Necrotizing Enterocolitis is Associated with Elevated Iron Levels and Toxic Lipid Radicals in Neonatal Intestinal Tissue

Authors:
Alex Frickenstein, Aslan Massahi, Catherine Hunter, Chase Calkins, Cody Dalton, Grant Gershner, Heather Grubbs

Body of Abstract:
Purpose: Necrotizing enterocolitis (NEC) is a devastating disease that continues to plague neonates in neonatal intensive care units, despite over sixty years of research. Ferroptosis, a recently discovered version of programmed cell death similar to Apoptosis, is characterized by the accumulation of lipid radicals and iron. While Ferroptosis has been illustrated in NEC, but its role has yet to be defined. We hypothesize that patients with active NEC will have higher levels of iron compared to other groups. We also hypothesize that patients with active NEC will have higher levels of the toxic lipid radicals 4-hydroxyl-2-hexenal (4-HHE) and 4-hydroxynonenal (4-HNE), which are seen in oxidative stress.

Methods: For iron analysis, intestinal tissue was collected from control patients, patients with a history of NEC, and patients with active NEC. The tissue underwent acid digestion, and a subsequent colorimetric assay was performed using a bathophenanthroline-based colorimetric assay. Using the same patients, new tissue samples were prepared and analyzed using inductively coupled plasma mass spectrometry (ICP-MS). For lipid radical analysis, enteroids were grown from similar cohorts and underwent NEC induction. These were then harvested, processed, and analyzed using western blotting for 4HHE and 4HNE. Statistical analysis was then performed using One- or Two-way ANOVA and Student’s t-test as appropriate.

Results: Patients with active NEC had significantly elevated levels of iron compared to control (p=0.0029) and those with a history of NEC (p=0.0008). There was no significant difference between control patients and those with a history of NEC (p=0.6699). This pattern was consistent for ICP-MS. Patients with NEC had significantly higher levels of iron compared to control patients (p=0.015). Enteroids who underwent NEC induction had significantly elevated levels of 4-HHE (p=0.0114). While these enteroids had decreased levels of 4-HNE compared to the control, this difference did not reach statistical significance.

Conclusion: Patients with active NEC had significantly higher levels of iron compared to controls and those who had recovered from NEC. This was found true using two different modes of iron level quantification. Additionally, NEC enteroids had significantly elevated levels of 4-HHE. In line with our lab’s previous research, this strongly implies that Ferroptosis plays a role in the pathology of NEC. In line with our lab’s previous research, this strongly implies that Ferroptosis plays a role in the pathology of NEC.