Association of vitamin D with fatty acids in pregnancy

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Association of Vitamin D with Fatty Acids in Pregnancy A. Nandi , N. Wadhwani , K. Randhir , G. Wagh , S.R. Joshi PII: DOI: Reference:

S0952-3278(19)30124-3 https://doi.org/10.1016/j.plefa.2019.102030 YPLEF 102030

To appear in:

Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA)

Received date: Accepted date:

15 June 2019 5 November 2019

Please cite this article as: A. Nandi , N. Wadhwani , K. Randhir , G. Wagh , S.R. Joshi , Association of Vitamin D with Fatty Acids in Pregnancy, Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA) (2019), doi: https://doi.org/10.1016/j.plefa.2019.102030

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Highlights 

Lower maternal and cord serum 25-hydroxyvitamin D [25(OH)D] levels in women with preeclampsia as compared to normotensive control women



Lower maternal plasma levels of polyunsaturated fatty acids (PUFA) but higher levels of total saturated fatty acids (SFA) and total monounsaturated fatty acids (MUFA) in preeclampsia group



Maternal 25(OH)D levels were positively associated with maternal PUFA but negatively associated with maternal total SFA and total MUFA

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Association of Vitamin D with Fatty Acids in Pregnancy A. Nandi1, N. Wadhwani1, K. Randhir1, G. Wagh2, S.R. Joshi1* 1

Mother and Child Health, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be University), Pune Satara Road, Pune-411043, India

2

Department of Obstetrics and Gynaecology, Bharati Medical College and Hospital, Bharati Vidyapeeth (Deemed to be University), Pune Satara Road, Pune-411043, India

Address for Correspondence: Dr. Sadhana Joshi, Professor and Head, Mother and Child Health, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be University), Pune Satara Road, Pune 411043, India Tel: (020) 24366920 E-mail: [email protected]

Abbreviations list:

25(OH)D: 25-hydroxyvitamin D, 1,25(OH)2D: 1,25-dihydroxyvitamin D, AA: arachidonic acid, ACOG: The American College of Obstetricians and Gynaecologists, ALA: alphalinolenic acid, BMI: body mass index, BP: blood pressure, DGLA: dihomo-gamma-linolenic acid,

DHA:

docosahexaenoic

acid,

DPA:

docosapentaenoic

acid,

EDTA:

ethylenediaminetetraacetic acid, EIA: Enzyme Immunoassay, EPA: eicosapentaenoic acid, GLA: gamma linolenic acid, LA: linoleic acid, LCPUFA: long chain polyunsaturated fatty acids, MUFA: monounsaturated fatty acids, MYR: myristic acid, MYRO: myristoleic acid, NC: normotensive control, OLE: oleic acid, PAL: palmitic acid, PALO: palmitoleic acid, PE: preeclampsia, SFA: saturated fatty acids, STE: stearic acid

2

Summary Vitamin D and long chain polyunsaturated fatty acids (LCPUFA) are known to play a role in regulating inflammation. The present study explores the association of maternal vitamin D and fatty acids in pregnancy. This study includes 69 normotensive control (NC) and 50 women with preeclampsia (PE). Maternal and cord serum 25-hydroxyvitamin D [25(OH)D] levels were lower in women with PE. Maternal plasma LCPUFA levels were lower while saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) were higher in women with PE. Maternal 25(OH)D levels were negatively associated with maternal systolic and diastolic blood pressure. Maternal 25(OH)D levels were positively associated with maternal LCPUFA and negatively associated with maternal SFA and MUFA. This study for the first time demonstrates an association of maternal vitamin D with fatty acid levels in pregnancy. Our results suggest that vitamin D and fatty acids may work in concert to regulate fetal growth. 1.

Introduction Preeclampsia is a pregnancy specific syndrome majorly characterised by the new onset of

hypertension after 20 weeks of gestation and can be accompanied with new onset of one or more of the following: proteinuria, thrombocytopenia, renal insufficiency, impaired liver function, pulmonary edema and cerebral and visual symptoms [1]. It affects 2-10 % of the pregnancies worldwide [2] and is a leading cause of maternal and fetal morbidity and mortality [3]. In India, its incidence is reported to be around 8-10% among pregnant women [4]. Abnormal placentation leading to improper uterine and placental perfusion are the major causes of preeclampsia [5]. Poor utero-placental perfusion leads to oxidative stress, endothelial dysfunction and hypertension [6]. The pathophysiology of preeclampsia includes excessive maternal immune response to trophoblasts followed by excessive endothelial activation and hyper inflammation [7]. Several nutrients are known to be involved in placental inflammatory

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response and endothelial activation and may therefore play a key role in the pathogenesis of preeclampsia [8]. Among the various nutrients, deficiency of vitamin D in pregnancy is reported to increase the risk of development of preeclampsia [9][10]. During pregnancy, vitamin D plays an important role in immunomodulation at the fetal maternal interface [11], angiogenesis [12], and has anti-inflammatory roles [13].

Deficiency of this vitamin may contribute to impaired

extravillous trophoblast invasion during placentation [14]. However, the pathogenic mechanism through which vitamin D influences the risk of preeclampsia is not completely understood. In addition to vitamin D, fatty acids including polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) and are also known to be involved in the regulation of endothelial function and oxidative stress [15] in pregnancy [16]. Long chain polyunsaturated fatty acids (LCPUFA) such as docosahexaenoic acid (DHA), an omega-3 fatty acid and arachidonic acid (AA), an omega-6 fatty acid, play a crucial role in pregnancy and are required for normal fetal growth and development [17]. The omega-3 LCPUFA have anti-inflammatory properties and can limit oxidative damage in placenta [18]. In contrast, omega-6 LCPUFA, in particular AA gives rise to the proinflammatory prostanoids and leukotrienes [19]. An abnormal ratio of omega-3 and omega-6 LCPUFA can influence inflammatory and oxidative pathways resulting in abnormal placentation [20]. Both vitamin D [21] and fatty acids[22] have a role in regulating inflammation, oxidative stress, and endothelial function, processes that are known to be altered in preeclampsia [23]. In our earlier studies, we have demonstrated that mothers with preeclampsia have altered fatty acid levels and metabolism [24][25][26][27], increased homocysteine [28] and higher oxidative stress levels [24][29]. We also observed that, altered LCPUFA levels in preeclampsia were

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associated with increased levels of homocysteine [30], a well known risk factor for preeclampsia. We recently hypothesised that; maternal vitamin D deficiency will alter fatty acid metabolism and production of fatty acid metabolites by influencing homocysteine production in the one carbon cycle and increasing oxidative stress [31]. Our recent animal study demonstrates that maternal deficiency of vitamin D influences fatty acid metabolism and alters fatty acid levels [32]. Furthermore, we also observed that maternal vitamin D deficiency increases the ratio of vasoconstrictor and vasodilator lipid metabolites of LCPUFA by increasing homocysteine and oxidative stress levels [33]. In view of this, we propose that maternal vitamin D and fatty acid status will together contribute to the pathophysiology of preeclampsia. The present study was therefore undertaken to explore the association of vitamin D and fatty acid status in women with preeclampsia. 2. Material and methods 2.1. Subjects The present cross-sectional study was carried out at the Department of Obstetrics and Gynecology at Bharati Medical College and Hospital, Pune, India. A total of 119 pregnant women [69 normotensive control (NC) and 50 women with PE] were recruited at delivery. Pregnant individuals with a singleton pregnancy, aged 18–35 years were enrolled. Pregnant women with other complications (i.e. chronic hypertension, seizure disorder, type 1 or type 2 diabetes mellitus, renal disease, or liver disease), smoking, and drug or alcohol use were excluded from the study. The NC group included women delivering at term (total gestation ≥37 weeks and baby weight ≥2.5 kg) with no medical or obstetric complications. Preeclampsia was diagnosed according to the ACOG (The American College of Obstetricians and Gynecologists)

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guidelines i.e. a blood pressure (BP) of ≥140/90 mm Hg on two occasions after 20 weeks of gestation in a pregnant woman with a normal blood pressure previously and proteinuria (>1+ on a dipstick test or 300 mg or more per 24 hours) [1]. Diagnosis was confirmed by repeated blood pressure measurements at 6-hour interval. In absence of proteinuria, new onset of hypertension accompanied by new onset of any of the following: thrombocytopenia, renal insufficiency, impaired liver function, pulmonary edema, cerebral or visual symptoms were considered as preeclampsia [1]. This study includes women with preeclampsia delivering both at term and preterm. Gestational age was calculated from the last menstrual period and confirmed by routine ultrasonography. Ethical approval was taken from the Institutional Ethics Committee of Bharati Vidyapeeth Medical College, Bharati Vidyapeeth (Deemed to be University) to conduct this study and written consent was taken from all participants. 2.2. Maternal and neonatal characteristics Maternal characteristics such as age, body mass index (BMI) and clinical information such as systolic and diastolic BP, gestation and parity were recorded at the time of delivery. Infant birth weight, length and head and chest circumference were recorded according to standard methods. 2.3. Blood sample collection and processing Maternal venous blood sample (10 mL) was collected at delivery and umbilical cord blood (10 mL) was obtained immediately post-partum. Half of the maternal and cord blood samples were collected into ethylenediaminetetraacetic acid (EDTA) coated tube and another half of the blood samples were collected in non EDTA coated tubes. The EDTA coated tube containing blood samples were centrifuged at 2000 rpm for 30 min to separate plasma and

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erythrocytes fractions. The blood samples in the non EDTA coated tubes were kept at room temperature for 30 minutes to separate serum from coagulated blood. These blood samples were then centrifuged at 2500 rpm to get serum. The plasma, erythrocyte and serum fractions were collected and immediately stored at -80°C for further analysis. 2.4.

Analysis of 25-hydroxyvitamin D [25(OH)D] levels Serum 25(OH)D levels were determined by the Enzyme Immunoassay (EIA) method,

using AC-57SF1, 25- Hydroxy Vitamin Ds EIA kit (AC-57SF1, IDS, Boldon, UK). 25(OH)D levels were expressed as ng/ml. 25(OH)D levels were considered to be optimal if the levels were 30–100 ng/ml, insufficient if in the range of 21–29 ng/ml and deficient if ≤20 ng/ml [34]. 2.5. Estimation of maternal plasma homocysteine levels Maternal

plasma

homocysteine

levels

were

analysed

using

Chemiluminescent Microparticle Immuno Assay technology (Abbott Diagnostics, Abbott Park, Ill., USA). The plasma homocysteine levels are expressed as μmol/L. 2.6.

Analysis of fatty acids The estimation of fatty acids from maternal as well as cord blood plasma and erythrocyte

fractions were performed according to the modified original method of Fisk et al., 2014 [35]. Briefly, lipids were extracted from the plasma and erythrocyte fractions using the solvent mixtures of chloroform and methanol.

Methanolic–sulphuric acid was used for

transesterification of the fatty acids in extracted samples. Fatty acid methyl esters were separated using Perkin- Elmer Gas Chromatograph (SD 2330, 30-m capillary column, Supelco, PA, USA). Fatty acid methyl ester standards (Sigma, USA) were used for the identification of individual fatty acids. The following 15 fatty acids were analysed i.e. saturated fatty acids

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(SFA) [myristic acid (MYR), palmitic acid (PAL) and stearic acid (STE)], monounsaturated fatty acids (MUFA) [myristoleic acid (MYRO), palmitoleic acid (PALO), oleic acid (OLE)], omega-3 fatty acids [alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), DHA and omega-3 docosapentaenoic acid (n-3 DPA)] and omega-6 fatty acids [linoleic acid (LA), gamma linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), AA and omega-6 docosapentaenoic acid (n-6 DPA)]. Each fatty acid was expressed as g/100 g fatty acids. 2.7. Statistical analysis All the data were analysed using SPSS/PC+ package (Version 20, Chicago IL). Values are reported as mean ± SD. Independent t-test test was used to compare mean values of the various parameters between NC and PE women. Correlation between variables was studied using Pearson’s correlation analysis. Categorical variables were compared using Chi-square test. Statistical significance was considered at p <0.05. The samples numbers were variable for different parameters due to inadequate sample volume. 3. Results 3.1. Maternal and neonatal characteristics Table-1 shows the maternal and neonatal characteristics. Compared to NC women, women with PE were older (p<0.05), had a higher BMI (p<0.01) and had a higher systolic and diastolic BP (p<0.01). Greater proportions of women with PE were nulliparous and delivered their baby by caesarean delivery. Infants born to women with PE had lower birth weight, length, head circumference and chest circumference (p<0.01 for all) as compared to the NC women (Table-1). 3.2. Maternal and cord serum 25(OH)D levels

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Women from both the groups i.e. NC and PE were deficient in both maternal and cord serum 25(OH)D levels (vitamin D levels < 20 ng/ml). A large proportion of women i.e. 94 % in the PE group while 76% in the NC group were deficient in the maternal 25(OH)D levels. In case of cord 25(OH)D levels, 98% of women with PE and 85.2% of NC women were deficient. Women with PE had lower maternal and cord serum 25(OH)D levels (p<0.01 for both) as compared to the NC women (Table-2). 3.3. Maternal plasma homocysteine levels Maternal plasma homocysteine levels were higher (p<0.01) in the PE group (10.75 ± 6.75 μmol/L (n=47)) as compared to NC group (7.71 ± 2.83 μmol/L (n=58)) 3.4. Maternal plasma fatty acid levels (g/100g of fatty acids) in NC and PE women Table-3 shows the maternal plasma fatty acid profile in the NC women and women with PE. The levels of MUFA and SFA were higher (p<0.05 for both) whereas the levels of PUFA were significantly lower (p<0.05) in PE as compared to NC women. The levels of omega-3 fatty acids were similar between both the groups. The levels of LA and total omega-6 fatty acids were lower (p<0.05 for both) while GLA was higher (p<0.05) in women with PE as compared to NC women. However, the levels of omega-6 fatty acids like AA were similar in both the groups. 3.5. Cord plasma fatty acid levels (g/100g of fatty acids) in NC and PE women Table-4 shows the cord plasma fatty acid profile in NC women and women with PE. The omega-6 and omega-3 fatty acids, SFA, MUFA and PUFA did not show any difference between the groups. 3.6. Maternal erythrocyte fatty acid levels (g/100g of fatty acids) in NC and PE women

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Table-5 shows the maternal erythrocyte fatty acid profile in NC women and women with PE. The levels of LA were lower and AA were higher (p<0.05 for both) in women with PE as compared to NC women. Other omega-6 and omega-3 fatty acids, SFAs, MUFAs and PUFAs were similar in both the groups. 3.7.

Cord erythrocyte fatty acid levels (g/100g of fatty acids) in NC and PE women Table-6 shows the cord erythrocyte fatty acid profile in NC women and women with PE.

The levels of LA (p<0.05), AA and total omega-6 fatty acids (p<0.01 for both) were higher while DGLA (p<0.05) and DPA omega-6 (p<0.01) were lower in women with PE as compared to NC women. The levels of ALA, DPA omega-3 and total omega-3 fatty acids were higher (p<0.01 for all) in women with PE as compared to NC women. The levels of EPA and DHA were similar in both the groups. The levels of PUFA were higher (p<0.01) in the PE group as compared to NC group. SFAs and MUFAs did not show any changes in both the groups. 3.8. Association of maternal 25(OH)D with cord 25(OH)D levels There was a positive association of maternal 25(OH)D levels with cord 25(OH)D levels in the whole cohort (r=0.814, p<0.001, n=97), NC group (r=0.795, p<0.001, n=51) and also in the PE group (r=0.829, p<0.001, n=46). 3.9. Association of maternal 25(OH)D levels with maternal and neonatal characteristics Maternal 25(OH)D levels were negatively associated with maternal BMI (r= -0.272, p= 0.005, n= 105), systolic (r=-0.295, p=0.001, n=119) and diastolic BP (r= -0.311, p= 0.001, n= 119) whereas, positively associated with maternal gestation age (r= 0.277, p= 0.002, n= 119) in the whole cohort. Maternal 25(OH)D levels were not found to be associated with neonatal characteristics such as birth weight, length, head and chest circumference.

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3.10. Association of maternal and cord 25(OH)D levels with maternal plasma homocysteine levels Maternal serum 25(OH)D levels were negatively associated with maternal plasma homocysteine levels in the whole cohort (r= -0.236, p= 0.015, n= 105). Cord serum 25(OH)D levels were negatively associated with maternal plasma homocysteine levels in the whole cohort (r=-0.291, p=0.005, n=90) and in women with PE (r=0.342, p=0.025, n=43). 3.11. Association of maternal 25(OH)D levels with maternal plasma fatty acid levels Maternal 25(OH)D levels were negatively associated with maternal plasma MYR (r=0.214, p=0.003, n=100), PAL (r=-0.205, p=0.041, n=100) and total SFA (r=-0.244, p=0.014, n=100) in the whole cohort. A negative association of maternal 25(OH)D levels was also observed with OLE (r=-0.251, p=0.012, n=100) and total MUFA levels (r=-0.284, p=0.004, n=100). In contrast, maternal 25(OH)D levels were positively associated with LA (r=0.238, p=0.017, n=100), total omega-6 fatty acids (r=0.306, p=0.002, n=100) and PUFA (r=0.312, p=0.002, n=100) in the whole cohort. 3.12.

Association of maternal 25(OH)D levels with cord plasma fatty acid levels

Maternal 25(OH)D levels were negatively associated with cord plasma MYRO (r=-0.233, p=0.032, n=85) while positively associated with cord plasma DGLA (r=0.306, p=0.004, n=85) in the whole cohort. In the NC group maternal 25(OH)D levels was negatively associated with cord plasma MYRO (r=-0.364, p=0.015, n=44). In the PE group maternal 25(OH)D levels were positively associated with cord plasma STE (r=0.309, p=0.049, n=41) and DGLA (r=0.357, p=0.022, n=41). 3.13.

Association of cord 25(OH)D levels with cord plasma fatty acid levels

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No significant association was observed between cord 25(OH)D levels and cord plasma fatty acid levels in the whole cohort as well as in the NC group. However, in the PE group, cord 25(OH)D levels were positively associated with cord plasma levels of MYR (r=0.290, p=0.034, n=54) and STE (r=0.275, p=0.044, n=54). 3.14. Association of maternal 25(OH)D with maternal erythrocyte fatty acid levels Maternal 25(OH)D levels were positively associated with maternal erythrocyte levels of LA (r=0.230, p=0.054, n=71)

in the whole cohort. The maternal 25(OH)D levels were

negatively associated with maternal erythrocyte levels of OLE (r=-0.432, p=0.014, n=32) and MUFA (r= -0.443, p=0.011, n=32) in the NC group. 3.15. Association of maternal 25(OH)D with cord erythrocyte fatty acid levels There was a positive association observed between maternal 25(OH)D levels and cord erythrocyte levels of DGLA in the whole cohort (r=0.228, p=0.041, n=81) and PE group(r=0.341, p=0.042, n=36). The maternal 25(OH)D levels were negatively associated with cord erythrocyte MYR (r=-0.409, p=0.013, n=36) and MYRO (r=-0.367, p=0.027, n=36) in the PE group. 3.16. Association of cord 25(OH)D with cord erythrocyte fatty acid levels There was a positive association between cord 25(OH)D levels and cord erythrocyte PUFA levels in the NC group (r=0.306, p=0.030, n=50) . 4. Discussion To the best of our knowledge, this study for the first time reports the association of 25(OH)D levels with fatty acid levels in pregnant women. Some key findings of our study are (1) Women with PE had markedly lower maternal and cord serum 25(OH)D levels compared to NC women; (2) Lower maternal plasma levels of PUFA but higher levels of total SFA and

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total MUFA in the PE group; (3) Higher levels of cord erythrocyte PUFA in women with PE; (5) Maternal 25(OH)D levels were negatively associated with maternal weight, systolic and diastolic BP but positively associated with gestational age (6) Maternal and cord 25(OH)D levels were negatively associated with maternal homocysteine levels; (7) Maternal 25(OH)D levels were positively associated with maternal plasma LA, omega-6 fatty acids, PUFA and negatively associated with maternal plasma total SFA and total MUFA. In the present study, we observed lower levels of 25(OH)D in women with PE as compared to NC women, although, both the NC and PE groups were deficient in 25(OH)D levels. Levels of 25(OH)D lesser than 10 ng/ml (25 nmol/L) were considered as severe deficiency by various studies [36][37][38]. Based on this classification the PE women in our study were severely deficient in 25(OH)D levels. It is reported that a 10 ng/mL increase in 25OHD levels decreases the risk of severe PE by 63% [39]. Bodnar and co-workers studied a large cohort with 717 PE women and 2,986 NC women, and concluded that maternal vitamin D deficiency is a clear risk factor for the development of severe PE. They found that maternal 25(OH)D levels ≥ 50 nmol/L (20 ng/ml) reduces the risk of severe PE development by 40% in comparison to women with 25(OH)D <50 nmol/L [40]. Two prospective cohorts in Canada found that 25(OH)D concentrations <30 nmol/L in early pregnancy led to a greater risk of developing PE when compared to concentrations > 50 nmol/L [41]. It is known that fetus is dependent on the mother for its 25(OH)D supply; and the lower cord levels observed in the preeclampsia group may be due to lower maternal levels. Reports suggest that, although the active form of vitamin D i.e. 1,25-dihydroxyvitamin D (1,25(OH)2D) does not cross the placental tissue, its inactive precursor 25(OH)D readily crosses the tissue to the fetal compartment and gets converted to the active form i.e. 1,25(OH)2D with the help of

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vitamin D metabolising enzymes [42]. It has been suggested that 1,25(OH)2D modulates antiinflammatory effects and influences immunological tolerance during pregnancy [43]. In our study, we observed altered maternal and cord plasma and erythrocyte fatty acid profile in PE as compared to NC group. We observed lower maternal plasma levels of LA, and PUFA in the PE group as compared to NC group. Our findings are similar to a study by Wang et al., which also reports lower maternal plasma levels of LA and PUFA in women with PE as compared to normal pregnant subjects [44]. The lower maternal plasma PUFA levels observed may either result from dysregulated synthesis; alterations in their storage and mobilization; or dietary unavailability [45]. Furthermore, increased oxidative stress in preeclampsia could also play a role in lowering the levels of PUFA since it is known to cause peroxidative degradation of PUFA [46]. In this study we observed higher levels of PAL and total SFA levels in women with preeclampsia. The increased levels of PAL could be linked with increased adipose and hepatic tissues (the main sites of fatty acid metabolism) endoplasmic reticulum (ER) stress [47]. A cell culture study with placental trophoblasts has also shown that PAL can increase ER stress in placenta [48]. Furthermore, ER stress is known to be a contributing factor for oxidative stress in preeclampsia[49]. We also observed higher levels of maternal plasma MUFA levels in the PE group as compared to the NC group. A prospective study of three population based cohort reports that higher circulating concentrations of MUFA levels increases cardiovascular disease (CVD) risk [50]. Furthermore, it is well known that women with preeclampsia are at an increased risk of developing cardiovascular disease [51]. However, a meta-analysis has shown that, effects of MUFA on cardiovascular risk factors are ambiguous [52].

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Women with PE had significantly lower maternal erythrocyte levels of LA and higher levels of AA compared to NC women. This result may be attributed to increased conversion of LA to AA in the PE group. The higher levels of AA observed may increase the production of pro-inflammatory eicosanoids as it is known that AA is a regulator of inflammation and can control cardiometabolic function by cyclooxygenase, lipooxygenase and cytochrome P-450 (CYP) pathways [53][54]. These pathways are further known to be altered in PE [55][56][57]. In the present study, we observed higher levels of cord erythrocyte LA, AA, total omega-6 fatty acids, ALA, and PUFA in the PE group. These altered levels of cord fatty acids may possibly be due to increased placental uptake, transport and metabolism of fatty acids in women with preeclampsia. In the present study we observed that maternal serum 25(OH)D levels were negatively associated with systolic and diastolic BP in the whole cohort. Similar observations were reported by Adela et al., where in the third trimester of pregnancy, serum 25(OH)D levels were negatively associated with SBP and DBP [58]. Reports indicate that 1,25(OH)2D can function as a suppressor of renin biosynthesis to regulate the renin–angiotensin system which is known to play a central role in the regulation of BP [59][43]. In our study we observed that both maternal and cord 25(OH)D levels were positively associated with gestational age. A recent meta-analysis of randomized controlled trials (RCT) and observational studies reports that maternal vitamin D deficiency increases the risk of preterm birth [60]. One possible explanation for this association may be attributed to the protective effect of vitamin D in reducing intrauterine infection and inflammation [61]. The present study demonstrates that 25(OH)D levels were positively associated with total PUFA levels. Vitamin D is well known for its anti-inflammatory and immune regulatory properties [62]. PUFA are also known play protective role against inflammation and

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endothelial dysfunction [15]. PUFA like AA have been shown to play an important role in homeostatic regulation of vitamin D and parathyroid hormone (PTH) during bone growth [63]. PUFA are derived from phospholipids which are known to be linked with one carbon cycle by the phosphatidylethanolamine methyltransferase (PEMT) pathway [64]. Our recent animal study has shown that maternal vitamin D deficiency increases homocysteine levels by reducing the expression of cystathionine beta synthase (CBS) enzyme in transsulfuration pathway of the one carbon cycle [33]. Higher levels of homocysteine increases oxidative stress which further influence PUFA metabolism and production of PUFA metabolites [32][33]. Similarly, in the present human study we observed that lower maternal levels of vitamin D were associated with higher levels homocysteine. Our earlier human studies reports that maternal homocysteine is negatively associated with DHA levels [30]. A recent human study has indicated that serum homocysteine levels can affect AA metabolism [65]. In the present study neither AA nor DHA were found to be associated with vitamin D levels. A possible explanation for this may be may be due to the conversion of these PUFA to their eicosanoid metabolites. Therefore, further studies are needed to examine the eicosanoid metabolites of PUFA. We also demonstrated a negative association of vitamin D with SFA and MUFA levels. SFA are known to be inversely associated with endothelial vasodilatory function and increases cardiovascular risk by increasing coagulation and inflammation in healthy individuals [66]. It is often stated that MUFA (oleic acid) is anti-inflammatory and may have beneficial effect on endothelial function [67][68], although its effect on inflammatory processes in humans are modest [69]. One limitation of this study is that we did not analyse various inflammatory markers. 5. Conclusion

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To summarize, the present study simultaneously examined the levels of both vitamin D and fatty acids in women with PE and report lower levels of vitamin D and PUFA in women with preeclampsia. We also report a positive association of maternal vitamin D with PUFA and a negative association with SFA and MUFA. In this study, it is difficult to attribute a causal effect of vitamin D on various fatty acids. Future studies need to be undertaken with vitamin D supplementation during pregnancy to confirm the effect of vitamin D on fatty acid status. Nevertheless, our findings suggest that vitamin D and fatty acids work in concert with each other to regulate inflammatory pathways in preeclampsia. Conflicts of interest statement The authors declare that they have no conflicts of interest to disclose.

Acknowledgement The authors acknowledge University Grant Commission (UGC), Government of India for providing Anindita A. Nandi the Senior Research Fellowship (SRF). The authors are thankful to all the staff members and nurses of Bharati Hospital for their help in sample collection.

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Table-1: Maternal and neonatal characteristics Variables Maternal characteristics Age (years) BMI (kg/m2) Systolic BP(mmHg) Diastolic BP(mmHg) Gestational age (weeks) Gravida n(%) Primigravida Multigravida Parity n(%) Nulliparity Multiparity Type of delivery n(%) Normal Caesarean Education n(%) Illiterate Primary Secondary Higher secondary Undergraduate Graduate Neonatal characteristics Birth weight (kg) Birth length (cm) Head circumference (cm) Chest circumference (cm)

NC (n=69)

PE (n=50)

25.84±4.34 26.34±3.7 115.14±6.41 74.20±5.53 38.83±0.86

27.88±4.81* 31.16±5.91** 147.18±15.74** 95.92±10.067** 35.50±2.98**

19(38.0) 31(62.0)

20(40.0) 30(60.0)

29(42.0) 40(58.0)

26(52.0) 24(48.0)

43(66.2) 22(33.8)

9(19.6) 37(80.4)

2(2.9) 5(4.9) 20(29.4) 15(22.1) 2(2.9) 28(43.1)

2(2.9) 4(5.8) 16(32.0) 9(18.0) 2(4.0) 21(42.8)

2.93±0.28 47.03±2.82 33.91±3.57 32.28±1.99

2.17±0.76** 43.93±4.5** 31.30±2.49** 28.55±3.78**

Values are expressed as mean ± SD. NC, normotensive control; PE, preeclampsia; BMI, body mass index; BP, blood pressure; n, number of subjects; **p < 0.01 and *p < 0.05 as compared to NC.

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Table-2: Maternal and cord serum 25(OH)D levels in NC and PE women Parameters Maternal serum 25(OH)D level (ng/ml)

NC (n=69) 15.03 ± 9.9 NC (n=61) 12.18 ± 7.74

Cord serum 25(OH)D level (ng/ml)

PE (n=50) 8.34± 8.08** PE (n=60) 8.48± 4.41**

Values are expressed as mean ± SD. 25(OH)D; 25-hydroxyvitamin D, NC, normotensive control; PE, preeclampsia; n, number of subjects; **p < 0.01 as compared to NC.

Table-3: Maternal plasma fatty acid levels (g/100g of fatty acids) in NC and PE Fatty acids NC group (n=51) PE group (n=49) (g/100g fatty acids) 1.07 ± 0.37 0.282 ± 0.03 MYR 0.05 ± 0.05 0.05 ± 0.05 MYRO 28.64 ±2.28 29.8 ±2.68* PAL 1.81 ±0.87 2.16±1 PALO 5.57 ±1.03 5.5 ±1.05 STE 15.64±2.03 16.43 ±3.03 OLE 32.02 ±5.96 29.78 ±4.91* LA 0.19 ±0.11 0.24 ±0.13* GLA 0.35 ±0.21 0.43±0.28 ALA 1.50 ±0.41 1.49 ±0.35 DGLA 5.66±2.07 5.74 ±1.39 AA 0.11 ±0.1 0.11 ±0.08 EPA 0.53 ±0.27 0.56 ±0.22 DPA omega-6 0.13±0.08 0.15± 0.08 DPA omega-3 1.05 ±0.56 1.04 ±0.40 DHA 1.65±0.75 1.73 ±0.66 Total omega-3 40.63±7.48 37.80 ±4.28* Total omega-6 30.61 ±20.90 25.63 ±11.54 Omega-6: Omega-3 35.27±2.44 36.39±2.75* SFA 17.62 ±2.28 18.68 ±2.99* MUFA 42.28 ±7.24 39.54 ±4.16* PUFA Values are expressed as mean ± SD. NC, normotensive control; PE, preeclampsia; n, number of subjects; *p < 0.05 **p < 0.01 as compared to NC. Total omega-3 fatty acids: [Alpha-linolenic acid (ALA) + Eicosapentaenoic acid (EPA) + omega-3 Docosapentaenoic acid (n-3 DPA), Docosahexaenoic acid (DHA)], Total omega-6

29

fatty acids: [Linoleic acid (LA) + Gamma linolenic acid (GLA) + Dihomo-gamma-linolenic acid (DGLA) + Arachidonic acid (AA) + Omega-6 docosapentaenoic acid (n-6 DPA), Total monounsaturated fatty acids (MUFAs): [Myristoleic acid (MYRO) + Palmitoleic acid (PALO) + Oleic acid (OLE)], Total saturated fatty acids (SFAs): [Myristic acid (MYR) + Palmitic acid (PAL) + Stearic acid (STE)].

Table-4: Cord plasma fatty acid levels (g/100g of fatty acids) in NC and PE Cord Plasma Fatty acids (g/100g NC (n=53) PE (n=54) fatty acids) 1.26 ± 0.43 1.24 ± 0.43 MYR 0.12 ± 0.17 0.12 ± 0.14 MYRO 27.80 ±1.66 28.41 ±2.02 PAL 2.21±0.65 2.48±0.76* PALO 9.65 ±1.08 9.24 ±1.14 STE 17.50±2.16 18.2 ±3.08 OLE 11.89 ±5.26 12.16±3.72 LA 0.22 ±0.18 0.19 ±0.13 GLA 0.16 ±0.11 0.17±0.09 ALA 2.47 ±0.72 2.08 ±0.67* DGLA 12.31±2.14 12.38 ±2.14 AA 0.09 ±0.05 0.11 ±0.07 EPA 1.16 ±0.39 0.91 ±0.29* DPA omega-6 0.12±0.07 0.15± 0.09* DPA omega-3 1.80 ±0.71 1.86 ±0.71 DHA 2.18±0.79 2.29 ±0.85 Total omega-3 28.07±4.22 27.72 ±3.47 Total omega-6 14.70 ±6.26 13.76 ±5.46 Omega-6: Omega-3 38.67±1.99 38.89±2.30 SFAs 19.83 ±2.52 20.80 ±3.43 MUFAs 30.24 ±4.28 30.01 ±3.77 PUFAs Values are expressed as mean ± SD. NC, normotensive control; PE, preeclampsia; n, number of subjects; *p < 0.05 **p < 0.01 as compared to NC. Total omega-3 fatty acids: [Alpha-linolenic acid (ALA) + Eicosapentaenoic acid (EPA) + omega-3 Docosapentaenoic acid (n-3 DPA), Docosahexaenoic acid (DHA)], Total omega-6 fatty acids: [Linoleic acid (LA) + Gamma linolenic acid (GLA) + Dihomo-gamma-linolenic acid (DGLA) + Arachidonic acid (AA)+ Omega-6 docosapentaenoic acid (n-6 DPA), Total monounsaturated fatty acids (MUFA): [Myristoleic acid (MYRO) + Palmitoleic acid (PALO) + Oleic acid (OLE)], Total saturated fatty acids (SFA): [Myristic acid (MYR) + Palmitic acid (PAL) + Stearic acid (STE)].

30

Table-5: Maternal erythrocyte fatty acid levels (g/100g of fatty acids) in NC and PE Maternal RBC Fatty acids (g/100g fatty NC (n=51) acids) 0.53 ± 0.31 MYR 0.02 ± 0.01 MYRO 27.95 ±3.14 PAL 0.32 ±0.18 PALO 10.11 ±1.20 STE 12.69±2.15 OLE 13.37 ±2.45 LA 0.05 ±0.05 GLA 0.17 ±0.11 ALA 1.51 ±0.42 DGLA 11.33±2.19 AA 0.15 ±0.10 EPA 1.07 ±0.46 DPA omega-6 0.81±0.32 DPA omega-3 2.43 ±0.84 DHA 3.56±1.09 Total omega-3 27.35±3.06 Total omega-6 8.36 ±2.64 Omega-6: Omega-3 38.597±3.55 SFAs 13.23 ±2.12 MUFAs 30.91 ±3.35 PUFAs Values are expressed as mean ± SD. NC, normotensive control; PE, of subjects; *p < 0.05 **p < 0.01 as compared to NC.

PE (n=50) 0.51 ± 0.28 0.03 ± 0.1 27.92 ±2.69 0.38±0.26 10.23 ±0.99 12.63 ±2.5 12.11 ±2.61* 0.07 ±0.07 0.14±0.12 1.50 ±0.39 12.51 ±2.51* 0.13 ±0.08 1.09±0.54 0.79± 0.36 2.34 ±0.75 3.44 ±0.93 27.28 ±2.74 8.44 ±2.25 38.66±3.04 13.24 ±2.51 30.73 ±3.17 preeclampsia; n, number

Total omega-3 fatty acids: [Alpha-linolenic acid (ALA) + Eicosapentaenoic acid (EPA) + omega-3 Docosapentaenoic acid (n-3 DPA), Docosahexaenoic acid (DHA)], Total omega-6 fatty acids: [Linoleic acid (LA) + Gamma linolenic acid (GLA) + Dihomo-gamma-linolenic acid (DGLA) + Arachidonic acid (AA)+ Omega-6 docosapentaenoic acid (n-6 DPA), Total monounsaturated fatty acids (MUFA): [Myristoleic acid (MYRO) + Palmitoleic acid (PALO) + Oleic acid (OLE)], Total saturated fatty acids (SFA): [Myristic acid (MYR) + Palmitic acid (PAL) + Stearic acid (STE)].

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Table-6: Cord erythrocyte fatty acid levels (g/100g of fatty acids) in NC and PE Cord RBC Fatty acids (g/100g fatty acids) NC (n=51) PE (n=56) 0.34 ± 0.19 0.31 ± 0.15 MYR 0.01 ± 0.001 0.01 ± 0.004 MYRO 29.57 ±1.73 29.92 ±1.60 PAL 0.43 ±0.13 0.40±0.16 PALO 11.33 ±0.92 11.02 ±0.99 STE 6.12±0.78 5.95 ±0.91 OLE 5.13 ±0.79 5.55 ±0.93* LA 0.07 ±0.06 0.1 ±0.07 GLA 0.12±0.04 0.15±0.07** ALA 2.47 ±0.33 2.29 ±0.36* DGLA 17.74±1.93 19.39 ±1.49** AA 0.07 ±0.11 0.08 ±0.09 EPA 2.12 ±0.60 1.75±0.66** DPA omega-6 0.28±0.12 0.38± 0.15** DPA omega-3 3.36 ±0.86 3.55 ±0.94 DHA 3.82±0.91 4.15 ±1.07** Total omega-3 27.52±1.91 29.08 ±1.74** Total omega-6 7.20 ±2.10 7 ±1.62 Omega-6: Omega-3 41.24±1.93 41.25±1.55 SFAs 6.71 ±0.88 6.55 ±1.03 MUFAs 31.35 ±2.12 33.24 ±1.80** PUFAs Values are expressed as mean ± SD. NC, normotensive control; PE, preeclampsia; n, number of subjects; *p < 0.05 **p < 0.01 as compared to NC. Total omega-3 fatty acids: [Alpha-linolenic acid (ALA) + Eicosapentaenoic acid (EPA) + omega-3 Docosapentaenoic acid (n-3 DPA), Docosahexaenoic acid (DHA)], Total omega-6 fatty acids: [Linoleic acid (LA) + Gamma linolenic acid (GLA) + Dihomo-gamma-linolenic acid (DGLA) + Arachidonic acid (AA) + Omega-6 docosapentaenoic acid (n-6 DPA), Total monounsaturated fatty acids (MUFA): [Myristoleic acid (MYRO) + Palmitoleic acid (PALO) + Oleic acid (OLE)], Total saturated fatty acids (SFA): [Myristic acid (MYR) + Palmitic acid (PAL) + Stearic acid (STE)].

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GRAPHICAL ABSTRACT

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