title: A liquid chromatography mass spectrometry method for the measurement of cystathionine β-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 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.
  
  
  CONCLUSIONS
  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.affiliation: Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands. d.smith@vumc.nl
authors.forename: Desirée E C
authors.initials: DE
authors.lastname: Smith
authors.forename: Marisa I S
authors.initials: MI
authors.lastname: Mendes
authors.forename: Leo A J
authors.initials: LA
authors.lastname: Kluijtmans
authors.forename: Mirian C H
authors.initials: MC
authors.lastname: Janssen
authors.forename: Yvo M
authors.initials: YM
authors.lastname: Smulders
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Blom
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D003541:Cystathionine beta-Synthase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D006712:Homocystinuria / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D008715:Methionine / Q000378:metabolism / Q000633:toxicity; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002457:Cell Extracts; D008715:Methionine; D003541:Cystathionine beta-Synthase