Analysis of Arginine Metabolism Using LC-MS and Isotopic Labeling
Arginine metabolism is linked to several important metabolic processes, and reprogramming of arginine metabolism occurs in various physiological and pathological conditions. Here we describe a method, using a LC-MS-based metabolomics and 15N4-arginine tra
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roduction Arginine metabolism is connected to several important metabolic pathways including nitric oxide (NO) production, the urea cycle, polyamine synthesis, and creatine synthesis (Fig. 1). It plays a major role in the functions of immune cells [1–3], and alterations in arginine metabolism have been linked to various diseases, such as cancer [4–7], vascular dysfunction [8, 9], and asthma [10]. In myeloid cells, arginase (ARG) and nitric oxide synthase (NOS) are differentially regulated by type 1 and type 2 cytokines, and the reprogramming of arginine metabolism is critical for inflammation regulation [1, 2]. CD8+ T cells have increased survival capacity and antitumor activity when l-arginine levels are increased, further exemplifying the connection between immune cells and arginine [3]. Additionally, a number of cancers including melanoma, hepatocellular carcinoma, prostate cancer, and renal cell carcinoma are deficient in argininosuccinate synthase (ASS) and auxotrophic for arginine [4]. Therefore, lowering arginine availability has become an effective strategy to treat these cancers [5–7]. The relevance of arginine metabolism goes beyond immune cell function and cancer. For instance, changes in arginine metabolism can affect the production of nitric oxide, which is important in vasodilation of Angelo D’Alessandro (ed.), High-Throughput Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1978, https://doi.org/10.1007/978-1-4939-9236-2_13, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Fig. 1 Murine arginine metabolism pathway. Metabolites detected using described method are indicated in red. Those metabolic reactions whose fluxes are determined using described method are indicated in blue: NOS nitric oxide synthase, ARG arginase, ASS argininosuccinate synthase, ASL argininosuccinate lyase, OTC ornithine transcarbamylase, OAT ornithine aminotransferase, ODC ornithine decarboxylase, GATM glycine amidinotransferase, CitEx citrulline export, OrnEx ornithine export, and ArgIn Arginine import. Arginine nitrogens are color coded to track their transfer to downstream metabolites
coronary arterioles and regulation of hyperreactive airways in asthma [8–10]. Analysis of metabolites and metabolic fluxes in arginine metabolism enables a quantitative understanding of how this pathway is reprogrammed in different physiological and pathological conditions and thus can bring insight into the specific roles of arginine metabolism in cell function and disease pathophysiology. Here we describe a protocol to quantitatively measure the levels of arginine metabolism intermediates using a liquid chromatography-mass spectrometry (LC-MS)-based method and to determine the fluxes of arginine metabolism reactions using 15N tracer-based metabolic flux analysis [11]. Specifically, cells are cultured in the presence of 15N4-arginine until pseudo-steady state is reached; at this point, the incorporation of labeled nitrogen into downstream metabolites quantitatively r
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