dihydrosphingosine-1-phosphate and lysophosphatidic-acid

Itching and infiltration of immune cells are important hallmarks of atopic dermatitis (AD). Although various studies have focused on peripheral mediator-mediated mechanisms, systemic mediator-mediated mechanisms are also important in the pathogenesis and development of AD. Herein, we found that intradermal injection of lysophosphatidic acid (LPA), a bioactive phospholipid, induces scratching responses by Institute of Cancer Research mice through LPA1 receptor- and opioid μ receptor-mediating mechanisms, indicating its potential as a pruritogen. The circulating level of LPA in Naruto Research Institute Otsuka Atrichia mice, a systemic AD model, with severe scratching was found to be higher than that of control BALB/c mice, probably because of the increased lysophospholipase D activity of autotaxin (ATX) in the blood (mainly membrane associated) rather than in plasma (soluble). Heparan sulfate proteoglycan was shown to be involved in the association of ATX with blood cells. The sequestration of ATX protein on the blood cells by heparan sulfate proteoglycan may accelerate the transport of LPA to the local apical surface of vascular endothelium with LPA receptors, promoting the hyperpermeability of venules and the pathological uptake of immune cells, aggravating lesion progression and itching in Naruto Research Institute Otsuka Atrichia mice.

Topics: Animals; Blood Cells; Cell Membrane; Chromatography, Liquid; Hypersensitivity; Inflammation; Lysophospholipids; Male; Mass Spectrometry; Mice; Mice, Inbred BALB C; Phosphoric Diester Hydrolases; Protein Binding; Pruritus; Skin; Solubility; Sphingosine

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are ubiquitous lipid messengers found in the blood and most cell types. Both lysophospholipids are ligands of G protein-coupled receptors and mediate important physiological processes. Moreover, lysophospholipids are potential biomarkers for various diseases, including atherosclerosis and cancer. Because existing methodologies are of limited value for systematic evaluations of S1P and LPA in clinical studies, we developed a fast and simple quantification method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS).. Sphingoid base 1-phosphates and LPA species were quantified in negative-ion mode with fragments of m/z 79 and 153, respectively. The internal standards LPA 17:0 and [(13)C(2)D(2)]S1P were added before butanol extraction. Application of hydrophilic-interaction chromatography allowed coelution of analytes and internal standards with a short analysis time of 2.5 min.. Comparison of butanol extraction with a frequently used extraction method based on strong acidification of human plasma revealed artificial formation of LPA from lysophosphatidylcholine with the latter method. Validation according to US Food and Drug Administration guidelines showed an overall imprecision (CV) of <12% and a limit of detection <6 nmol/L for all lysophospholipid species. Concentrations of S1P and sphinganine 1-phosphate (SA1P) in EDTA-containing plasma were stable for 24 h at room temperature, whereas LPA concentrations increased substantially over this period.. Our validated LC-MS/MS methodology for quantifying LPA, S1P, and SA1P features simple sample preparation and short analysis times, therefore providing a valuable tool for diagnostic evaluation of these lysophospholipids as biomarkers.

Topics: Chromatography, Liquid; Humans; Lysophospholipids; Reproducibility of Results; Sphingosine; Tandem Mass Spectrometry