Location: Children's Nutrition Research Center
2023 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, we continued to investigate the relationships between the bacteria in our gut (digestive or gastrointestinal tract) and diet. Diet can greatly influence the types and numbers of bacteria in the gut. Similarly, the types and numbers of bacteria in the gut can influence health by either increasing or reducing the amount of inflammation in the body. Low grade inflammation has been shown to contribute to such conditions as heart disease. Our work this year has shed more light on how gut bacteria can change the types of hormones in the body by metabolizing hormones in different ways. We have been using an advanced technique called whole shotgun sequencing to identify specific types of bacteria. Traditional methods can only identify bacteria that belong to larger groups (for example, identifying a crowd of people as opposed to an individual in a crowd). In addition, we use an advanced technique called metabolomics that allows us to measure products produced by the bacteria. These products are called metabolites.
It is known that children who have unexplained belly pain are more likely to be girls. Unexplained belly pain affects 10-15% of school-age children around the world and the same proportion of adults as well. In adults, those affected with belly pain also are more likely to be women. There is some evidence that girls and women may be more sensitive to painful stimuli than males. Many children with belly pain appear to be sensitive to certain types of carbohydrates (sugars) in their diet. On the other hand, certain dietary fibers appear to be able to prevent belly pain. Overall, we know that children and adults in the United States do not eat enough fiber in general.
Given that females appear more sensitive to painful stimuli and are more likely to have unexplained belly pain, we investigated if the types of gut bacteria and the metabolites they produce could be responsible. Importantly, we wanted to determine if these factors could result in boys responding differently from girls to fiber in their diet.
We analyzed stool samples for bacteria and metabolites, diet records (what the children had been eating), and diaries recording if the child had been having belly pain and if so, how much. The records came from three studies. In one study children had been given either extra dietary fiber or a placebo for six weeks. In the other two studies we followed children for two weeks as they ate a regular diet.
We discovered that when extra fiber was provided, only the boys had less pain; the girls' belly pain did not change. Similarly, when the children on their own ate the recommended amount of fiber, the boys had less pain. There was evidence that the girls had even more pain. Analysis of the gut bacteria identified a specific bacteria that was increased by dietary fiber in the boys but not the girls. This bacteria has been associated with the production of a metabolite that can reduce inflammation. We are analyzing the metabolites but there is some indication that the metabolites may differ as well.
Objective 3 was accomplished with two distinct clinical studies. Objective 3A compared four supplementary foods of differing carbohydrate and protein compositions in the treatment of moderate acute malnutrition (MAM) and their effects on gut permeability and intestinal microbiome. These studies provided novel data as to how dairy and vegetable ingredients affect intestinal permeability, the fecal microbiome configuration, and the fecal metabolome in young children.
Environmental enteric dysfunction (EED) is prevalent in malnourished children and is characterized by increased permeability, chronic inflammation. Our goal was to see if milk protein and/or carbohydrate will improve intestinal permeability. A high precision piece of equipment called the high-performance liquid chromatography was used to quantify urine lactulose (sugar) content. We found that there were no differences in the percentage of lactulose excretion after four weeks of receiving different supplementary foods and 75% of children had abnormal (greater than 0.2%) lactulose excretion after four weeks of supplementary feeding. Thus, none of the diets helped to heal the leaky guts.
Researchers also evaluated fecal microbiome composition since it is altered in cases of severe acute malnutrition and there is some evidence that specific gut bacteria can help children recover from malnutrition. Our goal was to see if milk protein and/or carbohydrate will repair microbiome composition in cases where there is an imbalance in the microbiota. Specialized (16s rRNA) analyses were done using multiamplicon variable regional DNA sequencing. We found that there were no significant differences after 4 weeks of supplementary feeding. The foods did not alter the bacteria in the stool.
Further, we conducted a study looking at the fecal metabolome, which are all the metabolites present in the cells, since diets can alter the metabolic profile of the microbiome. We aimed to determine if milk protein and/or sugar would alter the fecal metabolic profile, activating beneficial pathways for weight gain. Fecal samples were run with a liquid chromatography–mass spectrometry (mass spec) using different columns chosen to collect data on the full range of polarity and chemical configuration. A peak detected by mass spec is called a feature. There may be multiple features for each metabolite and multiple metabolites may contribute to the same feature. Data from our analyses revealed 10,579 features, reduction of this set of features by median absolute deviation left 2,888 unique features. Peaks associated with each feature were refined using quality control-based signal correction to remove batch effect (dose of metabolites in samples) and correct signal drift. This method has been widely used in untargeted metabolomics. Comparisons were made between children consuming milk protein vs. soy protein and whey permeate (lactose) vs. soy carbohydrates using parametric statistical tests. Since milk powder is clinically superior, it was compared to soy. Sixteen features were present in greater abundance in children consuming milk protein. These were ceramides (sphingolipids), amino acids and oligopeptides and keto acids. 106 features were present in greater abundance among children consuming soy. Most of these features were flavones, particularly isoflavones. Flavonoids have antimicrobial activity, perhaps important to plant immunity. Twenty-two features among lactose consumers showed greater amounts of nucleosides, dipeptides, amides and amines - all basic biological building blocks. This suggests that lactose is used by microbes to synthesize a variety of necessary nutrients.
From these studies in Sub-objective 3A we can conclude that: (1) ready-to-use supplementary food (RUSF) with milk protein and milk carbohydrate led to greater weight gain than RUSF made with vegetable protein and vegetable carbohydrate; (2) there were no differences in intestinal permeability between food groups and this is likely not the avenue by which milk is beneficial, 75% of children continued to have pathologically elevated intestinal permeability despite 4 weeks of supplementary feeding with high-quality foods; (3) there were no differences in the fecal microbial taxonomy as determined by 16S rRNA sequencing between groups (beta-diversity), nor evidence for differences in within-sample richness (alpha-diversity); and (4) metabolomic analysis revealed subtle, but not substantial differences in metabolic profile between food groups. Dramatically different diets result in very similar fecal microbial and metabolomic compositions.
For Sub-objective 3B a urinary test for cowpea consumption was sought with untargeted metabolomics, a method which detects and measures all organic molecules present in urine. A group of 20 pregnant women and 20 children were fed prescribed amounts of cowpea for 15 days. A combination of metabolites in urine accurately measures how much cowpea was consumed. The biomarker is 3 microbial metabolites, 3 lipids, 3 phytochemicals and 1 amino acid. Four local varieties of cowpea were used in this study.
Accomplishments
1. Boys and girls respond differently to dietary fiber (carbohydrate). Fiber in the diet is beneficial yet Americans eat much less than the recommended amount. Whether fiber is beneficial to specific individuals is less clear. To investigate whether boys and girls respond differently to fiber in the diet, researchers at the Children’s Nutrition Research Center in Houston, Texas, studied the relationship between fiber intake and the development of belly pain. They discovered that increasing fiber intake led to less pain in school-age boys but not girls. Fiber intake in boys increased the presence of a gut bacteria associated with less inflammation but did not in girls. These results shed light on how fiber can be beneficial in boys with belly pain and also highlight the great importance of examining potential differences between the sexes when studying the relationships between diet, the gut bacteria, and health.
