Generation of C-Labeled Inositol and Inositol Phosphates by Stable Isotope Labeling Cell Culture for Quantitative Metabolomics.

Inositol and inositol phosphates (IPx) are central metabolites. Their accurate quantitative analysis in complex biological samples is challenging due to lengthy sample preparation procedures, sample losses by strong adsorption to surfaces, and unpredictable matrix effects. Currently, UC-inositol and UC-IPx are not available from commercial sources. In this study, we developed a method that is capable of generating UC-inositol and UC-IPx. An inositol-independent cell line L929S was cultured in inositol-free medium supplemented with UC-glucose. Inositol contamination in FBS was observed as the critical parameter for labeling efficiency (LE). A balance between cell growth and LE was achieved by adopting a two-step labeling strategy. In the first step, a LE of 90% could be obtained by normal cell growth in the long-term. Cells were then cultured in a second step in ultra-labeling medium for improved LE for a short duration before harvesting. The generated UCanalogs were of high isotopic purity (>99%). Utilized as internal standards spiked before sample preparation in biological applications, UCanalogs can effectively compensate sample loss during sample preparation as well as the matrix effect during electrospray ionization. An exemplary pharmacological study was conducted with phospholipase C inhibitor and activator to document the great utility of the prepared stable isotope-labeled internal standards in elucidating the PLC-dependent IP code. UCIPx are used as internal standards to generate quantitative profiles of IPx in HeLa cell samples after treatment with PLC inhibitor and activator. This established method generating UCanalogs is cost-effective, robust, and reproducible, which can facilitate quantitative studies of inositol and IPx in biological scenarios.