Albumin Thiolation and Oxidative Stress Status in Patients with Aortic Valve Stenosis

Figures

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  Figure 1. Representative chromatograms of different HSA proteoforms. Serum was obtained from (A) patients with aortic valve stenosis before valve replacement, (B) patients undergoing a second valve replacement, and (C) controls.

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  Figure 2. HSA proteoforms in patients and control subjects. Percentages of (A) HSA-Cys, (B) HSA-Gly, (C) HSA-SH were measured in serum obtained from patients with valvular disease before valve replacement (Valv repl, n = 36), patients requiring a second valve replacement to a degenerated bioprosthetic valve (Redo valv repl, n = 8), and healthy donors (Controls n = 10) by LC–mass spectrometry. Data are expressed as mean ± SEM. * p < 0.05; **** p < 0.0001 by one-way ANOVA followed by Tukey’s post hoc test.

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  Figure 3. Correlation between HSA-Cys and free sulfhydryl groups and levels of free sulfhydryl groups in patients and control subjects. (A) HSA-Cys was measured in serum of all participants to the study by LC–mass spectrometry. Free sulfhydryl groups were detected in serum using Ellman’s reagent. (B) Free sulfhydryl groups detected in serum obtained from patients with valvular disease before valve replacement (Valv repl, n = 36), and healthy donors (Controls n = 10). Data are expressed as mean ± SEM. * p < 0.05.

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  Figure 4. Analysis of free sulfhydryl groups in plasma treated with ASA. Blood obtained from healthy donors was incubated in vitro at 37 °C for 120 min in the presence or the absence of ASA. After centrifugation, plasma was collected, and free sulfhydryl groups were measured using Ellman’s reagent. n = 4. * p < 0.05 by paired Student’s t-test.

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  Figure 5. HSA proteoforms in serum of patients with aortic valve stenosis after in vitro treatment with NAC or its amide derivative AD4/NACA. Serum obtained from patients before valve replacement was incubated at 37 °C for 60 min in the presence of NAC or AD4/NACA. Levels of (A) HSA-Cys and (B) HSA-SH were measured by LC–mass spectrometry. n = 6. **** p < 0.0001 by one-way ANOVA followed by Dunnett’s post hoc test.

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  Figure 6. Analysis of serum free sulfhydryl groups after in vitro treatment with NAC or its amide derivative AD4/NACA. Serum of patients before valve replacement was incubated at 37 °C for 60 min in the presence of NAC or AD4/NACA. Free sulfhydryl groups were measured using Ellman’s reagent. n = 6. * p < 0.05; *** p < 0.001 by ANOVA followed by Dunnett’s post hoc test.

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  Figure 7. Antioxidant activity of serum after in vitro treatment with NAC or its amide derivative AD4/NACA. The serum of patients before valve replacement was incubated at 37 °C for 60 min in the presence of NAC or AD4/NACA. Antioxidant activity was detected by DCF fluorescence. (A) A representative curve of fluorescence of DCF (λex = 485 nm, λem = 532 nm) in the presence of AAPH as a generator of radical species. Data are expressed as arbitrary units of fluorescence (AFU) of DCF. (B) Effect of NAC and AD4/NACA on the oxidation of DCFH. The fluorescence of DCF was measured after 180 min after the start of the reaction. Data are expressed as % of fluorescence. n = 6. **** p < 0.0001 by ANOVA followed by Dunnett’s post hoc test.

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  Figure 8. Feedback mechanisms that might perpetuate oxidative stress in patients with valvular disease. After the initiation of valvular inflammation and stenosis, mechanosensitive feedback pathways may provide the coupling link that promotes further valvular interstitial cell differentiation, valvular inflammation, and oxidative stress.