Vitamin C is usually considered to act as an antioxidant, protecting NO from inactivation by reactive oxygen species (ROS). However, we and others have observed that, under some conditions, vitamin C inactivates NO (de Saram et al., 2002; Schrammel et al., 2000). One potential mechanism for this is the generation of superoxide anion (O₂-, which inactivates NO) by vitamin C from trace concentrations of iron or other transition metals. In this study we examined chemical interactions between authentic NO, vitamin C and iron (Fe³⁺) using electrochemical measurement of NO. We also studied effects of vitamin C on relaxation of rabbit aortic rings to authentic NO. The potential role of O₂- was investigated using the O₂- scavengers superoxide dismutase (SOD) and Tiron.

NO in solution was measured under constant stirring at 23°C using a Clark-type NO electrode, calibrated by adding KNO₂ to KI/H₂SO₄ solution. The initial rate of NO oxidation was measured in the presence and absence of vitamin C (100 µmol/L) alone or in combination with Fe³⁺ (25 µmol/L), SOD (500 U/ml), Tiron (10 mmol/L) or the specific iron chelator diethylenetriaminepentaacetic acid (DTPA, 0.1 mmo/L). Thoracic aortic rings (2 mm) obtained from New Zealand white male rabbits (2-2.5 Kg) were mounted in organ baths containing oxygenated Krebs solution at 37°C. Responses were recorded isometrically. Rings were constricted with phenylephrine to 80% maximum tension and then relaxed with NO to obtain control relaxation responses. Rings were then washed out and incubated with vitamin C (0.1-10 mmol/L, 15 min) ± Fe³⁺ ± Tiron or SOD and contraction then relaxation to NO repeated. As a positive control relaxation responses to NO were also examined in the presence of the O₂- generator pyrogallol (10 µmol/L) ± SOD.

Vitamin C (100 µmol/L) enhanced the rate of NO oxidation in solution from 0.8±0.1 to 7.4±0.3 nmol/L/sec (P<0.001, n=4). Additional Fe³⁺ (25 µmol/L) augmented the effect of vitamin C (rate of oxidation: 24±1.2 nmol/L/sec, P<0.001, n=4) but DTPA and SOD incompletely reversed the effect of vitamin C on NO oxidation. In organ bath experiments vitamin C (0.1-10 mmol/L, 15 min) produced a concentration-dependent attenuation of relaxation to NO with a parallel shift to a higher concentration range of the dose-response curve of 1.8±0.2 log units at the highest dose (n=5, P<0.001). Fe³⁺ did not augment the inhibitory effect of vitamin C (100 µmol/L) on relaxation to NO. Neither SOD nor Tiron reversed the effect of vitamin C (10 mmol/L) on relaxation to NO, (EC₅₀ -5.8±0.2 vs. -5.9±0.1 log units for vitamin C alone and vitamin C plus Tiron respectively, n=4). In contrast pyrogallol inhibited relaxation to NO with a parallel shift to a higher concentration range of the dose response curve of 0.97±0.1 log units (n=2) and this inhibition was abolished in the presence of SOD.

Transition metal ion driven generation of O₂- may contribute to inactivation of NO by vitamin C. However, the failure of iron chelation and O₂- scavengers to fully reverse effects of vitamin C suggests an additional reaction, possibly involving the ascorbyl radical.

de Saram K. et al. (2002) Br. J. Pharmacol. 135: 1044-1050.
Schrammel A. et al. (2000) Cardiovasc. Res. 47: 602-608.