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Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach

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Abstract

Introduction

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD+ and its related metabolites (hereafter, the NAD+ metabolome) represents an important indicator of cellular function.

Objectives

A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD+ metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS).

Methods

The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode.

Results

Quantification of 17 metabolites in the NAD+ metabolome in U251 human astroglioma cells could be achieved. Changes in NAD+ metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated.

Conclusion

This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.

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Acknowledgements

The authors would like to thank the Mark Wainwright Analytical Centre in conjunction with the Bioanalytical Mass Spectrometry Facility for providing use of the TSQ Vantage bench-top mass spectrometer, analytical grade reagents and data processing software. NB is recipient of the ARC DECRA Fellowship.

Funding

This study was funded by Mark Wainwright Analytical Centre Research Grant (No. 2016).

Author information

Authors and Affiliations

  1. Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia

    Sonia Bustamante

  2. Faculty of Medicine, School of Psychiatry, Centre for Healthy Brain Ageing, University of New South Wales Sydney, Sydney, Australia

    Tharusha Jayasena, Perminder Singh Sachdev & Nady Braidy

  3. Faculty of Medicine, School of Women’s and Children’s Health, University of New South Wales Sydney, Sydney, Australia

    Dulama Richani & Robert Bruce Gilchrist

  4. Department of Pharmacology, School of Medical Sciences, University of New South Wales Sydney, Sydney, NSW, 2052, Australia

    Lindsay E. Wu & David A. Sinclair

  5. Department of Genetics, Paul F. Glenn Center for the Biology of Aging, Harvard Medical School, Boston, MA, 02115, USA

    David A. Sinclair

  6. Neuropsychiatric Institute, Euroa Centre, Prince of Wales Hospital, Sydney, Australia

    Perminder Singh Sachdev

  7. UNSW School of Psychiatry, NPI, Euroa Centre, Prince of Wales Hospital, Barker Street, Randwick, Sydney, NSW, 2031, Australia

    Nady Braidy

Authors
  1. Sonia Bustamante
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  2. Tharusha Jayasena
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  3. Dulama Richani
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  4. Robert Bruce Gilchrist
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  5. Lindsay E. Wu
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  6. David A. Sinclair
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  7. Perminder Singh Sachdev
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  8. Nady Braidy
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Correspondence to Nady Braidy.

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Conflict of interest

LW and DS are non-paid directors and shareholders of MetroBiotech NSW Pty Ltd, whose patents concern the use of NAD+ elevating agents to treat disease and are unrelated to nicotinamide (WO2014059034 and Australian provisional patent application number 2013904415). DS is a shareholder and paid consultant to GlaxoSmithKline, a shareholder and scientific advisor to OvaScience, and a shareholder and director to MetroBiotech NSW Pty Ltd and MetroBiotech LLC. None of these interests relate to the present work. The authors confirm that they have no restrictions on the sharing of data or materials related to this work. This does not alter the authors’ adherence to Metabolomics policies on sharing data and materials.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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Bustamante, S., Jayasena, T., Richani, D. et al. Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach. Metabolomics 14, 15 (2018). https://doi.org/10.1007/s11306-017-1310-z

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