Division of Biology, California Institute of Technology, Pasadena, California, United States of America.
PLoS One. 2010 Aug 24;5(8):e12353. doi: 10.1371/journal.pone.0012353.
Drosophila melanogaster has recently emerged as a useful model system in which to study the genetic basis of regulation of fat storage. One of the most frequently used methods for evaluating the levels of stored fat (triglycerides) in flies is a coupled colorimetric assay available as a kit from several manufacturers. This is an aqueous-based enzymatic assay that is normally used for measurement of mammalian serum triglycerides, which are present in soluble lipoprotein complexes. In this short communication, we show that coupled colorimetric assay kits cannot accurately measure stored triglycerides in Drosophila. First, they fail to give accurate readings when tested on insoluble triglyceride mixtures with compositions like that of stored fat, or on fat extracted from flies with organic solvents. This is probably due to an inability of the lipase used in the kits to efficiently cleave off the glycerol head group from fat molecules in insoluble samples. Second, the measured final products of the kits are quinoneimines, which absorb visible light in the same wavelength range as Drosophila eye pigments. Thus, when extracts from crushed flies are assayed, much of the measured signal is actually due to eye pigments. Finally, the lipoprotein lipases used in colorimetric assays also cleave non-fat glycerides. The glycerol backbones liberated from all classes of glycerides are measured through the remaining reactions in the assay. As a consequence, when these assay kits are used to evaluate tissue extracts, the observed signal actually represents the amount of free glycerols together with all types of glycerides. For these reasons, findings obtained through use of coupled colorimetric assays on Drosophila samples must be interpreted with caution. We also show here that using thin-layer chromatography to measure stored triglycerides in flies eliminates all of these problems.
黑腹果蝇最近已成为研究脂肪储存调控遗传基础的有用模式生物系统。评估果蝇中储存脂肪（甘油三酯）水平最常用的方法之一是一种偶联比色法，该方法有几个制造商提供试剂盒。这是一种基于水的酶促测定法，通常用于测量哺乳动物血清中的甘油三酯，这些甘油三酯存在于可溶性脂蛋白复合物中。在本简讯中，我们表明偶联比色法试剂盒不能准确测量果蝇中储存的甘油三酯。首先，当用类似于储存脂肪的组成的不溶性甘油三酯混合物或用有机溶剂从果蝇中提取的脂肪进行测试时，它们无法给出准确的读数。这可能是由于试剂盒中使用的脂肪酶无法有效地从不溶性样品中的脂肪分子上切下甘油头部基团。其次，试剂盒测量的最终产物是醌亚胺，它在与果蝇眼色素相同的波长范围内吸收可见光。因此，当从粉碎的果蝇中提取的物质进行测定时，大部分测量的信号实际上是由于眼色素。最后，比色法测定中使用的脂蛋白脂肪酶也会切割非脂肪甘油酯。从所有甘油酯类中释放出的甘油骨架通过测定中的其余反应进行测量。因此，当这些测定试剂盒用于评估组织提取物时，观察到的信号实际上代表了游离甘油以及所有类型的甘油酯的量。由于这些原因，通过在果蝇样品上使用偶联比色法获得的发现必须谨慎解释。我们还在此表明，使用薄层层析法测量果蝇中储存的甘油三酯可以消除所有这些问题。
