131 - Nitro-Fatty Acid Pharmacokinetics in the Adipose Tissue Compartment

Abstract

Metabolic and redox reactions of nitric oxide and nitrite induce the nitration of biomolecules such as proteins, nucleic acids and unsaturated fatty acids. Nitrated unsaturated fatty acids (NO2-FAs) promote adaptive and anti-inflammatory cell signaling responses as a result of an electrophilic character that supports post-translational protein modifications. By reversibly adducting nucleophilic amino acids such as cysteine, NO2-FAs induce salutary enzymatic and transcriptional regulatory protein responses. NO2-FAs are found endogenously in a broad range of living organisms, become elevated in concentration during metabolic and inflammatory stress and at present, synthetic NO2-FA homologs are being tested in humans as a drug strategy for treating kidney disease. A unique pharmacokinetic profile is expected for electrophilic fatty acids because of an ability to undergo both Michael addition with biological nucleophiles and esterification into complex lipids. We now report new methods for the qualitative and quantitative analysis of complex lipids containing esterified NO2-FAs. This new capability supports the more complete definition of the distribution and metabolism of electrophilic fatty acid derivatives in tissue lipid compartments. Herein we report via quantitative whole-body autoradiography analysis of rats gavaged with radiolabeled 10-nitro-[14C]oleic acid (10-NO2-[14C]OA). This NO2-FA preferentially accumulates in adipose tissue over two weeks. To define the metabolism and incorporation of NO2-FA and their metabolites in adipose tissue lipids, adipocyte cultures were supplemented with 10-NO2-OA, nitro-stearic acid (NO2-SA), nitro-conjugated-linoleic acid (NO2-CLA) and nitro-linolenic acid (NO2-LnA). Then, quantitative high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis was performed on adipocyte neutral and polar lipid fractions, both before and after acidic hydrolysis of esterified fatty acids. Electrophilic nitroalkene derivatives preferentially incorporated in the monoacyl- and diacyl-glyceride than in the triacylglyceride lipid pools of adipocytes, while the non-electrophilic NO2-SA showed an opposite distribution. This differential distribution profile was confirmed in vivo in the adipose tissue of NO2-OA-treated mice. This pattern of electrophilic nitroalkene deposition lends new insight into the unique pharmacokinetics and pharmacologic actions displayed by this chemically-reactive class of endogenous signaling mediators and synthetic drug candidates.