Validation of enhanced luminol-H202-horseradish peroxidase chemiluminescent assay

master's thesis

• Author: Maja Raknić
• Mentor: Marijana Zovko Končić (mentor)
• Committee member: Marijana Zovko Končić
• Committee member: Dubravka Vitali Čepo
• Committee member: Lovorka Vujić
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmacognosy); Zagreb
• Defense date and country: 2015-09-30, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Slobodni radikali definirani su kao bilo koji atom, molekula ili ion s nesparenim elektronom. Samim time, oni su izrazito nestabilni i kemijski vrlo aktivni. Gubitkom ravnoteže između slobodnih radikala i antioksidansa, dolazi do stanja oksidacijskog stresa koje rezultira promjenom i oštećenjem mnogih fizioloških funkcija. Svrha ovog istraživanja bila je ustanoviti postoji li mogućnost primjene poboljšanog luminol-H2O2-peroksidaza kemiluminiscentnog testa za određivanje antioksidativne aktivnosti poznatih antioksidansa, askorbinske kiseline i Troloxa. Analizirane su tri koncentracije peroksidaze, među kojima je 50000 puta razrijeđena peroksidaza (konačne koncentracije 0.97 μU/ml) pokazala najbolje rezultate prilikom ispitivanja s 44 μM vodikovim peroksidom. Baždarni pravci za oba ispitivana antioksidansa pokazali su uvjerljivu korelaciju između koncentracija antioksidansa i vrijednosti površine ispod krivulja (AUC). Značajna linearna korelacija također je primijećena pri usporedbi koncentracije i vremena odgode početka reakcije, iako su vrijednosti koeficijenta korelacije u tom slučaju bile niže. Iz dobivenih rezultata može se zaključiti da bi ovaj validirani kemiluminiscentni test bio koristan u ispitivanju antioksidativne aktivnosti tvari.

Abstract (english)

Free radicals reactive oxygen species and reactive nitrogen species are generated by the human body by various endogenous systems, exposure to different physiochemical conditions or pathological states. A balance between free radicals and antioxidants is necessary for proper physiological function. If free radicals overwhelm the body's ability to regulate them, a condition known as oxidative stress ensues. The purpose of this study was to establish whether the enhanced luminol-H2O2- horseradish chemiluminescent assay could be used to determine antioxidant activity of reputed antioxidants, ascorbic acid and Trolox. The optimization of enhanced chemiluminescent assay was conducted. Among three concentrations of horseradish peroxidase, the 50000 fold diluted POD (with end concentration of 0.97 μU/ml) showed the best results when consolidated with 44 μM hydrogen peroxide. Both Trolox and ascorbic acid linear curves showed compelling connection between antioxidant concentration and AUC values. Noticeable linear correlation was also recognized when antioxidant concentration and time delay were compared, although CV values were a bit lower in this case. Based on given results we can conclude that validated enhanced chemiluminescent assay could be a valuable addition to the research of antioxidant activity.

• Keywords: validacija; luminol; Trolox; vitamin C; peroksidaza; kemiluminiscentni test
• Keywords (english): validation; luminol; Trolox; vitamin C; horseradish peroxidase; chemiluminescent assay
• Language: english
• URN:NBN: urn:nbn:hr:163:851697
• 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: Access restricted to students and staff of home institution
• Created on: 2016-02-22 14:27:16