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Clinical Nutrition 30 (2011) 60e64

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Clinical Nutrition

journal homepage: http: / /www.elsevier .com/locate/clnu

Original Article

Association of omega-3 fatty acids and homocysteine concentrationsin pre-eclampsiaq

Asmita Kulkarni a, Savita Mehendale b, Hemlata Pisal a, Anitha Kilari a, Kamini Dangat a,Satyajeet Salunkhe b, Vaishali Taralekar b, Sadhana Joshi a,*aDept of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune-Satara Road, Pune 411043, Maharashtra, IndiabDept of Obstetrics and Gynaecology, Bharati Medical College Hospital, Bharati Vidyapeeth University, Pune-Satara Road, Pune 411043, Maharashtra, India

a r t i c l e i n f o

Article history:Received 17 November 2009Accepted 14 July 2010

Keywords:HomocysteineVitamin B12FolateDHAPre-eclampsia

q Conference presentation: Accepted for Young scie“National Conference of Nutrition society of IndiaHyderabad, India).* Corresponding author. Tel.: þ91 20 24366929/31;

E-mail address: srjoshi62@gmail.com (S. Joshi).

0261-5614/$ e see front matter � 2010 Elsevier Ltd adoi:10.1016/j.clnu.2010.07.007

s u m m a r y

Background & aims: The present study examines the associations of folic acid, vitamin B12 and omega-3fatty acids and increased homocysteine which are implicated in the pathology of pre-eclampsia.Methods: 49 Pre-eclamptic and 57 normotensive women were recruited at Bharati hospital, Pune, India.Plasma folate, vitamin B12, homocysteine and erythrocyte omega-3 and omega-6 fatty acids wereanalyzed.Results: Homocysteine concentrations were higher in pre-eclamptic than in normotensive women(14.28� 7.31 vs. 11.03� 4.38 mmol/l, p< 0.01) despite similar levels of folic acid and vitamin B12. In thepre-eclamptic group, plasma folate levels were positively associated with erythrocyte omega-6 fattyacids (p< 0.05) while erythrocyte docosahexaenoic acid levels were negatively associated with plasmahomocysteine levels (p< 0.01).Conclusions: Our study provides evidence for the associations of altered omega-3 fatty acids especiallydocosahexaenoic acid and the resultant increased homocysteine concentrations in pre-eclampsia. Futurestudies need to examine if docosahexaenoic acid supplementation during pregnancy reduces homo-cysteine levels and ameliorates the risk of developing pre-eclampsia.

� 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

1. Introduction

Epidemiological studies have demonstrated a relationshipbetween pre-eclampsia and an increased risk of maternal coronaryheart disease and metabolic syndrome in later life.1 The incidenceof pre-eclampsia and rates of adverse pregnancy outcomes is highin developing countries like India making it an important healthissue. However, despite being the leading cause of maternal deathand perinatal morbidity, the mechanisms responsible for thepathogenesis of pre-eclampsia have not been completely under-stood2 and therefore there is a pressing need to better understandthemechanisms of the disease, with the ultimate goal of preventingthis disorder.3

Reduced levels of circulating folate during pregnancy are knownto be associated with increased risks of preterm delivery, infant low

ntist Award (junior category),” (19e20 November 2009,

fax: þ91 20 24366929.

nd European Society for Clinical N

birth weight and foetal growth retardation.4 Defects in one-carbonmetabolism are suggested to be at the heart of intrauterineprogramming of adult disease.5 Folate and vitamin B12 are themajor determinants of one-carbon metabolism in which S-adeno-syl methionine (SAMe) is formed6 which helps to maintain methylgroup supply for various macromolecules like DNA, neurotrans-mitters, proteins and membrane phospholipids.7 Our own andother animal studies have shown that maternal folic acid supple-mentation alters brain essential polyunsaturated fatty acid levelsespecially omega-3 fatty acids.8,9 Omega-3 fatty acids are essentialfor foetal growth and our recent studies in pregnancy have wellestablished the role of docosahexaenoic acid (DHA) in pregnancyoutcome.10e13

Previous epidemiological studies relating maternal plasmafolate, vitamin B12, and homocysteine concentrations to pre-eclampsia risk show a relationship of serum homocysteine withpre-eclampsia.14,15 However, there are other studies refuting anassociation.16 This may be attributed to differences in patient pop-ulation, study design, laboratory techniques and disease definition.

Recent studies have shown that Indians are deficient in vitaminB12 which is responsible for increased homocysteine concentra-tions.17 Elevated plasma homocysteine has been implicated in

utrition and Metabolism. All rights reserved.

A. Kulkarni et al. / Clinical Nutrition 30 (2011) 60e64 61

vascular changes compatible with atherosclerosis and endothelialdysfunction similar to the vascular changes of the placenta in pre-eclampsia.18

Endothelial dysfunction induced by homocysteine elevationsmay also be mediated by the generation of reactive oxygenspecies.19 Our recent study in pre-eclamptic women has shownincreased oxidative stress in pre-eclamptic women leading todecreased plasma levels of DHA.10 It is, therefore, of importance tounderstand the role of homocysteine in the pathology of pre-eclampsia by understanding the associations of folic acid, vitaminB12 and omega-3 fatty acids.

The present study examines the levels of key micronutrients(folic acid, vitamin B12) and omega-3 fatty acids, vital componentsof one-carbon metabolism and resultant homocysteine concen-trations in pre-eclamptic women and compares it with normo-tensive women. Both normotensive and pre-eclamptic womenwere extremely well matched for dietary and lifestyle patterns withno smoking, drug or alcohol use to reduce confounds to intake andmetabolism of these key components.

2. Patients and methods

This study was conducted at the Dept. of Obstetrics andGynaecology, Bharati Hospital, Pune during the year 2007e2008.This study was conducted with the understanding and the consentof each subject and was approved by Institutional EthicalCommittee. Written informed consent was obtained from eachsubject. A total number of 106 pregnant women (57 normotensivewomen and 49 pre-eclamptic with singleton pregnancy) wererecruited at term for this prospective study. Womenwere excludedfrom the study if there was evidence of other pregnancy compli-cations like multiple gestation, chronic hypertension, type I or typeII diabetesmellitus, seizure disorder, renal or liver disease. Pregnantwomen with alcohol or drug abuse were also excluded from thestudy. The normotensive group consisted of pregnant women withno medical or obstetrical complications. All women were routinelygiven iron tablets as per the National Prophylaxis programme. Noneof the participants was on vitamin supplements (folic acid andvitamin B12) either during pregnancy or preconception.

Pre-eclampsia was defined by systolic and diastolic bloodpressures greater than 140 and 90 mmHg respectively withpresence of proteinuria (>1þ or 300 mg/24 h) in a dipstick test.Edema was present in some cases. Blood pressure was measuredin the left arm with a mercury sphygmomanometer. Pre-eclampsia was confirmed by repeated recording of the bloodpressure with an interval of 6 h. Gestational age was calculatedby last menstrual period and then confirmed by ultrasound. Bothpre-eclamptic and normotensive women were from the lowersocioeconomic group and had similar level of education, parityand lifestyle.

2.1. Dietary assessments

A food frequency questionnaire was used to estimate thefrequency of intake of foods rich in folic acid, vitamin B12 andomega-3 fatty acids. These foods were identified using ‘NutritiveValues of Indian Foods’.20 The questionnaire consisted of 17 foodgroups and a number of foods (approximately 10) were listed undereach of the food groups. The frequency of intake of foods wasrecorded on an eight-point scale from ‘never’ to ‘thrice daily’.Monthly scores were calculated for each food item. For example, anitem consumed once a week has a score of 4 while that consumeddaily has a score of 30. All participants had limited access to foodsfortified with folic acid and vitamin B12.

2.2. Sample collection, processing and storing

2.2.1. Blood samples10 ml of venous blood was collected into the ethylenediamine

tetra-acetic acid (EDTA) vials just before delivery. All blood sampleswere immediately layered on histopaque (a density gradientobtained from SigmaeAldrich) and centrifuged at 2000 rpm for30 min to separate the plasma and erythrocytes. The erythrocyteswere washed 3 times with normal saline. The plasma and eryth-rocytes aliquots were stored at �80 �C until further analysis.

2.2.2. Biochemical estimationsAll biochemical analyses were performed at laboratories sepa-

rate from subject recruitment sites. Investigators were blinded tosubject identity (normotensive vs. pre-eclamptic) which was indi-cated by a code number maintained by the clinical staff untilanalysis was completed. The blood samples were always collectedat the same time from pre-eclamptic and normotensive controls,stored at �80

�C and analyses were done in matched pairs every 3

months. Unfortunately, red cell total lipids were not analyzed.

2.2.3. Fatty acid analysisThe procedure for fatty acid analysis used in our studywas revised

from the original method of Manku et al.21 and was reported by usearlier in separate studies10e12 was done using the gas chromato-graph. The data included is on only critically important essentialpolyunsaturated fatty acids (EPUFA) and major fatty acids. Theomega-3 fatty acids included alpha linolenic acid, eicosapentaenoicacid and docosahexaenoic acid while omega-6 fatty acids includedlinoleic acid, gammalinolenic acid, di-homo-gammalinolenic acid,docosapentaenoic acid and arachidonic acid.

2.2.4. Folate, vitamin B12 and homocysteine estimationsFolate and vitamin B12 were estimated by the fluorescence

polarization immunoassay (Abbott Diagnostics).22 Since the folatein plasma is known to be unstable with long storage times, theblood samples were always collected around the same time fromnormotensive and pre-eclamptic women and analyses were donein matched pairs. Homocysteine estimation was performed by themicro particle enzyme immunoassay method (Abbott Diagnostics,Abbott Park, IL).23 We have reported these procedures earlier.24

2.3. Statistical analysis

Values are mean� SD. The data were analyzed using SPSS/PCþpackage (Version 11.0, Chicago IL). Skewed variables (maternalarachidonic acid and docosahexaenoic acid) were transformed tonormality using the following transformations: log to the base e(ln). Mean values of the various parameters were compared usingthe Student ‘t’ test for significance p< 0.05. The extent of linearrelationship between several variables was studied using partialcorrelation method after adjusting for maternal age, body massindex, duration of gestation as possible confounders.

3. Results

Table 1 shows the characteristics of the subjects. The frequencyof consumption of both folate (green leafy vegetables) and vitaminB12 rich foods (non vegetarian food, dairy products) was similar inboth the groups. 95% pre-eclamptic and 92% normotensive womenconsumed green leafy vegetables more than once per week. 10% ofwomen of normotensive group and 2% of women from pre-eclamptic group ate non vegetarian foods more frequently i.e. morethan every alternate day. However, the portion sizes of non vege-tarian foods were very small and included meat, fish and eggs.

Table 1Maternal characteristics of normotensive and pre-eclamptic women.

Mean� SD

Normotensive (n¼ 57) Pre-eclamptic (n¼ 49)

Age (yr) 22.5� 2.9 23.7� 3.7BMI (kg/m2) 20.5� 6.3 22.6� 7.7Income (INR) 5016� 3961 5451� 3255Education (grade) 10� 3 10� 3BP systolic (mmHg) 124� 6 149� 14**BP diastolic (mmHg) 79� 5 98� 10**Gestation (wks) �37 �37

**p< 0.01.

A. Kulkarni et al. / Clinical Nutrition 30 (2011) 60e6462

Dairy product consumption was similar in both the groups. 82%normotensive and 87% pre-eclamptic women consumed dairyproducts more than every alternate day. Dairy products includedwhole milk plus milk products (milk in tea and other beverages,yoghurt, buttermilk, ghee, ice cream and other milk basedpreparations).

3.1. Circulating micronutrients and homocysteine concentrations

Table 2 shows circulating micronutrient levels in normotensiveand pre-eclamptic women. Plasma folic acid levels were similarbetween normotensive and pre-eclamptic women. Althoughvitamin B12 levels were higher in pre-eclamptic women ascompared to normotensive, the difference was not statisticallysignificant. However, homocysteine levels were significantly higherin pre-eclamptic women as compared to normotensive (p< 0.01).

3.2. Erythrocyte docosahexaenoic acid (DHA) and arachidonic acid(AA) concentrations

As reported by us earlier10 erythrocyte DHA concentrations inthe present study were also lower in the pre-eclamptic women ascompared to normotensive women (p< 0.05). There was nostatistical difference in arachidonic acid, omega-3 and omega-6fatty acids between groups (Table 2).

3.3. Associations of folic acid and vitamin B12 with erythrocyte fattyacids

After adjusting for age, BMI and gestational age there was noassociation of folic acid or plasma vitamin B12 with erythrocyteomega-3 fatty acids in both normotensive as well as pre-eclampticgroups.

Table 2Circulating levels of micronutrient in normotensive and pre-eclamptic women.

Mean� SD

Normotensive(n¼ 57)

Pre-eclamptic(n¼ 49)

Plasma folic acid (ng/ml) 8.45� 4.88 9.42� 5.51<10 ng/ml, n (%) 66.66 48.04

Plasma vitamin B12 (pg/ml) 146.35� 70.28 163.02� 77.09<150 pg/ml, n (%) 64.26 38.88

Plasma homocysteine (mmol/l) 11.03� 4.38 14.28� 7.27**>10 mmol/l, n (%) 47.37 71.43

Erythrocyte DHA (g/100 g fatty acids) 3.87� 0.91 3.45� 1.05*Erythrocyte AA (g/100 g fatty acids) 14.83� 2.30 15.48� 2.80Erythrocyte omega-3 fatty acids

(g/100 g fatty acids)4.40� 0.94 4.09� 1.04

Erythrocyte omega-6 fatty acids(g/100 g fatty acids)

28.02� 3.67 29.15� 3.80

*p< 0.05; **p< 0.01, DHA e docosahexaenoic acid; AA e arachidonic acid.

3.4. Associations of folic acid and B12 with homocysteineconcentrations

There was a negative association of plasma folic acid withplasma homocysteine concentration (r¼�0.344; p¼ 0.03; n¼ 38)only in the pre-eclamptic group. However plasma vitamin B12 didnot show any association with plasma homocysteine concentra-tions in either group.

3.5. Associations of erythrocyte DHA with homocysteineconcentrations

After adjusting for age, BMI and gestation, erythrocyte omega-3levels (r¼�0.453; p¼ 0.003; n¼ 38) were negatively associatedwith plasma homocysteine concentrations in the pre-eclampticgroup (Fig. 1). Similarly, erythrocyte DHAwas negatively associatedwith plasma homocysteine concentrations (r¼�0.505; p¼ 0.001;n¼ 38). However, such associations were not seen in the normo-tensive group.

4. Discussion

This study is novel and significant since: 1) The pre-eclampticand normotensive women shared common ethnicity, dietarypatterns and lifestyle in addition to being matched for age, socio-economic and educational status, which are critical to reduceconfounds on the key components of one-carbon metabolism; 2)The study simultaneously analyzed the key causal components,plasma folic acid, vitamin B12 and DHA and the consequentialcomponents, homocysteine to explain better the mechanisms ofaltered one-carbon metabolism in this unique cohort.

Our data indicate no change in plasma folic acid and vitamin B12

levels, in pre-eclamptic women as compared to normotensivewomen. Similar findings are reported by others in pre-eclampticwomen.14,19 In contrast, some studies have reported lower plasmafolic acid levels in pre-eclamptic women than in normotensive.15,25

The levels of folic acid in our cohort are similar to those reportedearlier.14 In contrast, the levels of vitamin B12 in our cohort are

Fig. 1. Association between plasma homocysteine and erythrocyte omega-3 fatty acidlevels in pre-eclamptic group.

A. Kulkarni et al. / Clinical Nutrition 30 (2011) 60e64 63

significantly lower than those reported.14 This is consistent withsuggestion that B12 levels in Indian population are generally lowdue to vegetarian diet.17

In our study, increased levels of homocysteine were seen in pre-eclamptic women as compared to normotensive women. Similarincrease has been reported by Makedos et al.14 In contrast, othersdid not find an increase in homocysteine concentrations in pre-eclamptic women.19 This has been attributed to racial differences inhomocysteine levels where reports suggest that the black womenwith pre-eclampsia have higher homocysteine concentrations thanwhite women with pre-eclampsia.25 The level of homocysteine inour study in normotensive women was slightly higher than thatreported by Makedos et al.,14 although the level in pre-eclampticwomen was comparable.

Our results suggest an increase in homocysteine levels with nochange in plasma folate and vitamin B12 levels in pre-eclampticwomen at the time of delivery. Our findings are in confirmationwith other reported studies wherein homocysteine levels aresignificantly elevated in pre-eclamptic patients with no vitamindeficiencies observed.14 We have previously reported that reducedantioxidants and increased oxidative stress lead to impairedessential polyunsaturated fatty acid levels like DHA which may bethe key factor involved in the development of pre-eclampsia.10,11 Inthis cohort also erythrocyte DHA levels were reduced confirmingour earlier findings. One of the mechanisms proposed for thedeleterious effects of homocysteine is its ability to generate reactiveoxygen species thereby producing oxidative stress. Homocysteinecan rapidly auto-oxidize in circulation to form homocysteine(oxidized disulfide form) and hydrogen peroxide (H2O2), therebygenerating oxidative stress.26 In our study, erythrocyte DHA levelsshowed strong negative association with homocysteine in pre-eclamptic women. Bohles et al. have reported a negative associa-tion between maternal plasma homocysteine levels and theerythrocyte phospholipid DHA concentrations of their offspring.27

A high homocysteine concentration decreases glutathione peroxi-dase activity and reduces tissue concentrations of glutathione sincehomocysteine is a precursor of glutathione.28 Glutathione helps toregenerate stores of other antioxidants like vitamin C and E andprotects both mother and foetus from the damaging effects of freeradicals and oxidative stress.29 This has implications in pre-eclamptic women where our earlier studies have shown that pre-eclamptic women have lower levels of vitamins E and C.10

Increased homocysteine levels can result from one or more keyconstituents of one-carbon cycle. The transfer of methyl group from5-MTHF to homocysteine requires vitamin B12 and results in thesynthesis ofmethionine.Methionine is converted into S-adenosyl-L-methionine (SAM). Methyl groups from SAM are transferred byphosphatidyl ethanolamine-N-methyltransferase (PEMT) to etha-nolamine in a series of steps that convert it to phosphatidylcholineand produce homocysteine.7 PEMTmay therefore play an importantrole in determining homocysteine concentrations. Membranephospholipids are major methyl group acceptors and reduced DHAlevels may result in diversion of methyl groups towards DNA ulti-mately resulting in DNAmethylation as we have recently describedin one-carbon metabolic pathway (Kale et al., 2009). DNA methyl-ation is critical for developmental changes in gene regulation, andchanges that take place during this critical period may result inaltered imprinting of genes which will be transferred to the nextgeneration. In viewof this, it would therefore be relevant to examinethe methylation patterns in pre-eclamptic women.

Our study provides evidence for the associations of maternalomega-3 fatty acids especially DHA and resultant homocysteineconcentrations. One limitation of our study is we have examinedthe levels of the micronutrients and homocysteine concentrationsat the end of pregnancy. It would be relevant to examine the

associations between omega-3 fatty acids and homocysteineconcentrations early in pregnancy and whether homocysteine risein early pregnancy can be used as a biomarker for pre-eclampsia.Future studies need to examine if docosahexaenoic acid supple-mentation during pregnancy reduces homocysteine levels andameliorates the risk of developing pre-eclampsia.

Conflict of InterestThere are no conflicts of interest to disclose.

Author agreement

All authors have made substantial contributions and finalapproval of the conceptions, drafting, and final version. All authorshave met the criteria for authorship as established by the Inter-national Committee of Medical Journals Editors, believe that thepaper represents honest work, and are able to verify the validity ofthe results reported.

Acknowledgement

The authors would like to thank all the subjects for volunteeringin this study and nurses of Bharati hospital who helped in collectingthe samples.

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