Autoxidation of ascorbate

The kinetic results reported by Jameson and Blackburn (11,12) for the copper catalyzed autoxidation of ascorbic acid are substantially different from those of Taqui Khan and Martell (6). The former could not reproduce the spontaneous oxidation in the absence of added catalysts when they used extremely pure reagents. These results imply that ascorbic acid is inert toward oxidation by dioxygen and earlier reports on spontaneous oxidation are artifacts due to catalytic impurities. In support of these considerations, it is worthwhile noting that trace amounts of transition metal ions, in particular Cu(II), may cause irreproducibilities in experimental work with ascorbic acid (13). While this problem can be eliminated by masking the metal ion(s), the masking agent needs to be selected carefully since it could become involved in side reactions in a given system. 

Iron(III)-catalyzed autoxidation of ascorbic acid has received considerably less attention than the comparable reactions with copper species. Anaerobic studies confirmed that Fe(III) can easily oxidize ascorbic acid to dehydroascorbic acid. Xu and Jordan reported two-stage kinetics for this system in the presence of an excess of the metal ion, and suggested the fast formation of iron(III) ascorbate complexes which undergo reversible electron transfer steps (21). However, Bansch and coworkers did not find spectral evidence for the formation of ascorbate complexes in excess ascorbic acid (22). On the basis of a combined pH, temperature and pressure dependence study these authors confirmed that the oxidation by Fe(H20)g+ proceeds via an outer-sphere mechanism, while the reaction with Fe(H20)50H2+ is substitution-controlled and follows an inner-sphere electron transfer path. To some extent, these results may contradict with the model proposed by Taqui Khan and Martell (6), because the oxidation by the metal ion may take place before the ternary oxygen complex is actually formed in Eq. (17). 

In alkaline solution (pH 11), the complex is present as a p-oxo dimer and ascorbic acid is fully deprotonated. In the absence of oxygen, kinetic traces show the reduction of Fe(III) to Fe(II) with a reaction time on the order of an hour at [H2A] =5xlO-3M. The product [Fen(TPPS)] is very sensitive to oxidation and is quickly transformed to Fe(III) when 02 is added. This leads to a specific induction period in the kinetic traces which increases with increasing [02]. The net result of the induction period is the catalytic two-electron autoxidation of ascorbic acid in accordance with the following kinetic model (23) ... 

The presence of ascorbic acid as a co-substrate enhanced the rate of the Ru(EDTA)-catalyzed autoxidation in the order cyclohexane < cyclohexanol < cyclohexene (148). The reactions were always first-order in [H2A]. It was concluded that these reactions occur via a Ru(EDTA)(H2A)(S)(02) adduct, in which ascorbic acid promotes the cleavage of the 02 unit and, as a consequence, O-transfer to the substrate. While the model seems to be consistent with the experimental observations, it leaves open some very intriguing questions. According to earlier results from the same laboratory (24,25), the Ru(EDTA) catalyzed autoxidation of ascorbic acid occurs at a comparable or even a faster rate than the reactions listed in Table III. It follows, that the interference from this side reaction should not be neglected in the detailed kinetic model, in particular because ascorbic acid may be completely consumed before the oxidation of the other substrate takes place. 

The main features of the copper catalyzed autoxidation of ascorbic acid were summarized in detail in Section III. Recently, Strizhak and coworkers demonstrated that in a continuously stirred tank reactor (CSTR) as well as in a batch reactor, the reaction shows various non-linear phenomena, such as bi-stability, oscillations and stochastic resonance (161). The results from the batch experiments can be suitably illustrated with a two-dimensional parameter diagram shown in Pig. 5. 

Dunn JA, Ahmed MU, Murtiashaw MH, Richardson JM, Walla MD, Thorpe SR and Baynes JW (1990) Reaction of ascorbate with lysine and protein under autoxidizing conditions formation of N-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate. Biochemistry 29, 10964-10970. 

The autoxidation of ascorbate, a cosubstrate of dopamine P-monooxygenase, induces the degradation of most proteins including catalase and dopamine p-monooxygenase, but with the exception of (Cu,Zn)-SOD. Catalase protects dopamine P-monooxy-genase and is therefore generally added in the assay systems . The apparent activation or rather the stabilization of the enzyme (6.5 pg) by small amounts of catalase (3.1 pg) was enhanced by native but not by boiled SOD (100 pg) and also by similar amounts of serumalbumin (100 pg) or of boiled catalase (65 pg)... 

The effect of SOD points to the intervention of Oj in the autoxidation of ascorbate. Proteins in large amounts could react with the strong oxidizing agent formed in this Udenfriend s system , thus protecting the enzyme. The ineffectiveness of boiled SOD could be due to its amino-acid composition (See Sect. 4.1.2). While O did not inactivate dopamine P-monooxygenase the rate pf inactivation in the presence of... 

Catalysis of the Autoxidation of Ascorbic Acid by Metal Ions and Metal Chelates... 

Metal-Ion-Catalyzed Autoxidation. Figure 1 illustrates the variation of the first-order rate constants for the autoxidation of ascorbic acid by molecular oxygen with the concentration of the Cu (II) ion, which is present in catalytic (i.e. low) concentrations (8). The linear relationship indicates second-order behavior [first order in ascorbic acid and first order in Cu(II)]. The catalytic effect of Cu(II) is also seen to decrease... 

A reaction mechanism for the metal-ion-catalyzed autoxidation of ascorbic acid, involving the formation of an intermediate ternary ascorbate-metal ion-dioxygen complex, is illustrated in Scheme 2. Although the bonding between the metal ion and the dioxygen in the intermediate... 

Figure 3. Variation of second-order rate constants for the Fe(lll) catalyzed autoxidation of ascorbic acid as a function of oxygen concentration at -log [H ] values of A, 3.85 B, 3.45 C, 3.00 t = 25°C = O.IOM...

The work described here on the Cu(II)- and Fe(III)-catalyzed autoxidation of ascorbic acid has been extended to catalytic systems involving vanadyl (12) and uranyl (13) ions. On the basis of the results described above it would seem that there are potentially many other metal ions that are capable of undergoing redox reactions with the ascorbate ion, and that may function as catalysts in the autoxidation of ascorbic acid. Analogous mechanisms may also apply to systems involving metal-ion catalysis of ascorbate oxidation in which the primary oxidant is a reagent other than molecular oxygen. 

Figure 4, Variation of rate constants for the autoxidation of ascorbic acid as a function of concentration of Cu(ll) chelates at 25°C and - log [H ] of 3.45 EDTA = ethylenediaminetetraacetic acid HEDTA = hy-droxyethylethylenediaminetetraacetic acid NT A = nitrilotriacetic acid HIMDA = hydroxyethyliminodiacetic acid IMDA = iminodiacetic acid.

Figure 6. Dependence of second-order rate constants for Fe(lll)-chelate-catalyzed autoxidation of ascorbic acid on hydrogen ion concentration at 25°C. Abbreviations are those given in Figures 4 and 5.

Scheme 3. Proposed mechanism for metal-chelate-catalyzed autoxidation of ascorbic acid.

Kate Laws for Metal-Ion- and Metal-Chelate-Catalyzed Autoxidation of Ascorbic Acid... 

Recently Jameson and Blackburn (14,15,16) have suggested an alternate mechanism for the copper-catalyzed autoxidation of ascorbic acid, involving the formation of a binuclear Cu(II) complex (17) of the ascorbate anion, and the subsequent formation of an intermediate peroxo type Cu( II)-dioxygen-ascorbate complex (18). Their kinetic data suggested a variety of rate behavior depending on the nature of the supporting electrolyte. Formula 17, which was postulated for nitrate... 

Ascorbic acid reacts with 02" generated from the xanthine oxidase system and may play a role against 02" mediated toxicity. Ascorbic acid quenches the hydroxyl radical (40), Ascorbic acid may protect against free radicals in the lung because ascorbic acid is found in the fluid (39). The toxicity of ozone and oxygen may also be reduced by ascorbic acid (39). Carbon tetrachloride mortality in rats is lowered by ascorbic acid. Autoxidation of ascorbic acid did not generate 02". Reduced glutathione reacts with dehydroascorbic acid (VII) and recycles ascorbic acid. 

An example of a reaction in which the reduced form of the metal ion is converted back to its higher oxidation state by molecular oxygen, is the autoxidation of ascorbic acid by copper(II) (206, 207). The probable course of the reaction is as follows The ascorbate ion forms an intermediate copper(II) chelate which undergoes an internal oxidation-reduction reaction, thereby forming a copper(I) semiquinone chelate. Dissociation of the relatively unstable copper(I) chelate occurs and the copper(I) ion is oxidized by molecular oxygen and the semiquinone is oxidized by molecular oxygen or copper(II) (160). 

The inhibition of the autoxidation of ascorbic acid in the presence of sucrose, dextrose, levulose, and corn sirup, has been studied by various workers (47, 50, 60, 77, 91, 96, 97,109). The autoxidation can be reduced by 10 to 90%, depending upon the concentration of sugar, the pH, and the amount of copper present. Shamrai 97) and Joslyn and Miller (50) attribute the reduced rate of the autoxidation of ascorbic acid at pH of 4 and above to the complexing action of copper by sugars. Copper is an effective oxidation catalyst even at concentrations as low as 1 p.p.m. 

The first model proposed for the drug monooxygenase stem in liver microsomes was described by Udenfnend et al. ) and since then referred to as the Udenfriend system . It consists of ferrous iron, EDTA and ascorbate which are incubated aerobically in an aqueous buffer of pH 5—8. Aromatic compounds were hydroxylated in this mixture to phenols and the substitution pattern pointed to an electrophilic mechanism. Breslow and Lukens argued that the Udenfriend system was not different from the Fenton stem since the autoxidation of ascorbate provides hydrogen peroxide, which by the reduction with ferrous ions forms hydroxyl radicals as hydroxylating agent ... 

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