Journal
ANALYTICAL BIOCHEMISTRY
Volume 508, Issue -, Pages 118-123Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2016.06.002
Keywords
Urinary homocysteine thiolactone; Protein N-linked and S-linked homocysteine; HPLC; Ferritin; Major urinary protein
Funding
- National Science Center [2012/07/B/NZ7/01178, 2013/09/B/NZ5/02794, 2013/11/B/NZ1/00091]
- American Heart Association [12GRNT9420014]
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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 mu L 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 beta-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease. (C) 2016 Elsevier Inc. All rights reserved.
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