Background: Novel noninvasive methods for evaluating disease activity in patients with inflammatory bowel disease (IBD) are needed to reduce patient burden of traditional assessments, which can involve lab sample collections for blood and stool tests, bowel preparation, and additional appointments. Such noninvasive techniques can alleviate many challenges associated with traditional lab, imaging, and endoscopic testing. We report a noninvasive method to identify biomarkers in exhaled breath condensate (EBC) collected using a home-based tool to determine disease status in patients with ulcerative colitis (UC) and Crohn’s disease (CD). This approach may offer significant advantages, including timely results and can be particularly suitable for populations facing challenges accessing healthcare.
Methods: EBC samples were collected from 29 patients with IBD, including 15 patients with active (n=6) and inactive (n=9) UC, and 14 patients with active (n=7) and inactive (n=7) CD. Disease activity was determined by treating physician via clinical/endoscopic assessment the day of collection. Following an overnight fast, patients exhaled into an RTube^TM (Respiratory Research) surrounded by a pre-prepared cooling sleeve that allows for rapid condensation for 4 minutes. EBC samples were then temporarily stored at -80°C. Once all samples were collected, we extracted metabolites with HPLC-grade methanol and performed data acquisition via LC-MS/QTOF. To compare the findings obtained from EBC, selected metabolites in patients with active and inactive IBD are profiled by analyzing plasma (reflecting systemic metabolism) in an independent cohort using LC-MS/QTOF.
Results: Patients with active IBD exhibited a distinct breath metabolite profile (Fig. A, E), with decreased arachidonic acid metabolites in active vs. inactive IBD (FDR<0.05). In patients with active UC, steroid biosynthesis pathways (FDR<0.05) were downregulated, compared to patients with inactive UC. In addition, patients with active UC had reduced primary antioxidant glutathione (GSH, p<0.05) while its derivative analog, ophthalmic acid (OPH) was increased (p<0.01) (Fig.B-C, AUC 0.900). These findings align with the finding that showed increased oxidized GSH in plasma from patients with active UC (Fig.D). Patients with active CD had significantly increased β-hydroxybutyrate (BHB, p<0.01, Fig.F), which was also observed in the plasma samples from an independent cohort (Fig.H). BHB demonstrated a strong discriminatory ability for detecting active vs inactive CD (AUC 0.911, Fig.G).
Conclusion: Differential metabolite patterns in active vs. inactive IBD reveal BHB and OPH (combined with GSH) in EBC as possible biomarkers for IBD disease activity. This novel noninvasive and accessible approach has the potential to improve outcomes for IBD patients by facilitating timely assessment and more targeted treatment.

<b>Figure</b>: <b>Multimodal Analysis Reveals Distinct Metabolic Signatures in Active vs. Inactive States of Crohn's Disease (CD) and Ulcerative Colitis (UC) in EBC.</b> Partial Least Squares Discriminant Analysis (plSDA) plot illustrates the metabolic separation between active and inactive phases of UC (A) and CD (E). Volcano plot highlights differentially expressed metabolites in UC (B) and CD (F) during active and inactive states (FC>1.5, p<0.05). ROC biomarker analysis of OPH (C) and BHB (G) reveals robust discriminatory ability. D) Oxidized glutathione of plasma reflects the deprivation of GSH and increase of OPH in EBC of patients with active UC. (H) Concordant elevation of plasma BHB levels in patients with active CD corroborates the findings observed in EBC. <i>*p<0.05</i> vs Inactive. Abbreviations: EBC: exhale breath condensate; OPH: ophthalmic acid; BHB: beta-hydroxybutyric acid; ROC: receiver operating characteristic; AUC: area under the ROC curve.

Figure: Multimodal Analysis Reveals Distinct Metabolic Signatures in Active vs. Inactive States of Crohn's Disease (CD) and Ulcerative Colitis (UC) in EBC. Partial Least Squares Discriminant Analysis (plSDA) plot illustrates the metabolic separation between active and inactive phases of UC (A) and CD (E). Volcano plot highlights differentially expressed metabolites in UC (B) and CD (F) during active and inactive states (FC>1.5, p<0.05). ROC biomarker analysis of OPH (C) and BHB (G) reveals robust discriminatory ability. D) Oxidized glutathione of plasma reflects the deprivation of GSH and increase of OPH in EBC of patients with active UC. (H) Concordant elevation of plasma BHB levels in patients with active CD corroborates the findings observed in EBC. *p<0.05 vs Inactive. Abbreviations: EBC: exhale breath condensate; OPH: ophthalmic acid; BHB: beta-hydroxybutyric acid; ROC: receiver operating characteristic; AUC: area under the ROC curve.

Hyunsu Jang

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