Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice.
Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2-20 μL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine β-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease.
International Center for Public Health, Department of Microbiology, Biochemistry & Molecular Genetics, Rutgers-New Jersey Medical School, Newark, NJ 07103, USA; Department of Biochemistry and Biotechnology, University of Life Sciences, Poznań, Poland; Institute of Bioorganic Chemistry, Poznań, Poland. Electronic address: jakubows@rutgers.edu.
Hieronim
H
Jakubowski
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003541:Cystathionine beta-Synthase / Q000652:urine*; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000652:urine*; D051379:Mice; D016482:Urinalysis / Q000379:methods*
D006710:Homocysteine; C007957:homocysteine thiolactone; D003541:Cystathionine beta-Synthase
Ferritin; HPLC; Major urinary protein; Protein N-linked and S-linked homocysteine; Urinary homocysteine thiolactone