Fluorogenic substrates for high-throughput measurements of endothelial lipase activity

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluo- rescent Bodipy-labeled substrates that can be used in ho- mogeneous,ultra-high-throughputkineticassaysthatmeasure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles con- taining Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum.—Mitnaul, L. J., J. Tian, C. Burton, M-H. Lam, Y. Zhu, S. H. Olson, J. E. Schneeweis, P. Zuck, S. Pandit, M. Anderson, M. M. Maletic, S. T. Waddell, S. D. Wright,C.P.Sparrow,andE.G.Lund.Fluorogenicsubstrates for high-throughput measurements of endothelial lipase activity. J. Lipid Res. 2007. 48: 472-482.