biochemical issues in estimation of cytosolic free nadnadh ratio在胞质自由nadnadh估计比生化问题.pdfVIP

Biochemical Issues in Estimation of Cytosolic Free NAD/ NADH Ratio . . Feifei Sun , Chunyan Dai , Jiansheng Xie, Xun Hu* Key Laboratory for Cancer Intervention and Prevention, Zhejiang Provincial Key Laboratory of Molecular Biology in Medical Sciences, China National Ministry of Education, Cancer Institute, School of Medicine, Zhejiang University, The Second Affiliated Hospital, Hangzhou, Zhejiang, China 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