Abstract
Context: Exogenous nitrogen oxides must be made bioavailable to sustain normal physiology because nitric oxide synthase (NOS) deficient mice are viable. In the stomach, S-nitrosoglutathione (GSNO) is formed from ingested nitrite and high levels of airway glutathione (GSH) that are cleared and swallowed. However, gastric GSNO may be broken down by nutrients like ascorbic acid (AA) before it is absorbed.Objective: To study the effect of AA on GSNO formation and stability.Materials and methods: GSH and nitrite were reacted with or without 5 mM AA or Resiston (5 mM AA with retinoic acid and α-tocopherol). GSNO was measured by reduction/chemiluminescence and HPLC. AA and reduced thiols were measured colorimetrically. O-Nitrosoascorbate and AA were measured by gas chromatography–mass spectrometry (GC–MS).Results: GSNO was formed in saline and gastric samples (pH ∼4.5) from physiological levels of GSH and nitrite. Neither AA nor Resiston decreased [GSNO] at pH >3; rather, they increased [GSNO] (0.12 ± 0.19 μM without AA; 0.42 ± 0.35 μM with AA; and 0.43 ± 0.23 μM with Resiston; n = 4 each; p ≤ 0.05). However, AA compounds decreased [GSNO] at lower pH and with incubation >1 h. Mechanistically, AA, but not dehydroascorbate, increased GSNO formation; and the O-nitrosoascorbate intermediate was formed.Conclusions: AA, with or without other antioxidants, did not deplete GSNO formed from physiological levels of GSH and nitrite at pH >3. In fact, it favoured GSNO formation, likely through O-nitrosoascorbate. Gastric GSNO could be a NOS-independent source of bioavailable nitrogen oxides.
Highlights
Proteins and peptides that have been modified to form S-nitrosothiol bonds are involved in guanylate cyclase (GC)-independent signalling by nitrogen oxides, though S-nitrosylation affects GC-dependent processes (Mayer et al 1998)
Aerobic reduction/chemiluminescence method that was used for nitrosocompound detection could not completely distinguish between GSNO and O-nitrosoascorbate (Gow et al 2002) and the total amount of nitrosocompounds was measured; but the presence of the intermediate was confirmed by GC–MS
We conclude that strong biological reducing agents in the form of AA and antioxidant vitamins do not deplete the beneficial GSNO formed in human gastric contents at physiological gastric pH; they can augment it via transnitrosation from O-nitrosoascorbic acid to GSH
Summary
Protein S-nitrosylation, the post-translational modification of a cysteine thiol by a nitric oxide (NO) group, is involved in a broad spectrum of cell signalling effects (Gow et al 2002; Gaston, Singel, et al 2006; Paige et al 2008; Foster et al 2009). We have identified a reaction in the gastric mucosa that can lead to increased formation of the endogenous, clinically beneficial S-nitrosothiol, S-nitrosoglutathione (GSNO), in vivo. This reaction is augmented, not inhibited, by ascorbic acid (AA) at gastric pH
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