In vitro measurement of ATX activity against LDL-associated substrates

Four hundred nanograms of isolated human LDL free of EDTA (Lee Biosolutions, Maryland Heights, MO; 360-10) were incubated at 37°C for 4 h with 2 ng/μl recombinant human ATXβ in assay buffer [100 mM Tris (pH 8.0), 400 mM NaCl, 5 mM MgCl₂, 5 mM CaCl₂, 10 uM CoCl₂] modified from (35) and samples processed for MS measurements of LPA. The ATX dependence of increases in LPA was determined by including a potent ATX inhibitor, 1 μM PF-8380, in the incubations (Sigma-Aldrich, St. Louis, MO; SML0715). LPC (15:0; 1 mM) was used as an exogenous substrate to monitor activity of the recombinant human ATXβ, and activity was quantified by MS measurement of 15:0 LPA. In other experiments, endogenous ATX activity in mouse plasma was measured by incubating heparin-collected plasma from LDLr^−/− mice fed the high-fat Western diet for 8 weeks at 37°C for 12 h and then fractionating the plasma by size-exclusion chromatography and quantifying LPA species associated with each fraction by MS. Albumin bound-LPA was prepared by preincubating fatty acid-free BSA (Sigma-Aldrich; A8806) with 17:0 LPA for 4 h at 37°C. This LPA-BSA complex was then incubated with or without isolated human LDL overnight. These incubated plasma preparations were then fractionated by size-exclusion chromatography to separate LDL from albumin; lipids were extracted from the fractions and 17:0 LPA quantified by MS.