Lipolytic activity, assay

Fatty acids released by lipases can be determined quantitatively by TLC, GC and HPLC. TLC methods are used in conjunction with densitometric methods or autoradiographic methods using radiolabelled TAG. Many GC and HPLC methods that have been outlined earlier are widely used to isolate and quantify FFAs in lipolytic assays. Additionally a method using p-nitrophenyllaurate as a substrate was described by Maurich et al. (1991) who quantified activity by the release of p-nitrophenol. Veeraragavan (1990) used a RP-HPLC method with triolein as the substrate. Triolein was emulsified in buffer with the aid of a surface active agent and the lipase added under controlled conditions. Lipolytic activity was measured by the release of oleic acid and quantified by absorbance at 208 nm. 

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. 

The microbial lipolytic activity is determined by comparing the rate at which a solution of microbial lipase powder hydrolyzes a substrate of olive oil emulsion with the rate at which a solution of microbial lipase F.I.P. standard hydrolyzes the same substrate under the same conditions. It should be clearly stated that a lipase of microbial origin is involved to prevent confusion with the assay for pancreatic lipase. 

Administration of heparin has frequently been tried in EHL. Whereas no significant effect on plasma lipids was reported by Kuo et al. (1959) and in a heparin unresponsive subject with fat-induced hyperlipemia by Furman et al. (1961), the latter authors demonstrated significant improvement in plasma lipid levels of two other patients with carbohydrate-induced hyperlipemia. One might anticipate unresponsiveness to long term heparin administration with low postheparin lipolytic activity found with the in vitro assay of Fredrickson et al. (1963) (see Table 1). Such a correlation is not necessarily present as evidenced by the response in a child with the fat-induced variety, whom we have observed (Kinsell and Schlierf 1965). 

Methods. Lipolytic activity measurements using tri- C-oleoylglycerol and methylumbelliferyl esters in enzyme assays, or by measurement of total lipid and free fatty acid (FFA) levels were as described recently Oxygen uptake by aqueous suspensions of wholemeal, bran, etc. correlates very closely (r 0.96 P < 0.001) with FFA content and has been used as a rapid and simple routine method for measuring deterioration of such materials during storage... 

The diagnosis of primary hyperlipoproteinaemia can usually be confirmed, after exclusion of secondary causes, by an investigation of medical history, analysis (by electrophoresis and determination of blood lipids) of lipoprotein patterns and screening of near relatives. Further ambiguities may be removed by such procedures as the measurement of post-heparin plasma lipolytic activity or assay of LDL receptor function in cultured fibroblasts or blood lymphocytes. 

In addition to membrane-bound PLAiS, mammals have cytosolic PLAjS. Cytosolic PLAi activities have been studied in various tissues including heart, brain and testis. PA-PLAi has been purified from brain [45] and testes [39, 46]. Like other lipolytic enzymes, PLAi is affected by the assay conditions. Using a mixed micelle system Glomset and his colleagues [46] found that a 110 kDa enzyme from testes preferentially hydrolyzed phosphatidic acid. They cloned the PA-PLAi from bovine [16]. This PLAi lacks sequence similarity to type I PLAi, lysophospholipase, LCAT, and triacylglycerol lipases. 

Continuous assays are rarely described for lipases, but are frequently described for esterases. Similar to lipases, esterases hydrolyze ester bonds. However, in contrast to lipases, their substrates are water-soluble and thus water-soluble fluorescent substrates can be used to measure their enzymatic activity. Some of these water-soluble substrates have been proposed for the measurement of lipolytic ac-... 

Acid CEH has an optimum pH of 4.5 and is located within lysosomes (Fig. 1). Like many lipolytic enzymes, it is water soluble whereas its substrate is not. For this reason, devising a reliable assay method is difficult, and results should be viewed with caution [26,35]. Recently developed conditions foimd to yield satisfactory linearity with both time of incubation and enzyme concentration are 12.7 jitM cholesteryl oleate, dispersed in 1.27 mM egg PC 50 mM acetate buffer 2.0 mM sodium taurocholate 0.005% digitonin pH 3.9 [36]. In aortic cells these conditions gave an apparent of 1.5 /iM for cholesteryl oleate. It should be noted, however, that the nature, physical form, and molecular organization of the CE and associated molecules can have a critical effect on the activity of any preparation of CEH [37]. Rat liver acid CEH, for example, shows K s of 15.3, 14.3, and 7.3 jaM for cholesteryl oleate when the latter is present in vesicles, micelles, and emulsions, respectively [35]. 

We synthesized fluorogenic alkyldiacylglycerols and carboxylic acid esters that are useful substrates for the fast and accurate determination of activity and stereopreference of lipolytic enzymes in aqueous systems and organic solvents. The respective lipids can also be used for the discrimination of lipase and esterase activities. The continuous fluorescence assay can be performed using a cheap fluorometer or, for the analysis of many different samples at a time, a fluorescence plate reader... 

A great number of methods are availaible for assaying activities of lipolytic enzymes(l). The most commonly used techniques include titrimetric, radioactive, or optical methods. Most of these techniques are time-consuming and/or suffer from several shortcomings, such as poor reproducibility, lack of sensitivity, or the production of radioactive waste. Here, we describe the synthesis and application of a new class of fluorogenic substrates that are useful substrates for the fast and accurate determination of activity and stereoselectivity of lipolytic enzymes in aqueous media and organic solvents (4, 5)... 

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.

Background Serum biomarkers for exocrine pancreatic injury in humans and preclinical species traditionally include amylase and lipase. In terms of assay methodology, serum amylase and lipase use an activity-based endpoint, which utilizes the substrate (e.g., diacylglycerol is the lipolytic substrate for serum lipase) to detect the serum activity levels. The assays have been used for over a decade in diagnostic... 

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