Phytosphingosine is a characteristic component of the glycolipids in the vertebrate intestine

Sphingoids in the intestinal lipids of an agnatha, a chondrichthyes, two osteichthyes, three amphibia, three reptiles and two avian species were analyzed by reversed phase high performance liquid chromatography. The glycolipid fraction of all the samples studied contained 4-D-hydroxysphinganine as the major component together with sphingosine and sphinganine. While the trihydroxy base was not found in their sphingomyelin fraction. The trihydroxy base was considered to be a characteristic component of the intestinal glycolipids for the vertebrates in general. Its concentration in the intestinal tissue had little correlation with the food habitat of the animals.     

Studies on the mechanism of 3-ketosphinganine synthetase.

The biosynthesis of sphinganine and 4-D-hydroxysphinganine was studied in rat liver microsomes and whole cells of yeast (Hansenula ciferri). It was shown in both cases that the condensation of [2,3,3-2H3]serine and palmitic acid yielded long chain bases containing only two deuterium atoms, both of which were located on the terminal (C-1) carbon atom by combined gas-liquid chromatography/mass spectrometry. When the reaction with the liver microsomal system was carried out in 2H2O with the protium species of serine, the sphinganine contained a deuterium atom on C-2. These results suggest that the synthesis of 3-ketosphinganine involves the replacement of the alpha-hydrogen atom and the carboxyl group of serine by a proton from the medium and a palmitoyl group, rather than a previously proposed mechanism in which the alpha-hydrogen of serine is retained. Some stereochemical requirements of 3-ketosphinganine synthetase are discussed.     

Evaluation of sphinganine and sphingosine as human breast cancer chemotherapeutic and chemopreventive agents

No comparative study of the effects of sphingolipid metabolites on proliferation and differentiation in normal human breast epithelial cells versus stem cells and tumorigenic cells has been reported. The purpose of this study was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases (sphingosine and sphinganine) using a novel cell culture system of normal human breast epithelial cells (HBEC) developed from breast tissues of healthy women obtained during reduction mammoplasty (Type I HBEC with stem cell characteristics and Type II HBEC with basal epithelial cell phenotypes) and transformed tumorigenic Type I HBEC. The results show that sphinganine inhibited the growth and induced apoptosis of transformed tumorigenic Type I HBEC more potently than sphingosine (IC(50) for sphinganine 4 microM; sphingosine 6.4 microM). Both sphinganine and sphingosine at high concentrations (8-10 lM) arrested the cell cycle at G(2)/M. Sphinganine inhibited the growth and caused death of Type I HBEC more strongly than sphingosine. In comparison, Type II HBEC (normal differentiated cells) were less sensitive to the growth-inhibitory effects of sphingoid bases than Type I HBEC (stem cells) or transformed tumorigenic Type I HBEC, suggesting that sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05, 0.1, and 0.5 microM) that are below the growth-inhibitory range, sphingoid bases induced differentiation of Type I HBEC to Type II HBEC, as detected morphologically and via expression of a tumor suppressor protein, maspin, which is a marker of Type II HBEC. Thus, sphingoid bases may function as chemotherapeutic as well as chemopreventive agents by preferentially inhibiting cancer cells and eliminating stem cells from which most breast cancer cells arise.     

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.     

Modulation of sphingolipid biosynthesis in primary cultured neurons by long chain bases

Sphingolipid biosynthesis was studied in cultured murine cerebellar cells in the absence and presence of exogenous sphingosine homologues with different alkyl chain lengths (12, 18, and 24 carbon atoms). Labeling of cells with [14C]serine for 24 h indicated that endogenous sphingosine biosynthesis with incorporation of radiolabeled serine was inhibited by these long chain bases (0.5-50 microM) in a concentration-dependent manner; the inhibition was fully reversible after removal of the long chain bases from the culture medium. Metabolic labeling of neurons with [14C]galactose provided strong evidence that the cells were able to use the exogenous sphingosine homologues, irrespective of their alkyl chain length, as substrates for the biosynthesis of glycosphingolipids. When the biosynthetically inert sphingoid, azidosphingosine (5-50 microM), was fed to the cells, de novo sphingosine and glycosphingolipid biosynthesis were both strongly inhibited.     

Quantitative analysis of free sphingoid bases in the brain and spinal cord tissues by high-performance liquid chromatography with a fluorescence detection

The o-phthaldialdehyde precolumn derivatives of psychosine, sphinganine and sphingosine extracted from brain and spinal cord tissues were determined by high-performance liquid chromatography–fluorescence detection. This method was developed with the purpose of detecting an endogenous amount of psychosine, sphingosine and sphinganine using small aliquots of brain tissues and spinal cord in rats. These sphingolipid bases were extracted in various ratios of chloroform–methanol and several pH values. Recovery of the method is about 81% in 12 ng/tube (final volume, 320 μl), 90–95% in 45 ng/tube of sphingosine and sphinganine within 2–12% relative standard deviation. Detection limits of these sphingoid bases were about 0.05 pmol/mg brain tissue. In the forebrain, brainstem and spinal cord of rats at three different ages of postnatal days (PND) 1, PND 13 and 6 months old, the endogenous concentrations of psychosine, sphingosine and sphinganine were determined. From these results, this method is suitable for the determination of sphingoid bases in small aliquot of brain and spinal cord tissues.     

Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases

The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase(PcSYR2), as well as structural genes for dihydroceramide Δ(4)-desaturase (PcDES1) and sphingolipid Δ(8)-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts. In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240mgL(-1) triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890mgkg(-1) triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications.     

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.     

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.     

Preparation of the tritiated molecular forms of gangliosides with homogeneous long chain base composition

Gangliosides G_M2, G_M1 and G_D1b were radiolabelled at C-6 of the terminal galactose orN-acetylgalactosamine by the galactose oxidase/[³H]NaBH₄ method; gangliosides G_M2, G_M1, Fuc-G_M1 and G_D1a were radiolabelled at C-3 of the long chain base by the 2,3-dichloro-5,6-dicyanobenzoquinone/[³H]NaBH₄ method.By application of an original HPLC procedure, eight different molecular species were prepared from each labelled ganglioside. Each of these species was characterized by the presence of one of the following long chain bases:erythro C18 sphingosine,threo C18 sphingosine,erythro C18 sphinganine,threo C18 sphinganine,erythro C20 sphingosine,threo C20 sphingosine,erythro C20 sphinganine andthreo C20 sphinganine.From G_D1b only the species containing theerythro forms of long chain bases were obtained.The individual molecular species were more than 99% homogeneous and had a radiopurity better than 99%. The molecular species of the same ganglioside, radiolabelled at C-3 of the long chain base, had identical specific radioactivity, namely 1.17, 1.25, 0.85 and 1.28 Ci/mmol for G_M2, G_M1, Fuc-G_M1 and G_D1a respectively. The molecular species of the same ganglioside, radiolabelled at C-6 of terminal galactose orN-acetylgalactosamine, had similar specific radioactivity, namely 1.34–1.40, 1.44–1.51, 1.37–1.44 Ci/mmol for G_M2, G_M1 and G_D1b respectively.     

Differential labelling of sphingolipids by [3H]serine and ([3H]methyl)-methionine in fish leukocytes

Long chain bases are constituents of all sphingolipids and their biosynthesis is presumed to occur via the initial condensation of serine with palmitoyl-CoA. The biosynthesis of phytosphingosine, a long chain base containing three hydroxyl groups, has been less studied than sphingosine but is assumed to occur by hydroxylation of sphinganine. We report in this paper that the label from ([3H]methyl)-methionine is preferentially incorporated into phytosphingosine bases of neutral glycosphingolipids, whereas the label from [3H]serine is mainly incorporated into the sphingoid base of sphingomyelin. These results show that in fish leukocytes the biosynthesis of individual sphingoid bases and their downstream sphingolipid products follow different pathways of metabolism. Our observations suggest that in fish leukocytes the synthesis of the constitutive long chain bases of sphingomyelin and complex glycosphingolipids is coordinately regulated and may be localized in separate compartments.     

Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase

This paper describes a simultaneous analytical method for the measurement of sphingoid base 1-phosphates and sphingoid bases from a variety of biological samples. This method consists of two steps of sample pretreatment: the enzymatic dephosphorylation of sphingoid base 1-phosphates by alkaline phosphatase (APase) and the subsequent analysis of o-phthalaldehyde (OPA) derivatives of the liberated sphingoid bases by HPLC. By introducing C17-sphingosine 1-phosphate and C17-sphingosine as internal standards, not only phytosphingosine 1-phosphate, sphingosine 1-phosphate, and sphinganine 1-phosphate but also phytosphingosine, sphingosine, and sphinganine present in a sample could be quantified in 12 min on a C18 reversed-phase column with a simple mobile phase of acetonitrile:deionized distilled water (90:10, v/v). With this HPLC method, we could reproducibly analyze the levels of sphingoid base 1-phosphates over a broad range of concentrations from 0.5 to 100.0 pmol from various biological samples including serum, cultured cells, and rat tissue homogenates. The conversion of sphingoid base 1-phosphates into sphingoid bases increased the stability of the OPA adducts. Thus, this indirect measurement of sphingoid base 1-phosphates increased the sensitivity and reproducibility of the method. This HPLC method was also used to measure the changes in the levels of sphingoid base 1-phosphates in cultured cells after treatment with 1,25-(OH)2D3, a sphingosine kinase activator, or with fumonisin B1, a sphinganine N-acyltransferase inhibitor.     

Modulation of the free sphingosine levels in human neutrophils by phorbol esters and other factors

Because free long-chain bases have been recently found to have potent pharmacological effects when added to neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623) and other cell types, the levels in human neutrophils were measured by high-performance liquid chromatography. Sphingosine was the major free long-chain base in freshly isolated cells and ranged from 13 to 101 pmol/10(7) cells for different donors (mean +/- S.E. of 50 +/- 5, n = 17). Upon incubation at 37 degrees C, there was a time-dependent increase in free sphingosine (57 +/- 8% in 1 h, n = 17), but no change was seen at 4 or 25 degrees C. The sphingosine was apparently derived from more complex sphingolipids because little (less than 1%) could be accounted for by new synthesis from [14C]serine. Greater increases in free sphingosine were obtained when neutrophils were incubated with serum, plasma, or serum lipoproteins (about 2-fold higher than for cells incubated alone). In contrast, agonists such as phorbol 12-myristate 13-acetate, A23187, arachidonic acid, low concentrations (10 nM) of N-formyl-methionyl-leucyl-phenylalanine, and opsonized zymosan either decreased the amount of free sphingosine or blunted the time-dependent increase. This may be due to enhanced removal of free sphingosine because phorbol 12-myristate 13-acetate-treated cells exhibited an increased conversion of exogenously added [3H]sphinganine to ceramides. Endogenous sphingosine was approximately one-tenth the level found in neutrophils when exogenous long-chain bases were added to inhibit protein kinase C. Hence, depending on the subcellular localization of the endogenous versus exogenous long-chain bases, the amounts of free sphingosine in neutrophils might be sufficient to affect the function of these cells.     

Phytosphingosine in intestinal glycolipids of the rat fetus

The intestinal glycolipids of rat fetuses contained two major long-chain bases, sphingosine and phytosphingosine. The occurrence of phytosphingosine in glucosylceramide and GM3 was lower at 17 days of gestation than at birth. The base composition of GD3 remained stable and consisted mainly of sphingosine.     

Preparation of 13C-labeled ceramide by acetic acid bacteria and its incorporation in mice

We prepared 2-hydroxypalmitoyl-sphinganine (dihydroceramide) labeled with a stable isotope by culturing acetic acid bacteria with ¹³C-labeled acetic acid. The GC/MS spectrum of the trimethylsilyl derivative of ¹³C-labeled dihydroceramide gave molecular ions with an increased mass of 12–17 Da over that of nonlabeled dihydroceramide. The fragment ions derived from both sphinganine base and 2-hydroxypalmitate were confirmed to be labeled with the stable isotope in the spectrum. Therefore, ¹³C-labeled dihydroceramide can be an extremely useful tool for analyzing sphingolipid metabolism. The purified [¹³C]dihydroceramide was administered orally to mice for 12 days, and the total sphingoid base fractions in various tissues were analyzed by GC/MS. The spectrum patterns specific to ¹³C-labeled sphingoids were detected in the tissues tested. Sphinganine pools in skin epidermis, liver, skeletal muscle, and synapse membrane in brain were replaced by [¹³C]sphinganine at about 4.5, 4.0, 1.0, and 0.3%, respectively. Moreover, about 1.0% of the sphingosine pool in the liver was replaced by [¹³C]sphingosine, implying that exogenous dihydroceramide can be converted to sphingosine. These results clearly indicate that ingested dihydroceramide can be incorporated into various tissues, including brain, and metabolized to other sphingolipids.     

狗小肠鞘氨醇糖脂中长链碱组成的研究

 利用气相色谱、气相色谱-质谱联用等方法测定了狗小肠鞘氨醇糖脂中的长链碱组成。其主要的长链碱为鞘氨醇(Sphingosine)、异鞘氨醇(isosphingosine)、二氢鞘氨醇(Sphinganine)和植物鞘氨酸(Phyto-sphingosine)。一共分离出十三个鞘氨醇糖脂。在唯一的五糖基神经酰胺中异鞘氨醇是主要成份。在一个一糖基神经酰胺中植物鞘氨醇是主要成份。植植物鞘氨酸也是两个二糖基神经酰胺和一个三糖基神酰胺的主要长链碱。说明它不仅存于植物体内。     

Reactivity of Sphingosine Base in Enzymatic Synthesis of Ceramide

The reactivity of sphingosine bases as substrates in the enzymatic synthesis of ceramide was in decreasing order of erythro-4-sphingenine, erythro-sphinganine, threo-4-sphingenine and thero-sphinganine in the microsomal fraction obtained from chicken liver. Unsaturated base-containing ceramide was enzymatically formed from 4-sphingenine and saturated base-containing ceramide from sphinganine. The formation of unsaturated base-containing ceramide proceeded similar to that of saturated base-containing ceramide. The data explain in part, the fact that 4-sphingenine is generally superior to sphinganine as the constituent sphingosine base in sphingolipids, the biochemical derivatives of ceramide.     

Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst

The neutrophil oxidative burst is characterized by increased cellular O2 consumption due to the activation of a membrane-associated superoxide-generating NADPH-oxidase. The response is triggered by a variety of stimuli, including opsonized zymosan, formylmethionylleucinephenylalanine (FMLP), arachidonate, short-chain diacylglycerols, and phorbol myristate acetate (PMA). We herein demonstrate that incubation of cells with sphinganine or sphingosine blocks or reverses activation by these agonists. The inhibition is reversible, does not affect cell viability, and does not affect another complex cell function, phagocytosis. Inhibitory concentrations of sphinganine did not significantly affect cytoplasmic calcium levels or FMLP-generated calcium transients. Structural requirements for inhibition of the oxidative burst include a long aliphatic chain and an amino-containing head-group, and there is modest specificity for the native (erythro) isomer of sphinganine. Inhibition involves stimulus-induced activation mechanisms rather than a direct effect on the NADPH oxidase, since sphinganine did not inhibit NADPH-dependent superoxide generation in isolated membranes containing the active enzyme. Activation by FMLP, diacylglycerol, PMA, opsonized zymosan, and arachidonate was blocked by the same concentrations of sphinganine, indicating that these agonists share a common inhibited step. Three lines of evidence indicate that this step involves protein kinase C. First, in a micelle system and in platelets, long-chain bases are inhibitors of this enzyme (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). Second, sphinganine blocks PMA-stimulated incorporation of 32PO4 into neutrophil proteins. Third, sphinganine inhibits the binding of [3H]phorbol dibutyrate to its cellular receptor, known to be protein kinase C. We suggest that long-chain bases function as physiologic modulators of cellular regulatory pathways involving protein kinase C.     

Sphinganine in Sphingomyelins of Tumors and Mouse Regenerating Liver

Contents of sphingenine (sphingosine) and sphinganine were studied in sphingomyelins of transplantable mouse tumors (hepatoma-22, melanoma B16, Lewis lung carcinoma, intestine carcinoma) and rat nephroma RA. The content of sphinganine was increased in sphingomyelins of hepatoma-22 and nephroma RA compared to sphingomyelins of liver and kidneys. Significant contents of sphinganine were also found in sphingomyelins of other studied tumors. The content of sphinganine in regenerating mouse liver (30 h after hepatectomy) was normal. The data suggest that disorders should exist in biosynthesis of sphingoid bases in tumors but not in normal rapidly proliferating tissue.     

Differential labelling of sphingolipids by [H]serine and ([H]methyl)-methionine in fish leukocytes

Long chain bases are constituents of all sphingolipids and their biosynthesis is presumed to occur via the initial condensation of serine with palmitoyl-CoA. The biosynthesis of phytosphingosine, a long chain base containing three hydroxyl groups, has been less studied than sphingosine but is assumed to occur by hydroxylation of sphinganine. We report in this paper that the label from ([³H]methyl)-methionine is preferentially incorporated into phytosphingosine bases of neutral glycosphingolipids, whereas the label from [³H]serine is mainly incorporated into the sphingoid base of sphingomyelin. These results show that in fish leukocytes the biosynthesis of individual sphingoid bases and their downstream sphingolipid products follow different pathways of metabolism. Our observations suggest that in fish leukocytes the synthesis of the constitutive long chain bases of sphingomyelin and complex glycosphingolipids is coordinately regulated and may be localized in separate compartments.