Research output: Contribution to journal › Article › peer-review
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.
In: International Journal of Molecular Sciences, Vol. 19, No. 12, 3906, 12.2018.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - NAD metabolome analysis in human cells using 1 H NMR spectroscopy
AU - Shabalin, Konstantin
AU - Nerinovski, Kirill
AU - Yakimov, Alexander
AU - Kulikova, Veronika
AU - Svetlova, Maria
AU - Solovjeva, Ljudmila
AU - Khodorkovskiy, Mikhail
AU - Gambaryan, Stepan
AU - Cunningham, Richard
AU - Migaud, Marie E.
AU - Ziegler, Mathias
AU - Nikiforov, Andrey
N1 - Funding Information: Funding: This work was supported by the Russian Science Foundation (grant No. 16-14-10240). Publisher Copyright: © 2018 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2018/12
Y1 - 2018/12
N2 - 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.
AB - 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.
KW - Human cells
KW - NAD metabolome
KW - NMR spectroscopy
KW - Vitamin B3
UR - http://www.scopus.com/inward/record.url?scp=85058324244&partnerID=8YFLogxK
U2 - 10.3390/ijms19123906
DO - 10.3390/ijms19123906
M3 - Article
C2 - 30563212
AN - SCOPUS:85058324244
VL - 19
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
SN - 1422-0067
IS - 12
M1 - 3906
ER -
ID: 73949594