The determination of homocysteine-thiolactone in human plasma

Grazyna Chwatko, Hieronim Jakubowski

Research output: Contribution to journalArticlepeer-review

94 Scopus citations

Abstract

The thioester homocysteine-thiolactone, a reactive metabolite of homocysteine, has been implicated in human cardiovascular disease. However, data on the levels of homocysteine-thiolactone in humans are limited, mostly due to a lack of facile and reliable assays. Here we describe a sensitive assay for the determination of plasma homocysteine-thiolactone and demonstrate its utility with a cohort of 60 healthy human subjects. Plasma homocysteine-thiolactone is first separated from macromolecules by ultrafiltration and then selectively extracted with chloroform/methanol. Further purification of plasma homocysteine-thiolactone is achieved by high-performance liquid chromatography on a cation exchange microbore column. The detection and quantification is by monitoring fluorescence after postcolumn derivatization with o-phthaldialdehyde. The limit of detection is 0.36 nM. Using this assay, homocysteine-thiolactone concentrations in plasma from normal healthy human subjects (n = 60) were found to vary from zero to 34.8 nM, with an average of 2.82 ± 6.13 nM. In 29 of the 60 human plasma samples analyzed, homocysteine-thiolactone levels were below the detection limit. Homocysteine-thiolactone represented from 0 to 0.28%, on average 0.023 ± 0.05%, of plasma total homocysteine.

Original languageEnglish (US)
Pages (from-to)271-277
Number of pages7
JournalAnalytical Biochemistry
Volume337
Issue number2
DOIs
StatePublished - Feb 15 2005

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Keywords

  • HPLC
  • Homocysteine-thiolactone
  • Human plasma

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