nutrients

Article
Impact of Maternal Lifestyle and Dietary Habits during
Pregnancy on Newborn Metabolic Profile
Ilaria Cicalini 1,2 , Samanta Moffa 3 , Maria Lucia Tommolini 1,2 , Silvia Valentinuzzi 1,3 , Mirco Zucchelli 1,2 ,
Ines Bucci 1,4 , Piero Chiacchiaretta 1,2 , Antonella Fontana 3 , Luca Federici 1,2 , Vincenzo De Laurenzi 1,2 ,
Piero Del Boccio 1,3 , Claudia Rossi 1,2, * and Damiana Pieragostino 1,2

1 Center for Advanced Studies and Technology (CAST), “G. d’Annunzio” University of Chieti-Pescara,
66100 Chieti, Italy; [email protected] (I.C.); [email protected] (M.L.T.);
[email protected] (S.V.); [email protected] (M.Z.); [email protected] (I.B.);
[email protected] (P.C.); [email protected] (L.F.); [email protected] (V.D.L.);
[email protected] (P.D.B.); [email protected] (D.P.)
2 Department of Innovative Technologies in Medicine and Dentistry, “G. d’Annunzio” University of
Chieti-Pescara, 66100 Chieti, Italy
3 Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, 66100 Chieti, Italy;
[email protected] (S.M.); [email protected] (A.F.)
4 Department of Medicine and Aging Science, “G. d’Annunzio” University of Chieti-Pescara, 66100 Chieti, Italy
* Correspondence: [email protected]; Tel.: +39-0871-541290

Abstract: Expanded newborn screening (NBS) is a preventive program that allows for the early
identification of over 40 congenital endocrine-metabolic diseases by analyzing dried blood spot
samples collected from the newborn’s heel within 48–72 h of birth. The determination of amino acids
and acyl-carnitines by Flow Injection Analysis Tandem Mass Spectrometry (FIA-MS/MS) may also
highlight metabolic alterations resulting from external factors, such as maternal nutrition. In the
present study, we developed a questionnaire to investigate the eating habits of 109 women during
pregnancy and statistically correlated the results from the investigation on dietary habits with the
Citation: Cicalini, I.; Moffa, S.;
data obtained by the NBS laboratory of Abruzzo region (Italy). Parameters such as smoking, physical
Tommolini, M.L.; Valentinuzzi, S.;
activity, and the intake of iodized salt, drugs, and supplements were analyzed. This study aimed to
Zucchelli, M.; Bucci, I.;
Chiacchiaretta, P.; Fontana, A.;
highlight how maternal lifestyle, diet, and drug intake during pregnancy may affect the neonatal
Federici, L.; De Laurenzi, V.; et al. metabolic profile, possibly generating false positive or false negative results in the NBS test. The
Impact of Maternal Lifestyle and results pointed out how the knowledge of maternal nutrition and lifestyle may also be precious in
Dietary Habits during Pregnancy on preventing misinterpretations of the neonatal metabolic profile, thereby reducing unnecessary stress
Newborn Metabolic Profile. Nutrients for newborns and their parents and limiting costs for the health system.
2023, 15, 2297. https://doi.org/
10.3390/nu15102297 Keywords: newborn screening; metabolic profiling; mass spectrometry; dietary habits; lifestyle;
Academic Editor: Christopher
nutrition
Papandreou

Received: 17 April 2023

Revised: 8 May 2023 1. Introduction
Accepted: 11 May 2023
Expanded Newborn Screening (NBS) is a preventive medicine program whose goal is
Published: 13 May 2023
to identify infants at risk for over 40 inherited endocrine-metabolic diseases caused by spe-
cific enzymatic defects, which determine the accumulation of toxic metabolic intermediates
and which, if not detected promptly at birth, can compromise the patient’s life with severe
Copyright: © 2023 by the authors.
and often irreversible damage. The analysis is performed on dried blood spot (DBS) samples
Licensee MDPI, Basel, Switzerland. obtained by pricking the newborn’s heel generally within 48–72 h of birth and by collecting
This article is an open access article and letting dry the drops of whole blood on a special filter paper. To date, the Italian panel
distributed under the terms and of diseases object of the expanded NBS includes congenital hypothyroidism (CH), cystic
conditions of the Creative Commons fibrosis (CF), galactosemia, biotinidase deficiency (BTD), aminoacidopathies (AA), urea
Attribution (CC BY) license (https:// cycle disorders (UCD), organic aciduria (OA) and fatty acid oxidation disorders (FAOD),
creativecommons.org/licenses/by/ for a total of over than 40 inborn errors of metabolism (IEMs). The advantage offered by the
4.0/). NBS in terms of time is guaranteed by the use of tandem mass spectrometry (MS/MS), a

Nutrients 2023, 15, 2297. https://doi.org/10.3390/nu15102297 https://www.mdpi.com/journal/nutrients

Nutrients 2023, 15, 2297 2 of 11

versatile, specific, and sensitive technology that is becoming essential for high-throughput
analysis. In fact, in a single analytical run of approximately 1 min, it provides a semi-
quantitative picture of different metabolites (amino acids, acyl-carnitines, succinyl-acetone)
as markers of the diseases of interest [1,2]. Moreover, this targeted metabolomic approach
ensures excellent analytical precision and facilitates the identification and quantification of
metabolites [3]. In particular, the MS-based NBS strategy allows the diagnostic process to be
undertaken early and to promptly start the necessary treatments, thus improving the child’s
health and changing the natural history of the disease. It is worth remembering that various
maternal and neonatal factors can affect the interpretation of NBS results. Interestingly,
there is ample evidence that the metabolic results of the NBS test can be influenced by
maternal lifestyle, nutrition, pharmacological treatments, and diseases, as well as neonatal
clinical variables such as prematurity, the type of feeding, parenteral nutrition, sepsis,
and distress [4–6]. In these cases, the metabolic alterations at expanded NBS could be a
transitory condition generating false positive results. The most consistent false positives
are associated with the suspect of propionic and methyl-malonic acidemia, where various
predisposing factors are considered, such as sepsis, hypoxic-ischemic encephalopathy, drug
intoxication due to maternal exposure, and other congenital disorders of metabolism [7].
Often, the altered profile of C3 (propionyl-carnitine), the C3/C2 (C3/acetyl-carnitine) ra-
tio, C16: 1OH\C17 (3-hydroxy-hexadecenoylcarnitine) and methionine is attributable to
maternal vitamin B12 deficiencies due to poor diets or being totally free of animal prod-
ucts [4], untreated pernicious anemia or nutrient absorption dysfunctions (e.g., gastric
bypass). Vitamin B12 deficiency in pediatric populations can predispose increased plasma
homocysteine levels, an emerging risk factor for cardiovascular disease, low bone mineral
density predisposing to osteoporosis, neural tube defects, intrauterine growth retardation,
autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) [8]
while in pregnant women there is a risk of premature birth, preeclampsia, and spontaneous
abortion [9]. Therefore, it is clear that maternal nutrition and lifestyle can play a crucial
role in the growth, health, and well-being of the fetus [10].
The infant’s feeding can, in turn, alter the metabolite profile. In fact, nutritional
supports rich in amino acids can cause false positives: total parenteral nutrition is often
able to return false alterations to mimic congenital hyperphenylalaninemia [11,12] or
other AA.
Another factor that determines the alteration of metabolites at screening may be the
intake of drugs during pregnancy and breastfeeding. It is well known that some antibiotics,
such as ampicillin and cefotaxime, can induce an increase in the level of acyl-carnitines (C5,
C14: 1, and C16: 1-OH) due to the presence of the pivaloyl functional group [3].
Taken together, all these aspects highlight the importance of post-analytical interpre-
tive tools to resolve the issue of false-positive results at newborn screening for IEMs by
using the information on clinical variables potentially associated with abnormal outcomes.
On the basis of this evidence, in this study, we realized a questionnaire to investigate
the eating habits of 109 women during pregnancy and statistically correlated the results
from the investigation with around 80 metabolic parameters, including metabolic ratios,
obtained by the NBS laboratory of Abruzzo region, Italy. More precisely, we investigated
factors related to lifestyle (physical activity and smoking), dietary habits during pregnancy
(including iodized salt consumption), and also the intake of drugs and supplements (pro-
gesterone, folic acid) and demonstrated their influence on the metabolic profile of newborns.
These factors can eventually overlap with pathological conditions and possibly generate
false positives and/or false negatives in NBS results, supporting the importance of an
adequate nutritional status during pregnancy. Our approach, through a better knowledge
of maternal nutrition and lifestyle, aims at improving our understanding of the neonatal
metabolic profile and avoiding misinterpretations, thus allowing a reduction in unnecessary
stress for newborns and their families and limiting the costs of the health system.
Nutrients 2023, 15, 2297 3 of 11

2. Materials and Methods

2.1. Routinely Newborn Screening Analysis
Dried blood spot (DBS) samples for NBS were punched out into 3.2 mm disks to
perform a flow injection-tandem mass spectrometry analysis (FIA-MS/MS) for the detection
of over 40 IEMs, including AAs, urea cycle, organic acid, and fatty acid oxidation disorders.
Four 3.2 mm DBS disks were used for immunofluorimetric assays by a Genetic Screening
Processor GSP® Instrument (Perkin Elmer Life and Analytical Sciences, Turku, Finland) to
test for congenital hypothyroidism (CH), cystic fibrosis (CF), galactosemia and biotinidase
deficiency, respectively, and the fifth DBS disk was employed for FIA-MS/MS analysis.
For the latter, the DBS disk (of approximately 3–3.2 µL whole blood) was extracted by
adding 125 µL of extraction solutions containing an internal standard provided by the
NeoBase 2 Non-Derivatized MSMS Kit (Perkin Elmer Life and Analytical Sciences, Turku,
Finland) for 30 min at 45 ◦ C at 700 rpm. After the incubation step, 100 µL of the solution
was transferred onto a clear plate for the next determination of 14 AAs, 35 acylcarnitines
(ACs), free carnitine, and succinylacetone by FIA-MS/MS.
The MS/MS system consists of RenataDX™ Screening Systems (Waters Corporation,
Milford, MA, USA) as a fully FIA-MS/MS IVD system for high-throughput analysis. The
system components include the 3777C IVD Sample Manager, ACQUITY™ UPLC™ I-Class
IVD Binary Solvent Manager, and Xevo™ TQD IVD Mass Spectrometer (Waters Corpo-
ration, Milford, MA, USA). The system operates in the positive electrospray ionization
mode with multiple reaction monitoring (MRM) acquisitions. A total of 10 µL were in-
jected into the ion source, and the run time was 1.3 min, injection-to-injection, as already
described [2]. Data were finally processed by the MassLynx™ (IVD) Software V4.2 with
IonLynx™ Application Manager (Waters Corp.). Mass spectrometry parameters, including
MS/MS transitions, cone potentials, and collision energies for each metabolite and its
relative internal standard, are fully listed in Table S1 of the Supplementary Materials [4,11].

2.2. Questionnaire
A questionnaire was created to obtain information about the lifestyle and nutrition
of women during pregnancy. Significant data were then collected from the volunteer
women in post-partum periods from the 9 Birth Points in the Abruzzo region (Chieti,
Vasto, Lanciano, L’Aquila, Sulmona, Sant’Omero, Teramo, Pescara, and Avezzano). In total,
109 women participated in the study. They were contacted by telephone and received the
questionnaire via smartphone or email. The mothers of newborns who tested positive for
the pathologies covered by the expanded NBS as well as mothers affected by diseases that
are the object of NBS were not included in the study.
The aim of the questionnaire was to obtain information about the lifestyle (physi-
cal activity and smoking) and nutritional habits followed by women during pregnancy
(Table S2). In particular, lifestyle, smoking and alcohol intake, the type of diet followed
(Mediterranean; Lacto-ovo-vegetarian; Lacto-vegetarian; Ovo-vegetarian; Lacto-ovo-pesco-
vegetarian; Vegan; Other types), and the frequency of consumption of each food class
(white and red meat, cooked cured meats, raw cured meats, fish, eggs, fresh and aged
cheeses, yogurt or milk or milk substitutes, cereals, legumes, vegetables, fresh and dried
fruit, nervine drinks and sweets) were of interest. Moreover, it was also decided to examine
the intake of drugs and/or the treatment of particular previous pathologies and dietary
support supplements (Vitamin A; Vitamin E; Vitamin K; Vitamin C; Vitamin B12; Other
Vitamins of group B; Calcium; Iron; Phosphorus; Magnesium; Omega-3; Probiotics; Antiox-
idants; Proteins/Amino acids; Vegetable supplements), as well as the feeding modalities of
the newborn (breast, artificial, mixed and/or total parenteral nutrition).
The questionnaire, as developed in the electronic format on the Google Forms platform,
is available at the following link: https://docs.google.com/forms/d/19fRGZf58IcP4fJS0
1ei6vGKQprv1VxW33x9K9wUmGeE/edit?usp=drivesdk.
Nutrients 2023, 15, 2297 4 of 11

2.3. Matrix Preparation

A single Excel matrix was prepared containing the information extrapolated from the
cards where the newborn’s blood sample was collected, the responses of the mothers to
the questionnaire, and the values of amino acids and acylcarnitines obtained by tandem
mass spectrometry. A score was assigned to each answer given by the mothers in the
questionnaire, as reported in Table S2 of the Supplementary Materials. Factors from the
questionnaire were selected from time to time and were statistically correlated to the
metabolite concentrations of each newborn.

2.4. Statistical Analysis

A statistical correlation between the concentrations of amino acids and acylcarnitines
obtained by MS/MS analysis and the responses of interest given by the mothers in the
questionnaire was performed. The correlation analyses were processed by GraphPad
Prism (GraphPad software, Inc., Boston, MA, USA) and Metaboanalyst 4.0. Supervised
multivariate analysis was performed using Sparse PLS-DA (Sparse Partial Least Squares–
Determination Analysis), and univariate analysis was performed using the t-test and
ANOVA. Correlation analysis was performed on the basis of the questionnaire responses
and metabolite levels measured in infants who were clustered according to biochem-
ical criteria (amino acids, urea cycle metabolites, short-, medium-, long-chain acylcar-
nitines and odd-chain acylcarnitines) using the Python sklearn library available online at
https://scikit-learn.org/stable/.

3. Results
In our study, among all the data collected, some neonatal metabolic parameters
correlated with the maternal factors addressed by the questionnaire. In the following,
the results obtained by the correlation analysis of the metabolites significantly modulated
in relation to physical activity, the use of iodized salt in the diet, and smoking during
pregnancy are described.

3.1. Correlation Analysis

3.1.1. Physical Activity
The first factor analyzed was physical activity. The segregation of the total matrix as
a function of variables relating to maternal physical activity during pregnancy through
sPLS-D statistical analysis shows a scatter plot in which individuals are reported as dots
of different colors depending on the amount of physical activity performed. In particular,
the red dots (51%) indicate a lack of training, and the blue ones (49%) represent physical
activity, as shown in Figure 1A,B.
The univariate statistical analysis by t-test and the elimination of the outliers high-
lighted a significant difference in the levels of glutamine (GLN) and glutamate (GLU) in
the metabolic profile of newborns with active mothers during pregnancy (GLN p < 0.001;
GLU p < 0.01), as represented in Figure 1C. More precisely, in this comparison, a higher
concentration of GLN and GLU were observed by the MS/MS screening analysis of DBS
samples from newborns with mothers who were physically active during pregnancy.

3.1.2. Use of Iodized Salt in the Diet

The segregation of the total matrix as a function of the consumption of iodized salt
during pregnancy by sPLS-DA statistical analysis produced an undefined separation
between the two groups. Figure 2A,B shows the scatter plot with dots of different colors
depending on the maternal habit for the consumption of iodized salt during pregnancy.
Nutrients
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Figure 1. (A,B)
Figure 1. (A,B)PLS-DA
PLS-DAscattered
scatteredanalysis:
analysis:selection
selectionandandclustering
clusteringof of
variables relating
variables to physical
relating to physical
activity:
activity: sedentary
sedentarymothers
mothersininred,
red,active
activemothers
mothersininblue.
blue.(C)
(C)Dots
Dotsplot showing
plot showing thethe
concentrations
concentrations
µmol/L for GLN and
in µmol/L and GLU
GLU in innewborns
newbornswithwithmothers
motherswho whodiddidnot
not exercise
exercise (in(in red)
red) andandwhowho
exercised(in
exercised (inblue)
blue)during
duringpregnancy
pregnancy (GLN:
(GLN: p =p 0.0060;
= 0.0060; GLU:
GLU: p =p0.0302).
= 0.0302).
(*) (*)
p <p0.05
< 0.05
andand
(**)(**)
p <p0.01.
< 0.01.

3.1.2.InUse
particular,
of Iodizedthe Salt
intake of iodized
in the Diet salt is represented with blue dots (68%), while the
non-intake is shown with red dots (32%). Moreover, the univariate statistical analysis for a
The segregation of the total matrix as a function of the consumption of iodized salt
comparison between the use of iodized or non-iodized salt in the gestational period allowed
during pregnancy by sPLS-DA statistical analysis produced an undefined separation be-
us to highlight statistically significant lower levels of free carnitine (C0) in the metabolic
tween the two groups. Figure 2A,B shows the scatter plot with dots of different colors
profile of newborns from mothers who took iodized salt (p < 0.001), as highlighted in
Figure 2C. on the maternal habit for the consumption of iodized salt during pregnancy.
depending
In particular, the intake of iodized salt is represented with blue dots (68%), while the
non-intake
3.1.3. Smokingis shown with red dots (32%). Moreover, the univariate statistical analysis for
a comparison
In addition, between
anotherthefactor
use of iodized was
analyzed or non-iodized
smoking. By salt in the gestational
segregating period
the matrix as a al-
lowed usof
function tosmoking
highlightduring
statistically significant
pregnancy, lower levels
the sPLS-DA of free analysis
statistical carnitineshowed
(C0) in the met-
a fair
abolic profile
separation of newborns
between the two from
groups:mothers who
mothers took iodized
without saltcigarette
a habit of (p < 0.001), as highlighted
smoking during
in Figure 2C.
pregnancy in red (91%) and mothers with this habit in blue (9%), as depicted in Figure 3A,B.
Nutrients 2021,15,
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12

Figure2.2.(A,B)
Figure (A,B)PLS-DA
PLS-DAscattered
scatteredanalysis:
analysis:selection
selectionand
andclustering
clusteringof of variables
variables relating
relating to the
to the useuse
of
of iodized salt in the diet: maternal intake of non-iodized salt during pregnancy in red,
iodized salt in the diet: maternal intake of non-iodized salt during pregnancy in red, maternal intake maternal
intake
of of iodized
iodized salt during
salt during pregnancy
pregnancy in blue. in blue.
(C) Dots(C) Dots
plot plot showing
showing the concentrations
the concentrations in μmol/L
in µmol/L for C0
for C0 in newborns with maternal non-intake of iodized salt (in red) and maternal intake of iodized
in newborns with maternal non-intake of iodized salt (in red) and maternal intake of iodized salt (in
salt (in blue) during pregnancy (C0: p = 0.00169). (**) p < 0.01
blue) during pregnancy (C0: p = 0.00169). (**) p < 0.01.

3.1.3.Keeping
Smoking in mind the statistical limitations due to the fact that the cohort of smokers was
In addition,
far lower than that another factor analyzed
of non-smokers, was
univariate smoking.
statistical By segregating
analysis the matrix
in Figure 3 panel as a
C pointed
function
out someofacylcarnitines,
smoking during pregnancy,
i.e., the sPLS-DA statistical
ocatadecenoylcarnitine (C18:1), analysis showed a fair sep-
octadecadienoylcarnitine
(C18:2)
arationand tetradecenoylcarnitine
between (C14:1), as
the two groups: mothers statistically
without significant
a habit features
of cigarette (C14:2
smoking and
during
C18:2 p < 0.001;
pregnancy in redC18:1 p =and
(91%) 0.0392).
mothers with this habit in blue (9%), as depicted in Figure
3A,B.
3.2. Cluster Analysis
The quantified metabolites were normalized and grouped into 7 clusters: Cluster
1 containing amino acids, Cluster 2 containing urea cycle amino acids, and metabolites,
Cluster 3 containing C0, C2 and C4, Cluster 4 containing the odd acylcarnitines C3 and
C5 and the C3/C2 and C3/C16 ratios, Cluster 5 containing the sum of hydroxylated and
dicarboxylic acylcarnitines, Cluster 6 containing the medium chain acylcarnitines and
finally Cluster 7 containing long and very long chain acylcarnitines. A correlation analysis
was carried out between the groups of metabolites quantified in the DBS of newborns
with some maternal food habits and lifestyles as well as with some neonatal features.
The correlation analysis revealed a significant correlation between Cluster 4 of odd-chain
acylcarnitines and some information relating to the newborn’s gestational age, weight, and
use of antibiotics (R2 = 0.40 and F-statistic = 2.54 × 10−13 ) (Figure 4A). This first result
demonstrated how the prematurity of the newborn affected the levels of some metabolites,
such as acylcarnitine C5 and the C3/C16 ratio, resulting in a significant modulation as
Nutrients 2023, 15, 2297 7 of 11

reported in Figure S1 of the Supplementary Materials. Cluster 5 showed a good correlation

between the frequency of consumption of nervine drinks in pregnancy and the levels of
hydroxylated and dicarboxylic acylcarnitines in the newborn, with R2 = 0.24 and F-statistic
= 1.16 × 10−7 (Figure 4B). Finally, this correlation analysis showed that Cluster 7 of long-
Nutrients 2021, 13, x FOR PEER REVIEW 7 of 12
chain acylcarnitines was significantly correlated with the frequency of taking supplements
2 − 6
during pregnancy, with R = 0.18 and F-statistic = 5.06 × 10 (Figure 4C).

selection and
Figure 3. (A,B) PLS-DA scattered analysis: selection and clustering
clustering of
of variables
variables relating
relating to
to smoking:
smoking:
non-smoker mothers in red, smoker mothers in blue. blue. (C) Dots plot shows the concentrations in
µmol/L for
µmol/L for C14:2, C18:1
C18:1 and
andC18:2
C18:2ininnewborns
newbornswithwithmothers
mothers without
without a habit
a habit of cigarette
of cigarette smoking
smoking (in
(in red) and for mothers with this habit (in blue) during pregnancy. (C14:2: p = 0.0015, C18:1:
red) and for mothers with this habit (in blue) during pregnancy. (C14:2: p = 0.0015, C18:1: p = 0.0392, p =
0.0392, and C18:2: p = 0.0032). (*) p < 0.05 and (**)
and C18:2: p = 0.0032). (*) p < 0.05 and (**) p < 0.01. p < 0.01.

Keeping in mind the statistical limitations due to the fact that the cohort of smokers
was far lower than that of non-smokers, univariate statistical analysis in Figure 3 panel C
pointed out some acylcarnitines, i.e., ocatadecenoylcarnitine (C18:1), octadecadi-
enoylcarnitine (C18:2) and tetradecenoylcarnitine (C14:1), as statistically significant fea-
tures (C14:2 and C18:2 p < 0.001; C18:1 p = 0.0392).

3.2. Cluster Analysis

 in Figure S1 of the Supplementary Materials. Cluster 5 showed a good correlation between
the frequency of consumption of nervine drinks in pregnancy and the levels of hydrox-
ylated and dicarboxylic acylcarnitines in the newborn, with R2 = 0.24 and F-statistic = 1.16
× 10−7 (Figure 4B). Finally, this correlation analysis showed that Cluster 7 of long-chain
Nutrients 2023, 15, 2297 acylcarnitines was significantly correlated with the frequency of taking supplements8dur-
of 11
ing pregnancy, with R2 = 0.18 and F-statistic = 5.06 × 10−6 (Figure 4C).

Figure 4. (A)
Figure 4. (A) Correlation
Correlationanalysis
analysisbetween
between cluster
cluster 4 (quantified
4 (quantified oddodd chain
chain acylcarnitines
acylcarnitines in new-
in newborns)
borns) and maternal responses to the questionnaire. The best features are represented by gestational
and maternal responses to the questionnaire. The best features are represented by gestational age,
age, newborn weight, and antibiotic use. (B) Correlation analysis between cluster 5 (quantified hy-
newborn weight, and antibiotic use. (B) Correlation analysis between cluster 5 (quantified hydroxy-
droxylated and dicarboxylic acylcarnitines in newborns) and maternal responses to the question-
lated and
naire. Thedicarboxylic
best feature acylcarnitines
is representedinbynewborns) and maternal
the frequency of takingresponses to the during
nervine drinks questionnaire. The
pregnancy.
bestCorrelation
(C) feature is represented by the frequency
analysis between of taking nervine
cluster 7 (quantified, drinks
long, and veryduring
long pregnancy. (C) Correla-
chain acylcarnitines in
tion analysisand
newborns) between cluster
maternal 7 (quantified,
responses long, and very long
to the questionnaire. chainfeature
The best acylcarnitines in newborns)
is represented by the and
fre-
quency
maternalofresponses
taking supplements during pregnancy.
to the questionnaire. The best feature is represented by the frequency of taking
supplements during pregnancy.
It must be pointed out that all the metabolites analyzed, both in their absolute value
It must be pointed
and individually, outwithin
were all that allthe
thereference
metabolites analyzed,
limits used in both in theiranalysis
the routine absolutefor
value
the
and individually, were all within the reference limits used in the routine
diagnosis of related metabolic diseases. Furthermore, no significance emerged from the analysis for
the diagnosis of related metabolic diseases. Furthermore, no significance emerged
univariate statistical analysis except for the odd chain acylcarnitines C5 and C3/C16 ratio, from
the univariate
which statistical modulated
was significantly analysis except for thetoodd
in relation chain acylcarnitines
gestational age but not C5 and C3/C16
to maternal nu-
ratio, which
trition was significantly modulated in relation to gestational age but not to maternal
and lifestyle.
nutrition and lifestyle.
4. Discussion
4. Discussion
Many authors have already discussed how the expansion of NBS has highlighted a
Many authors have already discussed how the expansion of NBS has highlighted
number of “side effects”, even when considering the significant advantages achieved in the
a number of “side effects”, even when considering the significant advantages achieved
diagnosis and treatment of IEMs [4–6,13]. Lanpher B et al., in their review, gave a signifi-
in the diagnosis and treatment of IEMs [4–6,13]. Lanpher B et al., in their review, gave
cant description of the importance of metabolite flux and of the integration of phenotypic
a significant description of the importance of metabolite flux and of the integration of
characterization with environmental, genetic, and biochemical factors. They clarified the
phenotypic characterization with environmental, genetic, and biochemical factors. They
crucial role of this integration in leading to new diagnostic and therapeutic approaches
clarified the crucial role of this integration in leading to new diagnostic and therapeutic
[14]. In this view, it is worth remembering that a metabolic profile is not exclusively
approaches [14]. In this view, it is worth remembering that a metabolic profile is not
exclusively influenced by the genome but also by a variety of clinical variables as well as
external factors that may have an impact on it. Actually, it is well known that alterations
in specific markers by MS/MS NBS allow for the identification of affected patients, but
this may also be a consequence of maternal defects, nutritional deficiency, partial enzyme
deficiency, late-onset forms of the disease, heterozygosis variants, prematurity, and the use
of drugs [4–6].
In the present study, we investigated a possible correlation between maternal lifestyle
and dietary habits during pregnancy and the newborn metabolic profile, using the data
obtained by the NBS laboratory of Abruzzo Region, Italy. As regards physical activity, high
concentrations of GLN in women who exercised during pregnancy suggested a mobilization
of amino acids in the muscles and a probable role of GLU and GLN in the detoxification
processes. At the same time, an optimized function of the immune system was also
described in newborns born by physically active mothers [15]. GLU in high quantities could
be used partly for GLN formation, which is essential in synthesis and immunocompetence
processes and for transamination reactions. Furthermore, it could be used partly to enter
the Krebs cycle as an energy substrate in the form of α-ketoglutarate [16,17].
Nutrients 2023, 15, 2297 9 of 11

Maternal iodized salt consumption has been associated with lower levels of neonatal
C0 in our study. Iodine is necessary for thyroid hormone synthesis, and a severe deficiency
causes hypothyroidism, and a higher iodine intake is often required during pregnancy [18].
Carnitine is a quaternary ammonium compound. It transports fatty acids across the
mitochondrial membrane as acyl carnitine esters so that these can be metabolized in the
beta-oxidation of fatty acids. Carnitine determines good cardiac function and a reduction in
oxidative stress, inflammation, and necrosis processes. The relationship between carnitine
and thyroid function comes from clinical studies showing the antagonistic thyroid hormone
action effects of C0 [19–22] and a positive correlation between urinary carnitine with serum
thyroxin concentration [23]. In our study, C0 was lower in newborns whose mothers
used iodized salt during pregnancy (Figure 2C). It is challenging to explain these data,
mainly because we do not have maternal TSH and thyroid hormone levels since it does
not represent a routine investigation in NBS. However, we did carry out a correlation
between the levels of free carnitine in the newborn with their TSH values, measured
contextually after about 48 h of life, finding no correlation, as reported in Figure S2 of the
Supplementary Materials. To the best of our knowledge, there are no studies evaluating
the relation of the neonatal carnitines profile and maternal thyroid function. A really
recent study addressed the correlation between maternal thyroid hormones, TSH, and C0
with birth weight showing a positive correlation between maternal C0, TSH, and thyroid
hormones and a negative association between C0, free thyroxine, and birth weight [24].
Following this questionnaire, in our study, a significant decrease in the levels of C0 was
revealed in the metabolic profile of newborns whose mothers (68% of participants) took
iodized salt during pregnancy. In this context, when focusing on iodine intake, it would
be also interesting to evaluate the geographical origin (sea areas or mountain ones) of
mothers [25] and the condition of hypothyroidism, hyperthyroidism, or euthyroidism. As
regards smoking, acylcarnitines, such as C18: 1, C18: 2, and C14: 1, were all higher in
the metabolic profiling of newborns from pregnant smokers. It has been already reported
that cigarette smoking causes a depletion of ascorbic acid, which is a cofactor for the
enzymes trimethyl-lysine hydroxylase and gamma-butyrobetaine hydroxylase involved in
the synthesis of carnitine [26,27]. From this point of view, we would expect that cigarette
smoking may lead to a reduction in unsaturated carnitines and an increase in hydroxylated
ones. The reversal of this trend we observed in the newborns’ metabolic profiling would
make us speculate on a possible compensatory effect: an adaptive response that the fetus
acquires in response to the oxidative stress induced by smoking in the mother’s body. To
support and eventually confirm this hypothesis, it would be essential to use a larger cohort.
Finally, from the correlation analyses obtained by clustering the metabolites quantified
in the newborns, some interesting correlations emerged, above all relating to the frequency
of the intake of supplements and the frequency of consuming nervine drinks during
pregnancy. As discussed above, when looking at each metabolite separately, no significance
emerged, with the exception of the odd chain acylcarnitines, which are statistically related
to the condition of the relative prematurity of the newborn.
Post-analytical interpretive tools, often applied by NBS programs worldwide, may
resolve the issue of false-positive screens for IEMs by using the information on a variety
of clinical variables potentially associated with abnormal results, such as birth weight,
gestational age, gender, time at blood collection, antibiotic and/or cortisone treatment,
and type of nutrition. In this way, the maternal nutritional information and lifestyle
collected by our questionnaire could be helpful in the management of newborn clinical
outcomes in the post-analytical stage to potentially minimize false-positive results, thus
enabling a personalized medicine approach. We are aware that this must be considered as
a preliminary study, based on a limited number of participants, while also considering that
our region was very small and mother participation was on a voluntary basis. Nevertheless,
achieving a higher number of participants over time would be necessary to provide a
post-analytical tool that is suitable for supporting clinical applications in NBS laboratories.
Nutrients 2023, 15, 2297 10 of 11

5. Conclusions
In this study, by correlating the data obtained from the NBS laboratory of the Abruzzo
region in Italy with the results of the questionnaire we developed, it was possible and
interesting to investigate the impact of mothers’ lifestyles and dietary habits during preg-
nancy on newborns’ metabolic profile. Of note, physical activity, the use of iodized salt,
consumption of supplements and nervine drinks, as well as smoking were all found to
correlate with specific metabolites among those investigated in newborns. However, we
recognize that it is important to confirm these correlations in a larger cohort and be able
to achieve a global and complete knowledge of newborns and mothers. In doing so, it
would be possible to reconsider the newborn metabolic profile by expanding NBS in view
of the maternal lifestyle and dietary habits for an improved understanding and a better
diagnostic evaluation.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/nu15102297/s1, Figure S1. C5 and C3/C16 ratio univariate
statistical analysis performed by Metaboanalyst based on data from Cluster 3, considering gestational
week (g.w.) and antibiotics treatment in newborn. Figure S2. Pearson correlation between DBS
TSH levels (µU/mL) and DBS free carnitine C0 levels (µM). R squared is 0.00011, p-value 0.91 (not
significant). Table S1. Mass spectrometry parameters used for FIA-MS/MS analysis on RenataDXTM
Screening Systems by NeoBase™ 2 Non-derivatized MSMS kit. For each analyte, MRM transition,
cone voltage (V) and collision energy (eV) are shown. The internal standards (ISs) are reported in
bold. Table S2. Questionnaire.
Author Contributions: Writing—original draft preparation, formal analysis, I.C.; writing, data
curation, and investigation, S.M.; methodology and validation, M.L.T., S.V. and M.Z.; investigation,
visualization, and review, I.B., A.F., L.F. and P.D.B.; formal analysis, P.C.; resources and supervision,
V.D.L.; writing—review and editing, conceptualization, C.R.; review and editing, supervision, D.P.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Ethics Committee approval N. 21 30 September 2021
(protocol code 0000992).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data presented in this study are available in Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest.

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