PUFA, isolation

In 1987, Corey reported an effective process for the isolation of DHA (7) from fish oil containing a mixture of PUFAs by iodolactonization of 7, forming the y-lactone 106, which could then be isolated and converted back to 7 [81]. Later, Kuklev and coworkers used fish oil containing 35% DHA (7) as the starting material for the synthesis of aU-d5 -3,6,9,12, 15-octadecapentaenoic acid (107) [82], a PUFA isolated from the microalgae Gymnodinium kowalevskii (Scheme 3.28). Methanolysis of the iodolactone 106 under basic conditions afforded epoxy ester 108, which could be cleaved with periodic... 

Studies conducted by Barenghi eta.1. (1990) and Lodge etal. (1993) independently have demonstrated the facile, multicomponent analysis of a wide range of PUFA-derived peroxidation products (e.g. conjugated dienes, epoxides and oxysterols) in samples of oxidized LDL by high-field H-NMR spectroscopy. Figure 1.9 shows the applications of this technique to the detection of cholesterol oxidation products (7-ketocholesterol and the 5a, 6a and 5/3,60-epoxides) in isolated samples of plasma LDL pretreated with added coppcr(Il) or an admixture of this metal ion with H2O2, an experiment conducted in the authors laboratories. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Although LOX from soybean seed is the best characterized of plant LOXs, this enzyme is present in a wide variety of plant and animal tissues (Liavonchanka and Feussner, 2006). The enzyme occurs in a variety of isoenzymes, which often vary in their optimum pH and in product and substrate specificity. Given the occurrence of multiple LOX isoenzymes in soybean leaves and the proposed roles of these enzymes in the plant metabolism, it is possible that individual isoenzymes play specific functions (Feussner and Wasternack 2002). The molecular structure of soybean LOX is the most reported, and four isoenzymes have been isolated (Baysal and Demirdoven 2007). Soy isoenzyme 1 produces 9- and 13-hydroperoxides (1 9) when the enzyme acts on free PUFA at pH 9.0, its optimum pH (Lopez-Nicolas and others 1999). Soy isoenzyme 2 acts on triglycerides as well as free PUFA leading to 9- and 13-hydroperoxide... 

The most logical material for the analysis of PUFA and plasmalogens are the erythrocytes. Fatty acids can also be quantitated in plasma. The plasmalogens are also easily detectable in homogenates of cultured fibroblasts and may add to the definitive diagnosis of patients with a generalised or isolated peroxisomal dysfunction. 

To extend this unique fungal ability of fatty acid transformation, several kinds of PUFA biosynthesis mutants of M. alpina 1S-4 have been isolated through... 

Changes in EFA status affect the activity of several membrane-associated enzymes and proteins 68-71 Reduced adenyl cyclase activity occurred in EFA-deficient animals,72 while in animals supplemented with n-6 or n-3 fatty acids, increased adenyl cyclase activity was seen in cardiac membranes.73,74 However, the opposite effect has been reported in other membranes, possibly reflecting differences in initial fatty acid composition.75 n-3 PUFAs have been shown to activate membrane Ca-ATPase and inhibit Na, K-ATPase in isolated basolateral membranes from rat duodenal enterocytes76 and inhibit both Ca-ATPase and Na,K-ATPase activity in synaptosomal membranes isolated from rat cerebral cortex.77 EFAs have the ability to modify neuronal Ca-ATPase activity... 

Figure 11. The GLC C20 region of a menhaden omega-3 PUFA concentrate (ethyl ester) (a) before and (b) after heat treatment at 220° C, and (c) the 20 5 region of an artifact concentrate isolated by AgN03 column chromatography. Peaks A-E refer to artifacts formed after heat treatment. Analysis on a SUPELCOWAX-10 fused-silica capillary column operated isothermally at 195X1. Note that components B-E fall into the region where several 22 1 isomers may be found (cf. Figs. 5 and 7). From (77).

The first elongase involved in PUFA biosynthesis was isolated from the fungus M. alpine [242]. The specific enzyme participating in the elongation of GLA and SDA, was identified employing a cDNA yeast expression library made from the fungus. After isolation of a... 

Apart from the above mentioned enzymes, there also exist a few enzymes involved in PUFA metabolic pathways that have recently been found. One of them is a novel n-3 fatty acid desaturase, isolated from an EPA-rich fungus, Saprolegnia diclina [248]. The gene was isolated by PCR amplification from a fungus cDNA library and then, expressed in S. cerevisiae, which was cultured in the presence of several FA as substrates. The study showed that the recombinant protein could exclusively desaturase C20 n-6 PUFAs, with preference for AA which was converted into EPA. This represents a completely novel and different activity from any organism previously described, and its potential for use in EPA production in transgenic oilseed crops has been outlined. 

The feeding of an EPA-free source of supplementary DHA (from algae) to human volunteers indicated a metabolic retroconversion of DHA to EPA (Conquer and Holub, 1996, 1997). Previous animal and in vitro studies in isolated rat liver cells have demonstrated that DHA can be retroconverted to EPA, and that this retroconversion is a peroxisomal function (Schlenk et al., 1969 Gronn et al., 1991). Studies in isolated rat liver cells by Schlenk et al., 1969 have also indicated that the resultant EPA can be chain-elongated to DPA (22 5n-3) for subsequent esterification into cellular lipids. An acyl-CoA oxidase has been identified as the enzyme responsible for the chain shortening of DHA in the peroxisomal beta-oxidation of PUFA in human fibroblasts (Christensen et al., 1993). The aforementioned in vivo human studies have estimated the extent of retroconversion of DHA to EPA to be approximately 10% (Conquer and Holub, 1996, 1997). 

ISOLATION OF PUFA VIA LIPASE-CATALYZED SELECTIVE ESTERIFICATION... 

Synthesis of a structured TAG with PUFA confined to either the l-(3-) or 2-position requires 1,3-selective lipases to interesterify a TAG using FFA or fatty acid esters (reaction 1), or to transesterify two different populations of TAG molecules (Fig. 3). Alternatively, a two-step approach can be employed, where a TAG is first subjected to 1,3-selective lipolysis, resulting in 2-MAG that is carefully isolated, then crystallized, to prevent acyl migration. The second step is 1,3-selective esterification of the 2-MAG.f " Such an approach results in a 72-85% yield and >95%... 

Lipases have also been employed to isolate tocopherols and sterols, important antioxidants from deodorizer distillate, a by-product formed during the deodoriza-tion step of seed oil purification, by hydrolyzing MAG and DAG in the distillate, and by esterifying sterols into sterol esters. The latter serves as antioxidant in non-polar food products such as margarine (e.g., Benacol from the Raisio Group, Finland and Take Control from Unilever, Englewood Cliffs, New Jersey, U.S.A.). PUFA-sterol esters would provide the benefits of essential fatty acid intake and antioxidant protection. 

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