Discovery and characterization of signaling molecules from tetracosahexaenoic acid

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Docosahexaenoic acid (22:6n-3; DHA) is an omega-3 polyunsaturated fatty acid that is highly abundant in the brain, where it is thought to regulate many important processes. Through a series of desaturations and elongations, largely occurring in the liver, alpha-linolenic acid (18:3n-3) ALA is converted to DHA. One especially interesting, and sometimes confusing, biochemical step is that ALA, upon a few steps, is converted to tetracosahexaenoic acid (24:6n-3;THA) before one round of beta-oxidation removes two carbons producing DHA. We, and others, have been a bit perplexed by the apparent complexity of this pathway and think that some of the issues may arise by not examining the pathway kinetically. Thus, the overall objective of this grant proposal is to understand if THA is a short lived and committed precursor to DHA, as is commonly accepted, or if THA is a product of DHA and/or is possibly itself a signaling molecule. Our focus will be on the liver for synthesis studies as that is thought to be the major site of synthesis, although other tissues will be examined, while we will focus on the brain for signaling studies. The first specific objective of this grant is to quantify and compare the rate of synthesis of DHA to THA with the rate of synthesis of THA to DHA in vivo using our established and newly developed kinetic models in rodents. The first model requires a 5 minute intravenous infusion of labelled THA and DHA (which we have sourced) in the unanaesthetized, free-living rat. The second approach to this question is similar, but labelled THA and DHA are infused for 3 hours and the appearance of their “products” are measured in the blood and applied in a kinetic model that determines their synthesis/secretion rate. Comparing these two kinetic models will not only achieve these objectives, but will give us insight into why tissue levels of THA are low by testing if THA is extensively secreted from the liver. The second part of this grant is to identify and quantify THA in the brain and to test if it is enzymatically converted into molecules that could be involved in signaling. Our second objective will focus on the brain and will determine if THA is present in common fatty acid signaling pathways. To do this, we have developed sensitive LC- and GC-MS methods to measure THA and its candidate signaling derivatives. We will now apply these methods to detect if THA is released from neuronal membranes upon ischemia into the unesterified fatty acid pool, a phenomenon common among signaling molecules. We will also use these techniques to identify if THA is enzymatically converted to molecules that act as signaling molecules similar to DHA and arachidonic acid (ARA, 20:4n-6). We will also perform these experiments in wildtype mice and in mice lacking critical enzymes for the synthesis of the signaling molecules.