The major premise for this proposal is that humans require a dietary source of choline. Choline is a dietary component essential for the structural integrity and signaling functions of cell membranes; it is the major source of methyl-groups in the diet, and it directly affects cholinergic neurotransmission, transmembrane signaling and lipid transport/metabolism. The recent recommendations on dietary reference intakes made by the Institute of Medicine included estimates of the amount of choline required by humans. This report noted that choline is important for normal function, and that it is likely that humans require dietary choline. However, they noted that evidence in humans is insufficient to make accurate estimations of this requirement, and that """"""""...research that could provide such human data is assigned the highest priority."""""""" We propose studies that will begin to address these questions. In this 71 day repeated measure, within subject study with graded repletion, adult men, adult premenopausal and postmenopausal women will be admitted to the General Clinical Research Center and fed a standard diet containing a known control amount of choline (550 mg/choline/2500 Kcal diet; 35 Kcal/kg body weight) with the RDA for folate and methionine for 10 days (baseline). They then are placed on an experimental diet (no choline-low folate/methionine; or no choline-normal folate/methionine) for 21 days. Possible biomarkers for dietary choline status will be assessed including: plasma concentrations of choline and its metabolites, RBC choline, liver choline metabolites, total body choline by isotopic dilution, methionine oxidation rate, urinary choline and betaine excretion, serum alanine aminotransferase, hepatic steatosis, plasma homocysteine, folate and methionine, mass resonance spectroscopy of liver and physical examination. Isotopic labeling studies will be used to assess rates of de novo choline biosynthesis from S-adenosylmethioinine and phosphatidylethanolamine. If no functional effects of choline deficiency are observed at 21 days, the choline devoid diet will be continued until such effects occur, or until another 21 days has been completed (experience in males suggests that this will not be needed). At the end of the depletion period subjects will be switched to a diet containing 25 percent control amount of choline (and low folate/methionine; or normal folate/methionine) for 10 days. If the above biomarkers indicate that depleted choline status persists after 10 days (biomarkers are stable and depleted)- subjects would then be switched to a diet containing 50 percent control amount of choline (and low folate/methionine; or normal folate/methionine) for 10 days. If biomarkers do not indicate repletion, then subjects would then be switched to a diet containing 75 percent control amount of choline (and low folate/methionine; or normal folate/methionine) for 10 days. If not repleted, then subjects would be fed a diet containing 100 percent control amount of choline (and low folate/methionine; or normal folate/methionine) until repleted. Subjects who experience grade 2 toxicity will be immediately switched to the control diet and followed until they return to normal.
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