RationaleFatty acid esters of hydroxy fatty acids (FAHFAs) are recently discovered endogenous lipids with outstanding health benefits. FAHFAs are known to exhibit antioxidant, antidiabetic and anti‐inflammatory properties. The number of known long‐chain FAHFAs in mammalian tissues and dietary resources increased recently because of the latest developments in high‐resolution tandem mass spectrometry techniques. However, there are no reports on the identification of short‐chain fatty acid esterified hydroxy fatty acids (SFAHFAs).MethodsIntestinal contents, tissues, and plasma of rats fed with high‐fat diet (HFD) and normal diet (ND) were analyzed for fatty acids, hydroxy fatty acids, and FAHFAs using ultra‐high‐performance liquid chromatography (UHPLC) and linear trap quadrupole‐Orbitrap mass spectrometry (LTQ Orbitrap MS) with negative heated electrospray ionization.ResultsUntargeted analysis of total lipid extracts from murine samples (male 13‐week‐old WKAH/HKmSlc rats) led to the identification of several new SFAHFAs of acetic acid or propanoic acid esterified long‐chain (>C20)‐hydroxy fatty acids. Furthermore, MS3 analysis revealed the position of the hydroxyl group in the long‐chain fatty acid as C‐2. The relative amounts of SFAHFAs were quantified in intestinal contents and their tissues (Cecum, small intestine, and large intestine), liver, and plasma of rats fed with HFD and ND. The large intestine showed the highest abundance of SFAHFAs with a concentration range from 0.84 to 57 pmol/mg followed by the cecum with a range of 0.66 to 28.6 pmol/mg. The SFAHFAs were significantly altered between the HFD and ND groups, with a strong decreasing tendency under HFD conditions.ConclusionsIdentification of these novel SFAHFAs can contribute to a better understanding of the chemical and biological properties of individual SFAHFAs and their possible sources in the gut, which in turn helps us tackle the role of these lipids in various metabolic diseases.

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.

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Lysophosphatidylethanolamines (LysoPEs) are the partial hydrolysis products of phosphatidylethanolamine. Despite the unique in vitro bioactivities of LysoPEs, there are limited reports on the pathophysiological role for LysoPEs in the serum, due to the lack of sensitive analytical methods for determination of each molecular species in clinical samples. Herein, we developed a highly sensitive quantitative method to profile the serum LysoPE species by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM). The internal standard (IS), chemically synthesized in-house and the lineup of seven major LysoPE species were used in this study. The limits of detection and quantification for each LysoPE species ranged within 0.5-3.3 pmol/mL and 1.0-5.0 pmol/mL, respectively. The combined concentration of LysoPEs in the serum from healthy subjects (n=8) and the patients with non-alcoholic fatty liver diseases (NAFLD) including simple steatosis (SS, n=9) and non-alcoholic steatohepatitis (NASH, n=27) were 18.030 ± 3.832, 4.867 ± 1.852, and 5.497 ± 2.495 nmol/mL, respectively. The combined and individual concentration of LysoPEs, except for LysoPE 18:0, significantly decreased in the patients with NAFLD compared with those for the healthy subjects. However, no significant difference was observed between the SS and NASH groups. Our proposed LC-MS/MS method is valid and has advantages of small sample volume, high sensitivity and simultaneous absolute quantitation for multiple molecular species. This method may enable diagnostic evaluation and elucidation of yet uncovered pathophysiological role for LysoPEs.

High fat diet (HFD)-induced obesity is a primary risk factor for serious health problems. Although much research has been performed at the genomic level, lipidomic studies were limited. In this study, we aim to obtain the comprehensive profile of circulating plasma lipids, which are altered in rodent rat obesity by untargeted liquid chromatography-mass spectrometry. Rats were fed with HFD for 8 weeks have the increased body weight, liver and adipose tissue weight. The analysis results revealed that polyunsaturated fatty acids (PUFAs) and their corresponding phosphatidylcholine, phosphatidylinositol, and phosphatidylserine were significantly decreased in rats fed with HFD. In contrast, less unsaturated and ether type phosphatidylglycerols were increased. The triacylglycerides (TAGs) having saturated FA were increased in HFD condition, whereas TAGs having PUFA were decreased. The levels of many plasma lipids were altered, interestingly PUFAs derived lipids were negatively associated with obesity signifies the importance of PUFAs enriched diet to overwhelm obesity associated diseases.