A LC-MS-based workflow for measurement of branched fatty acid esters of hydroxy fatty acids.
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ABSTRACT:
SUBMITTER: Zhang T
PROVIDER: S-EPMC4797065 | biostudies-literature | 2016 Apr
REPOSITORIES: biostudies-literature
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Project description:A recently discovered class of endogenous mammalian lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), possesses anti-diabetic and anti-inflammatory activities. Here, we identified and validated carboxyl ester lipase (CEL), a pancreatic enzyme hydrolyzing cholesteryl esters and other dietary lipids, as a FAHFA hydrolase. Variants of CEL have been linked to maturity-onset diabetes of the young, type 8 (MODY8), and to chronic pancreatitis. We tested the FAHFA hydrolysis activity of the CEL MODY8 variant and found a modest increase in activity as compared with that of the normal enzyme. Together, the data suggest that CEL might break down dietary FAHFAs.
| S-EPMC5056623 | biostudies-literature
Project description:Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4-7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80-90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.
| S-EPMC9242854 | biostudies-literature
Project description:Background: Fatty acid esters of hydroxy fatty acids (FAHFAs) are a group of fatty acids with potential anti-inflammatory and anti-diabetic effects. The blood levels of FAHFAs and their regulation in humans have hardly been studied. Objective: We aimed to investigate serum FAHFA levels in well-characterized human cohorts, to evaluate associations with age, sex, BMI, weight loss, diabetic status, and diet. Methods: We analyzed levels of stearic-acid-9-hydroxy-stearic-acid (9-SAHSA), oleic-acid-9-hydroxy-stearic-acid (9-OAHSA) and palmitic-acid-9-hydroxy-palmitic-acid (9-PAHPA) as well as different palmitic acid-hydroxy-stearic-acids (PAHSAs) by HPLC-MS/MS with the use of an internal standard in various cohorts: A cohort of different age groups (18-25y; 40-65y; 75-85y; Σ n = 60); severely obese patients undergoing bariatric surgery and non-obese controls (Σ n = 36); obese patients with and without diabetes (Σ n = 20); vegetarians/vegans (n = 10) and omnivores (n = 9); and young men before and after acute overfeeding with saturated fatty acids (SFA) (n = 15). Results: Omnivores had substantially higher FAHFA levels than vegetarians/vegans [median (25th percentile; 75th percentile) tFAHFAs = 12.82 (7.57; 14.86) vs. 5.86 (5.10; 6.71) nmol/L; P < 0.05]. Dietary overfeeding by supplementation of SFAs caused a significant increase within 1 week [median tFAHFAs = 4.31 (3.31; 5.27) vs. 6.96 (6.50; 7.76) nmol/L; P < 0.001]. Moreover, obese patients had lower FAHFA levels than non-obese controls [median tFAHFAs = 3.24 (2.80; 4.30) vs. 5.22 (4.18; 7.46) nmol/L; P < 0.01] and surgery-induced weight loss increased 9-OAHSA level while other FAHFAs were not affected. Furthermore, significant differences in some FAHFA levels were found between adolescents and adults or elderly, while no differences between sexes and between diabetic and non-diabetic individuals were detected. Conclusions: FAHFA serum levels are strongly affected by high SFA intake and reduced in severe obesity. Age also may influence FAHFA levels, whereas there was no detectable relation with sex and diabetic status. The physiological role of FAHFAs in humans remains to be better elucidated. Trial Registration: All studies referring to these analyses were registered in the German Clinical Trial Register (https://www.drks.de/drks_web/) with the numbers DRKS00009008, DRKS00010133, DRKS00006211, and DRKS00009797.
| S-EPMC8310931 | biostudies-literature
Project description:Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs' plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.
| S-EPMC7278747 | biostudies-literature
Project description:Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.
| S-EPMC7196635 | biostudies-literature
Project description:Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with anti-diabetic and anti-inflammatory effects. Identification of FAHFAs is challenging due to both the relatively low abundance of these metabolites in most biological samples and the significant structural diversity arising from the co-occurrence of numerous regioisomers. Ultimately, development of sensitive analytical techniques that enable rapid and unambiguous identification of FAHFAs is integral to understanding their diverse physiological functions in health and disease. While a battery of mass spectrometry (MS) based methods for complex lipid analysis has been developed, FAHFA identification presents specific challenges to conventional approaches. Notably, while the MS2 product ion spectra of [FAHFA - H]¯ anions afford the assignment of fatty acid (FA) and hydroxy fatty acid (HFA) constituents, FAHFA regioisomers are usually indistinguishable by this approach. Here, we report the development of a novel MS-based technique employing charge inversion ion/ion reactions with tris-phenanthroline magnesium complex dications, Mg(Phen)32+, to selectively and efficiently derivatize [FAHFA - H]¯ anions in the gas phase, yielding fixed-charge cations. Subsequent activation of [FAHFA - H + MgPhen2]+ cations yield product ions that facilitate the assignment of FA and HFA constituents, pinpoints unsaturation sites within the FA moiety, and elucidates ester linkage regiochemistry. Collectively, the presented approach represents a rapid, entirely gas-phase method for near-complete FAHFA structural elucidation and confident isomer discrimination without the requirement for authentic FAHFA standards.
| S-EPMC7477303 | biostudies-literature
Project description:Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived oxylipins with potential physiological relevance in inflammatory processes as well as in maintaining an intact skin barrier. Due to the high number of possible TriHOME isomers with only subtle differences in their physicochemical properties, the stereochemical analysis is challenging and usually involves a series of laborious analytical procedures. We herein report a straightforward analytical workflow that includes reversed-phase ultra-HPLC-MS/MS for rapid quantification of 9,10,13- and 9,12,13-TriHOME diastereomers and a chiral LC-MS method capable of resolving all sixteen 9,10,13-TriHOME and 9,12,13-TriHOME regio- and stereoisomers. We characterized the workflow (accuracy, 98-120%; precision, coefficient of variation ?6.1%; limit of detection, 90-98 fg on column; linearity, R2 = 0.998) and used it for stereochemical profiling of TriHOMEs in bronchoalveolar lavage fluid (BALF) of individuals with chronic obstructive pulmonary disease (COPD). All TriHOME isomers were increased in the BALF of COPD patients relative to that of smokers (P ? 0.06). In both COPD patients and smokers with normal lung function, TriHOMEs with the 13(S) configuration were enantiomerically enriched relative to the corresponding 13(R) isomers, suggesting at least partial enzymatic control of TriHOME synthesis. This method will be useful for understanding the synthetic sources of these compounds and for elucidating disease mechanisms.
| S-EPMC6168300 | biostudies-literature
Project description:Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous lipids with antidiabetic and anti-inflammatory activities. Interest in these lipids is due to their unique biological activites and the observation that insulin-resistant people have lower palmitic acid esters of hydroxystearic acid (PAHSA) levels, suggesting that a FAHFA deficiency may contribute to metabolic disease. Rigorous testing of this hypothesis will require the measurement of many clinical samples; however, current analytical workflows are too slow to enable samples to be analyzed quickly. Here we describe the development of a significantly faster workflow to measure FAHFAs that optimizes the fractionation and chromatography of these lipids. We can measure FAHFAs in 30 min with this new protocol versus 90 min using the older protocol with comparable performance in regioisomer detection and quantitation. We also discovered through this optimization that oleic acid esters of hydroxystearic acids (OAHSAs), another family of FAHFAs, have a much lower background signal than PAHSAs, which makes them easier to measure. Our faster workflow was able to quantify changes in PAHSAs and OAHSAs in mouse tissues and human plasma, highlighting the potential of this protocol for basic and clinical applications.
| S-EPMC5962352 | biostudies-literature
Project description:Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are an important family of endogenous lipids, possessing antidiabetic and anti-inflammatory functions. Therefore, analysis of FAHFAs in biological samples obtained under healthy and disease states can uncover underlying mechanisms of various relevant disorders (e.g., diabetes and autoimmune diseases). Up to now, due to their extremely low abundance, the determination of the changed levels of these species is still a huge challenge, even though great efforts have been made by utilizing liquid chromatography-tandem mass spectrometry with or without derivatization. Herein, we described a novel method for analysis of FAHFAs present in lipid extracts of biological examples after solid-phase extraction and chemical derivatization with one authentic FAHFA specie as an internal standard based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The approach possessed marked sensitivity, high specificity, and broad linear dynamic range of over 3 orders without obvious matrix effects. Moreover, after chemical derivatization, the molecular masses of FAHFAs shift from an overlapped region with ceramide species to a new region without overlaps, removing these contaminating signals from ceramides, and thereby reducing the false results of FAHFAs. Finally, this novel method was successfully applied for determining FAHFAs levels in varieties of representative biological samples, including plasma from lean and overweight/obese individuals of normoglycemia, and tissue samples (such as liver and white adipose tissue from diabetic (db/db) mice). We revealed significant alterations of FAHFAs in samples under patho(physio)logical conditions compared to their respective controls. Taken together, the developed method could greatly contribute to studying altered FAHFA levels under a variety of biological/biomedical conditions, and facilitate the understanding of these lipid species in the patho(physio)logical process.
| S-EPMC7384334 | biostudies-literature
Project description:Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function.
| S-EPMC8306801 | biostudies-literature