To measure the protein digestibility and absorption by minimally invasive stable isotope-based methods and to assess the effect of amino acid based intervention on Environmental Enteric Dysfunction.

Environmental enteric dysfunction (EED) refers to an incompletely defined syndrome of inflammation, reduced absorptive capacity, and reduced barrier function in the small intestine. It is widespread among children and adults in low- and middle-income countries. Several factors such as environmental and nutritional deficiencies including chronic infections and other diseases can cause EED. With recent development on stable isotope-based technique to measure protein quality, it was observed that plant-based foods have low protein digestibility of indispensable amino acids (IAAs) in infants from low and middle-income countries (LMICs). This raises yet another important question about EED, which is a major contributor of stunting among these children and has been found to be associated with growth faltering. However, it is not clear if EED could contribute to low digestibility of plant protein in this population. Therefore, there is a need to study and test the applicability of combined minimally invasive stable isotope-based protocols for the protein digestion and absorption in infants and assess the effect of amino acids-based intervention on EED. We aim to study the use of dual stable isotope test (DSIT) for the digestion and absorption of amino acids of mung bean protein in 18-36 months infants (with growth faltering) and to evaluate the use of 13C-Sucrose Breath Test (13C-SBT) to assess nutrient absorption as an indicator of intestinal function in the context of EED. Finally, we also aim to use an amino acid-based intervention on the recommended nutrient intake for this age group, to assess its role in ameliorating EED, and to generate new data on the complex interplay of protein metabolism with EED. This study is a multicentric randomized control trial for a Coordinated Research Project (CRP) titled ‘The Efficacy of Amino Acid Supplementation in Treating Environmental Enteric Dysfunction Among Children at Risk of Malnutrition’ including participants from Ghana, India, Malawi, Morocco, Peru, Zambia, and Philippines. Each study site will contribute data from 60 children, for a total of 420 children. The intervention will be provided in coded sachets, distributed as per convenient for local custom to best ensure compliance with a minimum of three supervised feeds per week to ensure supplement is consumed at least periodically by the child and to assess compliance. Protocol treatment will start within two weeks of enrollment to the study. Following the baseline study assessments, each participant will receive the standard complementary foods (In the control group; n=30), or standard complementary foods supplemented with IAAs (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, in the intervention group; n=30). The IAA dose to be consumed daily provides x1.5 the EAR for each IAA for children 18-36 months old, based on the FAO requirements.For each participant, tests to be conducted at study enrollment will be spread over two days. Similarly, at the end of the study (day 28), as summarized below. Height & Weight-The first anthropometric measurement will be taken at the screening visit (study day 0), because the child’s weight is needed to calculate the dosage for the 13C-SBT and DSIT tests on days 1 and 2. The second anthropometric assessment will be made on study day 32 (after the intervention period). Questionnaires- Questionnaires will be used to collect demographic and socio-economic information. In addition, questionnaires will be used to assess basic dietary data (dietary diversity). 13C-SBT-On study day, 13C-sucrose breath test will be used to assess intestinal sucrase activity following previously published protocols. Briefly, at the start of the study period (time=0), 13C12-sucrose (0.4mg/kg) will be given with 100mL water followed by breath sample collection into an Exetainer tube every 20mins over 240mins (4hrs). DSIT-First, to complete the DSIT, a single blood sample is collected before the start of the study and the time is noted. A mini meal consisting of a local complementary food will be prepared with a cereal/legume blend containing no milk. Immediately prior to the DSIT, the mini meal for the child will be split into 9 portions (8 to be consumed by the child and 1 to be kept for laboratory analysis). The dual stable isotope tracer mixes of U-13C spirulina (10mg/kg) and 2H-AA mix (1.25mg/kg) will be added separately to each portion. The 9th mini meal potion will be kept for analysis. The sample will be frozen to -80°C, lyophilized, and then ground to fine flour and stored for further analysis. Before the start of the study period (immediately before time=0), two breath samples will be collected about 15min apart (time of collection noted). At the start of the study period (time=0), NaH13CO3 (3?mol/kg) will be added to the prime portion i.e., the first 3 mini meal portions (portions 1-3) together. Subsequently, each mini-meal 4-8 is given at hourly intervals for up to 5hrs. Three further blood samples will be collected at 5, 5½ and 6hrs after the commencement of the study (if possible), or a single blood sample collected at 5hrs will be used. Throughout the DSIT, a breath sample will be collected into an Exetainer tube every 20mins over 360mins (6hrs). L/R-On the study day, the lactulose-rhamnose test (L/R) will be alongside the DSIT test. A single urine sample is collected before the start of the study and the time noted. At the start of the study period (time=0), lactulose (1g) and rhamnose (0.2g) will be given with 100mL water. All urine passed up to 5hrs is collected and pooled and the total volume will be noted. Stool samples–On study day 0 (baseline) and 28 (endline), participant caretakers will be asked to provide a stool sample from their children for the assessment of intestinal inflammation (myeloperoxidase and neopterin). Stool samples will be transported at 2-8°C and immediately stored (-80°C). Blood samples-On study days 0 (baseline) and 28 (endline), venous blood will be collected and immediately centrifuged and stored (-80°C) for the determination of plasma markers of intestinal function (LPSBP and iFABP). Breath 13CO2 will be analyzed by isotope ratio mass spectrometry (IRMS). Urine samples (2mL) are stored (-20°C) for analysis of lactulose:rhamnose ratio (L:R) and glycylsarcosine excretion by GCMS. Blood plasma will be collected and aliquoted for assessment of protein digestive capacity (0.5mL; 13C AA’s, 13C9/13C6 Phe) and inflammatory markers (0.1mL; LPSBP, Myo, Neo, IFABP). In addition, urinary metabolomics will also be performed to assess the effect of intervention on EED (only in Indian site).