基于GC-MS技术的蹄叶炎奶牛血浆代谢谱分析

【目的】利用气相色谱/质谱联用技术(gas chromatograph tandem mass spectrometer technology,GC-MS)对奶牛血浆进行代谢组学分析,以明确奶牛发生蹄叶炎时血浆中代谢物的变化,进一步揭示其发病机制。【方法】根据蹄叶炎奶牛的临床症状和活动量情况,选取蹄叶炎患病组S和健康对照组C奶牛各10头,采集血浆样品,经衍生化处理后,利用GC-MS方法对两组奶牛的血浆进行全部代谢物检测。用SIMCA-P 11.5软件进行主成分分析(principal component analysis,PCA)和偏最小二乘法分析(supervised partial least squares-discriminant analysis,PLS-DA),对蹄叶炎组奶牛和健康组奶牛检测数据进行模式识别分析,筛选差异代谢物,并利用生物信息学方法进行代谢通路富集分析。为进一步验证代谢组学的试验结果,检测了与差异代谢通路相关的脂质代谢指标和抗氧化能力指标。【结果】利用GC-MS方法,在奶牛血浆中共检测到242种代谢物,通过多元统计分析结合t-检验筛选出37种差异代谢物(VIP1,P0.05),其中在蹄叶炎奶牛血浆中3种物质含量上调(FC1),分别是氨基氧乙酸、油酸、乳糖,34种物质含量下调(FC1),包括脂肪酸、氨基酸等。经代谢通路富集分析,发现变化显著(P0.05)的代谢通路包括脂肪酸生物合成通路,甘氨酸/丝氨酸/苏氨酸代谢通路,不饱和脂肪酸生物合成通路,嘌呤代谢通路,甲烷代谢通路,苯丙氨酸代谢通路和氰基氨基酸代谢通路。在验证试验中,发现两组奶牛血浆中脂质代谢相关指标差异显著,且蹄叶炎患病牛血浆抗氧化能力显著低于健康牛,与代谢组学结果一致。【结论】利用GC-MS技术分析了蹄叶炎患病牛与健康奶牛血浆代谢谱的变化,发现两组间代谢物存在显著差异,并筛选出了37种差异代谢物,而这些差异分子可能成为奶牛蹄叶炎早期诊断或群体监测的潜在生物标记物。本结果全面揭示了奶牛蹄叶炎发生后血浆代谢物的代谢规律,为进一步阐明其发病机制提供理论依据。

Plasma Metabolic Profiling Analysis of Dairy Cows with Laminitis Based on GC-MS

【Objective】In order to discover the difference between the cows suffered with laminitis and healthy cows, metabonomics analysis based on gas chromatograph tandem mass spectrometer technology (GC-MS) was utilized in the study.【Method】Twenty plasma samples were collected from cows with laminitis and healthy groups. Plasma metabolic profiling was obtained by GC-MS after derivatization. Multivariate statistics including principal component analysis (PCA) and supervisedpartial least squares-discriminant analysis (PLS-DA) were conducted to screen the changed metabolites. Bioinformatics was analyzed to identify the significantly changed metabolic pathways.【Result】A total of 242 variables were detected and 37 compounds were significantly different between healthy and laminitis-suffered cows of which 3 were up-regulated including aminooxyacetic acid, lactose and oleic acid and 34 were down-regulated in laminitis group involved amino acid and fatty acid. The main changed metabolic pathways were fatty acid biosynthesis, Gly, Ser, Thr metabolism, biosynthesis of unsaturated fatty acids, purine metabolism, methane metabolism, cyanoamino acid metabolism and phenylalanine metabolism. Results of the the experiment of validation showed that the indexes related to fatty acid metabolism and antioxidation were significantly different between healthy cows and sick cows. 【Conclusion】 Based on GC-MS, this study revealed the changed regularity of metabolism in plasma with laminitis, and the discovered different metabolites would be potential biomarkers in occurrence and development process of laminitis, which can lay a theoretical foundation for further understanding of the pathogenesis, early diagnosis and therapy of laminitis in dairy cows.