Heneicosanoic acid

Total FAs were analyzed in food, blood, and gut tissues of experimental mice. Samples (20 μL or 20 mg) were extracted using the method of Bligh and Dyer [11 (link)], and a known amount of pentadecanoic acid (Sigma-Aldrich) and heneicosanoic acid (Sigma-Aldrich) was added as internal standard. After the extraction, the organic layers were combined and evaporated under a stream of nitrogen at 30 °C then dissolved in 1 mL of ethanol. Sample preparation was based on the method of Metcalfe et al. [12 ] as described previously [13 (link)]. Samples in ethanol were treated with 0.1 mL of 50% aqueous KOH, tubes were purged with nitrogen, and then heated to 75 °C for 1 h. After cooling, nonsaponifiable lipids were removed by extraction with 3 mL of hexane. The aqueous layer was acidified and extracted with 3 mL of hexane, and the hexane layer was removed and dried under a stream of nitrogen. The residue was treated with 0.5 M methanolic NaOH at 100 °C for 5 min. After cooling, samples were treated with 1 mL of 14% BF₃ in methanol (Sigma-Aldrich), flushed with Ar, and then heated to 100 °C for 5 min. After cooling, samples were treated with 4 mL of hexane and 4 mL of saturated, aqueous NaCl, and the hexane layer was transferred to a new tube, dried under a stream of nitrogen, then dissolved with hexane (200 μL for blood samples or 1000 μL for tissue samples).

Samples for fatty acid methyl ester (FAME) analyses were collected when lipid accumulation reached its peak, normally after 3–4 days of nutrient starvation. A total of 4 mL of microalgal culture was collected and centrifuged at 8,000 × g for 5 min. Biomass was collected and freeze-dried for 30 min. Lipids in the microalgal pellet were hydrolyzed and methyl-esterified in 300 μL of a 2% H₂SO₄ in methanol solution for 2 h at 80°C. Prior to the reaction, 50 μg of heneicosanoic acid provided by Sigma, USA was added as internal standard. After the esterification step, 300 μL of 0.9% (w/v) NaCl solution and 300 μL of hexane were added and mixed for 20 s. To separate the phase, samples were then centrifuged at 16,000 × g for 3 min. A total of 1 μL of the hexane layer was injected into an Agilent 6890 gas chromatograph coupled to a 5975 MSD mass spectrometer. The running conditions were followed using Agilent’s RTL DBWax method as described previously (Lim et al., 2012 (link)).

All HPLC solvents were mass spectrometry grade (Carlo Erba Reagents, Italia). The internal standard for cholesterol and oxysterols quantification was cholesterol-2,2,3,4,4,6-d₆ (SIGMA-ALDRICH) for fatty acids internal standards were heneicosanoic acid (C21:0) and uniformly labeled 13C-linoleic acid (C18:2) (SIGMA-ALDRICH). Phospholipid standards: C13:0 lysophosphatidylcholines (LysoPC); C25:0 phosphatidylcholines (PC); C12:0 sphingomyelin (SM); 12:0–13:0 phosphatidylserine (PS); 12:0–13:0 phosphatidylinositol (PI); 12:0–13:0 phosphatidylglycerol (PG); 12:0–13:0 phosphatidic acid (PA); 12:0–13:0 phosphatidylethanolamine (PE); C12 ceramide (Cer); glucosyl (β) C12 ceramide (GC); lactosyl (β) C12 ceramide (LacCer); C17 mono-sulfo galactosyl(β) ceramide (D18:1/17:0; GalCer) were purchased from Avanti Polar Lipids.

Samples for FAME analysis were collected when lipid accumulation reached its peak, after 3 days of starvation. A total of 4 mL of microalgal culture was collected and centrifuged at 8000 × g for 5 min. Biomass was collected and dried by a vacuum pump for 30 min. Lipids in the microalgal pellet were hydrolyzed and methyl-esterified in 300 μL of a 2% H₂SO₄ and methanol solution for 2 h at 80°C. Prior to the reaction, 50 μg of heneicosanoic acid (Sigma, USA) was added as internal standard. After the esterification step, 300 μL of 0.9% (w/v) NaCl solution and 300 μL of hexane were added and mixed for 20 s. To separate the phase, samples were centrifuged at 16,000 × g for 3 min. A total of 1 μL of hexane layer was injected into an Agilent 6890 gas chromatograph (GC) coupled to a 5975 MSD mass spectrometer (MS). The running conditions were described by Agilent's RTL DBWax method (Brown, 1991 (link)).

Pentadecanoic acid (C_15:0), palmitic acid (C_16:0), palmitoleic acid (C_16:1), heptadecanoic acid (C_17:0), stearic acid (C_18:0), oleic acid (C_18:1), linoleicacid (C_18:2), linolenic acid (C_18:3), nonadecanoic acid (C_19:0), arachidonic acid (C_20:4), heneicosanoic acid (C_21:0), docosahexaenoic acid (C_22:6), L-α-phosphatidylinositol sodium salt (PI, PI(16:0/18:2)), 9-aminoacridine (9-AA), 1,8-bis(dimethylamino)naphthalene (DMAN), and N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate (sodium salt) (PA(18:1(9Z)/18:1(9Z))), 1,2-di-(9E-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:1(9E)/18:1(9E))), 1,2-di-(9E-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9E)/18:1(9E))) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (PG(16:0/16:0)) were purchased from Avanti Polar lipids (Alabaster, AL, USA). GO was from Nanjing JCNANO Technology Co., Ltd (Nanjing, China). HPLC-grade isopropanol and hexane were from Fisher Scientific (Pittsburg, PA, USA). Ultrapure water was purified by a Milli-Q system (Millipore, MA, USA).