A New Stable Isotope Method Enables the Simultaneous Measurement of Nucleic Acid and Protein Synthesis In Vivo in Mice

Manuscript received 29 January 1998. Initial reviews completed 3 April 1998. Revision accepted 21 May 1998.

USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030

We developed a method based on the incorporation of ¹³C₂-units derived from [U-¹³C]glycine that allows the simultaneous quantification of tissue protein and RNA synthesis in vivo. Two groups of 26 mice were fed diets containing a high (HF diet) or a low quantity of fiber (LF diet). After 6 d, [U¹³C]glycine was added to the diet and groups of four mice were killed after 2, 4, 6, 8, 12 and 24 h. Hepatic and intestinal mucosal free and RNA-bound purine nucleosides were extracted and enzymically degraded to allantoin. Allantoin was degraded to glyoxylate, which was then reductively aminated to glycine, which contains the two ¹³C-atoms incorporated via de novo synthesis. Ingestion of the HF diet was associated with significantly (P < 0.05) higher rates of total RNA synthesis in both the liver ( HF diet, 29%/d; LF diet, 21%/d) and mucosa (HF diet, 27%/d; LF diet, 17 %/d). The mean rates of RNA synthesis in each tissue were significantly (P < 0.01) lower than the respective rates of protein synthesis (liver, 67%/d; mucosa, 74%/d). The isotopic enrichment of the free purine nucleotide pool increased rapidly and exponentially, but the steady-state value was substantially (P < 0.001) lower than that of the RNA-bound purines. The results suggest that the free nucleotide pool consists of two kinetically distinct compartments, one of which is small and has a rapid rate of turnover. This, we propose, acts as the RNA precursor pool. The other is large, has a low rate of turnover and, we believe, is the pool of adenosine triphosphate involved in cellular energetics.

The Journal of Nutrition Vol. 128 No. 9 September 1998, pp. 1562-1569
Copyright ©1998 by the American Society for Nutritional Sciences

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