Acevedo L D, Holloway H W, Rapoport S I, Shetty H U
Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Mass Spectrom. 1997 Apr;32(4):395-400. doi: 10.1002/(SICI)1096-9888(199704)32:4<395::AID-JMS487>3.0.CO;2-#.
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.
本文描述了一种用于体外研究神经元摄取肌醇的气相色谱（GC）/质谱方法。将来自二倍体和16三体小鼠（唐氏综合征动物模型）胎儿的培养皮质神经元与生理浓度的十六氘代肌醇孵育2 - 40分钟。洗涤后的细胞裂解，将异肌醇（内标）加入到细胞内物质中，该物质包含细胞摄取的标记肌醇以及内源性未标记的肌醇。样品蒸发至干，分析物转化为乙酸酯衍生物。通过毛细管GC分离各组分，并监测离子阱质谱仪中化学电离产生的标记肌醇的m/z 379离子以及肌醇和异肌醇的m/z 373离子。在孵育2 - 40分钟内实现了对神经元摄取的氘标记肌醇以及内源性肌醇的定量。在这些神经元中，标记肌醇的摄取在长达20分钟内呈线性，且依赖于Na +。这种非放射性同位素方法用于证明相对于对照神经元，16三体小鼠的皮质神经元摄取肌醇的速率显著增加（40%）。肌醇摄取增加与以下证据一致：肌醇转运蛋白基因位于人类21号染色体和小鼠16号染色体上，并且成年唐氏综合征个体的脑脊液和16三体小鼠胎儿的大脑中肌醇浓度升高。
