The determination of homocysteine-thiolactone in biological samples.
Homocysteine-thiolactone, a cyclic thioester of homocysteine, is synthesized by methionyl-tRNA synthetase in all cell types. A new assay for the determination of homocysteine-thiolactone in biological samples is described. The assay involves separation of homocysteine-thiolactone from macromolecules by ultrafiltration. Homocysteine-thiolactone is further purified and quantified by high-pressure liquid chromatography either on a reverse phase or a cation exchange micro-bore column. The detection and quantitation are obtained by monitoring the absorbance at 240 nm, a maximum in a UV spectrum of homocysteine-thiolactone. The sensitivity of detection is 5 pmol. This assay has been applied to bacteria (Escherichia coli and Mycobacterium smegmatis), the yeast Saccharomyces cerevisiae, cultured human vascular endothelial cells, and human plasma. The data support the conclusion that homocysteine-thiolactone is a ubiquitous metabolite whose levels are directly related to homocysteine levels.
Jakubowski
Hieronim
H
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark 07101, USA. jakubows@umdnj.edu
D002138:Calibration; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D020102:Mycobacterium smegmatis / Q000378:metabolism; D012680:Sensitivity and Specificity
D006710:Homocysteine; C007957:homocysteine thiolactone