Fathi Fariba, Brun Antonio, Rott Katherine H, Falco Cobra Paulo, Tonelli Marco, Eghbalnia Hamid R, Caviedes-Vidal Enrique, Karasov William H, Markley John L
Biochemistry Department, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706, USA.
Metabolites. 2017 Nov 16;7(4):61. doi: 10.3390/metabo7040061.
Metabolites present in liver provide important clues regarding the physiological state of an organism. The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small quantity of liver tissue. We extracted the tissue with a methanol/chloroform/water mixture and isolated the polar metabolites from the methanol/water layer and the non-polar metabolites from the chloroform layer. Following drying, we re-solubilized the fractions for analysis with a 600 MHz NMR spectrometer equipped with a 1.7 mm cryogenic probe. In order to evaluate the feasibility of this protocol for metabolomics studies, we analyzed the metabolic profile of livers from house sparrow () nestlings raised on two different diets: livers from 10 nestlings raised on a high protein diet (HP) for 4 d and livers from 12 nestlings raised on the HP diet for 3 d and then switched to a high carbohydrate diet (HC) for 1 d. The protocol enabled the detection of 52 polar and nine non-polar metabolites in ¹H NMR spectra of the extracts. We analyzed the lipophilic metabolites by one-way ANOVA to assess statistically significant concentration differences between the two groups. The results of our studies demonstrate that the protocol described here can be exploited for high-throughput screening of small quantities of liver tissue (approx. 100 mg wet mass) obtainable from small animals.
肝脏中存在的代谢物为了解生物体的生理状态提供了重要线索。本研究的目的是评估一种基于核磁共振（NMR）的高通量分析方法，用于分析少量肝脏组织中的极性和非极性代谢物。我们用甲醇/氯仿/水混合物提取组织，从甲醇/水层中分离出极性代谢物，从氯仿层中分离出非极性代谢物。干燥后，我们将这些馏分重新溶解，以便用配备1.7毫米低温探头的600兆赫核磁共振光谱仪进行分析。为了评估该方法在代谢组学研究中的可行性，我们分析了以两种不同饮食饲养的家麻雀雏鸟肝脏的代谢谱：10只雏鸟在高蛋白饮食（HP）下饲养4天的肝脏，以及12只雏鸟在高蛋白饮食下饲养3天，然后改为高碳水化合物饮食（HC）1天的肝脏。该方法能够在提取物的¹H NMR光谱中检测到52种极性代谢物和9种非极性代谢物。我们通过单因素方差分析对亲脂性代谢物进行分析，以评估两组之间具有统计学意义的浓度差异。我们的研究结果表明，本文所述的方法可用于对从小动物获得的少量肝脏组织（约100毫克湿重）进行高通量筛选。
