Diminished Citrulline‐Arginine‐Nitric Oxide Production Rates are Associated with Necrotizing Enterocolitis Incidence in Premature Pigs

Abstract

Necrotizing enterocolitis (NEC) is a major gastrointestinal disease in premature infants that is associated with formula feeding and intestinal hypoxia. Low arginine availability in these infants has been linked to NEC since arginine is the sole precursor of nitric oxide (NO), a critical mediator of vasodilation, blood flow and tissue oxygenation. Arginine availability is dependent on its de novo production from gut‐derived citrulline and the action of the rate‐limiting enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) in the gut and other tissues. However, arginine availability is balanced by arginine catabolism to either NO by NO synthase 2&3 (NOS2&3) or to ornithine by arginase 1&2 (Arg1&2). We hypothesized that citrulline‐arginine‐NO production rates are limiting in premature pigs, which predisposes them to NEC. Cesarean‐derived preterm (90% term) and term (98–100% term) pigs were provided TPN for 24 h, followed by enteral formula for 48 h to induce NEC. Intravenous infusions of [13C6]arginine, [15N‐ureido]citrulline and [15N18O3]nitrate were provided throughout feeding to quantify in vivo citrulline‐arginine‐NO kinetics. Preterm and term pig tissues were also collected at birth, and we report that intestinal expression of the arginine producing genes ASS and ASL were lower in preterm vs term pigs (P<0.05). However, the expression of arginine catabolizing genes NOS2&3 and Arg1&2 in gut were not affected by gestational age at birth. Intestinal gene expression was further assessed after formula feeding, and Arg2 and NOS3 expression were increased in preterm vs term pigs, while ASS expression remained low in preterm pigs after feeding (P<0.05). Histological scoring of fed pigs showed that NEC incidence was 90% in preterm pigs, whereas term pigs did not get NEC. Importantly, the rates of arginine‐citrulline‐NO flux showed that preterm pigs produced ~40–50% less citrulline, arginine and NO than term pigs regardless of feeding mode (P<0.05). To better assess NO metabolism we also quantified its oxidation products, nitrate and nitrite. Nitrate flux was ~60% lower in preterm pigs during TPN (P<0.05), and all pigs exhibited a drastic reduction in nitrate flux after the introduction of enteral formula. Strikingly, there was a marked supraphysiological nitrite surge in plasma prior to and immediately after oral feed initiation in preterm piglets that was absent in term pigs. Moreover, preterm pigs with higher post‐feeding plasma nitrite surges were NEC‐resistant (P<0.05). Our results suggest that depressed intestinal blood flow and oxygenation that are implicated in NEC may be linked to the diminished capacity to produce citrulline‐arginine‐NO necessary for vasodilation. These findings provide a rationale for arginine supplementation of preterm infants to overcome the limited capacity for arginine and NO production that predisposes NEC etiology.Support or Funding InformationGrant Funding Source: USDA/ARS