A liquid chromatography mass spectrometry method for the measurement of cystathionine beta-synthase activity in cell extracts

Abstract

Background: In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine beta-synthase (CBS) a sensitive analytical method is necessary. Methods: An electrospray LC-MS/MS method preceded by a solid phase extraction step was developed for the measurement of CBS activity in cell extracts. Nonafluoropentanoic acid was used as an ionpair to provide the underivatized cystathionine the desired retention on a C18 column. Results: A detection limit of 50pmol cystathionine/h/mg protein was achieved. In fibroblasts, intra- and inter-assay CVs for the CBS activity were 5.2% and 14.7%, respectively. A K(m) value of 8mumol/L for homocysteine, and 2.5mumol/L for serine was calculated. In fibroblasts wildtype, heterozygous, and homozygous CBS activity ranges measured were 8.5-27.0, 4.2-13.4, 0.0-0.7nmol/hxmg protein, respectively. The method was applied to a study where rats were fed 2 diets. Increase of dietary methionine (7.7 versus 3.8mg/kg methionine) significantly increased the CBS activity in rat liver lysates from a median of 58.0 to a median of 71.5 (P=0.037)nmol/hxmg protein. In a lymphoblasts cell culture experiment, the addition of Hcy to the culture media increased the activity of CBS 3 fold. Conclusion: This LC-MS/MS is able to diagnose CBS deficiency at the enzyme level, and can accurately measure the effect diets or therapy might have on the CBS activity in a variety of cell types.