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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2012-12, Vol.911, p.186-191
• Main Authors: Smith, Desirée E.C., Mendes, Marisa I.S., Kluijtmans, Leo A.J., Janssen, Mirian C.H., Smulders, Yvo M., Blom, Henk J.
• Format: Article
• Language: English
• Subjects: Animals; cell culture; Cell Extracts - chemistry; Cells, Cultured; Chromatography, Liquid - methods; culture media; cystathionine; cystathionine beta-synthase; Cystathionine beta-Synthase - analysis; Cystathionine beta-Synthase - genetics; Cystathionine beta-Synthase - metabolism; Cystathionine β-synthase; detection limit; diet; diet therapy; Diets; Enzymes; Female; Fibroblasts; Fibroblasts - enzymology; Fibroblasts - metabolism; heterozygosity; homocysteine; Homocystinuria; Homocystinuria - chemically induced; Homocystinuria - genetics; Homocystinuria - metabolism; homozygosity; Humans; Hydrogen-Ion Concentration; Linear Models; Liquid chromatography; liver; Liver - cytology; Liver - enzymology; Liver - metabolism; mass spectrometry; Mathematical analysis; Methionine; Methionine - metabolism; Methionine - toxicity; Proteins; Rats; Rats, Wistar; Reproducibility of Results; Sensitivity and Specificity; serine; solid phase extraction; Tandem mass spectrometry; Tandem Mass Spectrometry - methods; Therapy
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2012.10.041

► A straightforward LC–MS/MS method for the determination of CBS enzyme activity. ► The method can be applied to different cell types. ► The method can be used for diagnosis of cystathionine β-synthase deficiency. ► The method can be used to study effects of treatment of homocystinuria. ► The method can be used to study effects of diet on mild hyperhomocystenemia. 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 Km 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.