Novel methods for carbon and nitrogen determinations in biological samples and for the bacterial biosynthesis of carbon-14-vitamin B12

The elemental composition of compounds, especially of carbon and nitrogen, is of considerable interest in many areas of science. Accurate and precise carbon measurements have become even more important as Accelerator Mass Spectrometry (AMS) has been increasingly used for nutrition metabolism studies to follow the fate of a 14C-labeled nutrient or xenobiotic. A method was developed for simultaneous carbon and nitrogen measurements in biological samples and utilizes an internal standardization and isotope ratio mass spectrometry (IRMS). Carbon demonstrated linearity (r = 0.9989) over the range of atom % 13C values from close to natural abundance at 1.1 to a large degree of enrichment at 10.4. The slope of the line is 0.812x +/- 0.0191 and the y-intercept was 0.176 +/- 0.120. Nitrogen also demonstrated linearity (r = 0.9999) over the range of atom % 15N values from close to natural abundance at 0.369 to a large degree of enrichment at 3.56. The slope of the line is 0.948 +/- 0.00183 and the y-intercept was 0.0267 +/- 0.00355.; A second project was the biosynthesis of 14C-vitamin B 12 to be used as a radioactive tracer for future human metabolism studies using AMS. The biosynthesis of 14C-vitamin B12 in Salmonella typhimurium is performed in a one-pot procedure. Fermentation is conducted with aeration and agitation in a single flask for 24 hours in the dark at 37°C with shaking at 250 RPM. Dimethylbenzimidazole (DMB) is used by the bacteria as a precursor to the imidazole moiety of the vitamin B12 molecule. 14C-DMB was synthesized from 14C-formic acid and dimethylphenylenediamine, and the resulting product added to the fermentation flask. The 14C-vitamin B12 was purified by HPLC and the identification confirmed through comparison of UV/vis spectra and LC/MS spectra with those of cyanocoblamin standards. The total mass yield of vitamin B12 produced was 25 mug (18.6 nmoles) and 880 nCi, corresponding to a specific activity of 47.3 mCi/mmol.