Multiple Infusion Start Time Mass Spectrometry Imaging of Dynamic SIL-Glutathione Biosynthesis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

Due to the high association of glutathione
metabolism perturbation
with a variety of disease states, there is a dire need for analytical
techniques to study glutathione kinetics. Additionally, the elucidation
of microenvironmental effects on changes in glutathione metabolism
would significantly improve our understanding of the role of glutathione
in disease. We therefore present a study combining a multiple infusion
start time protocol, stable isotope labeling technology, infrared
matrix-assisted laser desorption electrospray ionization, and high-resolution
accurate mass–mass spectrometry imaging to study spatial changes
in glutathione kinetics across in sectioned mouse liver tissues. After
injecting a mouse with the isotopologues [2-13C,15N]-glycine, [1,2-13C2]-glycine, and [1,2-13C2,15N]-glycine at three different
time points, we were able to fully resolve and spatially map their
metabolism into three isotopologues of glutathione and calculate their
isotopic enrichment in glutathione. We created a tool in the open-source
mass spectrometry imaging software MSiReader to accurately compute
the percent isotope enrichment (PIE) of these labels in glutathione
and visualize them in heat-maps of the tissue sections. In areas of
high flux, we found that each label enriched an approximate median
of 1.6%, 1.8%, and 1.5%, respectively, of the glutathione product
pool measured in each voxel. This method may be adapted to study the
heterogeneity of glutathione flux in diseased versus healthy tissues.