The central goal of this thesis was to generate a better understanding of the role of 4-hydroxyacids in vivo. This work spans disciplines to gain a more fundamental understanding of the origins of endogenous 4-hydroxyacids, and the eventual metabolic fate(s) of this class of molecules. These molecules can be produced endogenously, such as with 4-hydroxy-2-(E)-nonenal (more commonly known as 4-HNE), which is derived from the peroxidation (LPO). Alternatively they can come from exogenous sources, such as drugs of abuse including ¿¿¿¿-hydroxybutyric acid (GHB) and ¿¿¿¿-hydroxypentanoic acid (GHP). Despite the ubiquitous nature of these molecules in vivo, we discovered that very little was known about their eventual metabolic fates.
Our main experimental tools for the majority of the studies detailed in this thesis were isotopically labeled 4-HNE and other 4-hydroxyacids, which were synthesized with 2H and 13C labels at strategic positions. These compounds were subsequently perfused in a live rat liver and led to our finding of a highly evolved catabolic pathway for 4-hydroxyacids. A number of analytical tools including LC-MS/MS, GC/MS and 31P NMR were used to characterize the metabolic intermediates. A key finding of this work was that 4-hydroxyacid catabolism can proceed via two parallel pathways that involve either a phosphorylation and isomerization of the C-4 hydroxyl or a ¿¿¿¿-oxidation/¿¿¿¿-oxidation sequence. This is the first report on the catabolism of this class of biological molecules. We were also able to quantify the differential catabolic flux of 4-HNE (via 4-hydroxyacids) down the two parallel pathways.
In addition to the aforementioned catabolism/recycling of 4-hydroxyacids, we also utilized many of our analytical methods to answer other questions important to LPO and general metabolism. For example, we were able to better define the enantioselectivity of glutathionylation of 4-HNE, 4-ONE, and other 4-HAE derivatives by developing a highly sensitive LC-MS/MS method. Using the isotopic chemistries developed, we were able to define new pathways for the catabolism of the smallest 4-hydroxyacid (which differs from longer-chain members), GHB. Finally, we were able to expand our understanding of 4-HNE, by studying how this molecule modulates oxidative stress in the macrophage. We identified a negative feedback loop in biological oxidant formation that is regulated by 4-HNE. This model stands in contrast to the accepted model in the field that views 4-HNE as a cytotoxic xenobiotic derived from physiology gone awry. This thesis is focused on defining a more comprehensive understanding of both the origins, and fates of 4-hydroxyacids and other LPO products. The signaling in conjunction with the metabolism presented in this thesis argues for a more fundamental role for the 4-HNE, and the formation of LPO products does not necessarily represent aberrant physiology in itself. Rather, the production and elimination of LPO products is a carefully controlled component of physiology, and the aforementioned disease states are indicative of a perturbation of this normal homeostasis.