Ispitivanje stabilnosti glutationa u uzorcima plazme i krvi

master's thesis

• Title (english): Testing of glutation stability in serum and plasma samples
• Author: Vesna Vujeva
• Mentor: Ana-Marija Domijan (mentor)
• Committee member: Ana-Marija Domijan (predsjednik povjerenstva)
• Committee member: Petra Turčić (član povjerenstva)
• Committee member: Marko Gerić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2019-06-05, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Reducirani glutation (GSH) tripeptid je cisteina, glutaminske kiseline i glicina te predstavlja najvažniji neenzimatski antioksidans u organizmu. Cilj ovoga istraživanja bio je optimizirati metodu mjerenja GSH u ljudskoj krvi i plazmi te ispitati stabilnost GSH prilikom pohranjivanja uzoraka. Uzorci krvi i plazme pribavljeni su od dobrovoljnog davatelja krvi. Ispitivanje stabilnosti GSH provedeno je tako da je jedan uzorak podijeljen u četiri epruvete. U prvom uzorku plazme ili krvi koncentracija GSH izmjerena je odmah. Dio je uzoraka pohranjen u inkubatoru (na 37 °C u vlažnoj atmosferi s 5% CO2), a preostala po dva uzorka nakon sakupljanja pospremljeni su u zamrzivač na -20 °C na 8 i 30 dana. Koncentracija GSH izmjerena je pomoću Ellmanovog reagensa (5,5'-ditiol-(2-nitrobenzojeva kiselina), DTNB), spektrofotometrijski pri valnoj duljini  = 412 nm i sobnoj temperaturi. Reakcija tiolne skupine GSH s DTNB-om odvija se na sobnoj temperaturi i pH = 7,4 pri čemu nastaje žuti produkt 5'-tio-2-nitrobenzojeva kiselina (TNB). Mjerenja su provedena na UV-Vis spektrofotometru, a koncentracija GSH određena je pomoću molarnog apsorpcijskog koeficijenta (ε) koji iznosi 14,15 x 10-3 M-1 cm-1.
Ispitivanja su pokazala da je centrifugiranje plazme, prije spektrofotometrijskog određivanja GSH optimalno kako bi se uklonio dio interferencija koje bi mogle reagirati sa DTNB reagensom i odredila koncentracija GSH. Za određivanje koncentracije GSH u krvi potrebno je krv tretirati s 5 % klorovodičnom kiselinom kako bi se uklonile interferencije i omogućilo određivanje GSH. Nakon pohranjivanja uzorka krvi na 37 °C u vlažnoj atmosferi tijekom 24 sata uočen je statistički značajan porast koncentracije GSH i u plazmi i u krvi te se može zaključiti da su ovi uvjeti potakli sintezu GSH u krvnim stanicama. U uzorcima plazme i krvi pohranjenima -20°C tijekom 8 i 30 dana nisu uočene statistički značajne razlike u koncentraciji GSH. Stoga se može zaključiti da prilikom pohranjivanja uzoraka plazme i krvi na -20 °C do 30 dana ne dolazi do razgradnje GSH.

Abstract (english)

Reduced glutathione (GSH) is cysteine, glutamic acid and glycine tripeptide. It is the most important non-enzymatic antioxidant in the body. The aim of this study was to optimize the GSH measurement method in human blood and plasma samples, and to investigate GSH stability when storing samples. Blood and plasma samples were obtained from volunteer blood donor. To test GSH stability blood or plasma sample was divided into four tubes. In the first sample, concentration of GSH was measured immediately. Part of the samples were stored in the incubator (at 37 ° C in a humid atmosphere with 5% CO2) and the remaining samples were frozen at -20 ° C and kept for 8 or 30 days. GSH concentration was measured using an Ellman reagent (5,5'-dithiobis- (2-nitrobenzoic acid), DTNB spectrophotometrically at wavelength λ = 412 nm on room temperature. The reaction of thiol group GSH with DTNB was carried out at room temperature and pH 7.4, resulting in a yellow product of 5'-thio-2-nitrobenzoic acid (TNB). The measurements were performed on UV-Vis spectrophotometer and the GSH concentration was determined by a molar absorption coefficient (ε) which is 14.15 x 10-3 M-1 cm-1.
Tests have shown that centrifugation of plasma prior to spectrophotometric determination of GSH is optimal in order to remove interfering molecules and to determine the actual GSH concentration. Blood sample needs to be treated with 5% chlorodhydrogen acid, to remove possible interfering molecules with the reagents, and to reliably measure GSH. Storing plasma and blood sample at 37 ° C and the humid atmosphere after 24 hours showed statistically significant increase in GSH level in blood and plasma, probably due to GSH synthesis in blood cells. Storing blood and plasma samples at -20 ° C for 8 or 30 days did not affect the level of the GSH. Therefore can be concluded that during storage of plasma and blood samples at -20 °C for 30 days GSH was not degraded.

• Keywords: glutation; antioksidansi; oksidacijski stres; stabilnost GSH; DTNB; biološki uzorak
• Keywords (english): glutathione; antioxidants; oxidative stress; GSH stability; DTNB; biological sample
• Language: croatian
• URN:NBN: urn:nbn:hr:163:932719
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-10-01 10:51:19