TY - JOUR
T1 - The determination of homocysteine-thiolactone in human plasma
AU - Chwatko, Grazyna
AU - Jakubowski, Hieronim
N1 - Funding Information:
This work was supported by grants from the American Heart Association and the National Science Foundation.
PY - 2005/2/15
Y1 - 2005/2/15
N2 - 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.
AB - 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.
KW - HPLC
KW - Homocysteine-thiolactone
KW - Human plasma
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U2 - 10.1016/j.ab.2004.11.035
DO - 10.1016/j.ab.2004.11.035
M3 - Article
C2 - 15691507
AN - SCOPUS:13244287981
SN - 0003-2697
VL - 337
SP - 271
EP - 277
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -