• Archives of Pharmacal Research
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• v.29 no.11
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• pp.1049-1054
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• 2006

Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; $100\;{\mu}M\;of\;C_{17}-Sph\;and\;30\;{\mu}g$ protein of F9-12 cells lysate in 20 min. Sphingosine analog $C_{17}-Sph$ was efficiently phosphorylated by Sphk activity ($K_{m}:67.08\;{\mu}M,\;V_{max}\;:1507.5\;pmol/min/mg$). New product $C_{17}-S1P$ was separated from S1P in reversed-phase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while $20\;{\mu}M$ of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.

• Natural Product Sciences
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• v.17 no.4
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• pp.315-320
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• 2011

Routinely applicable HPLC assay procedures for the ganglioside content in various deer antler preparations were established through the creation of a UV-absorbing chromophoric substance - trans-${\alpha},{\beta}$-unsaturated-hexadecene-aldehyde - from the sphingosine moiety in ganglioside molecules by two step chemical reactions. In order to guarantee the assay's accuracy and sensitivity, the HPLC-assay procedure adopted internal reference procedures by mixing cis-${\alpha},{\beta}$-unsaturated-hexadecene aldehyde[V] or cis-3-heptadecene- 1,2-diol[IV] to assay samples. The internal reference compound [IV] or [V] was synthesized in our laboratory starting from mannitol-diacetonide through three or four step organic reactions. This new HPLC-assay procedure was successfully applied to deer antler extracts with good dose-dependent calibration curves at the picomole level of gangliosides.

• Molecular & Cellular Toxicology
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• v.3 no.4
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• pp.273-281
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• 2007

Ceramide is involved in cell death as a lipid mediator of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine ($C_{17}$ ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acidfree bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and measured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSAceramide complex leads to the increased deacylation of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples.

• Archives of Pharmacal Research
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• v.22 no.3
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• pp.294-299
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• 1999

Precolumn orthophthaldehyde (OPA) labeling method of sphingoid bases, sphingosine and sphinganine, was investigated to obtain high fluorescent detectability. In order to improve the fluorescent yield, we investigated the optimal solubility of sphingoid bases for five pre-incubation solvents by incorporating the heating procedure before OPA derivatization. The pre-incubation in ethanol prominently increased the fluorescent peak height of OPA derivative for each sphingoid bases in high performance liquid chromatography. About tenfold increase of detectability was archived by pre-incubating lipid extracts pellets in ethanol at $60^{\circ}C$ for 30 min. Optimal derivatization was performed in 30 min at ambient temperature and the fluorescent intensity of OPA derivative was stable for two weeks at $4^{\circ}C$. The detection limit of sphingosine was 0.1 pmol as injected amount. This method was applied to the determination of cellular sphingosine and sphinganine in various human lung cancer cells. This OPA procedure was prospective to be useful for quantitating the amount of sphingoid bases in other cancer cells.

• Biomolecules & Therapeutics
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• v.16 no.2
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• pp.95-99
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• 2008

Apoptosis is essential for a variety of pathophysiological progress. Apoptosis induction by various agents changes cellular morphology, DNA content and lipid membrane composition. Recently, sphingosine 1-phosphate (S1P) is avidly released from not only platelets and erythrocytes but vascular endothelium. Here we established S1P releasing cells by deleting S1P lyase (F9-12 cells). We observed apoptosis induction by the treatment of fumonisin B1 (FB1) in F9-12 cells but not in F9 wild-type cells. We measured high amounts of accumulated S1P and dihydroS1P (DHS1P) in FB1-induced apoptotic F9-12 cells. We also showed DHS1P release in an early stage of the apoptosis induction by FB1 but not by phorbol 12-myristate 13-acetate (PMA)-induced apoptosis, suggesting differential apoptotic processes.