Bloomfield, Frank H.; van Zijl, Pierre L.; Bauer, Michael K.; Harding, Jane E.
Amino acids are essential precursors for many processes involved in fetal growth. Amniotic fluid contains many nutrients, including large amounts of amino acids (1). In the human, it has been calculated that the nitrogen content of amniotic fluid is sufficient to provide up to 15% of the nitrogen requirements of the late-gestation fetus (2). Human fetuses that are unable to swallow amniotic fluid, for example, secondary to an esophageal atresia, have been reported to be smaller than controls, especially when born near to term (3,4). Swallowed amniotic fluid may thus provide a paraplacental route of nutrition in the late-gestation human fetus. However, the importance of swallowed amniotic fluid for fetal growth in other animals is less clear, with some studies reporting a reduction in fetal size when fetal swallowing is prevented (5–7), and some reporting no effect (8,9). Although amniotic fluid amino acid concentrations have been reported in the human (10,11), cow (12), and pig (13), they have not been reported previously in the sheep. Therefore, the first aim of this study was to describe the concentrations of amino acids in fetal blood and amniotic fluid of late-gestation sheep.
The role of amniotic fluid as a paraplacental route of fetal nutrition may become more important in the fetus with intrauterine growth restriction (IUGR) secondary to placental insufficiency. Reduced transplacental transfer of amino acids has been demonstrated both in vivo and in vitro in ovine and human fetuses with established IUGR (14–18). Circulating fetal concentrations of the branched chain amino acids valine, leucine, and isoleucine are most consistently reduced (19,20), although other essential amino acids such as threonine and arginine may also be reduced (21). In contrast, circulating concentrations of some amino acids such as proline and glycine are increased in IUGR fetuses (20,21). However, as far as we are aware, amino acid concentrations in amniotic fluid of fetuses with IUGR has not been reported in any species. The second aim of this study was therefore to describe the effects of IUGR induced by placental embolization on fetal blood and amniotic fluid amino acid concentrations in late-gestation sheep.
One amino acid of particular interest is glutamine. In the mature gut, glutamine can be used as a metabolic fuel (22) and is an important source of metabolic substrates for other anabolic pathways. In adult animals who have undergone bowel resection, glutamine reduces bacterial translocation across the gut (23) and promotes the compensatory trophic response of the remaining gut, especially when given in conjunction with growth hormone or insulin-like growth factor-I (IGF-1) (24,25). In the postsurgical human, glutamine supplementation of parenteral nutrition has been reported to improve immune status and shorten hospital stays (26). Preterm infants who develop necrotizing enterocolitis (NEC) are reported to have lower circulating glutamine concentrations for 7 days before the development of the disease (27). Addition of glutamine to intravenous nutrition has been advocated to prevent NEC (28), although a metaanalysis has not found a beneficial effect (29). Furthermore, in IUGR, the gut is often severely affected (30), and IUGR infants are at higher risk of developing NEC (31). However, the role of both circulating and amniotic glutamine in the growth and metabolism of the fetal gut is unknown. The third aim of this study was therefore to determine whether glutamine is taken up by the gut of normal and IUGR fetal sheep in utero.
Swallowed amniotic fluid is essential for normal development of the gut (5,8,32,33). Various growth factors in amniotic fluid, such as IGF-1 and epidermal growth factor, have also been reported to have a trophic effect on the fetal gut (5,9,34,35). Although Trahair et al. found that a short-term luminal infusion of IGF-1 failed to prevent the gut atrophy seen in fetal sheep after esophageal ligation (36), we previously reported that a chronic luminal infusion of a low dose of IGF-1 does prevent gut atrophy after esophageal ligation (5). We also demonstrated, in the same cohort of animals that we report in this article, that a low dose of IGF-1 given once daily into the amniotic fluid rather than directly into the gut lumen reverses the gut atrophy seen after placental embolization (37). Furthermore, there are reports of synergistic effects of glutamine and IGF-1 on gut growth (25), and also of increased luminal glutamine transport with IGF-1 treatment in a cultured intestinal cell model (38). The final aim of this study was therefore to determine whether intraamniotic IGF-1 treatment affected gut uptake of glutamine or any other amino acid in fetal sheep made IUGR by placental embolization.
MATERIALS AND METHODS
Approval for the experiment was obtained from the institutional animal ethics committee. Twenty-nine ewes carrying singleton fetuses underwent surgery at 110 days' gestation (term = 145 days). Using halothane anesthesia and aseptic technique, the maternal abdomen was opened and the fetus accessed via an hysterotomy. Polyvinyl catheters were placed in the fetal femoral artery and vein via the tarsal vessels. A fetal laparotomy was performed in the left lower quadrant, and a loop of small bowel was exteriorized. The portal vein was catheterized via an arcuate mesenteric vein using a polyvinyl catheter with a silicone tip to reduce the risk of perforation of this thin-walled vessel. The fetal abdomen was closed in two layers. Four amniotic fluid catheters were attached to the fetal skin, one to either side of the fetal neck, and one to each fetal hindlimb. The main uterine arteries supplying each uterine horn were catheterized via an arterial branch near the tip of each horn. Catheters were also placed in the maternal carotid and femoral arteries and the jugular vein. At surgery, the ewe received 5 mL streptopen (250 mg procaine penicillin/250 mg dihydrostreptomycin sulphate; Pittman Moore Ltd., Upper Hutt, New Zealand) intramuscularly, and the fetus received 80 mg gentamicin in the amniotic fluid.
The ewes were housed in individual cages with free access to water and ad libitum quantities of a standard laboratory diet of chaffage and pelleted stock feed.
At the end of the experiment (131 days' gestation), ewes were killed with an overdose of phenobarbitone. The uterus was removed intact from the ewe, and the fetal fluids were collected and weighed to obtain an estimate of amniotic fluid volume. The placement of catheters was checked, and the fetus was dissected and weighed. Only animals in which the tip of the portal vein catheter lay within the portal vein, proximal to its union with the umbilical vein, are reported in this study.
Animals were randomly assigned to one of three groups: control (n = 9), growth-restricted treated with saline (n = 9), and growth-restricted treated with IGF-1 (n = 11). Growth restriction was induced between 114 and 119 days' gestation by repetitive embolization of the uteroplacental bed with 20 μm to 50 μm polystyrene microspheres (Superose 12 diluted 1:100; Pharmacia Biotech AB, Uppsala, Sweden) as described previously (37).
From 120 to 130 days' gestation, saline-treated fetuses received 2 mL saline as a once-daily bolus injection into the amniotic fluid, and IGF-1–treated fetuses received 20 μg recombinant human IGF-1 (Pharmacia and Upjohn, Peapack, NJ, U.S.A.) mixed with 2 mL fresh amniotic fluid. This dose was calculated to increase amniotic fluid IGF-1 concentrations approximately fivefold (5,37). Control fetuses were untreated.
Plasma and amniotic fluid samples were collected simultaneously from maternal and fetal artery and fetal portal venous and amniotic catheters every 2 to 3 days, immediately before embolization or treatment. Samples were collected between 08:00 and 09:00, just before the ewes were given fresh food. Baseline samples were collected after the ewes had recovered from surgery but before any intervention. Blood was stored at −80°C until assayed for urea and amino acids. Amino acids and urea were measured in amniotic fluid and portal venous and fetal arterial blood at four time points: before embolization (baseline sample; day 114), at the completion of embolization but before treatment was commenced (day 120), midway through treatment (day 125), and at the completion of treatment (day 131). The concentrations of individual amino acids in blood and amniotic fluid obtained from the last sample (immediately before postmortem) were summed to obtain the total measured amino acid concentration. The available nitrogen pool in amniotic fluid at the end of the experiment was calculated by multiplying the nitrogen units for each amino acid (amino acid concentration x number of nitrogen units in each molecule) by the amniotic fluid volume obtained at postmortem and obtaining the sum for all measured amino acids. This was converted to grams of nitrogen.
Arterial blood for gas analysis was collected on ice and rapidly analyzed on a blood gas analyzer (Chiron M845 blood gas analyzer; Chiron Corp., Emeryville, CA, U.S.A.). Blood oxygen content was calculated from hemoglobin concentration, oxygen saturation, and oxygen tension (39).
Samples of blood and amniotic fluid for measurement of urea and amino acids were deproteinized in duplicate by tungstate precipitation. Urea was measured by a 96-well microplate enzyme-linked colorimetric assay using urease (Sigma, St Louis, MO, U.S.A.) (40). The intraassay and interassay coefficients of variation were 5.34% and 7.50%, respectively.
Blood and amniotic fluid amino acid concentrations were determined by a novel high-performance liquid chromatography (HPLC) method. Thirty microliters of blood or amniotic fluid was deproteinized in duplicate by tungstate precipitation, with the addition of an internal standard (15 μmol/L norvaline, Sigma) to the sulfuric acid. Supernatants were stored at −80°C until analysis.
Before chromatography, amino acids in each sample were rendered fluorescent by derivatization with the Waters AccQ.Tag method (Waters Associates, Milford, MA, U.S.A.). In brief, 20 μL of the supernatant was mixed with 60 μL borate buffer (Waters catalogue number WAT052875) in a glass tube. Twenty microliters of 10 mmol/L AccQ-fluor reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; Waters catalogue number WAT052875) in acetonitrile was then added and instantly mixed. This mixture was transferred to an HPLC injection vial, which was incubated at 55°C for 10 minutes. Ten microliters of sample was then injected onto the HPLC column.
For quality control, samples of fetal blood were included at the precipitation step, and samples of maternal blood supernatant were included at the derivatization step.
The HPLC system consisted of a Waters 2690 Alliance separation module, a 300 × 3.9-mm C18 Nova-Pak column (Waters) at 37°C, and a Waters 474 fluorescence detector set at Ex 250 nm Em 395 nm, gain 100.
The mobile phase consisted of a buffer (80 mmol/L sodium acetate, 3 mmol/L triethylamine, 1 mg/L [2.67 μmol/L] disodium calcium ethylenediamine tetraacetic acid) at pH 6.43 (obtained with addition of orthophosphoric acid) run with a complex gradient of acetonitrile from 0% to 16% over 115 minutes.
Data were captured directly by computer with Waters Millennium32 software. A standard curve for each amino acid was generated from four standards injected in duplicate. Each standard contained 15 μmol/L norvaline and each amino acid to be measured in one of four concentrations. The concentrations of individual amino acids in each standard were different, as different amino acids are present in different concentrations in blood and amniotic fluid. The standard curve was generated by plotting the concentration of the amino acid to be measured on the x-axis against the ratio of the area of the peak of that amino acid to the area of the norvaline peak on the y-axis. The amino acid:norvaline peak ratio in the sample was read against the standard curve to determine the amino acid concentration in the sample.
Recovery of a select group of amino acids from blood and amniotic fluid (glycine, glutamine, glutamate, alanine, arginine, leucine, and isoleucine) was checked over a range of 20, 50, 100, and 200 μmol/L above endogenous concentrations. Minimum recoveries were 90% to 96% for blood and 93% to 100% for amniotic fluid. The mean intraassay and interassay coefficients of variation for all amino acids in fetal controls (thus including both precipitation and derivatization steps) were 2.6% and 8.4%, respectively.
After surgery, but before any other intervention (baseline samples), there were no significant differences between the three treatment groups for any of the parameters measured (analysis of variance). Therefore, as all animals had been treated identically to this point, animals were grouped together for analysis of baseline amino acid concentrations in blood and in amniotic fluid. Differences in baseline amino acid concentrations between blood and amniotic fluid were assessed by two-tailed paired t test. Both embolized groups were treated in an identical manner, and there were no differences in the effect of embolization on fetal blood gases, growth, or endocrine status. These data have been reported previously (37). Therefore, the effect of embolization on amino acid concentrations in blood and amniotic fluid was assessed by paired t test in all embolized animals comparing baseline amino acid concentrations with those at the end of embolization. Concentrations from these two time points were also compared in control animals to identify changes that may be ontological rather than caused by embolization. The effect of treatment on amino acid concentrations was assessed by repeated-measures analysis of variance, with the Games-Howell post hoc test applied to results with a significant interaction. Arteriovenous concentration differences between portal venous and fetal arterial samples were identified by paired t tests. If a positive arteriovenous difference was detected, indicating uptake of that amino acid by the gut, the metabolite:oxygen quotient for the gut for that amino acid was calculated from the following formula EQUATION
where Δsubstrate and ΔO2 are the umbilical arteriovenous concentration differences ([A-V]) in molar units measured simultaneously, and k is the number of moles of oxygen required for complete oxidation of 1 mole of substrate. For glutamine, k = 4.5. Changes in [A-V] across the gut with embolization were assessed by paired t test comparing the preembolization and postembolization values. The effect of treatment on [A-V] was analyzed by repeated-measures analysis of variance.
Bonferroni correction was applied to statistical analyses investigating effects on individual amino acids to account for repeated testing. Significance for these analyses was taken at P < 0.002. Nonparametric data were log-transformed before analysis.
The effects of embolization and IGF-1 treatment on fetal growth, gut histology, fetal metabolism, and IGF-1 concentrations have been reported previous (37). Briefly, embolization resulted in fetal hypoxia, hypoglycemia, and polycythemia (Table 1). At postmortem examination, embolized fetuses demonstrated asymmetric growth restriction (Table 1). Fetal weight was reduced by 21% and fetal gut weight by 26%, but brain/body weight ratio was increased by 20%. IGF-1 treatment increased amniotic fluid IGF-1 concentrations threefold to fivefold. Circulating IGF-1 concentrations were not increased with treatment, and were in fact lower in IGF-1–treated animals (Table 1). IGF-1 treatment had no effect on fetal weight, but gastrointestinal weight in IGF-1–treated animals was not different from controls, in contrast to the reduction observed in saline-treated animals.
Amino Acid Concentrations in Blood and Amniotic Fluid of the Late-Gestation Fetal Sheep
Concentrations were higher in fetal arterial blood than in amniotic fluid for 10 of the 23 amino acids measured (Fig. 1). Only serine, alanine, and methylhistidine were present in higher concentrations in amniotic fluid than in fetal arterial blood, with serine present in millimolar amounts.
Effect of Embolization on Blood and Amniotic Fluid Amino Acid Concentrations
Embolization increased fetal blood concentrations of glycine, alanine, and asparagine and decreased blood concentrations of serine, glutamine, and methylhistidine (Fig. 2A). Amniotic fluid concentrations of alanine were increased and serine were reduced by embolization (Fig. 2B). There was no effect of embolization on serine/glycine ratios in blood or amniotic fluid. Embolization reduced the total measured amino acid concentration in blood by 13% (P < 0.001;Fig. 2C) and in amniotic fluid by 15% (P < 0.05;Fig. 2C). The total nitrogen content in amniotic fluid was reduced by 37% in embolized fetuses compared with unembolized controls (4,282 ± 431 vs. 6,808 ± 1,656 nitrogen units [60 mg ± 6 mg vs. 95 mg ± 23 mg nitrogen];P < 0.05), presumably due in part to a 28% reduction in amniotic fluid volume in embolized fetuses (976 mL ± 100 mL vs. 1,350 mL ± 265 mL;P = 0.1).
Gut Glutamine Uptake Before and After Embolization
Glutamine was taken up by the fetal gut, with a baseline systemic–portal difference of 98.5 ± 33.7 μmol/L (P < 0.0001). The baseline glutamine:oxygen quotient across the fetal intestine was 0.65 ± 0.12, indicating that up to 65% of the oxygen consumption of the gut could be accounted for by glutamine oxidation. Citrulline was released by the gut into the portal circulation, with a systemic–portal difference of −17.6 ± 1.5 μmol/L (P < 0.0001). There were no significant [A-V] differences for any of the other measured amino acids at any point in the study. There was no significant systemic–portal difference in urea concentration at baseline. Embolization did not significantly affect glutamine uptake by the gut, the glutamine:oxygen quotient from blood across the gut, or citrulline release by the gut (data not shown). Embolization did not result in an [A-V] difference of urea across the gut.
Effect of IGF-1 Treatment on Amino Acid Concentrations and Gut Glutamine Uptake
There were no statistically significant effects of intraamniotic IGF-1 treatment on amino acid concentrations in blood or amniotic fluid. IGF-1 treatment did not affect the total measured amino acid concentration in blood or amniotic fluid and did not significantly change the available pool of measured amniotic fluid amino acids (data not shown).
However, IGF-1 treatment reduced the systemic–portal concentration difference for glutamine by 15% (P < 0.05;Fig. 3A) and the glutamine:oxygen quotient for the gut from blood by 15% (0.65 ± 0.06 to 0.55 ± 0.09;P < 0.1). The negative systemic–portal difference for citrulline (Fig. 3B) and the citrulline:oxygen quotient did not change. Circulating urea concentrations were not significantly different between groups during embolization (Table 1) or during treatment (control, 5.18 ± 0.24; saline, 5.12 ± 0.23; IGF-1, 5.73 ± 0.25 mM), but IGF-1 treatment resulted in the appearance of a significant negative A-V difference in urea across the gut, indicating urea output by the gut (P < 0.05;Fig. 3C). Glutamine concentrations in amniotic fluid decreased by 29% with IGF-1 treatment, compared with 10% in saline-treated animals and no change in controls, but this was not statistically significant (P < 0.1;Fig. 3D). The serine/glycine ratio in amniotic fluid decreased by 53% in IGF-1–treated animals (P < 0.05;Fig. 3E), but only by 10% and 16% in saline and control animals (not statistically significant). In blood, there was no significant change in serine/glycine ratios in any group over the treatment period (Fig. 3F).
In this study we describe for the first time amniotic fluid amino acid concentrations in the late-gestation fetal sheep and the effect of IUGR caused by placental embolization on both circulating and amniotic fluid amino acid concentrations. Furthermore, we report the first demonstration of fetal gut uptake of circulating glutamine. The methodology we used for measuring amino acid concentrations is relatively straightforward and very reliable. The values we obtained for circulating amino acid concentrations are comparable with those reported previously in late-gestation fetal sheep (41–43).
Amino Acids in Amniotic Fluid
Serine, glycine, glutamine, valine, methyl-lysine, threonine, alanine, taurine, and lysine were present in the highest concentrations in amniotic fluid, with values of 200 μmol/L or greater. This is in broad agreement with findings from studies in the fetal calf (12), pig (13), and human (44–46), although in none of these species was serine present in the highest concentration nor in concentrations greater than circulating serine concentrations. Our finding that glycine was present in significantly lower concentrations in amniotic fluid than in blood is also consistent with previous reports (44), although concentrations in the two fluids were not compared statistically in those studies.
A possible explanation for the differences between blood and amniotic fluid concentrations of serine and glycine is metabolism of these amino acids by the fetal gut and/or placenta. Although there are no data in the fetus, the mature rat intestine converts serine to glycine via serine hydroxymethyltransferase (47). A similar interconversion of serine to glycine occurs in the placenta of the fetal lamb (48). Glycine is then decarboxylated to serine by the fetal liver. Thus, serine and glycine are involved in a placental–hepatic carbon shuttle in the fetal lamb, with the net result being the transfer of methyl groups derived from glycine oxidation within the fetal liver to the placenta (49,50). Either, or both, of these sites of serine-glycine metabolism could result in discordant blood and amniotic fluid concentrations. The fetus has a high metabolic demand for glycine. When nutrient supply is precarious, such as occurs with placental damage after embolization, glycine supply may become potentially limiting on fetal growth (51). Studies in pregnant women suggest that glycine supply may be potentially limiting even in normal pregnancies (52). Thus, the decrease in serine/glycine ratio observed in amniotic fluid in IGF-1–treated fetuses could reflect an attempt to increase or preserve fetal glycine, either by increasing serine to glycine conversion within the placenta or by reducing glycine to serine conversion within the fetal compartment.
Effects of Embolization
The changes in amino acid concentrations that we report with the onset of IUGR suggest that the amniotic fluid amino acid concentrations do not simply reflect filtered fetal plasma, nor are they likely to be solely caused by the reduced circulating insulin concentrations observed in embolized fetuses (37). Hypoinsulinemia has been reported to cause a more generalized reduction in amino acid concentrations (53,54) rather than selective changes as we report here.
Embolization increased alanine concentrations in fetal blood, consistent with other reports in growth-restricted fetal sheep (55), guinea pigs (56), and humans (57), although normal alanine concentrations have also been reported in IUGR fetuses (21,58). We also describe elevated alanine concentrations in the amniotic fluid of embolized fetuses. There are no previous reports of amniotic fluid amino acid concentrations in IUGR fetuses in any species. Tracer studies in fetal sheep suggest that most of the placental–fetal alanine flux is secondary to alanine production in the placenta, rather than a direct maternal–fetal transfer of alanine (59). The elevated concentrations observed in growth-restricted fetuses may therefore be caused either by increased placental release, for example, from increased breakdown of branched-chain amino acids, or from decreased fetal use, perhaps as a consequence of impaired gluconeogenesis in the IUGR fetus. In IUGR in the sheep, the placenta has been reported to increase use of branch-chain amino acids as a fuel, perhaps conserving other fuels for the fetus (60). Reduced gluconeogenic enzyme activity has been reported in IUGR rats (61) and guinea pigs (62) and is suggested in IUGR human neonates by an impaired glucose increase after alanine infusion (63). Further work is needed to elucidate the importance and mechanisms of the changes in blood and amniotic fluid alanine concentrations in this paradigm.
Concentrations of the branched-chain amino acids were not reduced by embolization in either amniotic fluid or fetal blood in our study, consistent with findings in previous studies of maternal fasting in sheep (43). However, in human pregnancies complicated by IUGR, fetal circulating concentrations of branched chain amino acids are reduced (21), and placental transport measured by fetal/maternal leucine enrichment is also reduced (15,19). In sheep, branched chain amino acids are taken up by the placenta in late gestation and converted to keto acids, which are either metabolized further by the placenta or released to the fetus (64,65). During maternal fasting (64), or when placental growth is restricted by carunclectomy (60), placental uptake of branched chain amino acids is increased, and in the latter case this may be at the expense of the fetus. Embolization may limit this process by reducing the amount of viable placental tissue, thus sparing fetal supplies of branched-chain amino acids in our study. However, it is also possible that adaptations that may occur after chronic placental insufficiency may not have had time to develop during the relatively short window following embolization that we have studied in this paradigm.
Glutamine Metabolism by the Fetal Gut
In postnatal life, glutamine is an important metabolic fuel for the gut in all species studied (22). Our finding of glutamine uptake by the fetal gut suggests that this may also be true before birth. Further studies including measurements of intestinal blood flow and tracer uptakes will give a clearer indication of the magnitude of glutamine uptake by the fetal gut. However, the glutamine:oxygen metabolic quotient we measured in this study is very high, suggesting that more than 50% of the oxidative demands of the fetal gut could be met by glutamine oxidation. However, this is probably not the major role of glutamine in the gut. Indeed, an experiment in isolated rat intestine demonstrated that there is no decrease in oxidative rate when the intestine is perfused with a glutamine-free infusate, suggesting that glutamine is probably not essential for oxidative metabolism in the gut (66). There are other potential metabolic fates of glutamine in the gut, such as in the synthesis of nucleotides, amino sugars, and hexosamines. Hexosamines are important precursors for macromolecules involved in tight junction formation and for surface barrier molecules that inhibit passage of bacteria across the mucosal surface. In adult animals who have undergone bowel resection, glutamine reduces bacterial translocation across the gut (23). In the postsurgical human, glutamine supplementation of parenteral nutrition has also been reported to improve immune status and shorten hospital stays (26).
Several studies have investigated the role of enteral glutamine in reducing the incidence of neonatal gastrointestinal-related morbidity (29). In a recent cohort study of preterm infants, those who went on to develop NEC had significant reductions in circulating glutamine and arginine concentrations for 7 days before the onset of NEC (27). Addition of glutamine to intravenous nutrition has been advocated to prevent NEC (28), although a metaanalysis has not found a beneficial effect (29). Furthermore, in IUGR, the gut is often severely affected (30), and infants born IUGR are at increased risk of developing NEC (31). Our findings that the fetal gut takes up circulating glutamine, and that IUGR induced by embolization reduces circulating glutamine concentrations, raise the possibility that reduced gut glutamine supply may contribute to the increased vulnerability of the gut in IUGR infants. However, it should be noted that the IUGR in this study was of late onset and begun at a point in gestation at which the fetal gut is already well developed, although not mature (67). Severe human IUGR is usually of early onset, and this may have implications for the function and structure of the gut. The effect of early-onset IUGR on fetal gut function and glutamine metabolism requires further investigation.
Effects of IGF-1 Treatment
Intraamniotic IGF-1 treatment did not increase circulating IGF-1 concentrations in this study (37), and it is therefore not surprising that circulating amino acid concentrations were not altered. The relatively large volume of amniotic fluid at this gestation (approximately three times the circulating blood volume) means that substantial changes in amino acid metabolism would have to occur before changes could be detected in amniotic fluid amino acid concentrations. However, intraamniotic (and thence fetal enteral) IGF-1 treatment did significantly reduce the arteriovenous difference for glutamine across the gut and the gut glutamine:oxygen quotient in embolized animals without changing these parameters for citrulline. Furthermore, a significant negative arteriovenous difference for urea appeared with IGF-1 treatment, indicating urea production by the gut. This is in contrast to the traditional view that only hepatocytes are capable of ureagenesis, but is consistent with a report that enterocytes of developing piglets can also produce urea (68). Amniotic fluid contains increasing quantities of ammonia throughout gestation (13,69) (our unpublished data), and ureagenesis by the gut may provide a useful means of dealing with this toxic compound.
We previously reported that there was increased gut growth in these IGF-1–treated animals, with an increase in crypt mitoses (37). These data suggest that intraamniotic IGF-1 treatment increased gut uptake and use of amino acids, and the finding of a negative arteriovenous difference for urea with IGF-1 treatment would support this. However, we did not detect a positive arteriovenous concentration difference with IGF-1 treatment for any amino acid other than glutamine, and the glutamine [A-V] was decreased. Although we cannot exclude the possibility that uptake of amino acids below the limit of detection of our method may have occurred, this would only represent a small amino-nitrogen uptake, which would be insufficient to account for the change in urea output or gut growth. We therefore conclude that IGF-1 treatment may have stimulated gut uptake of amino acids from amniotic fluid. Glutamine concentrations in amniotic fluid decreased by nearly 30% with IGF-1 treatment, although this was not statistically significant, and with the known selective use of glutamine by the gut, this would seem to be the most likely candidate. It was not possible to measure uptake of amino acids from the gut lumen in this study, as intestinal blood flow and luminal amino acid concentrations were not measured. This information could be obtained by tracer methodology, and we plan to investigate this further.
The trophic effect of IGF-1 on the gut may lead to an increased uptake of luminal nutrients in the fetus simply by virtue of the increased surface area of the gut. Alternatively, there may be a direct effect on glutamine metabolism, either by an effect on the enzymes or on the enterocyte uptake. Both IGF-1 and epidermal growth factor have been reported to have direct effects on stimulating transport of glutamine across the gut lumen (38,70). Epidermal growth factor has also been shown to increase the activity of mucosal glutaminase and glutamine synthase when given subcutaneously to adult parenterally fed male rats (71). Elucidation of the interaction between IGF-1 and glutamine, and the effect on gut growth in our experimental paradigm require further investigation.
An alternative explanation for the reduced glutamine arteriovenous difference in blood across the gut with IGF-1 treatment could be that luminal IGF-1 increased intestinal blood flow via nitric oxide production (72). This would result in a reduced arteriovenous concentration difference without a change in uptake of glutamine or in the glutamine:oxygen quotient. However, this explanation seems unlikely, as the glutamine:oxygen quotient decreased by a similar degree as the arteriovenous concentration difference for glutamine, and a change in blood flow would not explain the increased gut growth, nor the production of urea by the gut. Furthermore, there was no change in the arteriovenous difference in citrulline, which might be expected if there were changes in blood flow alone.
In summary, we have described amino acid concentrations in the blood and amniotic fluid of the late-gestation fetal sheep and the changes that occur with IUGR induced by uteroplacental embolization. We demonstrate for the first time that the fetal gut also uses glutamine as a fuel and releases citrulline, possibly arising from the conversion of glutamine. We report that intraamniotic therapy with IGF-1 affects glutamine uptake by the fetal gut and have previously demonstrated that this treatment increases gut growth (37).
Our data therefore suggest the possibility that administration of IGF-1, perhaps with amino acid supplements, into amniotic fluid may provide a route for supplementation of the IUGR fetus. As circulating amino acid concentrations were not affected, this may lead to a net gain in nitrogen supply to the fetus. We did not measure placental uptake in this study, but previous experiments in healthy fetuses suggest that enteral administration of amino acids does result in a net gain for the fetus (73). An improvement in gut growth and integrity before birth in IUGR fetuses may also be beneficial in increasing food tolerance, reducing the risk of gut-acquired infections, and possibly in reducing the incidence of NEC in a population that is at high risk for all of these perinatal complications.
1. Lind T. The biochemistry of amniotic fluid. In: Fairweather DVI, Eskes TKAB, eds. Amniotic fluid: Research and clinical application. Amsterdam: Elsevier/North-Holland Biomedical, 1978: 59–81.
2. Pitkin RM, Reynolds WA. Fetal ingestion and metabolism of amniotic fluid protein. Am J Obstet Gynecol 1975; 123:356–61.
3. Blakelock R, Upadhyay V, Kimble R, et al. Is a normally functioning gastrointestinal tract necessary for normal growth in late gestation? Pediatr Surg Int 1998; 13:17–20.
4. Szendrey T, Danyi G, Czeizel A. Etiological study on isolated esophageal atresia. Hum Genet 1985; 70:51–8.
5. Kimble RM, Breier BH, Gluckman PD, et al. Enteral IGF-1 enhances fetal growth and gastrointestinal development in oesophageal ligated fetal sheep. J Endocrinol 1999; 162:227–35.
6. Mulvihill SJ, Albert A, Synn A, et al. In utero supplemental fetal feeding in an animal model: Effects on fetal growth and development. Surgery 1985; 98:500–5.
7. Jacobs DG, Wesson DE, Mago-Cao H, et al. Effect of esophageal ligation on the growth of fetal rabbits. J Pediatr Gastroenterol Nutr 1989; 8:245–51.
8. Trahair JF, Harding R. Restitution of swallowing in the fetal sheep restores intestinal growth after midgestation esophageal ligation. J Pediatr Gastroenterol Nutr 1995; 20:156–61.
9. Trahair JF, Sangild PT. Fetal organ growth in response to oesophageal infusion of amniotic fluid, colostrum, mild or gastrin-releasing peptide: A study in fetal sheep. Reprod Fertil Dev 2000; 12:87–95.
10. Wirtschafter ZT. Free amino acids in human amniotic fluid, fetal and maternal serum. Am J Obstet Gynecol 1958; 76:1219–25.
11. Velázquez A, Rosado A, Bernal A, et al. Amino acid pools in the feto-maternal system. Biol Neonate 1976; 29:28–40.
12. Baetz AL, Hubbert WT, Graham CK. Developmental changes of free amino acids in bovine fetal fluids with gestational age and the interrelationships between the amino acid concentrations in the fluid compartments. J Reprod Fertil 1975; 44:437.
13. Wu G, Bazer FW, Tou W. Developmental changes of free amino acid concentrations in fetal fluids of pigs. J Nutr 1995; 125:2859–68.
14. Glazier JD, Cetin I, Perugino G, et al. Association between the activity of the system A amino acid transporter in the microvillous plasma membrane of the human placenta and severity of fetal compromise in intrauterine growth restriction. Pediatr Res 1997; 42:514–9.
15. Marconi AM, Paolini CL, Stramare L, et al. Steady state maternal-fetal leucine enrichments in normal and intrauterine growth-restricted pregnancies. Pediatr Res 1999; 46:114–9.
16. Jansson T, Persson E. Placental transfer of glucose and amino acids in intrauterine growth retardation: Studies with substrate analogs in the awake guinea pig. Pediatr Res 1990; 28:203–8.
17. Ross JC, Fennessey PV, Wilkening RB, et al. Placental transport and fetal utilization of leucine in a model of fetal growth retardation. Am J Physiol 1996; 270:E491–503.
18. Paolini CL, Marconi AM, Ronzoni S, et al. Placental transport of leucine, phenylalanine, glycine, and proline in intrauterine growth-restricted pregnancies. J Clin Endocrinol Metab 2001; 86:5427–32.
19. Cetin I, Corbetta C, Sereni LP, et al. Umbilical amino acid concentrations in normal and growth-retarded fetuses sampled in utero by cordocentesis. Am J Obstet Gynecol 1990; 162:253–61.
20. Cetin I, Marconi AM, Bozzetti P, et al. Umbilical amino acid concentrations in appropriate and small for gestational age infants: A biochemical difference present in utero. Am J Obstet Gynecol 1988; 158:120–6.
21. Economides DL, Nicolaides KH, Gahl W, et al. Plasma amino acids in appropriate and small-for-gestational age infants. Am J Obstet Gynecol 1989; 161:1219–27.
22. Windmueller HG. Glutamine utilization by the small intestine. Adv Enzymol 1982; 53:202–37.
23. Li YS, Li JS, Jiang JW, et al. Glycyl-glutamine-enriched long-term total parenteral nutrition attenuates bacterial translocation following small bowel transplantation in the pig. J Surg Res 1999; 82:106–11.
24. Zhou X, Li YX, Li N, et al. Glutamine enhances the gut-trophic effect of growth hormone in rat after massive small bowel resection. J Surg Res 2001; 99:47–52.
25. Ziegler TR, Mantell MP, Chow JC, et al. Gut adaptation and the insulin-like growth factor system: Regulation by glutamine and IGF-1 administration. Am J Physiol 1996; 271:G866–75.
26. Powell-Tuck J. Total parenteral nutrition with glutamine dipeptide shorted hospital stays and improved immune status and nitrogen economy after major abdominal surgery. Gut 1999; 44:155.
27. Becker RM, Wu G, Galanko JA, et al. Reduced serum amino acid concentrations in infants with necrotizing enterocolitis. J Pediatr 2000; 137:785–93.
28. Neu J, Roig JC, Meetze WH, et al. Enteral glutamine supplementation for very low birth weight infants decreases morbidity. J Pediatr 1997; 131:691–9.
29. Tubman TRJ, Thompson SW. Glutamine supplementation for preventing morbidity in preterm infants (Cochrane Review). The Cochrane Library. Oxford: Update Software, 2000.
30. Trahair JF, DeBarro TM, Robinson JS, et al. Restriction of nutrition in utero selectively inhibits gastrointestinal growth in fetal sheep. J Nutr 1997; 127:637–41.
31. Beeby PJ, Jeffery H. Risk factors for necrotising enterocolitis: The influence of gestational age. Arch Dis Child 1992; 67:432–5.
32. Trahair JF, Harding R, Bocking AD, et al. The role of ingestion in the development of the small intestine in fetal sheep. Q J Exp Physiol 1986; 71:99–104.
33. Mulvihill SJ, Stone MM, Fonkalsrud EW, et al. Trophic effect of amniotic fluid on fetal gastric development [Abstract]. Gastroenterology 1985; 88:A1511.
34. Trahair JF, Wing SJ, Quinn KJ, et al. Regulation of gastrointestinal growth in fetal sheep by luminally administered insulin-like growth fator-I. J Endocrinol 1997; 152:29–38.
35. Weaver LT, Gonnella PA, Israel EJ, et al. Uptake and transport of epidermal growth factor by the small intestinal epithelium of the fetal rat. Gastroenterology 1990; 98:828–37.
36. Trahair JF, Wing SJ, Horn JL. Failure of short-term luminal IGF-1 to protect against atrophy in a model of esophageal atresia. J Pediatr Surg 1995; 30:1564–70.
37. Bloomfield FH, Bauer MK, van Zijl PL, et al. Amniotic IGF-1 supplements improve gut growth but reduce circulating IGF-1 in growth-restricted fetal sheep. Am J Physiol Endocrinol Metab 2002; 282:E259–69.
38. Karinch AM, Pan M, Lin CM, et al. Glutamine metabolism in sepsis and infection. J Nutr 2001; 131:2535S–8S.
39. Gull I, Charlton V. Effects of antipyrine on umbilical and regional metabolism in late gestation in the fetal lamb. Am J Obstet Gynecol 1993; 168:706–13.
40. Kerscher L, Ziegenhorn J. Urea. In: Bergmeyer HU, ed. Methods of enzymatic analysis. Weinheim: Verlag Chemie, 1984:444–53.
41. Slater JS, Mellor DJ. Concentrations of free amino acids in maternal and fetal plasma from conscious catheterised ewes during the last five weeks of pregnancy. Res Vet Sci 1979; 26:296–301.
42. Lemons JA, Adcock EWI, Jones Jr, MD et al. Umbilical uptake of amino acids in the unstressed fetal lamb. J Clin Invest 1976; 58:1428–34.
43. Lemons JA, Reyman D, Schreiner RL. Fetal and maternal amino acid concentrations during fasting in the ewe. J Pediatr Gastroenterol Nutr 1984; 3:249–55.
44. A'Zary E, Saifer A, Schneck L. The free amino acids in maternal and fetal extracellular fluids collected during early pregnancy. Am J Obstet Gynecol 1973; 116:854–66.
45. Levy HL, Montag PP. Free amino acids in human amniotic fluid: A Quantitative study by ion-exchange chromatography. Pediatr Res 1969; 3:113–20.
46. Mesavage WC, Suchy SF, Weiner DL, et al. Amino acids in amniotic fluid in the second trimester of gestation. Pediatr Res 1985; 19:1021–4.
47. Kikuchi G, Hiraga K, Yoshida T. Role of the glycine cleavage system in glycine and serine metabolism in various organs. Biochem Soc Trans 1980; 8:504–6.
48. Marconi AM, Battaglia FC, Meschia G, et al. A comparison of amino acid arteriovenous differences across the placenta and liver in the fetal lamb. Am J Physiol 1989; 257:E909–15.
49. Cetin I, Fennessey P, Sparks JW, et al. Fetal serine fluxes across fetal liver, hindlimb and placenta in late gestation. Am J Physiol 1992; 263:E786–93.
50. Cetin I, Fennessey PV, Quick Jr, AN et al. Glycine turnover and oxidation and hepatic serine synthesis from glycine in fetal lambs. Am J Physiol 1991; 260:E371–8.
51. Bennet FI, Jackson AA. Glycine is not formed through the amino transferase reaction in human or rat placenta. Placenta 1998; 19:329–31.
52. Jackson AA, Persaud C, Werkmeister G, et al. Comparison of urinary 5-L-oxoproline (L-pyroglutamate) during normal pregnancy in women in England and Jamaica. Br J Nutr 1997; 77: 183–196.
53. Silver M, Fowden AL, Taylor PM, et al. Blood amino acids in the pregnant mare and fetus: The effects of maternal fasting and intrafetal insulin. Exp Physiol 1994; 79:423–33.
54. Phillips AF, Rosenkrantz TS, Lemons JA, et al. Insulin-induced alterations in amino acid metabolism in the fetal lamb. J Dev Physiol 1990; 13:251–9.
55. Robinson JS, Kingston EJ, Jones CT, et al. Studies on experimental growth retardation in sheep: The effect of removal of endometrial caruncles on fetal size and metabolism. J Dev Physiol 1979; 1:379–98.
56. Lafeber H, Jones CT, Rolph T. Some of the consequences of the intra-uterine growth retardation. In: Visser HKA, ed. Nutrition and growth of the fetus and infant. The Hague: Martinus Nijhoff, 1979:43–62.
57. Yu K. Umbilical and maternal amino acid concentrations in appropriate and small for gestational age infants. Zhonghua Yi Xue Za Zhi 1992; 72:453–55.
58. Cetin I, Marconi AM, Corbetta C, et al. Fetal amino acids in normal pregnancies and in pregnancies complicated by intrauterine growth retardation. Early Hum Dev 1992; 29:183–6.
59. Timmerman M, Chung M, Wilkening RB, et al. Relationship of fetal alanine uptake and placental alanine metabolism to maternal plasma alanine concentration. Am J Physiol 1998; 275:E942–50.
60. Owens JA, Owens PC, Robinson JS. Experimental fetal growth retardation: Metabolic and endocrine aspects. In: Gluckman PD, Johnston BM, Nathanielsz PW, eds. Advances in fetal physiology: Reviews in honour of G.C. Liggins. Ithaca: Perinatology Press, 1989:263–86.
61. Pollak A, Susa JB, Stonestreet BS, et al. Phosphoenolpuruvate carboxykinase in experimental intrauterine growth retardation in rats. Pediatr Res 1979; 13:175–7.
62. Rolph T, Jones CT. Delayed development of gluconeogenic capacity and the appearance of hypoglycaemia in the newborn guinea-pig after intra-uterine growth restriction. J Dev Physiol 1982; 4:1–21.
63. Mestyan J, Schultz K, Horvath M. Comparative glycaemic responses to alanine in normal term and small-for-gestational-age infants. J Pediatr 1974; 85:276–8.
64. Liechty EA, Kelley J, Lemons JA. Effect of fasting on uteroplacental amino acid metabolism in the pregnant sheep. Biol Neonate 1991; 60:207–14.
65. Smeaton TC, Owens JA, Kind KL, et al. The placenta releases branched-chain keto acids into the umbilical and uterine circulations in the pregnant sheep. J Dev Physiol 1989; 12:95–9.
66. Plauth M, Raible A, Vieillard-Baron D, et al. Is glutamine essential for the maintenance of intestinal function? A study in the isolated perfused rat small intestine. Int J Colorectal Dis 1999; 14:86–94.
67. Trahair JF, Harding R. Development of the gastrointestinal tract. In: Thorburn GD, Harding R, eds. Textbook of fetal physiology. Oxford: Oxford University, 1994:219–35.
68. Wu G. Urea synthesis in enterocytes of developing pigs. Biochem J 1995; 312:717–23.
69. Garcia MV, Martin-Barrientos J, Medina JM. Maternal-fetal relationship in ammonia metabolism during late gestation in the rat. Biol Neonate 1988; 53:315–20.
70. Wang HT, Miller JH, Iannoli P, et al. Intestinal adaptation and amino acid transport following massive enterectomy. Front Biosci 1997; 2:e116–22.
71. Zhang GX, Lai JH, Jia TW, et al. Effect of epidermal growth factor on glutamine metabolic enzymes in small intestine and skeletal muscle of parenterally fed rats. Nutrition 1997; 13:652–5.
72. Schini-Kerth VB. Dual effects of insulin-like growth factor-I on the constitutive and inducible nitric oxide (NO) synthase-dependent formation of NO in vascular cells. J Endocrinol Invest 1999; 22:82–8.
73. Charlton VE, Reis BL. Effects of gastric nutritional supplementation on fetal umbilical uptake of nutrients. Am J Physiol 1981; 241:E178–85.
© 2002 Lippincott Williams & Wilkins, Inc.