BIOLOGY OF REPRODUCTION 71, 901–908 (2004) Published online before print 12 May 2004. DOI 10.1095/biolreprod.104.029645
Maternal Nutrient Restriction Reduces Concentrations of Amino Acids and Polyamines in Ovine Maternal and Fetal Plasma and Fetal Fluids1 Hyukjung Kwon,3 Stephen P. Ford,4 Fuller W. Bazer,3 Thomas E. Spencer,3 Peter W. Nathanielsz,4,5 Mark J. Nijland,4,5 Bret W. Hess,4 and Guoyao Wu2,3 Department of Animal Science and Center for Animal Biotechnology and Genomics,3 Texas A&M University, College Station, Texas 77843 Center for the Study of Fetal Programming and Department of Animal Science,4 University of Wyoming, Laramie, Wyoming 82071 Department of Obstetrics and Gynecology,5 New York University School of Medicine, New York, New York 10016 ABSTRACT
INTRODUCTION
Amino acids and polyamines are essential for placental and fetal growth, but little is known about their availability in the conceptus in response to maternal undernutrition. We hypothesized that maternal nutrient restriction reduces concentrations of amino acids and polyamines in the ovine conceptus. This hypothesis was tested in nutrient-restricted ewes between Days 28 and 78 (experiment 1) and between Days 28 and 135 (experiment 2) of gestation. In both experiments, ewes were assigned randomly on Day 28 of gestation to a control group fed 100% of National Research Council (NRC) nutrient requirements and to an nutrient-restricted group fed 50% of NRC requirements. Every 7 days beginning on Day 28 of gestation, ewes were weighed and rations adjusted for changes in body weight. On Day 78 of gestation, blood samples were obtained from the uterine artery and umbilical vein for analysis. In experiment 2, nutrient-restricted ewes on Day 78 of gestation either continued to be fed 50% of NRC requirements or were realimented to 100% of NRC requirements until Day 135. Fetal weight was reduced in nutrient-restricted ewes at both Day 78 (32%) and Day 135 (15%) compared with controls. Nutritional restriction markedly reduced (P , 0.05) concentrations of total a-amino acids (particularly serine, arginine-family amino acids, and branched-chain amino acids) and polyamines in maternal and fetal plasma and in fetal allantoic and amniotic fluids at both mid and late gestation. Realimentation of nutrient-restricted ewes increased (P , 0.05) concentrations of total a-amino acids and polyamines in all the measured compartments and prevented intrauterine growth retardation. These novel findings demonstrate that 50% global nutrient restriction decreases concentrations of amino acids and polyamines in the ovine conceptus that could adversely impact key fetal functions. The results have important implications for understanding the mechanisms responsible for both intrauterine growth retardation and developmental origins of adult disease.
Intrauterine growth retardation (IUGR) occurs in response to maternal undernutrition in humans, sheep, pigs, and rats [1–4]. Nutrient restriction in pregnant women may result from low dietary intake of nutrients owing to either a limited supply of food or severe nausea and vomiting known as hyperemesis gravidarum [5]. Epidemiological studies in humans suggest that alterations in fetal nutrition and endocrine status may result in developmental adaptations that permanently change the structure, physiology, and metabolism of the offspring, thereby predisposing individuals to cardiovascular, metabolic, and endocrine diseases in adult life [6]. Maternal nutrient restriction can also occur in domestic animals [7, 8]. For example, unsupplemented grazing ewes lose a significant amount of body weight during pregnancy, and their health, fetal growth, and lactation performance are compromised [7]. Thus, studies of the mechanisms responsible for IUGR brought about by maternal nutrient restriction have important implications for both human medicine and animal agriculture. Amino acids play a vital role in the development of the conceptus (embryo/fetus and associated placental membranes). In addition to serving as building blocks for tissue protein synthesis, amino acids function as antioxidants, regulators of hormone secretion, major fuels for fetal growth, and cell signaling molecules [9, 10]. Furthermore, amino acids are essential precursors for the synthesis of nonprotein substances with biological importance, including nitric oxide, polyamines, neurotransmitters, amino sugars, purine and pyrimidine nucleotides, creatine, carnitine, porphyrins, melatonin, melanin, and sphingolipids [11, 12]. For example, nitric oxide, a product of arginine catabolism, plays a crucial role in regulating placental angiogenesis and fetalplacental blood flows during gestation [13–15]. Polyamines (polycationic molecules) regulate gene expression, signal transduction, ion channel function, and DNA and protein synthesis, as well as cell proliferation, differentiation, and function [10]. Surprisingly, little is known about the effect of maternal nutrient restriction on concentrations of amino acids and polyamines in the conceptus of any species. The sheep is a widely used animal model for studying fetal and placental development [14–17]. We have recently reported striking changes in concentrations of both amino acids [18] and polyamines [19], as well as polyamines and nitric oxide synthesis [19, 20] in the ovine conceptus during pregnancy. In the present study, we used an established ovine model of IUGR [21] to test the hypothesis that ma-
amino acids, nutrition, pregnancy, sheep Supported, in part, by grants from USDA/NRI 2001-02259 and 200102166, the Texas A&M University Life Science Program, the University of Wyoming BRIN P20, RR16474, and NIH HD 21350. 2 Correspondence: Guoyao Wu, Department of Animal Science, Room 212, Kleberg Building, Texas A&M University, 2471 TAMU, College Station, TX 77843-2471. FAX: 979 845 6057; e-mail:
[email protected] 1
Received: 11 March 2004. First decision: 2 April 2004. Accepted: 4 May 2004. Q 2004 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
901
902
KWON ET AL.
ternal nutrient restriction reduces the availability of amino acids and polyamines in the conceptus. MATERIALS AND METHODS
Animals Two series of experiments were conducted with multiparous ewes of mixed breeding. In both experiments 1 and 2, maternal nutrient restriction (50% of National Research Council [NRC] nutrient requirements) was carried out between Days 28 and 78 of gestation, as described previously [21]. Briefly, the diet consisted of a pelleted beet pulp (93.5% dry matter, 79.7% total digestible nutrient, 10% crude protein) and a mineral-vitamin mixture. On Day 20 of gestation, 16 ewes were weighed so that individual diets could be calculated on a metabolic body weight basis (weight0.75). On Day 21 of gestation, all ewes were placed in individual pens and provided all nutrients that met NRC maintenance requirements for early gestation. On Day 28 of gestation, ewes were assigned randomly to a control group (n 5 8) fed 100% of NRC nutrient requirements or to a nutrient-restricted group (n 5 8) fed 50% of NRC nutrient requirements. Every 7 days beginning on Day 28 of gestation, ewes were weighed and rations adjusted for changes in body weight. On Day 45 of gestation, the number of fetuses carried by each ewe was determined by ultrasonography (Ausonics Microimager 1000 sector scanning instrument; Ausonics Pty Ltd., Sydney, Australia). At Day 78 of gestation, each ewe in experiment 1 was administered an overdose of sodium pentobarbital (Abbott Laboratories, Abbott Park, IL) and exsanguinated. After the tip of the gravid uterine horn was exposed, blood samples were withdrawn from the uterine artery and umbilical vein into cooled heparinized tubes. The tubes were immediately centrifuged at 48C and 3000 3 g for 10 min to obtain plasma. Allantoic and amniotic fluids were obtained through the amniochorion and chorioallantoic membranes, respectively [18]. All plasma and fetal fluid samples were stored at 2808C until analyzed. At Day 78 of gestation in experiment 2, nutrient-restricted ewes either continued to be fed 50% of NRC nutrient requirements (n 5 5) or were realimented to 100% of NRC nutrient requirements (n 5 8). At Day 135 of gestation, blood samples were obtained from the uterine artery and umbilical vein of all ewes, and allantoic and amniotic fluids were collected as described for experiment 1. This study was approved by the University of Wyoming Animal Care and Use Committee.
Analysis of Amino Acids Plasma and fetal fluids were analyzed for amino acids, as described previously [18, 22]. Briefly, amino acids, except for proline, were determined by HPLC methods involving precolumn derivatization with ophthaldialdehyde. The values for total cysteine in plasma and fluids represent free cysteine plus one-half cystine. Proline was measured using an HPLC method involving precolumn derivatization with 9-fluorenylmethyl chloroformate. Amino acids in samples were quantified on the basis of known amounts of standards (Sigma Chemical Co., St. Louis, MO) using Millenium-32 Software (Waters, Milford, MA).
Analysis of Polyamines Plasma and fetal fluids were analyzed for polyamines by an ion-pairing HPLC method involving precolumn derivatization with o-phthaldialdehyde [23, 24]. Putrescine, spermidine, and spermine in samples were quantified on the basis of known amounts of standards (Sigma) using Millenium-32 Software (Waters).
Statistics Analyses Statistical comparisons between the two groups of ewes killed on Day 78 of gestation were performed using the Student unpaired t-test. Statistical comparisons among the three groups of ewes killed on Day 135 of gestation were completed using a one-way ANOVA. Post hoc analysis was performed with a Tukey test for highly significant differences. All statistical analyses were performed using the SAS V8.2 program for Windows (SAS Institute Inc., Cary, NC), and significance was accepted when P # 0.05. Data are presented as mean 6 standard error of the mean (SEM).
RESULTS
Maternal Undernutrition Between Days 28 and 78 of Gestation
At Day 78 of gestation, fetuses from control ewes were heavier (P , 0.05) than fetuses from nutrient-restricted ewes, averaging 326.4 6 20.0 g and 221.7 6 7.9 g (n 5 8), respectively. Table 1 summarizes concentrations of amino acids in maternal and fetal plasma at Day 78 of gestation. Glycine was the most abundant amino acid in both maternal and fetal plasma. Compared with control ewes, maternal nutrient restriction between Days 28 and 78 of gestation increased (P , 0.01) plasma concentration of glycine, but decreased (P , 0.05) plasma concentrations of most amino acids (arginine, citrulline, cysteine, glutamine, isoleucine, leucine, ornithine, phenylalanine, proline, serine, and valine) in both maternal and fetal plasma. Nutrient restriction also decreased (P , 0.05) concentrations of lysine, methionine, threonine, tryptophan, and tyrosine in maternal plasma, but had no effect (P . 0.05) on these amino acids in fetal plasma. Maternal nutrient restriction had no effect (P . 0.05) on concentrations of b-alanine, asparagine, aspartate, glutamate, histidine, and taurine in either maternal or fetal plasma. Concentrations of total a-amino acids were 8.8% and 8.3% lower (P , 0.01), respectively, in plasma from nutrient-restricted ewes and their fetuses when compared with control ewes. At Day 78 of gestation, concentrations of total polyamines (putrescine plus spermidine plus spermine) were 3.1 6 0.25 and 2.6 6 0.14 mmol/L (n 5 8, P , 0.05) in maternal plasma of control and nutrient-restricted ewes, and were 4.5 6 0.28 and 3.2 6 0.21 mmol/L (n 5 8, P , 0.05) in fetal plasma of control and nutrient-restricted ewes, respectively. Concentrations of amino acids in allantoic and amniotic fluids at Day 78 of gestation are summarized in Table 2. Alanine, citrulline, glutamine, glycine, and serine represented 73% and 76% of total a-amino acids in the allantoic fluid of control and nutrient-restricted ewes, respectively. Compared with control ewes, maternal nutrient restriction between Days 28 and 78 of gestation increased (P , 0.01) concentrations of glycine more than two-fold but decreased (P , 0.05) concentrations of many amino acids (arginine, citrulline, glutamine, isoleucine, leucine, proline, serine, and valine; Table 2) and polyamines (Table 3) in both allantoic and amniotic fluids. Nutrient restriction had no effect (P . 0.05) on concentrations of cysteine, lysine, and taurine in allantoic fluid, but decreased (P , 0.05) concentrations of these amino acids in amniotic fluid; the opposite was observed for ornithine. Concentrations of alanine, balanine, asparagine, aspartate, glutamate, histidine, methionine, phenylalanine, threonine, tryptophan, and tyrosine in allantoic and amniotic fluids did not differ (P . 0.05) between control and nutrient-restricted ewes. At Day 78 of gestation, maternal nutrient restriction had no effect (P . 0.05) on concentrations of total a-amino acids in allantoic fluid, but decreased (P , 0.05) concentrations of total aamino acids by 5% in amniotic fluid. Maternal Undernutrition Between Days 28 and 135 of Gestation
At Day 135 of gestation, weights of fetuses from control and nutrient-restricted realimented ewes were similar (P . 0.05), averaging 4782.0 6 165.1 g (n 5 8) and 4639.6 6 236.4 g (n 5 8), respectively, whereas weights of the fetuses of ewes that were continuously nutrient-restricted
903
NUTRIENT RESTRICTION AND AMINO ACIDS IN PREGNANCY TABLE 1. Amino acid concentrations (mmol/L) in ovine maternal and fetal plasma at Day 78 of gestation.* Uterine arterial plasma Amino acid Ala b-Ala Arg Asn Asp Cit Cys Gln Glu Gly His Ile Leu Lys Met Orn Phe Pro Ser Taurine Thr Trp Tyr Val Total a-AA
Control† 229 11 130 21 11 175 168 219 69 454 49 65 97 102 21 53 48 111 75 68 71 31 52 164 2411
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
Fetal umbilical plasma
Restricted‡
8 0.6 3 0.9 0.8 3 3 7 2 17 2 1.4 4 3 0.6 2 2 4 2 3 2 2 2 3 21
200 10 100 19 11 130 139 156 65 669 46 46 71 83 17 37 37 78 54 63 57 23 38 125 2200
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
7 0.5 4a 0.8 0.7 7a 5a 3a 2 2a 1.5 1.4a 3a 2a 0.3a 1.5a 1.6a 3a 2a 3 2a 0a 1a 4a 41a
Control† 405 86 223 97 16 211 186 437 72 499 55 77 158 189 63 114 118 194 418 13 333 49 126 195 4215
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
Restricted‡
16 5 11 4 0.9 9 4 13 4 15 2 2 5 6 2 5 6 4 16 8 9 1.3 8 5 49
421 83 157 80 14 140 170 349 73 772 58 64 128 175 57 95 100 153 323 119 305 51 118 164 3865
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
17 3 5a 7 0.7 4a 5b 10a 4 15a 3 2a 4a 7 2 3a 6b 4a 16a 7 10 1.7 4 4a 38a
* Data are means 6 SEM; n 5 8. AA, amino acids. † Ewes were fed 100% of NRC nutrient requirements between Days 28 and 78 of gestation. ‡ Ewes were fed 50% of NRC nutrient requirements between Days 28 and 78 of gestation. a P , 0.01 vs. control. b P , 0.05 vs. control.
TABLE 2. Amino acid concentrations (mmol/L) in ovine fetal fluids at Day 78 of gestation.* Allantoic fluid Amino acid Ala b-Ala Arg Asn Asp Cit Cys Gln Glu Gly His Ile Leu Lys Met Orn Phe Pro Ser Taurine Thr Trp Tyr Val Total a-AA
Control† 4183 6 1049 6 813 6 374 6 136 6 2541 6 536 6 4431 6 150 6 3308 6 221 6 150 6 553 6 1076 6 65 6 386 6 385 6 735 6 4296 6 1383 6 795 6 118 6 103 6 414 6 25 768 6
272 34 24 27 9 126 15 264 9 145 20 9 31 44 3 28 21 23 186 82 47 3 5 20 596
Amniotic fluid Restricted‡ 3905 6 256 1033 6 65 653 6 23a 338 6 23 145 6 11 1534 6 121a 469 6 34 3218 6 133a 151 6 6 7129 6 408a 200 6 19 119 6 4a 453 6 19b 1025 6 47 70 6 3 293 6 12b 361 6 14 591 6 30a 3480 6 176a 1208 6 82 765 6 26 111 6 6 93 6 6 349 6 14b 25 449 6 556
* Data are means 6 SEM; n 5 8. AA, amino acids. † Ewes were fed 100% of NRC nutrient requirements between Days 28 and 78 of gestation. ‡ Ewes were fed 50% of NRC nutrient requirements between Days 28 and 78 of gestation. a P , 0.01 vs. control. b P , 0.05 vs. control.
Control†
Restricted‡
43 6 2 31 6 1 30 6 2 16 6 0.8 6.6 6 0.4 36 6 1.7 42 6 2 76 6 4 11 6 0.6 79 6 3 4.6 6 0.3 5 6 0.3 39 6 2 54 6 3 4.4 6 0.2 82 6 6 27 6 1.6 43 6 1.8 95 6 3 92 6 3 27 6 1.8 4.8 6 0.4 5.5 6 0.4 25 6 1.4 753 6 1
37 6 2.2 28 6 1.8 23 6 0.9a 14 6 1 5.8 6 0.3 23 6 1.1a 34 6 2b 49 6 2a 9.5 6 0.6 180 6 8a 4.2 6 0.3 3.7 6 0.2a 29 6 1a 46 6 2b 4 6 0.2 73 6 3 24 6 1.3 35 6 1.3a 65 6 3a 82 6 3b 25 6 1.9 4.4 6 0.2 5 6 0.3 20 6 0.9b 714 6 11b
904
KWON ET AL.
TABLE 3. Polyamine concentrations (nmol/L) in ovine fetal fluids at Day 78 of gestation.* Allantoic fluid Control†
Polyamine Putrescine Spermidine Spermine Total
1469 1544 1655 4668
6 6 6 6
Amniotic fluid Restricted‡
49 53 47 144
951 1015 994 2961
6 6 6 6
Control†
33a 52a 62a 121a
760 1753 1120 3622
6 6 6 6
Restricted‡
20 73 72 133
547 1104 753 2404
6 6 6 6
37a 65a 20a 61a
* Data are means 6 SEM; n 5 8. † Ewes were fed 100% of NRC nutrient requirements between Days 28 and 78 of gestation. ‡ Ewes were fed 50% of NRC nutrient requirements between Days 28 and 78 of gestation. a P , 0.01 vs. control.
(4047.0 6 175.5 g; n 5 5) were reduced (P , 0.05) when compared with control ewes. Concentrations of amino acids in maternal and fetal plasma at Day 135 of gestation are summarized in Table 4. Maternal nutrient restriction between Days 28 and 135 of gestation markedly reduced (P , 0.05) concentrations of most amino acids (alanine, arginine, asparagine, citrulline, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, taurine, threonine, tryptophan, tyrosine, and valine) in both maternal and fetal plasma. Concentrations of b-alanine, aspartate, glutamate, glycine, and serine in maternal plasma, and concentrations of b-alanine and aspartate in fetal plasma did not differ (P . 0.05) between control and nutrient-restricted ewes. Concentrations of total a-amino acids were 28% and 34% lower (P , 0.05), respectively, in maternal and fetal plasma of ewes fed 50% of NRC nutrient requirements between Days 28 and 135 of gestation when compared with control ewes. Realimentation of underfed ewes beginning on Day 78 of gestation increased (P , 0.05) concentrations of several
amino acids (alanine, cysteine, methionine, proline, taurine, and tryptophan) in both maternal and fetal plasma by Day 135 of gestation compared with ewes underfed between Days 28 and 135 of gestation. Realimentation of underfed ewes completely restored concentrations of alanine and asparagine in maternal plasma and of alanine, asparagine, histidine, taurine, and tyrosine in fetal plasma to values in control ewes. However, in both maternal and fetal plasma, concentrations of most amino acids remained lower (P , 0.05) in realimented ewes in comparison with control ewes. In maternal and fetal plasma, concentrations of total a-amino acids in realimented ewes were 10%–16% higher (P , 0.01) than those in underfed ewes, but were 21%–23% lower (P , 0.01) than those in control ewes. At Day 135 of gestation, concentrations of total polyamines (putrescine plus spermidine plus spermine) were 3.4 6 0.19 (n 5 8), 2.5 6 0.13 (n 5 5), and 2.9 6 0.17 mmol/ L (n 5 8) in maternal plasma of control, nutrient-restricted, and realimented ewes, and were 4.3 6 0.20 (n 5 8), 2.9 6 0.11 (n 5 5), and 3.6 6 0.15 mmol/L (n 5 8) in fetal
TABLE 4. Amino acid concentrations (mmol/L) in ovine maternal and fetal plasma at Day 135 of gestation.* Maternal plasma Amino acid Ala b-Ala Arg Asn Asp Cit Cys Gln Glu Gly His Ile Leu Lys Met Orn Phe Pro Ser Taurine Thr Trp Tyr Val Total a-AA
Control† 172 34 184 39 10 150 149 329 86 565 47 83 112 139 27 41 46 135 88 118 87 37 57 132 2713
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
5a 3 8a 2a 1 8a 4a 19a 3 22 2a 4a 5a 6a 1a 3c 2a 4a 4 9a 5a 2a 3a 6a 52a
Restricted‡ 138 31 97 26 9 83 63 244 113 515 28 67 66 85 17 31 27 64 104 72 44 19 37 75 1952
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
6b 1 2b 2b 0.3 2b 3c 6b 7 29 1b 2b 4c 3c 0.6c 2b 2c 6c 12 2b 6c 2c 1b 5b 39c
Fetal plasma Realimented§ 194 6 4a 29 6 2 113 6 6b 36 6 3a 10 6 1 79 6 5b 93 6 4b 241 6 19b 89 6 6 497 6 25 31 6 2b 70 6 2b 80 6 3b 98 6 4b 21 6 1b 33 6 2b 36 6 2b 85 6 6b 97 6 7 108 6 8a 57 6 4b 26 6 2b 42 6 3b 87 6 5b 2138 6 37b
Control† 485 205 309 103 21 239 186 881 71 1052 73 147 211 200 67 143 153 309 629 221 515 71 184 357 6407
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
19a 17 18a 3a 1 9a 8a 35a 4a 49a 4a 3a 8a 9a 2a 8a 9a 9a 31a 11a 26a 4a 8a 14a 140a
Restricted‡
Realimented§
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
448 6 16a 198 6 12 182 6 12b 88 6 5a 19 6 1 150 6 7b 118 6 6b 726 6 29b 63 6 3b 829 6 45b 69 6 4a 119 6 4b 161 6 8b 149 6 8b 52 6 2b 93 6 5b 113 6 5b 212 6 11b 406 6 15b 187 6 17a 358 6 20b 63 6 2b 163 6 12a 254 6 12b 4943 6 101b
374 192 224 69 18 145 100 753 46 635 54 106 130 140 38 73 112 163 328 79 360 49 119 220 4255
26b 24 22b 5b 1 9b 3c 25b 4c 40c 5b 3b 13b 7b 6c 3c 12b 5c 30c 5b 33b 5c 10b 9b 72b
* Data are means 6 SEM. AA, amino acids. † Ewes were fed 100% of NRC nutrient requirements between Days 28 and 135 of gestation (n 5 8). ‡ Ewes were fed 50% of NRC nutrient requirements between Day 28 and 135 of gestation (n 5 5). § Ewes were fed 50% of NRC nutrient requirements between Days 28 and 78 of gestation and realimented to 100% of NRC nutrient requirements between Days 78 and 135 of gestation (n 5 8). a,b,c Means with different superscripts are different (P , 0.05).
905
NUTRIENT RESTRICTION AND AMINO ACIDS IN PREGNANCY TABLE 5. Amino acid concentrations (mmol/L) in ovine fetal fluids at Day 135 of gestation.* Allantoic fluid Amino acid Ala b-Ala Arg Asn Asp Cit Cys Gln Glu Gly His Ile Leu Lys Met Orn Phe Pro Ser Taurine Thr Trp Tyr Val Total a-AA
Control† 1075 6 56ab 1044 6 49 670 6 44a 260 6 16 182 6 13 592 6 35a 393 6 18a 1036 6 50b 215 6 16a 1447 6 65b 341 6 20a 167 6 8a 258 6 11a 493 6 36a 197 6 12a 152 6 11a 136 6 11a 590 6 26a 19 814 6 556a 1010 6 51 494 6 28b 165 6 7a 222 6 10a 407 6 16a 29 304 6 615a
Restricted‡ 1342 6 75a 1162 6 139 272 6 28c 274 6 12 143 6 11 214 6 18c 196 6 17c 1469 6 117a 155 6 10b 3103 6 373a 264 6 12b 108 6 7b 151 6 6b 287 6 17b 106 6 4c 99 6 2b 105 6 4b 275 6 12c 3110 6 157c 984 6 69 1087 6 85a 86 6 3c 180 6 7b 229 6 15c 13 255 6 373c
Amniotic fluid Realimented§ 774 6 110b 1041 6 46 476 6 36b 285 6 17 153 6 9 318 6 20b 265 6 15b 798 6 74c 202 6 12a 1299 6 22b 358 6 18a 103 6 5b 150 6 7b 266 6 25b 129 6 5b 89 6 5b 100 6 6b 371 6 18b 14 438 6 1591b 958 6 78 486 6 83b 112 6 6b 182 6 18b 304 6 9b 23 168 6 526b
Control† 194 162 186 37 22 178 127 386 69 326 26 29 50 55 22 25 27 141 771 127 201 15 44 78 2988
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
8a 11 8a 2a 1a 8a 4a 14a 4a 14a 2a 2a 2a 2a 2a 1a 2a 5a 43a 10 13a 1a 2a 4a 86a
Restricted‡
Realimented§
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
114 6 11b 144 6 13 95 6 13b 32 6 3a 17 6 1b 43 6 5b 87 6 5b 121 6 8b 50 6 4b 240 6 18b 18 6 1b 14 6 1b 31 6 2b 28 6 2b 17 6 1b 14 6 1b 13 6 1b 82 6 5b 365 6 31b 103 6 10 99 6 6b 13 6 1a 20 6 1b 50 6 4b 1620 6 70b
71 149 39 17 15 22 50 86 47 184 12 10 17 19 16 13 9 54 205 102 64 9 13 28 998
2c 8 4c 2b 2b 3c 4c 8c 3b 6c 1c 1c 1c 2c 1b 1b 1c 3c 4c 8 4c 1b 1c 2c 22c
* Data are means 6 SEM. AA, amino acids. † Ewes were fed 100% of NRC nutrient requirements between Days 28 and 135 of gestation (n 5 8). ‡ Ewes were fed 50% of NRC nutrient requirements between Days 28 and 135 of gestation (n 5 5). § Ewes were fed 50% of NRC nutrient requirements between Days 28 and 78 of gestation and realimented to 100% of NRC nutrient requirements between Days 78 and 135 of gestation (n 5 8). a,b,c Means with different superscripts are different (P , 0.05).
plasma of control, nutrient-restricted, and realimented ewes, respectively. Concentrations of total polyamines in realimented ewes were higher (P , 0.05) than those in nutrientrestricted ewes, but were lower (P , 0.05) than those in control ewes. Table 5 summarizes concentrations of amino acids in allantoic and amniotic fluids on Day 135 of gestation. Maternal nutrient restriction between Days 28 and 135 of gestation markedly reduced (P , 0.05) concentrations of most amino acids (arginine, citrulline, cysteine, glutamate, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, taurine, tryptophan, tyrosine, and valine) in both allantoic and amniotic fluids. The decrease in allantoic fluid serine (84%) was the most striking. Surprisingly, concentrations of alanine, glutamine, glycine, and threonine in allantoic fluid increased (P , 0.01) in nutrient-restricted ewes compared with control ewes. Concentrations of alanine, b-alanine, asparagine, aspartate, and taurine in allantoic fluid or concentrations of b-alanine in amniotic fluid did not differ (P . 0.05) between control and nutrient-restricted ewes. Concentrations of total a-amino acids were 55% and 67% lower (P , 0.01), respectively, in allantoic and amniotic fluids of nutrient-restricted ewes when compared with control ewes. Realimentation of nutrient-restricted ewes beginning on Day 78 of gestation increased (P , 0.05) concentrations of arginine, citrulline, cysteine, histidine, proline, serine, tryptophan, and valine in both allantoic and amniotic fluids in comparison with nonrealimented ewes. However, in these fetal fluids, concentrations of most amino acids in allantoic and amniotic fluids remained lower (P , 0.05) in realimented ewes in comparison with control ewes. Realimentation of underfed ewes completely restored, to values for control ewes, con-
centrations of glutamate and histidine in allantoic fluid and of asparagine and tryptophan in amniotic fluid. In both allantoic and amniotic fluids, concentrations of total a-amino acids (Table 5) and polyamines (Table 6) in realimented ewes were higher (P , 0.01) than those in nutrient-restricted ewes, but were lower (P , 0.01) than those in control ewes. DISCUSSION
This is the first report of changes in concentrations of amino acids and polyamines in the ovine conceptus in response to precisely controlled maternal nutrient restriction. Although maternal and fetal plasma concentrations of amino acids in human IUGR have been documented, it is not known whether these changes were directly caused by maternal nutrient restriction or other environmental factors acting on a self-selected genetic population [25–29]. There are five major findings from this study: 1) 50% global nutrient restriction of pregnant ewes for both the first half of gestation and throughout gestation until just prior to term leads to IUGR, which can be reversed by realimentation during the second half of gestation; 2) maternal nutrient restriction markedly reduced concentrations of arginine-family amino acids and branched-chain amino acids in maternal and fetal plasma and in fetal fluids; 3) serine exhibited the most striking decrease in allantoic fluid (84%) and amniotic fluid (73%) by late gestation; 4) concentrations of polyamines in maternal and fetal plasma and in fetal allantoic and amniotic fluids were reduced in nutrient-restricted ewes compared with control ewes; and 5) realimentation of underfed ewes increased concentrations of total a-amino acids and
906
KWON ET AL.
TABLE 6. Polyamine concentrations (nmol/L) in ovine fetal fluids at Day 135 of gestation.* Allantoic fluid Polyamine Putrescine Spermidine Spermine Total
Control† 423 723 946 2092
6 6 6 6
17a 28a 66a 38a
Restricted‡ 152 236 303 691
6 6 6 6
16c 21c 23c 42c
Amniotic fluid Realimented§ 267 517 647 1431
6 6 6 6
17b 20b 31b 38b
Control† 396 589 2787 3773
6 6 6 6
18a 21a 76a 84a
Restricted‡ 131 164 821 1116
6 6 6 6
10c 12c 43c 52c
Realimented§ 273 383 1697 2353
6 6 6 6
11b 25b 93b 78b
* Data are means 6 SEM. † Ewes were fed 100% of NRC nutrient requirements between Days 28 and 135 of gestation (n 5 8). ‡ Ewes were fed 50% of NRC nutrient requirements between Days 28 and 135 of gestation (n 5 5). § Ewes were fed 50% of NRC nutrient requirements between Days 28 and 78 of gestation and realimented to 100% of NRC nutrient requirements between Days 78 and 135 of gestation (n 5 8). a,b,c Means with different superscripts are different (P , 0.05).
polyamines in maternal and fetal plasma and in fetal fluids in association with compensatory fetal growth. The marked decrease in all essential amino acids in maternal plasma (except for histidine at Day 78 of gestation) at both mid and late gestation in response to maternal nutrient restriction is in contrast to the reported elevation in concentrations of most essential amino acids in maternal plasma in women with IUGR of unknown etiology [28]. Concentration of urea (a major nitrogenous product of protein catabolism [30]) is reduced in maternal and fetal plasma of nutrient-restricted ewes [21], suggesting a decrease in amino acid catabolism by the whole body and/or possibly enhanced recycling of urea-nitrogen into the rumen to support microbial protein synthesis. Despite this mechanism of nitrogen conservation, the mobilization of maternal protein reserve (mainly from skeletal muscle) in ewes fed 50% of NRC requirements during the first half of gestation and throughout gestation did not appear to sufficiently compensate for the decreased supply of metabolizable protein from diet. Our finding that concentrations of total a-amino acids decreased to similar extents in both maternal and fetal plasma of underfed ewes supports the view that placental transport is a major mechanism responsible for fetal amino acid homeostasis [31–33]. Each amino acid has its own unique and tissue-specific metabolic pathways [9, 12]. Consistent with this notion, maternal undernutrition differentially affected concentrations of amino acids in maternal and fetal plasma. For example, at Day 78 of gestation, the concentration of glycine in both maternal and fetal plasma of nutrient-restricted ewes increased, but concentrations of most amino acids decreased compared with control ewes. Despite the elevated fetal glycine levels, serine concentration was markedly reduced in fetal plasma and fluids by maternal nutrient restriction. The uterus derives serine from maternal plasma, but there is little transplacental transport of serine to the ovine fetus owing to its extensive catabolism by uteroplacental tissues [16, 34]. Thus, in fetal lambs large amounts of serine are synthesized from glycine and N5,N10-methylenetetrahydrofolate as well as from 3-phosphoglycerate (an intermediate of glycolysis) and glutamate [16, 35]. Reduction in serine synthesis as a result of maternal nutrient restriction may indicate a decrease in liver and/or kidney function, which would have long-term metabolic and cardiovascular consequences later in life [36]. Serine is a major glucogenic amino acid in humans [9] and ewes [37]. It also plays an important role in one-carbon unit metabolism essential for 29-deoxythymidylate synthesis and methylation of biomolecules [38]. In addition, serine participates in the synthesis of phosphatidylserine and ceramide (signaling molecules) [9]. All of these events are critical for cell me-
tabolism and function. Therefore, reduced availability of serine in the conceptus of underfed ewes may impair the synthesis of glucose, DNA, and protein, thereby contributing to IUGR. Another interesting finding of this study is that concentrations of branched-chain amino acids and arginine-family amino acids were consistently decreased in all of the measured compartments of underfed ewes at mid and late gestation. Likewise, IUGR in humans is associated with reduced fetal plasma concentrations of branched-chain amino acids [25–28] and arginine [33]. A reduced availability of branched-chain amino acids would impair placental and fetal glutamine synthesis [39]. In support of this view, glutamine concentrations were markedly reduced in ovine fetal plasma at both mid and late gestation. This may have an important impact on fetal growth, because glutamine is a major fuel for the growing fetus, a primary transporter of carbon and nitrogen among fetal organs, and an essential substrate for DNA and aminosugar syntheses [10, 32]. In addition, decreased arginine availability would reduce endothelial nitric oxide synthesis [40], and therefore placental-fetal blood flows, which in turn would decrease the transfer of nutrients and oxygen from maternal to fetal blood. Amniotic fluid is essential for fetal growth and development [41]. Interestingly, we have shown that glutamine and polyamines are abundant in ovine amniotic fluid [18, 19]. The swallowing of amniotic fluid provides a source of rich nutrients for utilization by the fetal intestine and other tissues [42], and therefore the prevention of amniotic fluid entry into the small intestine by esophageal ligation results in IUGR in sheep [43]. Amniotic fluid is derived from both the fetus (kidneys, lungs, and epidermis) and fetal placental blood vessels [41]. Thus, a fall in concentrations of glutamine and polyamines in maternal and fetal plasma resulted in a decrease in their availability in fetal amniotic fluid. The latter would contribute to retarded growth of the small intestine and other tissues in fetuses of nutrient-restricted ewes [21]. Available evidence shows that increasing maternal plasma levels of amino acids can increase their availability in fetal plasma and fluids [32]. Remarkably, realimentation of nutrient-restricted ewes increased serine concentrations in allantoic fluid four-fold compared with restricted ewes, suggesting an increase in intrafetal synthesis of serine. However, in all of the measured compartments, concentrations of total a-amino acids and polyamines remained lower in realimented ewes compared with control ewes. There are several possible explanations for these findings. First, utilization of dietary amino acids for tissue protein synthesis in realimented ewes increases during compensatory growth
NUTRIENT RESTRICTION AND AMINO ACIDS IN PREGNANCY
of both the mother and the fetus. Second, because nutrientrestricted ewes exhibit compensatory growth of placentomes in response to realimentation during late gestation [44], an increased amount of amino acids would be directed toward placentomal protein synthesis, thereby reducing their transfer from maternal to fetal blood. Third, arginine, ornithine, and methionine, as well as proline and glutamine (precursors of ornithine), are important substrates for the synthesis of putrescine, spermidine, and spermine [11], and therefore the reduced availability of these amino acids in fetal plasma and fluids would contribute to a decrease in polyamine synthesis by placental, uterine, and fetal tissues [19]. Whatever the mechanisms, our results indicate that realimentation of nutrient-restricted ewes increased the availability of amino acids (substrates for the synthesis of protein and other biologically important molecules) and polyamines (key regulators of DNA and protein synthesis as well as cell function) in the conceptus, which prevented IUGR brought about by maternal undernutrition. In conclusion, concentrations of total a-amino acids (particularly serine, arginine-family amino acids, and branched-chain amino acids) and polyamines were severely reduced in maternal plasma, fetal plasma, and fetal allantoic and amniotic fluids of nutrient-restricted ewes at both mid and late gestation. Realimentation of underfed ewes beginning from mid-gestation increased concentrations of total a-amino acids and polyamines in all of the measured compartments and prevented IUGR. These findings establish a foundation for further studies to define the roles of amino acids and polyamines in the prevention and treatment of IUGR brought about by maternal nutrient restriction. ACKNOWLEDGMENT We thank research personnel in our laboratories for technical assistance.
REFERENCES 1. Widdowson EM. Prenatal nutrition. Ann NY Acad Sci 1977; 300: 188–196. 2. Mellor DJ. Nutritional and placental determinants of fetal growth rate in sheep and consequences for the newborn lamb. Br Vet J 1983; 139: 307–324. 3. Osgerby JC, Wathes DC, Howard D, Gadd TS. The effect of maternal undernutrition on ovine fetal growth. J Endocrinol 2002; 173:131– 141. 4. Wu G, Pond WG, Ott T, Bazer FW. Maternal dietary protein deficiency decreases amino acid concentrations in fetal plasma and allantoic fluid of pigs. J Nutr 1998; 128:894–902. 5. Snell LH, Haughey BP, Buck G, Marecki MA. Metabolic crisis: hyperemesis gravidarum. J Perinat Neonat Nurs 1998; 12:26–37. 6. Barker DJP, Clark PM. Fetal undernutrition and disease in later life. Rev Reprod 1997; 2:105–112. 7. Thomas VM, Kott RW. A review of Montana winter range ewe nutrition research. Sheep Goat Res J 1995; 11:17–24. 8. Chaturvedi OH, Bhatta R, Santra A, Mishra AS, Mann JS. Effect of supplemental feeding of concentrate on nutrient utilization and production performance of ewes grazing on community rangeland during late gestation and early lactation. Asian Aust J Anim Sci 2003; 16: 983–987. 9. Stipanuk MH, Watford M. Amino acid metabolism. In: Stipanuk MH (ed.), Biochemical and Physiological Aspects of Human Nutrition. Philadelphia: W.B. Saunders; 2000:233–286. 10. Flynn NE, Meininger CJ, Haynes TE, Wu G. The metabolic basis of arginine nutrition and pharmacotherapy. Biomed Pharmacother 2002; 56:427–438. 11. Wu G, Morris SM Jr. Arginine metabolism: nitric oxide and beyond. Biochem J 1998; 336:1–17. 12. Wu G, Self JT. Amino acids: metabolism and functions. In: Pond WG, Bell AW (eds.), Encyclopedia of Animal Science. New York: Marcel Dekker. DOI: 10.1081/E-EAS 120019779.
907
13. Reynolds LP, Redmer DA. Angiogenesis in the placenta. Biol Reprod 2001; 64:1033–1040. 14. Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol Regul Integr Comp Physiol 1997; 272:R441–R463. 15. Bird IM, Zhang LB, Magness RR. Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function. Am J Physiol Regul Integr Comp Physiol 2003; 284:R245–R258. 16. Moores RR Jr, Carter BS, Meschia G, Fennessey PV, Battaglia FC. Placental and fetal serine fluxes at midgestation in the fetal lamb. Am J Physiol Endocrinol Metab 1994; 267:E150–E155. 17. Wallace JM. Nutrient partitioning during pregnancy: adverse gestational outcome in overnourished adolescent dams. Proc Nutr Soc 2000; 59:107–117. 18. Kwon H, Spencer TE, Bazer FW, Wu G. Developmental changes of amino acids in ovine fetal fluids. Biol Reprod 2003; 68:1813–1820. 19. Kwon H, Wu G, Bazer FW, Spencer TE. Developmental changes in polyamine levels and synthesis in the ovine conceptus. Biol Reprod 2003; 69:1626–1634. 20. Kwon H, Wu G, Meininger CJ, Bazer FW, Spencer TE. Developmental changes in nitric oxide synthesis in the ovine placenta. Biol Reprod 2004; 70:679–686. 21. Vonnahme KA, Hess BW, Hansen TR, McCormick RJ, Rule DC, Moss GE, Murdoch WJ, Nijland MJ, Skinner DC, Nathanielsz PW, Ford SP. Maternal undernutrition from early to mid gestation leads to growth retardation, cardiac ventricular hypertrophy and increased liver weight in the fetal sheep. Biol Reprod 2003; 69:133–140. 22. Wu G, Davis PK, Flynn NE, Knabe DA, Davidson JT. Endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in postweaning growing pigs. J Nutr 1997; 127:2342– 2349. 23. Wu G, Pond WG, Flynn SP, Ott TL, Bazer FW. Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation. J Nutr 1998; 128:2395–2402. 24. Wu G, Flynn NE, Knabe DA. Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets. Am J Physiol Endocrinol Metab 2000; 279:E395–E402. 25. Cetin I, Corbetta C, Sereni LP, Marconi AM, Bozzetti P, Pardi G, Battaglia FC. Umbilical amino acid concentrations in normal and growth-retarded fetuses sampled in utero by cordocentesis. Am J Obstet Gynecol 1990; 162:253–261. 26. Cetin I, Marconi AM, Bozzetti P, Sereni LP, Corbetta C, Pardi G, Battaglia FC. Umbilical amino acid concentrations in appropriate and small for gestational age infants: a biochemical difference present in utero. Am J Obstet Gynecol 1988; 158:120–126. 27. Cetin I, Marconi AM, Corbetta C, Lanfranchi A, Baggiani AM, Battaglia FC, Pardi G. Fetal amino acids in normal pregnancies and in pregnancies complicated by intrauterine growth retardation. Early Hum Dev 1992; 29:183–186. 28. Cetin I, Ronzoni S, Marconi AM, Pergino G, Corbetta C, Battaglia FC, Pardi G. Maternal concentrations and fetal-maternal concentration differences of plasma amino acids in normal and intrauterine growthrestricted pregnancies. Am J Obstet Gynecol 1996; 174:1575–1583. 29. Economides DL, Nicolaides KH, Gahl W, Bernadini I, Evans MI. Plasma amino acids in appropriate and small-for-gestational age infants. Am J Obstet Gynecol 1989; 161:1219–1227. 30. Scholljegerdes EJ, Ludden PA, Hess BW. Site and extent of digestion and amino acid flow to the small intestine in beef cattle consuming limited amounts of forage. J Anim Sci 2004; 82:1146–1156. 31. Ford SP. Control of blood flow to the gravid uterus of domestic livestock species. J Anim Sci 1995; 73:1852–1860. 32. Bell AW, Ehrhardt RA. Regulation of placental nutrient transport and implications for fetal growth. Nutr Res Rev 2002; 15:211–230. 33. Bajoria R, Sooranna SR, Ward S, D’Souza S, Hancock M. Placental transport rather than maternal concentration of amino acids regulate fetal growth in monochorionic twins: implications for fetal origin hypothesis. Am J Obstet Gynecol 2001; 185:1239–1246. 34. Narkewicz MR, Jones G, Thompson H, Kolhouse F, Fennessey PV. Folate cofactors regulate serine metabolism in fetal ovine hepatocytes. Pediatr Res 2002; 52:589–594. 35. Narkewicz MR, Moores RR Jr, Battaglia FC, Frerman FF. Ontogeny of serine hydroxymethyltransferase isoenzymes in fetal sheep liver, kidney, and placenta. Mol Genet Metab 1999; 68:473–480. 36. McMillen IC, Adams MB, Ross JT, Coulter CL, Simonetta G, Owens JA, Robinson JS, Edwards LJ. Fetal growth restriction: adaptations and consequences. Reproduction 2001; 122:195–204. 37. Clark MG, Filsell OH, Jarrett IG. Gluconeogenesis in isolated intact
908
38. 39. 40. 41.
KWON ET AL.
lamb liver cells. Effects of glucagons and butyrate. Biochem J 1976; 156:671–680. Snell K, Fell DA. Metabolic control analysis of mammalian serine metabolism. Adv Enzyme Regul 1990; 30:13–32. Self JT, Spencer TE, Johnson GA, Hu J, Bazer FW, Wu G. Glutamine synthesis in the developing porcine placenta. Biol Reprod 2004; 70: 1444–1451. Wu G, Meininger CJ. Regulation of nitric oxide synthesis by dietary factors. Annu Rev Nutr 2002; 22:61–86. Ross MG, Nijland MJM. Development of ingestive behavior. Am J Physiol Regul Integr Comp Physiol 1998; 274:R879–R893.
42. Sagawa N, Nishimura T, Ogawa M, Inouge A. Electrogenic absorption of sugars and amino acids in the small intestine of human fetus. Membr Biochem 1979; 2:393–404. 43. Trahair JF, Harding R. Restitution of swallowing in the fetal sheep restores intestinal growth after midgestation esophageal obstruction. J Pediatr Gastroenterol Nutr 1995; 20:156–161. 44. Gardner DS, Ward JW, Giussani DA, Fowden AL. The effect of a reversible period of adverse intrauterine condition during late gestation on fetal and placental weight and placentome distribution in sheep. Placenta 2002; 23:459–466.
