Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing.

Autor: Lin P; Center for Environmental and Systems Biochemistry, Dept. of Toxicology and Cancer Biology, Markey Cancer Center, University of Kentucky, Lexington, KY, USA., Sledziona J; Department of Biological Chemistry and Pharmacology, The Ohio State University, 1060 Carmack Rd, Columbus, OH, 43210, USA; The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA., Akkaya-Colak KB; Department of Biological Chemistry and Pharmacology, The Ohio State University, 1060 Carmack Rd, Columbus, OH, 43210, USA; The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA., Mihaylova MM; Department of Biological Chemistry and Pharmacology, The Ohio State University, 1060 Carmack Rd, Columbus, OH, 43210, USA; The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA., Lane AN; Center for Environmental and Systems Biochemistry, Dept. of Toxicology and Cancer Biology, Markey Cancer Center, University of Kentucky, Lexington, KY, USA. Electronic address: andrew.lane@uky.edu.
Jazyk: angličtina
Zdroj: Analytica chimica acta [Anal Chim Acta] 2024 May 15; Vol. 1303, pp. 342511. Date of Electronic Publication: 2024 Mar 21.
DOI: 10.1016/j.aca.2024.342511
Abstrakt: Background: Mammalian cells both import exogenous fatty acids and synthesize them de novo. Palmitate, the end product of fatty acid synthase (FASN) is a substrate for stearoyl-CoA desaturases (Δ-9 desaturases) that introduce a single double bond into fatty acyl-CoA substrates such as palmitoyl-CoA and stearoyl-CoA. This process is particularly upregulated in lipogenic tissues and cancer cells. Tracer methodology is needed to determine uptake versus de novo synthesis of lipids and subsequent chain elongation and desaturation. Here we describe an NMR method to determine the uptake of 13 C-palmitate from the medium into HCT116 human colorectal cancer cells, and the subsequent desaturation and incorporation into complex lipids.
Results: Exogenous 13 C 16 -palmitate was absorbed from the medium by HCT116 cells and incorporated primarily into complex glycerol lipids. Desaturase activity was determined from the quantification of double bonds in acyl chains, which was greatly reduced by ablation of the major desaturase SCD1.
Significance: The NMR approach requires minimal sample preparation, is non-destructive, and provides direct information about the level of saturation and incorporation of fatty acids into complex lipids.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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