2062

The Journal of Maternal-Fetal and Neonatal Medicine, 2012; 25(10): 2062–2065© 2012 Informa UK, Ltd.ISSN 1476-7058 print/ISSN 1476-4954 onlineDOI: 10.3109/14767058.2012.683895

Objective: To prospectively investigate cord blood concentrations of intestinal fatty acid-binding protein-[I-FABP, a useful marker in the early detection of necrotizing enterocolitis-(NEC)] in full-term intrauterine-growth-restricted-(IUGR, associated with NEC, regardless of gestational age) and appropriate-for-gestational-age-(AGA) pregnancies. We also aimed to determine cord blood I-FABP concentrations in IUGR cases with abnormal versus normal antenatal Doppler results and investigate a possible association with feeding intolerance or NEC. Methods: I-FABP concentrations were determined by ELISA in 154 mixed arterio-venous cord blood samples from IUGR (n = 50) and AGA (n = 104) singleton full-term infants. Results: Cord blood I-FABP concen-trations did not differ between IUGR and AGA groups, as well as between IUGR infants with normal versus abnormal (however, lacking absent/ reversed end-diastolic umbilical artery flow) antenatal Doppler results. No infant presented with feeding intolerance or NEC. Customized centiles were lower in IUGR infants with abnormal versus normal antenatal Doppler results (p < 0.001). Conclusions: Full-term IUGR infants present with normal cord blood I-FABP concentrations and do not seem to be at higher risk for developing feeding intolerance or NEC, including those with compromised fetal perfusion.

Keywords: Necrotizing enterocolitis, feeding intolerance, Doppler studies, fetus, neonate

IntroductionFatty acid binding proteins (FABPs) comprise a group of cyto-plasmic small molecular mass proteins with high organ specificity, which are rapidly released into the systemic circulation upon cell damage [1]. Intestinal fatty acid-binding protein (I-FABP) consti-tutes up to 2% of the cytoplasmic protein content of the mature enterocyte [1] and is predominantly present in the small intestine [2]. Upon death of the enterocyte, its cytoplasmic contents are liberated into the circulation, and a rise in plasma I-FABP concen-tration has been demonstrated both in animal models [3] and in a variety of human intestinal diseases [2,4], including necro-tizing enterocolitis (NEC) [5,6]. Therefore, several studies have supported the use of circulating, as well as urinary concentrations of I-FABP as a marker of mucosal damage in NEC, predicting the extent and severity of intestinal involvement [5–8], even before disease onset [8,9].

NEC reflects a spectrum of intestinal pathologic entities generated by different mechanisms. Thus, term and preterm NEC are considered to be two different disease entities [10,11]. In term infants, the disease is more often associated with ischemia secondary to perinatal stressors, including intrauterine growth restriction (IUGR) caused by placental insufficiency [11].

However, the association of NEC and IUGR remains contro-versial. In this respect, several studies indicated that the decreased intrauterine vascular supply to the gastrointestinal tract, due to blood flow redistribution to the vital organs, predisposes to NEC development in the IUGR newborn, regardless of gestational age [11–13], especially in the most severely affected cases of absent or reversed end-diastolic flow in the umbilical artery (AREDF) [14,15]. Furthermore, older and most recent reports showed that full-term IUGR neonates present with intestinal structural atrophy and altered bacterial colonization [16–18], probably predisposing them to feeding intolerance and NEC in early post-natal life. However, other reports have shown no increased risk of NEC in IUGR infants [19,20]. The heterogeneity of IUGR infants may contribute to the discrepancies in the potential association between NEC and IUGR.

The objective of this prospective study was to evaluate and compare, for the first time to our knowledge, cord blood I-FABP concentrations in asymmetric, well-characterized IUGR and appropriate for gestational age full-term infants, as well as in IUGR cases with normal versus abnormal antenatal Doppler results. Furthermore, we aimed to investigate the possible associa-tion of cord blood I-FABP concentrations with potential feeding intolerance or occurrence of NEC. Lastly, this study provides cord blood I-FABP reference values in healthy full-term infants, and explores potential relationship between the above concentra-tions and a variety of maternal and fetal anthropometric/clinical variables.

Materials and methodsApproval of the study was obtained by the Ethics Committee of our teaching hospital and informed consent was acquired from all recruited mothers before cord blood sampling. From July 2009 to December 2009, one hundred and fifty four Caucasian women were included in a prospective study. During this time period 50 asymmetric IUGR singleton, full-term infants (birth weight ≤ 5th customized centile) were consecutively born and the AGA

Cord blood intestinal fatty acid-binding protein (I-FABP) in full-term intrauterine growth restricted pregnancies

Despina D. Briana1, Sofia Liosi1, Dimitrios Gourgiotis2, Maria Boutsikou1, Stavroula Baka1, Antonios Marmarinos2, Dimitrios Hassiakos1 & Ariadne Malamitsi-Puchner1

1Neonatal Division, 2nd Department of Obstetrics and Gynecology, Athens University Medical School, Athens, Greece and 2Research Laboratories, 2nd Department of Pediatrics, Athens University Medical School, Athens, Greece

Correspondence: Ariadne Malamitsi-Puchner, MD, Neonatal Division, 2nd Department of Obstetrics and Gynecology, Athens University Medical School, 19 Soultani Street, 10682 Athens, Greece. Tel: +30 6944443815, Fax: + 30 2107233330. E-mail: amalpu@aretaieio.uoa.gr or amalpu@tellas.gr

The Journal of Maternal-Fetal and Neonatal Medicine

2012

25

10

2062

2065

© 2012 Informa UK, Ltd.

10.3109/14767058.2012.683895

1476-7058

1476-4954

08September2011

04March2012

28March2012

I-FABP in IUGR

D. D. Briana et al.

J M

ater

n Fe

tal N

eona

tal M

ed D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Vir

gini

a on

10/

03/1

2Fo

r pe

rson

al u

se o

nly.

I-FABP in IUGR 2063

© 2012 Informa UK, Ltd.

singleton, full-term baby, born simultaneously or immediately before/afterwards was enrolled as control (totally n = 104).

The Gestation Related Optimal Weight computer-generated program was used to calculate the customized centile for each pregnancy, taking into consideration significant determinants of birth weight, such as maternal height and booking weight, ethnic group, parity, gestational age and gender [21]. Gestational age was determined, according to the date of the last menstrual period and was confirmed by early antenatal ultrasound. Birth weight was measured with an electronic scale.

Fetal blood flow pulsatility was measured based on obstetric decisions, including evaluation of suspected IUGR. The latter fetuses were closely observed by Doppler studies every 10–15 days from the 32nd gestational week and onwards. Only blood flow pulsatility measurements of the umbilical, uterine and cerebral artery, performed within seven days before birth were acceptable for the study [22]. The last measurement before birth was used for the analysis. Flow velocity waveforms were analyzed by the pulsatility index (PI), defined as the difference between peak systolic and end diastolic value, divided by the time average velocity [22,23]. A pathological fetal perfusion was defined by a PI of uterine arteries and umbilical artery above the 90th percen-tile for the corresponding gestational age and by a PI of middle cerebral artery below the 10th percentile for the corresponding gestational age of a normal group [24].

The timing of delivery was decided on the basis of clinical assessment, ultrasound fetal growth, Doppler velocimetry, biophysical profile and computerized cardiotocographic analysis.

Six of the 50 mothers with IUGR offspring presented with preeclampsia, five presented with pregnancy-induced hyperten-sion, 15 suffered from various diseases, such as iron-deficient anemia (3 cases), severe type I DM (5 cases) and hypothyroidism (7 cases). The remaining 24 women were smoking >10 cigarettes/day during the whole duration of pregnancy.

Amniotic fluid was diminished in all IUGR cases, a finding compatible with placental insufficiency [25]. For the evaluation of the amniotic fluid, the largest fluid column on the vertical plane was assessed and was defined as diminished if <2 cm. Placental weights were reduced [26], ranging from 310 to 450 g.

In the AGA group, mothers were healthy and were either non-smokers or abstained from smoking during pregnancy. Placentas were normal in appearance and weight [26], ranging from 480 to 621 g.

Tests for congenital infections were negative in all women and their offspring had no symptoms of intrauterine infection, signs of major congenital/chromosomal abnormalities or anomalies of the gastrointestinal tract. One- and five-minute Apgar scores were in all infants ≥ 8. All neonates were breastfed (colostrum was supplied early after birth, according to the department protocol) and they all adapted well to extrauterine life with no signs of peri-natal asphyxia, respiratory distress or polycythemia. No infant presented with feeding intolerance or NEC [27].

Demographic data of included IUGR and AGA infants and their mothers are presented in Table I.

After double clamping of the umbilical cord, mixed arte-riovenous cord blood (reflecting the fetal state) was collected in pyrogen-free tubes. Serum was separated by centrifugation and was kept frozen at −80°C until assay. The determination of serum I-FABP concentrations was performed by ELISA (Human I-FABP, Hycult Biotech, Uden, the Netherlands). The minimum detectable concentrations, inter- and intra-assay coefficients of variation were 47 pg/ml, 3.6%, and 6.3%, respectively.

Statistical analysis

I-FABP data were not normally distributed (Kolmogorov–Smirnov test). Variables regarding birth-weight, maternal and gestational age presented with normal distribution. Independent samples T test was used to detect differences in normally distributed variables between IUGR cases and AGA controls. Otherwise, non-parametric Mann–Whitney U-test was applied. Pearson’s chi square test was used to detect differences, concerning gender, mode of delivery and parity between groups. Pearson’s or Spearman correlation coefficient was applied, where appropriate, to detect any positive or negative correlations. A p < 0.05 was considered statistically significant. Statistical analysis was performed, by using SPSS 11.5.

ResultsSerum I-FABP values are shown in Figure 1. No significant differences in cord blood I-FABP concentrations were observed between IUGR and AGA groups [median (range): 267.89 pg/ml (63.13–966.5) and 264.19 pg/ml (28.7–1481.7), respectively].

As already noted, no infant presented with feeding intolerance or NEC.

In the IUGR group, compromised fetal perfusion was recorded in 12 cases. In 8 cases respective information was lacking. In the remaining 30 cases, PI values of the uterine and umbilical arteries were found to be in the upper physiological limits for the corre-sponding gestational age, while Doppler studies of the middle cerebral arteries showed resistance to be in the lower physiolog-ical limits for gestational age [22–24], indicating the initiation of blood flow redistribution process, in order to spare vital organs (brain, heart and adrenals). However, no fetus presented with AREDF in the umbilical artery.

No statistically significant differences were detected in cord blood I-FABP concentrations between IUGR cases with abnormal versus normal antenatal Doppler results (mean ± SD: 360.11 ± 244.41 pg/ml versus 285.12 ± 128.76 pg/ml). IUGR infants with abnormal antenatal Doppler results presented with lower centiles,

Table I. Demographic data for appropriate-for-gestational-age (AGA) and intrauterine growth restricted (IUGR) neonates and their mothers.

AGA casesMean ± SD/median (range)

IUGR casesMean ± SD/median (range)

p Value

Birthweight (g) 3280 ± 290.28 2507 ± 264.5 <0.001Birthweight Centile

43.5 [20–89] 3.0 [0–5] <0.001

Gestational age (weeks)

38.98 ± 1.07 38.36 ± 1.27 NS

Maternal age (years)

30.10 ± 5.0 32.06 ± 4.53 0.02

Gender n (%) 0.038 Male 63 (60.6) 21 (42) Female 41 (39.4) 29 (58)Mode of delivery n (%)

0.001

Vaginal 76 (73.1) 22 (44) Caesarean section

28 (26.9) 28 (56)

Parity n (%) NS Primigravida 72 (69.2) 31 (62) Other 32 (30.8) 19 (38)NS: non significant.

J M

ater

n Fe

tal N

eona

tal M

ed D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Vir

gini

a on

10/

03/1

2Fo

r pe

rson

al u

se o

nly.

2064 D. D. Briana et al.

The Journal of Maternal-Fetal and Neonatal Medicine

as compared with those with normal Doppler results (mean ± SD: 1.17 ± 1.85 and 3.43 ± 1.36, respectively, p < 0.001).

In both groups, the effect of maternal age, parity, gestational age, customized centile, birthweight and gender on I-FABP concentrations was not significant.

DiscussionThe results of this study indicate that full-term asymmetric IUGR infants present with normal cord blood I-FABP concentrations and do not seem to be at higher risk of developing feeding intoler-ance or NEC, including those with pathological fetal perfusion. Furthermore, as expected, the customized centiles of infants with abnormal antenatal Doppler results were lower, compared to those with normal ones, reflecting the severity of IUGR.

However, in the present study, no fetus presented with AREDF in the umbilical artery, a situation urging for immediate delivery, usually preterm, and commonly associated with NEC [14,15]. Moreover, it should be noted that all included infants were breastfed, according to the department policy, which is based on the established knowledge that formula consistently decreases tissue-specific enzyme activities and increases NEC sensitivity [28].

Current evidence suggests that I-FABP is liberated into the circulation after intestinal mucosal injury [1,3], as it happens in NEC. When intestinal ischemia is limited to a period of less than 2 h, only the villi are affected, while the crypt cells remain intact [29]. Since I-FABP is mainly expressed in the villi and not in the crypt [9,30], its circulating concentrations are considered an early and sensitive marker of intestinal ischemia [5,6,8]. Furthermore, both animal and human studies have shown that serum I-FABP levels correlate with the phases of histologic mucosal injury after intestinal ischemia-reperfusion [1,3].

IUGR caused by placental insufficiency is characterized by blood flow redistribution to the vital organs, while other organs,

including the gastrointestinal tract, are deprived from sufficient blood flow [31]. As a consequence, IUGR infants are thought to have impaired gut function after birth, which may result in intes-tinal disturbances, ranging from temporary intolerance of enteral feeding to full blown NEC, regardless of gestational age [12,14].

Thus, an association among IUGR, increased umbilical artery resistance and gastrointestinal morbidity has been observed in preterm infants [14,15]. However, when birth weight and gesta-tional age at delivery were controlled for, increased umbilical artery resistance was not an independent predictor of NEC [32,33]. In accordance, a previous report in term IUGR infants indicated that abnormal Doppler velocimetry (but not in the form of AREDF in the umbilical artery) is not associated with increased risk for NEC or mortality [14]. Additionally, Doppler studies of the fetal mesenteric circulation have shown increased vascular resistance as a late sign of fetal blood flow redistribution, but were not supportive of a greater likelihood of NEC [34,35].

On the other hand, alterations in intestinal development, such as structural atrophy and impaired nutrient absorption and utilization have been associated with IUGR at term [16–18]. Additionally, most recent studies in animal models showed that IUGR neonates seem to present with enhanced bacterial adhe-sion, as well as with signs of intestinal inflammation in the immediate postnatal period [16,17]. As a consequence, intestinal trophic responses to enteral food introduction are altered, prob-ably contributing to feeding intolerance or NEC.

By contrast, a recent study demonstrated that IUGR at term does not predispose to NEC or compromised postnatal intestinal adaptation [20]. In this respect, the gastrointestinal tract of term IUGR neonates does not seem to have a reduced capacity to tolerate enteral nutrition, as assessed by intestinal structure, func-tion, and NEC sensitivity [20].

The results of the present study show that full-term asym-metric IUGR infants present with normal cord blood I-FABP concentrations and do not seem to be at higher risk of developing feeding intolerance or NEC, including those with abnormal ante-natal Doppler results. Additional studies are needed to further elucidate the association between NEC and IUGR and focus on the critical transition to neonatal life to identify relevant triggers in predisposed neonates.

Declaration of Interest: The authors report no declarations of interest.

References 1. Pelsers MM, Hermens WT, Glatz JF. Fatty acid-binding proteins as

plasma markers of tissue injury. Clin Chim Acta 2005;352:15–35. 2. Derikx JP, Vreugdenhil AC, Van den Neucker AM, Grootjans J, van

Bijnen AA, Damoiseaux JG, van Heurn LW, et al. A pilot study on the noninvasive evaluation of intestinal damage in celiac disease using I-FABP and L-FABP. J Clin Gastroenterol 2009;43:727–733.

3. Gollin G, Marks C, Marks WH. Intestinal fatty acid binding protein in serum and urine reflects early ischemic injury to the small bowel. Surgery 1993;113:545–551.

4. Wiercinska-Drapalo A, Jaroszewicz J, Siwak E, Pogorzelska J, Prokopowicz D. Intestinal fatty acid binding protein (I-FABP) as a possible biomarker of ileitis in patients with ulcerative colitis. Regul Pept 2008;147:25–28.

5. Edelson MB, Sonnino RE, Bagwell CE, Lieberman JM, Marks WH, Rozycki HJ. Plasma intestinal fatty acid binding protein in neonates with necrotizing enterocolitis: a pilot study. J Pediatr Surg 1999;34:1453–1457.

6. Guthmann F, Börchers T, Wolfrum C, Wustrack T, Bartholomäus S, Spener F. Plasma concentration of intestinal- and liver-FABP in

Figure 1. Box and whiskers plots of cord blood intestinal fatty acid-binding protein (I-FABP) concentrations in appropriate for gestational age (AGA) and intrauterine growth restricted (IUGR) groups. Each box represents the median concentration with the interquartile range (25th and 75th percentiles). The upper and lower whiskers represent the range.

J M

ater

n Fe

tal N

eona

tal M

ed D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Vir

gini

a on

10/

03/1

2Fo

r pe

rson

al u

se o

nly.

I-FABP in IUGR 2065

© 2012 Informa UK, Ltd.

neonates suffering from necrotizing enterocolitis and in healthy preterm neonates. Mol Cell Biochem 2002;239:227–234.

7. Evennett NJ, Hall NJ, Pierro A, Eaton S. Urinary intestinal fatty acid-binding protein concentration predicts extent of disease in necrotizing enterocolitis. J Pediatr Surg 2010;45:735–740.

8. Mannoia K, Boskovic DS, Slater L, Plank MS, Angeles DM, Gollin G. Necrotizing enterocolitis is associated with neonatal intestinal injury. J Pediatr Surg 2011;46:81–85.

9. Lieberman JM, Sacchettini J, Marks C, Marks WH. Human intestinal fatty acid binding protein: report of an assay with studies in normal volunteers and intestinal ischemia. Surgery 1997;121:335–342.

10. Lin PW, Nasr TR, Stoll BJ. Necrotizing enterocolitis: recent scientific advances in pathophysiology and prevention. Semin Perinatol 2008;32:70–82.

11. Martinez-Tallo E, Claure N, Bancalari E. Necrotizing enterocolitis in full-term or near-term infants: risk factors. Biol Neonate 1997;71:292–298.

12. Gilbert WM, Danielsen B. Pregnancy outcomes associated with intrauterine growth restriction. Am J Obstet Gynecol 2003;188:1596–9; discussion 1599.

13. Andrews DA, Sawin RS, Ledbetter DJ, Schaller RT, Hatch EI. Necrotizing enterocolitis in term neonates. Am J Surg 1990;159:507–509.

14. Bhatt AB, Tank PD, Barmade KB, Damania KR. Abnormal Doppler flow velocimetry in the growth restricted foetus as a predictor for necrotising enterocolitis. J Postgrad Med 2002;48:182–5; discussion 185.

15. Malcolm G, Ellwood D, Devonald K, Beilby R, Henderson-Smart D. Absent or reversed end diastolic flow velocity in the umbilical artery and necrotising enterocolitis. Arch Dis Child 1991;66:805–807.

16. D’Inca R, Gras-Le Guen C, Che L, Sangild PT, Le Huërou-Luron I. Intrauterine growth restriction delays feeding-induced gut adaptation in term newborn pigs. Neonatology 2011;99:208–216.

17. D’Inca R, Kloareg M, Gras-Le Guen C, Le Huërou-Luron I. Intrauterine growth restriction modifies the developmental pattern of intestinal structure, transcriptomic profile, and bacterial colonization in neonatal pigs. J Nutr 2010;140:925–931.

18. Baserga M, Bertolotto C, Maclennan NK, Hsu JL, Pham T, Laksana GS, Lane RH. Uteroplacental insufficiency decreases small intestine growth and alters apoptotic homeostasis in term intrauterine growth retarded rats. Early Hum Dev 2004;79:93–105.

19. Friedman SA, Schiff E, Kao L, Sibai BM. Neonatal outcome after preterm delivery for preeclampsia. Am J Obstet Gynecol 1995;172:1785–8; discussion 1788.

20. Che L, Thymann T, Bering SB, LE Huërou-Luron I, D’inca R, Zhang K, Sangild PT. IUGR does not predispose to necrotizing enterocolitis or compromise postnatal intestinal adaptation in preterm pigs. Pediatr Res 2010;67:54–59.

21. Gardosi J, Chang A, Kalyan B, Sahota D, Symonds EM. Customised antenatal growth charts. Lancet 1992;339:283–287.

22. Baschat AA, Galan HL, Bhide A, Berg C, Kush ML, Oepkes D, Thilaganathan B, et al. Doppler and biophysical assessment in growth restricted fetuses: distribution of test results. Ultrasound Obstet Gynecol 2006;27:41–47.

23. Acharya G, Wilsgaard T, Berntsen GK, Maltau JM, Kiserud T. Reference ranges for serial measurements of umbilical artery Doppler indices in the second half of pregnancy. Am J Obstet Gynecol 2005;192:937–944.

24. Ott WJ. Diagnosis of intrauterine growth restriction: comparison of ultrasound parameters. Am J Perinatol 2002;19:133–137.

25. Chamberlain PF, Manning FA, Morrison I, Harman CR, Lange IR. Ultrasound evaluation of amniotic fluid volume. I. The relationship of marginal and decreased amniotic fluid volumes to perinatal outcome. Am J Obstet Gynecol 1984;150:245–249.

26. Burkhardt T, Schäffer L, Schneider C, Zimmermann R, Kurmanavicius J. Reference values for the weight of freshly delivered term placentas and for placental weight-birth weight ratios. Eur J Obstet Gynecol Reprod Biol 2006;128:248–252.

27. Bell MJ, Ternberg JL, Feigin RD, Keating JP, Marshall R, Barton L, Brotherton T. Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging. Ann Surg 1978;187:1–7.

28. Lucas A, Cole TJ. Breast milk and neonatal necrotising enterocolitis. Lancet 1990;336:1519–1523.

29. Robinson JW, Mirkovitch V. The recovery of function and microcirculation in small intestinal loops following ischaemia. Gut 1972;13:784–789.

30. Sacchettini JC, Hauft SM, Van Camp SL, Cistola DP, Gordon JI. Developmental and structural studies of an intracellular lipid binding protein expressed in the ileal epithelium. J Biol Chem 1990;265:19199–19207.

31. Baschat AA. Fetal responses to placental insufficiency: an update. BJOG 2004;111:1031–1041.

32. McCowan LM, Harding JE, Stewart AW. Umbilical artery Doppler studies in small for gestational age babies reflect disease severity. BJOG 2000;107:916–925.

33. Adiotomre PN, Johnstone FD, Laing IA. Effect of absent end diastolic flow velocity in the fetal umbilical artery on subsequent outcome. Arch Dis Child Fetal Neonatal Ed 1997;76:F35–F38.

34. Coombs RC, Morgan ME, Durbin GM, Booth IW, McNeish AS. Abnormal gut blood flow velocities in neonates at risk of necrotising enterocolitis. J Pediatr Gastroenterol Nutr 1992;15:13–19.

35. Korszun P, Dubiel M, Breborowicz G, Danska A, Gudmundsson S. Fetal superior mesenteric artery blood flow velocimetry in normal and high-risk pregnancy. J Perinat Med 2002;30:235–241.

J M

ater

n Fe

tal N

eona

tal M

ed D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Vir

gini

a on

10/

03/1

2Fo

r pe

rson

al u

se o

nly.