Overcoming Sample Matrix Effect in Quantitative Blood Metabolomics Using Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry

Blood is widely used for discovery metabolomics to search for disease biomarkers. However, blood sample matrix can have a profound effect on metabolome analysis, which can impose an undesirable restriction on the type of blood collection tubes that can be used for blood metabolomics. We investigated the effect of blood sample matrix on metabolome analysis using a high-coverage and quantitative metabolome profiling technique based on differential chemical isotope labeling (CIL) LC-MS. We used ¹²C-/¹³C-dansylation LC-MS to perform relative quantification of the amine/phenol submetabolomes of four types of samples (i.e., serum, EDTA plasma, heparin plasma, and citrate plasma) collected from healthy individuals and compare their metabolomic results. From the analysis of 80 plasma and serum samples in experimental triplicate, we detected a total of 3651 metabolites with an average of 1818 metabolites per run (<i>n</i> = 240). The number of metabolites detected and the precision and accuracy of relative quantification were found to be independent of the sample type. Within each sample type, the metabolome data set could reveal biological variation (e.g., sex separation). Although the relative concentrations of some individual metabolites might be different in the four types of samples, for sex separation, all 66 significant metabolites with larger fold-changes (FC ≥ 2 and <i>p</i> < 0.05) found in at least one sample type could be found in the other types of samples with similar or somewhat reduced, but still significant, fold-changes. Our results indicate that CIL LC-MS could overcome the sample matrix effect, thereby greatly broadening the scope of blood metabolomics; any blood samples properly collected in routine clinical settings, including those in biobanks originally used for other purposes, can potentially be used for discovery metabolomics

Metabonomic Profiles Discriminate Hepatocellular Carcinoma from Liver Cirrhosis by Ultraperformance Liquid Chromatography–Mass Spectrometry

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and usually develops in patients with liver cirrhosis (LC). Biomarkers that discriminate HCC from LC are important but are limited. In the present study, an ultraperformance liquid chromatography–mass spectrometry (UPLC–MS)-based metabonomics approach was used to characterize serum profiles from HCC (<i>n</i> = 82), LC (<i>n</i> = 48), and healthy subjects (<i>n</i> = 90), and the accuracy of UPLC–MS profiles and alpha-fetoprotein (AFP) levels were compared for their use in HCC diagnosis. By multivariate data and receiver operating characteristic curves analysis, metabolic profiles were capable of discriminating not only patients from the controls but also HCC from LC with 100% sensitivity and specificity. Thirteen potential biomarkers were identified and suggested that there were significant disturbances of key metabolic pathways, such as organic acids, phospholipids, fatty acids, bile acids, and gut flora metabolism, in HCC patients. Canavaninosuccinate was first identified as a metabolite that exhibited a significant decrease in LC and an increase in HCC. In addition, glycochenodeoxycholic acid was suggested to be an important indicator for HCC diagnosis and disease prognosis. UPLC–MS signatures, alone or in combination with AFP levels, could be an efficient and convenient tool for early diagnosis and screening of HCC in high-risk populations

Risk-oriented banking supervision: approaches to the assessment of general systemic risks

The last change introduced by the Basel Committee into the Basic principles of banking supervision is their focusing on the assessment and management of banking risks. As we see, banks are at the center of attention of the banking supervision in accordance with the recommendations of the Basel II. However, in our opinion, for the smooth functioning of the economic system more profound problems have to be solved, with the possibilities of banks' bankruptcies and general systemic risks to be addressed. The goal of the article is the development of a conception for the assessment of general economic risks of the banking system as part of a risk-focused banking supervision

<i>In vivo</i> antileukemic activity of DIM in U937 xenografts.

<p>20 NOD/SCID mice were inoculated with U937 cells (2×10⁶ cells/mouse, i.p.) and randomly divided into two groups (10/group) for treatment with DIM (50 mg/kg, i.p., daily, five times per week) or with vehicle control solvent. (a) Average tumor volume in vehicle control mice and mice treated with 50 mg/kg DIM. <i>P</i><0.01, significantly different compared with vehicle control by Student's <i>t</i>-test. (b) body weight changes of mice during the 20 days of study. (c) At the 20 days after DIM treatment, the tumors were excised and subjected to H&E staining for determination of pathological evaluation, TUNEL assay for determination of apoptosis, and immunohistochemical staining to determine Cleavage-caspase-3 immunoreactivity. Original magnification ×400. (d) After treatment with DIM, tumor tissues were sectioned and subjected to immunohistochemistry using anbibodies as indicated.</p

Effects of PI3K inhibitor, LY294002 (LY) and genetic activation of Akt on apoptosis induced by DIM.

<p>U937 cells were pretreated with 20 µM of LY for 1 h, followed by the addition of 40 µM of DIM for 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and LY were significantly greater than those for cells treated with DIM alone by Student's <i>t</i>-test; <i>p</i><0.01. (b–c) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (d) U937 cells were stably transfected with an empty vector (pcDNA3.1), Akt-CA, and Akt-DN. Cells were treated with 80 µM of DIM for 24 h, after which apoptosis was analysed using Annexin V/PI assay. **Values for Akt-CA cells treated with DIM were significantly decreased compared to those for pcDNA3.1 cells by Student's <i>t</i>-test, <i>p</i><0.01. (e–f) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated.</p

Inhibition of JNK significantly protect cells from DIM-induced apoptosis.

<p>U937 cells were pretreated with 10 µM of JNK inhibitor, SP600125 (SP), for 1 h, followed by the addition of 80 µM of DIM for 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and SP were significantly less than those obtained for cells treated with DIM alone by Student's t-test, <i>p</i><0.01. (b–c) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated. (d) U937 cells were transfected with JNK1 siRNA oligonucleotides or controls and incubated for 24 h at 37°C, after which cells were treated with 80 µM of DIM for 24 h. Apoptosis was determined using the Annexin V/PI assay. **Values for cells treated with DIM after transfection with JNK1 siRNA were significantly decreased compared to those for control cells treated with DIM by Student's t-test; <i>p</i><0.01. Total cellular extracts were prepared and subjected to Western blot analysis using antibodies against JNK1.</p

DIM induces apoptosis in U937, Jurkat, and HL-60 cells, and in AML blast samples, but not in normal bone marrow mononuclear cells.

<p>(a) U937, Jurkat, and HL-60 cells were treated with 80 µM DIM for 24 h, after which apoptosis was determined by annexin V/PI staining and flow cytometry. ** Values for cells treated with DIM were significantly increased compared to values in control cells by Student's t-test, <i>p</i><0.01. (b) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (c–d) Blasts from 15 patients with AML and normal bone marrow mononuclear cells were treated with 80 µM DIM for 24 hours, apoptosis was determined by annexin V/PI staining and flow cytometry.</p

DIM induces apoptosis, caspase activation, downregulation of Mcl-1, upregulation of p21, inactivation of Akt, and activation of JNK in U937 human leukemia cells in dose- and time-dependent manners.

<p>U937 cells were treated with various concentrations of DIM as indicated for 12 h and 24 h or treated with 80 µM DIM for 1, 3, 6, 9, 12, and 24 h. (a) Cells were washed twice with PBS and stained with Annexin V/propidium iodide (PI), and apoptosis was determined using flow cytometry. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations. The values obtained from annexin V/PI assays represent the mean ± SD for three separate experiments. (b–d) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated.</p

Effects of inhibition of caspases by Z-VAD-FMK on apoptosis, expression of Mcl-1 and p21, and phosphorylation of Akt and JNK.

<p>U937 cells were pretreated with the caspase inhibitor Z-VAD-FMK (20 µM) for 1 h, followed by treatment with 80 µM DIM for 12 h and 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and Z-VAD-FMK were significantly reduced compared to values obtained for DIM alone by Student's t-test, <i>p</i><0.01. (b–d) Total protein extracts were prepared and subjected to Western blot assay using antibodies as indicated.</p

Return of the Metabolic Trajectory to the Original Area after Human Bone Marrow Mesenchymal Stem Cell Transplantation for the Treatment of Fulminant Hepatic Failure

Our recent study first demonstrated that human bone marrow mesenchymal stem cell (hBMSC) transplantation could prevent death from fulminant hepatic failure (FHF) in pigs. To further clarify the metabolic mechanism of hBMSC transplantation in FHF, the plasma collected from FHF pigs that received transplantation of hBMSCs was examined using metabolic analysis to identify the key molecular markers that regulate recovery. The results showed that obvious metabolic disturbance occurred during FHF, whereas the hBMSC transplantation group showed less severe liver injury. The metabolic trajectory returns to its original state at week 3 following the hBMSC transplantation. In total, the concentration of 26 metabolites, including conjugated bile acids, phosphatidylcholines, lysophosphatidylcholines, fatty acids, amino acid and sphingomyelin, are significantly different between the FHF group and the hBMSC transplantation group. Moreover, the time course of changes in the metabolites corresponded with that of the biochemical and histological analyses. Real-time PCR further confirmed that the gene expression of phospholipase A1, lecithin-cholesterol acyltransferase and lysophosphatidylcholine acyltransferase 1 decreased significantly, whereas that of phospholipase A2 remained stable, which explains the decrease of the phosphatidylcholines and lysophosphatidylcholines. These novel results have revealed a metabolic mechanism for the hBMSC transplantation in FHF, which could lead to the future development of treatment strategies for stem cell therapies