Ascorbic acid (vitamin C) is usually considered to be an antioxidant, protecting nitric oxide (NO) from inactivation by superoxide anion (O₂^-). However, ascorbic acid also generates O₂^- in oxygenated solutions containing trace concentrations of transition metal ions (Rietjens et al., 2002). This partially accounts for its paradoxical pro-oxidant effects. We investigated whether this or an alternative mechanism explains inhibitory effects of ascorbic acid on relaxation of rabbit aortic rings to authentic NO (de Saram et al., 2002). We examined chemical interactions between NO, ascorbic acid and ferric iron (Fe³⁺) using electrochemical measurement of NO. The potential role of O₂^- was investigated using 4,5-dihydroxy-1,3-benzene-disulfonic acid ( Tiron) and superoxide dismutase (SOD), intra- and extracellular O₂^- scavengers respectively.

NO in solution was measured under constant stirring at 23 °C using a Clark-type NO electrode. The initial rate of NO decay was measured in the presence and absence of ascorbic acid (0.1-10 mmol/L) alone or in combination with Fe³⁺ (25 m mol/L), SOD (500 U/mL) 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. Rings were constricted with phenylephrine to 80% maximum tension and then relaxed with authentic NO . Rings were washed out and incubated with ascorbic acid (0.1-10 mmol/L, 15 min) ± Fe³⁺± Tiron or SOD and contraction then relaxation to NO repeated. Electron paramagnetic resonance (EPR) was used to measure ascorbyl radical formation in solutions of ascorbic acid (1 mmol/L) both in the absence and presence of NO (400 m mol/L) or the NO donor diethylamine NONOate (DEA-NONOate, 800 µmol/L). Data are expressed as means ± SEM.

Ascorbic acid (0.1-10 mmol/L) increased the rate of NO decay from 1.45±0.26 (baseline) to 15.4±1.6 nmol/L/s (ascorbic acid, 10 mmol/L, P<0.0001, n=10). Effects of physiological ascorbic acid (0.1 mmol/L) were augmented by Fe³⁺ (19 ± 2.0 nmol/L/sec, 25 µmol/L, P<0.001) and inhibited by SOD (500 U/mL) and DTPA (0.1 mmol/L) (each P<0.01, n=6). By contrast ascorbic acid (10 mmol/L) inactivated NO in a manner independent of DTPA or SOD. Ascorbic acid (0.1-10 mmol/L) inhibited relaxation of pre-contracted rabbit aortic rings to NO causing a parallel shift to a higher concentration range of the concentration-response curve, increasing logEC₅₀ from –7.4 ± 0.1 (control) to –5.9 ± 0.1 (ascorbic acid, 10 mmol/L, P<0.0001, n=8). Such inhibitory effects were not augmented by Fe³⁺ and not reversed by Tiron (10 mmol/L) or SOD (500 U/mL). EPR studies demonstrated a qualitative increase in ascorbyl radical intensity following addition of NO or DEA-NONOate to ascorbic acid. Transition metal driven generation of O₂^- contributes to inactivation of NO by ascorbic acid. However, these data suggest an additional direct reaction between physiologically relevant concentrations of ascorbic acid and NO, independent of O₂^- and Fe³⁺, with potentially diverse biological consequences.

Rietjens, I. et al. (2002) Environ. Toxicol. Pharmacol.11, 321-333.
de Saram, K. et al. (2002) Br. J. Pharmacol.135, 1044-1050.