Rt 2560 capillary column

RBC total lipids were extracted with chloroform/methanol/water (2:2:1.8; v:v:v) according to the Bligh and Dyer method. Nonadecanoic acid (C19:0; Sigma-Aldrich) was used as internal standard. Each sample was then dried under nitrogen and methylated at 70°C through a 1 h-incubation with 0.5 ml of 0.1 mol/l KOH in methanol, followed by a 15 min-incubation in 0.2 ml of 1.2 mol/l HCl in methanol. The fatty acid methyl esters (FAME) were then extracted by 1 ml hexane and separated by gaz chromatography (Schneider et al., 2012 (link)). The chromatograph (GC Trace-1310, Thermo Quest, Italy) was equipped with a RT2560 capillary column (100 m × 0.25 mm internal diameter, 0.2 μm film thickness; Restek) and a flame ionization detector (FID, Thermo Quest). The carrier gas used was H₂ at constant pressure (200 kPa). The FID was continuously flowed by H₂ (35 ml/min) and air (350 ml/min) and kept at a constant temperature of 255°C. The temperature program was as follows: an initial temperature of 80°C, which increased at 25°C/min up to 175°C, a holding temperature of 175°C during 25 min, a new increase at 10°C/min up to 205°C, a holding temperature of 205°C during 4 min, a new increase at 10°C/min up to 215°C, a holding temperature of 215°C during 25 min, a last increase at 10°C/min up to 235°C and a final holding temperature of 235°C during 10 min (Ferain et al., 2016 (link)).

The method used was adapted from Mellery et al. [41 (link)]. DHAO sample (200 mg) was weighed and converted to fatty acid methyl esters (FAME) via a two-step methylation with nonadecanoic acid (C19: 0, Sigma-Aldrich) used as internal standard. First, the oil was methanolysed with 10 mL of 0.1 M KOH in methanol at 70 °C for 60 min, then cooled to room temperature. Second, an acid-catalysed methanolysis was carried out by adding 4 mL of 1.2 M HCl in methanol and incubating at 70 °C for 20 min. The FAME were then extracted by addition of hexane and Milli-Q water. The FAME were separated and quantified by running samples in a GC Trace 1310 (ThermoQuest, Milan, Italy) gas chromatograph equipped with an As Triplus autosampler (Thermo Electron, Milan, Italy), an RT2560 capillary column of 100 m × 0.25 mm internal diameter (Restek, Bellefonte, PA, USA) and a flame ionisation detector set at 255 °C. Fatty acids in the samples were identified and quantified by comparison of retention time and peak area with those of pure FAME standards (Larodan, Solna, Sweden). The data were processed using the Chromquest 5.0 data processing software (ThermoFisher Scientific, Waltham, MA, USA) and results are expressed as mg/g of oil.

For IMF analysis, the tissue samples of the LT muscle (30 g) were freeze-dried and pulverized. Thereafter, an examination of the IMF content of LT muscle tissue samples was conducted using the ether extraction method according to the Association of Official Analytical Chemists regulations [34 ]. The expression of IMF content was reported as g of lipid in 100 g of muscle tissue. After the extraction of lipids, they were converted into FA methyl esters (FAMEs) using the method described in a previously published study [5 (link)]. The FA composition was evaluated with the method established in our previous study [15 (link)] by using a gas chromatography-flame ionization detector (GC-FID, SCION 456-GC, Bruker Daltonics, Fremont, CA, USA). Separation was performed on an RT-2560 capillary column (RESTEK, Bellefonte, PA, USA). The FAMEs were identified and quantified with a 37-component standard FAME Mix (RESTEK). Individually, FA composition values were reported as g per 100 g of total FAs. Subsequently, sums of SFAs (C12:0 + C14:0 + C16:0 + C18:0 + C20:0), MUFAs (C16:1n-7 + C18:1n-9 + C20:1n-9), and n-6 polyunsaturated FAs (PUFAs) (C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6) were estimated. Lastly, ratios of MUFAs to SFAs, MUFAs to n-6 PUFAs, and n-6 PUFAs to SFAs were then calculated.