Nuclear magnetic resonance (NMR)-based 13C tracing is widely used in medicine, metabolomics, and environmental research. Here, we introduce a 2D 1H–1H (12C/13C) “in-phase/opposite-phase” (IP/OP) TOCSY experiment that uniquely generates 2D subspectra discriminating 13C–13C, 12C–13C, and 12C–12C connectivity. The sequence was demonstrated first on a standard mixture of 50/50 13C-phenylalanine and 1-13C-glucose, followed by an in vivo ethanol fermentation using brewer’s yeast (Saccharomyces cerevisiae) with 1-13C-glucose. Finally, incorporation of 13C in marine copepods (Tigriopus californicus) was monitored ex vivo. Copepods were analyzed at natural abundance and again on 7 days of feeding >98% 13C-enriched green algae. The sequence successfully identified three carbon pools: intact fragments or molecules from the 13C diet (13C–13C), intact fragments from pre-existing biomass (12C–12C), and new bond formation between 12C and 13C molecules during metabolite turnover (12C–13C). Molecules involved in osmotic regulation, alanine, proline, glycine, choline, betaine, and TMAO, were particularly abundant in the 12C–13C pool, suggesting rapid turnover by combining 13C food with existing 12C biomass. This was supported by a quantitative 1D 1H–(12C/13C) IP/OP experiment, measuring average enrichment of the six osmolytes at 22.8 ± 0.1% 13C on day 7. A complementary “singlet-only” experiment quantified glycine at 27.4 ± 0.2% 13C and betaine at 3.7 ± 0.4% 13C, enabling detection of molecules without scalar couplings. In summary, the 2D 1H–1H IP/OP TOCSY simultaneously identified pre-existing and newly synthesized molecules, while 1D experiments provide quantitative support, offering a sensitive framework to study carbon dynamics, preservation, and transformation in complex in vivo and ex vivo processes.
Steiner et al. (Sat,) studied this question.