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.