ª 2014 by the Academy of Nutrition and Dietetics.

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Research and Professional Briefs

Egg n-3 Fatty Acid Composition ModulatesBiomarkers of Choline Metabolism in Free-LivingLacto-Ovo-Vegetarian Women of Reproductive AgeAllyson A. West, PhD; Yun Shih, MS, RD; Wei Wang, PhD; Keiji Oda, MS; Karen Jaceldo-Siegl, DrPH; Joan Sabaté, MD, DrPH;Ella Haddad, DrPH; Sujatha Rajaram, PhD; Marie A. Caudill, PhD, RD; Bonny Burns-Whitmore, DrPH, RD

ARTICLE INFORMATION

Article history:Accepted 5 December 2013

Keywords:CholineEggPhosphatidylethanolamineN-methyltransferase (PEMT)Docosahexaenoic acid (DHA)Trimethylamine oxide (TMAO)

Copyright ª 2014 by the Academy of Nutritionand Dietetics.2212-2672/$36.00http://dx.doi.org/10.1016/j.jand.2014.02.012

ABSTRACTThe lacto-ovo-vegetarian (LOV) dietary regimen allows eggs, which are a rich sourceof choline. Consumption of eggs by LOV women may be especially important duringpregnancy and lactation when demand for choline is high. The aim of this singleblind, randomized, crossover-feeding study was to determine how near-daily eggconsumption influenced biomarkers of choline metabolism in healthy LOV womenof reproductive age (n¼15). Because long-chain n-3 fatty acids could influencecholine metabolism, the effect of n-3eenriched vs nonenriched eggs on cholinemetabolites was also investigated. Three 8-week dietary treatments consisting of sixn-3eenriched eggs per week, six nonenriched eggs per week, and an egg-free controlphase were separated by 4-week washout periods. Choline metabolites were quantifiedin fasted plasma collected before and after each treatment and differences in post-treatment choline metabolite concentrations were determined with linear mixedmodels. The n-3eenriched and nonenriched egg treatments produced different cholinemetabolite profiles compared with the egg-free control; however, response to the eggsdid not differ (P>0.1). Consumption of the n-3eenriched egg treatment yielded higherplasma free choline (P¼0.02) and betaine (P<0.01) (vs egg-free control) concentrations,whereas consumption of the nonenriched egg treatment yielded borderline higher(P¼0.06) plasma phosphatidylcholine (vs egg-free control) levels. Neither egg treatmentincreased levels of plasma trimethylamine oxide, a gut-floraedependent oxidativecholine metabolite implicated as a possible risk factor for cardiovascular disease. Overallthese data suggest that egg fatty-acid composition modulates the metabolic use ofcholine.J Acad Nutr Diet. 2014;-:---.

CHOLINE IS A QUATERNARY AMINE MOLECULE USEDto produce the ubiquitous phospholipid phosphati-dylcholine (PC) via the cytidine diphosphate (CDP)-choline biosynthetic pathway (Figure). Choline may

also be acetylated to form the neurotransmitter acetylcho-line or oxidized to the methyl donor betaine. Dimethylgly-cine is produced when betaine donates a methyl group tomethionine (Figure). Sphingomyelin, a choline metabolite,is derived from PC and is a component of lipid membranesand lipoproteins.1 PC synthesized via the phosphatidyletha-nolamine N-methyltransferase (PEMT) pathway providesa de novo source of choline (see the Figure); however,exogenous dietary choline is required to meet somaticrequirements.2

The Adequate Intake level for premenopausal women is425 mg/day,2 although it is estimated that <10% of Americanwomen achieve this recommendation.3 Women with dietsthat restrict consumption of choline-rich animal source foodsmay be particularly vulnerable to choline intakes belowcurrent guidelines. The lacto-ovo-vegetarian (LOV) dietary

regimen prohibits intake of some choline-rich foods such asred meat and poultry, but allows consumption of eggs, anexcellent dietary source of choline.4 Therefore, eggs maybe used to enhance choline intake among LOV women ofreproductive age, which is of particular importance duringpregnancy and lactation when the demand for cholineis high.5

The current study is an extension of a feeding study6 inwhich the consumption of n-3 fatty-acideenriched eggs,nonenriched eggs, and walnuts was compared with regard toserum lutein.6 The aim of this research was to determinethe effect of near-daily egg consumption vs egg restriction oncirculating choline metabolites in free-living LOV women ofreproductive age. The influence of n-3eenriched vs non-enriched eggs on biomarkers of choline metabolism was alsoinvestigated secondary to recent reports of a striking rela-tionship between the n-3 fatty acid docosahexaenoic acid(DHA) and PC metabolism.7,8 Finally, due to associations be-tween trimethylamine oxide (TMAO), a gut-floraedependentoxidative choline metabolite, and cardiovascular disease

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Figure. Diagram illustrating the relationships of choline metabolic products. Choline may be used to synthesize phosphatidyl-choline (PC) via the cytidine diphosphate (CDP)-choline pathway or oxidized to betaine. A labile methyl group from betaine may betransferred to homocysteine to form dimethylglycine and methionine, a precursor of the universal methyl donor S-adenosylme-thionine (SAM). PC can also be synthesized endogenously via the phosphatidylethanolamine N-methyltranferase (PEMT) pathwayand used to generate free choline. PC molecules produced by the CDP-choline and PEMT pathways are also used for very-low-density lipoprotein (VLDL) synthesis. 5-methyl-THFolate¼5-methyltetrahydrofolate. THFolate¼tetrahydrofolate. PE¼phospha-tidylethanolamine; SAH¼S-adenosylhomocysteine.

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risk,9,10 pre- and posttreatment plasma TMAO concentrationswere quantified.

STUDY PARTICIPANTS AND METHODSStudy ParticipantsHealthy LOV adults recruited primarily from the southernCalifornia area completed a multistage screening process, as

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previously described by Burns-Whitmore and colleagues.6

Participant inclusion criteria of the original study were aged21 to 90 years, no existing medical condition, no dietarysupplement use, nonsmoking, nonalcohol using, LOV for�3 months before study enrollment, and willing to followstudy protocols.6 A three-stage screening process thatincluded a self-disclosure on the initial screening question-naire, a food frequency questionnaire, and an in-person

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interview regarding dietary practices confirmed adherence toa LOV dietary regimen among recruits. Of 26 recruited, 20individuals completed the study protocol (n¼15 premeno-pausal women, n¼1 postmenopausal woman, and n¼4men).6 Because the present study focused on women ofreproductive age, only samples collected from the 15 LOVwomen of reproductive age were used.All study participants signed a written informed consent,

approved by the Institutional Review Boards at Loma LindaUniversity, Loma Linda, CA, and California State PolytechnicUniversity, Pomona.

Study DesignThis was a randomized, crossover study conducted in free-living participants with three 8-week treatment arms and4-week washout intervals separating treatment periods.6

Each participant was randomly assigned to one of sixpossible sequences that included all three treatment phases.The 8-week treatment phases included consumption ofsix n-3 fatty-acideenriched eggs/week, consumption of sixnonenriched eggs/week, and an egg-free control phase dur-ing which walnuts were consumed in place of the eggs.6

Participants were asked to limit consumption of eggs andwalnuts for 2 weeks before starting the study and wereblinded to the type of egg (n-3eenriched vs nonenriched)they received. Eggs (n-3eenriched and nonenriched) camefrom chickens fed vegetarian flaxseed-based and soy-basedfeed, respectively, and were all from farms located in south-ern California.6

Study participants were counseled by a registered dietitiannutritionist every 2 weeks during treatment periods at theLoma Linda University campus when they picked up theirtreatment food.6 Participants were instructed to not consumeeggs other than those provided by the study. In addition,participants were asked to avoid consumption of largeamounts of soy products, dark leafy green vegetables, andfoods rich in n-3 fatty acids (ie, flaxseed/linseed, canola, orflax oil) during the study periods; however, they were notasked to restrict intake of these foods during the 4-weekwashout periods. Participants noted deviations from the di-etary protocol as well as medicines used in a personal dietdiary.Study compliance was assessed for each participant with

personal diet diaries and three 24-hour dietary recalls perstudy treatment that were conducted by telephone onrandomly assigned days. Although primarily employed togauge compliance, the 24-hour recalls were also used togenerate average dietary intakes of energy, carbohydrates,protein, fat, choline, and folate. Diet information from the24-hour recalls was analyzed with the Nutrition Data System-Research software (2004, Nutrition Coordinating Center,Department of Epidemiology, University of Minnesota) andwas updated with choline and betaine data using the Nutri-tion Data System-Research 2007 database.Covance Inc quantified the fatty-acid composition and total

choline content of the treatment eggs and walnuts used inthis study.Before and after each study treatment, 12-hour fasting

blood draws were performed. After collection, blood sampleswere immediately centrifuged at 1,500g for 15 minutes at 4�Cand stored at e80�C until analysis.

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Choline Metabolite QuantificationPlasma choline, betaine, and dimethylglycine levels weremeasured using the method of Holm and colleagues,11 withmodifications.12 PC and sphingomyelin levels were deter-mined using the method developed by Koc and colleagues,13

with modifications.14 Plasma TMAO was quantified using themethod developed by Wang and colleagues,9 with modifi-cations.15 Briefly, deuterium-labeled internal standards wereadded to each sample and subsequently analyzed using liquidchromatography tandem mass spectrometry. The sampleswere run in batches, with each batch containing six samplesand pooled plasma as control. The six samples were obtainedbefore and after each of the three treatments, and all analyseswere done in duplicate. The intraassay coeffients of variation(CVs) for choline, betaine, PC, sphingomyelin, and TMAOwere <6% and the intraassay CV for dimethylglycine was<15%. All interassay CVs were <15%.

Statistical AnalysisKruskal-Wallis tests were used to detect differences betweenpretreatment baseline metabolite concentrations. Posttreat-ment metabolite concentrations were compared using linearmixed models, which included baseline concentration ascovariate, dietary treatment (ie, n-3eenriched egg, non-enriched egg, and egg-free control) and sequence (ie, order oftreatment) as fixed effects, and subject ID as a random effect.Sidak corrections were made for multiple comparisons whenthe treatment effect was significant (P<0.05). Results arepresented as model-generated estimated means�standarderror unless noted otherwise. Data were analyzed with SPSSversion 21 (2012, IBM-SPSS Statistics) with the exception ofthe 24-hour dietary recall data, which was analyzed with SASversion 9.2 (2009, SAS Institute, Inc).

RESULTSSubject CharacteristicsThe average age and body mass index of the study partici-pants were 35.7�12.9 years and 23.7�4.7, respectively. Theaverage time for which the subjects were LOV before joiningthe study was 16.0�13.4 years. The self-reported ethnic/racialmakeup of participants was eight white non-Hispanic (53%),four Hispanic (27%), two Asian (13%), and one Caribbean(South American Indian and African) (7%).

Nutrient Content of Study Dietary TreatmentsThe lipid and choline content of the dietary treatments asquantified by Covance Inc are reported in Table 1. Measuredamounts were similar to the values contained in the NDS-Rdatabase for both the eggs (112.7 mg/large egg); thus, thesepreestablished values were used in dietary analysis.

Dietary Analysis from the 24-Hour RecallsAverage daily intake of energy, carbohydrate, protein, and fatdid not vary (P¼0.11 to 0.86) by treatment group (Table 2). Incontrast, cholesterol intake was greater (P<0.001) during theegg treatments vs the egg-free control phase (Table 2).Total choline intake was greater (P<0.001) during the egg

treatments (vs the egg-free phase), but was not different(P¼0.99) between the n-3eenriched and control egg treat-ments. The average daily intake of total choline throughout

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Table 1. Nutrient contents of the three dietary treatmentsa used in a study to determine how near-daily egg consumptioninfluenced biomarkers of choline metabolism in free-living lacto-ovo-vegetarian women of reproductive ageb

Nutrient

n-3eEnriched egg(50 g egg[w1 large egg)

Nonenriched egg(50 g egg[w1 large egg)

Egg-freecontroltreatment

Total choline (mg) 138 126 5.0

18:2 n-6 linoleic acid (g) 2.77 2.09 12.1

20:4 n-6 arachidonic acid (g) 0.23 0.25 0.02

Total n-6 (g) 3.02 2.38 14.4

18:3 n-3 alpha linolenic acid (g) 0.47 0.07 2.95

20:5 n-3 eicosapentaenoic acid (g) <0.02 <0.02 —

22:6 n-3 docosahexaenoic acid (g) 0.24 0.11 —

Total n-3 (g) 0.74 0.18 2.95

Saturated fat (g) 4.17 4.12 1.68

Monounsaturated fatty acid (g) 5.15 5.60 2.79

Polyunsaturated fatty acid (g) 3.60 2.46 14.3

Total fat (g) 13.5 12.7 19.0

aValues measured by Covance Inc.bData were reported without standard deviations and could not be analyzed for differences.

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the study (ie, w265 mg/day during the egg treatments andw170 mg/day during the egg-free control phase) was lowerthan the Adequate Intake of 425 mg/day. Notably, dailynutrient intakes were very similar during the egg treatmentphases (Table 2), implying that metabolic differencesobserved between the egg treatment groups were due to thetreatment (ie, composition of the egg) rather than anotherdietary component.

Table 2. Comparison of the average daily nutrient intake calculaparticipant (N¼15) during each treatment used in a study to deterof choline metabolism in free-living lacto-ovo-vegetarian women

Intake per dayn-3 Enriched egg(n[15)

Nonen(n[15

�������������������mean�sta

Energy (kcal) 1,675�200 2,044�Carbohydrate (g) 254�34 300�Protein (g) 56�10 78�Total fat (g) 58�6 61�Cholesterol (mg) 244�27y 216�Total choline (mg) 263�19y 265�Betaine (mg) 183�32 255�Folate (mg) 465�123 671�aAnalyzed with Nutrition Data System-Research (NDS-R) software (2004, Nutrition Coordinatinganalyzed using NDS-R 2007 (Regents of the University of Minnesota).bData were analyzed with mixed models that included treatment, diet sequence, and randomcThe women consumed each 8-wk treatment in random order with a 4-wk washout period byzValues in the same row with dissimilar superscript letters (y, z) are significantly different (P<

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Plasma Choline and MetabolitesPlasma metabolite concentrations did not differ (P>0.05)among the dietary treatments at baseline (Table 3). However,at the end of the 8-week regimens, plasma free choline(P¼0.02), betaine (P<0.01), and PC (P¼0.05) varied by dietarytreatment (Table 4). Consumption of the n-3eenriched eggtreatment yielded greater concentrations of plasma freecholine (P¼ 0.02) and betaine (P< 0.01) vs the egg-free

ted from three randomly administered 24-h recalls from eachmine how near-daily egg consumption influenced biomarkersof reproductive ageabc

riched egg)

Egg-free control(n[15)

TreatmentP

ndard error�������������������!

195 1,632�197 0.22

33 230�34 0.29

10 55�10 0.11

6 62�6 0.87

27y 51�27z <0.001

19y 169�19z <0.001

31 192�31 0.20

120 470�122 0.36

Center, Division of Epidemiology, University of Minnesota). Choline and betaine were

subject effect using SAS version 9.2 (2009, SAS Institute).etween treatments.0.05).

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Table 3. Comparison of the baseline plasma choline metabolite and trimethylamine oxide concentrations before consumingn-3eenriched egg, nonenriched egg, and egg-free control dietary treatments used in a study to determine how near-dailyegg consumption influenced biomarkers of choline metabolism in free-living lacto-ovo-vegetarian women of reproductive agea

Metabolite (mmol/L)n-3eEnriched eggtreatment (n[15)

Nonenriched eggtreatment (n[15)

Egg-free controltreatment (n[15)

TreatmentP

���������������������median (95% CI)

���������������������!Choline 8.5 (7.4-8.9) 8.0 (6.4-8.7) 8.7 (6.8-10.0) 0.74

Betaine 32.3 (27.8-40.0) 38.2 (29.1-51.2) 38.7 (30.0-45.6) 0.22

Phosphatidylcholine 1,343 (1,209-1,460) 1,254 (1,080-1,423) 1,427 (1,205-1,551) 0.33

Dimethylglycine 2.3 (1.9-2.9) 2.6 (2.2-2.9) 2.8 (2.1-3.2) 0.75

Sphingomyelin 429 (354-497) 403 (352-506) 430 (372-475) 0.94

Trimethylamineoxide

1.6 (1.1-2.2) 1.9 (1.6-3.3) 1.7 (1.1-2.2) 0.23

aData were analyzed with Kruskal-Wallis tests with treatment as grouping variable using SPSS version 21 (2012, IBM-SPSS Statistics).

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control; however, no differences (P¼0.27 to 0.30) in thesemetabolites were detected between the nonenriched eggand egg-free control treatments (Table 4). Consumption ofthe nonenriched egg treatment tended to yield greater(P¼0.06) plasma PC vs the egg-free control; however, nodifference (P¼0.93) in plasma PC was detected between then-3eenriched egg and egg-free control treatments (Table 4).None of the dietary treatments altered plasma levels ofdimethylglycine (P¼0.17) or sphingomyelin (P¼0.17)(Table 4).

Plasma TMAOPlasma TMAO concentrations did not differ among the di-etary treatments at baseline (P¼0.23) (Table 3) or after thedietary treatments (P¼0.91) (Table 4).

Table 4. Comparison of plasma choline metabolite and trimethynonenriched egg, and egg-free control dietary treatments used iinfluenced biomarkers of choline metabolism in free-living lacto-

Metabolite (mmol/L)n-3 Enriched egg(n[15)

None(n[1

��������model generated estim

Choline 8.4�0.4y 8.0

Betaine 46.7�3.1y 39.6

Phosphatidylcholine 1,371�58 1,513

Dimethylglycine 3.2�0.2 2.8

Sphingomyelin 457�16 441

Trimethylamine oxide 2.3�0.3 2.1

aData were analyzed with mixed models that included posttreatment status as dependent variablcomparisons were adjusted with Sidak correction.bBorderline difference (P¼0.06) between nonenriched egg and egg-free control treatments.yzDissimilar superscript letters within a row (y, z) indicate pairwise difference at P<0.05.

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DISCUSSIONThe results of this study show that regular egg consumption(vs egg restriction) alters biomarkers of choline metabolismamong LOV women. Notably, compared with the egg-freecontrol, consumption of n-3eenriched eggs increased circu-lating concentrations of free choline and betaine, whereasconsumption of nonenriched eggs tended to increase plasmaPC (Table 4). Because eggs from both dietary treatmentscontained similar amounts of choline, the differential effectsof the egg treatments on the choline metabolite profile mayarise from differences in their fatty-acid profiles.Eggs enriched in n-3 fatty acids contain a higher quantity

of DHA compared with nonenriched eggs and the egg-free control diet (Table 1). DHA has been shown to in-crease PEMT substrate (ie, containing polyunsaturated fattyacid),16,17 which may in turn increase the amount of PC

lamine oxide estimates after 8-wk n-3eenriched egg,n a study to determine how near-daily egg consumptionovo-vegetarian women of reproductive ageab

nriched egg5)

Egg-free control(n[15)

TreatmentP

ated mean�standard error��������!

�0.4yz 7.4�0.4z 0.02

�3.1yz 34.5�3.0z <0.01

�59 1,334�59 0.05

�0.2 2.7�0.2 0.17

�17 413�16 0.17

�0.3 2.3�0.3 0.91

e and baseline status as covariate using SPSS version 21 (2012, IBM-SPSS Statistics). Multiple

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produced via the PEMT pathway.18 Enhanced PC productionvia PEMT would be expected to reduce PC synthesis by theCDP-choline pathway,19 thereby sparing choline for itsoxidation to betaine, and increase the endogenous produc-tion of choline following hydrolysis of PEMT-PC to freecholine. Therefore, the data presented herein suggest that byupregulating PC synthesis via the PEMT (vs the CDP-choline)pathway, the n-3eenriched eggs increased the availability ofcholine for its oxidation to the methyl donor betaine. Becauseprevious studies have shown depletion of betaine during thesecond half of pregnancy,15,20 consumption of eggs enrichedwith n-3 fatty acid may be a nutrition-related strategy toincrease circulating concentrations of these important methyldonors. Nonetheless, additional studies are needed to rigor-ously test this hypothesis and to determine whether dietarysources of DHA low in choline would elicit a similar response.Results from the current study also suggest that the fatty-

acid profile of eggs (ie, n-3eenriched vs nonenriched)distinctly influences lipid metabolism. Specifically, comparedwith the egg-free control, plasma PC (a primary componentof circulating lipoproteins19) tended to be greater afterconsumption of the nonenriched egg regimen, but notafter consumption of the n-3eenriched egg regimen(Table 4). Long chain n-3 fatty acids may decrease very-low-density lipoprotein (VLDL) cholesterol secretion by reducingtriglyceride synthesis,21 stimulating degradation of apolipo-protein B,21 and/or inhibiting VLDL cholesterol formation.22

Prior reports have been mixed with respect to the influenceof egg consumption (n-3eenriched and nonenriched) onplasma lipid fractions23-25; however, these findings suggestthat the long-chain n-3 fatty-acid content of the enrichedeggs countered the stimulatory effect that egg consumptionhadonVLDL secretion among LOVwomen in the current study.TMAO has recently been identified as a possible risk factor

for cardiovascular disease9,10,26 and can be formed fromvarious dietary substrates, including choline,27 PC,10 andcarnitine.26 Upon consumption, gut bacteria convert thesecompounds to trimethylamine, which is then absorbed viaenterohepatic circulation and converted to TMAO by liverflavin monooxygenases. A new study10 reported that con-sumption of two hard-boiled eggs (ie, a PC challenge) tran-siently increases plasma TMAO levels. However, an earlierstudy demonstrated that only fish consumption (whichcontains large amounts of TMAO itself) resulted in substan-tially higher urinary TMAO levels; consumption of eggs,meats, fruits, vegetables, or grains did not increase urinaryTMAO.27 In the present study, posttreatment fasted plasmaconcentrations of TMAO did not differ among dietary treat-ments, suggesting that regular egg consumption (ie, 1 egg/day)among healthy women of reproductive age would not beexpected to elevate TMAO.

LimitationsBecause 24-hour recalls are subject to memory loss and bias,a limitation of this study is the use of self-reported 24-hourrecalls for calculation of average daily nutrient intakes. Inaddition, dietary restriction during treatment periods (ie,asking participants to avoid eggs, nuts, and select otherfoods) may have altered normal consumption. Nonetheless,these limitations are partially offset by each study participantserving as her own control because dietary patterns are

6 JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

more stable over time within an individual than betweenindividuals.Small sample size (n¼15) is an additional limitation.

However, the present research limited its focus to datagenerated from 15 premenopausal women of the 20 totalstudy participants because of the importance of cholinenutriture in women of reproductive age5 as well as notabledifferences in choline metabolism by sex and age.2,28 Thus,the homogeneity of this group (ie, sex, age, and diet) in-creases its generalizability to similar populations.

CONCLUSIONSNear-daily egg consumption (vs egg restriction) alters bio-markers of choline metabolism among LOV women withoutincreasing plasma TMAO concentrations. Compared with theegg-free control women, circulating concentrations of plasmafree choline and betaine were greater after consumption ofeggs enriched with the long-chain n-3 fatty acid, whichsuggests such foods may be especially beneficial duringpregnancy when choline-derived methyl donors becomedepleted.

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2. Institute of Medicine, Food and Nutrition Board. Dietary ReferenceIntakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12,Pantothenic Acid, Biotin, and Choline. Washington, DC: NationalAcademies Press; 1998.

3. Jensen H, Batres-Marquez S, Carriquiry A, Schalinkske K. Choline inthe diets of the U.S. population: NHANES, 2003-2004. FASEB J.2007;21:lb219.

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5. Caudill MA. Pre- and postnatal health: Evidence of increased cholineneeds. J Am Diet Assoc. 2010;110(8):1198-1206.

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14. Abratte CM, Wang W, Li R, Moriarty DJ, Caudill MA. Folate intake andthe MTHFR C677T genotype influence choline status in youngMexican American women. J Nutr Biochem. 2008;19(3):158-165.

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21. Davidson MH. Mechanisms for the hypotriglyceridemic effect ofmarine omega-3 fatty acids. Am J Cardiol. 2006;98(4A):27I-33I.

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AUTHOR INFORMATIONA. A. West is a postdoctoral associate and M. A. Caudill is a professor, Division of Nutritional Science, Cornell University, Ithaca, NY. Y. Shih is arenal dietitian, DaVita Dialysis Center, Montclair, CA; at the time of the study, she was a graduate student, Department of Human Nutrition andFood Science, California State Polytechnic University, Pomona. W. Wang is a lecturer, Department of Animal and Veterinary Science, K. Oda is astatistician and instructor, Department of Epidemiology, Biostatistics, and Population Medicine, K. Jaceldo-Siegl is an assistant research professor,J. Sabaté is a professor, E. Haddad is an associate professor, and S. Rajaram is an associate professor, Department of Nutrition, all at Loma LindaUniversity, Loma Linda, CA. B. Burns-Whitmore is a professor, Department of Human Nutrition and Food Science, California State PolytechnicUniversity, Pomona.

Address correspondence to: Bonny Burns-Whitmore, DrPH, RD, Department of Human Nutrition and Food Science, California State PolytechnicUniversity, 3801W Temple Ave, Pomona, CA 91768. E-mail: bburnswhitmo@csupomona.edu

STATEMENT OF POTENTIAL CONFLICT OF INTERESTNo potential conflict of interest was reported by the authors.

FUNDING/SUPPORTThis study was funded by a fellowship grant from the American Egg Board and the Agriculture Research Institute at California State PolytechnicUniversity, Pomona.

ACKNOWLEDGEMENTSThe authors thank the American Egg Board and the Agriculture Research Institute at California State Polytechnic University, Pomona, for sup-porting this study and for their dedication to scientific research.

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