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Lactate and intestinal fatty acid binding protein as essential biomarkers in neonates with necrotizing enterocolitis: ultrasonographic and surgical considerations

Open AccessPublished:April 23, 2020DOI:https://doi.org/10.1016/j.pedneo.2020.03.015

      Background

      Necrotizing enterocolitis (NEC) is a neonatal disease with its pathogenesis still not well understood, although it is hypothesized to be related to decreased perfusion of the intestinal wall. The current study aimed to evaluate the plasma lactate levels and assess the validity of plasma and urinary intestinal fatty acid binding protein (I-FABPp and I-FABPu/Cru respectively) in NEC.

      Methods

      The study included 55 neonates with variable Bell's stages who were comparable with 23 matched controls. Colorimetric assays of plasma lactate and ELISA assays of I-FABP in both serum and urine of the included neonates have been performed.

      Results

      There were significantly higher median levels of I-FABPp, I-FABPu and lactate among cases (2.84 ng/ml, 1.74 ng/g creat. and 32.34 mg/dl, respectively) compared with controls (0.16 ng/ml, 0.60 ng/g creat. and 15.33 mg/dl, respectively) with p ˂ 0.05 for all. I-FABPp at cut-off point >3.24 ng/ml showed 90% sensitivity, 72% specificity, PPV = 52.6%, NPP = 94.7%, while for I-FABPu (at cut-off point > 2.93 ng/g creat.) those values were 90%, 92%, 81.8% and 95.8% respectively, in discriminating stage IIIA from stage II with p = 0.001. In predicting surgical NEC, I-FABPp at the cut-off point of 6.95 ng/ml revealed 75% sensitivity, 100% specificity, PPV = 100%, NPP = 95%, while for I-FABPu (cut-off point>4.13 ng/g creat.) they were 100%, 76.19%, 44.4 %and 100%, p = 0.04.

      Conclusion

      s: In addition to clinical judgment, sonographic data and plasma lactate, I-FABPp was shown to be a specific marker for early identification of surgical NEC, while I-FABPu could be more useful for differentiating Bell's stage II from stage III.

      Key Words

      1. Introduction

      Necrotizing enterocolitis (NEC) is a serious neonatal disease with inflammation and necrosis of the gastrointestinal tract.
      • Neu J.
      • Walker W.A.
      Necrotizing enterocolitis.
      Its cause is still not well understood and though it is related to decreased intestinal wall perfusion and ischemia especially in immature guts with disruption of the intestinal barrier, activation of the inflammatory mediators, and enabling of bacterial passage.
      • Staryszak J.
      • Stopa J.
      • Kucharska-Miąsik I.
      • Osuchowska M.
      • Guz W.
      • Bła˙z W.
      Usefulness of ultrasound examinations in the diagnostics of necrotizing enterocolitis.
      Multiple risk factors have been implicated in its development, such as preterm birth, umbilical catheterization, perinatal asphyxia, early enteral feeding in preterm newborns, and congenital heart defects.
      • Staryszak J.
      • Stopa J.
      • Kucharska-Miąsik I.
      • Osuchowska M.
      • Guz W.
      • Bła˙z W.
      Usefulness of ultrasound examinations in the diagnostics of necrotizing enterocolitis.
      Plasma lactate level is an indicator for the normal metabolism of the intestinal flora. Progressive ischemic injury to the mesentery with mucosal damage and the changes in the homeostasis of the intestinal flora lead to bacterial translocation and may cause an increase in the serum lactate levels.
      • Oner E.
      • Günalp M.
      • Evirgen O.
      • Hayırlı E.N.
      • Polat O.
      • Yesilkaya I.
      Prognostic value of intestinal fatty acid binding protein, L-lactate and D-dimer levels, and their combination in the early stage of acute mesenteric ischemia confirmed with histological outcomes: an experimental study.
      Fatty acid-binding protein (FABP) is a small (14–15 kDa) intracellular protein,
      • Benkoe T.M.
      • Mechtler T.P.
      • Weninger M.
      • Pones M.
      • Rebhandl W.
      • Kasper D.C.
      Serum levels of interleukin-8 and gut-associated biomarkers in diagnosing necrotizing enterocolitis in preterm infants.
      which increases in conditions related to inflammation and ischemia and has an important role in protecting cells from the side effects of fatty acids.
      • Niewold T.A.
      • Meinen M.
      • van der Meulen J.
      Plasma intestinal fatty acid binding protein (I-FABP) concentrations increase following intestinal ischemia in pigs.
      Intestinal FABP (I-FABP) is located in the gastric epithelial cells and in the intestinal mucosa,
      • Glatz J.F.
      • van der Vusse G.J.
      Cellular fatty acid-binding proteins: their function and physiological significance.
      and it can be measured in plasma (I-FABPp) and urine (I-FABPu).
      • Schurink M.
      • Scholten I.G.
      • Kooi E.M.
      • Hulzebos C.V.
      • Kox R.G.
      • Groen H.
      • et al.
      Intestinal fatty acid-binding protein in neonates with imminent necrotizing enterocolitis.
      Urine collection may be less invasive and more feasible than blood sampling, especially in extreme low birth weight (ELBW) and very low birth weight (VLBW) infants with NEC.
      • Robinson J.R.
      • Rellinger E.J.
      • Hatch L.D.
      • Weitkamp J.H.
      • Speck K.E.
      • Danko M.
      • et al.
      Surgical necrotizing enterocolitis.
      Early identification of developing NEC remains a challenging issue, as the current laboratory and radiology tests lack sufficient accuracy power.
      • Hällström M.
      • Koivisto A.M.
      • Janas M.
      • Tammela O.
      Laboratory parameters predictive of developing necrotizing enterocolitis in infants born before 33 weeks of gestation.
      The incidence of surgical necrotizing enterocolitis (NEC) has not decreased significantly over the past decade. Pneumoperitoneum and clinical deterioration (despite maximal medical therapy) are the most common indications for operative treatment.
      • Robinson J.R.
      • Rellinger E.J.
      • Hatch L.D.
      • Weitkamp J.H.
      • Speck K.E.
      • Danko M.
      • et al.
      Surgical necrotizing enterocolitis.
      ,
      • Villamil V.
      • Fernández-Ibieta M.
      • Gilabert Ubeda M.A.
      • Aranda García M.J.
      • Ruiz Pruneda R.
      • Sánchez Morote J.M.
      • et al.
      Correlation between pneumoperitoneum and surgical findings and morbidity and mortality in newborns with necrotising enterocolitis.
      Therefore, the current study aimed to evaluate the role of I-FABPp and I-FABPu as biomarkers for early diagnosis of necrotizing enterocolitis in neonates, in addition to serum lactate levels and ultrasonographic findings. Also, the study aimed to compare and correlate the plasma and urinary I-FABP2 levels with various ultrasonographic findings among neonates with NEC.

      2. Patients and methods

      2.1 Study design and participants

      The current prospective cohort study has been conducted with 55 neonates (preterm or full-term) of both sexes with various NEC stages admitted to the Neonatal Intensive Care Unit, Pediatrics Department, South Valley University Hospitals, Qena, Egypt, in addition to 23 healthy neonates attending the NICU for routine clinical follow-up, age- and sex-matched with included cases, who were selected as a control group. The study was approved by the Ethics committee of the Faculty of Medicine, South Valley University, Qena, Egypt, and it was conducted in accordance with the Declaration of Helsinki. Informed written consent has been obtained from the parents of every included neonate. The study duration was one year from January 1st, 2018 to December 30th, 2018.

      2.2 Patients' selection criteria

      Neonates with NEC were categorized according to Modified Bell's system which was first proposed by Bell et al. as stage I (suspected), II (proven) or III (advanced),
      • Bell M.J.
      • Ternberg J.L.
      • Feigin R.D.
      • Keating J.P.
      • Marshall R.
      • Barton L.
      • et al.
      Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging.
      and later modified by Walsh and Kliegman in 1986.
      • Walsh M.C.
      • Kliegman R.M.
      Necrotizing enterocolitis: treatment based on staging criteria.
      In this modified scheme, stages are further subdivided into A or B depending on the radiographic findings: stage IA or B (normal or intestinal dilation), stage IIA (ileus, pneumatosis intestinalis) or IIB (portal venous gas), stage IIIA (ascites) or IIIB (pneumoperitoneum).
      • Staryszak J.
      • Stopa J.
      • Kucharska-Miąsik I.
      • Osuchowska M.
      • Guz W.
      • Bła˙z W.
      Usefulness of ultrasound examinations in the diagnostics of necrotizing enterocolitis.
      ,
      • Bell M.J.
      • Ternberg J.L.
      • Feigin R.D.
      • Keating J.P.
      • Marshall R.
      • Barton L.
      • et al.
      Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging.
      Neonates who presented with pneumoperitoneum by x-ray as in Bell's scoring IIIB or who had any pervious GIT operation or proved to have another diagnosis. Those who had incomplete data, or with congenital anomalies or metabolic error disorders, or neonates whose parents refused to participate in the study, were excluded from the study.

      2.3 Clinical evaluation of the included neonates

      Detailed medical history was taken focusing on the following: postnatal age of the patients, gestational age, age of onset of the symptoms, obstetric history as pre-eclampsia, DM and chorioamnionitis, and time and type of first feeding. Full clinical examination was done for the included patients with special concern with the abdominal examination. The examination included general examination, anthropometric measurements (weight, length, head circumference), Down score, and cardiac, chest, and neurological examination.

      2.4 Investigatory battery

      2.4.1 Imaging tools

      Plain x-ray abdomen AP view in the supine position, using Philips medical systems (D-22335, Hamburg, Germany) was used for all included patients on admission to assess the presence of abnormal bowel dilatation, intramural gas, portal venous gas, separation of bowel loops and fixed appearance of bowel loops on serial films.
      Abdominal ultrasonography including gray scale and color Doppler examination using GE LOGIQ P6 ultrasound machine, USA; using convex and linear transducers (3.4 and 11.8 MHz) was administered to all study patients. Gray-scale abdominal US was used to assess the presence of peritoneal fluids, peritoneal air, portal venous gas, bowel wall thickening (2.7 mm or greater), bowel wall thinning (1.0 or lesser), increased bowel wall echogenicity, peristalsis, and pneumatosis intestinalis (echogenic spots). Color Doppler US examination was done to evaluate the perfusion state of the intestinal wall and mesenteric vessels, indicating early increased vascularity (hyperperfusion) giving (Y) shaped pattern and late hypoperfusion of the necrotic bowel loops or even complete loss of perfusion, Fig. 1.
      Fig. 1
      Fig. 1Imaging data of an included preterm with necrotizing enterocolitis, +/−33 weeks, 1.3 Kg, severe abdominal distension on clinical exam (A). Abdominal radiograph & serial film (Antro-posterior view) shows mild dilatation of bowel loops (B & C). Ultrasonographic serial examination (D & E): decreased peristalsis, wall-thickening with intramural gas echogenicity & free intra-peritoneal fluid. On doppler examination, decreased perfusion was noted (F).
      The final decision regarding the presence or absence of intestinal necrosis was based on the US and Doppler findings (portal venous gas, ascites, hypoperfusion) of which the surgeon was aware when making decisions on the possible need for surgery. Neonates having necrotic intestinal segment upon laparotomy underwent resection and diversion.

      2.4.2 Laboratory workup

      In addition to the routine laboratory complete blood counts (using Cell Dyn 1800-Abbott diagnostics, Germany), C-reactive protein (using the semi-quantitative latex agglutination test, AVITEX CRP kits; Catalog No. OD023; supplied by Omega Diagnostics, UK) and serum electrolytes (sodium, potassium and ionized calcium, using Dimension® EXL™ 200 Integrated Chemistry System, Germany), 4-ml venous blood samples and 2 ml of urine were collected from every included neonate at time of diagnosis of suspected or definite NEC stage after clinical and imaging evaluation. Two ml of blood samples were placed on fluorinated tubes and the remaining 2 ml were evacuated into EDTA containing tubes; both samples were then centrifuged at 3500 rpm for 10 min, and the separated plasma was transferred into 1 ml cryotubes and stored at −80 °C for later biochemical analysis. Urine samples were collected using urine collecting bags, evacuated into 1 ml cryotubes and stored at −80 °C for later biochemical analysis of I-FABPu which was expressed as ratio to urinary creatinine (ng/g creat.). At the time of assay of I-FABPu, urine samples were incubated at 37 °C for 15 min and were then centrifuged at 3500 rpm for 5 min and supernatants were used for the biochemical assays.
      Biochemical assays included: A) plasma lactate level, which was measured using commercially available colorimetric assay kit supplied by Spectrum Diagnostics, Cairo, Egypt, Catalog No: 274001, using spectrophotometer (Chem-7, Erba Diagnostics Mannheim GmbH, Germany); and plasma and urinary I-FABP levels, which were measured using commercially available ELISA assay kit supplied by Chongqing Biospes Co. Ltd, China with catalog number BYEK2440, using microplate ELISA reader (EMR-500, USA).

      2.5 Statistical analysis

      Data entry and data analysis were done using SPSS version 19 (Statistical Package for Social Science). Data were presented as a number, percentage, the mean and standard deviation for parametric data, and the median and inter-quartile range for non-parametric data. Chi-square test and Fisher exact test were used to compare qualitative variables. Mann–Whitney U test was used to compare between two quantitative variables and Kruskal-Wallis test was used to compare more than two quantitative variables for non-parametric data. Spearman correlation test was done to measure the correlation between quantitative variables in case of non-parametric data. Medcalc Program was used to calculate sensitivity, specificity, positive and negative predictive values. P-value was considered statistically significant when <0.05.

      3. Results

      3.1 Baseline characteristics of the included neonates

      The current study was conducted on 55 neonates with necrotizing enterocolitis (35 males and 20 females). Their mean post-natal age was 16.85 ± 6.34 days and their mean gestational age was 33.38 ± 3.39 weeks. For 23 healthy controls (15 males and 8 females) matched with the study patients for gestational age, post-natal age and sex, with mean gestational age was 35.30 ± 3.86 weeks and post-natal age was 14.30 ± 6.41 days. The included neonates with NEC were consecutive neonates with suspected NEC (55); after clinical and imaging investigations, 35 of them were proved definite NEC on admission, while the remaining 20 neonates recovered and did not progress into definite NEC.

      3.2 Possible associated risk factors for NEC among the included cases

      Among the neonates with NEC, male-to-female ratio was 1.75. Forty cases (72.7%) were preterm and 15 (27.3%) were full term. Forty-five cases (81.8%) were delivered by cesarean section and the remaining 18.2% (10 cases) were delivered via normal vaginal delivery. Birth anoxia was present in 5 cases (9.1%). Maternal history of pre-eclampsia was present in 15 cases (27.3%), diabetes mellitus in 11 cases (20%), with chorioamnionitis and irradiation in 6 cases (10.9%) each. Regarding the feeding type, breast feeding was present in 9.1% (5 neonates), while 31 neonates (56.4%) were artificially fed and the remaining 19 neonates (34.5%) had history of mixed feeding (both breast and artificial milk).

      3.3 Clinical and imaging data of the included neonates with NEC

      According to the Modified Bell's system, the study neonates with NEC were classified into the following subgroups; stage Ia, Ib, IIa, IIb and IIIa (each included 10 cases except for stage IIb with 15 patients).
      All cases (100%) exhibited signs of feeding intolerance in the form of vomiting, gastric residuals and abdominal distension. Eight patients (14.5%) had audible intestinal sounds, 18 patients (32.7%) had abdominal tenderness and 8 patients (14.5%) had abdominal cellulitis. Constipation was present in 39 cases (70.9%). Twenty-five (45.4%) cases had lethargy and weak reflexes. The study cases showed significantly lower mean ± SD diastolic blood pressure and significantly higher Down score (36.24 mmHg ±4.82,2.24 ± 0.98 respectively) in comparison to the controls (41.30 mmHg ±5.19, 0.70 ± 0.67, respectively), (p ˂0.05 for both).
      As regards the ultrasonographic and Doppler findings of the study cases, intestinal wall thickening was present in all cases (100%), intramural gas was frequent in 27 cases (49.1%), 10 cases (18.2%) had good bowel perfusion and 15cases (27.3%) revealed presence of peristalsis. Portal venous gas was detected in 19 cases (34.5%) and ascites was present in 16 cases (29.1%) (Fig. 1).

      3.4 Routine laboratory investigations of study groups

      There were statistically significant lower total calcium, hemoglobin levels and platelet counts (9.00 mg/dl ± 1.22, 13.73 g/dl ± 2.60 and 174.04 ˟103/μl ± 141.81, respectively) with significantly higher white blood cell counts (10.01 ˟103/μl ± 3.61) among cases when compared with the controls (10.14 mg/dl ± 0.82, 16.20 g/dl ± 1.48, 429.40 ˟103/μl ± 76.67 and 6.90˟103/μl ± 2.23, respectively), with p <0.05 for all. CRP was positive in 25.5% (14) of cases.

      3.5 Plasma lactate, I-FABPp and I-FABPu levels among the studied groups

      There were significantly higher median levels of I-FABPp, I-FABPu and lactate among cases (2.84 ng/ml, 1.74 ng/g creat. and 32.34 mg/dl, respectively) when compared with the controls (0.16 ng/ml, 0.60 ng/g creat. and 15.33 mg/dl, respectively) with p ˂0.05 for all (Table 1).
      Table 1Comparison of the plasma I-FABP, urinary I-FABP and blood lactate levels among the included cases versus controls.
      Biochemical markersControls (n = 23)Cases (n = 55)P-value
      I-FABPp (ng/ml)0.000∗
      Median (Range)0.16 (0.030.34)2.84 (0.128.20)
      I-FABPu (ng/g creat.)0.000∗
      Median (Range)0.60 (0.210.78)1.74 (0.476.68)
      Lactate (mg/dl)0.000∗
      Median (Range)15.33 (10.2318.70)32.34 (25.0855.14)
      N.B: P value > 0.05 insignificant, ∗P < 0.05 significant. I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein.
      In comparison of the plasma I-FABP, urinary I-FABP and blood lactate levels among the study neonates with necrotizing enterocolitis, increased with the progress of the NEC stages in a statistically significant manner with the lowest median levels at stage IA and the highest median levels at stage III-A, with p <0.05 for all (Table 2).
      Table 2Comparison of the plasma I-FABP, urinary I-FABP and blood lactate levels among the included neonates with necrotizing enterocolitis according to Modified Bell's system stages and the control group.
      Biochemical markersNeonates with NEC n = 55Controls n = 23
      Stage I-A n = 10Stage I–B n = 10Stage II-A n = 10Stage II-B n = 15Stage III-A n = 10P-value
      I-FABPp (ng/ml)0.000∗
      Median (Range)0.27 (0.120.45)0.28 (0.130.52)2.30 (1.802.95)3.24 (3.106.95)6.50 (1.208.20)0.16 (0.030.34)
      I-FABPu (ng/g creat)0.000∗
      Median (Range)0.65 (0.470.75)1.30 (1.201.60)1.71 (1.501.97)2.93 (2.715.88)5.08 (0.786.68)0.60 (0.210.78)
      Lactate (mg/dl)0.000∗
      Median (Range)28.1 (27.130.1)31.5 (25.134.5)33.4 (28.443.7)32.3 (30.045.0)55.1 (30.055.1)15.33 (10.2318.7)
      N.B: P value > 0.05 insignificant, ∗P < 0.05 significant. I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein. Kruskal-Wallis test was used.
      Additionally, there were significantly positive correlations between I-FABPp and I-FABPu(r = 0.913, p = 0.000), I-FABPp and lactate (r = 0.611, p = 0.000) and between I-FABPu and lactate (r 0.682, p = 0.000) (Table 3). Non-significant correlations could be found between both I-FABPp and I-FABPu with gestational age, birth body weight or weight at onset of symptoms among the study cases, p <0.05 for all (Table 4).
      Table 3Correlations between I-FABPp, I-FABPu and plasma lactate levels among the included neonates with necrotizing enterocolitis.
      Biochemical parametersI-FABPp (ng/ml)I-FABPu (ng/g creat)
      I-FABPu (ng/g creat)r-value0.913
      P-value0.000∗
      Lactate (mg/dl)r-value0.6110.682
      P-value0.000∗0.000∗
      N.B: P value > 0.05 insignificant, ∗P < 0.05 significant. I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein.
      Table 4Correlations of I-FABPp and I-FABPu levels with gestational age, birth weight and weight at onset of symptoms among the included neonates with necrotizing enterocolitis.
      VariablesI-FABPp (ng/ml)I-FABPu (ng/g creat)
      r-valueP-valuer-valueP-value
      Gestational age (weeks)−0.0320.8160.0850.535
      Birth weight (Kg)0.0470.7350.1720.209
      Weight at onset of symptoms (Kg)0.0510.7110.1620.236
      N.B: P value > 0.05 insignificant. I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein.
      There were non-significant differences in the median values of I-FABPp and I-FABPu levels among the study cases with positive CRP vs. those who were negative for CRP, with p <0.05 for all, Table .5. The median levels of I-FABPp and I-FABPu were significantly higher among cases with positive ultrasonographic findings (portal venous gas 6.50 ng/ml and 2.93 ng/g creat., respectively) and ascites (4.50 ng/ml and 3.63 ng/g creat., respectively) when compared with cases who did not exhibit positive ultrasonographic findings (portal venous gas 2.90 ng/ml and 1.74 ng/g creat., respectively) and ascites (3.10 ng/ml and 2.93 ng/g creat., respectively), with p ˂0.05 for all, with non-significant differences as regards plasma lactate levels with p ˃0.05 for all (Table 6).
      Table 5Comparison of I-FABPp and I-FABPp levels among the included neonates with necrotizing enterocolitis in terms of CRP.
      Biochemical markersNeonates with NEC (n = 55)P-value
      Negative CRP (n = 41)Positive CRP (n = 14)
      I-FABPp (ng/ml)0.428
       Median2.002.95
       Range0.137.780.128.20
      I-FABPu (ng/ml)0.249
       Median1.501.80
       Range0.476.130.546.68
      N.B: P value > 0.05 insignificant, I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein.
      Table 6Plasma I-FABP, urinary I-FABP and blood lactate levels in relation to ultrasonographic findings among neonates with necrotizing enterocolitis.
      Ultrasonographic findings (n = 35)I-FABPp (ng/ml)I-FABPu (ng/g creat)Lactate (mg/dl)
      Portal venous gasYes

      19 (54.3%)
      Median (Range)6.50 (3.248.20)2.93 (2.716.68)32.34 (30.0055.14)
      No

      16 (45.7%)
      2.90 (1.207.78)1.74 (0.785.88)38.48 (28.4045.00)
      P-value0.000∗0.001∗0.701
      AscitesYes

      16 (45.7%)
      Median (Range)4.50 (1.208.20)3.63

      0.786.68))
      44.10 (30.0055.14)
      No

      19 (54.3%)
      3.10 (1.806.95)2.93 (1.505.88)33.44 (28.4045.00)
      P-value0.040∗0.035∗0.243
      N.B: P value > 0.05 insignificant, ∗P < 0.05 significant. I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein.
      As regards the performance characteristics of I-FABPp and I-FABPu in discriminating stage IIIA from stage II, I-FABPp at cut-off point >3.24 ng/ml showed 90% sensitivity, 72% specificity, positive predictive value (PPV) = 52.6%, negative predictive value (NPP) = 94.7% with AUC = 0.768, while for I-FABPu, performance characteristics at cut-off point> 2.93 ng/g creat. were 90%, 92%, 81.8 and 95.8, respectively with AUC = 0.864 (Fig. 2).Regarding the performance characteristics of I-FABPp and I-FABPu in predicting NEC with impending perforation (negative outcome), I-FABPp at cut-off point 6.95 ng/ml revealed 75% sensitivity, 100% specificity, PPV = 100%, NPP = 95% with AUC = 0.881, while for I-FABPu at cut-off point>4.13 ng/g creat., results were 100%, 76.19%, 44.4% and 100 %with AUC = 0.821 (Fig. 2).
      Fig. 2
      Fig. 2Receiver Operating Characteristic (ROC) curves for: A) I-FABPp and B) I-FABPu, respectively in neonates for predicting NEC progression and severity, I-FABPu vs I-FABPp 0.001∗. C) I-FABPp and D) I-FABPu, respectively in neonates for predicting surgical NEC, -FABPp vs I-FABPu 0.04∗.
      Eight cases were surgically explored who exhibited positive ultrasonographic findings in addition to the clinical judgment; 2 cases were stage II-B (one of them revealed full bowel wall necrotic intestinal segment without actual perforation, i.e. impending perforation); and 6 cases were stage–III–A (five of them confirmed to have impending intestinal perforation).There were higher ranges for both I-FABPp and I-FABPu levels among NEC with intestinal necrosis after exploration (5.71–8.22 ng/ml and 4.41–6.72 ng/g creat., respectively) than NEC without intestinal necrosis after exploration (1.23–4.51 ng/ml and 0.81–5.12 ng/g creat., respectively).

      4. Discussion

      Necrotizing enterocolitis (NEC) is a common gastrointestinal disorder of newborns with high morbidity and mortality.
      • Rees C.M.
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      Neurodevelopmental outcomes of neonates with medically and surgically treated necrotizing enterocolitis.
      Abdominal ultrasound is more sensitive for NEC diagnosis than conventional X-ray in identification of free fluid in the abdominal cavity and in determination of the thickness and the perfusion of the intestinal loops.
      • Bohnhorst B.
      Usefulness of abdominal ultrasound in diagnosing necrotising enterocolitis.
      Our data showed that intramural gas was frequent in 49.1% of the included cases, and portal venous gas was detected in 34.5%, while ascites was present in 29.1%. Yang et al.
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      Diagnostic value of intestinal fatty-acid-binding protein in necrotizing enterocolitis: a systematic review and meta-analysis.
      investigated the role and accuracy of abdominal US in diagnosing NEC and disease severity, they reported that abdominal US showed significantly higher detection rates of portal venous gas and dilatation of the intestine than plain abdominal X-ray film, and the surgery/death group had significantly higher detection rates of dilatation of intestine, bowel wall thickening, peritoneal effusion and free intraperitoneal air compared with the medically treated group.
      Plasma lactate and intestinal IFABP are two surrogate markers of evaluating intestinal mucosal integrity and barrier function, especially in cases with intestinal ischemia.
      • Acosta S.
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      Current status on plasma biomarkers for acute mesenteric ischemia.
      ,
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      D-Lactate and intestinal fatty acid-binding protein are elevated in serum in patients with acute ischemic stroke.
      Lactate is considered a bacterial by-product, so its elevation is thought to indicate enteric bacterial translocation.
      • Demir I.E.
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      Beyond lactate: is there a role for serum lactate measurement in diagnosing acute mesenteric ischemia?.
      Acute intestinal ischemia, reperfusion, and bacterial colonization were associated with failure of the mucosal barrier, increasing the permeability of the intestinal wall and allowing lactate to enter the portal circulation with increased plasma lactate levels in both portal and systemic blood.
      • Nielsen C.
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      • Erlandsen E.J.
      • Mortensen F.V.
      D-lactate as a marker of venous-induced intestinal ischemia: an experimental study in pigs.
      Garcia et al.
      • Garcia J.
      • Smith F.R.
      • Cucinell S.A.
      Urinary D-lactate excretion in infants with necrotizing enterocolitis.
      found that urinary lactate level increased in infants with NEC with increased enteric bacterial activity in those cases. Recently, Lei et al.
      • Lei G.
      • Zhang J.
      • Wang X.
      • Chen M.
      Plasma D-lactate levels in necrotizing enterocolitis in premature infants.
      examined levels of plasma lactate in premature infants affected by NEC, finding them to increase significantly early in direct proportion to the overall extent of intestinal disease, but sensitivity and specificity were not reported. In the current study, plasma lactate was found to be elevated among the included neonates with NEC when compared with the controls.
      I-FABP constitutes up to 2% of the cytoplasmic protein content of the mature enterocyte.
      • Pott J.
      • Hornef M.
      Innate immune signalling at the intestinal epithelium in homeostasis and disease.
      This small cytosolic protein is released into the bloodstream after cell disruption upon loss of enterocyte membrane integrity in bowel wall ischemia,
      • Relja B.
      • Szermutzky M.
      • Henrich D.
      • Maier M.
      • de Haan J.J.
      • Lubbers T.
      • et al.
      Intestinal-FABP and liver-FABP: novel markers for severe abdominal injury.
      ,
      • Girish T.U.
      • Hegde A.
      Intestinal fatty acid binding protein (I-FABP) as a marker for acute intestinal ischemia.
      and it can pass the glomerular filter.
      • van de Poll M.C.
      • Derikx J.P.
      • Buurman W.A.
      • Peters W.H.
      • Roelofs H.M.
      • Wigmore S.J.
      • et al.
      Liver manipulation causes hepatocyte injury and precedes systemic inflammation in patients undergoing liver resection.
      As a consequence, plasma and urinary i-FABP levels may reflect the extent of intestinal epithelial cell damage. Therefore, the plasma I-FABP (I-FABPp) and urinary I-FABP (I-FABPu) are promising biomarkers in early diagnosis of NEC.
      • Yang G.
      • Wang Y.
      • Jiang X.
      Diagnostic value of intestinal fatty-acid-binding protein in necrotizing enterocolitis: a systematic review and meta-analysis.
      Our results revealed significantly higher I-FABP levels in NEC neonates than in the control group, which was in line with many investigators.
      • Benkoe T.M.
      • Mechtler T.P.
      • Weninger M.
      • Pones M.
      • Rebhandl W.
      • Kasper D.C.
      Serum levels of interleukin-8 and gut-associated biomarkers in diagnosing necrotizing enterocolitis in preterm infants.
      ,
      • Abdel-Haie O.M.
      • Behiry E.G.
      • Abd Almonaem E.R.
      • Ahmad E.S.
      • Assar E.H.
      Predictive and diagnostic value of serum intestinal fatty acid binding protein in neonatal necrotizing enterocolitis (case series).
      ,
      • Ng E.W.
      • Poon T.C.
      • Lam H.S.
      • Cheung H.M.
      • Ma T.P.
      • Chan K.Y.
      • et al.
      Gut-associated biomarkers L-FABP, I-FABP and TFF3 and LIT score for diagnosis of surgical necrotizing enterocolitis in preterm infants.
      We also noticed that I-FABPp and I-FABPu levels increased according to Bell's staging at the time of diagnosis of NEC. These data were in accordance with other studies.26,29Also, there were significant positive correlations between IFABPp and IFABPu and between lactate with both IFABPp and IFABPu. In agreement with our findings, Schurink et al.
      • Schurink M.
      • Scholten I.G.
      • Kooi E.M.
      • Hulzebos C.V.
      • Kox R.G.
      • Groen H.
      • et al.
      Intestinal fatty acid-binding protein in neonates with imminent necrotizing enterocolitis.
      emphasized the importance of such biomarkers as indicators of enterocyte damage and compromised intestinal barrier function (increased permeability) that were induced by ischemia and hypoperfusion.
      Radiological findings were the most powerful predictors of the need for surgical intervention including the following; persistent dilation of the bowel loops, evidence of portal venous gas, bowel wall thickening, absent peristalsis and echogenic-free fluid or focal fluid collection. Many authors stated that air in the portal system is one of the indicators of surgical necessity as this is associated with the degree of intestinal necrosis.
      • He Y.
      • Zhong Y.
      • Yu J.
      • Cheng C.
      • Wang Z.
      • Li L.
      Ultrasonography and radiography findings predicted the need for surgery in patients with necrotising enterocolitis without pneumoperitoneum.
      • Kosloske A.M.
      • Musemeche C.A.
      • Ball Jr., W.S.
      • Ablin D.S.
      • Bhattacharyya N.
      Necrotizing enterocolitis: value of radiographic findings to predict outcome.
      • Kosloske A.M.
      Indications for operation in necrotizing enterocolitis revisited.
      • Dos Santos I.G.G.
      • Mezzacappa M.A.
      • Alvares B.R.
      Radiological findings associated with the death of newborns with necrotizing enterocolitis.
      Our findings revealed significantly higher I-FABPp and I-FABPu among neonates with NEC who exhibited positive sonographic findings (portal venous gas and/or ascites) than those who did not, indicating the informative value of such biomarkers regarding diagnosis and severity of NEC.
      Various studies revealed that the levels of IFABP were a helpful marker of evaluating intestinal necrosis.
      • Girish T.U.
      • Hegde A.
      Intestinal fatty acid binding protein (I-FABP) as a marker for acute intestinal ischemia.
      ,
      • Abdel-Haie O.M.
      • Behiry E.G.
      • Abd Almonaem E.R.
      • Ahmad E.S.
      • Assar E.H.
      Predictive and diagnostic value of serum intestinal fatty acid binding protein in neonatal necrotizing enterocolitis (case series).
      ,
      • Vermeulen Windsant I.C.
      • Hellenthal F.A.
      • Derikx J.P.
      • Prins M.H.
      • Buurman W.A.
      • Jacobs M.J.
      • et al.
      Circulating intestinal fatty acid-binding protein as an early marker of intestinal necrosis after aortic surgery: a prospective observational cohort study.
      We plotted a ROC curve for both serum and urinary I-FABP as a marker for diagnosis of NEC, discriminating stage- II from stage-III with equal sensitivity (90% for each), but with higher specificity for I-FABPu (92%) than I-FABPp. The characteristic performances of both assays in the prediction of surgical NEC revealed that I-FABPp (100%) was more specific than I-FABPu (76.19%). Several studies found plasmatic concentrations of I-FABP to be a specific marker for early identification of severe NEC (Bell's stage III), they were less useful for differentiating initial Bell's stages.
      • Edelson M.B.
      • Sonnino R.E.
      • Bagwell C.E.
      • Lieberman J.M.
      • Marks W.H.
      • Rozycki H.J.
      Plasma intestinal fatty acid binding protein in neonates with necrotizing enterocolitis: a pilot study.
      ,
      • Guthmann F.
      • Börchers T.
      • Wolfrum C.
      • Wustrack T.
      • Bartholomäus S.
      • Spener F.
      Plasma concentration of intestinal- and liver-FABP in neonates suffering from necrotizing enterocolitis in healthy preterm neonates.
      A multicenter study conducted by Heida et al.
      • Heida F.H.
      • Hulscher J.B.
      • Schurink M.
      • Timmer A.
      • Kooi E.M.
      • Bos A.F.
      • et al.
      Intestinal fatty acid-binding protein levels in necrotizing enterocolitis correlate with extent of necrotic bowel: results from a multicenter study.
      demonstrated that both plasma and urine I-FABP levels were strongly associated with the length of bowel resection in newborns with surgical NEC, supporting the hypothesis that increased I-FABP levels correspond with the extent of necrotic tissue. Additionally, Evennett et al.
      • Evennett N.J.
      • Hall N.J.
      • Pierro A.
      • Eaton S.
      Urinary intestinal fatty acid-binding protein concentration predicts extent of disease in necrotizing enterocolitis.
      concluded that I-FABPu concentration could predict the extent of disease in necrotizing enterocolitis.

      5. Conclusions

      The current study provides additional evidence regarding the utility of serum and urinary I-FABP levels, when combined with clinical and imaging data, as a useful informative marker for early diagnosis and prediction of disease severity in NEC and in early recognition of impending perforation, thus decreasing the mortality associated with perforated NEC among neonates.

      6. Study limitations

      Its small sample size and lack of serial measurements of the studied biomarkers among the included cases were the main study limitations.

      Funding

      The current research has been funded by the authors themselves.

      Declaration of Competing Interest

      No potential conflict of interest was reported by the authors.

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