Tricosanoic acid

DHA-95E, an ethyl all cis-4,7,10,13,16,19-docosahexaenoic acid, was obtained from Harima Chemicals (Tokyo, Japan). Tricosanoic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid and other fatty acids, phytic acid, bipyridine were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other materials of the highest grade were obtained commercially.

The following fatty acid and fatty acid methyl ester standards were purchased from Sigma Aldrich (Gillingham, Dorset, UK): tricosanoic acid (C23:0), behenic acid (C22:0), cis-11,14-eicosadienoic acid (C20:2 Δ11,14), heptadecanoic acid (C17:0), cis-10-pentadecanoic acid (C15:1Δ10), pentadecanoic acid methyl ester (C15:0) (PDA), palmitic acid methyl ester (C16:0), palmitoleic acid methyl ester (C16:1Δ9), cis-10-heptadecanoic acid methyl ester (C17:1Δ10), stearic acid methyl ester (C18:0), elaidic acid methyl ester (t-C18:1Δ9), oleic acid methyl ester (C18:1Δ9), linolelaidic acid methyl ester (t-C18:2 Δ9, 12) linoleic acid methyl ester (C18:2 Δ9,12), arachidic acid methyl ester (C20:0), linolenic acid methyl ester (C18:3 Δ9,12,15), heneicosanoic acid methyl ester (C21:0), all-cis-11,14,17-eicosatrienoic acid methyl ester (C20:3 Δ11,14,17), arachidonic acid methyl ester (C20:4 Δ5,8,11,14), all-cis-13,16-docosadienoic acid methyl ester (C22:2 Δ13,16), lignoceric acid methyl ester (C24:0), nervonic acid methyl ester (C21:1 Δ15), docosahexanoic acid methyl ester (C22:6 Δ4,7,10,13,16,19) (DHA), all-cis-8,11,14-eicosaterinoic acid methyl ester (C20:3 Δ8,11,14), erucic acid methyl ester (C22:1 Δ13). All solvents used (methanol n-hexane and dichloromethane) were CHROMASOLV HPLC grade (Sigma Aldrich, UK). Hydrogen chloride (1.25 M) in methanol was purchased from Fluka Analytical (UK).

The method described by Lewis, Nichols (33) was used for the direct transesterification of fungal biomass and as transesterification method prior to GC analysis of the Folch and Bligh extracts. Biomass or dried extracts were dissolved in 2 mL methanol/chloroform/HCl (10:1:1, v/v). Biomass was homogenized with a glass rod. The internal standards, tridecanoic acid (C13:0, Sigma Aldrich, USA) and tricosanoic acid (C23:0, Sigma Aldrich, USA) were dissolved in hexane and added to a final amount of 1.0 and 0.7 mg per sample, respectively. Another 1 mL of methanol/chloroform/HCl (10:1:1, v/v) was added to the extraction tube. The sample was vortexed for 30 sec and incubated at 90°C for 1 hour. Samples were cooled down and 1mL water was added. FAMEs were extracted with 2 x 2mL hexane/chloroform (4:1, v/v) after phase separation by centrifugation (2800 g, 10min). The extract was collected in a pre-weighted tube and the solvent was evaporated under a steam of nitrogen. FAMEs were dissolved in 1 mL hexane with 0.01% butylated hydroxytoluene (BHT, Sigma-Aldrich, USA) and subsequently transferred into a GC vial, which was stored at -20°C until analysis.