The determination of homocysteine-thiolactone in human plasma.
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.
Chwatko
Grazyna
G
Department of Microbiology & Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, NJ 07103, USA.
Jakubowski
Hieronim
H
D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D006262:Health; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000097:blood / Q000302:isolation & purification; D006801:Humans; D015394:Molecular Structure; D012680:Sensitivity and Specificity
D006710:Homocysteine; C007957:homocysteine thiolactone