title: Application of stable isotope tracer combined with mass spectrometric detection for studying myo-inositol uptake by cultured neurons from fetal mouse: effect of trisomy 16.
abstract: A gas chromatographic (GC)/mass spectrometric method for studying myo-inositol uptake by neurons in vitro is described. Cultured cortical neurons from fetuses of diploid and trisomy 16 mouse (animal model for Down syndrome) were incubated with a physiological concentration of hexadeuterated myo-inositol for 2-40 min. Washed cells were lysed and scyllo-inositol (internal standard) was added to the intracellular material 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 myo-inositol and scyllo-inositol generated by chemical ionization in an ion trap mass spectrometer were monitored. Quantitation of the deuterium-labeled myo-inositol taken up by the neuron along with endogenous myo-inositol was achieved for 2-40 min of incubation. The labeled myo-inositol uptake was linear for up to 20 min and was Na+ dependent in these neurons. This non-radioisotope method was used to demonstrate a significant (40%) increase in the rate of myo-inositol uptake by cortical neurons from the trisomy 16 mouse relative to control neurons. An increased myo-inositol uptake is consistent with evidence that the myo-inositol transporter gene is on both human chromosome 21 and mouse chromosome 16, and that myo-inositol concentrations are elevated in cerebrospinal fluid from adult Down syndrome individuals and brains from the fetal trisomy 16 mouse.
authors.affiliation: Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Acevedo
authors.forename: H W
authors.initials: HW
authors.lastname: Holloway
authors.forename: S I
authors.initials: SI
authors.lastname: Rapoport
authors.forename: H U
authors.initials: HU
authors.lastname: Shetty
mesh_terms: D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D004171:Diploidy; D004314:Down Syndrome / Q000235:genetics / Q000378:metabolism; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007294:Inositol / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014314:Trisomy
chemical_list: D007294:Inositol; D000254:Sodium-Potassium-Exchanging ATPase