Vitamin C: Electron Emission, Free Radicals and Biological Versatility

Emission of e_aq⁻ and Ascorbic Free Radicals

Vitamin C belongs to the group of antioxidant vitamins, the action of which is based on the emission of e_aq⁻ in aqueous media (3, 10). In organisms, this process can, for instance, be enzymatically induced. It has been simulated by irradiation with monochromatic UV-light (λ=254 nm, E=4.85 eV/hν) in airfree, aqueous media (pH~7), in which chlorethanol was used as scavenger for the ejected e_aq⁻, where the quantum yield (Q) of Cl⁻ equals that of e_aq⁻.

(Eq1)

(Eq2)

The obtained yield of e_aq⁻ as a function of ascorbate concentration determined under these conditions, is shown in Figure 1. The e_aq⁻ represents the basic form of the H atom.

A deeper insight into the reaction mechanisms of free radicals of vitamin C has been obtained by pulse radiolysis (12, 13) and by laser flash photolysis methods (14).

Exhaustive pulse radiolysis studies of vitamin C in aqueous solution delivered a better understanding of the involved free radical's reaction mechanisms (15-17). In aqueous solution, OH-attack on ascorbate (AH⁻) results in the formation of an ascorbate radical (AH^•): (Eq3) Details of characteristic spectroscopic and kinetic data of AH• transients are given in Table I.

At the absorption maxima obtained, different reaction rate constants for formation and decay of AH^• radicals indicate the formation of different ascorbate radicals. This means that OH-attack takes place on different positions of the ascorbate molecule. Hence, the measured absorption spectrum of AH^• radical is superimposed.

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Figure 1.

Yield of ejected e_aq⁻ (mol l⁻¹) as a function of ascorbate concentration (mol l⁻¹) in airfree aqueous solution (pH~7) obtained by absorption of monochromatic UV-light (λ=254 nm, E=4.85 eV hν⁻¹). UV-intensity: I=1×10²⁰ hν l⁻¹

In order to have the ascorbate radical (AH^•) exclusively generated by e_aq⁻ emission, azide radicals (N₃^•) produced in N₂O-saturated solution were used as a specific reactant. The following reactions take place (3, 10, 18).

(Eq4)

(Eq5)

(Eq6)

(Eq7)

The AH^• radical obtained by this method showed only two absorptions maxima at 300 nm (ε=2360 l mol⁻¹ s⁻¹) and at λ=360 nm (ε=3670 l mol⁻¹ s⁻¹), with different ε values as compared to those given in Table I.

The transient spectrum obtained by this method is assumed to represent the AH^• radical generated by e_aq⁻ emission.

Scavenger for Oxidizing Species

The strong tendency of ascorbate to eject e_aq⁻ under formation of ascorbate free radicals classifies it as a very potent electron donor. By reaction of e_aq⁻ with O₂ in aerated solutions, peroxyl radicals are formed (19), which posses a powerful ability to kill bacteria and inactivate viruses. This property is valid to some extent also for the antioxidant vitamins E and β-carotene.

(Eq8)

(Eq9)

Ozone also reacts with e_aq⁻, generating strong oxidizing transients: (Eq10) (Eq11)

As a consequence of e_aq⁻ emission, vitamin C becomes a neutral free radical (AH^•), whereas vitamin E (α-tocopherol) and β-carotene (β-car) in such case are converted into the corresponding radical cations, namely into E^•+ and β-car^•+, respectively. All antioxidant vitamins react with reducing and oxidizing species, generating very active secondary transients. Some kinetic data for vitamin C are given in Table II for illustration.

The most reactive oxidizing species are the OH radicals. Their preferred reaction sites are double bonds of biological molecules, mostly at ortho- and para-positions, but to some extent also at meta- and ipso-positions of the molecules. The OH radicals can also split off a H atom or electron from substrate molecules. Vitamin C and related compounds are very efficient scavengers of the attacking species, as indicated by the data shown in Table II. This subject matter is discussed in more detail elsewhere (19).

Protection of Cell Membranes

Ascorbate plays an important role in the protection of cell membranes. Cell membranes (7-10 nm thickness) consist of 20-25% proteins and lipids as well as of 2-5% vitamins (C, E and β-carotene) and water. The structure of the membrane depends on its origin, and on the specific function of the cell. The cell membranes in organisms are constantly being attacked by oxidizing free radicals (OH, O₂^•−, ROO^• etc.), which are generated also in the human organism by enzymes. It was established by pulse radiolysis that membrane degradation is efficiently avoided by an electron cascade transfer within the above mentioned antioxidant vitamins (3). The following reactions proceed e.g.: (Eq12) (Eq13) (Eq14) (Eq7) These reactions are very fast, having rate constants of k>2×10⁹ l mol⁻¹ s⁻¹ (3).

The resulting main product, DHA is found to be an even better electron donor than to AH⁻ (see later).

DHA is not stable in aqueous solution and decomposes into about 50 products (20, 21).

Radiation Protection by Vitamin C

Using Escherichia coli (AB1157), as well as cultured cells (SCCVII), as a model for in vitro experiments, it was established that vitamin C acts as a radiation-protecting agent (22). The obtained ΔD37 (Gy)-values derived from survival curves, representing the N/N₀ ratio as a function of absorbed radiation dose (Gy), serves as a measure of the protecting effect, where, N₀=number of bacteria colonies before and N=number after the exposure to vitamin C. The D₃₇ (Gy) value represents the reaction dose (Gy) corresponding to N/N₀=0.37 and ΔD₃₇ (Gy)=D37 (sampe) – D37 (buffer). The radiation-protecting effect is predicated on the primarily acting radicals of water radiolysis, under the given conditions. The experimental results for E. coli are summarized in Table III.

Obviously, the highest radiation-protecting effect was observed in N₂O, in which OH radicals were predominant since AH⁻ acts as a very strong OH-scavenger (see Table I). Similar results were also obtained using SCCVII cells as an alternate in vitro model (22).

Synergistic Effect of Vitamin C on Mitomycin C

It has been experimentally found that vitamin C, as a potent e_aq⁻ donor, can significantly affect the activity of cytostatics. This effect is demonstrated on mitomycin C (MMC) and on sanazole.

MMC, based on its peculiar chemical structure (aziridine, carbamate and quinone cytotoxic groups on the same pyrrole nucleus, 1.2 α-indolic), belongs to the drugs having preferential toxicity towards hypoxic cells. MMC can be activated enzymatically (NADPH-cytochrome P-450 reductase) (23), chemically or by ionizing radiation (3, 24, 25) to yield MMC-quinone or hydroquinone. Both forms of MMC lead to the formation of a covalent cross-link adduct with DNA, which stops the proliferation of tumor cells. The same effect is observed by the action of MMC metabolites, generated by attacks of OH, O₂^•− or other oxidizing species (26).

It has been demonstrated by in vitro experiments (model: E coli AB1157) that under γ-irradiation of aerated, aqueous solution (pH~7.4), vitamin C acts as a powerful provider of e_aq⁻, forming also MMC^•− transients (Table IV). As expected, vitamin C used solely, demonstrates its ability to protect against oxidation (see Table IV: ΔD₃₇ (Gy)=85). However, in combination with MMC, it causes a very strong increase of MMC cytostatic properties by the formation of MMC-quinones.

Synergistic Effect of DHA on MMC

The survival curves (N/N0 ratio as a function of γ-radiation dose) observed in in vitro experiments (E coli AB1157) demonstrated a very strong effect on the cytostatic property of MMC in mixtures with DHA. Obviously DHA, as well as its numerous degradation products, are efficient e_aq⁻ donors, which leads to increased MMC^•− formation. Some typical results are given in Table V.

Vitamin C Synergism with Sanazole

Sanazole (AK-2123) acts as sensitizer for various cytostaticas, e.g. MMC (28). It possesses the property to enhance the chemical sensitivity of tumors and has a rather low toxicity.

The effect of vitamin C on sanazole efficiency has been investigated in the frame of in vitro experiments (E. coli AB1157) under irradiation with γ-ray in three test media: airfree, aerated and saturated with N₂O at pH~7.4 (29). Under these conditions, information was obtained about the specific action of the primary radicals generated by water radiolysis. The obtained results, expressed ΔD₃₇ (Gy) values which were derived from the corresponding survival curves and are presented in Table VI.

The ΔD₃₇ (Gy) values given in Table VI clearly demonstrate that independent of the nature of the free radicals, vitamin C very strongly affects the efficiency of sanazole. This is based on its capability to act as efficient radical scavenger (Table II) as well as a potent electron donor. These features are, so far, also valid for vitamin C degradation products including DHA, however, the reaction mechanism in this case is not yet completely understood.

Regeneration of Hormones

It has been experimentally shown that hormones and phytohormones can emit e_aq⁻ when excited into singlet state in aqueous or in water-alcohol solution (30, 31). The emitted e_aq⁻ retains the specific frequency of the corresponding mother molecule (32). By electron transfer from a given hormone, via the brain, to other hormones or to a biological system, communication is established (32, 33). The transients generated as a sequence of e_aq⁻ emission from a hormone under given conditions can induce the formation of cancerons cells, undergo degradation, or in the presence of a potent electron donor, such as vitamin C, they can be regenerated (33, 36).

The regeneration process of hormones by electron transfer from vitamin C to a hormone transient can be realized only within the life-time (status nascendi state) of the given transient (33-36). Since the hormones emit, as well as react with, e_aq⁻ (rate constants, k~10⁹-10¹⁰ l mol⁻¹ s⁻¹) (11) and can transfer electrons to other systems, they can also be classified as electron mediators in the organism (37). The regeneration process of hormones, for instance by vitamin C, and hormone degradation (e.g. caused by free radicals in the organism) are in permanent competition. Therefore, an adequate consumption of vitamin C is very recommendable.

The regeneration of hormone transients depends on several factors, including hormone molecular structure, concentration, rate constant of reaction with e_aq⁻ and with ascorbate (Table II) and pH of the medium (33-37). The regeneration process of some hormones by electron transfer from vitamin C are given as examples in Table VII.

It should be mentioned that the hormones at >10⁻⁷ mol l⁻¹ tend to form associates (36), similar to vitamin C. This fact favorizes electron transfer, even in the presence of air.