7

Vitamin C: From Molecular Actions to Optimum Intake

Sebastian J. Padayatty, Rushad Daruwala, Yaohui Wang, Peter K. Eck, Jian Song, Woo S. Koh, and Mark Levine
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

I. A.

DEFICIENCY OF VITAMIN C Introduction

Vitamin C (ascorbic acid, ascorbate) is a water-soluble vitamin found widely in plants. Deciency results in scurvy, a disease with an insidious onset, but fatal results. Vitamin C deciency is now uncommon, although this was not always true. Scurvy was widespread until recent times, especially in northern latitudes whenever fruits and vegetables were scarce, and was endemic in many parts of Europe. More dramatic was its widespread occurrence whenever small or large bodies of men depended on stored rations, whether during military campaigns or ocean voyages. Large-scale fatalities from scurvy were often the limiting factor in these expeditions. It was so common during sea voyages that scurvy came to be regarded as the dread of sailors. After many false leads, it became clear that consumption of fruits and vegetables could prevent and cure this disease. However, preventive measures were only tfully adopted by the merchant ships and navies. The widespread provision of antiscorbutic food, particularly citrus fruits, eventually eradicated this disease among sailors. Even so, scurvy was widespread among troops as recently as World War I. In fact, this was the impetus responsible for the studies that led to the identication of the antiscorbutic principle.

B.

Scurvy and the Discovery of Ascorbic Acid

The earliest recorded descriptions of scurvy are probably bleeding from the gums and skin, described in Egyptian hieroglyphs circa 3000 bc. Hippocrates described the disease in 500 bc (1). Military campaigns from the Crusades to the Napoleonic wars, the American Civil War, and even World War I, were stymied by widespread and often fatal scurvy among troops. The explorer Robert Scott and his companions suffered from scurvy on the way back from the

Copyright 2002 by Taylor & Francis Group, LLC

South Pole and probably perished from it. Most sea voyages until recent times fell victim to it, despite early experience showing that it could be prevented by simple measures. For example, in 1600, 105 out of a total crew of 424 on the ships of the East India Company died of scurvy on the way to the Cape of Good Hope. However, none aboard the commanders ship died. He carried with him bottled lime juice and gave three teaspoons of it to any sailor with signs of scurvy. Convincing evidence that fresh fruits and vegetables could prevent scurvy was gradually accepted after James Lind published his Treatise on Scurvy, in 1753 (2). Even then, scurvy was thought to be caused by many factors, including cold climate, dampness, lack of fresh air, foggy weather, and other unhealthy climatic or living conditions, in addition to the lack of fruits and vegetables. In the late 18th century, the Royal Navy made it mandatory to issue 1 oz. of lemon juice daily to every sailor after 2 weeks at sea, but this was enforced only in 1804. The term limey (for a British sailor) comes from the obligatory provision of lemon (after lime) juice to all sailors of the Royal Navy. Sailors in the merchant navies continued to suffer from scurvy for years later. Following the outbreak of scurvy during World War I, it was shown that germinating, but not dry, cereals and legumes were effective against scurvy in monkeys and guinea pigs. In 1928, Albert Szent-Gyorgyi isolated a six-carbon reducing substance from ox adrenals (3), oranges, and cabbages. In 1932, he (4) and C. C. King (5) showed this substance to be the antiscorbutic principle. Albert Szent-Gyorgyi named it ascorbic acid and was awarded the Nobel Prize in 1937.

C.

Symptoms and Signs of Scurvy

Although vitamin C is concentrated in many tissues, these tissue stores are easily depleted. Lind reported the onset of the disease in sailors after 1 1/2 month at sea (2). The early symptoms are weakness, fatigue, listlessness, and lassitude. These were noted by Lind and conrmed others (6,7). Physical signs follow: these include perifollicular hyperkeratosis; erythema and purpura; bleeding into the skin, subcutaneous tissues, muscles, and joints; breakdown of wounds; swollen and friable gums; fever; and confusion. Untreated, scurvy is fatal. Although this disease is now rare, subclinical vitamin C deciency may be common (8), especially because the rst symptoms of deciency are unremarkable and nonspecic. The more serious manifestations of scurvy, although rarely seen in clinical practice, serve to remind us to focus research on areas where vitamin C may have important roles. The only proved function of vitamin C is the prevention of scurvy. Evidence suggests vitamin C may have many other functions in the body, albeit at concentrations higher than that required to prevent scurvy. However, whether vitamin C confers clear health benets other than to prevent scurvy remains contentious. New data describing vitamin C absorption, bioavailability, doseconcentration relation, urinary excretion, cellular transport, tissue accumulation, and recycling throw light on the possible roles of this vitamin in human physiology and pathology.

II. A.

PROPERTIES AND FUNCTIONS OF VITAMIN C Ascorbic Acid as a Vitamin

Vitamin C (ascorbic acid, ascorbate) is a six-carbon lactone. Most animals synthesize it from glucose in the liver (mammals) or kidneys (birds and reptiles). Several species of animals, scattered throughout the evolutionary tree, are unable to synthesize vitamin C. These include human and nonhuman primates, guinea pigs, Indian fruit bats, bulbuls, and some sh. Primates (9) and guinea pigs (10) lack the terminal enzyme in the biosynthetic pathway, gulonolactone

Copyright 2002 by Taylor & Francis Group, LLC

oxidase. In the human, the gene encoding this enzyme has extensive mutations, so that there is no protein product (11). For humans, the inability to synthesize ascorbic acid makes this otherwise ubiquitous chemical a vitamin. Other animals unable to synthesize vitamin C usually obtain sufcient amounts from their largely plant diet but, similar to humans, will rapidly develop scurvy when fed on processed diets in captivity (12). Vitamin C is synthesized by plants from several precursors and is abundant in leaves and, in particular, the chloroplast (13). It may play a role in photosynthesis, stress resistance, and plant growth and development (14).

B.

Ascorbate Is an Electron Donor in Chemical Reactions

Ascorbate is an electron donor, and this property accounts for its known and postulated functions. As an antioxidant or reducing agent, it sequentially donates two electrons from the C2C3 double bond, forming the intermediate free radical semidehydroascorbic acid (ascorbate free radical) (Fig. 1). The ascorbate free radical is unstable (105 s), but is relatively unreactive with other compounds to form potentially harmful free radicals, and can be reversibly reduced to ascorbate (15). These properties make ascorbate an ideal electron donor. Semidehydroascorbic acid, being unstable, undergoes further oxidation to form the more stable product, dehydroascorbic acid (DHA), which can exist in more than one structural form (see

Figure 1

Ascorbic acid metabolism. (From Ref. 136.)

Copyright 2002 by Taylor & Francis Group, LLC

Fig. 1), but only a few minutes at physiological pH. DHA can be reduced back to ascorbate by glutathione, with formation of glutathione disulde (16,17) or by enzymatic reduction mediated by at least three distinct proteins. If not reduced, DHA undergoes ring rupture and is irreversibly hydrolyzed to 2,3-diketogulonic acid. Diketogulonic acid is metabolized to xylose, xylonate, lyxonate, and oxalate, the last being a clinically signicant end product of ascorbate metabolism. Although carbons from ascorbate contribute to expired carbon dioxide in some animals, this probably does not occur in humans (18,19). Molecular oxygen, with or without trace metals (iron, copper), superoxide, hydroxyl radical, and hypochlorous acid, all can oxidize ascorbic acid to DHA in biological systems.

C.

Ascorbate Is a Cofactor for Enzymes

Ascorbate serves as a cofactor for eight different enzymes in mammals, and an additional three in yeast (Table 1) (20,21). It is assumed that scurvy is a result of impairment of these enzyme actions. Thus, the many signs related to wound dehiscence and friable gums may reect impaired collagen synthesis. However, there is no experimental evidence that directly link the signs and symptoms of scurvy with specic enzyme actions.

D.

Nonenzymatic Functions of Vitamin C

Vitamin C may have nonenzymatic functions, owing to its redox potential and free radical intermediate, and may be an electron donor in many intracellular and extracellular reactions (see Table 1). Intracellularly, vitamin C might act as an antioxidant to regulate gene expression, regulate mRNA translation, or prevent oxidant damage to intracellular proteins (37,38). Extracellular vitamin C might also be protective against oxidants and oxidant-mediated damage. Many studies have described that vitamin C prevents low-density lipoprotein (LDL) oxidation in vitro (41,42). Although high LDL is a risk factor for atherosclerosis, it is atherogenic only when oxidized. It is possible that antioxidants inhibit LDL oxidation. In vitro, vitamin C inhibits metal-catalyzed LDL oxidation, possibly by quenching aqueous free radicals. Ascorbic acid protects LDL from oxidation at concentrations above 50 M/L (43,44). Another potential protective mechanism is indirect, as vitamin C can regenerate oxidized -tocopherol (vitamin E) in LDL. Whether vitamin C has these effects in vivo is unknown. Although cells in vessel walls may be affected by vitamin C in vivo, its action may be independent of its effect on metal-catalyzed LDL oxidation in vitro. This is because the high metal concentrations and the relatively long times needed to induce oxidation in vitro are unlikely to occur in the intact organism, especially as the relevant cations (iron, copper) in vivo are tightly bound to proteins. Furthermore, the oxidant itself may not be present at sufcient concentrations for these reactions to occur clinically. Additional effects of extracellular vitamin C in atherosclerosis could be due to its effects on adhesion of monocytes to endothelium or aggregation of platelets and leukocytes (45). Vitamin C may quench oxidants that leak from activated neutrophils or macrophages (46) that, in turn, may damage supporting tissues, such as collagen or surrounding broblasts (47). Although laboratory data show a protective role for vitamin C in atherosclerotic heart disease, epidemiological data are inconsistent (4850). Vitamin C may be the primary antioxidant in plasma for quenching aqueous peroxyl radicals and lipid peroxidation products (39). It is preferentially oxidized before other antioxidants in plasma, including uric acid, tocopherols, and bilirubin. However, all the foregoing studies on the antioxidant actions of vitamin C were conducted in vitro. These oxidationreduction reactions may not specically require vitamin C in vivo. Thus, it is unknown whether such effects demonstrated in vitro are relevant in vivo. Because ascorbic acid can donate electrons to

Copyright 2002 by Taylor & Francis Group, LLC

Table 1 Known and Postulated Functions of Vitamin Ca Cofactor for enzymes Enzymes Known roles Mammalian Prolyl 4-hydroxylase Prolyl 3-hydroxylase Lysyl hydroxylase Trimethyllysine hydroxylase -Butyrobetaine hydroxylase Dopamine -monooxygenase Peptidyl-glycine -amidating monooxygenase 4-Hydroxyphenylpyruvate dioxygenase Fungi Deoxyuridine 1 -hydroxylase Thymine 7-hydroxylase Pyridine deoxyribonucleoside 2 hydroxylase Reducing agent Small intestine Antioxidant Cells Function of enzyme Ref.

Collagen hydroxylation

2224

Carnitine biosynthesis Norepinephrine biosynthesis Amidation of peptide hormones Tyrosine metabolism

25,26 27,28 29,30 21,31

Reutilization pathways for pyrimidines or the deoxyribose moiety of deoxynucleosides

32,33

Promote iron absorption

34,35

Postulated roles

Plasma Stomach

Regulate gene expression and mRNA translation, prevent oxidant damage to intracellular proteins Quench aqueous peroxyl radicals and lipid peroxidation products Prevent formation of N -nitroso compounds

3638

39 40

a Its function as a cofactor for eight different enzymes in mammals and a further three in yeast are fairly

well characterized. The postulated functions are all nonenzymatic and are based on the fact that vitamin C is a reducing agent. Postulated functions have been demonstrated in vitro; their relevance in vivo is unclear. Supporting data from animal or human experiments are sparse.

form semidehydroascorbic acid without the formation of reactive and harmful intermediaries, it remains the most potent water-soluble antioxidant in the body. Its physiological role in the intact organism relative to oxidation reactions is as yet uncertain. Vitamin C can quench reactive oxygen metabolites in the stomach or duodenum, and prevent the formation of N -nitroso compounds that are mutagenic. In normal subjects, the concentration of vitamin C in gastric juice is three times higher than that of plasma (51). These properties make it an attractive candidate for the prevention of gastric cancer (52). However, gastric juice vitamin C concentrations are normal in patients at risk for familial gastric cancer (53). Ascorbic acid content is low in the gastric juice of patients with atrophic gastritis and Helicobacter pylori infection, a condition associated with gastric cancer. Eradication of the bacteria increases gastric ascorbic acid secretion (54). Whether this suggested antioxidant

Copyright 2002 by Taylor & Francis Group, LLC

action has any signicance in vivo is unclear. Formation of nitrosamines in the gastrointestinal tract can be reduced by foods high in ascorbic acid. This effect may be due to ascorbic acid as well as other chemicals in food. Whether this has any clinical benet is unknown (40). Although high vitamin C dietary intake correlates with reduced gastric cancer risk (55), it is not certain what confers protection: vitamin C itself or other components of foods, particularly fruits and vegetables, that also happen to contain vitamin C. At doses of 2060 mg, vitamin C promotes iron absorption in the small intestine (34,35). Absorption of soluble nonorganic iron is increased by vitamin C, which might keep iron in a reduced form. Amounts necessary for enhancing iron absorption are found in foods that are good sources of the vitamin. Although the effects of supplemental vitamin C on increased iron absorption have been shown in many (56,57), but not all studies (58,59), its effect on raising hemoglobin concentration is modest at best (57,60), particularly when studied under real-life conditions (61). Many of the studies on the effects of vitamin C on iron absorption and improvement in hematological parameters were conducted over short periods on small numbers of patients. Clinical trials, however, cannot detect signicant changes in hematocrit or hemoglobin concentrations unless they are long-term studies with sufcient statistical power.

III. A.

PHYSIOLOGY OF VITAMIN C Tissue Distribution of Vitamin C

Ascorbic acid is widely distributed in the human and animals and is concentrated in many organs. The highest concentrations are found in adrenal and pituitary glands at 30400 mg/ 100 g of tissue. Liver, spleen, pancreas, kidney, brain, and lens contain 1050 mg/100 g (62). Assuming that 1 g of tissue is approximately equal to 1 mL, and since the molecular weight of ascorbic acid is 176, it is evident that many organs contain ascorbic acid in millimolar concentrations. By virtue of its mass, liver is the largest store of vitamin C. The choroid plexus actively secretes ascorbate into the cerebrospinal uid, from where it is taken up and concentrated by many parts of the brain (63). Animal studies in the guinea pig (64) and other animals (62) show that tissue concentrations are related to intake, so that higher intake results in a higher tissue concentration. These studies were done before accurate ascorbic acid assays were available. Because it is labile, rapid loss can occur in isolated organs if samples are not processed rapidly. Therefore, the tissue concentrations of vitamin C reported in the literature may be inaccurate. Nevertheless, these studies indicate the wide range in ascorbic acid tissue concentration and its selective uptake by specic organs (6567). The reason that many of these tissues concentrate vitamin C is unknown; it is also concentrated by white blood cells. These have been studied extensively owing to their easy availability and a possible link between vitamin C and infection. Neutrophils, lymphocytes, and monocytes have 1.34 mM internal concentrations of ascorbic acid (20- to 30-fold higher than plasma) (19). Mechanisms of ascorbic acid transport, described in the following section, were characterized based in part on studies of neutrophils (68).

B.

Transport and Accumulation of Vitamin C

1. Vitamin C Recycling in Neutrophils Neutrophils transport ascorbic acid from plasma and the extracellular milieu, probably utilizing the vitamin C transporter hSVCT2. When neutrophils are activated, superoxide and other oxidants are formed. Some oxidants diffuse out of the cell, and oxidize ascorbate to DHA. DHA enters neutrophils by glucose transporters, presumably the facilitative glucose transporters

Copyright 2002 by Taylor & Francis Group, LLC

GLUT1 and GLUT3. Once inside neutrophils, DHA is immediately reduced to ascorbic acid by the glutathione-dependent protein glutaredoxin (Fig. 2). Glutathione is oxidized by glutaredoxin to glutathione disulde, and glutathione is regenerated by reducing equivalents from NADPH by the pentose shunt. Oxidation of extracellular ascorbate and its reduction intracellularly back to ascorbate is termed ascorbate recycling. It is likely that ascorbate recycling protects the neutrophil and surrounding tissues from oxidative damage by oxidants generated during

Figure 2 A model of dehydroascorbic acid and ascorbate transport and recycling in human neutrophils: Ascorbate and dehydroascorbic acid are transported differently (46,69,70,74,76,137). The ascorbate transporter (open circle), probably SVCT2, transports ascorbate and probably maintains millimolar concentrations of ascorbate inside neutrophils (46,76,98). With activation, neutrophils secrete reactive oxygen species that oxidize extracellular ascorbate to dehydroascorbic acid. Dehydroascorbic acid is rapidly transported by glucose transporter isoforms GLUT1 and GLUT3 (open diamond). Intracellular dehydroascorbic acid is immediately reduced to ascorbate. In neutrophils, glutaredoxin is responsible for most intracellular reduction (138). As a result of dehydroascorbic acid transport and reduction, as much as tenfold higher ascorbate internal concentrations are achieved compared with activity of the ascorbate transporter alone. The proposed mechanism of reduction could require glutathione, NADPH, and the enzymes shown (138). Abbrev: AA, ascorbate; DHA, dehydroascorbic acid; GRX, glutaredoxin; GSH, reduced glutathione; GSSG, oxidized glutathione; 6-PGD, 6-phosphogluconate dehydrogenase; GRD, glutathione reductase. (From Ref. 16.)

Copyright 2002 by Taylor & Francis Group, LLC

neutrophil activation and phagocytosis. Extracellular oxidants are quenched by ascorbic acid, which might protect surrounding tissue from oxidative damage from oxidants generated by neutrophil activation, and as a consequence, DHA is formed. DHA is then transported and reduced within the neutrophil, so that additional ascorbate is available to quench intracellular oxidants generated by neutrophil activation. For neutrophils, the most potent activators of ascorbate recycling are bacteria (Fig. 3). Ascorbate recycling is induced by gram-positive and gram-negative bacteria as well as Candida albicans (68). However, bacteria lack mechanisms for ascorbate and DHA uptake, do not recycle ascorbate, and cannot use these mechanisms for oxidant protection. Thus, ascorbate recycling is a protective mechanism that is specic for the host, in this case humans, and is not utilized by pathogens, meaning bacteria and Candida. It was predicted that ascorbate recycling would not occur in patients whose neutrophils do not make oxidants, as in chronic granulomatous disease (Fig. 4). Neutrophils from these patients have defective superoxide generation. Because superoxide generation is necessary for generating reactive oxidant, it was predicted and found that neutrophils from these patients cannot recycle ascorbate. This specically is because, in the absence of oxidants, extracellular

Figure 3 Induction of ascorbate recycling by different microorganisms (68): Neutrophils were incubated with 100 M of ascorbate for 45 min with the indicated microorganism: neutrophils (effector/target) ratios for the following microorganisms: Escherichia coli CP9 () or CP922 (); Enterococcus faecalis (); Moraxella catarrhalis (); Klebsiella oxytoca (); Acinetobacter baumannii (); and Candida albicans (). Neutrophils incubated with ascorbate and no microorganisms are indicated by () at the lower left hand corner of the gure. Intracellular ascorbate was measured by scintillation spectrometry and is shown as millimoles (mM) (left axis) and nanomoles (nmol per milligram of protein (right axis). (From Ref. 68.)

Copyright 2002 by Taylor & Francis Group, LLC

Figure 4 Ascorbate recycling in chronic granulomatous disease neutrophils: Chronic granulomatous disease neutrophils were incubated with 100 M ascorbate ( and ) for 45 min or 300 M dehydroascorbic acid ( and ) for 5 min, and the indicated target/effector ratios for E. coli CP9 ( and ) or E. coli CP922 ( and ) were determined. Intracellular ascorbate was measured by HPLC and is shown as mM/mg (left axis) and nmol/mg protein (right axis). (From Ref. 68.)

ascorbic acid is not oxidized to DHA, which, therefore, is not available for transport by glucose transporters into neutrophils. When DHA is provided to neutrophils from patients with chronic granulomatous disease, DHA transport and reduction occurs efciently. 2. Vitamin C Transporters Two sodium-dependent vitamin C transporters, SVCT1 and SVCT2, have been identied (69,70). Properties of each transporter were measured using the Xenopus laevis oocyte expression system (Fig. 5). Both carrier proteins couple the transport of 2 Na+ :1 ascorbate. Kinetic analyses indicate that SVCT1 is a low-afnity (KM 237 M), high-velocity (Vmax 15.8 pmol/min per oocyte) transporter (69). SVCT2 has a tenfold higher afnity for ascorbate (KM 23 M), but exhibits a lower rate of uptake (Vmax 0.2 pmol/min per ve oocytes). Northern blot analyses show that SVCT1 is primarily localized to the epithelium of the small intestine and kidney, consistent with a role in intestinal absorption and renal reabsorption of ascorbate. Other organs known to accumulate ascorbic acid, such as liver, ovary, and prostate, also have high levels of SVCT1. SVCT2 has a more general distribution, with mRNA found in most tissues, including the brain, retina, placenta, spleen, small intestine, and gonads (71,70). Neither of these transporters transport DHA (69). The gene for human (h)SVCT1 has been mapped (71) to chromosome 5q23 (72) and that of hSVCT2 to chromosome 20p12.3 (72).

Copyright 2002 by Taylor & Francis Group, LLC

Figure 5 Functional expression of hSVCT1 and hSVCT2 in Xenopus oocytes: Oocytes were incubated in buffer containing 100 M [14 C]ascorbate (AA), 500 M dehydroascorbic acid (DHA), 2 mM unlabeled AA, choline, and [14 C]ascorbate. Sham oocytes were injected with sterile water. (From Ref. 69.)

Copyright 2002 by Taylor & Francis Group, LLC

3. DHA Transporters Tissues transport DHA 10- to 20-fold faster than ascorbate. DHA is transported by facilitative glucose transporters GLUT1 and GLUT3 (7375). GLUT1 and 3 transport DHA with an afnity similar to that for glucose (Fig. 6) (74). GLUT2, GLUT5, and SGLT1 do not transport DHA, and no glucose transporters transport ascorbic acid. DHA transport is inhibited by glucose and glucose analogues (74,76). Based on the interaction of DHA with some glucose transporters, it is possible that diabetes adversely affects ascorbate recycling in neutrophils. Other substances, such as endotoxin (77) and transforming growth factor beta (TGF- ) (78), also may have effects on DHA transport, but these remain uncertain.

Figure 6 DHA transport by glucose transporter isoforms GLUT1 and GLUT3: Xenopus oocytes expressing individual glucose transporter isoforms GLUT1 and GLU3 were tested for [14 C]DHA transport activity. Oocytes were incubated with the concentrations shown of [14 C]DHA () or 2-[3 H]deoxyglucose (), depending on the transport protein being tested, and the internalized radioactivity in individual oocytes was quantied. [14 C]DHA () transport into sham water-injected oocytes is also shown. (From Ref. 74.)

Copyright 2002 by Taylor & Francis Group, LLC

4. Studies of Vitamin C and DHA Transporters Using Xenopus laevus Oocytes The individual transporters that transport ascorbic acid and DHA can be studied singly or in combination using oocytes of the tropical frog X. laevis. mRNA for the appropriate transporter is microinjected into the surgically isolated oocyte, and the oocyte incubated in culture medium for 24 days. The transport proteins are translated from the injected mRNA, synthesized, and translocated to the oocyte membrane, where they are functionally active. When these oocytes are incubated with the appropriate substance, it is transported and concentrated in the oocyte, analogous to events in the human cell. These studies have demonstrated the specicity and the dynamics of each of the transporters for ascorbic acid and DHA and greatly increased our understanding of vitamin C transport (69,73,74). 5. Ascorbate Recycling as a General Mechanism for Ascorbate Accumulation The contribution of ascorbate recycling to ascorbate accumulation in many tissues is unknown. DHA is not present in plasma of healthy persons, although it is uncertain whether DHA is found in plasma of ill patients. It is possible that DHA forms locally in the extracellular milieu, and that it is transported by glucose transporters and reduced internally to ascorbate. Ascorbic acid oxidizes to DHA outside of oxidant-generating cells, such as neutrophils. It is unknown whether low rates of ascorbic acid oxidation to DHA occur in the extracellular milieu of other tissues, and also whether such DHA contributes substantially to ascorbate accumulation in these tissues. It is possible for ascorbate recycling to occur, to some degree, in many tissues because most tissues contain GLUT1, GLUT3, or GLUT4, and glutaredoxin is also widely distributed. Because DHA formation is essential for recycling to occur, and because it is unknown if and how much DHA formation occurs locally, the contribution of ascorbate recycling to ascorbate accumulation remains to be determined. Others have postulated that DHA transport is responsible for most of the ascorbate accumulated, especially in the brain and in tumors (79,80). Just as for ascorbate recycling, the mechanism of its accumulation depends on which substrates are available as well as which transporters are present. It is unrealistic to infuse completely unphysiological amounts of DHA into animals and then conclude that DHA transport is the major mechanism of ascorbate accumulation (80).

C.

Biochemical Function in Relation to Concentration

Many experiments have shown varying biochemical roles of vitamin C, such as its role as cofactors for enzymes involved in collagen, carnitine, catecholamine, or peptide hormone synthesis, and perhaps in neutrophil function. However, we still do not know whether these functions are related to particular plasma or tissue concentrations of vitamin C. It is possible that many of these functions take place at maximal rates in vivo at low concentrations of vitamin C, perhaps as low as those resulting from doses of vitamin C that are just adequate to prevent scurvy. For example, catecholamine synthesis can occur efciently as ascorbic acid is regenerated in chromafn granules, where it is a cosubstrate for norepinephrine synthesis. The kinetics of this regenerating mechanism are such that reduction rates achieve Vmax in situ (81). However, many other signicant ndings hint at a higher optimum plasma concentrations for vitamin C. For example, the Vmax of vitamin C transporter hSVCT2 is 70 M. LDL oxidation in vitro is inhibited by ascorbic acid at 4050 M. As described in detail later, plasma vitamin C concentrations in humans are tightly controlled at approximately 70 M, and circulating white blood cells also saturate at this concentration. That physiological mechanisms involving vitamin C function optimally at 5070 M plasma concentrations may indirectly indicate some benet to the organism if this plasma concentration is sustained.

Copyright 2002 by Taylor & Francis Group, LLC

D.

Steady-State Plasma Concentration in Relation to Dose

To study the physiology, absorption, bioavailability, and the renal threshold of vitamin C, it is necessary to attain and maintain steady-state plasma concentrations of this vitamin. In the absence of a steady-state level, that is if plasma concentrations are decreasing or increasing, reliable measurements of doseconcentration relations and other dose- or concentration-related physiological functions cannot be made. 1. Vitamin C Assay and Sample Processing For these experiments to be successfully carried out, the rst prerequisite is a reliable method to measure ascorbic acid. It is now possible to measure it with a high degree of accuracy and precision using high-performance liquid chromatography (HPLC) with coulometric electrochemical detection (82,83). Optimal sample collection and handling are equally important, as ascorbate is labile and easily oxidized. Blood samples have to be carefully drawn to avoid hemolysis as ascorbate is oxidized in the presence of hemoglobin. In whole blood ascorbic acid is stable for 2448 h if the sample is immediately placed on ice and then refrigerated, as long as no hemolysis occurs. Once the sample is centrifuged to remove red cells, white cells, and platelets, processing must not be interrupted. The sample is deproteinized, the precipitated protein removed by centrifugation, and the supernatant frozen at 80 C. The sample should be thawed immediately before assay. Under such careful conditions, the assay will reliably reect plasma concentration. In healthy persons, all of the vitamin is present in the plasma in the form of ascorbic acid, and there is no detectable DHA in the circulation (84). There is still no direct assay for DHA. DHA is measured indirectly after reduction to ascorbic acid. Minute amounts of DHA, if present in the plasma, cannot be measured accurately by this method, as DHA will be masked by the much larger quantities of ascorbic acid present. Ascorbic acid is found in the plasma in a free formit is not bound to plasma protein (84).

E.

Vitamin C Intake Versus Plasma Concentration: Results of Dietary Surveys

When plasma ascorbic acid concentrations are correlated with estimated daily vitamin C intake derived from the number of fruit or vegetable servings per week, no relation is seen between the two (unpublished data). This is probably because of the inaccuracy of dietary surveys (85) and the inability of subjects to recall exactly what they ate and how much in the preceding days. Factors other than recall may also play a rolethe type of fruits and vegetables eaten will have different amounts of vitamin C, the method of preparation may entail varying loss of vitamin C, and bioavailability might differ depending on the exact mix of diet consumed. The best way to eliminate these variables and study vitamin C doseconcentration relations is with precisely controlled intakes of known amounts of vitamin C.

F.

DepletionRepletion Studies

Depletionrepletion studies of vitamin C have been carried out for half a century. The early studies by the Royal Air Force (U. K.) showed that the dose of vitamin C necessary to prevent scurvy is probably less than 10 mg. The studies also concluded that body stores of vitamin C were sufcient to prevent scurvy for several months. Flaws of these studies are that dietary intake and vitamin C content were based on recall, without quantitation. A study conducted on prisoners (U. S.), in which four male prisoners were fed a vitamin C-free diet through a nasogastric tube, supported the nding that physical signs (what a physician nds on examination) of scurvy could be prevented by 10 mg of vitamin C daily (7,86). These ndings were

Copyright 2002 by Taylor & Francis Group, LLC

conrmed in a further ve prisoners (87,88) and, for many years, formed the basis of the U. S. Recommended Dietary Allowance (RDA). The prisoner studies, however, indicated that body stores of vitamin C were much less than previously believed, and could prevent scurvy for less than 6 weeks. The prisoner studies unfortunately also suffer from defects (89). Vitamin C assays were unreliable and imprecise, the number of subjects were few and, in the prisoners study, the diet was decient in not one, but many nutrients. Data collection was incomplete, as some of the prisoners escaped during the study. Other depletion studies carried out in outpatient settings demonstrated the ease with which plasma concentrations of ascorbic acid fell (9097), consistent with Linds observations more than 200 years ago. The 1996 National Institutes of Health (NIH) study is the only one that determined steadystate concentration as a function of dose and used many different doses (19). This study used a repletiondepletion design with seven healthy men who were admitted to the NIH Clinical Center for an average period of 145 days (range 116180) (Fig. 7). The subjects consumed

Figure 7 A schematic diagram of the 1996 NIH inpatient depletionrepletion study of seven healthy men. Note that the gure is not drawn to scale. The results of the study are presented in Table 2. To reduce inpatient time in the depletion phase, subjects were placed on a 60-mg vitamin C diet as outpatients for 3 weeks before admission. This led to a fall in plasma ascorbic acid concentration from 67 17.6 M (mean SD) at the time of prerecruitment screening to 23.0 6.9 M at the time of admission. After admission to the NIH Clinical Center, subjects were started on an inpatient vitamin C-free diet, containing < 5 mg/day of vitamin C. Blood samples were drawn fasting, in the morning, for vitamin C measurements daily, or several times per week. On this inpatient depletion diet, plasma vitamin C concentration fell to 6.9 2 M/L, at a rate of 1.3 0.5 M/L per day (100). Repletion, or supplementation, was started at this stage, with an initial dose of 30 mg daily in two divided doses until a steady-state concentration was reached. Bioavailability studies were carried out, and blood and white cell samples collected for vitamin C measurements. Subjects were given vitamin C in the following doses, and steady-state levels were attained at each dose: 30, 60, 100, 200, 400, 1000, and 2500 mg. The subjects were studied for a mean period of 145 days (range 116180). (From Ref. 19.)

Copyright 2002 by Taylor & Francis Group, LLC

a diet containing less than 5 mg vitamin C daily, but with all other nutrients in adequate amounts. Blood samples were drawn fasting, in the morning, for vitamin C measurements daily or several times per week. When plasma levels of vitamin C fell to between 5 and 10 M, the depletion phase was terminated. At this level, all subjects exhibited lassitude, but no signs of scurvy. It has to borne in mind that fatigue also was described by Lind in his treatise on scurvy in 1753 as the early symptom of impending scurvy. In the repletion or supplementation phase, subjects were commenced on graduated increases in vitamin C intake until they reached steady-state for each dose. The starting dose was 15 mg twice daily. On this dose (30 mg/day), most subjects reached steady-state levels in approximately 1 month. The steady-state level was dened as at least ve consecutive measurements in which vitamin C concentrations of plasma samples obtained over at least 7 days had a SD of 10% or less: 85% of steady-state calculations were based on six or more plasma samples (per patient). An example of steadystate level for a single subject at the 60-mg dose is shown in Figure 8. At the steady-state level, bioavailability studies were performed, as will be described later. Blood samples were obtained for vitamin C measurements and isolation of neutrophils, and other white blood cells were obtained by apheresis (98,99). Subjects were then given the next higher dose of vitamin C (60 mg/day) until a new steady-state level was reached. Again, bioavailability studies were carried out, and blood and white cell samples collected for vitamin C measurements. In this fashion, patients were sampled for daily vitamin C doses of 30, 60, 100, 200, 400, 1000, and 2500 mg. Vitamin C was administered in fasted state, as pure vitamin C in water with pH adjusted to 6.5. After the initiation of a vitamin C-free diet, plasma vitamin C concentrations fell to 6.9 2 M/L, at a rate of 1.3 0.5 M/L per day (100). Mean plasma steady-state concentrations attained at each of the vitamin C doses is given in Table 2. Different subjects required differing periods of time to deplete and to reach steady-state at each dose (Fig. 9). The underlying reasons for this interindividual variability are unknown. When the mean plasma steady-state concentrations for all subjects are plotted against dose, a sigmoidal doseconcentration curve results (Fig. 10). At the 30-mg dose, there is very little increase in plasma vitamin C concen-

Figure 8 Steady-state fasting plasma ascorbic acid concentrations for a single subject at the 60-mg dose. Steady-state concentration was dened as ve consecutive measurements in which vitamin C concentrations of plasma samples obtained over at least 7 days had a SD of 10% or less. (From Ref. 19.)

Copyright 2002 by Taylor & Francis Group, LLC

Table 2 Steady-State Plasma Concentrationa of Vitamin C in an Inpatient RepletionDepletion Study Steady-state plasma concentration of vitamin C Oral dose (mg) 0 (depletion nadir) 30 60 100 200 400 1000 2500 Plasma concentration (M) mean (SD) 7.6 8.7 24.8 56 65.8 70 76.9 85 (1.6) (1.7) (14.1) (4.5) (7.3) (6.9) (5.3) (5.4)

a Seven healthy men consumed a diet containing < 5 mg vitamin

C daily. Subjects were given vitamin C as daily oral doses of 30, 60, 100, 200, 400, 1000, and 2500 mg until a steady-state level was attained for each dose. Steady-state concentration was dened as at least ve consecutive plasma measurements obtained over at least 7 days, with a SD of 10% or less. Samples were obtained in the morning in the fasted state. Source: Ref. 19.

Figure 9 The relation between oral doses of vitamin C, inpatient time, and the fasting plasma ascorbic acid concentrations for each of the seven healthy men in the NIH inpatient depletionrepletion study. Different subjects required differing periods of time to deplete and to reach a steady-state level at each dose. (From Ref. 19.)

Copyright 2002 by Taylor & Francis Group, LLC

Figure 10 The relation between oral doses of vitamin C and the mean fasting steady-state plasma ascorbic acid concentration in seven healthy men from the NIH inpatient depletionrepletion study. The daily doses of vitamin C were 30, 60, 100, 200, 400, 1000, and 2500 mg. At the 30-mg dose, there is very little increase in plasma vitamin C concentration. Between 30 and 100 mg, there is a large increase, with the mean vitamin C concentration increasing from 8.7 to 56 M. Further increase in oral intake results in relatively smaller increases in plasma concentration, so that a daily intake of 2500 mg produces only 85 M. The doseconcentration curve is sigmoidal, with its steep portion between 30 and 100 mg of vitamin C daily. (From Ref. 19.)

tration. Between 30 and 100 mg, there is a large increase, with mean vitamin C concentrations increasing from 8.7 to 56 M. Further increase in oral intake results in relatively smaller increases in plasma concentrations, so that a daily intake of 2500 mg produces only 85 M. This steep portion of the sigmoid curve thus lies between 30 and 100 mg of vitamin C daily. As a consequence, small changes in intake in this range will result in large changes in plasma concentrations (19). This is of great public health importance, as well as in the study of vitamin C physiology, because accurate control over intake is necessary for meaningful experiments. The foregoing data show that plasma is saturated at 1000 mg/day, a dose at which the plasma concentration was approximately 80 M. Intracellular vitamin C concentrations in circulating blood cells (neutrophils, monocytes, and lymphocytes) also exhibit steep dose concentration curves, but these cell saturate before plasma (Fig. 11). This is because they actively transport and concentrate vitamin C from the plasma, and the Vmax s for these transporters are 6070 M. 1. Diet Used for DepletionRepletion Studies Previous studies used narrow vitamin C dose ranges or designs in which dietary intake of vitamin C could not be strictly controlled. As is apparent from the NIH study, the vitamin C doseconcentration relation is sigmoidal, so that small changes in oral intake produce a large change in plasma concentrations at low doses. Therefore, a consistently rigorous diet is essential to obtain meaningful data (85). In the NIH study, volunteers were admitted as inpatients for 46 months to ensure strict dietary control (19). A new diet was designed ensuring vitamin

Copyright 2002 by Taylor & Francis Group, LLC

Figure 11 Intracellular ascorbic acid concentrations (mM) in circulating immune cells as a function of dose (mg/day) in seven healthy men in the NIH depletionrepletion study: Neutrophils (), monocytes (), and lymphocytes () were isolated when the subjects were at steady state for each dose. Numbers in parenthesis ( ) at each dose indicate the number of volunteers from whom neutrophils were obtained; numbers in brackets [ ] at each dose indicate the number of volunteers from whom lymphocytes and monocytes were obtained. (From Ref. 19.)

C intake of less than 5 mg/day, but at the same time providing a varied fare (100). Patients could choose their diet from a computerized menu consisting of vitamin C-free foods. When the selected food contained vitamin C, it was weighed to ensure that daily intake of vitamin C was kept at less than 5 mg. All food consumed was weighed so that accurate daily amount of intake for each patient was known for vitamin C, calories, carbohydrates, fat, protein, and 18 vital nutrients. Nutrients that might possibly have been decient were supplemented by daily vitamin and mineral tablets. The daily intake of vitamin C for all subjects from this vitamin C-decient depletion diet was 3.87 0.64 (mean SD) mg over the entire study period.

G.

Bioavailability of Oral Vitamin C

The amount of vitamin C that has to be taken orally for optimum physiological function partly depends on its bioavailability. Oral consumption does not equate with availability to the tissues. A substance can be altered or destroyed in the gastrointestinal tract, be bound or otherwise made unabsorbable, destroyed by intestinal mucosal cells or by rst-pass metabolism in the liver, or simply pass out of the gut unabsorbed. Previous studies of vitamin C bioavailability have compared oral absorption with urine excretion, and studied relative bioavailability by comparing vitamin C in food with that in supplements, but these studies were not performed at steady-state concentrations (97,101104). True bioavailability studies are more demanding and have to be done at a steady-state level for that dose. At steady-state, plasma and tissues are in equilibrium relative to the vitamin C dose. This avoids selective uptake of the vitamin by the liver or other tissues, producing misleadingly low plasma levels. Vitamin C is administered orally and plasma concentrations measured

Copyright 2002 by Taylor & Francis Group, LLC

at frequent intervals. The same dose is administered intravenously at a different time (usually the next day). Again plasma concentrations are measured. Except for substances that are modied to their active form by rst-pass hepatic metabolism, intravenous administration ensures complete bioavailability, as all the administered substance reaches the peripheral circulation. The areas under the curve (AUC) for oral administration (AUCpo ) and for intravenous administration (AUCiv ) are calculated (Fig. 12). True bioavailability is given by the AUCpo /AUCiv ratio. These calculations showed that bioavailability for oral vitamin C is approximately 100% for 200 mg, 73% for 500 mg, and 49% for 1250 mg (Fig. 13) (19). Although these bioavailability data appear convincing, they suffer from some uncertainties. This is because bioavailability using the AUC calculations is accurate only if the test substance has a constant volume of distribution and a constant rate of clearance. As described earlier, vitamin C is distributed differently between plasma and blood cells. Many other compartments into which vitamin C is distributed, such as other tissues, cerebrospinal uid, and so on, will have yet other distributions. Neither is excretion linear, as renal excretion starts only above the renal threshold, which occurs at a plasma concentration of approximately 60 M. These objections are particularly relevant for doses less than 200 mg, and at these doses the AUC method cannot be used to calculate bioavailability. A mathematical model was developed to account for the nonlinearity in clearance and volume of distribution (105). By using this model, bioavailability was calculated to be 80% for 100 mg and 46% for 1250 mg. Values for bioavailability using these methods are shown in Table 3.

Figure 12 Schematic diagram of the method used to determine bioavailability of ascorbic acid: Bioavailability is determined by calculating the ratio of the areas under the curves (AUCs) of plasma concentrations, following oral () or intravenous () administration of the same dose of vitamin C on successive days. AUC is calculated by the linear trapezoidal method. The study is carried out at steady-state concentrations for that dose.

Copyright 2002 by Taylor & Francis Group, LLC

Figure 13 Ascorbic acid bioavailability in plasma: The upper gure displays bioavailability in a single subject for a 200-mg dose. The lower gure displays bioavailability for a single subject at the 1250-mg dose. For each dose ascorbic acid was administered at 0 time (8 am) orally (). The resulting plasma concentrations are shown for the times indicated. Baseline is indicated by a dashed line with large spaces (- - - -). After 24 h, the same dose was given intravenously and samples were taken for the time indicated (). Baseline is indicated by a dashed line with small spaces (----). For oral doses, samples taken before 0 time and between 13 and 24 h are not shown for clarity. AUCs were calculated using the linear trapezoidal method. Bioavailability was the ratio of the area of the oral dose (AUCpo ) divided by the area of the intravenous dose (AUCiv ). AUC after the curve returned to baseline was assumed to equal zero. (From Ref. 19.)

These values were based on vitamin C administered as an aqueous solution on an empty stomach (fasted state). As the bioavailability for vitamin C is close to 100% when given in a chemically pure form, any alteration in bioavailability can mean only a decrease in bioavailability. It is not known what the bioavailability of vitamin C is when it is present in food or when administered as a supplement with food. Bioavailability may be substantially less than in its pure form as some food material might contain sequestered vitamin C, making it unavailable for absorption. Alternatively, other components of food, such as glucose (106) or avonoids (107), might theoretically interfere with its absorption. Knowledge of bioavailability of vitamin C from food is critical to formulating recommendations for optimum dietary intake (108,109). Such information is currently unavailable.

H.

Urinary Excretion of Vitamin C

As in bioavailability studies, accurate studies of urinary excretion can be done only at a steady state for that dose. The studies that previously demonstrated saturable tubular resorptive mechanisms were not done at steady-state levels. The prisoners study stated that urinary

Copyright 2002 by Taylor & Francis Group, LLC

Table 3 Bioavailability of Vitamin C in 7 Healthy Men at Steady State Bioavailability of ascorbic acida Method using area under curve Dose (mg) 15 30 50 100 200 500 1250 Mean % (SD) 112 (25) 73 (27) 49 (25) Method using multicompartment mathematical model Median (%) 89 87.3 58 80 72 63 46

a Subjects were given vitamin C as daily doses of 30, 60, 100, 200, 400,

1000, and 2500 mg until a steady-state level was attained for each dose. Bioavailability for 15 mg was determined at the nadir (that is at the end of depletion, but before starting repletion with vitamin C). Note that for some doses, the amount of vitamin C used for determining bioavailability was slightly different from the dose used to attain steady-state. Thus, when steady-state concentration was obtained for the 60-mgdaily dose, bioavailability for 50 mg was determined. Bioavailability using area under curves could not be determined when vitamin C did not have a constant volume of distribution or a constant rate of clearance. This is accounted for in the multicompartment mathematical model used to determine bioavailability. Source: Refs. 19, 105.

excretion at oral doses occurred above 60 mg, but no data are available. Other studies have used a narrow-dose range or failed to use controlled vitamin C doses before measuring vitamin C excretion (110114). Some of these studies also suffered from imprecise assays. Studies of renal excretion carried out at steady-state levels for different doses of vitamin C show that no vitamin C appears in the urine until the oral dose is 100 mg, corresponding to a plasma concentration of approximately 60 M. At higher doses, vitamin C appears in the urine in increasing amounts (Fig. 14). However, bioavailability falls with increasing oral doses, so that smaller and smaller amounts of vitamin C is absorbed. For example, when 1250 mg of vitamin C is given orally, less than half of it is absorbed, and this amount (approximately 600 mg) is excreted in the urine (see Fig. 14). If vitamin C is administered intravenously, renal excretion can be studied without the confounding effects of bioavailability. After intravenous administration, virtually all of the administered dose is excreted at 500 and 1250 mg (see Fig. 14B) (19). Vitamin C is not protein bound. It is, therefore, presumably ltered at the glomerulus and reabsorbed in the renal tubules. Reabsorption probably occur in a concentrationdependent manner, so that once the transport mechanism is saturated, vitamin C will be excreted in the urine. Whether additional mechanisms, such as active secretion, exist for the excretion of vitamin C is unknown. Vitamin C excretion may be similar to that of glucose, which appears in the urine when the ability of the kidney to reabsorb it is overwhelmed. In patients with renal failure, supplemental ascorbic acid can accumulate in the body, and large doses may produce hyperoxalemia (115120), but there may not be a good correlation between the

Copyright 2002 by Taylor & Francis Group, LLC

Figure 14 Ascorbic acid excretion as a function of single vitamin C doses: Ascorbic acid excretion in urine was determined after administration of single doses of vitamin C given either orally () or intravenously (). Urine was collected during determination of ascorbic acid bioavailability for each dose. The collection time for oral sampling was 24 h, and the collection time for intravenous sampling was 910 h, the intervals required to be certain plasma ascorbate returned to baseline. (Inset A) Ascorbic acid excretion for 15100 mg single oral () or intravenous () doses of vitamin C. x Axis indicates vitamin C dose and y axis indicates ascorbic acid excretion in the urine (mg). (Inset B) Fractional excretion (the fraction of the dose excreted) of ascorbic acid: Urine samples were collected after intravenous administration of single doses of vitamin C for bioavailability sampling. The minimum amount of ascorbate excreted was 0.4 mg. Fractional excretion was not determined for any dose of vitamin C administered orally, because of decreasing bioavailability at doses > 200 mg. x Axis indicates vitamin C dose and y axis indicates fractional excretion, dened as ascorbate excreted in urine (mg) divided by the dose administered intravenously. (From Ref. 19.)

plasma concentrations of ascorbate and oxalate (120). More commonly, patients with chronic renal failure lose ascorbic acid during dialysis. Its concentrations can fall by nearly half during a single dialysis session (120124), so that these patients have chronically low plasma vitamin C concentrations (125,126) unless intake is adequate (127).

I.

Food Sources of Vitamin C

Fruits containing large amounts of vitamin C include strawberries, papaya, oranges, kiwi, cantaloupe, grapefruit, mango, and honeydews. Vegetables rich in vitamin C are broccoli, brussel sprouts, red or green pepper, tomato, cabbage, potato, sweet potato, cauliower, snow peas, and kale. Fruit juices such as orange juice, tomato juice, grapefruit juice, and fortied juices are also rich sources of vitamin C.

Vitamin C is widely distributed in plant foods.

Five servings of a variety of fruits and vegetables per day will provide 210280 mg of vitamin C, but consumption restricted to a narrow selection of fruits and vegetables may not provide

Copyright 2002 by Taylor & Francis Group, LLC

the same amount of the vitamin (128). Vitamin C content varies to some extent depending on the season. Vitamin C may also be lost during food transportation and storage, or during cooking.

J.

Vitamin C Intake in the United States

Several dietary surveys have examined vitamin C consumption in the U. S. population. The National Health and Nutrition Examination Survey (NHANHS) 3 study found that the median intake of vitamin C for males 20 years old or more, is 84 mg and for females 20 years old or more, it is 73 mg. Approximately 30% of adults consume less than 2.5 servings of fruits and vegetables per day. The estimated vitamin C intake is lower among many population subgroups including children (129,130). In one study of 9- and 10-year-old girls, approximately one-fourth had vitamin C ingestion less than the RDA of 45 mg for their age (131). Results of a survey of Latino children were that fewer than 15% ingested the recommended intake of fruits and vegetables (132). Data from the earlier nutritional survey NHANES 2 have been examined extensively. The analyses indicated that 2030% of U. S. adults ingested less than 60 mg daily of vitamin C (129,130,133).

K.

Recommended Dietary Allowance for Vitamin C

Recently U.S. RDAs for vitamin C were increased from 60 mg/day to 75 mg/day for women and 90 mg/day for men (134). These recommendations are based on the recent ndings concerning the vitamin C intake necessary to saturate neutrophils. As a result of recent research into the varied actions of vitamin C, a wealth of biochemical, molecular, epidemiological, and clinical data have become available. From these data, the following criteria can serve as sound foundation for recommendations on optimum vitamin C intake. Dietary availability Steady-state concentrations in plasma in relation to dose Steady-state concentrations in tissue in relation to dose Bioavailability Urine excretion Adverse effects Biochemical and molecular function in relation to vitamin concentration Benecial effects in relation to dose; direct effects and epidemiological observations Prevention of deciency When these criteria are applied to the Food and Nutrition Boards classication guidelines, the Dietary Reference Intakes, the Recommended Dietary Allowance has been proposed by us to be 100120 mg daily (135). It is necessary to note that this value depends on the data selected for the new dietary reference intake classication, the Estimated Average Requirement. Adequate Intake, another new dietary reference intake classication, is used when data are considered insufcient for a recommended dietary allowance. Adequate Intake for vitamin C was estimated to be 200 mg daily, to be obtained from ve servings of fruits and vegetables. The last new classication, Tolerable Upper Intake, is proposed to be less than 1 g of vitamin C daily (135). Physicians can tell patients now that ve servings of fruits and vegetables provide sufcient vitamin C intake for healthy persons and are benecial in preventing cancer, and that 1 g or more of vitamin C might have adverse consequences in some persons.

Copyright 2002 by Taylor & Francis Group, LLC

REFERENCES
1. Clemenston CAB. Classical scurvy: a historical review. In: Vitamin C. Vol. 1. Boca Raton, FL: CRC Press, 1989. 2. Lind J. The true causes of the disease, from observations made upon it, both at sea and land. In: Stewart CP, Guthrie D, eds. Linds Treatise on Scurvy. Bicentenary Volume. Edinburgh: Edinburgh University Press, 1953:69112. 3. SzentGyorgyi A. Observations on the function of peroxidase systems and chemistry of the adrenal cortex: description of a new carbohydrate derivative. Bioichem J 1928; 22:1387. 4. Svirbely JL, SzentGyorgyi A. The chemical nature of vitamin C. Biochem J 1932; 26:865870. 5. King CG, Waugh WA. The chemical nature of vitamin C. Science 1932; 75:357. 6. Lind J. The diagnostics, or signs. In: Stewart CP, Guthrie D, eds. Linds Treatise on Scurvy. Bicentenary Volume. Edinburgh: Edinburgh University Press, 1953:113132. 7. Hodges RE, Baker EM, Hood J, Sauberlich HE, March SC. Experimental scurvy in man. Am J Clin Nutr 1969; 22:535548. 8. Johnston CS, Thompson LL. Vitamin C status of an outpatient population. J Am Coll Nutr 1998; 17:366370. 9. Ohta Y, Nishikimi M. Random nucleotide substitutions in primate nonfunctional gene for l-gulonogamma-lactone oxidase, the missing enzyme in l-ascorbic acid biosynthesis. Biochim Biophys Acta 1999; 1472:408411. 10. Nishikimi M, Kawai T, Yagi K. Guinea pigs possess a highly mutated gene for l-gulono-gammalactone oxidase, the key enzyme for l-ascorbic acid biosynthesis missing in this species. J Biol Chem 1992; 267:2196721972. 11. Nishikimi M, Fukuyama R, Minoshima S, Shimizu N, Yagi K. Cloning and chromosomal mapping of the human nonfunctional gene for l-gulono-gamma-lactone oxidase, the enzyme for l-ascorbic acid biosynthesis missing in man. J Biol Chem 1994; 269:1368513688. 12. Ratterree MS, Didier PJ, Blanchard JL, Clarke MR, Schaeffer D. Vitamin C deciency in captive nonhuman primates fed commercial primate diet. Lab Anim Sci 1990; 40:165168. 13. Wheeler GL, Jones MA, Smirnoff N. The biosynthetic pathway of vitamin C in higher plants. Nature 1998; 393:365369. 14. Smirnoff N. Ascorbic acid: metabolism and functions of a multi-faceted molecule. Curr Opin Plant Biol 2000; 3:229235. 15. Buettner GR, Moseley PL. EPR spin trapping of free radicals produced by bleomycin and ascorbate. Free Radic Res Commun 1993; 19:S89S93. 16. Rumsey SC, Levine M. Absorption, transport, and disposition of ascorbic acid in humans. Nutr Biochem 1998; 9:116130. 17. Winkler BS, Orselli SM, Rex TS. The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic Biol Med 1994; 17:333349. 18. Baker EM, Halver JE, Johnsen DO, Joyce BE, Knight MK, Tolbert BM. Metabolism of ascorbic acid and ascorbic-2-sulfate in man and the subhuman primate. Ann NY Acad Sci 1975; 258:7280. 19. Levine M, ConryCantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, Park JB, Lazarev A, Graumlich JF, King J, Cantilena LR. Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci USA 1996; 93:37043709. 20. Levine M. New concepts in the biology and biochemistry of ascorbic acid. N Engl J Med 1986; 314:892902. 21. Englard S, Seifter S. The biochemical functions of ascorbic acid. Annu Rev Nutr 1986; 6:365406. 22. Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem 1995; 64:403434. 23. Kivirikko KI, Myllyla R. Post-translational processing of procollagens. Ann NY Acad Sci 1985; 460:187201. 24. Peterkofsky B. Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy. Am J Clin Nutr 1991; 54:1135S1140S. 25. Rebouche CJ. Ascorbic acid and carnitine biosynthesis. Am J Clin Nutr 1991; 54:1147S1152S. 26. Dunn WA, Rettura G, Seifter E, Englard S. Carnitine biosynthesis from gamma-butyrobetaine and from exogenous protein-bound 6-N -trimethyl-l-lysine by the perfused guinea pig liver. Effect of ascorbate deciency on the in situ activity of gamma-butyrobetaine hydroxylase. J Biol Chem 1984; 259:1076410770.

Copyright 2002 by Taylor & Francis Group, LLC

27. Levine M, Dhariwal KR, Washko PW, Butler JD, Welch RW, Wang YH, Bergsten P. Ascorbic acid and in situ kinetics: a new approach to vitamin requirements. Am J Clin Nutr 1991; 54:1157S 1162S. 28. Kaufman S. Dopamine-beta-hydroxylase. J Psychiatr Res 1974; 11:303316. 29. Eipper BA, Milgram SL, Husten EJ, Yun HY, Mains RE. Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains. Protein Sci 1993; 2:489497. 30. Eipper BA, Stoffers DA, Mains RE. The biosynthesis of neuropeptides: peptide alpha-amidation. Annu Rev Neurosci 1992; 15:5785. 31. Lindblad B, Lindstedt G, Lindstedt S. The mechanism of enzymic formation of homogenisate from p-hydroxyphenylpyruvate. J Am Chem Soc 1970; 92:74467449. 32. Wondrack LM, Hsu CA, Abbott MT. Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2 -hydroxylase activities in Rhodotorula glutinis. J Biol Chem 1978; 253:65116515. 33. Stubbe J. Identication of two alpha-ketoglutarate-dependent dioxygenases in extracts of Rhodotorula glutinis catalyzing deoxyuridine hydroxylation. J Biol Chem 1985; 260:99729975. 34. Hallberg L, Brune M, RossanderHulthen L. Is there a physiological role of vitamin C in iron absorption? Ann NY Acad Sci 1987; 498:324332. 35. Hallberg L. Wheat ber, phytates and iron absorption. Scand J Gastroenterol Suppl 1987; 129:73 79. 36. Hitomi K, Tsukagoshi N. Role of ascorbic acid in modulation of gene expression. Subcell Biochem 1996; 25:4156. 37. Toth I, Roger JT, McPhee JA, Elliott SM, Abramson SL, Bridges KR. Ascorbic acid enhances ironinduced ferritin translation in human leukemia and hepatoma cells. J Biol Chem 1995; 270:2846 2852. 38. Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Chem Res Toxicol 1997; 10:485494. 39. Frei B, Stocker R, England L, Ames BN. Ascorbate: the most effective antioxidant in human blood plasma. Adv Exp Med Biol 1990; 264:155163. 40. Helser MA, Hotchkiss JH, Roe DA. Inuence of fruit and vegetable juices on the endogenous formation of N -nitrosoproline and N -nitrosothiazolidine-4-carboxylic acid in humans on controlled diets. Carcinogenesis 1992; 13:22772280. 41. Gokce N, Frei B. Basic research in antioxidant inhibition of steps in atherogenesis. J Cardiovasc Risk 1996; 3:352357. 42. Jialal I, Fuller CJ, Huet BA. The effect of alpha-tocopherol supplementation on LDL oxidation. A doseresponse study. Arterioscler Thromb Vasc Biol 1995; 15:190198. 43. Jialal I, Vega GL, Grundy SM. Physiologic levels of ascorbate inhibit the oxidative modication of low density lipoprotein. Atherosclerosis 1990; 82:185191. 44. Jialal I, Fuller CJ. Effect of vitamin E, vitamin C and beta-carotene on LDL oxidation and atherosclerosis. Can J Cardiol 1995; 11:97G103G. 45. Weber C, Erl W, Weber K, Weber PC. Increased adhesiveness of isolated monocytes to endothelium is prevented by vitamin C intake in smokers. Circulation 1996; 93:14881492. 46. Washko PW, Wang Y, Levine M. Ascorbic acid recycling in human neutrophils. J Biol Chem 1993; 268:1553115535. 47. Mukhopadhyay CK, Chatterjee IB. Free metal ion-independent oxidative damage of collagen. Protection by ascorbic acid. J Biol Chem 1994; 269:3020030205. 48. Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willett WC. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 1993; 328:14441449. 49. Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary heart disease in man. N Engl J Med 1993; 328:14501456. 50. Gey KF. Cardiovascular disease and vitamins. Concurrent correction of suboptimal plasma antioxidant levels may, as important part of optimal nutrition, help to prevent early stages of cardiovascular disease and cancer, respectively. Bibl Nutr Dieta 1995; 52:7591. 51. Rathbone BJ, Johnson AW, Wyatt JI, Kelleher J, Heatley RV, Losowsky MS. Ascorbic acid: a factor concentrated in human gastric juice. Clin Sci 1989; 76:237241. 52. Correa P. Human gastric carcinogenesis: a multistep and multifactorial processFirst American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cancer Res 1992; 52:67356740.

Copyright 2002 by Taylor & Francis Group, LLC

53. Sobala GM, Schorah CJ, Pignatelli B, Crabtree JE, Martin IG, Scott N, Quirke P. High gastric juice ascorbic acid concentrations in members of a gastric cancer family. Carcinogenesis 1993; 14:291292. 54. Sobala GM, Schorah CJ, Shires S, Lynch DA, Gallacher B, Dixon MF, Axon AT. Effect of eradication of Helicobacter pylori on gastric juice ascorbic acid concentrations. Gut 1993; 34:10381041. 55. Byers T, Guerrero N. Epidemiologic evidence for vitamin C and vitamin E in cancer prevention. Am J Clin Nutr 1995; 62:1385S1392S. 56. Davidsson L, Walczyk T, Morris A, Hurrell RF. Inuence of ascorbic acid on iron absorption from an iron-fortied, chocolate-avored milk drink in Jamaican children. Am J Clin Nutr 1998; 67:873877. 57. Hunt JR, Mullen LM, Lykken GI, Gallagher SK, Nielsen FH. Ascorbic acid: effect on ongoing iron absorption and status in iron-depleted young women. Am J Clin Nutr 1990; 51:649655. 58. Stack T, Aggett PJ, Aitken E, Lloyd DJ. Routine l-ascorbic acid supplementation does not alter iron, copper, and zinc balance in low-birth-weight infants fed a cows-milk formula. J Pediatr Gatroenterol Nutr 1990; 10:351356. 59. Harju E, Lindberg H. Ascorbic acid does not augment the restoration effect of iron treatment for empty iron stores in patients after gastrointestinal surgery. Am Surg 1986; 52:463466. 60. Rhode BM, Shustik C, Christou NV, MacLean LD. Iron absorption and therapy after gastric bypass. Obes Surg 1999; 9:1721. 61. Hunt JR, Gallagher SK, Johnson LK. Effect of ascorbic acid on apparent iron absorption by women with low iron stores. Am J Clin Nutr 1994; 59:13811385. 62. Hornig D. Distribution of ascorbic acid, metabolites and analogues in man and animals. Ann NY Acad Sci 1975; 258:103118. 63. Rebec GV, Pierce RC. A vitamin as neuromodulator: ascorbate release into the extracellular uid of the brain regulates dopaminergic and glutamatergic transmission. Prog Neurobiol 1994; 43:537 565. 64. Keith MO, Pelletier O. Ascorbic acid concentrations in leukocytes and selected organs of guinea pigs in response to increasing ascorbic acid intake. Am J Clin Nutr 1974; 27:368372. 65. Hornig D, Weber F, Wiss O. Tissue distribution of labelled material in vitamin C-decient guinea pigs after intravenous injection of (1-14 C) ascorbic acid or (1-14 C) dehydroascorbic acid. Int J Vitam Nutr Res 1972; 42:511523. 66. Chinoy NJ. Ascorbic acid levels in mammalian tissues and its metabolic signicance. Comp Biochem Physiol A 1972; 42:945952. 67. Horning D, GalloTorres HE, Weiser H. Tissue distribution of labelled ascorbic acid in normal and hypophysectomized rats. Int J Vitam Nutr Res 1972; 42:487496. 68. Wang Y, Russo TA, Kwon O, Chanock S, Rumsey SC, Levine M. Ascorbate recycling in human neutrophils: induction by bacteria. Proc Natl Acad Sci USA 1977; 94:1381613819. 69. Daruwala R, Song J, Koh WS, Rumsey SC, Levine M. Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett 1999; 460:480484. 70. Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen XZ, Wang Y, Brubaker RF, Hediger MA. A family of mammalian Na+ -dependent l-ascorbic acid transporters. Nature 1999; 399:7075. 71. Wang Y, Mackenzie B, Tsukaguchi H, Weremowicz S, Morton CC, Hediger MA. Human vitamin C (l-ascorbic acid) transporter SVCT1. Biochem Biophys Res Commun 2000; 267:488494. 72. Stratakis CA, Taymans S, Daruwala R, Song J, Levine M. Mapping of the human genes (SLCA23A2 and SLCA23A1) coding for vitamin C transporters 1 and 2 (SVCT1 and SVCT2), to 5q23 and 20p12, respectively. J Med Genet 2000; 37:E20. 73. Vera JC, Rivas CI, Fischbarg J, Golde DW. Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature 1993; 364:7982. 74. Rumsey SC, Kwon O, Xu GW, Burant CF, Simpson I, Levine M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J Biol Chem 1997; 272:1898218989. 75. Rumsey SC, Daruwala R, Al-Hasani H, Zarnowski M, Simpson IA, Levine M. Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes. J Biol Chem 1997; 272:1898218989. 76. Welch RW, Wang Y, Crossman A Jr, Park JB, Kirk KL, Levine M. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol Chem 1995; 270:1258412592.

Copyright 2002 by Taylor & Francis Group, LLC

77. Padh H, Aleo JJ. Ascorbic acid transport by 3T6 broblasts. Regulation by and purication of human serum complement factor. J Biol Chem 1989; 264:60656069. 78. Dixon SJ, Wilson JX. Adaptive regulation of ascorbate transport in osteoblastic cells. J Bone Miner Res 1992; 7:675681. 79. Agus DB, Gambhir SS, Pardridge WM, Spielholz C, Baselga J, Vera JC, Golde DW. Vitamin C crosses the bloodbrain barrier in the oxidized form through the glucose transporters. J Clin Invest 1997; 100:28422848. 80. Agus DB, Vera JC, Golde DW. Stromal cell oxidation: a mechanism by which tumors obtain vitamin C. Cancer Res 1999; 59:45554558. 81. Dhariwal KR, Shirvan M, Levine M. Ascorbic acid regeneration in chromafn granules. In situ kinetics. J Biol Chem 1991; 266:53845387. 82. Levine M, Wang Y, Rumsey SC. Analysis of ascorbic acid and dehydroascorbic acid in biological samples. Methods Enzymol 1999; 299:6576. 83. Rumsey SC, Wang Y, Levine M. Ascorbic acid and dehydroascorbic acid analyses in biological samples. In: Song WO, Beecher GR, eds. Modern Analytical Methodologies on Fat and Water Soluble Vitamins. New York: John Wiley & Sons, 2001 (in press). 84. Dhariwal KR, Hartzell WO, Levine M. Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum. Am J Clin Nutr 1991; 54:712716. 85. Hegsted DM. Truly quantitative dietary studies have stringent requirements. Am J Clin Nutr 1997; 66:14771479. 86. Baker EM, Hodges RE, Hood J, Sauberlich HE, March SC. Metabolism of ascorbic-1-14 C acid in experimental human scurvy. Am J Clin Nutr 1969; 22:549558. 87. Hodges RE, Hood J, Canham JE, Sauberlich HE, Baker EM. Clinical manifestations of ascorbic acid deciency in man. Am J Clin Nutr 1971; 24:432443. 88. Bake EM, Hodges RE, Hood J, Sauberlich HE, March SC, Canham JE. Metabolism of 14 C- and 3 H-labeled l-ascorbic acid in human scurvy. Am J Clin Nutr 1971; 24:444454. 89. Hodges RE. Whats new about scurvy? Am J Clin Nutr 1971; 24:383384. 90. Block G, Mangels AR, Patterson BH, Levander OA, Norkus EP, Taylor PR. Body weight and prior depletion affect plasma ascorbate levels attained on identical vitamin C intake: a controlled-diet study. J Am Coll Nutr 1999; 18:628637. 91. Leggott PJ, Robertson PB, Rothman DL, Murray PA, Jacob RA. The effect of controlled ascorbic acid depletion and supplementation on periodontal health. J Periodontol 1986; 57:480485. 92. Leggott PJ, Robertson PB, Rothman DL, Murray PA, Jacob RA. Response of lingual ascorbic acid test and salivary ascorbate levels to changes in ascorbic acid intake. J Dent Res 1986; 65:131134. 93. Holloway DE, Hutton SW, Peterson FJ, Duane WC. Lack of effect of subclinical ascorbic acid deciency upon antipyrine metabolism in man. Am J Clin Nutr 1982; 35:917924. 94. Blanchard J, Conrad KA, Watson RR, Garry PJ, Crawley JD. Comparison of plasma, mononuclear and polymorphonuclear leucocyte vitamin C levels in young and elderly women during depletion and supplementation. Eur J Clin Nutr 1989; 43:97106. 95. Leggott PJ, Robertson PB, Jacob RA, Zambon JJ, Walsh M, Armitage GC. Effects of ascorbic acid depletion and supplementation on periodontal health and subgingival microora in humans. J Dent Res 1991; 70:15311536. 96. Blanchard J. Depletion and repletion kinetics of vitamin C in humans. J Nutr 1991; 121:170176. 97. Mangels AR, Block G, Frey CM, Patterson BH, Taylor PR, Norkus EP, Levander OA. The bioavailability to humans of ascorbic acid from oranges, orange juice and cooked broccoli is similar to that of synthetic ascorbic acid. J Nutr 1993; 123:10541061. 98. Washko P, Rotrosen D, Levine M. Ascorbic acid transport and accumulation in human neutrophils. J Biol Chem 1989; 264:1899619002. 99. Bergsten P, Amitai G, Kehrl J, Dhariwal KR, Klein HG, Levine M. Millimolar concentrations of ascorbic acid in puried human mononuclear leukocytes. Depletion and reaccumulation. J Biol Chem 1990; 265:25842587. 100. King J, Wang Y, Welch RW, Dhariwal KR, ConryCantilena C, Levine M. Use of a new vitamin C-decient diet in a depletion/repletion clinical trial. Am J Clin Nutr 1997; 65:14341440. 101. Piotrovskij VK, Kallay Z, Gajdos M, Gerykova M, Trnovec T. The use of a nonlinear absorption model in the study of ascorbic acid bioavailability in man. Biopharm Drug Dispos 1993; 14:429 442. 102. Gregory JFD. Ascorbic acid bioavailability in foods and supplements. Nutr Rev 1993; 51:301303.

Copyright 2002 by Taylor & Francis Group, LLC

103. Sacharin R, Taylor T, Chasseaud LF. Blood levels and bioavailability of ascorbic acid after administration of a sustained-release formulation to humans. Int J Vitam Nutr Res 1977; 47:6874. 104. Vinson JA, Bose P. Comparative bioavailability to humans of ascorbic acid alone or in a citrus extract. Am J Clin Nutr 1988; 48:601604. 105. Graumlich JF, Ludden TM, ConryCantilena C, Cantilena LR Jr, Wang Y, Levine M. Pharmacokinetic model of ascorbic acid in healthy male volunteers during depletion and repletion. Pharm Res 1997; 14:11331139. 106. Washko P, Levine M. Inhibition of ascorbic acid transport in human neutrophils by glucose. J Biol Chem 1992; 267:2356823574. 107. Park JB, Levine M. Intracellular accumulation of ascorbic acid is inhibited by avonoids via blocking of dehydroascorbic acid and ascorbic acid uptakes in HL-60, U937 and Jurkat cells. J Nutr 2000; 130:12971302. 108. Clydesdale FM, Ho CT, Lee CY, Mondy NI, Shewfelt RL. The effects of postharvest treatment and chemical interactions on the bioavailability of ascorbic acid, thiamin, vitamin A, carotenoids, and minerals. Crit Rev Food Sci Nutr 1991; 30:599638. 109. Mayersohn M. Vitamin C bioavailability. J Nutr Sci Vitaminol (Tokyo) 1992; Spec:446449. 110. Kallner A, Hartmann D, Hornig D. Steady-state turnover and body pool of ascorbic acid in man. Am J Clin Nutr 1979; 32:530539. 111. Kallner A, Hartmann D, Hornig D. On the absorption of ascorbic acid in man. Int J Vitam Nutr Res 1977; 47:383388. 112. Mitch WE, Johnson MW, Kirshenbaum JM, Lopez RE. Effect of large oral doses of ascorbic acid on uric acid excretion by normal subjects. Clin Pharmacol Ther 1981; 29:318321. 113. Wagner ES, Lindley B, Cofn RD. High-performance liquid chromatographic determination of ascorbic acid in urine: effect on urinary excretion proles after oral and intravenous administration of vitamin C. J Chromatogr 1979; 163:225229. 114. Blanchard J, Conrad KA, Garry PJ. Effects of age and intake on vitamin C disposition in females. Eur J Clin Nutr 1990; 44:447460. 115. Tomson CR, Channon SM, Parkinson IS, McArdle P, Qureshi M, Ward MK, Laker MF. Correction of subclinical ascorbate deciency in patients receiving dialysis: effects on plasma oxalate, serum cholesterol, and capillary fragility. Clin Chim Acta 1989; 180:255264. 116. Balcke P, Schmidt P, Zazgornik J, Kopsa H, Haubenstock A. Ascorbic acid aggravates secondary hyperoxalemia in patients on chronic hemodialysis. Ann Intern Med 1984; 101:344345. 117. Ono K. Secondary hyperoxalemia caused by vitamin C supplementation in regular hemodialysis patients. Clin Nephrol 1986; 26:239243. 118. Ono K. The effect of vitamin C supplementation and withdrawal on the mortality and morbidity of regular hemodialysis patients. Clin Nephrol 1989; 31:3134. 119. Pru C, Eaton J, Kjellstrand C. Vitamin C intoxication and hyperoxalemia in chronic hemodialysis patients. Nephron 1985; 39:112116. 120. Rolton HA, McConnell KM, Modi KS, Macdougall AI. The effect of vitamin C intake on plasma oxalate in patients on regular haemodialysis. Nephrol Dial Transplant 1991; 6:440443. 121. Allman MA, Truswell AS, Tiller DJ, Stewart PM, Yau DF, Horvath JS, Duggin GG. Vitamin supplementation of patients receiving haemodialysis. Med J Aust 1989; 150:130133. 122. Sullivan JF, Eisenstein AB. Ascorbic acid depletion during hemodialysis. JAMA 1972; 220:1697 1699. 123. Sullivan JF, Eisenstein AB. Ascorbic acid depletion in patients undergoing chronic hemodialysis. Am J Clin Nutr 1970; 23:13391346. 124. Bohm V, Tiroke K, Schneider S, Sperschneider H, Stein G, Bitsch R. Vitamin C status of patients with chronic renal failure, dialysis patients and patients after renal transplantation. Int J Vitam Nutr Res 1997; 67:262266. 125. Papastephanidis C, Agroyannis B, TzanatosExarchou H, Orthopoulos B, Koutsicos D, Frangos Plemenos M, Kallitsis M, Yatzidis H. Re-evaluation of ascorbic acid deciency in hemodialysed patients. Int J Artif Organs 1987; 10:163165. 126. Wang S, Eide TC, Sogn EM, Berg KJ, Sund RB. Plasma ascorbic acid in patients undergoing chronic haemodialysis. Eur J Clin Pharmacol 1999; 55:527532. 127. Shah GM, Ross EA, Sabo A, Pichon M, Bhagavan H, Reynolds RD. Ascorbic acid supplements in patients receiving chronic peritoneal dialysis. Am J Kidney Dis 1991; 18:8490. 128. Johnston CS. Recommendations for vitamin C intake. JAMA 1999; 282:2118; discussion 2119.

Copyright 2002 by Taylor & Francis Group, LLC

129. Koplan JP, Annest JL, Layde PM, Rubin GL. Nutrient intake and supplementation in the United States (NHANES II). Am J Public Health 1986; 76:287289. 130. Patterson BH, Block G, Rosenberger WF, Pee D, Kahle LL. Fruit and vegetables in the American diet: data from the NHANES II survey. Am J Public Health 1990; 80:14431449. 131. Simon JA, Schreiber GB, Crawford PB, Frederick MM, Sabry ZI. Dietary vitamin C and serum lipids in black and white girls. Epidemiology 1993; 4:537542. 132. Basch CE, Zybert P, Shea S. 5-A-DAY: dietary behavior and the fruit and vegetable intake of Latino children. Am J Public Health 1994; 84:814818. 133. Murphy SP, Rose D, Hudes M, Viteri FE. Demographic and economic factors associated with dietary quality for adults in the 198788 Nationwide Food Consumption Survey. J Am Diet Assoc 1992; 92:13521357. 134. Food and Nutrition Board; Panel on Dietary Antioxidants and Related Compounds. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Beta Carotene, and other Carotenoids. Washington DC: National Academy Press, 2000:95185. 135. Levine M, Rumsey SC, Daruwala R, Park JB, Wang Y. Criteria and recommendations for vitamin C intake. JAMA 1999; 281:14151423. 136. Washko PW, Welch RW, Dhariwal KR, Wang Y, Levine M. Ascorbic acid and dehydroascorbic acid analyses in biological samples. Anal Biochem 1992; 204:114. 137. Dyer DL, Kanai Y, Hediger MA, Rubin SA, Said HM. Expression of a rabbit renal ascorbic acid transporter in Xenopus laevis oocytes. Am J Physiol 1994; 267:C301306. 138. Park JB, Levine M. Purication, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identication as glutaredoxin. Biochem J 1996; 315:931938.

Copyright 2002 by Taylor & Francis Group, LLC