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.

Introduction Diabetes mellitus is a serious metabolic disorder causing multiple organ damage in human.However, the lipidomic profiles in different organs and their associations are rarely studied in either diabetic patients or animals. ObjectivesTo evaluate and compare the characteristics of lipid species in serum and multiple tissues in a diabetic mouse model. MethodsSemi-quantitative profiling analyses of intact and oxidized lipids were performed in serum and multiple tissues from a diabetic mouse model fed a high fat diet and treated with streptozotocin by using LC/HRMS and MS/MS. The total content of each lipid class, and the tissue-specific lipid species in all tissue samples were determined and compared by multivariate analyses. ResultsThe diabetic mouse model displayed characteristic differences in serum and multiple organs: the brain and heart showed the largest reduction in cardiolipin, while the kidney had the most remarkable alterations in triacylglycerol. Interestingly, the lipidomic differences also existed between different regions of the same organ: triacylglycerol species with shorter fatty acyl chains decreased in renal medulla but increased in cortex; cardiolipin species with highly polyunsaturated fatty acyls decreased only in atrium but not in ventricle.Importantly, diabetes caused an accumulation of lipid hydroperoxides, suggesting that oxidative stress was induced in all organs except for the brain during the development of diabetes. ConclusionThese findings provided novel insight into the organ-specific relationship between diabetes and lipid metabolism, which might be useful for evaluating not only diabetic tissue injury but also the effectiveness of diabetic treatments.

An in vitro study was conducted to investigate the effect of malate or fumarate on fermentation characteristics, and production of conjugated linoleic acid (CLA) and methane (CH 4 ) by rumen microbes when incubated with linolenic acid (α-C 18:3 ). Sixty milligrams of α-C 18:3 alone (LNA), or α-C 18:3 with 24 mM malic acid (M-LNA) or α-C 18:3 with 24 mM fumaric acid (F-LNA) were added to the 150 ml culture solution consisting of 75 ml strained rumen fluid and 75ml McDougall's artificial saliva. Culture solution for incubation was also made without malate, fumarate and α-C 18:3 (Control). Two grams of feed consisting of 70% concentrate and 30% ground alfalfa (DM basis) were also added to the culture solution of each treatment. In vitro incubation was made anaerobically in a shaking incubator up to 12 h at 39°C. Supplementation of malate (M-LNA) or fumarate (F-LNA) increased pH at 6 h (p<0.01) and 12 h (p<0.001) incubation times compared to control and linolenic acid (LNA) treatments. Both malate and fumarate did not influence the ammonia-N concentration. Concentration of total VFA in culture solution was higher for M-LNA and F-LNA supplementation than for control and LNA treatments from 6 h (p<0.040) to 12 h (p<0.027) incubation times, but was not different between malate and fumarate for all incubation times. Molar proportion of C 3 was increased by F-LNA and M-LNA supplementation from 6 h (p<0.0001) to 12 h (p<0.004) incubation times compared to control and LNA treatments. No differences in C 3 proportion, however, were observed between M-LNA and F-LNA treatments. Accumulated total gas production for 12h incubation was increased (p<0.0002) by M-LNA or F-LNA compared to control or LNA treatment. Accumulated CH 4 production for 12 h incubation, however, was greatly reduced (p<0.0002) by supplementing malate or fumarate compared to the control, and its production from M-LNA or F-LNA treatment was smaller than that from LNA treatment. Methane production from LNA, M-LNA or F-LNA treatment was steadily lower (p<0.01 -p<0.001) from 3 h incubation time than that from the control, and was also lower for M-LNA or F-LNA treatment at incubation times of 6 h (p<0.01) and 9 h (p<0.001) than for LNA treatment. Methane production from LNA, however, was reduced (p<0.01 -p<0.001) from 3 h to 9 h incubation times compared to the control. Both malate and fumarate increased concentration of trans11-C 18:1 from 3 h to 12 h incubation (p<0.01), cis9,trans11-CLA up to 6 h incubation (p<0.01 -p<0.01), trans10,cis12-CLA at 3 h (p<0.05) and 12 h (p<0.01), and total CLA for all incubation times (p<0.05) compared to corresponding values for the α-C 18:3 supplemented treatment (LNA). In conclusion, malate and fumarate rechanneled the metabolic H 2 pathway to production of propionate and CLA, and depressed the process of biohydrogenation and methane generation. Linolenic acid alone would also be one of the optimistic alternatives to suppress the CH 4 generation.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with promising physiological functions in mammals. We previously introduced a new type of lipids to this family called short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs), branching specific to the C2 carbon of a long-chain fatty acid (≥C20). In this study, we discovered a homologous series of SFAHFAs comprising C16–C26 hydroxy fatty acids esterified with short-chain fatty acids (C2–C5) in mouse colon contents. The detected SFAHFAs were characterized by high-resolution mass spectrometry with MSn analysis. The double-bond position of monounsaturated SFAHFAs was determined by the epoxidation reaction of samples with m-chloroperoxybenzoic acid and their MSn analysis. Further, the measurement of SFAHFA concentration in the colon contents of mice infected with influenza A/Puerto Rico/8/34 (H1N1; PR8) virus revealed a significant increase in their levels compared to native control. A strong correlation was observed between hydroxy fatty acid and SFAHFAs. Detection, characterization, and profiling of these new SFAHFA levels in relation with pandemic H1N1; PR8 influenza virus will contribute to the in-depth study of their function and metabolism.