Proteomics and Metabolomics Shared Resource, Duke University, Durham, North Carolina 27710, United States.
Department of Neurology, Duke University, Durham, North Carolina 27710, United States.
J Am Soc Mass Spectrom. 2021 Jun 2;32(6):1448-1458. doi: 10.1021/jasms.1c00055. Epub 2021 May 24.
Immune-based metabolic reprogramming of arginine utilization in the brain contributes to the 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 (CN) arginine) and analysis methods using capillary electrophoresis high-resolution accurate-mass mass spectrometry for isotope tracing of metabolic products of arginine. We developed a pseudoprimed infusion-dosing regimen, using repeated injections, to achieve a steady state of uniformly labeled arginine in 135-195 min post bolus dose. Incorporation of stable isotope labeled carbon and nitrogen from uniformly labeled arginine into a host of downstream metabolites was measured 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 whole brain and plasma after 285 min. Preliminary demonstration 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.
基于免疫的精氨酸代谢重编程在大脑中有助于阿尔茨海默病（AD）相关的神经元病变。为了实现我们基于该通路活性对 AD 小鼠模型基因型、年龄和性别进行长期区分的目标，我们在这里描述了一种新的剂量（使用均标记的（CN）精氨酸）和分析方法，该方法使用毛细管电泳高分辨率精确质量质谱法用于精氨酸代谢产物的同位素示踪。我们开发了一种伪引物输注剂量方案，使用重复注射，在推注剂量后 135-195 分钟内实现均标记精氨酸的稳态。使用来自尾巴的连续采样干血斑测量稳定同位素标记的碳和氮从均标记精氨酸掺入宿主下游代谢物中的情况。除了干血斑时间过程样本外，还在 285 分钟后测量整个大脑和血浆中与精氨酸相关的代谢物的总同位素掺入量。该技术的初步演示确定了在散发性阿尔茨海默病（APOE4/huNOS2）小鼠模型中雄性和雌性小鼠之间精氨酸代谢物中同位素掺入的差异。本文描述的技术将允许在小鼠基因型、年龄、性别或药物治疗之间对精氨酸途径活性进行区分，以阐明该途径对阿尔茨海默病的贡献。
