Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Ian A. Blair

Second Advisor

E. J. Petersson

Abstract

LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR THE STUDY OF ARACHIDONIC ACID METABOLITES

Xiaojing Liu

Ian A. Blair, PhD

Arachidonic acid (AA) oxidation metabolism has been an important research topic for decades, and numerous oxidation products as well as enzymes involved in AA metabolism together with their downstream metabolites have been identified, although unknown pathways still remain to be characterized. In the present a study a new AA metabolite, 11-oxo-eicosatetraenoic acid (ETE), generated from a major product of cyclooxygenase (COX-2), 11(R)-hydroxyeicosatetraenoic acid (HETE), through 15-hydroxyprostaglandine dehydrogenase (15-PGDH)-mediated oxidation. 11-Oxo-ETE was found to have an anti-proliferative effect on human umbilical vein endothelial cells (HUVECs), with a similar IC50 to a well- known anti-inflammatory mediator, 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2). It was also found that 11-oxo-ETE could be inactivated through the glutathione-S-transferase (GST)/glutathione (GSH) pathway by forming a GSH adduct, or by undergoing reduction to 11(S)-HETE through metabolism by unknown reductases.

AA is also subject to non-enzymatic oxidation due to its bis-allylic hydrogen atoms and four double bonds. Chiral LC-MS methods were used to distinguish the enzymatic and non-enzymatic products by monitoring the relevant products that were formed. Non-enzymatic pathways generate racemic mixtures, while enzymatic pathways are normally highly stereoselective. It is known that both enzymatic and non-enzymatic oxidation of AA followed by decomposition of the resulting oxidized intermediates, can generates short-chain aldehydes such as 4-hydroperoxy-2(E)-nonenal (HPNE), 4-hydroxy-2(E) nonenal (HNE), and 4-oxo-2(E)-nonenal (ONE). However, it was discovered that the endoperoxide product of COX-2, PGG2, is a major contributor to the formation of these aldehydes. Moreover, PGG2 and other hydroperoxides generate HPNE or HNE with different chiral characteristics. From COX-2 expressing human colorectal adenocarcinoma epithelial cell line (LoVo) cell lysate, 4(S)-HNE was detected with high enantiomeric purity indicating that the primary source was prostaglandin G2 (PGG2) and not 15- hydroperoxyeicosatetraenoic acid (HPETE) as had been previously suggested.

Finally, through studies of 11(R)-HPETE and 15-HPETE decomposition, a new carboxylate-containing aldehyde, 11-oxoundeca-5,8-dienoic acid (OUDE) was identified. OUDE contains the -terminus of the lipid hydroperoxide precursor and is analogous to HNE, which is derived from the -terminus. Nuclear magnetic resonance spectroscopy (NMR) together with chiral liquid chromatography-mass spectrometry (LC-MS) analysis showed that OUDE was a mixture of two double bond isomers. It has been proposed that the two isomers are generated as a result of a rearrangement reaction subsequent to the formation of OUDE. In addition, , by monitoring the chirality of the HNE that was formed from 11(R)-HPETE and 15-HPETE decomposition, a homolytic mechanism for HNE formation has been proposed.

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