20.500.12556/DKUM-19203 DOLOČEVANJE SESTAVE MAŠČOBNIH KISLIN IN NJIHOVIH PROFILOV V MEMBRANAH RDEČIH KRVNIH CELIC BOLNIKOV Z RAZLIČNIMI BOLEZNIMI Z METODO PLINSKE KROMATOGRAFIJE DETERMINATION OF RED CELL MEMBRANE FATTY ACID PROFILES IN PATIENTS WITH DIFFERENT DISEASES WITH GAS CHROMATOGRAPHY V doktorski disertaciji sem primerjala sestave maščobnih kislin in njihove karakteristične profile izoliranih fosfolipidov membran rdečih krvnih celic bolnikov z različnimi boleznimi z namenom, da bi preverila hipotezo, da so razlike med profili maščobnih kislin pomembne pri prepoznavanju različnih bolezenskih stanj oziroma motenj v metabolizmu. V študijo sem vključila vzorce krvi 272 bolnikov, od tega 135 bolnikov s kronično vnetno črevesno boleznijo (60 bolnikov s Crohnovo črevesno boleznijo in 75 bolnikov z drugimi vnetnimi črevesnimi boleznimi), 18 bolnikov s črevesnimi polipi, 52 bolnikov z astmo, 53 bolnic z miomi na maternici ter 14 vzorcev krvi žensk, ki miomov nimajo. 70 zdravih ljudi, ki niso oboleli za nobeno od naštetih bolezni, je predstavljalo kontrolno skupino. Izbira bolnikov je bila naključna in neodvisna od njihovih prehranjevalnih navad ali prehranskih dodatkov lipidov. Uporabila sem robustno in natančno analizno metodo ekstrakcije lipidov, separacije lipidnih razredov in izolacije fosfolipidov na silikagel trdni fazi (SPE-Si), ki sem jo izboljšala in optimizirala. Izolaciji rdečih krvnih celic je sledila metoda postopne ekstrakcije tekoče-tekoče, s katero sem povečala izkoristek izoliranih celokupnih lipidov, ki sem jih z ekstrakcijo na trdni fazi ločila na nepolarne lipide in polarne fosfolipide. Optimizacijo izolacije fosfolipidov na trdni fazi sem izvedla z vzporednim preskušanjem in uporabo tankoplastne kromatografije (TLC). Umiljenju fosfolipidne frakcije je sledila esterifikacija v metanolni raztopini borovega trifluorida. Sestavo metilnih estrov maščobnih kislin (FAME) fosfolipidov rdečih krvnih celic sem določala s plinsko kromatografijo (GC) s plamensko ionizacijskim detektorjem (FID). V membranah rdečih krvnih celic sem določila tudi dva dimetil acetala (heksadekanal in oktadekanal dimetil acetal; 16:0 DMA in 18:0 DMA), ki sta merilo oksidacijske prizadetosti celičnih membranskih fosfolipidnih plazmalogenov, ki so pomembni lipidni antioksidanti. S kemometrijskimi in statističnimi metodami sem karakterizirala vzorce glede na sestavo in vsebnost maščobnih kislin in dimetil acetalov. Študija je potrdila, da obstajajo pri opisanih bolnikih razlike v sestavi maščobnih kislin, kar bi lahko uporabili za diagnostične presejalne teste, kjer bi maščobne kisline prevzele vlogo bioloških označevalcev bolezni. The dissertation presented here aims to compare the fatty acid composition and their characteristic profiles derived from erythrocyte phospholipids of patients with different diseases and to test the hypothesis that the changes in their profiles might be relevant to various diseases and metabolic abnormalities. The study sample consisted of 272 patients, among them 135 with inflammatory bowel disease (60 patients with Crohn disease and 75 patients with other inflammatory bowel diseases), 18 with colon adenomas, 52 with asthma, 53 with uterine leiomyoma, 14 with verified absence of uterine leiomyoma. 70 blood samples without any of mentioned diseases were used as a control group. A robust and reliable analytical method, additionally improved and optimised, was used for the lipid extraction, separation of lipid classes and isolation of phospholipids by silica solid phase (SPE-Si). After the isolation of erythrocytes from blood samples, the method of sequential liquid-liquid extraction was used to yield more isolated total lipids, which were then separated by solid phase extraction into non polar lipids and polar phospholipids. The phospholipid SPE isolation was optimised by thin layer chromatography (TLC). After the saponification of phospholipid fraction, the esterification process followed with boron trifluoride methanol reagent. The fatty acid methyl ester (FAME) composition of the total red blood cell phospholipid fraction was analyzed by gas chromatography (GC) with flame ionization detector (FID). Additionally, two fatty aldehyde dimethyl acetals (hexadecanal and octadecanal dimethyl acetals; 16:0 DMA and 18:0 DMA) derived from erythrocyte membrane plasmalogen phospholipids were also determined. Plasmalogens act as targets for oxidants and are major lipophilic antioxidants. The resulting fatty acids and plasmalogen linked dimethyl acetals were evaluated by chemometrics and statistical methods. The study suggests differences in membrane fatty acid profiles in patients with different diseases which might have a potential for diagnostics and population screening for diseases, where fatty acids may play role as biomarkers for diseases. rdeče krvne celice eritrociti celične membrane fosfolipidi maščobne kisline profili maščobnih kislin plazmalogeni plinska kromatografija tankoplastna kromatografija metoda glavnih osi klasifikacija red blood cell human erythrocytes cell membranes phospholipids fatty acids fatty acid profiles plasmalogens gas chromatography thin layer chromatography principal component analysis classification true true false Z. Cencič Kodba] Slovenski jezik Angleški jezik Doktorska disertacija 2011-06-27 15:54:09 2011-07-01 14:59:56 2022-04-13 15:10:56 2023-12-25 03:11:40 2011 0 [Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 543.544:577.115:612.111(043.3) 256841472 URN:SI:UM:DK:HWLBTCT5 DR_Cencic_Kodba_Zdenka_2011.pdf DR_Cencic_Kodba_Zdenka_2011.pdf 1 6200504FCE78090F7F2CBFE387BE2884 9aef9e37c723ba88a81bee19bbe7810af6c51b6a67bd1a82c7b81b270cfee6ef 512b1099-7c05-11eb-bb7a-00155d0001ca 20.500.12556/dkum/3207cf63-2579-44bf-8a1c-f31f017b05e0 https://dk.um.si/Dokument.php?lang=slv&id=23130 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0