Prilagodba neenimske metode s Ellmanovim reagensom za određivanje glutationa u tkivima štakora

master's thesis

• Title (english): Adjustment of non-enzymatic method with Ellman's reagent for determination of glutathione in rat tissues
• Author: Sandra Orešković
• Mentor: Branimir Šimić (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Toxicology); Zagreb
• Defense date and country: 2007-05-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Food Technology

Abstract

Najvažniji neenzimski antioksidans je glutation (GSH) – tripeptid glutamata, cisteina i glicina. U organizmu ima višestruke uloge od kojih su najbitnije detoksikacija ksenobiotika i antioksidativna obrana reproduktivnih organa. Kao stanični biomarker, vrlo je značajan u identificiranju oksidativnog stresa u organizmu. Razvijene su mnoge metode za njegovo određivanje, a najpoznatije su enzimske metode po Ellmanu. U ovom radu prilagođavala se metoda čija je osnova neenzimska reakcija s Ellmanovim reagensom, a koja je prikladna za izvođenje u jednostavnim laboratorijskim uvjetima i s manjim brojem uzoraka. Količina GSH u uzorku određivala se spektrofotometrijski. Potvrđena je izvrsna linearnost u mjernom području do 200 nmol, a preciznost višekratnim mjerenjima više uzoraka unutar pokusa (%v = 2,47–4,23%) i između pokusa (%V = 1,27–3,08%). Točnost metode provjeravala se metodom dodatka standarda u uzorke tkiva štakora s iskorištenjem od 97–103% te metodom promjene volumena dodanog uzorka. Određena je količina GSH u tkivu testisa (2,98 ± 0,104 nmol GSH/mg tkiva), epididimisa (1,54 ± 0,104 nmol GSH/mg tkiva) i prostate (0,99 ± 0,100 nmol GSH/mg tkiva). Ispitani su uvjeti čuvanja supernatanta tkiva štakora i utvrđeno je da čuvanjem pri –20 °C ne dolazi do značajnih promjena količina GSH u supernatantu tijekom 21 dana.

Abstract (english)

Most important non-enzymatic antioxidant is glutathione (GSH) – glutamate, cysteine and glycine tripeptide. It plays multiple roles in organism, of which the most important are detoxification of xenobiotics and antioxidative defence of reproductive organs. As a cell biomarker, it is important in identification of oxidative stress. Among numerous methods for determination of glutathione concentration the most used is Ellman’s assay. In this work glutathione concentration was determined by non-enzymatic reaction with Ellman's reagent using spectrophotometric method, which is suitable for use in laboratories that can't provide assays with the large number of samples. The following features were examined: linearity and sensitivity in the useful range of detection (3.12–200 nmol), within-assay precision (%v = 2.47–4.23%) and inter-assay (%V = 1.27–3.08%) precision. Accuracy of the method was checked by recovery method, where standard was added in supernatants of rat tissues. Recovery was in range of 97–103%. Among the reproductive rat tissues, the highest content of GSH have been found in testicle (2.98 ± 0.104 nmol GSH/mg tissue sample) followed by those in the epididimys (1.54 ± 0.104 nmol GSH/mg sample) and prostate (0.99 ± 0.100 nmol GSH/mg sample). Keeping supernatants at temperature of –20 °C for 21 days as the tested tissue storage conditions, results showed no any decrease in quantities of GSH in the tested sample tissues.

• Keywords: glutation; neenzimsko određivanje GSH; epididimis; prostata; testis
• Keywords (english): glutathione; non-enzymatic GSH determination; epididimys; prostate; testicle
• Language: croatian
• URN:NBN: urn:nbn:hr:159:203580
• Study programme: Title: Food Technology; specializations in: Nutrition; Course: Nutrition; Study programme type: university; Study level: graduate; Academic / professional title: diplomirani inženjer prehrambene tehnologije; smjer nutricionizam (diplomirani inženjer prehrambene tehnologije; smjer nutricionizam)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-09-06 12:41:51