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Lipid composition of the spinal cord in the fruit bat Rousettus aegyptiacus
Comp. Biochem. Physiol. Vol. 75B, No. 3, pp. 441444, 1983 Printed in Great Britain.
0305-0491/8353.00+ .00 (el 1983 Pergamon Press Ltd
LIPID COMPOSITION OF THE SPINAL CORD IN THE FRUIT BAT R O U S E T T U S A E G Y P T I A C U S J. VAN DER WESTHUYZEN MRC Brain Metabolism Research Group, Department of Haematology, School of Pathology of the South African Institute for Medical Research and the University of the Witwatersrand, P.O. Box 1038, Johannesburg 2000, South Africa (Received 8 November 1982)
Abstract--l. Spinal cords were removed from ten Egyptian fruit bats Rousettus aegyptiacus and the lipids analysed. 2. The major phosphatides were choline glycerophosphatide (32.3~o), ethanolamine glycerophosphatide (29.3~o), serine glycerophosphatide (15.2~o) and phosphatidylinositol (8.4~o). Sphingomyelin accounted for 13.8~o of the phospholipid. 3. Glycosphingolipids amounted to 43.2mol/100mol lipid phosphorus, plasmalogens 32.5mol/ 100 mol P and cholesterol 159.5 mol/100 mol P. 4. The fatty acid composition of whole cord, sphingomyelin and non-hydroxy cerebroside were determined. 5. The results were compared with data from other species.
INTRODUCTION Neural tissues are particularly rich in lipids due to the high lipid content of myelinated nerve fibres, especially in the white matter of the vertebrate nervous system. Brain and myelin lipid composition has been studied in a variety of species (Cuzner et al., 1965) including man (Sun, 1973), but the spinal cord has been infrequently examined. While the nervous system of bats has been studied from the functional, electrophysiological and histological points of view (Schneider, 1966; Henson, 1970) lipids have not been studied. This represents a gap in our knowledge, since of all the mammals, the order Chiroptera is surpassed only by the rodents in terms of variety and numbers. The Egyptian fruit bat R o u s e t t u s aegyptiacus and the other fruit bats are classified into one family, the Pteropidae of the suborder Megachiroptera. R o u s ettus has been described as having one of the highest brain to body weight ratios among the bats, nearly identical to the progressive insectivors, and exceeding the lowest encephalized primate (Pirlot and Stephan, 1970). In the present report, the lipid composition of the spinal cord and the fatty acid composition of sphingolipids are examined. MATERIALS AND METHODS Animals Groups of the Egyptian fruit bat Rousettus aegyptiacus caught in the wild, were maintained in avaries on a diet of fresh peeled fruit and water. The diets were supplemented fortnightly with intramuscular vitamin Bj2 (0.5/Jg cyanocobalamin/100 g body weight) and 0.2 ml per bat of an oral vitamin preparation (Abidec, Parke-Davis). The latter dose contained 100IU vitamin D, 0.25mg thiamin, 0.1mg riboflavin, 0.12 mg vitamin B6, 0.4 mg vitamin A, 0.12 mg niacin and 12.5 mg ascorbic acid. Ten healthy adult animals of both sexes with a mean body weight of 1 3 8 + 6 g (mean __+SEM) were used in this study.
441
Experimental procedure Animals were killed by exsanguination and weighed. The spinal cords were rapidly removed, weighed and homogenised in chloroform-methanol 2:1 (v/v) for lipid analysis. The cords provided 233 ___11 mg of tissue for analysis. Lipid analysis Total lipids were extracted using the method of Folch et al. (1957) in solvents containing and antioxidant 2,6-di-tertbutyl-4-methyl-phenol (0.005~o). Phospholipids and sphingolipids were separated by onedimensional TLC on glass plates coated with 0.5 mm silica gel G using a solvent system consisting of chloroform/ methanol/acetic acid/water (60:30:8.4:3.6, v/v). Spots were identified by comparison with known standards (Sigma) and spraying with specific sprays (van der Westhuyzen et al., 1981). For phospholipid estimations, spots visualised in iodine vapour were scraped from the plate and eluted using the solvent systems of Cuzner and Davison (1967). Phospholipid phosphorus was then determined by the colorimetric method of Raheja et al. (1973). Glycosphingolipids (Svennerholm, 1956), plasmalogens (Williams et al., 1962) and cholesterol (Crawford, 1958) were estimated by established techniques. Fatty acid analyses were carried out on whole spinal cord tissue (for total ester-linked fatty acids) or on separated sphingolipids. Lipids were separated by one-dimensional TLC as described above and visualised by spraying with 0.05~o Rhodamine 6G in methanol. Fatty acid methyl esters were prepared by the method of Morrison and Smith (1964). Methanolysis was continued for 9 min in the case of total ester-linked fatty acids (glycerides, phospholipids and cholesterol) and for 75min for amide-linked fatty acids (sphingomyelin and non-hydroxy cerebroside). Methyl esters were separated on a 10~o SP 222 PS column (6 ft x 0.25 in ID., Supelco Inc., Bellefonte, PA, USA) in a Packard Becker 417 chromatograph fitted with a flame ionisation detector. A temperature gradient of 166-172°C at 0.2°C/min and a nitrogen flow rate of 30ml/min was employed. Fatty acids were identified by comparing retention times with those of known standards and checked on log of retention time versus carbon number plots. Peak areas were computed using an integrator (Autolab Mini-
I¢.-
442
J. VAN DER W E S T H U Y Z E N
grator, Spectra-Physics, USA) and checked by triangulation. Results were expressed as mean + SEM.
RESULTS
The lipid composition of spinal cord tissue is shown in Table 1. The major phospholipids were choline glycerophosphatide and ethanolamine glycerophosphatide (phosphatidylethanolamine and its plasmalogen), followed by serine glycerophosphatide (phosphatidylserine and its plasmalogen) and phosphatidylinositol. Sphingomyelin represented nearly 14Yo of the total phosphorus. Plasmalogens represented approximately one third of the total phospholipid present on a molar basis. Examination of TLC plates after lipid separation and staining with iodine showed that plasmalogens were present mainly in the ethanolamine form with some serine phosphatide also present. The molar ratio of cholesterol:phospholipid:glycosphingolipid was 159.5:100:43.2. The fatty acid composition of spinal cord lipids is shown in Table 2. With the method of preparation employed, fatty acids of whole cord includes acids occurring in ester linkage (i.e. limited to glycerides, phospholipids and cholesterol) and specifically excludes the fatty acid amides of sphingolipids. The principle fatty acids were 18: 1, 16:0, 18:0 and 22:6. Polyunsaturated fatty acids comprised 18.3~o of the total, while negligible amounts of fatty acids > C24 were found. The principle fatty acids of sphingomyelin (Table 2) were 18:0, followed by the long chain fatty acids 24:0 plus 24:1. Polyunsaturated fatty acids comprised only 3.6Yo of the total, while long chain fatty acids > C24 amounted to 35.3~o. Similarly, the fatty acids =>C24 accounted for 46.6Yo of the total nonhydroxy fatty acids of cerebroside (Table 2). Polyunsaturated fatty acids amounted to only 7.1 ~o of the total.
DISCUSSION Most of the studies of central nervous system lipids have been in brain tissue rather than spinal cord tissue, due no doubt to the relatively easier accessibility of the brain, as well as providing a larger amount of tissue. The amount of tissue becomes an important factor in small animals: in the bat the ratio of brain to cord weight is 9:1 (2.1 g:230 mg). Nevertheless, ample spinal cord tissue was removed from the bat for lipid analysis. A comparison of the molar phospholipid composition of bat cord, 31-day old rat spinal cord (Ramsey and Fischer, 1978) and white matter of adult rabbit cord (Ishibe and Yamamoto, 1979) showed notable differences. Ethanolamine glycerophosphatide comprised 60.1~o of the phospholipid in the rat, and 40.2~ in rabbit white matter (29.3Yo in bat cord). Choline glycerophosphatide on the other hand, made up 17.4Yo in the rat and 19.3~ in rabbit white matter (32.3Yo in the bat). Serine glycerophosphatide and phosphatidylinositol together comprised 14.9~ of rat cord phospholipid and 15.1yo in the rabbit white matter (23.6yo in the bat cord). Sphingomyelin in the rat cord (7.7yo) was considerably less than in rabbit white matter (18.9~) and bat spinal cord (13.8~). Relative to total phospholipid, the molar concentration of glycosphingolipid (primarily galactolipid) in the bat spinal cord (43.2 mol/100 mol phosphorus) was similar to that of rabbit cord white matter (47.1 mol/100 molP; Ishibe and Yamamoto, 1979). However, the molar concentration of cerebroside in mature sheep cord (Patterson et aL, 1971), was considerably higher (95.3 mol/100 mol P) than in rabbit white matter (Ishibe and Yamamoto, 1979). The concentration of cholesterol in bat spinal cord (159.5 mol/100 mol P) was similar to that of neonatal piglets (153mol/100molP; Patterson et al., 1972) and mature sheep cord (156 mol/100 mol P; Patterson et al., 1971), but higher than in white matter of rabbit spinal cord (114.5 mol/100 mol P; Ishibe and Yamamoto, 1979).
Table h Lipid composition of spinal cord tissue of Rousettus Lipid Total phospholipid
I00
Ethanolamine glycerophosphatide
29.3 ± 1.0
Choline glycerophosphatide
32.3 ± l.O
Serine glycerophosphatide
15.2 ± 0.4
Phosphatidylinositol Sphingomyelin Phosphatidic acid plus origin material
8.4 ± 0.8 13.8 ± 0.8 l.l ± 0.3
Glycosphingolipids
43.2 ± 2.4
Plasmalogens
32.5 ± 1.7
Cholesterol
159.5 ± 2.5
Results are expressed as mol/100 mol lipid phosphorus (means_ SEM. n = 10).
Lipids of bat spinal cord
443
Table 2. Fatty acid composition of spinal cord lipids, sphingomyelin and non-hydroxycerebroside Fatty acid
Whole cord I
Sphingomyelin
Cerebroside
15:0
0.2 -+ 0
0.I -+ 0.I
0.4 ± 0
16:0
23.2 -+ 0.4
4.7 ± 1.0
10.6 ,+ 1.3
16:l
1.6 ,+ 0.2
O.l ± O.l
1.2 ± 0.4
17:0
O.l ,+ 0
0.7 ,+ 0.2
2.3 ± 0.3
17:l
1.0 ,+ O.l
ND
0.5 ± O.l
18:0
15.0 -+ 0.2
39.1 ,+ 2.0
12.2 ,+ 0.8
18:l
34.6 ,+ 0.4
1.2 ,+ 0.5
9.1 ± 0.8
19:0
0.5 ,+ 0
0.7 -+ 0.2
0.5 ,+ 0.2
20:0
0.2 -+ 0
7.2 ± 0.7
2.2 -+ 0.2
20:I
3.S ± O.l
ND
0.9 ,+ O.l
20:2
ND
0.5 -+ O.l
0.9 ,+ 0.2
20:3
0.5 ± O.l
0.2 ,+ O.l
0.4 + 0
20:4
2.6 ± O.l
0.6 -+ 0.2
0.8 -+ 0.3
1.0 ,+ 0
3.8 ± 0.4
3.7 ± 0.4
22:0, 22:1 22:2
ND
1.8 -+ 0.4
4.1 ,+ 0.8
23:0
ND
3.3 '+ 0.3
ND
23:1
ND
ND
0.3 ± O.l
22:4
2.8 ± O.l
0.5 ,+ O.l
0.6 + O.l
22:5
4.0 ± 0.2
ND
ND
24:0, 24:1
0.8 ,+ 0.2
31.5 ± 2.4
38.0 ± 2.6
22:6
8.4 ,+ 0.3
0.I ± 0
0.3 ± O.l
25:0, 25:1
ND
2.7 -+ 0.3
5.3 ± 0.3
26:0, 26:1
NO
l.l ,+ 0.I
3.2 ± 0.6
35.3 ,+ 2.3
46.6 ± 3.4
24
0.8 _+ 0.2
Results are expressed as areas per cent + SEM (n = 10). SEMs less than 0.05 are given as zero. ±Total fatty acids occurring in ester-linkage. ND, not detectable. The fatty acid composition of rat spinal cord lipids (Fehling et al., 1978) was fairly similar to that of the bat. The major fatty acids of the rat were 18:1 (36.19/o of the total vs 34.6~o in the bat), 18:0 (16.9% vs 15.0~o), 20:1 (16.7 vs 3.8~o), 16:0 (11.9% vs 23.2%) and 20:4 (4.29/o vs 2.6%). In the rat, more 22:5 than 22:6 was found (8.0 and 2.4~) while in the bat spinal cord the reverse applied (4.0 and 8.4%). Considerably more unsaturated fatty acids were found in the rat (70.9%) compared with the bat cord (59.3%). The level of polyunsaturated fatty acids was identical (18.1 vs 18.3% in the bat). Compared to the fruit bat, the non-hydroxy fatty acid composition of cerebrosides from adult pig spinal cord (Sweasey et al., 1976) showed a slightly higher percentage of C24 fatty acids (43.0 vs 38.0~) and a considerably higher percentage of C25 plus C26 acids (21.2 vs 8.5~). In the adult pig, there were much higher levels of 20:0 and 22:0 and lower levels of 16:0, 18:0 and 18:1. With respect to interspecies differences, it should be mentioned that a major source of variation would be the ratio of gray to white matter which have substantially different lipid compositions (Cuzner et al., 1965; Ishibe and Yamamoto, 1979). Thus brain cBP75/3(B~E
and spinal cord compositions differ significantly. In addition, allowances should also be made for differences in technique, and the age of the animal, when viewing minor differences between species. While the lipid composition of bat spinal cord differs in some respects from other species, there are many similarities. Many reports have shown that the myelin composition of many species tends to show a similar pattern (Cuzner et al., 1965). This suggests that the nervous system specialisations of the bat (echo location, flight and senses) do not lie in its gross brain or spinal cord composition, but on the structural/functional relationships which have developed and which are uniquely expressed in this group of animals. Acknowledgements--Professor J. Metz and Dr R. Cantrill are thanked for helpful discussions. Supported in part by grants from the South African Medical Research Council.
REFERENCES
Crawford N. (1958) An improved method for the determination of free and total cholesterol using the ferric chloride reaction. Clin. Chim. Acta 3, 357-367.
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J. VAN DERWESTHUYZEN
Cuzner M. L. and Davison A. N. (1967) Quantitative thin layer chromatography of lipids. J. Chromat. 27, 388-397. Cuzner M. L., Davison A. N. and Gregson N. A. (1965) The chemical composition of vertebrate myelin and microsomes. J. Neurochem. 12, 469~481. Fehling C., J~igerstad M., A,kesson B., Axelsson J. and Brun A. (1978) Effects of vitamin Bt: deficiency on lipid metabolism of the rat liver and nervous system. Br. J. Nutr. 39, 501-513. Folch J., Lees M. and Sloane Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Henson O. W. (1970) The central nervous system. In Biology o f Bats (Edited by Wimsatt W. A.), Vol. 2, pp. 58 152. Academic Press, New York. Ishibe T. and Yamamoto A. (1979) Regional differences in lipid composition in rabbit nervous tissue. J. Neurochem. 32, 1665-1670. Morrison W. R. and Smith L. M. (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res. 5, 600-608. Patterson D. S. P., Sweasey D. and Harding J. D. J. (1972) Lipid deficiency in the central nervous system of Landrace piglets affected with congenital tremor AIII, a form of cerebrospinal hypomyelinogenesis. J. Neurochem. 19, 2791 2799. Patterson D. S. P., Sweasey D. and Hebert C. N. (1971) Changes occurring in the chemical composition of the central nervous system during foetal and post-natal development of the sheep. J. Neurochem. 18, 2027-2040.
Pirlot P. and Stephan H. (1970) Encephalization m Chiroptera. Can. J. Zool. 48, 433--444. Raheja R. K., Kaur C., Singh A. and Bhatia I. S. (1973) New colorimetric method for the quantitative estimation of phospholipids without acid digestion. J. Lipid Res. 14, 695-697. Ramsey R. B. and Fischer V. W. (1978) Effect of l-aminocyclopentane-l-carboxylic acid (cycloleucine) on developing rat central nervous system phospholipids. J. Neurochem. 30, 447~,57. Schneider R. (1966) Das Gehirn yon Rousettus aegyptiacus (Geoffroy E. 1810). Ahh. senckenb, naturforsch. Ges. 513, I 160. Sun G. Y. (1973) Phospholipids and acyl groups in subcellular fractions from human cerebral cortex. J. Lipid Res. 14, 656,663. Svennerholm L. (1956) The quantitative estimation of cerebrosides in nervous tissue. J. Neurochem. I, 42 53. Sweasey D., Patterson D. S. P. and Glancy E. M. (1976) Biphasic myelination and the fatty acid composition of cerebrosides and cholesterol esters in the developing central nervous system of the domestic pig. J. Neurochem. 27, 375--380. van der Westhuyzen J., Cantrill R. C., Fernandes-Costa 1~'. and Metz J. (1981 ) Lipid composition of the brain in the vitamin BL,-deficient fruit bat (Rousettus aegyptiacus) with neurological impairment. J. Neurochem. 37, 543 -549. Williams J. N., Anderson C. E. and Jasik A. D. (1962) A sensitive and specific method for plasmalogens and other cnol ethers..I. Lipid Res. 3, 378 381.
0305-0491/8353.00+ .00 (el 1983 Pergamon Press Ltd
LIPID COMPOSITION OF THE SPINAL CORD IN THE FRUIT BAT R O U S E T T U S A E G Y P T I A C U S J. VAN DER WESTHUYZEN MRC Brain Metabolism Research Group, Department of Haematology, School of Pathology of the South African Institute for Medical Research and the University of the Witwatersrand, P.O. Box 1038, Johannesburg 2000, South Africa (Received 8 November 1982)
Abstract--l. Spinal cords were removed from ten Egyptian fruit bats Rousettus aegyptiacus and the lipids analysed. 2. The major phosphatides were choline glycerophosphatide (32.3~o), ethanolamine glycerophosphatide (29.3~o), serine glycerophosphatide (15.2~o) and phosphatidylinositol (8.4~o). Sphingomyelin accounted for 13.8~o of the phospholipid. 3. Glycosphingolipids amounted to 43.2mol/100mol lipid phosphorus, plasmalogens 32.5mol/ 100 mol P and cholesterol 159.5 mol/100 mol P. 4. The fatty acid composition of whole cord, sphingomyelin and non-hydroxy cerebroside were determined. 5. The results were compared with data from other species.
INTRODUCTION Neural tissues are particularly rich in lipids due to the high lipid content of myelinated nerve fibres, especially in the white matter of the vertebrate nervous system. Brain and myelin lipid composition has been studied in a variety of species (Cuzner et al., 1965) including man (Sun, 1973), but the spinal cord has been infrequently examined. While the nervous system of bats has been studied from the functional, electrophysiological and histological points of view (Schneider, 1966; Henson, 1970) lipids have not been studied. This represents a gap in our knowledge, since of all the mammals, the order Chiroptera is surpassed only by the rodents in terms of variety and numbers. The Egyptian fruit bat R o u s e t t u s aegyptiacus and the other fruit bats are classified into one family, the Pteropidae of the suborder Megachiroptera. R o u s ettus has been described as having one of the highest brain to body weight ratios among the bats, nearly identical to the progressive insectivors, and exceeding the lowest encephalized primate (Pirlot and Stephan, 1970). In the present report, the lipid composition of the spinal cord and the fatty acid composition of sphingolipids are examined. MATERIALS AND METHODS Animals Groups of the Egyptian fruit bat Rousettus aegyptiacus caught in the wild, were maintained in avaries on a diet of fresh peeled fruit and water. The diets were supplemented fortnightly with intramuscular vitamin Bj2 (0.5/Jg cyanocobalamin/100 g body weight) and 0.2 ml per bat of an oral vitamin preparation (Abidec, Parke-Davis). The latter dose contained 100IU vitamin D, 0.25mg thiamin, 0.1mg riboflavin, 0.12 mg vitamin B6, 0.4 mg vitamin A, 0.12 mg niacin and 12.5 mg ascorbic acid. Ten healthy adult animals of both sexes with a mean body weight of 1 3 8 + 6 g (mean __+SEM) were used in this study.
441
Experimental procedure Animals were killed by exsanguination and weighed. The spinal cords were rapidly removed, weighed and homogenised in chloroform-methanol 2:1 (v/v) for lipid analysis. The cords provided 233 ___11 mg of tissue for analysis. Lipid analysis Total lipids were extracted using the method of Folch et al. (1957) in solvents containing and antioxidant 2,6-di-tertbutyl-4-methyl-phenol (0.005~o). Phospholipids and sphingolipids were separated by onedimensional TLC on glass plates coated with 0.5 mm silica gel G using a solvent system consisting of chloroform/ methanol/acetic acid/water (60:30:8.4:3.6, v/v). Spots were identified by comparison with known standards (Sigma) and spraying with specific sprays (van der Westhuyzen et al., 1981). For phospholipid estimations, spots visualised in iodine vapour were scraped from the plate and eluted using the solvent systems of Cuzner and Davison (1967). Phospholipid phosphorus was then determined by the colorimetric method of Raheja et al. (1973). Glycosphingolipids (Svennerholm, 1956), plasmalogens (Williams et al., 1962) and cholesterol (Crawford, 1958) were estimated by established techniques. Fatty acid analyses were carried out on whole spinal cord tissue (for total ester-linked fatty acids) or on separated sphingolipids. Lipids were separated by one-dimensional TLC as described above and visualised by spraying with 0.05~o Rhodamine 6G in methanol. Fatty acid methyl esters were prepared by the method of Morrison and Smith (1964). Methanolysis was continued for 9 min in the case of total ester-linked fatty acids (glycerides, phospholipids and cholesterol) and for 75min for amide-linked fatty acids (sphingomyelin and non-hydroxy cerebroside). Methyl esters were separated on a 10~o SP 222 PS column (6 ft x 0.25 in ID., Supelco Inc., Bellefonte, PA, USA) in a Packard Becker 417 chromatograph fitted with a flame ionisation detector. A temperature gradient of 166-172°C at 0.2°C/min and a nitrogen flow rate of 30ml/min was employed. Fatty acids were identified by comparing retention times with those of known standards and checked on log of retention time versus carbon number plots. Peak areas were computed using an integrator (Autolab Mini-
I¢.-
442
J. VAN DER W E S T H U Y Z E N
grator, Spectra-Physics, USA) and checked by triangulation. Results were expressed as mean + SEM.
RESULTS
The lipid composition of spinal cord tissue is shown in Table 1. The major phospholipids were choline glycerophosphatide and ethanolamine glycerophosphatide (phosphatidylethanolamine and its plasmalogen), followed by serine glycerophosphatide (phosphatidylserine and its plasmalogen) and phosphatidylinositol. Sphingomyelin represented nearly 14Yo of the total phosphorus. Plasmalogens represented approximately one third of the total phospholipid present on a molar basis. Examination of TLC plates after lipid separation and staining with iodine showed that plasmalogens were present mainly in the ethanolamine form with some serine phosphatide also present. The molar ratio of cholesterol:phospholipid:glycosphingolipid was 159.5:100:43.2. The fatty acid composition of spinal cord lipids is shown in Table 2. With the method of preparation employed, fatty acids of whole cord includes acids occurring in ester linkage (i.e. limited to glycerides, phospholipids and cholesterol) and specifically excludes the fatty acid amides of sphingolipids. The principle fatty acids were 18: 1, 16:0, 18:0 and 22:6. Polyunsaturated fatty acids comprised 18.3~o of the total, while negligible amounts of fatty acids > C24 were found. The principle fatty acids of sphingomyelin (Table 2) were 18:0, followed by the long chain fatty acids 24:0 plus 24:1. Polyunsaturated fatty acids comprised only 3.6Yo of the total, while long chain fatty acids > C24 amounted to 35.3~o. Similarly, the fatty acids =>C24 accounted for 46.6Yo of the total nonhydroxy fatty acids of cerebroside (Table 2). Polyunsaturated fatty acids amounted to only 7.1 ~o of the total.
DISCUSSION Most of the studies of central nervous system lipids have been in brain tissue rather than spinal cord tissue, due no doubt to the relatively easier accessibility of the brain, as well as providing a larger amount of tissue. The amount of tissue becomes an important factor in small animals: in the bat the ratio of brain to cord weight is 9:1 (2.1 g:230 mg). Nevertheless, ample spinal cord tissue was removed from the bat for lipid analysis. A comparison of the molar phospholipid composition of bat cord, 31-day old rat spinal cord (Ramsey and Fischer, 1978) and white matter of adult rabbit cord (Ishibe and Yamamoto, 1979) showed notable differences. Ethanolamine glycerophosphatide comprised 60.1~o of the phospholipid in the rat, and 40.2~ in rabbit white matter (29.3Yo in bat cord). Choline glycerophosphatide on the other hand, made up 17.4Yo in the rat and 19.3~ in rabbit white matter (32.3Yo in the bat). Serine glycerophosphatide and phosphatidylinositol together comprised 14.9~ of rat cord phospholipid and 15.1yo in the rabbit white matter (23.6yo in the bat cord). Sphingomyelin in the rat cord (7.7yo) was considerably less than in rabbit white matter (18.9~) and bat spinal cord (13.8~). Relative to total phospholipid, the molar concentration of glycosphingolipid (primarily galactolipid) in the bat spinal cord (43.2 mol/100 mol phosphorus) was similar to that of rabbit cord white matter (47.1 mol/100 molP; Ishibe and Yamamoto, 1979). However, the molar concentration of cerebroside in mature sheep cord (Patterson et aL, 1971), was considerably higher (95.3 mol/100 mol P) than in rabbit white matter (Ishibe and Yamamoto, 1979). The concentration of cholesterol in bat spinal cord (159.5 mol/100 mol P) was similar to that of neonatal piglets (153mol/100molP; Patterson et al., 1972) and mature sheep cord (156 mol/100 mol P; Patterson et al., 1971), but higher than in white matter of rabbit spinal cord (114.5 mol/100 mol P; Ishibe and Yamamoto, 1979).
Table h Lipid composition of spinal cord tissue of Rousettus Lipid Total phospholipid
I00
Ethanolamine glycerophosphatide
29.3 ± 1.0
Choline glycerophosphatide
32.3 ± l.O
Serine glycerophosphatide
15.2 ± 0.4
Phosphatidylinositol Sphingomyelin Phosphatidic acid plus origin material
8.4 ± 0.8 13.8 ± 0.8 l.l ± 0.3
Glycosphingolipids
43.2 ± 2.4
Plasmalogens
32.5 ± 1.7
Cholesterol
159.5 ± 2.5
Results are expressed as mol/100 mol lipid phosphorus (means_ SEM. n = 10).
Lipids of bat spinal cord
443
Table 2. Fatty acid composition of spinal cord lipids, sphingomyelin and non-hydroxycerebroside Fatty acid
Whole cord I
Sphingomyelin
Cerebroside
15:0
0.2 -+ 0
0.I -+ 0.I
0.4 ± 0
16:0
23.2 -+ 0.4
4.7 ± 1.0
10.6 ,+ 1.3
16:l
1.6 ,+ 0.2
O.l ± O.l
1.2 ± 0.4
17:0
O.l ,+ 0
0.7 ,+ 0.2
2.3 ± 0.3
17:l
1.0 ,+ O.l
ND
0.5 ± O.l
18:0
15.0 -+ 0.2
39.1 ,+ 2.0
12.2 ,+ 0.8
18:l
34.6 ,+ 0.4
1.2 ,+ 0.5
9.1 ± 0.8
19:0
0.5 ,+ 0
0.7 -+ 0.2
0.5 ,+ 0.2
20:0
0.2 -+ 0
7.2 ± 0.7
2.2 -+ 0.2
20:I
3.S ± O.l
ND
0.9 ,+ O.l
20:2
ND
0.5 -+ O.l
0.9 ,+ 0.2
20:3
0.5 ± O.l
0.2 ,+ O.l
0.4 + 0
20:4
2.6 ± O.l
0.6 -+ 0.2
0.8 -+ 0.3
1.0 ,+ 0
3.8 ± 0.4
3.7 ± 0.4
22:0, 22:1 22:2
ND
1.8 -+ 0.4
4.1 ,+ 0.8
23:0
ND
3.3 '+ 0.3
ND
23:1
ND
ND
0.3 ± O.l
22:4
2.8 ± O.l
0.5 ,+ O.l
0.6 + O.l
22:5
4.0 ± 0.2
ND
ND
24:0, 24:1
0.8 ,+ 0.2
31.5 ± 2.4
38.0 ± 2.6
22:6
8.4 ,+ 0.3
0.I ± 0
0.3 ± O.l
25:0, 25:1
ND
2.7 -+ 0.3
5.3 ± 0.3
26:0, 26:1
NO
l.l ,+ 0.I
3.2 ± 0.6
35.3 ,+ 2.3
46.6 ± 3.4
24
0.8 _+ 0.2
Results are expressed as areas per cent + SEM (n = 10). SEMs less than 0.05 are given as zero. ±Total fatty acids occurring in ester-linkage. ND, not detectable. The fatty acid composition of rat spinal cord lipids (Fehling et al., 1978) was fairly similar to that of the bat. The major fatty acids of the rat were 18:1 (36.19/o of the total vs 34.6~o in the bat), 18:0 (16.9% vs 15.0~o), 20:1 (16.7 vs 3.8~o), 16:0 (11.9% vs 23.2%) and 20:4 (4.29/o vs 2.6%). In the rat, more 22:5 than 22:6 was found (8.0 and 2.4~) while in the bat spinal cord the reverse applied (4.0 and 8.4%). Considerably more unsaturated fatty acids were found in the rat (70.9%) compared with the bat cord (59.3%). The level of polyunsaturated fatty acids was identical (18.1 vs 18.3% in the bat). Compared to the fruit bat, the non-hydroxy fatty acid composition of cerebrosides from adult pig spinal cord (Sweasey et al., 1976) showed a slightly higher percentage of C24 fatty acids (43.0 vs 38.0~) and a considerably higher percentage of C25 plus C26 acids (21.2 vs 8.5~). In the adult pig, there were much higher levels of 20:0 and 22:0 and lower levels of 16:0, 18:0 and 18:1. With respect to interspecies differences, it should be mentioned that a major source of variation would be the ratio of gray to white matter which have substantially different lipid compositions (Cuzner et al., 1965; Ishibe and Yamamoto, 1979). Thus brain cBP75/3(B~E
and spinal cord compositions differ significantly. In addition, allowances should also be made for differences in technique, and the age of the animal, when viewing minor differences between species. While the lipid composition of bat spinal cord differs in some respects from other species, there are many similarities. Many reports have shown that the myelin composition of many species tends to show a similar pattern (Cuzner et al., 1965). This suggests that the nervous system specialisations of the bat (echo location, flight and senses) do not lie in its gross brain or spinal cord composition, but on the structural/functional relationships which have developed and which are uniquely expressed in this group of animals. Acknowledgements--Professor J. Metz and Dr R. Cantrill are thanked for helpful discussions. Supported in part by grants from the South African Medical Research Council.
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