Progress 01/15/24 to 01/14/25
Outputs
Target Audience:Results generated from this research will be relevant to the overall United States (US) population as data generated provide information on habitual diet and its impact on the gut microbiome, iron status and iron absorption and on how these factors interact with one another. Findings generated from the study will be presented to scientific audiences at national conferences and published in peer reviewed journals. Undergraduate and graduate students will be involved in the implementation of this research and the clinical metabolic studies will provide educational opportunities for these groups. Findings will be released in lay language to communicate key findings to the public. We are studying a relatively young population and addressing the impact of common food components on the gut microbiome. Characterizing interactions between diet and gut health in these younger ages provide data that will help identify modifiable factors that can be targeted to improve health and thereby prevent disease. Changes/Problems:Overall, our study progressed smoothly this year without major unexpected challenges. However, our previous post-doc researcher on this project terminated her work with us due to medical illness. We have successfully identified another new post-hoc researcher expertise in the gut microbiome, who will join us in June. In the meantime, we do expect to have more challenges in recruiting vegetarian participants given the lower prevalence of this dietary pattern in our community, but we are confident we will recruit the necessary volunteers over the next reporting period. We will do this by promoting our study during campus events and classes, distributing flyers on bulletin boards, contacting all active student clubs, posting our flyers on campus displays, and engaging local communities, including vegetarian restaurants and grocery stores. Through these strategies, we aim to enhance awareness and to encourage participation in this study. What opportunities for training and professional development has the project provided?In 2024, our laboratory engaged 24 undergraduate research assistants, who contributed to participant recruitment through campus tabling, flyer distribution, and monitoring the study email. They also assisted with data entry for anthropometric, dietary, and biochemical metrics and received training in anion exchange chromatography, blood sample digestion, and iron isolation. Our laboratory group recruited two new doctoral students and three new master's students. These students have gained valuable training while supporting the clinical studies, participant recruitment, mass spectrometric analyses of isotopic enrichment, and fecal sample processing. One of the new doctoral students will work on this project for her dissertation. While our previous post-doc had to leave the project due to a medical issue, we have successfully identified a new post-doctoral researcher, who will join the group to oversee the 16S and microbial metagenomic studies. She received her doctorate working with our co-investigator, Dr. Elizabeth Johnson, and is positioned to rapidly undertake the necessary analyses. We continued weekly journal club meetings over the past year, offering undergraduate and graduate students opportunities to discuss recent publications relevant to the FeMicrobiome project and providing a forum for them to develop their academic communication skills. Each student presented at least twice during the year, fostering critical engagement and professional growth. How have the results been disseminated to communities of interest?Data was presented at the 2024 USDA annual meeting by Dr. Xu. We have submitted an abstract to ASN 2025 that will be presented in May of 2025 in Orlando, FL. Zhang, YK, Xu Y, Johnson E, O'Brien KO. Iron Absorption and Storage in Plant-Based vs. Omnivorous Diets: Foundations for Exploring Dietary Iron Types and Gut Microbiota Interactions. Submitted 2025; American Society of Nutrition Meeting. Orlando, FL What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will finish the remaining clinical studies and finish all analyses of biospecimens and dietary data. We will analyze baseline participant characteristics and interpret the dietary data to assess dietary quality and estimate heme and non-heme iron intake in our sample groups. To enhance our heme content estimation database, we will add standardized heme content data for eggs, which will help us explore potential correlations between egg consumption and ferritin levels in vegetarian participants. We plan to submit the first manuscript from this study discussing the dietary patterns between the vegetarian and non-vegetarian participants. We will continue analyzing biomarkers and performing mass spectrometric analyses. We aim to complete the quantification of serum hepcidin and initiate analysis of serum erythropoietin and erythroferrone levels, the latter two proteins play critical roles in hepcidin regulation, offering more insights into iron absorption and regulation within our samples. With fecal samples, we will finalize the fecal sample digestions and analysis of total fecal iron concentrations, and we will also complete fecal DNA extraction and initiate 16S rRNA gene sequencing for the whole sample set once the recruitment complete to characterize microbial composition differences among vegetarians and non-vegetarians.
Impacts
What was accomplished under these goals?
We have made significant progress over the second year of this project. We have completed the clinical study for both male and female non-vegetarian groups (n=64). Among non-vegetarians, two participants were excluded because they failed to collect a fecal sample within the appropriate time window after completing their first visit. One participant was excluded because they later revealed that they were not fasted when they ingested the tracer. Finally, one female participant was excluded because the phlebotomist could not obtain a blood sample. We also have recruited 35 of the 60 vegetarian individuals (10 men and 25 women). Upon the quality assessment of our collected dietary data, we have excluded 8 vegetarian participants (7 female and 1 male) due to hidden animal food intake in their food frequency questionnaires. Anthropometrics and iron status analyses have been completed in all recruited participants to date. In total, 9 participants were found to be anemic (including 1 vegetarian male, 1 vegetarian female and 7 non-vegetarian females). We have also completed the assessment of iron status (serum ferritin (SF), soluble transferrin receptor (sTfR) and hepcidin in all recruited participants to date. Total body iron was calculated using the SF and sTfR data. The male vegetarian participants demonstrated a significantly lower amount of storage iron when compared to the non-vegetarian males. Smaller but non-significant differences were observed among the female participants. Data on dietary intake has been collected using the ASA24 and the DHQIII surveys. All recruited participants successfully completed these assessments. We have estimated heme and non-heme iron intake based on the database our group previously compliedto estimate the heme Fe content among various animal-based foods. Surprisingly, we foundtotal iron intake per day was significantly higher among vegetarian participants in the study, especially for female vegetarians. Further, the vegetarian group had a better diet quality score (HEI score) compared to the non-vegetarian group, with more prominent differences observed between the vegetarian and non-vegetarian male groups. To evaluate iron absorption, each participant received an oral dose of 7 mg of 57Fe to measure red blood cell iron incorporation (a proxy of iron absorption). We have completed the iron absorption measures using magnetic sector thermal ionization mass spectrometry in 80 participants, and the remaining blood samples are currently being processed. From analyses to date, male vegetarians exhibited a significantly greater iron absorption than male non-vegetarians as would be expected based on their significantly lower iron status. No significant differences were observed between the females in relation to diet group. As expected, SF was inversely correlated with Fe absorption in both non-vegetarians (p < 0.001, R2=0.38) and vegetarians (p < 0.05, R2=0.42). To prepare for the fecal microbiome analyses we have standardized the fecal DNA extraction protocol and successfully obtained pure high concentration fecal DNA from the 12 pilot samples analyzed. The doctoral student working on this project successfully completed a Cornell training course on 16s rRNA gene sequencing. We are currently conducting a pilot 16s sequencing round with 12 fecal DNA samples being analyzing.
Publications
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:Results generated from this research will be relevant to the overall United States (US) population as data generated provide information on habitual diet and its impact on the gut microbiome, iron status and iron absorption and on how these factors interact with one another. Findings generated from the study will be presented to scientific audiences at national conferences and published in peer reviewed journals. Undergraduate and graduate students will be involved in the implementation of this research and the clinical metabolic studies will provide educational opportunities for these groups. Findings will be released in lay language to communicate key findings to the public. We are studying a relatively young population and addressing the impact of common food components on the gut microbiome. Characterizing interactions between diet and gut health in these younger ages provide data that will help identify modifiable factors that can be targeted to improve health and thereby prevent disease. Changes/Problems:We have not experienced any changes in the approach as detailed in the grant submission. What opportunities for training and professional development has the project provided?We currently have 28 undergraduate students working in our laboratory for credit as research assistants. These students are involved in many aspects of the study. They help us with recruiting by tabling on campus, distributing fliers, monitoring our study email, and entering screening, dietary, and biochemical data. Several of our undergraduates are trained in anion exchange chromatography and they assist with blood digestion and iron isolation from samples. We also have four doctoral students and a postdoctoral fellow who contribute to this research and gain professional and training opportunities by assisting with this project. These individuals assist with clinical studies, recruitment, and mass spectrometric analysis of isotopic enrichment. One of the graduate students will be working on this study for her dissertation project. In addition, for the past year, Dr. Clara Park, an Associate Professor, from Chonnam National University in Gwangju, Korea has been working in our laboratory as a visiting scientist. This study is also benefiting Dr. Clara Park's professional development as she learns about stable isotope studies, mineral metabolism, and the gut microbiome. We have a weekly journal club that is attended by all laboratory personnel. Many of the studies discussed are related to this research project. All members of the laboratory group make presentations at this journal club over the course of the semester. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Over the next reporting period we plan to continue recruiting theremaining study participants and complete the clinical studies needed to measure iron absorption and collect the fecal and other biospecimens. We will continue to analyze the blood biomarkers and undertake the mass spectrometric analysis of the iron isotopic enrichment in blood as these samples are collected. We will also begin digesting aliquots of the fecal samples to measure total iron concentrations. The animal product foods ingested will be entered and the heme content of these foods will be quantified using our compiled tables from the published literature. Preliminary absorption and iron status data will be submitted for the annual American Society of Nutrition meeting.
Impacts
What was accomplished under these goals?
We have made significant progress over the first year of this project. All clinical protocols are established and working, and laboratory analyses are progressing as planned. Stable iron isotope was purchased, prepared for dosing, and certified for sterility and isotopic enrichment. All IRB approvals were obtained, recruitment ads were developed and approved to be used in paper format, on social websites and sent to campus list serves. Recruitment was initiated and volunteer interest in the project has been excellent. We have completed studies in 27 individuals to date; 21 of whom were habitually ingesting a diet containing animal products (8 men and 13 women) and 6 were habitually ingesting a plant-based diet (3 men and 3 women). Race and ethnic composition were self-reported and representative of the diversity that is evident on our campus. Body mass index and hemoglobin were measured on the day the iron absorption study was completed. Subject characteristics are provided below. Of the 27 individuals recruited, 5 women were found to be anemic. We are evaluating iron status of study participants based on serum ferritin (SF), soluble transferrin receptor (sTfR) and hepcidin. Total body iron will be calculated using the SF and sTfR data. Biochemical analysis of the serum collected to date has been initiated. Data on dietary intake is being collected using the ASA24 and the DHQIII. Subjects have had no problems completing these assessments even with the extended time that is needed to fill out these surveys. We have been working to compile and publish a detailed review of the heme content of meats as determined using chemical analysis following the Hornsey method. Using this database, we will directly estimate the heme content of the diet. Collection of the fecal microbiome samples has progressed as planned and participants have not had any issues following the detailed protocol they are provided for these collections. We have met with the director of our core center to discuss the fecal metagenomic analyses and have developed a work plan for this process. They have recommended that we send samples for analysis in larger batches than we had initially planned. To prepare for this we are working to standardize the DNA extraction process. Each participant receives an oral dose of 7 mg of 57Fe to measure red blood cell iron incorporation (a proxy of iron absorption). We have extracted iron from the blood collected from the participants studied to date and have begun to analyze the isotopic enrichment of these samples using thermal ionization mass spectrometry. Enrichment in the 7 samples analyzed to date confirms that dosing requirements are optimal and average absorption in samples evaluated to date was 29.8% in those ingesting a plant-based diet (n=3) and 23.4% in those ingesting a diet containing animal products / heme (n=4). Absorption data from the first 7 participants also found markedly higher iron absorption in the anemic participants compared to the non-anemic participants: 57.7% (n=2) vs.13.5% (n=5) respectively. We currently have a list of eligible volunteers that were not able to complete the study before Cornell's winter break. We will begin enrolling the remaining participants this month when classes resume.
Publications
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