Lipids alkaline hydrolysis

In the experience of our laboratory, compared to methods involving extraction of total lipids, alkaline hydrolysis can give better recovery of FFA, at least in certain types of samples particularly powders (baby formula, soup, drinks), probably because of the strong association of lipid with other components and possibly the presence of lipid in a microencapsulated form. If the form (free or esterified) in which the fatty acids occur in the food is unknown, then it may be advisable to hydrolyze the sample so that all fatty acids are in the free form, and then use an appropriate derivatization method (see Section 2.3.3), although hydrolysis could also be done after hpid extraction. 

Alkaline hydrolysis (saponification) has been used to remove contaminating lipids from fat-rich samples (e.g., pahn oil) and hydrolyze chlorophyll (e.g., green vegetables) and carotenoid esters (e.g., fruits). Xanthophylls, both free and with different degrees of esterification with a mixture of different fatty acids, are typically found in fruits, and saponification allows easier chromatographic separation, identification, and quantification. For this reason, most methods for quantitative carotenoid analysis include a saponification step. 

An alternative procedure involves the release of the fatty acids by alkaline hydrolysis (saponification) by refluxing the extracted sample with dilute alcoholic potassium hydroxide for 1 h. After cooling, adding water and acidifying, the fatty acids are extracted into diethyl ether. The methyl esters can then be prepared by treatment with diazomethane, which may also be used directly on free fatty acids. Saponification is less satisfactory, because it is a lengthy procedure and often results in the loss of lipid components. 

Retinoids The challenge in fat-soluble vitamins analysis is to separate them from the lipid fraction that contains interferents. Alkaline hydrolysis, followed by LLE, is widely applied to remove triglycerides. This technique converts the vitamin A ester to all-trani-retinol. A milder process, which does not hydrolyze vitamin A ester, is alcoholysis carried out with metha-nolic KOH solution under specific conditions that favor alcoholysis rather than saponification. A more accurate explanation of this technique is reported in the book Food Analysis by FIPLC [409]. For some kind of matrices a simple liquid extraction can be sufficient with [421-423] or without [424,425] the purification... 

There are basically two mechanisms to convert the fatty acids in a complex lipid to fatty acid methyl esters (FAMEs) methylation following hydrolysis of the fatty acids from the complex lipids, or direct transesterification. The first mechanism involves saponification (alkaline hydrolysis) in which the ester bond is cleaved between the fatty acid and the glycerol moiety (e.g., triacylglycerols and phospholipids) under heat and in the presence of an alkali (usually sodium hydroxide), followed by methylation performed in the presence of an acidic catalyst in methanol. Direct transesterification is usually a one-step reaction involving alkaline or acidic catalysts. 

Saponification. Before solvent is added for extraction, saponification (alkaline hydrolysis) is a step used in most extractions of tocopherols and tocotrienols. It should be noted that acetate forms of tocopherols or tocotrienols in a sample are changed to free tocopherols and tocotrienols after saponification. This process breaks down the ester bonds of lipids and sample matrices as well. In most extraction procedures, a 60% to 80% (w/v) aqueous solution of KOH is used to perform the saponification. The volume of KOH required varies according to the amount of lipid contained in the sample. Also, ethanol is needed to stabilize the saponified solution and prevent the precipitation of soap material. Usually, the ratio of KOH, ethanol, and fat (in sample) during saponification is 3 (g) 15 (ml) 1 (g), respectively (Ball, 1988). The ratio may need to be adjusted based on the types of fats in the sample. Although ethanol concentration has no effect on the extraction of a-tocopherol by hexane, a concentration above 30% may cause lower recoveries of other tocopherols (Ueda and Igarashi, 1990). For most food samples, saponification for 30 min at70°C is sufficient. 

Methods of extracting the fat-soluble vitamin from food matrices include alkaline hydrolysis, enzymatic hydrolysis, alcoholysis, direct solvent extraction, and supercritical fluid extraction of the total lipid component. 

Hie first preparation of an LPS derivative which retained immunostimulatory activity yet had significantly reduced toxicity was made in 1979 by Ribi et al. (1979) who found that mild acid hydrolysis of LPS reduced the pyrogenicity without affecting tumor inhibition activity. Uiis reduced-toxicity product was shown to be the 4/-monophosphoryl derivative of lipid-A (MPL ) (Qureshi et al., 1982). Ribi s group later demonstrated that further alkaline hydrolysis of MPL resulted in de-acylation at the 3 position, producing a molecule with even lower toxicity... 

Triglycerides are hydrolyzable into their component fatty adds and glycerol. They are espedally susceptible to alkaline hydrolysis. If KOH or NaOH is used, the process is saponification and the products, sodium and potassium salts of fatty adds, are called soaps. In the human organism, triglycerides are hydrolyzed by various esterases called lipases. These enzymes are quite spedfic, and they do not necessarily remove all three fatty add molecules from a triglyceride molecule. Thus, pancreatic lipase, the main lipid digestive enzyme of the small intestine, catalyzes the removal of fatty acids from positions 1 and 3 only. 

Alkaline hydrolysis (e.g., the Schmidt-Thann-hauser procedure, which employs 1 N KOH at 37°C for 24 hr) cleaves most ester bonds but leaves the amide groups in sphingolipids intact. Alcoholic alkali may be necessary with lipid mixtures that do not disperse readily into aqueous solutions. Potassium hydroxide is preferable because it is more soluble in alcohol than NaOH similarly, potassium salts of fatty acids are more soluble than their equivalent sodium salts. 

Alkaline hydrolysis (saponification) can be used to remove interfering lipids and chlorophylls present in plant and food samples, such as... 

Acid hydrolysis is effectively performed using hydrochloric acid (6M) and heating at 85 °C, followed by dilution with water and extraction of sterols with a mixture of heptane/diethyl ether (1 1). Saponification (alkaline hydrolysis) of the lipids is commonly obtained by hydrolyzing the samples in alkaline alcoholic solutions (treatment with potassium hydroxide under reflux for 30 min to 3h) (Goad and Akihisa, 1997 Nystrom et al., 2009). For a more detailed example of hydrolysis procedures, see Nystrom et al. (2009). 

Extraction parameters such as solvent type, mixture ratios, metal ion concentration, pH of the aqueous phase, extraction time, and temperature influence the recovery of extracted lipids and must be validated to ensure reliable results. For example, the recovery of the acidic lipids PA and phosphatidylglycerol (PG) can be less than 30% in classic Folch and Bligh Dyer extraction, where these lipids can become bound to proteins tightly (17). Lipids bound to proteins covalently are only released under appropriate conditions, which depend on the type of lipid-protein linkage. For example, ceramides bound to protein of the comi-fled envelop in the human skin (18) can be extracted after mild alkaline hydrolysis of the ester linkage between hpid and protein. Special conditions are required for extraction of more polar lipids such as gangliosides, lysophospholipids and lysosphin-golipids, or phosphatidylinositol-phosphates. 

The principal lipid constituent of the tubercle bacilli was a wax-like substance. Anderson obtained this by chloroform extraction of fat-free bacilli. The preliminary removal of lipids with alcohol and other solvents served to dissolve the free fatty acids, neutral fat and phospha-tides. The crude wax was fractionated with alcohol or acetone. The purified derivative was a white amorphous powder containing 0.41% nitrogen and 0.77% phosphorus. The substance was very resistant to acid hydrolysis. Continuous treatment with acidified alcohol resulted in very slow hydrolysis. Alkaline hydrolysis proceeded very rapidly, with the formation of ether-soluble components together with a water-soluble polysaccharide. 

Alkaline hydrolysis of forskolin is inhibited in a lipid emulsion.550 Indomethacin in an oil-water gel is solubilized in the surfactant aggregates and stabilized against acid- and base-catalyzed hydrolysis.551... 

Wittig reactions then afforded the unsaturated phenolic lipids after demethylation and alkaline hydrolysis as depicted in Schemes 5b and c. 

The ability of PI synthetase to use 5-deoxy-5-fluoro-myo-inositol (4) as a substrate was confirmed by use of a radiolabeled compounds as shown in Figure 7. PI synthetase incorporated the analog into lipid in a time-dependent manner. The incorporation was absolutely dependent on the presence of CDP-diglyceride and was inhibited by the presence of myo-inositol (1) in the incubation mixture, as expected for PI synthetase. Chromatography of the reaction mixture revealed that a single radiolabeled product was formed with a mobility similar to, but distinct from, that of PI. Subsequent analysis has shown that the product is converted to a water-soluble form on mild alkaline hydrolysis and yields 5-deoxy-5-fluoro-myo-inositol (4) on treatment with phospholipase D, in agreement with the formation of phosphatidyl-5-deoxy-5-fluoro-myo-inositol as the product (data not shown). Determination of the absolute structure of these phospholipids awaits large-scale enzymatic synthesis, isolation of the product, and studies by mass spectrometry and NMR spectroscopy. 

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