Abstract
Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL's hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface.
Highlights
Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans
We hypothesized that proper presentation of the PED-A1substrate to the EL enzyme is required for determining EL activity in the mouse plasma
Because the active dimer of EL is attached to the endothelial cell surface via reversible binding to heparan sulfate proteoglycans [27], a bolus of heparin administered intravenously was used to release EL into the plasma compartment; this method is routine for LPL and HL activity measurements [28]
Summary
Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans This regulatory function is critically dependent on EL’s hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. Radiometric assays using 3H-trioleoyl glycerol or 14C-labeled phosphatidylcholine as substrates in emulsions have been extensively used for determining plasma EL activity and studying EL biology [24] These assays, which require separation of the released labeled fatty acids, are time consuming and have a low throughput. We describe development of a novel homogeneous PLA1 assay for plasma EL, which employs a commerciallyavailable fluorogenic phospholipid analog, the PED-A1 substrate The changes in both EL protein levels and PLA1 activity in plasma, with time after heparin injection, were
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