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Inter-Alpha Inhibitor Protein Level in Neonates PredictsNecrotizing 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, nonspecific abdominal disorders.Study design This was a prospective observational study of neonates in the neonatal intensive care unit. NECwas diagnosed according to Bells staging criteria. The nNeonates in the control group had a nonspecificabdominal disorder, but no radiographic evidence of NEC and no disease progression. All neonates with radio-graphically confirmed NEC were included. Plasma IaIp levels were quantitated by enzyme-linked immunosorbentassay.Results Seventeen neonates had confirmed NEC, and 34 neonates had nonspecific abdominal disorders that im-proved 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 significantly lower in the NEC groupcompared with the control group (137 38 mg/L; 95% confidence interval [CI], 118-157 mg/L vs 258 53 mg/L;

95% CI, 238-277 mg/L; P

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tween September 2007 and January 2009 with InstitutionalReview Board approval. During the study period, a total of1817 neonates were admitted to the NICU. Of these, 136

had a birth weight

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and subsequent radiographs performed 12-24 hours latershowed no progression.

Mean IaIp level was significantly lower in the confirmed

NEC group compared with control group (137 38 mg/L;95% confidence interval [CI], 118-157 mg/L vs 258 53mg/L; 95% CI, 238-277 mg/L; P

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in IL-6, C-reactive protein, calprotectin, procalcitonin, andintestinal fatty acidbinding protein levels13,14,18-23 havebeen examined, but clinical studies are limited, and the re-

sults have not demonstrated sufficient reliability.4 Becauseno current method has been shown to be both sensitiveand specific in identifying neonates with definite NEC earlyin the course of disease, we examined IaIp as a candidate mol-ecule.

The IaIps include a family of structurally related serine

protease inhibitors found at relatively high concentrationsin human plasma.5 Unlike other inhibitor molecules, thisfamily of inhibitors is composed of a combination of cova-lently linked polypeptide chains. IaIps play roles in inflam-

mation, wound healing, and cancer metastasis.7,24-28 They

are known to inhibit several serine proteases, including tryp-sin, human leukocyte elastase, plasmin, cathepsin G, andgranzyme K. On forming a stable complex with TSG-6 (apossible ligand of IaIp), the inhibitory activity of IaIp towardplasmin is enhanced significantly.6 Plasmin is a serine prote-ase involved in the activation of matrix metalloproteinases

that are part of the proteolytic cascade associated with in-flammation. The liver is the major source of heavy and lightchains of IaIp.26

The protective effects of exogenously administered IaIpcan be explained through inhibition of destructive serineproteases such as elastase, plasmin, cathepsin G, granzyme

K, and furin.7 The inhibitory activity of IaIp againstneutrophil-induced elastase activity might be of considerablepathophysiological importance in severe sepsis.29,30 The inhi-bition of granzyme K may contribute to apoptotic signaling

by cytotoxic T cells in severe sepsis. Inhibition of plasmin ac-tivity attenuates plasmin-mediated activation of matrix met-

alloproteinases, which may cause tissue injury in systemicinflammatory states.6 IaIps are also potent inhibitors offurin,31,33 an endogenous cell membraneassociated serineendoprotease that plays a role in the partial proteolytic acti-vation of various bacterial toxins, including Pseudomonasexotoxin A, diphtheria toxin, lethal toxin, and edema toxinformation byBacillus anthracis, as well as a variety of othermicrobial exotoxins.31,33 IaIps also operate through down-regulation of proinflammatory cytokines such as TNF-

a and IL-6,26 and by blocking excess complement activationand generation of circulating C5a.31,32 Administration of IaIp

(30 mg/kg) to 2-day-old mice within hours of inducing neo-

natal sepsis with either Escherichia coli or group B beta hemo-lytic streptococci was found to significantly improvesurvival.34

We and others have shown that in adult patients with sep-sis, plasma IaIp levels are decreased significantly (by 20%

90%) and inversely correlated with unfavorable outcome.5

We previously studied a cohort of newborn neonates of 24-42 weeks gestational age.8 We measured IaIp levels in umbil-ical cord blood, postnatal blood, and samples obtained at thetime of evaluation for sepsis and found that IaIp was pro-

duced 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 signifi-

cantly decreased in neonatal sepsis. In a larger study of theirdiagnostic utility, we found that IaIp level is a more reliablediagnostic marker for neonatal sepsis than other currently

available tests.9 Moreover, in several adult and newborn an-imal models of sepsis, IaIp treatment to normalize the de-creased systemic levels has been demonstrated tosignificantly decrease sepsis-related mortality.29,30

In summary, in the present study we have demonstratedthat IaIp levels are significantly decreased in patients with

NEC stage II/III compared with a control group with nonspe-cific abdominal disorders. We hypothesize that IaIp is in-volved in the pathogenesis of NEC. As one of the criticalacute phase reactants during the host response to inflamma-

tion and tissue injury, IaIp appears to rapidly decrease as themolecule is consumed during the inflammatory processand rapidly excreted through the kidneys. IaIp level may bea useful, sensitive biomarker for detecting NEC. The abilityto detect NEC in the early stages will allow initiation of ap-propriate 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.

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