Determination of sphingosine-1-phosphate lyase activity by gas chromatography coupled to electron impact mass spectrometry

Reina, Ester; Camacho, Luz; Casas, Josefina; Veldhoven, Paul P. Van; Fabriás, Gemma

doi:10.1016/j.chemphyslip.2012.01.001

Cited by 13 publications

(11 citation statements)

References 27 publications

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“…The detection limit established in the current assay is similar to ones reported for using a semicarbazide derivatization assay (Berdyshev et al, 2011). The LOQ is lower than previously reported values of 287 fmol which was determined from perflurobenzyloxime-derivatized ( 2E )-hexadecenal (Reina et al, 2012). Linearity of response for S1P (shown in Figure 3B) also shows excellent linearity within the same concentration range as tested for ( 2E )-hexadecenal.…”

Section: Resultscontrasting

confidence: 72%

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A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

Suh

Eltanawy

Rangan

et al. 2016

Chemistry and Physics of Lipids

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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 50 fmol, 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.

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Section: Resultscontrasting

confidence: 72%

“…Recovery across different matrices was excellent and showed greater than 84.5 ± 4.9% yield (data not shown) and was on par with other assays (Reina et al, 2012)…”

Section: Resultsmentioning

confidence: 73%

A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

Suh

Eltanawy

Rangan

et al. 2016

Chemistry and Physics of Lipids

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“…The first MS-based method was developed by Berdyshev et al (26) to characterize the S1PL activity in mouse liver microsomal extracts ( K M = 5.7 μM, V max = 0.171 pmol/min/μg). A similar GC-electron ionization MS method used pentafluorobenzoyloxime derivitization of the 2E-HEX to analyze the S1PL in mouse embryonic fibroblasts ( K M = 6 μM, V max = 0.374 pmol/min/μg) (27). Novartis has recently identified the human S1PL as an attractive target for the development of therapeutics and developed a high throughput MS-based method to screen for novel inhibitors (11).…”

Section: Discussionmentioning

confidence: 99%

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei

McLean

Marles‐Wright

Custódio

et al. 2017

Journal of Lipid Research

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Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

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“…Although it has been unequivocally confirmed that THI decreases SPL activity in vivo, the inhibitory mechanism is partly still controversial because THI did not show any inhibitory activities on SPL in vitro. For example, it has been reported so far that THI failed to inhibit SPL in several cell-free assays [6,9,10]. SPL inhibition can also be estimated using cell-based assays in which the enzyme reaction is monitored by accumulation of S1P or dihydroS1P (dhS1P).…”

Section: Introductionmentioning

confidence: 99%

Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency

et al. 2014

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Caramel food colorant 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) causes lymphopenia in animals through sphingosine 1-phosphate lyase (SPL) inhibition. However, this mechanism of action is partly still controversial because THI did not inhibit SPL in vitro either in cell-free or in cell-based systems. It is thought that the in vitro experimental conditions which have been used so far were not suitable for the evaluation of SPL inhibition, especially in case of cell-based experiments. We speculated that the key factor might be the coenzyme pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 (VB6), because media used in cell-based assays usually contain an excess amount of VB6 which leads to the activation of SPL. By the use of VB6-deficient culture medium, we could regulate apo- (without PLP) and holo- (with PLP) SPL enzyme in cultured cells, resulting in the successful detection of SPL inhibition by THI. Although the observed inhibitory effect was not as strong as that of 4-deoxypyridoxine (a VB6 analog SPL inhibitor), these findings may be useful for further understanding the mechanism of action of THI.

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Determination of sphingosine-1-phosphate lyase activity by gas chromatography coupled to electron impact mass spectrometry

Cited by 13 publications

References 27 publications

A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei

Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency

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