A pilot case-control study of urine metabolomics in preterm neonates with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of gastrointestinal morbidity and mortality in preterm neonates. The aim of this pilot study was to explore using metabolomics alternations in the urine metabolites related to NEC that could possibly serve as diagnostic biomarkers of the disease. Urine samples were prospectively collected at the day of initial evaluation for NEC from 15 diseased preterm neonates (five Bell's stage I and ten stage II/III) and an equal number of matched controls. Urine metabolic profiles were assessed using non-targeted nuclear magnetic resonance spectroscopy and targeted liquid chromatography-tandem mass spectrometry monitoring 108 metabolites. Multivariate statistical models with data from either analytical approach showed clear separation between the metabolic profiles of neonates with NEC and controls. Twenty-five discriminant metabolites were identified belonging to amino and organic acids, sugars and vitamins. A number of metabolite combinations were found to have an excellent diagnostic performance in detecting neonates developing NEC. Our results show that the metabolic profile of neonates with NEC differs significantly from that of controls, making possible their separation using urine metabolomic analysis. Nevertheless, whether the small set of significant metabolites detected in this investigation could be used as early diagnostic biomarkers of NEC should be validated in larger studies.

Non-invasive assessment of oxidative stress in preterm infants.

Preterm newborns have an immature antioxidant defense system and are especially susceptible to oxidative stress. Resuscitation, mechanical ventilation, intermittent hypoxia and apneic episodes require frequently oxygen supplementation which leads to oxidative stress in preterm newborns. The consequences of oxidative damage are increased short and long-term morbidities, neurodevelopmental impairment and increased mortality. Oxidative stress biomarkers are determined in blood samples from preterm children during their stay in neonatal intensive care units especially for research purposes. However, there is a tendency towards reducing invasive and painful techniques in the NICU (Neonatal Intensive Care Unit) and avoiding excessive blood extractions procedures. In this paper, it has been described some studies that employed non-invasive samples to determine oxidative stress biomarkers form preterm infants in order to perform a close monitoring biomarker with a significant greater predictive value. Among these methods we describe a previously developed and validated high-performance liquid chromatography tandem mass spectrometry method that allow to accurately determine the most reliable biomarkers in biofluids, which are non-invasively and painlessly obtained.

Prostaglandin E-major urinary metabolite as a noninvasive surrogate marker for infantile necrotizing enterocolitis.

BACKGROUND: Early definitive diagnosis of necrotizing enterocolitis (NEC) based on Bell's staging criteria is difficult because there are few observable changes on abdominal imaging and blood chemistry tests at the onset of the disease. PURPOSE: To investigate whether prostaglandin E-2 major urinary metabolite (PGE-MUM) can be a useful surrogate marker reflecting the disease state and severity of NEC in infants. METHODS: Infants were enrolled in this study between January 2014 and December 2016. NEC diagnosis was based on Bell's staging criteria > Stage II or necrotic bowel observed at surgery. After diagnosis, PGE-MUM level was measured and compared with that of the other disease and healthy infant groups. RESULTS: Median PGE-MUM value was highest in the NEC group (576 [65-3672] µg/g•Cre/BSA × 1000), followed by the other disease group (94 [57-296] µg/g•Cre/BSA × 1000) and the healthy infant group (19 [10-44] µg/g•Cre/BSA × 1000) (sensitivity: 92.3%, specificity: 81.5%, accuracy: 85.0%; p < 0.01). PGE-MUM level correlated with improved status of NEC, length of necrotic intestine, and Bell's staging criteria. CONCLUSIONS: PGE-MUM level may be a useful surrogate biomarker reflecting the disease state of NEC. The method of urine sample collection is also advantageous, being noninvasive for infants. This is the first study reporting PGE-MUM level in NEC. TYPE OF STUDY: Study of diagnostic test. LEVEL OF EVIDENCE: LEVEL II.

Antibiotic Treatment Preventing Necrotising Enterocolitis Alters Urinary and Plasma Metabolomes in Preterm Pigs.

Necrotising enterocolitis (NEC) is a serious gut inflammatory condition in premature neonates, onset and development of which depend on the gut microbiome. Attenuation of the gut microbiome by antibiotics can reduce NEC incidence and severity. However, how the antibiotics-suppressed gut microbiome affects the whole-body metabolism in NEC-sensitive premature neonates is unknown. In formula-fed preterm pigs, used as a model for preterm infants, plasma and urinary metabolomes were investigated by LC-MS and 1H NMR, with and without antibiotic treatment immediately after birth. While it reduced the gut microbiome density and NEC lesions as previously reported, the antibiotic treatment employed in the current study affected the abundance of 44 metabolites in different metabolic pathways. In antibiotics-treated pigs, tryptophan metabolism favored the kynurenine pathway, relative to the serotonin pathway, as shown by specific metabolites. Metabolites associated with the gut microbiome, including 3-phenyllactic acid, 4-hydroxyphenylacetic acid, and phenylacetylglycine, all from phenylalanine, and three bile acids showed lower levels in the antibiotics-treated pigs where the gut microbiome was extensively attenuated. Findings in the current study warrant further investigation of metabolic and developmental consequences of antibiotic treatment in preterm neonates.

The relationship of red blood cell transfusion to intestinal mucosal injury in premature infants.

OBJECTIVE: To determine the incidence of intestinal mucosal injury before and after transfusions in premature infants. STUDY DESIGN: Urine was collected throughout the hospital stay of 62 premature infants and specimens obtained within 24h before and after transfusion were assayed for intestinal fatty acid binding protein (iFABP). A urinary iFABP:creatinine ratio (iFABPu:Cru) of 2.0pg/nmol was considered elevated. RESULT: Forty-nine infants were transfused. iFABPu:Cru was elevated following 71 (75.6%) of 94 transfusions for which urine was available. In 51 (71.8%) of these, iFABPu:Cru was also elevated prior to the transfusion. Among four cases of transfusion-associated NEC, iFABPu was elevated following every sentinel transfusion and prior to three of them. CONCLUSION: Subclinical intestinal mucosal injury is frequent following blood transfusions in premature infants and, when present, usually precedes transfusion. This suggests that transfusion may not be a primary mediator of intestinal injury so much as anemia and its associated conditions. LEVEL OF EVIDENCE: Prognosis study/level 3.

Urinary Intestinal Fatty Acid-Binding Protein Can Distinguish Necrotizing Enterocolitis from Sepsis in Early Stage of the Disease.

Necrotizing enterocolitis (NEC) is severe disease of gastrointestinal tract, yet its early symptoms are nonspecific, easily interchangeable with sepsis. Therefore, reliable biomarkers for early diagnostics are needed in clinical practice. Here, we analyzed if markers of gut mucosa damage, caspase cleaved cytokeratin 18 (ccCK18) and intestinal fatty acid-binding protein (I-FABP), could be used for differential diagnostics of NEC at early stage of disease. We collected paired serum (at enrollment and week later) and urine (collected for two days in 6 h intervals) samples from 42 patients with suspected NEC. These patients were later divided into NEC (n = 24), including 13 after gastrointestinal surgery, and sepsis (n = 18) groups using standard criteria. Healthy infants (n = 12), without any previous gut surgery, served as controls. Both biomarkers were measured by a commercial ELISA assay. There were no statistically significant differences in serum ccCK18 between NEC and sepsis but NEC patients had significantly higher levels of serum and urinary I-FABP than either sepsis patients or healthy infants. Urinary I-FABP has high sensitivity (81%) and specificity (100%) and can even distinguish NEC from sepsis in patients after surgery. Urinary I-FABP can be used to distinguish NEC from neonatal sepsis, including postoperative one, better than abdominal X-ray.

Metabolomics in Newborns.

Metabolomics is the quantitative analysis of a large number of low molecular weight metabolites that are intermediate or final products of all the metabolic pathways in a living organism. Any metabolic profiles detectable in a human biological fluid are caused by the interaction between gene expression and the environment. The metabolomics approach offers the possibility to identify variations in metabolite profile that can be used to discriminate disease. This is particularly important for neonatal and pediatric studies especially for severe ill patient diagnosis and early identification. This property is of a great clinical importance in view of the newer definitions of health and disease. This review emphasizes the workflow of a typical metabolomics study and summarizes the latest results obtained in neonatal studies with particular interest in prematurity, intrauterine growth retardation, inborn errors of metabolism, perinatal asphyxia, sepsis, necrotizing enterocolitis, kidney disease, bronchopulmonary dysplasia, and cardiac malformation and dysfunction.

Changes in plasma and urinary nitrite after birth in premature infants at risk for necrotizing enterocolitis.

BACKGROUND: Plasma nitrite serves as a reservoir of nitric oxide (NO) bioactivity. Because nitrite ingestion is markedly lower in newborns than adults, we hypothesized plasma nitrite levels would be lower in newborns than in adults, and that infants diagnosed with necrotizing enterocolitis (NEC), a disease characterized by ischemia and bacterial invasion of intestinal walls, would have lower levels of circulating nitrite in the days prior to diagnosis. METHODS: Single blood and urine samples were collected from 9 term infants and 12 adults, 72 preterm infants every 5 d for 3 wk, and from 13 lambs before and after cord occlusion. RESULTS: Nitrite fell 50% relative to cord levels in the first day after birth; and within 15 min after cord occlusion in lambs. Urinary nitrite was higher in infants than adults. Plasma and urinary nitrite levels in infants who developed NEC were similar to those of preterm control infants on days 1 and 5, but significantly elevated at 15 and 20 d after birth. CONCLUSION: Plasma nitrite falls dramatically at birth while newborn urinary nitrite levels are significantly greater than adults. Acute NEC is associated with elevated plasma and urinary nitrite levels.

Urine biomarkers for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in premature neonates. Despite decades of investigation, treating clinicians are still not able to determine which premature infants are at greatest risk of developing NEC and which of the affected infants will develop severe NEC requiring operation. A biomarker is a specific molecular indicator that can be used to identify or measure the progress of a disease. Many potential biomarkers have been studied for their potential relevance to NEC. Those showing promise include C-reactive protein, intestinal fatty acid-binding protein, platelet-activating factor and many others. None to date have achieved sufficient predictive value to be clinically useful. Distinguishing between the specific changes in NEC and the non-specific inflammation of sepsis has proven challenging. Urine is a particularly attractive site for potential biomarkers. It can be collected readily and non-invasively, and it is a rich source of both proteins and peptides. Preliminary work has revealed some promising biomarkers of NEC in urine. Combined with clinical data, they have been shown to be highly predictive in small series of patients. Advances in high-throughput molecular analysis have opened the door to finding biomarkers that may meaningfully improve the outcome of infants at risk for NEC.

Intestinal fatty acid-binding protein as a diagnostic marker for complicated and uncomplicated necrotizing enterocolitis: a prospective cohort study.

BACKGROUND: Early NEC symptoms are non-specific and diagnostic tests lack discriminative power. Intestinal fatty acid-binding protein (I-FABP), mainly located in small bowel enterocytes, is released into the blood following NEC-associated enterocyte disruption. Aim of this prospective cohort trial was to determine the diagnostic value of I-FABP measured in plasma (I-FABPp) and urine (I-FABPu) for the presence of NEC, to evaluate I-FABP levels during NEC development, and to assess its prognostic value for the progression from suspected to complicated disease. METHODS: Between 2010 and 2012 we prospectively enrolled neonates with suspected NEC. We measured I-FABP levels eight-hourly from onset of suspected NEC for at least 48 hours, or until surgery. NEC diagnosis was confirmed radiologically or during operation. We defined NEC as complicated if it resulted in surgery and/or death. We determined disease course and diagnostic I-FABP cut-off points. RESULTS: The study comprised 37 neonates (24M, 13F), gestational age 28 (24-36) weeks, birth weight 1190 (570-2,400) grams. We found significantly higher I-FABPp and I-FABPu levels in NEC patients (n = 22) than in patients with other diagnoses (n = 15). Cut-off values for diagnosing NEC were 9 ng/mL I-FABPp and 218 ng/mL I-FABPu, with corresponding likelihood ratios (LRs) of 5.6 (95% CI 0.89-35) and 5.1 (95% CI 0.73-36), respectively. I-FABP levels were highest in the first eight hours after symptom onset and gradually decreased over time. Cut-off values for complicated disease were 19 ng/mL I-FABPp and 232 ng/mL I-FABPu, with LRs of 10 (95% CI 1.6-70) and 11 (95% CI 1.6-81), respectively. CONCLUSIONS: Both plasma and urinary I-FABP levels specifically identify NEC in preterm infants prior to appearance of diagnostic radiological signs suggestive for NEC. Moreover, serial I-FABP measurements accurately predict development of complicated disease.

Intestinal fatty acid-binding protein levels in Necrotizing Enterocolitis correlate with extent of necrotic bowel: results from a multicenter study.

BACKGROUND: Intestinal fatty acid-binding protein (I-FABP) is considered as a specific marker for enterocyte damage in necrotizing enterocolitis (NEC). OBJECTIVE: The purpose of this study was to evaluate the association of plasma and urinary I-FABP levels with the extent of macroscopic intestinal necrosis in surgical NEC. METHODS: We combined data from prospective trials from two large academic pediatric surgical centers. Nine and 10 infants with surgical NEC were included, respectively. Plasma and urinary of I-FABP at disease onset were correlated with the length of intestinal resection during laparotomy. RESULTS: Median length of bowel resection was 10cm (range 2.5-50) and 17cm (range 0-51), respectively. Median I-FABP levels were 53ng/mL (range 6.3-370) and 4.2ng/mL (range 1.1-15.4) in plasma in cohort 1 respectively cohort 2 and 611ng/mL (range 3-23,336) in urine. The length of bowel resection significantly correlated with I-FABP levels in plasma (Rho 0.68; p=0.04 and Rho 0.66;p=0.04) and in urine (Rho 0.92; p=0.001). CONCLUSION: This 'proof of concept' study demonstrates that plasma and urine I-FABP levels at disease onset was strongly associated with the length of intestinal resection in surgical NEC. This offers further evidence that I-FABP levels are a promising biomarker for assessing intestinal necrosis in infants with advanced NEC.

Intestinal fatty-acid binding protein and metronidazole response in premature infants.

OBJECTIVES: In premature infants with suspected intra-abdominal infection, biomarkers for treatment response to antimicrobial therapy are lacking. Intestinal fatty acid-binding protein (I-FABP) is specific to the enterocyte and is released in response to intestinal mucosal injury. I-FABP has not been evaluated as a surrogate marker of disease response to antimicrobial therapy. We examined the relationship between metronidazole exposure and urinary I-FABP concentrations in premature infants with suspected intra-abdominal infection. STUDY DESIGN: We conducted an intravenous metronidazole pharmacokinetic study, collecting ≤3 urine samples per infant for I-FABP concentration measurements. We analyzed the relationship between I-FABP concentrations and measures of metronidazole exposure and pharmacokinetics, maturational factors, and other covariates. RESULTS: Twenty-six samples from 19 premature infants were obtained during metronidazole treatment. When analyzed without regard to presence of necrotic gastrointestinal disease, there were no significant associations between predictor variables and I-FABP concentrations. However, when the sample was limited to premature infants with necrotic gastrointestinal disease, an association was found between average predicted metronidazole concentration and I-FABP concentration (p = 0.006). CONCLUSION: While a predictive association between urinary I-FABP and metronidazole systemic exposure was not observed, the data suggest the potential of this endogenous biomarker to serve as a pharmacodynamic surrogate for antimicrobial treatment of serious abdominal infections in neonates and infants.

Early detection of impending necrotizing enterocolitis with urinary intestinal fatty acid-binding protein.

BACKGROUND: Necrotizing enterocolitis (NEC) is diagnosed after the development of feeding intolerance and characteristic physical and imaging findings. Earlier detection of a subclinical prodrome might allow for the institution of measures that could prevent or attenuate the severity of the disease. OBJECTIVES: We sought to determine whether urinary intestinal fatty acid-binding protein (iFABPu) might be elevated prior to the first clinical manifestations of NEC. METHODS: Urine was collected daily from 62 infants of a gestational age of 24-28 weeks. Based on clinical, imaging and operative findings, subjects were determined to have Bell stage 2 or 3 NEC. In all the subjects with NEC and in 21 age-matched controls, iFABPu was determined using an ELISA, and was expressed in terms of its ratio to urinary creatinine (Cr), i.e. iFABPu/Cru. Receiver operating characteristic (ROC) curves were constructed to define the predictive value of iFABPu/Cru for impending NEC in the days prior to the first clinical manifestations. RESULTS: Five subjects developed NEC (stage 2: n = 3 and stage 3: n = 2). The day before the first clinical manifestation of NEC, a ROC curve showed that an iFABPu/Cru >10.2 pg/nmol predicted impending NEC with a sensitivity of 100% and a specificity of 95.6%. iFABPu/Cru did not predict NEC 2 days prior to the first sign of disease. CONCLUSIONS: An elevated iFABPu was a sensitive and specific predictor of impending NEC 1 day prior to the first clinical manifestations. iFABPu screening might identify infants at a high risk and allow for the institution of measures that could ameliorate or prevent NEC.

Evaluation of sodium deficit in infants undergoing intestinal surgery.

BACKGROUND: Sodium is a critical growth factor for children. Severe deficits cause growth impairment and cognitive dysfunction. Both the diagnosis and risk of sodium depletion in children undergoing intestinal surgery are poorly understood. METHODS: With IRB approval, children undergoing intestinal surgery (2009-2012) who had a urine sodium measurement were retrospectively reviewed. Sodium deficits were defined: urine sodium <30 mmol/L and <10 mmol/L were deficient and severely deficient, respectively. Demographics, weight changes, and intake (sodium, fluid, and nutritional) were tabulated. Data were analyzed using regression analysis and Mann Whitney U tests. RESULTS: Thirty-nine patients, 51.3% female, with a gestational age of 32.2 weeks and weight of 1.43 kg were identified. The most common diagnoses were NEC (38.5%), intestinal atresia (20.5%), and isolated perforation (10.3%). Sodium deficiency was documented in 36/39 (92%) and 92.9% for those in continuity. Severe deficiency occurred in 64%. Urine sodium was significantly correlated with weight gain (p=0.002). Weight gain in patients with urine sodium <30 mmol/L was significantly decreased vs. those ≥30 mmol/L (+0.58 g/d vs. +21.6 g/d, p=0.016). CONCLUSION: In this population, sodium depletion is common in children undergoing intestinal surgery, even when the colon is in continuity. Correction of the sodium deficit to achieve urine sodium >30 mmol/L is associated with improved weight gain.

Intestinal fatty acid-binding protein in neonates with imminent necrotizing enterocolitis.

BACKGROUND: Intestinal fatty acid-binding protein (I-FABP) is a promising marker for necrotizing enterocolitis (NEC). It can be measured in plasma (I-FABPp) and urine (I-FABPu). Data on the best way to measure I-FABP (in plasma or urine) and the necessity of simultaneous measurement of the urinary creatinine concentration to correct for physiological variations in urine concentration are not available. This holds also true for the reciprocal relation between I-FABPp, I-FABPu and other more conventional laboratory parameters. OBJECTIVES: To evaluate the above-mentioned correlations of I-FABP measurements in neonates with suspected NEC. METHODS: All neonates with suspected NEC were prospectively included. I-FABPp and I-FABPu were analyzed at regular intervals during the first 24 h after onset of symptoms. Correlation and agreement were assessed between these and other parameters (i.e., IL-6, WBC, platelet count, CRP, pH and lactate). RESULTS: Included were 24 boys, 13 girls [median (range) GA 28 weeks (24-36), median birth weight 1,190 g (570-2,400)]. I-FABPu correlated strongly with I-FABPp (r 0.80, p < 0.001) with an adequate agreement. A very strong correlation between I-FABPu and I-FABPu/urine creatinine ratio (r 0.98, p < 0.001) existed. Correlations between I-FABPp/u and conventional parameters were moderate to strong until 8 h after onset of symptoms. CONCLUSION: In neonates with suspected NEC, I-FABPu correlates strongly with I-FABPp, offering an opportunity to choose the most appropriate way of measuring I-FABP. Calculating urinary IFABP/creatinine ratio seems redundant. Moderately strong correlations between I-FABPu and IL-6, WBC and lactate were found.

Non-invasive serum amyloid A (SAA) measurement and plasma platelets for accurate prediction of surgical intervention in severe necrotizing enterocolitis (NEC).

OBJECTIVE: To evaluate the value of biomarkers to detect severe NEC. SUMMARY BACKGROUND DATA: The time point of surgery in necrotizing enterocolitis (NEC) is critical. Therefore, there is a need for markers that detect severe NEC, because clinical signs of severe NEC often develop late. This study evaluated the value of biomarkers reflecting intestinal cell damage and inflammation to detect severe NEC. METHODS: 29 neonates with NEC were included. Two definitions of moderate versus severe NEC were analyzed: medical NEC (n = 12) versus surgical or fatal NEC (n = 17); and Bell stage II NEC (n = 13) versus stage III NEC (n = 16). Urinary intestinal fatty acid binding protein (I-FABP), serum amyloid A (SAA), C3a and C5a, and fecal calprotectin were measured. C-reactive protein (CRP), white blood cell count (WBC) and platelet count data were measured in blood. RESULTS: In both definitions of moderate versus severe NEC, urinary SAA levels were significantly higher in severe NEC. A cut-off value of 34.4 ng/ml was found in surgical NEC versus medical NEC (sensitivity, 83%; specificity, 83%; LR+, 4.88 (95% CI, 1.37-17.0); LR-, 0.20 (95% CI, 0.07-0.60)) at diagnosis of NEC and at one day prior to surgery in neonates who were operated later on. Combination of urinary SAA and platelet count increased the accuracy, with a sensitivity, 94%; specificity, 83%; LR+, 5.53 (95% CI, 1.57-20.0); and LR-, 0.07 (95% CI, 0.01-0.48). CONCLUSION: Urinary SAA is an accurate marker in differentiating severe NEC from moderate NEC; particularly if combined with serum platelet count.

Urinary intestinal fatty acid binding protein predicts necrotizing enterocolitis.

Necrotizing enterocolitis, characterized by sudden onset and rapid progression, remains the most significant gastrointestinal disorder among premature infants. In seeking a predictive biomarker, we found intestinal fatty acid binding protein, an indicator of enterocyte damage, was substantially increased within three and seven days before the diagnosis of necrotizing enterocolitis.

A novel urine peptide biomarker-based algorithm for the prognosis of necrotising enterocolitis in human infants.

OBJECTIVE: Necrotising enterocolitis (NEC) is a major source of neonatal morbidity and mortality. The management of infants with NEC is currently complicated by our inability to accurately identify those at risk for progression of disease prior to the development of irreversible intestinal necrosis. We hypothesised that integrated analysis of clinical parameters in combination with urine peptide biomarkers would lead to improved prognostic accuracy in the NEC population. DESIGN: Infants under suspicion of having NEC (n=550) were prospectively enrolled from a consortium consisting of eight university-based paediatric teaching hospitals. Twenty-seven clinical parameters were used to construct a multivariate predictor of NEC progression. Liquid chromatography/mass spectrometry was used to profile the urine peptidomes from a subset of this population (n=65) to discover novel biomarkers of NEC progression. An ensemble model for the prediction of disease progression was then created using clinical and biomarker data. RESULTS: The use of clinical parameters alone resulted in a receiver-operator characteristic curve with an area under the curve of 0.817 and left 40.1% of all patients in an 'indeterminate' risk group. Three validated urine peptide biomarkers (fibrinogen peptides: FGA1826, FGA1883 and FGA2659) produced a receiver-operator characteristic area under the curve of 0.856. The integration of clinical parameters with urine biomarkers in an ensemble model resulted in the correct prediction of NEC outcomes in all cases tested. CONCLUSIONS: Ensemble modelling combining clinical parameters with biomarker analysis dramatically improves our ability to identify the population at risk for developing progressive NEC.