Development of an Inhibition-Based Colorimetric Method For Glutathione Determination

Year 2024, Volume: 9 Issue: 1, 24 - 33, 30.04.2024

Erhan Canbay , Utkan Ceylan , Zeynep İlay Topçu , Zeynep Çelik Canbay , Özge Çakar , Ebru Demirel Sezer

https://doi.org/10.58854/jicm.1412375

Abstract

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.

Keywords

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

GSH Tayini için İnhibisyon Temelli Kolorimetrik Bir Yöntemin Geliştirilmesi

Abstract

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.

Keywords

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

Ethical Statement

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

Supporting Institution

Özel Ege Lisesi

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