Department of Pediatrics, Case Western Reserve University School of Medicine, Rainbow Babies and Children's Hospital, Cleveland, Ohio 44106
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein and nitrogen
(N) accretion by the mother is a major adaptive response to pregnancy
in humans and animals to meet the demands of the growing conceptus.
Quantitative changes in whole body N metabolism were examined during
normal pregnancy by measuring the rates of leucine N
(QN) and carbon
(QC) kinetics
with the use of
[1-13C,15N]leucine.
Rate of synthesis of urea was measured by
[15N2]urea
tracer. Pregnancy-related change in total body water was quantified by
H2[18O]
dilution, and respiratory calorimetry was performed to quantify substrate oxidation. A significant decrease in the rate of urea synthesis was evident in the 1st trimester (nonpregnant 4.69 ± 1.14 vs. pregnant 3.44 ± 1.11 µmol · kg1 · min
1;
means ± SD, P < 0.05). The lower
rate of urea synthesis was sustained through the 2nd and 3rd
trimesters. QN
was also lower in the 1st trimester during fasting; however, it reached
a significant level only in the 3rd trimester (nonpregnant 166 ± 35 vs. 3rd trimester 135 ± 16 µmol · kg
1 · h
1;
P < 0.05). There was no significant
change in QC
during pregnancy. A significant decrease in the rate of transamination
of leucine was evident in the 3rd trimester both during fasting and in
response to nutrient administration (P < 0.05). The rate of deamination of leucine was correlated with the
rate of urea synthesis during fasting
(r = 0.59, P = 0.001) and during feeding
(r = 0.407, P = 0.01). These data show that
pregnancy-related adaptations in maternal N metabolism are evident
early in gestation before any significant increase in fetal N
accretion. It is speculated that the lower transamination of
branched-chain amino acids may be due to decreased availability of N
acceptors such as
-ketoglutarate as a consequence of resistance to
insulin action evident in pregnancy.
leucine; stable isotopes; nitrogen accretion
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE METABOLIC ADAPTATIONS observed during pregnancy in humans and animals are aimed to provide the nutrient and energy requirements for both the mother and the growing conceptus. Because glucose is the primary source of energy for the fetus and is a significant contributor to maternal energy metabolism, changes in maternal glucose metabolism appear to occur in parallel with the increasing demands of pregnancy and conceptus (7, 26, 28, 34). Similarly, protein/nitrogen accretion by the mother for the synthesis of new maternal and fetal tissue necessitates changes in maternal protein turnover as well as nitrogen (N) excretion starting early in gestation. Previous studies using balance data in human pregnancy have shown that N retention by the mother throughout pregnancy occurs in excess of the theoretical protein cost, supporting the concept that maternal N gain as lean body mass is a significant component of protein accretion over and above that deposited in the fetus and products of conception (27). The physiological mechanisms associated with the N accretion of pregnancy have not been studied in detail.
Rates of urea synthesis and rates of excretion of urinary urea N have
been employed to quantify catabolism and oxidation of protein late in
gestation in humans and animals. These data show that in the latter
part of pregnancy, in both humans and animals, the rate of synthesis of
urea and its rate of excretion in urine are lower when compared with
nonpregnant women, both during fasting and in response to amino acid
load (4, 16, 29, 36). The mechanism for the lower rate of urea
synthesis during pregnancy is not clear. It could be the consequence of
decreased delivery of ureogenic substrates to the liver or due to
decreased activity of urea cycle (29). Although pregnancy hormones such
as progesterone and estrogen have been shown to have suppressive
effects on the activity of the urea cycle enzymes (39), a significant
decrease in the plasma concentration of circulating amino acids has
also been observed early in gestation, both in humans and animals (18, 29, 30). The decreased concentration of -amino-N, by decreasing the
hepatic delivery or ureogenic substrate, would be expected to result in
decreased rate of urea synthesis. In the present study, we have
quantified longitudinally the rate of urea synthesis through pregnancy
in normal women, starting early in gestation with the use of stable
isotopic tracer
[15N2]urea.
Because branched-chain amino acids are the major source of N for the
ureogenic amino acids alanine and glutamine (21, 23), we have also
quantified the rates of transamination of leucine and related these to
changes in urea synthesis as gestation progresses. Direct carcass
analysis and other indirect data suggest increased N accretion by the
mother as lean body mass early in pregnancy, even before significant N
accretion by the fetus (25, 44). Because N accretion has been
associated with significant changes in rates of whole body turnover of
protein, kinetics of leucine were also quantified as a measure of the
dynamic aspects of whole body protein metabolism. In addition, changes
in total body water (TBW) during pregnancy were measured with the use
of H2[18O]
to quantify changes in lean body mass. The studies were aimed at
examining the hypothesis that conservation of N is an important component of N accretion and that it will manifest as decreased rate of
urea synthesis and a decrease in the rate of leucine transamination. Furthermore, these changes will be apparent early in gestation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Leucine and urea kinetics studies were performed during pregnancy in normal, healthy subjects. Ten subjects were studied in the 1st trimester, twelve in the 2nd trimester, and eight in the 3rd trimester. Seven subjects could be studied serially throughout the pregnancy. The reasons for attrition included premature delivery, lack of interest, and pregnancy-related problems. However, inclusion of their data as reported here did not have any significant impact on the conclusions of the study. The data on longitudinally studied subjects (n = 6) are displayed (see Figs. 2-4). The data on the seventh subject were outside the range of the others because of unexplained reasons (see RESULTS). In addition, she delivered a large-for-gestational-age infant at term gestation weighing 4,261 g. Seven nonpregnant women were studied as controls.
All subjects were healthy, were not receiving any medications other than vitamin supplements, and had no medical complications related to pregnancy. Their glucose tolerance tests at 26-28 wk of gestation were normal. The nonpregnant subjects were studied between day 8 and 15 of menstrual cycle. They were not receiving any medications, including oral contraceptives, and did not have a family history of diabetes. The clinical characteristics of the study subjects are presented in Table 1. The protocol was approved by the Institutional Review Board for Investigation in Humans. Written informed consent was obtained from each subject and her husband after the procedure was fully explained.
|
All subjects were instructed to consume a diet consisting of a minimum of 75 g protein/day for 7 days before the study. Actual intake was monitored by interview and estimated from self-recorded diaries (Table 1) and then calculated by a dietician using Diet Planner software (13). As shown, there was no significant difference in the weight-specific calorie intake between the nonpregnant and the pregnant subjects before the study. Each subject consumed an average of 2,000-2,200 kcal and 90-100 g protein per day.
Subjects were asked to come to the General Clinical Research Center in the morning after an overnight fast of 10 h. Two 22-gauge cannulas were placed in superficial veins on the dorsum of the hands, one for the infusion of isotopic tracers and the other for obtaining blood samples. The sampling site was kept patent by infusing isotonic saline solution at 10 ml/h and was kept warm to obtain arterialized blood samples.
[15N2]urea,
99% 15N;
[1-13C,15N]leucine,
99% 13C and 99%
15N;
NaH[13C]O3,
99% 13C; and
H2[18O],
99% 18O, were obtained from Merck
(Dorvall, PQ, Canada). The tracers for intravenous administration were
dissolved in sterile isotonic saline, sterilized by Millipore
filtration, and tested for sterility and pyrogenicity as previously
described (29). After basal blood and breath samples were obtained, the
tracers were infused according to prime constant rate infusion
technique. For
[15N2]urea,
the prime was 2.0 mg/kg and the infusion rate was 0.2 mg · kg1 · h
1.
For
[1-13C,15N]leucine,
the prime was 5 µmol/kg and the infusion rate was 5 µmol · kg
1 · h
1.
To achieve an early steady state in the
CO2/bicarbonate pool, a priming
dose of 10 mg of
NaH[13C]O3
was also administered at the start of the study. In addition, an
accurately weighed dose (2-2.5 g) of
H2[18O]
was given orally to estimate the TBW. The subjects continued the fast
for the next 165 min. The response to a mixed nutrient load was
evaluated by giving oral Ensure Plus (Ross Laboratories, Columbus, OH)
at a rate of 35 ml every 30 min for the next 3 h. This dose of Ensure
Plus corresponds to 101 kcal and 3.7 g protein per hour or 2,424 kcal/day and 89 g protein/day. The study design is displayed in Fig.
1.
|
Arterialized blood samples were obtained at 30-min intervals during the
1st hour and every 15 min for the rest of the study. Blood samples were
centrifuged immediately, and plasma was stored at 70°C until
analysis. In addition, breath samples for
13C analysis were collected every
30 min by having the subject breathe through a one-way valve (3, 22).
Respiratory calorimetry measurements were performed intermittently
throughout the study using an open-circuit system described previously
(3, 22). The rates of oxygen consumption
(
O2) and
CO2 production
(
CO2) were measured at
hourly intervals by placing a ventilated hood over the subject's head.
Recordings were obtained for a period of at least 15 min. The analyzer
was calibrated using gravimetrically measured standard mixtures of
oxygen and CO2. The accuracy and precision of the respiratory calorimetry system were checked by measuring the respiratory quotient of absolute alcohol and were within
2% of the expected value.
The effect of Ensure Plus feeding without isotopic tracers on 13C enrichment of breath CO2 was examined in three nonpregnant and six pregnant women ranging from 12 to 34 wk of gestation. Feeding of Ensure Plus for 3 h resulted in an average increase in 13C enrichment of breath CO2 by 0.0016% (SD: ±0.0009%). There was no significant difference between the pregnant and nonpregnant groups or within the pregnant group with advancing gestation. Because the change in 13C enrichment of CO2 was not significant, no correction was made for this change in calculating leucine oxidation during Ensure administration.
Analytic procedures. Plasma glucose and urea N concentrations were measured by glucose oxidase and urease methods, respectively, on respective commercial analyzers (Beckman Instruments, Fullerton, CA). Plasma insulin levels were measured by double-antibody RIA. The lowest detectable level by this assay is 2 µU/ml. These analytic techniques have been reported from our laboratory previously (26, 29).
Mass spectrometric analysis.
The mass spectrometric methods for the measurement of isotopic
enrichment of leucine, -ketoisocaproic acid (KIC), and urea have
been described from this laboratory (16, 19, 40, 42). Leucine and urea
were separated from the plasma by preparatory ion exchange
chromatography on a mini column. An
N-acetyl,
N-propyl ester derivative of leucine
was prepared according to the method of Adams (1) with certain
modifications (16). A Hewlett-Packard model 5985 gas
chromatography-mass spectrometry system was utilized. Methane chemical
ionization was used, and mass-to-charge ratios (m/z)
216, 217, and 218, representing the mono- and di-labeled leucine, were
monitored using the selected ion monitoring software. To quantify the
15N enrichment of leucine,
m/z
128 and
m/z
129 were also monitored in the electron-impact mode (35). From these
data, the enrichments of
[1-13C,15N]leucine
and total [13C]leucine
were calculated as described previously (11, 35). Norleucine was used
as internal standard to measure plasma leucine levels. A
trifluoroaceto-hydroxypirimidine derivative of urea was prepared, and
the 15N enrichment was quantified
in the electron impact mode by monitoring of
m/z
153 and
m/z
155 (42). The 13C enrichment of
plasma KIC was measured using a quinoxalone derivative (19). Standard
solutions of known enrichments were run along with the unknowns to
correct for analytic variations.
13C enrichment of the
CO2 in expired air was measured
after separation of the CO2 by
cryogenic distillation in vacuum, as previously described (22).
18O enrichment of the expired
CO2 was also measured at the same time as 13C measurements with the
use of an isotope ratio mass spectrometer (16).
Calculations.
O2 and
CO2 were calculated by
multiplication of the gradients across the face with the flow rate of
air and application of Haldane transformation (22). The measured
O2 and
CO2 were corrected to the
standard temperature and pressure. The rate of appearance of leucine
(Ra) was calculated from the
tracer dilution during steady state (28).
Ra = I × [(Ei/Ep)
1], where I is the rate of infusion of the tracer
(µmol · kg
1 · min
1)
and Ei and
Ep represent the enrichments of
infusate and of plasma leucine at steady state, respectively. Leucine
carbon flux
(QC) was
calculated using plasma
[13C]KIC, and leucine
N flux (QN) was
calculated using
[1-13C,15N]leucine
enrichment during isotopic steady state (11, 35). Isotopic steady state
was determined by visual inspection of the data. Enrichment data
between 135 and 160 min and between 315 and 360 min were used to
calculate the turnover rate. The coefficient of variation for the
enrichment data for leucine and KIC in individual subjects was between
3 and 5%, and the slope was not different from zero.
Statistics.
All data are reported as means ± SD. The data were analyzed with
the SPSS/PC+ version 4.0 Statistical Package for the Social Sciences
(Chicago, IL). The Mann-Whitney U test
and Wilcoxon's signed rank test were used for unpaired and paired
interval level data, respectively. For patients followed longitudinally
during pregnancy, their interval level data were first analyzed by the Friedman test. If the overall P value
was <0.05, the post hoc pair-wise comparisons were then analyzed by
Wilcoxon's signed rank test. In the regression analyses, outliers were
identified through the examination of the Studentized residuals
(outliers defined as greater than 3.16 or less than 3.16),
Mahalanobis' distance, and Cook's distance. By examination of the
plot of the residuals, the assumption of equal variance and a linear
model were met in each of the analyses. Statistical significance was defined a priori as a P value < 0.05 (2 tail).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pregnant and nonpregnant women were similar in age, body weight, and body mass index (Table 1). All subjects gained weight appropriately during pregnancy. The serially studied subjects, those who completed all the studies, delivered at term gestation. Their infants were normal and appropriate for gestational age (birth weight 3,307 ± 564 g). With the increase in body weight through gestation, there was a corresponding increase in tracer-measured TBW. However, the fraction of body weight represented by body water did not change with advancing gestation (Fig. 2).
|
The plasma concentrations of glucose, urea N, leucine, and insulin during fasting and during feeding (Ensure Plus) are displayed in Table 2. As shown, the plasma glucose concentration during fasting was slightly lower in pregnant subjects compared with nonpregnant subjects. In response to feeding, there was a significant increase in plasma glucose concentration in all subjects. Plasma insulin concentration during fasting was slightly higher during pregnancy (not significant), particularly during the 3rd trimester, and there was a significant (P < 0.02) increase in insulin levels in response to feeding in all subjects. Plasma urea N concentration was significantly lower (P < 0.003) in early gestation compared with the nonpregnant group and remained significantly lower throughout pregnancy. In response to feeding, there was a significant decrease in plasma urea N concentration in the nonpregnant subjects. Plasma leucine levels, also displayed in Table 2, were slightly lower during pregnancy and increased in response to feeding.
|
Urea synthesis during pregnancy. The rate of urea synthesis during fasting in the 1st trimester of pregnancy was significantly less (P < 0.05) compared with nonpregnant subjects (Table 3). With advancing gestation, there was a further decrease in urea synthesis during fasting. Feeding did not result in any significant change in the rate of urea synthesis in all subjects.
|
Leucine carbon and N kinetics. The rate of appearance of leucine N (QN) was calculated from the enrichment of leucine (m+2) species, whereas QC was calculated from the enrichment of KIC (m+1) species in the plasma. During fasting, QN was less during pregnancy compared with nonpregnant state in the 1st and 3rd trimesters; however, the differences were statistically significant only in the 3rd trimester (P < 0.05). QN was higher during the 2nd trimester compared with the 1st and 3rd trimesters. In contrast to QN, QC was unchanged throughout pregnancy compared with nonpregnant controls. In addition, there was no difference in the fraction of leucine C-1 decarboxylated between nonpregnant or pregnant women nor was there any significant impact of advancing gestation on leucine oxidation. There was a trend toward decrease in fractional rate of oxidation with advancing gestation. The calculated rate of nonoxidative disposal of leucine carbon was not affected by pregnancy.
In the serially studied subjects (Fig. 3), there was no significant change in QN or QC between the 1st and 3rd trimesters during fasting. However, there was an increase in QN during the 2nd trimester when compared with the 1st trimester. There was a statistically significant decrease in the fraction of leucine carbon turnover oxidized (C/QC) between the 1st and 2nd trimesters (18.8 ± 2.9%, 1st trimester; 16.1 ± 1.6%, 2nd trimester; P < 0.05) and between the 1st and 3rd trimesters (16.2 ± 2.7%, 3rd trimester; P < 0.05). The changes in transamination of leucine in serially studied subjects are displayed in Fig. 4. As with the whole group, XO and XN were lower in the 1st trimester compared with nonpregnant subjects. There was no significant change in XN and XO from the 1st to the 3rd trimester. The fraction of leucine reaminated (XN/XO) was significantly reduced with advancing gestation when all the data were examined by Friedman two-way ANOVA (P < 0.05).
|
|
|
Respiratory calorimetry.
As described previously, there was no change in weight-specific
O2 or
CO2 values
during pregnancy. The respiratory exchange ratio was less in the 1st
trimester compared with nonpregnant subjects and was higher in the 3rd
trimester compared with the 1st trimester and nonpregnant controls
(Table 5). In response to
feeding, there was an increase in
O2 and
CO2 as well as respiratory
exchange ratio. All the observations were similar among the four groups
in the fed state (data not shown).
|
Correlations. The XO of leucine was significantly correlated with the rate of urea synthesis, both during fasting (r = 0.589, P = 0.0001, y = 93.6 + 1.78x; Fig. 5) and in response to feeding (r = 0.407, P = 0.012). Removal of one outlier in the fasting data improved the correlation (r = 0.736). A significant correlation was also observed between the XN of leucine and synthesis of urea during fasting (r = 0.66, P = 0.0004) but not during feeding. Interestingly, there was no correlation between rate of decarboxylation (oxidation) of leucine (C) and rate of urea synthesis or between calculated rates of oxidation of protein using leucine or urea method.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The significant results of the present study can be summarized as follows: 1) commensurate with the increase in body weight during pregnancy, there is a proportional increase in TBW and hence lean body mass; 2) there is a decrease in the rate of synthesis of urea early in gestation that is sustained throughout the pregnancy; 3) a decrease in QN is evident early in gestation, whereas the QC does not change with pregnancy; 4) a significant correlation was evident between the XO of leucine and synthesis of urea. These data are significant in that, for the first time, they point to the mechanism of N conservation, i.e., lower rate of urea synthesis and lower rate of transamination of branched-chain amino acids during pregnancy, and show that conservation of N occurs early in gestation before any significant growth in fetal mass. Thus these adaptive changes may be aimed at the accretion of N (protein) by the mother as lean body mass, which may be a necessary anticipatory adaptation to pregnancy and fetal N requirements.
Estimates of TBW have been used to quantify lean body mass to relate changes in protein and energy metabolism. Previous estimates of TBW in human pregnancy using either [2H]2O or H2[18O] are similar to those reported here (16, 25, 44). All of these show no significant change in the fraction of body weight represented by TBW during pregnancy. However, calculation of lean body mass from TBW alone using the conventional equations for nonpregnant adults may cause errors because of changes in hydration index of maternal tissue during pregnancy and because of the contribution of fetal tissues, with much higher content of water, to these measurements (8, 31, 44). Use of newer hydration index would have resulted in small change in estimated lean body mass. For these reasons, we elected not to calculate lean body mass from these data. Nevertheless, because there was no change in the fraction of body weight represented by TBW, expression of the kinetic data per unit lean body mass would have had minimal impact on the overall results. Finally, these changes in TBW during pregnancy do suggest that accretion of lean body mass (protein) is a significant component of increase in body weight by the mother during pregnancy and that this protein accretion starts early in gestation.
Although a lower concentration of plasma urea N during pregnancy has been consistently reported in both human and animal studies, few studies have quantified the rates of urea synthesis in vivo. Studies in animals, either in vivo or of isolated perfused liver preparation, have shown a decreased rate of urea synthesis in response to an amino acid or protein load in pregnancy compared with nonpregnant state (4, 36). Data in human pregnancy show a decreased or unchanged rate of urea N synthesis and excretion during the 3rd trimester (16, 20, 29). Our previous data showed a lower rate of urea synthesis in both normal and diabetic women during fasting in the 3rd trimester compared with postpartum. Forrester et al. (20) have quantified rates of urea production in normal pregnant women in Jamaica throughout the day using [15N2]urea tracer as intermittent intravenous dose. The dilution of the tracer was measured in urinary urea. In addition, the dietary protein and caloric intakes of their study subjects were higher throughout pregnancy compared with nonpregnant controls. These authors reported no change in urea production between pregnant and nonpregnant women. However, the rate of urea synthesis was significantly less during the 3rd trimester when compared with the 1st trimester. It should be underscored that their study represents a sum of both fasting and fed state, the data having been obtained throughout the day. In addition, the impact of intermittent tracer dose on calculated rates of urea synthesis remains unknown. In our study, we examined the responses during fasting and in response to nutrient intake after rigid dietary preparation and constant-rate tracer infusion. The protein and caloric intakes were those recommended for the pregnant and nonpregnant women. These data are the first to show a decreased rate of urea synthesis, or conservation of N, in the 1st trimester of pregnancy, much before there is any significant fetal N accretion. An almost 30% lower rate of urea synthesis, when compared with nonpregnant controls, was evident in the 1st trimester, and a 45% lower rate was evident in the 3rd trimester.
The dynamic aspects of protein turnover in pregnancy have been quantified using labeled leucine, phenylalanine, or glycine tracers to calculate change in rates of synthesis and breakdown of whole body protein. The impact of the use of different isotopically labeled amino acid tracers and related problems have been discussed (5). Studies in pregnant rats using [1-14C]leucine tracer showed a decrease in weight-specific rate of leucine turnover and an increase in oxidation of leucine between day 17 and 20 of gestation (33). However, there was no change in the amount of leucine incorporated into proteins. In contrast, studies in normal human subjects during the 3rd trimester of pregnancy, using [1-13C]leucine tracer, showed no significant change in leucine kinetics between 20 and 40 wk of gestation (16). When compared with nonpregnant women, there was a significant decrease in leucine turnover during pregnancy. However, these kinetics were calculated on the basis of enrichment of plasma leucine rather than that of KIC. Thompson and Halliday (41) quantified protein turnover during pregnancy in six subjects with the use of [13C]leucine tracers and by measuring the dilution in plasma KIC. When expressed in relation to total body weight, the calculated rate of protein turnover did not change during pregnancy. All of these data are similar to those reported here, showing no change in the QC during pregnancy. Of significance, these rates were similar to those in the nonpregnant women. In addition, the QC values during feeding were also similar in pregnant and nonpregnant women (Table 3). There was a small decrease in the fraction of leucine decarboxylated (C/QC) during pregnancy, and it was statistically significant in the serially studied subjects (Fig. 3). These data suggest that, as with urea synthesis, N conservation by the mother is also evident in the lower rate of decarboxylation of leucine. However, the magnitude of change estimated by leucine decarboxylation was less than that measured by change in rate of urea synthesis, and there was no correlation between the rates of decarboxylation of leucine and urea synthesis.
Because deamination of branched-chain amino acids in the muscle is considered the major source for alanine and glutamine N (21, 23), which in turn are the sources for urea N, we hypothesized that the decreased rate of urea N synthesis will be associated with decreased QN. This hypothesis was confirmed by the lower QN early in pregnancy. In addition, a strong correlation was observed between the rates of urea synthesis and rate of deamination of leucine (Fig 5). These are the first estimates of leucine N kinetics in human pregnancy and provide a mechanistic explanation for the decreased rate of urea N synthesis. A small decrease in glycine N turnover was also seen by DeBenoist et al. (14) in pregnant women in late gestation.
Branched-chain -ketodehydrogenase complex is the rate-limiting
enzyme involved in the irreversible loss of leucine. No significant change in the rate of decarboxylation of leucine was observed in the
present study or reported in other states of protein accretion (growth), such as puberty, growth hormone therapy, or newborn infant
(2, 15, 38). A decreased rate of urea synthesis has also been observed
after growth hormone therapy (12). Of interest, pregnancy, puberty,
neonate, and growth hormone therapy are characterized as states of
insulin resistance, specifically in relation to glucose uptake. The
mechanism of the decrease in transamination and its impact on leucine
metabolism and protein synthesis remain speculative. Because the
equilibrium constant for most transaminase reactions is close to unity,
transamination is a freely reversible process with no net transfer of
N. However, the transfer of N may be regulated by the availability of
the N acceptor, in this instance
-ketoglutarate, which in turn may be regulated by changes in anaplerotic carbon flux into the
tricarboxylic acid cycle induced by insulin resistance. Evidence
presented by Hedden and Buse (24) in in vitro muscle preparation
suggests that an increase in N acceptors, e.g., by increasing pyruvate, results in decreased rate of protein synthesis, possibly by decreasing the intracellular leucine pool and increasing the KIC pool. Such a
situation should also lead to lower rates of glutamine and possibly alanine synthesis, as has been reported after growth hormone therapy (6). Whether such reduction in glutamine turnover also occurs in
pregnancy needs to be examined.
In human studies, the contribution of the fetus to the overall whole
body measurements in the mother, as described here, cannot be
separated. The observed changes in maternal N/protein metabolism are
likely to be minimally influenced by the quantitative N requirements of
the fetus, since the estimated fetal amino acid requirements for N
accretion and energy (oxidation) cannot explain the observed changes in
the N metabolism of the mother. First, the decrease in urea synthesis
and in leucine N kinetics occurred before any significant fetal N
accretion in the 1st trimester. Second, the changes in
QN during the 2nd
and 3rd trimesters did not parallel changes in N accretion by the
fetus. In fact, there was a small increase in
QN in the 2nd
trimester (Table 3). Estimates of amino acid balance across the
umbilical circulation and estimates of N accretion and amino acid
oxidation by the fetus in human studies suggest fetal N uptake to be
~450 mg
N · kg1 · day
1
(9, 32). Of this, only 120 mg
N · kg
1 · day
1
represent N accretion by the human fetus (10, 45). In relation to the
body weight of the mother, these represent an N uptake by the fetus of
<1 mg N · kg maternal body
wt
1 · h
1
and a urea production rate by the fetus of 0.2 mg
N · kg maternal body
wt
1 · h
1,
resulting in an almost negligible contribution to the maternal N
metabolism.
In summary, data from the present study show that during human
pregnancy, there is a significant reduction in the rate of urea
synthesis early in gestation. The decrease in urea synthesis is
correlated with a lower rate of transamination of leucine. It is
speculated that the lower transamination of branched-chain amino acids
may be the consequence of decreased availability of N acceptors
(-ketoglutarate). The latter, in turn, may be related to the
resistance to insulin action (glucose uptake) often manifest in states
of N accretion and growth, e.g., puberty or after growth hormone
therapy.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the staff of the General Clinical Research Center at University Hospitals of Cleveland for help in the conduct of these studies. The administrative support of Joyce Nolan is gratefully appreciated.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants P50-HD-11089 and RR-00080.
Address for reprint requests: S. C. Kalhan, Rainbow Babies and Children's Hospital, 11100 Euclid Ave., Cleveland, OH 44106-6010.
Received 19 November 1997; accepted in final form 12 May 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, R. F.
Determination of amino acid profiles in biological samples by gas chromatography.
J. Chromatogr.
95:
189-212,
1974[Medline].
2.
Arslanian, S. A.,
and
S. C. Kalhan.
Correlations between fatty acid and glucose metabolism. Potential explanation of insulin resistance of puberty.
Diabetes
43:
908-914,
1994[Abstract].
3.
Assel, B.,
K. Rossi,
and
S. Kalhan.
Glucose metabolism during fasting through human pregnancy: comparison of tracer method with respiratory calorimetry.
Am. J. Physiol.
265 (Endocrinol. Metab. 28):
E351-E356,
1993
4.
Beaton, G. H.
Urea formation in the pregnant rat.
Arch. Biochem. Biophys.
67:
1-9,
1957.
5.
Bier, D. M.
Intrinsically difficult problems: the kinetics of body proteins and amino acids in man.
Diabetes Metab. Rev.
5:
111-132,
1989[Medline].
6.
Biolo, G.,
F. Iscra,
G. Toigo,
B. Ciocchi,
R. Situlin,
A. Gullo,
and
G. Guarnieri.
Effects of growth hormone administration on skeletal muscle glutamine metabolism in severely traumatized patients: preliminary report.
Clin. Nutr. (Edinb.)
16:
89-91,
1997.
7.
Catalano, P. M.,
E. D. Tyzbir,
R. R. Wolfe,
J. Calles,
N. M. Roman,
S. B. Amini,
and
E. A. H. Sims.
Carbohydrate metabolism during pregnancy in control subjects and women with gestational diabetes.
Am. J. Physiol.
264 (Endocrinol. Metab. 27):
E60-E67,
1993
8.
Catalano, P. M.,
W. W. Wong,
N. M. Drago,
and
S. B. Amini.
Estimating body composition in late gestation: a new hydration constant for body density and total body.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E153-E158,
1995
9.
Cetin, I.,
C. Corbetta,
L. P. Sereni,
A. M. Marconi,
P. Bozzetti,
G. Pardi,
and
F. C. Battaglia.
Umbilical amino acid concentrations in normal and growth-retarded fetuses sampled in utero by cordocentesis.
Am. J. Obstet. Gynecol.
162:
253-261,
1990[Medline].
10.
Cetin, I.,
A. M. Marconi,
P. Bozzetti,
L. P. Sereni,
C. Corbetta,
G. Pardi,
and
F. C. Battaglia.
Umbilical amino acid concentrations in appropriate and small for gestational age infants: a biochemical difference present in utero.
Am. J. Obstet. Gynecol.
158:
120-126,
1988[Medline].
11.
Cheng, K. N.,
P. J. Pacy,
F. Dworzak,
G. C. Ford,
and
D. Halliday.
Influence of fasting on leucine and muscle protein metabolism across the human forearm determined using L-[1-13C,15N]leucine as the tracer.
Clin. Sci. (Colch.)
73:
241-246,
1987[Medline].
12.
Dahms, W. T.,
R. P. Owens,
S. C. Kalhan,
D. S. Kerr,
and
R. K. Danish.
Urea synthesis, nitrogen balance, and glucose turnover in growth hormone-deficient children before and after growth hormone administration.
Metabolism
38:
197-203,
1989[Medline].
13.
Dare, D.,
and
M. Kelly.
Diet Planner User's Guide. San Francisco: Regents of the Univ. of California General Clinical Research Center, 1994.
14.
DeBenoist, B.,
A. A. Jackson,
J. S. E. Hall,
and
C. Persaud.
Whole-body protein turnover in Jamaican women during normal pregnancy.
Hum. Nutr. Clin. Nutr.
39C:
167-179,
1985[Medline].
15.
Denne, S. C.,
and
S. C. Kalhan.
Leucine metabolism in human newborns.
Am. J. Physiol.
253 (Endocrinol. Metab. 16):
E608-E615,
1987
16.
Denne, S. C.,
D. Patel,
and
S. C. Kalhan.
Total body water measurement in normal and diabetic pregnancy: evidence for maternal and amniotic fluid equilibrium.
Biol. Neonate
57:
284-291,
1990[Medline].
17.
Denne, S. C.,
D. Patel,
and
S. C. Kalhan.
Leucine kinetics and fuel utilization during a brief fast in human pregnancy.
Metabolism
12:
1249-1256,
1991.
18.
Felig, P.,
Y. J. Kim,
V. Lynch,
and
R. Hendler.
Amino acid metabolism during starvation in human pregnancy.
J. Clin. Invest.
51:
1195-1202,
1972[Medline].
19.
Fernandes, A. A.,
S. C. Kalhan,
F. G. Njorge,
and
G. S. Matousek.
Quantitation of branched-chain -keto acids as their N-methylquinoxalone derivatives: comparison of O- and N-alkylation versus -silylation.
Biomed. Environ. Mass Spectrom.
13:
569-581,
1986[Medline].
20.
Forrester, T.,
A. V. Badaloo,
C. Persaud,
and
A. A. Jackson.
Urea production and salvage during pregnancy in normal Jamaican women.
Am. J. Clin. Nutr.
60:
341-346,
1994[Abstract].
21.
Garber, A. J.,
I. E. Karl,
and
D. M. Kipnis.
Alanine and glutamine synthesis and release form skeletal muscle. II. The precursor role of amino acids in alanine and glutamine synthesis.
J. Biol. Chem.
10:
836-843,
1976.
22.
Glamour, T. S.,
A. J. McCullough,
P. J. J. Sauer,
and
S. C. Kalhan.
Quantification of carbohydrate oxidation by respiratory gas exchange and isotopic tracers.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E789-E796,
1995
23.
Haymond, M. W.,
and
J. M. Miles.
Branched chain amino acids as a major source of alanine nitrogen in man.
Diabetes
31:
86-89,
1982[Abstract].
24.
Hedden, M.,
and
M. G. Buse.
Leucine transamination rates may affect protein synthesis in muscle.
In: Developments in Biochemistry. Metabolism and Clinical Implications of Branched-Chain Amino Acids and Ketoacids, edited by M. Walser,
and J. Williamson. Amsterdam: Elsevier/North Holland, 1981, vol. 18, p. 233-238.
25.
Hopkinson, J. M.,
N. F. Butte,
K. J. Ellis,
W. W. Wong,
M. R. Puyau,
and
E. O. Smith.
Body fat estimation in late pregnancy and early postpartum: comparison of two-, three-, and four-component models.
Am. J. Clin. Nutr.
65:
432-438,
1997[Abstract].
26.
Kalhan, S.,
K. Rossi,
L. Gruca,
E. Burkett,
and
A. O'Brien.
Glucose turnover and gluconeogenesis in human pregnancy.
J. Clin. Invest.
100:
1775-1781,
1997
27.
Kalhan, S. C.
Protein metabolism in pregnancy.
In: Principles of Perinatal-Neonatal Metabolism (2nd ed.), edited by R. M. Cowett. New York: Springer-Verlag, 1998.
28.
Kalhan, S. C.,
L. J. D'Angelo,
S. M. Savin,
and
P. A. J. Adam.
Glucose production in pregnant women at term gestation. Sources of glucose for human fetus.
J. Clin. Invest.
63:
388-394,
1979[Medline].
29.
Kalhan, S. C.,
K. Tserng,
C. Gilfillan,
and
L. J. Dierker.
Metabolism of urea and glucose in normal and diabetic pregnancy.
Metabolism
31:
824-833,
1982[Medline].
30.
Kerr, G. R.
The free amino acids of serum during development of Macaca mulatta. II. During pregnancy and fetal life.
Pediatr. Res.
2:
493-500,
1968[Medline].
31.
Lederman, S. A.
Variability of water content of lean tissue of pregnant and non pregnant rats and its effect on body fat estimation.
Am. J. Clin. Nutr.
37:
663-668,
1983[Abstract].
32.
Lemons, J. A.,
E. W. Adcock III,
M. D. Jones, Jr.,
M. A. Naughton,
G. Meschia,
and
F. C. Battaglia.
Umbilical uptake of amino acids in the unstressed fetal lamb.
J. Clin. Invest.
58:
1428-1434,
1976[Medline].
33.
Ling, P. R.,
B. R. Bistrian,
G. L. Blackburn,
and
N. Istfan.
Effect of fetal growth on maternal protein metabolism in postabsorptive rat.
Am. J. Physiol.
252 (Endocrinol. Metab. 15):
E380-E390,
1987[Abstract].
34.
Marconi, A. M.,
E. Davoli,
I. Cetin,
A. Lanfranchi,
G. Zerbe,
R. Fanelli,
P. V. Fennessey,
G. Pardi,
and
F. C. Battaglia.
Impact of conceptus mass on glucose disposal rate in pregnant women.
Am. J. Physiol.
264 (Endocrinol. Metab. 27):
E514-E518,
1993
35.
Matthews, D. E.,
D. M. Bier,
M. J. Rennie,
R. H. T. Edwards,
D. Halliday,
D. J. Millward,
and
G. A. Clugston.
Regulation of leucine metabolism in man: a stable isotope study.
Science
214:
1129-1131,
1981[Medline].
36.
Metzger, B. E.,
F. S. Angnoli,
and
N. Freinkel.
Effect of sex and pregnancy on formation of urea and ammonia during gluconeogenesis in the perfused rat liver.
Horm. Metab. Res.
2:
367-368,
1970[Medline].
37.
Nair, K. S.,
C. G. Ford,
K. Ekberg,
E. Fernqvist-Forbes,
and
J. Wahren.
Protein dynamics in whole body and in splanchnic and leg tissue in type I diabetic patients.
J. Clin. Invest.
95:
2926-2937,
1995[Medline].
38.
Rizza, R. A.,
L. J. Mandarino,
and
J. E. Gerich.
Effects of growth hormone on insulin action in man. Mechanisms of insulin resistance, impaired suppression of glucose production, and impaired stimulation of glucose utilization.
Diabetes
31:
663-669,
1982[Abstract].
39.
Roberge, A.,
R. Charbonneau,
and
L. Berlinguet.
Variation of the enzymes of the urea cycle and aspartate transcarbamylase in liver of pregnant rats.
Can. J. Biochem.
45:
1371-1374,
1967[Medline].
40.
Shoup, V. A.,
and
S. C. Kalhan.
Stabilization of -ketoisocaproic acid in serum and plasma.
Clin. Chem.
35:
495-496,
1989[Medline].
41.
Thompson, G. N.,
and
D. Halliday.
Protein turnover in pregnancy.
Eur. J. Clin. Nutr.
46:
411-417,
1987.
42.
Tserng, K. Y.,
and
S. C. Kalhan.
Gas chromatography/mass spectrometric determination of [15N]urea in plasma and application to urea metabolism study.
Anal. Chem.
54:
489-491,
1982[Medline].
43.
Tserng, K. Y.,
and
S. C. Kalhan.
Calculation of substrate turnover rate in stable isotope tracer studies.
Am. J. Physiol.
245 (Endocrinol. Metab. 8):
E308-E311,
1983
44.
Van Raaij, J. M. A.,
M. E. M. Peek,
S. H. Vermaat-Miedema,
C. M. Schonk,
and
J. G. A. J. Hautvast.
New equations for estimating body fat mass in pregnancy from body density or total body water.
Am. J. Clin. Nutr.
48:
24-29,
1988[Abstract].
45.
Widdowson, E. M.,
D. A. T. Southgate,
and
E. N. Hey.
Body composition of the fetus and infant.
In: Nutrition and Metabolism of the Fetus and Infant, edited by H. K. A. Visser. London: Martinus Nijhoff, 1979, p. 169-177.