Detection o f Glycosphingolipids in Small Samples o f Human Tissue*f

Transcription

1 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, Vol. 2, No. 4 Copyright , Institute for Clinical Science Detection o f Glycosphingolipids in Small Samples o f Human Tissue*f GLYN D A W SO N, Ph.D. Departments of Pediatrics and Biochemistry Joseph P. Kennedy Jr. Mental Retardation Research Center University of Chicago Chicago, IL Introd uction Glycosphingolipids are minor components of cell membranes but have important biological properties. The structures of the major glycosphingolipids are given in table I. Cellular metabolism of these compounds is grossly altered when cells are transformed by DNA-viruses12 15 (with fundamental implications for understanding the biochemistry of cancer) or when normal cells contain an inactive mutant lysosomal glycosyl hydrolase These latter inborn errors of metabolism are usually referred to as the sphingolipidoses. Although they are rare, they are invariably fatal and constitute a significant pediatric problem. Because of their inherited nature, the development of an assay system for the biochemical aberration involved enables one to detect pre-symptomatic homozygotes, heterozygote carriers and normal siblings. This greatly facilitates genetic counselling and possible remedial therapy, and also allows one to control the disease by making a pre-natal diagnosis. In certain * Presented at the Applied Seminar on the Clinical Pathology of the Lipids, November, f This investigation was supported by USPHS Mental Retardation Research Center Grant HD and Grant ( RR-305) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. 274 of these diseases, for example Tay-Sachs disease, GMi-gangliosidosis, metachromatic leukodystrophy and Gaucher s disease, the mutant glycosyl hydrolase can be detected in very small tissue samples by its inability to produce a chromagen from the appropriate commercially-available synthetic glycoside (table I I ). However, in other related diseases such as globoid cell leukodystrophy and lactosylceramidosis, it is necessary to use the radioactive-labelled natural substrate which is not commercially available. In some cases, chemical detection of the accumulating glycosphingolipid may be simpler and more reliable. Characterization of the accumulating glycosphingolipid not only corroborates enzymic data but, in many of the diseases where the biochemistry is not understood, it is the only way in which a new disease can be characterized. A combination of silicic acid, thin-layer and gas-liquid chromatographic techniques enables quantitative glycosphingolipid assays to be carried out on small amounts of blood, urine or biopsied tissue. Previously, such chemical diagnoses were only achieved by examining the large amounts of tissue available post-mortem. M aterials and Methods Heparinized blood samples (4 to 8 ml) were centrifuged foi 60 minutes at 3,000

2 DETECTION OF GLYCOSPHINGOLIPIDS 275 TABLE I C h e m i c a l C o m p o s i t i o n o p t h e M a j o r G l y c o s p h i n g o l i p i d s Nom enclature S tru c tu re Disease in w hich i t accumulates Ceram ide c h 3 ( c h 2 ) 12 c h = c h - c h o h - c h n h [ c o - ( c h 2 ) ] c h 2 o h F ä r b e r ' s G L -la glc -ceram ide Gaucher * s GL -lb gal-ceram ide Krabbe' s G L -2 a g a l- g lc -c e r a m id e L a c t o s y lc e r a m id o s is GL-2b ga l-gal-ceram id e F a b ry1s G L -lbs SOgH -gal-ceram ide Metachrom atic leukodystrophy G L-3a g a l - g a l - g l c - c e r amide F a b r y 's G L-4a g a ln A c -g a l-g a l-g lc - c e r a m id e S a n d h o f f 1s t o galn Ac -^N AN Aj-gal-glc -ceram ide g a l-g a ln A c -^ N A N A j-g a l-g lc -c e ra m id e Tay-Sachs; S a n d h o ff's G ^ - g a n g l i o s i d o s i s A b b re via tions : g lc, glucose ; g a l, ga la ctose ; galnac, N -acetylgalactosam ine; NANA, N -acetylneuram inic acid ; SO3H, s u lf a t e. marrow aspirate was treated in the same manner. Urine (24 hr collection) was cen- trifuged at 10,000 rpm, the supernatant set rpm to separate plasma and erythrocytes; leukocytes were discarded and the red cells washed twice with 0.15 M NaCl. Bone TABLE II C h a r a c t e r i s t i c s o f t h e M a j o r S p h i n g o l i p i d o s e s Major Characteristics Typical onset Age of: death Neurological Visceral Accumulating sphingolipid Missing enzyme Disease Gray matter Gaucher1s Infantile Hepatosplenomegaly, GL-la 3-glucosidase* foam cells, high Adult acid phosphatase GL-la P-glucosidase* Lactosylceramidosis Moderate hepatosplenomegaly, foam cells, dementia, blindness 2? + + GL-2a GL-2a P-galactosidase Fabry's Skin lesions, recurrent pain, kidney failure GL-2b GL-3a Cü-galactosidase* Sandhoff's Cherry red spot in maculae; hepatosplenomegaly GL-4a GfC All Hexosaminidases (A and B)* Tay-Sachs Gfti-gangliosidosis Cherry red spot, dementia, blindness Coarse features, bony abnormalities, hepatosplenomegaly GM2 Hexosaminidase A* or - Total p-galactosidase* (Gj^-p-galactosidase) Niemann-Pick Hepatosplenomegaly, foam cells, cherry red spot Sphingomyelin Sphingomyelinase White matter Metachromatic leukodys trophy Normal early development, ataxia, slow degeneration or - GL-lbS Sulfatase* Krabbe1s leukodystrophy Rapid neurological degeneration. Globoid cells in brain * These enzymes can be assayed with commercially available synthetic substrates GL-lb GL-lb ß-galactosidase

3 276 DAWSON QUANTITATIVE ISOLATION OF GLYCOSPHINGOLIPIDS BLOOD (2-4 m l) TISSUE (100 mg) 300 VOLS C:M (2:1) FILTER, RE-EXTRACT ADD 20% WATER UPPER PHASE ALKALINE METHANOLYSIS. DIALYSIS GANGLIOSIDES I TLC MONO-) * DI- SIALOGANGLIOSIDES T R I-) ACID (1.0 N HCI) METHANOLYSIS RE-N-ACETYLATE VISUALIZE WITH IODINE ; SCRAPE GELJELUTE WITH C:M:H20 (1 0 0 :4 0-6}? ADD MANNITOL LOWER PHASE SILICIC ACID CHROMATOGRAPHY I A:M (9 :1 ) CHCIS, CHjOH { GLYCOSPHINGOLIPIDS +PL ALKALINE METHANOLYSIS DIALYSIS GLYCOSPHINGOLIPIDS I TLC /C 110\ M 40 4, 'H 20 6 ' NEUTRAL GLYCOSPHINGOLIPIDS+ GM3 PHOSPHOLIPIDS 2-D TLC CHOLESTEROL P - ETHANOL AMINE STEROID ESTERS P-SERINE GLYCERIDES P - INOSITOL FATTY ACIDS P-CHOLINE SPHINGOMYELIN + OTHER PHOSPHOLIPIDS SPHINGOSINE BASES FATTY ACIDS METHYL ESTERS ALL MAY BE ESTIMATED BY GLC F i g u r e 1. Scheme for the quantitative isolation of glycosphingolipids from small amounts of tissue. Abbreviations: NL, neutral lipids; PL, phospholipids; A:M> acetone:methanol; P- ethanolamine etc., phosphatidylethanolamine, etc.; 2-D TLC, two-dimensional thin-layer chromatography; GLC, gas-liquid chromatography. aside for enzymic and mucopolysaccharide analysis, and the sediment lyophilized. In cases where delivery of a 24 hr urine sample was a problem, the urine sediment was collected in filter paper (this may be attached to the child in the same manner as a pediatric bag ) and the dried filter paper mailed to this laboratory. Liver and brain biopsies were homogenized in 10 volumes of 0.15 M NaCl prior to extraction of the lipids. Total lipids were extracted from each sample or tissue homogenate by the addition of choloroform-methanol (2:1)10 and fractionated21 into neutral lipids (cholesterol, steroid esters, triglycerides, and fatty acids), glycosphingolipids, and phospholipids on a 4 g column of silicic acid according to the scheme shown in figure 1. The glycosphingolipid fraction, eluted with 100 ml of acetone-methanol (9:1), was subjected to alkaline methanolysis,20 neutralized, and exhaustively dialyzed against distilled water.3 Individual glycosphingolipids were separated by thin-layer chromatography (TLC) on silica gel, using the solvent system chloroform-methanolwater (110:40:6), and visualized by iodine vapor (figure 2).* The glycosphingolipid bands were scraped and the eluted mate- * Diazo Projection Paper ( 8 -J " X 11", Cat. #1919A, B. K. Elliot Co., 3661 Stafford St., Pittsburgh, PA 15204) may be placed over the stained thin layer plate or paper electrophoretogram, etc. which is illuminated from below. An ordinary horizontal slide or X-ray viewer is ideal for this purpose. After about 10 minutes, the paper is quickly transferred to a 5 liter beaker containing 5 ml of concentrated ammonia solution. The ammonia gas reacts instantly with any of the remaining diazo to give blue spots on a light background. This color does not fade when stored in a folder and gives an accurate, permanent record of the separation achieved.

4 27 DETECTION OF GLYCOSPHINGOLIPIDS BLOOD GLYCOSPHINGOLIPIDS G lc -C e r Pigment L a c -C e r G a l-l a c -C e r Globoside Tay-Sachs Control ( R.L ) Pooled Tay-Sachs Control Tay-Sachs (G.H) (J. I.V.) (G.S) F i g u r e 2. Thin-layer chromatographic separation of neutral erythrocyte glycosphingolipids from controls and patients with Tay-Sachs disease. The solvent system used was chloroform-methanol-water (1 1 0:40:6). METHYL GLYCOSIDES - MAJOR MONOSIALOGANGLIOSIDE ISOLATED FROM ERYTHROCYTES OF A PATIENT WITH SAN-FILIPPO SYNDROME 3 % OV AT 27MIN. DETECTOR RESPONSE TMSi TIME (MIN.) 3. Gas-liquid chromatographic separation of TMSi monosaccharide methyl glyco sides from human erythrocyte G mi. Conditions are described in the text. Abbreviations: F, furanose ring form; a-p, alpha pyranose ring form; fi-p, beta pyranose ring form. Mannitol is the internal standard. F ig u r e

5 278 DAWSON TMSi METHYL GLYCOSIDES" MAJOR MONOSIALOGANGLIOSIDE ISOLATED FROM HUMAN BRAIN 3% OV AT 2 /MIN. TIME (MIN.) F ig u r e 4. Gas-liquid chromatographic separation of TMSi monosaccharide methyl glycosides from human brain Gmi. Conditions are described in the text. rial subjected to acid methanolysis (1.0 N HC1 in dry methanol at 80 C for 16 hours); a known amount of the internal standard (mannitol, 0.02 to 0.20 / moles) was added prior to methanolysis. The methanolysate was neutralized with solid silver carbonate, re-n-acetylated with 6 drops of acetic anhydride and left for 16 hours at room temperature. The dried residue was trimethylsilylated2 20 and the TMSi methyl glycosides separated on a 6 ft column of 3 percent OV-1 by temperature-programming from 160 to 240 at 2 per minute (figure 3). The upper phase of the chloroformmethanol-water (2:1:0.6) partition, usually referred to as the ganglioside fraction, was dried, subjected to mild alkaline hydrolysis, and exhaustively dialyzed. After an initial TLC separation in chloroformmethanol-2.5 N NH4OH (60:40:9), the TLC plate was dried and re-chromatographed in the same solvent. Gangliosides (glycosphingolipids containing sialic acid and hexosamine) were analyzed in the same manner as neutral glycosphingolipids. Results E r y t h r o c y t e s Normal human erythrocytes contain five major glycosphingolipids (figure 2); Glccer (G L-la), Lac-cer (GL-2a), Gal-lac-cer (GL-3), Globoside (GL-4), and hematoside (GM3), the latter partitioning between the upper and lower phases of the Folch10 extraction. They were identified by their Rf values on thin-layer chromatographic plates and by the ratio of galactose: glucose : N-acetylgalactosamine: sialic acid. All these sugars are found in brain Gmi (figure 4); galactose gives three peaks, glucose and GalNAc, two peaks, and sialic acid, one peak. The results of quantitative analyses are given in table III. Analysis of erythrocyte glycosphingolipids is of diagnostic value in Gaucher s disease21 (after splenectomy) and lactosylceramidosis,6 but its use has not been reported in other glycosphingolipidoses. Analysis of erythrocyte glycosphingolipids from patients with Tay- Sachs disease showed the trihexosylceramide band to be virtually absent (figure

6 DETECTION OF GLYCOSPHINGOLIPIDS 279 T A B L E I I I G l y c o s p h i n g o l i p i d L e v e l s i n S o m e N o r m a l H u m a n T i s s u e s Glycosphingolipid Erythrocytes Plasma Urine Sediment* Kidney Liver Spleen Gray Matter White Matter GL-la ( no4.es/100 ml tmoles/100 g. fresh weight GL-lb <1.0 < GL-2a <1.0 GL-2b GL-lbS GL GL M <1.0 <1.0 M2 M1 g D3 GDla/b GTla < * Results are expressed as «nolee/g. dry weight. 2). The small amount of glycosphingolipid in this region is the asialo form of the GM2 ganglioside which accumulates in the brain of these patients, rather than the GL-3 found in normal red cells. This finding was in eight cases of Tay-Sachs disease, but the simplicity of the enzymic assay17 makes this chemical assay rather impractical for the routine diagnosis of Tay-Sachs disease. The Tay-Sachs ganglioside (GM2 ) in erythrocytes could not be detected in any of the eight cases. Although visceral tissue probably contains trace amounts of the gangliosides associated with nervous tissue, they have never been adequately characterized apart from GM3 and its disialo form GD3 (both of which do not contain hexosamine). Small amounts have been reported in erythrocytes22 and we have isolated substantial amounts (1.0 / moles per 100 ml packed cells) from a patient with mucopolysaccharidosis III ( Sanfilippo s disease). This ganglioside contained Gal :Glc:GlcIVAc: NANA in the ratio of 2:1:1:1 (figure 3) and may be compared to the major ganglioside of the brain, GMi (figure 4) where the ratio is the same but the hexosamine is N-acetylgalactosamine. The metabolic defect in the Sanfilippo syndrome has not yet been elucidated. Pl a sm a In Gaucher s disease,21 lactosylceramidosis6 and Fabry s disease,21 plasma GL-la, GL-2a, and GL-3, respectively, show a 2 to 3-fold elevation over normal values (table III). In each disease the elevation is specific for the one glycosphingolipid and is of diagnostic value. B o n e M arro w Results with erythrocytes and plasma can be duplicated in bone marrow samples. Further, in diseases where the bone marrow contains high concentration of the accumulating material in the form of foam cells (Gaucher s disease, lactosylceramidosis, Niemann-Pick disease, GMigangliosidosis, and possibly Sandhoff s disease), it should be possible to isolate and characterize this accumulating material and arrive at an unambiguous diagnosis.

7 280 DAWSON TMSi METHYL GLYCOSIDES! DISIALOHEMATOSIDE (Goa) ISOLATED FROM HUMAN AMNIOTIC FLUID (J.Y.) 3 % 0V AT 2 / MIN. TIME (MIN.) F ig u r e 5. Gas-liquid chromatographic separation of TMSi monosaccharide methyl glycosides from human amniotic fluid G ds. Conditions ate described in the text. U rine Sedim ent Urine sediment can be regarded as somewhat analogous to a kidney biopsy and thin-layer chromatographic analysis of urinary glycosphingolipids can be used to diagnose Fabry s disease, metachromatic leukodystrophy, lactosylceramidosis, Sandhoff s disease and Gaucher s disease (the latter only after splenectomy).8 A normal 24 hr urine sediment contains very low levels of the glycosphingolipids associated with kidney diseases. It does not appear to be of value in the diagnosis of Tay- Sachs disease, Gm -gangliosidosis or globoid cell leukodystrophy. U rin e The urine from patients with GMi-gangliosidosis contains large amounts of a keratosulfate-like material Less than 1.0 ml of dialyzed urine is required for gas-liquid chromatographic analysis of this glycoprotein. Galactose and ZV-acetylglucosamine are present in almost equimolar amounts together with mannose and trace amounts of fucose and sialic acid. Normal urine contains negligible amounts of glycoprotein; the only purification necessary is simple dialysis. There is evidence that mucopolysaccharide metabolism is affected in other sphingolipidoses, and this technique may be of more general diagnostic value. Since uronic acids can be detected by GLC, it is also applicable to diagnosis of Hurler s, Hunter s, Sanfilippo s disease, and other mucopolysaccharidoses. A m n io t ic F lu id Neutral glycosphingolipids are virtually undetectable in the 5 to 10 ml samples usually obtained by amniocentesis, but two sialo-glycosphingolipids, GM3 and GD3, were found in equal amounts (0.2 / moles per 100 ml). The glycosphingolipid GD 3 often migrates as two bands on thin-layer chromatography, and these can be mis-identified as GM2 and Gmi if brain glycosphingolipids are used for comparison. The use of gas-liquid chromatography (GLC) for the identification of GD3 (figure 5) eliminates any ambiguity. This is important in the pre-natal diagnosis of diseases such as Gjn-gangliosidosis. In a pregnancy at risk for GMi-gangliosidosis a high level of GD 3 was found (1.0 / moles per 100 ml) in amniotic fluid, which looked like an ele-

8 DETECTION OF GLYCOSPHINGOLIPIDS L IV E R GLYCOSPHINGOLIPIDS GL-la GL-la GL-2a GL-3 G M3 GM3 GL-2a GL-4 GL-3 GL-F1 GL-4 GL-F2 GM3 CONTROL SOLVENT SYSTEM a -FUCOSI DOSI S PATIENT C - M N NH40H ( 6 0 : 4 0 = 9 ) C-M-H-0 (11:4:0-6) F i g u r e 6. Thin-layer chromatographic separation o f liver neutral glycosphingolipids from a patient with fucosidosis. The solvent system used was chloroform-me'hanol-2.5 N NH4OH ( ) and the control glycosphingolipids were derived from 1.0 g of liver. The right-hand chromatogram shows the separation of liver neutral glycosphingolipids in the solvent system chloroform-methanol-water ( ). GL F1 is the glycosphingolipid subsequently identified as the H-antigen. vation of GMi as judged by thin-layer chro matography. The /J-galactosidase level in cultured amniotic fluid cells was normal, there was no evidence of keratosulfate-like material (which should be readily detect able), and the child did not have the dis ease. Provided suitable precautions are taken, glycosphingolipid analysis should provide useful corroborative evidence in the pre-natal diagnosis of ganglioside stor age diseases; at present there are no re ports to confirm or deny this. L iv e r B io p s y The liver is the major storage organ of the body and hepatic accumulation of gly- cosphingolipid is characteristic of most of the storage diseases.3 The normal level of glycosphingolipids in the liver is quite low (table III) which makes chemical diag nosis of sphingolipidoses a simple matter in many cases. A liver biopsy from a pa tient with a rare neurovisceral storage dis ease called fucosidosis was recently ex amined.914 The patient was the fourth case thus far reported, and enzymic assays on the liver showed the characteristic total absence of «-L-fueosida.se activity toward the synthetic substrate p-nitrophenyl <*-Lfucoside.12 Since fucose is a constituent of glycoproteins, keratosulfate and some immunologically active glycosphingolipids, it

9 282 DAWSON TABLE IV L a c t o s y l c e r a m i d e L e v e l s i n P a t i e n t ( S. H. ) C o m p a r e d t o N o r m a l I n d i v i d u a l s T issu e examined Degree o f e le v a tio n o f lactosylceram ide (x normal) B io p sy Autopsy E ryth ro c yte s 3... Plasma 4 Bone marrow ce lls 2... U rin e sediment 3 S k in f ib r o b la s t s 2 L iv e r 7 7 G ray matter W hite m atter Lymph node Spleen... 4 A drenal gland... 7 Kidney... 3 was of interest to determine the nature of the material which was causing the severe hepatomegaly and mental retardation. A small piece of biopsied liver (0.063 g fresh weight) was obtained and the glycosphingolipids were isolated. It would not be possible to reliably detect the glycosphingolipids in such a small amount of normal liver, but in the fucosidosis liver sample, a major band was seen in the pentaglycosylceramide region on thin-layer chromatography (figure 6). Gas-liquid chromatographic analysis showed this glycosphingolipid (2.8 / moles per g fresh weight) to contain Fucose:Gal:Glc:GlcIVAc in the ratio 1:2:1:1,4 suggesting a structure compatible with that of the H-antigen.13 The patient was blood group AB and the H- antigen is a common catabolite of these two blood group-active glycosphingolipids. B r a i n In the case of severe, terminal, neurological disease, a brain biopsy can be used for diagnosis by microscopic and biochemical methods. All the neurological diseases shown in table II can be diagnosed by chemical identification of the accumulating glycosphingolipid. It can be seen in table III that the glycosphingolipid composition of brain is different from that of visceral tissue. The ability to diagnose neurological diseases from small samples of fluid or skin has obviated the necessity of carrying out brain biopsies in many of the sphingolipidoses. Application of These Techniques to the Diagnosis of a New Disease L a c t o s y l c e r a m i d o s i s In September, 1969, a 3-year-old negro female (S.H.) was admitted to the University of Chicago Wyler Children s Hospital with a suspected storage disease. Cerebral ataxia was prominent and the child soon lost all motor skills and ceased to have contact with environment; no cherry red spot was seen. Liver and spleen were somewhat enlarged and foam cells were prominent in the bone marrow. Gaucher s disease, Gm-gangliosidosis or Niemann-Pick disease were considered as likely diagnoses but the clinical characteristics and biochemical tests eliminated them from consideration. 8 Examination of erythrocytes, plasma, bone marrow, urine sediment, and fibroblasts cultured from a skin biopsy and liver biopsy revealed a consistent and specific two to seven-fold elevation of lactosylceramide in each tissue (table IV). By analogy to other glycosphingolipid storage diseases it was suspected that a catabolic enzyme deficiency was responsible for the elevation of lactosylceramide (G L-2a). This was confirmed enzymically in liver and cultured fibroblasts (table V) by measuring the amount of 3H-galactose liberated from GL-2a labelled specifically at C- 6 of the terminal galactose. 19 Activity in the patient s tissue was 10 to 15 percent of normal and fibroblasts cultured from the patient s skin had 50 percent of normal activity. 7»8 This was good suggestive evidence that the disease was genetic in nature and that a structural gene mutation was probably involved. The child died from a respiratory infection at the age of 4-ii years. An analysis of glycosphingolipid levels in gray matter, white matter, kidney, adrenal gland, lymph node, liver, and spleen obtained at autopsy confirmed the analyses on the biopsy samples (table IV ). The elevation of lactosylceramide ranged from forty-fold in gray matter to four-fold in spleen and in gray matter spleen and liver was accompanied by smaller ancillary elevations of glucosylceramide (G L -la) and hematoside ( Gnu). From consideration of the age of onset and amount of residual enzyme activity (10 to 15 percent), this case is probably

10 DETECTION OF GLYCOSPHINGOLIPIDS 283 the juvenile form of lactosylceramidosis. Since no other cases of lactosylceramidosis have been well documented, one can only speculate that the infantile form (total lactosylceramidase deficiency) is a lethal mutant. Sum m ary The diagnosis and complete characterization of a hitherto unknown sphingolipidosis from small samples of tissue and fluid, available from a living patient,5 6 7 indicated the usefulness of this quantitative technique. Further, a second sphingolipidosis, fucosidosis, was chemically characterized for the first time using a g liver biopsy as the sole source of tissue.4 In both of these diseases, complementary enzymatic studies were essential in determining if the lesion was catabolic or anabolic. Once a disease has been chemically characterized, detection of the inactive mutant enzyme is often the simplest method of diagnosis. However, the number of synthetic substrates that are commercially available is somewhat restricted; because of their broad specificity, it is often possible to obtain ambiguous results. Quantitative analysis of individual glycosphingolipids, while possibly more time-consuming, is less ambiguous and gas chromatographs are available in all large laboratories. In TABLE V L a c t o s y l c e r a m i d e G a l a c t o s y l H y d r o l a s e L e v e l s i n P a t i e n t (S.H.), H e r P a r e n t s a n d C o n t r o l s Sam ple % o f n o rm a l a c t i v i t y * L iv e r N orm al 100 S.H. 17 F i b r o b l a s t s N orm al 100 S.H. 11 S.H. m other 48 S.H. f a t h e r 55 F abry hem izygote 98 F abry heterozygote 105 G a u c h e r hom ozygote 140 * N orm al a c t i v i t y i n l i v e r was 231 cpm/mg. p r o t e in / h r., and i n f i b r o b l a s t s 1100 cpm/ mg. p r o t e in / h r. the storage diseases where the inherited defect remains to be elucidated, the identification of abnormal amounts of glycosphingolipid material is the first critical step. Finally, the same basic gas-liquid chromatographic procedure can be applied to glycoproteins,2 mucopolysaccharides, polysaccharides and other macromolecules of biological interest. Acknowledgments Thanks are extended to Dr. A. O. Stein, University of Chicago, for providing control samples and samples from the patient with Lactosylceramidosis; to Drs. B. W. Volk and L. Schneck, Kingsbrook Jewish Medical Center, New York, and to Dr. H. J. Grossman, Illinois State Pediatric Institute, Chicago, for samples of blood from patients with Tay-Sachs disease; to Dr. J. W. Spranger, University of Kiel, Germany, for providing the liver biopsy from the patient with fucosidosis; to Dr. R. Matalon, University of Chicago, for growing the skin fibroblasts; and to Mr. John Oh for technical assistance. References 1. B r a d y, R. O.: Metabolic disorders of sphingolipid metabolism in man. Chem. Phys. Lipids 5: , C l a m p, J. R., D a w s o n, G., a n d H o u g h, L.: The simultaneous estimation of 6 -deoxy-lgalactose (L-fucose), D-mannose, D-galactose, 2-acetamido-2-deoxy-D-glucose ( N-acetyl-Dglucosamine) and JV-acetyl-neuraminic acid (sialic acid) in glycopeptides and glycoproteins. Biochim. Biophys. Acta 148: , D a w s o n, G.: Glycosphingolipid abnormalities in liver from patients with glycosphingolipid and mucopolysaccharide storage diseases. Sphingolipids, Sphingolipidoses and Allied Disorders, Volk, B. W. and Aronson, S. M., eds. Plenum Publishing Co., New York, D a w s o n, G. a n d S p h a n g e r, J. W.: Fucosidosis: a glycosphingolipidosis. New Engl. J. Med. 2S5.122, D a w s o n, G. a n d S t e i n, A. O.: Lactosylceramidosis: Catabolic enzyme defect of glycosphingolipid metabolism. Science 170: , D a w s o n, G. a n d S t e i n, A. O.: Lactosylceramidosis: characterization of a new glycosphingolipidosis. Lipid Storage Diseases, Bernsohn, J. and Grossman, H. J., eds., Academic Press, New York, pp , D a w s o n, G., M a t a l o n, R., a n d S t e i n, A. O.: Lactosylceramidosis: Lactosylceramide galactosyl hydrolase deficiency and accumulation of lactosylceramide in cultured skin fibroblasts. J. Pediat. 79: , 1971.

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