The goal of the proposed study is to develop an improved assessment method of human folate status using stable isotope dilution methodology. Total body folate reserves will be estimated from the ratio of methyltetrahydrofolic acid-d4/mthyltetrahydrofolic acid (MTHFA-d4/MTHFA) in a blood specimen drawn at an appropriate time (pseudoequilibration + approximately 4 days) after ingesting a 4 mg dose of [2', 3', 5', '6'2H4] folic acid (folic acid-dm). Pseudoequilibration of MTHFA-d4/MTHFA in plasma will be determined from the pattern and appearance and disappearance of MTHFA-d4 in plasma. The proposed assessment method will initially be developed and validated in rats where total body stores of folate can be varied by controlling diet folate, and verified by direct analysis of body tissues. In experiment 1, weanling rats will be fed a folate free amino acid-based diet supplemented with 125, 500, 1000, 2000 or 4000 mug folic acid/kg for approximately 3 weeks. Then each rat will receive a small dose of folic acid-d4 by gavage an be killed 3, 6, 9, 12, 18, 24, 30, 36 hours or 2, 3, 4, 6, 8, 12 or 16 days later. Plasma, livers and carcasses will be analyzed for total folate (after hog kidney conjugase treatment) by L casei and ratios of MTH-d4/MTHFA by mass spectroscopy. The time needed for pseudoequilibration of plasma MTHFA- d4/MTHFA and the amount of labeled folate taken up by the tissues will be determined. In experiment 2, additional rats are red and given folic acid- d4 as described above but, these will be killed at the same time (approximately 4 days after pseuodequilibration as determined in experiment 1). Ratios of MTHFA-d4/MTHFA in plasma and liver, and total carcass folate (total body reserves) will be measured. In experiment 3 total body folate reserves will be regressed on plasma MTHFA-d4/MTHFA ratios as predictors, using logarithmic transformation of the independent variables as necessary to achieve approximate normality, homoscedasticity and goodness of fit. Goodness of fit and model assumptions will be checked by normal probability and residual plots. Stepwise regression will be used to find best predictors of total body folate reserves among the transformed response variables. Sufficient rats will be studied for a co- efficient of variation of approximately 11% for total body folate reserves. In experiment 4, healthy adults wholes regular folate intake ranges from less 50 to greater than 450% of their US RDA for folate will receive a single 4 mg bolus of folic acid-d4 and provide serial blood and complete urine for 2 weeks. Plasma MTHFA-d4/MTHFA will be measured as in experiment 1. Urine acetamidobenzoyl-d4- glutamate/acetamidobenzoylglutamate ratios will be determined. Correlation between dietary folate intake (which reflects body reserves) and isotopomer ratio in plasma and/or urine will validate the principle of isotope dilution for assessing folate reserves. Determining precise factors to convert isotopomer ratios to total body reserves will be proposed in a renewal of this application. A reliable method for assessing folate status is needed because of evidence linking marginal folate status to neural tube defects, cancer and ineffective hematopoiesis.
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