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Preparation of some labeled glycosphingolipids by catalytic addition of tritium
Chemistry and Physics o f Lipids 13 (1974) 447-452
© NORTH-HOLLANDPUBLISHINGCOMPANY
P R E P A R A T I O N OF SOME L A B E L E D G L Y C O S P H I N G O L I P I D S BY CATALYTIC ADDITION OF TRITIUM J.L. DI CESARE and M.M. RAPPORT Division o f Neuroscience, New York State Psychiatric Institute and Department o f Biochemistry, College o f Physicians and Surgeons, Columbia University, New York, N. Y. 10032, USA
Three experiments are described in which seven tritium-labeled glycosphingolipidswere prepared by catalytic addition of tritium gas to unsaturated centers. These compounds include glucosyl ceramide, galactosyl ceramide, lactosyl ceramide (cytolipin H), N-acetylgalactosaminyl 031 ~ 3) galactosyl (c~l~ 3) galactosyl (/31~ 4) glucosyl ceramide (cytolipin R), and three species of gangliosides: GM1 (G4), GM3 (G6) , and GD1a (G3).
I. Introduction The use of radioactively labeled compounds in studying the biochemical properties of compounds is now common practice. Three methods are currently favored for introducing tritium into glycosphingolipids. In the first, a labelled precursor is used to prepare the compound biosynthetically [ 1]. In the second, the label is introduced into the carbohydrate moiety by oxidation with galactose oxidase followed by reduction with tritiated borohydride [2, 3]. In the third, the label is introduced into the hydrophobic residues by catalytic addition of tritium gas [4, 5]. We have had several experiences with the latter method that may be of value to others attempting to label their compounds in this way, and it is the purpose of this short communication to describe them. Addition of tritium was carried out by the New England Nuclear Corporation, Boston, Mass. The charge for carrying out this procedure is substantial ($350 in 1972); since the quantity of material so treated is not related to the charges imposed and since many of the compounds are available only in very small amounts, the cost of labeling by tritium addition is not an unimportant consideration. We have solved this problem in part by subjecting mixtures of compounds to tritium addition, selecting as components of the mixtures those glycosphingolipids that can be easily separated by chromatography.
448
J.L. Di Cesare,M.M. Rapport, Tritium-labeledglycosphingolipids
II. Materials and methods
Glycosphingolipids used in this study were homogeneous by thin layer chromatography. The lipids were isolated from animal tissues by conventional chromatographic methods. Their sources were as follows: glucosyl ceramide from human (Gaucher) spleen; galactosyl ceramide from bovine spinal cord; lactosyl ceramide from bovine spleen; cytolipin R from rat lymphosarcoma; GM3 ganglioside from rat spleen; and GMI and GD1a gangliosides from bovine brain. Thin layer chromatography was carried out on plates of silica gel G (20 cm × 20 cm), 0.5 mm thick for preparative chromatography and 0.25 mm thick for analytical chromatography. Column chromatography was carried out on silicic acid (Unisil; Clarkson Chemical Co,, Williamsport, Pa.). Radioactivity was determined by scintillation counting with a Packard 3320 Counter using Bray solution [6]. Quenching corrections were determined by the channels ratio method. Radio purities of final products were determined by TLC. At least 95% of the radioactivity was recovered from areas corresponding to those occupied by the appropriate standards.
III. Results A. Tritium addition.
Three experiments were carried out. In the first a suspension of 5.0 mg of cytolipin H and 5.0 mg of cytolipin R in 2.5 ml of isopropanol-ethyl acetate-water (1 : 1 : 0.5) was stirred overnight at room temperature with 25 mg of platinum catalyst in the presence of 5 Ci of tritium gas. In the second, this treatment was applied to a solution of 11.3 mg of galactosyl ceramide and 10.8 mg of G Dla ganglioside in 2.5 ml of the same solvent. In the third, it was applied to a solution of 6.4 mg of glucosyl ceramide, 5.4 mg of GM1 ganglioside and 5.0 mg of GM3 ganglioside in 2.5 ml of the same solvent. In each case the solvent was removed, and the residue was taken up in 5 ml of chloroform-methanol (1 : 1) and evaporated twice to remo~'e labile tritium. The residue was then dissolved in 10 ml of chloroform-methanol (1 : 1). The procedure to this point was carried out by the New England Nuclear Corporation. B. Purification o f cytolipin H and Cytolipin R
To the solution, 5 ml of chloroform was added followed by 3 ml of 0.1 M KC1. After shaking, the phases were separated. The lower phase, after washing 3 times with "pure solvents upper phase", contained 1000 ~tCi of activity. The lipids were separated by column chromatography (Unisil, 10 g), loading the column with a lipid solution in chloroform-methanol (95 : 5) and eluting with 250 ml portions of
J.L. Di Cesare,M.M. Rapport, Tritium-labeledglycosphingolipids
449
chloroform-methanol, 95 : 5, 90 : 10, 85 : 15, 80 : 20, and 67 : 33. Cytolipin H was eluted with chloroform-methanol (90 : 10) and cytolipin R with chloroformmethanol (80 : 20 to 67 : 33). The cytolipin H fraction (3.9 mg) was homogeneous by analytical TLC. The cytolipin R fraction was recrystallized from 1 ml of methanol, and additional compound was obtained from the mother liquor by preparative TLC. Total recovery, 4.2 mg. Specific activities, based on analysis of long chain base [7], were cytolipin H, 420/ICi/mg and cytolipin R, 760 pCi/mg.
C. Purification of galactosyl ceramide and GD1a ganglioside To the 12 ml of solution in chloroform-methanol (1 : 1), 6 ml of chloroform was added, followed by 3.6 ml of water. The upper and lower phases were separated and the lower phase was extracted 3 times with "pure solvent upper phase."
1. GD1a ganglioside The combined upper phase and extracts were dialyzed against running tap water for 1 day and against 3 changes of deionized water (4 1.) for 1 day (activity, about 500 ~tCi). The dialyzed ganglioside was then subjected to chromatography on silicic acid (7 g) in chloroform-methanol (90 : 10) and eluted with chloroform-methanol (90 : 10,225 ml; 67 : 33,250 ml) and chloroform-methanol-water (61 : 32 : 7, 250 ml). The ganglioside was eluted with the last solvent and recrystallized from methanol to give 1.9 mg of tritiated product, homogeneous by TLC, and having a specific activity of 200 btCi/mg. The mother liquor yielded additional material (0.5 mg).
2. Galactosyl ceramide The lower phase was evaporated to dryness and the residue was subjected to chromatography on silicic acid (10 g), eluting with chloroform (250 ml), and chloroform-methanol (95 : 5,150 ml; 90 : 10, 150 ml). This caused the galactosyl ceramide to separate into 2 fractions: the first was eluted with chloroform-methanol (95 : 5; normal fatty acid fraction) and the second with chloroform-methanol (90 : 10; hydroxy fatty acid fraction). The solvents were removed and the residues were purified by a sequence of 3 steps: preparative TLC, recrystallization from methanol, and preparative TLC again. The glactosyl ceramides with normal and hydroxy fatty acids weighed 3.2 and 3.0 mg, respectively, and had specific activities of 150/aCi/mg and 390 gCi/mg, respectively.
D. Purification of glucosyl ceramide, GM1 and GM3 gangliosides The sample was evaporated to dryness, and the residue was fractionated on a column of silicic acid (11 g) as follows. The portion soluble in 5 ml of chloroform
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J.L. Di Cesare, M.M. Rapport, Tritium-labeledglycosphingolipids
was applied to the column, which was then eluted with chloroform (300 ml). The insoluble portion was dissolved in chloroform-methanol (95 : 5, 5 ml) and applied to the column. Eluting solvents were 300 ml portions of chloroform-methanol (95 : 5; 90 : 10; 67 : 33; and 50 : 50), methanol, and finally methanol water 50 : 50. TLC analysis showed that glucosyl ceramide was eluted with chloroformmethanol (95 : 5) and both gangliosides were eluted together with chloroformmethanol (50 : 50). A plot of radioactivity vs. fraction number for the glucosyl ceramide fraction showed two well-separated peaks, the second much smaller in area than the first. Material in the first peak, glucosyl ceramide with normal fatty acid chains, was recrystallized from methanol and again purified by preparative TLC to give 2.8 mg with a specific activity of 220/aCi/mg. 1. GM3 and GM1 gangliosides The material eluted with chloroform-methanol (50 : 50) was dried and then taken up in chloroform-methanol (80 : 20) and subjected to chromatography on silicic acid (13 g) eluting with chloroform-methanol (80 : 20,300 ml; 67 : 33, 300 ml; and 50 : 50,300 ml). GM3 ganglioside was present in the 67 : 33 eluate, whereas GM1 ganglioside was present in the 50 : 50 eluate. The weights of GM3 and GM1 were 1.9 and 1.5 mg, respectively, with specific activities of 850/~Ci/mg and 23/~Ci/mg, respectively.
IV. Discussion
The several methods of labeling glycosphingolipids with tritium are very different in convenience, in general applicability, and in the quality of the product obtained. The biosynthetic method is least convenient and produces compounds with the lowest specific activities, but the compounds themselves are clearly most closely related to those present in tissue. The galactose oxidase oxidation-sodium borohydride reduction method yields compounds with intermediate specific activities, but is limited to molecules which are effective substrates for galactose oxidase. The method of tritium addition is the most general and the most convenient, since the difficult step involving chemical reaction with very highly radioactive material can be performed commercially. Compounds with very high specific activities are obtained. The major limitation is that the products, having all of their unsaturated centers removed, may display physical and chemical properties that differ in some degree from those of the natural products. In the study of Seyama et al [4] five labeled compounds were prepared of which only two, galactosyl ceramide and lactosyl ceramide, corresponded to materials we are describing in this report. One of these, lactosyl ceramide was shown to migrate on silicic acid columns somewhat differently from the natural product, a difference that was no longer seen when the
J.L. Di Cesare, M.M. Rapport, Tritium-labeled glycosphingolipids
451
double bonds of the natural product were saturated [4]. Therefore, some caution needs to be exercised in the use of glycosphingolipids labeled by tritium addition in interpreting the data for its relevance to the behavior of the natural products. This will, of course, depend on the particular use to which they are put, whether as substrates for enzymes [1, 3], or for isotope dilution experiments to determine the concentrations of glycosphingolipids in tissue [4], or to study the incorporation of glycosphingolipids into cell membranes [8]. The importance of describing these experiments is that experience with tritium addition to glycosphingolipids is still limited and subject to variations that are not clearly understood. For example, in the preparation of labeled asialoGM1 ganglioside, 400 mg of glycolipid in 10 ml of chloroform-methanol-water (64 : 32 : 4) was treated with 1 Ci of tritium gas using palladium black. When this solvent and catalyst were used for labeling a mixture of cytolipin H and cytolipin R, a successful result was not obtained. The New England Nuclear Corporation then suggested the solvent and catalyst described here. The yields of glycosphingolipids are clearly dependent on the scale of operation. In this report of Seyama et al. [4] yields of purified compounds were not presented since carrier lipid was added before purification. Our studies indicate that it is feasible to carry out such labeling on amounts of 5 mg of a single glycosphingolipid and still obtain sufficient product to work with, despite destruction during tritium addition and subsequent mechanical losses during purification~ The degree of loss during tritium addition is difficult to determine, but our results suggest that gangliosides are more susceptible to destruction than neutral glycosphingolipids, since the yields were poorer. It is perhaps worth recalling that the labeling of cytolipin H previously reported [5] was obtained as a by-product of tritium addition t o , asialoGM1 ganglioside, indicating that specific linkages can be broken without extensive alteration of internal structure. Seyama et al. [4] have called attention to the instability of glycosphingolipids highly labeled with tritium, citing as an example experience obtained with a ceramide tetrasaccharide. This instability is probably very different for compounds having different chemical structures and probably accounts for the very large differences in specific activities found in the purified products,' even when these were prepared simultaneously.
Acknowledgements These studies were supported in part by grants from the National Science Foundation (GB-36937) and by the National Institute of Mental Health (MH-10315). We wish to thank Ms. Anthea D'Orel for assistance in the preparation of this manuscript.
452
J.L. Di Cesare, M.M. Rapport, Tritium-labeled glycosphingolipids
References [1] [2] [3] [4] [5] [6] [7] [8]
E.H. Kolodny, R.O. Brady, J.M. Quirk and J.N. Kanfer, J. Lipid Res. 11 (1970) 144 A.K. Hajra, D.M. Bowen, Y. Kishimoto and N.S. Radin, J. Lipid Res. 7 (1966) 379 Y. Suzuki and K. Suzuki, J. Lipid Res. 13 (1972) 687 Y. Seyama, T. Yamakawa and T. Komai, J. Biochem. 64 (1968) 487 S. Gatt and M.M. Rapport, Biochem. J. 101 (1966) 680 G.A. Bray, Anal. Biochem. 1 (1960) 279 C.J. Lauter and E.G. Trams, J. Lipid Res. 3 (1962) 136 M.E. King, D.W. King, L. Graf and M.M. Rapport, Fed. Proc. 33 (1974) 629 Abs
© NORTH-HOLLANDPUBLISHINGCOMPANY
P R E P A R A T I O N OF SOME L A B E L E D G L Y C O S P H I N G O L I P I D S BY CATALYTIC ADDITION OF TRITIUM J.L. DI CESARE and M.M. RAPPORT Division o f Neuroscience, New York State Psychiatric Institute and Department o f Biochemistry, College o f Physicians and Surgeons, Columbia University, New York, N. Y. 10032, USA
Three experiments are described in which seven tritium-labeled glycosphingolipidswere prepared by catalytic addition of tritium gas to unsaturated centers. These compounds include glucosyl ceramide, galactosyl ceramide, lactosyl ceramide (cytolipin H), N-acetylgalactosaminyl 031 ~ 3) galactosyl (c~l~ 3) galactosyl (/31~ 4) glucosyl ceramide (cytolipin R), and three species of gangliosides: GM1 (G4), GM3 (G6) , and GD1a (G3).
I. Introduction The use of radioactively labeled compounds in studying the biochemical properties of compounds is now common practice. Three methods are currently favored for introducing tritium into glycosphingolipids. In the first, a labelled precursor is used to prepare the compound biosynthetically [ 1]. In the second, the label is introduced into the carbohydrate moiety by oxidation with galactose oxidase followed by reduction with tritiated borohydride [2, 3]. In the third, the label is introduced into the hydrophobic residues by catalytic addition of tritium gas [4, 5]. We have had several experiences with the latter method that may be of value to others attempting to label their compounds in this way, and it is the purpose of this short communication to describe them. Addition of tritium was carried out by the New England Nuclear Corporation, Boston, Mass. The charge for carrying out this procedure is substantial ($350 in 1972); since the quantity of material so treated is not related to the charges imposed and since many of the compounds are available only in very small amounts, the cost of labeling by tritium addition is not an unimportant consideration. We have solved this problem in part by subjecting mixtures of compounds to tritium addition, selecting as components of the mixtures those glycosphingolipids that can be easily separated by chromatography.
448
J.L. Di Cesare,M.M. Rapport, Tritium-labeledglycosphingolipids
II. Materials and methods
Glycosphingolipids used in this study were homogeneous by thin layer chromatography. The lipids were isolated from animal tissues by conventional chromatographic methods. Their sources were as follows: glucosyl ceramide from human (Gaucher) spleen; galactosyl ceramide from bovine spinal cord; lactosyl ceramide from bovine spleen; cytolipin R from rat lymphosarcoma; GM3 ganglioside from rat spleen; and GMI and GD1a gangliosides from bovine brain. Thin layer chromatography was carried out on plates of silica gel G (20 cm × 20 cm), 0.5 mm thick for preparative chromatography and 0.25 mm thick for analytical chromatography. Column chromatography was carried out on silicic acid (Unisil; Clarkson Chemical Co,, Williamsport, Pa.). Radioactivity was determined by scintillation counting with a Packard 3320 Counter using Bray solution [6]. Quenching corrections were determined by the channels ratio method. Radio purities of final products were determined by TLC. At least 95% of the radioactivity was recovered from areas corresponding to those occupied by the appropriate standards.
III. Results A. Tritium addition.
Three experiments were carried out. In the first a suspension of 5.0 mg of cytolipin H and 5.0 mg of cytolipin R in 2.5 ml of isopropanol-ethyl acetate-water (1 : 1 : 0.5) was stirred overnight at room temperature with 25 mg of platinum catalyst in the presence of 5 Ci of tritium gas. In the second, this treatment was applied to a solution of 11.3 mg of galactosyl ceramide and 10.8 mg of G Dla ganglioside in 2.5 ml of the same solvent. In the third, it was applied to a solution of 6.4 mg of glucosyl ceramide, 5.4 mg of GM1 ganglioside and 5.0 mg of GM3 ganglioside in 2.5 ml of the same solvent. In each case the solvent was removed, and the residue was taken up in 5 ml of chloroform-methanol (1 : 1) and evaporated twice to remo~'e labile tritium. The residue was then dissolved in 10 ml of chloroform-methanol (1 : 1). The procedure to this point was carried out by the New England Nuclear Corporation. B. Purification o f cytolipin H and Cytolipin R
To the solution, 5 ml of chloroform was added followed by 3 ml of 0.1 M KC1. After shaking, the phases were separated. The lower phase, after washing 3 times with "pure solvents upper phase", contained 1000 ~tCi of activity. The lipids were separated by column chromatography (Unisil, 10 g), loading the column with a lipid solution in chloroform-methanol (95 : 5) and eluting with 250 ml portions of
J.L. Di Cesare,M.M. Rapport, Tritium-labeledglycosphingolipids
449
chloroform-methanol, 95 : 5, 90 : 10, 85 : 15, 80 : 20, and 67 : 33. Cytolipin H was eluted with chloroform-methanol (90 : 10) and cytolipin R with chloroformmethanol (80 : 20 to 67 : 33). The cytolipin H fraction (3.9 mg) was homogeneous by analytical TLC. The cytolipin R fraction was recrystallized from 1 ml of methanol, and additional compound was obtained from the mother liquor by preparative TLC. Total recovery, 4.2 mg. Specific activities, based on analysis of long chain base [7], were cytolipin H, 420/ICi/mg and cytolipin R, 760 pCi/mg.
C. Purification of galactosyl ceramide and GD1a ganglioside To the 12 ml of solution in chloroform-methanol (1 : 1), 6 ml of chloroform was added, followed by 3.6 ml of water. The upper and lower phases were separated and the lower phase was extracted 3 times with "pure solvent upper phase."
1. GD1a ganglioside The combined upper phase and extracts were dialyzed against running tap water for 1 day and against 3 changes of deionized water (4 1.) for 1 day (activity, about 500 ~tCi). The dialyzed ganglioside was then subjected to chromatography on silicic acid (7 g) in chloroform-methanol (90 : 10) and eluted with chloroform-methanol (90 : 10,225 ml; 67 : 33,250 ml) and chloroform-methanol-water (61 : 32 : 7, 250 ml). The ganglioside was eluted with the last solvent and recrystallized from methanol to give 1.9 mg of tritiated product, homogeneous by TLC, and having a specific activity of 200 btCi/mg. The mother liquor yielded additional material (0.5 mg).
2. Galactosyl ceramide The lower phase was evaporated to dryness and the residue was subjected to chromatography on silicic acid (10 g), eluting with chloroform (250 ml), and chloroform-methanol (95 : 5,150 ml; 90 : 10, 150 ml). This caused the galactosyl ceramide to separate into 2 fractions: the first was eluted with chloroform-methanol (95 : 5; normal fatty acid fraction) and the second with chloroform-methanol (90 : 10; hydroxy fatty acid fraction). The solvents were removed and the residues were purified by a sequence of 3 steps: preparative TLC, recrystallization from methanol, and preparative TLC again. The glactosyl ceramides with normal and hydroxy fatty acids weighed 3.2 and 3.0 mg, respectively, and had specific activities of 150/aCi/mg and 390 gCi/mg, respectively.
D. Purification of glucosyl ceramide, GM1 and GM3 gangliosides The sample was evaporated to dryness, and the residue was fractionated on a column of silicic acid (11 g) as follows. The portion soluble in 5 ml of chloroform
450
J.L. Di Cesare, M.M. Rapport, Tritium-labeledglycosphingolipids
was applied to the column, which was then eluted with chloroform (300 ml). The insoluble portion was dissolved in chloroform-methanol (95 : 5, 5 ml) and applied to the column. Eluting solvents were 300 ml portions of chloroform-methanol (95 : 5; 90 : 10; 67 : 33; and 50 : 50), methanol, and finally methanol water 50 : 50. TLC analysis showed that glucosyl ceramide was eluted with chloroformmethanol (95 : 5) and both gangliosides were eluted together with chloroformmethanol (50 : 50). A plot of radioactivity vs. fraction number for the glucosyl ceramide fraction showed two well-separated peaks, the second much smaller in area than the first. Material in the first peak, glucosyl ceramide with normal fatty acid chains, was recrystallized from methanol and again purified by preparative TLC to give 2.8 mg with a specific activity of 220/aCi/mg. 1. GM3 and GM1 gangliosides The material eluted with chloroform-methanol (50 : 50) was dried and then taken up in chloroform-methanol (80 : 20) and subjected to chromatography on silicic acid (13 g) eluting with chloroform-methanol (80 : 20,300 ml; 67 : 33, 300 ml; and 50 : 50,300 ml). GM3 ganglioside was present in the 67 : 33 eluate, whereas GM1 ganglioside was present in the 50 : 50 eluate. The weights of GM3 and GM1 were 1.9 and 1.5 mg, respectively, with specific activities of 850/~Ci/mg and 23/~Ci/mg, respectively.
IV. Discussion
The several methods of labeling glycosphingolipids with tritium are very different in convenience, in general applicability, and in the quality of the product obtained. The biosynthetic method is least convenient and produces compounds with the lowest specific activities, but the compounds themselves are clearly most closely related to those present in tissue. The galactose oxidase oxidation-sodium borohydride reduction method yields compounds with intermediate specific activities, but is limited to molecules which are effective substrates for galactose oxidase. The method of tritium addition is the most general and the most convenient, since the difficult step involving chemical reaction with very highly radioactive material can be performed commercially. Compounds with very high specific activities are obtained. The major limitation is that the products, having all of their unsaturated centers removed, may display physical and chemical properties that differ in some degree from those of the natural products. In the study of Seyama et al [4] five labeled compounds were prepared of which only two, galactosyl ceramide and lactosyl ceramide, corresponded to materials we are describing in this report. One of these, lactosyl ceramide was shown to migrate on silicic acid columns somewhat differently from the natural product, a difference that was no longer seen when the
J.L. Di Cesare, M.M. Rapport, Tritium-labeled glycosphingolipids
451
double bonds of the natural product were saturated [4]. Therefore, some caution needs to be exercised in the use of glycosphingolipids labeled by tritium addition in interpreting the data for its relevance to the behavior of the natural products. This will, of course, depend on the particular use to which they are put, whether as substrates for enzymes [1, 3], or for isotope dilution experiments to determine the concentrations of glycosphingolipids in tissue [4], or to study the incorporation of glycosphingolipids into cell membranes [8]. The importance of describing these experiments is that experience with tritium addition to glycosphingolipids is still limited and subject to variations that are not clearly understood. For example, in the preparation of labeled asialoGM1 ganglioside, 400 mg of glycolipid in 10 ml of chloroform-methanol-water (64 : 32 : 4) was treated with 1 Ci of tritium gas using palladium black. When this solvent and catalyst were used for labeling a mixture of cytolipin H and cytolipin R, a successful result was not obtained. The New England Nuclear Corporation then suggested the solvent and catalyst described here. The yields of glycosphingolipids are clearly dependent on the scale of operation. In this report of Seyama et al. [4] yields of purified compounds were not presented since carrier lipid was added before purification. Our studies indicate that it is feasible to carry out such labeling on amounts of 5 mg of a single glycosphingolipid and still obtain sufficient product to work with, despite destruction during tritium addition and subsequent mechanical losses during purification~ The degree of loss during tritium addition is difficult to determine, but our results suggest that gangliosides are more susceptible to destruction than neutral glycosphingolipids, since the yields were poorer. It is perhaps worth recalling that the labeling of cytolipin H previously reported [5] was obtained as a by-product of tritium addition t o , asialoGM1 ganglioside, indicating that specific linkages can be broken without extensive alteration of internal structure. Seyama et al. [4] have called attention to the instability of glycosphingolipids highly labeled with tritium, citing as an example experience obtained with a ceramide tetrasaccharide. This instability is probably very different for compounds having different chemical structures and probably accounts for the very large differences in specific activities found in the purified products,' even when these were prepared simultaneously.
Acknowledgements These studies were supported in part by grants from the National Science Foundation (GB-36937) and by the National Institute of Mental Health (MH-10315). We wish to thank Ms. Anthea D'Orel for assistance in the preparation of this manuscript.
452
J.L. Di Cesare, M.M. Rapport, Tritium-labeled glycosphingolipids
References [1] [2] [3] [4] [5] [6] [7] [8]
E.H. Kolodny, R.O. Brady, J.M. Quirk and J.N. Kanfer, J. Lipid Res. 11 (1970) 144 A.K. Hajra, D.M. Bowen, Y. Kishimoto and N.S. Radin, J. Lipid Res. 7 (1966) 379 Y. Suzuki and K. Suzuki, J. Lipid Res. 13 (1972) 687 Y. Seyama, T. Yamakawa and T. Komai, J. Biochem. 64 (1968) 487 S. Gatt and M.M. Rapport, Biochem. J. 101 (1966) 680 G.A. Bray, Anal. Biochem. 1 (1960) 279 C.J. Lauter and E.G. Trams, J. Lipid Res. 3 (1962) 136 M.E. King, D.W. King, L. Graf and M.M. Rapport, Fed. Proc. 33 (1974) 629 Abs