Immune-based metabolic reprogramming in the brain, causing arginine starvation and accumulation of neurotoxic polyamines, progressively leads to neuronal pathology associated with Alzheimer’s disease (AD). To enable our long-term goals of differentiation of AD mouse model genotypes, ages, and sexes based on activity of this pathway, we describe here the novel dosing (using uniformly labeled (13C615N4) arginine) and analysis methods using capillary electrophoresis high-resolution accurate-mass mass spectrometry (CE-HRAM-MS) for isotope tracing of metabolic products of arginine. We developed a pseudo-primed infusion-dosing regimen, using repeated injections, to achieve a steady state of uniformly labeled arginine in 135-195 minutes post bolus dose. Incorporation of stable isotope labeled carbon and nitrogen from uniformly labeled arginine into a host of downstream metabolites was measured in vivo in mice using serially-sampled dried blood spots from the tail. In addition to the dried blood spot time course samples, total isotope incorporation into arginine-related metabolites was measured in the brain and plasma after 285 minutes. Preliminary demonstration of the utility of the technique identified differences isotope incorporation in arginine metabolites between male and female mice in a mouse-model of sporadic Alzheimer’s disease (APOE4/huNOS2). The technique described herein will permit arginine pathway activity differentiation between mouse genotypes, ages, sexes or drug treatments in order to elucidate the contribution of this pathway to Alzheimer’s disease.

Capillary Electrophoresis - High Res Mass Spectrometry analysis of a series of dried blood spot, plasma, and brain samples from animals which had been dosed with stable-isotope labeled arginine. CE-MS and Skyline analysis allowed isotope tracing through a number of metabolic fates of Arginine.

Mouse Brain, Plasma, and dried blood spots.