Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation
Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation
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DOI：
10.1006/abio.2001.5375
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发表时间：
2001-11-15
影响因子：
2.9
通讯作者：
Hellerstein, MK
中科院分区：
文献类型：
--
作者：
Neese, RA;Siler, SQ;Hellerstein, MK
直到最近才出现测量人类 DNA 合成速率以及细胞增殖速率的方法。我们（D. C. Macallan、C. A. Fullerton、R. A. Neese、K. Haddock、S. S. Park 和 M. K. Hellerstein, 1998, Proc. Natl. Acad. Sci. USA 95, 708-713）最近开发了一种稳定同位素质谱技术，通过标记嘌呤的脱氧核糖 (dR) 部分来测量 DNA 合成通过从头核苷酸合成途径脱氧核糖核苷酸。然而，最初的分析方法有其局限性。在这里，我们描述了提高方法产量、稳定性、灵敏度和重现性的技术改进。使用 LC18 SPE 柱直接从 DNA 水解产物中分离嘌呤脱氧核糖核苷、脱氧腺苷 (dA)。开发了两种衍生物用于分析单独的 dA（无碱基）的 dR 部分，即醛腈三乙酸酯衍生物和还原戊糖四乙酸酯 (PTA) 衍生物。与之前的 dA 衍生物相比，PTA 衍生物尤其表现出更高的稳定性（几周后不会降解）、更强的 GC/MS 信号以及低得多的同位素比率丰度灵敏度（即，质量同位素异构体丰度对注入质谱仪源的材料量的依赖性较小）。从而避免了对复杂的、多维丰度校正标准曲线的需要。使用 PTA 衍生物，体内水分 (H2O)-H-2 富集度为 2.2-2.8% 的啮齿动物完全翻转细胞的 DNA 的 dR 富集率为 9.0-9.5%，并且需要少于 1.0 微克 DNA（约 2 x 10(5) 个细胞）进行可重复分析。总之，这些方法学的进步使得能够在人类和实验动物体内测量稳定同位素掺入 DNA 并计算细胞增殖和死亡率，并且细胞数量更少，重现性更高，劳动力更少。可以预见这种方法的许多应用。 (C) 2001 年学术出版社。
Methods for measuring rates of DNA synthesis, and thus cell proliferation, in humans had not been available until recently. We (D. C. Macallan, C. A. Fullerton, R. A. Neese, K. Haddock, S. S. Park, and M. K. Hellerstein, 1998, Proc. Natl. Acad. Sci. USA 95, 708-713) recently developed a stable isotope-mass spectrometric technique for measuring DNA synthesis by labeling the deoxyribose (dR) moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway. The original analytic approach had limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentose-tetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after several weeks), greater GC/MS signal, and much less abundance sensitivity of isotope ratios (i.e., less dependence of mass isotopomer abundances on the amount of material injected into the mass spectrometer source), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with (H2O)-H-2 enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1.0 mug DNA (ca. 2 x 10(5) cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned. (C) 2001 Academic Press.