Figure 8. Chronic vascular oxidative stress promotes isoketal-adduct formation.

(A and B) Stable isotope dilution multiple reactions monitoring MS analysis of isoketal-lysine-lactam (IsoK-Lys) adducts in aortas of 9-month-old animals. Representative LC/MS chromatographs from pooled samples for each group (A). The left panel shows chromatographs for IsoK-Lys in samples, while the right panel shows chromatographs for [¹³C₆¹⁵N₂] internal standards in the same samples. Mann-Whitney tests were employed for analysis. The bar graph in B shows mean data. (C) Fix-perfused mouse aortas were embedded in paraffin and subjected to IHC for isoketal staining. Images are ×20 magnification. Scale bars: 100 μm. (D–I) Intracellular staining for isoketals in spleen macrophages (D and E), DCs (F and G), and monocytes (H and I). These data were analyzed using 1-way ANOVA, n = 6. *P < 0.05; **P < 0.01. (J) DCs isolated from 9-month-old WT and tg^sm/p22phox mice were exposed to weak acid to elute peptides presented on MHC-I complexes. The presence of isoketal-adducted peptides in the eluent was visualized by dot blot. Isoketal adducts were present in 3 of 4 tg^sm/p22phox mice but not in WT mice (see Supplemental Figure 5C for full uncut gel).