Využití HPLC/ED k monitorování některých klinicky významných sirných sloučenin

Mgr. Soňa Sechovcová

Doctoral thesis

Use of HPLC/ED to monitoring some clinically important sulphur compounds

Abstract:

Cilem teto prace bylo (a) vyvinout vhodnou metodu pro stanoveni endogennich hladin kyseliny lipoove, (b) ov..it, zda je tato metoda vhodna i pro stanoveni hladin kyseliny lipoove po suplementaci, (c) ur.it cytotoxicitu kyseliny lipoove na erytrocyty, (d) ov..it vyznam homocysteinu jako biomarkeru v klinicke praxi, ziskat obecny p.ehled o celkovem metabolismu methioninu u pacient. s onemocn.nim koronarnich arterii a ov..it okam.ity vliv perkutanni koronarni intervence na hladiny methioninu a jeho metabolit.. (a) Cilem bylo vyvinout vhodnou metodu pro stanoveni hladin volne kyseliny lipoove. Jedna se o vysokou.innou kapalinovou chromatografii s elektrochemickou detekci. Vzorky krve byly ziskany od 40 dobrovolnych darc., kte.i spl.ovali podminky pro zapsani do registru darc.. Tito darci nebyli suplementovani kyselinou lipoovou. P.i stanoveni kyseliny lipoove v plasm. byly vzorky krve odst.ed.ny a ziskana plasma byla deproteinovana acetonitrilem. Po odst.ed.ni nasledovala extrakce na pevne fazi a kyselina lipoova byla eluovana z kolonky methanolem. Eluat byl odpa.en do sucha v atmosfe.e dusiku. Odparek byl rozpu.t.n ve sm.si acetonitrilu a deionizovane vody, po filtraci byl vzorek davkovan na analytickou kolonu s vazanou reverzni fazi. Limit detekce a kvantifikace teto metody byl 1,85 a 5,18 nmol/l. Dosahli jsme ni..i hodnoty meze detekce v porovnani s p.edchozimi studiemi (2,4 a 4,9 nmol/l), ktere se zabyvaly stanovenim volne kyseliny lipoove u nesuplementovanych jedinc.. Nicmen. i p.esto jsme hladiny kyseliny lipoove v plasm. u v.ech 40 dobrovolnych darc. nedetekovali. Vysledky na.i studie ukazuji, .e endogenni koncentrace volne kyseliny lipoove v plasm. jsou pravd.podobn. ni..i ne. 1,85 nmol/l. (b) Tato metoda byla vyu.ita k ov..eni hladin kyseliny lipoove v plasm. u suplementovanych dobrovolnych darc. a je tak vhodna pro studie zabyvajici se stanovenim hladin kyseliny lipoove u suplementovanych pacient.. (c) Byl sledovan vliv kyseliny lipoove na .ivotaschopnost (viabilitu) erytrocyt.. Vzorky krve byly ziskany od dobrovolnych nesuplementovanych darc.. Krev byla odst.ed.na p.i 750 x g, separovane erytrocyty byly celkem 3x promyty fosfatovym pufrem, nasledn. na.ed.ny Ringerovym roztokem a davkovany do 12- ti jamkovych kultiva.nich desti.ek. K suspenzi erytrocyt. byly p.idany r.zn. na.ed.ne roztoky kyseliny lipoove. Inkubace probihala 24 a 48 hodin p.i 37 ??C. K stanoveni viability bun.k byla pou.ita metoda vylu.ovani trypanove mod.i. Nami testovane koncentrace kyseliny lipoove (1, 5 a 10 ?Ęmol/l) nezp.sobily ani po 48 hodinove inkubaci statisticky vyznamne zm.ny ve viabilit. ani po.tu erytrocyt. oproti kontrole. Take .adna z testovanych vysokych koncentraci (50, 100, 250 ?Ęmol/l) kyseliny lipoove nezp.sobila statisticky vyznamne zm.ny ve viabilit. erytrocyt.. Na po.et erytrocyt. m.ly vysoke koncentrace statisticky vyznamny vliv ji. po 24 hodinove inkubaci. (d) Hladiny celkoveho homocysteinu, cysteinu, methioninu a cysteinyl-glycinu v plasm. byly stanoveny metodou vysokou.inne kapalinove chromatografie s coulometrickou detekci. D.vodem byla rychla p.iprava vzorku bez nutnosti derivatizace a ni..i finan.ni naroky oproti jinym metodam. Vzorky krve byly ziskany od 68 pacient. s prokazanym onemocn.nim koronarnich arterii, kte.i byli rozd.leni do t.i skupin dle stupn. zava.nosti onemocn.ni. Nebyly nalezeny .adne statisticky vyznamne rozdily v prevalenci diabetu, pre-diabetu a onemocn.ni ledvin mezi sledovanymi skupinami, rovn.. mezi ku.aky, neku.aky .i pacienty s normalnim a zvy.enych krevnim tlakem. Byly zji.t.ny signifikantni rozdily v hladinach albuminu, celkoveho cholesterolu, LDL-cholesterolu a stupn. indexu aterogenity u sledovanych skupin pacient.. Byly zji.t.ny statisticky vyznamn. zvy.ene hladiny homocysteinu u pacient. s akutni formou onemocn.ni v porovnani s pacienty trpici chronickou formou tohoto onemocn.ni. Na.e vysledky podporuji hypotezu, .e homocystein nelze vyu.it pro v.asnou diagnostiku stenozy v koronarnich arteri

Abstract:

The aims of this thesis are: (a) the development of a suitable method for the determination endogenous levels of lipoic acid, (b) the verification of a possibility to use the method for determining the lipoic acid levels after supplementation, (c) the determination of lipoic acid cytotoxicity for erythrocytes, and (d) to verify the importance of homocysteine as a biomarker in clinical practice. Moreover, the aim is to obtain a general overview about the whole methionine metabolism for patients, suffering from coronary artery disease at different degrees of severity, as well as?n to verify the immediate effect of a percutaneous coronary intervention on the methionine level and its metabolites. (a) The aim was to develop a suitable method in order to determine the endogenous levels of free lipoic acid using a high-performance liquid chromatography method with a very sensitive coulometric detection. Plasma samples were collected from 40 voluntary blood donors. This group was non-supplemented with lipoic acid and met the preconditions in for being included to the blood donorsˇ? registry. A blood sample was collected into a tube containing ethylene diamintetraacetic acid as an anticoagulant and the obtained plasma was deproteinized with acetonitrile. A solid phase extraction was used for the extraction. The samples were eluted with methanol and the eluate was evaporated to dryness using a gentle nitrogen stream. The residue was re-dissolved in a mixture of acetonitrile/water. After filtration, the sample was injected into an analytical column with reversed-phase. The limit of detection and the limit of quantification values were 1.85 and 5.18 nmol/l. The limit of detection was better than in previous studies: 2.4 nmo/l and 4.9 nmol/l respectively. However, the levels of LA in the plasma of the 40 non-supplemented voluntary blood donors were not detectable in all cases. The results of our study show that endogenous concentrations of LA are less than1.85 nmol/l. (b) This method was used in order to determine lipoic acid levels in the plasma of supplemented voluntary blood donors. This method is suitable for further studies in order to determine lipoic acid levels in patients after supplementation. (c) The effect of lipoic acid on the viability of erythrocytes was monitored. Plasma samples were collected from voluntary blood donors, non-supplemented with lipoic acid. The venous blood was centrifuged at 750 x g; the separated erythrocytes were washed three times with phosphate buffer, diluted with Ringer's solution, and injected into 12-well culture plates. Lipoic acid solutions, which had previously been diluted in different rations, were added to the erythrocyte suspension. The incubation time was 24 and 48 hours at 37˘XC. For the determination of the cell viability the Tryphan blue exclusion test was used. The concentrations of lipoic acid (1.5 and 10 Łgmol/l) did not cause a statistically significant change regarding the viability or number of erythrocytes after the 24 and 48-hour incubation time. The high concentrations of lipoic acid (50, 100, 250 Łgmol/l) did neither cause a statistically significant change regarding the viability or the number of erythrocytes after the 24 and 48 hour incubation time. On the other hand, the high concentrations of lipoic acid had a statistically significant effect on the number of erythrocytes already after the 24-hour incubation time. (d) We measured the total plasma level of homocysteine, cysteine, methionine and cysteinyl-glycine using high-performance liquid chromatography with coulometric detection. Blood samples were obtained from 68 patients, suffering from coronary artery disease. The patients were divided into three groups according to the severity of their disease. We did not find any significant differences in the prevalence of diabetes, pre-diabetes and kidney diseases between the selected groups. We did not find any significant differences between the number of smokers and hypert

Keywords

cytotoxicita, elektrochemická detekce, erytrocyty, homocystein, kyselina lipoová, onemocnění koronárních artérií, perkutánní koronární intervence, plasma, vysokoúčinná kapalinová chromatografie

Language used: Czech

Date on which the thesis was submitted / produced: 6. 7. 2018

Accessible from:: 31. 12. 2999

Thesis defence

• Date of defence: 14. 9. 2018

University of Pardubice

Faculty of Chemical Technology

Doctoral programme / field:
Analytical Chemistry / Analytical Chemistry