Pharmacologic ascorbic acid concentrations
selectively kill cancer cells: Action as a pro-drug
to deliver hydrogen peroxide to tissues
Qi Chen*†, Michael Graham Espey‡, Murali C. Krishna‡, James B. Mitchell‡, Christopher P. Corpe*, Garry R. Buettner§,
Emily Shacter†, and Mark Levine*¶*Molecular and Clinical Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,Bethesda, MD 20892; ‡Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; §Free Radical
and Radiation Biology Program, University of Iowa, Iowa City, IA 52242-1101; and †Laboratory of Biochemistry, Center for Drug Evaluation
and Research, Food and Drug Administration, Bethesda, MD 20892
Communicated by J. E. Rall, National Institutes of Health, Bethesda, MD, August 2, 2005 (received for review June 1, 2005)

Human pharmacokinetics data indicate that i.v. ascorbic acid(ascorbate) in pharmacologic concentrations could have an unanticipated role in cancer treatment. Our goals here were to test
whether ascorbate killed cancer cells selectively, and if so, todetermine mechanisms, using clinically relevant conditions. Cell
death in 10 cancer and 4 normal cell types was measured by using
1-h exposures. Normal cells were unaffected by 20 mM ascorbate,
whereas 5 cancer lines had EC50 values of <4 mM, a concentration
easily achievable i.v. Human lymphoma cells were studied in detailbecause of their sensitivity to ascorbate (EC50 of 0.5 mM) andsuitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis兾necrosis. Cell death was independent of metalchelators and absolutely dependent on H2O2 formation. Cell deathfrom H2O2 added to cells was identical to that found when H2O2
was generated by ascorbate treatment. H2O2 generation wasdependent on ascorbate concentration, incubation time, and thepresence of 0.5–10% serum, and displayed a linear relationshipwith ascorbate radical formation. Although ascorbate addition to
medium generated H2O2, ascorbate addition to blood generated
no detectable H2O2 and only trace detectable ascorbate radical.
Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug forformation of H2O2, and that blood can be a delivery system of the
pro-drug to tissues. These findings give plausibility to i.v. ascorbic
acid in cancer treatment, and have unexpected implications fortreatment of infections where H2O2 may be beneficial.
cell death 兩 ascorbate radicalA

scorbic acid (vitamin C, ascorbate) has a controversial history
in cancer treatment (1). Observational reports describedascorbate, given in pharmacologic doses of 10 g daily, as effectivein treating some cancers and in improving patient well-being (2–4).
Subsequently, the same dose had no effect on patient well-being
and survival in two double-blind placebo-controlled trials, andascorbate was discarded as a treatment modality (5, 6). Recent
clinical evidence, however, indicates that the role of ascorbate incancer treatment should be examined anew (7). The originallyreported observational studies used i.v. and oral ascorbate, but thesubsequent double-blind placebo-controlled studies used only oral
ascorbate. It was not recognized that the route of ascorbate
administration might produce large differences in plasma concentrations. Recent pharmacokinetics studies in men and women showthat 10 g of ascorbate given i.v. is expected to produce plasma
concentrations of nearly 6 mM, which are ⬎25-fold higher thanthose concentrations from the same oral dose (7–9). As much as a
70-fold difference in plasma concentrations is expected between
oral and i.v. administration, depending on dose. Despite inconsistencies, some in vitro studies showed that ascorbate killed cancer
cells, although mechanisms and physiologic relevance were unclear
13604 –13609 兩 PNAS 兩 September 20, 2005 兩 vol. 102 兩 no. 38(10–12). Complementary and alternative medicine practitioners
worldwide currently use ascorbate i.v. in some patients, in part
because there is no apparent harm (13–15).
Given its potential safety and benefit, there is merit in investigating i.v. ascorbate as a possible novel cancer treatment modality.
It is essential first to learn whether ascorbate acts as an anticancer
agent in vitro, and if so, by what mechanisms. Our goals were toaddress the following: Does ascorbate in pharmacologic concentrations kill cancer cells, but not normal cells, using conditions thatmimic i.v. use and a clinically relevant time course? Is action
dependent on extracellular ascorbate, intracellular ascorbate, orboth? If effective, what are the mechanisms? Can ascorbate bedelivered to tissues without harm? Are there implications for otherdiseases?
We studied ascorbate at physiologic (0.1 mM) and pharmacologic (0.3–20 mM) concentrations using 1-h incubations to mimicclinical i.v. use (7–9). The data showed that pharmacologic concentrations of ascorbate killed cancer but not normal cells, that cell
death was dependent only on extracellular but not intracellular
ascorbate, and that killing was dependent on extracellular hydrogen
peroxide (H2O2) formation with ascorbate radical as an intermediate. Ascorbate generated detectable levels of H2O2 in extracellular medium in the presence of trace serum protein but not inwhole blood. The findings indicate that ascorbate at pharmacologic
concentrations in blood may be a pro-drug for H2O2 delivery totissues, with major therapeutic implications.
Materials and Methods
Cells and Reagents. Human Burkitt’s lymphoma cells (JLP-119)
were obtained and studied as described in ref. 16. Other cell lines
were purchased from American Type Culture Collection and weregrown at 37°C in 5% CO2兾95% air in recommended media
containing 10% FBS (GIBCO). Human lymphocytes and monocytes were isolated by apheresis (17) from at least six healthy
subjects and used immediately. Ascorbic acid was always buffered
to pH 7.0 with sodium hydroxide and prepared immediately before
use. Dehydroascorbic acid was freshly prepared (18). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
was purchased from Molecular Probes and bacto-agar was from
Difco. Other reagents, enzymes, and media were from generalcommercial sources.
Cell Death. Nuclear staining with Hoechst 33342 (Hoechst Pharmaceuticals) and propidium iodide (PI) was used for morphologicalAbbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI,
propidium iodide.
¶To whom correspondence should be addressed at: Molecular and Clinical Nutrition

Section, National Institutes of Health, Building 10, Room 4D52, MSC-1372, Bethesda,
MD 20892-1372. E-mail: markl@mail.nih.gov.
© 2005 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0506390102

Quantitative Procedures. Catalase activity was determined by using

Amplex Red (Molecular Probes) (22). Glutathione was detected byusing 5,5⬘-dithio bis-2-nitrobenzoic acid, and glutathione peroxidase activity was measured by a coupled reaction with glutathione
reductase (Cayman Chemical, Ann Arbor, MI), according to the
manufacturer’s instructions.
Ascorbate radical in culture media and blood was detected byusing electron paramagnetic resonance (23, 24). Spectrometer (E9series, Varian) settings were as follows: microwave power, 20 mW;
modulation amplitude, 1.0 G; time constant, 0.25 s; scan range, 4 ⫻10 G; and scan time, 4 min. Radical quantitation was performed byusing 3-carboxyproxyl as a standard (23).
Because ascorbate interferes with most peroxidase-based detection methods, H2O2 was measured by using a Clark-type oxygen
electrode (5兾6 Oxygraph, Gilson Medical Electronics, Middleton,
WI). Oxygen evolution was measured upon introduction of catalase: 2H2O2 3 2H2O ⫹ O2. Calibration was performed with freshly
prepared solutions of H2O2 (10–200 ␮M) (25).
Ascorbate was measured by HPLC with coulometric electrochemical detection (26). Protein was determined by using bicinchoninic acid (27). Cell volumes were determined by using a
Coulter Multisizer II cell counter. Intracellular ascorbate concentrations were calculated by converting cell protein to a measured
intracellular volume (18).
Results
Effects of Ascorbic Acid in Pharmacologic Concentrations on Survival
of Tumor and Normal Cells. We first investigated whether ascorbate

in pharmacologic concentrations selectively affected the survival ofcancer cells by studying nine cancer cell lines, four normal cell types,
and clinically relevant conditions. Clinical pharmacokinetics analyses show that pharmacologic concentrations of plasma ascorbate,from 0.3 to 15 mM, are achievable only from i.v. administration (7).
These concentrations are cleared within hours by renal filtrationand excretion. In contrast, plasma ascorbate concentrations frommaximum possible oral doses cannot exceed 0.22 mM because oflimited intestinal absorption, which is bypassed with i.v. administration (7–9). To mimic potential clinical i.v. use, tested cells wereincubated for 1 h with either pharmacologic ascorbate concentrations (0.3–20 mM) or a high physiologic concentration (0.1 mM) as
control. Once ascorbate was removed, cell survival was determinedby nuclear staining or MTT after 24 h (Fig. 1A). For five of the ninecancer cell lines, ascorbate concentrations causing a 50% decreasein cell survival (EC50 values) were less than 5 mM, a concentration
Chen et al.

Fig. 1. Effects of pharmacologic ascorbic acid concentrations on cancer andnormal cells. Concentrations in this and all figures indicate final concentrations.
(A) EC50 values of ascorbate in human and mouse cancer cells and normal humancells. All cells were treated with ascorbate for 1 h, washed, and recultured without
ascorbate. EC50 values were determined 18 –22 h later by using Hoechst兾PI for
human Burkitt’s lymphoma cells (JLP119), MTT and Hoechst兾PI for normal lymphocytes and monocytes, and MTT for all other cells (see Materials and Methods).
(B) Colony formation of cancer cells in soft agar after a 1-h treatment with 5 mM
ascorbate. Surviving fraction, expressed in log scale, indicates the number oftreated colonies compared with matched untreated control cells.

easily achievable from i.v. infusion (7). All tested normal cells wereinsensitive to 20 mM ascorbate.
Colony formation assays were used as an additional means todetermine cell survival (21). Four cancer cell lines were incubatedwith 5 mM ascorbate or untreated media for 1 h. Cells were diluted
and plated and growth assessed after 14 days (Fig. 1B). All four
untreated cell lines grew in soft agar, whereas three of four exposed
to ascorbate displayed at least 99% growth inhibition.
Effects of Ascorbic Acid on Death of Human Lymphoma Cells. Humanlymphoma cells (JLP-119) were studied in detail to determine theeffects of ascorbate on cell death. Lymphoma cells were selected
because of their sensitivity to ascorbate (Fig. 1 A), the suitability ofthese cells for nuclear staining to characterize the mode of cell
death (16, 19, 28), and the report of a positive clinical response oflymphoma to i.v. ascorbate (14) (unpublished work). Cells wereincubated for 1 h with 0.1–5 mM ascorbate and washed, andHoechst兾PI nuclear staining was performed 18 h later to determine
the amount and type of cell death (Fig. 2A). Ascorbate induced
concentration-dependent cell death, which was nearly 100% at 2
mM. As ascorbate concentration increased, the pattern of deathchanged from apoptosis to pyknosis兾necrosis, a pattern suggestiveof H2O2-mediated cell death (19). We determined the time necessary for cell death after exposure to 2 mM ascorbate for 1 h (Fig.
2B). Apoptosis occurred by 6 h after exposure, and cell death by
pyknosis was ⬇90% at 14 h after exposure. In contrast to lymphoma
cells, there was little or no killing of normal lymphocytes andmonocytes by ascorbate (Fig. 2C).
The roles of intracellular versus extracellular ascorbate in causing
cell death were examined, using ascorbate and its oxidized productdehydroascorbic acid. Ascorbate is transported into cells as such bysodium-dependent transporters, whereas dehydroascorbic acid istransported into cells by glucose transporters and then immediately
reduced internally to ascorbate (29). By using either external
ascorbate or external dehydroascorbic acid, lymphoma cells were
loaded to equal internal concentrations of ascorbate over 1 h (data
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assessment of apoptosis, necrosis, and pyknosis兾necrosis by fluorescence microscopy as described in ref. 19. Briefly, 2.5 ⫻ 105 cells
per ml were incubated with ascorbate or H2O2 for 1 h, washed with
PBS, and suspended in fresh media. After 18–22 h, at least 200 cells
were stained with Hoechst兾PI and visualized under fluorescencemicroscopy.
MTT was used as a screening assay and performed as describedin ref. 20. Cells in 96-well plates were treated with ascorbate (0.1–20mM) for 1 h, washed, and incubated for an additional 24 h. The
EC50 value was the concentration that reduced survival by 50%.
For colony formation on soft agar plates, cells were treated with
5 mM ascorbate for 1 h, washed, and plated. A two-layer agar
system was used, and colonies were visualized after 10–14 days (21).
To determine the effects of red blood cells on ascorbate-induced
cell death, red blood cells were prepared by centrifugation of
heparinized human blood at 500 ⫻ g for 30 min. Human Burkitt’s
lymphoma cells at 2.5 ⫻ 105 cells per ml were mixed with red blood
cells, 25% or 50% hematocrit. Cell mixtures were treated with 2
mM ascorbate for 1 h. Lymphoma cells were recovered by using
Vacutainer CPT tubes (Becton Dickinson) according to the manufacturer’s instructions. After washing, lymphoma cells were returned to fresh medium and assessed after 18 h by nuclear staining
as above.

Similar to most cultured cells, lymphoma cells contain no ascorbate unless the vitamin is added to the extracellular medium (datanot shown) (17). In contrast, excepting red blood cells, all cells invivo or acutely isolated contain ascorbate, usually in millimolar
concentrations. We investigated whether the prior presence of
intracellular ascorbate affected death mediated by extracellular
ascorbate. Lymphoma cells were preloaded with physiologic concentrations of ascorbate to produce millimolar intracellular concentrations, similar to normal lymphocytes (8, 9). Their response toexternal ascorbate was compared with unloaded cells (Fig. 2E).
Whether or not intracellular ascorbate was preloaded, extracellular
ascorbate induced the same amount and type of death. Taken
together, the data in Fig. 2 A–E indicate that extracellular ascorbate
in pharmacologic concentrations mediates death of lymphoma cells
by apoptosis and pyknosis兾necrosis, independently of intracellular
ascorbate.
Mechanism of Ascorbate-Mediated Cell Killing. To determine theFig. 2. Effects of ascorbic acid on human Burkitt’s lymphoma cells. Cells weretreated for 1 h, washed, and recultured without ascorbate. Amounts and types ofcell death were determined 18 –22 h later by nuclear staining with Hoechst兾PI.
Types of cell death: necrosis (black), pyknosis兾necrosis (gray), early apoptosis(blue), and late apoptosis (red). (A) Amount and type of cell death as a functionof external ascorbate concentration. (B) Time course and type of cell death after
1 h external ascorbate (2 mM). (C) Cell death as a function of external ascorbate
concentration in human Burkitt’s lymphoma cells (⽧), normal lymphocytes (■),
and normal monocytes (Œ). (D) Cell death as a function of external ascorbate (⽧)
or dehydroascorbic acid (䊐) concentrations (1-h incubation). (E) Type and amountof cell death with 2 mM ascorbate treatment, in cells previously loaded to contain
3 mM ascorbate (right), compared with unloaded cells (left).

not shown). Despite similar intracellular ascorbate concentrations
under both conditions, cells died only when ascorbate was present
externally (Fig. 2D).
13606 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0506390102

mechanism of ascorbate-mediated lymphoma cell death, we tested
the effects of the membrane-impermeant H2O2-scavenger catalase,
the membrane-permeant H2O2-scavenger tetrakis (4-benzoic acid)meso-substituted manganoporphyrin (MnTBAP) (30), and thethiol-reducing agent Tris (2-carboxyethyl) phosphine hydrochloride(TCEP) (31). We also tested whether adventitious transition metals
were responsible, by using the membrane impermeant chelator
diethylenetriamine-pentaacetic acid (DTPA) (32) and the membrane permeant chelator N,N⬘-bis(2-hydroxybenzyl)ethylenediamine-N,N⬘-diacetic acid (HBED) (33–35) (Fig. 3A). The H2O2
scavengers were completely protective, identifying H2O2 as theeffector species mediating pharmacologic ascorbate-induced cell
death. The effect of ascorbate was not due to chelatable, trace
redox-active metals, because the two chelators had no effect on
preventing death. Superoxide dismutase was not protective (datanot shown), consistent with its action in producing but not degrading H2O2 (36).
Because these data implicated H2O2 in cell killing, we added
H2O2 to lymphoma cells and studied death patterns using nuclear
staining (19, 28). The death patterns found with exogenous H2O2
exposure were similar to those found with ascorbate. For bothascorbate and H2O2, death changed from apoptosis to pyknosis兾
necrosis as concentrations increased (Fig. 3B).
As a specific test of ascorbate action, the amount of H2O2 formedin the presence of ascorbate was measured by using an oxygen
electrode. We compared the effects on cell death of H2O2 amounts
formed in the presence of ascorbate to effects from exogenously
added H2O2. H2O2 generated by ascorbate oxidation and exogenously added H2O2 produced cell death curves that were indistinguishable (Fig. 3C).
Sensitivity to direct exposure to H2O2 was greater in lymphoma
cells compared with normal lymphocytes and normal monocytes(Fig. 3D), consistent with the cytotoxicity pattern found above withpharmacologic ascorbate exposure. Taken together, these data areconsistent with the conclusion that extracellular ascorbate induced
cell death by formation of H2O2.
We investigated whether activities of intracellular H2O2-removal
systems correlated with ascorbate-mediated cell death, for all cells
studied. There was no association between the EC50 for ascorbatemediated cell death and intracellular glutathione concentrations,catalase activity, or glutathione peroxidase activity (data notshown).
Mediators and Inhibitors of H2O2 Generation. H2O2 concentrations
generated by ascorbate were similar with tumor cells, normal cells,
or in medium without cells (data not shown), as measured by using
an oxygen electrode as above. H2O2 generation was dependent ontime, ascorbate concentration, and the presence of trace amountsof serum in media (Fig. 4 A and B).
Based on these data, the most cogent explanation of ascorbate
Chen et al.

Fig. 3. Extracellular ascorbate kills human Burkitt’s lymphoma cells by generating H2O2. Cell death determined and symbolized as in Fig. 2; H2O2 measured byoxygen electrode (see Materials and Methods). (A) Effects of reactive oxygen
species quenchers兾scavengers, reducing agent, and metal chelators on ascorbatemediated cell death. The following (final concentrations) were preincubatedwith cells for 30 min before exposure to ascorbate (2 mM): catalase (100 ␮g兾ml);
tetrakis (4-benzoic acid) meso-substituted manganoporphyrin (MnTBAP) (100␮M); Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (500 ␮M); diethylenetriamine-pentaacetic acid (DTPA) (1 mM); and N,N⬘-bis(2-hydroxybenzyl)ethylenediamine-N,N⬘-diacetic acid (HBED) (50 ␮M). (B) Type and amount of cell death
as function of added H2O2 (final concentrations). (C) Cell death as a function ofadded H2O2 for 1 h (⽧) or mean H2O2 concentration generated by 0.2–2 mMascorbate during a 1-h incubation (‚). (D) Cell death in human Burkitt’s lymphoma cells (⽧), normal lymphocytes (■), and normal monocytes (Œ) as functionof added H2O2 (final concentrations).

action in forming H2O2 is that the first step is ascorbate oxidation
to its radical. We measured H2O2 concentration as a function of
ascorbate radical concentration and found a linear relationship(Fig. 4C). These data imply that ascorbate radical is a surrogate
marker for H2O2 formation.
For ascorbate to be useful clinically, it should increase theChen et al.

steady-state concentration of H2O2 in the extracellular milieu but
not in blood. We predicted that steady-state concentrations of H2O2
generated by ascorbate oxidation would be undetectable in bloodfor several reasons. First, if any ascorbate radical is generated inblood, only very low concentrations are expected, and such concentrations should be lower than that needed to form detectablesteady-state concentrations of H2O2 (37). Second, whatever H2O2
is generated should be removed by glutathione peroxidase andcatalase within red blood cells, because H2O2 is membrane permeable (38–41). These predictions were explored in the following
experiments. First, ascorbate (0–10 mM) was added to whole bloodand to medium, and ascorbate radical was measured by electron
paramagnetic resonance. Ascorbate radical in whole blood was notdetectable when ascorbate concentrations were ⬍3 mM and waspresent at minimal concentrations thereafter. In contrast, there wasrobust ascorbate radical generation in medium, a surrogate forextracellular fluid (Fig. 5A). Second, as direct tests, H2O2 concentrations were measured under the following conditions: In whole
blood in the presence of varying concentrations of ascorbate, inwhole blood after exogenous H2O2 addition, and in medium withvarying concentrations of ascorbate (Fig. 5B). H2O2 was notdetected in whole blood under either condition, even in thepresence of far higher added concentrations than could be generPNAS 兩 September 20, 2005 兩 vol. 102 兩 no. 38 兩 13607

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Fig. 4. Enhancing factors for ascorbate-mediated H2O2 generation in cell
culture medium. H2O2 was measured by oxygen electrode, and ascorbate
radical was measured by electron paramagnetic resonance. (A) H2O2 formation as function of time and ascorbate concentration: 0.2 mM (⫻), 0.5 mM (Œ),
1 mM (䊐), and 2 mM (⽧). (B) H2O2 formation as a function of the percentage
of FBS for 1 h (2 mM ascorbate). (C) H2O2 formation as a function of ascorbate
radical formation (0.2–2 mM ascorbate, 1-h incubation).

Fig. 5. Human blood inhibits H2O2 and ascorbate radical generation fromascorbate. Ascorbate radical was measured by electron paramagnetic resonance, H2O2 was measured by oxygen electrode, and cell death was measured
and displayed as in Fig. 2. (A) Ascorbate radical formation as function of
ascorbate concentrations added to blood (⽧) or medium (Œ). (B) H2O2 generated by ascorbate concentrations added to blood (⽧) or medium (Œ) (1-hincubation), and H2O2 measured in blood immediately after the addition ofindicated concentrations (‚). (C) Human Burkitt’s lymphoma cell death in thepresence or absence of red blood cells (RBC) at 25% or 50% hematocrit (HCT)(2 mM ascorbate, 1-h treatment).

ated by ascorbate oxidation. Control formation of H2O2 as afunction of ascorbate concentration in medium proceeded as
expected. These data indicate that even if ascorbate radical wasformed in blood and H2O2 was generated, it would be immediately
scavenged to concentrations below detection limits. Based on these
data, an additional functional experiment was conducted, based on
the prediction that blood would protect tumor cells from ascorbatemediated cell death. Lymphoma cells were incubated in the presence or absence of red blood cells, with and without added
ascorbate. Red blood cells completely protected lymphoma cells
from ascorbate-mediated cell death (Fig. 5C). Taken together,
these data indicate that ascorbate cannot generate sustainable
H2O2 concentrations in whole blood. The data are consistent withthe hypothesis that ascorbate in pharmacologic concentrations is a
pro-drug for H2O2 generation in the extracellular milieu but not inblood.
Discussion
Our data show that ascorbic acid selectively killed cancer but notnormal cells, using concentrations that could only be achieved by i.v.
13608 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0506390102

administration and conditions that reflect potential clinical use.
The effect was due only to extracellular and not intracellular
ascorbate, consistent with clinical i.v. dosing. Ascorbate-mediatedcell death was due to protein-dependent extracellular H2O2 generation, via ascorbate radical formation from ascorbate as theelectron donor. Like glucose, when ascorbate is infused i.v., theresulting pharmacologic concentrations should distribute rapidly inthe extracellular water space (42). We showed that such pharmacologic ascorbate concentrations in media, as a surrogate forextracellular fluid, generated ascorbate radical and H2O2. In contrast, the same pharmacologic ascorbate concentrations in whole
blood generated little detectable ascorbate radical and no detectable H2O2. These findings can be accounted for by efficient andredundant H2O2 catabolic pathways in whole blood (e.g., catalase
and glutathione peroxidase) relative to those in media or extracellular fluid (38–41). The totality of the data are consistent with theinterpretation that ascorbic acid administered i.v. in pharmacologic
concentrations may serve as a pro-drug for H2O2 delivery to theextracellular milieu, but without H2O2 accumulation in blood.
Although it is possible that H2O2 might accumulate in blood, thiswould occur only under specific conditions that reflect on thegeneral safety of i.v. ascorbate. Ascorbate administered i.v. is likely
to be safe in most patients, with virtually no toxicity compared tomost currently available cancer chemotherapeutic agents. The
occurrence of one predicted complication, oxalate kidney stones, iscontroversial (13). In patients with glucose-6-phosphate dehydrogenase deficiency, i.v. ascorbate is contraindicated because it causes
intravascular hemolysis (13). The mechanism of this previously
unexplained observation is now straightforward, based on the
results here. H2O2 generated in blood is normally removed bycatalase and glutathione peroxidase within red blood cells, withinternal glutathione providing reducing equivalents. The electron
source for glutathione is NADPH from the pentose shunt, via
glucose-6-phosphate dehydrogenase. If activity of this enzyme isdiminished, the predicted outcome is impaired H2O2 removalcausing intravascular hemolysis, the observed clinical finding.
Ascorbate as a potential cancer therapeutic agent has a controversial and emotionally charged past (1, 3–6). Clinical observational
studies reported possible benefit in selected patients, but doubleblind placebo-controlled studies reported no benefit, and ascorbate
was discarded as a potential therapy by conventional practitioners.
Only recently has it been understood that the discordant clinicalfindings can be explained by previously unrecognized fundamental
pharmacokinetics properties of ascorbate (7). In vitro effects ofascorbate on death and survival of cell lines have been reported, butthere are multiple experimental concerns. For example, reports
compared an experimental condition to that with no ascorbate atall (43, 44), but such a condition has had unclear physiologic
relevance, because ascorbate outside and inside cells is always
present unless there is severe scurvy. It was unclear whether
observed effects were due to extracellular or intracellular ascorbate,or both (12, 43–46). Some experiments have used widely varying
incubation times and ascorbate concentrations that have had nocorresponding clinical context, making interpretation difficult.
H2O2 generation by ascorbate oxidation in culture media wasvariously interpreted as artifact (47, 48), even though chelators had
no effect (49), or reported to mediate damage internally due todiminished intracellular ascorbate, but using an H2O2 assay inwhich ascorbate could interfere (43, 44).
The experiments presented here provide a clear clinical context
for ascorbate action. Conditions were selected to reflect peak
ranges of i.v. ascorbate concentrations, which clinically might last a
few hours at most, depending on the infusion rate (7). Intracellulartransport of ascorbate is tightly controlled in relation to extracellular concentration (8, 9, 29). Intravenous ascorbate infusion is
expected to drastically change extracellular but not intracellularconcentrations (8, 9). For i.v. ascorbate to be clinically useful inkilling cancer cells, pharmacologic but not physiologic extracellular
Chen et al.

concentrations should be effective, independent of intracellular
ascorbate concentrations. This was what was observed here. The
experiments here provide a cohesive explanation for ascorbate
action in generating H2O2 outside cells, without H2O2 accumulation in blood, leading to the conclusion that ascorbate at pharmacologic concentrations in blood is a pro-drug for H2O2 delivery totissues.
We observed that H2O2 generation was independent of metalchelators and dependent on at least 0.5% extracellular protein. The
responsible proteins were between 10 and 30 kDa (data not shown).
It is reasonable that extracellular milieu contains these proteins,
given that extracellular milieu protein is as much as 20% of serum
protein, and favors lower-molecular-weight proteins (50). Although
identities of the proteins responsible are unknown, we postulatethat they may have redox-active metal centers. While chelators may
marginally affect these metals, they could participate in the oxidation of ascorbate when it is at pharmacologic concentrations, withsubsequent formation of superoxide and H2O2 (34). It is also
possible that in vivo, cell membranes and their associated proteins
could harbor metals accessible to extracellular fluid and could react
similarly. In either case, ascorbate, an electron-donor in suchreactions, ironically initiates pro-oxidant chemistry and H2O2 formation (34, 51).
It is unknown why ascorbate, via H2O2, killed some cancer cells
but not normal cells. There was no correlation with ascorbateinduced cell death and glutathione, catalase activity, or glutathione
peroxidase activity. The data here showed that ascorbate initiated
H2O2 formation extracellularly, but H2O2 targets could be either
intracellular or extracellular, because H2O2 is membrane permeant(38, 52). For example, extracellular H2O2 might target membrane
lipids, forming hydroperoxides or reactive intermediates that arequenched or repaired in normal cells but not in sensitive cancer
cells. In sensitive but not resistant cancer cells, intracellular H2O2
could target DNA, DNA repair proteins, or mitochondria becauseof diminished superoxide dismutase activity (53). New insights may
follow from future studies of a very broad range of tumor cells orfrom microarray analysis of resistant and sensitive cells derived
from the same genetic lineage.
H2O2, as the product of pharmacologic ascorbate concentrations,has potential therapeutic uses in addition to cancer treatment,
especially in infections. H2O2 is a potent mammalian antimicrobial
defense mechanism (54). Neutrophils generate H2O2 from superoxide, in turn formed by NADPH oxidase-catalyzed reduction ofmolecular oxygen. There may be particular therapeutic applicationin patients with chronic granulomatous disease who have diminished superoxide production (55). Old observational animal experiments, although uncontrolled, suggest that i.v. ascorbate is effective
in some viral infections (56, 57). This finding is also consistent within vitro experiments, in which H2O2 is toxic to hepatitis C (58). Useof ascorbate as an H2O2-delivery system against sensitive pathogens, viral or bacterial, has substantial clinical implications thatdeserve rapid exploration.
To proceed clinically in potential treatment of infectious diseases
and cancer, clear safety documentation of i.v. ascorbate administration is necessary. More than 100 patients have been described,
presumably without glucose-6-phosphate dehydrogenase deficiency, who received 10 g or more of i.v. ascorbate with no reported
adverse effects other than tumor lysis (3, 4, 15, 59). However, thesedescriptions lack formal safety documentation. Complementary
and alternative medicine practitioners worldwide currently use
ascorbate i.v. in doses as high as 70 g over several hours (14, 15, 59).
Because i.v. ascorbate is easily available to people who seek it, aphase I safety trial in patients with advanced cancer is justified andunderway.

1. Padayatty, S. J. & Levine, M. (2000) J. Am. Coll. Nutr. 19, 423–425.
2. Cameron, E. & Campbell, A. (1974) Chem. Biol. Interact. 9, 285–315.
3. Cameron, E. & Pauling, L. (1976) Proc. Natl. Acad. Sci. USA 73, 3685–3689.
4. Cameron, E. & Pauling, L. (1978) Proc. Natl. Acad. Sci. USA 75, 4538–4542.
5. Creagan, E. T., Moertel, C. G., O’Fallon, J. R., Schutt, A. J., O’Connell, M. J., Rubin, J. &Frytak, S. (1979) N. Engl. J. Med. 301, 687–690.
6. Moertel, C. G., Fleming, T. R., Creagan, E. T., Rubin, J., O’Connell, M. J. & Ames, M. M.
(1985) N. Engl. J. Med. 312, 137–141.
7. Padayatty, S. J., Sun, H., Wang, Y., Riordan, H. D., Hewitt, S. M., Katz, A., Wesley, R. A.
& Levine, M. (2004) Ann. Intern. Med. 140, 533–537.
8. Levine, M., Conry-Cantilena, C., Wang, Y., Welch, R. W., Washko, P. W., Dhariwal, K. R., Park,J. B., Lazarev, A., Graumlich, J., King, J., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 3704–3709.
9. Levine, M., Wang, Y., Padayatty, S. J. & Morrow, J. (2001) Proc. Natl. Acad. Sci. USA 98,
9842–9846.
10. Bram, S., Froussard, P., Guichard, M., Jasmin, C., Augery, Y., Sinoussi-Barre, F. & Wray,
W. (1980) Nature 284, 629–631.
11. Leung, P. Y., Miyashita, K., Young, M. & Tsao, C. S. (1993) Anticancer Res. 13, 475–480.
12. Sakagami, H., Satoh, K., Hakeda, Y. & Kumegawa, M. (2000) Cell. Mol. Biol. 46, 129–143.
13. Levine, M., Rumsey, S. C., Daruwala, R. C., Park, J. B. & Wang, Y. (1999) J. Am. Med. Assoc.
281, 1415–1423.
14. Riordan, N. H., Riordan, H. D. & Casciari, J. J. (2000) J. Orthomol. Med. 15, 201–203.
15. Riordan, H. D., Hunninghake, R. B., Riordan, N. H., Jackson, J. J., Meng, X., Taylor, P.,
Casciari, J. J., Gonzalez, M. J., Miranda-Massari, J. R., Mora, E. M., et al. (2003) P. R. HealthSci. J. 22, 287–290.
16. Lee, Y. & Shacter, E. (1997) J. Clin. Invest. 89, 4480–4492.
17. Bergsten, P., Amitai, G., Kehrl, J., Dhariwal, K. R., Klein, H. G. & Levine, M. (1990) J. Biol.
Chem. 265, 2584–2587.
18. Washko, P. W., Wang, Y. & Levine, M. (1993) J. Biol. Chem. 268, 15531–15535.
19. Lee, Y. J. & Shacter, E. (1999) J. Biol. Chem. 274, 19792–19798.
20. Heeg, K., Reimann, J., Kabelitz, D., Hardt, C. & Wagner, H. (1985) J. Immunol. Methods
77, 237–246.
21. Leone, A., Flatow, U., VanHoutte, K. & Steeg, P. S. (1993) Oncogene 8, 2325–2333.
22. Zhou, M., Diwu, Z., Panchuk-Voloshina, N. & Haugland, R. P. (1997) Anal. Biochem. 253,
162–168.
23. Buettner, G. R. (1990) Free Radical Res. Commun. 10, 5–9.
24. Buettner, G. R. & Kiminyo, K. P. (1992) J. Biochem. Biophys. Methods 24, 147–151.
25. Long, L. H., Evans, P. J. & Halliwell, B. (1999) Biochem. Biophys. Res. Commun. 262, 605–609.
26. Washko, P. W., Hartzell, W. O. & Levine, M. (1989) Anal. Biochem. 181, 276–282.
27. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D.,
Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, 76–85.
28. Englert, R. P. & Shacter, E. (2002) J. Biol. Chem. 277, 20518–20526.
29. Corpe, C. P., Lee, J. H., Kwon, O., Eck, P., Narayanan, J., Kirk, K. L. & Levine, M. (2005)
J. Biol. Chem. 280, 5211–5220.
30. Day, B. J., Fridovich, I. & Crapo, J. D. (1997) Arch. Biochem. Biophys. 347, 256–262.

31. Burns, J. A., Butler, J. C., Moran, J. & Whitesides, G. M. (1991) J. Org. Chem. 56, 2648–2650.
32. Buettner, G. R. (1986) Free Radical Res. Commun. 1, 349–353.
33. L’Eplattenier, F., Murase, I. & Martell, A. E. (1967) J. Am. Chem. Soc. 89, 837–843.
34. Halliwell, B. (1990) Free Radical Res. Commun. 9, 1–32.
35. Samuni, A. M., Afeworki, M., Stein, W., Yordanov, A. T., DeGraff, W., Krishna, M. C.,
Mitchell, J. B. & Brechbiel, M. W. (2001) Free Radical Biol. Med 30, 170–177.
36. Fridovich, I. (1995) Annu. Rev. Biochem. 64, 97–112.
37. Wang, X., Liu, J., Yokoi, I., Kohno, M. & Mori, A. (1992) Free Radical Biol. Med. 12, 121–126.
38. Chance, B., Sies, H. & Boveris, A. (1979) Physiol. Rev. 59, 527–605.
39. Brown, J. M., Grosso, M. A., Terada, L. S., Beehler, C. J., Toth, K. M., Whitman, G. J.,
Harken, A. H. & Repine, J. E. (1989) Am. J. Physiol. 256, H584–H588.
40. Guemouri, L., Artur, Y., Herbeth, B., Jeandel, C., Cuny, G. & Siest, G. (1991) Clin. Chem.
37, 1932–1937.
41. Motoyama, S., Saito, S., Inaba, H., Kitamura, M., Minamiya, Y., Suzuki, H., Saito, R.,
Kamata, S., Nakae, H. & Ogawa, J. (2000) Liver 20, 200–208.
42. Frossard, M., Blank, D., Joukhadar, C., Bayegan, K., Schmid, R., Luger, A. & Muller, M.
(2005) Diabet. Med. 22, 56–60.
43. Han, S. S., Kim, K., Hahm, E. R., Lee, S. J., Surh, Y. J., Park, H. K., Kim, W. S., Jung, C. W.,
Lee, M. H., Park, K., et al. (2004) J. Cell. Biochem. 93, 257–270.
44. Park, S., Han, S. S., Park, C. H., Hahm, E. R., Lee, S. J., Park, H. K., Lee, S. H., Kim, W. S.,
Jung, C. W., Park, K., et al. (2004) Int. J. Biochem. Cell Biol. 36, 2180–2195.
45. Koh, W. S., Lee, S. J., Lee, H., Park, C., Park, M. H., Kim, W. S., Yoon, S. S., Park, K., Hong,
S. I., Chung, M. H., et al. (1998) Anticancer Res. 18, 2487–2493.
46. Calderon, P. B., Cadrobbi, J., Marques, C., Hong-Ngoc, N., Jamison, J. M., Gilloteaux, J.,
Summers, J. L. & Taper, H. S. (2002) Curr. Med. Chem. 9, 2271–2285.
47. Arakawa, N., Nemoto, S., Suzuki, E. & Otsuka, M. (1994) J. Nutr. Sci. Vitaminol. (Tokyo)
40, 219–227.
48. Sestili, P., Brandi, G., Brambilla, L., Cattabeni, F. & Cantoni, O. (1996) J. Pharmacol. Exp.
Ther. 277, 1719–1725.
49. Clement, M. V., Ramalingam, J., Long, L. H. & Halliwell, B. (2001) Antioxid. Redox. Signal.
3, 157–163.
50. Weinberger, A. & Simkin, P. A. (1989) Semin. Arthritis Rheum. 19, 66–76.
51. Buettner, G. R. & Jurkiewicz, B. A. (1996) Radiat. Res. 145, 532–541.
52. Antunes, F. & Cadenas, E. (2000) FEBS Lett. 475, 121–126.
53. Oberley, L. W. (2001) Antioxid. Redox. Signal. 3, 461–472.
54. Babior, B. M. (2000) Am. J. Med. 109, 33–44.
55. Heyworth, P. G., Cross, A. R. & Curnutte, J. T. (2003) Curr. Opin. Immunol. 15, 578–584.
56. Edwards, W. C. (1968) Vet. Med. Small. Anim. Clin. 63, 696–698.
57. Leveque, J. I. (1969) Vet. Med. Small. Anim. Clin. 64, 997–999.
58. Choi, J., Lee, K. J., Zheng, Y., Yamaga, A. K., Lai, M. M. & Ou, J. H. (2004) Hepatology
39, 81–89.
59. Riordan, N. H., Riordan, H. D., Meng, X., Li, Y. & Jackson, J. A. (1995) Med. Hypotheses
44, 207–213.

Chen et al.

PNAS 兩 September 20, 2005 兩 vol. 102 兩 no. 38 兩 13609

MEDICAL SCIENCES

This work was supported in part by the Intramural Research Programsof the National Institute of Diabetes and Digestive and Kidney Diseases
and the National Cancer Institute (National Institutes of Health Grant
Z01 DK 54506).