Determination of glutamine and alpha-ketoglutarate concentration and specific activity in plasma using...

A method is described for measuring glutamine (GLN) and alpha-ketoglutarate (KG) concentration and specific activity (SA) using high-performance liquid chromatography (HPLC). Plasma GLN and KG are separated on miniature ion-exchange columns. KG is derivatized with O-phenylene diamine, the derivative is extracted in ethyl acetate, dried, and dissolved in pH 7 phosphate buffer. The isolated GLN is enzymatically converted to KG and analysed as such. Derivatized samples are stable for weeks at -20 degrees C. Samples are injected onto a reversed-phase HPLC column. Absolute standards are injected to determine the nmol content of unknown samples. alpha-Ketoadipate and [3H]-glutamine are used as internal standards to quantitate KG and GLN concentrations, respectively. Collection of the entire peak of interest permits determination of the radioactivity in the GLN and KG peaks; this together with the determination of the nanomoles injected permits the calculation of the SA. Typical precision is 3.5 and 4.6% for GLN and KG concentrations and 5.3 and 3.3% for GLN and KG SA, respectively. Analysis time is ca. 7 min. Using this method, the turnover rate of GLN carbon was determined during a 5-h infusion of L-[U-14C]glutamine in a human subject.