Colorimetric measurement of triglycerides cannot provide an accurate measure of stored fat content in Drosophila.
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.
Al-Anzi
Bader
B
Division of Biology, California Institute of Technology, Pasadena, California, United States of America. alanzi@caltech.edu
Zinn
Kai
K
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D003124:Colorimetry / Q000379:methods*; D004331:Drosophila melanogaster / Q000378:metabolism*; D008297:Male; D014280:Triglycerides / Q000378:metabolism*
D014280:Triglycerides
Cell Metab. 2009 Dec;10(6):481-90
19945405
Nat Rev Genet. 2002 Aug;3(8):589-600
12154382
Pediatr Res. 2009 Feb;65(2):132-7
18978647
PLoS One. 2009 Jul 10;4(7):e6211
19593430
Curr Biol. 2003 Apr 29;13(9):790-5
12725740
J Lipid Res. 2007 Nov;48(11):2514-20
17717377
Aging Cell. 2008 Aug;7(4):478-90
18485125
Neuron. 2009 Aug 13;63(3):329-41
19679073
BMC Genomics. 2010 May 11;11:297
20459830
Cell Metab. 2005 May;1(5):323-30
16054079
Curr Biol. 2003 Apr 1;13(7):603-6
12676093
J Lipid Res. 1996 May;37(5):1153-61
8725166
J Biol Chem. 1975 May 10;250(9):3414-8
1168195
Cell Metab. 2010 May 5;11(5):427-37
20417156
Cell. 2010 Jan 8;140(1):148-60
20074523
Lipids. 1974 Aug;9(8):548-53
4473126