这项工作是由俄亥俄州立大学食品卫生发现主题和食品科学与技术在美国俄亥俄州立大学的部门支持。作者要感谢俄亥俄州立大学的NMR设施和宾夕法尼亚州立大学的核磁共振设备。

1. NMR样品制备注意:注意，使用前请咨询所有相关的材料安全数据表（MSDS）。在样品制备中使用的氘代氯仿（CDCL 3）是有毒的。进行样品制备，包括使用通风橱和个人保护设备的时，请使用所有的适当的安全实践（安全眼镜，手套，实验室外套，全长裤，封闭趾鞋）。 <ol><li> 1个 H和13个 C样品的制备 <ol><li>用注射器从膳食胶囊提取鱼油的120μL（〜110毫克），并将其放置在一个4姆·格拉斯小瓶中。记录鱼油的重量。 </li><li>溶样<ol><li>溶解约120μL鱼油中含有被用作1 H和13角 C化学位移的参考四甲基硅烷（TMS）的0.01％CDCL 3的500μL。 注:TMS是使用ð仅化学位移校准（见步骤编号2.2.1.2.7和2.2.2.2.7），而不是定量（见步骤编号2.2.1.3和2.2.2.3）的目的。 </li><li>制备2,6- 二叔丁基-4-甲基苯酚（BHT）的储备溶液中，如果量化以mg / g的需要，通过将BHT的约220毫克和15毫克铬（III），乙酰丙酮的（铬（ ACAC）3）在20ml CDCL 3的含TMS的0.01％。使用储备溶液的500微升溶解100毫克（±10毫克）的鱼油。 </li></ol></li><li>油（这需要几秒钟）溶解后，转移所有的溶液直接进入一个高品质5毫米NMR管和附连的盖。制备样品之后分析24个小时内的样本。 </li></ol></li></ol> 2. NMR仪器制备注:注意，提防强磁场的通过NMR仪器产生的存在可以影响医疗器械并实现了一套蚁如起搏器和外科假体，以及电子项目，如信用卡，手表等时使用非屏蔽磁体所执行的分析的附加警告是必需的。两个NMR仪器被用于获取1 H和13 C NMR谱的;分光计在850.23 MHz和213.81 MHz的分别用于1 H和13个 C核，操作，装配有三重共振氦冷却逆（TCI）5毫米探针和光谱仪在500.20 MHz和125.77 MHz的1 H和13 C操作核，分别配备有观察到宽波段（BBO）氮冷却5毫米探针。 （见材料列表），在25±0.1ºC进行所有实验和光谱通过一个标准的NMR数据分析采集和处理软件程序包进行处理。 <ol><li> 用于获取NMR谱制备 注:1 H和13 C NMR谱可以在不从仪器中取出样品因此获得的。 <ol><li>插入NMR管插入一个微调涡轮机（参见材料列表）。 </li><li>放置旋转器和一个分级深度计的顶部的管，轻轻地按压管的顶部，直至其底部接触轨距的底部。 </li><li>放置NMR样品中SampleCase的打开位置。注意样品被放置在槽号。 </li><li>加载在NMR样品，返回到控制计算机和类型"SX＃"，其中＃是在SampleCase保持你的样品槽中。 </li><li>等待CDCL 3的氘信号出现在锁定窗口画面上。如果没有自动出现，键入"lockdisp"。一旦氘信号是可见的，类型的命令行上的"锁定"，并且为了使用CDCL 3氘共振锁定样品从溶剂的列表中选择"CDCL 3"。 注:氘小号如果先前的用户所使用的不同的溶剂ignal可能不会出现。用户应该等待该样品是向下的指标，然后锁定。 </li><li>键入"bsmsdisp"在命令行中，以确保纺纱不活跃。如果"旋转"按钮为绿色，单击它停用纺纱。 </li><li>键入"新建"命令来创建一个新的数据集。该数据在"名称"选项卡，在"EXPNO"选项卡中的实验数设置输入一个名称。在"PROCNO"选项卡使用数字"1"。在"实验"选项卡，点击"选择"，然后选择"质子"参数文件。写实验的标题在"标题"选项卡。点击"确定"。 </li><li>在命令行键入"getprosol"，以获得用于当前NMR探针和溶剂的标准参数。 </li><li>重复步骤2.1.7 13 C，选择"C13IG"脉冲序列中的"实验"标签用于1D的13 C逆克ated分离实验。 </li><li>在命令行键入"getprosol"，以获得用于当前NMR探针和溶剂的标准参数。 </li><li>键入命令"灵魂"来执行自动调谐和探针的匹配为碳和质子核。 </li><li>用于NMR信号进行一维梯度匀场实现高度均匀的磁场，并且因此最佳线形状。 <ol><li>使用标准的自动过程来1D匀场，简单地通过顺序地执行这些命令"曲topshim 1dfast SS"，"曲topshim tuneb SS"，并且在命令行上"去topshim报告"。 </li></ol></li></ol></li><li> 参数优化 <ol><li>的90°脉冲校准<ol><li>创建1小时（见2.1.7步骤和2.1.8）新的数据集。 </li><li>在命令行上的命令"paropt"开始自动化程序用于校准90°PULSE。选择脉冲持续时间，P1，作为参数进行修改。 </li><li>开始"2"作为微秒p1的初始值，输入"2"μs的增量，并执行"16"的实验。 </li><li>创建一个新的数据的13 C设置（见步骤2.1.9）并重复此过程13个 C核（参见步骤2.2.1.2和2.2.1.3）。 </li></ol></li><li>通过零位法16 1小时测量的T 1个测量 注:空方法使用反转恢复脉冲序列，由延迟（TAU）由180°脉冲的后续的，以允许松弛沿着z轴和最终的90°脉冲，其产生可观察到的横向磁化。 <ol><li>创建1小时（见2.1.7步骤和2.1.8）新的数据集。 </li><li>输入"PULPROG t1ir1d"脉冲序列改变到反转恢复实验。 </li><li>输入在comman以下命令d线的设置以ppm光谱宽度，RF发射器的中心，扫描伪扫描的次数和数据点"SW 8"的数量，"O1P 3.8"，"NS 2"，"DS的数量2&quot; 和 "TD 64K"。 </li><li>类型"P1（值）"，并作为由脉冲校准确定为90°脉冲输入持续时间值（见步骤2.2.1），并输入"P2（值）"为180°脉冲（对于180°的持续时间值脉冲是90°脉冲持续时间乘以二）。 </li><li>通过键入"D1 10"循环延迟设置为一个非常大的值，如10秒。 </li><li>通过在命令行键入"D7 10ms的"设定的tau到短值，如10毫秒。 </li><li>设置使用命令"RGA"为RG的自动计算接收机增益（RG）到适当的值。 </li><li>通过键入命令"ZG"运行的频谱。 </li><li>通过键入"EFP"执行傅立叶变换在命令行。 </li><li>通过在命令行键入命令"APK"执行自动相位校正。如果需要额外的相位调整，以进一步提高频谱，点击中的"进程选项卡"，然后单击"调节相位"图标进入相位校正模式。 <ol><li>通过拖动鼠标，直到所有的信号在负吸收模式使用零阶（0）和一阶（1）的相位校正的图标。申请并通过点击"返回并保存"按钮退出相位校正模式保存相位修正值。 </li></ol></li><li>增加的tau，直到所有的峰是正或零化通过重复步骤2.2.2.6-2.2.2.9。为了确定T 1值，简单地划分在峰值零化与LN2的tau蛋白值。 </li></ol></li><li>通过零位法16 13℃下测定T 1个测量<ol><li>创建一个新的数据对13 C中（参见步骤2.1.9） </li><li>类型"PULPROG t1irpg"改变脉冲序列的反转恢复实验碳核。 </li><li>在命令行设置以ppm光谱宽度，RF发射器的中心，扫描的次数，虚设扫描次数和数据点的数量上的以下命令:"SW 200"，"O1P 98" ， "NS 8"， "DS 2" 和 "TD 64K"。 </li><li>类型"P1（值）"，并作为由脉冲校准确定为90°脉冲输入持续时间值（见步骤2.2.1）和类型"P2（值）"为180°脉冲（持续时间值是90°脉冲持续时间乘以二）。 </li><li>通过键入"D1 100"循环延迟设置为一个非常大的值，如100秒。 </li><li>集的tau到短值，如100毫秒通过在命令行键入"D7 100毫秒"。 </li><li>设置接收地ER增益（RG）到适当的值使用命令"RGA"为RG的自动计算。 </li><li>通过键入命令"ZG"运行的频谱。 </li><li>通过在命令行中键入"EFP"执行傅立叶变换。 </li><li>通过在命令行中输入命令"APK"执行自动相位校正。如果需要额外的相位调整，以进一步提高频谱，点击"调节相位"图标和用于零阶（0）和第一阶相（1）校正的相位校正的图标。 <ol><li>而在零阶和第一阶相位校正的图标点击，拖动鼠标，直到所有的信号在负吸收模式。申请并通过点击"返回并保存"按钮退出相位校正模式保存相位修正值。 </li></ol></li><li>增加的tau，直到所有的峰是正或零化通过重复步骤2.2.3.6-2.2.3.9。为了确定的T 1值，简单地划分在峰值零化与LN2的tau蛋白值。 </li></ol></li></ol></li><li> 一维（1D）。NMR谱 <ol><li> 1 H-NMR谱 <ol><li> NMR数据的采集 <ol><li>转到步骤2.1.7创建的1个 H数据集和通过在命令行键入"PULPROG ZG"使用标准的"脉冲获取"脉冲序列" ZG"。 </li><li>在命令行上下面的命令来设置，以ppm光谱宽度，RF发射器的中心，扫描的次数，虚设扫描的数量，数据点的数目和脉冲持续时间为90°脉冲角: "SW 8"， "3.8 O1P"， "NS 2"， "DS 2"， "TD 64K"和"P1（如通过脉冲校准测定）"（见步骤2.2.1）。 注:32K数据点可用于500 MHz仪器上。 </li><li>通过键入"D1 7S"或"D1 787-9"，分别在命令行上设置的7个S为500 MHz仪器上或9号为850 MHz仪器上的松弛延迟。 </li><li>设置使用命令"RGA"为RG的自动计算接收机增益（RG）到适当的值。 </li><li>键入"digmod baseopt"以获取与改善基线频谱。 </li><li>通过在命令行键入脉冲获取命令"ZG"开始采集。 </li></ol></li><li> NMR数据的处理 <ol><li>在命令行键入"SI 64K"应用零填充和所述真实频谱的大小设置为64K。 </li><li>通过在命令行键入"磅0.3"，以用线展宽0.3赫兹的因子前，傅立叶变换应用的加权函数（指数衰减）设置谱线增宽参数至0.3赫兹。 </li><li>通过在命令中键入"EFP"执行傅立叶变换线。 </li><li>通过在命令行中输入命令"APK"执行自动相位校正。如果需要额外的相位调整，以进一步提高频谱，点击中的"进程选项卡"，然后单击"调节相位"图标和用于零阶（0）和一阶（1）的相位校正的相位校正图标。 <ol><li>而在零阶和第一阶相位校正的图标点击，拖动鼠标，直到所有的信号都是正的吸收模式。申请并通过点击"返回并保存"按钮退出相位校正模式保存相位修正值。 </li></ol></li><li>通过键入命令"ABS N"时整合申请基线校正的多项式四阶函数。 注意:这确保了与最小强度平坦的光谱基线。 </li><li>由TMS以ppm报告化学位移（δ= 0）。点击校准（"CALIB。斧是"）的图标，并且将光标与上TMS NMR信号（峰最接近于0）的顶部上的红线。左键点击并输入‘0’。 </li></ol></li><li> NMR数据分析 <ol><li>整合的光谱区从δ1.1至在δ4.98δ0.6以及峰，δ5.05和δ5.81使用"整合"图标（"进程"标签下）和高亮（"定义新的区域"）的图标。通过积分右键单击并拖动。 注意:如果有需要关注的区域，点击高亮显示图标停用左键点击并拖动鼠标以放大的区域。调节阈值强度，如果需要的话使用鼠标按钮。再次点击高亮显示图标，使整合功能激活，然后移动到下一个高峰。 <ol><li>正常化有关显示的积分值以上的积分，以100通过右击的总和S的信号下，选择"归一化的积分之和"。输入框中的值"100"，然后单击"返回并保存"退出一体化模式。 </li></ol></li><li>当使用BHT作为内部标准，在整合δ6.98峰值和设定的积分等于BHT的每0.5毫升储备溶液的毫摩尔。 </li><li>整合的感兴趣的峰（参见步骤2.3.1.3.1）从峰值的每一侧延伸10赫兹，在可能的情况。 </li><li>进行到以类似的方式执行13 C-NMR谱的采集和处理。 </li></ol></li></ol></li><li> 13 C-NMR谱 <ol><li> NMR数据的采集 <ol><li>转至13 C数据集，并通过在命令行键入"PULPROG zgig"使用逆门控去耦脉冲序列" zgig"。 注意:要运行碳实验与标准的宽带decouPLED的脉冲序列，在命令行键入"PULPROG zgpg"。 </li><li>在命令行上下面的命令来设置，以ppm光谱宽度，RF发射器的中心，扫描的次数，虚设扫描的数量，数据点的数目和脉冲持续时间为90°脉冲角: "SW 200"， "95 O1P"， "NS 16" "DS 2"， "TD 64K" 和 "P1（如通过脉冲校准测定）"（见步骤2.2.1.4）。 </li><li>通过键入"D1 35S"或"D1 45S"，分别在命令行上设置的35秒为500 MHz仪器上或45秒为850 MHz仪器上的松弛延迟。当使用BHT，弛豫延迟应该是在500 MHz仪器上50秒和60秒在850兆赫的仪器。 </li><li>设置使用命令"RGA"为RG的自动计算接收机增益（RG）到适当的值。 </li><li>在命令行键入"digmod baseopt"来获取光谱瓦特第i个改进的基线。 </li><li>通过在命令行键入脉冲获取命令"ZG"开始采集。 </li></ol></li><li> NMR数据的处理 <ol><li>在命令行键入"SI 64K"应用零填充和所述真实频谱的大小设置为64K。 </li><li>通过在命令行键入"磅1.0"，以用线展宽1.0赫兹的因子前，傅立叶变换应用的加权函数（指数衰减）设置谱线增宽参数为1.0赫兹。 </li><li>通过在命令行中键入"EFP"执行傅立叶变换。 </li><li>通过在命令行中输入命令"APK"执行自动相位校正。如果需要额外的相位调整，以进一步提高频谱，点击中的"进程选项卡"，然后单击"调节相位"图标和用于零阶（0）和第一阶相（1）修正的相位校正图标。 <ol><lI&gt;虽然点击零阶和第一阶相位校正图标，拖动鼠标，直到所有的信号都是正的吸收模式。申请并通过点击"返回并保存"按钮退出相位校正模式保存相位修正值。 注:对于记录在214兆赫（850兆赫的仪器）的频率相关的错误（一阶）的校正的拉莫尔频率碳谱可以是具有挑战性的并且对于缺乏经验的用户的因大的偏共振效应耗时的90°脉冲。 </li></ol></li><li>通过在命令行键入命令"ABS N"时整合申请基线校正的多项式四阶函数。 </li><li>由TMS以ppm报告化学位移（δ= 0）。点击校准（"CALIB。轴"）的图标，并且将光标与NMR信号的顶部红线被引用。左键点击并输入"0"。 </li></ol></li><li> NMR数据分析 <ol><li>整合的光谱区从δ175使用"整合"图标（"进程"标签下）和高亮〜δ171（"定义新区域"）的图标。通过积分右键单击并拖动。 注意:如果有需要关注的区域，点击高亮显示图标停用左键点击并拖动鼠标以放大的区域。再次点击高亮显示图标，使整合功能激活，然后移动到下一个高峰。 <ol><li>这样做对信号下出现的整数值，右键点击设置积分为100，然后选择"校准电流积分"。输入框中的值"100"，然后单击"返回并保存"，退出一体化模式。 </li></ol></li><li>当使用BHT作为内标，在δ151.45整合的峰值和设定的积分等于每0.5 ml的储备溶液的BHT的毫摩尔。 </li><li>整合的延伸从所述峰值的每侧5赫兹感兴趣的峰（参见步骤2.3.2.3.1）。 </li></ol></li></ol></li></ol></li></ol>

<ol>
	<li>Simopoulos, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+ratio+of+omega-6/omega-3+essential+fatty+acids.">The importance of the ratio of omega-6/omega-3 essential fatty acids.</a> Biomed. Pharmacother. 56 (8), 365-379 (2002).</li><li>Goodnight, S. H., Harris, W. S., Connor, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+dietary+omega+3+fatty+acids+on+platelet+composition+and+function+in+man:+a+prospective,+controlled+study.">The effects of dietary omega 3 fatty acids on platelet composition and function in man: a prospective, controlled study.</a> Blood. 58 (5), 880-885 (1981).</li><li>Harper, C., Jacobsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+omega-3+fatty+acids+and+the+prevention+of+coronary+heart+disease.">Usefulness of omega-3 fatty acids and the prevention of coronary heart disease.</a> Am. J. Cardiol. 96 (11), 1521-1529 (2005).</li><li>Kremer, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high-dose+fish+oil+on+rheumatoid+arthritis+after+stopping+nonsteroidal+antiinflammatory+drugs.+Clinical+and+immune+correlates.">Effects of high-dose fish oil on rheumatoid arthritis after stopping nonsteroidal antiinflammatory drugs. Clinical and immune correlates.</a> Arthritis and Rheumatol. 38 (8), 1107-1114 (1995).</li><li>Malasanos, T., Stackpoole, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+effects+of+omega-3+fatty+acids+in+diabetes+mellitus.">Biological effects of omega-3 fatty acids in diabetes mellitus.</a> Diabetes Care. 14, 1160-1179 (1991).</li><li>Han, Y., Wen, Q., Chen, Z., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Methods+Used+for+Microalgal+Lipid-Content+Analysis.">Review of Methods Used for Microalgal Lipid-Content Analysis.</a> Energ. Procedia. 12, 944-950 (2011).</li><li>Guill&#233;n, M., Ruiz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1H+nuclear+magnetic+resonance+as+a+fast+tool+for+determining+the+composition+of+acyl+chains+in+acylglycerol+mixtures.">1H nuclear magnetic resonance as a fast tool for determining the composition of acyl chains in acylglycerol mixtures.</a> Eur. J. Lipid Sci. Technol. 105, 502-507 (2003).</li><li>Sacchi, R., Medina, I., Aubourg, S. P., Addeo, F., Paolillo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+nuclear+magnetic+resonance+rapid+and+structure+specific+determination+of+&#969;-3+polyunsaturated+fatty+acids+in+fish+lipids.">Proton nuclear magnetic resonance rapid and structure specific determination of &#969;-3 polyunsaturated fatty acids in fish lipids.</a> J. Am Oil Chem Soc. 70, 225-228 (1993).</li><li>Igarashi, T., Aursand, M., Hirata, Y., Gribbestad, I. S., Wada, S., Nonaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondestructive+quantitative+acid+and+n-3+fatty+acids+in+fish+oils+by+high-resolution+1H+nuclear+magnetic+resonance+spectroscopy.">Nondestructive quantitative acid and n-3 fatty acids in fish oils by high-resolution 1H nuclear magnetic resonance spectroscopy.</a> J. Am. Oil Chem. Soc. 77, 737-748 (2000).</li><li>Plans, M., Wenstrup, M., Saona, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Infrared+Spectroscopy+for+Characterization+Dietary+Omega-3+Oil+Supplements.">Application of Infrared Spectroscopy for Characterization Dietary Omega-3 Oil Supplements.</a> J. Am. Oil Chem. Soc. 92, 957-966 (2015).</li><li>Jian-hua, C. I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+Spectrum+Detection+of+Fish+Oil+DHA+Content+Based+on+Empirical+Mode+Decomposition+and+Independent+Component+Analysis.">Near-infrared Spectrum Detection of Fish Oil DHA Content Based on Empirical Mode Decomposition and Independent Component Analysis.</a> J Food Nutr Res. 2 (2), 62-68 (2014).</li><li>Millen, A. E., Dodd, K. W., Subar, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+vitamin,+mineral,+nonvitamin,+and+nonmineral+supplements+in+the+United+States:+The+1987,+1992,+and+2000+National+Health+Interview+Survey+results.">Use of vitamin, mineral, nonvitamin, and nonmineral supplements in the United States: The 1987, 1992, and 2000 National Health Interview Survey results.</a> J. of Am. Diet Assoc. 104 (6), 942-950 (2004).</li><li>Dwyer, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+developing+analytical+and+label-based+dietary+supplement+databases+at+the+NIH+office+of+dietary+supplements.">Progress in developing analytical and label-based dietary supplement databases at the NIH office of dietary supplements.</a> J. Food Compos. Anal. 21, S83-S93 (2008).</li><li>Monakhova, Y. B., Ruge, I., Kuballa, T., Lerch, C., Lachenmeier, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+coenzyme+Q10+in+food+supplements+using+1H+NMR+spectroscopy.">Rapid determination of coenzyme Q10 in food supplements using 1H NMR spectroscopy.</a> Int. J. Vitam. Nutr. Res. 83 (1), 67-72 (2013).</li><li>Monakhova, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardless+1H+NMR+determination+of+pharmacologically+active+substances+in+dietary+supplements+and+medicines+that+have+been+illegally+traded+over+the+internet.">Standardless 1H NMR determination of pharmacologically active substances in dietary supplements and medicines that have been illegally traded over the internet.</a> Drug Test. Anal. 5 (6), 400-411 (2013).</li><li>Berger, S., Braun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 200 and more NMR experiments: a practical course. , (2004).</li><li>Knothe, G., Kenar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+fatty+acid+profile+by+1H-NMRspectroscopy.">Determination of the fatty acid profile by 1H-NMRspectroscopy.</a> Eur. J. Lipid Sci. Technol. 106, 88-96 (2004).</li><li>Sacchi, R., Medina, J. I., Aubourg, S. P., Paolillo, I. G. L., Addeo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+High-Resolution+13C+NMR+Analysis+of+Lipids+Extracted+from+the+White+Muscle+of+Atlantic+Tuna+(Thunnus+alalunga).">Quantitative High-Resolution 13C NMR Analysis of Lipids Extracted from the White Muscle of Atlantic Tuna (Thunnus alalunga).</a> J. Agric. Food Chem. 41 (8), 1247-1253 (1993).</li><li>Dais, P., Misiak, M., Hatzakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+marine+dietary+supplements+using+NMR+spectroscopy.">Analysis of marine dietary supplements using NMR spectroscopy.</a> Anal. Methods. 7 (12), 5226-5238 (2015).</li><li>Pickova, J., Dutta, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+Oxidation+in+Some+Processed+Fish+Products.">Cholesterol Oxidation in Some Processed Fish Products.</a> J. Anal. Oil Chem. Soc. 80 (10), 993-996 (2003).</li><li>Siddiqui, N., Sim, J., Silwood, C. J. L., Toms, H., Iles, R. A., Grootveld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+analysis+of+encapsulated+marine+oil+supplements+using+high-resolution+1H+and+13C+NMR+techniques.">Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C NMR techniques.</a> J. of Lipid Rsrch. 44 (12), 2406-2427 (2003).</li><li>Sua&#180;rez, E. R., Mugford, P. F., Rolle, A. J., Burton, I. W., Walter, J. A., Kralovec, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C-NMR+Regioisomeric+Analysis+of+EPA+and+DHA+in+Fish+Oil+Derived+Triacylglycerol+Concentrates.">13C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates.</a> J. Am. Oil Chem. Soc. 87, 1425-1433 (2010).</li><li>Youlin, X. A., Moran, S., Nikonowiczband, E. P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Z-restored+spin-echo+13C+1D+spectrum+of+straight+baseline+free+of+hump,+dip+and+roll.">Z-restored spin-echo 13C 1D spectrum of straight baseline free of hump, dip and roll.</a> Magn. Reson. Chem. 46, 432-435 (2008).</li><li>Tengku-Rozaina, T. M., Birch, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+distribution+of+fatty+acids+on+hoki+and+tuna+oil+triglycerides+by+pancreatic+lipase+and+13C+NMR+analysis.">Positional distribution of fatty acids on hoki and tuna oil triglycerides by pancreatic lipase and 13C NMR analysis.</a> Eur. J. Lipid Sci. Technol. 116 (3), 272-281 (2014).</li><li>Berry, S. E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triacylglycerol+structure+and+interesterification+of+palmitic+and+stearic+acid-rich+fats:An+overview+and+implications+for+cardiovascular+disease.">Triacylglycerol structure and interesterification of palmitic and stearic acid-rich fats:An overview and implications for cardiovascular disease.</a> Nutr. Res. Rev. 22 (1), 3-17 (2009).</li><li>Hunter, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+effects+of+dietary+fatty+acids+as+related+to+their+position+on+triglycerides.">Studies on effects of dietary fatty acids as related to their position on triglycerides.</a> Lipids. 36, 655-668 (2001).</li><li>Vlahov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regiospecific+analysis+of+natural+mixtures+of+triglycerides+using+quantitative+13C+nuclear+magnetic+resonance+of+acyl+chain+carbonyl+carbons.">Regiospecific analysis of natural mixtures of triglycerides using quantitative 13C nuclear magnetic resonance of acyl chain carbonyl carbons.</a> Magnetic Res. in Chem. 36, 359-362 (1998).</li></ol>

作者什么都没有透露。

修改和策略疑难解答 光谱质量 。 NMR信号，从而NMR谱的分辨率的线宽是高度依赖于匀场，这是用于磁场的均匀性的优化的方法。对于常规分析，1D匀场是足够的，并不需要3D匀场，因为它是通过NMR人员定期进行。 D 2 O中（90:10）:如果不是这种情况下，3D匀场必须被分析之前，使用含有0.6毫升氧化氢的样品进行的。为了实现更好更快匀场，样品需要在射...

在饮食Ñ-3脂肪酸的消费已被证明是对几个条件有利如心脏疾病1，2，3，炎性疾病4和糖尿病5。西方饮食被认为是穷人的N-3脂肪酸，因此建议鱼油补充剂的消费，提高n个 -6 / N -3平衡消费者的营养1。尽管鱼油补充剂的消费，最近越来越多，问题仍然是关于安全性，真实性，以及一些产品的质量。鱼油补充剂的快速和准确的成分分析是必不可少的，以正确评估这些商业产品的质量，保证消费者的安全。 鱼油补充评估的最常见方法s为气相色谱法（GC）和红外光谱（IR）。虽然这些都是高度敏感的方法，他们从几个缺点遭受6。 GC分析是因为单独的化合物的分离和衍生化，需要7和分析8,9期间可能发生的脂质氧化耗时（4-8小时）。虽然IR光谱可以是定量，需要一个预测模型，使用偏最小二乘回归（PLSR）构造，但也有例外，其中IR带可以归因于一个单一的化合物10。 PLSR需要大量的样品，从而增加了分析11的时间的分析。出于这个原因，存在的新的分析方法，使大量的鱼油样品的准确，快速分析发展的越来越大的兴趣。组织，如OFFI在健康（NIH）和美国食品和药物管理局（FDA）全国学院膳食补充剂（ODS）的CE与官方分析化学师协会（AOAC）合作开发这些新方法12，13。 一个用于筛选和多组分基质，如膳食补充品的评价最有前途的分析方法，是核磁共振（NMR）光谱14,15。 NMR光谱法有几个优点:它是一种非破坏性的和定量技术，它需要极少的无样品制备，并且其特征在于优良的精确度和再现性。此外，NMR光谱是一种环境友好的方法，因为它仅使用少量的溶剂。 NMR光谱法的主要缺点是其相对低的灵敏度相比其他analytiCAL方法，但是，例如更强的磁场，各种直径的低温探针，先进的数据处理，和通用的脉冲序列和技术在仪表最近的技术进步增加了灵敏度高达nM范围内。虽然核磁共振仪器是成本高，核磁共振波谱仪的长寿命和NMR的许多应用降低了分析的，从长远来看成本。这个详细的视频协议的目的是帮助本领域从业者新避免与1个 H和13个 C NMR光谱分析鱼油补充剂的相关联的缺陷。

<table><tbody><tr><td>Avance III 850 NMR instrument</td><td>Bruker</td><td></td><td></td></tr><tr><td>Avance III 500 NMR instrument</td><td>Bruker</td><td></td><td></td></tr><tr><td>TCI 5 mm probe</td><td>Bruker</td><td></td><td>Helium cooled inverse (proton deetected) NMR probe featuring three independent channels (&lt;sup&gt;1&lt;/sup&gt;H, &lt;sup&gt;13&lt;/sup&gt;C, &lt;sup&gt;15&lt;/sup&gt;N)</td></tr><tr><td>BBO prodigy 5 mm probe</td><td>Bruker</td><td></td><td>Nitrogen cooled observe (X-nuclei detected) probe, featuring two channels; one for &lt;sup&gt;1&lt;/sup&gt;H and &lt;sup&gt;19&lt;/sup&gt;F detectionand one for X-nuclei (covering from &lt;sup&gt;15&lt;/sup&gt;N to &lt;sup&gt;31&lt;/sup&gt;P)</td></tr><tr><td>Spinner turbin</td><td>Bruker</td><td></td><td>NMR spinners are made by polymer materials and they have a rubber o-ring to hold the NMR tube securely in place</td></tr><tr><td>Topspin 3.5</td><td>Bruker</td><td></td><td></td></tr><tr><td>deuterated chloroform</td><td>Sigma-Aldrich</td><td>&amp;nbsp;865-49-6</td><td>99.8 atom % D, contains 0.03 TMS</td></tr><tr><td>2,6-Di-&lt;em&gt;tert&lt;/em&gt;-butyl-4-methylphenol</td><td>Sigma-Aldrich</td><td>&amp;nbsp;128-37-0</td><td>purity &gt;99%</td></tr><tr><td>Fish oil samples</td><td></td><td></td><td></td></tr><tr><td>NMR tubes</td><td>New Era</td><td>NE-RG5-7</td><td>5mm OD Routine &amp;ldquo;R&amp;rdquo; Series NMR Sample Tube</td></tr><tr><td>BSMS</td><td>Bruker</td><td></td><td>Bruker Systems Management System; control system device</td></tr></tbody></table>

nmr spectroscopy as a robust tool for the rapid evaluation of the lipid profile of fish oil supplements

西方饮食同n-3脂肪酸差，因此建议鱼油补充剂的消费，以增加这些必需营养素的摄入量。这个工作的目的是利用高分辨率1个 H和13利用两个不同的NMR仪器C NMR光谱法证实包封的鱼油补充剂的定性和定量分析;一个500兆赫和850兆赫的仪器。两个质子（1个 H）和碳（13 C）NMR谱可用于定量测定鱼油补充剂的主要成分。在鱼油补充剂脂质的定量是通过适当的NMR信号的积分在相关1D光谱来实现。通过1 H和13 C NMR得到的结果彼此一致，尽管在两个核和两个仪器之间的分辨率和灵敏度的差异。 1 H-NMR报价SA的快速分析相比13 C NMR，作为频谱可被记录在小于1分钟，而相比之下，13点 C NMR分析，它从10分钟持续到一小时。 13 C NMR谱，然而，更加丰富。它可以用于单个脂肪酸更大数量提供定量数据，并可以被用于确定在甘油骨架的脂肪酸的位置分布。两者的核可以提供仅在一个实验中的定量信息，而无需纯化或分离步骤。的磁场的强度主要影响:1个 H NMR谱由于相对于13 C NMR其较低的分辨率，但是，即使是更低的成本NMR仪器可以有效地应用为食品工业和质量控制实验室的标准方法。

1 H和13个 C NMR光谱收集使用两个NMR仪器市售鱼油补充剂; 850 MHz和500 MHz光谱。这些光谱可用于定量测定鱼油的组分，如二十二碳六烯酸（DHA）和二十碳五烯酸（EPA），以及其它化合物，如正 -1酰基链的和营养的重要指标，如正 -6 / N -3比。所述定量可即使不使用内标来进行，然而，定量结果必须表示为相对摩尔百分比。当数据需要以绝对?...

Watch this Scientific Journal Video about NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements at JoVE.com

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

这里，高分辨率1 H和13 C核磁共振（NMR）光谱被用作包封的鱼油补充剂的定量和定性分析的快速和可靠的工具。

核磁共振作为鱼油补充剂的血脂的快速评价一个强大的工具

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

nmr-spectroscopy-as-robust-tool-for-rapid-evaluation-lipid-profile

Research

JoVE Journal

Chemistry

25.9K 次观看.  The Ohio State University. 这里，高分辨率1 H和13 C核磁共振（NMR）光谱被用作包封的鱼油补充剂的定量和定性分析的快速和可靠的工具。 

视频: NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

脂肪酸组分和含量微藻分析

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

科研

环境科学

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

化学

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

生物化学

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Determination of Total Lipid and Lipid Classes in Marine Samples

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix.
If seawater has been sampled, the first step usually involves separation into operationally defined &#39;dissolved&#39; and &#39;particulate&#39; factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement.
Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).

This protocol is for the determination of lipids in seawater and biological specimens. Lipids in filtrates are extracted with chloroform or mixtures of chloroform and methanol in the case of solids. Lipid classes are measured by rod thin-layer chromatography with flame ionization detection and their sum gives the total lipid content.

海洋样品中总脂质和脂质类别的测定

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

生物学

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

大鼠脑组织被提取物的高分辨核磁共振代谢组学分析

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

神经科学

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

啮齿动物组织的霰弹枪脂质组学

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

代谢物的浓度从低密度浮游社区的环境代谢，利用核磁共振光谱

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

神经系统疾病中的脂质组学和转录组学

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...