School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Anal Bioanal Chem. 2019 May;411(13):2891-2904. doi: 10.1007/s00216-019-01742-0. Epub 2019 Mar 19.
Folates are typically present in polyglutamyl form in organisms. In traditional extraction methods, polyglutamyl folates are hydrolyzed to monoglutamates, sacrificing valuable information. To advance folate metabolism research, we developed an accurate, sensitive, and reproducible extraction method for polyglutamyl folate species in maize, the main crop in most parts of the world. Twelve folates, including six polyglutamyl folates, were simultaneously determined in maize for the first time using high-performance liquid chromatography-tandem mass spectrometry. The glutamation states of the folates were protected by boiling, which inactivated the native conjugases. α-Amylase and protease were added to obtain better recoveries and decrease difficulties in centrifugation and filtration. The recoveries (n = 5) of six polyglutamyl folates were between 80.5 and 101%. All calibration curves showed good linear regression (r ≥ 0.994) within the working range. The instrumental limits of detection and quantitation ranged from 0.070 to 2.4 ng/mL and 0.22 to 8.0 ng/mL, respectively. Intra- and inter-day precision was below 7.81% and 11.9%, respectively (n = 5). Using this method, changes in poly- and monoglutamyl folates during maize germination were determined for the first time. The results suggest that folates were largely synthesized as germination initiated, and 5-methyltetrahydrofolate was the most abundant species. Tetraglutamyl 5-methyltetrahydrofolate contributed more than 50% of the 5-methyltetrahydrofolate species. Inverse changes in contents of 5,10-methenyltetrahydrofolate, and 10-formyl folic acid, monoglutamate, and diglutamate of 5-formyltetrahydrofolate were also observed, indicating potential regulation. Additionally, polyglutamyl folates in sweet potatoes were determined using this method, indicating its applications in starchy crops.
叶酸通常以多聚谷氨酸的形式存在于生物体中。在传统的提取方法中，多聚谷氨酸叶酸会水解为单谷氨酸盐，从而损失了有价值的信息。为了推进叶酸代谢研究，我们开发了一种准确、灵敏、可重现的玉米中多聚谷氨酸叶酸提取方法，玉米是世界上大部分地区的主要作物。我们首次使用高效液相色谱-串联质谱法同时测定了玉米中的 12 种叶酸，包括 6 种多聚谷氨酸叶酸。通过煮沸保护叶酸的谷氨酸状态，从而使天然结合酶失活。添加α-淀粉酶和蛋白酶以获得更好的回收率，并减少离心和过滤的困难。6 种多聚谷氨酸叶酸的回收率（n=5）在 80.5%至 101%之间。所有校准曲线在工作范围内均显示出良好的线性回归（r≥0.994）。仪器检测限和定量限范围分别为 0.070 至 2.4ng/mL 和 0.22 至 8.0ng/mL。日内和日间精密度分别低于 7.81%和 11.9%（n=5）。使用该方法，首次确定了玉米发芽过程中多聚和单谷氨酸叶酸的变化。结果表明，随着发芽的开始，叶酸大量合成，5-甲基四氢叶酸是最丰富的物种。四谷氨酸 5-甲基四氢叶酸对 5-甲基四氢叶酸物种的贡献超过 50%。5,10-亚甲基四氢叶酸、10-甲酰基叶酸、5-甲酰基四氢叶酸的单谷氨酸盐和二谷氨酸盐含量的含量也发生了相反的变化，表明存在潜在的调节作用。此外，还使用该方法测定了甘薯中的多聚谷氨酸叶酸，表明其在淀粉作物中的应用。
Zotero 插件