All catheter
positions were confirmed by direct examination at necropsy. The data
contained in this report were normalized, where appropriate, to fetal
weight at necropsy [3.23 ± 0.14 (SE) kg] to allow
comparison of these data with other published data. We did not,
however, correct for fetal growth (estimated to be ~8% of original
fetal weight) (31) over the 2
days of this study because the actual daily rate of growth was unknown. However, our inability to
correct for fetal weight could not have influenced the conclusions of
this study, because its crossover design allowed separation of those
experimental effects due to hypoxia from those due to the duration of
the study (such as fetal growth). This study was approved by the
University of Utah Institutional Animal Care and Use Committee.
Experimental design.
This study protocol includes 2 days of experimentation on each animal
(Fig. 1). The first protocol
(day 1) included measurements made
during control conditions and then during decreased fetal oxygen
availability. Following a washout period (
36 h), a second experiment
(day 2) was done with the
experimental order reversed (i.e., measurements were made during
decreased oxygen availability and then during control conditions). This
experimental design was chosen so that the effects of decreased fetal
oxygen availability could be separated from those due to the duration
or order of the experiment. Day 1 experiments were initiated by starting an infusion of
[1-14C]leucine at
~5.5 × 106 dpm/min into
the fetal inferior vena cava catheter. This infusion was continued for
180 min before blood samples were taken. This duration is sufficient
for leucine specific activity to become constant (25). After
[1-14C]leucine had
been infused for 2 h, an intravenous infusion of antipyrine (24 mg/ml
in sterile 0.9% saline) was started at 0.2 ml/min to measure umbilical
blood flow. One hour later, blood was drawn simultaneously from the
fetal umbilical vein and aorta for analysis of whole blood contents of
glucose, antipyrine, oxygen, leucine, and
-ketoisocaproate as well
as of radioactivity as 14CO2
and [1-14C]leucine. In
addition, a sample of arterial plasma was obtained for analysis of
-ketoisocaproate specific activity. A second similar set of blood
samples was drawn ~15 min later. Data obtained from these two samples
were averaged before subsequent calculations were made. As the second
set of samples was obtained, arterial blood was also taken for analysis
of hematocrit, pH,
PCO2 and PO2. After
each set of blood samples, the fetus was transfused with an amount of
maternal blood equal to that removed from the fetus.

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Fig. 1.
Outline of experimental design. On day
1, control measurements (arrowheads) were made after 3 h of [1-14C]leucine
infusion and 1 h of antipyrine (Apy) infusion. After these samples were
taken, ewes were made hypoxic by allowing them to breathe ~9%
oxygen. One hour after this change in condition,
[1-14C]leucine
infusion was restarted, followed 2 h later by reinitiation of Apy
infusion. A 2nd set of blood samples identical to the 1st was taken
after Apy infusion had continued for 1 h. On day
2 protocol was similar except that decreased maternal
inspired oxygen concentration began the protocol and, after the 1st set
of blood samples was taken, 60 min were allowed for maternal and fetal
recovery before radioactive leucine and antipyrine infusions were
begun.
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After the first set of two blood samples was obtained, the
[1-14C]leucine and
antipyrine infusions were stopped and fetal oxygen availability was
diminished by delivering 7.6-9.8%
O2 and 3-4% CO2 to a bag enclosing the ewe's
head. During this and all subsequent periods of hypoxia, maternal
fraction of inspired O2
(FIO2) was continuously monitored. Blood for measurement of fetal pH and blood
gases was drawn every 30 min. One hour after the start of hypoxia, the
[1-14C]leucine
infusion was restarted, followed 2 h later by a restart of the
antipyrine infusion. After an additional hour of both infusions, a
second set of two fetal blood samples, identical to the first, was
obtained. On the second experimental day (day
2), to reverse the order of the experiment, the ewe
was allowed to breathe an inspired oxygen concentration identical to
that used to induce hypoxemia on day
1. Fetal
[1-14C]leucine and
antipyrine infusions were begun at 60 and 180 min, respectively, after
the induction of maternal hypoxemia, again at rates identical to those
on day 1. After the first set of fetal blood samples had been drawn, the
[1-14C]leucine and
antipyrine infusions were stopped, and the mother was allowed to
breathe room air. Sixty minutes later, we restarted the fetal
[1-14C]leucine
infusion, followed after an additional 2 h by the antipyrine infusion.
After an additional hour of both infusions, blood samples identical to
those of the first set were again obtained.
Chemical analysis.
The methodology for measurement of fetal whole blood concentrations of
antipyrine, leucine,
-ketoisocaproate, leucine whole blood
radioactivity, fetal plasma specific activity of
-ketoisocaproate and blood
14CO2
has previously been described (25). Whole blood glucose concentration
was measured by the glucose oxidase method (Sigma Chemical, St. Louis,
MO). Methods for measurement of whole blood concentrations of lactate
and
-amino nitrogen-containing substances have also been previously
described (20). Maternal inspired oxygen concentration was measured by
direct analysis (MiniOX O2 monitor; Catalyst Research, Owings Mills, MD). Blood oxygen
concentration was calculated from measurements of oxygen saturation and
hemoglobin content (Radiometer Copenhagen Hemoximeter, OSM 3). Blood
gases and pH were measured at 39°C with standard electrodes
(Radiometer Copenhagen ABL30 Acid Base Analyzer). Hematocrits were
measured using the microhematocrit technique.
Calculations.
Umbilical blood flow was calculated by the steady-state diffusion
method. The fetal availability of oxygen and other substrates was
calculated as the umbilical blood flow times the umbilical venous
oxygen or substrate content. Fetal oxygen uptake was calculated by the
Fick principle (umbilical blood flow times the umbilical venoarterial
oxygen concentration difference). Fetal oxygen extraction was the fetal
oxygen uptake divided by fetal oxygen availability.
We have previously described the methods used to calculate fetal
protein metabolism (21, 25). Briefly, the total fetal leucine flux
(i.e., the sum of all fates for leucine in the fetus) was calculated as
the leucine infusion rate corrected for transumbilical loss of tracer
divided by the specific activity of plasma
-ketoisocaproate
The
specific activity of
-ketoisocaproate was used to approximate
intracellular leucine specific activity (13), presumed to be the site
of leucine use. The transumbilical loss of tracer leucine was
where
dpmLeu-UA and
dpmLeu-UV are the radioactivities
of leucine in the umbilical artery and vein, respectively. The rate of
leucine decarboxylation was estimated by measuring the transumbilical loss of radioactive CO2 and
dividing that quantity by the specific activity of plasma
-ketoisocaproate noted above
where
14CO2-UA
and
14CO2-UV
are the radioactivities in CO2 in
the umbilical artery and vein, respectively. Fetal protein synthesis (i.e., nonoxidative disposal) was
Because
of the rapid interconversion of leucine and
-ketoisocaproate,
its transamination product, total fetal leucine uptake was calculated
as the sum of uptake of both substances. Specifically, fetal uptake of
leucine and
-ketoisocaproate was calculated as
where
[Leu]UV and
[Leu]UA are leucine
concentrations in the umbilical vein and artery, respectively, and
[KIC]UV and
[KIC]UA are the
respective concentrations of
-ketoisocaproate in the umbilical vein
and artery. The rate of accretion of leucine into fetal protein was
measured as the difference between uptake of leucine and
-ketoisocaproate and the rate of decarboxylation.
Finally,
the rate of fetal protein breakdown was calculated as the rate of
leucine use for protein synthesis minus its rate of accretion into
protein, a quantity equivalent to the difference between total leucine
flux and uptake
Statistics.
Data are expressed as means ± SE. Analysis of variance was used to
test for differences among dependent variables such as FIO2,
pH, and blood gases at various times during the protocol. Analysis of
variance for crossover experiments was used to test for the effects of
decreased fetal oxygen availability as separate from the effects of the
duration of the experiment. This analysis allows calculation of a
single difference between control and experimental conditions that can
be ascribed specifically to decreased oxygen availability rather than
the order of the experiment. Comparison with the
t distribution was used to find whether such differences were significant
(P < 0.05).
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RESULTS |
Maternal
FIO2
and fetal arterial blood gases during hypoxia.
Analysis of maternal inspired oxygen concentration during maternal
hypoxemia revealed a rapid decrease in maternal inspired FIO2
to ~9% when first measured after 30 min, with no significant
variation throughout the remainder of the experimental protocol (Fig.
2). In response to the decrease in maternal
inspired oxygen concentration, fetal arterial
PO2 fell
from initial values of 19.6 ± 0.6 and 20.0 ± 0.4 mmHg on
days 1 and
2, respectively, to 11.4 ± 1.1 and
11.1 ± 0.4 mmHg on days 1 and
2, respectively (P < 0.001) by 30 min (Fig. 2).
There were no significant changes in fetal
PO2 after
30 min of decreased inspired maternal oxygen concentration.

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Fig. 2.
Maternal inspired oxygen concentration ( ) and fetal
PO2 ( )
during hypoxic conditions on day 1 (A) and day
2 (B) of experiment.
Values are means ± SE. If no SE bar is evident, SE is within
symbol.
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Maternal hypoxia decreased fetal pH within 60-90 min (Fig.
3). After 60 min (day
1) or 90 min (day
2) fetal pH remained constant for the duration of the
hypoxia. Because there were no consistent changes in fetal
PCO2 during
maternal hypoxia (results not shown), there was no evidence of
respiratory acidosis. Measurement of fetal base excess confirmed that
the acidosis was metabolic. On day 1,
base excess was significantly lower than baseline by 90 min of hypoxia,
after which no further significant changes occurred (Fig. 3). On
day 2, base excess was significantly
negative by 60 min of hypoxia, and no further significant changes
occurred after 120 min.

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Fig. 3.
Fetal pH ( ) and base excess ( ) during experimental periods.
Values are means ± SE. Significant differences
(P < 0.05) from: * prehypoxic
values; final values.
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Fetal hematocrit, pH, and blood gas values during sampling periods.
Fetal hematocrit rose 4% (average for both study days;
P < 0.05; Table
1). As expected, decreased maternal
inspired oxygen concentration resulted in a 42% decrease in fetal
arterial
PO2 (P < 0.001). In contrast, 4 h of
decreased maternal inspired oxygen concentration had no significant
effect on fetal
PCO2. Fetal
pH, however, decreased (P < 0.001).
As would be expected given decreased pH in the presence of normal
PCO2, there
was increased metabolic acidosis [base excess decreased on both
study days (P < 0.001; Table
1)].
Fetal oxygenation.
Hypoxia increased umbilical blood flow by 17%
(P < 0.001; Table
2). Fetal arterial oxygen concentration
also decreased (65%) in response to decreased maternal inspired oxygen
concentration. Indeed, the decrease in fetal blood oxygen concentration
was sufficient that fetal oxygen availability (the product of umbilical
venous oxygen concentration and umbilical blood flow) fell by 43%
(P < 0.001) despite the above noted
increase in umbilical blood flow. Hypoxia decreased the venoarterial
concentration difference for oxygen across the umbilical circulation by
30% (P < 0.001), a decrease that
resulted in a 16% decrease in fetal oxygen uptake (umbilical blood
flow times umbilical oxygen venoarterial difference) during hypoxia
(P < 0.01). This decrease in fetal
oxygen uptake was, however, less severe than the decrease in fetal
oxygen availability, so that fetal oxygen extraction increased during
hypoxia (P < 0.001).
Fetal oxidative substrate concentrations and availability.
Hypoxia had no effect on fetal arterial glucose concentration (Table
3). The availability of glucose to the
fetus through the umbilical vein, however, did increase slightly (14%;
P < 0.025), primarily because of the
above noted increase in umbilical blood flow. In contrast to glucose
concentration, fetal lactate concentration markedly increased during
reduced fetal oxygen availability (P < 0.001). Fetal lactate concentration increased not only in the umbilical artery but also in the umbilical vein (results not shown). The marked increase in umbilical venous lactate concentration was
primarily responsible for a similar increase in fetal lactate availability (P < 0.001; Table 3).
In response to decreased maternal inspired oxygen concentration there
was a 22% increase in blood arterial amino nitrogen concentration
(P < 0.001). Increased fetal amino
nitrogen concentration and the increase in umbilical blood flow noted
above increased fetal amino nitrogen availability by 35%
(P < 0.001; Table 3).
Fetal leucine kinetics.
Decreased fetal oxygen availability increased fetal arterial leucine
concentration by 37% (Table 4). Fetal
umbilical venous leucine concentration also rose, although to a lesser
extent (31%). Consequently, there was a significant decrease in the
umbilical venoarterial blood concentration difference for leucine, a
decrease that was sufficient to account for a decrease (53%) in
leucine uptake (Table 4) even though umbilical blood flow increased. Because of the rapid equilibration between leucine and its
transamination product,
-ketoisocaproate, transplacental uptake of
this substance represents an additional source of leucine uptake. Fetal
umbilical arterial and venous
-ketoisocaproate concentrations
increased by 100 and 90%, respectively, in response to hypoxia.
Despite these concentration changes, the umbilical venoarterial
difference for
-ketoisocaproate was unaffected by hypoxia, as was
the transplacental uptake of
-ketoisocaproate (Table 4). However,
because
-ketoisocaproate uptake represented only 9 and 17% of total
leucine carbon uptake during control and hypoxic conditions,
respectively, total leucine uptake reflected the effect of hypoxia on
leucine uptake and fell by 48% during hypoxia.
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Table 4.
Fetal blood leucine and -ketoisocaproate concentrations
and transplacental uptake during control and hypoxic conditions
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To measure other aspects of the leucine kinetics,
[1-14C]leucine was
infused into the fetus at 127,900 ± 19,100 dpm · min
1 · kg
1.
During control conditions, between 23% (29,400 ± 3,900 dpm · min
1 · kg
1;
day 1) and 21% (27,200 ± 10,300 dpm · min
1 · kg
1;
day 2) of this radioactivity was
lost across the umbilical circulation to the placenta. During hypoxic
conditions, this percentage increased so that on day
1, 45% (57,100 ± 9,000 dpm · min
1 · kg
1)
and on day 2, 35% (44,900 ± 10,000 dpm · min
1 · kg
1)
left the fetal compartment. This transumbilical leucine loss was
subtracted from the radioactivity infused before the total leucine flux
was calculated. Corrected infusion rates were, therefore, 98,500 ± 8,700 dpm · min
1 · kg
1
and 70,800 ± 11,900 dpm · min
1 · kg
1
during control and hypoxic conditions on day
1, respectively, and 83,000 ± 8,300 dpm · min
1 · kg
1
and 100,700 ± 8,800 dpm · min
1 · kg
1
during hypoxic and control conditions on day
2, respectively.
The specific activity of
-ketoisocaproate, the transamination
product of leucine, reached constant specific activity before blood
sampling occurred (180 min) in each experiment (Fig.
4). During decreased fetal oxygen
availability,
-ketoisocaproate specific activity increased
(P < 0.001; Table
5) compared with control conditions.
Because
-ketoisocaproate specific activity increased while the
infused radioactivity retained in the fetus decreased, total leucine
flux fell by 32% during reduced fetal oxygen availability. Fetal
leucine decarboxylation, an index of the flux of leucine to fates other
than protein synthesis, also fell by 30%
(P < 0.001; Table 5).

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Fig. 4.
Fetal plasma -ketoisocaproate specific activity during control and
hypoxic conditions on day 1 (A) and day
2 (B). Values are
means ± SE. No differences were found by paired
t-test analysis between 1st (1) and
2nd (2) samples on either day for either control ( ) or hypoxia ( )
experiments.
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Fetal protein metabolism.
Fetal nonoxidative leucine disposal, an index of the use of leucine for
fetal protein synthesis, decreased by 32%
(P < 0.001) during decreased fetal
oxygen availability (Table 5 and Fig. 5).
Fetal protein breakdown was also significantly decreased during decreased fetal oxygen availability but to a lesser extent (22% decrease; P < 0.001). Because fetal
protein synthesis decreased to a greater extent than fetal protein
breakdown, fetal protein accretion (the difference between synthesis
and a breakdown) fell markedly (62% decrease;
P < 0.001; Table 5 and Fig. 5).

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Fig. 5.
Use of leucine for protein synthesis, return from protein breakdown,
and accretion into fetal proteins during control and hypoxic
conditions. Data summarize both study days. *Differences between
control and hypoxic conditions significant at
P < 0.001.
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DISCUSSION |
This study was performed to find whether restriction of fetal oxygen
availability sufficient to constrain fetal oxygen consumption affected
not only fetal protein synthesis but fetal proteolysis as well. A
corollary to this purpose was to find whether fetal protein accretion,
the balance between fetal protein synthesis and proteolysis, was
affected by such a decrease in fetal oxygen availability. Lowering
maternal inspired oxygen concentration to ~9% decreased fetal oxygen
availability enough to limit fetal oxygen consumption. Under such
conditions, both the uptake of leucine across the placenta and fetal
oxidation of leucine decreased. In addition, not only did the use of
leucine for fetal protein synthesis also diminish (32%), but the
return of leucine to the intracellular pool from protein breakdown
decreased as well (by 22%). Finally, because fetal protein synthesis
decreased more than protein breakdown, fetal protein accretion, an
important component of fetal growth, diminished.
There are both strengths and limitations to this study. First, we
allowed the ewes 6 days to recover from surgery before any experiments
were performed so that fetal metabolism was minimally affected by
operative or experimental stress. Others (7) have shown that fetal
nitrogen balance, although negative 2 h after surgery, was positive
within 4 days. More importantly, the rate of leucine accretion into
fetal proteins during control measurements is no different from that of
normally growing fetal lambs not subjected to surgery (18). Second, we
employed an experimental design (i.e., crossover) that is specifically
designed to separate factors related to either the duration or order of
the experiment from those related to the experimental condition itself.
Thus maternal or fetal stress, diurnal variation, or blood sampling could not have accounted for the effects attributed to hypoxia. Third,
we limited our sampling to <3% of fetal blood volume, a volume shown
to have minimal effect on fetal homoeostasis (33), and replaced sampled
blood with maternal blood to maintain fetal blood volume. We did
notice, however, a slight increase in fetal hematocrit during hypoxia.
The 4% increase in fetal hematocrit that occurs during hypoxia
corresponds to a decrease in fetal blood volume of 4% (4). Such a
decrease has been previously reported during fetal hypoxia (4) and may
be either a direct effect of hypoxia or secondary to increased fetal
norepinephrine concentration (24). Finally, this experiment does not
address the effects of longer-term deprivation of fetal oxygen supply. Whether the changes in fetal leucine and protein metabolism described in this report persist as the duration of oxygen deprivation is continued remains a subject for further study.
The methods used in this experiment to measure fetal leucine kinetics
and protein metabolism have met the conditions to appropriately make
such measurements (2). First, the most fundamental assumption made in
this experiment is that the specific activity of plasma
-ketoisocaproate measures the specific activity of the precursor for
each of the intracellular fates of leucine. Such uniformity of specific
activity through each of the intracellular leucine pools seems
unlikely. Nevertheless, given that those latter amino acid pools could
be sampled only if the animal were killed, an experimental design that
would preclude experiments such as this, plasma
-ketoisocaproate
specific activity has been shown to be a reasonable and reproducible
surrogate with which to measure intracellular leucine specific activity
(13). Second, we used the constant infusion method to measure leucine
kinetics, a method that assumes that the tracer specific activity has
reached steady state. To meet this condition, we showed no difference
between specific activities in the first and second blood samples for either condition on both experimental days (Fig. 4). Third,
CO2 fixation into fetal
bicarbonate pools is insignificant (34). Consequently, we have not
corrected the loss of labeled CO2
across the umbilical circulation for such retention. Fourth, other
investigators (15, 17) have calculated fetal leucine disposal rate, a
different flux from the total fetal leucine flux defined in this
report. Fetal leucine disposal rate has three major components: the
rate of leucine decarboxylation, the rate at which leucine enters fetal protein synthesis, and the flux of leucine into the placenta from the
fetus (15). Thus fetal leucine disposal rate differs from the total
fetal leucine flux of this report by the flux of leucine into the
placenta from the fetus. Our methods for calculating quantities such as
leucine uptake, decarboxylation, and use for protein synthesis are,
however, identical to theirs. Finally, we assume that the loss of
radioactivity from the fetus is irreversible. Actually, various
measurements show that between 0 and 6% of the infused tracer leucine
(17, 21, 25) returns to the fetus from the mother as labeled
-ketoisocaproate. Consequently, minimal, if any, underestimation of
fetal total leucine flux or the quantities derived from it, protein
synthesis and protein breakdown, occurred consequent to reuptake of
tracer.
A number of methods, including reduction of maternal inspired oxygen
concentration (14), maternal anemia (28), and restriction of placental
(3) or umbilical blood flow (30), have been used to decrease the
availability of oxygen to the fetus. Unfortunately, some of these
methods decrease not only oxygen availability but fetal availability of
other substrates as well (30). In such cases, it is difficult to decide
which variable (i.e., reduced availability of oxygen, glucose, or amino
acids) is the most direct cause of the effects described. To avoid this
problem, we wanted fetal oxygen availability decreased, but the
availability of the other substrates important to fetal metabolism
(glucose, lactate, and amino acids) either maintained constant or
increased. We defined the availability of a given substrate to the
fetus as that quantity transported into the fetus through the umbilical
vein (in
µmol · kg
1 · min
1),
a quantity that comes from two sources. A portion of each of these
substrates is the net amount acquired by the umbilical circulation from
the placenta. The remainder is that quantity in the umbilical artery as
it comes from the fetus to the placenta. Fetal oxygen availability fell
because both fetal oxygen uptake across the placenta and the quantity
of oxygen remaining in the umbilical arterial circulation fell. In
contrast, although fetal glucose availability was unaffected by
decreased maternal inspired oxygen concentration, fetal lactate and
amino acid availability rose, primarily because fetal arterial
concentrations rose, and therefore more remained in the umbilical
arterial circulation. Thus these experiments describe effects of
decreased oxygen availability on fetal leucine kinetics in the presence
of normal or increased availability of the other fetal metabolic
substrates.
The fetal need for oxidative substrate is normally met by the continual
transplacental uptake of three substances: glucose, lactate, and amino
acids (1). Amino acid uptake in general, and tyrosine uptake in
particular, are diminished during decreased fetal oxygen availability
(19, 20). In the present experiment, leucine uptake also decreased
during hypoxia. Transplacental transport of amino acids employs
specific amino acid transporters located in both the microvillous and
the basal membranes of the syncytiotrophoblast (27). Leucine transport
at both membranes uses an active, carrier-mediated transporter that
includes an ATPase (27). It is tempting, therefore, to postulate that
decreased intracellular ATP concentrations resulting from inadequate
availability of oxygen directly affect such transporters. However, it
seems more likely that the process is multifactorial. For example,
metabolic acidosis, noted during hypoxia, also decreases fetal leucine
uptake (23). Consequently, the mechanism by which decreased leucine
transport occurs in response to hypoxia remains to be defined.
Once leucine has entered the fetal compartment, it has two major fates,
either decarboxylation (and further use as oxidative substrate) or use
for protein synthesis. In this study, leucine decarboxylation
diminished when fetal oxygen availability was decreased. In contrast,
another study of amino acid oxidation during reduced fetal oxygen
availability found that tyrosine decarboxylation was unaffected (19).
Thus some variability exists in the response of this aspect of amino
acid metabolism to reduced fetal oxygen availability. The decreased
rate of leucine oxidation in the present study cannot be attributed to
the effects of acidosis, because leucine oxidation is increased rather
than decreased by metabolic acidosis (23). In addition, the decrease in
fetal leucine oxidation is unlikely to be due to changes in leucine
concentration, because the increased leucine concentrations that occur
during decreased fetal oxygen availability should have increased rather
than decreased leucine decarboxylation (21).
Decreased fetal oxygen availability also affected several aspects of
fetal protein metabolism. First, the use of leucine for protein
synthesis decreased by 32% during decreased fetal oxygen availability.
In previous experiments, the use of
[1-14C]tyrosine for
protein synthesis decreased to 39% of the value obtained in normoxia.
In adults, the depletion of intracellular ATP by hypoxia may directly
diminish protein synthesis (5). This experiment neither supports nor
refutes this idea. Finally, the possibility that increased
concentration of leucine during reduced fetal oxygen availability
affected fetal protein synthesis is unlikely, because increases in
leucine concentration stimulate, rather than decrease, protein
synthesis (6).
This study is the first to measure the effect of reduced fetal oxygen
availability on fetal protein breakdown. Because breakdown of most
proteins is a process requiring energy in the form of intracellular ATP
(26), we hypothesized that fetal proteolysis would diminish if oxygen
availability were decreased sufficiently to limit oxygen consumption, a
condition met by the present experiment. Again, however, whether
intracellular ATP depletion is the primary reason for the 22% decrease
in the rate of fetal proteolysis during decreased fetal oxygen
availability remains unclear. Leucine decreases proteolysis in adults
(29), so the possibility that increased fetal leucine concentration
contributed to the suppression of proteolysis remains a distinct
possibility.
The relationship between effects on protein synthesis and breakdown
becomes especially important in fetal life given the exquisite sensitivity of fetal protein accretion to changes in these two variables. In this experiment, fetal protein synthesis decreased more
than breakdown. Consequently, fetal protein accretion, the difference
between synthesis and breakdown, decreased by 62%. This decrease in
fetal protein accretion in response to 4 h of decreased fetal oxygen
availability shows, in the absence of a significant change in fetal
body composition, that 4 h of reduced fetal oxygen availability affects
fetal growth. Obviously, the change in fetal weight that occurs over 4 h could not be measured given the inaccessibility of the live fetus to
accurate weighing. However, in other experiments, decreased fetal
oxygen availability has been caused by hypobaric hypoxia (14),
prolonged reduction in uterine blood flow (3), and maternal anemia
(28), and, in each of these experiments, between 7 and 21 days of
decreased fetal oxygen availability decreased fetal weight.
Changes in protein metabolism such as those just described have
important implications for fetal oxidative metabolism. The change in
leucine use for protein synthesis during hypoxia, 3.0 µmol
leucine · kg
1 · min
1
(or 45 µmol · kg
1 · min
1
peptide bonds synthesized) would require 30 µmol · kg
1 · min
1
less oxygen consumption during hypoxia than during normoxic conditions (9). A similar calculation for protein breakdown based on a decrease of
1.6 µmol · kg
1 · min
1
in leucine return from proteolysis (24 µmol · kg
1 · min
1
peptide bonds cleaved) would require 4 µmol · kg
1 · min
1
less oxygen during hypoxia than during normoxia. The total metabolic cost of protein turnover is, therefore, 34 µmol · kg
1 · min
1
less oxygen under hypoxic than during normoxic conditions, a difference
accounting for ~72% of the decrease in fetal oxygen consumption
during hypoxia (47 µmol · kg
1 · min
1).
The effect of hypoxia on fetal protein breakdown may have resulted in
even larger energy savings because, at least in turtle hepatocytes, not
only does protein breakdown decrease during hypoxia but it becomes
partially independent of ATP (16). Finally, there are other
energy-requiring processes not measured in this experiment, such as
cellular uptake of amino acids, RNA synthesis, DNA synthesis (11), and
fetal breathing (12), that could have decreased sufficiently to account
for the rest of the decrease of fetal oxygen needs that occurred during
hypoxia.
Whether the effects of decreased fetal oxygen availability on fetal
protein metabolism are directly attributable to decreased intracellular
energy state or are secondary to other changes in fetal metabolism
remains undefined. In this experiment, fetal pH fell while lactate and
amino acid concentrations increased. Other studies make metabolic
acidosis an unlikely explanation for the effects of decreased oxygen
availability on fetal leucine and protein kinetics (23). However, the
effects of increased lactate and amino acid concentrations on these
variables remain unstudied. However, some of the changes in the fetal
hormonal milieu seem unlikely modulators of the reported effects on
leucine kinetics. Even though a modest decrease in fetal insulin
concentration occurs during fetal hypoxemia (32), fetal insulin
concentration does not affect protein synthesis (21). Although
increased insulin concentration can reduce fetal proteolysis, larger
concentration increases than expected during fetal hypoxia are needed
for this effect. Fetal hypoxia increases fetal cortisol concentration
(12), but such changes should not affect fetal protein synthesis (22) and would increase rather than decrease protein breakdown (22). However, increased fetal catecholamine concentration, which occurs during fetal hypoxemia (12), causes a constellation of effects on fetal
leucine kinetics and protein metabolism similar to those of decreased
oxygen availability (24). Consequently, some, or maybe even all, of the
effects reported here could be due to increased fetal catecholamine
concentrations. One may as easily hypothesize, however, that the
effects of norepinephrine are consequent to decreased peripheral blood
flow in the fetus, an effect that could decrease intracellular ATP
concentrations, at least in those tissues poorly perfused (8). Thus the
effects of norepinephrine could be due to tissue ATP depletion rather
than the converse. In any case, whether increased fetal catecholamine
concentration is required to modulate the effects of decreased fetal
oxygen availability on fetal leucine kinetics and protein metabolism
will require further study.
In summary, reduced availability of oxygen to the fetus decreases both
transplacental uptake of leucine and fetal leucine oxidation. The use
of leucine for fetal protein synthesis is also reduced, as is the
return of leucine from protein breakdown, although to a lesser degree.
As a result of these changes in fetal protein synthesis and breakdown,
fetal protein accretion, an important component of fetal growth, is
diminished by 4 h of decreased fetal oxygen availability. In addition,
decreases in fetal protein synthesis and breakdown, which both require
intracellular expenditure of ATP, decrease fetal energy
needs sufficiently to account for most of the decrease in oxygen
consumption.
 |
ACKNOWLEDGEMENTS |
The author thanks John Sweeley and Yiting Yang for their excellent
technical assistance and Drs. David P. Carlton, Diane E. Lorant, and
Ronald S. Bloom for their critical review of the manuscript.
 |
FOOTNOTES |
This research was supported by National Institutes of Child Health and
Human Development Grant RO1-HD-27455.
Address for reprint requests: J. R. Milley, Dept. of Pediatrics,
Division of Neonatology, Univ. of Utah School of Medicine, 50 N. Medical Dr., Salt Lake City, UT 84132.
Received 27 June 1997; accepted in final form 9 January 1998.
 |
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