311

ARTICLE

I

Lipid Composition of the Pineal Organ from Rainbow Trout

(Oncorhynchusmykiss) R.J. Henderson a,*, M.V. Bell a, M.To Park a, J.R. Sargent a and J. Falcon b aN.E.R.C. Unit of Aquatic Biochemistry, Department of Biological and Molecular Science, University of Stirling, Stirling FK9 4LA, Scotland, United Kingdom and bLaboratoire de Neurobiologie et de Neuroendocrinologie Cellulaires, URA CNRS 290, Faculte des Sciences, 86022 Poitiers Cedex, France

The lipid composition of the pineal organ from the rainbow trout (Oncorhynchus mykiss) was determined to establish whether the involvement of this organ in the control of circadian rhythm~ is reflected by specific adaptations of lipid composition. Lipid comprised 4.9% of the tissue wet weight and triacylglycerols were the major lipid class present (47% of total Hpid). Phosphatidylcholine (PC) was the principal polar lipid, and smaller proportions of other phospholipids and cholesterol were also present. Plasmalogens contributed 11% of the ethanolsmlne glycerophospholipids (EGP). No cerebrosides were detected. The fatty acid composition of triacylglycerols was generally similar to that of total lipids in w h i c h saturated, m o n o u n s a t u r a t e d and polyunsaturated fatty acids (PUFA) were present in almost equal proportions. Each of the polar lipid classes had a specific fatty acid composition. With the exception of phosphatidylinositol (PI), in which 20:4n-6 comprised 27.4% of the total fatty acids, 22.~n-3 was the principal PUFA in all lipid classes. The proportion of 20:5n-3 never exceeded 6.9% of the fatty acids in any lipid class. The p r e d o m i n a n t molecular species of PC were 16d)/22:6n-3 and 16d)/18:1, which accounted for 33.2 and 28.5%, respectively, of the total molecular species of this phospholipid. P h o s p h a t i d y l e t h a n o l a m i n e (PE) contained the highest level of di-22.~n-3 (13.0%) of any phospholipid.'There was also 4.9% of this molecular species in phosphatidylserine (PS) and 4.1% in PC. In PE, the species 16:0/22.~, 18:1/22.~ and 18d)/22.~ totalled 45.1%, while in PS 18d}/22:6 accounted for 43.9% of the total molecular species. The most a b u n d a n t molecular species of PI was 18.~/20:4n-6 (37.8%). The lipid composition of the pineal organ of trout, and particularly the molecular species composition of PI, is more similar to the composition of the retina than that of the brain. Lipids 29, 311-317 (1994).

In fish, as in all vertebrates, the retina of the eye and the pineal organ of the brain are essential components of the circadian system that measures the period and phase of the daily light-dark cycle and ultimately controls rhythmic processes (1,2). In keeping with this role, the pineal organ offish contains photoreceptor cells that display close cyto-funetional analogies with the photore*To whom correspondenceshould be addressed. Abbreviations: EGP, ethanolAmine glycerophospholipids;HPLC, high-performance liquid chromatography; HPTLC, high-performance thin-layer chromatography;PC, phosphatidylcholine;PE, phosphatidylethan01amine; PG, phosphatidylglycerol;PI, phosphatidylinositol; PS, phosphatidylserine;PUFA, polyunsaturated fatty acids~ SM, sphing0myelin;TAG, triacylglycerols.Molecular species are abbreviated as follows:e.g., 16:0/22:6PC is 1-palmitoyl2-docosahexaenoyl-sn-glycero-3-phosphocholine.

Copyright 9 1994 by AOCS Press

ceptors of the retina (3,4). The photoreceptor cells from both tissues translate the light-dark information into a neural output of excitatory neurotransmitter (5) and a neurohormonal output in the form of melatonin (3). In addition to being multimessenger cells, the photoreceptor cells of the pineal organ are also multieffectors and can transduce information supplied by other eternal (such as temperature) or internal (catechol-amines, adenosine, steroids) factors. Whereas retinal melatonin acts preferentially in an autocrineJparacrine manner, the melatonin secreted by the pineal contributes largely to the circulating levels and may be involved in the control of seasonal events, particularly reproduction, in vertebrates (3,6-8). The structural phospholipids of the retina and brain of vertebrates, including fish, are known to contain high levels of the polyunsaturated fatty acid (PUFA) 22:6n-3 (9,10). Within the retina, the 22:6n-3 is apparently concentrated in the membranous outer segments of the photoreceptor rod cells (11), and these cells isolated from frog retina exhibit a selective uptake of 22:6n-3 in vitro (12). A requirement for 22:6n-3 in the visual process has been demonstrated in studies with newborn primates and preterm human infants, which have shown that the visual acuity is affected by deprivation of 22:6n-3 during postnatal development of the infant (13). Peroxidation of 22:6n-3 is one of the primary events observed in inherited or traumatically induced photoreceptor degeneration (9,14). Di-22:6n-3 phospholipids are known to be major components of rod outer segment membranes in frog and rat (15,16). Recent analyses of the phospholipids of the brain and retina of trout and cod have shown that di22:6n-3 molecular species are major constituents of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in these tissues and that this is particularly true of the retina, where the amounts are considerably higher than those found in terrestrial mammals (17,18). The importance of 22:6n-3 in the visual process of fish is also indicated by the observation that the proportion of di-22:6n-3 in the phospholipid of the developing eyes of herring larvae increases with age as the rods are recruited into the photoreceptor population (19). The results of these nutritional studies and the presence of large amounts of di-22:6 phospholipid in the photoreceptors of the retina suggest that 22:6no3 has an essential and unique role in the primary events associated with the absorption and transduction of photons. This might be of crucial importance for the photoperiodic control of the production of messages, such as melatonin.

UPIDS, Vol. 29, no. 5 (1994)

312

R.J. HENDERSONETAL. By analogy with the retina, it is probable that 22:6n-3 is a major PUFA of the lipids in the photoreceptor cells of the fish pineal organ. To examine this hypothesis, we determined in the present study the detailed lipid composition of the trout pineal with particular attention to the PUFA content and molecular species of phospholipids. As far as we are aware, the detailed lipid composition of the pineal organ from fish has not been reported previously and only a limited amount of information is available on the lipids of the organ from mammals (20,21). Information gained from the analysis of the trout pineal organ is of basic importance as mammalian pinealocytes are accepted as being phylogenetically derived from the fish pineal photoreceptor cells (2). MATERIALS AND METHODS

Fish and pineal organs. Rainbow trout (Oncorhynchus mykiss) of average weight 800 g were obtained from a commercial fish farm (Pisciculture Bellet, Angouleme, France) where they had been maintained under natural conditions of water temperature and photoperiod. One hundred fish were killed by decapitation. Pineal organs were removed immediately from the fish, frozen in liquid nitrogen and stored at -80~ until taken for analysis. Chemicals and solvents. Phospholipase C from Bacillus cereus was purchased from Boehringer Corporation (London) Ltd. (Lewes, East Sussex, England). Oxalyl chloride and anthracene 9-carboxylic acid were supplied by Aldrich Chemical Co. (Gillingham, Dorset, England). All other chemicals and biochemicals were purchased from Sigma (Poole, Dorset, England), and solvents of high-performance liquid chromatography (HPLC) grade were obtained from Rathburn Chemicals (Walkerburn, Peeblesshire, Scotland). Lipid extraction and analysis. After thawing, the pineal organs were weighed and transferred to a Teflonin-glass homogenizer. The organs were homogenized in 37 mL of chloroform/methanol (2:1, vol/vol) essentially as described by Christie (22) to extract lipids. Solvent was removed under a stream of nitrogen and the resulting lipid extract desiccated overnight under vacuum in a preweighed tube. The tube and contents were then reweighed to obtain the weight of the lipid extract which was redissolved in chloroform/methanol (2:1, vol/vol) and stored under an atmosphere of nitrogen at -70~ between analyses. To establish the lipid class composition, aliquots of lipid extract were subjected to high-performance thinlayer chromatography (HPTLC) alongside authentic standards using hexane/diethyl ether/glacial acetic acid (80:20:2, by vol) as the developing solvent for the separation of neutral lipid classes and methyl acetato/propan-2-ol/chloroform/methanol/0.25% (wt/vol) aq. KC1 (25:25:25:10:9, by vol) for the separation of polar lipids. To confirm which polar lipid classes were present, aliquots of total lipid were also subjected to two-dimensional HPTLC. The polar lipid developing solvent systom described previously was used for development in the first dimension and chloroform/acetone/methanol/ acetic acid/water (10:4:2:2:1, by vol) was employed for LIPIDS, Vol. 29, no. 5 (1994)

development in the second dimension. Developed chromatograms were visualized with copper acetate in phosphoric acid (23). Lipid class composition was quantitated by double-development HPTLC coupled with scanning densitometry, as described elsewhere (24). Estimates of the relative amounts of the plasmalogen and the diacyl forms of ethanolamine glycerophospholipids (EGP) were obtained by acid hydrolysis of the isolated EGP in situ on an HPTLC plato followed by chromatography and quantitative phosphate staining as described by Bell and Dick (25). For the analysis of fatty acid composition, individual lipid classes were separated by two-dimensional TLC on 20 • 20 cm glass plates coated with silica gel G 60 (0.25 mm thick) using the solvent systems described here. The separated classes were visualized by spraying the chromategram with 0.1% 2',7'-dichlorofluorescein in methanol containing 0.01% butylated hydroxytoluene and by viewing under ultraviolet light. Triacylglycerols (TAGs) were purified by redeveloping the chromatogram in the reverse direction of the second development using hexane/diethyl ether/glacial acetic acid (80:20:2, by vol) after removal of the individual polar lipid classes. The fatty acids of the separated lipid classes were converted to their methyl esters on the adsorbent by acid-catalyzed transestorification (22). An aliquot of total lipid was also subjected to the same procedure. The resulting fatty acid methyl esters were purified by HPTLC and recovered from the adsorbent with hexane/diethyl ether (1:1, vol/vol). Fatty acid methyl esters were analyzed on a Packard 439 gas chromatograph equipped with a fused silica capillary column (50 m • 0.22 mm i.d.) coated with FFAP phase (S.G.E., Milton Keynes, United Kingdom). Sample application was by on-column injection, and hydrogen was used as the carrier gas. During the course of an analysis, the oven temperature was programmed to increase from 50 to 225~ Samples were also analyzed using an Omegawax 250 fused silica column (30 m • 0.25 mm i.d., Supelchem U.K. Ltd., Essex, United Kingdom) with the oven temperature programmed from 50 to 260~ Fatty acid components were identified by reference to a well-characterized fish oil fatty acid mixture, and the unsaturated nature of components was confirmed by re-analysis of samples after catalytic hydrogenation over PtO 2. The separated components were quantitatod using a recording integrator linked to the chromatograph. Analysis of molecular species. A 500-lag portion of total lipid was separated into the component phospholipids by HPTLC alongside 20 lag of a cod retina total lipid standard using methyl acetate/propan-2-ol/chloroform/methanol/0.25% (wt/vol) aq. KC1 (25:25:25:10:9, by vol) as the developing solvent. The standard spots and the edge of the bands of pineal organ lipids were visualized by exposure to iodine vapor, and the bands of adsorbent, containing PC, PS, phosphatidylinositol (PI) and EGP, were scraped from the plato. The phospholipids were hydrolyzed on the silica with phospholipase C using a two-phase system of 1 mL diethyl ether and I mL of 0.1 M sodium borate buffer, pH 7.5 at room tern-

313

LIPID COMPOSITION OF TROUT PINEAL ORGAN p e r a t u r e u n d e r n i t r o g e n for 2 h (26). A t t h e e n d o f t h e incubation period, 1,2-diradylglycerols were extracted, dried down under nitrogen and finally desiccated under v a c u u m for 1 l~ 9 - A n t h r o y l c h l o r i d e w a s p r e p a r e d f r o m 9 - a n t h r a c e n e c a r b o x y l i c a c i d a n d o x a l y l c h l o r i d e a s des c r i b e d b y G o t o et al. (27). T h e d i r a d y l g l y c e r o l s w e r e der i v a t i z e d a n d p u r i f i e d b y a m o d i f i c a t i o n of t h e m e t h o d o f T a k a m u r a a n d K i t o (28) a s d e s c r i b e d e l s e w h e r e (19). The 1-O-alk-l'-enyl-2-acyl derivatives were removed d u r i n g t h e f i n a l H P T L C p u r i f i c a t i o n s t e p . T h e 1,2-diacyl-3-anthrocyl-sn-glycerols were separated by HPLC at 19-20"C o n a n O D S c o l u m n (25 x 0.46 cm, 5 ~ m p a r t i c l e size; B e c k m a n I n s t r u m e n t s U . I ~ L t d . , H i g h W y c o m b e , Buckinghamshire, United Kingdom) using a Pye Unicam PU4010 pump (Pye Unicam Ltd., Cambridge, England) and two isocratic solvent systems, methanol/ p r o p a n - 2 - o l (4:1, vol/vol) a t a flow r a t e of 1.0 m L / m l n , a n d a c e t o n i t r i l e / p r o p a n - 2 - o l (7:3, vol/vol) a t a flow r a t e of 1.0 m L / m i n a s d e s c r i b e d b y T a k a m u r a a n d K i t o (28). PeAk.q w e r e d e t e c t e d u s i n g a W a t e r s 470 s c a n n i n g fluor e s c e n c e d e t e c t o r ( M i l l i p o r e U K Ltd., E d i n b u r g h , S c o t l a n d ) w i t h e x c i t a t i o n a n d e m i s s i o n w a v e l e n g t h s o f 360 a n d 460 urn, r e s p e c t i v e l y , a n d q u a n t i f i e d u s i n g a S h i madzu CR3A recording integrator (Anachem, Luton, United Kingdom). Peaks were identified by their relat i v e r e t e n t i o n t i m e u s i n g 16:0/22:6n-3 a s a r e f e r e n c e p e a k . D i - d o c o s a h e x a e n o y l g l y c e r o l (di-22:6n-3; N u - C h e k P r e p , E l y s i a n , M N ) w a s a l s o a v a i l a b l e for d i r e c t comp a r i s o n o f r e t e n t i o n t i m e s , a s w e r e a r a n g e o f s a m p l e s of k n o w n c o m p o s i t i o n f r o m p r e v i o u s s t u d i e s (17,18,29). Each sample was chromatographed three times in each solvent system and the standard deviations calculated. Where final peak areas were calculated by subtraction, the standard deviations of the contributing peaks were a d d e d to give t h e f i n a l error.

RESULTS T h e l i p i d c o n t e n t a n d l i p i d c l a s s c o m p o s i t i o n of t r o u t p i n e a l o r g a n a r e p r e s e n t e d i n T a b l e 1. A r o u n d 4.9% of t h e w e t w e i g h t of t h e p i n e a l o r g a n s w a s lipid, of w h i c h alm o s t h a l f (47%) w a s i n t h e f o r m of TAG. P C w a s t h e m a j o r p o l a r l i p i d p r e s e n t (16.5% of t o t a l lipid), followed

TABLE 1 L i p i d s of Trout P i n e a l Organ: L i p i d Class Composition a Lipid class Cholesteryl esters Triacylglycerols Free fatty acids Cholesterol Diacylglycerols EthanolAmlne glycerophospholipids Phosphatidylglycerol Phosphatidylinositel Phosphatidylserine Phosphatidylcholine Sphingomyelin aValues are means • SD of three determinations.

% Total lipid 3.3 • 47.0 • 3.4 • 8.7 • 1.5 • 9.9 • 1.4 • 3.2 • 3.4 • 16.5 • 1.7 •

0.2 2.1 0.6 0.4 0.1 0.6 0.1 0.3 0.3 0.8 0.1

b y E P G (9.9%). N o c h o l i n e p l a s m a l o g e n s w e r e d e t e c t e d , w h e r e a s e t h a n o l s m i n e p l a s m a l o g e n s a c c o u n t e d for 11% of t h e t o t a l E P G fraction. P I a n d P S e a c h a c c o u n t e d for less t h a n 5% of t h e t o t a l lipid, a n d b o t h p h o s p h a t i d y l glycerol (PG) a n d s p i n g o m y e l i n (SM) w e r e p r e s e n t a t l e s s t h a n 2%. No c a r d i o l i p i n or c e r e b r o s i d e s w e r e d e t e c t e d . P a l m i t i c a c i d (16:0) c o m p r i s e d 23.7% of t h e f a t t y a c i d s in t h e t o t a l l i p i d s a n d w a s t h e m o s t a b u n d a n t f a t t y a c i d i n t h e p i n e a l o r g a n (Table 2). T h e m o n o u n s a t u r a t e d 18:1n-9 a n d p o l y u n s a t u r a t e d 22:6n-3 a c c o u n t e d for 17.6 a n d 12.4%, r e s p e c t i v e l y , of t h e t o t a l f a t t y acids. O v e r a l l , saturated, monounsaturated and polyunsaturated fatty a c i d s a c c o u n t e d for s i m i l a r p r o p o r t i o n s o f t h e t o t a l l i p i d f a t t y acids. T h e f a t t y a c i d c o m p o s i t i o n o f T A G s (Table 2) w a s g e n e r a l l y s i m i l a r to t h a t o f t h e t o t a l l i p i d a l t h o u g h t h e p r o p o r t i o n of 18:2n-6 (12.8%) w a s n o t a b l y h i g h e r , a n d t h a t of 16:0 lower, t h a n i n t o t a l lipids.

TABLE 2 Fatty Acid Composition (wt%) of Total Lipid and Lipid Classes from Trout P i n e a l O r g a n a Acyl chain Total lipid TAG PC EGP 14:0 15:0 16:ODMA 16:0 16:1n-9 16:1n-7 17:0 18:0DMA 18:ln-9DMA 18:ln-7DMA 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1n-ll 22:1n-9 22:4n-6 22:5n-6 22:5n-3 22:6n-3 24:1n-9 Unidentified

2.9 0.9 -23.7 -5.8 0.6 ---7.2 17.6 4.0 9.3 1.2 2.5 0.6 0.4 1.6 -0.9 3.5 1.7 0.3 0.1 -1.2 12.4 0.7 0.9

3.5 0.3 -17.4 -8.0 0.4 ---4.2 18.5 3.7 12.8 2.0 3.1 1.2 0.2 0.5 0.2 1.4 3.9 2.0 0.3 0.3 0.2 1.8 12.6 0.1 1.4

0.6 0.3 -34.0 0.6 1.3 0.3 ---5.4 18.3 2.4 1.8 -0.4 0.4 0.3 2.0 -0.3 4.6 ---0.2 1.3 24.6 -0.9

0.2 0.9 0.6 10.7 -0.7 0.3 0.4 2.2 0.7 10.2 10.3 3.9 3.4 0.3 1.4 0.9 0.4 7.2 -0.6 4.8 ---0.4 2.2 36.0 0.4 0.7

Total saturated 35.3 25.8 40.6 23.3 Total monounsaturated 32.6 35.7 23.0 19.6 Total PUFA 31.2 37.1 35.5 56.2 Total n-3 19.2 21.9 30.8 43.9 Total n-6 12.0 15.2 4.7 12.3 n-3/n-6 1.60 1.44 6.55 3.57 aEGP, ethanolamine glycerophospholipids; PC, phosphatidylcholine; TAG, triacylglycerols; PUFA, polyunsaturated fatty acids; DMA, dimethylacetals. LIPIDS, Vol. 29, no. 5 (1994)

314

R.J. HENDERSON E T A L . TABLE 3 Fatty Acid Composition Pineal Organ a

m o n o u n s a t u r a t e d fatty acids and PUFA which comprised 44.1 a n d 37.1%, respectively, of the total f a t t y acids in this phospholipid (Table 3). The levels of 16:1n-7 and 18:1n-7 (17.0 and 10.2%) in P G were higher t h a n in a n y other lipid class, and, of all the phospholipids P G h a d the highest content of 18:2n-6. In SM more t h a n h a l f (58.2%) of the f a t t y acids were m o n o u n s a t u r a t e d , m a i n l y due to the presence of a very high proportion of 24:1 (45.9%). PUFA comprised only 8.9% of the SM f a t t y acids (Table 3). In all lipid classes, 20:5n-3 was p r e s e n t in small a m o u n t s and n e v e r exceeded 6.0% of the component f a t t y acids. T h e long c h a i n monoenoic f a t t y acid 2 2 : 1 n - l l observed in total lipid was concentrated in TAG, where it accounted for around 2% of the f a t t y acids. Of all the lipid classes, TAG also contained the highest level of 14:0. The principal molecular species of PC, PE, PS and PI are presented in Table 4. Two molecular species predominated in PC, namely 16:0/22:6n-3 and 16:0/18:1, which accounted for 33.2 and 28.5%, respectively, of the total molecular species of this phospholipid. Di-PUFA, dis a t u r a t e d and di-monounsaturated species each comprised less t h a n 6% of the total PC and the content of monounsaturated-PUFA species totalled 4.9%, within which 18:1/22:6n-3 was the major component. Molecular species containing 22:6n-3 were particularly a b u n d a n t in PE. Di-22:6n-3 comprised 13.0% of the total, a n d 16:0/22:6 and 18:1/22:6 were both present at levels of more t h a n 15%. In PS, 18:0/22:6n-3 accounted for almost h a l f (43.9%) of the total molecular species, a n d 16:0/22:6n-3 was the only other species present a t a level of greater t h a n 10%. The most a b u n d a n t molecular species of PI was 18:0/20:4n-6 which accounted for 37.8%. Another species containing 20:4n-6, 16:0/20:4n-6, comprised 14.0% of the m o l e c u l a r species of PI a n d 18:0/20:5n-3 accounted for 10.8%, the highest level for a species containing 20:5n-3 observed in a n y of the phospholipids examined. in

(wt%) of Lipid

Acyl chain

PI

14:0 15:0 16:0 16:1n-9 16:1n-7 17:0 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1n-ll 22:1n-9 22:4n-6 22:5n-6 22:5n-3 22:6n-3 24:0 24:1n-9 Unidentified

0.3 0.4 14.0 . -0.7 33.8 5.3 1.3 1.5 0.1 0.2 0.2 0.3 27.4 . 0.1 5.7 . -0.5 -0.4 7.0 --

0.4 0.4

Classes

PS

0.3 0.7 3.0 . . 9.3 0.4 38.2 5:5 2.6 1.4 0.4 1.2 0.4 -1.0 . . -1.3 . . -0.9 0.4 3.0 29.4

from Trout

PG

SM

0.8 0.3 10.7 . 17.0 0.5 5.4 15.5 10.2 8.8 0.9 1.4 1.1 1.0 1.8 . 0.5 1.6 . -0.9 -0.7 19.8

0.6 0.4 19.2

---

---

0.6

1.1

0.5 0.6 9.5 8.9 1.9 2.2 -0.4 1.0 1.0 0.5 0.2 0.9 0.6 --0.2 2.9 0.8 45.9 1.3

Total saturated 49.2 42.6 17.7 31.6 Total monounsaturated 7.2 18.6 44.1 58.2 Total PUFA 43.2 38.2 37.1 8.9 Total n-3 13.3 34.1 23.5 4.2 Total n-6 29.9 4.1 13.6 4.7 n-3/n-6 0.45 8.32 1.73 0.89 aPG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; PUFA, polyunsaturated fatty acids.

DISCUSSION Each of the polar lipid classes h a d a specific f a t t y acid composition. In PC, 16:0 and 22:6n-3 were the principal components and together accounted for nearly 60% of the total f a t t y acids p r e s e n t in this phospholipid (Table 2). I n contrast, 16:0 comprised only 10.7% of the f a t t y acids in EGP, w h e r e a s the level of 22:6n-3 (36%) was the highest observed in a n y lipid class. As a consequence, the total content of PUFA in E G P (56.2%) was the highest of all lipid classes. Only the E G P fraction produced dimethyl acetals by t r a n s m e t h y l a t i o n of 1-Oa l k - l ' - e n y l linked e t h e r chains. PI was unique a m o n g the lipid classes in t h a t 20:4n-6 was a m a j o r component and accounted for 27.4% of the f a t t y acids, w h e r e a s 22:6n-3 c o m p r i s e d only 7.0% (Table 3). As a consequence, the overall ratio of n-3 to n-6 PUFA in PI was the lowest of a n y lipid class. The s a t u r a t e d f a t t y acid 18:0 was also a m a j o r fatty acid in PI. PS was characterized by a high content of 18:0 and 22:6n-3, coupled with a low content of 16:0. PG was rich LIPIDS, Vol. 29, no. 5 (1994)

Although the pineal organ is a n adjunct of the brain, it is k n o w n to h a v e evolved from a well-differentiated photoreceptive organ t h a t is frequently considered to be a functional third eye in lower v e r t e b r a t e s (3). In fact, to date, the properties established for the pineal photoreceptors h a v e been extended to the retinal photoreceptors a n d vice versa (3). The m a j o r difference between the pineal and retina relates to the neuronal organization, which is simple in the pineal b u t complex in the retina. Thus, pineal photereceptors m a k e contact with second order neurons t h a t send their axons to b r a i n centers. Retinal photorecepters, on the other hand, are in contact with bipolar cells that, in turn, communicate with ganglion cells a n d n u m e r o u s i n t e r n e u r o n s t h a t are present (horizontal, a m a c r i n e , i n t e r p e x i f o r m cells). Consequently, the ratio of photerecepters to other neurons is m u c h higher in the pineal t h a n in the retina, and the lipid composition of the pineal organ can be expected to reflect t h a t of the photorecepter cells.

315 LIPID COMPOSITION

OF TROUT

PINEAL

ORGAN

TABLE 4 M o l e c u l a r Species of PhosphoHpids from Trout P i n e a l O r g a n a Species

Di-PUFA 20:5/22:6 22:6/22:6 22:5/22:6 Saturated/PUFA 14:0/22:6 16:0/20:5 16:0/22:6 16:0/20:4 16:0/22:5 18:0/20:5 18:0/22:6 18:0/20:4 18:0/22:5 Monounsaturated/PUFA 16:1/22:6 18:1/20:5 18:1/22:6 18:1/20:4 18:1/22:5 Di-saturated 16:0/16:0 Saturated/monouns aturated 16:0/18:1 16:0/18:1 Di-monounsaturated 18:1/18:1 20:1/18:1

PC

PE

0.6 • 0.0 4.1 • 0.2 1.0 • 0.1 1.2 • 6.7 • 33.2 • 1.5 • 2.1 • 1.2 • 3.7 • 0.5 • 1.5 •

PS

1.0 _+0.0 13.0 • 0.3 4.5 • 0.3

PI

0.3 • 0.0 4.9 • 0.0 2.4 • 0.1

0.3 • 0.0 0.1 • 0.0 0.2 • 0.1 4.1 • 0.2 10.3 _+0.5 14.0 • 0.3 0.6 • 0.2 10.8 _+0.1 7.1 • 0.5 37.8 • 1.0 0.4 • 0.1

0.1 0.4 1.6 0.2 0.3 0.2 0.2 0.1 0.2

3.8 • 4.2 • 20.1 • 2.4 • 1.9 • 3.7 • 9.6 • 2.7 • 0.6 •

0.1 0.1 0.1 0.1 0.1 0.1 0.3 0.1 0.2

0.7 • 0.2 1.8 • 0.3 15.0 • 0.2 1.8 • 0.1 1.1 • 0.1 3.7 • 0.3 43.9 -+ 0.2 0.6 • 0.1 4.7 • 0.1

0.5 • 0.1 0.9 • 0.1 2.9 • 1.7 0.6 • 0.1

0.9 • 3.7 • 15.4 • 2.6 • 1.1 •

0.1 0.1 0.4 0.1 0.1

0.7 -+ 0.1 6.3 • 1.0 1.1 • 0.1 1.3 • 0.1

0.2 • 0.7 • 0.6 • 4.1 • -

0.9 • 0.1

0.1 • 0.0

1.1 • 0.1

-

28.5 • 1.0 2.5 • 0.1

1.2 _+ 0.1 0.2 • 0.1

2.8 _+ 0.0 0.9 • 0.0

6.9 • 0.3 0.3 • 0.0

0.8 • 0.1 -

0.6 • 0.1 -

1.2 • 0.1 0.3 • 0.0

0.6 • 0.0 -

0.0 0.0 0.5 0.2

aValues are mol% and are means of triplicate determinations • 1 SD. It was assumed that the most saturated fatty acids were located on the sn-1 position. Molecular species containing minor fatty acids or fatty acid isomers were not resolved from the major components. Molecular species containing 18:2n-6 co-elute with those containing 22:5n-3 and 22:5n-6. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositel; PS, phosphatidylserine; PUFA, polyunsaturated fatty acids.

T h e l i p i d c o n t e n t of t h e t r o u t p i n e a l (4.9% o f w e t w e i g h t ) is a l m o s t e x a c t l y h a l f w a y b e t w e e n t h a t f o u n d for t h e b r a i n a n d r e t i n a (6.8 a n d 3.1%, r e s p e c t i v e l y ) o f t h e s a m e s p e c i e s (10). H o w e v e r , t h e level o f T A G i n t h e l i p i d o f t h e p i n e a l (47%) is c o n s i d e r a b l y h i g h e r t h a n t h e 8.4 a n d 30% f o u n d i n t h e l i p i d o f t h e b r a i n a n d r e t i n a , b u t i s n e v e r t h e l e s s c l o s e r to t h e l a t t e r . T h e l e v e l s of T A G i n b r a i n s a n d r e t i n a of t r o u t a r e k n o w n to b e h i g h e r t h a n i n t h e s a m e t i s s u e s f r o m cod (10). A n i n f l u e n c i n g f a c t o r may be the fact that the trout examined were farmed fish. I t is w e l l k n o w n t h a t f a r m e d fish, i n c l u d i n g t r o u t , have higher lipid contents in their flesh than their wild c o u n t e r p a r t s (30). W h e t h e r t h e l i p i d c l a s s c o n t e n t of n e u r a l t i s s u e s differs b e t w e e n w i l d a n d f a r m e d fish o r w h e t h e r h i g h T A G l e v e l s a r e a specific f e a t u r e of t h e l i p i d s o f t r o u t n e u r a l t i s s u e s r e m a i n s to b e e s t a b l i s h e d . A n o t a b l e f e a t u r e o f t h e b r a i n of f i s h a n d a n i m a l s i n g e n e r a l is t h e h i g h p r o p o r t i o n of E G P i n t h e t o t a l l i p i d (10,31). I n b o t h cod a n d t r o u t , t h e E G P / P C r a t i o i n b r a i n l i p i d is a p p r o x i m a t e l y 1:1 (10). T h e l e v e l o f E G P i n t h e p i n e a l l i p i d is n o t a b l y l o w e r t h a n t h a t of PC, t h e E G P / P C r a t i o b e i n g 0.6:1. T h i s v a l u e is c l o s e r to t h e 0.7:1 o b s e r v e d for t h e r a t i o o f t h e s e t w o p h o s p h o l i p i d s i n

t h e r e t i n a of b o t h t r o u t a n d cod (10). T h e e t h a n o l a m i n e p l a s m a l o g e n c o n t e n t of t h e p i n e a l is m u c h l o w e r t h a n t h a t o f f i s h b r a i n ( 3 6 - 3 8 % of E G P ) (25) a n d c l o s e r to t h a t o f r e t i n a (<5% o f E G P ; Bell, M.V., u n p u b l i s h e d d a t a ) . T h e a b s e n c e o f c e r e b r o s i d e s i n t h e l i p i d s o f t h e p i n e a l is also m o r e t y p i c a l of r e t i n a t h a n b r a i n , a s is t h e low a m o u n t o f e t h a n o l a m i n e p l a s m a l o g e n s (31). T h e a b sence of cardiolipin in significant amounts in pineal l i p i d s is u n u s u a l a s t h i s l i p i d c l a s s is a c o m m o n compon e n t of m i t e c h o n d r i a l m e m b r a n e s a n d h a s b e e n f o u n d i n l i p i d e x t r a c t e d f r o m b o t h f i s h r e t i n a a n d b r a i n (10). T h e t o t a l l i p i d a n d c o m p o n e n t l i p i d c l a s s e s of t h e p i n e a l o r g a n s , w i t h t h e e x c e p t i o n of SM, w e r e c h a r a c t e r i z e d b y h i g h c o n t e n t s of P U F A . N e v e r t h e l e s s , t h e cont e n t of P U F A i n t h e t o t a l l i p i d of t r o u t o r g a n (31.2%) is l o w e r t h a n t h e c o r r e s p o n d i n g v a l u e s o f 41.4 a n d 40.6% r e p o r t e d for t h e b r a i n a n d r e t i n a , r e s p e c t i v e l y , of t h e s a m e s p e c i e s (10), a n d is c o n s i d e r a b l y l e s s t h a n t h e P U F A c o n t e n t (43.6%) of t o t a l l i p i d f r o m r a t p i n e a l (20). T h e d i s t r i b u t i o n of f a t t y a c i d s a m o n g t h e v a r i o u s l i p i d c l a s s e s c o n f o r m s to w e l l e s t a b l i s h e d p a t t e r n s . F o r e x a m ple, t h e l o n g - c h a i n m o n o e n o i c f a t t y a c i d 2 2 : 1 n - l l occ u r r e d o n l y i n TAG. T h e a b s e n c e o f t h i s f a t t y acid, w h i c h

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316

R.J. HENDERSONETAL. originates in calanoid zooplankton, is a common feature of fish phospholipids (32). The PUFA 18:2n-6 was also confined to TAG. It is notable that the total lipid of pineal gland contains higher levels of this fatty acid than brain in the rat (20), although it remains to be established whether the 18:2n-6 is specifically associated with TAG. The presence of high levels of 16:0 in PC is a characteristic feature of this phospholipid, including that extracted from trout brain and retina (10) and bovine pineal organ (21). The EGP of neural tissues are known to be specifically enriched in 22:6n-3 (31), and this was also a notable feature observed in the present study of the EGP from the trout pineal. The value for 22:6n-3 in EGP (36%) is slightly higher than the content in EGP from trout brain (34%), but less than that of the retina EGP (46.3%) of the same species (10). Interestingly, the 22:6n-3 contents of PC, PI and PS found here in the pineal are all intermediate between those found in the brain and in the retina. The PI of the pineal organ conformed to the well established pattern in fish tissues, whereby PI has a higher content of 20:4n-6 than other phospholipids and is consequently characterized by a low ratio of n-3 to n-6 PUFA (32). The actual content of 20:4n-6 in the pineal PI (27.4% of total fatty acids) is markedly higher than that reported for trout brain (10.2%) and retina (14.8%) and for the same tissues of cod (4.4 and 12.5%, respectively) (10). The results suggest that in terms of the 20:4n-6 content of PI the pineal exceeds retina which, in turn, exceeds brain. In the rat, the level of 20:4n-6 in the total lipids from the pineal organ is also higher than that found in brain lipids (20). This is consistent with the idea according to which phototransduction might activate phospholipase A2 with the subsequent formation of eicosanoids (33). An involvement of eicosanoids derived from 20:4n-6 in the light-dependent control of melatonin production is therefore indicated. Consistent with the overall fatty acid composition of EGP, PE contained the highest level (13.0%) of di-22:6 molecular species of the phospholipids examined. However, the value was less than that observed previously in trout brain (14.9%), and considerably less than that in trout retina (41.3%) (17). Likewise, di-22:6 molecular species of PC and PS were less abundant in the pineal than the retina, and only pineal PC had a higher di-22:6 content than the corresponding phospholipids from brain. The other molecular species were all as expected, with 16:0/22:6 and 16:0/18:1 dominating PC, 16:0/22:6, 18:1/22:6 and 18:0/22:6 each comprising 9.6-20.1% of PE, and 18:0/22:6 being the predominant PS species. In contrast, PI from fish is known to be relatively impoverished with respect to 22:6n-3 containing molecular species, while C20 PUFA species are abundant (17,18). In trout, retinal PI was predominantly 18:0/20:4n-6 (40.1%) and brain PI 18:0/20:5n-3 (42.3%) (17). This tissue specificity in the molecular species composition of PI was confirmed in cod, in which 18:0/20:4n-6 was the predominant species in liver and roe (36.7 and 49.1%, respectively) and 18:1/20:4n-6 was the next most abundant (18,29). In brain, 18:0/20:5n-3 and 18:1/20:5n-3 comprised over half the PI (18) with arachidonyl species LIPIDS, Vol. 29, no. 5 (1994)

totalling only 15.3%, whereas in retina 16:0/22:6, 18:0/20:4, 18:0/22:6 and 18:0/20:5 were the most abundant species, in that order, totalling 84.5% of PI (18). The PI from trout pineal organ thus closely resembles that of trout retina with 18:0/20:4n-6 predominant. The 18:0/20:5n-3 species so characteristic of PI from trout and cod brain comprised only 10.8% of trout pineal PI. Thus, although it is situated adjacent to the brain and is very closely associated with the brain, the pineal is almost identical to retina in terms of PI composition. Furthermore, no 18:1/24:1 was found in the PC of the pineal organ, whereas this molecular species comprises between 9 and 13% offish brain PC (17,18). Although the total lipid of the trout pineal organ has a lower content of 22:6n-3 than that found in brain or retina, the level is nevertheless still higher than that usually observed in other non-neural tissues, such as liver or muscle (32). Furthermore, the 22:6n-3 content of individual phospholipids is similar to that found in the retina and brain. Consequently, the lipid composition of the pineal organ displays features typical of nervous tissues. Di-22:6n-3 molecular species may be unique to photoreceptor membranes as these are abundant in rod outer segments of many animal species and have recently also been found, albeit in smaller amounts, in the all-cone retina of larval herring (19). The outer segments of the pineal photoreceptors, which correspond to cone-like cells, are not as well developed compared to those of the retinal photoreceptor (3), and the overall amount of outer segment membrane per photoreceptor cell is probably considerably less in the pineal than in the retina. This might explain why the high levels of di22:6n-3 molecular species which characterize the PC, and especially PS and PE, of trout and cod retina (17,18) were not observed in the trout pineal. In conclusion, the present study demonstrates for the first time that, in terms of lipid composition, the pineal resembles the retina more closely than the brain or other tissues. Photoreceptor membranes can now be isolated from a population of purified pineal cells (34) and analysis of their component lipids may disclose specific adaptations in lipid composition. This opens interesting perspectives for the study of the light-dependent signal production by these cells. RERRENCES

1. Menaker, M. (1985) in Photoperiodism, Melatonin and the Pineal (Evered, D., and Clark, S., eds.) pp. 78-92, Pitman, London. 2. Collin, J.P., Voisin, R, Falcon, J., Faure, J.R, Brisson, P., and DeFaye, J.R. (1989)Arch. Histol. Cytol. 52, 441-449. 3. Falcon, J., Thibault, C., Begay, V., Zachmann, A., and Collin, J.-P. (1992) in Rhythms in Fishes (Ali, M.A., ed.) pp. 167-198, Plenum Press, New York. 4. McNulty,J.A. (1984) Cell Tissue Res. 238, 565-575. 5. Meissel, H., and Ekstrom, P. (1988) Neuroscience 25, 1071-1076. 6. Hastings, M.H., Herbert, J., Martensz, N.D., and Roberts,A.C. (1985) in Photoperiodisrn, Melatonin and the Pineal (Evered, D., and Clark, S., eds.) pp. 55-77, Pitman, London. 7. Bromage, N., Jones, J., Randall, C., Thrush, M., Davies, B., Springate, J., Duston, J., and Barker, G. (1992) in The Rainbow Trout (Gall, G.A., ed.) pp. 141-166, Elsevier, Amsterdam.

317

LIPID COMPOSITION OF TROUT PINEAL ORGAN 8. Nayak, P.K., and Singh, T.P. (1987) J. Pineal Res. 4, 387-396. 9. Fliesler, S.J., and Anderson, R.E. (1983) Prog. Lipid. Res. 22, 79-131. 10. Tocher, D.R., and Harvie, D.G. (1988) Fish Physiol. Biochem. 5, 229-239. 11. Wiegand, R.D., and Anderson, R.E., (1983) Exp. Eye Res. 37, 159-173. 12. Rodriguez de Turco, E.B., Gordon, W.C., and Bazan, N.G. (1991) J. Neurosci. 11, 3667-3678. 13. Neuringer, M.D., Connor, W.E., Lin, D.S., Barstad, L., and Luck, S. (1986) Proc. Natl. Acad. Sci. USA 83, 4021-4025. 14. De La Paz, M.A., and Anderson, R.E. (1992) Invest. Opthal. Vis. Sci. 33, 2091-2096. 15. Louie, K , Wiegand, R.D., and Anderson, R.E. (1988) Biochemistry 27, 9014-9020. 16. Stinson, A.M., Wiegand, R.D., and Anderson, R.E. (1991) Exp. Eye Res. 52, 213-218. 17. Ben, M.V., and Tocher, D.R. (1989) Biochem. J. 264, 909-915. 18. Bell, M.V., and Dick, J.R. (1991) Lipids 26, 565-573. 19. Bell, M.V., and Dick, J.R. (1993) J. Mar. Biol. Ass. U.K. 73, 679-688. 20. Sarda, N., Gharib, A., Croset, M., Moliere, P., and Lagarde, M. (1991) Biochim. Biophys. Acta 1081, 75-78. 21. Basinka, J., Sastry, P.S., and Stancer, H.C. (1969) J. Neurochem. 16, 707-714.

22. Christie, W.W. (1982) Lipid Analysis, 2nd edn., pp. 51-122, Pergamon Press, Oxford. 23. Fewster, M.E., Burns, B.J., and Mead, J.F. (1969) J. Chromatogr. 43, 120-126. 24. Olsen, R.E., and Henderson, R.J. (1989) J. Exp. Mar. Biol. Ecol. 129, 189-197. 25. Bell, M.V., and Dick, J.R., (1993) Lipids 28, 19-22. 26. Renkonen, O. (1965) J. Am. Oil Chem. Soc. 42, 298-304. 27. Goto, J., Goto, N., Shamsa, F., Saito, M., Komatsu, S., Suzaki, I~, and Nambara, T. (1983) Analytica Chimica Acta 147, 397-400. 28. ~ k a m u r a , H , and Kito, M. (1991) J. Biochem. 109, 436-439. 29. Bell, M.V. (1989) Lipids 24, 585-588. 30. van Vliet, T., and Katan, M.B. (1990) Am. J. Clin. Nutr. 51, 1-2. 31. Sastry, P.S. (1985) Prog. Lipid Res. 24, 69-176. 32. Henderson, R.J., and Tocher, D.R. (1987) Prog. Lipid Res. 26, 281-347. 33. Redburn, D.A., and Pasantes-Morales, H. (1989) Neurol. Neurobiol. 49, 1-310. 34. Begay, V., Falcon, J., Thibault, C., Ravault, J.P., and Collin, J.P.J. (1992)Neuroendocrinol. 4, 337-345. [Received November 19, 1993, and in revised form March 23, 1994; Revision accepted March 23, 1994]

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