Improved HPLC method for total plasma homocysteine detection and quantification

• Authors: Wojciech Sawuła , Zyta Banecka-Majkutewicz , Leszek Kadziński , Joanna Jakóbkiewicz-Banecka , Grzegorz Węgrzyn , Walenty Nyka , Bogdan Banecki
• Languages of publication: EN

Abstracts

EN

Recent clinical research has pointed at hyperhomocysteinemia as an independent risk factor in a number of cardiovascular and neurological diseases. We have improved a chromatographic method of total plasma homocysteine measurements in order to obtain higher sensitivity, reliability and reproducibility. The method demonstrates excellent linearity (R = 0.999), range (< 2-100 µM), precision (instrumental RSD 0.06 and method RSD 1.17), accuracy (recovery of 99.92 and RSD 1.27), reproducibility, quantification limit and ruggedness (e.g. pH from 2.0 to 2.5). Because even a small increase in homocysteine level can be a significant risk factor of cardiovascular diseases, such a precise method is required. The constructed method allows the measurement of plasma pyridoxal phosphate, PLP, the co-enzyme form of vitamin B6, on the same column and similar reagents. The developed method has been successfully applied to measure both total plasma and serum homocysteine in a group of acute stroke patients.

Keywords

EN

homocysteine measurement; stroke risk factors; HPLC; validation

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2008
• Volume: 55
• Issue: 1
• Pages: 119-126

Dates

published

2008

received

2007-09-12

revised

2007-11-20

accepted

2007-12-11

(unknown)

2008-01-16

Contributors

author

Wojciech Sawuła

• Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland

author

Zyta Banecka-Majkutewicz

• Department of Neurology, Medical University of Gdańsk, Gdańsk, Poland

author

Leszek Kadziński

• Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland

author

Joanna Jakóbkiewicz-Banecka

• Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland

author

Grzegorz Węgrzyn

• Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland

author

Walenty Nyka

• Department of Neurology, Medical University of Gdańsk, Gdańsk, Poland

author

Bogdan Banecki

• Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland

References

• Accinni R, Campolo J, Bartesaghi S, De Leo G, Lucarelli C, Cursano CF, Parodi O (1998) High-performance liquid chromatographic determination of total plasma homocysteine with or without internal standards. J Chromatogr A 828: 397-400.
• Bald E, Kaniowska E, Chwatko G, Glowacki R (2000) Liquid chromatographic assessment of total and protein-bound homocysteine in human plasma. Talanta 50: 1233-1243.
• Brosnan JT, Jacobs RL, Stead LM, Brosnan ME (2004) Methylation demand: a key determinant of homocysteine metabolism. Acta Biochim Polon 51: 405-413.
• Deitrick CL, Katholi RE, Huddleston DJ, Hardiek K, Burrus L (2001) Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5'-phosphate in human plasma. J Chromatogr B Biomed Sci Appl 751: 383-387.
• Finkelstein JD (1990) Methionine metabolism in mammals. J Nutr Biochem 1: 228-237.
• Finkelstein JD, Martin JJ (2000) Homocysteine. Int J Biochem Cell Biol 32: 385-389.
• Frick B, Schrocksnadel K, Neurauter G, Wirleitner B, Artner-Dworzak E, Fuchs D (2003) Rapid measurement of total plasma homocysteine by HPLC. Clin Chim Acta 331: 19-23.
• Garcia AJ, Apitz-Castro R (2002) Plasma total homocysteine quantification: an improvement of the classical high-performance liquid chromatographic method with fluorescence detection of the thiol-SBD derivatives. J Chromatogr B Analyt Technol Biomed Life Sci 779: 359-363.
• Gilfix BM, Blank DW, Rosenblatt DS (1997) Novel reductant for determination of total plasma homocysteine. Clin Chem 43: 687-688.
• Iciek M, Chwatko G, Lorenc-Koci E, Bald E, Włodek L (2004) Plasma levels of total, free and protein bound thiols as well as sulfane sulfur in different age groups of rats. Acta Biochim Polon 51: 815-824.
• Kelly PJ, Kistler JP, Shih VE, Mandell R, Atassi N, Barron M, Lee H, Silveira S, Furie KL (2004) Inflammation, homocysteine, and vitamin B6 status after ischemic. stroke. Stroke 35: 12-15.
• Lievers KJA, Boers GHJ, Verhoef P, Heijer M, Kluijtmans LA, Put NM, Trijbels FJ, Blom HJ (2001) A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk. J Mol Med 79: 522-528.
• Mayer EL, Jacobsen DW, Robinson K (1996) Homocysteine and coronary atherosclerosis. J Am Coll Cardiol 27: 517-527.
• Minniti G, Piana A, Armani U, Cerone R (1998) Determination of plasma and serum homocysteine by high-performance liquid chromatography with fluorescence detection. J Chromatogr A 828: 401-405.
• Moriyama Y, Okamura T, Kajinami K, Iso H, Inazu A, Kawashiri M, Mizuno M, Takeda Y, Sakamoto Y, Kimura H, Suzuki H, Mabuchi H (2002) Effects of serum B vitamins on elevated plasma homocysteine levels associated with the mutation of methylenetetrahydrofolate reductase gene in Japanese. Atherosclerosis 164: 321-328.
• Nekrassova O, Lawrence NS, Compton RG (2003) Analytical determination of homocysteine: a review. Talanta 60: 1085-1095.
• Pietrzik K, Bronstrup A (1998) Vitamins B12, B6 and folate as determinants of homocysteine concentration in the healthy population. Eur J Pediatr 157: S135-S138.
• Refsum H, Ueland PM (1998) Recent data are not in conflict with homocysteine as a cardiovascular risk factor. Curr Opin Lipidol 9: 533-539.
• Selhub J, Jaques PF, Rosenberg IH, Rogers G, Bowman BA, Gunter EW, Wright JD, Johnson CL (1999) Serum total homocysteine concentrations in the third national health and nutrition examination survey (1991-1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med 131: 331-339.
• Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'Agostino RB, Wilson PW, Wolf PA (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med 346: 476-483.
• Sun CF, Haven TR, Wu TL, Tsao KC, Wu JT (2002) Serum total homocysteine increases with the rapid proliferation rate of tumor cells and decline upon cell death: a potential new tumor marker. Clin Chim Acta 321: 55-62.