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