BACKGROUND: In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine β-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 8μmol/L for homocysteine, and 2.5μmol/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/h×mg 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/h×mg 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.
BACKGROUND: In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine β-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 8μmol/L for homocysteine, and 2.5μmol/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/h×mg 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/h×mg 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.
Authors: Marisa I S Mendes; Henrique G Colaço; Desirée E C Smith; Rúben J J F Ramos; Ana Pop; Silvy J M van Dooren; Isabel Tavares de Almeida; Leo A J Kluijtmans; Mirian C H Janssen; Isabel Rivera; Gajja S Salomons; Paula Leandro; Henk J Blom Journal: J Inherit Metab Dis Date: 2013-08-23 Impact factor: 4.982
Authors: Andrew A M Morris; Viktor Kožich; Saikat Santra; Generoso Andria; Tawfeg I M Ben-Omran; Anupam B Chakrapani; Ellen Crushell; Mick J Henderson; Michel Hochuli; Martina Huemer; Miriam C H Janssen; Francois Maillot; Philip D Mayne; Jenny McNulty; Tara M Morrison; Helene Ogier; Siobhan O'Sullivan; Markéta Pavlíková; Isabel Tavares de Almeida; Allyson Terry; Sufin Yap; Henk J Blom; Kimberly A Chapman Journal: J Inherit Metab Dis Date: 2016-10-24 Impact factor: 4.982
Authors: Soraia Poloni; Fernanda Sperb-Ludwig; Taciane Borsatto; Giovana Weber Hoss; Maria Juliana R Doriqui; Emília K Embiruçu; Ney Boa-Sorte; Charles Marques; Chong A Kim; Carolina Fischinger Moura de Souza; Helio Rocha; Marcia Ribeiro; Carlos E Steiner; Carolina A Moreno; Pricila Bernardi; Eugenia Valadares; Osvaldo Artigalas; Gerson Carvalho; Hector Y C Wanderley; Johanna Kugele; Melanie Walter; Lorena Gallego-Villar; Henk J Blom; Ida Vanessa D Schwartz Journal: Mol Genet Genomic Med Date: 2018-01-20 Impact factor: 2.183