Isotopic labeling of heme and assorted metabolites by¹³C diethyl succinate,¹³C glucose or¹³C glutamine, demonstrates that glutamine is strongly used as a precursor for heme but succinate is not. (A-C) Bar graphs show the experimentally determined labeling of heme, isolated from differentiating MEL cells after treatment with the indicated labeled metabolite and show the distribution of ¹³C-provided mass (“M”) for isolated hemes. In these figures, M represents heme detected with no ¹³C incorporated into the 34 carbons of heme (carbons are all ¹²C): M+1 is heme with 1 atom of ¹³C incorporated; M+2 is heme with 2 ¹³C carbons, etc. The green bars represent the control values from samples treated with unlabeled metabolite and show the natural distribution of ¹³C incorporation. (A) The source of ¹³C is succinate as supplied by DES (yellow bars). Carbons derived from DES are only poorly used in heme biosynthesis. (B) The source of ¹³C label is glucose (black bars). The bar graph shows that some carbons from labeled glucose are incorporated into heme. (C) The source of ¹³C is glutamine (blue bars), which is converted to α-KG by deamination prior to KDH-driven synthesis of succinyl-CoA for ALA synthesis. Glutamine is shown here to be a superior precursor for heme biosynthesis, and isolated hemes are found to contain up to 23 carbon atoms derived from labeled glutamine. (D) The distribution of label originating from ¹³C glucose, ¹³C glutamine, and ¹⁵N glutamine into glutamine and glutamate are shown. (E) The distribution of label originating from ¹³C glucose, ¹³C glutamine, and ¹⁵N glutamine into the TCA cycle intermediates malate and fumarate are shown. (F) The distribution of label originating from ¹³C glucose, ¹³C glutamine, and ¹⁵N glutamine into proline is shown. Each labeling experiment was carried out in triplicate.