Song Pingping, Kuryatov Alexander, Axelsen Paul H
School of Biological Engineering, Guizhou Medical University, Guiyang, 550025, Guizhou, China.
Department of Pharmacology, Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, 19104, USA.
AMB Express. 2020 Mar 11;10(1):45. doi: 10.1186/s13568-020-00981-0.
Docosahexaenoic acid (DHA) enriched in brain can yield many important degradation products after the attack of hydroxyl radicals, which is known to serve as a nutraceutical and neuroprotective effects. Oxidative stress is a commonly observed feature of Alzheimer's disease (AD). Therefore, uniformly radiolabeled DHA plays an important role in studying the oxidative fate of DHA in vivo and vitro. However, carbon isotope labeled DHA isn't commercially available now. The heterotrophic microalgae Crypthecodinium cohnii (C. cohnii) has been identified as a prolific producer of DHA. In this study, the growth rate and DHA production in C. cohnii were optimized in a new defined media, and the biosynthesis of U-C-DHA from U-C-glucose and U-C-DHA from U-C-glucose were analyzed by HPLC-MS/MS. Approximately 40 nmoles of U-C-DHA with higher isotopic purity of 96.8% was produced in a 300 μL batch, and ~ 0.23 μCi of U-C-DHA with significant specific activity of 5-6 Ci/mol was produced in a 300 μL batch. It was found that C. cohnii had the optimal growth and DHA accumulation at 25 °C in this defined media (C/N = 10). An efficient protocol for the biosynthesis of U-C-DHA and U-C-DHA were set up firstly, which provides the basic support for the analysis of oxidative degradation products of DHA in AD.
富含于大脑中的二十二碳六烯酸（DHA）在羟基自由基攻击后会产生许多重要的降解产物，已知其具有营养保健和神经保护作用。氧化应激是阿尔茨海默病（AD）常见的一个特征。因此，均匀放射性标记的DHA在研究DHA在体内和体外的氧化命运中起着重要作用。然而，碳同位素标记的DHA目前尚无商业供应。异养微藻寇氏隐甲藻（C. cohnii）已被确定为DHA的高产生产者。在本研究中，在一种新的限定培养基中优化了寇氏隐甲藻的生长速率和DHA产量，并通过高效液相色谱-串联质谱法（HPLC-MS/MS）分析了从U-C-葡萄糖合成U-C-DHA以及从U-C-葡萄糖合成U-C-DHA的生物合成过程。在300μL批次中产生了约40纳摩尔同位素纯度较高（96.8%）的U-C-DHA，在300μL批次中产生了约0.23微居里比活度显著为5 - 6居里/摩尔的U-C-DHA。发现在这种限定培养基（C/N = 10）中，寇氏隐甲藻在25℃时具有最佳生长和DHA积累。首先建立了一种高效的U-C-DHA和U-C-DHA生物合成方案，这为分析AD中DHA的氧化降解产物提供了基础支持。
