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—Gangliosides and allied neutral glycosylceramides were isolated from human infant (2-24 months of age) cerebral cortex and white matter. The individual glycolipids were separated quantitatively by a combination of column and thin-layer chromatographic methods on silica gel, DEAE-cellulose and Sephadex G-25. In cerebral cortex GD1a and GM1 were the major fractions and constituted more than 70 per cent of the total gangliosides. The concentrations of neutral glycolipids, except for galactosylceramides, were very low: lactosylceramide and glucosylceramide comprised 30 and 5 nmol/g wet weight, respectively. In white matter their concentrations were 10 times higher. The ganglioside concentration was only 50 per cent of that in cerebral cortex: the difference was accounted for mainly by the much lower content of the major di- and trisialogangliosides. Stearic acid was the predominant fatty acid of all brain gangliosides. GM3, and GD3 had a considerable content of the very long-chain fatty acids, C22-C24, particularly in the white matter. Glucosylceramide and lactosylceramide had almost identical fatty acid patterns between each other in cerebral cortex and white matter. In the cerebral cortex stearic acid and in the white matter the very long-chain acids predominated. d20:1 Sphingosine comprised more than 20 per cent of total sphingosine in all the gangliosides of the Gl- and G2-series. GM3, and GD3 like lactosylceramide contained significantly less of d20:1 sphingosine. The findings suggest the existence of separate compartments for the biosynthesis of the gangliosides. Glucosylceramides and lactosylceramides of white matter have the same ceramide composition as the galactosylceramides with normal fatty acids and are thus unlikely to be intermediates in the metabolism of the major brain gangliosides which have a completely different fatty acid composition.

Methane chemical ionization spectra of acetylated and perdeutero-acetylated ceramides, cerebrosides, and ceramide dihexosides have been analyzed and compared with electron impact ionization spectra of the same compounds. Abundant fragment ions for the loss of acetic acid from the protonated molecular ion in C.I. spectra readily enable the determination of the molecular weights of the principal species of sphingolipid mixtures. Complete structures can be elucidated by the combination of E.I. and C.I. mass spectra.

Tumour tissue from three patients operated on for renal carcinoma were analyzed for sphingolipids. Tissue from all three tumours contained substantial amounts of sphingomyelin. Two of them had very low amounts of ceramide and glycosphingolipids and were not analyzed in detail. The third and most malignant tumour had sphingolipids in the following concentrations (mg/g dry weight; literature data for normal total kidney in parentheses): ceramide 2.7 (0.6), sphingomyelin 6.7 (6.5), monoglycosylceramide i.i (0.22), diglycosylceramide 3.2 (0.53), triglycosyl-ceramide less than 0.2 (1.2), tetraglycosylceramide not detected (1.8), sulphatide not detected (0.58), ganglioside 1.7 (0.3). The following molecular species or components found in normal kidney sphingolipids were not found in the tumour: galactosylceramide, 2-hydroxy fatty acids, phytosphingosine. The ganglioside was identified as a hematoside by a newly developed mass spectrometric method for intact glycolipids. The biological significance of the findings and their relationship to tumour malignancy cannot be decided.

The biosynthesis of ceramides by mouse brain microsomes from sphingosine and stearoyl or lignoceroyl CoA was investigated. Microsomes from Quaking and normal mice showed similar activities when the acyl donor was stearate, but the Quaking mice exhibited about 55% less activity than normals when the acyl donor was lignocerate. This is interpreted to support the previous conclusion that two acyl transferases are involved in the synthesis of the nonhydroxy ceramides. It would appear that the lack of the lignoceroyl transferase is a specific aspect of the Quaking disorder. The composition of the components of cerebrosides from the brains of normal and Quaking mice was determined. An increased proportion of dihydrosphingosine and glucocerebroside was observed in the lipid from the mutants. A relative lack of the very long chain nonhydroxy acids was also observed, in confirmation of previous observations. The acyltransferase catalyzing stearoyl sphingosine formation was found to be unstable when incubated without substrates for 30 min, but considerable stabilization by sphingosine was observed.

Summary Embryonic chicken brain preparations catalyze the transference of sialyl groups from CMP-sialic acid to the exogenous acceptor galactosyl-N-acetylgalactosaminyl- (N-acetylneuraminyl)2-galactosylglucosylceramide (GDIb) to form a trisialoganglioside. Partial removal of sialyl groups by acid hydrolysis of the synthesized trisialoganglioside yielded a labeled disialoganglioside indicating that at least a part of the sialyl groups incorporated were not in the terminal position of a chain of sialyl groups. This result is compatible with the possibility that a substantial part of the trisialoganglioside synthesized was the isomer N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)2-galactosylglucosylceramide (GTIb).

A particulate fraction was prepared from kidneys, lyophilized, and coated with ceramide. After incubation with UDP-[14C]Gal and appropriate cofactors, the major radioactive component in the lipid fraction was galactosylceramide (Gal-Cer). If UDP-Glc was substituted for UDP-Gal, the product was glucosylceramide (Glc-Cer). Specific activity for Gal-Cer synthesis was present at comparable levels in kidney preparations from males or females at early ages. However, between 20 and 30 days of age there was a 5-fold increase in activity in preparations from male kidney, followed by a slight decline. In contrast, Gal-Cer synthesis in kidney preparations from female mice decreased slowly from 10 to 70 days of age. Despite the low levels of Gal-Cer in kidney, enzymatic activity for its synthesis was high, 0.8 nmole per mg of protein per hour in 30-day-old males. This was 40% of the specific activity for Gal-Cer synthesis in brains of the same animals. Glc-Cer synthesis by kidney followed a developmental pattern markedly different from that for Gal-Cer formation. There was an increase in activity throughout the time period studied (10 to 64 days of age), but activity was most rapid between 30 and 40 days of age. Activity in preparations of female kidney followed a similar pattern but was lower than that of preparations from males at 10 days of age and increased less rapidly. In kidneys of mature male animals, the specific activity for Glc-Cer synthesis was twice that of the females.

Mass spectra of the permethyl derivatives of a series of glycosphingolipids have been recorded. Fragments containing one, two and in some cases three sugar moieties were detected for the polyglycosyl compounds, several of these being of high intensity. The utility of such ions in establishing the position of hexosamine in an oligosaccharide chain was demonstrated. Low molecular weight gangliosides gave peaks corresponding to permethylated sialic acid under certain experimental conditions, permitting identification of N-acetyl- and N-glycoclylneuraminic acids. Glass sample containers appeared to catalyze destruction of these units. Peaks corresponding to the entire ceramide unit were detected but only in a few instances did these reflect the true ceramide composition. Specific fragments derived from the fatty acids and long-chain bases, respectively, were shown to be useful for qualitative identification of these components.

Activity of lactosyl ceramide β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) was found to be extremely low in enzyme preparations from liver, brain, and cultured skin fibroblasts from patients with Krabbe's disease. Leukocytes from one set of parents had enzyme levels approximately half those measured in control leukocytes. The low activity observed for this galactolipid hydrolase is the fourth enzymatic deficiency noted for this genetic disease. Beta-galactosidase activity toward galactocerebroside, psychosine, and monogalactosyl diglyceride is also low in patients with Krabbe's disease. Other lysosomal enzymes measured were found to be in the normal range. This enzymatic defect may provide a better explanation for the pathological and chemical findings previously reported for this syndrome.

Sphingolipids were found to dominate in the glycolipids from the cell envelope of baker's yeast. A relatively large quantity of ceramides was detected. Among the several complex phosphosphingolipids described, ceramide-(P-inositol)2-mannose was the main component. About 55% of long-chain bases in sphingolipids consisted of C18-phytosphingosine (4D-hydroxysphinganine). Other bases, found in decreasing concentrations, were C20-phytosphingosine, C20-dehydrophytosphingosine, C18-dihydrosphingosine (sphinganine) and C19-dihydrosphingosine. The presence of sterol glycosides, sulpholipids, cerebrosides and acylglucoses was demonstrated.

Galactosyl ceramide and lactosyl ceramide β-galactosidase activity was measured in extracts of previously frozen human brain. Using the assay methods described here, both galactosyl ceramide and lactosyl ceramide gave Michaelis constants of about 20 μM. Evidence from these studies indicates that at least a major portion of human brain lactosyl ceramide β-galactosidase activity is identical to galactosyl ceramide β-galactosidase activity. The evidence includes the following: co-purification by all methods tried, identical heat denaturation pattern under various conditions, same response to chemical inhibitors or stimulators, competitive inhibition with either substrate by the other, and severe deficiency of both galactosyl ceramide and lactosyl ceramide β-galactosidase activity in all tissues tested from patients with Krabbe's disease. Liver, brain, fibroblast, leukocyte and spleen extracts from patients with GM1 gangliosidosis had normal ability to degrade both galactosyl ceramide and lactosyl ceramide.

By use of galactose oxidase (EC 1.1.3.9), followed by reduction with tritiated sodium borohydride, the surface structures of transformed 3T3 and NIL cells, under ordinary growth conditions, were characterized by ( i ) deletion of the normally existing glycoprotein label and ( ii ) appearance or increase of a new glycoprotein label. NIL cells had a galactoprotein label with molecular weight 200,000 that was deleted in NIL cells transformed by polyoma virus. 3T3 cells had a glycoprotein label with molecular weight of 30,000 that was lost after transformation. Glycoproteins of transformed 3T3 cells, with molecular weight 105,000, and those of transformed NIL cells, with molecular weight 85,000, were not labeled in normal confluent cells, but became labeled after trypsin treatment. The label in glycolipids was quantitatively different in normal and transformed cells. The labeling pattern in glycoprotein and glycolipids of transformed NIL and 3T3 cells became similar to that of nontransformed cells when contact responses of transformed cells became conspicuous when cells were cultured in the presence of dextran sulfate or dibutyryl cyclic adenosine monophosphate, or in medium in which glucose was replaced with galactose.

The purpose of this investigation was to establish levels of cerebrosides in human plasma as a guide in investigating cerebroside levels in destructive and demyelinating central nervous system disorders. As an increase in plasma galactosylceramide may accompany myelin breakdown, this test may serve as a means of monitoring diseases such as multiple sclerosis. Cerebrosides and lactosylceramides were analyzed in 50-ml. pooled plasma samples from patients with nonneurologic diseases. Particular attention was given to the determination of the proportions of galactosylceramide in the cerebroside fraction; the normal levels were 6–12%. Gas–liquid chromatography and mass spectrometry confirmed that this glycolipid was galactosylceramide and established the fatty acid composition. It was also confirmed that plasma cerebrosides consist of both glucosylceramide and galactosylceramide with normal and hydroxy fatty acids.

Direct inlet mass spectrometry has been performed on different derivatives of a hematoside (a triglycosylceramide of a tumour) and the major monosialoganglioside of brain (a pentaglycosyl-ceramide). As a confirmation of earlier results it was shown that trimethylsilyl derivatives gave information on ceramide structure (fatty acids and long-chain bases) but no specific information on carbohydrate structure. Fully methylated derivatives on the other hand, not analyzed before, gave in addition to ceramide fragments, specific ions for the sialic acid as well as carbohydrate sequence and branching. Using these derivatives molecular ions were not obtained for the brain ganglioside. However, by reduction of the methylated derivatives with LiA1H 4 (amide groups of ceramide and amino sugars were reduced to the corresponding amines) and trimethylsilylation of the converted sialic acid ester group, molecular weight ions were obtained for both gangliosides. In addition very strong peaks were found for the complete carbohydrate plus the fatty acid, of importance for the determination of the type and exact ratio of sugars, and also the fatty acid composition of the molecules. Ions were also obtained for a conclusive information on carbohydrate sequence and branching. It is concluded that a combined mass spectrometric use of methylated and methylated plus reduced ganglioside derivatives affords structural information on the complete molecules, which will be of considerable help in the characterization of gangliosides on a microscale.

The chemistry with regard to synthesis and structural analyses of long chain bases are summarized and pathways for the synthesis of their radioactively labelled optical isomers are outlined. Aided by labelled substrates, the implication of the stereochemistry of the sphingosine bases in their catabolic and anabolic reactions and the metabolism of complex sphingolipids have been investigated.

— Gangliosides were isolated from plaque tissue and normal appearing white matter of multiple sclerosis (MS) brain. All four plaques showed decreased ganglioside concn relative to normal human white matter on a wet wt basis, but significant elevation in terms of dry wt. The wet wt and dry wt concn of MS white matter gangliosides showed smaller but statistically significant decreases below normal. Thin-layer patterns of the plaques showed several departures from normal white matter, including decrease of G4 and G5, and complete loss of G7 (sialosylgalactosylceramide). Most of the plaques had significant elevation of G2A and G3A along with increases of the slower-migrating polysialogangliosides. An additional ganglioside was present between G2 and G2A which was not seen in normal white matter. The TLC pattern of MS white matter gangliosides was essentially normal. The evidence for a general decrease of acidic lipids within normal appearing white matter is discussed.

SummaryStearic acid, galactose and ceramide digalactoside have been γ-irradiated in the solid state. Decarboxylation of stearic acid readily occurs to produce heptadecane (G = 6·4) and traces of hexadecane. For stearic acid −G is 11·5 and for galactose is 14; the values are the same in air and in vacuo.Ceramide digalactoside on irradiation gives small amounts of penta and tridecane (G ∼ 9 × 10−3). The −G values for disappearance of the stearyl and palmityl moieties are 0·17 and 0·13 respectively. There is also evidence for breakdown of the trans olefinic double bond of the sphingosyl residue. Scission of the β-glycosidic bond results in the formation of galactose (G = 1·9). For the galactose residues −G is 3·7.

A highly purified preparation of bovine hypertrophie thyroid nuclei was isolated by means of an adapted method of Widnell, C. C. and Tata, J. R. (1964) Biochem. J. 92, 313–317, followed by a centrifugation step in a zonal rotor. The total lipid content amounted to 2.1 pg/nucleus, approximately 90% of it being phospholipids. 60% of the total neutral lipid fraction was cholesterol. The phospholipid pattern showed 57% phosphatidylcholine, 22% phosphatidylethanolamine, 12% phosphatidylserine + phosphatidylinositol and 2% sphingomyelin. No glycolipids could be detected.

1. The lipid composition of the salt (nasal) gland of eider duck and herring gull, an organ with a high transport capacity for sodium ions, was characterized in detail. 2. The three major lipids of both animals were cholesterol, ethanolamine phosphoglycerides and choline phosphoglycerides. The major fatty acids of ethanolamine phosphoglycerides were stearic and arachidonic acid, and the major fatty acids of choline phosphoglycerides were palmitic, oleic and arachidonic acid. Small amounts of plasmalogens were found. 3. Other phospholipids identified were sphingomyelins, diphosphatidylglycerols, inositol phosphoglycerides and serine phosphoglycerides. 4. The major glycosphingolipids were sulphatides, with similar structure for the two animals. A sulphate group was identified in position 3 of galactose, bound to ceramide mainly containing the C20 homologue of sphingosine and saturated 2-d-hydroxy C22, C23 and C24 fatty acids. No branched-chain or trihydroxy bases were present. 5. An earlier unknown glycolipid, xylopyranosylceramide, was found in small amounts in herring gull but not in eider duck. Glucosyl-and galactosylceramides were present in both animals. 6. The ratio of Na+-K+-dependent ATPase and sulphatides was similar to ratios found for other tissues with increased capacity for the transport of sodium ions. No other lipid resembled sulphatides in this respect.

1. 1. Total monoglycosylceramide fractions were prepared from mucosa of bovine rennet stomach, duodenum, jejunum-ileum and colon ascendens. 2. 2. The lipids were characterized by thin-layer chromatography and mass spec trometry of intact molecules and by gas chromatography of carbohydrates after methanolysis. 3. 3. All regions contained galactosyi- and glucosylceramides. The amount of glucosylceramides was about 5 times higher in rennet stomach than colon ascendens. 4. 4. Trihydroxy base-containing species were present only in duodenum and jejunum-ileum. 5. 5. Galactosylceramide with sphingosine and 2-hydroxyhexadecanoic acid was exclusively present in colon ascendens. The corresponding glycosylceramide was not found.

— Isolated neuronal cell bodies and astroglia of young (15–20-day-old) rat brains were both found to contain small concentrations of a variety of glycosphingolipids, including glucosylceramide, galactosylceramide, sulphatide, dihexosylceramide and gangliosides. These sphingolipids, plus sphingomyelin, were isolated, quantitated and their fatty acid and long chain base patterns determined. These data were compared to similar data obtained on these lipids isolated from whole brain and myelin of rats of the same age range. Glucosylceramide was found in an amount equal to galactosylceramide in neurons, and accounted for 35 per cent of the total monohexosylceramide in astroglia. Dihexosylceramide was present in nearly the same amount as sulphatide in both cell types. The sphingolipids of each cell type had characteristic fatty acid patterns. Generally the whole brain fatty acid patterns resembled those of astroglial lipids rather than neuronal lipids. In no case did the cell sphingolipid fatty acids resemble those of myelin. However, the galactosylceramide and sulphatides of both cells had unsubstituted and α-hydroxy acids, both of which had appreciable quantities of C24 acids. The ganglioside fatty acids of each cell type were similar and not unusual, but were quite different from those of glucosylceramide and dihexosylceramide; the latter having appreciable quantities of 16:0 and acids longer than 18:0. The ganglioside patterns of these cells were similar and only slightly different from that of whole brain. Long chain bases of sphingolipids were mainly C18-sphingosine in both cell types, and those of ganglioside and sphingomyelin contained small amounts of C20-sphingosine.

Trihexosylceramide isolated from rat spleen was shown by methylation to consist mainly of a molecular species in which two galactosyl residues are linked 1→ 3. Thus a compound of this structure occurs normally in rat spleen. This fact is consistent with the previously reported biosynthesis in vitro of galactopyranosyl-α-(1→3)-galactopyranosyl-β-(1→4)-glucopyranosyl-β-(1→1)-ceramide in the presence of galactosyltransferases from rat spleen and bone marrow.

1. After injection of [6-3H]glucosamine into 8-day-old rats it was found that all the major brain gangliosides and their sialyl groups were labelled at essentially the same rate, except the hematoside, which was the least labelled. In 18-day-old rats it was found that the two major gangliosides with the sialyl (2→8)-sialyl linkage, and their sialyl groups were more labelled than the hematoside, the Tay–Sachs ganglioside, the other two major gangliosides and their respective sialyl groups. 2. No difference was found in any of the cases studied between the specific radioactivities of the neuraminidase-resistant and -labile sialyl groups belonging to the same ganglioside. The same was found for the specific radioactivities of the galactosyl groups proximal and distal to the ceramide moiety of total brain gangliosides from rats injected with [U-14C]glucose. From this it was concluded that partial turnover of the ganglioside molecule does not occur. 3. A model for the synthesis of gangliosides is presented that accounts for results from previous experiments in vitro and the lack of precursor–product relationships observed in experiments in vivo.

The diglycosylceramide which is present only in trace amounts in the kidney glycosphingolipids of normal adult female C57/BL mice but in substantial amounts in the kidney glycosphingolipids of female mice previously treated with testosterone is identified as digalactosylceramide. The increase in the amount of this compound in the kidneys of female mice treated with testosterone is the result of a stimulation by the hormone of the synthesis of the precursor, galactosylceramide, and the monoglycosylceramide fraction from the kidneys of female mice treated with testosterone contained double the proportion of galactosylceramide in the same fraction from the kidneys of normal female mice. Evidence is presented which suggests that testosterone controls the synthesis of galactosylceramide via ceramide and UDP galactose through its influence on the activity of the galactosyltransferase.

Correction of the Enzymic Defect in Cultured Fibroblasts from Patients with Fabry's Disease: Treatment with Purified α-Galactosidase from Ficin

A case of GM 1 gangliosidosis in a 3-month-old cat is described. The clinical, chemical and histopathological features closely resemble those seen in this condition in man. There is an increase in GM 1 ganglioside and ceramide tetrahexoside in the brain and liver. There is also storage of an extremely labile substance in the liver and to a lesser extent in the perivascular pericytes of the cerebral white matter. Membranous cytoplasmic bodies are seen in neurones and glia, but large cytoplasmic bodies are the more common type of glial inclusion. There is a paucity of myelin and oligodendrocytes in the cortical white matter.

SummaryCeramide, an N-acyl derivative of sphingosine bases, has been isolated for the first time from bovine milk in the free form and its constitution studied. Sixteen component fatty acids were found, among which C23:0(38·1%), C24:0(29·5%), C22:0(17·9%) and C16:0(7·2%) were predominant. Seven component long-chain bases were detected. The principal bases were C18-sphingosine (35·0%) and C16-sphingosine (31·6%).

1. 1. Glycosphingolipids were extracted from rat intestinal mucosa, brush border membranes, and isolated microvillus membranes and analyzed both qualitatively and quantitatively. 2. 2. The major glycosphingolipids in rat small intestinal mucosa were monohexosylceramide, trihexosylceramide and a ganglioside, containing neuraminidase-sensitive N -glycolyl sialic acid. Three additional fractions, (Complexes 1, 2 and 3), two of which contained fucose, were also identified. 3. 3. The distribution of glycosphingolipids was proportionately similar in the plasma membrane except for the appearance of substantial dihexosylceramide in two of three preparations. Gas-liquid Chromatographic analysis of sugars in monohexosylceramide, trihexosylceramide and in Complexes 1, 2 and 3 revealed almost identical sugar composition for corresponding membrane and mucosal fractions. Therefore membrane glycosphingolipids do not differ substantially from mucosal glycosphingolipids. 4. 4. The microvillus membrane contained four times as much glycosphingolipid expressed as percentage of total lipid, as did the intestinal mucosa. The relative enrichment of the membrane with glycosphingolipid was greater than with cholesterol. 5. 5. A minimum of 19.1 % of microvillus membrane lipid is made up by glycosphingolipid. This concentration is much higher than levels previously reported for extraneural mammalian plasma membrane.

Analyses were made of the glycosphingolipid composition of human platelets before and after treatment with thrombin. Within 10 min after incubation was initiated, there was approximately a two-fold increase in the concentration of hematoside. Concomitantly, the level of lactosylceramide was decreased about three-fold, and there was also a slight diminution in the ceramide levels of these cells. Platelets treated with phenylmethyl-sulfonyl fluoride and thrombin did not exhibit such an effect. These results suggest that lactosylceramide and ceramide are utilized as precursors in the biosynthesis of hematoside and that this conversion accompanies the aggregation of human platelets.

1. 1. A detailed analysis is given of the lipids extractable from the fused cerebral, thoracic and abdominal ganglia of 6-day-old larvae of the housefly, Musca domestica. Larvae from two strains were examined, one susceptible and one resistant to the action of cylodiene insecticides, but no differences in their lipid compositions were apparent. 2. 2. The major lipid fractions present in the nervous tissues, are free cholesterol triglycerides and phospholipids in the ratio of 1·0 : 1·3 : 7·6. 3. 3. The pattern of phospholipids is similar to that found in extracts of whole larvae except that there is a larger proportion of ceramidephosphorylethanolamine and a smaller proportion of polyglycerophosphatides in the nervous tissue. 4. 4. Not more than 4 per cent of the phospholipids of the nervous tissue are plasmalogens or alkylether phospholipids. 5. 5. There is no evidence that polyphosphoinositides are concentrated in the nervous tissue. 6. 6. The lipids of the nervous system contain a higher proportion of C-18 acids than do the lipids extracted from whole larvae. Acids with an odd-number of carbon atoms become incorporated into the nerve lipids but to a lesser extent than into the lipids of the whole larva. 7. 7. The present results are compared with those previously reported for mammalian and other invertebrate nervous tissue.

HYDROLYSIS OF THE GLYCEROPHOSPHATIDES 61 Phospho[ipase A 61 Phospho[ipase C 67 Phosphoiipase D 68 Lysophospholipase 68 CDP-diglyceride hydrolase 69

1. 1. Sphingomyelins of whole bovine kidney (14.4 mg/g dry weight) have been isolated and characterized in detail. 2. 2. After selective N-demethylation and gas chromatography and mass spectrometry the polar part was shown to be N,N-dimethylphosphorylethanolamine, in accordance with an original phosphorylcholine structure. No phosphonate analogue was found. 3. 3. The fatty acids were straight-chain, mainly saturated C16, C18, C22, C23 and C24 acids. The major unsaturated acid was C24cis-monoene. Small amounts of trans-monoenic acids were also present. 4. 4. The long-chain bases had a very complex pattern (about 30 molecular species). By a series of improved methods the bases were converted to dinitrophenyl derivatives, fractionated and structurally characterized. 5. 5. Three groups of bases were found by thin-layer chromatography; saturated dihydroxy bases, unsaturated dihydroxy bases and saturated trihydroxy bases, in the approximate ratio of 2:20:3. 6. 6. The paraffin chain distribution was similar for the three groups of bases; with C16–C20 bases, dominated by C18 homologues, and with unusually high amounts of C17 bases. No evidence for bases below C16 was found. 7. 7. By thin-layer chromatography, infrared spectroscopy and optical rotation measurement the geometry and configuration were assigned for the major part of the three groups of bases. Thus the major saturated dihydroxy base was dihydrosphingosine ( d -erythro-1,3-dihydroxy-2-aminooctadecane), the major unsaturated dihydroxy base sphingosine ( d -erythro-1,3-dihydroxy-2-amino-4-trans-octadecene), and the major trihydroxy base phytosphingosine ( d -ribo-I,3,4-trihydroxy-2-aminooctade-cane). 8. 8. 10–15% of the bases were branched-chain bases, with a methyl branch in Position 2 or 3 from the methyl end. The major bases had the branch in the same position (16) from the polar end irrespective of total chain length. 9. 9. A minor dienic base was identified as eryth-I,3-dihydroxy-2-amino-4, 14-octadecadiene, probably identical with an earlier described base of human plasma sphingomyelin. 10. 10. Ceramides and sphingomyelins were prepared from bovine kidney cortex, medulla and papilla. Their concentration decreased from the cortex to the papilla. 11. 11. The medium paraffin chain length of long-chain bases increased from the cortex to the papilla. 12. 12. The relative amounts of branched long-chain bases decreased from the cortex to the papilla. 13. 13. The relative amounts of trihydroxy bases were higher in the medulla than in the cortex or papilla.

Free ceramides were isolated from human platelets. Their structures were unequivocally determined by gas-liquid chromatography-mass spectrometry of the trimethylsilyl ether derivatives. The major components were N-(palmitoyl) sphingosine, N-(stearoyl) sphingosine, N-(eicosanoyl) sphingosine, N-(docosanoyl) sphingosine, N-(tetracosanoyl) sphingosine, and N-(tetracosenoyl) sphingosine. Sphinganine-and sphingadienine-containing ceramides as well as ceramides containing other unsaturated acids were also present. The amount of ceramides was determined by quantitative gas-liquid chromatography, using radioactive ceramide as internal standard and synthetic crystalline ceramides for comparison of peak areas. The concentration of ceramides was found to be 1.31 micro g/10(9) platelets or 0.47 micro g/mg of platelet protein.

— The levels of cerebrosides in neural tissues of adult mice were determined by densitometry of cerebroside spots on charred thin-layer chromatograms of washed total lipid extracts. Values for brain, spinal cord and peripheral nerves were 9·2, 33·0 and 36·9 mg/g of tissue, respectively. In adult Quaking mice these values were 6·4, 24 and 35 % of normal, respectively. Normal levels in brain, spinal cord and peripheral nerve of 21-day-old mice were 3·10, 13·5 and 17·8 mg/g, respectively. In 21-day-old Quaking mice the levels were reduced to 16,21 and 57% of normal, respectively. Biosynthesis of psychosine (galacto-sylsphingosine) by homogenates of Quaking brain, spinal cord and peripheral nerve, respectively, was 18, 24 and 42% of the normal rates at 21 days after birth and 16, 66 and 60% of the normal rates at 94 days. Our results suggest a quantitative relationship between the rate of formation of psychosine in vitro and the rate of accumulation of cerebrosides. in vivo. Biosynthesis of lactosylceramide was not reduced in homogenates of brain and spinal cord from Quaking mice. Cerebroside levels in normal and Quaking spinal cord and in normal brain increased 2- to 3-fold after 21 days of age, but in Quaking brain there was little or no increase.

Glial and neuronal cell preparations were made from young rat cerebrum and assayed for 3 enzymes involved in sphingolipid metabolism. A galactosyltransferase which makes galactocerebroside, a primary component of myelin, was found in all cell types examined, at fairly similar levels of activity. The same distribution of activities was found for the β-galactosidase which hydrolyzes galactocerebroside. It is suggested that the very low levels of galactocerebroside found in neurons are the result of an inability of neurons to form the lipoidal cerebroside precursor, hydroxy ceramide, or a cerebroside-binding protein. The glucosyltransferase which makes glucocerebroside, an intermediate in ganglioside biosynthesis, was found only in neurons. This may be a new marker enzyme for neurons, in contrast to other brain cells. Since gangliosides are found in non-neuronal membranes, it appears likely that they (or some intermediate in biosynthesis) are transferred from neurons.

Interest in sphingolipid metabolism has increased rapidly during the past decade, and many of the steps involved in the biosynthesis and metabolic degradation of sphingolipids are now known. In this review these studies are critically examined. Emphasis has been placed on the in vitro studies with cell-free systems, since these represent the groundwork for further purification and characterization of the enzyme systems involved. Experimental problems specific to this field of study, and the manner in which these may affect interpretation of experimental results, are discussed.

— The fatty acid composition of cerebrosides, sulphatides and ceramides has been determined at 20 days postpartum in the brains of Quaking mutant mice and of littermate controls. There was a significant deficit in the proportion of long-chain fatty acids (C22-C24) affecting both normal and a-hydroxy fatty acids of the cerebrosides. The proportion of normal but not the a-hydroxy long-chain fatty acids of the sulphatides was also decreased. Striking and disproportionate deficits of the C24:1 and C24 h:1 fatty acids of cerebrosides, sulphatides and ceramides characterized the brain of the Quaking mutant, and an increased proportion of C23 h:O fatty acid was found in the cerebrosides and sulphatides of the brain of this mutant. We compared these data with findings on the Jimpy mutant which has been examined by the same techniques. The deficiency of long-chain fatty acids which was found in the cerebrosides and sulphatides of both mutants was less extensive but more selective in the Quaking mutant.

Mass spectra of trimethylsilyl derivatives of a series of homogeneous cerebrosides have been recorded. The compounds studied had a d -galactopyranose in β-glycosidic linkage to different ceramides (containing dihydroxy or trihydroxy long-chain base and normal or hydroxy fatty acid). The composition of some fragments was established by high resolution analysis. Molecular ions were found for all compounds ( m/e 863–1265). Fragments of the silylated hexose were of relatively low intensity (at m/e 451, and 451 minus trimethylsilanol at m/e 361). Specific fragments of medium intensity were obtained from primary cleavage of the long-chain base and of a hydroxy fatty acid. Two rearrangement ions were specific for the fatty acid. Fragments of the base plus fatty acid were also obtained. Trimethylsilyl derivatives should therefore be useful in the analysis of long-chain bases and fatty acids and combinations thereof in natural mixtures of cerebrosides.