Project Summary/AbstractNecrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. Despiteadvances in neonatal practice its pathophysiology is poorly understood treatments are mainly supportive andno predictive test is available. In experimental NEC we found that bile acids (BAs) are a crucial component ofdisease pathogenesis. In infants preliminary data suggest that day-to-day fluctuations of total fecal BAs are abetter predictor of NEC than overall levels. In Specific Aim 1 using prospectively collected fecal specimensfrom multiple NICUs we will define the characteristics of BA levels and composition in premature infants thatdevelop NEC and in an independent cohort with blinded analyses test the predictive ability of BAs in NEC.Preliminary studies suggest infants that develop NEC are have extreme variability in fecal BA levels and pulsa-tile exposure to hydrophobic BAs is more damaging to intestinal epithelium than continuous exposure. In Spe-cific Aim 2 we will test the hypothesis that in neonatal intestinal epithelium pulsatile exposure to BAs is morecytotoxic than continuous exposure. Although bacteria have been suspected of playing a critical role in NECnone have been definitively linked to disease development. However gut bacteria play an important role inconverting 1o BAs to their more hydrophobicand cytotoxic2o forms through their ability to dehydroxylate ordeconjugate BAs. Preliminary data suggest bacteria capable of these transformations are more abundant infeces taken from infants that develop NEC compared to those that did not develop disease. In Specific Aim 3we will test the hypothesis that premature infants that develop NEC have increased bacteria capable of dehy-droxylating and deconjugating primary BAs resulting in increased BA hydrophobicity. In addition germ-freeneonatal mice will be populated with bacteria that can dehydroxylate or deconjugate BAs to test the hypothesisthat bacterial conversion of BAs to more cytotoxic forms is an initiator of NEC. Completion of these aims willelucidate the mechanisms of action of BAs in NEC and could lead to future development of the first predictivetest for this devastating disease.