Furanoid Fatty Acids from Fish Lipids 1 ROBERT L. GLASS, THOMAS P. KRICK, DONALD M. SAND, CURTIS H. RAHN, and HERMANN SCHLENK, Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 551 01 and The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 ABSTRACT
INTRODUCTION
Fatty acids, recently reported as constituents of certain fish lipids, were identified to be derivatives of furan (furanoid fish fatty acids). 12,15-Epoxy1 3,14-dimethyleicosa-12,14-dienoic acid is predominant among the furan acids and is associated with bis-homologs in regard to chain length. Monomethyl acids, such as 12,1 5-epoxy-13-methyleicosa-12,14d i e n o i c , are present in appreciable amounts. The structures were concluded from oxidative degradations, from mass spectrometry of methyl esters o f the novel acids and fatty acids derived from them by opening the ring, and from nuclear magnetic resonance, infrared, and Raman spectra. The results from chemical procedures a n d f r o m spectrometric methods were in agreement with those obtained with authentic methyl 9,12epoxyoctadeca-9,11-dienoate. The number of substituents at the furan ring greatly influences hydrogenation, hydrogenolysis, and hydrolysis reactions of the ring.
A new series of fatty acids recently has been discovered by Glass and coworkers (1) as constituents of lipids from northern pike (Esox lucius) and other fish species. They are found primarily in the liver and testes lipids, and wide seasonal fluctuations o f their amounts have been observed. For example, at spawning time of northern pike in early spring, the new acids are 90% of the acids bound in cholesteryl esters. Altogether, eight memebers of the new series of acids have been detected so far. Their similarities in thin layer chromatography (TLC), gas liquid chromatography (GLC), and mass spectrometry (MS) indicated close structural relationships (1). It now has been established that they have a furan ring in common and it is expedient to use the term furanoid fatty acids for them, abbreviated F 1 to F 8. Abbreviations previously used (1), P or PMFA for pike or pike male fatty acids, respectivety, are too narrow, as these acids have since been detected in many 1Scientific Journal Series 9154, Agricltural Experiment Station, University of Minnesota, St. Paul, MN other fish and, at low levels, in some female specimens (Glass, et al., unpublished data). A S5101; Hormel Institute Publication No. 749. TABLE I Structures of Furanoid Acids from Fish HOOC-(CH2)m [ ~ O ~ R1
(CH2)n-CH3 R2
Compound a
Empirical formula
m
n
R1
R2
F1 F2 F3 F4 F5 F6 F7b E8 Reference c
C18H3003 C19H320 3 C20H3403 C20H3403 C21 H3603 C22H3803 C23H40 O3 C24H4203 C18H3003
8 8 8 10 10 10 12 12 7
2 4 4 2 4 4 4 4 5
CH 3 CH3 CH 3 CH3 CH3 CH3 CH3 CH3 H
CH3 H CH3 CH3 H CH3 H CH3 H
aThe esters are numbered in the sequence of gas liquid chromatography retention times. bR 1 = CH3 is suggested for F 7 by inference from F2 and F 5. c9,12-Epoxyoctadeca-9,11-dienoic acid, semisynthetic from ricinoleic acid (3). 695
696
R.L. GLASS, T.P. KRICK, D.M. SAND, C.H. RAHN, AND H. SCHLENK
furanoid fatty acid also has been described from Exocarpus cupressiformis seed oil (2). The furanoid acids, F1 to F8, represent a homologous series, with F3 and F 4 being isomers. F 6 was the predominant furanoid acid in all materials and was used in most of the structure investigations. The formula of this acid, C22H3sO3, had been verified by high resolution MS, and elemental analysis was in agreement (1). According to the results, the new fatty acids from fish lipids contain a furan ring with 3 or 4 substituents. Specific structures as listed in Table I are proposed for F 1 to F s . For example, furanoid fish acid F 6 is 12,15-epoxy13,14-dimethyleicosa- 12,14-dienoic acid. EXPERIMENTAL PROCEDURES Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out on layers of Silica Gel H (E.C. Merck, D a r m s t a d t , Germany) with hexane:diethyl ether:acetic acid (85:15:1) as developing solvent. In argentation chromatography, the adsorbent contained 5% AgNO3. The plates were activated at 110 C for 16 hr. The mixture of F esters, as well as pure F 6 and pure F s esters, gave, with 50% H2SO 4 + K2C207, a red color on the chromatograms which appeared sooner than the red color of cholesterol or its esters. The semisynthetic reference compound, 9,12-epoxyoctadeca-9,11dienoate (3), gave a yellow-orange stain prior to charring. Gas Liquid Chromatography
Gas liquid chromatography conditions were (a) for analysis of fatty acid methyl esters and fatty alcohol acetates or trimethylsilyl ethers: 10% Silar 10C on Gas Chrom Q, 100/120 mesh (Applied Science Labs, Inc., State College, PA), in an aluminum column, 3.2 mm internal diameter (ID) and 240 cm long, at 190 C; (b) for preparative separation of F methyl esters: 6% SE-30 (Fisher Scientific Co., Chicago, IL) on Chromosorb W 60/80 mesh (Johns Manville, Celite Division, New York, NY), in a column, 7.9 mm ID and 1 8 0 c m long, at 212 C; (c) for analysis of short chain monoesters: as for fatty acid methyl esters, but at 105 C; (d) for analysis of diesters: 15% HI-EFF-2BP (ethylene glycol succinate) on Gas Chrom P, 100/120 mesh (Applied Science Labs) in a column, 3.2 mm ID and 180 cm long, at 180 C. Furanoid Fish Acid Methyl Esters
According to previously described procedures (1), lipids were extracted from tissues in a Waring Blendor by chloroform:methanol (2: 1) LIPIDS, VOL. 10, NO. 11
and recovered as usual. Alkaline methanolysis yielded the methyl esters which were subjected to hydrogenation in chloroform for 20 min b y H 2 at atmospheric pressure with PrO2 catalyst. Under these conditions, only the common fatty acid methyl esters were hydrogenated, and they were removed by crystallization as urea complexes (1). GLC and MS showed that F methyl esters were not changed by these procedures. Unsaturated straight chain fatty esters also can be removed by argentation-TLC, where the F esters migrate closely behind saturated fatty esters which are in the mixtures. Individual F esters were obtained by GLC after the above purification or enrichment. Minor amounts of saturated straight chain est e r s did not interfere with GLC of the F esters. However, F3 and F4 methyl esters have similar retention times so that their preparative separation was not efficient. A reference compound, methyl 9,12-epoxyoctadeca-9,11-dienoate, was prepared from ricinoleic acid according to published procedures (3). Hydrogenations
Hydrogenation of F6 methyl ester with 10% Pd on charcoal (ICN-K&K Laboratories, Inc., Plainview, NY) in chloroform yielded a product which had, in TLC, an approximate Rf value of 0.6. However, GLC indicated that this was a mixture of at least two compounds, I and II, in about equal amounts. They emerged as well separated peaks with equivalent chain length (ECL) values (4) 24.2 (I) and 25.7 (II) on Silar 10C, while the F 6 ester had ECL 24.8. MS indicated the uptake of 4 H for each fraction, and their fragmentation patterns were not distinguishable. Therefore, tetrahydro F6-I and II must be stereoisomers, but each of them may still represent a mixture of stereoisomers. When hydrogenating F 6 methyl ester with PrO2 (Engelhardt Industries, Inc., Chemical Division, Newark, NJ) in acetic acid, the ratio of I and II was greatly changed in favor of the latter, so that tetrahydro F6-I represented
INTRODUCTION
Fatty acids, recently reported as constituents of certain fish lipids, were identified to be derivatives of furan (furanoid fish fatty acids). 12,15-Epoxy1 3,14-dimethyleicosa-12,14-dienoic acid is predominant among the furan acids and is associated with bis-homologs in regard to chain length. Monomethyl acids, such as 12,1 5-epoxy-13-methyleicosa-12,14d i e n o i c , are present in appreciable amounts. The structures were concluded from oxidative degradations, from mass spectrometry of methyl esters o f the novel acids and fatty acids derived from them by opening the ring, and from nuclear magnetic resonance, infrared, and Raman spectra. The results from chemical procedures a n d f r o m spectrometric methods were in agreement with those obtained with authentic methyl 9,12epoxyoctadeca-9,11-dienoate. The number of substituents at the furan ring greatly influences hydrogenation, hydrogenolysis, and hydrolysis reactions of the ring.
A new series of fatty acids recently has been discovered by Glass and coworkers (1) as constituents of lipids from northern pike (Esox lucius) and other fish species. They are found primarily in the liver and testes lipids, and wide seasonal fluctuations o f their amounts have been observed. For example, at spawning time of northern pike in early spring, the new acids are 90% of the acids bound in cholesteryl esters. Altogether, eight memebers of the new series of acids have been detected so far. Their similarities in thin layer chromatography (TLC), gas liquid chromatography (GLC), and mass spectrometry (MS) indicated close structural relationships (1). It now has been established that they have a furan ring in common and it is expedient to use the term furanoid fatty acids for them, abbreviated F 1 to F 8. Abbreviations previously used (1), P or PMFA for pike or pike male fatty acids, respectivety, are too narrow, as these acids have since been detected in many 1Scientific Journal Series 9154, Agricltural Experiment Station, University of Minnesota, St. Paul, MN other fish and, at low levels, in some female specimens (Glass, et al., unpublished data). A S5101; Hormel Institute Publication No. 749. TABLE I Structures of Furanoid Acids from Fish HOOC-(CH2)m [ ~ O ~ R1
(CH2)n-CH3 R2
Compound a
Empirical formula
m
n
R1
R2
F1 F2 F3 F4 F5 F6 F7b E8 Reference c
C18H3003 C19H320 3 C20H3403 C20H3403 C21 H3603 C22H3803 C23H40 O3 C24H4203 C18H3003
8 8 8 10 10 10 12 12 7
2 4 4 2 4 4 4 4 5
CH 3 CH3 CH 3 CH3 CH3 CH3 CH3 CH3 H
CH3 H CH3 CH3 H CH3 H CH3 H
aThe esters are numbered in the sequence of gas liquid chromatography retention times. bR 1 = CH3 is suggested for F 7 by inference from F2 and F 5. c9,12-Epoxyoctadeca-9,11-dienoic acid, semisynthetic from ricinoleic acid (3). 695
696
R.L. GLASS, T.P. KRICK, D.M. SAND, C.H. RAHN, AND H. SCHLENK
furanoid fatty acid also has been described from Exocarpus cupressiformis seed oil (2). The furanoid acids, F1 to F8, represent a homologous series, with F3 and F 4 being isomers. F 6 was the predominant furanoid acid in all materials and was used in most of the structure investigations. The formula of this acid, C22H3sO3, had been verified by high resolution MS, and elemental analysis was in agreement (1). According to the results, the new fatty acids from fish lipids contain a furan ring with 3 or 4 substituents. Specific structures as listed in Table I are proposed for F 1 to F s . For example, furanoid fish acid F 6 is 12,15-epoxy13,14-dimethyleicosa- 12,14-dienoic acid. EXPERIMENTAL PROCEDURES Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out on layers of Silica Gel H (E.C. Merck, D a r m s t a d t , Germany) with hexane:diethyl ether:acetic acid (85:15:1) as developing solvent. In argentation chromatography, the adsorbent contained 5% AgNO3. The plates were activated at 110 C for 16 hr. The mixture of F esters, as well as pure F 6 and pure F s esters, gave, with 50% H2SO 4 + K2C207, a red color on the chromatograms which appeared sooner than the red color of cholesterol or its esters. The semisynthetic reference compound, 9,12-epoxyoctadeca-9,11dienoate (3), gave a yellow-orange stain prior to charring. Gas Liquid Chromatography
Gas liquid chromatography conditions were (a) for analysis of fatty acid methyl esters and fatty alcohol acetates or trimethylsilyl ethers: 10% Silar 10C on Gas Chrom Q, 100/120 mesh (Applied Science Labs, Inc., State College, PA), in an aluminum column, 3.2 mm internal diameter (ID) and 240 cm long, at 190 C; (b) for preparative separation of F methyl esters: 6% SE-30 (Fisher Scientific Co., Chicago, IL) on Chromosorb W 60/80 mesh (Johns Manville, Celite Division, New York, NY), in a column, 7.9 mm ID and 1 8 0 c m long, at 212 C; (c) for analysis of short chain monoesters: as for fatty acid methyl esters, but at 105 C; (d) for analysis of diesters: 15% HI-EFF-2BP (ethylene glycol succinate) on Gas Chrom P, 100/120 mesh (Applied Science Labs) in a column, 3.2 mm ID and 180 cm long, at 180 C. Furanoid Fish Acid Methyl Esters
According to previously described procedures (1), lipids were extracted from tissues in a Waring Blendor by chloroform:methanol (2: 1) LIPIDS, VOL. 10, NO. 11
and recovered as usual. Alkaline methanolysis yielded the methyl esters which were subjected to hydrogenation in chloroform for 20 min b y H 2 at atmospheric pressure with PrO2 catalyst. Under these conditions, only the common fatty acid methyl esters were hydrogenated, and they were removed by crystallization as urea complexes (1). GLC and MS showed that F methyl esters were not changed by these procedures. Unsaturated straight chain fatty esters also can be removed by argentation-TLC, where the F esters migrate closely behind saturated fatty esters which are in the mixtures. Individual F esters were obtained by GLC after the above purification or enrichment. Minor amounts of saturated straight chain est e r s did not interfere with GLC of the F esters. However, F3 and F4 methyl esters have similar retention times so that their preparative separation was not efficient. A reference compound, methyl 9,12-epoxyoctadeca-9,11-dienoate, was prepared from ricinoleic acid according to published procedures (3). Hydrogenations
Hydrogenation of F6 methyl ester with 10% Pd on charcoal (ICN-K&K Laboratories, Inc., Plainview, NY) in chloroform yielded a product which had, in TLC, an approximate Rf value of 0.6. However, GLC indicated that this was a mixture of at least two compounds, I and II, in about equal amounts. They emerged as well separated peaks with equivalent chain length (ECL) values (4) 24.2 (I) and 25.7 (II) on Silar 10C, while the F 6 ester had ECL 24.8. MS indicated the uptake of 4 H for each fraction, and their fragmentation patterns were not distinguishable. Therefore, tetrahydro F6-I and II must be stereoisomers, but each of them may still represent a mixture of stereoisomers. When hydrogenating F 6 methyl ester with PrO2 (Engelhardt Industries, Inc., Chemical Division, Newark, NJ) in acetic acid, the ratio of I and II was greatly changed in favor of the latter, so that tetrahydro F6-I represented
