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Research ArticleAbnormal Biomarkers of Homocysteine Metabolism
inNeonates with Conotruncal Heart Defects
Piotr Surmiak, MaBgorzata Baumert, andMagdalena Paprotny
Department of Neonatology, School of Medicine in Katowice,
Medical University of Silesia, Katowice, Poland
Correspondence should be addressed to Piotr Surmiak;
[email protected]
Received 25 January 2017; Revised 4 May 2017; Accepted 28 June
2017; Published 27 July 2017
Academic Editor: Betti Giusti
Copyright © 2017 Piotr Surmiak et al. This is an open access
article distributed under the Creative Commons Attribution
License,which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly
cited.
Objectives. The etiology of conotruncal heart defects (CHD)
remains unknown; however relation between homocysteine,
folatelevels, and congenital heart disease was found. With this
perspective in mind, the aim of the study was to investigate
biomarkers ofhomosyteine metabolism pathway in mothers and their
neonates with CHD. Material and Methods. Forty-three pairs of
mothersand their neonates with CHD and forty pairs of mothers and
neonates with nonconotruncal heart defects (non-CHD)
wereenrolled.The control group (CG) consisted of fifty-nine pairs
ofmothers and their healthy neonates. For estimating the plasma
totalhomocysteine (tHcy), serum folates, and cobalamin levels,
mothers’ venous blood samples and umbilical cord blood were taken
inall groups. Results. We observed higher tHcy levels in newborns
with CHD in comparison to their mothers and to neonates
withnon-CHD. Cobalamin levels were significantly lower in neonates
with CHD compared to other children. Folates and cobalaminlevels
were lower in CHD mothers compared to their children. Conclusions.
Elevated homocysteine levels in neonates with CHDand folate
metabolism disturbances in their mothers were noticed.The observed
differences in homocysteine and cobalamin levelsbetween neonates
with CHD suggest the influence of various agents disturbing
homocysteine metabolic pathways.
1. Introduction
Themost common problem of contemporary perinatology isa high
number of congenital anomalies of which conotruncalheart defects
(CHD) are one of the common defects inthe developmental period [1,
2]. Conotruncal heart defectsrepresent an anatomically
heterogeneous group of cardiacmalformations affecting the outflow
tract of the ventricles andthe arterial pole of the heart.
According to current knowledge,more than 80%of congenital defects
with recognized etiologyare stimulated by genetic and environmental
factors [2]. Chiefamong the aforementioned influences was a
deficiency ofessential microelements and vitamins in the
preconceptionperiod, such as the B-vitamins (i.e., folic acid and
cobalamin).Some reports suggested that folates play a key role
inutilization reactions of many biogenic compounds,
includinghomocysteine [3, 4].
Recent studies suggested that maternal hyperhomocys-teinemia is
an independent risk factor for congenital heartdefects, by
interfering with the development of conotruncalseptum of the heart
[5, 6].
The aim of our study was to evaluate the differences intotal
homocysteine (tHcy), folate, and cobalamin levels ofmaternal and
umbilical cord blood samples in groups ofchildren with congenital
heart defects.
2. Material and Method
A prospective, case-control study was conducted in theDepartment
of Neonatology at the Medical University ofSilesia in Katowice
between January 2012 and January 2015.The study was approved by the
Ethics Committee of theMedical University of Silesia (nr
KNW/0022/KB1/25/13).
Among 1,271 childbirths in our unit at the time of thestudy we
enrolled 83 neonates (6.5%) with prenatally diag-nosed congenital
heart defects.
The control group (CG) is comprised of 59 pairs of
healthymothers residing in the unit during the study and
theirhealthy, full-term newborns.
All mothers from the study group underwent at leasttwo prenatal
examinations in which the congenital heartdefects were diagnosed.
Congenital heart defects in neonates
HindawiBioMed Research InternationalVolume 2017, Article ID
7404397, 5 pageshttps://doi.org/10.1155/2017/7404397
https://doi.org/10.1155/2017/7404397
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2 BioMed Research International
were confirmed by echocardiography performed as soon aspossible
after delivery.
The exclusion criteria comprised neonates with chromo-somal
aberrations, complex congenital malformations, new-borns
frommultiple pregnancies, and neonates with evidenceof congenital
infections, as well as those that were born tomothers with clinical
chorioamnionitis. We also excludedmothers who administered
medications during pregnancy,which could possibly affect
homocysteine and vitamin Bmetabolism (i.e., folate antagonists,
antiepileptic drugs, oralcontraceptives, barbiturates, and
levodopa) within the periodof six months before conception, as well
as pregnant womensuffering from hypertension, thromboembolic
diseases, kid-ney, and heart defects. Neonates from the control
groupunderwent a cranial ultrasound and an
echocardiographicexamination after delivery.
All included mothers and their children with prenatallydiagnosed
congenital heart defect were divided into twosubgroups: with
conotruncal heart defects (CHD, 𝑛 = 43)and nonconotruncal heart
defects (non-CHD, 𝑛 = 40). CHDgroup included neonates with
diagnosed Persistent TruncusArteriosus, PTA (𝑛 = 13, 30.1%),
Tetralogy of Fallot, TOF(𝑛 = 11, 25.6%), Interrupted Aortic Arch,
IAA (𝑛 = 6, 13.9%),and Double Outlet Right Ventricle, DORV (𝑛 = 5,
11.6%).
Nonconotruncal heart defects (non-CHD) comprisedchildren with
Atrial Septum Defect, ASD (𝑛 = 16, 40.0%),Ventricular Septum
Defect, VSD (𝑛 = 15, 37.5%), andAtrioventricular Septal Defect,
AVSC (𝑛 = 9, 22.5%). Allcongenital anomalies were reported to the
Polish Registry ofCongenital Malformations.
2.1. Laboratory Performances. During childbirth, 5 millilitersof
maternal blood was collected from ulnar vein and 5 ml ofblood from
umbilical artery from the placental side.
All blood samples were collected in EDTA-containingtubes,
centrifuged for 10 minutes (2500 rotations/min) andstored at −70∘C
until full analysis had beenmade.The follow-ing was assessed:
folate, cobalamin, and total homocysteineconcentrations. Folate and
cobalamin in serum were deter-mined with the aid of microparticle
enzyme immunoassay(MEIA), using ABBOTT reagent sets in an
immunochemicalanalyzer (AxSYM). Total homocysteine concentration
inplasmawas determined by an immunochemicalmethodwiththe
fluorescence polarization immunoassay (FPIA) using anIMx analyzer
and special ABBOTT sets. All procedures wererecommended by the
ABBOTT company which producestesting sets used in our
examination.
2.2. Statistical Analysis. Results were analyzed
statisticallywith the certified program STATISTICA 10 (StatSoft
PolskaInc.). The distribution of the data was analyzed by
theShapiro-Wilk test. Results were presented as means andstandard
deviations or as percentiles of the total. Baselinecharacteristics
and biomarkers of homocysteine metabolismbetween all study groups
were compared using Kruskal-Wallis test, 𝑈Mann–Whitney test, or chi
square tests.
The association between variables was measured by aSpearman’s
rank correlation test. For all the statistical proce-dures 𝑝 value
< 0.05 was considered to be significant.
Umbilical cord bloodExtreme values
Mothers’ venous bloodExtreme values
CHD Non-CHD ControlsGroup
0
5
10
15
20
25
30
35
40
Tota
l hom
ocys
tein
e lev
el (u
mol
/l)
∗
∗
∗
Figure 1: Maternal and umbilical cord blood total
homocysteineconcentrations in conotruncal heart defects (CHD),
nonconotrun-cal heart defects (non-CHD), and controls (CG). Results
presentedasmeans and standard deviations aswell as 95% confidence
intervalsand extreme values. ∗Extreme values.
3. Results
Mothers and their neonates from all investigated groupswere
comparable to controls with respect to demographic-perinatal
characteristics, presented in Table 1.
We have noticed that daily dietary supplementation offolic acid
(0.4mg) was taken by 86.0% (𝑛 = 37) of mothersfrom CHD group, 87.5%
(𝑛 = 35) of mothers from non-CHD group, and 89.8% (𝑛 = 53) from
controls. However,those results were not statistically significant
(𝑝 = 0.3). Weobserved no relevant differences in creatinine levels
in allinvestigated mothers (CHD 0.75mg/dl, non-CHD 0.72mg/dl, CG
0.74mg/dl, 𝑝 = 0.7).
3.1. Total Homocysteine Levels. In our study, we
observedsignificantly higher tHcy levels in umbilical cord blood
innewborns with CHD compared to their mothers.
Significantdifferences in tHcy concentrations were observed in
umbili-cal cord blood between CHD compared to non-CHD groups.We
also noticed higher umbilical cord tHcy levels in CHDneonates
compared to controls.
However, we observed no significant differences in tHcylevels in
neonates with non-CHD in comparison to theirmothers and to
controls.
We found no relevant differences in tHcy between moth-ers in all
investigated groups.
All results are presented on Figure 1 and Table 2.
3.2. Folate Levels. No differences were shown in umbilicalcord
folate levels in all investigated children. However, we
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BioMed Research International 3
Table 1: Demographic characteristics of investigated neonates
and their mothers.
Variable Conotruncal heart defects(𝑛 = 43)Nonconotruncal heart
defects
(𝑛 = 40)Controls(𝑛 = 59) 𝑝 value
Mothers(i) Age (years) 26 [4.3] 29 [5.8] 31 [5.4] 0.5(ii)
Primigravida [𝑛,%] 26, 60.5% 27, 67.5% 35, 59.3% 0.4(iii) Dietary
supplementation of folic acid [𝑛,%] 37, 86.0% 35, 87.5% 53, 89.8%
0.3(iv) Delivery mode: caesarean section [𝑛,%] 26, 60.5% 22, 55.0%
30, 50.8% 0.1Newborns(i) Gender female [𝑛,%] 20, 46.5% 23, 57.5%
30, 50.8% 0.7(ii) Gestational age (weeks) 38.5 [3.6] 37 [2.7] 39.5
[4.2] 0.6(iii) Birth weight (g) 3090 [620] 3313 [840] 3230 [750]
0.4(iv) Head circumference (cm) 34.5 [2.0] 34.5 [1.5] 34.0 [2.5]
0.1(v) Body length (cm) 53.0 [1.5] 54.5 [2.5] 53.5 [1.5] 0.5(vi)
Apgar 1st min [𝑛,%]
0–3 pts 2, 4.7% 1, 2.3% 0 0.64–7 pts 5, 11.6% 8, 20.0% 8,
13.6%8–10 pts 36, 83.7% 32, 80.0% 51, 86.4%
(vii) Apgar 5th min [𝑛,%]0–3 pts 0 0 0 0.54–7 pts 2, 4.7% 6,
15.0% 5, 8.5%8–10 pts 41, 95.3% 34, 85.0% 54, 81.5%
Results are shown as mean and standard deviation [SD] or
percentile; 𝑝 value from Kruskal-Wallis test or chi square
test.
Table 2: Total homocysteine (tHcy), folate, and cobalamin levels
in umbilical cord blood andmothers’ venous blood samples in all
investigatedgroups.
Variable Conotruncal heart defects(𝑛 = 43)
Nonconotruncal heart defects(𝑛 = 40)
Controls(𝑛 = 59) p value
Umbilical cord blood(i) tHcy level [𝜇mol/l] 12.6 [2.4] 7.7 [2.3]
8.1 [2.6] 0.01(ii) Folate level [ng/ml] 13.7 [4.1] 14.3 [2.7] 15.0
[1.3] 0.2(iii) Cobalamin level [pg/ml] 300.5 [95.7] 425.1 [120.7]
325.4 [105.2]
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4 BioMed Research International
Umbilical cord bloodExtreme values
Mothers’ venous bloodExtreme values
CHD Non-CHD ControlsGroup
0
5
10
15
20
25
30
35
40
Folat
e lev
el (n
g/m
l)
∗
Figure 2: Maternal and umbilical cord blood folate
concentrationsin conotruncal heart defects (CHD), nonconotruncal
heart defects(non-CHD), and controls (CG). Results presented as
means andstandard deviations as well as 95% confidence intervals
and extremevalues. ∗Extreme values.
4. Discussion
In this study, we analyzed biomarkers of the
folate-dependenthomocysteine pathway metabolism in neonates with
con-genital heart defects and their mothers. We noticed
elevatedhomocysteine levels in umbilical cord blood in neonatesborn
with conotruncal heart defects in comparison to new-borns with
nonconotruncal heart defects. However, somestudies indicate
maternal elevated homocysteine level asa main risk factor for
congenital heart defects in theiroffspring [7, 8]. We did not
observe such differences inhomocysteine levels between investigated
mothers. Perhapsthose differences are the result of the
proportionally smallgroup of participants in our study.
Additionally, our studydemonstrated decreased cobalamin levels in
neonates withcongenital heart defects and their mothers compared to
con-trols. Some studies presented cobalamin and folate deficiencyin
pregnancy complicated by congenital anomaly, which maysuggest
inadequate daily folic acid and cobalamin dietarysupplementation
during pregnancy [9, 10]. Different resultsare presented by Hobbs
et al., where vitamin B-12 andfolic acid concentrations did not
differ significantly betweenmothers with congenital heart defect
and control subjects[8]. However, other authors suggested that
congenital heartanomalies are associated with low maternal folate
as well aswith hyperhomocysteinemia [11, 12]. Authors revealed
thatcobalamin and folate administration may help to reduce
thedevelopment of cardiac malformations [13, 14].
The etiology of conotruncal heart diseases is complex,with both
environmental and genetic causes. It has been
Umbilical cord bloodExtreme values
Mothers’ venous bloodExtreme values
0
100
200
300
400
500
600
700
Cob
alam
in le
vel (
pg/m
l)
CHD Non-CHD ControlsGroup
Figure 3: Maternal and umbilical cord blood cobalamin
concen-trations in conotruncal heart defects (CHD), nonconotruncal
heartdefects (non-CHD), and controls (CG). Results presented as
meansand standard deviations as well as 95% confidence intervals
andextreme values.
well documented that hyperhomocysteinemia, which is
oftenaccompanied by the defects of folic acid metabolism,
isassociated with the occurrence of congenital defects, and itseems
to be an independent risk factor of conotruncal heartdefects
[15–17].
Based on our results, we postulated that mothers’ homo-cysteine
levels had no direct influence on the developmentof conotruncal
heart defects. Thus, we suggested that hyper-homocysteinemia and
decreased folate levels observed inumbilical cord bloodmay be
associated with the disturbancesin homocysteine pathway metabolism
in newborns withCHD. Zhao et al. found in children with CHD a gene
muta-tion coding for an enzyme, which plays an important rolein the
homocysteine remethylation process [18]. It is prob-able that, in
fetuses with CHD, the excess homocysteine ismetabolized by
remethylation, as we discovered a decreasedconcentration of folic
acid in children with CHD comparedto those with non-CHD.
According to Solanky et al., remethylation of Hcy tomethionine
using methyl donation from folate is the preva-lent pathway in the
human placenta, indicating a markedreliance on folate availability
[19]. Consequently, vitamin Bdeficiency in mothers whose offspring
have congenital mal-formations additionally causes a disturbance in
the mainpathway of Hcy remethylation in the placenta and results
inan increased transfer of Hcy from maternal to fetal
circu-lation.
The basis for the observed abnormal metabolic profileamong
neonates with conotruncal heart defects and their
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BioMed Research International 5
mothers cannot be defined without further analysis of rele-vant
genetic and environmental factors.Therefore, confirma-tion by
future prospective multicentre cohorts is needed.
5. Conclusions
Elevated homocysteine levels in neonates with conotruncalheart
defects and folate metabolism disturbances in theirmothers were
noticed.
The observed differences in homocysteine and cobalaminlevels
between neonates with congenital heart defects suggestthe influence
of various agents disturbing homocysteinemetabolic pathways.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
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