Qualitative and Quantitative Study of Glycosphingolipids in Human Milk and Bovine Milk Using High Performance Liquid Chromatography-Data-Dependent Acquisition-Mass Spectrometry.

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography-tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9-17.4 µg Crb/mL and 1.3-3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8-12.0 and 14.3-16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

Metabolomic and Lipidomic Profiling of Bone Marrow Plasma Differentiates Patients with Monoclonal Gammopathy of Undetermined Significance from Multiple Myeloma.

Oncogenic drivers of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) such as c-MYC have downstream effects on intracellular metabolic pathways of clonal plasma cells (PCs). Thus, extracellular environments such as the bone marrow (BM) plasma likely have unique metabolite profiles that differ from patients with MGUS compared to MM. This study utilized an untargeted metabolite and targeted complex lipid profiling of BM plasma to identify significant differences in the relative metabolite levels between patients with MGUS and MM from an exploratory cohort. This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles such as decreased branch chain amino acids (BCAAs) and increased catabolism of tryptophan to the active kynurenine metabolite 3-hydroxy-kynurenine between patients with MGUS and MM. A decrease in the total levels of complex lipids such as phosphatidylethanolamines (PE), lactosylceramides (LCER) and phosphatidylinositols (PI) were also detected in the BM plasma samples from MM compared to MGUS patients. Thus, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between patients with MGUS and MM. This may provide insight into the possible differences of the intracellular metabolic pathways of their clonal PCs.

Analysis of sphingolipid composition in human vitreous from control and diabetic individuals.

OBJECTIVE: Sphingolipids have a fundamental role in many cellular processes, and they have been implicated in insulin resistance and Diabetes Mellitus (DM) and its complications, including diabetic retinopathy (DR). Little is known about how bioactive sphingolipids relate to retinopathies in human DM. In this study, we analyzed the sphingolipid composition of type 2 diabetic (T2DM) and non-diabetic human vitreous samples. METHODS: We conducted an observational study on post-mortem human vitreous samples from non-diabetic (Controls; n = 4; age: 71.6 ±â€¯11.0 years, mean ±â€¯SD) and type 2 diabetic (T2DM; n = 9; age: 67.0 ±â€¯9.2 years) donors to identify changes in sphingolipid composition. Samples were analyzed by a triple quadrupole mass spectrometer and individual sphingolipid species were identified and quantified using established protocols. RESULTS: The total quantity (pmol/mg) of ceramide (Cer), lactosylceramide (Lac-Cer), and sphingomyelin (SM) were increased in type 2 diabetic vitreous samples. Among individual species, we found a general trend of increase in the longer chain species of ceramides, hexosylceramides (Hex-Cer), Lac-Cer, and SM. CONCLUSIONS: This study shows the presence of measurable levels of sphingolipids in human vitreous. The results indicate changes in sphingolipid composition in the vitreous due to type 2 diabetes, which could be connected to the disease pathologies of the retina, retinal vessels, vitreous and the surrounding tissues.

Carbohydrate-containing molecules as potential biomarkers in colon cancer.

Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.

Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons.

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.

E-LDL and Ox-LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages.

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages.

Lactosylceramide in lysosomal storage disorders: a comparative immunohistochemical and biochemical study.

Immunohistochemical studies of the presence of lactosylceramide (LacCer) in lysosomal storage disorders (LSDs) were done using anti-LacCer monoclonal antibody of the CDw 17 type (clone MG-2). No sign of an association between LacCer and the lysosomal system in normal cells was observed, except for histiocytes active in phagocytosis. A comparative study of a group of LSDs showed a general tendency for LacCer to increase in storage cells in Niemann-Pick disease type C (NPC), and types A and B, GM1 gangliosidosis, acid lipase deficiency, glycogen storage disease type II and mucopolysaccharidoses. LacCer accumulated in storage cells despite normal activity of relevant lysosomal degrading enzymes. The accumulation of LacCer displayed variability within storage cell populations, and was mostly expressed in neurons in NPC. An absence of the increase in LacCer in storage cells above control levels was seen in neuronal ceroid lipofuscinoses (neurons and cardiocytes) and in Fabry disease. Gaucher and Krabbe cells showed significantly lower levels, or even the absence, of LacCer compared with control macrophages. Results of immunohistochemistry were corroborated by semiquantitative lipid thin-layer chromatography (TLC). It is suggested that different associations of LacCer with the lysosomal storage process may reflect differences in glycosphingolipid turnover induced by the storage-compromised lysosomal/endosomal system.

Ceramides and glycosphingolipids in maturation process: leukemic cells as an experimental model.

Leukemic cells were used as experimental material to demonstrate changes in the content of GSLs during the development and maturation of neutrophils. The most abundant cellular GSL is LacCer. An elevation in the LacCer level occurs twice during the maturation process: initially, on formation of azurophil granules, and subsequently, (a more significant rise) on formation of specific granules. The formation of the latter is accompanied by an increase in the level of GalGalCer. During the maturation of myeloblasts, there is a simultaneous growth in the content of LacCer and GM3 as well as that of their common precursors, that is, free ceramides. Like other tumor cells, GM3 rich myeloblasts in the peripheral blood from patients with AML are characterized by shedding of gangliosides. The quantitative Cer/GlcCer ratio in these cells seems to be advantageous for the efficacy of chemotherapy in the induction of apoptosis. Myelo- and metamyelocytes achieve the highest level of GSLs. Their entry into the full maturity stage is accompanied by a decrease in the level of GSLs. Patterns of GSLs expression change greatly during development and maturation. However, with respect to the composition and content of GSLs, there are no significant differences between normal and leukemic mature neutrophils. At each stage of the development and maturation of myelogenous leukemic cells, as well as in normal mature neutrophils, there occurs the synthesis of the same molecular species both free ceramides and ceramide portions of LacCer, precursor of more complex GSLs.

Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.

Sphingolipids participate in membrane structure and signaling in neuronal cells, and an emerging strategy for control of gliomas is to inhibit growth and/or induce apoptosis using ceramide and ceramide analogs. Nonetheless, some sphingolipids (ceramides and sphingosine) induce and others (sphingosine 1-phosphate) inhibit apoptosis; therefore, when testing putative anti-cancer agents, it is critical to obtain precise knowledge of the types and quantities of not only the test compounds, but also their effects on endogenous species. Combination of liquid chromatography and tandem mass spectrometry affords a "metabolomic" profile of all of the intermediates of ceramide biosynthesis (3-ketosphinganine, sphinganine and dihydroceramides) and the direct products of ceramide metabolism (sphingomyelins and monohexosylceramides as well as sphingosine and sphingosine 1-phosphate). This method has been applied to four human glioma cell lines (LN18, LN229, LN319 and T98G), and differences in the amounts and types of sphingolipids were found. For example, LN229 and LN319 have approximately twice the sphingosine 1-phosphate of LN18 and T98G; LN229 and LN319 have more monohexosylceramides than lactosylceramides, whereas the opposite is the case for LN18 and T98G; and the fatty acyl chain distributions of the sphingolipids differ among the cell lines. The ability to obtain this type of "metabolomic" profile allows studies of how anti-cancer agents (especially sphingolipids and sphingolipid analogs) affect the amounts of these bioactive species, and may lead to a better understanding of the abnormal phenotypes of gliomas.

Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils.

This study is focused on the functional significance of neutrophil lactosylceramide (LacCer)-enriched microdomains, which are involved in the initiation of a signal transduction pathway leading to superoxide generation. Treatment of neutrophils with anti-LacCer antibody, T5A7 or Huly-m13, induced superoxide generation from the cells, which was blocked by PP1, a Src kinase inhibitor; wortmannin, a phosphatidylinositol-3 kinase inhibitor; SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor; and H7, an inhibitor for protein kinase C. When promyelocytic leukemia HL-60 cells were differentiated into neutrophilic lineage by dimethyl sulfoxide (DMSO) treatment, they acquired superoxide-generating activity but did not respond to anti-LacCer antibodies. Density gradient centrifugation revealed that LacCer and Lyn were recovered in detergent-insoluble membrane (DIM) of neutrophils and DMSO-treated HL-60 cells. However, immunoprecipitation experiments indicated that LacCer was associated with Lyn in neutrophils but not in DMSO-treated HL-60 cells. Interestingly, T5A7 induced the phosphorylation of Lyn in neutrophils but not in DMSO-treated HL-60 cells. Moreover, T5A7 induced the phosphorylation of p38 MAPK in neutrophils. T5A7-induced Lyn phosphorylation in neutrophil DIM fraction was significantly enhanced by cholesterol depletion or sequestration with methyl-beta-cyclodextrin or nystatin. Collectively, these data suggest that neutrophils are characterized by the presence of cell surface LacCer-enriched glycosphingolipid signaling domain coupled with Lyn and that the ligand binding to LacCer induces the activation of Lyn, which may be suppressibly regulated by cholesterol, leading to superoxide generation through the phosphatidylinositol-3 kinase-, p38 MAPK-, and protein kinase C-dependent signal transduction pathway.

Glycolipid analysis of different tissues and cerebrospinal fluid in type II Gaucher disease.

The lipid composition or the liver, spleen, brain, cerebellum and cerebrospinal fluid of a Gaucher disease type II patient who died at the age of 5 months was examined. The glycolipid analysis demonstrated a marked increase of total amounts not only in the peripheral tissues but also in the brain cerebellum and cerebrospinal fluid, with a prevalence of glucosylceramide. A reduction in gangliosides was observed in all the analysed tissues with a relative increase of GD3 in the nervous tissue. The fatty acid composition of glucosylceramide showed a prevalence of stearic acid in the central nervous system, while in the peripheral tissues palmitic acid was prevalent. This result suggests a different origin of the glucosylceramide stored in different tissues. The generalized reduction of gangliosides and their modified distribution together with the central nervous system GD3 increment represent a new observation. These data could be useful in the effort to clarify the pathophysiological mechanism of brain damage in neuronopathic Gaucher disease.

Identification of GM3 as a marker of therapy-resistant periradicular lesions.

The purpose of this study was to analyze the profile of glycosphingolipids (GSLs) in periradicular lesions refractory to endodontic treatment. Sixteen periapical lesions were removed surgically from patients (experimental group) and compared with 10 samples of periodontal ligament removed from extracted intact third molars (control group). After the GSLs extraction and purification procedures were performed the neutral and acidic GSL fractions were analyzed by high-performance thin-layer chromatography and quantified by densitometry. Data reported herein show that: (i) tissues in the experimental group presented about twice as much GSLs as the control group; (ii) lesion tissues express lactoneotetraosylceramide, and lactofucopentaosyl (IV) ceramide, whereas these neutral GSLs are absent in normal tissues; and (iii) normal tissues express GT1b, whereas lesions cells do not express this ganglioside. In contrast lesion tissues express GM3, which is conspicuously absent in normal tissues.

Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria.

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three-fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long-chain bases found in free ceramides were d18:1 sphingosine, d18:0 3-ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3-ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long-chain base of glucosylceramide of outer membranes.

Localization of verotoxin receptors in nervous system.

We use immunohistochemistry to show the existence of verotoxin receptor in small sensory neurons in DRG of human, rabbit, rat and mouse. In capillary in nervous system, the verotoxin receptor exists in human and rabbit, but the receptor could not be demonstrated in rat and mouse, by this method. The receptors in sensory neuron of rat and in capillary in rabbit brain are determined as galactosylglobotriaosylceramide (GalGb3) and globotriaosylceramide (Gb3,), respectively. Although verotoxin was reported to bind to glycolipid receptors that possess the terminal disaccharide Galalpha1-4Galbeta (galactobiose), the binding to toxin to galabiosylceramide was half of that of GalGb3 which has galactobiose internally.

Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle.

Membrane vesicles were prepared from rabbit skeletal muscle, separated by sucrose density gradient centrifugation and characterized by their specific marker enzymes, ligand binding, and ion flux activities. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M). Their glycosphingolipid compositions were analyzed by biochemical and immunochemical methods with specific antibodies (TLC immunostaining) and characteristic patterns were obtained from respective membrane fractions, expressed on a protein basis. Glucosylceramide, the main neutral glycosphingolipid of rabbit muscle, was found in SL and TT fractions, whereas SR and Tr/M vesicles lack this compound. Lactosylceramide was selectively recovered in the SR1 fraction. GM3(Neu5Ac), the main ganglioside in rabbit muscle, was found to account for 64% in the SL, 13% in the TT, 7% in the SR1, 3% in the SR2 and 13% in the Tr/M fractions. IV3Neu5Ac-nLcOse4Cer was mostly abundant in SL and decreased in the order SL > TT, Tr/M > SR1, SR2. IV6Neu5Ac-nLcOse4Cer was only detected in the SL and Tr/M fractions in noteworthy quantities. Ganglioseries gangliosides GM1, GD1a, GD1b and GT1b displayed homogeneous distribution patterns in each membrane preparation. They were expressed only in small amounts but mainly in SL, TT and Tr/M vesicles and to less extent in SR1 and SR2 fractions. The presence of GM3(Neu5Ac) in the SL as well as on subcellular level was confirmed in transverse muscle cryosections by means of indirect immunofluorescence microscopy. The SL was brightly stained, but considerable intracellular fluorescence was observed as expected from the biochemical analyses. Thus, the neutral GSL and ganglioside expression of the SL and the intracellular membraneous network is different in skeletal muscle both in terms of quantitative and qualitative GSL composition as demonstrated in details by means of biochemical and immunochemical techniques. The modulatory functions of GM3 and gangliosides of the neolacto- and ganglio-series towards the voltage dependent Ca(2+)-channel, largely preponderant in the triads-containing Tr/M fraction, is the subject of the accompanying paper (J. Müthing, U. Maurer, and S. Weber-Schürholz, Carbohydr. Res., 307 (1998) 147-157).

Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells.

Neutral glycosphingolipids and gangliosides were isolated from 3.7 x 10(9) primary bovine aortic endothelial cells and structurally characterized by immunological and chemical methods. Glucosyl- and lactosylceramide were detected as the main neutral glycosphingolipids (28% and 40% of total orcinol stain, respectively); LcOse3Cer and nLcOse4Cer were expressed to somewhat minor amounts (16% and 10% of total orcinol stain, respectively), and nLcOse6Cer occurred only in trace quantities. No neutral glycosphingolipids of the ganglio-series (GgOse3Cer and GgOse4Cer) and the globo-series (GbOse4Cer and the Forssman antigen) have been detected; only traces of GbOse3Cer were identified by TLC immunostaining. Positive CD15 bands obtained by TLC overlay with anti-Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-R antibody indicated the presence of lipid bound Lewisx antigen, whereas the isomeric Lewis(a) structure (Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-R) was not detectable. G(M3) substituted with Neu5Gc and Neu5Ac in a 2:1 ratio was the major ganglioside comprising about 95% within the whole ganglioside fraction. G(M3)-structures were further characterized by FAB-MS and GC-MS of the native compounds and their permethylated derivatives. C18-sphingosine was the only long chain base, whereas variation occurred due to C(24:0,24:1) and C16 fatty acids. Terminally alpha2-3 sialylated neolacto-series gangliosides with nLcOse4- and nLcOse6Cer (<5% of total resorcinol stain) were found in almost equal quantities, whereas no alpha2-6 sialylated counterparts were detected. Fucosylated gangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis[x], sialyl Lewisa, and VIM-2 antigen) and sulfoglucuronylneolacto series structures with HNK-1 epitope were not detectable in the acidic glycosphingolipid fraction by TLC immunostaining. Gangliotetraose-type gangliosides G(M1) and G(D1a) (<1 % of total resorcinol stain) as well as traces of G(D1b) and G(T1b) have been distinctly identified by combined choleragenoid-TLC-immunostaining and previous neuraminidase treatment. The expression of dominant glycosphingolipids lactosylceramide and G(M3)(Neu5Gc) was proved by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The results provide the basis for investigation of the role of glycosphingolipids as cell surface antigens of cellular interaction as well as receptors for blood components and macromolecules of the extracellular matrix.

Glycosphingolipids in human pancreatic juice. Unique fatty acid compositions of glucosylceramides and lactosylceramides.

Glycosphingolipids in human pancreatic juice were isolated and purified by DEAE and silica gel column chromatographs, and further by HPLC on silica gel and reversed-phase columns. The structures of the glycosphingolipids were determined to be glycosylceramides and lactosylceramides by means of fast atom bombardment mass spectrometry, 1H-NMR spectroscopy, and component analysis involving GLC-mass spectrometry. The ceramide portions of the glucosylceramides consist of palmitic, tetracosanoic, and alpha-hydroxy tetracosanoic fatty acids, and d18:1 and t18:0 sphingosines. The ceramide portions of the lactosylceramides consist of palmitic, teracosanoic, tetracosenoic, and tetracosadienoic fatty acids, and d18:1 sphingosine. The fatty acid compositions are different from the free fatty acid compositions of serum and pancreatic juice. The predominance of saturated, unsaturated, and hydroxy tetracosanoic fatty acid is quite unique, and the possibility that these glycosphingolipids may play a specific role in pancreatic juice deserves consideration. These glycosphingolipids were documented to be major components of the pancreatic juice from 15 patients with a variety of pancreatic diseases.

A monoclonal antibody reactive with terminal lactotriaosyl residue-containing oligosaccharides and its application to characterizing cell surface expression of the glyco-epitopes in COS-1 cells.

A new monoclonal antibody (MoAb), designated JF12, reactive strongly with Lc3 (GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->Cer) but only slightly with nLc5 (GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->Cer) has been prepared after immunization of Balb/c mice with Lc3. By the flowcytometrical analyses of COS-1 cells, which was strongly stained with JF12, the reactivity completely disappeared in the confluent condition upon harvesting by the protease treatments of the cell surface. In the sparse condition, however, the cells still retained JF12 reactivity in spite of the protease treatments. This strongly suggests that the expression of terminal GlcNAc-containing glycoconjugates on the surface of COS-1 cells may be dramatically modulated by protease-sensitive membranous components being dependent upon the cell density.

Compensatory occurrence of IV2Fucalpha,II3NeuAcalpha-Gg4Cer and human fetal antigen Lc4Cer in small cell carcinomas of the human lung.

By means of a thin-layer chromatography immunostaining procedure involving a human monoclonal anti-Lc4Cer antibody, which was established by hybridizing murine myeloma cells and human lymphocytes from a cancer patient, Lc4Cer was proven to be a fetal antigen of human lung and to be a cancer-related antigen in small cell carcinomas of human lung, but not of other lung cancers, i.e., large cell carcinomas, adenocarcinomas, and squamous carcinomas. With the simultaneous detection of IV2Fuc alpha,II3NeuAc alpha-Gg4Cer with rabbit anti-IV2Fuc alpha,II3NeuAc alpha-Gg4Cer antiserum, the expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer was found to be compensatory and, consequently, small cell lung carcinomas could be classified into Lc4Cer- and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer-expressing types, L-SCLC and F-SCLC, respectively, which were detected in four and 27 of 31 patients' tissues and in one and three of four nude mouse-transplanted small cell lung carcinoma tissues, respectively. The compensatory expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer in small cell carcinomas indicated that different metabolic pathways for glycosphingolipids were activated to give the distinct glycosphingolipid compositions in the two types of small cell lung carcinomas.