Mass spectrometry-based study defines the human urine nitrolipidome

Area of Science:

• Biochemistry
• Human Physiology
• Analytical Chemistry

Background:

• Nitrated fatty acids (NO2-FA) are endogenous signaling molecules implicated in digestion and inflammation.
• Despite known signaling roles, the endogenous pool and detection of NO2-FA remain incompletely characterized.
• A systematic approach is required to define the endogenous NO2-FA profile in humans.

Purpose of the Study:

• To screen for and characterize endogenous NO2-FA in human urine, the primary excretion route.
• To identify and confirm novel nitrated fatty acid species.
• To re-evaluate the presence of previously reported nitrated fatty acids in humans.

Main Methods:

• Urine samples from healthy human volunteers were analyzed for NO2-FA.
• High-resolution mass spectrometry and fragmentation analysis were employed for identification and confirmation.
• Comparison with synthetic nitrated standards and metabolic studies using mouse hepatocytes were performed.

Main Results:

• Only isomers of conjugated linoleic and linolenic acid were found to be nitrated in human urine.
• Several novel nitrated species were identified, primarily nitrated conjugated linolenic acid (NO2-CLnA).
• Previously reported nitrated omega-3s, oleic, arachidonic, and linolenic acids were not confirmed, necessitating re-evaluation.

Conclusions:

• Nitrated conjugated linolenic acid (NO2-CLnA) isomers represent a significant portion (39%) of urinary NO2-FA.
• NO2-CLnA isomers are electrophilic and participate in Michael addition reactions, suggesting a role in cell signaling.
• The findings highlight the importance of NO2-CLnA and warrant re-evaluation of other reported NO2-FA in human biological systems.