Placental transport of leucine, phenylalanine, glycine, and proline in intrauterine growth-restricted pregnancies.

L-[1-13C]Leucine, [1-13C]glycine, L-[1-13C]phenylalanine, and L-[1-13C]proline were infused as a bolus into the maternal circulation of seven appropriate for gestational age at 30.3 +/- 3.0 wk and 7 intrauterine growth-restricted pregnancies at 26.5 +/- 1.0 wk gestation to investigate placental transport in vivo. Umbilical venous samples were obtained at the time of in utero fetal blood sampling at 450 +/- 74 sec from the bolus injection. In normal pregnancies the fetal/maternal (F/M) enrichment ratios for leucine (0.76 +/- 0.06) and phenylalanine (0.77 +/- 0.06) were higher (P < 0.01) than the F/M ratios for glycine (0.18 +/- 0.04) and proline (0.22 +/- 0.02). This suggests that these two essential amino acids rapidly cross the placenta in vivo. Compared with the essentials, both glycine and proline had significantly lower F/M enrichment ratios, which were not different from each other. The results support the hypothesis that amino acids with high affinity for exchange transporters cross the placenta most rapidly. In intrauterine growth-restricted pregnancies, the F/M enrichment ratio was significantly lower (P < 0.01) for L-[1-13C]leucine (0.76 +/- 0.06 vs. 0.48 +/- 0.07) and for L-[1-13C]phenylalanine (0.77 +/- 0.06 vs. 0.46 +/- 0.07) compared with appropriate for gestational age pregnancies reflecting impaired transplacental flux. The F/M enrichment ratio did not differ for [1-13C]glycine (0.18 +/- 0.04 vs. 0.17 +/- 0.03), and L-[1-13C]proline (0.22 +/- 0.02 vs. 0.18 +/- 0.04).

A multiple infusion start time (MIST) protocol for stable isotope studies of fetal blood.

A new approach utilizing multiple infusion start times for two stable isotopes of leucine was applied to seven pregnancies in order to assess equilibration times for isotopic studies when a single fetal blood sample is available. Two infusates, one containing l -[1-(13)C]-leucine and the other l -[5,5,5-D3]-leucine, were given as a primed constant infusion in the maternal circulation at fetal blood sampling (FBS). In five patients l -[1-(13)C]-leucine infusion was started at time zero (T(0)) whereas l -[5,5,5-D3]-leucine infusion began 30 min later, and both were continued until the umbilical sample was obtained at 149.7+/-8.8 min. In order to assure non-steady state conditions, in two patients the first infusion started at T(0)and the second 17 and 6 min before FBS was performed at 115 and 154 min, respectively. The fetal/maternal ratio for l -[5,5,5-D3]-leucine over the fetal/maternal ratio for l -[1-(13)C]-leucine was 0.98+/-0.03, indicating steady state conditions for both infusions for the first six patients. In the last patient the ratio was 0.51, indicative of non-steady state conditions for the shortest infusion time. Our results show that a single fetal sample can provide data for fetal amino acid enrichments reflecting multiple time points. Leucine steady state is achieved 20 min after a primed continuous infusion both in the maternal and fetal circulations.

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

Under normal physiological conditions, essential amino acids (EA) are transported from mother to fetus at different rates. The mechanisms underlying these differences include the expression of several amino acid transport systems in the placenta and the regulation of EA concentrations in maternal and fetal plasma. To study the relation of EA transplacental flux to maternal plasma concentration, isotopes of EA were injected into the circulation of pregnant ewes. Measurements of concentration and molar enrichment in maternal and fetal plasma and of umbilical plasma flow were used to calculate the ratio of transplacental pulse flux to maternal concentration (clearance) for each EA. Five EA (Met, Phe, Leu, Ile, and Val) had relatively high and similar clearances and were followed, in order of decreasing clearance, by Trp, Thr, His, and Lys. The five high-clearance EA showed strong correlation (r(2) = 0.98) between the pulse flux and maternal concentration. The study suggests that five of the nine EA have similar affinity for a rate-limiting placental transport system that mediates rapid flux from mother to fetus, and that differences in transport rates within this group of EA are determined primarily by differences in maternal plasma concentration.

Estimation of doubly labeled water energy expenditure with confidence intervals.

Bivariate regression is used to estimate energy expenditure from doubly labeled water data. Two straight lines are fitted to the logarithms of the enrichments of oxygen-18 and deuterium simultaneously as a bivariate regression, so that the correlations between the oxygen and deuterium regression coefficients can be estimated. Maximum likelihood methods are used to extend bivariate regression to unbalanced situations caused by missing observations and to include replicate laboratory determination from the same urine samples, even if one of the replicates is missing. Use of maximum likelihood allows the determination of a confidence interval for the energy expenditure based on the log likelihood surface rather than use of the propagation of variance methods for nonlinear transformations. The model is extended to include the subject's deviations from the two lines as a bivariate continuous-time first-order autoregression to allow for serial correlation in the observations. The analysis of data from two subjects, one without apparent serial correlation and one with serial correlation, is presented.

Estrogens in intrahepatic cholestasis of pregnancy.

OBJECTIVE: To determine whether estrogen production and excretion are impaired in gravidas with intrahepatic cholestasis. METHODS: Plasma and urine samples were collected from 13 women from the United States and Chile at 35-38 weeks' gestation with mild (n = 9) or severe (n = 4) intrahepatic cholestasis of pregnancy. Urinary and plasma steroid levels from women with cholestasis were compared with levels from 27 normal pregnant women within the same gestational age range. Urinary concentrations of dehydroepiandrosterone (DHEA), estrone (E1), estradiol (E2), estriol (E3), estetrol, progesterone, and 16-hydroxy-pregnenolone were measured by gas chromatography mass spectrometry, and plasma concentrations of DHEA sulfate, progesterone, unconjugated E1, unconjugated E2, unconjugated E3, sulfated E3 derivatives, glucuronidated E3 derivatives, and total E3 were measured by radioimmunoassay. RESULTS: Compared with normal pregnant women, women with cholestasis had significantly lower plasma levels of estrogens and DHEA sulfate, the precursor to placental estrogen production synthesized by the fetal adrenal gland (Hotelling-Lawley trace = 0.81; F4,19 = 3.9; P = .02). The mean plasma DHEA sulfate, unconjugated E2, unconjugated E3, and total E3 concentrations were 0.271, 10.21, 9.80, and 99.53 ng/mL, respectively, in women with cholestasis compared with 0.802, 18.98, 16.28, and 145.07 ng/mL for controls. CONCLUSION: Fetal adrenal production of DHEA sulfate, and in response, downstream placental production of estrogens, was compromised by intrahepatic cholestasis of pregnancy.

Improved stable isotope dilution-gas chromatography-mass spectrometry method for serum or plasma free 3-hydroxy-fatty acids and its utility for the study of disorders of mitochondrial fatty acid beta-oxidation.

BACKGROUND: Disorders of fatty acid oxidation (FAO) are difficult to diagnose, primarily because in many of the FAO disorders measurable biochemical intermediates accumulate in body fluids only during acute illness. Increased concentrations of 3-hydroxy-fatty acids (3-OH-FAs) in the blood are indicative of FAO disorders of the long- and short-chain 3-hydroxy-acyl-CoA dehydrogenases, LCHAD and SCHAD. We describe a serum/plasma assay for the measurement of 3-OH-FAs with carbon chain lengths from C(6) to C(16). METHODS: We used stable isotope dilution gas chromatography-mass spectrometry (GC-MS) with electron impact ionization and selected ion monitoring. Natural and isotope-labeled compounds were synthesized for the assay. RESULTS: The assay was linear from 0.2 to 50 micromol/L for all six 3-OH-FAs. CVs were 5-15% at concentrations near the upper limits seen in healthy subjects. In 43 subjects, the medians (and ranges) in micromol/L were as follows: 3-OH-C(6), 0.8 (0.3-2.2); 3-OH-C(8), 0.4 (0.2-1.0); 3-OH-C(10), 0.3 (0.2-0.6); 3-OH-C(12), 0.3 (0.2-0.6); 3-OH-C(14), 0.2 (0.0-0.4); and 3-OH-C(16), 0.2 (0.0-0.5). 3-OH-FAs were increased in infants receiving formula containing medium chain triglycerides. Two patients diagnosed with LCHAD deficiency showed marked increases in 3-OH-C(14) and 3-OH-C(16) concentrations. Two patients diagnosed with SCHAD deficiency showed increased shorter chain 3-OH-FAs but no increases in 3-OH-C(14) to 3-OH-C(16). CONCLUSION: Measuring blood concentrations of the 3-OH-FAs with this assay may be a valuable tool for helping to rapidly identify deficiencies in LCHAD and SCHAD and may also provide useful information about the status of the FAO pathway.

Steady state maternal-fetal leucine enrichments in normal and intrauterine growth-restricted pregnancies.

The aim of this study was to compare the fetal/maternal (F/M) leucine-enrichment ratio in normal (AGA) and intrauterine growth-restricted (IUGR) pregnancies at the time of fetal blood sampling (FBS). A maternal primed-constant infusion of L-[1-13C]-leucine was given in six AGA and 14 IUGR pregnancies, divided into three groups according to the pulsatility index (PI) of the umbilical artery and to fetal heart rate (FHR): group 1 (normal FHR and PI, four cases); group 2 (normal FHR and abnormal PI, five cases); and group 3 (abnormal FHR and PI, five cases). Maternal arterialized samples were taken at time zero and every 20 min for 125+/-7 min. Umbilical venous samples were obtained after 114+/-42 min of infusion. Under steady state conditions, there was a significant linear relationship between maternal leucine disposal rate and maternal leucine concentration. The comparison of fetal to maternal leucine enrichment showed a progressive dilution of the fetal enrichment relative to the mother between AGA and IUGR of group 1 (0.89 versus 0.78, p < 0.02), group 2 (0.71, p < 0.001), and group 3 (0.62, p < 0.001), and also among the three IUGR groups. The F/M leucine molar percent enrichment (MPE) ratio showed a positive correlation with the umbilical venous oxygen content and an inverse correlation with fetal lactate concentration. We conclude that the dilution in the fetal/maternal leucine-enrichment ratio correlates with the severity of growth restriction and reflects decreased transplacental leucine flux and/or increased protein breakdown within the fetoplacental compartments.

Relationship of fetal alanine uptake and placental alanine metabolism to maternal plasma alanine concentration.

Uterine and umbilical uptakes of alanine (Ala) were measured in 10 ewes before (control) and during intravenous infusion of Ala, which increased maternal arterial Ala concentration from 115 +/- 14 to 629 +/- 78 microM (P < 0.001). In 8 of these ewes, placental Ala fluxes were traced by constant intravenous infusion of L-[3,3,3-2H3]Ala in the mother and L-[1-13C]Ala in the fetus. Rates are reported as micromoles per minute per kilogram fetus. Ala infusion increased uterine uptake (2.5 +/- 0.6 to 15.6 +/- 3.1, P < 0.001), umbilical uptake (3.1 +/- 0.5 to 6.9 +/- 0.8, P < 0.001), and net uteroplacental utilization (-0.7 +/- 0.8 to 8.6 +/- 2.7, P < 0.01) of Ala. Control Ala flux to fetus from mother (Rf,m) was much less than the Ala flux to fetus from placenta (Rf,p) (0.17 +/- 0.04 vs. 5. 0 +/- 0.6). Two additional studies utilizing L-[U-13C]Ala as the maternal tracer confirmed the small relative contribution of Rf,m to Rf,p. During maternal Ala infusion, Rf,m increased significantly (P < 0.02) but remained a small fraction of Rf,p (0.71 +/- 0.2 vs. 7.3 +/- 1.3). We conclude that maternal Ala entering the placenta is metabolized and exchanged for placental Ala, so that most of the Ala delivered to the fetus is produced within the placenta. An increase in maternal Ala concentration increases placental Ala utilization and the fetal uptake of both maternal and placental Ala.

Protein balance in the first week of life in ventilated neonates receiving parenteral nutrition.

BACKGROUND: Protein intake is frequently delayed in ill neonates because of concerns about their ability to metabolize substrates. OBJECTIVE: We aimed to determine the factors affecting protein balance in ventilated, parenterally fed newborns during the first week of life. DESIGN: Leucine kinetic studies were performed in 19 neonates by using the [1-(13)C]leucine tracer technique after 24 h of a stable total parenteral nutrition (TPN) regimen. TPN intakes were prescribed by rotating attending physicians, enabling assessment of protein metabolism over a range of clinically used nutrient intakes. RESULTS: Mean (+/-SD) birth weight was 1.497 +/- 0.779 kg, gestational age at birth was 30.3 +/- 4.0 wk, and age at study was 3.9 +/- 1.4 d. Amino acid intakes (AAIs) ranged from 0.0 to 2.9 g x kg(-1) x d(-1). Based on leucine kinetic data, protein balance was calculated as the difference between protein synthesis and catabolism. By multiple regression analysis, AAI was the only predictor associated independently with protein balance (P < 0.01); energy intake, lipid intake, glucose intake, birth weight, and gestational age were not. Both leucine oxidation and nonoxidative leucine disposal rates were significantly correlated with leucine intake (P < 0.0005 and P < 0.01, respectively). Of the 12 infants with AAIs > 1 g x kg(-1) x d(-1), only 1 infant was significantly catabolic (protein balance <-1 g x kg(-1) x d(-1)). There was no evidence of protein intolerance as determined by elevated creatinine (69 +/- 31 micromol/L), plasma urea nitrogen (6.7 +/- 2.53 mmol/L), or metabolic acidosis (pH: 7.36 +/- 0.05). CONCLUSIONS: Ill neonates can achieve a positive protein balance in the first days of life without laboratory evidence of protein toxicity.

Elevated intakes of zinc in infant formulas don not interfere with iron absorption in premature infants.

BACKGROUND: Zinc and iron may share common pathways for absorption and compete for uptake into mucosal cells. We determined whether elevated ratios of zinc to iron would interfere with erythrocyte incorporation of iron in premature infants both during and between feeds. METHODS: In the first experiment, five premature infants (<2500 g birth weight) were enrolled, once receiving full oral feeds by nasogastric tube. They received either high (1200 microg/kg, ratio 4:1) or low (300 microg/kg, ratio 1:1) doses of oral zinc sulfate, together with 300 microg/kg oral 58Fe as chloride in saline with 10 mg/kg vitamin C, between designated feeding periods. Each infant served as its own control and randomly received either high or low doses of zinc or iron and then the alternate dose after 2 weeks. In the second experiment, nine additional premature infants were assigned to the same zinc:iron intake protocol except zinc and iron were given with usual oral feeds (premature formula or human milk) equilibrated before feeding. Iron absorption was measured by the erythrocyte incorporation of 58Fe. RESULTS: High doses of zinc given between feeds significantly inhibited erythrocyte incorporation of iron. 58Fe incorporation (%) with the 1:1 ratio of zinc:iron intake was 7.5 (5.7, 10; geometric mean, -I SD, +1 SD). The percentage of 58Fe incorporation on the 4:1 ratio of zinc:iron intake was 3.6 (2.6, 5.1). Given with feeds, the percentage of 58Fe incorporation on low zinc:iron intake was 7.0 (2.6, 19). Finally, the percentage of 58Fe incorporation on high zinc:iron intake was 6.7 (2.5, 19). CONCLUSION: Elevated intakes of zinc do not interfere with erythrocyte incorporation of iron in premature formulas.

Lipophilic siderophores of Mycobacterium tuberculosis prevent cardiac reperfusion injury.

Reperfusion injury, which occurs upon the reintroduction of blood flow to an ischemic organ, is responsible for considerable damage in heart attacks and strokes. However, no treatment for reperfusion injury is currently available. A major cause of reperfusion injury is the iron-mediated generation of hydroxyl radical (.OH). In this study we have explored the capacity of novel iron chelators called "exochelins" to prevent reperfusion injury. Exochelins, siderophores of Mycobacterium tuberculosis, are unique iron chelators because they are lipid soluble, and hence able to enter cells rapidly. In the iron-free state, exochelins prevented .OH formation. Desferri-exochelins prevented oxidative injury to cultured cardiac myocytes, and did so more rapidly and effectively than the nonlipid soluble iron chelator deferoxamine. The capacity of various desferri-exochelins to protect myocytes from oxidative injury varied directly with their solubility in lipid. Infused into isolated rabbit hearts during reperfusion after a period of ischemia, desferri-exochelins dramatically improved systolic and diastolic left ventricular function, preserved coronary flow, reduced release of the cardiac enzyme lactic dehydrogenase, and reduced myocardial concentrations of .OH metabolites. Thus, highly diffusible desferri-exochelins block injury caused by .OH production and have potential for the treatment of reperfusion injury.

Placental transport of threonine and its utilization in the normal and growth-restricted fetus.

Placental transport and fetoplacental utilization of threonine (Thr) were compared at 130 +/- 1 days gestational age between seven control ewes (C) and six ewes in which intrauterine growth restriction (IUGR) had been induced by exposure to high ambient temperature from 33 +/- 1 to 112 +/- 2 days of gestation. The fluxes were measured using simultaneous intravenous infusions of L-[1-13C]Thr into the mother and L-[U-14C]Thr into the fetus. The IUGR group had less fetal weight (1.27 +/- 0.14 vs. 3.10 +/- 0.10 kg, P < 0.01) and placental weight (120 +/- 17 vs. 295 +/- 14 g, P < 0.01) than the C group. The direct flux of maternal Thr into the fetal systemic circulation was less in the IUGR fetuses, both relative to fetal weight (1.40 +/- 0.19 vs. 2.19 +/- 0.18 mumol.min-1.kg fetus-1, P = 0.0107) and placental weight (1.5 +/- 0.2 vs. 2.3 +/- 0.2 mumol.min-1.100 g placenta-1, P = 0.0187). In both groups, there was excretion of CO2 produced from fetal Thr. The rate of CO2 production from fetal plasma Thr carbon by fetus plus placenta was reduced in the IUGR group (1.50 +/- 0.23 vs. 2.86 +/- 0.32 mumol.min-1.kg fetus-1, P = 0.0065). We conclude that the flux of maternal Thr into the IUGR fetus is markedly reduced because of a reduction in placental mass and because of a weight-specific reduction in Thr placental transport. The reduced flux is routed into fetal Thr accretion via a decrease in fetal Thr oxidation.

Leucine metabolism in chronically hypoglycemic hypoinsulinemic growth-restricted fetal sheep.

We measured leucine flux rates during infusions of L-[1-14C]- and L-[1-1C]leucine in fetal sheep exposed to maternal insulin-induced hypoglycemia over the last 8 wk (40%) of gestation to determine effects of chronic glucose deficiency and hypoglycemia on fetal leucine metabolism. Compared with control fetuses (C, n = 5), hypoglycemic fetuses (HG, n = 8) weighed less (C, 3.43 +/- 0.07 kg; HG, 2.32 +/- 0.24 kg), had lower plasma glucose (C, 1.04 +/- 0.02 mM; HG, 0.59 +/- 0.01 mM), insulin (C, 48 +/- 6 pM; HG, 12 +/- 6 pM), and leucine concentrations (C, 195.6 +/- 8.3 microM; HG, 140.8 +/- 15.0 microM), lower rates of net leucine uptake (C, 4.2 +/- 0.6 mumol.min-1.kg-1; HG, 2.1 +/- 0.4 mumol.min-1.kg-1) and leucine flux into protein accretion (C, 2.8 +/- 0.2 mumol.min-1.kg-1; HG, 0.6 +/- 0.1 mumol.min-1.kg-1), and an increased rate of leucine release from protein breakdown (C, 1.1 +/- 0.1 mumol.min-1.kg-1; HG, 3.3 +/- 0.2 mumol.min-1.kg-1) (P < 0.05 for all). Plasma leucine disposal, flux into protein synthesis, and oxidation were not different between groups. We conclude that adaptations of fetal leucine metabolism to long-term hypoglycemia and decreased glucose apply represent diminished leucine uptake and increased leucine release from protein breakdown, which are associated with decreased incorporation of leucine into protein accretion and a slower rate of fetal growth.

Serine and glycine metabolism in hepatocytes from mid gestation fetal lambs.

Using stable isotopes of serine, glycine, and glutamine, the metabolism of serine and glycine was investigated in primary hepatocytes from six mid-gestation fetal lambs (mean gestational age = 81 +/- 6 d, normal gestation = 145 d). Serine production was 6.84 +/- 1.22 mumol/24 h/mg of DNA and exceeded serine utilization (3.76 +/- 1.44 mumol/24 h/mg of DNA) with a resultant net increase in medium serine of 2.58 +/- 1.70 mumol/24 h/mg of DNA. In contrast, glycine production (6.84 +/- 1.16 mumol/24 h/mg of DNA) was less than glycine utilization (12.10 +/- 1.78 mumol/24 h/mg of DNA) with a net decline in medium glycine of -5.44 +/- 2.03 mumol/24 h/mg of DNA. Of the serine produced, 50.4 +/- 4.3% was derived from glycine via the action of serine hydroxymethyltransferase (SHMT) and the glycine cleavage enzyme complex (GCS). Increasing the medium serine concentration resulted in an increase in serine utilization and sparing of the utilization of other amino acids. Biosynthesis of glycine from serine accounts for only 18.1 +/- 5.6% of glycine production, and this percentage is not affected by changes in medium serine concentration. Using 2.5-[15N2]glutamine as the tracer, an estimated 18 +/- 7% of serine production was derived from transamination reactions. The specific activity of both cytosolic and mitochondrial SHMT was constant for the duration of the cultures. We conclude that, in mid-gestation fetal ovine hepatocytes, there is net production of serine (with glycine as the primary metabolic source of this serine biosynthesis) and net glycine utilization. These data suggest that flux through SHMT and GCS accounts for 50% of serine biosynthesis in mid-gestation fetal ovine hepatocytes. The sparing of the utilization of other amino acids by serine suggests that serine a conditionally essential amino acid for the mid-gestation fetal liver.

Placental transport and fetal utilization of leucine in a model of fetal growth retardation.

Placental transport and fetal utilization of leucine were studied at 130 days of gestation in six control ewes and in seven ewes in which intrauterine growth retardation (IUGR) had been induced by exposure to heat stress. Leucine fluxes were measured during simultaneous intravenous infusion of L-[1-13C]leucine into the mother and L-[1-14C] leucine into the fetus. In the IUGR group, the following leucine fluxes, expressed as micromol/min/kg fetus, were reduced compared with control: net uterine uptake (3.44 vs. 8.56, P<0.01), uteroplacental utilization (0.0 vs. 4.7, P<0.01), fetal disposal rate (6.4 vs. 8.9, P<0.001), flux from placenta to fetus (5.0 vs. 7.1, P<0.01), direct transport from mother to fetus (1.6 vs. 3.4, P<0.01), flux from fetus to placenta (1.5 vs. 3.2, P<0.001), and oxidation of fetal leucine by fetus plus placenta (2.1 vs. 3.2, P<0.02). Uterine uptake, uteroplacental utilization, and direct transport were also significantly reduced per gram placenta. We conclude that maternal leucine flux into the IUGR placenta is markedly reduced. Most of the reduced flux is routed into fetal metabolism via a decrease in placental leucine utilization and a decrease in the leucine flux from fetus to placenta.

Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells.

Serine hydroxymethyltransferase (SHMT) is the primary enzyme in the interconversion of serine and glycine. The roles of mitochondrial and cytosolic SHMT in the interconversion of serine and glycine were determined in two Chinese hamster ovary (CHO) cell lines that both contain cytosolic SHMT but either have (CHOm+) or lacK (CHOm-) mitochondrial SHMT. Mitochondrial SHMT activity was significantly reduced in CHOm- (0.24 +/- 0.11 nmol/min per mg of mitochondrial protein) compared with CHOm+ (3.21 +/- 0.66 nmol/min per mg of mitochondrial protein; P = 0.02) cells, whereas cytosolic SHMT activity was similar in CHOm- and CHOm+ cells (1.09 +/- 0.31 and 1.53 +/- 0.12 nmol/min per mg of cytosolic protein respectively; P = 0.57). In CHOm+ and CHOm- cells, the relative flux of glycine to serine measured with either [1-13C]- or [2-13C]-glycine was similar (CHOm-: 538 +/- 82 nmol/24 per mg of DNA; CHOm+: 616 +/- 88 nmol/24 h per mg of DNA; P = 0.42). In contrast, the relative flux of serine to glycine measured with [1-13C]serine was low in CHOm- cells (80 +/- 28 nmol/24 h per mg of DNA) compared with CHOm+ cells (3080 +/- 320 nmol/24 h per mg of DNA; P = 0.0001). The rate of glycine production determined by [1-(13)C]glycine dilution was lower in CHOm- (1200 +/- 200 nmol/24 h per mg of DNA) than CHOm+ (10200 +/- 1800 nmol/24 h per mg of DNA; P = 0.03) cells, whereas glycine utilization was similar in the two cell lines. Serine production was similar in the two cell lines but serine utilization was lower in CHOm- (3800 +/- 1200 mu mol/24 h per mg of DNA) than CHOm+ (6600 +/- 1000 nmol/24 h per mg of DNA; P = 0.0002) cells. Increasing the serine concentration in the medium resulted in an increase in glycine production in CHOm+ but not in CHOm- cells. Intracellular studies with [1-13C]serine confirm the findings of decreased glycine production from serine. In CHO cells there is partitioning of intracellular serine and glycine metabolism. Our data support the hypothesis that mitochondrial SHMT is the primary pathway for serine into glycine interconversion.

Pathways of serine and glycine metabolism in primary culture of ovine fetal hepatocytes.

Previous in vivo studies in the ovine fetus have demonstrated net serine production by the fetal liver, a pattern not seen in the adult sheep. The goal of this study was to determine the major metabolic pathways responsible for fetal hepatic serine production by using stable isotope methodology in primary culture of late gestation ovine fetal hepatocytes. Specifically selected tracers of glycine were added to individual cultures, with production of labeled serine determined after 24 h of incubation. When [1-13C1]glycine or [2-13C1]glycine was used as the initial tracer, serine enrichment at 24 h indicated that approximately 30% of serine production comes from glycine. Quantitative comparison of serine enrichment from these two tracers suggests that serine synthesis from glycine occurs via the combined action of the glycine cleavage enzyme system (GCE) and serine hydroxymethyltransferase (SHMT). Using [1,2-13C2(15)N1]glycine as the tracer, there was no significant increase in M + 2 glycine in the medium over 24 h, suggesting no reversible transamination of glycine, and therefore no significant movement of glycine through the glyoxalate pathway. These data demonstrate that in primary culture of fetal ovine hepatocytes, approximately 30% of serine biosynthesis is derived from glycine, primarily via the combined action of GCE and SHMT.

Plasma disposal rate and hepatic alanine metabolism in pregnant and nonpregnant rabbits.

To better define the glucose-alanine relationship and hepatic alanine metabolism during pregnancy, glucose and alanine turnover rates (TR) and simultaneous hepatic alanine uptake were determined in nine term pregnant (P) and 12 nonpregnant (NP) rabbits who had undergone chronic catheterization of both the systemic and hepatic circulations. [U-14C3]Alanine and [3-3H1]glucose were used as tracers. Whole animal [U-14C3]alanine TR was significantly greater in P than in NP animals (42.7 +/- 1.7 versus 31.9 +/- 1.8 mumol.min-1, respectively, p < 0.001). However, there was no difference when the alanine TR was compared on a weight-specific basis (11.3 +/- 0.6 versus 9.6 +/- 0.6 mumol.min-1.kg-1). The glucose TR was higher in P compared with NP rabbits on both an absolute (122.3 +/- 6.2 versus 79.8 +/- 5.8 mumol.min-1, p < 0.001) and a weight-specific basis (32.9 +/- 1.4 versus 24.0 +/- 1.9 mumol.min-1.kg-1, p < 0.005). Although the fraction of plasma glucose derived from plasma alanine in the whole animal was similar in P and NP animals (16.7% +/- 1.4 versus 13.9 +/- 0.7%, respectively), the absolute amount of plasma glucose derived from plasma alanine was greater in P rabbits (20.8 +/- 1.8 versus 11.2 +/- 1.2 mumol.min-1, p < 0.0005). In addition, the percentage of plasma alanine being converted to plasma glucose was greater in P than in NP animals (48.1 +/- 2.6 versus 34.6 +/- 2.0%, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo placental transport of glycine and leucine in human pregnancies.

L-[1-13C]Glycine and L-[1-13C]leucine were infused as a bolus into 12 pregnant patients carrying normal fetuses before fetal blood sampling at gestational ages ranging from 20 to 37 wk. Maternal venous samples were obtained every 2-3 min for 15 min after the bolus infusion. Fetal samples were obtained from the umbilical vein within 15 min of the bolus. Amino acid plasma enrichments (molar percent enrichment) were determined by gas chromatography-mass spectroscopy and their concentrations by ion exchange chromatography. The ratios of glycine and leucine transfer were assessed from fetal/maternal enrichment ratios for each amino acid. We now report that over the gestational age range of 20-37 wk, under relatively undisturbed fetomaternal conditions (fetal blood sampling), human placental glycine transfer is limited, with a glycine/leucine ratio = 0.16 +/- 0.02. We hypothesize that, in human pregnancies, the relative rates of in vivo transplancental transport of amino acids can be assessed indirectly utilizing fetal blood sampling and stable isotope methodology. The application of this approach to leucine and glycine demonstrates that the transfer of leucine is rapid (demonstrable in seconds), whereas that of glycine is more limited.