Unbiased metabolomics analysis of heme-loaded macrophages reveals enrichment for metabolites associated with glycolysis and the PPP. (A) Volcano plot representing the changes in BMDM metabolism after 6 hours of 10 µM heme loading, as measured by unbiased mass spectrometry–based metabolomics. Log2 values of the fold change of individual metabolites are plotted on the x-axis, and –log2 of the P value of a Welch’s t test between control and heme-loaded BMDMs is plotted on the y-axis (n = 5). (B) Changes in heme metabolite abundance in heme-loaded BMDMs. Metabolites in red text are increased in heme-loaded BMDMs relative to vehicle control with corresponding plots for the individual metabolites involved in heme catabolism (n = 5). (C) Plots representing individual metabolites involved in the glycolysis pathway (n = 5). (D) Plots representing individual metabolites involved in the PPP (n = 5). (E) Pathway analysis summary of glycolysis and the PPP in heme-loaded BMDMs. Metabolites in red text are increased in heme-loaded BMDMs relative to vehicle control. Data are mean ± standard deviation (SD) of relative abundance measurements for control and heme-loaded BMDMs (n = 5). Statistical significance between 2 groups was determined by Welch’s unpaired t test. *P < .05; **P < .01; ***P < .001. See also supplemental Figure 1.