Did the loss of endogenous ascorbate accelerate the evolution of Anthropoidea and Homo sapiens?
J. Challem
The Nutrition Reporter, PO Box 5505, Aloha, Oregon 97006
Harman theorized that free-radical reactions led to the first life on Earth approximately 3.5 billion years ago, and their ability to randomly cause genetic mutations may have promoted the evolution of life. Because of the potentially destructive effects of radicals, nearly all animals evolved to manufacture large amounts endogenous ascorbate (vitamin C), a powerful scavenger of hydroxyl radicals.
Approximately 45 million years ago, a mutation occurred in an evolutionary ancestor of Anthropoidea, which now includes Homo sapiens. This mutation damaged the gene that codes for L-gulono lactone oxidase, the terminal step in endogenous ascorbate production. All surviving descendants inherited this genetic defect, making them dependent on dietary ascorbate. However, the amount of ascorbate obtainable through the diet, even among primates living in the jungle, falls short of the amount typically produced endogenously per kg of body weight. Stone theorized that while H. sapiens lost the ability to manufacture endogenous ascorbate, the species did not lose the requirement for large amounts of ascorbate.
What were the evolutionary consequences of losing the ability to manufacture endogenous ascorbate? It is theorized that large numbers of radicals would have remained unquenched through normal metabolic processes. Furthermore, other endogenous antioxidants, such as superoxide dismutase and urate, are not interchangeable with ascorbate, so they would not fully compensate for the loss of ascorbate. The large number of radicals resulting from the loss of endogenous ascorbate would have increased oxidative stress and the frequency of mutations. While some of these mutations would have been somatic, others would have been inheritable, creating a rich genetic palette that other evolutionary forces could select for or against, leading eventually to H. sapiens.
The role of radicals in fostering the evolution of Anthropoidea and H. sapiens is supported by their role in creating mutations in general. For example, radical-induced mutations cause many cancers and contribute to the aging process. Conversely, studies have shown that ascorbate and other antioxidants can prevent DNA mutations.
Current dietary recommendations for ascorbate (60 mg/day) are substantially lower than the amount generally manufactured by mammals (~184.2 mg/kg/day). Furthermore, the oxidative stress associated with many diseases may increase ascorbate requirements. (Among ascorbate producers, ascorbate production is up regulated during stress.) Under conditions of chronically high oxidative stress and low ascorbate intake, H. sapiens may be continuing to evolve at a faster rate relative to species producing endogenous ascorbate. If this theory is correct, large amounts of dietary or supplemental ascorbate would reduce the number of mutations, decrease the risk of cancer, and slow the evolution of H. sapiens.
Iron overload and vitamin C: Pro- or anti-oxidant?
Balz Frei
Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR
Vitamin C (ascorbate) can act as an antioxidant or a pro-oxidant in vitro, depending on the absence or presence, respectively, of redox active transition metal ions. We performed a number of studies to investigate the (patho)physiological significance of the proposed pro oxidant activity of ascorbate in the presence of iron-overload, using F2-isoprostanes as an in vivo marker of lipid oxidative damage. We found that F2-isoprostane levels were increased in the plasma of premature infants compared to adult controls, but not different between those infants with non-protein bound iron in their plasma (138 ± 51 pg/ml, n = 19) and those without (126 ± 41 pg/ml, n = 10). When excess iron was added to adult plasma, non-protein bound iron became detectable and endogenous ascorbate was rapidly oxidized. However, there was no detectable lipid peroxidation as long as ascorbate was present at > 10% of its initial concentration. Furthermore, when iron was added to plasma devoid of ascorbate, lipid hydro peroxides were formed immediately, whereas endogenous and exogenous ascorbate delayed the onset of iron-induced lipid peroxidation in a dose-dependent manner. To confirm and extend these observations, we performed an in vivo study using guinea pigs fed high or low doses of vitamin C (50 or 0.5 mg/d for 4 wks) with or without prior iron-loading (1.5 g Fe-dextran/kg body weight, i.p., or dextran only). We found that those guinea pig cohorts fed high levels of vitamin C had significantly lower hepatic F2-isoprostane levels (control: 5.9 ± 2.2 and iron loaded: 4.9 ± 2.6 ng/g tissue) than those fed low levels of vitamin C (control: 7.6 ± 2.8 and iron loaded: 9.4 ± 2.6 ng/g), p < 0.05. Similar observations were made with respect to plasma levels of F2-isoprostanes. Our data indicate that vitamin C protects against oxidative damage under physiologic conditions, independent of iron status, and that iron loading per se does not cause lipid oxidative damage in vivo. There exists no evidence for a pro-oxidant effect of ascorbate in the presence iron-overload in vivo or in physiological matrices such as plasma.
Antioxidant interactions between vitamin E and coenzyme Q: Role of superoxide
Valerian E. Kagan
Departments of Environmental and Occupational Health and Pharmacology, University of Pittsburgh, Pittsburgh, PA 15238, USA
Vitamins E and coenzyme Q are the two major free radical scavengers in membrane lipid bilayers where they can act independently or in concert to provide for antioxidant protection. We have previously characterized mitochondrial and microsomal electron-transport dependent vitamin E recycling in which coenzyme Q played a prominent role.
Our recent studies using purified human recombinant NADPH cytochrome P450 oxidoreductase (OR) as well as purified plasma membrane coenzyme Q reductase (PMCQR) demonstrated that coenzyme Q mediates recycling of vitamin E in a superoxide-driven reaction. We found that OR reduced phenoxyl radicals of vitamin E and its homologues (e.g., radicals of 2,2,5,7,8-pentamethyl-6-hydroxy chromane) in a NADPH-dependent reaction both directly and via co enzyme Q/superoxide-driven mechanisms. The superoxide-driven recyling of vitamin E by OR/NADPH was almost completely inhibited by SOD.
PMCQR catalyzed NADH/coenzyme Q0-dependent reduction of phenoxyl radicals generated by lipoxygenase/linoleic acid from Trolox, a water-soluble homologue of vitamin E. Characteristic ESR spectra of Trolox phenoxyl radicals were not observable in the presence of NADH/coenzyme Q and PMCQR. Typical spectra of coenzyme Q0 semiquinone radicals were detectable instead. Trolox radical signals reappeared in the spectra after complete consumption of NADH. The reduction of phenoxyl radicals occurred through their interactions with reduced coenzyme Q0 (or its semiquinone radical). Both superoxide-driven reduction and direct PMCQR-mediated reduction of coenzyme Q0 was involved in the recycling of Trolox from its phenoxyl radicals as evidenced by a significant (more than 50%) inhibitory effect of Cu,Zn-SOD.
We conclude that OR and PMCQR-catalyzed reduction of vitamin E phenoxyl radicals by a superoxide-driven reduction of coenzyme Q represents an important free radical reductase activity that may significantly contribute to antioxidant protection of cells against oxidative stress.
Norman I. Krinsky
Department of Biochemistry, School of Medicine and the Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111, USA
For many years, the most promising action of carotenoids was assumed to be their relationship to a decreased risk of chronic degenerative diseases, particularly some cancers. This action was based on extensive epidemiological data indicating that diets rich in orange and yellow fruits and vegetables, the source of carotenoids in our diet, reduced the risk of cancers such as lung, cervical and esophageal. However, the results of several large intervention trials have cast doubt on the direct effectiveness of supplemental b-carotene. It may be time to consider other actions of carotenoids that may be related to the protection seen with fruit and vegetable consumption.
The biological actions of carotenoids can be classified as primary, due to the intact molecule, or secondary, due to metabolites or breakdown products. The primary actions of carotenoids play major roles in nature. Among these are those associated with light absorption and subsequent transfer of energy in photosynthetic antennae systems, light absorption leading to an attenuation of irradiation, which may play a role in the primate macula, cis-trans isomerization, which plays a role in photosynthetic reaction centers, and antioxidation, due either to quenching of singlet oxygen or radical reactions. Recent studies have suggested that dietary carotenoids show significant differences in their ability to react with radicals or transfer electrons.
The secondary actions due to metabolites and breakdown products are expanding as we learn more about the biological activity of these breakdown products.
Supported by NIH R01CA66914
Catherine Rice-Evans
International Antioxidant Research Centre, UMDS-Guyís Hospital, London SE1 9RT, United Kingdom
This presentation will address, firstly, the screening of flavonoids and hydroxycinnamates for potential use as antioxidants in counteracting damage mediated by reactive oxygen and nitrogen species and, secondly, the interpretation of the structural considerations which underline their efficacy as antioxidants.
The bioactivity of an antioxidant can be assessed by determining:
€ the direct free radical scavenging activity as a hydrogen- or electron-donor, dependent on the reduction potentials of the specific groups and the structural chemistry of the molecule;
€ the efficacy as a chain-breaking antioxidant in scavenging lipid peroxyl radicals, dependent on the partition coefficient and the reduction potential;
€ the ability to chelate transition metal ions, and prevent free radical formation, as compared with the ability to reduce iron or copper and promote free radical formation, dependent on specific structural characteristics of the molecule;
€ the scavenging of reactive nitrogen species and whether nitration or oxidation is the fate of the antioxidant;
€ the interaction with other antioxidants, depending on the position of the redox couple in the hierarchy of reduction potentials.
Examples will be drawn from the major flavonoid constituents of wines and teas, the catechin/gallates, vegetables, the flavonols and their glycosides and the major phenolic constituents of fruit, chlorogenic, p-coumanic and ferulic acids.
Pycnogenol®: Effects on the redox antioxidant network and nitrogen monoxide (NO) metabolism
Lester Packer, Fabio Virgili, Hirotsugu Kobuchi, and Elaine Cossins
Department of Molecular and Cell Biology, University of California at Berkeley
Pycnogenol® is a distinctive complex of more than 40 bioflavo noids and organic acids extracted from the bark of the French maritime pine tree. Flavonoid containing phytochemicals, owing to the number and localization of phenolic hydroxyl groups attached to ring structures, display strong antioxidant activity and have been demonstrated to have a beneficial effect on the circulation. Among various plant extracts we tested for antioxidant capacity, Pycnogenol® was found the most efficient in vitro scavenger of the reactive oxygen species HO. and O2.. Pycnogenol® may act in the redox antioxidant network at the interface between ascorbate and lipophilic antioxidants such as tocopherols and tocotrienols. Using a model system of ascorbate/ascorbate oxidase coupled with ESR detection, Pycnogenol® was found to be remarkably effective in extending ascorbyl radical lifetime, indicating that it can play an important role in the cellular antioxidant network. The participation of Pycnogenol® in the antioxidant network and its capacity to increase overall antioxidant cell capacity is corroborated by its ability both to regenerate tocopheryl radical (vitamin E radical) during LDL oxidation and to spare a-tocopherol in cultured cells under oxidative stress conditions.
Another important property of flavonoid-containing mixtures is the modulation of NO (nitric oxide) metabolism in inflammation. We found that Pycnogenol® modulates the large and sustained production of NO of cultured rat macrophages activated by bacterial lipopolysaccharide (LPS) and Interferon-g (IFN-g), showing a stimulatory effect at very low concentrations, and then inhibiting cell NO production at higher concentrations. This effect was found to be due to the combination of different biological activities: i) a direct antioxidant scavenging activity of NO, ii) modulation of the activity of the inducible form of nitric oxide synthase (iNOS) and iii) inhibition of expression of iNOS-mRNA expression. These findings help to explain the biological activity of Pycnogenol® as a therapeutic agent in various human disorders associated either with oxidative stress or dysfunction of NO production.
EGb 761: Antioxidant properties and cell regulation
Sashwati Roy, Hirotsugu Kobuchi, Chandan K. Sen, Kishorchandra Gohil, Savita Khanna and Lester Packer
Membrane Bioenergetics Group, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200
EGb 761, a standardized extract prepared from the leaves of Ginko biloba , has proven to be beneficial in pathologies related to cerebral and circulatory disorders. Such effects of EGb 761 extract have been substantiated with placebo-controlled double- blind clinical trials. Mechanisms underlying the beneficial effects of EGb 761 is only partially understood. One of the main mechanism seems related to the antioxidant properties of EGb 761 which appear to be due to the synergistic action of its major constituents such as flavonoids, terpenoids and the organic acids. EGb 761 directly scavenges hydroxyl, superoxide, peroxyl and nitric oxide radicals in vitro. EGb 761 also protects against free radical mediated damage in biological model systems, including ischemia-reperfusion injury of organs and oxidative modification of low density lipoprotein. To elucidate the molecular mechanisms underlying the protective effects of EGb 761 in vascular and cerebral we investigated various aspects of cell regulation. Nitric oxide metabolism and cell adhesion processes play important role in vascular and cerebral disorders. EGb 761 inhibited nitric oxide production in macrophages by inhibiting nitric oxide synthase (NOS) activity and regulating inducible-NOS gene expression. Agonist-in duced cell adhesion molecule expression in human endothelial cells and lymphocyte-endothelial cell adherence was down-regulated by pretreatment of cells with EGb761. EGb 761 was also protective against etoposide-induced apoptosis in rat thymocytes. Exposure of human endothelial cells to EGb 761 resulted in modulation of several mRNA transcripts identified by differential display of mRNA. Identification and characterization of such EGb 761 sensitive genes are currently in process. The regulatory effects of EGb 761 on nitric oxide metabolism, cell adhesion processes, apoptosis and gene expression may be implicated in the beneficial effect of the extract in brain and circulatory disorders.
José Viña
Department of Physiology, School of Medicine, University of Valencia, Valencia, Spain
The role of mitochondria in the generation of oxidative stress associated with aging was postulated some twenty years ago. Several studies had shown, using isolated mitochondria, that mitochondrial function is impaired in aging. We will report experiments showing for the first time, the impairment of mitochondrial function within intact cells of old animals. We have used, both a metabolic approach -the study of specific metabolic pathways that involve both cytosol and mitochondria- and a flow cytometric approach. Mitochondria from old animals have a lower membrane potential and produce more pero xides than those from young ones. Specific transport systems such as the one for malate are impaired with aging. Other important mitochondrial functions like oxidative phosphorilation are also affected. These changes may be due to oxidation of key molecules such as mitochondrial DNA, which is affected in aging.
Mitochondria are also affected in apoptotic cells. Some common features of apoptotic cells and of cells from old animals include an increased mitochondrial peroxide production, oxidation of glutathione and oxidation of mitochondrial DNA. However, a relationship between aging and apoptosis has not been established.
Antioxidants protect against oxidative stress associated with aging. However not all antioxidants are efficient under all circumstances. Indeed some antioxidants may have a prooxidant effect "in vivo". Thus, although antioxidants have a beneficial effect in the protection against aging, care should be taken to test each specific antioxidant in its potential protective effect against age-associated impairment in molecular, metabolic, and physiological functions.