The Journal of , Vol. 66, No . 2. ;959

Cerebral and Visceral in a Case of Tay-Sachs Disease

By TAMOTSU TAKETOMI and NARIKO KAWAMURA

(From the Department of Biochemistry,Institute of Adaptation Medicine, Shinshu University, Malsumoto)

(Received for publication, December 23, 1968)

Cerebral and visceral glycolipids of a patient with the Tay-Sachs disease were investigated in the present study. The disease was found to be characterized by exces sive accumulation of Tay-Sachs (Go) in the brain , but any storage of gan glioside was not detected in other visceral organs such as the spleen, kidney or liver. Tay-Sachs was also present only in the Tay-Sachs brain. It was found that only a trace amount of glucocerebroside and a small amount of lactoside were present in the brain with the Tay-Sachs disease. The fatty acid composition of ceramide lactoside, globoside or ganglioside (Go) in the Tay-Sachs brain was different from that of and , being composed mostly of stearic acid. Tay- Sachs ganglioside (Go) contained mostly C,8-, besides small amounts of C8- dihydrosphingosine and CO-sphingosine. Moreover, it was noted that ceramide dihexo side showing two bands on thin layer chromatography was the major glycolipids in the Tay-Sachs spleen glycolipids and that the Tay-Sachs liver contained mostly ceramide trihexoside and hematoside. A normal neonatal human brain was used for comparison. Contrary to the Tay-Sachs ganglioside, the amounts of major in the normal neonatal human brain were roughly in the order of GII>GI -GIII.

In contrast to the well known fact that brain, but not in the spleen, kidney and liver. large amounts of the so-called Tay-Sachs It was noted that a pattern of silicic acid ganglioside (Go) accumulate in the brain of a column chromatogram of this cerebral glyco patient with the infantile amaurotic idiocy was very similar to that from another (the Tay-Sachs disease), it has been recently patient with the same disease, which has found that gangliosides similar to the normal previously been reported by MAKITA and major gangliosides (Gr) accumlate in the brain, YAMAKAWA(3). An additional storage of spleen, liver, kidney, etc. of a patient with the the so-called Tay-Sachs globoside was also so-called " generalized ( I )". found in this brain. For reference and com Also, SANDHOFFet al. (2) reported an excep parison, the fresh normal brain of a male tional case of the Tay-Sachs disease with neonatal infant who died of atelectasis 2 days visceral involvement. In the present investi after birth was similarly analyzed. In the gation, the authors attempted to isolate glyco present paper, the results of these investiga lipids from the brain as well as from the tions are described in detail. spleen, kidney and liver of a patient with the

Abbreviations used;TLC: Thin layer chromatography,GLC : Gas-liquid chromatography,TMS: Trimethylsilyl,EGS : Ethylene glycol succinate. Tay-Sachs disease. An abnormal accumula MATERITALS AND METHODS tion of ganglioside (Go) was found in the Extraction and Isolation of Glycolipids-Fresh frozen tissues of the brain (40g), spleen (33g), kidney (40g) Abbreviations used; and liver (200g) of a female patient who died of the TLC: Thin layer chromatography, Tay-Sachs disease at the age of 11 months were kindly GLC : Gas-liquid chromatography, supplied by the Department of Pediatrics, Nihon TMS: Trimethylsilyl, University. The tissues were extracted with 20 volumes EGS : Ethylene glycol succinate. of chloroform : methanol (2 : 1, v/v) at room tem- 165 166 T. TAKETOMI and N. KAWAMURA

peraturc for a few minutes in a Waring blendor. The viously reported by the authors (8), GLC was car extract was filtered through a Buchner funnel and ried out with a U-shaped glass column (4 ft. long and evaporated to dryness in vacuo. The residual material 1/8 in. I.D., packed with 3.8%% SE-30 on F & M's was referred to as the total . Approximate Diatoport S) at 160°C. Fatty acid constituents of amount of the total lipid in each tissue was as follows: various glycolipids were determined by GLC after brain, 2.8g; spleen, 1.1 g; kidney, 1.4g and liver, methanolysis with 5 % methanolic HCI. The fatty 9.2 g. The total lipid was treated twice with acetone. acid methyl esters were analyzed with a U-shaped The acetone insoluble fraction was then treated twice glass column (6 ft. long and 1/8in. I.D., packed with with diethyl-ether. The yields of the diethyl ether 15,'c EGS on Chromosorb W) at 180°C. Sphingosine insoluble fractions, i.e., crude , were as bases of glycolipids were determined by a slight modi follows: brain, 620.8 mg ; spleen, 44.8 mg ; kidney, fication of GAYER and SWEELEY'Smethod (9) and 104.5 mg and liver, 140 mg. Each of these diethyl CARTER and GAVER's method (10). GLC of TMS- ether insoluble materials was subjected to silicic acid: derivatives of sphingosine bases was carried out with Hyflo Super Cel (2: 1, w/w) column chromatography a U-shaped glass column (4ft. long and 1/8in. I.D., and separated into several fractions. Elution packed with 3.8% SE-30 on F & M's Diatoport S) at was carried out with stepwise increasing concentrations 210-220°C. The chemical linkage of sugar moieties of methanol in chloroform. Ten or 20 m1 of the of glycolipids was determined by the procedure de effluent were collected in each tube, and their aliquots scribed in the previous report (8). The methylhexo were used for estimation of hexose content. The sides from methylated sugars were identified by GLC fresh normal brain of a male neonatal infant 'oho using 5% neopentyl glycol-succinate on a Chromosorb died of atelectasis 2 days after birth (kindly supplied W AW column (4 ft. long and 1/8 in. I.D., U-shaped by the Department of Pathology, Institute of Medical glass) at 170-180°C. Science, University of Tokyo) was extracted with 20

volumes of chloroform : methanol (2: 1, v/v) as already RESULTS described. The extract was separated into two phases Chromatography of Cerebral Glycolipids of Tay after the addition of one-fifth volume of water follow- Sachs Disease-The diethyl ether insoluble frac ing the procedure of FOLCHand his coworkers (4). tion of the brain of a patient with the Tay The upper phase containing water soluble gangliosides was collected and put into a cellophane tubing. The Sachs disease was separated into four fractions tubing was dialyzed against distilled water for several days and the inner solution was lyophilized. The crude ganglioside thus obtained was subjected to col umn chromatography to be separated into different kinds of gangliosides. The lower phase was evaporated to dryness and the residue was treated with acetone. The acetone-insoluble fraction was then treated with diethyl ether. The diethyl ether insoluble fraction was also subjected to column chromatography as described above. Analytical ProceduresHexose was determined by ROE'S method (5) with a slight modification using the anthrone reagent, hexosamine by GATT and BER- MAN'smodification (6) of Elson-Morgan's method and FIG. 1. Silicic acid : Hyflo Super Cel (2 : 1, sialic acid by the method of 1 AMAKAWAand his co- -/w; 30 g) column chromatography of the cerebral workers (7) with a little modification according to glycolipids of a patient with Tay-Sachs disease. Klenk and Langerbein. Thin-layer chromatography 279.5 mg of the diethyl ether-insoluble fraction was carried out on Silica Gel G (Merck) using as was loaded on a column (22 mm I.D.) and eluted solvent systems chloroform: methanol: water, 65: 25: 4 with 50 ml of chloroform, 200 ml each of a mix and 60 : 35 : 8, by volume. The infrared spectra of ture of chloroform : methanol (9 : 1, 8 : 2, 6 : 4, glycolipids were taken by a Hitachi infrared spectro 4 : 6, 2 : 8, v/v) and 200 ml of methanol . 20 ml photometer, model EPI-2 using a KBr-tablet. The fractions were collected in each tube . 0.3 ml ratio of galactose to in glycolipid was deter- aliquot of each tube was used for assay of hexose mined by gas-liquid chromatography (GLC) of tri by the anthrone reagent. The molar ratio of methylsilyl ether (TMS) derivatives of methyl hexosides galactose to glucose in each fraction determined after methanolysis with 5% methanolic HCI, as pre- by GLC is also shown. Glycolipids in Tay-Sachs Disease 167 of glycolipids by silicic acid column chromato shown in Fig. 2 (Plate B). Sulfatide was also graphy, as shown in Fig. 1. At first, these easily detected by the infrared spectrum on fractions were identified by thin layer chro the basis of sulfate group absorption at 1250 matography (TLC) and the molar ratio of cm-1 and 820 cm-1. Fr-III eluted with chloro galactose to glucose in each fraction was form: methanol (6: 4, v/v) seemed to be iden- determined by GLC of TMS-derivatives of methyl hexosides. As shown in Fig. 2, Fr-I eluted with chloroform: methanol (9: 1, v/v) contained cerebroside. Particularly, the first portion of Fr-I seemed to be kerasine and the last portion to be phrenosine. This frac tion seemed to contain only a trace amount of glucocerebroside judging from the molar ratio of galactose to glucose as shown in Fig. 3. Fr-II eluted with chloroform: methanol (8: 2, v/v) consisted mainly of sulfatide and a small amount of ceramide lactoside which was clearly detected by applying a relatively large amount of this fraction to TLC, as

1 2 3 4 Plate (B); 1 Ceramide lactoside. 2 : Cerebroside (kerasine, phrenosine). 3 : Fr-II [ceramide lactoside (upper spot) and sulfatide (lower spot)]. 4 : Sulfatide. The plate was developed with chloroform : methanol : water (60 : 25 : 4, by volume) for 50 min at room temperature.

1 2 3 4 5 6 Fin. 2. Thin layer chromatograms of cere bral glycolipids of a patient with Tay-Sachs dis ease on Silica Gel G. Plate (A); 1 : The first portion of Fr-I (kerasine). 2 : The last portion of Fr-I (phrenosine). 3 : Fr-II (ceramide lactoside and sulfatide). 4 : Fr-III (Tay-Sachs globoside). 5 : Fr-IV [Tay-Sachs ganglioside (Go)].6 : Tay-Sacks vanglioside (Go) from another FIG. 3. Gaschromatograms of TMS-deriva patient with Tay-Sachs disease obtained by tives of methyl hexosides after methanolysis of Makita and Yamakawa. Fr-I of Tay-Sachs brain glycolipids (1) and Fr-I The plate was developed with chloroform: of normal neonatal human brain glycolipids (2). methanol : water (60 : 35 : 8, by volume) for 60 Galactose was the major sugar component and min at room temperature. Spots were detected only a trace amount of glucose was detected in by spraying the plate with the anthrone reagent these fractions. followed by heating at 120°C for 10 min. 168 T. TAKETOMI and N. KAWAMURA

tical with the Tay-Sachs globoside report- and separated into four fractions as shown ed by MAKITA and YAMAKAWA (3). This in Fig. 6. The molar ratio of galactose to fraction contained 18.0% hexosamine. Fr-IV glucose in each fraction was determined by eluted with chloroform: methanol (4:6, v/v) GLC. The results obtained are also shown contained a large amount of the Tay-Sachs in Fig. 6. As shown in Fig. 5, the thin layer ganglioside (Go). This fraction contained chromatograms of the fractions showed that 14.8% hexosamine and 22.0% sialic acid. Fr-A corresponds to G5, Fr-B to G,, Fr-C to Chromatography of Glycolipids from Neonatal GII and Fr-D to Gin. Particularly, methyl Human Brain-A chloroform: methanol (2: 1, hexosides after methanolysis of the permeth

v/v) extract of the neonatal human brain was ylated sample of Fr-C was subjected to separated into lower and upper phases by the GLC. As shown in Fig. 7, it was found that partition dialysis according to FOLCH and his peak A corresponds to methyl ƒÀ-2, 3, 6-tri-O- coworkers (d ). The lower phase was evapo methylglucoside, peak B to methyl ƒÀ-2, 4, 6- rated to dryness and the residue was succes tri-O-methylglucoside, and peak C to methyl sively treated with acetone and diethyl ether. ƒ¿-2, 3, 6-tri-O-methylglucoside and methyl ƒ¿- The diethyl ether insoluble fraction was 2, 4, 6-tri-O-methylgalactoside. This result sug

subjected to silicic acid column chromato gested that the sugar moiety of Fr-C may

graphy and separated into two fractions as possess a linkage similar to that of sugar shown in Fig. 4. As shown in Fig. 5, Fr-I moieties of Gn proposed by KUHN and seemed to be composed of kerasine and WIEGANDT ( 11 ). The infrared spectrum of phrenosine, but glucocerebroside seemed to present only in a trace amount judged from the ratio of galactose to glucose by GLC (Fig. 3) and Fr-II contained sulfatide and a small amount of ceramide lactoside, as just mentioned above for the Tay-Sachs brain glycolipids. The upper phase was dialyzed against distilled water and lyophilized. The crude gangliosides thus obtained were applied to silicic acid column chromatography

1 2 3 4 5 6 7

FIG. 5. Thin layer chromatograms of neo- natal human brain glycolipids. 1 : The first portion of Fr-I (kerasine). FIG. 4, Silicic acid : Hyflo Super Cel (2: 1, 2 : The last portion of Fr-I (phrenosine). w/w ; 60g) column chromatography of neonatal 3 : Fr-II [ceramide lactoside (upper spot) and human brain glycolipids. 400 mg of crude glyco sulfatide (lower spot)]. lipids were loaded on a column (32 min I.D.) and 4 : Fr-A (G0). eluted with 200 ml of chloroform, 200 ml each of 5 : Fr-B (G,). a mixture of chloroform : methanol (9 : 1, 8 : 2, 6 : Fr-C (GII). 6 : 4, 4 : 6, v/v) and 200 ml of methanol. 20 ml 7 : Fr-D (Gill). of effluent was collected in each tube. 1.0 ml The plate was developed with chloroform: aliquot of each tube was used for estimation of methanol : water (60 : 35 : 8, by volume) for 60 hexose content using the anthrone reagent. min at room temperature. Glycolipids in Tay-Sachs Disease 169 this fraction is shown in Fig. 8. lipid fraction of the Tay-Sachs spleen was Tay-Saeh,s Spleen Clycnlijpids--A crude glyco- separated into four fractions by silicic acid column chromatography, as shown in Fig. 9. Based on the ratio of galactose to glucose and the thin layer chromatogram (Fig. 10) of each fraction, it was suggested that Fr-I con tained glucocerebroside and only a trace amount of galactocerebroside, Fr-II ceramide dihex oside showing two bands on TLC, Fr-III was a mixture of ceramide dihexoside and ceramide trihexoside, and Fr-IV consisted mostly of globoside. At any rate, it was found that no kind of gangliosides aecumlated in the Tay-

Fir.. 6. Silicic acid : Hyflo Super Cel (2 : 1, w/w ; 105g) column chromatography of neonatal human brain gangliosides. 840 mg of crude gan

gliosides were loaded on a column (32mm I.D.) and eluted with 200 ml each of a mixture of chloroform : methanol (9 : 1, 8 : 2, 7 : 3, 6 : 4,

5: 5, 4: 6, 3: 7, 2 : 8, v/v) and 200 ml of methanol. 20 ml of effluent was collected in each tube. 0.5 ml aliquot in each tube was used for estimation of hexose content using the anthrone reagent

(-•œ-). Each 0.3 ml aliquot in each tube, after FIG. 7. Separation of methyl hexosides from tube No. 40, was used for assay of sialic acid with the methylated sample of Fr-C by GLC.

the Bial reagent (-: -) and hexosamine with Peak A : Methyl ƒÀ-2, 3, 6-tri-O-methylglucoside.

the Ehrlich reagent (--•œ--). The molar ratio of Peak B : Methyl 3-2, 4, 6-tri-O-methylgalactoside.

galactose to glucose in each fraction determined Peak C : Methyl ƒ¿-2, 3, 6-tri-O-methylglucoside and by GLC is also shown. methyl ƒ¿-2, 4, 6-tri-O-methylgalactoside.

FIG. 8. Infrared spectrum of Fr-C obtained by using a KBr-tablet. 170 T. TAKETOMI and N. KAWAMURA

Sachs spleen. insoluble fraction of the Tay-Sachs kidney Tap-Sacks Kidniy Glycoliiids-Silicic acid yielded three fractions of glycolipids, as shown column chromatography of a diethyl ether in Fig. 11. Fr-I eluted with chloroform : meth anol (9: 1, v/v) was assumed to contain two

FIG. 9. Silicic acid : Hyflo Super Cel (2 : 1, w/w ; 6g) column chromatography of Tay-Sachs spleen glycolipids. 44.8 mg of crude glycolipids Fin. 11. Silicic acid: Hyflo Super Cel (2: 1, were loaded on a column (13 mm 1. D.) and eluted w/w ; 9g) column chromatography of Tay-Sachs with 50 ml of chloroform, 100 ml each of mixtures kidney glycolipids. 104.5 mg of crude kidney of chloroform : methanol (9 : 1, 8 : 2, 7 : 3, 6 : 4, glycolipids were loaded on a column (13 mm I.D.) 5:5, 2:8) and 100 ml of methanol. 10 ml of and eluted with 50 ml of chloroform, 100 ml each effluent was collected in each tube. 0.5 ml aliquot of mixtures of chloroform : methanol (9 : 1, 8 : 2, 6 : 4, 4 : 6, 2 : 8) and 100 ml of methanol. 10 ml of each tube was used for estimation of hexose content using the anthrone reagent. The molar of effluent was collected in each tube. 0.3 ml ratio of galactose to glucose in each fraction de aliquot of each tube was used for estimation of hexose content by using the anthrone reagent. termined by GLC is also shown. The molar ratio of galactose to glucose in each fraction determined by GLC is also shown.

1 2 3 4 5 1 2 3 4 Fin. 10. Thin layer chromatograms of Tay- Fin. 12. Thin layer chromatograms of Tay- Sachs spleen glycolipids on Silica Gel G. Sachs kidney glycolipids on Silica Get G. 1 Fr-II (ceramide dihexoside). 1 Fr-I (glucocerebroside, galactocerebroside) . 2 : Fr-I (glucocerebroside). 2 : The first portion of Fr-II (ceramide dihexo 3 1, Fr-III (ceramide drhexoside and ceramide tri- side, sulfatide). 4 hexoside). 3 : The last portion of Fr-II (sulfatide, ceramide 5 : Fr-IV (globoside). trihexoside). The plate was developed with chloroform : 4 : Fr-III (globoside). methanol : water (60 : 25 : 4, by volume) for 50 Solvent system ; min at room temperature. Chloroform : methanol : water (60: 25: 4, by volume) . Glycolipids in Tay-Sachs Disease 171 kinds of ceramide monohexoside, galacto galactosyl(1-4)glucosyl ceramide. Storage of cerebroside and glucocerebroside in the ratio ganglioside was not detected in the Tay-Sachs of 2: 1, as judged from the molar ratio of galactose to glucose determined by GLC. Fr- II eluted with chloroform : methanol (8: 2, v/v) was also assumed to contain ceramide dihexoside, sulfatide and ceramide trihexoside.

Particularly, as shown in Fig. 12, the first portion of this fraction consisted of ceramide dihexoside and sulfatide, and the last portion contained sulfatide and ceramide trihexoside.

Fr-III eluted with chloroform: methanol

(6: 4, v/v) was considered to be globoside. It contained 14.5% hexosamine. The ratio FIG. 14. Silicic acid: Hyflo Super Cel (2: 1, of galactose to glucose in this fraction was w/w ; 15 g) column chromatography of Tay-Sachs

determined to be 2: 1. To elucidate the liver glycolipids. 140 mg of crude glycolipids were

chemical linkage of sugar moieties of this frac loaded on a column (22 mm I.D.) and eluted with 50m1 of chloroform, 150ml each of mixtures of tion, methyl hexoside after methanolysis of the chloroform : methanol (9 : 1, 8 : 2, 7 : 3, 6 -.4, permethylated sample was subjected to GLC. 5 : 5, 4 : 6, 2 : 8) and finally with 200 ml of As shown in Fig. 13, it was found that peak methanol. 10 ml of effluent was collected in each A corresponded to methyl 3-2, 3, 6-tri-O-meth tube. 0.5 ml aliquot of each tube was used for ylgalactoside, peak B to methyl /3-2, 3, 6- estimation of hexose content. The molar ratio of tri-O-methylglucoside, peak C to methyl /3- galactose to glucose in Fr-III and Fr-V determined 2, 4, 6-tri-O-methylgalactoside, and peak D to by GLC is also shown. ƒ¿-anomers of peaks A, B and C. Thus, this

fraction was inferred to have the chemical

structure of globoside type characterized as

N - acetylgalactosaminyl (1-3) galactosyl (1-> 4)

FIG. 15. Thin layer chromatograms of Tay- Sachs liver glycolipids on Silica Gel G.

FIG. 13. Separation of methyl hexosides from 1 : Fr-II (ceramide dihexoside). methylated sample of Fr-Ill by GLC. 2 : Fr-III (ceramide trihexoside). PeakA: Methyl /3-2, 3, 6-tri-O-methylgalactoside. 3 : Fr-IV (globoside). Peak B : Methyl /3-2, 3, 6-tri-O-methylglucoside. 4 : Fr-V (hematoside). Solvent system ; PeakC : Methyl /3-2, 4, 6-tri-O-methylgalactoside. PeakD: ƒ¿-Anomers of Peak A, Peak B and Peak C. Chloroform : methanol: water (65: 25: 4, by volume). 172 T. TAKETOMI and N. KAWAMURA

kidney. Fr-V were the main fractions of liver glyco

Tay-Sachs Liver Glycolipids-A crude glyco lipids and the other fractions were present

lipid fraction of the Tay-Sachs liver was only in trace amounts. TLC (Fig. 15) of separated into five fractions by silicic acid each fraction showed that Fr-II might be column chromatography (Fig. 14). Fr-II and identical with ceramide dihexoside, Fr-III

with ceramide trihexoside, Fr-IV with glo

boside and Fr-V with hematoside. The ratio

of galactose to glucose in Fr-III was 2: 1.

The gaschromatogram (Fig. 16) of methylhexo

sides after methanolysis of the permethylated

sample of Fr-III showed that peak A cor- responded to methyl ƒ¿- and ƒÀ-2,3,4,6-tetra-

0-meth ylgalactosides, peak B to methyl 3-

2, 3, 6-tri-O-methylgalactoside, peak C to

FIG. 16. Separation of methyl hexosides from methylated sample of Fr-III by GLC. PeakA: Methyl ƒ¿- and ƒÀ-2, 3, 4, 6-tetra-O-methyl- FIG. 17. Separation of methyl hexosides from galactosides. methylated sample of Fr-V by GLC. Peak B : Methyl 13-2, 3, 6-tri-O-methylgalactoside . PeakA: Methyl 13-2, 3, 6-tri-O-methylglucoside. PeakC : Methyl (3-2, 3, 6-tri-O-methylglucoside. Peak B : Methyl ,0-2, 4, 6-tri-O-methylgalactoside . PeakD: ƒ¿-Anomers of Peak B and Peak C. PeakC: ƒ¿-Anomers of Peak A and Peak B .

TABLE I Fatty acid compositionof cerebral glycolipids of a patient with Tay-Sachs disease and a normal neonatal human. Glycolipids in Tay-Sachs Disease 173

TABLE II Sphingosine base compositionsof cerebral glycolipids of a patient with Tay-Sachs disease and a normal neonatal human.

methyl ƒÀ-2, 3, 6-tri-O-methylglucoside and peak (Go) occupied a high proportion of cerebral D to ƒ¿-anomers of peaks B and C. Thus, glycolipids. It was noted that the elution pat- the chemical structure of this fraction was tern of cerebral glycolipids in silicic acid deduced to be galactosyl(1-4)galactosyl(1>4) chromatography was very similar to that of glucosyl ceramide. Fr-V contained galactose the glycolipids obtained from another patient and glucose in the ratio of 1 : 1 and 20.0% with the Tay-Sachs disease reported by MAKITA sialic acid. As shown in Fig. 17, the gas and YAMAKAWA(3). The Tay-Sacks glo chromatogram of methyl glycosides from the beside was also present in this brain. SUZUKI methylated sample of Fr-V showed that peak and CHEN (12) reported that more than half A corresponded to methyl ~-2,3,6-tri-O- of the ceramide monohexoside in the gray methylglucoside, peak B to methyl ƒÀ-2, 4, 6- matter was glucocerebroside in the Tay-Sachs tri-O-methylgalactoside and peak C to ƒ¿- disease and "generalized gangliosidosis ", and anomers of peaks A and B. Thus, Fr-V was in both diseases ceramide lactoside was present found to be identical with hematoside. at higher levels than normal. The presence Fatty Acid and Sphingosine Base Compositions of ceramide lactoside in the brains of Tay of Cerebral Glycolipids of a Patient with Tay- Sachs patients was also reported by SVEN- Sachs Disease and a Normal Neonatal Minion- NERHOLM(13). However, in the present in The fatty acid compositions of cerebral glyco vestigation, it was noted that a trace amount lipids are summarized in Table I. These of glucocerebroside and a small amount of glycolipids may be classified into two types ceramide lactoside were present in the brain according to the fatty acid compositions. of a Tay-Sachs patient, as well as in the Cerebroside and sulfatide belong to the first normal neonatal human brain. The amounts type and ceramide lactoside, Tay-Sachs glo of major ganglioside present in the normal boside and ganglioside (Go) to the second neonatal human brain seemed to be in the type. The first type consisted of relatively order of G,,>G,>G,II. It was also noted long chain fatty acids and the second type that a small amout of the Tay-Sachs ganglio mainly of stearic acid. As shown in Table side (Go) was definitely present in the normal II, Go contained a relatively smaller amount neonatal brain. It was a remarkable fact of C18-dihydrosphingosine compared with that the ganglioside constituents of the Tay- cerebroside and sulfatide. It was also noted Sachs brain were entirely different from that Go contained 6.4% of C,-sphingosine. those of the normal neonatal brain. Fur thermore, it was found that stearic acid was DISCUSSION the predominant fatty acid in ceramide In the case of the Tay-Sachs disease, it lactoside, globoside and ganglioside (Go) in has been found that only Tay-Sachs ganglioside the Tay-Sachs brain as well as in the major 174 T. TAKETOMI and N. KAWAMURA gangliosides of the normal neonatal human course of this research. We are also grateful to Prof. brain. As far as we analyzed the visceral N. Yoshikura, in the Department of Pediatrics, Nihon glycolipids of the spleen, kidney and liver, University for supplying the tissue samples of a Tay- accumulation of gangliosides was not found Sachs patient and to Assist. Prof. T. Aoyama in the at all. However, it was interesting that two Department of Pathology, Institute of Medical Science, additional components of ceramide trihexoside University of Tokyo for supplying us the tissue samples of the neonatal human brain. and hematoside occupied high proportions in the normal neonatal human liver glyco lipids. At any rate, these findings suggest REFERENCES that this state of affairs of Tay-Sachs disease (1) J. O'Brien, M .B. Stern, B.H. Landing, J.K. may be due to a specific metabolic disorder O'Brien, and G. Donnel, Am. J. Disease Children, of ganglioside in the nervous system. The 109, 338 (1965) (2) K. Sandhoff, U. Andreae, and H. jatzkewitz, present investigation on the Tay-Sachs brain Life Sciences, 7, 283 (1968) glycolipids has shown that the presence of (3) A. Makita and T. Yamakawa, Japan J. Exp. glucocerebroside and ceramide lactoside in Med., 33, 361 (1963) the Tay-Sachs brain is of minor significance (4) J. Folch, M. Lees, and G.H. 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