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Biochemical issues in estimation of cytosolic free NAD/NADH ratio - PubMed

Biochemical issues in estimation of cytosolic free NAD/NADH ratio

Feifei Sun et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Intracellular lactate concentration and L/P ratio are highly labile in Bcap-37 cells.

Bcap-37 cells were incubated in complete RPMI-1640 medium containing 12 mM glucose and supplemented with 6 mM glucose every 24 hours. At the indicated time point, intracellular lactate and pyruvate, glucose consumption and lactate generation by Bcap-37 cells, and cell growth were monitored. (A) Intracellular lactate; (B) Intracellular pyruvate; (C) Intracellular L/P ratio; (D) Cytosolic free NAD/NADH ratio erroneously estimated from the corresponding L/P ratio; (E) Cell proliferation curves; (F) Glucose consumption; (G) Lactate generation; (H) L/G ratio (the generated lactate divided by the consumed glucose between 2 time points). Data are mean±SD. Data were confirmed by 2 independent experiments.

Figure 2
Figure 2. The effect of extracellular lactate concentration on intracellular lactate concentration and intracellular L/P ratio in Bcap-37 cells.

Bcap-37 cells were incubated in complete RPMI-1640 medium containing 12 mM glucose supplemented with 20 mM lactate. At the indicated time point, intracellular lactate and pyruvate were measured. (A) Intracellular lactate; (B) Intracellular pyruvate; (C) Intracellular L/P ratio. Data are mean ± SD. Data were confirmed by 2 independent experiments.

Figure 3
Figure 3. Cytosolic free NAD/NADH ratio estimated at the conversion at equilibrium in Bcap-37 cells.

Bcap-37 cells were incubated in complete RPMI-1640 medium containing 12 mM glucose supplemented with or without lactate (x axis). After 24-hour incubation, glucose consumption and lactate generation by Bcap-37 cells, cell growth, and intracellular lactate and pyruvate were measured. (A) Cell proliferation; (B) Glucose consumption; (C) Lactate generation; (D) L/G ratio; (E) Intracellular pyruvate; (F) Intracellular lactate; (G) Intracellular L/P ratio; (H) Cytosolic free NAD/NADH ratio estimated from the corresponding L/P ratio(see corresponding text). Data are mean±SD. Data were confirmed by 3 independent experiments.

Figure 4
Figure 4. The effect of oxygen levels on intracellular concentration of lactate and pyruvate in Hela cells.

Hela cells were incubated in complete RPMI-1640 medium containing 12 mM glucose. After 24-hour incubation, glucose consumption and lactate generation by Hela cells, cell growth, and intracellular lactate and pyruvate were measured. (A) Cell proliferation; (B) Glucose consumption; (C) Lactate generation; (D) L/G ratio; (E) Intracellular pyruvate; (F) Intracellular lactate; (G) Intracellular L/P ratio; (H) Cytosolic free NAD/NADH ratio erroneously estimated from the corresponding L/P ratio. Data are mean±SD. Data were confirmed by 2 independent experiments.

Figure 5
Figure 5. The effect of oxygen levels on cytosolic free NAD/NADH ratios in Hela cells.

Hela cells were incubated in complete RPMI-1640 medium containing 12 mM glucose supplemented with 20 mM lactate under oxygen levels between 21% to 1%. After 24-hour incubation, glucose consumption and lactate generation by Hela cells, cell growth, and intracellular lactate and pyruvate were measured. (A) Cell proliferation; (B) Glucose consumption; (C) Lactate generation; (D) L/G ratio; (E) Intracellular pyruvate; (F) Intracellular lactate; (G) Intracellular L/P ratio; (H) Cytosolic free NAD/NADH ratio estimated from the corresponding L/P ratio (see description in text). Data are mean±SD. Data were confirmed by 2 independent experiments.

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