Materials and Methods

The following materials were of the origin indicated. Itaconic acid was from the Eastman Kodak Company and mesaconic acid from the Aldrich Chemical Company, Inc.(both were recrystallized before use). Citraconic acid was obtained from the HM Chemical Company, Ltd. nL-Citramalic acid was kindly provided by Dr. HA Barker, and CY4-pyruvic acid was obtained from the Volk Radio-Chemical Company. cu-Hydroxyglutaric acid was prepared by catalytic hydrogenation of cr-ketoglutaric acid in the presence of PtOz. Hydrogenation was carried out in glacial acetic acid solution and the solvent removed under reduced pressure. The residue was dissolved in moist ethyl acetate (2); crystals formed upon addition of acetone to the solution and had a melting point of 71-72”(value reported in literature, 72-73’). CY4-itaconic acid was prepared as previously described (1). Cl4-mesaconic acid was obtained by boiling C14-itaconic acid with 10% NaOH for 7 hours (3). The solution was treated with Amberlite IRC-50 (H+ form) to remove Na+. C14-itaconic and the formed CY4-mesaconic and citraconic acids were then separated by chromatography on Dowex 1. The fractions of the mesaconic acid region (1) were combined and lyophilized. The compound had a melting point of 201-202 “and showed the same melting point when mixed with authentic mesaconic acid. Itaconyl-, mesaconyl-, citramalyl-, citraconyl-, and cr-hydroxyglutaryl-CoA were prepared by the mixed anhydride method (4). It should be emphasized that these CoA derivatives comprise structural isomers (in

* Supported in part by grants from the National Institutes of Health. United States Public Health Service and the Life Insurance Medical Research Fund. A detailed report, including fractionation of the extract of rat liver mitochondrial acetone powder, is included in the thesis submitted by Shu-Fang Wang in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of Wisconsin, 1960. 1 Itaconic acid is methylene succinic acid; mesaconic is methylfumaric acid; citraconic is methylmaleic acid; citramalic is (I-methylmalic acid.