The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Summary

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Abstract

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Introduction

Gas chromatography (GC) is a well-established technique used to identify and quantify the incorporation of fatty acids into lipid pools and cell membranes^1,2 during supplementation or physiological conditions such as obesity (and related diseases such as diabetes) or pregnancy^3-5. It is also suitable for analyzing the types and quantities of fats in foods. This is useful when characterizing experimental diets, as well as ensuring that the food industry complies with regulations. For example, GC can be used to confirm the identity and quantity of fatty acids within a product such as a dietary supplement to ensure that labeling is correct and regulations are adhered to^6,7.  Analysis of fatty acids can provide valuable insights into lipid metabolism in health and disease, the impact of dietary change, and the effect of changes in physiological state⁸. Use of GC to study samples during pregnancy has provided important information on changes in fatty acid and complex lipid homeostasis³.

In advance of the chromatographic separation, lipids are typically extracted from the sample using the solubility of lipids in solvent mixtures of chloroform and methanol. Sodium chloride is added to facilitate the separation of the mixture into aqueous and organic lipid containing phases^9,10. Complex lipid classes of interest can be separated from the total lipid extract by solid phase extraction (SPE). This separation technique elutes lipid classes based upon their polarity or binding affinity. Triacyglycerols (TAG) and cholesteryl esters (CE) are eluted first as a combined fraction, further classes, phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and non-esterified fatty acids (NEFA) are eluted by increasing the polarity of the eluting solvent. The separation of TAG from CE exploits the binding of TAG only to a fresh SPE cartridge, allowing CE to be eluted. TAG can then be eluted by increasing the polarity of the eluting solvent^9,10. This method allows multiple samples to be separated simultaneously with a higher yield than is achieved with thin layer chromatography, which means that relatively small sized samples (e.g. <100 µl plasma or serum, <100 mg tissue) can be analyzed^11,12.

GC is a well-established technique first described in the 1950s; it was suggested that the mobile phase in the then liquid-liquid systems could be replaced with vapor. It was initially used for petroleum analysis but rapidly expanded into other areas such as amino acid analysis and lipid biochemistry, which is still of major interest. Advances in GC equipment and technology such as the development of capillary columns from the previously used packed columns has led to our current techniques in which fatty acids are able to be separated more efficiently at lower temperatures resulting in GC being routinely used to identify and quantify fatty acids in a wide range of investigations¹³.

GC requires fatty acids to be derivatized in order that they may become sufficiently volatile to be eluted at reasonable temperatures without thermal decomposition. This usually involves the substitution of a functional group containing hydrogen to form esters, thioesters or amides for analysis. Methyl esters are commonly studied derivatives, which are produced by methylation. In this method the ester bonds in complex lipids are hydrolyzed to release free fatty acids, which are transmethylated to form fatty acid methyl esters (FAME). The resulting profile of FAME, determined by GC, is referred to as the fatty acid composition and may be easily compared between different experimental groups^9,10. The technique allows both the proportions of individual fatty acids and their concentrations to be measured.

In addition to the use of GC for analyzing fatty acids in nutrition studies and within the food industry, the technique can be used across a wide range of analytical fields. For example, environmental analyses using GC include measuring water contamination by insecticides and soil analyses measuring chlorobenzene content. In toxicology, GC has also been used to identify illegal substances in urine and blood samples of individuals; such a sports performance enhancers¹² and the ability to separate complex mixtures of hydrocarbons makes this technique popular in the petroleum industry for petrochemical analysis¹².

Pregnancy is associated with significant changes to the fatty acid composition of maternal tissues, specifically in the content of omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFA)³.  In the current study, we exemplify the use of GC in the measurement of fatty acids by describing its use in the analysis of the fatty acid composition of liver tissue taken from virgin and pregnant rats fed low and high fat diets with different oil sources. The experimental diets provided here were a low fat soybean oil based diet, a high-fat soybean oil-based diet (130.9 g total fat/kg total fat) or a high-fat linseed oil-based diet (130.9 g total fat/kg diet), provided for 20 days. The full nutrient and fatty acid composition of these diets have been described previously¹⁴. The soybean oil diets are rich in linoleic acid (18:2n-6) and contain some α-linolenic acid (18:3n-3) while the linseed oil diet is rich in α-linolenic acid. These high-fat diets represent different rations of linoleic to α-linolenic acids (rations of 8:1 and 1:1, respectively). The method for isolation of individual lipid classes and analysis by GC is well established and validated, and has been published previously¹⁰ but without the detailed technical description found herein.

Protocol

1. Animal Procedures

1. All animal work should be carried out in accordance with the Home Office Animals (Scientific Procedures) Act (1986).
2. Mate Wistar rats aged 10 weeks old by monogamous breeding, and confirm pregnancy by the appearance of a vaginal plug. Record this as day 1 of gestation, and commence experimental diet. For virgin females, house each rat individually, and commence experimental diet.
3. After feeding the experimental diets for 20 days euthanize rats by CO₂ asphyxiation followed by cervical dislocation.
4. Use dissection forceps and scissors to expose the abdominal cavity and excise the liver by cutting the ligaments, which connect the liver to the diaphragm, anterior wall of the abdomen, stomach and duodenum. Wash the liver in PBS and freeze in liquid nitrogen before storing at -80 °C.

2. Preparation of a Total Lipid Extract⁹

1. Add molecular sieves (to fill 1/10 of solvent container) to all solvents to create ‘dry’ solvents. Carry out all solvent work within a fume hood.
2. Cut approximately 100 mg frozen liver and weigh. Place the tissue into a tube in an ice bucket and add 0.8 ml ice cold 0.9% NaCl. Homogenize the tissue.
3. Add internal standards dissolved in 1 ml/mg of dry chloroform: methanol (2:1, v/v) containing butylated hydroxytoluene (BHT; 50 mg/l) as anti-oxidant. For 100 mg of rat liver add 100 μg of CE standard (Cholesteryl heptadecanoate 17:0). Caution: Chloroform and BHT are hazardous.
4. Add 5.0 ml dry chloroform: methanol (2:1, v/v) containing BHT (50 mg/L).
5. Add 1.0 ml 1 M NaCl, mix thoroughly by vortexing until mixture looks uniform. Samples can be capped and stored at -20 °C at this stage for up to a week.
6. Centrifuge at 1,000 x g for 10 min, low brake at room temperature.
7. Collect lower phase using glass Pasteur pipette, transfer to new screw cap glass tube and dry under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week.

3. Separation of Lipid Classes by Solid Phase Extraction (SPE)¹⁰

1. Connect the SPE tank to a vacuum pump and place aminopropyl silica SPE cartridge on the tank.
2. Place new screw-cap glass tube labeled TAG and CE in the tank rack under the column to collect first fraction.
3. Dissolve the total lipid extract in 1.0 ml dry chloroform and vortex.
4. Apply sample to the column using a glass Pasteur pipette and allow to drip through into the screw-cap tube under gravity. When no further drips fall, remove the remaining liquid by vacuum.
5. Elute the TAG and CE fraction under vacuum, wash the column with 2 x 1.0 ml washes of dry chloroform.
6. When all liquid is removed, dry the TAG and CE fraction under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week.
7. Place new screw-cap glass tube labeled PC into the tank tray under the column.
8. Elute the PC fraction under vacuum with the addition of 2 x 1.0 ml dry chloroform: methanol (60:40, v/v) until all liquid is removed from the column.
9. Remove and dry PC fraction under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week.
10. Place a new screw-cap glass tube labeled PE into the tank tray and elute PE fraction with the addition of 1.0 ml dry methanol under vacuum.
11. Remove and dry PE fraction under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week.
12. Place new screw-cap glass tube labeled NEFA into the tank tray and elute NEFA fraction under vacuum by the addition of 2 x 1.0 ml washes of dry chloroform: methanol: glacial acetic acid (100:2:2, v/v/v). Caution: Glacial acetic acid is hazardous. 
13. Remove collected NEFA fraction and dry under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week.
14. Place a new aminopropyl silica SPE cartridge on the SPE tank and place a screw-cap glass tube in the tank tray under the cartridge to collect waste.
15. Wash the column with 3 washes of dry hexane under vacuum and then a final 1.0ml wash under gravity. Do not allow the cartridge to become dry (turn the cartridge column channels to a closed position when hexane level is close to the cartridge matrix). Caution: Hexane is hazardous.
16. Replace waste tube with new screw-cap glass tube labeled CE.
17. Dissolve the dried TAG and CE fraction (prepared in step 3.6) in 1.0 ml of dry hexane and vortex. Apply this to the column using a glass Pasteur pipette and allow to drip through under gravity.
18. When no further drips fall, remove the remaining liquid under vacuum.
19. Under vacuum, wash the column with 2 x 1.0 ml washes of dry hexane to elute CE and dry collected fraction under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week.
20. Place new screw-cap glass tube labeled TAG in the tank tray and elute TAG with the addition of 2 x 1.0 ml washes of dry hexane: methanol: ethyl acetate (100:5:5) under vacuum.
21. Dry collected fraction under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week. Caution: Ethyl acetate is hazardous.

4. Preparation of FAME from CE¹⁰

1. Add 0.5 ml of dry toluene to the separated CE fraction (collected in step 3.19) and vortex. Caution: Toluene is hazardous.
2. Prepare methylation reagent (dry methanol with 2% (v/v) H₂SO₄), of which 1.0 ml is required per sample. Dispense volume of dry methanol into glass or suitable plastic container with lid and add the required amount of H₂SO₄ dropwise then mix by inversion. Caution: Sulfuric acid is hazardous.
3. Add 1.0 ml of the methylating reagent to the samples dissolved in dry toluene, cap the tubes securely, and mix gently.
4. Heat the samples for 2 hr at 50 °C.
5. After 2 hr remove tubes from heat. Once cool add 1.0 ml neutralizing solution (0.25 M KHCO₃ 0.5M K₂CO₃). Caution: Potassium bicarbonate and potassium carbonate are hazardous.
6. Add 1.0 ml dry hexane and vortex.
7. Centrifuge at 250 x g for 2 min, low brake at room temperature.
8. Collect upper phase, which contains the FAME, and transfer into a new non screw-cap disposable glass tube.
9. Dry the collected FAME under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week.

5. Removal of Free Cholesterol Contamination from CE FAME¹⁴

(Free Cholesterol can contaminate the sample; see Figure 1 for example chromatograph traces with and without free cholesterol removal).

1. Place a waste tube into SPE tank and place a silica gel SPE cartridge onto the tank.
2. Wash column with 3 x 1 ml washes of dry hexane under vacuum and 1 x 1 ml under gravity.
3. Remove waste washes and add new waste tube into tank.
4. Dissolve CE FAME in 1 ml dry hexane, vortex.
5. Apply to the column using a glass Pasteur pipette and allow to drip through under gravity.
6. Wash column with 3 x 1 ml washes of hexane under vacuum.
7. Remove waste washes and place new non screw cap tube into tank labeled CE FAME.
8. Elute the CE FAME with 2 x 1 ml dry hexane: diethyl ether (95:5 v/v) washes.
9. Dry under nitrogen at 40 °C. Samples can be capped and stored at -20 °C at this stage for up to a week. Caution: Diethyl ether is hazardous.

6. Transfer of FAME into GC Auto Sampler Vial

1. Add 75 μl dry hexane to sample, vortex, and transfer to a GC auto sample vial.
2. Add a further 75 μl dry hexane to sample, vortex and transfer to the same GC auto sample vial. Samples can be capped and stored at -20 °C at this stage for up to a month.

7. Analysis Using the Gas Chromatograph¹⁴

1. Analyze FAME on a gas chromatograph. Example set up: 30 m x 0.25 μm x 0.25 mm BPX-70 fused silica capillary column with temperature protocol:
   Initial temperature 115 °C, hold 2 min, ramp 10 °C/min to 200 °C, hold 18.5 min, ramp 60 °C/min to 245 °C, hold 4 min.
   Column: Helium gas, flow rate 1.0, pressure 14.6 and velocity 29.
   Injector: Temperature = 300 °C.
   Detector: Hydrogen flow 40.0, air flow 184.0, make up gas Helium, flow 45.0, temperature = 300 °C.
2. Set split ratio as appropriate (e.g. 25:1 for CE FAME analysis).
3. Determine the area under each peak using appropriate software and identify FAME by comparison with standards. See Figure 2 for example chromatograms.
4. Use the area under the peak data to calculate the contribution of individual fatty acids as a percentage of total fatty acids.
5. Calculate absolute concentrations of fatty acids by dividing the area of internal standard by the amount added. Divide the area of each fatty acid by this result to obtain absolute concentrations of each fatty acid within the amount of tissue used.

Results

The success of this method is dependent on following the protocol precisely and on using clean solvents and reagents in order to reduce ‘noise’ and contamination that can appear on a chromatogram. Contaminated samples are more challenging to analyze, lowering the accuracy of the area under the curve calculations. If the protocol is followed successfully a chromatogram with clear symmetrical, well defined peaks and with minimal background noise should be obtained as illustrated in Figure 3. If...

Discussion

Gas chromatography is an accurate technique to use for fatty acid analysis, and its high reproducibility deems this technique suitable for clinical analyses. Appropriate GC columns must be used to enable identification of fatty acids of interest, with available columns having variations in the polarity of the stationary phase, column length and internal diameter. The use of a fused silica capillary column in this method of analysis provides good thermal stability and high reproducibility of retention times due to it...

Materials

Name	Company	Catalog Number	Comments
Methanol	Fisher Scientific	M/4056/17	'CAUTION' Fumes - HPLC Grade
Chloroform	Fisher Scientific	C/4966/17	'CAUTION' Fumes - HPLC Grade
BHT	Sigma- Aldrich	W218405	'CAUTION' Dust fumes - Anhydrous
NaCl	Sigma- Aldrich	S9888	Anhydrous
Hexane	Fisher Scientific	H/0406/17	'CAUTION' Fumes - HPLC Grade
Glacial acetic acid	Sigma- Aldrich	695084	'CAUTION' Burns - 99.85%
Sulfuric acid	Sigma- Aldrich	339741	'CAUTION' Burns - 99.999%
Potassium carbonate	Sigma- Aldrich	209619	99% ACS Reagent grade
Potassium bicarbonate	Sigma- Aldrich	237205	99.7% ACS Reasgent grade
Ethyl acetate	Fisher Scientific	10204340	'CAUTION' Fumes - 99+% GLC SpeciFied
Toluene	Fisher Scientific	T/2300/15	'CAUTION' Fumes
Diethyl ether	Sigma- Aldrich	309966	'CAUTION' Fumes
Nitrogen (oxygen free) cylinder	BOC	44-w	'CAUTION' Compressed gas - explosion risk
Aminopropyl silica SPE cartridges	Agilent	12102014	Cartridge - Bead mass 100 mg
Silica gel SPE cartidges	Agilent	14102010	Cartridge - Bead mass 100 mg
Molecular seives	Sigma- Aldrich	334324	Pellets, AW-300, 1.6 mm
Glass Pasteur pipettes	Fisher Scientific	FB50251