Flow Infusion Electrospray Ionization High-Resolution Mass Spectrometry of the Chloride Adducts of Sphingolipids, Glycerophospholipids, Glycerols, Hydroxy Fatty Acids, Fatty Acid Esters of Hydroxy Fatty Acids (FAHFA), and Modified Nucleosides

2020 ◽  
pp. 69-76
Author(s):  
Paul L. Wood ◽  
Randall L. Woltjer
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acids ◽  
Fatty Acid ◽  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
Hydroxy Fatty Acids ◽  
Modified Nucleosides ◽  
Fatty Acid Esters ◽  
Acid Esters ◽  
Resolution Mass

scholarly journals A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 118 ◽  
Author(s):  
Magdalena Topolska ◽  
Fernando Martínez-Montañés ◽  
Christer S. Ejsing
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acid ◽  
High Resolution ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
High Resolution Mass Spectrometry ◽  
Enzymatic Process ◽  
Product Specificity ◽  
High Resolution Mass ◽  
Resolution Mass

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of de novo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.

scholarly journals Sequestration of 9-Hydroxystearic Acid in FAHFA (Fatty Acid Esters of Hydroxy Fatty Acids) as a Protective Mechanism for Colon Carcinoma Cells to Avoid Apoptotic Cell Death

Cancers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 524 ◽  
Author(s):  
Juan P. Rodríguez ◽  
Carlos Guijas ◽  
Alma M. Astudillo ◽  
Julio M. Rubio ◽  
María A. Balboa ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cell Lines ◽  
Hydroxy Fatty Acids ◽  
Fatty Acid Esters ◽  
Protective Mechanism ◽  
Hydroxystearic Acid ◽  
Colorectal Tumors ◽  
Wide Range ◽  
Acid Esters

Hydroxy fatty acids are known to cause cell cycle arrest and apoptosis. The best studied of them, 9-hydroxystearic acid (9-HSA), induces apoptosis in cell lines by acting through mechanisms involving different targets. Using mass spectrometry-based lipidomic approaches, we show in this study that 9-HSA levels in human colorectal tumors are diminished when compared with normal adjacent tissue. Since this decrease could be compatible with an escape mechanism of tumors from 9-HSA-induced apoptosis, we investigated different features of the utilization of this hydroxyfatty acid in colon. We show that in colorectal tumors and related cell lines such as HT-29 and HCT-116, 9-HSA is the only hydroxyfatty acid constituent of branched fatty acid esters of hydroxyfatty acids (FAHFA), a novel family of lipids with anti-inflammatory properties. Importantly, FAHFA levels in tumors are elevated compared with normal tissue and, unlike 9-HSA, they do not induce apoptosis of colorectal cell lines over a wide range of concentrations. Further, the addition of 9-HSA to colon cancer cell lines augments the synthesis of different FAHFA before the cells commit to apoptosis, suggesting that FAHFA formation may function as a buffer system that sequesters the hydroxyacid into an inactive form, thereby restricting apoptosis.

scholarly journals A Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of Free Hydroxy Fatty Acids in Cow and Goat Milk

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3947 ◽  
Author(s):  
Maroula G. Kokotou ◽  
Christiana Mantzourani ◽  
Asimina Bourboula ◽  
Olga G. Mountanea ◽  
George Kokotos
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acids ◽  
Liquid Chromatography ◽  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
Goat Milk ◽  
Cow Milk ◽  
Hydroxystearic Acid ◽  
Resolution Mass

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.

scholarly journals Distinct biological activities of isomers from several families of branched fatty acid esters of hydroxy fatty acids (FAHFAs)

2021 ◽  
Vol 62 ◽  
pp. 100108
Author(s):  
Pratik Aryal ◽  
Ismail Syed ◽  
Jennifer Lee ◽  
Rucha Patel ◽  
Andrew T. Nelson ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Biological Activities ◽  
Hydroxy Fatty Acids ◽  
Fatty Acid Esters ◽  
Acid Esters

scholarly journals Branched Fatty Acid Esters of Hydroxy Fatty Acids Are Preferred Substrates of the MODY8 Protein Carboxyl Ester Lipase

Biochemistry ◽  
2016 ◽  
Vol 55 (33) ◽  
pp. 4636-4641 ◽  
Author(s):  
Matthew J. Kolar ◽  
Siddhesh S. Kamat ◽  
William H. Parsons ◽  
Edwin A. Homan ◽  
Tim Maher ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Hydroxy Fatty Acids ◽  
Fatty Acid Esters ◽  
Acid Esters

scholarly journals Study of the Royal Jelly Free Fatty Acids by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS)

Metabolites ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 40 ◽  
Author(s):  
Maroula G. Kokotou ◽  
Christiana Mantzourani ◽  
Rodalia Babaiti ◽  
George Kokotos
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acids ◽  
Liquid Chromatography ◽  
High Resolution ◽  
Free Fatty Acids ◽  
Royal Jelly ◽  
High Resolution Mass Spectrometry ◽  
High Resolution Mass ◽  
Resolution Mass

The lipidome of royal jelly (RJ) consists of medium-chained (8–12 carbon atoms) free fatty acids. We present herein a liquid chromatography-high resolution mass spectrometry (HRMS) method that permits the determination of RJ fatty acids and at the same time the detection of suspect fatty acids. The method allows for the direct quantification of seven free fatty acids of RJ, avoiding any derivatization step. It was validated and applied in seven RJ samples, where the major RJ fatty acid trans-10-hydroxy-2-decenoic acid (10-HDA) was found to vary from 0.771 ± 0.08 to 0.928 ± 0.04 g/100 g fresh RJ. Four additional suspect fatty acids were simultaneously detected taking advantage of the HRMS detection.

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