Razvoj i validacija biokemijskih indikatora funkcije kožne barijere

doctoral thesis

• Title (english): Development and validation of biochemical markers of the skin barrier function
• Author: Irena Đapić; MBZ: 313134
• Mentor: Ivone Jakaša (mentor); MBZ: 300874
• Mentor: Renata Kobetić (komentor)
• Committee member: Snežana Miljanić (predsjednik povjerenstva)
• Committee member: Ivone Jakaša (član povjerenstva); MBZ: 300874
• Committee member: Renata Kobetić (član povjerenstva)
• Committee member: Branka Marinović (član povjerenstva); MBZ: 224655
• Committee member: Sanda Rončević (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2014-11-18, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

U održanju funkcije kožne barijere važnu ulogu imaju prirodni čimbenici zadržavanja vlage (engl. natural moisturizing factors, NMF) koji sudjeluju u regulaciji pH i hidrataciji rožnatog sloja kože te međustanični lipidi koji predstavljaju barijeru pri transportu tvari kroz kožu. Cilj ovog rada bio je razviti pouzdane i selektivne LC-UV i LC-MS metode za određivanje biokemijskih indikatora za procjenu funkcije kožne barijere, slobodnih masnih kiselina, aminokiselina i ceramida u rožnatom sloju izuzetom adhezivnim vrpcama. Razvijena je RP-LC-UV metoda za određivanje komponenata NMF-a, histidina, piroglutaminske kiseline, trans- i cis-urokanske kiseline i tirozina u rožnatom sloju kože te međustaničnoj tekućini. Ekstrakcija komponenata NMF-a s amonijakom u odnosu na tradicionalnu ekstrakciju jakim bazama omogućila je kraće vrijeme obrade uzorka zaobilazeći dodatni korak neutralizacije s jakom kiselinom. Analitička metoda za određivanje slobodnih masnih kiselina u rožnatom sloju kože temelji se na detekciji 3-aciloksimetil-N-metilpiridinij jodid (NMP) derivata primjenom RP-LC-ESI-MS i praćenju višestrukih tranzicijskih reakcija. Utjecaj matrice na analizu slobodnih masnih kiselina minimiziran je kvantifikacijom uz zamjenski (surogat) analit i unutarnju standardizaciju izotopno obilježenim NMP derivatom C19:0. Sadržaj slobodnih masnih kiselina oslobođenih iz adhezivne vrpce procijenjen je iz korelacije s C12:0 koji nije detektiran u rožnatom sloju. Razvijene analitičke metode pokazale su se dovoljno osjetljive i reproducibilne za istovremeno određivanje NMF-a odnosno slobodnih masnih kiselina dugih i vrlo dugih lanaca u normalnoj koži i koži oštećenoj uslijed djelovanja unutarnjih i okolišnih faktora. Ceramidne klase izolirane su iz humanog epidermisa optimiranim višestrukim ekstrakcijskim postupcima. Sintetski ceramidi III, IIIB i VI okarakterizirani su primjenom LC-ESI-MS tehnike.

Abstract (english)

Natural moisturizing factors (NMF) play important role in the regulation of pH and hydration of the stratum corneum and the intercellular lipids form a barrier to transport of substances through the skin. The primary aim of this thesis was to develop reliable and selective LC-UV and LC-MS methods for the determination of biomarkers of skin barrier function including free amino acids, free fatty acids and ceramides obtained by tape stripping technique. Sensitive and robust RP-LC-UV method for determination of NMF components, histidine, pyroglutamic acid, trans- and cis-urocanic acid and tyrosine in the stratum corneum and interstitial fluid was developed. Extraction of NMF components with ammonia avoids a time consuming neutralization step as required with extraction procedures using strong base or acid. The method for determination of free fatty acids (FFAs) in the stratum corneum is based on detection of 3-acyloxymethyl-1-methylpyridinium iodide (NMP) derivatives in positive ionization mode using RP-LC-ESIMS and multiple reactions monitoring. To overcome the matrix effect, FFAs in the human stratum corneum were quantified by surrogate analyte method and internal standardization using isotope labelled derivatives of FFAs. The content of FFAs originated from the adhesive tape was calculated from the correlation with C12:0 signal not detected in the stratum corneum. Developed LC-UV and LC-ESI-MS methods showed to be reproducible and sensitive enough for simultaneous detection and quantification of NMF or eight long and very long chain FFAs in the normal and compromised human stratum corneum. Ceramide classes were isolated from human epidermis by multiple extraction steps. Synthetic ceramides III, IIIB and VI were characterized by LC-ESI-MS technique.

• Keywords: biokemijski indikatori funkcije kožne barijere; kožna barijera; LC-ESI-MS; LC-UV; NMF; slobodne masne kiseline; surogat analit
• Keywords (english): biomarkers of the skin barrier function; free fatty acids; LC-ESI-MS; LC-UV; NMF; skin barrier; surrogate analyte
• Language: croatian
• URN:NBN: urn:nbn:hr:217:774167
• Study programme: Title: Doctoral study in chemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija (doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija)
• Type of resource: Text
• Extent: 166 str. ; 30 cm
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-02-14 14:42:37