UPLC-Q-TOF/MS-Based Plasma and Urine Metabolomics Contribute to the Diagnosis of Sepsis.

In this study, we aimed to identify potential metabolic biomarkers that can improve the diagnostic accuracy of sepsis. Sixty-six patients including 30 septic and 36 nonsepsis patients from an intensive care unit were recruited. The global plasma and urine metabolomic profiles were determined by ultraperformance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry-based methodology. The risk factors, including both traditional physiological indicators and metabolic biomarkers, were investigated by binary logistic regression analysis and used to build a least absolute shrinkage and selection operator (Lasso) regression model to evaluate the ability of diagnosis. Fifty-five metabolites in plasma and 11 metabolites in urine were identified through orthogonal projections to latent structures discriminant analysis (OPLS-DA). Among them, ten (PE (20:4(5Z, 8Z, 11Z, 14Z)/P-18:0), harderoporphyrinogen, chloropanaxydiol, (Z)-2-octenal, N1,N8-diacetylspermidine, 1-nitroheptane, venoterpine, α-CEHC, LysoPE (20:0/0:0), corticrocin) metabolites were identified as risk factors. The Lasso regression model incorporating these ten metabolic biomarkers and five traditional physiological indicators displayed better differentiation than the traditional model, represented by the elevated area under receiver operating characteristic curve (AUROC) from 96.80 to 100.0%. Furthermore, patients with septic shock presented a significantly lower level of PE-Cer (d16:1(4E)/19:0). This study suggests that metabolomic profiling could be an effective tool for sepsis diagnosis.

Ultraperformance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry-Based Metabolomics and Lipidomics Identify Biomarkers for Efficacy Evaluation of Mesalazine in a Dextran Sulfate Sodium-Induced Ulcerative Colitis Mouse Model.

This study aims to identify biomarkers for evaluating the therapeutic efficacy of mesalazine on ulcerative colitis by metabolomics and lipidomics. A dextran sulfate sodium-induced mouse model was used. The disease status was assessed by a disease activity index, the TNF-α level of colon was measured by an enzyme-linked immunosorbent assay, and the pathological changes of colon tissue was examined by hematoxylin-eosin staining. Serum metabolomics and lipidomics analysis based on ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were applied to decipher the metabolic profile changes. Multivariate analysis was applied to differentiate the metabolites of controls, models, and mesalazine-treated mice. By the receiver operating characteristic (ROC) analysis, 40 differential metabolites with an area under curve (AUC) >0.80 were screened out between control and model groups. Among them, four potential biomarkers (palmitoyl glucuronide, isobutyrylglycine, PC (20:3 (5Z, 8Z, 11Z)/15:0) and L-arginine) had a signficantly reversed level of peak areas in the mesalazine group, and three of them were closely correlated with mesalazine efficacy by linear regression analysis. Furthermore, metabolic pathway analysis revealed several dysregulated pathways in colitis mice, including glycerophospholipid metabolism, pyrimidine metabolism, linoleic acid metabolism, arginine biosynthesis, etc. This study indicates that serum metabolomics is a useful approach that can noninvasively evaluate the therapeutic effect and provide unique insights into the underlying mechanism of mesalazine.

UPLC-QTOF-MS-Based Plasma Lipidomic Profiling Reveals Biomarkers for Inflammatory Bowel Disease Diagnosis.

Identification of new biomarkers may help in the early diagnosis of inflammatory bowel disease (IBD). In this study, ultrahigh-performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to analyze the untargeted lipidomics and compare plasma lipid profiles between IBD patients and control subjects. The principal component analysis and partial least-squares-discriminant analysis were carried out to distinguish IBD patients from control subjects. Using univariate and multivariate analysis, 55 significantly different metabolites from five lipid classes, fatty acyls (n = 19), glycerophospholipids (n = 5), prenol lipids (n = 10), sphingolipids (n = 2), and sterol lipids (n = 19) were identified. Forty-four of the 55 metabolites were analyzed by receiver operating characteristic (ROC) curve and area under curve (AUC) of >0.80. After validation in an independent cohort, IBD patients were differentiated from the control subjects by significantly altered plasma level of palmitic acid, 7alpha, 25-dihydroxycholesterol, 20-hydroxyeicosatetraenoic (HETE)-d6, (+/-)5,6-epoxy-eicosatrienoic acid (EpETrE), docosahexaenoic acid (DHA), 9-heptadecylenic acid, lactucaxanthin, α-carotene, traumatic acid, and neoquassin with both sensitivity and specificity above 80%. Pathway analysis suggested that IBD dysregulation was related to the biosynthesis of primary bile acid, the metabolism of arachidonic acid, the metabolism of sphingolipid, fatty acid elongation, and glycerophospholipid metabolism. Our results suggest that the lipidomic profiling of patients plasma could be a potential method for IBD diagnosis.

Detection of chloramphenicol in chicken, pork and fish with a molecularly imprinted polymer-based microtiter chemiluminescence method.

In this study, 4-nitrotoluene (NT) was used as dummy template to synthesize a molecularly imprinted polymer that was highly specific for chloramphenicol. The polymer was coated in the wells of 96-well microplates as recognition reagent to develop a chemiluminescence method. The analyte solution and an enzyme-labelled hapten were added into the wells to perform competition, and the light signal was induced with a highly efficient luminol-H2O2-4-(imidazol-1-yl)phenol system. Then, the optimized method was used to determine chloramphenicol in meat (chicken, pork and fish), and the limit of detection (LOD) was 5.0 pg g-1. Furthermore, the polymer-coated plate could be reused four times, and one test could be finished within 20 min. The recoveries from the standard fortified blank meat samples were in the range of 71.5-94.4%. Therefore, this method could be used as a useful tool for routine screening the residue of chloramphenicol in meat samples.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Pharmacokinetic and metabolic studies of Vortioxetine in rats using ultra high performance liquid chromatography with tandem mass spectrometry.

Vortioxetine is a multimodal antidepressant that has been recently utilized globally. Vortioxetine hemi-hydrochloride is a novel salt that was previously reported in our research. However, the pharmacokinetics of this salt and the metabolites of Vortioxetine in vivo remain unknown. In this study, the pharmacokinetics of the Vortioxetine hemi-hydrochloride salt is explored in rats through a newly developed ultra-performance liquid chromatography with tandem mass spectrometry method. In addition, ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry was used to identify the metabolites of Vortioxetine in vivo. The results demonstrate that after a single, 3 mg/kg oral dose, the maximum concentration for the Vortioxetine hemi-hydrochloride salt is 14.63 ± 4.00 ng/mL, and is attained in 1.00∼4.00 h. The area under the plasma concentration-time curve from time 0 to 24 h is 67.30 ± 23.78 ng·h·mL-1 . Additionally, 29 metabolites were identified after the oral administration of 10 mg/kg, including 17 metabolites in the plasma, nine in the urine, and 12 in the feces. Eleven metabolites were novel. The major metabolic pathways include methylation, hydroxylation, oxidation, and glucuronidation. In conclusion, this study provides insight for further development of the Vortioxetine hemi-hydrochloride salt.

Suppression of glioblastoma growth and angiogenesis through molecular targeting of methionine aminopeptidase-2.

Methionine aminopeptidases (MetAPs) have been pharmacologically linked to cell growth, angiogenesis, and tumor progression, which make it an attractive target for cancer therapy. We investigated MetAP2's biological role in glioblastoma (GBM), an aggressive tumor characterized by massive neovascularization. We examined the effect of anti-MetAP2 RNA interference on proliferation and angiogenesis in GBM cell line. The biological effects of MetAP2 knockdown were assessed by comparing the proliferation, tumorigenecity, and angiogenesis of parental cells and MetAP2 knockdown cells. We generated MetAP2 knockdown cells using lentiviral short hairpin RNAs against MetAP2 in SNB19 GBM cells, which normally express high levels of MetAP2. MetAP2 knockdown cells were less proliferative and less tumorigenic when compared to the parental cells. MetAP2 knockdown decreased vascular endothelial growth factor (VEGF) secretion and expression at the mRNA and protein levels. Decreased VEGF expression in MetAP2 knockdown cells correlated very well with decreased vessel formation in a tube formation assay. We showed that VEGF suppression in MetAP2 knockdown cells was mediated by the von Hippel-Lindau protein. In in vivo animal studies using an intracranial SNB19 tumor model, MetAP2 knockdown also reduced the tumor growth rate and angiogenesis, which in turn prolonged the survival of mice in xenograft model. Our results show that MetAP2 regulates angiogenesis in GBM and identify MetAP2-specific substrates that may serve as candidates for clinical assay development.

Miltirone protects human EA.hy926 endothelial cells from oxidized low-density lipoprotein-derived oxidative stress via a heme oxygenase-1 and MAPK/Nrf2 dependent pathway.

BACKGROUND: Oxidized low-density lipoprotein (ox-LDL) is an underlying cause of endothelial dysfunction, which is an early event in the pathogenesis of atherosclerosis. In our previous study, we established an ARE-driven luciferase reporter system and screened out several potential Nrf2 activators from Salvia miltiorrhiza Bunge. PURPOSE: Since miltirone showed the most potent ARE-driven luciferase activity, the aim of this study was to test the protective role of miltirone against oxidative stress in endothelial cell and to investigate the underlying mechanistic signaling pathways. STUDY DESIGN/METHOD: In the present study, miltirone increased the expression of nuclear translocation and transcriptional activities of NF-E2-related factor 2 (Nrf2), which led to augmented expression of antioxidant-response element (ARE)-dependent heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO1). Inhibition of Nrf2/HO-1 by RNA interference abolished miltirone-induced cytoprotective effects against ox-LDL, which suggested that Nrf2 and the downstream expression of HO-1 are required for the functional effects of miltirone. Ox-LDL-stimulated mitogen-activated protein kinase activation, ROS production, and miltirone dramatically inhibited synthesis of ROS, as well as decreased SOD and glutathione S-transferase (GST) in human EA.hy926 endothelial cells. RESULTS: Miltirone-induced Nrf2 and HO-1 expression was related to mitogen-activated protein kinase (MAPK) pathways. The activation of MAPK was partially dependent on the phosphorylation of the c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways, but not P38 MAPK signaling. However, miltirone-induced Nrf2/HO-1 expression can only be effectively blocked by JNK inhibitor SP600125. CONCLUSION: Our findings reveal that miltirone exerts protective functions on endothelial cells in response to ox-LDL-induced oxidative stress, and does so via Nrf2/HO-1, which provides novel insights into the antioxidant capacity of miltirone.

Ultra-high performance liquid chromatography with ultraviolet and tandem mass spectrometry for simultaneous determination of metabolites in purine pathway of rat plasma.

It has been proved that the purine metabolic pathway has been implicated in various biological disorders including gout, diabetes, coronary heart diseases, and neurodegenerative diseases. The analysis of the purine metabolic pathway in organisms reveals important alterations under different physiological and pathological conditions, which contributes to the pathological study, diagnosis, and therapy of related diseases. In the present study, an ultra-high performance liquid chromatography with ultraviolet and tandem mass spectrometry (UHPLC-UV-MS/MS) method was developed for conducting the comprehensive analysis of the metabolite profiles of the purine pathway in rat plasma through a single analysis. The purine metabolites including adenosine-5'-monophosphate, guanosine-5'-monophosphate, adenosine, inosine, guanosine, inosine-5'-monophosphate, deoxyadenosine, deoxyguanosine, deoxyinosine, xanthine, hypoxanthine, and uric acid, were separated and quantified in the short running time of 10min. After rapid chromatographic separation achieved by an Agilent Zorbax SB-Aq column, high concentration of uric acid and the remaining purine metabolites at lower levels were respectively detected by ultraviolet detector and triple quadruple mass spectrometry within a single analysis. The proposed method was validated by applying charcoal-stripped plasma as a matrix and it was proved to be linear (R2>0.982), accurate (with a relative error for accuracy <±15% and the relative standard deviation for intra- and inter-run precision <11%) and reproducible (with a matrix effect ranging between 86.49% and 111.44% with a maximum RSD of 8.69%). As a result, the method was successfully applied to the quantification of the endogenous purine metabolites in rat plasma. It was found that the concentration levels of the purine metabolites may keep a physiological balance as an integrated system in normal individuals and the concentration level of uric acid in rat plasma was 64µM which was more than 200 times greater than the other purine metabolites. The established method and the measurement of the concentration of these purines in normal rat plasma may help the investigation of the action mechanisms between purine disorders and related diseases.