title: Bioconversion of α-linolenic acid into n-3 long-chain polyunsaturated fatty acid in hepatocytes and ad hoc cell culture optimisation.
abstract: 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.
authors.affiliation: School of Life and Environmental Sciences, Deakin University, Victoria, Australia.
authors.forename: Ramez
authors.initials: R
authors.lastname: Alhazzaa
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Sinclair
authors.forename: Giovanni M
authors.initials: GM
authors.lastname: Turchini
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D003470:Culture Media / Q000378:metabolism; D004281:Docosahexaenoic Acids / Q000378:metabolism; D015118:Eicosapentaenoic Acid / Q000378:metabolism; D015525:Fatty Acids, Omega-3 / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D007700:Kinetics; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D053858:Metabolic Networks and Pathways; D051381:Rats; D015203:Reproducibility of Results; D013997:Time Factors; D017962:alpha-Linolenic Acid / Q000378:metabolism*
chemical_list: D003470:Culture Media; D015525:Fatty Acids, Omega-3; D005231:Fatty Acids, Unsaturated; D017962:alpha-Linolenic Acid; D004281:Docosahexaenoic Acids; D015118:Eicosapentaenoic Acid; C026219:docosapentaenoic acid
references.citation: J Lipid Res. 2007 Jul;48(7):1592-8
references.pmid: 17409318
references.citation: Cell Metab. 2007 Jun;5(6):415-25
references.pmid: 17550777
references.citation: J Nutr. 1998 Feb;128(2 Suppl):427S-433S
references.pmid: 9478042
references.citation: J Am Coll Nutr. 1986;5(2):183-211
references.pmid: 2873160
references.citation: Prog Lipid Res. 2010 Apr;49(2):186-99
references.pmid: 20018209
references.citation: Mol Cell Biochem. 1992 Nov 18;117(2):107-18
references.pmid: 1336810
references.citation: J Allergy Clin Immunol. 1984 Sep;74(3 Pt 2):430-6
references.pmid: 6088616
references.citation: J Lipid Res. 1963 Apr;4:151-9
references.pmid: 14168145
references.citation: PLoS One. 2013;8(2):e57463
references.pmid: 23460861
references.citation: Mar Biotechnol (NY). 2009 Sep-Oct;11(5):627-39
references.pmid: 19184219
references.citation: Diabetes Metab. 2003 Nov;29(5):478-85
references.pmid: 14631324
references.citation: J Nutr. 2012 Aug;142(8):1449-55
references.pmid: 22739372
references.citation: J Biol Chem. 1966 Nov 25;241(22):5213-9
references.pmid: 5927998
references.citation: Br J Nutr. 2002 Oct;88(4):355-63
references.pmid: 12323085
references.citation: J Biol Chem. 1982 Dec 25;257(24):14968-72
references.pmid: 7174678
references.citation: Br J Nutr. 2009 Jul;102(1):69-81
references.pmid: 19123959
references.citation: J Nutr. 1997 Jan;127(1):146-52
references.pmid: 9040558
references.citation: J Lipid Res. 2001 Apr;42(4):581-6
references.pmid: 11290830
references.citation: Biochim Biophys Acta. 1970 Jun 9;210(1):15-27
references.pmid: 5456034
references.citation: Nutr Metab (Lond). 2009 Feb 19;6:8
references.pmid: 19228394
references.citation: Biochem J. 2000 Sep 15;350 Pt 3:765-70
references.pmid: 10970790
references.citation: Biochim Biophys Acta. 1999 Apr 19;1438(1):63-72
references.pmid: 10216281
references.citation: Prostaglandins Leukot Essent Fatty Acids. 2009 Feb-Mar;80(2-3):85-91
references.pmid: 19269799
references.citation: Reprod Nutr Dev. 1994;34(6):539-68
references.pmid: 7840871
references.citation: Prostaglandins Leukot Essent Fatty Acids. 2011 Sep-Oct;85(3-4):155-61
references.pmid: 21807486
references.citation: Food Chem. 2013 Nov 15;141(2):1424-32
references.pmid: 23790934
references.citation: J Lipid Res. 2005 Jul;46(7):1474-83
references.pmid: 15834128
references.citation: J Agric Food Chem. 2011 Aug 10;59(15):8423-34
references.pmid: 21707030
references.citation: Prog Lipid Res. 1982;21(1):1-45
references.pmid: 6287500
references.citation: J Lipid Res. 1995 Mar;36(3):552-63
references.pmid: 7775867
references.citation: PLoS One. 2012;7(10):e47813
references.pmid: 23110104
references.citation: J Biol Chem. 1968 Sep 10;243(17):4440-51
references.pmid: 4971695
references.citation: PLoS One. 2011;6(12):e29662
references.pmid: 22216341
references.citation: Mol Cell Biochem. 1995 Feb 23;143(2):99-105
references.pmid: 7596353
references.citation: Prostaglandins Leukot Essent Fatty Acids. 2007 Nov-Dec;77(5-6):251-61
references.pmid: 18060754
references.citation: J Biol Chem. 1957 May;226(1):497-509
references.pmid: 13428781
references.citation: J Nutr. 2013 Jan;143(1):12-6
references.pmid: 23173174
references.citation: Lipids. 2007 Nov;42(11):1065-71
references.pmid: 17701238
references.citation: Prostaglandins Leukot Essent Fatty Acids. 2013 Jan;88(1):53-60
references.pmid: 22521090
references.citation: Curr Opin Lipidol. 2008 Jun;19(3):242-7
references.pmid: 18460914
references.citation: Prostaglandins Leukot Essent Fatty Acids. 2013 Jan;88(1):139-46
references.pmid: 22515943
references.citation: J Nutr. 1996 Sep;126(9):2199-208
references.pmid: 8814208
references.citation: J Lipid Res. 2001 Aug;42(8):1257-65
references.pmid: 11483627
references.citation: Comp Biochem Physiol A Mol Integr Physiol. 2013 Jun;165(2):243-53
references.pmid: 23524056
references.citation: Prog Lipid Res. 2009 Nov;48(6):355-74
references.pmid: 19619583
references.citation: Am J Clin Nutr. 1991 Sep;54(3):438-63
references.pmid: 1908631
references.citation: Lipids. 2002 Aug;37(8):733-40
references.pmid: 12371743
references.citation: J Nutr. 1993 Jul;123(7):1313-9
references.pmid: 8100576
references.citation: Lipids. 2001 Sep;36(9):975-9
references.pmid: 11724470
references.citation: Am J Clin Nutr. 1995 Feb;61(2):320-4
references.pmid: 7840069
references.citation: Pediatr Res. 1999 Jan;45(1):87-93
references.pmid: 9890614
references.citation: Biochem J. 1972 Apr;127(2):375-85
references.pmid: 4342556
references.citation: Pharmacol Ther. 2012 Apr;134(1):82-106
references.pmid: 22252140
references.citation: Lipids. 1991 May;26(5):374-80
references.pmid: 1832732
references.citation: J Nutr. 2006 Jan;136(1):83-7
references.pmid: 16365063
references.citation: J Lipid Res. 2005 Apr;46(4):706-15
references.pmid: 15654130