title: Stable isotope dilution mass spectrometric assay for PRPP using enzymatic procedures.
abstract: 5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. A method for the measurement of PRPP in erythrocytes was designed, which is based on the determination of [(13)C(5)]glutamate derived from [(13)C(5)]glutamine following the utilization of PRPP by the action of amidophosphoribosyltransferase. The present study describes a gas chromatographic-mass spectrometric method for determination of [(13)C(5)]glutamate using [(13)C(2)]glutamate as an internal standard. The methods involved purification by anion-exchange chromatography using a BondElut SAX and derivatization with isobutyl chlorocarbonate in water-methanol-pyridine. Quantitation was performed by selected ion monitoring of the protonated molecular ions in the chemical ionization mode. The intra-day reproducibility in the amounts of [(13)C(5)]glutamate determined was in good agreement with the actual amounts added in erythrocytes. A linear relationship was found between the amount of PRPP added and the amount of [(13)C(5)]glutamate formed from [(13)C(5)]glutamine using amidophosphoribosyltransferase.
authors: Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. sinohara@toyaku.ac.jp
authors: Y
authors: Y
authors: Shinohara
authors: Y
authors: Y
authors: Suzuki
authors: H
authors: H
authors: Hasegawa
authors: M
authors: M
authors: Nakamura
authors: T
authors: T
authors: Nishiyama
authors: A
authors: A
authors: Hiratsuka
authors: K
authors: K
authors: Ichida
mesh_terms: D000582:Amidophosphoribosyltransferase / Q000378:metabolism*; D002247:Carbon Isotopes; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D007201:Indicator Dilution Techniques; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010754:Phosphoribosyl Pyrophosphate / Q000032:analysis*
chemical_list: D002247:Carbon Isotopes; D005973:Glutamine; D010754:Phosphoribosyl Pyrophosphate; D000582:Amidophosphoribosyltransferase