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NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modificati...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321329/ https://www.ncbi.nlm.nih.gov/pubmed/30563212 http://dx.doi.org/10.3390/ijms19123906 |
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author | Shabalin, Konstantin Nerinovski, Kirill Yakimov, Alexander Kulikova, Veronika Svetlova, Maria Solovjeva, Ljudmila Khodorkovskiy, Mikhail Gambaryan, Stepan Cunningham, Richard Migaud, Marie E. Ziegler, Mathias Nikiforov, Andrey |
author_facet | Shabalin, Konstantin Nerinovski, Kirill Yakimov, Alexander Kulikova, Veronika Svetlova, Maria Solovjeva, Ljudmila Khodorkovskiy, Mikhail Gambaryan, Stepan Cunningham, Richard Migaud, Marie E. Ziegler, Mathias Nikiforov, Andrey |
author_sort | Shabalin, Konstantin |
collection | PubMed |
description | Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that (1)H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome. |
format | Online Article Text |
id | pubmed-6321329 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-63213292019-01-07 NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy Shabalin, Konstantin Nerinovski, Kirill Yakimov, Alexander Kulikova, Veronika Svetlova, Maria Solovjeva, Ljudmila Khodorkovskiy, Mikhail Gambaryan, Stepan Cunningham, Richard Migaud, Marie E. Ziegler, Mathias Nikiforov, Andrey Int J Mol Sci Article Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that (1)H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome. MDPI 2018-12-06 /pmc/articles/PMC6321329/ /pubmed/30563212 http://dx.doi.org/10.3390/ijms19123906 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Shabalin, Konstantin Nerinovski, Kirill Yakimov, Alexander Kulikova, Veronika Svetlova, Maria Solovjeva, Ljudmila Khodorkovskiy, Mikhail Gambaryan, Stepan Cunningham, Richard Migaud, Marie E. Ziegler, Mathias Nikiforov, Andrey NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy |
title | NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy |
title_full | NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy |
title_fullStr | NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy |
title_full_unstemmed | NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy |
title_short | NAD Metabolome Analysis in Human Cells Using (1)H NMR Spectroscopy |
title_sort | nad metabolome analysis in human cells using (1)h nmr spectroscopy |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321329/ https://www.ncbi.nlm.nih.gov/pubmed/30563212 http://dx.doi.org/10.3390/ijms19123906 |
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