Bile Acid Profiling by Liquid Chromotography-Tandem Mass Spectrometry in Neonates with Necrotizing Enterocolitis

Author(s):
Anne Roberts, Children’s Hospital Los Angeles; Anastasia Kalli, Thermo Fisher Scientific; Xiaowei Fu, Children’s Hospital Los Angeles; Henri Ford, Children’s Hospital of LA, University of SC; Christopher P. Gayer, Children’s Hospital Los Angeles

Background:

Necrotizing enterocolitis (NEC) is a severe intestinal disorder of premature infants. Intestinal bacteria convert liver-generated primary bile acids into more toxic secondary metabolites. Animal models suggest that secondary bile acids can induce intestinal damage similar to NEC. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) can be used to measure the concentration of specific bile acid metabolites in a clinical setting.

Hypothesis:

We hypothesize that LC-MS/MS is a useful tool for measuring bile acids clinically in patients with NEC. Furthermore, we hypothesize that patients with NEC will have elevated levels of secondary bile acids in serum.

Methods:

Serum samples were collected from patients with NEC at the time of diagnosis for up to 10 weeks. Using 50uL of serum, we isolated bile acids using methanol extraction followed by centrifugation. The supernatant was dried under a nitrogen stream and re-suspended in methanol. LC was performed in MRM mode over a 12-minute elution time on a Kinetex C18 column. Nineteen bile acid standards were optimized. Two deuterized internal standards and their calibration curves were used to calculate sample concentrations.

Results:

A total of 7 patients with NEC were included, three treated surgically and four treated medically. In patients successfully treated medically, no secondary bile acids were identified in serum during the two weeks of treatment. In the patient who failed medical management, we identified increased secondary bile acids for 4 days leading up to surgical intervention. These metabolites disappeared after successful treatment. In the patients treated surgically, normal primary bile acids levels increased post-resection corresponding to clinical recovery.

Conclusions:

LC-MS/MS is effective at measuring, monitoring and quantifying bile acid profiles in patients with NEC with only 50uL of serum. Our results indicate that secondary bile acids are elevated in the serum of patients with NEC. Post-treatment, this is replaced by normal primary bile acids. LC-MS/MS may be a useful clinical tool in the diagnosis of NEC, where bacterial changes lead to production of harmful bile acid metabolites.