title: A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity.
abstract: A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)- d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection.
authors.affiliation: Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
authors.forename: Jung H
authors.initials: JH
authors.lastname: Suh
authors.affiliation: Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
authors.forename: Abeer
authors.initials: A
authors.lastname: Eltanawy
authors.affiliation: Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
authors.forename: Apoorva
authors.initials: A
authors.lastname: Rangan
authors.affiliation: Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States. Electronic address: jsaba@chori.org.
authors.forename: Julie D
authors.initials: JD
authors.lastname: Saba
mesh_terms: D000446:Aldehyde-Lyases / Q000172:deficiency / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D057075:Enzyme Assays / Q000379:methods*; D006835:Hydrazones / Q000138:chemical synthesis / Q000737:chemistry; D007201:Indicator Dilution Techniques; D007554:Isotopes; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015394:Molecular Structure
chemical_list: D006835:Hydrazones; D007554:Isotopes; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase)
keywords: (2E)-Hexadecenal; 2-Hydrazinoquinoline; Sphingolipids; Sphingosine phosphate lyase; Sphingosine-1-phophate; sphingosine phosphate lyase