Elevated Coefficient of Variation in Total Fecal Bile Acids Precedes Diagnosis of Necrotizing Enterocolitis

Abstract

Accumulation of bile acids (BAs) may mediate development of necrotizing enterocolitis (NEC). Serial fecal samples were collected from premature infants with birth weight (BW) ≤ 1800 g, estimated gestational age (EGA) ≤ 32 weeks, and <30 days old prior to initiation of enteral feeding. Nine infants that developed Bell's Stage ≥ II NEC were matched with control infants based on BW, EGA, day of life (DOL) enteral feeding was initiated and DOL of the first sample. From each subject, five samples matched by DOL collected were analyzed for BA levels and composition. Fifteen individual BA species were measured via LC-MS/MS and total BA levels were measured using the Diazyme Total Bile Acid Assay kit. No statistically significant differences in composition were observed between control and NEC at the level of individual species (p = 0.1133) or grouped BAs (p = 0.0742). However, there was a statistically significant difference (p = 0.000012) in the mean coefficient of variation (CV) between the two groups with infants developing NEC having more than four-fold higher mean CV than controls. Importantly, these variations occurred prior to NEC diagnosis. These data suggest fluctuations in total fecal BA levels could provide the basis for the first predictive clinical test for NEC.protective responses 20 , and the concomitant dysregulation of hepatic BA transporters 21 during development of NEC. This novel paradigm encompasses major disease-associated findings and risk factors: 1) the more premature an infant, the more likely they will develop NEC 10,11 . Many of the key processes of BA homeostasis are not fully developed in neonates, but reach maturity at weaning [22][23][24][25][26][27] . 2) Formula-fed preemies are 6-10 times more likely to develop NEC 12 and have higher fecal BA levels than breast-fed preemies 28 . Further, formula feeding is required to develop experimental NEC 29,30 . 3) The majority of BA reclamation occurs in the ileum and colon, the primary sites of NEC injury 31 , and ileal BA levels increase 24-hours before inflammation or histological damage in experimental NEC 32 . 4) Bacterial colonization is required for both development of NEC and formation of secondary BAs. While no specific pathogen has been conclusively associated with NEC 33-43 , disease cannot be developed in germ free conditions 13,14 and pneumatosis intestinalis, the most pathognomonic radiological finding of NEC, is thought to be caused by bacterial overgrowth 44 . Bacteria are also required for formation of more toxic, hydrophobic BAs 45-47 , which are higher in both experimental 18 and human NEC 48 .To further characterize how BAs are dysregulated in human NEC, we prospectively collected fecal samples from premature infants and determined BA levels and composition from matched-pairs diagnosed with NEC versus those without NEC. Contrary to a previous study 48 , we found no statistically significant differences in BA composition between control and NEC. However, there was a statistically significant difference (p = 0.000012) in the mean...

Lipid Composition, Digestion, and Absorption Differences among Neonatal Feeding Strategies: Potential Implications for Intestinal Inflammation in Preterm Infants

“…Comparisons of fecal excretion of bile salts with diet have demonstrated highest BA loss in infants fed a high LCPUFA, soy-based formula, lower loss in infants fed bovine-based formula, and lowest loss in infants fed exclusively HM [ 194 ]. Alternatively, Halpern et al demonstrated, if high levels of bile acids are not excreted through the feces, but rather taken up by ileal enterocytes, they may be inappropriately retained within the intestinal epithelium, resulting in mucosal damage in rodents very similar to that seen in human NEC [ 195 , 196 , 197 ].…”

The inhibition of enterocyte proliferation by lithocholic acid exacerbates necrotizing enterocolitis through downregulating the Wnt/β‐catenin signalling pathway

“…It has been reported that DCA is closely related to NEC by regulating cell proliferation or the BA transporter 27,28 . Similarly, LCA has been found to be elevated in intestinal bile salts from patients with NEC 29 . However, the role and mechanisms of LCA in NEC remains clear.…”

Perinatal inflammation and adverse outcomes of the intestine and liver

“…In chapter 5, total ileal BA concentrations remained unchanged at different time points after IA LPS administration. In line, in a more recent study in which NEC infants were compared to matched controls, no differences in total fecal BAs nor differences in BA species were found (64). However, in the month prior to NEC diagnosis, a difference in the mean coefficient of variation of total BAs was detected when compared to controls (64).…”

“…However, in the month prior to NEC diagnosis, a difference in the mean coefficient of variation of total BAs was detected when compared to controls (64). This latter finding indicates that fluctuations in fecal BA levels were measured, which suggests that the neonatal ileum may be especially sensitive to pulsatile exposure to BAs in the first month, potentially contributing to NEC development (64).…”

“…Plant sterols, also called phytosterols, are dietary constituents present in vegetable oils, nuts, grains and fruit (59) that are increasingly valued for their immune regulatory properties (60). Plant sterols are found in amniotic fluid (61,62) and human breast milk (63)(64)(65)(66), both fluids that protect against NEC (67,68). Studies in rodent inflammatory bowel disease models and in human volunteers showed that plant sterols possess anti-inflammatory properties (69)(70)(71).…”