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ß-Aminoisobutyric Acid, a New Probe for the Metabolism of DNA and RNA in Normal and Tumorous Tissue

Departments of Clinical Chemistry, Pathology, and Surgery, Sundby Hospital, Copenhagen, Denmark [H. R. N., K-E. S., K. N.], and Department of Microbiology, University of Colorado Medical Center, Denver, Colorado 80220 [B. S. B., R. W., E. B.]

ß-Aminoisobutyric acid, a catabolite of thymine metabolism, originates from the thymine of both DNA and transfer RNA. The ß-aminoisobutyric acid originating from the thymine of DNA and transfer RNA can be distinguished by specific labeling of the former with ¹⁴C formate and of the latter with methionine-methyl-³H. Therefore, it can serve as a probe for the metabolism of each macromolecule. In an animal with a rapidly growing tumor (Novikoff hepatoma), the excretion of ß-aminoisobutyric acid diminishes, probably because of the salvage of thymine and its reutilization for DNA synthesis. Such a salvage path is suggested by the increased presence of tritium-labeled thymine in the DNA of the tumor.

AIB³ is a catabolic product that is normally excreted in low levels in human urine (1), and this excretion is elevated in many patients with urothelial tumors (9). Until recently, the origin of this metabolic product was thought to be the thymine of DNA. However, since thymine is present in low levels in tRNA as well, it seemed of interest to ascertain whether AIB may also stem from this source. In a cooperative study in our 2 laboratories, it was determined that the urinary AIB indeed has a dual origin in the normal rat (8). Such a demonstration could be accomplished by taking advantage of the known difference in the pathway of synthesis of the thymine of DNA and of tRNA. The former is synthesized from the 1-carbon pool, with formyltetrahydrofolic acid as the intermediate donor (4). This thymine can, therefore, be labeled by formate-¹⁴C. On the other hand, it was shown by Mandel and Borek (5) that the thymine of tRNA is synthesized by the direct addition of an intact methyl group to uracil which had been previously incorporated into the macromolecule. The methyl donor in this reaction is S-adenosylmethionine (3), and the methyl group of the thymine of tRNA can, therefore, be readily labeled with tritium. If the tritiated methyl group should enter the 1-carbon pool, most of its tritium would be exchanged and diluted.

With the availability of such specific markers for DNA and tRNA, a number of hitherto moot questions in the metabolism of these macromolecules can be approached. In this communication we report that AIB is derived, in part, from the thymine of tRNA in normal rats and rats bearing slow-growing Morris hepatomas. However, in rats with the rapidly growing Novikoff hepatoma, excretion of AIB is lower than in the animal prior to the tumor implant. This finding confirms an observation made in vitro by Ferdinandus et al. (2) in their studies of enzyme activities in tumor tissues. They had reported a marked decline in the degradation of thymine via the dihydrothymine and AIB pathway in rapidly growing tumors. Weber has suggested a salvage pathway for thymine in rapidly growing tumors. Our findings of the diminished excretion of AIB in rats bearing Novikoff hepatoma confirm this salvage pathway in the whole animals. However, the diminshed excretion of AIB with both labels implies a salvage pathway of thymine from both DNA and RNA with the possible reincorporation of the thymine into DNA. Such a pathway is implied by data obtained from analysis of 5-methylcytosine and thymine in isolated DNA of the tumor and compared with the same components isolated from the DNA of hepatectomized normal livers.

³ The abbreviation used is: AIB, ß-aminoisobutyric acid.

¹ Supported by Grant CA-14047-02 from the NIH, National Cancer Institute, and by the Danish Cancer Society.

² Supported by Grant j.nr. 512-2115 and -2125.

Received 1/14/74. Accepted 3/ 4/74.

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