title: Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.
abstract: Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.
authors.affiliation: Institute of Inherited Metabolic Disorders--1st Faculty of Medicine, Charles University in Prague, Praha, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Krijt
authors.forename: Jana
authors.initials: J
authors.lastname: Kopecká
authors.forename: Aleš
authors.initials: A
authors.lastname: Hnízda
authors.forename: Stuart
authors.initials: S
authors.lastname: Moat
authors.forename: Leo A J
authors.initials: LA
authors.lastname: Kluijtmans
authors.forename: Philip
authors.initials: P
authors.lastname: Mayne
authors.forename: Viktor
authors.initials: V
authors.lastname: Kožich
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods / Q000592:standards; D002138:Calibration; D016022:Case-Control Studies; D002853:Chromatography, Liquid; D003541:Cystathionine beta-Synthase / Q000172:deficiency* / Q000378:metabolism*; D004795:Enzyme Stability; D006712:Homocystinuria / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods / Q000592:standards; D010949:Plasma / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D011732:Pyridoxal Phosphate; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase
references.citation: Thromb Haemost. 2000 Apr;83(4):554-8
references.pmid: 10780316
references.citation: J Biol Chem. 2004 Jul 16;279(29):29871-4
references.pmid: 15087459
references.citation: Anal Chem. 2004 Sep 15;76(18):5345-53
references.pmid: 15362891
references.citation: Arch Biochem Biophys. 1982 Feb;213(2):695-707
references.pmid: 6803676
references.citation: Am J Hum Genet. 1985 Jan;37(1):1-31
references.pmid: 3872065
references.citation: Blood. 1993 Jun 15;81(12):3404-13
references.pmid: 8507876
references.citation: Arch Biochem Biophys. 2005 Jan 1;433(1):144-56
references.pmid: 15581573
references.citation: J Inherit Metab Dis. 2006 Apr-Jun;29(2-3):397-404
references.pmid: 16763908
references.citation: J Pediatr. 2009 Mar;154(3):431-7
references.pmid: 18950795
references.citation: Mol Genet Metab. 2010 Jan;99(1):1-3
references.pmid: 19819175
references.citation: J Inherit Metab Dis. 2011 Feb;34(1):39-48
references.pmid: 20490928