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J.P. Dillon, Z. Wang, D.C. Paik, L.V. Del Priore, E.R. Gaillard; Detection of Nitro–Adducts by LC/MS in Aged Human Bruch's Membrane . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1399.
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© ARVO (1962-2015); The Authors (2016-present)
Sources of human nitrite exposure to Bruch's membrane include smoking and inflammation, both well known to be implicated in the development of AMD. We have recently observed that nitrite modification of basement membrane proteins can have deleterious effects on overlying retinal pigment epithelial cells. Thus, the present work was undertaken in order to determine if evidence of tissue nitration exists in older human Bruch's membrane.
Samples of human Bruch's membrane/choroid complex were isolated using standard techniques from globes obtained through the Illinois Eye Bank. Absence of free nitrite was confirmed colorimetrically. The samples were acid hydrolyzed in 6N HCl and analyzed by LC/MS (ThermoElectron, LCQ Advantage, Surveyor) for the presence of nitro–adducts, including 3–nitro–tyrosine. The instrument consists of a Surveyor LC with PDA detector and a quadrupole ion trap mass analyzer with an electrospray ion source. A Synergi Max C12 RP column (150 X 4.6 mm) was used. The LC mobile phase was ACN balanced with H2O (both containing 0.05% acetic acid) with the following gradients: 1–10% ACN for 50 mins, 10–60% ACN for 30 mins, 60–100% ACN for 20 mins and a flow rate 0.2 mL/min.
The 3–nitro–tyrosine (3nt) peak (M+1=m/z 227) was identified at ca. 52 min in both Bruch's membrane samples (2 of 3 from individuals over age 70) as well as standard 3nt (Sigma). Selected Reaction monitoring (SRM) specific for 3nt was used for positive identification (i.e. monitoring for m/z 227 with fragment m/z 181 representing loss of a nitro=46). Tandem fragmentation spectrum (MS/MS) indicated losses of 17 (–H2O) and 46 (–NO2) from the parent ion. In order to quantitate the levels of 3nt, a calibration curve was obtained from area calculations of the SRM scans using commercially available 3nt. The regression equation was 282952902x + 4468958. Using this equation, the amount of 3nt present in BM was estimated to be approximately 25 nanograms (110 pmoles) per 3cm diameter tissue sample.
Our data suggests that there is nitration present within Bruch's membrane harvested from humans over 70. To our knowledge this is the first chemical/molecular evidence that tissue nitration occurs in aged human Bruch's membrane and may indicate that reactive nitrogen species play a role in the pathogenesis of AMD.
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