Analytical Approaches for the Quantitation of Redox-active Pyridine Dinucleotides in Biological Matrices

Somogyi, Anna; Horvai, George; Csala, Miklós; Tóth, Blanka

doi:10.3311/ppch.9470

Cited by 6 publications

(5 citation statements)

References 37 publications

(135 reference statements)

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“…Its high selectivity, sensitivity and accuracy make HPLC-ESI-MS/MS technique suitable for efficient and precise quantitative analysis of various cellular compounds of low molecular weight and of similar structures in biological matrices [33]. Here, we demonstrate the development of a reverse-phased HPLC-ESI-MS/MS method for the simultaneous determination of a wide range of ceramides and DAGs in cultured cells.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Somogyi

Donkó

Sarnyai

et al. 2020

Period. Polytech. Chem. Eng.

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A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well.

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Section: Discussionmentioning

confidence: 99%

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Somogyi

Donkó

Sarnyai

et al. 2020

Period. Polytech. Chem. Eng.

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“…NAD + and NADP + are coenzymes involved in redox reactions in eukaryotic cells, including cellular metabolism and various processes. – The reduced forms, NADH and NADPH, respectively, maintain cellular redox homeostasis and modulate biological events. – Imbalances or deficiencies in these redox couples are associated with pathological disorders. , They are also involved in several redox reactions, such as energy metabolism, gluconeogenesis, oxidative phosphorylation, glycolysis, and the tricarboxylic acid cycle, as well as various cellular processes, including calcium homeostasis, oxidative stress, gene repair, mitochondrial functions, embryonic development, immune function, transcription, cell cycle, cell death, cellular senescence, hypoglycemia, apoptosis, hypoxia, and virus infections. – Detecting NAD(P)H concentrations is crucial for understanding physiological developments and pathological mechanisms. Assessing NAD(P)H levels in live cells provides valuable insights into cellular redox homeostasis and modulates biological events.…”

Section: Introductionmentioning

confidence: 99%

“…1−3 Imbalances or deficiencies in these redox couples are associated with pathological disorders. 4,5 They are also involved in several redox reactions, such as energy metabolism, gluconeogenesis, oxidative phosphorylation, glycolysis, and the tricarboxylic acid cycle, as well as various cellular processes, including calcium homeostasis, oxidative stress, gene repair, mitochondrial functions, embryonic development, immune function, transcription, cell cycle, cell death, cellular senescence, hypoglycemia, apoptosis, hypoxia, and virus infections. 1−5 Detecting NAD(P)H concentrations is crucial for understanding physiological developments and pathological mechanisms.…”

Section: ■ Introductionmentioning

confidence: 99%

Highly Sensitive Cyanine Dyes for Rapid Sensing of NAD(P)H in Mitochondria and First-Instar Larvae of Drosophila melanogaster

Arachchige

Dwivedi

Jaeger

et al. 2023

ACS Appl. Bio Mater.

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We have developed two highly sensitive cyanine dyes, which we refer to as probes A and B. These dyes are capable of quick and sensitive sensing of NAD(P)H. The dyes were fabricated by connecting benzothiazolium and 2,3dimethylnaphtho[1,2-d]thiazol-3-ium units to 3-quinolinium through a vinyl bond. In the absence of NAD(P)H, both probes have low fluorescence and absorption peaks at 370 and 400 nm, correspondingly. This is because of their two electron-withdrawing acceptor systems with high charge densities. However, when NAD(P)H reduces the probes' electron-withdrawing 3-quinolinium units to electron-donating 1,4-dihydroquinoline units, the probes absorb at 533 and 535 nm and fluoresce at 572 and 586 nm for A and B correspondingly. This creates well-defined donor−π− acceptor cyanine dyes. We successfully used probe A to monitor NAD(P)H levels in live cells during glycolysis, under hypoxic conditions induced by CoCl 2 treatment and after treatment with cancer drugs, including cisplatin, camptothecin, and gemcitabine. Probe A was also employed to visualize NAD(P)H in Drosophila melanogaster first-instar larvae. We observed an increase in NAD(P) H levels in A549 cancer cells both under hypoxic conditions and after treatment with cancer drugs, including cisplatin, camptothecin, and gemcitabine.

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“…Contingent on the subcellular pool (for example, cytosol, mitochondria, or ER), the phosphorylated or nonphosphorylated pyridine redox ratios can differ logarithmically (>10 4 ) (Somogyi et al. ). And, accordingly, the ratios therein dictate the subcellular metabolic processes in each domain (Hosios and van der Heiden ).…”

Section: Introductionmentioning

confidence: 99%

“…In vitro experimental approaches must approximate extant eukaryotic pyridine nucleotide concentrations, and their ratios, in disparate tissue organelles. Contingent on the subcellular pool (for example, cytosol, mitochondria, or ER), the phosphorylated or nonphosphorylated pyridine redox ratios can differ logarithmically (>10 4 ) (Somogyi et al 2016). And, accordingly, the ratios therein dictate the subcellular metabolic processes in each domain (Hosios and van der Heiden 2018).…”

Section: Introductionmentioning

confidence: 99%

Pyridine nucleotide regulation of hepatic endoplasmic reticulum calcium uptake

2019

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Pyridine nucleotides serve an array of intracellular metabolic functions such as, to name a few, shuttling electrons in enzymatic reactions, safeguarding the redox state against reactive oxygen species, cytochrome P450 (CYP) enzyme detoxification pathways and, relevant to this study, the regulation of ion fluxes. In particular, the maintenance of a steep calcium gradient between the cytosol and endoplasmic reticulum ( ER ), without which apoptosis ensues, is achieved by an elaborate combination of energy–requiring ER membrane pumps and efflux channels. In liver microsomes, net calcium uptake was inhibited by physiological concentrations of NADP . In the presence of 1 mmol/L NADP , calcium uptake was attenuated by nearly 80%, additionally, this inhibitory effect was blunted by concomitant addition of NADPH . No other nicotinamide containing compounds ‐save a slight inhibition by NAADP ‐hindered calcium uptake; thus, only oxidized pyridine nucleotides, or related compounds with a phosphate moiety, had an imposing effect. Moreover, the NADP inhibition was evident even after selectively blocking ER calcium efflux channels. Given the fundamental role of endoplasmic calcium homeostasis, it is plausible that changes in cytosolic NADP concentration, for example, during anabolic processes, could regulate net ER calcium uptake.

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Analytical Approaches for the Quantitation of Redox-active Pyridine Dinucleotides in Biological Matrices

Cited by 6 publications

References 37 publications

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Highly Sensitive Cyanine Dyes for Rapid Sensing of NAD(P)H in Mitochondria and First-Instar Larvae of Drosophila melanogaster

Pyridine nucleotide regulation of hepatic endoplasmic reticulum calcium uptake

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