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UNIVERSIDADE FEDERAL DO RIO DE JANEIRO 
INSTITUTO DE QUÍMICA 
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA DE ALIMENTOS 
 
 
 
 
 
VANESSA DE ARAUJO GOES 
 
 
 
 
 
 
 
DIETARY ANTIOXIDANTS AND MATERNAL REDOX STATE IN THE CONTEXT 
OF GESTATIONAL DIABETES MELLITUS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
RIO DE JANEIRO 
2018 
 
VANESSA DE ARAUJO GOES 
 
 
 
 
 
 
 
DIETARY ANTIOXIDANTS AND MATERNAL REDOX STATE IN THE CONTEXT 
OF GESTATIONAL DIABETES MELLITUS 
 
 
 
 
 
Master dissertation presented as part of the 
requirements to obtain Master degree in Food 
Science at the Instituto de Química, Universidade 
Federal do Rio de Janeiro. 
 
Research supervisor: Tatiana El-Bacha Porto, DSc 
 
 
 
 
 
 
 
 
 
 
 
RIO DE JANEIRO 
2018 
CIP - Catalogação na Publicação
Elaborado pelo Sistema de Geração Automática da UFRJ com os
dados fornecidos pelo(a) autor(a).
G598d
Goes, Vanessa de Araujo
 Dietary antioxidants and maternal redox state in
the context of gestational diabetes mellitus /
Vanessa de Araujo Goes. -- Rio de Janeiro, 2018.
 79 f.
 Orientador: Tatiana El Bacha Porto.
 Dissertação (mestrado) - Universidade Federal do
Rio de Janeiro, Instituto de Química, Programa de Pós
Graduação em Ciência de Alimentos, 2018. 
 1. Diabetes Mellitus Gestacional. 2.
Antioxidantes. 3. Homeostase Redox. 4. Função
placentária. I. Porto, Tatiana El Bacha, orient.
II. Título.
VANESSA DE ARAUJO GOES 
DIETARY ANTIOXIDANTS AND MATERNAL REDOX STATE IN THE CONTEXT 
OF GESTATIONAL DIABETES MELLITUS 
Master dissertation presented as part of the 
requirements to obtain Master degree in Food 
Science at the Instituto de Química, Universidade 
Federal do Rio de Janeiro. 
Approved: 28/06/2018. 
________________________________________________________________________ 
Tatiana El-Bacha Porto, DSc 
Universidade Federal do Rio de Janeiro 
________________________________________________________________________ 
Patricia Coelho de Velasco, DSc 
Universidade Federal do Rio de Janeiro 
________________________________________________________________________ 
Anderson Junger Teodoro, DSc 
Universidade Federal do Estado do Rio de Janeiro 
ACKNOWLEDGMENTS 
A Vida 
Aos meus pais Antônio Tomé e Maria Helena pela vida, amizade e amor incondicional 
Ao meu filho Julien por sua compreensão, generosidade, paciência, companheirismo, 
amizade, amor, alegria … 
Ao meu amor Cláudio 
Aos amigos queridos 
A amiga e orientadora brilhante, sempre presente, Tatiana 
Aos colegas pesquisadores guerreiros 
Sem vocês a realização desse trabalho não teria sido possível 
Muito obrigada 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
“A sabedoria é um paradoxo. O homem que mais sabe é aquele que mais 
reconhece a vastidão da sua ignorância!” 
 
Nietszche 
 
 
RESUMO 
A Diabetes Mellitus Gestacional (DMG) é o principal distúrbio metabólico que ocorre na gestação. 
Esta patologia está associada a respostas pró-inflamatórias e pró-oxidantes que comprometem a 
função placentária, levando a desfechos adversos tanto para a mãe quanto para o feto. A nutrição 
materna tem papel essencial na função placentária e no desenvolvimento fetal. Evidências indicam 
que alguns micronutrientes e compostos bioativos desempenham papéis importantes na atenuação 
dos desfechos indesejáveis. O objetivo deste estudo foi investigar a associação entre antioxidantes 
dietéticos, estado redox materno e desfechos neonatais no contexto da DMG por meio de uma 
coorte prospectiva de gestantes na Maternidade Escola da UFRJ/RJ, onde 23 gestantes foram 
selecionadas e estratificadas em grupos não DMG (n=15) e DMG (n=8). Foram coletados dados 
sóciodemográficos, dietéticos e de desfechos neonatais, assim como amostras de sangue materno, 
nos 2º e 3º trimestres gestacionais. O estado redox materno foi avaliado a partir da capacidade 
antioxidante total (CAT) do plasma através dos métodos FRAP e ORAC e correlacionada com a 
ingestão dietética de micronutrientes antioxidantes e de polifenólicos, assim como com os 
desfechos neonatais comprimento, peso ao nascer e perímetro cefálico. Nossos resultados 
mostraram que as características sóciodemograficas e relacionadas aos desfechos neonatais foram 
semelhantes nos 2 grupos, assim como a ingestão de carboidratos, lipídios, proteínas, 
micronutrientes e polifenólicos. Pontualmente, a ingestão de carotenoides foi maior no 3º trimestre 
quando comparado ao 2º trimestre somente no grupo não-DMG. A CAT aumentou ao longo da 
gestação considerando o grupo todo, podendo estar relacionada a um processo fisiológico deste 
estado. Fatores dietéticos que parecem estar associados ao aumento da CAT foram a ingestão de 
selênio e zinco no 3º trimestre no grupo DMG, e de vitamina C no 2º trimestre no grupo não-DMG. 
Em relação aos desfechos neonatais, a ingestão de Vitamina E foi relevante pois apresentou 
correlação positiva com peso e comprimento ao nascer e perímetro cefálico, quando considerado o 
grupo todo. A CAT no 2º trimestre também se correlacionou positivamente com comprimento ao 
nascer quando considerado o grupo todo e o grupo não-DMG. Em conclusão, poucas associações 
foram observadas entre o consumo dietético de antioxidantes e a homeostase redox materna e os 
desfechos neonatais. Embora novas investigações sejam necessárias, selênio, zinco, vitamina C e E, 
carotenoides, especialmente b-caroteno, estão entre os componentes dietéticos que merecem 
atenção durante o aconselhamento nutricional de gestantes. Adicionalmente, dado que em cada 
trimestre gestacional observou-se particularidades acerca do impacto da dieta sobre os parâmetros 
estudados, os diferentes estágios da gestação devem ser um ponto central na investigação dos 
mecanismos de ação dos nutrientes e compostos dietéticos sobre os desfechos neonatais. Palavras-
chave: Diabetes Mellitus Gestacional; Antioxidantes; Homeostase Redox; Desfechos neonatais 
 
ABSTRACT 
The Gestational Diabetes Mellitus (GDM) is the main metabolic disorder that occurs during 
pregnancy. This disease is associated with pro-inflammatory and pro-oxidant responses that 
compromise placental function leading to maternal and fetal adverse outcomes. Maternal nutrition 
plays an essential role on placental function and fetal development. Evidences indicate that some 
micronutrients and bioactive compounds plays important roles in the attenuation of adverse 
outcomes. The aim of this study was to investigate the association between dietary antioxidants, 
maternal redox state and neonatal outcomes in the context of GDM through a pregnant women 
prospective cohort in Maternidade Escola from UFRJ/RJ, where 23 pregnant women were selected 
and stratified in non GDM (n=15) and GDM (n=8) groups. Sociodemographic, dietetic and neonatal 
outcomes data, as well as maternal blood samples were collected in the 2nd and 3rd gestational 
trimesters. Maternal redox state was evaluated from plasma total antioxidant capacity (TAC) 
through FRAP and ORAC methods and correlated with dietary intake of antioxidant micronutrients 
and bioactive compounds as well as the neonatal outcomes, birth length, weight and cephalic 
perimeter. Our results showed that the sociodemographic and neonatal outcomes characteristics 
were similar between both groups as well as carbohydrates, lipids, proteins micronutrients and 
polyphenols intake. Precisely, carotenoids intake was higher in the 3rd trimester when compared to 
the 2nd trimester considering only the non-GDM group. The TAC increased throughout pregnancy 
when considering the whole group, what could be related to a physiological process inherent to this 
state. Dietetic factors that could be related to TAC increasement were selenium and zinc intake in 
the 3rd trimester in GDM group and vitamin C in the 2nd trimester in the non-GDM group. 
Concerning neonatal outcomes, vitamin E intake was relevantbecause it presented positive 
correlation with birth weight and length and cephalic perimeter, when considered the whole group. 
The 2nd trimester TAC was also positively correlated with birth length, when considering the whole 
group and the non GDM group. In conclusion, few associations were observed between dietetic 
antioxidant intake and maternal redox homeostasis and neonatal outcomes. Even though further 
investigations are necessary, selenium, zinc, vitamin C and E, carotenoids, especially β carotene, 
are among the dietetic components that deserves attention during pregnant women nutritional 
counseling. Additionally, since in each gestational trimester was observed particularities concerning 
dietetic impact on the studied parameters, the different pregnancy stages should be a central point in 
the investigations of the nutrients and bioactive compounds mechanisms of action on neonatal 
outcomes. Key-words: Gestational Diabetes Mellitus; Antioxidants; Redox homeostasis; Neonatal 
outcomes 
 
 
 
LIST OF FIGURES 
 
Figure 1. Physiological pro diabetogenic state in pregnancy............................................ 18 
Figure 2. Nutrient transport across the placenta………………………………………… 22 
Figure 3. Classification of antioxidants……………………………………………….… 28 
Figure 4. Flavonols intake ratio from non-GDM and GDM pregnant women…….......... 45 
Figure 5. Plasma antioxidant capacity of pregnant women…………………………...… 46 
Figure 6. Correlation between selenium intake and 2nd and 3rd trimesters TAC from pregnant 
women…………………………………………………………………………..………... 47 
Figure 7. Correlation analyses between selenium intake ratio (3T:2T) and 3rd trimester TAC 
from pregnant women………………………….……...………………………..………... 47 
Figure 8. Correlation analyses between zinc intake and 2nd and 3rd trimesters TAC from 
Pregnant women………..………………………………………………………..………...48 
Figure 9. Correlation analyses between tocopherol intake and 2nd and 3rd trimesters TAC from 
pregnant women …………………………………………………………………………. 49 
Figure 10. Correlation analyses between ascorbic acid intake and 2nd and 3rd trimesters TAC 
from pregnant women ……………….………………………………………….....………50 
Figure 11. Correlation analyses between total carotenoid intake and 2nd and 3rd trimesters 
TAC from pregnant women ……………………………………………………….…….. 51 
Figure 12. Correlation analyses between b-carotene intake and 2nd and 3rd trimesters TAC 
from pregnant women ………………………………………………….……..…............. 52 
Figure 13. Correlation analyses between total polyphenol intake ratio (3T:2T) and 3rd 
trimester TAC from pregnant women …………………………………………………… 53 
Figure 14. Correlation analyses between total flavonoids intake ratio (3T:2T) from pregnant 
women ……………………................................................................................................ 54 
Figure 15. Correlation analyses between lignan intake and 2nd and 3rd trimesters TAC from 
pregnant women ……………………………………………………..……………………55 
Figure 16. Correlation analyses between lignan intake ratio (3T:2T) and 3rd trimester TAC 
from pregnant women……................................................................................................. 55 
Figure 17. Correlation analyses between tocopherol intake ratio (3T:2T) and neonatal 
 outcomes from the cohort study at ME/UFRJ…………………………………………….56 
Figure 18. Correlation analyses between antioxidant capacity and birth weight from 
the cohort study at ME/UFRJ………………………......……………………………….…57 
 
Figure 19. Correlation analyses between antioxidant capacity and birth length from 
the cohort study at ME/UFRJ………………………......…………………………………58 
Figure 20. Correlation analyses between antioxidant capacity and cephalic perimeter from 
the cohort study at ME/UFRJ………………………......…………………………………59 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LIST OF TABLES 
 
Table 1. Characteristics of pregnant women and newborns..................................................39 
Table 2. Energy, macronutrients and fibers intake………………………………………... 40 
Table 3. Mineral intake………………………………………….………………………….41 
Table 4. Vitamin intake………………………………………….…………………………42 
Table 5. Antioxidant micronutrients intake…………………………………...………........43 
Table 6. Polyphenols intake ………………………………………….…………………….44 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LIST OF ANNEXES 
 
ANNEX 1. Dietary recomendations intake 
ANNEX 2. Certificate of presentation for ethic appreciation (CAAE) 
ANNEX 3. Study design fluxogram 
ANNEX 4. Free and informed consent form 
ANNEX 5. Baseline (Research protocol) 
ANNEX 6. 24h recall (24h R) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LIST OF ABBREVIATIONS 
 
AGEs – Advanced glycation end products 
BMI – Body mass index 
DM2 – Type 2 diabetes mellitus 
DNA – Deoxyribonucleic acid 
ER – Endoplasmatic reticulum 
FA – Fatty acids 
FFA – Free fatty acids 
FRAP – Ferric reducing ability of plasma 
GDM – Gestational diabetes Mellitus 
GLUT 1 – Glucose transporter type 1 
GPx – Glutathione peroxidase 
GSH – Glutathione 
H2O2 – Hydrogen peroxide 
IR – Insulin receptor 
MAMs – Mitochondria Endoplasmic reticulum associated membranes 
ME-UFRJ – Maternidade Escola da Universidade Federal do Rio de Janeiro 
MSM – Multiple source method 
MVM – Microvillous plasma membrane 
NADPH – Nicotinamide adenine dinucleotide phosphate oxidase 
O2˚ - Oxygen singlet 
OH˚ – Hydroxyl radical 
ORAC – Oxygen radical absorbance capacity 
PUFA – Polyunsaturated fatty acid 
ROS – Reactive oxygen species 
RNS – Reactive nitrogen species 
SFA – Saturated fatty acid 
SOD – Superoxide dismutase 
TAC – Total antioxidant capacity 
TNF-a – Alfa Tumor necrosis factor 
USDA – United States Department of Agriculture 
24H-R – 24 hour recall 
 
 
SUMMARY 
	
1. INTRODUCTION…………………………………………………………………..……17 
1.1. Physiological and metabolic adaptations in pregnancy…………………………….....…17 
1.2. Gestational Diabetes Mellitus (GDM)…………………………………………..…...….18 
1.3. The pathophysiology of GDM………………………………….……………….….……20 
1.3.1. The placenta in GDM……………………………………………………..........…......20 
1.3.2. Mitochondria and Endoplasmic reticulum (ER) stress in GDM……………………...23	
1.3.3. Redox state and oxidative stress in pregnancy and GDM…………………………....24 
1.4. Dietary antioxidants in pregnancy and in GDM……………………………………....…26 
1.4.1. Dietetic bioactive compounds in GDM………………………...……………….....…29 
2. JUSTIFICATIVE / HYPOTHESIS………….………………...………………………..31 
3. OBJECTIVES…………………………………………………………………………....32 
4. METHODS……………………………………………………………………………….33 
4.1. Study design……………………………………………………………………………..33 
4.2. Sociodemographic, Anthropometric and Medical data………………………………….34 
4.3. Dietary data…………………...…………………………………………………………34 
4.4. Biological material and neonatal outcomes data…………….…………………………..35 
4.5. Maternal total antioxidant capacity (TAC)……………………………...………………35 
4.5.1. Ferric reducing ability of plasma (FRAP)……………………………………………..35 
4.5.2. Oxygen radical absorbance capacity (ORAC)……………………………………..….36 
4.6. Statistical analyses………………………………………………………………………36 
4.7. Financial support…………………………………………………………………….…..37 
5. RESULTS………………………………………………………………………….……...38 
5.1. Sociodemographic, Anthropometric data and Neonatal outcomes…………….………..38 
5.2. Dietary intake……………………………………………………………………………39 
5.2.1. Macronutrients, Fibers and Energy intake…………………………………………….39 
5.2.2. Micronutrient intake………………………………………………..........…………….40 
5.2.3. Antioxidant micronutrient intake…………………………………………….......…….42 
5.3. Bioactive and Polyphenols intake………………………………………………………..43 
5.3.1. Carotenoids and Polyphenols intake…………………...…………….......…………….43 
5.3.2. Changes in dietary intake throughout pregnancy…………………………………...….44 
5.4. Total antioxidant capacity (TAC)…………………………….…………………………45 
5.4.1. Antioxidant capacity and Micronutrient intake……………………………………….465.4.2. Maternal antioxidant capacity and Bioactive compounds intake…………….……….50 
5.4.3. Maternal antioxidant capacity and Neonatal outcomes……………………….………56 
6. DISCUSSION…………………………………………………………………………….60 
6.1. Pregnant women characterization: low income, Obesity-GDM and Diet………………60 
6.2. Dietary intake – Macronutrients, Energy and Fiber and associations with TAC and 
Neonatal outcomes……………………………………...……………………………………60 
6.2.1. Micronutrients, Antioxidant intake and associations with TAC and Obstetric 
outcomes………………………………………………………………………………..……62 
6.2.2. Bioactive compounds intake and associations with TAC and Neonatal outcomes…...65 
7. CONCLUDING REMARKS………………………………………………………...….66 
REFERENCES……………………………………………………………………….......…69 
 
 17 
1. INTRODUCTION 
 
1.1. Physiological and metabolic adaptations in pregnancy 
 
During pregnancy, there is a wide array of physiological and metabolic adaptations 
(Figure 1) which sustain fetal development and growth and also prepare for lactation and 
the care of the newborn. These adaptations are mainly mediated by maternal and placental 
hormones and growth factors, such as insulin, prolactin, human chorionic gonadotropin, 
human placental lactogen, placental growth hormones and steroid hormones. Insulin 
plays a central role in the gestational adaptations (MOUZON; LASSANCE, 2015). In 
early gestation, the increase in insulin secretion by 60%, with no alteration in insulin 
sensitivity, stimulates lipogenesis and reduces fatty acid oxidation, promoting maternal 
fat accretion. In contrast, during late gestation, insulin sensitivity decreases by 45 – 70%, 
mobilizing maternal energy reserves, which is necessary to support fetal growth 
(FREEMARK, 2006). In combination with these hormonal adaptations, there is a rise in 
a-Tumor Necrosis Factor (TNF-a) production, free cortisol and leptin and a fall in plasma 
adiponectin that facilitates the emergence of maternal insulin resistance which constitute 
a pro-diabetogenic state. In the majority of pregnancies, this condition is counter-
regulated by an up regulation of maternal insulin production (2-fold increase in the third 
trimester) as a consequence of the expansion of pancreatic b-cells, driven by placental 
lactogens and prolactin. On the other hand, if any of these factors fail to respond, actual 
diabetogenic condition might develop, which constitutes gestational diabetes mellitus 
(NEWBERN; FREEMARK, 2011). 
 18 
 
Figure 1. Physiological pro diabetogenic state in pregnancy. Hormones like placental growth, 
progesterone, lactogen and also inflammatory cytokines and adipokines are produced by the placenta to 
mediate pregnancy adaptations. In the 1st half of pregnancy, the increase in insulin secretion plays a central 
role, stimulating lipogenesis and reducing fatty acid oxidation, promoting maternal fat accretion. 
Posteriorly, in the 2nd half of pregnancy, insulin sensitivity decreases promoting lipolysis, mobilizing 
maternal energy to support fetal growth establishing the physiological pro diabetogenic state. Adapted 
from MOUZON; LASSANCE, 2015. 
 
1.2. Gestational Diabetes Mellitus (GDM) 
 
Gestational Diabetes Mellitus (GDM) can be defined as a condition of glucose 
intolerance, insulin resistance and hyperglycemia diagnosed during pregnancy (ADA, 
2016). GDM is the most common metabolic disorder of pregnancy with increasing 
prevalence worldwide. The world incidence is estimated to affect 3 to 30 % of pregnant 
women depending on the population studied and the diagnostic criteria used (ADA, 
2011). The prevalence in Brazil is estimated to be around 18 % (NEGRATO et al., 2016). 
16.2 % of live births have some form of hyperglycaemia in pregnancy and an estimated 
85.1 % is due to GDM (OGURTWOVA et al., 2017). 
GDM is a multifactorial disease associated with both genetic (<10 % of the cases) and 
non-genetic environmental factors such as diet, physical inactivity and obesity. The latter 
is the most important environmental factor that is associated with GDM. 
Women with GDM present an increase in low grade-inflammation, which is 
characteristic of pregnancies without adverse outcomes, resulting in higher TNF-α and 
Interleukine-6 levels in serum (SANTANGELO et al., 2016). In addition, the 
hyperglycemic milieu is associated with oxidative stress (CLAPÉS; FERNÁNDEZ; 
↑ Placental lactogen
↑ Prolactin
Beta cells expansion
↑ Insulin
Placental growth hormone/ progesterone / Lactogen
↑ Inflamatory cytokines
↑ Adipokines (Leptin, Resistin)
Insulin resistance
↑ Blood glucose
↑ Lipolysis
1st half of pregnancy ⇾ increase of energy reserves
2nd half of pregnancy ⇾ availability of these reserves to the fetus
ADIPOSE TISSUE
PLACENTA
MUSCULAR TISSUE
PANCREAS
 19 
SUÁREZ, 2013; HUNT; SMITH; WOLFF, 1990). The pro-inflammatory and pro-
oxidant state of GDM induces alterations in placental structure and function resulting in 
numerous short and long term adverse pregnancy outcomes for both the mother and the 
child. In the short term, for the mother, GDM is associated with hypertensive disorders 
during pregnancy, cesarean section delivery and lower rates of breastfeeding. Poor 
glycemic control could increase the incidence of stillbirths and miscarriage. In the long 
term, there is an increased risk of developing metabolic complications like type 2 diabetes 
mellitus (DM2), cardiovascular diseases and metabolic syndrome. For infants born from 
GDM mothers, there are increased risk for macrosomia, congenital anomalies, neonatal 
hypoglycemia, hyperbilirubinemia, shoulder dystorcia, a higher percentage of body fat 
and metabolic intrauterine programming (YESSOUFOU; MOUTAIROU, 2011). 
Macrosomia, defined as birth weight above 4 kg, is the main adverse outcome on the 
offspring, being a result of an increased placental transport of glucose and other nutrients 
from the mother to the fetus. Maternal hyperlipidemia and hyperglycemia during diabetic 
pregnancy has been shown to be one of the predisposing factors for this condition. This 
state is perpetuated in macrosomic offspring and persists with age, being linked to insulin 
resistance, excessive lipogenesis and hyperinsulinemia, resulting in an increase of fat 
synthesis and body size (YESSOUFOU; MOUTAIROU, 2011). In the long term, the 
offspring also have increased risk of developing metabolic complications such as obesity 
and DM2 in later life (CASTILLO-CASTREJON; POWELL, 2017; HASTIE; LAPPAS, 
2014). The process by which a stimulus during fetal development induces long term 
impacts on fetus had been described as “fetal programming” by Hales and Barker 
(HALES; BARKER, 2001). This concept is related to the interplay between an 
individual’s genetic background and the adverse intrauterine environment including 
maternal diet, obesity and pregnancy complications that results in structural, metabolic 
and epigenetic permanent changes that impact the risk for later life chronic disease. 
Epigenetics refers to the changes in the biochemical structure of Deoxyribonucleic acid 
(DNA) which include among others, DNA methylation, histone modification and non-
coding Ribonucleic acid processes, that alter gene expression (SMITH; RYCKMAN, 
2015). 
 
 
 
 
 20 
1.3.The pathophysiology of GDM 
 
Being a multifactorial disease, the pathophysiology of GDM is still not well 
elucidated. It is known to be related to a non-maternal adaptation to the physiological and 
metabolic changes that occur during pregnancy and also to placental dysfunction 
(HUYNH et al., 2015). Alterations in the molecular regulators of β-cell mass and function 
during pregnancy could lead to its secretory impairment resulting in a defective 
adaptation. This condition may exist before pregnancy, and the same can be applied to 
placental dysfunction (ERNST et al., 2011). Some of the risk factors associated to GDM 
such as maternal age and obesity could also be implicated. Recently, Wu etal (2016) 
associated genetics with GDM as well as epigenetic modification of placental DNA, 
independently of other risk factors. More studies are needed to elucidate whether 
abnormal global DNA methylation is involved in the pathogenesis of GDM or a 
consequence of this disease (REICHETZEDER et al., 2016). Hyperglycaemia, through 
various mechanisms, plays a central role in the pathogenesis of GDM resulting in 
dysregulation of many physiological processes like blood-flow, vascular permeability and 
angiogenesis, immune and inflammatory activation, reactive oxygen species (ROS) 
production, generating many of the complications of diabetes (COUGHLAN et al., 2004; 
RADAELLI et al., 2003). 
 
1.3.1. The placenta in GDM 
 
The placenta is a transient organ that exists exclusively during pregnancy. The 
placenta acts as a natural barrier between the maternal and fetal blood circulations and 
fulfills a wide range of endocrine and transport functions, being a crucial regulator of fetal 
nutrition, gas exchange and maternal immune tolerance. Due to its location, between 
mother and fetus circulation, the placenta is a target for both maternal and fetal metabolic 
alterations associated with pregnancy pathologies (GAUSTER et al., 2012). The 
hyperglycemic environment of GDM, depending on the degree of glucose control, may 
disturb placental development and/or its function which may be associated with fetal 
complications. Placental angiogenesis and vasodilatation are crucial for placental 
function. Those processes are regulated by angiogenic associated factors including 
vascular endothelial growth factor and fibroblast growth factor-2. It has been shown that 
women with GDM present decrease concentration of these factors resulting in fetus-
 21 
placenta endothelial dysfunction (ZHOU et al., 2016). In general, the placenta of poorly 
controlled diabetic women is enlarged and plethoric and many histopathological changes 
have been described such as villous immaturity, alterations in villous branching, villous 
edema, fibrinoid necrosis. All these histological alterations seem to be associated to a 
reduction in the maternal-fetal blood diffusion which negatively affects both fetal 
oxygenation and transplacental nutrient supply (JAUNIAUX; BURTON, 2006). 
Fetal growth is directly related to nutrient availability and the placenta’s ability to 
transport these nutrients into fetal circulation. Placental nutrient transport is dependent on 
placental size, morphology, nutrient transporter availability/capacity and uterus and fetus-
placental blood flow. Alterations in the expression and activity of placental nutrient 
transporters is implicated in cases of restricted and excessive fetal growth (BRETT et al., 
2014), which is frequently observed in women with GDM. 
Placental nutrient transporters (Figure 2) are localized to the syncytiotrophoblast, the 
multinucleated epithelial barrier comprised of the microvillous plasma membrane 
(MVM) facing the maternal circulation and basal plasma membrane directed toward the 
fetal circulation (CASTILLO-CASTREJON; POWELL, 2017). 
 
 
 
 
 
 
 
 22 
 
Figure 2. Nutrient transport across the placenta. Glucose is transported across the MVM and BM 
primarily by GLUT1. The accumulative transporters, System A, mediate the uptake of small neutral amino 
acids. Amino acids are transported across the BM towards the fetal capillary by System L facilitated 
transporters (LAT2, 3 and 4) and exchangers (X). LPL and EL hydrolyze maternal (TG) into FFA that cross 
the MVM through FATPs, FAT/CD36 and FABPpm. FFAs are trafficked through the cytosol via FABPs 
and across the BM by FATPs and FAT/CD36. Abbreviations: MVM - microvillous membrane; BM - basal 
membrane; GLUT - glucose transporter; LAT - large neutral amino acid transport; TG - triglycerides; LPL 
- lipoprotein lipase; EL - endothelial lipase; FFA - fatty acid; FAT/CD36 - fatty acid translocase; FATP - 
fatty acid transport protein; FABP - fatty acid binding protein; FABPpm - plasma membrane fatty acid 
binding protein; X - exchangers (BRETT et al, 2014). 
 
Even though there is limited and controversial information concerning alterations in 
placental nutrient transport in pregnancies complicated by GDM, Catillo-Castrejon and 
Powell (2017) review, point out that there is an overall increase in glucose, amino acids 
and fatty acids (FA) uptake by the placenta, and this is related to increased delivery to the 
fetus (CASTILLO-CASTREJON; POWELL, 2017). Regarding glucose transport, 
Gaither et al (1999) pointed out to an upregulation of glucose transporter type 1 (GLUT-
1), the main glucose transporter isoform, which is highly abundant in the 
syncytiotrophoblast plasma membrane (GAITHER; QURAISHI; ILLSLEY, 1999). 
There is the hypothesis that placental glucose transporters are sensitive to regulation by 
nutrient availability mainly during early pregnancy where the number of GLUT-1 
transporters per membrane area in basal membrane vesicles increases (JANSSON et al., 
2001). The activity of amino acid transporters in placentas from diabetic women has not 
been well stablished and the available data are conflicting as well, however Jansson et al 
(2002) demonstrated that amino acid transport mediated by system A is increased in the 
syncytiotrophoblast MVM in association with diabetes during pregnancies (JANSSON et 
 23 
al, 2002). Additionally, it has been shown that leptin, a hormone that is usually increased 
in pregnancies complicated by GDM, stimulates system A amino acid uptake in primary 
villous fragments from the placenta (VERSEN-HOYNCK et al., 2009). Moreover, there 
are evidences suggesting an increased placental capacity to deliver FA to the fetus in 
GDM pregnancies. Placental transfer of lipids may be increased due to a rise of maternal 
fetal concentration gradient of free fatty acids (FFA) and triacylglycerol. Lipoprotein 
lipase activity in MVM from diabetic pregnancies was found to be increased as well as 
the expression of liver fatty acid binding protein in pre gestation diabetic and GDM 
placentas (MAGNUSSON et al., 2004). Ultimately, GDM is associated with negative 
alterations in placental development and function mainly based on changes on the micro-
anatomical and/or molecular level (GAUSTER et al., 2012). Placental damage plays an 
important role in determining the various adaptations and development changes in the 
fetus impacting on short and long term gestational outcomes (DU et al., 2016), therefore 
it is important to investigate its physiology and possible interventions to improve its 
structure and function in the context of GDM. 
 
1.3.2. Mitochondria and endoplasmatic reticulum (ER) stress in GDM 
 
The mitochondria and the ER are tubular network structures with multiple distinct 
essential functions. Mitochondria provide essential signaling for cellular homeostasis, 
acting on energy metabolism, on the control cell death and ROS production, in the 
synthesis and catabolism of metabolites (HOLLAND et al., 2017; MARCHI; 
PATERGNANI; PINTON, 2014). 
The ER synthetizes many secretory proteins, lipids, and membrane phospholipids and 
act in concert with mitochondria, to regulate and control mediators of cell death 
(BRAND; NICHOLLS, 2011). These two organelles bind tight together at specific 
contact sites termed mitochondria-ER associated membranes (MAMs), an interface with 
distinct biochemical properties which provides a platform for the regulation of different 
processes fundamental for cellular metabolism. It is a crossroad of several hormonal and 
nutrient regulated signaling pathways in metabolic tissues. Besides calcium transfer, 
regulation of mitochondrial fission, autophagy, inflammasome formation and lipid 
synthesis (RIEUSSET, 2018; MARCHI; PATERGNANI; PINTON, 2014) recent studies 
point out to MAMs role in the control of glucose homeostasis through insulin signaling 
and as aglucose sensor adapting to cellular bioenergetics (RIEUSSET, 2018). 
 24 
GDM is characterized by hyperglycemia and hypoxia leading to a state of low grade 
inflammation, elevated circulating FFA and advanced glycation end products (AGEs), all 
of which are involved in the production of pro-inflammatory cytokines which impair 
insulin signaling in peripheral tissues, particularly adipose tissue, in pregnant women 
(LAPPAS, 2014; CARE, 2004). 
Alterations in insulin action and secretion are associated with mitochondrial and ER 
stress (RIEUSSET, 2018; CHANG et al., 2015). Hyperglycemia induces an 
overproduction of superoxide (O2-˚) by mitochondria that inhibits glucose-6-phosphate 
dehydrogenase, enzyme required for providing reducing equivalents to the antioxidant 
defense system resulting in enhanced sensitivity to oxidative stress associated with 
intracellular ROS (ROLO; PALMEIRA, 2006). Mitochondria and ER are the most 
vulnerable organelles to hyperglycemia and hypoxia, being susceptible to damage by 
ROS, which may result in alterations in their structure and function. Meng et al (2015) 
observed architectural disruption of the mitochondria and dilation of ER cisternae in the 
villous tissues of the placenta from GDM women suggesting that those changes are likely 
responsible for the impairment of placental function being related to pregnancy 
complications and adverse gestational outcomes. Mitochondrial dysfunction, ER stress, 
and oxidative stress have been proposed as mechanisms to explain the link between 
inflammation and insulin resistance through the nucleotide-binding and oligomerization 
pyrin domain-containing protein 3 receptor inflammasome activation (RHEINHEIMER 
et al., 2017) which induces the pro-inflammatory cytokine Interleukine 1𝛽 secretion. A 
recent study demonstrated a relationship between ER-mitochondria miscommunication 
and hepatic insulin resistance (TUBBS et al., 2014). Since oxidative stress is a key factor 
in the pathophysiology of GDM being present in both mitochondria and ER dysfunction 
(NICOLSON, 2013; YUNG et al., 2007), the use of dietetic antioxidant might be a 
strategy for attenuate the complications associated to this condition. 
 
1.3.3. Redox state and oxidative stress in pregnancy and GDM 
 
A pregnancy without any adverse outcomes is considered a state of enhanced 
oxidative stress since ROS and reactive nitrogen species (RNS) are potent signaling 
molecules that are crucial to all pregnancy stages from implantation to labor, including 
placental development and function. In pathological pregnancies, such as in GDM, the 
pro-oxidative state is exacerbated due to insufficient antioxidant defense, which results 
 25 
in overproduction of ROS and RNS, which could lead to placental dysfunction and 
adverse effects to the mother and the fetus (ULLAH et al., 2016; SPADA et al., 2014; 
LAPPAS et al., 2011). Oxidative stress is stablished when there is an unbalance between 
pro-oxidants effects and the capacity of the body to handle with oxidative damage, which 
mainly include oxidation of cellular proteins, lipids, and DNA, leading to loss-of-function 
of these biomolecules. Antioxidants are substances that are able to oxidize (by reducing 
the reactive specie) and to remain stable at this oxidation state. In general, good 
antioxidants act in low concentrations. According to this definition, Vitamin C and 
Vitamin E are important dietary antioxidants. Vitamin C acts as a scavenger of hydroxyl 
radical (OH•) and Vitamin E acts as a chain breaking antioxidant, reducing the lipid 
peroxyl radical, blocking the propagation of fatty acid peroxidation. Additionally, there 
are anti-oxidant enzymes such as catalase, which reduces hydrogen peroxide (H2O2) to 
water, superoxide dismutase (SOD), which reduces oxygen singlet (O2•) to H2O2 and 
glutathione peroxidase (GPx), which also reduces H2O2 to water. The latter reaction 
involves the oxidation of glutathione (GSH), the most important, in quantitative terms, 
intracellular antioxidant. All antioxidant enzymes have a transition metal in its structure. 
The mitochondrial isoform of SOD is manganese-dependent and the cytosolic isoform is 
zinc and copper-dependent. GPx belongs to the family of Seleno-enzymes, highlighting 
the central importance of dietary selenium as potent antioxidant nutrient (VALKO et al., 
2007). 
As mentioned previously, in GDM, hyperglycemia induces oxidative stress through 
several pathways like the polyol and hexosamine pathway, formation of AGEs, activation 
of protein kinase C and enhanced ROS production in the mitochondria (LAPPAS et al., 
2011). Pregnancy is characterized by an altered inflammatory profile with a fine balance 
between pro- and anti-inflammatory cytokines needed for normal development however, 
inflammatory processes like GDM may alter this balance compromising normal 
development. GDM has been linked to the down-regulation of adiponectin and anti-
inflammatory cytokines like interleukin-4 and interleukin-10 and up-regulation of leptin 
and pro-inflammatory cytokines implicated in insulin resistance like IL-6 and TNF-a 
(CHALLIS et al., 2009). Oxidative stress induces inflammation and vice versa. 
Inflammatory cytokines activate phagocytic nicotinamide adenine dinucleotide 
phosphate oxidase (NADPH) increasing ROS production. TNF-a, interleukin-1, 
lipopolysaccharide stimulate cyclooxygenase-1 production of ROS (DRÖGE, 2002). 
 26 
There are suggestive evidences that in GDM, oxidative stress is exacerbated because of 
an increased production of inflammatory mediators by the placenta and the mother, which 
has been shown to cause endothelial dysfunction, which relates to alterations in 
angiogenesis, decreased proliferation and activation of apoptosis, and mitochondrial 
dysfunction in trophoblasts (GAUSTER et al., 2012; BURTON; JAUNIAUX, 2011; 
LAPPAS et al., 2011). Besides that, there is an association between the activation of the 
immune system and the development of insulin resistance caused by the cross-talk 
between inflammatory (TNF-a) and metabolic (insulin receptor (IR)/insulin receptor 
substrates) signaling cascades (SHOELSON; LEE; YUAN, 2003). Therefore, one might 
conclude that dietary antioxidants are paramount for the success of pregnancy and also 
for influencing long and short term health effects of both the mother and the baby, 
particularly in the context of an exacerbated pro-oxidant milieu. In this scenario, diet 
should be addressed as a strategy for improving pregnancy outcomes in the context of 
GDM (MISTRY; WILLIAMS, 2011). 
 
1.4. Dietary antioxidants in pregnancy and in GDM 
 
The physiological adaptations that occur during pregnancy influence the nutritional 
needs of pregnant women. Energy intake should increase to account not only for the 
increased maternal and fetal metabolism but also for fetal and placental growth 
(KOMINIAREK; RAJAN, 2017). 
The recommendations for daily micronutrient intake for pregnant women in Brazil 
are based on the Dietary Reference Intake (IOM, 2006; ANNEX 1). Generally in Brazil, 
a daily multivitamin is recommended preconception and during pregnancy. Increased 
intake of folic acid and iron is recommended to support both maternal tissue and fetal 
growth. Iron needs nearly double and folic acid, due to its role in the 1 Carbon 
metabolism, is necessary to support rapid cell proliferation and growth, nucleotide 
synthesis and placental development (TSERGA et al., 2017). 
Maternal nutrition plays an essential role on fetal development and growth and 
influences the health of the offspring at later stages of life (RAMAKRISHNAN et al., 
2012; ABU-SAAD; FRASER, 2010). Inadequate maternal diet results in placental 
insufficiency which impairs fetal development (BELKACEMI et al., 2010; BELL; 
EHRHARDT, 2002). Nutrition is the major intrauterine environmental factor that alters 
expression of the fetal genomeand have lifelong consequences (BARKER, 1997). 
 27 
Oxidative stress is an essential factor in GDM pathophysiology where elevated glucose 
levels is associated with increased production of ROS (WU; TIAN; LIN, 2015; 
JAUNIAUX; POSTON; BURTON, 2006). As mentioned, some micronutrients function 
as antioxidants or as essential cofactors for antioxidant enzymes, such as selenium, zinc, 
vitamin A, C and E (Figure 3). When the supply of dietetic antioxidant is limited, 
exaggerated oxidative stress might occur, resulting in adverse pregnancy outcomes 
(MISTRY; WILLIAMS, 2011). Therefore, dietetic micronutrient inadequacy could be 
involved in different ways in the establishment of oxidative stress and in the complication 
of pregnancies related to this pro-oxidative state (BURTON e JAUNIAUX, 2011). A few 
studies have addressed the relationship between selenium nutritional status, glucose 
metabolism and oxidative stress. Al-Saleh et al. (2007) showed that women with GDM 
presented lower plasma concentration of selenium when compared to healthy subjects. 
Similar findings were described by Hawkes et al. (2004), which observed an inverse 
correlation between serum selenium and hyperglycemia in GDM pregnancies. Since 
plasma or serum selenium is one of the biomarkers related to selenium nutritional status, 
these results indicated that poor selenium status might be involved in the alterations 
related to GDM. 
Zinc is a mineral that presents essential function as a prosthetic group, regulator 
of gene expression, participates in the immune function, to name a few. Due to its ability 
to compete with transition metals as iron and copper for binding sites on the cell 
membranes and also as a cofactor of the extracellular antioxidant enzyme SOD, it may 
prevent OH˚ and O2˚ production (BRAY; BETTGER, 1990). As described for selenium, 
Bo et al. (2005) observed that the serum concentration of zinc from pregnant women was 
negatively associated with gestational hyperglycemia, indicating a possible role of 
inadequate zinc nutritional status in the complications of GDM. 
 28 
 
Figure 3. Classification of antioxidants. Antioxidants classified into two major groups: enzymatic and 
nonenzymatic antioxidants. Abbreviations: SOD – superoxide dismutase; EGCG – Epigallocatechin 
Gallate (BUNACIU; ABOUL-ENEIN; FLESCHIN, 2012). 
 
The importance of antioxidants vitamins in ameliorating the oxidative stress 
outcomes in GDM pregnancies is still poorly elucidated and, at present, there are not 
many studies evaluating if supplementing maternal diet with dietetic antioxidants would 
be effective in decreasing adverse outcomes. Richter et al., (2012) conducted a study with 
a rat model of hypoxic pregnancy where maternal treatment with vitamin C decreased 
heat shock protein 70 and 4-hydroxynonenal, both markers of oxidative and ER stress, in 
placental tissue as well as increased birth weight. Another study using the cohen diabetic 
rat model showed a decrease in lipid peroxidation and an increased activity of SOD, 
which were related to the attenuation of both fetal and placental oxidative damage 
(ORNOY et al., 2009). Cederberg et al., (2001) in a study supplementing the diet of 
diabetic rats with vitamin C and E, concluded that this combined antioxidant treatment 
decreased fetal malformation and diminished tissue damage related to oxygen radical 
(CEDERBERG; SIMÁN; ERIKSSON, 2001). Muriel et al., (2016), suggested that 
vitamin C and E supplementation could be associated with reduced risk of preterm 
deliveries, low birth weight, stillbirth and neonatal deaths and low Apgar score 
 29 
(MURIEL; SUSHAMA; CARDOSO, 2016). The mechanisms by which vitamins C and 
E decrease the burden of pregnancy complications associated to hyperglycemic stress are 
related to their role in the regulation of intracellular redox homeostasis, as in the 
mitochondria and in the ER, in both cytosolic and membranes compartments (MANDL; 
SZARKA; BÁNHEGYI, 2009; SCHAFF, 2005). Additionally, vitamins C and E seemed 
to attenuate ER stress caused by hyperglycemia in the placental cell line BeWo by 
mechanisms other than their antioxidant role (YUNG et al., 2016). Vitamin A also have 
high antioxidant potential due to its ability to scavenge free radicals and related species, 
even though it remains considerably unexplored, compared with other antioxidants such 
as C and E (MEERZA et al., 2016; ROEHRS et al.,2009). Vitamin A supplementation 
has been reported to be successful in reducing antioxidant enzymes, catalase and 
glutathione reductase, activities in diabetic mice (MEERZA et al., 2016). Another study 
found that the level of retinol in pregnant diabetic women was significantly lower than in 
the control group that may be due to the reduced antioxidant defenses in GDM women 
(KEKMAT et al., 2014). Therefore, it is important to consider maternal dietary intake of 
food sources of micronutrients as a part of the treatment of GDM. 
 
1.4.1. Dietetic bioactive compounds in GDM 
 
Bioactive compounds are phytochemicals present as natural constituents in plant 
based food such fruit, vegetable, whole grain, cereal, legume, tea, coffee, wine and cocoa 
that provide health benefits (BIESALSKI et al., 2009). Among those compounds are the 
polyphenols, a complex class of compounds having a phenolic ring in their structure. 
They can be classified on the basis of the numbers of phenol rings they contain and the 
structural elements that bind these rings (SANTANGELO et al., 2016). The main classes 
of polyphenols are flavonoids, phenolic acids, stilbenes and lignans. Flavonoids are the 
most abundant class and include different subclasses, that is, flavonols, flavones, 
flavanones, anthocyanidins and isoflavones. Due to its chemical structure, polyphenols 
feature multiple activities, interacting with many metabolic pathways and cellular 
components. Among those activities are anti-hyperglycemic, antioxidants and anti-
inflammatory effects. Several studies are indicating the link between polyphenol intake 
and health promotion and metabolic diseases prevention (BAHADORAN, ZAHRA; 
MIRMIRAN, PARVIN; AZIZI, 2013; RAHMAN; BISWAS; KIRKHAM, 2006). 
Regular intake of bioactive compounds through diet seems to be beneficial, however 
 30 
further investigations are needed to confirm their effects in pregnancy (BAHADORAN, 
ZAHRA; MIRMIRAN, PARVIN; AZIZI, 2013). 
Although not specifically concerning GDM and mostly derived from in vitro or 
animal, a few studies support that dietary polyphenols have beneficial actions in 
complications related to this disease like insulin signaling, hyperglycemia and insulin 
resistance as well as oxidative stress and inflammation (SIN OH; JUN, 2014; 
HANHINEVA et al., 2010). 
A study with human placenta explants showed that punicalagin, the major polyphenol 
in the pomegranate pulp, facilitated syncytiotrophoblast differentiation and increased 
cytotrophoblast proliferation (CHEN et al., 2016). In another study from the same group 
punicalagin reduced oxidative stress in vivo and in vitro and attenuated apoptotic cell 
death in villous explants and human trophoblast cell line (CHEN et al., 2012). 
Another class of bioactive compounds are the carotenoids, richly colored molecules 
present in mainly fruits and vegetables in the human diet. α-Carotene, β-carotene, β-
cryptoxanthin, lutein, zeaxanthin, and lycopene are the most common dietary carotenoids 
(YOUNG; LOWE, 2001). Studies associating dietary carotenoids with diabetes are scarce 
and inconsistent but there are a few studies suggesting that they might be beneficial in 
diabetes by reducing oxidative stress (WESOŁOWSKA et al., 2017; DI TOMO et 
al.,2012; YOUNG; LOWE, 2001). Carotenoids are involved in the scavenging of ROS 
like O2˚ and peroxyl radicals as well as in the deactivation of molecules involved in the 
generation of O2˚ (YOUNG; LOWE, 2001). Di Tomo etal., (2012) observed, in human 
umbilical vein endothelia cells exposed to physiological concentrations of both β-
carotene and lycopene a significant reduction of inflammatory response by down-
regulation of TNF-α expression, due to their reducing activity which caused a decrease in 
ROS and nitrotyrosine generation and the maintenance of nitric oxide bioavailability (DI 
TOMO et al., 2012). Despite the existence of a few studies, the molecular mechanisms 
and targets of how these bio-compounds act are still unknown. Then, it’s of crucial 
importance to better understand the mechanisms governing the dietary impact on the 
metabolic system in GDM (SANTANGELO et al., 2016). 
Accordingly, ensuring adequate supply of micronutrients and bioactive compounds 
for pregnant women, might be a good strategy for reducing pregnancy complications. We 
hypothesize that it could be a promising therapeutic intervention for diabetes-associated 
adverse pregnancy outcomes. Nutritional counseling should be considered the first line 
of treatment of GDM and understanding the exact connections between maternal intake 
 31 
of dietary antioxidants and the gestational outcomes in this context is paramount for 
translational nutrition and to personalized dietary recommendations. 
 
2 – JUSTIFICATIVE / HYPOTHESIS 
 
Gestational Diabetes Mellitus is the most common metabolic disorder which 
occur during pregnancy and its prevalence is increasing worldwide (CHEN et al., 2014). 
This disease is associated with pro-inflammatory and pro-oxidants responses that 
compromise placental function leading to short and long term maternal and fetal adverse 
outcomes (MYATT; MALOYAN, 2016). 
It is a consensus that dietetic nutrients and bioactive compounds with antioxidants 
and anti-inflammatory properties have an important role in the prevention and treatment 
of these diseases. However, the exact roles that these dietary components play in maternal 
redox state, on placental function and consequently in pregnancy outcomes in GDM 
remains to be determined. The first-line treatment of GDM worldwide have been insulin 
(ACOG, 2017; ADA, 2017) which, controversially has been related to adverse outcomes 
(BROWN et al., 2017a). A dietary treatment results in satisfactory levels of blood glucose 
in 90% of women with GDM (BROWN et al, 2017b). Improving knowledge in the 
therapeutic potential of nutrition beyond glycemic control, is of great value. 
The reduction of maternal and child mortality and nutrition improvement are 
among the greatest world healthy challenges (WHO, 2016). Therefore, it is extremely 
important to investigate the relation between antioxidants and other dietetic components 
present in brazilian pregnant women diet and the pro oxidant and pro inflammatory 
maternal and placental conditions in GDM context. 
Our hypothesis is that the improvement of maternal redox homeostasis through 
the consumption of dietetic antioxidants as ascorbic acid, tocopherol, selenium, zinc, 
carotenoids and polyphenols could attenuate placental dysfunction and consequently 
adverse gestational outcomes in the context of GDM. 
 
 
 
 
 
 
 32 
3. OBJECTIVES 
 
The aim of the present study is to investigate the association between dietary 
antioxidants and maternal redox state in the context of GDM. In order to achieve our goal, 
the following aspects will be investigated throughout pregnancy: 
 
ü Maternal intake of the antioxidant micronutrients vitamins C and E, selenium, zinc; 
ü Maternal intake of carotenoids and polyphenols; 
ü Maternal Plasma Total Antioxidant Capacity; 
ü Correlations between maternal total antioxidant capacity and dietary antioxidants; 
ü Correlations between gestational outcomes with dietary intake of antioxidants and 
maternal redox homeostasis. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 33 
4. METHODS 
 
4.1.Study design 
 
This is an ongoing prospective cohort study of pregnant women carried out since July 
2017 at the Maternidade Escola from the Universidade Federal do Rio de Janeiro (ME-
UFRJ). This research project was approved by the Research Ethics Committee (CEP) 
from the ME-UFRJ and registered at the Research Ethics National Council (CONEP), 
under the nº. 66949217.0.0000.5275 (Certificate of presentation for ethic appreciation-
CAAE) (ANNEX 2). 
Data are collected in 3 different times throughout pregnancy: 24th-28th, 32nd-36th 
gestational weeks and at delivery as showed in the study design flow chart (ANNEX 3). 
Considering pregnancy as a very dynamic metabolic period, the outset or first recognition 
of GDM around 2nd or 3rd trimester and the logistic of prenatal at ME-UFRJ, it was 
stablished the collection of maternal blood around 24th-28th and 32nd-36th gestational 
weeks, in the 2nd and 3rd trimesters, respectively. Retrospective data relative to the first 
trimester of gestation is being obtained in their medical records. 
Eligibility criteria for enrolment in the study were: (a) Signed and informed consent 
(ANNEX 4); (b) age between 18 and 45 years old; (c) up to 24th gestational week; (d) free 
of chronic and non-transmissible diagnosed diseases prior to pregnancy (e.g., 
hypertension, DM2); (e) free of infectious diseases; (f) singleton pregnancy and (g) 
intention to delivery at the ME-UFRJ. Exclusion criteria were (a) usual smokers and (b) 
pre-pregnancy body mass index (BMI) < 18,5 Kg/m2. 
Participants follow up was made mainly through whatsapp messages, telephonic 
contacts and personal contact eventually during the routine prenatal visits at ME-UFRJ. 
Up until now, a total of 35 pregnant women enrolled in the study, where 23 (66 %) had 
their 2nd and 3rd trimester blood samples. Therefore, in the present study, all results that 
will be presented are related to the data from this universe of 23 women since we had 
segment losses as described next: abandoned without notifying (3); not having 3rd blood 
collected by the time of analyzes (9); abortion, prematurity (3); delivery in another 
maternity (1); no delivery by the time of data analyzes (5); From those 23 women, 9 (47 
%) had already delivered and their obstetric outcomes were included in the data analysis. 
The diagnosis of GDM was done according to the criteria proposed by the American 
Diabetes Association (ADA, 2016): fasting blood glucose ≧92 mg/dL; blood glucose 
 34 
after 1h of oral glucose load ≧180 mg/dL; or blood glucose after 2h of oral glucose load 
≧153 mg/dL. From these 23 women, 15 (65 %) had uncomplicated pregnancies (non-
GDM group) and 8 (25 %) were diagnosed with GDM. The majority of GDM diagnostics 
were done between 24th and 28th gestational week. 
 
4.2. Sociodemographic, anthropometric and medical data 
 
The sociodemographic, anthropometric and medical data was collected using a 
baseline questionnaire (ANNEX 5) and applied by a trained researcher in the recruitment 
day and also by consultation of the participant’s medical records. Anthropometric data 
included: pre-gestational and actual weight, height, pre-gestational BMI. Medical data 
included actual and previous reproductive and gestational history, family medical history. 
Information regarding sleep habits, intestinal function, use of medication was also 
collected. 
 
4.3.Dietary data 
 
Dietary records were collected with two 24 hour recall (24h-R) (ANNEX 6) in the 
24th-28th and two in the 32nd-36th gestational weeks. In both segments, the first 24h-R was 
obtained in person and the second by telephone. This strategy was already validated and 
it is a useful approach to access dietary intake of Brazilian pregnant women (BARBIERI 
et al., 2015). 
Dietetic data was processed using the software DietBox, which uses the tabela 
brasileira de composição de alimentos (UNICAMP/NEPA, 2011), Instituto brasileiro de 
geografia e estatística (BRASIL/IBGE, 2011), United States department of agriculture(EUA/USDA, 2017) and Tucunduva (PHILIPPI, 2012) databases to retrieve the total 
intake of nutrients. Is noteworthy that our study pioneered the dietary analysis of the 
consumption of carotenoids and polyphenols in the context of GDM in Brazil. For the 
determination of carotenoids, the following tools were used: Tabela brasileira de 
composição de Carotenóides em Alimentos –Ministério do Meio ambiente 
(RODRIGUES-AMAYA et al., 2008) and the United States Department of Agriculture 
database (EUA/USDA, 2017) for α and β Carotene, β Cryptoxanthin, lutein, zeaxanthin 
and lycopene (BHAGWAT et al., 2016); And for polyphenols intake it was used the 
 35 
USDA flavonoid database (BHAGWAT et al., 2015); the USDA Isoflavone database 
(BHAGWAT et al., 2008); the USDA Proanthocyanidin database (BHAGWAT et al., 
2004) and the Phenol explorer 3.6 for phenolic acids and Lignans (ROTHWELL et al., 
2013). Dietary intake was then analyzed by the Multiple Source Method (MSM) validated 
by (BARBIERI et al., 2015). The MSM is a statistical method to estimate regular food 
consumption that uses at least two different inputs (like two 24h-R or 24h-R plus a FFQ), 
which identifies the sporadic or usual nutrient intake using one of the inputs as a co-
variable (HARTTIG et al., 2011). 
 
4.4.Biological material and neonatal outcomes data 
 
Fasting blood samples were collected by trained professionals in the 24th-28th and 
32nd-36th gestational weeks, corresponding to the 2nd and 3rd trimester respectively. 
Approximately 8 mL of blood was collected in 2 vacutainer tubes EDTA 
(Ethylenediamine tetraacetic acid). Almost immediately, samples were then centrifuged 
for 1,500 rpm for 15 minutes and 0,5 mL plasma were aliquoted in cryotubes and 
immediately frozen in liquid nitrogen and transported to the Universidade Federal do Rio 
de Janeiro (UFRJ) where they were stored at -80ºC until analyses. 
The obstetric outcomes data were obtained by consultation of participant’s medical 
records after delivery and included Apgar score, birth weight, length and cephalic 
perimeter at birth. 
 
4.5. Maternal total antioxidant capacity 
 
The maternal total antioxidant capacity (TAC), was evaluated in the plasma using 
two different methods: Ferric reducing ability of plasma (FRAP) and Oxygen radical 
absorbance capacity (ORAC). 
 
4.5.1 Ferric reducing ability of plasma (FRAP): 
 
The antioxidant capacity by the FRAP method was determined according to Benzie 
and Strain, 1999 (BENZIE; STRAIN, 1999). This method is based on the capacity of the 
sample to promote the reduction of the complex Fe (III)-TPTZ (orange) to the complex 
Fe (II)-TPTZ (dark blue) in acid medium, which is then quantified at 595 nm in a 
 36 
spectrophotometer. 1.8 mL of FRAP solution which is composed by ferric chloride (III), 
acetate buffer (pH3.6) and 2,3,4-tris (2-pyridyl)-S triazine (TTPZ) was warmed to 37º C 
and a reagent blank reading was taken at 595 nm; 0.1 mL of freeze dried plasma sample 
resuspended in Phosphate Buffer Saline (PBS) was added, in triplicate, along 0.1 mL of 
destilled water. Absorbance was read regularly during the monitoring period of 8 minutes 
in a spectrophotometer model 340 Sequoia-Turner TM. The change in absorbance 
between the final reading selected and the blank reading was calculated for each sample 
and related to an absorbance of a ferrous sulphate standard (solution tested in parallel) 
curve (500; 1,000; 1,500 and 2,000 µM). The results were expressed in µM of ferrous 
sulphate per L of sample. 
 
4.5.2. Oxygen radical absorbance capacity (ORAC) 
 
The ORAC method (PRIOR et al., 2003) evaluates the antioxidant activity of a sample 
through its ability to inhibit of the oxidation of a fluorescence probe induced by the 
peroxil radical. 0.1 mL of freeze dried plasma samples were diluted in PBS 3,000, 2,000, 
1,000, 700, 400, 100-fold. 0.01 mL of diluted samples were then added, in duplicate, to a 
microplate following the addition of 0.120 mL fluorescein (used as the fluorescent probe) 
and 0.060 mL AAPH (2,2’-azobis (2-amidinopropane) dihydrochloride (the oxidizing 
agent). The microplate containing the samples and the buffer phosphate was incubated 
for 3 hours at 37º C. Fluorescence was measured at 485 nm excitation and 535 nm 
emission. The area under the curve (AUC) was calculated from time zero until the end 
the of the reading, with a 30 s-interval between measurements. The prevention of 
fluorescein oxidation, measured by the decay of fluorescence, indicates the antioxidant 
capacity of the sample. A calibration curve with Trolox (6-hydroxy-2,3,7,8-
tetramethylchroman-2-carboxylic acid), x to y concentration was done and the results 
were expressed in µmoles Trolox equivalent /g sample. 
 
4.6. Statistical analyses 
 
To compare the intake of energy, nutrient and polyphenols between the non-GDM 
and GDM and to evaluate the differences within gestational trimesters, two-way analysis 
of variance (ANOVA) was used, with a Sidak post-test for multiple comparisons. 
Similarly, differences in TAC were evaluated by ANOVA, to identify the differences 
 37 
between groups and the TAC throughout pregnancy. The associations between nutrients 
intake, obstetric outcomes and maternal redox homeostasis were evaluated by Pearson 
correlation. Differences were considered statistically significant when p value < 0.05. 
GraphPad Prism 7.0 software (GraphPad Software, Inc) was used in all statistical 
analyses. Values were represented by mean and standard deviation. 
 
4.7. Financial support 
 
This research project is sponsored by FAPERJ (Fundação Carlos Chagas Filho de 
Amparo à Pesquisa do Estado do Rio de Janeiro), CNPq (Conselho Nacional de 
Desenvolvimento Tecnológico e Científico) and Academy of Medical Sciences, UK. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 38 
5. RESULTS 
 
5.1. Sociodemographic, anthropometric data and neonatal outcomes 
 
A total of 35 pregnant women were recruited to the study. 12 of them did not complete 
all 3 phases for different reasons, such as missing blood samples, not answering messages 
or phone calls or have delivered in another maternity. 23 pregnant women completed the 
study, where 15 (65 %) were healthy and did not develop GDM (non-GDM) and 8 (35 
%) developed GDM. Since this is an ongoing cohort, by the time of the completion of 
this study, there were 14 deliveries, 9 from the non-GDM and 5 from the GDM group. 
The characteristics of the mothers and the newborns according to study group and 
gestational trimester are shown in Table 1. It can be seen that there were no significant 
differences between groups in any of the parameters. An important observation is the fact 
that the majority of women from both groups were overweight or obese and in the GDM 
group 75 % had BMI above 25 kg/m2. The prevalence of pre-conception smoking and 
alcohol consumption were very similar between groups as well as average income and 
formal education, with a higher prevalence of women who attended the University in the 
GDM group, although not statistically different. Regarding the neonatal outcomes, as 
observed with the sociodemographic and anthropometric data, there were no significant 
differences between groups, and all outcomes were within the range of normality 
according to NCHS/WHO, (1996) (DE ONIS; YIP, 1996; Table 1). 
 
 
 
 
 
 
 
 
 
 
 
 
 39 
Table 1. Characteristics of pregnant women and newborns according to study group and 
gestational trimester from the cohort study at the Maternidade Escola/Universidade Federal 
do Rio de Janeiro1 
Pregnant women Non GDMa (n=15) GDMb (n=8) 
Maternal age (years) 28.4 ±5.4 33 ± 6.9 
Pre gestational BMI (Kg/m2)c 
 18-25 (eutrophic) 
 ≥ 25 (overweight) 
 ≥ 30 (obesity) 
27.9 ± 6.3 
40 % 
27 % 
33 % 
29 ± 4.2 
25 % 
37.5 % 
37.5 % 
Pre-conception smoking 33 % 37 % 
Pre-conceptionalcohol consumption 73 % 62 % 
Formal Education 
 Elementary school 
 High school 
 University 
 
100 % 
73 % 
13 % 
 
100 % 
62 % 
37 % 
Income (R$) 3455,9 ± 2704 3904.6 ± 1803.7 
Gestational outcomes Non GDM (n=9) GDM (n=5) 
Gestational age at birth (Weeks) 39.8 ± 0.5 37 ± 0.5 
Weight (g) 3,274 ± 435 3,037 ± 314 
Length (cm) 47.6 ± 1.3 47.6 ± 1 
Cephalic perimeter (cm) 
Apgar score – first minute 
Apgar score – fifth minute 
34 ± 1.7 
8.7 ± 0.7 
9 ± 0.5 
33 ± 1.4 
9 ± 0.5 
9 ± 0.5 
1Maternal age, pre-gestational and gestational outcomes are represented as average ± SD. aNon-GDM: 
women without Gestational Diabetes Mellitus; bGDM: women diagnosed with Gestational Diabetes 
Mellitus according to American Diabetes Association (2016) at 24 weeks of pregnancy; cWHO 
classification (2000). 
 
5.2. Dietary intake 
 
5.2.1. Macronutrients, fibers and energy intake 
 
Dietary intake was evaluated in the 2nd (24 - 28 gestational week) and in the 3rd (34 
- 35 gestational week) trimesters. The average intake of carbohydrate, protein and lipid 
was within the reference values (IOM, 2006) in both groups and trimesters (Table 2). On 
the other hand, 40 % and 25 % of women in the non-GDM and GDM groups respectively, 
had insufficient polyunsaturated fatty acid (PUFA) intake in the 2nd trimester. 
Additionally, monounsatured fatty acid (MUFA) intake was inadequate in the 2nd 
trimester in 60 % and 50 % in the non-GDM and GDM groups respectively, and around 
100% in the 3rd trimester in both groups. Saturated fatty acid (SFA) intake, was above the 
recommended value and fiber intake was below recommendation (IOM, 2006; Table 2). 
Added sugar intake was around 4% of total energy in both groups and trimesters being 
within the recommendation. The daily intake of added sugar was not set but is 
recommended to be no more than 10% of total energy intake (IOM, 2006). When 
comparing the intake between trimesters within each group, there was a significant 
 40 
decrease in energy, carbohydrate and sugar in the 3rd trimester when comparing to the 2nd 
trimester only in the non-GDM group with exception of the energy decrease that was 
observed in both groups. Lastly, the intake of energy, macronutrients and fiber was 
similar between groups in both trimesters. 
 
Table 2. Energy, macronutrients and fibers intake (average ± SD)1 from pregnant women in the non-
GDM and GDM groups at the 2nd and 3rd trimester which participated in the cohort study at the 
Maternidade Escola/Universidade Federal do Rio de Janeiro. 
1Estimated values obtained by 24 h Dietary recalls, analyzed by the Multiple Source Method; 2DRI: Dietary 
Reference Intakes for pregnant women (IOM, 2006): macronutrient intake expressed as Acceptable Macronutrient 
Distribution Range (AMDR) and fiber intake expressed as Recommended Dietary Allowance (RDA); 3Inadequacy 
based on Estimated Average Requirement (EAR) (IOM, 2006) *statistically different from 2nd trimester, same 
group: ap=0.0105; bp=0.0016; cp= 0.0063; dp=0.0479; ep=0.0497; two-way ANOVA, repeated measures; (-) = no 
reference value stablished; n/a: not applicable; SFA: Saturated fatty acid; MUFA: Monounsaturated fatty acid; 
PUFA: Polyunsaturated fatty acid. 
 
5.2.2. Micronutrient intake 
 
Regarding minerals, the intake of manganese, phosphorus and sodium was 
adequate for the majority of the women. Manganese intake was insufficient in 1/3 of 
women from the non-GDM in the 2nd trimester (IOM, 2006; Table 3). Calcium, iron, 
 
 
Dietary Intake DRI2 
(Quantity/day) 
Inadequacy 
(Prevalence %)3 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Gestational Trimester Gestational Trimester 
2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 
Energy 
(kcal) 
 
2,282.8 
± 63.5 
 
2,138.7*, a 
± 183 
2,285.9 
± 87.3 
2,039.8*, b 
± 166.7 - n/a n/a n/a n/a 
Carbohydrate 
(% energy) 
 
56.4 ± 8.0 54*, c ± 3.9 53.6 ± 7.7 53.4 ± 1.1 45 – 65 % 7 0 13 0 
Protein 
(% energy) 
 
15.9 ± 3.3 16.9 *, d ± 2.8 16.8 ± 3.7 17 ± 1.9 10 – 35 % 0 0 0 0 
Lipids 
(% energy) 
 
29.7 ± 3.6 29.6 ± 2.3 30 ± 3.9 30.5 ± 1.2 20 – 35 % 0 0 0 0 
SFA 
(% energy) 
 
10.9 ± 0.2 
 
10 ± 2.0 
 
11 ± 0.2 
 
10.6 ± 1.4 
 
≦10 % 0 0 0 0 
MUFA 
(% energy) 
 
9.3 ± 0.9 
 
9.0 ± 0.2 
 
 
9.5 ± 1.2 
 
 
9 ± 0.2 
 
10-15 % 60 93 50 100 
PUFA 
(% energy) 
 
5.4 ± 1.8 
 
5.8 ± 0.8 
 
 
5.2 ± 0.9 
 
 
5.2 ± 0.6 
 
5 – 10 % 40 0 25 0 
Fiber 
(g) 22.8 ± 3.2 23.7 ± 4.6 24.3 ± 3.2 25.3 ± 5.7 28 g
 93 80 87 75 
Sugar 
(% energy) 4.1 ± 1.08 3.7
*, e ± 0.09 3.7 ± 0.9 3.9 ± 0.08 ≦10 % 0 0 0 0 
 41 
magnesium and potassium intake, on the other hand, was below the reference value in the 
majority of the volunteers. The prevalence of inadequacy for calcium was above 60 % in 
the non-GDM group in both trimesters and in the GDM group above 50 %. Despite the 
fact that the intake of dietary iron was insufficient for almost 100 % of the women in both 
groups, all volunteer took iron supplements systematically. Over 80 % of the women in 
both groups presented insufficient intake of magnesium (Table 3). 
 
 Table 3. Mineral intake (average ± SD)1 from pregnant women in the non-GDM and GDM groups 
at 2nd and 3rd trimester which participated in the cohort study at the Maternidade 
Escola/Universidade Federal do Rio de Janeiro. 
 1Estimated values obtained by 24h Dietary recalls, analyzed by the Multiple Source Method; 2DRI: Dietary 
Reference Intakes for pregnant women (IOM, 2006): micronutrient intake expressed as Recommended 
Dietary Allowance (RDA); *Adequate Intakes (AI); 3Inadequacy based on Estimated Average Requirement 
(EAR) (IOM, 2006). 
 
Vitamins A, B1, B2, B3, B12 intake was adequate in both groups and trimesters, while 
the intake of vitamins D, B6 and Folate was below the reference values (IOM, 2006) 
(Table 4). Vitamin D had a prevalence of inadequacy of 100 % in both groups and 
trimesters. The same was observed for folate with exception of the third trimester in the 
non GDM group that had 93% of prevalence of inadequacy. Despite the fact that the 
intake of dietary folate was insufficient for almost 100 % of the women in both groups, 
all volunteers took folate supplements systematically. 
 
Minerals 
Dietary Intake DRI2 
(quantity/day) 
Inadequacy 
(Prevalence %)3 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Gestational Trimester Gestational Trimester 
 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 
Calcium 
(mg) 
 
687 
± 270.3 
703 
±320.8 
805.2 
± 252.5 
746.2 
± 239.4 1,000 mg
 80 67 50 62 
Iron 
(mg) 
 
11.7 ± 2.0 14 ± 5.0 
12.4 ± 
1.6 12.5 ± 3.1 27 mg 100 93 100 100 
Magnesium 
(mg) 
 
225.9 
± 43 
227 
± 49.8 
256 
± 52.7 
242.9 
±60.7 350 – 360 mg 93 87 80 80 
Manganese 
(mg) 
 
2.9 ± 2 6.3 ± 9 3.3 ± 0.9 3.6 ± 0.9 2 mg * 33 7 0 0 
Phosphorus 
(mg) 
 
1,056 
± 251.3 
1,134.4 
± 327.3 
1,227.9 
± 325.6 
1,136.2 
± 296 700 mg 0 0 0 0 
Potassium 
(g) 
 
2.4 
± 621 
2.1 
±501.5 
2.7 
± 475.3 
2.4 
± 635 4.7 g * 100 100 100 100 
Sodium 
(g) 
 
2.1 
± 57.4 
2.2 
± 994.2 
2.1 
± 55.5 
2 
± 713 1.5 g * 0 0 0 0 
 42 
 Table 4. Vitamins intake (average ± SD)1 from pregnant women in the non-GDM and GDM groups at 2nd 
and 3rd trimester which participated in the cohort study at the Maternidade Escola/Universidade Federal 
do Rio de Janeiro. 
 1Estimated values obtained by 24h Dietary recalls, analyzed by the Multiple Source Method; 2DRI: Dietary 
Reference Intakes for pregnant women (IOM, 2006); 2DRI: Dietary Reference Intakes for pregnant women 19 – 30 
and 31 – 50 years (IOM, 2006): micronutrient intake expressed as Recommended Dietary Allowance (RDA); 
*Adequate Intakes (AI); 3Inadequacy based on Estimated Average Requirement (EAR) (IOM, 2006). 
 
5.2.3 Antioxidant micronutrient intake 
 
 Concerning the intake of antioxidants, the following were evaluated: vitamins C 
and E, selenium and zinc. The prevalence of inadequacy of vitamin E wasthe highest 
among the dietary antioxidants evaluated, around 90 % in both groups. Zinc intake had 
an inadequacy of nearly 50%. Concerning vitamin C, the prevalence of inadequacy was 
slightly higher in the 2nd trimester in the GDM group, around 40 % vs 20 % in the non-
GDM and similar in both groups in the 3rd trimester, around 50 %. Selenium was the only 
dietary antioxidant that showed 100 % of adequacy in the diet (IOM, 2006). When 
comparing antioxidants intake between non-GDM and GDM women, there was no 
significant differences in both trimesters (Table 5). 
 
 
Vitamins 
Dietary Intake DRI2 
(quantity/day) 
Inadequacy 
(Prevalence %)3 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Gestational Trimester Gestational Trimester 
 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 
Vitamin A 
(µg) 
 
984.5 
± 255 
1,011.2 
± 69.4 
1,181.5 
± 379.4 
979.8 
± 43.7 770 µg 0 0 0 0 
Vitamin D 
(µg) 
 
2.8 ± 1.2 2.9 ± 1 3.7 ± 1.5 3.2 ± 1.3 15 µg 100 100 100 100 
Vitamin B1 
(mg) 
 
1.4 ± 0.1 1.4 ± 0.4 1.4 ± 0.1 1.3 ± 0.4 1.4 mg 13 47 0 50 
Vitamin B2 
(mg) 
 
1.7 ± 0.3 1.9 ± 0.8 1.8 ± 0.4 1.7 ± 0.6 1.4 mg 7 13 0 13 
Vitamin B3 
(mg) 
 
19.7 ± 2.2 20.9 ± 6.9 19 ± 1.5 18 ± 3.5 18 mg 0 20 0 13 
Vitamin B6 
(mg) 
 
1.7 ± 0.2 1.7 ± 0.6 1.6 ± 0.1 1.6 ± 0.3 1.9 mg 13 33 25 38 
Vitamin B9 
(µg) 
 
158 ± 6 229.5 ± 199.6 160 ± 6.8 225.5 ± 121.6 600 µg 100 93 100 100 
Vitamin B12 
(µg) 4 ± 2.4 4.7 ± 3.5 5.7 ± 4.0 3.6 ± 1.3 2.6 µg 27 20 0 13 
 43 
Table 5. Antioxidant micronutrients intake (average ± SD)1 of pregnant women in the non-GDM and 
GDM groups at 2nd and 3rd trimester which participated in the cohort study at the Maternidade 
Escola/Universidade Federal do Rio de Janeiro. 
1Estimated values obtained by 24h Dietary recalls, analyzed by the Multiple Source Method; 2DRI: Dietary 
Reference Intakes for pregnant women (IOM, 2006): micronutrient intake expressed as Recommended 
Dietary Allowance (RDA); 3Inadequacy based on Estimated Average Requirement (EAR) (IOM, 2006). 
 
5.3 Bioactive compounds intake 
 
5.3.1. Carotenoids and polyphenols intake 
 
The intake of polyphenols and carotenoids was also analyzed to increment the 
discussion on the association of dietary antioxidants and gestational outcomes in the 
context of GDM. The intake of total polyphenols and total flavonoids was similar between 
groups in both trimesters (Table 6). Additionally, the intake of isoflavones was 
significantly higher in the 3rd trimester compared to the 2nd trimester in both groups (Table 
6). The same was observed with total carotenoids and b-carotene although only in the 
non-GDM group. On the other hand, lignans intake in the 3rd trimester was 1/3 of that 
observed in the 2nd trimester in the non-GDM group (Table 6). 
 
 
 
 
 
 
 
Antioxidant 
Micronutrients 
 
Dietary Intake 
DRI2 
(quantity/day) 
Inadequacy 
(Prevalence %)3 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Non GDM 
(n=15) 
GDM 
(n=8) 
 Gestational Trimester Gestational Trimester 
 2 3 2 3 2 3 2 3 
Vitamin C 
(mg) 
 
123 
± 67 
174.8 
± 325.6 
122.9 ± 
79.6 
186 
± 94.7 85 mg
 20 47 38 50 
Vitamin E 
(mg) 
 
9.2 
± 3 
9.7 
± 3.8 
8.5 
± 2.1 
7.2 
± 2.2 15 mg
 87 80 87 100 
Selenium 
(µg) 
 
79.3 
± 2.3 
84.9 
± 10.5 
80 
± 2.2 
82 
± 14.5 60 µg
 0 0 0 0 
Zinc 
(mg) 
 
10 
± 4.3 
10.9 
± 3.8 
10.5 
± 2.8 
10.2 
± 2.5 11 mg
 47 40 38 50 
 44 
Table 6. Carotenoids and Polyphenols intake (average ± SD)1 of pregnant 
women in the non-GDM and GDM groups at 2nd and 3rd trimester which 
participated in the cohort study at the Maternidade Escola/Universidade 
Federal do Rio de Janeiro. 
1Estimated values obtained by 24h Dietary recalls, analyzed by the Multiple 
Source Method; *statistically different from the 2nd trimester, same group; 
#statistically different when compared to non-GDM, two-way ANOVA, 
repeated measures. ap<0.0001; bp=0.0059; cp=0.0543; dp<0.0001; ep<0.0001. 
 
5.3.2. Changes in dietary intake throughout pregnancy 
 
 We next compared the intake ratio (3rd trimester:2nd trimester) of all nutrients and 
dietary compounds described above between groups in an attempted to identify changes 
in dietary habits that might be related to the neonatal outcomes. When analyzing the 
intake ratio of energy, macro and micronutrients, we observed no significant differences 
between groups (data not shown). However, concerning the bioactive compounds, GDM 
women presented a flavonols intake ratio 175 % higher when compared to non-GDM 
women (Figure 4). This result reflects the fact that flavonols intake increased, although 
not significantly, in the 3rd trimester compared to the 2nd trimester in women with GDM. 
 
 
Food component 
Dietary Intake 
Non GDM 
(n=15) 
GDM 
(n=8) 
 Gestational Trimester Gestational Trimester 
 2nd 3rd 2nd 3rd 
Total carotenoids 
(mg) 
 
5.7 ± 4 18.4*, a ± 2 11.4 ± 10.4 19.6 ± 2.6 
 b-carotene 
(mg) 
 
2.2 ± 1.4 6.0*, b ± 4.9 3.9 ± 4 5.2 ± 3.2 
Total polyphenols 
(mg) 
 
477 ± 182.4 428 ± 307.4 470.8 ± 159.4 
 
435.4 ± 266.6 
 
Phenolic acids 
(mg) 
 
355.2 ± 211.2 324 ± 310.7 343.6 ± 177.2 361.9 ± 177.8 
Lignans 
(mg) 
 
28.8 ± 22.3 10.3*, c± 18.4 29.7 ± 25 16.4 ± 15.4 
Total flavonoids 
(mg) 
 
123 ± 6.4 106 ± 76.5 126.3 ± 4.9 142.7 ± 111.4 
Anthocyanidins 
(mg) 
 
58.7 ± 22.6 73.8 ± 56 73.8 ± 23.4 57 ± 39.5 
Flavonols 
(mg) 
 
3 ± 0.8 2.3 ± 3.4 3.34 ± 0.2 5 ± 3.5 
Isoflavones 
(mg) 
 
 
0.13 ± 0.05 
 
 
0.27*, d ± 0.06 
 
 
0.12 ± 0.03 
 
0.29*, e ± 0.1 
Proanthocyanidin 
(mg) 31.4
 ± 2.7 30 ± 16 31.5 ± 2 35.4 ± 30.3 
 45 
 
Figure 4. Flavonols intake ratio from non GDM and GDM pregnant women from the cohort study 
at the Maternidade Escola/Universidade Federal do Rio de Janeiro. n=15 Non GDM group and n=8 
GDM group. Paired t test. *statistically significant; Non GDM: women without Gestational Diabetes 
Mellitus; GDM: women diagnosed with Gestational Diabetes Mellitus according to ADA (2016) 
classification. ◼ Non GDM group; ▲GDM group 
 
5.4. Total antioxidant capacity (TAC) 
 
Considering our hypothesis, that there might have an association between dietetic 
antioxidants intake and the redox homeostasis in this population, we evaluated maternal 
plasma total antioxidant capacity (TAC) between groups and trimesters to look for 
associations. When comparing the TAC between trimesters in the groups, we observed a 
higher AC in the 3rd trimester when compared to the 2nd considering the whole group of 
volunteers (Figure 5A) and when comparing non-GDM and GDM groups 2nd vs 3rd 
trimesters using two-way ANOVA (Figure 5B). There were no differences between 2nd 
and 3rd trimesters TAC within each group. Additionally, the change in antioxidant 
capacity throughout pregnancy was similar in non-GDM and GDM women by evaluating 
the ratio 3rd:2nd trimester (Figure 5C). 
 
 
 
 
 
 
 
 
0
2
4
6
Fl
av
on
ol
s
In
ta
ke
Ra
tio
(3
T:
2T
)
Non-GDM GDM
*
 46 
 
Figure 5. Plasma antioxidant capacity of pregnant women from the cohort study at the Maternidade 
Escola/Universidade Federal do Rio de Janeiro. 2nd and 3rd trimesters in the whole (A), in the non-GDM 
and GDM groups (B) and the antioxidant capacity ratio 3rd:2nd between groups (C). n=23 whole group, 
n=15 Non GDM group and n=8 GDM group. (A) * p< 0.05, compared to 2nd trimester, Student’s t test; (B) 
& p=0.0227, two-way anova, repeated measures, 2nd vs 3rd trimesters. Non GDM: women without 
Gestational Diabetes Mellitus; GDM: women diagnosed with Gestational Diabetes Mellitus according to 
ADA (2016) classification. In (A)● Whole group second trimester; ○ whole group third trimester; In (B) 
◼ Non GDM group second trimester; ◻ Non GDM group third trimester; ▲GDM group second trimester 
and △ GDM group third trimester. In (C) ◼ Non GDM group; ▲GDM group; FRAP method. 
 
5.4.1. Antioxidant capacity and micronutrient intake 
 
Correlation analyses were performed in an attempt