Arginine supplementation in prevention of necrotizing enterocolitis in the premature infant: an updated systematic review

Necrotizing enterocolitis (NEC) is the most common acquired gastrointestinal emergency in premature infants. It is characterized by ischemic necrosis of the intestinal mucosa, inflammation, invasion of enteric gas-forming organisms, and dissection of gas into the muscularis and portal venous system [1]. NEC occurs in 1–3 per 1000 live births and 1–7.7% of admissions to neonatal intensive care units (NICU) [2]. The mortality of NEC varies based on the birth weight of the affected infant and the NEC Stage (I, II, III) and ranges from 20-30%, with the greatest mortality among infants requiring surgical intervention [3, 4]. The pathogenesis of NEC remains elusive; however, it is likely the result of a multifactorial process in a susceptible host. Of particular interest is the role played by intestinal vascular resistance in the development of NEC [5‐7]. Hypoxic-ischemic injury is thought to play a significant role [8]. Mesenteric blood flow in neonates may decline in the presence of extreme hypoxia and severe abdominal distension [9, 10]. The resulting increased mesenteric vascular resistance can lead to reduced intestinal oxygen extraction and subsequent mesenteric acidemia [9]. Mucosal injury is seen initially, which may result in mucosal necrosis with ulceration and tissue sloughing [8]. Reperfusion-induced tissue damage after a hypoxic-ischemic event can produce ongoing injury to the intestinal mucosa via cytotoxic vascular endothelial cell damage and cytotoxic effects on cells of oxygen free radicals [8, 11‐15]. NEC is a complex and multifactorial disease. Various clinical studies revealed that inflammatory mediators especially TNFα, IL-1, platelet activating factor, and nitric oxide (NO), produced by enterocytes and macrophages may play a role in the pathogenesis of NEC [16].

Nitric oxide (NO) plays an important role in maintaining baseline vasodilator tone [17]. It is the principal inhibitory neurotransmitter in the gastrointestinal system inducing gut smooth muscle relaxation, and helps regulate mucosal blood flow, maintenance of mucosal integrity, and intestinal barrier function [18‐20]. A number of animal model studies of bowel injury have demonstrated that inhibition of NO increases the area of intestinal damage [5, 20‐24]. NO is synthesized from the amino acid L-arginine by NO synthases (NOS) [17, 25]. Continuous intravenous infusion with L-arginine markedly reduced intestinal injury in a neonatal pig model of NEC [26]. Plasma arginine concentrations are decreased in premature infants with NEC [27, 28].

A Cochrane review of the role of L-arginine based on one study showed a reduction of NEC in premature neonates [29]. However, due to the small number of neonates in that study and without further evidence from other RCTs, the role of prophylactic L-arginine did not become a common practice in modern NICUs [30]. There is one more study published since the previous review [31]. The primary objective of this systematic review was to use all available data, including those from recently published randomized trials, to evaluate the effectiveness of supplemental L-arginine versus placebo in the prevention of necrotizing enterocolitis in preterm infants.

The search strategy of the Cochrane Neonatal Review Group was used. The systematic review reporting follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [32].

Twenty-one studies were identified as being potentially relevant to this systematic review (Figure 1). Seventeen studies were excluded as they did not meet all the inclusion criteria. Two of the remaining four studies were systematic reviews by the same author but published in different versions of the Cochrane library based on one randomized, controlled trial without any revision. The remaining two studies were analyzed and data were compiled (Table 1).

The analysis of this updated systematic review of the RCTs conducted in premature infants receiving L-arginine as a prophylactic agent for prevention of NEC showed a statistically significant reduction in the incidence of stage II and III NEC (P = 0.02) (NNT = 9) and all stages of NEC (P = 0.001) (NNT = 5) in preterm infants supplemented with L-arginine compared to those in the placebo group. Given the NNT for both stage II and III NEC and all stages of NEC, this certainly makes the prospect of L-arginine having a role in the routine care of premature neonates an interesting one. In addition, with the absence of significant side effects and a reduction in both medical and surgical NEC, L-arginine may have a prominent role in upcoming years.

Our results demonstrate a statistically significant reduction in the incidence of stage II and III NEC. This was initially suggested by Amin et al.; however, did not reach statistical significance (P = 0.077) [30]. The results from Polycarpou et al. were also non-significant [31]. The significant P-value that was found in our study is likely in large part due to the increased proportion of cases of stage III NEC in Polycarpou et al. compared to Amin et al. and the larger sample size achieved from pooling the studies. As such, our results support Amin et al.’s previous findings that were suggestive of a reduction in the incidence of NEC [30]. Polycarpou et al.’s study did not show statistical significance for all stages of NEC, but did note a statistically significant reduction in the incidence of stage III NEC [31].

There were no statistically significant differences between both groups with respect to secondary outcomes (Table 3). Regarding IVH, Polycarpou et al. did not exclude neonates with IVH grades III and IV, as they were in Amin et al.’s study [31, 30]. As such, a prominent difference was noted in the proportion of infants with grades III and IV IVH when data were compared: 4% of Amin et al.’s total sample vs. 25% of Polycarpou et al.’s total sample [30, 31]. When the data were combined and compared against placebo, a statistically significant difference was not found (P = 0.64).

We demonstrate in our systematic review that L-arginine supplemented infants in one RCT for prevention of NEC did not have any difference in the long term neurodevelopmental outcomes at the age of 36 months of corrected age compared with those who received placebo [34].

NO plays a key role in intestinal epithelial injury in NEC. Ford and his co-investigators have established the role of iNOS-derived NO in NEC and also found an upregulation of iNOS mRNA and protein in infants undergoing laparotomy for NEC, as compared to infants those were undergoing for resection of intestine due to other reasons [6]. NO is an endothelial-derived relaxing factor – a potent, short-lived vasodilator. NO also modulates various physiological processes including tissue homeostasis, neurotransmission, and inflammation. Nitric oxide is a product of NO synthase (NOS) which converts arginine and oxygen into NO and citrulline. There are three isomers of NOS and each coded by different genes. Endothelial NOS (eNOS) and neuronal NOS (nNOS) isoforms are expressed at low levels and these enzymes produce a small amount of NO. Both of these isoforms are activated by calmodulin. The third isoform is calcium-independent and is known as inducible NOS (iNOS) and binds to calmodulin with a very good affinity. iNOS isoforms are produced at high levels during periods of inflammation. During expression of iNOS, there is further production of NO in nanomolar to micromolar concentrations. The reaction of NO with superoxide leads to the production of peroxynitrite, a potent oxidant. These molecules further lead to cytopathic effects and result in enterocyte apoptosis or necrosis, impairment of enterocyte proliferation, and epithelium restitution through enterocyte migration. Tissue injury and repair initiates the inflammatory cascade, leading to the classical picture of NEC [6, 35, 36].

The limited de novo arginine production capacity in neonates makes arginine an essential amino acid in early life. In these two studies, L-arginine in premature infants was supplemented with the intention of increasing NO synthesis with the rationale that NO’s role as a vasodilator would be protective to the gut through prevention of ischemic injury [30, 31]. Interestingly, while only a fraction of arginine metabolism enters the NOS pathway to produce NO, it appears as though this small proportion of the overall body arginine lends substantially to the prevention of intestinal ischemia, likely via regulating mesenteric blood flow.

The strengths of this updated systematic review are the inclusion of a recent trial, increased power based on sample size, and detailed subgroup analyses. The current analysis provides evidence in the favor of prophylactic use of L-arginine in premature infants to prevent NEC.

This review included only two small RCTs with a small number of subjects. The limitation of the two included studies was overcome by conducting this systematic review. Additionally, one of the two studies was underpowered. Despite this, statistically significant reductions in the incidence of stage II and III NEC, as well as all stages of NEC, were noted, with p-values of less than 0.05.

Our study revealed that L-arginine has a significant role in reducing the incidence of medical and surgical NEC in modern NICUs without impact on long-term neurodevelopmental outcomes at 36 months of corrected age. However, in the absence of large multi-centre, randomized, controlled trials, the use of supplemental L-arginine in an effort to prevent necrotizing enterocolitis in preterm neonates has not become routine practice.

KM: MD, Neonatal-Perinatal Medicine Fellow, University of British Columbia, Children’s and Women’s Health Centre of British Columbia, Vancouver, British Columbia, Canada

AL: MD, Paediatric Allergy & Immunology Fellow, University of British Columbia, Children’s and Women’s Health Centre of British Columbia, Vancouver, British Columbia, Canada

HA: MBBS, FRCPC, Staff Neonatologist, Director, NICU, South Health Campus, Alberta Health Services; Associate Professor, Department of Pediatrics, University of Calgary, Calgary, Alberta, Canada

HS: MD, FCPS (Ped). Third-year Neonatal-Perinatal Medicine Fellow, University of Calgary, Foothills Medical Centre, Calgary, Alberta, Canada

HLR: MLIS, BA, Liaison Librarian, Clinical Medicine, Health Sciences Library, Health Sciences Centre, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada

AKL: MBBS, MD, DM, MSC, Staff Neonatologist, Foothills Medical Centre, Alberta Health Services, Clinical Epidemiologist; Assistant Professor, Department of Pediatrics and Department of Community Health Services, Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada