Combined Supplementation of Choline and Docosahexaenoic...

Research ArticleCombined Supplementation of Choline andDocosahexaenoic Acid during Pregnancy EnhancesNeurodevelopment of Fetal Hippocampus

Huban Thomas Rajarethnem1 Kumar Megur Ramakrishna Bhat1

Malsawmzuali Jc1 Siva Kumar Gopalkrishnan2

Ramesh Babu Mugundhu Gopalram3 and Kiranmai Sesappa Rai3

1Department of Anatomy Kasturba Medical College Manipal University Manipal India2Department of Physiology Kasturba Medical College Manipal University Manipal India3Department of Physiology Melaka-Manipal Medical College Manipal University Manipal India

Correspondence should be addressed to Kiranmai Sesappa Rai hodphysiogmailcom

Received 5 August 2016 Revised 6 November 2016 Accepted 4 December 2016 Published 22 January 2017

Academic Editor Changiz Geula

Copyright copy 2017 HubanThomas Rajarethnem et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Choline is an essential nutrient for humans which plays an important role in structural integrity and signaling functionsDocosahexaenoic acid (DHA) is a polyunsaturated fatty acid highly enriched in cell membranes of the brain Dietary intake ofcholine or DHA alone by pregnant mothers directly affects fetal brain development and function But no studies show the efficacyof combined supplementation of choline and DHA on fetal neurodevelopment The aim of the present study was to analyze fetalneurodevelopment on combined supplementation of pregnant dams with choline and DHA Pregnant dams were divided intofive groups normal control [NC] saline control [SC] choline [C] DHA and C + DHA Saline choline and DHA were givenas supplements to appropriate groups of dams NC dams were undisturbed during entire gestation On postnatal day (PND)40 brains were processed for Cresyl staining Pups from choline or DHA supplemented group showed significant (119901 lt 005)increase in number of neurons in hippocampus when compared to the same in NC and SC groups Moreover pups from C + DHAsupplemented group showed significantly higher number of neurons (119901 lt 0001) in hippocampus when compared to the same inNC and SC groups Thus combined supplementation of choline and DHA during normal pregnancy enhances fetal hippocampalneurodevelopment better than supplementation of choline or DHA alone

1 Introduction

Human brain development begins in the third gestationalweek and is a protracted process that depends on thedifferentiation of neural progenitor cells and continues untolate adolescence impacting brain functions for the entirelifespan [1] Hippocampus is a part of the brain which formsthe limbic system and is important for emotional learning[2] The role of the hippocampus in relational memory isthat it receives multiple inputs to create and allows forstorage of representations of the associations among theconstituent elements of scenes and events [3] This function

of hippocampus is important for the long-term memoryin cortical regions of the brain The hippocampus developsreciprocal connections with cortex through medial temporallobe The hippocampal formation consists of the dentategyrus cornu ammonis (CA) regions and the subiculumThe dentate gyrus receives afferent connections from theentorhinal cortex which act as an input region The cornuammonis regions of the hippocampus are comprised mainlyof pyramidal cells [4ndash6] The entorhinal cortex and the CA1region of the hippocampus establish reciprocal connectionsvia the subiculum [7] This reciprocal connection involvestwo pathways one between the dentate gyrus and CA3

HindawiNeurology Research InternationalVolume 2017 Article ID 8748706 9 pageshttpsdoiorg10115520178748706

2 Neurology Research International

region of the hippocampus and the other between the CA1region of the hippocampus and subiculum The subiculumintern sends an efferent input back to the entorhinal cortex[8] The hippocampus in humans develops during the lasttrimester and the volume of hippocampus is fully maturedat 15 months of age whereas in rats hippocampus developsduring embryonic [E] days E11ndashE17 [9]

Neurodevelopment of fetal brain is influenced by variousdietary nutrients including essential nutrients such as cholineand docosahexaenoic acid (DHA) Choline is the majorsource of methyl groups in the diet it is needed for the struc-tural integrity and signaling functions of cell membranesCholine concentration directly affects cholinergic neuro-transmission transmembrane signaling and lipid transportas well as metabolism [10] Dietary choline concentrationinfluences neural tube closure hippocampal developmentapoptotic signaling in neurons and in liver cells hepatictransport of lipoproteins and hepatic carcinogenesis [11]Moreover choline and DHA are related by the influenceof phosphatidylethanolamine-N-methyltransferase (PEMT)that catalyzes the biosynthesis of phosphatidylcholine [Ptd-Cho] from phosphatidylethanolamine which is enriched inlong-chain polyunsaturated fatty acids such as arachidonicacid and docosahexaenoic acid [12 13] Observations fromrecent studies indicate that DHA is necessary for neurode-velopment neurotransmission and protecting the brain fromoxidative stress These functions explain the important roleof DHA within the core of neural membranes [14] DHAmaintains the activity ofNa+K+ATPasewhich is the key cellmembrane enzyme that draws out energy from ATP to drivethe cellular sodium pump which is necessary to controls theelectrical impulses between the cells [15] Phosphatidylserine(PS) is an important component of the cell membrane and isvital for cell survival its concentration in brain is regulatedby DHA and thereby DHA influence the cognitive process[16] DHA deficiency causes reduction in brain PS whichaffects cell signaling for survival through enzymes such asNa+K+ ATPase and by calcium uptake Calcium is one ofthe multifaceted and most common among cell-signalingsystems and is regulated by DHA [17] DHA regulate avast array of cellular functions including function of themitochondria activation of gene neurotransmitter releasedeveloping brain and neuronmaturation andmigration [18]

Alternately various studies show that choline deficiencyduring fetal development reduces proliferation andmigrationof neuronal precursor cells in the mouse fetal hippocampusand these changes are associated with modifications inthe protein levels of some cell cycle regulators [19] Whenrodents are exposed to choline deficiency during their fetaldevelopment they showdecreased cell division and increasedapoptosis in their hippocampus [20ndash22] Choline-deficientdiets fed to dams increased the release of the followingmarkers and regulators of neural proliferation and differen-tiation namely TGF 1 a growth factor calretinin markerfor early neuronal differentiation p27Kip1 known for cyclin-dependent kinase inhibitor and TOAD-64 which is themarker for neuronal differentiation in the fetal brain [23ndash25] Various animal studies have clearly shown that DHAdeficiency during embryogenesis and subsequently during

Table 1 Rat pellet feed contents obtained from VRK NutritionalSolutions Pune having the following ingredients

Number Contents values1 Crude protein 21852 Crude fat 4853 Crude fibre 3154 Calcium 1105 Phosphorus 0516 Total ash 5807 Carbohydrates 6500

Table 2

Number Fatty acids Diet (g100 g)1 C 161 0812 C 180 0173 C 181 0674 C 182 2035 C 183 0166 C 201 0127 Total 396

lactation could not be fully corrected later in life [26] DHAstatus during birth has long-term impact andDHAdeficiencywas found to affect the behaviors in children until 7 yearsof age [27] The present study was designed to analyze theefficacy of either combined or individual supplementation ofcholine and DHA during gestation on fetal hippocampal anddentate gyrus neurodevelopment in rats

2 Materials and Methods

The present study was carried out in accordance with guide-lines laid by Manipal University Institutional Animal EthicsCommittee (IAECKMC3212) Manipal India Laboratoryrats of Wistar strain weighing approximately 200 g werehoused in Central Animal Research Facility Manipal Uni-versity Manipal Rats were housed in polypropylene cagescontaining sterile paddy husk as bedding and maintainedunder standard controlled lab conditions with temperaturesranging 23 plusmn 2∘C humidity (50 plusmn 5) and 12 h light-darkcycle All animals were fed with rat pellet feed obtainedfrom (with choline content 1mgkg feed) VRK NutritionalSolutions VRKrsquos ldquoScientistrsquos Choicerdquo Laboratory AnimalDiets Pune ndash 411037 (Table 1) and allowed free access towater ad libitum Female rats in estrus cycle were identifiedand allowed for mating with male rats at 2 1 ratio The firstday of gestation was determined by the vaginal smear testOnce pregnancy was confirmed pregnant female rats wereisolated and provided with nesting material and allowed tolitter naturally

Rat pellet feed fatty acid composition was analyzed byNational Institute of Nutrition Indian Council of MedicalResearch Hyderabad India (Table 2)

Neurology Research International 3

60

40

20

0

NC SC C DHA C + DHA

Mea

n nu

mbe

r of C

A1

neur

al ce

lls

Groups (n = 6)

ab

lowast

lowastlowastlowast

Figure 1 Comparison of numbers of neural cells in CA1 region in 5 120583 thick hippocampal sections Mean number of neural cells plusmnStandard Error from 250 120583m area of CA1 region of hippocampus from either side in normal control (NC) saline control (SC) choline (C)docosahexaenoic acid (DHA) and choline + docosahexaenoic acid (C + DHA) age matched group of rats One-way ANOVA followed byTukeyrsquos post hoc test ldquolowastrdquo NC versus supplemented groups ldquoardquo C supplemented group versus C + DHA group and ldquobrdquo DHA supplementedgroup versus C + DHA group lowastab119901 lt 005 amp lowastlowastlowast119901 lt 0001

3 Study Design

Pregnant dams confirmed by vaginal smear test were dividedinto five groups (1) normal control [NC] (2) saline control[SC] (3) choline [C] (4) docosahexaenoic acid [DHA]and (5) choline and DHA [C + DHA] groups Pregnantdams from NC group were undisturbed in their home cageand were provided with normal animal feed and waterad libitum Pregnant dams from saline control group weresupplemented with saline throughout the period of gestation[from embryonic day 0 (E0) to delivery] choline group weresupplemented with choline chloride throughout the periodof gestation [choline chloride 98 Extra Pure obtained fromLoba Chemie Laboratory Reagents and Fine Chemicals]dissolved with distilled water (46mmolkgday of choline)[28] DHA group were supplemented with DHA throughoutthe period of gestation [soft gelatin capsules containing300mg docosahexaenoic acidcapsule were obtained fromNouveau Medicament (P) Ltd Chennai] as such from thecapsules (400mgday of DHA) [29] and choline + DHAgroup were supplemented with both choline and DHAthroughout the period of gestation Supplementation of thecomponents stated previously was given to the rats by feedingneedles Following gestation dams were allowed to delivernaturally and pups from each group of damsweremaintainedundisturbed in their home cages for a period of 40 days

Pups on the 40th day were anaesthetized by ether andthe brain was perfused transcardially with saline followed by10 formalin Brain was excised and processed for paraffinsectioning 300 sections of 5120583 thickness of the entire hip-pocampus were obtained One section30 hippocampal sec-tions was serially selected and a total of 10 sections from eachrat were stained with Cresyl Violet staining method Sectionswere observed under light microscope at 40x magnificationNeural cell count was carried out by another experimenter

blind to the study after coding the slides 250 120583m area wasrandomly selected in CA1 CA3 and CA4 subregions as wellas upper blade of dentate gyrus from each of the selected5 120583 thick hippocampal section and unbiased quantificationwas done manually using ocular micrometer scale Datawere analyzed using one-way ANOVA followed by Tukeyrsquosmultiple comparison tests and expressed asmeanplusmn SEMwiththe significance level at 119901 lt 005 using statistical softwareGraph pad prism version 503

4 Results

PND 40 rats supplemented prenatally with either cholineor DHA alone show significant (lowast119901 lt 005) increase inthe number of neural cells when compared with the samein age matched normal control and saline control group ofrats [Figures 1 and 2] However PND 40 rats supplementedprenatally with choline + DHA showed significantly (lowastlowastlowast119901 lt0001) higher number of neural cells when comparedwith thesame in agematched normal control and saline control groupof rats [Figures 1 and 2] Additionally significantly highernumber [ab119901 lt 005] of hippocampal neural cells in CA1subregion was observed when compared with the same inage matched rat groups supplemented with either choline orDHA alone [Figures 1 and 2]

PND 40 rats supplemented prenatally with either cholineor DHA alone shows significantly (lowast119901 lt 005) increasednumber of neural cells in the CA3 region of hippocampuswhen compared to the same in age matched normal controland saline control group of rats However PND 40 ratssupplemented prenatally with both choline + DHA showedsignificantly (lowastlowastlowast119901 lt 0001) higher number of neural cells inCA3 region when compared with the same in age matchednormal control and saline control group of rats Additionally


NC SC C

DHA C + DHA

Figure 2 CA1 region representative photomicrographs of CA1 region from 5 120583 thick hippocampal sections viewed by 40x magnificationshowing Cresyl Violet stained neurons from all experimental groups of PND 40 rats Note rats supplemented with combined choline andDHA show significant increase in the number of neural cells in CA1 region

significantly higher number [ab119901 lt 005] of hippocampalneural cells were observed in CA3 subregion when comparedwith the same in agematched rat groups supplemented eithercholine or DHA alone [Figures 3 and 4]

PND 40 rats supplemented prenatally with either cholineor DHA alone shows significant (lowast119901 lt 005) increasenumber of neural cells in the CA4 region of hippocampuswhen compared with the same in age matched normalcontrol and saline control group of rats However PND40 rats supplemented prenatally with combined choline +DHA showed significantly (lowastlowastlowast119901 lt 0001) higher numberof neural cells in the CA4 region of hippocampus whencompared with the same in age matched normal control andsaline control group of rats Additionally significantly highernumber [ab119901 lt 005] of hippocampal neural cells wereobserved in CA4 subregion when compared with the samein age matched rat groups supplemented with either cholineor DHA alone [Figures 5 and 6]

PND 40 rats supplemented prenatally with either cholineor DHA alone show significant (lowast119901 lt 005) increase in thenumber of DG neural cells when compared to the same inage matched normal control and saline control group of ratsHowever PND 40 rats supplemented prenatally with com-bined choline + DHA showed significantly (lowastlowastlowast119901 lt 0001)higher number of DG neural cells when compared with thesame in agematched normal control and saline control groupof rats Additionally significantly higher number [ab119901 lt005] of DG neural cells were observed when compared to

the same in agematched rat groups supplemented with eithercholine or DHA alone [Figures 7 and 8]

5 Discussion

The results of the present study showed that PND 40 ratssupplemented prenatally with choline alone showed signifi-cant increase in the number of hippocampal neural cells inthe following subregions CA1 CA3 CA4 and DG whencompared with the same in age matched normal controland saline control group of rats This result is consistentwith previous studies that show rodents receiving cholinesupplements resulting in alterations in hippocampus [20 22]Dietary choline content during brain development influencesthe neurodevelopment of hippocampus and thus cholinesupplementation during pregnancy elicits a major improve-ment in memory performance of the offspring [30] In thepresent study choline was supplemented during the entiregestational period A study conducted by Craciunescu et alin 2003 shows that supplementing choline during embryonicdays 12ndash17 enhances mitosis of the progenitor cell in devel-oping hippocampus [22] Choline is an essential nutrientand is a precursor for many important compounds such asacetylcholine phospholipids and the methyl donor betainePerinatal supplementation of choline in rodents enhancesspatial and temporal cognition which continues across thelifespan [31] Thus there may be high demand for cholineduring prenatal and neonatal periods which stimulates rapid


NC0

50

100

150

SC C DHA C + DHAGroups (n = 6)

a

blowast

lowastlowastlowast

Mea

n nu

mbe

r of C

A3

neur

al ce

lls

Figure 3 Comparison of neural cells in CA3 region of 5 120583 thick hippocampal sections Mean number of neural cells plusmn Standard Error from250 120583m area of CA3 region of hippocampus from either side in normal control (NC) saline control (SC) choline (C) docosahexaenoic acid(DHA) and choline + docosahexaenoic acid (C + DHA) age matched group of rats One-way ANOVA followed by Tukeyrsquos post hoc test ldquolowastrdquoNC versus supplemented groups ldquoardquo C supplemented group versus C + DHA group and ldquobrdquo DHA supplemented group versus C + DHAgroup lowastab119901 lt 005 amp lowastlowastlowast119901 lt 0001

NC SC C

DHA C + DHA


neurogenesis and synaptogenesis [32 33] Various studiesindicate that choline deficiency during pregnancy affectscognitive function in offspring as prenatal choline availabilityenhances the attention and spatial memory of the offspring[34] The need for choline is increased during lactation as itis found that human milk has 15 to 2mM choline moiety perliter [35] Plasma choline concentrations in humans and othermammals are much higher at birth than those in adults [36]

Additionally in the present study PND 40 group of ratsprenatally supplemented with DHA also shows a significantincrease in the number of hippocampal neural cells inthe following subregions CA1 CA3 CA4 and DG whencompared to the same in age matched normal control andsaline control group of rats Studies show that DHA avail-ability significantly alters neurodevelopment in hippocampusand synaptic function Furthermore DHA deficiency during


Mea

n nu

mbe

r of C

A4

neu

ral c

ells

60

40

20

0

NC SC C DHA C + DHAGroups (n = 6)

ab

lowast

lowastlowastlowast


NC SC C

DHA C + DHA


development also resulted in marked decreases of synapsinsand NMDA receptor subunit NR2A particularly in CA3region of hippocampus with concomitant impairment oflong-term potentiation [37] ReducedDHA is interconnectedwith impairments in behavioral and cognitive function [38]

DHA uptake by the brain depends upon serum lev-els of DHA and its precursors which depend upon bothdietary intake and liver biosynthesis Dietary content ofDHA markedly influences brain DHA content [39] DHAdeficiencies during gestation alter the neurodevelopment



Mea

n nu

mbe

r of D

G n

eura

l cel

ls

ab

lowast

lowastlowastlowast

50

100

150

0

Figure 7 Comparison of number of neural cells in DG region of 5 120583 thick hippocampal sections Mean number of neural cells plusmnStandard Error from 250 120583m area of DG region of hippocampus from either side in normal control (NC) saline control (SC) choline (C)docosahexaenoic acid (DHA) and choline + docosahexaenoic acid (C + DHA) age matched group of rats One-way ANOVA followed byTukeyrsquos post hoc test ldquolowastrdquo NC versus supplemented groups ldquoardquo C supplemented group versus C + DHA group and ldquobrdquo DHA supplementedgroup versus C + DHA group lowastab119901 lt 005 amp lowastlowastlowast119901 lt 0001

NC SC C

DHA C + DHA

Figure 8 DG region representative photomicrographs of DG region from 5 120583 thick hippocampal sections viewed by 40x magnificationshowing Cresyl Violet stained neurons from all experimental groups of PND 40 rats Note rats supplemented with combined choline andDHA show significant increase in the number of neural cells in DG region

and can lead to later disease and brain malfunction [40]DHA deficiency during fetal development leads to decreasedDHA levels in neural tissue and is associated with deficitsin psychomotor development [41] reading skills [42] visualacuity [43] problem solving [44] and attention [45]

Importantly results of the present study also show thatPND 40 rats supplemented prenatally with both choline +DHA showed significantly higher number of hippocampalneural cells in all subregions that were assessed and analyzed[CA1 CA3 CA4 and DG] when compared to the same in


age matched normal control and saline control group of ratsMoreover importantly PND40 rats supplemented prenatallywith both choline + DHA persistently showed significantlyhigher number of hippocampal neural cells in all subregions[CA1 CA3 CA4 and DG] when compared to the samein age matched rat groups given either choline or DHAalone Thus it is evident that combined supplementationof both choline and DHA is more efficacious in enhancinghippocampal neurodevelopment than supplementing thesenutrients separately

DHA metabolism and choline metabolism are linked bythe enzyme phosphatidylethanolamine-N-methyltransferase(PEMT) [12] which catalyzes de novo biosynthesis of phos-phatidylcholine (PtdCho) by methylation of PtdEtn enrichedwith DHA [46] This enzyme prefers species of PtdEtn thatcontain long-chain polyunsaturated fatty acids such as DHA[47] thereby forming DHA-enriched species of PtdCho inmembranes especially in the brain Available PtdCho andPtdEtn are also used to form phosphatidylserine (PtdSer)by serine base exchange enzymes [48] Thus availability ofboth choline and DHA during neurodevelopment allowsfor increased laying down of membranes a prerequisitefor increased neural cell formation in all subregions ofhippocampus and dentate gyrus

6 Conclusion

The present study demonstrates the combined efficiency ofsupplementation with choline and DHA in diet on hip-pocampal neurodevelopment The study also emphasizes therequirements of both choline and DHA for better enhance-ment of fetal brain developmental rather than individualsupplementation of any one of the above stated dietarynutrients

Competing Interests

There is no conflict of interests among all coauthors regardingdoing this study or publishing it

Acknowledgments

The authors are thankful to Kasturba Medical College andManipal University for supporting and providing infrastruc-ture for the research study

References

[1] J Stiles and T L Jernigan ldquoThe basics of brain developmentrdquoNeuropsychology Review vol 20 no 4 pp 327ndash348 2010

[2] C Raineki P J Holman J Debiec M Bugg A Beasley andR M Sullivan ldquoFunctional emergence of the hippocampus incontext fear learning in infant ratsrdquoHippocampus vol 20 no 9pp 1037ndash1046 2010

[3] N J Cohen J Ryan C Hunt L Romine T Wszalek andC Nash ldquoHippocampal system and declarative (Relational)memory summarizing the data from functional neuroimagingstudiesrdquo Hippocampus vol 9 no 1 pp 83ndash98 1999

[4] D G Amaral ldquoEmerging principles of intrinsic hippocampalorganizationrdquoCurrent Opinion in Neurobiology vol 3 no 2 pp225ndash229 1993

[5] R Insausti D G Amaral and W M Cowan ldquoThe entorhinalcortex of the monkey II Cortical afferentsrdquo Journal of Compar-ative Neurology vol 264 no 3 pp 356ndash395 1987

[6] W L Suzuki and D G Amaral ldquoPerirhinal and parahippocam-pal cortices of the macaque monkey cortical afferentsrdquo Journalof Comparative Neurology vol 350 no 4 pp 497ndash533 1994

[7] M P Witter and D G Amaral ldquoEntorhinal cortex of themonkey V Projections to the dentate gyrus hippocampus andsubicular complexrdquo Journal of Comparative Neurology vol 307no 3 pp 437ndash459 1991

[8] F M Benes ldquoMyelination of cortical-hippocampal relays dur-ing late adolescencerdquo Schizophrenia Bulletin vol 15 no 4 pp585ndash593 1989

[9] D Rice and S Barone Jr ldquoCritical periods of vulnerability forthe developing nervous system evidence from humans andanimal modelsrdquo Environmental Health Perspectives vol 108supplement 3 pp 511ndash533 2000

[10] S H Zeisel and J K Blusztajn ldquoCholine and human nutritionrdquoAnnual Review of Nutrition vol 14 pp 269ndash296 1994

[11] S H Zeisel K A Da Costa P D Franklin et al ldquoCholine anessential nutrient for humansrdquoThe FASEB Journal vol 5 no 7pp 2093ndash2098 1991

[12] K-A da Costa K S Rai C N Craciunescu et al ldquoDietarydocosahexaenoic acid supplementation modulates hippocam-pal development in the Pemtminusminus mouserdquo Journal of BiologicalChemistry vol 285 no 2 pp 1008ndash1015 2010

[13] C J DeLong Y-J Shen M J Thomas and Z Cui ldquoMoleculardistinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylationpathwayrdquo Journal of Biological Chemistry vol 274 no 42 pp29683ndash29688 1999

[14] S M Innis ldquoDietary (n-3) fatty acids and brain developmentrdquoJournal of Nutrition vol 137 no 4 pp 855ndash859 2007

[15] N Turner P L Else and A J Hulbert ldquoDocosahexaenoicacid (DHA) content of membranes determinesmolecular activ-ity of the sodium pump implications for disease states andmetabolismrdquo Naturwissenschaften vol 90 no 11 pp 521ndash5232003

[16] N Salem Jr B Litman H-Y Kim and K Gawrisch ldquoMecha-nisms of action of docosahexaenoic acid in the nervous systemrdquoLipids vol 36 no 9 pp 945ndash959 2001

[17] S Chalon S Delion-Vancassel C Belzung et al ldquoDietary fishoil affects monoaminergic neurotransmission and behavior inratsrdquo Journal of Nutrition vol 128 no 12 pp 2512ndash2519 1998

[18] M Sergeeva M Strokin and G Reiser ldquoRegulation of intracel-lular calcium levels by polyunsaturated fatty acids arachidonicacid and docosahexaenoic acid in astrocytes possible involve-ment of phospholipase A2rdquo Reproduction Nutrition Develop-ment vol 45 no 5 pp 633ndash646 2005

[19] M D Niculescu C N Craciunescu and S H Zeisel ldquoDietarycholine deficiency alters global and gene-specific DNA methy-lation in the developing hippocampus of mouse fetal brainsrdquoThe FASEB Journal vol 20 no 1 pp 43ndash49 2006

[20] C D Albright A Y Tsai C B Friedrich M-H Mar and SH Zeisel ldquoCholine availability alters embryonic development ofthe hippocampus and septum in the ratrdquo Developmental BrainResearch vol 113 no 1-2 pp 13ndash20 1999


[21] C D Albright M-H Mar C B Friedrich E C Brown and SH Zeisel ldquoMaternal choline availability alters the localizationof p15Ink4B and p27Kip1 cyclin-dependent kinase inhibitorsin the developing fetal rat brain hippocampusrdquo DevelopmentalNeuroscience vol 23 no 2 pp 100ndash106 2001

[22] C N Craciunescu C D Albright M-H Mar J Song and SH Zeisel ldquoCholine availability during embryonic developmentalters progenitor cell mitosis in developing mouse hippocam-pusrdquo Journal of Nutrition vol 133 no 11 pp 3614ndash3618 2003

[23] C D Albright A Y Tsai M-H Mar and S H ZeiselldquoCholine availability modulates the expression of TGF1205731 andcytoskeletal proteins in the hippocampus of developing ratbrainrdquoNeurochemical Research vol 23 no 5 pp 751ndash758 1998

[24] C D Albright C B Friedrich E C Brown M-H Mar and SH Zeisel ldquoMaternal dietary choline availability alters mitosisapoptosis and the localization of TOAD-64 protein in thedeveloping fetal rat septumrdquoDevelopmental Brain Research vol115 no 2 pp 123ndash129 1999

[25] C D Albright D F Siwek C N Craciunescu et al ldquoCholineavailability during embryonic development alters the localiza-tion of calretinin in developing and agingmouse hippocampusrdquoNutritional Neuroscience vol 6 no 2 pp 129ndash134 2003

[26] D Li H S Weisinger R S Weisinger et al ldquoOmega 6 toomega 3 fatty acid imbalance early in life leads to persis-tent reductions in DHA levels in glycerophospholipids in rathypothalamus even after long-term omega 3 fatty acid reple-tionrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol74 no 6 pp 391ndash399 2006

[27] L Krabbendam E Bakker G Hornstra and J van OsldquoRelationship between DHA status at birth and child problembehaviour at 7 years of agerdquo Prostaglandins Leukotrienes andEssential Fatty Acids vol 76 no 1 pp 29ndash34 2007

[28] J D Thomas J S Biane K A OrsquoBryan T M OrsquoNeill andH D Dominguez ldquoCholine supplementation following third-trimester-equivalent alcohol exposure attenuates behavioralalterations in ratsrdquo Behavioral Neuroscience vol 121 no 1 pp120ndash130 2007

[29] T Sakamoto M Cansev and R J Wurtman ldquoOral sup-plementation with docosahexaenoic acid and uridine-51015840-monophosphate increases dendritic spine density in adult gerbilhippocampusrdquo Brain Research vol 1182 no 1 pp 50ndash59 2007

[30] S H Zeisel ldquoCholine needed for normal development ofmemoryrdquo Journal of the American College of Nutrition vol 19S5 pp 528Sndash531S 2000

[31] J A Lamoureux W H Meck and C L Williams ldquoPrenatalcholine availability alters the context sensitivity of Pavlovianconditioning in adult ratsrdquo Learning amp Memory vol 15 no 12pp 866ndash875 2008

[32] R A Waterland and K B Michels ldquoEpigenetic epidemiologyof the developmental origins hypothesisrdquo Annual Review ofNutrition vol 27 pp 363ndash388 2007

[33] T M Nafee W E Farrell W D Carroll A A Fryer and KM K Ismail ldquoReview article epigenetic control of fetal geneexpressionrdquo BJOG vol 115 no 2 pp 158ndash168 2008

[34] C Signore P M Ueland J Troendle and J L Mills ldquoCholineconcentrations in human maternal and cord blood and intelli-gence at 5 y of agerdquoThe American Journal of Clinical Nutritionvol 87 no 4 pp 896ndash902 2008

[35] MQHolmes-McNaryW-L ChengM-HMar S Fussell andS H Zeisel ldquoCholine and choline esters in human and rat milkand in infant formulasrdquo American Journal of Clinical Nutritionvol 64 no 4 pp 572ndash576 1996

[36] S H Zeisel M F Epstein and R JWurtman ldquoElevated cholineconcentration in neonatal plasmardquo Life Sciences vol 26 no 21pp 1827ndash1831 1980

[37] D Cao K Kevala J Kim et al ldquoDocosahexaenoic acidpromotes hippocampal neuronal development and synapticfunctionrdquo Journal of Neurochemistry vol 111 no 2 pp 510ndash5212009

[38] S M Innis ldquoDietary (n-3) fatty acids and brain developmentrdquoThe Journal of Nutrition vol 137 no 4 pp 855ndash859 2007

[39] G-Y Diau A T Hsieh E A Sarkadi-Nagy VWijendran P WNathanielsz and J T Brenna ldquoThe influence of long chainpolyunsaturate supplementation on docosahexaenoic acid andarachidonic acid in baboon neonate central nervous systemrdquoBMCMedicine vol 3 article no 11 2005

[40] R L Blaylock ldquoDHA supports brain development and protectsneurological functionrdquo Life Extension Magazine pp 44ndash552008

[41] C L Jensen R G Voigt T C Prager et al ldquoEffects ofmaternal docosahexaenoic acid intake on visual function andneurodevelopment in breastfed term infantsrdquoAmerican Journalof Clinical Nutrition vol 82 no 1 pp 125ndash132 2005

[42] A J Richardson J R Burton R P Sewell T F Spreckelsenand P Montgomery ldquoDocosahexaenoic acid for reading cog-nition and behavior in children aged 7-9 years a randomizedcontrolled trial (the DOLAB study)rdquo PLoS ONE vol 7 no 9Article ID e43909 2012

[43] E E Birch D R Hoffman R Uauy D G Birch and CPrestidge ldquoVisual acuity and the essentiality of docosahex-aenoic acid and arachidonic acid in the diet of term infantsrdquoPediatric Research vol 44 no 2 pp 201ndash209 1998

[44] P Willatts J S Forsyth M K Dimodugno S Varma andM Colvin ldquoEffect of long-chain polyunsaturated fatty acids ininfant formula on problem solving at 10 months of agerdquo TheLancet vol 352 no 9129 pp 688ndash691 1998

[45] J Colombo K N Kannass D J Shaddy et al ldquoMaternal DHAand the development of attention in infancy and toddlerhoodrdquoChild Development vol 75 no 4 pp 1254ndash1267 2004

[46] N D Ridgway and D E Vance ldquo[43] Phosphatidyleth-anolamine N-methyltransferase from rat liverrdquo Methods inEnzymology vol 209 pp 366ndash374 1992

[47] C J DeLong Y-J Shen M J Thomas and Z Cui ldquoMoleculardistinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylationpathwayrdquo The Journal of Biological Chemistry vol 274 no 42pp 29683ndash29688 1999

[48] J E Vance ldquoThematic Review Series Glycerolipids Phos-phatidylserine and phosphatidylethanolamine in mammaliancells twometabolically related aminophospholipidsrdquo Journal ofLipid Research vol 49 no 7 pp 1377ndash1387 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


region of the hippocampus and the other between the CA1region of the hippocampus and subiculum The subiculumintern sends an efferent input back to the entorhinal cortex[8] The hippocampus in humans develops during the lasttrimester and the volume of hippocampus is fully maturedat 15 months of age whereas in rats hippocampus developsduring embryonic [E] days E11ndashE17 [9]

Neurodevelopment of fetal brain is influenced by variousdietary nutrients including essential nutrients such as cholineand docosahexaenoic acid (DHA) Choline is the majorsource of methyl groups in the diet it is needed for the struc-tural integrity and signaling functions of cell membranesCholine concentration directly affects cholinergic neuro-transmission transmembrane signaling and lipid transportas well as metabolism [10] Dietary choline concentrationinfluences neural tube closure hippocampal developmentapoptotic signaling in neurons and in liver cells hepatictransport of lipoproteins and hepatic carcinogenesis [11]Moreover choline and DHA are related by the influenceof phosphatidylethanolamine-N-methyltransferase (PEMT)that catalyzes the biosynthesis of phosphatidylcholine [Ptd-Cho] from phosphatidylethanolamine which is enriched inlong-chain polyunsaturated fatty acids such as arachidonicacid and docosahexaenoic acid [12 13] Observations fromrecent studies indicate that DHA is necessary for neurode-velopment neurotransmission and protecting the brain fromoxidative stress These functions explain the important roleof DHA within the core of neural membranes [14] DHAmaintains the activity ofNa+K+ATPasewhich is the key cellmembrane enzyme that draws out energy from ATP to drivethe cellular sodium pump which is necessary to controls theelectrical impulses between the cells [15] Phosphatidylserine(PS) is an important component of the cell membrane and isvital for cell survival its concentration in brain is regulatedby DHA and thereby DHA influence the cognitive process[16] DHA deficiency causes reduction in brain PS whichaffects cell signaling for survival through enzymes such asNa+K+ ATPase and by calcium uptake Calcium is one ofthe multifaceted and most common among cell-signalingsystems and is regulated by DHA [17] DHA regulate avast array of cellular functions including function of themitochondria activation of gene neurotransmitter releasedeveloping brain and neuronmaturation andmigration [18]

Alternately various studies show that choline deficiencyduring fetal development reduces proliferation andmigrationof neuronal precursor cells in the mouse fetal hippocampusand these changes are associated with modifications inthe protein levels of some cell cycle regulators [19] Whenrodents are exposed to choline deficiency during their fetaldevelopment they showdecreased cell division and increasedapoptosis in their hippocampus [20ndash22] Choline-deficientdiets fed to dams increased the release of the followingmarkers and regulators of neural proliferation and differen-tiation namely TGF 1 a growth factor calretinin markerfor early neuronal differentiation p27Kip1 known for cyclin-dependent kinase inhibitor and TOAD-64 which is themarker for neuronal differentiation in the fetal brain [23ndash25] Various animal studies have clearly shown that DHAdeficiency during embryogenesis and subsequently during

Table 1 Rat pellet feed contents obtained from VRK NutritionalSolutions Pune having the following ingredients

Number Contents values1 Crude protein 21852 Crude fat 4853 Crude fibre 3154 Calcium 1105 Phosphorus 0516 Total ash 5807 Carbohydrates 6500

Table 2

Number Fatty acids Diet (g100 g)1 C 161 0812 C 180 0173 C 181 0674 C 182 2035 C 183 0166 C 201 0127 Total 396

lactation could not be fully corrected later in life [26] DHAstatus during birth has long-term impact andDHAdeficiencywas found to affect the behaviors in children until 7 yearsof age [27] The present study was designed to analyze theefficacy of either combined or individual supplementation ofcholine and DHA during gestation on fetal hippocampal anddentate gyrus neurodevelopment in rats

2 Materials and Methods

The present study was carried out in accordance with guide-lines laid by Manipal University Institutional Animal EthicsCommittee (IAECKMC3212) Manipal India Laboratoryrats of Wistar strain weighing approximately 200 g werehoused in Central Animal Research Facility Manipal Uni-versity Manipal Rats were housed in polypropylene cagescontaining sterile paddy husk as bedding and maintainedunder standard controlled lab conditions with temperaturesranging 23 plusmn 2∘C humidity (50 plusmn 5) and 12 h light-darkcycle All animals were fed with rat pellet feed obtainedfrom (with choline content 1mgkg feed) VRK NutritionalSolutions VRKrsquos ldquoScientistrsquos Choicerdquo Laboratory AnimalDiets Pune ndash 411037 (Table 1) and allowed free access towater ad libitum Female rats in estrus cycle were identifiedand allowed for mating with male rats at 2 1 ratio The firstday of gestation was determined by the vaginal smear testOnce pregnancy was confirmed pregnant female rats wereisolated and provided with nesting material and allowed tolitter naturally

Rat pellet feed fatty acid composition was analyzed byNational Institute of Nutrition Indian Council of MedicalResearch Hyderabad India (Table 2)


60

40

20

0

NC SC C DHA C + DHA

Mea

n nu

mbe

r of C

A1

neur

al ce

lls

Groups (n = 6)

ab

lowast

lowastlowastlowast


3 Study Design




4 Results




NC SC C

DHA C + DHA






5 Discussion



NC0

50

100

150


a

blowast

lowastlowastlowast

Mea

n nu

mbe

r of C

A3

neur

al ce

lls


NC SC C

DHA C + DHA





Mea

n nu

mbe

r of C

A4

neu

ral c

ells

60

40

20

0


ab

lowast

lowastlowastlowast


NC SC C

DHA C + DHA






Mea

n nu

mbe

r of D

G n

eura

l cel

ls

ab

lowast

lowastlowastlowast

50

100

150

0


NC SC C

DHA C + DHA







6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















60

40

20

0

NC SC C DHA C + DHA

Mea

n nu

mbe

r of C

A1

neur

al ce

lls

Groups (n = 6)

ab

lowast

lowastlowastlowast


3 Study Design




4 Results




NC SC C

DHA C + DHA






5 Discussion



NC0

50

100

150


a

blowast

lowastlowastlowast

Mea

n nu

mbe

r of C

A3

neur

al ce

lls


NC SC C

DHA C + DHA





Mea

n nu

mbe

r of C

A4

neu

ral c

ells

60

40

20

0


ab

lowast

lowastlowastlowast


NC SC C

DHA C + DHA






Mea

n nu

mbe

r of D

G n

eura

l cel

ls

ab

lowast

lowastlowastlowast

50

100

150

0


NC SC C

DHA C + DHA







6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC SC C

DHA C + DHA






5 Discussion



NC0

50

100

150


a

blowast

lowastlowastlowast

Mea

n nu

mbe

r of C

A3

neur

al ce

lls


NC SC C

DHA C + DHA





Mea

n nu

mbe

r of C

A4

neu

ral c

ells

60

40

20

0


ab

lowast

lowastlowastlowast


NC SC C

DHA C + DHA






Mea

n nu

mbe

r of D

G n

eura

l cel

ls

ab

lowast

lowastlowastlowast

50

100

150

0


NC SC C

DHA C + DHA







6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC0

50

100

150


a

blowast

lowastlowastlowast

Mea

n nu

mbe

r of C

A3

neur

al ce

lls


NC SC C

DHA C + DHA





Mea

n nu

mbe

r of C

A4

neu

ral c

ells

60

40

20

0


ab

lowast

lowastlowastlowast


NC SC C

DHA C + DHA






Mea

n nu

mbe

r of D

G n

eura

l cel

ls

ab

lowast

lowastlowastlowast

50

100

150

0


NC SC C

DHA C + DHA







6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mea

n nu

mbe

r of C

A4

neu

ral c

ells

60

40

20

0


ab

lowast

lowastlowastlowast


NC SC C

DHA C + DHA






Mea

n nu

mbe

r of D

G n

eura

l cel

ls

ab

lowast

lowastlowastlowast

50

100

150

0


NC SC C

DHA C + DHA







6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Mea

n nu

mbe

r of D

G n

eura

l cel

ls

ab

lowast

lowastlowastlowast

50

100

150

0


NC SC C

DHA C + DHA







6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















6 Conclusion


Competing Interests


Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Combined Supplementation of Choline and Docosahexaenoic...

Documents

Transcript of Combined Supplementation of Choline and Docosahexaenoic...