Kinetic study on the reaction of sodium nitrite with neurotransmitters secreted in the stomach

Abstract

Nitroso-compounds are potentially mutagenic and carcinogenic compounds due to their ability to alkylate DNA bases. One of the most common sources of human exposure to nitroso-compounds is their formation in the acidic environment of the stomach by the reaction between electron-rich molecules present in the lumen and sodium nitrite ingested in the diet. To date, the formation of nitroso-compounds by the reaction of nitrite with food components has been investigated in depth, but little attention has been paid to substances secreted in the stomach, such as dopamine or serotonin, whose reaction products with nitrite have proven mutagenic properties. In this article, we present a kinetic study with UV-visible spectroscopy of the nitrosation reactions of both molecules, as well as of L-tyrosine, the amino-acid precursor of dopamine. We determined the kinetic parameters and reaction mechanisms for the reactions, studying the influence of the reactants concentration, pH, temperature, and ionic strength on the reaction rate. In all cases, the favoured reaction product was a stable nitroso-compound. Serotonin, the molecule whose product was the most mutagenic, underwent two consecutive nitrosation reactions. These findings suggest that additional biological research is needed to understand how this reaction alters the function of these neurotransmitters as well as the potentially toxic effects they may have once nitrosated.

Figure 1

Substrates studied in this paper.

Figure 2

Influence of the acidity of the medium on the observed rate constant k_obs. Clockwise from the top left: tyrosine, dopamine, and the second and first reactions of serotonin. The black line shows the fit of the experimental data to the rate equation derived from the proposed mechanisms. Tyrosine: [Tyr]₀ = 7.7·10⁻⁴ M, [Nit] = 0.01—0.03 M, T = 25.0 °C, I = 0.20 M. Dopamine: [DA]₀ = [Nit]₀ = 6.0·10⁻⁴—2.02·10⁻³ M, T = 20.0 °C, I = 0.2 M. Serotonin: [5-HT]₀ = 1.31·10⁻⁴ M, [Nit] = 3.02·10^–3 M, T = 20.0 °C, I = 0.24 M.

Figure 3

Proposed mechanism for the C-nitrosation of tyrosine and dopamine. The first step is the formation of the nitrosonium/nitrosacidium ion, which subsequently attacks tyrosine (top) or dopamine (bottom).

Figure 4

ΔH^‡/ΔS^‡ isokinetic relationship for the C-nitrosation reactions of tyrosine (Tyr), dopamine (DA), serotonin (5-HT (2)) and other nitrosatable substrates: phenol (ph), m-cresol (mc), o-cresol (oc), 2,3-dimethylphenol (d23p), 2,6-dimethylphenol (d26p), 3,5-dimethylphenol (d35p), o-chlorophenol (ocp), o-bromophenol (obp) and tyramine (tra)^{, ,} .

Figure 5

Kinetic profile of the nitrosation reactions of serotonin at different pHs and their fit to Eq. (9). The total absorbance, A₃₇₁, is shown with a grey line, the absorbance of nitrososerotonin (A_B) with a dashed black line, and the absorbance of dinitrososerotonin (A_C) with a solid black line. [5-HT]₀ = 1.31·10⁻⁴ M, [Nit] = 3.02·10⁻³ M, T = 20.0 °C.

Figure 6

Proposed mechanism for the first nitrosation of serotonin: the N-nitrosation of the nitrogen of the indole ring of the molecule.

Figure 7

Influence of the pH and nitrite concentration on the r_Subs/r_Phe ratio (Eq. (15)) of the nitrosation reactions of dopamine (left) and serotonin (first nitrosation in the middle, second at the right). The number closer to each isoline is the z parameter.