A novel method for measuring human lipoprotein lipase and hepatic lipase activities in postheparin plasma

Abstract

The objective of this study was to establish a new lipoprotein lipase (LPL) and hepatic lipase (HL) activity assay method. Seventy normal volunteers were recruited. Lipase activities were assayed by measuring the increase in absorbance at 546 nm due to the quinoneine dye. Reaction mixture-1 (R-1) contained dioleoylglycerol solubilized with lauryldimethylaminobetaine, monoacylglycerol-specific lipase, glycerolkinase, glycerol-3-phosphate oxidase, peroxidase, ascorbic acid oxidase, and apolipoprotein C-II (apoC-II). R-2 contained Tris-HCl (pH 8.7) and 4-aminoantipyrine. Automated assay of lipase activities was performed with an automatic clinical analyzer. In the assay for HL 1 LPL activity, 160 ml R-1 was incubated at 37°C with 2 ml of sample for 5 min, and 80 ml R-2 was added. HL activities were measured under the same conditions without apoC-II. HL and LPL activities were also measured by the conventional isotope method and for HL mass by ELISA. Lipase activity detected in a 1.6 M NaCl-eluted fraction from a heparin-Sepharose column was enhanced by adding purified apoC-II in a dose-dependent manner, whereas that eluted by 0.8 M NaCl was not. Postheparin plasma-LPL and HL activities measured in the present automated method had high correlations with those measured by conventional activity and mass methods. This automated assay method for LPL and HL activities is simple and reliable and can be applied to an automatic clinical analyzer.-Imamura, S., J. Kobayashi, K. Nakajima, S. Sakasegawa The diagnosis of LPL and HL deficiency is based on the lack of their respective enzyme activities in postheparin plasma (PHP) from affected individuals (1). The conventionally available method for measuring LPL and HL activity has been the use of 3 H-or 14 C-labeled trioleoyl glycerol as substrate in the presence of 1 M NaCl or the anti-LPL monoclonal antibody, 5D 2 (10, 11); remaining activity is considered to be HL activity under these conditions. However, this assay procedure is complicated and is not suitable for routine clinical or research work. Methods for measuring LPL or HL protein mass by ELISA in PHP have been developed by several groups, including ours, in the last two decades (10-15). These methods have provided considerable evidence for understanding how the amount of LPL or HL protein changes in variable metabolic disorders. To diagnose LPL or HL deficiency, it is essential to determine the absence of the respective lipase activity in PHP, because there are patients with normal mass and lipase activity for each (1). Very recently, we reported a new and simple assay

Myocardial FFA metabolism during rest and atrial pacing in humans

“…We infused a small amount of heparin to maintain patency of the arterial line in each study. The amount of heparin used to mobilize lipoprotein lipase, which would increase FFA concentration, is ϳ60 U/kg, or 4,400 units for the average subject in this study (16). We infused only 15 U/h, and therefore, we did not observe changes in FFA concentration outside the normal increase with duration of fasting.…”

Lipoprotein lipase does not increase significantly in the postprandial plasma

“…The pre-heparin LPL and HTGL mass were determined with a highly sensitive and specific ELISA [20,21]. The LPL and HTGL activities were determined with the automated assay [22]. Plasma samples were kept frozen at −80°C until analysis.…”

“…The LPL and HTGL activities were determined by an automated activity assay [22] for measuring the increase in absorbance at 546 nm by means of a quinoneine dye. Reaction mixture-1 (R-1) contained dioleoylglycerol solubilized with lauryldimethylaminobetaine, monoacylglycerol-specific lipase, glycerolkinase, glycerol-3-phosphate oxidase, peroxidase, ascorbic acid oxidase and apolipoprotein C-II (apoC-II).…”

Cardiac production of B-type natriuretic peptide is inversely related to the plasma level of free fatty acids in obese individuals - possible involvement of the insulin resistance -

“…Thus, the results of this study may not be generalizable to other populations. And finally, low dose heparin was used for cardiac catheterization and this might have affected FFA levels [46]. However, we addressed the issue of relationship…”