The Function of Ascorbic Acid through Occam’s Razor: What We Know, What We Presume and What We Hope For

Abstract

After being identified as the anti-scurvy factor vitamin C, ascorbic acid (AsA) became the subject of an astonishing amount of research. Many studies confirmed that AsA is essential to plants and animals, however, the molecular mechanisms by which AsA exerts its action are only in part understood. Much attention has been given to the so-called antioxidant function of AsA, but the concept of antioxidant is in itself rather vague and, at least in some studies, proper quantitative approaches to assess the actual relevance of AsA as an antioxidant are lacking. On the other hand, in the last few decades more and more research focused on the specific function of AsA as a regulatory co-factor of 2-oxoglutarate-dependent dioxygenases, a large class of enzymes catalyzing an array of different and apparently unrelated reactions, all sharing a complex mechanism based on the integration of relevant pieces of molecular information. The present contribution aims to critically discuss available evidence in support of current hypotheses on AsA function.

1. Introduction

One hundred years ago, the 29-years old scientist Albert von Szent-Györgyi was conducting observations on the biological oxidation of the adrenal cortex. After exploring different plant and animal systems, he found in turnip extracts a putative carbohydrate that inhibited the peroxidase reaction and named it hexuronic acid [1]. At the time, he would have been probably surprised to know that the identification of hexuronic acid as the anti-scurvy factor vitamin C [2] would have earned him the Nobel prize in Physiology or Medicine in 1937. Ever since those pioneer years, hexuronic acid, soon renamed ascorbic acid (AsA) to make clear also in the name its essence as the anti-scurvy factor, has been receiving much attention from researchers worldwide, and tens of thousands of studies have been published, reporting on many different aspects of this interesting molecule. In parallel with the scientific literature, vitamin C encountered unprecedented popularity: today a simple google search of “vitamin C” returns over 210 million hits. Finding one’s way in this ocean of information, sometimes contradicting, is not easy. It is perhaps necessary to recapitulate the main steps of this long story and tell facts from opinions, if possible. Already in its early years, AsA showed some kind of a “dual nature”, acting both as a reducing agent (antioxidant) and a protectant from the dreadful disease known as scurvy [3, 4, 5]. The aim of this contribution is to present evidence in support of the hypothesis that the two “faces” of AsA coexist and are both parts of an efficient signal transduction mechanism allowing living organisms to sense their environment and successfully adapt to variable conditions.

2. Vitamin C and scurvy

What is vitamin C, and where does this odd name come from? To answer this question we have to go back to the work of Kazimierz Funk, a pioneer biochemist who studied the etiology of some “deficiency disorders” caused by the lack of key nutritional factors [6]. Funk hypothesized that each of those disorders is due to a diet deficient in a specific “vital amine”. In 1912, Funk named each of those (still hypothetical) factors after the first letters of the alphabet. Vital amines changed their name in vitamines and then vitamins, when it was clear enough that, with the exception of a few belonging to the B complex, they are not amines. In Funk’s nomenclature, vitamin C is the one preventing the disease known as scurvy. Of course, Funk had no idea of the chemical nature of the factor. He only suggested that a deficiency in this unknown molecule caused the disease, and, conversely, the uptake of vitamin C could cure the disease. Over the centuries scurvy has killed millions, and especially sailors in the long voyages of exploration that occurred between the late XV and mid-XIX centuries [7]. Scurvy causes a variety of symptoms, which hardly seem to relate (Figure 1).

Figure 1.

Partial list of scurvy-related symptoms.

Interestingly, the symptoms appear progressively, the first one being usually lethargy, followed by neurological disorders and impaired vision. Subsequent signs of the disease include bleeding, spots, swelling gums, bone fractures, problems in wound healing, and even old scars and fractures re-open and re-break [8]. The identification of the anti-scurvy factor by Szent-Györgyi opened the way to a new line of research, aimed at understanding not only the pathogenesis of scurvy but also the mechanisms by which AsA can prevent and cure it. It took, however, almost three decades before the riddle of the biochemical function of AsA could be, at least in part, solved.

It is worth mentioning that most animals do not need AsA supplementation in their diet, because they can produce it by means of a well-characterized biosynthetic pathway [9]. Humans and a few other mammals, as well as some birds and fishes, have lost the capability to synthesize AsA because of the loss of function of the gene coding for the terminal enzyme in the pathway, l-gulono-1,4-lactone oxidase (GULO) [10]. This is the cause of our dependency on external AsA sources (mostly plant-derived food, and primarily fleshy fruits) for our survival.

What is the function of AsA in cell metabolism? A classical reverse genetic approach, frequently used when trying to identify the function of a gene or metabolite, is based on the knocking out of that gene (or of a gene regulating the biosynthesis of the metabolite of interest) and the characterization of the resulting phenotype [11]. In the case of AsA, we do not have to look too far to find the perfect experimental model: all organisms, including humans, who are unable to synthesize AsA are knockout mutants in the GULO gene, and the resulting phenotype is clear and known for centuries. This phenotype is scurvy.

3. The biochemistry behind scurvy

In the search for a common origin of at least some of the many symptoms observed in scurvy, collagen-related disorders caught the eye of early investigators. Fractures, bleeding, and poor wound healing, all relate to the malfunctioning of tissues (bones, blood vessels, skin) containing a substantial amount of different collagen forms [12, 13]. One striking feature of collagens is the presence in the polypeptide chain of hydroxyproline (hypro), and hydroxylysine, two unusual amino acids which are not incorporated as such during the protein synthesis process, but result from the post-translational modification of selected proline and lysine residues catalyzed by the enzymes prolyl-4-hydroxylase (P4H) and lysyl hydroxylase [14, 15]. Hydroxylated residues have an essential role in collagen stabilization, and underhydroxylated collagen is not functional at all. Collagen underhydroxylation was observed in scurvy animals, a condition reversed by AsA administration [16]. Eventually, AsA involvement in the reaction catalyzed by P4H was ascertained [17, 18]. Still, some doubts occurred about the actual role of AsA in P4H-mediated catalysis. It should be considered that 2-ODDs share a complex catalytic mechanism requiring, besides a specific substrate, molecular oxygen, 2-oxoglutarate, Fe²⁺ and AsA [19]. Some studies suggested that AsA is required to keep iron in the reduced state [20], implying that any reducing agent able to reduce Fe³⁺ to Fe²⁺ could replace AsA, but this opinion is contradicted by the high specificity of AsA as the anti-scurvy factor in vivo.

Although AsA involvement in the reactions catalyzed by collagen hydroxylases explains many scurvy symptoms, some others (fatigue, neurological symptoms, impaired vision) are definitely not related to collagen malfunctions. This issue remained unsolved until it became clear that collagen hydroxylases share their catalytic mechanism with a vast array of enzymes, within the large class of 2-oxoglutarate-dependent dioxygenases (2-ODDs) [21]. Checking the long list of 2-ODDs, and comparing it with scurvy symptoms, potential candidates can be spotted, whose inactivation is likely to be caused by AsA deficiency, in analogy to what is observed with collagen hydroxylases. As an example, fatigue and lethargy are the consequences of impaired energy metabolism, possibly caused by impaired activity of two enzymes involved in carnitine biosynthesis: ε-N-trimethyllysine hydroxylase and γ-butyrobetaine hydroxylase [22]. Indeed, AsA deficiency affects carnitine synthesis [23]. It is reasonable to assume that also the remaining scurvy symptoms are somehow related to the inactivation of the other 2-ODDs. A partial list of enzymes using AsA for their catalytic activity is reported in the EBI (European Bioinformatics Institute) CoFactor database [24]. Eighteen out of the 20 enzymes reported, belong to the 2-ODD class.

4. Integration of different signals by 2-ODDs: HIF prolyl hydroxylases and TET dioxygenases

As mentioned above, the unusual catalytic mechanism of 2-ODDs requires, besides the substrate undergoing the specific modification, the cooperation of four different co-substrates, namely O₂, 2-oxoglutarate, Fe²⁺, and AsA. From an evolutionary point of view, it is unlikely that such a complex mechanism developed just by chance. Unexpectedly, further studies on an apparently unrelated subject brought novel information about 2-ODD-mediated catalysis in general, and more specifically about the role of AsA. Starting from the observation that low-oxygen (hypoxic) conditions induce nuclear expression of a novel Hypoxia Inducible Factor (HIF) orchestrating downstream cellular responses [25], an interesting oxygen-sensing system was identified in human cells (Figure 2). The oxygen level is critical to activate a prolyl-hydroxylase, different from the collagen-related one. When two prolines in the HIF protein are modified to hydroxyprolines (hypro) by a highly specific HIF-prolyl hydroxylase (HPH), the HIF protein binds another factor known as the Von Hippel Lindau protein (pVHL), targeting the complex to ubiquitination and degradation. On the other hand, if oxygen is not available, the hydroxylation cannot take place, and the unhydroxylated HIF moves to the nucleus, where it acts as a transcriptional regulator of several genes involved in the hypoxic response [26]. The three main investigators (Semenza, Kaelin, and Ratcliffe) who deciphered this elegant mechanism received the 2019 Nobel prize in Physiology or Medicine.

Figure 2.

The mechanism of oxygen sensing mediated by the Hypoxia-Inducible Factor 1α. Left: in the presence of oxygen, the HIF Prolyl Hydroxylase (HPH) catalyzes the conversion of two critical proline (pro) residues into hydroxyproline (hypro). Hydroxylated HIF1α binds the von Hippel–Lindau protein (pVHL) and the resulting complex is ubiquitinated and degraded in the proteasome. Right: If oxygen is not available, no hydroxylation of HIF1α occurs, and the transcription factor moves to the nucleus, activating transcriptional responses to hypoxia.

Although at the very beginning the HIF mechanism was mainly considered an oxygen-sensing mechanism, some studies tried to assess whether the remaining co-substrates also modulate HIF hydroxylation and the consequent hypoxic response. It was immediately clear that both AsA and Fe²⁺ cooperate with oxygen in the regulation of the response [27]. High AsA, by favoring the hydroxylation reaction, targets the HIF factor to degradation thus preventing the transcriptional cascade [28]. A new group of oxygen-sensing thiol dioxygenases, operating with a mechanism functionally very similar to the one observed in the HIF signaling module, has been recently characterized [29].

If in the past some doubts had been expressed about the specificity of AsA in the reaction catalyzed by collagen prolyl hydroxylase (see above), in the case of HIF prolyl hydroxylases sound experimental data confirm beyond any reasonable doubt that different reducing agents (such as glutathione, or dithiothreitol) cannot take over [30, 31]. Therefore, AsA is not just a generic reducing agent required to keep iron in the reduced state [20]; on the contrary, it appears tailored to the needs of HIF prolyl hydroxylase (and, by extension, of many other 2-ODDs), in conjunction with the other co-substrates.

At first sight, the four co-substrates may seem an odd combination of unrelated molecules, but a careful examination suggests that three out of the four co-substrates bring relevant molecular information regarding energy metabolism: 2-oxoglutarate, as a key intermediate of the Krebs cycle; oxygen, as the obvious respiratory substrate; iron, not only for its involvement in respiration but also for oxygen transport (hemoglobin) [32, 33]. In this perspective, the contribution of AsA to the potential signaling-related content of 2-ODDs appears less clear and will be further discussed below (Section 8). The fascinating hypothesis that the co-substrates can coordinate in parallel all the many different 2-ODDs operating in a cell [34] needs further experimental support, but at least provides a tentative explanation to the surprising complexity of the 2-ODD catalytic mechanism.

Another striking discovery stressing the outstanding importance of dioxygenases in the regulation of cellular responses was the identification of another group of 2-ODDs involved in gene expression. DNA methylation is a widespread mechanism for the epigenetic regulation of gene expression. The hydroxylase activity catalyzed by Ten Eleven Translocation 1 (TET1) converts methylcytosine into hydroxymethylcytosine [35]. The same enzyme also catalyzes further steps toward demethylation and the consequent modulation of gene expression [36]. It has been demonstrated that TET activity is strictly AsA-dependent [37], opening new and unexpected perspectives into our understanding of AsA biological function. Epigenetic modulation is essential to implement the developmental program of any organism. The involvement of AsA in the regulation of gene expression has been widely investigated, in relation to stem cell and cancer research [38, 39, 40, 41].

Besides TET hydroxylases, epigenetic regulation by AsA occurs by means of histone demethylases characterized by the Jumonji domain [42]. In this case, the methylation of lysine residues in histone proteins is removed by a specific dioxygenase, thus modulating gene expression.

5. Is AsA the king of antioxidants?

The reader probably noticed that, when discussing the effects of AsA deficiency, the word ‘antioxidant’ was never mentioned. If we just follow the direct way of analyzing the phenotype of gulo knockout mutants unable to synthesize AsA, the main information we get is that AsA is specifically required by relevant enzymes belonging to the 2-ODD class. This might appear surprising, considering that AsA is mainly known for its antioxidant properties. A quick recap of the main events that determined the popularity of antioxidants in general, and of AsA in particular, is given below.

The use of the word ‘antioxidant’ changed over time [43]. It was originally used to indicate antioxygenic substances and inhibitors of enzymes catalyzing oxidative reactions [44], then progressively assumed the current meaning of molecules able to scavenge reactive oxygen species (ROS) [45]. In the current usage, ROS include the molecules resulting from univalent dioxygen reduction (also named reactive oxygen intermediates: hydrogen peroxide, superoxide anion, hydroxyl radical) [46] plus singlet oxygen and ozone, but also organic hydroperoxides and other oxidizing molecules are sometimes considered part of the ROS family [47]. Due to their high reactivity as electron acceptors, ROS can potentially damage several cell components (lipids, proteins, nucleic acids) [48]. ROS quenching occurs when a suitable reducing agent donates its electrons [43]. The best-known physiological antioxidants, i.e. reducing agents able to react with ROS, are AsA, glutathione (GSH) and tocopherols. In addition, plant secondary metabolism produces a number of molecules with reducing properties and putative antioxidant function. Beside the antioxidant molecules mentioned above, aerobic organisms developed several antioxidant enzymes: superoxide dismutases (SOD), catalases (CAT), glutathione peroxidases [49] peroxiredoxins [50] and, in plants only, also AsA peroxidases [51].

It is not surprising that the discovery of this potentially powerful army of anti-ROS weapons induced us to think that ROS must be kept under control [52]. Unfortunately, the popularized and oversimplified version of the complex ROS/antioxidant interactions has led to the wrong conclusion that ROS are always dangerous and must therefore be entirely scavenged by antioxidants. This shallow, pervasive concept deeply influenced the general public, and unfortunately also some researchers who lost contact with updated scientific literature.

The common opinion that antioxidants are the key to a long and healthy life dates back to the 1970s and 1980s, when Linus Pauling (who had been awarded the Nobel Prize twice, in Chemistry and for Peace) became a fervent communicator of this new concept [53]. The main assumption of his theory was that high AsA doses (18 g daily or more!) can counteract many diseases (from the common cold to cancer) and delay aging. Pauling’s conclusions were mainly based on his own experience and on the limited clinical evidence obtained by Ewan Cameron, a physician who had been using AsA in the treatment of cancer with apparently good results. Pauling’s claims opened the way to the view that antioxidant supplements are always good for human health irrespective of the amount taken: the more the better. It is worth noting that the main international agencies suggest a daily intake of vitamin C in the order of 80–90 mg per day [54].

Of course, it is undeniable that AsA, with a redox potential of 0.28 V [55], is an excellent reducing agent and an efficient antioxidant. As already mentioned, the fortunate encounter of Szent-Györgyi with his hexuronic acid occurred because it inhibited the peroxidase reaction [1]: a typical “antioxidant job”. On the other hand, the opinion that harmful ROS must be totally removed is not consistent with many experimental observations, which could be summarized in three categories. First, enzymes “deliberately” producing ROS are known for a long time: NADPH oxidases occur in many organisms and use NADPH-reducing power to produce ROS. Second, ROS in turn activate several forms of communication, including defense response and epigenetic control of lifespan extension [56, 57, 58, 59]. Third, as a corollary to the previous point, some reports established that improving ROS removal beyond a certain threshold has negative effects on the lifespan of different organisms [60]. In addition, recent studies on AsA reactivity showed that it reacts preferentially with iron and copper, rather than ROS [61, 62]. All this concurred with the definition of a totally new vision of ROS/antioxidant dynamics, overriding the simplistic “good antioxidant/bad ROS” duality [63, 64, 65, 66, 67]. Aerobic organisms produce ROS, but rather than finding ways to erase them, cell metabolism implemented new resources in two different directions: on one side developing antioxidants to control ROS accumulation in some specific cell locations or developmental stages; on the other side using ROS generated by fluctuating environmental conditions as an opportunity to drive cell responses.

6. AsA in plants: coping with oxygen and beyond

Excess oxygen conditions (hyperoxia) increase the risk of ROS overproduction. Hyperoxic conditions typically occur in the lungs of animals [68], but also in plant green tissues, where oxygen is produced at high rates in the photosynthetic process [69]. Photosynthetic organisms have developed, in addition to SOD, CAT and other antioxidant enzymes, also a family of AsA-specific peroxidases present in multiple isoforms in most cell compartments, possibly by duplication of the original chloroplastic forms [70]. Interestingly, accurate studies have ascertained that AsA peroxidases are not simply scavenging enzymes, but their catalytic activity is calibrated so that hydrogen peroxide can still act as a molecular signal [71, 72].

Plants produce AsA using a complex biosynthetic (Smirnoff-Wheeler) pathway that evolved in photosynthetic organisms [73]. In difference to AsA synthesizing animals, the last step in the pathway is catalyzed by a mitochondrial enzyme (l-galactono-1,4-lactone dehydrogenase). Notably, all plants must synthesize their own AsA in order to survive, because they cannot take it from other organisms. The only case described of a plant unable to synthesize AsA is the Arabidopsis vtc2/vtc5 double mutant, which is not viable if not supplemented with AsA [74]. Plant mutants with relatively low AsA content (about 30–50% of the wild type) generally show increased sensitivity to environmental stresses [75, 76]. The expression of pathogenesis-related proteins and, in general, of defense-related genes is also induced by AsA deficiency [77, 78].

In plants, 2-ODDs are the second most represented class of enzymes [19, 79]. A large number of them are involved in the synthesis and/or the catabolism of plant hormones and growth regulators, including auxins [80], gibberellins [81], ethylene [82], abscisic acid [83], strigolactones [84]. Cysteine oxidases in plants perform an oxygen-sensing mechanism similar to the animal HIF-mediated pathway [85, 86, 87]. A novel dioxygenase using directly AsA (and not 2-oxoglutarate) for an unusual modification of cytosine has been recently observed in the green algae Chlamydomonas reinhardtii [88]. All this information from plants confirms that AsA functions not only as an antioxidant, but also as a regulator of many signaling routes through the action of different dioxygenases. As compared to animals, plants developed even more specialized antioxidants (e.g. AsA peroxidases), but their action is in equilibrium with ROS production, which is required for the activation of crucial signaling pathways [89]. A schematic representation of AsA centrality in different aspects of plant cell signaling and regulation is given in Figure 3.

Figure 3.

The central role of ascorbate (AsA) in plant cell signaling. The last step in AsA biosynthesis is catalyzed by the mitochondrial enzyme l-Galactono-1,4-lactone dehydrogenase (l-GalLDH). Reactive Oxygen Species (ROS) are formed in mitochondria, chloroplasts and as a consequence of different stress conditions. ROS (red dots) can be reduced by AsA in uncatalyzed reactions, and hydrogen peroxide can be removed in the reaction catalyzed by AsA peroxidases (AsA px) located in different cell compartments and organelles. AsA is also specifically used by 2-oxoglutarate-dependent dioxygenases (2-ODDs), including enzymes involved in the synthesis and/or catabolism of plant hormones (ABA: abscisic acid; GA: gibberellin; strigo: stigolactones). AsA oxidation produces dehydroascorbic acid (DHA, in the most stable dimeric form). In the nucleus, AsA cooperates in the regulation of the activity of Ten-Eleven-Translocation (TET) methylcytosine dioxygenases, a class of enzymes involved in the epigenetic control of gene expression.

7. A rewarding double play: the two faces of AsA

A continuing challenge for all organisms is the real-time adaptation to ever-changing environmental conditions. Specific external/internal cues must be sensed, and the resulting signal transduced to the corresponding response regulator. For example, in two-component systems, observed in bacteria and several eukaryotes, but not in humans, the signal is transduced by a chain of phosphorylation events [90]. Another common form of signal transduction is ensured by redox regulation using thiol-disulfide exchanges [91, 92]. In both cases, the signal is rapidly and efficiently transduced to a transcriptional regulator activating some general and some specific responses in a cascade mechanism. Many stress and pathological conditions induce ROS production, but different forms of stress, or their combination, result in distinct signatures of ROS levels [93], increasing the specificity of the response.

AsA is oxidized by the direct interaction with ROS and, in plants, also by the reaction catalyzed by AsA peroxidases. In stress conditions, when high ROS production occurs, AsA is converted to its oxidized form dehydroascorbic acid (DHA) via the dismutation of the short-lived ascorbate free radical (AFR, also known as monodehydroascorbate) [94]. The more AsA is oxidized, the less AsA is available for the reactions catalyzed by 2-ODDs. This is indirectly confirmed by the observed correlation between the inactivation of some 2-ODD coding genes and higher AsA content in Arabidopsis insertion mutants [95]. In this perspective, AsA is at the same time the sensor of stress conditions and the activator of downstream responses via 2-ODDs. In other words, AsA antioxidant function, rather than just a way to remove ROS, should also be considered the first step in the activation of a multi-directional signaling module.

8. If AsA is so much important, why did we lose AsA biosynthetic capability?

We are all aware that vitamin C is not only important for our health but even essential for our own survival. It cannot be fully substituted by any other antioxidant, and it has a specific role in the reactions catalyzed by several dioxygenases. With all this in mind, the loss of the AsA biosynthetic capability that occurred independently in some animal lineages is a mind-boggling paradox. Is there any possible advantage in the loss-of-function of the gene coding for the last step in AsA biosynthesis? The mutations occurring in the GULO gene [10] have been conserved in primates, but reversed in some bats and birds, suggesting that the capability to synthesize AsA is a neutral trait [96]. According to a different interpretation, the advantage of losing GULO activity is that the mechanism of the enzyme yields hydrogen peroxide, limiting the beneficial effect of AsA production [73]. An additional hypothesis is advanced, based on the informational content that AsA can bring to the mechanism of 2-ODDs, and the trade-off between stress response and lifespan [97]. As mentioned above, 2-ODDs require 2-oxoglutarate, oxygen, iron, and AsA. While the involvement of the former three co-substrates is easily associated with energy metabolism (Section 4, above), the contribution of AsA in terms of molecular information is not as clear. However, as discussed in Section 7, the peculiar reactivity of AsA, and more specifically its interaction with different oxidants, makes it the perfect proxy for evaluating general stress conditions occurring in a given cell or cell compartment. The example of the HIF1α transcription factor (Figure 2) is especially relevant and useful to understand this point. The HIF1α transcription factor is constitutively expressed, but it becomes active exclusively when the hydroxylation of its proline residues (depending of co-substrate availability) does NOT take place. Like this, the transcriptional response is very fast: HIF is already there and immediately operative. The response is needed whenever there is no oxygen, not enough energy, and the cell is under heavy stress conditions, resulting in AsA depletion. A combination of these conditions variably induces the response. A similar mechanism, in which ligand depletion activates the transcriptional response, has been recently reported in plants [98].

A mechanism in which a multiple anti-stress response is activated by the absence of selected co-substrates usually associated with the effector (transcription factor) has several advantages. The proxies corresponding to different (variable) clues integrate directly at the effector site, producing a graded response. In this model, the co-substrates set the “meters” of the response (Figure 4). Many possible intermediate conditions would lie between the two extremes reported in the scheme. However, the quick response to unfavorable conditions also brings a burden to the organism if growth is reduced, and all available resources are diverted to defense. In the case of chronic stress, and the consequent continuous activation of the defenses, the lifespan is shortened, and early senescence occurs. This has been observed, for example, in the case of plants with lower AsA content [77]. The model also accommodates the sometimes conflicting observations on the effects of antioxidants on aging and health conditions: an adequate AsA supply is not a guarantee of good health per se, because it must be integrated with other signals. This also means that the huge amount of literature showing that antioxidants have a positive effect on the health of both animals and plants, the effect invariably explained with the simplistic assumption that more antioxidants “kill” harmful ROS, is correct in the observations, but misled in the interpretation.

Figure 4.

A tentative model explaining the role of ascorbate (AsA) in putative signaling modules activating stress-responsive genes, in analogy with the HIF1α mechanism.

The hypothesis outlined above also helps in tentatively answering the overwhelming question in the title of this section: why losing AsA biosynthesis? If AsA content is critical to accurately set the “meter” of the response mediated by some regulatory 2-ODDs, “homemade” AsA could alter the baseline of the response. On the contrary, an AsA pool totally depending on the dietary intake provides full information on whether a non-synthesizing individual takes (or not) enough AsA with available food, helping in setting the proper levels of housekeeping stress responses. In years of personalized medicine, the possibility that an organism adjusts its own defenses à la carte is quite intriguing and deserves further investigation. In terms of trade-off, losing AsA biosynthetic capability might have resulted in the advantage of accurately setting the alert level for each individual.

A possible testing place for the model of AsA-dependent regulation of basal defense levels comes from the emerging field of ecological immunology. The concept that AsA improves immune defenses is universally accepted, and some studies support this claim, usually explaining the beneficial effects of AsA on the immune system with its antioxidant function [99]. Ecological immunology analyzes different aspects of the immune response, including how stress conditions differently influence immunity in different individuals [100]. The energy cost of immune responses is key to understanding why acute stress conditions can suppress part of defenses diverting energy resources to the activation of “first-line” responses [101]. The relationship between energy and stress and their integration as the driver of complex immune responses resembles the situation described in the model sketched in Figure 4.

9. Conclusions

The aim of the present contribution is to provide the reader with some hints on how to make one’s way in the labyrinth of available information on AsA. What we know for sure is that AsA can prevent and cure the disease known as scurvy, which is caused by the inactivation of some enzymes belonging to the large class of dioxygenases. The reducing properties of AsA, and its ability to react with ROS are also undeniable facts. We know that AsA is involved in the signaling module regulated by the HIF1α transcription factor. In addition, many reports confirm the beneficial effects of AsA under diverse conditions, while a few other express some doubts about its efficacy. It is often presumed that any positive effect of AsA is due to its capability to remove reactive oxygen species, but this opinion is challenged by an increasing number of reports, showing that the ROS/antioxidant dynamics are much more complex than we thought only a few decades ago. Eventually, future research on AsA should take into due consideration the possibility that AsA acts as a unique regulator of multiple cell responses, especially in relation to variable environmental conditions. This novel approach will hopefully open a new chapter in the long but still mysterious story of vitamin C.

Acknowledgments

The author gratefully acknowledges funding from the University of Bari Aldo Moro.

Notes

The illustrations reported here are original drawings created with BioRender.com.