Single-tube biosynthesis and extraction of U-13C and U-14C arachidonic acid from microcultures of Mortierella alpina for in vivo pharmacology and metabolic tracing studies

Abstract

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-¹³C) or radioactive carbon-14 (U-¹⁴C) isotopes over singly labeled isotopic versions. Polyunsaturated fatty acids (PUFAs) represent one classic example where uniform ¹³C or ¹⁴C isotopic enrichment of the hydrocarbon backbone has numerous technical, metabolic and pharmacological advantages. PUFAs are usually produced in fungi or algae and uniform ¹³C or ¹⁴C enrichment of the hydrocarbon chain is achieved by feeding the microorganism a suitable U-¹³C or U-¹⁴C substrate. Previous literature methods describing the biosynthesis of U-¹³C or U-¹⁴C 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-¹³C and U-¹⁴C arachidonic acid (AA), a standard PUFA, from microcultures of the soil fungus Mortierella alpina. To produce U-¹³C-AA, a suspension of fungal spores and mycelial fragments was directly inoculated and grown into submerged cultures in a medium composed of U-¹³C-glucose and NaNO₃ as the respective and only sources of carbon and nitrogen. The total ¹³C enrichment of AA was in excess of 95% and the percentage of U-¹³C-AA was in the range of 60-70%. These values have not been surpassed by previously reported methods. To produce U-¹⁴C-AA, the procedure was modified to limit the radiotoxic effects of ¹⁴C on fungal growth. Submerged cultures were initially grown on common ¹²C-glucose. Then, following glucose depletion, the biomass was collected and immediately cultured on U-¹⁴C-glucose. This approach is unprecedented in reported literature and has significantly limited the radiotoxic effects of ¹⁴C on the microorganism. Biomass transfer from ¹²C to ¹⁴C 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 ¹⁴C product. The specific activity of ¹⁴C enriched AA was estimated at 864 Ci/mol (range of 708-1020 Ci/mol) suggesting 69.2% (range of 56.7-81.7%) ¹⁴C 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-¹³C and U-¹⁴C-AA are comparable to literature methods and sufficient for small scale in vitro and in vivo pharmacological studies.

Keywords: Arachidonic acid; Metabolic tracers; Mortierella alpina; Polyunsaturated fatty acids; Radioisotope labeling; Stable isotope labeling.