Naslov
High performance liquid chromatography analysis of S-adenosylmethionine and S-adenosylhomocysteine in fibroblasts with S-adenosylhomocysteine hydrolase deficiency

Autori
Majić, Željka ; Lovrić, Mila ; Bilić, Karmen ; Zekušić, Marija ; Škaričić, Ana ; Petković Ramadža, Danijela ; Fumić, Ksenija ; Ćuk, Mario ; Barić, Ivo

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of abstracts 13th International Chromatography School, Zagreb, 2012 / - , 2012

Skup
13th International Chromatography School

Mjesto i datum
Zagreb, Hrvatska, 18.06.2012. - 19.06.2012

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
HPLC; S-adenosylmethionine; S-adenosylhomocysteine; fibroblasts; S-adenosylhomocysteine hydrolase deficiency

Sažetak
S-adenosylhomocysteine hydrolase deficiency (SAHH) is a rare disease, described for the first time in 2004. SAHH is one of the enzymes in methionine cycle, in which it catalyzes the hydrolysis of S- adenosyhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy). AdoHcy is formed by transmethylation processes in which CH3 group is transfered from S-adenosylmethionine (AdoMet) to numerous methyl acceptor molecules using methyltransferases (MT). Ratio of AdoMet/AdoHcy concentration is called methylation index of the cell. Methylation index is considered to be one of the key players in disease development by inhibiting metyltransferases and leading to misregulated methylation of molecules within cell. The purpose of this work is to determine methylation index in fibroblast cell culture with S-adenosylhomocysteine hydrolase deficiency. Fibroblasts cultivated in 75 cm2 flasks were washed in phosphate buffer solution. Immediately after washing, 5 ml of 0.6 N perchloric acid with N-6-methyladenosine as an internal standard was added to cells. Cell debris was collected into tubes by scraping. The tubes were shaken thoroughly, placed on ice and centrifuged (4 000 x g, 10 min at +4 °C). The supernatant was adjusted to pH 5.5 by adding 2 M K2CO3/1 M KH2PO4 and then centrifuged. Supernatant was applied onto solid- phase extraction column (BondElut LRC-PBA). Elution of AdoHcy, AdoMet and internal standard was performed by using 0.1 M HCl and the eluate was analysed by high performance liquid chromatography (MN CC 250/4.6 NUCLEODUR C18 Gravity 5 m) with UV-detection (258 nm). The mobile phase consisted of solvent A (50 mM natrium dihydrogenphosphate, 8 mM heptanesulfonic acid sodium salt with addition of o-phosphoric acid, pH 3, 0) and solvent B (methanol). High performance liquid chromatography was previously used for AdoMet/AdoHcy ratio analysis in whole blood. Here, we demonstrate that AdoMet/AdoHcy ratio can be measured not only in whole blood, but also in cells, which could be useful biomarker at cellular level for the diagnosis of S-adenosylhomocysteine hydrolase deficiency. Our results suggest that the AdoMet/AdoHcy ratio is significantly lower in SAHH deficient fibroblast cells when compared to control.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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