Single-tube biosynthesis and extraction of U-C and U-C arachidonic acid from microcultures of Mortierella alpina for in vivo pharmacology and metabolic tracing studies.

Heavy and radioisotope labeling are commonly used methods to trace the pharmacological activity and metabolic fate of a biochemical or pharmaceutical in vivo. Recent years witnessed increased demand for molecules uniformly labeled with heavy carbon-13 (U-C) or radioactive carbon-14 (U-C) isotopes over singly labeled isotopic versions. Polyunsaturated fatty acids (PUFAs) represent one classic example where uniform C or C isotopic enrichment of the hydrocarbon backbone has numerous technical, metabolic and pharmacological advantages. PUFAs are usually produced in fungi or algae and uniform C or C enrichment of the hydrocarbon chain is achieved by feeding the microorganism a suitable U-C or U-C substrate. Previous literature methods describing the biosynthesis of U-C or U-C fatty acids reported variable isotopic enrichments that were less than anticipated and suffered from inconsistent growth of the microorganism due to radiotoxicity. In the present study, a single-tube method is described for the biosynthesis and extraction of U-C and U-C arachidonic acid (AA), a standard PUFA, from microcultures of the soil fungus Mortierella alpina. To produce U-C-AA, a suspension of fungal spores and mycelial fragments was directly inoculated and grown into submerged cultures in a medium composed of U-C-glucose and NaNO as the respective and only sources of carbon and nitrogen. The total C enrichment of AA was in excess of 95% and the percentage of U-C-AA was in the range of 60-70%. These values have not been surpassed by previously reported methods. To produce U-C-AA, the procedure was modified to limit the radiotoxic effects of C on fungal growth. Submerged cultures were initially grown on common C-glucose. Then, following glucose depletion, the biomass was collected and immediately cultured on U-C-glucose. This approach is unprecedented in reported literature and has significantly limited the radiotoxic effects of C on the microorganism. Biomass transfer from C to C substrates was timed to keep an uninterrupted supply of carbon required to sustain the microorganism in the fatty acid synthesis mode and suppress β-oxidation, a metabolic status that is prerequisite for enhanced isotopic purity of the C product. The specific activity of C enriched AA was estimated at 864 Ci/mol (range of 708-1020 Ci/mol) suggesting 69.2% (range of 56.7-81.7%) C enrichment along the AA hydrocarbon backbone. The present method used a single tube for microbial culture and lipid extraction to minimize manipulative losses and oxidative degradation of the labeled products. Production cost is comparatively cheaper to custom labeling and yields of U-C and U-C-AA are comparable to literature methods and sufficient for small scale in vitro and in vivo pharmacological studies.