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Lipid hydroperoxides (LOOH) are the initial products of the peroxidation of unsaturated lipids and play a crucial role in lipid oxidation due to their ability to decompose into free radicals and cause adverse effects on human health. Thus, LOOHs are commonly considered biomarkers of oxidative stress-associated pathological conditions. Despite their importance, the sensitive and selective analytical method for determination is limited, due to their low abundance, poor stability, and low ionizing efficiency. To overcome these limitations, in this study, we chemically synthesized eight fatty acid hydroperoxides (FAOOH), including FA 18:1-OOH, FA 18:2-OOH, FA 18:3-OOH, FA 20:4-OOH, FA 20:5-OOH, FA 22:1-OOH, FA 22:6-OOH as analytes, and FA 19:1-OOH as internal standard. Then, they were chemically labeled with 2-methoxypropene (2-MxP) to obtain FAOOMxP by one-step derivatization (for 10 min). A selected reaction monitoring assisted targeted analytical method was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). The MxP-labelling improved the stability and enhanced the ionization efficiency in positive mode. Application of reverse-phase chromatography allowed coelution of analytes and internal standards with a short analysis time of 6 min. The limit of detection and quantification for FAOOH ranged from 0.1-1 pmol/mu L and 1-2.5 pmol/mu L, respectively. The method was applied to profile total FAOOHs in chemically oxidized human serum samples (n = 5) and their fractions of low and high-density lipoproteins (n = 4). The linoleic acid hydroperoxide (FA 18:2-OOH) and oleic acid hydroperoxide (FA 18:1-OOH) were the most abundant FAOOHs in human serum and lipoproteins. Overall, our validated LC-MS/MS methodology features enhanced detection and rapid separation that enables facile quantitation of multiple FAOOHs, therefore providing a valuable tool for determining the level of lipid peroxidation with potential diagnostic applications.

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a–/– mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

Seaweeds are a good source of bioactive lipids and are known for their nutritional benefits, making them a valuable food source. Despite their dietary significance and nutritional importance, there are limited reports on comprehensive lipidome analysis of lipids with antioxidant properties. Therefore, this study aimed to compare the lipid profiles of five commonly consumed Japanese dietary seaweeds using non-targeted liquid chromatography/mass spectrometry (LC/MS). A total, of 304 molecular species from four major lipid classes were detected and characterized by MS/MS analysis. Multivariate statistical analysis revealed distinct lipid molecular compositions in kombu and sea mustard compared to hijiki, mozuku, and laver seaweeds. Kombu has been shown to contain large amounts of antioxidants, such as polyunsaturated fatty acids (PUFAs), and a high health promotion index compared to other seaweeds. Hierarchical cluster correlations indicated the predominance of glycerophospholipids (GPs) and glycerolipids (GLs) in sea mustard and kombu. As a result, dietary seaweeds have great potential as antioxidants and health-promoting foods for human consumption due to their high levels of PUFA-rich GPs and GLs. Unsaturated triacylglycerols are predominant in hijiki, whereas other health-beneficial lipids, such as monogalactosyldiacylglycerol and sulfoquinovosyl diacylglycerols, are predominant in sea mustard. This study provides a detailed characterization of lipids and their comparative fingerprints in seaweeds, demonstrating the potential use of dietary seaweeds in biotechnological and industrial applications involving the development of functional food products.

Short-chain fatty acids (SCFAs) are the end products of the fermentation of complex carbohydrates by the gut microbiota. Although SCFAs are recognized as important markers to elucidate the link between gut health and disease, it has been difficult to analyze SCFAs with mass spectrometry technologies due to their poor ionization efficiency and high volatility. Here, we present a novel and sensitive method for the quantification of SCFAs, including C2-C6 SCFAs and their hydroxy derivatives, by liquid chromatography/tandem mass spectrometry (LC-MS/MS) upon N,N-dimethylethylenediamine (DMED) derivatization with a run time of 10 min. Moreover, the quantification method of DMED-derivatized SCFAs in intestinal contents using isotope-labeled internal standards was also established. The method validation was performed by analyzing spiked intestinal samples; the limits of detection and quantification of SCFAs with this method were found to be 0.5 and 5 fmol, respectively; the recovery was greater than 80% and good linearity (0.9932 to 0.9979) of calibration curves was obtained over the range from 0.005 to 5000 pmol/mu L; the intraday and interday precisions were achieved in the range of 1-5%. Furthermore, the validated method was applied to analyze SCFAs in the cecum and colon contents of mice infected with the influenza virus. The results showed that the concentration of most of the SCFAs tested here decreased significantly in a time-dependent manner after the infection, suggesting a possibility that SCFAs in intestinal samples could be used as severe disease markers. Overall, we here successfully developed a simple, fast, and sensitive method for SCFA analysis by LC-MS/MS combined with DMED derivatization. The method for the quantification of SCFAs will be a useful tool for both basic research and clinical studies.

Fish is an important nutrition source because its lipids, which are rich in e3-3 fatty acids, are beneficial for human health. However, studies focusing on their detection, composition, and nutritional value are limited. In this study, we applied a non-targeted lipidomic approach based on ultra-high performance liquid chromatography coupled with linear-ion trap-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap-MS) to comprehensively profile, compare, and detect unknown lipids in eleven types of dietary fish. A total of 287 molecular species from five major lipid classes were characterized by MS/MS analysis. Multivariate principal component analysis revealed the distinct lipid composition in shishamo smelt and Japanese sardine compared to other fish types. The assessment of nutritional indices based on the levels of free fatty acid suggested that among the eleven fish types, shishamo smelt is highly beneficial for health. Further, lipids such as N-acyl lysophosphatidylethanolamine were detected and characterized for the first time in fish fillets. Hierarchical cluster correlations indicated the predominance of glycerophospholipids (GPs) and sphingolipids in sardine, whereas fatty acyls and triacylglycerols (TAGs) were predominant in shishamo smelt. The high levels of polyunsaturated fatty acid-enriched GPs and TAGs in dietary fish endow it with great potential as a health-promoting food for human consumption. This study offers a comprehensive analysis of lipids and their compositions in fish fillets, demonstrating their potential use in the nutritional assessment of functional foods.

Fish is an important nutrition source because its lipids, which are rich in e3-3 fatty acids, are beneficial for human health. However, studies focusing on their detection, composition, and nutritional value are limited. In this study, we applied a non-targeted lipidomic approach based on ultra-high performance liquid chromatography coupled with linear-ion trap-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap-MS) to comprehensively profile, compare, and detect unknown lipids in eleven types of dietary fish. A total of 287 molecular species from five major lipid classes were characterized by MS/MS analysis. Multivariate principal component analysis revealed the distinct lipid composition in shishamo smelt and Japanese sardine compared to other fish types. The assessment of nutritional indices based on the levels of free fatty acid suggested that among the eleven fish types, shishamo smelt is highly beneficial for health. Further, lipids such as N-acyl lysophosphatidylethanolamine were detected and characterized for the first time in fish fillets. Hierarchical cluster correlations indicated the predominance of glycerophospholipids (GPs) and sphingolipids in sardine, whereas fatty acyls and triacylglycerols (TAGs) were predominant in shishamo smelt. The high levels of polyunsaturated fatty acid-enriched GPs and TAGs in dietary fish endow it with great potential as a health-promoting food for human consumption. This study offers a comprehensive analysis of lipids and their compositions in fish fillets, demonstrating their potential use in the nutritional assessment of functional foods.

Seaweeds are a good source of bioactive lipids and are known for their nutritional benefits, making them a valuable food source. Despite their dietary significance and nutritional importance, there are limited reports on comprehensive lipidome analysis of lipids with antioxidant properties. Therefore, this study aimed to compare the lipid profiles of five commonly consumed Japanese dietary seaweeds using non-targeted liquid chromatography/mass spectrometry (LC/MS). A total, of 304 molecular species from four major lipid classes were detected and characterized by MS/MS analysis. Multivariate statistical analysis revealed distinct lipid molecular compositions in kombu and sea mustard compared to hijiki, mozuku, and laver seaweeds. Kombu has been shown to contain large amounts of antioxidants, such as polyunsaturated fatty acids (PUFAs), and a high health promotion index compared to other seaweeds. Hierarchical cluster correlations indicated the predominance of glycerophospholipids (GPs) and glycerolipids (GLs) in sea mustard and kombu. As a result, dietary seaweeds have great potential as antioxidants and health-promoting foods for human consumption due to their high levels of PUFA-rich GPs and GLs. Unsaturated triacylglycerols are predominant in hijiki, whereas other health-beneficial lipids, such as monogalactosyldiacylglycerol and sulfoquinovosyl diacylglycerols, are predominant in sea mustard. This study provides a detailed characterization of lipids and their comparative fingerprints in seaweeds, demonstrating the potential use of dietary seaweeds in biotechnological and industrial applications involving the development of functional food products.

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.

Influenza remains a world-wide health concern, causing 290,000-600,000 deaths and up to 5 million cases of severe illnesses annually. Noticing the host factors that control biological responses, such as inflammatory cytokine secretion, to influenza virus infection is important for the development of novel drugs. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and has essential biological functions in inflammation. However, the kinetic effects of influenza virus infection on physiological S1P levels and their signaling in multiple tissues remain unknown. In this study, we utilized a mouse model intranasally infected with 50 or 500 plaque forming units (PFU) of A/Puerto Rico/8/34 (H1N1; PR8) virus to investigate how S1P levels and expression of its regulating factors are affected by influenza virus infection by the liquid-chromatography/mass spectrometry and real-time PCR, respectively. The S1P level was significantly high in the plasma of mice infected with 500 PFU of the virus than that in control mice at 6 day-post-infection (dpi). Elevated gene expression of sphingosine kinase-1 (Sphk1), an S1P synthase, was observed in the liver, lung, white adipose tissue, heart, and aorta of infected mice. This could be responsible for the increased plasma S1P levels as well as the decrease in the hepatic S1P lyase (Sgpl1) gene in the infected mice. These results indicate modulation of S1P-signaling by influenza virus infection. Since S1P regulates inflammation and leukocyte migration, it must be worth trying to target this signaling to control influenza-associated symptoms.

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.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are novel endogenous lipids with important physiological functions in mammals. We previously identified a new type of FAHFAs, named short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs), with acetyl or propyl esters of hydroxy fatty acids of carbon chains, C >= 20. However, sensitive determination of SFAHFAs is still a challenge, due to their high structural similarity and low abundance in biological samples. This study employs one-step chemical derivatization following total lipid extraction using 2-dimethylaminoethylamine (DMED) for enhanced detection of SFAHFAs. The labeled extracts were subjected to ultrahigh performance liquid chromatography coupled to linear ion trap quadrupole-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap MS). Our results demonstrated that the detection sensitivities of SFAHFAs increased after DMED labeling, and is highly helpful in discovering six additional novel SFAHFAs in the cecum and colon contents of WKAH/HKmSlc rats fed with normal and high-fat diet (HFD). The identified DMED labeled SFAHFAs were characterized by their detailed MS/MS analysis, and their plausible fragmentation patterns were proposed. The concentrations of SFAHFAs were significantly reduced in the cecum of HFD group compared to the control. Hence, the proposed method could be a promising tool to apply for the enhanced detection of SFAHFAs in various biological matrices, which in turn facilitate the understanding of their sources, and physiological functions of these novel lipids.

Rationale Fatty 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). Methods Intestinal 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. Results Untargeted 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, MS(3)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. Conclusions Identification 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.

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 of 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 concentrations 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 concentrations 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 the as-yet uncovered pathophysiological role of LysoPEs.