Walnuts and Colon Health

Study Overview
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Brief Summary
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The purpose of this research study is to examine whether adding walnuts to your diet can have a beneficial effect on the gut bacteria population, inflammatory markers in the blood, and the tissue that lines the inside of the colon.

Detailed Description
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This is a 29-day dietary intervention study where participants will be asked to consume 2 ounces of walnuts daily for 21 days, and at the end of the study period they will come in for a routine colonoscopy. After being informed about the study and potential risks, participants giving written informed consent will first start a 7-day wash-out period where they will be asked to avoid foods high in ellagitannins for the duration of the study. In addition, participants will be asked to complete food and activity questionnaires, a walnut consumption log, and two sets of 3-day dietary records during their participation in the study. Participants will also be asked to provide three urine samples, two blood samples, and two stool samples at multiple time points, and 8-10 colon tissue specimens (biopsies) will be collected during their colonoscopy procedure for the purposes of this study.

Official Title
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Microbiota, Metabolites and Colon Neoplasia

Conditions
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Colorectal Cancer, Diet Habit

Intervention / Treatment
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* Other: Walnuts


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Individuals in one of the following groups: Men and women between the ages of 40 to 75 years old, who are scheduled to undergo a routine screening or surveillance colonoscopy for colorectal cancer (CRC) and have a family history of CRC in a first degree relative diagnosed with CRC under the age of 65 years, or Men and women between the ages of 45 to 75 years old, who are referred for colonoscopy following a positive fecal immunochemical test (FIT) and have not had a high quality colonoscopy in the past 3 years, or Men and women between the ages of 45 to 75 years old, who have a personal history of colon polyps and are scheduled to undergo a surveillance colonoscopy Willing and able to provide written informed consent for study participation Willing to consume 2 ounces of walnuts daily for 3 weeks Willing to avoid intake of ellagic acid/ellagitannin-rich foods (e.g., pomegranates, hazelnuts, pistachios, strawberries, raspberries, blackberries, oak-aged wines, and other food on a list given by researchers) and fermented dairy products containing viable Bifidobacteria or Lactobacilli) Willing to stop taking dietary supplements, including probiotics Willing to have 2 separate blood draws, as well as urine and stool collections Willing to comply with all study requirements Exclusion Criteria: Individual has a personal history of CRC, or a history of any malignancy (other than skin cancer) within the past 5 years Individual meets the Amsterdam criteria for Lynch Syndrome or has a history of familial adenomatous polyposis (FAP) Individual has been treated with immunosuppressive agents or systemic steroids, excluding inhalers, at least two weeks prior to the Screening Visit and for the duration of the study Use of antibiotics at least one month prior to the Screening Visit and for the duration of the study Patients with severe medical illness or those at high risk for anesthesia, as determined by good clinical practice Current evidence or previous history of ulcerative colitis or Crohn's disease Colonoscopy performed for reasons other than screening or surveillance for CRC HIV infection, chronic viral hepatitis Allergy to walnuts or hypersensitivity to tree nuts Peri-menopausal women with any chance or plan of pregnancy Individuals with blood coagulation disorders or on anti-coagulant therapy Any other condition that, in the opinion of the PI, might interfere with study objectives No race/ethnicity, language or gender exclusions for this study

Ages Eligible for Study
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Minimum Age: 40 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Walnut Consumption<br>Following enrollment, participants will start a 7-day wash-out period where they will be asked to avoid foods and beverages high in ellagitannins. These include pomegranates, hazelnuts, pistachios, walnuts (besides the samples given by the researchers), strawberries, raspberries, blackberries, oak-aged wines and spirits; a full list of foods and beverages to avoid will be provided. Then, participants will consume 2 ounces of walnuts daily with their usual diet while continuing to avoid ellagitannins for 21 days prior to their routine colonoscopy. | Other: Walnuts<br>* Participants consume 2 ounces of walnuts daily for 21 days<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Bacterial composition and taxonomy changes in the fecal microbiome | Bacterial composition and taxonomy changes in the fecal microbiome will be assessed using 16 Svedberg unit (16S) ribosomal ribonucleic acid (rRNA) sequencing, at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 
| Bacterial diversity changes and strain-level variations in the fecal microbiome | Bacterial diversity/abundance changes and strain-level variations in the fecal microbiome will be assessed using metagenomic shotgun sequencing at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 
| Bacterial gene expression profile changes in the fecal microbiome | Bacterial gene expression profile changes in the fecal microbiome will be assessed using metagenomic ribonucleic acid (RNA) sequencing at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 
| Urolithin levels in urine | Urolithin levels will be measured in urine by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry at day 0 (baseline/pre-washout), day 7 (post-washout/pre-walnut supplementation) and day 29 (post-walnut supplementation/end of study). | Day 0, Day 7 and Day 29 | 
| Association of urolithin levels with presence (and type) of colonic lesions | Baseline urolithin levels measured in the urine by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry will be associated with the presence (and type) of colonic lesions (e.g., advanced adenomas (AAs) or sessile serrated adenoma/polyps (SSA/Ps)) detected during the colonoscopy procedure at the end of the study (day 29). | Day 29 | 
| Correlation of urolithin levels with fecal microbiome composition | Detailed statistical analyses will be used to correlate urolithin formation with the composition of the fecal microbiome at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 
| Correlation of colonic lesion gene expression with urolithin production | Colonic lesion (AAs and SSA/Ps) biopsies obtained at the end of the study (day 29) during the colonoscopy procedure will undergo DNA sequence-based analysis to determine gene expression profiling. These results will be compared to urinary urolithin levels measured at baseline to establish correlations between urolithin production and colorectal cancer risk markers. | Day 29 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Short-chain fatty acid composition in stool | Short-chain fatty acids in freeze-dried stool samples will be measured using differential mobility separation at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 
| Bile acid metabolism in stool | Primary and secondary bile acids in freeze-dried stool samples will be analyzed using standard liquid chromatography-mass spectrometry methods at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 
| Inflammatory markers in blood | Markers of systemic inflammation associated with adenoma risk will be measured in plasma using commercial enzyme-linked immunoassay (ELISA) kits at day 7 (post-washout/pre-walnut supplementation) and at day 29 (end of study). | Day 7 and Day 29 | 
| Correlation of dietary behavior with presence (or absence) of colonic polyps | Overall diet quality and habits documented on brief food questionnaires will be analyzed by NutritionQuest at day 7 (post-washout/pre-walnut supplementation). Dietary behavior data will be correlated with the presence or absence of colonic polyps detected during the colonoscopy procedure at the end of the study (day 29). | Day 7 and Day 29 | 
| Correlation of dietary behavior with fecal microbiome composition and diversity | Dietary behavior data documented on 3-day dietary records will be analyzed by Nutrition Data System for Research software. This data will be correlated with fecal microbiome composition and diversity at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 
| Correlation of dietary behavior with urolithin production | Dietary behavior data documented on 3-day dietary records will be analyzed by Nutrition Data System for Research software. This data will be correlated with urolithin levels at day 7 (post-washout/pre-walnut supplementation) and at day 28 (post-walnut supplementation). | Day 7 and Day 28 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Walnuts, Colorectal cancer, Microbiome, Urolithin, Diet habit, Inflammatory markers, Colon inflammation