Centre for Human Metabolomics, Faculty of Natural and Agricultural Sciences, North-West University (Potchefstroom Campus), Private Bag X6001, Potchefstroom, South Africa.
Metabolomics. 2018 May 21;14(6):74. doi: 10.1007/s11306-018-1372-6.
The analysis of limited-quantity samples remains a challenge associated with mouse models, especially for multi-platform metabolomics studies. Although inherently insensitive, the highly specific characteristics of nuclear magnetic resonance (NMR) spectroscopy make it an advantageous platform for global metabolite profiling, particularly in mitochondrial disease research.
Show method equivalency between a well-established standard operating protocol (SOP) and our novel miniaturized H-NMR method.
The miniaturized method was performed in a 2 mm NMR tube on a standard 500 MHz NMR spectrometer with a 5 mm triple-resonance inverse TXI probe at room temperature.
Firstly, using synthetic urine spiked with low (50 µM), medium (250 µM) and high (500 µM) levels (n = 10) of nine standards, both the SOP and miniaturized method were shown to have acceptable precision (CV < 15%), relative accuracy (80-120%), and linearity (R > 0.95), except for taurine. Furthermore, statistical equivalence was shown using the two one-sided test. Secondly, pooled mouse quadriceps muscle extract was used to further confirm method equivalence (n = 3), as well as explore the analytical dynamics of this novel approach by analyzing more-concentrated versions of samples (up to 10× concentration) to expand identification of metabolites qualitatively, with quantitative linearity. Lastly, we demonstrate the new technique's application in a pilot metabolomics study using minute soleus muscle tissue from a mouse model of Leigh syndrome using Ndufs4 KO mice.
We demonstrate method equivalency, supporting our novel miniaturized H-NMR method as a financially feasible alternative to cryoprobe technology-for limited-quantity biological samples in metabolomics studies that requires a volume one-tenth of the SOP.
在小鼠模型中，分析有限量的样本仍然是一个挑战，特别是对于多平台代谢组学研究。尽管核磁共振（NMR）光谱本身不灵敏，但它具有高度特异性，是进行全局代谢物分析的有利平台，特别是在研究线粒体疾病方面。
展示一种成熟的标准操作方案（SOP）和我们新的小型化 H-NMR 方法之间的方法等效性。
在室温下，使用标准的 500MHz NMR 光谱仪和 5mm 三共振反向 TXI 探头，在 2mm NMR 管中进行小型化方法。
首先，使用合成尿液（n=10），用低（50µM）、中（250µM）和高（500µM）水平（n=10）的 9 种标准品进行掺杂，结果表明，SOP 和小型化方法都具有可接受的精密度（CV<15%）、相对准确性（80-120%）和线性度（R>0.95），除了牛磺酸。此外，通过双单边检验证明了统计学等效性。其次，使用混合的小鼠四头肌提取物进一步确认方法等效性（n=3），并通过分析更浓缩的样品版本（高达 10 倍浓度）来探索该新方法的分析动力学，从而在定性上扩展代谢物的鉴定，同时保持定量线性。最后，我们使用 Ndufs4 KO 小鼠的 Leigh 综合征小鼠模型的微小比目鱼肌组织进行了一项代谢组学研究，展示了新技术的应用。
我们证明了方法等效性，支持我们新的小型化 H-NMR 方法作为一种经济可行的替代方案，用于代谢组学研究中需要冷冻探针技术的有限量生物样本，该方法所需的样本量为 SOP 的十分之一。
