α-Eleostearic acid-containing triglycerides for a continuous assay to determine lipase sn-1 and sn-3 regio-preference

Lipases are essentially described as sn-1 and sn-3 regio-selective. Actually few methods are available to measure this lipase regio-selectivity, moreover they require chiral chromatography analysis or specific derivations which are discontinuous and time consuming. In this study we describe a new, convenient, sensitive and continuous spectrophotometric method to screen lipases regio-selectivity using synthetic triglycerides (TG) containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) either at the sn-1 position [1-α-eleostearoyl-2,3-octadecyl-sn-glycerol (sn-EOO)] or at the sn-3 position [1,2-octadecyl-3-α-eleostearoyl-sn-glycerol (sn-OOE)] and coated onto the wells of microtiter plates. A non-hydrolysable ether bond, with a non UV-absorbing alkyl chain, was introduced at the other sn positions to prevent acyl chain migration during TG synthesis or lipolysis. The synthesis of TG containing α-eleostearic acid was performed from S-glycidol in six steps to obtain sn-EOO and in five steps to sn-OOE. The α-eleostearic acid conjugated triene constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the TG harboring it. The lipase activity on coated sn-EOO or sn-OOE was measured by the increase in the absorbance at 272nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. Human and porcine pancreatic lipases, guinea pig pancreatic lipase related protein 2, Thermomyces lanuginosus lipase, Candida antarctica lipase A and Candida antarctica lipase B were all used to validate the assay. This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases.