Tohoku J. exp. Med., 1966, 88 , 277-388

Glycolipids Isolated from the Spleen of Gaucher's Disease

Akira Makita, Chiyuki Suzuki and Zensaku Yosizawa Department of Biochemistry(Prof. Z. Yosizawa), Tohoku UniversitySchool of Medicine,Sendai and Tasuke Konno Department of Pediatrics (Prof. Ts. Arakawa), Tohoku UniversitySchool of Medicine,Sendai

Gyyeolipids of Gaueher's spleens (three cases) were examined for their content and chemical characterization, comparing with normal control. Apparent accumulation of glucocerebroside was found in all cases. In a case, however, a faster-moving glycolipid fraction than cerebroside on a thin-layer chromatogram, traces of which were detected also in other cases and

normal control, was found in a certain amount. From this fraction, a fatty acid ester of glucocerebroside was isolated. Two-thirds of fatty acid of this fraction were composed of the acids below C20. Hematoside which increased in a certain extent, had the same chemical structure, N-acetylneuraminoyl-(2•¨3)-galactosyl-(1•¨4)-glucosyl-ceramide, as that of normal human spleen. Other glycolipids separated from Gaucher's spleens showed no significant variation in amounts and were all identical in chemical character with those found already in normal spleen.

Gaucher's disease, one of sphingolipidoses, has been well characterized by the accumulation of glycolipids in the reticuloendothelial system of various organs, especially of the spleen. In 1924, the deposited lipid in Gaucher's spleen was shown by Liebl to be cerebroside (cerasine). Since Aghion2 and Halliday et al.3 presented evidence that the accumulated cerebroside contains predominantly glucose, instead of galactose of cerebroside in central nervous system, many discussions4-7 have been focused on the constituent hexose of Gaucher's cerebroside. Recent investigations,8,9 however, established that the hexose moiety of cerebroside deposited in Gaucher's spleen was ex clusively glucose. In addition, the chemical structure10,11of Gaucher's cerebroside was confirmed to be identical with that of brain galactocerebroside except dif ferences of the species of its hexose and fatty acid. On the other hand, Makita and Yamakawa12 revealed that cerebroside isolat ed from non-Gaucher's spleen also contained predominantly glucose. Therefore,

Received for publication, January 12, 1966. 277 278 A. Makita et al. they assumed that physiological glucocerebroside was accumulated abnormally in Gaucher's spleen. Recently, increased formation of other glycolipids in Gaucher's spleen as well as glucocerebroside was reported. That is, Philippart et al.13 showed the accumulation of hematoside-type mucolipid and ceramide dihexoside in addition to glucocerebroside in this disease. Brady et al.14 reported, moreover, the deficiency of an enzyme catalyzing conversion of glucocerebroside to ceramide in Gaucher's spleen. The present communication reports the results of the study on glycolipids of three cases of Gaucher's spleen, comparing with those of non-Gaucher spleens. In one case (acute infantile cerebral form) of these Gaucher's spleens, a cere broside-fatty acid ester and hematoside were found to be accumculated. Chemical structures of these compounds are proposed by the results of gas-chromatography on the methylated glycosides.

MATERIALS AND METHODS

The spleens were obtained surgically from 3 patients with Gaucher's disease.

Case 1, an 1-year-old female with Gaucher's disease of acute cerebral form, kept frozen (128 g); Case 2, a 14-year-old male, of non-cerebral form, kept frozen (51 g,); and Case 3 , a 6-month-old male, of cerebral form, kept in formalin (11 g). As normal control, pooled seven spleens (82 g) from those who died at the age of 1-5 years were used.

For quantitative isolation of glycolipids, the procedure of the preparation used earlier15 was partly modified by employing Folch's method.16 Typical preparation of glycolpids was carried out as follows: A portion (128 g) of the spleen in Case 1 was homogenized and extracted with 20 volumes (vow) of chloroform-methanol (2:1, v/v). The filtered extract was evaporated to dryness.

The dried residue was extracted again with chloroform-methanol (2:1, v/v) to remove an insoluble material. This lipid mixture (6.4g, total lipids) was dissolved in hexane and then dialyzed against hexane through thin-rubber membrane.17

Outer hexane was changed twice. The non-dialyzable complex lipids were subjected to mild alkaline hydrolysis* in a mixture of each 100 ml of aqueous

0.6 N KOH and chloroform at 37•Ž for 1 hour under continuous stirring. After cooling and acidifying the reaction mixture to about pH 4 with 6 N HCl, each 100 ml of chloroform and methanol were added and shaken vigorously. When clear separation into two phases was not obtained, additional amount of methanol was introduced. By partition, the lower phase was separated and then dialyzed against water. The non-dialyzable fraction was freed from chloroform by

* In Cases 2 and 3, a portion of the complex lipids, without alkaline treatment , was chromatographed directly on silicic acid by elution with 4% methanol in chloroform (96C-M), by which esterified glycolipids should be eluted. Glycolipids in Gaucher's Spleens 279 evaporation, lyophilyzed and dialyzed against hexane , as mentioned above, to remove free fatty acids . Furthermore, from the upper phase, small amount of glycolipids was obtained by the same method, and combined with the main crude sphingolipid fraction. A portion (1.1 g) of the substance (2.7 g, crude sphingolipid fraction) thus obtained was applied to a column of silicic acid (Fig. 1). Glycolipids in the eluates were checked by anthrone reaction as well as

Fig. 1. Silicic acid column chromatogram of crude sphingolipids of Gau her's spleen

(Case 1). Circles indicate the mobility of glycolipids on TLC developed with solvent I (chloroform-methanol-water, 65:25:4, v/v), detected by anthrone sulfuric reagent. Abbreviations: FG, faster glycolipid fraction; CMH, cerebroside; CDH, ceramide dihexoside; CTH, ceramide trihexoside; Glob, globoside; ML ‡U, hematoside. 90C-M indicates 90% chloroform in methanol.

thin-layer chromatography (TLC) compared with the authentic compounds. By the first silicic acid chromatography, only sialic acid-containing

glycolipid (ML ‡U) was obtained homogeneously as glycolipid on TLC (Fig. 1). This ML ‡U was further purified by using a column of diethylaminoethyl (DEAE-)

cellulose18 to remove sphingomyelin. Other fractions containing two glycolipids

were chromatographed successively on silicic acid or Florisil in a small scale, as

described previously. 15 After one chromatographic treatment on silicic acid, 87

95% (w/w) of lipids was recovered. The amounts of the fractions (Frs. I-V of Fig. 1) obtained by the first silicic acid chromatography were estimated by weighing. As the glycolipid preparations were still contaminated with some phospholipids, the real amounts of the glycolipids were calculated by subtraction of the weight of phospholipids (P value•~25). Then, the amount of each 280 A. Makita et al .

Fig. 2. Chromatograms of faster

glycolipid fraction. Faster-glyco lipid fraction (95 mg) was applied

to a column prepared from a slurry of silicic acid (8 g) and Hyflosupercel (4 g), eluting with 100 ml of each solvent (2-1). TLC

(2-2) was developed by solvent ‡U . 1, Gaucher's cerebroside; 2, bovine brain cerebroside which was obtained from phospholipid containing cerebroside fraction

by the alkaline treatment em

ployed in the present study. Glycolipids in Gaucher's Spleens 281 glycolipid was determined from the proportion obtained by the final chromato graphic separation. Reprecipitation of lipid with acetone from the concentrated effiuents was avoided in all experiments described here, because a little loss of some glycolipids occurred by this procedure. Compositions of hexoses19 and fatty acids in glycolipids were determined by means of gas-chromatography (GLC). Based on the ratio of galactose to glucose thus obtained, the amount of hexose was estimated using anthrone and thionalide20 reagents. Other analytical methods15 and procedures of methyla tion followed by GLC of methylated glycosides21were described earlier, respectively. Chemical characterization of isolated compounds was carried out mainly in Case 1, and in the other cases the glycolipids separated were identified on TLC and by hexose compositions on GLC.

RESULTS AND DISCUSSION

1. Cerebroside fatty acid ester. By the first chromatography (Fig. 1) of crude

sphingolipids, a faster-moving glycolipid fraction (faster glycolipids) than cere broside on TLC appeared in Fr. I (Fig. 1) which contained cerebroside. This frac tion was subjected to Florisil chromatography by the elution with 96% chloroform in methanol (96C-M), 70C-M and 50C-M. The glycolipid fraction eluted with

96C-M was again dialyzed against hexane through rubber membrane. The non-dialyzable portion revealed to contain at least three glycolipids other than

cerebroside by TLC in solvent ‡U (chloroform-methanol-water, 85:15:1.5, v/v).

Therefore, it was rechromatographed on silicic acid (Fig. 2). A pure glycolipid (35 mg, Fr. ‡U of Fig. 2) was isolated by the elution with

98 C-M. This material showed an absorption at 1,730 cm-1 (ester carbonyl) in

addition to those of cerebroside by infrared spectral analysis (Fig. 3).

Chemical character: It was slightly yellow and partly soluble in acetone.

Reactions of Liebermann-Burchard for cholesterol and Bial for sialic acid were negative. This compound had m.p. 104?-107?; [ƒ¿]18589-4.7? (c, 2.4; in pyridine);

P, nil; ester value, 0.99 ,uequiv. /µmole (Snyder and Stephan's method22); hexose

(as glucose), (16.9%) (thionalide reagent20). Glucose was comprised in 98% of total hexose and the remaining was galactose. Elemental analysis: C, 72.41; H, 11.95; N, 1.35. Diarachidyl-cerebroside,

C64H123O9N (1049), requires C, 73.21; H, 11.73; N, 1.33; hexose, 17.2 On the bases of these data, this compound is most likely to be fatty acid ester

of cerebroside. As the fatty acid composition in Table I indicates, the faster

glycolipid fraction involved in this substance contained larger proportions of

palmitic and stearic acids than those of cerebroside. This type of compound has not yet been found in Gaucher's and normal

spleens. However, from the other cases (Cases 2 and 3) of Gaucher's spleens in 282 A. Makita et al. which the mild alkaline hydrolysis was avoided, and in even normal control, the faster glycolipid fraction was also obtained in very small amounts but failed to purify further because of lesser amounts of the starting materials.

Fig. 3. Infrared spectra of cerebroside (1) and cerebroside-fatty acid ester (2) isolated from Gaucher's spleen, in KBr.

From bovine brain, however, the isolation of similar substances containing predominantly galactose23,24 has so far been reported. Norton and Brotz23 isolated three specimens of fatty acid ester of galactocerebroside in which the positions of ester linkage were not determined. Their melting points (150?, glycolipid C; 102?, glycolipid A) and infrared absorptions showed reasonable agreement with those of our compound. On the other hand, Kochetkov et al.24 reported the isolation of a compound tentatively suggested as 3-O-alkyl cerebroside. This compound was resistant to alkaline hydrolysis but its infrared spectrum showed the difference from our material in the absence of absorption of ester carbonyl. Since our substance, which was obtained by the method of the mild alkaline treatment, still contained ester linkage in the pure state, it might be relatively stable in the mild alkaline condition employed in this experiment. However, other substances included in the faster glycolipid fraction in Case 1 were not to isolate. It might be due to their meager amounts or unstable characters to this treatment. Although the reason of the contradictory findings is obscure, the real amounts of this fraction in the intact state of Case 1 may be higher than that reported here. Glycolipids in Gaucher's Spleens 283

Methylation study: Eleven mg of the cerebroside-fatty acid ester were methylated for 30 hours, as described previously,21 affording the methylated

derivative which still indicated the presence of small amount of free OH (near

3350 cm-1). Gas-chromatogram (2 m-column with 5% Ucon LB-550 on Gas Chrom

LCH, at 210ß) of methylated methyl glycosides obtained from this compound

revealed the presence of methyl-2,3,4-tri-O-methylglucosides [RGi, relative

retention time to ƒÀ-methyl-tetra-O-methylglucoside=1.52 (ƒÀ) and 1.86 (ƒ¿)] and

methyl-tetra-0-methylglucosides [RGl=1.00(ƒÀ) and 1.13 (ƒ¿)], in the ratio of 5:4, respectively. Provided that the latter was derived from deacylation of the

cerebroside-fatty acid ester during methylation procedure, it might be assumed

that the ester formed-fatty acid is located at C-6 of glucose, such as gluco

cerebroside-6'-fatty acid ester. Further confirmation of this structure was

impossible, because of lack of the purified material.

2. Hematoside (ML-‡U). This substance was white and amorphous powder.

Chemical analysis: Hexose, 26.4%; galactose/glucose=1.05. Sialic acid, 23.5%, as N-acetylenuraminic acid. Hexosamine, nil; P. 0.02%; [ƒ¿]20589-9.5? (c,

2.4; in pyridine). The fatty acid composition is listed in Table 1.

Structural study: After the permethylation of this material (35 mg) followed

by methanolysis, methyl-2,3,6-tri-O-methylglucosides [RGai, relative retention time

to methyl-tetra-0-methylgalactoside,=1.67 (ƒÀ) and 2.08 (ƒ¿)] and -2, 4, 6-tri-0

- methylgalactosides [RGal=1.89 (ƒÀ) and 2.08(ƒ¿)] were detected by GLC (3 m-column

with 5% neopentylglycol succinate on Microsorb-W).

On the other hand, ML (76 mg) was hydrolyzed with 0.1 N H2SO4 at 90?

for 1 hour, and then dialzyed against water. After removal of SO4-2 and conentration of the dialyzable portion, N-acetylneuraminic acid solely was

detected on paper chromatogram.25 However, 2% of glycolyl derivative were

also estimated by the Klenk and Uhlenbruck's method.26 From the non-dialyz

able portion (56 mg), a glycolipid (20 mg) corresponding to cermaide dihexoside

on TLC was purified by Florisil column by the elution with 50 C-M. This

glycolipid gave [ƒ¿]24589- 10.5? (c, 1.2; in pyridine) and was confirmed to be ceramide lactoside by the methylation study resulting in methyl-tetra-O-methylagalctoside

(RGal=1.00) and -2, 3, 6-tri-O-methylglucosides [RGal=1.67(ƒÀ) and 2.08(ƒ¿)] on

gas-chromatogram. Based on these findings, the structure of ML ‡U is proposed to be N-acetyl

neuraminoyl-(2•¨•@ 3)-galactosyl-(1•¨4)-glucosyl-cermaide, identical with that of

normal human spleen27, and of equine28,29 and canine29,30 red cell hematosides,

except N-acyl moiety of sialic acid. The equine one was solely N-glycolyl and

the canine29 was a mxiture of N-acetyl (73%) and N-glycolyl (27%).

Since four glycolipids to be described below were identical with those already reported, only brief descriptions about these compounds would be made. 284 A. Makita et al.

3. Cerebroside. Hexose, 20.2%; glucose was exclusively detected by GLC, in three cases of Gaucher's spleens. On the other hand, 91% of glucose and 9% of galactose were determined in the normal control. Elemental analysis: C, 68.71; H, 11.21; N, 1.89. [ƒ¿]20589-10.4? (c. 2.6; in pyridine). The fatty acid composition is listed in Table 1.

TABLE 1. Fatty acid composition of cerebroside fatty acid ester, cerebroside and hematoside of Gaucher's spleen

* C24, includes both lignoceric and nervonic acids. ** Determined on the faster glycolipid fraction.

4. Cermmide dihexoside. Galactose/glucose=0.9. 5. Cermaide trihexoside. Galactose/glucose=1.8. 6. Globoside. Galactose/glucose=1.8. The lower values of hexosamine (10.3%) and hexose (36.2%) than those expected for globoside were observed in this preparation, because of the contamination with some phospholipids. On TLC, this compound gave the same mobility as that of globoside (Globoside I)31 of human kindey,15 differing from that of Forssman hapten32 of the equine spleen. Thin-layer chromatogram of the purified glycolipids described above is shown in Fig. 4. Quantitative Survey of Glycolipidsfrom Gaucher's and Normal Spleens Compositions of glycolipids from Gaucher's spleen and normal control are shown in Table 2. As has been well established by many workers, the amount of glucocerebroside markedly increases in all cases of Gaucher's spleen, from 33 to 75 times as much as normal control, expressed in terms of mg/g wet tissue. The amounts (mg/g wet tissue) of ceramide di and trihexosides and globoside showed no significant deviation between Gaucher's and normal spleens, though an accumulation of ceramide dihexoside13 in Gaucher's spleens was reported. Suomi and Agranoff33 obtained, using hexose oxidases, similar values to our results in Gaucher's spleens for glucocerebroside (13.1-28.9) and ceramide lactoside (0.37-1.12), expressed as mg/g wet tissue. On the other hand, certain amounts of hematoside accumulated in our cases. Glycolipids in Gaucher's Spleens 285

Fig. 4. Thin-layer chromatogram of the glycolipids isolated from Case 1 of Gaucher's spleen, developed with solvent I (see Fig. 1) for 50 min. 1, Cerebroside-fatty acid ester; 2, cerebroside; 3, ceramide dihexoside; 4, ceramide trihexoside; 5, globoside; 6, hematoside.

Philippart et al.13 presented a remarkable increase (about 10 times as much as normal control) of this substance based on the sialic acid determination on lipid extracts. The faster-glycolipid fraction comprising cerebroside-fatty acid ester was estimated only in Case 1. As to such glycolipids of Gaucher's spleen, no report was yet available. 286 A. Makita et al.

TABLE2. Glycolipid composition of the spleens from Gaucher's disease and normal control

1) Expressed in mg/g wet tissue. 2) Expressed in per cent in total glycolipids.

Acknowledgments

The authors are indebted to Prof. Ts. Arakawa (Deparatment of Pediatrics) for his encouragement. Thanks are also due to Miss. N. Kawamura of Department of Chemistry, the Institute for Infectious Diseases, the University of Tokyo, for the elemental tinalysis.

References

1) Lieb, H. Cerebrosidspeicherung bei Splenomegalie, Typus Gaucher. Z. physiol. Chem., 1924. 140, 305-313. 2) Aghion, H. La maladie de Gaucher dans l'enfance. These de Paris, 1934. 3) Halliday, N., Duel, H.J. Jr., Tragermon, L.J. & Ward, W.E. On the isolation of a glucose-containing cerebroside from spleen in of Gaucher's disease. J. biol. Chem., 1940, 132, 171-180. 4) Klenk, E. Beitrage zur Chemie der Lipoidosen. Z. physiol. Chem., 1940, 267, 128 144. 5) Lieb, H. Der Zucker im Cerebrosida der Milz bei der Gaucher-Krankheit. Z. physiol. Chem., 1941, 271, 211-213. 6) Klenk, E. & Rennkamp, F. Der Zucker im Cerebrosid der Milz bei tier Gaucher Krankheit. Z. physiol. Chem., 1942, 272, 280-282. 7) Devor, A.W. Carbohydrate residue, in spleen cerebrosides. Arch. Biochem. Biophys., 1954, 50, 217-218. 8) Rosenberg, A. & Chargaff, E. A reinvestigation of the cerebroside deposited in Gaucher's disease. J. biol. Chem., 1958, 233, 1323-1326. 9) Agranoff, B.W., & Radin, N. W. Enzymic oxidation of cerebrosides: Studies on Gaucher's disease. Biochim. biophys. Acta, 1962, 57, 194-196. 10) Marinetti, G.V., Ford, T. & Stotz, F. The structure of cerebrosides in Gaucher's Glyoolipids in 0auoher's Spleens 287

disease. J. Lipid Research, 1960, 1, 203-207. 11) Carter, H.E., Rothfus, J.A. & Gigg, R. Biochemistry of the sphingolipids: ?. Conversion of cerebrosides to ceramides and sphingosine; Structure of Gaucher cerebroside. J. Lipid Research, 1961, 2, 228-234. 12) Makita, A. & Yamakawa, T. Biochemistry of organ glycolipids: I. Ceramide oligohexosides of human, equine and bovine spleens. J. Biochem., 1962, 51, 124-133. 13) Philippart, M. & Menkes, J.H. Isolation and characterization of the main splenic

glycolipids in Gaucher's disease: Evidence for the site of metabolic block. Biochem. biophys. Res. Commun., 1964, 15, 551-555. Philippart, M., Rosenstein, B. &Menkes, J.H. Isolation and characterization of the main splenie glycolipids in the normal organ and in Gaucher's disease: Evidence for the site of metabolic block. J. Neuropath. exp. Neurol., 1965, 24, 290-303. 14) Brady, R.O., Kanfer, J.N. & Shapiro, D. Metabolism of glucocerebrosides ‡U.

Evidence of an enzymatic deficiency in Gaucher's disease. Biochem. biophys. Res. Commun., 1965, 18, 221-225. 15) Makita, A. Biochemistry of organ glycolipids ‡U. Isolation of human kidney

glycolipids. J. Biochem., 1964, 55, 269-276. 16) Folch-Pi, J., Lees, M. & Sloane-Stanley, G.H. A simple method for the isolation and

purification of total lipids from animal tissues. J. biol. Chem., 1957, 226, 497-509. 17) Van Beers, G.J., Delongh, H. & Boldingh, J. Essential Fatty Acid, ed. by Sinclair, H.M., Butterworths Scientific Publishers, London, 1958, p. 43, . 18) Rouser, G., Bauman, A.J., Kritchevsky, G., Heller, D. & O'Brien, J.S. Quantitative chromatographic fractionation of complex lipid mixtures of brain. J. Amer. Oil Chemists' Soc., 1961, 38, 544-555. 19) Yamakawa, T. & Ueta, N. Gas-liquid chromatography of carbohydrates. Japan. J. exp. Med., 1964, 34, 37-51. 20) Masamune, H. & Sakamoto, M. Biochemical studies on carbohydrates CLXXIX. Spectrophotometric determination of hexoses by the aid of thionalide. Tohoku J. exp. Med., 1956, 63, 345-355. 21) Makita, A. & Yamakawa, T. Biochemistry of organ glycolipids ‡V, The structures of human kidney cerebroside sulfuric ester, ceramdie dihexoside and ceramide trihexosie. J. Biochem., 1964, 55, 365-370. 22) Snyder, F. & Stephan, N. A simplified spectrophotometric determination of ester

groups in lipids. Biochim. biophys. Acta, 1959, 34, 244-245. 23) Norton, W.T. & Brotz, M. New Galactolipids of Brain: A monoalkyl-monoacyl

glyceryl galactoside and cerebroside fatty acid esters. Biochem. biophys. Res. Commun., 1963, 12, 198-203. 24) Kochetkov, N.K., Zhukova, I.G. & Glukhoded, I.S. Sphingoplasmalogens. A new type of sphinoglipids. Biochim. Biophys. Acta, 1963, 70, 716-719.

25) Svennerholm, E. & Svennerholm. L. Quantitative paper partition chromatography of silaic acids. Nature, 1958, 182, 1154-1155. 26) Klenk, E. & Uhlenbruek, G. Uber die Abspaltung von N-Glykolyl-neuraminasaure (P - Sialinsiiure aus dem Schweine-Submaxillarismucin durch das "Receptor Destroying Enzyme". Z. physiol. Chem., 1957, 307, 266-271.

27) Svennerholm. L. Isolation of the major ganglioside of human spleen. Acta chem. stand., 1963, 17, 860-862. 28) Klenk, E. & Padberg, G. Uber die Ganglioside von Pferdeerythrocyten. Z.

physiol. Chem., 1962, 327, 249-255. 29) Handa, S. & Yamakawa, T. Chemistry of lipids of posthemolytic residue or stroma of erythrocytes ?. Chemical structure and chromatographic behaviour of hematosides obtained from equine and dog erythrocytes. Japan. J. exp. Med., 1964, 34, 293-304. 288 A. Makita et al.

30) Klenk, E. & Heuer, K. Ganglioside of dog erythrocytes. Deut. Z. Verdau. u. Stoffwechselkr., 1960, 20. 180-193. 31) Yamakawa, T. Nishimura, S. & Kamimura, M. The chemistry of lipdis of posthemolytic residues or stroma of erythrocytes ?. Further studies on human red cell glycolipids. Japan. J. exp. Med., 1965, 35, 201-207. 32) Makita, A., Suzuki, C. & Yosizawa, Z. Chemical and immunological characterization of Forssman hapten isolated from equine organs. J. Biochem., 1966 (in press). 33) Suomi, W.D. & Agranoff, B.W. Lipids of the spleen in Gaucher's disease. J. Lipid .Res., 1965, 6, 211-219.