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The Oxidative Metabolic Pattern of Mouse Hepatoma C₉₅₄ as Studied with C¹⁴-labeled Acetates, Propionate, Octanoate, and Glucose^*

G. W. Brown, Jr., J. Katz and I. L. Chaikoff

( Department of Physiology, University of California School of Medicine, Berkeley, Calif.)

1. The incorporation of isotope from acetate-1-C¹⁴, acetate-2-C¹⁴, propionate-1-C¹⁴, octanoate-1-C¹⁴, pyruvate-2-C¹⁴, and from uniformly labeled glucose into respiratory CO₂ and various intermediary products was studied with slices of normal mouse liver, host liver, and with the hepatoma C₉₅₄.

2. A chromatographic-autoradiographic method was employed to demonstrate and identify about twelve or more labeled intermediates. Radioactive areas of the chromatograms, as indicated by autoradiograms, were assayed with a thin end-window Geiger tube.

3. In both liver and hepatoma, octanoate-1-C¹⁴ was utilized in preference to acetate-1-C¹⁴ as judged by (a) conversion to C¹⁴O₂; (b) over-all incorporation into nonvolatile compounds; (c) incorporation into individual intermediary products; and (d) incorporation into acetoacetate.

4. Major C¹⁴ products (comprising more than 5 per cent of the nonvolatile residues) for the hepatoma incubated with octanoate-1-C¹⁴ were, in generally decreasing order of isotope incorporation, glutamate, di- and tricarboxylic acids (taken as a group), ß-hydroxybutyrate, lactate, aspartate, and glutamine. Essentially the same pattern was found when acetate-1-C¹⁴ was the substrate. Quite striking was the value of the ratio of glutamate-C¹⁴ to glutamine-C¹⁴, which was 6.7 for hepatoma, as compared with 0.6 for host liver. The hepatoma incorporated relatively larger amounts of isotope into di- and tricarboxylic acids, glutamate, and aspartate than did the liver.

5. Normal and host liver incorporated isotope from labeled 2-carbon fragments into glucose, but hepatoma failed to carry out this incorporation to any significant extent. The hepatoma was more active than was host liver, however, in utilization of glucose.

6. Two-carbon fragments from acetate were shown to interact with those from octanoic acid in the hepatoma and to participate in the ketogenic activity of this short-chain fatty acid.

7. Alanine and lactate contained relatively more isotope when acetate-2-C¹⁴ was employed instead of acetate-1-C¹⁴, with hepatoma. The explanation for this lies in the dynamics of the tricarboxylic acid cycle, the intermediates of which become more heavily labeled with the methyl carbon than with the carboxyl carbon of a 2-carbon fragment.

8. Propionate-2-C¹⁴ was converted by the hepatoma to (in decreasing order) lactate, alanine, glutamate, aspartate, glutamine, and several unidentified compounds. Little or no isotope was converted to glucose.

9. Presumptive evidence that the tricarboxylic acid cycle operates in the hepatoma was provided by (a) oxidation of labeled 2-carbon fragments to C¹⁴O₂; (b) incorporation of isotope from 2-carbon fragments into compounds of the cycle and to those arising from them by ancillary processes; (c) inhibition by malonate; (d) depression in the oxidation of labeled acetate by short chain fatty acids which are thought to be inhibitors of the tricarboxylic acid cycle; and (e) the pattern of isotope labeling similar to that of liver, in which the cycle is known to operate.

10. The pattern of labeling of various intermediates observed when C¹⁴-labeled compounds are incubated with slices of hepatoma differs quantitatively from the pattern observed with host or normal liver. The observations made here on quantitative differences in the hepatoma C₉₅₄ reflect its neoplastic state, but such differences from the liver tissue do not lend themselves to a clear-cut decision regarding the specific loci for the metabolic derangement(s) associated with neoplasia.

^* Aided by a grant from the California Division of the American Cancer Society and by Cancer Research Funds of the University of California.

Present address: National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md.

Present address: Institute for Medical Research, Cedars of Lebanon Hospital, Los Angeles, Calif.

Received 12/15/55.