Anajirih, Nuha
(2019)
Endocannabinoid hydrolases in human blood fractions.
PhD thesis, University of Nottingham.
Abstract
Despite extensive studies of the cardiovascular effects of cannabinoids and evidence that endocannabinoid levels in the blood can be influenced by diet and metabolic state, there has been no investigation of the activities of endocannabinoid hydrolases (fatty acid amide hydrolase, FAAH, and monoacylglycerol lipase, MAGL, which hydrolyse anandamide, AEA, and 2-arachidonoylglycerol, 2AG, respectively), their variation in human blood, and which methods can be used to monitor them. This thesis had the aim of identifying the activity levels of these enzymes in human blood fractions and the influence of clotting processes.
To characterise enzyme activities, multiple assay styles (a radiometric assay, activity-based protein profiling, ABPP, and a spectrophotometric assay) were utilized with blood vessels and blood samples from Wistar rats. Functional expression of MAGL and FAAH in these samples was measured using the Gold Standard technique of the radiometric assay; the results of which agreed broadly with ABPP. By contrast, the spectrophotometric assay used for MAGL activity detection was not suitable for enzyme detection using complex samples. In rat mesenteric arteries, MAGL was not affected, whereas FAAH activity increased with age of the donor animals, which suggests that FAAH may either be a potential therapeutic target or biomarker for vascular dysfunction associated with age. The activity levels of these enzymes were higher in circulating blood compared to vascular tissue, suggesting a potentially greater influence on circulating endocannabinoid levels.
In a human study, overnight fasting blood samples were obtained on three different occasions from healthy volunteers of both genders within the University of Nottingham (n=18). Studies provided evidence that MAGL and FAAH activities were measurable and reproducible in healthy human blood. Platelets contained a higher proportion of MAGL activity while erythrocytes contained a higher proportion of FAAH activity compared to other blood fractions, which predicts their roles; MAGL may be involved in the platelet aggregation process, while FAAH may be involved in the turnover of erythrocytes. There was no difference in the level of MAGL and FAAH activities when comparing activity over the three visits for individual donors or between male and female donors. The stability of enzyme activities within an individual suggests that the activity level of these enzymes could be used as a biomarker in the future. However, these studies identified that enzyme activity of isolated platelets and erythrocytes degraded after short term storage at -80 oC, but was retained after storage in liquid nitrogen. Clearly, this has implications for the archiving of material for subsequent enzyme analysis.
A second human study collected blood samples on one occasion from eight volunteers to investigate the influence of clotting processes. Clot formation was initiated by addition of thrombin and enzyme activity was measured in the supernatant layer following centrifugation at low speed equivalent to that used to separate serum. The clotting process altered enzyme distribution, where FAAH in the serum-like fraction could be liberated from either platelets or erythrocytes, while MAGL activity was not present in the supernatant layer and was only marginally reduced by the direct proteolytic effects of thrombin. ABPP was able to detect multiple serine hydrolase enzymes with higher activity such as MAGL, but not those with marginal activity, such as FAAH, in these blood fractions.
Finally, the functional impact of extended inhibition of MAGL and FAAH activities on proliferation and migratory activity were investigated with the MCF-7 breast adenocarcinoma cell line using MTT and cell exclusion assays, respectively. Although MCF-7 cells were found to express FAAH, but not MAGL, inhibition of FAAH activity did not have any functional impact on proliferation or migration of these cells. Thus, FAAH inhibition could be considered not useful as a therapeutic target for breast cancer.
These findings provide evidence, for the first time, of the reproducible detection of MAGL and FAAH activities in human blood samples, how their functional expression changes during normal patho/physiological processes such as clotting, and how neither longer term FAAH nor MAGL inhibition has any effects on MCF-7 cell proliferation or migration. This research sets the foundation for future clinical studies using the expression of these enzymes as a potential biomarker for dysfunction or disease.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Alexander, Stephen
O'sullivan, Saoirse |
| Keywords: |
Endocannabinoid, MAGL, FAAH and blood cells. |
| Subjects: |
Q Science > QP Physiology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
57303 |
| Depositing User: |
Anajirih, Nuha
|
| Date Deposited: |
26 Apr 2022 09:30 |
| Last Modified: |
26 Apr 2022 09:31 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/57303 |
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