Lyophilized fecal short-chain fatty acid and electrolyte determination by capillary electrophoresis with indirect UV detection for assessment of pediatric inflammatory bowel disease

Abstract

Short-chain fatty acids (SCFAs) and electrolytes are major constituents of human feces involved in maintaining gastrointestinal homeostasis that underlie complex diet, host and microbiome interactions. Reliable quantification of SCFAs and electrolytes is challenging given the heterogeneity of stool specimens from pediatric patients with diarrhea-predominate inflammatory bowel disease (IBD). Herein, we introduce two validated methods for determination of 3 SCFAs and 5 electrolytes consistently quantified from fecal extracts when using capillary electrophoresis with indirect UV detection (CE-iUV), where concentrations are normalized to total dried weight (mmol/kg d.w.). Lyophilization facilitates sample handling and extraction of heterogeneous stool specimens (∼ 15 mg) from a cohort of children with Crohn’s disease (CD, n = 12) and ulcerative colitis (UC, n = 10) treated with exclusive enteral nutrition (EEN) or corticosteroid (CS) therapy to induce remission, respectively. Good technical precision (mean CV = 13 %, n = 14) and accuracy (recovery from 84 to 116%) is demonstrated for SCFAs and electrolytes from freeze dried stool extracts using a modified Bligh-Dyer protocol with low micromolar detection limits (∼ 2–15 μM). Fecal butyrate is 2.6-fold higher in CD as compared to UC patients (effect size = 1.51; p = 0.00291), and there is a strong co-linearity between fecal butyrate and acetate (r = 0.835) unlike propionate, which is correlated with fecal calprotectin (r = 0.517), a protein biomarker of intestinal inflammation. Also, a longitudinal study of matching stool samples collected from a sub-set of IBD patients revealed about a 7-fold enrichment in magnesium and calcium following 4 weeks of EEN as compared to baseline (F > 4.1 ; p < 0.05) unlike the CS treatment arm with no changes in other fecal SCFAs and electrolytes, including sodium, potassium, and ammonium. CE-iUV enables rapid fecal SCFA and electrolyte determination as required for new insights into the role of gut dysbiosis in IBD, as well as treatment monitoring of nutritional interventions that stabilize the disease course in affected children.