Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major
arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix
and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes
and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been
shown to be an effective cytoprotective agent to some cell types against oxidative damage by
reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in
the presence of hypochlorite (HOCl). Although a considerable amount of work has been
published on the composition of neopterin in atherosclerotic plaques, very little is known
about the variation of 78NP and other oxidative biomarkers across the length of the carotid
and femoral and their contribution to plaque progression, which was researched in this work.
Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced
into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8-
dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •-
aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7-
ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and
neopterin was also investigated in this study.
It was shown that carotid plaques significantly vary from femoral plaques, in the levels and
range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker
concentrations between plaques but also between sections of an individual plaque. Femoral
plaques on the other hand showed lower amounts of biomarkers with very little variation in
biomarker concentrations. High variation with pterin concentrations and other biomarkers
suggests dynamic and active changes in inflammation within the plaque. Collectively, it was
observed that every plaque was unique with respect to its composition and correlations
between the biomarkers.
Though shown to be a well-known antioxidant and a radical scavenger, there is no published
literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how
it enters the cells could explain the difference in its protective ability of different cell types
Abstract
xxviii
against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin
will provide insights about its protection of monocyte/macrophage cell death which could
potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is
produced from guanosine, a nucleoside that is transported using specialized nucleoside
transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside
transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin
transport.
It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside
transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major
transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1
cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human
monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH
mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that
nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular
protection against oxidative stress.
Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is
critical aspect to assess disease progression. The current C18 HPLC method used in our
laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile
(ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also
variable at times giving inconsistent measurement of neopterin so the manganese oxide
(MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another
option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to
neopterin.
A new method using strong cation exchange (SCX) column was developed for a precise,
sensitive neopterin assay which got rid of the ACN interference completely. The MnO2
method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum
or plaque homogenates. Electrochemical detection was also found to be very unreliable and
inconsistent.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/9224 |
| Date | January 2013 |
| Creators | Janmale, Tejraj Vijaykumar |
| Publisher | University of Canterbury. School of Biological Sciences |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Tejraj Vijaykumar Janmale, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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