Flame ionization detector

Of the total cohort, we had 23 samples available for SCFA quantification (NW = 12 [GDM-NW = 5, No GDM-NW = 7; EWG-NW = 4, No EWG-NW = 8] and OW/OB = 11 [GDM-OW/OB = 6, No GDM-OW/OB = 5; EWG-OW/OB = 5, No EWG-OW/OB = 6]). Infant stool samples were weighed and diluted to equivalency with acidified water (pH 2.5) containing 1 mmol ethyl-butyric acid per liter as an internal standard. Samples were sonicated for 10 min, incubated at room temperature for 10 min, and then centrifuged at 10,000 × g for 10 min at room temperature. Supernatant was collected, re-centrifuged, and stored at −80°C until analysis, as previously (12 (link), 45 (link)). Samples were analyzed using a 6890 series gas chromatograph with flame ionization detector (Agilent). Samples were injected at a 10:1 split ratio; the inlet was held at 228°C and the transfer line was held at 230°C. Separation of SCFAs was achieved on a 30m TG-WAX-A column (0.25-mm ID, 0.25-mm film thickness; Thermo Scientific) by using a temperature program as previously (45 (link)). Acetate, propionate, and butyrate were quantified using standards of commercially purchased compounds and samples were adjusted for extraction efficiency differences by normalizing to the internal standard.

BVL or BVL-7 was methylated by adding 1:1 hexane and 14% boron trifluoride/methanol and heated at 100°C for an hour. Fatty acids methyl esters were analyzed by a fully automated 6890N Network Gas Chromatography equipped with a flame-ionization detector (Agilent Technologies, Palo Alto, CA). Individual fatty acid was determined by retention time using a reference standard, GLC461 (Nu-Chek Prep, Elysian, MN).

Fatty acid composition of serum, anterior tibialis, quadriceps, epididymal fat pad and liver was measured by gas chromatography and flame ionization detection of fatty acid methyl esters. Tissue lipids were extracted with chloroform/methanol (2:1, vol/vol) and sonication. Extracted lipids were hydrolyzed with 0.5 N methanolic NaOH, and fatty acids were methylated with 10% (w/w) methanolic boron trifluoride. The solvent was removed, and the residue was reconstituted in isooctane and analyzed on a 7890A gas chromatograph equipped with a 30 m × 0.25 mm × 0.15 mm DB-225 column and flame ionization detector (Agilent Technologies, Palo Alto, CA, USA).^{27 (link)} Sample peaks were identified by comparison with authentic standards (Nu-Chek-Prep Inc., Elysian, MN, USA). Results of fatty acid methyl ester analysis values were determined by weight percentage reports based on the retention times and peak responses of authentic standards (not detected less than 50 ng per peak).

Methyl esterification of the cis‐ and trans‐fatty acids was performed according to Zribi et al. (2014). Cis‐ and trans‐fatty acid compositions were analyzed using the Agilent 7890N GC instrument coupled with a flame ionization detector (Agilent Technologies Inc.). A nonpolar fused‐silica capillary column (HP‐88, 100 m × 0.25 mm × 0.20 μm) was used for gas chromatography (GC) analysis. Helium was used as a carrier gas at a flow rate of 1.5 ml/min. A split ratio was set as 1:30. The temperature program was as follows: Initial temperature set at 120°C was on hold for 1 min, after which it was increased to 180°C at 20°C/min; next, the speed was set as 5°C/min, and finally, the column temperature was increased to 220°C and held for 5 min. Both the detector and injector port temperatures were maintained at 260°C. The fatty acids were identified from retention time comparison versus mixed standards run under the same conditions. They were quantified according to percentage peak area ratio. The final results were expressed as the percentages of individual fatty acids.

Fatty acid profiles of mouse diets and tail and colon tissues were analyzed by gas chromatography (GC), as described previously [18 (link),97 (link)]. Briefly, tissue or food samples were ground to powder under liquid nitrogen and subjected to total lipid extraction and fatty acid methylation by 14% boron trifluoride (BF3)-methanol reagent (Sigma-Aldrich) at 100 °C for 1 h. Fatty acid methyl esters were analyzed using a fully automated HP5890 gas chromatography system equipped with a flame-ionization detector (Agilent Technologies, Palo Alto, CA, USA). The fatty acid peaks were identified by comparing their relative retention times with the mixed commercial standards (NuChek Prep, Elysian, MN, USA), and the area percentage for all resolved peaks were analyzed by using a PerkinElmer M1 integrator. The fatty acids examined as total n-6 PUFA with GC include: Linoleic acid (C18:2n6), Gamma-linolenic acid (C18:3n6), Eicosadienoic acid (C20:2n6), Dihomo-gamma-linolenic acid (C20:3n6), Arachidonic acid (C20:4n6), Docosadienoic acid (C22:2n6), Adrenic acid (C22:4n6) and Docosapentaenoic acid (22:5n6). The fatty acids examined as total n-3 PUFA with GC include: α-Linolenic acid (C18:3n3), Eicosatrienoic acid (C20:3n3), Eicosapentaenoic acid (C20:5n3), Docosapentaenoic acid (C22:5n3), and Docosahexaenoic acid (C22:6n3).

The concentrations of the short chain fatty acids (SCFAs), including acetate, propionate, butyrate, isobutyrate and n-valerate, and isovalerate in the cecal content were determined as follows. Two milliliters of supernatant were prepared by reconstituting all cecal content of each mouse in 0.01 M PBS followed by centrifugation at 9000 g for 5 min at 4 °C. The supernatant was acidified with a 1/10 volume of 50% H₂SO₄ and extracted with ethyl ether. The concentrations of SCFAs were determined in the organic phase using an Agilent 6890 N gas chromatograph (Agilent Technologies, Wilmington, DE, USA) equipped with a polar HP-FFAP capillary column (0.25 mm × 0.25 mm × 30 m) and flame ionization detector (Agilent Technologies, Wilmington, DE, USA). Helium was used as the carrier gas. The initial oven temperature was 120 °C, which was maintained for 16 min and then raised to 122 °C at 5 °C / min, increased to 250 °C at 30 °C / min, and held at this temperature for 3 min. The detector temperature was 270 °C, and the injector temperature was 260 °C. Data handling was performed with an Agilent ChemStation (version G2070AA, Agilent Technologies, Wilmington, DE, USA).