La maladie coronarienne est l'une des principales causes de morbi-mortalité parmi les patients atteints d'une insuffisance rénale terminale (IRT). Le rôle de l'urémie comme facteur de risque indépendant de morbimortalité cardiovasculaire est incomplètement compris et pourrait impliquer une calcification des vaisseaux plus importante. Il semble également que les antiagrégants plaquettaires inhibant le récepteur P2Y12 à l'ADP comme le clopidogrel ou le prasugrel soient moins efficaces dans leur capacité à inhiber l'agrégation plaquettaire selon le degré d'insuffisance rénale chronique. Le cas d'un patient dialysé avec maladie coronarienne ayant présenté deux resténoses sur stent malgré un traitement de clopidogrel nous a conduit à une réflexion sur son efficacité dans ces conditions et à une revue de la littérature sur le niveau de preuve dans la prescription des antiagrégants plaquettaires lors de l'IRT. Les propriétés pharmacocinétiques de ces traitements sont potentiellement perturbées à différents niveaux (absorption, distribution, et métabolisme) pouvant conduire à une efficacité moindre qui a été décrite dans la littérature pour le clopidogrel et le prasugrel. Le ticagrélor semble donc être le traitement de choix dans cette indication chez le patient en IRT et sa prescription pourrait, à l'avenir, être quantifiée grâce au profil métabolomique des cytochromes P450 avant de débuter le traitement

Alhazzaa, Ramez

Sinclair, Andrew J.

Turchini, Giovanni M.

PLoS ONE

PubMed

This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 18:3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20:5n-3 (EPA) production from 50 µM 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 125µM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22:5n-3 (DPA) and 22:6n-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% (Km) of the theoretical maximal (Vmax = 3654 µmol.g−1 of cell protein.hour−1) Fads2 activity on ALA can be achieved with 81 µM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation) 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

Sinclair, A.

Turchini, G.

espace@Curtin

English

Bioconversion of α-linolenic acid into n-3 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 18:3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20:5n-3 (EPA) production from 50 µM 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 125 µM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22:5n-3 (DPA) and 22:6n-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% (Km) of the theoretical maximal (V max = 3654 µmol.g(-1) of cell protein.hour(-1)) Fads2 activity on ALA can be achieved with 81 µM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation) 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

Ramez Alhazzaa

Andrew J Sinclair

Giovanni M Turchini

Directory of Open Access Journals

Bioconversion of α-linolenic acid into n-3 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 18:3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20:5n-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 22:5n-3 (DPA) and 22:6n-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% (Km) of the theoretical maximal (Vmax = 3654 mmol.g21 of cell protein.hour21) Fads2 activity on ALA can be achieved with 81 mM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation) 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

Deakin Research Online

Bioconversion of α-Linolenic Acid into n-3 Long-Chain polyunsaturated fatty acid in hepatocytes and ad hoc cell culture optimisation

Andrew J. Sinclair

Giovanni M. Turchini

Crossref

Bioconversion of α-Linolenic Acid into n-3 Long-Chain Polyunsaturated Fatty Acid in Hepatocytes and Ad Hoc Cell Culture Optimisation

<div>This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 18∶3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20∶5n-3 (EPA) production from 50 µM 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 125µM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22∶5n-3 (DPA) and 22∶6n-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% (Km) of the theoretical maximal (Vmax = 3654 µmol.g−1 of cell protein.hour−1) Fads2 activity on ALA can be achieved with 81 µM initial ALA. Interestingly, the apparent activity of Elovl2 (20∶5n-3 elongation) 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.</div

Ramez Alhazzaa (457549)

Andrew J. Sinclair (457550)

Giovanni M. Turchini (382598)

FigShare

Bioconversion of α-Linolenic Acid into n-3 Long-Chain Polyunsaturated Fatty Acid in Hepatocytes and Ad Hoc Cell Culture Optimisation

https://figshare.com/articles/Bioconversion_of_Linolenic_Acid_into_n_3_Long_Chain_Polyunsaturated_Fatty_Acid_in_Hepatocytes_and_Ad_Hoc_Cell_Culture_Optimisation/797461