Fatty acid methylation kit

Arabidopsis seedlings (∼150 mg FW) at 10 DAG was used for the analysis of fatty acids, most of which are components of membrane lipids (mainly phospholipids and glycolipids) (Ohlrogge & Browse, 1995 (link)). The fatty acids were methylated and extracted using fatty acid methylation kit (Nacalai Tesque) following the manufacturer’s instructions. The fatty acid compositions were determined using an Agilent 6890 gas chromatograph (Agilent Technologies) equipped with a DB-23 column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies). Nonadecanoic methyl ester (C19:0) was used as the internal standard.

The fatty acid compositions present in the aorta and sera of ApoEKO and ApoEST2DKO mice were quantified utilizing gas chromatography–mass spectrometry (GC-MS), employing an Agilent 7890B/7000D instrument (Agilent Technologies, Santa Clara, CA, USA). A total of 10 mg of aortic tissue and 50 μL of serum were subjected to methylation using a fatty acid methylation kit (Nacalai Tesque Inc., Kyoto, Japan). The resultant product was then introduced onto a Varian capillary column (DB-FATWAX UI, Agilent Technologies, Santa Clara, CA, USA).
For fatty acid separation, the CP-Sil 88 FAME capillary column was employed (with dimensions of 100 m × an inner diameter of 0.25 mm × membrane thickness of 0.20 μm; Agilent Technologies). The temperature of the column was initially set at 100 °C for a duration of 4 min, followed by a gradual increase of 3 °C per minute until reaching 240 °C, where it was maintained for 7 min. Sample injection was performed in split mode with a split ratio of 5:1. Each fatty acid methyl ester was then detected in the selected ion-monitoring mode. All obtained results were subsequently standardized against the peak height of the C17:0 internal standard [23 (link)].

Plasma fatty acid compositions were analyzed using the modified method reported by Monguchi et al. [36 (link)]. Briefly, total fatty acid extraction from 25 µL of plasma and methyl-ester derivatization were performed using the Fatty Acid Methylation kit (Nacalai Tesque, Inc., Kyoto, Japan) per the manufacturer’s instructions. Methyl-ester-derivatized fatty acids were reconstituted with 200 µL of hexane for subsequent analysis.
To analyze the hepatic fatty acid composition, liver samples (200 mg) were homogenized with 1 mL of 50 mM sodium acetate, then extracted using the chloroform-methanol mixture described above. Subsequently, aliquots of the extracts (20 mg liver equivalent) were methyl-ester-derivatized and reconstituted with 200 µL of hexane.
Fatty acids were analyzed using a gas chromatography–flame ionization detector (GC-2010, Shimadzu Co., Kyoto, Japan). The SUPELCOWAXTM 10 capillary column (30 m length × 0.32 mm inner diameter × 0.25-μm film thickness; Sigma-Aldrich, St. Louis, MO, USA) was used to separate the fatty acids. The column oven temperature was elevated from 170 °C to 225 °C, and the separated fatty acid methyl ester was detected using the flame ionization detector. The standard mixture of methyl ester fatty acids (Supelco 37 Component FAME Mix) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

The composition of fatty acids in murine adipose tissue was assessed using GC/MS, Agilent 7890B/7000D (Agilent Technologies, Santa Clara, CA). Briefly, the adipose tissue sample (15 mg) was methylated using the fatty acid methylation kit (Nacalai Tesque, Kyoto, Japan). The samples were loaded onto a capillary column, Vf-5 ms (30 m × 0.25 mm [inner diameter] × 0.25 μm [membrane thickness]; Agilent Technologies). The column temperature was maintained at 80 °C for 3 min, then increased gradually by 25 °C/min to 190 °C, by 3 °C/min to 220 °C and by 15 °C/min to 310 °C, which was held there for 4.6 min. The sample was injected in the split mode with a split ratio of 5:1. Each fatty acid methyl ester was detected in the selected ion monitoring mode. All results were normalized to the peak height of the C17:0 internal standard.

Fecal lipids were extracted as described by Folch and Lees [28 (link)]. Methyl esterification of fatty acids (palmitic acid, stearic acid, oleic acid, and linoleic acid) was carried out using a Fatty Acid Methylation Kit (Nacalai Tesque, Kyoto, Japan), and fatty acids were analyzed on an Agilent 7890 GC-MS with CP-Sil 88 for FAME column (Agilent, Palo Alto, CA, USA). The initial oven temperature of 100 °C was increased to 240 °C (3 °C/min) and maintained for 15 min. One microliter of sample was injected into the GC/MS system. Fecal total bile acids were also determined using a Total Bile Acids Test Wako Kit (Fujifilm Wako Pure Chemical Corporation) after ethanol extraction.

Since serum fatty acid composition is greatly influenced by the most recent meal [18 (link),19 (link)], blood samples of the participants were collected in the morning after an overnight fasting in a tube containing disodium ethylenediaminetetraacetate. First, fatty acids in the serum lipids, including glycerides, phospholipids, free fatty acids, and cholesterol esters, were methylated using a fatty acid methylation kit (Nacalai Tesque, Kyoto, Japan). Gas chromatography–mass spectrometry (GC-MS) (5975C inert XL MSD; Agilent) was used for measuring the concentrations of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), palmitoleic acid (C16:1 omega-7), oleic acid (C18:1 omega-9), linoleic acid (C18:2 omega-6), alpha- and gamma-linolenic acid (C18:3), arachidonic acid (C20:4 omega-6), eicosapentaenoic acid (C20:5 omega-3), and docosahexaenoic acid (C22:6 omega-3). The instrument was equipped with a capillary column (0.25 µm × 30 m × 0.25 mm, VF-WAXms; Agilent). Nonadecanoic acid (C19:0) (Sigma-Aldrich, St. Louis, MO, USA) was used as the internal standard for the following reasons: (1) it is chemically stable, (2) it elutes close to the fatty acids analyzed in this study, (3) it separates almost completely from all components in the sample, and (4) it is virtually absent in human blood [20 (link)].

For fatty acid analysis by GC-FID, lipid samples were extracted from tissues by the method of Bligh and Dyer. C23:0 (Supelco n-Tricosanoic acid, Sigma–Aldrich) was added to the extracted samples as an internal standard. Then, the fatty acids were methylated with the Fatty Acid Methylation Kit (Nacalai Tesque), and purified using the Fatty Acid Methyl Ester Purification Kit (Nacalai Tesque) following the manufacturer's instructions. Fatty acid methyl ester samples were characterized with GC-2010 Plus system (Shimadzu) equipped with an FID. The flow rate of carrier gas (He) was set at 45 cm/s linear velocity. The temperature of the injection unit and the detector were 240°C and 250°C, respectively. The oven temperature was initiated at 140°C, then raised to 200°C at a rate of 11°C/min, then increased to 225°C at a rate of 3°C/min, and finally elevated to 240°C at a rate of 20°C/min and held at this temperature for 5 min. The injection volume was 2 μl in the split injection mode. For separation, a capillary column (FAMEWAX, 30 m, 0.25 mm ID, 0.25 μm; Restek Corporation, Bellefonte, PA) was used. Fatty acid methyl esters were identified and quantified using a mixture of fatty acid methyl ester standards (Supelco 37 Component FAME Mix and DPA (n-3) from Sigma–Aldrich; DPA (n-6) from Nu-Chek Prep, Inc., Elysian, MN; DTA (n-6) from Cayman) for calibration.