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Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells

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Abstract

Mitochondrial toxicity has been a serious concern, not only in preclinical drug development but also in clinical trials. In mitochondria, there are several distinct metabolic processes including fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), and each process contains discrete but often intimately linked steps. Interruption in any one of those steps can cause mitochondrial dysfunction. Detection of inhibition to OXPHOS can be complicated in vivo because intermediate endogenous metabolites can be recycled in situ or circulated systemically for metabolism in other organs or tissues. Commonly used assays for evaluating mitochondrial function are often applied to ex vivo or in vitro samples; they include various enzymatic or protein assays, as well as functional assays such as measurement of oxygen consumption rate, membrane potential, or acidification rates. Metabolomics provides quantitative profiles of overall metabolic changes that can aid in the unraveling of explicit biochemical details of mitochondrial inhibition while providing a holistic view and heuristic understanding of cellular bioenergetics. In this paper, we showed the application of quantitative NMR metabolomics to in vitro myotube cells treated with mitochondrial toxicants, rotenone and antimycin A. The close coupling of the TCA cycle to the electron transfer chain (ETC) in OXPHOS enables specific diagnoses of inhibition to ETC complexes by discrete biochemical changes in the TCA cycle.

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Abbreviations

NMR:

Nuclear magnetic resonance

FID:

Free induction decay

WET:

Water suppression enhanced through T1 effects

DSS-d6:

2,2-dimethyl-2-silapentane-5-sulfonate sodium, or sodium 3-(trimethylsilyl)-1-propanesulfonate

DMSO:

Dimethyl sulfoxide

LC–MS:

Liquid chromatography-mass spectrometer

MRS:

Magnetic resonance spectroscopy

TCA:

Tricarboxylic acid

ETC:

Electron transfer chain

PCr:

Phosphocreatine

ATP:

Adenosine triphosphate

ADP:

Adenosine diphosphate

NADH:

Reduced nicotinamide adenine dinucleotide

NAD+ :

Nicotinamide adenine dinucleotide

FADH2 :

Reduced flavin adenine dinucleotide

FAD+ :

Flavin adenine dinucleotide

CoQ or Q:

Coenzyme Q, or ubiquinone

CoQH2 :

Ubiquinol

OXPHOS:

Oxidative phosphorylation

OCR:

Oxygen consumption rate

ECAR:

Extracellular acidification rate

MPT:

Mitochondrial permeability transition

LDH:

Lactate dehydrogenase

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Acknowledgments

We would like to thank Dr. Oded Shaham for his skilled preparation of the cell samples, and helpful suggestion of data analyses, and Drs. Eric Schadt, Vamsi Mootha, and Jun Zhu for facilitating the project. We feel grateful to Jill Williams for her superb artistic touch to Fig. 1. We appreciate Drs. Steven Pitzenberger and Frank Sistare for their critical reading of the manuscript.

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Authors and Affiliations

  1. Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA, 19486, USA

    Qiuwei Xu, Heather Vu, Liping Liu & William H. Schaefer

  2. Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, NJ, 07065, USA

    Ting-Chuan Wang

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  1. Qiuwei Xu
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  2. Heather Vu
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  3. Liping Liu
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  4. Ting-Chuan Wang
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Correspondence to Qiuwei Xu.

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Xu, Q., Vu, H., Liu, L. et al. Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells. J Biomol NMR 49, 207–219 (2011). https://doi.org/10.1007/s10858-011-9482-8

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