chemicallytechFine Chemical TechnologiesТонкие химические технологии2410-65932686-7575MIREA – Russian Technological University (RTU MIREA).10.32362/2410-6593-2023-18-1-65-74chemicallytech-1933Research ArticleANALYTICAL METHODS IN CHEMISTRY AND CHEMICAL TECHNOLOGYАНАЛИТИЧЕСКИЕ МЕТОДЫ В ХИМИИ И ХИМИЧЕСКОЙ ТЕХНОЛОГИИDetermination of possible microRNA-markers of cobalt abuse by real-time qPCR using hypoxia signaling pathway panelsОпределение возможных микроРНК-маркеров злоупотребления препаратами кобальта методом ПЦР в реальном времени с использованием панелей сигнального пути гипоксииhttps://orcid.org/0000-0003-3424-0582ПостниковП. В.PostnikovP. V.

Постников Павел Викторович, к.х.н., начальник отдела допингового контроля

105005, Москва, Елизаветинский переулок, д. 10, стр. 1)

Scopus Author ID 57021610900

Pavel V. Postnikov, Cand. Sci. (Chem.), Head of the Doping Control Department

10-1, Elizavetinskii per., Moscow, 105005

Scopus Author ID 57021610900

postnikov@dopingtest.ruhttps://orcid.org/0000-0003-0938-9609РадусФ. В.RadusF. V.

Радус Федор Валерьевич, ассистент кафедры аналитической химии им. И.П. Алимарина

119571, Москва, пр-т Вернадского, д. 86

Fedor V. Radus, Assistant, I.P. Alimarin Department of Analitical Chemistry

86, Vernadskogo pr., Moscow, 119571

radus20@mail.ruhttps://orcid.org/0000-0002-3582-0012ЕфимоваЮ. А.EfimovaYu. A.

Ефимова Юлия Александровна, к.х.н., доцент кафедры аналитической химии им. И.П. Алимарина

119571, Москва, пр-т Вернадского, д. 86

Scopus Author ID 25228417800

Yuliya A. Efimova, Cand. Sci. (Chem.), Assistant Professor, I.P. Alimarin Department of Analitical Chemistry

86, Vernadskogo pr., Moscow, 119571

Scopus Author ID 25228417800

efimova_yulia@bk.ruhttps://orcid.org/0000-0002-0423-7801ПронинаИ. В.ProninaI. V.

Пронина Ирина Валерьевна, к.б.н., главный специалист отдела допингового контроля; старший научный сотрудник лаборатории патогеномики и транскриптомики

105005, Москва, Елизаветинский переулок, д. 10, стр. 1

125315, Балтийская ул., д. 8

Scopus Author ID 8161867200, ResearcherID G-3951-2014

Irina V. Pronina, Cand. Sci. (Chem.), Main Specialist, Doping Control Department, National Anti-Doping Laboratory (Institute); Senior Researcher, Pathogenomics and Transcriptomics Laboratory

10-1, Elizavetinskii per., Moscow, 105005

8, ul. Baltiiskaya, Moscow, 125315

Scopus Author ID 8161867200, ResearcherID G-3951-2014

zolly_sten@mail.ruНациональная антидопинговая лаборатория (Институт) Московского государственного университета им. М.В. Ломоносова (НАДЛ МГУ)РоссияNational Anti-Doping Laboratory (Institute), M.V. Lomonosov Moscow State University (NADL MSU)Russian FederationМИРЭА – Российский технологический университет (Институт тонких химических технологий им. М.В. Ломоносова)РоссияMIREA – Russian Technological University (M.V. Lomonosov Institute of Fine Chemical Technologies)Russian FederationНациональная антидопинговая лаборатория (Институт) Московского государственного университета им. М.В. Ломоносова (НАДЛ МГУ); Научно-исследовательский институт общей патологии и патофизиологииРоссияNational Anti-Doping Laboratory (Institute), M.V. Lomonosov Moscow State University (NADL MSU); Institute of General Pathology and PathophysiologyRussian Federation20232003202318165–74Copyright © Postnikov P.V., Radus F.V., Efimova Y.A., Pronina I.V., 20232023Постников П.В., Радус Ф.В., Ефимова Ю.А., Пронина И.В.Postnikov P.V., Radus F.V., Efimova Y.A., Pronina I.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.finechem-mirea.ru/jour/article/view/1933

Objectives. Cobalt mimics the state of hypoxia to prevent degradation of the alpha subunit of hypoxia-inducible factor, resulting in an increase in blood oxygen capacity and endurance. Athletes can use this property to gain competitive advantage. Nowadays, direct methods of inductively coupled plasma mass spectrometry and liquid chromatography-tandem mass spectrometry are used to determine total cobalt levels in the body. However, the World Anti-Doping Agency is yet to establish a maximum allowable threshold concentration of this element in biofluids. The lack of clear identification criteria complicates the interpretation of the obtained results for the purposes of doping control. In this regard, the present work proposes a new approach for the indirect determination of possible cobalt abuse based on changes in the expression levels of miRNAs involved in the regulation of hypoxia signaling pathways. Here, the aim is to identify possible microRNA markers whose expression does not depend on exercise-induced hypoxia, but changes markedly when taking cobalt preparations.Methods. MicroRNA isolation was performed from blood plasma samples using the PAXgene Blood miRNA Kit. Quantitative real-time polymerase chain reaction (PCR) was performed on CFX96 Bio-Rad (USA) analyzer using miScript® SYBR® Green PCR Kits and panels for studying the expression profiles of mature microRNAs of the hypoxia signaling pathway miScript® miRNA PCR Array.Results. Based on the statistical analysis of the data, it was found that the expression of hsa-miR-15b-5p in the blood plasma of the subjects does not depend on physical activity, but increases when taking cobalt preparations.Conclusions. The difference in expression levels during anaerobic exercise-induced hypoxia and cobalt-induced hypoxia makes hsa-miR-15b-5p a potential candidate to be a marker of erythropoiesis-stimulating agent abuse.

Цели. Кобальт имитирует состояние гипоксии, препятствуя деградации альфасубъединицы гипоксия-индуцируемого фактора, что приводит к увеличению кислородной емкости крови и может использоваться спортсменами в качестве допинга для получения конкурентных преимуществ. На сегодняшний момент для определения общего кобальта в организме используют прямые методы масс-спектрометрии с индуктивно связанной плазмой, жидкостной хроматографиитандемной масс-спектрометрии, однако Всемирным антидопинговым агентством не установлена максимально допустимая пороговая концентрация этого элемента в биожидкостях. Отсутствие четких критериев идентификации осложняет интерпретацию полученных результатов. В связи с этим, в данной статье впервые предлагается подход по косвенному определению возможных злоупотреблений кобальтом для целей допинг-контроля, основанный на изменении уровней экспрессии микроРНК, задействованных в регуляции сигнального пути гипоксии. Цель исследования заключалась в поиске возможных микроРНК-маркеров, экспрессия которых не зависит от гипоксии, вызванной физическими нагрузками, но заметно изменяется при приеме препаратов кобальта.Методы. Выделение микроРНК из образцов плазмы крови проводили при помощи набора PAXgene Blood miRNA Kit. Количественную полимеразную цепную реакцию в реальном времени проводили на амплификаторе CFX96 Bio-Rad (США) с помощью наборов miScript® SYBR® Green PCR Kit и панелей для исследования профилей экспрессии зрелых микроРНК сигнального пути гипоксии Hypoxia Signaling Pathway miScript® miRNA PCR Array.Результаты. На основании статистического анализа данных было установлено, что экспрессия hsa-miR-15b-5p в плазме крови испытуемых не зависит от физической нагрузки, но возрастает при приеме препаратов кобальта.Выводы. Разница в уровнях экспрессии при гипоксии, вызванной анаэробной физической нагрузкой, и имитацией гипоксии за счет применения кобальта делает hsa-miR-15b-5p потенциальным претендентом на роль маркера злоупотребления данным эритропоэзстимулирующим агентом.

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