Hindawi Publishing Co po a ion
Spec oscopy: An In e na ional Jou nal
Volume 27 (2012), Issue 5-6, Pages 515–523
doi:10.1155/2012/128367
Can Bio luids Me abolic P o ilingHelp o
Imp o e Heal hca e du ing P egnancy?
Gonc¸alo G ac¸a,1S´
ıl ia O. Diaz,1Joana Pin o,1An ´
onio S. Ba os,2Iola F. Dua e,1
B ian J. Good ellow,1Eul´
alia Galhano,3C is ina Pi a,3Ma ia do C´
eu Almeida,3
Isabel M. Ca ei a,4and Ana M. Gil1
1CICECO Depa men o Chemis y, Uni e si y Campus o San iago, Uni e si y o A ei o,
3810-193 A ei o, Po ugal
2QOPNA Resea ch Uni , Depa men o Chemis y, Uni e si y Campus o San iago,
Uni e si y o A ei o, 3810-193 A ei o, Po ugal
3Ma e nidade Bissaya Ba e o, Cen o Hospi ala de Coimb a, 3000 Coimb a, Po ugal
4Cy ogene ics and Genomics Labo a o y, Facul y o Medicine, Uni e si y o Coimb a and
Fo ensic Science Cen e (CENCIFOR), Coimb a, Po ugal
Co espondence should be add essed o Ana M. Gil, [email p o ec ed]
Copy igh © 2012 Gonc¸alo G ac¸a e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion
License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly
ci ed.
Abs ac . This pape desc ibes a me abonomics s udy o 2nd imes e bio luids (amnio ic luid, ma e nal u ine, and blood
plasma), in an a emp o co ela e bio luid me abolic changes wi h suspec ed/diagnosed e al mal o ma ions (FM) and
ch omosomal diso de s as well as wi h la e occu ing ges a ional diabe es melli us (GDM), p e e m deli e y (PTD), and
p ema u e up u e o memb anes (PROM). The global biochemical pic u e gi en by he h eesome o bio luids should enable
he de ini ion o po en ial disease signa u es and un eil po en ial me aboli e ma ke s o clinical use in p edic i e p ena al
diagnos ics. Resul s show ha ela i ely s ong me abolic dis u bances accompany FM, e lec ed in all h ee bio luids and
hus sugges ing he in ol emen o bo h e al and ma e nal me abolisms. Rega ding GDM, amnio ic luid and ma e nal u ine
seem po en ial good media o de ec ea ly me abolic changes, and PTD subjec s show small me aboli e changes in he same
bio luids, unde going wo k being ocused on plasma composi ion. Ch omosomal diso de s show an in e es ingly ma ked e ec
on ma e nal u ine, whe eas no s a is ically ele an ea ly changes ha e been obse ed o PROM subjec s. In e es ingly, in he
case o FM and ch omosomal diso de s, ma e nal bio luids show some sensi i i y o diso de ype, o example, o cen al
ne ous sys em mal o ma ions and isomy 21, espec i ely. These esul s show he use ulness o bio luid me abonomics o
p obe o e all me abolic dis u bances in ela ion o p ena al diso de s.
Keywo ds: P ena al heal h, p egnancy, diagnosis, diabe es, p e e m, isomy, amnio ic luid, u ine blood, me abolomics,
me abonomics, NMR, mul i a ia e analysis
1. In oduc ion
P ena al diagnos ic me hods include a a ie y o p ocedu es, wi h emphasis on ou ine ul asound
es ing, o en in combina ion wi h ma e nal se um ma ke s, o de ec ion o ch omosomal diso de s
516 Spec oscopy: An In e na ional Jou nal
and e al mal o ma ions. Fo highe isk p egnancies, diagnosis o ch omosomal anomalies o speci ic
gene ic diseases is based on cho ionic illus sampling, amniocen esis and co docen esis, in asi e
p ocedu es which s ill ca y some isk o misca iage, in ec ion, o p ema u e bi h [1]. In addi ion,
diso de s such as ges a ional diabe es melli us (GDM), p eeclampsia (PE), p e e m deli e y (PTD),
small o ges a ional age (SGA), o in au e ine g ow h es ic ion (IUGR) may pose a signi ican isk o
mo he and e us bu canno easily be p edic ed by he p esen ly a ailable me hods.
This pape desc ibes he use o me abonomics, mainly based on nuclea magne ic esonance
(NMR) spec oscopy, as a holis ic analy ical s a egy in he ques o new and ea lie bioma ke s o
bo h e al and ma e nal heal h. In his con ex , p e ious epo s o NMR o amnio ic luid (AF) ha e
add essed, a a p elimina y le el, e al ma u i y [2,3], ges a ional age [4,5], and se e al diso de s, o
example, PE, e al mal o ma ions (FM), GDM, PTD, and p ema u e up u e o memb anes (PROM)
[2,6–8]. Me aboli e p o iling o u ine and blood has been, howe e , less de eloped, in spi e o he g ea
po en ial o hese bio luids o less-in asi e diagnosis me hods. In his con ex , some s udies on blood
ha e ocused on PE, employing bo h NMR [9,10] and ul a high pe o mance liquid ch oma og aphy
and mass spec ome y (UPLC-MS) [11]. He eby, a me abonomics s udy o 2nd imes e bio luids (AF,
ma e nal u ine, and blood plasma) is p esen ed, in an a emp o co ela e bio luid me abolic changes
wi h suspec ed/diagnosed FM and ch omosomal diso de s, and wi h GDM, PTD, and PROM de eloped
la e in p egnancy. The global biochemical pic u e gi en by he h eesome o bio luids should enable
he de ini ion o po en ial disease signa u es and un eil po en ial me aboli e ma ke s o clinical use in
p ena al diagnos ics and p ognos ics.
2. Expe imen al
2.1. Samples
Amnio ic luid (AF), u ine, and blood plasma samples we e collec ed a he ime o amniocen esis (14–
25 ges a ional weeks, g.w.), o p egnan women aged >35 o based on medical his o y, and s o ed
a −80◦C un il analysis. All p egnancies we e ollowed un il bi h, e en ually de ining sample g oups
acco ding o hei clinical ou come: con ols, FM, p ediagnos ic GDM ( o women diagnosed wi h GDM
la e in hei p egnancy), p e-PTD ( o women who ga e bi h p io o 37g.w.), p e-PROM ( o women
who had p elabo up u e o memb anes a e 37g.w.), and ch omosomal diso de s (gene ally diagnosed
ca. 2 weeks a e amniocen esis). O he diso de s we e no conside ed a his s age due o insu icien
sample numbe s. Table 1 lis s sample numbe s, and de ailed sample p epa a ion o analysis is desc ibed
elsewhe e [8,12].
2.2. NMR Spec oscopy
All 1D and 2D NMR spec a we e eco ded on a B uke A ance DRX 500 spec ome e equipped wi h
an ac i ely shielded g adien uni wi h a maximum g adien s eng h ou pu o 53.5G/cm, a 300 K.
The de ailed desc ip ion o he acquisi ion condi ions and pa ame e s employed is desc ibed elsewhe e
[8,12].
Spec oscopy: An In e na ional Jou nal 517
Table 1: Lis o 2nd imes e amnio ic luid, u ine, and blood plasma samples collec ed.
Condi ion AF U ine Blood plasma
Con ols∗82 25 20
Fe al mal o ma ions (FM)∗27 29 27
P ediagnos ic ges a ional diabe es melli us
(GDM)∗27 29 14
P e e m deli e y (PTD)∗12 19 6
P eeclampsia (PE) 5 5 2
P ema u e up u e o memb anes (PROM)∗34 38 18
In au e ine g ow h es ic ion (IUGR) 6 5 4
Ch omosomal diso de s (CD)∗10 (1) 25 (9) 23 (8)
∗G oups wi h su icien sample numbe s o allow mul i a ia e analysis o be pe o med, a his s age. Numbe s in b acke s
indica e numbe o isomy 21 cases.
2.3. Chemome ics
NMR da a we e p ocessed o chemome ics using no maliza ion o o al spec al a ea and di e en
a iable scaling me hods o compa ison pu poses (uni a iance, Pa e o and cen e ed scaling). P incipal
componen analysis (PCA) [13] and pa ial leas squa es disc iminan analysis (PLS-DA) [14] and i s
o hogonal a ian (O-PLS-DA) [15] we e applied o he da a, using SIMCA-P 11.5 (Ume ics, Ume˚
a,
Sweden). Mon e-Ca lo c oss- alida ion (MCCV) was also applied, along wi h spec al in eg a ion o
ele an signals.
3. Resul s and Discussion
Gi en he sample numbe s shown in Table 1, only he esul s ob ained o he highligh ed g oups a e
p esen ed below. Figu e 1 shows ypical 1D s anda d 1H NMR spec a o 2nd imes e AF and ma e nal
blood plasma and u ine, illus a ing he complex na u e o he da a, which comp ises many ens hund eds
o signals, g ea ly o e lapped in some spec al egions. As expec ed, i is clea ly seen ha he plasma
1H NMR spec um is domina ed by b oade lipid esonances, whe eas bo h AF and u ine spec a show
a p edominance o na ow esonances. In he case o AF and plasma, a se o addi ional 1D expe imen s
has been ca ied ou (no shown) in an a emp o e eal u he spec al in o ma ion: he Ca -Pu cell-
Meiboom-Gill (CPMG) expe imen which eco ds only he na owe signals om as umbling, ha
is, smalle me aboli es, and a di usion-edi ed expe imen which eco ds he b oad p o ile o slowe
umbling molecules such as p o eins and lipids. The weal h o composi ional in o ma ion p esen in 1H
NMR spec a has been ansla ed in o a lis o me aboli es p esen in each bio luid, h ough a ange o 2D
NMR expe imen s (homonuclea and he e onuclea co ela ions and J- esol ed) [16] and compa ison o
spec al p o iles wi h hose ound in spec al da abases (B uke Bio e code da abase and o he da abases
a ailable online [17,18]).
In his way, a me aboli e p o ile has been es ablished o each bio luid [7,8,12] as ypical o
2nd imes e con ols (heal hy p egnancies h oughou ) and se ing as a e e ence o he de ec ion
o e en ual spec al p o ile changes ela ed o each diso de , in o de o iden i y s a is ically ele an
518 Spec oscopy: An In e na ional Jou nal
19 8765432
(ppm)
×8
(a)
198765432
(ppm)
×8
(b)
198765432
(ppm)
×4
(c)
Figu e 1: Typical s anda d 1H NMR spec a o 2nd imes e (a) amnio ic luid, (b) ma e nal blood
plasma, and (c) ma e nal u ine.
diso de signa u es. This has been ca ied ou wi h he aid o mul i a ia e analysis o he 1HNMR
1D spec a, and Figu e 2(a) shows a PLS-DA sco es plo o he 1H NMR spec a o amnio ic luid
co esponding o con ols and FM g oups. The appa en g oup sepa a ion sugges s ha some di e ences
exis be ween he spec al p o iles ep esen a i e o he wo g oups; howe e , his mus be e alua ed and
in e p e ed wi h ca e, employing bo h MCCV alida ion and con i ma ion o spec al changes h ough
spec al inspec ion and, when possible, signal in eg a ion and uni a ia e analysis (P alue <0.05). In
he case shown, he s a is ical ele ance o he g oup sepa a ion obse ed is exp essed by a su icien ly
high Q2 alue (0.38) and classi ica ion a e (89%), as gi en by MCCV alida ion [8]. In e p e a ion o
he unde lying o igins o g oup sepa a ion was based on he co esponding loadings plo (Figu e 2(b)),
which shows posi i e and nega i e peaks, co esponding o me aboli es mo e concen a ed in con ols
and in he FM g oup, espec i ely. In his way, changed le els we e no ed in 23 me aboli es p esen
in he AF o mal o med e uses: inc eased asco ba e, c ea inine, glycop o eins, glycine, glu amine,
lac a e, me hionine, se ine, succina e, and h eonine along wi h dec eased α-oxoiso ale a e, alanine,
phenylalanine, glucose, isoleucine, leucine, py u a e, y osine, aline, and 4 unknowns [8]. Addi ional
UPLC-MS wo k on amnio ic luid [19] has de ec ed inc eases in ca ni ine, py oglu ama e, and polyol
con en s. The me aboli e changes no ed a e consis en wi h enhanced glycolysis, possibly unde e al
Spec oscopy: An In e na ional Jou nal 519
LV1
LV2
−40
−20
−20
−15
−10
−5
0
0
5
10
15
20
20
R2X=0.14 R2Y=0.758 Q2=0.383
(a)
Leucine
Succina e
Loadings [1]
Chemical shi (ppm)
1e−04
0e+00
−1e−04
Valine
Lac a e
Lac a e
N-Ace yl
Glu amine
Glu amine
Glycine
C ea inine
Th eonine
Glucose
1432
1.06 ppm
α-Oxoiso ale a e
(b)
Figu e 2: PLS-DA sco es and LV1 loadings o NMR o AF o con ols and FM g oups.
hypoxia (o igina ing enhanced usage o glucose and lac a e inc ease) and educed use o he espi a o y
chain pa hway (consis en wi h a succina e inc ease). In ela ion o amino acids, he dec eases in
glucogenic amino acids (alanine, isoleucine, glu ama e, me hionine, phenylalanine, y osine, aline)
may indica e hei enhanced use in gluconeogenesis o he eplenishmen o glucose, whe eas inc eases
in glycine, glu amine, se ine, and h eonine sugges he possible in ol emen o hese amino acids
in o he pa hways. Fo ins ance, a highe glu amine le el sugges s e al kidney unde de elopmen ,
which is also consis en wi h he obse ed accumula ion o glycop o eins. Glycine and se ine inc eases
sugges a dis u bance in olic acid pool egula ion, wi h possible e lec ions in amino acid and choline
me abolisms. O he indica o s ela e o he ma ked dec eases in leucine and α-oxoiso ale a e, which
may indica e a highe demand o p o ein syn hesis in mal o med e uses. Finally, he ma ked inc ease
in asco ba e may also e lec amino acid and/o nucleo ide suga biosyn hesis al e a ions since his
compound is in ol ed in suga in e con e sion pa hways. These me abolic dis u bances ha e been
suppo ed by he pa allel analysis o ma e nal plasma and u ine [12], which has p o ided u he
e idence o hypoxia and enhanced lipid oxida ion, oge he wi h choline me abolism dis u bance. In
addi ion, p elimina y esul s on plasma sugges a me abolic-based sepa a ion o cen al ne ous sys em

520 Spec oscopy: An In e na ional Jou nal
0
25
25
50
50
R2X=0.498 R2Y=0.428 Q2=0.269
−50
−50
−25
−25
LV1
LV2
0
(a)
0
50
100
050
100
R2X=0.156 R2Y=0.912 Q2=0.278
−50
−50
−100
−100
LV1
LV2
(b)
0
10
20
01020
R2X=0.128 R2Y=0.807 Q2=0.229
LV1
LV2
−20
−20
−10
−10
(c)
Figu e 3: Compa ison o PLS-DA sco es ob ained o (a) amnio ic luid, (b) blood plasma, and (c) u ine
co esponding o con ol and p ediagnos ic GDM g oups.
mal o ma ion cases om he emaining ypes (ca diac, u ogeni al, so issues, and pulmona y), hus
opening in e es ing possibili ies as o he speci ici y o di e en bio luids o di e en mal o ma ion
ypes.
A simila s a egy has been applied o he emaining diso de g oups conside ed in his s udy:
p ediagnos ic GDM, p e-PTD, p e-PROM, and ch omosomal diso de s. The PLS-DA sco e plo s
ob ained o each o he h ee bio luids co esponding o con ol and p ediagnos ic GDM g oups
e lec a weake me abolic impac o his diso de s, exp essed by he lowe Q2 alues (Figu e 3).
In e es ingly, he esul s ha e shown ha a small inc ease in glucose is appa en in AF [8], bu no
in he co esponding plasma and u ine, a he ime o sample collec ion, ha is, se e al weeks be o e
clinical diagnosis [12]. In addi ion, changes in 9 u he me aboli es ha e been no ed in AF (c ea inine,
ace a e, o ma e, glu amine, glycine, p oline, glyce ophosphocholine (GPC), se ine, and au ine) along
wi h be aine and ime hylamine-N-oxide in blood and 7 me aboli es in u ine (3-hyd oxyiso ale a e,
Spec oscopy: An In e na ional Jou nal 521
Table 2: Summa y o he impac o diso de s on he me aboli e p o iling o 2nd imes e amnio ic luid,
u ine, and blood plasma.
Diso de FM P ediagnos ic GDM P e-PTD P e-PROM Ch omosomal diso de s
Bio luid
Amnio ic luid +++ ++ + + +
Ma e nal blood plasma +++∗+ab +∗∗
Ma e nal u ine +++ ++ + b++∗∗
aInsu icien sample numbe s a his s age, bno me aboli e changes no ed, ∗di e en mal o ma ion ypes, and ∗∗ ch omosomal
diso de s a e appa en ly dis inguishable, + small impac , ++ medium impac , +++ la ge impac .
2-hyd oxyisobu y a e, choline, N-me hyl-2-py idone-5-ca boxamide (2PY), N-me hyl-nico inamide
(NMND), and 2 unknowns). In spi e o he low magni ude o hese changes and he low p edic i e
alue o he models ob ained so a [8,12], small me abolic a ia ions seem o cha ac e ise women
who will de elop GDM la e on in p egnancy, pa icula ly a he le els o e al insulin managemen and
nucleo ide and amino acid me abolisms.
In ela ion o p e-PTD subjec s ( esul s no shown), allan oin (possible ma ke o oxida i e
s ess) has been ound o be aised in AF along wi h 4 dec eased me aboli es (alanine, ci a e, myo-
inosi ol, and 1 unknown) [8]. In addi ion, ma e nal u ine shows aised le els o 3 me aboli es (2-
hyd oxyisobu y a e, choline, and 1 unknown), sugges ing ea ly dis u bances in choline and amino
acid/gu mic o lo a me abolisms, al hough no excluding a possible ela ionship o BMI alues [12].
In ela ion o ch omosomal diso de s, i was in e es ing o no e ha lipids seem o play an impo an
ole as a e lec ion o me abolic dis u bances, as iewed h ough blood lipop o ein p o iles and a u ine
choline inc ease. Fu he mo e, i became appa en ( esul s no shown) ha he plasma lipid p o iles
may be sensi i e o ch omosomal diso de ype, wi h isomy 21 samples ha ing been dis inguished
om o he diso de s [12]. Finally, p e-PROM subjec s ha e shown no signi ican changes in bio luid
composi ion, wi h he excep ion o a ain sugges ion o amino acid me abolism dis u bance, as iewed
h ough AF [8].
4. Conclusion
Table 2 summa ises he esul s desc ibed abo e, indica ing ela i ely s ong me abolic dis u bances
accompanying e al mal o ma ions, e lec ed in all h ee bio luids (AF, ma e nal u ine, and plasma)
and hus sugges ing he in ol emen o bo h e al and ma e nal me abolisms. Rega ding GDM, AF, and
ma e nal u ine seem o be po en ially good media o de ec small ea ly me abolic changes ha ake
place p io o he clinical diagnosis o inc eased blood glucose. P e-PTD subjec s show small me aboli e
changes in he same bio luids, unde going wo k being ocused on plasma composi ion. Ch omosomal
diso de s show an in e es ingly ma ked e ec on ma e nal u ine, along wi h weake a ia ions no ed in
AF and blood plasma. In he case o he e ogeneous g oups such as FM and ch omosomal diso de s,
he ma e nal bio luids ha e shown some sensi i i y o diso de ype, o example, o cen al ne ous
sys em mal o ma ions and isomy 21, espec i ely. Finally, no s a is ically ele an changes ha e been
522 Spec oscopy: An In e na ional Jou nal
obse ed o p e-PROM subjec s, al hough a sugges ion o amino acid dis u bance has been egis e ed
o amnio ic luid.
These esul s show he use ulness o bio luid me abonomics o p obe o e all me abolic dis u b-
ances in ela ion o p ena al diso de s. This wo k may be pu sued along se e al a enues, one in ol ing
he use o mo e sophis ica ed mul i a ia e analysis me hods o ind concomi an and in e connec ed
me aboli e changes, ei he be ween da ase s (e.g., NMR and MS) o be ween di e en bio luids.
Fu he mo e, new de elopmen s should include a comp ehensi e ollow-up s udy h oughou he whole
p egnancy pe iod, so ha ime cou se in o ma ion may be ob ained and a mo e comple e me abolic
pic u e may be o med o p egnancy i sel and he ele an diso de s a ec ing i .
Acknowledgmen s
Funding is acknowledged om he Eu opean Regional De elopmen Fund h ough he Compe i i e
Fac o s Thema ic Ope a ional P og amme and om he Founda ion o Science and Technology (FCT),
Po ugal ( esea ch P ojec PTDC/QUI/66523/2006 and esea ch G an s SFRH/BD/41869/2007 and
SFRH/BD/64159/2009). The Po uguese Na ional NMR Ne wo k (RNRMN), suppo ed wi h FCT unds
is also acknowledged, and he au ho s a e g a e ul o M. Sp aul, B uke BioSpin, Ge many, o p o iding
access o spec al NMR da abases.
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