Molecular Species of Free Ceramides in Aspergillus oryzae

Free ceramides isolated from A. oryzae were fractionationed into three groups by thin-layer chromatography on silica gel G according to degree of hydroxylation of the molecules. Each group was converted to trimethylsilyl ether derivatives, which were analyzed by gas-liquid chromatography-mass spectrometry for the structure of the molecular species. As a result, the representative molecular species of ceramides were characterized as N-lignoceroyl-phytosphingosine (1%), N-2-hydroxylignoceroyl-phytosphingosine (31%) and N-2,3-dihydroxylignoceroyl-phytosphingosine (31%).     

Ceramides, sphinganine, sphingosine and acid sphingomyelinases in the human umbilical cord blood.

Ceramides, sphingosine, sphinganine, as well as Zn (++)-dependent and Zn (++)-independent acid sphingomyelinase are present in the plasma of adults. The aim of the present study was to examine the concentrations of these compounds and activities of both enzymes in the umbilical cord blood in humans. Twenty-two women with uncomplicated term pregnancy volunteered for the study. Blood was taken from the umbilical cord artery and from the antecubital vein of the mother immediately after delivery. Free ceramides were isolated by thin layer chromatography, and their fatty acids were identified and quantified by gas-liquid chromatography. Free sphingosine and sphinganine concentrations were determined using high-performance liquid chromatography. Acid Zn (++)-dependent and Zn (++)-independent sphingomyelinase activity was measured using sphingomyelin [choline-methyl-14C] as a substrate. We found that the compounds examined are present in the umbilical cord blood. The total fatty acid-containing ceramide concentrations in fetal blood were lower than in mother's blood. The mean sphingosine and sphinganine concentrations in the fetal and maternal serum were similar. The examined enzymes were present in the fetal serum, and their mean activity did not differ from that in the mother. In conclusion, we have shown the presence of ceramides, sphingosine and sphinganine and both isoforms of acid sphingomyelinase in the human fetal cord blood. They are most likely the product of the fetus itself.     

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.     

Free sphingosines in porcine epidermis

Sphingosines and phytosphingosines serve as intermediates in the synthesis of ceramides and glucosylceramides, which are prominent components of mammalian epidermis. In the present study, we have investigated the possibility that free sphingoid bases also may be present in epidermal tissue. Samples of pig epidermis were trypsinized to separate the stratum corneum from the unkeratinized portion of the epidermis. After drying, the lipids were extracted and analyzed by thin-layer chromatography using ninhydrin to detect free amino groups. Both the stratum corneum and the unkeratinized epidermal material contained a ninhydrin-positive material with the same mobility as the sphingosine standard. Quantitation of the chromatograms by photodensitometry indicated that free sphingosine bases account for 0.44% by weight of the total stratum corneum lipid and 0.09% of the lipid in the viable portion of the epidermis. To further identify this material, it was treated with 1-fluoro-2,4-dinitrobenzene, which resulted in the production of an intensely yellow N-2,4-dinitrophenyl derivative with the same mobility as N-2,4-dinitrophenylsphingosine on thin-layer chromatography. Oxidation of the isolated dinitrophenyl derivative with lead tetraacetate produced a mixture of aldehydes which were analyzed by gas-liquid chromatography. This analysis indicates that the free sphingoid bases from the stratum corneum consist of a mixture of mainly 16- through 20-carbon sphingenines and sphinganines, the most abundant components being d17:0, d17:1, d18:1 and d20:1. The production of these free sphingosine bases may be significant in regulating epidermal differentiation.     

Stimulation of erythroblast maturation in vitro by sphingolipids

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.     

A new procedure for synthesis of 3-keto derivatives of sphingolipids and its application for study of fatty acid composition of brain ceramides and cerebrosides containing dihydrosphingosine of sphingosine

3-Keto derivatives were prepared in good yield by the oxidative procedure with 2,3-dichloro-5,6-dicyanobenzoquinone from N-acetyl sphingosine, N-palmitoyl sphingosine, N-lignoceroyl sphingosine, and N-lignoceroyl psychosine. None of these 3-keto derivatives, except the one from N-acetyl sphingosine, have been previously reported. Ceramides were isolated from a calf brain and reacted with 2,3-dichloro-5, 6-dicyanobenzoquinone. Ceramides containing sphingosine (4-sphingenine) were converted to 3-keto derivative, while those containing dihydrosphingosine (sphinganine) remained intact under these conditions. The 3-keto ceramides were then separated from the ceramides containing dihydrosphingosine by preparative thin layer chromatography. Similarly cerebrosides from the same calf brain were oxidized and fractionated to 3-ketocerebrosides (from cerebrosides containing sphingosine) and unreacted cerebrosides (cerebrosides containing dihydrosphingosine). The fatty acid composition of these four sphingolipids were determined. Both the ceramides and the cerebrosides containing sphingosine had more unsaturated fatty acids than the corresponding dihydrosphingosine-containing compounds. The ratio of C₁₆-C₂₀ fatty acids to C₂₂-C₂₆ acids was higher in the ceramides containing sphingosine than in ceramides containing dihydrosphingosine, while the ratio was reversed in cerebrosides. The possible precursor-product relationship among these lipids is discussed.     

Separation and identification of ceramides derived from human plasma sphingomyelins

Sphingomyelins from human blood plasma have been converted into ceramides by enzymatic hydrolysis with phospholipase C. After acetylation the ceramides were fractionated by thin-layer chromatography on silica gel containing silver nitrate. Four main fractions obtained by this method were subsequently converted to di-O-trimethylsilyl ether derivatives and separated by gas-liquid chromatography on 1% OV-1. 2-11 components could be distinguished in each of the four fractions. The major fractions emerging from the gas chromatograph were analyzed by mass spectrometry and their main molecular species were identified. Two of the gas chromatographic fractions contained essentially pure molecular species, namely N-tetracosenoyl sphingosine and N-tetracosenoylsphinga-4, 14-dienine.     

Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes

Stratum corneum ceramides play an essential role in the barrier properties of skin. However, their structure-activity relationships are poorly understood. We investigated the effects of acyl chain length in the non-hydroxy acyl sphingosine type (NS) ceramides on the skin permeability and their thermotropic phase behavior. Neither the long- to medium-chain ceramides (8-24 C) nor free sphingosine produced any changes of the skin barrier function. In contrast, the short-chain ceramides decreased skin electrical impedance and increased skin permeability for two marker drugs, theophylline and indomethacin, with maxima in the 4-6C acyl ceramides. The thermotropic phase behavior of pure ceramides and model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesterol sulfate was studied by differential scanning calorimetry and infrared spectroscopy. Differences in thermotropic phase behavior of these lipids were found: those ceramides that had the greatest impact on the skin barrier properties displayed the lowest phase transitions and formed the least dense model stratum corneum lipid membranes at 32°C. In conclusion, the long hydrophobic chains in the NS-type ceramides are essential for maintaining the skin barrier function. However, this ability is not shared by their short-chain counterparts despite their having the same polar head structure and hydrogen bonding ability.     

A method for the quantitative determination of neutral glycosphingolipids in urine sediment

A method is described for the isolation and quantitation of six neutral glycosyl ceramides from human urinary sediment. Total lipids were extracted from sediments of 24-hr urine collections, and the glycosyl ceramides were isolated by silicic acid column chromatography followed by thin-layer chromatography. Methanolysis of the individual glycosyl ceramides yielded methyl glycosides which were quantitated as the trimethylsilyl ethers by gas-liquid chromatography. By this technique, the submicromolar concentrations of six glycosyl ceramides in normal subjects and in individuals with Fabry's disease, an hereditary glycosphingolipid storage disease, were determined. Trihexosyl ceramide (galactosyl-galactosylglucosyl ceramide) and a digalactosyl ceramide accumulated in the urinary sediment of patients with Fabry's disease.     

Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum

Long-chain (sphingoid) bases may serve as another category of "lipid second messenger" because they inhibit protein kinase C and affect multiple cellular functions. Free sphingosine has been found in rat liver (Merrill, A. H., Jr., Wang, E., Mullins, R. E., Jamison, W. C. L., Nimkar, S., and Liotta, D. C. (1988) Anal. Biochem. 171, 373-381); hence, this study determined if liver plasma membranes contain free long-chain bases and have the ability to form them from endogenous enzymes and substrates. Isolated plasma membranes contained 0.45 nmol of sphingosine/mg of protein which, based on the recovery of the membranes, was equivalent to 3.5 +/- 1.2 nmol/g of liver and at least half of the total free sphingosine in liver. When the membranes were incubated at 37 degrees C, the amount increased at an initial rate of 5-25 pmol/min/mg, resulting in a 2-3-fold increase over an hour. Sphingosine formation required divalent cations, was optimal at neutral to alkaline pH, and was temperature-dependent. Activities with these characteristics were not identified in microsomes or lysosomes (lysosomal activities with acidic pH optima were detected, however); hence, they appear to reflect a separate plasma membrane system. Sphingosine formation was stimulated by ceramides either added exogenously or formed endogenously by treating the membranes with sphingomyelinase (but not endoglycoceramidase). Sphingomyelin hydrolysis to ceramide was also observed during incubation of the plasma membranes alone. Some of the properties of this system resembled the neutral sphingomyelinase and ceramidase activities of liver. While the physiological significance of this endogenous sphingosine is not known, this system has the appropriate subcellular location to provide sphingosine as a participant in signal transduction.     

Identification of a xylose-containing cerebroside in the salt gland of the herring gull

A pentose-containing cerebroside has been identified in the salt gland of the herring gull, using mass spectrometry of acetyl and trimethylsilyl derivatives. A detailed interpretation of the spectra allowed a conclusion concerning the major long-chain base (the C(20) homolog of sphingosine) and the major fatty acids (C(22)-C(25) 2-hydroxy fatty acids), using reference spectra of synthetic galactosylceramides. A six-membered glycose ring (aldopyranose) was demonstrated by mass spectrometry of the acetyl derivative of periodic acid-oxidized and sodium borodeuteride-reduced pentosylceramide. By gas-liquid chromatography and mass spectrometry of methanolysis products, the pentose was shown to be identical with xylose. The procedures were applied to 25-50 micro g of glycolipid.     

Apoptosis occurs via the ceramide recycling pathway in human HaCaT keratinocytes

Keratinocytes contain abundant ceramides compared to other cells. However, studies on these cells have mainly focused on the barrier function of ceramide, while their other roles, such as those in apoptosis or cell cycle arrest, have not been well addressed. In this study, we investigated the apoptosis-inducing effect of exogenously added cell-permeable ceramides in HaCaT keratinocytes. We found that N-hexanoyl sphingosine (C6-ceramide) induced apoptosis efficiently through the accumulation of long chain ceramides. On the other hand, N-acetyl sphingosine (C2-ceramide) induced neither apoptosis nor accumulation of long chain ceramides. We also found that exogenously added C6-ceramide was hydrolyzed to sphingosine and then reacylated in long chain ceramides (ceramide recycling pathway), but that C2-ceramide was not hydrolyzed and thus not recycled. We propose that this is the basis for the chain length-specific heterogeneity observed in ceramide-induced apoptosis in these cells. These results also imply that keratinocytes utilize exogenous sphingolipids or ceramides to coordinate their own ceramide compositions.     

Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry

A rapid, simple, and reliable method has been developed for the characterization and quantitation of ceramide molecular species directly from chloroform extracts of biological samples by electrospray ionization tandem mass spectrometry (ESI/MS/MS). By exploiting the differential fragmentation patterns of deprotonated ceramide ions, individual 2-hydroxy and nonhydroxy ceramide molecular species were readily identified by ESI/MS/MS with the neutral loss of fragments of mass 256.2 and 327.3 which correspond to sphingosine derivatives. The ions generated from the neutral loss of 256.2 (i.e., [M - H - 256.2](-)) are unique for ceramides with N-acyl sphingosine with the 18-carbon homolog. However, the sensitivity for nonhydroxy ceramides in ESI/MS/MS with the neutral loss of 256.2 is approximately threefold higher than that for 2-hydroxy ceramides. The ions resulting from the neutral loss of 327.3 (i.e., [M - H - 327.3](-)) are specific for 2-hydroxy ceramides. Additionally, all ceramides including both 2-hydroxy and nonhydroxy forms can be confirmed and accurately quantitated by ESI/MS/MS with the neutral loss of 240.2 after correction for (13)C isotope factors. This methodology demonstrated a 1000-fold linear dynamic range and a detection limit at the subfemtomole range and was applied to directly quantitate ceramide molecular species in chloroform extracts of biological samples including brain tissues and cell cultures.     

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.     

The hydrophobic mismatch determines the miscibility of ceramides in lipid monolayers

The organization of lipids within membranes strongly depends on the interaction with other lipid and protein molecules. Sphingolipids comprise a structurally diverse family, the ceramides being some of the simplest members. Although small chemical modifications of ceramide structure, such as varying the N-acyl chain length, lead to a complex polymorphism of this lipid, only long acyl chain ceramides have usually been studied and their properties became a putative hallmark for all ceramides. In this work, we studied the mixing behavior of C10:0 Cer, which has the N-acyl chain shorter than that of the sphingosine acyl chain and displays an expanded to condensed phase transition at 25mNm(-1) at 24°C, with ceramides N-acylated with longer fatty acyl chains C12:0, C14:0 and C18:0. The N-acyl chain length determined the miscibility of ceramides in Langmuir monolayers, as it was ascertained by the dependence of the mean molecular area, perpendicular dipole moment, surface topography and film thickness with the mixture composition. We found that, as the hydrophobic mismatch in ceramides increased complete miscibility, partial or complete immiscibility can occur.     

Ceramide composition of the psoriatic scale

This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, ω-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains α-OH fatty acids and sphingosines; (5) Cer[AP], which contains α-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alterarion is due to a deranged water bioavailability, associated with psoriaris.     

Increasing ceramides sensitizes genistein-induced melanoma cell apoptosis and growth inhibition

The aim of the current study is to investigate the effect of ceramides on genistein-induced anti-melanoma effects in vitro. We found that exogenously added cell-permeable short-chain ceramides (C6) dramatically enhanced genistein-induced growth inhibition and apoptosis in cultured melanoma cells. Genistein treatment only induced a moderate intracellular ceramides accumulation in B16 melanoma cells. Two different agents including 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a ceramide glucosylation inhibitor, and the sphingosine kinase-1 (SphK1) inhibitor II (SKI-II), a sphingosine (ceramides precursor) phosphorylation inhibitor, both facilitated genistein-induced ceramides accumulation and melanoma cell apoptosis. Co-administration of ceramide (C6) and genistein induced a significant Akt inhibition and c-jun-NH(2)-kinase (JNK) activation, caspase-3 cleavage and cytochrome c release. Caspase-3 inhibitor z-DVED-fmk, JNK inhibitor SP 600125, or to restore Akt activation by introducing a constitutively active form of Akt (CA-Akt) diminished ceramide (C6) and genistein co-administration-induced in vitro anti-melanoma effect. Our study suggests that increasing cellular level of ceramides may sensitize genistein-induced anti-melanoma effects.     

Combined gas chromatography-chemical ionization mass spectrometry of sphingolipids. I. Glucosyl sphingosine,ceramides and cerebrosides of the spleen in Gaucher's disease

Trimethylsilylated glucosyl sphingosine, ceramides and glucocerebrosides were analysed by combined gas chromatography (GC)-chemical ionization (CI) mass spectrometry. Isobutane, methane and ammonia were used as reactant gases for chemical ionization. Essentially the same fragment ions were detected in the spectra with the different reactant gases.Purified glucocerebrosides isolated from the spleen of a patient with Gaucher's disease were clearly separated into their 8 molecular species by gas chromatography. Three other minor components were detected by spectrometry, and these 11 molecular species of glucocerebrosides from the spleen of the patient with Gaucher's disease have been analysed.The ceramides obtained by periodate oxidation and then alkaline reduction of the glucocerebrosides were also separated into 11 molecular species by GC-CI mass spectrometry.Molecular weight could be determined using the major fragment ion of m/e (M⁺?90) in the spectra of ceramides and cerebrosides. The chemical ionization method is useful for structural analyses and determination of the molecular species of sphingoglycolipids.     

Ceramide in nitric oxide inhibition of glioma cell growth. Evidence for the involvement of ceramide traffic

The treatment of C6 glioma cells with the nitric oxide donor, PAPANONOate ((Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate), resulted in a dose-dependent inhibition of cell proliferation. This was associated to a rapid and significant increase of ceramide levels and was mimicked by treatments that augment cellular ceramide. Metabolic experiments with radioactive sphingosine, serine, and choline showed that nitric oxide strongly reduced the utilization of ceramide for the biosynthesis of both sphingomyelin and glucosylceramide. Nevertheless, nitric oxide did not modify the activity of different enzymes of ceramide metabolism. The possibility that nitric oxide impairs the availability of ceramide for sphingolipid biosynthesis was then investigated. The metabolism of N-hexanoyl-[(3)H]sphingosine demonstrated that nitric oxide did not affect the biosynthesis of N-hexanoyl-[(3)H]sphingolipids but inhibited the metabolic utilization of long chain [(3)H]ceramide, synthesized in the endoplasmic reticulum (ER) from the recycled [(3)H]sphingosine. Moreover, results obtained with fluorescent ceramides, brefeldin A, ATP depletion, as well as in a ceramide transport assay indicate that nitric oxide impairs the traffic of ceramide from ER to Golgi apparatus. All this supports that, in glioma cells, the modulation of ceramide traffic can contribute to the regulation of its intracellular levels and participate in the nitric oxide-activated signaling pathway involved in the control of cell proliferation.     

Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide.

Ceramide is a pivotal molecule in signal transduction and an essential structural component of the epidermal permeability barrier. The epidermis is marked by a high concentration of ceramide and by a unique spectrum of ceramide species: Besides the two ceramide structures commonly found in mammalian tissue, N-acylsphingosine and N-2-hydroxyacyl-sphingosine, six additional ceramides differing in the grade of hydroxylation of either the sphingosine base or the fatty acid have been identified in the epidermis. Here we report on the characterization of an IgM-enriched polyclonal mouse serum against ceramide. In dot blot assays with purified epidermal lipids the antiserum bound to a similar extent to N-acyl-sphingosine (ceramide 2), N-acyl-4-hydroxysphinganine (ceramide 3), and N-(2-hydroxyacyl)-sphingosine (ceramide 5), whereas no specific reaction was detected with glycosylceramides, sphingomyelin, free sphingosine, phospholipids, or cholesterol. In contrast, a monoclonal IgM antibody, also claimed to be specific for ceramide, was shown to bind specifically to sphingomyelin and therefore was not further investigated. In thin-layer chromatography immunostaining with purified lipids a strong and highly reproducible reaction of the antiserum with ceramide 2 and ceramide 5 was observed, whereas the reaction with ceramide 1 and ceramide 3 was weaker and more variable. Ceramide 2 and ceramide 5 were detected in the nanomolar range at serum dilutions of up to 1:100 by dot blot and thin-layer immunostaining. In thin-layer chromatography immunostaining of crude lipid extracts from human epidermis, the antiserum also reacted with N-(2-hydroxyacyl)-4-hydroxysphinganine (ceramide 6) and N-(2-hydroxyacyl)-6-hydroxysphingosine (ceramide 7). Furthermore, the suitability of the antiserum for the detection of endogenous ceramide by immunolight microscopy was demonstrated on cryoprocessed human skin tissue. Double immunofluorescence labeling experiments with the anti-ceramide antiserum and the recently described anti-glucosylceramide antiserum (Brade et al., 2000, Glycobiology 10, 629) showed that both lipids are concentrated in separate epidermal sites. Whereas anti-ceramide stained the dermal and basal epidermal cells as well as the corneocytes, anti-glucosylceramide staining was concentrated in the stratum granulosum. In conclusion, the specificity and sensitivity of the reagent will enable studies on the subcellular distribution and biological functions of endogenous ceramide.