Lipase Assays

Henriksen EL, Petersen PH, Beck-Nielsen H, Horder M (2001) Calibration, specificity and trueness of a postheparin plasma lipoprotein lipase assay. Clin Chem Lab Med 39 263-269... 

Aside from the time required to prepare reagents, the least amount of time is required per lipase assay by the spectrophotometric method, and the greatest amount of time is required per assay for the titrimetric method. Although all assays are described as requiring up to 30 min for the reaction mixture to be subsampled, time savings can be realized by subsampling more frequently over a shorter period of time, as long as one obtains valid initial rate data. Thus, for all assays, the time involved to run the lipase reaction can be normalized to be the same at -10 to 15 min. The difference in time requirements for the protocols becomes embedded in sample workup procedures. 

Kwon, D.Y. and Rhee, J.S. 1986. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Am. Oil Chem. Soc. 63 89-92. 

An exhaustive account of 73 commercially available lipase preparations, comparing activities using six hydrolytic and five esterification assay substrate systems. Illustrates the wide range of activities between lipases and for a given lipase assayed by different protocols. 

Transesterification, fatty acid analysis of lipids, 437, 439 Triacetin, lipase assays, 378 Triacylglycerol acylhydrolase, 371, 375, 378. See also Lipases Triacylglycerols, 432 Tributyrin, lipase assays, 378 Trichloroacetic acid (TCA) solubility index for protein hydrolysis, 152 in TBARS determination, 548-550 Trienes, conjugated, determination of, 515-517, 523-524, 526, 528 Trifluoroacetic acid (TFA), for determination of neutral sugars, 721-722, 724-725, 729-730... 

Scheme 1.5 Fluorescence lipase assay on a solid support.

E682 Tetrault, G. (1990). Serum lipase assayed on Ektachem 700 is not specific for pancreatitis in patients with elevated amylase or lipase. Clin. Chem. 36, 1123, Abstr. 800. 

EN102 Guidi, A.J., Jablonski, E. and Yeo, K..-T. (1992). Evaluation of the Raichem lipase assay on the Hitachi 717 chemistry system, Clin, Chem. 38, 1053, Abstr. 

A new lipase assay has been developed and used to screen the Aventis compound library. Several classes of hits have been elucidated - one of which, the oxadiazo-lones, has been selected for a chemical optimization program. Computer-assisted experimental design has enabled optimization wifh respect to activity and selectivity. Small potent inhibitors were obtained that are drug-like and follow fhe Li-pinsky rules of five, which are often useful for a first theoretical prediction of oral bioavailability [54]. Ongoing pharmacological trials will show if lipase inhibitors can successfully treat diabetes and afherosclerosis. 

R. Gupta, P. Rathi, N. Gupta, and S. Bradoo, Lipase assays for conventional and molecular screening an overview, Biotechnol. Appl. Biochem., 2003, 37, 63-71. 

With method D, the inhibitor was previously dissolved into the substrate before carrying out the lipase assay (Fig. 9.5 D). This inhibition method was used with the pH-stat, rnonomolecular films and oil drop techniques. Lipase hydrolytic activity was then continuously recorded as described below. 

After a few minutes of incubation with orlistat, the HPL initial activity measured on both tributyrin (Fig. 9.13) and PSO (data not shown) emulsions was drastically reduced. The lipolytic activity gradually increased with time, however, reaching a steady state regime after a lag period of a few minutes. This reactivation phenomenon is probably due to a partial deacylation, during the lipase assay, of the covalent HPL-orlistat complex. 

In the lipase assay system, when the (E, THLc, E-THLoj and E-THL02) mixture was added to a TAG emulsion, the weakef complex (E-THLoj) was hydrolyzed, explaining the reactivation process as revealed by the existence of lag times. The hydrolysis of this weak complex was enhanced when bile salts were present in the assay medium, probably due to the formation of a mixed orlistat/lipase/bile salt complex. The fact that the reactivation of HPL did not reach 100% of its initial value (before incubation with THL) at the end of the kinetic experiments (Fig. 9.13) might be attributable to the existence of a second fraction of lipase molecules, which is covalently and irreversibly inhibited by orlistat (E-THL02). The coexistence of two different forms of orlistat covalently bound to HPL may be due to the inhibitor molecule being differently oriented in the catalytic cavity of the lipase molecule. The existence in the catalytic cleft of lipases of two different orientations in the case of molecules of substrate analogues was previously reported [113, 114]. 

Lipoprotein Lipase, Assay of, in vivo and in vitro (Korn). 7 145... 

Figure 2. Principle of the fluorescence lipase assay. The fluorogenic substrate contains a fluorophore and a fluorescence quencher. Release of the quencher fatty acid by a lipolytic enzyme leads to a continuous increase in fluorescence intensity from which enzyme activity can be determined.

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