A method of increasing glutathione levels in mammalian cells comprising administering an oral bolus of encapsulated pharmaceutically stabilized glutathione in a rapidly dissolving formulation to a mammal on an empty stomach. Pharmaceutical formulations including glutathione are also disclosed.

Pharmaceutical preparations of glutathione and methods of administration thereof

Superoxide (O2-) is the compound obtained when oxygen is reduced by one electron. For a molecule with an unpaired electron, O2- is surprisingly inert, its chief reaction being a dismutation in which it reacts with itself to form H2O2 and oxygen. The involvement of O2- in biological systems was first revealed by the discovery in 1969 of superoxide dismutase, an enzyme that catalyzes the dismutation of O2-. Since then it has been found that biological systems produce a bewildering variety of reactive oxidants, all but a few arising ultimately from O2-. These oxidants include O2- itself, H2O2 and alkyl peroxides, hydroxyl radical and other reactive oxidizing radicals, oxidized halogens and halamines, singlet oxygen, and peroxynitrite. These various oxidants are able to damage molecules in their environment, and are therefore very dangerous. They are thought to participate in the pathogenesis of a number of common diseases, including among others malignancy, by their ability to mutate the genome, and atherosclerosis, by their capacity for oxidizing lipoproteins. Their properties are put to good use, however, in host defense, where they serve as microbicidal and parasiticidal agents, and in biological signalling, where their liberation in small quantities results in redox-mediated changes in the functions of enzymes and other proteins.

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Superoxide: a two-edged sword

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) activity and content in the rat ovary were measured at various stages of the estrous cycle, and the enzyme protein in the ovary was localized by immunohistochemistry. The cyclic change of ovarian 3 alpha-HSD activity towards 5 alpha-dihydrotestosterone (5 alpha-DHT) as a substrate was characterized by two peaks. The first peak occurred at 0800 h on proestrus; then the reductase activity decreased and reached its minimum at 2000 h on proestrus. Thereafter, it gradually increased, reaching the second peak (170% of the value at 2000 h on proestrus) at noon of estrus. Quantitative analysis by immunoblotting revealed that the alteration in 3 alpha-HSD content in the rat ovary during the estrous cycle was essentially similar to that in 5 alpha-DHT reductase activity. Changes in the reductase activities towards 5 alpha-androstane-3,17-dione and 5 alpha-DHT and in the dehydrogenase activity towards androsterone in the ovary were entirely different from those in the 5 alpha-DHT reductase activity and 3 alpha-HSD content; on the other hand, the change in carbonyl reductase activity towards p-nitroacetophenone was similar to changes in 5 alpha-DHT reductase activity and 3 alpha-HSD content. Therefore, p-nitroacetophenone may be a useful substrate, instead of 5 alpha-DHT, for detection of 3 alpha-HSD activity at a high sensitivity, since the p-nitroacetophenone reductase activity was 10-fold higher than the 5 alpha-DHT reductase activity. The enzyme was primarily localized in the granulosa cells and CL cells. At 2000 h on proestrus, however, the overall intensity of immunostaining in the granulosa cells of the Graafian follicles was markedly diminished. In addition, immunoreactivity in the CL cells at 0800 h on estrus was observed only in the cells outlining the CL in some cases.

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Cyclic change in 3 alpha-hydroxysteroid dehydrogenase in rat ovary during the estrous cycle.

Characterization of multiple acid phosphatases in bovine liver cytosol and lysosome. Inactivation of cytosolic enzymes by disulfides and its redox regulation by thioltransferase

Characterization of bovine liver cytosolic 3α-hydroxysteroid dehydrogenase and its aldo-keto reductase activity

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50), purified to homogeneity from rat liver, was strongly inactivated by incubation with a disulfide such as GSSG, L-cystine or L-cystamine, as well as an SH-reagent such as DTNB (5,5'-dithiobis(2-nitrobenzoic acid)), NEM (N-ethylmaleimide) or iodoacetic acid. The inactivation advanced with incubation time. Coenzyme (NADP+) completely protected the enzyme from this inactivation by disulfides, but neither of the substrates (androsterone and benzenedihydrodiol) did. The activity of inactivated enzyme was restored by treatment with thiols such as DTT (dithiothreitol) or GSH. In the GSH/GSSG redox buffer, the enzyme existed in an equilibrium between active (reduced) and inactive (oxidized) forms.

Modulation of 3 alpha-hydroxysteroid dehydrogenase activity by the redox state of glutathione.

Rat liver cytosolic 3α-hydroxysteroid dehydrogenase (EC 11150) (dihydrodiol dehydrogenase) which can catalyze the conversion between androsterone and androstanedione in the presence of NADP(H) has also been shown to catalyze the oxidation of benzenedihydrodiol to catechol (Penning et al, 1984, 1985; Hara et al, 1988) From the facts that can oxidize the trans-dihydrodiol of polycyclic aromatic hydrocarbons such as benzo(a)pyrene and benzo(a)anthracene, it has been suggested that dihydrodiol dehydrogenase plays an important role in the detoxification of the polycyclic aromatic hydrocarbon through the effective suppression in the formation of the ultimate carcinogenic anti-diol epoxides (Penning et al, 1984, 1985)

Kazuhiko Shinagawa

Papers

Modulation of 3 alpha-hydroxysteroid dehydrogenase activity by the redox state of glutathione.

Study on dihydrodiol dehydrogenase (II) modulation of dihydrodiol dehydrogenase activity by biological disulfides.