The determination of homocysteine–thiolactone in human plasma

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.