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 [13C5]glutamate derived from [13C5]glutamine following the utilization of PRPP by the action of amidophosphoribosyltransferase. The present study describes a gas chromatographic-mass spectrometric method for determination of [13C5]glutamate using [13C2]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 [13C5]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 [13C5]glutamate formed from [13C5]glutamine using amidophosphoribosyltransferase.
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