APPLICATION OF STABLE-ISOTOPE TRACER COMBINED WITH MASS-SPECTROMETRICDETECTION FOR STUDYING MYOINOSITOL UPTAKE BY CULTURED NEURONS FROM FETAL MOUSE - EFFECT OF TRISOMY-16

A gas chromatographic (GC)/mass spectrometric method for studying myo- inositol uptake by neurons in vitro is described. Cultured cortical ne urons from fetuses of diploid and trisomy 16 mouse (animal model for D own syndrome) were incubated with a physiological concentration of hex adeuterated myo-inositol for 2-40 min. Washed cells were lysed and scy llo-inositol (internal standard) was added to the intracellular materi al which contained labeled myo-inositol taken up by the cells as well as the endogenous, unlabeled myo-inositol. The samples were evaporated to dryness and the analytes were converted into acetate derivatives. The components were separated by capillary GC, and the m/z 379 ion for labeled myo-inositol and the m/z 373 ion for mya-inositol and scyllo- inositol generated by chemical ionization in an ion trap mass spectrom eter were monitored. Quantitation of the deuterium-labeled mya-inosito l taken up by the neuron along with endogenous myo-inositol was achiev ed for 2-40 min of incubation. The labeled myo-inositol uptake was lin ear for up to 20 min and was Na+ dependent in these neurons. This non- radioisotope method was used to demonstrate a significant (40%) increa se in the rate of myo-inositol uptake by cortical neurons from the tri somy 16 mouse relative to central neurons. An increased myo-inositol u ptake is consistent with evidence that the myo-inositol transporter ge ne is on both human chromosome 21 and mouse chromosome 16, and that my o-inositol concentrations are elevated in cerebrospinal fluid from adu lt Down syndrome individuals and brains from the fetal trisomy 16 mous e. (C) 1997 by John Wiley & Sons, Ltd.