A LC-MS–based workflow for measurement of branched fatty acid esters of hydroxy fatty acids

Zhang, Tejia; Chen, Shili; Syed, Ismail; Ståhlman, Marcus; Kolar, Matthew J.; Homan, Edwin A.; Chu, Qian; Smith, Ulf; Borén, Jan; Kahn, Barbara B.; Saghatelian, Alan

doi:10.1038/nprot.2016.040

Cited by 65 publications

(111 citation statements)

References 32 publications

(52 reference statements)

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“…We have an established method to separate and analyze these different families and isomers using reversed-phase chromatography, 15 however, this method is unable to resolve stereoisomers. The first step in determining the stereochemistry of natural FAHFAs required us to develop a method capable of separating the different isomers.…”

Section: Resultsmentioning

confidence: 99%

“…One caveat with this method is that liquid chromatography conditions required to separate the stereoisomers use formic acid, a mobile phase modifier typically reserved for positive mode ionization, but 9-PAHSA detection by mass spectrometry requires negative mode ionization. In our initial tests we used a mobile phase that was similar to our established FAHFA measurement method, which includes 0.01% ammonium hydroxide, 15 however, this could not achieve base-line resolution of the stereoisomers (Supporting Info, Fig S2 ). It is important to note that with the presence of 0.1% formic acid, we observed a 25-fold reduction in the sensitivity of our detection due to the impaired ionization of the 9-PAHSA (Supporting Info, Fig S2 ).…”

Section: Resultsmentioning

confidence: 99%

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Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation

et al. 2017

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Lipids have fundamental roles in the structure, energetics, and signaling of cells and organisms. The recent discovery of fatty acid esters of hydroxy fatty acids (FAHFAs), lipids with potent anti-diabetic and anti-inflammatory activities, indicates that our understanding of the composition of lipidome and the function of lipids is incomplete. The ability to synthesize and test FAHFAs was critical in elucidating the roles of these lipids, but these studies were performed with racemic mixtures, and the role of stereochemistry remains unexplored. Here, we synthesized the R- and S- palmitic acid ester of 9-hydroxystearic acid (R-9-PAHSA, S-9-PAHSA). Access to highly enantioenriched PAHSAs enabled the development of a liquid chromatography-mass spectrometry (LC-MS) method to separate and quantify R- and S-9-PAHSA, and this approach identified R-9-PAHSA as the predominant stereoisomer that accumulates in adipose tissues from transgenic mice where FAHFAs were first discovered. Furthermore, biochemical analysis of 9-PAHSA biosynthesis and degradation indicate that the enzymes and pathways for PAHSA production are stereospecific, with cell lines favoring the production of R-9-PAHSA and carboxyl ester lipase (CEL), a PAHSA degradative enzyme, selectively hydrolyzing S-9-PAHSA. These studies highlight the role of stereochemistry in the production and degradation of PAHSAs and define the endogenous stereochemistry of 9-PAHSA in adipose tissue. This information will be useful in the identification and characterization of the pathway responsible for PAHSA biosynthesis, and access to enantiopure PAHSAs will elucidate the role of stereochemistry in PAHSA activity and metabolism in vivo.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation

et al. 2017

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“…Lipids were extracted from 80–100mg WAT tissue or 80–100μL serum, and targeted mass spectrometry (for PAHSA measurement) and untargeted lipidomic analysis (to determine relative fatty acid abundance) were performed and analyzed as described (Yore et al, 2014; Zhang et al, 2016). …”

Section: Methodsmentioning

confidence: 99%

Absence of Carbohydrate Response Element Binding Protein in Adipocytes Causes Systemic Insulin Resistance and Impairs Glucose Transport

et al. 2017

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Summary Lower adipose-ChREBP and de novo lipogenesis (DNL) are associated with insulin resistance in humans. Here we generated adipose-specific ChREBP knockout (AdChREBP KO) mice with negligible sucrose-induced DNL in adipose tissue (AT). Chow-fed AdChREBP KO mice are insulin-resistant with impaired insulin action in liver, muscle and AT, and increased AT inflammation. HFD-fed AdChREBP KO mice are also more insulin-resistant than controls. Surprisingly, adipocytes lacking ChREBP display a cell-autonomous reduction in insulin-stimulated glucose transport which is mediated by impaired Glut4 translocation and exocytosis, not lower Glut4 levels.. AdChREBP KO mice have lower levels of palmitic acid esters of hydroxy stearic acids (PAHSAs) in serum and AT. 9-PAHSA supplementation completely rescues their insulin resistance and AT inflammation. 9-PAHSA also normalizes impaired glucose transport and Glut4 exocytosis in ChREBP KO adipocytes. Thus, loss of adipose-ChREBP is sufficient to cause insulin resistance potentially by regulating AT glucose transport and flux through specific lipogenic pathways.

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“…Branched fatty acid esters of hydroxyl fatty acids (FAHFA) species, consisting of four FAs and four HFAs in varying combinations, were reported and palmitic acid hydroxy stearic acid (PAHSA) has been positively correlated to insulin sensitivity in adipose tissue and serum of humans [91,92]. The role of PAHSA as biomarker and potential therapeutic strategy to prevent IR and T2D is currently discussed.…”

Section: Lipidomics In Metabolic Disease Researchmentioning

confidence: 99%

Lipidomics—Reshaping the Analysis and Perception of Type 2 Diabetes

Markgraf

Al-Hasani

Lehr

2016

IJMS

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As a consequence of a sedentary lifestyle as well as changed nutritional behavior, today’s societies are challenged by the rapid propagation of metabolic disorders. A common feature of diseases, such as obesity and type 2 diabetes (T2D), is the dysregulation of lipid metabolism. Our understanding of the mechanisms underlying these diseases is hampered by the complexity of lipid metabolic pathways on a cellular level. Furthermore, overall lipid homeostasis in higher eukaryotic organisms needs to be maintained by a highly regulated interplay between tissues, such as adipose tissue, liver and muscle. Unraveling pathological mechanisms underlying metabolic disorders therefore requires a diversified approach, integrating basic cellular research with clinical research, ultimately relying on the analytical power of mass spectrometry-based techniques. Here, we discuss recent progress in the development of lipidomics approaches to resolve the pathological mechanisms of metabolic diseases and to identify suitable biomarkers for clinical application. Due to its growing impact worldwide, we focus on T2D to highlight the key role of lipidomics in our current understanding of this disease, discuss remaining questions and suggest future strategies to address them.

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A LC-MS–based workflow for measurement of branched fatty acid esters of hydroxy fatty acids

Cited by 65 publications

References 32 publications

Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation

Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation

Absence of Carbohydrate Response Element Binding Protein in Adipocytes Causes Systemic Insulin Resistance and Impairs Glucose Transport

Lipidomics—Reshaping the Analysis and Perception of Type 2 Diabetes

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