Identification of differential metabolic characteristics between tumor and normal tissue from colorectal cancer patients by gas chromatography–mass spectrometry

Colorectal cancer (CRC) is one of the most common human malignancies and encompasses cancers of the colon and rectum. Although the gold‐standard colonoscopy screening method is effective in detecting CRC, this method is invasive and can result in severe complications for patients. The purpose of this study was to determine differences in metabolites between CRC and matched adjacent nontumor tissues from CRC patients, to identify potential biomarkers that may be informative and developed screening methods. Metabolomic analysis was performed on clinically localized CRC tissue and matched adjacent nontumor tissue from 20 CRC patients. Unsupervised analysis, supervised analysis, univariate analysis and pathway analysis were used to identify potential metabolic biomarkers of CRC. The levels of 25 metabolites in CRC tissues were significantly altered compared with the matched adjacent nontumor tissues. Four metabolites (lactic acid, alanine, phosphate and aspartic acid) demonstrated good area under the curve of receiver‐operator characteristic with acceptable sensitivities and specificities, indicating their potential as important biomarkers for CRC. Alterations of amino acid metabolism and enhanced glycolysis may be major factors in the development and progression of CRC. Lactic acid, alanine, phosphate, and aspartic acid could be effective diagnostic indicators for CRC.     

Determination of proline in human serum by a robust LC‐MS/MS method: application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification

Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC‐MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l ‐proline‐¹³C₅,¹⁵ N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose‐1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra‐ and inter‐day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p‐value = 0.011). We report here a simple, specific and reproducible LC‐MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer. Copyright © 2014 John Wiley & Sons, Ltd.     

Urinary levoglucosan as a biomarker for residential wood smoke exposure

As part of a randomised controlled residential intervention study, levoglucosan (LG) was investigated as a biomarker for wood smoke exposure. This study was conducted among 33 children living in homes that used wood stoves for residential heating. Indoor fine particulate matter (PM_2.5) concentrations and corresponding urine samples from participants were collected during pre- and a post-intervention winter sampling periods. Interventions included the installation of an air filtration unit and a wood stove change out. Homes and children assigned to a placebo filter served as the control condition. Results showed a strong reduction in indoor PM_2.5 among the air filter homes (≈58% reduction), whereas the wood stove change out homes did not have a significant PM_2.5 reduction from pre- to post-intervention observations. Children living in the air filter homes did not show a corresponding reduction in urinary LG concentrations. Further analysis did not show an association between overall changes in indoor PM_2.5 concentrations and changes in urinary LG concentrations. These findings suggest that urinary LG is not a reliable indicator of wood smoke exposure in residential wood heating settings.     

基于双醛基功能化离子液体构建电化学免疫传感器

合成了含双醛基的离子液体,此离子液体一端的醛基与修饰在电极表面的氨基发生共价键作用,将离子液体修饰在电极表面,另一端的醛基可用来固定抗体,构建电化学免疫传感器,实现对心肌肌钙蛋白I(cTnI)的检测。离子液体通过共价键作用固定在电极表面,不仅减少了从电极表面向检测溶液的渗透,提高传感器的稳定性,而且还可以直接固定抗体,不需要使用其他交联试剂;同时,离子液体可增强传感界面的导电性,提高传感器的灵敏度。在优化的实验条件下,传感器的线性范围为0.1~40 ng/mL,检出限为0.06 ng/mL。     

Heartbeat sensors under pressure: a new method for assessing hyperbaric physiology

Non-invasive heartbeat sensors to measure the cardiac activity of crustaceans have been adapted for use under hyperbaric conditions. Able to record data continuously over long timescales, these sensors can collect high-resolution data on the physiological state of an organism up to a tested limit of 300 atm. Using this technique, heart rate was recorded in a juvenile of the sublittoral spider crab, Maja brachydactyla (Decapoda: Majidae), when subjected to hydrostatic pressures of 1, 50, 100, and 150 atm for periods of 30 min. Heart rate increases with pressure until 100 atm (one-way repeated measures ANOVA: F _{(4, 25)}=154.76, p<0.001). However, the significant decrease in the mean heart rate from 137.07 bpm at 100 atm to 118.40 bpm at 150 atm (t-test: t=4.581, d.f.=10, p<0.001) indicates a mechanistic limit in the cardiac response of this species to pressures beyond 100 atm. This method could be potentially applied to any marine invertebrate with a neurogenic heart.     

PSAQ™ standards for accurate MS–based quantification of proteins: from the concept to biomedical applications

Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable‐isotope‐labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ?) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here. Copyright © 2012 John Wiley & Sons, Ltd.     

Fabrication of a Colorimetric Carcino-Embryonic Antigen Sensor Using High-Activity DNAzyme as a Catalytic Label

Detection of biomarkers in a biologically complex mixture remains a major challenge. Herein, an ultrasensitive colorimetric sandwich sensor for carcino-embryonic antigen (CEA) detection is introduced. The DNAzyme was tethered to biotinylated monoclonal antibodies (McAbs) which serve as the sensing element to recognize the target protein and was then introduced on to the CEA-McAbs assembled micro plate. The CEA was captured in a sandwich assay by the McAbs. The peroxidase-like DNAzyme catalyzed the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), which generated a blue-green colorimetric signal. This method detected CEA in a serum-containing medium at a concentration as low as 10 nM. This strategy is a promising tool for bioanalytical and clinical applications.     

Critical aspects of urine profiling for the selection of potential biomarkers using UPLC-TOF-MS

Biomarker selection through the metabolomics approach involves the acquisition of nontargeted metabolic profiles. In this study, some critical factors that may affect this process were investigated using urine test samples and a UPLC‐TOF system. Repeated injections of a single sample demonstrated that the percentage of undetected and poorly repeatable measurements (intensity RSD > 15%) decreased from 22.5 to 5.8% and from 32.9 to 14.7%, respectively, as the scan time was increased up to 0.6 s (approximately 11 data points per peak). An additional critical factor was identified in the presence of broad concentration differences between the samples; the application of a dilution scheme that minimized these differences reduced the number of missing values in the final datasets by 36%. The impact of missing values was further investigated in the study of two groups of samples produced by using a spike as artificial marker. Missing values weakened the models used for the interpretation of the metabolic profiles, and greatly hindered the identification of possible markers. Finally, a simple strategy for an effective analysis of urine samples was outlined; it proved to limit the need for the post‐acquisition elaboration of the data. The same strategy can easily be adapted to other matrices. Copyright © 2011 John Wiley & Sons, Ltd.     

Serum metabolomics as a novel diagnostic approach for gastrointestinal cancer

Conventional tumor markers are unsuitable for detecting carcinoma at an early stage and lack clinical efficacy and utility. In this study, we attempted to investigate the differences in serum metabolite profiles of gastrointestinal cancers and healthy volunteers using a metabolomic approach and searched for sensitive and specific metabolomic biomarker candidates. Human serum samples were obtained esophageal (n = 15), gastric (n = 11), and colorectal (n = 12) cancer patients and healthy volunteers (n = 12). A model for evaluating metabolomic biomarker candidates was constructed using multiple classification analysis, and the results were assessed with receiver operating characteristic curves. Among the 58 metabolites, the levels of nine, five and 12 metabolites were significantly changed in the esophageal, gastric and colorectal cancer patients, respectively, compared with the healthy volunteers. Multiple classification analysis revealed that the variations in the levels of malonic acid and l ‐serine largely contributed to the separation of esophageal cancer; gastric cancer was characterized by changes in the levels of 3‐hydroxypropionic acid and pyruvic acid; and l ‐alanine, glucuronoic lactone and l ‐glutamine contributed to the separation of colorectal cancer. Our approach revealed that some metabolites are more sensitive for detecting gastrointestinal cancer than conventional biomarkers. Our study supports the potential of metabolomics as an early diagnostic tool for cancer. Copyright © 2011 John Wiley & Sons, Ltd.