Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments

<div><p>Stable isotope labeling is the state of the art technique for <i>in vivo</i> quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated ²H₂-glucose (D₂-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D₂O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D₂-glucose and D₂O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both <i>in vitro</i> and murine <i>in vivo</i> labeling experiments using identical protocols with both D₂-glucose and D₂O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D₂-glucose <i>in vivo</i> were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D₂-glucose and D₂O confirmed this problem, particularly in the case of short term D₂-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D₂-glucose and slightly increased estimates made using D₂O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique.</p>