Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.
Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.
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DOI：
10.1371/journal.pcbi.1004355
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发表时间：
2015-10
影响因子：
4.3
通讯作者：
Asquith B
中科院分区：
文献类型：
--
作者：
Ahmed R;Westera L;Drylewicz J;Elemans M;Zhang Y;Kelly E;Reljic R;Tesselaar K;de Boer RJ;Macallan DC;Borghans JA;Asquith B
稳定同位素标记是人体淋巴细胞动力学体内定量的最新技术。它是许多开创性研究的核心，特别是在HIV-1和白血病的背景下。然而，在不同的研究中估计的淋巴细胞增殖率之间存在显著差异。值得注意的是，氘代2 H2-葡萄糖（D2-葡萄糖）标记研究始终比氘代水（D2 O）标记研究产生更高的增殖估计值。这阻碍了我们对免疫功能的理解，并削弱了我们对这项重要技术的信心。这些差异是否由两种化合物之间的基本生化差异和/或研究中的方法学差异引起尚不清楚。D2-葡萄糖和D2 O标记实验从未在相同的实验条件下由同一组进行过;因此，不可能对这两种技术进行直接比较。我们试图解决这个问题。我们使用相同的方案用D2-葡萄糖和D2 O进行体外和鼠体内标记实验。这表明，两种化合物之间的内在差异不会导致增殖速率估计值的差异，但体内使用D2-葡萄糖进行的估计值容易因高度可变的血糖富集而难以标准化。对使用D2-葡萄糖和D2 O进行的三项已发表的人体研究的分析证实了这个问题，特别是在短期D2-葡萄糖标记的情况下。校正归一化中的这些不准确性降低了使用D2-葡萄糖进行的增殖速率估计值，并且略微增加了使用D2 O进行的估计值;因此使来自两种方法的估计值显著接近，并且突出了使用该技术时可靠归一化的重要性。稳定同位素标记用于量化人类活细胞增殖和死亡的速率。它是许多开创性研究的核心，特别是在病毒感染如HIV-1和白血病方面。然而，不同的标记（氘代水或氘代葡萄糖）对细胞增殖和损失的速率产生不同的估计;这阻碍了我们的理解，削弱了我们对这一重要技术的信心。我们进行了体外和体内实验以及对现有数据的新分析，以直接比较两种标签。这表明，差异的主要来源在于难以评估氘代葡萄糖的可用性。我们协调已发表的研究，并提供建议，以避免在未来的这个问题。
Stable isotope labeling is the state of the art technique for in vivo 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 in vitro and murine in vivo 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 in vivo 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. Stable isotope labeling is used to quantify the rate at which living cells proliferate and die in humans. It has been central to a number of seminal studies, particularly in viral infections such as HIV-1, and leukemia. However, different labels (deuterated water or deuterated glucose) yield different estimates for the rate of cell proliferation and loss; this hampers our understanding and weakens our confidence in this important technique. We performed in vitro and in vivo experiments as well as a new analysis of existing data to directly compare the two labels. This reveals that a major source of the discrepancy lies in the difficulty of assessing deuterated glucose availability. We reconcile published studies and provide recommendations to avoid this problem in the future.