Oxygen

by Nick Lane

  Such a diverse array of functions lends vitamin C an aura of magic. Yet in each of these cases, vitamin C behaves in exactly the same way at the molecular level, as repetitively as flipping a coin, even if the outcomes are opposed. To see how, let us take a single example in a little more detail.

  Collagen synthesis illustrates not only how vitamin C works, but also throws light on its antioxidant properties, as well as its more dangerous side.

  Collagen can only be manufactured in the presence of molecular oxygen (as we noted in Chapter 4). Oxygen is needed, along with vitamin 2 Chronic granulomatous syndrome is an unpleasant condition caused by a mutation in the gene coding for the enzyme NADH oxidase, which generates oxygen free radicals in activated neutrophils. People with the syndrome cannot kill bacteria efficiently and develop chronic suppurating granulomas (abscesses) in the skin, lymph nodes, lung, liver and bones.

  They also suffer frequently from opportunistic infections. The disease usually develops in childhood and can be treated to some extent with antibiotics.

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  C, to modify some of its amino acids after they are incorporated into the final protein. The amino acids are hydroxylated, which means that they have an additional hydroxyl (–OH) group attached to them. This hydroxylation enables cross-links to be formed between the individual collagen chains, first to form triple-chain collagen molecules, and then to link these molecules into thicker collagen fibrils. These cross-links are responsible for the tremendous tensile strength of collagen. Without vitamin C

  and oxygen, collagen cross-links cannot form and the connective tissues are weakened. Not only this, but non-hydroxylated collagen is retained inside the cells that make it, rather than being exported for building purposes. It is also less stable, more sensitive to heat, and more readily broken down by digestive enzymes. Jelly made from scorbutic collagen would be a sorry sight at a tea party.

  The mechanism of collagen hydroxylation betrays the secret of vitamin C: it is an electron donor. The oxygen atom in the hydroxyl group comes from molecular oxygen. To attach this oxygen, a single electron must be added to each of the two atoms in molecular oxygen.

  Because electrons tend to double up as pairs, few compounds can give up a single electron without becoming unstable and reactive themselves.

  Metals such as iron and copper, which exist in several stable oxidation states, can do so, as can vitamin C. In biological reactions, vitamin C

  always donates electrons. This and no more. Having said that, it is not profligate with its electrons; under physiological conditions it is most likely to offer them to iron or copper.3 In the process of collagen synthesis, this is exactly what happens. Vitamin C donates an electron to iron, which is embedded at the core of the enzymes that carry out the hydroxylation reaction. The iron thrusts this electron onto oxygen, which can now be attached to amino acids in collagen. In the process, iron is oxidized to the biologically inactive form (Fe3+), until it receives an electron back from vitamin C.

  The role of vitamin C in this case is therefore to regenerate the biologically active form of iron by passing an electron to the oxidized form.

  The hydroxylating enzyme acts as a kind of merry-go-round that uses iron to attach oxygen to amino acids. By providing iron with electrons, vitamin C provides the motive force that keeps the merry-go-round turning.

  The triumvirate of iron (or copper), vitamin C and oxygen is at the 3 In more technical terms, vitamin C has a relatively neutral reduction potential: it donates electrons to strong oxidants, rather than thrusting them onto weak oxidants, so does not interfere with normal workings of the cell.

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  heart of virtually all the physiological actions of vitamin C. A total of at least eight enzymes use vitamin C as a cofactor. All of these enzymes contain iron or copper at their core. All attach oxygen atoms to amino acids, using the iron or copper. All use vitamin C to regenerate active iron or copper. Essentially the same reaction also accounts for the ability of vitamin C to promote iron absorption in the intestine. In this case, vitamin C

  donates an electron to oxidized iron, converting it into the soluble form that can be absorbed.

  Why is vitamin C used so extensively as an electron donor? There are two main reasons. First, vitamin C is very soluble in water, so it can be concentrated in confined spaces surrounded by membranes (which are made of lipids impermeable to vitamin C). The synthesis of noradrenaline from dopamine, for example, takes place in small membrane-bounded spaces, or vesicles, within cells of the cortex of the adrenal glands. The vitamin C concentration inside these vesicles reaches about 100 times that of blood plasma. As vitamin C is consumed by the enzyme dopamine mono-oxygenase, electrons are passed across the vesicle membrane (via an iron-containing protein, cytochrome b 651), to regenerate vitamin C

  within the vesicles. Thus, for periods of days or weeks, the intracellular vitamin C needed for physiological tasks can be ‘insulated’ from changes in plasma levels caused by variations in diet, and maintained at the ideal levels for a particular reaction.

  The second reason that vitamin C is used as an electron donor is that the reaction product is fairly stable and unreactive. When vitamin C gives up an electron, it becomes a free radical called the ascorbyl radical. By free-radical standards, the ascorbyl radical is not very reactive. Its structure is stabilized by electron delocalization — the resonance effect first described by Linus Pauling in the late 1920s. This means that vitamin C can block free-radical chain reactions by donating an electron, while the reaction product, the ascorbyl radical, does not perpetuate the chain reaction itself.

  Despite its slow reactivity, the ascorbyl radical usually gives up a second electron to produce dehydroascorbate. This molecule is unstable and needs to be caught quickly if it is not to break down spontaneously and irrevocably, and be lost from the body. The continual seeping loss of vitamin C in this way accounts for our need to replenish body pools by daily intake. Even so, we can minimize losses by recycling dehydroascorbate.

  Several different enzymes bind dehydroascorbate to regenerate vitamin C.

  These enzymes usually take two electrons from a small peptide called

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  glutathione, and transfer them to dehydroascorbate. Because a pair of electrons are transferred, the regeneration of vitamin C does not produce free-radical intermediates.

  The repetitive, coin-flip action of vitamin C is thus to donate single electrons (it donates two of them to form dehydroascorbate). It is regenerated from dehydroascorbate by receiving two electrons from glutathione. This cycle explains not only the enzyme-cofactor effects of vitamin C, but also its antioxidant activity. Even though vitamin C usually gives up electrons to iron or copper, other molecules in search of a single electron can extract one from vitamin C. These include many free radicals, which of course are defined as atoms or molecules with a single unpaired electron (see Chapter 6).

  When a free radical reacts, it usually snatches an electron from the reactant, turning it into a free radical. This in turn will steal a single electron from another nearby molecule. A chain reaction ensues until two free radicals react together, effectively neutralizing each other, or alternatively, until an unreactive free-radical product is formed. Free radicals are said to be ‘quenched’ by vitamin C, because the free-radical product —

  the ascorbyl radical — is so unreactive. As a result, free-radical chain reactions are terminated. Lipid-soluble vitamin E (␣-tocopherol) works in the same way, in membranes rather than in solution, often in cooperation with vitamin C at the interface between membranes and the cytosol (the watery ground substance of the cytoplasm that surrounds the intracellular organelles). When vitamin E reacts with a free radical, it too produces a poorly reactive (resonance-stabilized) free-radical product, called the

  ␣-tocopheryl radical. Tocopheryl radicals can be reconverted into vitamin E using electrons fro
m vitamin C.

  As I intimated at the beginning of this chapter, these simple, repetitive reactions conceal a hidden danger — the darker side of vitamin C. We have noted the link between iron, vitamin C and oxygen. When vitamin C interacts with iron and oxygen, it is acting as an electron donor, but not as an antioxidant. Quite the contrary. By regenerating the active form of iron inside an enzyme, vitamin C aids and abets the addition of oxygen —

  in other words, it helps to oxidize the substrate. Thus many of the beneficial actions of vitamin C are actually pro-oxidant actions, not antioxidant actions at all.

  When iron is bound to the core of a protein this is safe enough — the iron is restrained in the manner of a horse wearing blinkers, and kept

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  focused on a narrowly defined task. But it is a different matter when the iron is in solution. Now the iron is free to react in an uncontrolled way.

  We saw the danger coming in Chapter 6. Remember the Fenton reaction?

  Iron reacts with hydrogen peroxide to produce brutal hydroxyl radicals and the oxidized, inactive form of iron. Hydroxyl radicals react immediately with their nearest neighbours to initiate free-radical chain reactions.

  These damaging chain reactions can only be started when active iron is available, and so they eventually peter out after the active iron is used up.

  We saw that superoxide radicals are dangerous because they can regenerate active iron, perpetuating the Fenton reaction. They do this by donating electrons. Vitamin C can also donate electrons to regenerate active iron. In principle, then, there is no reason why vitamin C should not exacerbate free-radical damage rather than acting as an antioxidant. Theoretically, it can act as a pro-oxidant as well as an antioxidant.

  The possibility that vitamin C could act as a pro-oxidant is not just theoretical. A common test for the potency of an antioxidant actually depends on vitamin C to promote injury. The method begins by stimulating free-radical chain reactions in a preparation of cell membranes, then measures how well antioxidants can stop the process in its tracks. The chain reactions are started using a concoction of iron and vitamin C — iron to catalyse the reaction, and vitamin C to regenerate the active iron. If this situation were ever to take place in the body, the effect could be catastrophic.

  Two questions spring to mind. Does vitamin C ever act as a pro-oxidant in the body to cause damage? And if not, why not? What stops it from doing so? Few subjects have raised so many hackles among normally placid researchers, and the answers are by no means certain. Nonetheless, ambiguities aside, the issues raised by even the possibility of pro-oxidant effects give us a perspective on antioxidant ‘networking’ in cells — and also on why Linus Pauling and Ewan Cameron might just have been right about the anti-cancer activity of vitamin C after all.

  I should say that there is very little evidence that vitamin C ever acts as a pro-oxidant in people. There are a few indications that the body is aware of the danger, however. In particular, we control our blood plasma levels of vitamin C carefully. Even if we take mega-doses of vitamin C, plasma levels hardly rise. There are two main controls on vitamin C levels in blood plasma — absorption and excretion. The absorption of vitamin C from the intestine falls off drastically the larger the dose. Mega-doses

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  have a laxative effect and cause diarrhoea.4 Some advocates of mega-dose vitamin C even recommend ‘titrating to bowel tolerance’ — eating nearly enough to cause diarrhoea — to ensure that the maximum possible amount is absorbed. It doesn’t work. Less than 50 per cent of a 1-gram dose is absorbed from the intestine, and most of this is then excreted in the urine. Being freely soluble, vitamin C is filtered by the kidneys, and not all of it is reabsorbed unless an acute shortage leaves the body grasping at straws. Excretion in the urine starts at doses of about 60 to 100

  milligrams daily and builds up from there. Nearly all of a 500 milligram dose is excreted in this way. In fact, blood and body pools are saturated by about 400 milligrams of vitamin C each day. It doesn’t matter how much extra you take, you won’t increase your body levels.

  While this information is valuable in itself, the fact that the body controls vitamin C levels so tightly suggests that there might be a problem if it did not. Although nobody ever proved that vitamin C is toxic in this way, we should appreciate that neither has anybody proved that vitamin C works as an antioxidant in the body. We know that it can act as an antioxidant, and probably does, but we are far from the kind of rigorous proof that a rocket scientist might demand. Here, for example, is Baltz Frei, current head of the Linus Pauling Institute, writing in 1999: “the current evidence is insufficient to conclude that antioxidant vitamin supplementation materially reduces oxidative damage in humans.” So what about the other side of the coin, actual bodily harm? If the potential toxicity of vitamin C relates to its interactions with iron, then the first place to look for danger is a disease in which iron metabolism is disrupted in some way.

  One such condition is iron overload. Rather surprisingly, we have no specific mechanism for removing excess iron from the body, save menstrual bleeding or the shedding of gut-lining cells. The rate of absorption of iron therefore needs to be regulated tightly. The inherited form of iron overload, called haemochromatosis, stems from a failure of the normal mechanism for regulating iron absorption in the intestine. Too much iron is absorbed, and in time — 40 years or more, depending on diet — this exceeds the body’s capacity to store it safely. Free iron appears in the blood stream. The effect is devastating. Without treatment, the victim can 4 It is conventional to view the laxative property of vitamin C as a non-harmful side-effect —

  nothing in comparison with the side-effects of many pharmaceutical drugs — but it may be wiser to view the laxative effect as a deliberate physiological response on the part of the body to potentially toxic levels of vitamin C.

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  expect to suffer from liver failure (caused by cirrhosis or cancer), weight loss, skin pigmentation, joint inflammation, diabetes and heart failure.

  Nor is this a rare condition. In western populations, the prevalence is nearly 0.5 per cent, making it one of the most common genetic disorders.5

  In theory, vitamin C can affect people with iron overload in two different ways. First, it might increase the rate of iron absorption from the intestine. Although there is no evidence that mega-doses of vitamin C

  cause iron overload in healthy subjects, it is not at all clear how they affect iron levels in people with haemochromatosis. Second, vitamin C

  might convert excess iron into the active form that can catalyse free-radical reactions. Whether this might tip the balance between vitamin C

  acting as an antioxidant or a pro-oxidant in people with haemochromatosis is unknown. Most evidence suggests that it does not, although a few case reports have claimed a detrimental effect. One unfortunate young man in Australia, for example, took high doses of vitamin C for a year before being admitted into hospital with severe heart failure. He died eight days later, diagnosed at autopsy with haemochromatosis. The doctors concluded that the progression of the disease might well have been accelerated by his excessive intake of vitamin C.

  But let us consider the risk from another point of view. If there is a dark side of vitamin C, then it might offer an advantage in the treatment of cancer. We know that vitamin C can kill tumour cells grown in the test tube, and that the way in which it does so is dependent on supplies of iron and oxygen. Vitamin C can also kill malarial parasites at certain points in their life cycle, when they are actively accumulating iron from haemoglobin. Is this perhaps an explanation for the Pauling and Cameron findings? It is certainly plausible. The cores of large tumours are often composed of dead or dying cells, which release iron as they degenerate.

  The fine control of iron metabolism in normal cells may also be lost in cancer cells, which are chaotic and oppor
tunistic in their behaviour. In addition, radiotherapy and chemotherapy cause transient plasma iron overload, which presumably derives partly from the tumour itself. For all 5 Iron overload can also result from the medical treatment of other conditions, notably the thalassaemias, which are caused by an inability to produce haemoglobin at the normal rate.

  Unless treated with regular blood transfusions, patients with thalassaemia major die in childhood. On the other hand, given regular transfusions, many thalassaemia patients eventually develop iron overload.

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  these reasons, iron is likely to be present in tumours at a higher concentration than in normal tissues. In the presence of oxygen and vitamin C, then, tumours might conceivably be placed under enough oxidative stress to kill them.

  If so, why were Cameron’s results so hard to reproduce? In an incisive reappraisal of the anti-cancer effects of vitamin C in the Canadian Medical Association Journal in 2001, Mark Levine and Sebastian Padayatty (also at the NIH) argued that the route of administration holds the key. Pauling and Cameron infused vitamin C intravenously, while the Mayo clinic, in attempting to replicate their data, used high oral doses. When given orally, the low rate of absorption and the high rate of excretion maintain virtually constant plasma levels. Intravenous dosing, on the other hand, side-steps absorption altogether, while the kidneys take some time to remove vitamin C from the blood. For short periods, then, blood levels of vitamin C may reach 50 times their normal saturation point; and this might make all the difference. Padayatty and Levine called for rigorous new trials to test the possibility.