title: Biochemical issues in estimation of cytosolic free NAD/NADH ratio.
abstract: Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al reported a method to estimate this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chemical equilibrium. Numerous studies used L/P ratio to estimate the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equilibrium but not verifying how near it was. In addition, it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equilibrium; (2) that cytosolic free NAD/NADH could be accurately estimated by L/P only when the conversion is at or very close to equilibrium otherwise a calculation error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estimation of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P.
authors.affiliation: Key Laboratory for Cancer Intervention and Prevention, China National Ministry of Education, Cancer Institute, School of Medicine, Zhejiang University, The Second Affiliated Hospital, Hangzhou, Zhejiang, China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Sun
authors.forename: Chunyan
authors.initials: C
authors.lastname: Dai
authors.forename: Jiansheng
authors.initials: J
authors.lastname: Xie
authors.forename: Xun
authors.initials: X
authors.lastname: Hu
mesh_terms: D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011773:Pyruvates / Q000378:metabolism
chemical_list: D011773:Pyruvates; D009243:NAD; D019344:Lactic Acid