Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice.

Autor: Jakubowski H; 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.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2016 Sep 01; Vol. 508, pp. 118-23. Date of Electronic Publication: 2016 Jun 09.
DOI: 10.1016/j.ab.2016.06.002
Abstrakt: 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.
(Copyright © 2016 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: