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.
Min
Jung-Kee
JK
College of Pharmacy, Chungbuk National University, Chongju 361-763, South Korea.
Yoo
Hwan-Soo
HS
Lee
Eun-Young
EY
Lee
Woo-Jin
WJ
Lee
Yong-Moon
YM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002264:Carboxylic Acids / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D037341:Fumonisins; D018922:HL-60 Cells; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry / Q000187:drug effects; D008246:Lysophospholipids; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000494:pharmacology; D013723:Teratogens / Q000494:pharmacology; D014018:Tissue Distribution; D009764:o-Phthalaldehyde / Q000737:chemistry / Q000378:metabolism
D002264:Carboxylic Acids; D037341:Fumonisins; D008246:Lysophospholipids; D013723:Teratogens; C060506:sphingosine 1-phosphate; C056933:fumonisin B1; D009764:o-Phthalaldehyde; D013250:Steroid Hydroxylases; C406322:vitamin D 1-alpha hydroxylase; D000469:Alkaline Phosphatase; D013110:Sphingosine