Enteral and intravenous supplementation of arginine and citrulline fail to prevent necrotizing enterocolitis in preterm neonatal pigs

Authors: VONDEROHE, CAITLIN - Children'S Nutrition Research Center (CNRC); MANCEBO, J - Baylor College Of Medicine; MELENDEZ HEBIB, VALERIA - Children'S Nutrition Research Center (CNRC); STOLL, BARBARA - Children'S Nutrition Research Center (CNRC); DIDELIJA, INKA - Children'S Nutrition Research Center (CNRC); ELEFSON, SARAH - Children'S Nutrition Research Center (CNRC); MOHAMMAD, M - Children'S Nutrition Research Center (CNRC); EARLS, B - Baylor College Of Medicine; GUTHRIE, GREG - Children'S Nutrition Research Center (CNRC); Burrin, Douglas

Submitted to: Journal of Parenteral and Enteral Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2025
Publication Date: 2/1/2026
Citation: Vonderohe, C., Mancebo, J.G., Melendez Hebib, V., Stoll, B., Didelija, I., Elefson, S., Mohammad, M., Earls, B., Guthrie, G., Burrin, D.G. 2026. Enteral and intravenous supplementation of arginine and citrulline fail to prevent necrotizing enterocolitis in preterm neonatal pigs. Journal of Parenteral and Enteral Nutrition. 50(2):252-260. https://doi.org/10.1002/jpen.70031.
DOI: https://doi.org/10.1002/jpen.70031

Interpretive Summary: Necrotizing enterocolitis (NEC) is one of the most devastating diseases that can affect preterm babies. Unfortunately, there are limited treatment and prevention strategies for NEC, and many of these strategies do not work very well or are limited in availability. Therefore we decided to try a novel approach, adding the amino acids arginine and citrulline to the diets of preterm piglet models of preterm babies. Arginine has been studied closely as a strategy to improve recovery in patients with critical illness because it can be broken down into a molecule, nitric oxide, that is involved in the immune system and maintaining blood flow. Unfortunately, arginine is broken down very quickly after being fed or infused. Citrulline can be transformed into arginine, and therefore nitic oxide, but is not broken down as quickly as arginine. We not only added citrulline and arginine to the diet, but also provided it intravenously, to preterm pigs, and measured how many pigs developed NEC. In this case, we found that giving any kind of citrulline or arginine did not effectively prevent NEC. This strategy was so ineffective that we believe that these amino acids are not being broken down into nitric oxide, but another molecule that may be making the preterm pigs more likely to develop NEC.

Technical Abstract: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants with a morality rate that approaches 50%. Arginine has been widely studied in the field of clinical nutrition as a supplement for patients experiencing critical illness because it can be metabolized into nitric oxide (NO), an important agent for supporting immunity and microcirculation. Citrulline has received less attention but can be metabolized into arginine and has a much longer plasma half-life than arginine. We used the highly translational preterm pig model to determine the effect of intravenous and enteral supplementation of arginine and citrulline on NEC incidence in preterm neonates. A total of 67 pigs were delivered by cesarean section on day 105/115 (analogous to 30 weeks gestation in humans) and allocated to 6 treatments: preterm infant formula (CTL), donor human milk (DHM), formula with arginine (OG ARG) or citrulline (OG CIT), and formula with intravenous arginine (IV ARG) or citrulline (IV CIT). Pigs were monitored for clinical signs associated with NEC and tissue was collected for later analysis. NEC diagnosis and severity was quantified using previously validated gross and histologic scales. Enteral and intravenous supplementation of arginine and citrulline did not impact NEC incidence or severity. NEC incidence tended (P<0.07) to be lower in the DHM pigs. NEC incidence was highest in the CTL (60%), IV ARG (64%), and OG CIT (62.5%) pigs. Citrulline and arginine supplementation are not feasible or safe nutritional strategies to prevent NEC in preterm neonates.