Quantitative analysis of sulfatides in RAW 264.7 cells treated with Kdo 2 ‐Lipid A

Utilizing the uniform protocols established by the LIPID MAPS Consortium ( www.lipidmaps.org ), RAW 264.7 cells were treated with Kdo 2 ‐Lipid A, a defined endotoxin that activates macrophages via TLR‐4, then the sulfatide amounts and types were analyzed using liquid chromatography, electrospray tandem mass spectrometry. Sulfatides are detected and quantified by multiple reaction monitoring analysis in negative ion mode using an API 4000 quadrupole linear ion trap (varying the collision energy from 80 eV to 130 eV for the internal standard, a C12:0 homolog from Avanti Polar lipids, and the cellular sulfatides with C16:0 to 26:0 ceramide backbones). Although RAW 264.7 cells contain essentially no endogenous sulfatide, there is a large increase beginning approximately 12 h after Kdo2‐Lipid A addition. As best we have been able to ascertain, this is the first finding of induction of sulfatide biosynthesis by macrophage activation, and it will be interesting to determine if the increase in sulfatides has an impact on macrophage function. This work was supported by NIH grant GM069338 (LIPID MAPS Consortium).