Division of Molecular Structure, Medical Research Council National Institute for Medical Research , The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
Anal Chem. 2013 Dec 17;85(24):12046-54. doi: 10.1021/ac403111s. Epub 2013 Nov 22.
The accurate measurement of metabolite concentrations in miniscule biological sample volumes is often desirable, yet it remains challenging. In many cases, the starting analyte volumes are imprecisely known, or not directly measurable, and hence absolute metabolite concentrations are difficult to calculate. Here, we introduce volume determination using two standards (VDTS) as a general quantitative method for the analysis of polar metabolites in submicrolitre samples using (1)H NMR spectroscopy. This approach permits the back calculation of absolute metabolite concentrations from small biological samples of unknown volume. Where small sample volumes are also variable, VDTS can improve multivariate chemometric analysis. In this context, principal component analysis (PCA) yielded more logically consistent and biologically insightful outputs when we used volume-corrected spectra, calculated using VDTS, rather than probabilistic quotient normalization (PQN) of raw spectra. As proof-of-principle, the VDTS-based method and PCA were used to analyze polar metabolites in the hemolymph (blood) extracted from larvae of the very small but widely used genetic model organism Drosophila. This analysis showed that the hemolymph metabolomes of males and females are markedly different when larvae are well fed. However, gender-specific metabolomes tend to converge when larval dietary nutrients are restricted. We discuss the biological implications of these surprising results and compare and contrast them to previous analyses of Drosophila hemolymph and mammalian blood plasma. Together, these findings reveal an interesting and hitherto unknown sexual dimorphism in systemic Drosophila metabolites, clearly warranting further biological investigation. Importantly, the VDTS approach should be adaptable to many different analytical platforms, including mass spectrometry.
准确测量微小生物样本中代谢物浓度通常是可取的，但仍然具有挑战性。在许多情况下，起始分析物体积的精度未知，或者无法直接测量，因此难以计算绝对代谢物浓度。在这里，我们介绍了使用两个标准品进行体积测定（VDTS），作为使用（1）H NMR 光谱分析亚微升样品中极性代谢物的通用定量方法。这种方法允许从小体积未知的生物样本中反算绝对代谢物浓度。在小样本体积也存在差异的情况下，VDTS 可以改善多元化学计量分析。在这种情况下，当我们使用 VDTS 计算的体积校正光谱而不是原始光谱的概率商归一化（PQN）进行主成分分析（PCA）时，PCA 产生了更符合逻辑和更具生物学洞察力的输出。作为原理验证，基于 VDTS 的方法和 PCA 用于分析从小型但广泛使用的遗传模式生物果蝇幼虫提取的血淋巴（血液）中的极性代谢物。该分析表明，当幼虫得到良好喂养时，雄性和雌性的血淋巴代谢组明显不同。然而，当幼虫的饮食营养受到限制时，特定于性别的代谢组趋于收敛。我们讨论了这些令人惊讶的结果的生物学意义，并将其与之前对果蝇血淋巴和哺乳动物血浆的分析进行了比较和对比。这些发现共同揭示了系统果蝇代谢物中有趣且迄今未知的性别二态性，显然需要进一步的生物学研究。重要的是，VDTS 方法应该适用于许多不同的分析平台，包括质谱。
