Biosynthesis of uniformly carbon isotope-labeled docosahexaenoic acid in Crypthecodinium cohnii

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-¹³C-DHA from U-¹³C-glucose and U-¹⁴C-DHA from U-¹⁴C-glucose were analyzed by HPLC-MS/MS. Approximately 40 nmoles of U-¹³C-DHA with higher isotopic purity of 96.8% was produced in a 300 μL batch, and ~ 0.23 μCi of U-¹⁴C-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-¹³C-DHA and U-¹⁴C-DHA were set up firstly, which provides the basic support for the analysis of oxidative degradation products of DHA in AD.