Lipases triglyceride determination

Consumer acceptance of milk is strongly determined by its sensory characteristics. The development of off-flavor in milk as a result of lipolysis can reduce the quality of milk. The enzymatic release, by milk lipase, of free fatty acids (FFA) from triglycerides causes a flavor defect in milk described as rancid . Triglycerides in milk contain both long chain and short chain fatty acids, which are released at random by milk lipase. The short chains FFA, like butyric acid, are responsible for the off-flavor. 

H. Uzawa, Y. Nisbida, H. Ohrui, H. Meguro, A New Approach to Determine the Stereospecificity in Lipase Catalyzed Hydrolysis Using Circular Dichroism (CD) Lipases Produce Optically Active Diglycerides from Achiral Triglycerides , Biochem. Biophys. Res. Commun. 1990, 168, 506-511. 

Enzymatic hydrolysis is a nondestructive alternative to saponification for removing triglycerides in vitamin K determinations. For the simultaneous determination of vitamins A, D, E, and K in milk- and soy-based infant formulas and dairy products fortified with these vitamins (81), an amount of sample containing approximately 3.5-4.0 g of fat was digested for 1 h with lipase at 37°C and at pH 7.7. This treatment effectively hydrolyzed the glycerides, but only partially converted retinyl palmitate and a-tocopheryl acetate to their alcohol forms vitamin D and phyllo-quinone were unaffected. The hydrolysate was made alkaline in order to precipitate the fatty acids as soaps and then diluted with ethanol and extracted with pentane. A final water wash yielded an organic phase containing primarily the fat-soluble vitamins and cholesterol. 

Potentiometry is another useful method for determining enzyme activity in cases where the reaction liberates or consumes protons. This is the so-called pH-stat method. pH is kept constant by countertitration, and the amount of acid or base required is measured. An example of the use of this method is the determination of lipase activity. The enzyme hydrolyzes triglycerides and the fatty acids formed are neutralized with NaOH. The rate of consumption of NaOH is a measure of the catalytic activity. 

Uzawa, H., Nishida, Y., Ohrui, H., and Meguro, H. 1990. A new approach to determine the stereospecificity in lipase catalysed hydrolysis using circular dichroism (CD) lipases produce optically active diglycerides from achiral triglycerides. Biochem. Biophys. Res. Comm., 168, 506-511. 

Villeneuve, P., Pina, M., Montet, D., and Graille, J. 1995. Determination of lipase specificities through the use of chiral triglycerides and their racemics. Chem. Phys. Lipids, 76,109-113. 

In either method the equilibrium of the dehydrogenase reaction, which hes to the left, is displaced in the forward direction by working at pH 9.8 and by trapping the dihydroxyacetone (phosphate) with hydrazine. Triglyceride concentrations can also be determined by one of these methods. The triglycerides are hydrolyzed, either with lipase or non-enzymatically, and the glycerol concentration is determined. 

Lipids such as triglycerides were determined with lipoprotein lipase and phospholipids with phospholipase D. In the case of triglycerides, good linearity between 0.05-10 mM (tributyrin) and 0.1-5 mM (triolein) was obtained, whereas for phospholipids linearity was obtained between 0.03-0.19 mM [41]. 

Fig. 8.3 Aliquots of EL, HL and LPL medium were assayed for triglyceride lipase and phospholipase activities [16]. The ratio of triglyceride lipase to phospholipase activity was then determined.

Thus, triglycerides have been determined with lipoprotein lipase (EC 3.1.1.34) immobilized on CPG with a pore size of 2000 A. The assay buffer was 0.1 M Tris buffer, pH 8.0, containing 0.5% Triton X-100. The linear response was 0.05-10 mM for tributyrin and 0.1-5 mM for triolein [24]. 

Fatty acids are absorbed from the bloodstream by adipocytes. Using glycerol-3-phosphate, produced as a by-product of glycolysis, triglycerides are synthesized. Triglycerides are constantly being hydrolyzed and resynthesized in adipocytes. The rates of hydrolysis and synthesis are determined by lipases that are under hormonal control. 

Lipases catalyze the hydrolytic cleavage of triglycerides into fatty acids and glycerol, or into fatty acids and mono or di-glycerides, at oil interfaces in nature. However, this hydrolytic reaction can be reversed and transformed into reactions of esterification (inter or transesterification), alcoholysis or aminolysis by engineering the medium polarity or the water content of the medium. Therefore, substrates for lipases can be esters, like the natural triglyceride substrates in hydrolytic reactions or, if the reaction is reversed, carboxylic acids, alcohols, amines or esters. The reaction medium not only determines the direction of the reaction (hydrolytic or synthetic), but also determines the solubility and stability of lipase substrates. Therefore, lipase activity and selectivity are strongly influenced by reaction medium. 

To throw some light upon mechanisms by which dietary fiber could influence plasma lipoproteins, heart and adipose tissue lipoprotein lipase activities were measured after feeding some of the dietary fiber containing diets. This key enzyme in lipoprotein metabolism is one of the most important factors determining plasma triglyceride and plasma HDL concentrations (16). 

Rates of lipase reaction can therefore be measured by determining either (a) the rate of disappearance of the substrate, the triglyceride, or (b) the rate of production of the fatty acids. Determination of the rate of diglyceride, monoglyceride, or glycerol produetion is theoretically possible, but is practically difficult and seldom carried out. 

The oil is obtained by pressing or solvent extraction. The refined oil is used for edible purposes. It is also widely used as a drying oil because of its nonyellowing characteristics and as a component of alkyd resins. Both the seed and the oil are free of nicotine. The meal is rich in protein and is used in cattle feed. The triglyceride composition determined by silver nitrate TLC and lipase hydrolysis is detailed in Table 3.128. 

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