title: Enzymatic diagnosis of homocystinuria by determination of cystathionine-ß-synthase activity in plasma using LC-MS/MS.
abstract: BACKGROUND
  Cystathionine β-synthase (CBS) is released into plasma from organs expressing this enzyme. Decreased plasma CBS activity has been demonstrated in CBS-deficient patients with 16 different genotypes. The aim of this study was to determine plasma CBS activity in patients carrying 11 additional genotypes using two LC-MS/MS methods. Patients and methods CBS activity was measured in EDTA or heparin plasma using either a previously described or a newly developed LC-MS/MS method optimized for analysis of the reaction product, 3,3-(2)H2-cystathionine, as its butyl ester derivative. We analyzed plasma samples from 26 CBS-deficient patients with known genotypes and 57 controls.
  
  
  RESULTS
  We developed a new LC-MS/MS method for simple and sensitive determination of CBS activity. Plasma CBS activity was low (i.e., 0.001-0.036 of the multiples of median control values, MoM) in patients homozygous for the prevalent Hispanic mutation c.572C>T (p.T191M) but was highly elevated (2.95 MoM) in a single patient homozygous for the c.1330G>A (p.D444N) mutation. Patients with the remaining nine genotypes exhibited decreased activities (0.00-0.22 MoM), which did not overlap with the controls (0.29-2.10 MoM).
  
  
  CONCLUSIONS
  The determination of CBS activity in plasma is a rapid and non-invasive procedure for detecting a subgroup of CBS-deficient patients with distinct genotypes.
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Facultad de Ciencias, Universidad Autónoma Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28049 Madrid, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Alcaide
authors.affiliation: Institute of Inherited Metabolic Disorders, Charles University in Prague, 1st Faculty of Medicine and General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Krijt
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Facultad de Ciencias, Universidad Autónoma Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28049 Madrid, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Ruiz-Sala
authors.affiliation: Institute of Inherited Metabolic Disorders, Charles University in Prague, 1st Faculty of Medicine and General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors.forename: P
authors.initials: P
authors.lastname: Ješina
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Facultad de Ciencias, Universidad Autónoma Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28049 Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Ugarte
authors.affiliation: Institute of Inherited Metabolic Disorders, Charles University in Prague, 1st Faculty of Medicine and General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors.forename: V
authors.initials: V
authors.lastname: Kožich
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Facultad de Ciencias, Universidad Autónoma Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28049 Madrid, Spain. Electronic address: bmerinero@cbm.csic.es.
authors.forename: B
authors.initials: B
authors.lastname: Merinero
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid; D003540:Cystathionine / Q000097:blood*; D003541:Cystathionine beta-Synthase / Q000097:blood* / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005838:Genotype; D006712:Homocystinuria / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D006720:Homozygote; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010375:Pedigree; D053719:Tandem Mass Spectrometry
chemical_list: D003540:Cystathionine; D003541:Cystathionine beta-Synthase
keywords: Cystathionine ß-synthase activity; Homocystinuria; LC-MS/MS; Pyridoxine response