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Title: DEVELOPMENT OF A METHOD FOR QUANTITATING SPHINGOID BASE 1-PHOSPHATES IN BLOOD SPOTS

Author
item Riley, Ronald
item Zitomer, Nicholas
item GELINEAU VAN WAES, J - Creighton University

Submitted to: Society of Toxicology
Publication Type: Abstract Only
Publication Acceptance Date: 11/1/2010
Publication Date: 3/6/2011
Citation: Riley, R.T., Zitomer, N.C., Gelineau Van Waes, J. 2011. DEVELOPMENT OF A METHOD FOR QUANTITATING SPHINGOID BASE 1-PHOSPHATES IN BLOOD SPOTS. Society of Toxicology. Toxicological Sciences 115(S1):311.

Interpretive Summary: Red blood cells (RBC) accumulate, store and release sphingoid base 1-phosphates,important ligands for the extracellular receptors S1P1-5. The ability of RBC to accumulate these bioactive lipids is because, with the exception of sphingosine kinase, the enzymes responsible for metabolizing sphingosine (So) and sphinganine (Sa) are absent from RBC. In addition, the enzymes responsible for degrading So- and Sa-1-phosphates (So 1-P and Sa 1-P)are lacking in RBC. Thus, sphingoid bases and sphingoid base analogs such as FTY720 are taken up by RBC and are phosphorylated. In mice treated with fumonisin B1, significant elevation of Sa 1-P in RBC is detected soon after exposure. The half life of phosphorylated sphingoid bases in RBC is short based on the rapid elimination of the phosphorylated sphingoid base analog FTY720. The purpose of this IRB approved study was to develop an analytical method to measure sphingoid bases and sphingoid base 1-P in blood using blood spots collected on FTA® elute micro cards. The FTA® card was chosen because it is impregnated with bactericidal and viricidal chemicals and blood collected on these cards are exempt from shipping restrictions. Blood was collected from finger sticks and allowed to dry at room temperature over night and then stored desiccated at -20oC. An 8 mm core (17.5 µl of blood) was removed from the cards and extracted with acetonitrile/water 5% formic acid. Sa 1-P and So 1-P were easily detected in extracts using liquid chromatography-electro spray ionization-mass spectrometry and levels were proportional to the blood volume of the spot and also the UV (A275) absorbance of the extracts. Sa 1-P and So 1-P were stable for at least 1 month after collection when stored desiccated at -20oC. Extraction efficiencies ranged from 60% to 80% and the limit of detection was less than 2 pmol. The method will be used to study the effects of fumonisin exposure on Sa 1-P levels in human studies in Guatemala.

Technical Abstract: Red blood cells (RBC) accumulate, store and release sphingoid base 1-phosphates,important ligands for the extracellular receptors S1P1-5. The ability of RBC to accumulate these bioactive lipids is because, with the exception of sphingosine kinase, the enzymes responsible for metabolizing sphingosine (So) and sphinganine (Sa) are absent from RBC. In addition, the enzymes responsible for degrading So- and Sa-1-phosphates (So 1-P and Sa 1-P)are lacking in RBC. Thus, sphingoid bases and sphingoid base analogs such as FTY720 are taken up by RBC and are phosphorylated. In mice treated with fumonisin B1, significant elevation of Sa 1-P in RBC is detected soon after exposure. The half life of phosphorylated sphingoid bases in RBC is short based on the rapid elimination of the phosphorylated sphingoid base analog FTY720. The purpose of this IRB approved study was to develop an analytical method to measure sphingoid bases and sphingoid base 1-P in blood using blood spots collected on FTA® elute micro cards. The FTA® card was chosen because it is impregnated with bactericidal and viricidal chemicals and blood collected on these cards are exempt from shipping restrictions. Blood was collected from finger sticks and allowed to dry at room temperature over night and then stored desiccated at -20oC. An 8 mm core (17.5 µl of blood) was removed from the cards and extracted with acetonitrile/water 5% formic acid. Sa 1-P and So 1-P were easily detected in extracts using liquid chromatography-electro spray ionization-mass spectrometry and levels were proportional to the blood volume of the spot and also the UV (A275) absorbance of the extracts. Sa 1-P and So 1-P were stable for at least 1 month after collection when stored desiccated at -20oC. Extraction efficiencies ranged from 60% to 80% and the limit of detection was less than 2 pmol. The method will be used to study the effects of fumonisin exposure on Sa 1-P levels in human studies in Guatemala.