Reduction of dehydroascorbic acid

Figure 12. Ascorbic acid-dehydroascorbic acid redox system (a) oxidation of ascorbate to semidehydroascorbic acid, (b) disproportionation of semidehydroascorbic acid, and (c) reduction of dehydroascorbic acid [From (100), with permission].

I. Dehydroascorbic Acid Determination by Reduction. A disadvantage of the dichlorophenolindophenol reduction method is that the dehydroascorbic acid can be determined only after its reduction to ascorbic acid. The reducing agent used may interfere in the subsequent reduction of the dye. Since ascorbic acid exists mainly in the reduced form in most tissues, this is not a serious disadvantage for the reduction method in the usual analysis. The reduction of dehydroascorbic acid to ascorbic acid is usually performed vi ith HaS by treatment for 15 min between pH 1.2 and 4.7 (R20). The completeness of the reduction was checked by the dinitrophenylhydrazine method. 

Reduction of dehydroascorbic acid is one of the steps of a potential electron transport system in plants involving ascorbic add oxidase as the terminal oxidation reaction (Eqs. 19). 

The complexity of the redox processes involving vitamin C have not prevented numerous studies of the oxidation of L-ascorbic acid and some on the oxidation and reduction of dehydroascorbic acid and other oxidation products. One of the reasons for the great interest in this subject is that the role of vitamin C in living systems is certainly connected to its oxidation-reduction behaviour and this may ultimately be the key to understanding the biological mechanisms of the actions of vitamin C. In this. section we will examine the oxidation and reduction of vitamin C by a wide variety of reagents, with emphasis on the behaviour of L-ascorbic acid since this is the most studied compound in this respect. 

The ascorbic acid is oxidized by ascorbic acid oxidase in the flour dough to dehydroascorbic acid. In turn, the reduction of dehydroascorbic acid is coupled with the oxidation of protein thiol groups by the enzyme dehydroascorbic acid reductase known to be in wheat (133). 

Whether coenzyme I was concerned in the earlier experiments was not determined and has not mnce been determined. The evidence from these studies was suggestive of the participation of ascorbic acid as a respiratory carrier. It was, however, only suggestive and not conclusive, for the authors did not demonstrate either with their lactate dehydrogenase or hexose diphosphate systems the direct reduction of dehydroascorbic acid to ascorbic acid. 

Further evidence of like character in support of the participation of coenzyme I in reactions associated with the reduction of dehydroascorbic acid has been advanced by Waygood (1950). Cell-free extracts of wheat seedlings were found to contain a malic dehydrogenase enzyme, reducing coenzyme I, as well as ascorbic oxidase and peroxidase enzymes. When to such extracts malic acid, coenzyme I, and ascorbic acid were added, together with a fixative for the oxalacetate formed in the reaction, the system absorbed oxygen in excess of that required for the complete oxidation of ascorbic acid. In this system methylene blue could replace ascorbic acid. 

The rate of the uncatalyzed reaction is, however, too slow to be of much consequence, the half-time period for the reduction of dehydroascorbic acid by GSH at physiological temperatures and at pH values and in concentrations usually found in vivo being of the order of 15 minutes, whereas under the same conditions the conversion of DHA to 2,3-diketo-gulonic acid has a half life of only 2 minutes (Ball, 1937). 

The electrochemical reduction of dehydroascorbic acid monophenylhydrazone gave scorbaminic acid more easily than did chemical methods, and electrooxidation was found to be an efficient method of converting ascorbic acid to its dehydro-derivative.A further electrochemical study examined the redox reaction of tris(2-deoxy-2-L-ascorbyl)amine. ... 

Plants have an enzyme system linking the oxidation of,glutathione to the reduction of dehydroascorbic acid. A similar enzyme may occur in animal tissues, although in this case a facile non-en matic reaction could possibly account for the observed activity. The plant eni me provides a... 

The reduction of dehydroascorbic acid by glutathione directly and in a non-enzymatic fashion has been suggested (Borsook et al., 1937). However, such stud-... 

The Cambridge workers were initially engaged in a study of the oxidation of ascorbic acid by O2 in the presence of the plant juices. During the course of this study they noted that addition of GSH apparently prevented the oxidation of ascorbic acid. Further analysis showed that the added GSH was itself oxidized to GSSG under these circumstances. The rate of the GSH oxidation was equal to the rate of the oxidation of ascorbic acid in the absence of added GSH. These observations were explained by the additional observation that the reduction of dehydroascorbic acid by GSH proceeded four times as fast as the oxidation of ascorbic acid by O2. In the presence of GSH, the dehydroascorbic acid (formed by the action of ascorbic-acid oxidase) was reduced as fast as it was formed. Under these circumstances the ascorbic-acid system apparently catalyzed the oxidation of GSH by O2, and the GSH protected the ascorbic acid from oxidation. 

The conclusions of Hopkins and Morgan were questioned by Kertesz (46), who reported that he could not confirm the experimental findings. Kohman and Sanborn (10) showed, however, that the fresh juice of peas and beans catalyzed the reduction of dehydroascorbic acid by GSH. Subsequent work by Crook and Hopkins (47) and by Crook (41) confirmed and extended the original observations of the Cambridge workers. Ascorbic-acid reductase was separated from ascorbic-acid oxidase with which it had initially been associated, and it was shown to be a distinct enzyme. Its existence is no longer questioned. 

The chemical reduction of dehydroascorbic acid by GSH occurs very readily at slightly alkaline pH s. It is slow at pH 6.0, but the reaction rate increases markedly with increasing pH. 

Parrot and Gazave (53) have reported that addition of catechol increases the extent of reduction of dehydroascorbic acid by GSH. Their observations appear to require confirmation. If they are correct, it would seem that catechol can participate in the reaction under consideration. 

It is hoped that further work on the plant mitochondria preparations studied by Young and Conn (25) may help to clarify the possible role of GSH in plant respiration. Preliminary experiments have shoAvn that the mitochondria from avocados catalyze oxidation of ascorbic acid by O2, and that GSH is also oxidized by O2 when a trace of ascorbic acid is added to the particles. Some evidence has also been obtained that the particles catalyze a reduction of dehydroascorbic acid by citrate in the presence of TPN and GSH. Further work on these systems is in progress. Experiments are also in progress to determine whether oxidative phosphorylations occur during reactions of the type described. 

A reduction of dehydroascorbic acid (DHA) by sulfhydryl compounds occurs in the absence of enzyme. Pfankuch (19) reported on the presence, in potato extracts, of a heat-labile component that catalyzed this reaction. Hopkins and his collaborators (20, 21, 22) studied an enzyme, present in various plant juices, which markedly accelerated the reaction between GSH and DHA. Mapson and Goddard (17) coupled the reaction with GSSG reductase. Thus a complete hydrogen-transfer system from the substrate to oxygen, catalyzed by enzymes that occur in some plants, can be written as follows ... 

Ascorbic acid and dehydroascorbic acid have been determined by reversed-phase h.p.l.c., post-column reduction of dehydroascorbic acid to ascorbic acid with dithiothreitol, reaction of excess reagent with JV-ethylmaleimide, and electrochemical detection. Ascorbic acid and its 2-phosphate were determined by h.p.l.c. on an aminopropyl bonded-phase silica column. Dehydroascorbic acid could also be determined by the increase in the ascorbic acid content after reduction with dithiothreitol The method was applied to raw apple and potato to which these compounds are added to prevent browning. ... 

The reversible oxidation of ascorbic acid is the basis of its physiological activity. The reduction of dehydroascorbic acid, stable at pH 2.5-5.5 at 4°C for days, can be easily achieved by cysteine. ... 

L 0dum. pH optimum of the reduction of dehydroascorbic acid by dithioeryiriol. Scand J Lab Invest 53 367—371, 1993. 

The rate of reduction of dehydroascorbic acid to ascorbic acid by H2S is related to the pH of the solution. Roe et cd. (23) obtained complete reduction at pH ranges from 4.7 to 1.2. Below pH 1.2 the completeness of the reduction was sharply diminished. When the H2S was introduced as fine bubbles by being passed through a sintered glass tube, complete reduction of dehydroascorbic acid was obtained in 5 minutes at pH 3.5, and in 15 minutes at pH 2.0. Bessey (1) i owed that no significant loss of ascorbic acid in H S solution occurred at pH 3.5 and 25°C. in 4 hours. It is not desirable to treat for too long with HjS, however, as losses in ascorbic acid occur upon standing in H2S solution. 

Sawamura, M., Ooishi, S., and Li, Z., Reduction of dehydroascorbic acid by sodium hydro-sulfide and liquid chromatographic determination of vitamin C in citrus juices, J. Sci. FoodAgric., 53, 279-281, 1990. 

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