GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O Isotopes
Nitrite (O=N-O<sup>−</sup>, NO<sub>2</sub><sup>−</sup>) and nitrate (O=N(O)-O<sup>−</sup>, NO<sub>3</sub><sup>−</sup>) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-05-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/28/11/4281 |
| _version_ | 1797597078958899200 |
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| author | Dimitrios Tsikas |
| author_facet | Dimitrios Tsikas |
| author_sort | Dimitrios Tsikas |
| collection | DOAJ |
| description | Nitrite (O=N-O<sup>−</sup>, NO<sub>2</sub><sup>−</sup>) and nitrate (O=N(O)-O<sup>−</sup>, NO<sub>3</sub><sup>−</sup>) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (<sup>●</sup>NO). <sup>●</sup>NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of <sup>●</sup>NO synthases. It is considered that the autoxidation of <sup>●</sup>NO in aqueous solutions and in O<sub>2</sub>-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO<sup>●</sup>) intermediates. In aqueous buffers, endogenous <i>S</i>-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., <i>S</i>-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., <i>S</i>-nitrosoglutathione, GSNO) may be formed during the autoxidation of <sup>●</sup>NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O<sup>−</sup> + H<sup>+</sup>; p<i>K</i><sub>a</sub><sup>HONO</sup>, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of <sup>●</sup>NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (<sup>14</sup>NO<sub>2</sub><sup>−</sup>) and labeled nitrite (<sup>15</sup>NO<sub>2</sub><sup>−</sup>) and RSNO (RS<sup>15</sup>NO, RS<sup>15</sup>N<sup>18</sup>O) performed in pH-neutral aqueous buffers of phosphate or t<i>ris</i>(hydroxyethylamine) prepared in unlabeled (H<sub>2</sub><sup>16</sup>O) or labeled H<sub>2</sub>O (H<sub>2</sub><sup>18</sup>O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography–mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of <sup>●</sup>NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl<sub>2</sub> accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating <sup>18</sup>O from H<sub>2</sub><sup>18</sup>O into the SNO group. In aqueous buffers prepared in H<sub>2</sub><sup>18</sup>O, synthetic peroxynitrite (ONOO<sup>−</sup>) decomposes to nitrite without <sup>18</sup>O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS<sup>15</sup>NO and H<sub>2</sub><sup>18</sup>O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of <sup>●</sup>NO and hydrolysis of RSNO. |
| first_indexed | 2024-03-11T03:01:42Z |
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| id | doaj.art-76086c64328241a2b6772b22c4f6c14c |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-11T03:01:42Z |
| publishDate | 2023-05-01 |
| publisher | MDPI AG |
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| series | Molecules |
| spelling | doaj.art-76086c64328241a2b6772b22c4f6c14c2023-11-18T08:14:28ZengMDPI AGMolecules1420-30492023-05-012811428110.3390/molecules28114281GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O IsotopesDimitrios Tsikas0Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, GermanyNitrite (O=N-O<sup>−</sup>, NO<sub>2</sub><sup>−</sup>) and nitrate (O=N(O)-O<sup>−</sup>, NO<sub>3</sub><sup>−</sup>) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (<sup>●</sup>NO). <sup>●</sup>NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of <sup>●</sup>NO synthases. It is considered that the autoxidation of <sup>●</sup>NO in aqueous solutions and in O<sub>2</sub>-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO<sup>●</sup>) intermediates. In aqueous buffers, endogenous <i>S</i>-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., <i>S</i>-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., <i>S</i>-nitrosoglutathione, GSNO) may be formed during the autoxidation of <sup>●</sup>NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O<sup>−</sup> + H<sup>+</sup>; p<i>K</i><sub>a</sub><sup>HONO</sup>, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of <sup>●</sup>NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (<sup>14</sup>NO<sub>2</sub><sup>−</sup>) and labeled nitrite (<sup>15</sup>NO<sub>2</sub><sup>−</sup>) and RSNO (RS<sup>15</sup>NO, RS<sup>15</sup>N<sup>18</sup>O) performed in pH-neutral aqueous buffers of phosphate or t<i>ris</i>(hydroxyethylamine) prepared in unlabeled (H<sub>2</sub><sup>16</sup>O) or labeled H<sub>2</sub>O (H<sub>2</sub><sup>18</sup>O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography–mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of <sup>●</sup>NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl<sub>2</sub> accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating <sup>18</sup>O from H<sub>2</sub><sup>18</sup>O into the SNO group. In aqueous buffers prepared in H<sub>2</sub><sup>18</sup>O, synthetic peroxynitrite (ONOO<sup>−</sup>) decomposes to nitrite without <sup>18</sup>O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS<sup>15</sup>NO and H<sub>2</sub><sup>18</sup>O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of <sup>●</sup>NO and hydrolysis of RSNO.https://www.mdpi.com/1420-3049/28/11/4281autoxidationderivatizationGC-MSnitric oxidepentafluorobenzyl bromidestable isotopes |
| spellingShingle | Dimitrios Tsikas GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O Isotopes Molecules autoxidation derivatization GC-MS nitric oxide pentafluorobenzyl bromide stable isotopes |
| title | GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O Isotopes |
| title_full | GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O Isotopes |
| title_fullStr | GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O Isotopes |
| title_full_unstemmed | GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O Isotopes |
| title_short | GC-MS Studies on Nitric Oxide Autoxidation and <i>S</i>-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using <sup>15</sup>N and <sup>18</sup>O Isotopes |
| title_sort | gc ms studies on nitric oxide autoxidation and i s i nitrosothiol hydrolysis to nitrite in ph neutral aqueous buffers definite results using sup 15 sup n and sup 18 sup o isotopes |
| topic | autoxidation derivatization GC-MS nitric oxide pentafluorobenzyl bromide stable isotopes |
| url | https://www.mdpi.com/1420-3049/28/11/4281 |
| work_keys_str_mv | AT dimitriostsikas gcmsstudiesonnitricoxideautoxidationandisinitrosothiolhydrolysistonitriteinphneutralaqueousbuffersdefiniteresultsusingsup15supnandsup18supoisotopes |