STABLE ISOTOPE LABELLED FOLATE--KEY TO NUTRITION

拟议研究的目标是开发一种改进的评估方法。 使用稳定同位素稀释法对人体叶酸状况进行评估。总计 体内的叶酸储量将从以下比例估计 甲基四氢叶酸/甲基四氢叶酸(MTHFA-d4/MTHFA) 在适当的时间抽取血液样本(假平衡+ 大约4天)在摄入4毫克剂量的[2‘，3’，5‘，’6‘2H4]之后 叶酸(叶酸-dm)甲基四氢呋喃-d4/甲基四氢呋喃的假平衡 血浆将根据图案和外观确定，并 MTHFA-d4在血浆中的消失。拟议的评估方法将 最初是在大鼠身上开发和验证的，在那里 叶酸可以通过控制饮食叶酸来改变，并通过直接验证 身体组织的分析。在实验1中，断奶的大鼠将被喂以 添加125、500、1000、2000的无叶酸氨基酸基础日粮 或4000杯叶酸/公斤，持续约3周。然后每只老鼠都会 接受小剂量叶酸-d4灌胃被杀死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 将对身体进行总叶酸分析(在猪肾之后 共轭酶处理)和甲基四氢呋喃/甲基四氢呋喃的质量比 光谱学。血浆MTHFA假平衡所需的时间- D4/MTHFA和组织吸收的标记叶酸的量将是 下定决心。在实验2中，额外的大鼠是红色的，并给予叶酸- D4如上所述，但它们将同时被杀死 (如中所确定的，大约在伪证合格后4天 实验1)。血浆和肝脏中MTHFA-d4/MTHFA的比值和总 将测量屠体叶酸(身体总储备)。在实验3 体内总叶酸储备将与血浆MTHFA-d4/MTHFA倒退 以比率作为预测因子，使用对数变换的独立性 为达到近似正态、同方差所需的变量 以及身体健康的好处。将检查拟合优度和模型假设 通过正态概率和残差曲线图。逐步回归将是 用于在以下各项中找到身体总叶酸储备的最佳预测因子 转换后的响应变量。将对足够的大鼠进行研究，以便联合 总叶酸的变异系数约为11% 预备队。在实验4中，健康的成年人全部正常摄入叶酸 范围从低于美国RDA的50%到超过450%的叶酸意愿 接受一次4毫克的叶酸-d4注射，并提供连续的血液和 完全排尿2周。血浆MTHFA-d4/MTHFA的测量方法如下 实验1.尿乙酰氨基苯甲酰-d4- 将测定谷氨酸/乙酰氨基苯甲酰谷氨酸的比例。相关性 膳食叶酸摄入量(反映身体储备)和 血浆和/或尿液中的同种异构体比率将验证 用于评估叶酸储量的同位素稀释法。确定精确的 将同质异构体比率换算为身体总储备量的系数将为 在本申请的续期中提出。一种可靠的评估方法 叶酸状况是必要的，因为有证据表明边缘叶酸状况 神经管缺陷、癌症和无效的造血。

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.