Ex vivo imaging and analysis of ROS generation correlated with microglial activation in rat model with acute neuroinflammation induced by intrastriatal injection of LPS.

Neuroinflammation and oxidative stress are hallmarks of neurodegenerative diseases. Microglia, the major important regulators of neuroinflammation, are activated in response to excessive generation of reactive oxygen species (ROS) from damaged cells and resulting in elevated and sustained damages. However, the relationship between microglia and ROS-regulatory system in the early stages of neuroinflammation prior to the appearance of neuronal damages have not been elucidated in detail. In this study, we analyzed the time-dependent changes in ROS generation during acute neuroinflammation in rats that were given an intrastriatal injection of lipopolysaccharide (LPS). We evaluated the effects of minocycline, an anti-inflammatory antibiotic, and N,N'-dimethylthiourea (DMTU), a radical scavenger, to understand the correlation between activated microglia and ROS generation. Ex vivo fluorescence imaging using dihydroethidium (DHE) clearly demonstrated an increased ROS level in the infused side of striatum in the rats treated with LPS. The level of ROS was changed in time-dependent manner, and the highest level of ROS was observed on day 3 after the infusion of LPS. Immunohistochemical studies revealed that time-dependent changes in ROS generation were well correlated to the presence of activated microglia. The inhibition of microglial activation by minocycline remarkably reduced ROS levels in the LPS-injected striatum, which indicated that the increased ROS generation caused by LPS was induced by activated microglia. DMTU decreased ROS generation and resulted in remarkable inhibitory effect on microglial activation. This study demonstrated that ROS generation during acute neuroinflammation induced by LPS was considerably associated with microglial activation, in an intact rat brain. The results provides a basis for understanding the interaction of ROS-regulatory system and activated microglia during neuroinflammation underlying neurodegenerative diseases.

Metabolomic profiling of urine-derived extracellular vesicles from rat model of drug-induced acute kidney injury.

Extracellular vesicles (EVs) are lipid bilayer particles that are released by various cells and provide a real-time snapshot of the state of these cells in tissue in a noninvasive manner. EVs contain components, including mRNA, miRNAs, proteins, and metabolites. Therefore, EVs hold promise for the discovery of liquid biopsy-based biomarkers for disease diagnosis. In the present study, metabolome analysis of urine EVs in rats with kidney injury caused by cisplatin and puromycin aminonucleoside was performed using liquid chromatography/mass spectrometry to identify candidate biomarkers that reflect the type and extent of injury in drug-induced nephrotoxicity. A total of 396 metabolites were detected in urine EVs, of which 65 were identified as potential biomarkers in urine EVs of drug-induced nephrotoxicity. Pathway analysis revealed that these metabolites may reflect changes occurring within damaged cells during kidney injury, suggesting that metabolomics of urine EVs could be a useful informative tool.

Fragmentation study of tryptophan-derived metabolites induced by electrospray ionization mass spectrometry for highly sensitive analysis.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is interfaced with electrospray ionization (ESI), which generally produces intact gas-phase ions of biomolecules. However, ESI induces the fragmentation of tryptophan-derived metabolites, which are known to act as neurotransmitters and psychoactive drugs. Tryptophan-derived metabolites undergo N-Cα bond dissociation during ESI, producing a fragment ion with a spiro[cyclopropane-indolium] backbone. Fragmentation is suppressed by the presence of an α-carboxyl group and the modification of amino groups. In particular, tryptamine and serotonin, which lack such functional groups, produce more intense fragment-ion signals than protonated molecules. The multiple reaction monitoring (MRM)-based quantitative analysis of tryptamine and serotonin used the fragment ions produced from in-source collision-induced dissociation as the precursor ions, which improved the signal-to-noise ratio of the resulting spectra. The present method allows for the quantitative analysis of tryptamine and serotonin with high sensitivity.

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and <1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis.

In-Source Fragmentation of Phenethylamines by Electrospray Ionization Mass Spectrometry: Toward Highly Sensitive Quantitative Analysis of Monoamine Neurotransmitters.

Electrospray ionization mass spectrometry (ESI-MS) is widely used to analyze biomolecules, which are usually detected as protonated and cation-adducted molecules in the positive-ion mode. However, phenethylamine derivatives, which are known as neurotransmitters and psychoactive drugs, undergo the protonation and subsequently lose NH3 during ESI. As a result, intense fragment-ion signals are observed in their ESI-MS spectra, which hamper the unambiguous identification of phenethylamine derivatives. To understand the mechanism of the loss of NH3 from these phenethylammoniums, the fragmentations of model 4-substituted phenethylamines were investigated and the fragment ions were identified as spiro[2.5]octadienyliums. Fragmentation was enhanced by the presence of electron-donating groups, and most substituted phenethylamines generated spiro[2.5]octadienyliums as fragment ions during ESI-MS, except those with strong electron-withdrawing groups. The quantitative analysis of phenethylamines by liquid chromatography tandem mass spectrometry is typically performed by multiple reaction monitoring using protonated molecules as the precursor. In contrast, the conversion of precursor ions from the protonated molecules into the spiro[2.5]octadienylium fragment improved the signal-to-noise ratio, allowing the quantitative analysis of phenethylamines with high sensitivity and accuracy.

18F-Labeled dihydromethidine: positron emission tomography radiotracer for imaging of reactive oxygen species in intact brain.

Dihydromethidine (DHM) labeled with 18F at the para position of the peripheral benzene ring was designed as a positron emission tomography (PET) radiotracer for non-invasive imaging of reactive oxygen species (ROS). This compound readily crosses the blood-brain barrier and is oxidized by ROS, and the oxidation product is retained intracellularly. PET imaging of ROS-producing rat brain microinfused with sodium nitroprusside identified specific brain regions with high ROS concentrations. This tracer should be useful for studies of the pathophysiological roles of ROS, and in the diagnosis of neurodegenerative diseases.

A Simple Ex Vivo Semiquantitative Fluorescent Imaging Utilizing Planar Laser Scanner: Detection of Reactive Oxygen Species Generation in Mouse Brain and Kidney.

OBJECTIVE: Oxidative stress plays an important role in the onset of many neuronal and peripheral disorders. We examined the feasibility of obtaining semiquantitative fluorescent images of reactive oxygen species (ROS) generation in mouse brain and kidney utilizing a planar laser scanner and dihydroethidium (DHE). METHODS: To investigate ROS generation in brain, sodium nitroprusside was injected into the striatum. Dihydroethidium was injected into the tail vein. After DHE injection, tissue slices were analyzed utilizing a planar laser scanner. For kidney study, cis-diamminedichloroplatinum [II] (cisplatin) was intraperitoneally administrated into mice. RESULTS: Clear and semiquantitative fluorescent images of ROS generation in the mouse brain and kidney were obtained. Furthermore, the fluorescence intensity was stable and not affected by fading. Sodium nitroprusside induced approximately 6 times the fluorescence accumulation in the brain. Cisplatin caused renal injury in all mice, and in comparison with control mice, more than 10 times fluorescence accumulation was observed in the renal medulla with tubular necrosis and vacuolization. CONCLUSIONS: We successfully obtained ex vivo semiquantitative fluorescent images of ROS generation utilizing a planar laser scanner and DHE. This simple method is useful for ROS detection in several ROS-related animal models and would be applicable to a variety of biochemical processes.

Isotope Corrected Chiral and Achiral Nontargeted Metabolomics: An Approach for High Accuracy and Precision Metabolomics Based on Derivatization and Its Application to Cerebrospinal Fluid of Patients with Alzheimer's Disease.

We propose a chiral metabolomics approach based on a data-dependent MS/MS analysis (DDA) using high-resolution quadrupole-time-of-flight mass spectrometry (Q-TOFMS) and 13C-isotope coded derivatization (ICD) reagents, i.e., iDMT-( S)-A and iDMT-( S)-PO. The advantage of the method is the correction of all detected derivatives by parallel derivatization of the isotope-coded and noncoded reagents. The automatic data analysis platform using an MSDIAL and ICD discrimination program, called DINA, was also developed and used for the data analysis process. As a result, a 0.5-2.0% (d-/l-isomer) variation of the isomers was correctly recognized in the automatic data analysis step. Both the semiquantitative comparison and identification efficiency were improved as a result of the high resolution/accuracy of the MS and MS/MS spectra derived from the DDA analysis. This method was used for biomarker discovery in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD). Twenty-four biomarker candidates were successfully determined, including 8 chiral ones.

High-Throughput Bioanalysis of Bevacizumab in Human Plasma Based on Enzyme-Linked Aptamer Assay Using Anti-Idiotype DNA Aptamer.

We propose a highly selective, sensitive, accurate, and high-throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A-HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader. The calibration curve of bevacizumab ranged from 0.05 to 5.0 µg/mL, and showed a good correlation coefficient ( r2 = 1.000). The limit of detection was 2.09 ng/mL. We also demonstrated both the possibility of highly sensitive detection using luminol chemiluminescence and the repeated use of affinity plates. The proposed method is applicable for planning optimal therapeutic programs and for an evaluation of the biological equivalencies in the development of biosimilars.

Anti-Idiotype DNA Aptamer Affinity Purification⁻High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab.

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification⁻high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 µg/mL and showed good correlation coefficients (r² > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 µg/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.

Assessment of Anticancer Drug Effects on Pancreatic Cancer Cells under Glucose-Depleted Conditions Using Intracellular and Extracellular Amino Acid Metabolomics.

Previously, we developed a method to evaluate states of cells treated with anticancer drugs via the comprehensive analysis of amino acids, termed amino acid metabolomics. In the present study, we evaluated the effects of the anticancer drugs, gemcitabine hydrochloride and pyrvinium pamoate, on the proliferation of a pancreatic cancer cell line (PANC-1) under hypoglycemic conditions using amino acid metabolomics. Intracellular and extracellular amino acid profiles of PANC-1 were determined by hydrophilic interaction chromatography-tandem mass spectrometry with simple pretreatment. Changes to the drugs' anticancer effects resulting from glucose starvation conditions were presented in score plots obtained from principal component analyses. In particular, the analysis of intracellular amino acids was found to be the superior approach because the results allowed a clearer assessment of the cell state. Further, orthogonal partial least squares discriminant analysis was performed to search for amino acid candidates that discriminate with anticancer drug-treated PANC-1 cells. We identified several amino acids that might be able to distinguish the drug-treated group from the control group. These results might provide a better understanding of the mechanisms underlying cell responses such as drug resistance or austerity. The present study is the first to evaluate the efficacy of anticancer drugs under glucose starvation based on the analysis of the variation of extracellular and intracellular amino acid profiles in vitro.

Caffeine Suppresses the Activation of Hepatic Stellate Cells cAMP-Independently by Antagonizing Adenosine Receptors.

During liver injury, hepatic stellate cells (HSCs) are activated by various cytokines and transdifferentiated into myofibroblast-like activated HSCs, which produce collagen, a major source of liver fibrosis. Therefore, the suppression of HSC activation is regarded as a therapeutic target for liver fibrosis. Several epidemiological reports have revealed that caffeine intake decreases the risk of liver disease. In this study, therefore, we investigated the effect of caffeine on the activation of primary HSCs isolated from mice. Caffeine suppressed the activation of HSC in a concentration-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator, had no effect on the caffeine-induced suppression of HSC activation. None of the isoform-selective inhibitors of phosphodiesterase1 to 5 affected changes in the morphology of HSC during activation, whereas CGS-15943, an adenosine receptor antagonist, inhibited them. Caffeine had no effect on intracellular cAMP level or on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. In contrast, caffeine significantly decreased the phosphorylation of Akt1. These results suggest that caffeine inhibits HSC activation by antagonizing adenosine receptors, leading to Akt1 signaling activation.

The great importance of normalization of LC-MS data for highly-accurate non-targeted metabolomics.

The non-targeted metabolomics analysis of biological samples is very important to understand biological functions and diseases. LC combined with electrospray ionization-based MS has been a powerful tool and widely used for metabolomic analyses. However, the ionization efficiency of electrospray ionization fluctuates for various unexpected reasons such as matrix effects and intraday variations of the instrument performances. To remove these fluctuations, normalization methods have been developed. Such techniques include increasing the sensitivity, separating co-eluting components and normalizing the ionization efficiencies. Normalization techniques allow simultaneously correcting of the ionization efficiencies of the detected metabolite peaks and achieving quantitative non-targeted metabolomics. In this review paper, we focused on these normalization methods for non-targeted metabolomics by LC-MS.

Dried Saliva Spot (DSS) as a Convenient and Reliable Sampling for Bioanalysis: An Application for the Diagnosis of Diabetes Mellitus.

This paper proposes the dried saliva spot (DSS) as a convenient sampling technique for bioanalysis. The analytical method with the DSS was used for the determination of D,L-lactic acid (D,L-LA) and the D/L ratio of diabetic patients and prediabetic persons for the simple screening of the disease. The D,L-LA in the DSS was labeled with a chiral reagent (DMT-3(S)-Apy) for carboxylic acids and determined by UPLC-ESI-MS/MS. The limits of detection (signal-to-noise ratio (S/N) = 3) for the DSS analysis were on the amol level (∼30 amol). Because good stability, recovery, accuracy, and precision of the D,L-LA for the DSS method was also obtained from the proposed procedure, the DSS method was applied to the determination of the D- and L-isomers of LA of diabetic patients, and prediabetic and healthy persons. The D/L-LA ratio by the present DSS method and the HbA1c value in blood were well-correlated to the serious diabetic patients, whereas the relation in the prediabetic persons was not very good. The reason seems to be due to the rough saliva sampling, and not to the DSS method, because strict regulation was not requested for the prediabetic and healthy persons. In order to have a successful DSS analysis, the stability of the target molecule, the detection sensitivity to the target molecule, and the validated determination method are important.

Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2) > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus.

Simultaneous and rapid determination of gefitinib, erlotinib and afatinib plasma levels using liquid chromatography/tandem mass spectrometry in patients with non-small-cell lung cancer.

A simultaneous, selective, sensitive and rapid liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of gefitinib, erlotinib and afatinib in 250 µL samples of human blood plasma. Diluted plasma samples were extracted using a liquid-phase extraction procedure with tert-butyl methyl ether. The three drugs were separated by high-performance liquid chromatography using a C18 column and an isocratic mobile phase running at a flow rate of 0.2 mL/min for 5 min. The drugs were detected using a tandem mass spectrometer with electrospray ionization using imatinib as an internal standard. Calibration curves were generated over the linear concentration range of 0.05-100 nm in plasma with a lower limit of quantification of 0.01 or 0.05 nm for all compounds. Finally, the validated method was applied to a clinical pharmacokinetic study in patients with nonsmall-cell lung cancer (NSCLC) following the oral administration of afatinib. These results indicate that this method is suitable for assessing the risks and benefits of chemotherapy in patients with NSCLC and is useful for therapeutic drug monitoring for NSCLC treatment. As far as we know, this is the first report on LC-MS/MS method for the simultaneous quantification of NSCLC tyrosine kinase inhibitor plasma concentrations including afatinib. Copyright © 2015 John Wiley & Sons, Ltd.

Uric acid quantification in fingernail of gout patients and healthy volunteers using HPLC-UV.

The presence of elevated uric acid (UA) levels is a sign of gout, that is, hyperuricemia. In this study the monitoring of the UA levels in less-invasive biological samples, such as the human fingernail, is suggested for the diagnosis and therapy of gout. Twenty-six healthy volunteers (HV) and 22 gout patients (GP) were studied. The UA was extracted from human fingernail samples, then separated on an Inertsil ODS-3 column (250 × 4.6 mm i.d., 4.0 µm, GL Sciences) by isocratic elution using methanol-74 mm phosphate buffer (pH 2.2) 2:98 (v/v). A UV detector was used to monitor the samples at 284 nm. Using the developed method, different UA concentrations were found in the GP and HV. When comparing the concentrations from GP with those from HV, a statistically significant correlation was observed between the UA (p < 0.01). In this study, the UA was confirmed as a potential biomarker for the diagnosis and therapy of gout. We have developed a novel sensitive, and simple method for the determination of UA in the fingernails of GP and HV. The human fingernail may serve as a noninvasive biosample for the diagnosis of gout. Copyright © 2016 John Wiley & Sons, Ltd.

Principal component analysis of molecularly based signals from infant formula contaminations using LC-MS and NMR in foodomics.

BACKGROUND: The challenge in developing analytical assessment of unexpected excess contaminations in infant formula has been the most significant project to address the widespread issue of food safety and security. Foodomics based on metabolomics techniques provides powerful tools for the detection of tampering cases with intentional contaminations. However, the safety and risk assessments of infant formula to reveal not only the targeted presence of toxic chemicals, but also molecular changes involving unexpected contaminations, have not been reported. In this study, a huge amount of raw molecularly based signals from infant formula was analysed using reversed phase and hydrophilic interaction chromatography with time-of-flight MS (LC-MS) and (1) H nuclear magnetic resonance (NMR) and then processed by a principal component analysis (PCA). RESULTS: PCA plots visualised signature trends in the complex signal-data batches from each excess contamination of detectable chemicals by LC-MS and NMR. These trends in the different batches from a portion of excess chemical contaminations such as pesticides, melamine and heavy metals and out-of-date products can be visualised from spectrally discriminated infant formula samples. CONCLUSION: PCA plots provide possible attempts to maximise the covariance between the stable lot-to-lot uniformity and excess exogenous contaminations and/or degradation to discriminate against the molecularly based signals from infant formulas. © 2015 Society of Chemical Industry.

Profiling of chiral and achiral carboxylic acid metabolomics: synthesis and evaluation of triazine-type chiral derivatization reagents for carboxylic acids by LC-ESI-MS/MS and the application to saliva of healthy volunteers and diabetic patients.

Novel triazine-type chiral derivatization reagents, i.e., (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) and (S)-4,6-dimethoxy-N-(pyrrolidin-3-yl)-1,3,5-triazin-2-amine (DMT-1(S)-Apy), were developed for the highly sensitive and selective detection of chiral carboxylic acids by UPLC-MS/MS analysis. Among the synthesized reagents, DMT-3(S)-Apy was a more efficient chiral reagent for the enantiomeric separation of chiral carboxylic acids in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The DMT-3(S)-Apy was used for the determination of 13 carboxylic acids in human saliva of healthy volunteers and diabetic patients. Various biological carboxylic acids including chiral carboxylic acids, and mono- and di-carboxylic acids were clearly identified in the saliva of healthy persons and diabetic patients. The concentrations of carboxylic acids detected in the saliva of diabetic patients were relatively higher than those in the healthy persons. Furthermore, the concentration of D-lactic acid (LA) and the ratio of D/L-LA in the diabetic patients were significantly higher than those in the healthy persons. The low ratio of D/L-LA in healthy persons was also identified to be independent of age and sex. These results suggest that the determination of the D/L-LA ratio in saliva might be applicable for the diagnosis of diabetes. Based on these observations, DMT-3(S)-Apy seems to be a useful chiral derivatization reagent for the determination not only of chiral carboxylic acids but also achiral ones. In conclusion, the proposed method using DMT-3(S)-Apy is useful for the carboxylic acid metabolomics study of various specimens.

Automatic analyzer for highly polar carboxylic acids based on fluorescence derivatization-liquid chromatography.

A sensitive, versatile, and reproducible automatic analyzer for highly polar carboxylic acids based on a fluorescence derivatization-liquid chromatography (LC) method was developed. In this method, carboxylic acids were automatically and fluorescently derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride by adopting a pretreatment program installed in an LC autosampler. All of the DBD-PZ-carboxylic acid derivatives were separated on the ODS column within 30 min by gradient elution. The peak of DBD-PZ did not interfere with the separation and the quantification of all the acids with the exception of lactic acid. From the LC-MS/MS analysis, we confirmed that lactic acid was converted to an oxytriazinyl derivative, which was further modified with a dimethoxy triazine group of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). We detected this oxytriazinyl derivative to quantify lactic acid. The detection limits (signal-to-noise ratio = 3) for the examined acids ranged from 0.19 to 1.1 µm, which correspond to 95-550 fmol per injection. The intra- and inter-day precisions of typical, highly polar carboxylic acids were all <9.0%. The developed method was successfully applied to the comprehensive analysis of carboxylic acids in various samples, which included fruit juices, red wine and media from cultured tumor cells.