Chromatography, Gas-Liquid

Fatty acid profiles were analyzed in whole retina, liver, and plasma from Fat1⁺/TG2⁺, Fat1^–/TG2⁺, Fat1⁺/TG2^–, and Fat1^–/TG2^– mice of C57BL/6J strains. One single retina was taken for each analysis. Each plasma sample was obtained by centrifuging the heparinized blood at 2,000x g in EGTA-containing tubes to obtain approximately 100 µl plasma. For whole retina and plasma, total lipids were extracted following the method of Bligh and Dyer [30 (link)]. The tissues were extracted in chloroform:methanol:water (1:1:1) and the chloroform phase collected. The remaining aqueous phase was extracted once again with chloroform, keeping the chloroform:methanol:water ratios at 1:1:1. As before, the chloroform phase was collected and combined with the chloroform phase from the initial extraction. The combined chloroform phases were then extracted with chloroform:methanol:water (3:48:47) and the aqueous phase discarded. The remaining purified lipid extract was stored under nitrogen. For liver, total lipids were extracted following the method of Folch et al. [31 (link)]. The tissues (n=3–4 mice per group) were homogenized in 4 ml of chloroform:methanol (2:1). Proteins in the homogenate were pelleted by centrifugation at 1,000x g for 10 min and the lipid extract was removed. The pellet was washed twice with 1 ml of chloroform:methanol 1:1 and the washes were combined with the lipid extract. The lipid extract was then washed with 0.2 volumes of 1 mM DTPA(aq) followed by 0.2 volumes of chloroform:methanol:water (3:48:47), each time discarding the aqueous phase. The resulting purified lipid extract was dried under nitrogen and re-suspended in a known volume of toluene. Purified lipid extracts from plasma and liver were resolved into individual lipid classes using one-dimensional thin-layer chromatography. Briefly, an aliquot of each extract was spotted onto 10×20 cm Silica Gel 60 plates (EM Science, Gibbstown, NJ). Lipid classes were resolved hexane:etyhy ether: glacial acetic acid (70:30:2.3, by vol) acid mobile phase, and plates were stained with 0.05% (wt/vol) 2,7-dichlorofluorescein in 75% (vol/vol) methanol. Fatty acids of purified lipid extracts of whole retina and of scraped thin-layer chromatography spots from plasma and liver extracts were derivatized to form fatty acid methyl esters and analyzed using gas-liquid chromatography. The fatty acid compositions were determined by injecting 3 µl of each sample at 250 °C with a split ratio of 20:1 (10:1 for whole retina) onto a (30 m long x 0.32 mm I.D.) DB-225 capillary column (J&W Scientific, Folsom, CA) in an Agilent 6890N gas chromatograph with model 7683 autosampler (Agilent Technologies, Wilmington, DE). The column temperature was programmed to begin at 160 °C, ramped to 220 °C at 1.33 °C/min, and held at 220 °C for 18 min. Hydrogen carrier gas was allowed to flow at 1.6 ml/min, and the flame ionization detector temperature was set to 270 °C. The chromatographic peaks were integrated and processed with ChemStation® software (Agilent Technologies). FAMES were identified by comparison of their relative retention times with authentic standards (NU-CHEK PREP, Elysian, MN) and relative mole percentages were calculated.

The monosaccharide composition of BM-related samples was achieved as alditol acetates [73 (link)] by gas liquid chromatography (GLC) analysis. Briefly, 1 mg of a sample was hydrolysed by 1 mL of 2 M trifluoroacetic acid (TFA) at 120 °C for 2 h. Methanol was added to the system, followed by evaporation to dryness in order to remove TFA, and the obtained substance became a hydrolysate, followed by its reaction with a solution of 0.25 M NaBH₄ in 1 M ammonia (0.5 mL at 20 °C for 1 h). After neutralisation with 10% acetic acid in methanol, followed by evaporation, 0.5 mL of pyridine and 0.5 mL of acetic anhydride were added to the sample (kept at 100 °C for 45 min). All standard sugars (glucose, xylose, arabinose, mannose, rhamnose, fucose, galactose) were converted to their acetylated derivatives, according to the methods described above. The alditol acetates were analysed on a DB-5 capillary column (30 m × 0.32 nm, 0.25 μm) (Agilent, Santa Clara, CA, USA), by using a GC-2010 chromatograph (Shimadzu, Kyoto, Japan). The injection volume was 1 μL; the split ratio was 10:1; the carrier gas was ultra-pure nitrogen; and the temperature of the detector was 270 °C. The temperature gradient was 160 °C (1 min) to 250 °C at 7 °C·min⁻¹.
The fatty acids of the BM-related samples were determined as fatty acid methyl esters (FAMEs) [74 (link)] using a GC-2010 instrument (Shimadzu, Kyoto, Japan) equipped with a DB-5 capillary column (Agilent, Santa Clara, CA, USA). The temperature gradient was 130 °C (3 min) to 250 °C at 4 °C·min⁻¹. The retention times of the analysed peaks in the samples were compared with those of a standard Bacterial Acid Methyl Ester (BAME) mix (Sigma–Aldrich, St. Louis, MO, USA).

The treatment diets were freeze-dried before analysis and samples were sent to Nofima BioLab, Fyllingsdalen, Norway for analyses of fat, protein, dry matter, ash, fibre and fatty acid profiles as described previously [22 (link)]. In short, crude fat was analysed by the method of Bligh and Dyer [23 (link)] and fatty acids methyl esters were analysed by gas liquid chromatography with the AOCS Official Method Ce 1b-89 [24 ]. Nitrogen was analysed using the Kjeldahl method (Kjeltech Auto Analyser, Tecator, Höganäs, Sweden) and crude protein calculated as N × 6.25 [25 (link)]. Crude fibre was analysed by a modified version of ISO 5498 based on acid and alkaline hydrolysis. Dry matter was determined by drying at 105 °C until constant weight, and ash levels were obtained after flame combustion and incineration at 550 °C.