Bioconversion of alpha Linolenic Acid into n3 Long Chain Polyunsaturated Fatty Acid in Hepatocytes and Ad Hoc Cell Culture Optimisation

This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 183n  3 (ALA) bioconversion into n  3 long chain polyunsaturated fatty acid, and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 205n  3 (EPA) production from 50 mM ALA initial concentration was achieved after 3 days of incubation. Next, it was established that a gradual increase in the ALA concentration from 0 up to 125mM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 225n  3 (DPA) and 226n  3 (DHA) in 3 day incubations. Of interest, ALA bioconversion products were observed in the culture medium. Therefore, in vitro experiments disregarding the medium fatty acid content are underestimating the metabolism efficiency. The novel application of the fatty acid mass balance (FAMB) method on cell culture system (cells with medium) enabled quantifying the apparent enzymatic activities for the biosynthesis of n  3 LC  PUFA. The activity of the key enzymes was estimated and showed that, under these conditions, 50 per cent (Km) of the theoretical maximal Fads2 activity on ALA can be achieved with 81 mM initial ALA. Interestingly, the apparent activity of Elovl2  was the slowest amongst other biosynthesis steps. Therefore, the possible improvement of Elovl2 activity is suggested toward a more efficient DHA production from ALA. The present study proposed and described an ad hoc optimised cell culture conditions and methodology towards achieving a reliable experimental platform, using FAMB, to assist in studying the efficiency of ALA bioconversion into n  3 LC  PUFA in vitro. The FAMB proved to be a powerful and inexpensive method to generate a detailed description of the kinetics of n  3 LC  PUFA biosynthesis enzymes activities in vitro. (Authors abstract)

Quantifying the endogenous conversion of 183n  3 (ALA) into n3 long chain PUFA is not yet optimised and still surrounded with confusion. Species specific, tissue specific and other intrinsic factors appear to affect this bioconversion as physiological state and pathological conditions in vertebrates. Liver is known to be the major organ for lipid metabolism, where hepatocytes contain the necessary enzymes for the elongation and desaturation of ALA to 205n  3 (EPA) and 22:6n  3 (DHA). Therefore, hepatocytes have been used regularly in PUFA metabolism studies. Numerous reports have inferred the kinetics of fatty acid (FA) metabolism by analysing FA composition in tissues after controlled feeding experiments, while other studies used in vivo, ex vivo or in vitro approaches with labelled FA. A whole body in vivo FA mass balance (FAMB) method has enabled the estimation of the overall capacity of an organism to metabolise FA. It is envisaged that combining the advantage of results reproducibility obtained from a cell line platform with the FAMB approach could provide a detailed insight on the efficiency of ALA bioconversion into EPA and DHA. In the present study, mammalian hepatocytes were utilised in order to establish an optimised experimental platform for investigating ALA conversion into n  3 LC  PUFA in vitro. The objectives were to determine the most effective duration and concentration of ALA to be converted into n  3 LC  PUFA in hepatocytes and to estimate the apparent enzymatic activities through this pathway by implementing FAMB on the whole flask. In the present study, FaO cells with 50 mM of ALA added recorded a peak of EPA at 3 days, and this was mainly incorporated into the phospholipid fraction of the cells. A novel observation of this study was that significant amounts of ALA bio converted products were found to be exported by the cells into the culture medium. Discarding, or not considering, the medium FA content, can affect the interpretation of results and the understanding of the dynamics of the bioconversion pathways itself, as n  3 LC  PUFA in the medium are reported to have direct feedback on ALA metabolism. Therefore, considering the entire flask FA composition (cells with their culture medium), seems to be necessary for the accurate assessment of the bioconversion dynamics in vitro. The efficiency of ALA bioconversion to EPA and DHA in hepatocytes has been commonly attributed to enzyme affinity, substrate availability and transcriptional factors in experiments assessing FA metabolism in the cells alone, but the presence of bioconversion products is also known to have direct effects. In fact, competition between ALA and other n  3 LC  PUFA has been suggested to limit DHA accumulation in hepatocyte membrane in vitro, and increased availability of n  3 LC  PUFA in medium is known to down regulate the transcription rate of enzymes involved in n  3 LC  PUFA biosynthesis. Therefore, it is suggested that EPA appearing from ALA could be responsible for slowing down the subsequent steps of n  3 LC  PUFA production, with the above mentioned feedback mechanisms.  In the present study, the maximal EPA production in FaO cells added with 50 mM of ALA was recorded at 72 h, and therefore 3 days incubation duration was selected for studying the optimal ALA concentration. However, it should be noted that in the following two days, FA composition of cells was not static, and actually, during days 4 and 5 a reduction of EPA and an increase in DPA and DHA levels were apparent, clearly suggesting that a longer time period would have been required for allowing the complete bioconversion of ALA up to DHA. In the present study, it was clearly shown that, amongst all the bioconversion steps, the fastest and more efficient one was the elongation (Elovl5) of SDA to ETA, followed by the D  5 desaturation (Fads1) of ETA to EPA, then the D 6 desaturation (Fads2) of ALA to SDA, and eventually the elongation (Elovl2 plus Elovl5) of EPA to DPA was the slowest recorded.  Therefore, a key outcome of the present study was that it was clearly shown that rather than the existence of a single rate limiting step affecting the overall pathway, a combination of different level of efficiency in each enzymatic step is responsible for the production of n  3 LC  PUFA biosynthesis. It should be noted that the amount of product generated by an enzyme is not only relative to the activity (velocity) of the enzyme itself, but also the time available for this reaction. Accordingly, the elongation of EPA was the slowest recorded step in n  3 LC  PUFA biosynthesis, and because of this limited DPA production, it was not possible to record any specific trend in its further bioconversion towards the final production of DHA.

Two important observations: i) cell culture based studies aiming at assessing DHA production should consider longer incubation time, and ii) further investigations are warranted towards a better understanding of Elovl2, as the possible slowest step in the production of DHA from ALA in hepatocytes. It has been suggested that rat Elovl2, expressed in yeast, controls DHA synthesis from EPA or DPA. The application of FAMB method in lipid metabolism research has increased in the last few decades as a practical and accurate alternative to costlier analysis. However, specific variability of an individual sample would need to be to be taken into consideration, if the method was to be applied to different animals. In culturing established cell lines, maintaining defined equipment and materials of the same high quality guarantees reproducible and reliable results. Therefore, fewer variations are expected to influence FAMB computations when applied on cell culture flasks compared with living animals. The current study has demonstrated FAMB method as a powerful, informative and inexpensive tool for FA metabolism research in vitro and to generate detailed description for the kinetics of n  3 LC  PUFA biosynthesis enzymes activities. (Editors comments)