Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 (cid:14)9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 6 Glycerophospholipid metabolism DOUGLAS R. TOCHER NERC Unit of Aquatic Biochemistry, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, Scotland, U.K. I. Introduction II. Biosynthesis, turnover and catabolism III. Digestion, absorption and transport 1. Digestion and absorption 2. Transport 2.1. Transport from intestine to liver 2.2. Transport between liver and extra-hepatic tissues 2.3. Vitellogenin IV. Composition 1. Content 2. Head group composition 3. Fatty acyl composition 3.1. Total glycerophospholipids 3.2. Glycerophospholipid classes 4. Molecular species 5. Dietary effects 6. Adaptation to environmental factors 6.1. Temperature 6.2. Salinity and hydrostatic pressure V. Roles 1. Structural roles 2. Metabolic roles 2.1. Eicosanoid metabolism 2.1.1. Species and tissue distribution of eicosanoids in fish 2.1.2. Range of eicosanoids in fish 2.1.3. Stimuli for production of eicosanoids 2.1.4. Fatty acid precursors of eicosanoids 2.1.5. Glycerophospholipid sources of precursor fatty acids 2.1.6. Functions of eicosanoids 2.2. The phosphoinositide cycle 2.3. Other metabolism 2.3.1. Protein kinase C 2.3.2. Platelet-activating factor 3. Nutritional roles 3.1. Embryonic development 3.2. Larval diet VI. Conclusions and perspectives VII. References
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`120 D.R. Tocher I. Introduction Glycerophospholipids are the major class of complex lipids characterized by a glycerol backbone with one of the primary hydroxyl groups (sn-3) esterified to phosphoric acid (Fig. 1). The secondary hydroxyl group in glycerophospholipids (sn- 2) is always esterified to a long-chain fatty acid, which in the majority of instances is monounsaturated or polyunsaturated. The sn-1 hydroxyl group is most commonly esterified to another fatty acid, generally saturated or monounsaturated, forming the diacyl glycerophospholipids (or phosphoglycerides). However, the sn-1 position can also contain a long aliphatic chain in c/s adS-unsaturated ether linkage in the case of the plasmalogens (alk-l-enyl acyl derivatives), or a saturated aliphatic chain in simple ether linkage (alkyl acyl derivatives) (Fig. 1). Phosphatidic acid (PtdA), a quantitatively minor glycerophospholipid, is nonethe- less an important intermediate in the biosynthesis of glycerophospholipids and can be regarded as the simplest. In PtdA the glycerol backbone is esterified to two fatty acids and phosphoric acid (Fig. 1). The quantitatively important glyeerophospho- lipids contain nitrogenous bases, esterified to the phosphoric acid, such as choline (phosphatidylcholine, PtdCho), ethanolamine (phosphatidylethanolamine, PtdEtn) and serine (phosphatidylserine, PtdSer) or polyalcohols, such as inositol (phos- phatidylinositol, Ptdlns) or glycerol (phosphatidylglycerol) (Fig. 1). Cardiolipin CH2-O-H CH2-O-CO-RI I I CH-O-H CH-O-CO-R2 I I CH2-O-H CH2-O-H Glycerol Diacylglycerol (DAG) CH2-O-CO-RI CH2-O-CO-RI I I CH-O-CO-R2 CH-O-CO-R2 I I CH2-O-P-O-H CH2-O-P-O-X Phosphatidic acid (PtdA) Diacyl glycerophospholipid CH2-O-R1 CH2-O-C-C-R1 I I CH-O-CO-R2 CH-O-CO-R2 I I CH2-O-P--O-X CH2-O-P-O-X 1-O-alkyl-2-acyl-glycerophospholipid 1-O-alk- 1 '-enyl-2-acyl-glycerophospholipid R1 -- Long-chain aliphatic group, usually saturated or monounsaturated R2 - Long-chain aliphatic group, usually polyunsaturated or monounsaturated P -- PO2H X - choline (-CH2CH2 N+(CH3)3), ethanolamine (-CH2CH2NH~), serine (-CH2 CTI(NH +)COO-), myo-inositol (-C6 H 11 O5), glycerol (-CH2CH(OH)CH2OH) or phosphatidylglycerol. Fig. 6.1. Basic structures of the glycerophospholipids, their precursors and head groups.
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`Glycerophospholipid metabolism 121 (diphosphatidylglycerol) is formed when the phosphate groups of two phospha- tidic acid molecules are bridged by a third glycerol moiety esterified at the 1 and 3 positions. All the glycerophospholipids are important components of biological membranes and are not found in high concentrations elsewhere in the cell. Glycerophospholipids, phosphoglycerides and phospholipids are notsynonymous terms although the terminologies are still used in a confused manner today. Not all glycerophospholipids are phosphoglycerides, as the term phosphoglyceride should, strictly speaking, be reserved for the diacyl derivatives alone and should not include the ether-linked derivatives. Similarly, not all phospholipids are glycerophospho- lipids. For instance, sphingomyelin contains phosphorus and so is a phospholipid but the phosphoric acid is esterified to a sphingosine backbone and not glycerol and so is correctly termed a sphingolipid. This chapter deals exclusively with the metabolism of the major classes of glycerophospholipids (PtdCho, PtdEtn, PtdSer and Ptdlns), including the ether-linked derivatives, although, due to the dearth of information on the metabolism of the ether-linked derivatives in fish, the focus will be on the diacyl derivatives. I have endeavored to maintain this nomen- clature throughout this chapter, but there are many instances where the use of phospholipid rather than glycerophospholipid has been more appropriate in the discussion of previous work and therefore for simplicity the term 'phospholipid' is often used. II. Biosynthesis, turnover and catabolism The pathways of glycerophospholipid biosynthesis have not been extensively studied or elucidated in fish 83. However, the existing evidence strongly suggests that the same pathways operate in fish as in mammals. Holub et al. 1~176 demonstrated the existence of glycerol-3-phosphate acyltransferase in the liver of rainbow trout (On- corhynchus mykiss). When liver microsomes were incubated with sn-[U-14C]glycerol - 3-phosphate in the presence of activated fatty acid, palmitoyl-CoA, 77% of the radioactivity was recovered in total glycerophospholipids with the remainder re- covered in neutral lipids. PtdA and lysoPtdA were also labeled, supporting the conclusion that glycerophospholipid and lipid biosynthesis in general proceeded via a PtdA intermediate in fish. The presence of cytidine diphosphate (CDP)-choline-l,2-diacylglycerol choline phosphotransferase has been demonstrated in the microsomes of trout liver 1~ and brain and liver from goldfish (Carassius auratus) 129. The synthesis of Ptd- Cho from 14C-CDP-choline and 1,2-diacylglycerol (diolein) in the presence of Mg2+established that the CDP-choline pathway for the biosynthesis of PtdCho, as studied in detail in mammals, also operated in fish 101'129. There have been few studies in fish to fully characterize the biosynthetic pathways for PtdCho, PtdEtn, PtdSer, PtdIns and cardiolipin or the pathways, known in mammals, for intercon- version between the glycerophospholipids. However, in a recent study, the de novo pathways of glycerophospholipid biosynthesis were investigated in trout hepatocytes and the activities of CDP-choline and CDP-ethanolamine phosphotransferases,
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`122 D.R. Tocher PtdEtn-methyltransferase (PtdEtn ~ PtdCho) and PtdSer-decarboxylase (PtdSer PtdEtn) were demonstrated 9~ In mammals, ether-linked glycerophospholipids are formed solely via the dihy- droxyacetonr phosphate pathway (see ref. 194). Briefly, fatty alcohol is formed by the NADPH reduction of fatty acyl-CoA. Fatty alcohol then reacts with fatty acyldihydroxyacetone phosphate to form alkyldihydroxyaeetone phosphate which is then reduced, specifically with NADPH, to form alkylglycerophosphate. The enzymes responsible for the synthesis and reduction of alkyldihydroxyacetone phos- phate are located in the peroxisomes. Reaction with fatty acyl-CoA, removal of phosphate and reaction with CDP-base results in the formation of alkyl glyc- erophospholipid. Alk-l-enyl acylglycerophospholipids (plasmalogens) are formed by oxidation of the corresponding alkyacylglycerophospholipid by a microsomal enzyme requiring NADPH and molecular oxygen. Little of the above pathway has been characterized in fish, but the available evidence from studies with spiny dogfish (Squalus acanthias) appears to suggest that the biosynthesis of ether-linked glycerophospholipids in fish is via a pathway similar to that outlined above (see ref. 194). A review of muscle lipase activities in various fish species indicated that the cat- alytic hydrolysis of phospholipids was primarily under the control of phospholipases A1 and A2 205. Intracellular phospholipase A activities have been demonstrated in muscle tissue from rainbow trout 112, pollock (Gadus pollachius) 11, winter flounder (Pseudopleuronectes americanus) z~ and Atlantic cod (Gadus morhua) 48. Neas and Hazel 154-156 studied the activity of phospholipase A2 towards PtdCho in the micro- somes of trout liver. Activity of phospholipase C has been demonstrated directly in isolated olfactory cilia from the channel catfish (Ictalurus punctatus) 39 and has been implicated indirectly in other tissues by the demonstration of a phosphoinosi- tide cycle (see section V.2.2) 1~176 but it appears that phospholipasr D activity has not been investigated in fish. Holub et al. ~02 showed that trout liver microsomes also contained acylCoA: 1-acyl-sn-glycero-3-phosphorylcholine acyltransferase ac- tivity. Therefore, enzymes required for partial catabolism of glycerophospholipids and for the reacylation of lyso-glycerophospholipids, and thus for the turnover of glycerophospholipids, have been demonstrated in fish. Catabolism of ether-linked glycerophospholipids hinges on the cleavage of the ether bond. Enzymic cleavage of the O-alkyl bond has been demonstrated in fish 194. The cleavage is considered to occur in two steps, whereby the alkyl bond is first oxidized to alk-l-enyl via a reaction involving NADPH, molecular oxygen and a pteridine cofactor, before cleavage to generate fatty aldehyde 194. The above enzyme system has yet to be directly studied in fish, and so it is not known if it will also cleave plasmalogens. Plasmalogenases, as described in mammalian brain, do not appear to have been studied in fish 194. The specificities of the enzymes involved in both de novo synthesis of the glyc- erophospholipids and in the turnover processes of deacylation/reacylation with respect to both head group and fatty acyl chains have important consequences in maintaining the normal glycerophospholipid class composition, the fatty acyl dis- tribution among the glyeerophospholipids, and in the adaptation to environmental
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`Glycerophospholipid metabolism 123 changes. Some of the more direct enzyme studies, discussed above, addressed this problem in relation to environmental temperature 1~176 However, there has been a considerable amount of data obtained from more indirect studies of the effects of environment on glycerophospholipid metabolism and this is summarized later (see section IV.6). III. Digestion, absorption and transport I. Digestion and absorption Depending upon the precise nature of the diet, a significant and potentially large and consistent portion of the lipid component in the natural food of fishes will be biomembrane lipids, primarily glycerophospholipids. Unfortunately, the lack of a discrete pancreas in most teleost species has hampered studies on intesti- nal lipolysis in fish. In consequence, even less is known about the digestion and absorption of dietary glycerophospholipids than is known about the biosyn- thetic pathways. There are virtually no studies on the intestinal digestion of glycerophospholipids in fish, but it could be presumed that the mechanisms are similar to those in mammals. Therefore dietary glycerophospholipids are presum- ably digested by pancreatic or intestinal phospholipases resulting in the forma- tion of 1-acyl lyso-glycerophospholipids and free fatty acids that are absorbed by the intestinal mucosal cells 96'198. Mankura et al. studied the hydrolysis of L-1- palmitoyl-2-[1-14C]arachidonyl-3-sn-glycerophosphatidylcholine by carp hepatopan- creas preparations TM. They found phospholipase A2 activity distributed in all the subcellular fractions, although the highest activity was located in the 10,000 g su- pernatant. The activity was dependent upon Ca 2+ and bile salt, consistent with a pancreatic enzyme, but had a conflicting acidic pH optimum of 5.0 TM. Whether this phospholipase activity reflects an intestinal activity or an intracellular phospholipase is, therefore, unclear. Recent work has suggested that cod pyloric caeca/pancreas contains a single, bile salt-activated, lipase activity with a wide substrate speci- ficity including triacylglycerols, steryl esters, fatty acid methyl esters and carboxyl esters 79,8~ Whether this enzyme is also active towards phospholipid and whether cod intestine actually lacks phospholipase A2 activity is unclear. The concentration of lyso-glycerophospholipids is very low in fish plasma and so it has also been assumed that the majority of lysophospholipid is re-esterified within the intestinal mucosa before export into the circulatory system. However, 14 studies on the incorporation of [1- C]palmitate and [U-14C]L-glycerol-3-phosphate into lipids in carp (Cyprinus carpio) intestinal homogenates in the presence of CTP, CDP-choline and CDP-ethanolamine showed that glycerophospholipid biosynthe- sis proceeded via PtdA and diacylglycerol (DAG) intermediates 11~ Therefore, mechanisms may exist in fish intestinal mucosa for the synthesis of glycerophos- pholipids from moieties more degraded than lyso-glycerophospholipids. Iijima and coworkers l~ have also studied the absorption of radioactivity from [l:4C]dioleoyl PtdCho force-fed to carp. At 20-28 h after dosing, radioactivity in the lipids of
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`124 D.R. Tocher plasma lipoproteins was primarily associated with triacylglycerols followed by Ptd- Cho and free fatty acids l~ The radioactivity associated with the sn-1 position of PtdCho was more than twice that associated with the sn-2 position 1~ These data support the hypothesis that the majority of glycerophospholipids are digested and absorbed v/a 1-acyl lyso-glycerophospholipid intermediates with re-estedfication before export from the intestinal cells. Z Transport There are several reviews that between them cover the area of lipid transport in fish very thoroughly 16,66,n6,2~ This section will give an overview of the subject particularly focusing on the role of glycerophospholipids in lipoprotein structure and function without major consideration of the apoprotein constituents. 2.1. Transport from intestine to liver As in mammals, glycerophospholipids are transported from the intestine in the form of lipoproteins. The re-esterification reactions occur primarily in the endoplasmic reticulum leading to the production of chylomicron-like and very low density lipoprotein (VLDL)-like particles in the lumen as observed in carp 162, tench (Tinca tinca) 163 and trout 1s,2~176 Studies in trout showed that lipid load and degree of unsaturation affected the relative production of the lipoproteins, with high dietary lipid and polyunsaturated fatty acids (PUFA) leading to the production of larger chylomicrons, whereas high dietary saturated fatty acids resulted in the production of smaller VLDL particles 2~ The effects of glycerophospholipid content and composition on the production of intestinal lipoproteins has not been studied. However, as trout chylomicrons contain about 8% phospholipid and trout VLDL contains approximately 21% phospholipid ~,67,69,211, it is possible that variable proportions of dietary phospholipid could be accommodated by varying the relative proportions of the intestinal lipoproteins produced. In mammals, the intestinal lipoproteins are transported from the intestine almost exclusively v/a the lymphatic system. It appears that in fish such as trout and tench the majority of the intestinal lipoproteins are similarly transported via the lymphatic system 31,32,163,21~ before appearing in the circulatory system 47'204,212 and delivery to the liver. In carp, however, it was shown that intestinal lipoproteins may be transported exclusively and directly to the liver v/a the portal system 162. This pathway may also operate for a portion of intestinal lipoproteins in other fish such as trout and tench. 2. 2. Transport between liver and extra-hepatic tissues Due to their amphipathic nature, glycerophosholipids are integral components of the plasma lipoproteins responsible for the transport of the neutral lipid classes such as triacylglycerol and steryl esters that are insoluble in aqueous solvents. As well as chylomicrons and VLDL, mammalian plasma also contains low density lipopro- teins (LDL), intermediate density lipoproteins (IDL) and high density lipoproteins (HDL) 13~ The lipoproteins vary in size and structure, as well as in protein'lipid
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`Glycerophospholipid metabolism 125 ratios and in the relative proportions of the different lipid classes leading to the density differences which have been used traditionally to separate and classify the different types 13~ Fish plasma contains a similar range of lipoproteins 16,66,2~ Al- though there are differences in detail, the general size, structure and composition of the plasma lipoproteins are comparable throughout the vertebrates, including fish 46. In most fish virtually all glycerophospholipid is transported in the plasma in the form of lipoprotein but analysis of an albumin-like protein from carp plasma showed that it contained 22% lipid of which 15% was phospholipid, predominantly PtdCho 151. This accounted for approximately 50% of the total plasma lipid in carp, with the remainder being transported via the lipoproteins ~5~ Total phospholipids account for 8, 21, 25 and 29% of the total weight of chylomi- crons, VLDL, LDL and HDL, respectively 47,67,69,211. However, as a percentage of the total lipids, the proportion of total phospholipids in trout lipoproteins ranges from under 9% in chylomicrons to 23% in VLDL, 35% in LDL and 53% in HDL 47'67'69'211. Similar levels of phospholipids were found in serum VLDL, LDL and HDL from Pacific sardine (Sardinops caerulea) 125, HDL from pink salmon (On- corhynchus gorbusha) 158 and HDL from chum salmon (Oncorhynchus keta) 8. Most of the above studies were performed with fed fish. Iijima et al. 1~ compared the lipid composition of carp plasma lipoproteins under starved and fed conditions. The carp lipoproteins contained proportionally more phospholipid than trout, salmon or sardine lipoproteins with the total lipid from VLDL, LDL and HDL containing 30, 58 and 82% phospholipid, respectively, in fed fish 1~ The proportion of phos- pholipid in the total lipid was not significantly altered by starvation in the carp 1~ In a later study, lijima et al. l~ studied the absorption and transport of radioactivity from [1-14C]dioleoyl PtdCho fed to carp. At 20-28 h after dosing, radioactivity was primarily associated with HDL followed by LDL and VLDL 1~ Chylomicrons are produced exclusively in the intestine, but although some VLDL can also be synthesized in the gut as described above, the majority of VLDL in the plasma is synthesized in the liver, at least in rainbow trout ~aa,2~ The major enzymes of lipoprotein metabolism and remodelling, including lipoprotein lipase (LPL) and hepatic lipase (HL), have been shown in trout and cod tissues 34,36. Lecithin:cholesterol acyl transferase (LCAT), a plasma enzyme which catalyzes the esterification of cholesterol using fatty acid from PtdCho, has been demonstrated in the plasma of trout 35, carp 11a and char (Salvelinus alpinus) 57. Lecithin:alcohol acyltransferase, which catalyzes the transfer of an acyl group from PtdCho to long- chain alcohols has been shown in carp plasma 139 and may be, at least partially, responsible for the surprisingly high level of circulating wax ester reported in that species 139,14~ In addition, intermediate density lipoprotein (IDL), a fraction with density between that of VLDL and LDL has been fractionated from trout serum 14. The presence of the whole spectrum of lipoproteins, including IDL, and the enzymes described above strongly suggests that lipoprotein remodelling processes, as characterized in mammals, also occur in fish. Therefore, triacylglycerols in chylomicrons and VLDL are hydrolyzed by LPL and HL at tissue sites with the hydrolysis products being absorbed. Excess surface constituents, including phospholipids, 'bud off' as nascent HDL particles (similar to HDL3), which can
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`126 D.R. Tocher also be secreted by the liver. HDL3 can take up free cholesterol from peripheral tissues which is then esterified by the action of LCAT resulting in the production of mature HDL (HD~). Remnants of ehylomierons and VLDL hydrolysis can be taken up by the liver, but further action by LPL and HL leads to the formation of LDL via IDL. As in mammals, the relative proportions of the plasma lipoproteins in fish can vary from species to species, but is a constant characteristic of each species depend- ing upon dietary status. In trout, HDL is the predominant class ranging from 0.5-2.3 g/dl, followed by LDL (0.2-1.1 g/dl) and then VLDL (0.1-0.7 g/dl) 47,67,69,211. HDL was also the main lipoprotein class in carp 6, sea bass (Dicentrarchus labrax) 193, pink salmon (O. gorbusha) 158, chum salmon ls2 and channel catfish 136. HDL appeared to be absent from the plasma of carp 15~ The relative amounts of HDL, LDL and VLDL vary with age, nutrition and sexual cycle 66. In common with most vertebrates, PtdCho appears to be the predominant glycerophospholipid class in fish lipoproteins 46,158. However, the precise phos- pholipid class composition in fish plasma lipoproteins has been rarely reported in the above studies. Similarly, the precise fatty acid compositions of individ- ual glycerophospholipid classes have not been extensively studied. The fatty acid compositions of total lipids and total phospholipids have been reported. Fish lipoproteins generally contain higher levels of PUFA, particularly (n-3)PUFA, than the corresponding mammalian lipoproteins ~i,nT.158,211,2n. In trout the phospholipid fractions from all lipoproteins were particularly rich in 16:0 and 22:6n-3, and total (n-3)PUFA was higher and total (n-6)PUFA lower, in phospholipids in comparison with triacylglycerols 47'67'69'127. However, the levels of (n-3)PUFA in the cholesteryl ester fractions of LDL and HDL exceeded those of the phospholipids 47, 67, 69,127 . The exact fatty acid composition of the plasma lipoproteins were affected by diet, both acutely, particularly with chylomierons and VLDL after a meal, and chronically as seen with essential fatty acid-deficiency in trout 7~ The apoprotein compositions of fish lipoproteins are similar to mammalian lipoproteins with the major apoproteins being apoprotein A (I and II) in HDL, apoprotein B in LDL and mixtures of apoproteins B, C, and E in VLDL and A, B and C in chylomicrons 6,13,15,16,'ui'47,136,ls8,lsS'211. There are few direct studies on the functions of the different apoproteins in fish, but it is likely that they have the same metabolic functions such as receptor binding (apoproteins B and E) and enzyme activation (AI and LCAT; CII and LPL) as in mammalian systems ~37. Consistent with this, it was shown that trout adipose tissue LPL was activated by the apoprotein fraction of trout HDL (mainly AI and C) ss. There are virtually no reports of studies on the uptake of phospholipid from the plasma lipoproteins in fish. However, by analogy with the system characterized in mammals, phospholipids can probably be taken up into the tissues by two or three main mechanisms 137. Probably the most important pathway, quantitatively, is receptor-mediated endocytosis via B/E and E receptors. These are important pathways for LDL (apo B), VLDL- and ehylomieron-remnants (apo B and E) and HDL (apo E, especially in HDL1, an apo E-rich variant). These receptors are found on various tissues including liver. In mammals the precise tissue distribution
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`Glycerophospholipid metabolism 127 varies between different species. Probably all the lipoproteins, but particularly LDL and HDL, can be taken up by tissues via non-specific pinocytosis. For instance, in liver approximately 30% of LDL uptake is via a non-receptor-mediated pathway. Finally, it may be that surface components of VLDL and chylomicrons, including phospholipids, may be taken up or exchanged via direct interaction with the endothelial cell membranes in the tissues. 2.3. Vitellogenin (very high density lipoprotein) Another lipoprotein class in fish important in the transport of phospholipids is vitellogenin which is only found in mature oviparous females or estrogen- injected fish 43,55,166,217,248. Vitellogenin has a density higher than HDL and has been designated very high density lipoprotein I (VHDL 1) 149 and contains about 80% protein and 20% lipid in rainbow trout 43,7~ sea trout (Salmo trutta) 166 and goldfish 61,1~ The lipid is predominantly phospholipid (about 65-70% of total lipid) 7~176 and rich in (n-3)PUFA 7~ particularly 22:6n-3, which accounts for 20% of total fatty acids in trout vitellogenin 127. There are few data on the detailed class compositions of fish vitellogenins. Vitellogenin is synthesized in the liver and is transported to the ovary during the first stage of oogenesis termed vitellogenesis 247,248. Vitellogenin is taken up intact by receptor-mediated micropinocytosis 2~ into the developing oocytes where it is cleaved into a phosphate- rich protein, phosvitin and a lipid-rich protein, lipovitellin 111,2~ Lipovitellin in trout eggs was composed of 77% protein and 23% lipid with a lipid class composition similar to HDL 212. Cod roe lipovitellin had higher lipid content than the trout at over 40% with 70% of the lipid being glycerophospholipids 226. Of the total glycerophospholipids in cod lipovitellin, 67% was PtdCho and 22% was PtdEtn with 4% each of PtdSer and PtdIns 226. During the early stages of vitellogenesis, VLDL in the plasma may also be increased in response to estrogen 187, and may also be taken up into the developing oocytes by receptor-mediated endocytosis 248, at least in eggs with high triacylglyc- erol content and lipid droplets. Recently it has been confirmed that winter flounder contains a further high density lipoprotein (VHDL II), originally called Pk A as its density and relationship to vitellogenin was unknown 216, that is taken up in vivo by the ovary of viteUogenic females 149. 114 Composition The net result of the many metabolic pathways discussed in Sections II and III is the composition of glycerophospholipids in the tissues. The metabolism is a very dynamic situation, of course, but the glycerophospholipid composition is more stable provided the environmental conditions and diet are reasonably constant. There are many papers reporting the glycerophospholipid composition of fish tissues, and the effects of diet and environmental factors. A comprehensive review of these areas is beyond the scope of this article. The reader is directed to some other reviews that cover various aspects of glycerophospholipid composition in
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`128 D.R. Tocher fish 1-5'96'194'198. This section contains some generalizations that can be made about fish glycerophospholipid composition focusing on some features that have been of particular interest in our own laboratory. 1. Content It is difficult to generalize about the glycerophospholipid content of fish tissues for several reasons. One of the major reasons is that studies have tended to report contents in a variety of ways, such as mg/g tissue or as percentage of weight of tissue, in both cases either wet or dry weights, or as percentage of total lipids. Irrespective of the method used, nutritional status of the fish will have an effect as variation in neutral lipid content will influence the results obtained. This is complicated by tissue differences. Other than adipose tissue, some fish, such as cod and halibut (Hippoglossus hippoglossus) store significant amounts of lipid in the liver, whereas in others, such as mackerel (Scomber scombrus) and capelin (Mallotus villosus), deposits of lipid between skin and muscle can account for a large proportion of the fish's total reserves 96. In snakehead (Channa sp.) fillet, eviscerated body of the guppy (Poecilia retie. ulata) and carp muscle, total phospholipids accounted for 21-26% of the total lipid s6,96. Phospholipids were present in goldfish muscle mitochondria at almost 2 mg/g muscle e42. In several studies on trout, phospholipids ranged from approx- imately 45-75% of the total lipid in liver 9~. In chum and coho (Oncorhynchus kisutch) salmon livers, phospholipids were consistently less than 40 and 30%, re- spectively, whereas in parr and smolt cherry salmon (Oncorhynchus masou) livers, phospholipids predominated 96. About 58% of the total lipid in goldfish intestinal tissue was phospholipid 144. It was reported in two studies on goldfish brain that phospholipids accounted for 3.9% of the tissue weight 129, and 47% of the total lipid 189. In contrast, total lipid from trout and cod brains was approximately 75% total polar lipids, predom- inantly glycerophospholipids 2~. In sea bass on a variety of diets, the proportion of phospholipids in the brain varied between 65 and 71% 17s. Phospholipids in coelacanth (Latimeria chalumnae) brain amounted to over 41 mmol/g wet weight of tissue 223. In a study of many marine fish including elasmobranchs and teleosts, total phosphorus in the brains varied between 460 and 2650 mg/g fresh weight 123. Total polar lipids, predominantly glycerophospholipids, made up 60 and 82% of the total lipid in retinal tissue from trout and cod 228. However in whole eyes from guppy, phospholipids were only 28% of the total lipid s6. Relatively high levels of polar lipids, predominantly glyeerophospholipids, are generally associated with fish eggs containing relatively low lipid contents 96. There- fore, the roe of some species of marine fish including cod, herring (Clupea haren- gus), haddock (Melanogrammus aeglefinus), whiting (Merlangus merlangus) and saithe (Pollachius virens) were relatively rich in phospholipids, which accounted for 61-72% of the total lipid 23~ However, in sand eel (Ammodytes lancea) roe which was richer in lipid and contained distinct oil globules, total lipid contained only 23% 23o phospholipid .
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`Glycerophospholipid metabolism 129 2. Head group composition The class composition of glycerophospholipids in fish tissues can be far more easily generalized than content. PtdCho is almost invariably the major glycerophospho- lipid class with PtdEtn almost invariably being the second most abundant. PtdCho accounted for between 47 and 84% and PtdEtn accounted for between 16 and 34%, of the total phospholipids in a range of tissues including muscle, liver, gill and intestines from various fish species 96. This is a similar situation to that in mammals, and also like mammals, the main exception to this is brain tissue where the per- centage of PtdEtn can exceed that of PtdCho, as reported for brains from cod and trout 228, coelacanth 223, rohu (Labio rohita) 2~ and a range of elasmobranchs and teleosts 13s. However, other studies have found PtdCho exceeding PtdEtn in brain tissues from pike (Esox lucius) and carp 153, hake (Merluccius hubbsi) and sea bass (Acanthustius brasilianus) 12 and a range of fishes from the Caribbean 123. PtdSer ranged from 2 to 9%, and Ptdlns ranged from 3 to 8% in several tissues from various species of fish 86,88,96,128,129. The levels of PtdSer exceed those of Ptdlns in fish neural tissues 96,123'129,153 and trout brush border membranes 12a, whereas the opposite was true in trout gills 88 and liver 86, goldfish liver 129 and the roes from several marine fish 23~ Polyphosphoinositides, Ptdlns4P and Ptdlns4,5P2, constituted 1.0 and 0.9 mol% of the total phospholipids in the lesser spotted dogfish (Scyliorhinus canicula) rectal gland 29,2~ and were also found in cod gills 3~ Cardiolipin ranged from 3 to 4% in trout liver and gills to over 8% in trout intestinal brush border membranes 86,88,128. As in mammals, relatively high levels (6-11%) of cardiolipin were associated with mitochondria in goldfish gill 5a and muscle 242. Many aspects of ether-linked glycerides including the glycerophospholipid deriva- tives in marine animals, including fish, were the subject of recent reviews 45,194. Plas- malogens (alkenylacyl derivatives) were detected in small amounts in total hepatic lipid of the spiny dogfish 138. In the non-myelinated olfactory nerve of the garfish (Lepisosteus osseus), ethanolamine, choline and