Breath Gases, Gut Flora, and Immunity in Children With Constipation

Correlation Study on End-expiratory NO, H2S, H2, CH4, Intestinal Flora and Immune Function in Children With Constipation

Constipation is a common problem in children that can affect their daily life, school performance, and social activities. Researchers have found that the bacteria living in our intestines (called "gut microbiome") may play an important role in constipation. However, we still do not fully understand exactly how these bacteria affect constipation in children.

This study aims to explore whether the gases children breathe out-including nitric oxide (NO), hydrogen sulfide (H₂S), hydrogen (H₂), and methane (CH₄)-can tell us something about the bacteria in their guts. By comparing children with constipation to healthy children, we hope to find out:

Whether the gases in their breath are different Whether the types of bacteria in their guts are different Whether there is a relationship between breath gases and gut bacteria If we find clear connections, this could lead to a simple, non-invasive breath test that doctors can use to understand a child's gut health without the need for more invasive procedures.

Study Overview

• Status: Active, not recruiting
• Conditions: Constipation - Functional

Detailed Description

Childhood functional constipation (FC) is a multifactorial syndrome with a global prevalence of approximately 9.5% among pediatric populations, accounting for up to 29% of functional gastrointestinal disorders. It significantly affects physical health, academic performance, and psychosocial development, with approximately 25% of affected children continuing to experience gastrointestinal symptoms into adulthood. While conventional factors such as diet, fluid intake, and psychological stress have been implicated, the precise pathophysiological mechanisms remain incompletely understood.

Emerging evidence highlights the critical role of the gut microbiome in regulating intestinal motility and visceral sensitivity through the microbiota-gut-brain axis. Microbial metabolites-including short-chain fatty acids (SCFAs), serotonin, bile acids, and gases (methane, hydrogen, hydrogen sulfide)-serve as key signaling molecules that influence enteric neurotransmission, smooth muscle contraction, and gastrointestinal transit time. Notably, hydrogen (H₂) and methane (CH₄) are exclusively of microbial origin (produced via anaerobic fermentation of carbohydrates by intestinal flora), while hydrogen sulfide (H₂S) is generated both endogenously and by sulfate-reducing bacteria. Nitric oxide (NO), though primarily endogenously produced, may also reflect intestinal inflammatory status and microbial metabolic activity.

The integration of breath gas analysis and metagenomic profiling offers a promising, non-invasive approach to elucidate host-microbe interactions in FC. However, systematic evaluations linking these four exhaled gases (NO, H₂S, H₂, CH₄) to the taxonomic and functional composition of the gut microbiome in constipated children remain scarce. This study is designed to bridge this knowledge gap, providing mechanistic insights and potentially establishing breath testing as a surrogate marker for gut dysbiosis in pediatric constipation.

• Study Type: Observational
• Enrollment (Estimated): 200

Contacts and Locations

Study Locations

• China
  • Shanghai Municipality
    • Shanghai, Shanghai Municipality, China, 200025
      • Ruijin Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

Children aged 6 to 14 years recruited from the outpatient clinic of the Department of Pediatrics at Ruijin Hospital (North Campus), Shanghai, China. The study population includes two groups: (1) children diagnosed with functional constipation according to Rome criteria, and (2) healthy children undergoing routine health check-ups with no chronic or acute digestive disorders. All participants must have no recent (within 4 weeks) exposure to antibiotics, probiotics, or medications affecting gastrointestinal motility. Informed consent is obtained from the legal guardian of each participant.

Description

Inclusion Criteria:

• Children aged 6 to 14 years, male or female.
• Legal guardian provides written informed consent.
• No use of antibiotics, probiotics, lactulose, antacids, or gastrointestinal prokinetic agents within 4 weeks prior to enrollment.
• For the constipation group: Clinical diagnosis of functional constipation according to Rome criteria, defined as meeting at least two of the following criteria for two consecutive months: (a) fewer than two bowel movements per week; (b) at least one episode of fecal incontinence per week; (c) history of massive fecal retention or posturing related to fecal retention; (d) history of painful or difficult defecation; (e) presence of a large fecal mass in the rectum; (f) passage of stools large enough to obstruct the toilet. Additionally, no enema or acute enteritis within 2 weeks prior to enrollment.
• For the healthy control group: No chronic digestive diseases or acute illnesses; no enema or acute enteritis within 2 weeks prior to enrollment.

Exclusion Criteria:

• Use of antibiotics, probiotics, lactulose, antacids, or gastrointestinal prokinetic agents within 4 weeks prior to enrollment (if not already excluded at screening).
• Clinically significant abnormalities in liver, kidney, neurological, respiratory, or coagulation function as determined by the investigator.
• Unstable vital signs.
• Presence of other underlying systemic diseases (e.g., metabolic, endocrine, or organic gastrointestinal disorders).
• Any other condition that, in the investigator's opinion, makes the participant unsuitable for enrollment.

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort
Constipation Group; Children aged 6 to 14 years diagnosed with functional constipation according to Rome criteria (at least two of the following for two consecutive months: fewer than two bowel movements per week; at least one episode of fecal incontinence per week; history of massive fecal retention or posturing related to fecal retention; history of painful or difficult defecation; presence of a large fecal mass in the rectum; passage of stools large enough to obstruct the toilet). Participants have not used antibiotics, probiotics, lactulose, antacids, or prokinetic agents within 4 weeks prior to enrollment, and have not received enema or had acute enteritis within 2 weeks. Intervention: Non-interventional; participants undergo a one-time breath test (end-expiratory NO, H₂S, H₂, CH₄), stool collection (within 2 days), and blood draw (2 mL).
Control Group; Healthy children aged 6 to 14 years with no chronic digestive diseases or acute illnesses, recruited from routine health check-up visits. Participants have not used antibiotics, probiotics, lactulose, antacids, or prokinetic agents within 4 weeks prior to enrollment. Intervention: Non-interventional; participants undergo a one-time breath test (end-expiratory NO, H₂S, H₂, CH₄), stool collection (within 2 days), and blood draw (2 mL).

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
End-expiratory concentrations and peak values of H2	Comparison of the fasting end-expiratory concentrations (ppm) and peak values of hydrogen (H2) between children with functional constipation and healthy controls. Breath samples are collected during a single morning session after an overnight fast using a validated portable breath analyzer.	Measured at baseline (single time point) after an overnight fast, during a single methane-hydrogen breath test session.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
End-expiratory concentrations and peak values of H2S	Comparison of the fasting end-expiratory concentrations (ppm) and peak values of hydrogen sulfide (H2S) between children with functional constipation and healthy controls. Breath samples are collected during a single morning session after an overnight fast using a validated portable breath analyzer.	Measured at baseline (single time point) after an overnight fast, during a single methane-hydrogen breath test session.
End-expiratory concentration and peak value of methane (CH₄)	Comparison of fasting end-expiratory CH₄ concentration (ppm) and peak values between children with functional constipation and healthy controls. CH₄ is exclusively produced by methanogenic archaea in the gut and is not metabolized by mammalian cells. Elevated CH₄ levels (e.g., ≥10 ppm) are associated with prolonged colonic transit time and increased constipation severity. The analysis will compare intergroup differences in both baseline concentration and maximum peak values during the breath test.	Measured at baseline (single time point) after an overnight fast, during a single methane-hydrogen breath test session.
End-expiratory concentration and peak value of nitric oxide (NO)	Comparison of fasting end-expiratory NO concentration (ppb) and peak values between children with functional constipation and healthy controls. End-expiratory NO reflects systemic and intestinal inflammatory status. Alterations in NO levels may indicate mucosal barrier dysfunction or low-grade inflammation associated with constipation. The analysis will compare intergroup differences in both baseline concentration and maximum peak values.	Measured at baseline (single time point) after an overnight fast, during a single methane-hydrogen breath test session.

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Fecal metabolomic profiles	Identification and comparison of untargeted fecal metabolomic profiles via LC-MS/MS between constipated children and healthy controls. Differential metabolites and enriched KEGG metabolic pathways will be identified.	Within 2 days after the breath test (baseline)
serum gastrointestinal hormone levels	Comparison of serum levels of gastrointestinal hormones (e.g., motilin) and routine blood parameters between the two groups. Blood samples (2 mL) are drawn at enrollment and processed via ELISA and automated hematology analyzers.	At the single study visit (baseline)

Collaborators and Investigators

• Sponsor: Ruijin Hospital

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2026
• Primary Completion (Estimated): December 31, 2027
• Study Completion (Estimated): December 31, 2027

Study Registration Dates

• First Submitted: August 17, 2026
• First Submitted That Met QC Criteria: August 27, 2026
• First Posted (Actual): September 1, 2026

Study Record Updates

• Last Update Posted (Actual): September 1, 2026
• Last Update Submitted That Met QC Criteria: August 27, 2026
• Last Verified: August 1, 2026

More Information

Terms related to this study

• Other Study ID Numbers: C001

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: Individual participant data (IPD) will not be shared publicly due to the following reasons: (1) This is a single-center observational study with a relatively small sample size (N=200), and the data are collected specifically for the exploratory objectives of this protocol; (2) The study involves pediatric participants (ages 6-14), and the informed consent form, approved by the institutional ethics committee, does not include provisions for sharing individual-level data with external investigators; (3) The data include sensitive genomic (metagenomic sequencing) and metabolomic profiles, which may carry re-identification risks; (4) The research team lacks the infrastructure and funding to support data anonymization, transfer, and external curation at this time. Summary-level results will be disseminated through peer-reviewed publications and conference presentations.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No