LC-MS based serum metabonomic analysis for renal cell carcinoma diagnosis, staging, and biomarker discovery

A LC-MS based method, which utilizes both reversed-performance (RP) chromatography and hydrophilic interaction chromatography (HILIC) separations, has been carried out in conjunction with multivariate data analysis to discriminate the global serum profiles of renal cell carcinoma (RCC) patients and healthy controls. The HILIC was found necessary for a comprehensive serum metabonomic profiling as well as RP separation. The feasibility of using serum metabonomics for the diagnosis and staging of RCC has been evaluated. One-hundred percent sensitivity in detection has been achieved, and a satisfactory clustering between the early stage and advanced-stage patients is observed. The results suggest that the combination of LC-MS analysis with multivariate statistical analysis can be used for RCC diagnosis and has potential in the staging of RCC. The MS/MS experiments have been carried out to identify the biomarker patterns that made great contribution to the discrimination. As a result, 30 potential biomarkers for RCC are identified. It is possible that the current biomarker patterns are not unique to RCC but just the result of any malignancy disease. To further elucidate the pathophysiology of RCC, related metabolic pathways have been studied. RCC is found to be closely related to disturbed phospholipid catabolism, sphingolipid metabolism, phenylalanine metabolism, tryptophan metabolism, fatty acid beta-oxidation, cholesterol metabolism, and arachidonic acid metabolism.     

Determination of clomipramine by flow‐injection analysis with acidic potassium permanganate–formic acid chemiluminescence detection

A sensitive and simple chemiluminescent (CL) method for the determination of clomipramine has been developed by combining the flow‐injection analysis (FIA) technique, which is based on the CL intensity generated from the redox reaction of potassium permanganate (KMnO₄)–formic acid in sulphuric acid (H₂SO₄) medium. Under the optimum conditions, the linear range for the determination of clomipramine was 0.04–4 µg/mL, with a correlation coefficient of 0.9988 (n = 10) and a detection limit of 0.008 µg/mL (3σ), and the relative standard deviation (RSD) for 2.0 µg/mL clomipramine (n = 11) is 1.26%. The proposed method has been successfully applied to the determination of the studied clomipramine in pharmaceutical preparations. The possible reaction mechanism is discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Electrochemiluminescence of palmatine being oxidized by electrogenerated hydroxyl radical and its analytical application

A strong electrochemiluminescence (ECL) of palmatine in NaOH medium was observed at a vaseline‐impregnated graphite anode. The ECL production could be described as follows: hydroxyl radical (OH^?) was generated via the oxidation of hydroxyl group (OH^‐) in NaOH medium, and the formed OH^? subsequently oxidized palmatine base converted from palmatine in NaOH medium to the excited state oxypalmatine (oxypalmatine*). As the oxypalmatine* went back to its ground state, a stronger chemiluminescence was produced. Based on the ECL of palmatine, an ECL method for the determination of palmatine was proposed. An ECL signal of palmatine in NaOH solution was obtained by applying direct current of 15 mA to the vaseline‐impregnated graphite anode. The ECL intensity was rectilinear with palmatine concentration in the range of 8.0 × 10^?7 to 2.0 × 10^?5 mol l^?1 and the limit of detection (signal‐to‐noise = 3) was 3 × 10^?7 mol l^?1. The proposed method was applied to the determination of palmatine in pharmaceutical preparations. Copyright © 2010 John Wiley & Sons, Ltd.     

Bladder cancer determination via two urinary metabolites: a biomarker pattern approach

The purpose of this study was to use metabonomic profiling to identify a potential specific biomarker pattern in urine as a noninvasive bladder cancer (BC) detection strategy. A liquid chromatography-mass spectrometry based method, which utilized both reversed phase liquid chromatography and hydrophilic interaction chromatography separations, was performed, followed by multivariate data analysis to discriminate the global urine profiles of 27 BC patients and 32 healthy controls. Data from both columns were combined, and this combination proved to be effective and reliable for partial least squares-discriminant analysis. Following a critical selection criterion, several metabolites showing significant differences in expression levels were detected. Receiver operating characteristic analysis was used for the evaluation of potential biomarkers. Carnitine C9:1 and component I, were combined as a biomarker pattern, with a sensitivity and specificity up to 92.6% and 96.9%, respectively, for all patients and 90.5% and 96.9%, respectively for low-grade BC patients. Metabolic pathways of component I and carnitine C9:1 are discussed. These results indicate that metabonomics is a practicable tool for BC diagnosis given its high efficacy and economization. The combined biomarker pattern showed better performance than single metabolite in discriminating bladder cancer patients, especially low-grade BC patients, from healthy controls.     

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.     

Mechanism of the mesenchymal-epithelial transition and its relationship with metastatic tumor formation

Cancer metastasis consists of a sequential series of events, and the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are recognized as critical events for metastasis of carcinomas. A current area of focus is the histopathological similarity between primary and metastatic tumors, and MET at sites of metastases has been postulated to be part of the process of metastatic tumor formation. Here, we summarize accumulating evidence from experimental studies that directly supports the role of MET in cancer metastasis, and we analyze the main mechanisms that regulate MET or reverse EMT in carcinomas. Given the critical role of MET in metastatic tumor formation, the potential to effectively target the MET process at sites of metastasis offers new hope for inhibiting metastatic tumor formation.     

MtDNA haplogroups M7 and B in southwestern Han Chinese at risk for acute mountain sickness

We conducted a case-control study to investigate the association of mitochondrial DNA (mtDNA) haplogroups with acute mountain sickness (AMS) in Han Chinese from southwestern (SW) China. Pearson's chi-square test or Fisher's exact test revealed significant reduction of mtDNA haplogroups D and M9, while a significant increase of haplogroup M7 in AMS subjects compared with non-AMS subjects. The multivariate logistic regression analysis after adjustment for body mass index (BMI), a risk factor of AMS in the present study, showed that both D and M9 were associated with significantly decreased risk of AMS, while M7 was associated with a significantly increased risk of AMS (OR=0.605, p=0.000; OR=0.037, p=0.001, and OR=2.419, p=0.001, respectively). In addition, further analysis stratified by the AMS severities indicated that haplogroup B was correlated with a 2.41-folds increased risk of developing severe AMS (95%C.I=1.288-4.514, p=0.006). Our findings provide evidence that, in SW Han Chinese, mtDNA haplogroups D and M9 are related to individual tolerance to AMS, while haplogroups M7 and B are risk factors for AMS.     

A missense mutation in PPARD causes a major QTL effect on ear size in pigs

Chinese Erhualian is the most prolific pig breed in the world. The breed exhibits exceptionally large and floppy ears. To identify genes underlying this typical feature, we previously performed a genome scan in a large scale White Duroc × Erhualian cross and mapped a major QTL for ear size to a 2-cM region on chromosome 7. We herein performed an identical-by-descent analysis that defined the QTL within a 750-kb region. Historically, the large-ear feature has been selected for the ancient sacrificial culture in Erhualian pigs. By using a selective sweep analysis, we then refined the critical region to a 630-kb interval containing 9 annotated genes. Four of the 9 genes are expressed in ear tissues of piglets. Of the 4 genes, PPARD stood out as the strongest candidate gene for its established role in skin homeostasis, cartilage development, and fat metabolism. No differential expression of PPARD was found in ear tissues at different growth stages between large-eared Erhualian and small-eared Duroc pigs. We further screened coding sequence variants in the PPARD gene and identified only one missense mutation (G32E) in a conserved functionally important domain. The protein-altering mutation showed perfect concordance (100%) with the QTL genotypes of all 19 founder animals segregating in the White Duroc × Erhualian cross and occurred at high frequencies exclusively in Chinese large-eared breeds. Moreover, the mutation is of functional significance; it mediates down-regulation of β-catenin and its target gene expression that is crucial for fat deposition in skin. Furthermore, the mutation was significantly associated with ear size across the experimental cross and diverse outbred populations. A worldwide survey of haplotype diversity revealed that the mutation event is of Chinese origin, likely after domestication. Taken together, we provide evidence that PPARD G32E is the variation underlying this major QTL.     

An improved approach for extraction and high-performance liquid chromatography analysis of paraquat in human plasma

A simple, sensitive, reliable and economical HPLC method for quantifying paraquat concentration in human plasma has been developed, using diethyl paraquat as an internal standard. The drugs were extracted from the sample and separated on Xtimate C18 column with a mobile phase of 15% acetonitrile in 0.1M orthophosphoric acid containing SDS (150 mg/l). The pH of the mobile phase was adjusted to 3 with triethylamine and the detection wavelength was 256 nm for both paraquat and the internal standard. The average extraction recoveries were 91.9%. Good linearity (R(2)=0.9984) was observed throughout the range of 0.02-10 μg/ml in 0.5 ml plasma. The overall accuracy of this method was 97.6-107.3% and the lower limit of detection was 0.01 μg/ml. The intra- and inter-day variations were lower than 3.65% and 2.64%, respectively. We used this method to examine the paraquat concentrations of 53 patients with acute paraquat intoxication of whom 26 (49.1%) survived. In conclusion, this method was suitable for quantification of paraquat plasma concentration in toxicological samples. It was helpful in both assessing the severity of intoxication and predicting the outcome of paraquat poisoning.