Use of stable isotope-labeled fatty acids to measure desaturase activities with negative chemical ionization GC-MS

IF 3.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Youenn Launay , Iwan Jan , Vincent Ciesielski , Lydie Hue , Mélodie Succar , Léa Fret , Thomas Guerbette , Karima Begriche , Philippe Legrand , Daniel Catheline , Manuel Vlach , Vincent Rioux
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引用次数: 0

Abstract

Fatty acid desaturases are key enzymes in lipid metabolism. They introduce double bonds between defined carbons of the fatty acyl chain and catalyze rate-limiting steps in the biosynthesis of polyunsaturated fatty acids. For decades, in vitro desaturase activities have been determined by using radiolabeled fatty acids as substrates, incubated with tissue or cell fractions containing membrane-bound desaturases. However, handling radioactivity is being increasingly complicated due to safety and regulatory concern. Radiolabeled fatty acids are also expensive and many of them are not commercially available. There is therefore a crucial need to develop new methods. Although methods using unlabeled fatty acids as substrates have recently been validated, they are well suited for large tissue samples and did not achieve the same sensitivity as the radioactive ones. Here, we show that negative chemical ionization GC-MS on stable isotope-labeled fatty acids, derivatized to pentafluorobenzyl esters, now offers this opportunity, because of its high sensitivity in the selected ion monitoring mode. By using this simple and affordable improved method, we measured the kinetic parameters of mouse liver Δ6-desaturase for its two main substrates (C18:2 n-6 and C18:3 n-3; 10–13 µM). Moreover, this method enabled to compare Δ5-desaturase apparent Km values (19–22 µM) for its two main substrates (C20:3 n-6 and C20:4 n-3). Finally, we re-evaluated the controversial effect of freezing on desaturase activities by using both frozen rat tissues and cryopreserved human hepatocytes. This safe, reliable and sensitive method may be applied to other enzymatic activities involving fatty acids (elongation, hydroxylation) in miniaturized samples.
利用负化学电离 GC-MS 测量稳定同位素标记的脂肪酸的去饱和酶活性。
脂肪酸去饱和酶是脂质代谢中的关键酶。它们在脂肪酰基链的特定碳之间引入双键,并催化多不饱和脂肪酸生物合成过程中的限速步骤。几十年来,体外去饱和酶活性的测定一直是以放射性标记的脂肪酸为底物,与含有膜结合去饱和酶的组织或细胞组分进行孵育。然而,出于安全和监管方面的考虑,处理放射性越来越复杂。此外,放射性标记的脂肪酸价格昂贵,而且很多都无法在市场上买到。因此,亟需开发新的方法。虽然使用未标记脂肪酸作为底物的方法最近得到了验证,但这些方法非常适合大量组织样本,而且无法达到与放射性方法相同的灵敏度。在这里,我们展示了以五氟苄基衍生化的稳定同位素标记脂肪酸为底物的负化学电离气相色谱-质谱(GC-MS),由于其在选择离子监测模式下的高灵敏度,现在提供了这一机会。利用这种简单而经济的改进方法,我们测量了小鼠肝脏Δ6-去饱和酶对其两种主要底物(C18:2 n-6 和 C18:3 n-3;10-13 µM)的动力学参数。此外,这种方法还能比较Δ5-去饱和酶对其两种主要底物(C20:3 n-6 和 C20:4 n-3)的表观 Km 值(19-22 µM)。最后,我们利用冷冻的大鼠组织和冷冻保存的人类肝细胞,重新评估了冷冻对去饱和酶活性的争议性影响。这种安全、可靠和灵敏的方法可用于微型样本中涉及脂肪酸(伸长、羟基化)的其他酶活性。
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来源期刊
Chemistry and Physics of Lipids
Chemistry and Physics of Lipids 生物-生化与分子生物学
CiteScore
7.60
自引率
2.90%
发文量
50
审稿时长
40 days
期刊介绍: Chemistry and Physics of Lipids publishes research papers and review articles on chemical and physical aspects of lipids with primary emphasis on the relationship of these properties to biological functions and to biomedical applications. Accordingly, the journal covers: advances in synthetic and analytical lipid methodology; mass-spectrometry of lipids; chemical and physical characterisation of isolated structures; thermodynamics, phase behaviour, topology and dynamics of lipid assemblies; physicochemical studies into lipid-lipid and lipid-protein interactions in lipoproteins and in natural and model membranes; movement of lipids within, across and between membranes; intracellular lipid transfer; structure-function relationships and the nature of lipid-derived second messengers; chemical, physical and functional alterations of lipids induced by free radicals; enzymatic and non-enzymatic mechanisms of lipid peroxidation in cells, tissues, biofluids; oxidative lipidomics; and the role of lipids in the regulation of membrane-dependent biological processes.
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