title: Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism.
abstract: We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify pentose phosphate pathway intermediates (triose-3-phosphates, tetrose-4-phosphate, pentose-5-phosphate, pentulose-5-phosphates, hexose-6-phosphates and sedoheptulose-7-phosphate (sed-7P)) in bloodspots, fibroblasts and lymphoblasts. Liquid chromatography was performed using an ion pair loaded C(18) HPLC column and detection of the sugar phosphates was carried out by tandem mass spectrometry using an electron ion spray source operating in the negative mode and multiple reaction monitoring. Reference values for the pentose phosphate pathway intermediates in blood spots, fibroblasts and lymphoblasts were established. The method was applied to cells from patients affected with a deficiency of transaldolase. The transaldolase-deficient cells showed an increased concentration of sedoheptulose-7-phosphate. (Bloodspots: 5.19 and 5.43 micromol/L [0.49-3.33 micromol/L]; fibroblasts 7.43 and 26.46 micromol/mg protein [0.31-1.14 micromol/mg protein]; lymphoblasts 16.03 micromol/mg protein [0.61-2.09 micromol/mg protein].) The method was also applied to study enzymes of the pentose phosphate pathway by incubating fibroblasts or lymphoblasts homogenates with ribose-5-phosphate or 6-phosphogluconate and the subsequent analysis of the formed sugar phosphates.
authors.affiliation: Metabolic Unit of the Department of Clinical Chemistry, VU University Medical Centre, Amsterdam, The Netherlands.
authors.forename: Mirjam M C
authors.initials: MM
authors.lastname: Wamelink
authors.forename: Eduard A
authors.initials: EA
authors.lastname: Struys
authors.forename: Jojanneke H J
authors.initials: JH
authors.lastname: Huck
authors.forename: Birthe
authors.initials: B
authors.lastname: Roos
authors.forename: Marjo S
authors.initials: MS
authors.lastname: van der Knaap
authors.forename: Cornelis
authors.initials: C
authors.lastname: Jakobs
authors.forename: Nanda M
authors.initials: NM
authors.lastname: Verhoeven
mesh_terms: D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D008661:Metabolism, Inborn Errors / Q000175:diagnosis / Q000378:metabolism; D010427:Pentose Phosphate Pathway; D012016:Reference Values; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013403:Sugar Phosphates / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D014153:Transaldolase / Q000378:metabolism; D014174:Transketolase / Q000378:metabolism
chemical_list: D013403:Sugar Phosphates; D014174:Transketolase; D014153:Transaldolase