G.H. Gershner1,2, C.M. Dalton1,2, K.B. Snyder1,2, H.C. Grubbs1,2, A. Massahi1,2, C.L. Calkins1,2, C.J. Hunter1,2 1University Of Oklahoma College Of Medicine, Pediatric Surgery, Oklahoma City, OK, USA 2University Of Oklahoma College Of Medicine, Surgery, Oklahoma City, OK, USA
Introduction:
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, 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 there will be elevated levels of the toxic lipid radicals 4-hydroxyl-2-hexenal (4-HHE) and 4-hydroxynonenal (4-HNE), which are seen in oxidative stress.
Methods:
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 to evaluate iron levels. Additionally, enteroids were grown from similar cohorts and underwent NEC induction. These were then harvested, processed, and analyzed using mass spectrometry lipidomics to evaluate lipid profiles. Statistical analysis was then performed using One-Way ANOVA and Student's t-test as appropriate.
Results:
Patients with active NEC had significantly elevated levels of iron compared to control (p=0.0135) and those with a history of NEC (p=0.0053). There was no significant difference between control patients and those with a history of NEC (p=0.5854). 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. 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.