Key words: Brain lipid; Steliate gailglion; Fin nerve; Lipid composition; Phospho~i~id; Fatty acid; (Squid nervous tissue)

ave studied the li

ditiiow cardioli

transport [I]. Understan anises for these processes has so Ear retied heavdy

* Present addresses: Department of Physiology, iUniversity of Tokushima, School of Medicine, Tokushima 770, Japan.

* * Present address: Department of Pediatrics, Utiversity of Tokushima, School of Medicine, Tokushima 710, Axpan.

Correspondence: Y. Kishimoto, Kennedy Institute, 707 North bundfes of smal! caliber fibers csmprisirg the 51

Broadway, Baltimore, MD 21205, U.S.A. nerve.

OOO5-2740/87/$03.50 0 1981 Elsevier Science Publishers B.V. (

79

Materials and Methods

Materials. Squids (Loligo paelei), obtained dur- ing the summer months at the Marine Biological Laboratories, Woods Hole, MA, were decapitated and brains (optic and cerebral lobes), fin nerves and stellate ganglia were removed, lyophilized and stored at -20” C until used. TLC plates pre- coated with silica gel G were purchased from Analtech. Two types of silica gel were used for column chromatography; Unisil, 100-200 mesh, and sihcic acid, 325 mesh, purchased from Clark- son Chemical Co. and Bio-Rad, respectively. All other common chemicals and glass-redistilled solvents were reagent grade or better and were purchased from general suppliers. Chloroform contained 1% ethanol as preservative.

Extraction of lipids. Lyophilized tissues were homogenized with 10 ~01s. of water and then with chloroform/methanol (1: 1) (lo-times the volume of the homogenate) and centrifuged. The residues were rehomogenized with the same volume of the same solvent and centrifuged. Chloroform was added to the combined extracts to make the ratio of chloroform to methanol 2 : 1 and the solution was washed with one-fifth vol. of 0.1 M KC1 according to Folch et al. [5]. The lipid-containing lower layer was then evaporated to dryness.

TLC-densitometric analysis of total lipids. A por- tion of total lipids without fractionation was analyzed by TLC on silica gel G plates with a mixture of chloroform/methanol/2 M NH,OH (24 : 7 : 1). To quantify lipids, the plate was sprayed with a solution of cupric sulfate in phosphoric acid [6] and then heated, and the color intensity was measured with a Shimadzu Model CS-930 Densitometer as described previously [7].

Fractionation of total lipids. For the characteri- zation of individual brain lipids, particulate frac- tion (after removal of cytosol from whole homo- genate) was obtained from Dr. Ron Vale. Total lipids extracted from 41.4 g of lyophilized material were fractionated by silica gel column chromatog- raphy as described previously [7] with modifica- tion. The total lipids, 10.0 g, dissolved in 50 ml of chloroform, were chromatographed on a 3.6 cm i.d. X 58 cm column containing 280 g of Bio-Rad silicic acid. The column was first eluted with 500 ml of chloroform, followed by 2 1 of chloroform/

methanol (99: 1) and then 3 1 of acetone. The column was further eluted sequentially with 700 ml, 600 ml, 500 ml and 1 1 of a mixture of chloroform/methanol in the ratio of (4: 1, 2 : 1, 1 : 1 and 1 : 4), respectively, and finally with 1 1 of methanol.

Cholesterol and free fatty acids in the chloro- form eluate were further purified from a portion of this fraction by another column chromatogra- phy (Unisil), followed by preparative TLC on a silica gel G plate with hexane/ether/acetic acid (90 : 10 : 1) as the eluting solvent. Most of the cardiolipin, along with some phosphatidic acid, was present in chloroform/methanol (99 : 1) and was isolated from this fraction by preparative TLC as described previously [7]. Phosphatidyl- ethanolamine, phosphatidylcholine, phosphatid- ylserine and phosphatidylinositol were included in fractions eluted with acetone, and chloroform/ methanol (4 : 1, 2 : 1, 1: 1 and 1 : 4) and methanol. Equal portions of each fraction were pooled and the above phospholipids were isolated from the pooled fraction by preparative TLC, also as previ- ously described [7]. All the sphingomyelin was found in the fractions eluted by chloroform/ methanol (1 : 4) and methanol. To further purify sphingomyelin, equal portions of these fractions were combined and subjected to mild alkaline methanolysis [S] and then sphingomyelin was iso- lated by preparative TLC as described previously [9]. Ceramide 2aminoethylphosphonate was iso- lated from the fractions eluted with chloroform/ methanol (2 : 1 and 1: 1) by treatment similar to that described above for the isolation of sphingomyelin. All isolated phospholipids were stored at - 20 o C in the presence of 0.05% butyl- ated hydroxytoluene (Sigma Chemical Co.) until analysis.

Structural analysis of individual lipids. A portion of phospholipid purified by preparative TLC was methanolyzed with methanolic-HCl as described previously [lo]. Liberated fatty acid methyl esters and dimethylacetals of aldehydes were extracted with hexane, purified by preparative TLC, and analyzed by gas chromatography-mass spectrome- try (GC-MS) as described previously [7]. For gas chromatographic analysis of polyunsaturated fatty acids, a capillary column, 10 m x 0.53 mm, con- taining carbowax 20 M as 1.33 p film thickness

tissues. As desctib

ceratide 2-atinoeth

81

In addition to these commonly found lipids, two relatively minor fast moving lipids were de- tected in brain and in lesser amounts in stellate ganglia on the TLC scan (Fig. 1). By comparison with respective standards on TLC, the faster run- ning lipid was identified as cardiolipin and the slower running lipid consisted of free fatty acids.

Further characterization of lipids in brain (cerebral

and optic lobes)

Total lipids (10.0 g), extracted from the par- ticulate fractions after centrifugation at 100000 X

g for 1 h from homogenates of combined cerebral and optic lobes (41.4 g dry weight), were fractionated by silica gel column chromatography. Cholesterol (1.67 g) was the single major com- pound present in the chloroform fraction. GC-MS analysis of cholesterol isolated from the chloro- form fraction by preparative TLC showed that at least 95% was cholesterol. Several minor peaks in this chromatogram were not identical with cholestanol or desmosterol and were not char- acterized. Small amounts of free fatty acids were also present in the chloroform fraction as identi- fied by their co-migration with standard palmitic acid on TLC. Methyl esters produced from the free fatty acids were analyzed by GC-MS. Nearly 70% of these esters were polyunsaturated. These included 20 : 4, 20 : 5 and 22 : 6, all n - 3 isomers.

The acetone eluate contained little glycolipid. The material obtained in this fraction was treated with mild alkaline methanolysis [8] and further purified by chromatography on a Unisil column. The acetone fraction from the second Unisil col- umn was benzoylated and desulfated and the product was analyzed by HPLC [12]. The chro- matogram revealed a single peak with a retention time near that of perbenzoylated glucocerebroside from shrimp nerve. From the size of the peak, only traces of this tentatively identified gluco- cerebroside were present in squid brain.

The remaining lipids in squid brain were all phospholipids. The presence of cardiolipin was confirmed by its co-migration with an authentic standard in two different TLC systems and by color development with molybdenum blue reagent [13]. Its fatty acids were mostly saturated or monounsaturated (Table II).

Phosphatidylethanolamine, phosphatidylcho-

line, phosphatidylserine and phosphatidylinositol were similarly identified. These lipids were treated with phospholipase A, and fatty acid moieties in the C-l and C-2 positions of glycerol were de- termined separately (Table II). Phosphatidyl- ethanolamine, phosphatidylserine and phosphatid- ylinositol were enriched with polyunsaturated fatty acids, mainly 20 : 4, 20 : 5, and 22 : 6 (all n-3 iso- mers) usually in the C-2 position. Fatty acids in the C-l positions of these phospholipids, as well as fatty acids in both positions of phosphatidyl- choline, were mostly saturated or monodis- aturated, such as 16: 0, 18:0, 18: 1 and 20: 1. Only phosphatidylethanolamine contained detec- table amounts of plasmalogens. When the isolated phosphatidylethanolamine was exposed to HCl vapor and subjected to TLC, as described previ- ously [7], about 3% was judged to be plasmalogen. The composition of the liberated aldehyde moiety (determined by GC-MS) included mainly saturated and monounsaturated isomers with C&-C,, chain length.

Sphingomyelin and ceramide 2-aminoethyl- phosphonate were identified by TLC analysis of mild alkali stable product by comparison with authentic standards. A known standard of the latter compound, purified from fresh water bivalve (Carbicula sandai) lipids was obtained from Drs. Hori and Sugita. After acid hydrolysis [14], the presence of 2-aminoethylphosphonate was con- firmed by its reaction with ninhydrin and by comparison with authentic 2aminoethylphospho- nate (purchased from Sigma Chemical Co.). The fatty acid compositions of two sphingolipids, sphingomyelin and ceramide 2aminoethylphos- phonate (Table II), were very similar to each other but different from the above-mentioned glycero- phospholipids. These fatty acids are highly saturated, mainly with 16 : 0 and 18 : 0. Sphingoid base, derived from both sphingomyelin and ceramide 2aminoethylphosphonate, appears to have an unusual structure and is being analyzed.

Discussion

The overall lipid compositions of brain (optic and cerebral ganglia), stellate ganglia, and fin nerves were similar to each other. In agreement with previous studies [15-181, cholesterol, phos-

82

TABLE II

FATTY ACID AND FATTY ALDEHYDE COMPOSITION OF PHOSPHOLIPIDS IN SQIJID BRAIN

IndividuaI glycerophosphohpids, except cardiolipin, were treated with phosphohpase A, and fatty acids and lysophosphohpids

obtained were separated. The fatty acids were converted to methyl esters, analyzed and methyl esters were simihuly analyzed (C-l).

The product from lysophosphatidylethanolamine was fractionated to fatty acid methyl esters and dimethykcetals (from aIdehyde

moiety of plasmaIogen). The composition of the latter is given under PL. Cardiohpin, sphingomyehn and ceramide 2-

aminoethylphosphonate were directly methanolyzed and the methyl esters obtained were analyzed. Details are given in the text. CL,

cardiolipin; PE, phosphatidylethanolamine; PL, etbanolamine plasmalogen; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidyhnositol; SM, sphingomyehn; CAEP, ceramide 2-aminoethylphosphonate. Each fatty acid methyl ester and dimethyi-

acetal homolog is shown by carbon number followed by number of double bond. AlI polyenes are tentativeiy identified as II ---3

isomers.

Carbon chain Individual lipids

CL PE PL PC PS PI SM CAEP

Gl c-2 C-l c-2 C-l G2 C-l c-2 ~-_

16:0 24 28 3 15 80 26 14 11 9 3 41 37

17:o

18:0

18:l

19:o

20:o

2O:l

20:4

20:5

22~6

2 _a 4 3 1 - 2 - 3 - 2 3

14 34 5 58 5 4 35 10 47 5 48 56 18 6 - 24 5 44 7 6 5 - - -

1 1 3 - - 1 23 3 10 2 1 1 - - - - - 2 2

22 15 - - 6 15 13 7 13 - - - 8 8 _ - 3 - 2 - 3 1 - - 4 2 33 _ 4 3 18 9 82 - I 7 2 56 - - 6 1 45 1 7 - _

a Less than 1% of total.

phatidylethanolamine and phosphatidylchohne were the major lipids, whereas phosphatidylserine, phosphatidylinositol and sphingomyelin were pre- sent in smaller amounts. Squid brain contained less ethanolamine plasmalogen than did the other two invertebrate nervous systems [7,9]. Small amounts of cardiolipin and free fatty acids were found in squid brain and stellate ganglia, but not in fin nerve.

Significant quantities of a phosphonolipid, ceramide 2-aminoethylphosphonate are widely distributed among invertebrate tissues [19], includ- ing the whole body of Loligo edulis [20]. Together with previous report on the occurrence of this lipid in the nervous system of Apiysia kurodai [21], its finding in squid brain suggests wide distri- bution of sphingophosphonolipids in invertebrate nervous systems. Although the function of this lipid remains to be investigated, an interesting correlation between spbingophosphonolipids and membrane excitability was suggested in ciliary movement of Paramecium [22].

Fatty acid compositions of squid brain phos-

pholipids were determined for the first tune. These lipids were highly enriched in polyunsaturated fatty acids, which comprised 90, 63, and 89% of fatty acids in the /3 positions of phosphatidyl- ethanolamine, phosphatidylserine and pho@hatid- ylinositol, respectively. Fatty acids in (Y positions of these lipids as well as total fatty acids in other glycerophospholipids contained only relatively small amounts of polyenes. This specific locdi- zation of polyunsaturated fatty acids in glycerophospholipids resembled that found in mammalian tissues 1231. The abundance of poly- enes in squid nerve was in sharp contrast with the lipids from shrimp [9] and earthworm nerve 171, which did not contain even traces of polyun- saturated fatty acids. On the other hand, the squid brain lipid did not contain very long chain (C22-C26) fatty acids which are enriched in shrimp and earthworm nerve lipids (monounsaturated and saturated).

Fatty acid compositions of sphingomyelin and ceramide 2arninoethylphosphonate were. similar, and distinctly different from those of glycerophos-

pholipids. Thus, the similarity in the fatty acids suggests a possible metabolic relationship between the two sphingophospholipids, possibly sharing the same ceramide pool for their synthesis. There was also a significant difference in the fatty acids of sphingomyelin in squid brain from those of shrimp and earthworm nerves [7,9]. Like myelin from mammalian brain [24], sphingomyelin in shrimp and earthworm was characterized by very long chain fatty acids. Only C,,-C,, fatty acids were present in squid brain sphingomyelin. As previously reported by McMurray et al. [16], galactocerebrosides and sulfatides, major glyco- lipids in myelinated nervous tissue [25] were not present in squid. Glucocerebrosides, which were present in large quantities in crustacean nerves [26] and in smaller quantities in other protostomes [27], were found only in trace quantities in squid brain.

Polyunsaturated fatty acids present in high con- centrations in squid nervous system lipids were not detected in earthworm [7] and shrimp [9] nervous system lipids. The striking differences among relatively close protostome invertebrates may be related to diet, acclimatization to environ- ment, or differences in need for biologically active product prostaglandins and leukotrienes. The polyunsaturated fatty acids in squid nerves may be of nutritional origin, because squid mostly re- lies on fish, rich in polyenes, as a nutritional source. Whether the polyunsaturated fatty acids are exogenous or endogenous, the role of these fatty acids of invertebrate animals requires clarifi- cation.

Acknowledgements

A portion of this investigation was performed in the laboratory of Dr. Nicholas Ingoglia at Woods Hole Marine Biological Laboratories. We are grateful to his support and helpful discussions. We also thank Drs. Ron Vale and Bruce Schnapp of National Institutes of Health for collecting squid brain preparations, Drs. Taro Hori and Mutsumi Sugita, Shiga University, Japan, for giv- ing us ceramide-2arninoethylphosphonate, Mrs. Ann Moser for her assistance in gas chromatogra- phy-mass spectrometry, and Dr. Pamela Talalay and Janice White for assistance in preparing this

83

manuscript. This investigation was supported by Grant BNS8314337 from the National Science Foundation and NS13559, and NS13980 from the National Institutes of Health, U.S. Public Health Service.

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