STABLE ISOTOPE LABELLED FOLATE--KEY TO NUTRITION

这项研究的目的是发展一种改进的评估方法 用稳定同位素稀释法测定人体叶酸水平。总 身体叶酸储备将估计的比例 甲基四氢叶酸-d4/甲基四氢叶酸 在适当时间抽取的血液样本中（假平衡+ 给药后约4天）给予4 mg剂量的[2'、3'、5'、6'2H4] 叶酸（叶酸-DM）。MTHFA-d4/MTHFA在小鼠中的假平衡 将根据图案和外观确定血浆， MTHFA-d4在血浆中消失。拟议的评估方法将 最初在大鼠中开发和验证，其中全身储存 叶酸可以通过控制饮食叶酸而变化，并通过直接 分析身体组织。在实验1中，断奶大鼠将被喂食 叶酸游离氨基酸为基础的饮食补充125，500，1000，2000 或4000 μ g叶酸/kg持续约3周。然后每只老鼠 通过管饲法接受小剂量的叶酸-D4并被杀死3，6，9，12， 18、24、30、36小时或2、3、4、6、8、12或16天后。血浆，肝脏 并且将分析尸体的总叶酸（在猪肾之后 和MTH-d4/MTHFA的质量比 谱血浆MTHFA伪平衡所需的时间- d4/MTHFA和组织摄取的标记叶酸的量将被 测定在实验2中，额外的大鼠是红色的，并给予叶酸- d4如上所述，但是，这些将同时被杀死 （如图1所示，假去平衡后约4天） 实验1）。血浆和肝脏中MTHFA-d4/MTHFA的比值，以及总量 将测量尸体叶酸盐（全身储备）。在实验3中 总身体叶酸储备将回归血浆MTHFA-d4/MTHFA 比率作为预测因子，使用独立 达到近似正态性、同方差性所需的变量 和拟合度。将检查拟合优度和模型假设 通过正态概率和残差图。逐步回归将是 用来寻找最好的预测全身叶酸储备之间的 转换的响应变量。将对足够的大鼠进行研究， 总叶酸的变异效率约为11% 储备在实验4中，健康成年人完全定期摄入叶酸 范围从小于50到大于450%的美国RDA叶酸将 接受叶酸-D4的单次4mg推注，并提供连续的血液， 2周内完全排尿。血浆MTHFA-d4/MTHFA将按照以下方法测量： 实验1.尿乙酰氨基苯甲酰-d4- 将测定谷氨酸盐/乙酰氨基苯甲酰谷氨酸盐的比率。相关性 膳食叶酸摄入量（反映身体储备）和 血浆和/或尿液中的同位素异构体比率将验证以下原理： 同位素稀释法用于评估叶酸储量。测定精密度 将同位素比例转换为全身储备的因素将是 在本申请的续期中提出。一种可靠的评估方法 需要补充叶酸，因为有证据表明， 神经管缺陷、癌症和无效造血。

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.