Fig. 4.

Effect of extracellular catalase on ROS and HbNO formation in human RBCs.

A-B) Effect of catalase on ROS formation (A) in total erythrocytes assayed by DCFDA (1 μmol/L) fluorescence; and on HbNO concentrations (B) assayed by EPR in human RBCs (50% haematocrit) pre-incubated with Spermine-NONOate (50 μmol/L; 45 min). Non-PEGylated catalase was pre-incubated under venous O₂ level (4% O₂; 45 min), then H₂O₂ (10 μmol/L; or vehicle) added and fluorescence measured every 5 min. (A); or erythrocytes were frozen every 15 min up to 45 min for low-temperature EPR spectroscopy (B).

C) Typical EPR spectra recorded in human RBC samples frozen at 30 min after pre-treatment with 50 μmol/L of Spermine-NONOate, without (a–c) or with (b–d) catalase (45 min at 4% of O₂), and exposed to vehicle (a–b) or H₂O₂ (c–d). The hyperfine structure (hfs) of the HbNO EPR signal is shown by the arrows.

D) Erythrocyte HbNO concentrations quantified from EPR signals of RBCs (50% of haematocrit in isotonic buffer) pre-incubated with antioxidant enzymes SOD or catalase at 21% of O₂ and then, exposed to graded concentrations of Spermine-NONOate for 45 min at 1% of O₂ before freezing for the low-temperature EPR assay.

Data in A, B and D are shown as mean values ± SEM and treated using a mixed model statistical analysis with Dunnett's adjustment for multiple comparisons; *P < 0.05 ^$P < 0.01; n = 3–6 different RBC preparations.