De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [¹⁵N]alanine

Abstract

The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N‐labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of l‐[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.

Measuring DNA Synthesis Rates with [1-13C]Glycine

“…As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13 C/ 12 C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones.…”

“…Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1- 13 C]glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer.…”

“…The comprehensiveness of detection, chromatographic resolution, sensitivity, signal/noise, and quantitative abilities of CRIMS were compared with radioactivity monitoring (RAM) and in no case was RAM superior. With HPLC/CRIMS, stable isotopes such as 13 C and 15 N can be comprehensively detected and quantitative patterns of drug metabolism from biological fluids can be produced that mirror the results when 14 C is used. Therefore, stable isotopes may be substituted for radioisotopes in studies of metabolism where the ability of the latter approach to detect a label independent of the structures in which the label appears has been the primary reason for continuing to use a hazardous tracer.…”

“…An IRMS is designed to accept gaseous samples, such as CO 2 , ionize this gas with high efficiency, and transmit the appropriate ions to a multiple collector that monitors two or three ions at the same time. The use of IRMS allows measurements of natural variations in the abundance of 13 C and 15 N 3 and extends the range of tracer incorporation orders of magnitude below what is accomplished with a conventional GC/MS or HPLC/MS system. 4,5 Before CRIMS, coupling chromatography to an IRMS involved a combustion interface.…”

“…Folate is involved in de novo thymidine biosynthesis. As will be described, it is important that dT is not labeled by the tracer glycine molecule, because the 13 C isotope ratio of dT will serve as a control for any enrichment found in deoxyguanosine or deoxyadenosine. Therefore, our first experiments used [1- 13 C]glycine as the purine precursor.…”