Preučevanje metabolizma bisfenola AP in vitro in razvoj bioanalizne metode za njegovo merjenje v krvni plazmi

Šarman - Gaberšek, Zala (avtor), Trontelj, Jurij (mentor)

Bisfenoli sodijo med kemijske spojine, ki delujejo kot motilci endokrinega sistema, saj lahko posnemajo ali zavirajo delovanje naravnih hormonov. Med najbolj znanimi predstavniki je bisfenol A (BPA). Uporabljajo se predvsem pri izdelavi polikarbonatne plastike in epoksi smol. Dolgotrajna izpostavljenost tovrstnim snovem ima na človeški organizem toksične in škodljive učinke ter lahko vodi v razvoj številnih bolezni, zato vse več držav omejuje in prepoveduje uporabo BPA v različnih izdelkih. Uporabljati so se začeli strukturno podobni analogi BPA, med katerimi je tudi bisfenol AP (BPAP). Čeprav veljajo za varnejše alternative, pa so razpoložljivi podatki o metabolizmu in toksikokinetiki še neraziskani in omejeni. Večina do sedaj preučevanih bisfenolov se prvenstveno presnavlja s konjugacijo z glukuronsko kislino ali s sulfatom. Zato je bil namen našega dela raziskati II. fazo metabolizma BPAP v pogojih in vitro ter razviti bioanalizno metodo za merjenje BPAP in identificiranih poglavitnih metabolitov v plazmi podgan, ki so prejemale BPAP. Izvedli smo inkubacije BPAP s podganjo S9 frakcijo pri različnih koncentracijah substrata in spremljali nastanek konjugiranih metabolitov. Rezultati so pokazali, da S9 frakcija hitro in učinkovito tvori predvsem glukuronidni konjugat BPAP, medtem ko je bilo sulfatiranje zanemarljivo. Glukuronidiranje smo dodatno preučili še z uporabo podganjih jetrnih mikrosomov, ki zagotavljajo večjo encimsko aktivnost in ponovljivost. Na podlagi eksperimentalnih podatkov smo izračunali encimske kinetične parametre in z uporabo ustreznih faktorjev ekstrapolirali očistek na celotna jetra. Razvili in validirali smo bioanalizno metodo LC-MS/MS za kvantifikacijo BPAP v bioloških vzorcih na osnovi izbranih zahtev iz smernice ICH M10. Validacija je obsegala oceno ključnih parametrov, kot so selektivnost, točnost, ponovljivost, spodnja meja kvantifikacije (LLOQ), integriteta razredčitve in učinek matrice. V nadaljevanju smo razvito metodo uporabili za analizo plazemskih vzorcev podgan, ki so bile peroralno izpostavljene znanemu odmerku BPAP. V plazmi podgan smo odkrili zelo nizke koncentracije BPAP, ki so bile na meji zaznave (0,8 nM) ali pod njo. Določili pa smo značilno višje in dobro merljive koncentracije BPAP-glukuronida (54 nM ± 0,9), kar se dobro ujema z našimi rezultati in vitro metaboličnih poskusov, ki so pokazali hitro pretvorbo BPAP v njegov glukuronid. Pridobljeni podatki predstavljajo pomembno osnovo za nadaljnje ocenjevanje varnosti BPAP kot nadomestka BPA.

• Jezik: Slovenski jezik
• Ključne besede: bisfenol AP, glukuronidiranje, validacija, LC-MS/MS
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-175875
• COBISS.SI-ID: 257297923
• Datum objave v RUL: 12.11.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Investigation of in vitro metabolism of bisphenol AP and development of a bioanalytical method for its quantitation in blood plasma

Izvleček

Bisphenols are categorized as endocrine-disrupting chemicals (EDCs) as they can mimic or inhibit the function of natural hormones. One of the most studied representatives is bisphenol A (BPA). These substances are primarily used in the production of polycarbonate plastics and epoxy resins. Long term exposure to these agents has toxic and harmful effects on the human body and may lead to the development of various diseases. As a result, an increasing number of countries are restricting or banning their use. In response, structurally similar BPA analogues, such as bisphenol AP (BPAP), have been proposed as potential alternatives. However, despite their presumed greater safety, data on the metabolism and toxicokinetics of these compounds remain poorly explored. The aim of this master theses was to study the in vitro metabolism of BPAP, concentrating particularly on phase II conjugation reactions – glucuronidation and sulfation. In our study, BPAP was incubated with rat liver S9 fraction with different substrate concentrations, and the formation of conjugated metabolites was monitored. The results suggested that BPAP was rapidly and efficiently glucuronidated in the S9 fraction, meanwhile the sulfation was less significant. Therefore, further glucuronidation studies were conducted using liver microsomes, which provide higher enzymatic activity and repeatability. Based on the experimental data, we calculated the kinetic parameters and extrapolated the liver clearance using appropriate scaling factors. Additionally, an LC-MS/MS bioanalytical method for the quantification of BPAP in biological samples was developed and validated according to ICH M10 guidelines. Validation comprised assessment of accuracy, precision, selectivity, lower limit of quantification (LLOQ), dilution integrity, and matrix effect. The developed method was applied to the analysis of plasma samples from rats orally exposed to a known dose of BPAP. In rat plasma, we found very low concentrations of BPAP or even below the limit of detection (0,8 nM). In contrast, we determined much higher concentrations of BPAP-glucuronide (54 nM ± 0,9), which is in a good agreement with our in vitro BPAP metabolism study showing rapid formation of BPAP glucuronide. The results provide an important base for further evaluation of BPAP safety as a potential BPA substitute.

• Ključne besede: bisphenol AP, glucuronidation, validation, LC-MS/MS