Research Project: Intestinal Microbiome and Childhood Feeding

Location: Children's Nutrition Research Center

2024 Annual Report

Objectives
Objective 1: Compare the effects of inulin and fructo-oligosaccharides against a maltodextrin placebo in obese children, using a double-blind randomized controlled trial, to study weight loss, fecal microbiota and their functions. Objective 2: removed due to investigator departure. Objective 3: Conduct a clinical trial adding black-eyed peas to diets of young children at risk for stunting. Determine efficacy in reducing stunting and analyze fecal sample to understand potential mechanisms by which the food supplement ameliorates stunting. Assess dietary compliance in a novel, quantitative manner using a urinary biomarker for black eyed peas. Currently quercetin and ferulic acid are candidates for this biomarker.

Approach
A number of pressing nutritional issues face the US and other nations. Over 20% of children throughout the world are obese with even more children overweight; both associated with diabetes and heart disease. Given the importance of the gut bacteria in our general health and weight control, we will test via various sample analyses a dietary supplement (prebiotic) that selectively enhances the growth and activity of bacteria associated with leanness. We anticipate that this prebiotic will reduce the risk of overweight and obesity in children. There is a lack of molecular and metabolic biomarkers of existing nutritional therapy limiting the ability to appropriately and adequately assess the utility of dietary supplementation. We will perform serial measures of DNA methylation and tissue metabolites to identify suitable biomarkers of nutritional deficit and recovery. In addition, stunting affects about 23% of all children under 5 years of age globally. Most of these children are in Africa and south Asia and the consequences include lower economic productivity, decreased cognition and more diabetes and hypertension. Similar to obesity, the microbiome is implicated as a cause of stunting and new treatments are needed. We will determine using biomarker analysis if legume supplements can extend their benefits to children in West Africa.

Progress Report
For Objective 1, our goal was to better understand the relationships between diet and the bacteria in our gut (digestive or gastrointestinal tract) and how their interaction can impact our health. What we eat and drink can greatly influence the types and numbers of bacteria in our gut. Similarly, the types and numbers of gut bacteria can greatly influence our health. The bacteria are considered a “hidden organ” because of their ability to impact many of the processes that occur in our bodies. For example, the types and number of gut bacteria greatly impact the immune system (e.g., they can increase or decrease the amount of inflammation in our body), food digestion (e.g., increase or decrease the amount and type of nutrients that are absorbed), drug absorption and metabolism (e.g., or decrease the effectiveness), brain function (there is two-way communication between the brain and the gut bacteria), and the heart and vascular system (e.g., certain bacteria in combination with diet can increase the risk of developing hardening of the arteries (atherosclerosis)). Certain types of dietary fiber have shown to impact the types and numbers of gut bacteria. Dietary fiber is not broken down by enzymes in our small intestine, so we have evolved to rely upon bacteria in the colon to break down fiber. Most evidence suggests that dietary fiber increases the number and types of bacteria that are associated with health. Thus, the USDA sets a recommendation for the amount of dietary fiber that we should eat. Dietary fiber is considered a prebiotic, a substance that induces the growth or activity of beneficial gut bacteria. The breakdown products of dietary fiber produced by gut bacteria can have beneficial effects. However, some dietary fibers can induce symptoms (gas that is associated with belly pain and bloating) in susceptible children and adults. We previously showed that the administration of a certain type of fiber (psyllium) could reduce belly pain symptoms in children who often get belly pain of unknown cause. Belly pain affects approximately 1 out of 10 children worldwide. More recently we have shown that the benefit of psyllium was only seen in boys and girls. To confirm our findings, we evaluated the diets of both healthy children and those with belly pain. Both groups of children kept diaries that recorded what they ate, whether they had belly pain, and how often they had bowel movements and whether they had normal, hard, or loose bowels. We also determined what types of bacteria were present in the children’s guts. In both healthy boys and those with belly pain, as more dietary fiber was consumed, the less likely they were to report belly pain. In contrast, in girls there was no relationship between dietary fiber intake and the report of belly pain. In fact, in girls who normally reported problems with belly pain, those who ate more than the recommended intake of fiber reported more pain episodes and more days on which they had pain compared with the girls who ate less than the recommended amount of fiber. Surprisingly, in none of the above studies did the dietary fiber intake appear to impact the stool consistency (hard vs soft). Using advanced techniques to evaluate the bacteria in the gut, we compared the gut bacterial population in boys and girls. Overall, the number and types of bacterial species that were present was similar between the boys and girls. However, we did find an increased abundance of a specific bacteria (Actinomyces turicensis) in girls compared with boys. We are investigating how these specific bacteria may be involved in the sex-differential response to dietary fiber. We also are investigating the role of hormones, given how boys and girls differ in their hormone profiles. There is some evidence that sex hormones can influence the gut bacterial population both in terms of its makeup and the function of bacteria. Objective 3 aimed to achieve two main goals: 3A) To compare four supplementary foods with varying carbohydrate and protein compositions in treating moderate acute malnutrition (MAM). This included studying their effects on gut permeability (how easy things pass through the lining of the stomach and intestines) and intestinal microbiome (community of bacteria and other microbes that live in the intestines). The objective also sought to provide new insight into how dairy and vegetable ingredients impact intestinal permeability, microbiome composition, and the fecal metabolome (chemicals and substances found in stool) in young children. 3B) To evaluate dietary compliance in a new, quantitative way using a urinary biomarker for black-eyed peas. For Sub-objective 3A, a study was conducted in rural Pujehun, Sierra Leone, from September 2020 to March 2022, involving 1,067 children who were randomly assigned to one of four groups. Among these children, 413 were eligible for stool and urine collection after one month to assess gut permeability, fecal microbial composition, and fecal metabolites. All collected samples were transported to the U.S. for analysis. This year stool samples were analyzed to determine the types and quantities of bacteria present, as well as how the overall bacterial population combined. This analysis, termed 16S rRNA, included assessing beta diversity and bacterial taxa counts. An assessment of biological molecules in the stool was also conducted. Urine samples were analyzed for lactulose excretion, a marker of gut leakiness. For Sub-objective 3B, in 2019 20 pregnant women (over 18 years) and 20 children (9-21 months) participated in our study by consuming measured amounts of cowpea in increasing quantities every five days, starting with no cowpea and ending with a large quantity (75g for children and 260g for women). Urine samples and blood spots were collected during each 5-day period. All samples were transported to the U.S. for processing. Samples underwent metabolite detection via ultra-performance liquid chromatography tandem mass spectrometry in positive and negative ion mode. A sample of each bean was collected and transported for food metabolome analysis. Metabolite profiling was performed on the food to show molecules that would be present in the food ingredients themselves. Through this process 10 candidate biomarkers for cowpea consumption were identified, providing insight into how cowpea strengthens human nutrition. In addition, the blood spots underwent untargeted metabolomics and showed a significant increase in amino acids, phytochemicals, and lipids. Biomarker data from the dried blood spots was compared with the urine data with a few additional noteworthy candidate biomarkers.

Accomplishments
1. Boys and girls have different reactions to dietary fiber. Americans consume far less fiber than recommended and it is unclear if fiber is beneficial to specific individuals more so than others. To confirm our previous finding that increasing dietary fiber intake reduced the amount of belly pain in boys but not girls, researchers at the Children’s Nutrition Research Center in Houston, Texas, evaluated the dietary fiber intake in healthy children and those who often had belly pain. We found that in healthy boys and those with frequent belly pain, the more dietary fiber consumed, the less pain the boys had while in contrast, in healthy girls and those with frequent belly pain, there was no relationship between the amount of dietary fiber intake and belly pain. When we examined only children with frequent belly pain, boys who ate more than the recommended amount of dietary fiber had less belly pain than those who did not eat at least the recommended amount. In contrast, in girls with belly pain, those who ate more than the recommended amount of dietary fiber had more belly pain than the girls who ate less than the recommended amount. These novel findings highlight the importance of sex in response to dietary nutrients such as fiber and the need for additional studies to understand what factors underlie these sex differences.

2. A reliable measurement for cowpea consumption is possible. Legumes, particularly cowpeas, are indigenous crops in sub-Saharan Africa and contain larger quantities of protein than cereals but at present, there is no reliable, convenient way to determine the quantity of cowpea, to combat childhood malnutrition and growth faltering. This limits the ability of researchers to assess the effectiveness of activities that promote legume consumption as well as compliance to nutritional education efforts. Researchers associated with the Children's Nutrition Research Center in Houston, Texas, aimed to identify a suite of metabolic consumption biomarkers by collecting and analyzing urine and dried blood spots from children and pregnant women in Northern Ghana that consumed four cowpea varieties. Ten candidate biomarkers were identified for cowpea consumption which included phytochemicals unique to plants and other biomarkers that tell us the effect of cowpea in human metabolism and provides insight into how cowpeas enhance human nutrition. These results support a suite of key metabolites for dietary legume and cowpea-specific food exposure of global health importance and will permit a reliable measure of cowpea consumption in the future.