The value of hydrogen and methane breath test in predicting colorectal polyps


 BackgroundTo investigate the value of hydrogen and methane breath test in predicting colorectal polyps. Methods382 patients were enrolled in this study. They were divided into colorectal polyps group and Non polyps group as control. Hydrogen and methane breath test was used in both groups for small intestinal bacteria overgrowth (SIBO) diagnosis. All patients were examined by colonoscopy to investigate colorectal polyps. Diamine oxidase, D-lactate and Lipopolysaccharides (LPS) were measured in all patients to evaluate the intestinal barrier function.Results 1) The age of colorectal polys group is significantly higher than control group (P=0.000); the prevalence of colorectal polyps increases with age. 2) The prevalence of colorectal polys in male is significantly higher than female (P=0.004); 3) Lactuloses breath test (LBT) was used for small intestinal bacteria overgrowth (SIBO) diagnoses. Prevalence of hydrogen dominate SIBO and methane dominate SIBO in colorectal polys group were significantly higher than control group (P = 0.000, 0.013);4) The prevalence of constipation was higher in colorectal polys group than control group (P=0.023);5) The peoples who had high hydrogen production peoples showed less intestinal barrier function damage and lower LPS level than peoples who had low hydrogen production (P=0.029, 0.049);6) The sensitivity and specificity of SIBO is 75.5% and 51.0% respectively in SIBO diagnosis.ConclusionThe occurrence of colorectal polyp increase with age, the year 46 is a cut off age for peoples need colonoscopy examination. Male has a higher prevalence than female. Intestinal hydrogen may play a role in prevent intestinal mucosa damage via its antioxidant function. SIBO shows a positive relationship with colorectal polyp. Hydrogen and methane breath test is a useful mass prescreening method for colorectal polyp risk.


Abstract Background
To investigate the value of hydrogen and methane breath test in predicting colorectal polyps.
Methods 382 patients were enrolled in this study. They were divided into colorectal polyps group and Non polyps group as control. Hydrogen and methane breath test was used in both groups for small intestinal bacteria overgrowth (SIBO) diagnosis. All patients were examined by colonoscopy to investigate colorectal polyps.
Diamine oxidase, D-lactate and Lipopolysaccharides (LPS) were measured in all patients to evaluate the intestinal barrier function.

Results
1) The age of colorectal polys group is signi cantly higher than control group (P=0.000); the prevalence of colorectal polyps increases with age.
2) The prevalence of colorectal polys in male is signi cantly higher than female (P=0.004);

3) Lactuloses breath test (LBT) was used for small intestinal bacteria overgrowth (SIBO) diagnoses.
Prevalence of hydrogen dominate SIBO and methane dominate SIBO in colorectal polys group were signi cantly higher than control group (P = 0.000, 0.013); 4) The prevalence of constipation was higher in colorectal polys group than control group (P=0.023); 5) The peoples who had high hydrogen production peoples showed less intestinal barrier function damage and lower LPS level than peoples who had low hydrogen production (P=0.029, 0.049); 6) The sensitivity and speci city of SIBO is 75.5% and 51.0% respectively in SIBO diagnosis.

Conclusion
The occurrence of colorectal polyp increase with age, the year 46 is a cut off age for peoples need colonoscopy examination. Male has a higher prevalence than female. Intestinal hydrogen may play a role in prevent intestinal mucosa damage via its antioxidant function. SIBO shows a positive relationship with colorectal polyp. Hydrogen and methane breath test is a useful mass prescreening method for colorectal polyp risk.

Background
The colorectal polyps are the small clumps of proliferated cells on the colorectal mucosal. The person over 50 has a high risk to develop colorectal polyps. Most colorectal polyps are harmless, but over the time, some of them may develops into colorectal cancer. It is important to examine the colorectal polyps and remove them before they develop to cancerous. Screening tests can help nd early stage colorectal cancer before it shows symptoms, to give a good chance of recovery.
The prevalence of colorectal polyps varies widely from different genders, ages, and races. Lieberman and colleagues reported the asymptomatic black men and women are more likely to have one or more polyps > 9 mm compared to whites [1] . The differences were especially striking among women. Wang and colleagues reported the prevalence of asymptomatic colorectal polyps is 27.4% in Taiwan, the prevalence of hyperplastic polyps and adenomatous polyps was 11.1% and 16.1%, respectively [2] . They reported man had higher percentage adenomatous polyp than women (71.9% vs. 28.1%, P = 0.017).
Since the colorectal polyps are often no symptoms, it is hardly for asymptomatic peoples to looking for examination before they are in the later stages of the colorectal cancer which may fetal to life. Colonoscopy examination is an expensive and uncomfortable method to screen colorectal polyps and cancer. It is important to nd a noninvasive and less expensive method to screen the peoples in the high risk persons.
The increased evidences showed the gut microbiome may play crucial role in carcinogenesis. Hydrogen and methane are the speci c metabolite of intestinal bacteria fermentation. Hydrogen and methane breath test is a widely used as noninvasive technique to evaluate small intestinal bacteria overgrowth (SIBO) and methane-producing organisms in colorectal intestine.
We use lactulose hydrogen and methane breath test (LBT) to evaluate the small intestinal bacterial overgrowth and constipation in all patients. All subjects intestinal mucosal were examined by colonoscopy to evaluate the intestinal barrier condition. We also evaluate the prevalence polys with age and gender.

Study design
We retrospectively reviewed patients in our hospital during the period from July 2017 to February 2019. 382 patients were recruited in this study (213 males and 169 females). The age range of subjects is from 22 to 92, average age is 57±14 years. All patients signed informed consent before the test. The study was approved by the ethics committee of the First A liated Hospital of Guangdong Pharmaceutical University.
All patients were tested by LBT and colonoscopy. The age, gender, LBT result, colonoscopy examination ndings, intestinal barrier function and patients' history were recorded. Patients with colorectal polyps were in the colorectal polyps group and those with normal colorectal mucosa were in control group. All experiments were conducted in accordance with relevant guidelines and regulations.

Criteria for patient selection
Inclusion criteria: Colorectal polyp group: colorectal polyps (including in ammatory, proliferative, adenomatous, etc.) were observed under colonoscopy and con rmed by pathological biopsy.
Normal colorectal mucosa group: no colorectal polyp or other intestinal lesions were observed under colonoscopy.
Exclusion criteria: (1) patients with acute intestinal infection; (2) antibiotics used in recent 4 weeks; (3) patients with severe heart, lung, brain and other diseases who are unable to tolerate colonoscopy examination; (4) patients at risk of hypoglycemia; (5) patients younger than 18 years old.

SIBO examination
The LBT was tested based upon North American Consensus' suggestion. Quintron BreathTracker SC model equipment was used to measure concentration of hydrogen, methane, and carbon dioxide. All patients were fast 12 hours and brushed teeth before tests. They took 10 g lactulose solution in warm water after taking fast breath test. Breath samples were collected in every 30 minutes until nished the test at the 150 minute after taking lactulose solution. No drink, food, exercise, and sleep was allowed during the tests.
SIBO criteria was based upon North American Consensus', suggestion. SIBO positive was diagnosed if breath hydrogen increased ≥20 ppm, or methane increased ≥10 ppm above fasting baseline withing 90 minutes. SIBO was also diagnosed as positive if the combined value of hydrogen and methane increased ≥15 ppm above the fasting baseline within 90 minutes based upon manufacture's suggestion.

Intestinal barrier function examination
Intestinal barrier function test: The test instruments and kits were purchased from Beijing Zhongsheng Jinyu Diagnostic Technology Co., Ltd. Blood samples were taken after 8 hours fast and storage in 4 6℃ refrigerator for no more than 4 hours. Diamine oxidase, D-lactate and LPS were measured according to the instruction provided by the manufacturer. Patients' symptoms and signs were recorded 48 hours before the test. The normal reference value of diamine oxidase is ≤ 10U/ L, D-lactate ≤ 15mg / L, LPS is ≤ 20U/ L. Any value above these numbers is considered as intestinal barrier function damage.

Statistical methods
All data were analyzed by using IBM spss22.0 statistical software. T test was used for comparison of two independent data. Four grid C 2 test was used to compare the qualitative data, P < 0.05 (bilateral) was considered as statistically signi cant.
Result 382 patients were enrolled in this retrospective study. All patients were conducted LBT for SIBO and examined by using colonoscopy for colonrectal polyps. Most of subjects had basic diseases (such as hypertension, type 2 diabetes, hyperlipidemia, fatty liver cirrhosis, functional constipation, chronic super cial gastritis, gastroesophageal re ux disease, peptic ulcer).
213 patients showed normal intestine mucosa. 169 patients had colorectal polyp (52 patients had in ammatory polyp, 29 had hyperplastic polyp, and 88 had adenomatous polyp). 136 polyps were ≤ 1.0cm, 25 polyps were between 1.0cm and 2.0cm, 8 polyps were ≥ 2.0cm. 71 patients had single polyp, 98 patients had multiple polyp. 114 patients had polyp on the left side colon (including sigmoid colon, descending colon, and rectum) and 55 patients had polyp on other parts of the colon.

Prevalence of polyp in age and gender
The prevalence of colorectal polyp was signi cantly correlated with age. The average age of the colorectal polyp group was higher than control group (P=0.000). The prevalence of the colorectal polyp was increased with age (P=0.000). Male showed higher colorectal polyp prevalence than female (P=0.004). The data present in the table 1 and table 2 Based upon small intestine hydrogen and methane production we divided the subjects into the following groups: SIBO+: total LBT positive patients from both groups.
SIBO-: total LBT negative patients from both groups.
The prevalence of total SIBO patients, SIBO in high hydrogen production, and SIBO in high methane production in colorectal polyp group are signi cantly higher than that in control group. The data present on table 3. There is no signi cant correlation between SIBO and all types of polyps (including in ammatory polyp, hyperplastic polyp, adenomatous polyp). This nding suggests that the prevalence of SIBO is not correlation with the pathological classi cation of colorectal polyps.

Correlation between colorectal polyp and constipation
We evaluated the correlation between colorectal polyps and constipation. The constipation was diagnosed according to the "Rome IV" criteria. The patients who had diarrhea had been excluded from this study. The colorectal polyp group showed higher constipation prevalence than control group (P=0.023). The data present in table 4. HMPG: The high methane production group includes all the patients whose breath methane was over 10 ppm above fasting baseline within 90 minutes.
LMPG: The low methane production group includes all the patients whose breath methane was less than 10 ppm above fasting baseline within 90 minutes.

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HHPG: The high hydrogen production group includes all the patients whose breath hydrogen was over 20 ppm above fasting baseline within 90 minutes.
LHPG: The low hydrogen production group includes all the patients whose breath methane was less than 20 ppm above fasting baseline within 90 minutes.
In this study, HHPG showed less intestinal barrier function damage than LHPG (c 2 =4.758 P=0.029). There is no signi cant difference between HMPG and LMPG in intestinal barrier function change (P=0.188).
The correlation data between breath hydrogen/methane and intestinal barrier function present in Table 5. The LPS level of HHPG is signi cantly lower than that of LHPG (t=1.974, P=0.049). The DAO and D-lac shows no difference between these two group patients. The data resent in table 6. We divided the SIBO positive patients into three groups based upon their hydrogen and methane production, then evaluated the sensitivity and speci city of each group. The data present in the table 7.  [2] . We suggest the year 46 is a cut off age for colonoscopy examination. A suitable mass prescreen method is required before colonoscopy examination.
Adenomatous polyp is the precursor lesions for colorectal cancer. We found 88 patients had adenomatous polyp in total 382 patients (23.3%). This nding agreed with Giacosa and colleagues' study [3] . They reported adenoma prevalence is more than 25% in colonoscopy studies.

Correlation between colorectal polyp and SIBO
In recent years, increasing studies have been revealed the occurrence and development of colorectal polyp have close relationship with the change of intestinal ora. Chen and colleagues [4] compared the intestinal mucosa bacterial composition between normal colorectal individuals and colorectal adenoma individuals by molecular ngerprint and clone sequencing technology. Their result showed a signi cant difference between these two populations. They suggested adherent bacteria might play an important role in adenoma and colorectal cancer development. The changes of the intestinal ora composition and structure of the adherent bacterial community might contribute to the occurrence and development of adenomatous polyp. Mangifesta and colleagues [5] used 16S rRNA gene sequencing revealed the composition of intestinal polyp microbial community. In a meta-analysis, it reported in addition to genetics, diet, lifestyle, obesity, drinking and smoking, intestinal micro ora played a very important role in the occurrence of colorectal cancer and colorectal polyp. Intestinal ora and its metabolites might be involved in the initiation or development of colorectal polyp or tumors through various mechanisms, including the production of free oxygen radicals and other gene toxins, phenolic compounds, indoles, etc.
We found SIBO had a correlation with polys (P=0.010), either hydrogen dominate SIBO or methane dominate SIBO had signi cant correlation with colorectal poplys (P=0.000, 0.013). Our nding suggested intestinal Methanogenic bacteria might play a role in colorectal polyp development. We didn't evaluate the correlation between SIBO and colorectal polyp pathological types, diameter, size, number and location. This is the rst study to investigate the correlation between SIBO and colorectal polyps.
We found that the incidence of constipation in the colorectal polyp group was signi cantly higher than control group (29.5% vs 18.1%, P=0.023), and the colorectal polyp group showed higher methane production than control group(71.0% vs, 58.7%, P=0.013). Waqar and Rehan [6] reported methane prolonged oro-cecal transit time (OCTT) and raised the intestine contraction amplitude, slowing peristalsis, resulting in constipation. Liu [7] reported that constipation can signi cantly increase the incidence of colorectal polyps, but did not signi cantly increase the incidence of colorectal cancer even it is a risk factors for colorectal cancer. Comstock and colleagues [8] reported in ammatory cytokines level in patients with colorectal polyp was higher than that in patients without colorectal polyp. Their nding suggested colorectal polyp are associated with chronic intestinal in ammation. The increase of cytokines and the decrease of antioxidants will lead to oxidative stress, delay gastrointestinal motility and lead to SIBO.

Hydrogen may protect intestinal mucosal from in ammation
In this study, we found LPS level of HHPG is signi cantly lower than LHPG (t=1.974, P=0.049). LPS is known as lipoglycans and endotoxins, found in the outer membrane of gram-negative bacteria in intestine. Less LPS level in blood plasma can be considered less intestinal mucosa damage in HHPG than LHPG. Bilinski and colleagues [9] reported neoplastic colon polyp displaying increased acute and chronic in ammation comparing to normal and adjacent non-dysplastic colonic mucosa. The adenoma polyp' size correlated with degree of acute and chronic in ammation. It is well known that oxidative stress plays an important role in intestine in ammation and can lead to damages of the intestinal mucosa and bacterial metabolite invasion, such as LPS. Hydrogen has the functions of selective antioxidant, anti-in ammatory, inducing gene expression and gas signal modulation. Chen and colleagues reported [4] molecular hydrogen was a potent antioxidant and able to protect organs from oxidative stress injuries. Lactulose administration signi cantly induced hydrogen production might help to prevent the development of dextran sodium sulfate-induced colitis and alleviate oxidative stress in the colon. Our nding shows agreement with their reports. The persons who produced higher hydrogen had less intestinal mucosa damage. Hydrogen breath test may have potential role in evaluate the intestinal mucosa in ammation.

The value of hydrogen and methane breath in prescreening colorectal polys
It is important to nd a convenience method for mass prescreening the people who has risk of colorectal polyp and cancer development. We evaluate the colorectal polyp prediction value of LBT SIBO positive patients based upon their hydrogen and methane production. The result showed hydrogen dominate SIBO had a low sensitivity and speci city. It may not a good method in predict the occurrence of colorectal polyp. The Methane dominate SIBO has a moderate sensitivity and speci city, may also not a proper method to the occurrence of colorectal polyp. The SIBO patients who had both high hydrogen and methane had 75.5% sensitivity and 51.0% speci city in predict colorectal polyp. It suggests high hydrogen and methane LBT may have a predict value in colorectal polyps mass prescreening before colonoscopy examination.
Summarize the ndings in this study, colorectal polyp has a correction with constipation and high methane production. The high methane excretion on breath test is association with clinical constipation. The North America Consensus suggested breath test is useful in the diagnosis of methane associated constipation. Our study con rmed the relationship of high methane production, constipation, and colorectal polyp. Based upon our knowledge this is the rst study to evaluate the predict value of hydrogen and methane breath test in colorectal polyp occurrence.
Hydrogen and methane breath test is a noninvasive and less expensive test. It shows a clinical value in predict colorectal polyp for mass prescreening. The ndings from this study suggest hydrogen and methane breath test may help to reveal the colorectal polyp in the early stage and help in colorectal cancer prevention. It may also have a value in helping reduce medical care expenses and save medical resources.

Conclusion:
The prevalence of colorectal polyp increases with age. Male has a higher prevalence than female in the same age group. Intestinal hydrogen may play a role in prevent intestinal barrier function damage via its antioxidant function. Both hydrogen and methane dominated SIBO have a correlation with colorectal polyp. Hydrogen and methane breath test has value for mass prescreening in discovering the colorectal polyp in the early stage and cancer prevention.
This is a retrospective study. The random controlled double blind study needs to be done in a large scale to re-evaluate the ndings from this study. Further studies need to be done to investigate the impact of hydrogen on intestine in ammation, intestinal permeability, LPS, and colorectal polyp.