Determination of lipoprotein lipase activity using a novel fluorescent lipase assay

Abstract

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.

Fig. 1.

Characterization of lipoprotein lipase (LPL) activity with the EnzChek substrate. Fluorescence increase with time was determined using a fixed amount of LPL (175 ng/100 μl) and increasing amounts of the EnzChek substrate at 37°C in 100 μl total volume in the presence of 0.15 M NaCl, 20 mM Tris-HCl, pH 8.0, 0.0125% Zwittergent, and 1.5% FA-free BSA. Background substrate hydrolysis was deducted from each measurement. Initial rate was plotted against substrate concentration. Each data point is the mean of triplicate determinations.

Fig. 2.

Evaluation of the upper limit of the LPL concentration range that gives linear fluorescence versus time measurements. Reactions were carried out under the conditions described in Fig. 1 using a fixed EnzChek substrate concentration (0.62 μM) and increasing concentrations of LPL (35 ng to 1,109 ng). Background substrate hydrolysis was deducted from all measurements. Each data point is the mean of triplicate determinations.

Fig. 3.

Comparison of LPL activities using the EnzChek and 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) (DGGR) substrates. Initial rates were obtained using 9 min, single-point fluorescence measurements at increasing substrate concentrations. The EnzChek assay was carried out under the conditions described in Fig. 1, the DGGR assay as described by Panteghini, Bonora, and Pagani (19). Background substrate hydrolysis was deducted from all measurements. Data were analyzed by linear regression. The quantities y (y = zx, where y = relative fluorescent units (RFU), x = LPL concentration (ng/100 μl), z = slope of line) and R² (R = the sample correlation coefficient) are given for each assay type. Each data point is the mean of triplicate determinations. The experiment was carried out three times with similar results.

Fig. 4.

The EnzChek substrate assay allows measurement of activation of LPL by apolipoprotein C-II (apoC-II) and inhibition by tetrahydrolipstatin (THL). The assay was carried out as described in Fig. 1 using 0.62 μM EnzChek substrate and 175 ng of bovine LPL, with human apoC-II (A) and THL (B) concentrations as indicated. Background substrate hydrolysis was deducted from all measurements. Each data point is the mean of triplicate determinations ± SD, the latter too small to be visible. The experiment was carried out three times with similar results.

Fig. 5.

Measurement of human LPL activity using the EnzChek assay. Conditioned human LPL medium (25 μl) was mixed with control medium (25 μl) or conditioned human angiopoietin-like protein-4 (ANGPTL4) medium (25 μl) and held on ice for 30 min prior to addition to LPL activity assays measured using: (A), the EnzChek assay as described in Fig. 1 with 0.62 μM EnzChek substrate, or (B), the radiometric triolein assay as described in Materials and Methods. Background substrate hydrolysis was deducted from each measurement. LPL activity is expressed as a percentage of the activity measured in the absence of ANGPTL4. Values are the mean ± SD of triplicate repeats.* P < 0.05.

Fig. 6.

Measurement of triglyceride lipase activities in mouse plasma. Pre- and postheparin plasma samples (1 μl) were incubated on ice for 30 min with one of the following: PBS, ANGPTL4-N, antibodies anti-mEL IgG or control IgG, prior to analysis, as described in Fig. 1 using 0.62 μM EnzChek substrate. The final concentrations in the assays of ANGPTL4-N and each antibody were 40 nM and 20 μg/ml, respectively. Background substrate hydrolysis was deducted from each measurement. Free FAs released are the mean ± SD of triplicate repeats. * P < 0.001. This experiment was repeated three times, and similar results were obtained.