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. 2025 Mar 20;15(6):e5246.
doi: 10.21769/BioProtoc.5246.

HS-GC-MS Method for the Diagnosis of IBD Dynamics in a Model of DSS-Induced Colitis

Olga Yu Shagaleeva  1 , Daria A Kashatnikova  1   2 , Dmitry A Kardonsky  1 , Boris A Efimov  1   3 , Viktor A Ivanov  1 , Svetlana V Smirnova  2 , Yana A Zorkina  4 , Elizaveta A Vorobjeva  1 , Artemiy S Silantiev  1 , Viktoriia D Kazakova  1 , Irina V Kolesnikova  1 , Maria I Markelova  5 , Anna A Vanyushkina  6 , Andrei V Chaplin  1   3 , Tatiana V Grigoryeva  5 , Natalya B Zakharzhevskaya  1
Affiliations

HS-GC-MS Method for the Diagnosis of IBD Dynamics in a Model of DSS-Induced Colitis

Olga Yu Shagaleeva et al. Bio Protoc. .

Abstract

Inflammatory bowel disease (IBD) is highly prevalent globally and, in the majority of cases, remains asymptomatic during its initial stages. The gastrointestinal microbiota secretes volatile organic compounds (VOCs), and their composition alters in IBD. The examination of VOCs could prove beneficial in complementing diagnostic techniques to facilitate the early identification of IBD risk. In this protocol, a model of sodium dextran sulfate (DSS)-induced colitis in rats was successfully implemented for the non-invasive metabolomic assessment of different stages of inflammation. Headspace-gas chromatography-mass spectrometry (HS-GC-MS) was used as a non-invasive method for inflammation assessment at early and remission stages. The disease activity index (DAI) and histological method were employed to assess intestinal inflammation. The HS-GC-MS method demonstrated high sensitivity to intestine inflammation, confirmed by DAI and histology assay, in the acute and remission stages, identifying changes in the relative content of VOCs in stools. HS-GC-MS may be a useful and non-invasive method for IBD diagnostics and therapy effectiveness control. Key features • Experiments performed in vivo to better control DSS-induced colon damage and to aid the study of IBD development in humans. • Optimized for the following organisms: Wistar rats and C57BL/6 mice. • An easily assessed disease activity index (DAI) (weight loss, stool consistency, degree of fecal occult blood) and histopathological examination are suggested for additional IBD confirmation. • Enables VOC testing with relatively small stool samples.

Keywords: DSS-induced colitis; Headspace–gas chromatography–mass spectrometry (HS–GC–MS); IBD; Metabolomic profile; Volatile organic compounds (VOCs).

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Conflict of interest statement

Competing interestsThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1.
Figure 1.. General workflow for colitis modelling, stool sampling, and rat euthanasia
Figure 2.
Figure 2.. Examples of three different types of rat stools
Figure 3.
Figure 3.. Dynamics of the disease activity index (DAI) in each group
Figure 4.
Figure 4.. Stages of rat dissection.
(A) Fixing the rat on the plate. (B) Skin incision and fixation of the skin flaps. (C) Opening of the abdominal cavity. (D) Visualization of the intestine. (E) Incision of the mesentery. (F) Improvement of access to the colon. (G) Extraction of the colon. Dotted line: cutting line. * Xiphoid process. I: Caecum. II: Anus. III: Rectum. IV: Mesentery.
Figure 5.
Figure 5.. Fine surgical tools for preparing animal colons for histology for the Swiss roll technique
Figure 6.
Figure 6.. Preparing animal colons for histology with the Swiss roll technique.
(A) Isolated colon (I: cecum; II: anus). (B) Colonic fecal cleansing. (C) Cleaned colon. (D) Colon without the cecum. (E) Colon dissection. (F) Fixing a toothpick to the anus. (G) Starting to make a roll. (H) Finishing the roll. (I) Fixing the roll with sewing thread. (J) Finished Swiss roll.
Figure 7.
Figure 7.. Histological cassette with an insert that fixes the specimen
Figure 8.
Figure 8.. Histopathological evaluation of the colon of each group of rats.
Data are presented as individual means or mean ± SD for each group. *p < 0.025; ***p < 0.0005.
Figure 9.
Figure 9.. Colon tissue stained with H&E (10×) in different phases of inflammation.
(A) Healthy colon wall of a control-group rat. (B) Damaged rat colon from the DSS7 group. Disturbed mucosal architecture, isolated goblet cells, and marked inflammatory infiltration. (C) Rat colon from the DSS14 group. Signs of restoration of the mucosal architecture and appearance of crypts.
Figure 10.
Figure 10.. GCMS-QP2020 device and GC–MS process.
(A) Gas chromatography–mass spectrometry equipment. (B) Gas phase formation reaction from the sample.
Figure 11.
Figure 11.. Examples of GC–MS spectra of a stool sample
Figure 12.
Figure 12.. Heatmap of stable metabolites (log transformation) identified in the sodium dextran sulfate (DSS) and control groups over time
Figure 13.
Figure 13.. Boxplots comparing the concentrations of 10 stable, detected metabolites in the experimental and control groups on day 0 evaluated by Mann–Whitney tests with Bonferroni correction for multiple comparisons
Figure 14.
Figure 14.. Boxplots comparing the concentrations of the four most stable detected metabolites between the two groups and across different days of the experiment evaluated by Mann–Whitney tests with Bonferroni correction for multiple comparisons
Figure 15.
Figure 15.. Comparison of the metabolomic profile data of sodium dextran sulfate (DSS) group rats on days 7 and 14 with the control group.
A. PCA, OPLS-DA. B. Heatmap.
Figure 16.
Figure 16.. Changes in the average concentration of six consistently detected metabolites in the sodium dextran sulfate (DSS) group compared to the control group
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