C17 sphingosine

Manufactured by Avanti Polar Lipids

Sourced in United Kingdom, United States

C17-sphingosine is a long-chain sphingoid base used as an internal standard for the analysis of sphingolipids. It serves as a reference compound for the identification and quantification of related lipid species.

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Lab products found in correlation

Close spelling variants of this product

C17-sphingosine-1-phosphate (6 citations) C17-d-erythro-sphingosine (C17-Sph) (2 citations) D-erythro-sphingosine (C17 base) (2 citations)

27 protocols using c17 sphingosine

Extraction and Quantification of Sphingolipids

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To measure sphingolipids, total lipids were extracted as previously described (19 (link)). Briefly, cell pellets were re-suspended in chloroform/methanol/water (10:5:1; v/v/v), followed by addition of internal standards containing 0.5 nmoles of C12:0 ceramide, C17-sphingosine, C17-sphinganine, C25:0-ceramide and C12:0-sphingomyelin from Avanti Polar Lipids (Alabaster, AL). After tip sonication, samples were incubated at 48 °C overnight. The next day, 100 μl of aliquots were taken out and dried under N₂ for measurement of total phospholipids (Wako Chemicals, Neuss, Germany). The rest samples were added with 75 μl of 1M KOH in methanol, sonicated for 30 min, then incubated at 37 °C for 2 h and dried in a nitrogen evaporator.

WANG Y., PARK N.Y., JANG Y., MA A, & JIANG Q. (2015). Vitamin E γ-tocotrienol inhibits cytokine-stimulated NF-κB activation by induction of anti-inflammatory A20 via stress adaptive response due to modulation of sphingolipids. Journal of immunology (Baltimore, Md. : 1950), 195(1), 126-133.

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Quantification of sphingolipid metabolites

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All samples were stored at -80°C until analysis. The content of S1P, SA1P, sphingosine, sphinganine, and ceramide was determined as described previously in detail [26] . Briefly, lipids were extracted from samples in the presence of internal standards (C 17 -sphingosine and C 17 -S1P, Avanti Polar Lipids, Alabaster, AL). An aliquot of the lipid extract was transferred to a fresh tube with pre-added N-palmitoyl-D-erythro-sphingosine (C17 base) (a kind gift of Dr Z. Szulc, Medical University of South Carolina) as an internal standard, and then subjected to alkaline hydrolysis to deacylate ceramide to sphingosine. The amount of S1P and SA1P was determined indirectly after dephosphorylation to sphingosine and sphinganine, respectively, with the use of alkaline phosphatase (bovine intestinal mucosa, Sigma). Free sphingosine and sphinganine, dephosphorylated sphingoid bases, and sphingosine released from ceramide were then converted to their o-phthalaldehyde derivatives and analyzed using a HPLC system (ProStar, Varian Inc., Palo Alto, CA) equipped with a fluorescence detector and C18 reversed-phase column (Varian Inc. OmniSpher 5, 4.6´150mm). The isocratic eluent composition of acetonitrile (Merck, Darmstadt, Germany): water (9: 1, v/v) and a flow rate of 1 mL/min were used. Column temperature was maintained at 30°C.

Książek M., Baranowska U., Chabowski A, & Baranowski M. (2018). Arteriovenous Sphingosine-1-Phosphate Differences Across Selected Organs of the Rat. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 45(1).

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Ceramide Quantification in Salivary Glands

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Ceramide content in the salivary glands was assessed using the method described by Garbowska et al. [23 (link)]. Briefly, a small portion of the chloroform phase was transferred to a fresh tube containing C17 sphingosine (Avanti Polar Lipids, UK) used as an internal standard. The organic phase containing ceramide was then hydrolyzed at 90°C for 60 minutes in the solution of 1 M KOH in 90% methanol. The obtained sphingosine was subsequently analyzed using the high-performance liquid chromatography (HPLC) technique. N-Palmitoylsphingosine (Avanti Polar Lipids, UK) was applied as a standard for the preparation of the calibration curve. The assessed amount of ceramide was adjusted with respect to the level of free sphingosine in the sample.

Żendzian-Piotrowska M., Ziembicka D.M., Łukaszuk B, & Kurek K. (2020). Impact of Acute Pancreatic Injury on Sphingolipid Metabolism in the Salivary Glands. BioMed Research International, 2020, 6403482.

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Ceramide Quantification via HPLC

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A portion of lipids extracts, contained in the chloroform phase, was transferred to another tube containing C17-sphingosine (Avanti Polar Lipids, UK) as an internal standard. The organic phase containing ceramide was then hydrolyzed (in the solution of 1 M KOH in 90% methanol) for 60 min at the temperature of 90°C. The liberated sphingosine was subsequently analyzed using HPLC (high performance liquid chromatography). N-palmitoyl-sphingosine (Avanti Polar Lipids, UK) was used as a standard for preparation of the calibration curve. The content of ceramide was adjusted for the concentration of free sphingosine measured in the same sample.

Kurek K., Mikłosz A., Łukaszuk B., Chabowski A., Górski J, & Żendzian-Piotrowska M. (2015). Inhibition of Ceramide De Novo Synthesis Ameliorates Diet Induced Skeletal Muscles Insulin Resistance. Journal of Diabetes Research, 2015, 154762.

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Ceramide Derivatives Analysis by HPLC

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The ceramide derivatives were measured according to the method described by Min et al. [26 (link)]. Prior to samples homogenization and ultrasonication, internal standards (C17-sphingosine and C17-S1P (Avanti Polar Lipids, USA)) were added. The sphingoid bases were converted to their o-phthalaldehyde derivatives and analyzed on a HPLC system (ProStar, Varian, Inc., USA) equipped with a fluorescence detector and C18 reversed-phase column (Varian, Inc., OmniSpher 5, 4.6 mm × 150 mm).

Kurek K., Wiesiołek-Kurek P., Piotrowska D.M., Łukaszuk B., Chabowski A, & Żendzian-Piotrowska M. (2014). Inhibition of Ceramide De Novo Synthesis with Myriocin Affects Lipid Metabolism in the Liver of Rats with Streptozotocin-Induced Type 1 Diabetes. BioMed Research International, 2014, 980815.

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Sphingolipid Quantification Protocol

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The contents of ceramide, sphinganine and sphingosine were determined as described previously in detail by Baranowski et al. (2008) (link). Briefly, all the samples, apart from incubation media, were homogenized by ultrasonication and lipids were extracted in the presence of internal standard (10 pmol of C17-sphingosine, Avanti Polar Lipids). An aliquot of the lipid extract was transferred to a fresh tube with pre-added 40 pmol of N-palmitoyld-erythro-sphingosine (C17 base) as an internal standard and then subjected to alkaline hydrolysis to deacylate ceramide to sphingosine. The free sphinganine, as well as sphingosine released from ceramide were then converted to their o-phthalaldehyde derivatives and analyzed using a HPLC system. Before the sphingolipid analysis, the protein content was measured.

Harasim-Symbor E., Konstantynowicz-Nowicka K, & Chabowski A. (2016). Additive effects of dexamethasone and palmitate on hepatic lipid accumulation and secretion. Journal of molecular endocrinology, 57(4).

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Sphingolipid Extraction and Quantification

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To measure sphingolipids, total lipids were extracted as previously described [19 (link)]. Briefly, cell pellets were re-suspended in methanol/chloroform /water (10:5:1 [v/v/v]), followed by addition of internal standards containing 0.5 nmoles of C12:0-ceramide (Cer), C17-sphingosine, C17sphinganine, C25:0-Cer, and C12:0-sphingomyelin from Avanti Polar Lipids (Alabaster, AL). After tip sonication, samples were incubated at 48°C overnight. The next day, 100 μl aliquots was taken out and dried under N₂ for measurement of total phospholipids (Wako Chemicals, Neuss, Germany). For the rest of the samples, 75 μl of 1 M KOH in methanol was added, sonicated for 30 min, and then incubated at 37°C for 2 h and dried in a nitrogen evaporator.

YANG C, & JIANG Q. (2018). Vitamin E δ-tocotrienol inhibits TNF-α-stimulated NF-κB activation by up-regulation of anti-inflammatory A20 via modulation of sphingolipid including elevation of intracellular dihydroceramides. The Journal of nutritional biochemistry, 64, 101-109.

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Lipidomic Analysis of Cell Extracts

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Cell pellets were spiked with internal standards (C17-sphingosine Cayman Chemical, Ann Arbor, MI, USA; C17-S1P, Avanti Polar Lipids, Alabaster, AL, USA) and lipids were extracted using a published method (Couttas et al., 2014 (link)). Liquid chromatography tandem-mass spectrometry was performed at PhenoSwitch Bioscience, Inc. (Sherbrooke, QC, Canada). Liquid chromatography gradient was performed using 0.2% v/v formic acid in water and with 0.2% v/v formic acid with 10 mM ammonium formate in methanol on a reversed phase Halo PFP column (Advance Materials Technology, Wilmington, DE, USA), which was maintained at 50°C. Acquisition was performed with an ABSciex TripleTOF 5600 (Sciex, Foster City, CA, USA) equipped with an electrospray interface. Quantification was done using the area under the curve with the MuliQuant software (Sciex). See Supplementary Methods and Supplementary Table 1 for details.

Gendron D.R., Lecours P.B., Lemay A.M., Beaulieu M.J., Huppé C.A., Lee-Gosselin A., Flamand N., Don A.S., Bissonnette É., Blanchet M.R., Laplante M., Bourgoin S.G., Bossé Y, & Marsolais D. (2017). A Phosphorylatable Sphingosine Analog Induces Airway Smooth Muscle Cytostasis and Reverses Airway Hyperresponsiveness in Experimental Asthma. Frontiers in Pharmacology, 8, 78.

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Overexpression and Knockdown of Band3 for C17-Sphingosine-1-Phosphate Assay

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To overexpress Band3, we cloned human Band3 cDNA obtained from Flexi ORF Clone (Promega, Co., Madison, WI) into a CMV shuttle vector (Stratagene, La Jolla, CA). Then, we transfected human Band3-coding plasmids or GFP-coding plasmids into K562 cells or HEK293 cells using Lipofectamine LTX with Plus Reagent (15338100; Invitrogen Co., Carlsbad, CA), according to the manufacturer’s protocol. To knockdown Band3, we transfected siRNA against human Band3 or control siRNA at 10 nM (sc-42735, sc-37007; Santa Cruz Biotechnology) into K562 cells utilizing Lipofectamine RNAiMAX (12778–075; Invitrogen Co.); 48 hours later, we treated the cells (K562 cells: 2–4 x 10⁶ / 1 mL, HEK293 cells: around 80% confluency) with 10 μM C₁₇-sphingosine (860654P; Avanti Polar Lipids, Alabaster, AL) at 37°C for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% fatty acid-free BSA (A8806; Sigma-Aldrich Co.) and incubated the cells at 37°C for another 20 minutes. The supernatants and cells were then collected and subjected to the C₁₇S1P assay.

Kurano M., Nishikawa M., Kuma H., Jona M, & Yatomi Y. (2017). Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes. PLoS ONE, 12(5), e0177543.

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Quantification of Plasma Sphingolipids

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The levels of ceramide, sphingosine (SFO), sphinganine (SFA), and sphingosine-1-phosphate (S1P) were determined as previously described [39 (link)]. Briefly, lipids were extracted from 250 μL of plasma in the presence of internal standards (10 pmol C17-sphingosine and 30 pmol C17-S1P, Avanti Polar Lipids). An aliquot of the lipid extract was transferred to a fresh tube with pre-added 40 pmol N-palmitoyl-Derythro-sphingosine (C17 base; a kind gift of Dr. Z. Szulc, Medical University of South Carolina) as an internal standard, and then subjected to alkaline hydrolysis to deacylate ceramide to SFO. The amount of S1P was determined indirectly after dephosphorylation to SFO, with the use of alkaline phosphatase (bovine intestinal mucosa, Fluka). Free SFO, dephosphorylated sphingoid bases, and SFO released from ceramide were then converted to their o-phthalaldehyde derivatives and analyzed using a HPLC system equipped with a fluorescence detector and C18 reversed-phase column (Varian Inc., OmniSpher 5, Palo Alto, United States; 4.6 × 150 mm). The isocratic eluent composition of acetonitrile (Merck, Darmstadt, Germany):water (9:1, v/v) and a flow rate of 1 mL/min were used.

Wątek M., Piktel E., Barankiewicz J., Sierlecka E., Kościołek-Zgódka S., Chabowska A., Suprewicz Ł., Wolak P., Durnaś B., Bucki R, & Lech-Marańda E. (2019). Decreased Activity of Blood Acid Sphingomyelinase in the Course of Multiple Myeloma. International Journal of Molecular Sciences, 20(23), 6048.

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