Back to post

[1] Evidence type: review
Vitamine—vitamin. The early years of discovery
Louis Rosenfeld
Clinical Chemistry Vol. 43, Issue 4 April 1997
“In 1911, Casimir Funk isolated a concentrate from rice polishings that cured polyneuritis in pigeons. He named the concentrate “vitamine” because it appeared to be vital to life and because it was probably an amine. Although the concentrate and other “accessory food substances” were not amines, the name stuck, but the final “e” was dropped. ”

[2] Evidence type: review
The Genetics of Vitamin C Loss in Vertebrates
Drouin, Guy, Jean-Rémi Godin, and Benoît Pagé.
Current Genomics 12.5 (2011): 371–378. PMC. Web. 19 Dec. 2016.
“Vitamin C (ascorbic acid) plays important roles as an anti-oxidant and in collagen synthesis. These important roles, and the relatively large amounts of vitamin C required daily, likely explain why most vertebrate species are able to synthesize this compound. Surprisingly, many species, such as teleost fishes, anthropoid primates, guinea pigs, as well as some bat and Passeriformes bird species, have lost the capacity to synthesize it. Here, we review the genetic bases behind the repeated losses in the ability to synthesize vitamin C as well as their implications. In all cases so far studied, the inability to synthesize vitamin C is due to mutations in the L-gulono-γ-lactone oxidase (GLO) gene which codes for the enzyme responsible for catalyzing the last step of vitamin C biosynthesis. The bias for mutations in this particular gene is likely due to the fact that losing it only affects vitamin C production. Whereas the GLO gene mutations in fish, anthropoid primates and guinea pigs are irreversible, some of the GLO pseudogenes found in bat species have been shown to be reactivated during evolution. The same phenomenon is thought to have occurred in some Passeriformes bird species. Interestingly, these GLO gene losses and reactivations are unrelated to the diet of the species involved. This suggests that losing the ability to make vitamin C is a neutral trait.”

[3] Evidence type: observation
Ascorbate synthesis-dependent glutathione consumption in mouse liver
Bánhegyi Gábor,Csala Miklós,Braun László,Garzó Tamás and Mandl József
FEBS Letters, 381, doi: 10.1016/0014-5793(96)00077-4 (1996)
“Ascorbic acid and glutathione are involved in the antioxidant defense of the cell. Their connections and interactions have been described from several aspects: they can substitute each other [1], dehydroascorbate can be reduced at the expense of GSH [2] and glutathione depletion results in the stimulation of ascorbate synthesis [3]. In ascorbate-synthesising animals, the formation of ascorbate from gulonolactone catalysed by microsomal gulonolactone oxidase is accompanied by the stoichiometric consumption of O2 and production of the oxidant hydrogen peroxide [4]. Metabolism of hydrogen peroxide by glutathione peroxidase requires reduced glutathione. Therefore, we supposed that synthesis of ascorbate should decrease the intracellular glutathione level. To prove our hypothesis, experiments were undertaken to investigate the effect of ascorbate synthesis stimulated by the addition of gulonolactone on the oxidation of GSH in isolated mouse hepatocytes and liver microsomal membranes.”

“In this paper, a new connection between ascorbate and GSH metabolism is described. Our data show that the synthesis of ascorbate leads to consumption of GSH, the other main intracellular antioxidant (Fig. 1). We suppose that the formation of hydrogen peroxide is underlying the increased GSH consumption. First, oxidation of GSH caused by increased ascorbate synthesis was prevented by the addition of catalase in microsomal membranes (Table 1). Second, inhibition of glutathione peroxidase by mercaptosuccinate moderated the gulonolactone-dependent glutathione consumption in microsomes (Table 2). Third, the inhibition of catalase by aminotriazole deepened the ascorbate synthesis-dependent GSH depletion in isolated hepatocytes (Table 3). This interaction may be one of the causes why primates and some other species have lost their ascorbate-synthesising ability. This event occurred in the ancestors of primates about 70 million years ago, owing to mutation(s) in the gulonolactone oxidase gene [14]. Despite the well-known benefits [15] of ascorbate, the mutation(s) had to be advantageous, as this metabolic error did not remain an enzymopathy affecting only a minority of the population, but spread widely amongst the species (and individuals) of primates and became exclusive [16]. There is no explanation for this unexpected outcome. Based on these analytical data, the following conceptual evolutionary hypothesis can be outlined: in the tropical jungle of the Cretaceous Period, when exogenous ascorbate was abundant [17,18], the loss of gulonolactone oxidase activity could have proved to be advantageous. It saved the reduced GSH, the main defence system against oxidants, while the access to ascorbate was not hindered. Later, the evolutionary gains of these periods allowed the conservation of the genetic disorder manifested in the loss of ascorbate synthesis.”

[4] Evidence type: experiment
Vitamin C enters mitochondria via facilitative glucose transporter 1 (Glut1) and confers mitochondrial protection against oxidative injury.
KC S, Cárcamo JM, Golde DW.
FASEB J. 2005 Oct;19(12):1657-67.
Reactive oxygen species (ROS)-induced mitochondrial abnormalities may have important consequences in the pathogenesis of degenerative diseases and cancer. Vitamin C is an important antioxidant known to quench ROS, but its mitochondrial transport and functions are poorly understood. We found that the oxidized form of vitamin C, dehydroascorbic acid (DHA), enters mitochondria via facilitative glucose transporter 1 (Glut1) and accumulates mitochondrially as ascorbic acid (mtAA). The stereo-selective mitochondrial uptake of D-glucose, with its ability to inhibit mitochondrial DHA uptake, indicated the presence of mitochondrial Glut. Computational analysis of N-termini of human Glut isoforms indicated that Glut1 had the highest probability of mitochondrial localization, which was experimentally verified via mitochondrial expression of Glut1-EGFP. In vitro mitochondrial import of Glut1, immunoblot analysis of mitochondrial proteins, and cellular immunolocalization studies indicated that Glut1 localizes to mitochondria. Loading mitochondria with AA quenched mitochondrial ROS and inhibited oxidative mitochondrial DNA damage. mtAA inhibited oxidative stress resulting from rotenone-induced disruption of the mitochondrial respiratory chain and prevented mitochondrial membrane depolarization in response to a protonophore, CCCP. Our results show that analogous to the cellular uptake, vitamin C enters mitochondria in its oxidized form via Glut1 and protects mitochondria from oxidative injury. Since mitochondria contribute significantly to intracellular ROS, protection of the mitochondrial genome and membrane may have pharmacological implications against a variety of ROS-mediated disorders.

[5] Evidence type: non-human animal experiment
Adaptive regulation of ascorbic acid synthesis in rat-liver extracts. Effect of x-irradiation and of dietary changes
Stirpe F, Comporti M, Caprino G.
Biochem J. 1963 Feb;86:232-6.
“Effect of starvation and subsequent feeding. The effect of starvation was then investigated, and it appeared that a 24 hr. period of starvation was enough to decrease the synthesis of ascorbic acid (Table 2). Since Caputto et al. (1958) had shown that the maximum effect of vitamin-E deficiency on the synthesis of ascorbic acid was reached as shortly as 3-4 days after deprivation, the possibility was considered that the effect of starvation was actually due to lack of vitamin E. This was discounted by giving starved animals enough vitamin E to prevent formation of peroxides; there was no effect on the synthesis of ascorbic acid. The effect of starving was quickly reversed by feeding the rats again for 24 hr.”

“Effect of omission of carbohydrates from the diet and of administration of precursors: The effect of starvation could be attributed either to the stress or to the lack of some dietary components. A strong impairment of the synthesis of ascorbic acid was observed in rats given a carbohydrate-free diet for 24 hr., whereas values significantly higher but still below normal ones were obtained by giving this same diet for 6 days (Table 3). Rats on this ration had a lower content of ascorbic acid in the liver, but showed an enhanced excretion of ascorbic acid in the urine. Since carbohydrates are precursors of ascorbic acid in the rat, this observation led to the hypothesis of an adaptive response of the enzyme system to lack of substrates, and evidence was sought by giving glucuronolactone to rats. Administration of glucuronolactone did not affect the rate of synthesis in normal rats, but caused a moderate but significant enhancement in starved animals. However, a similar enhancement followed the administration of an equal amount of glucose. All rats receiving glucuronolactone had a higher liver content and an enhanced urinary excretion of ascorbic acid.”

[6] Evidence type: non-human animal experiment
Ascorbic acid synthesis is stimulated by enhanced glycogenolysis in murine liver
Braun L1, Garzó T, Mandl J, Bánhegyi G.
FEBS Lett. 1994 Sep 19;352(1):4-6.
“The role of the hepatic glycogen content in ascorbic acid synthesis was investigated in isolated mouse hepatocytes. The cells were prepared from fed or 48 h-starved mice and the ascorbic acid content was measured in the suspension (cells+medium). After 48 h starvation hepatocytes did not contain measurable amounts of glycogen. The initial concentration of ascorbic acid was lower in the suspension of glycogen-depleted hepatocytes compared to the fed controls (Fig. 1) and only a moderate synthesis could be observed under both nutritional conditions. The effects of dibutyryl CAMP and glucagon on ascorbate synthesis were examined. Glucagon or dibutyryl cyclic AMP caused a stimulation of ascorbic acid synthesis in hepatocytes from fed mice, while in hepatocytes from 48 h starved animals ascorbic acid production was not increased significantly by the two agents (Fig. 1). The addition of glucose and gluconeogenic precursors to the incubation medium did not result in a significant increase in ascorbic acid production (Fig. 1). In another series of experiments glucose and ascorbic acid production of the cells was measured simultaneously. The rate of glucose production (in the absence of gluconeogenic precursors mainly via glycogenolysis) and ascorbic acid synthesis showed a close correlation (r = 0.9091) (Fig. 2). As ascorbic acid synthesis and glycogenolysis seemed to be connected, we examined the effect on ascorbic acid synthesis of various agents known to increase glycogenolysis. The al agonist phenylephrine, the protein phosphatase inhibitor okadaic acid and vasopressin all increased the rate of ascorbic acid production in isolated hepatocytes prepared from fed mice similarly to glucagon (Table 1).
“Glycogenolysis was stimulated by the in vivo addition of glucagon. Glucagon elevated the blood glucose level of mice by 50%; at the same time a more than fifteenfold increase of plasma ascorbic acid concentration could be observed (Table 2). The concentration of ascorbic acid in the liver was also increased, indicating a stimulated hepatic synthesis (Table 2).”
Glycogen content is considered to be a sensitive marker showing the actual metabolic state of the liver. Observations described in this paper suggest that ascorbic acid synthesis in murine liver is tightly connected with the glycogen pool; the source of ascorbic acid is glycogen. The following results gained in isolated hepatocytes support this assumption: first, in hepatocytes isolated from glycogen-depleted animals the ascorbic acid level as well as the rate of synthesis is lower than that in hepatocytes from control fed mice (Fig. 1); second, different glycogen-mobilizing agents acting via different mechanisms enhance ascorbic acid production in hepatocytes from fed but not from fasted animals (Fig. 1, Table 1); third, addition of glucose to hepatocytes prepared from glycogen-depleted mice failed to increase the formation of ascorbic acid (Fig. 1). The results gained under in vitro conditions in isolated hepatocytes were confirmed by in vivo experiments: a single i.p. injection of glucagon elevated both the plasma and liver ascorbic acid levels within 15 min (Table 2). ”

“The finding that the source of ascorbate production is glycogenolysis is in according with the fact that liver and kidney -the main sites of glycogen storage – are responsible for the ascorbic acid supply in most animal species [2]. The increased hepatic ascorbic acid production after glucagon administration can be explained as a compensatory mechanism of the missing intake of ascorbate, i.e. adaptation of ascorbic acid supply from external to internal sources. Considering the fifteenfold elevation of plasma ascorbate levels, in the light of recent findings concerning the effect of ascorbate on insulin secretion [18] and on the calcium channels in pancreatic beta cells [19] it might be also regarded as a possible intercellular messenger. ”

[7] Evidence type: experiment
Role of the antioxidant ascorbate in hibernation and warming from hibernation.
Drew KL, Tøien Ø, Rivera PM, Smith MA, Perry G, Rice ME.
Comp Biochem Physiol C Toxicol Pharmacol. 2002 Dec;133(4):483-92.
“During hibernation plasma ascorbate concentrations w(Asc)px were found to increase 3–5 fold in two species of ground squirrels, AGS and 13-lined ground squirrels (TLS); S. tridecemlineatus and cerebral spinal fluid (CSF) ascorbate concentration w(Asc)CSFx doubled in AGS (CSF was not sampled in TLS) (Drew et al., 1999). During arousal, however, when oxygen consumption peaks and the generation of reactive oxygen species is thought to be maximal, plasma ascorbate concentrations progressively decrease to levels typical for euthermic animals (Fig. 3).”

[8] Evidence type: observation
The membrane Transport of ascorbic acid
George Mann and Pamela Newton
Ann N Y Acad Sci. 1975 Sep 30;258:243-52.
“We have formulated two hypotheses. The first proposes that the transport of ascorbate across cell membranes may be impaired by glucose. The second proposes that the transport of ascorbate in certain tissues is facilitated by insulin. If either hypothesis is valid, those species requiring exogenous ascorbate would be in double jeopardy if they were also hyperglycemic. Carbohydrate intolerance resulting from either a lack of or a resistance to insulin is common in Western man. Gore et al. have shown with electron microscopy that the vascular lesion of scurvy involves collagenous structures in the basement membranes, and this is also the site of the lesion in diabetic microangiopathy. These hypotheses, which propose that the intracellular availability of dehydroascorbate (DHA), the transportable form of vitamin C, would be impaired in certain tissues by either hyperglycemia or lack of insulin, suggest that diabetic microangiopathy, the main complication of human diabetes, may be a consequence of local ascorbate deficiency. The laboratory investigations described here deal with the first and somewhat simpler of these hypotheses: Glucose will impair the transport of dehydroascorbate into cells. The data collected show that D-glucose does inhibit the transport of dehydroascorbate into human red blood cells, a noninsulin-dependent tissue. Trials with other sugars show a hierarchy of sugars that inhibit transport, suggesting that DHA and D-glucose share a carrier mechanism.”

[9] Evidence type: review
Hyperglycemia-induced ascorbic acid deficiency promotes endothelial dysfunction and the development of atherosclerosis
Price KD, Price CS, Reynolds RD.
Atherosclerosis. 2001 Sep;158(1):1-12.
“Hyperglycemia-induced ascorbic acid deficiency
Vitamin C is a derivative of glucose and Mann [138] proposed that the structural similarity between these two molecules may account for many of the complications of diabetes. Glucose has been shown to inhibit vitamin C transport in several mesenchymal cell types, including endothelial cells [139], mononuclear leukocytes [140], neutrophils [141,142], fibroblasts [143,144], and erythrocytes [145]. Facilitative glucose transporters (GLUTs) bind dehydroascorbic acid and are thought to be the primary transporters of vitamin C in mammalian cells [146]. After transport, dehydroascorbic acid is quickly reduced to ascorbic acid. Glucose competitively inhibits the uptake of dehydroascorbic acid but does not affect ascorbic acid transport. Ascorbic acid is transported by a family of membrane-bound proteins that are Na+-dependent and whose function is not directly inhibited by elevated extracellular concentrations of glucose [146,147]. This latter system is prevalent in bulk-transporting epithelia (e.g. kidney and small intestine) and have been recently isolated in both human [148] and rat [149] biological systems. Many cell types, of course, [150,151] express both transport systems.
High blood glucose concentrations mimic the conditions of vitamin C deficiency. Acute hyperglycemia, for example, impairs endothelium-dependent vasodilation in healthy humans [152], an effect which can be reversed by acute administration of vitamin C [153]. Ascorbic acid plays an important role in extracellular matrix regulation and has a stimulatory effect on sulfate incorporation in mesangial cell and matrix proteoglycans; high glucose concentrations have been shown to impair this effect [154]. Endothelial surface proteoglycans help prevent thrombus formation and also inhibit smooth-muscle growth [1]. High glucose concentrations also have been shown to inhibit the stimulatory effect of ascorbic acid on collagen and proteoglycan synthesis in cultured fibroblasts [114]. Moreover, a high concentration of glucose can induce the expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells [155]. Endothelial cells express these and other membrane-bound proteins to enable leukocyte adhesion and transmigration across the endothelium during an inflammatory response. Atherosclerosis is one such inflammatory response.
Experimental and clinical studies suggest that latent scurvy is characterized by IGT [16,24] and diabetes mellitus is a disease complex characterized by impaired glucose and vitamin C metabolism [27,28]. Diabetic patients are prone to hyperglycemia, prolonged wound healing, infection, increased synthesis of cholesterol, decreased liver glycogen, and notably, diffuse vascular disease. All of these findings are consistent with latent scurvy [16]. Diabetic platelets have been shown to have low intracellular ascorbic acid concentrations and display hypercoagulability [156]. Long-term vitamin C administration has beneficial effects on glucose and lipid metabolism in aged NIDDM patients [157]. It has also been suggested that vitamin C consumption above the RDA may provide important health benefits for individuals with IDDM [158]. This latter recommendation is supported by recent evidence. For example, mesenchymal cells from patients with IDDM have an impaired uptake of dehydroascorbic acid that persists in culture [159] and ascorbic acid has been shown to prevent the inhibition of DNA synthesis induced by high glucose concentrations in cultured endothelial cells [160]. Diabetic patients have been observed to have a lowered ascorbic acid/dehydroascorbic acid plasma ratio, indicating a decreased vitamin C status [161]. Therefore, diabetic patients may benefit from vitamin C supplementation to alleviate multiple physiologic and metabolic impairments in a variety of cell types.”

[10] Evidence type: review
Vitamin C as an antioxidant: evaluation of its role in disease prevention
Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta A, Dutta SK, Levine M.
J Am Coll Nutr. 2003 Feb;22(1):18-35.
“Problems in Demonstrating Antioxidant Benefit of Vitamin C in Clinical
“Studies Despite epidemiological and some experimental studies, it has not been possible to show conclusively that higher than anti-scorbutic intake of vitamin C has antioxidant clinical benefit. This is despite the fact that vitamin C is a powerful antioxidant in vitro. It is of course possible that the lack of antioxidant effect of vitamin C in clinical studies is real. It seems more likely that vitamin C has antioxidant or other benefits. Detection of these benefits has remained elusive due to the vicissitudes of experimental design. Vitamin C may be a weak antioxidant in vivo, or its antioxidant actions may have no physiological role, or its role may be small. The oxidative hypothesis is unproven, and oxidative damage may have a smaller role than anticipated in some diseases. Further, antioxidant actions of vitamin C may occur at relatively low plasma vitamin C concentrations. Thus additional clinical benefits that occur at higher vitamin C concentrations may be difficult to demonstrate. Although all these are possible explanations, it seems unlikely that these are the real reasons for the lack of detectable effects of vitamin C in clinical studies. Many factors may contribute to the failure so far to demonstrate clear antioxidant benefits of vitamin C in clinical studies. The antioxidant actions of vitamin C may be specific to certain reactions or occur only at specific locations. In either case, beneficial effects can be shown only in disorders where such reactions or sites are the focus of disease process. There may be many different antioxidants that are active at the same time. In the face of such redundancy, only multiple antioxidant deficiencies will have detectable clinical effects. Antioxidant deficiency may have to be of long duration for accumulated damage to be noticeable. Antioxidant effects may be of importance only in those with oxidant stress. Thus, normal subjects or those with mild disease may have no need for high antioxidant concentrations. In a way analogous to the effect of acetaminophen on fever, antioxidants may have no effect in the absence of marked oxidant stress. A further problem is presented by the sigmoidal dose concentration curve for vitamin C. Small changes in oral intake of vitamin C produce large changes in plasma vitamin C concentrations. This makes it difficult to conduct controlled studies such that the plasma vitamin C concentrations of the control and study groups differ sufficiently to have physiological meaning.”

[11] Evidence type: review
Vitamin C: the known and the unknown and Goldilocks
Padayatty SJ, Levine M
Oral Dis. 2016 Sep;22(6):463-93. doi: 10.1111/odi.12446. Epub 2016 Apr 14.
(Emphasis mine)
“Collagen hydroxylation
“Common symptoms of scurvy include wound dehiscence, poor wound healing and loosening of teeth, all pointing to defects in connective tissue (Crandon et al, 1940; Lind, 1953; Hirschmann and Raugi, 1999). Collagen provides connective tissue with structural strength. Vitamin C catalyzes enzymatic (Peterkofsky, 1991) posttranslational modification of procollagen to produce and secrete adequate amounts of structurally normal collagen by collagen producing cells (Kivirikko and Myllyla, 1985; Prockop and Kivirikko, 1995). Precollagen, synthesized in the endoplasmic reticulum, consists of amino acid repeats rich in proline. Specific prolyl and lysyl residues are hydroxylated, proline is converted to either 3-hydroxyproline or 4-hydroxyproline, and lysine is converted to hydroxylysine. The reactions catalyzed by prolyl 3-hydroxylase, prolyl 4- hydroxylase, and lysyl hydroxylase (Peterkofsky, 1991; Prockop and Kivirikko, 1995; Pekkala et al, 2003) require vitamin C as a cofactor. Hydroxylation aids in the formation of the stable triple helical structure of collagen, which is transported to the Golgi apparatus and eventually secreted by secretory granules. In the absence of hydroxylation, secretion of procollagen decreases (Peterkofsky, 1991) and it probably undergoes faster degradation. However, some hydroxylation can occur even in the absence of vitamin C (Parsons et al, 2006). Secreted procollagen is enzymatically cleaved to form tropocollagen that spontaneously forms collagen fibrils in the extracellular space. These fibrils form intermolecular collagen cross-links, giving collagen its structural strength. Independent of its effects on hydroxylation, ascorbate may stimulate collagen synthesis (Geesin et al, 1988; Sullivan et al, 1994). Collagen synthesis may be decreased in scorbutic animals (Peterkofsky, 1991; Kipp et al, 1996; Tsuchiya and Bates, 2003). Reduced collagen cross-links may be a marker of vitamin C deficiency in the guinea pig (Tsuchiya and Bates, 2003) but this may not be specific to vitamin C deficiency. Although many features of human scurvy appear to be due to weakening of connective tissue, it has not been shown that these lesions are due to defective collagen synthesis.”

[12] Evidence type: non-human animal experiment
Glutathione ester delays the onset of scurvy in ascorbate-deficient guinea pigs
J Mårtensson, J Han, O W Griffith, and A Meister
Proc Natl Acad Sci U S A. 1993 Jan 1; 90(1): 317–321.
“Guinea pigs given an ascorbate-deficient diet gained weight through day 14, but gained at a slower rate than the control animals,and then lost weight (Table 1, group A).The animals given GSH ester (group B)gained more weight than those of group A, and the weight gain during days 10-14 was =70% of the control group. Animals in group A became obviously sick after about day 17. They could not walk and moved very little, apparently immobilized by fractures of the hind legs and by swelling of the joints of the extremities, which were tender and had periosteal hematomas. Radiography showed major fractures of the femur in two animals. Animals in group A died or were sacrificed on day 21 or 22. Animals in group B (GSH ester)did not have fractures or hematomas; 75% of these animals were indistinguishable by general appearance from controls. Histological study showed significant loss of osteoid material from long bones in group A,whereas most animals in group B had no decrease of osteoid material (Fig.1)or only a moderate decrease. In a separate experiment, several animals comparable to those of group B were kept for 40 days and showed no significant signs of scurvy (tender swollen joints,fractures);they exhibited some weight loss.”

[13] Evidence type: in vitro experiment
GSH is required to recycle ascorbic acid in cultured liver cell lines
Li X, Qu ZC, May JM.
Antioxid Redox Signal. 2001 Dec;3(6):1089-97.
“Liver is the site of ascorbic acid synthesis in most mammals. As human liver cannot synthesize ascorbate de novo, it may differ from liver of other species in the capacity or mechanism for ascorbate recycling from its oxidized forms. Therefore, we compared the ability of cultured liver-derived cells from humans (HepG2 cells) and rats (H4IIE cells) to take up and reduce dehydroascorbic acid (DHA) to ascorbate. Neither cell type contained appreciable amounts of ascorbate in culture, but both rapidly took up and reduced DHA to ascorbate. Intracellular ascorbate accumulated to concentrations of 10-20 mM following loading with DHA. The capacity of HepG2 cells to take up and reduce DHA to ascorbate was more than twice that of H4IIE cells. In both cell types, DHA reduction lowered glutathione (GSH) concentrations and was inhibited by prior depletion of GSH with diethyl maleate, buthionine sulfoximine, and phenylarsine oxide. NADPH-dependent DHA reduction due to thioredoxin reductase occurred in overnight-dialyzed extracts of both cell types. These results show that cells derived from rat liver synthesize little ascorbate in culture, that cultured human-derived liver cells have a greater capacity for DHA reduction than do rat-derived liver cells, but that both cell types rely largely on GSH- or NADPH-dependent mechanisms for ascorbate recycling from DHA.”

[14] Evidence type: non-human animal experiment
The ketogenic diet increases mitochondrial glutathione levels
Jarrett SG, Milder JB, Liang LP, Patel M.
J Neurochem. 2008 Aug;106(3):1044-51. doi: 10.1111/j.1471-4159.2008.05460.x. Epub 2008 May 5.
“The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague-Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of beta-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H2O2 production and H2O2-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H2O2 production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.”

[15] Evidence type: non-human animal experiment
Acute oxidative stress and systemic Nrf2 activation by the ketogenic diet
Milder JB, Liang LP, Patel M.
Neurobiol Dis. 2010 Oct;40(1):238-44. doi: 10.1016/j.nbd.2010.05.030. Epub 2010 May 31.
“The mechanisms underlying the efficacy of the ketogenic diet (KD) remain unknown. Recently, we showed that the KD increased glutathione (GSH) biosynthesis. Since the NF E2-related factor 2 (Nrf2) transcription factor is a primary responder to cellular stress and can upregulate GSH biosynthesis, we asked whether the KD activates the Nrf2 pathway. Here we report that rats consuming a KD show acute production of H(2)O(2) from hippocampal mitochondria, which decreases below control levels by 3 weeks, suggestive of an adaptive response. 4-Hydroxy-2-nonenal (4-HNE), an electrophilic lipid peroxidation end product known to activate the Nrf2 detoxification pathway, was also acutely increased by the KD. Nrf2 nuclear accumulation was evident in both the hippocampus and liver, and the Nrf2 target, NAD(P)H:quinone oxidoreductase (NQO1), exhibited increased activity in both the hippocampus and liver after 3 weeks. We also found chronic depletion of liver tissue GSH, while liver mitochondrial antioxidant capacity was preserved. These data suggest that the KD initially produces mild oxidative and electrophilic stress, which may systemically activate the Nrf2 pathway via redox signaling, leading to chronic cellular adaptation, induction of protective proteins, and improvement of the mitochondrial redox state.”

[16] Evidence type: review
Glutathione-ascorbic acid antioxidant system in animals
Meister A
J Biol Chem. 1994 Apr 1;269(13):9397-400.
“Ascorbate and GSH have actions in common and can spare each other under appropriate experimental conditions; this redundancy reflects the metabolic importance of such antioxidant activity.
[Sorry, this paper is hard to quote. It’s free. Go look. :-)]

[17] Evidence type: review
Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: A hypothesis
Bruce N. Ames, Richard Cathcart, Elizabeth Schwiers, and Paul Hochstein
Proc. NatL Acad. Sci. USA Vol. 78, No. 11, pp. 6858-6862, November 1981 Biochemistry
“During primate evolution, a major factor in lengthening life-span and decreasing age-specific cancer rates may have been improved protective mechanisms against oxygen radicals. We propose that one of these protective systems is plasma uric acid, the level of which increased markedly during primate evolution as a consequence of a series of mutations. Uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. We show that, at physiological concentrations, urate reduces the oxo-heme oxidant formed by peroxide reaction with hemoglobin, protects erythrocyte ghosts against lipid peroxidation, and protects erythrocytes from peroxidative damage leading to lysis. Urate is about as effective an antioxidant as ascorbate in these experiments. Urate is much more easily oxidized than deoxynucleosides by singlet oxygen and is destroyed by hydroxyl radicals at a comparable rate. The plasma urate level in humans (about 300 ILM) is considerably higher than the ascorbate level, making it one of the major antioxidants in humans. Previous work on urate reported in the literature supports our experiments and interpretations, although the findings were not discussed in a physiological context.”

[18] Evidence type: review
Uric acid and oxidative stress.
Glantzounis GK, Tsimoyiannis EC, Kappas AM, Galaris DA.
Curr Pharm Des. 2005;11(32):4145-51.
“It has been proposed that UA may represent one of the most important low-molecular-mass antioxidants in the human biological fluids [23-26]. Ames et al. proposed in the early eighties that UA can have biological significance as an antioxidant and showed, by in vitro experiments, that it is a powerful scavenger of peroxyl radicals (RO2.), hydroxyl radicals (.OH) and singlet oxygen [23]. The authors speculated that UA may contribute to increased life-span in humans by providing protection against oxidative stress-provoked ageing and cancer. UA is an oxidizable substrate for haem protein/H2O2 systems and is able to protect against oxidative damage by acting as an electron donor [27]. Apart from its action as radical scavenger, UA can also chelate metal ions, like iron and copper, converting them to poorly reactive forms unable to catalyse free-radical reactions [28-30].”
[…] “A randomized placebo-controlled double-blind study has evaluated the effects of systemic administration of 100 mg UA, in healthy volunteers compared with vitamin C 1000 mg [45]. A significant increase in serum free radical scavenging capacity from baseline was observed during UA and vitamin C infusion – but the effect of UA was substantially greater. No adverse reactions to UA administration were reported. Another clinical study indicated a significant inverse relationship between serum UA concentrations and oxidative stress during acute aerobic exercise [46], while an increase in muscle allantoin levels was detected [32]. The authors concluded that ROS are formed in human skeletal muscle during intense sub-maximal exercise and urate is used as a local antioxidant. Another clinical trial involving healthy young men showed that 50 and 80 km marches led to 25 % and 37 % rises, respectively, in plasma levels of UA, probably due to increases in the metabolic rate and consequently pyrimidine nucleotide metabolism [47]. A randomized double-blind placebo controlled crossover study evaluated the free radical properties of UA in healthy volunteers [48]. UA (0.5 g in 250 ml of 0.1 % lithium carbonate / 4 % dextrose vehicle or vehicle alone as control) was given to subjects who then performed high intensity aerobic exercise for 20 min to induce oxidative stress. A single high-intensity exercise caused oxidative stress (as reflected by increased plasma F2- isoprostanes) immediately after exercise and recovery. Administration of UA increased circulating UA concentrations, which increased serum free radical scavenging capacity and reduced the exercise-induced increases in plasma F2-isoprostanes. The authors concluded that the antioxidant properties of UA are of physiological consequence and support the view that UA has potentially important free radical scavenging effects in vivo.”

[19] Evidence type: observational
Urate and ascorbate: their possible roles as antioxidants in determining longevity of mammalian species.
Cutler RG
Arch Gerontol Geriatr. 1984 Dec;3(4):321-48.
“Urate has been shown to be a major antioxidant in human serum and was postulated to have a biological role in protecting tissues against the toxic effects of oxygen radicals and in determining the longevity of primates. This possibility has been tested by determining if the maximum lifespan potentials of 22 primate and 17 non-primate mammalian species are positively correlated with the concentration of urate in serum and brain per specific metabolic rate. This analysis is based on the concept that the degree of protection a tissue has against oxygen radicals is proportional to antioxidant concentration per rate of oxygen metabolism of that tissue. Ascorbate, another potentially important antioxidant in determining longevity of mammalian species, was also investigated using this method. The results show a highly significant positive correlation of maximum lifespan potential with the concentration of urate in serum and brain per specific metabolic rate. No significant correlation was found for ascorbate. These results support the hypothesis that urate is biologically active as an antioxidant and is involved in determining the longevity of primate species, particularly for humans and the great apes. Ascorbate appears to have played little or no role as a longevity determinant in mammalian species.”

[20] Evidence Type: review
Glucose Hysteresis as a Mechanism in Dietary Restriction, Aging and Disease
Charles V. Mobbs, Jason Mastaitis, Minhua Zhang, Fumiko Isoda, Hui Cheng, and Kelvin Yen
Interdiscip Top Gerontol. 2007; 35: 39–68.

(emphasis mine)

Glucose Oxidation Favors Complex I, Lipid/Amino Acid Oxidation Favors Complex II

“The significance of the shift in source of carbon atoms for oxidation produced by dietary restriction may be that the oxidation of lipids and amino acids depends much more on mitochondrial complex II than on (free-radical generating) complex I, whereas glucose oxidation depends much more on complex I than on complex II. When glucose is broken down by glycolysis, the only reducing equivalents it makes are in the form of NADH. When the final carbon product of glucose, pyruvate, is metabolized in the Krebs cycle, almost all the reducing equivalents are produced in the form of NADH, except for one step at complex II (succinate dehydrogenase) that makes (then oxidizes) FADH2. Ultimately the metabolism of one molecule of glucose produces an NADH: FADH2 ratio of 5:1 [53, p. 20]. In contrast, when lipids are broken down by β-oxidation (fatty acid counterpart to glycolysis), an equal number of NADH and FADH2 molecules are formed. When the lipid-derived carbons are metabolized in the Krebs cycle, reducing equivalents are produced in the ratio of 3 NADH molecules per FADH2 molecule. Therefore ultimately lipid metabolism yields an NADH:FADH2 ratio of about 2:1 [53, p. 38] or even more if the fatty acid contains enough carbon atoms. For example, when one molecule of palmitate is oxidized, it produces 15 molecules of FADH2 and 31 molecules of NADH, which are ultimately oxidized to produce a net total of 129 ATP molecules. In contrast, production of the same number of ATP molecules from glucose would entail producing then oxidizing 8.66 FADH2 and 43.3 NADH molecules. Amino acid oxidation also proceeds by a similar 2-step mechanism yielding an NADH:FADH2 ratio between that of lipids and that of glucose, the precise number depending on the specific amino acid. The significance of this shift in the NADH:FADH2 ratio is that NADH is oxidized only at mitochondrial complex I, whereas FADH2 is oxidized only at complex II [53, p. 17]. Thus palmitate oxidation entails utilizing complex II at roughly twice the (FADH2-dependent) rate as glucose oxidation entails. Therefore shifting away from glucose utilization toward lipid and amino acid utilization would be expected to substantially reduce the production of reactive oxygen species, without necessarily reducing ATP production. As described below, other beneficial effects also occur as a result of this altered pattern of glucose fuel use, including a shift toward producing antioxidizing NADPH and increased protein and lipid turnover, which reduces the accumulation of oxidized protein and lipids.”

[21] Evidence type: experiment
Ketogenic diet decreases oxidative stress and improves mitochondrial respiratory complex activity.
Greco T, Glenn TC, Hovda DA, Prins ML
J Cereb Blood Flow Metab. 2016 Sep;36(9):1603-13
“Mechanisms of ketogenic improvement
“As mentioned previously, it is thought that much of the KD’s improvement in cellular metabolism and neuroprotection is through its ability to act as an alternative substrate. Here, we show rather that it first acts in an antioxidant manner to reverse mitochondrial dysfunction. Both Complex I and II–III are inhibited in CCISTD mice at 6 h post-injury. Increased production of lactate is a reflection of impairment of oxidative phosphorylation as well as an attempt to maintain ATP concentrations and cellular membrane potential through increased glycolytic output.13 While Complex I activity returns to sham levels by 24 h, Complex II–III activity remains inhibited. ONOO has been shown to not only inhibit Complex II–III, but also Complex V40 and suggests that the observed decline in ATP production in PND35 animals [13] is due in part to impaired Complex III and/or V activity. In addition to inhibition of mitochondrial complexes, decomposition products of ONOO increase the amount of lipid peroxidation leading to thiol linkages and pore formation in the inner membrane ultimately uncoupling the mitochondria. Although Complex I activity is inhibited in CCI-KD animals, Complex II–III activity is not. This will continue to allow electron flow through the respiratory chain and production of ATP. KD not only has antioxidant properties, but may provide substrates beyond Acetyl-CoA. The reaction of AcAc with Succinyl-CoA produces succinate, and animals either fed a KD or infused with ßOHB show a significant increase in succinate concentrations [41,42]. Other groups have also shown that KD increases Complex II activity (succinate dehydrogenase activity) [43]. By increasing Complex II activity and its substrate, KD is able to maintain mitochondrial membrane potential and ATP production and prevent bioenergetic failure. At 24 h post-injury, KD is likely to exert its effects through three mechanisms: (1) continued direct and indirect ROS/RNS scavenging, (2) increased Complex II activity and (3) increased acetyl-CoA and succinate.”

[22] Evidence type: experiment
[I cannot access the original experiment, but it is referred to here in the documents used by the RDA] Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids
The National Academies Press. 2000
“Overall, while evidence suggests that vitamin C deficiency is linked to some aspects of periodontal disease, the relationship of vitamin C intake to periodontal health in the population at large is unclear. Beyond the amount needed to prevent scorbutic gingivitis (less than 10 mg/day) (Baker et al., 1971), the results from current studies are not sufficient to reliably estimate the vitamin C requirement for apparently healthy individuals based on oral health endpoints.”
Baker EM, Hodges RE, Hood J, Sauberlich HE, March SC, Canham JE. 1971. Metabolism of 14C- and 3H-labeled L-ascorbic acid in human scurvy. Am J Clin Nutr 24:444–454.

[23] Evidence type: observation
Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans.
Carr AC, Frei B.
Am J Clin Nutr. 1999 Jun;69(6):1086-107.
The current recommended dietary allowance (RDA) for vitamin C for adult nonsmoking men and women is 60 mg/d, which is based on a mean requirement of 46 mg/d to prevent the deficiency disease scurvy. However, recent scientific evidence indicates that an increased intake of vitamin C is associated with a reduced risk of chronic diseases such as cancer, cardiovascular disease, and cataract, probably through antioxidant mechanisms. It is likely that the amount of vitamin C required to prevent scurvy is not sufficient to optimally protect against these diseases. Because the RDA is defined as “the average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all healthy individuals in a group,” it is appropriate to reevaluate the RDA for vitamin C. Therefore, we reviewed the biochemical, clinical, and epidemiologic evidence to date for a role of vitamin C in chronic disease prevention. The totality of the reviewed data suggests that an intake of 90-100 mg vitamin C/d is required for optimum reduction of chronic disease risk in nonsmoking men and women. This amount is about twice the amount on which the current RDA for vitamin C is based, suggesting a new RDA of 120 mg vitamin C/d.