A Trail of Research from Lipoic Acid to α-Keto Acid Dehydrogenase Complexes

In this article I shall retrace a trail of research that began with the isolation and characterization of a microbial growth factor and led to elucidation of the structure, function, and regulation of -keto acid dehydrogenase complexes. The high points of this trail are presented below. Isolation and Characterization of Lipoic Acid This trail of discovery started in the spring of 1949, about 6 months after I joined the faculty of the Department of Chemistry at the University of Texas. At that time I started working on the isolation of a factor that replaced acetate in the growth medium for certain lactic acid bacteria. Research on the “acetate-replacing factor” was initiated by Esmond Snell and associates at the University of Wisconsin and then at the University of Texas. I inherited this project in the spring of 1949. We established that this factor is widely distributed in animal, plant, and microbial cells and that liver is a rich source. The factor is tightly bound to liver protein and is released by proteolysis or by acid hydrolysis. At that time pharmaceutical companies were processing large amounts of pork and beef liver to obtain extracts suitable for treatment of pernicious anemia. The active principle was shown later to be vitamin B12. Fresh liver was extracted with warm water, and the residual liver proteins and fatty material were dried and sold as an animal feed supplement. Arrangements were made with Eli Lilly and Co. to obtain liver residue, and we developed procedures for extracting and purifying the acetatereplacing factor. We progressed to the point of being able to process about 6 pounds of liver residue at a time. A 16,000–50,000-fold purification was achieved. In the late 1940s and early 1950s several other groups were trying to isolate factors that were similar to, if not identical with, the acetate-replacing factor. These factors included the “pyruvate oxidation factor” of I. C. Gunsalus and associates that was necessary for oxidation of pyruvate to acetate and carbon dioxide by Streptococcus faecalis cells grown in a synthetic medium. Gunsalus was also collaborating with Eli Lilly and Co. In the fall of 1950, the Lilly Research Laboratories merged the two separate collaborations to facilitate isolation of the acetate-replacing/pyruvate oxidation factor. The Lilly group adapted and scaled up isolation procedures developed by us. Instead of processing 6-pound batches of liver residue at a time, they were able (using commercial equipment) to process 250-pound batches. Concentrates of the factor that were 0.1–1% pure were sent to my laboratory for further processing. I obtained the first pale yellow crystals of the factor, about 3 mg, on or about March 15, 1951, a truly memorable occasion. It was partially characterized and given the trivial name -lipoic acid (1). The isolation involved a 300,000-fold purification. A total of 30 mg of crystalline lipoic acid was eventually isolated. We estimated that 10 tons of liver residue were processed to obtain this small amount of the pure substance. And to think that I was processing about 6 pounds of liver residue at a time, convinced that I would eventually isolate the pure material. We established that lipoic acid is a cyclic disulfide, either 6,8-, 5,8-, or 4,8-dithiooctanoic acid. That the correct structure is 6,8-dithiooctanoic acid (1,2-dithiolane-3-valeric acid) was established by synthesis of DL-lipoic acid, first achieved by E. L. R. Stokstad and associates at Lederle THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 42, Issue of October 19, pp. 38329–38336, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.