The Mystery of Vitamin C

Also known as ascorbic acid, vitamin C is a small carbohydrate molecule first identified in the 1920s by Albert von Szent Györgyi, who discovered that it was able to prevent and cure scurvy. Scurvy is a pathological life-threatening condition suffered by people who do not have access to fruits or vegetables for long periods of time. A decade earlier, Kazimierz Funk had prepared a list of nutritional factors, called vitamins, whose deficiencies cause severe diseases in humans. In his list, Funk used the letter "C" to designate a factor still unidentified, but known to prevent scurvy. Later on, Szent Györgyi and Haworth chemically identified "C" as ascorbic acid, and named it so because ascorbic means "anti-scurvy." Over the next century, what we now know as vitamin C became one of the most popular drugs in human history.

Why is this molecule so well-known? Apart from its deficiency causing scurvy in humans, vitamin C is also vitally important to other species. Neither animals nor plants can live without vitamin C, and it is therefore surprising that some animals (some fishes and birds, and a few mammals, including guinea pigs and humans) have lost the capability to produce it over the course of evolution. How did this happen?

In each step of a biosynthetic pathway, a substrate is converted into a product in a reaction catalysed by an enzyme. The product of the first reaction becomes the substrate of the second one, and the steps of the pathway run sequentially until the final product is produced (Figure 1). In 1957, the famous biochemist Albert Lehninger studied vitamin C biosynthesis in animals, and realized that, unlike many species, such as cats and dogs, which can biosynthesize their own vitamin C supply, humans are unable to do so. Human cells cannot perform the crucial last step of vitamin C biosynthesis, the conversion of l-gulono-g-lactone into ascorbic acid, which is catalysed by the enzyme gulonolactone oxidase. As a follow-up to Lehninger's work several years later, Nishikimi and co-workers observed that the gene that codes for gulonolactone oxidase is actually present in humans, but is not active due to the accumulation of several mutations that turned it into a non-functional pseudogene (Nishikimi & Yagi 1991). Notably, not only all humans, but also gorillas, chimps, orangutans, and some monkeys have this inborn genetic flaw, meaning that the loss of vitamin C biosynthesis must have occurred first in one of our primate ancestors. But how can something so crucial for survival be eliminated through the course of evolution? Typically, we expect that positive traits should be retained during evolution, and as vitamin C is beneficial, how would natural selection remove such a crucial biosynthetic capability? Indeed, individuals carrying the mutation(s) in the gene encoding gulonolactone oxidase should have had less chance of surviving and reproducing. However, the opposite occurred, and those who had lost vitamin C biosynthesis survived. How can we explain this apparent paradox?

Figure 1: The diversity of biosynthetic pathways for ascorbate and its analogs

Abbreviations: Ara/AraL, arabinose/arabinonolactone; Gal/GalA/GalL, galactose/galacturonic acid/galactonolactone; L-GalDH, galactose dehydrogenase; L-GalLDH, galactonolactone dehydrogenase; D-GalUR, galacturonic acid reductase; Glc/GlcA/GlcL, glucose/glucuronic acid/gluconolactone; GulL, gulonolactone; L-GulO, gulonolactone oxidase; GDP, guanosine diphosphate; Man, mannose; MeGalA, methyl D-galacturonic acid; NDP, nucleoside diphosphate; UDP, uridine diphosphate.

© 2003 Bob Crimi. All rights reserved.

Figure Detail

To better understand how and why the loss of vitamin C occurred, we need to understand the benefits of it. Scurvy is a deadly disease that occurs in vertebrates that are unable to synthesize vitamin C when their diet does not include fresh fruit and vegetables, rich sources of the vitamin. Historically, this disease killed many sailors, who did not have such perishable foods available during their long voyages at sea. Scurvy takes some time to develop in a human with a vitamin-C-free diet, and when it does it can show a range of symptoms. These include lassitude, neurological dysfunction, and, more commonly, dramatic defects in blood vessel and bone integrity. These latter symptoms are the most easily recognized because they cause skin spots, bleeding of gums, and loose teeth, as well as bone and cartilage fragility.

In the late 1950s, the new tools of biological chemistry allowed the identification of another essential role for vitamin C that helped explain these fundamentally disabling symptoms. In 1962, through analysis of the radioactivity incorporated into collagen using a tritiated version of the amino acid proline, Stone and Meister discovered that vitamin C is used as a co-substrate by peptidyl-prolyl hydroxylase, an enzyme that catalyzes the selective modification of proline to hydroxyproline. This modification is essential for proper collagen folding. Consequently, the lack of vitamin C results in the formation of non-functional collagen in blood vessels and bones, which accounts for most of the severe bone and blood vessel related symptoms. The variety of scurvy symptoms beyond those stemming from collagen defects occur because vitamin C is also a co-substrate for multiple enzymes involved in biosynthesis, including the synthesis of dopamine, an important neurotransmitter, and carnitine which helps mitochondria keep in pace with the demand for energy production. Lack of these two compounds helps explain the neurological dysfunction and lassitude symptoms of scurvy.

Nowadays, scurvy is not as widespread as it used to be, although cases still occur among people with unhealthy eating habits. However, vitamin C became quite popular in the 20^th century, not for its role in the prevention of scurvy, but for its potent "antioxidant" function. The identification of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide ions, as molecules that are potentially harmful for biological membranes and other cell components, has intensified interest in things that are anti-ROS, known as antioxidants. These compounds are able to react with dangerous oxidants and keep cells and tissues healthy. Indeed, vitamin C is one of the best physiological non-toxic antioxidants because it is so efficient: it reacts with many different kinds of ROS. There is a common misconception that antioxidants are always beneficial, when rather they are complex molecules that are part of intricate systems ensuring proper cell function. Regardless, because of the integral role that vitamin C plays inside a cell, be it antioxidant or co-substrate, preserving its biosynthesis should have been a selective advantage. Why did humans lose this ability?

Like humans, other animals unable to synthesize vitamin C can always find a supply of it in other organisms that synthesize it on their own, namely plants. Humans who consume regular portions of fresh fruit (or tablets, which are less effective) will avoid the consequences of not making their own vitamin C. Inclusion of vitamin C in the human diet explains why our non-synthesizing ancestors did not become extinct, as they found this an effective compensation for the mutations in the gulonolactone oxidase gene. However, biochemists speculate that there may have been some concurrent advantage of this mutation that caused it to persist and spread in the human lineage. For instance, since one of the products of the reaction catalysed by gulonolactone oxidase is hydrogen peroxide, Halliwell suggested in 2001 that the loss of biosynthesis balanced the "cost" of production, since the advantage of producing one vitamin C molecule would be lost by the production of this reactive oxygen species (Halliwell 2001).

More recently, Grano and De Tullio proposed another hypothesis, based on the studies by Knowles et al. In 2003, Knowles et al. demonstrated that vitamin C regulates a key stress-induced transcription factor called Hypoxia Inducible Factor 1α (HIF1α), a protein that, when activated, regulates the expression of hundreds of stress-related genes. Notably, the activation of HIF1α occurs in the absence of adequate oxygen or vitamin C supply. Grano and De Tullio therefore proposed that organisms that have lost vitamin C biosynthesis have an advantage: they can finely regulate HIF1α activation on the basis of the dietary intake of vitamin C (Grano & De Tullio 2007). When vitamin C supply is sufficient, the HIF transcription factor is less active than in conditions of vitamin C deficiency. In other words, the lack of vitamin C biosynthesis allows our bodies to know more about our nutritional status and consequently set the proper baseline of HIF1α expression. It is like a sensitive titration system.

There is a third yet still unexplored possibility. We know from other studies that pseudogenes are not inert, but can have a significant role in epigenetic control of gene expression (Poliseno et al. 2010). Could this also apply to the human gulonolactone oxidase pseudogene? Time (and much research) will tell.

Vitamin C, initially identified as the factor preventing the disease known as scurvy, became very popular for its antioxidant properties. Vitamin C is an important co-substrate of a large class of enzymes, and, among other things, regulates gene expression by interacting with important transcription factors. We still do not know why humans lost the capability of synthesizing vitamin C. This event probably had evolutionary significance.