Ascorbic Acid: An Essential Micronutrient Provided by Plants
INTRODUCTION
In 1753 James Lind published his Treatise on the Scurvy, in which he described his research on the curative effects of citrus fruits on scurvy, the devastating disease that plagued sailors of the era. However, it was not until 1932 that L-ascorbic acid, the agent responsible for the prevention of scurvy, was purified and chemically synthesized. Today, ascorbic acid is a well-known antioxidant and enzyme cofactor with many roles in human health. Humans, unable to synthesize this micronutrient, depend on obtaining the majority via a diet that includes plants. Despite this, it was only recently that the plant ascorbic acid biosynthetic pathway was unraveled.
AFR AND DHA
Chemically, ascorbic acid can be oxidized to the relatively unreactive ascorbate free radical (AFR). The loss of a second electron produces dehydroascorbate (DHA). Ascorbate free radicals can also be disproportionate to form DHA and ascorbic acid. Both AFR and DHA can be reduced to ascorbic acid in mammalian cells by a number of systems, including glutaredoxin, thioredoxin reductase, AFR reductase, NADPH-dependent DHA reductase, and NADH: ascorbate radical oxidoreductase. Extracellular AFR can be reduced back to ascorbic acid with the use of intracellular ascorbic acid.
These chemical properties of ascorbic acid make it an ideal antioxidant. By donation of an electron, it can reduce (and therefore detoxify) highly reactive oxygen intermediates such as singlet oxygen, superoxide, and hydroxyl radicals, forming instead fairly non-reactive AFR or DHA, both of which can be enzymatically recycled back to the fully reduced ascorbic acid. In addition, ascorbic acid can reduce oxidized forms of a-tocopherol (vitamin E), maintaining this membrane antioxidant in its active state.
ASCORBIC ACID AND HUMAN HEALTH
Oxidative damage is thought to be one of the leading factors contributing to the degenerative processes that result in conditions such as aging cardiovascular disease and cancer. However, in the prevention of cardiovascular disease, the role of ascorbic acid is difficult to differentiate from the overall role of a healthy diet and lifestyle.[4] Ascorbic acid has been shown to decrease oxidative DNA damage. People with low ascorbic acid diets risk elevated DNA damage via oxidation. In contrast, supplementation of healthy subjects with additional ascorbic acid results in no change in the level of oxidized DNA. It is thought that the current US RDA for ascorbic acid may be the level at which the maximum benefit for protection against DNA damage is achieved.
However, those who either do not meet the U.S. RDA or have lifestyles that are known to decrease serum ascorbic acid levels (such as smoking) would most likely realize decreased damage to their DNA via supplementation. Protection of DNA against oxidative damage has led to the suggestion that ascorbic acid is involved in cancer prevention. However, the main role of ascorbic acid in the prevention of gastric cancer may be due to the vitamin’s inhibition of nitrosamine production rather than via detoxification of reactive oxygen species.
Ascorbic acid is also a cofactor of many dioxygenases and it is a deficiency in this activity that leads to scurvy. Ascorbic acid reduces prosthetic metal ions and also keeps other cofactors such as tetrahydrobiopterin in a reduced state. In addition to its well-known role in collagen biosynthesis, ascorbic acid also acts as a cofactor for enzymes involved in carnitine, progesterone, oxytoxin, catecholamine, and nitric oxide synthesis and has been shown to improve vasodilation by enhancing the synthesis of NO in endothelial cells.
The current U.S. RDA for ascorbic acid is 90 mg/day. In a 1994–95 USDA-sponsored survey, only 63.5% of Americans were meeting 100% of the U.S. RDA for ascorbic acid (two-day average). However, supplementation with single high daily doses of ascorbic acid may not be effective at increasing the intracellular pool of ascorbic acid as an expression of SVCT1 (the Na2+-dependent transporter that facilitates uptake of dietary ascorbic acid in epithelial cells) decreases substantially when cells are exposed to high levels of ascorbic acid.[8] This finding may help explain the lack of consensus regarding the role of single daily supplemental doses of ascorbic acid in the prevention of degenerative disease.
ASCORBIC ACID PATHWAYS
Despite the dependence of humans on plants for meeting ascorbic acid requirements, the plant ascorbic acid biosynthetic pathway was only recently determined. In 1998, Wheeler and Smirnoff presented evidence for a plant pathway via intermediates that included D-mannose and L-galactose.[9] Prior to this seminal paper, two different plant ascorbic acid pathways were proposed, one analogous to the animal pathway, and the other quite different, involving the osones glucose and sorbosone. Evidence for the pathway similar to animals rested primarily on the fact that plants harbor a mitochondrial-localized L-galactono-1,4-lactone dehydrogenase with similarity to a Lgulono-g-lactone oxidase in the animal pathway. This plant enzyme can convert exogenously supplied L-galactono-1,4-lactone to ascorbic acid.
The second pathway took into account evidence that (unlike the animal pathway) inversion of the carbon skeleton in the final product (ascorbic acid) relative to that in the primary substrate, D-glucose, does not occur in plants, however, there is little evidence for the proposed enzymatic activities necessary for the conversion of these osone intermediates into ascorbic acid (reviewed in Ref. 10). Wheeler and Smirnoff presented two key findings that reconcile the existence of the L-galactono-1,4-lactone dehydrogenase (similar to the animal pathway) and the non-inversion of the glucose carbon skeleton in plants. The first was the demonstration that L-galactono-1,4-lactone is produced in plants from L-galactose by an L-galactose dehydrogenase. Secondly, they found that plants synthesize L-galactose very efficiently from D-mannose, most likely via a GDP-D-mannose-3,5-epimerase. The ascorbic acid biosynthetic pathway was constructed from this data.
Early supportive evidence for this biosynthetic pathway came from analysis of the Arabidopsis ascorbic acid-deficient mutant, vtc1. The Vtc1 gene was found to encode a GDP-mannose pyrophosphorylase, the activity that catalyzes the generation of GDP-mannose substrate for the aforementioned epimerase. Potato lines expressing an antisense copy of this pyrophosphorylase gene have diminished ascorbic acid levels, independent confirmation of the role of this enzyme in ascorbic acid biosynthesis.
In addition to the GDP-mannose pyrophosphorylase gene (Vtc1), several additional genes involved in plant ascorbic acid biosynthesis have been identified. The L-galactono-1,4-lactone dehydrogenase gene has been cloned from several plant species including cauliflower, sweet potato, and tobacco (reviewed in Ref. 10). In addition, annotated sequences with high similarity to known L-galactono-1,4-lactone dehydrogenase sequences are found in the Arabidopsis thaliana genomic database. The peptide sequence of a purified GDP-mannose-3,5- epimerase from Arabidopsis led to the identification of the epimerase gene.[13] Using a similar strategy, the Smirnoff lab has cloned the L-galactose dehydrogenase gene from Arabidopsis. In addition to the ascorbic acid-deficient Arabidopsis mutant vtc1, three other VTC alleles have been identified by virtue of mutant alleles negatively affecting ascorbic acid synthesis.
To date, one of these (Vtc2) has been cloned although the role of the Vtc2 gene product in ascorbic acid biosynthesis is yet to be determined. In the future, it will be theoretically possible to engineer plants to produce elevated levels of ascorbic acid as the plant ascorbic acid biosynthetic pathway is better understood, and genes encode key rate-limiting enzymes in the pathway have been identified. In fact, transgenic tobacco has been described that overexpressing the rat L-gulono-1,4- lactone oxidase enzyme (which may have the same activity as plant L-galactono-1,4-lactone dehydrogenase) and accumulating somewhat elevated levels of ascorbic acid.
CONCLUSION
Although scurvy in the modern world is quite rare and occurs at <10 mg/day of ascorbic acid, it is clear that ascorbic acid has many roles beyond that of a cofactor in collagen biosynthesis, and obtaining at least the U.S. RDA for ascorbic acid is beneficial. Given the fact that many do not meet this minimum RDA, engineering edible plants to produce more of this essential vitamin may lead to improvements in health. Consuming elevated levels of a vitamin such as ascorbic acid via ‘‘functional foods’’ would also result in the consumption of other beneficial phytochemicals, a benefit that would not be achieved via a vitamin supplement.
The introduction of such plants may also reduce the commercial dependence on sulfites as antibrowning agents. Alternatively, crop plants engineered to have increased levels of ascorbic acid may be indirectly beneficial to human health in that such crops may be resistant to environmental stresses that generate ROS and thus produce higher yields, especially in inhospitable environments. As mentioned above, several of the genes in the plant ascorbic acid biosynthetic pathway have been cloned, and research on engineering plants with elevated levels of ascorbic acid is currently underway.