Electrochemical flow injection approach for routine screening of lipase activity in pancreatic preparations.

A state-of-the-art strategy for the determination of lipase activity in pancreatic preparations using flow injection analysis (FIA) with electrochemical detection (FIA-ED) is described. The procedure is based on the enzymatic reaction of a specific substrate (1,3-dilinoleoyl-glycerol) with lipase from porcine pancreas and determination of enzymatically formed linoleic acid (LA) at +0.4 V by applying a cobalt (II) phthalocyanine-multiwalled carbon-nanotubes modified carbon paste electrode (Co(II)PC/MWCNT/CPE). In order to get a high-performance analytical method, sample preparation, flow system, and electrochemical conditions were optimized. Under optimized conditions the lipase activity of porcine pancreatic lipase was calculated to be 0.47 units per mg lipase protein based on the definition that 1 unit hydrolyses 1 microequivalent linoleic acid from 1,3-dilinoleoyl-glycerol per 1 min at pH 9 and 20 °C (kinetic measurement: 0-25 min). Moreover, the developed procedure was shown to be easily adaptable for the fixed-time assay (incubation time 25 min) as well. In this case, linear correlation between flow signal and lipase activity was found in the range from 0.8 to 18 U L-1. LOD and LOQ were determined to be 0.3 U L-1 and 1 U L-1, respectively. The kinetic assay was further preferred for the determination of lipase activity in commercially available pancreatic preparations. The lipase activities of all preparations obtained by the present method were found to be in good correlation with those obtained by the titrimetric method and declared by manufacturers.

Voltammetric lipase activity assay based on dilinolein and a modified carbon paste electrode.

In this work, a novel electrochemical assay for characterizing both lipases and lipase inhibitors as well as for the determination of lipase activity is described. It is based on a carbon paste electrode, modified with cobalt(II)phthalocyanine, and multi-walled carbon nanotubes (MWCNTs). As reaction media, a sodium borate buffer was used (0.1 M, pH 9). The measurements were carried out in a batch system using differential pulse voltammetry (DPV) and 1,3-dilinolein as standard substrate. The activity assay showed a linearity for porcine pancreas lipase activity in a range between 20 and 300 U L-1 (per min) with a limit of detection (LOD) of 7 U L-1 and a limit of quantification (LOQ) of 20 U L-1. The kinetic behavior of the lipase reaction was investigated, resulting in a KM value of 0.29 mM. The applicability of the activity assay could be shown by investigating the activity of lipases from Aspergillus oryzae and Candida rugosa, and the results were confirmed by a reference method. The inhibitory effects were characterized with Orlistat.