A new stable isotope method enables the simultaneous measurement of nucleic acid and protein synthesis in vivo in mice.
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.
Perez
J F
JF
USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.
Reeds
P J
PJ
D000818:Animals; D002247:Carbon Isotopes; D002482:Cellulose / Q000008:administration & dosage; D004043:Dietary Fiber / Q000008:administration & dosage; D005260:Female; D005998:Glycine / Q000008:administration & dosage* / Q000032:analysis; D006038:Glyoxylates / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008813:Mice, Inbred ICR; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis; D011684:Purine Nucleosides / Q000032:analysis / Q000096:biosynthesis*; D012313:RNA / Q000032:analysis*
D002247:Carbon Isotopes; D004043:Dietary Fiber; D006038:Glyoxylates; D011506:Proteins; D011684:Purine Nucleosides; D012313:RNA; D002482:Cellulose; C031150:glyoxylic acid; D005998:Glycine