Analyzing Fatty Acids in Fish and Human Cells with LC–MS

Scientists from the Institute of Marine Research in Bergen, Norway created a new system for comparing resolvins in human and fish cells. Information about the technique, which is based on liquid chromatography–mass spectrometry (LC–MS) methods, was published in the Journal of Chromatograhy B (1).

The fatty acid α-linolenic acid is the metabolic precursor of the polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Mammals, including humans, cannot synthesize the fatty acid alpha-linolenic acid (ALA) from the beginning, and are therefore dependent on dietary ALA for the endogenous production of EPA and DHA. Many fish species also require dietary ALA, but the ability to elongate and desaturate ALA into EPA and DHA is very limited in humans and marine fish, or even non-existent in some marine fish species; as such, dietary EPA and DHA are needed to meet physiological demands.

Resolvins are lipid mediators that are vital for resolving inflammatory processes in living organisms. Several studies in mammals have shown that resolvins promote tissue repair while helping to restore physiological homeostasis in murine wound healing models of peritonitis, mucosal injury, and corneal injury, as well as in a human intestinal epithelial cell model (2–5). Resolvins have also been shown to alleviate both inflammatory and neuropathic pain by modulating the activity of immune and glial cells, thus contributing to inflammation resolution in the nervous system. However, studies on resolvins’ effects on fish health are scarce.

In this research, a LC–MS method was validated for resolvins E (RvE) and D (RvD) released into culture media by salmon head kidney cells and human peripheral blood mononuclear cells exposed to ALA, EPA, and DHA. Assay performance was evaluated using fish and human cell culture media, demonstrating acceptable results that followed international guidelines. Assay performance was evaluated using both fish and human cell culture media, demonstrating acceptable results according to international guidelines. Key performance parameters included selectivity, range (0.5–50 ng/mL), linearity (R² = 0.98–0.99), limits of detection (∼0.02–0.09 ng/mL), limits of quantification (∼0.08–0.3 ng/mL), and accuracy (∼97–109 %).

The validated method enabled a reliable cross-species comparison, revealing that the DHA → RvD conversion is the predominant pathway in both fish and human cells. In contrast, the EPA → RvE and ALA-indirect pathways (ALA → EPA/DHA → RvE/RvD) contributed minimally to both species. This hints that the contributions of the indirect enzymatic pathway ALA → EPA → DHA to the production of resolvins is very low and similar in both salmon and human cells.

Direct conversion DHA → RvD1–5 was declared the dominant enzymatic pathway of resolving biosynthesis in fish and human cells, outperforming both EPA and ALA. Surprisingly, EPA supplementation produced little activation of the EPA → RvE1 enzymatic pathway, especially in humans. Meanwhile, the study also revealed that producing E-series resolvins could be promoted by a retroconversion pathway DHA → EPA that was the main source of RvE1.

The capacity to produce resolvins is species-dependent, but the mechanisms behind resolvin production are the same. For example, salmon cells biosynthesize resolvins at significantly higher rates than human cells, using the same direct enzymatic pathways, more specifically, DHA for production of D-series resolvins, and DHA-to EPA back-loop for E-series resolvins.

References

(1) Araujo, P.; Espe, M.; Holen, E.; Austgulen, M. H.; et al. Cross-Species Comparison of Resolvin E and Resolvin D Biosynthesis: Quantification by Liquid Chromatography Mass Spectrometry in Fish and Human Cells Exposed to Alpha-Linolenic Acid, Eicosapentaenoic Acid, and Docosahexaenoic Acid. J. Chromatogr. B 2025, 1264, 124729. DOI: 10.1016/j.jchromb.2025.124729

(2) Bannenberg, G. L.; Chiang, N.; Ariel, A.; Arita, M. Molecular Circuits of Resolution: Formation and Actions of Resolvins and Protectins. J. Immunol. 2005, 174 (7), 4345–4355. DOI: 10.4049/jimmunol.174.7.4345

(3) Colgan, S. P. Resolvins Resove to Heal Mucosal Wounds. Proc. Natl. Acad. Sci. U.S.A. 2020, 117 (20), 10621–10622. DOI: 10.1073/pnas.2005652117

(4) Zhang, Z. Technology-Adjusted National Carbon Accounting for a Greener Trade Pattern. Energy Econ. 2018, 73, 274–285. DOI: 10.1016/j.eneco.2018.05.025

(5) Storniolo, C. E.; Pequera, M.; Vilariño, A.; Moreno, J. J. Specialized Pro-Resolvin Mediators Induce Cell Growth and Improve Wound Repair in Intestinal Epithelial Caco-2 Cell Cultures. Prostaglandins Leukot. Essent. Fat. Acids 2022, 187, 102520. DOI: 10.1016/j.plefa.2022.102520