Quantification of Fatty Acids in Mammalian Tissues by Gas Chromatography–Hydrogen Flame Ionization Detection

[Abstract] In mammalian organisms, fatty acids (FAs) exist mostly in esterified forms, as building blocks of phospholipids, triglycerides, and cholesteryl esters, while some exist as non-esterified free FAs. The absolute quantification of FA species in total lipids or in a specific lipid class is critical in lipid-metabolism studies. To quantify FAs in biological samples, gas chromatography–hydrogen flame ionization detection (GC-FID)-based methods have been used as highly robust and reliable techniques. Prior to GC-FID analysis, FAs need to be derivatized to volatile FA methyl esters (FAMEs). The derivatization of unsaturated FAs using classical derivatization methods that rely on high reaction temperature requires skill; consequently, the quantification results are often unreliable. The recently available FA-methylation procedure rapidly and reliably derivatizes a variety of FA species, including poly-unsaturated FAs (PUFAs). To analyze FAs in mammalian tissue samples, lipid extraction and fractionation are also critical for robust analysis. In this report, we describe a whole protocol for the GC-FID-based FA quantification of mammalian tissue samples, including lipid extraction, fractionation, derivatization, and quantification. The protocol is useful when various FAs, especially unsaturated FAs, need to be reliably quantified.


Procedure
The entire experimental workflow described in this protocol is shown in Figure 1. The blue arrow is followed when determining only total FAs, while the green arrows are followed when determining FAs in neutral-lipid and/or phospholipid fractions. tissues are cryogenically pulverized. The pulverized tissue is directly subjected to the derivatization procedure (blue path) when only total FA quantification is required. To quantify FAs in neutral-lipid (NL) and phospholipid (PL) fractions, as well as total lipids (total), the total lipids are extracted from the pulverized tissue using the Bligh-Dyer method, fractionated by solid-phase extraction, and then derivatized (green route).
A. Tissue pulverization 1. Place frozen mouse tissue (~50 mg or less) and a stainless-steel crusher into a 2.0-ml pulverizing tube with a stainless steel inner lid, and cool in liquid nitrogen.
3. If only total FA analysis is required, continue to Procedure D. When phospholipid and neutral lipid fractions also need to be analyzed, continue to Procedure B.
B. Bligh-Dyer total lipid extraction from pulverized tissue (Bligh and Dyer, 1959) 1. Add 800 µl of methanol to the tube containing the pulverized tissue and the crusher.
3. Remove crusher from the tube with forceps. 6 www.bio-protocol.org/e3613 10. Centrifuge the glass test tube at 1,000 x g at room temperature using the swing rotor for 10 min.
11. Transfer the lower (organic) phase to a new glass test tube (16 x 125 mm).
12. For full lipid recovery, add 1.0 ml of chloroform to the remaining aqueous phase, vortex for 2 min, centrifuge for 10 min, collect the lower phase, and combine it with the first extract. 13. Evaporate the glass test tube containing the extracts to dryness using a vacuum evaporator.
This step typically takes ~1 h for ~50 mg of tissue from Procedure A, although this depends on the tissue type and evaporator performance.
14. Add 1 ml of chloroform and vortex until the extracts are dissolved.  14. Continue to Procedure F. E. Derivatizing lipids dissolved in organic solvents 1. Evaporate the samples to be derivatized (i.e., total lipids, neutral lipids, and phospholipids, prepared in Procedure C) to dryness using a vacuum evaporator.
Note: This step is critical for quantitative accuracy. Pre-wetting should be carried out until no further liquid drips are observed from the pipette tip.

Vortex for 20 s.
Note: Use test tube caps for vortexing and incubation.   6. If a low FAME concentration is expected then continue to Procedure G. Otherwise continue to Procedure H.
G. Concentrating FAMEs 1. Evaporate the FAME solution prepared in Procedure F using a vacuum evaporator. This step will take 10-15 min. Extended evaporation after dryness will lose shorter-chain FAMEs and therefore should be avoided; this is significant for FAMEs shorter than C16. 2. Set the helium carrier gas to a linear velocity of 45 cm/s.
3. Analyze the standards (std1 to std4, and the DTA/DPA FAME mix, Recipes 6-10) by injecting a 2-µl volume at a split ratio of 1:25 onto the FAMEWAX column. The oven-temperature program summarized in Table 1 will give good separation (Figure 2).
Note: The cis and trans FAME isomers are combined throughout data processing for C18:1n-9 and C18:2n-6 as they are not resolved in this protocol. This is practical for mammalian tissue samples that contain only cis FA isomers.
4. Analyze unknown samples with the same GC method (Figure 3). The split ratio and injection volume can be adjusted according to the sample concentration. 9 www.bio-protocol.org/e3613

Data analysis
1. In the Shimadzu GC Solution software, prepare a compound table for quantification using a linear calibration curve (Curve Fit Type: 'Linear'), by filling the retention times and calibration levels of the 27 target FAMEs (24 FAMEs are from the Supelco 37-component FAME mix, and three additional FAMEs are from the DTA/DPA FAME mix) ( Figure 4A, red box). Retention times can be filled according to the peak results obtained during standard analyses. Calibration levels (concentrations) can be set as shown in Table 2.  and **C18:2n-6 FAMEs are expressed as sums of cis and trans isomers, as these isomers are not separated by GC (see Figure 2). 2. Process the std1 to std4 data files to generate linear calibration curves with the 'force origin' option, which forces the Y-intercept to be zero. Check that linear responses are observed for the 24 FAMEs ( Figure 4A, blue box).

FAME
3. Change 'Curve Fit Type' from 'Linear' to 'Manual RF (Linear)' in the GC Solution software ( Figure   4B). This allows the user to modify slope (response factor, RF) values manually. Copy the RF value of C23:0 FAME to those for the DTA/DPA FAMEs to calibrate them as C23:0 FAMEequivalents ( Figure 4B, arrow). This approximation is practical and valid due to the  4. Process data files for unknown samples using the prepared calibration settings. 5. Check that all peaks are correctly detected; if not, correct either manually or by modifying the peak-detection parameters. 6. Copy-and-paste the raw quantification data from GC Solution into an MS Excel worksheet and calculate the absolute amounts of FA in the original sample ( Table 3). As the raw quantification data are the FAME concentrations in the GC vials, the absolute amounts of the FAMEs in the sample can be obtained by proportional calculations, using the known amount (i.e., 10 µg) of the C23:0 FA spiked into the samples: Amount of FAME X (µg) = Amount of C23: 0 FA (µg) Conc. of C23: 0 FAME (µg/ml) × Molecular weight of C23: 0 FAME Molecular weight of C23: 0 FA × Conc. of FAME X (µg/ml) Amount of FAME X (µg) = 10 Conc. of C23: 0 FAME (µg/ml) × 368.637 354.610 × Conc. of FAME X (µg/ml) The FAME amounts can then be converted into FA amounts using the molecular weights of the  This table uses the quantification results for E18.5 mouse small intestine as an example. Raw quantification data from the GC Solution software, showing FAME concentrations in the GC vial (µg/ml), can be converted into FAME amounts (µg) by proportional calculations, which adjust the amount of C23:0 FAME in the sample to 10.396 µg (equivalent to 10 µg of C23:0 FA). FAME amounts can be further converted into FA amounts using molecular-weight ratios. Values shown in red are constants that are not affected by the various samples. When a part of a sample is subjected to FAME derivatization, further proportional calculations are necessary to determine the amounts of FA in the initial sample.  7. Perform proportional calculations to determine the amounts of FA in the initial sample. The amounts should be multiplied by 2.5 (= 1.0/0.4) for samples prepared using Procedure C (in which 0.4 ml from the total 1.0 ml was analyzed).

Notes
With the exception of pipette tips, do not use plasticware (tubes and containers) for organic solvents, as the quantification of several FAMEs will fail due to components leaching from the plasticware.
For the same reason, do not transfer reagents from the Fatty Acid Methylation Kit into plastic containers before use. To volumetrically measure organic solvents accurately, pipettes should be pre-wetted with the solvent until no further drips are observed. This takes more than 10 aspiration/ejection cycles.
For safety, organic solvents should be used in a fume hood or a draught chamber. Wear lab coats and use protective glasses and gloves. a. Label four empty 0.5-ml microtubes as 'std1', 'std2', 'std3', and 'std4'.
b. Place 30 µl of the Supelco 37-component FAME mix in the std1 tube. Dispense 30 µl of dichloromethane into the std2, std3, and std4 tubes. Use a pre-wetted pipette for accuracy. c. Transfer 10 µl of std1 into std2 with a pre-wetted pipette and vortex (1/4 diluted).
e. Transfer std1 to std4 to glass GC vial inserts. Label the GC vials as 'std1' to 'std4'. The concentrations of the FAME standards in each vial are listed in Table 2.
Note: Do not store the std1 to std4 tubes for extended times as they will become contaminated with plasticware components.
b. Dilute the ~10 mg/ml stock with dichloromethane to prepare a ~1 mg/ml solution.
c. Store at -30 °C 9. DPA (C22:5n-3) FAME (~1 mg/ml stock solution) a. Evaporate ~100 µl of a 10-mg/ml solution of C22:5n-3 FAME in heptane to dryness under a gentle stream of nitrogen gas and reconstitute with 1 ml of dichloromethane b. Store at -30 °C 10. DTA and DPA FAME mix a. Mix equal volumes (100 µl each) of the C22:4n-6, C22:5n-6, and C22:5n-6 FAME solutions in a 0.5-ml microtube b. Store at -30 °C Note: The exact concentrations in this vial are not critical, as the solution is used only to determine retention times.