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Abstract INTRODUCTION:
Environmental enteropathy (EE) is a widespread inflammatory condition effecting the gastrointestinal tract. It is characterised by morphological changes to the gut barrier, which impairs absorption, as well as reduced levels of various cell types (e.g., goblet cells, Paneth cells, and lymphocytes).[1] The presence of EE is linked to undernutrition and in children leads to stunted growth, diminished cognitive development, and reduced vaccine response. It is estimated that chronic undernutrition is a contributory cause in at least one-third of childhood mortality (< 5 years) in low- and middle-income countries (LMICs).[2] Inflammation induced by environmental or infectious challenges are associated with up-regulation of inducible nitric oxide synthase (iNOS) and increased nitric oxide (NO) release. NO acts as an anti-inflammatory signalling molecule with over-expression at pro-inflammatory epithelial surfaces, regulating the activity of important immune cells, such as macrophages and lymphocytes.[3] NO production often correlates with increased levels of pro-inflammatory cytokines, e.g., TNF-α and IL-6, indicating broad inflammatory stimulus,[4] although localising tissue specific NO production, particularly in the gut, remains elusive. NO production is driven by sequential oxidisation of L-arginine by iNOS to form L-citrulline and NO. NO is highly lipid soluble and is readily absorbed by tissues. With a relatively short half-life, this molecule acts in a paracrine fashion.[5] NO is oxidised to nitrite (NO2-), and ultimately through to the stable form nitrate (NO3-). Ultimately the kidneys filter and excrete NO3- into the urine. In this study, we used an oral 15N2-arginine bolus with a short time course (3 h) urine collection to interrogate proximal small intestinal NO production as a proxy for gut inflammation in EE. The excreted 15N-labelled nitrate is used as an indirect measure of 15NO production from 15N2-arginine.
OBJECTIVES:
The study has three primary objectives: 1) Use a 15N2-arginine tracer to monitor 15NO production in the small intestine. 2) Develop a novel Orbitrap Exploris™ Isotope Solution (OEIS) mass spectrometry method for analysis of NO3- isotopologues in human urine from an EE cohort. 3) Determine the relationship between urinary 15NO3- with markers of gut dysfunction, damage and inflammation in EE.
METHODS:
Samples were collected as part of the GI Tools study from a cohort of 80 Zambian adults with EE,[6] who underwent an array of biomarker assessments, including intestinal fatty acid binding protein (iFABP) and lactulose/rhamnose ratio (LRR), and intestinal biopsy to assess gut barrier morphometry and extent of epithelial damage associated with EE. Baseline urine was collected prior to oral administration of 15N2-(guanidineimino)-arginine (fixed dose: 1.667 mg). A subsequent urine sample was collected at 180 mins. Urines were diluted (1:100) in ACN:MeOH (75:25, v/v) + 0.2% formic acid. Samples were analysed by in-flow injection on a ThermoFisher Scientific Vanquish Neo HPLC coupled to an Orbitrap Exploris™ 480 Isotope Solutions mass spectrometer system with 20 min acquisition period for NO3- isotopologues (61-65 m/z) in negative ion mode (RF Lens (%): 70; Normalised AGC Target (%): 70; microscans: 5). Each sample injection was bracketed by an isotopically calibrated NO3 standard (USGS35) to produce δ15N-values relative to the internationally accepted reference scale for 15N, δ15AIR = 0 per mil (‰). Δ15NO3-, defined as 180 min δ15NO3- – baseline δ15NO3-, was calculated and used to show enrichment of nitrate in the urine.
RESULTS:
15NO3- enrichment was measured in 37 participants (mean = 86.2‰, SD = 71.5‰) indicating sufficient transfer of tracer from 15N2-arginine to urinary 15NO3- for OEIS analysis. The data were correlated with measurements relating to gut barrier integrity and inflammation, both morphometry and plasma markers. Urinary δ15NO3- enrichment showed significant positive correlation with iFABP (r = 0.44; p = 0.007) and negative correlation with epithelial surface area (ESA) (r = -0.42; p = 0.019).
CONCLUSION/DISCUSSION:
This simple, non-invasive test demonstrates the potential of urinary 15NO3- as a marker for localised inflammation in the proximal small intestine when 15N2-arginine is administered. Further work is ongoing to collect urinary 15NO3 data from a high socioeconomic status Zambian comparator group. The results of this additional analysis will give greater insight into the utility of urinary 15NO3- to assess gut health in environmental enteropathy. OEIS presents a simple to use sample workflow and instrumental platform to analyse urinary 15NO3- enrichment from low-level tracer studies.
REFERENCES:
1. Kummerlowe, C., et al., Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation. Sci Transl Med, 2022. 14(660): p. eabi8633.
2. Prendergast, A. and P. Kelly, Enteropathies in the developing world: neglected effects on global health. Am J Trop Med Hyg, 2012. 86(5): p. 756-63.
3. Iwata, M., et al., The protective role of localized nitric oxide production during inflammation may be mediated by the heme oxygenase-1/carbon monoxide pathway. Biochem Biophys Rep, 2020. 23: p. 100790.
4. Soufli, I., et al., Overview of cytokines and nitric oxide involvement in immuno-pathogenesis of inflammatory bowel diseases. World J Gastrointest Pharmacol Ther, 2016. 7(3): p. 353-60.
5. Yang, T., A.N. Zelikin, and R. Chandrawati, Progress and Promise of Nitric Oxide-Releasing Platforms. Adv Sci (Weinh), 2018. 5(6): p. 1701043.
6. Phiri, T.N., et al., Novel gastrointestinal tools (GI Tools) for evaluating gut functional capacity in adults with environmental enteropathy in Zambia and Zimbabwe: A cross-sectional study protocol. F1000Research, 2025. 13.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
