Figure 4. Comparison of the absolute number of Elovl2 and Elovl5 mRNA template copies / ng of total RNA in rat liver and heart. Elovl2 was detected in heart at ,10 copies / ng of total RNA. All measurements were performed in duplicate and the results are the mean 6 S.E.M. (n = 3). doi:10.1371/journal.pone.0029662.g004 was responsible. It is clear now that DPA does not inhibit SDA elongation because they are substrates for different enzymes, Elovl2 and Elovl5, respectively. However, we confirm that EPA does not appear to inhibit SDA elongation by Elovl5. The sequential reactions of EPARDPAR24:5n-3 performed by Elovl2 presented difficulties for examining possible competition between EPA and DPA (Fig. 8). Attempts were made to examine EPA/DPA competition, but results were uninterpretable due to DPA being both product and substrate with the same enzyme. Use of labelled EPA was considered, but was discounted due to similar concerns with interpretation of the results. However, the dose response of EPA with Elovl2 provided insights into the one enzyme dual reaction processes. With increasing EPA concentra- tions up to 400 mM, DPA increased proportionately whereas the subsequent product, 24:5n-3, reached a plateau at 200 mM EPA. This could reflect competition from EPA for the second reaction, DPAR24:5n-3. However, the simpler explanation is that the Elovl2 second reaction, DPAR24:5n-3, may be saturated at a lower substrate concentration than the first reaction, EPARDPA. This explanation is supported by the demonstration of apparent saturation of the reaction DPAR24:5n-3 using DPA as the exogenous substrate. Possible saturation of the Elovl2 second reaction in the absence of saturation of the first reaction provides an additional explanation for the increase of DPA but not DHA after certain concentrations/intakes of ALA have been exceeded. These conclusions are contingent in part on the expressed elongases being rate limiting for the process of elongation which requires the Elovl enzyme and three additional enzymes. The Elovl enzyme catalyses the initial condensation reaction while the enzymes required for the subsequent reduction-dehydration- reduction steps necessary for complete elongation are supplied by the yeast [10]. However, it has been shown that the condensation reaction is the rate-limiting step in elongation, even in a yeast heterologous expression system [10]. The rat Fads2 was active with the conventional D6-desaturase C18 and C24 PUFA substrates, but does not have D5- or D8- desaturase activity. This was unlike the baboon Fads2 which had D6- and D8-desaturase activity [11]. However, our findings with Fads2 are in agreement with previous studies that confirmed lack of D5-desaturase activity with the rat enzyme expressed in yeast or the mouse enzyme expressed in primary cultures of rat hepatocytes and in CHO cells, although these studies did not report activity values and did not examine C24 PUFA substrates [12,13]. A later study indirectly examined the rat D6-desaturase activity towards Role of Elongase Reactions in LCPUFA Synthesis Figure 5. The rat Fads2 substrate specificity. Recombinant S. cerevisiae expressing Fads2 were grown in the presence of 100 mM of various PUFA substrates. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to desaturation product was calculated as [product/(product + substrate)]6100. The product contained one more double bond than the substrate. The results are the means 6 S.D. of triplicate incubations. Values with different symbols are significantly different from each other. doi:10.1371/journal.pone.0029662.g005 the C24 substrates by expressing the enzyme in COS-7 cells [14]. The transfected cells were supplemented with DPA or 22:4n-6, relying on the endogenous cellular elongases to produce the D6- desaturase substrates 24:5n-3 and 24:4n-6 [14]. This study suggested that the rat D6-desaturase was more active towards ALA than 24:5n-3 although it is difficult to compare D6-desaturase activity when ALA was supplied exogenously but 24:5n-3 was formed endogenously. Fads2 has been accepted as a control point in the production of DHA from ALA. Fads2 is an essential enzyme because DHA was not present or substantially reduced in Fads2 knockout mice [15,16,17]. The contribution of Fads2 in controlling the synthesis of DHA is two-fold. It is rate-limiting for conversion of ALA to EPA [1,2,3] and it also converts 24:5n-3, the product of Elovl2 activity, to 24:6n-3. Thus, there is the potential for competitive C18 and C24 n-3 substrate interactions and this could limit one or both reactions. Although it has been suggested there is competition between the C18 and C24 n-3 substrates for Fads2, it had not been investigated directly [7,8]. The Fads2 dose response with ALA and 24:5n-3 was used to select a series of 24:5n-3/ALA ratios to examine competition. In ALA dietary studies with rats or humans where ALA is readily detectable in membrane phospholipids, 24:5n-3 is not detectable or barely detectable. Therefore, we examined ratios with ALA as the predominant fatty acid as we considered this would reflect in vivo ratios more closely compared with ratios in the other direction. Our study demonstrated that increasing concentrations of ALA can inhibit the conversion of 24:5n-3. This directly supports suggestions that the competition between ALA and 24:5n-3 may explain the decreasing DHA levels after certain intakes of ALA are exceeded [7,8]. The present study indicates that Elovl2 is another potential control point, additional to that of Fads2. The scheme in Fig. 8 places the elongases according to their substrate selectivities. Although both Elovl2 and Elovl5 could catalyse EPA to DPA PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29662