Here we hypothesise that pancreatic lipase activity can CDK and cancer be inhibited by alginates and that the extent can be modulated to a different degree dependent on the structural characteristics of alginate used. Well characterised alginates from
both sources (bacteria and seaweed) were used in this study, including alginates that were enzymatically modified. All alginate samples were kindly provided by Technostics Limited (Hull, UK) (Table 1). The bile acids (deoxycholate sodium salt and taurodeoxycholate sodium salt) were both purchased from Fluka (Buchs, Switzerland). The lipase, colipase and orlistat (tetrahydrolipstatin), tris(hydroxymethyl)-methylamine, 1,2 Di-o-lauryl-rac-glycero-3-(glutaric GSK2118436 manufacturer acid 6-methyl resorufin ester) (DGGR), sodium acetate, calcium chloride and acetone were all purchased from Sigma–Aldrich (Poole, UK). The olive oil was purchased from a local supermarket (Cooperative Foods, UK) and the aluminium oxide was purchased from Fisher Scientific (Loughborough, UK). The lipase activity assay was a modified version of the method developed by Panteghini, Bonora, and Pagani (2001). The assay was comprised of three solutions; solution 1, solution
2 and the lipase solution. Solution 1; Tris buffer (50 mmol/l, pH 8.4 at 23 °C), 1 mg/l of colipase and 1.8 mM deoxycholate sodium salt. Solution 2; acetate buffer (18 mmol/l, pH 4.0 at 23 °C) 72 mM
taurodeoxycholate sodium salt, 0.1 mM calcium chloride and 0.24 mM DGGR. Solution 2 was mixed with a magnetic stirrer at 500 rpm and 4 °C overnight. The lipase solution contains 1 g/l of porcine pancreatic lipase in deionised water, where 1 mg contains 60 U of lipase activity (where one unit will hydrolyse 1.0 microequivalent of fatty acid from a triglyceride in one hour at pH 7.4 using triacetin). A 4 mg/ml stock solution of each polymer was prepared by slowly adding lyophilised biopolymer to the vortex formed by vigorously stirring solution 1 on a magnetic stirrer. The resulting stock solution (4 mg/ml) was then further diluted with solution 1 to achieve 1 and 0.25 mg/ml samples. This achieved a concentration of 3.43, 0.86 and 0.21 mg/ml, to respectively in the reaction mixture. Two controls were used in the assay, an inhibition control (100% inhibition) and a lipase control (0% inhibition). The inhibition control contained 0.025 mg/ml orlistat added to solution 1 and the lipase control was the standard reaction with no inhibitors or biopolymers. All solutions were stored at 4 °C for up to 24 h. The assay was set up over two 96 well microplates. The first contained 15 μl of solution 2 in every well. The second plate contained 180 μl of solution 1, or a concentration of biopolymer in solution 1.