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Cys34 thiol group of human serum albumin : possibilities and importance of determination in clinical practise

dc.contributor.advisorMandić, Ljuba
dc.contributor.otherVrvić, Miroslav
dc.contributor.otherDimitrijević-Srećković, Vesna
dc.creatorJovanović, Vesna
dc.date.accessioned2016-09-18T07:44:33Z
dc.date.available2016-09-18T07:44:33Z
dc.date.issued2013-12-28
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=3893
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:13070/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=45320463
dc.identifier.urihttp://nardus.mpn.gov.rs/123456789/6545
dc.description.abstractHumani serum albumin (HSA) ima više fizioloških funkcija, jedna od njih je odbrana organizma od oksidativnog i/ili karbonilnog stresa. Istaknuta funkcija je posledica prisustva jedne slobodne tiolne grupe ostatka Cys34 na površini njegovih molekula. Određivanje sadržaja HSA-SH grupa bi moglo biti pogodno za procenu stepena oksidativnog i/ili karbonilnog stresa u različitim patološkim stanjima. Da bi našlo primenu u kliničkoj praksi, u prvom koraku potrebno je izolovati HSA iz seruma ili plazme, a potom odrediti sadržaj tiolnih grupa. Ispitivanjem pouzdanosti metode izolovanja HSA afinitetnom hromatografijom sa Cibacron Blue (CB) iz seruma zdravih osoba i dijabetičara, i kvantifikacije tiolne grupe, ustanovljeno je da je sadržaj HSA-SH grupa veći za 6 do 33 % od ukupnog sadržaju tiola seruma, odnosno da je tačnost metode mala (recovery sadržaja HSA-SH grupa od 113,6 do 130,1%). Utvrđena diskrepancija nije posledica uslova izolovanja (zapremina uzorka, karakteristike matriksa, protok kroz kolonu i vreme potrebno za izolovanje HSA i određivanje sadržaja SH grupa), prisustva pufera za izolovanje i drugih proteina u izolovanom HSA (čistoće od 88,4 do 91,4 %), uticaja proteina, malih molekula i jona prisutnih u serumu na određivanje HSA-SH, već selekcije HSA molekula od strane CB prema njihovoj konformaciji. Promena konformacije (utvrđena emisionom fluorescentnom spektroskopijom i CD), odnosno selekcija zavisi od vrste i broja molekula masnih kiselina (Mk) vezanih za HSA. Osim toga, priroda i broj Mk vezanih za HSA doprinose povećanju reaktivnosti HSA-SH grupa: konstanta brzine pseudo prvog reda za reakciju HSA-SH grupe s DTNB u CB-vezanoj-HSA frakciji (21.74x10-3 s-1) je veća u odnosu na nevezanu- HSA frakciju (11.2x10-3 s-1), kao i u grupi dijabetičara (n=20) (20.9x10-3 s-1) u odnosu na kontrolnu grupu (n=17) (12.9x10-3 s-1). Recovery vrednosti sadržaja Cys34-SH grupa u CBvezanim- HSA frakcijama (od 98,5 do 101,7 %), dobijene nakon odmašćivanja HSA, potvrdile su da metoda izolovanja HSA afinitetnom hromatografijom nije pogodna za određivanje sadržaja HSA-SH grupa za kliničke svrhe. U cilju postizanja veće pouzdanosti u izvođenju zaključaka o promenama sadržaja HSA-SH grupa u različitim patološkim stanjima, optimizovana je metoda izolovanja HSA iz seruma dvostepenim taloženjem sa amonijum-sulfatom (AS) (zasićenje AS od 54 % u prvom i od 70 % u drugom koraku), sa prinosom HSA od 69,7 ± 4,4 %. Način taloženja HSA (sa čvrstim AS-om ili zasićenim rastvorom AS-a) i uklanjanje AS-a iz izolovanog preparata, ne utiče značajno na čistoću izolovanog HSA, u kojem je pored monomera (zastupljenost 91,9 ± 3,6 %) dokazano (SDS PAG elektroforezeom i imunoblotom) prisustvo dimera HSA (oko 10 %), tako de se predloženim postupkom izoluje HSA čistoće oko 100 %. Predložena metoda je jednostavna, brza i jeftina, što je važno za kliničku praksu. Omogućava precizno (RSD 3,2 %), tačno (recovery vrednost 101,2 ± 2,0 %, za opseg fizioloških vrednosti od 0,25 do 0,75 mol-SH/mol HSA) i pouzdano (doprinos HSASH ukupnom sadržaju tiola u serumu kod zdravih osoba iznosi 83,2 ± 5,3 %) određivanje sadržaja HSA-SH grupa. Predložena metoda može da se primeni i za izolovanje HSA iz plazme (ne postoji statistički značajna razlika između sadržaja Cys34-SH grupa HSA izolovanog iz plazme ili seruma (0.574 ± 0.026; 0.570 ± 0.028 mol-SH/mol HSA, resp.). Mogućnost i značaj određivanja sadržaja HSA Cys34-SH grupe kao markera u kliničkoj praksi, proverena je određivanjem sadržaja HSA-SH grupa i ukupnih tiola u grupi: obolelih od tipa 2 dijabetesa (n=23) i kontrolnoj grupi (n=17); kod trudnica sa (n=15) i bez preeklampsije (n=15). U oba patološka stanja, kod pacijenata sa tipom 2 dijabetesa i sa preeklampsijom, dobijeno je statistički značajno smanjenje (p<0.05) sadržaja HSA-SH grupa i ukupnih tiola seruma u odnosu na odgovarajuće kontrolne grupe. Određivanje sadržaja HSA Cys34-SH grupa se može primenjivati kao marker procene karbonilnog stresa u ovim patološkim stanjima. Pored toga, nađeno je da se reaktivnost tiolne grupe Cys34 ostatka u ovim stanjima menja, što ima važne implikacije na mogućnost modulacije reaktivnosti (antioksidativnih svojstava) tiolne grupe pomoću suplemenata (na primer. masnih kiselina).sr
dc.description.abstractHuman serum albumin (HSA) has a multitude of physiological functions, and one of them is to defend the organism against oxidative and/or carbonyl stress. This prominent function is enabled by the presence of single free Cys34 thiol group at the surface of HSA molecules. Determination of HSA-SH group content could be a suitable parameter for oxidative and/or carbonyl stress level assesemnet in different pathological states. In order to become aplicable in clinical practice, it is necessary to isolate HSA from serum or plasma in the first, and then to determine the content of thiol group. Monitoring of the reliability of affinity chromatography with Cibacron Blue (CB) for HSA isolation from serum of healthy and diabetic persons, and thiol group quantification, showed that HSA-SH group content was higher from 6 to 33% than the total serum thiols content, i.e. that the accuracy of this method is low (HSA-SH group content recovery ranging from 113.6 to 130.1%). This discrepancy is not caused by the isolation conditions (sample volume, matrix charateristics, column flow and the time needed for HSA isolation and SH group content determination), presence of buffer for HSA isolation and other proteins in isolated HSA preparation (purity range from 88.4 to 91.4%), influence of proteins, small molecules and ions present in the serum on the HSA-SH content determination, but the selection of HSA molecules by CB according to their conformations. The change in conformation (determined by emmission flourescent spectroscopy and CD), and accordingly the selection, depend on the type and the number of fatty acid (FA) molecules bound to HSA. Besides that, the nautre and the number of HSA bound FAs contribute to the increase in HSA-SH group reactivity: pseudo-first order rate constant for the reaction HSA-SH group with DTNB in CB bound fraction (21.74x10-3 s-1) is higher compared to unbound HSA fraction (11.2x10-3 s-1), as well as in diabetic group (n=20) (20.9x10-3 s-1) compared to the controls (n=17) (12.9x10-3 s-1). Recovery values for Cys34-SH group content in CB bound HSA fractions (from 98,5 to 101,7 %), acquired after HSA defatting, confirmed that isolation of HSA by affinity chromatography is not suitable for determination of HSA-SH group content in clinical practice. A two step ammonium sulphate (AS) precipitation method for HSA isolation from serum (AS saturation from 54% in first step and 70% in second step), with HSA yield of 69.7±4.4%, has been optimised in order to make more reliabile conclusions about the changes in HSA-SH group content in different pathological states. HSA precipitation method (with solid AS or saturated AS solution) and the removal of AS from isolated preparation, has no significant influence on the purity of isolated HSA, where the presence of HSA dimer (about 10 %) was detected (by SDS PAG electrophoresis and immunoblot) along with the HSA monomer (91.9%±3.6% abundance), so the purity of isolated HSA can be regarded as 100%. The proposed method is simple, fast and low cost, which is of greate importance for good clinical practice. It allows for precise (RSD 3,2%), accurate (with 101,2±2% recovery for physiological values from 0,25 to 0,75 mol -SH/mol HSA) and reliable (HSA-SH contribution to the total serum thiols content in healthy persons is 83,2 ± 5,3 %) determination of HSA-SH group content. In addition, proposed method could be aplicable for isolation of HSA from plasma samples (there is no significant statistical differrence between the HSA Cys34-SH content isolated form plasma and serum (0.574 ± 0.026; 0.570 ± 0.028 mol-SH/mol HSA, resp.). The possibility and the importance of HSA Cys34-SH content determination as a marker in clinical practice, was tested by the determination of HSA-SH and total thiols content in group of: patients with diabetes mellitus type II (n=23) and control group (n=17); pregnant women with (n=15) and without preeclampsia (n=15). In both pahtological states, in diabetic patients and pregnant women with preeclampsia, there is a statistically significant decrease (p<0.05) in HSA-SH group content as well as in total serum thiols content compared to appropriate controls. Determination of HSA Cys34- SH content could be applied as a marker for carbonyl stress level assesement in these pthological states. In addition, it was found that the reactivity of Cys34 thiol group in these pathological states changes, which is an important implication for possible modulation of its reactivity (antioxidant properties) by supplements (eg. fatty acids).en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Београду, Хемијски факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172049/RS//
dc.rightsAutorstvo-Nekomercijalno-Bez prerade 3.0 Srbija (CC BY-NC-ND 3.0)
dc.sourceУниверзитет у Београдуsr
dc.subjectpouzdanost određivanja tiolne grupe Cys34 HSAsr
dc.subjectHSA Cys34 thiol group determination reliabilityen
dc.subjectreaktivnost Cys34 tiolne grupesr
dc.subjectizolovanje HSA iz seruma i plazmesr
dc.subjectafinitetna hromatografijasr
dc.subjectmetoda dvostepenog taloženjasr
dc.subjectmasne kiseline vezane za HSAsr
dc.subjectkarbonilni stressr
dc.subjectdijabetessr
dc.subjectpreeklampsijasr
dc.subjectCys34 thiol group reactivityen
dc.subjectHSA isolation from serum and plasmaen
dc.subjectaffinity chromatographyen
dc.subjecttwo-step precipitation methoden
dc.subjectHSA bound fatty aciden
dc.subjectcarbonyl stressen
dc.subjectdiabetesen
dc.subjectpreeclampsiaen
dc.titleTiolna grupa Cys34 humanog serum-albumina : mogućnosti i značaj određivanja u kliničkoj praksisr
dc.titleCys34 thiol group of human serum albumin : possibilities and importance of determination in clinical practiseen
dc.typeThesis
dcterms.abstractМандић, Љуба; Врвић, Мирослав; Димитријевић-Срећковић, Весна; Јовановић, Весна;


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