Measurement of the phospholipase activity of endothelial lipase in mouse plasma

Abstract

This article is available online at http://www.jlr.orgIn evaluating plasma EL function, measurement of EL enzymatic activity is more informative than quantifi cation of EL concentration by immunoassay because the latter does not differentiate between active noncovalent homodimer and inactive monomer ( 15 ), active full-length enzyme and inactive proteolytically cleaved enzyme ( 16,17 ), and the presence or absence of the endogenous plasma inhibitor of EL,18 ).Since the discovery of EL in 1999 ( 19, 20 ), substantial efforts have been devoted to developing an EL phospholipase A1 assay. Of those currently used, the nonradiolabeled assays ( 21-23 ) are suitable for screening inhibitors of EL in the test tube. However, there is no published data regarding their use for assaying EL in plasma. Radiometric assays using 3 H-trioleoyl glycerol or 14 C-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. Furthermore, they are not specifi c for EL due to the presence in plasma of HL and LPL. All three lipases belong to the triacylglycerol (TG) lipase family and can hydrolyze both TGs and phospholipids although with very different preferences for the two types of substrates ( 25,26 ). For these reasons, there is a need for an easy-touse assay specifi c for plasma EL activity. Of note, the major activity of purifi ed EL in the test tube against substrates in emulsions is as a phospholipase A1 (PLA1) ( 23 ). This activity is much greater than its activity against the TGs in emulsions. Therefore, a phospholipase A1 assay for EL acting in plasma (i.e., not purifi ed) will be more sensitive than a TG lipase assay.Here, we describe development of a novel homogeneous PLA1 assay for plasma EL, which employs a commerciallyavailable fl uorogenic phospholipid analog, the PED-A1 substrate. The changes in both EL protein levels and PLA1 activity in plasma, with time after heparin injection, were 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 specifi c for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fl uorescent substrate in combination with an EL neutralizing antibody. The specifi city 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. Endothelial lipase (EL), which hydrolyzes HDL phospholipids and thu...

Plasma Activity of Endothelial Lipase Impacts High-Density Lipoprotein Metabolism and Coronary Risk Factors in Humans

“…The present study successfully established a direct method for assessing the plasma human EL-specific phospholipase activity using a combination of IP and a fluorogenic phospholipid substrate. The principle of this assay has recently been reported and validated for the measurement of the murine plasma EL activity 25) . In the present study, furthermore, we depleted native IgG using pretreatment of plasma samples with Protein A prior to EL-IP, as plasma is abundant in native IgG and may interfere with the IP process.…”

Angiopoietin-like 3 inhibition of endothelial lipase is not modulated by angiopoietin-like 8

“…EL activity assays-Phospholipase activity was monitored using the EnzChek Phospholipase A1 assay kit (ThermoFisher) as described previously (26). EL (final concentration of ~10 μg/ml) and ANGPTL proteins (final concentrations: 0-4000 ng/ml ANGPTL3; 0-210 ng/ml ANGPTL8) were combined and incubated at 37°C (or at 4°C or 22°C for temperature studies) for 30 minutes (unless otherwise indicated).…”

Furin Is the Primary in Vivo Convertase of Angiopoietin-like 3 and Endothelial Lipase in Hepatocytes

“…To directly assess the EL-specific activity in Fur-hKO plasma versus that in WT plasma, we used a commercially available fluorogenic substrate and a neutralizing mouse EL antibody to measure specifically the EL activity as recently reported (42). The phospholipase activity was determined in pre-and postheparin plasma from WT and Fur-hKO mice of 2 months of age, in the presence or absence of a mouse EL antibody (Fig.…”