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The authors have retracted this article [1] because after publication they became aware that the equine urine samples analysed for loxoprofen in this study were in fact equine plasma samples. Therefore the results and conclusions of this article cannot be relied upon. All authors agree to this retraction.

Loxoprofen is a non-steroidal anti-inflammatory drug of the 2-arylpropionic acid type, which has used to treat musculoskeletal disorders in the horse racing industry. However, it has also used illicitly to mask clinical signs of inflammation and pain in racehorses. Thus, its accurate analysis has become an important issue in horse doping laboratories. In this study, an analytical method of loxoprofen was developed as tert-butyldimethylsilyl (TBDMS) derivative by gas chromatography-mass spectrometry (GC-MS). Characteristic fragment ions of [M-15], [M-57], and [M-139] permitted the accurate and selective detection of loxoprofen. Under optimal conditions, this method showed good linearity (r ≥ 0.999) in the range of 10-500 ng/mL, repeatability (% relative standard deviation = 5.6-8.5), and accuracy (% relative error = - 0.3-0.9) with a detection limit of 1.0 ng. When applied to the analysis of loxoprofen in tablet and patch products, loxoprofen was positively identified as TBDMS derivative by GC-MS. The present method provided rapid and accurate determination of loxoprofen in patch and tablet products. Levels of loxoprofen were highest in equine urine at 0.5 and 1 h after oral administration with single dose (3 mg/kg) to three horses, and then rapidly reduced to below the lower limit of quantification at 24 h. Therefore, the present method will be useful for the pharmacokinetic study and doping tests for loxoprofen and other similar acidic drugs in horses.

More than 50% of sepsis cases are associated with pneumonia. Sepsis is caused by infiltration of bacteria into the blood via inflammation, which is triggered by the release of cell wall components following lysis. However, the regulatory mechanism of lysis during infection is not well defined. Mice were infected with Streptococcus pneumoniae D39 wild-type (WT) and lipase mutant (ΔlipA) intranasally (pneumonia model) or intraperitoneally (sepsis model), and survival rate and pneumococcal colonization were determined. LipA and autolysin (LytA) levels were determined by qPCR and western blotting. S. pneumoniae Spd_1447 in the D39 (type 2) strain was identified as a lipase (LipA). In the sepsis model, but not in the pneumonia model, mice infected with the ΔlipA displayed higher mortality rates than did the D39 WT-infected mice. Treatment of pneumococci with serum induced LipA expression at both the mRNA and protein levels. In the presence of serum, the ΔlipA displayed faster lysis rates and higher LytA expression than the WT, both in vitro and in vivo. These results indicate that a pneumococcal lipase (LipA) represses autolysis via inhibition of LytA in a sepsis model.

Introduction: Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease with heterogeneous clinical manifestations mediated by immune dysregulation., Objectives: We aimed to analyze the metabolomic differences in free fatty acids (FFAs) between patients with SLE and healthy controls (HCs)., Methods: In this study, the levels of 24 FFAs, as their tert-butyldimethylsilyl derivatives, in the plasma of 41 patients with SLE and 41 HCs, were investigated using gas chromatography with mass spectrometry in selected-ion monitoring mode., Results: The results showed that patients with SLE and HCs had significantly different levels of 13 of the 24 FFAs. The levels of myristic, palmitoleic, oleic, and eicosenoic acids were significantly higher, whereas the levels of caproic, caprylic, linoleic, stearic, arachidonic, eicosanoic, behenic, lignoceric, and hexacosanoic acids were significantly lower in patients with SLE, than in the HCs. In the partial-correlation analysis of the FFA profiles and markers of disease activity of SLE, several metabolic markers correlated with SLE disease activity., Conclusions: Our results provide a comprehensive understanding of the relationship between FFAs and markers of SLE disease activity. Thus, this approach has promising potential for the discovery of metabolic biomarkers of SLE.

Osteopenia is characterized by bone loss and deterioration of trabecular bone, which leads to osteoporotic fractures. This disease is highly prevalent in industrialized areas and is associated with exposure to endocrine disrupting chemicals (EDCs). Diisononyl phthalate (DINP) is one of these EDCs and is mainly used as a plasticizer in flexible polyvinyl chloride (PVC) products. Although it is well known that exposure to DINP is harmful to humans, no studies have been reported concerning its contribution to osteopenia. Therefore, in this study, we injected DINP (2, 20, and 200mg/kg) into C3H/HeN mice for 6weeks and found that the uterus weight, bone (femur and tibia) weight, and bone length of the DINP-exposed mice were reduced compared to those of the SHAM group. On the other hand, body weight, the serum alkaline phosphatase (ALP) and inorganic phosphorus (IP) levels in the DINP treated mice were increased compared with those of the SHAM group. The tartrate-resistant acid phosphatase (TRAP) activity (bone resorption marker) was increased and the bone alkaline phosphatase (BALP) activity was lowered by the treatment with DINP as compared with the SHAM group. Furthermore, the microarchitecture of the femur and tibia in the intact mice was destroyed by the DINP injection. The tissue volume (TV), bone volume (BV), BV/TV, bone surface (BS), BS/TV, trabecular thickness (Tb.Th), and trabecular number (Tb.N) were reduced and the trabecular pattern factor (Tb.Pf), structure model index (SMI), and trabecular separation (Tb.Sp) were increased by the DINP injection. The bone mineral density (BMD) of the femur and tibia was lower in the DINP group than in the SHAM group. These results indicate that DINP contributes to an increased risk of osteopenia via destruction of the microarchitecture and enhancement of osteoclast activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Diisononyl phthalate (DINP), a member of the phthalate family, is used to plasticize polyvinyl chloride (PVC). This chemical is known to enhance airway inflammation in the OVA-induced asthma model (adjuvant effects) and aggravate allergic dermatitis. Moreover, DINP enhances the production of interleukin-4 in activated CD4 + T cells. However, the effect of DINP itself on the differentiation of naïve CD4 + T cells into T helper cells (Th1/Th2) in vitro and allergic asthma in vivo has not yet been studied. In this study, DINP was shown to suppress the polarization of Th1 and enhance the polarization of Th2 from naïve CD4 + T cells in vitro. Also, DINP induced allergic asthma via the production of IL-4, IL-5, IgE and IgG1 and the reduction of IFN-γ and IgG2a. Finally, we confirmed that exposure to DINP induces the infiltration of inflammatory cells and PAS positive cells and increases the expression of caspase-1 and caspase-3 in asthmatic mice. In conclusion, we suggest that DINP as an environmental pollutant or endocrine disruptor (ECD) induces asthma via the modulation of the Th1/Th2 equilibrium and production of Th2 mediated cytokines and immunoglobulin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The liquid chromatographic enantiomer separation of various chiral acids as 1-naphthylamides was performed using several chiral stationary phases (CSPs). The CSPs used in this study were six covalently bonded and four coated type CSPs derived from amylose and cellulose derivatives as chiral selectors. The degree of enantioseparation is affected by the structure of chiral acids and the CSPs used, which have different chiral selectors and types of immobilization. For the enantiomer resolution of chiral acids as 1-naphthylamide derivatives, the performance of the coated type Lux Cellulose-1 was superior to those of the other CSPs, except for 2-aryloxypropionic acid derivatives. Owing to the strong ultraviolet absorbance of the 1-naphthyl group, the convenient analytical method developed and validated in this study could be expected to be very useful for the enantiomer separation of various chiral acids as 1-naphthylamide derivatives using polysaccharide-derived CSPs.

Metabolomic analysis of urinary polyamines (PAs) from rat exposed to 915 MHz radiofrequency identification (RFID) signal for 8 h/day for 2 weeks was performed by gas chromatography-mass spectrometry as N-ethoxycarbonyl/N-pentafluoropropionyl derivatives. Large alterations in nine PA levels including four aliphatic and five acetylated PAs were monitored in sham-exposed and RFID-exposed groups. Total PA and urinary levels of N (1)-acetylputrescine, N (1)-acetylcadaverine, putrescine, cadaverine, N (1)-acetylspermidine, N (8)-acetylspermidine, spermidine and spermine were reduced, whereas N (1)-acetylspermine was significantly increased after sham and RFID exposure compared with those before exposure. Their levels were normalized to the corresponding group means before exposure and then plotted into star symbol patterns. N (1)-Acetylspermine after RFID exposure was 54 % higher compared to the level before RFID exposure, while it was elevated by only 17 % in the sham group. The results suggest that 915 MHz RFID exposure may induce metabolic disturbance of PA. It may also elevate spermidine/spermine acetyltransferase (SSAT) activity. Thus, the present metabolic profiling combined with star pattern recognition method might be useful for understanding the complexity of biochemical events after exposure to RFID signal.

Purpose: We investigated the effects of whole-body exposure to the 915 MHz radiofrequency identification (RFID) on melatonin biosynthesis and the activity of rat pineal arylalkylamine N-acetyltransferase (AANAT)., Materials and Methods: Rats were exposed to RFID (whole-body specific absorption rate, 4 W/kg) for 8 h/day, 5 days/week, for weeks during the nighttime. Total volume of urine excreted during a 24-h period was collected after RFID exposure. Urinary melatonin and 6-hydroxymelatonin sulfate (6-OHMS) was measured by gas chromatography-mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. AANAT enzyme activity was measured using liquid biphasic dif-13 fusion assay. Protein levels and mRNA expression of AANAT was 14 measured by Western blot and reverse transcription polymerase 15 chain reaction (RT-PCR) analysis, respectively., Results: Eight hours of nocturnal RFID exposure caused a significant reduction in both urinary melatonin (p = 0. 003) and 6-OHMS (p = 0. 026). Activity, protein levels, and mRNA expression of AANAT were suppressed by exposure to RFID (p < 0. 05)., Conclusions: Our results suggest that nocturnal RFID exposure can cause reductions in the levels of both urinary melatonin and 6-OHMS, possibly due to decreased melatonin biosynthesis via suppression of Aanat gene transcription in the rat pineal gland.

Purpose: The apnea-hypopnea index (AHI) is closely related with the severity of daytime sleepiness, but excessive daytime sleepiness (EDS) is not presented on all patients with obstructive sleep apnea (OSA). It is unclear why daytime sleepiness is not always present in OSA patients even if their sleep is disrupted from the perspective of polysomnographic findings. This study aimed to analyze the correlation between sleepiness and urine metabolites of neurotransmitters involved in the arousal system., Methods: On the basis of AHI in polysomnography, 49 consecutive OSA patients were included. According to their Epworth sleepiness scale (ESS), 23 non-sleepy patients (ESS <11) and 26 sleepy patients (ESS ≥11) were included. Urine samples were collected before and after polysomnography and analyzed by gas chromatography-mass spectrometry with selective ion monitoring. Six metabolites of dopamine, norepinephrine and serotonin were analyzed., Results: The dopamine metabolites, homovanillic acid (r = 0.366, P = 0.017) and 3,4-dihydroxyphenylacetic acid (DOPAC; r = 0.584, P < 0.0001), were significantly correlated with ESS after adjusting for age, sex, body mass index, AHI, sleep efficiency and total sleep time. A two-by-two table analysis revealed that the overnight increase in the urine DOPAC was more frequent in sleepy patients while its decrease was more frequent in non-sleepy patients (odds ratio = 3.54, P = 0.032)., Conclusion: Urine dopamine metabolites may identify sleepy patients with OSA. In particular, the overnight change of urine DOPAC may indicate OSA patients with EDS.

A novel derivatization method was developed for the simultaneous determination of six acidic metabolites of catecholamine and serotonin by gas chromatography-mass spectrometry (GC-MS). The metabolites were converted to O-ethoxycarbonyl/tert-butyldimethylsilyl (EOC/TBDMS) derivatives for the direct GC-MS analysis in selected ion monitoring mode. Their mass spectral pattern as EOC/TBDMS derivatives showed characteristic fragment ions of [M - 15](+) and [M - 57](+), which permitted rapid and accurate structural confirmation of acidic metabolites. The present method was linear (r ≥ 0.998), reproducible (percentage relative standard deviation = 1.0-10.0) and accurate (% relative error = -9.7-9.8) with detection limits of 0.001-4.7 ng/mL. When applied to human urine samples, the method allowed simultaneous determination of six acidic metabolites of catecholamine and serotonin., (Copyright © 2012 John Wiley & Sons, Ltd.)

Chiral crown ethers have been widely used in the resolution of various chiral compounds containing a primary amino group. Covalently bonded chiral stationary phases derived from (18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) were developed in our groups and utilized for the resolution for several types of analytes. By use of NMR spectroscopy, chiral discrimination studies were performed for α-amino acids and their esters using 18-C-6-TA. Here, advances in the development and application of chiral stationary phases and chiral solvating agents using 18-C-6-TA for enantiomer resolution are described in relationship to recent chiral recognition mechanism studies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Mesenchymal stem cells (MSCs) have the potential to promote brain repair and improve recovery following stroke. We investigated changes in free fatty acids (FFAs) following intravenous human MSC (hMSC) transplantation into rats that had undergone transient middle cerebral artery occlusion (MCAo)., Methods: Rats were subjected to 2-hours MCAo, followed by intravenous transplantation of hMSC or phosphate-buffered saline (PBS) at one day after MCAo. All rats were sacrificed 5 days after MCAo. Metabolic profiling of free fatty acids (FFAs) level was assessed in plasma and brain from control rats (n=8), PBS-treated MCAo rats (n=6), and hMSC-treated MCAo rats (MCAo+hMSC, n=6)., Results: The levels of some FFAs in plasma and brain samples of the MCAo and MCAo+hMSC groups were significantly different from those of the control group. The percentage composition of myristic acid in plasma and those of myristic acid, linoleic acid, and eicosenoic acid in brain tissues of the MCAo+hMSC group were significantly reduced compared to those in the untransplanted MCAo group., Conclusion: Our metabolic approach has provided insights into understanding the complexity of biochemical and physiological events that occur in ischemic brain injury and the transplantation effects of MSCs in stroke.

Background Aims: Because of their ability to differentiate and widespread availability, human mesenchymal stromal cells (hMSC) are often used as a clinical therapeutic tool. However, the factors that determine the quality and viability of hMSC are not well understood., Methods: We evaluated the viability of hMSC over time using flow cytometry analysis (FACS) and transmission electron microscopy (TEM) to determine if morphologic changes occurred in hMSC. In addition, we conducted gene expression prof ling using an Affymetrix Human Genome U133 Plus 2.0 Array., Results: FACS analysis revealed that 83% and 76% of the cells were viable in sterilized phosphate-buffered saline (PBS) after 6 h and 12 h, respectively. TEM data revealed that the total number of cells with healthy chromatins or a few cytosolic vacuoles was significantly reduced over time. We then conducted gene expression profiling using a microarray, which revealed changes in the expression of 2949 functional genes. Specifically, among the total of 50,000 gene probes evaluated, the expression levels of apoptosis and stress-related genes were significantly increased over time., Conclusions: The results of this study suggest that the viability of hMSC decreases after disassociation from the culture dish and time is an essential factor when considering hMSC as a potential source for stem cell-based direct transplantation.

Adenoviral vectors are among the most promising vectors available for human gene therapy. However, the use of recombinant adenoviral vectors, including replicationcompetent adenovirus (RCA), raises a variety of safety concerns in relation to the development of new therapies based on gene therapy. To examine how organic compounds change in rat plasma following the injection of adenovirus, beta-galactosidase expressing recombinant adenovirus (designated rAdLacZ) or RCA, we investigated the content of fatty acids (FAs), which are important biochemical indicators in pathological conditions. Pattern recognition analysis on the level of FAs in rat plasma is described for the visual discrimination of adenovirus infection groups from normal controls. Plasma FAs from four control rats (normal group), and from four rats with rAdLacZ infection and six rats with RCA infection (the two abnormal groups), were examined by gas chromatography-mass spectrometry in selected ion monitoring modes as their tert-butyldimethylsilyl derivatives. In total, 20 FAs were positively detected and quantified. The results of the Studentos t-test on the normal mean of two abnormal groups, the levels of three FAs (p< 0.05) from rAdLacZ group and eleven FAs (p < 0.05) from RCA group were significantly different. When star symbol plotting was applied to the group mean values of 20 FAs after normalization to the corresponding normal mean values, the resulting eicosagonal star patterns of the two infected groups were distorted into similar shapes, but were distinguishable from each other. Thus, these approaches will be useful for screening and monitoring of diagnostic markers for the effects of infection following the use of adenoviral vectors in gene therapy.

Solvent extractive two-phase menthoxycarbonyl (MnOC) derivatization was combined with trimethylsilyl (TMS) reaction for enantioseparation of beta-blockers by gas chromatography employing achiral DB-5 and DB-17 dual-columns of different polarity. beta-Blockers in alkaline solution were vortex-mixed with menthyl chloroformate present in dichloromethane to be extracted as diastereomeric N-MnOC derivatives. The subsequent O(N)-TMS reaction allowed complete enantioseparations of two beta-blockers and partial separations of five as N-MnOC/O(N)-TMS derivatives in a single analysis. The temperature-programmed retention index sets were characteristic of each derivative, facilitating chiral discrimination of each enantiomer.