A critical examination of substoichiometric isotope dilution analysis using thymidine and leucine

Abstract

Isotope dilution analysis is used to determine the specific activity of radiolabelled precursors such as 3H-thymidine and 3H-leucine incorporated into DNA or protein in environmental samples. However, it is shown theoretically, experimentally, and using the results of published studies that the method can be misleading when one of its many requirements is not met. First, on the basis of our current understanding of thymidine metabolism, it is not only possible, but probable, that endogenous thymidine synthesis will not remain constant at different concentrations of external thymidine addition. Changes in the rate of thymidine biosynthesis will significantly bias the results of the standard analysis. Yet it is very difficult or impossible to know whether changes have occurred in any particular case. Second, published results show evidence of stimulation of total thymidine incorporation rate at higher thymidine concentrations. These changes in incorporation rate will also bias the conclusions drawn from the assay. Third, impurities common to heavily labelled precursors can produce misleading results. Finally, the current statistical methodology of isotope dilution experiments can be greatly improved with little or no extra effort. It is suggested that, if possible, isotopes should be used empirically to derive conversion factors based on incubations; theoretical calculations based on isotope dilution experiments are not always trustworthy.