FISHERIES SCIENCE 2002; 68: 236–238 Short Paper Fatty acid composition of two species of Japanese freshwater sponges Heterorotula multidentata and Spongilla alba Noriko U SATA,1a Masaki KANENIWA,1* Yoshiki MASUDA,2 Yasuhiro ANDO3 AND Haruka IIDA4 1Marine Biochemistry Division, National Research Institute of Fisheries Science, Yokohama, Kanagawa 236-8648, 2Department of Biology, Kawasaki Medical School, Kurashiki, Okayama 701-0192, 3Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041-8611 and 4Coastal Fisheries and Aquaculture Division, National Research Institute of Fisheries Science, Yokosuka, Kanagawa 238-0316, Japan KEY WORDS: demospongic acids, fatty acid, Japanese freshwater sponges. Demospongic fatty acids are distinctive fatty acids acetals. The FAME were separated using prepara- contained in the lipids of marine and freshwater tive thin-layer chromatography (PTLC) on silica gel sponges.1–10 These fatty acids have a long hydro- 60G plates (Merck, Darmstadt, Germany) with a carbon chain with 5,9-diene and often have a mixture of n-hexane and diethyl ether (85 : 5 v/v), further double bond in the terminal part of the whereas dimethyl acetals were separated with molecule. Many studies have been done on demo- toluene.13 spongic acids in marine sponges.1–7 Recently, the Gas liquid chromatography (GLC) analysis of presence of three demospongic acids (5,9–26 : 2, the FAME was performed using a Shimadzu GC14A 5,9,19–26 : 3, and 5,9,20–27 : 3) in the Japanese (Shimadzu Seisakusho Co., Kyoto, Japan), with a marine sponge Halichondria panicea were identi- flame ionization detector on a fused-silica capil- fied.11 Only a few investigations of demospongic lary column coated with Omegawax 320 (30 m ¥ acids in freshwater sponges have been reported,8–10 0.32 mm i.d.). The carrier gas was helium. The and no study has dealt with Japanese freshwater column temperatures were programmed from sponges. In the present report, we present the 180∞C to 240∞C (2∞C/min), and the injector results of a detailed study of the fatty acids present and detector temperature was 260∞C. Peak area in two species of Japanese freshwater sponges, percentages were obtained with a Shimadzu inte- Heterorotula multidentata and Spongilla alba. grator C-R6A (Shimadzu Seisakusho Co.). Compo- The freshwater sponge H. multidentata was nents of each peak on the gas chromatogram were collected from the Doro River, Nagano prefecture, identified by comparison of the retention time Japan, and S. alba was collected from Lake Shinji, with authentic specimens (Supelco, Bellefonte, Shimane prefecture, Japan. Total lipids (TL) were PA, USA) and from FAME prepared from the lipids extracted from the sponge colonies according to of the marine sponge H. panicea.11 A gas chro- the procedure of Bligh and Dyer.12 The lipid con- matogram of FAME from the sponge H. multiden- tents of the two species of sponges examined were tata is shown in Fig. 1. Five peaks (A–E in Fig. 1) due 0.6% for H. multidentata and 0.5% for S. alba. To to the presence of very long chain fatty acids were determine the composition of fatty acids in TL, detected, which could not be identified by authen- treatment with 5% HCl–methanol of TL afforded tic specimens. Two peaks, D and E, were identified fatty acid methyl esters (FAME), as well as dimethyl as 5,9-26:2 and 5,9,19-26:3 by agreement with the retention times of those components present in the marine sponge H. panicea. *Corresponding author: Tel: 81-45-788-7657. Fax: 81-45-788- For the structural determination of peaks A, B, 5001. Email: [email protected] and C, the FAME of H. multidentata were fraction- aPresent address: Department of Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa ated depending upon their degrees of unsaturation 223-8522, Japan. by PTLC on AgNO3-impregnated silica gel 60 plates Received 31 October 2000. Accepted 14 March 2001. (AgNO3–TLC) with a mixture of n-hexane and ethyl Fatty acids of Japanese freshwater sponges FISHERIES SCIENCE 237 Fig. 1 Gas chromatogram of the fatty acid methyl esters from the total lipids of the freshwater sponge Heteroro- Fig. 2 MS/MS spectra of m/z 418 and m/z 446 ions of tula multidentata. The chromatogram was obtained on pyrrolidides from the fraction 2 obtained by prepara- an Omegawax 320 column (180–240∞C at 2∞C/min). tive thin-layer chromatography on AgNO3-impregnated silica gel 60 plates. Table 1 Fatty acid composition of the lipids from [25 : 2 (M + H)+], and m/z 446 [26 : 2 (M + H)+] (in the Japanese freshwater sponges (wt%) dienoic fraction containing three compounds B, C AgNO3–TLC fraction of H. multidentata and D), respectively. FAB-MS/MS spectra of each Fr. 1 ion were measured to determine the position of H. multi- Sat. & Fr. 2 Fr. 3 Fr. 4 Fatty acids S. alba dentata mono. Di Tri Tetra double bonds. MS/MS spectra of ions at m/z 418 14 : 0 1.2 1.3 3.0 ND ND ND and m/z 446 are shown in Fig. 2. Tomer et al. have 16 : 0 1.5 2.8 7.0 ND ND ND reported that large fragment peaks due to cleavage 16 : 1n-7 1.1 2.6 6.6 ND ND ND 18 : 0 1.2 4.1 10.6 ND ND ND of the carbon–carbon bond allylic to the double 18 : 1n-9 0.6 2.3 5.9 ND ND ND bond have been observed in the FAB MS/MS of 18 : 1n-7 2.7 3.6 9.4 ND ND ND 15 18 : 2n-9 5.2 3.5 1.6 6.8 5.7 ND unsaturated fatty acids. Prominent fragment 18 : 2n-6 0.2 1.1 ND 3.4 ND ND peaks at m/z 126, m/z 180, and m/z 234 were 18 : 3n-6 0.6 1.2 1.1 ND 4.1 ND 18 : 3n-3 0.4 1.0 ND ND 3.6 ND observed in both spectra and these fragment peaks 18 : 4n-3 0.8 0.5 ND ND ND 4.7 20 : 0 2.3 1.3 2.9 ND ND ND were due to the allylic cleavage of 5,9-double 20 : 4n-6 0.5 2.6 ND ND 6.6 5.9 bonds. MS/MS spectrum of ions at m/z 420 in the 20 : 4n-3 0.1 0.2 ND ND ND 0.9 20 : 5n-3 2.8 9.6 1.6 ND ND 69.8 saturated and monoenoic fraction showed promi- 22 : 5n-3 1.9 0.7 1.0 ND ND 1.9 nent fragment peaks at m/z 294 and m/z 348, and 24 : 1n-7 6.7 8.7 19.0 ND ND ND 5,9-24 : 2 14.0 8.7 ND 34.4 0.8 ND these fragment peaks were due to the C17–C18 5,9-25 : 2 6.7 2.7 ND 11.1 ND ND double bond. Mass spectrometeric analyses of the 5,9-26 : 2 12.1 8.1 ND 34.8 ND ND 5,9,19-26 : 3 8.1 13.5 ND ND 62.0 ND pyrrolidide derivatives established that the com- Others 29.2 19.8 30.2 9.4 17.1 16.8 ponents A, B, and C were fatty acids that have the 24 : 1n-7, 5,9-24 : 2, and 5,9-25 : 2 structures, Sat. and mono., Saturated and monoenoic; Di, dienic; Tri, trienoic; Tetra, tetraenoic or more unsaturated; ND, not detected. respectively, and peak D was confirmed as 5,9- 26 : 2. Infrared spectra of FAME of the saturated and monoenoic, dienoic, and trienoic fractions acetate (85 : 15 v/v). The fatty acid compositions obtained by AgNO3–TLC were measured using a of each fraction were analyzed by GLC. The JASCO FT/IR-410 spectrometer (JASCO Co., Tokyo, peaks were concentrated in the saturated and Japan). The infrared spectra of FAME of these three monoenoic fraction (A), dienoic fraction (B–D), fractions showed no bands near 970 cm–1, which is and trienoic fraction (E) (Table 1). characteristic of a trans-ethylenic bond. The struc- The structures of these fatty acid components ture of 18 : 2n-9 was confirmed as a pyrrolidide were determined by mass spectrometric analyses derivative by gas chromatography–mass spec- of their pyrrolidides prepared using the method of trometry (GC-MS; Shimadzu QP-5000, ionization Andersson et al.14 The pyrrolidide derivatives were energy of 70 eV, source temperature 260∞C, column subjected to fast atom bombardment mass spec- temperature 235∞C) with a CPB-20M capillary trometry (FAB-MS; JEOL JMX-SX102 mass spec- column (25 m ¥ 0.22 mm i.d.). trometer, positive mode; JEOL Co. Ltd, Tokyo, The fatty acid composition of TL obtained from Japan) using m-nitrobenzyl alcohol as a matrix. two Japanese freshwater sponges is shown in Table FAB-MS of the pyrrolidides from each fraction, 1. The predominant fatty acids (> 5% of the total which was obtained by AgNO3–TLC, showed ions fatty acids in TL in one or each species) were 18 : at m/z 420 [24 : 1 (M + H)+] (in the saturated and 2n-9, 20 : 5n-3, 24 : 1n-7, 5,9-24 : 2, 5,9-25 : 2, 5,9-26 monoenoic fraction containing A as the major : 2, and 5,9,19-26 : 3. Summation of these seven component), m/z 418 [24 : 2 (M + H)+], m/z 432 fatty acids made up 50% of the total fatty acids.