Sebastian J. Padayatty

Vitamin C as an Antioxidant: Evaluation of Its Role in Disease Prevention

Vitamin C in humans must be ingested for survival. Vitamin C is an electron donor, and this property accounts for all its known functions. As an electron donor, vitamin C is a potent water-soluble antioxidant in humans. Antioxidant effects of vitamin C have been demonstrated in many experiments in vitro. Human diseases such as atherosclerosis and cancer might occur in part from oxidant damage to tissues. Oxidation of lipids, proteins and DNA results in specific oxidation products that can be measured in the laboratory. While these biomarkers of oxidation have been measured in humans, such assays have not yet been validated or standardized, and the relationship of oxidant markers to human disease conditions is not clear. Epidemiological studies show that diets high in fruits and vegetables are associated with lower risk of cardiovascular disease, stroke and cancer, and with increased longevity. Whether these protective effects are directly attributable to vitamin C is not known. Intervention studies with vitamin C have shown no change in markers of oxidation or clinical benefit. Dose concentration studies of vitamin C in healthy people showed a sigmoidal relationship between oral dose and plasma and tissue vitamin C concentrations. Hence, optimal dosing is critical to intervention studies using vitamin C. Ideally, future studies of antioxidant actions of vitamin C should target selected patient groups. These groups should be known to have increased oxidative damage as assessed by a reliable biomarker or should have high morbidity and mortality due to diseases thought to be caused or exacerbated by oxidant damage.

Vitamin C Pharmacokinetics: Implications for Oral and Intravenous Use

Context Clinical studies of vitamin C as a potential anticancer agent have produced inconsistent results despite in vitro evidence that high concentrations kill cancer cells. Contribution Pharmacokinetic studies in healthy persons, using a depletion-repletion design, show that intravenous administration can achieve 70-fold higher blood levels of vitamin C than the highest tolerated oral dose. Cautions Although this study provides better understanding of the pharmacokinetic issues involved in research on vitamin C, it provides no evidence that vitamin C has any effect on cancer cells and cannot be used to support its clinical use for therapeutic purposes. The Editors Vitamin C in gram doses is taken orally by many people and administered intravenously by complementary and alternative medicine practitioners to treat patients with advanced cancer (1, 2). After oral intake, vitamin C plasma concentrations are tightly controlled at 70 to 85 mol/L for amounts (as much as 300 mg daily) that can be obtained from food (3, 4). However, concentrations achieved by higher pharmacologic doses are uncertain. Despite poor rationale, vitamin C in gram doses was proposed as an anticancer agent decades ago (5). Unblinded studies with retrospective or nonrandom controls reported clinical benefit from oral and intravenous vitamin C administered to patients with terminal cancer at a dosage of 10 g daily (1, 6, 7). Placebo-controlled trials in patients with cancer reported no benefit from oral vitamin C at a dosage of 10 g daily (8, 9), and vitamin C treatment was judged ineffective (10). However, in vitro evidence showed that vitamin C killed cancer cells at extracellular concentrations higher than 1000 mol/L (11, 12), and its clinical use by some practitioners continues. We recognized that oral or intravenous routes could produce substantially different vitamin C concentrations (13). We report here that intravenous doses can produce plasma concentrations 30- to 70-fold higher than the maximum tolerated oral doses. These data suggest that the role of vitamin C in cancer treatment should be reexamined, and insights from vitamin C pharmacokinetics can guide its clinical use. Methods Pharmacokinetic Studies in Healthy Persons The study was approved by the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. After we obtained written informed consent, 17 healthy volunteers (7 men, 10 women; age, 19 to 27 years) were studied as inpatients by using a depletion-repletion study design (3, 4). Participants were hospitalized for 3 to 6 months and consumed a vitamin C-deficient diet containing less than 0.005 g of vitamin C per day. At plasma vitamin C concentrations less than 8 mol/L, persons were depleted without signs of scurvy. Vitamin C, 0.015 g twice daily, was then administered orally until participants achieved a steady state for this dose (0.03 g daily). Participants received successive oral daily vitamin C doses of 0.03 g, 0.06 g, 0.1 g, 0.2 g, 0.4 g, 1.0 g, and 2.5 g until a steady state was achieved for each dose. Bioavailability sampling was conducted at a steady state for vitamin C doses of 0.015 g, 0.03 g, 0.05 g, 0.1 g, 0.2 g, 0.5 g, and 1.25 g. For each bioavailability sampling, vitamin C was administered in the fasting state. After oral administration, blood samples were collected at 0, 15, and 30 minutes and at 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 19, 22, and 24 hours (3, 4). After intravenous administration at 250 mg/min, blood samples were collected at 0, 2.5, 5, 10, 15, and 30 minutes and at 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, and 10 hours. Data obtained from bioavailability samplings were used to determine peak plasma and urine vitamin C concentrations. Pharmacokinetic Modeling We used data from 7 men to construct a unique 3-compartment vitamin C pharmacokinetic model with parameters describing saturable absorption, tissue distribution, and renal excretion and reabsorption (14). This model was used to predict peak plasma and urine vitamin C concentrations attained when pharmacologic doses of the vitamin are administered. For intravenous administration, it was assumed that vitamin C was infused at a rate of 1 g/min, and urine output was 100 mL/h. Vitamin C Assay Vitamin C was measured by using high-performance liquid chromatography with coulometric electrochemical detection (3, 4, 15). Statistical Analysis We compared plasma vitamin C concentration curves (against either dose or time) by repeated-measures analyses of variance (ANOVA). In addition to the repeating factor (dose or time), other factors considered were sex and route of administration. In the comparison of routes of administration at multiple doses, in which sex not only was an important factor itself but also had an important interaction with route, separate ANOVA were determined for men and women to assess the importance of route of administration. Analyses were performed by using DataDesk, version 5 (1995) (Data Description, Inc., Ithaca, New York). Role of the Funding Source The funding source had no role in the design, conduct, and reporting of the study or in the decision to submit the manuscript for publication. Results When 1.25 g of vitamin C was given intravenously, plasma concentrations were significantly higher than when the vitamin was given orally (P < 0.001 by repeated-measures ANOVA) (Figure 1). In addition, plasma concentrations were significantly higher over all doses (P < 0.001 by repeated-measures ANOVA) with intravenous compared with oral administration (Figure 1, inset). At the highest dose of 1.25 g, mean peak values from intravenous administration were 6.6-fold higher than mean peak values from oral administration. When all doses were considered, peak plasma vitamin C concentrations increased with increasing intravenous doses, whereas peak plasma vitamin C concentrations seemed to plateau with increasing oral doses. Urine vitamin C concentrations were higher for the same dose given intravenously compared with that administered by the oral route. At the highest dose of 1.25 g, peak urine concentrations from intravenous administration were approximately 3.5 times higher than from oral administration (data not shown). Figure 1. Plasma vitamin C concentrations in healthy volunteers after intravenous or oral administration of vitamin C. Inset: The 3-compartment vitamin C pharmacokinetic model that we developed predicted that a single oral dose of 3 g, the maximum tolerated single dose, produced a peak plasma concentration of 206 mol/L (Figure 2, top). Peak predicted concentration after a single 1.25-g oral dose was slightly lower at 187 mol/L. For 200 mg, an amount obtained from vitamin C-rich foods, peak predicted concentration was approximately 90 mol/L. Plasma concentrations for all of these amounts returned to steady-state values, approximately 70 to 85 mol/L, after 24 hours. With 3 g given orally every 4 hours, the maximum tolerable (6), peak predicted plasma concentration was approximately 220 mol/L (Figure 2, top). By contrast, after intravenous administration, predicted peak plasma vitamin C concentrations were approximately 1760 mol/L for 3 g, 2870 mol/L for 5 g, 5580 mol/L for 10 g, 13 350 mol/L for 50 g, and 15 380 mol/L for 100 g (Figure 2, bottom). Doses of 60 g given intravenously are used for cancer treatment by complementary and alternative medicine practitioners (2). Predicted peak urine vitamin C concentrations were as much as 140-fold higher after intravenous administration compared with oral administration (data not shown). Figure 2. Predicted plasma vitamin C concentrations in healthy persons after oral ( top ) or intravenous ( IV ) ( bottom ) administration of vitamin C. Discussion Our data show that vitamin C plasma concentrations are tightly controlled when the vitamin is taken orally, even at the highest tolerated amounts. By contrast, intravenous administration bypasses tight control and results in concentrations as much as 70-fold higher than those achieved by maximum oral consumption. Both findings have clinical relevance. Vitamin C oral supplements are among the most popular sold, and gram doses are promoted for preventing and treating the common cold, managing stress, and enhancing well-being (1). Our data show that single supplement gram doses produce transient peak plasma concentrations that at most are 2- to 3-fold higher than those from vitamin C-rich foods (200 to 300 mg daily). In either case, plasma values return to similar steady-state concentrations in 24 hours. Because differences in plasma concentrations from supplements and from food intake are not large, supplements would be expected to confer little additional benefit, a finding supported by available evidence (16, 17). However, consumption of fruits and vegetables, which contain vitamin C, is beneficial for unknown reasons (16, 17). On the basis of current knowledge and the pharmacokinetics presented here, physicians should advise their patients to consume fruits and vegetables, not vitamin C supplements, to obtain potential benefits. Just as important, our data show that intravenous administration of vitamin C produces substantially higher plasma concentrations than can be achieved with oral administration of vitamin C. This difference was previously unrecognized and may have treatment implications. Case series published by Cameron, Campbell, and Pauling (l, 6, 7) have been controversial. In these series, several hundred patients with terminal cancer treated with 10 g of vitamin C intravenously for 10 days and then 10 g orally indefinitely were compared with more than 1000 retrospective and prospective controls. Patients treated with vitamin C survived 150 to 300 days longer than controls (1, 6, 7). Other researchers reported benefit consisting of increased survival, improved well-being, and reduced pain (1). All of these studies

A new recommended dietary allowance of vitamin C for healthy young women

The recently released Recommended Dietary Allowance of vitamin C for women, 75 mg daily, was based on data for men. We now report results of a depletion–repletion study with healthy young women hospitalized for 186 +/− 28 days, using vitamin C doses of 30–2,500 mg daily. The relationship between dose and steady-state plasma concentration was sigmoidal. Only doses above 100 mg were beyond the linear portion of the curve. Plasma and circulating cells saturated at 400 mg daily, with urinary elimination of higher doses. Biomarkers of endogenous oxidant stress, plasma and urine F2-isoprostanes, and urine levels of a major metabolite of F2-isoprostanes were unchanged by vitamin C at all doses, suggesting this vitamin does not alter endogenous lipid peroxidation in healthy young women. By using Food and Nutrition Board guidelines, the data indicate that the Recommended Dietary Allowance for young women should be increased to 90 mg daily.

Phase I clinical trial of i.v. ascorbic acid in advanced malignancy

BACKGROUND Ascorbic acid is a widely used and controversial alternative cancer treatment. In millimolar concentrations, it is selectively cytotoxic to many cancer cell lines and has in vivo anticancer activity when administered alone or together with other agents. We carried out a dose-finding phase I and pharmacokinetic study of i.v. ascorbic acid in patients with advanced malignancies. PATIENTS AND METHODS Patients with advanced cancer or hematologic malignancy were assigned to sequential cohorts infused with 0.4, 0.6, 0.9 and 1.5 g ascorbic acid/kg body weight three times weekly. RESULTS Adverse events and toxicity were minimal at all dose levels. No patient had an objective anticancer response. CONCLUSIONS High-dose i.v. ascorbic acid was well tolerated but failed to demonstrate anticancer activity when administered to patients with previously treated advanced malignancies. The promise of this approach may lie in combination with cytotoxic or other redox-active molecules.

Intravenously administered vitamin C as cancer therapy: three cases

Early clinical studies showed that high-dose vitamin C, given by intravenous and oral routes, may improve symptoms and prolong life in patients with terminal cancer. Double-blind placebo-controlled studies of oral vitamin C therapy showed no benefit. Recent evidence shows that oral administration of the maximum tolerated dose of vitamin C (18 g/d) produces peak plasma concentrations of only 220 μmol/L, whereas intravenous administration of the same dose produces plasma concentrations about 25-fold higher. Larger doses (50–100 g) given intravenously may result in plasma concentrations of about 14 000 μmol/L. At concentrations above 1000 μmol/L, vitamin C is toxic to some cancer cells but not to normal cells in vitro. We found 3 well-documented cases of advanced cancers, confirmed by histopathologic review, where patients had unexpectedly long survival times after receiving high-dose intravenous vitamin C therapy. We examined clinical details of each case in accordance with National Cancer Institute (NCI) Best Case Series guidelines. Tumour pathology was verified by pathologists at the NCI who were unaware of diagnosis or treatment. In light of recent clinical pharmacokinetic findings and in vitro evidence of anti-tumour mechanisms, these case reports indicate that the role of high-dose intravenous vitamin C therapy in cancer treatment should be reassessed.

Vitamin C: A Concentration-Function Approach Yields Pharmacology and Therapeutic Discoveries

A concentration-function approach to vitamin C (ascorbate) has yielded new physiology and pharmacology discoveries. To determine the range of vitamin C concentrations possible in humans, pharmacokinetics studies were conducted. They showed that when vitamin C is ingested by mouth, plasma and tissue concentrations are tightly controlled by at least 3 mechanisms in healthy humans: absorption, tissue accumulation, and renal reabsorption. A 4th mechanism, rate of utilization, may be important in disease. With ingested amounts found in foods, vitamin C plasma concentrations do not exceed 100 μmol/L. Even with supplementation approaching maximally tolerated doses, ascorbate plasma concentrations are always <250 μmol/L and frequently <150 μmol/L. By contrast, when ascorbate is i.v. injected, tight control is bypassed until excess ascorbate is eliminated by glomerular filtration and renal excretion. With i.v. infusion, pharmacologic ascorbate concentrations of 25-30 mmol/L are safely achieved. Pharmacologic ascorbate can act as a pro-drug for hydrogen peroxide (H(2)O(2)) formation, which can lead to extracellular fluid at concentrations as high as 200 μmol/L. Pharmacologic ascorbate can elicit cytotoxicity toward cancer cells and slow the growth of tumors in experimental murine models. The effects of pharmacologic ascorbate should be further studied in diseases, such as cancer and infections, which may respond to generation of reactive oxygen species via H(2)O(2).

Vitamin C transporter Slc23a1 links renal reabsorption, vitamin C tissue accumulation, and perinatal survival in mice

Levels of the necessary nutrient vitamin C (ascorbate) are tightly regulated by intestinal absorption, tissue accumulation, and renal reabsorption and excretion. Ascorbate levels are controlled in part by regulation of transport through at least 2 sodium-dependent transporters: Slc23a1 and Slc23a2 (also known as Svct1 and Svct2, respectively). Previous work indicates that Slc23a2 is essential for viability in mice, but the roles of Slc23a1 for viability and in adult physiology have not been determined. To investigate the contributions of Slc23a1 to plasma and tissue ascorbate concentrations in vivo, we generated Slc23a1-/- mice. Compared with wild-type mice, Slc23a1-/- mice increased ascorbate fractional excretion up to 18-fold. Hepatic portal ascorbate accumulation was nearly abolished, whereas intestinal absorption was marginally affected. Both heterozygous and knockout pups born to Slc23a1-/- dams exhibited approximately 45% perinatal mortality, and this was associated with lower plasma ascorbate concentrations in dams and pups. Perinatal mortality of Slc23a1-/- pups born to Slc23a1-/- dams was prevented by ascorbate supplementation during pregnancy. Taken together, these data indicate that ascorbate provided by the dam influenced perinatal survival. Although Slc23a1-/- mice lost as much as 70% of their ascorbate body stores in urine daily, we observed an unanticipated compensatory increase in ascorbate synthesis. These findings indicate a key role for Slc23a1 in renal ascorbate absorption and perinatal survival and reveal regulation of vitamin C biosynthesis in mice.

Reevaluation of Ascorbate in Cancer Treatment: Emerging Evidence, Open Minds and Serendipity

Some clinicians and alternative therapy practitioners advocate megadose intravenous and oral ascorbate treatment of cancer. Randomized control studies using oral ascorbate showed no benefit. Recent data show that intravenous but not oral administration of ascorbate can produce millimolar plasma concentrations, which are toxic to many cancer cell lines. We propose that ascorbate treatment of cancer should be reexamined by rigorous scientific scrutiny in the light of new evidence.

Standard-Dose vs High-Dose Multivitamin Supplements for HIV

1. Fergusson DA, Hébert P, Hogan DL, et al. Effect of fresh red blood cell transfusions on clinical outcomes in premature, very low-birth-weight infants: the ARIPI randomized trial. JAMA. 2012;308(14):1443-1451. 2. Bell EF, Strauss RG, Widness JA, et al. Randomized trial of liberal versus restrictive guidelines for red blood cell transfusion in preterm infants. Pediatrics. 2005; 115(6):1685-1691. 3. Singh R, Visintainer PF, Frantz ID III, et al. Association of necrotizing enterocolitis with anemia and packed red blood cell transfusions in preterm infants. J Perinatol. 2011;31(3):176-182. 4. Marik PE, Sibbald WJ. Effect of stored-blood transfusion on oxygen delivery in patients with sepsis. JAMA. 1993;269(23):3024-3029. 5. Department of Health and Human Services; Office of the Assistant Secretary for Health. The 2009 national blood collection and utilization survey report. http: //www.hhs.gov/ash/bloodsafety/nbcus/index.html. Accessed October 19, 2012.

Vitamin and Mineral Supplements in the Primary Prevention of Cardiovascular Disease and Cancer

TO THE EDITOR: Fortmann and colleagues meta-analysis (1) showed that vitamin and mineral supplementation provides little benefit and does not aid cognition (2) or prevent cardiovascular events (3). The authors concluded that supplements have no value. However, data constraints limit interpretation to a narrower conclusion: Supplementation in well-nourished persons does not affect the end points studied but may have other benefits or abolish signs and symptoms of unrecognized deficiencies, which are surprisingly common. Many studies of vitamin supplements are flawed, including Fortmann and colleagues review, because vitamin concentrations at enrollment are usually not measured. It is predictable that study populations include those with low concentrations of vitamins, subclinical deficiencies, or both and others who are vitamin replete. These groups are distinguishable only if baseline, and preferably postsupplementation, vitamin concentrations are measured. Without measurement, assuming that all participants are vitamin and mineral replete is unsafe. Benefits in one group may be hidden by no effect in the other. Measurement of vitamin concentrations before and after supplementation will allow distinct effects to be recognized. For example, 22% and 7% of U.S. adults are considered vitamin C insufficient (serum vitamin C concentrations <28 mol/L) or deficient (<11 mol/L), respectively (4). Only those with concentrations less than 5 mol/L may exhibit scurvy. In contrast, vitamin C concentrations of approximately 50 to 60 mol/L are predictable in persons consuming the recommended dietary allowance of vitamin C; additional vitamin C intake increases concentrations only modestly at best among these persons. To put the problem in perspective, studies to test antihypertensive therapy would not be done without measuring blood pressure at enrollment. To treat everyone regardless of blood pressure would be illogical. However, this strategy has been persistently pursued in evaluating vitamin supplements. Vitamin and mineral supplements may benefit persons with marginal deficiencies. For example, patients with plasma vitamin C concentrations less than 20 mol/L report lassitude, a symptom that precedes scurvy (5). However, lassitude is common and nonspecific, and marginal vitamin C deficiency is difficult to recognize. Marginal vitamin and mineral deficiencies, even without obvious clinical disease, may decrease quality of life or have long-term adverse effects. This factor may be an adequate reason for supplement use, even if it does not prevent diseases other than deficiency states. Meticulous attention to the design and conduct of clinical trials, many study participants, and meta-analyses cannot compensate for fundamental physiologic oversights about vitamin doses and concentrations. Perhaps vitamin and mineral supplementation has little value in the general population, but these studies are inadequate to show it.