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分析生物化学科学中可用的内容列表

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甘油三酯的脂肪酸分析：通过气相色谱法制备脂肪酸甲酯。

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文章历史：2015年10月8日收到，2015年11月8日收到了修改后的表格，2015年11月9日被接受，2015年11月30日上传到网上。

摘要：建立脂肪酸甲酯的气相色谱（GC）分析方法。三甘醇与甲醇CH3ONA在含极性溶剂的己烷中在室温下混合10秒。在这些条件下，三油酸甘油酯转化为油酸甲酯，平均产率为99.3%。这一过程给出了GC测定的脂肪酸组成的可靠和可重复的数据。2015 Elsevier公司保留所有权利。

关键词：脂肪酸分析； 脂肪酸甲酯； 三酰基甘油； 气相色谱法；

在生物界中，膜和储存脂类的成分是各自分开的。这些甘油脂质的物理、化学和生理、营养特性主要由它们的脂肪酸组成决定[1,2]。气相色谱分析法（GC）是最常用的测定脂肪酸组成的方法[3,4]。在GC分析之前，甘油酯的酰基残基经过酸或碱催化剂，转化为脂肪酸甲酯（FAMES）。目前已经提出了各种方法并用于制备FAMEs，但许多方法需要加热并且耗时[3,4]。 在温和的温度条件下的一些快速方法已有报道[5e7]。利用一个1996年研发的方法[6]，甘油磷脂可在室温条件下15秒生成，且在连续摇晃下花费2分钟将TGs完全转化为FAMEs。然而，在广泛的医学领域[8]、营养科学[9]、农业[10]和食品工业[11]中，大量脂质的样品需要更快速地从TG中合成脂肪酸甲酯，而无需持续搅拌2分钟。脂质代谢学领域也需要它，它正在开始实现其代谢性疾病研究革命性的希望[12]。我们发现丙酮在由己烷和甲醇组成的溶剂体系中加入，明显的促进了TG的碱催化的甲醇分解。与目前的快速方法[6]相比，酯交换反应所需的反应时间已缩短到不到十分之一秒。在这里，我们提出了一种科学方案，为了TGs的脂肪酸组成做GC分析，制备FAMES。在室温条件下，由于TGs对甘油类脂类的酯类转化最具有抗性，故在室温下以甘油三油酸酯为主要研究对象。以下是优化过的实验报告：通过用甲醇稀释25％（w / w，4.37M）CH3ONa（SigmaeAldrich）制备甲醇CH3ONa（2M）。在小玻璃试管（16.5times;105mm）中放入1ml含有20mg或更少的TGs和0.5ml丙酮的己烷。向该脂质溶液中加入75ml 2M CH3ONa并摇晃。 在10秒内完成甲醇分解，并用1ml 0.5M乙酸终止反应。从产量的角度来看，在这些步骤中管连续摇晃是合乎需要的，但也不是非做不可。为了便于操作，将75ml的2M CH3ONa加入TG溶液（1.5ml）中，然后将管摇晃2或3秒。在6到8S后，用1毫升0.5 M乙酸停止反应。总反应时间约为10秒。推荐从加入2 M CH3ONA到终止0.5 M乙酸的操作时间最迟应在30秒或1分钟内。在不同的试管中，用1ml水进行洗涤含有FAMES的上部有机层，可以省略该洗涤步骤。用薄层色谱（TLC）和GC分析法对FAMES的己烷溶液进行分析，或在20℃下储存2周再进行GC分析。

TLC表明，目前的甲烷分解过程的反应产物几乎全部由FIX组成，伴随少量的游离脂肪酸（图1）。75毫升或2毫升或更多浓度的甲醇CH3ONA溶液需要提供，以完全转化TG（图1A和B）。在所有反应产物中检测到少量游离油酸。TG甲醇分解反应中间体二甘酰甘油（DG）和单酰基甘油（MG），在碱浓度较低（0.E1.5mu;m）的反应物、以及在体积较小（10e50ml）的反应产物2mu;m的甲醇CH3ONA中保留。大量的TG在没有丙酮的己烷/甲醇溶液中10秒后并没有变化，但加入丙酮后明显的促进了反应（图1C）。1.0毫升己烷和0.5毫升丙酮的混合溶液使TG完全转化成FAME，而过量丙酮增加游离脂肪酸副产物。 反应5秒后仍保留少量TG，DG和MG（图1D）。 这些甘油酯在7或8秒后减少，在该反应体系中，10秒的反应时间足以中生成FAME。在TG的甲醇分解中，逐步的发生了三个可逆反应，在之前碱催化酯交换反应的动力学的情况已有报道[13]。

一些不是丙酮的有机溶剂，也加速了反应。叔丁基甲基醚的效力较丙酮低，但可以代替丙酮。甲苯和乙酸甲酯的效应明显低于丙酮。丙酮在一般条件下二聚，进一步生成水，从而降低了FAMES的产率。因此，含有丙酮的反应混合物必须及时处理。虽然TGS也在没有己烷的情况下转化为FAMES，但己烷的存在降低了游离脂肪酸或其他副产物的形成。试剂级己烷实际上是无水的，而丙酮和叔丁基甲基醚中含有少量的水。有可能是由后两种溶剂取代己烷，导致大量游离脂肪酸的形成。NaOH和KOH也作为甲醇分解的催化剂，但释放的游离脂肪酸比CH3ONa要多。这些碱金属氢氧化物在甲醇中溶解生成水，形成甲醇离子。此外，试剂级KOH（85％，w / w）是KOH和KOH一水合物的混合物; 因此，甲醇KOH比甲醇的NaOH产生更多的游离脂肪酸。TG（20 mg）可在反应体系中进行完全的酯转化反应，尽管100mg的TG也几乎完全转化为FAME，但反应后只有极少量的TG和部分的甘油酯被留了下来。高产量是用酸催化甲烷的优势之处[14]，与TG不同，甘油磷脂容易与甲醇反应形成FAME，无论丙酮的存在如何（参考线上补充资料图S1），如前所述[6]。水强烈抑制TG的反应。

图1(A)甲醇CH3ONa浓度对TG甲醇分解的影响。(B) TG甲醇分解对2 M甲醇CH3ONa的影响。(C)丙酮对TG甲醇分解的影响。(D) TG的甲醇分解时间过程。典型反应混合物由10 mg甘油三油酸酯、1 ml己烷、0.5 ml丙酮、75 ml 2 M甲醇CH3ONa组成。MG没有从原点移动，硅胶薄层色谱板的来源点主要是MG。TLC的溶剂体系是己烷/叔丁基甲醚/乙酸（85∶15∶0.4，V/V/V）。脂质通过使用50%（W/W）的硫酸，并在137℃下进行加热15分钟。FFA，游离脂肪酸；S，标准脂质；O，原产地。

表1通过不同方法由TG制备的FAME的产率

注意，在本方法中，TG底物甘油三油酸酯基催化剂是CH3ONA，但同样检查NAOH和KOH作为催化剂的有效性。带有火焰离子化检测器的毛细管柱设备用于测定GC，为SUPELCOWAX10，(0.53mm*30m,1.0 薄膜厚度)，柱温为等温225℃，每个收率值为四次室温测试的平均值。

在内部的标准下，十七酸甲酯测定三油酸甘油酯产生的油酸甲酯产率，并将它们与三种常规方法（表1）进行比较。获得的最佳产率为99.8％，这是通过常规方法2（Nacalai Tesque的试剂盒，目录号06482-04）来进行的，其中包括两个反应步骤。在本方法中催化剂为CH3ONA的平均产率大于99%，比常规方法1（14）和3（6）所得到的平均产率要好。当CH3ONa被NaOH或KOH取代时，比起CH3ONa时的产量从 99.3%各自降低到98%或97%，表1还表明，对于酸催化的甲醇分解来说，需要比碱催化的甲醇分解更高的温度和更长的反应时间，并且在温和的温度条件下，在类似的产率下，本方法在已知的碱性甲烷化反应中拥有更快的反应速率。

通过比较由三种常规方法和本方法确定的鱼油脂肪酸组成（参见表S1和补充资料中的图S2），对该方法的准确性和有效性进行了评估，这些脂肪酸的组成彼此非常相似。在本方法和常规方法1和2之间发现了一些小但显着的脂肪酸组成差异。这两种常规方法包括酸催化剂，以及可以由酯化脂肪酸部分和游离脂肪酸形成的脂肪酸甲酯。这可能反映在脂肪酸的总成分中。本方法与传统方法3中脂肪酸组成无显著差异，该方法以碱催化甲醇分解为基础。通过本方法测定小鼠/大鼠商业食品的丸状产品中甘油酯的脂肪酸组成，对由不同操作者制备的FAME的数据的可靠性（参见补充材料中的表S2）进行评估。经8位操作员独立制备的脂肪酸组成分别显示，并没有明显的差异，表明了数据的高可靠性。

目前的方法将有助于高通量分析含有甘油脂类的脂肪酰基残基，就方便性和再现性而言，这是最主要的脂类。它可用于农业、日常、海鲜产品中的脂质、脂肪和油中的组织脂肪酸分析或血甘油脂的分析。

致谢

特此感谢Kouhei Yamamoto（大阪府立大学）对GC分析的有利建议。我们也感谢ASTE（SHIO MiyaK[HORBA]）、Kaoru Ikehara和Rei Naito的成员在样品制备方面的帮助。

附录B补充资料

与本文相关的补充数据可以查阅http://dx.doiOr/101016/j.ab.2015.119009。

参考文献

[1] H. Okuyama, Y. Ichikawa, Y. Sun, T. Hamazaki, W.E.M. Lands, Prevention of Coronary Heart Disease: from the Cholesterol Hypothesis to u6/u3 Balance, Karger, Basel, Switzerland, 2007.

[2] L. Hodson, C.M. Skeaff, B.A. Fielding, Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake, Prog. Lipid Res. 47 (2008) 348e380.

[3] W.W. Christie, Lipid Analysis, third ed., Oily Press, Bridgwater, UK, 2003.

[4] Cyberlipid Center,

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Analytical Biochemistry

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Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

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Article history: Received 8 October 2015 Received in revised form 8 November 2015 Accepted 9 November 2015 Available online 30 November 2015

Keywords: Fatty acid analysis Fatty acid methyl ester Triacylglycerol Gas chromatography

Abstract: A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH3ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC. copy; 2015 Elsevier Inc. All rights reserved.

components of membrane and storage lipids, respectively, in the biosphere. The physical, chemical, and physiological/nutritional properties of these glycerolipids depend primarily on their fatty acid compositions [1,2]. Gas chromatography (GC) is the most widely used method for determination of fatty acid composition [3,4]. Prior to GC analysis, acyl residues of glycerolipids are converted to fatty acid methyl esters (FAMEs) using an acid or base catalyst. Various methods have been proposed and used for the preparation of FAMEs, but many require heating and are timeconsuming [3,4]. Some rapid methods under mild temperature conditions have been reported [5e7]. Using a method developed in 1996 [6], glycerophospholipids can be derivatized within 15 s at room temperature, whereas it takes 2 min under continuous vortexing for the complete conversion of TGs to FAMEs. However, more rapid synthesis of FAMEs from TGs without constant agitation for 2 min is now required for huge numbers of lipid samples in extensive fields of medicine [8], nutritional science [9], agriculture [10], and the food industry [11]. It is also needed in the field of lipid metabolomics, which is beginning to deliver on its promise to revolutionize metabolic disease research [12]. We found that the addition of acetone to solvent systems composed of hexane and methanol markedly promoted base-catalyzed methanolysis of TGs. Compared with a current rapid method [6], the reaction time required for transesterification has been reduced to less than one tenthd10 s. Here, we present protocols to prepare FAMEs for GC analysis of fatty acid compositions of TGs. The formation of FAME was mainly investigated with glyceryl trioleate on the second time scale at room temperature because TGs are the most resistant to transesterification of glycerolipid classes. The following is the optimized protocol. Methanolic CH3ONa (2 M) was prepared by diluting 25% (w/w, 4.37 M) CH3ONa (SigmaeAldrich) with methanol. In a small glass test tube (16.5 105 mm) were placed 1 ml of hexane containing 20 mg or less of TGs and 0.5 ml of acetone. To the lipid solution was added 75 ml of 2 M CH3ONa with vortexing. Methanolysis was completed within 10 s, and the reaction was terminated with 1 ml of 0.5 M acetic acid. Continuous vortexing of the tubes during these steps is desirable from the viewpoint of yield, but it is not indispensable. For ease of operation, 75 ml of 2 M CH3ONa was added to the TG solution (1.5 ml), and then the tube was immediately vortexed for 2 or 3 s. After 6e8 s, the reaction was stopped with 1 ml of 0.5 M acetic acid. The total reaction time was approximately 10 s. It is recommended that the operation time from the addition of 2 M CH3ONa to termination with 0.5 M acetic acid should be within 30 s or 1 min at the latest. The upper organic layer containing FAMEs was washed with 1 ml of water in different test tubes, but this washing step could be omitted. The hexane solution of FAMEs was analyzed by thin-layer chromatography (TLC) and GC or stored at 20 C for up to 2 weeks until GC analysis.

TLC showed that the reaction products of the present methanolysis procedure were composed almost entirely of FAME accompanied by small amounts of free fatty acid (Fig. 1). A volume of 75 ml or more of 2 M or higher concentrations of methanolic CH3ONa solution added is required for complete conversion of TG (Fig. 1A and B). Small amounts of free oleic acid were detected in all of the reaction products. Diacylglycerol (DG) and monoacylglycerol (MG), which are reaction intermediates of TG methanolysis, remained in the reaction products for lower concentrations (0.5e1.5 M) of the base and also in those for smaller volumes (10e50 ml) of 2 M methanolic CH3ONa. A considerable amount of TG remained unchanged after 10 s in the hexane/methanol solution without acetone, but the addition of acetone markedly promoted the reaction (Fig. 1C). The mixed solution of 1.0 ml of hexane and 0.5 ml of acetone gave complete conversion of TG to FAME, whereas excess of acetone increased free fatty acid as a byproduct. Small amounts of TG, DG, and MG remained after 5 s of reaction (Fig. 1D). These glycerides were diminished after 7 or 8 s, and the reaction time of 10 s was sufficient for production of FAME in this reaction system. In methanolysis of TGs, three stepwise and reversible reactions occur, and the kinetics of the base-catalyzed transesterification was reported previously [13].

Some organic solvents other than acetone also accelerated the reaction. tert-Butyl methyl ether was somewhat less effective than acetone, but it could be used instead of acetone. Toluene and methyl acetate were markedly inferior to acetone. Acetone dimerizes under basic conditions and then further generates water, which reduces the yields of FAMEs. The rea

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