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SCIENTI IC RePoR S | 7: 16267 | DOI:10.1038/s41598-017-15809-4
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Simila i ies be ween acylca ni ine
p o iles in la ge o ges a ional age
newbo ns and obesi y
Paula Sánchez-Pin os1, Ma ia-Jose de Cas o1, I ia Roca1, Segundo Ri e2, Miguel López3,4 &
Ma ia-Luz Couce1
La ge o ges a ional age (LGA) newbo ns ha e an inc eased isk o obesi y, insulin esis ance, and
me abolic synd ome. Acylca ni ine p o iles in obese child en and adul s a e cha ac e ized by inc eased
le els o C3, C5, and ce ain medium-chain (C12) and long-chain (C14:1 and C16) acylca ni ines. C2 is
also inc eased in insulin- esis an s a es. In his 1-yea obse a ional s udy o 2514 newbo ns (246 LGA
newbo ns, 250 small o ges a ional age (GA) newbo ns, and 2018 app op ia e o GA newbo ns), we
analyzed and compa ed pos na al acylca ni ine p o iles in LGA newbo ns wi h p o iles desc ibed o
obese indi iduals. Acylca ni ine analysis was pe o med by andem mass spec ome y on d ied-blood
spo s collec ed on day 3 o li e. LGA newbo ns had highe le els o o al sho -chain acylca ni ines
(p < 0.001), C2 (p < 0.01) and C3 (p < 0.001) acylca ni ines, and all C12, C14, and C16 acylca ni ines
excep C12:1. They also had a highe endency owa ds ca ni ine insu iciency (p < 0.05) and ca ni ine
de iciency (p < 0.001). No signi ican di e ences we e obse ed be ween LGA newbo ns bo n o
mo he s wi h o wi hou a his o y o ges a ional diabe es. This no el s udy desc ibes a pos na al
acylca ni ine p o ile in LGA wi h highe le els o C2, C3, o al acylca ni ines, and o al sho -chain
acylca ni ines ha is cha ac e is ic o childhood and adul obesi y and linked o an unheal hy me abolic
pheno ype.
La ge o ges a ional age (LGA) is de ined as a bi h weigh abo e he 90 h pe cen ile o he co esponding ges-
a ional age (GA)1. The ma e nal ac o s mos closely associa ed wi h LGA a e ma e nal obesi y, excessi e ges a-
ional weigh gain, ma e nal ges a ional diabe es melli us (GDM), p eges a ional obesi y, and ma e nal s ess2–9.
Fe al ac o s associa ed wi h LGA consis p ima ily o gene ic o ch omosomal diso de s. LGA p e alence is
es ima ed a be ween 4.6% and 15.3%10 and is in luenced by e hnici y, wi h highe a es ound in child en bo n o
A ican Ame ican and non-Hispanic Asian Ame ican women in U.S. s udies11,12.
Excessi e e al g ow h has nega i e consequences ha ex end beyond he neona al pe iod and hese include
medium- and long- e m neu ological, beha io al, and ca dio ascula impac s13–16. LGA newbo ns a e also a an
inc eased isk o obesi y17–20, me abolic synd ome21,22, and insulin esis ance23 in la e li e. They ha e also been
ound o ha e ele a ed lep in and as ing insulin and homeos asis model assessmen (HOMA) index le els24
du ing childhood, in addi ion o ele a ed adiponec in le els25, despi e p e ious epo s o he con a y om
o he s udies o insulin s a es. The isk o obesi y in LGA newbo ns inc eases wi h co-occu ence o ma e nal
o e weigh /obesi y o diabe es melli us26.
Dys egula ion o a y acid oxida ion and subsequen lipo oxici y play an impo an ole in he pa hophysi-
ology o obesi y-induced insulin esis ance27,28. Analysis o acylca ni ine p o iles by andem mass spec ome y
(MS/MS) in d ied-blood spo s has been used o in es iga e a y acid oxida ion al e a ions in obesi y and ype 1
and ype 2 diabe es melli us in bo h human and animal models29–32. A ecen sys ema ic e iew, howe e , ailed o
iden i y a consis en me aboli e p o ile in GDM33.
1Diagnosis and T ea men o Congeni al Me abolic Diseases Uni (UDyTEMC). Neona ology Se ice. Depa men
o Pedia ics. Hospi al Clínico Uni e si a io. Uni e si y o San iago de Compos ela. Ins i u e o Clinical Resea ch o
San iago de Compos ela (IDIS). CIBERER, San iago de Compos ela, Spain. 2Neona ology Uni . Uni e si y Hospi al
Miguel Se e , Za agoza, Spain. 3Neu Obesi y G oup. Depa men o Physiology, CIMUS. Uni e si y o San iago
de Compos ela. Ins i u e o Clinical Resea ch o San iago de Compos ela (IDIS), San iago de Compos ela, 15782,
Spain. 4CIBER Fisiopa ología de la Obesidad y Nu ición (CIBERobn), San iago de Compos ela (IDIS), 15706, Spain.
Co espondence and eques s o ma e ials should be add essed o P.S.-P. (email: [email p o ec ed])
Recei ed: 25 Ap il 2017
Accep ed: 23 Oc obe 2017
Published: xx xx xxxx
OPEN
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SCIENTI IC RePoR S | 7: 16267 | DOI:10.1038/s41598-017-15809-4
The aim o his s udy was o cha ac e ize pos na al plasma acylca ni ine p o iles in a coho o LGA newbo ns.
As a seconda y ou come, we analyzed and compa ed he acylca ni ine inge p in o LGA in an s bo n o mo he s
wi h and wi hou ges a ional diabe es (LGA-GDM and LGA-noGDM espec i ely).
Resul s
Gene al cha ac e is ics o he s udy popula ion. In o al, 2514 newbo ns (1362 males and 1152
emales) we e included o e he 1-yea s udy pe iod. None o hem me he exclusion c i e ia. The e we e 2302
ull- e m newbo ns and 212 p e e m newbo ns, wi h a medium GA o 39 weeks and he ollowing an h opome -
ic cha ac e is ics: mean bi h weigh , 3225 ± 591 g; mean bi h leng h, 49.05 ± 2.47 cm; and mean head ci cun-
e ence, 34.3 ± 1.66 cm. In he p e e m g oup, he espec i e measu emen s we e 2127 ± 644 g, 44.03 ± 3.88 cm,
and 31.13 ± 2.42 cm. The dis ibu ion acco ding o bi h weigh pe cen ile was 9% o small o GA (SGA) new-
bo ns (250/2514), 80.2% o app op ia e o GA (AGA) newbo ns (2018/2514), and 9.7% o LGA newbo ns
(246/2514). All bi h measu emen s we e highe in LGA newbo ns: weigh , 4118 ± 234 g; leng h, 51.95 ± 1.36 cm;
and head ci cum e ence, 35.97 ± 1.36 cm. Only one p e e m newbo n included in he s udy was classi ied as LGA.
A low diag am o he coho is shown in Supplemen a y Figu e 1.
The pe cen age o newbo ns in he se e e LGA g oup (>97 h pe cen ile) was 3% (75/2514), which is iden ical
o he pe cen age o newbo ns in he se e e SGA g oup (<3 d pe cen ile).The e we e hus 2364 newbo ns in he
AGA(3-97) g oup, which con ained newbo ns wi h a bi h weigh ≥3 d pe cen ile and ≤97 h pe cen ile.
Nine pe cen (246/2514) o he newbo ns we e bo n o mo he s wi h a his o y o GDM; 81.4% o he mo h-
e s ecei ed die a y ea men and 18.6% equi ed insulin ea men . The b eakdown o he LGA g oup was as
ollows: 42 LGA-GDM newbo ns and 204 LGA-noGDM newbo ns. No signi ican di e ences we e obse ed
be ween he mo he s in hese subg oups o ei he age o obs e ic como bidi ies (Supplemen a y Table1).
Acylca ni ine p o iles. All acylca ni ine alues a e exp essed as medians and he co esponding 95% con-
idence in e al is gi en in he ables. Compa ed wi h AGA newbo ns, LGA newbo ns had highe le els o FC,
TC, AC (p < 0.01), ACm, ACl, and, in pa icula , ACs (p < 0.01). As shown in Table1, LGA newbo ns had
he highes AC/FC a io (0.795) and he lowes FC/TC a io (0.557). This was close o he cu o o neona al
ca ni ine insu iciency ( AC/FC > 0.83) and ca ni ine de iciency (FC/TC < 0.54), sugges ing educed ca ni ine
s o age in his g oup (SGA, 0.603; AGA, 0.578; and LGA, 0.557) (p < 0.001). Sepa a e analysis o he a ious
acylca ni ines showed ema kably highe le els o C2 and C3 in LGA newbo ns (p < 0.01), al hough plasma
concen a ions o he majo i y o sho - and long-chain acylca ni ines we e also highe in he LGA g oup. The
C8/C2 a io was conside ably lowe in he LGA g oup (0.0078 s. 0.0097 o he AGA g oup, p < 0.01). The mos
signi ican di e ences be ween LGA and AGA newbo ns a e summa ized in Fig.1.
Al hough se e e LGA newbo ns had highe le els o FC, TC, AC, ACs, ACm, and ACl, he only signi i-
can di e ence compa ed wi h AGA(3-97) newbo ns we e highe le els o C3 (p < 0.05) (Table2). In addi ion, no
inc ease in ca ni ine de iciency o insu iciency ma ke s was obse ed in his g oup.
The compa ison be ween LGA-GDM and LGA-noGDM newbo ns e ealed ha a his o y o ges a ional dia-
be es was associa ed wi h highe le els o FC, TC, and sho -chain acylca ni ines, including p opionylca ni ine
(LGA-GDM: 3.33 µmol/L; LGA-noGDM: 2.69 µmol/L), and lowe le els o medium- and long-chain acylca ni-
ines, al hough he di e ences we e no signi ican (Table3). Analysis o he indi idual acylca ni ines e ealed no
ema kable di e ences be ween he wo subg oups.
Discussion
Dis u bances in e al nu i ion, such as in au e ine g ow h es ic ion (IUGR) and mac osomia, can impac
heal h du ing adolescence and adul hood13–16,34 and a e isk ac o s o la e o e weigh 17–20,35,36. Animal s udies
ha e shown ha e al o e nu i ion esul s in inc eased adiposi y in newbo ns, leading o insulin esis ance sim-
ila o ha seen in cases o pos na al o e nu i ion37. Gi en he high wo ldwide p e alence o child obesi y and
o e weigh 38, imp o ed knowledge o me abolic homeos asis in highe - isk subg oups, such as LGA newbo ns,
is essen ial o iden i ying possible ea men a ge s.
Recen in es iga ions ha e iden i ied abno mali ies in he me abolic p o iles o obese adul s and child en,
such as inc eased plasma concen a ions o b anched-chain amino acids (BCAAs) ( aline, leucine, and isoleu-
cine), BCAA me abolism byp oduc s (e.g. alanine, glu ama e/glu amine), C3 and C5 acylca ni ines, and a oma ic
amino acids (phenylalanine and y osine)29,39–44. The inc ease in BCAA and sho -chain acylca ni ine concen-
a ions is linked o ele a ed p o ein in ake45, and le els a e posi i ely co ela ed wi h adiposi y46,47 and s ongly
associa ed wi h IR29,32,44,48. The me abolic en i onmen in LGA is less well-known. Highe le els o adipokines49,
BCAAs, and o he me aboli es, such as alanine, glu amine, h eonine, ci ic acid, glyce ol, and glucose, ha e been
de ec ed in co d-blood samples o LGA newbo ns50, and simila indings ha e been epo ed o myo-inosi ol
le els in u ine samples51.
Ou s is he i s s udy o cha ac e ize acylca ni ine p o iles in LGA newbo ns. In line wi h he esul s o obesi y
s udies in child en41,43 and adul s29,31, ou da a show ha LGA newbo ns ha e inc eased pos na al le els o C3, a
p oduc o mi ochond ial BCAA ca abolism, and in pa icula o isoleucine and aline ca abolism29. BCAAs ac
as signaling molecules o nu i ional s a us52. Inc eased concen a ions in obese and insulin- esis an humans
may be caused by down- egula ion o BCAA oxida ion enzymes in adipose issue53,54, as has been obse ed in ani-
mal models wi h gene ic o die -induced obesi y55. P opionylca ni ine is a ca ni ine conjuga e o p opionyl-CoA,
which has been iden i ied as a po en ial subs a e o odd-chain a y acid syn hesis56. Mo eo e , C3 and C5 le -
els a e p omising bioma ke s o disc imina ing me abolic wellness in obese indi iduals57, as highe le els ha e
been obse ed in me abolically unheal hy indi iduals, independen ly o body mass index58. Consis en wi h hese
obse a ions, he LGA newbo ns in ou s udy had highe C3 (p < 0.001) and C5 le els han AGA newbo ns, and
he concen a ions o C3 we e e en highe han hose obse ed in obese child en41,43 and adul s29. This could
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SCIENTI IC RePoR S | 7: 16267 | DOI:10.1038/s41598-017-15809-4
e lec an unheal hy me abolic s a us in LGA newbo ns wi h po en ial complica ions in la e li e. The LGA g oup
also had highe C2 plasma le els, and hese we e pa icula ly e iden in se e e LGA newbo ns. C2 may play a ole
in insulin esis ance h ough i s in e ac ion wi h ace yl-CoA ia ca ni ine ace yl-CoA ans e ase. This enzyme
seems o ac as a posi i e egula o o o al body glucose ole ance and muscle ac i i y o py u a e dehyd oge-
nase59. C2 le els a e inc eased in p ediabe ic s a es60, diabe es melli us30,32,60, and me abolic synd ome32, and a e
signi ican ly and posi i ely co ela ed wi h HbA1c le els30. In e es ingly, he le els in he LGA g oup we e highe
han hose epo ed o diabe ic adul pa ien s30,32,60.
One o he mos no able di e ences be ween LGA and AGA newbo ns in ou s udy was he signi ican ly
highe ACs le els in he LGA g oup (p < 0.0001). This obse a ion is consis en wi h epo s o inc eased ACs in
obesi y, impai ed glucose ole ance, and diabe es melli us61.
The acylca ni ine pa e n o inc eased AC, ACs, C2, and C3 le els was mo e e iden in he SGA g oup, sup-
po ing p e ious obse a ions in animal models62 and neona al s udies63, and sugges ing an impai ed a y acid
me abolism in bo h e al g ow h diso de s.
In ag eemen wi h he p o ile desc ibed o o e weigh adul s29 and child en64, LGA newbo ns also showed
highe ( hough no signi ican ly so) concen a ions o medium- and long-chain acylca ni ines han AGA
newbo ns.
A s ong co ela ion was ecen ly demons a ed be ween ca ni ine and body composi ion65. Al hough we
obse ed sligh ly highe plasma concen a ions o ee ca ni ine in LGA newbo ns, ou da a also showed a
g ea e endency owa ds ca ni ine de iciency (p < 0.05) and insu iciency (p < 0.001) in his g oup. Inc eases
in AC/FC a io p ecede a dec ease in o al plasma ca ni ine and indica e low issue bioa ailabili y o FC66. The
highe FC le els in LGA con as wi h he ca ni ine deple ion epo ed o die -induced obesi y67. Ne e heless,
SGA (n: 250) AGA (n:2018) LGA (n:246) SGA s AGA AGA s LGA
Median Range Median Range Median Range P195% CI P295%CI
FC 33.58 8.27–80.24 27.96 7.12–100.56 28.20 9.23–80.16 6.8e−83.73, 6.72 NS −0.90, 1.75
TC 57.57 17.00–118.44 49.01 20.05–135.89 51.20 23.26–114.70 2.09e−75.68, 10.36 NS 0.34, 4.47
AC 22.16 8.72–55.98 20.49 7.95–69.41 22.34 11.83–49.65 0.0003 1.17, 2.88 0.0003 1.11, 2.77
ACs
C2
C3
C3:1
C4
C4-OH
C5
C5:1
C5-OH
16.58
13.33
2.76
0.001
0.002
0.276
0.251
0.028
0.153
6.16–50.20
4.06–43.78
0.51–10.06
0–0.553
0–1.632
0–0.911
0.03–1.048
0–0.263
0.007–0.43
14.29
11.50
2.19
0.009
0.002
0.260
0.176
0.026
0.151
5.19–62.23
4.19–58.85
0.46–9.83
0–1.75
0–1.677
0–1.747
0–1.578
0–0.806
0–0.888
15.82
12.83
2.57
0.007
0.276
0.270
0.179
0.026
0.148
7.47–41.43
5.35–36.48
0.77–8.78
0–0.493
0–1.526
0.045–1.963
0.018–1.09
0–0.302
0–0.713
4.86e−7
0.0002
5.71e−10
NS
8.73e−4
NS
3.94e−16
NS
NS
1.78, 3.2
1.05, 2.22
0.41, 0.70
−4.0e−5, 1.5e−5
0.038, 0.078
−0.009, 0.025
0.062, 0.095
−5.9e−5, 0.003
−0.005, 0.011
0.0001
0.007
4.42e−7
NS
NS
NS
NS
NS
NS
0.91, 2.23
0.66, 1.73
0.27, 0.54
−2.9e−5, 5.2e−5
−0.009, 0.012
−0.012, 0.023
−0.01, 0.011
−0.001, 0.002
−0.005, 0.011
ACm
C6
C6–OH
C8
C8:1
C10
C10:1
C10:2
C5DC
C4DC
C12
C12:1
C12:2
0.770
0.063
0.061
0.121
0.217
0.114
0.106
0.061
0.08
0.280
0.137
0.083
0.027
0.171–2.61
0–0.663
0–0.244
0–0.361
0–0.675
0–0.482
0–0.414
0–0.296
0–0.332
0.04–0.834
0–0.628
0–0.38
0–0.153
0.805
0.040
0.050
0.110
0.180
0.128
0.101
0.052
0.067
0.332
0.152
0.093
0.026
0.109–4.98
0–0.731
0–0.479
0–2.394
0–0.683
0–0.966
0–0.79
0–0.414
0–0.395
0–1.063
0–1.789
0–0.502
0–0.277
0.773
0.524
0.054
0.111
0.203
0.120
0.099
0.050
0.063
0.334
0.159
0.090
0.025
0.202–7.057
0–0.349
0–0.403
0–0.46
0–0.581
0–0.988
0–0.753
0–0.395
0–0.296
0.11–1.106
0.03–4.391
0–0.461
0–0.197
NS
NS
NS
NS
NS
NS
NS
NS
NS
1.06e−9
0.002
NS
NS
−0.021, 0.05
9.4e–6, 0.013
−0.002, 0.008
−0.002, 0.014
0.012, 0.039
−0.019, 0.002
−0.006, 0.011
0.002, 0.015
0.003, 0.015
−0.071, −0.039
−0.029, −0.005
−0.021, −0.008
−0.001, 0.004
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
−0.032, 0.038
−1.8e−6, 0.008
−0.006, 0.004
−0.014, 0.001
−0.006, 0.021
−0.015, 0.003
−0.008, 0.008
−0.007, 0.003
−0.007, 0.004
−0.015, 0.02
−0.013, 0.066
−0.01, 0.005
−0.001, 0.004
ACl
C14
C14:1
C14:2
C14–OH
C14:1–OH
C16
C16:1
C16–OH
C16:1–OH
C18
C18:1
C18:2
C18–OH
C18:1–OH
C18:2–OH
4.558
0.277
0.132
0.081
0.035
0.041
2.454
0.162
0.037
0.056
1.468
1.938
0.350
0.033
0.096
0.214
1.79–18.08
0.03–0.733
0.03–0.628
0–0.708
0–0.23
0–0.251
0.87–9.949
0–0.455
0–0.169
0–0.330
0.44–6.857
0.61–9.213
0.07–1.716
0–0.175
0.004–0.31
0.02–0.808
5.223
0.270
0.127
0.060
0.030
0, 040
3.018
0.168
0.042
0.055
1.591
1.904
0.244
0.036
0.096
0.221
1.13–19.51
0.04–1.138
0–0.813
0–1.151
0–0.259
0–0.377
0.29–12.787
0–0.851
0–0.342
0–0.346
0.29–5.744
0.3–6.953
0.02–1.263
0–0.283
0–0.502
0.002–0.956
5.503
0.277
0.125
0.060
0.036
0.044
3.252
0.183
0.043
0.054
1.630
1.916
0.234
0.036
0.090
0.211
2.22–16.02
0.09–1.05
0–0.976
0–0.754
0–0.154
0–0.407
1.06–10.793
0.01–0.795
0–0.224
0–0.316
0.368–4.932
0.738–5.051
0.041–1.105
0–0.158
0.007–0.395
0–0.932
4.01e−7
NS
NS
2.5e−2
NS
NS
2.1e12
NS
NS
NS
NS
NS
6.8e−17
NS
NS
NS
–0.837, −0.446
−0.01, 0.017
−0.004, 0.013
0.012, 0.026
−0.001, 0.004
−0.004, 0.003
−0.655, −0.402
−0.016, 0.004
−0.006, 0.001
−0.002, 0.005
−0.181, 0.002
−0.002, 0.126
0.085, 0.12
−0.005, 0.004
−0.011, 0.004
−0.028, 0.001
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.06, 0.476
−0.004, 0.024
−0.01, 0.006
−0.004, 0.008
−0.001, 0.005
−0.001, 0.006
0.09, 0.367
−0.004, 0.026
−0.003, 0.003
−0.004, 0.004
−0.044, 0.116
−0.026, 0.131
−0.02, 0.012
−0.002, 0.003
−0.009, 0.005
−0.031, 0.008
AC/FC 0.657 0.31–1.651 0.728 0.246–2.42 0.795 0.332–1.634 0.005 –0.101, −0.03 0.036 0.019, 0.094
FC/TC 0.603 0.37–0.761 0.578 0.292–0.803 0.557 0.38–0.751 0.014 0.011, 0.033 0.0001 −0.031,
−0.009
Table 1. Acylca ni ine p o iles (µmol/L) acco ding o bi h weigh based on he 10 h and 90 h pe cen iles. p1,
compa ison be ween SGA and AGA newbo ns; p2, compa ison be ween AGA and LGA newbo ns; 95% CI,
95% con idence in e al; FC, ee ca ni ine; TC, o al ca ni ine; AC, o al acylca ni ines; ACs, o al sho -chain
acylca ni ines; ACm, o al medium-chain acylca ni ines; ACl, o al long-chain acylca ni ines; SGA, small o
ges a ional age; AGA, app op ia e o ges a ional age; LGA, la ge o ges a ional age; NS, p no signi ican .
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me abolomic s udies o obesi y ha e also shown highe le els o ca ni ine in obese child en43. Ca ni ine insu i-
ciency in ou coho appea s o be un ela ed o an ena al exposu e o GDM. This is ele an gi en he p oposed
causa i e ole o ca ni ine insu iciency in mi ochond ial dys unc ion and obesi y- ela ed impai men s in glucose
ole ance68. I is also consis en wi h epo s ha documen ha GDM in p egnan women does no nega i ely
a ec he e iciency o he ca ni ine sys em69. We did no ind any pos na al di e ences be ween acylca ni ine
p o iles in LGA-GDM and LGA-noGDM newbo ns in ou s udy, al hough he o me had highe concen a ions
o FC, TC, AC and ACs. In line wi h his obse a ion, highe FC and TC le els ha e been epo ed in p egnan
women wi h GDM e sus heal hy p egnan women a 30–33 weeks o ges a ion69. We do no conside ha he
absence o signi ican di e ences be ween he LGA-GMD and LGA-no GDM g oups is due o sample size, as he
minimum de ec able e ec sizes o he samples used in each compa ison (wi h 5% signi icance and 80% s a is-
ical powe ) we e 0.2 o AGA s LGA and AGA s SGA (Table1), 0.25 o SGA s LGA (Table1), 0.33 o AGA
s LGA (Table2), and 0.48 o LGA-noGMD s LGA-GMD (Table3). This means ha , e en in he wo s -case
scena io (Table3), we a e able o de ec ue be ween-g oup di e ences o highe han 50% o he SD, which a e
conside ed medium e ec sizes70.
Ou indings desc ibe a pos na al acylca ni ine p o ile in LGA newbo ns ha is cha ac e is ic o obesi y
and associa ed wi h he de elopmen o insulin esis ance and p ediabe ic s a es, suppo ing he iew ha ea ly
imbalance in me abolic homeos asis in LGA newbo ns could con ibu e o dele e ious e ec s in he long e m.
Iden i ica ion o his p o ile, linked o an unheal hy me abolic pheno ype, in he pos na al pe iod could help o
es ablish ea ly die a y in e en ion and ollow-up o educe he isk o o e weigh and me abolic synd ome in
la e li e.
Pa ien s and Me hods
S udy design. The acylca ni ine p o iles o LGA newbo ns we e de e mined in a 1-yea obse a ional s udy
app o ed by he Resea ch E hics Commi ee o Galicia, Spain ( egis y numbe 2015/315). The p ocessing o clin-
ical da a o esea ch pu poses a he beginning o he s udy and he s udy p o ocol complied wi h he p inciples
o he Helsinki Decla a ion o 1964, as e ised in Oc obe 2013 in Fo aleza, B azil.
Figu e 1. Main di e ences be ween la ge o ges a ional age (LGA) and app op ia e o ges a ional age (AGA)
newbo ns.
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Pa ien s. This s udy was conduc ed a Hospi al Clínico Uni e si a io de San iago de Compos ela, a e ia y
hospi al in no h-wes Spain. All newbo ns bo n in o e e ed o ou hospi al du ing he i s 48 hou s o li e
o e he pe iod o 1 yea (2015) we e included in he s udy. In o med consen was ob ained om pa en s o
legal gua dians. In an s wi h an es ablished diagnosis o an inbo n e o o me abolism known o al e acylca ni-
ine p o iles (p ima y sys emic ca ni ine de iciency, mi ochond ial a y acid ß-oxida ion de ec s, ß-ke o hiolase
de iciency, p opionic, me hylmalonic o iso ale ic acidemia, 3-me hylc o onyl-CoA ca boxylase de iciency,
3-hyd oxy-3-me hylglu a yl-CoA lyase de iciency, o 3-me hylglu aconic acidu ia) we e excluded om he anal-
ysis. The ollowing a iables we e eco ded a bi h: sex, ges a ional age, weigh (g), leng h (cm), head ci cum e -
ence (cm), his o y o GDM, and ea men s ecei ed (die a y and/o insulin ea men ).
Newbo ns we e classi ied in o he ollowing g oups acco ding o hei bi h weigh : AGA (≥10 h pe cen ile
and ≤90 h pe cen ile o bi h weigh o GA), SGA (<10 h pe cen ile o bi h weigh o GA), and LGA (>90 h
pe cen ile o bi h weigh o GA). Fo addi ional analyses we also classi ied newbo ns wi h a bi h weigh g ea e
han he 97 h pe cen ile o GA in o a se e e LGA g oup.
Bi h weigh pe cen iles and z-sco es o GA we e calcula ed using he online nu i ional assessmen ool o
he Spanish Socie y o Gas oen e ology, Hepa ology and Nu i ion (www.gas oin .es), which is based on Spanish
neona al g ow h cu es71.
GDM was de ined acco ding o he c i e ia es ablished in he 2016 Guidelines o he Ame ican Diabe es
Associa ion72 using he wo-s ep diagnos ic s a egy: 1) i plasma glucose is ≥140 mg/dL (7.8 mmol/L) in he
1-hou glucose loading- es , p egnan women mus 2) unde go a glucose ole ance es (adminis a ion o 100 g
o glucose a e 8 hou s o as ing wi h subsequen sequen ial blood sampling). Diagnosis is con i med when wo
o mo e o he ollowing glucose c i e ia a e ul illed: as ing, ≥105 mg/dL (5.8 mmol/L); 1 hou , ≥190 mg/dL
(10.6 mmol/L); 2 hou s, ≥165 mg/dL (9.2 mmol/L); and 3 hou s, ≥145 mg/dL (8.0 mmol/L).
SGA (n:75) AGA(3–97) (n:2364) LGA (n:75) SGA VS AGA AGA VS LGA
Median Range Median Range Median Range P195%CI P295% CI
FC 35.43 14.22–64.09 28.31 7.12–100.56 28.22 11.58–73.43 0.009 4.01,9.29 NS −2.29, 2.685
TC 62.31 25.14–105.97 50.12 17.00–135.89 52.33 25.14–114.70 1.72e−68.046, 16.168 NS −2.361, 5.609
AC 25.29 10.92–55.98 21.07 7.95–69.41 23.07 13.16–49.65 0.0001 2.607, 5.995 NS 0.131, 3.203
ACs
C2
C3
C3:1
C4
C4–OH
C5
C5:1
C5–OH
19.41
14.60
3.67
0.002
0.443
0.337
0.489
0.032
0.175
8.02–50.20
5.84–43.78
1.00–10.06
0–0.128
0.03–1.53
0–0.911
0.128–1.048
0–0.189
0.007–0.431
14.51
11.67
2.22
0.008
0.302
0.266
0.179
0.026
0.151
5.19–62.23
4.06–58.85
0.461–9.83
0–1.75
0–1.677
0–1.963
0–1.578
0–0.806
0–0.888
16.24
12.95
2.853
0.011
0.269
0.293
0.161
0.028
0.167
8.23–41.43
5.35–36.48
0.93–5.62
0–0.154
0.10–1.34
0.045–0.978
0.018–1.09
0–0.108
0.032–0.713
9.16e−9
0.012
4.61e−8
NS
9.66e−5
NS
1.30e−15
NS
NS
3.472, 6.357
1.651, 3.981
1.034, 1.732
−3.7e−5, 1.8e−5
0.094, 0.179
0.034, 0.097
0.239, 0.349
6.3e−6, 0.011
0.004, 0.034
NS
NS
0, 015
NS
NS
NS
NS
NS
NS
0.278, 2.745
0.171, 2.12
0.228, 0.76
−4.8e−5,
2.3e−5
−0.049, 0.011
−0.019, 0.044
−0.023, 0.013
−0.003, 0.006
0.0002, 0.031
ACm
C6
C6–OH
C8
C8:1
C10
C10:1
C10:2
C5DC
C4DC
C12
C12:1
C12:2
0.688
0.06
0.051
0.119
0.163
0.113
0.097
0.045
0.08
0.221
0.125
0.073
0.025
0.171–1.471
0–0.268
0–0.193
0–0.361
0–0.594
0.006–0.314
0–0.251
0–0.167
0–0.332
0.04–0.596
0–0.387
0.006–0.181
0–0.114
0.762
0.05
0.057
0.116
0.195
0.126
0.102
0.052
0.068
0.329
0.152
0.092
0.026
0.109–7.057
0–0.731
0–0.479
0–2.394
0–0.683
0–0.988
0–0.79
0–0.414
0–0.395
0–1.063
0–4.391
0–0.502
0–0.277
0.779
0.052
0.063
0.123
0.218
0.133
0.110
0.067
0.07
0.325
0.157
0.09
0.029
0.376–3.108
0–0.349
0–0.403
0.026–0.46
0.039–0.453
0–0.284
0–0.252
0–0.23
0–0.178
0.149–1.106
0.047–1.554
0–0.461
0–0.115
NS
NS
NS
NS
NS
NS
NS
NS
NS
4.86e−8
4.79e−6
0.0007
NS
−0.121, 0.003
−6.7e−5, 0.025
−0.013, 0.005
−0.008, 0.018
−0.043, 0.005
−0.03, −0.001
−0.02, 0.011
−0.015, 0.004
−0.001, 0.021
−0.12, −0.068
−0.062, −0.03
−0.031, −0.012
−0.003, 0.007
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
−0.028, 0.087
−0.005, 0.013
−0.005, 0.013
−0.006, 0.02
−0.01, 0.034
−0.017, 0.013
−0.017, 0.014
5.1e−6, 0.022)
−0.007, 0.013
−0.033, 0.026
−0.021, 0.092
−0.014, 0.016
−2.8e−5, 0.011
ACl
C14
C14:1
C14:2
C14–OH
C14:1–OH
C16
C16:1
C16–OH
C16:1–OH
C18
C18:1
C18:2
C18–OH
C18:1–OH
C18:2–OH
4.47
0.261
0.133
0.081
0.03
0.038
2.24
0.167
0.034
0.055
1.450
2.02
0.378
0.028
0.076
0.225
2.239–7.095
0.047–0.658
0.038–0.46
0–0.592
0–0.105
0–0.132
0.871–4.328
0–0.441
0–0.169
0–0.162
0.583–2.751
0.989–3.7
0.089–0.991
0–0.113
0.014–0.274
0.039–0.797
5.54
0.272
0.128
0.061
0.035
0.042
2.996
0.169
0.042
0.055
1.580
1.90
0.250
0.037
0.096
0.219
1.139–19.51
0.037–1.138
0–0.976
0–1.151
0–0.259
0–0.407
0.294–12.78
0–0.851
0–0.342
0–0.346
0.29–6.857
0.3–9.213
0.022–1.716
0–0.283
0–0.502
0–0.956
5.98
0.266
0.127
0.069
0.038
0.047
3.30
0.176
0.044
0.055
1.67
1.90
0.266
0.039
0.101
0.240
3.045–11.326
0.126–0.977
0–0.939
0–0.729
0–0.259
0–0.184
1.655–6.529
0.05–0.795
0–0.167
0.009–0.316
0.518–3.038
0.987–3.809
0.041–0.567
0–0.158
0.019–0.224
0.027–0.831
2.86e−8
NS
NS
NS
NS
NS
9.39e−12
NS
NS
NS
NS
NS
1.31e−6
0.016
NS
NS
−1.13, −0.482
−0.02, 0.019
0.01, 0.02
0.008, 0.03
−0.008, 0.002
−0.01, −0.001
−0.95, −0.524
−0.025, 0.011
−0.01, −0.002
−0.005, 0.007
−0.177, 0.033
−0.073, 0.21
0.093, 0.16
−0.013, −0.004
−0.02, −4.3e−4
−0.04, 0.018
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
−0.046, 0.648
−0.022, 0.028
−0.015, 0.015
−0.007, 0.017
−0.004, 0.007
0.001, 0.013
0.007, 0.459
−0.007, 0.031
−0.004, 0.006
−0.006, 0.006
−0.078, 0.148
−0.054, 0.225
−0.023, 0.042
−0.001, 0.008
−0.005, 0.02
−0.031, 0.039
AC/FC 0.693 0.314–1.58 0.724 0.246–2.42 0.776 0.431–1.634 NS −0.096, 0.042 NS −0.014, 0.112
FC/TC 0.591 0.388–0.761 0.580 0.292–0.803 0.563 0.38–0.699 NS −0.008, 0.032 NS −0.038, 0.001
Table 2. Acylca ni ine p o iles (µmol/L) acco ding o bi h weigh based on 3 d and 97 h pe cen iles. p1,
compa ison be ween se e e SGA and AGA(3–97) g oups; p2, compa ison be ween AGA(3–97) and se e e LGA
g oups; 95% CI, 95% con idence in e al; FC, ee ca ni ine; TC, o al ca ni ine; AC, o al acylca ni ines;
ACs, o al sho -chain acylca ni ines; ACm, o al medium-chain acylca ni ines; ACl, o al long-chain
acylca ni ines; SGA, small o ges a ional age; AGA, app op ia e o ges a ional age; LGA, la ge o ges a ional
age; NS, p no signi ican .
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Based on he co esponding obs e ic his o y o ges a ional diabe es, LGA newbo ns we e sub-classi ied as
LGA-GDM (co-occu ence o ges a ional diabe es) o LGA-noGDM (absence o GDM).
Tandem mass spec ome y and s udy pa ame e s. Analyses o ee ca ni ine and acylca ni ines
we e pe o med on a andem mass spec ome e coupled o a iple quad upole analyze (ESI-MS/MS API 2000;
Applied Biosys ems Sciex, To on o, Canada) ollowing an es ablished me hodology73.
The pla es we e p epa ed using he ollowing p o ocol. The pape blood sample disks and pa e ns we e
cu using a BSD 700 au oma ic d ill (BSD ech., B isbane, Aus alia) and hand d ills ollowed by mic opla e
placemen and addi ion o each well o me hanol. Acylca ni ines we e pu i ied wi h me hanol and s able
iso ope-labeled pa e ns we e used o de e mine hei espec i e concen a ions. The acylca ni ines we e hen
ex ac ed by o ex shaking o 25 minu es. Subsequen ly, all he me hanol was ans e ed o ano he pla e o
dis il he blood disks and hen e apo a ed in a gas ex ac o . The acylca ni ines we e de i a ized wi h bu anol
o hei bu yl-es e s in an acid medium o inc ease he selec i i y o he echnique. This was done wi h he
addi ion o 3N HCl in n-bu anol ollowed by hea ing a 65 ± 5 °C o 20 minu es and cooling o 5 minu es in a
eeze . Excess bu anol was e apo a ed o d yness and once he e apo a ed pla es we e a oom empe a u e, a
new solu ion was p epa ed wi h 100 μL o he mobile phase o he ch oma og aph (ace oni ile: wa e , 1:1). The
pla es we e hen co e ed wi h oil, o exed o 5 minu es, and inally analyzed (p ecu so m/z 120–280 amu).
The eagen s we e p epa ed using wa e pu i ied wi h a Milli-Q sys em (Millipo e) and he mobile phase was
composed o ace oni ile (LiC osol Me ck, e . 00030) and o mic acid 0.005% (Me ck, e .02264).
A comp ehensi e analysis o acylca ni ine p o iles was conduc ed by MS/MS using d ied-blood spo s
collec ed on he hi d day o li e o expanded newbo n sc eening. We analyzed: sho -chain acylca ni ines:
ace yl- (C2), p opionyl- (C3), p openyl- (C3:1), C4-, 3-OH-bu y yl- (C4-OH), C5- and iglyl-ca ni ine (C5:1);
medium-chain acylca ni ines: C6-, 3-hyd oxy-hexanoyl- (C6-OH), C8-, oc enoyl- (C8:1), me hylmalonyl-
(C4DC), C10-, decenoyl- (C10:1), decadienoyl- (C10:2), C12- and dodecenoyl-ca ni ine (C12:1); and
long-chain acylca ni ines: C14-, my is oleyl- (C14:1), hyd oxymy is oyl- (C14-OH), C16-, hexadecenoyl-
(C16:1), 3-hyd oxi-hexadecanoyl- (C16-OH), 3-hyd oxypalmi oleylc- (C16:1-OH), C18-, oleyl- (C18:1),
linoleyl- (C18:2), hyd oxyoleyl- (C18:1-OH) and 3-hyd oxy-linoleyl-ca ni ine (C18:2-OH). I should be no ed
ha he analy ical me hod employed does no allow o he di e en ia ion o isoba ic acylca ni ines. The
ollowing pa ame e s we e also assessed: o al sho -chain acylca ni ines ( ACs), o al medium-chain acyl-
ca ni ines ( ACm), o al long-chain ( ACl) acylca ni ines ( he sum o sho -, medium-, and long-chain acyl-
ca ni ines, espec i ely), o al acylca ni ines ( AC) ( he sum o all acylca ni ines s udied); o al ca ni ine (TC),
de ined as he sum o ee ca ni ine (FC) and o al acylca ni ines ( AC), FC/TC a io ( alues <0.54 in neona es
a e sugges i e o ca ni ine de iciency), and AC/FC a io ( alues >0.83 in newbo ns a e indica i e o ca ni ine
insu iciency)74. We also e alua ed h ee acylca ni ine a ios ypically included in neona al sc eening: C8/C2,
C8/C10, and FC/C16.
S a is ical analyses. Da a we e analyzed using he R s a is ical package ( e sion 3.2.1; R P ojec o
S a is ical Compu ing). Sample no mali y was assessed using he Kolmogo o -Smi no es . ANOVA was used o
compa e no mally dis ibu ed da a, and he K uskal–Wallis es was used o compa e non-no mally dis ibu ed
da a. Quali a i e a iables we e compa ed using Fishe ’s exac es . No mal samples wi h unknown a iance we e
compa ed using S uden ’s - es , while non-no mally dis ibu ed da a we e compa ed using he Wilcoxon ank
es . Finally, he p- alues ob ained we e adjus ed using Bon e oni co ec ion. Only adjus ed p- alues < 0.05 we e
conside ed s a is ically signi ican .
Re e ences
1. Das, U. G. & Sysyn, G. D. Abno mal e al g ow h: in au e ine g ow h e a da ion, small o ges a ional age, la ge o ges a ional age.
Pedia . Clin. N. Am. 51, 639–54 (2004).
2. Pe low, J. H., Mo gan, M. A., Mon gome y, D., Towe s, C. V. & Po o, M. Pe ina al ou come in p egnancy complica ed by massi e
obesi y. Am. J. Obs e . Gynecol. 167, 958–62 (1992).
LGA-noGDM (n:204) LGA-GDM (n:42)
P95% CIMedian Range Median Range
FC 27.93 9.235–80.164 30.17 19.11–58.364 NS −5.81, 0.93
TC 50.48 23.265–114.703 54.17 37.47–103.696 NS −9.16, 1.13
AC 22.29 11.831–49.653 23.37 17.241–45.332 NS −3.88, 0.64
ACs 15.65 7.474–41.438 17.11 11.224–36.76 NS −3.69, −0.05
ACm 0.77 0.202–7.057 0.807 0.483–1.557 NS −0.10, 0.06
ACl 5,56 2.228–16.023 5.081 2.879–9.113 NS −0.16, 0.88
AC/FC 0.809 0.332–1.634 0.777 0.505–1.624 NS −0.09, 0.11
FC/TC 0.553 0.38–0.751 0.563 0.381–0.664 NS −0.03, 0.02
Table 3. Acylca ni ine pa e n (µmol/L) in LGA newbo ns acco ding o ma e nal his o y o ges a ional
diabe es. GDM, ges a ional diabe es melli us; FC, ee ca ni ine; TC, o al ca ni ine; AC, o al acylca ni ines;
ACs, o al sho -chain acylca ni ines; ACm, o al medium-chain acylca ni ines; ACl, o al long-chain
acylca ni ines; SGA, small o ges a ional age; AGA, app op ia e o ges a ional age; LGA, la ge o ges a ional
age; NS, p no signi ican ; 95% CI, 95% con idence in e al.
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SCIENTI IC RePoR S | 7: 16267 | DOI:10.1038/s41598-017-15809-4
3. Voh , B. R., McGa ey, S. T. & Coll, C. G. E ec s o ma e nal ges a ional diabe es and adiposi y on neona al adiposi y and blood
p essu e. Diabe es Ca e 18, 467–75 (1995).
4. Djelan ik, A. A., Kuns , A. E., ande Wal, M. F., Smi , H. A. & V ijko e, T. G. Con ibu ion o o e weigh and obesi y o he
occu ence o ad e se p egnancy ou comes in a mul i-e hnic coho : popula ion a ibu i e ac ions o Ams e dam. BJOG 119,
283–90 (2012).
5. Oya zo, C. e al. Ad e se pe ina al ou comes a e he Feb ua y 27 h 2010 Chilean ea hquake. J. Ma e n. Fe al Neona al Med. 25,
1868–73 (2012).
6. Blackwell, S. C. e al. Rela ionship be ween excessi e ges a ional weigh gain and neona al adiposi y in women wi h mild ges a ional
diabe es melli us. Obs e . Gynecol. 128, 1325–1332 (2016).
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Acknowledgemen s
D Lopez has ecei ed unding om he Eu opean Communi y’s Se en h F amewo k P og amme (FP7/2007-
2013) unde g an ag eemen no. 281854 - he ObERS ess p ojec , Xun a de Galicia (2015-CP079 and 2016-
PG068), MINECO co- unded by FEDER (SAF2015-71026-R and BFU2015-70454-REDT/Adipoplas ).
Au ho Con ibu ions
Sánchez-Pin os P. and Couce M.L. designed he s udy, e iewed he publica ions included in he sys ema ic
e iew, con ibu ed o he acquisi ion and analysis o he da a, and d a ed he manusc ip . De Cas o M.J.
con ibu ed o he acquisi ion o he da a. Roca I. pa icipa ed in he analysis and in e p e a ion o he da a. Ri e S.
and López M. pa icipa ed in he c i ical e iew o he manusc ip . All he au ho s ead and app o ed he inal
manusc ip .
Addi ional In o ma ion
Supplemen a y in o ma ion accompanies his pape a h ps://doi.o g/10.1038/s41598-017-15809-4.
Compe ing In e es s: The au ho s decla e ha hey ha e no compe ing in e es s.
Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
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