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Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 5600™ Mass
Shuang Deng1, David Scott1, Douglas Myers2
1Department of Pathology and Laboratory Medicine, Children's Mercy Hospitals and Clinics, 2401 Gillham Road, Kansas City, MO, 64108, USA.
Abstract:
Triosephosphate isomerase (TPI) is a glycolytic enzyme which catalyzes the interconversion between glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). TPI deficiency results in accumulation of DHAP in human red blood cells and other tissues. The disease is characterized by congenital hemolytic anemia, and progressive neuromuscular dysfunction. The laboratory diagnosis is generally made by measurement of TPI activity in RBCs. Measurement of DHAP can be useful in further confirmation and follow-up of the disease. We developed HPLC/TOF-MS method for quantitation of DHAP in RBCs. The method involves simple protein precipitation, reverse phase C8 column chromatography, ion pairing with tributylamine, and long run time of 50 min to separate the two isomers (G3P and DHAP).
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