PhD Oral Exam - Oluwatosin Racheal Kuteyi, Chemistry

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Oxylipins are lipid mediators involved in inflammation, immunity, and oxidative stress. Accurately measuring oxylipins in biospecimens is analytically challenging due to their poor stability and susceptibility to both enzymatic and non-enzymatic reactions during sampling, storage, and transportation. Recently, in vivo solid-phase microextraction (SPME) has been introduced as an effective method for direct sampling and extraction of oxylipins from biological tissues and fluids. In vivo SPME protects oxylipins from enzymatic reactions by preventing the co-extraction of proteins onto the device, thereby eliminating enzymatic conversion in biological fluids and tissues after collection. However, it is not known how the storage in the SPME coating affects oxylipin stability and susceptibility to non-enzymatic reactions such as autoxidation, hydrolysis, and isomerization, which may significantly contribute to oxylipin degradation. The objective of this thesis was to evaluate the stability of oxylipins on SPME devices post-extraction and investigate whether the use of antioxidants, such as butylated hydroxytoluene (BHT), is useful to minimize degradation processes. The appropriate procedures to pre-load antioxidant(s) onto SPME devices during conditioning step, or load antioxidants(s) post-extraction during the rinse step were successfully developed and are compatible with in vivo SPME workflows. Finally, degradation products of selected unstable oxylipins were comprehensively mapped and it was determined whether they interfere with the analysis of stable oxylipins.

To investigate the stability of oxylipins on SPME devices, oxylipins were extracted from standard solutions or citrated human plasma samples using hydrophobic lipophilic balance (HLB) SPME devices and analyzed by C18 liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Oxylipin stability, with or without BHT, was evaluated by comparing test conditions to control samples (0-T), using ANOVA and acceptance criteria of 80-120% of the control samples. Out of 49 oxylipins tested during a 3-freeze-and-thaw (3-FT) cycles stability study, only arachidonic acid (AA) and prostaglandin E2 (PGE2) were found unstable after extraction from a standard solution. After extraction of oxylipins from spiked and non-spiked human plasma, 13-hydroxydocosahexaenoic acid (13-HDHA), 11-hydroxydocosahexaenoic acid (11-HDHA), 9-oxooctadecadienoic acid, and 9-hydroxyoctadecatrienoic acid were unstable. 4-hydroxydocosahexaenoic acid, 13-oxo-octadecadienoic acid, 5-oxo-eicosatetraenoic acid, 15-hydroxy-eicosatetraenoic acid (15-HETE), and 11,12-epoxy- eicosatetraenoic acid were unstable when stored on SPME devices at room temperature (RT) for up to 18 days. Next, pre- and post-extraction loading method for BHT was successfully developed to investigate its ability to minimize autooxidation during storage of SPME devices. BHT loading successfully improved stability for AA and PGE2 during 3-FT cycle stability study. For oxylipins extracted from standard solutions, all oxylipins were found to be stable when using BHT post-extraction loading, whereas only 11-prostaglandinF2α was unstable when BHT was pre-loaded onto SPME devices. In the spiked human plasma samples, some oxylipins were identified as unstable after both pre- and post-extraction loading of BHT, including 16-hydroxydocosahexaenoic acid, 13-HDHA, 11-HDHA, and 12-hydroxyeicosatetraenoic acid. Other unstable oxylipins were identified for each approach: 8-iso-15R-prostaglandin F2α, prostaglandinF2α, 14,15-dihydroxyeicosatrienoic acid, 5-hydroxyeicosatetraenoic acid, 13-hydroxyoctadecadienoic acid, AA, and linoleic acid were unstable when BHT was pre-loaded, while leukotriene B4, 9-hydroxyeicosatetraenoic acid, and docosahexaenoic acid were unstable when BHT was loaded post-extraction during rinse step.

Lastly, all of the above unstable oxylipins were subjected to forced degradation studies including photooxidation (365 nm for 5 and 7 days), copper sulphate oxidation, and elevated temperatures (37°C and 50°C for 3 days) to map their degradation pathways and systematically assess whether degradation products of unstable oxylipins and their precursors interfere with the accurate measurement of stable oxylipins. In conclusion, this is the first study to characterize the stability of oxylipins on SPME devices, demonstrating how SPME can effectively improve stability during sample storage, handling, and shipping. Importantly, these stability results also show how the degradation of unstable oxylipins can impact the accurate measurement of stable oxylipins, thus representing an under-appreciated source of error during the measurement. This study also provides important novel insight into major degradation products of PUFAs and selected unstable oxylipins.