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Poster Presentations
Day 2, June 23（Mon.）
Room P (Maesato East, Foyer, Ocean Wing)
- 2P-PM-22
Development of Targeted LC-MS/MS Platform for Precise NADHX Detection
(Waseda Univ.)
oKodai Takahashi, Kazuki Nakajima, Nobuhito Goda
ATP-dependent (S)-NAD(P)H-hydrate dehydratase (NAXD) is a crucial enzyme in the NAD repair system that regenerates NADH, an essential electron donor in metabolic redox reactions. NAD+ and its related metabolic pathways connect cellular metabolism and the expression of genes responsible for adipogenesis. Silencing of the Naxd gene attenuated differentiation-induced lipid accumulation. To investigate the relationship between NADHX and adipogenesis, we attempted to detect NADHX using an LC-MS system. However, NADHX remained undetectable. To demonstrate the toxicity of NADHX, we have enhanced the LC-MS system and developed a highly sensitive LC-MS/MS platform for precise NADHX quantification. A purification system was established, allowing us to isolate NADHX and identify its characteristic product ions. Using purified NADHX, we constructed calibration curves, enabling accurate comparisons of NAD-related metabolite levels. Optimization of solvent conditions and analytical parameters further refined detection capabilities, increasing sensitivity from 20 pmol in conventional LC-MS system to 1 pmol with LC-MS/MS platform. Enhanced sensitivity enabled NADHX detection in wild-type cells, unveiling its potential toxicity. The development of this experimental platform will be the way for uncovering novel cytotoxicity and pathological mechanisms driven by NADHX accumulation.