pmedpharmФармация и фармакологияPharmacy & Pharmacology2307-92662413-2241Pyatigorsk Medical and Pharmaceutical Institute - branch of Volgograd State Medical Univer10.19163/2307-9266-2024-12-1-49-62pmedpharm-1450Research ArticleОРИГИНАЛЬНАЯ СТАТЬЯRESEARCH ARTICLEРазработка методики количественного определения оксида азота (NO) в тканях крыс с применением высокоэффективной жидкостной хроматографии и масс-спектрометрииDevelopment of a method for quantitative determination of nitric oxide (NO) in rat tissues based on high-performance liquid chromatography and mass spectrometryhttps://orcid.org/0000-0002-1792-7414ПоповН. С.PopovN. S.

кандидат фармацевтических наук, доцент кафедры фармакологии и клинической фармакологии, заведующий научно-исследовательской лабораторией, ФГБОУ ВО Тверской ГМУ Минздрава России. 

170100, Россия, г. Тверь, ул. Советская, д. 4.

Candidate of Sciences (Pharmacy), Associate Professor of the Department Of Pharmacology and Clinical Pharmacology, head of the Research Laboratory of Tver State Medical University.

4, Sovietskaya Str., Tver, Russia, 170100.

ns.popov@mail.ruhttps://orcid.org/0009-0003-8692-0878ГавриленкоД. А.GavrilenkoD. A.

ассистент кафедры химии ФГБОУ ВО Тверской ГМУ Минздрава России.

170100, Россия, г. Тверь, ул. Советская, д. 4.

assistant at the Department of Chemistry of Tver State Medical University.

4, Sovietskaya Str., Tver, Russia, 170100.

dimirdome@gmail.comhttps://orcid.org/0000-0002-9339-7603БарановМ. С.BaranovM. S.

доктор химических наук, старший научный сотрудник научно-исследовательской лаборатории химии лекарственных субстанций НИИ трансляционной медицины, ФГАОУ ВО РНИМУ им. Н.И. Пирогова Минздрава России. 

117997, Россия, г. Москва, ул. Островитянова, д. 1

Doctor of Sciences (Chemistry), senior researcher at the Research Laboratory of the Chemistry of Medicinal Substances of Research Institute of Translational Medicine, Pirogov Russian National Research Medical University. 

1, Ostrovityanov Str., Moscow, Russia, 117997.

baranovmikes@gmail.comhttps://orcid.org/0000-0002-5744-7060БалабаньянВ. Ю.BalabanyanV. Yu.

доктор фармацевтических наук, доцент, ведущий научный сотрудник научно-исследовательской лаборатории химии лекарственных субстанций НИИ трансляционной медицины, ФГАОУ ВО РНИМУ им. Н.И. Пирогова Минздрава России.

117997, Россия, г. Москва, ул. Островитянова, д. 1

Doctor of Sciences (Pharmacy), Associate Professor, leading researcher at the Research Laboratory of the Chemistry of Medicinal Substances of Research Institute of Translational Medicine, Pirogov Russian National Research Medical University. 

1, Ostrovityanov Str., Moscow, Russia, 117997.

bal.pharm@mail.ruФедеральное государственное бюджетное образовательное учреждение высшего образования «Тверской государственный медицинский университет» Министерства здравоохранения Российской Федерации.РоссияTver State Medical UniversityRussian FederationФедеральное государственное автономное образовательное учреждение высшего образования «Российский национальный исследовательский медицинский университет имени Н.И. Пирогова» Министерства здравоохранения Российской Федерации.РоссияPirogov Russian National Research Medical UniversityRussian Federation2024080520241214962Copyright © Попов Н.С., Гавриленко Д.А., Баранов М.С., Балабаньян В.Ю., 20242024Попов Н.С., Гавриленко Д.А., Баранов М.С., Балабаньян В.Ю.Popov N.S., Gavrilenko D.A., Baranov M.S., Balabanyan V.Y.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.pharmpharm.ru/jour/article/view/1450

Количественная оценка продукции оксида азота (NO) в тканях организма является актуальной задачей фармакологии и биохимии. Изучение физиологических процессов, протекающих с участием NO, а также метаболизма и фармакодинамики фармакологических средств из группы донаторов NO требует внедрения точных и воспроизводимых методик количественного определения данного метаболита в биологических средах.

Цель

Цель. Разработка ВЭЖХ-МС/МС методики количественного определения оксида азота в различных тканях крыс.

Материалы и методы

Материалы и методы. Непрямое количественное определение NO было основано на оценке уровня более стабильных метаболитов: нитритов и нитратов, которые извлекали из тканей крыс путем гомогенизации с водой. Восстановление нитратов до нитритов осуществляли с помощью нитрат редуктазы. В основе дериватизации нитритов использовали реакцию с реактивом Грисса. Полученный азокраситель определяли с помощью ВЭЖХ-МС/МС с использованием аналитической колонки Agilent InfinityLab Poroshell 120 EC-C18 4,6×100 мм, 2,7 мкм. Общее время хроматографического анализа составило 12 минут, время удерживания аналита составило 6,1 минут. Аналитический диапазон методики составил 0,1–100,0 нмоль (в пересчете на нитрит) на 1 мл плазмы или гомогената ткани.

Результаты

Результаты. Разработанная биоаналитическая методика была валидирована по следующим параметрам: селективность, матричный эффект, степень извлечения, перенос пробы, линейность аналитического диапазона, нижний предел количественного определения (НПКО), внутри- и межсерийная точность и прецизионность, стабильность на всех этапах анализа. Для апробации методики было проведено определение содержания NO в плазме, головном мозге, сердце, аорте и легких крыс.

Заключение

Заключение. Разработанная биоаналитическая ВЭЖХ-МС/МС-методика полностью соответствует валидационным требованиям. Метрологические характеристики методики позволяют с высокой точностью оценить продукцию NO в различных тканях крыс, что, несомненно, представляет актуальным и востребованным в исследовании патологических процессов, а также механизма действия фармакологических средств из группы донаторов NO.

A quantitative assessment of nitric oxide (NO) production in body tissues is an urgent problem in pharmacology and biochemistry. The study of physiological processes occurring with the participation of NO, as well as the metabolism and pharmacodynamics of pharmacological agents from the group of NO donors, requires the introduction of accurate and reproducible methods for the quantitative determination of this metabolite in biological media.

The aim of the study was to develop the HPLC-MS/MS methods for the quantitative determination of NO in various tissues of rats.

Materials and methods

Materials and methods. The indirect NO quantification was based on estimation of the level of more stable metabolites: nitrites and nitrates extracted from rat tissues by homogenization with water. The reduction of nitrates to nitrites was carried out using nitrate reductase. The derivatization of nitrites was based on a reaction with Griess reagent. The resulting azo dye was determined by HPLC-MS/MS using an Agilent InfinityLab Poroshell 120 EC-C18 4.6×100 mm, 2.7 μm analytical column. The total chromatographic analysis time was 12 minutes, and the analyte retention time was 6.1 minutes. The analytical range of the method was 0.1–100.0 nmol (in terms of nitrite) per 1 ml of plasma or tissue homogenate.

Results

Results. The developed a bioanalytical method was validated according to the following parameters: a selectivity, a matrix effect, a recovery degree, a sample transfer, an analytical range linearity, a lower limit of quantification (LLOQ), an intra- and inter-assay accuracy and precision, and a stability at all the stages of the analysis. To test the method, the NO content in the plasma, brain, heart, aorta and lungs of rats was determined.

Conclusion

Conclusion. The developed bioanalytical HPLC-MS/MS methods fully meets the validation requirements. The metrological characteristics of the technique make it possible to highly accurately estimate the NO production in various tissues of rats, which is undoubtedly relevant and in demand in the study of pathological processes as well as the mechanism of action of pharmacological agents from the group of NO donors.

ВЭЖХ-МС/МСхроматографиямасс-спектрометрияэндотелиальный релаксирующий фактороксид азотаHPLC-MS/MSchromatographymass spectrometryendothelial relaxing factornitric oxideДанное исследование было выполнено в рамках государственного задания, номер госрегистрации – 124020900020-4.This study was carried out as part of a state assignment, the state registration number is 124020900020-4.ReferencesIgnarro L.J. Nitric oxide is not just blowing in the wind // Br J Pharmacol. – 2019. – Vol. 176, No. 2. – P. 131. DOI: 10.1111/bph.14540Ignarro LJ. Nitric oxide is not just blowing in the wind. Br J Pharmacol. 2019;176(2):131–4. DOI: 10.1111/bph.14540Lundberg J.O., Weitzberg E. Nitric oxide signaling in health and disease // Cell. – 2022. – Vol. 185, No. 16. – P. 2853–2878. DOI: 10.1016/j.cell.2022.06.010Lundberg JO, Weitzberg E. Nitric oxide signaling in health and disease. Cell. 2022;185(16):2853–78. DOI: 10.1016/j.cell.2022.06.010Zweier J.L., Ilangovan G. Regulation of nitric oxide metabolism and vascular tone by cytoglobin // Antioxid Redox Signal. – 2020. – Vol. 32, No. 16. – P. 1172–1187. DOI: 10.1089/ars.2019.7881Zweier JL, Ilangovan G. Regulation of Nitric Oxide Metabolism and Vascular Tone by Cytoglobin. Antioxid Redox Signal. 2020;32(16):1172–87. DOI: 10.1089/ars.2019.7881Gupta R.M., Libby P., Barton M. Linking regulation of nitric oxide to endothelin-1: The Yin and Yang of vascular tone in the atherosclerotic plaque // Atherosclerosis. – 2020. – Vol. 292. – P. 201–203. DOI: 10.1016/j.atherosclerosis.2019.11.001Gupta RM, Libby P, Barton M. Linking regulation of nitric oxide to endothelin-1: The Yin and Yang of vascular tone in the atherosclerotic plaque. Atherosclerosis. 2020;292:201–3. DOI: 10.1016/j.atherosclerosis.2019.11.001Enayati M., Schneider K.H., Almeria C., Grasl C., Kaun C., Messner B., Rohringer S., Walter I., Wojta J., Budinsky L., Walpoth B.H., Schima H., Kager G., Hallström S., Podesser B.K., Bergmeister H. S-nitroso human serum albumin as a nitric oxide donor in drug-eluting vascular grafts: Biofunctionality and preclinical evaluation // Acta Biomater. – 2021. – Vol. 134. – P. 276–288. DOI: 10.1016/j.actbio.2021.07.048Enayati M, Schneider KH, Almeria C, Grasl C, Kaun C, Messner B, Rohringer S, Walter I, Wojta J, Budinsky L, Walpoth BH, Schima H, Kager G, Hallström S, Podesser BK, Bergmeister H. S-nitroso human serum albumin as a nitric oxide donor in drug-eluting vascular grafts: Biofunctionality and preclinical evaluation. Acta Biomater. 2021;134:276–88. DOI: 10.1016/j.actbio.2021.07.048Shvetsova A.A., Borzykh A.A., Selivanova E.K., Kiryukhina O.O., Gaynullina D.K., Tarasova O.S. Intrauterine Nitric Oxide Deficiency Weakens Differentiation of Vascular Smooth Muscle in Newborn Rats // Int J Mol Sci. – 2021. – Vol. 22, No. 15. – P. 8003. DOI: 10.3390/ijms22158003Shvetsova AA, Borzykh AA, Selivanova EK, Kiryukhina OO, Gaynullina DK, Tarasova OS. Intrauterine Nitric Oxide Deficiency Weakens Differentiation of Vascular Smooth Muscle in Newborn Rats. Int J Mol Sci. 2021;22(15):8003. DOI: 10.3390/ijms22158003Grigorieva M.E., Myasoedov N.F., Lyapina L.A. Participation of Nitric Oxide in the Realization of Hemostatic Effects of Glyproline Peptides // Dokl Biochem Biophys. 2022. – Vol. 506, No. 1. – P. 177–180. DOI: 10.1134/S1607672922050052Grigorieva ME, Myasoedov NF, Lyapina LA. Participation of Nitric Oxide in the Realization of Hemostatic Effects of Glyproline Peptides. Dokl Biochem Biophys. 2022;506(1):177–80. DOI: 10.1134/S1607672922050052Norooznezhad A.H., Mansouri K. Endothelial cell dysfunction, coagulation, and angiogenesis in coronavirus disease 2019 (COVID-19) // Microvasc Res. – 2021. – Vol. 137. – P. 104188. DOI: 10.1016/j.mvr.2021.104188Norooznezhad AH, Mansouri K. Endothelial cell dysfunction, coagulation, and angiogenesis in coronavirus disease 2019 (COVID-19). Microvasc Res. 2021;137:104188. DOI: 10.1016/j.mvr.2021.104188Wallace J.L. Nitric oxide in the gastrointestinal tract: opportunities for drug development // Br J Pharmacol. – 2019. – Vol. 176, No. 2. – Р. 147–154. DOI: 10.1111/bph.14527Wallace JL. Nitric oxide in the gastrointestinal tract: opportunities for drug development. Br J Pharmacol. 2019;176(2):147–54. DOI: 10.1111/bph.14527Sanders K.M., Ward S.M. Nitric oxide and its role as a non-adrenergic, non-cholinergic inhibitory neurotransmitter in the gastrointestinal tract // Br J Pharmacol. – 2019. – Vol. 176, No. 2. – P. 212–227. DOI: 10.1111/bph.14459Sanders KM, Ward SM. Nitric oxide and its role as a non-adrenergic, non-cholinergic inhibitory neurotransmitter in the gastrointestinal tract. Br J Pharmacol. 2019;176(2):212–27. DOI: 10.1111/bph.14459Tripathi M.K., Kartawy M., Amal H. The role of nitric oxide in brain disorders: Autism spectrum disorder and other psychiatric, neurological, and neurodegenerative disorders // Redox Biol. – 2020. – Vol. 34. – P. 101567. DOI: 10.1016/j.redox.2020.101567Tripathi MK, Kartawy M, Amal H. The role of nitric oxide in brain disorders: Autism spectrum disorder and other psychiatric, neurological, and neurodegenerative disorders. Redox Biol. 2020;34:101567. DOI: 10.1016/j.redox.2020.101567Ghasemi M., Mayasi Y., Hannoun A., Eslami S.M., Carandang R. Nitric oxide and mitochondrial function in neurological diseases // Neuroscience. – 2018. – Т. 376. – Р. 48–71. DOI: 10.1016/j.neuroscience.2018.02.017Ghasemi M, Mayasi Y, Hannoun A, Eslami SM, Carandang R. Nitric Oxide and Mitochondrial Function in Neurological Diseases. Neuroscience. 2018;376:48–71. DOI: 10.1016/j.neuroscience.2018.02.017Tewari D., Sah A.N., Bawari S., Nabavi S.F., Dehpour A.R., Shirooie S., Nabavi S.M. Role of nitric oxide in neurodegeneration: function, regulation, and inhibition // Curr Neuropharmacol. – 2021. – Vol. 19, No. 2. – P. 114–126. DOI: 10.2174/1570159X18666200429001549Tewari D, Sah AN, Bawari S, Nabavi SF, Dehpour AR,Shirooie S, Braidy N, Fiebich BL, Vacca RA, Nabavi SM. Role of Nitric Oxide in Neurodegeneration: Function, Regulation, and Inhibition. Curr Neuropharmacol. 2021;19(2):114–26. DOI: 10.2174/1570159X18666200429001549Green S.J. Covid-19 accelerates endothelial dysfunction and nitric oxide deficiency // Microbes Infect. – 2020. – Vol. 22, No. 4. – P. 149. DOI: 10.1016/j.micinf.2020.05.006Green SJ. Covid-19 accelerates endothelial dysfunction and nitric oxide deficiency. Microbes Infect. 2020;22(4-5):149–50. DOI: 10.1016/j.micinf.2020.05.006Xu S., Ilyas I., Little P.J., Li H., Kamato D., Zheng X., Weng J. Endothelial dysfunction in atherosclerotic cardiovascular diseases and beyond: from mechanism to pharmacotherapies // Pharmacological Reviews. – 2021. – Vol. 73, No. 3. – P. 924–967. DOI: 10.1124/pharmrev.120.000096Xu S, Ilyas I, Little PJ, Li H, Kamato D, Zheng X, Luo S, Li Z, Liu P, Han J, Harding IC, Ebong EE, Cameron SJ, Stewart AG, Weng J. Endothelial Dysfunction in Atherosclerotic Cardiovascular Diseases and Beyond: From Mechanism to Pharmacotherapies. Pharmacol Rev. 2021;73(3):924–67. DOI: 10.1124/pharmrev.120.000096Cetin M., Erdogan T., Kiris T., Ozyildiz A.G., Ergul E., Durakoglugil E., Cicek Y. Endothelial dysfunction, subclinical atherosclerosis and LDL cholesterol are the independent predictors of left atrial functions in hypertension // Int J Cardiovasc Imaging. – 2020. – Vol. 36, No. 1. – P. 69–77. DOI: 10.1007/s10554-019-01699-2Çetin M, Erdoğan T, Kırış T, Özyıldız AG, Ergül E, Durakoğlugil E, Durak H, Kalaycıoğlu E, Çiçek Y. Endothelial dysfunction, subclinical atherosclerosis and LDL cholesterol are the independent predictors of left atrial functions in hypertension. Int J Cardiovasc Imaging. 2020;36(1):69–77. DOI: 10.1007/s10554-019-01699-2Feelisch M. The use of nitric oxide donors in pharmacological studies // Naunyn-Schmiedeberg’s Archives of Pharmacology. – 1998. – Vol. 358, No. 1. – P. 113–122. DOI: 10.1007/PL00005231Feelisch M. The use of nitric oxide donors in pharmacological studies. Naunyn-Schmiedeberg’s Archives of Pharmacology. 1998;358(1):113–22. DOI: 10.1007/PL00005231Sun J., Zhang X., Broderick M., Fein H. Measurement of nitric oxide production in biological systems by using Griess reaction assay // Sensors. – 2003. – Vol. 3, No. 8. – P. 276–284. DOI: 10.3390/s30800276Sun J, Zhang X, Broderick M, Fein H. Measurement of nitric oxide production in biological systems by using Griess reaction assay. Sensors. 2003;3(8):276–84. DOI: 10.3390/s30800276Ricart-Jane D., Llobera M., Lopez-Tejero M.D. Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method // Nitric Oxide. – 2002. – Vol. 6, No. 2. – P. 178–185. DOI: 10.1006/niox.2001.0392Ricart-Jané D, Llobera M, López-Tejero MD. Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method. Nitric Oxide. 2002;6(2):178–85. DOI: 10.1006/niox.2001.0392Yucel A.A., Gulen S., Dincer S., Yucel A.E., Yetkin G.I. Comparison of two different applications of the Griess method for nitric oxide measurement // J Exp Integr Med. – 2012. – Vol. 2, No. 1. – P. 167. DOI: 10.5455/jeim.200312.or.024Yucel AA, Gulen S, Dincer S, Yucel AE, Yetkin GI. Comparison of two different applications of the Griess method for nitric oxide measurement. J Exp Integr Med. 2012;2(1):167. DOI: 10.5455/jeim.200312.or.024Hunter R.A., Storm W. L., Coneski P.N., Schoenfisch M.H. Inaccuracies of nitric oxide measurement methods in biological media // Analytical chemistry. – 2013. – Vol. 85, No. 3. – P. 1957–1963. DOI: 10.1021/ac303787pHunter RA, Storm WL, Coneski PN, Schoenfisch MH. Inaccuracies of nitric oxide measurement methods in biological media. Anal Chem. 2013;85(3):1957–63. DOI: 10.1021/ac303787pAziz H.A., Moustafa G.A.I., Abbas S.H., Derayea S.M., Abuo G.E.D.A.A. New norfloxacin/nitric oxide donor hybrids: Synthesis and nitric oxide release measurement using a modified Griess colorimetric method // Eur J Chem. – 2017. – Vol. 8, No. 2. – P. 119–124. DOI: 10.5155/eurjchem.8.2.119-124.1549Aziz HA, Moustafa GAI, Abbas SH, Derayea SM, Abuo GEDAA. New norfloxacin/nitric oxide donor hybrids: Synthesis and nitric oxide release measurement using a modified Griess colorimetric method. Eur J Chem. 2017;8(2):119–24. DOI: 10.5155/eurjchem.8.2.119-124.1549Minkler P., Stoll M., Ingalls S., Yang S., Kerner J., Hoppel C.L. Quantification of carnitine and acylcarnitines in biological matrices by HPLC electrospray ionization–mass spectrometry // Clin Chem. – 2008. – Vol. 54. – P. 1451–1462. DOI: 10.1373/clinchem.2007.099226Minkler PE, Stoll MS, Ingalls ST, Yang S, Kerner J, Hoppel CL. Quantification of carnitine and acylcarnitines in biological matrices by HPLC electrospray ionization-mass spectrometry. Clin Chem. 2008;54(9):1451–62. DOI: 10.1373/clinchemГуманова Н.Г. Оксид азота, его циркулирующие метаболиты NOx и их роль в функционировании человеческого организма и прогнозе риска сердечно-сосудистой смерти (часть I) // Профилактическая медицина. – 2021. – Т. 24, № 9. – С. 119–125. DOI: 10.17116/profmed202124091102Gumanova NG. Nitric oxide and its circulating NOx metabolites, their role in human body functioning and cardiovascular death risk prediction (part I). Russian Journal of Preventive Medicine. 2021;24(9):102–9. DOI: 10.17116/profmed202124091102. RussianFang W., Jiang J., Su L., Shu T., Liu H., Lai S., Wang J. The role of NO in COVID-19 and potential therapeutic strategies // Free Radic Biol Med. – 2021. – Vol. 163. – P. 153–162. DOI: 10.1016/j.freeradbiomed.2020.12.008Fang W, Jiang J, Su L, Shu T, Liu H, Lai S, Ghiladi RA, Wang J. The role of NO in COVID-19 and potential therapeutic strategies. Free Radic Biol Med. 2021;163:153–62. DOI: 10.1016/j.freeradbiomed.2020.12.008Попов Н.С., Балабаньян В.Ю., Колгина Н.Ю., Петров Г.А., Донсков С.А., Атаджанов И.Б. Количественное определение циклического гуанозинмонофосфата (ц-ГМФ) в тканях крыс с помощью жидкостной хроматографии и тандемной масс-спектрометрии // Фармакокинетика и фармакодинамика. – 2023. – № 3. – С. 28–38. DOI: 10.37489/2587-7836-2023-3-28-38Popov NS, Balabanyan VYu, Kolgina NYu, Petrov GA, Donskov SA, Atadzhanov IB. Quantitative determination of cyclic guanosine monoposphate (c-GMP) in rat tissues using liquid chromatography and tandem mass spectrometry. Pharmacokinetics and Pharmacodynamics. 2023;(3):28–38. DOI: 10.37489/2587-7836-2023-3-28-38. RussianInfante T., Costa D., Napoli C. Novel insights regarding nitric oxide and cardiovascular diseases // Angiology. – 2021. – Vol. 72, No. 5. – P. 411–425. DOI: 10.1177/00033197209792Infante T, Costa D, Napoli C. Novel Insights Regarding Nitric Oxide and Cardiovascular Diseases. Angiology. 2021;72(5):411–25. DOI: 10.1177/0003319720979243Idrizaj E., Traini C., Vannucchi M.G., Baccari M.C. Nitric oxide: from gastric motility to gastric dysmotility // Int J Mol Sci. – 2021. – Vol. 22, No. 18. – P. 9990. DOI: 10.3390/ijms22189990Idrizaj E, Traini C, Vannucchi MG, Baccari MC. Nitric Oxide: From Gastric Motility to Gastric Dysmotility. Int J Mol Sci. 2021;22(18):9990. DOI: 10.3390/ijms22189990Yaguchi J., Yaguchi S. Evolution of nitric oxide regulation of gut function // Proc Natl Acad Sci U S A. – 2019. – Vol. 116, No. 12. – P. 5607–5612. DOI: 10.1073/pnas.1816973116Yaguchi J, Yaguchi S. Evolution of nitric oxide regulation of gut function. Proc Natl Acad Sci USA. 2019;116(12):5607–12. DOI: 10.1073/pnas.1816973116Morales-Medina J.C., Aguilar-Alonso P., Di Cerbo A., Iannitti T., Flores G. New insights on nitric oxide: Focus on animal models of schizophrenia // Behavioural Brain Research. – 2021. – Vol. 409. – P. 113304. DOI: 10.1016/j.bbr.2021.113304Morales-Medina JC, Aguilar-Alonso P, Di Cerbo A, Iannitti T, Flores G. New insights on nitric oxide: Focus on animal models of schizophrenia. Behav Brain Res. 2021;409:113304. DOI: 10.1016/j.bbr.2021.113304Cyr A.R., Huckaby L.V., Shiva S.S., Zuckerbraun B.S. Nitric oxide and endothelial dysfunction // Crit Care Clin. – 2020. – Vol. 36, No. 2. – P. 307–321. DOI: 10.1016/j.ccc.2019.12.009Cyr AR, Huckaby LV, Shiva SS, Zuckerbraun BS. Nitric Oxide and Endothelial Dysfunction. Crit Care Clin. 2020;36(2):307–21. DOI: 10.1016/j.ccc.2019.12.009AbdelAziz E.Y., Tadros M.G., Menze E.T. The effect of metformin on indomethacin-induced gastric ulcer: Involvement of nitric oxide/Rho kinase pathway // Eur J Pharmacol. – 2021. – Vol. 892. – P. 173812. DOI: 10.1016/j.ejphar.2020.173812AbdelAziz EY, Tadros MG, Menze ET. The effect of metformin on indomethacin-induced gastric ulcer: Involvement of nitric oxide/Rho kinase pathway. Eur J Pharmacol. 2021;892:173812. DOI: 10.1016/j.ejphar.2020.173812Cieślik P., Kalinowski L., Wierońska J.M. Procognitive activity of nitric oxide inhibitors and donors in animal models // Nitric Oxide. – 2022. – Vol. 119. – P. 29–40. DOI: 10.1016/j.niox.2021.12.003Cieślik P, Kalinowski L, Wierońska JM. Procognitive activity of nitric oxide inhibitors and donors in animal models. Nitric Oxide. 2022;119:29–40. DOI: 10.1016/j.niox.2021.12.003Yuyun M.F., Ng L.L., Ng G.A. Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy? // Microvasc Res. – 2018. – Vol. 119. – P. 7–12. DOI: 10.1016/j.mvr.2018.03.012Yuyun MF, Ng LL, Ng GA. Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy? Microvasc Res. 2018;119:7–12. DOI: 10.1016/j.mvr.2018.03.012

The authors declare that there are no conflicts of interest present.