Multi-omics analysis supports a gut-liver-urine framework and identifies urinary sulfated bile acids as biomarkers of parenteral nutrition-associated cholestasis

Parenteral nutrition-associated cholestasis (PNAC) is a major complication in very preterm infants receiving prolonged parenteral nutrition (PN) and is typically diagnosed only after conjugated hyperbilirubinemia develops. Noninvasive biomarkers reflecting disturbances in the gut-liver axis remain limited. This study aimed to identify biomarkers associated with PNAC using an integrated multi-omics approach. In this prospective cohort study, 81 very preterm infants (< 30 weeks’ gestation) receiving prolonged PN were enrolled, including 20 who developed cholestasis and 61 age- and time-matched controls. Urine and stool samples were collected during PN exposure, predominantly when PNAC occurred. Multi-omics profiling included bile acid quantification in urine and feces using ultra-performance liquid chromatography-tandem mass spectrometry, urinary metabolomics using proton nuclear magnetic resonance spectroscopy, and stool microbiome profiling using 16 S ribosomal RNA sequencing. Logistic regression and receiver operating characteristic analyses evaluated associations and discriminatory performance. Infants who developed cholestasis showed alterations across microbiome composition, fecal bile acids, urinary bile acids, and urinary metabolites. Urinary bile acids demonstrated the strongest discriminatory performance among all omics layers. Several urinary sulfated bile acids, including taurolithocholic acid-3-sulfate and taurochenodeoxycholic acid-3-sulfate, were strongly associated with disease risk. A composite score based on the top three urinary bile acids achieved an area under the receiver operating characteristic curve of 0.950, outperforming the corresponding urinary metabolites (0.894), fecal bile acids (0.734), and microbiome features (0.641). Cross-omics analysis revealed coordinated associations among gut microbial features, fecal bile acids, urinary bile acids, and urinary metabolites, supporting an integrated gut-liver-urine framework. Urinary sulfated bile acids represent robust biomarkers associated with PNAC in very preterm infants and may serve as a noninvasive test for PNAC during PN exposure.