Bile Acid Profiling Reveals Distinct Signatures in Undernourished Children with Environmental Enteric Dysfunction

Xueheng Zhao, Kenneth D R Setchell, Rong Huang, Indika Mallawaarachchi, Lubaina Ehsan, Edward Dobrzykowski Iii, Junfang Zhao, Sana Syed, Jennie Z Ma, Najeeha T Iqbal, Junaid Iqbal, Kamran Sadiq, Sheraz Ahmed, Yael Haberman, Lee A Denson, Syed Asad Ali, Sean R Moore, Xueheng Zhao, Kenneth D R Setchell, Rong Huang, Indika Mallawaarachchi, Lubaina Ehsan, Edward Dobrzykowski Iii, Junfang Zhao, Sana Syed, Jennie Z Ma, Najeeha T Iqbal, Junaid Iqbal, Kamran Sadiq, Sheraz Ahmed, Yael Haberman, Lee A Denson, Syed Asad Ali, Sean R Moore

Abstract

Background: Intestinal inflammation and malabsorption in environmental enteric dysfunction (EED) are associated with early childhood growth faltering in impoverished settings worldwide.

Objectives: The goal of this study was to identify candidate biomarkers associated with inflammation, EED histology, and as predictors of later growth outcomes by focusing on the liver-gut axis by investigating the bile acid metabolome.

Methods: Undernourished rural Pakistani infants (n = 365) with weight-for-height Z score (WHZ) < -2 were followed up to the age of 24 mo and monitored for growth, infections, and EED. Well-nourished local children (n = 51) were controls, based on consistent WHZ > 0 and height-for-age Z score (HAZ) > -1 on 2 consecutive visits at 3 and 6 mo. Serum bile acid (sBA) profiles were measured by tandem MS at the ages of 3-6 and 9 mo and before nutritional intervention. Biopsies and duodenal aspirates were obtained following upper gastrointestinal endoscopy from a subset of children (n = 63) that responded poorly to nutritional intervention. BA composition in paired plasma and duodenal aspirates was compared based on the severity of EED histopathological scores and correlated to clinical and growth outcomes.

Results: Remarkably, >70% of undernourished Pakistani infants displayed elevated sBA concentrations consistent with subclinical cholestasis. Serum glycocholic acid (GCA) correlated with linear growth faltering (HAZ, r = -0.252 and -0.295 at the age of 3-6 and 9 mo, respectively, P <0.001) and biomarkers of inflammation. The proportion of GCA positively correlated with EED severity for both plasma (rs = 0.324 P = 0.02) and duodenal aspirates (rs = 0.307 P = 0.06) in children with refractory wasting that underwent endoscopy, and the proportion of secondary BA was low in both undernourished and EED children.

Conclusions: Dysregulated bile acid metabolism is associated with growth faltering and EED severity in undernourished children. Restoration of intestinal BA homeostasis may offer a novel therapeutic target for undernutrition in children with EED. This trial was registered at clinicaltrials.gov as NCT03588013.

Keywords: UHPLC-MS/MS; bile acid; biomarkers; endoscopy; environmental enteric dysfunction (EED); stunting; undernutrition.

© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.

Figures

FIGURE 1
FIGURE 1
Serum BA profiles in Pakistani undernourished and healthy children aged between 3 and 6 mo, and 9 mo. (A–B) Serum BA profile in Pakistani undernourished children and healthy controls aged 3–6 (n = 339 undernourished and 50 healthy controls) and 9 (n = 295 undernourished and 47 healthy controls) mo. On the left, total BA profiles were plotted (Dashed line indicates 8 μM an upper limit of normal range), and on the right 7 BA subspecies (out of 15 quantified BA) with >1% of the total BA pool plotted. *P <0.05, **P <0.01, ***P <0.001. (C) Scheme of biosynthesis pathway and metabolic transformations of BA in humans. BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; G, glycine; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GUDCA, glycoursodeoxycholic acid; LCA, lithocholic acid; T, taurine; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; UDCA, ursodeoxycholic acid.
FIGURE 2
FIGURE 2
Association of BA subspecies and correlation to cytokines of Pakistani undernourished and healthy children aged between 3 and 6 mo, and 9 mo. (A–B) Correlation network analysis of serum BA subspecies concentration at the age of 3–6 mo (n = 389), and 9 mo (n = 342). GLCA and TLCA were not included in the 3–6 mo plot due to their extremely low concentration in the sample. (C) Correlation of GCA (%) in serum and proinflammatory cytokines (n = 384 and 339 aged 3–6 mo and 9 mo, respectively). (D) Conditional random forest (CRF) model identified the top 5 important BA in predicting height-for-age Z score at 24 mo (linear growth) using the 9 mo data set consisted of 277 Pakistani children. BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; GLCA, glycolithocholic acid; GUDCA, glycoursodeoxycholic acid; LCA, lithocholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; TLCA, taurolithocholic acid; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.
FIGURE 3
FIGURE 3
Histopathological analysis of EED children and association to plasma and duodenal aspirate BA composition. (A) Representative hematoxylin and eosin staining (H&E) duodenal histology from an undernourished EED child. Arrowhead indicates villous blunting, short arrow indicates Paneth cell, and long arrow indicates intramucosal Brunner gland hyperplasia. (B) Correlation of percent secondary BA and GCA in plasma and duodenal aspirates (n = 63 EED children) with histopathological EED scores. (C) BA profile associates with clinical investigation blood test results in Pakistani EED children. Pearson correlation of GCA and secondary BA (%) in duodenal aspirate (n = 39) and plasma (n = 55) to clinical investigation lab results of Pakistani EED children. *P <0.05. BA, bile acid; EED, environmental enteric dysfunction; GCA, glycocholic acid; MCH, mean corpuscular hemoglobin concentration; RBC, red blood cell count; WBC, white blood cell count.
FIGURE 4
FIGURE 4
Correlation of plasma and duodenal aspirate GCA and secondary BA and their potential role as a disease-relevant signature in EED. (A) Correlation of percent GCA and secondary BAs in plasma and duodenal aspirates of EED children (n = 35). (B) Schematic illustration of BA signatures in undernourished EED children. BA, bile acid; EED, environmental enteric dysfunction; GCA, glycocholic acid.

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