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Selective and Inexpensive Determination of Homocysteine
Authors: Žáková Pavla | Kanďár Roman | Skalický Jiří | Kovařík Jakub | Ventura Karel
Year: 2003
Type of publication: ostatní - přednáška nebo poster
Name of source: Atherosklerosa, hyperhomocysteinemie
Publisher name: Společnost patologické a klinické fyziologie ČLS JEP
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cze Selektivní a levné stanovení homocysteinu homocysteine, ion-paired reversed phase HPLC, electrochemical detection, penicillamine, 1,4-dithioerytritol
eng Selective and Inexpensive Determination of Homocysteine Introduction. In the clinical laboratory homocysteine is measured by a variety of techniques, inclunding imunoassays and HPLC with pre- or postcolumn derivatization. The aim of our work was to develop a selective and inexpensive method for determination of total homocysteine in biological samples without derivatization. Materials and method. The red blood cells releases homocysteine, after sampling. The release is time and temperature dependent. Therefore, collect the blood on ice and centrifuge as soon as is necessary. The most recommended is EDTA or heparin as anti-coagulated reagents. We compared concentrations of total homocysteine in citrate plasma (blood was kept at room temperature and was centrifuged 2 hours after sampling) versus in EDTA plasma (blood was centrifuged direct after sampling). We did not register significantly different between these sampling methods. Homocystein circulates mostly as dissulfides complexed with albumin. Therefore, the determination of total plasma homocysteine include a reduction step and proteins precipitation step before separation. We used 1,4-dithioerytritol as reduction reagent. Sample preparation is simple and rapid, without time-consuming derivatization. We separated homocysteine chromatographically using ion-paired reversed-phase HPLC. Penicillamine is appropriate internal standard for this method. We detected reduced homocysteine with ESA Coulochem II 5100A electrochemical detector (USA), using ESA 5010 coulometric cell. An ESA 5020 Guard cell was instaled between the pump and ESA autosampler. Results. Analytical performance of this method is satisfactory with intra-assay CV 2.8 %, inter-assay CV 7.1 % and 97 % recovery. The lowest reproducible detected amount was 0.5 microM. We established reference values in EDTA plasma of blood donors aged 22-35 years. The reference range was 6.4 +/- 1.5 microM for women and 7.1 +/- 1.9 microM for men. We determined of total homocysteine in 70 plasma samples of blo homocysteine, ion-paired reversed phase HPLC, electrochemical detection, penicillamine, 1,4-dithioerytritol

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