- Email: [email protected]
Lipids of cartilaginous fish: Composition of ether and ester glycerophospholipids in the muscle of four species of shark
Comp. Biochem. Physiol. Vol. 113B, No. 2, pp. 305-312, 1996 Copyright © 1996 Elsevier Science Inc.
ISSN 0305-0491/96/$26.00 SSDI 0305-0491(95)02020-L
ELSEVIER
Lipids of Cartilaginous Fish: Composition of Ether and Ester Glycerophospholipids in the Muscle of Four Species of Shark Bo-Young Jeong,* Toshiaki Ohshima, "PHideki Ushioi- and Chiaki Koizumi-f "DEPARTMENT OF FOOD SCIENCE, GYEONGSANGNATIONAL UNIVERSITY, TONGYEONG, GYEONGNAM, 650-160, KOREA AND tDEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY, THE TOKYO UNIVERSITYOF FISHERIES, KONaN 4, MINATO-KU, TOKYO 108, JAPAN
ABSTRACT. The levels and hydrocarbon chain distributions of alk-l'-enyl-acyl, alkyl-acyl and diacyl subclasses of choline glycerophospholipid (GPC) and ethanolamine glycerophospholipid (GPE) in the muscles of four species of shark were investigated. The prominent subclasses in the EGP fraction were 1-O-alk-1 '-enyl-2acyl-sn-glycero-3-phosphoethanolamine (alk-l'-enyl-acyl-GPE) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (diacyl-GPE), accounting for 24.8% to 54.4% and 40.8% to 69.4%, respectively, of the total glycerophospholipids. In the GPC fraction, 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) was the most prominent subclass, ranging from 73.2% to 94.7% in the total glycerophospholipids. The levels of l-O-alkyl-2acyl-sn-glycero-3-phosphoethanolamine (alkyl-acyl-GPE), -phosphocholine (alkyl-acyl-GPC) and alk-l'-enylacyl-GPC were below 15% in the corresponding subclasses. The prominent alk-l'-enyl and acyl chains of the alk-l'-enyl-acyl-GPE were 16:0 (42.3 to 63.4%), 18:1 (10.0 to 30.0%) and 18:0 (5.7 to 10.9%) alk-1'-enyl chains, and 22:6n-3 acyl chain (48.8 to 75.7%). In the alkyl-acyl-GPE fraction, the prominent alkyl and acyl chains were similar to those of the alk-l'-enyl-acyl-GPE, but the levels of alkyl and acyl chains with 16 carbons were higher. The hydrocarbon chain compositions of alk-l'-enyl-acyl- and alkyl-acyl-GPC were also similar to those of the alkyl-acyl-GPE. The prominent acyl chains of diacyl-GPE and -GPC were 18:0, 16:0 and 22:6n-3, but the GPE fraction was richer in 18:0 acyl chain and the GPC fraction was richer in 16:0. These characteristics in alk-l'-enyl and alkyl chains of ether glycerophospholipids from shark muscles suggested that these muscles are suitable as sources of dietary ether-linked lipids for a certain disease that prints syndrome of deficiency in ether-linked lipids. COMPBIOCHEMPHYSIOL113B, 305-312, 1996. KEY WORDS. Ether lipids, plasmalogens, alkyl chains, alk-l'-enyl chains, cartilaginous fish, shark muscle
INTRODUCTION Marine organisms, especially invertebrates, are rich in ether glycerophospholipids (13,25). Recently, Sugiura et al. (28) reported that most species examined in various phyla other than Arthropoda contained large amounts of 1-O-alk-1 '-enyl2-acyl-sn-glycero-3-phosphocholine (alk-l'-enyl-acyl-GPC) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and that the levels of both ether glycerophospholipids were markedly high in the lower phyla. Koizumi and coworkers (10,11,14) also found high levels of ether glycerophospholipids in Pacific oyster wet organs, sea urchin gonads and ascidian muscle and viscera, and revealed the detailed hydrocarbon chain composition of the lipids. A small amount of ether glycerophospholipids is also reported in the skipjack Gyrrespondence to: T. Ohshima, Department of Food Science and Technology, The Tokyo University of Fisheries, Konan 4, Minato-ku, Tokyo 108, Japan. Tel. 03-5463-0613; Fax 03-5463-0627. Received 10 January 1995; revised 27 June 1995; accepted 29 June 1995.
white muscle (18,19) and in the Atlantic cod muscle (22). In this context, we have found high levels of ether glycerophospholipids in the muscles of certain species of shark. Although l-O-alkyl-2-acetyl-GPC has been reported to have a precise function as a platelet activating factor, physiological roles for other analogues of glycerophospholipid subclasses are still obscure. In certain genetic diseases such as a Zellweger cerebrohepatorenal syndrome, ether-linked lipids are deficient almost completely in the tissues of the patients (9). On the other hand, it is well known that dietary etherlinked lipids are incorporated into membrane ether-linked lipids through intestinal membrane cells, and further incorporated into plasmalogens in these tissues (1,3). These findings suggest that shark muscle might be a good source for dietary ether-linked lipids for patients suffering from ether-linked lipids deficiency syndrome. To clarify this speculation, we investigated, in this study, the lipid class levels and hydrocarbon chain distributions of glycerophospholipids in the muscles of four species of shark.
306
Jeong et al.
MATERIALS AND METHODS Anima/s
and alkyl-acyl-GPE and -GPC, and those in the sn-1 and sn-2 positions of diacyl-GPE and -GPC were quantified after
Four species of shark, blue shark Prionance glauca, thresher shark Alopias vulpinus, estuary shark Chacharhinus plumbeus and mackerel shark Lamna ditropis, were used in this study. These specimens were caught in the Pacific Ocean, off Choushi City, Chiba Prefecture, Japan, in April and May, 1993. Three separated portions of white muscles, weighing 200 g each, were taken from the dorsal parts of one specimen of each shark species within 24 hr after catch, and stored at - 80°C until used for analyses. The analysis of phospholipids of each shark's white muscles was repeated twice. The dark muscle of the mackerel shark was also analyzed in triplicate, since the muscle of this species is richer in dark muscle compared to the other three species. The contents of the glycerophospholipid subclasses were calculated from the amounts of the fatty acids (14).
conversion in methyl esters by gas-liquid chromatography (GLC) using 23:0 fatty acid of more than 99% purity (NuChek-Prep, Elysian, MN, U.S.A.) as an internal standard (6).
The FAMEs were analyzed by GLC using a Shimadzu GC 12APF or GC8APF instrument equipped with a fused silica open tubular column coated with a polar phase (SUPELCOWAX-10, 0.25/,m in film thickness, 30 m × 0.25 mm i.d., Supelco Japan, Ltd., Tokyo) and a flame ionization detector (FID). The injector and FID were held at 250°C. The column oven was programmed from 170 to 230°C at l°C/min.
Lipid Extraction and Fractionation
Determination of Alk. 1 '-enyl and Alkyl Chains Distributions
The shark muscle was minced and extracted with chloroform/ methanol according to the Bligh and Dyer procedure (2). The ethanolamine glycerophospholipid and choline glycerophospholipid classes in the extracted total lipids were separated by column chromatography on Silica Gel 60 (Merck, Darmstadt, Germany) according to the method of Hanahan et al. (8).
The alk-1 '-enyl and alkyl chains of 1-O-alk-1 '-enyl-2,3-TMSglycerol and 1-O-alkyl-2,3-TMS-glycerol derivatives were determined by GLC using a Shimadzu GC8APF instrument (Shimadzu, Kyoto, Japan) equipped with a SUPELCOWAX-10 fused silica open tubular column (30 m × 0.25 mm i.d., Supelco Japan Ltd., Tokyo). The injector and FID were held at 250°C and column temperature was programmed from 160 to 230°C at 4°C/min (18).
Quantitation of Phospholipid Classes Quantitative analysis of phospholipid classes was carried out in duplicate for each lipid sample using Chromarod S-IIl and an Iatroscan MK-5 TLC-FID analyzer (Dia-Iatron Co., Tokyo, Japan) according to the method of Ohshima et al.
(17). Der/~at/zat/on for Determination of Alk.1 '-enyl, Alkyl and Acyl Chains Distribution The GPE and GPC fractions were each hydrolyzed with phospholipase C (from Bacillus cereus, Boehringer Mannheim, Mannheim, Germany) to obtain the corresponding 1,2diradylglycerols (7). These were purified by preparative TLC on silicic acid and then acetylated (24). The acetylated glycerols were separated into subclasses by preparative TLC on silicic acid with petroleum ether/diethyl ether/acetic acid (85 : 15 : 1, v/v/v). The isolated and purified acetylglycerols were saponified to 1-O-alk-l'-enyl-glycerols and 1-O-alkylglycerols, followed by extraction with diethyl ether and conversion to the corresponding trimethylsilyl (TMS) ether derivatives, i.e. 1-O-alk-l'-enyl-2,3-TMS-glycerol and 1-Oalkyl-2,3-TMS-glycerol (18). After acidification, the sn-2 fatty acids liberated from the 1-O-alk-l'-enyl-2-acylglycerols and 1-O-alkyl-2-acylglycerols were recovered with diethyl ether. The fatty acids in the sn-2 positions of alk-l'-enyl-acyl-
Determination of Fatty Acid Distribution in Alk-1 '.enyl.acyl., Alkyl.acyl. and D/acyl-GPC and GPE
RESULTS
Lipid Class Contents and Alk-I '.enyl.acyl, Alkyl.acyl and Diacyl Glycerophospholipids Composition The lipid class contents of the shark muscle of the four species are shown in Fig. 1. The total lipids (TL) contents of the muscles of P. glauca, A. vulpinus and C. plumbens were about 0.6%, and were lower compared to those of L. ditropis white (2.7%) and dark (1.7%) muscles. The glycerophospholipids (PL) comprised >90% of the TL in the white muscle of P. glauca, A. vulpinus and C. plumbens, but were only 40% of the TL in the white muscle of L. ditropis. The major PL classes in all samples were GPC (50.8 to 70.0% of phospholipids) and GPE (15.7 to 37.2% of phospholipids). The composition of phospholipid subclasses is shown in Fig. 2. The major subclasses in the GPE of all shark muscles examined were alk-l'-enyl-acyl-GPE and diacyl-GPE, although the lipids of P. glauca contained a small amount of alkyl-acyl-GPE. The major subclass in GPC was diacyl-GPC, and alk-1 '-enyl-acyl- and alkyl-acyl-GPC were found as minor components. The level of alkyl.acyl-GPC was low in any of the muscles of the sharks, although this glycerophospholipid subclass is usually found in equal amounts to that of the alk-1 'enyl-acyl-GPE.
Glycerophospholipids of Cartilaginous Fish
307
3
~ 2.5
Neutrallipid
~
DPG
GPC
~
GPI
r~
"~
2
g 1.5 or) r.c) el)
~
1
° ,,,,~
0.5
0 P. glauca
A. vulpinus
C. plumbens
L. ditropis White Dark
FIG. 1. Lipid class contents of the shark muscles. GPC, choline glycerophospholipid; EGP, ethanolamine glycerophospholipid; SPM, sphingomyelin; DPG, diphosphatidyl glycerol; GPI, inositol glycerophospholipid; LGPC, choline lysoglycerophospholipid; GPS, serine glycerophospholipid.
A 100 90 80 70 60 50 40 30 20 10 0
B 100 90 80 70 60 ~ 50 ~
4o-~ 30 ~ 20 10 P. glauca
C. plumbens L. ditropis A. vulpinus White Dark
•
AIk-l'-enyl-acyl
P. glauca
[]
C, plumbens L. ditropis A. vulpinus White Dark
Alkyl-acyl
[]
Diacyl
FIG. 2. Subclass composition of ethanolamine (A) and choline (B) glycerophospholipids of the shark muscles. Alk.l'-enyl-acyl, 1-O-alk-l'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine and .choline; 1,2-diacyl, 1,2-diacyl-sn-3-phosphoethanolamine and -choline.
308
Alk. 1 '.enyl, Alkyl and Acyl Chains
Distribution of Glycerophospholipid Subclasses The hydrocarbon chain compositions of alk-l'-enyl-acyl, alkyl-acyl and diacyl subclasses of GPE in the shark muscles are shown in Tables 1 to 3. For the alk-1 '-enyl-acyl-GPE of any of the shark muscles (Table 1), 16:0, 18:1 and 18:0 alk-l'-enyl chains in the sn-1 position were prominent; particularly the level of 16:0 alk-l'-enyl chain was high, amounting to 42.3 to 63.9. The profiles of acyl chains in the sn-2 position were different among the species of shark. However, in all shark muscles, 22:6n-3 was commonly the most prominent acyl chain, ranging from 48.8 to 75.7%. From the results of analyses of these shark muscle glycerophospholipids, the most prominent molecular species in the alk-l'-enyl-acyl-GPE would be expected to be 16:0 alk-1'-enyl-22:6n-3. The alkyl chain compositions of the alkyl-acyl-GPE were quite simple in the all shark muscles (Table 2); most prominent alkyl and acyl chains were 16:0, 16: 1, 18:0 and 18:1 alkyl chains, and 22:6n-3 and 16:0 acyl chains, respectively. The level of 16:0 alkyl chain was especially high in the alkyl chains, ranging from 46.7 to 48.9% in all four species. From these results, the most prominent molecular species of the alkyl-acyl-GPE is presumably 16:0 alkyl-22:6n-3 and 16:0 alkyl- 16 : 0. For the diacyl-GPE (Table 3), most prominent acyl chains in both the sn-1 and the sn-2 positions were very simple; 22:6n-3 and 18:0, accounting respectively for 23.5 to 32.5% and 18.0 to 39.0%. Therefore, the major molecular species was expected to be 18:0-22:6n-3. The hydrocarbon chain compositions of alk-l'-enyl-acyl, alkyl-acyl and diacyl subclasses in the shark muscle GPC are shown in Tables 4-6. For the alk-l'.enyl-acyl-GPC (Table 4), most prominent alk-l'-enyl chains were 16:0, 18: 1, 16:1 and 14:0. The levels of 16:0 alk-1 '-enyl chain were especially high in this lipid subclass of all four shark, accounting for 51.1 to 78.5% of these chains. The compositions of acyl chains in the sn-2 position were different from species to species, while 22:6n-3 and 16:0 were found to be relatively important among the prominent acyl chains. These characteristic profiles of the alk-l'-enyl-acyl-GPC were similar to those of the alkyl-acyl-GPE. From these results, the prominent molecular species of the alk-l'-enyl-acyl-GPC can be predicted to be 16 : 0 alk- 1'-enyl chain-22 : 6n-3. As shown in Table 5, the level of 16 : 0 alkyl chain of the alkyl-acyl-GPC was the highest of the alkyl chains, accounting for 24.4 to 34.3%. Other prominent alkyl chains were 16 : 1, 18 : 1 an 14 : 0. The prominent acyl chains of the alkylacyl-GPC were 22 : 6n-3 and 16 : 0. Therefore, the most probable molecular species are 16:0 alkyl chain-22:6n-3 and 16:0 alkvl chain-16 : 0. The prominent fatty acyl chains in the sn-1 and the sn-2 positions of diacyl-GPC were 16:0, 22:6n-3, 18:0 and 18: ln-9 (Table 6). The level of 16:0 of the diacyl-GPC was about 3-fold higher than that of the diacyl-GPE. The level of 22 : 6n-3 was much lower compared to that of the diacyl-GPE.
Jeong et al.
The major molecular species of the diacyl-GPC would be expected to be 16:0-16:0, 16:0-22:6n-3, 18:0-22:6n-3 and 18 : ln-9-22 : 6n-3. DISCUSSION The present study demonstrates that the levels of ether glycerophospholipids in the muscle of four sharks were higher than those of muscles of bony fish, including those of bonito Katsuwonus pelamis (3.6 to 7.6%) (18,19) and Atlantic cod Gadus morhua (22). However, the levels of ether glycerophospholipids of invertebrates are much higher than those of the shark muscles. The proportion of alk-l'-enyl-acyl-GPE in the shark muscle phospholipids was lower than those of marine invertebrates, including the Pacific oyster (59.8%) (10,14), ascidian (69.0%) (11), and annelida, mollusca, platyhelminthes, and porifera (53.3 to 88.7%) (13,28). High levels of alk-l'-enyl-acyl-GPE can be found in other marine organisms including marine worms (21), abalone (4), snails and bivalves (12), sponges (26), and several species of invertebrates (5). In mammals, high levels of ether glycerophospholipids exist in specific organs such as heart (20,27) and particularly brain (15,16). The alk-l'-enyl and acyl chains compositions were similar to those of the bonito white muscle (18), thus being different from those of marine invertebrates. In the latter the prominent acyl chains of alk-l'-enyl-acyl-GPE, for example in the Pacific oyster wet organs (10,14), in the sea urchin gonads and in the ascidian muscle and viscera (11), were 20:5n-3, 22 : 2NMID (non-methylene-interrupted diene) and 20 : 4n-6. The alk-l'-enyl chain distribution was similar to that of the bonito white muscle lipids (18), but definitely different from lipids of certain marine invertebrates in which 18:0 alk-l'enyl chain was the prominent (10,14,28). The alkyl chain profiles of the alkyl-acyl-GPC in the shark muscles were very similar to those of the Pacific oyster (10,14) and of the bonito white muscle (18), but different from those of the sea urchin gonads and the ascidian muscle (11). When 1-O-heptadecyl-rac-glycerol was fed to rats, 46 to 60% incorporation of the Cl7-moiety in the sn. 1 position (this includes a carbon in the glycerol moiety) of ethanolamine plasmalogens accumulated in most tissues of the rats, however, the plasmalogen level in the tissues remained unchanged (3). The alkyl glyceryl ether level increased in liver, kidney and lung of rats to which 1-O-alkyl-2,3-diacetyl-sn-glycerol was fed (1). Therefore, it is possible to say that dietary alkyl glycerol ethers are absorbed, converted to plasmalogens in an intestinal tissue, and transported to other tissues where they are metabolized and deposited as membrane plasmalogens (3). However, the incorporation rate of ether lipids depends on the chain ler~gth of hydrocarbon chain; the lipids with saturated and monounsaturated alkyl chain length between C14 and CI8 are only incorporated into glycerol ether lipids in an intestinal tissue (1,3,23,29). The present study revealed that shark muscle glycerophospholipids have alkyl and alk-l'-enyl
309
Glycerophospholipids of Cartilaginous Fish
TABLE 1. P r o m i n e n t a l k - l ' e n y i a n d acyl c h a i n c o m p o s i t i o n ( o v e r 1%) in t h e sn-1 a n d sn-2 p o s i t i o n s o f a l k - l ' , e n y l , a c y l G P E in s h a r k m u s c l e s ( m e a n - SD, w t %)
Hydrocarbon chain sn- 1 position (alk- l'-enyl 14:0 16:0 16:1 17:0 iso 18:0 18: la b 18:1b b
Mackerel Blue chain) 1.48 ± 42.3 --3.17 ± 1.53 ± 10.5 ± 30.0 ± 4.31 ±
Thresher
Estuary
Ordinary
Dark
0.05 0.36 0.08 0.10 0.11 0.32 0.03
3.74 60.4 2.65 2.81 8.02 9.98 3.43
± -+ + + -+ -+ -+
0.03 0.12 0.07 0.02 0.02 0.01 0.05
0.58 + 0.00 58.2 -+ 0.03 tr a 2.93 -+ 0.01 10.9 -+ 0.03 13.1 -+ 0.05 5.89 -+ 0.02
2.59 63.9 3.49 2.23 5.33 11.7 4.49
± ± ± ± + __. -4-
0.06 0.33 0.01 0.20 0.04 0.09 0.04
1.08 57.3 1.34 3.55 9.33 12.7 4.54
-+ -+ + -+ ± ± ±
0.03 0.07 0.10 0.06 0.20 0.22 0.07
sn-2 position (acyl chain) 16:0 6.56 ± 0.23 16:ln-7 2.92 + 0.07 18:0 2.03 ± 0.03 18:1n-9 8.65 ± 0.22 20:4n-6 3.70 ± 0.04 20:5n-3 6.85 ± 0.04 22:5n-3 8.31 ± 0.21 22:6n-3 48.8 ± 0.79
1.23 0.40 0.43 2.10 2.60 0.40 10.8 75.7
+ -4-+ ± ± -+ -+ -+
0.13 0.02 0.01 0.07 0.04 0.03 0.26 0.48
2.02 1.23 0.73 8.04 9.65 3.10 4.99 64.9
6.55 1.86 1.80 7.46 4.43 0.93 9.53 52.4
+ _ -+ ± ± ±
0.15 0.06 0.00 0.05 0.03 0.01 0.11 0.54
2.83 _+ tr 0.79 --_ 9.38 ± 16.0 ± 3.69 ± 1.96 + 55.0 -+
0.24
+ + ± ± ± ± ± -+
0.18 0.26 0.01 0.08 0.08 0.03 0.44 0.33
0.04 0.55 0.15 0.03 0.04 1.25
~The "tr" denotes below 1%. bGeometric isomers.
TABLE 2. P r o m i n e n t a l k y l a n d a c y l c h a i n c o m p o s i t i o n ( o v e r 1%) i n t h e sn.1 a n d sn.2 p o s i t i o n s o f a l k y l - a c y l G P E in s h a r k m u s c l e s ( m e a n -+ SD, w t %)
Hydrocarbon chain
Mackerel Thresher
Estuary
sn-1 position (alkyl chain) 14:0 15:0 iso 15:0 16:0 16:1 17:0 iso 17:0 anteiso 17:0 18:0 18:1 c 18:1 c 18:V
7.64 1.85 1.38 46.9 11.9 2.01 1.10 1.71 3.16 4.05 7.65 3.59
± ± -+ -+ -+ _+ _+ -+ ± -+ ± ±
0.15 0.00 0.09 0.32 0.10 0.20 0.11 0.08 0.02 0.13 0.12 0.07
1.09 -+ a -46.8 ± 7.53 ± 1.97 ± 1.58 1.06 +10.2 -+ -11.1 -+ 5.57 +
sa-2 position (acyl chain) 14:0 16:0 16:1n-9 16:ln-7 17:0 18:0 18:1n-9 20:3n-3 20:4n-6 22:5n-3 22:6n-3
1.65 10.4 1.32 1.42 1.22 6.62 4.31 2.77 1.58 16.8 43.9
_+ +-+ + -+ + -+ ± -+ +
0.13 0.04 0.06 0.03 0.03 0.02 0.02 0.03 0.04 0.04 0.33
~'The "--" denotes not detected. bThe "tr" denotes below 1%. cOeometric isomers.
0.01
Ordinary
Dark
0.12 0.11
4.58 + 0.18 1.10 + 0.02 tr b 48.9 -+ 0.25 11.9 + 0.06 1.62 _+ 0.02 1.27 - 0.01 tr 2.26 +_ 0.02 2.37 ± 0.02 16.7 + 0.15 5.14 + 0.17
3.18 1.26 1.46 46.7 9.82 2.47 1.83 1.09 4.65 4.17 12.6 5.63
7.28 -+ 0.02 22.3 ± 0.17 7.01 ± 0.31 -1.64 -+ 0.01 9.29 -+ 0.03 11.0 -+ 0.05 -3.39 + 0.08 5.89 ± 0.01 23.7 ± 0.08
5.31 -+ 0.28 14.4 ± 0.57 tr 15.2 ± 0.67 tr 5.50 + 0.06 6.76 + 0.09 -0.96 + 0.05 8.91 + 0.29 32.2 + 1.21
1.96 +-- 0.01 10.5 + 0.11 4.28 + 0.09 -tr 4.65 + 0.04 7.71 + 0.67 2.11 +_ 0.03 7.45 -+ 0.19 1.92 -+ 0.15 50.9 + 0.15
0.07 0.01 0.04 0.04 0.02 0.03
+ + + ---+ -+ + +-+ + + -+
0.16 0.05 0.31 0.24 0.18 0.21 0.21 0.06 0.09 0.06 0.05 0.16
310
Jeong et al.
TABLE 3. Prominent acyl chain composition (over 1%) in the sn-1 and sn-2 positions of diacyl-GPE in shark muscles (mean _+ SD, wt %) Mackerel Acyl chain
Blue
16:0 18:0 18:1n-9 18:1n-7 20:ln-ll 20:1n-9 20:4n-6 20:5n-3 22:5n-3 22:6n-3
6.29 28.5 8.19 3.71 1.18 1.89 2.85 3.40 5.61 30.6
± ± ± ± ± ± ± ± ± +
Thresher
0.06 0.17 0.05 0.02 0.01 0.03 0.01 0.01 0.03 0.15
9.10 35.7 2.81 5.07
1.21 2.13 3.23 32.5
± 0.37 ± 0.49 ± 0.05 ± 0.16 tr ~ ± 0.03 ± 0.00 tr ± 0.14 ± 0.89
Estuary 6.12 35.3 4.21 7.27
4.87 1.06 4.11 23.5
± ± ± ± tr tr m ± ± ±
Ordinary
0.03 0.67 0.11 0.18
16.2 18.0 5.97 9.25
0.02 0.03 0.15 0.84
1.17 5.27 1.97 3.90 28.6
± ± ± ± tr ± ± ± ± +
Dark
0.62 0.24 0.09 0.19
5.67 39,0 4.27 2.76
0.01 0.09 0.03 0.15 1.05
8.59 2.23 1.95 28.9
± ± ± +_ tr tr ± +_ ± ±
0.12 0.34 0.08 0.07
0.05 0.02 0.03 0.47
aThe "tr" denotes below 1%.
TABLE 4. Prominent alk-l'-enyl and acyl chain composition (over 1%) in the sn-1 and sn-2 positions of alkenylacyl GPC in shark muscles (mean +-- SD, wt %) Hydrocarbon chain sn-1 position 14:0 15:0 iso 15:0 16:0 16:1 17:0 iso 18:1 b 18:1 b
Mackerel Blue
Thresher
Estuary
Ordinary
Dark
(alk-l'-enyl chain) 5.18 ± 0.23 1.23 ± 0.04 tr a 51.1 ± 0.72 9.35 ± 0.17 tr 14.7 ± 0.21 2.84 ± 0.23
11.6 1.75 2.52 60.1 6.66 1.11 4.66 1.51
± ± ± ± ± ± ± +_
0.04 0.02 0.08 0.08 0.17 0.10 0.09 0.02
1.42 ± tr tr 73.8 ± 2.42 ± 1.14 ± 8.64 +3.99 ±
4.07 2.17 20.6 6.89 5.57 1.64 6.26 13.4 2.33 2.05 6.81 5.39 16.3
2.29 1.67 13.8 5.45 2.94
± ± ± ± ± tr ± ± tr tr tr ± ±
0.13 0.07 0.35 0.27 0.10
1.76 + tr 7.44 ± 2.79 ± 2.79 ± tr 2.94 ± 5.41 ± tr 12.8 ± 6.69 ± 8.63 ± 45.0 ±
0.03
0.33 0.02 0.04 0.04 0.02
5.99 1.75 1.47 57.6 8.12 1.23 7.09 2.57
± ± ± ± ± ± ± ±
0.11 0.10 0.25 1.05 0.39 0.17 0.04 0.03
2.30 ± tr 1.60 ± 78.5 ± 1.98 ± 2.36 ± 3.23 ± 1.69 ±
4.75 2.97 28.0 5.55 2.37 1.09 9.37 16,7 1.25 2.17 1.29 4.17 15.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.05 0.70 0.19 0.05 0.09 0.23 1.84 0.16 0.12 0.02 0.08 0.27
1.83 ± tr 9.88 ± 2.29 ± 1.60 ± tr 2.71 ± 20.6 ± tr 9.92 ± 6,57 ± 2,55 ± 37.3 ±
0.05 0.06 1.74 0.31 0.38 0.21 0.22
sn-2 position (acyl chain) 14:0 15:0 16:0 16:1n-9 16:1n-7 17:0 18:0 18:1n-9 18:2n-6 20:4n-6 20:5n-3 22:5n-3 22:6n-3 ~The "tr" denotes below 1%. bGeometric isomers.
± ± ± ± ± ± ± ± ± ± ± ± ±
0.51 0.11 0.31 0.32 0.39 0.06 0.05 0.14 0.04 0.03 0.10 0.21 0.56
4.77 4.24
11.7 40.3
0.04 0.09
0.14 0.72
0.20 0.02 0.03 0.03 0,02 0.04 0.04 0.05 0.03 0.10
0.06 0.49 0.07 0.21 0.01 2.56 0.34 0.21 0.19 1.89
311
Glycerophospholipids of Cartilaginous Fish
TABLE 5. P r o m i n e n t a l k y i c h a i n c o m p o s i t i o n s ( o v e r 1%) i n t h e sn-1 a n d sn-2 p o s i t i o n s o f a l k y l . a c y l G P C i n s h a r k m u s c l e s ( m e a n -+ SD, w t % )
Hydrocarbon chain
Mackerel Blue
Thresher
Estuary
Ordinary
Dark
sn-1 position (alkyl chain) 14:0 15:0 16:0 16:1 16:1 18:0 18:1 a 18:1 a 18:1 a
11.4 1.45 34.3 6.40 16.5 1.45 3.05 20.1 1.73
-+ -+ ± + -+ -+ + ± ±
0.48 0.04 0.02 0.07 0.02 0.04 0.07 0.47 0.05
-+ 0.28 ± 0.05 -+ 0.43 -+ 0.22 + 0.25 tr b 2.61 -+ 0.09 6.51 -+ 0.07 1.32 - 0.02
6.19 1.03 30.1 4.87 25.8
sn-2 position (acyl chain) 14:0 16:0 16:1n-9 16:1n-7 17:0 18:0 18:1n-9 18:1n-7 18:2n-6 20:4n-6 20:5n-3 22:5n-3 22:6n-3
4.45 24.1 3.45 1.78 1.39 6.03 13.4 1.61 2.68 2.61 6.12 5.35 17.4
-+ + -+ ± -+ ± ± -+ -+ -+ -+ ±
0.10 0.12 0.11
2.31 16.6 10.1 2.03
5.31 -+ 0.68 24.8 -+ 0.04 6.21 _+ 0.68 tr 1.57 ± 0.02 8.33 -+ 0.04 8.07 ± 0.02 tr 1.15 _+ 0.00 5.77 -+ 0.03 2.62 _+ 0.06 6.03 _+ 0.05 19.5 ± 0.13
0.09 0.03 0.02 0.07 0.12 0.04 0.01 0.03 0.05 0.11
23.5 2.67 24.4 4.53 22.9
4.85 3.68
2.29 1.02 8.62 34.0
+ -+ ± --tr ± ± tr tr ± ± ± ±
0.01 0.04 0.05 0.03 0.03 0.02
0.06 0.05 0.14 0.34
± ± + -+ -+ tr 2.66 ± 10.6 ± 2.06 -+
0.06 0.01 0.21 0.05 0.17 0.07 0.12 0.22
16.5 1.72 28.8 1.37 18.5
± _+ _+ -+ _+ tr 2.88 ± 12.9 _+ 1.67 +
0.15 0.07 0.62 0.54 0.10
3.68 37.0 2.23 1.77 1.25 22.9 11.5 2.37
0.13 0.78 0.03 0.06 0.04 0.84 1.67
2.21 0.95 1.74 7.44
+ -+ _+ -+ ± _+ ± ± tr _+ ± -+ ±
9.18 2.51 27.3 2.26 27.1
± _+ ± _+ + tr 1.48 _+ 7.97 ± 2.45 _+
0.17 0.11 0.08
0.07 0.08 0.33 0.06 0.74 0.02 0.11 0.13
1.05 _+ 0.08 10.7 -+ 0.85 1.45 -+ 0.08 tr tr 1.97 _+ 0.07 9.78 -+ 0.56 tr tr 14.6 -+ 0.10 9.29 ± 0.06 3.36 _+ 0.17 42.8 + 1.71
0.29 0.07 0.10 0.04 0.24
aGeometric isomers. bThe "tr" denotes below 1%.
TABLE 6. P r o m i n e n t acyl c h a i n c o m p o s i t i o n ( o v e r 1%) in t h e sn.1 and sn-2 p o s i t i o n s o f d i a c y l . G P C in s h a r k m u s c l e s ( m e a n -+ SD, w t %)
Mackerel Acyl chain 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 20:4n-6 20:5n-3 22:4n-6 22:5n-3 22:6n-3
Blue 28.5 3.67 8.22 20.9 6.08 1.29 3.58
+ 0.15 -+ 0.01 + 0.04 +_ 0.12 -+ 0.06 --- 0.02 -+ 0.03 __a 3.53 + 0.37 8.33 _+ 0.07
~The "--" denotes not detected.
Thresher 25.9 1.59 7.81 6.52 5.51 2.66 2.41 1.46 7.96 28.2
_+ _+ _+ _+ _+ _+ _+ -+ _+ _+
0.03 0.00 0.01 0.01 0.01 0.01 0.01 0.00 0.12 0.10
Estuary 27.2 1.05 11.5 7.15 6.19 6.94 3.98 7.02 4.63 15.9
+ + _+ -+ + _+ -+ +_
0.16 0.01 0.03 0.01 0.01 0.01 0.01 0.04 0.04 0.13
Ordinary 48.7 7.04 4.24 8.16 3.19 3.97 4.88 0.31 2.05 10.3
_+ ± -+ + -4-+ ± -+ _+ _+
0.33 0.07 0.03 0.20 0.02 0.05 0.06 0.01 0.04 0.17
Dark 28.7 1.35 14.4 10.6 5.05 8.14 5.76 0.47 2.28 15.0
_+ 0.72 _+ 0.12 -+ 0.08 ___ 0.09 - 0.09 + 0.09 -+ 0.08 -+ 0.01 + 0.06 _+ 0.41
312
chains of carbon number between C14 and C18. Thus, this means the ether lipids have the potential to become a good source of ether-linked lipids, which are effective to improve the levels of membrane plasmalogens of the tissues of the patients suffering from certain genetic diseases such as a Zellweger cerebrohepatorenal syndrome. This work was supported financially in part by a Grant-in-Aid for Foreign Visiting Scholars Research from the Ministry of Education, Science and Culture of Japan. A Visiting Scholarship from the Japanese Promotion of Scientific Research to B.Y.J. is also acknowledged. Thanks are also ren&red to A. Takiguchi, Chiba Prefectural Fisheries Experimental Station, for providing shark specimens.
References 1. Blank, M. L.; Cress, E. A.; Smith, Z. L.; Snyder, F. Dietary supplementation with ether-linked lipids and tissue lipid composition. Lipids 26:166-169;1991. 2. Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911918;1959. 3. Das, A. K.; Holmes, R. D.; Wilson, G. N.; Hajra, A. K. Dietary ether lipid incorporation into tissue plasmalogens of humans and rodents. Lipids 27:401-405;1992. 4. De Koning, A. J. Phospholipids of marine origin IV. The abalone (Haliotis m/dae) J. Sci. Food Agric. 17:460-464;1966. 5. Dembitskii, V. M. Plasmalogens in phospholipids of marine invertebrates. Biol. Morya 5:86-90;1979. 6. Einig, R. G.; Ackman, R. G. Omega-3 PUFA in marine oil products. J. Am. Oil Chem. Soc. 64:499;1987. 7. Hanahan, D. J. ; Vercamer, R. The action of lecithinase D on lecithin. The enzymatic preparation of D-1,2-dipalmitolein and D-1,2-dipalmitin. J. Am. Chem. Soc. 76:1804-1806;1954. 8. Hanahan, D. J.; Dittmer, J. C.; Warashina, E. A column chromatographic separation of classes of phospholipides. J. biol. Chem. 228:685-700;1957. 9. Heymans, H. S. A.; Schutgens, R. B. H.; Tan, R.; van den Vosch, H. ; Tager, J. M. Severe plasmalogen deficiency in tissues of infants without peroxisomes (Zellweger syndrome). Nature 306:69-70; 1986. 10. Jeong, B. Y.; Ohshima, T.; Koizumi, C. Lipids, molecular species of 1-O-alk- l'-enyl-2-acyl, 1-O-alkyl-2-acyl- and 1,2-diacyl glycerophospholipids in Japanese Oyster Crassostrea gigas (Thunberg). 25:624-632;1990. 11. Jeong, B. Y.; Ohshima, T.; Koizumi, C. Hydrocarbon chain distribution of 1-O-alk-l'-enyl-2-acyl, 1-O-alkyl-2-acyl and 1,2diacyl glycerophospholipids in the muscle and viscera of the ascidian Halocynthia roretzi and in the gonad of the sea urchin Strongylocentrotus intermedius. (Submitted to Lipids. ; 1995.) 12. Joh, Y. G.; Hata, M. Studies on the lipids of abalone. II. The aldehyde composition of plasmalogen from abalone and some marine molluscs. Bull. Korean Fish. Soc. 12:181-189;1979.
Jeong et al.
13. Joseph, J. D. Lipid composition of marine and estuarine invertebrates. Part I1: Mollusca. Prog. Lipid Res. 21:109-153;1982. 14. Koizumi, C.; Jeong, B. Y.; Ohshima, T. Fatty chain composition of ether and ester glycerophospholipids in the Japanese oyster Crassostrea ~gas (Thunberg). Lipids 25:363-370;1990. 15. MacBrinn, M. C.; O'Brien, J. S. Lipid composition of optic nerve myelin. J. Neurochem. 16:7-12;1969. 16. O'Brien, J. S.; Sampson, E. L. Lipid composition of the normal human brain-gray matter, white matter, and myelin, (IV) fatty acid and fatty aldehyde composition of the major brain lipids in normal human gray matter, white matter, and myelin. J. Lipid Res. 6:545-551;1965. 17. Ohshima, T.; Ratnayake, W. M. N.; Ackman, R. G. Cod lipids, solvent systems and the effect of fatty acid chain length and unsaturation on lipid class analysis by Iatroscan TLC-FID. J. Am. Oil Chem. Soc. 24:363-370;1987. 18. Ohshima, T. ; Wada, S.; Koizumi, C. 1-O-alk- 1'-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids in white muscle of bonito Enthynnus pelamis (Linnaeus). Lipids 24:363-370;1989a. 19. Ohshima, T.; Wada, S.; Koizumi, C. Molecular species of 1-Oalk-l'-enyl-2-acyl glycerophospholipids of bonito white muscle. Nippon Suisan Gakaishi 55:885-890;1989b. 20. Panganamala, R. V.; Horrocks, L. A.; Geer, J. C. ; Cornwell, D. G. Positions of double bonds in the monounsaturated alk-1enyl groups from the plasmalogen of human heart and brain. Chem. Phys. Lipids 6:97-102;1971. 21. Pocock, D. M. E.; Marsden, J. R.; Hamilton, J. G. The presence of alkenyl and alkyl glycerides, and choline and ethanolamine plasmalogens in a marine worm. Comp. Biochem. Physiol. 30: 133-136;1969. 22. Ratnayake, W. M. N.; Timmins, A.; Ohshima, T.; Ackman, R. G. Mass spectra of fatty acid derivatives, of isopropylidenes of novel glyceryl ethers of cod muscle and of phenolic acetates obtained with the Finnigan Mat Ion Trap Detector. Lipids 21: 518-524;1986. 23. Reichwald, I.; Mangold, H. K. Nutr. Metab. Suppl. 21:198201;1977. 24. Renkonen, O. Individual molecular species of phospholipids III. Molecular species of ox-brain lecithins. Biochim. Biophys. Acta 125:298-309; 1966. 25. Sargent, J. R. Ether linked glycerides in marine animals. In Marine Biogenic Lipids, Fats and Oils (Edited by Ackman, R. G.), Vol. I, Boca Raton, Florida: CRC Press; 1989: pp. 175-197. 26. Smith, G. M.; Djerassi, C. Phospholipid studies of marine organisms: 14. Ether lipids of the sponge Tethya aurantia. Lipids 22:236-240; 1987. 27. Spener, F.; Mangold, H. K. Composition of alkoxylipids of human heart and aorta. J. Lipid Res. 10:609-613;1969. 28. Sugiura, T. ; Fukuda, T. ; Miyamoto, T. ; Waku, K. Distribution of alkyl and alkenyl ether-linked phospholipids and plateletactivating factor-like lipid in various species of invertebrates. Biochim. Biophys. Acta 1126:298-308;1992. 29. Weber, N. Metabolism of orally administered rac-l-O-[l'-14C] dodecylglycerol in mice. J. Lipid Res. 26:1412-1420;1985.
ISSN 0305-0491/96/$26.00 SSDI 0305-0491(95)02020-L
ELSEVIER
Lipids of Cartilaginous Fish: Composition of Ether and Ester Glycerophospholipids in the Muscle of Four Species of Shark Bo-Young Jeong,* Toshiaki Ohshima, "PHideki Ushioi- and Chiaki Koizumi-f "DEPARTMENT OF FOOD SCIENCE, GYEONGSANGNATIONAL UNIVERSITY, TONGYEONG, GYEONGNAM, 650-160, KOREA AND tDEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY, THE TOKYO UNIVERSITYOF FISHERIES, KONaN 4, MINATO-KU, TOKYO 108, JAPAN
ABSTRACT. The levels and hydrocarbon chain distributions of alk-l'-enyl-acyl, alkyl-acyl and diacyl subclasses of choline glycerophospholipid (GPC) and ethanolamine glycerophospholipid (GPE) in the muscles of four species of shark were investigated. The prominent subclasses in the EGP fraction were 1-O-alk-1 '-enyl-2acyl-sn-glycero-3-phosphoethanolamine (alk-l'-enyl-acyl-GPE) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (diacyl-GPE), accounting for 24.8% to 54.4% and 40.8% to 69.4%, respectively, of the total glycerophospholipids. In the GPC fraction, 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) was the most prominent subclass, ranging from 73.2% to 94.7% in the total glycerophospholipids. The levels of l-O-alkyl-2acyl-sn-glycero-3-phosphoethanolamine (alkyl-acyl-GPE), -phosphocholine (alkyl-acyl-GPC) and alk-l'-enylacyl-GPC were below 15% in the corresponding subclasses. The prominent alk-l'-enyl and acyl chains of the alk-l'-enyl-acyl-GPE were 16:0 (42.3 to 63.4%), 18:1 (10.0 to 30.0%) and 18:0 (5.7 to 10.9%) alk-1'-enyl chains, and 22:6n-3 acyl chain (48.8 to 75.7%). In the alkyl-acyl-GPE fraction, the prominent alkyl and acyl chains were similar to those of the alk-l'-enyl-acyl-GPE, but the levels of alkyl and acyl chains with 16 carbons were higher. The hydrocarbon chain compositions of alk-l'-enyl-acyl- and alkyl-acyl-GPC were also similar to those of the alkyl-acyl-GPE. The prominent acyl chains of diacyl-GPE and -GPC were 18:0, 16:0 and 22:6n-3, but the GPE fraction was richer in 18:0 acyl chain and the GPC fraction was richer in 16:0. These characteristics in alk-l'-enyl and alkyl chains of ether glycerophospholipids from shark muscles suggested that these muscles are suitable as sources of dietary ether-linked lipids for a certain disease that prints syndrome of deficiency in ether-linked lipids. COMPBIOCHEMPHYSIOL113B, 305-312, 1996. KEY WORDS. Ether lipids, plasmalogens, alkyl chains, alk-l'-enyl chains, cartilaginous fish, shark muscle
INTRODUCTION Marine organisms, especially invertebrates, are rich in ether glycerophospholipids (13,25). Recently, Sugiura et al. (28) reported that most species examined in various phyla other than Arthropoda contained large amounts of 1-O-alk-1 '-enyl2-acyl-sn-glycero-3-phosphocholine (alk-l'-enyl-acyl-GPC) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and that the levels of both ether glycerophospholipids were markedly high in the lower phyla. Koizumi and coworkers (10,11,14) also found high levels of ether glycerophospholipids in Pacific oyster wet organs, sea urchin gonads and ascidian muscle and viscera, and revealed the detailed hydrocarbon chain composition of the lipids. A small amount of ether glycerophospholipids is also reported in the skipjack Gyrrespondence to: T. Ohshima, Department of Food Science and Technology, The Tokyo University of Fisheries, Konan 4, Minato-ku, Tokyo 108, Japan. Tel. 03-5463-0613; Fax 03-5463-0627. Received 10 January 1995; revised 27 June 1995; accepted 29 June 1995.
white muscle (18,19) and in the Atlantic cod muscle (22). In this context, we have found high levels of ether glycerophospholipids in the muscles of certain species of shark. Although l-O-alkyl-2-acetyl-GPC has been reported to have a precise function as a platelet activating factor, physiological roles for other analogues of glycerophospholipid subclasses are still obscure. In certain genetic diseases such as a Zellweger cerebrohepatorenal syndrome, ether-linked lipids are deficient almost completely in the tissues of the patients (9). On the other hand, it is well known that dietary etherlinked lipids are incorporated into membrane ether-linked lipids through intestinal membrane cells, and further incorporated into plasmalogens in these tissues (1,3). These findings suggest that shark muscle might be a good source for dietary ether-linked lipids for patients suffering from ether-linked lipids deficiency syndrome. To clarify this speculation, we investigated, in this study, the lipid class levels and hydrocarbon chain distributions of glycerophospholipids in the muscles of four species of shark.
306
Jeong et al.
MATERIALS AND METHODS Anima/s
and alkyl-acyl-GPE and -GPC, and those in the sn-1 and sn-2 positions of diacyl-GPE and -GPC were quantified after
Four species of shark, blue shark Prionance glauca, thresher shark Alopias vulpinus, estuary shark Chacharhinus plumbeus and mackerel shark Lamna ditropis, were used in this study. These specimens were caught in the Pacific Ocean, off Choushi City, Chiba Prefecture, Japan, in April and May, 1993. Three separated portions of white muscles, weighing 200 g each, were taken from the dorsal parts of one specimen of each shark species within 24 hr after catch, and stored at - 80°C until used for analyses. The analysis of phospholipids of each shark's white muscles was repeated twice. The dark muscle of the mackerel shark was also analyzed in triplicate, since the muscle of this species is richer in dark muscle compared to the other three species. The contents of the glycerophospholipid subclasses were calculated from the amounts of the fatty acids (14).
conversion in methyl esters by gas-liquid chromatography (GLC) using 23:0 fatty acid of more than 99% purity (NuChek-Prep, Elysian, MN, U.S.A.) as an internal standard (6).
The FAMEs were analyzed by GLC using a Shimadzu GC 12APF or GC8APF instrument equipped with a fused silica open tubular column coated with a polar phase (SUPELCOWAX-10, 0.25/,m in film thickness, 30 m × 0.25 mm i.d., Supelco Japan, Ltd., Tokyo) and a flame ionization detector (FID). The injector and FID were held at 250°C. The column oven was programmed from 170 to 230°C at l°C/min.
Lipid Extraction and Fractionation
Determination of Alk. 1 '-enyl and Alkyl Chains Distributions
The shark muscle was minced and extracted with chloroform/ methanol according to the Bligh and Dyer procedure (2). The ethanolamine glycerophospholipid and choline glycerophospholipid classes in the extracted total lipids were separated by column chromatography on Silica Gel 60 (Merck, Darmstadt, Germany) according to the method of Hanahan et al. (8).
The alk-1 '-enyl and alkyl chains of 1-O-alk-1 '-enyl-2,3-TMSglycerol and 1-O-alkyl-2,3-TMS-glycerol derivatives were determined by GLC using a Shimadzu GC8APF instrument (Shimadzu, Kyoto, Japan) equipped with a SUPELCOWAX-10 fused silica open tubular column (30 m × 0.25 mm i.d., Supelco Japan Ltd., Tokyo). The injector and FID were held at 250°C and column temperature was programmed from 160 to 230°C at 4°C/min (18).
Quantitation of Phospholipid Classes Quantitative analysis of phospholipid classes was carried out in duplicate for each lipid sample using Chromarod S-IIl and an Iatroscan MK-5 TLC-FID analyzer (Dia-Iatron Co., Tokyo, Japan) according to the method of Ohshima et al.
(17). Der/~at/zat/on for Determination of Alk.1 '-enyl, Alkyl and Acyl Chains Distribution The GPE and GPC fractions were each hydrolyzed with phospholipase C (from Bacillus cereus, Boehringer Mannheim, Mannheim, Germany) to obtain the corresponding 1,2diradylglycerols (7). These were purified by preparative TLC on silicic acid and then acetylated (24). The acetylated glycerols were separated into subclasses by preparative TLC on silicic acid with petroleum ether/diethyl ether/acetic acid (85 : 15 : 1, v/v/v). The isolated and purified acetylglycerols were saponified to 1-O-alk-l'-enyl-glycerols and 1-O-alkylglycerols, followed by extraction with diethyl ether and conversion to the corresponding trimethylsilyl (TMS) ether derivatives, i.e. 1-O-alk-l'-enyl-2,3-TMS-glycerol and 1-Oalkyl-2,3-TMS-glycerol (18). After acidification, the sn-2 fatty acids liberated from the 1-O-alk-l'-enyl-2-acylglycerols and 1-O-alkyl-2-acylglycerols were recovered with diethyl ether. The fatty acids in the sn-2 positions of alk-l'-enyl-acyl-
Determination of Fatty Acid Distribution in Alk-1 '.enyl.acyl., Alkyl.acyl. and D/acyl-GPC and GPE
RESULTS
Lipid Class Contents and Alk-I '.enyl.acyl, Alkyl.acyl and Diacyl Glycerophospholipids Composition The lipid class contents of the shark muscle of the four species are shown in Fig. 1. The total lipids (TL) contents of the muscles of P. glauca, A. vulpinus and C. plumbens were about 0.6%, and were lower compared to those of L. ditropis white (2.7%) and dark (1.7%) muscles. The glycerophospholipids (PL) comprised >90% of the TL in the white muscle of P. glauca, A. vulpinus and C. plumbens, but were only 40% of the TL in the white muscle of L. ditropis. The major PL classes in all samples were GPC (50.8 to 70.0% of phospholipids) and GPE (15.7 to 37.2% of phospholipids). The composition of phospholipid subclasses is shown in Fig. 2. The major subclasses in the GPE of all shark muscles examined were alk-l'-enyl-acyl-GPE and diacyl-GPE, although the lipids of P. glauca contained a small amount of alkyl-acyl-GPE. The major subclass in GPC was diacyl-GPC, and alk-1 '-enyl-acyl- and alkyl-acyl-GPC were found as minor components. The level of alkyl.acyl-GPC was low in any of the muscles of the sharks, although this glycerophospholipid subclass is usually found in equal amounts to that of the alk-1 'enyl-acyl-GPE.
Glycerophospholipids of Cartilaginous Fish
307
3
~ 2.5
Neutrallipid
~
DPG
GPC
~
GPI
r~
"~
2
g 1.5 or) r.c) el)
~
1
° ,,,,~
0.5
0 P. glauca
A. vulpinus
C. plumbens
L. ditropis White Dark
FIG. 1. Lipid class contents of the shark muscles. GPC, choline glycerophospholipid; EGP, ethanolamine glycerophospholipid; SPM, sphingomyelin; DPG, diphosphatidyl glycerol; GPI, inositol glycerophospholipid; LGPC, choline lysoglycerophospholipid; GPS, serine glycerophospholipid.
A 100 90 80 70 60 50 40 30 20 10 0
B 100 90 80 70 60 ~ 50 ~
4o-~ 30 ~ 20 10 P. glauca
C. plumbens L. ditropis A. vulpinus White Dark
•
AIk-l'-enyl-acyl
P. glauca
[]
C, plumbens L. ditropis A. vulpinus White Dark
Alkyl-acyl
[]
Diacyl
FIG. 2. Subclass composition of ethanolamine (A) and choline (B) glycerophospholipids of the shark muscles. Alk.l'-enyl-acyl, 1-O-alk-l'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine and .choline; 1,2-diacyl, 1,2-diacyl-sn-3-phosphoethanolamine and -choline.
308
Alk. 1 '.enyl, Alkyl and Acyl Chains
Distribution of Glycerophospholipid Subclasses The hydrocarbon chain compositions of alk-l'-enyl-acyl, alkyl-acyl and diacyl subclasses of GPE in the shark muscles are shown in Tables 1 to 3. For the alk-1 '-enyl-acyl-GPE of any of the shark muscles (Table 1), 16:0, 18:1 and 18:0 alk-l'-enyl chains in the sn-1 position were prominent; particularly the level of 16:0 alk-l'-enyl chain was high, amounting to 42.3 to 63.9. The profiles of acyl chains in the sn-2 position were different among the species of shark. However, in all shark muscles, 22:6n-3 was commonly the most prominent acyl chain, ranging from 48.8 to 75.7%. From the results of analyses of these shark muscle glycerophospholipids, the most prominent molecular species in the alk-l'-enyl-acyl-GPE would be expected to be 16:0 alk-1'-enyl-22:6n-3. The alkyl chain compositions of the alkyl-acyl-GPE were quite simple in the all shark muscles (Table 2); most prominent alkyl and acyl chains were 16:0, 16: 1, 18:0 and 18:1 alkyl chains, and 22:6n-3 and 16:0 acyl chains, respectively. The level of 16:0 alkyl chain was especially high in the alkyl chains, ranging from 46.7 to 48.9% in all four species. From these results, the most prominent molecular species of the alkyl-acyl-GPE is presumably 16:0 alkyl-22:6n-3 and 16:0 alkyl- 16 : 0. For the diacyl-GPE (Table 3), most prominent acyl chains in both the sn-1 and the sn-2 positions were very simple; 22:6n-3 and 18:0, accounting respectively for 23.5 to 32.5% and 18.0 to 39.0%. Therefore, the major molecular species was expected to be 18:0-22:6n-3. The hydrocarbon chain compositions of alk-l'-enyl-acyl, alkyl-acyl and diacyl subclasses in the shark muscle GPC are shown in Tables 4-6. For the alk-l'.enyl-acyl-GPC (Table 4), most prominent alk-l'-enyl chains were 16:0, 18: 1, 16:1 and 14:0. The levels of 16:0 alk-1 '-enyl chain were especially high in this lipid subclass of all four shark, accounting for 51.1 to 78.5% of these chains. The compositions of acyl chains in the sn-2 position were different from species to species, while 22:6n-3 and 16:0 were found to be relatively important among the prominent acyl chains. These characteristic profiles of the alk-l'-enyl-acyl-GPC were similar to those of the alkyl-acyl-GPE. From these results, the prominent molecular species of the alk-l'-enyl-acyl-GPC can be predicted to be 16 : 0 alk- 1'-enyl chain-22 : 6n-3. As shown in Table 5, the level of 16 : 0 alkyl chain of the alkyl-acyl-GPC was the highest of the alkyl chains, accounting for 24.4 to 34.3%. Other prominent alkyl chains were 16 : 1, 18 : 1 an 14 : 0. The prominent acyl chains of the alkylacyl-GPC were 22 : 6n-3 and 16 : 0. Therefore, the most probable molecular species are 16:0 alkyl chain-22:6n-3 and 16:0 alkvl chain-16 : 0. The prominent fatty acyl chains in the sn-1 and the sn-2 positions of diacyl-GPC were 16:0, 22:6n-3, 18:0 and 18: ln-9 (Table 6). The level of 16:0 of the diacyl-GPC was about 3-fold higher than that of the diacyl-GPE. The level of 22 : 6n-3 was much lower compared to that of the diacyl-GPE.
Jeong et al.
The major molecular species of the diacyl-GPC would be expected to be 16:0-16:0, 16:0-22:6n-3, 18:0-22:6n-3 and 18 : ln-9-22 : 6n-3. DISCUSSION The present study demonstrates that the levels of ether glycerophospholipids in the muscle of four sharks were higher than those of muscles of bony fish, including those of bonito Katsuwonus pelamis (3.6 to 7.6%) (18,19) and Atlantic cod Gadus morhua (22). However, the levels of ether glycerophospholipids of invertebrates are much higher than those of the shark muscles. The proportion of alk-l'-enyl-acyl-GPE in the shark muscle phospholipids was lower than those of marine invertebrates, including the Pacific oyster (59.8%) (10,14), ascidian (69.0%) (11), and annelida, mollusca, platyhelminthes, and porifera (53.3 to 88.7%) (13,28). High levels of alk-l'-enyl-acyl-GPE can be found in other marine organisms including marine worms (21), abalone (4), snails and bivalves (12), sponges (26), and several species of invertebrates (5). In mammals, high levels of ether glycerophospholipids exist in specific organs such as heart (20,27) and particularly brain (15,16). The alk-l'-enyl and acyl chains compositions were similar to those of the bonito white muscle (18), thus being different from those of marine invertebrates. In the latter the prominent acyl chains of alk-l'-enyl-acyl-GPE, for example in the Pacific oyster wet organs (10,14), in the sea urchin gonads and in the ascidian muscle and viscera (11), were 20:5n-3, 22 : 2NMID (non-methylene-interrupted diene) and 20 : 4n-6. The alk-l'-enyl chain distribution was similar to that of the bonito white muscle lipids (18), but definitely different from lipids of certain marine invertebrates in which 18:0 alk-l'enyl chain was the prominent (10,14,28). The alkyl chain profiles of the alkyl-acyl-GPC in the shark muscles were very similar to those of the Pacific oyster (10,14) and of the bonito white muscle (18), but different from those of the sea urchin gonads and the ascidian muscle (11). When 1-O-heptadecyl-rac-glycerol was fed to rats, 46 to 60% incorporation of the Cl7-moiety in the sn. 1 position (this includes a carbon in the glycerol moiety) of ethanolamine plasmalogens accumulated in most tissues of the rats, however, the plasmalogen level in the tissues remained unchanged (3). The alkyl glyceryl ether level increased in liver, kidney and lung of rats to which 1-O-alkyl-2,3-diacetyl-sn-glycerol was fed (1). Therefore, it is possible to say that dietary alkyl glycerol ethers are absorbed, converted to plasmalogens in an intestinal tissue, and transported to other tissues where they are metabolized and deposited as membrane plasmalogens (3). However, the incorporation rate of ether lipids depends on the chain ler~gth of hydrocarbon chain; the lipids with saturated and monounsaturated alkyl chain length between C14 and CI8 are only incorporated into glycerol ether lipids in an intestinal tissue (1,3,23,29). The present study revealed that shark muscle glycerophospholipids have alkyl and alk-l'-enyl
309
Glycerophospholipids of Cartilaginous Fish
TABLE 1. P r o m i n e n t a l k - l ' e n y i a n d acyl c h a i n c o m p o s i t i o n ( o v e r 1%) in t h e sn-1 a n d sn-2 p o s i t i o n s o f a l k - l ' , e n y l , a c y l G P E in s h a r k m u s c l e s ( m e a n - SD, w t %)
Hydrocarbon chain sn- 1 position (alk- l'-enyl 14:0 16:0 16:1 17:0 iso 18:0 18: la b 18:1b b
Mackerel Blue chain) 1.48 ± 42.3 --3.17 ± 1.53 ± 10.5 ± 30.0 ± 4.31 ±
Thresher
Estuary
Ordinary
Dark
0.05 0.36 0.08 0.10 0.11 0.32 0.03
3.74 60.4 2.65 2.81 8.02 9.98 3.43
± -+ + + -+ -+ -+
0.03 0.12 0.07 0.02 0.02 0.01 0.05
0.58 + 0.00 58.2 -+ 0.03 tr a 2.93 -+ 0.01 10.9 -+ 0.03 13.1 -+ 0.05 5.89 -+ 0.02
2.59 63.9 3.49 2.23 5.33 11.7 4.49
± ± ± ± + __. -4-
0.06 0.33 0.01 0.20 0.04 0.09 0.04
1.08 57.3 1.34 3.55 9.33 12.7 4.54
-+ -+ + -+ ± ± ±
0.03 0.07 0.10 0.06 0.20 0.22 0.07
sn-2 position (acyl chain) 16:0 6.56 ± 0.23 16:ln-7 2.92 + 0.07 18:0 2.03 ± 0.03 18:1n-9 8.65 ± 0.22 20:4n-6 3.70 ± 0.04 20:5n-3 6.85 ± 0.04 22:5n-3 8.31 ± 0.21 22:6n-3 48.8 ± 0.79
1.23 0.40 0.43 2.10 2.60 0.40 10.8 75.7
+ -4-+ ± ± -+ -+ -+
0.13 0.02 0.01 0.07 0.04 0.03 0.26 0.48
2.02 1.23 0.73 8.04 9.65 3.10 4.99 64.9
6.55 1.86 1.80 7.46 4.43 0.93 9.53 52.4
+ _ -+ ± ± ±
0.15 0.06 0.00 0.05 0.03 0.01 0.11 0.54
2.83 _+ tr 0.79 --_ 9.38 ± 16.0 ± 3.69 ± 1.96 + 55.0 -+
0.24
+ + ± ± ± ± ± -+
0.18 0.26 0.01 0.08 0.08 0.03 0.44 0.33
0.04 0.55 0.15 0.03 0.04 1.25
~The "tr" denotes below 1%. bGeometric isomers.
TABLE 2. P r o m i n e n t a l k y l a n d a c y l c h a i n c o m p o s i t i o n ( o v e r 1%) i n t h e sn.1 a n d sn.2 p o s i t i o n s o f a l k y l - a c y l G P E in s h a r k m u s c l e s ( m e a n -+ SD, w t %)
Hydrocarbon chain
Mackerel Thresher
Estuary
sn-1 position (alkyl chain) 14:0 15:0 iso 15:0 16:0 16:1 17:0 iso 17:0 anteiso 17:0 18:0 18:1 c 18:1 c 18:V
7.64 1.85 1.38 46.9 11.9 2.01 1.10 1.71 3.16 4.05 7.65 3.59
± ± -+ -+ -+ _+ _+ -+ ± -+ ± ±
0.15 0.00 0.09 0.32 0.10 0.20 0.11 0.08 0.02 0.13 0.12 0.07
1.09 -+ a -46.8 ± 7.53 ± 1.97 ± 1.58 1.06 +10.2 -+ -11.1 -+ 5.57 +
sa-2 position (acyl chain) 14:0 16:0 16:1n-9 16:ln-7 17:0 18:0 18:1n-9 20:3n-3 20:4n-6 22:5n-3 22:6n-3
1.65 10.4 1.32 1.42 1.22 6.62 4.31 2.77 1.58 16.8 43.9
_+ +-+ + -+ + -+ ± -+ +
0.13 0.04 0.06 0.03 0.03 0.02 0.02 0.03 0.04 0.04 0.33
~'The "--" denotes not detected. bThe "tr" denotes below 1%. cOeometric isomers.
0.01
Ordinary
Dark
0.12 0.11
4.58 + 0.18 1.10 + 0.02 tr b 48.9 -+ 0.25 11.9 + 0.06 1.62 _+ 0.02 1.27 - 0.01 tr 2.26 +_ 0.02 2.37 ± 0.02 16.7 + 0.15 5.14 + 0.17
3.18 1.26 1.46 46.7 9.82 2.47 1.83 1.09 4.65 4.17 12.6 5.63
7.28 -+ 0.02 22.3 ± 0.17 7.01 ± 0.31 -1.64 -+ 0.01 9.29 -+ 0.03 11.0 -+ 0.05 -3.39 + 0.08 5.89 ± 0.01 23.7 ± 0.08
5.31 -+ 0.28 14.4 ± 0.57 tr 15.2 ± 0.67 tr 5.50 + 0.06 6.76 + 0.09 -0.96 + 0.05 8.91 + 0.29 32.2 + 1.21
1.96 +-- 0.01 10.5 + 0.11 4.28 + 0.09 -tr 4.65 + 0.04 7.71 + 0.67 2.11 +_ 0.03 7.45 -+ 0.19 1.92 -+ 0.15 50.9 + 0.15
0.07 0.01 0.04 0.04 0.02 0.03
+ + + ---+ -+ + +-+ + + -+
0.16 0.05 0.31 0.24 0.18 0.21 0.21 0.06 0.09 0.06 0.05 0.16
310
Jeong et al.
TABLE 3. Prominent acyl chain composition (over 1%) in the sn-1 and sn-2 positions of diacyl-GPE in shark muscles (mean _+ SD, wt %) Mackerel Acyl chain
Blue
16:0 18:0 18:1n-9 18:1n-7 20:ln-ll 20:1n-9 20:4n-6 20:5n-3 22:5n-3 22:6n-3
6.29 28.5 8.19 3.71 1.18 1.89 2.85 3.40 5.61 30.6
± ± ± ± ± ± ± ± ± +
Thresher
0.06 0.17 0.05 0.02 0.01 0.03 0.01 0.01 0.03 0.15
9.10 35.7 2.81 5.07
1.21 2.13 3.23 32.5
± 0.37 ± 0.49 ± 0.05 ± 0.16 tr ~ ± 0.03 ± 0.00 tr ± 0.14 ± 0.89
Estuary 6.12 35.3 4.21 7.27
4.87 1.06 4.11 23.5
± ± ± ± tr tr m ± ± ±
Ordinary
0.03 0.67 0.11 0.18
16.2 18.0 5.97 9.25
0.02 0.03 0.15 0.84
1.17 5.27 1.97 3.90 28.6
± ± ± ± tr ± ± ± ± +
Dark
0.62 0.24 0.09 0.19
5.67 39,0 4.27 2.76
0.01 0.09 0.03 0.15 1.05
8.59 2.23 1.95 28.9
± ± ± +_ tr tr ± +_ ± ±
0.12 0.34 0.08 0.07
0.05 0.02 0.03 0.47
aThe "tr" denotes below 1%.
TABLE 4. Prominent alk-l'-enyl and acyl chain composition (over 1%) in the sn-1 and sn-2 positions of alkenylacyl GPC in shark muscles (mean +-- SD, wt %) Hydrocarbon chain sn-1 position 14:0 15:0 iso 15:0 16:0 16:1 17:0 iso 18:1 b 18:1 b
Mackerel Blue
Thresher
Estuary
Ordinary
Dark
(alk-l'-enyl chain) 5.18 ± 0.23 1.23 ± 0.04 tr a 51.1 ± 0.72 9.35 ± 0.17 tr 14.7 ± 0.21 2.84 ± 0.23
11.6 1.75 2.52 60.1 6.66 1.11 4.66 1.51
± ± ± ± ± ± ± +_
0.04 0.02 0.08 0.08 0.17 0.10 0.09 0.02
1.42 ± tr tr 73.8 ± 2.42 ± 1.14 ± 8.64 +3.99 ±
4.07 2.17 20.6 6.89 5.57 1.64 6.26 13.4 2.33 2.05 6.81 5.39 16.3
2.29 1.67 13.8 5.45 2.94
± ± ± ± ± tr ± ± tr tr tr ± ±
0.13 0.07 0.35 0.27 0.10
1.76 + tr 7.44 ± 2.79 ± 2.79 ± tr 2.94 ± 5.41 ± tr 12.8 ± 6.69 ± 8.63 ± 45.0 ±
0.03
0.33 0.02 0.04 0.04 0.02
5.99 1.75 1.47 57.6 8.12 1.23 7.09 2.57
± ± ± ± ± ± ± ±
0.11 0.10 0.25 1.05 0.39 0.17 0.04 0.03
2.30 ± tr 1.60 ± 78.5 ± 1.98 ± 2.36 ± 3.23 ± 1.69 ±
4.75 2.97 28.0 5.55 2.37 1.09 9.37 16,7 1.25 2.17 1.29 4.17 15.0
± ± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.05 0.70 0.19 0.05 0.09 0.23 1.84 0.16 0.12 0.02 0.08 0.27
1.83 ± tr 9.88 ± 2.29 ± 1.60 ± tr 2.71 ± 20.6 ± tr 9.92 ± 6,57 ± 2,55 ± 37.3 ±
0.05 0.06 1.74 0.31 0.38 0.21 0.22
sn-2 position (acyl chain) 14:0 15:0 16:0 16:1n-9 16:1n-7 17:0 18:0 18:1n-9 18:2n-6 20:4n-6 20:5n-3 22:5n-3 22:6n-3 ~The "tr" denotes below 1%. bGeometric isomers.
± ± ± ± ± ± ± ± ± ± ± ± ±
0.51 0.11 0.31 0.32 0.39 0.06 0.05 0.14 0.04 0.03 0.10 0.21 0.56
4.77 4.24
11.7 40.3
0.04 0.09
0.14 0.72
0.20 0.02 0.03 0.03 0,02 0.04 0.04 0.05 0.03 0.10
0.06 0.49 0.07 0.21 0.01 2.56 0.34 0.21 0.19 1.89
311
Glycerophospholipids of Cartilaginous Fish
TABLE 5. P r o m i n e n t a l k y i c h a i n c o m p o s i t i o n s ( o v e r 1%) i n t h e sn-1 a n d sn-2 p o s i t i o n s o f a l k y l . a c y l G P C i n s h a r k m u s c l e s ( m e a n -+ SD, w t % )
Hydrocarbon chain
Mackerel Blue
Thresher
Estuary
Ordinary
Dark
sn-1 position (alkyl chain) 14:0 15:0 16:0 16:1 16:1 18:0 18:1 a 18:1 a 18:1 a
11.4 1.45 34.3 6.40 16.5 1.45 3.05 20.1 1.73
-+ -+ ± + -+ -+ + ± ±
0.48 0.04 0.02 0.07 0.02 0.04 0.07 0.47 0.05
-+ 0.28 ± 0.05 -+ 0.43 -+ 0.22 + 0.25 tr b 2.61 -+ 0.09 6.51 -+ 0.07 1.32 - 0.02
6.19 1.03 30.1 4.87 25.8
sn-2 position (acyl chain) 14:0 16:0 16:1n-9 16:1n-7 17:0 18:0 18:1n-9 18:1n-7 18:2n-6 20:4n-6 20:5n-3 22:5n-3 22:6n-3
4.45 24.1 3.45 1.78 1.39 6.03 13.4 1.61 2.68 2.61 6.12 5.35 17.4
-+ + -+ ± -+ ± ± -+ -+ -+ -+ ±
0.10 0.12 0.11
2.31 16.6 10.1 2.03
5.31 -+ 0.68 24.8 -+ 0.04 6.21 _+ 0.68 tr 1.57 ± 0.02 8.33 -+ 0.04 8.07 ± 0.02 tr 1.15 _+ 0.00 5.77 -+ 0.03 2.62 _+ 0.06 6.03 _+ 0.05 19.5 ± 0.13
0.09 0.03 0.02 0.07 0.12 0.04 0.01 0.03 0.05 0.11
23.5 2.67 24.4 4.53 22.9
4.85 3.68
2.29 1.02 8.62 34.0
+ -+ ± --tr ± ± tr tr ± ± ± ±
0.01 0.04 0.05 0.03 0.03 0.02
0.06 0.05 0.14 0.34
± ± + -+ -+ tr 2.66 ± 10.6 ± 2.06 -+
0.06 0.01 0.21 0.05 0.17 0.07 0.12 0.22
16.5 1.72 28.8 1.37 18.5
± _+ _+ -+ _+ tr 2.88 ± 12.9 _+ 1.67 +
0.15 0.07 0.62 0.54 0.10
3.68 37.0 2.23 1.77 1.25 22.9 11.5 2.37
0.13 0.78 0.03 0.06 0.04 0.84 1.67
2.21 0.95 1.74 7.44
+ -+ _+ -+ ± _+ ± ± tr _+ ± -+ ±
9.18 2.51 27.3 2.26 27.1
± _+ ± _+ + tr 1.48 _+ 7.97 ± 2.45 _+
0.17 0.11 0.08
0.07 0.08 0.33 0.06 0.74 0.02 0.11 0.13
1.05 _+ 0.08 10.7 -+ 0.85 1.45 -+ 0.08 tr tr 1.97 _+ 0.07 9.78 -+ 0.56 tr tr 14.6 -+ 0.10 9.29 ± 0.06 3.36 _+ 0.17 42.8 + 1.71
0.29 0.07 0.10 0.04 0.24
aGeometric isomers. bThe "tr" denotes below 1%.
TABLE 6. P r o m i n e n t acyl c h a i n c o m p o s i t i o n ( o v e r 1%) in t h e sn.1 and sn-2 p o s i t i o n s o f d i a c y l . G P C in s h a r k m u s c l e s ( m e a n -+ SD, w t %)
Mackerel Acyl chain 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 20:4n-6 20:5n-3 22:4n-6 22:5n-3 22:6n-3
Blue 28.5 3.67 8.22 20.9 6.08 1.29 3.58
+ 0.15 -+ 0.01 + 0.04 +_ 0.12 -+ 0.06 --- 0.02 -+ 0.03 __a 3.53 + 0.37 8.33 _+ 0.07
~The "--" denotes not detected.
Thresher 25.9 1.59 7.81 6.52 5.51 2.66 2.41 1.46 7.96 28.2
_+ _+ _+ _+ _+ _+ _+ -+ _+ _+
0.03 0.00 0.01 0.01 0.01 0.01 0.01 0.00 0.12 0.10
Estuary 27.2 1.05 11.5 7.15 6.19 6.94 3.98 7.02 4.63 15.9
+ + _+ -+ + _+ -+ +_
0.16 0.01 0.03 0.01 0.01 0.01 0.01 0.04 0.04 0.13
Ordinary 48.7 7.04 4.24 8.16 3.19 3.97 4.88 0.31 2.05 10.3
_+ ± -+ + -4-+ ± -+ _+ _+
0.33 0.07 0.03 0.20 0.02 0.05 0.06 0.01 0.04 0.17
Dark 28.7 1.35 14.4 10.6 5.05 8.14 5.76 0.47 2.28 15.0
_+ 0.72 _+ 0.12 -+ 0.08 ___ 0.09 - 0.09 + 0.09 -+ 0.08 -+ 0.01 + 0.06 _+ 0.41
312
chains of carbon number between C14 and C18. Thus, this means the ether lipids have the potential to become a good source of ether-linked lipids, which are effective to improve the levels of membrane plasmalogens of the tissues of the patients suffering from certain genetic diseases such as a Zellweger cerebrohepatorenal syndrome. This work was supported financially in part by a Grant-in-Aid for Foreign Visiting Scholars Research from the Ministry of Education, Science and Culture of Japan. A Visiting Scholarship from the Japanese Promotion of Scientific Research to B.Y.J. is also acknowledged. Thanks are also ren&red to A. Takiguchi, Chiba Prefectural Fisheries Experimental Station, for providing shark specimens.
References 1. Blank, M. L.; Cress, E. A.; Smith, Z. L.; Snyder, F. Dietary supplementation with ether-linked lipids and tissue lipid composition. Lipids 26:166-169;1991. 2. Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911918;1959. 3. Das, A. K.; Holmes, R. D.; Wilson, G. N.; Hajra, A. K. Dietary ether lipid incorporation into tissue plasmalogens of humans and rodents. Lipids 27:401-405;1992. 4. De Koning, A. J. Phospholipids of marine origin IV. The abalone (Haliotis m/dae) J. Sci. Food Agric. 17:460-464;1966. 5. Dembitskii, V. M. Plasmalogens in phospholipids of marine invertebrates. Biol. Morya 5:86-90;1979. 6. Einig, R. G.; Ackman, R. G. Omega-3 PUFA in marine oil products. J. Am. Oil Chem. Soc. 64:499;1987. 7. Hanahan, D. J. ; Vercamer, R. The action of lecithinase D on lecithin. The enzymatic preparation of D-1,2-dipalmitolein and D-1,2-dipalmitin. J. Am. Chem. Soc. 76:1804-1806;1954. 8. Hanahan, D. J.; Dittmer, J. C.; Warashina, E. A column chromatographic separation of classes of phospholipides. J. biol. Chem. 228:685-700;1957. 9. Heymans, H. S. A.; Schutgens, R. B. H.; Tan, R.; van den Vosch, H. ; Tager, J. M. Severe plasmalogen deficiency in tissues of infants without peroxisomes (Zellweger syndrome). Nature 306:69-70; 1986. 10. Jeong, B. Y.; Ohshima, T.; Koizumi, C. Lipids, molecular species of 1-O-alk- l'-enyl-2-acyl, 1-O-alkyl-2-acyl- and 1,2-diacyl glycerophospholipids in Japanese Oyster Crassostrea gigas (Thunberg). 25:624-632;1990. 11. Jeong, B. Y.; Ohshima, T.; Koizumi, C. Hydrocarbon chain distribution of 1-O-alk-l'-enyl-2-acyl, 1-O-alkyl-2-acyl and 1,2diacyl glycerophospholipids in the muscle and viscera of the ascidian Halocynthia roretzi and in the gonad of the sea urchin Strongylocentrotus intermedius. (Submitted to Lipids. ; 1995.) 12. Joh, Y. G.; Hata, M. Studies on the lipids of abalone. II. The aldehyde composition of plasmalogen from abalone and some marine molluscs. Bull. Korean Fish. Soc. 12:181-189;1979.
Jeong et al.
13. Joseph, J. D. Lipid composition of marine and estuarine invertebrates. Part I1: Mollusca. Prog. Lipid Res. 21:109-153;1982. 14. Koizumi, C.; Jeong, B. Y.; Ohshima, T. Fatty chain composition of ether and ester glycerophospholipids in the Japanese oyster Crassostrea ~gas (Thunberg). Lipids 25:363-370;1990. 15. MacBrinn, M. C.; O'Brien, J. S. Lipid composition of optic nerve myelin. J. Neurochem. 16:7-12;1969. 16. O'Brien, J. S.; Sampson, E. L. Lipid composition of the normal human brain-gray matter, white matter, and myelin, (IV) fatty acid and fatty aldehyde composition of the major brain lipids in normal human gray matter, white matter, and myelin. J. Lipid Res. 6:545-551;1965. 17. Ohshima, T.; Ratnayake, W. M. N.; Ackman, R. G. Cod lipids, solvent systems and the effect of fatty acid chain length and unsaturation on lipid class analysis by Iatroscan TLC-FID. J. Am. Oil Chem. Soc. 24:363-370;1987. 18. Ohshima, T. ; Wada, S.; Koizumi, C. 1-O-alk- 1'-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids in white muscle of bonito Enthynnus pelamis (Linnaeus). Lipids 24:363-370;1989a. 19. Ohshima, T.; Wada, S.; Koizumi, C. Molecular species of 1-Oalk-l'-enyl-2-acyl glycerophospholipids of bonito white muscle. Nippon Suisan Gakaishi 55:885-890;1989b. 20. Panganamala, R. V.; Horrocks, L. A.; Geer, J. C. ; Cornwell, D. G. Positions of double bonds in the monounsaturated alk-1enyl groups from the plasmalogen of human heart and brain. Chem. Phys. Lipids 6:97-102;1971. 21. Pocock, D. M. E.; Marsden, J. R.; Hamilton, J. G. The presence of alkenyl and alkyl glycerides, and choline and ethanolamine plasmalogens in a marine worm. Comp. Biochem. Physiol. 30: 133-136;1969. 22. Ratnayake, W. M. N.; Timmins, A.; Ohshima, T.; Ackman, R. G. Mass spectra of fatty acid derivatives, of isopropylidenes of novel glyceryl ethers of cod muscle and of phenolic acetates obtained with the Finnigan Mat Ion Trap Detector. Lipids 21: 518-524;1986. 23. Reichwald, I.; Mangold, H. K. Nutr. Metab. Suppl. 21:198201;1977. 24. Renkonen, O. Individual molecular species of phospholipids III. Molecular species of ox-brain lecithins. Biochim. Biophys. Acta 125:298-309; 1966. 25. Sargent, J. R. Ether linked glycerides in marine animals. In Marine Biogenic Lipids, Fats and Oils (Edited by Ackman, R. G.), Vol. I, Boca Raton, Florida: CRC Press; 1989: pp. 175-197. 26. Smith, G. M.; Djerassi, C. Phospholipid studies of marine organisms: 14. Ether lipids of the sponge Tethya aurantia. Lipids 22:236-240; 1987. 27. Spener, F.; Mangold, H. K. Composition of alkoxylipids of human heart and aorta. J. Lipid Res. 10:609-613;1969. 28. Sugiura, T. ; Fukuda, T. ; Miyamoto, T. ; Waku, K. Distribution of alkyl and alkenyl ether-linked phospholipids and plateletactivating factor-like lipid in various species of invertebrates. Biochim. Biophys. Acta 1126:298-308;1992. 29. Weber, N. Metabolism of orally administered rac-l-O-[l'-14C] dodecylglycerol in mice. J. Lipid Res. 26:1412-1420;1985.