Humans are unable to synthesise L-ascorbic acid (L-AA, ascorbate, vitamin C), and are thus entirely dependent upon dietary sources to meet needs. In both plant and animal metabolism, the biological functions of L-ascorbic acid are centred around the antioxidant properties of this molecule. Considerable evidence has been accruing in the last two decades of the importance of L-AA in protecting not only the plant from oxidative stress, but also mammals from various chronic diseases that have their origins in oxidative stress. Evidence suggests that the plasma levels of L-AA in large sections of the population are sub-optimal for the health protective effects of this vitamin. Until quite recently, little focus has been given to improving the L-AA content of plant foods, either in terms of the amounts present in commercial crop varieties, or in minimising losses prior to ingestion. Further, while L-AA biosynthesis in animals was elucidated in the 1960s, 1 it is only very recently that a distinct biosynthetic route for plants has been proposed. 2 The characterisation of this new pathway will undoubtedly provide the necessary focus and impetus to enable fundamental questions on plant L-AA metabolism to be resolved. This review focuses on the role of L-AA in metabolism and the latest studies regarding its bio- synthesis, tissue compartmentalisation, turnover and catabolism. These inter-relationships are considered in relation to the potential to improve the L-AA content of crops. Methodology for the reliable analysis of L-AA in plant foods is briefly reviewed. The concentrations found in common food sources and the effects of processing, or storage prior to consumption are discussed. Finally the factors that determine the bioavailability of L-AA and how it may be improved are considered, as well as the most important future research needs. # 2000 Society of Chemical Industry

Plant L‐ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.

Vitamin C. Biosynthesis, recycling and degradation in mammals.

The ability to synthesize ascorbic acid is absent in the insects, invertebrates, and fishes. The biosynthetic capacity started in the kidney of amphibians, resided in the kidney of reptiles, became transferred to the liver of mammals, and finally disappeared from the guinea pig, the flying mammals, monkey, and man. A similar transition in the biosynthetic ability was observed in the branched evolution of the birds.

https://science.sciencemag.org/content/sci/182/4118/1271.full.pdf

Evolution and the biosynthesis of ascorbic acid

Although ascorbic acid is an important water-soluble antioxidant and enzyme cofactor in plants and animals, humans and some other species do not synthesize ascorbate due to the lack of the enzyme catalyzing the final step of the biosynthetic pathway, and for them it has become a vitamin. This review focuses on the role of ascorbate in various hydroxylation reactions and in the redox homeostasis of subcellular compartments including mitochondria and endoplasmic reticulum. Recently discovered functions of ascorbate in nucleic acid and histone dealkylation and proteoglycan deglycanation are also summarized. These new findings might delineate a role for ascorbate in the modulation of both pro- and anti-carcinogenic mechanisms. Recent advances and perspectives in therapeutic applications are also reviewed. On the basis of new and earlier observations, the advantages of the lost ability to synthesize ascorbate are pondered. The increasing knowledge of the functions of ascorbate and of its molecular sites of action can mechanistically substantiate a place for ascorbate in the treatment of various diseases.

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1476-5381.2009.00282.x

Vitamin C: update on physiology and pharmacology

The requirement of ascorbic acid (vitamin C ) is a common property of living organisms, and it has long been considered that all animals except the guinea pig, monkey, and man can synthesize this vitamin. The classic method for determining the ability of an animal to synthesize ascorbic acid is to feed it a scorbutogenic diet for a prolonged period and to observe the appearance of the scurvy syndrome. Obviously, the method is laborious and time-consuming. Also, the onset of the scorbutic syndrome depends on the ascorbic-acid-retention capacity of the animal. For example, whereas the guinea pigs can be made scorbutic in about 3 weeks, it takes 3 to 4 months to produce scurvy in man. Since the discovery of the technique for studying ascorbic acid synthesir in vitro,1-8 the task has become much simpler. In this technique, the tissue homogenates or the subcellular fractions are incubated with precursors of ascorbic acid and the amount of the vitamin formed is estimated. Using the in vitro method, we have examined the ascorbic acid synthesizing abilities of different species of animals in the phylogenetic tree, and the results are given below.

Synthesis and some major functions of vitamin C in animals.

The enzyme system catalysing the conversion of D-glucuronolactone and L-gulonolactone into Lascorbic acid in animal tissues has been previously reported to be entirely located in the microsomal fractions ofratand goat-liver homogenates (Burns, Peyser & Moltz, 1956; Chatterjee, Ghosh, Ghosh & Guha, 1957a, b; 1958a, b). The kidney tissue has been found to be the site of this conversion in amphibian, reptilian and some of the avian species examined (Roy & Guha, 1958), and in chick kidney the enzyme system concerned has also been found to be located in the microsomes. Isherwood (1953) stated that mitochondria from rat liver catalyse the transformation of L-gulonolactone into L-ascorbic acid. However, our experiments indicate that the observed activity of mitochondria is probably due to their contamination by microsomes. Potassium cyanide greatly accelerates this synthesis from Dglucuronolactone but the synthesis from L-gulonolactone does not require cyanide or any other added factor (Chatterjee et al. 1958 b). Subsequently the conversion of L-gulonolactone into L-ascorbic acid by rat-liver microsomes has been found to be greatly accelerated by boiled rat-liver supernatant or a metal-binding agent such as sodium pyrophosphate, oco'-dipyridal and 8-hydroxyquinoline (Chatterjee, Chatterjee, Ghosh, Ghosh & Guha, 1958c). The microsomal enzyme concerned in the oxidation of L-gulonolactone to L-ascorbic acid is inhibited by heavy-metal ions (Hg2+, Cu2+ and Zn2+) and reversibly by p-chloromercuribenzoate, indicating the involvement of some essential thiol groups in the enzyme (Chatterjee et al. 1958c). It has already been reported that the microsomes can convert only the lactone forms of the precursors, namely D-glucuronolactone and Lgulonolactone. The sodium salts of the corresponding free acids are not acted on (Chatterjee et al. 1957b; 1958 a, b). The soluble supernatant has been found to inhibit the rate of conversion of L-gulQnolactone into L-ascorbic acid to a great extent and, though L-gulonate is not converted by the microsomes alone, it leads to the formation of L-ascorbic acid when soluble supernatant is added to the system. The factor in the supernatant which inhibits the rate of microsomal synthesis of ascorbic acid from L-gulonolactone but, curiously, helps in

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Biological synthesis of L-ascorbic acid in animal tissues: conversion of L-gulonolactone into L-ascorbic acid.

Simpson, S. A. & Tait, J. F. (1952). Endocrinology, 50, 150. Simpson, S. A. & Tait, J. F. (1953). Mem. Soc. Endocrin. 2,9. Southcott, C. M., Bandy, H. E., Newsom, S. E. & Darrach, M. (1956). Canad. J. Biochem. Phy8iol. 34, 913. Speirs, R. S. & Meyer, R. K. (1951). Endocrinology, 48, 316. Vogt, M. (1955). J. Physiol. 130, 601. Vogt, M. (1957). Ciba Foundation Colloq. Endocrin. 11, 193. Weichselbaum, T. E. & Margraf, H. W. (1955). J. clin. End,ocrin. Metab. 15, 970. Wilson, H. & Fairbanks, R. (1955). Arch. Biochem. Biophys. 54, 441. Zaffaroni, A. & Burton, R. B. (1951). J. biol. Chem. 193, 749.

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Biological synthesis of l-ascorbic acid in animal tissues: conversion of d-glucuronolactone and l-gulonolactone into l-ascorbic acid

In exploring the mechanism of biosynthesis of ascorbic acid by the rat, King and his coworkers (Musulin, Tully, Longenecker & King, 1939; Longenecker, Musulin, Tully & King, 1939; Longenecker, Fricke & King, 1940) observed that rats treated with Chloretone (1:1:1-trichloro-2methylpropan-2-ol) excreted more ascorbic acid than did the controls. By employing Chloretonized rats and isotopically labelled precursors they (Jackel, Mosbach, Burns & King, 1950; Horowitz, Doerschuk & King, 1952; Horowitz & King, 1953a, b) presented evidence for the following route of the synthesis: D-Glucose -+ D-glucurono-y-lactone -* L-ascorbic acid. Subsequently, Isherwood, Chen & Mapson (1954) and Mapson, Isherwood & Chen (1954) came to the conclusion from an extensive series of experiments that in plant and animal systems respectively the following sequences were involved: (1) D-Galactose -+ D-galacturonic acid methyl ester -+ L-galactono-y-lactone -+ L-ascorbic acid. (2) D-Glucose -+ D-glucurono-y-lactone -+ Lgulono-y-lactone --. L-ascorbic acid. Hassan & Lehninger (1956) reported that ratliver extracts containing the microsomes and soluble supernatant could convert D-glucuronolactone, L-gulonolactone and also the corresponding free acids into L-ascorbic acid in the presence of cofactors. In experiments previously carried out in this Laboratory it had been observed that rat-liver tissue incubated with sodium pyruvate showed a higher ascorbic acid value than did the controls incubated without the substrate (Roy, Roy & Guha, 1946). But in no experiment was the value so obtained higher than the original ascorbic acid content of the tissue before incubation, and it was possible therefore that pyruvic acid was not a direct precursor. In a search for a system in which a net synthesis over and above the original tissue ascorbic acid level takes place, Chatterjee, Ghosh, Ghosh & Guha (1957a) found, in later experiments, that a marked synthesis of ascorbic acid occurred in homogenates of rat-liver tissue, containing potassium cyanide, from D-glucurono-ylactone, the increase in ascorbic acid value being sometimes two to three times that originally present. No other substrate, including sodium Dglucuronate and sodium pyruvate, could be so converted into ascorbic acid. Subsequently, it was observed that L-gulonolactone, but not sodium L-gulonate, could also act as the substrate and this conversion took place in the absence of cyanide. Liver tissues of all the mammals investigated could bring about this cyanide-stimulated synthesis, but homogenates of the brain (rat), kidney (rat and goat) or adrenal gland (cow) were ineffective in this respect. Extracts from cow and goat livers were found to be the most active, and goat liver, being more freshly available, was chosen for further studies on the enzyme system concemed. The enzyme system was separated from goat-liver homogenates and obtained in the microsomes and the soluble fraction; it could be further concentrated from the extract by precipitation with ammonium sulphate (Chatterjee, Ghosh, Ghosh & Guha, 1958a). The present paper deals with detailed studies of this enzyme preparation.

Effect of cyanide on the biosynthesis of ascorbic acid by an enzyme preparation from goat-liver tissue.

http://science.sciencemag.org/content/sci/126/3274/608.full.pdf

J. J. Ghosh

Papers

Biological synthesis of L-ascorbic acid in animal tissues: conversion of L-gulonolactone into L-ascorbic acid.

Biological synthesis of l-ascorbic acid in animal tissues: conversion of d-glucuronolactone and l-gulonolactone into l-ascorbic acid

Effect of cyanide on the biosynthesis of ascorbic acid by an enzyme preparation from goat-liver tissue.

Effect of cyanide on biosynthesis of ascorbic acid in vitro.

Identification of 2-keto-L-gulonolactone as an intermediate in the biosynthesis of L-ascorbic acid