Biosynthesis of uniformly labeled 13C- and 14C-arachidonic acid in Mortierella alpina

Abstract

Arachidonic acid (ARA) is one of the most abundant polyunsaturated fatty acids (PUFAs) in the mammalian brain. Many enzymatically- and nonenzymatically-produced metabolic products have important and potent pharmacological properties. However, uniformly isotope labeled forms of ARA are not commercially available for studying the metabolic fates of ARA. This study describes a simple and efficient protocol for the biosynthesis of U-¹³C-ARA from U-¹³C-glucose, and U-¹⁴C-ARA from U-¹⁴C-glucose by Mortierella alpina. The protocols yield approximately 100nmol quantities of U-¹³C-ARA with an isotopic purity of 95% from a 500μl batch volume, and approximately 2μCi quantities of U-¹⁴C-ARA with an apparent specific activity in excess of 1200Ci/mol from a 250μl batch volume.

Keywords: Arachidonic acid; Carbon isotope labeling; Micro-scale culture; Mortierella alpina.

Fig. 1

Production in 5 gm/L ¹³C-glucose media. Averages of three separate batches ± standard deviations, (a) glucose consumption and growth curve, (b) U-¹³C-ARA yield and % ¹³C labels in all of recovered isotopes.

Fig. 2

Production in 0.1 gm/L ¹⁴C-glucose media. Averages of three separate batches ± standard deviations, (a) glucose consumption and growth curves, (b) ¹⁴C activity in ARA and % ¹⁴C Activity ARA activity in total recovered activity.