Razvoj analiznih metod za oddaljeno spremljanje aktivnosti bolezni bolnikov s kronično vnetno črevesno boleznijo

Penko, Petra (avtor), Vovk, Tomaž (mentor), Drobne, David (komentor)

Kronična vnetna črevesna bolezen (KVČB) je dolgotrajna bolezen, ki prizadene predvsem sluznico črevesja. Za bolezen je značilen nepredvidljiv potek z izmenjujočimi se obdobji poslabšanja in remisije, ko so simptomi začasno odsotni. Povečano vnetje v aktivni fazi KVČB vodi do povečanega obsega oksidativnega stresa v telesu, kar povečuje obseg lipidne peroksidacije in s tem povišanje kazalnikov kot je malondialdehid (MDA). Ta torej lahko služi kot pomemben kazalnik aktivnosti bolezni. Namen magistrske naloge je bil razviti, optimizirati in validirati zanesljivo metodo za kvantitativno določanje koncentracije MDA v posušenih krvnih madežih (DBS). Razvito metodo smo temeljili na že obstoječi metodi ekstrakcije MDA iz DBS vzorcev, ki smo jo združili z drugo že razvito metodo za določanje MDA v plazemskih vzorcih. Metodi smo združili in ustrezno prilagodili za potrebe naše raziskave, nato pa smo jo poskušali optimizirati. Tekom optimizacije smo raziskali štiri potencialne dejavnike (čas stresanja vzorca v ekstrakcijskem mediju, vpliv stresanja ali izpostavitve vzorca ultrazvočnim valovom, vpliv dodajanja različnih razmerij volumnov ekstrakcijskega topila in vpliv koncentracije derivatizacijskega reagenta). Kot najbolj optimalno se je izkazalo enkratno 30 minutno stresanje DBS vzorcev v 200 µl ekstrakcijskega medija na stresalniku, nato pa derivatizacija z 2,4-dinitrofenilhidrazinom s koncentracijo 1 mg/ml. Poleg same optimizacije ekstrakcije smo izvedli tudi test vpliva volumna krvi v DBS vzorcih na odziv MDA in test stabilnosti DBS vzorcev, kjer smo raziskali še vpliv hematokrita. Linearna odvisnost odziva MDA od volumna krvi v DBS vzorcih je bila potrjena, izvedeni testi stabilnosti pa so potrdili, da so vzorci MDA nestabilni pri povišanih temperaturah, relativno stabilni na sobni temperaturi, najbolj stabilni pa v primeru hrambe na hladnem. Ugotovili smo, da tudi hematokrit vpliva na dobljene rezultate. Tekom raziskave smo izvajali tudi meritve MDA v DBS in plazemskih vzorcih bolnikov s KVČB. Koncentracijo MDA v DBS vzorcih smo primerjali z izmerjenim koncentracijami MDA v plazemskih vzorcih in ugotovili, da trendi padanja in naraščanja koncentracij med temi vzorci niso primerljivi. Na podlagi rezultatov zaključujemo, da bi bilo smiselno v prihodnje dodatno preučiti vpliv volumna krvi in hematokrita na koncentracije MDA DBS vzorcih. Poleg tega bi bilo smiselno ponovno raziskati korelacijo med koncentracijami MDA v DBS in plazemskimi vzorci bolnikov, s konstantnimi pogoji hrambe vzorcev.

• Jezik: Slovenski jezik
• Ključne besede: Kronična vnetna črevesna bolezen, malondialdehid, mikrovzorčenje, posušeni krvni madeži
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-170121
• Datum objave v RUL: 02.07.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of analytical methods for remote monitoring disease activity in patients with inflammatory bowel disease

Izvleček

Chronic inflammatory bowel disease (IBD) is a long-term condition that primarily affects the digestive mucosa of the intestines. It is characterized by an unpredictable course with alternating periods of flare ups and remission, during which symptoms are temporarily absent. Increased inflammation during the active phase of the disease leads to elevated oxidative stress in the body, which in turn enhances the extent of lipid peroxidation and increases the concentration of biomarker malondialdehyde (MDA). Thus, MDA can serve as an important biomarker of disease activity. The aim of this master's thesis was to develop, optimize, and validate a reliable method for the quantitative determination of malondialdehyde concentration in dried blood spots (DBS). The developed method was based on an existing protocol for extracting MDA from DBS, which we combined with another previously established method for determining MDA in plasma samples. The methods were merged and appropriately adjusted for the needs of our study, after which we attempted to optimize the procedure. During the method optimization, we adjusted four potential factors: shaking time of the DBS sample in the extraction medium, the effect of shaking versus ultrasonic exposure, the impact of adding different volume ratios of extraction solvent, and the concentration of derivatization reagent. The most optimal conditions were found to be a single 30-minute shaking of the DBS samples in 200 µl of extraction medium on a shaker, followed by derivatization using 2,4-dinitrophenylhydrazine (DNPH) at a concentration of 1 mg/ml. In addition to optimizing the extraction method, we performed a linearity test of MDA response relative to blood volume in DBS samples and a sample stability test, where we also examined the effect of blood haematocrit. Linearity of the MDA concentration relative to blood volume in DBS samples was confirmed, while the stability tests showed that MDA in DBS samples is unstable at elevated temperatures, relatively stable at room temperature, and most stable when stored in freezers. We also determined that haematocrit influences the results. During the study, we performed measurements of MDA in DBS samples and plasma samples from patients with IBD. We found that the trends of MDA concentration fluctuations between plasma and DBS samples were not comparable. Based on this, we conclude that future studies should further examine the influence of blood volume and haematocrit on MDA concentration in DBS samples. Additionally, it would be beneficial to explore the relation between MDA concentration trends in DBS and plasma samples under consistent sample storage conditions.

• Ključne besede: Inflammatory bowel disease, malondialdehyde, microsampling, dried blood spots