Haemostasis pp 383-386 | Cite as

Testing for Hyperhomocysteinemia in Subjects with a History of Thromboembolic Events Using HPLC Technique

  • Jonas Denecke
Part of the Methods in Molecular Biology book series (MIMB, volume 992)


An elevated homocysteine level is a well-known thrombophilic risk factor. Determination of total plasma homocysteine therefore is an integrated part of the diagnostic setting after thromboembolic events; about 5–7% of the population do have elevated homocysteine levels. Some forms of hyperhomocysteinemia are treatable; thus a standardized and reliable diagnostic setting has to be at hand. HPLC analysis is widely available in routine diagnostic laboratories. We use the fluorogenic reagent SBD-F to derivate with plasma homocysteine after release of the amino acid from homo- and heterodimers and protein bond using TBP. Separation is performed using a c18 reverse-phase column with aqua and acetonitrile as solvent. Due to continuous release of homocysteine from blood cells centrifugation and separation of plasma within 30 min after venous puncture are crucial for reproducible results.

Key words

Homocysteine Thromboembolic risk factors HPLC 


  1. 1.
    D’ Angelo A, Coppola A, Madonna P, Fermo I, Pagano A, Mazzola G, Galli L, Cerbone AM (2000) The role of vitamin B12 in fasting hyperhomocysteinemia and its interaction with the homozygous C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene. A case-control study of patients with early-onset thrombotic events. Thromb Haemost 83:563–570Google Scholar
  2. 2.
    Gaustadnes M, Rudiger N, Rasmussen K, Ingerslev J (2000) Intermediate and severe hyperhomocysteinemia with thrombosis: a study of genetic determinants. Thromb Haemost 83:554–558PubMedGoogle Scholar
  3. 3.
    Desouza C, Keebler M, McNamara DB, Fonseca V (2002) Drugs affecting homocysteine metabolism: impact on cardiovascular risk. Drugs 62:605–616PubMedCrossRefGoogle Scholar
  4. 4.
    Chen P, Poddar R, Tipa EV, Dibello PM, Moravec CD, Robinson K, Green R, Kruger WD, Garrow TA, Jacobsen DW (1999) Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease. Adv Enzyme Regul 39:93–109PubMedCrossRefGoogle Scholar
  5. 5.
    McCully KS (1996) Homocysteine and vascular disease. Nat Med 2:386–389PubMedCrossRefGoogle Scholar
  6. 6.
    Thirup P, Ekelund S (1999) Day-to-day, postprandial, and orthostatic variation of total plasma homocysteine. Clin Chem 45:1280–1283PubMedGoogle Scholar
  7. 7.
    Jacobsen DW (1998) Homocysteine and vitamins in cardiovascular disease. Clin Chem 44:1833–1843PubMedGoogle Scholar
  8. 8.
    Araki A, Sako Y (1987) Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr 422: 43–52PubMedCrossRefGoogle Scholar
  9. 9.
    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 779:359–363PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2013

Authors and Affiliations

  • Jonas Denecke
    • 1
  1. 1.Department of PaediatricsUniversity Hospital Hamburg-EppendorfHamburg-EppendorfGermany

Personalised recommendations