High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver.
High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver.
复制标题
高效液相色谱法测定大鼠肝脏中天然存在的叶酸衍生物的分布。
DOI：
10.1016/0003-2697(84)90502-5
复制
发表时间：
1984
影响因子：
2.9
通讯作者：
Horne,DW
中科院分区：
文献类型：
--
作者：
Wilson,SD;Horne,DW
已经开发了允许提取和定量大鼠肝脏中不稳定的、还原的叶酸衍生物的程序。这些程序需要在100°C下用2%(W V)抗坏血酸钠、0.2m 2-巯基乙醇、pH 7.85提取肝脏叶酸。提取液用共轭酶水解叶酸多谷氨酸，反相离子对高效液相色谱分离得到单谷氨酸，用干酪乳杆菌进行微生物检测。高效液相色谱纯化的标准衍生物的实验表明，四氢蝶酰谷氨酸(H4PteGlu)、10-甲酰基-H4PteGlu、5-甲酰基-H4PteGlu、5-甲基-H4PteGlu和蝶酰谷氨酸(PteGlu)具有良好的稳定性。在此条件下，H2PteGlu的回收率约为56%，约27%转化为PteGlu；5，10-亚甲基-H4PteGlu定量回收为H4PteGlu。这些程序被应用于测量自然产生的叶酸辅助因子在大鼠肝脏中的分布。结果表明，大鼠肝脏叶酸组成如下：5-甲基-H4PteGlu，37.2%；H4PteGlu，32.7%；10-甲酰基-H4PteGlu，22.6%；5-甲酰基-H4PteGlu，7.7%。用[~3H]PteGlu注射剂进行的实验表明，所有肝脏的叶酸与放射测定法和干酪乳杆菌测定的比放射性相同，表明干酪乳杆菌对所有在大鼠肝脏中检测到的叶酸的生长反应是相同的。
Procedures which allow extraction and quantitation of labile, reduced folic acid derivatives in rat liver have been developed. These procedures entail extraction of hepatic folates at 100°C in 2% (w v ) sodium ascorbate, 0.2 m 2-mercaptoethanol, pH 7.85. The extract was treated with conjugase to hydrolyze folate polyglutamates and reverse-phase, ion-pair high-performance liquid chromatography was used to separate the resulting monoglutamates which were measured by microbiological assay using Lactobacillus casei. Experiments with HPLC-purified standard derivatives, so treated, showed excellent stability of tetrahydropteroylglutamic acid (H4PteGlu), 10-formyl-H4PteGlu, 5-formyl-H4PteGlu, 5-methyl-H4PteGlu, and pteroylglutamic acid (PteGlu). Under these conditions, approximately 56% of H2PteGlu was recovered unchanged while about 27% was converted to PteGlu; 5,10-methylene-H4PteGlu was quantitatively recovered as H4PteGlu. These procedures were applied to the task of measuring the distribution of naturally occurring folate cofactors in rat liver. These results indicated that rat liver folates have the following compositions: 5-methyl-H4PteGlu, 37.2%; H4PteGlu, 32.7%; 10-formyl-H4PteGlu, 22.6%; and 5-formyl-H4PteGlu, 7.7%. Experiments with [3H]PteGlu injection showed that all hepatic folates had the same specific radioactivity as determined by radioassay and L. casei assay, indicating that L. casei exhibited the same growth response to all the folates detected in rat liver.