GSH Tayini için İnhibisyon Temelli Kolorimetrik Bir Yöntemin Geliştirilmesi

Öz

Amaç: Başlıca hücresel antioksidanlardan biri olan glutatyon (GSH, L-γ-glutamyl-L-cysteinyl-glycine), tüm canlı organizmalarda reaktif oksijen türlerinin (ROS) detoksifikasyonu için sülfhidril grupları içeren önemli bir protein olmayan hücre içi fizyolojik antioksidandır. GSH eksikliğinin kardiyovasküler, bağışıklık ve yaşlanma hastalıkları, artrit ve diyabet dahil olmak üzere birçok insan hastalığı ile ilişkili olduğu gösterilmiştir. Bu nedenle, biyolojik sıvılarda GSH tayini için doğru, güvenilir ve hassas bir yöntemin geliştirilmesi, tıpta ve biyokimyasal araştırmalarda GSH homeostazının anlaşılması için gereklidir. Gereç ve Yöntem: Bu çalışmada, glutatyon (GSH) tayini için çıplak gözle tespit edilebilen çok ucuz, pratik, hızlı, hassas ve oldukça spesifik bir kolorimetrik yöntem geliştirilmiştir. Yöntem, horseradish peroksidazın (HRP) GSH tarafından inhibisyonuna dayanmaktadır. Glutatyon konsantrasyonu arttıkça, 4-klorofenol, H2O2 ve 4-aminoantipirinden (4-AAP) oluşan pembe renkli bir bileşik, HRP tarafından katalize edilen reaksiyon sonucunda ayrışır ve böylece rengin yoğunluğu azalır. Bulgular: Geliştirilen yöntemin doğrusal aralığı 15,6-1000 mM arasında bulunurken, yöntemin gün içi ve günler arası tekrarlanabilirlik % varyasyon katsayısı değerleri %15’in altında bulunmuştur. Potansiyel interferans maddelerinin geliştirilen metot üzerindeki etkisi test edilmiş ve sistein dışında herhangi bir interferansa rastlanmamıştır. Sistein, GSH için yanıtı %10 oranında artırmıştır. Geliştirilen yöntem ticari serum örneklerinde GSH tayini için kullanılmış ve %91-106 arasında sonuçlar elde edilmiştir. Sonuç: Sonuç olarak, bu çalışmada GSH tayini için çok basit, ucuz ve benzersiz bir kolorimetrik yöntem geliştirilmiştir.

Anahtar Kelimeler

• Glutatyon
• Yabani turp peroksidazı
• 4-AAP
• İnhibisyon temelli

Destekleyen Kurum

Özel Ege Lisesi

Etik Beyan

Çalışma kapsamında gerçek örnek denemeleri ticari olarak satın alınan serum örneklerinde yapılmıştır. Bu nedenle herhangi bir etik kurul izni gerekmemektedir.

Development of an Inhibition-Based Colorimetric Method For Glutathione Determination

Öz

Objectives: Glutathione (GSH, L-γ-glutamyl-L-cysteinyl-glycine), one of the major cellular antioxidants, is an important non-protein intracellular physiological antioxidant with sulphhydryl groups for detoxification of reactive oxygen species (ROS) in all living organisms. GSH deficiency has been shown to be associated with many human diseases, including cardiovascular, immune and ageing diseases, arthritis and diabetes. Therefore, the development of an accurate, reliable and sensitive method for the determination of GSH in biological fluids is essential for the understanding of GSH homeostasis in medicine and biochemical research Material and Methods: In this study, a very inexpensive, practical, rapid, sensitive, and highly specific colorimetric method for the determination of glutathione (GSH) that can be detected by the naked eye was developed. This method is based on the inhibition of horseradish peroxidase (HRP) by GSH. As the concentration of glutathione increases, a pink coloured compound consisting of 4-chlorophenol, H2O2 and 4-aminoantipyrine (4-AAP) decomposes as a result of the reaction catalyzed by HRP, thus reducing the intensity of the colour. Results: While the linear range of the developed method was found to be between 15.6-1000 mM, the intra- and inter-day repeatability % coefficient of variation values of the method were less than 15%. The effect of potential interfering substances on the developed method was tested, and no interference was found, except for cysteine. Cysteine increased GSH response by 10%. The developed method was used for the determination of GSH in commercial serum samples, and results were obtained between 91-106%. Conclusion: In conclusion, this study has developed a very simple, inexpensive and unique colourimetric method for the determination of GSH.

Anahtar Kelimeler

• Glutathione
• Horse radish peroxidase
• 4-AAP
• Inhibition based

Kaynakça

1. Bansal A, Celeste Simon M. Glutathione metabolism in cancer progression and treatment resistance. The Journal of cell biology. 2018;217(7): 2291–2298. https://doi.org/10.1083/JCB.201804161.
2. Malla JA, Umesh RM, Yousf S, Mane S, Sharma S, Lahiri M, et al. A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels. Angewandte Chemie (International ed. in English). 2020;59(20): 7944–7952. https://doi.org/10.1002/ANIE.202000961.
3. Mayer RJ, Ofial AR. Nucleophilicity of Glutathione: A Link to Michael Acceptor Reactivities. Angewandte Chemie International Edition. 2019;58(49): 17704–17708. https://doi.org/10.1002/ANIE.201909803.
4. Huang X, Xia F, Nan Z. Fabrication of FeS 2 /SiO 2 Double Mesoporous Hollow Spheres as an Artificial Peroxidase and Rapid Determination of H 2 O 2 and Glutathione. Cite This: ACS Appl. Mater. Interfaces. 2020;12: 46539–46548. https://doi.org/10.1021/acsami.0c12593.
5. Singh S, Ghosh S, Pal VK, Munshi M, Shekar P, Tumkur D, et al. Antioxidant nanozyme counteracts HIV-1 by modulating intracellular redox potential. EMBO Molecular Medicine. 2021;13(5): e13314. https://doi.org/10.15252/EMMM.202013314.
6. Appala RN, Chigurupati S, Appala RVVSS, Selvarajan KK, Mohammad JI. A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells. Scientifica. 2016;2016. https://doi.org/10.1155/2016/6897890.
7. Kasprzyk W, Koper F, Flis A, Szreder D, Pamuła E, Bogdał D, et al. Fluorescence assay for the determination of glutathione based on a ring-fused 2-pyridone derivative in dietary supplements. Analyst. 2021;146(6): 1897–1906. https://doi.org/10.1039/D0AN02245D.
8. Herzog K, Ijlst L, van Cruchten AG, van Roermund CWT, Kulik W, Wanders RJA, et al. An UPLC-MS/MS Assay to Measure Glutathione as Marker for Oxidative Stress in Cultured Cells. Metabolites. 2019;9(3). https://doi.org/10.3390/METABO9030045.

9. Tang Z, Zhang H, Ma C, Gu P, Zhang G, Wu K, et al. Colorimetric determination of the activity of alkaline phosphatase based on the use of Cu(II)-modulated G-quadruplex-based DNAzymes. Microchimica Acta. 2018;185(2). https://doi.org/10.1007/S00604-017-2628-Y.
10. Peng B, Zhou J, Xu J, Fan M, Ma Y, Zhou M, et al. A smartphone-based colorimetry after dispersive liquid–liquid microextraction for rapid quantification of calcium in water and food samples. Microchemical Journal. 2019;149. https://doi.org/10.1016/j.microc.2019.104072.
11. Chen Y, Fu G, Zilberman Y, Ruan W, Ameri SK, Zhang YS, et al. Low cost smart phone diagnostics for food using paper-based colorimetric sensor arrays. Food Control. 2017;82: 227–232. https://doi.org/10.1016/j.foodcont.2017.07.003.
12. Kojima T, Nakane A, Zhu B, Alfi A, Nakano H. A simple, real-time assay of horseradish peroxidase using biolayer interferometry. Bioscience, Biotechnology, and Biochemistry. 2019;83(10): 1822–1828. https://doi.org/10.1080/09168451.2019.1621156.
13. Lindgren A, Ruzgas T, Gorton L, Csöregi E, Bautista Ardila G, Sakharov IY, et al. Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer. Biosensors and Bioelectronics. 2000;15(9–10): 491–497. https://doi.org/10.1016/S0956-5663(00)00110-X.
14. Zhu Z, Wang J, Munir A, Zhou HS. Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized on bamboo shaped carbon nanotubes/chitosan matrix. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2011;385(1–3): 91–94. https://doi.org/10.1016/j.colsurfa.2011.05.047.
15. Trinder P. Determination of Glucose in Blood Using Glucose Oxidase with an Alternative Oxygen Acceptor. Annals of Clinical Biochemistry: International Journal of Laboratory Medicine. 1969;6(1): 24–27. https://doi.org/10.1177/000456326900600108.
16. Varadaraju C, Tamilselvan G, Enoch I, Selvakumar PM. Phenol Sensing Studies by 4-Aminoantipyrine Method–A Review. Organic & Medicinal Chemistry International Journal. 2018;5(2): 46–52. https://doi.org/10.19080/OMCIJ.2018.05.555657.
17. Li JJ, Qiao D, Yang SZ, Weng GJ, Zhu J, Zhao JW. Colorimetric determination of cysteine based on inhibition of GSH-Au/Pt NCs as peroxidase mimic. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2021;248: 119257. https://doi.org/10.1016/J.SAA.2020.119257.
18. Ren S, Zhou F, Xu C, Li B. Simple method for visual detection of glutathione S-transferase activity and inhibition using cysteamine-capped gold nanoparticles as colorimetric probes. Gold Bulletin. 2015;48(3–4): 147–152. https://doi.org/10.1007/S13404-015-0171-3/FIGURES/7.
19. Martínez GA, Civello PM, Chaves AR, Aón MC. Characterization of peroxidase-mediated chlorophyll bleaching in strawberry fruit. Phytochemistry. 2001;58(3): 379–387. https://doi.org/10.1016/S0031-9422(01)00266-7.
20. Wang Q, Liu Y, Campillo-Brocal JC, Jiménez-Quero A, Crespo GA, Cuartero M. Electrochemical biosensor for glycine detection in biological fluids. Biosensors and Bioelectronics. 2021;182: 113154. https://doi.org/10.1016/J.BIOS.2021.113154.
21. Tekin S, Seven E. Assessment of serum catalase, reduced glutathione, and superoxide dismutase activities and malondialdehyde levels in keratoconus patients. Eye 2021 36:10. 2021;36(10): 2062–2066. https://doi.org/10.1038/s41433-021-01753-1.
22. Xu H, Wang Y, Huang X, Li Y, Zhang H, Zhong X. Hg2+-mediated aggregation of gold nanoparticles for colorimetric screening of biothiols. Analyst. 2012;137(4): 924–931. https://doi.org/10.1039/C2AN15926K.
23. Shamsipur M, Safavi A, Mohammadpour Z. Indirect colorimetric detection of glutathione based on its radical restoration ability using carbon nanodots as nanozymes. Sensors and Actuators B: Chemical. 2014;199: 463–469. https://doi.org/10.1016/J.SNB.2014.04.006.
24. Zou H, Yang T, Lan J, Huang C. Use of the peroxidase mimetic activity of erythrocyte-like Cu1.8S nanoparticles in the colorimetric determination of glutathione. Analytical Methods. 2017;9(5): 841–846. https://doi.org/10.1039/C6AY03034C.
25. Wu D, Li G, Chen X, Qiu N, Shi X, Chen G, et al. Fluorometric determination and imaging of glutathione based on a thiol-triggered inner filter effect on the fluorescence of carbon dots. Microchimica Acta. 2017;184(7): 1923–1931. https://doi.org/10.1007/S00604-017-2187-2/FIGURES/5.
26. Yuan L, Guo W, Fu Y, Zhang Z, Wang P, Wang J. A rapid colorimetric method for determining glutathione based on the reaction between cobalt oxyhydroxide nanosheets and 3,3′,5,5′-Tetramethylbenzidine. Microchemical Journal. 2021;160: 105639. https://doi.org/10.1016/J.MICROC.2020.105639.
27. Jia R, Jin K, Zhang J, Zheng X, Wang S, Zhang J. Colorimetric and fluorescent detection of glutathione over cysteine and homocysteine with red-emitting N-doped carbon dots. Sensors and Actuators B: Chemical. 2020;321: 128506. https://doi.org/10.1016/J.SNB.2020.128506.
28. Wang M, Wang J, Ma N, Yu S, Kong J, Zhang X. A novel colorimetric detection of glutathione based on stable free radical TEMPO oxidation of 3,3′,5,5′−tetramethylbenzizine (TMB) via Copper(II) acetylacetonate catalysis. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2023;285: 121875. https://doi.org/10.1016/J.SAA.2022.121875.