Reproducibility of Crude Oil Spectra Obtained with Ultrahigh Resolution Mass Spectrometry.

In this study, the reproducibility of crude oil analyzed with (+) atmospheric pressure photoionization ultrahigh resolution mass spectrometry was evaluated. Three sets of data were obtained at intervals of approximately a month for a span of three months. For each monthly data set, four oil samples were analyzed with four replicates in 1 day. The obtained 48 spectra were processed to examine the reproducibility of the class, double bond equivalent (DBE), and individual peak distributions. The reproducibility of the relative abundance was better than that of the absolute abundance. The distributions of major classes were consistent within the three sets with a less than 1% relative standard deviation (RSD). The DBE distribution for each data set was reproducible within 1% RSD, whereas the DBE distributions for the combined data sets had RSD values of 1%-6%. The RSD values were higher for minor components, suggesting that care must be taken in the use of minor values for quantitative or semiquantitative evaluation. The relative abundances of individual peaks in the major classes were reproducible within 1%-3% RSD for each data set. However, the RSD values of the combined data sets were over 10%, even for abundant peaks. The smaller RSD of the class and DBE distributions than that of individual peaks for combined data sets strongly suggest that variations observed from individuals were caused by random errors. The data presented in this study provide guidelines for evaluating petroleomic data obtained in the laboratory at different times or laboratories.

Metformin ameliorates acetaminophen hepatotoxicity via Gadd45ß-dependent regulation of JNK signaling in mice.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure. Prolonged c-Jun N-terminal kinase (JNK) activation plays a central role in APAP-induced liver injury and growth arrest, and DNA damage-inducible 45 beta (Gadd45ß) is known to inhibit JNK phosphorylation. Metformin has recently been shown to have hepatoprotective effects. The aim of the present study is to investigate whether metformin mitigates APAP-induced hepatotoxicity and to ascertain the molecular basis of this effect. METHODS: We used APAP- and/or metformin-treated Gadd45ß knockout (KO) mice and wild type (WT) C57BL/6J control mice. Primary mouse hepatocytes were isolated from WT and Gadd45ß KO mice were used for in vitro study. RESULTS: Metformin pretreatment protected against APAP toxicity with decreased liver damage, and inhibited APAP-induced prolonged hepatic JNK phosphorylation in WT mice. Gadd45ß expression was increased after APAP treatment, and the expression of Gadd45ß was further enhanced by metformin. The effects of metformin on APAP-induced liver injury and JNK phosphorylation were abolished in Gadd45ß KO mice. Notably, subtoxic doses of APAP caused cell death and sustained JNK phosphorylation in Gadd45ß-deficient primary hepatocytes. In parallel, APAP increased mortality, severe liver injury, and JNK activation in Gadd45ß KO mice. Interestingly, metformin administered after APAP treatment protected against APAP-evoked hepatotoxicity in WT mice, but not in Gadd45ß KO mice. CONCLUSIONS: This study is the first to demonstrate that metformin shows protective and therapeutic effects against APAP overdose-evoked hepatotoxicity via Gadd45ß-dependent JNK regulation. Metformin would be a promising therapeutic strategy for treatment of APAP overdose.

The protective role of NAD(P)H:quinone oxidoreductase 1 on acetaminophen-induced liver injury is associated with prevention of adenosine triphosphate depletion and improvement of mitochondrial dysfunction.

An overdose of acetaminophen (APAP) causes hepatotoxicity due to its metabolite, N-acetyl-p-benzoquinone imine. NAD(P)H: quinone oxidoreductase 1 (NQO1) is an important enzyme for detoxification, because it catabolizes endogenous/exogenous quinone to hydroquinone. Although various studies have suggested the possible involvement of NQO1 in APAP-induced hepatotoxicity, its precise role in this remains unclear. We investigated the role of NQO1 against APAP-induced hepatotoxicity using a genetically modified rodent model. NQO1 wild-type (WT) and knockout (KO) mice were treated with different doses of APAP, and we evaluated the mortality and toxicity markers for cell death caused by APAP. NQO1 KO mice showed high sensitivity to APAP-mediated hepatotoxicity (as indicated by a large necrotic region) as well as increased levels of nitrotyrosine adducts and reactive oxygen species. APAP-induced cell death in the livers and primary hepatocytes of NQO1 KO mice, which was accompanied by an extensive reduction in adenosine triphosphate (ATP) levels. In accordance with this ATP depletion, cytosolic increases in mitochondrial proteins such as apoptosis-inducing factor, second mitochondria-derived activator of caspases/DIABLO, endonuclease G, and cytochrome c, which indicate severe mitochondrial dysfunction, were observed in NQO1 KO mice but not in WT mice after APAP exposure. Severe mitochondrial depolarization was also greater in hepatocytes isolated from NQO1 KO mice. Collectively, our data suggest that NQO1 plays a critical role in protection against energy depletion caused by APAP, and NQO1 may be useful in the development of therapeutic approaches to effectively diminish the hepatotoxicity caused by an APAP overdose.

Isotope ratios of total C, N, and S in particulate matter simultaneously calibrated by mixed USGS and IAEA reference materials.

This study presents a method for calibrating the isotope ratios of the total carbon, nitrogen, and sulfur in particulate matter (PM) collected from the Seoul metro using an elemental analyzer-isotope ratio mass spectrometer (EA-IRMS). Mixtures of isotope reference materials (MRMs) from the U.S. Geological Survey (USGS) and International Atomic Energy Agency (IAEA) reference materials formed an input dataset for generalized least squares (GLS) regression to yield calibration lines. The analytical method proposed in this study enabled the measurement of stable isotope ratios of total carbon, nitrogen, and sulfur simultaneously. Results showed good linearity and repeatability for carbon and nitrogen isotopes, but poor results for sulfur isotopes due to peak broadening. Reference values with uncertainties for the isotope ratios of total carbon, nitrogen, and sulfur were determined for the collected PM, demonstrating twice as much uncertainty as that of the USGS and IAEA reference materials. Homogeneity was the biggest uncertainty source for the calibrated values.

Particulate Matter Induces Oxidative Stress and Ferroptosis in Human Lung Epithelial Cells.

Numerous toxicological studies have highlighted the association between urban particulate matter (PM) and increased respiratory infections and lung diseases. The adverse impact on the lungs is directly linked to the complex composition of particulate matter, initiating reactive oxygen species (ROS) production and consequent lipid peroxidation. Excessive ROS, particularly within mitochondria, can destroy subcellular organelles through various pathways. In this study, we confirmed the induction of ferroptosis, an iron-dependent cell death, upon exposure to an urban PM using RT-qPCR and signaling pathway analysis. We used KRISS CRM 109-02-004, the certified reference material for the analysis of particulate matter, produced by the Korea Research Institute of Standards and Science (KRISS). To validate that ferroptosis causes lung endothelial toxicity, we assessed intracellular mitochondrial potential, ROS overproduction, lipid peroxidation, and specific ferroptosis biomarkers. Following exposure to the urban PM, a significant increase in ROS generation and a decrease in mitochondrial potential were observed. Furthermore, it induced hallmarks of ferroptosis, including the accumulation of lipid peroxidation, the loss of antioxidant defenses, and cellular iron accumulation. In addition, the occurrence of oxidative stress as a key feature of ferroptosis was confirmed by increased expression levels of specific oxidative stress markers such as NQO1, CYP1B1, FTH1, SOD2, and NRF. Finally, a significant increase in key ferroptosis markers was observed, including xCT/SLC7A11, NQO1, TRIM16, HMOX-1, FTL, FTH1, CYP1B1, CHAC1, and GPX4. This provides evidence that elevated ROS levels induce oxidative stress, which ultimately triggers ferroptosis. In conclusion, our results show that the urban PM, KRISS CRM, induces cellular and mitochondrial ROS production, leading to oxidative stress and subsequent ferroptosis. These results suggest that it may induce ferroptosis through ROS generation and may offer potential strategies for the treatment of lung diseases.

Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are required for steroidogenesis in testicular Leydig cells.

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

Sizing by weighing: characterizing sizes of ultrasmall-sized iron oxide nanocrystals using MALDI-TOF mass spectrometry.

We present a rapid and reliable method for determining the sizes and size distributions of <5 nm-sized iron oxide nanocrystals (NCs) using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). MS data were readily converted to size information using a simple equation. The size distribution obtained from the mass spectrum is well-matched with the data from transmission electron microscopy, which requires long and tedious analysis work. The size distribution obtained from the mass spectrum is highly resolved and can detect size differences of only a few angstroms. We used this MS-based technique to investigate the formation of iron oxide NCs, which is not easy to monitor with other methods. From ex situ measurements, we observed the transition from molecular precursors to clusters and then finally to NCs.

Identification of structurally diverse alkaloids in Corydalis species by liquid chromatography/electrospray ionization tandem mass spectrometry.

RATIONALE: Alkaloids with significant therapeutic effects are the main active constituents of Corydalis (C.) species. There are several kinds of alkaloids in C. species associated with diverse alkaloid metabolism in plants, but they are rarely identified. This study aimed to identify diverse alkaloids in C. species by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). METHODS: Several types of alkaloids were extracted from C. species using ultrasonication with 70% CH(3)OH, and the extract was partitioned at pH 2 and 12. Separation of alkaloids was achieved by C18 high-performance liquid chromatography (HPLC), and MS/MS analysis was conducted by electrospray ionization triple-quadrupole mass spectrometry. For further confirmation, LC/Fourier transform ion cyclotron resonance (FTICR)-MS was used to obtain accurate mass data and gas chromatography (GC)/MS combined with trimethylsilyl derivatization was applied for identification of the minor alkaloids. RESULTS: Thirty-three alkaloids among three different C. species were successfully separated and identified by LC/ESI-MS/MS and LC/FTICR-MS. Structural assignment of individual alkaloids was performed according to MS/MS spectral patterns. For further confirmation, accurate mass data of alkaloids by LC/FTICR-MS were obtained within 5 ppm and the GC/MS data for the trimethylsilyl alkaloids were also obtained. Among 33 alkaloids identified from this study, 13 alkaloids were reported for the first time in the investigated C. species. CONCLUSIONS: The LC/ESI-MS/MS technique was effective in obtaining structural information and yielded diagnostic ions for diverse alkaloids. Based on the identified 33 alkaloids, marker compounds were suggested for the three C. species with different geographic origins. This study may also be useful for elucidating unknown alkaloids in herbal medicines.

Seasonal variations of the airborne microbial assemblages of the Seoul subway, South Korea from 16S and ITS gene profiles with chemical analysis.

In this study, we determined the seasonal airborne microbial diversity profiles at SMRT stations by sequencing the 16S rRNA and ITS. Particulate matter samples were collected from air purifiers installed in the platform area of the SMRT subway stations. Three stations that included the most crowded one were selected for the sampling. The sampling was done at each season during 2019. After extracting the total DNA from all seasonal samples, PCR was performed with Illumina overhang adapter primers for the V3-V4 region of the 16S rRNA gene and ITS2 region of the ITS gene. The amplified products were further purified, and sequencing libraries were made. Sequencing was carried with the Illumina Miseq Sequencing system (Illumina, USA) followed by in-depth diversity analyses. The elemental composition of the particulate matter samples collected from the different subway stations were obtained using a WD-XRF spectrometer. The SMRT microbiome showed extensive taxonomic diversity with the most common bacterial genera at the subway stations associated with the skin. Overall, the stations included in this study harbored different phylogenetic communities based on α- and ß-diversity comparisons. Microbial assemblages also varied depending upon the season in which the samples were taken and the station. Major elements present at the subway stations were from aerosols generated between wheels and brake cushions and between the catenaries and the pantographs. This study shows that the microbial composition of the SMRT subway stations comes from a diverse combination of environmental and human sources, the season and the lifestyle of commuters.

Contribution of the peroxisomal acox gene to the dynamic balance of daumone production in Caenorhabditis elegans.

Dauer pheromones or daumones, which are signaling molecules that interrupt development and reproduction (dauer larvae) during unfavorable growth conditions, are essential for cellular homeostasis in Caenorhabditis elegans. According to earlier studies, dauer larva formation in strain N2 is enhanced by a temperature increase, suggesting the involvement of a temperature-dependent component in dauer pheromone biosynthesis or sensing. Several naturally occurring daumone analogs (e.g. daumones 1-3) have been identified, and these molecules are predicted to be synthesized in different physiological settings in this nematode. To elucidate the molecular regulatory system that may influence the dynamic balance of specific daumone production in response to sudden temperature changes, we characterized the peroxisomal acox gene encoding acyl-CoA oxidase, which is predicted to catalyze the first reaction during biosynthesis of the fatty acid component of daumones. Using acox-1(ok2257) mutants and a new, robust analytical method, we quantified the three most abundant daumones in worm bodies and showed that acox likely contributes to the dynamic production of various quantities of three different daumones in response to temperature increase, changes that are critical in C. elegans for coping with the natural environmental changes it faces.

Activation of NAD(P)H:quinone oxidoreductase 1 prevents arterial restenosis by suppressing vascular smooth muscle cell proliferation.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (betaL) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. betaL significantly reduced the neointimal formation induced by balloon injury. betaL also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. betaL increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the betaL-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by betaL is mediated by LKB1 in the presence of NQO1. Taken together, these results show that betaL inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs.

Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone.

Pheromones are cell type-specific signals used for communication between individuals of the same species. When faced with overcrowding or starvation, Caenorhabditis elegans secrete the pheromone daumone, which facilitates communication between individuals for adaptation to adverse environmental stimuli. Daumone signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle with distinctive adaptive features and extended life. Because daumone is a key regulator of chemosensory processes in development and ageing, the chemical identification of daumone is important for elucidating features of the daumone-mediated signalling pathway. Here we report the isolation of natural daumone from C. elegans by large-scale purification, as well as the total chemical synthesis of daumone. We present the stereospecific chemical structure of purified daumone, a fatty acid derivative. We demonstrate that both natural and chemically synthesized daumones equally induce dauer larva formation in C. elegans (N2 strain) and certain dauer mutants, and also result in competition between food and daumone. These results should help to elucidate the daumone-mediated signalling pathway, which might in turn influence ageing and obesity research and the development of antinematodal drugs.

Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis.

Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.

High dissolved organic radiocarbon in precipitation during winter and its implication on the carbon cycle.

Radiocarbon (14C) analysis is a powerful tool for tracing carbon in the global carbon cycle. Precipitation is a component of the global carbon cycle through which dissolved organic carbon (DOC) enters terrestrial and aquatic ecosystems from the atmosphere. In previous studies, the Δ14C of DOC in rain or snow was negative indicating an input of relatively old organic carbon including fossil fuels, with only a few positive values up to +108‰ showing the signal of recent photosynthesis. However, here we report surprisingly high Δ14C-DOC in bulk precipitation, more than 1000‰ in Seoul, South Korea, especially when the Northwesterly wind blows during winter. In contrast, Δ14C of particulate organic carbon (POC) in bulk precipitation was negative, indicating that the sources of POC and DOC were different. Although the sources of the high Δ14C-DOC are not clear and future studies on them are required, the relatively high Δ14C-DOC in a nearby headwater stream suggests that precipitation DOC has the potential to affect the local carbon cycle, and that stream DOC derived from terrestrial ecosystems could be older than previously thought. The analysis of Δ14C-DOC of precipitation in many other locations is necessary to understand how long carbon stays in terrestrial ecosystems.

A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry.

D-erythro-Sphingosine is known to be phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate. With the importance of sphingosine-1-phosphate in biological functions being made evident by recent research, a selective and convenient method of assay to measure sphingosine kinase activity is required. Here we developed a new sphingosine kinase assay using murine teratocarcinoma mutant F9-12 cells and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with direct infusion. Sphingosine-1-phosphate in the crude extract of enzyme reaction mixture was selectively characterized and quantitated using precursor ion scanning for [PO(3)](-) in the negative electrospray ionization mode. The method was successfully validated for an activator and an inhibitor of sphingosine kinase. Direct quantitation of S1P without the use of radioactive reagents, chemical derivatization, and extensive chromatographic separation enables simplified assay for sphingosine kinase activity at the cellular system level, and the use of a structural analog as an internal standard provides robustness to the assay.

Development of a Certified Reference Material (NMIJ CRM 7203-a) for Elemental Analysis of Tap Water.

A certified reference material (CRM), NMIJ CRM 7203-a, was developed for the elemental analysis of tap water. At least two independent analytical methods were applied to characterize the certified value of each element. The elements certified in the present CRM were as follows: Al, As, B, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Rb, Sb, Se, Sr, and Zn. The certified value for each element was given as the (property value ± expanded uncertainty), with a coverage factor of 2 for the expanded uncertainty. The expanded uncertainties were estimated while considering the contribution of the analytical methods, the method-to-method variance, the sample homogeneity, the long-term stability, and the concentrations of the standard solutions for calibration. The concentration of Hg (0.39 µg kg-1) was given as the information value, since loss of Hg was observed when the sample was stored at room temperature and exposed to light. The certified values of selected elements were confirmed by a co-analysis carried out independently by the NMIJ (Japan) and the KRISS (Korea).

Optimized precursor ion selection for labile ions in a linear ion trap mass spectrometer and its impact on quantification using selected reaction monitoring.

The fragmentation of fragile ions during the application of an isolation waveform for precursor ion selection and the resulting loss of isolated ion intensity is well-known in ion trap mass spectrometry (ITMS). To obtain adequate ion intensity in the selected reaction monitoring (SRM) of fragile precursor ions, a wider ion isolation width is required. However, the increased isolation width significantly diminishes the selectivity of the channels chosen for SRM, which is a serious problem for samples with complex matrices. The sensitive and selective quantification of many lipid molecules, including ceramides from real biological samples, using a linear ion trap mass spectrometer is also hindered by the same problem because of the ease of water loss from protonated ceramide ions. In this study, a method for the reliable quantification of ceramides using SRM with near unity precursor ion isolation has been developed for ITMS by utilizing alternative precursor ions generated by in-source dissociation. The selected precursor ions allow the isolation of ions with unit mass width and the selective analysis of ceramides using SRM with negligible loss of sensitivity. The quantification of C18:0-, C24:0- and C24:1-ceramides using the present method shows excellent linearity over the concentration ranges from 6 to 100, 25 to 1000 and 25 to 1000 nM, respectively. The limits of detection of C18:0-, C24:0- and C24:1-ceramides were 0.25, 0.25 and 5 fmol, respectively. The developed method was successfully applied to quantify ceramides in fetal bovine serum.

An indole derivative protects against acetaminophen-induced liver injury by directly binding to N-acetyl-p-benzoquinone imine in mice.

AIMS: Acetaminophen (APAP)-induced liver injury is mainly due to the excessive formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) through the formation of a reactive intermediate, N-acetyl-p-benzoquinone imine (NAPQI), in both humans and rodents. Here, we show that the indole-derived synthetic compound has a protective effect against APAP-induced liver injury in C57Bl/6 mice model. RESULTS: NecroX-7 decreased tert-butylhydroperoxide (t-BHP)- and APAP-induced cell death and ROS/RNS formation in HepG2 human hepatocarcinoma and primary mouse hepatocytes. In mice, NecroX-7 decreased APAP-induced phosphorylation of c-Jun N-terminal kinase (JNK) and 3-nitrotyrosine (3-NT) formation, and also protected mice from APAP-induced liver injury and lethality by binding directly to NAPQI. The binding of NecroX-7 to NAPQI did not require any of cofactors or proteins. NecroX-7 could only scavenge NAPQI when hepatocellular GSH levels were very low. INNOVATION: NecroX-7 is an indole-derived potent antioxidant molecule, which can be bound to some types of radicals and especially NAPQI. It is well known that the NAPQI is a major intermediate of APAP, which causes necrosis of hepatocytes in rodents and humans. Thus, blocking NAPQI formation or eliminating NAPQI are novel strategies for the treatment or prevention of APAP-induced liver injury instead of GSH replenishment. CONCLUSION: Our data suggest that the indole-derivative, NecroX-7, directly binds to NAPQI when hepatic GSH levels are very low and the NAPQI-NecroX-7 complex is secreted to the blood from the liver. NecroX-7 shows more preventive and similar therapeutic effects against APAP-induced liver injury when compared to the effect of N-acetylcysteine in C57Bl/6 mice.

A strategy for enrichment of the bioactive sphingoid base-1-phosphates produced by Hypericum perforatum L. in a balloon type airlift reactor.

An efficient enrichment method using immobilized metal affinity chromatography (IMAC) was developed for selective extraction of bioactive sphingoid base-1-phosphates (SB1Ps) from adventitious roots of Hypericum perforatum cultured in bioreactor. The phosphate-selective IMAC enrichment coupled with LC-MS/MS enabled sensitive analysis of low-abundance SB1Ps present in the root biomass, which would not be feasible otherwise due to severe interferences from complex biological matrices. The time-dependent growth rate and production of SB1Ps from adventitious roots were investigated. The level of phytosphingosine-1-phosphate, which was found to be the major SB1Ps, reached a maximum amount of 635.6pmolpergram of dry weight after 3weeks of culture and decreased between 3 and 5weeks of culture subsequently. On the other hand, sphingosine-1-phosphate and sphinganine-1-phosphate were present at levels of 18.91 and 73.15pmolpergram of dry weight, respectively, after a week of culture and their level decreased thereafter.