Margaric acid

One hundred µl of plasma (n = 6 in each group and sex), labyrinthine area and liver samples randomly chosen from each litters, were stored at –20°C in chloroform/methanol. Lipids corresponding to 400 mg of placental and hepatic tissue were extracted with CHCl₃-MeOH [31] (link). The phospholipids were separated from the non-phosphorous lipids on silica acid cartridges. The phospholipid fraction, mostly representative of the membrane lipids, and the neutral lipid fraction, representative of the storage lipids were transmethylated with Boron trifluoride methanol 7% (Sigma-Aldrich) [32] (link). The methyl esters of phospholipid or neutral FA were analyzed by gas chromatography coupled to FID (Gas Chromatograph 3900 Varian) on an Econo-Cap EC-WAX capillary column using heptadecanoic acid (margaric acid, C17:0) as internal standard [33] (link). Results were expressed as g/l of plasma and mg/g of placenta or liver.

Lipids in the FF supernatant were extracted with methyl-tert-butyl-ether as described by Matyash et al.[27 (link)]. Lipid fractions were separated using SPE-columns [28 (link),29 (link)]. Total plasma lipid extracts were dissolved in chloroform (1.0 ml) and applied to an aminopropyl silica column (pasteur pipette containing 100 mg aminopropyl silica gel) under gravity. Cholesteryl-esters and TGs were eluted with chloroform (1.0 ml and 0.5 ml), combined, dried under N₂ and dissolved in 1.0 ml hexane. Non-esterified FAs were eluted with diethyl ether/acetic acid (100:2; 1.0 ml and 0.5 ml) and PLs with 1 ml methanol/chloroform (6:1) followed by 0.5 ml 0.05 M sodium acetate in methanol/chloroform (6:1). Cholesteryl-esters and TGs were further separated on a pre-packed 100 mg aminopropyl column (Varian). The CHE and TG fractions were loaded in 1 ml hexane and the CHE fraction was eluted with hexane (1.0 ml and 0.5 ml). Triglycerides were eluted with hexane/chloroform/ethyl acetate (100:5:5; 1.0 ml and 0.5 ml). Fatty acids in lipid extracts were methylated using a basic followed by an acid methylation step. Toluene (2 ml) containing the internal standard (C13:0) and methanolic NaOH (2 ml) was added and the mixture was incubated at 70°C (60 min) followed by 30 min at 50°C after addition of methanolic HCl (3 ml), prepared by dissolving 10 ml acetyl chloride in 50 ml methanol. Fatty acid methyl esters (FAME) were extracted with hexane. Fatty acids in the TG fraction were methylated as described by Chouinard et al.[30 (link)] whereas NEFAs were methylated by an acid methylation step only. Fatty acids in PL and CHE were methylated using a basic followed by an acid methylation step.
Composition analyses of the FAs were conducted using a gas chromatograph (HP 7890A, Agilent Technologies, Diegem, Belgium) equipped with a 75-m SP-2560™ capillary column (i.d., 0.18 mm, film thickness, 0.14 μm; Supelco Analytical, Bellefonte, PA) and a flame ionization detector. The oven temperature program was 70°C before being raised to 175°C (50°C/min) for 13 min, after which it was raised to 215°C (5°C/min) for 25 min. The inlet temperature was 250°C and the detector temperature 255°C. Various control materials were used: BR2/BR3, (Larodan Fine Chemicals, Malmö, Sweden), Supelco® 37 (Supelco Analytical, Bellefonte, PA), PUFA-3 (Matreya LLC, Pleasant Gap, PA) and GLC463 (NU-CHEK-PREP Inc., Elysian, MN, USA). Fatty acids for which no standards were commercially available were identified by order of elution according to Precht et al.[31 (link)] and Kramer et al.[32 (link)].
The FA analysis generated data on the concentrations of lauric acid (12:0), myristic acid (14:0), pentadecaenoic acid (15:0), palmitic acid (16:0), palmitoleic acid n-7 (16:1 n-7), palmitoleic acid n-9 (16:1 n-9), margaric acid (17:0), stearic acid (18:0), oleic acid (18:1 n-9), vaccenic acid n-11 (18:1 n-11), linoleic acid (18:2 n-6), arachidic acid (20:0), gamma-linolenic acid (18:3 n-6), alfa-linolenic acid (18:3 n-3), eicosenoic acid (20:1), eicosadienoic acid (20:2), di-homo-gamma-linolenic acid (20:3 n-6), arachidonic acid (20:4 n-6), eicosapentaenoic acid (20:5 n-3), docosatetraenoic acid (22:4 n-6), docosapentaenoic acid n-6 (22:5 n-6), docosapentaenoic acid n-3 (22:5 n-3), docosahexaenoic acid (22:6 n-3). For each FA measurement, both the absolute (μmol/l) and the relative concentration (mol%) was determined. Fatty acids were attributed to the PL, TG, CHE or NEFA fraction.

Individual FAs within the same class (saturated FAs, monounsaturated FAs [MUFAs], polyunsaturated FAs [PUFAs], n-3, n-6, n-7, and n-9) were summed as follows to quantify total FA levels: total saturated FAs: lauric acid + myristic acid + pentadecanoic acid + palmitic acid + margaric acid + stearic acid + arachidic acid; total MUFAs: palmitoleic acid + heptadecanoic acid + oleic acid + gadoleic acid + erucic acid + nervonic acid; total PUFAs: ALA + stearidonic acid + 11,14,17-eicosatrienoic acid (ETE) + EPA + DPA + DHA + LA + GLA + DGLA + ARA + adrenic acid + osbond acid + 5,8,11-eicosatrienoic acid; total n-3 PUFAs: ALA + stearidonic acid + ETE + EPA + DPA + DHA; total n-6 PUFAs: LA + GLA + DGLA + ARA + adrenic acid + osbond acid; total n-7 FAs: palmitoleic acid + heptadecanoic acid; total n-9 FAs: oleic acid + gadoleic acid + erucic acid + nervonic acid + 5,8,11-eicosatrienoic acid.
Ratios of product/substrate FAs were used as in vivo activity markers for the following desaturases and elongases: stearoyl-CoA desaturase-16 (SCD16): palmitoleic acid/palmitic acid [88 (link)]; stearoyl-CoA desaturase-18 (SCD18): oleic acid/stearic acid [53 (link)]; delta-6-desaturase (D6D): GLA/LA [88 (link)]; delta-5-desaturase (D5D): ARA/DGLA [88 (link)]; elongase-6 (ELOVL6): stearic acid/palmitic acid [89 (link)]; elongase-5 (ELOVL5): DGLA/GLA [89 (link)]; elongase-2 (ELOVL2): adrenic acid/ARA [90 (link)].