The characterization of sphingolipids from neurons and astroglia of immature rat brain

Abstract— 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.     

The fatty acid composition of sphingolipids from bovine CNS axons and myelin

Abstract— Cerebrosides, sulphatides and sphingomyelin were isolated from bovine CNS myelin and from myelin-free axons derived from myelinated axons. The fatty acid composition of each sphingolipid was determined by gas-liquid chromatography of the fatty acid methyl esters. In each case the fatty acids of the axonal sphingolipids were of shorter average chain length than those from the corresponding myelin lipids. These differences, however, were small and the fatty acids of the axonal cerebrosides and sulphatides were similar in average chain length to those reported previously for bovine myelin. The principal unsubstituted acid of both cerebroside and sulphatide from axons was 24: 1, with the total long chain acids (> C₁₈) amounting to 80 and 85 per cent, respectively. The corresponding figures for myelin galactolipids were 94 and 95 per cent long chain acids. The principal α-hydroxy acid of both axonal galactolipids was 24 h:0, with cerebroside having 80 per cent and sulphatide 92 per cent long chain acids, compared to the figures of 87 and 97 per cent for the corresponding myelin lipids. In axonal sphingomyelin the major acid was 18:0 (compared to 24:1 in myelin) and the long chain acids were 61 per cent of the total vs 76 per cent of the total for myelin sphingomyelin. The non-identity of axonal and myelin sphingolipid fatty acids substantiates the belief that they are intrinsic axonal constituents. These findings do not rule out the possibility of a close metabolic relationship between the sphingolipids of the axon and its myelin sheath.     

FATTY ACIDS AND LONG CHAIN BASES OF VERTEBRATE BRAIN GANGLIOSIDES

Abstract— Fatty acid and long-chain base composition of gangliosides from brains of animals belonging to various vertebrate classes (fishes, amphibia, reptiles, birds and mammals) was studied by gas-liquid chromatography. Stearic acid was found to be the main fatty acid everywhere. Brain gangliosides of cold-blooded animals contain lesser amounts of stearic and higher amounts of palmitic and monoenoic acids as compared to those of mammals. Long-chain bases were separated as trimethylsilyl derivatives. Large amounts (23-48 per cent) of long chain bases with 20 carbon atoms were found in brain gangliosides of mammals while in those of cold-blooded animals they constitute 3-12 per cent of the total. The comparison of the composition of gangliosides with that of cerebrosides and sulphatides indicates, that the fatty acid and long chain base composition of gangliosides from mammalian brain differs from that of other glycosphingolipids, whereas in brains of cold-blooded animals they are much more similar.     

Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C_18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C_18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration.     

Brain lipids of a case of juvenile Niemann-Pick disease

—Lipids of frontal lobe grey and white matter were examined in parallel from a normal and a diseased child (M. Niemann-Pick), both nine years of age. In the grey matter of the pathological case the following changes, although small, were found: a slight increase in all phospholipids and decreased values for nervonic acid in cerebrosides and for hydroxy fatty acids in sulphatides. White matter seemed much more affected by the disease: water content was about 6 per cent higher which corresponds to an approx. 20 per cent loss of dry substance compared with the normal brain. Further increases were observed in ‘ganglioside’ fraction and in all phosphatides. Cerebroside and sulphatide levels appeared decreased owing to destruction of myelin. In all of the glycerophosphatides oleic acid portions were lowered whereas in sphingolipids mainly nervonic acid values were reduced. Aldehyde content of both tissues seemed lowered in the disease, however, changes in composition were observed only in white matter, where the stearaldehyde portion of ethanolamine glycerophospholipid increased at the expense of palmitaldehyde and oleinaldehyde.     

GANGLIOSIDES OF HUMAN MYELIN: SIALOSYLGALACTOSYLCERAMIDE (G7) AS A MAJOR COMPONENT

Abstract— Gangliosides were isolated from purified human myelin in a yield of 62 μg of lipid-bound sialic acid per 100 mg of dry myelin. Sialosylgalactosyl ceramide (G₇) was found to be a major component of the ganglioside fraction, amounting to 15 per cent of the total sialic acid. It accounted for 10 per cent of lipid-bound sialic acid in adult human white matter, making it the third most abundant ganglioside on a molar basis. These results were obtained with an improved method for isolating total gangliosides in high yield, by employing DEAE-Sephadex column chromatography. Myelin from other mammalian species had considerably less G₇, and there were also indications of maturational changes. Both 2-hydroxy and unsubstituted fatty acids were components of the ceramide unit, in a ratio of 3:2, respectively. The overall fatty acid pattern was very similar to that for myelin cerebroside and sulphatide. Long-chain bases included only C₁₈ species, with sphingosine predominating (>90 per cent). These observations suggest a metabolic relationship between G₇ and either cerebroside or sulphatide.     

Characterization of neutral sphingolipids from chicken erythrocytes

The neutral sphingolipids from chicken erythrocytes were characterized. The total concentration of neutral sphingolipids was found to be 480 nmol/g of dry stroma. They were isolated and purified by droplet counter-current chromatography, Iatrobeads column chromatography, and preparative thin-layer chromatography. The major neutral sphingolipids were free ceramide, ceramide monohexoside, ceramide dihexoside, and ceramide pentahexoside, which represented 43%, 23.5%, 10.0%, and 3.6% of the long chain bases, respectively. Thus, free ceramide was the most abundant neutral sphingolipid in chicken erythrocytes. Ceramide monohexoside was composed of more galactosylceramide than glucosylceramide. Galabiosylceramide was found in the ceramide dihexoside fraction together with lactosylceramide. Ceramide pentahexoside was a Forssman glycolipid. There were two groups of neutral sphingolipids; one had mainly C16 fatty acid and the other had C22 and C24 fatty acids. In both groups sphingosine (d18:1) was predominant as a long chain base. 2-Hydroxy-C16 fatty acid was a major component of one of the ceramide monohexosides.     

PELIZAEUS-MERZBACHER DISEASE: BRAIN LIPID AND FATTY ACID COMPOSITION

Abstract— Biochemical analysis of the leukodystrophy brain from a case of Pelizaeus-Merzbacher disease, classical type, was performed. A decrease in the amount of solid material present was found. The lyophilized brain weight was reduced to 76% of normal with a slightly greater decrease in the amount of extractable lipid. Total myelin was diminished to 7% of normal. Among specific lipids plasmalogens were present in slightly lowered amounts. Cerebrosides and sulphatides were drastically reduced to 8% of normal, whereas sphingomyelin was less severely affected. Fatty acids from phospholipids were close to normal, only enols being slightly diminished. Analysis of pure cerebrosides and sulphatides revealed that the a-hydroxylated compounds as well as very long chain fatty acids (over C₁₈, especially C₂₃ to C₂₆) were greatly reduced. For chain lengths over C₁₈, the ratio of leukodystrophy fatty acid to normal fatty acid was close to 10%. The defect in very long chain fatty acids is estimated at 99.2% in total brain.
Thus, we have found a marked decrease in the amount of very long chain fatty acids and a less marked decrease in sphingolipids. The reduced amount of these acids appears to be partially offset by an increase in the amount of medium-chain fatty acids in sphingolipids. We conclude that one aspect of Pelizaeus-Merzbacher disease may be a defect in the synthesis of myelin very long chain fatty acids (as these acids are far much reduced than any other myelin molecule).     

Rapid turnover of sphingosine synthesized de novo from [14C]serine by Chinese hamster ovary cells

It has been hypothesized that complex sphingolipids may serve as another "lipid second messenger" system via their hydrolysis to free sphingosine, which inhibits protein kinase C and affects multiple cellular functions. To investigate sphingolipid turnover, Chinese hamster ovary cells were pulse labelled with [14C]serine and the [14C]sphingosine in cellular sphingolipids was determined over time. Much of the radiolabelled sphingosine was initially seen in ceramides and was incorporated into sphingomyelin during the 5-hour chase. A major portion of the radiolabel that was initially seen in other sphingolipids disappeared over time. Overall, about half of the total long-chain bases made during this pulse were degraded within 2 to 5 h, depending on the method of analysis. Hence, a substantial portion of the sphingosine synthesized de novo by these cells is turned over fairly quickly. Since the doubling time of these cells is 12 h, this rapid turnover may reflect the remodelling of the cell surface, or the utilization of the free sphingosine derived from sphingolipid turnover, as part of the control of cell growth and division.     

Cerebrosides of<Emphasis Type="Italic"> Candida lipolytica</Emphasis> yeast

Candida lipolytica yeast was grown batchwise on glucose medium. Cerebrosides were isolated from the sphingolipid fraction of total lipids using column chromatography and separated into two compounds by high-performance thin-layer chromatography. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterised by a predominance of saturated fatty acids and by a high proportion of fatty acids with 16 carbon atoms. The dominant fatty acid was h16:0. The principal long-chain base components of both cerebroside species were trihydroxy bases, 18- and 20-phytosphinosine. The unique characteristic of cerebrosides was the presence of a high proportion of sphingosine (one-fourth of the total long-chain bases), which is a common characteristic of mammalian sphingolipids and rarely occurs in yeast cerebrosides. The ceramide moiety profile of cerebrosides is similar to that of epidermal ceramides, which implies a possibility for their application in care cosmetics.     

Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s)

The de novo biosynthesis of sphinganine and sphingosine was studied using LM cells incubated with [14C] serine in serum-free media. Most of the radiolabeled long-chain bases were initially found in dihydroceramides (as sphinganine) and the proportion appearing in complex sphingolipids (as sphingosine) increased over time. Since free long-chain bases were not detected (although formation of 3-ketosphinganine, the first condensation product of serine and palmitoyl-CoA, could be demonstrated in vitro), it appears that the first step is rate-limiting for dihydroceramide biosynthesis. The kinetics suggested that after N-acyl-sphinganines were formed they were dehydrogenated to N-acylsphingosines. No evidence was found for the formation in vivo or in vitro of the putative intermediates of the direct biosynthesis of sphingosine from sphinganine (i.e. 3-ketosphingosine and free sphingosine). The conversion of N-acylsphinganines to N-acyl-sphingosines was confirmed by incubating cells with [14C] serine followed by unlabeled serine, which resulted in a rapid increase in the sphingosine-to-sphinganine ratio in amide-linked sphingolipids during the chase. These findings are most consistent with a pathway for long-chain base biosynthesis in which N-acyl-sphinganines are first synthesized by LM cells and the 4-trans-double bond is added to this or subsequent products, as opposed to the most cited pathway wherein sphingosine is made directly from sphinganine.     

Sphingolipid uptake by cultured cells: complex aggregates of cell sphingolipids with serum proteins and lipoproteins are rapidly catabolized

Human fibroblasts, rat neurons, and murine neuroblastoma cells, cultured in the presence of fetal calf serum, were fed with [1-(3)H]sphingosine to radiolabel sphingolipids. The fate of cell sphingolipids, the release of sphingolipids in the culture medium, the interaction of sphingolipids with the proteins and lipoproteins of fetal calf serum, and the fate of sphingolipids taken up by the cells were investigated. For this latter purpose, the culture medium containing radioactive sphingolipids was delivered to nonlabeled cells. The presence of tritium at position 1 of sphingosine allowed us to follow the extent of sphingolipid catabolism by measuring the production of radioactive phosphatidylethanolamine and proteins by recycling the radioactive ethanolamine formed during sphingosine catabolism and the production of tritiated water. We confirmed that in cells the recycling of sphingosine occurred to a high extent and that only a minor portion of cell sphingolipids was catabolized to the small fragments of ethanolamine and water. Cell sphingolipids were released in the culture medium, where they formed large lipoproteic aggregates at a rate of about 12% per day. Released sphingolipids were taken up by the cells and catabolized to the sphingosine and then to ethanolamine, and recycling of sphingosine was not observed. This suggests that in the presence of fetal calf serum in the culture medium, exogenous sphingolipids directly reach the lysosomes, were they are entirely catabolized. Thus, the trafficking of sphingolipids from cells to the extracellular environment and from this to other cells does not allow the modification of the plasma membrane composition.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

THE FATTY ACID COMPOSITION OF PHOSPHOLIPIDS AND GLYCOLIPIDS IN JIMPY MOUSE BRAIN

Abstract— Phospholipids and sphingolipids from brains of normal and Jimpy mice were isolated in a pure form by thin-layer chromatographic procedures. The fatty acid composition of the major phospholipids, i.e. ethanolamine glycerophospholipids, serine glycerophospholipids, choline glycerophospholipids and inositol glycerophospholipids, as well as sphingomyelin, cerebrosides and sulphatides was determined by gas-liquid chromatography. A specific fatty acid pattern for each of the four glycerophospholipids was found. The fatty acid composition of inositol glycerophospholipid, which has not previously been studied in mouse brain, was characterized by a high concentration of arachidonic acid. After 16 days of age, fatty acid analysis showed definite differences between the phospholipids from normal and mutant brains. A small increase of polyunsaturated fatty acids in glycerophospholipids of ethanolamine, serine and choline from the Jimpy central nervous system was found, which has been explained by the myelin deficiency. Sphingomyelin, cerebrosides and sulphatide analyses showed a wide distribution of saturated and mono-unsaturated fatty acids in both normal and mutant mice. A reduction in the amount of long-chain fatty acids was demonstrated in mutant brain sphingolipids; in sulphatides and cerebrosides, the amount of non-hydroxy fatty acids was reduced to a greater extent than in sphingomyelin. The distribution of fatty acids in sphingolipids from the myelin and microsomal fractions was also investigated in both types of mice. Cerebrosides were characterized by a high content of long-chain fatty acids in myelin as well as in microsomes. Sulphatides and sphingomyelin, on the other hand, showed a higher content of medium-chain fatty acids in microsomes than in myelin. In the mutant brain, the amount of long-chain fatty acids was reduced in both subcellular fractions. The deviation from normal in the pattern of fatty acid distribution in Jimpy brain is discussed in relation to the current concepts of glycolipid biosynthesis.     

Recycling of sphingosine is regulated by the concerted actions of sphingosine-1-phosphate phosphohydrolase 1 and sphingosine kinase 2

In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C(6)-ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B(1), an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.     

Efflux of sphingoid bases by P-glycoprotein in human intestinal Caco-2 cells

The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 microM digoxin for 48 h caused up-regulation of multidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.     

Inhibition of Ca2+ release channel (ryanodine receptor) activity by sphingolipid bases: mechanism of action

Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca2+ release channel. Three lipids, N,N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [3H]ryanodine binding by greatly increasing the rate of dissociation of bound [3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [3H]ryanodine binding. These bases also decreased the activity of the Ca2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO4 and C6 to C18 ceramides of sphingosine had no significant effect on [3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4-5 decreased the ability of the sphingolipid bases to inhibit [3H]ryanodine binding 2-3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca2+ release channel and as a class possess a common mechanism of inhibition.     

High-performance liquid chromatographic determination of sphinganine and sphingosine in serum and urine of subjects from an endemic nephropathy area in Croatia

Endemic nephropathy (EN) is a chronic renal disease present as an endemic in Brodska Posavina, Croatia. The aim of the study was to assess the possible role of fumonisins, i.e., mycotoxins produced by Fusarium moniliforme, as causative agents for EN. Fumonisins inhibit ceramide synthase, the enzyme of de novo synthesis of sphingolipids, which leads to an increase in the sphinganine/sphingosine ratio. In the present study, a modified method has been used for the determination of the sphinganine/sphingosine ratio in human serum and urine of healthy subjects and EN patients from the endemic area. Free sphingoid bases, sphinganine and sphingosine, were obtained by base hydrolysis. Afterwards, precolumn ortho-phthaldialdehyde derivatisation, HPLC separation and quantification by fluorescence detection were performed. The results thus obtained pointed to a sphingolipid metabolism impairment, which may have been induced by fumonisins or fumonisin-like mycotoxins. As statistically significant differences were recorded in the subjects not yet affected with EN, an impairment in the metabolism of sphingolipids might be considered as an early indicator of EN.     

The total syntheses of D-erythro-sphingosine, N-palmitoylsphingosine (ceramide), and glucosylceramide (cerebroside) via an azidosphingosine analog.

The total synthesis of D-erythro-sphingosine (9) was performed by a chirospecific method starting from D-galactose via an azidosphingosine intermediate to give highly homogeneous (>99.9% C18:1) sphingosine base (9) which contained no observable olefin isomerization by product and was demonstrated to be optically pure by a novel method utilizing Mosher's acid. Ceramide (10) was prepared from this sphingosine (9) with highly homogeneous (99.8% C16:0) palmitic acid by two methods. The cerebroside glucosylceramide (23) was the next sphingolipid in this series to be synthesized in a highly homogeneous form. These three sphingolipids are currently being used for biophysical studies of the structures of their hydrated bio-molecular assemblies.     

BIOSYNTHESIS AND BIODEGRADATION OF RAT BRAIN GANGLIOSIDES STUDIED IN VIVO

Abstract— Metabolic relationships between the four major brain gangliosides, GM1, GD1a, GDlb and GT1 were studied in vivo . Labelled acetate and glucosamine were injected intracerebrally into 6–12-day-old rats and the radioactivities of the cerebral gangliosides were analysed. Radioactivity from [³H]acetate was determined in sialic acid, sphingosine and stearic acid and from [1-¹⁴C]glucosamine in hexosamine and sialic acid. The gangliosides were labelled in proportion to their pool size. In 6 day-old rats the labelling was approx. 30 per cent lower in the sialidase-stable sialyl group than in the labile one. When the brain gangliosides were labelled in 12-day-old rats, however, the specific activities of sialidase-labile and stable sialyl groups were the same at 0.5 months after the injection of precursors and disappeared at the same rate. The results indicate that at the age of 6 days a small pool of monosialogangliosides exists, which is converted to di- and trisialogangliosides. The degradation of gangliosides was studied by following the radioactivities in sphingosine and stearic acid from 2 to 6 months after the injection of labelled acetate. The specific activities of sphingosine and stearic acid decreased simultaneously at the same rate in all the four major gangliosides. The specific activity of stearic acid was the same in total brain lipids as in gangliosides. The half-lives for the degradation of the gangliosides were age-dependent and estimated to 60 days in adult rats. They were much shorter in younger rats but no reliable figures could be determined.