P158

Background

Opisthorchiasis is a form of foodborne trematodiasis which is caused by liver flukes. It has been shown that a chronic Opisthorchiasis infection increases a risk of cholagiocarcinoma of liver. It is commonly believed that a gradual change of homeostasis in a parasite microenvironment (bile) leads to liver fluke-induced cancer. Nevertheless, no systematic, analytically driven studies confirming this hypothesis have been published yet. The restricted access to clinical material and extreme complexity of the biological matrix (bile) both are the important “rate limiting factors” for a progress in the field. Here we present for the first time a cross-platform mass spectrometric analysis of bile juice collected from the patients with cholangiocarcinoma-associated diseases. We show that an effective analysis of such complex biological matrix as bile juice requires a combination of orthogonal analytical platforms (e.g. RPLC–MS and HILIC–MS) maximizing coverage of the metabolic space.

Materials and methods

28 patients with O. felineus infection and 30 negative controls were included in the study. The infection status was confirmed using microscopy analysis of the bile. Bile samples were collected from the gallbladder using sterile puncture, directly frozen and stored at −80 °C until analysis. The samples were randomized and organized into the acquisition blocks consisting of the samples and quality controls (QC). Experiments were carried out with a Dionex Ultimate 3000 LC system (Thermo Scientific/Dionex, The Netherlands) equipped with a Dual Gradient Separation pump allowing for parallel LC analysis, and hyphenated to an Impact UHR-qTOF mass analyzer (Bruker Daltonics, Germany). Reversed-phase experiments (RPLC) were performed with an UHPLC BEH Shield RP18 column 100 × 2.1 mm, 1.7 μm (Waters) and HILIC experiments with a Luna HILIC column (Phenomenex, The Netherlands) of 100 $\times$ 2.00 mm, 3 μ m. RPLC data were acquired in ESI positive mode and HILIC in negative mode, respectively. The data acquisition rate was set to 1 Hz over a mass range of m/z 50–1000. The LC–MS data files were aligned by using the in-house developed alignment algorithm MS-Align 2 tool (www.ms-utils.org/msalign2).

Results

After the data prepressing, which includes alignment, noise filtering and peak picking two data matrixes costing of 412 features (metabolites) for RPLC and 428 ones for HILIC were generated. To evaluate a degree of similarity between the two data matrixes the RV coefficient (a multivariate extension of correlation coefficient) was used. The coefficient has flattened at 0.58 showing that despite a strong overlap between the datasets there is a substantial number of the “platform specific” metabolites. Those structures will certainly be missed if a single platform strategy is applied.

Conclusion

Here we present for the first time a cross-platform mass spectrometric analysis of bile juice collected from the patients cholangiocarcinoma-associated diseases. We show that a combination of the two platforms greatly improves the coverage of the metabolome and as such should be a firstchoice for exploratory studies of the complex biological matrixes.

Work was conducted with the application of the Tomsk regional common use center technical equipment acquired thanks to a grant of the Russian Ministry of the Agreement No. 14.594.21.0001 (RFMEFI59414X0001). This project is supported by “The Tomsk State University Academic D.I. Mendeleev Fund Program” under Grant (No. 18.1.52.2015).