The online version of this article (doi:10.1186/1476-9255-9-37) contains supplementary material, which is available to authorized users.
No conflicting interests.
Kim, R and Lee, D performed the experiments. Suh, JH designed the study, performed mass spectrometry analysis, interpreted data, and wrote the manuscript. All authors read and approved the final manuscript.
Shifts in intracellular arginine (Arg) and sulfur amino acid (SAA) redox metabolism modulate macrophage activation, polarization and phenotype. Despite their importance in inflammation and redox regulatory pathways, comprehensive analysis of these metabolic networks was not previously possible with existing analytical methods.
The Arg/thiol redox LC-MS/MS metabolomics assay permits simultaneous assessment of amino acids and derivative products generated from Arg and SAA metabolism. Using this assay, LPS-induced changes in macrophage amino acid metabolism were monitored to identify pathway shifts during activation and their linkage to cellular redox regulation.
Metabolite concentrations most significantly changed after treatment of a macrophage-like cell line (RAW) with LPS for 24 hrs were citrulline (Cit) (48-fold increase), ornithine (Orn) (8.5-fold increase), arginine (Arg) (66% decrease), and aspartic acid (Asp) (73% decrease). The ratio Cit + Orn/Arg + Asp (CO/AA) was more sensitive to LPS stimulation than other amino acid ratios commonly used to measure LPS-dependent inflammation (e.g., SAM/SAH, GSH/GSSG) and total media NOx. The CO/AA ratio was also the first ratio to change significantly after LPS treatment (4 hrs). Changes in the overall metabolomic profile over time indicated that metabolic pathways shifted from Arg catabolism to thiol oxidation.
Simultaneous quantification of Arg and SAA metabolic pathway shifts following LPS challenge of macrophage indicate that, in this system, the Arg-Citrulline/NO cycle and arginase pathways are the amino acid metabolic pathways most sensitive to LPS-challenge. The cellular (Cit + Orn)/(Arg + Asp) ratio, which summarizes this pathway, was more responsive to lower concentrations of LPS and responded earlier than other metabolic biomarkers of macrophage activation including GSH redox. It is suggested that the CO/AA ratio is a redox- independent early biomarker of macrophage activation. The ability to measure both the CO/AA and GSH-redox ratios simultaneously permits quantification of the relative effects of LPS challenge on macrophage inflammation and oxidative stress pathways. The use of this assay in humans is discussed, as are clinical implications.
Additional file 1:Table S1. Mass spectrometric setting used in this study. (DOC 87 KB)12950_2012_253_MOESM1_ESM.doc
Additional file 2:Figure S1. Effects of iodoacetamide (IAM) pretreatment on cellular arginine (Arg), citrulline (Cit) and ornithine (Orn) concentrations. The effects of iodoacetamide pretreatment on intracellular Arg (Panel A), Cit (Panel B) and Orn (Panel C) concentration are shown. RAW cells (2 × 106) were left untreated (control) or treated with 0.1, 1, 10, 100 and 1000 ng/ml LPS (LPS) for 24 hrs at 37°C. For extraction without IAM, cells were harvested and washed with phosphate buffered saline and immediatedly treated with perchloric acid. For extraction with IAM, cells were pre-incubated with IAM solution for 1 hr prior to perchloric acid treatment. Metabolite concentrations were normalized to protein concentrations. Results show no significant differences between cells treated with or without IAM. (PDF 58 KB)12950_2012_253_MOESM2_ESM.pdf
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- A new metabolomic assay to examine inflammation and redox pathways following LPS challenge
Jung H Suh
Robert Y Kim
Daniel S Lee
- BioMed Central
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