Comparison of leucine and dispensable amino acid kinetics between ...

1 downloads 0 Views 119KB Size Report
Jun 23, 2010 - Anura V Kurpad, Pratibha Dwarkanath, Tinku Thomas, Arun Mhaskar, Annamma Thomas, Rita Mhaskar, and Farook Jahoor. ABSTRACT.
Comparison of leucine and dispensable amino acid kinetics between Indian women with low or normal body mass indexes during pregnancy1–3 Anura V Kurpad, Pratibha Dwarkanath, Tinku Thomas, Arun Mhaskar, Annamma Thomas, Rita Mhaskar, and Farook Jahoor ABSTRACT Background: Evidence suggests that in women with a normal to high body mass index (BMI; in kg/m2), the extra amino acids needed during pregnancy are met through reduced oxidation. It is not known whether a woman with a low BMI can make this adaptation successfully. Objective: The objective was to measure and compare leucine kinetic parameters and alanine-nitrogen, glutamine amide-nitrogen, and glycine and cysteine fluxes in Indian women with a low and normal BMI in early and midpregnancy. Design: Fasted- and fed-state kinetics were measured by infusing 1-[13C]leucine, [2H2]cysteine, [2H2]glycine, [5-15N]glutamine, and [15N]alanine in groups of 10 women with a low BMI (,18.5) and 10 women with a normal BMI (18.5–25) in the first and second trimesters of pregnancy. Results: Leucine, glutamine, glycine, and cysteine fluxes were faster in women with a low BMI in both trimesters, but there was no difference in alanine flux between groups. This difference was explained in the first trimester by a higher proportion of fat-free mass in low-BMI women. Leucine oxidation and percentage of dietary leucine oxidized were higher in low-BMI women in both trimesters, but nonoxidative disposal was not different between groups. Conclusions: Although they use dietary protein less efficiently, low-BMI women maintain net protein synthesis at the same rate as do normal-BMI women and produce similar quantities of labile nitrogen for the de novo synthesis of other dispensable amino acids such as glycine and cysteine. The extra amino acids required for increased maternal protein synthesis during pregnancy are provided by an overall decrease in amino acid catabolism in women with normal or low BMI. Am J Clin Nutr 2010;92:320–9. INTRODUCTION

The prevalence of low birth weight (LBW) is high in Indian infants (1) and is a significant contributor to neonatal morbidity and mortality (2). Most LBW infants in India and in most developing countries are a result of intrauterine growth retardation and a compromised maternal nutritional status (3, 4). Evidence that short teenage mothers with lower body weights and body mass indexes (BMIs; in kg/m2) have lighter placentas and smaller infants (5) suggests that underweight mothers are not capable of providing adequate nutrients, especially energy and protein, for growth of the reproductive tissues and the fetus. However, the metabolic mechanisms linking maternal weight and fetal growth are not fully understood.

320

Some consistent findings are that protein synthesis is higher in healthy pregnant women in the second and third trimesters than in the first trimester (6) and that urea production and excretion are higher in healthy pregnant women than in nonpregnant women (7–9). These findings, together with an almost identical pattern of change in leucine transamination (7), suggest that the extra amino acids required for increased maternal protein synthesis are provided by an overall decrease in amino acid catabolism. However, with potentially low protein stores, as can be assumed from earlier studies in Indians with a low BMI who had lower ratios of muscle mass to viscera in their fat-free mass (10), it is not known whether pregnant women with a low BMI can make these adaptations successfully. Furthermore, for the same reasons, it is also possible that the availability of the dispensable amino acids will be compromised in pregnant woman with a low BMI if there is insufficient labile amino nitrogen to support their de novo synthesis. The dispensable amino acids have an important role in intermediary metabolism: for example, besides being a neurotransmitter and a one-carbon donor, glycine is a precursor for the formation of purines, porphyrins, creatine, glutathione, and, through its interconversion to serine, phospholipids, and cysteine (11). Cysteine is the rate-limiting precursor for synthesis of glutathione (12), the primary intracellular antioxidant. Alanine and glutamine, as primary carriers of nitrogen in the body, play pivotal roles linking amino acid, glucose, and protein metabolism, and their fluxes would reflect the availability of labile nitrogen for de novo amino acid synthesis. Because pregnant women with a low BMI may have low protein stores, the present study aimed first to determine whether protein (represented by leucine) turnover and catabolism rates 1

From St John’s Research Institute (AVK, PD, and TT) and the Department of Obstetrics and Gynecology (AM, AT, and RM), St John’s National Academy of Health Sciences, Bangalore, India, and the US Department of Agriculture, Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX (FJ). 2 Supported by the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement no. 58-6250-6001. 3 Address correspondence to AV Kurpad, Division of Nutrition, St John’s Research Institute, St John’s National Academy of Health Sciences, Bangalore 560034, India. E-mail: [email protected]. Received January 13, 2010. Accepted for publication May 25, 2010. First published online June 23, 2010; doi: 10.3945/ajcn.2010.29205.

Am J Clin Nutr 2010;92:320–9. Printed in USA. Ó 2010 American Society for Nutrition

LOW-BMI PREGNANCY AND AMINO ACID KINETICS

were lower in these women than in women with a normal BMI during pregnancy and, second, to test the hypothesis that in the fasted state, pregnant women with low BMI would have slower fluxes of glutamine amide-nitrogen and alanine-nitrogen, which indicates the decreased availability of labile nitrogen for the de novo synthesis of other dispensable amino acids such as glycine and cysteine. Therefore, leucine kinetics and the fluxes of cysteine, glycine, the amide-nitrogen moiety of glutamine, and the amino-nitrogen moiety of alanine were measured in the first 2 trimesters of pregnancy in pregnant women with a low BMI (18.5) or a normal BMI (.18.5 and 25.0) in the fasted and fed states. SUBJECTS AND METHODS

The study was conducted from December 2006 to July 2007 at the obstetrics ward of St John’s Medical College Hospital, India, which caters to patients of diverse socioeconomic status. The experimental protocols were approved by the Institutional Ethical Review Board of St John’s Medical College Hospital and by the Institutional Review Board for Human Subject Research of Baylor College of Medicine & Affiliated Hospitals. A written consent was obtained from each study subject at enrollment. Subjects Pregnant women at ,13 wk of gestation were eligible for the study. Women with multiple pregnancies, those with a clinical diagnosis of chronic illness (eg, diabetes mellitus, hypertension, heart disease, thyroid disease, and epilepsy), and those who tested positive for hepatitis B surface antigen, HIV, or syphilis were excluded. Twenty subjects, 10 with a low BMI (BMI  18.5) and 10 with a normal BMI (BMI . 18.5 and  25.0) were enrolled for the study after screening. Two subjects were excluded during the course of study; one because of an abortion at 14 wk and the other because of signs of epilepsy at 20 wk of gestation. At recruitment, routine antenatal tests were carried out. Folic acid, iron, and calcium supplements and tetanus toxoid were given as routine antenatal care. Anthropometric measures and habitual food intakes At recruitment, the age and obstetric history were recorded for each subject. Gestational age was assessed from the last menstrual period and confirmed by ultrasound within 2 wk of the initial visit. Maternal weight and height were recorded to the nearest 0.1 kg and 0.1 cm just before each tracer experiment. Gestational weight gain (GWG) was calculated per week between measurements. Mid-upper arm circumference was measured to the nearest 0.1 cm, and skinfold thicknesses were measured at 3 sites (biceps, triceps, and subscapular) to the nearest 0.2 mm by using skinfold calipers (Holtain, Crymych, United Kingdom). The skinfold-thickness measurement was used for the prediction of body density by using prediction equations (13), from which the percentage body fat was estimated in the first trimester (14). At birth, the infants were measured to the nearest 0.01 kg on a standard beam scale balance. LBW was defined as birth weight ,2.5 kg, and intrauterine growth retardation was defined as a birth weight below the 10th percentile for the gestational age (15).

321

A food-frequency questionnaire was administered at each trimester visit to obtain information on the habitual dietary intake for the preceding 3 mo. The food-frequency questionnaire was adapted from that developed for the urban middle class residing in South India (16) and was validated against 24-h food recalls that were obtained thrice during each trimester of pregnancy in 100 subjects. For energy intakes, the correlation between the methods was significant at all trimesters (r = 0.4, 0.5, and 0.5 in first, second, and third trimesters, respectively; P , 0.001). Tracer-infusion protocol The tracer-infusion protocol consisted of 2 parts: a fasted state of 4 h and a fed state of 5 h. In the fed state, the subjects were given small hourly meals, which provided 1/12th of their habitual daily protein and energy, based on 1.35 times their basal metabolic rate. The meals were made with a powdered dietary supplement (Ensure; Abbott Health Care Private Ltd, Mumbai, India), skimmed milk powder, beet sugar, bread, and butter. The meals consisted of 65% carbohydrate, 15% protein, and 20% fat and provided ’65 lmol leucine  kg21  h21. The actual dietary intake of each amino acid was calculated by using food-composition tables and the manufacturer’s published composition. For the low-BMI group, in the first and second studies, the meals provided a mean (6SE) of 11.1 6 0.6 and 12.6 6 0.7 lmol cysteine  kg21  h21, 43.3 6 1.0 and 46.9 6 1.1 lmol glycine  kg21  h21, 38.3 6 1.1 and 41.6 6 1.2 lmol alanine  kg21  h21, and 99.5 6 11 and 113 6 13 lmol glutamine  kg21  h21 in the first and second trimester studies, respectively. For the normal-BMI group, the mini meals provided a mean (6SE) of 11.6 6 0.3 and 12.7 6 0.4 lmol cysteine  kg21  h21, 45.8 6 0.5 and 51.8 6 0.8 lmol glycine  kg21  h21, 41.8 6 0.8 and 47.2 6 0.9 lmol alanine  kg21  h21, and 114 6 6.4 and 125 6 7.4 lmol glutamine  kg21  h21 in the first and second trimester studies, respectively. All subjects were studied on 2 occasions, at the end of the first (12 6 1 wk of gestation) and second (24 6 1 wk of gestation) trimesters. The women were not willing to come in for an assessment in the third trimester; therefore, this time point was not studied. Subjects were admitted to the obstetrics ward in the evening and finished dinner based on their habitual diet before 1900. About 3 h later, an intravenous catheter (Jelco, 22G; Medex Medical Ltd, Lancashire, United Kingdom) was inserted into the antecubital vein of one arm for the infusion of isotopes, whereas another catheter was inserted into the dorsal vein of the contralateral hand for drawing blood samples and kept patent with a slow saline drip. A warm blanket around the hand, maintained at 60–65°C, was used to arterialize the venous blood collected; the extent of arterialization was assessed by measuring hemoglobin saturation in a test sample; this was usually .95%. Sterile solutions of NaH13CO3, [1-13C]leucine, [2H2] cysteine, [2H2]glycine, [5-15N]glutamine, and [15N]alanine (Cambridge Isotope Laboratories, Woburn, MA) were prepared in sterile isotonic saline. After baseline breath and blood samples were collected, a primed (4 lmol/kg) continuous infusion of [1-13C]leucine was started and maintained at a rate of 4 lmol  kg21  h21. Primed, continuous infusions of [15N]alanine and [15N]glutamine (prime = 6 lmol/kg, infusion = 6 lmol  kg21  h21), [2H2]cysteine (prime = 1 lmol/kg, infusion = 1 lmol  kg21  h21), and [2H2]glycine (prime = 4 lmol/kg, infusion = 4

322

KURPAD ET AL

lmol  kg21  h21) were also started. The bicarbonate pool was also primed with 0.5 lmol NaH13CO3/kg at the start. After a 4-h isotope infusion (the fasted state), the subjects were given small hourly meals for the remaining 5 h (fed state), and the enrichments of different tracers at the end of each metabolic period are presented in Figure 1. This pattern of early feeding was necessary because all of the women did not want to spend the additional next day in the obstetrics ward and wished to return home in the morning, because they had families to care for. In addition, although there are distinct circadian patterns of hormone secretion, human studies have shown no circadian effect on energy expenditure and protein oxidation (17) and that the overriding regulatory influence on protein utilization is the availability of amino acids, rather than insulin (18). Blood and breath samples were collected at 10-min intervals during the last 0.5 h of the fasted and fed states. Total carbon dioxide excretion was determined with an open-circuit indirect calorimeter (MetaMax, Cortex, Germany) during the last hour of the fasted and fed states. At the end of the infusion, the subjects were given breakfast and discharged.

tific, Crewe, United Kingdom), with monitoring ions at m/z ratios of 44 and 45. Plasma amino acid concentrations were measured by reversed-phase HPLC on a Hewlett-Packard 1090 HPLC equipped with a model HP 1046A fluorescence detector (Hewlett-Packard, Avondale, PA). Plasma a-keto-isocaproic acid (aKICA) isotopic enrichment was measured by negative chemical ionization gas chromatography–mass spectrometric analysis of its pentafluorobenzyl derivative by selectively monitoring ions at m/z 129–130. Plasma amino acids were isolated by ionexchange (Dowex 200·) chromatography, and their isotopic enrichments were determined by negative chemical ionization gas chromatography–mass spectrometric analysis of their heptafluorobutyramide derivative. Dithiothreitol, 0.075 mL of a 10-mmol/L solution, was added to the derivatization mixture to convert cystine to cysteine. Ions were selectively monitored at m/z ratios of 535–537 for cysteine, 293–295 for glycine, 307– 308 for alanine, and 346–347 for glutamine. In all these assays, a Hewlett-Packard 5890 quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA) was used.

Calculations Laboratory analyses Blood samples were drawn into 5-mL syringes and transferred into EDTA-coated anticoagulant tubes and centrifuged at 4°C for 15 min at 1200 · g. Plasma was removed and stored at 280°C. The breath samples were analyzed for 13C abundance in carbon dioxide by gas-isotope-ratio mass spectrometry (Europa Scien-

Total amino acid flux (Q) was calculated as follows:       Q lmol  kg 2 1  h 2 1 ¼ I 3 Tr Trinf Tr Trpl

ð1Þ

where Tr/Trinf is the tracer/tracee ratio of the infusate, I is the tracer infusion rate (lmol  kg21  h21), and Tr/Trpl is the tracer/

FIGURE 1. Mean (6SD) net tracer-to-tracee molar ratios above baseline [molar percentage excess (MPE)] for plasma a-keto-isocaproic acid (A; solid and open squares for normal and low BMI, respectively), alanine (A; solid and open circles for normal and low BMI, respectively), glycine (B; solid and open circles for normal and low BMI, respectively), cysteine (B; solid and open squares for normal and low BMI, respectively), and glutamine (C; solid and open circles for normal and low BMI, respectively) in fasted (2.5–3 h) and fed (7.5–8 h) states in pregnant women with a normal or low BMI at the end of the first (A1, B1, and C1) and second (A2, B2, and C2) trimesters.

323

LOW-BMI PREGNANCY AND AMINO ACID KINETICS TABLE 1 Characteristics of pregnant women with a normal and low BMI at recruitment Normal BMI (n = 9)

Low BMI (n = 9)

Age (y) 23.7 6 3.61 22.9 6 3.0 Gestational age at recruitment (wk) 11.2 6 2.1 10.5 6 1.8 Gestational age in the second trimester (wk) 23.7 6 0.9 23.5 6 0.7 Primiparous (n) 4 7 Weight (kg) 50.9 6 6.3 38.8 6 4.22 Height (cm) 154.2 6 9.6 153.0 6 4.0 BMI (kg/m) 21.4 6 1.3 16.5 6 1.22 Sum of 3 skinfold thicknesses (mm) 39.6 6 10.4 22.7 6 4.62 Body fat (%) 27.0 6 4.0 18.7 6 3.12 Fat-free mass (kg) 37.1 6 4.5 31.3 6 2.82 Gestational weight gain (kg/wk) 0.33 6 0.13 0.43 6 0.09 Hemoglobin (g/dL) 12.3 6 1.1 12.0 6 1.8 Physical activity level 1.5 6 0.1 1.4 6 0.2 Birth weight (g) 2534 6 575 2653 6 365 1 2

of breath carbon dioxide, and EpaKICA is the plasma isotopic enrichment of aKICA. Nonoxidative leucine disposal (NOLD), that is, leucine used for protein synthesis, was calculated as Q minus leucine oxidation (Leuoxd). Leucine balance (Leubal), an index of net protein synthesis (fed state) or loss (fasted state), was calculated as the difference between leucine intake and leucine oxidation. For example, in the fed state Leubal ðlmol  kg 2 1  h 2 1 Þ ¼ ðdietleucine þ intravenous½13 CleucineÞ 2 Leuoxd

ð4Þ

The percentage of dietary leucine intake oxidized was calculated as follows: Leuoxd =ðdietleucine þ intravenous½13 CleucineÞ 2 100

ð5Þ

Mean 6 SD (all such values). P , 0.001 (independent t test).

Statistical methods tracee ratio of aKICA, cysteine, glycine, alanine, or glutamine in plasma. Endogenous amino acid flux was only calculated in the fasted state (Qendofast), ie, amino acids derived from protein breakdown and de novo synthesis (in the case of dispensable amino acids) were calculated as the difference between total amino acid flux and intravenous tracer amino acid intake. For example, endogenous fasted leucine flux is calculated as follows:  Qendofast lmol  kg 2 1  h 2 1 ¼ Q 2 fintravenous½13 C 2 leucineðlmol  kg 2 1  h 2 1 Þg ð2Þ In the case of leucine, the following terms were additionally calculated. Leucine oxidation (Leuoxd), an index of protein catabolism, was calculated as follows: Leuoxd ðlmol  kg 2 1  h 2 1 Þ ¼ ðV CO2 =0:78Þ 3 ECO2 =EpaKICA

ð3Þ

where VCO2 is the rate of carbon dioxide excretion in the breath, 0.78 adjusts for the fraction of carbon dioxide produced that is recovered in expired air, ECO2 is the plateau isotopic enrichment

The data are presented as arithmetic means 6 SD, unless otherwise specified. The anthropometric and dietary intake variables were analyzed between groups by using an independent Students t test and between trimesters by using a paired Student’s t test. The metabolic variables were analyzed by using mixed-models analysis of variance. This model, for amino acid flux, or leucine oxidation, NOLD, and balance, included the group (low and normal BMIs), metabolic phase (fasted and fed state), trimester (first and second), and all interactions between the 3 factors with metabolic phase and trimester considered as repeated factors. Post hoc comparisons were performed for significant interactions by using Tukey’s test. The kinetics data were normalized for weight because the 2 groups were significantly different in weight and because the weight-specific data could be compared with earlier data from the literature. Additionally, whole-body data (using weight as a covariate) as well as FFM-specific values (except for trimesterbased effects, because FFM was not estimated in the second trimester) were analyzed by using the same model, because body weights were significantly different between the groups. For percentage dietary leucine oxidized (which was only measured in the fed state), the same mixed model was used without metabolic phase. Bivariate correlations between birth weight or

TABLE 2 Maternal habitual dietary intakes in women with a low or normal BMI in the first and second trimesters1 Normal BMI (n = 9) Intake 21

21

Energy (kJ  kg  d ) Protein (g  kg21  d21) Carbohydrate (g  kg21  d21) Fat (g  kg21  d21) Leucine (mg  kg21  d21)

Low BMI (n = 9)

First

Second

First

6 6 6 6 6

6 6 6 6 6

6 6 6 6 6

143 0.9 5.7 0.8 72

28 0.2 1.3 0.2 15

151 1.1 5.5 1.0 83

47 0.4 1.7 0.4 27

207 1.5 7.6 1.5 116

Second 2

66 0.53 2.32 0.63 383

221 1.5 8.3 1.5 119

6 6 6 6 6

592 0.42 2.52 0.42 302

All values are means 6 SDs. Data were analyzed by using an independent t test for comparisons of the low- and normal-BMI groups in the same trimester. There were no significant differences between the first and second trimesters within groups. 2,3 Significantly different from the same trimester in the normal-BMI group (independent t test): 2P , 0.05, 3P , 0.01. 1

324

KURPAD ET AL

TABLE 3 Plasma amino acid concentrations in pregnant women with a low or normal BMI at the end of the first and second trimesters1 Normal BMI (n = 9) Plasma amino acid concentrations

Low BMI (n = 9)

First

Second

First

Second

73 6 20 98 6 21

70 6 19 95 6 24

73 6 23 107 6 34

78 6 40 110 6 44

231 6 98 225 6 86

232 6 61 200 6 79

238 6 78 245 6 68

242 6 53 252 6 45

372 6 157 496 6 109

401 6 66 450 6 85

449 6 165 513 6 142

462 6 166 579 6 183

459 6 87 571 6 141

523 6 146 538 6 174

497 6 147 524 6 139

449 6 134 530 6 136

2

Leucine (lmol/L) Fasted Fed Glycine (lmol/L) Fasted Fed Alanine (lmol/L)2 Fasted Fed Glutamine (lmol/L) Fasted Fed 1 2

All values are means 6 SDs. Plasma cysteine concentrations were not measured. Data were analyzed by using a mixed-model ANOVA. Significant main effect of metabolic phase (fasted compared with fed, P , 0.01), but no main effect of trimester or BMI and interaction effects.

GWG and leucine kinetic variables were examined for the entire data set by using Pearson’s correlation coefficient. Two-sided P values of 0.05 indicated significance for tests of interactions and main effects. The data were analyzed by using the PROC MIXED module in SAS version 9.1.3 (SAS Institute Inc, Cary, NC). RESULTS

Anthropometric measures and dietary intakes The mean anthropometric indexes of the subjects are summarized in Table 1. There were no differences between the groups in their habitual physical activity level, weekly GWG, or birth weight of the infants. The habitual dietary intakes of the subjects in both trimesters are summarized in Table 2. In general, the body weight–normalized macronutrient intakes of the low-BMI group were significantly higher than those of the normal-BMI group. However, there were no significant changes in intakes from the first to the second trimester for either group. The protein:energy ratio of the diet was between 11% and 12% for both groups in both trimesters. The low-BMI group had a higher leucine intake because of its generally higher cereal and protein intakes. Leucine kinetics Plasma leucine concentrations did not change from the first to the second trimesters, were not significantly different between groups, but were significantly lower in the fasted state than in the fed state (P = 0.0004; Table 3). Leucine flux was lower in the normal-BMI group (P = 0.004) and, with regard to metabolic state, was higher in the fed state (P , 0.0001). In addition, there was a significant interaction effect between trimester and BMI group (P = 0.023), such that in the low-BMI group in the first trimester, leucine flux (in both fasted and fed state) was significantly higher (P = 0.009) than in the normal-BMI group. The percentage changes in leucine flux between trimesters in the fasted and fed states were 11% and 7% in the normal-BMI group and 0% and 23% in the low-BMI group, respectively. Endogenous fasted leucine flux followed similar trends. The percentage change in fasted endogenous leucine flux between trimesters was 12% in the normal-BMI group, whereas it was 1% in the low-BMI group (Table 4). Across trimesters and BMI groups,

leucine oxidation was higher in the fed state (P , 0.0001). There was a significant interaction effect between metabolic phase and BMI group (P = 0.0196), such that leucine oxidation was significantly lower in the normal-BMI group (P = 0.04) than in the lowBMI group in the fed state. The percentage leucine intake that was oxidized was lower in the second trimester by ’34% and 32% than in the first trimester in the normal- and low-BMI groups, respectively; therefore, the efficiency of protein utilization increased significantly in the second trimester. With respect to NOLD, there was a significant increase in the second trimester (P = 0.029), and, although the group interaction was not significant, the increase in NOLD from the first to the second trimester tended to be greater in the normal-BMI group. Leucine balance increased significantly across trimesters in both groups (P = 0.003) and was higher in the fed state (P , 0.0001). When whole-body data (using weight as a covariate) were analyzed, the BMI group–based differences in leucine kinetic parameters disappeared; however, all the trimesterbased (first compared with second trimesters) and metabolic state (fasted compared with fed) differences persisted. Similar findings occurred when FFM-specific data were analyzed in the first trimester (Table 4). Statistically significant correlations between leucine kinetic parameters, such as oxidation, NOLD, and flux in the first trimester and birth weight and maternal weekly GWG are shown in Table 5.

Alanine-nitrogen and glutamine amide-nitrogen kinetics Alanine and glutamine flux were significantly higher in the fed state than in the fasted state (P , 0.0001 and P = 0.005, respectively; Table 6), whereas glutamine flux was higher in the low-BMI group (P = 0.0005), specifically, in the first trimester (P = 0.003). In addition, in the low-BMI group, glutamine flux in the first trimester was significantly higher than in the second trimester (in both the fasted and fed states; P = 0.013). Similar differences existed for both fasted alanine and glutamine endogenous flux. When whole-body flux rates were examined with weight as a covariate, glutamine flux remained higher (P = 0.002) in the second trimester. FFM-specific glutamine flux was higher in the low-BMI group (P = 0.04) in the first trimester (Table 6). The difference in metabolic state also remained

325

LOW-BMI PREGNANCY AND AMINO ACID KINETICS TABLE 4 Maternal leucine kinetics in the low- and normal-BMI groups at the end of the first and second trimesters1 Normal BMI (n = 9) Leucine kinetics Total flux Fasted (lmol  kg21  h21)2 Fed (lmol  kg21  h21)2 Fasted (lmol  kg FFM21  h21)3 Fed (lmol  kg FFM21  h21)3 Endogenous flux Fasted (lmol  kg21  h21)4 Fasted (lmol  kg FFM21  h21) Oxidation Fasted (lmol  kg21  h21)5 Fed (lmol  kg21  h21)5 Fasted (lmol  kg FFM21  h21)3 Fed (lmol  kg FFM21  h21)3 Total flux oxidized (%) Fasted6 Fed6 Diet intake oxidized (%)7 NOLD Fasted (lmol  kg21  h21)8 Fed (lmol  kg21  h21)8 Fasted (lmol  kg FFM21  h21) Fed (lmol  kg FFM21  h21) Balance Fasted (lmol  kg21  h21)9 Fed (lmol  kg21  h21)9

First 102.5 124.2 140.6 170.8

6 6 6 6

Second 11.0 15.4 18.0 27.9

98.5 6 11.0 135.2 6 17.9

First 6 6 6 6

Second

112.9 6 7.6 131.9 6 13.2 — —

117.2 147.5 144.5 182.3

14.0 11.0 14.0 15.6

116.7 6 7.5 143.3 6 8.1 — —

108.9 6 7.6 —

113.2 6 14.0 139.5 6 14.0

112.7 6 7.5 —

10.1 11.6 13.3 15.9

11.0 6 3.4 29.1 6 10.2 — —

16.7 46.3 20.5 59.0

15.4 8.5 17.4 8.7

12.5 6 3.4 39.5 6 11.6 — —

14.2 6 9.0 28.9 6 9.2 59.0 6 19.1

9.8 6 3.3 22.0 6 7.1 43.6 6 14.0

13.9 6 12.0 31.6 6 6.8 70.5 6 14.4

11.0 6 3.0 27.5 6 7.8 53.0 6 14.0

6 6 6 6

101.9 6 8.8 102.8 6 13.1 — —

6 6 6 6

104.2 6 7.6 103.8 6 11.3 — —

14.6 35.8 20.0 49.1

87.9 88.4 120.6 121.7

6 6 6 6

Low BMI (n = 9)

12.9 16.9 20.9 28.2

210.6 6 10.1 25.1 6 12.1

27.0 6 3.4 37.4 6 9.4

100.5 101.1 123.9 123.3

6 6 6 6

16.7 14.5 20.9 18.7

212.7 6 15.4 19.7 6 10.0

28.5 6 3.4 34.6 6 9.7

1 All values are means 6 SDs. Fat-free mass (FFM)–specific leucine kinetics are presented only for the first trimester, because this measurement was only available for the first trimester. NOLD, nonoxidative leucine disposal. Data were analyzed by using mixed-model ANOVA. 2 Significant main effect of metabolic phase (fasted compared with fed, P , 0.0001) and BMI (low BMI compared with normal BMI, P , 0.05) but no main effect of trimester. Significant interaction effect between trimester and BMI group (P , 0.05); total leucine flux in first trimester was significantly lower in the normal-BMI than in the low-BMI group (P , 0.05); other 2-factor and 3-factor interactions were not significant. 3 Significant main effect of metabolic phase (fasted compared with fed, P , 0.001) but no main effect of BMI. 4 Significant main effect of BMI (low BMI compared with normal BMI, P , 0.05) but no main effect of trimester. Significant interaction effect between trimester and BMI group (P , 0.05); fasted endogenous flux in first trimester was significantly lower in the normal-BMI than in the low-BMI group (P , 0.05). 5 Significant main effect of metabolic phase (fasted compared with fed, P , 0.0001), but main effects of trimester and BMI were not significant. Significant interaction effect between metabolic phase and BMI group (P , 0.05): leucine oxidation in the fed phase was significantly lower in the normalBMI than in the low-BMI group (P , 0.05); other 2-factor and 3-factor interactions were not significant. 6 Measured for per kg body weight data only. Significant main effect of metabolic phase (fasted compared with fed, P , 0.0001) and trimester (first compared with second trimester, P , 0.05) but no main effects of BMI and interaction effects. 7 Measured for per kg body weight data only. Significant effect of trimester (first compared with second trimester, P , 0.005) but no main effect of BMI or interaction effects. 8 Significant effect of trimester (first trimester compared with second trimester, P , 0.05) but no main effect of metabolic phase or BMI, and no interaction effects. 9 Measured for per kg body weight data only. Significant effect of metabolic phase (fasted compared with fed, P , 0.0001) and trimester (first compared with second trimester, P , 0.005) but no main effect of BMI. Significant interaction effect between metabolic phase and trimester (P , 0.005): leucine balance in the fed state was significantly lower in the first trimester than in the second trimester (P , 0.005); other 2-factor and 3-factor interactions were not significant.

significantly higher in the fed state for both alanine and glutamine. Plasma alanine concentrations were significantly higher in the fed state than in the fasted state (P = 0.007), but there were no differences between groups or trimesters. There were also no significant differences in glutamine concentrations between groups, trimesters, or metabolic state (Table 3).

Cysteine and glycine kinetics Cysteine flux rates were higher in the low-BMI group (P = 0.02) in the second trimester (P = 0.01) and in the fasted state (P ,

0.0001). For glycine flux, the main effects of BMI and trimester were significant (Table 7). Glycine flux in the low-BMI group was higher (P = 0.01) and first trimester was higher (P = 0.01). The endogenous fasted glycine flux showed similar differences as the total fasted glycine flux. When whole-body flux rates were examined with weight as a covariate or with FFM-specific data in the first trimester (Table 7), the effect of metabolic state remained significant for both cysteine (P , 0.001) and glycine (P = 0.005). In addition, whole-body cysteine flux was higher in the normal-BMI group (P = 0.046) and in the second trimester (P , 0.001). Plasma glycine concentrations did not change from

326

KURPAD ET AL

TABLE 5 Significant correlations between birth weight (g) or gestational weight gain (kg/wk) between the first and second trimesters with different leucine kinetic parameters in the first trimester1 Leucine parameters

Pearson’s correlation (r)

P

20.59 0.52 0.59

0.01 0.03 0.01

Birth weight Fasted state Oxidation NOLD Balance Fed state NOLD Gestational weight gain Fasted state Total flux Endogenous flux 1

0.49

0.041

0.48 0.48

0.04 0.04

NOLD, nonoxidative leucine disposal.

the first to the second trimesters and was not different between groups or metabolic states (Table 3). DISCUSSION

The results of this study showed a decrease in weight-specific leucine oxidation with increases in NOLD and leucine balance from the first to the second trimesters in both groups of women, which indicated that the extra amino acids required for increased maternal protein synthesis during pregnancy are provided by an

overall decrease in amino acid catabolism in women with normal or low BMIs. Thus, our a priori hypothesis that the low-BMI group may not adapt to the requirements of pregnancy was rejected. Indeed, given their higher protein intake but similar flux rates, it is possible that the low-BMI group up-regulated protein turnover to a greater extent than did the normal-weight group in early pregnancy. We did not study nonpregnant women, because our specific aim was to determine whether there was a difference in amino acid kinetics between the BMI groups at similar times during pregnancy, assuming that the normal BMI women represented a control state. In earlier studies in normal-weight pregnant women, lower urea production and excretion were reported (7–9) along with a progressive decrease in the proportion of dietary nitrogen converted to urea (8). This, together with a similar decrease in leucine transamination and percentage leucine flux oxidized (7), suggest that the amino acids required for increased maternal protein synthesis are provided by an overall reduction in amino acid catabolism. Until now it was not known whether the underweight pregnant woman with a low BMI and body protein stores could also make these adaptations successfully. The present study corroborates similar findings by others (6–9, 19), indicating that the low-BMI group also provides the amino acids required for pregnancy by decreasing amino acid catabolism as pregnancy progressed, even though their leucine oxidation rates were significantly higher than those of the normal-BMI group. In both groups, this translated to a greater efficiency of utilization of

TABLE 6 Alanine nitrogen and glutamine amide nitrogen fluxes in pregnant women with low and normal BMIs at the end of the first and second trimesters1 Normal BMI (n = 9) Amino acid kinetics Alanine flux Fasted (lmol  kg21  h21)2 Fed (lmol  kg21  h21)2 Fasted (lmol  kg FFM21  h21)3 Fed (lmol  kg21  h21)3 Endogenous alanine flux Fasted (lmol  kg21  h21) Fasted (lmol  kg FFM21  h21) Glutamine flux Fasted (lmol  kg21  h21)4 Fed (lmol  kg21  h21)4 Fasted (lmol  kg FFM21  h21)5 Fed (lmol  kg FFM21  h21)5 Endogenous glutamine flux Fasted (lmol  kg21  h21)6 Fasted (lmol  kg FFM21  h21)

First

Second

First

293.1 6 47.4 380.5 6 57.5

350.5 425.2 431.0 524.1

76.1 71.8 84.4 82.0

307.0 6 53.7 379.9 6 35.4

284.8 6 51.1 390.4 6 74.9

287.1 6 47.4

344.5 6 76.1 423.6 6 84.6

301.0 6 53.7

247.5 6 22 256.3 6 28

285.5 325.0 351.8 400.7

6 6 6 6

35.1 43.4 34.6 47.5

260.7 6 22.6 279.5 6 25.4

241.5 6 21.8

279.5 6 35.1 344.4 6 34.8

254.7 6 22.6

241.6 257.6 331.3 352.7

6 6 6 6

24.6 16.1 39.8 26.8

235.6 6 24.6 323.0 6 39.6

6 6 6 6

Second

51.1 40.1 75.1 75.2

290.8 398.1 398.7 546.9

6 6 6 6

Low BMI (n = 9)

1 All values are means 6 SDs. Fat-free mass (FFM)–specific kinetics are presented only for the first trimester, because this measurement was only made in the first trimester. Data were analyzed by using a mixed-model ANOVA. 2 Significant main effect of metabolic phase (fasted compared with fed, P , 0.001) but no main effect of trimester or BMI. 3 Significant main effect of metabolic phase (fasted compared with fed, P , 0.001) but no main effect of BMI. 4 Significant main effect of BMI (low BMI compared with normal BMI, P , 0.05), metabolic phase (fasted compared with fed, P , 0.0005), or trimester (first compared with second trimester, P , 0.05). Significant interaction effect between trimester and BMI group (P = 0.02); in the low-BMI group, total glutamine flux was significantly higher (P = 0.013) in the first trimester than in the second trimester; total glutamine flux was significantly higher (P = 0.003) in the low-BMI group than in the normal-BMI group in the first trimester; and other 2-factor and 3-factor interactions were not significant. 5 Significant main effect of metabolic phase (fasted compared with fed, P , 0.0001) and BMI (low BMI compared with normal BMI, P , 0.05). 6 Significant main effect of BMI (low BMI compared with normal BMI, P , 0.05) and trimester (first compared with second trimester, P , 0.05). Significant interaction effect between trimester and BMI group (P = 0.005); endogenous glutamine flux was significantly higher (P = 0.0004) in the low-BMI group than in the normal BMI group in the first trimester; for the low-BMI group, flux was significantly lower in the second trimester than in the first trimester (P = 0.0005).

327

LOW-BMI PREGNANCY AND AMINO ACID KINETICS TABLE 7 Cysteine and glycine fluxes in pregnant women with low and normal BMIs at the end of the first and second trimesters1 Normal BMI (n = 9) Amino acid kinetics Cysteine flux Fasted (lmol  kg21  h21)2 Fed (lmol  kg21  h21)2 Fasted (lmol  kg FFM21  h21)3 Fed (lmol  kg FFM21  h21)3 Endogenous cysteine flux Fasted (lmol  kg21  h21)4 Fasted (lmol  kg FFM21  h21) Glycine flux Fasted (lmol  kg21  h21)5 Fed (lmol  kg21  h21)5 Fasted (lmol  kg FFM21  h21)6 Fed (lmol  kg FFM21  h21)6 Endogenous glycine flux Fasted (lmol  kg21  h21)2 Fasted (lmol  kg FFM21  h21)

First

Second

First

36.3 6 2.7 31.7 6 2.2

37.4 32.4 46.2 40.0

4.8 3.2 5.9 3.6

38.1 6 4.3 33.6 6 3.1

30.8 6 3.9 42.3 6 6.1

35.3 6 2.7

36.4 6 4.8 45.0 6 5.9

37.1 6 4.3

6 6 6 6

60.6 63.0 82.7 88.4

294.9 6 27.1 294.9 6 35.1

365.9 390.0 449.8 480.0

6 6 6 6

70.8 61.0 73.1 62.7

334.2 6 45.2 342.6 6 35.7

313.8 6 60.0 429.5 6 82.6

290.9 6 27.1

361.9 6 70.8 444.8 6 73.2

330.2 6 45.2

317.8 337.1 435.0 461.8

6 6 6 6

Second

3.9 3.2 6.2 5.1

31.8 28.7 43.6 39.4

6 6 6 6

Low BMI (n = 9)

All values are means 6 SDs. Fat-free mass (FFM)–specific kinetics are presented only for the first trimester, because this measurement was only made for the first trimester. Data were analyzed by using a mixed-model ANOVA. 2 Significant main effect of BMI (low BMI compared with normal BMI, P , 0.05), metabolic phase (fasted compared with fed, P , 0.0005), and trimester (first compared with second trimester, P , 0.05). 3 Significant main effect of metabolic phase (fasted compared with fed, P , 0.001) but no main effect of BMI. 4 Significant main effect of BMI (low BMI compared with normal BMI, P , 0.05) but no main effect of trimester. 5 Significant main effect of BMI (low BMI compared with normal BMI, P , 0.05) and trimester (first compared with second trimester, P , 0.05) but no main effect of metabolic state. 6 Significant main effect of metabolic phase (fasted compared with fed, P , 0.01) but no main effect of BMI. 1

dietary leucine for protein synthesis in the second trimester, as was found earlier (7, 8). Whereas the efficiency tended to be lower in the low-BMI group, it was compensated for by their spontaneous consumption of 50% more dietary protein than the normal-BMI group in both trimesters (Table 2). It is therefore a distinct possibility that this relatively inefficient use of protein by the mothers with a low BMI could lead to lower lean tissue deposition as pregnancy progresses, if they do not have ready access to more food. It has been shown that maternal protein accretion occurs in early pregnancy, even before there is significant growth of the fetus (20, 21). The correlation of both infant birth weight and GWG with leucine kinetic parameters such as oxidation, NOLD, and flux in the first but not the second trimester suggests that early changes in maternal protein metabolism are more critical for a successful pregnancy and are probably important predictors of pregnancy outcome. The women with a low BMI were also capable of producing alanine nitrogen at the same rate as in women with a normal BMI in both the first and second trimesters of pregnancy, whereas producing glutamine amide-nitrogen at a faster rate, in both the fed and fasted states, in the first trimester. The faster protein breakdown rate in the low-BMI group in the first trimester is a logical explanation for their faster glutamine amide-nitrogen flux at this time, which suggests that the availability of labile nitrogen to support the de novo synthesis of other dispensable amino acids was greater in this group of women early in pregnancy. Because alanine and glutamine are 2 primary providers of nitrogen for the de novo synthesis of dispensable amino acids (22, 23), and because birth weight outcomes were similar between the groups, it is suggested that the women with a low BMI were

producing sufficient quantities of labile nitrogen to support the syntheses of other dispensable amino acids necessary for the synthesis and deposition of new maternal and fetal tissues. In healthy men and women it has been reported that glutamine and alanine contribute carbon equally (’5% each) to the glucose produced in the fasted state (24). Hence, the higher glutamine flux of the low-BMI group suggests an ample supply of proteinderived carbon to support gluconeogenesis, more so in the fasted state when it has been reported that 42% of glutamine carbon and 25% of alanine carbon are derived from amino acids released from protein breakdown (24). This may explain our earlier finding (25) that gluconeogenesis and glucose production were similar in fasted pregnant women with low or normal BMI. Although it can be argued that we measured fluxes of alanine nitrogen and glutamine amide-nitrogen in the present study, evidence in the literature indicates that alanine and glutamine carbon fluxes are usually higher in fasted human subjects and that measured nitrogen fluxes were remarkably close to the values reported by others for fasted healthy subjects (22, 24, 26). The glycine results in the low-BMI group were very similar to those of glutamine, which suggests that an adequate supply (compared with control subjects) was available in early pregnancy for this critical amino acid that is both a substrate for proteins and peptides. Glycine is also a precursor for biomolecules that are necessary for the laying down of new maternal and fetal tissues, particularly in later gestation, when, for example, most glycine-rich collagen is synthesized (27). However, in the present study, glycine flux tended to decrease at the end of the second trimester in both groups in a fashion comparable with that in adolescent girls at risk of delivering LBW

328

KURPAD ET AL

infants, who have also been shown to have a lower glycine flux in later pregnancy than control subjects, in whom glycine flux tended to increase (28). Cysteine flux was also higher in the low-BMI group, particularly in the second trimester; however, unlike glutamine and glycine, flux was lower in the fed state. This suggests that de novo cysteine synthesis was not sufficiently stimulated to sustain total flux when its release from protein breakdown was diminished. This may be due to the fact that cysteine, although it is categorized as a dispensable amino acid, has to receive its sulfur from the indispensable amino acid methionine during synthesis. Because of the marked reduction in protein breakdown in the fed state, a distinct possibility was that cysteine synthesis was not sufficiently increased to maintain total flux because of the insufficient availability of methionine. Whereas a low maternal BMI and low GWG are thought to be the important determinants of LBW (1–4), our present finding of similar infant birth weights between the 2 groups indicates that women with a low BMI who have adequate access to energy and protein can adapt successfully to the demands of pregnancy. This could be for 2 reasons. First, all of the women in this study had access to optimal health care and good nutrition. Second, sufficient amino acids were available for the successful laying down of maternal reproductive tissues and early fetal protein accretion, particularly in the fasted state. The ability of the low-BMI group to have a net protein synthesis, positive balance, and weekly GWG similar to the normal-BMI group, despite a higher amino acid oxidation rate, suggests that they had sufficient amino acids to satisfy protein accretion, fetal energy utilization, and increased maternal gluconeogenesis. This latter finding may have important implications concerning the supplemental feeding of pregnant women with a low BMI early in pregnancy. From these results we conclude that the extra amino acids and labile nitrogen needed for increased protein and de novo amino acid synthesis in pregnancy are obtained through a common mechanism of increased protein breakdown and decreased oxidation in both normal-weight and underweight women. The former was more marked in the women with a low BMI, who had higher protein intakes but a lower efficiency of utilization of dietary and endogenously produced leucine. These changes in protein and amino acid metabolism emphasize the importance of an early and adequate intake of dietary protein in pregnancy. We are grateful to the nursing staff of the obstetrics and gynecology ward at St John’s Medical College Hospital for their care of the subjects and to Margaret Frazer and Melanie Del Rosario for their excellent work analyzing the samples. The authors’ responsibilities were as follows—AVK, PD, and FJ: involved in the study design, data collection, data analysis, data interpretation, and writing of the manuscript; TT: involved in the analysis and interpretation of the data and the writing of the manuscript; and AM, AT, and RM: provided the study subjects and the facilities for this collaborative research. None of the authors had any conflict of interest with the funding agency or any other conflicts of interest.

3.

4.

5.

6.

7.

8.

9.

10.

11. 12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

22.

REFERENCES 1. Chhabra P, Sharma AK, Grover VL, Aggarwal OP. Prevalence of low birth weight and its determinants in an urban resettlement area of Delhi. Asia Pac J Public Health 2004;16:95–8. 2. Bang AT, Reddy HM, Bang RA, Deshmukh MD. Why do neonates die in rural Gadchiroli, India? (Part II): estimating population attributable

23.

24.

risks and contribution of multiple morbidities for identifying a strategy to prevent deaths. J Perinatol 2005;25:S35–43. de Onis M, Blossner M, Villar J. Levels and patterns of intrauterine growth retardation in developing countries. Eur J Clin Nutr 1998;52: S5–15. Administrative Committee on Coordination/Sub-Committee on Nutrition. Low birthweight. A report based on the International Low Birth weight Symposium and Workshop held June 14–17, 1999 in Dhaka, Bangladesh. Nutrition Policy Paper 18. Geneva, Switzerland: Administrative Committee on Coordination/Subcommittee on Nutrition, 2000. Thame M, Wilks RJ, McFarlane-Anderson N, Bennett FI, Forrester TE. Relationship between maternal nutritional status and infant’s weight and body proportions at birth. Eur J Clin Nutr 1997;51:134–8. Duggleby SL, Jackson AA. Protein, amino acid and nitrogen metabolism during pregnancy: how might the mother meet the needs of her fetus. Curr Opin Clin Nutr Metab Care. 2002;5:503–9. Kalhan SC, Rossi KQ, Gruca LL, Super DM, Savin SM. Relation between transamination of branched-chain amino acids and urea synthesis: evidence from human pregnancy. Am J Physiol 1998;275:E423–31. Forrester T, Badaloo AV, Persaud C, Jackson AA. Urea production and salvage during pregnancy in normal Jamaican women. Am J Clin Nutr 1994;60:341–6. Duggleby SL, Jackson AA. Higher weight at birth is related to decreased maternal amino acid oxidation during pregnancy. Am J Clin Nutr 2002; 76:852–7. Kurpad AV, Regan MM, Raj T, et al. Lysine requirement of chronically undernourished adult Indian subjects, measured by the 24h indicator amino acid oxidation and balance technique. Am J Clin Nutr 2003;77: 101–8. Jackson AA. The glycine story. Eur J Clin Nutr 1991;45:59–65. Badaloo A, Reid M, Forrester T, Heird WC, Jahoor F. Cysteine supplementation improves the erythrocyte glutathione synthesis rate in children with severe edematous malnutrition. Am J Clin Nutr 2002;76: 646–52. Durnin JVGA, Wormersely J. Estimates of total body fat from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years. Br J Nutr 1974;32:77–9. Siri WE. Body composition from the fluid spaces and density: analysis of methods. In: Brozek J, Henschel A, eds. Techniques for measuring body composition. Washington, DC: National Academy of Sciences, National Research Council, 1961:223–44. World Health Organization. Physical status: the use and interpretation of anthropometry. Report of a WHO Expert Committee. Geneva, Switzerland: World Health Organization, 1995. Vaz M, Bharathi AV, Muthayya S, Kurpad AV. Food frequency questionnaire-based estimates of compliance to ATP III (National Cholesterol Education Programme). Recommended diets in the middleclass adult population of Bangalore city. J Assoc Physicians India 2009; 57:443–6. Holmba¨ck U, Forslund A, Forslund J, et al. Metabolic responses to nocturnal eating in men are affected by sources of dietary energy. J Nutr 2002;132:1892–9. Millward DJ, Fereday A, Gibson NR, Pacy PJ. Post-prandial protein metabolism. Baillieres Clin Endocrinol Metab 1996;10:533–49. Duggleby SL, Jackson AA. Relationship of maternal protein turnover and lean body mass during pregnancy and birth length. Clin Sci (Lond) 2001;101:65–72. Hopkinson JM, Butte NF, Ellis KJ, Wong WW, Puyau MR, Smith EO. Body fat estimation in late pregnancy and early postpartum: comparison of two-, three-, and four component models. Am J Clin Nutr 1997;65: 432–8. Van Raaij JMA, Peek MEM, Vermaat-Miedema SH, Schonk CM, Hautvast JGAJ. New equations for estimating body fat mass in pregnancy from body density or total body water. Am J Clin Nutr 1988;48: 24–9. Darmaun D, Matthews DE, Bier DM. Glutamine and glutamate kinetics in humans. Am J Physiol 1986;251:E117–26. Lopez HW, Morendras C, Morand C, Demigne C, Remesy C. Opposite fluxes of glutamine and alanine in the splanchnic area are an efficient mechanism for nitrogen sparing in rats. J Nutr 1998;128:1487–94. Nurjhan N, Bucci A, Perriello G, et al. Glutamine: a major gluconeogenic precursor and vehicle for interorgan carbon transport in man. J Clin Invest 1995;95:272–7.

LOW-BMI PREGNANCY AND AMINO ACID KINETICS 25. Dwarkanath P, Kurpad AV, Muthayya S, et al. Glucose kinetics and pregnancy outcome in Indian women with low and normal body mass indices. Eur J Clin Nutr 2009;63:1327–34. 26. Yang RD, Mathews DE, Bier DM, Wen ZM, Young VR. Response of alanine metabolism in humans to manipulation of dietary protein and energy intakes. Am J Physiol 1986;250:E39–46.

329

27. Meier P, Teng C, Battaglia FC, Meschia G. The rate of amino acid nitrogen and total nitrogen accumulation in the fetal lamb. Proc Soc Exp Biol Med 1981;167:463–8. 28. Thame M, Fletcher H, Baker T, Jahoor F. Comparing the in vivo glycine fluxes of adolescent girls and adult women during early and late pregnancy. Br J Nutr (Epub ahead of print 25 March 2010).