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Vitamin C

Its Chemistry and Biochemistry

Royal Society of Chemistry

Introduction

Everyone has heard of vitamin C. There can be few simple organic molecules which have excited such universal interest. At least part of the reason for this has been the general interest in the beneficial effects of all vitamins and other trace substances on human health which has developed in recent years along with concern on the effects of other substances, particularly additives, on those who consume food containing them. We know that vitamins are essential to our well-being and because of this they have excited an interest and curiosity which has resulted in many of them being attributed with disease-healing and health-giving properties which they could not possibly have, Vitamin C has itself been said to have almost magical properties by some writers and it is useful to get a picture of the chemistry and biochemistry of this enigmatic compound.

Vitamin C is different. It is different from the other vitamins and we shall see in the course of this book that its chemistry and biochemistry single it out amongst molecules in many important ways, Vitamin C is ubiquitous. It is found throughout the plant and animal kingdoms, where its roles are often not known or are poorly understood. The synthetic vitamin is very widely used as a food additive and therefore has an E number (E300). However, unlike many other additives, few people would object to its presence in foods. There is no doubt that its anti-oxidant properties confer stability on foods to which it has been added.

Vitamin C has been the subject of frequent controversy, even before its nature had been established. Its role (as a constituent of fruits and vegetables) in the cure and prevention of scurvy was widely debated for hundreds of years. Its very existence was doubted by many even as recently as the the beginning of the twentieth century. There were quarrels over who was the first to discover it. Even today there is much controversy about the exact role of the vitamin in human health and there is not even agreement over the amount of the vitamin which needs to be consumed for optimum well being, with various authorities recommending amounts varying from 30 mg to 10 g per day. The role in the relief of cold symptoms, in the improvement of quality of life for cancer patients and in other medical areas are all topics for intense discussion. The biochemistry of L-ascorbic acid in mammals is very poorly understood, so that it is not even clear what the biochemical role of the vitamin is in such systems. Although the chemical structure of L-ascorbic acid has been unequivocally established by single crystal X-ray diffraction, the structure of its very important two-electron oxidation product, dehydroascorbic acid, has not been finally established, since it has not yet proved possible to isolate crystals, or indeed the pure compound, as a solid.

Vitamin C is chemically the simplest of the vitamins and for this reason was among the first to be isolated, characterised, and purified and to have its structure determined. *More vitamin C is produced industrially than any other vitamin, or indeed all the other vitamins put together. It is one of the few pure chemical compounds which is taken routinely by human beings in gram quantities (a possible challenger is sugar). I t appears to have no harmful effects even in these large amounts and it is a medicine which it is a pleasure to take, especially in the form of fruit or vegetables.

It may be thought that the chemistry of this simple molecule would no longer hold any surprises after the vast amount of research that has been carried out over the years. However, conferences on aspects of vitamin C chemistry still attract large numbers of workers in the field and new aspects of the chemistry are always being revealed. The reason for the continued interest in the chemistry of L-ascorbic acid lies in the fact that despite it being such a simple molecule, its ene-diol structure provides it with a highly complex chemistry. Thus it has a very complicated redox chemistry involving comparatively stable radical intermediates which is heavily modified by the acidic properties of the molecule. It has been known for many years that L-ascorbic acid is easily oxidised by dioxygen. Although the first product of this process is dehydroascorbic acid, which still has antiscorbutic properties, the further oxidation by oxygen produces compounds which are not readily converted back to L-ascorbic acid, and the vitamin is effectively destroyed. The mechanisms of the reactions involved are still largely unknown, although they have been widely studied. There has been much recent work on the interactions of vitamin C with metal ions, particularly transition metal ions. This has unearthed a rich vein of chemistry involving L-ascorbic acid as both a redox companion and as a complexing agent; indeed the reaction of L-ascorbic acid with oxygen and other oxidising agents is catalysed by transition metal ions, especially copper(II), so that sometimes solutions are stabilised by the addition of EDTA, which complexes the metal ions and arrests the catalysis. It appears that vitamin C may not always act alone in its biochemical processes, but may act synergistically with other substances, of which vitamin E may be a typical example.

Research into vitamin C chemistry appears to have reached a kind of steady state. In the years 1969, 1979, and 1989, there were about the same number of papers published each year on aspects of the chemistry of L-ascorbic acid. Thus the extent of the work on this compound has been remarkably constant over the past twenty years and there is no sign of a diminution of interest yet.

The development of analytical techniques to detect and determine vitamin C has been crucial in the understanding of the presence and stability of the compounds in nature. At first, biological techniques were used and these gradually became replaced by chemical methods which were more sensitive, more selective, and easier to carry out. Today the analysis is largely centred around the use of high-performance liquid chromatography and there are many successful methods available. However, the technique is still limited by the fact that detection of dehydroascorbic acid in the presence of L-ascorbic acid still has a comparatively low sensitivity by virtually all detection techniques. This makes the determination of amounts of dehydroascorbic acid in plants and animals much more difficult than for L-ascorbic acid. The same applies to further oxidation products and there is a need for further work in the development of detection methods for these compounds and to investigate the extent and kinetics of oxidation of L-ascorbic acid in fruits and vegetables by determining the amounts of all oxidation products and the oxidation pathways by which these products are formed.

We are familiar with the use of ascorbic acid in pharmaceutical preparations. Although it is often found as the pure compound, in most cases it is present with a wide variety of other substances, which are often present simply to make the vitamin more palatable. However, much ascorbic acid is used for less well-known purposes. Much research has been carried out on the effects of ascorbic acid on various aspects of plant growth. It has been found to have effects upon germination and root growth. Spraying with ascorbic acid has been found to be effective in the protection of plants against the worst effects of ozone in the atmosphere produced by photolytic action on polluted air, particularly in big cities. *Most domestic and farm animals are able to synthesise their own vitamin C, but even so this is sometimes reinforced by additional ascorbic acid. Fish are unable to synthesise ascorbic acid and the results of' a vitamin C deficiency in fish are collectively known as 'broken back syndrome'. Thus, fish with a low dietary intake of vitamin C commonly suffer from distortions of the vertebral column, impaired collagen production, poor growth, and other symptoms. The widespread increase in aquaculture has meant that large amounts of ascorbic acid are used in the breeding and rearing offish.

When L-ascorbic acid is used in the food industry, we can broadly divide the applications into two categories.

(1) It is frequently used as an additive to foods where it enhances the nutritious qualities. It may be added to restore loss of vitamin due to the food processing or to increase the natural amount of the vitamin present. In either case the term nutrification has been used to describe its addition. Thus, L-ascorbic acid is added to fruit juice to fortify that which is naturally present or it may be added to artificial fruit drinks to improve taste and the nutritiousness of the drink.

(2) L-Ascorbic acid may also be used as a food additive in circumstances where it is not expected to provide any increase in the nutritious nature of the food, but where it is present to prevent oxidation, as a preservative, to increase acidity, as a stabiliser, or as a flour improver. It is very often used as an additive for all these purposes.

Red meat has its characteristic colour because of the myoglobin and similar iron complexes which are present. This red colour is enhanced by the addition of nitrite as part of the curing process. This is due to the reaction of nitrogen monoxide with the myoglobin. The addition of ascorbic acid to meat also improves colour, flavour and odour, as well as lowering the amount of nitrite which has to be used in curing. Almost as a side effect it has been found that ascorbic acid, alone or in co-operation with tocopherol when used in meat curing, inhibits the formation of nitroso compounds, which are believed to be carcinogenic, while not interfering significantly with the inhibition by nitrite of the very dangerous Clostridium botulinum micro-organism.

L-Ascorbic acid is very widely used in bread baking, where it is present as a 'flour improver'. In practice, this means that the addition of L-ascorbic acid improves the bread texture and the size of the resulting loaf, the dough has greater elasticity, increased gas retention, and improved water absorption. Furthermore the addition means that storage time can be saved, since flour to which L-ascorbic acid has been added behaves rather like flour which has been matured over time. It is comforting to know that the products of' the decomposition of L-ascorbic acid in bread making are carbon dioxide, L-threonic acid, and 2,3-diketogulonic acid and NOT oxalic acid! In many countries, it is the only flour improver which is allowed. Where others are used, it is interesting that they are all oxidising agents such as potassium bromate. L-Ascorbic acid is the only reducing agent used for such purposes. The exact mode of action of the vitamin C is still a mystery. It is clear that during the dough mixing operation all the L-ascorbic acid is converted into dehydroascorbic acid and this remains stable in the mixture. Perhaps the best-known process using L-ascorbic acid as an additive in bread making is the so-called Chorleywood process.

Some countries have a legal maximum for the amount of L-ascorbic acid used as a flour improver. These may be as high as 200 mg kg-1 in Canada, or as low as 20 mg kg-1 in Uruguay. Many other countries, however, such as the United Kingdom leave the amount added to Good Manufacturing Practice.

Other areas where L-ascorbic acid has found uses in industrial processes are, for example, in polymerisation reactions, in photographic developing and printing, in metal technology, and even in intravaginal contraceptives. Most of these applications involve the use of the reducing properties of L-ascorbic acid in some way.

L-Ascorbic acid is found all over the plant world, often in quite large quantities and distributed throughout the plant. The biochemistry of vitamin C in plants is very poorly understood. A view which seems to be accepted generally is that in some way L-ascorbic acid is merely a secondary product of plant metabolism. It seems curious that such a ubiquitous compound in plants should be there almost incidentally as a by-product of other processes, though it is fortunate for those creatures that have lost their ability to synthesise the vitamin that it is so!

There is evidence that tartaric acid in grapes has L-ascorbic acid as a major precursor. Thus, immature grapes fed with L-ascorbic acid labelled with 14C at C-1 were found to have 72ο

Nothing emphasises the importance of vitamin C to human beings more than the effect of being without it for a relatively short time. Just a few months' deprivation produces the particularly unpleasant and ultimately fatal disease, scurvy. It is hard for us today to appreciate the fear with which this mysterious disease was regarded, particularly by seafarers in the middle ages. Although it was only one disease among many which afflicted sailors, it seemed to flare up for no apparent reason, particularly on long sea voyages, which were becoming more and more common from the fourteenth century. It was not uncommon to lose more than half the crew on such a journey. Vasco da Gama lost half his complement of men when he first rounded the Cape between 1497 and 1499 and scurvy continued to take its toll of sea travellers for four hundred years after this time.

What is this disease? A 'textbook' definition would be something like: A disease which produces haemorrhaging into tissues, bleeding gums, loose teeth, anaemia and general weakness. However, contemporary descriptions of individual cases bring home to us the unpleasantness of scurvy. Thus Thomas Stevens wrote from a ship travelling from Lisbon to Goa in 1579:

'... their gums wax great, and swell, ... their legs swell, and all the body becometh sore, and so benumbed, that they can not stir hand nor foot, and they die for weakness, or fall into fluxes and agues, and die thereby....'

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Excerpted from Vitamin C by Michael B. Davies, John Austin, David A. Partridge. Copyright © 1991 The Royal Society of Chemistry. Excerpted by permission of Royal Society of Chemistry.
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