Albert Szent-Györgyi: Vitamin C identification

Article (PDF Available)inBiochemical Journal 400(3) · October 2006with 1,524 Reads 
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
DOI: 10.1042/BJ2006c005
Cite this publication
Abstract
Today, our nutritional need for vitamin C (ascorbic acid) is well known and understood by the general public. As a dietary supplement, it is the vitamin with the largest commercial volume; some 108 kg (>US$600 million in the global market) are sold each year as tablets, a component of multivitamin products and an addition to many foods and drinks with the intention of promoting health. However, it can also be added to products simply to enhance the sales appeal. It can appear in unexpected places, such as chewing gum and sweets, personal care products and even in pet and animal feed (ascorbic acid is not considered to be a vitamin for animals other than humans). The demand for vitamin C is growing fast and new production facilities that are coming online in China are restructuring the manufacturing and supply of this product.
Biochemical Journal classic papers
special feature
The isolation and identification of ascorbic acid as
vitamin C was one of the most important advances
that lead to improving human nutrition in the 20th
Century. The delicate and seemingly fickle nature
of this versatile reducing agent was one of the
reasons that its isolation and the subsequent iden-
tification of its chemical nature presented such a
major challenge. Professor Albert Szent-Györgyi
was a key contributor to all aspects of the research
that lead to the identification of vitamin C in the
1920s and 1930s. The Biochemical Journal pub-
lished four key papers
1–4
from his laboratory, and
these provided a wide range of insights into the
basic chemistry of ascorbic acid and its identity as
vitamin C, as well as the large-scale isolation of
vitamin C from Hungarian red peppers
4
.
By 1928, it was known that the adrenal cortex
was somehow connected to biological oxidations;
however, the nature of this connection could not
be found. Szent-Györgyi found this connection
by studying the juice of turnips, the roots of which
contain significant quantities of the enzyme per-
oxidase. Peroxidase reduces hydrogen peroxide to
water and in turn oxidizes an appropriate sub-
strate. He demonstrated that there was a previ-
ously unrecognized reducing factor in the juice
and that it was the preferred reducing agent for the
enzyme. Other substrates would not be oxidized
until this reducing factor was exhausted. He went
on to demonstrate that a reducing factor that
behaved in a similar fashion was present in onion,
leek, cabbage, orange, lemon, grapefruit and apple.
Szent-Györgyi then demonstrated that the
The Biochemist — October 2006.
©
2006 Biochemical Society
31
Albert Szent-Györgyi:
Today, our nutritional need for vitamin C (ascorbic acid) is well known and understood by the
general public. As a dietary supplement, it is the vitamin with the largest commercial volume;
some 10
8
kg (>US$600 million in the global market) are sold each year as tablets, a component
of multivitamin products and an addition to many foods and drinks with the intention of
promoting health. However, it can also be added to products simply to enhance the sales appeal.
It can appear in unexpected places, such as chewing gum and sweets, personal care products and
even in pet and animal feed (ascorbic acid is not considered to be a vitamin for animals other
than humans). The demand for vitamin C is growing fast and new production facilities that are
coming online in China are restructuring the manufacturing and supply of this product.
Garry R. Buettner and
Freya Q. Schafer
(University of Iowa, USA)
vitamin C identification
Professor Albert Szent-Györgyi.
Reproduced with the permission of
the Nobel Foundation.
adrenal gland has a high level of a reducing factor
with these same properties. He devised a method
to isolate this substance and, using kilogram
quantities of gland, was able to obtain relatively
pure crystals of the ‘reducing factor’ (approxi-
mately 300 mg/kg of gland). He was the able to
begin the characterization of this substance. In his
“Discovery consists of seeing what
everybody has seen and thinking
what nobody has thought”.
for a test of its antiscorbutic (anti-scruvy) proper-
ties using guinea pigs. He found that hexuronic acid
(1 mg/day) provided complete protection against
scurvy and, indeed, that hexuronic acid is vitamin
C
2,3
. He shared this large supply with all researchers
working on vitamin C, including the leading carbo-
hydrate chemist of that era, Walter Norman
Haworth at the University of Birmingham. In
Haworth’s laboratory the definitive determination
of this substance’s structure was accomplished. To
confirm the structure, the compound was also syn-
thesized; this was the first artificial synthesis of a
vitamin. It is interesting to note that the last line of
the Banga and Szent-Györgyi paper
4
“No patents
were taken out for the process.”
With this large supply it was then possible to
obtain a very pure, single-component preparation
of hexuronic acid. This allowed a definitive animal
test to be undertaken and indeed the antiscorbutic
properties were found to be due to hexuronic acid,
not to any possible contaminant in the preparation
from adrenal glands.
With the definitive structure and the solid evidence
that hexuronic acid was vitamin C, Szent-Györgyi
and Haworth re-named it “a-scorbic” acid, because it
prevented scorbutus (scurvy).
The 1937 Nobel committee honoured discoveries
about vitamins. The Nobel Prize for Physiology or
Medicine was awarded to Szent-Györgyi “for his
discoveries in connection with the biological com-
bustion processes, with especial reference to vitamin
C and the catalysis of fumaric acid.” Norman
Haworth and Paul Karrer, shared that year’s prize in
chemistry. Haworth was awarded for his work on
carbohydrates because “he has, above all, made clear
the chemical structure of vitamin C” and Karrer “for
brilliant investigations on carotenoids and flavins, as
well as on vitamins A and B
2
”.
Many laboratories at that time worked on the
isolation and identification of vitamin C, but it is
first paper in The Biochemical Journal
1
he reported
the following properties of this reducing factor:
1. It is highly oxidizable.
2. The oxidation is catalysed by OH
as well as
metals such as iron and copper.
3. It has an acidic hydrogen atom and an equiva-
lent mass of 178.
4. Its molecular mass is 178±3;
5. It appeared to be a lactone with the chemical
formula C
6
H
8
O
6
and was given the name
hexuronic acid*.
6. Hexuronic acid could be isolated from plants
(e.g. oranges and cabbage), linking the observa-
tions on the reducing factor in plants and ani-
mals.
7. It is oxidized both reversibly and irreversibly.
8. It provides two hydrogen atoms (two equiva-
lents) as a reversible reducing agent.
9. The reversible oxidation product of hexuronic
acid is reduced by glutathione and other thiols.
10.The reversible oxidation product is ‘intensely’
reduced by animal tissues.
This paper
1
laid the foundation for much of the
chemistry and biochemistry of vitamin C that we
know today.
Szent-Györgyi had only a limited supply of
hexuronic acid, as the isolation from adrenal glands
was arduous and produced only milligram amounts
of the substance. Nonetheless, enough was available
The Biochemist — October 2006.
©
2006 Biochemical Society
32
Biochemical Journal classic papers
special feature
The structure of ascorbic acid.
The recycling of vitamin C as discovered by Professor Szent-Györgyi. One of the many important discoveries
presented in the 1928 paper
1
was that the reversible oxidation product of ascorbate could be reduced by
glutathione. This concept was new and could explain how so little of the vitamin could do so much in an organism.
*This work was done at Cambridge upon the invitation Sir Frederick
Gowland Hopkins, the eminent British biochemist. It was Hopkins who
urged him to publish these results in the
Biochemical Journal
. This
required that the substance be named. Szent-Györgyi jokingly suggested
calling it “Ignose” — from ‘ignosco’ (I don’t know) and ‘-ose’ to
indicate a sugar. However, the editor rejected this suggestion as well as
Szent-Györgyi’s second choice, ‘Godnose’. The editor proposed ‘hexuronic
acid’ (using ‘hex’ to indicate the six carbon atoms). The University of
Cambridge awarded Szent-Györgyi a PhD in biochemistry at the end of
1927 for this work.
Biochemical Journal classic papers
special feature
clear that the first isolation and characterization of
ascorbic acid during Szent-Györgyi’s doctoral
research paved the way for much of the progress
made by the research community. His large-scale
isolation of the compound and his sharing of it with
other researchers was an incredible catalyst for fast
progress in the understanding of this fickle vitamin.
Some 75 years later, vitamin C still offers many
challenges to researchers. We still struggle with its
instability, are learning the details of its biochemi-
cal functions, are dealing with controversy because
we do not understand fully its pro- and anti-oxi-
dant actions, as well as all of its biochemical func-
tions and are learning new aspects of its potential
medical applications.
It will continue to be a subject of controversy and
research for many years to come and many of
its mysteries remain to be uncovered. These classic
papers in the Biochemical Journal
1–4
provided the
foundation for the research on vitamin C in the
1920s and 1930s
5–7
, as well as for continuing work
in the modern era
8–13
.
The Biochemist — October 2006.
©
2006 Biochemical Society
33
1. Szent-Györgyi, A. (1928) CLXXIII. Observations on the function of peroxidase systems and the chemistry
of the adrenal cortex. Description of a new carbohydrate derivative. Biochem. J. 22, 1387–1409
2. Svirbely, J.L. and Szent-Györgyi,A. (1932) CV. The chemical nature of vitamin C. Biochem. J. 26,865–870
3. Svirbely, J.L. and Szent-Györgyi,A. (1933) XL. The chemical nature of vitamin C. Biochem. J. 27,279–285
4. Banga, I. and Szent-Györgyi, A. (1934) CCXIV. The large scale preparation of ascorbic acid from
Hungarian pepper. Biochem J
.
28, 1625–1628
5. Haworth,W.H., Hirst, E.L. and Reynolds R.J.W. (1932) Letters to the editor on: hexuroinc acid as the
antiscorbutic factor. Nature (London) 129,576–577
6. Haworth,W.H. and Hirst, E.L. (1933) Synthesis of ascorbic acid. Chem. Ind. (London) 52, 645–647
7. Szent-Györgyi, A. and Haworth,W.H. (1933) Hexuronic acid (ascorbic acid) as the antiscorbutic factor.
Nature (London) 131,24
8. Anon. (1988) The identification of vitamin C, an historical summary. J. Nutr. 118, 1290–1293
9. Buettner, G.R. (1988) In the absence of catalytic metals, ascorbate does not autoxidize at pH 7:
ascorbate as a test for catalytic metals. J. Biochem. Biophys. Meth
.
16, 20–40
10. Asard, H., May, J.M. and Smirnoff, N. (eds) (2004) Vitamin C: Function and Biochemistry in Animals and
Plants
.
BIOS Scientific Publishers, London.
11. Packer, L. and Fuchs, J. (1997) Vitamin C in Health and Disease. Marcel Dekker, New York
12. Davis, M.B., Austin, J. and Partridge, D.A. (1991) Vitamin C: its Chemistry and Biochemistry. The Royal
Society of Chemistry, Cambridge
13. Chen, Q., Espey, M.G., Krishna, M.C. et al. (2005) Pharmacologic ascorbic acid concentrations selectively kills cancer
cells: action to deliver hydrogen peroxide to tissues. Proc. Natl.Acad. Sci. USA 102, 13604-–3609
References
Garry R. Buettner is a Professor in the
Free Radical and Radiation Biology
Program in the Department of
Radiation Oncology at The University
of Iowa. His interest in vitamin C
began when he was a postdoctoral fel-
low at The University of Iowa under
the supervision of Dr Larry Oberley.
Research on ascorbate has been a
component of his research pro-
gramme for over two decades.After a
brief period of teaching chemistry at
Wabash College, Crawfordsville, IN,
he became a Senior Fellow at the NIEHS and a Fulbright Scholar at the GSF
Research Institute in Munich, Germany. He joined The University of Iowa in
1988 where he set up the Electron Spin Resonance Core Facility. His
research has focused on basic mechanisms in free radical biology.
email: garry-buettner@uiowa.edu
Websites that have excellent further reading on the life and career
of Professor Albert Szent-Györgyi and vitamin C
The Nobel Prize website:http://nobelprize.org/medicine/laureates/1937/
The National Institutes of Health profile of Professor Szent-Györgyi:http://profiles.nlm.nih.gov/WG/
An excellent overview of the historical aspects of vitamin C:http://www.cambridge.org/us/books/kiple/vitaminc.htm
Freya Q. Schafer is a Research
Scientist in the Free Radical and
Radiation Biology Program at The
University of Iowa. She received
her degree in human biology in 1993
at the University of Ulm in Germany.
She became involved in free radical
biology during her postdoctoral
studies at New Jersey Medical
College and the University of Maine.
Her interest in ascorbate is in
understanding its role as a biological
reducing agent, with respect to its
antioxidant action as well as the pro-oxidant actions of ascorbate.
Her interests range from fundamental aspects of redox biology to the
teaching of science and career development.
email: freya-schafer@uiowa.edu
The Albert Lasker Award 1954
presented by the American Heart
Association to Albert Szent-Györgyi.
  • ... GSH is the most abundant low molecular thiol in plants, discovered in 1926 (Hunter and Eagles 1926), and later associated with powerful antioxidant functions against lipid peroxides and ROS in general. AsA, in turn, was discovered by Szent-Gyorgyi by the same time (Buettner and Schafer 2006) and later identified as vitamin C. It is considered the terminal small molecule antioxidant in biological systems (Sharma and Buettner 1993), acting as a natural reductant of free radical species. ...
    Chapter
    Full-text available
    Plants confront fluctuating and in some cases intense environmental conditions, such as changes in irradiation, water availability, extreme temperatures, mineral nutrient accessibility, and air pollutants exposition among others. In order to face abiotic stress situations, the redox buffer capacity, mainly represented by ascorbic acid (AsA) and glutathione (GSH) pools, is involved in growth–stress responses crossroad. These compounds are associated in a set of reactions known as AsA-GSH cycle. The main function of the AsA-GSH cycle originally observed was the detoxification of reactive oxygen species (ROS) in different subcellular compartments such as chloroplast, mitochondria, or cytosol. More recently, the crucial participation of the AsA-GSH cycle in the optimization of photosynthesis was established. In addition, these antioxidants are considered essential components of cell signaling pathways triggering adaptive plant responses. The role of AsA-GSH cycle is analyzed regarding the ability of plants to overcome some selected abiotic stress situations.
  • ... Since the discovery of ascorbic acid (ASA), the number of its known biological and physiological functions is constantly expanding. All known functions of ASA are due to its action as an electron donor, in which the ability to donate one or two electrons makes it an excellent water-soluble reducing agent and donor antioxidant (Buettner and Schafer, 2006). The literature implicating ASA in the prevention of chronic and neurodegenerative diseases as AD remains controversial (Bowman, 2012). ...
    Article
    Full-text available
    Few studies have been carried out to assess the neurotoxic effect of aluminum (Al) on the aquatic creatures. This study aims to evaluate the neurotoxic effects of long term Al exposure on the Nile catfish (Clarias gariepinus) and the potential ameliorative influence of ascorbic acid (ASA) over a 180 days exposure period. Forty eight Nile catfish were divided into four groups: control group, placed in clean water, ASA exposed group (5mg/L), AlCl3 received group (28.96μg/L; 1/20 LC50), and group received AlCl3 concomitantly with ASA. Brain tissue was examined by using flow cytometry to monitor the apoptotic cell population, HPLC analysis for the quantitative estimation of brain monoamine neurotransmitters [Serotonin (5-HT), Dopamine (DA), Norepinephrine (NE)]. The amino acid neurotransmitters [serum Taurine, Glycine, Aspartate and Glutamine and brain gamma aminobutyric acid (GABA)] levels were assessed, plus changes in brain tissue structure using light microscopy. The concentration of Al in both brain tissue and serum was determined by using atomic absorption spectrophotometery. The Al content in serum and brain tissue were both elevated and Al exposure induced an increase in the number of apoptotic cells, a marked reduction of the monoamine and amino acids neurotransmitters levels and changes in tissue morphology. ASA supplementation partially abolished the effects of AL on the reduced neurotransmitter, the degree of apoptosis and restored the morphological changes to the brain. Overall, our results indicate that, ASA is a promising neuroprotective agent against for Al- induced neurotoxicity in the Nile catfish.
  • ... In the 80 years since the discovery of vitamin C (ascorbic acid, AscH 2 ; ascorbate, AscH − ) [1,2], the number of its known biological functions is continually expanding. Because of the ease of oxidation of ascorbate, gaining the first understanding of its role as the antiscorbutic vitamin was a major challenge. ...
    Article
    Since the discovery of vitamin C, the number of its known biological functions is continually expanding. Both the names ascorbic acid and vitamin C reflect its antiscorbutic properties due to its role in the synthesis of collagen in connective tissues. Ascorbate acts as an electron-donor keeping iron in the ferrous state thereby maintaining the full activity of collagen hydroxylases; parallel reactions with a variety of dioxygenases affect the expression of a wide array of genes, for example via the HIF system, as well as via the epigenetic landscape of cells and tissues. In fact, all known physiological and biochemical functions of ascorbate are due to its action as an electron donor. The ability to donate one or two electrons makes AscH(-) an excellent reducing agent and antioxidant. Ascorbate readily undergoes pH-dependent autoxidation producing hydrogen peroxide (H(2)O(2)). In the presence of catalytic metals this oxidation is accelerated. In this review, we show that the chemical and biochemical nature of ascorbate contribute to its antioxidant as well as its prooxidant properties. Recent pharmacokinetic data indicate that intravenous (i.v.) administration of ascorbate bypasses the tight control of the gut producing highly elevated plasma levels; ascorbate at very high levels can act as prodrug to deliver a significant flux of H(2)O(2) to tumors. This new knowledge has rekindled interest and spurred new research into the clinical potential of pharmacological ascorbate. Knowledge and understanding of the mechanisms of action of pharmacological ascorbate bring a rationale to its use to treat disease especially the use of i.v. delivery of pharmacological ascorbate as an adjuvant in the treatment of cancer.
  • Article
    Full-text available
    Background To measure ascorbic acid concentration in aqueous humor of patients with cataract after oral or intravenous vitamin C supplementation. Methods Forty-two eyes of 42 patients with senile cataract who underwent uncomplicated cataract surgery were enrolled. Patients (n = 14 each) were administered oral vitamin C (2 g), intravenous vitamin C (20 g) or no treatment (control group) on the day before surgery. Samples of aqueous humor (0.1 cm³) were obtained by anterior chamber aspiration at the beginning of surgery and stored at −80 °C. Ascorbic acid concentration in aqueous humor was measured by high-pressure liquid chromatography. Results The mean age at surgery was 62.5 years, with no difference among the three groups. The mean ± standard deviation concentrations of ascorbic acid in aqueous humor in the control and oral and intravenous vitamin C groups were 1347 ± 331 μmol/L, 1859 ± 408 μmol/L and 2387 ± 445 μmol/L, respectively. Ascorbic acid concentration was significantly lower in the control than in the oral (P < 0.01) and intravenous (P < 0.001) vitamin C groups and was significantly higher in the intravenous than in the oral vitamin C group (P < 0.05). Conclusions Ascorbic acid concentration in aqueous humor is increased by systemic vitamin C supplementation, with intravenous administration being more effective than oral administration.
  • Article
    Full-text available
    Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.
  • Article
    Szent-Gyorgyi isolated hexuronic acid in 1927, but it was not identified as vitamin C until 1932. For that matter, lactochrome (riboflavin was isolated from milk by Blyth in 1879, β-carotene was isolated from carrots by Willstatter and Leder in 1910, nicotinic acid was isolated from yeast by Funk in 1913 and phthiocol was isolated by Anderson from tubercle bacilli prior to 1939. None of these authors gets credit for discovering a vitamin. There is a very good reason for this: a substance must be biologically tested before it becomes recognized as a vitamin. Hexuronic acid was tested by Svirbely, who carried the idea to Szent-Gyorgyi as recorded by Szent-Gyorgyi. Svirbely got the idea, and the instruction and experience in carrying out the test, from C. G. King and brought them to Szent-Gyorgyi. King followed a separate pathway to the identification of vitamin C. He used lemon juice. Like Szenty-Gyorgyi, he was led astray on hexuronic acid by Zilva. King and his group clearly isolated vitamin C from lemon juice and assayed their fractions at every step. In 1931, Smith and King noted the similarity of vitamin C to hexuronic acid 'isolated by Szent-Gyorgyi'. The letter of March 15 from King to Svirbely constitutes the only tangible evidence for Moss's claim that King on March 15, was still confused as to the identity of vitamin C. (The rest of Moss's case rests mainly on querulous, anecdotal complaints by Szent-Gyorgyi.) Moss alleges that King's statement of 1953 'is clearly wrong. By 15 March King still had a great deal of work to do on it'. This allogation is based on King's statement in the letter that 'the product appears to be identical with S-G's product, but further chemical work will have to be done before one can be sure', which is in accordance with conventional biochemical criteria requiring synthesis as ultimate proof of identity. The identification of vitamin C is one of the strangest episodes in the history of vitamins. Synthesis of vitamin C followed promptly as announced by Haworth, Hirst and their collaborators and by Reichstein, Grussner and Oppenauer. The advance of knowledge, the availability of synthetic vitamin C and improvements in food supply brought the threat of scurvy to an end. The dispute concerning isolation of vitamin C was discussed many years ago, including the summary in 1937 by G.J. Cox. The dispute has now been inaccurately redescribed by Moss, and as a result, King has been accused of 'shamelessly jumping the gun' in sending the note to Science published April 1, 1932. Moss claims that a letter to Svirbely by King on March 15, shows that King was 'still holding up a note to Science' and that 'it is hard to avoid the conclusion that King received Svirbely's letter, realized that the time for hesitation has passed and quickly sent off his own contribution to Science'. The evidence is that King prepared his note for Science long before he submitted it and that he held it up until he could resolve Rygh's claims. Why should Moss object to King's 'sending off his own contribution to Science' at any time? It was King's right to do so whenever he chose. He had been writing and publishing on the vitamin C problem for several years starting in 1927, and he had trained Svirbely (who had unexpectedly jumped ship to Szent-Gyorgyi's lab, possibly with the purpose of 'scooping' King on the identification of vitamin C). Waugh had crystallized vitamin C from lemon juice, and it was King's privilege to send his results of the preceding several months to Science either before or after receiving the letter from Svirbely. It seems that King's statement that he had submitted his paper to the ASBC a few weeks before he heard of Svirbely's results is incorrect. But it seems certain that King's manuscript to Science had been prepared before King heard from Svirbely about the manuscript to Nature. It should be remembered that King stated in Science that vitamin C from lemon juice 'is apparently identical with the hexuronic acid described by Szent-Gyorgyi', thus making it obvious that King wished to give Szent-Gyorgyi credit. In contrast, Szent-Gyorgyi and Svirbely consistently avoided mentioning the work in King's lab, or that Svirbely had been trained there and that he had taken the vitamin C problem to Szent-Gyorgyi's lab. In the note to Nature, Svirbely did not list his 1931 publication in the references. From Moss's antipathy toward King, one would think that King, rather than Szent-Gyorgyi, had collared the Nobel prize. King's own accounts of the incidents were published in 1953 and 1979. Ironically, in the New York Times Review of Books, a reviewer, G. Johnson, states that 'As [Moss] studied Szent-Gyorgyi's past, he learned that the scientist tended to make grossly exaggerated claims about his research. 'He was an amoral person'.' Yet Moss has relied extensively on Szent-Gyorgyi in his account of the identification of vitamin C.
  • Article
    Trace amounts of adventitious transition metals in buffer solutions can serve as catalysts for many oxidative processes. To fully understand what role these metals may play it is necessary that buffer solutions be 'catalytic metal free'. We demonstrate here that ascorbate can be used in a quick and easy test to determine if near-neutral buffer solutions are indeed 'catalytic metal free'. In buffers which have been rendered free of catalytic metals we have found that ascorbate is quite stable, even at pH 7. The first-order rate constant for the loss of ascorbate in an air-saturated catalytic metal free solution is less than 6 X 10(-7) s-1 at pH 7.0. This upper limit appears to be set by the inability to completely eliminate catalytic metal contamination of solutions and glassware. We conclude that in the absence of catalytic metals, ascorbate is stable at pH 7.
  • Article
    Full-text available
    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, to determine 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 EC(50) values of <4 mM, a concentration easily achievable i.v. Human lymphoma cells were studied in detail because of their sensitivity to ascorbate (EC(50) of 0.5 mM) and suitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis/necrosis. Cell death was independent of metal chelators and absolutely dependent on H(2)O(2) formation. Cell death from H(2)O(2) added to cells was identical to that found when H(2)O(2) was generated by ascorbate treatment. H(2)O(2) generation was dependent on ascorbate concentration, incubation time, and the presence of 0.5-10% serum, and displayed a linear relationship with ascorbate radical formation. Although ascorbate addition to medium generated H(2)O(2), ascorbate addition to blood generated no detectable H(2)O(2) 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 for formation of H(2)O(2), 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 for treatment of infections where H(2)O(2) may be beneficial.