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Colorimetric measurement of triglycerides cannot provide an accurate measure of stored fat content in Drosophila.

Al-Anzi B, Zinn K - PLoS ONE (2010)

Bottom Line: 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.We also show here that using thin-layer chromatography to measure stored triglycerides in flies eliminates all of these problems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, United States of America. alanzi@caltech.edu

ABSTRACT
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.

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The coupled colorimetric assay is unsuitable for analysis of insoluble triglyceride mixtures.Lard (A), butter (B), and extracted fly fat (C) do not produce linear increases in quinoneimine absorption as more of each substance is assayed. Both the Stanbio Triglyceride Liquicolor Kit (blue bars) and the Sigma TR0100 kit (yellow bars) were used. The pink bars (Calculated) indicate the signal that would be observed if the samples could be measured accurately. Error bars are standard deviations of four different replicates for a given sample type. The butterfat and lard samples were sonicated and boiled as in ref. [5]. The extracted fly fat samples were resuspended without sonication and boiling, because sonicated and boiled samples generated even smaller signals (data not shown). Lard (D), butter (E), and extracted fly fat (F) all produce signals that increase with increasing amounts of either substance when assayed by the TLC method. The arrows indicate the location of the triglyceride band on the TLC assay. Asterisks denote T-test statistical significance: *; P<0.05–0,005, **; P<0.005–0.0005, ***; P<0.0005.
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pone-0012353-g001: The coupled colorimetric assay is unsuitable for analysis of insoluble triglyceride mixtures.Lard (A), butter (B), and extracted fly fat (C) do not produce linear increases in quinoneimine absorption as more of each substance is assayed. Both the Stanbio Triglyceride Liquicolor Kit (blue bars) and the Sigma TR0100 kit (yellow bars) were used. The pink bars (Calculated) indicate the signal that would be observed if the samples could be measured accurately. Error bars are standard deviations of four different replicates for a given sample type. The butterfat and lard samples were sonicated and boiled as in ref. [5]. The extracted fly fat samples were resuspended without sonication and boiling, because sonicated and boiled samples generated even smaller signals (data not shown). Lard (D), butter (E), and extracted fly fat (F) all produce signals that increase with increasing amounts of either substance when assayed by the TLC method. The arrows indicate the location of the triglyceride band on the TLC assay. Asterisks denote T-test statistical significance: *; P<0.05–0,005, **; P<0.005–0.0005, ***; P<0.0005.

Mentions: Partially purified Drosophila fat was obtained by crushing 200 flies in 2 mL of chloroform [19]. The homogenate was gently agitated for 1 hour at room temperature and spun down in an Eppendorff centrifuge at maximum speed for 15 min. 1.5 mL of the choloroform layer was taken and allowed to evaporate to dryness at room temperature. The resulting pellet was dissolved in 100 µL of 2∶1 chloroform/methanol, and the fat yield was estimated using the TLC assay with a known concentration of lard as a standard. For colorimetric measurements of the fly fat preparation, a measured amount of the solution was taken, and the chloroform/methanol solvent was allowed to evaporate, after which the samples were resuspended in PBT and assayed same as butter and lard samples. We assayed samples that were either resuspended only by vortexing (data shown in Fig. 1), or by sonication and boiling as described above (data not shown). Surprisingly, stronger colorimetric assay signals were observed when sonication and boiling were omitted.


Colorimetric measurement of triglycerides cannot provide an accurate measure of stored fat content in Drosophila.

Al-Anzi B, Zinn K - PLoS ONE (2010)

The coupled colorimetric assay is unsuitable for analysis of insoluble triglyceride mixtures.Lard (A), butter (B), and extracted fly fat (C) do not produce linear increases in quinoneimine absorption as more of each substance is assayed. Both the Stanbio Triglyceride Liquicolor Kit (blue bars) and the Sigma TR0100 kit (yellow bars) were used. The pink bars (Calculated) indicate the signal that would be observed if the samples could be measured accurately. Error bars are standard deviations of four different replicates for a given sample type. The butterfat and lard samples were sonicated and boiled as in ref. [5]. The extracted fly fat samples were resuspended without sonication and boiling, because sonicated and boiled samples generated even smaller signals (data not shown). Lard (D), butter (E), and extracted fly fat (F) all produce signals that increase with increasing amounts of either substance when assayed by the TLC method. The arrows indicate the location of the triglyceride band on the TLC assay. Asterisks denote T-test statistical significance: *; P<0.05–0,005, **; P<0.005–0.0005, ***; P<0.0005.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927426&req=5

pone-0012353-g001: The coupled colorimetric assay is unsuitable for analysis of insoluble triglyceride mixtures.Lard (A), butter (B), and extracted fly fat (C) do not produce linear increases in quinoneimine absorption as more of each substance is assayed. Both the Stanbio Triglyceride Liquicolor Kit (blue bars) and the Sigma TR0100 kit (yellow bars) were used. The pink bars (Calculated) indicate the signal that would be observed if the samples could be measured accurately. Error bars are standard deviations of four different replicates for a given sample type. The butterfat and lard samples were sonicated and boiled as in ref. [5]. The extracted fly fat samples were resuspended without sonication and boiling, because sonicated and boiled samples generated even smaller signals (data not shown). Lard (D), butter (E), and extracted fly fat (F) all produce signals that increase with increasing amounts of either substance when assayed by the TLC method. The arrows indicate the location of the triglyceride band on the TLC assay. Asterisks denote T-test statistical significance: *; P<0.05–0,005, **; P<0.005–0.0005, ***; P<0.0005.
Mentions: Partially purified Drosophila fat was obtained by crushing 200 flies in 2 mL of chloroform [19]. The homogenate was gently agitated for 1 hour at room temperature and spun down in an Eppendorff centrifuge at maximum speed for 15 min. 1.5 mL of the choloroform layer was taken and allowed to evaporate to dryness at room temperature. The resulting pellet was dissolved in 100 µL of 2∶1 chloroform/methanol, and the fat yield was estimated using the TLC assay with a known concentration of lard as a standard. For colorimetric measurements of the fly fat preparation, a measured amount of the solution was taken, and the chloroform/methanol solvent was allowed to evaporate, after which the samples were resuspended in PBT and assayed same as butter and lard samples. We assayed samples that were either resuspended only by vortexing (data shown in Fig. 1), or by sonication and boiling as described above (data not shown). Surprisingly, stronger colorimetric assay signals were observed when sonication and boiling were omitted.

Bottom Line: 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.We also show here that using thin-layer chromatography to measure stored triglycerides in flies eliminates all of these problems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, United States of America. alanzi@caltech.edu

ABSTRACT
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.

Show MeSH