Fatty acid substrates encountered

Lipogenesis is controlled by a number of mechanisms, including allosteric effectors, covalent modification, and availability of substrate. Pyruvate is an excellent potential precursor for fatty acids, particularly in the liver. One of the difficulties encountered is that pyruvate can proceed to acetyl CoA in the mitochondrion, however, acetyl CoA in the mitochondrion will not directly produce fatty-acid synthesis because this process occurs in the cytosol. 

The conversion of a monounsaturated fatty acid to acetyl-GoA requires a reaction that is not encountered in the oxidation of saturated acids, a cis-trans isomerization (Figure 21.9). Successive rounds of P-oxidation of oleic acid (18 1) provide an example of these reactions. The process of P-oxidation gives rise to unsaturated fatty acids in which the double bond is in the trans arrangement, whereas the double bonds in most naturally occurring fatty acids are in the cis arrangement. In the case of oleic acid, there is a cis double bond between carbons 9 and 10. Three rounds of P-oxidation produce a 12-carbon unsaturated fatty acid with a cis double bond between carbons 3 and 4. The hydratase of the P-oxidation cycle requires a trans double bond between carbon atoms 2 and 3 as a substrate. A cis-trans isomerase produces a trans double bond between carbons 2 and 3 from the cis double bond between carbons 3 and 4. From this point forward, the fatty acid is metabolized the same as for saturated fatty acids. When oleic acid is P-oxidized, the first step (fatty acyl-GoA dehydrogenase) is skipped, and the isomerase deals with the cis double bond, putting it into the proper position and orientation to continue the pathway. 

A soluble extract was obtained by Lynen and Tada (1961) fromP. patulum that required acetyl-CoA and malonyl-CoA plus NADPH for activity synthesis of 6-methylsalicylic acid, therefore, involves a reductive step to remove an oxygen function. These substrates are identical to those encountered in fatty acid synthesis, and this has led to the proposal that a multienzyme complex similar to fatty acid synthetase might be involved (Lynen and Tada, 1961 Lynen, 1961). Active thiol sites associated with this enzyme were also implicated in binding the intermediates by thioester linkage. The general mechanism applicable to the two synthetases, therefore, appears to be very similar, and the importance of their relationship to each other may be stressed. 

Nearly all analysts then are going to make use of polar stationary phases for the major proportion of their work. Any attempt at a comprehensive account of what has been achieved with columns of this type would be only slightly more readable than a telephone directory. I have therefore elected to show what can be accomplished with a "standard" 25 m WCOT column of fused silica coated with Carbowax 20M , and with a similar column coated with a highly polar phase CP-Sil 84 . As the first substrate for analysis, pig testis (obtained from an abattoir) lipids were selected, as the fatty acids have been well-characterised and have been used as an external standard in the Hormel Institute for some years [392,393] they contain a wide range of fatty acids of the (n-6) series, encountered typically in animal tissues. The second substrate is cod liver oil (obtainable from any pharmacy), which has also been well-characterised and is used as an external standard in the analysis of lipids of marine origin [15] it contains many different fatty acids, and especially those of the (n-3) family. Both of these materials were used by the author in studies of the efficacy of picolinyl ester derivatives of fatty acids for identification by GC-mass spectrometry (see Chapter 7), so all the main components have been identified unequivocally [173,184]. 

Most natural fatty acids are unsaturated. In addition, as discussed before (section 3.1.3) most double bonds are cis and, in polyunsaturated fatty acids, are methylene interrupted (three carbons apart). When unsaturated fatty acids are /3-oxidized, two problems may be encountered - the unsaturated acids have cis double bonds and these may be at the wrong position for )8-oxidation. The German biochemist, Stoffel, has shown that an isomerase exists to convert the cis-3 compound into the necessary trans-l-fatty acyl-CoA. The isomerase will also act with trans-3 substrates, though at lower rates. Once over this obstacle, -oxidation can again continue to eliminate a further two carbons and then dehydrogenation to produce a 2-tranSyA-cis-decadienoyl-CoA from linoleoyl-CoA. The discovery of a 2,4-dienoyl-CoA... 

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