methionine, homocysteine, one carbon metabolism and fetal growth

Methionine, homocysteine, one carbon metabolismand fetal growth

Satish C. Kalhan & Susan E. Marczewski

Published online: 15 March 2012# Springer Science+Business Media, LLC 2012

Abstract Methionine and folate are the key components ofone carbon metabolism, providing the methyl groups fornumerous methyl transferase reactions via the ubiquitousmethyl donor, s-adenosyl methionine.Methioninemetabolismis responsive to nutrient intake, is regulated by several hor-mones and requires a number of vitamins (B12, pyridoxine,riboflavin) as co-factors. The critical relationship betweenperturbations in the mother’s methionine metabolism and itsimpact on fetal growth and development is now becomingevident. The relation of folate intake to fetal teratogenesis hasbeen known for some time. Studies in human pregnancyshow a continuous decrease in plasma homocysteine, and anincrease in plasma choline concentrations with advancinggestation. A higher rate of transsulfuration of methionine inearly gestation and of transmethylation in the 3rd trimesterwas seen in healthy pregnant women. How these processes areimpacted by nutritional, hormonal and other influencesin human pregnancy and their effect on fetal growth hasnot been examined. Isocaloric protein restriction in pregnantrats, resulted in fetal growth restriction and metabolicreprogramming. Isocaloric protein restriction in the non-

pregnant rat, resulted in differential expression of a numberof genes in the liver, a 50% increase in whole body serinebiosynthesis and high rate of transmethylation, suggestinghigh methylation demands. These responses were associatedwith a significant decrease in intracellular taurine levels inthe liver suggesting a role of cellular osmolarity in theobserved metabolic responses. These unique changes inmethionine and one carbon metabolism in response to phys-iological, nutritional and hormonal influences make theseprocesses critical for cellular and organ function and growth.

Keywords Methionine . Homocysteine . Fetus . Growth .

One-carbon . Folate

1 Introduction

Fetal growth, and its regulation in humans and animal models,has been related to the nutritional and hormonal interactionsbetween the mother, the placenta and the fetus. Of specialimportance is the delivery of micro- and macronutrients fromthe mother to the fetus, control of these processes by specifictransporters, growth factors and by the fetal and maternalendocrine milieu. A large body of data exists examining theinfluence of maternal nutrient status, or the impact of nutrientrestriction and supplement on maternal and fetal metabolic andendocrine responses and on fetal growth (reviewed in [1–3]).The role of specific nutrients, e.g. glucose (and other sub-strates) in diabetes, and placental insufficiency in pregnancyrelated disorders, on fetal growth and the birth weight of theneonate has been reported [4–8]. Most of these studies haveexamined the direct contribution of the nutrient, such as glu-cose or amino acids, to fetal mass. However, some aminoacids, for example methionine, not only contribute to the fetal(protein) mass, but also, because of their unique role in

S. C. KalhanDepartment of Molecular Medicine, Cleveland Clinic LernerCollege of Medicine of Case Western Reserve University,9500 Euclid Avenue,Cleveland, OH 44195, USA

S. C. Kalhan (*) : S. E. MarczewskiDepartment of Pathobiology, NE-40,Lerner Research Institute, Cleveland Clinic,9500 Euclid Avenue,Cleveland, OH 44195, USAe-mail: [email protected]

S. E. Marczewskie-mail: [email protected]

Rev Endocr Metab Disord (2012) 13:109–119DOI 10.1007/s11154-012-9215-7

metabolism, may impact fetal growth. The metabolism ofmethionine, along with folate, (one carbon pool) influences alarge number of processes that directly or indirectly effect cellproliferation and gene expression, particularly in the growingorganism.Methionine may also impact nutrient transport by itseffect (via homocysteine) on the uteroplacental vasculature. Inthis review, we will discuss the metabolism of methionine inthe human mother and newborn, and the potential effects ofnutrient modifications on methionine metabolism. Recent datafrom studies in human pregnancy show unique changes in themetabolism of methionine with advancing gestation and arapid increase in the transsulfuration of methionine in theneonate soon after birth. In addition, studies in animals showthat adaptive responses in the metabolism of methionine andone carbon pool occur rapidly in response to changes in dietaryprotein content. These data underscore the sensitivity of onecarbon metabolism to physiological and pathological influen-ces that may impact the growing organism. The possiblemechanisms of alterations in methionine and homocysteinemetabolism in the mother that may influence fetal growth arediscussed.

2 Methionione metabolism

2.1 Metabolism of methionine in-vivo

Methionine, an essential or indispensable amino acid and acomponent of all proteins, is also the immediate source of themethyl (one carbon) groups required for the methylation ofnucleic acids, protein, biogenic amines, and phospholipids etc.[9]. The metabolism of methionine or members of the “acti-vated”methionine cycle is closely entwined with that of folateand the two together participate in the transfer of methyl

groups of serine and glycine for various methyltransferasereactions (Fig. 1). Since the methionine and folate cycles areubiquitously present in every cell in the body and participatein key metabolic reactions, in DNA synthesis and by methyl-ation of DNA in gene expression, perturbation in its metabo-lism either by nutrient deficiency, or by nutrient, hormonaland environmental interactions can have profound impact onthe cell function, metabolism, growth and proliferation. Thismay have its greatest impact on the growing embryo and thefetus. Previous studies in human and animals have largelyfocused on the effects of folate deficiency on growing embryoprimarily for its terratogenic (neural tube defect) effects andon the effects of homocysteine on maternal and placentalvascular biology. Recent data, however, show that changesin nutrient intake, particularly dietary protein restriction, canhave major impact on one-carbon transfers. These changesmay lead to altered methylation of DNA and acetylation ofhistones and to epigenetic changes in the growing embryo,which may affect fetal growth and cause long term morbidityin the offspring.

The metabolism of methionine in-vivo, and its regulation,in humans and in animal models, has been reviewed exten-sively [10, 11]. As shown in Fig. 1, the metabolism of methi-onine consists of two components: 1) the ubiquitousmethionine cycle, variously called the transmethylation cycleor the “activated” methionine cycle, and 2) the transsulfura-tion pathway. The methionine transmethylation cycle involvesthe transfer of the methyl group from the folate dependentone-carbon pool or from (non-vitamin dependent) betaine, tovarious methyl acceptors and is catalyzed by several methyl-transferase reactions. Serine and glycine are the primary con-tributors of methyl groups to the one carbon pool [12, 13]. Theintermediates in the methionine cycle are s-adenosyl methio-nine (SAM), the universal bioactive methyl donor, s-adenosyl

Methionine

SAM

SAH

Homocystine

MS(B12)

THF

5CH3THF

5, 10-CH2THF

Purines

Serine

Glycine

Pyrimidines

Methyltransferase

X-CH3

ProteinsLipidsDNA – Epigenetics

Protein modificationNF-κB – inflammationEndothelial damageThrombosis

CβS(B6)

Serine

Cystathionine

CγL

Cysteine

Taurine

Cellular Osmolarity

GSH

ROS

X

Betaine

BHMT

DMG Glycine

Sarcosine

GNMT

Fig. 1 Methionine and folatecycles in-vivo and potentialmechanism of impaired fetalgrowth (bold italics). Discussedin detail in text. THF:tetrahydro folate, MS:methionine synthase, BHMT:betaine homocysteine methyltransferase, SAM:s-adenosylmethionine, SAH:s-adenosylhomocysteine,GNMT: glycine n methyltransferase, CβS: cystathioninebeta synthase, CγL:cystathionine gamma lyase,GSH: glutathione, ROS:reactive oxygen species

110 Rev Endocr Metab Disord (2012) 13:109–119

homocysteine (SAH) and homocysteine. Vitamin B-12 is acofactor for methionine synthase, the enzyme responsible forthe folate dependent methylation of homocysteine. Methio-nine is catabolized via the transsulfuration pathway, presentonly in liver, pancreas, intestine, kidney and possibly in thebrain. This pathway involves the condensation of homocys-teine with serine to form cystathionine, catalyzed by cysta-thionine ß-synthase (CßS). Cystathionine is then converted tocysteine, α-ketobutyrate and ammonia by cystathionine γ-lyase (CɣL). The carbon skeleton of methionine enters theTCA cycle as propionyl CoA formed by the decarboxylationof α-ketobutyrate while the sulfylhydryl group condenses withserine to form cysteine. Cysteine is the precursor for taurine,as well as a component of the tripeptide, glutathione, the majorintracellular antioxidant. Methionine metabolism is regulatedby the nutrient and hormonal status of the organism. It issignificant to note that three vitamins, folate, B-12 and pyri-doxine are directly involved in the metabolism of methionine.In addition, insulin and glucagon exert their effect on thisprocess directly by regulating the transsulfuration cascade orthe remethylation of homocysteine via methionine synthaseand indirectly by their effect on whole body protein turnover[14–18].

2.2 Methionine metabolism in human pregnancy

Few studies have examined the changes in methionine andone carbon metabolism in human pregnancy. The ones thathave, are primarily limited to measurements of plasma con-centration of methionine, homocysteine and methyl donorssuch as choline and betaine in healthy pregnant women andthose with complications of pregnancy [19–25]. These datashow that the concentration of homocysteine in plasma is

lower in healthy pregnant women, compared with the non-pregnant women, and that there is a linear decrease inconcentration of homocysteine with advancing gestation.[20, 21]. Interestingly, a gestation related decrease in theplasma B12 concentration was also observed, while there wasno significant change in the levels of folate in the plasma [21].Since homocysteine in the plasma is bound to proteins, specif-ically albumin, the decrease in the total homocysteine concen-tration parallels the decrease in plasma albumin concentrationwith advancing gestation. Molloy et al. [23] have shown thatthe level of folate in the plasma of the mother is the primedeterminant of the plasma homocysteine concentration.Homocysteine is transported across the placenta by the neutralamino acid transporters, primarily by the system L transporteragainst a concentration gradient. Studies using in-vitro systemsshow that homocysteine competes with endogenous aminoacids for the transporter activity [26–28].

Our group has quantified the kinetics of methionine, its rateof appearance, transmethylation and transsulfuration in healthywomen with advancing gestation, using various stable isotopictracers of methionine and by GC-mass spectrometric methods[29]. As shown in Table 1, in association with the decrease intotal α-amino nitrogen concentration, the concentration ofmethionine in the plasma was lower in pregnant women ascompared with that in the non-pregnant women. The rate ofappearance (Ra) of methionine was not significantly differentamongst the pregnant and non-pregnant subjects. However, aswas seen for other essential amino acids, (i.e. phenylalanine)there was a significant positive relationship between the rate ofappearance of methionine and gestational age (r²00.15, P00.02), suggesting a gestation related increase in whole bodyrate of protein turnover. Of particular interest was a higher rateof transsulfuration of methionine in the first trimester and a

Table 1 Methionine kinetics during fasting in human pregnancy

Non-pregnant 1st trimester 2nd trimester 3rd trimester p

Plasma methionine 31.00±1.95 24.25±4.05 25.20±3.37 23.10±3.92 0.001

Ra (m1 tracer) 21.12±4.90 18.25±2.73 20.53±4.36 22.66±4.96 NS(8) (10) (5) (10)

Transsulfuration 2.16±0.61 3.45±0.76 2.92±0.49 2.56±0.91 0.004(8) (10) (4) (9)

Remethylation 6.16±6.82 5.68±6.09 b 8.06±9.92 17.04±8.65d a 0.021(7) (8) (4) (10)

Transmethylation 8.18±6.84 9.04±5.70 b 10.98±9.44 19.21±9.58 a 0.067(7) (8) (4) (9)

Ra Rate of appearance; () 0 n. All data are mean ± SD; units are, plasma methionine micromoles.l¯¹, all others micromoles.kg−1 .h−1

p: Kruskal-Wallis 1-way ANOVA

m1 tracer: [1-13 C] methionine

a p<0.05 compared with non-pregnant subjects by Mann–Whitney U Testb p<0.05 with third trimester subjects by Mann–Whitney U Test

Data from reference [29]

Rev Endocr Metab Disord (2012) 13:109–119 111

higher rate of transmethylation in the third trimester of preg-nancy. The physiological significance of these changes is notclear. A high rate of transsulfuration would provide for a highrate of synthesis of cysteine, taurine and glutathione, while ahigh rate of transmethylation would meet the methylationdemands [30]. However, the organ systems in the mother orin the growing conceptus that place such unique demands fortranssulfuration and transmethylation at specific periods ingestation have not been identified. In this context, the hepatictranssulfuration pathway is not active in the human fetusbecause cystathionine γ-lyase is absent in fetal liver and a highrate of transsulfuration may in part be related to fetal demandsfor cysteine [31, 32]. It also should be stressed that transsulfu-ration is the catabolic pathway of methionine, and an increasein transsulfuration will result in an obligatory requirement fordietary methionine in early pregnancy. This is consistent withthe high rate of neural tube defects in women with lowerdietary intake of methionine [33, 34] and the demonstratedfetal growth retardation in animal models of restricted methi-onine intake during pregnancy [35].

2.3 Protein energy intake and methionine metabolism

The effect of change in the quantity of dietary protein onmethionine metabolism continues to be of interest becauseof methionine’s role as the source of methyl groups viaSAM and for its possible contribution to plasma/systemichomocysteine concentration, the former being responsiblefor numerous methylation reactions involved in biologicalprocesses such as the synthesis of creatine, catecholamine,phosphotidyl ethanolamine (required for VLDL export bythe liver) and methylation of DNA and consequent regula-tion of gene expression and epigenetics. Plasma homocys-teine concentrations have been associated with vascular andthrombophilic morbidities and cardiovascular disease [36].

High protein and methionine (20% of energy) intake inoverweight subjects did not affect the plasma concentrationof homocysteine during fasting when compared with thelower protein (15% of energy) group [37]. A small increasein postprandial and not fasting plasma homocysteine levelswas observed when healthy subjects were placed on a highprotein diet for 8 days [38]. These data are not surprising,since in healthy individuals, who are in nitrogen balance, themetabolic fate of excess protein is transamination of the aminogroups and the disposal of carbon via oxidation in the citricacid cycle or its conversion to glucose. Since transsulfurationis the catabolic pathway for methionine, it does not participatein significant transamination, excess methionine from the dietwill result in high rate of transsulfuration and higher rate ofdisposal of homocysteine generated by the elevated methio-nine load [39]. Such was the case in newborn babies whoreceived methionine that was greater than their requirementfor protein synthesis, in the parenteral nutrient amino acid

mixture[39]. Data from studies with rats have shown that highprotein intake is associated with increased expression andactivity of cystathionine ß-synthase in the liver, consistentwith the increased need for the disposal of the excess methi-onine [40]. Thus, in healthy individuals, the excess proteinload is rapidly disposed of and the plasma concentration ofmethionine and homocysteine is maintained in a narrow rangeby the balance between the transsulfuration and transmethy-lation pathways of homocysteine and the overall homeostasisof protein metabolism [41]. Only in disease states when thetranssulfuration pathway is impaired, such as in patients withcystathionine ß-synthase deficiency (homocysteinuria), orwhen remethylation of homocysteine is impaired due todefects in folate metabolism or a folate or B-12 deficiency, ahigh protein diet is likely to cause hyperhomocysteinemia andrelated consequences.

In contrast, protein restriction or protein malnutrition, inthe presence of isocaloric energy intake, results in a uniquemetabolic pattern in both humans and animals. It is importantto underscore that isocaloric energy intake prevents an in-crease in protein breakdown and adipose tissue lipolysis as-sociated with starvation or energy restriction [42]. Althoughdata from studies with humans are confounded by a host ofother factors, some important inferences can be drawn. In anespecially intriguing study, Ingenbleek and colleagues [43,44] showed that subclinical protein malnutrition was associ-ated with hyperhomocysteinemia. Using serum levels oftransthyretin as a marker of protein malnutrition, they ob-served that homocysteine levels correlated negatively withtransthyretin levels. Of significance, plasmamethionine levelsdid not change with the worsening protein nutrition statuswhile the levels of other essential amino acids decreasedprogressively. This suggests an acquired defect of transsulfu-ration pathway aimed at preserving methionine homeostasis.A marked decrease in the concentration of both essential andnon-essential amino acids was observed in this study and in anumber of other studies of protein malnutrition in infants andadults [45–47]. Alterations in the patterns of change of indi-vidual amino acids have been observed with progressiveprotein malnutrition, although these data are difficult to inter-pret due to the associated stress, infection etc. A recent studyby our group using rats demonstrated that isocaloric proteinrestriction for 7 to 10 days, was associated with significantdifferential expression of a number of genes involved in cellcycle, cell differentiation, transcription, transport, and othermetabolic processes in the liver [48].

Genes that are involved in the biosynthesis of serine andfatty acid oxidation were markedly increased while thoseinvolved in urea synthesis and fatty acid synthesis were mark-edly down-regulated in the livers of protein-restricted rats.The plasma and liver free amino acid concentration followingisocaloric protein restriction are shown in Table 2. The plasmaconcentrations of serine and glycine were markedly increased


in the protein-restricted animals, but there were no significantchange in the levels of taurine and methionine. The concen-tration of free glycine and serine in the liver was increased inthe protein-restricted animals, while that of taurine was mark-edly lower, suggesting a rapid efflux of taurine. Tracer isotopestudies showed that the rate of appearance of serine wassignificantly increased as a result of isocaloric protein restric-tion. (Table 3). In addition, the rate of transmethylation ofmethionine, as measured by the rate of appearance of methi-onine using methyl labeled tracer, and the methylation poten-tial (SAM/SAH ratio) was significantly increased. As shownby others [40], the activity of the enzymes in the transsulfura-tion cascade was decreased, however, the rate of transsulfura-tion measured using isotopic tracers was not affected byisocaloric protein restriction. Thus isocaloric protein restric-tion causes profound changes in one-carbon transfer in theliver within a short period. The adaptive responses to pro-longed protein restriction in non-pregnant animals and during

pregnancy remain to be examined. We speculated [48] thatthese changes in one-carbon metabolism are related to thehigh methylation demands [49] placed on the organism andmediated possibly by change in the cellular osmolarity asevidenced by the efflux of the intracellular taurine.

These studies show unique adaptation in methioninemetabolism during pregnancy in healthy women and neo-nates. In addition, characteristic responses in one carbonmetabolism to isocaloric protein restriction are evident innon-pregnant state in humans and animals. The adaptiveresponses in methionine metabolism during pregnancycould potentially be perturbed by modest nutrient inadequa-cies that are insufficient to cause classical deficiency syn-drome and could impact maternal adaptation to pregnancy,fetal growth and development.

3 One-carbon metabolism and fetal growth

The potential mechanism/s by which alterations in folate andmethionine cycles may impact fetal growth are identified inthe Fig. 1. Micronutrient (folate, B12, B6) deficiencies orinsufficiency could influence fetal growth by their effect onfolate/methionine metabolism in the mother and the fetus.Alterations in folate metabolism either due to dietary insuffi-ciency or due to gene polymorphism could impact purine andpyrimidine metabolism, DNA synthesis and/or cell prolifera-tion; and affect fetal growth and result in teratogenesis andcongenital malformations.

The vascular endothelial damage associated with elevatedhomocysteine levels has been discussed extensively in litera-ture and its effect on pregnancy related disorders has beenreported. Thus, perturbations in the one-carbon metabolisminduced by marginally low protein intake and micronutrientinsufficiency rather than classical deficiency may be the

Table 2 Free amino acid in plasma and liver of protein restricted rats

Plasma (milliemoles/L) Liver (millimoles/kg)

Serine Glycine Taurine Methionine Serine Glycine Taurine Methionine

LP 0.53 * 1.00 § 0.083 0.05 2.37 ** 4.44 • 2.02 † 0.08

(0.05) (0.076) (0.023) (0.003) (0.38) (0.48) (0.43) (0.006)

NP 0.23 0.87 0.083 0.05 0.72 2.93 6.43 0.09

(0.03) (0.07) (0.01) (0.004) (0.11) (0.33) (0.375) (0.015)

All data are mean ± (SEM); NP normal protein; LP low protein

* p00.001

§ p00.007

** p<0.002

• p<0.01

† p0<0.002


Table 3 Effect of dietary protein restriction on one-carbon metabolismin non-pregnant rat

Serine Ra Methionine Ra TS SAM/SAH Cysteineμmoles/100 g.h

μmoles/100 g.h

μmoles/100 g.h

μmoles/g.tissueweight

LP 96.1 8.5 6.9 5.2 1.66

(15.1) (1.8) (1.6) (0.25) (0.19)

NP 60.1 6.3 4.8 3.5 2.15

(11.9) (1.5) (0.8) (0.40) (0.08)

P00.066 P00.03 ns P00.015 P00.004

Ra Rate of appearance; () 0 n. All data are mean ± (SEM)

NP normal protein; LP low protein; TS transsulfuration

SAM s-adenosyl methionine; SAH s adenosyl homocysteine



primary contributor to impaired fetal growth and associatedlong term consequences in the offspring.

3.1 Role of homocysteine

Homocysteine, a sulfur containing amino acid is the deme-thylated product of methionine and an intermediate in theactivated methionine cycle. It does not participate in proteinsynthesis. Within the cell, it can either be methylated to formmethionine, irreversibly enter the transsulfuration pathway,or be released into the extracellular compartment (Fig. 1).Thus the changes in plasma concentration of homocysteineare related to alterations in its cellular metabolism leading toits accumulation in the cell and consequently its release.Extremely high plasma concentrations of homocysteine areseen in patients with inborn errors of transsulfuration, i.e. acystathionine ß-synthase defect, causing homocysteineuria.In addition, a change in the rate of methylation of homo-cysteine as a result of folate or B12 deficiency or due tomethylenetetrahydrofalate reductase polymorphism result-ing in a decrease in enzyme activity, may also cause mod-erate increase in plasma levels of homocysteine [50, 51].Homocysteine in the plasma is bound to proteins, mostlyalbumin. Therefore, a decrease in the plasma albumin con-centration, such as in pregnancy, would be associated with alower total homocysteine concentration.

The relationship between plasma homocysteine concen-trations during pregnancy and fetal growth remains unclear.Whether elevated homocysteine levels are simply a biomarkerof altered metabolism or a cause of pregnancy related compli-cations, or of perturbations in fetal growth, remains to beestablished experimentally. Based on the data in literaturethe following can be inferred: [1] Elevated levels of homo-cysteine during pregnancy have been associated withpregnancy-related disorders such as pre-eclampsia, early preg-nancy losses, abruptio- placenta, venous thrombosis etc. andhave been attributed to the effect of homocysteine on vascularendothelial cell function, increased pro-oxidant activity andthrombo embolic activity [20, 25, 52, 53]. These disorders ofpregnancy could impact fetal growth by their effect on pla-cental growth and function and consequently nutrient trans-port across the placenta. [2] Elevated homocysteine maysimply be the result of micro (folate, B12,pyridoxine) ormacro (protein) nutrient deficiency. Homocysteine is thus abiomarker of the primary change in nutrient state and theimpact on fetal growth may be the consequence of perturba-tions in the maternal and fetal metabolism [53]. [3] Higherhomocysteine levels may also suggest intracellular accumula-tion of SAH, and therefore a decrease in SAM\SAH ratio andlower cellular methylation potential [54, 55]. A change ingenomic methylation caused by change in cellular methyla-tion status could have profound effect on cell growth, prolif-eration, and interfere with other cellular functions. These

changes could potentially impact growth of the developingembryo and the fetus and result in the long term or permanenteffects in the offspring.

3.2 Folate and B12

The relationship between the micronutrient status (folate, B12, pyridoxine) of the mother during pregnancy and thebirth weight of the infant has been examined in a numberof studies from different parts of the world [54–61]. Variousmeasures of dietary intake of the specific nutrient, theirplasma levels or red blood cell levels, or the concentrationof homocysteine in the plasma were quantified at differenttimes during gestation [57–60]. It is important to underscorethat poor micronutrient intake is often associated with adiminished intake of total calories and macronutrients [57].The mandatory fortification of folate in the diet in somecountries, and supplemental use of micronutrients in generalpopulation has complicated dietary estimations. In addition,birth weight is a crude outcome measure of complex pro-cesses and is influenced by not only the nutrient status of themother but also by a number of other factors such as mother’santhropometry, bodymass index, race and ethnicity, the lengthof gestation, sex of the baby, smoking, endocrine factors and ahost of environmental toxins etc. Therefore, an examination ofthe association between the specific micronutrient status of themother and the infant’s birth weight requires statistical adjust-ment for the confounding variables and multiple logistic re-gression analysis. These adjustments can be difficult and maynot be robust enough to elicit the effect size. Even with thesepossible limitations, the data from several studies show apositive correlation between red blood cell or plasma folatelevels and infant birth weight [57–63], and reviewed by Scholland Johnson [56]. In contrast, Nilsen et al. [62] did not findany relation in a well-nourished Norwegian population ofpregnant women between dietary folate, and plasma folateduring the second trimester of pregnancy and infant birth size.A similar lack of relationship between dietary folate intakeand birth weight, even in the presence of low intake, wasreported in studies of a Japanese population [61]. However,they did not measure plasma or RBC levels of folate. Inanother study from Japan, Takimoto et al. [60] did not findany correlation between plasma folate throughout the preg-nancy and infant’s birth weight. In summary, these data showeither a positive or no correlation between plasma or RBCfolate concentration through pregnancy and infants’ birthweight. Because of the established relationship between lowfolate status during the periconceptional period and birthdefects, such as neural tube defects, and because of its rolein synthesis and repair of DNA, and in transfer of methylgroups to homocysteine as methyl donor, a number of studieshave examined the relation between maternal folate status andfetal growth. However, recent evidence suggests that vitamin


B12 may have an important role in fetal growth and its lateconsequences particularly in populations with marginal die-tary B12 intake. Refsum and colleagues [64] observed that47% of the vegetarian Asian Indian subjects had cobalaminedeficiency (total serum cobalamine <150 pmol/L) and about75% showed evidence of cobalamine deficiency in the form ofelevated total homocysteine andmethyl malonic acid levels. Asimilarly high incidence of low cobalamine status was ob-served in the pregnant subjects of the PuneMaternal Nutritionstudy ([65], also see Yajnik in this issue). In addition, theoffspring of the mothers with low vitamin B12 at 18 weeksof gestation had higher HOMA-R at age 6 years [65]. Highermaternal erythrocyte folate at 28 weeks predicted higher off-spring adiposity and an elevated HOMA-R. The offspring ofmothers with a combination of high folate and low vitaminB12 concentrations were the most insulin resistant. These dataare of scientific, as well as of public health importance. Theinteraction between folate and B12 is complex and not fullyunderstood. Similar to the data of Yajnik et al., [65] resultsfrom studies using older adults show that those with low B12status and high plasma folate levels were anemic and hadgreater cognitive impairment [66]. In addition, these subjectshad higher plasma total homocysteine and methyl malonicacid concentrations, suggesting diminished enzymatic func-tion of B12 in the presence of high folate levels. The mecha-nism for this interaction is as yet unclear and has beendiscussed [67–69]. The commonly accepted hypothesis of“folate trap”, i.e. trapping of folate due to lower activity ofmethionine synthase does not fully explain this interaction[70].

3.3 Dietary protein

Observational studies, as well as intervention trials duringpregnancy in humans, have suggested a correlation betweenprotein intake and the size of baby at birth [71–74]. Kramerand Kakuma [75] reviewed the effect of energy and proteinintake on the outcome of pregnancy and suggested that bothhigh and low protein intake were associated with a decreasein birth weight. In 13 trials of 4,665 women, a balancedenergy/protein supplement was associated with a modestincrease in maternal weight gain and in mean birth weight.Themechanism of these effects on the neonatal birth weight oron maternal metabolism was not examined in these studies.This is in contrast to the large body of data in rodents showingthat isocaloric protein restriction of varying duration duringpregnancy causes intrauterine growth restriction, and long-term consequences such as hypertension, type 2 diabetes,pancreatic dysfunction, shortened lifespan, alterations in ami-no acid transport, and alterations in the circadian physiologyin the offspring [76–82]. The detailed phenotype and potentialmechanisms involved in these effects have been discussed inseveral excellent reviews [83–87]. Rees and colleagues [88]

reported that maternal protein restriction resulted in hyper-methylation of DNA in the fetal liver. Subsequent studies haveshown changes in the expression of a number of genes in-volved in metabolism, substrate and nutrient transport and theIGF axis in specific organs, such as placenta, liver, kidney andthe hypothalamus [81, 89–94]. These have the effect of notonly altering the growth and development of the organism, butalso altering its function both immediately and in the longterm (programming). The specific change in maternal metab-olism that leads to these changes in the fetus has not beendelineated. Several groups have examined the maternal met-abolic responses, specifically alterations in the concentrationof amino acids, and the endocrine response to dietary proteinrestriction in pregnant rats and mice. These data are difficult tocompare because of significant differences in the dietary regi-mens, such as the quantity of dietary protein, and timing ofrestriction of protein in relation to early or late pregnancy. It isalso critical to consider whether the animals were pair-fed,since the low protein group could compensate for the reducedprotein content of the diet by increasing overall food intake.Data from a longitudinal study of pair-fed pregnant rats fed a6% casein based diet showed a distinct pattern of circulatingamino acids [95]. The total α-amino nitrogen was unchangedearly in pregnancy in the protein-restricted group, but wasincreased significantly in later gestation. This increase inamino nitrogen in the blood was primarily due to an increasein non-essential amino acids, primarily serine, glycine andglutamine [95]. These changes were associated with a lowerconcentration of plasma urea and an increase in whole bodyenergy consumption. The high rate of energy consumptionmay be related to the possible elevated rate of protein synthe-sis in the liver suggested by the changes in the translationinitiation factors in the liver. A significant increase in theconcentration of glycine in the plasma was also reported byRees et al. [96], who, in addition, observed a significantdecrease in the levels of plasma threonine in response toprotein restriction. The metabolic significance of changes inthreonine is unclear. Petrie and colleagues [97] also observedmarked increase in the concentration of homocysteine inserum early in pregnancy (day 3 and 4) in both mice and ratsfed a protein-restricted diet for 2 weeks prior to conception. Inaddition, protein restriction was associated with lower expres-sion of elongase and desaturase in the maternal liver andhigher levels of both leptin and insulin in the blood [98].The effect of these changes on the maternal, placental andfetal physiology requires further investigations. The changesin plasma glycine, serine and homocysteine suggest a role forperturbations in one carbon transfer as a potential contributorto the fetal growth restriction. This hypothesis is supported bya number of studies where the provision ofmethyl donors, likeglycine and folate, have been shown to prevent epigeneticmodifications of hepatic gene expression, ameliorate theprogramming effects in the fetus of protein-restricted animals


[99–103], and cause epigenetic variation and DNA methyla-tion in the offspring.

4 Conclusions

The mechanism/s involved in the impaired fetal growth andthe long term consequences to the offspring caused by pertur-bation in maternal one carbon metabolism remain to be de-fined. The current data show that dietary protein restriction,and changes in micronutrient status, insufficient to causeclassical deficiency, can have profound impact on one carbonmetabolism. However, the adaptation by the mother to suchnutrient insult has not been studied in detail. It is likely to be acomplex interaction between the direct effects of micronutri-ent status, and its interaction with protein intake, on methio-nine/homocysteine metabolism in the mother and the growingembryo. These will have specific impacts on various biolog-ical functions in the mother, fetus and the placenta. Onlycarefully conducted and detailed studies, using animal modelsand where possible humans, both in vivo and in vitro, willfully define the mechanism involved in the observed alter-ations in one carbon metabolism, fetal growth and long termprogramming.

Acknowledgements Our sincere thanks to all our colleagues whohelped in and did many of the studies cited from the authors’ labora-tory. We apologize to the authors whose work was not cited in themanuscript. This was simply related to the constraint of space and notto the importance of the work. The work from the author’s lab wassupported by grants from the National Institutes of Health HD11089,RR00080, HD042154 and DK079937. We thank Manoa Hui for herhelp in preparing this manuscript. We also thank Dr Richard Hansonfor his critique of the manuscript.

References

1. Murphy VE, Smith R, Giles WB, Clifton VL. Endocrine regula-tion of human fetal growth: the role of the mother, placenta, andfetus. Endocr Rev. 2006;27(2):141–69.

2. Fowden AL, Forhead AJ. Endocrine regulation of feto-placentalgrowth. Horm Res. 2009;72(5):257–65.

3. Godfrey KM. Maternal regulation of fetal development andhealth in adult life. Eur J Obstet Gynecol Reprod Biol. 1998;78(2):141–50.

4. HAPO Study Cooperative Research Group. Hyperglycemia andadverse pregnancy outcome (HAPO) study: associations withneonatal anthropometrics. Diabetes. 2009;58(2):453–9.

5. Riskin-Mashiah S, Younes G, Damti A, Auslender R. First-trimester fasting hyperglycemia and adverse pregnancy out-comes. Diabetes Care. 2009;32(9):1639–43.

6. Higgins M, Mc Auliffe F. A review of maternal and fetal growthfactors in diabetic pregnancy. Curr Diabetes Rev. 2010;6(2):116–25.

7. Resnik R. Intrauterine growth restriction. Obstet Gynecol. 2002;99(3):490–6.

8. Pardi G, Marconi AM, Cetin I. Placental-fetal interrelationship inIUGR fetuses—a review. Placenta. 2002;23(Suppl A):S136–41.

9. Brosnan JT, Brosnan ME. The sulfur-containing amino acids: anoverview. J Nutr. 2006;136(6 Suppl):1636S–40S.

10. Stipanuk MH. Sulfur amino acid metabolism: pathways for pro-duction and removal of homocysteine and cysteine. Annu RevNutr. 2004;24:539–77.

11. Selhub J. Homocysteine metabolism. Annu Rev Nutr. 1999;19:217–46.

12. Gregory 3rd JF, Cuskelly GJ, Shane B, Toth JP, Baumgartner TG,Stacpoole PW. Primed, constant infusion with [2H3]serine allowsin vivo kinetic measurement of serine turnover, homocysteineremethylation, and transsulfuration processes in human one-carbon metabolism. Am J Clin Nutr. 2000;72(6):1535–41.

13. Lamers Y, Williamson J, Theriaque DW, Shuster JJ, Gilbert LR,Keeling C, Stacpoole PW, Gregory 3rd JF. Production of 1-carbon units from glycine is extensive in healthy men and women.J Nutr. 2009;139(4):666–71.

14. Jacobs RL, Stead LM, Brosnan ME, Brosnan JT. Hyperglucago-nemia in rats results in decreased plasma homocysteine andincreased flux through the transsulfuration pathway in liver. JBiol Chem. 2001;276(47):43740–7.

15. Ratnam S, Maclean KN, Jacobs RL, Brosnan ME, Kraus JP,Brosnan JT. Hormonal regulation of cystathionine beta-synthaseexpression in liver. J Biol Chem. 2002;277(45):42912–8.

16. Kalhan SC. Metabolism of methionine in vivo: impact of preg-nancy, protein restriction, and fatty liver disease. Nestle NutrWorkshop Ser Pediatr Program. 2009;63:121–31. discussion131–3, 259–68.

17. Kalhan SC. Fatty acids, insulin resistance, and protein metabo-lism. J Clin Endocrinol Metab. 2009;94(8):2725–7.

18. Gelfand RA, Barrett EJ. Effect of physiologic hyperinsulinemiaon skeletal muscle protein synthesis and breakdown in man. JClin Invest. 1987;80(1):1–6.

19. Cattaneo M. Homocysteine and the risk of intrauterine growthretardation. Clin Chem. 2003;49(9):1432–3.

20. Eskes TK. Homocysteine and human reproduction. Clin ExpObstet Gynecol. 2000;27(3–4):157–67.

21. Velzing-Aarts FV, Holm PI, Fokkema MR, van der Dijs FP,Ueland PM, Muskiet FA. Plasma choline and betaine and theirrelation to plasma homocysteine in normal pregnancy. Am J ClinNutr. 2005;81(6):1383–9.

22. Friesen RW, Novak EM, Hasman D, Innis SM. Relationship ofdimethylglycine, choline, and betaine with oxoproline in plasmaof pregnant women and their newborn infants. J Nutr. 2007;137(12):2641–6.

23. Molloy AM, Mills JL, Cox C, Daly SF, Conley M, Brody LC,Kirke PN, Scott JM, Ueland PM. Choline and homocysteineinterrelations in umbilical cord and maternal plasma at delivery.Am J Clin Nutr. 2005;82(4):836–42.

24. Seghieri G, Breschi MC, Anichini R, De Bellis A, Alviggi L,Maida I, Franconi F. Serum homocysteine levels are increased inwomen with gestational diabetes mellitus. Metabolism. 2003;52(6):720–3.

25. Patrick TE, Powers RW, Daftary AR, Ness RB, Roberts JM.Homocysteine and folic acid are inversely related in black womenwith preeclampsia. Hypertension. 2004;43(6):1279–82.

26. Jansson T. Novel mechanism causing restricted fetal growth: doesmaternal homocysteine impair placental amino acid transport? JPhysiol. 2009;587(Pt 17):4123.

27. Tsitsiou E, Sibley CP, D'Souza SW, Catanescu O, Jacobsen DW,Glazier JD. Homocysteine transport by systems L, A and y+Lacross the microvillous plasma membrane of human placenta. JPhysiol. 2009;587(Pt 16):4001–13.

28. Tsitsiou E, Sibley CP, D'Souza SW, Catanescu O, Jacobsen DW,Glazier JD. Homocysteine is transported by the microvillous


plasma membrane of human placenta. J Inherit Metab Dis.2011;34(1):57–65.

29. Dasarathy J, Gruca LL, Bennett C, Parimi PS, Duenas C,Marczewski S, Fierro JL, Kalhan SC. Methionine metabolism inhuman pregnancy. Am J Clin Nutr. 2010;91(2):357–65.

30. Stead LM, Jacobs RL, Brosnan ME, Brosnan JT. Methylationdemand and homocysteine metabolism. Adv Enzym Regul.2004;44:321–33.

31. Levonen AL, Lapatto R, Saksela M, Raivio KO. Human cysta-thionine gamma-lyase: developmental and in vitro expression oftwo isoforms. Biochem J. 2000;347(Pt 1):291–5.

32. Kalhan SC, Bier DM. Protein and amino acid metabolism in thehuman newborn. Annu Rev Nutr. 2008;28:389–410.

33. Shoob HD, Sargent RG, Thompson SJ, Best RG, Drane JW,TocharoenA. Dietarymethionine is involved in the etiology of neuraltube defect-affected pregnancies in humans. J Nutr. 2001;131(10):2653–8.

34. Shaw GM, Velie EM, Schaffer DM. Is dietary intake of methio-nine associated with a reduction in risk for neural tube defect-affected pregnancies? Teratology. 1997;56(5):295–9.

35. Rees WD, Hay SM, Cruickshank M. An imbalance in the methi-onine content of the maternal diet reduces postnatal growth in therat. Metabolism. 2006;55(6):763–70.

36. Loscalzo J. Homocysteine-mediated thrombosis and angiostasisin vascular pathobiology. J Clin Invest. 2009;119(11):3203–5.

37. Haulrik N, Toubro S, Dyerberg J, Stender S, Skov AR, Astrup A.Effect of protein and methionine intakes on plasma homocysteineconcentrations: a 6-mo randomized controlled trial in overweightsubjects. Am J Clin Nutr. 2002;76(6):1202–6.

38. Verhoef P, van Vliet T, Olthof MR, Katan MB. A high-proteindiet increases postprandial but not fasting plasma total homocys-teine concentrations: a dietary controlled, crossover trial inhealthy volunteers. Am J Clin Nutr. 2005;82(3):553–8.

39. Thomas B, Gruca LL, Bennett C, Parimi PS, Hanson RW, KalhanSC. Metabolism of methionine in the newborn infant: response tothe parenteral and enteral administration of nutrients. Pediatr Res.2008;64(4):381–6.

40. Yamamoto N, Tanaka T, Noguchi T. The effect of a high-proteindiet on cystathionine beta-synthase activity and its transcript levelsin rat liver. J Nutr Sci Vitaminol (Tokyo). 1996;42(6):589–93.

41. Pacy PJ, Price GM, Halliday D, Quevedo MR, Millward DJ.Nitrogen homeostasis in man: the diurnal responses of proteinsynthesis and degradation and amino acid oxidation to dietswith increasing protein intakes. Clin Sci (Lond). 1994;86(1):103–16.

42. Felig P, Owen OE, Wahren J, Cahill Jr GF. Amino acid metabo-lism during prolonged starvation. J Clin Invest. 1969;48(3):584–94.

43. Ingenbleek Y, Hardillier E, Jung L. Subclinical protein malnutritionis a determinant of hyperhomocysteinemia. Nutrition. 2002;18(1):40–6.

44. Ingenbleek Y, Young VR. The essentiality of sulfur is closelyrelated to nitrogen metabolism: a clue to hyperhomocysteinaemia.Nutr Res Rev. 2004;17(2):135–51.

45. Holt Jr LE, Snyderman SE, Norton PM, Roitman E, Finch J. Theplasma aminogram in kwashiorkor. Lancet. 1963;2(7322):1342–8.

46. Snyderman SE, Holt Jr LE, Nortn PM, Roitman E, PhansalkarSV. The plasma aminogram. I. influence of the level of proteinintake and a comparison of whole protein and amino acid diets.Pediatr Res. 1968;2(2):131–44.

47. Arroyave G, Wilson D, De Funes C, Behar M. The free aminoacids in blood plasma of children with kwashiorkor and maras-mus. Am J Clin Nutr. 1962;11:517–24.

48. Kalhan SC, Uppal SO, Moorman JL, Bennett C, Gruca LL,Parimi PS, Dasarathy S, Serre D, Hanson RW. Metabolic and

genomic response to dietary isocaloric protein restriction in therat. J Biol Chem. 2011;286(7):5266–77.

49. Brosnan JT, Jacobs RL, Stead LM, Brosnan ME. Methylationdemand: a key determinant of homocysteine metabolism. ActaBiochim Pol. 2004;51(2):405–13.

50. Perla-Kajan J, Twardowski T, Jakubowski H. Mechanisms ofhomocysteine toxicity in humans. Amino Acids. 2007;32(4):561–72.

51. Medina M, Urdiales JL, Amores-Sanchez MI. Roles of homocys-teine in cell metabolism: old and new functions. Eur J Biochem.2001;268(14):3871–82.

52. Vollset SE, Refsum H, Irgens LM, Emblem BM, Tverdal A,Gjessing HK, Monsen AL, Ueland PM. Plasma total homocys-teine, pregnancy complications, and adverse pregnancy out-comes: the hordaland homocysteine study. Am J Clin Nutr.2000;71(4):962–8.

53. Picciano MF. Is homocysteine a biomarker for identifying womenat risk of complications and adverse pregnancy outcomes? Am JClin Nutr. 2000;71(4):857–8.

54. Fux R, Kloor D, Hermes M, Rock T, Proksch B, Grenz A, DelabarU, Bucheler R, Igel S, Morike K, Gleiter CH, Osswald H. Effectof acute hyperhomocysteinemia on methylation potential oferythrocytes and on DNAmethylation of lymphocytes in healthymale volunteers. Am J Physiol Ren Physiol. 2005;289(4):F786–92.

55. Yi P, Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ.Increase in plasma homocysteine associated with parallelincreases in plasma S-adenosylhomocysteine and lymphocyteDNA hypomethylation. J Biol Chem. 2000;275(38):29318–23.

56. Scholl TO, Johnson WG. Folic acid: influence on the outcome ofpregnancy. Am J Clin Nutr. 2000;71(5 Suppl):1295S–303S.

57. Rao S, Yajnik CS, Kanade A, Fall CH, Margetts BM, JacksonAA, Shier R, Joshi S, Rege S, Lubree H, Desai B. Intake ofmicronutrient-rich foods in rural Indian mothers is associatedwith the size of their babies at birth: Pune maternal nutritionstudy. J Nutr. 2001;131(4):1217–24.

58. Pagán K, Hou J, Goldenberg RL, Cliver SP, Tamura T. Mid-pregnancy serum homocysteine and B-vitamin concentrationsand fetal growth. Nutr Res. 2002;22:11330–41.

59. Relton CL, Pearce MS, Parker L. The influence of erythrocytefolate and serum vitamin B12 status on birth weight. Br J Nutr.2005;93(5):593–9.

60. Takimoto H, Mito N, Umegaki K, Ishiwaki A, Kusama K, Abe S,Yamawaki M, Fukuoka H, Ohta C, Yoshiike N. Relationshipbetween dietary folate intakes, maternal plasma total homocys-teine and B-vitamins during pregnancy and fetal growth in Japan.Eur J Nutr. 2007;46(5):300–6.

61. Watanabe H, Fukuoka H, Sugiyama T, Nagai Y, Ogasawara K,Yoshiike N. Dietary folate intake during pregnancy and birthweight in Japan. Eur J Nutr. 2008;47(6):341–7.

62. Nilsen RM, Vollset SE, Monsen AL, Ulvik A, Haugen M, MeltzerHM, Magnus P, Ueland PM. Infant birth size is not associated withmaternal intake and status of folate during the second trimester inNorwegian pregnant women. J Nutr. 2010;140(3):572–9.

63. Kordas K, Ettinger AS, Lamadrid-Figueroa H, Tellez-Rojo MM,Hernandez-Avila M, Hu H, Wright RO. Methylenetetrahydro-folate reductase (MTHFR) C677T, A1298C and G1793Agenotypes, and the relationship between maternal folate in-take, tibial length and infant size at birth. Br J Nutr.2009;102(6):907–14.

64. Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, OrningL, Guttormsen AB, Joglekar A, Sayyad MG, Ulvik A, Ueland PM.Hyperhomocysteinemia and elevated methylmalonic acid indicate ahigh prevalence of cobalamin deficiency in Asian Indians. Am JClin Nutr. 2001;74(2):233–41.


65. Yajnik CS, Deshpande SS, Jackson AA, Refsum H, Rao S, FisherDJ, Bhat DS, Naik SS, Coyaji KJ, Joglekar CV, Joshi N, LubreeHG, Deshpande VU, Rege SS, Fall CH. Vitamin B12 and folateconcentrations during pregnancy and insulin resistance in theoffspring: the pune maternal nutrition study. Diabetologia.2008;51(1):29–38.

66. Morris MS, Jacques PF, Rosenberg IH, Selhub J. Folate andvitamin B-12 status in relation to anemia, macrocytosis, andcognitive impairment in older Americans in the age of folic acidfortification. Am J Clin Nutr. 2007;85(1):193–200.

67. Carmel R. Does high folic acid intake affect unrecognized cobal-amin deficiency, and how will we know it if we see it? Am J ClinNutr. 2009;90(6):1449–50.

68. Smith AD. Folic acid fortification: the good, the bad, and thepuzzle of vitamin B-12. Am J Clin Nutr. 2007;85(1):3–5.

69. Selhub J, Morris MS, Jacques PF. In vitamin B12 deficiency,higher serum folate is associated with increased total homocys-teine and methylmalonic acid concentrations. Proc Natl Acad SciU S A. 2007;104(50):19995–20000.

70. Scott JM, Weir DG. The methyl folate trap. A physiological re-sponse in man to prevent methyl group deficiency in kwashiorkor(methionine deficiency) and an explanation for folic-acid inducedexacerbation of subacute combined degeneration in perniciousanaemia. Lancet. 1981;2(8242):337–40.

71. Kramer MS. Effects of energy and protein intakes on pregnancyoutcome: an overview of the research evidence from controlledclinical trials. Am J Clin Nutr. 1993;58(5):627–35.

72. Kramer MS. Balanced protein/energy supplementation in preg-nancy. Cochrane Database Syst Rev. 2000;2(2):CD000032.

73. Sloan NL, Lederman SA, Leighton J, Himes JH, Rush D. Theeffect of prenatal dietary protein intake on birth weight. Nutr Res.2001;21:129–39.

74. Godfrey K, Robinson S, Barker DJ, Osmond C, Cox V. Maternalnutrition in early and late pregnancy in relation to placental andfetal growth. BMJ. 1996;312(7028):410–4.

75. Kramer MS, Kakuma R. Energy and protein intake in pregnancy.Cochrane Database Syst Rev. 2003;4(4):CD000032.

76. Snoeck A, Remacle C, Reusens B, Hoet JJ. Effect of a lowprotein diet during pregnancy on the fetal rat endocrine pancreas.Biol Neonate. 1990;57(2):107–18.

77. Dahri S, Snoeck A, Reusens-Billen B, Remacle C, Hoet JJ. Isletfunction in offspring of mothers on low-protein diet during ges-tation. Diabetes. 1991;40 Suppl 2:115–20.

78. Fernandez-Twinn DS, Wayman A, Ekizoglou S, Martin MS,Hales CN, Ozanne SE. Maternal protein restriction leads tohyperinsulinemia and reduced insulin-signaling protein expres-sion in 21-mo-old female rat offspring. Am J Physiol RegulIntegr Comp Physiol. 2005;288(2):R368–73.

79. Chen JH, Martin-Gronert MS, Tarry-Adkins J, Ozanne SE. Mater-nal protein restriction affects postnatal growth and the expression ofkey proteins involved in lifespan regulation in mice. PLoS One.2009;4(3):e4950.

80. Sutton GM, Centanni AV, Butler AA. Protein malnutrition duringpregnancy in C57BL/6J mice results in offspring with alteredcircadian physiology before obesity. Endocrinology. 2010;151(4):1570–80.

81. Kwong WY, Miller DJ, Ursell E, Wild AE, Wilkins AP, OsmondC, Anthony FW, Fleming TP. Imprinted gene expression in the ratembryo-fetal axis is altered in response to periconceptional ma-ternal low protein diet. Reproduction. 2006;132(2):265–77.

82. Chisari AN, Giovambattista A, Perello M, Spinedi E. Impact ofmaternal undernutrition on hypothalamo-pituitary-adrenal axis andadipocyte functions in male rat offspring. Endocrine. 2001;14(3):375–82.

83. Hales CN. Metabolic consequences of intrauterine growth retar-dation. Acta Paediatr Suppl. 1997;423:184–7. discussion 188.

84. Ozanne SE, Hales CN. Early programming of glucose-insulinmetabolism. Trends Endocrinol Metabol. 2002;13(9):368–73.

85. Warner MJ, Ozanne SE. Mechanisms involved in the develop-mental programming of adulthood disease. Biochem J. 2010;427(3):333–47.

86. Godfrey KM, Gluckman PD, Hanson MA. Developmentalorigins of metabolic disease: life course and intergenerationalperspectives. Trends Endocrinol Metabol. 2010;21(4):199–205.

87. Hochberg Z, Feil R, Constancia M, Fraga M, Junien C, Carel JC,Boileau P, Le Bouc Y, Deal CL, Lillycrop K, Scharfmann R,Sheppard A, Skinner M, Szyf M, Waterland RA, Waxman DJ,Whitelaw E, Ong K, Albertsson-Wikland K. Child health, devel-opmental plasticity, and epigenetic programming. Endocr Rev.2011;32(2):159–224.

88. Rees WD, Hay SM, Brown DS, Antipatis C, Palmer RM. Mater-nal protein deficiency causes hypermethylation of DNA in thelivers of rat fetuses. J Nutr. 2000;130(7):1821–6.

89. McNeil CJ, Hay SM, Rucklidge GJ, Reid MD, Duncan GJ, ReesWD. Gene and protein expression profiles in the foetal liver of thepregnant rat fed a low protein diet. Genes Nutr. 2009;4(3):189–94.

90. Mortensen OH, Olsen HL, Frandsen L, Nielsen PE, Nielsen FC,Grunnet N, Quistorff B. Gestational protein restriction in mice haspronounced effects on gene expression in newborn offspring’s liverand skeletal muscle; protective effect of taurine. Pediatr Res.2010;67(1):47–53.

91. Strakovsky RS, Zhou D, Pan YX. A low-protein diet duringgestation in rats activates the placental mammalian amino acidresponse pathway and programs the growth capacity of offspring.J Nutr. 2010;140(12):2116–20.

92. Chen JH, Tarry-Adkins JL, Matharu K, Yeo GS, Ozanne SE.Maternal protein restriction affects gene expression profiles inthe kidney at weaning with implications for the regulation ofrenal function and lifespan. Clin Sci (Lond). 2010;119(9):373–84.

93. El-Khattabi I, Gregoire F, Remacle C, Reusens B. Isocaloricmaternal low-protein diet alters IGF-I, IGFBPs, and hepatocyteproliferation in the fetal rat. Am J Physiol Endocrinol Metab.2003;285(5):E991–E1000.

94. Gomez-Angelats M, Ruiz-Montasell B, Felipe A, Marin JJ,Casado FJ, Pastor-Anglada M. Effect of protein malnutrition onneutral amino acid transport by rat hepatocytes during develop-ment. Am J Physiol. 1995;268(2 Pt 1):E368–74.

95. Parimi PS, Cripe-Mamie C, Kalhan SC. Metabolic responses toprotein restriction during pregnancy in rat and translation initia-tion factors in the mother and fetus. Pediatr Res. 2004;56(3):423–31.

96. Rees WD, Hay SM, Buchan V, Antipatis C, Palmer RM. Theeffects of maternal protein restriction on the growth of the ratfetus and its amino acid supply. Br J Nutr. 1999;81(3):243–50.

97. Petrie L, Duthie SJ, Rees WD, McConnell JM. Serum concen-trations of homocysteine are elevated during early pregnancy inrodent models of fetal programming. Br J Nutr. 2002;88(5):471–7.

98. Torres N, Bautista CJ, Tovar AR, Ordaz G, Rodriguez-Cruz M,Ortiz V, Granados O, Nathanielsz PW, Larrea F, Zambrano E.Protein restriction during pregnancy affects maternal liver lipidmetabolism and fetal brain lipid composition in the rat. Am JPhysiol Endocrinol Metab. 2010;298(2):E270–7.

99. Wolff GL, Kodell RL, Moore SR, Cooney CA. Maternal epige-netics and methyl supplements affect agouti gene expression inAvy/a mice. FASEB J. 1998;12(11):949–57.

100. Jackson AA, Dunn RL, Marchand MC, Langley-Evans SC. In-creased systolic blood pressure in rats induced by a maternal low-


protein diet is reversed by dietary supplementation with glycine.Clin Sci (Lond). 2002;103(6):633–9.

101. Boujendar S, Reusens B, Merezak S, Ahn MT, Arany E, Hill D,Remacle C. Taurine supplementation to a low protein diet duringfoetal and early postnatal life restores a normal proliferation andapoptosis of rat pancreatic islets. Diabetologia. 2002;45(6):856–66.

102. Lillycrop KA, Phillips ES, Jackson AA, Hanson MA, BurdgeGC. Dietary protein restriction of pregnant rats induces and folicacid supplementation prevents epigenetic modification of hepaticgene expression in the offspring. J Nutr. 2005;135(6):1382–6.

103. Cooney CA, Dave AA, Wolff GL. Maternal methyl supplementsin mice affect epigenetic variation and DNA methylation ofoffspring. J Nutr. 2002;132(8 Suppl):2393S–400S.


methionine, homocysteine, one carbon metabolism and fetal growth

Documents