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 regul....

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 th....

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 contaminati.......

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

List of materials used in this article

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