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Assay of methylumbelliferyl-α-galactosidase activity (MUM-α-Galase) in plasma reflects the activity of ceramide trihexosidase and (or) ceramide digalactosidase, which are deficient in Fabry's disease. Intending to institute this assay routinely, we have evaluated substrate stability and concentration, sample stability, heat-inactivation conditions, and normal values. The heat-labile isoenzyme(s) deficient in plasma from patients with Fabry's disease must either be assayed immediately after sampling or the plasma must be stored at or below -50 °C. This heat-labile fraction can be inactivated by incubating a plasma aliquot at 45 °C for 45 min. Normal adult ranges for total and heat-labile MUM-α-Galase are 8.9-31.7 and 7.4-27.4 nmoles of MUM per hour per milliliter of EDTA-treated plasma.

The purified β-toxin (sphingomyelinase C) of Staphylococcus aureus released water-soluble organic phosphorus from aqueous dispersions of sphingomyelin, and bovine and human erythrocyte ghosts in the presence of Mg 2+ , in amounts indicating extensive sphingomyelin degradation. Extensive ultrastructural changes were evident in both human and bovine freeze-etched ghosts after sphingomyelinase C treatment. Pools of apparently solid particle-free material, possibly the ceramide product of hydrolysis, accumulated in the hydrophobic region of the bilayer. These observations can be explained in terms of a membrane in which sphingomyelin is preferentially located in the outer half of the bilayer. This staphylococcal toxin should prove useful in further studies on the distribution of phospholipids in various membranes and their relationship to membrane function.

N-(n-Hexyl)-O-glucosylsphingosine completely inhibited β-glucosidase in cultured rat astrocytoma (RGC-6) and mouse neuroblastoma cell strains (NB41A and NB2a), creating a model for the infantile Gaucher nervous system. The inhibitor, at a concentration of 5 µg per ml, caused a 5-fold elevation of glucosylceramide in RGC-6 and a 6-fold elevation in NB41A cells. Under these conditions the growth rates of cells were unaffected. Other lysosomal enzymes were only depressed to approximately 80% of normal with the exception of β-glucuronidase (37% of normal); such reductions would not be anticipated to cause abnormal pathology and none was observed. Studies with glucosyl[14C]ceramide (labeled in the stearoyl moiety) showed that the glycolipid was rapidly taken up from the medium. In the presence of the β-glucosidase inhibitor the uptake of glucosylceramide was reduced in NB2a, NB41A, and RGC-6 cells, and in the latter two cell strains no subsequent catabolism could be detected. Studies with more complex glycolipids, such as gangliosides, showed that they were readily taken up and metabolized and that catabolism was not affected by the inhibitor. This was interpreted as evidence for two β-glucosidases, one which utilizes free glucosylceramide and is deficient in Gaucher's disease and the other which acts as an integral part of a multienzyme complex for the degradation of complex glycolipids and has normal activity in Gaucher's disease. Results indicated that specific catabolic reactions may be inhibited by substrate analogs to simulate the pathology of certain genetic diseases in neurons and glia in tissue culture.

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 synthesis of phospholipids and glycolipids during the cell mitotic cycle of an established hamster line, NIL, has been studied. Cells were synchronized with excess thymidine and mitotically harvested by shaking. Cells were radioactively labeled for 4 h with palmitate, glucosamine, or galactose. Lipids were analyzed by thin-layer chromatography. As cells progressed through the mitotic cycle, incorporation into phospholipids increased but the fraction represented by each remained constant. Similarly, ceramide monohexoside, dihexoside, and hematoside were labeled equally in all phases. Ceramide trihexoside and tetrahexoside were labeled only during G1 and S. Ceramide pentahexoside (the Forssman antigen) shows density-dependent synthesis, accumulation, and reactivity. Ceramide pentahexoside was labeled during all phases of the mitotic cycle but the rate of incorporation decreased in S and G2. The total amount of lipid assayed immunologically in cell extracts gradually increased. Exposure of the Forssman antigen in untreated or trypsin-treated cells was studied using binding of chemically labeled antiForssman antiserum. The amount of antigen detected in trypsinized cells increased during G1 and early S but then remained constant. Mitotic cells exposed all detectable antigen. As cells progressed through the mitotic cycle, a large fraction of the Forssman antigen became cryptic.

Neurospora crassa strains labeled uniformly with ^(32)P_i and [^3H]inositol exhibit at least six phospholipid components containing ^3H when separated by paper chromatography. One of the major components is phosphatidylinositol. Other components, which account for 40 to 60% of the lipid-extractable ^3H in various strains, are stable to mild alkaline methanolysis and appear to be sphingolipids with equivalent amounts of inositol and phosphorus. The major phosphosphingolipid was purified by means of differential solubility and by column chromatography on porous silica beads. This substance contains equivalent amounts of hydroxysphinganine and hydroxytetracosanoic acid and 2 eq each of myoinositol, phosphorus, and sodium. Alkaline degradation yielded 2 eq of inositol monophosphate and periodate degradation gave a C-15 fragment. The elemental composition of this compound also fits the formulation, (inositol-P)_2-ceramide. A [^3H]inositol pulse-chase experiment carried out with an inositol-requiring mutant in exponential growth shows labeled inositol accumulating in the sphingolipid accompanied by decreased labeling in phosphatidylinositol and the acid-soluble fraction. These changes also occur when the chase is carried out during inositol starvation suggesting that degradation of phosphatidylinositol and formation of sphingolipid occurs in the absence of growth. A neutral glycosphingolipid was also obtained as a by-product of the phospholipid purification. This substance is provisionally formulated as the ceramide tetrahexoside: [(gal)_3glu]-N-hydroxytetracosonyl-hydroxysphinganine.

Conditions have been determined for the benzoylation of ceramides containing nonhydroxy and hydroxy fatty acids, and a high performance liquid chromatography system for the separation and measurement of these derivatives has been devised that is capable of good resolution and high sensitivity. These methods have been used to determine quantitatively the levels of ceramides in human tissues, and in serum and urine, and to demonstrate elevated amounts of ceramide in Farber's disease urine and tissues.

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.

Hepatic lipids of a patient with Histiocytosis X who had clinically hemolytic jaundice, intermittent fever and growth retardation and also showed histopathologically remarkable proliferation of enlarged reticular cells in liver, spleen, lymph node and bone marrow were isolated and analyzed. It was found that free fatty acids and lyso-glycerophospholipids markedly increased, whereas glycerophospholipids decreased. There was no increase in concentration of cholesterol and cholesterol esters. Relatively higher amounts of glucosyi ceramide, galactosyl ceramide, digalactosylglucosyl ceramide and hematoside seemed to be due to noticeable proliferation of enlarged reticular cells in liver. It was noted that neutral lipids and glycerophospholipids except free fatty acids gave lower percentages of unsaturated fatty acids. The fatty acid compositions of various sphingolipids contained more than 70% of longer chain fatty acids from C20 to C24.

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.

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.

About one-third of the nondeacylatable phospholipids in Saccharomyces cerevisiae have been shown to be a group of sphingolipids with a single phosphoinositol moiety. This group of lipids was obtained richly concentrated in high yield by several simple steps involving differential solubility. Four components of this group were further purified by liquid chromatography on base-treated porous silica beads. The major component of this group proved to be the novel substance, inositol phosphorylceramide, the ceramide consisting of hydroxysphinganine and a hydroxy C-26 fatty acid. Two other inositol phosphorylceramides were purified which differed from the major component in their ceramide moieties. Two glycolipids are minor members of this group of monoinositol-containing sphingolipids and one of these was purified, and had a composition consistent with its formulation as a mannosylinositol phosphorylceramide. The major inositol phosphorylceramide could be cleaved quantitatively by alkali to give an inositol monophosphate and could be cleaved by periodate to give a C-15 fragment. Periodate treatment followed by borohydride reduction and HCl hydrolysis gave a significant yield of phosphoserinol. These facts suggest strongly that the phosphoinositol moiety is attached to position 1 of the hydroxysphinganine. Data obtained with cells uniformly labeled with [3H]-inositol suggest that all significant ( g 1% lipid 3H) inositol-containing lipids in yeast have now been chemically characterized.

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 conversion of 14C-labeled fatty acylsphingosine and phosphatidyl[14C]choline to sphingomyelin could be demonstrated in lyophilized microsomes from mouse liver. Radioactive CDP-choline did not act as a choline donor. No cofactors were needed, although CDP-choline and P-choline exerted some stimulatory action. The unnatural ceramide, acetyl threo-sphingosine, could be converted to the corresponding sphingomyelin but this reaction required Mn2+ and CDP-choline. Evidence from isotopic trapping experiments was interpreted to indicate that the P-choline transfer from lecithin to ceramide did not go through free CDP-choline, choline, or P-choline. The transferase was found in kidney, lung, liver, spleen, and heart (in decreasing order of activity) but not in brain. The enzyme was inhibited by diglyceride and lysolecithin.

Radioactive glucocerebroside was formed when purified calf spleen β-glucosidase was incubated in the presence of 4-methylumbelliferyl-β-D-glucoside and (14C) labeled ceramide. The product was identified by cochromatography on thin layer plates and carrier dilution and crystallization to constant specific activity. The radioactive glucocerebroside was also a substrate for hydrolysis by this same β-glucosidase preparation employed for its synthesis resulting in the liberation of labeled ceramide. Neither methyl-β-D-glucoside nor free D-glucose were effective in producing radioactive glucocerebroside when incubated with labeled ceramide in this system.

Glucosylsphingosine has been isolated for the first time as a natural constituent from Gaucher's spleen. On thin-layer chromatography, it migrates with authentic glucosylsphingosine, yielding a positive color reaction with ninhydrin for the amino group and with alpha-naphthol-sulfuric acid for the carbohydrate residue. N-Acylation with palmitic acid gave rise to glucosylceramide, which was cleaved by purified glucosylceramide: beta-glucosidase to ceramide. Gas-liquid chromatography of the trimethylsilyl derivative showed a retention time similar to authentic glucosylsphingosine. Gas-liquid chromatographic analysis of the trimethylsilyl derivatives after methanolysis revealed the presence of only glucose and C(18)-sphingosine. Mass spectral data further supported the structural identity with glucosylsphingosine.

Four glycosphingolipids were isolated from rabbit aorta, plasma, and red blood cells. They were identified, by thin-layer chromatography and by quantitative analysis of hexose and fatty acid, as cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The rabbits had been maintained on a normal diet or on one of three high cholesterol diets for 180 days. The quantities of the glycosphingolipids and their fatty acid distributions were determined, and comparisons were made between the control and experimental animals. Aorta and plasma glycosphingolipids were more affected by the high cholesterol diets than were those from red blood cells. The effects on aorta and plasma glycosphingolipids were similar. The amount of cerebroside was increased in aorta and plasma in all animals in the experimental groups. The amount was also increased in red blood cells in rabbits from two of the experimental groups. The average fatty acid chain length was greater in the lipids from the experimental animals than in those from the control animals for all measured glycosphingolipids from aorta. The average chain length was also greater in cerebrosides from the experimental animals from all three tissues. Probably the most notable differences in the experimental animals were the increased 24:1/24:0 ratios and the increased concentrations of 24:2. These increases occurred in nearly all samples from plasma and aorta, but not in red blood cells. There was also an increase of total unsaturated fatty acids in aorta cerebrosides from the experimental animals. Except for the increase in 24:2, lard generally caused more deviation from normal than did cottonseed oil when the level of cholesterol in the diet was 1%.

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.

A lipid factor previously isolated from leukocytes and found to stimulate basophilic erythroblast formation in an in vitro system of incubated rabbit bone marrow cells has been analyzed by thin-layer chromatography, gas-liquid chromatography, and gas-liquid chromatography-mass spectrometry. The biologically active components are sphingosine ceramides of tetracosanoic and dehydrotetracosanoic acids. Tests of a series of related ceramides show a high degree of structural specificity for the C(24)-V-acyl compounds with significant but markedly lower activity of the C(22) analog. Commercially available sphingomyelin shows activity comparable to that of the tetracosanoic acid ceramide. Sphingosine and tetracosanic acid supplied in equimolar amounts have negligible activity. The results, in the context of other findings, suggest a possible supportive role of plasma ceramides and sphingomyelins in red cell maturation.

Gas chromatography-mass spectrometric identification of partially methylated aminosugars has been developed: (a) various kinds of O-methylated 2-deoxy-2-(N-methyl)-acetamidohexitols were prepared from partially O-(1-methoxy)-ethylated 2-deoxy-2-acetamidohexoses, and their gas chromatography-mass spectrometric patterns were determined; (b) permethylated glycolipids gave a satisfactory yield of 2-deoxy-2-N-methyl-amidohexoses by acetolysis with 0.5 n sulfuric acid in 95% acetic acid, followed by aqueous hydrolysis; (c) the resulting partially methylated aminosugars and neutral sugars were analyzed after borohydride reduction and acetylation according to the procedure of Lindberg and associates (Bjorndal, Lindberg and Svennson, 1967; Bjorndal, Hellerqvist, Lindberg and Svensson, 1970). This method was successfully applied to analysis of aminosugar linkages in blood group B-active ceramide pentasaccharide from rabbit erythrocytes and in Forssman antigen of equine spleen. The structure of blood group B-active glycolipid of rabbit erythrocyte was found to be Galα1 → 3Galβ1 → 4G1cNAcβ1 → 3Ga11 → 4Glc → Cer, and that of Forssman antigen to be GaNAcα1 → 3GalNAcβ1 → 3Galα1 → 4Ga11 → 4Glc → Cer.

A method is described for the qualitative and quantitative estimation of four neutral glycosyl ceramides from human plasma and erythrocytes. Total lipids extracted from 50 ml of plasma or packed erythrocytes were separated by silicic acid chromatography into neutral lipids, a fraction of mixed glycolipids that was eluted with acetone-methanol 9:1, and phospholipids. After mild alkali-catalyzed methanolysis to remove contaminants from the crude fraction of glycolipids, individual glycosyl ceramides were isolated by preparative thin-layer chromatography. The oligosaccharide portions of these lipids were characterized by cleavage with methanolic hydrogen chloride and gas chromatography of the O-trimethylsilyl methyl glycosides. It was possible to study the composition of the carbohydrate and sphingolipid base fractions in the same gas chromatographic analysis. With mannitol as an internal standard for gas chromatographic estimation of glucose, concentrations of each of the glycosyl ceramides were determined with a precision of about 10%. Recoveries of the lipids from plasma varied with the complexity of the oligosaccharide moiety and ranged from 94% with glucosyl ceramide to 71% with globoside. Concentrations of the four glycosyl ceramides in plasma and in erythrocytes were determined for samples from young, healthy males. Amounts of glycolipid as low as 0.1 micromole can be determined conveniently by this procedure.

An enzyme which catalyzes the transfer of galactose from UDP-galactose to galNAc-gal-glc-ceramide is described. The enzyme is found mainly in the nervous tissue of tadpole (Taylor and Kollros stage 17), adult frog, adult and 8 day old rat. The enzymic activity is localized in the 11,500 xg, 20,000 xg and 100,000 xg particles. The UDP-galactose: galNAc-gal-glc-ceramide from the particles by treatment with sodium desoxylcholate and Triton X-100. The pH optimum for the solubilized enyme is between 6.8 and 7.0 in cacodylate buffer, and the Km is 4.25 ×10−5 M. The enzymic reaction is proportional to time for 4 hr and to the amount of protein added. The product of the transferase reaction, using galNAc-gal-glc-ceramide. A pathway for the biosynthesis of brain gangliosides requiring UDP-galactose: galNAc-gal-glc-ceramide galactosyl transferase is proposed.

Two thin-layer chromatography systems for the separation of glycosphingolipids from total lipid extracts of erythrocytes were developed. By system I (Silica Gel HR-magnesium silicate (4:1)/tetrahydrofuran-water (5:1)), ceramide hexosides were separated from all other lipids and fractionated into mono-, di- and triglycosyl ceramides. By system II (Kieselguhr G, impregnated with a 0.60 M boric acid-0.15 M disodium tetraborate buffer (pH 7.9)/chloroform-methanol-water (65:25:4)), complex glycosphingolipids (mucolipids) were separated from all other lipids and fractionated. The red cells of nine different mammals showed no species variation in the qualitative composition of the ceramide hexosides but a marked variation in the mucolipid composition as well as in the concentration of the glycosphingolipids.

β-N-Acetylhexosaminidase A and B were partially purified from normal human liver using DEAE-cellulose column chromatography. Hexosaminidase B was also purified from the livers of patients who had died of Tay-Sachs disease. The hexosaminidase fractions were tested for their ability to hydrolyze the amino sugar moiety of synthetic substrates and of three amino sugar-containing glycolipids, GA2, globoside, and GM2. Hexosaminidase A and B had similar ability to catalyze the hydrolysis of synthetic N-acetylglycosaminides. The hexosaminidase B from the liver of a Tay-Sachs disease patient had a slightly higher Michaelis constant toward the same substrates as the control hexosaminidase B. Radioactively labeled GA2, N-acetyl-[1-14C] galactosaminyl (β 1 → 4)-galactosyl (β 1 → 4)-glucosyl (β 1 → 1)-ceramide was prepared biosynthetically. Hexosaminidase A and B from normal liver had identical Michaelis constants for the hydrolysis of GA2; whereas, the hexosaminidase B from a Tay-Sachs disease patient possessed a higher Michaelis constant. Globoside was hydrolyzed by both hexosaminidase A and B, although hexosaminidase B has a significantly lower Michaelis constant than the hexosaminidase A. Under the conditions of incubation neither hexosaminidase A nor B catalyzed the hydrolysis of the N-acetylgalactosaminyl residue from ganglioside GM2. However, in the presence of a neuraminidase preparation from Clostridium perfringens plus hexosaminidase A or B at pH 3.8 there was significant enzymatic hydrolysis of GM2 to ceramide lactose. Both GM2 and globoside were found to be excellent competitive inhibitors of GA2 hydrolysis by hexosaminidase B. These results are discussed with respect to the accumulation of these glycolipids in two GM2 ganglioside-storage diseases.

The concentration of sphingomyelin, gangliosides, glycosaminoglycans, and glycoprotein carbohydrate was elevated in brain tissue from a case of Niemann-Pick's Disease. Liver presented increased levels of cholesterol, sphingomyelin, phospholipids, gangliosides, and lactosyl ceramide. The urinary sediment contained a high concentration of sphingomyelin. The gangliosides GM3, GM2, and GD3 were elevated in brain tissue. The elevation of ganglioside GD3 was especially pronounced in liver. The two-fold increase in glycoprotein carbohydrate was due to increased levels of glycoprotein material bearing non-dialyzable, higher molecular weight, heteropolysaccharide units containing NANA, fucose, N-acetylglucosamine, galactose and mannose. These polysaccharides also differed in composition inasmuch as they appeared to be deficient in the linear external chains consisting of —N-acetylglucosaminegalactose-NANA that are attached to the internal mannose-rich core of the heteropolysaccharide unit. There was no increase in the concentration of glycoprotein material bearing the dialyzable heteropolysaccharide units of lower molecular weight and which contain predominately mannose and N-acetylglucosamine. Brain glycosaminoglycans showed a two-fold elevation. The substances which accumulated in tissues in this case of Niemann-Pick's Disease are constituents of the plasma membrane, suggesting that the block in the degradation of sphingomyelin may have the secondary effect of impeding the catabolism of other membrane constituents.

The terminal galactosyl residue of ceramide trihexoside of human erythrocytes and BHK fibroblasts and that of the ceramide trihexoside derived from "globoside" of human erythrocytes were hydrolyzed and totally converted to ceramide lactoside by an α-galactosidase isolated from fig. The enzyme showed a strict substrate specificity for α-galactosides and was completely freed from β-galactosidase activity. With the sequential application of glycosidases, the carbohydrate sequence and anomeric linkages of glycosphingolipids have been simultaneously determined. The presence of α-anomeric (equatorial) proton in these glycolipids was supported by a doublet at δ 4.6 ppm (J = 2 Hz) in the nuclear magnetic resonance spectrum of a trimethylsilyl derivative and by an absorption at 850 cm-1 in the infrared spectra. From these results and the results of methylation study, the structures of ceramide trihexoside and of globoside were determined, respectively, galactopyranosyl-α-(1 → 4)-galactopyranosyl-β-(1 → 4)glucopyranosyl-(1 → 1)ceramide, and 2-acetamido-2-deoxy-galactopyranosyl-β-(1 → 3)galactopyranosyl-α-(1 → 4)galactopyranosyl-β-(1 → 4)glucopyranosyl(1 → 1)ceramide.

A galactosyltransferase that catalyzes the synthesis of galactocerebrosides (galactosylceramide) containing α-hydroxy fatty acids linked to the amino group of the sphingosine moiety has been isolated from 19- to 20-day-old embryonic chicken brain. The enzyme catalyzes the following reaction: UDP-galactose-14C + ceramide → 14C-galactocerebroside (+ UDP) Several ceramides (N-acylsphingosines) were tested as galactose acceptors, including those containing C16 and C18 saturated and unsaturated fatty acids, but the only active ceramide preparation was one containing a mixture of the α-hydroxy fatty acids 2-hydroxy C18:0 (97%) and 2-hydroxy C18:1 (2%). The reaction was stimulated by Mg++, Mn++, and Ca++. UDP-Galactose-14C could not be replaced by 14C-labeled galactose, galactose-1-P, or ADP-galactose; the Km values were 1.1 x 10-4 and 0.4 x 10-4 m for ceramide and UDP-galactose, respectively. The galactosyltransferase that catalyzes the synthesis of cerebroside is different from two other galactosyltransferases previously obtained from embryonic chicken brain, which are concerned with two steps in the synthesis of gangliosides. The UDP-galactose:ceramide galactosyltransferase activity was low in homogenates of brains from young chick embryos; in the period between 15 and 20 days of embryonic development, there was a rapid increase in activity (per mg of protein). Maximal activity was detected in homogenates from 19- to 20-day-old embryos, and was 10- to 20-fold greater than the activity in young embryonic brain. The results are in accord with the suggestion that α-hydroxy fatty acid-containing cerebrosides are synthesized via the following pathway: sphingosine → ceramide → cerebroside.

A particulate preparation from embryonic chicken brain was previously described that catalyzed the transfer of sialic acid from CMP-N-acetylneuraminic acid to galactosyl → glucosyl → ceramide to give a monosialoganglioside, sialyl → galactosyl → glucosyl → ceramide (Reaction A). In the present studies, two monosialogangliosides served as acceptors in similar reactions, giving the corresponding disialogangliosides (Reactions B and C). Reaction B yields the major disialoganglioside of human brain; the acceptor is a ceramide-pentasaccharide containing a terminal galactose residue, and the sialic acid is transferred to this galactose unit. Reaction C involves the synthesis of N-acetylneuraminyl → N-glycolylneuraminyl → galactosyl → glucosyl → ceramide, where the terminal sialic acid is linked at C-8 to the N-glycolylneuraminic acid. The sialyl → sialyl unit occurs in brain tri- and tetrasialogangliosides, and in the minor disialogangliosides. Reaction B represents the last step in the synthesis of the major disialogangliosides from ceramide according to a proposed pathway, while Reaction C represents a branch in the pathway, and may be a key step in the synthesis of the minor disialogangliosides, and of the tri- and tetrasialogangliosides. Kinetic studies indicate that Reactions A, B, and C are catalyzed by different sialyltransferases in the particulate fraction.

Concentrations of four neutral glycosyl ceramides were determined in plasma and erythrocytes from nine hemizygous patients with Fabry's disease (a hereditary glycolipid lipidosis), from the sister of one of the patients, and from the heterozygous mother of another one. The concentration of a trihexosyl ceramide, galactosylgalactosylglucosyl ceramide, was elevated in plasma from the patients about threefold above the normal mean level, and the amount of this lipid was also increased in plasma from the two female relatives. The concentrations of glucosyl ceramide and lactosyl ceramide in plasma were slightly less than normal in the affected males, while globoside or a similar tetrahexosyl ceramide was slightly higher than normal. All but one of the Fabry patients had significantly less globoside in the red cells, as compared with the normal range, although the red cell concentration of trihexosyl ceramide was normal in all of the patients. Thus the concentration of the lipid that accumulates in the tissues in Fabry's disease is elevated in plasma but not in red cells, whereas in Gaucher's disease the accumulating lipid (glucosyl ceramide) is elevated in both plasma and red cells.

A glucosyltransferase that catalyzes the synthesis of glucosylceramide (glucocerebroside) from ceramide (N-acylsphingosine) and UDP-glucose was isolated from 13- to 14-day-old embryonic chicken brain. The reaction is the following: Ceramide + UDP-glucose → glucosylceramide (+ UDP) Ceramide was the most effective lipid acceptor, and a variety of potential donors could not replace UDP-glucose. The Km values for ceramide and UDP-[14C]glucose were 0.08 mm and 0.12 mm, respectively. Metal ions did not stimulate the reaction, and it was not inhibited by ethylenediaminetetraacetate. The particulate preparation of UDP-glucose:ceramide glucosyltransferase showed an absolute requirement for detergent, and optimal activity was obtained with a mixture of Cutscum and Triton X-100. The glucosyltransferase was detectable in brains from chick embryos as early as 7 days old; maximal activity was detected between 13 and 17 days of embryonic development. The enzyme catalyzes the first step in the synthesis of gangliosides.

Splenic cholesterol, and glucosyl, lactosyl, trihexosyl, and sialyl lactosyl ceramides were studied in developing normal rats from birth to 96 days of age. Total lipid, extracted from pooled organs for each age group in the study, were subjected to mild alkaline hydrolysis and separated into purified glycolipid fractions in high yield, by a series of silicic acid column and thin-layer chromatographic procedures. Enzymatic and colorimetric methods were applied to quantitative analysis of each splenic glycosphingolipid fraction, and the fatty acids were analyzed by gas–liquid chromatography. Glycosphingolipid content in the total spleen increased over the period of the study. The most rapid increase occurred during the third and fourth weeks. There was also a 2.5-fold change in the concentration (per gram of tissue) of glucosyl and sialyl lactosyl ceramides, the principal glycosphingolipids of rat spleen. In contrast, cholesterol concentration increased only slightly. A precisely equimolar ratio of glucosyl and sialyl lactosyl ceramide was observed throughout all stages of splenic development. Fatty acid compositions of these two lipid fractions were always significantly different.

The tenth case of Farber's lipogranulomatosis is reported. The clinical and pathologic features closely resemble those previously described except that in this patient the macula was also involved, there was nerve cell loss and neuronal storage in the cerebral cortex, and there was a strong family history of rheumatoid arthritis. The foam cells within the granulomas as well as certain of the neurons contained periodic acid-Schiff positive cytoplasmic material with the histochemical properties of an acidic glycolipid. Biochemical studies demonstrated an excess of gangliosides, roughly in proportion to the accumulation of the periodic acid-Schiff-positive material. The excess gangliosides were found to consist mainly of hematoside, the visceral ganglioside. Lymph nodes, liver, kidney and lung contained a ten- to sixtyfold excess of free ceramide, and similar very high levels of free ceramide were present in a subcutaneous nodule. In rats, subcutaneous injection of ceramide produced granulomas which resembled those found in the patients with the lipogranulomatosis syndrome. It is suggested that the lipogranulomatosis syndrome represents an inborn error of lipid metabolism. Its enzymatic basis is as yet undetermined.

Hammarstrom (6) has described a method to prepare ceramides by direct coupling of the long-chain bases and fatty acids in the presence of a mixed carbodiimide. No 0-acyl derivatives are formed. Yields of ceramide were 60-75% based on long-chain base and 30-37.5% based on fatty acid. N-Hydroxysuccinimide esters of fatty acids are stable crystalline compounds which are easily prepared in high yield (7). They have been used for the synthesis of N-acyl amino acids (7) and fatty acyl co~ (8). A~ described in this report, they can also be used for the direct Nacylation of sphingenine or sphinganine. The reaction proceeds in high yield from small amounts of starting material. The product ceramides are pure after one recrystallization. Materials. trans-2-Hexadecenoic acid was synthesized by the method of Shapiro, Segal, and Flowers (9). CERAMIDES have been implicated as intermediates in the Palmitic, stearic, and lignoceric acids were purchased biosynthesis of sphingomyelin and cerebrosides and are from Sigma Ckmical CO., St. Louis, Mo. [1-'4c1important constituents of all sphingolipids. '4C-labeled Palmitic and [ 1 -'4C]stearic acids were purchased from ceramides are useful as substrates for enzyme studies and New England Nuclear, Boston, Mass. N-Hydroxystudies of sphingolipid metabolism in the whole animal. succinimide (Aldrich Chemical CO., Milwaukee, Wis.) Some previously reported syntheses of ceramides was recrystallized from ethyl acetate before use. Dihave used appropriate fatty acid chlorides to acylate cyclohexylcarbodiimide was obtained from Pierce Chembenzoyl sphingenine or psychosine (1, 2). The inter- ical C0.j Rockford, 111. DL-Sphinganine (DL-erJ'thrO-173mediate compounds formed are then hydrolyzed to dihydroxy-2-aminooctadecane) was from Nutritional Bioproduce the desired ceramide. Direct acylation of chemical Corp., Cleveland, Ohio. DL-Sphingenine (DLsphingenine or sphinganine by an acid chloride can er~thro-trans-l,3-dih~drox~-2-amino-4-octadecene)

Phytoglycolipid has been isolated for the first time from plant leaves (Phaseolus vulgaris). The purified product (almost identical with the phytoglycolipid isolated from flax seed) was a ceramide attached through phosphate diester linkage to an oligosaccharide, which consisted of the usual trisaccharide unit (inositol, hexuronic acid, hexosamine) to which were attached mannose, galactose, and arabinose. The major fatty acids were the saturated 2-hydroxy C(22), C(24), and C(26) acids; the major long-chain bases were dehydrophytosphingosine (d-ribo-1,3,4-trihydroxy-2-amino-8-trans-octadecene) (53%) and phytosphingosine (d-ribo-1,3,4-trihydroxy-2-amino-octadecane) (32%). A ceramide and a cerebroside were also isolated. In the ceramide the major fatty acids and the major long-chain bases were the same as in the phytoglycolipid. In the cerebroside, the fatty acid composition was similar to that in the ceramide and phytoglycolipid, but the long-chain bases consisted of dehydrophytosphingosine and phytosphingosine (7:1) with a substantial amount of unidentified long-chain base. The sugar component was glucose.

The galactoside bond in cerebroside was found to be cleaved by an enzyme in rat and pig brain. Emulsified stearoyl-1% psychosine was used as the substrate and the extent of cleavage was studied by isolating and counting the stearoyl sphingosine (ceramide) formed. The reaction products, cer- amide and galactose, were characterized by column and thin- layer chromatography. Cerebroside containing gala~tose-~H was also used to show liberation of galactose. Cholic acid was found to be required for activation of the enzyme, which has a pH optimum of 4.5. Similar cerebrosidase activity was found in spleen, kidney, and lung of rat; liver and heart showed very slight activity. The partially purified enzyme from pig brain also formed ceramide from ceramide lactoside, ceramide glucoside, and cerebronoyl psychosine. The enzyme was active toward o- nitrophenyl galactoside and could be fractionated by Sephadex chromatography into a fraction active toward the nitrophenyl galactoside only and a fraction active toward both this sub- strate and ceramide galactoside. Human spleen, normal and Gaucher, exhibited cerebrosidase activity.

Homogenates of aortae of rats, rabbits, dogs, guinea pigs and of human umbilical arteries were investigated for sphingomyelin cholinephosphohydrolase activity, using biosynthetically labelled, rat liver sphingomyelin. The products of hydrolysis were identified as ceramide and phosphorylcholine. The enzymic activity was found to be dependent on the presence of Triton, had a pH optimum of 5.1 and remained in the supernatant of homogenates prepared in 0.4% Triton even after centrifugation for 1 h at 100000 ×g. The arterial enzyme hydrolyzed both endogenous and exogenous sphingomyelin to the same extent. The species investigated varied in the sphingomyelin cholinephosphohydrolase activity of their aortae (relative to the DNA content) in the ascending order: rabbit < guinea pig < dog < human (umbilical) artery < rat. The possible role of the change in sphingomyelin cholinephosphohydrolase activity of arteries in the proneness to accumulate sphingomyelin with age and atherosclerosis is discussed.

An enzyme that hydrolyzes sphingomyelin to ceramide and phosphorylcholine has been purified from the growth medium of Clostridium perfringens. The activity of the enzyme is stimulated about 2-fold by magnesium chloride and by diethyl ether. The activity of the enzyme is completely inhibited by 10-3 m ethylenediaminetetraacetic acid or 10-3 m calcium chloride. Lysolecithin and dipalmitoyl lecithin are hydrolyzed at about 10% of the rate of sphingomyelin. No hydrolysis of phosphatidylserine, phosphatidyl-ethanolamine, or phosphatidylinositol is detected. The enzyme does not catalyze the exchange of phosphorylcholine-14C into sphingomyelin.

Studies have been continued on the synthesis of glycolipids by the NIL 2 line of hamster cells. Several clones were isolated from this line. These clones vary in morphology, saturation density, and glycolipid composition. Contrary to expectation there was no correlation between saturation density and complexity of the glycolipid pattern. In fact, the clone with the highest saturation density was the only one to show the complete set of glycolipids found previously in NIL 2 cells. All untransformed NIL clones show an increase in the level of “higher” (more than two saccharides/ceramide) glycolipids as the cells approach confluence. In the line containing all three “higher” neutral glycolipids, all three increased as cells approached saturation density. In the line containing only hematoside, this compound increased at confluence. Cells transformed by hamster sarcoma virus or polyoma virus showed no density-dependent glycolipid synthesis.

Column chromatography (on cellulose, silicic acid, and Florisil) and thin-layer chromatography were em- ployed for the separation and purification of lipid fractions from normal and Gaucher spleens. A new hydrolysis procedure, followed by paper chromatography, was used for identification of sugar moieties. A nonhydrolytic combined colorimetric pro- cedure, with anthrone and orcinol, was used for the estimation of glucose and galactose separately in glycolipids. The limita- tions of this method were examined. Spleens from two control subjects and three patients with Gaucher's disease have been examined in detail. In all Gaucher spleens, the predominant feature was the massive acccumulation of glucocerebroside ; neutral ceramide oligohexoside levels were probably within the normal range, as were other neutral lipids and phospholipids. In one case examined for gangliosides. these were increased twentyfold. One Gaucher spleen, in which others had reported that the stored "cerebroside" contained predominantly lactose as the saccharide moiety, has been examined in detail and it has been established that the stored material was, in fact, glucocerebro- side, ceramide lactoside levels not being significantly elevated. In a further nine cases glucose was the major sugar detected in the splenic lipids.

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.

The optimal conditions for the incorporation of acetate-1-14C and palmitic acid-1-14C into platelet lipids have been described. In buffer incubations with acetate there was a sharp pH optimum at 6.8; in plasma incubations, there was a broad pH optimum between 6.8-7.4. Maximal incorporation of acetate occurred at a final concentration of 1.5 mmoles/liter. In buffer, no labeled lipids were released from platelets into the medium. In plasma, 40% of newly formed lipids was recovered in the plasma. 75% of the incorporated acetate could be recovered in ceramide, lecithin, and free fatty acids. Platelet fatty acids were formed both by de novo synthesis and chain elongation. The fatty acids formed by de novo synthesis exchanged with plasma free fatty acids. In buffer incubations no turnover of newly labeled lipids occurred, but in the plasma incubations exchange of newly labeled lecithin with plasma lipids was demonstrable. Palmitic acid-1-14C added to plasma was incorporated into platelet lipids. The distribution among the lipid classes of palmitate taken up from plasma was the same as that of palmitate formed intracellularly by de novo synthesis.

—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.