Triolein emulsions

The LPL catalytic assay measures the hydrolysis of a [14C[- or [3H]-triolein emulsion producing the 14C- or 3H -labeled free oleic acid [6]. The 14C- or 3H-labeled oleic acid is isolated by a selective extraction procedure and its radioactivity is determined by liquid scintillation counting [40]. Lipase activity is calculated as nanomoles of oleic acid released per minute per milliliter of postheparin plasma [41]. 

Ten microliters of each sample will be added to 500 pi of the triolein emulsion in triplicate and the HL-catalyzed triglyceride hydrolysis will be allowed to proceed for 1 h at 28°C. The reaction is stopped by adding 5.33 ml of methanolichloroform heptane (56 50 4 by volume) and vigorous shaking. 

Porcine lipase exhibits a series of unusual properties (142). The most interesting one, directly connected with its structure, is certainly its specific interactions with emulsified esters. When an aqueous solution of lipase at pH 5 is mixed with an excess of a triolein emulsion and when the cream is separated by centrifugation, no lipase can be detected in the clear aqueous lower phase. All activity is in the cream. But it returns at once into water when the emulsion is broken (145). This simple experiment suggests that lipase is adsorbed at the oil/water interface of the emulsion and that this interface may be the normal site of its action. Such an assiunption is confirmed by a series of experiments with substrates and inhibitors. 

Fig. 15. Exclusive action of porcine lipase on emulsified esters (146). Ordinates activity in perj cent of maximal activity on triolein emulsion. Abscissas lower axis, substrate amounts expressed in fractions of saturation for the solutions (on the left of the vertical dotted line) or in multiples of saturation for the emulsions (on the right of the line) upper axis, interfacial area expressed in 10 X cm in 100 ml. White circles, impure lipase containing some esterases. Black triangles, purified lipase. The substrate is triacetin on the left and methylbutyrate on the right. 

Fig. 16. Electrophoretic separation of the lipase and esterase activities of porcine pancreas (146, 147). Starch columns equilibrated with 0.025 M acetate buffer, pH 5.25. The activities of the fractions have been determined (o) on emulsions of triolein and tributyrin (black circles), methyl oleate, methyl laurate, and p-nitro-phenyllaurate (black triangles), (b) On solutions of methyl butyrate and p-nitro-phenylacetate (crosses). White circles and dotted line, protein background. Figures along the first peak give the specific activity (lipase) of some fractions, determined against triolein emulsion. Ordinates and abscissas are the same as in Fig. 14.

Topical pancreatic lipase substrates like tributyrin and triolein emulsions are hydrolyzed by carboxylester lipase in the presence of bile salt but slowly—at a rate lower than 3% and 0.5%, respectively, of that observed with the lipase-colipase complex. On the other hand, the positional specificity is not restricted all three sn positions of triglycerides can be split by the carboxylester lipase. While long-chain phospholipids are resistant, short-chain phospholipids are readily attacked by carboxylester lipase [40]. The low substrate specificity of carboxylester lipase makes possible an essential role for this enzyme in the hydrolysis of triglycerides containing certain esterified polyunsaturated fatty acids, such as eicosapentaenoic, arachidonic, or linoleic acids [41], and which may be resistant to attack by pancreas lipase (see p. 190). 

Lymphatic uptake of the lipid soluble dye Sudan blue and Vitamin A acetate from triolein-in-water emulsions requires the presence of both bile salts and phosphatidylcholine [237]. However, the overall contribution of lymphatic uptake for these two solutes is very small. In the absence of sodium taurocholate in rats with bile hstulae the administration of 20 ml polysorbate 80 did not stimulate lymphatic transport, although absorption from a 4 % polysorbate 80 solution is equivalent to that for the triolein emulsions in intact amounts, but slightly more rapid. 

Multivesicular Liposomes Kim and his colleages described a method for the preparation of cell size liposomes with high encapsulation efficiency the so-called multivesicular liposomes (Kim et al., 1983). The lipid phase consists of a combination of amphiphatic lipids and a small amount of triglycerides (triolein or trioctanoin) dissolved in chloroform-diethyl ether (1 1). The aqueous phase is slowly added to the organic phase and after vigorous shaking a water-ip-lipid emulsion is formed (Fig. 2A-B). Via a narrow Pasteur pipet the emulsion is subsequently added to a sucrose solution. 

Emulsion for each 1 ml of final suspension, add to the tube or vial from Step 1 above, 10 pmol of phosphatidylcholine, and 5 pmol of cholesterol from the stock solutions, plus 50 pmol (48 pi) of triolein (or other oil, see above) and dry with a N2 stream at the... 

Arimoto, I., Matsumoto, C., Tanaka, M., Okuhira, K., Saito, H., and Handa, T. (1998), Surface composition regulates clearance from plasma and triolein lipolysis of lipid emulsions, Lipids, 33,773-779. 

Figure 11 Hydrolysis of an artificial triolein/phospholipid/cholesteiol oleate emulsion with either pancreatic phospholipase A2 (PLA2) and/or pancreatic carboxylester lipase (CEL) in the presence of 10 mM bile salt. Closed squares PLA2 + CEL open squares CEL alone. Hydrolysis is monitored using tritiated tracers. (From Ref. 61.)...

Myocardial LPL activity was estimated in post-heparin perfusate ( heparin-releasable ) and in acetone/ether-dried ground tissue powders ( residual-tissue ) by using a H-labelled triolein substrate emulsion and counting radioactivity in evolved fatty acids extracted in methanol/chloroform/heptane. ... 

Sarda and Desnuelle (6) showed more recently that methyl butyrate was hydrolyzed by pure pancreatic lipase, provided the ester was present in an emulsion, but that the hydrolysis was very much slower than that of tributyrin. Similarly, methyl oleate is hydrolyzed by lipase at only one thirtieth the rate of triolein (6). 

Fig. 1. Variation of the rate of hydrolysis of simple glycerides with the chain length of the fatty acid. Each experiment was carried out on an emulsion with an optimum inteifacial area. Hydrolysis rates are expressed as a percentage of the rate of hydrolysis of triolein (241).

Fic. 8. Variation of the rate of lipase hydrolysis of triolein and tributyrin with the interfacial area of the substrate emulsions. 0=triolein, B=tributyrin (6),... 

Stern and Shapiro (1954) incubated the intact mesenteric fat of starved rats with serum and observed a deerease in the total serum esterified fatty acid content but no decrease in the phospholipid or cholesterol ester fraction. When mesenteric adipose tissue was incubated with sodium stearate-1-C or sodium oleate, a significant uptake could be demonstrated and the fatty acids that disappeared from the medium could be recovered in good yield inside the tissue. Of the stearate absorbed, 20-30% was found in the tissue in a combined form, presumably in ester linkage. Stern and Shapiro (1954) also showed that adipose tissue can absorb fatty acids from emulsions of triolein, sorbitan-monolaurate, sorbitan-monopalmitate, and... 

Serum levels of this enzyme increase up to four-fold in acute pancreatitis. It can be estimated by hydrolysis of olive oil emulsion and titration of the liberated fatty acids [479 —480] or radiochemically using triolein labelled with l or C and solvent extraction of the reaction mixture [481-482]. 

Trioleoylglycerol (triolein) and olive oil are commonly used substrates, and stable emulsions may be readily obtained for use as substrate preparations. Enzyme activity can be monitored by a pH stat or analysis of released fatty acids (Brockerhoff and Jensen, 1974). The sensitivity of low-level activities may be increased with radioactive substrates. Tributyrin is commonly used because of its ease of dispersion in water, but it cannot be assumed that tributyrin hydrolysis is necessarily a measurement of true lipase activity. Triacetin is water-soluble, and although often used for lipase determinations this practice is not recommended. (A major commercial enzyme supplier markets wheat germ lipase but states on the containers that the preparation will not hydrolyze olive oil—it is, however, active on triacetin.)... 

Cohen, Morgan, and Hofmann obtained gastric juice by tube from 73 normal human subjects, and they said their samples were less than 3% contaminated with duodenal juice. They measured the lipolytic activity of their samples against that of triolein emulsified with sodium taurocholate at pH 6, having previously found a broad optimum between pH 2 and 6 with that substrate. They said the pH optimum was lower with shorter-chain triglycerides as substrates. When trioctanoin was the substrate, lipolysis was inhibited by added octanoate. This, they said, is in part explained by the fact that hydrolysis of the substrate was incomplete, for octanoate can accumulate at the oil-water interface of the emulsion. Hydrolysis of triglycerides of shorter chain length was more complete, for the liberated fatty acid could diffuse more rapidly into the aqueous phase. 

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