levels of key enzymes of methionine-homocysteine metabolism in

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 731962 8 pageshttpdxdoiorg1011552013731962

Research ArticleLevels of Key Enzymes of Methionine-HomocysteineMetabolism in Preeclampsia

Alejandra Peacuterez-Sepuacutelveda1 Pedro P Espantildea-Perrot1 Ximena Fernaacutendez B1

Veroacutenica Ahumada1 Vicente Bustos1 Joseacute Antonio Arraztoa1 Aneta Dobierzewska1

Horacio Figueroa-Diesel2 Gregory E Rice3 and Sebastiaacuten E Illanes2

1 Department of Obstetrics and Gynecology Faculty of Medicine Universidad de los Andes San Carlos de Apoquindo7620001 Santiago Chile

2 Clinical Perinatal Unit Clınica Davila 8431657 Santiago Chile3The University of Queensland Centre for Clinical Research RBWH Campus Herston Brisbane QLD 4029 Australia

Correspondence should be addressed to Sebastian E Illanes sillanesuandescl

Received 8 May 2013 Revised 13 July 2013 Accepted 17 July 2013

Academic Editor Kotaro Kitaya

Copyright copy 2013 Alejandra Perez-Sepulveda et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Objective To evaluate the role of key enzymes in the methionine-homocysteine metabolism (MHM) in the physiopathology ofpreeclampsia (PE) Methods Plasma and placenta from pregnant women (32 controls and 16 PE patients) were analyzed afterinformed consent Protein was quantified by western blot RNA was obtained with RNA purification kit and was quantified byreverse transcritase followed by real-time PCR (RT-qPCR) Identification of the C677T and A1298C methylenetetrahydrofolatereductase (MTHFR) single-nucleotide polymorphisms (SNPs) andA2756Gmethionine synthase (MTR) SNPwas performed usingPCR followed by a high-resolution melting (HRM) analysis S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH)were measured in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLCMSMS) The SNPassociation analysis was carried out using Fisherrsquos exact test Statistical analysis was performed using a Mann-Whitney test ResultsRNA expression of MTHFR and MTR was significantly higher in patients with PE as compared with controls Protein SAM andSAH levels showed no significant difference between preeclamptic patients and controls No statistical differences between controlsand PE patients were observed with the different SNPs studied Conclusion The RNA expression of MTHFR and MTR is elevatedin placentas of PE patients highlighting a potential compensation mechanism of the methionine-homocysteine metabolism in thephysiopathology of this disease

1 Introduction

Preeclampsia (PE) is a pregnancy-specific disease defined bynew-onset hypertension and proteinuria after 20 weeks ofgestation It is present in around 5ndash10of all pregnantwomenworldwide [1] and is associated with an increased risk ofmaternal and fetal morbidity and mortality and recently ithas been associated with an increased risk of later-life deathdue to cardiovascular disease for both mother and child [2ndash4]

Several factors have been suggested to be involved in theetiology of PE but there is consensus that the first step in

the pathogenesis of this disease is a defective trophoblasticinvasion early in pregnancy [5] which leads to reducedplacental perfusion and hypoxia [6] Nevertheless it has beenproposed that a hypoxic environment is a necessary conditionfor the invasive phenotype of trophoblast cells [7] Morerecently Lee et al have described that 2-methoxyestradiol(2-ME) a natural metabolite of 17-120573-estradiol synthesized byCathechol-O-Methyltransferase (COMT) induces the differ-entiation of the endovascular cytotrophoblast cells into itsinvasive phenotype in the presence of hypoxia [8] Kanasakiet al have reported low levels of circulating 2-ME during

2 BioMed Research International

the third trimester of pregnancy in patients that would laterdevelop PE as compared with controls [9] and our groupfound the same trend during the early first trimester ofpregnancy [10] With COMT being responsible for methy-lating 2-Hydroxyestradiol (2-HE) into 2-ME it has beenproposed that the low activity or expression of this enzymecould be involved in the pathogenesis of PE The normalexpression of COMT in placentae of patients with PE [11] andthe conflicting results regarding the presence of SNPs thatmay reduce the activity of this enzyme in these patients [10]force us to search other explanations [12] The Methionine-homocysteine metabolism (MHM) the process responsiblefor supplying COMT with the methyl group necessary for 2-ME synthesis could be an alternative if it is somehow alteredbeing unable to supply enough methyl groups to sustainadequate concentrations of 2-ME

It has been shown that alterations in the methionine-homocysteine metabolism (MHM) may be related to thesystemic damage that leads to the classical clinical pictureof PE [13 14] In fact in normal pregnant women plasmaconcentrations of homocysteine are low in the first trimesterof pregnancy and reach their lowest level in the secondhalf of gestation [15 16] whereas hyperhomocysteinemiahas been described in women who would develop PE [17ndash20] Homocysteine is biosynthesized by the MHM in aprocess involving the donation of a methyl group by S-adenosylmethionine (SAM) which in turn gets convertedinto S-adenosylhomocysteine (SAH) a precursor of homo-cysteine Through the enzyme methionine synthase (MTR)homocysteine may be remethylated into methionine using5-methyltetrahydrofolate (5-Methyl-THF) as a cosubstrateMethylenetetrahydrofolate reductase (MTHFR) is the cata-lyst for the conversion of 510-methylenetetrahydrofolate into5-methylTHF Methionine can then be converted into SAMonce more to restart the cycle [21] SAM acts as the principalmethyl donor within cells These methyl groups are keyfactors in DNA methylation that might control for examplecellular proliferation cellular migration differentiation cell-to-cell recognition and other important processes such asthe production of 2-ME through COMT

Low levels of SAM may be caused by the presence ofsingle-nucleotide polymorphisms (SNPs) in the genes thatcode for the enzymes involved in SAM metabolism such asMTR and MTHFR [22] Hill et al recently described thatMTHFR modulates the availability of SAM and the minorldquoTrdquo allele of the MTHFR C677T SNP has been associatedwith reduced MTHFR activity resulting in a decrease inSAM production [23] Furness et al suggested that thematernal and placental MTR A2756G allele is an importantrisk factor for the development of uteroplacental insufficiency[24] though they did not link it to SAM levels and attributedthe uteroplacental insufficiency directly to the enzyme

We hypothesize that changes in the levels of MTR andMTHFR by the abovementioned SNPs have a negative effecton SAM levels which when lowered contribute to thedevelopment of PEThe purpose of this study was to evaluatethe bioavailability of SAM as well as of MTR and MTHFRtheir respective SNPs and their relation with preeclampsia

2 Methods

21 Study Design A prospective cohort study was conductedin the Obstetrics and Fetal Medicine Unit from Hospital Par-roquial de San Bernardo Santiago Chile Sixteen preeclamp-tic patients and 32 controls were recruited from this cohortBoth groups consisted of women with singleton gestationand none of them took multivitamins or aspirin duringpregnancy Maternal anthropometric data and blood sampleswere collected at 11ndash14-week gestation and at 32ndash36 weeks PEwas defined as hypertension (arterial pressure (AP) higheror equal to AP 14090mmHg on two occasions separatedby 6 h or higher or equal to 160110mmHg) that occurredafter 20 weeks of gestation in women with previously normalblood pressure accompanied by proteinuria (300mg24 h)Controls who do not differ in racial origin from PE patientswere healthy subjects without pregnancy complications orchronic medical problems

Written informed consent was obtained from the womenwho agreed to participate in the study which was approvedby Hospital Parroquial de San Bernardo and the Universidadde los Andes Ethics Committee

22 Tissue and Blood Sample Collection Placental tissueswere obtained within 15min of delivery from all of thesubjects Placental samples (asymp1 cm3) were taken from astandardized location a placental cotyledon between cordinsertion and placental border midway avoiding tissue fromareas showing placental infarcts The tissue was washedin ice-cold physiologic solution (NaCl 09) to removematernal blood contamination Samples were processed andstored at minus80∘C until use for RNA extraction SNP analysisand protein quantification Blood was collected into edeticacid (EDTA) Vacutainer tubes at 11ndash14 weeks and at 32ndash34 weeks of gestation placed in 16mL DNAseRNAse-freeEppendorf tubes and centrifuged at 1300 g for 10min atroom temperatureThe supernatant was transferred to a fresh16mL tube and centrifuged oncemore at 7200 g for 10min atroom temperatureThe supernatant was transferred to a freshtube and stored at minus80∘C

23 Protein Quantification A sim5mg section from a pla-cental tissue sample (spanning from the maternal to fetalface) was homogenized in a 16mL Eppendorf tube usingradioimmunoprecipitation assay (RIPA) lysis buffer (50mMTris-HCl pH 88 150mM NaCl 05 sodium deoxycholate01 Sodium Dodecyl Sulfate (SDS) 1 Tergitol-type NP-40) with protease inhibitor (cOmplete Mini Roche) Thehomogenate was centrifuged at 25800 g for 30min at 4∘CThe supernatantwas carefully transferred to a different 16mLEppendorf and stored at minus20∘C Total protein amount inthe supernatant was quantified using BCA Protein Assay Kit(Thermo Scientific) and spectrophotometer NanoDrop 2000(Thermo Scientific) according tomanufacturerrsquos instructions20120583g of total protein was loaded for each sample in 12polyacrylamide gels that were additionally corrected usingCoomassie Blue staining and densitometric analysis Aninternal control was prepared by pooling 4 different samples

BioMed Research International 3

(3 controls and 1 PE sample) and was loaded into all gels toallow normalization of data and intergel comparison

Protein samples were electrophorised in 12 SDS-polyacrylamide gels and transferred to nitrocellulose mem-branes (Biorad Laboratories Inc) Membranes were blockedin 5 (wv) nonfat dry milk and incubated overnight at4∘C with 1 1000 diluted specific monoclonal primary anti-bodies for MTHFR (Abcam noab55530) and MTR (Abcamno66039) Secondary antibody incubation was performedfor 2 hours at room temperature for both genes using a 1 1000diluted peroxidase IgG conjugated anti-mouse secondaryantibody (Kirkegaard and Perry Laboratories Inc) Specificbands were detected using the ECL Western Blotting Sub-strates (Thermo Scientific) Blots were scanned and resultsfrom densitometric analysis of gels were shown as arbitraryunits

24 SAM and SAH Quantification Plasma concentrations ofSAM and SAH were measured through high-performanceliquid chromatography-tandem mass spectrometry (HPLC-MSMS) at Innolab Ltda in Santiago Chile Briefly 500120583L ofplasma was vortexed with 312 120583L perchloric acid at 100mlLand then centrifuged for 10min at 13000 rpm Samples werethen transferred to a glass tube and 140 120583L phosphate buffer(pH 115) was added along with 1mL H

2O Strata X columns

were conditioned with 1mL methanol and 750 120583L lauric acidat 10mmolL in 01molL NaOH and 1mL H

2O Samples

were loaded onto columns and washed with 700120583LH2OThe

column was eluted with 1mL ethanol at 01 formic acid andthe elate was dried under a N

2at 40∘C The samples were

reconstituted with 200mL of mobile phase and analyzed inthe API 5000 Mass Spectrometer

25 RNA Quantification Total placental RNA was isolatedwith MasterPure Complete DNA and RNA Purification Kit(Epicentre Biotechnologies) according tomanufacturerrsquos pro-cedures Total nucleic acid extraction was followed by DNaseI treatment to remove DNA contamination The amount andquality of RNAwere assessed usingNanoDrop 2000 (ThermoScientific) at 260280 with all samples having values between18 and 20 Reverse transcriptase PCR was performed on1 120583L of total RNA to obtain complementary DNA usingRandom Primers (Promega) For gene detection real-timePCR (qPCR)was performedwith the StratageneMx3000 ForMTHFR forward primer 51015840-ATCACTTGCCCCATCGTC-31015840 reverse primer 51015840-CATCGTTGTCTTTGATTGGCTC-31015840with probe sequence 51015840-ATCACGTCCTTGATCTCCTGT-GGC-31015840 For MTR forward primer 51015840-ATCTCATCTGGA-ATAAAGACCCTG-31015840 reverse primer 51015840-TTCACAAGG-GCATACTCAAGG-31015840 with probe sequence 51015840-CAAGGC-ACAGGAGGGAAGAAAGTCAT-31015840 To quantify the geneexpression of each enzyme a 7-point standard curve (fg120583L)made from a synthetic oligo of the sequence of interest wasrun with the samples Sample concentration was extrapolatedfrom the lineal regression equation of the standard curve usedand results are shown in fg120583L plusmn SEM

26 MTHFR and MTR Genotyping In order to determinethe presence of placental MTHFR and MTR SNPs tissue

DNA was isolated from samples using MasterPure DNApurification kits (Cat no MC85200 Epicentre Biotechnolo-gies Madison USA) following the manufacturerrsquos instruc-tions After extraction DNA samples were stored at minus20∘CMutation screening for all three SNPs was performed byqPCR followed by high resolution melting analysis Thistechnique allows us to differentiate between possible geno-types (both wild type and mutated homocygote and hete-rocygote) through the differences in melting temperature ofeach haplotype given by the differences in the base pairing(see Figure 1 in supplementary material available onlineat httpdxdoiorg1011552013731962) The primers usedfor each SNP are MTHFR C677T sense 51015840-TGAGGCTGA-CCTGAAGCACTTGAA-31015840 antisense 51015840-ATGTCGGTG-CATGCCTTCACAAAG-31015840MTHFRA1298C sense 51015840-CTT-TGGGGAGCTGAAGGACTACTAC-31015840 antisense 51015840- CAC-TTTGTGACCATTCCGGTTTG-31015840 MTR A2756G sense 51015840-GGATGAATACTTTGAGGAAATCATGG-31015840 antisense 51015840-TGTTTCTACCACTTACCTTGAGAG-31015840 Conditions forthe PCR reactions were as follows preincubation at 95∘C for10min followed by 45 cycles of denaturation at 95∘C for 10 s15 s at 62∘C annealing 10 s at 72∘C for extension followedby 60 s denaturation at 95∘C 60 s at 40∘C ending with a65ndash95∘C at 005∘Cs high-resolution curve All samples wererun in duplicate using the LightCycler Nano (Roche) and themelting curves were analyzed using the LightCycler NanoSoftware 10 (Roche)

27 SNP Association Analysis The SNP association analysiswith PE including the three possible genotypes was carriedout using a Fisherrsquos exact statistic test The relationshipbetween the SNPs and PE was also tested separately for thesubgroups of preeclamptic women against control womenusing a dichotomous method (positive-SNP versus negative-SNP) This was done to analyze whether carrying the SNPswas associated with the development of the disease and aFisherrsquos exact test was used for this purpose A threshold of119875 = 005 was set for statistical significance of all computedanalyses

28 Statistical Analysis Statistical analysis was performedusing a Mann-Whitney test to establish significant statisticaldifferences in parameters between controls and cases con-cerning mRNA and protein amounts while Fisherrsquos exacttest was carried out to establish significant differences amongSNPs Values are expressed as mean plusmn SEM Significantdifferences were considered when 119875 lt 005

3 Results

31 Clinical Characteristics of the Population The clinicalcharacteristics of the total group of patients enrolled in thisstudy are shown in Table 1 At 11ndash14 weeks of gestationwomen who developed PE later in gestation showed asignificantly higher systolic and diastolic arterial pressure(119875 = 0003 and 119875 = 0005 resp) than controls as wellas in maternal weight and Body Mass Index (BMI) (119875 =0026 and 119875 = 001 resp) Gestational age at delivery wasalso significantly different between controls and preeclampsia


Table 1 Clinical characteristics of controls and preeclampsia patients at 11ndash14 weeks of gestation

Control patients (119899 = 32) Preeclampsia patients (119899 = 16) 119875 valueMaternal age (years) 255 plusmn 12 299 plusmn 19 00835Gestation age at delivery (weeks) 393 plusmn 02 366 plusmn 10 lt00001lowastlowastlowast

At 11ndash14 weeksSystolic pressure (mmHg) 1073 plusmn 19 1214 plusmn 36 00029lowastlowast

Diastolic pressure (mmHg) 647 plusmn 14 723 plusmn 38 00053lowast

Maternal weight (kg) 643 plusmn 22 751 plusmn 44 00263lowast

Body mass index (kgm2) 254 plusmn 09 304 plusmn 17 00099lowast

Values are given as mean plusmn SEM Statistical significance was assessed using MannWhitney test lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001

Table 2 Frequencies of genotypes of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) gene in controlsand preeclamptic patients

Genotypes Control patients (119899 = 32) Preeclampsia patients (119899 = 16) P value OR (95 Conf interval)MTHFR C677T

Wild Type 9 (281) 3 (188) 0725 169 [0389ndash7400]Heterozygotehomozygote 23 (719) 13 (812)

MTHFR A1268CWild Type 4 (125) 6 (375) 0063 0238 [0055ndash1022]Heterozygotehomozygote 28 (875) 10 (625)

MTR A2756GWild Type 16 (500) 10 (625) 0542 06 [0176ndash2046]Heterozygotehomozygote 16 (500) 6 (375)

MTHFR C677T wild type (CC) heterozygote (CT) and homozygote (TT) MTHFR A1268C wild type (AA) heterozygote (AC) homozygote (CC)MTR A2756G wild type (AA) heterozygote (AG) homozygote (GG) Data show the number of control and preeclamptic patients with or without thepolymorphism Statistical significance was assessed using Fisherrsquos exact test

patients (119875 lt 00001) No significant differences wereobserved in maternal age

32 Placental Frequency of MTHFR and MTR Polymor-phisms In order to determine the placental presence ofthe C677T A1298C MTHFR and A2756G MTR gene poly-morphisms and their association with PE developmentwedetermined the genotype of placental tissue from controland preeclamptic women Table 2 shows the frequencies ofthe C677T MTHFR A1298C MTHFR and A2756G MTRpolymorphisms There was no significant difference betweenSNP frequencies when comparing controls and PE patients(MTHFR C677T control 729 versus PE 813 119875 = 0725MTHFR A1268C control 875 versus PE 625 119875 = 0064MTR A2756G control 500 versus PE 375 119875 = 0542)

33 MTHFR MTR RNA Levels MTHFR and MTR RNAexpression was measured in term placentas from all patientsWe used RT-PCR followed by qPCR to calculate the relativeconcentrations of the different RNA As shown in Figure 1women who developed PE had significantly higher RNAexpression for MTHFR and MTR (MTHFR 1817 plusmn 526(SEM) versus 3899 plusmn 1389 fg120583L 119875 = 002 MTR 2100 plusmn479 versus 9713 plusmn 3040 fg120583L 119875 = 00002)

34 MTHFR and MTR Protein Levels Protein content wasmeasured in placental tissue from all patients in this studyto determine if there was any association between altered

protein content and the development of PE Figure 2 showsthe relative protein content for MTHFR and MTR withno significant differences between controls and PE patients(MTHFR 152plusmn019 (SEM) versus 158plusmn026 arbitrary units(au) 119875 = 044 MTR 087 plusmn 005 versus 080 plusmn 005 au119875 = 055)

35 SAM and SAH Levels Metabolite concentrations weremeasured in maternal serum samples obtained 4ndash6 weeksbefore delivery from all patients in the study to establish therelationship between SAHproduced and SAMavailable in PEpatients and control Figure 3 shows the amount of SAM andSAH with no significant differences between control and PEpatients (SAM 7536plusmn669 (SEM) versus 1041plusmn1434 ngmL119875 = 008 SAH 2358 plusmn 115 versus 2660 plusmn 207 ngmL119875 = 032 SAHSAM 043plusmn006 versus 034plusmn006 119875 = 016)

4 Discussion

In this study we tested if the development of PE wasconnected to an alteration in the enzymes involved in theMHM As early as 11ndash14 weeks of gestation the PE cohortdisplayed significantly higher systolic and diastolic bloodpressure (119875 gt 005) as well as significantly higher maternalweight andBMI (119875 gt 005) (Table 1) and all displayed clinicalfeatures that have been described previously as predictorsof PE or related to the development of this disease [25ndash27]The case and control cohorts used in this study are thuswell characterized and defined with difference between them


Control PE 0

20

40

60M

THFR

RN

A le

vels

(fg120583

L)

lowast

(a)

Control PE 0

50

100

150

MTR

RN

A le

vels

(fg120583

L)

lowastlowast

(b)

Figure 1 Elevated levels of mRNA for (a) MTHFR and (b) MTR in women who developed PE Levels were measured by RT-PCR followedby qPCR Values are plusmn SEM from 32 control and 16 PE patients lowast119875 lt 005 lowastlowast119875 lt 001

Control PE 00

05

10

15

20

MTH

FR p

rote

in le

vels

(au

)

(a)

Control PE 00

02

04

06

08

10M

TR p

rote

in le

vels

(au

)

(b)

Figure 2 (a) MTHFR and (b) MTR protein levels analyzed from placental tissue by western blot Values are plusmn SEM from 32 controls and 16PE patients

Control PE0

10

20

30

40

SAH

leve

ls (n

gm

L)

(a)

Control PE0

50

100

150

SAM

lev

els (n

gm

L)

(b)

Figure 3 (a) SAM and (b) SAH levels obtained form maternal serum 4ndash6 weeks prior to delivery Amounts measured by HPLC-MSMSValues are plusmn SEM from 32 controls and 16 PE patients


not only present in the third trimester but also in the firsttrimester of pregnancy

Recently we reported that low 2-ME plasma concen-trations at 11ndash14 weeks of gestation are associated with thedevelopment of preeclampsia but these low concentrationscannot be explained by abnormal expression of COMT [11]and there are conflicting results regarding the presence ofSNPs that could reduce the activity of COMT in patientswith PE In this study we tested the hypothesis that theselow concentrations were associated with an alteration inthe MHM key enzymes andor SAM and SAH plasmaconcentrations and PE development

First we evaluated if there was an association between thepresence of placentalMTHFR andMTR functional polymor-phisms and the development of PE Analysis through PCRfollowed by high-resolution melting revealed no significantdifference in the frequency of SNPs between PE patients andthe control group Robertson et al however conducted asystematic review of 25 studies (119899 = 11 183) to investigatethe association between inherited thrombophilias and therisk of adverse pregnancy outcomes including PE Theyreported that the risk of PE was significantly higher inthe presence of MTHFR homozygosity (MTHFR TT) theMTHFR C677T SNP had an odds ratio of 137 (95 CI 107ndash176) for developing PE [28] The reduced number of patientsin our cohort could explain this difference but until a largerstudy that tests the penetration of these SNPs in our normalpopulation is carried out wewill be unable to give a definitiveanswer

Second we evaluated if there was a difference in RNAand protein content for these key enzymes of the MHM thatmay be associated with the development of PE While RNAexpression of MTHFR and MTR was significantly higherin placental tissue obtained from PE patients there was nodifference in protein content for these two enzymes betweenthe studied groups This may be explained by the time atwhich the samples were collected RNA and protein havingdifferences in synthesis time While RNA is increased theprotein content could remain low and when RNA is trans-lated into protein RNA may decrease while protein contentgradually increasesThus though the RNA expression provedto be significantly different the change may have not beenyet reflected in protein content or a regulating step waspresent not during transcription but during translation of theRNA into protein [29] Also it has been shown that small-interference RNA (siRNA) andor micro-RNA (miRNA) areother possible mechanisms to regulate translation process[30] More studies are needed to elucidate if altered siRNAor miRNA expressions are involved in differential RNA-protein levels observed between PE and control patientsNevertheless the high expression of RNA of these enzymesin patients with PE reported here could be reflecting acompensatory mechanism that for example ensures normalMHM enzyme levels to maintain the role of the MHM cyclein pregnancy physiology

Finally we measured the plasma concentrations of SAMand SAH in order to determine if there was an alteration inthese key metabolites in PE patients compared with controlsWith the observed higher mRNA expression we expected

decreased concentrations of SAM and increased SAH and2-ME levels No differences however were identified in theconcentrations of SAM or SAH between PE patients and thecontrol groups One possible explanation for this result isthe origin of our cohort The Chilean population has folicacid supplemented in the diet and most pregnant womentake additional folic acid supplementation (our patients werenot the exception) thus providing adequate amounts ofmethionine for conversion into SAM The measurement ofplasma folic acid concentrations may have provided furthersupport to this explanation The observation that SAMconcentrations are not reduced in patients that develop PEhowever suggests that other processes contribute to reducing2-ME

The data obtained in this study indicate that MHM isnot altered in women who subsequently develop PE Never-theless 2-ME concentrations are decreased as early as 11ndash14weeks of gestation [10] When interpreting the data howeverit must be taken into account that protein and RNA contentas well as the presence of SNPs were analyzed in placentawhile the SAM and SAH were measured in maternal plasmaIt is possible that thematernal aspect of the cycle is sustainingadequate concentrations of SAM and SAH Nevertheless itis likely that another pathway(s) account for the lower levelsof 2-ME in preeclamptic patients 2-ME is a natural metabo-lite of 17-120573-estradiol therefore reduced substrate availabil-ity andor conversion to 2ME may contribute to reducedbiosynthesis The aromatase pathway is responsible for theconversion of androgens into estrogens more specificallytestosterone and androstenedione into 17-120573-estradiol andestrone respectively [31] 17-120573-estradiol is hydroxylated at the2-position by the cytochrome p450 resulting in the synthesisof 2-HE that subsequently undergoes O-methylation of thecatechol ring by COMT to ultimately result in 2-ME Ithas also been suggested that aromatase is the rate-limitingenzyme in the production of 2-ME [32] making it a potentialkey enzyme to study in the development of PE as alterationsin its bioavailability may affect the levels of 2-ME Furtherstudies will be needed to test this hypothesis

5 Conclusion

In conclusion this study shows that women who developPE are clinically distinguishable at 11ndash14 weeks of gestationKey enzyme RNA expression is increased in preeclampticpatients but this change is not reflected at the protein contentSNP frequencies as well as SAM and SAH concentrationsdisplay no difference between women with PE and controlsThese results highlight a potential role of the MHM as acompensation mechanism in the presence of low levels of 2-ME

Ethical Approval

This study was approved by the Universidad de los AndesEthics Committee on August 23 2010 (Reference NumberEC0810)


Disclosure

Thispaper corresponds to an original studywith unpublisheddata that is not being submitted for publication elsewhere atthe same time

Conflict of Interests

There are no conflicts of interest for the authors

Authorsrsquo Contribution

Alejandra Perez-Sepulveda designed the main aspects ofthe study and conceived and designed experiments Shealso performed experiments and analyzed data She par-ticipated in the writing of the paper Sebastian E Illanescontributed to elaborate the initial idea about the study aswell as to design experiments He also participated in dataanalysis and the writing of the paper Pedro P Espana-PerrotXimena Fernandez B and Aneta Dobierzewska performedexperiments and analyzed data and also participated inthe writing of the paper Horacio Figueroa-Diesel and JoseAntonio Arraztoa contributed to the analysis of data andalso participated in the writing of the paper Gregory ERice participated in the analysis of the data and writing ofthe paper Vicente Bustos and Veronica Ahumada helpedperform experiments

Acknowledgments

The authors wish to acknowledge the staff of the Biologyof Reproduction Laboratory that helped in the collectionand order of detailed information which made this studypossible This paper is supported by FONDECYT 1110883

References

[1] L Duley ldquoThe Global Impact of Pre-eclampsia and EclampsiardquoSeminars in Perinatology vol 33 no 3 pp 130ndash137 2009

[2] L Bellamy J-P Casas A D Hingorani and D J WilliamsldquoPre-eclampsia and risk of cardiovascular disease and cancer inlater life systematic review and meta-analysisrdquo British MedicalJournal vol 335 no 7627 pp 974ndash977 2007

[3] SDMcDonald AMalinowskiQ Zhou S Yusuf andP J Dev-ereaux ldquoCardiovascular sequelae of preeclampsiaeclampsia asystematic review and meta-analysesrdquo American Heart Journalvol 156 no 5 pp 918ndash930 2008

[4] M L Mongraw-Chaffin P M Cirillo and B A CohnldquoPreeclampsia and cardiovascular disease death prospectiveevidence from the child health and development studiescohortrdquo Hypertension vol 56 no 1 pp 166ndash171 2010

[5] I Rauramo and M Forss ldquoEffect of exercise on placental bloodflow in pregnancies complicated by hypertension diabetesor intrahepatic cholestasisrdquo Acta Obstetricia et GynecologicaScandinavica vol 67 no 1 pp 15ndash20 1988

[6] R Pijnenborg L Vercruysse and I Brosens ldquoDeep placenta-tionrdquoBest Practice and Research vol 25 no 3 pp 273ndash285 2011

[7] F Rodesch P Simon C Donner and E Jauniaux ldquoOxygenmeasurements in endometrial and trophoblastic tissues during

early pregnancyrdquo Obstetrics and Gynecology vol 80 no 2 pp283ndash285 1992

[8] S B Lee A P Wong K Kanasaki et al ldquoPreeclampsia 2-Methoxyestradiol induces cytotrophoblast invasion and vascu-lar development specifically under hypoxic conditionsrdquo Ameri-can Journal of Pathology vol 176 no 2 pp 710ndash720 2010

[9] K Kanasaki K Palmsten H Sugimoto et al ldquoDeficiencyin catechol-O-methyltransferase and 2-methoxyoestradiol isassociated with pre-eclampsiardquo Nature vol 453 no 7198 pp1117ndash1121 2008

[10] A Perez-Sepulveda M J Torres F J Valenzuela R LarraınH Figueroa-Diesel et al ldquoLow 2-methoxyestradiol levels at thefirst trimester of pregnancy are associatedwith the developmentof pre-eclampsiardquo Prenat Diagn vol 32 no 11 pp 1053ndash10582012

[11] K Palmer B Saglam C Whitehead O Stock M Lappas andS Tong ldquoSevere early-onset preeclampsia is not associated witha change in placental catechol O-methyltransferase (COMT)expressionrdquo American Journal of Pathology vol 178 no 6 pp2484ndash2488 2011

[12] A Perez-Sepulveda P P Espana-Perrot E R Norwitz and SE Illanes ldquoMetabolic pathways involved in 2-methoxyestradiolsynthesis and their role inpreeclampsiardquo Reproductive SciencesIn press

[13] E Lopez-Quesada M A Vilaseca and J M Lailla ldquoPlasmatotal homocysteine in uncomplicated pregnancy and in pree-clampsiardquoEuropean Journal of Obstetrics Gynecology and Repro-ductive Biology vol 108 no 1 pp 45ndash49 2003

[14] V ShenoyKKanasaki andRKalluri ldquoPre-eclampsia connect-ing angiogenic and metabolic pathwaysrdquo Trends in Endocrinol-ogy and Metabolism vol 21 no 9 pp 529ndash536 2010

[15] R J L M Cikot R P M Steegers-Theunissen C M GThomas T M De Boo H M W M Merkus and E A PSteegers ldquoLongitudinal vitamin and homocysteine levels innormal pregnancyrdquo British Journal of Nutrition vol 85 no 1pp 49ndash58 2001

[16] MMMurphy JM Scott J MMcPartlin and J D Fernandez-Ballart ldquoThe pregnancy-related decrease in fasting plasmahomocysteine is not explained by folic acid supplementationhemodilution or a decrease in albumin in a longitudinal study1-3rdquoAmerican Journal of Clinical Nutrition vol 76 no 3 pp 614ndash619 2002

[17] R W Powers R W Evans A K Majors et al ldquoPlasmahomocysteine concentration is increased in preeclampsia andis associated with evidence of endothelial activationrdquo AmericanJournal of Obstetrics and Gynecology vol 179 no 6 pp 1605ndash1611 1998

[18] J Wang B J Trudinger N Duarte D E Wilcken and X LWang ldquoElevated circulating homocyst(e)ine levels in placentalvascular disease and associated pre-eclampsiardquo British Journalof Obstetrics and Gynaecology vol 107 no 7 pp 935ndash938 2000

[19] R DrsquoAnna G Baviera F Corrado R Ientile D Granese andN C Stella ldquoPlasma homocysteine in early and late pregnanciescomplicatedwith preeclampsia and isolated intrauterine growthrestrictionrdquo Acta Obstetricia et Gynecologica Scandinavica vol83 no 2 pp 155ndash158 2004

[20] G Makedos A Papanicolaou A Hitoglou et al ldquoHomocys-teine folic acid and B12 serum levels in pregnancy complicatedwith preeclampsiardquo Archives of Gynecology and Obstetrics vol275 no 2 pp 121ndash124 2007


[21] M FodingerWHHorl andG Sunder-Plassmann ldquoMolecularbiology of 510-methylenetetrahydrofolate reductaserdquo Journal ofNephrology vol 13 no 1 pp 20ndash33 2000

[22] P Sharma R D Senthilkumar V Brahmachari et al ldquoMiningliterature for a comprehensive pathway analysis a case studyfor retrieval of homocysteine related genes for genetic andepigenetic studiesrdquo Lipids in Health and Disease vol 5 article1 2006

[23] L D Hill T P York J P Kusanovic et al ldquoEpistasis betweenCOMT and MTHFR in maternal-fetal dyads increases risk forpreeclampsiardquo PLoS ONE vol 6 no 1 Article ID e16681 2011

[24] D L F FurnessM F Fenech Y T Khong R Romero andG ADekker ldquoOne-carbon metabolism enzyme polymorphisms anduteroplacental insufficiencyrdquoAmerican Journal of Obstetrics andGynecology vol 199 no 3 pp 276e1ndash276e8 2008

[25] L C Y Poon N A Kametas C Valencia T Chelemen and KH Nicolaides ldquoHypertensive disorders in pregnancy screeningby systolic diastolic and mean arterial pressure at 11ndash13 weeksrdquoHypertension in Pregnancy vol 30 no 1 pp 93ndash107 2011

[26] S Sohlberg O Stephansson S Cnattingius and A-K Wik-strom ldquoMaternal body mass index height and risks ofpreeclampsiardquo American Journal of Hypertension vol 25 no 1pp 120ndash125 2012

[27] A Syngelaki F E Bredaki E Vaikousi N Maiz and K HNicolaides ldquoBody mass index at 11-13 weeksrsquo gestation andpregnancy complicationsrdquo Fetal Diagnosis andTherapy vol 30no 4 pp 250ndash265 2011

[28] L Robertson O Wu P Langhorne et al ldquoThrombophilia inpregnancy a systematic reviewrdquo British Journal of Haematologyvol 132 no 2 pp 171ndash196 2006

[29] C Vogel and E M Marcotte ldquoInsights into the regulation ofprotein abundance from proteomic and transcriptomic analy-sesrdquo Nature Reviews Genetics vol 13 no 4 pp 227ndash232 2012

[30] B Zhang Q Wang and X Pan ldquoMicroRNAs and their regula-tory roles in animals and plantsrdquo Journal of Cellular Physiologyvol 210 no 2 pp 279ndash289 2007

[31] I Czajka-Oraniec and E R Simpson ldquoAromatase research andits clinical significancerdquoEndokrynologia Polska vol 61 no 1 pp126ndash134 2010

[32] A Hertig P Liere N Chabbert-Buffet et al ldquoSteroid profilingin preeclamptic women evidence for aromatase deficiencyrdquoAmerican Journal of Obstetrics and Gynecology vol 203 no 5pp e471ndashe1479 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the third trimester of pregnancy in patients that would laterdevelop PE as compared with controls [9] and our groupfound the same trend during the early first trimester ofpregnancy [10] With COMT being responsible for methy-lating 2-Hydroxyestradiol (2-HE) into 2-ME it has beenproposed that the low activity or expression of this enzymecould be involved in the pathogenesis of PE The normalexpression of COMT in placentae of patients with PE [11] andthe conflicting results regarding the presence of SNPs thatmay reduce the activity of this enzyme in these patients [10]force us to search other explanations [12] The Methionine-homocysteine metabolism (MHM) the process responsiblefor supplying COMT with the methyl group necessary for 2-ME synthesis could be an alternative if it is somehow alteredbeing unable to supply enough methyl groups to sustainadequate concentrations of 2-ME

It has been shown that alterations in the methionine-homocysteine metabolism (MHM) may be related to thesystemic damage that leads to the classical clinical pictureof PE [13 14] In fact in normal pregnant women plasmaconcentrations of homocysteine are low in the first trimesterof pregnancy and reach their lowest level in the secondhalf of gestation [15 16] whereas hyperhomocysteinemiahas been described in women who would develop PE [17ndash20] Homocysteine is biosynthesized by the MHM in aprocess involving the donation of a methyl group by S-adenosylmethionine (SAM) which in turn gets convertedinto S-adenosylhomocysteine (SAH) a precursor of homo-cysteine Through the enzyme methionine synthase (MTR)homocysteine may be remethylated into methionine using5-methyltetrahydrofolate (5-Methyl-THF) as a cosubstrateMethylenetetrahydrofolate reductase (MTHFR) is the cata-lyst for the conversion of 510-methylenetetrahydrofolate into5-methylTHF Methionine can then be converted into SAMonce more to restart the cycle [21] SAM acts as the principalmethyl donor within cells These methyl groups are keyfactors in DNA methylation that might control for examplecellular proliferation cellular migration differentiation cell-to-cell recognition and other important processes such asthe production of 2-ME through COMT

Low levels of SAM may be caused by the presence ofsingle-nucleotide polymorphisms (SNPs) in the genes thatcode for the enzymes involved in SAM metabolism such asMTR and MTHFR [22] Hill et al recently described thatMTHFR modulates the availability of SAM and the minorldquoTrdquo allele of the MTHFR C677T SNP has been associatedwith reduced MTHFR activity resulting in a decrease inSAM production [23] Furness et al suggested that thematernal and placental MTR A2756G allele is an importantrisk factor for the development of uteroplacental insufficiency[24] though they did not link it to SAM levels and attributedthe uteroplacental insufficiency directly to the enzyme

We hypothesize that changes in the levels of MTR andMTHFR by the abovementioned SNPs have a negative effecton SAM levels which when lowered contribute to thedevelopment of PEThe purpose of this study was to evaluatethe bioavailability of SAM as well as of MTR and MTHFRtheir respective SNPs and their relation with preeclampsia

2 Methods

21 Study Design A prospective cohort study was conductedin the Obstetrics and Fetal Medicine Unit from Hospital Par-roquial de San Bernardo Santiago Chile Sixteen preeclamp-tic patients and 32 controls were recruited from this cohortBoth groups consisted of women with singleton gestationand none of them took multivitamins or aspirin duringpregnancy Maternal anthropometric data and blood sampleswere collected at 11ndash14-week gestation and at 32ndash36 weeks PEwas defined as hypertension (arterial pressure (AP) higheror equal to AP 14090mmHg on two occasions separatedby 6 h or higher or equal to 160110mmHg) that occurredafter 20 weeks of gestation in women with previously normalblood pressure accompanied by proteinuria (300mg24 h)Controls who do not differ in racial origin from PE patientswere healthy subjects without pregnancy complications orchronic medical problems

Written informed consent was obtained from the womenwho agreed to participate in the study which was approvedby Hospital Parroquial de San Bernardo and the Universidadde los Andes Ethics Committee

22 Tissue and Blood Sample Collection Placental tissueswere obtained within 15min of delivery from all of thesubjects Placental samples (asymp1 cm3) were taken from astandardized location a placental cotyledon between cordinsertion and placental border midway avoiding tissue fromareas showing placental infarcts The tissue was washedin ice-cold physiologic solution (NaCl 09) to removematernal blood contamination Samples were processed andstored at minus80∘C until use for RNA extraction SNP analysisand protein quantification Blood was collected into edeticacid (EDTA) Vacutainer tubes at 11ndash14 weeks and at 32ndash34 weeks of gestation placed in 16mL DNAseRNAse-freeEppendorf tubes and centrifuged at 1300 g for 10min atroom temperatureThe supernatant was transferred to a fresh16mL tube and centrifuged oncemore at 7200 g for 10min atroom temperatureThe supernatant was transferred to a freshtube and stored at minus80∘C

23 Protein Quantification A sim5mg section from a pla-cental tissue sample (spanning from the maternal to fetalface) was homogenized in a 16mL Eppendorf tube usingradioimmunoprecipitation assay (RIPA) lysis buffer (50mMTris-HCl pH 88 150mM NaCl 05 sodium deoxycholate01 Sodium Dodecyl Sulfate (SDS) 1 Tergitol-type NP-40) with protease inhibitor (cOmplete Mini Roche) Thehomogenate was centrifuged at 25800 g for 30min at 4∘CThe supernatantwas carefully transferred to a different 16mLEppendorf and stored at minus20∘C Total protein amount inthe supernatant was quantified using BCA Protein Assay Kit(Thermo Scientific) and spectrophotometer NanoDrop 2000(Thermo Scientific) according tomanufacturerrsquos instructions20120583g of total protein was loaded for each sample in 12polyacrylamide gels that were additionally corrected usingCoomassie Blue staining and densitometric analysis Aninternal control was prepared by pooling 4 different samples





2O Strata X columns


2O Samples










3 Results






















4 Discussion



Control PE 0

20

40

60M

THFR

RN

A le

vels

(fg120583

L)

lowast

(a)

Control PE 0

50

100

150

MTR

RN

A le

vels

(fg120583

L)

lowastlowast

(b)


Control PE 00

05

10

15

20

MTH

FR p

rote

in le

vels

(au

)

(a)

Control PE 00

02

04

06

08

10M

TR p

rote

in le

vels

(au

)

(b)


Control PE0

10

20

30

40

SAH

leve

ls (n

gm

L)

(a)

Control PE0

50

100

150

SAM

lev

els (n

gm

L)

(b)










5 Conclusion


Ethical Approval



Disclosure






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2O Strata X columns


2O Samples










3 Results






















4 Discussion



Control PE 0

20

40

60M

THFR

RN

A le

vels

(fg120583

L)

lowast

(a)

Control PE 0

50

100

150

MTR

RN

A le

vels

(fg120583

L)

lowastlowast

(b)


Control PE 00

05

10

15

20

MTH

FR p

rote

in le

vels

(au

)

(a)

Control PE 00

02

04

06

08

10M

TR p

rote

in le

vels

(au

)

(b)


Control PE0

10

20

30

40

SAH

leve

ls (n

gm

L)

(a)

Control PE0

50

100

150

SAM

lev

els (n

gm

L)

(b)










5 Conclusion


Ethical Approval



Disclosure






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































4 Discussion



Control PE 0

20

40

60M

THFR

RN

A le

vels

(fg120583

L)

lowast

(a)

Control PE 0

50

100

150

MTR

RN

A le

vels

(fg120583

L)

lowastlowast

(b)


Control PE 00

05

10

15

20

MTH

FR p

rote

in le

vels

(au

)

(a)

Control PE 00

02

04

06

08

10M

TR p

rote

in le

vels

(au

)

(b)


Control PE0

10

20

30

40

SAH

leve

ls (n

gm

L)

(a)

Control PE0

50

100

150

SAM

lev

els (n

gm

L)

(b)










5 Conclusion


Ethical Approval



Disclosure






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control PE 0

20

40

60M

THFR

RN

A le

vels

(fg120583

L)

lowast

(a)

Control PE 0

50

100

150

MTR

RN

A le

vels

(fg120583

L)

lowastlowast

(b)


Control PE 00

05

10

15

20

MTH

FR p

rote

in le

vels

(au

)

(a)

Control PE 00

02

04

06

08

10M

TR p

rote

in le

vels

(au

)

(b)


Control PE0

10

20

30

40

SAH

leve

ls (n

gm

L)

(a)

Control PE0

50

100

150

SAM

lev

els (n

gm

L)

(b)










5 Conclusion


Ethical Approval



Disclosure






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























5 Conclusion


Ethical Approval



Disclosure






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Disclosure






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















levels of key enzymes of methionine-homocysteine metabolism in

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