Mass spectrometric approaches in discovering lipid biomarkers for COVID-19 by lipidomics: Future challenges and perspectives.

Coronavirus disease 2019 (COVID-19) has emerged as a global health threat and has rapidly spread worldwide. Significant changes in the lipid profile before and after COVID-19 confirmed the significance of lipid metabolism in regulating the response to viral infection. Therefore, understanding the role of lipid metabolism may facilitate the development of new therapeutics for COVID-19. Owing to their high sensitivity and accuracy, mass spectrometry (MS)-based methods are widely used for rapidly identifying and quantifying of thousands of lipid species present in a small amount of sample. To enhance the capabilities of MS for the qualitative and quantitative analysis of lipids, different platforms have been combined to cover a wide range of lipidomes with high sensitivity, specificity, and accuracy. Currently, MS-based technologies are being established as efficient methods for discovering potential diagnostic biomarkers for COVID-19 and related diseases. As the lipidome of the host cell is drastically affected by the viral replication process, investigating lipid profile alterations in patients with COVID-19 and targeting lipid metabolism pathways are considered to be crucial steps in host-directed drug targeting to develop better therapeutic strategies. This review summarizes various MS-based strategies that have been developed for lipidomic analyzes and biomarker discoveries to combat COVID-19 by integrating various other potential approaches using different human samples. Furthermore, this review discusses the challenges in using MS technologies and future perspectives in terms of drug discovery and diagnosis of COVID-19.

Sample pretreatment effects on isotopic compositions of oxygen and sulfur in BaSO4 derived from atmospheric sulfate.

RATIONALE: Preparation of BaSO4 from samples of atmospheric rain, snow, and aerosols has been used for δ18 O and δ34 S analyses. In the present study, we investigated the effect of various sample pretreatments on δ18 O and δ34 S values determined from a Na2 SO4 reagent solution and samples of atmospheric precipitation to improve assay time and cost efficiency. METHODS: BaSO4 was prepared from a Na2 SO4 solution by (a) evaporative concentration, (b) evaporation to dryness, (c) evaporation to dryness after adding HCl, and (d) evaporation to concentration after adding HCl, followed by cooling and then precipitation using a BaCl2 solution. To analyze the atmospheric precipitation samples for δ18 O, BaSO4 prepared from the samples was treated with diethylenetriaminepentaacetic acid (DTPA) and SO4 2- and separated chromatographically. The values of δ18 O and δ34 S were measured using a continuous-flow elemental analyzer coupled to an isotope-ratio mass spectrometer. RESULTS: The δ34 S values in BaSO4 precipitated from Na2 SO4 solution determined by methods (a)-(c) were consistent within precisions of ±0.5‰. The δ18 O values of methods (a) and (b) were consistent within ±0.2‰, whereas the δ18 O values of methods (c) and (d) increased with increasing HCl concentrations. Similar results were obtained from samples of atmospheric precipitations. The δ18 O values from DTPA-treated BaSO4 were consistent with those obtained by chromatographic separation within ±0.5‰. CONCLUSIONS: We found no significant differences in the effects of various pretreatments (acidification, heating, concentration, and drying) on δ18 O and δ34 S values in sulfate from samples of atmospheric precipitation and aerosols extracted as BaSO4 if HCl was not added to the sample before heating and BaSO4 was treated with DTPA for the δ18 O analysis.

Serum 25-hydroxyvitamin D3 Levels and Diabetes in a Japanese Population: The DOSANCO Health Study.

BACKGROUND: Both decreased insulin sensitivity and impaired insulin secretion are common in Asian populations with diabetes, in contrast to Western populations. There is limited evidence regarding the association between insulin response in diabetes in Asian populations and serum 25-hydroxyvitamin D3 (25[OH]D3) insufficiency. METHODS: The present cross-sectional study compared the prevalence of diabetes, defined as a fasting plasma glucose level ≥126 mg/dL and/or a HbA1c level ≥6.5%, among 480 participants aged 35-79 years not taking anti-diabetes medications, based on serum 25(OH)D3 levels. A logistic regression model was used to calculate the odds ratios for diabetes in each serum 25(OH)D3 group. Furthermore, this study examined the association between serum 25(OH)D3 levels and the index of homeostasis model assessment of insulin resistance (HOMA-IR) using a linear regression model. RESULTS: The prevalence of diabetes was 7.29% in the study population, and was higher in lower serum 25(OH)D3 quartile groups. The odds ratios for diabetes in the first, second, and third serum 25(OH)D3 quartile groups (25[OH]D3: ≤18.10, 18.11-22.90, and 22.91-28.17 ng/mL) were 4.02 (95% confidence interval [CI], 1.25-12.92), 2.50 (95% CI, 0.77-8.10), and 1.91 (95% CI, 0.60-6.09), respectively, with the fourth quartile group (⩾28.18 ng/mL) serving as the reference group, after adjusting for sociodemographic, lifestyle, physical and environmental factors. Serum 25(OH)D3 levels showed an inverse association with log-transformed HOMA-IR after adjusting for similar factors (standardized ß = -0.08; 95% CI, -0.14 to -0.02). CONCLUSION: Serum 25(OH)D3 levels were inversely associated with diabetes prevalence in a general Japanese population, with a slight inverse association between serum 25(OH)D3 levels and HOMA-IR.

A Pacific Oyster-Derived Antioxidant, DHMBA, Protects Renal Tubular HK-2 Cells against Oxidative Stress via Reduction of Mitochondrial ROS Production and Fragmentation.

The kidney contains numerous mitochondria in proximal tubular cells that provide energy for tubular secretion and reabsorption. Mitochondrial injury and consequent excessive reactive oxygen species (ROS) production can cause tubular damage and play a major role in the pathogenesis of kidney diseases, including diabetic nephropathy. Accordingly, bioactive compounds that protect the renal tubular mitochondria from ROS are desirable. Here, we aimed to report 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), isolated from the Pacific oyster (Crassostrea gigas) as a potentially useful compound. In human renal tubular HK-2 cells, DHMBA significantly mitigated the cytotoxicity induced by the ROS inducer L-buthionine-(S, R)-sulfoximine (BSO). DHMBA reduced the mitochondrial ROS production and subsequently regulated mitochondrial homeostasis, including mitochondrial biogenesis, fusion/fission balance, and mitophagy; DHMBA also enhanced mitochondrial respiration in BSO-treated cells. These findings highlight the potential of DHMBA to protect renal tubular mitochondrial function against oxidative stress.

Oxidized Low-Density Lipoproteins Trigger Hepatocellular Oxidative Stress with the Formation of Cholesteryl Ester Hydroperoxide-Enriched Lipid Droplets.

Oxidized low-density lipoproteins (oxLDLs) induce oxidative stress in the liver tissue, leading to hepatic steatosis, inflammation, and fibrosis. Precise information on the role of oxLDL in this process is needed to establish strategies for the prevention and management of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Here, we report the effects of native LDL (nLDL) and oxLDL on lipid metabolism, lipid droplet formation, and gene expression in a human liver-derived C3A cell line. The results showed that nLDL induced lipid droplets enriched with cholesteryl ester (CE) and promoted triglyceride hydrolysis and inhibited oxidative degeneration of CE in association with the altered expression of LIPE, FASN, SCD1, ATGL, and CAT genes. In contrast, oxLDL showed a striking increase in lipid droplets enriched with CE hydroperoxides (CE-OOH) in association with the altered expression of SREBP1, FASN, and DGAT1. Phosphatidylcholine (PC)-OOH/PC was increased in oxLDL-supplemented cells as compared with other groups, suggesting that oxidative stress increased hepatocellular damage. Thus, intracellular lipid droplets enriched with CE-OOH appear to play a crucial role in NAFLD and NASH, triggered by oxLDL. We propose oxLDL as a novel therapeutic target and candidate biomarker for NAFLD and NASH.

Identification of a ß-Carboline Alkaloid from Chemoselectively Derived Vanilla Bean Extract and Its Prevention of Lipid Droplet Accumulation in Human Hepatocytes (HepG2).

Targeting bioactive compounds to prevent lipid droplet accumulation in the liver, we explored an antioxidative extract from vanilla bean (Vainilla planifolia) after chemo-selective derivatization through heating and acid modification. The chemical analysis of vanilla bean extract through chemoselective derivatization resulted in the identification of sixteen compounds (34-50) using LC-MS/MS analysis. A ß-carboline alkaloid with a piperidine C-ring and a vanillin moiety at C-1 (34) was identified by molecular networking and diagnostic fragmentation filtering approaches. ß-carboline alkaloid 34 exhibited significant inhibitory activity of lipid droplet accumulation (LDAI) in oleic acid-loaded hepatocellular carcinoma HepG2 cells. The LDAI activity was associated with both activation of lipolysis and suppression of lipogenesis in the cells. The study indicates that crude plant extracts, following chemoselective derivatization, may contain bioactive compounds that could be beneficial in preventing hepatosteatosis and could serve as a source of lead compounds for drug development. This approach may be useful to investigate other mixtures of natural products and food resources.

Quantitative Evaluation on the Degradation Process of the Pulmonary Surfactant Monolayer When Exposed to Low-Level Ozone of Ambient Environment.

Ozone is a potent environmental oxidant with high chemical reactivity and is present in the ambient environment at a low level of a few tens of ppb. However, only limited information is known about low-level ozone's influence on the respiratory system. In the present study, we systematically investigated the degradation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which is one of the major components of the pulmonary surfactant (PS), enabling breath function of the lung exposed to low ambient-level ozone (40 ± 10 ppb). Using the liquid chromatography-mass spectrometry technique combined with the Langmuir-Blodgett approach, we first tracked the degradation process of POPC molecules by determining the degradation products during exposure to the ambient environment. As a result, we found that the POPC molecules can be readily degraded from the C═C moiety in 45 min, yielding an aldehyde-type product of 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (POnPC) and a trace amount of an acid-type one of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzPC), as well as a pair of secondary ozonide (SOZ) isomers. Furthermore, with prolonged exposure, the SOZ stayed constant but the yield of PAzPC significantly increased with the decrease in POnPC. The low-level ozone-induced oxidation mechanisms for unsaturated lipids are discussed based on the quantitative analyses of these experimental observations. The present results demonstrate that the ground-level ozone is strong enough to induce dramatic oxidation damage to the unsaturated lipids of the PS. These oxidized species may trigger the lung's inflammatory response and be used as biomarkers for oxidative stress and inflammation.

Determination of short-chain fatty acids by N,N-dimethylethylenediamine derivatization combined with liquid chromatography/mass spectrometry and their implication in influenza virus infection.

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/µL; 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.

Adverse effect investigation using application software after vaccination against SARS-CoV-2 for healthcare workers.

INTRODUCTION: The usefulness of smartphone-based application software as a way to manage adverse events (AEs) after vaccination is well known. The purpose of this study is to clarify the usefulness and precautions of employing a smartphone application for collecting AEs after the administration of Comirnaty®ï¸. METHODS: Healthcare workers (HCWs) who were vaccinated with Comirnaty®ï¸ were asked to register for the application software and to report AEs for 14 days after vaccination. AEs were self-reported according to severity. The software was set to output an alert in case of fever. RESULTS: The number of HCWs who received the first dose was 2,551, and 2,406 (94.3%) reported their vaccinations. 2,547 received the second dose, and 2,347 (92.1%) reported their vaccinations. With the first dose, the reporting rate stayed above 83.3% until the final day. On the other hand, that of the second dose decreased rapidly after 6 days. The most frequent symptom was "pain at injection site" (more than 70%). Severe AEs were 6.6% after the second dose, with 0.6% visiting a clinic. Many AEs peaked on the day after administration and disappeared within 1 week. There were few reports of fever. CONCLUSION: Smartphone applications can be used to collect information on AEs after vaccination. Application settings and dissemination are necessary to maintain the reporting rate of HCWs.

Antibody responses and SARS-CoV-2 infection after BNT162b2 mRNA booster vaccination among healthcare workers in Japan.

INTRODUCTION: Vaccine effectiveness against SARS-CoV-2 infections decreases due to waning immunity, and booster vaccination was therefore introduced. We estimated the anti-spike antibody (AS-ab) recovery by booster vaccination and analyzed the risk factors for SARS-CoV-2 infections. METHODS: The subjects were health care workers (HCWs) in a Chiba University Hospital vaccination cohort. They had received two doses of vaccine (BNT162b2) and a booster vaccine (BNT162b2). We retrospectively analyzed AS-ab titers and watched out for SARS-CoV-2 infection for 90 days following booster vaccination. RESULTS: AS-ab titer eight months after two-dose vaccinations had decreased to as low as 587 U/mL (median, IQR (interquartile range) 360-896). AS-ab titer had then increased to 22471 U/mL (15761-32622) three weeks after booster vaccination. There were no significant differences among age groups. A total of 1708 HCWs were analyzed for SARS-CoV-2 infection, and 48 of them proved positive. SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 1.8% and 4.0%, respectively, and were not significant. However, when restricted to those 20-29 years old, SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 2.9% and 13.6%, respectively (p = 0.04). After multivariate logistic regression, COVID-19 wards (adjusted odds ratio (aOR):2.9, 95% confidence interval (CI) 1.5-5.6) and those aged 20-49 years (aOR:9.7, 95%CI 1.3-71.2) were risk factors for SARS-CoV-2 infection. CONCLUSIONS: Booster vaccination induced the recovery of AS-ab titers. Risk factors for SARS-CoV-2 infection were HCWs of COVID-19 wards and those aged 20-49 years. Increased vaccination coverage, together with implementing infection control, remains the primary means of preventing HCWs from SARS-CoV-2 infection.

Sphingosine-1-phosphate interactions in the spleen and heart reflect extent of cardiac repair in mice and failing human hearts.

Sphingosine-1-phosphate (S1P) is a bioactive mediator in inflammation. Dysregulated S1P is demonstrated as a cause of heart failure (HF). However, the time-dependent and integrative role of S1P interaction with receptors in HF is unclear after myocardial infarction (MI). In this study, the sphingolipid mediators were quantified in ischemic human hearts. We also measured the time kinetics of these mediators post-MI in murine spleen and heart as an integrative approach to understand the interaction of S1P and respective S1P receptors in the transition of acute (AHF) to chronic HF (CHF). Risk-free 8-12 wk male C57BL/6 mice were subjected to MI surgery, and MI was confirmed by echocardiography and histology. Mass spectrometry was used to quantify sphingolipids in plasma, infarcted heart, spleen of mice, and ischemic and healthy human heart. The physiological cardiac repair was observed in mice with a notable increase of S1P quantity (pmol/g) in the heart and spleen significantly reduced in patients with ischemic HF. The circulating murine S1P levels were increased during AHF and CHF despite lowered substrate in CHF. The S1PR1 receptor expression was observed to coincide with the respective S1P quantity in mice and human hearts. Furthermore, selective S1P1 agonist limited inflammatory markers CCL2 and TNF-α and accelerated reparative markers ARG-1 and YM-1 in macrophages in the presence of Kdo2-Lipid A (KLA; potent inflammatory stimulant). This report demonstrated the importance of S1P/S1PR1 signaling in physiological inflammation during cardiac repair in mice. Alteration in these axes may serve as the signs of pathological remodeling in patients with ischemia.NEW & NOTEWORTHY Previous studies indicate that sphingosine-1-phosphate (S1P) has some role in cardiovascular disease. This study adds quantitative and integrative systems-based approaches that are necessary for discovery and bedside translation. Here, we quantitated sphinganine, sphingosine, sphingosine-1-phosphate (S1P) in mice and human cardiac pathobiology. Interorgan S1P quantity and respective systems-based receptor activation suggest cardiac repair after myocardial infarction. Thus, S1P serves as a therapeutic target for cardiac protection in clinical translation.

Analysis of serum lysophosphatidylethanolamine levels in patients with non-alcoholic fatty liver disease by liquid chromatography-tandem mass spectrometry.

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.

Docosahexaenoic Acid Esters of Hydroxy Fatty Acid Is a Novel Activator of NRF2.

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.

A mouse model of short-term, diet-induced fatty liver with abnormal cardiolipin remodeling via downregulated Tafazzin gene expression.

BACKGROUND: Cardiolipin (CL) helps maintain mitochondrial structure and function. Here we investigated whether a high carbohydrate diet (HCD) fed to mice for a short period (5 days) could modulate the CL level, including that of monolysoCL (MLCL) in the liver. RESULTS: Total CL in the HCD group was 22% lower than that in the normal chow diet (NCD) group (P < 0.05). The CL72:8 level strikingly decreased by 93% (P < 0.0001), whereas total nascent CLs (CLs other than CL72:8) increased (P < 0.01) in the HCD group. The total MLCL in the HCD group increased by 2.4-fold compared with that in the NCD group (P < 0.05). Tafazzin expression in the HCD group was significantly downregulated compared with that in the NCD group (P < 0.05). A strong positive correlation between nascent CL and total MLCL (r = 0.955, P < 0.0001), and a negative correlation between MLCL and Tafazzin expression (r = -0.593, P = 0.0883) were observed. CONCLUSION: A HCD modulated the fatty acid composition of CL and MLCL via Tafazzin in the liver, which could lead to mitochondrial dysfunction. This model may be useful for elucidating the relationship between fatty liver and mitochondrial dysfunction. © 2021 Society of Chemical Industry.

Comprehensive lipidomic profiling in serum and multiple tissues from a mouse model of diabetes.

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. OBJECTIVES: To evaluate and compare the characteristics of lipid species in serum and multiple tissues in a diabetic mouse model. METHODS: Semi-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. RESULTS: The 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 more alterations in triacylglycerol. Interestingly, the lipidomic differences also existed between different regions of the same organ: cardiolipin species with highly polyunsaturated fatty acyls decreased only in atrium but not in ventricle, while renal cortex showed longer fatty acyl chains for both increased and decreased triacylglycerol species than renal medulla. 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. CONCLUSIONS: These 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.

Identification of short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs) in a murine model by nontargeted analysis using ultra-high-performance liquid chromatography/linear ion trap quadrupole-Orbitrap mass spectrometry.

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, 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. 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.

Identification of novel biomarkers of hepatocellular carcinoma by high-definition mass spectrometry: Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry and desorption electrospray ionization mass spectrometry imaging.

RATIONALE: Hepatocellular carcinoma (HCC) is a highly malignant disease for which the development of prospective or prognostic biomarkers is urgently required. Although metabolomics is widely used for biomarker discovery, there are some bottlenecks regarding the comprehensiveness of detected features, reproducibility of methods, and identification of metabolites. In addition, information on localization of metabolites in tumor tissue is needed for functional analysis. Here, we developed a wide-polarity global metabolomics (G-Met) method, identified HCC biomarkers in human liver samples by high-definition mass spectrometry (HDMS), and demonstrated localization in cryosections using desorption electrospray ionization MS imaging (DESI-MSI) analysis. METHODS: Metabolic profiling of tumor (n = 38) and nontumor (n = 72) regions in human livers of HCC was performed by an ultrahigh-performance liquid chromatography quadrupole time-of-flight MS (UHPLC/QTOFMS) instrument equipped with a mixed-mode column. The HCC biomarker candidates were extracted by multivariate analyses and identified by matching values of the collision cross section and their fragment ions on the mass spectra obtained by HDMS. Cryosections of HCC livers, which included both tumor and nontumor regions, were analyzed by DESI-MSI. RESULTS: From the multivariate analysis, m/z 904.83 and m/z 874.79 were significantly high and low, respectively, in tumor samples and were identified as triglyceride (TG) 16:0/18:1(9Z)/20:1(11Z) and TG 16:0/18:1(9Z)/18:2(9Z,12Z) using the synthetic compounds. The TGs were clearly localized in the tumor or nontumor areas of the cryosection. CONCLUSIONS: Novel biomarkers for HCC were identified by a comprehensive and reproducible G-Met method with HDMS using a mixed-mode column. The combination analysis of UHPLC/QTOFMS and DESI-MSI revealed that the different molecular species of TGs were associated with tumor distribution and were useful for characterizing the progression of tumor cells and discovering prospective biomarkers.

Separating and Profiling Phosphatidylcholines and Triglycerides from Single Cellular Lipid Droplet by In-Tip Solvent Microextraction Mass Spectrometry.

The analysis of lipid droplets (LDs) by mass spectrometry at the single LD level is still an analytical challenge. In this work, we developed a novel technique termed in-tip solvent microextraction mass spectrometry for the separation and profiling of phosphatidylcholines and triglycerides within a single LD. This method has been successfully used to analyze LDs in mammalian cells and to compare the profiles of triglycerides and phosphatidylcholines in LDs induced at different conditions. Our method has the potential to be applied to such fields as fundamental lipid biology to further our understanding on the mechanisms of lipid production, lipid packaging, and their pathophysiological roles.

Interaction between LDL-mimetic liposomes and acid-treated carbon nanotube electrode during Cu2+-mediated oxidation.

Oxidation of low-density lipoproteins (LDL) causes atherosclerosis. Detection of oxidation of LDL-mimetic liposomes using an electrode might serve as a convenient tool in the search of antioxidants for the prevention of atherosclerosis. This report proposes a reaction mechanism between LDL-mimetic liposomes and an acid-treated carbon nanotube (CNT) electrode. Oxidation of the liposomes, mediated by Cu2+, was monitored by the change in electrode potential, and the fluorescence intensity generated by diphenyl-1-pyrenylphosphine (DPPP) as control. The electrode potential and fluorescence intensity increased concomitantly during oxidation, followed by a gradual decrease. Although the electrical potential peaked faster than the fluorescence intensity, addition of CNT to the DPPP reaction accelerated the latter, suggesting the role of CNT as an accelerator of liposome oxidation. Atomic force microscopy showed increased binding of liposomes to CNT along with liposomal deformation. Further, binding of Cu2+ to the liposome-bound CNT surface was observed by quartz crystal microbalance. In conclusion, the interaction of liposomes with Cu2+ and CNT surface explains the rapid response of the electrode in liposome oxidation.

Appendicular muscle mass and exercise/sports participation history in young Japanese women.

Background: It remains unclear how past exercise habits can affect the skeletal muscle mass in adulthood in Japanese populations. Aim: The purpose of the present investigation was to examine the association of appendicular muscle mass (AMM) and skeletal muscle mass index (SMI) with the history of exercise and/or physical activity participation in Japanese women. Subjects and methods: One hundred and twenty females, aged between 18 and 28 years old, participated in the present investigation. Using a dual-energy X-ray absorptiometry scanner, the appendicular lean soft tissue, which is considered as a measure of AMM, was evaluated. Skeletal muscle mass index (SMI) was also assessed. Furthermore, all subjects answered a physical activity questionnaire. Results: Exercise habits at 7 years of age or older positively affected the AMM and SMI. The results of the multiple regression analysis showed that exercise history at 16-18 years of age as well as the current status of exercise and/or physical activity participation was a significant predictor of SMI and AMM. Conclusions: These results indicate that not only the past history of participation in physical and/or sports activities but also the current status of daily physical activity and sports activity play an important role in maintaining appropriate SMI and AMM in young women.