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J Lipid Res. 2018 Mar;59(3):542-549. doi: 10.1194/jlr.M083030. Epub 2018 Jan 20.

Altered eicosanoid production and phospholipid remodeling during cell culture.

Author information

1
Department of Biochemistry, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.
2
Department of Pharmacology, Anschutz Medical Campus, University of Colorado Denver, Aurora, CO 80045.
3
Department of Pharmacology, Anschutz Medical Campus, University of Colorado Denver, Aurora, CO 80045 Robert.Murphy@ucdenver.edu.

Abstract

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B2, PGE2, and PGD2, and the 5-lipoxygenase pathway, leukotriene (LT)B4, LTC4, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB3, all trans-LTB3, LTC3, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.

KEYWORDS:

Mead acid; polyunsaturated fatty acid; volcano plot

PMID:
29353239
PMCID:
PMC5832926
[Available on 2019-03-01]
DOI:
10.1194/jlr.M083030

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