Conclusions

This large (n=114,384), prospective cohort study among women ages 50-79 years examined the relation of choline intake estimated from the 122-item WHI FFQ with incident

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CHD while considering micronutrient methyl donor intra-individual one-year variability. Choline was found to be associated with incident CHD in Whites and Blacks with a substantially greater effect within Blacks. Correction for reliability of the FFQ estimates demonstrated that measurement error would result in substantial attenuation of effect estimates. Correction for reliability increased the strength of association with a one SD difference of nutrient density choline by 7% in Whites (1.06 versus 1.13) and 13% in Blacks (1.26 versus 1.42).

Increased nutrient density choline intake was associated with incident CHD in Whites and Blacks with a substantially greater effect estimate within Blacks. The possible

differential effect by race/ ethnicity requires further evaluation and replication. The findings from this third such study of choline and incident CHD provides further evidence in support of a positive association; however, no study has examined the proportional reduction in incident CHD that would occur by modifying choline exposure. Because our study examined choline as calculated by NDSR that grouped free choline and esterified forms

(phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin) into one choline exposure variable, future studies should assess whether the association of choline and incident CHD varies by the form of dietary choline.

We further submit that future work should examine the attributable disease burden associated with increased intake of choline. The population-attributable risk estimates should be based on plausible levels of intake and not presume complete elimination of a choline intake. While choline can be biosynthesized de novo, the concentration of free choline in serum and tissues is heavily dependent on the dietary intake of choline,20-27 as humans can become depleted of choline and betaine.28, 29 Choline depletion can affect health status, as it

is a micronutrient that is essential for normal function of all cells9. Choline directly affects cholinergic neurotransmission 21, 30-32, and directly affects lipid transport from the liver. 9, 33-35 Correspondingly, the attributable disease burden should be assessed using estimates of

choline intake based on realistic and attainable lowering of the population distribution, using metrics such as a Potential Impact Fraction (PIF).36, 37

Since we only examined the relations between choline intake and a single cardiovascular disease manifestation, the relation of choline intake should be further investigated with other atherothrombotic endpoints such as ischemic stroke. Importantly, dietary choline and betaine deficiencies decrease S-adenosylmethionine (SAMe)

concentrations which results in DNA hypomethylation 38, 39 which may result in increased expression of oncogenes and an increased risk of DNA mutations, thus providing a basis for primary tumor growth and metastasis. Associations between DNA hypomethylation and colorectal,40 breast,41 and lung cancer 42, 43 have been reported. Thus, similar investigations may be warranted for the relation between choline and colorectal, breast, and lung cancers.

171 REFERENCES

1. Design of the women's health initiative clinical trial and observational study. The women's health initiative study group. Control Clin Trials. 1998;19:61-109 2. Patterson RE, Kristal AR, Tinker LF, Carter RA, Bolton MP, Agurs-Collins T.

Measurement characteristics of the women's health initiative food frequency questionnaire. Ann Epidemiol. 1999;9:178-187

3. Anderson GL, Manson J, Wallace R, Lund B, Hall D, Davis S, Shumaker S, Wang CY, Stein E, Prentice RL. Implementation of the women's health initiative study design. Ann Epidemiol. 2003;13:S5-17

4. Fleiss JL, Levin BA, Paik MC. Statistical methods for rates and proportions. Hoboken, N.J.: Wiley-Interscience; 2003.

5. Kleinbaum DG, Kupper LL, Morgenstern H. Epidemiologic research : Principles and

quantitative methods. Belmont, Calif.: Lifetime Learning Publications; 1982.

6. Cheng D, Branscum AJ, Stamey JD. Accounting for response misclassification and covariate measurement error improves power and reduces bias in epidemiologic studies. Ann Epidemiol. 2010;20:562-567

7. Bidulescu A, Chambless LE, Siega-Riz AM, Zeisel SH, Heiss G. Usual choline and betaine dietary intake and incident coronary heart disease: The atherosclerosis risk in communities (aric) study. BMC Cardiovasc Disord. 2007;7:20

8. Dalmeijer GW, Olthof MR, Verhoef P, Bots ML, van der Schouw YT. Prospective study on dietary intakes of folate, betaine, and choline and cardiovascular disease risk in women. Eur. J. Clin. Nutr. 2007

9. Zeisel SH, Blusztajn JK. Choline and human nutrition. Annu Rev Nutr. 1994;14:269- 296

10. Hollister LE, Jenden DJ, Amaral JR, Barchas JD, Davis KL, Berger PA. Plasma concentrations of choline in man following choline chloride. Life Sci. 1978;23:17-22 11. da Costa KA, Gaffney CE, Fischer LM, Zeisel SH. Choline deficiency in mice and

humans is associated with increased plasma homocysteine concentration after a methionine load. The American journal of clinical nutrition. 2005;81:440-444 12. Olthof MR, van Vliet T, Verhoef P, Zock PL, Katan MB. Effect of homocysteine-

lowering nutrients on blood lipids: Results from four randomised, placebo-controlled studies in healthy humans. PLoS Med. 2005;2:e135

13. McGregor DO, Dellow WJ, Robson RA, Lever M, George PM, Chambers ST. Betaine supplementation decreases post-methionine hyperhomocysteinemia in chronic renal failure. Kidney international. 2002;61:1040-1046

14. Schwab U, Torronen A, Toppinen L, Alfthan G, Saarinen M, Aro A, Uusitupa M. Betaine supplementation decreases plasma homocysteine concentrations but does not affect body weight, body composition, or resting energy expenditure in human subjects. The American journal of clinical nutrition. 2002;76:961-967

15. Danne O, Lueders C, Storm C, Frei U, Mockel M. Whole blood choline and plasma choline in acute coronary syndromes: Prognostic and pathophysiological

implications. Clinica chimica acta; international journal of clinical chemistry. 2007;383:103-109

16. LeLeiko RM, Vaccari CS, Sola S, Merchant N, Nagamia SH, Thoenes M, Khan BV. Usefulness of elevations in serum choline and free f2)-isoprostane to predict 30-day cardiovascular outcomes in patients with acute coronary syndrome. The American

journal of cardiology. 2009;104:638-643

17. Zeisel SH. Betaine supplementation and blood lipids: Fact or artifact? Nutr Rev. 2006;64:77-79

18. Fischer LM, Scearce JA, Mar MH, Patel JR, Blanchard RT, Macintosh BA, Busby MG, Zeisel SH. Ad libitum choline intake in healthy individuals meets or exceeds the proposed adequate intake level. The Journal of nutrition. 2005;135:826-829

19. Bidulescu A, Chambless LE, Siega-Riz AM, Zeisel SH, Heiss G. Repeatability and measurement error in the assessment of choline and betaine dietary intake: The atherosclerosis risk in communities (aric) study. Nutr J. 2009;8:14

20. Bligh J. The level of free choline in plasma. J Physiol. 1952;117:234-240

21. Haubrich DR, Wang PF, Chippendale T, Proctor E. Choline and acetylcholine in rats: Effect of dietary choline. Journal of neurochemistry. 1976;27:1305-1313

22. Wang FL, Haubrich DR. A simple, sensitive, and specific assay for free choline in plasma. Anal Biochem. 1975;63:195-201

23. Jacob RA, Pianalto FS, Henning SM, Zhang JZ, Swendseid ME. In vivo methylation capacity is not impaired in healthy men during short-term dietary folate and methyl group restriction. The Journal of nutrition. 1995;125:1495-1502

24. Institute of Medicine and National Academy of Sciences. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin b6, folate, vitamin b12, pantothenic acid,

173

25. Buchman AL, Dubin MD, Moukarzel AA, Jenden DJ, Roch M, Rice KM, Gornbein J, Ament ME. Choline deficiency: A cause of hepatic steatosis during parenteral nutrition that can be reversed with intravenous choline supplementation. Hepatology. 1995;22:1399-1403

26. Chawla RK, Wolf DC, Kutner MH, Bonkovsky HL. Choline may be an essential nutrient in malnourished patients with cirrhosis. Gastroenterology. 1989;97:1514- 1520

27. Buchman AL, Moukarzel A, Jenden DJ, Roch M, Rice K, Ament ME. Low plasma free choline is prevalent in patients receiving long term parenteral nutrition and is associated with hepatic aminotransferase abnormalities. Clin Nutr. 1993;12:33-37 28. Buchman AL, Ament ME, Sohel M, Dubin M, Jenden DJ, Roch M, Pownall H,

Farley W, Awal M, Ahn C. Choline deficiency causes reversible hepatic

abnormalities in patients receiving parenteral nutrition: Proof of a human choline requirement: A placebo-controlled trial. JPEN J Parenter Enteral Nutr. 2001;25:260- 268

29. Zeisel SH, Da Costa KA, Franklin PD, Alexander EA, Lamont JT, Sheard NF, Beiser A. Choline, an essential nutrient for humans. Faseb J. 1991;5:2093-2098

30. White HL, Cavallito CJ. Choline acetyltransferase. Enzyme mechanism and mode of inhibition by a styrylpyridine analogue. Biochim Biophys Acta. 1970;206:343-358 31. Blusztajn JK, Holbrook PG, Lakher M, Liscovitch M, Maire JC, Mauron C,

Richardson UI, Tacconi M, Wurtman RJ. "Autocannibalism" of membrane choline- phospholipids: Physiology and pathology. Psychopharmacol Bull. 1986;22:781-786 32. Zeisel SH. Dietary choline: Biochemistry, physiology, and pharmacology. Annu Rev

Nutr. 1981;1:95-121

33. Zeisel SH. Choline: Critical role during fetal development and dietary requirements in adults. Annu Rev Nutr. 2006;26:229-250

34. Kent C. Regulation of phosphatidylcholine biosynthesis. Progress in lipid research. 1990;29:87-105

35. Vance DE, Ridgway ND. The methylation of phosphatidylethanolamine. Progress in

lipid research. 1988;27:61-79

36. Vander Hoorn S, Ezzati M, Rodgers A, Lopez A, Murray C. Estimating attributable burden of disease from exposure and hazard data. Comparative quantification of health risks: Global and regional burden of disease attributable to selected major

37. Morgenstern H, Bursic ES. A method for using epidemiologic data to estimate the potential impact of an intervention on the health status of a target population. Journal

of community health. 1982;7:292-309

38. Dizik M, Christman JK, Wainfan E. Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet.

Carcinogenesis. 1991;12:1307-1312

39. Locker J, Reddy TV, Lombardi B. DNA methylation and hepatocarcinogenesis in rats fed a choline-devoid diet. Carcinogenesis. 1986;7:1309-1312

40. Bariol C, Suter C, Cheong K, Ku SL, Meagher A, Hawkins N, Ward R. The relationship between hypomethylation and cpg island methylation in colorectal neoplasia. Am J Pathol. 2003;162:1361-1371

41. Narayan A, Ji W, Zhang XY, Marrogi A, Graff JR, Baylin SB, Ehrlich M.

Hypomethylation of pericentromeric DNA in breast adenocarcinomas. Int J Cancer. 1998;77:833-838

42. Botto F, Seree E, el Khyari S, Cau P, Henric A, De Meo M, Bergeron P, Barra Y. Hypomethylation and hypoexpression of human cyp2e1 gene in lung tumors.

Biochem Biophys Res Commun. 1994;205:1086-1092