Serum metabolic profiling of rats infected with Clonorchis sinensis using LC-MS/MS method

Abstract

Background: Clonorchiasis is an important foodborne parasitic disease. The omics-based-techniques could illuminate parasite biology and further make innovations in the research for parasitic diseases. However, knowledge about the serum metabolic profiles and related metabolic pathways in clonorchiasis is very limited.

Methods: A untargeted ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) was used to profile the serum metabolites of rats at both 4 and 8 weeks post infection (wpi) with Clonorchis sinensis (C. sinensis). Additionally, multivariate statistical analysis methods were employed to identify differential metabolites. Next, serum amino acids and phosphatidylcholines (PCs) levels were determined by targeted metabolomics analysis.

Result: A total of 10530 and 6560 ions were identified in ESI+ and ESI- modes. The levels of phosphatidylcholines, glycerophosphocholine and choline were significantly changed, with the shift in lipid metabolism. Significant changes were also observed in amino acids (isoleucine, valine, leucine, threonine, glutamate and glutamine). Targeted analysis showed that BCAAs (isoleucine, valine, leucine) levels significantly increased at 4 wpi and decreased at 8 wpi; threonine was increased at 8 wpi, whereas glutamate and glutamine showed a decreasing trend at 8 wpi. Additionally, the level of 17 PCs were significantly changed in infected rats. Marked metabolic pathways were involved in clonorchiasis, including glycerophospholipid metabolism, alanine, aspartate and glutamate metabolism, histidine metabolism and pyrimidine metabolism.

Conclusion: These results show that C. sinensis infection can cause significant changes in the rat serum metabolism, especially in amino acids and lipids. The metabolic signature together with perturbations in metabolic pathways could provide more in depth understanding of clonorchiasis and further make potential therapeutic interventions.

Keywords: clonorchiasis; metabolic pathway; serum; targeted metabolomics; untargeted metabolomics.

Figure 1

Orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showing the separation between C. sinensis-infected and control groups at 4 and 8 wpi in ESI+ mode (A-C) and ESI− mode (D-F). The ellipses enclose the 95% confidence intervals estimated by the sample means and covariances of each group.

Figure 2

Differential metabolites were identified among different groups in ESI+ and ESI− modes. (A) Volcano plots showed significant differences among different groups. Each point in the map represents a metabolite. The size of the scatter represents the VIP value of the OPLS-DA model, and the larger the scatter, the larger the VIP value. (Red plot, up-regulated; Blue plot, down-regulated; Black plot, non-significant). (B) Heat maps representing the significantly changed metabolites between infected groups and the corresponding control groups. Each row represents data for a specific metabolite, and each column represents a rat (C. sinensis-infected or healthy control). Different colors correspond to the different intensity levels of metabolites. Red and blue colors represent increased and decreased levels of metabolites, respectively.

Figure 3

The concentrations of 14 significantly changed amino acids in serum of different groups. *P < 0.05, **P < 0.01, t-test. ns, no significant change.

Figure 4

ROC analysis of amino acids metabolites (A) and phosphatidylcholines (B) in infected and control groups.

Figure 5

The concentrations of 17 phosphatidylcholines in serum of different groups. *P < 0.05, **P < 0.01, t-test. ns, no significant change.

Figure 6

Pathway analysis of the identified differential metabolites in ESI+ mode and ESI− mode. Plots depict the pathway impacts of the key metabolites (x-axis) and the computed metabolic pathway as a function of –log (P) (y-axis) that different among 4 wpi vs. 4 con (A, D), 8 wpi vs. 8 con (B, E), and 8 wpi vs. 4 wpi (C, F).

Figure 7

Integrated metabolic networks of significantly altered metabolic pathways during C. sinensis infection. The black arrows indicate up-regulation and down-regulation. Horizontal lines indicate that the metabolites are no changed.