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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.

SUBMITTER: Sun F

PROVIDER: S-EPMC3343042 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Biochemical issues in estimation of cytosolic free NAD/NADH ratio.

Sun Feifei F Dai Chunyan C Xie Jiansheng J Hu Xun X

PloS one 20120503 5

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 ...[more]

PMID: 22570687

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Project description:BackgroundDuring sepsis, serve vascular dysfunctions lead to life-threatening multiple organ failure, due to vascular smooth muscle cells (VSMC) impairments, resulting in vasoplegia, hypotension and hypoperfusion. In addition, septic patients have an altered cell metabolism that leads to lactic acidosis. Septic patients suffering from lactic acidosis have a high risk of mortality. In addition, septic survivors are at risk of secondary vascular disease. The underlying mechanisms of whether and how lactic acidosis leads to the changes in VSMCs is not well understood. The aim of this study was to comprehensively investigate the effect of lactic acidosis on VSMCs and additionally compare the effects with those induced by pure acidosis and sodium lactate.MethodsPrimary human aortic smooth muscle cells (HAoSMCs) were treated for 48 h with lactic acidosis (LA_pH 6.8), hydrochloric acid (HCl_pH 6.8), sodium lactate (Na+-lactate_pH 7.4) and the respective controls (ctrl._pH 7.4; hyperosmolarity control: mannitol_pH 7.4) and comparatively analyzed for changes in (i) transcriptome, (ii) energy metabolism, and (iii) phenotype.ResultsBoth types of acidosis led to comparable and sustained intracellular acidification without affecting cell viability. RNA sequencing and detailed transcriptome analysis revealed more significant changes for lactic acidosis than for hydrochloric acidosis, with lactate being almost ineffective, suggesting qualitative and quantitative synergism of acidosis and lactate. Bioinformatic predictions in energy metabolism and phenotype were confirmed experimentally. Lactic acidosis resulted in strong inhibition of glycolysis, glutaminolysis, and altered mitochondrial respiration which reduced cellular ATP content, likely due to increased TXNIP expression and altered NAD+/NADH ratio. Hydrochloric acidosis induced significantly smaller effects without changing the NAD+/NADH ratio, with the ATP content remaining constant. These metabolic changes led to osteo-/chondrogenic/senescent transdifferentiation of VSMCs, with the effect being more pronounced in lactic acidosis than in pure acidosis.ConclusionsOverall, lactic acidosis exerted a much stronger effect on energy metabolism than pure acidosis, whereas lactate had almost no effect, reflecting the qualitative and quantitative synergism of acidosis and lactate. As a consequence, lactic acidosis may lead to acute functional impairments of VSMC, sustained perturbations of the transcriptome and cellular dedifferentiation. Moreover, these effects may contribute to the acute and prolonged vascular pathomechanisms in septic patients.

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Biochemical issues in estimation of cytosolic free NAD/NADH ratio.

Publications

Biochemical issues in estimation of cytosolic free NAD/NADH ratio.

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