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. Author manuscript; available in PMC: 2024 Jun 12.
Published in final edited form as: Methods Mol Biol. 2022;2468:271–281. doi: 10.1007/978-1-0716-2181-3_14

Lipid Extraction and Analysis

Henry H Harrison 1, Jennifer L Watts 1
PMCID: PMC11168205  NIHMSID: NIHMS1992326  PMID: 35320570

Abstract

Lipids are major components of cellular membranes and energy stores. Lipids contribute vital structural, energetic, and signaling functions. We have optimized methods to extract and analyze lipids from the nematode Caenorhabditis elegans based on standard methods. Here we describe a method to extract total lipids from C. elegans larvae, adults, or embryos. We describe a thin-layer chromatography method to separate major lipid classes and a gas chromatography method to analyze fatty acid composition from lipid extracts, lipid fractions, or directly from nematode larvae, adults, or embryos.

Keywords: Thin-layer chromatography, Lipid extraction, Phospholipids, Neutral lipids, Fatty acid methyl esters, Gas chromatography

1. Introduction

Changes in lipid composition and metabolism are associated with many biological processes in Caenorhabditis elegans, including neuronal function, aging, innate immunity, cell death, and embryonic development [1–5]. The small size of C. elegans has traditionally been a barrier to biochemical analysis. While single-worm or single-tissue lipid analysis is not possible, mass spectrometry based methods are sensitive enough to analyze fatty acid composition of populations of several hundred adult-staged C. elegans [6, 7].

The combination of chloroform and methanol is widely used for lipid extraction across many species of microorganisms, plants, and animals. The Folch method uses chloroform/methanol in a ratio of 2:1 [8], while the Bligh and Dyer method uses chloroform/methanol in a ratio of 1:1, with water provided in various amounts due to samples in aqueous solutions, such as nematodes washed off of growth plates [9]. More recently, the methyl tert-butyl ether extraction method describes an alternative extraction solvent that does not require the use of chloroform [10, 11]. Others have demonstrated that various lipid extraction methods are appropriate for C. elegans [7]. Our method, based on Bligh and Dyer, works well to extract a variety of phospholipids and neutral lipids from C. elegans.

Thin-layer chromatography of lipids uses a silica gel stationary phase and a mobile phase consisting of various organic solvents [12]. TLC is a somewhat “old fashioned” lipid separation technique, widely replaced by high performance liquid chromatography (HPLC) and mass spectrometry (MS) based lipidomics. However, using TLC to separate lipid classes prior to gas chromatography (GC) analysis offers several advantages, including low cost, convenience, and simplicity. Other lipid profiling techniques, such as “shotgun” lipidomics, in which raw lipid extracts are separated and analyzed using ion trap MS-MS and quadrupole-time of flight (Q-Tof) instruments, have been reviewed elsewhere and compared for their advantages and disadvantages for the analysis of C. elegans lipids [7].

For fatty acid composition analysis of whole worms, or for analysis of fatty acid composition in TLC-separated phospholipids, GC offers a sensitive and precise detection of a range of fatty acids, including cis/trans conformations and separation of isomers such as cis-vaccenic and oleic acid (18:1n-7 vs. 18:1n-9). Before injection on the GC, fatty acids associated with phospholipids and neutral lipids are converted to fatty acid methyl esters (FAMEs) which remain in the gas phase upon injection. GC coupled with a flame ionization detector can be used to separate and identify all the fatty acids in C. elegans. GC coupled with a single quad mass spectrometry detector (GC-MS) provides more certain species identification as well as the possibility for stable-isotope labeling experiments and 4,4-dimethyloxazoline (DMOX) derivatization to identify the double bond location [13–16].

The procedures described below allow for the separation of specific lipid types by polarity using TLC and GC. Depending on the solvent used, the TLC method separates various neutral lipid classes from polar lipids or separates individual phospholipids for further analysis.

2. Materials

2.1. Lipid Extraction

  1. Chloroform:methanol (1:1): In a glass bottle, add 50 mL Chloroform (Approx. 0.75% Ethanol as Preservative/HPLC) and 50 mL methanol (OmniSolv for HPLC Gradient Analysis, Spectrophotometry, and Gas Chromatography). Mix well and store at −20 °C.

  2. Hajra’s solution: 0.2 M H3PO4, 1 M KCl.

  3. Glass screw top tubes, large (16 mm × 125 mm).

  4. Glass screw tops, to fit large tubes.

  5. 9-inch Pasteur pipette.

  6. Glass screw top tubes, small (13 mm × 100 mm).

  7. Glass screw tops, to fit small tubes.

  8. Argon or Nitrogen gas.

2.2. Thin-Layer Chromatography

  1. TLC plates (Silica gel HL, 20 × 20 cm, 250 μm).

  2. TLC tank (27.0 cm × 26.5 cm × 7.0 cm).

  3. Cellulose filter paper (3MM CHR sheets, 15 cm × 17.5 cm).

  4. Developing solvent for TLC (neutral lipids): 80 mL hexane (95% n-hexane, HPLC-grade), 20 mL diethyl ether (anhydrous with BHT as inhibitor), 2 mL glacial acetic acid (aldehyde-free/sequencing).

  5. Developing solvent for TLC (phospholipids): 65 mL chloroform, 43 mL methanol, 3 mL water, 2.5 mL glacial acetic acid.

  6. Hamilton syringe 25 μL.

  7. Primuline spray: Dissolve 5 mg primuline in 100 mL of acetone/water (80/20, v/v).

  8. Glass sprayer.

  9. Internal standard. Pentadecanoic Acid, C15:0 (Nu-Chek Prep, N-15-A). Make stock solution of 2 mg/mL in hexane and dilute the solution to 0.1 M with hexane for the working stock. Store 2 mg/mL stock solution in a tightly sealed glass tube at −20 °C.

  10. Sulfuric acid methanol solution: 2.5% sulfuric acid in methanol. In a fume hood, using a glass pipette, slowly pipette 25 mL sulfuric acid into 1000 mL of methanol (see Note 1).

2.3. FAMEs and Gas Chromatography

  1. Glass screw top tubes, small (13 mm × 100 mm).

  2. Glass screw tops, to fit small tubes.

  3. Internal standard. Pentadecanoic Acid, C15:0. Make stock solution of 2 mg/mL in hexane and dilute solution to 0.1 mg/mL with hexane. Store stock solution tightly sealed at −20 °C.

  4. Sulfuric acid methanol solution (2.5% sulfuric acid in methanol). In a fume hood, using a glass pipette, slowly pipette 25 mL sulfuric acid into 1000 mL of methanol (see Note 1).

  5. Hexane (95% n-hexane, HPLC-grade).

  6. GC vials (2 mL crimp top vial).

  7. GC vial inserts.

  8. GC vial caps (11 mm aluminum caps with a PTFE/Silicone septa).

  9. 11 mm E-Z Crimper.

  10. GC-MS system equipped with a polar GC column (20 m × 0.25 mm).

2.4. NGM Agar Plates for Growth of C. Elegans

  1. Prepare 1 M stock solution of potassium phosphate buffer for use in Step 5 by mixing 132 mL of K2HPO4 (1 M) with 868 mL of KH2PO4 (1 M). Filter-sterilize.

  2. Dissolve 3 g/L NaCl and 2.5 g/L bacto peptone in H2O.

  3. Add 17 g/L agar (for 6- and 10-cm plates) or 10 g/L agar and 10 g/L agarose (for 14.5-cm plates).

  4. Autoclave; then cool the solution to 50 °C.

  5. Add the following components: 25 mL potassium phosphate buffer (prepared in step 1), 1 mL MgSO4 (1 M), 1 mL CaCl2 (1 M), 1 mL cholesterol (5 mg/mL in ethanol).

  6. Mix the solution well and pour into Petri dishes.

2.5. Alkaline Hypochlorite Method to Isolate C. elegans Embryos

  1. Prepare alkaline hypochlorite solution: 1.25 mL bleach, 0.25 mL 5 N NaOH, 3.5 mL water.

  2. Harvest gravid adult C. elegans from NGM agar plates by rinsing plates with sterile water or M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 mL 1 M MgSO4, H2O to 1 L) and collecting the worm suspension into a 15 mL conical bottom tube.

  3. Pellet the worms via centrifugation for 1 min at 1300 × g.

  4. Remove the supernatant and suspend worm pellet in 5 mL of alkaline hypochlorite solution.

  5. Mix the worms slowly so that they remain in suspension. Monitor worms under a stereomicroscope for dissolution of adults and release of embryos.

  6. After adult worm carcasses have dissolved, pellet embryos via centrifugation for 1 min at 1300 × g.

  7. Resuspend embryos in 10 mL sterile M9 buffer and centrifuge for 1 min at 1300 × g. Repeat M9 wash. Resuspend embryo pellet in 30–50 μL of water for FAMEs analysis.

3. Methods

3.1. Lipid Extraction from C. elegans

  1. Wash worms grown on 20–30 10 cm NGM agar plates (see Subheading 2.4), approximately 1000 worms/plate, with water into a large glass screw top test tube. Let worms settle on ice (adults), centrifuge gently to form a pellet (larvae), or isolate embryos from gravid adult worms using the alkaline hypochlorite method (see Subheading 2.5). Remove water with a glass Pasteur pipette and wash with water, letting worms settle on ice or centrifuging gently for larvae or embryos. Repeat wash if necessary. The wash solution should not be cloudy with suspended E. coli. Worms can be lyophilized if desired, but it is not necessary.

  2. Remove most of water from the worm pellet using a glass Pasteur pipet. The pellet should be 300–400 μL volume for worms, smaller for embryos. Quick freeze the pellet using liquid nitrogen and store the tube at −80 °C. Glass tubes with frozen worms or embryos can be stored at −80 °C for several weeks.

  3. Using a glass pipette, add 5 mL of ice cold (−20 °C) chloroform:methanol (CHCL3:CH3OH)(1:1) directly to the frozen pellet (see Note 2). Do not let the pellet thaw out before adding the cold CHCL3:CH3OH. Shake tube vigorously and incubate the tube overnight in the −20 °C freezer. If too much water is present in the sample, two phases will form. If this occurs, add another 5 mL of cold CHCL3:CH3OH (1:1). This should lead to a single-phase solution. Let worm pellets extract at −20 °C overnight.

  4. Use a 5 mL glass pipette to add 2.2 mL Hajra’s solution to each sample tube and shake vigorously. If 10 mL of CHCL3:CH3OH was used for the overnight extraction, then add 4.4 mL Hajra’s solution. Shake well and centrifuge for 1 min. at 12,000 × g using a clinical centrifuge. Solution will form two phases with the worm carcass pellet at the interface.

  5. The lipids will be in the lower phase, the chloroform layer. Remove the lower phase carefully using a glass Pasteur pipette to transfer most of the lower phase into a new glass tube.

  6. Re-extract the top phase with 0.5 mL chloroform (see Note 3). Centrifuge for 1 min at 12,000 × g. Remove the lower phase with a glass Pasteur pipette, combine this in the tube with the lower phase from the first extraction.

  7. Using an evaporator dryer with a heat block, set heat at 50 °C and evaporate under a stream of nitrogen or argon until only 100 μL of chloroform remains. Wash the sides of the tube with 300 μL chloroform and continue evaporation until 100 μL remains. Do not let the lipids evaporate to dryness.

  8. Bring the volume up to 200 μL or desired volume with chloroform. Flush tube with argon or nitrogen to remove air and store at −20 °C while preparing the TLC tank and the GC tubes.

  9. If lipids are to be separated using TLC with subsequent GC-MS analysis, remove three 10 μL aliquots into 3 glass tubes and place tubes at −20 °C. These will be used to determine the fatty acid composition of the “total lipid” samples by GC.

  10. The remaining lipid extract can be divided into 3 portions to separate on a TLC plate. Lipid extracts can be flushed with argon or nitrogen and stored at −20 °C for several weeks.

3.2. Thin-Layer Chromatography of C. elegans Lipid Extracts

  1. Preparation of TLC plates. For most consistent separation of lipids, bake the TLC plate in 110 °C oven for 1 h 15 min (±15 min max). This step is not typically necessary for neutral lipid separation in C. elegans but it improves the consistency of phospholipid separation.

  2. Mark the TLC plate using a dull pencil with as many lanes needed for samples, plus two 1 cm lanes for standards on both edges of plate. Make a mark for loading the samples several cm from the bottom. If desired, use a razor blade to scrape a straight line between lanes.

  3. Cut several sheets of cellulose filter paper to the size of the tank and place the paper in the tank.

  4. Add TLC solvent mixture to the TLC tank (see Note 4).

  5. Secure the lid to the TLC tank tightly. Let the solvent soak into the cellulose filter paper and allow the solvent to equilibrate the atmosphere in the tank for 30 min.

  6. Load the samples onto TLC plate using a Hamilton syringe. Load 30–40 μL of total lipid extract per lane (see Note 5).

  7. Gently and quickly, lower the plate into the TLC tank, ensuring that the samples are above the solvent level. Secure the lid tightly with tape or a heavy brick (see Note 6). Allow the solvent to run up the TLC plate until the solvent front is within 1–2 cm of the top of plate. When finished, remove the plate and allow the solvent to evaporate in the hood or dry the plate with a nitrogen or argon stream. Dispose of TLC solvent in a proper waste container.

  8. Instead of a one-solvent development, we routinely use a two-solvent development scheme to separate the major phospholipids and the major neutral lipid components of C. elegans on one plate. The TLC plate is first developed approximately 60% up the plate with chloroform:methanol:H2O: acetic acid (65:43:3:2.5) to separate phospholipids. The plate is removed from the TLC tank, air dried, and then developed in hexane: diethyl ether:acetic acid (80:20:2) to within 1–2 cm of the top of the plate (Fig. 1c).

  9. In a fume hood equipped with a pressurized airline, attach the glass sprayer to the air line and spray the plate lightly with primuline solution, ensuring to not saturate the plate (see Note 7).

  10. Visualize using a UV transilluminator (340 nm); lipids will appear as bright spots against the white silica of the plate (Fig. 1). Photograph the plate. Carefully mark the lipids of interest with a dull pencil, ensuring to not scrape the silica or look directly into UV light. Once bands of interest are identified, use a clean razorblade to carefully scrape the marked silica spot marked into labeled glass tubes for each lane, scraping and discarding extraneous silica between bands of interest. The blade should be held at a 45° angle and move toward you, allowing for precise scraping.

  11. Use weigh paper and a funnel to add the scraped silica to a glass screw top tube (13 × 100 mm) with screw cap.

  12. Prepare FAMEs from scraped silica samples as described below.

Fig. 1.

Fig. 1

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Separation of lipids from C. elegans using TLC. (a) Schematic of TLC plate developed with hexane: diethyl ether:acetic acid (80:20:2) to separate various neutral lipid species. (b) Schematic of TLC plate developed with chloroform:methanol:H2O: acetic acid (65:43:3:2.5) to separate phospholipids. (c) Photograph of TLC separation of C. elegans lipids developed in a two-solvent system. The plate was developed with chloroform:methanol:H2O: acetic acid (65:43:3:2.5) until solvent reached the white line. The plate was removed from the TLC tank, air dried, and then developed to the 1 cm below the top with hexane:diethyl ether:acetic acid (80:20:2). Lipid species were identified by comigration with known standards. PL: phospholipids; DAG: diacylglycerols; FFA: free fatty acids; TAG: triacylglycerols; SM: sphingomyelin; PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; PE: phosphatidylethanolamine; CL: cardiolipin

3.3. Preparation of Nematode Samples for Fatty Acid Methyl Esters (FAMEs)

  1. Harvest 200–1000 well-fed C. elegans from several 6 cm NGM plates, avoiding the bacterial lawn.

  2. Wash well-fed worms with 1–3 mL water into a glass tube (13 × 100 mm) with screw cap. Glass is preferred because plastic tubes can shed contaminants that will interfere with the FAMEs analysis (see Note 8).

  3. Place tubes in a bucket of ice and let worms settle for at least 5 min. Once the worms have settled to the bottom of the tube, carefully remove as much of the water and bacteria as possible (without disturbing the pellet) using a 9-inch Pasteur pipette. Wash again with water if visible bacteria are still present. During the final wash, remove as much water from the loose worm pellet as possible (see Note 9). Tubes may be stored at −20 °C for several days.

3.4. Preparation of Fatty Acid Methyl Esters (FAMEs)

This method to prepare fatty acid methyl esters (FAMEs) can be used to generate FAMEs directly from frozen or freshly harvested worm pellets, from lipid extracts, or from silica fractions scraped from TLC plates.

  1. For quantification of lipids separated from TLC plates, add 30 μL of 0.1 mg/mL C15:0 fatty acid internal standard to each sample tube, including the three total lipid samples that have been set aside.

  2. Add 1 mL of MeOH, 2.5% H2SO4 to each tube, cap tubes tightly with screw caps, and heat for 60 min in a 70 °C water bath (see Note 10).

  3. During incubation, check tubes periodically to make sure the methanol solution is not bubbling. Bubbling indicates a leak in the screw top and samples will eventually evaporate if tubes are not sealed properly.

  4. After 60 min, remove tubes and allow to cool at room temperature for 5 min.

  5. Add 0.2 mL hexane and 1.5 mL H2O to each tube. Higher volumes of hexane can be used for more concentrated lipid samples.

  6. Shake tubes vigorously.

  7. Centrifuge tubes in clinical centrifuge at 12,000 × g for 1 min.

  8. Carefully remove 80 μL of the upper hexane phase by pipetting it into a GC vial fitted with a glass GC vial insert (see Note 11).

  9. Cap the GC vials tightly using screw caps with Teflon liner or crimp caps.

3.5. GC-MS Separation of FAMEs

  1. Separate FAMEs on a SP-2380 or other polar column using a GC system equipped with an inert mass spectrometry detector and an autosampler. A flame ionizing detector also works well for FAMEs analysis, as long as peak retention times can be compared to authentic standards.

  2. Program the autosampler to inject 2 μL of sample.

  3. Program the GC oven. Initial temperature of 120°, hold for 1 min followed by an increase of 10°/min to 190° followed by an increase of 2°/min to 200°. This program takes 14 min per sample.

  4. The relative amounts of each fatty acid in the sample are calculated by dividing the corrected area of each peak by the total area of all peaks (Fig. 2).

Fig. 2.

Fig. 2

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Typical GC chromatogram of C. elegans FAMEs separated on a SP-2380 column showing the major fatty acid methyl ester peaks. Fatty acid nomenclature, x:y(nz), x = # of carbons, y = # of double bonds, nz = location of terminal double bond, z carbons from the methyl end of the fatty acid. 15:iso, 13-methyltetradecanoic acid; 17:iso, 15-methylhexanoic acid; 17Δ, cis-9,10-methylenehexadecanoic acid; DMA, dimethylacetal; 19Δ, cis-11-12-methylene octadecanoic acid, 20:0–18:3, sum of 20:0 and 18:3n6, peaks which do not resolve on our GC column under these running conditions

4 Notes

1.

The mixing of sulfuric acid and methanol is highly exothermic. In a fume hood, using a glass pipette, add the sulfuric acid very slowly to the methanol with constant stirring. Wear protective lab coat, gloves, and eye protection.

2.

From this point forward, always use glass tubes, pipettes, and bottles. Avoid plastic when working with lipids.

3.

If the bottom phase is cloudy, add 0.5 mL ethanol:benzene (9:1).

4.

To separate neutral lipids, use 80 mL hexane, 20 mL diethyl ether, 2 mL glacial acetic acid solvent mix (Fig. 1a). To separate phospholipids, use 65 mL chloroform, 43 mL methanol, 3 mL water, and 2.5 mL glacial acetic acid (Fig. 1b).

5.

To avoid saturating the silica with chloroform, use a Hamilton syringe to apply 10 μL at a time, drop by drop, evenly across the sample line and allow the chloroform to evaporate before applying the next 10 μL aliquot. Load at least three TLC lanes per lipid sample, these are technical replicates.

6.

A secure lid will maintain saturation of the atmosphere in the tank and prevent solvent evaporation.

7.

Other visualization methods to preserve lipids are also be used, including iodine crystals, dichlorofluoresceine, rhodamine G. Alternatively, it is common to use radioactive labeling of lipids and visualization using a phosphorimager [12].

8.

For consistency, harvest well-fed worms that have not yet cleared the E. coli lawn, as food runs out the fatty acid composition of nematodes changes.

9.

Water competes with the methylation reaction, leading to hydrolysis, rather than methylation of fatty acids, leading to reduced peak size or random noise in the chromatograph readout obscuring the fatty acid peaks of interest.

10.

This can be added directly to total lipid extracts, scraped silica from TLC plate, or freshly harvested worm pellets or frozen pellets. If worm samples were frozen, do not let samples in the tubes thaw, this leads to immediate destruction of lipids. Instead, add the 1 mL of 2.5% H2SO4 in MeOH, directly to the frozen pellet.

11.

Take care not to remove any of the aqueous phase; if this phase is injected into the GC-MS, it will destroy the GC column.

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