Inter-Alpha Inhibitor Protein Level in Neonates Predicts Necrotizing Enterocolitis

Hala Chaaban, MD, Michael Shin, BSc, Edward Sirya, BSc, Yow-Pin Lim, MD, PhD, Michael Caplan, MD, and James F. Padbury, MD Objectives To compare inter-alpha inhibitor protein (IaIp) levels in neonates with proven necrotizing enterocolitis
(NEC) and neonates with other, nonspecic abdominal disorders. Study design This was a prospective observational study of neonates in the neonatal intensive care unit. NEC was diagnosed according to Bells staging criteria. The nNeonates in the control group had a nonspecic abdominal disorder, but no radiographic evidence of NEC and no disease progression. All neonates with radiographically conrmed NEC were included. Plasma IaIp levels were quantitated by enzyme-linked immunosorbent assay. Results Seventeen neonates had conrmed NEC, and 34 neonates had nonspecic abdominal disorders that improved rapidly. Gestational age, postnatal age, weight, sex, maternal obstetric variables, rupture of membranes, and mode of delivery did not differ between the two groups. Mean IaIp level was signicantly lower in the NEC group compared with the control group (137 38 mg/L; 95% condence interval [CI], 118-157 mg/L vs 258 53 mg/L; 95% CI, 238-277 mg/L; P <.0001). Conclusions The nding of signicantly lower IaIp levels in neonates with NEC suggests that IaIp might be a useful, sensitive biomarker, allowing initiation of appropriate therapy and reducing antibiotic overuse in neonates with suspected but unproven NEC. Administration of IaIp may signicantly reduce the severity of systemic inammation and associated tissue injury. (J Pediatr 2010;157:757-61). ecrotizing enterocolitis (NEC) is an acute inammatory condition of the gastrointestinal tract resulting in intestinal necrosis, systemic sepsis, and multisystem organ failure found mostly in neonates born prematurely.1-3 Despite advances in the medical care of low birth weight neonates, the etiology of NEC remains elusive, and morbidity and mortality are still unacceptably high. Clinically, NEC poses major diagnostic challenges because early warning signs and symptoms are often nonspecic. The search for useful biomarkers based on cytokines or other mediators continues; so far, none has demonstrated sufcient sensitivity and specicity for clinical use.4 The inter-alpha inhibitor proteins (IaIps) compose a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma.5 Substantial evidence suggests that IaIps play an important regulatory role in systemic inammation.6 The normally high levels of circulating IaIps in plasma suggest the essential nature of these proteins. A complete absence of IaIps in a human subject has never been reported.7 Profound decreases in plasma IaIp levels have been reported in both adults and neonates with clinically proven sepsis, with levels correlated with disease severity and mortality.5,8-10 Our most recent study demonstrated that IaIps are a reliable diagnostic markers with high sensitivity, specicity, and predictive value in detecting neonatal sepsis.9 The marked decrease in plasma IaIp levels during sepsis and the concomitant increase in IaIp-related fragments in the urine demonstrate that these proteins are consumed and rapidly cleared from the systemic circulation during sepsis. Likewise, hepatic IaIp biosynthesis is down-regulated during severe inammation. As such, IaIps are considered typical negative acute-phase proteins.10 Because systemic host inammatory responses are strongly involved in NEC, we hypothesized that if inammatory responses are involved in the pathogenesis, then IaIp levels might be useful biomarkers for diagnosing NEC. Thus, we sought to examine IaIp levels in neonates with proven NEC and to compare these levels with those measured in neonates with other, nonspecic abdominal disorders.

Methods
This prospective, cohort-based observational study was carried out in the neonatal intensive care unit (NICU) at Women & Infants Hospital of Rhode Island beFrom the Department of Pediatrics, Women & Infants Hospital, Brown Medical School, Providence, RI (H.C., J.P.); ProThera Biologics, East Providence, RI (M.S., E.S., Y.-P.L.); and Department of Pediatrics, Evanston Hospital, Evanston, IL (M.C.) Supported in part by a grant from the National Institutes of Health, National Center for Research Resources (P20 RR018728). Y.-P.L. has equity in ProThera Biologics, where the IaIp protein assays used in these experiments were carried out. The other authors declare no conicts of interest.
0022-3476/$ - see front matter. Copyright 2010 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2010.04.075

CV IaIp NEC NICU

Coefcient of variation Inter-alpha inhibitor protein Necrotizing enterocolitis Neonatal intensive care unit

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tween September 2007 and January 2009 with Institutional Review Board approval. During the study period, a total of 1817 neonates were admitted to the NICU. Of these, 136 had a birth weight <1500 g. The control group comprised neonates who presented with nonspecic abdominal ndings, including feeding intolerance, increased gastric aspirate, abdominal distention, and abdominal tenderness. The initial clinical presentations were sufciently worrisome to warrant cessation of feedings, performance of abdominal radiographs, and blood sampling for complete blood count and blood cultures. An independent pediatric radiologist who was unaware of the group assignment interpreted all abdominal radiographs. Clinical manifestations and radiographic ndings were used to establish the stage of NEC according to the modied Bells staging criteria.10 In these criteria, stage II NEC is dened as radiographic ndings of peumatosis intestinalis with signicant intestinal dilation and ileus with or without portal vein gas or ascites, and stage III (advanced) is dened as additional ndings of ascites and/or bowel perforation. In stage I NEC, radiographic ndings are limited to mild intestinal dilation and dysmotility. All neonates identied with stage II or III NEC were included in the NEC group in the present study. Blood sampling was done in all neonates at the time of initial presentation and clinical evaluation. Plasma IaIp levels were measured quantitatively in residual blood using a competitive enzyme-linked immunosorbent assay with a monoclonal antibody against human IaIp (monoclonal antibody 69.26), as described previously.5,8,9 The assay has a sensitivity of 50 mg/L and a linear dynamic range of up to 750 mg/L. The intra-assay variability (coefcient of variation [CV]) is <3%, and the interassay CV is <7%. Clinical and demographic variables, including gestational age, postnatal age, weight, and sex, were recorded. All maternal medical and obstetrical variables were recorded, including hypertensive disease of pregnancy, prolonged rupture of membranes (dened as >24 hours), and mode of delivery. Statistical Analysis The two groups were compared using the unpaired t test, Mann-Whitney U test, or c2 test as appropriate. Statistical calculations were performed using Statistica (StatSoft, Tulsa, Oklahoma) and MedCalc (MedCalc, Mariakerke, Belgium).

Table I. Clinical characteristics of the study patients

Gestational age, weeks 27 (23-39) 29 (23-41) Age of onset, days 16 (4-165) 17 (1-89) Body weight, g 1360 (460-5000) 1200 (400-5400) Male sex, n (%) (77%) (47%) Maternal hypertension, n (%) 9 (26) 4 (23%) Prolonged rupture 4 (23.5%) 3 (8.8%) of membranes, n (%) C-section delivery, n (%) 21 12 1-minute Apgar #3, n (%) 1 (5.8%) 5 (14.7%) 5-minute Apgar #3, n (%) 0 (0%) 1 (2.9%) Respiratory distress 26 (76%) 13 (76%) syndrome, n (%) Patent ductus 4 (11) 3 (17) arteriosus, n (%) Intrauterine growth 8 (23%) 4 (23) retardation, n (%)

had received a transfusion within 48 hours of onset; the remaining neonates either received no transfusions or received remote transfusions early in the neonatal course. Clinical manifestations in the NEC group included gross blood in the stool (82%), abdominal distention (76%), and increased gastric residual (65%) (Table II). Seven neonates in the NEC group (41%) required increased respiratory support, 5 (29%) developed hypotension treated with uid boluses and vasopressors, and 3 (17%) developed disseminated intravascular coagulation, which was treated with transfusion of blood products. Also in the NEC group, 5 neonates (29%) had a positive blood culture (Staphylococcus epidermidis [n = 3] and Escherichia coli [n = 2]), 6 (35%) underwent placement of surgical drain and/or exploratory laparotomy, 5 (29%) developed a subsequent intestinal stricture, and 3 (17%) died. All initial abdominal radiographs in the NEC group were interpreted as abnormal. Radiographic ndings included pneumatosis intestinalis (n = 17) and intestinal perforation and/or ascites (n = 6). In the control group, the most common initial gastrointestinal manifestation was increased gastric residual (65%) and abdominal distention (59%). None of these neonates developed NEC. Their initial abdominal radiographs demonstrated unremarkable/nonspecic abdominal distension,

Results
The NEC group comprised 17 neonates with a conrmed diagnosis of NEC (Bell stage II or III). The control group comprised 34 neonates who presented with a nonspecic abdominal disorder that was worrisome enough to withhold feedings and obtain an abdominal radiograph, but improved rapidly (Table I). There were no signicant differences between the groups in terms of gestational age, birth weight, or other clinical variables. There was a tendency toward a higher proportion of males in the NEC group (P = .08). Among the neonates in the NEC group, only one
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Table II. Clinical presentation of the conrmed NEC group and the control group
Clinical presentation Gastric residual Abdominal distention Abdominal tenderness Bloody stool Vomiting Increased respiratory support Hypotension Disseminated intravascular coagulation NEC group (n = 17), n (%) 11 (64%) 13 (76%) 12 (70%) 14 (82%) 0 (0%) 7 (41%) 5 (29.5%) 3 (17%) Control group (n = 34), n (%) 25 (65.5%) 22 (59%) 2 (7%) 0 (0%) 7 (14%) 0 (0%) 0 (0%) 0 (0%)

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November 2010 and subsequent radiographs performed 12-24 hours later showed no progression. Mean IaIp level was signicantly lower in the conrmed NEC group compared with control group (137 38 mg/L; 95% condence interval [CI], 118-157 mg/L vs 258 53 mg/L; 95% CI, 238-277 mg/L; P <.0001) (Figure 1). There was no overlap between the IaIp values in the NEC and the control groups except in 1 patient in the NEC group, who presented with abdominal distention and gastric residuals with an initial IaIp level of 240 mg/dL (Figure 1). This patient had pneumatosis on the initial radiograph that disappeared on a subsequent lm within 12 hours. The patient was treated for NEC and recovered rapidly without complications. In the NEC group, mean IaIp level did not differ between those with stage II (132 29 mg/L; 95% CI, 114-150 mg/L; n = 12) and stage III NEC (138 34 mg/L; 95% CI, 95-181 mg/L; n = 4), and levels were similar in all patients with NEC regardless of the presence or absence of an associated positive blood culture (Figure 2).

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300

250

200

150

100

50 Negative Blood Culture Positive Blood culture

Discussion
NEC is an acute inammatory condition of the gastrointestinal tract resulting in intestinal necrosis, systemic sepsis, and multisystem organ failure. NEC is the most common neonatal gastrointestinal emergency1 and carries a relatively high mortality rate among affected neonates.3,4 The morbidity and mortality have not changed appreciably over the past several decades.1-4 Early warning signs and symptoms of NEC are nonspecic, often inconspicuous, and may be difcult to distinguish from other nonspecic abdominal disorders, such as gastrointestinal dysmotility and acute exacerbations of bronchopulmonary dysplasia.4 Thus, early identication of NEC in preterm neonates remains a major diagnostic challenge. To date, no reliable early biomarkers have demonstrated sufcient positive and negative predic-

Figure 2. IaIp levels in neonates with NEC with negative and positive blood cultures. There was no signicant difference in IaIp levels between the blood culture-positive neonates and the culture-negative neonates with proven NEC (142 31 mg/ L; 95% CI, 107-177 mg/L [n = 5] vs 141 28 mg/L; 95% CI, 112-170 mg/L [n = 12]; P = .96).

Figure 1. Plasma IaIp levels in neonates with conrmed NEC and control. IaIp was signicantly lower in the NEC group.

tive values to be clinically useful.4 Here we report that IaIp levels are signicantly lower in neonates with documented NEC compared with neonates later conrmed to have nonspecic abdominal disorders. With the exception of a single neonate, there was no overlap of the measured levels in the 2 groups. Although the precise etiology of NEC remains enigmatic, the pathogenesis appears to involve a pathway that includes the endogenous production of inammatory mediators involved in the development of intestinal injury.11 Endotoxin/lipopolysaccharide, platelet-activating factor, tumor necrosis factor (TNF)-a, interleukin (IL)-8, prostaglandins, leukotrienes, protease/antiprotease balance, and nitric oxide are thought to be involved in NEC pathogenesis.12-15 Recent studies suggested that an unbalanced proinammatory response contributes to the predilection toward NEC in animal studies and in preterm neonates.15-17 Up-regulation of proinammatory mediators occurs early in the inammatory response, and given that previous studies have demonstrated alterations in nuclear factor kB and IL-8 activation, these data suggest that identifying alterations in circulating levels as early biomarkers of NEC is possible.4,14 Along with contributing to the balance of the inammatory response, inhibitors of serine proteases (serpins) have been shown to protect against epithelial cell necrosis.15 Caenorhabditis elegans harboring a serpin knockout gene was shown to develop exaggerated cell death in response to hypotonic shock, cold exposure, and other forms of stress. These data suggest that lower antiprotease levels might not only be an early biomarker, but also could promote mucosal cell death and contribute to the pathophysiology of neonatal NEC. Differences
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Inter-Alpha Inhibitor Protein Level in Neonates Predicts Necrotizing Enterocolitis

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Vol. 157, No. 5 cantly decreased in neonatal sepsis. In a larger study of their diagnostic utility, we found that IaIp level is a more reliable diagnostic marker for neonatal sepsis than other currently available tests.9 Moreover, in several adult and newborn animal models of sepsis, IaIp treatment to normalize the decreased systemic levels has been demonstrated to signicantly decrease sepsis-related mortality.29,30 In summary, in the present study we have demonstrated that IaIp levels are signicantly decreased in patients with NEC stage II/III compared with a control group with nonspecic abdominal disorders. We hypothesize that IaIp is involved in the pathogenesis of NEC. As one of the critical acute phase reactants during the host response to inammation and tissue injury, IaIp appears to rapidly decrease as the molecule is consumed during the inammatory process and rapidly excreted through the kidneys. IaIp level may be a useful, sensitive biomarker for detecting NEC. The ability to detect NEC in the early stages will allow initiation of appropriate therapy and also may provide an objective means for reducing antibiotic overuse in neonates with suspected but unproven NEC. n
Submitted for publication Jan 24, 2010; last revision received Mar 30, 2010; accepted Apr 29, 2010. Reprint requests: James F. Padbury, MD, Department of Pediatrics, Women & Infants Hospital, 101 Dudley Street, Providence, RI 02905. E-mail: JPadbury@ wihri.org.

in IL-6, C-reactive protein, calprotectin, procalcitonin, and intestinal fatty acidbinding protein levels13,14,18-23 have been examined, but clinical studies are limited, and the results have not demonstrated sufcient reliability.4 Because no current method has been shown to be both sensitive and specic in identifying neonates with denite NEC early in the course of disease, we examined IaIp as a candidate molecule. The IaIps include a family of structurally related serine protease inhibitors found at relatively high concentrations in human plasma.5 Unlike other inhibitor molecules, this family of inhibitors is composed of a combination of covalently linked polypeptide chains. IaIps play roles in inammation, wound healing, and cancer metastasis.7,24-28 They are known to inhibit several serine proteases, including trypsin, human leukocyte elastase, plasmin, cathepsin G, and granzyme K. On forming a stable complex with TSG-6 (a possible ligand of IaIp), the inhibitory activity of IaIp toward plasmin is enhanced signicantly.6 Plasmin is a serine protease involved in the activation of matrix metalloproteinases that are part of the proteolytic cascade associated with inammation. The liver is the major source of heavy and light chains of IaIp.26 The protective effects of exogenously administered IaIp can be explained through inhibition of destructive serine proteases such as elastase, plasmin, cathepsin G, granzyme K, and furin.7 The inhibitory activity of IaIp against neutrophil-induced elastase activity might be of considerable pathophysiological importance in severe sepsis.29,30 The inhibition of granzyme K may contribute to apoptotic signaling by cytotoxic T cells in severe sepsis. Inhibition of plasmin activity attenuates plasmin-mediated activation of matrix metalloproteinases, which may cause tissue injury in systemic inammatory states.6 IaIps are also potent inhibitors of furin,31,33 an endogenous cell membraneassociated serine endoprotease that plays a role in the partial proteolytic activation of various bacterial toxins, including Pseudomonas exotoxin A, diphtheria toxin, lethal toxin, and edema toxin formation by Bacillus anthracis, as well as a variety of other microbial exotoxins.31,33 IaIps also operate through downregulation of proinammatory cytokines such as TNFa and IL-6,26 and by blocking excess complement activation and generation of circulating C5a.31,32 Administration of IaIp (30 mg/kg) to 2-day-old mice within hours of inducing neonatal sepsis with either Escherichia coli or group B beta hemolytic streptococci was found to signicantly improve survival.34 We and others have shown that in adult patients with sepsis, plasma IaIp levels are decreased signicantly (by 20% 90%) and inversely correlated with unfavorable outcome.5 We previously studied a cohort of newborn neonates of 2442 weeks gestational age.8 We measured IaIp levels in umbilical cord blood, postnatal blood, and samples obtained at the time of evaluation for sepsis and found that IaIp was produced endogenously at levels independent of maternal level, gestational age, and postnatal age and similar to the levels seen in adults.8 We also showed that IaIp levels were signi760

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