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 C18 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 μmol/L [0.49-3.33 μmol/L]; fibroblasts 7.43 and 26.46 μmol/mg protein [0.31-1.14 μmol/mg protein]; lymphoblasts 16.03 μmol/mg protein [0.61-2.09 μmol/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.
|Number of pages||8|
|Journal||Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences|
|Publication status||Published - 25 Aug 2005|