Nitrogen metabolism of asparagine and glutamate in Vero cells studied by (1)H/ (15)N NMR spectroscopy.

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. (15)N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with L: -[(15)N]glutamate, the (15)N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable (15)NH(+)(4) signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with L: -[2-(15)N]asparagine, the (15)N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with L: -[4-(15)N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of (15)NH(+)(4), showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.