Vitamin dehydroascorbate

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Absorption, Transport, and Excretion. The vitamin is absorbed through the mouth, the stomach, and predominantly through the distal portion of the small intestine, and hence, penetrates into the bloodstream. Ascorbic acid is widely distributed to the cells of the body and is mainly present in the white blood cells (leukocytes). The ascorbic acid concentration in these cells is about 150 times its concentration in the plasma (150,151). Dehydroascorbic acid is the main form in the red blood cells (erythrocytes). White blood cells are involved in the destmction of bacteria. 

Vitamin C (Figure 45-19) is a vitamin for human beings and other primates, the guinea pig, bats, passerine birds, and most fishes and invertebrates other animals synthesize it as an intermediate in the uronic acid pathway of glucose metabohsm (Chapter 20). In those species for which it is a vitamin, there is a block in that pathway due to absence of gulonolactone oxidase. Both ascorbic acid and dehydroascorbic acid have vitamin activity. 

In a recent study, serum ascorbate concentrations were significantly reduced in a group of elderly diabetic patients (w = 40, mean age 69 years) in comparison with an age-matched group of non-diabetic controls ( = 22, mean age 71 years), and this reduction was more pronounced in those patients with microangiopathy (Sinclair et al., 1991). Diabetic patients were shown to have a high serum dehydroascorbate/ascorbate ratio indicative of increased oxidative stress. Ascorbate deficiency was partially corrected by vitamin C supplementation, 1 g daily by mouth, but the obvious disturbance in ascorbate metabolism in the diabetic patients was accentuated, since serum ascorbate concentrations fell (after the initial rise) despite continued vitamin C supplementation (Fig. 12.3). 

Under stress conditions, such as cutting or light exposure, ascorbate oxidase has been described as promoting the transformation of ascorbic acid to dehydroascorbic acid (Wright and Kader 1997b). However, because ascorbic acid can be easily converted into dehydroascorbic acid, it is necessary to measure both ascorbic and dehydroascorbic acids to observe that the content of vitamin C was well preserved in fresh-cut fruit. 

GSH synthesis. GLUT1 transports dehydroascorbic acid, an oxidized form of vitamin C, to supply the retina with ascorbic acid [47],... 

Ascorbic acid or vitamin C is found in fruits, especially citrus fruits, and in fresh vegetables. Man is one of the few mammals unable to manufacture vitamin C in the liver. It is essential for the formation of collagen as it is a cofactor for the conversion of proline and lysine residues to hydroxyproline and hydroxylysine. It is also a cofactor for carnitine synthesis, for the conversion of folic acid to folinic acid and for the hydroxylation of dopamine to form norepinephrine. Being a lactone with two hydroxyl groups which can be oxidized to two keto groups forming dehydroascorbic acid, ascorbic acid is also an anti-oxidant. By reducing ferric iron to the ferrous state in the stomach, ascorbic acid promotes iron absorption. 

Because potatoes are good source of vitamin C, it is important to point out that irradiation does not adversely affect the vitamin C levels [23,30]. Although some ascorbic acid is converted into dehydroascorbic acid on irradiation, the latter is also biologically active. 

Since ascorbate reduces photooxidation of lipid emulsions and multivitamin preparations (see Figure 4) [19], Lavoie et al. [34] studied the formation of oxidative by-products of vitamin C in multivitamins exposed to light. They found that the loss of ascorbic acid in photoexposed multivitamin preparations was associated with the generation of products other than dehydroascorbate and 2,3-diketogulonic acid, which are the usual products of vitamin C oxidation. The authors showed that hydrogen peroxide at concentrations found in TPN solutions induced the transformation of dehydroascorbate into new, biologically active compounds that had the potential to affect lipid metabolism. They believe that these species have peroxide and aldehyde functions [35]. 

L-dehydroascorbic acid, are active forms of the vitamin. However, once the dehydro form is further oxidized to diketogiuconic acid, no vitamin C activity is retained. The thermal half life of dehydroascorbic acid is less than 1 minute at lOO C (pH 6), and 2 minutes at 7O C Vitamin C is also... 

However, its presence is not the only determinant of whether or not oxidative deterioration occurs. Olson and Brown (1942) showed that washed cream (free of ascorbic acid) from susceptible milk did not develop an oxidized flavor when contaminated with copper and stored for three days. Subsequently, the addition of ascorbic acid to washed cream, even in the absence of added copper, was observed to promote the development of an oxidized flavor (Pont 1952). Krukovsky and Guthrie (1945) and Krukovsky (1961) reported that 0.1 ppm added copper did not promote oxidative flavors in milk or butter depleted of their Vitamin C content by quick and complete oxidation of ascorbic acid to dehydroascorbic acid. Krukovsky (1955) and Krukovsky and Guthrie (1945) further showed that the oxidative reaction in ascorbic acid-free milk could be initiated by the addition of ascorbic acid to such milk. Accordingly, these workers and others have concluded that ascorbic acid is an essential link in a chain of reactions resulting in the development of an oxidized flavor in fluid milk. 

Dehydroascorbate, the oxidized form of vitamin C (Box 18-D) is also transported into cells by GLUT1 and GLUT3.409 A related transporter carries L-fucose into mammalian cells 410 Another facilitates the uptake of galactose in yeast.411... 

The biological functions of vitamin C appear to be related principally to its well-established reducing properties and easy one-electron oxidation to a free radical or two-electron reduction to dehydroascorbic acid. The latter is in equilibrium with the hydrated hemiacetal shown at the beginning of this box as well as with other chemical species.1 Vitamin C is a weak acid which also has metal complexing properties. 

Both forms are biologically active. Ascorbic acid (the reduced form) is relatively stable to heat however, dehydroascorbic acid (the oxidized form) is unstable. The lactone ring is easily hydrolyzed to diketogulonic acid, which has no antiscurvy activity. When fruits, vegetables, and other foods are heated, there is some loss of active vitamin C by conversion to diketogulonic acid. 

Ascorbic acid and dehydroascorbic acid have equivalent biological activities, whereas isoascor-bic acid has only 5% of the biological activity of AA (15,17,19). The absorption and metabolism of vitamin C were recently reviewed (17,20,21). 

This is one vitamin that most laboratories can measure. There are a number of old-fashioned approaches that use 2,6-dichloroindophenol in a titrimetric method such as AOAC 985.33. This works well in some systems but can give rise to false positive results if there are other reducing substances present. It will not detect dehydroascorbic acid (DHA) and so it may well underestimate the actual vitamin C activity if a product contains a significant level of DHA. However, even with for these shortcomings, it is often used as a quick and rough method. In the AOAC there is also a fluorometric method (AOAC 984.26) where ascorbic acid is oxidised to DHA and this is reacted with o-phenylenediamine to give a fluorometric compound which can be detected. This is a robust method that has general applicability. 

Compounds that can scavenge radicals are also referred to as antioxidants. The best known anti-oxidants are vitamin C and vitamin E. Vitamin C is L-ascorbate (7.8), a good reducing agent that prevents oxidation of other molecules. The oxidized form of L-ascorbate is L-dehydroascorbic acid (7.9). Vitamin E is a mixture of a-, [3-, y-, and 8-tocopherol (7.10a-d). Of these four compounds, a-tocopherol is the most effective. Vitamin E is lipid-soluble and has the ability to disrupt the chain reaction during lipid peroxidation (see Chapter 2, Section 1.9). 

The difference is two hydrogens. L-ascorbic acid is the reduced form of this vitamin L-dehydroascorbic acid is its oxidized form. The L indicates that each has the L configuration at carbon 5. 

The ease of oxidation of reduced ascorbic acid is the basis for a simple method of analysis by dye titration (58j. Ascorbic acid as it occurs in citrus juice is in the reduced form. When subjected to oxidation, ascorbic acid changes to the dehydro form. Dehydroascorbic acid has nearly the same physiological activity as the reduced form and is easily converted to the latter. Further oxidation of the dehydroascorbic acid converts it to 2,3-diketo-gulonic acid. This reaction is irreversible, and the oxidized product is devoid of biological activity. These reactions are shown in Figure 2. Nearly 90 percent or more of the vitamin C found in citrus juice and citrus products is in the reduced form (Table X) (59). 

Vitamin C (ascorbate) (Fig. 9.5) has the ability to act as a reducing agent, i.e. it will tend to reduce more reactive species. This ability to reduce Fe3+ to Fe2+may be important in promoting iron uptake in the gut. Oxidation of ascorbate by reaction with reactive oxygen species or reactive nitrogen species seems to lead to its depletion. In vitro, vitamin C can also exert pro-oxidant properties. Fe3+ can react with ascorbate to form Fe2+ and the semi-dehydroascorbate or ascorbyl radical. The latter can react with hydrogen peroxide to form Fe3+, the hydroxyl radical and a hydroxide anion. A key question with regard to the pro- or anti- oxidant effects of ascorbate may therefore be the availability of transition metal ions. Neurons main-... 

Analysis of vitamin C has been the topic of numerous papers. Most of the methods analyze L-ascorbic acid by HPLC, before and after reduction of the dehydroascorbic acid present.338 The concentration of the dehydroascorbic acid is calculated by subtraction. A later work describes a method, which combines iodometry with a voltammetric technique to detect the endpoint of the titration.339 The results are comparable to those obtained by HPLC and can be applied to vegetable and fruit samples. 

Dehydroascorbic Acid and Ascorbic Acid. The oxidized and reduced forms of vitamin C (dehydroascorbic acid and ascorbic acid, respectively) have redox characteristics that are similar to those for o-quinone/catechol systems.13 Al-... 

As aforementioned, derivatives of Vitamin C and their analogs can function as hyaluronidase inhibitors. In particular L-ascorbic acid-6-hexadecanoate is a potent inhibitor (A. Botski, et al., pers. commun.). Vitamin C itself, D-isoascorbic acid, and dehydroascorbic acid are also inhibitors 262 Thus, some of the ability of Vitamin C to enhance HA deposition may be attributed to its inhibiton of hyaluronidase. 

FIGURE 29.1 Pathways of the chain-breaking action of vitamin E in lipid peroxidation and its subsequent regeneration. LOOH lipid hydroperoxide, LOO lipid peroxyl radical, vitamin C ascorbate radical (semi-dehydroascorbate), vitamin E a-tocopheroxyl radical. The lipid peroxyl radical is reduced to lipid hydroperoxide by tocopherol. The resulting tocopheroxyl radical can be re-reduced by ascorbate. The thus formed ascorbate radical can be reduced to ascorbate by the NADH-dependent semidehydroascorbate reductase. 

One of the main problems of topical application of vitamin C is that it is extremly unstable, so hydrophilic derivatives like sodium ascorbyl phosphate and lipophilic esters with fatty acids were synthesized to improve stability.43,44 However, an efficient increase in vitamin C levels after topical application of different ascorbic acid derivatives including magnesium ascorbyl phosphate, ascorbyl-6-palmitate, and dehydroascorbic acid to porcine skin could not be shown.42... 

For example, when 02 is formed in the hydrophilic stage, vitamin C (18, L-ascorbic acid present in hydrophilic stage) assists the hydrogen atoms to form dehydroascorbic acid (19) via monodehydroascorbic acid, and hydrogen peroxide (eq. 1.9). 

Dehydroascorbic acid (but see later) and quinones, such as vitamin K and those formed enzymically from polyphenols, can also act as dicarbonyl compound in the reaction. 

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