Unlike the DGGR substrate, the absolute amount of alginate that was guluronate was not significant but as before the F(GG), F(GGG), and N(G > 1) still correlated significantly with lipase inhibition (Table 2). The adapted methods of Panteghini et al. (2001) and Vogel and Zieve (1963) are both effective for in vitro determination of pancreatic
lipase activity. There are drawbacks and advantages with both methods used in this paper. DGGR is a synthetic substrate whereas olive oil is a natural substrate, but being a natural substrate olive oil is a mixture of different triacylglycerol with varying acyl chain length, which will have differing affinity for the enzyme ( Jemel et al., 2009 and Rogalska http://www.selleckchem.com/products/wnt-c59-c59.html et al., 1990). The enzyme would also have to act on the substrate twice for there to be a detectable change in the optical density (OD), as diacylglycerol would not be solubilised and therefore not reduce the OD. This could explain the lower levels of inhibition seen using the olive oil as a substrate compared to the DGGR substrate which is only cleaved once. The two methods show relatively large error bars, which can be explained to some extent by the solubility of the substrate. This varied between the replicates however for each experiment the same substrate preparation has been used for the positive control, negative
control and the inhibition study. Both methods showed that alginates are effective inhibitors of pancreatic lipase, depending upon the structure. Alginates with a high G block content can inhibit lipase to a much greater extent than alginates CHIR99021 with high M block content. Therefore, it is possible to modulate the activity of pancreatic lipase to a varying degree depending upon the alginates used. Molecular weight of the alginates was not a determining factor of inhibition and neither was G protein-coupled receptor kinase viscosity as one of the best inhibitors, LFR5/60, has a viscosity of 6 mPas compared to a poor inhibitor, LF120L, which has a viscosity of 121 mPas (for 1% solution in phosphate buffered saline). There are several potential mechanisms for inhibition of lipase by alginate. Alginates have the potential to interact with both the
substrate and the enzyme itself. Alginates with a high G block content are known to interact with glycoprotein, specifically mucin measured by rheological assessment across a range of mucin: alginate ratios (Taylor et al., 2005a and Taylor et al., 2005b). It was hypothesised that alginate can interact with specific sites along the protein section of the glycoprotein, cross linking several mucin molecules together forming a gel (Taylor, Draget, Pearson, & Smidsrød, 2005). The G block content of the alginate was also key in the mucin interaction, as alginates with high mannuronate content would not interact and cross link the mucin molecules. Therefore showing that alginate can interact with protein and that G blocks are important for this interaction.