Potential Role of Serum Intestinal Fatty Acid-Binding Protein as a Marker for Early Prediction and Diagnosis of Necrotizing Enterocolitis in Preterm Neonates.

INTRODUCTION: Intestinal fatty acid-binding protein (I-FABP) is located in the apex of mature enterocytes and released into circulation; once the injury of enterocyte happens, its circulating levels are considered an early and sensitive marker of intestinal ischemia as in necrotizing enterocolitis (NEC); because of its small molecular weight, it can be detected in urine. AIMS: The aim was to study the usefulness of both serum and urine I-FABP in early diagnosis of NEC and to correlate the serum and urinary levels. SETTINGS AND
DESIGN: This study was case-control design.
METHODS: Simultaneous serum and urine samples obtained at the onset of symptoms, in 40 preterms with suspected NEC, with gestational age ± 27.70 weeks and birth weight ± 1.11 kg, i.e., 20 preterms diagnosed at Stage I, 12 preterms at Stage II, and 8 preterms at Stage III, were compared with age- and weight-matched preterms with no NEC. STATISTICAL ANALYSIS: The collected data were tabulated, coded, and then analyzed using the computer program Statistical Package for the Social Science (SPSS version 22).
RESULTS: Serum levels of I-FABP in NEC cases were significantly higher than the control group, with a mean of 6005.77 ± 6384.77 and 1480.79 ± 1276.48 pg/ml, respectively (P < 0.001). Urine levels of I-FABP in NEC cases were significantly higher than the control group, with a mean of 5009.22 ± 3941.64 and 2677.62 ± 2257.29 pg/ml, respectively (P = 0.04). Both serum and urine I-FABP levels not only in Stage II are significantly higher than Stage I but also in Stage III are significantly higher than Stage I and II (P < 0.001, P = 0.03, respectively), which showed significant positive correlation with stages of NEC (r = 0.618; P < 0.001; r = 0.306; P = 0.049, respectively). Both serum and urine I-FABP levels had a highly significant positive correlation with each other (r = 0.406 P < 0.0001). Receiving operating characteristic curve showed an area under the curve of 0.92 and 0.81 for serum and urine I-FABP, respectively.
CONCLUSIONS: Whether serum or urinary I-FABP is valuable in the diagnosis and prediction of NEC and strongly correlated with the disease severity and with each other. Copyright:

INTRODUCTION

Necrotizing enterocolitis (NEC) is a serious gastrointestinal disorder, considered the second most common cause of death in preterm infants, with a morbidity of 10%–15% and a mortality of 20%–40%.[1] Most commonly, it occurs in very low-birth-weight (BW) infants.[2] Prematurity,[3] enteral feeding, bacterial colonization, and ischemia of the gastrointestinal tract are risk factors for the development of NEC.[45]

It occurs in 1–3 per 1000 live births, and surgical treatment is necessary for 20%–40%. It remains an important cause of long-term morbidity, so early detection and proper management can improve clinical outcomes.[6]

The underlying mechanisms still poorly understood, in preterm neonates. Immaturity of the gut,[7] besides, a gut-derived sepsis, and increased intestinal permeability and inadequate intestinal immune responses play an important role in the pathology of NEC.[8]

NEC most frequently presents without warning and rapidly progresses to a condition requiring resection of bowel and/or death.[9]

NEC is difficult to predict and to be diagnosed during the initial symptoms, and because it has a sudden onset and rapid progress, it is difficult to treat.[10]

Early diagnosis is difficult because the initial symptoms are nonspecific and difficult to differentiate from sepsis and other gastrointestinal problems. Besides, current methods of diagnosis depend on the signs of already established or progressive disease.[11]

Because of the high mortality and morbidity associated with NEC and its fulminate course, and because early diagnosis is important for therapy and outcome, early detection is life-saving. This is possible through biomarkers that are increased before the clinical manifestations are established, thus giving time for early intervention and improving the management of NEC.[12]

The ideal biomarkers should reflect the main early steps in the pathogenesis of the disease. NEC is characterized by mucosal destruction. Therefore, identification of a noninvasive test that will reflect this destruction before its appearance on X-ray will be ideal.[6]

Fatty acid-binding proteins (FABPs) are a family of widely expressed lipid transport cytoplasmic proteins that have excellent organ specificity and are immediately delivered into the systemic circulation upon cell damage.[13]

Intestinal fatty acid-binding protein (I-FABP), a member of the FABPs family, is a water-soluble protein accounting for 2% of the cytoplasmic proteins in the mature enterocyte, with a small molecular weight of 14–15 kDa.[14]

I-FABP is expressed along the overall length of the small intestine, from the duodenum to the cecum; however, the majority is found to be abundant in the jejunum.[1]

Its highest concentration is found in the villus rather than crypt cells, located in the apex rather than the base of intestinal cells.[15]

Once the enterocyte is injured, the cytoplasmic content of I-FABP could be released into the systemic circulation. When intestinal ischemia is of a short time (<2 h), only the villi are affected, while the crypt cells remain intact. And because I-FABP is mainly present in the villi and not in the crypt. Hence, its circulating levels are considered an early and sensitive marker of intestinal ischemia.[16]

Therefore, theoretically, it indicates the intestinal epithelial cell injury in the very early phase; and because the intestinal epithelial barrier disruption occurs in the early steps of NEC pathogenesis, circulating I-FABP could be a useful plasma marker in the early stages of NEC.

Because of its small molecular weight, I-FABP could pass the glomerular filter and could be detected in urine, and because I-FABP could not be expressed in the urinary tract, I-FABP levels could reflect intestinal mucosal damage. Because its renal excretion is 28% and its half-life is 11 min, its level could reflect the extent of intestinal mucosal damage. Besides its release is cumulative, I-FABP could estimate the extent of intestinal damage, which is of great importance for management, including the time of surgery, antibiotic duration, and time of starting enteral feeding. Hence, urinary I-FABP is supposed to be a good biomarker for NEC.[17]

We aim to study the usefulness of both serum and urine I-FABP in the diagnosis and prediction of NEC in preterm neonates and to assess the relationship of their levels with each other and with NEC severity in premature infants.

Thus, a direct comparison between plasma and urinary gut barrier proteins in NEC infants is necessary for determining the usefulness of different types of specimen samples.

METHODS

Patients

This case–control study was carried out on preterm babies selected from the neonatal intensive care units (NICUs) of the Paediatric Department at Benha University Hospital and Benha Children Specialized Hospital from May 2017 to May 2018. Approval of the study protocol by the Ethical Scientific Committee of Benha University was obtained, and informed consent was obtained from the parents before enrollment in the study. A total of 200 preterm newborn babies aging <35 weeks and weighing <2 kg were enrolled in the study, to evaluate which of them will develop NEC.

Forty patients suspected of developing NEC were enrolled, with gestational age (GA) ranging from 25 to 29 weeks, with a mean of 27.70 ± 0.911 weeks, and BW ranging from 0.650 to 1.710 kg, with a mean of 1.11 ± 0.21 kg. These patients were later divided according to final diagnosis into NEC Stage I n = 20, Stage II n = 12, Stage III n = 8 as a case group.

Forty preterm babies with GA ranging from 25 to 33 weeks, with a mean of ± 29.55 weeks, and BW ranging from 0.780 to 1.655 kg, with a mean of ± 1.22 kg, who were admitted to the NICU with no evidence of sepsis, or systemic inflammatory response syndrome, and no signs of NEC at the time of sampling defined as control group. Exclusion criteria were full-term neonates, hypoxic-ischemic encephalopathy, congenital malformation, and fatal chromosomal defects or inborn errors of metabolism, with other causes of feeding intolerance.

For purposes of this study, the staging of Walsh and Kliegman was simplified; Stage I comprises Stages IA and IB, Stage II comprises Stages IIA and IIB, and Stage III comprises Stage IIIA and IIIB. If pneumatosis intestinalis was diagnosed on X-ray, the case was assigned to Stage II. Suspected NEC was defined as Bell Stage I, with only nonspecific symptoms, such as gastric retention, abdominal distension, mild ileus, temperature instability, apnea, lethargy, increased gastric residuals, abdominal distension, and occult blood in the stool. Neonates with definite NEC II or III had pneumatosis intestinalis. Neonates with Stage III had organ failure, peritonitis, ascites, or perforation and need laparotomy.

All babies included are subjected to full history-taking, full clinical examination, radiological studies for signs of NEC.

From complete blood count (CBC), C-reactive protein, blood culture, to rule out sepsis was done at the following time points for the case group: first 24 h of birth, at the time of diagnosis of NEC, and at the time of progression to a higher stage of the disease; and for the control group: first 24 h of birth.

I-FABP levels were measured in serum and urine using commercially available enzyme-linked immunosorbent assay (ELISA) kits. I-FABP levels were measured at the following two time points for cases group: at the time of diagnosis of NEC and at the time of progression of the disease to a higher stage.

Sample collection

Five milliliters of venous blood was withdrawn under complete aseptic conditions and divided as follows:

Two milliliters was collected in an EDTA vacutainer (violet cap), mixed up, and down gently to prevent clotting, which was used for CBC measuring, using a fully automated cell counter (Sysmex 5, Xs 800)

Three milliliters was collected in a serum separator tube; where samples allowed to clot for 30 min before centrifuged for 15 min at approximately 1000 rpm, and then the separated serum stored at −80°C till the time of assay.

Urine was aseptically collected using a urine bag or catheter if available (all samples suspected to be contaminated with the stool were discarded); then, samples are centrifuged to remove particulate matter and supernatant stored at −80C till the time of assay.

Enzyme-linked immunosorbent assay

The level of I-FABP was measured using human I-FABP ELISA (sandwich technique) kits provided by EIAab©, China (Wuhan EIAab Science Co., Ltd) (Catalog No. E0559 h), which is certified for both serum and urine. The assay was performed according to the manufacturer's instruction and the I-FABP concentration in serum presented as pg/ml.

Statistical analysis

The collected data were tabulated, coded, and then analyzed using the computer program Statistical Package for the Social Science (IBM SPSS statistics for windows, version 22.0. ,Armonk, NY:IBM Corp,USA).

Data were expressed as mean ± standard deviation, number, and percentage.

The Student's t-test was used to compare the mean of two groups of numerical (parametric) data. Analysis of variance was used to compare between more than two groups of numerical (parametric) data. For continuous nonparametric data, Mann–Whitney U-test was used for intergroup analysis. Pearson's correlation coefficient (r) test was used to correlate different parameters. Intergroup comparison of categorical data was performed by using the Chi-square test and Fisher's exact test. The receiving operating characteristic (ROC) curve analysis was used to determine the diagnostic power of each test.

A P < 0.05 was considered statistically significant(s), and P < 0.0001 was considered highly significant (HS) in all analyses.

RESULTS

During the study period, 40 preterm babies evaluated with NEC defined as the case group; 20 of them diagnosed at Stage I NEC (50%), 12 at Stage II NEC (30%), and 8 at Stage III NEC (20%) and 40 preterm babies of matched GA and weight with no signs of NEC defined as a control group [Table 1].

Table 1

Demographic characteristics of patients

Variable	NEC group (n=40)	Control group (n=40)	P
Gestational age (weeks) (mean±SD)	27.70±0.91	29.55±2.51	<0.001
Birth weight (kg) (mean±SD)	1.11±0.21	1.22±0.25	0.03
Male, n (%)	20 (50)	22 (55)	0.7
Female, n (%)	20 (50)	18 (45)
SVD, n (%)	4 (10)	10 (25)	0.08
C/S, n (%)	36 (90)	30 (75)

SD: Standard deviation, NEC: Necrotizing enterocolitis, SVD: spontaneous vaginal delivery, C/S: Cesarean section

A full blood count is done for all patients and showed a significant statistical difference in platelet count between NEC patients and the control group, where the mean platelet count in NEC and the control group was 164.88 ± 59.41 × 109/L 280.85 ± 115.44 × 109/L, respectively (P < 0.001).

The mean serum levels of I-FABP measured in NEC cases were 6005.77 ± 6384.77 pg/ml and the mean serum levels of I-FABP in the control group were 1480.79 ± 1276.48 pg/ml, which was significantly higher than the control group (P < 0.001).

Urine levels of I-FABP in NEC cases were 5009.22 ± 3941.64 pg/ml while urine concentrations of I-FABP in the control group were 2677.62 ± 2257.29 pg/ml, which was lower than those of the NEC group (P = 0.04) [Table 2].

Table 2

Levels of serum and urine intestinal fatty acid-binding protein in cases and control group

Variable	Mean±SD		P
	NEC group (n=40)	Control group (n=40)
Serum I-FABP	6005.77±6384.77	1480.79±1276.48	˂0.001
Urine I-FABP	5009.22±3941.64	2677.62±2257.29	0.04

I-FABP: Intestinal fatty acid-binding protein, SD: Standard deviation

Serum I-FABP levels, according to Bell classification, showed that the mean serum I-FABP Stage II is significantly higher than Stage I and the mean serum I-FABP in Stage III is significantly higher than Stage I and II (P < 0.001).

Urine I-FABP levels, according to Bell classification, showed that the mean serum I-FABP Stage II is significantly higher than Stage I and the mean serum I-FABP in Stage III is significantly higher than Stage I and II (P = 0.03) [Table 3].

Table 3

Levels of serum and urine intestinal fatty acid-binding protein in necrotizing enterocolitis cases according to Bill classification

Variable	Mean±SD			P
	Stage I (n=20)	Stage II (n=12)	Stage III (n=8)
Serum I-FABP pg/ml	1634.38±897.35	9910.08±8506.92	11,077.75±1041.84	<0.001
Urine I-FABP pg/ml	3923.75±3396.42	5142.57±4173.39	9522.88±4154.74	0.03

SD: Standard deviation, I-FABP: Intestinal fatty acid-binding protein

Serum I-FABP levels showed a high significant positive correlation with stages of NEC (r = 0.618; P < 0.001) [Figure 1]. Furthermore, urine levels showed a significant positive correlation with stages of NEC (r = 0.306; P = 0.049) [Figure 2].

Figure 1

Correlation between serum intestinal fatty acid-binding protein and necrotizing enterocolitis stages

Figure 2

Correlation between urine intestinal fatty acid-binding protein and s necrotizing enterocolitis stages

Serum I-FABP levels were correlated with urine I-FABP levels, and it was found a HS-positive correlation between serum I-FABP with urine I-FABP levels (r = 0.406; P < 0.0001) [Figure 3].

Figure 3

Correlation between serum intestinal fatty acid-binding protein with urine intestinal fatty acid-binding protein

ROC curve was constructed to study levels of both serum and urine I-FABP in the diagnosis of NEC where serum I-FABP showed an area under the curve (AUC) of 0.92 and urine I-FABP showed an AUC of 0.81 [Figure 4].

Figure 4

Receiving operating characteristic curve showing serum intestinal fatty acid-binding protein (area under the curve is 0.92) and urine intestinal fatty acid-binding protein (area under the curve is 0.81) in diagnosis of necrotizing enterocolitis

DISCUSSION

Early diagnosis and detection of patients who will develop NEC are still difficult because current laboratory and radiological methods have no enough differentiating power.[1819] Hence, there is a strong need for identification of new biomarkers, suitable for early diagnosis of NEC, which would give the chance for early intervention. FABPs are a family of widely distributed cytoplasmic proteins that have excellent organ specificity and are immediately delivered into the bloodstream upon the damage of cells.[13] As a member of the FABPs family, I-FABP is expressed in the intestine from the duodenum to the cecum and accounts for 2% of cytoplasmic proteins in mature enterocytes and could be measured in the blood and urine after injury of enterocytes.[14]

In our study, we found that the mean serum level of I-FABP in NEC patients is higher than the control group with a high statistical significance difference (P < 0.001) [Table 2].

In the first quantitative radioimmunoassay for human I-FABP,[20] it was reported that plasma I-FABP is higher in intestinal ischemia and might serve as a diagnostic marker for early intestinal mucosal compromise, as ischemia and loss of intestinal wall integrity are part of the pathogenesis of NEC, and I-FABP levels found higher in NEC patients. Our results corroborate their findings; however, in contrast to our findings, they were unable to detect I-FABP in urine, which may be attributed to the lower detection limit of the radioimmunoassay (1.9 ng/ml) while of ELISA assay detection limit is 0.1 ng/ml.

Many studies have compared plasma levels of I-FABP in preterm neonates developed NEC with healthy preterm neonates,[21] serum I-FABP with interleukin-8,[22] and plasma I-FABP with other gut barrier proteins (TFF3) in preterm neonates with NEC and with neonatal sepsis and with control patients (i.e., nonsepsis/non-NEC infants). In agreement with our findings, all reported that I-FABP levels were significantly higher I-FABP in NEC patients than control patients and patients with sepsis.[23]

Furthermore, we compared serum I-FABP levels, according to Bell staging at the time of diagnosis of NEC; we found that the mean serum I-FABP in Stage II is higher than in Stage I and the mean serum I-FABP in Stage III is higher than Stage I and Stage II, with a high statistical significance difference between stages of NEC patients (P < 0.001) Table 3.

Pearson's correlation coefficient test is used to correlate the serum level with disease stages, and serum level shows a highly statistically significant positive correlation with stages of NEC (r = 0.618, P = 0.000), indicating that the serum I-FABP level correlates with the severity of the disease [Figure 1].

In a study,[24] to determine if plasma I-FABP levels early in the course of NEC would differentiate patients by severity to know who had Stage III NEC and who had milder stages. I-FABP levels were measured by radioimmunoassay. They reported that at each time point, levels in Stage III subjects were significantly higher compared with the other stages while I-FABP levels undetectable in the majority of infants with Stage II NEC. In contrast to their findings, we found I-FABP levels detected in all stages and increasing with the highest in Stage III; this is due to the higher sensitivity of ELISA assay than radioimmunoassay.

Our results confirm the findings of another work[25] which reported the value of serum I-FABP in early diagnosis and predicting the severity of NEC. They reported that initial serum I-FABP levels were significantly higher in NEC patients than those of the control group. Further, reported I-FABP levels were related to the severity of NEC, where they found higher serum I-FABP levels at 72 h of suspicion of NEC in infants with Stage III NEC compared with Stage I or II NEC, but we found higher levels at the onset of symptoms and this may be related to the extent and severity of the NEC at the time of sampling.

In our study, levels of urine, I-FABP found higher in NEC patients, than the control group with a significant statistical difference (P = 0.04) [Table 2].

This corroborates the results of a study[26] done for the detection of intestinal mucosal cell damage in neonates with suspected NEC through daily urine I-FABP, and another study[27] measured fecal calprotectin (in the stool) and serum amyloid A in the urine combined with urinary I-FABP in NEC patients. Where reported that urinary I-FABP levels were significantly higher in neonates who developed NEC.

Similarly, it was reported that urinary I-FABP was elevated in all infants who developed NEC than control babies; even more, few control babies had a transient increase associated with episodes of feeding intolerance or sepsis.[28] This finding means that episodes of feeding intolerance or sepsis are associated with some degree of mucosal injury that usually resolves, as the villi heal, especially if feedings are advanced slowly.

In addition, we found urine I-FABP levels, according to Bell staging at the time of diagnosis of NEC, and found that the mean urine I-FABP levels in Stage II are higher than Stage I and Stage III are higher than Stage I and II with significant statistical significance difference between stages of NEC patients (P = 0.03) [Table 3].

Pearson's correlation coefficient test showed that urine level had a significant positive correlation with stages of NEC which reflects the severity of bowel injury (r = 0.306, P = 0.049), indicating that the urinary I-FABP level positively correlates with the severity of the disease [Figure 2].

In agreement with our work, another group[14] studied the relationship between urinary I-FABP and the extent of disease found at laparotomy and reported an increase in urinary I-FABP in infants with NEC than control, and infants with more severe NEC (multifocal) had significantly higher preoperative urinary I-FABP compared with those infants with focal disease.

Similarly, urinary I-FABP and claudin-3 and fecal calprotectin levels were compared concerning the severity of NEC.[29] In line with our findings, the authors reported that the median urinary I-FABP was significantly higher in neonates who developed NEC than in those with other diagnoses, and higher urinary I-FABP levels were found in neonates with severe NEC (NEC necessitating surgery or causing death) compared with neonates with conservatively managed NEC.

Although several studies report on I-FABP measured in the plasma[2124] and serum[25] and several others report on the use of urinary I-FABP measurements,[262729] no works compare or correlate simultaneous serum and urine levels in NEC patents.

Most of these studies reported high serum and urinary I-FABP in NEC than the control but without comparing or correlating both levels at the same time point. To the best of our knowledge, very few studies have reported on comparing I-FABP measured simultaneously in the serum and urine in preterm babies.

In our study, we correlated levels of both urine and serum I-FABP where we found a strong, statistically significant positive correlation between both levels (r = 0.406, P = 0.000) [Figure 3].

Our results corroborate findings of a study done as part of the prospective cohort “No NEC” trial in the Dutch Trial Registry,[30] where they found that 156 samples were obtained simultaneously (i.e., both plasma and urine), where reported a strong correlation was found between plasma I-FABP and urine I-FABP levels, measured during the first 24 h after onset of symptoms. In contrast to our study, they reported urine I-FABP values higher than plasma I-FABP values, but we found urine levels lower than serum levels, especially in Stage II and III; in addition to that our study is single sampling, this may be related to severity and extent of the disease at the time of the sample, or because of use of a different ELISA to measure I-FABP, where the lack of standardized I-FABP preparations and/or reference sera may affect the results and make the comparison between studies difficult.

The ROC curve constructed for both serum and urine levels showed that the AUC is for serum I-FABP is 0.92 and the AUC is for urine I-FABP is 0.81, indicating that they are very good diagnostic markers with high sensitivity and specificity in the diagnosis of NEC [Figure 4].

As a result, it was concluded that I-FABP levels can differentiate NEC patients from those with nonspecific symptoms, both serum I-FABP and urinary I-FABP correlated with the severity of the disease and differentiate surgical from nonsurgical cases.

In neonates with suspected NEC, urinary I-FABP correlates strongly with serum I-FABP, offering an opportunity to choose the most appropriate way of measuring I-FABP. Blood analysis has the advantage that a blood sample is always available. Because NEC could be a rapidly progressive disease, frequent blood sampling would be needed. Meanwhile, every blood sample means an extra burden for the (sick) baby (even in the presence of an arterial line), so noninvasive measurements are preferable.[31] Urine is a good alternative to blood sampling. Only in severe cases when urine output is affected, blood analysis will be superior.

Our findings provide further evidence of the association between elevated levels of I-FABP and NEC, suggesting that I-FABP may be useful as a diagnostic and predictive biomarker for earlier identification of NEC.

CONCLUSIONS

In preterm infants with NEC, the serum I-FABP level is significantly increased than in healthy preterm babies.

Serum I-FABP is strongly positively correlated with disease severity. It is a highly specific and sensitive protein for intestinal mucosal damage. ROC curve plotting demonstrates that it is an excellent test in the diagnosis of NEC.

Therefore, serum I-FABP can be a valid serologic biomarker for early prediction and diagnosis of NEC and determination of the severity of the disease in premature neonates.

Urinary I-FABP levels were significantly increased in preterm infants with NEC than in healthy preterm babies. And level is positively correlated with the severity of the disease.

Urinary I-FABP strongly correlates with serum I-FABP. Moreover, ROC curve plotting demonstrates that it is a good test in the diagnosis of NEC giving clinician opportunity to choose the most appropriate way of measuring I-FABP, especially when the clinical symptoms of NEC are nonspecific and when NEC can be confused with neonatal sepsis.

Whether serum I-FABP or urinary I-FABP is valuable in the diagnosis and prediction of NEC. Moreover, urinary I-FABP monitoring may become a routine component of the management of premature infants.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.